SK596A3 - Method in vitro of presence proof or quantity determining of soluble crosslinked desaabb fibrin polymers and set for carrying out this method - Google Patents

Abstract

The subject invention relates a method for the production of monoclonal antibodies. The method utilizes an immunized germfree animal. The invention also provides methods for the use of such monoclonal antibodies, and polyclonal antibodies derived from an immunized germfree animal, for in vitro and in vivo clinical diagnostics and therapeutics. Also, the subject invention provides a fibrin-specific monoclonal antibody.

Description

(57) Annotation:

The method for preparing monoclonal antibodies utilizes an immunized sterile animal. Also described are methods of using said monoclonal antibodies as well as methods of using polyclonal antibodies derived from immunized sterile animals for in vitro and in vivo clinical diagnosis and therapy. Further disclosed is a monoclonal antibody specific to fibrin.

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In-vitro method for detecting the presence or determination of the amount of soluble cross-linked DesAABB fibrin polymers and a kit for carrying out the method

Field of the invention

The invention relates to a process for the preparation of monoclonal antibodies. The method utilizes an immunized sterile animal. The invention also encompasses methods of using said monoclonal antibodies as well as methods of using polyclonal antibodies derived from immunized sterile animals for in vitro and in vivo clinical diagnosis and therapy. The invention further includes a fibrin-specific monoclonal antibody.

BACKGROUND OF THE INVENTION

Kohler and Milstein are recognized as the originators of techniques that have successfully led to the first hybridoma producing monoclonal antibodies (G. Kohler and C. Milstein, 1975, Nature 256, 495-497; 1976, Eur. J. Immunol., 6, 511) to 519). Fusion of antibody-producing cells (spleen B lymphocytes) with myeloma cells (malignant cells of primary bone marrow tumors) creates a hybrid cell line arising from a single fused cell hybrid (called a hybrid or a clone). Like lymphocytes, hybridomas have the ability to secrete one species of immunoglobulin; like myeloma cells, hybridomas have the ability to unrestrict cell division. The combination of these two features offers some advantages over conventional antisera.

Antisera derived from vaccinated animals are different mixtures of polyclonal antibodies that are repeatable and cannot always be prepared in the same quality. Monoclonal antibodies are highly specific immunoglobulins of one type. One type of immunoglobulin secreted by a hybridoma is specific to one and only one antigenic determinant or epitope on an antigen, a complex molecule that has multiple antigenic determinants. For example, if the antigen is a protein, the antigenic determinant may be one of a number of peptide sequences (generally 6 to 7 amino acids in length; M.Z.Atassi, 1980, Molec. Cell. Biochem., 32, 21-43) from the entire protein molecule. The monoclonal antibodies raised against one antigen may then differ from one another depending on the determinant that induced their formation; however, for any given hybridoma, all antibodies produced by it are the same. Further, the hybridoma cell line readily multiplies in vitro or in vivo and provides exceptionally high antibody concentrations.

The monoclonal antibody can be used as a probe to detect its antigen. Thus, monoclonal antibodies are used in in vitro diagnostics, for example in radioimmunoassay and enzyme-linked adsorbent analysis (ELISA), and in in vivo diagnostics, for example in in vivo imaging with a radiolabeled monoclonal antibody. The monoclonal antibody can also be used as a carrier to target drug delivery to an antibody antigen.

However, before a monoclonal antibody can be used for the above purposes, it is necessary to ensure that the monoclonal antibody has the ability to bind to the desired antigen, i.e., the target antigen. This is accomplished by sorting and selecting the prepared hybridomas to determine which hybridomas, or even some hybridomas, produce a monoclonal antibody that is capable of binding to a target antigen. This screening procedure is very difficult in that, for example, thousands of monoclonal antibodies must be screened before being detected by a hybrid that produces an antibody capable of binding to a target antigen. Accordingly, there is a need for a method of preparing monoclonal antibodies that increases the likelihood that a particular hybrid will produce an antibody to a target antigen.

SUMMARY OF THE INVENTION

The present invention provides a method for preparing monoclonal antibodies to an antigen, comprising:

(a) immunizing a sterile animal with said antigen, allowing antibody-forming cells to produce antibodies to said antigen, (b) removing at least a portion of said antibody-forming cells from said sterile animal, (c) generating a hybridoma by fusing one of said antibody-forming cells , with an immortal cell, wherein said hybrid is capable of producing a monoclonal antibody to said antigen, (d) multiplying said hybridoma, and (e) obtaining a monoclonal antibody produced by said hybridoma.

The invention also encompasses methods of using a monoclonal antibody or a polyclonal antibody obtained from a sterile animal. The invention also encompasses a fibrin-specific monoclonal antibody and methods of using said monoclonal antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 (A)

Determination of MH1 immunoreactivity with DesAABB fibrin monomer by affinity chromatography.

Figure 1 (B)

Comparative experiment to confirm the binding ability of the Sepharose - DesAABB matrix fibrin monomer using fibrinogen specific antibody 45J.

Figure 2

Immunoreactivity of uncrosslinked DesAABB fibrin polymers and crosslinked fibrin polymers in an in vitro procedure with soluble DesAABB fibrin polymers using MH1.

Figure 3

Formation of DesAA fibrin by the action of batroxobin on plasma fibrinogen.

Figure 4

Immunoreactivity of soluble DesAA fibrin polymers in an in vitro experiment with soluble DesAABB fibrin polymers using MHI.

Figure 5

Immunoreactivity of the MH1 antibody with fibrinogen when activated by factor XIII (factor XIIa).

Figure 6

Monitoring the progress of soluble DesAABB fibrin polymer in vitro using the soluble fibrin-MH1 assay.

Figure 7

Illustration of the purification of soluble fibrin from plasma using a Sepharose-MH1 column.

Figure 8

Determination of soluble DesAABB fibrin polymers in the blood of patients with chest pain and confirmed myocardial infarction.

EXAMPLES

Sterile animal

The present invention relates to the use of a sterile animal for the preparation of monoclonal antibodies. Sterile animals first developed at the end of the 19th century and are still widely used today. ¹

A sterile animal is a gnotobiot that lives in an environment free of any conceivable accompanying form of life, including bacteria, viruses, fungi, elements and other saprophytic or parasitic forms. Gnotobiot is an animal or strain derived by aseptic caesarean section, or by sterile egg-laying, which are tolerated and maintained by sterile technique under sterile isolator conditions and in which the composition of the accompanying fauna and flora, if present, is entirely defined by currently accepted methodology. (It should be noted that all mice are carriers of latent leukemogenic viruses and therefore mice in the present invention may be considered sterile despite the presence of said viruses).

The role of the sterile system is to prevent barriers against undesirable microbial contamination. In addition to mechanical barriers made of plastic, metal, rubber and glass that enclose animals, the system requires working barriers for air filtration, sterilization of food and water, and gloves handling, which forms an integral part of the entire barrier system. The insertion of materials into the insulator must also be carried out under sterile conditions.

It will be understood that any sterile animal may be used in the present invention. The most common sterile animals are mouse, pig, rat, rabbit, guinea pig, duck, sheep, primate and poultry. A mouse, especially a Balb / C mouse, is preferred.

Preparation, treatment and breeding of sterile animals

A large number of publications are devoted to the preparation, treatment and breeding of sterile animals. For example, Vostmann, B.S., Ed. , Gnotobiotics Standards and Guidlines for Breeding, Line and Management of Laboratory Animals, National Research Council, National Academy of Sciences, Vashington, D.C. 1970); Coates, M. E., et al., The Germfree Animal in Research, Academic Press, London, 1968; and Pleasants, J.R., Gnotobiotics, in: Handbook of Laboratory Animals Science, Vol. 1, Melby, E. C., et al., Eds., CRC Press, Boca Raton, Fla., 117, (1974), the disclosure of which is incorporated herein by reference.

The following is a summary derived from Vostmann B.S. , Ed. .

Gnobiotes: Standards and Guidlines for Breeding, Line and Management of Laboratory Animals, National Research Council, National Academy of Sciences, Vashington, D.C. , which describes the preparation, care and breeding of sterile rats and mice. It should be noted that the above preparation, breeding and treatment are similar for other animal species.

Room environment

Equipment, equipment and husbandry practices should be so designed and implemented as to ensure the best possible environmental control and optimal animal comfort and welfare. Cages, feeders and drinkers must be so designed and constructed that food and water are readily available and that cleaning and sterilization are readily accessible and efficient.

For extensive sterile activity areas are required:

1. working area for assembling and sterilizing insulators,

2. animal storage area in isolators; and

3. laboratory area for permanent control of the gnotobiotic environment.

The study and diet area should also be included in this kit.

The environment of the room in which the gnotobiotic insulators are located shall meet the standards laid down for the keeping of normal laboratory rodents. The building must be free of insects, walls and floors must be protected from moisture. The illumination shall be uniform and with the same light-dark cycle as it changes during the day and year. Ventilation shall remove any vapors caused by chemical sterilization and the climatic conditions shall be controlled as follows.

Temperature. A generally acceptable animal room temperature of 21 to 27 ° C may require a small narrowing of the interval so that the temperature of the insulators is maintained between 22 to 26 ° C.

Humidity. The relative humidity (RH) must be maintained at a level of 40 to 60% suitable for human well-being. However, when room air is used to ventilate insulators, a relative humidity of 40 to 50% is recommended.

Ventilation. The exchange of air in the room must be sufficient to remove any fumes resulting from chemical sterilization. 10 to 15 times the air change per hour is recommended. Full-face masks with fresh air ventilation shall be available in the premises to protect operators from exposure to hazardous levels of chemical vapor concentration.

Sci., 78: 47;

Remote-control

Sterile Devices (See Sacquiet, E., 1968, Equipment, Design and Management: General Technique of Maintaining Germ-free Animals, pp. 1 to 22, in: ME Coates, Ed., The Germfree Animals in Research, Academic Press, London Trexler, PC, 1968, Equipment Design and Management: Transport of Germ-free Animals and Current Developments in Equipment Design, pp. 23-35, in: ME Coates, Ed., The Germfree Animals in Research, Academic Press, London) .

