EP0220246A1 - DETECTION D'ANTICORPS DE l'ENCEPHALOMYELITE AVIENNE - Google Patents

DETECTION D'ANTICORPS DE l'ENCEPHALOMYELITE AVIENNE

Info

Publication number
EP0220246A1
EP0220246A1 EP86902775A EP86902775A EP0220246A1 EP 0220246 A1 EP0220246 A1 EP 0220246A1 EP 86902775 A EP86902775 A EP 86902775A EP 86902775 A EP86902775 A EP 86902775A EP 0220246 A1 EP0220246 A1 EP 0220246A1
Authority
EP
European Patent Office
Prior art keywords
antiserum
avian encephalomyelitis
sample
serum
avian
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP86902775A
Other languages
German (de)
English (en)
Other versions
EP0220246A4 (fr
Inventor
Ian James Smart
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
State of Victoria
Original Assignee
State of Victoria
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by State of Victoria filed Critical State of Victoria
Publication of EP0220246A1 publication Critical patent/EP0220246A1/fr
Publication of EP0220246A4 publication Critical patent/EP0220246A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses

Definitions

  • This invention relates to a process of immunosorbent analysis for the detection and measurement of specific avian antibodies and to antibody traps for use therein.
  • AEV avian encephalomyelitis
  • ELISA enzyme-linked immunosorbent assay
  • a liquid biological test sample is presented to an antibody trap in the form of a solid supporting substrate the surface of which has been treated to render it selectively abosrbent to the specific antibodies to be detected.
  • the adsorbed antibodies are then identified by, for example, coupling them to enzymes or radioactive tracers, which can subsequently be detected by conventional analytical techniques.
  • the adsorptive sites are provided by antigen corresponding to the antibody to be detected and the solid supporting substrate is, for example, a plastic strip or well.
  • the problem has been overcome by first coating the substrate with antibody for the antigen, which is readily absorbed onto the plastic to give a surface of reproducible uniformity. The required antigen is then adsorbed onto the treated substrate to complete the antibody trap.
  • the virus is first harvested from the yolk sacs 5 of innoculated eggs. Mammalian antibodies to AEV are then prepared drom the serum of animals immunised with the viral material extracted from the eggs.
  • a 5 .mammalian anti-avian encephalomyelitis antiserum comprising the immunoglobulin fraction of mammalian blood serum containing antibodies to avian encephalomyelitis which antiserum is substantially free of anti-chicken immunoglobulin.
  • the mammalian anti-avian encephalomyelitis antiserum is a goat antiserum.
  • immunoglobulin we mean three classes of immunoglobulin, IgG, IgA and IgM having molecular weights of about 155,000, 180,000 (monomeric) or 3000,00 (dimeric) and 5 900,000 respectively, and which have the function of anti-bacterial, anti-viral and anti-toxin antibodies.
  • the mammalian antibodies which we raise against chicken immunoglobulin would react with all three of these immunoglobulin classes.
  • a method of preparing an apparatus for use in a method for analysing a sample for avian encephalomyelitis which method includes (a) providing 5 (i) a solid support
  • the mammalian antiserum is a goat antiserum.
  • the method further includes (b') incubating the solid support for a period sufficient to allow the antibodies in the antiserum to become attached to the solid suppor , and (c 1 ) incubating the coated support for a period sufficient to allow the antigen to be absorbed onto the coated substrate.
  • an apparatus for enzyme-linked immunosorbent analysis including
  • the apparatus described above includes (i) a solid plastic support, (ii) a goat anti-avian encephalomyelitis antiserum coated on said support, and (iii)an avian encephalomyelitis antigen bound to said coated support.
  • a method of analysing a sample for avian encephalomyelitis virus which method includes (i) providing
  • the method of analysing a sample further includes
  • the general method of carrying out the analysis embodying the invention is as follows.
  • the partially purified anti-virus antiserum (mammalian anti-AEV) is coated into a solid plastic support.
  • a standard preparation of AEV antigen is bound onto the antiserum.
  • the test sample of serum is then reacted with the coated support. Any AEV antibody in the test serum will react with the bound AEV antigen. No non-specific binding of chicken antibody will occur because the anti-chicken immunoglobulin has been removed from the anti-virus antiserum.
  • the coated support is now treated with a suitable enzyme linked anti-chicken immunoglobulin which will bind specifically onto the previously bound antibodies from the test serum.
  • the amount of enzyme linked anti-chicken immunoglobulin bound is measured by an enzymic assay.
  • the enzymic assay thus gives a measure of the amount of AEV antibodies in the test serum.
