WO1998004918A1 - Detection of microbacteria - Google Patents

Abstract

The present invention is directed to methods and compositions for the detection of infection and disease due to members of the genus Mycobacterium. In particular, the present invention is well-suited to the detection and identification of patients with disease or infection due to M. tuberculosis or MAC.

Description

  
 



   DETECTION OF MYCOBACTERIA
FIELD OF THE INVENTION
 The present invention relates to the detection and diagnosis of infection and disease due to the mycobacteria. especially   Mycobacteriun?      tuberculo.si.s    and other mycobacteria commonly associated with disease in immunocompromised patients including those with acquired immunodeficiency syndrome (AIDS). The present invention is suited for rapid screening of large populations for the presence of M. tuberculosis carriers. as well as diagnosing and monitoring disease or infection in patients who present at healthcare or public health facilities.



  BACKGROUND OF THE INVENTION
 Organisms within the genus Mycobacterium include obligate parasites. saprophytes.



  and opportunistic pathogens. Most species are free-living in soil and water but for species such as M.   tuberctllosis    and   M.      Ieprae,    the causative agents of tuberculosis and leprosy respectively, the major ecological niche is the tissue of humans and other warm-blooded animals.



   Despite the fact that most mycobacteria do not cause disease. a relatively small group of organisms within the genus is responsible for a large percentage of human morbidity and mortality worldwide. Tuberculosis remains a major global health problem, with nearly one third of the world's population infected. Indeed. tuberculosis is the leading cause of death due to a single infectious agent. In addition. the World Health Organization estimates that worldwide, there are 8-10 million new cases and over 3 million deaths directly attributed to this disease reported worldwide (A. Kochi, The global tuberculosis situation and the new control strategy of the World Health Organization. Tubercle 72:1-6   [1991]).   



   M. tuberculosis is exceptionally easily transmitted. as it is carried in airborne particles termed "droplet nuclei," produced when a patient with active tuberculosis coughs. These particles are from 1-5   11    in size, and are readily suspended in air currents. Infection occurs when droplet nuclei are inhaled and reach the terminal airways of the new host's lungs.



  Usually, the host immune response limits the multiplication and spread of the organism.



  although some organisms may remain dormant. but viable, for many years post-infection.



  Individuals infected with M. tuberculosis but without disease, usually have a positive skin test   (i.e.. with purified protein derivative [PPD]), but are asymptomatic and generally not infectious. However. latently infected individuals have a   10%    risk for developing active tuberculosis at some point during their life: the risk is greatest within the first two years postinfection. For   HlV-positive    individuals, the risk is much greater. with the risk at   1()-15%    per year for progression to active disease (F.S. Nolte and B. Metchock. "Mycobacterium." in
Manual of Clinical   Microbioloy,    Sixth Edition, ASM Press: Washington, [1995],pp. 400437).



   Co-infection with human immunodeficiency virus (HIV) and M.   tuberculosis    has resulted in staggering increases in tuberculosis rates - as much as 200% in the past 4 years.



  particularly in impoverished countries with few rcsources available to control this epidemic.



  Yet even western industrialized countries have reported increases in tuberculosis rates of from   2    to 14% per year during the past decade (World Health Organization TB Programme. quotcd in "TB: A Global Emergency," WHO. 1994). These increases. coupled with the   emergence    of multi-drug-resistant strains. and the recognition that tuberculosis is one   HlV-related    opportunistic   infcction    which can be readily transmitted to HIV-uninfected persons, have focused the attention of phvsicians. researchers, and public health workers on issues related to tuberculosis control, particularly in terms of development of improved vaccines for tuberculosis prevention and improved tests for tuberculosis diagnosis.



   In the United States, aggressive approaches to tuberculosis control including isolation of patients in facilities such as sanitoria and the development of drugs   effective    against   At      tuberculosis    resulted in a steady decline in the incidence of tuberculosis until about   1985.   



  when the trend reversed and the reports of new tuberculosis cases began to increase. If the trend for the years 1980-1984 is used to calculate the number of expected cases. the Centers for Disease Control and Prevention (CDC) estimated that between 1985-1992. approximately   5 1.000    excess cases have accumulated (D.E. Snider et   also    "Global burden of tuberculosis."   in   
B.R. Bloom (ed.). Tuberculosis:   Pathogenesis.    Protection and Control, American Society for
Microbiology, Washington.   D.C.,      to994},    pp. 3-11).



   A number of contributory factors are likely to be responsible for the observed increase in tuberculosis cases, including the AIDS pandemic. immigration from areas with high endemicity of tuberculosis. general deterioration of the health care infrastructure. transmission in high-risk environments   (eg.,    homeless shelters), and the increase in the number of multidrug resistant   At      ruherculosis    strains (Nolte and Metchock.   Aziora.    at p. 400). Unless the effectiveness and availability of methods and drugs to detect and treat tuberculosis do not  substantially improve. it is expected that over 30 million deaths and 90 million new cases of tuberculosis will occur in the years between 1990-2000 (Snider   et      al.    at p. 10).



   Although it is the major cause, organisms other than   At    luberculosis are sometimes associated with tuberculosis in humans and other animals. These organisms are included in the "M. tuberculosis complex," which includes At bovis.   if      qfricanun.    and   At    microti. as well as   At    tuberculosis. M. hovis causes tuberculosis in cattle, humans and other primates.



  carnivores (e.g.. dogs and cats). swine, parrots, and some birds of prey. Human disease is virtually indistinguishable from that cause by M. tuberculosis. and is treated in a similar manner (Nolte and Metchock. supra. at 402). Similarities between   At      hoviv    and M.



     tuberculosis    led to the development of the bacillus of   Calmette-Guerin    (BCG) an attenuated form of M.   bovk.    as a vaccine against tuberculosis in many parts of the world (See e.g.. W.K.



  Joklik   et    al. (eds.). Zinsser   Microbiolosv,    18th ed.. Appleton-Century Crofts. Norwalk. CT, [1984], p.   564).The    human health problems associated with At bovis were largely responsible for the development of methods for the pasteurization of milk and the adoption of compulsory pasteurization in the early 1900s (See. C.O. Thoen, "Tuberculosis in wild and domestic mammals," in B.R. Bloom (ed.) Tuberculosis: Pathogenesis. Protection and Control.



  American Society for Microbiology, Washington. D.C.   [19941.    pages 157-162).   At    africanum has been reported from cases of tuberculosis in tropical Africa.   V      nlicrori    causes generalized tuberculosis in voles, and produces local lesions in such animals as guinea pigs. rabbits. and calves (Nolte and Metchock. supra at 402).



   Thus. M.   tuberculosis    is not the only respiratory pathogen of great public health concern. and neither is the   At    tuberculosis complex. Recent developments in the taxonomy and study of the mycobacteria have resulted in recognition of M.   avium    complex (MAC) organisms as the cause of disseminated disease in immunocompromised patients. in particular
AIDS patients. The two major species associated with MAC are   At    avium and   At    intracellulare. However, the MAC includes 28 serovars of these two distinct species.



  although three additional serovars of M scrofulaceum (i.e. M. avium-M. intracellulare-M.



  scrofulaceum complex) were previously included. Within the M. avium species, three subspecies have been proposed. based on phenotypic and   genotypic    characteristics (M.   avium    subspecies avium.   At    avium subspecies   paratuberculosis.    and   M.      avium    subspecies silvaticum) (M.-F. Thorel   et      al..    Numerical taxonomy of mycobactin-dependent mvcobacteria. emended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium.



  subsp. nov.   M      avium    subsp. paratuberculosis. subsp.   nov..    and Mycobacterium   a"iuni    subsp.  



  silvaticum subsp. nov., int. J. Syst.. Bacteriol.. 40:254-260 [1990]). As additional information is gathered on the genetic relationships among the mvcobacteria, it is likely that changes will occur in the taxonomy of these organisms.



   At avium. an important pathogen of poultry and swine was recognized as a cause of disease in chickens in the late 1800s. but was not recognized as a cause of human disease until 1943   (See.    Nolte and Metchock. supra, at 402).   M    intracellulare is usually associated with disease in swine and cattle. In addition to its veterinary significance, MAC is an important pathogen of immunocompromised patients. especially those with AIDS. Indeed.



  disseminated MAC infection is the most common opportunistic infection observed late in the course of HIV disease. It has been reported that the frequency of disease due to MAC rises from   3%    per year for individuals with CD4 counts of 100-200/ l to   39%    at CD4 counts of    < 10/p1    (S.D. Nightingale et al., "Incidence of   At    avium intracellulare complex bacteraemia in
HIV-positive patients." J. Infect. Dis.,   165:108-25    [1992]). Disease with MAC is characterized by fever. cachexia, hepatic dysfunction. and anemia. As with infection with M tuberculosis, infection with MAC may promote HIV expression. leading to accelerated HIV disease progression.



   Human acquisition of M. avium appears to occur via inhalation or ingestion of fresh water in which bacilli are concentrated (K.L. Fry   et    al., "Epidemiology of infection by nontuberculous mycobacteria. VI. Identification and use of epidemiologic markers for studies of Mycobacterium avium, M. intracellulare, and M. scrofulaceum." Am. Rev. Respir. Dis., 134:39-43[1986]). It is hypothesized that local infection is   followed    by hematogenous spread to organs of the reticuloendothelial system. It is here. in bone marrow and lymph nodes. that the number of MAC colony forming units (CFU) ultimately rise to levels several logs higher than are present in blood (D.

   Peterson et   al      At      avium    complex (MAC) disease in HIV- infected patients is a uniform infection of bone marrow that does not correlate with the level of infection in blood." Natl. Conf. Hum.   Retrovir.      Rel.    Pathogens 2:56   [1995]).    The observed mycobacteremia apparently represents "spillover" of bacilli from these heavily infected organs.



  Alternatively, MAC mycobacteremia may occur intermittently. arising from the   gastrointestinal    tract. without inevitably causing tissue infection.



   Recently. our understanding of the mycobacteria, including the recently described species (e.g., M. genavense) associated with disseminated disease in HIV-infected individuals.



  as well as the potential pathogens M. asiaticum,   V    haemophilum.   M      inalmoense.      V    shimoidei. and   M      celatuni    has greatly increased, largelv due to an increased interest in  opportunistic pathogens. especially those associated with disease in AIDS patients.



  Nonetheless. many problems remain unresolved. and reliable. rapid methods are needed for the detection of latent and active mycobacterial infections. especially in the case of AIDS patients.



  Treatment of Mvcobacterial Disease and infection
 The unique properties of the mvcobacterial cell wall. growth rates. and other factors have provided avenues as well as detours in our development of methods for detection and treatment of mycobacterial disease. The mycobacteria characteristicallv have cell walls with a high lipid content. including waxes such as mycolic acid. The properties of this waxy cell wall provide the "acid-fast" nature of the organisms, as once dye is taken into the cells. they are not easily decolorized. even with acid-alcohol. Thus. unlike most organisms. the mycobacteria are said to be "acid fast" and are often referred to as "acid-fast bacilli" or "AFB."
 The growth rate of the mycobacteria ranges from slow to very slow, with generation times ranging from two to 24 hours.

   Most isolates. including   At    tuberculosis require long incubation periods (i.e.. 4-8 weeks for traditional culture methods) under optimal conditions for growth to be easily visible in vitro. Once the organisms from a primary culture have grown, biochemical and other tests must be done in order to provide an identification. This is an unacceptably long time between sampling and a definitive identification of the organism causing disease in a patient.



     I he    slow growth rate and the pathogenic processes of   At      fllhe rczzlo.vi.s    contribute to problems encountered in treating tuberculosis patients. As most antimicrobial drugs work against actively growing cultures. the relatively metabolically inactive mycobacteria enclosed within relatively impermeable waxy cell walls are generally unaffected by most drugs commonly used to combat bacterial disease.



   The first line of drugs used against   At    tuberculosis include isoniazid   (INH),    rifampin.



  pyrazinamide. ethambutol, and streptomycin. The second line drugs include para-amino   salicyl ic    acid. ethionamide. cycloserine,   capromycin.    kanamycin. amikacin. ciprofloxacin.



     ofioxacin    and   rifabutin.    It is recommended that patients initially be treated with INH.



  rifampin.   ethambutol.    and pyrazinamide for 2 months. Those patients with fully drugsusceptible isolates then may be treated with INH and rifampin for an additional four months (American Thoracic Society and Centers for Disease Control. "Treatment of tuberculosis and  adults and children." Am. Rev. Respir. Dis.. 134:355-363 [1986]). Patients with isolates resistant to either   INI-I    or rifampin must be treated with alternative regimens for longer duration. In all cases. successful treatment of patients must continue long after acid-fast bacilli are no longer detected in sputum samples.



   Drug resistant   At      tuberculosis    strains of have become a serious concern worldwide. In a recent nationwide survey of drug resistance among tuberculosis cases reported   during    the first quarter of 1991. it was found that 14.9% had isolates that were resistant to at least one anti-tuberculosis drug, and 3.3% had isolates resistant to both INH and rifampin (Centers for
Disease Control. "National MDR-TB Task Force, national action plan to combat multidrugresistant tuberculosis." Morbid. Mortal. Wkly. Rept. 41:1-48 [1992]). This is of grave concern. as INH and rifampin are the most effective drugs in our arsenal against At   tuberculosis.   



   For MAC. the concerns are potentially even more significant. Strains of MAC have been reported to be intrinsically resistant to anti-tuberculosis drugs and many other antimicrobials due to failure of these drugs to penetrate the lipid-rich cell wall (N.   Kastop      et      "Enhancement    of drug susceptibility of Mycobacterium avium by inhibitors of cell envelope synthesis,"   Antimicrob.    Agents   Chemother..    34:759-764   [19901;    and N.   Rastog       et    al., "Simplified acetylcysteine-alkali digestion-decontamination procedure for isolation of mycobacteria from clinical specimens." J. Clin. Microbiol.,   25:1428-1438    [1987]). Indeed.



  optimal regimens for treatment of either chronic pulmonary disease or disseminated MAC infections in AIDS patients have not been defined. In addition, no therapeutic   regimen    has been shown to be of sustained clinical benefit for patients with disseminated MAC (Noite and
Metchock. supra, at p. 428). It is recommended that patients with   HIV    and 1100 CD4 cells be given prophylaxis against MAC that is to be continued for the patient's life. unless multidrug therapy becomes necessary due to disseminated disease (Centers for Disease Control.



  "Recommendations on prophylaxis and therapy for disseminated Mycobacterium   Oviun?    complex for adults and adolescents infected with human immunodeficiency virus." Morbid.



  Mortal. Wkly Rept.. 42(RR):14-20   [1993]).    Preventive therapy tor MAC can be problematic because of interactions with other drugs commonly used in HIV   infcction.    particularly the new protease inhibitors. Although no optimal treatment regimen has been defined for disseminated MAC. the U.S. Public   Health      Servicc Iask    Force on Prophylaxis and Therapy for MAC recommends that treatment continued for the lifetime of the patient, even if  improvement is noted; this treatment should include at least two chemotherapeutic agents. one of which should be azithromycin or clarithromycin.



   The situation is even potentially more grim if other Mycobacterium species are associated with disease in a patient. as almost all of the strains of the "rapid grower"   (i.e..    in vitro growth may be observed in as few a two days) Mycobactrium species (i.e..   NJ    chelonae.   At      /ortuitu)n,    M. abscessus, etc.) are resistant to the anti-tuberculosis drugs. Thus.



  prophylactic treatment of immunocompromised patients is problematic and treatment is dependent upon the results of antimicrobial susceptibility testing of patient isolates.



  Detection of Mycobacterial Disease and Infection
 The field of diagnostic and clinical microbiology has continued to evolve. and vet.



  there remains a general need for systems that provide rapid and reliable detection of disease and infection due to microorganisms such as   At    tuberculosis. Nontheless. the role of the clinical mycobacteriology laboratories cannot be underestimated in view of their potential contributions in controlling the spread of tuberculosis and non-tuberculosis disease through the timelv detection. isolation. identification. and determination of drug susceptibility of these organisms. There is a "new sense of urgency" regarding the reporting of acid-fast smear.



  cultures. and drug susceptibility results to phvsicians. prompted in large part to the emergence of multi-drug resistant strains of M. tuberculosis (Nolte and Metchock. supra. at p. 400).



  Traditionally, tuberculosis surveillance has involved the use of preliminary skin tests   (e,W,    tuberculin tests). with positives being further evaluated for active disease by radiographic analysis (i.e.. chest   X-rays).    and sputum cultures. Other samples are sometimes submitted to the laboratory for culture. including blood. bronchoalveolar lavage fluid, bronchial   washing.   



  gastric lavage fluids, urine, body fluids (e.g., cerebrospinal   [CSF1*    pleural. peritoneal.



  pericardial. etc.). tissues   (e.g.,    lymph nodes. skin. or other biopsy materials). abscess contents.



  aspirated fluids. skin lesions, and wounds.



   In contrast to the situation with tuberculosis, blood cultures are often used for the isolation of MAC from immunocompromised patients, especially those with AIDS. Positive
MAC blood cultures are generally. but not always. associated with clinical evidence of tissue infection. which typically can involve the bone marrow. liver. or lymph nodes (J.   Ilavlik      et    al., "I)isseminated   Mycobacteriwn      avium    complex infection: clinical identification and epidemiologic trends," J. infect. Dis.. 165:577-580   [19921,    FJ. Torriani   et    al., "Autopsy findings in AIDS patients with   Al.    avium complex bacteremia," J. Infect.

   Dis., 170:1601-5    [19941).    Thus. detection of MAC in tissue samples provides useful information on a patient's status. However. culturing of blood remains the most commonly used method for diagnosis of MAC infection. primarily because of the requirement for an invasive procedure to sample infected tissues.



   Culture Methods. Once a specimen has been received in the laboratory suspected of containing mycobacteria. the specimen will generally be stained and examined for the presence of   AEB.    Sputum and other "dirty" specimens are decontaminated and concentrated prior to staining and culturing. Specimens are inoculated onto either solid egg-based media (i.e., Lowenstein-Jensen agar). or liquid medium, in which growth is more rapid. Various commercial growth media systems and methods are available for detection of mycobacteria.



  including BACTEC (Becton-Dickinson Diagnostic Instrument Systems). Septi-Chek (Becton
Dickinson Microbiology Systems). and the   lsolator    system (Wampole). Automated detection methods   (e.g..    those based on production of radiolabelled   CO.,      turbiditv    or light) have been developed to identify cultures with microbial growth.   However.    growth of   At    tuberculosis and other mycobacterial species must be confirmed by other methods. Methods presently accepted for detection and identification of are described in great detail in various publications (see   e.g..    N. Master (section editor) Section 3. "Mycobacteriology." in   H.F.    Isenberg (editor in chief).

   Clinical   Microbiolov    Procedures Handbook. volume 1.   3.0 1-3 16 4      [1994j).   



   Immunological Methods. Methods for diagnosis of tuberculosis based on   immunologic    methods such as detection of delayed type hypersensitivity   (DT11)    skin responses. as well as the detection of anti-mycobacteria antibodies. or mycobacterial antigens have been studied. Historically, skin tests have been commonly used as indicators of infection with   At    tuberculosis. The tuberculin skin test. still commonly in use. was the first immunodiagnostic test developed for detection of tuberculosis. Problems with this test include its   inabilitv    to distinguish active disease from past sensitization. as well as its unknown predictive accuracy (D. Snider. "The tuberculin skin test." Am. Kev.   Kesp.      Dis..   



     125(Suppl.):108-118      [19821)    Even among healthy skin test reactors. the test cannot distinguish those individuals with continued latent infection (in whom there is a continued risk of   developing    active disease) from those in whom a protective immune response has eradicated that infection. In vitro tests to determine the cell-mediated responses to mycobacterial antigens have also been described. However. they are expensive.   technically     demanding to perform and interpret. and provide no additional data than are available from skin testing (See   e.g.,    Nolte and Metchock, supra. at p.. 426).



   Despite the fact that much effort has been devoted to development of serological tests for tuberculosis. these methods have not found widespread clinical use (E. Bardana.



  "Universal occurrence of antibodies to tubercle bacilli in sera from non-tuberculous and tuberculous individuals." Clin. Exp. Immunol., 13:65-77   [1973];    and T.M. Daniel and S.M.



  Debanne. "The serodiagnosis of tuberculosis and other mycobacterial disease by enzyme- linked immunosorbent assay," Am. Rev. Resp. Dis.. 158:678-680   [19871).    Nonetheless.



  perhaps because antibody detection methods are commonly used in the diagnosis of infectious disease. assay methods based on anti-mycobacterial antibody detection have been investigated.



   Antibody Detection. Several studies have focused on methods to determine the antimycobacterial antibody level in patients' sera. These studies have used a variety of antigen preparations. including crude extracts, purified native antigens. and recombinant proteins.