The complete elimination of microbes from the environment requires absolute barrier barriers. Successful operation of the insulator depends on continuous maintenance of the barrier. Two types of insulators are generally available, metal and plastic. Some metal units are built to withstand an internal water vapor pressure of 0.1406 MPa (20 psi). (See Reyniers, J.A., 1959, Construction and Operation of Sterile Animal Breeding,

Miyakawa, M. (1959):

Germfree Rearing Unit, Ann.

78:37). Other sterilizers are generally placed in a large autoclave for initial sterilization (see Gustafsson, B.E., 1959, Lighweight Stainless Systems for Rearing Germ-free Animals, Ann. N.Y. Acad. Sci., 78:17).

The most widely used unit currently is a flexible film insulator (see Trexler, P., C., L.I. Reynolds, 1957, Flexible Film Apparatus for Rearing and Use of Germfree Animals, Appl. Microbiol., 5: 406). It is usually made of flexible laminated vinyl, must be chemically sterilized and easily adaptable for a particular purpose. Another type, made of large nylon sleeves, closed on both

Ann.N.Y. Acad. The Miyakawa N.Y. Acad.

ends can be autoclaved. (See Lev, M.,

1962, An Autoclavable Plastic Unit for Rearing Animals under Germfree Conditions, J.Appl.Bacteriol. 25:30). Glass plexiglass insulators and disposable flexible film insulating units have also been developed. Many of them are so light that they can store two or three on the shelf, which means space saving.

A special roller is generally used to sterilize food and supplements into heat-sensitive insulators. It must be designed with a large filter area to allow air to be drawn off in a high-vacuum sterilizer (See Trexler,

P.C., 1963, An Isolator System for Contamination Control, Lab. Anim. Line 13: 572). Alternatively, a vent hose vented to the atmosphere may be attached to the cylinder; serves to remove air and condensation during sterilization without the use of vacuum (See Jaworski, N.E., and C.E. Miller,

1963, Refinement of Cylinder Techniques for Supplying Germfree Isolators, Lab. Anim. Line 13: 591).

sterilization

All equipment, food, bedding, water and air used in insulators must be absolutely sterile. The method and conditions used are determined by the nature of the individual cases.

Pressure steam is the best known method of sterilization. It is especially suitable for porous objects that are thermally stable. Any area where microbes can be anchored must be brought into direct contact with the steam. The exposure time is related to the temperature of the steam used. It is recommended that the worst accessible portion of the batch (middle of the package) be exposed to water vapor for at least 15 minutes at 121 ° C. Higher temperatures and shorter exposures can only be used after careful testing and ensuring sterility. Standard pack sizes and density of diet, bedding and other substances are essential to make sure that steam penetration will always be the same and predictable.

Dry heat is used to sterilize the air supplied to the insulator (see: Miyakawa, M., 1959, The Miyakawa Remote Control Germfree Rearing Unit, Ann. NY Acad. Sci. 78: 37; see Gustafsson, BE, 1959, Lighweight Stainless Systems for Rearing Germ-free Animals, Ann. NY Acad. Sci., 78:17).

In particular, peracetic acid (CH ^COOOH) is used for the sterilization of non-porous heat-sensitive materials, especially for flexible film units. Said acid is used in the form of a 2% solution together with a surfactant (see Trexler, L.I. Reynolds, 1957, Flexible Film Apparatus or Rearing and Use of Germfree Animals, Appl. Microbiol., 5: 406). In certain situations, other chemicals, such as hypochlorites, iodophores, or quaternary ammonium compounds in the liquid state on newborns induced by hysterectomy, or mercuric chloride, may be used to store eggs under sterile conditions prior to sitting.

Ethylene oxide (ETO) can be used to sterilize non-wettable and heat-sensitive objects. Sterilization time depends on temperature, humidity, pressure and ETO concentration. ETO can chemically react with bedding and dietary ingredients to form toxic or undesirable compounds. Because of the ETO's flammability and its toxicity, the use of ETO for sterilization is limited to commercially available gas mixtures containing no more than 20% ETO.

Glass fiber filters are commonly used to sterilize the supplied air. They must act as absolute filters.

Membrane filtration may be used in order to avoid thermal exposure to liquids, but provided that the membranes are absolute filters, such as filters that separate particles with a diameter greater than 0.22 microns.

Gamma rays or electron rays can be used to sterilize diets and in other special cases. Various doses ranging from 2.5 to 6.10 µL are used.

Inner environment

Temperature. The insulator's internal temperature is a function of room temperature and must be maintained between 22 and 26 ° C.

Humidity. Moisture condensation can occur in the insulator when the insulator is overloaded and / or with inadequate ventilation. The air entering the insulator must have less than 50% RH and preferably more than 40% RH.

Air inlet. The air in the insulator must be replaced 12 to 20 times per hour and the air must have an overpressure of 80 to 130 mm (3 to 5 inches) of water column. The air can be supplied from a central source or from individual blowers of each unit. A turbine-type air compressor is recommended for the central air supply system, as oil piston systems typically cause oil to be sprayed into the air ducts.

Air Diffusion Insulator (see Trexler, PC, 1968, Equipment Design and Management: Transport of germ-free animals and current developments in equipment design, pages 23 to 35, in: ME Coates, Ed., The germfree animals in research, Academic Press, London) does not interrupt ventilation even in the event of a power failure. However, this type has the disadvantage of a lower number of air changes per hour and the absence of protective overpressure to help prevent contamination with minor damage to the barriers.

Protection in danger. Within no more than a few minutes, the insulator shall be suitably provided with air pressure in the event of a power failure or mechanical failure. The collapse of unreinforced foil insulators may eventually result in asphyxiation of the animals, but the immediate danger is more that the animals are able to reach and damage the foil or gloves. This can be temporarily prevented by closing the air ducts with rubber plugs. Operation of the air sources of individual insulators requires an emergency spare energy source. The central air system must have a second turbine compressor as a spare air source.

Graphical recording of temperature and pressure is recommended. An audio-visual alarm system shall be installed in the central air system and shall be activated when the air pressure drops due to a power cut or mechanical failure. Similar warning systems shall indicate undesirable changes in the supply air temperature. In an insulator system with individual air sources, equipment with a continuous graphical record of the room environment is recommended.

Cage and interior fittings

Equipment. The basic list of insulator equipment may include a cage with safety hatch, water and food container, protective textile gloves for rubber gloves, a separate door basket or closing ring, long medical pliers with rubber tips, hemostats, scissors and towel, gauze swab, about 2 liter veterinary tool container, covered with about 4 liter diet container, spoon, culture tubes, paper bags and moisture resistant bags for used bedding.

Cages must be made of a smooth and corrosion-resistant material. They must not leak liquids and must be easy to sterilize. Acceptable materials are plastic, stainless steel and glass. Galvanized metals corrode over time and are not recommended as trace metal contamination may affect the results of the experiments.

The dimensions of the cages are usually limited by the size of the entrance.

y

The smallest area for female mice and garbage is 970 cm (50 in). Under different circumstances, a larger area will be needed per animal.

Table 1 shows the recommended animal floor area for mice and rats by weight group.

Table 1

Size! floor area, recommended for 1 animal in the behavior of mice and rats in the cage

category Weight! Area per animal Max.populácia number (G) cm ^z in 1 cage mice 1 to 10 40 40 2 10 to 15 50 30 3 15 to 25 75 20 4 nad 25 95 16 rats 1 to 50 95 50 2 50 to 100 110 50 3 100 to 150 125 40 4 150 to 200 150 40 5 200 to 300 185 30 6 over 300 260 25

Various recommendations

Control tests using freon are recommended to ensure the tightness of the barrier system.

Each unit should be provided with its own work record to chronologically record each procedure performed in that unit, starting with unit assembly and sterilization. Such records are conveniently stored on known hospital metal holders, indicated by the unit number. They should also include notes on routine maintenance, such as changing gloves. Multiplication records may also be kept in the same holder.

Due to the limited interior space of the insulators, paper and collapsible containers are recommended for food and bedding. The same material is recommended for moving animals between insulators connected by a sterile passage.

Ether must not be used inside the insulator, as static sparks may explode !.

As volatile and non-flammable anesthetic Fluothan (bromochlorotrifluoroethane).

is recommended

Nutrition, bedding and water

For a commercially produced food, its complete composition must be reported, listing all ingredients and their concentrations, including preservatives, antioxidants and other ingredients. The date of manufacture must be clearly indicated. The manufacturer must ensure that the food:

(1) has naturally occurring hormone activity only within normal allowable limits;

2. it does not contain additives containing drugs, hormones, antibiotics or other substances which may create abnormal physiological conditions or which may interfere with research procedures;

3. it does not contain salmonella, as evidenced by statistically selected samples;

4. it is not contaminated with rodents and insects;

5. it does not contain unprocessed pieces of meat or meat of fish which may contain pathogens.

Enrichment of nutrition

The nutrition of sterile animals must contain more than the normal requirements of certain nutrients in order to compensate for the loss of vitamins by sterilization (especially certain vitamins B, A and D) and a decrease in protein nutritional value (reduction of available lysine, methionine, arginine and tryptophan). The food must also contain ingredients that are produced by microbial synthesis in the gastrointestinal tract in normal animals (see Reddy, BS, BS Vostmann and JR Pleasants, 1968, Nutritionally Adequate Diets for Germ-free Aninals, pp. 87 to 111, in: ME Coates (Ed.), The Germfree Animal in Research, Academic Press, London). An example of such diets is L-485, not an expensive diet that has been widely tried (see Kellog, TF, and BSVostmann, 1969, Rat and Mouse Stock of L-485, Lab. Anim. Line) and can be commercially produced (see Table 2).

To compensate for the decrease in protein quality, supplementation with certain amino acids is preferable to increasing the total protein content. Increasing the total protein content of the diet leads to greater food intake and water excretion, causing humidification of the environment, and then only a limited number of animals can be kept in a certain size insulator.