  • test kit for analysing a sample, which test kit includes
  • test kit further includes
  • AEV Avian Encephalomyelitis Virus
  • the Van Rockel strain of AEV is innoculated into the yolk sacs of 7 day embryonating specific pathogen-free eggs using approximately 100 mean embryo infective doses per egg. After a further 12 days, the embryos are harvested and the AEV partially purified as described by Matsumoto M. and Murphy M.L. , "Avian Diseases" 1977, 2jL,300). After the extraction with trichloro-tri-fluoro ethane (as described by the above authors) the virus is precipitated in 4% polyethylene glycol 6000, resuspended in 0.1 M Tris-HCl pH8.0, 0.5 M NaCl, using 1 ml of this buffer for every embryo harvested. This virus preparation is then dialysed against this buffer for 3 days at 4°C.
  • Goat Antibody to Avian Encephalomyelitis Virus Goat antibody to AEV is prepared from the serum of goats immunised with the partially purified AEV preparation.
  • the immunisation protocol involves a primary immunisation with AEV virus emulsified in
  • Fruend's complete Adjuvant given intramuscularly. Subsequent injections comprise 2ml of AEV preparation, given subcutaneously at no less than monthly intervals. Blood samples are taken one week after the immunisation. The blood is taken into plain tubes and the serum separated from the clot. Goat IgG is prepared by caprylic acid fractionation of serum using a modification of a procedure described by Steinbuch M. and Andran R. ("Arch Biochem Biophys", 1969, 134, 279). In this procedure the pH of the serum is adjusted to pH 4.8 + 0.05 with IM acetic acid. Caprylic acid is added, dropwise with stirring,m using 6.8 gm of caprylic acid for every 100 ml serum.
  • the stirring is continued for a further 30 minutes, making sure that the temperature of the suspension does not fall below approximately 15 C.
  • the suspension is then centrifuged at 3000 g for 30 minutes, the - supernatent removed, filtered to remove fat, and neutralised with IM naOH.
  • This solution is then dialysed against phosphate buffered saline (PBS) for 3 days, maintaining the temperature above 15°C.
  • PBS phosphate buffered saline
  • Chicken IgG is prepared using the caprylic acid method described above.
  • Coat antibody to chicken IgG is prepared from the serum of goats immunised with this chicken IgG preparation.
  • the immunisation protocol is identical to that described above, except that 1-2 mg of chicken IgG is used for each immunisation.
  • Goat IgG is prepared as described above.
  • Affinity purified goat anti-chicken IgG is prepared from the goat IgG anti-chicken IgG using a column of sepharose 4B coupled to chicken IgG and the method described by Cripps et al ("J Immunol Methods", 1983, _57, 197) for affinity purifying human IgA and IgM.
  • a horse radish conjugate is prepared with affinity-purified Goat anti-chicken IgG antibody according to Wilson and Nakane.
  • Goat serum is prepared from goat blood collected from abbatoirs.
  • Goat IgG is prepared from this serum using the caprylic acid fractionation method described above.
  • the reference chicken serum is a pool of chicken serum prepared from adult chickens which have been 5 exposed to AEV. The amount of antibody against AEV in this pool is quantitated against a standard reference serum which is defined as containing 2 x 10 units of antibody activity per millilitre.
  • the quality control chicken serum is a dilution of field chicken serum in specific pathogen-free serum. This preparation contains sufficient antibody activity to bind approximately 50% of the conjugate bound by the highest concentration of reference
  • Antibody coated plates Add ELISA ANTIBODY to 5 ml of
  • Washing Solution Dulbecco's "A" phosphate buffered saline with 0.1% tween 20.
  • Reference serum dilutions Ref. 2: 0.5 ml Reference serum + 0.5 ml ELISA BUFFER
  • Ref. 4 0.1 ml Ref. 2 + 0.9 ml ELISA BUFFER
  • ELISA CONJUGATE 5 ml ELISA BUFFER 7.
  • Substrate solution the substrate recommended for use in this ELISA is repurified 5-aminosal-icylic acid (5-amino-2-hydroxybenzoic acid, Merck). This is prepared by mixing 9 g of 5-aminosalicylic acid, and 9 g of sodium eta bisulphite in 1 litre of distilled water at 80 C. To this suspension add 4 g of activated charcoal, stir for 5 minutes and then filter into a conical flask sitting in ice. After cooling the filtrate, the precipitate is collected on filter paper in a Buchner funnel and washed twice with 50 ml distilled water at 4°C. This precipitate is collected, dried and stored in the dark at room temperature.
  • 5-aminosal-icylic acid 5-amino-2-hydroxybenzoic acid, Merck
  • the substrate solution is prepared by dissolving 1 g of repurified 5-aminosalicylic acid in 1 litre 0.01 M sodium phosphate buffer pH 6.8 with 0.34 g of EDTA. The final pH of this solution varies between 5.9 and 6.0. This solution can be frozen in suitable sized aliquots for storage. For use the substrate solution is thawed, and 20 ul of 100 volume hydrogen peroxide added per 100 ml of subsrrate solution. 8. Test Sera : the test sera are all diluted 1:100 in ELISA Buffer (generally lOul test serum to 990ul ELISA Buffer). C. ASSAY PROCEDURE