  Immunoassays. including ELISAs and radioimmunoassays (RIA) have been used in many of these studies (See   e.g.,    E.G. Wilkins et   ell,    "A Rapid. Simple Enzyme Immunoassay for
Detection of Antibody to Individual Epitopes in the Serodiagnosis of   Tuberculosis."    Eur. J.



  Clin. Microbiol. Infect. Dis.. 10: 559-563 [1991]; R.G. Benjamin et   al..    "Serodiagnosis of
Tuberculosis Using the Enzyme-Linked Immunoabsorbent Assay (ELISA) of Antibody to
Mycobacterium tuberculosis Antigen 5Ú- . Amer. Rev. Respir. Dis.. 126:1013-1016 [1982]):
R. Maes et   al..    "Development of an Enzyme Immunoassay for the Serodiagnostic of
Tuberculosis and   Mycobacterioses."    Med. Microbiol. Immunol.. 178: 323-335 [1989]): S.B
Kalish   et    al., "Use of an Enzyme-Linked Immunosorbent Assay Technique in the Differential
Diagnosis of Active Pulmonary Tuberculosis in Humans." J. Infect. Dis., 147: 523-530 [1983]); E.

   Nassau   er      at.    "The Detection of Antibodies to   Mveoloucteriztm    tuberculosis by
Microplate Enzyme-Linked Immunosorbent Assay (ELISA)," Tubercle, 57: .67-70 [1976];   F.L.    Garcia-Carreno, "Enzyme Immunoassay Using BCG in Serodiagnosis of Pulmonary
Tuberculosis," J. Hyg.. 97: 483-487 [1986]; R. Hernandez   et    al "Sensitive Enzyme
Immunoassay for Early Diagnosis of Tuberculous Meningitis." J. Clin. Microbiol.,   20:533-535      119841;    J.A. McDonough et   at,    "Microplate and Dot   Immunoassavs    for the Serodiagnosis of
Tuberculosis." J. Lab Clin. Med..   120:318-322    [1992]; E. 

   Sada   et      at.    "An ELISA for the
Serodiagnosis of Tuberculosis Using a 30.00-Da Native Antigen of Mycobacterium   tubercuThsis."    J. Infect. Dis.. 162:928-931 [1990]: A. Mathai   et    al., "Rapid Diagnosis of  
Tuberculous Meningitis with a Dot Enzyme immunoassay to Detect Antibody in
Cerebrospinal Fluid." Eur. J. Clin. Microbiol. Infect. Dis.. 10:440-443 [1991]; M. Turneer et   till.    "Humoral Immune Response in Human Tuberculosis: Immunoglobulins G. A. and M
Directed against the Purified P32 Protein Antigen of Mycobacterium   hovis    Bacillus Calmette
Guerin." J. Clin.

   Microbiol..   26:1714-1719    [1988];   N.K.    Kaushik et   cii      Serodiagnosti be detected in cerebrospinal fluid and blood using a simple latex agglutination test (A.A. Gal et   al.     "The clinical laboratory evaluation of cryptococcal infections in the acquired immunodeficiency syndrome." Diagn. Microbiol. Infect. Dis.. 7:249-54 [1987].) In disseminated histoplasmosis. a heat stable polysaccharide antigen may be detected in blood.



  CSF. and urine   bv    radioimmunoassay (L.J. Wheat et   cit.    "Diagnosis of disseminated histoplasmosis bv detection of   Hisiopicisma    capsulatum antigen in serum and urine specimens." N. Engl. J. Med..   314:83-8    [1986]). In leprosy. serum levels of At leprae phenolic glycolipid I correlate with bacillary load at diagnosis and during therapy. ranging from 1 2 ng/ml in paucibacillary patients to as high as 8000 ng/ml in multibacillary patients   (D.B.    Young et   cuit,    "Detection of phenolic glycolipid I in sera from patients with lepromatous leprosy." J.

   Infect.   Dis.,    152:1078-81   [19851;).    However. the successes with these organisms have not been mirrored in diagnosis of tuberculosis and MAC disease. with the exception of one report for MAC. described below.



   Detection of Mycobacterial Antigens. Previous reports of antigen detection assays for rapid diagnosis of tuberculosis have been limited to examination of fluids obtained from the site of clinical disease. such as cerebrospinal fluid, sputum. or bronchoalveolar ravage fluid (E. Sada   et    al., Detection of mycobacterial antigens in cerebrospinal fluid of patients with tuberculous meningitis by enzyme-linked immunosorbent assay." Lancet 2:651-2 [1983]: 1.0. al Orainey et al., "Detection of mycobacterial antigens in sputum by an enzyme immunoassay." Eur. J. Clin. Microbiol. Infect. Dis.. 11:58-61 [1992]: R. Kansal   ci      cit.   



  "Detection of   mannophosphoinositide    antigens in sputum of tuberculosis patients   bv    dot enzyme immunoassay." Med. Microbiol. Immunol. Berl.. 180:73-8   119911:      MA.    Yanez er al., "Determination of mycobacterial antigens in sputum by enzyme immunoassay." J. Clin.



  Microbiol.. 23:822-5 [1986]; G.V. Kadival el al., "Radioimmunoassav of tuberculous antigen."
Indian J. Med. Res.. 75:765-70 [1982], A. Chandramuki   ci    al., "Detection of mycobacterial antigen and antibodies in the cerebrospinal fluid of patients with tuberculous meningitis." J.



  Med. Microbiol..20:239-247 [1985]; C.L. Cambiaso et al., "Immunological detection of mycobacterial antigens in infected fluids. cells and tissues   bv    latex agglutination - Animal model and clinical application." J.   linmunol.    Meth..   129:    9-14 [1990]). A number of other antigen assays have also been described (See   e.g..    T.M. Daniel. "Rapid diagnosis of tuberculosis: Laboratory techniques applicable in developing countries." Rev.   Infect.      Dis.,    2(Supplement 2): S471-S478   (11989]).     



   In other studies. the infected fluids were placed in liquid culture for a short period.



  and mvcobacterial products were detected in the culture medium (A. Raja et al.. "Specific detection of Mycobacterium tuberculosis in radiometric cultures by using an   immunnassay    for antigen   5."    J. Infect. Dis., 158:468-70 [1988]; A. Raja   ci      czl..    "The detection by immunoassay of antibody to mycobacterial antigens and mycobacterial antigens in bronchoalveolar lavage fluid from patients with tuberculosis and control subjects." Chest   94    133-137 [1988]; R.



  Schoningh R et   cii    Enzyme immunoassay for identification of heat-killed mycobacteria belonging to the   Mycobacteriuni    tuberculosis and Mycobacterium avium complexes and derived from early cultures" J. Clin. Microbiol.. 28:708-13 [1990]; A. Drowart   ci    al., "Detection of mycobacterial antigens present in short-term culture media using particle counting immunoassay." Am. Rev. Respir.   Dis..    147:1401-6 [1993]).   'fhe    detection threshold of these assays ranged from 1 ng to 1   pglml.    All used a polyclonal antiserum rather than monoclonal antibody for capture: several used the same serum for both capture and detection.



  In none of these reports were fluids remote from the site of infection studied. and in none could the assay identify subjects with latent infection.



   M.   taberculosis    Antigens. In contrast to the situation with many bacteria. the antigens of M. tuberculosis are a remarkably complex mixture of proteins polysaccharides. and lipids.



  The polysaccharide antigens share antigenic cross-reactivity with Nocardia. the   corynebacteria    and staphylococci. and generally do not elicit delayed type hypersensitivity (DTH) (Y.



  Yamamura et   cii..    "Biology of the mycobacterioses. Chemical and immunological studies on peptides and polysaccharides from tubercle bacilli," Ann. NY Acad. Sci..   154:88-97      [19681:    and S.D. Chaparas   ci    al., Comparison of lymphocyte transformation. inhibition of   macrnphage    migration and skin tests using dialyzable and nondialyzable tuberculin fractions from Mycobacterium   bovine      (BCG)."    J.

   Immunol..   107:14')-53      [1971]).    However. the protein antigens of the Mycobacterium elicit a DTH response. and stimulate lymphocyte blastogenic responses in both sensitized humans and guinea pigs   (L.F.    Affronti ci al., "Some early investigations of   Mveobacterium    tuberculosis," Am. Rev. Respir. Dis., 92:1-8   [1995j):    T.M.



  Daniel et al.. "Reactivity of purified proteins and polysaccharides from Mycobacterium tuberculosis in delayed skin test and cultured lymphocyte mitogenesis assays." Infect. Immun..



  9:44-7   [19741;    and S.D. Chaparas et al., "Tuberculin-active carbohydrate that induces inhibition of macrophage migration but not   lymphocyte    transformation." Science 170:637-9 [1970]).  



   The antigenic repertoire of At tuberculosis includes some proteins which are also found intracellularly, and some which appear uniquely as secreted proteins. As the technology to define these antigens has improved. their number has grown. In 1971. 11 antigens could be identified bv immunoprecipitation in culture filtrate (the spent medium of cultures of M.



  tuberculosis after the bacilli have been removed by filtration (B.W. Janicki   ci      al..    "A reference system for antigens of Mycobacterium tuberculosis," Am. Rev. Respir. Dis..



  104:602-4 [1971]). Antigen 5 (a 38 kD protein), and antigen 6 (a 30-32 kD protein later   tcrmed    alpha antigen. BCG 85B. and MPT59), were prominent. and appeared to have some   antigen    determinants which were restricted to   M.    tuberculosis   (T.M.    Daniel   ci      at.   



  "Immunobiology and species distribution of Mycobacterium tuberculosis antigen 5," Infect.



  Immun.,   24:77-82    [1979]; T.M. Daniel   ci      at,    "Demonstration   of a    shared epitope among mycobacterial antigens using a monoclonal antibody." Clin.   Exp.    Immunol.. 60:249-58   [19851:    and T.M. Daniel ci   at,    "Specificity of Mycobacterium tuberculosis antigen 5 determined with mouse monoclonal antibodies." Infect. Immun..   45:52-5      [1984J).    A decade later. Closs and colleagues identified as many as 50 distinct antigens using a system of crossed immunoelectrophoresis (0. Closs   ci      at,    "The antigens of Mycobacterium   hours.    strain BCG.



  studied by crossed immunoelectrophoresis: a reference system." Scand. J. Immunol..   12:249-    63 [1980]). The use of 2-D gel electrophoresis has increased this number to greater than 100.



  This growing number of potential antigens has presented a challenge in the development of diagnostic and treatment systems.



   MAC Antigens. In contrast to these reports in which only infected fluids were studied, a method was reported for detection of a MAC protein antigen in urine of AIDS patients with disseminated MAC infection (A.A. Sippola el   at.    "Mycobacterium avium antigenuria in patients with AIDS and disseminated   At    avium disease." J. Infect.   Dis..   



  168:466-8 [1993]). This assay utilized a goat antiserum ("K-II") which had been developed by immunization with   At      iniracellulare,    and which primarily recognized a 22.5 kD antigen of
M. intracellulare. M. avium and M. scrofulaceum, but not M. tuberculosis. Antigenuria was detected in clinical specimens using an assay in which supported nitrocellulose strips were dipped into voided urine samples. which were then probed with the antiserum. Antigenuria was detected in 7/11 patients with   At    avium-complex disease. and in 16/100 HIV+ controls.



  Two of the apparent false positive controls were subsequently found to have disseminated
M. avium infection.  



   Although this assay format was initially appealing in terms of its simplicity. it has several major shortcomings. including: the use of supported nitrocellulose strips hinders uniform washing of multiple samples; the binding of the target antigen to the paper is nonspecific; the assay cannot be used to detect non-protein antigens. as their binding to the paper may not be adequate. Also. the specificity of the assay is cntircly determined by that   of    the antiserum. It is therefore critical that the serum not cross-react with human proteins or antigens of other pathogens, an unrealistic expectation for this assay. Therefore. the advantages of these researchers' assay may be outweighed by its disadvantages and/or be inadequate to offer clinicians a substantial advantage when compared to the use of blood cultures for detection of mycobacteria.



  Effect of HIV Infection on Diagnosis of Mycobacterial Disease
 HIV infection alters the manifestations of tuberculosis. particularly in those patients with advanced HIV disease, in whom half have mycobacteremia and more than 75% have lymph node. hepatic. or bone marrow involvement. particularly those with low CD4 counts (B.E. Jones   ci    al., "Relationship of the manifestations of tuberculosis to CD4 cell counts in patients with human immunodeficiency virus infection," Am. Rev. Respir. Dis..   148:1292-    1297   [19931;    G.I. Santos   ci      al..    "Liver disease in patients with human immunodeficiency virus infection. Study of 100 biopsies," Rev. Clin. Esp., 193:115-8 [19931; A.D. Pithie   et      cit.   



  "Fine-needle extrathoracic lymph-node aspiration in   HlV-associated      sputum-negative    tuberculosis." Lancet 340:1504-5 [19921: and   P.1.    Barnes et al., "Tuberculosis in the   1990s."   
Ann. Intern. Med.. 119:400-10   [1993]   
   However.    HIV infection is accompanied by less radiographic evidence of pulmonary disease. fewer lung zones with cavitation, and reduced numbers of mycobacterial   colonv-    forming units in sputum (R.J. Brindle   ci      at.    "Quantitative   bacillarv    response to treatment in
HIV-associated pulmonary tuberculosis," Am. Rev. Respir. Dis.. 147:958-61 [1993]). Thus.



  the likelihood of diagnosis based on expectorated sputum is reduced in   l-llV-associated    tuberculosis. even though the total mycobacterial burden may be greater. This has led to the necessity of increased evaluation of specimens other than sputum. particularly tissues such as pleura. liver. and bone marrow. Patients with miliary or disseminated tuberculosis,   particularl     those with HIV infection. often require multiple biopsies prior to initiation of therapy.



   Again. despite the large number of studies. no antigen detection method has been developed to date which provides reliable. highly specific, and highly sensitive results.  



  especially for "dirty" samples such as sputum (See e.g., J.M. Grange, "The rapid diagnosis of paucibacillary tuberculosis," Tubercle, 70:1-4 11989]; and Nolte and Metchock. sllpra. at p.



     426).   



   Importantly. scrodiagnostic tests for tuberculosis. although potentially simple and inexpensive, have been especially hampered by poor sensitivity in   H1V-infected    persons in whom antibody responses are diminished (T.M. Daniel   et      at.    "Reduced sensitivity of tuberculosis serodiagnosis in patients with AIDS in Uganda," Tuber. Lung Dis.. 75:33-7   [1994]).    This is important because the patient populations that are dually infected with HIV and   M      tllherclulosis    is at greatest risk for developing active tuberculosis (M.E. Villarino   ci    al., "Management of persons exposed to multidrug-resistant tuberculosis." Morb.   Morn.      Wkly.   



     Rep..    41(RR-11):61-71 [1992]).



  Diagnosis of latent infection with M. tuberculosis
 Identification of individuals with latent   At      luberculosis    infection is a problem which cannot be addressed by current methods. The natural history of   At    tuberculosis infection is such that only 5-10% of individuals who are otherwise healthy will ever develop tuberculosis.



  As mentioned above. the risk of tuberculosis is highest in the first year following infection.



  but tuberculosis can occur as long as 50 years later. Thus. the infection   clearlv    is contained but not eradicated in many infected but healthy individuals. Presently, there is no reliable method to distinguish between individuals who are latently infected and those whose infections have been eradicated by a protective immune response. Both groups have positive skin tests with purified protein derivative   (PPD).    as the longevity of a positive skin test can reflect   immunologic    memory as well as persistent latent infection.



     'l'he    presence of immunosuppressive concurrent illnesses increases the likelihood of recrudescent disease in tuberculosis-infected persons. This is most strongly expressed in HIVco-infected persons. in whom the risk of re-activation of tuberculosis may be increased from 10 to 100 fold. Indeed. HIV has emerged as the most significant risk factor for the progression of latent tuberculosis to active disease (Snider et   al,    at p. 5: Selwyn   et    al., "A prospective study of the risk of tuberculosis among intravenous drug abusers with human immunodeficiency virus infection." N. Eng. J. Med.,   390:545-550    [1989]). However. skin testing becomes increasingly unreliable as an indicator of M. tuberculosis infection as the CD4 count declines.

   It thus remains difficult to efficiently target those individuals who would benefit most from preventive therapy.  



   Yet. identification of latently-infected persons is desirable for initiation of tuberculosis preventive therapy. Although such therapy   (dailv    doses of INH for nine months) can be administered to all skin test positive individuals. such therapy obviously would only benefit those subjects who ultimately would have developed tuberculosis. Thus at least 10 -20 subjects must be treated to prevent one case of tuberculosis. The other   9-1 9    treated subjects are subjected to risks associated with INH (e.g.. hepatitis. neuropathy) without any potential benefit. This problem is compounded by the present inability to monitor the effectiveness of preventive therapy Some individuals will fail preventive therapy because of poor compliance with   long-term    administration of INH.

   Others will fail because they were infected with an   IN H-resistant    isolate. At present, there is no method to determine whether preventive therapy has been successful in completely eradicating latent infection with   At      rttherculosis.   



   In sum. there is no entirely satisfactory method for diagnosis either active tuberculosis or latent mycobacterial infection. Direct examination of sputum or infected tissues is   insufliciently    sensitive (See   e.g.,    L.B.   Ileifets    and R.C. Good. "Current laboratory methods for the diagnosis of tuberculosis," in B.R. Bloom (ed.) Tuberculosis: Pathogenesis. Protection and Control. American Society for Microbiology, Washington. D.C. [1994]. pages 85-110).



  Traditional methods. including cultivation of the organism require the time and facilities for prolonged incubation. Assays based on DNA amplification are expensive and   technicalle    demanding. and may not be applicable fbr routine use in clinical laboratories outside of major medical centers in industrialized countries. The development of a simple, rapid. diagnostic test which does not rely on the growth of organisms   i)7      "i ttv.    but that is capable of   idcntif\'ing    individuals with latent. subclinical   M    tuberculosis infection. and   which    might predict the likelihood of subsequent relapse. would be of tremendous value for tuberculosis control programs worldwide.



  Monitoring of Therapy
 Many factors can adversely affect the response to anti-tuberculous therapy. These include primary drug resistance, patient non-compliance. malabsorption. adverse interactions with other medications. and other host factors. Inadequate treatment can lead to emergence of secondary drug resistance due to selective pressures on mycobacterial growth. In order to assess a patient's response to anti-tuberculosis therapy. patients must be monitored throughout their treatment regimen.  



   However. sputum cultures and   AFIB    smears return to negative   slowlv    during therapy.



  such that the proportion of samples which become negative are typically only 40% at 1 month. 80% at 2 months. and 90-95% at 3 months. Chest radiographs also improve slowly.



  and may not   significantly'    improve until 3 months of treatment. It thus is difficult to   identifv    promptly those patients who ultimately will fail any given tuberculosis treatment regimen.



  Better early indicators of success or failure clearly are needed.



   Mitchison has suggested that the early bactericidal activity of anti-tuberculosis regimens. as determined by quantitative sputum cultures. might predict the overall effectiveness of a treatment   rcgimen    (S.L. Chan et   cit    "The early bactericidal activity of   rifabutin    measured by sputum viable counts in Hong Kong patients with pulmonary tuberculosis." Tubercle 1992;33-8 [1992]; and A.J. Jindani   ci      1.    'The Early Bactericidal
Activity of Drugs in Patients with Pulmonary Tuberculosis." Am. Rev. Respir.   Dis..      121:939-    49 [1980]).

   He observed approximately a   10-3    drop in the number of viable   ,11    M. tuberculosis bacilli during the first 2 weeks of effective multi-drug therapy, and noted   Icsser    reductions with less effective regimens. He suggested that new drugs for tuberculosis might be evaluated in short term studies (1-2 weeks) using quantitative culture as an endpoint. However. this approach has not been widely accepted. largely because of difficulties of performing the assay in a standardized fashion, particularly with regard to   homogenization    of non-uniform sputum specimens. This lack of standardization precludes the use of this method in the clinical setting.



   Problems also exist in monitoring therapy for MAC infection in AIDS patients through serial blood cultures. An autopsy series of 44 patients with MAC bacteremia found that 13 (30%) had no histologic evidence of MAC disease (F.J. Torriani et   cit.    "Autopsy   findings    in
AIDS patients with   At    avium complex bacteremia." J. Infect. Dis.. 170:1601-5   11994]).   



  suggesting that transient bloodstream infection may occur and may be self-limited.



     Conversely.    other patients have only transiently or intermittently positive blood cultures in the face of high tissue burdens of   mvcobacteria    (C.A. Kemper   ci      cit.    "Transient bacteremia due to   At      avium    complex in patients with   AIDS."    J. Infect.   Dis..      170:488-93    [1994]). These observations suggest that sustained mycobacteremia may be a late event in the natural history of MAC infection. and that blood culture is not an adequate diagnostic or monitoring tool when used alone. However. access to the main site of infection (bone marrow. lymph node.



  or liver) requires an invasive diagnostic procedure which is not usually undertaken without prior attempts at diagnosis by non-invasive measures. These combined factors often lead to a  delay in diagnosis and initiation of therapy, and make it difficult to evaluate the response to therapy.