Table 2

Composition of L - 485 diet for rats and mice

Dietary component Quantity per 1 kg A child ground yellow corn grains 590 g soya flour (50% crude proteins) 300 Alfalfa flour 35 Corn oil (once refined) 30 NaCl 10 CaHPO ₄ .2 H ₂ 0 10 CaC0 ₃ 5 Lysine (food grade) 5 Methionine (food grade) 5 BHT (butylated hydroxytoluene) 0,125 mixture of trace minerals 0.25 Vitamin mixture A 26.000 IU ^D 3 1,000 IU E (tocopherol acetate) 225 mg Kn (menadione) riboflavin 90 30 Pantothenic acid 285 niacin 65 Choline chloride 2,000 B12 (0,1%, triturated in mannitol) 2 Thiamine hyarochloride 65 Pyridoxine hydrochloride 20 Folic acid 10 P-Aminobenzoic acid 50 Mixture of trace minerals (commercial) Mn as manganese oxide 65 mg Fe as ferrous carbonate 20 Cu as copper oxides 2 Zn as zinc oxide 15 Like potassium iodide 1.5 What about cobalt carbonate 0.6

Water Steam Sterilization (See Reddy, BS, BS Vostmann and JR Pleasants, 1968, Nutritionally Adequate Diets for Germ-free Aninals, pp. 87-111, in: ME Coates (Ed.), The Germfree Animal in Research, Academic Press, London). The procedure used will depend on the instrumentation. Three factors are of general importance:

A pre-sterilization vacuum of at least about 510 mm Hg (20 in. Hg) wherever possible. Pre-sterilization vacuum helps the vapor to penetrate the food in the cylinder resulting in the atmosphere. If the food cylinder is not vented, a vacuum of about 710 mm Hg (28 in. Hg) or greater is recommended.

2. Use of the shortest possible sterilization phase to ensure complete sterility along with safety, determined by equipment and operator experience. Temperatures are measured inside the food and must reach at least 121 ° C. At this temperature, the actual sterilization phase will last at least 15 minutes. At higher sterilization temperatures, the sterilization time will be proportionally shorter.

3. Post-sterilization vacuum will accelerate the drop in food temperature. This avoids unnecessary thermal degradation of the food ingredients. The design and operation of the equipment must be adequate to prevent leakage during the operation.

In the case of food sterilization, the aim is to prevent incomplete sterilization, but also to avoid unnecessary deterioration caused by excessive heating. Although some decrease in nutritional components cannot be avoided, quite good results can be achieved by setting the conditions:

(a) adjusting the technical parameters such as temperature, time and vacuum of pre-sterilization and post-sterilization, tablet size.

(b) the water content of the diet, to better utilize B vitamins merman, D.R. and B.S. Vostmann, Diets Sterilized for Germfree

The increase in water content results after sterilization (see Zim1963, Vitamin Stability in

Animals, J. Nutr. 79: 318).

For solid diets, a water content of up to 25%, or as high as tentatively determined, should not affect the quality of the food during storage. Variations in the water content of the diet require re-testing of the heating rate at which the diet reaches the sterilization temperature.

Radiation Sterilization (See Reddy, BS, BS Vostmann and JR Pleasants, 1968, Nutritionally Adequate Diets for germ-free animals, pp. 87 to 111, in: ME Coates (Ed.), The germfree animal in research, Academic Press, London ); Ley, F. J., J. Bleby, M.E. Coates and J. S. Peterson, 1969, Sterilization of laboratory animals using gamma radiation, Lab. Anim .. 3: 221).

Sterilization and dosimetry techniques depend on the equipment and type of dilution. Although radiation sterilization is generally considered to be less destructive to nutritional ingredients than heat sterilization, it is currently recommended that food be steam-sterilized.

Sterility tests

The use of Bacillus stearothermophilus spore strips is recommended for the continuous control of sterility using any sterilization procedure. The strips are placed inside the food. The isolator and the animals housed therein are then checked for microbiology. Such a systematic check is necessary to check for any accidental contamination caused by breakage of the insulator barriers or improper sterilization of the insulator and its contents. The control may be performed as described in Vostmann, B. S., Ed. , Gnotobiotes: Standards and Guidelines for Breeding Line and Management of Laboratory Animals, National Research Council, National Academy of Sciences, Vashington, D.C., 1970, p. 28 to 39).

Determination of nutritional losses by sterilization

As the usual test for the loss of vital nutrients, it is recommended to determine the acid extractable thiamine as an indicator of the use of thiamine added to the diet (see Vostmann, BS and PL Knight, 1960, The effect of methyl alcohol on thiamine to thiochrome, Experientia). 16: 500). Maintaining less than 25% of the extractable thiamine indicates a serious reduction in the nutritional value of food. With appropriate equipment and care, 50% or more can be maintained.

Solid food storage

Experiments with sterile animals are generally very expensive and it must therefore be ensured that nutrition with a significantly reduced nutritional value is never used. It is recommended that (a) the non-sterilized food should always be stored in the freezer and never for more than 1 month, and (b) the storage period of the sterilized food inside the insulator should be one week or less and never exceed 10 days.

litter

Bedding should be changed at least once a week. It is recommended that litter material is easy to sterilize and that animals do not eat very much. No toxic compounds must be formed when sterilizing litter. Bedding of pine (Pinus strobus) sawdust and wood shavings is recommended; lime and poplar shavings or crushed corn sticks are also acceptable. Kieselguhr products, cedar, bituminous and hardwoods are not recommended. Sterilization with ethylene oxide should not be used until the question of possible harmful compounds has been clarified.

Water

Drinking water must be sterilized. It can be autoclaved in square bottles, Mason containers or tanks connected to the unit. A small air space should be left in each container.

Basics of separation of gnotobions by Caesarean section

The success of each caesarean operation is partly based on the pregnancy being advanced to the deadline. This is particularly important in animals with a short gestational period where the fetus can gain up to 20% of its weight in the last 24 hours before farrowing. Timed mating is largely successful, but in animals with large litters (rats and mice) it may be useful to wait until the female has the first offspring before commencing the operation. In guinea pigs, the most preferred method is to select females for surgery by measuring pelvic enlargement and spacing (see Philips, B.P., P. A. Volfe and H. A. Gordon, 1959, Studies on Rearing the Guinea Pig Germ, Ann. N.Y. Acad. Sci., 78: 183).

The caesarean section must be placed in a sterile environment before the first inhalation. They can be removed directly from the mother by hysterectomy through a slit sterile barrier foil and placed in a sterile insulator, or hysterectomy through a germicidal seal of the sterile insulator. Conventional surgical preparation of a female prior to caesarean section involves removal of obdominal hair and cleansing and disinfection of the surgical site. Anesthesia is preferably performed by dislocating cervical vertebrae in rats and mice, although general or local abdominal moderate anesthesia may also be used without causing severe levels of fatal depression and mortality. In guinea pigs, intervention is generally carried out after the administration of sedatives and with local anesthesia.

The introduction of the baby by hysteroctomy through the berrier membrane requires special isolator equipment. Reyniers Stainless Steel Surgical Unit (see Reyniers, JA, 1965, Germfree Life Methodology (gnotobiotics) and experimental nutrition, pp. 458-466, in: Proc. 3rd Dormitory. Congr. Biochem., Bruxelles) has a built-in horizontal metal partition, that separates the upper and lower part of the unit. The partition has a circular opening, covered with a mylar plastic sheet to maintain the integrity of the upper compartment. The female ready for surgery is placed in the lower compartment with an abdominal pressed against mylar sheet.

All surgical instruments are in the upper compartment and the procedure is performed in this sterile field. The cut is conducted through plastic and leather through electrocautery or scalpel. For the cut of the skin, a self-sterilizing electrocautery knife is preferred. The edges of the skin and mylar are clamped together. The sterile gauze is placed on the edge to cover the edges of the cut of the skin and a warm disinfectant (benzalkonium chloride 1: 1000) is applied to the exposed strip before opening the posterior cavity. Particular attention and caution must be taken to ensure that cuts do not damage the viscera. Inserting a medical forceps or hemostat between the peritoneal wall and the viscera may be useful. The uterus is then opened and the cub removed. The fetal membranes are removed and the umbilical cord is clipped and separated. The chick is quickly dried and massaged to stimulate breathing. It is then moved to the rear caring unit. An additional portion of mylar may be secured through a circular surgical opening and the female procedure may be repeated 5 to 6 times without serious risk of contamination. Caesarean section can also be performed using a plastic insulator or sleeve as a surgical unit. The outer surface of the insulator floor is pre-sterilized and pressed against the abdomen of the animal and thus serves the same purpose as the mylar film as described above. After surgery, the slit in the plastic barrier is sealed with sterile tape and the surgery is repeated on another pregnant female.

Plastic isolators are more commonly used to deliver a baby by hysterectomy (see Foster, H., 1959, A Procedure for Obtaining the Nucleus Stock for a Pathogen-Free Animal Colony, Lab. Anim. Line 9: 135). The uterus is exposed aseptically and clamped close to the neck. The selected uterus is transferred to a sterile unit through a liquid germicidal plug. Immediately after transfer to the insulator, the young shall be removed as quickly as possible to prevent inhalation of amniotic fluid. It is normally dried and breathed before clamping and detaching the umbilical cord. The baby is then fed to the mother or is hand-raised.

Histeroctomy can be successfully used in mice, rats and pigs. In guinea pigs, however, hysterectomy is preferred because of the high mortality rate if more than two minutes elapse between separation of the mother's blood stream and removal within the insulator.

Gnotobiotic colonies reproduction systems

Blood genera

Reproduction by the Brother-Sister Crossing system used in normal reproduction may also be used in gnotobiotic colonies.

Nepokrvný chov

True arbitrary crossings include several crossbred siblings and first relatives. Although we avoid such crossbreeding in bloodless breeds, the resulting crossbreeding system does not minimize the proportion of inbreeding. Any system with minimal perfection may be employed (see Falconer, DC, 1967, Genetic aspects of breeding methods, pp. 72-76), in: The UFAQ Handbook on Line and Management of Laboratory Animals, 3rd ed., E. and S Livingstone, Ltd., London; National Research Council, Institute of Laboratory Animal Resources, 1969, A Guide to Genetic Standards for Laboratory Animals, National Academy of Science, Vashington, DC).