  • the quality control sample should record a value of approximately 15 AbU per 50ul.
  • Serum samples can be interpreted as follows. Interpretation Score
  • Each serum sample can be assessed individually as above or an overall flock index can be assessed as follows. The number of serum samples in each division is multiplied by the score for that group, and the total for the flock determined. This is expressed as a percentage of the potential maximum which is 3 x the number of serum samples.
  • the ELISA index can be interpreted as
  • ⁇ 30 flock susceptible Note It is not essential to use a plate reader to determine the absorbance value of each sample and determine the AbU as above.
  • the ELISA Index can be determined by classifying each serum as falling within each group by visually comparing the colour of each sample with those of the reference samples. These sera can be assigned a score and the ELISA index determined as above.
  • the wells of flat bottom microtitre trays were each coated with 50 ul of Goat IgG anti-AEV in 0.1 M sodium carbonate buffer, pH 9.5.
  • the antibody was adsorbed to the plastic surfaces for 18 hours at room temperature, after which the plates were stored at 4°C.
  • the wells were washed thrice with phosphate-buffered saline pH 7.2 with 0.1% Tween 20 (PBS-tween), and 50 ul of ELISA antigen diluted in ELISA buffer (0.1 M Tris-HCl pH 8.0, 0.5M NaCl, 0.002M EDTA, 0.05% tween 20, 5 x 10 M thimerosal) was added to each well and incubated for 1 hour at room temperature.
  • the relative concentrations of Goat anti-AEV and ELISA antigen were predetermined by chequerboard titration.
  • the wells were again washed thrice with PBS-tween and 50 ul serum samples, diluted 1:100 in ELISA buffer containing 1 mg/ml normal Goat IgG (ELISA buffer-G) were added, in duplicate, to the wells.
  • ELISA buffer-G normal Goat IgG
  • a reference curve was constructed by plotting the percentage bound in each reference sample against the logarithm of its antibody activity content, with the B ⁇ sample for the studies reported herein being defined as containing 1000 antibody units per 50 ul of a 1:100 dilution. Sample antibody activities were read off this curve and reported in this paper as antibody activity in 50 ul of the diluted serum. 5 The dose-response curve for the reference serum:
  • the relationship between the virus neutralization test and the ELISA was used to assess the ELISA for measurement of antibodies against AEV. A comparison of these two assays in the rank-orders given to a number of chicken
  • This ELISA has been demonstrated by comparison with virus neutralization, and by testing sera from specific-pathogen-free chickens which had been immunised with a variety of other viruses. Both the ELISA and virus neutralization tended to rank a number of sera in the same order in terms of antibody activity. Many of these sera were obtained from chickens shortly after a primary immunisation with AEV. Some of the differences between the two tests in terms of rank order of these sera may relate to the differential sensitivities of the two assay systems to different antibody populations, as it is known that the ELISA is particularly affected by the avidity of the antibody papulation (Butler et al, 1981).
  • Table 1 Relationship between Neutralisation Index and Antibody units (by ELISA) for the measurement of antibodies to avian encephalomyelitis .
  • a positive serum uas defined as one having greater than 10 antibody units per 50 ⁇ l of a 1:100 dilution of serum.
  • a positive serum uas defined as one having a neutralisation index of greater than 1.0.
  • Table 3 Measurement of antibodies to Avian encephalomyelitis virus, infectious bronchitis virus (IBV), Newcastle disease virus (NDU), and hae agglutinating avian adenovirus (HAV-) in the sera of specific-pathogen-free chickens - immunised with a variety of viruses.
  • IBV infectious bronchitis virus
  • NDU Newcastle disease virus
  • HAV- hae agglutinating avian adenovirus
  • Table 5 Within- and between-assay co-efficient of variation for three serum pools representing high-, mid-range, and low levels of antibody activity.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Antisérum de l'encéphalomyélite anti-avienne de mammifères comprenant la fraction d'immunoglobuline du sérum sanguin de mammifères contenant des anticorps de l'encéphalomiélite avienne, ledit antisérum étant substantiellement dénué d'immunoglobuline anti-volaille. L'antisérum est utilisé dans une analyse ELISA pour la détection de l'encéphalomyélite avienne.
EP19860902775 1985-04-26 1986-04-28 DETECTION D'ANTICORPS DE l'ENCEPHALOMYELITE AVIENNE. Withdrawn EP0220246A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPH032685 1985-04-26
AU326/85 1985-04-26