   In sum. despite advances in the detection of   At    tuberculosis and   other    mycobacteria.



  the need remains for a safe. reliable. easy-to-use system for the detection of infection with these organisms. as well as means for monitoring patients with disease or infection. In particular. there is an urgent need for useful methods to use samples such as urine and other fluids for the detection of infection and disease with Mycobacterium species.



  SUMMARY OF THE INVENTION
 The present invention provides a rapid method for the detection of disease and infection due to the mycobacteria. in particular   AJ    tuberculosis as well as MAC. The present invention is intended for detection of pulmonary mycobacterial disease or infection. and disseminated mvcobacterial disease. as well as localized infection with mycobacteria at sites other than the pulmonary area.



   The present invention provides kits for the detection of   MycoXcrclewrillm    in a test sample. comprising: a) a solid support: and b) a monoclonal antibody directed against a portion of alpha antigen immobilized on the solid support. In one embodiment. the kit further comprises a primary antibody, while in another embodiment the kit comprises a reporter antibody. in yet another embodiment the kit further comprises an amplifier system. It is also contemplated that the kit of the present invention comprise a primary antibody and reporter antibody. as well as an amplifier system.

   In a particularly preferred embodiment. the
Mycobacterium species detected is   Mvcohelcteriun?      Illhe rculosi.s.    In an alternative embodiment the   Mycobacterizirn    species detected is Mycobacterium   ctvilzm.   



   It is also contemplated that the kit of the present invention will include additional components. including, but not limited to. such items as an alpha antigen control. a diluent such as saline or water. as well as a plurality of alpha antigen samples with known concentrations of alpha antigen suitable for use in preparing a standard curve(s) for the determination of antigen concentration in the test   (i.e.,    patient) samples. Furthermore. in kits in which biotinylated antibodies are used. in one preferred embodiment. the primary antibody is preadsorbed with an avidin compound (i.e., strepavidin) coupled to biotinylated capture antibody. prior to use in the kit. It is further contemplated that the kit of the present invention comprise methods for   analvzing    samples using blots. including but not limited to
Western blots.

   These blotting kits may include additional reagents   such    as those listed above.  



  as well as reagents. including but not limited to, such as paper suitable for the blotting system used. and blocking solution.



   It is also contemplated that the antibodies of the kit of the present invention be prepared through the use of synthetic peptides as   immunogens.    In this embodiment. synthetic peptides of known sequence are used to induce the production of at least one of the antibodies used in the kit. It is also contemplated that the synthetic peptides be used as an immunogen while they are still attached to the beads used to prepare them. Thus. the present invention encompasses antigens of mycobacteria that are naturally occurring and harvested from samples such as sputum. urine or blood samples. as well as mycobacterial antigens that are produced synthetically for use in the production of antibodies for use in the kit. It is also contemplated that these synthetic peptides be used as antigens in the kit.

   In a preferred embodiment. the synthetic peptides correspond to the alpha antigen of   At      flJhercz(loxis    or MAC. Thus. the sequences of SEQ ID NOS:1-8 may be used in the form of synthetic peptides for use in the present invention. It is also contemplated that immune complexes will be tested using the kit of the present invention. In this embodiment. the immune complexes may be treated using methods known in the art to dissociate antibodies from antigens.



   It is also contemplated that the samples tested using the kit of the present invention will be obtained from individuals infected with one or more types of the human immunodeficiency virus. It is also contemplated that the kit will be used for monitoring the progression of therapy in individuals infected with   Mycobacteriurn.    in particular   M    tuberculosis and/or MAC. It is further contemplated that the kit will be used with samples from patients who are infected with   At    tuberculosis. as determined by skin test positivity, chest radiograph positivity. and/or sputum cultures containing   At    tuberculosis.

 

   The present invention also provides methods for the detection of Mycobacterium in a sample comprising: a) providing: i) a sample suspected of containing at least a portion of the alpha antigen of   Nfvcobcicterium    and ii) a monoclonal antibody directed against the port to the   antibody-antigten-antibody    sandwich to form an   anti body-antigen-antibody-antibody    sandwich. In a particularly preferred embodiment, the detecting   further    comprises adding an amplifier to said antibody-antigen-antibody-antibody sandwich.



   In one embodiment. the monoclonal antibody comprises a murine monoclonal antibody. In a   preferrcd    embodiment. the murine monoclonal antibody is biotinylated. In a particularly preferred embodiment, the primary antibody is preadsorbed with avidin   (e.g.,    streptavidin) coupled to biotinylated capture antibody, prior to use in the method of the present invention for the detection of   Alycobacteriurn    species. In yet another particularly preferred embodiment. the solid support is coated with avidin.



   In another embodiment. detection is achieved through use of such methods as enzyme immunoassay. radioimmunoassay, fluorescence immunoassay,   flocculation,    particle agglutination. and in situ chromogenic assay. It is not intended that the present invention be limited to any particular assay format.



   It is also contemplated that the method of the present invention will include additional components. including, but not limited to, such items as an alpha antigen control. a diluent such as saline or watcr, as well as a plurality of alpha antigen samples with known concentrations of alpha antigen suitable for use in preparing standard curve(s) for the determination of antigen concentration in the test   (it.,    patient) samples. Furthermore. in methods in which biotinylated antibodies are used. in a preferred embodiment. the primary antibody is preadsorbed with an avidin compound   (it.,    strepavidin) coupled to   biotinvlated    capture antibody prior to use in the method.

   It is further contemplated that the method of the present invention comprise methods for analyzing samples using blots. including but not limited to Western blots.   these    blotting methods may include additional reagents such as those listed above. as well as reagents, including but not limited to. such as paper suitable for the blotting system used, and blocking solution.



   It is also contemplated that the antibodies of the method of the present invention be prepared through the use of synthetic peptides as immunogens. In this embodiment. synthetic peptides of known sequence are used to induce the production of at least one of the antibodies used in the method. It is also contemplated that the synthetic peptides be used as an immunogen while they are still attached to the beads used to prepare them. Thus. the present invention encompasses antigens of mycobacteria that are naturally occurring and harvested from samples such as sputum. urine or blood samples, as well as mycobacterial antigens that are produced   synthctically    for use in the production of antibodies for use in the method. It is  also contemplated that these synthetic peptides be used as antigens in the kit.

   In a preferred embodiment. the synthetic peptides correspond to the alpha antigen of   At    tuberculosis or
MAC. Thus. the sequences of SEQ ID NOS:1-8 may be used in the form of synthetic peptides for use in the present invention.



   In one embodiment of the method. the   Mycobacteriuzn    detected by the method of the present invention is selected from the group consisting of   Mvcobcicictiuin      rlrherctrlosis.   



  Mycobacterium avium and Mycobacterium intracellulare. In one embodiment of the methods of the present invention the portion of said Mycobacterium alpha antigen is selected from the group comprising SEQ ID NOS: 1, 2. 3. 4. 5, 6. 7 and 8. Thus. it is contemplated that fragments of the alpha antigen. as well as the entire alpha antigen will be used in the methods of the present invention. It is also contemplated that immune complexes will be tested using the methods of the present invention. In this embodiment. the immune complexes may be treated using methods known in the art to dissociate antibodies from antigens.



   It is contemplated that any sample will be used in the method of the present invention.



  including but not limited to urine samples. sputum samples, blood samples. and serum samples. In the case of sputum samples. it is contemplated that most samples will be digested and/or decontaminated using standard methods prior to their analysis in the method of the present invention. It is also contemplated that some samples tested using the method of the present invention will be obtained from individuals infected with human immunodeficiency virus. including. but not limited to. HIV-I and HIV-2.



   It is also contemplated that the methods of the present invention will be used for monitoring the progression of therapy in individuals infected with Mycobacterium. in particular   At    tuberculosis and/or MAC. It is further contemplated that the methods will be used with samples from patients who are infected with   At    tuberculosis. as determined   bv    skin test positivity, chest radiograph positivity, and/or sputum cultures containing M.   tuberculosis.   



   The present invention also provides methods for the detection of mycobacterial antigen in a urine sample comprising a) providing: i) a urine sample suspected of containing at least   a    portion of alpha antigen of Mycobacterium; and ii) a monoclonal antibody directed against the portion of   Mvcobaderimn    alpha antigen; b) adding the urine sample to the monoclonal antibody under conditions such that the antibody binds to the Mycobacterium alpha antigen in the urine sample to form an   antibody-antigen    complex: and c) detecting the binding.



   The present invention also provides methods for the detection of Mycobacterium in a sample comprising: a) providing: i) a sample suspected of containing at least a portion of the  alpha antigen of Mycobacterium and ii) a monoclonal antibody directed against the portion of   Mycohacterium    alpha antigen: b) adding the sample to the monoclonal antibody under conditions such that the antibody binds to the   Mycobacierium    alpha antigen in the sample to form an   antibody-antigen    complex: and c) detecting the binding of said antigen and antibody.



   In a preferred embodiment of the method of the present invention, the detecting comprises adding the primary antibody to the antigen-antibody complex so that the primary antibody binds to the antigen to form an antibody-antigen-antibody sandwich. In another preferred embodiment of the method. the detecting further comprises adding a reporter reagent to the antibody-antigen-antibody sandwich to form an antibody-antigen-antibody-antibody sandwich. In a particularly preferred embodiment. the detecting further comprises adding an amplifier to said   antibodv-antigen-antibody-antibody    sandwich.



   In one embodiment, the monoclonal antibody comprises a murine monoclonal antibody. In a preferred embodiment. the murine monoclonal antibody is biotinylated. In a particularly preferred embodiment. the primary antibody is preadsorbed with avidin   (c.g..   



  streptavidin) coupled to biotinylated capture antibody prior to use in the method of the present invention for the detection of   Mycobacierium    species. in yet another particularly preferred embodiment. the solid support is coated with avidin.



   In another embodiment. detection is achieved through use of such methods as enzyme immunoassay, radioimmunoassay. fluorescence immunoassay, flocculation, particle agglutination. and in silu chromogenic assay. It is not intended that the present invention be limited to any particular assay format.



   It is also contemplated that the method of the present invention will include additional components. including, but not limited to. such items as an alpha antigen control. a   diluent    such as saline or water. as well as a plurality of alpha antigen samples with known concentrations of alpha antigen suitable for use in preparing standard curve(s) for the determination of antigen concentration in the test   (ie..    patient) samples. Furthermore. in methods in which biotinylated antibodies are used. in a preferred embodiment, the primary antibody is preadsorbed with an avidin compound   (i.e.,    strepavidin) coupled to biotinylated capture antibody prior to use in the method.

   It is further contemplated that the method of the present invention comprise methods for analyzing samples using blots. including but not limited to Western blots. These blotting methods may include additional reagents such as those listed above. as well as reagents. including but not limited to. such as paper suitable for the blotting system used. and blocking solution.  



   It is also contemplated that the antibodies of the method of the present invention be prepared through the use of synthetic peptides as immunogens. In this embodiment. synthetic peptides of known sequence are used to induce the production of at least one of the antibodies used in the method. It is also contemplated that the synthetic peptides be used as an immunogen while they are still attached to the beads used to prepare them. Thus. the present invention encompasses antigens of mycobacteria that are naturally occurring and harvested from samples such as sputum. urine or blood samples. as well as mycobacterial antigens that are produced synthetically for use in the production of antibodies for use in the method. It is also contemplated that these synthetic peptides be used as antigens in the kit.

   In a preferred embodiment. the synthetic peptides correspond to the alpha antigen of   M.    tuberculosis or
MAC. Thus. the sequences of SEQ ID   NOS: 1-8    may be used in the form of synthetic peptides for use in the present invention.



   In one embodiment of the method. the Mycobacterium detected by the method of the present invention is selected from the group consisting of Mycobacterium   tuberculosis.   



  Mycobacterium avium and Mycobacterium intracellulare. In one embodiment of the methods of the present invention. the portion of said Mycobacterium alpha antigen is selected from the group comprising SEQ ID NOS: 1. 2. 3, 4, 5, 6. 7 and 8. Thus, it is contemplated that fragments of the alpha antigen. as well as the entire alpha antigen will be used in the methods of the present invention. It is also contemplated that immune complexes present in urine samples will be tested using the methods of the present invention. In this embodiment. the immune complexes may be treated using methods known in the art to dissociate antibodies from antigens.



   It is also contemplated that some samples tested using the method of the present invention will be obtained from individuals infected with human immunodeficiency virus.



  including. but not limited to, HIV-I and HIV-2.



   It is also contemplated that the methods of the present invention will be used for monitoring the progression of therapy in individuals infected with Mycobacterium. in particular At   zuberculosis    and/or MAC. It is further contemplated that the methods will be used with samples from patients who are infected with At   tz,berciilosis,    as determined   bv    skin test positivity, chest radiograph positivity, and/or sputum cultures containing At   luliercluloisi.s.   



   The present invention also provides a composition comprising purified monoclonal antibody directed against a portion of   Mycobacteriun1    alpha antigen. In one embodiment. the monoclonal antibody is a murine monoclonal antibody. In an alternative embodiment. the  murine monoclonal antibody is directed against epitopes of the alpha antigen of   lld.   



     tuberculosis,      M.    avium or   M.      infracellulare.    In a particularly preferred embodiment. the murine monoclonal antibody is directed against At   tubercuThsis    epitopes. In yet another preferred embodiment. the alpha antigen used to produce the murine monoclonal antibody is comprised of SEQ ID NOS:I, 2, 3. 4, 5.   6.    7, or 8. It is also contemplated that the monoclonal antibody of the present invention will be produced through the use of synthetic peptides as antigens. In a preferred embodiment. the monoclonal antibody of the present invention is raised against SEQ ID NOS: 3.   4.    6 or 7.

   It is further contemplated that the monoclonal antibody be produced using any suitable methods for monoclonal antibody production including intrasplenic methods. cell culture. etc. It is not intended that the monoclonal antibody of the present invention be limited to an antibody produced using a particular method or produced using a particular animal species. It is also not intended that the antibodies used in the present invention be of a particular class of immunoglobulin.



  DESCRIPTION OF THE FIGURES
 Figure 1 shows Western blots for detection of alpha antigen purified from culture filtrates.



   Figure 2 is a graphical representation of the reaction used to produce a colored reaction product in the amplified ELISA test method.



   Figure 3 is a graph showing the results of an amplified ELISA test for detection of alpha antigen as described in Example 3.



   Figure 4 is a graph showing the detection of alpha antigen in spiked samples of human serum and urine.



   Figure 5 is a graph showing the results of a comparison between detection of   At      tuberculosi.v    by the BACTEC system and the amplified ELISA test method.



   Figure 6 is a graph showing the amplified ELISA results for urine samples from HIVnegative tuberculosis patients and controls.



   Figure 7 is a graph showing the amplified ELISA results   f'or    serum samples from
HIV-negative tuberculosis patients and controls.



   Figure 8 is a graph showing the amplified ELISA results for urine samples from HIVpositive tuberculosis patients and controls.



   Figure 9 shows Western blots of filtrates of M. tuberculosis.   M.      aviuin.    and   M.   



     smegmotis    using monoclonal TBC27 as the capture antibody.  



   Figure 10 is a graph showing the standard curves for alpha antigen amplified ELISA using culture filtrates of M. tuberculosis and M. avium.



   Figure 11 shows the urinary antigen values in HIV-negative subjects grouped according to their At tuberculosis and   M.    avium status.



   Figure 12 shows the urinary antigen values in   HIV-positive    subjects grouped according to their M.   tuberculosis    and M.   aviuni    status.



   Figure 13 shows the results of alpha antigen detection assays for sputum samples obtained from three patients. with one patient's results being shown in each of the three panels (i.e., Panels A. B. and C).



   Figure 14 shows the results of alpha antigen detection assays for sputum samples obtained from nine patients. with one   patients    results being shown in each of the nine panels   (i.      c..    Panels A-F).



   Figure 15 shows the results of alpha antigen detection assays for sputum samples obtained from one patient.



   Figure 16 shows Western blots for the detection of alpha antigen by three monoclonal antibodies.



   Figure 17 shows the results of alpha antigen detection assays from urine samples of
HIV-positive and HIV-negative individuals.



   Figure 18 shows a Western blot in which   5C5    was used to detect alpha antigen.



   Figure 19 shows the amino acid sequences of   M.    tuberculosis and   M.    avium alpha antigen.



   Figure 20 shows Western blots of   M.      aviuni    filtrate tested with murine antiserum directed against alpha antigen peptides.



   Figure 21 shows Western blots of M.   ctvium    and   NI.    tuberculosis culture filtrates tested with goat At intracellulare antisera. In this Figure. Panel 21A shows the results with Kris antiserum; Panel 21B shows the results with Kris antiserum adsorbed with   Ad.    tuberculosis: and Panel 21C shows the results with Jane antiserum.



   Figure 22 shows Western blots of M. avium and At tuberculosis culture filtrates tested with Kris antiserum and Kris antiserum adsorbed with M. tuberculosis. In this Figure. Panel 22A shows the results with the unabsorbed antiserum and Panel 22B shows the results with adsorbed antiserum.  



  GENERAL DESCRIPTION OF THE INVENTION
 Previous reports of antigen detection assays for direct diagnosis of tuberculosis have been limited to examination of fluids obtained from the site of clinical disease. such as cerebrospinal fluid, sputum. or bronchoalveolar lavage fluid. Furthermore. the range of the lower limit of detection of these assays was reported to be from 1 ng to 1   pg/ml.    In addition.



  the lower detection thresholds were only achieved in those assay which used a polyclonal antiserum for capture. Moreover, several used the same serum for both capture and detection.



  In none of these reports were fluids remote from the site of infection studied. None of these assays were able to identify subjects with latent infection. This is in contrast with the present invention. in which identification of a mycobacterial antigen in fluids remote from the site of infection in patients infected with   At    tuberculosis is possible.



   In the method of the present invention, a sandwich ELISA (enzymc-linked immuosorbent assay) with a monoclonal antibody for capture and a secondary antibody tor detection of bound monoclonal antibody is used. In a preferred embodiment. the signal from the assay is amplified using an recycling enzymatic method. which increases the signal over 500 fold. Special measures are included in order to reduce background noise. The   assav    offers numerous advantages as compared to existing methods for tuberculosis diagnosis. First.



  body fluids remote from the site of infection can be tested using the method of the present invention. The test can be used with infected body fluids. as well as urine and blood.



  Although urine and blood usually do not contain   At    tuberculosis. these sites may contain secreted products of the infection. Detection of these secreted products provides a clear advantage in attempts to diagnose a localized infection such as is usually the case in tuberculosis. It is a particularly important advantage in diagnosis of tuberculosis in children.



  from whom quality sputum specimens are difficult to collect and are generally not   available.   



  Detection of Alpha Antigen
 Alpha antigen is an abundantly-produced, secreted 30 kD mycobacterial protein involved in mycolic acid synthesis. During development of the present invention. it was determined that alpha antigen was a potentially useful indicator of mycobacterial disease. as it can account for up to 20% of the protein content of spent mycobacterial culture medium (A.



  Andersen   et      cuit,    "Proteins released from   Mycobacterizini    tuberculosis during growth." Infect.



  Immun.. 59:1905-10   [1991]).    In addition. homologous alpha proteins exist for most species of mycobacteria. including the   At      tiibercuThsis    and   M      aviiwi    complexes. However. it is   alse     contemplated that other antigens may be detected using the methods and compositions of the present invention.



     Thee    present invention provides rapid, reliable detection of mycobacterial antigens in such samples as body fluids (e.g.. urine. blood and sputum) for the detection of various mycobacterial species. as the antigens have both species specific and shared epitopes   (II.   



  Tasaka et   at,    "Specificity and distribution of alpha antigens of   Mycohacleri ln7    avium- intracellulare. Mycobacterium scrofulaceum, and related species of mycobacteria." Am. Rev.



  Respir. Dis..   1985:139:173-4      [19851).    Furthermore. the genes for alpha antigens of
M. tuberculosis, bovis, avium, and kansasii, have been cloned and sequenced (See e.g., K.



  Matsuo   ci      C    "Cloning and expression of the gene for the cross-reactive alpha antigen of
Mycobacterium kansasii." infect. Immun., 58:550-6   [1990];    K. Matsuo et al., "Cloning and expression of the Mycobacterium   hovis    BCG gene for extracellular alpha antigen." J
Bacteriol.. 170:3847-54 [1988]: N. Ohara et   at,    . "Cloning and sequencing of the gene for alpha antigen from M.   civimn    and mapping of B-cell epitopes." Infect. Immun..   61:1173-9      [1993]:    and L. DeWit   ci    al., "Nucleotide sequence of the 85B-protein gene of   At    bovis BCG and   At      tuberculosis:    DNA sequence." J.