Comparable conventional and gnotobiotic colonies

If it is desired to breed conventional and gnotobiotic colonies in order to compare them, then their mutually similar genetic composition can be ensured by using caesarean section in litters from a truly reproduced population of the normal colony to the gnotobiotic colony. This method appears to be suitable for those breeders who place more emphasis on non-gnobiotic rats and mice, but have the disadvantage of preventing the formation of a microbiological pedigree that would facilitate microbiological follow-up. Ideally, this could be done by using litters from certain crosses as a breeding base for a common colony, and by using other litters from each of said crosses as a breeding base for a gnotobiotic colony. If this procedure is carried out in every second or third generation, then the genetic composition of the two colonies becomes very similar, provided, of course, that the same crossing system is used in each of the colonies.

Alternatively, litters from a specific population of a gnotobiotic colony may be used to form or replace a conventional colony. The genetic consequences will be the same, provided the same procedures are used.

Record-keeping (See Volff, GL ,, 1967, Practical Matting Systems and Record-Keeping in Breeding Colony, pp. 97-113, in: The UFAV Handbook on the Line and Management of Laboratory Animals, 3rd ed., E. and S. Livingstone, Ltd., London).

Appropriate records shall be kept of the animals and the maintenance of the insulator. Animal records must express the effectiveness of the operation and the biological status of the animals. The isolator records must show the time sequence of facts relating to the insulator in order to assist in finding a barrier break if contamination is detected.

Antigenless animal

In a preferred embodiment of the present invention, it is preferred to breed a sterile animal on a chemically defined (CD), water-soluble, ultrafiltered, low molecular weight diet. Such a diet is considered to allow full control of nutritional intake and antigen uptake by the animal. Said diet is generally made entirely from ingredients that may be chemically defined, for example, from amino acids, simple sugars, fats, vitamins and minerals. For purposes of the present invention, the chemically defined diet comprises amino acids, simple sugars, fats, vitamins and minerals, and no other ingredients with a molecular weight greater than 10,000 Daltons. Thereafter, all components of the chemically definable diet are of low molecular weight and are naturally circulating components of animal nutrition. Therefore, it is believed that the components of the CD diet do not contribute to the immune response. Current literature indicates an animal that is kept on a CD diet as an antigen-free animal.

It is also preferred to use litter filter paper, as otherwise sterile animals consume litter, which could result in an immune response. Eating litter from filter paper is not considered to cause an immune response.

In specific CD diets for certain species, the components will be used in proportions and amounts to meet the known nutritional needs of those species. The composition and preparation of preferred diets for sterile animals are described below.

Table 3

Composition and preparation of chemically defined diet L 489E14Se

Component Quantity of the component in grams in

100 g ingredients

To 192 ml of Milli-Q water at 70 ° C is added:

Leucine 1.9

Phenylalanine 0.74

Isoleucine 1.08

Methionine 1.06

Tryptophan 0.37

Valín 1,23

Asparagine 0.91

Araganin hydrochloride 0.81

Threonine 0.74

Lysine Hydrochloride 1.77

Histidine hydrochloride 0.74

The solution is cooled to 45 ° C and:

Glycine 0.59

Proline 1.48

Serine 1.33

Alanine 0.59

Sodium glutamate 3.40

L-Tyrosine ethyl ester hydrochloride 0.62

Ferrous gluconate 0.05

Salts 35D ¹ 0.105

Sodium selenite 0.074

The solution is cooled to 5 ° C and:

Calcium glycerophosphate 5.22

Magnesium glycerophosphate 1.43

CaCl ₂ .2 H ₂ 0 0.185

Sodium chloride / potassium chloride mixture (containing 680 mg ΚΪ) 0,086

Vitamin B Vitamin B, 111E5 ^ 0.09

Vitamin B12 1.20 mg

Potassium acetate 1,85

To 108 mL of Milli-Q water was added at 70 ° C

D-dextrose, anhydrous 71.28

The solutions were cooled to 5 ° C and pooled for ultrafiltration.

The composition of the 35D salt mixture is shown in Table 7 ² Added in addition to the addition of sodium selenite in the 35D salt mixture ^ The vitamin B 111E5 composition is shown in Table 8 ⁴ Added in addition to the vitamin B-12 contained in the 111E5 vitamin mixture (see below) section examples).

This mixture was fed to mice or rats ad libitum.

Table 4

Composition of fat supplement LADEK 69E6

component Daily amount per adult * (0.385 ml) Purified soy triglyceride · '· 0.33 g retinyl palmitate 6.45 mg (11.7 I.U.) Cholecalciferol, 0.0288 mg (1.15 I.U.) 2-amboalfatokoferol 3.3 mg 2-amboalfatocoferol acetate 6.6 mg phylloquinone 72 mg

^L Composed of: 12 1, 24 55 8 * Mice in lactation receive twice the normal adult dose% palmitate 5% stearate% oleanu% linolan% linolean

For a more detailed description of CD diets, see Pleasants, J.R., et al. J. Nutr., 116, 1949-1964 (1986); Pleasants, J., et al., Germfree Research: Microflora Control and Its Application to Biomedical Sciences, B.S. Vostmann, Ed., P. 87, Lisss, New York (1985); Vostmann, B. S., et al., J. Nutr., 112, 552 (1982); and Pleasants, J.R., et al., J. Nutr, 100, 498 (1970), the disclosure of which is incorporated herein by reference.

Preparation of monoclonal antibody

The sterile animal is then used to prepare monoclonal antibodies. A sterile system can be used to prepare a monoclonal antibody, an animal can produce in antigens containing U.S. Pat.

antigen, which is a List of Examples It is believed for any non-sterile condition, however, that patent 3,935,074 shows that a sterile animal exhibits a much higher immune response to the antigen than non-sterile. Thus, the likelihood of capture of a B-lymphocyte that produces an antibody that is capable of binding to a specific antigen epitope can be increased. This is a major advantage of the present invention. In addition, the sterile system is considered to be particularly suitable for generating highly specific antibodies for those antigens having multiple epitopes.

The sterile animal may be immunized by conventional techniques. However, it is preferable to immunize at least three times a sterile animal with at least a three-week pause between each immunization and subsequent pre-vaccination. This increased level of immunization is considered necessary because it has been observed that after two immunizations, which is common, IgM-secreting B cells are still prevalent over preferred IgG-secreting B cells.

Somatic cells

Somatic cells, especially B cells, of a sterile animal that is capable of producing an antibody are suitable for fusion with the B cell myeloma line. Such antibody-forming cells that are in the stage of plasma blast splitting are preferably fused. Somatic cells can be derived from the lymph nodes, spleen, and peripheral blood of the best animals, the choice of lymphoid cells largely depending on their assessed feasibility in a particular fusion system. Somatic cells derived from the spleen are generally preferred. After hyperimmunization, sterile animals can be used as a source of antibody producing lymphocytes. Mouse lymphocytes give a higher percentage of stable fusions to the mouse myeloma lines described below. Antibody-producing cells from other sterile animals may also be used. The choice of a particular sterile animal depends on the choice of antigen; it is necessary that the sterile animal have a B cell from its B cell equipment which can produce an antibody to the antigen.

Immortal cells

For use in the preparation of hybridomas, specialized myeloma cell lines from lymphocyte tumors have been developed (G. Kohler and C. Milstein, 1976, Eur. J. Immunol., 6: 269-270). Cell lines have evolved for at least three reasons. The first reason is to facilitate the selection of fused myeloma cells. Usually this is done by using myelomas with an enzyme deficiency that causes them to grow in a certain selective medium, which promotes the growth of hybridomas. The second reason arises from the inherent ability of lymphocyte tumor cells to generate their own antibodies. The purpose of using monoclonal techniques is to obtain immortalized fused hybrid cell lines that produce the desired specific antibody itself, a genetically directed somatic cell component of the hybridoma. To suppress the production of tumor cell antibodies by a hybridoma, myeloma cell lines unable to produce light or heavy immunoglobulin chains, or those having an insufficient mechanism of antibody secretion, are used. A third reason for selecting these cell lines is their suitability and fusion efficiency.

Several myeloma cell lines may be used to prepare fused cell hybrids, including NS-1, X63-Ag8, NIS-Ag4 / 1, MPC11-45.6TG1.7, X63-Ag8.653, Sp2 / O-Agf14, FO and 194 /5XXO.Bu.1, all obtained from mice and 210 -.RCY3.Agl.2.3, obtained from rat (GJ Hanmerling and JF Kernly, eds., 1981, Monoclonal antibodies and hybridomas, in: JL Turk, ed., Research Monographs in Immunology, Vol. 3, Elsevier / North Holland Biomedical Press, New York).

merger

The method of preparing antibody-producing hybrids from spleen or lymph node cells and immortal cells generally comprises admixing somatic cells with immortal cells in a ratio which may vary from about 20: 1 to about 1: 1 in the presence of the agent (s) (chemical, viral or electrical) that promote cell membrane fusion. It is often preferred that the same animal species be used as a source of somatic and immortal cells for fusion. Fusion methods are described by Kohler and Milstein, 1975, Nature, 256: 495-497; 1976, Eur. J. Immunol., 6: 511-519), followed by Gefter et al. (1977, Somatic Calls Genet., 3: 231-236) and by Analysis et al. (1983, Immunology Today, 4, 72). The above researchers used reagents: Sendai virus and polyethylene glycol (PEG) to promote fusion.

Recently developed EBV transformation techniques (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pages 77-96) may also be utilized.

Isolation of clones and antibody detection

Fusions produce viable hybrids, usually at low frequency, about 1.10 ° to 12.10 °. Since the frequency of viable hybrids is low, there is a need to have a way to separate the fused cell hybrids from other unfused cells, especially myeloma unfused cells. There is also a need for a method for detecting hybridomas producing the desired antibody among other fused cell hybrids.

Fused cells are generally cultured in selective media, for example, in a HAT medium containing hypoxanthine, aminopterin and thymidine. The HAT environment permits hybrid cell proliferation and prevents the growth of unfused myeloma cells that would normally divide undefined. Aminopterin blocks de novo synthesis of purine and pyrimidine through inhibition of tetrahydrofolate production. The addition of thymidine bypasses the impediment to pyrimidine synthesis, while hypoxanthine is contained in the medium and has the task of inhibiting the cells to synthesize purine using the nucleotide reaction pathway.