Publications (2)

Publication Number Publication Date
EP0220246A1 true EP0220246A1 (fr) 1987-05-06
EP0220246A4 EP0220246A4 (fr) 1989-01-18

Family

ID=3771072

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19860902775 Withdrawn EP0220246A4 (fr) 1985-04-26 1986-04-28 DETECTION D'ANTICORPS DE l'ENCEPHALOMYELITE AVIENNE.

Country Status (2)

Country Link
EP (1) EP0220246A4 (fr)
WO (1) WO1986006382A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104407136B (zh) * 2014-12-07 2016-05-18 青岛易邦生物工程有限公司 一种鸡传染性脑脊髓炎活疫苗中病毒含量测定方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOLOGICAL ABSTRACTS, vol. 75, no. 3, 1983, page 1970, abstract no. 19308, Philadelphia, PA., US; A.A.S. AHMED et al.: "The serological detection of atibodies to avian encephalomyelitis virus", & AVIAN PATHOL., vol. 11, no. 2, 1982, pages 253-262 *
BIOLOGICAL ABSTRACTS, vol. 80, no. 2, 1985, page AB-548, abstract no. 14119, Philadelphia, PA., US; R. RICHTER et al.: "Comparative studies on the use of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the avian encephalomyelitis virus", & ZENTRALBL. VETERINAERMED. REIHE B, vol. 32, no. 2, 1985, pages 116-127 *
COMMONWEALTH AGRICULTURAL BUREAU ABSTRACTS, vol. 85, abstract no. 211017; T. IMADA et al.: "Detection of antibodies against avian encephalomyelitis virus by an enzyme-linked immunosorbent assay", & BULLETIN OF THE NATIONAL INSTITUTE OF ANIMAL HEALTH, no. 87, 1984, pages 23-31 *
See also references of WO8606382A1 *