   DNA Seq. Map., in press.   [1 9961).   



   At 30 kD. the alpha antigens may be too large to be filtered intact through the renal glomeruli, particularly when complexed with antibody, nonetheless. the present invention provides means to detect alpha antigens present in urine samples. While an understanding of the mechanism by which it is possible to detect the antigens in urine is not necessary to the successful practice of the invention. it is believed that cleavage of the protein may occur in vivo as a consequence of intracellular digestion by   proteolvtic    enzymes within the macrophages. in the circulation. or in the kidney   (e.g.,    bv brush border peptidases). and permit excretion of the antigen or portions thereot:

   Antibody epitopes of alpha antigens of   At    bovis.   M      leprac.    M. tuberculosis, and   At      ovizim    are largely restricted to the carboxyterminal half of the protein (Ohara   ci      at,    "Cloning and sequencing of the gene for alpha antigen from   At      ctvium    and mapping of B-cell epitopes," Infect. Immun..   61:1173-9    [1993]: and E. Filley et al., "Identification of an antigenic domain on Mycobacterium leprae protein antigen 85B. which is specifically recognized by antibodies from patients   with    leprosv." J
Infect. Dis.. 169:162-9 [1994]).

   Thus. fragments representing the N-terminal portion of   At    tuberculosis may be present in the urine. even if the native antigen is too large to be filtered by the glomeruli. It is contemplated that these fragments will be detected using the compositions and methods of the present invention.  



   The present invention provides numerous advantages over the methods previously and currently in use to detect infection and/or disease with At   lzlberculo.v.s    or MAC. For example. all of the previously reported tuberculosis antigen detection assays were applied only to infected fluids or to early cultures. the assays were considerably less sensitive than this assay. and in addition. relied upon an animal antiserum developed against a whole organism of another mycobacterial species (BCG) for detection. resulting in poor specificity.



  Furthermore. none of the previously described assays were applied to urine and blood samples.



   Also. the present invention detects products of the mycobacteria. rather than the host response to the organism. This provides an important advantage, as the host response is impaired in immunocompromised patients at highest risk of tuberculosis. Thus. tests such as those previously reported that are based on the host response are insufficiently sensitive in this population. In addition. the present invention permits detection of latent infection in individuals at highest risk of developing tuberculosis. This is significant. because once the infection is detected. preventive therapy can be initiated. This is not possible with the previously described methods.



   Also, unlike the previously described methods. the present invention can be used for early monitoring of anti-tuberculosis therapy. providing a useful tool in predicting which individuals might experience treatment failure with a particular regimen. Thus. the present invention can also be used in the rapid evaluation of new anti-tuberculosis therapies.



   Importantly for clinical diagnosis and public health tuberculosis clinics. the method of the present invention is rapid. requiring only 1-2 days to complete. as opposed to a minimum of 2-4 weeks for culture. Furthermore. the present invention can be performed during a single visit to the clinic. unlike the routine skin testing methods which require a return visit in order to observe and record the skin test results. Also importantly. the present invention provides an inexpensive method that is within the capabilities of existing clinical and public health mycobacteria laboratories. This is generally not true for methods that require specialized equipment and training   (e.g..    the polymerase chain reaction   tPCRi).    The test is very flexible, permitting the detection of various antigens or alpha antigen epitopes.

   The assay can be used to detect all secreted mycobacterial antigens by substituting a non-specific binding method for the capture antibody.



   In addition. the results obtained by the present invention for detection of   antigenuria    are much superior to those of such previous methods as that of Sippola et   al.    (A.A. Sippola   ci     al.,   "llvcobacteri2Jm      avium    antigenuria in patients with AIDS and disseminated   At      uvium    disease."   d.    Infect.   Dis..    168:466-8 [1993]). For example. as described in Example 16 of the present application. the   K-Il    antiserum was found by Western blot of   At    avium filtrate. to primarily recognize three antigens. One antigen was approximately 38-42 kD. and may be lipoarabinomannan (LAM). a mycobacterial polysaccharide.

   The second antigen is a 30 kD antigen that was determined to be alpha antigen. The third antigen was approximately 25 kD.



  This is significant for the reliable and reproducible detection of antigenuria. as these antigens differ considerably in terms of potential   species-specificity    Antibodies to polysaccharides reach high levels in mycobacterial infection; however. they generally are   broadlv    cross reactive and have limited diagnostic potential (S.D. Chaparas   et      al..    "Tuberculin-active carbohydrate that induces inhibition of macrophage migration but not lymphocyte transformation." Science 170:637-9 [1970]; and A. 

   Drowart   ci      cil      "lsoelectrophoretic    characterization of protein antigens present in mycobacterial culture filtrates and recognized by monoclonal antibodies directed against the Mycobacterium   hrjvis    BCG antigen 85 complex." Scand. J. Immunol.. 36:697-702 [1992]). It is contemplated that in addition to the alpha antigen. antigens such as the 25 kD antigen described in Example 16 will be detection in the present invention.



   In contrast with the previous studies, the present invention provides a more sensitive method fur the reliable detection of infection and/or disease due to   At  



   Biological samples may be animal. including human. fluid or tissue.   fuod    products and ingredients such as dairy items, vegetables, meat and meat by-products. and waste.



  Environmental samples include environmental material such as surface matter. soil. water. and industrial samples, as well as samples obtained from food and dairy processing instruments.



  *apparatus. equipment, disposable. and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.



   Whether biological or environmental. a sample suspected of containing microorganisms may (or may not) first be subjected to an enrichment means to create a "pure culture" of microorganisms. By "enrichment means" or "enrichment   trcatment."    the present invention contemplates (i) conventional techniques for isolating a particular microorganism of interest away from other microorganisms   bv    means of liquid. solid. semi-solid or any other culture medium and/or technique. and (ii) novel techniques for isolating particular microorganisms awav from other microorganisms. It is not intended that the present invention be limited   onl    to one enrichment step or type of enrichment means.

   For example. it is within the scope of the present invention following subjecting a sample to a conventional enrichment means. to subject the resultant preparation to further purification such that a pure culture of a stniin of a species of interest is produced. This pure culture may then be analyzed   bv    the medium and method of the present invention.



   As used herein. the term "culture" refers to any sample or specimen which is suspected of containing one or more microorganisms. "Pure cultures" are cultures in   which    the organisms present are only of one strain of a particular genus and species.   Fhis    is in contrast to "mixed cultures,"   which    are cultures in which more than one genus and/or species   ot    microorganism are present.



   As used herein. the term "organism" is used to refer to any species or type   ot    microorganism. including but not limited to bacteria. yeasts and other fungi. As used herein.



  the term fungi, is used in reference to eukaryotic organisms such as the molds and veasts.



  including dimorphic fungi.



   As used herein. the term "Mycobacterium" is used in reference to the genus
Mycobacterium. the only genus in the familv Mycobacteriaceae. As used herein. the term "mycobacteria" is used in reference to all of the organisms included within the genus
Mycobacterium. including all of the taxonomic levels lower than genus. including. but not limited to species. subspecies. strains. etc. As used herein.   .\1      tuterculoxis    is used in reference to all of the species included within the   "At      tzibet.cuh)sis    complex." including At  tuberculosis.   Af.    bovis.   V    microti.   At      cifricanuni,    and any other organism subsequently recognized as falling within this taxonomic group.



   As used herein. the terms "microbiological media" and "culture media." and "media" refer to any substrate for the growth and reproduction of microorganisms. "Media" may be used in reference to solid plated media which support the growth of microorganisms. Also included within this definition are semi-solid and liquid microbial growth systems including those that incorporate living host organisms, as well as any type of media.



   As used herein. the term "primary isolation" refers to the process of culturing organisms directly from a sample. Thus, primary isolation involves such processes as inoculating an agar plate from a culture swab. urine sample environmental sample. etc.



  Primary isolation may be accomplished using solid or semi-solid agar media. or in liquid. As used herein. the term "isolation" refers to any cultivation of organisms. whether it be primary isolation or any subsequent cultivation. including "passage" or "transfer"   of    stock cultures   of    organisms for maintenance and/or use.



   As used herein. the term "presumptive diagnosis" refers to a preliminary diagnosis which gives some guidance to the treating physician as to the etiologic organism involved in the patient's disease. Presumptive diagnoses are often based on "presumptive identifications." which as used herein refer to the preliminary identification of a microorganism based on observation such as colony characteristics, growth on primary isolation media, gram stain results. etc.



   As used herein. the term "definitive diagnosis" is used to refer to a final   diagnosis    in   which    the etiologic agent of the   paticnt's    disease has been identified. The term "definitive identification" is used in reference to the final identification of an organism to the genus and/or species level.



   As used herein. the terms "digestion" and "decontamination" are used in reference to the standard methods used by mycobacteriology laboratories to treat samples such as sputum prior to culturing or testing the samples.



   As used herein. the term "antibody" is used in reference to any immunoglobulin molecule that rcacts with a specific antigen. It is intended that the   tenn    encompass any    immunoglobulin (e.g., , IgG. IgM. IgA. IgE. IgD. etc.) obtained from any source (c.g . humans.   



  rodents. non-human primates. caprines. bovines. equines. ovines. etc.).



   As used herein. the term "antigen" is used in reference to any substance that is capable of reacting with an antibody. It is intended that this term encompass any antigen and   "immunogen"   (i.e.,    a substance which induces the formation of antibodies). Thus. in   all    immunogenic reaction. antibodies are produced in response to the presence of an antigen or portion of an antigen.



   As used herein. the terms "antigen fragment" and "portion of an antigen" are used in   reference    to a portion of an antigen. Antigen fragments or portions may occur in various sizes. ranging from a small percentage of the entire antigen to a large percentage, but not 100% of the antigen. However. in situations where at least a portion of an antigen is specified. it is contemplated that the entire antigen may be present. It is contemplated that antigen fragments or portions. may, but are not required to comprise and "epitope" recognized by an antibody. Antigen fragments or portions also may or may not be immunogenic.



   As used herein. the term "immunoassay" is used in reference to any method in which antibodies are used in the detection of an antigen. It is contemplated that a range of   immunoassav    formats be encompassed by this definition. including but not limited to direct   immunoassays.    indirect immunoassays. and "sandwich" immunoassays." A particularly preferred format is a sandwich enzyme-linked immunosorbent assay (ELISA). However. it is not intended that the present invention be limited to this format.

   It is contemplated that other formats. including radioimmunoassays   (RI A).    immunofluorescent assays   (I FA).    and other assay formats, including. but not limited to. variations on the ELISA method will be useful in the method of the present invention Thus. other antigen-antibody reaction formats may be used in the present invention. including but not limited to "flocculation"   (it..    a colloidal suspension produced upon the formation of antigen-antibodv complexes). "agglutination"   (i e.   



  clumping of cells or other substances upon exposure to antibody), "particle agglutination"   (it..    clumping of particles coated with antigen in the presence of antibody or the   clumping    of particles coated with antibody in the presence of antigen): "complement fixation" (i.e.. the use of complement in an antibody-antigen reaction method). and other methods commonly used in serology, immunology, immunocytochemistry,   histochemistry.    and related fields.



   As used herein, the term "cell staining" is used in reference to methods used to label or stain cells to enhance their   visualization.    This staining or labelling may be achieved through the use of various compounds. including but not limited to. fluorochromes. enzymes.



  gold. and iodine. It is contemplated that the definition encompasses such methods as   "/n      situ    chromogenic assays." in which a test   (it..    an assay) is conducted on a sample in   sites.    It is also contemplated that the in situ chromogenic assay will involve the use of an   immunoassav      (ie..    an ELISA).  



   As used herein. the term "capture antibody" refers to an antibody that is used to bind an antigen and thereby permit the recognition of the antigen by a subsequently applied antibody. For example. the capture antibody may be bound to a microtiter well and serve to bind   mycobacterial    antigens present in a sample added to the well. Another antibody (termcd the "primary antibody") is then used to bind to the antigen-antibody complex, in effect to form a "sandwich" comprised of   antibody-antigen-antibody    Detection of this complex can be performed by several methods. The primary antibody may be prepared with a label such as biotin. an enzyme. a fluorescent marker, or   radioactivity,    and may be detected directly using this label.

   Alternatively, a labelled "secondary antibody" or "reporter antibody" which recognizes the primary antibody may be added, forming a complex comprised of   antibody      antigen-antibody-anti body.    Again. appropriate reporter reagents are then added to detect the labelled antibody. Any number of additional antibodies may be added as desired. These antibodies may also be labelled with a marker. including. but not limited to an enzyme.



  fluorescent marker. or   radioactivity.   



   As used herein, the term "reporter reagent" or "reporter molecule" is used in reference to compounds which are capable of detecting the presence of antibody bound to antigen. For example. a reporter reagent may be a colorimetric substance which is attached to an enzymatic substrate. Upon binding of antibody and antigen. the enzyme acts on its substrate and causes the production of a color. Other reporter reagents include, but are not limited to fluorogenic and radioactive compounds or molecules. This definition also encompasses the use of biotin and avidin-based compounds   (e.g.,    including compounds but not limited to neutravidin and streptavidin) as part of the detection system. In one embodiment of the present invention. biotinylated antibodies may be used in the present invention in conjunction with avidin-coated solid support.



   As used herein the term "signal" is used in reference to an indicator that a reaction has occurred. for example. binding of antibody to antigen. It is contemplated that signals in the form of   radioactivity.    fluorogenic reactions. and enzymatic reactions will be used with the present invention. The signal may be assessed quantitatively as well as qualitatively.



   As used herein, the term "amplifier" is used in reference to a system which enhances the signal in a test method such as an ELISA.



   As used herein, the term "solid support" is used in reference to any solid material to which reagents such as antibodies. antigens, and other compounds may be attached. For example. in the ELISA method. the wells of microtiter plates often provide solid supports.  



  Other examples of solid supports include microscope slides. covcrslips. beads, particles. cell culture flasks. as well as many other items.



   As used herein. the term "kit" is used in reference to a combination of reagents and other materials. It is contemplated that the kit may include reagents such as capture antibody.



  reporter antibody. and amplifier.



  DETAILED DESCRIPTION OF THE INVENTION
 The present invention provides compositions and methods for the rapid detection of mycobacterial antigens in such samples as blood. serum, urine. sputum. and culture filtrates.



     '1herefore,    this invention provides significant advantages in the diagnosis. monitoring, and treatment of patients with   infcctions    and/or disease with such organisms as   ,.      tuberculosis    and MAC.



   In particular. the present invention provides methods and   compositions    tor an amplified ELISA method for the detection of the mycobacteria. In this method. two antibody preparations are used. namely a "capture" antibody and a "reporter" antibody (or "antibody pair"). The capture antibody is used to "capture" mycobacterial antigens present in a sample.



  in order to permit their detection upon the addition of the reporter antibody and amplifier system.



   In a particularly preferred embodiment. the present invention provides compositions and methods for the detection of alpha antigen present in patient samples or other specimens.



  It is contemplated that the   method    be used to detect either the antigen or specific epitopes within the alpha antigen. It is not intended that the invention be limited to particular   cpitopes    of the alpha antigen. Thus. it is also not intended that the present   invention    be limited to particular antibodies. For example. the capture antibody may be a monoclonal antibody directed against alpha antigen of   M.      tubercuThsis.    while the reporter antibody is a   polvclonal    or monoclonal antibody directed against an organism. such as   V      bovine    BC(;.



   Although embodiments have been described with some particularity. many modifications and variations of the preferred embodiment are possible without   deviating    from the invention.  



  EXPERIMENTAL
 The following examples are provided in order to demonstrate and further illustrate certain preferred   embodimcnts    and aspects of the present invention and are not to be construed as limiting the scope thereof.



   In the experimental disclosure which follows. the following abbreviations apply: eq (equivalents); M (Molar);   ttM    (micromolar); N (Normal); mol (moles); mmol (millimoles):   pLmol    (micromoles); nmol (nanomoles); g (grams); mg (milligrams);   ttg    (micrograms): ng (nanograms); pg (picograms); 1 or L (liters); ml (milliliters);   Cil    (microliters): cm (centimeters); mm (millimeters):  m (micrometers): nm (nanometers): OD (optical density);
OD492 (optical density at 492   nm):     C (degrees Centigrade); PPD (purified protein derivative of   Ail.    tuberculosis. Connaught Laboratories. Ontario. Canada); PBS (phosphate   buffered    saline.

   Sigma); CHAPS detergent (3[3(3-Cholamidopropyll)diethylammonio]-1-propanesulfonate); BSA (bovine serum albumin. Pierce); Tris (TRIZMA. Sigma): RPM (revolutions per minute): TBS (Tris buffered saline 0.05 M pH 8.0. Sigma). The following commercial enterprises or governmental agencies are identified as follows: Amersham (Amersham   I,ife   
Science. Arlington Hts. IL); ATCC (American Type Culture Collection. Rockville.   Marvland);   
Becton Dickinson (Becton Dickinson Diagnostic Systems   Instrumcnt    Systems. Sparks. MD;
Becton Dickinson Microbiology Systems. Cockeysville, MD); BioRad (BioRad,   Hercules.   



  CA);   Biowhittaker    (Walkersville. MD); Boehringer Mannheim (Indianapolis. IN): DAKO (DAKO. Carpinteria, CA); Difco (Difco Laboratories. Detroit. MI); Fisher (Fisher Scientific.



  Pittsburgh. PA); Isolator (Wampole Laboratories. Cranbury.   NJ):    NCBI (National   Ccnter    for
Biotechnology Information. Bethesda MD. http://www3.ncbi.nlm.nih.gov/Entrez/index.html):
NLM (National Library of Medicine. Bethesda MD): Pierce (Pierce. Rockford. IL): S & S (Schleicher  &  Schuell. Keene. NH); Sigma (Sigma Chemical Co., St. Louis. MO.): PGC
PGC Scientifics, Gaithersburgh, MD); Spectrum (Spectrum Medical Industries, Los Angeles.



  CA); U.S. Biochemical   (U.S.    Biochemical   Corp..    Cleveland.   OH);    Cambridge (Cambridge
Diagnostics. Cambridge, MA); and Molecular Devices (Molecular Devices. Menlo Park.   CA).   



  Unless otherwise indicated. all of the chemicals used in the following Examples were obtained from Sigma.  



   EXAMPLE 1
 Growth of Mycobacterium   txberculosis,    Preparation of Culture Filtrates,
 Purification of Alpha Antigen, and Confirmation by Western Blot
 In this Example, cultures of M. tuberculosis were grown in order to produce culture filtrates for detection of alpha antigen.   M      tuberculosis    strain H37Rv was obtained from
ATCC. Although ATCC   fit27294    was utilized in this Example, it is contemplated that other deposits of H37Rv (e.g., ATCC   tut25618.    35800. etc.) will be suitable for use in the present invention. Bacilli were cultured in Proskauer Beck medium in roller bottles at   370C.    at   r   
RPM. in a humidified atmosphere for 10 weeks.

   Bacilli were removed by filtration using filter paper with progressively smaller pore size filters, up to 0.2   p    (S  &  S). Ammonium sulfate (Sigma) was added to the liquid spent medium to a final concentration of 60% saturation at   3'1C.      'he    filtrate was   refrigerated    with constant magnetic stirring overnight. The resulting precipitate was sedimented at 2000 RPM. The liquid was removed. the precipitate resuspended in distilled water. and then was dialysed against a water bath using a   lOkD    cutoff   dialvsis    membrane (Spectrum).



   The precipitated protein was applied to a diethylaminoethyl cellulose column (Sigma).



  washed with 0.01 M phosphate buffer, and then eluted with a linear gradient of from ().01 M to 0.3 M sodium phosphate. Alpha antigen eluted at a salt concentration of 0.07 M. The antigen was concentrated by   lyophilization,    and then applied to a preparative isoelectric focusing cell (Rotofor chamber. Biorad), with 10 mg CHAPS detergent (Bio-Rad). 5 ml glycerol. and 1 ml pH 3-10 ampholytes (Bio-Rad), 1 ml pH 5-7   ampholvtes    (Bio-Rad) and a final volume of 55 ml. A constant power of 12 W was applied for 5 hr. The fractions containing alpha antigen were identified by SDS-PAGE and Western blot as described below.



  Selected fractions were then further fractionated by preparative SDS-PAGE using a PrepCell column (Bio-Rad) using a 10% acrylamide gel according to the manufacturer's recommendations. Protein fractions were collected from this column and were simultaneouslv concentrated and   dialvzed    using a cylindrical membrane device with a   l0kD    cutoff (Micro
ProDiCon. Spectrum). Fractions containing pure alpha antigen were identified   bv    SDS
PAGE. colloidal gold stain (Aurodye. Amersham), and Western blot.