The myeloma cells used are mutants depleted in phosphoribosylhypoxanthine transferase (HPRT) and thus cannot utilize said reaction pathway. By surviving hybrids, B cells supply genetic information to generate said enzyme. Because B cells alone have only a limited lifetime in culture (approximately two weeks), the only cells that can reproduce in the HAT environment are hybrids formed from myeloma and spleen cells.

The mixture of fused myelomas and B-lymphocytes is diluted with HAT and cultured in multiple wells of microtiter plates. This facilitates the screening of the antibodies secreted by the hybrids and prevents individual hybrids from outgrowing the others. After two to three weeks, when the hybrid clones become recognizable by microscope, the fluid from the individual wells containing the hybrid clones is tested for the production of the specific antibody.

The test must be sensitive, simple and fast. Assay techniques include radioimmunoassays, enzymatic immunoassays, cytotoxicity assays and plaque assays.

Cell proliferation and antibody production

When the desired fused cell hybrids are separated and cloned into individual antibody-producing cell lines, each cell line may be propagated by either of two conventional methods. A sample of hybridomas can be injected into a histocompatible animal of the same species as the donor of somatic and myeloma cells in the original fusion. The animal, after injection, develops tumors secreting a specific monoclonal antibody that results from fused cell hybrids. Animal body fluids, such as serum or ascites fluid, can serve to obtain monoclonal antibodies at high concentrations. Alternatively, individual cell lines may be propagated in vitro in laboratory culture flasks. A culture medium having a high concentration of a single specific monoclonal antibody can be treated by decanting, filtration or centrifugation.

Use of a monoclonal antibody

The monoclonal antibodies produced by the method of the invention may be used in any known technique, or in a technique that will be developed in the future that utilizes or will utilize a monoclonal antibody.

The main use of monoclonal antibodies is in immunoassay, which is a measure of antibody-antigen interaction. In general, immunoassays may be heterogeneous or homogeneous. In a homogeneous immunoassay, an immunological reaction usually requires a specific antibody, a labeled analytical reagent, and an assayed sample. The signal resulting from the label is adjusted, directly or indirectly, according to the binding of the antibody to the labeled reagent. The immunological reaction and detection are carried out in a homogeneous solution. Immunochemical labels that can be used include free radicals, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so on. The main advantage of homogeneous immunoassays is that the specific antibody does not need to be separated from the labeled environment of the analytical reagent.

In heterogeneous radioimmunoassays, the reagents are typically a sample, a specific antibody, and a means for generating a detectable signal. Generally, the sample is placed on a support, such as a plate or slide, and contacted with the antibody in the liquid phase. The support is then separated from the liquid phase and either the carrier or the liquid phase is analyzed by means of a signal from the device that produces the signal. The signal is related to the presence of the agent in the sample. The means for generating a recordable signal comprises the use of radiolabelled substances, fluorescent agents, enzymes, and the like. Examples of heterogeneous immunoassays are radioimmunoassays, immunofluorescence methods, enzyme immunoassays, and the like.

For a more detailed description and discussion of the above immunoanalytical techniques, see Enzyme-Immunoassay, by Edward T. Maggio, CRC Press, Inc., Boca Raton, Fla., 1980. See also, for example, U.S. Pat. U.S. Patent 3,690,834; 3,791,932; 3,817,837; 3,850,578; 3,853,987; 3,867,517; 3,901,654; 3,935,074; 3,984,533; 3,996,345 and 4,098,876. This selection is not intended to be complete.

Another major use of monoclonal antibodies is in-vivo imaging and therapy. Monoclonal antibodies may be labeled with radioactive compounds, for example, radioactive iodine, intravenously administered to a patient. The antibody may also be labeled with a magnetic label. NMR can then be used to detect the antigen. After localization of the antibody to the antigen, the antigen can be detected by emission tomography and nuclear scanning techniques to determine the position of the antigen.

As an example, the method of use is exemplified by suspending the purified monoclonal antibody in a suitable carrier, e.g., salt, with or without human albumin, and administering at appropriate doses intravenously, e.g., by continuous intravenous infusion over several hours, as reported by Miller et al. , in: Hybridomas in Cancer Diagnosis and Therapy (1982), which is incorporated herein by reference.

The monoclonal antibodies of the invention can be used in therapy. Antibodies with suitable biological properties are useful directly as therapeutic agents. Alternatively, the antibodies may bind to toxins to form an immunotoxin, or to a radioactive material or drug to form a radiopharmaceutical or drug. Methods for preparing immunotoxins and radicals from antibodies are widely known (see, for example, Cancer Treatment Reports (1984), 68: 317-328).

Polyclone antibodies derived from sterile animals can also be used for immunoassay and provide better results compared to polyclonal antibodies obtained from ordinary animals. Polyclone antibodies derived from a sterile animal can be prepared using a sterile animal as described above and by an immunization technique as described above, followed by separation ₍ polyclonal antibody from the animal by conventional techniques, for example, separation of serum from the animal).

Monoclonal antibody specific for fibrin

The homeostatic mechanism is a complex physiological response mechanism elicited to heal damage to ruptured vessels. Homeostasis is achieved through cooperative interactions between the damaged vessel wall, platelets and the coagulation system. The role of the coagulation system is to create an extensive fibrin network for stabilizing and anchoring the platelet plug to be formed on the subendothelial structure of the damaged vessel. The formation of insoluble fibrin matrix from circulating fibrinogen is the result of complex downstream reactions, culminating in the explosive formation of thrombin at the desired site. Coagulation is an enhanced process of a chain of enzymatic reactions in which proenzymes (clotting factors) are subsequently activated to active enzymes. There are several physiological mechanisms that control the process of fibrin polymerization to form a thrombus. These mechanisms include the thrombin inhibitor antithrombin III (ATIII), protein C, prostacyclin and various components of the fibrinolytic system such as tissue plasminogen activator (t-PA) and its fast acting inhibitor (PAI).

The homeostasis hypothesis proposed by Astrup in 1956 (Astrup, T., Blood, 11, 781-806 (1956)) suggests that there is a balance between fibrin formation (coagulation) and dissolution of fibrin (fibrinolysis). In normal, healthy condition, these functions are balanced. However, if the homeostatic process is disrupted, coagulation and fibrinolysis will manifest pathologically as thrombosis and bleeding. Clinical manifestations of pathological thrombosis or thrombotic disease are extremely diverse and include diffuse intravascular coagulation (DIC), deep vein thrombosis (DVT), arterial and venous thrombosis.

Thromboembolism and thrombotic complications of other vascular diseases (for example atherosclerosis) can result in major artery occlusions resulting in organ ischemia and accompanying life-threatening circumstances such as cerebrovascular accident (defeat), myocardial infarction and the like.

The fibrinolytic process is the conversion of an inactive zymogen, plasminogen into a proteolytic enzyme, plasmin, by the action of agents known as plasminogen activators. The molecular mechanism of physiological fibrinolysis is not fully solved, but it is known that during fibrin formation, plasminogen binds to fibrin where it can be activated by a plasminogen activator, for example ΐ-PA. In this way, plasmin is formed in the thrombus, where it is protected from inactivation by the major physiological inhibitor of plasmin, alpha2-antiplasmin.

By the action of plasmin, fibrinogen and fibrin break down into their degradation products. Fibrinogen degrades to fragments X and Y, and after further exposure to plasmin, to fragments D and E. Fibrin degrades to fragments X, Y, D and E from uncrosslinked fibrin and to crosslinked D-dimer, DD / E complex, Y dimer, YD-dimer and X oligomer of cross-linked fibrin.

Tests of thrombotic disease markers have until recently been conducted using both polyclonal antibodies by both the radioimmunoassay procedure and the latex agglutination type analysis. Gaffney has shown that these assays are extremely unreliable (Gaffney, P.J., Ann. N.Y. Acad. Sci., 408, 407-423, (1983)). Much more specific and sensitive is an immunoassay (such as an ELISA) that uses a monoclonal antibody and is becoming common practice in clinical laboratories. A limiting element of these diagnostic assays is the specificity and affinity of the particular monoclonal antibody used. The generation of highly specific antibodies to any of the potential indicators of homeostasis damage is hampered by the low level of indicator content and also by the antigenic relationship of a particular marker with its precursor, which is normally present in plasma at much higher concentrations than the actual marker. An example is the formation of complexes between enzymes and their inhibitors, for example thrombin-antithrombin III, plasmin-alpha2-antiplasmin, t-PA-PAI-1. The number of new antigenic sites resulting from said complex formation is very small, making the preparation of immunological probes (such as monoclonal antibodies) very difficult.

Similarly, the major problem associated with obtaining a monoclonal antibody to fibrin appears to be that fibrin and its physiological precursor fibrinogen are structurally and conformationally very similar. It has been found that when converting fibrinogen to fibrin, the covalent structure is retained to more than 98% (Plow, EF, et al., Semin. Thromb. Haemostat, 8, 36, (1982)) and therefore in fact only a small percentage of epitopes The fibrin molecule is composed of neoantigens (unique to fibrin). Many of the approaches that have been adapted to obtain fibrin * antibodies have focused on immunized animals with soluble fibrin fragments and synthetic peptides that mimic the affected neoantigenic fibrin sites. See Hui, K.Y., et al. Science 22: 1129-32 (1983).

Scheefers-Borchel, V., et al. , Proc. Natl. Acad. Sci. USA, 82, 7091-95 (1985); Elms, M. J., et al., Thromb. Haemost.

50, 591-94 (1983) and Kurdryk, B., et al., Mol. Immunol., 21, 89-94 (1984). It is believed that the binding sites of said antibodies are retained during the fibrin degradation process and therefore the compounds may also bind to fibrin degradation products.

The present invention represents a completely different approach and makes it possible to utilize intact fibrin antigen in conjunction with a higher immunological sensitivity of an antigen-free (AF) animal for the preparation of a fibrin-specific monoclonal antibody. For the purposes of the present invention, a fibrin-specific monoclonal antibody binds to fibrin and does not bind to fibrinogen, nor to fibrinogen degradation products or fibrin degradation products.