Also Published As

Publication number Publication date
EP0220246A4 (fr) 1989-01-18
WO1986006382A1 (fr) 1986-11-06

Similar Documents

Publication Publication Date Title
EP0216191B1 (fr) Essai immunologique pour antigènes de HTLV-III
Gosslau et al. Enzyme-linked immunosorbent microassay for quantification of specific antibodies to collagen type I, II, III
Grauballe et al. Optimized enzyme‐linked immunosorbent assay for detection of human and bovine rotavirus in stools: Comparison with electron‐microscopy, immunoelectro‐osmophoresis, and fluorescent antibody techniques
US4294817A (en) Method of fluoro immunoassay
US4332783A (en) Process for immunologic determination tests
EP0345462B1 (fr) Immunoessai pour des antigènes d'HIV-I utilisant des fragments de F(AB')2 comme sonde
DK165958B (da) Fremgangsmaade til isolering af htlv-iii proteiner, serologisk detektering af antistoffer til htlv-iii i serum fra aids- og prae-aids-patienter og detektering af htlv-iii infektioner ved immunoassays samt testsystem til brug ved fremgangsmaaden
Payne et al. Public Health Laboratory Service enzyme linked immunosorbent assay for detecting Toxoplasma specific IgM antibody.
Maiolini et al. A sandwich method of enzyme-immunoassay. II. Quantification of rheumatoid factor
Kelly et al. Qualitative testing for circulating immune complexes by use of zone electrophoresis on agarose.
Vaheri et al. Evaluation of solid‐phase enzyme‐lmmunoassay procedure in immunity surveys and diagnosis of rubella
Popow‐Kraupp Enzyme‐linked immunosorbent assay (ELISA) for mumps virus antibodies
Payne et al. Enzyme-linked immunosorbent assay (ELISA) using antibody class capture for the detection of antitoxoplasma IgM.
AU596404B2 (en) Competitive elisa for the detection of antibodies
Chiodi et al. Measles IgM antibodies in cerebrospinal fluid and serum in subacute sclerosing panencephalitis
US4814269A (en) Diagnostic testing for antibodies against microorganisms
Sandoval et al. Detection of antibodies against avian antigens in bronchoalveolar lavage from patients with pigeon breeder's disease: Usefulness of enzyme‐linked immunosorbent assay and enzyme immunotransfer blotting
CA2047742A1 (fr) Methode et trousse d'essai diagnostique pour la detection de l'anticardiolipide
EP0220246A1 (fr) DETECTION D'ANTICORPS DE l'ENCEPHALOMYELITE AVIENNE
CA1083956A (fr) Dosage immunologique des anticorps microbiens
US6379906B1 (en) Compositions and methods for detecting adult Taenia solium
Losso et al. Development of a particle concentration fluorescence immunoassay for the quantitative determination of IgG in bovine milk
EP0233048A2 (fr) Procédé pour la détection d'infection ou inflammation du faisceau urinaire
Smart et al. Measurement of antibodies to infectious avian encephalomyelitis virus by ELISA
Nielsen et al. Detection of immunoglobulin G antibodies to cytomegalovirus antigens by antibody capture enzyme-linked immunosorbent assay

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19870114

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

A4 Supplementary search report drawn up and despatched

Effective date: 19890118

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19890403

RIN1 Information on inventor provided before grant (corrected)

Inventor name: SMART, IAN, JAMES