   SDS polvacrylamide gel electrophoresis was performed using 10% acrylamide in the running gel. and 4% acrylamide in the stacking gel. in a   Mini-Protean    apparatus   (Biorad)    as suggested by the manufacturer. The protein to be electrophoresed was mixed with an equal  volume of reducing sample buffer (Tris 1.51 g, pH 6.75, 4% SDS. 20% glycerol. and 10   p1      2-mercaptoethanol    suspended to 100 ml in H,O), and boiled for 2 minutes prior to application to the gel. After electrophoresis. the sample was transferred to 0.2 It pore size nitrocellulose paper (S & S).



   Western blotting was performed by blocking nonspecific binding with 5% bovine fetal serum (BioWhittaker) and 5% bovine serum albumin (BSA. Sigma) in phosphate buffered saline   (Sigma).    The antibody was diluted from 1:10 to   1:5000,    as needed (i.e., depending upon the starting concentration), in wash buffer with   1%    BSA. and was incubated with the nitrocellulose blot overnight with constant rocking at 0.5 RPM at room temperature. The blot was washed twice for 5 minutes in PBS with Tween 20   0.1%    (Sigma). An alkaline phosphatase conjugated secondary antibody (Sigma) was diluted 1:500 in PBS with   1%    BSA and incubated for 6 hr with rocking at room temperature. The blot was washed twice as abovc.

   The blot was then added to 30 ml of a solution of 100 mM Tris. 100 mM NaCI. 5 mM MgCl2, with 50 mg nitroblue tetrazolium and 5 mg   5-bromo-4-cl1loro-'r-illdoyl    phosphate. Development was stopped by washing in water.



   These methods were satisfactory for purification of alpha antigen (85-B) from its related antigen 85-A, which differs only slightly in molecular size but more substantially by isoelectric point. A blot of   NJ      tuberculosis    alpha antigen purified by this approach is shown in Figure 1. In this Figure, lane "a" contained M. tuberculosis culture filtrate as the antigen and the stain on this lane was colloidal gold. Lane "b" of Figure 1 contained alpha antigen purified as described above and the stain was also colloidal gold. Lane "c" of Figure 1 contained alpha antigen identical to lane b and stained with   biotinvlated    TBC27.



   EXAMPLE 2
 Production of Murine Monoclonal Antibody
 The monoclonal antibody designated as TBC27 was produced for use in the ELISA method of the present invention. This monoclonal was developed by immunization with
At tuberculosis alpha antigen obtained using the methods of Example 1. Six week-old female   Balblc    mice (Charles River, MA) were immunized with 50  g alpha antigen in incomplete
Freund's adjuvant (DIFCO.   Detroit,    Ml). Twice. animals were boosted with additional 50   pg    of antigen at two week intervals, and sacrificed 2 weeks after the last boost. Spleen cells were fused to SP2/0 Ag 14 myeloma cells (ATCC) using polyethylene glycol (Sigma). and  were propagated in Dulbecco's modified Eagle medium (Sigma) with added hvpoxanthine.



     aminopterin.    and thymidine. Supernatants of wells with visible growth were tested for antibody by ELISA. The wells of Immulon 2 plates (PGC) were sensitized overnight   (i.e.,    12-18 hours), with 50   1l      M.    tuberculosis filtrate (10   ng/ml)*    and 25  l of 3% glutaraldehyde in 0.15 M sodium phosphate. The plates were washed with wash buffer (TBS with   0. 1%   
Tween-20). Supernatants from   hvbridoma    wells were added to the wells and allowed to incubate overnight at room temperature. with rocking at 5 RPM. The plates were washed with wash buffer.

   Antibody was detected using alkaline   phosphatase-conjugated    anti-mouse
Ig (Sigma) and alkaline phosphatase. as recommended by the manufacturer (Sigma). Wells found to be positive for antibody production were expanded in culture. through the use of standard methods for expansion of monoclonal antibody-producing clones. The supernatants were collected.   lyophilized.    and dialyzed. The antibody was biotinylated using the
ImmunoPure NHS LC-biotin kit (Pierce) and the manufacturer's recommendations.



   An immunoadsorbent column was prepared using ImmunoPure immobilized strepavidin (Pierce) according to the manufacturer's recommendations. Biotinylated TBC-27 was passed twice over the column. The column was regenerated between passes. according to the manufacturer's recommendations. The preadsorbed   Tl3C-97    was used in subsequent studies for the antigen detection ELISA. as described below.



   EXAMPLE 3
 Detection of Alpha Antigen in
 M.   tuberculosis    Culture Filtrates
 In this Example, an ELISA method (C.J. Stanley   ci      cit,      enzyme    amplification can enhance both the speed and the sensitivity of immunoassays." J. Immunol.   Meth.    83:89-95   [1985]:    and C.H. Self, "Enzyme amplification--a general method applied to provide an immunoassisted assay for placental alkaline phosphatase." J. Immunol. Meth.,   76:389-')3    [1985]) that substantially increases the speed and sensitivity of ELISA. was extensively modified for use for the detection of alpha antigen, including the addition of another step.

   In this method, an alkaline phosphatase conjugate was used to convert NADP   to NAL).      which    then entered a regenerating cycle leading to production of a colored formazan dye. A schematic of the reaction is shown in Figure 2. Its use in the alpha antigen assay has resulted in over a 1000 fold reduction in the lower limit of detection of the assay (i.e.. from 20   ng/ml.     



  to from 1 to 10 pg/ml). The samples used in this pilot study were culture filtrates obtained from cultures of   At    tuberculosis grown as described in Example 1.



   In this method, 100   til      biotin!rlated    TBC27 diluted 1:1000 in BSA blocker (Pierce) in
TBS was added to each well of neutravidin-precoated ELISA plates (commercially available from Pierce). After 2 hr. the plate was washed with wash buffer (TBS/Tween 20   0. 1%).   



  Further nonspecific binding was inhibited by adding 300   ttl    SuperBlock (Pierce) twice. as per the manufacturer's recommendations.



   Standards   (Af      tuberculosis    filtrate, with a 10% alpha antigen content as determined by densitometry of colloidal gold stained gels) and samples were diluted with equal volumes of an appropriate buffer depending on the nature of the experiment. For example. in assays comparing mycobacterial filtrates of different species, or of sputum samples, the diluent was
TBS. For serum or urine, the diluent was pooled serum or urine (respectively) obtained from a group of healthy individuals with no known TB exposure. no residence in a   TB    endemic area. no history of BCG vaccination. a PPD skin test reaction of   0    mm induration within the past vear. and no history of a reaction of greater than 0 mm in the past. 

   The diluted standards, and the samples. were then diluted with an equal volume of BSA blocker in TBS.



  One hundred   Cil    was then added to each well. Standards were tested in duplicate, and samples were tested in individual wells. The plate was incubated overnight on a rocking table at 5
RPM in a humidor. The plate was washed 3 times with washing buffer. 100   ptl    of 1:1000 anti-BCG rabbit serum (commercially available from DAKO) preabsorbed with biotinylated   TBC27    using an avidin column as described in Example 2. was added and allowed to incubate at room temperature overnight (i.e.,   1 standard curve relating concentration to optical density was generated using SoftMax computer software (Molecular Devices).



   Because background signal is often the limiting factor in amplified assays. several measures were undertaken to reduce background signal in this assay. First, the antiserum used for detection (anti-BCG rabbit serum) of the alpha antigen was reabsorbed by passing it over a streptavidin column to which biotinylated   TBC27      (ie..    the primary antibodv) had been coupled. This was done to remove nonspecific reactivity against mouse IgG as well as specific alpha-anti-idiotypic antibody. Second, a conjugated anti-rabbit-Ig was selected   which    had been depleted of reactivity with murine and human   IgG.    Third. biotin/avidin was used to fix the capture antibody to the plate; this in turn allowed for more vigorous washing protocols with a detergent-containing buffer.

   Fourth, particular blocking reagents (BSA Blocker and
SuperBlock. Pierce) were used. and albumin devoid of alkaline phosphatase   activitv    was selected (Piercc). Finally. several sources of NADP were tested to identify reliable sources of
NADP which contained the least amount of contaminating NAD. In multiple reproductions of this Example. it was determined that the best source of   NAI)P    was   Boehringer.    whereas that from Sigma was inferior.



   The results of this example are shown in Figure 3. Data in this figure are presented on a log-log scale to show its wide dynamic range. The horizontal axis reflects total protein content of   M    tuberculosis culture filtrate: of this. alpha antigen represented approximately 10%. as estimated by densitometric measurement of protein-stained gels. Thus. the lower limit of detection of the assay was approximately 1   pglml.   



   EXAMPLE 4
 Recovery of M. tuberculosis Alpha Antigen
 in Spiked Samples
 In this Example. the same methods as described in Example 3 were used for testing of serum or urine. with the exception that   M    tuberculosis culture filtrates were added to normal pooled normal human serum or urine. as described above. This experiment was conducted in order to determine whether alpha antigen from At   tuberciiThsis    filtrate could be detected in such samples. The results of this experiment are shown in Figure 4. As can be seen from
Figure 4. the sensitivity of the assay was not substantially affected   bv    either diluent   (i.c..     



  normal serum or urine). However. the study suggests that separate diluents for standards be used. depending upon the type of clinical sample.



   EXAMPLE 5
 Radiometric Detection of M. tuberculosis
 Compared to Alpha Antigen Detection
 Radiometric detection of growth of mycobacteria is the standard to which clinical laboratories are held for rapid diagnosis of tuberculosis by accrediting agencies such as the
Joint Commission on Accreditation of   Healthcare    Organizations (JCAHO). Thus. it was of interest to compare a commonly used radiometric method with the ELISA method of the present invention for the detection of   M.    tuberculosis.



   For clinical samples of   inflected    body fluids   (e.g.,    sputum.   etc.).    standard methods of specimen processing are used. including decontamination and concentration. These samples are then inoculated into special radiometric medium which contains   l4C-radiolabelled    palmitic acid. For the BACTEC system (Becton Dickinson), metabolic activity of any organisms in the culture bottle results in production   of'4CO,.    The gas above the liquid medium in the bottle is sampled daily and the   '4C0    content measured. The result is expressed as a growth index (GI). GI values above 20-30 are considered positive. Cultures with positive growth indices must then be examined using other methods in order to identify the organism(s) present.



   As in the previous Example, BACTEC 14B bottles supplied by the manufacturer were inoculated with from   104    to 10' colony forming units (CFU) of   At      tztbessrc2al0.sis    strain   H37rv.   



  Growth indices were measured daily as recommended by the manufacturer: simultaneous samples were collected for measurement of alpha antigen using the ELISA method of the present invention. On each day of measurement, 0.2 ml of medium was removed from the
BACTEC bottle for measurement of alpha antigen, and an equal volume of fresh medium was replaced. The results of this study are shown in Figure 5. In Figure 5. solid data points represent growth index (GI) values, while the open data points represent alpha antigen content. The lower limit of detection of alpha antigen in this experiment was 10 pg/ml.



  Clinical samples are generally scored as positive when growth indices reach 30. The time to positivity of the two assays is summarized in Table 1:  
TABLE 1. Comparison of BACTEC and Amplified ELISA
EMI42.1     


<tb> Inoculum <SEP> Days <SEP> to <SEP>    Gl > 30    <SEP> Alpha <SEP> Antigen <SEP> Days <SEP> to <SEP> Alpha
<tb>  <SEP> Concentration <SEP> at <SEP> Antigen <SEP>  > 10 <SEP> pg
<tb>  <SEP> GI=30
<tb>  <SEP> 104 <SEP> 5 <SEP> 61 <SEP>  < 4
<tb>  <SEP> 10  <SEP> 8 <SEP> 205 <SEP> 6
<tb>  <SEP>    102    <SEP> 12 <SEP> 68 <SEP> 8
<tb>  <SEP> 10' <SEP> 17 <SEP>  > 800 <SEP> 13
<tb> 
 Thus. regardless of inoculum size. growth was detected from 2 to 4 days earlier by the antigen detection assay of the present invention, as compared to radiometric assay.



   EXAMPLE 6
 Radiometric Detection of   M.    avium
 Compared to Alpha Antigen Detection
 In view of the importance of M.   avium.    the methods described in Example 5 above were used with a culture of M.   uvium    replacing the M.   Iuberculosis    culture used in the previous Example. This M. avium culture was a clinical isolate obtained from the blood of an
AIDS patient with disseminated M.   avium    infection. This culture was propagated in
Proskauer Beck medium as described above. with the exception that   1 N,    dextrose was added to the culture medium.



   It was found that the amplified ELISA assay system was substantially less sensitive   for    detection of M.   aviunt    alpha antigen than for M. tuberculosis Antigen was detected only simultaneously or within one day of positive GI values. Despite more rapid growth of
M. avium. antigen levels were less than   10%    of those found with   M.    tuberculosis. Table 2 shows the results observed in this experiment.  



  TABLE 2. M. avium Detection
EMI43.1     


<tb>  <SEP> Days <SEP> to <SEP>    A <SEP> p <SEP> a    <SEP> Antigen
<tb> Initial <SEP> CFU <SEP> Days <SEP> to <SEP> GI <SEP>  > 30 <SEP>     > 10    <SEP> pg/ml
<tb>  <SEP> 104 <SEP> 4 <SEP> 4
<tb>  <SEP> 10' <SEP> 5 <SEP> 5
<tb>  <SEP>    10'    <SEP> 6
<tb>  <SEP> 10' <SEP> 8 <SEP> 7
<tb> 
 Nonetheless. these results indicate that the present invention may be used for the detection of alpha antigen in culture filtrates.



   EXAMPLE 7
 Alpha Antigen Detection in   Urine    and Serum
 of HIV-Negative Tuberculosis Patients
 In this Example, urine and serum obtained from Ugandan tuberculosis patients not infected with   HI V.    as well as   tuberculosis-negative.    HIV-negative controls were tested using the amplified ELISA assay described in Example 3.



   These HIV-negative patients with tuberculosis had presented to the Uganda TB Control
Programme referral center at Mulago Hospital. Kampala. the clinical site of the Case Western
Reserve University TB Research Unit. Pulmonary tuberculosis was diagnosed on the basis of a compatible chest radiograph. a positive sputum AFB smear. and confirmatory growth of   At    tuberculosis on culture of sputum. These patients were determined to be   IIIV-1    uninfected by commercial serum ELISA test system (Cambridge). Urine and serum specimens were obtained for antigen detection assay prior to initiation of therapy for tuberculosis.



   Control subjects without tuberculosis were identified from among patients under treatment for conditions other than tuberculosis. at the Pulmonary Clinic at Mulago Hospital.



  Their diagnoses were asthma and chronic bronchitis. None of these patients were known or suspected to be HIV-infected. Given the prevalence of HIV infection in Kampala (approximately 8%) it is   unlikelv    that more than one subject in this group was HIV-infected.



     However.    in view of the prevalence of PPD skin test reactivity in Kampala (approximatelv 70% in HIV uninfected persons: reviewed in M.   Sehulzer    et al., "An estimate of the future size of the tuberculosis problem in sub-Saharan Africa resulting from   IIIV    infection." Tuber.



  Lung   Dis..    73:52-8   [1992],    and the published erratum which appears in Tuber. Lung Dis..  



  73(4):245-6 [1992]), it is likely that 14 of the subjects in this group had been infected with
M.   tuberculosis.    Assuming a lifetime risk of tuberculosis of 10% in At   tuberculosis-infected      HlV-negative    persons. and 70% in   M      tuberculosis-infected    HIV-positive persons. it is likely that approximately one subject in this cohort will develop tuberculosis in the future due to recrudescence of latent infection.



   The amplified ELISA test system of Example 3 was used to test serum and urine of these patients.   For    this experiment, cutoff values of 2   ng/ml    and 20 pg/ml for urine and sputum. respectively, were selected. At these values, the sensitivity and specificity of the assay with these specimens were determined. For serum samples from non-tuberculosis patients. 21 were found to be negative, while one was positive. For serum samples from tuberculosis patients,   1 3    were found to be positive, while seven were found to be negative.



  Thus. for serum samples. the sensitivity was 35%, and the specificity was   95%.    For urine samples from non-tuberculosis patients. 21 were found to be negative. and none were found to be positive. For urine samples from tuberculosis patients. eight were found to be negative, while   1 3    were found to be positive. Thus, for urine samples. the sensitivity was 62%. and the specificity was 100%. Figure 6 graphically shows the results with the urine. while Figure 7 shows the results with the serum samples from these patients.



   EXAMPLE 8
 Alpha Antigen Detection in Urine
 of HIV-Positive Tuberculosis Patients
 In this study. urine samples from two cohorts of HIV infected subjects were tested using the ELISA assay described in Example 3. The first cohort was composed of Ugandan patients and the second was composed of HIV patients from Cleveland. Ohio. The Ugandan
HIV-infected patients were diagnosed with tuberculosis based on the observation of a tuberculosis-compatible chest radiograph, a positive sputum AFB smear. and confirmatory growth of   M.      Ilbereldlosis    on culture of sputum. These patients were determined to be HIV-1 infected by the same commercial serum ELISA test described in Example 7.



   A control Ugandan group. composed of Ugandan   HI V-infected    patients without tuberculosis was identified in the course of evaluation and enrollment in tuberculosis preventive therapy studies in Uganda. These individuals had originally presented at anonymous HIV testing centers in Kampala, were found to be seropositive. and were referred  to the Case Western Reserve University tuberculosis center for possible   enrollment    in preventive therapy studies. These subjects had normal chest radiographs. and had negative sputum AFB smears and cultures. The mean CD4 count of this group was   360/p1.   



   The second cohort included HIV-infected patients with disseminated   At      aviuni    infection identified   through    the Cleveland AIDS Clinic or hospital ward. on the basis of a positive blood. bone marrow. or lymph node culture. Three individuals were being treated for their MAC disease at the time the urine cultures were collected.. although one individual was included in this study two weeks before the diagnosis was established. Three additional individuals were studied at the time of diagnosis but prior to initiation of therapy.



   A control group of HIV-infected subjects without MAC or tuberculosis were identified at similar locations in Cleveland. Several subjects had multiple blood cultures for suspected
M.   civizim    disease. but none were positive. The mean CD4 count of this group was   280/tLl.   



     Uhe    same   cutoff    (2 ng/ml) was used as in Example 7. The results tor detection of urinary alpha antigen in this example are shown in Figure 8. These results indicated that the sensitivity (77%) was increased from that in   HI V-uninfec ted    subjects in Example 7. Apparent false positives were observed in 22 of 25 HIV-infected Ugandans without tuberculosis.



  However. using the estimates in this group. it is likely that 18 were actually infected with M.



  tuberculosis, and 12 may develop tuberculosis due to recrudescence of latent infection. The positive values in this subgroup may therefore reflect production of alpha antigen by tubercle bacilli contained within granulomas.



   EXAMPLE 9
 Detection of Alpha Antigen in Urine
 Subjects for this example were identified as follows: patient with untreated pulmonary tuberculosis were identified on the basis of a positive sputum acid fast smear with culture confirmation. and a chest radiograph compatible with pulmonary tuberculosis at the
Tuberculosis Treatment Centre. Kampala. Uganda. All subjects   with    tuberculosis were tested   lor    HIV by ELISA. A 10% random sample of positives were confirmed by Western blot for   qualitv    assurance (using a commercial kit from BioRad). Although CD4 cell counts were not determined in this group. recent studies performed in a similar cohort (newly diagnosed   HIV    pulmonarv tuberculosis) at this site found a median CD4 cell count   of 943/p1..     



   HIV-infected Ugandans without tuberculosis were enrolled through their participation in a placebo control arm of a study of preventive therapy for tuberculosis in HlV-infccted persons. These subjects had chest films and sputum cultures at regular intervals which were negative for tuberculosis. The median CD4 cell count of this group was 355/ L.



   Ugandans without tuberculosis. and who were not known to be HIV-infected. were recruited through the Chest Clinic at Mulago Hospital. Kampala. This group consisted of patients with chronic bronchitis or asthma. normal chest radiographs. not known to be HIV seropositive. and never having had   M.      tuberculo.sis    identified in sputum. and not known to ever have been treated with any antituberculous drugs. Based on the prevalence of HIV seroreactivity and tuberculin skin test reactivity in Kampala. approximately   1 5    (70%) of these subjects were likely to PPD positive. and no more than 1-2 (10%) were likely to be HIV seropositive. Approximately half of the Ugandan population was vaccinated once with BCG in infancy.

   However. tuberculin skin test reactivity in Ugandan adults is not affected by the administration of BCG during infancy (i.e., as indicated bv the presence of a BCG scar): this is thought to reflect infection with   V    tuberculosis.



   HIV-infected persons at low risk for tuberculosis were recruited at University
Hospitals. Cleveland. Ohio. through the AIDS Clinical Trials Unit. The incidence of tuberculosis among American-born persons in the greater Cleveland area was   3.2/1 00.000    in 1994. one of the lowest among metropolitan areas in the United Stales. Subjects from this site were studied if they were not known to have disseminated MAC, tuberculosis. or a history of a positive tuberculin skin test. and had not emigrated from a   countrv    with a high risk of tuberculosis. and had not been given preventive therapy for tuberculosis.