Materials and procedures

The animals

Sterile BALB / cAnN mice were obtained from breeding sterile colonies (GF - germfree) mice at the University of Visconsin. GF conditions were provided when animals were transported to the research facility. Antigen free (AF) breeding was started by moving pregnant GF mice to isolators and replacing the original L 485 nutrition with natural ingredients (see

Pleasants, J. P., et al. J. Nutr. 116, 1949-1964 (1986)) for a chemically defined (CD) AF diet. Their offspring, which never came into contact with natural ingredient diets (NI), were weaned from breast milk in exchange for a CD diet and designated as the first AF generation. AF mice crossed in pairs until the female was definitely pregnant; then the male was removed to ensure that the female received her full daily supplemental fat dose. The pups were on breast milk until day 24 of age.

Location of animals

AF breeding animals were housed in pairs in one half of a conventional polycarbonate mouse cage 28 x 17.8 x 12.7 cm 2. The bottom was cut off and replaced with a fake stainless steel bottom. A flat stainless steel tray was attached to the ends of the plastic cage. The compartment protruded sufficiently over and behind the cage to suitably fasten the stainless steel hatch. The recessed hatch, which normally rests inside the cage, has turned to allow for a suitable upper space above the false bottom. Stainless steel sleeves of appropriate size were welded to the top of the hatch to serve as holders of 60 ml food bottles. Four stainless steel containers were welded to the sides of the bulkhead at the ends so that they were halfway between the false bottom and the top. (A cage image is published in Pleasants, JR, The Germfree System for Aging and Immunity, in: CRC Handbook of Immunology in Aging, (Kay, MM, BS Makinodan, T., eds.), Pp. 257-297, CRC Press , Boca Raton, F1, (1981). Diet bottles were made of brown glass. The diet and the water bottle had plastic caps with holes in their center. The bottles were filled, turned and placed in the holders. The fat supplement was measured daily into stainless steel containers. A plastic waste tray was placed under each cage.

Vhatman quality filter paper - ashless filter paper No. 41, purchased as cuttings (Sargent) - served as litter and at the same time as an indigestible fiber.

Velch). The litter was cut into strips. Uncut squares of paper were used for internal cleaning of the insulator. The paper was autoclaved for 25 minutes at 121 ° C or irradiated (4.5 Mrad) in plastic bags. All mice received enough paper to cover one end of the cage. The paper was replaced after getting wet, yellowed or dirty.

Cages were placed inside a 1.37 x 0.6 x 0.6 m 3 Trexler flexible insulator (Trexler, PC, Lab. Animal Line, 13, 572-581 (1963)), using conventional gnotobiotic technology (see Vostmann). , BS, Ed., Gnotobiotics Standards and Guide Lines for the Breeding, Line and Management of Laboratory Animals, National Research Council, National Academy of Sciences, Vashington, DC 1970). The insulators were kept in a room at 21 ° C with a 12 hour light / dark schedule.

25 mm diameter and 75.5 mm long tygon tubes were sealed to the top of the insulator and sealed with vinyl plugs at both ends. This allowed sterile filtration of diet, water and oil.

A child

Table 5 shows the dietary composition and the sequence of dissolution of the ingredients in ultrafiltered Mill-Q water (Millipore, MA, USA). Amino acids and dextrose were used in Sigma tissue culture quality. Vitamins were also from Sigma except for pure retinyl palmitate, provided by Hoffman-La Roche, Inc. (Nutley, NJ, U.S.A.). The other reagents have been certified by Fischer or in a quality equivalent to them. The completely water-soluble nutrient was filtered cold with an Amicon Diaflo TC3 ultrafilter using three 150 mm PmlO membranes (Amicon).

The ultrafiltration membranes had a molecular weight limited to 10,000 Dalts. The assembled ultrafiltration devices were pre-sterilized by washing with 0.5% sodium hypochlorite solution and then thoroughly washing with water. The ultrafiltered nutrition was stored at 4 ° C in sterile containers until use. Diet was introduced into AF isolators using a 0.2 μπ nylon (MSI) filter mounted in an autoclaved pressure filter holder having an inlet tubing inserted into the neoprene plug # 6. For this purpose, the upper vinyl plug of the tygon tube sealed on the top parts of insulator removed. The inside of the tube was sprayed with a sterilizing 2% peracetic acid solution containing 0.1% alkylaryl sulfonate. A filter holder plug was inserted in place of the top plug. After 20 minutes, the bottom plug (inside the insulator) was removed and the nutrient solution or water was filtered into the insulator under a pressure of 20 psi of nitrogen.

The composition of the fat supplement is given in Table 4. Soybean triglycerides were prepared from their methyl esters, which were distilled under vacuum in a temperature range, providing esters from palmitate to linoleate. Said esters were then esterified with glycerol to form mixed triglycerides. (Nu-Chek Prep., Elysian, MN.). Fat-soluble vitamins were added to the mixed triglycerides prior to filtration into the insulator. For filtration into the insulator, the fat supplement was preheated to 50 ° C, filtered by the same procedure as the water-soluble portion of the nutrition was filtered. Fat intake was dosed at 0.375 ml per day. An increased dose of fat supplement greatly reduced the infant mortality rate. The average litter frequency also increased compared to commonly kept animals. Females in lactation received twice the normal amount of fat supplement.

- 38 Table 5

Composition and preparation of chemically defined diet L 489ElSe

component Quantity in grams per 100 g of ingredients To 192 ml of Milli-Q water at 70 ° C was added: leucine 1.9 phenylalanine 0.74 isoleucine 1.08 methionine 1.06 tryptophan 0.37 valine 1.23 asparagine 0.91 Arganine hydrochloride 0.81 threonine 0.74 Lysine hydrochloride 1.77 Histidine hydrochloride 0.74 The solution was cooled to 45 ° C and added are: glycine 0.59 proline 1.48 serine 1.33 alanine 0.59 Sodium glutamate 3.40 L-Tyrosine ethyl ester hydrochloride 0.62 Ferrous gluconate 0.05 Salts 35D ¹ 0,105 Sodium selenite 0,074 The solution is cooled to 5 ° C and: Calcium glycerophosphate 5.22 Magnesium glycerophosphate 1.43 CaCl ₂ .2 H ₂ 0 0,185 The sodium chloride mixture was removed carbonate (containing 680 mg KI) '0.086 Vitamin B Vitamin Mixture, Mixture 111Ε5 ° 0,09 Vitamin Blz ^ 1,20 mg Choline chloride 0.31 Potassium acetate 1.85 To 108 ml of Milli-Q water was added at 70 ’C: D-dextrose, anhydrous 71.28

The solutions are cooled to 5 ° C and combined prior to ultrafiltration. The composition of the 35D salt mixture is shown in Table 7. ² Added in addition to the addition of sodium selenite contained in the 35D salt mixture. vitamin B-12 contained in vitamin E115 (see Examples below).

Table 6

Composition of fat supplement LADEK 69E6 «

component

Amount per adult daily dose * (0.375 ml)

Purified soy triglyceride 0,3 0.33 g

Retinyl palmitate 6.45 mg (11.7 I.U.)

Cholecalciferol 0,0288 mg (1,15 I.U.)

2-amboalfatocoferol 3.3 mg

2-amboalfatocoferol acetate 6.6 mg

Phyloquinone 72 mg * Mice in lactation receive twice the normal adult dose

Consisting of:

1.5% palmitate% stearate% olean% linolan% linolenane

Table 7

Composition of the 35D salt mixture

Salt Amount (per 300 ml diet (mg)) Mn (Chocoa) ₂ .4 H ₂ 0 55.4 ZnS0 ₄ . 7 H ₂ 0 40.6 Cu (CH ₃ CO ₂ ) ₂ . h ₂ o 3.7 Cr (CH ₃ C00) ₂ . H ₂ 0 2.5 NaF 2.1 SnS0 ₄ . 2 H ₂ 0 0.37 (NH ₄ ) ₆ Mo ₇ 0 ₂₄ .4 H ₂ 0 NiCl ₂ .3 H ₂ O 0.37 0.37 Co (CH ₃ COO) ₂ .4 H ₂ 0 0.11 For ₃ VO ₄ 0.22 Na ₂ SeO ₃ 0,096

Table 8

Composition of B-vitamin mixture 111E5

additive Amount (per 300 ml diet (mg)) Thiamine hydrochloride 1.23 Pyridoxine hydrochloride 1.54 biotin 0.25 Folic acid 0.37 Vitamin B 12 0.37 riboflavin 1.85 niacinamide 9.2 i-inositol 61.9 Calcium pantothenate 12.3

The water was ultrafiltered to Milli-Q and was filtered into the insulator in the same way as diet.

Ongoing microiological assessment (Monitoring) /

The antigen-free system was checked for microbiological contamination according to the recommendations of Vostmann, B.S., Ed. , Gnotobiotics Standards and Guidlines for Breeding,

Line and Management of Laboratory Animals, National Research Council, National Academy of Sciences, Washington, D.C. In short, diet and water-moistened swabs from inside the insulator were used to obtain fresh faecal samples directly from the mice. Samples were also taken from bowls placed under cages. The smears were also taken from the insulator walls, especially around the inlet openings. Two swabs were always taken. One set was tested by direct evaluation of t under a microscope for bacteria and fungi using Gram staining. A second set of swabs was used to detect microorganisms. Samples were cultured for three weeks before being evaluated as negative.

The microbiological evaluation was performed approximately every two weeks, or every few days for new entries into the isolators.

Preparation of monoclonal antibodies using mice without antigens

The above-mentioned antigen-free mice were used as lymphocyte donors in the preparation of monoclonal antibodies. Solutions of all antigens were prepared under sterile conditions under a laminar flow hood.

The following procedure was used to immunize AF mice. The respective antigen (25-50 micrograms) was dissolved in a sterile salt solution (100 microlitres) and emulsified with an equal volume of complete Freund's adjuvant (FCA). Before preparing the emulsion, interferon (1000 units) was added to the antigen solution. A sterile syringe and needle were used for each immunization. The syringes were transferred to the AF isolator through the inlet opening where they were sterilized by spraying with 2% peracetic acid solution. Repeated injections were administered using the same amount of antigen and substituting FCA with incomplete Freund's adjuvant. A total of three immunizations were given, every three weeks. The last injection (without adjuvant) was given 4 to 7 days prior to the fusion. All injections were administered intraperitoneally. On the fusion day, mice were removed from the isolator and immediately sacrificed by CO ₂ asphyxiation. Spleens were harvested and splenocytes fused to mouse myeloma cells (NS1) using conventional hybridoma technology.