   The other   III V-    related infectious diagnoses in this group included six patients with Pneumocystis   cnuril?ii    pneumonia   (PCP).    five patients with   cytomegalovirus    (CMV) infection. eight patients   infected    with herpes simplex virus or herpes zoster. five patients with candidiasis. one patient with toxoplasmosis. one patient with syphilis, one patient with   cryptococcosis.    two patients with tracheobronchitis due to Xanthomonas or Pseudomonas. and two patients with unspecified hepatitis.   'l'he    median CD4 cell count of this patient group was   1 70/ttl.   



   Subjects from this site identified with disseminated   At    avium infection (MAC) were diagnosed on the basis of positive blood or lymph node culture in the setting of a compatible clinical presentation. Two of the 7 subjects had simultaneous blood cultures which were positive and had not yet begun treatment for MAC.   Tl1e    median CD4 cell count of this group was   20/I.     



   Persons at low risk for HIV infection were identified among laboratory and university personnel. Subjects in this group who had not previously had a positive tuberculin skin test.



  and who had not been tested within the past year. were skin tested tested after urine collection. Information was collected from these subjects regarding BCG vaccination and   country    of origin. Subjects were excluded if they had been given preventive therapy for tuberculosis.



   Tuberculin skin testing was performed by the Mantoux method using   S    T.U.



  (tuberculin units) with PPD. Induration was read at 48 hr. Results were scored as positive if induration was    > 5    or 10 mm in HIV-infected and uninfected subjects. respectively. In   health   
American subjects. only a   0    mm response was read as negative. and subjects with reactions of from 1 to 9 mm were not included in the study.



   Dual color flow   cytometrv    was performed for enumeration of CD3+ and CD4+ cells.



     Growth    of   At    tuberculosis on culture was confirmed by standard methods. based on colonial morphology on Lowenstein-Jensen medium, nitrate and niacin positivity, and resistance to 5%   thiopencarboxyl ic    acid   hvdrazine.   



   Alpha antigen was detected by ELISA using a   biotinviated    monoclonal antibody   TBC27    as described in Example 3. A standard curve was generated using serial dilutions of
M. tuberculosis culture filtrate in which the alpha antigen content was determined by densitometric analysis of colloidal gold stained blots. in parallel with Western blot using   TBC27.    as described in Example 1. Pooled urine from PPD skin test-negative healthy subjects was used as a diluent for the standard.   V    tuberculosis filtrate was prepared by precipitation in ammonium sulfate of spent Proskauer   Beck    medium from 8 week cultures of   At      titbcrciilosis    strain   H37Rv.    as previously described in Example 1.

   V avium filtrate was similarly prepared with the exception that the spent medium was supplemented with   I    dextrose (i.e., as described in Example 6.



   The relative specificity of the capture monoclonal antibody for alpha antigen of   M      tuberculosis    when tested by western blot is shown in Figure 9. As shown in this Figure.



  there is a band of reduced intensity in the M. aviwn lane. as compared with the   V      tuberculosis    lane. Representative standard curves using culture filtrates of M.   tuberculosis    and
M. avium are shown in Figure 10. In Figure   10.    the horizontal axis reflects total protein content of the respective mycobacterial filtrates. In   V    tuberculosis filtrate. alpha antigen represents approximately 10% of total protein. as estimated by densitometric measurement of colloidal gold-stained blots. Thus. the threshold for detection of alpha antigen in this assay  was approximately 10 pg/ml. although in some experiments it was found to be as low as I pg/ml.

   The mid-range sensitivity of the assay for M. avium was found to be   l9-fold    less sensitive than that for M.   luberclIosis.    when the two filtrates were compared based on total protein content. Approximately half of this difference can be accounted by the reduced alpha antigen content of M. avium filtrate (5% instead of 10% of total protein).



   At high protein concentrations. the assay was found to become progressively less sensitive for   i      civizini    filtrate. approaching a 100-fold difference at 1  g/ml. In effect. this limited the maximum response to   V      avium    filtrate to the equivalent of 1-2   nglml   
M. tuberculosis alpha antigen. Additional specificity for   At      tuberculosis    may arise from differential secretion of proteins by the two species. in that following infection with equal numbers of organisms in Proskauer-Beck medium, growth of   V      civiirni    is more rapid. but results in a lower concentration of secreted protein.



     Urinary    alpha antigen levels were then evaluated in several groups of subjects.



  identified as follows, and represented in Figures 11 and 12. The results varied according to the TB and HIV status of the subjects. Log-transformed urinary antigen determinations   from    these subjects are presented in these figures. so that diff'erences between groups can be distinguished over a wide range of values. As shown in these   Isigures,    among HIVseronegative subjects. antigenuria was greatest in subjects with active untreated pulmonary tuberculosis. In this group, the median value was 2.24 alpha antigen (per mg/ml). Sixty-two percent of this group had values of 2 ng/ml or greater. Intermediate values were observed in two groups of subjects with latent infection with M. tuberculosis.

   These groups included   Ugandan    pulmonary clinic subjects with asthma and bronchitis. and   healthy    PPD-positive personnel from the American universitv cohort. None of the subjects in either group had received preventive therapy for tuberculosis. The degree of antigenuria did not differ between these two   groups    (median values of 79 and 50   pg/ml.    respectively'. in Ugandan and American subjects). Urinary antigen was undetectable in all but 2 of the skin test negative health
American subjects. Only I of 18 subjects had antigenuria of  > 2 ng/ml.



   Among all HIV-seronegative subjects. the median values differed significantly among those with active tuberculosis. those with latent   infcction.    and those who   hy    skin test were uninfected. using the Kruskal-Wallis analysis of variance of ranks   (p < .001).    The nonparametric ANOVA equivalent was used because the data failed a test of   normality.    All three possible pair wise comparisons also differed significantly (p < .05. using Dunn's method).  



   Antigenuria was greater in the corresponding groups of HIV-infected subjects. For example. in HIV-infected Ugandans with and without tuberculosis. median urinary antigen levels were 4.3 and 8.3 ng/ml. respectively (p < .001 for both when compared to corresponding
HIV-negative groups). The median urinary alpha antigen value in AIDS patients with disseminated MAC was 510 pg/ml: in those without MAC, it was 79 pg/ml. Antigenuria of  >    2      ngiml    was observed in only one subject without epidemiologic or culture evidence for
M. tuberculosis infection. Among all HIV-positive subjects. the median values differed by
Kruskal-Wallis rank ANOVA (p < .001). Pairwise comparisons showed differences between either Ugandan group and either Cleveland group (p < .05), but not in other comparisons.



   In HIV-seronegative persons. values of    > 2    ng/ml were diagnostic of active tuberculosis with a sensitivity of 62% and a specificity of 98%. Intermediate values (between 33   pg/in    and 2   ng/ml)    were consistent with either latent infection or active disease. These findings suggest that the mycobacteria in latently infected persons are not dormant, but rather are metabolicallv active. Thus, it is contemplated that the antigen detection assay may have potential application to distinguish those skin test positive individuals with viable   At    tuberculosis   from    those whose infection has been eradicated. either by preventive therapy or a protective immune response. This may allow for greater selectivity in application of preventive therapy for tuberculosis.



   No antigen could be detected in approximately 10% of Ugandan tuberculosis patients regardless of HIV status. Antibodies to other pathogens such as HIV can be detected in urine: it is possible that pretreatment of urine specimens to dissociate alpha antigen immune complexes might increase the sensitivity of the assay. Alternatively. it is possible that these patients may be infected with another member of the At tuberculosis complex such as   At    africanum. about which relatively little is known regarding alpha antigen genetics or biology.

 

   Urinary antigen levels in HIV-infected persons were. in general, higher than in HIVseronegative persons, and the conclusions which can be drawn from test results are somewhat more limited. Values of  > 2 ng/ml were found only in subjects with active tuberculosis. or with likely exposure to tuberculosis based on epidemiologic factors. This degree of antigenuria in HIV-infected persons may be used as an indication for either tuberculosis chemotherapy or preventive therapy. depending on the results of additional tests. In this application. the antigen assay had a sensitivity of 84% and a specificity of 98%.



   Unlike in   HlV-seronegative    subjects. the degree of antigenuria in   HI V-infected    subjects did not differ between those with latent infection   vs.    active disease. The reason for  this is not known. Many of the   HI V-positive    subjects who were studied had very high antigen levels ( > 10 ng/ml). Thus. it is preferred that the sample contain antigen levels that are less than or equal to 10   nglml.    It is contemplated that for samples with very high antigen   levels    (i.e.,  >  10 ng/ml). dilution of the sample to an antigen concentration lower than 10   nglml    may be desirable prior to analysis.

   Alternatively. it is possible that in dually-infected (IIIV and
M. tuberculosis) persons. the total mycobacterial burden may not be the main determinant of clinical disease. and that other factors. possibly   immunologic.    may be important in   detcrmining    whether the infection causes clinical illness.



   Values between 33 pg and 2 ng/ml were identified in many HIV-infected Cleveland subjects who were without either active tuberculosis or epidemiologic evidence suggesting tuberculosis exposure. These levels were significantly greater than in HIV seronegative PPD skin test negative controls. The etiology of these results is not certain, but it may reflect latent infection with to   A!vcobacteriiini      aviutn.    The apparent clustering of values in the range of 1-2 ng/ml in samples from these subjects is consistent with the behavior of the   assay    with
M. avium filtrate. Disseminated infection with organisms of the Mycobacterium avium intracellulare complex (MAC) is the most common opportunistic infection late in the course of HIV disease.

   The   frequents    of disease due to MAC rises from 3% per year for individuals with CD4 counts of   100-200/p1    to   39%    at CD4 counts of    < 10/p1.   



   EXAMPLE 10
 Detection of Alpha Antigen in Sputum
 In this Example. detection of alpha antigen was studied in sputum obtained from patients at the initiation of anti-tuberculosis therapy. In this study. multiple sequential expectorated sputum samples were obtained during the first two weeks of anti-tuberculosis therapy from a group of HIV-uninfected AFB smear-positive subjects who presented to the
Case Western Reserve University tuberculosis research unit site at Vittoria. Brazil. The sputum samples were homogenized by adding 10 mg N-acetvl cysteine (Sigma) and 10 3mm diameter glass beads (Fisher) to each 5-10 ml sputum sample. The samples were vortexed at room temperature for 1 minute. Aliquots of 200  l were frozen at -70 "C until analysis. A total of 37 pretreatment specimens was obtained from 13 subjects.

   Using the amplified
ELISA method described in Example 3. the mean alpha antigen concentration in the positive  specimens was determined to be   5 1    pg/ml. Every subject had at least one positive pretreatment specimen for a total of   28137    or 76% positive.



   Subsequent sputum specimens were also available on days 2. 4. 7, and 14 post initiation of treatment, from 10 patients. The alpha antigen concentrations in these subsequent specimens from these subjects are shown in Figures 13-15. In these Figures, the results from one patient are shown in each panel. For three patients. only the pre-treatment specimen was determined to positive. and all subsequent specimens were negative. The results from this group of patients are shown in Figure 13, with Figure 13. panel A. showing the results for "Subject   1.";    Figure 13. panel B. showing the results for "Subject   10.";    and Figure 13. panel
C. showing the results for "Subject 11." In these patients. the mean initial value obtained in the amplified ELISA was determined to be 27+ 13 pg/ml.

   The length of time alpha antigen was detectable in these specimens expressed in terms of half-life was determined to be 0.6   davs.   



   In nine of the 10 patients for which multiple sputum were tested. at least one subsequent specimen was positive. The results for these nine patients are shown in Figure 14.



  in panels A-F. with one patient's results being shown in each panel. As can be seen from this
Figure. this group of patients tended to have higher initial antigen concentration than the first group. although this difference did not reach statistical significance   (i.c..    the mean initial value was 41.4+11 pg/ml). The rate of disappearance of antigen from subsequent specimens from this group was found to be slower than in the first group. with a half-life of 5.6 davs.



   In one patient. the level   of    sputum alpha antigen rose during treatment. as shown in
Figure   15.    Simultaneous quantitative CFU rose more than 10 fold during this interval.



  Quantitative   CI:U    did not increase in any of the other subjects during this interval.



   These results indicate that the amplified ELISA method for detection of alpha antigen in sputum is suitable for detection of infection and/or disease with   At    tuberculosis.



  Importantly, these results indicate that this ELISA method provides significant information for monitoring the progress of tuberculosis patients.  



   EXAMPLE 11
 Production of Other Monoclonal Antibodies for
 Alpha Antigen Detection
 In addition to   TBC27    described and used in Example 3 above. other antibodies were produced for potential application in the purification and detection of alpha antigen of   At      tlzbelculosis.    These antibodies were developed by immunization six week-old female
Balb/c mice (Charles River, MA) using the method of intrasplenic immunization (P.C.



  Svalander   ei      cit.    "Intrasplenic immunization for production of monoclonal antibodies against mouse blastocysts." J. Immunol. Meth.. 105:221-7 [1987]). Mice were immunized with 1  g alpha antigen obtained from M. tuberculosis culture filtrate as described in Example 1. The portion of the nitrocellulose paper containing the alpha antigen was excised and the paper allowed to dry at room temperature. The paper was dissoived in DMSO (Sigma). It was then precipitated by the dropwise addition of sodium carbonate 0.05 M (Sigma). pH 9.6 The precipitated particles were then washed 3 times in 2 ml   RPMI-1640    medium (BioWhittaker) and suspended in a final volume of 1 ml. The immunization was performed by injection into the spleen. using a 25 gauge needle.

   Animals were boosted with additional I  g twice at two week intervals. and sacrificed 2 weeks after the last boost. Spleen cells were fused to SP2/0
Ag 14   myeloma    cells (ATCC) using polyethylene glycol (Sigma) and were propagated in
Dulbecco's modified   Eagle    medium (Sigma) with added hypoxanthine. aminopterin. and thymidine. Supernatants of wells with visible growth were tested for antibody by ELISA using plates sensitized with   V    tuberculosis filtrate as described in Example 2. Positive wells were expanded in culture. and the supernatants collected and   lyophilized.    and the   antibody    was   biotinvlated    using a biotinylation reagent kit (Pierce) according to the manufacturer's recommendations, all as described in Example 2.



   Several antibodies specific for M. tuberculosis alpha antigen were developed using the methods of this Example. including the antibody designated as "5C5." and "8G104.4." As shown in the Western blot in Figure 16. SCS and   8G10.4.4    reacted only with culture filtrates of M.   tuberculosis    and not   M.    smegmatis or M. avium. Thus, it is contemplated that these antibodies will also be useful in the amplified ELISA assay and provide increased specificity.



  However. with 5C5. cross-reactivity was observed. as described in Example 12.  



   EXAMPLE 12
 Use of 5C5 for Detection of
 Alpha Antigen in Urine
 In this Example. antibody   5C5    produced as described in Example 11 above was tested for its ability to detect   At      zlbercufoxis    alpha antigen in urine. The amplified ELISA method and patient groups described in Example 7 were used, with the exception that instead of using
TBC27, 5C5 was used (i.e.,the samples were retested with   5C5    in place of TBC27).



   The results of this study are shown in Figures 17 and 18. As shown in Figure 17, this antibody was capable of detecting antigen in urine. However. as shown in Figure 18. an apparent specificity to   M.      Iuberczllosis    as determined by Western blot   (sce    Figure 16) does not ensure success in the diagnosis of tuberculosis, as cross   reactivity    can arise. In Figure 18.



  lane 1 contained   M.      Ilhercul(7.si.s.    while lane 2 contained   M.    avium filtrate. As shown in
Figure 18. when biotinylated 5C5 was substituted for TBC27 in the alpha antigen ELISA. the test was of less value than the use of TBC27 for tuberculosis diagnosis. regardless of IIIV status (i.e.. the results were of no value for either the HIV-positive or   111V-negative    patient groups). The spurious results were in part due to unexpected cross reactivity of the antibody with proteins expressed on the surface of normal human monocytes. detected by immunohistochemistry of infected cells.



   EXAMPLE 13
 Use of Synthetic Peptides to Produce
 Monoclonals Specific for Alpha Antigen of M.   tuberculosis   
 In this Example. the possibility of using synthetic peptides to produce antibodies directed against M. tuberculosis alpha antigen was investigated. The amino acid sequences of the alpha antigens of   V      avium    and M. tuberculosis are compared in Figure 19 (SEQ ID NOS: 1 and 2). In this Figure, residues which differ between the two species are emphasized in bold face. As shown in this Figure. extensive identity is apparent (87%) between the alpha antigens of these two species.



   Upon inspection. potentially species-specific peptides were identified beginning at amino acid residues 147 and 229 of   M.    tuberculosis (SEQ ID NOS: 3 and 4). SEQ ID NO: 3 is comprised of the amino acid sequence "QWLSANRAVKPTGSAAI": while SEQ ID NO: 4 is comprised of the amino acid sequence "ERNDPTQQIPKLVAN."  
 These peptides were hypothesized as representing   scctions    that were specific to particular species of   Mvcohacterium.    Searches were performed using the Entrez database search engine at the National Center for Biotechnology Information at the National   Librarv    of
Medicine. This search engine is based on a program developed by S.F, Altschul termed   "BLASTP."    which is used to identify homologous sequences in the combined PDB,
SwissProt. PIR.

   GenPept. and GPupdate databases (S.F. Altschul   ci      cit.    "Basic local alignment search tool," J. Mol. Biol., 215:403-10 [1990]). A facility to search these databases for homologous sequences has been established by the National Library of Medicine. and was accessed by the Internet at URAL   http://www3.ncbi .nlm.nih.gov/Entrez/index. html.    The search engine allows the user to supply an animo acid sequence. It returns a list of all potentially homologous sequences which have been stored in its database.   The    engine is designed to   identify    sequences which are identical. which have similar amino acid residues in terms of charge or size. which have intervening inserted sequences. or which have deletions.



  However. in the comparison. the "expect" search parameter was increased from a default   ot'    10 to 1000 to increase the reporting of even marginal matches.



   As a control. a similar search was performed for a   1 7    amino acid intervening sequence "DQFIYAGSLSALLDPSQ" (SEQ ID NO: 5) , which begins at residue 181 of the   Al.      vim    antigen sequence. and represents a consensus region that is shared among the species of the
Mycobacterium genus. This sequence differs by one amino acid from the homologous stretch in the   At      tubercidosis    amino acid sequence. In the M.   herclglo.si.s    sequence, the first "D" is replaced with a "Q"   (i.e..the    entire sequence is   "QQFIYAGSLSAI,I DPSQ"    (SEQ ID NO: 8).



   The results of these three searches are shown in the following Tables. These data were divided into four tables. The first table (Table 3) shows the comparisons for strains of mycobacteria. The second table (Table 4) shows the comparisons for various potential human pathogens. The third table (Table 5) shows the comparisons for various human peptides. The fourth table (Table 6) shows the comparisons for peptides from other organisms.



   The data in these Tables are displayed as the percent identitv/homology between the target sequences and its best match in another species. Those   matches    with  >  65% identity or  >  80% homology are highlighted in bold. Items marked with an asterisk (*) required folding of at least one sequence for optimal alignment: the degree of homology of these matches is likely overestimated. No homologous sequences were identified for those entries left blank.  



  Table 3. Search Results for Peptides from Various
 Mycobacteria and Three Peptide Sequences    of Alpha Antigen of M. tuberculosis
 (% identity/%homology)   
EMI55.1     


<tb>  <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP> 229 <SEP> Amino <SEP> Acid <SEP> 181
<tb> M. <SEP> tuberculosis:
<tb>  <SEP> 85-B <SEP> 100/100 <SEP> 100/100 <SEP> 100/100
<tb>  <SEP> 85-A <SEP> 71/76 <SEP> 58/76 <SEP> 70/88
<tb>  <SEP> 85-C <SEP> 59/76 <SEP> 76/88 <SEP> 53/65
<tb> M. <SEP> bovis:

  
<tb>  <SEP> 85-B <SEP> 100/100 <SEP> 100/100 <SEP> 100/100
<tb>  <SEP> Bovis <SEP> antigen <SEP> 71/76 <SEP> 58/76 <SEP> 70/88
<tb>  <SEP> 85-A <SEP> 71/76 <SEP> 58/76 <SEP> 70/88
<tb>  <SEP> MPB51 <SEP> 53/71
<tb>   M.    <SEP> avium <SEP> 85-B <SEP> 47/82 <SEP>    58/82    <SEP> 100/100
<tb> M. <SEP> intracellulare <SEP> 58/88 <SEP> 70/82 <SEP> 94/100
<tb> Alpha <SEP> Antigen
<tb>   NI.    <SEP> kansasii <SEP> 85-B <SEP> 88/100 <SEP> 70/88 <SEP> 94/100
<tb>   NJ    <SEP> scrofulaceum <SEP> 64/94 <SEP> 82/88 <SEP> 94/100
<tb> Alpha <SEP> Antigen
<tb> M. <SEP> leprae:
<tb>  <SEP> 85-A <SEP> 52/88 <SEP> 58/76 <SEP> 70/76
<tb>  <SEP> 85-B <SEP> 70/88 <SEP> 70/76 <SEP> 81/93
<tb>  <SEP> 85-C <SEP> 59/76 <SEP> 76/88 <SEP> 53/65
<tb>   
 Table 4.