Use of an antigen-free animal system to prepare a monoclonal antibody specific for fibrin

The antigen-free system was used to elicit a monoclonal antibody specific for fibrin. The antibody is also highly specific and does not recognize either fibrinogen, nor fibrin decomposition products or fibrinogen decomposition products. The hybridoma cell line was prepared by fusing splenocytes from an antigen-free BALB / c mouse immunized with human fibrin and NS1 myeloma cells.

Immunization schedule

Three, eight week old female antigen-free mice were immunized with 33 micrograms of human fibrin preparation. The preparation consisted of a frozen fibrin sample, which was prepared as follows:

Human fibrinogen was converted to fibrin by thrombin and factor XIIa. The fibrin precipitate was then frozen in liquid nitrogen and mechanically crumbled to an exceptionally fine powder. A dispersion of this freeze-dried fibrin in a salt solution was prepared. A clear solution of cross-linked fibrin XL-Fn was obtained with a final concentration of 1 mg in 1 ml. 33 microliters of this fibrin antigen was used to immunize the animals. The volume of the antigen solution was adjusted to 100 microliters with a sterile saline solution and then emulsified with FCA as described above. Two booster immunizations were given at three-week intervals using the same level of antigen concentration in incomplete Freund's adjuvant as previously described. The last immunization was given 4 days before the fusion. The same level of antigen concentration was used, but the adjuvant was replaced with a salt solution.

Proof of presence and determination of antibody

Qualitative and quantitative monoclonal antibody assays were performed using enzyme-linked immunoadsorbent analysis (ELISA). These analyzes were performed using human fibrin immobilized on a 96 well PVC plate (Costar). Fibrin-coated assay plates were prepared by incubating 100 microliters of fibrinogen solution (Kabi, L-quality) (50 micrograms in 1 ml borate / salt buffer) overnight at 4 ° C. Unbound fibrinogen was removed by washing with PBS containing 0.05% Tween 80 (PBS-Tween).

The fibrinogen coating on each well of the plastic plate was converted to fibrin by incubation with 100 microliters of thrombin solution (10 µl units per ml) containing 2 mM CaCl 2 for 1 hour at 37 ° C. To evaluate the antibody, calibration graphs were constructed in a conventional manner using an antibody preparation that was homogeneous with SDS-PAGE.

To avoid non-specific binding, fibrin-coated plates were incubated with 1% BSA in PBS at pH 7.4. An antibody-containing solution (100 microliters) was then added and incubated at 37 ° C for 90 minutes. After each step of the procedure, the wells were effectively washed with PBS-Tween. Bound antibody was delegated by the addition of 1000-fold diluted alkaline phosphatase-conjugated anti-mouse antibody (Sigma) diluted in PBS with 1% BSA, pH 8.0.

Preparation of hybridomas - fusion

Mice after asphyxiation with CC> 2 were immediately splenectomized. Spleen cells from immunized mice were fused with fuzzoagent polyethylene glycol 4000 (3000-

3700). Cells were incubated in HAT selection medium in T flasks for 1 week. After this time, the cells were plated in 5 x 96 wells. Of these, 93 holes showed growth. Of these, 19 were fibrin antigen positive. One of these clones, F492D8 (later renamed MH1), produced an antibody that recognized the fibrin antigen and did not cross-react with fibrinogen. This particular MH1 clone was cloned three times (recloned) with limited dilution so that the third cloning phase was performed at a dilution of 1 cell per well. After cell stabilization, the cell was nourished in a serum-free environment and the cell line produced an antibody of about 7.5 mg / liter.

Purification of the monoclonal antibody

Tissue culture medium (4 liters of batch) was centrifuged to remove damaged cells prior to purification and filtered through a 0.8 micron nylon membrane to remove any particle debris. The hybridoma liquid medium (supernatant) was concentrated at 4 ° C to a volume of 50 ml using a 30,000 MW YM membrane type ultrafiltration system (Amicon). The buffer was diafiltrated to 20 mM 2 (N-morpholine) ethanesulfonic acid. (MES) pH 6 (Buffer A) according to the manufacturer's instructions. After further concentration to a final volume of 100 ml, the antibody solution was filtered through a 0.451 micron nylon membrane prior to further purification. The concentrated antibody solution was purified by liquid chromatography on a Vaters HPLC Chromatograph using a 7.75 mm x 10 cm ABx column (J.T.Baker, Phillipsburg, NJ, USA). The packed buffer A column was allowed to equilibrate. A sample (100 mL) was then fed at a flow rate of 1.0 mL per minute. After efficient washing of the column with buffer A, the antibody eluted from the column with a gradient system from buffer A to 100% buffer B (1M sodium acetate solution, pH 7) at a flow rate of 1 ml / min. Aliquots (2 ml each) were collected; those containing MAb (as determined by ELISA) were pooled and dialyzed against phosphate buffered saline (PBS) (20 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) and stored at -20 ° C at concentrations &Gt; 1 mg / ml. The ABx column was regenerated by washing for 5 minutes with 100% buffer B, then re-equilibrated with buffer A. The volume of buffer used was 15 times the volume of the column.

Determination of fibrin specificity

Initial determination of fibrin specificity was achieved by systematic testing of hybridoma supernatants, separately on fibrin and fibrinogen-coated microtiter plates. Only those cell lines that produced the antibody and which did not cross-react with fibrinogen were selected.

Further confirmation of fibrin specificity was performed by comparison with fibrinogen in solution, confirming that the antibody does not recognize fibrinogen in solution.

The control assay used to confirm the specificity of the antibody was performed according to the ELISA methodology described above with pre-incubation of the antibody with fibrinogen in solution. Briefly, the hybridoma supernatant was incubated at 37 ° C for 30 minutes with fibrinogen solutions at physiological concentration (4 mg / ml) containing BSA (10 mg / ml) to prevent non-specific binding of the antibody to fibrinogen. The fibrinogen / antibody solution was then transferred to wells of a microtiter plate already coated with fibrin. To prevent possible polymerization of fibrinogen by residual thrombin in the fibrin wells, GlyProArgPro (GPRP) was added to the fibrinogen inhibitor. The assay was then performed as a conventional ELISA for the antibody bound to the immobilized antigen. In all experiments in which the fibrin specificity of the MH1 antibody was tested, a second 45J antibody was also used as a control. Antibody 45J cross-reacts with both fibrin and fibrinogen.

Determination of cross-reactivity with fibrinogen

Immobilized fibrin and fibrinogen

The MH1 cell line produces a monoclonal antibody from mice that cross-reacts with fibrin when immobilized on the surface of a PVC microtiter assay plate (Table 9). Said antibody does not recognize fibrinogen immobilized on the plate in the same assay. As can be seen from the data in Table 9, there was a very significant increase in immunoreactivity when fibrinogen was converted to fibrin by thrombin. it

46 clearly indicates the manifestation or formation of neoepitope on the fibrin molecule. However, control antibody 45J clearly recognizes the epitope that is retained when converting fibrinogen to fibrin.

Comparative test with fibrin and fibrinogen

The fibrin specificity of the MH1 antibody was further demonstrated in a comparative assay in which the hybridoma supernatant was pre-incubated with a fibrinogen solution (final concentration 4 mg / ml) prior to ELISA analysis on fibrin-coated wells. Since a high level of fibrinogen (500 times the antibody concentration) was used in this assay, it was added to the BSA mixture (10 mg / ml final concentration). To prevent polymerization of fibrin by residual thrombin on fibrin-coated plates, GPRP peptide was added to the mixture. The results of this analysis show that the MH1 antibody does not recognize fibrinogen in solution (Table 12).

Assuming that 400 ng of fibrin is bound in each well of the microtiter assay plate, the fibrinogen level used in this particular comparative assay represents a 1000-fold excess compared to the bound fibrin antigen level. In addition, this means a 400-fold excess of antibody level in the tissue supernatant.

Table 9

Cross-reactivity of MH1 with fibrinogen and fibrin antigen

MAb ^A 405 _nm / 30 min fibrin f ibrinogen ΜΗ 1 0.90 0,025 45 J 1,65 ' 1.50

Table 10

Cross-reactivity of the MH1 antibody with fibrin in the presence of fibrinogen

MAb ^A 405 nm ^{/ 3} ° min + fibrinogen - fibrinogen ΜΗ 1 1.24 1.27 45 J 0,438 1.74

Determination of cross-reactivity with fibrin (ogen) degradation products

Fibrinogen degradation products (FDPs) were prepared by incubating fibrinogen with plasmin at 37 ° C for 10 minutes to 3 hours. Fibrinogenolysis was interrupted after the selected time by the addition of Trasylol (100 Kallikrein inhibition units / ml) and 20 ml epsilon-aminocaprylic acid (EACA).

Crosslinked fibrin degradation products (XLFDPs) were prepared by adding thrombin (4 NIH units / ml) to a solution of fibrinogen (5 mg / ml) in a buffered salt solution (TBS pH 7.4, 50 mM Tris HCl, 150 mM NaCl) containing 10 mM CaCl 2, plasminogen (0.25 mg / ml) and urokinase (50 IU / ml. The mixture was incubated for various long periods of time as described above for FDPs.

To determine cross-reactivity of antibodies to fibrin degradation products and fibrinogen degradation products, the respective degradation products were plated on microtiter plates and ELISA was performed in a conventional manner. The results in Table 11 indicate that the MH1 antibody does not cross react with any (plasmin generated) fibrin degradation product. As shown in Table 12, the antibody does not react with XL-fibrin degradation products. The same finding was achieved when Western immunoblotting analysis (VIBA) was performed.

From this, it can be concluded that said antibody recognizes an epitope of an intact fibrin molecule that is not present or obtainable on the surface of the precursor molecule, fibrinogen. Indeed, the epitope is degraded by the effect of plasmin on cross-linked fibrin, as can be expected from the results in Table 12.