   Search Results for
Various Human Pathogens and Three Peptide Sequences
 of Alpha Antigen of   M.      tuberclosis   
 (% identity/%homology)
EMI56.1     


<tb>  <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP> 229 <SEP> Amino <SEP> Acid <SEP> 181
<tb> Coxiella <SEP> trxB. <SEP>    spolIlE    <SEP> 35/59*
<tb> D-ala-D-ala <SEP> 41/76*
<tb> Carboxypeptidase
<tb> Aspergillus <SEP> P450 <SEP> 53 <SEP> 59/82*
<tb> E.

   <SEP> coli <SEP> tus <SEP>     <SEP> 35/53*    <SEP> 
<tb> Corynebacterium <SEP> | <SEP> | <SEP> 35/53
<tb>  <SEP> iron <SEP> protein
<tb> Simian <SEP> 59/82*
<tb> Paramyxovirus
<tb> Bacillus <SEP> cellulase <SEP> 41/65*
<tb> Bacillus <SEP>    # <SEP>     <SEP> endotoxin <SEP> 35/71
<tb> Human <SEP> Adenovirus <SEP> 53/76*
<tb> Human <SEP> Rotavirus <SEP> A <SEP> 59/59*
<tb> Human <SEP> Herpesvirus <SEP> 29/71
<tb> Hepatitis <SEP> C <SEP> 29/35*
<tb> Candida <SEP> p450 <SEP> 41/65
<tb> Saccharomyces <SEP> 53/82*
<tb> Yeast <SEP> DNA <SEP> helicase <SEP> 53/82*
<tb>   
 Table 5.

   Search Results for
Human Peptides and Three Peptide Sequences of M. tuberculosis
 (% identity/%homology)
EMI57.1     


<tb>  <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP> 229 <SEP> Amino <SEP> Acid <SEP> 181
<tb>   Tyrosine    <SEP> phosphatase <SEP> 41/76*
<tb> Testican <SEP> 65/71 <SEP> *
<tb> Isocitrate <SEP> 35/47
<tb> dehydrogenase
<tb> T-cell <SEP> receptor <SEP> 29/53
<tb> d <SEP> Chain
<tb> TEGT <SEP> (Testis <SEP> 53/59*
<tb> enhanced <SEP> gene
<tb> transcript)
<tb> Granulocyte <SEP> Colony <SEP> 41/53
<tb> Stimulating <SEP> Factor
<tb> 
 Table 6.

   Search Results for
Various Organisms and Three Peptide Sequences   
 of Alpha Antigen of M. tuberculosis
 (% identity/%homology)   
EMI57.2     


<tb>  <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP> 228 <SEP> Amino <SEP> Acid <SEP> 181
<tb> Simian <SEP> Herpesvirus <SEP>    59/76*    <SEP> 
<tb> Protein
<tb> Cladosporium <SEP> gag <SEP> 59/59
<tb> Protein
<tb> Arabidopisis <SEP> meri <SEP> 5 <SEP> 65/71*
<tb> Rice <SEP> Photosystem <SEP> II <SEP> 35/35*
<tb> Cyanophora <SEP> | <SEP> 47/47*
<tb>   Photosvstem    <SEP> II
<tb> Maize <SEP> Photosystem <SEP> 11 <SEP>    29/29*    <SEP> 
<tb> Wheat <SEP> Photosystem <SEP> 11 <SEP>    29/29*    <SEP> 
<tb> Shewanella <SEP> Fumarate <SEP> | <SEP> 47/59
<tb> Reductase
<tb> Rubus <SEP> 59/71*
<tb> Pyrophosphatase
<tb>   

  
 Table 6. Search Results for
Various Organisms and Three Peptide Sequences
 of Alpha Antigen of M. tuberculosis
 (% identity/%homology)
EMI58.1     


<tb>  <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP> 228 <SEP> Amino <SEP> Acid <SEP> 181
<tb> Mouse <SEP> Pax- <SEP> I <SEP> Protein <SEP> 41/41
<tb> Kluyveromyces <SEP> g6p <SEP> 41/51*
<tb>   I <SEP> somerase    <SEP> 
<tb> Cyanobacterium <SEP> ATP <SEP> | <SEP> 47/53
<tb> Synthetase
<tb> Alcaligenes <SEP> CnrB <SEP> 47/65
<tb> Caenorhabditis <SEP> 53/65 <SEP> 41/65
<tb> C14B9.5
<tb> Synechococcus <SEP> | <SEP> 47/53 <SEP> | <SEP> 29/41
<tb> Protein
<tb>   Metkvlophllus    <SEP> DCM <SEP> 47/65
<tb> Dehalogenase
<tb> Drosophila <SEP> .

   <SEP> 41/65 <SEP> 47/65
<tb> Xylano <SEP> Hydrolase <SEP>    41/65*    <SEP> 
<tb> Curcurbita <SEP> 59/59
<tb> Arabidopsis <SEP> 53/88*
<tb>   Gallus    <SEP> BRM <SEP> Protein <SEP> 35/53
<tb> Impatiens <SEP> Spot <SEP> Virus <SEP> . <SEP> 41/76
<tb> Phaseolus <SEP> Tonoplast <SEP> 41/71
<tb> 
 EXAMPLE 14
 Use of Synthetic Peptides to Produce
 Monoclonals Specific for Alpha Antigen of   M.      nvium   
 In this example, an alternative approach to identification of MAC-specific secreted antigens was designed based on identification of species-specific epitopes of characterized antigens. The amino acid sequences of alpha antigens of M. avium and At tuberculosis are  compared in Figure 19   (i.c.,    SEQ ID NOS: 1 and 2).

   As discussed in Example 13. extensive identity is apparent (87%).



   In this study, two candidate sequences were identified which might confer species specific responses to   At      crviurn    alpha antigen. The first. (SEQ ID NO: 6) with the sequence "SYLASNKGVKRTGNAAV." begins at residue 147. The second (SEQ ID NO: 7) . with the sequence   "QRNDPSLMIPELVGH."    begins at residue 229. To determine the potential for broad species specificity in these regions. searches were performed with these amino acid sequences using BLASTP to identify homologous sequences in the combined PDB,   SwissProt.   



  PIR. GenPept. and GPupdate databases at the NCBI as described for the above Example. The "expect" search parameter was increased from a default of 10 to 1000 to increase the reporting of even marginal matches.



   As a control. a similar search was performed for a   1 7    amino acid intervening sequence (SEQ ID NO: 5). with the sequence "DQFIYAGSLSALLDPSQ." which begins at residue 181 of the   V      civizini    alpha antigen sequence. and differs bv only one amino acid from the homologous   At      tuberculosis    sequence (SEQ ID NO: 8). As indicated above. this stretch represents a consensus region.



   The results of these three searches are   shown    in the following Tables. These data were divided into four tables. The first table (Table 7) shows the comparisons for strains of mycobacteria. The second table (Table 8) shows the comparisons for various potential human pathogens. The third table (Table 9) shows the comparisons for various human peptides. The fourth table (Table 10) shows the comparisons for peptides from other organisms.



   The data in these Tables are displayed as the percent   identity/homology    between the target sequences and its best match in another species. Those matches with  >  65% identity or  >  80% homology are highlighted in bold. Items marked with an asterisk (*) required folding of at least one sequence for optimal alignment: the degree of homology of these matches is likely overestimated. No homologous sequences were identified for those entries left blank.  



   Table 7. Search Results for Peptides from Various
Mycobacteria and Three Peptide Sequences of Alpha Antigen of M. avium
 (%   identity/ /Ohomology)   
EMI60.1     


<tb>  <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP>    229    <SEP> Amino <SEP> Acid <SEP> 181
<tb> M. <SEP> avium <SEP> Alpha <SEP> 100/100 <SEP>    1001100    <SEP> 100/100
<tb> Antigen
<tb> M. <SEP> intracellulare <SEP> 82/88 <SEP> 80/86 <SEP> 88/100
<tb> Alpha <SEP> Antigen
<tb> M. <SEP> hovis:

  
<tb>  <SEP> 85-A <SEP> 47/71 <SEP> 46/80 <SEP> 71/88
<tb>  <SEP> 85-B <SEP> 47/82 <SEP> 94/94
<tb>  <SEP> 85-B <SEP> 47/82 <SEP> 53/80 <SEP> 94/94
<tb>  <SEP> BCG <SEP> Antigen <SEP> 47/71 <SEP> 46/80 <SEP> 71/88
<tb>  <SEP> MPBS <SEP> 1 <SEP> 47/76 <SEP> 53/71
<tb>  <SEP> MPB70 <SEP> 41/53*
<tb>  <SEP>    /MPB8O    <SEP> 
<tb> M. <SEP>    karn'asii    <SEP> 85-B <SEP> 53/82 <SEP> 87/93 <SEP> 88/94
<tb> M. <SEP>    leprac:

      <SEP> 
<tb>  <SEP> 85-A
<tb>  <SEP> 65/76 <SEP> 76/82
<tb>  <SEP> 85-B <SEP> 47/76 <SEP> 53/66 <SEP> 88/94
<tb>  <SEP> 85-C <SEP> 64/76 <SEP> 53/73 <SEP> 59/71
<tb>  <SEP> MPT51-like <SEP> 53/71
<tb>  <SEP> Protein
<tb>  <SEP> ul740g <SEP> 47/59
<tb> M. <SEP> scrofulaceum <SEP> 71/76 <SEP> 66/93 <SEP> 88/94
<tb> Alpha <SEP> Antigen
<tb> M. <SEP> tuberculosis
<tb>  <SEP> 85-A <SEP> 47/71 <SEP> 46/80 <SEP> 71/88
<tb>  <SEP> 85-B <SEP> 47/82 <SEP> 53/80 <SEP> 100/100
<tb>  <SEP> 85-C <SEP> 64/82 <SEP> 53/73 <SEP>    r9/71    <SEP> 
<tb>   
 Table 8.

   Search Results for Other
Pathogens of AIDS Patients and Three Peptide Sequences
 of M.   nvium    Alpha Antigen
 (% identity/%homology)
EMI61.1     


<tb>  <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP> 229 <SEP> Amino <SEP> Acid <SEP> 181
<tb> Aeromonas <SEP>  -lactamase <SEP> 47/60
<tb>   Bacillus    <SEP> hypothetical <SEP> 53/73*
<tb> 49.5 <SEP> KD <SEP> Protein
<tb> Bacillus <SEP> L24 <SEP> Gene <SEP> 41/47
<tb> Product
<tb> Bacillus <SEP> ORFY <SEP> 53/73*
<tb>   Bcicillus    <SEP> Pbp <SEP> 5 <SEP> 53/73*
<tb> Candida <SEP>  -glucosidase <SEP> 53/65
<tb> Chlamydia <SEP> ItuA <SEP> Gene <SEP> 47/71*
<tb> Product
<tb>   C'lostridiuni    <SEP> 59/59*
<tb> Hypothetical <SEP> Protein
<tb> Enterococcus
<tb> Erythromycin <SEP> 41/65*
<tb> Resistance
<tb> Enterococcus <SEP> Serine 

  <SEP> 41/53
<tb> Protease
<tb> E. <SEP> coli <SEP> Glutaredoxin <SEP> 3 <SEP> 53/71*
<tb> (GRX <SEP> 3)
<tb> E. <SEP> coli <SEP> pap <SEP> Fimbrial <SEP> 29/59
<tb> Activator <SEP> Protein
<tb> E. <SEP> coli <SEP> Hypothetical <SEP> 53/59*
<tb> Protein <SEP> f83
<tb> E. <SEP> coli <SEP> Pantothenate <SEP> 40/53 <SEP> 
<tb> Permease
<tb> Haemophilus <SEP> Heme- <SEP> 47/59
<tb> Binding <SEP> Protein
<tb>   Haernophilzis    <SEP> HhdA <SEP> 47/70
<tb> Haemophilus <SEP> Preprotein <SEP> 47/70
<tb> Translocase
<tb>   
 Table 8.

   Search Results for Other
Pathogens of AIDS Patients and Three Peptide Sequences
 of M. avium Alpha Antigen
 (% identity/%homology)
EMI62.1     


<tb>  <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP> 229 <SEP> Amino <SEP> Acid <SEP> 181
<tb> Mycoplasma <SEP> genitalium <SEP> | <SEP> | <SEP> | <SEP> 65/71*
<tb> (random <SEP> genomic)

  
<tb> Neisseria <SEP> DTDP- <SEP> 41/59
<tb> Glucose-46-Dehydratase
<tb> Neisseria <SEP> meningitidis
<tb> UDP- <SEP> 41/59
<tb> Glucosyltransferase
<tb> Neisseria <SEP> meningitidis
<tb> UDP-Glucose-4- <SEP> 41/59
<tb> epimeras
<tb> Pseudomonas <SEP> 3-methyl
<tb> 2-oxobutanoate <SEP> 33/40
<tb>   Dehydrogenase    <SEP> 
<tb> Pseudomonas <SEP> Alkaline <SEP> 40/60
<tb> Phosphatase <SEP> H
<tb> Pseudomonas <SEP> putida <SEP> | <SEP> | <SEP> 33/40
<tb> bkdA2 <SEP> Protein
<tb>   Saccharoniyces    <SEP>    ORF    <SEP> 41/53
<tb> 233 <SEP> Gene <SEP> Product
<tb>   Sacchwvmyces    <SEP> 
<tb> Hypothetical <SEP> Protein <SEP>     <SEP> z3167*    <SEP> 
<tb> Ykl
<tb> Saccharomyces <SEP> Tubulin <SEP> 33/47
<tb> Suppressor
<tb> Saccharomyces
<tb> Ubiquitin-activating <SEP> 47/59
<tb> 

  Enzyme
<tb> Saccharomyces <SEP> 60/67*
<tb> (Unknown <SEP> Protein)
<tb>   Salmonella    <SEP> DTDP- <SEP> 47/70
<tb> Glucose <SEP> 46-Dehydratase
<tb> Schizosaccharomyces <SEP> 35/65
<tb> Hypothetical <SEP> 26.9 <SEP> kD
<tb>   
 Table 8. Search Results for Other
Pathogens of AIDS Patients and Three Peptide Sequences
 of M.

   avium Alpha Antigen
 (%   identityt%homology)   
EMI63.1     


 <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP> 229 <SEP> Amino <SEP> Acid <SEP> 181
<tb>  <SEP> Schizosaccharomyces <SEP> 47/53
<tb>  <SEP> (Unknown <SEP>    Protein)    <SEP> 
<tb>  <SEP> Shigella <SEP> DTDP-Glucose <SEP> 47/65
<tb>  <SEP>    46- <SEP> Dehydratase    <SEP> 
<tb>  <SEP>    Shigella    <SEP> rfbB <SEP> Protein <SEP> 47/65
<tb>  <SEP> Streptococcus <SEP> faecalis
<tb>  <SEP> 41/65*
<tb>  <SEP> plasmid <SEP> pAM
<tb>  <SEP> Streptococcus
<tb> 41/65*
<tb>  <SEP> agalactiae <SEP> MSL
<tb>  <SEP> Streptomyces <SEP> 3
 <SEP> Dehvdroquinate <SEP> 40/67*
<tb>  <SEP> Dehyrogenase
<tb>  <SEP> Streptomyces
<tb>  <SEP> Hypothetical <SEP> Protein <SEP> 47/53
<tb>  <SEP> 00929
<tb>  <SEP> Streptomyces <SEP> Phospho
 

  <SEP> N-acetyltransferase
<tb>  <SEP> Treponema <SEP> Flagellar <SEP> 35/59
<tb>  <SEP> Filament <SEP> Core <SEP> Protein
<tb>  <SEP> Variola <SEP> Major <SEP> Core <SEP> 53/65*
<tb>  <SEP> Protein <SEP> p4b
<tb>   
 Table 9. Search Results for
Human Peptides and Three Peptide Sequences
 of   M.    avium Alpha Antigen
 (% identity/%homology)
EMI64.1     


<tb>  <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP> 229 <SEP> Amino <SEP> Acid <SEP> 181
<tb> Band <SEP> 3 <SEP> Anion <SEP> 53/59*
<tb> Transport <SEP> Protein
<tb> Histidine <SEP> 47/47
<tb> Decarboxvlase
<tb>   Ig    <SEP> Kappa <SEP> Light <SEP> Chain <SEP> 47/59*
<tb> (VJC) 

  
<tb> MET <SEP> Gene <SEP> Product <SEP> 41/41
<tb> Protein-Tyrosine- <SEP> 47/60
<tb> Phosphatase
<tb> Protein <SEP> tyrosine <SEP> 35/47
<tb> phosphatase <SEP> PTPH1
<tb> T-cell <SEP> Factor <SEP> I <SEP> Splice <SEP> 40/47
<tb> Form <SEP> F
<tb> Transforming <SEP> Protein <SEP> 60/67*
<tb> (N-myc)
<tb> 
 Table 10. Search Results for
Various Organisms and Three Peptide Sequences
   of awl      aviunz    Alpha Antigen
 (%   identity/%homology)   
EMI64.2     


<tb>  <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP> 228 <SEP> Amino <SEP> Acid <SEP> 181
<tb> Arabidopisis <SEP> meri <SEP> 5 <SEP>  
 Table 10.

   Search Results for
Various Organisms and Three Peptide Sequences
 of M. avium Alpha Antigen
 (% identity/%homology)
EMI65.1     


<tb>  <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP> 228 <SEP> Amino <SEP> Acid <SEP> 181
<tb>  <SEP> Caenorhabditis <SEP> LIN-9
<tb> Protein
<tb> Caenorhabditis
<tb>   ZK637.6    <SEP> 
<tb> Dioscorea <SEP> Storage
<tb> Protein <SEP> 47/71
<tb> Dog <SEP>    Cytochrome    <SEP> C <SEP> 53/71*
<tb>  <SEP> Drosophila <SEP> Furin <SEP> 2 <SEP> 53/60
<tb> Drosophila <SEP> Homeobox
<tb>  <SEP> Protein <SEP> 41/53*
<tb> Drosophila <SEP> Kinesin- <SEP> 47/71
<tb> Like <SEP> Protein
<tb> Drosophila <SEP> Maternal
<tb> Tudor <SEP> Protein
<tb> Halobacterium
<tb> 47/47*
<tb> Ribosomal <SEP> Protein <SEP> S8
<tb> Hordeum <SEP> Lipid
<tb> 59/71*
<tb> Transfer <SEP> Protein
<tb>  <SEP> Horse <SEP> T-cell 

  <SEP> Antigen
<tb>  <SEP> CD2 <SEP> 60/80*
<tb>  <SEP> Impatiens <SEP> Virus <SEP> M <SEP> 41/76
<tb> Polyprotein
<tb> Lamb's <SEP> Quarters <SEP> *
<tb>  <SEP> Hypothetical <SEP> Protein <SEP> 47/65
<tb> Metalloproteinase <SEP> 65/76*
<tb> Inhibitor
<tb> Methanosarcina <SEP> vhtC
<tb> Gene <SEP> Product
<tb> Mouse
<tb> Metalloproteinase <SEP> 65/76*
<tb> Inhibitor <SEP> 1
<tb>  <SEP> Populous <SEP> cellulase <SEP> 43/59
<tb>   
 Table 10.

   Search Results for
Various Organisms and Three Peptide Sequences
 of M. avium Alpha Antigen
 (% identity/%homology)
EMI66.1     


<tb>  <SEP> Amino <SEP> Acid <SEP> 147 <SEP> Amino <SEP> Acid <SEP> 228 <SEP> Amino <SEP> Acid <SEP> 181
<tb> Rat <SEP> Dodecenoyl-CoA- <SEP> 41/59*
<tb> Delta <SEP> Isomerase
<tb> Rat <SEP> Lactogen <SEP> Receptor
<tb> 1 <SEP> 47/60
<tb> Rat <SEP> Prolactin <SEP> Receptor <SEP> 47/60
<tb> 2 <SEP> 0
<tb>   Rhodococcus    <SEP> 
<tb> Lipoprotein <SEP>     <SEP> 47/59    <SEP> 
<tb> Rice <SEP>    Cysteine <SEP> 47171    <SEP> 
<tb> Proteinase <SEP> Inhibitor <SEP> 47/71
<tb> Seal <SEP> Cytochrome <SEP> C <SEP> 53/71*
<tb> Spinacia
<tb> Phosphoglucomutase <SEP> 41/70
<tb>  <SEP> Strawberry <SEP> Virus <SEP> Coat
<tb> Protein <SEP> 47/53
<tb> Styela <SEP> Homeobox <SEP> 47/53*
<tb> Protein
<tb> 

  Stylonychia <SEP> DNA
<tb>  <SEP> 47/53
<tb> Polymerase <SEP> II
<tb>  <SEP>    Synechocysi <SEP>     <SEP> is <SEP> 41/53 <SEP> 53/67 <SEP> 41/53
<tb> Hypothetical <SEP> Protein
<tb> Tobacco <SEP> Superoxide
<tb>  <SEP> Dismutase <SEP> 47/71*
<tb>  <SEP> Tomato <SEP> Virus <SEP> M
<tb> Polyprotein <SEP> 41/65
<tb> Xenopus <SEP> Activin <SEP> Beta <SEP> B <SEP> 53/67 <SEP> 41/59
<tb> Subunit
<tb> 
 As shown in the above Tables, the potentially species-specific sequences generally established a high degree of similarity only with At intracellulare. a member of the   MAC    complex. and with   Avl.    scrofulaceum, an uncommon   pathogen.    Little similarity was noted with proteins of other species.  



   An additional directed search was performed to compare these same three At   aviurn    sequences with the PS I secreted antigen of the corynebacteria. which is a protein of approximately 30 kD. and which may be a member of the alpha antigen family (G.   JolifT      ci      cil,    "Cloning and nucleotide sequence of the cspl gene encoding PS 1. one of the two major secreted proteins of Corynebacterium   glutamicum:    the deduced N-terminal region of PS 1 is similar to the Mycobacterium antigen 85 complex." Mol. Microbiol..   6:2349-2362    [1992]).



  The same methods were used in making these comparisons as described above. The results of this study indicated only that there was only 24% identity and 40% homology. suggesting that cross-reactivity between the   V    avium alpha antigen and that of the corynebacteria was unlikely.



   EXAMPLE 15
 M.   avium    Alpha Antigen Peptide Sequences
 Based on the analysis performed in Examples 13 and 14. peptides were synthesized representing two potentially species-specific sequences of M. avium alpha antigen. The first of these sequences was "SYLASNKGVKRTGNAAV" (SEQ ID NO: 6). which begins at residue 147. The second sequence was "QRNDPSLHIPELVGH" (SEQ ID NO: 7), which begins at residue 229. The peptides were synthesized on 1   ti      polvstyrene    microparticles by the commercial MacroMolecular Structure Analysis Facility of the University of Kentucky.



     Lexington.    KY.



   In the synthesis of peptides. peptides are ordinarily cleaved from the solid support at the completion of the synthesis. However, for the purpose of immunization. allowing the peptides to remain on the beads may offer several advantages. First. the particles themselves have a modest adjuvant effect. and they may be injected intrasplenically directly (without use of nitrocellulose particles).



   Two groups of mice as described in Example 6. above were immunized with these peptides using both intrasplenic and conventional protocols as described in Example 6 above.



  One animal from each group was bled to test for development of serum antibody against the  peptides. One antibody preparation which recognized the native 30 kD alpha antigen and no other proteins in   Ad.    avium filtrate was obtained with immunization with the 229 peptide.



  Western blots of serum from these mice are shown in Figure 20. These Westerns were prepared in the same manner as described for Example 1.



   In addition to these preparations. it is contemplated that additional monoclonal and/or polyclonal antibodies be prepared that are useful in the present invention.



   EXAMPLE 16
 Comparison of the   Reactivity    of K-II Serum
 and Anti-Alpha Antigen Antibody
 In this example. a series of immunoabsorption studies were conducted to demonstrate that one of the antigens recognized bv the K-II (or "Kris") serum described by Sippola   ci    al.



  (Sippola   ci      al..    "Mycobacterium   aviutn    antigenuria in patients with AIDS and disseminated   At      civiuni    disease." J. Infect. Dis., 168:466-8 [1993]) is alpha antigen. as well as to show that the 22.5 kD to 25 kD protein present in   1M.      avium    filtrates, is not present in   A.    tuberculosis filtrates.



   To prepare the K-II serum. an adult goat was immunized by an initial injection of an emulsion of 1 ml incomplete Freund's adjuvant (Difco) 10   ing    heat-killed. dried   At    intracellulare serotype S (ATCC 35768). and 10 mg dried culture filtrate of M. intracellulare serotype 5. propagated and prepared as described above in Example I with glucose added to the medium. The animal was boosted twice with injection of the same   quantitv    of M.



  intracellulare culture filtrate.



   In this example. Western blots of culture filtrates of M.   aviuni    and M. tubrculosis were used to demonstrate the reactivity of the K-II antiserum. and the antibodies produced in
Example 2. The Western blot analysis was performed as described above in Example 1. The
K-II serum was either used diluted 1:5000 in TBS, or was prctreated by addition of   li)O     g
M.   tuberculosis    culture filtrate per S ml of diluted antiserum. incubated 4 hr at room temperature prior to use in Western blot. The results of this study are shown in the Western  blots of Figures 21 (panels A-C) and 22. As shown in these blots, an M. avium -specific protein recognized by the Kris ("K-II") serum migrated with a molecular size of approximately 25 kD.

   This results suggests a potential role for this protein in antigen detection studies for diagnosis of MAC infection and disease.



   Several   V      avium    protein antigens in this molecular weight range have been identified and cloned on the basis of homology with other species. These include a 27 kD   V      tuberculosis    lipoprotein (J. Nair et   al..    "Nucleotide sequence analysis and serologic characterization of a 27-kilodalton   Mycobacterium    intracellulare lipoprotein." Infect. Immun..



     61:1074-81[1993]),    and   22    and 19 kD proteins found in   V      tubcrcuThsis    and   V    leprae (D.P.



     
Harris et @ at, "Epitope specificity and isoforms of the mycobacterial 1 9-kilodalton antigen,"   
Infect. Immun., 62:2963-72 [1994]; R.J. Booth et al., "Homologs of Mycobacterium leprae 18-kilodalton and Mycobacterium tuberculosis 19-kilodalton antigens in other mycobacteria,"   Infect.    Immun..   61:1509-15    [1993]: and S.L. Morris   et      al    Isolation and characterization of recombinant lambda   ftll    bacteriophages expressing four different Mycobacterium intracellulare antigens." Infect. Immun.. 58:17-20 [1990]). However, the degree of homology with   M.      tuberculosis    in these antigens make it unlikely that any of these represent the MACspecific Kris antigen.



   EXAMPLE 17
 M. avium 25 kD Protein
 In this Example. the identity of the 25 kD protein identified in Example 16 above is investigated in order to determine its potential role in immunodiagnosis of MAC infection.



  The intraspelenic protocol described in Example 2 is used to produce monoclonal antibodies directed against this protein. and hybridomas are screened for reactivity against At   vlllm    filtrate. Those which score positive are then tested for lack of reactivity against
M.   tuberculosis    filtrate. Those which appear to be specific for   At      avium    are expanded in culture. biotinylated. and evaluated in the antigen detection assay. as described in Example 3.



  with the exception that these antibodies substitute for TBC-27.  



   In additional   expcriments.      K-Il    serum preadsorbed against the monoclonal antibody is substituted for the BCG antiserum. As   K-Il    serum is a goat antiserum, the   secondary    antibody used here is an anti-goat antibody, rather than the anti-rabbit antibody used in the previous Examples.



   These Examples clearly show that the amplified ELISA system of the present invention can be used to detect urinary mycobacterial antigen in patients with latent and active infection. The test is potentially valuable for diagnosis of tuberculosis because it can be applied to fluids remote from the site of active infection. It is rapid. inexpensive and technically straightforward, and thus has potential application in less-developed regions of the world where laboratory facilities and financial resources for tuberculosis control are limited.



  It has the added advantage that an additional clinic visit (for skin test reading) is not required.



  The assay may be useful in diagnosis of latent infection in persons in whom PPD skin testing is unreliable. as it detects microbial products rather than measuring the host response.   I:inally.   



  since the assay detects a secreted (rather than cell-associated) antigen. its decline during therapy of either latent of active infection may be a useful. early marker of treatment success.



  Lastly. this is the only method currently described which can potentially be used to   monitor    the effectiveness of preventive therapy.



   In summary. the present invention provides numerous advances and advantages over the prior art. including:   ( I)    much greater sensitivity, (2) much greater safety'. as there is no spillage. nor aerosolization of bacterial cultures during biochemical testing methods: and (3) the overall process for performing multiple tests is extremely simple and efficient. requiring very little labor on the part of the microbiologist. All of these advantages enhance the speed and accuracy of scoring test results in studies to detect   At      rNbcrcrriosis    infection and disease.  



   SEQUENCE LISTING (1) GENERAL INFORMATION:
 (i) APPLICANT: Wallis, Robert S.



   (ii) TITLE OF INVENTION: DETECTION OF MYCOBACTERIA
   (iii)    NUMBER OF SEQUENCES: 8
   (iv)    CORRESPONDENCE ADDRESS:
 (A) ADDRESSEE: MEDLEN  &  CARROLL, LLP
 (B) STREET: 220 Montgomery Street, Suite 2200
 (C) CITY: San Francisco
 (D) STATE: California
 (E) COUNTRY: United States of America
 (F) ZIP: 94104
 (v) COMPUTER   READABLE    FORM:
 (A) MEDIUM TYPE: Floppy disk
 (B) COMPUTER: IBM PC compatible
   (C)    OPERATING SYSTEM: PC-DOS/MS-DOS
 (D) SOFTWARE: PatentIn Release &num;1.0, Version &num;1.30
 (vi) CURRENT APPLICATION DATA:
 (A) APPLICATION NUMBER: US   08/690,347   
 (B) FILING DATE: 07-JUL-1996
   (C)    CLASSIFICATION:
 (viii) ATTORNEY/AGENT INFORMATION:
 (A) NAME: MacKnight, Kamrin T.



   (B) REGISTRATION NUMBER: 38,230
   (C)    REFERENCE/DOCKET NUMBER: CASE-02270
 (ix) TELECOMMUNICATION INFORMATION:
 (A) TELEPHONE: (415) 705-8410
 (B) TELEFAX: (415) 397-8338 (2) INFORMATION FOR SEQ ID NO:1:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 325 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: not relevant
 (D) TOPOLOGY: not relevant
 (ii) MOLECULE TYPE: peptide
 (xi) SEQUENCE DESCRIPTION:

   SEQ ID NO:1:
 Met Thr Asp Val Ser Arg Lys Ile Arg Ala Trp Gly Arg Arg Leu Met
 1 5 10 15
 Ile Gly Thr Ala Ala Ala Val Val Leu Pro Gly Leu Val Gly Leu Ala
 20 25 30   
 Gly Gly Ala Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val
 35 40 45   
 Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Val
 50 55 60
 Gln Phe Gln Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp
 65 70 75 80   
 Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp Ile Asn Thr Pro
 85 90 95     
 Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Ile Val Met Pro Val
 100 105 110
 Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly
 115 120 125
 Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu
 130 135 140
 Leu Pro Gln Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser
 145 150 155 160
 Ala Ala Ile Gly Leu Ser Met Ala Gly Ser Ser Ala Met 

    lie    Leu Ala
 165 170 175
 Ala Tyr His Pro Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu
 180 185 190
 Leu Asp Pro Ser Gln Gly Met Gly Pro Ser Leu Ile Gly Leu Ala Met
 195 200 205
 Gly Asp Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro Ser Ser
 210 215 220
 Asp Pro Ala Trp Glu Arg Asn Asp Pro Thr Gln Gln Ile Pro Lys Leu
 225 230 235 240
 Val Ala Asn Asn Thr Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro
 245 250 255
 Asn Glu Leu Gly Gly Ala Asn lie Pro Ala Glu Phe Leu Glu Asn Phe
 260 265 270
 Val Arg Ser Ser Asn Leu Lys Phe Gln Asp Ala Tyr Asn Ala Ala Gly
 275 280 285
 Gly His Asn Ala Val Phe Asn Phe Pro Pro Asn Gly Thr   H1s    Ser Trp
 290 295 300
 Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Gly Asp Leu Gln Ser
 305 310 315 320
 Ser Leu Gly Ala Gly
 325 (2) INFORMATION FOR SEQ ID NO:2:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH:

   330 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: not relevant
 (D) TOPOLOGY: not relevant
 (ii) MOLECULE TYPE: peptide
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
 Met Thr Asp Leu Ser Glu Lys Val Arg Ala Trp Gly Arg Arg Leu Leu
 1 5 10 15
 Val Gly Ala Ala Ala Ala Val Thr Leu Pro Gly Leu Ile Gly Leu Ala
 20 25 30
 Gly Gly Ala Ala Thr Ala Asn Ala Phe Ser Arg Pro Gly Leu Pro Val
 35 40 45
 Glu Tyr Leu Gln Val Pro Ser Ala Gly Met Gly Arg Asp Ile Lys Val
 50 55 60  
 Gln Phe Gln Ser Gly Gly Asn Gly Ser Pro Ala Val Tyr Leu Leu Asp
 65 70 75 80   
 Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp Ile Asn Thr Pro
 85 90 95   
 Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Val Ile Met Pro Val
 100 105 110
 Gly Gly Gln Ser Ser Phe Tyr Ala Asp Trp Tyr Gln Pro Ala Cys Gly
 115 120 125
 Lys Ala Gly Cys Ser Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu
 130 135 140
 Leu Pro 

  Ser Tyr Leu Ala Ser Asn Lys Gly Val Lys Arg Thr Gly Asn
 145 150 155 160   
 Ala Ala Val Gly Ile Ser Met Ser Gly Ser Ser Ala Met Ile Leu Ala
 165 170 175   
 Val Asn His Pro Asp Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu
 180 185 190
 Leu Asp Pro Ser Gln Gly Met Gly Pro Ser Leu Ile Gly Leu Ala Met
 195 200 205
 Gly Asp Ala Gly Gly Tyr Lys Ala Asp Ala Met Trp Gly Pro Ser Ser
 210 215 220
 Asp Pro Ala Trp Gln Arg Asn Asp Pro Ser Leu His Ile Pro Glu Leu
 225 230 235 240
 Val Gly His Asn Thr Arg Leu Trp Leu Tyr Cys Gly Asn Gly Thr Pro
 245 250 255
 Ser Glu Leu Gly Gly Ala Asn Met Pro Ala Glu Phe Leu Glu Asn Phe
 260 265 270
 Val Arg Ser Ser Asn Leu Lys Phe Gln Asp Ala Tyr Asn Gly Ala Gly
 275 280 285
 Gly His Asn Ala Val Phe Asn Phe Asn Ala Asn Gly Thr His Ser Trp
 290 295 300
 Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Pro Asp Leu Gln Gly
 305 310 315 320
 Thr 

  Leu Gly Ala Ser Pro Gly Gly Gly Gly
 325 330 (2) INFORMATION FOR SEQ ID   NO:3:   
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 17 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: not relevant
 (D) TOPOLOGY: not relevant
 (ii) MOLECULE TYPE: peptide
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
 Gln Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser Ala Ala
 1 5 10 15
 Ile   (2) INFORMATION FOR SEQ ID NO:4:
   (1)    SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 15 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: not relevant
 (D) TOPOLOGY: not relevant
 (ii) MOLECULE TYPE: peptide
 (xi) SEQUENCE DESCRIPTION:

   SEQ ID NO:4:
 Glu Arg Asn Asp Pro Thr Gln Gln Ile Pro Lys Leu Val Ala Asn
 1 5 10 15 (2) INFORMATION FOR SEQ ID   NO:5:   
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 17 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: not relevant
 (D) TOPOLOGY: not relevant
   (ii)    MOLECULE TYPE: peptide
 (xi) SEQUENCE DESCRIPTION: SEQ ID   NO:5:   
 Asp Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu Leu Asp Pro Ser
 1 5 10 15
 Gln (2) INFORMATION FOR SEQ ID   NO:6:   
   (1)    SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 17 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: not relevant
 (D) TOPOLOGY: not relevant
 (ii) MOLECULE TYPE: peptide
 (xi) SEQUENCE DESCRIPTION:

   SEQ ID   NO:6:   
 Ser Tyr Leu Ala Ser Asn Lys Gly Val Lys Arg Thr Gly Asn Ala Ala
 1 5 10 15
 Val (2) INFORMATION FOR SEQ ID   NO:7:   
   (1)    SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 15 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: not relevant
 (D) TOPOLOGY: not relevant
 (ii) MOLECULE TYPE: peptide
 (xi) SEQUENCE DESCRIPTION: SEQ ID   NO:7:   
 Gln Arg Asn Asp Pro ser Leu His Ile Pro Glu Leu Val Gly His
 1 5 10 15 (2) INFORMATION FOR SEQ ID   NO:8:   
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 17 amino acids  
 (B) TYPE: amino acid
 (C) STRANDEDNESS: not relevant
 (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu Leu Asp Pro Ser
 1 5 10 15
Gln 

Claims

CLAIMS What is claimed is: 1. A kit for the detection of Mycobacterium in a test sample, comprising: a) a solid support: and b) a monoclonal antibody directed against a portion of alpha antigen immobilized on said solid support.

2. The kit of Claim 1. further comprising a primary antibody.

3. The kit of Claim 2, further comprising a reporter antibody.

4. 'Uhe kit of Claim 3. further comprising an amplifier system.

5. The kit of Claim 1. wherein said Mycobacterium is Mycobacterium tuberculosis.

6. The kit of Claim 1. wherein said Mycobacterium is MvcohocteXrilzn? avium.

7. A method for detecting Mycobacterium in a sample comprising: a) providing: i) a sample suspected of containing at least a portion of' alpha antigen of said Mycobacterium and ii) a monoclonal antibody directed against a portion of Mycobacterium alpha antigen: b) adding said sample to said monoclonal antibody under conditions such that said antibody binds to said portion of said Mycobacterium alpha antigen in said sample to form an antibody antigen complex: and c) detecting said binding.

8. The method of Claim 7. wherein said detecting comprises adding a primary antibody to said antigen-antibodv complex so that said primarv antibody binds to said antigen to form an antibody-antigen-antibody sandwich.

9. The method of Claim 8. wherein said detecting further comprises adding a reporter reagent to said antibody-antigen-antibody sandwich to form an antibody-antigen-antibody-antibody sandwich.

10. The method of Claim 9. wherein said detecting further comprises adding an amplifier to said antibody-antigen-antibody-antibody sandwich.

11. The method of Claim 7, wherein said portion of said Mycobacterium alpha antigen is selected from the group comprising SEQ ID NOS: 1. 2. 3. 4. 5. 6.

7 and 8.

12. The method of Claim 7. wherein said Mycobacterium is selected from the group consisting of Mycobacterium tuberculosis, Mycohaclerlu 1 ai'iiini and Mycobacterium intracellulare.

13. The method of Claim 7, wherein said test sample is selected from the group consisting of urine samples. sputum samples. blood samples. and serum samples.

14. The method of Claim 7. wherein said monoclonal antibody' comprises a murine monoclonal antibody.

15. The method of Claim 14. wherein said murine monoclonal antibody is biotinylated.

16. The method of Claim 7, wherein said detecting is selected from the group consisting of enzyme immunoassay, radioimmunoassay. fluorescence immunoassay. flocculation, particle agglutination, and in situ chromogenic assay.

17. A method for detecting Mycobacterium in a urine sample comprising: a) providing: i) a urine sample suspected of containing at least a porition of alpha antigen of said Mycohaclerium; and ii) a monoclonal antibody directed against a portion of Mycobacterium alpha antigen: b) adding said urine sample to said monoclonal antibody under conditions such that said antibody binds to said portion of said iVycobacteriuni alpha antigen in said urine sample to form an antibody-antigen complex: and c) detecting said binding.

18. The method of Claim 17, wherein said detecting comprises adding a primary antibody to said antigen-antibody complex so that said primary antibody binds to said antigen to form an antibody-antigen-antibody sandwich.

19. The method of Claim 17. wherein said detecting further comprises adding a reporter reagent to said antibody-antigen-antibody sandwich to form an antibody-antigen-antibody-antibody sandwich.

20. The method of Claim 29. wherein said detecting further comprises adding an amplifier to said antibody-antigen-antibody-antibody sandwich.

21. The method of Claim 17. wherein said portion of said Mycobacterium alpha antigen is selected from the group comprising SEQ ID NOS: 1. 2. 3.4. 5. 6. 7 and 8.

22. The method of Claim 1 7. wherein said portion of said Mycohacteriunz is selected from the group consisting oi' Mycobacterium tuberculosis. Mycobacteriumn avium and Mycobacteriun intracellulare.

2;. The method of Claim 17, wherein said monoclonal antibody comprises a murine monoclonal antibody.

24. The method of Claim 23, wherein said murine monoclonal antibody is biotinylated.

25. The method of Claim 17, wherein said detecting is selected from the group consisting of enzyme immunoassay, radioimmunoassay. fluorescence immunoassay, flocculation. particle agglutination, and in situ chromogenic assay.