Accordingly, the MH1 antibody is a fibrin-specific antibody that can be defined as follows:

In the present invention, a fibrin-specific monoclonal antibody is a monoclonal antibody that:

(1) has a reactivity of less than 75%, preferably less than about 10%, of the fibrinogen cross-reactivity when used in an amount 1000 times greater than that of fibrin in the comparative tests of cross-reactivity with fibrin and fibrinogen as described above;

2. in the cross-reactive fibrin cross-reactivity assay and in the fibrin degradation product assay described above, the reactivity of the plasmin-reacted monoclonal antibody to plasmin for about 3 hours is less than 50%, and preferably less than 40%, of the reactivity a monoclonal antibody with fibrin at time zero, and

3. In the fibrinogen cross-reactivity assay and in the fibrinogen degradation product assay as described above, the reactivity of the fibrinogen monoclonal antibody treated with plasmin for about four hours is no greater than the reactivity of the fibrinogen monoclonal antibody at time zero.

The MH1 antibody was further characterized by determining its affinity for fibrin. Affinity was determined by Scatchard analysis (Frankel et al., Molecular Immunology 16: 101-106 (1979)) using the ^x1- labeled MH1 antibody. The Kp dissociation constant obtained was 6.7 x 10 6 M. The affinity shown is about 5,000 times that of ΐ-PA to fibrin.

It was also determined by VIBA that the MH1 antibody did not cross-react with the fibrinogen βα, β or gamma chains. It was also determined in the same analytical manner that pro49 MH1 antibody did not cross-react with fibrinogen Aa or β chains, even if they had been previously treated with thrombin. (The action of thrombin on fibrinogen causes the release of fibrinopeptide A from the Aa chain and fibrinopeptide B from the βb chain, thereby forming the α chain and the β chain of fibrin).

Table 11

Cross-reactivity of MH1 with fibrinogen degradation products

Reaction time with plasmin Λ 405 nm / 30 min (Min) ΜΗ 1 45 J 0 0.15 1,037 10 0.10 nm 20 0.11 1.02 40 0.11 0,410 60 ο, ϊο 0,330 240 0.09 0,300

Note: nm = not measured

Table 12

Cross-reactivity of antibody ΜΗ 1 with fibrin degradation products

Time of reaction with plasmin (hours) Λ 405 nm / 30 min ΜΗ 1 45 J 0 0,890 1.40 3 0, 354 1.0 5 0,310 0.77

In addition, the results in Table 11 show that control antibody 45J binds to fibrinogen and does not cross-react with fibrinogen degradation products. Accordingly, said monoclonal antibody is specific to fibrinogen and represents another aspect of the invention. A fibrinogen-specific monoclonal antibody can be immuno-analytically used to determine plasma fibrinogen levels in vitro. Antibody 45J was further characterized by determining that the 45J epitope is on the a chain of fibrinogen in the region from amino acid 206 to about 424, most likely in the region from amino acid 207 to 231. In this invention, a monoclonal antibody specific to fibrinogen is such. an antibody which, when determining cross-reactivity with fibrinogen and fibrinogen degradation products as described above, gives a reactivity of plasmin-treated monoclonal antibody to fibrinogen for about 40 minutes, less than 50% compared to reactivity of fibrinogen monoclonal antibody at time zero .

The 45J hybrid was prepared by conventional techniques using conventional Balb / c mice immunized with fibrin. However, fibrinogen may also be used as an antigen.

Determination of cross-reactivity with uncrosslinked fibrin and uncrosslinked fibrin clots

ELISA has shown that the MH1 antibody cross-reacts not only with cross-linked fibrin (XLFn) but also with non-cross-linked fibrin (NONXLFn). The ELISA was performed as follows:

1. 96-well microtiter plates were coated with 100 μΐ of fibrinogen solution (50 gg / ml in borate (pH 8.5) saline buffer) at 4 ° C overnight.

2. Crosslinked fibrin was formed in wells coated with fibrinogen by incubation with a solution of thrombin (10 NIH units / ml) in buffered (Tris) saline (TBS, pH 7.4, 50 mM Tris HCl, 150 mM NaCl) containing 2 mM CaCl 2 and 10 mM cysteine, for 1 hour at 37 ° C.

3. Uncrosslinked fibrin was formed in wells coated with fibrinogen by incubating for 1 hour at 37 ° C with a solution of thrombin (10 NIH units / ml) in phosphate buffer (pH 6.1) containing EDTA to a final concentration of 0.0125 M.

4. Bound antibody was determined by incubation with anti-mouse basic phosphatase conjugate. Bound conjugate was determined by the addition of an alkaline phosphatase substrate and the colorimetric reaction was monitored at 405 nM using an automated plate reader.

The results of the test are shown in Table 13; it can be concluded that antibody cross-reactivity with cross-linked fibrin is greater than with non-cross-linked fibrin. The simplest explanation is that the covalent crosslinking that is present in the crosslinked polymer structure serves as a closure or freezing of the conformation recognized by the antibody. Non-crosslinked types of conformations, although they arise, are not stabilized by the covalent bond of the polymer.

The antibody has also been shown to cross-react with both cross-linked and non-cross-linked precipitates in vitro. Crosslinked fibrin was prepared by incubating a fibrin solution (in Tris (50 mM, pH 7.4) with a salt solution containing CaCl ₂ (2 mM) and cysteine (10 mM)) with thrombin (10 NIH units / ml) for 3 hours at 37 ° C. C. Uncrosslinked fibrin was formed by incubating a solution of fibrinogen (3 mg / ml) in phosphate buffer (pH 6.1) containing EDTA with a final concentration of 0.0125 M with thrombin solution (10 NIH units / ml) for 3 hours at 37 ° C. The precipitates formed were washed and incubated at 37 ° C with 400 μΐ of a 1% BSA solution containing 175 µl of labeled MH1 antibody. Small aliquots of the solution were taken at various times and the amount of antibody consumed was determined by the gamma sensor.

Table 14 shows antibody consumption in both clots, uncrosslinked and crosslinked.

Table 13

Cross-reactivity of MH1 with cross-linked and non-cross-linked fibrin

Antibody concentration ng / ml crosslinked fibrin (absorbance Uncrosslinked fibrin at 405 nm) 100000 0,764 0,409 50 000 0,460 0,319 25 000 0,305 0,154 12 500 0,174 0,074 6 250 0,069 0,000

Table 14

Cross-reactivity of MH1 with crosslinked and uncrosslinked fibrin clot

clot % of consumption of the labeled antibody fibrin Mon 8 hours crosslinked 79% non-crosslinked 76%

Storage of hybridomas

MH1 and 45J were deposited with the American Type Culture Collection (ATCC) on June 9, 1988 and assigned HB 9739 and HB 9740 accession numbers. ATCC is at 12 301 Parklawn Drive, Rockville, MD 20852, USA. The MH1 antibody is an IgG-1 antibody with a kappa light chain and has been found to give a cross-reaction not only with human fibrin but also with rabbit fibrin. ¹

The present invention is not limited to the deposited hybridomas, which merely exemplify hybridomas that produce a fibrin-specific monoclonal antibody and a fibrinogen-specific monoclonal antibody, as mentioned above. Any cell line that is functionally equivalent is included within the scope of this invention. Functionally, it is understood that the antibody is capable of competing with the MH1 or 45J antibody for binding to the fibrin epitope to which the MH1 antibody binds, or to the fibrinogen epitope to which the 45J antibody binds. In addition, the term includes fibrin-specific non-clone antibodies and fibrinogen-specific monoclonal antibodies as defined herein that bind to a different epitope from that to which the MH1 antibody binds or to which the 45J antibody binds.

In vivo diagnosis and therapy using monoclonal antibodies specific for fibrin

Blood clot / vascular disease localization

The fibrin-specific monoclonal antibodies of the invention are capable of targeting fibrin clots or aggregated fibrin in vivo. Therefore, they can be used in humans to locate possible tissues or vascular disorders and to monitor vascular diseases. For this purpose, a fibrin-specific raonoclonal antibody is particularly preferred since said monoclonal antibody does not bind to fibrinogen, fibrin degradation products or fibrinogen degradation products. This lowers the background level of the assay, allowing much more accurate localization of the fibrin clot or aggregated fibrin.

Purified monoclonal antibodies are preferably used. Purification can preferably be accomplished by HPLC methods. Purification of monoclonal antibodies for administration in human medicine may be accomplished by precipitation with ammonium sulfate or sodium sulfate, followed by dialysis against brine and filter sterilization. Optionally, an immunoaffinity chromatography technique can be used to purify monoclonal antibodies.

After purification, the monoclonal antibodies may be labeled with radioactive compounds such as ¹²⁵ L, ¹³¹ L, 99 Tc, ¹¹¹ In, and administered intravenously to the patient.

The antibody may also be labeled with a magnetic probe. NMR can then be used to detect the precipitate. Upon localization of antibodies in a blood clot or aggregated fibrin, these agents can be detected by emission tomography and radio-nuclear scanning techniques to detect the site, for example, of a thrombus or a closed tumor fibrin envelope.

After purification, the monoclonal antibody is suspended in a suitable carrier, for example in saline, with or without human albumin. It is administered to a patient at a suitable dose. The monoclonal antibodies are preferably administered intravenously, for example by continuous infusion into a vein over several hours.

Treatment of vascular diseases with monoclonal antibody conjugates

The monoclonal antibodies of the invention can be used in conjunction with many pharmaceutical agents, such as cytotoxic agents and thrombolytic agents, for example t-PA, urokinase streptokinase, and other proteases that are capable of cleaving fibrin. This use is particularly preferred as fibrin-specific monoclonal antibodies of the invention allow effective use of said agents. None of the agents are then consumed for binding to fibrinogen, fibrin breakdown products, or fibrinogen breakdown products. For example, Bale, et al., 1980, Cancer Research, 40: 2965-2970; Ghose and Blair, 1978, J. Natl. Cancer Inst., 61 (3): 657-676; Gregoriadis, 1977, Nature, 265: 407-41, Gregoriadis, 1980, Pharmac. Ther., 10: 103-108; and Trouet et al., 1980, Recent Results Cancer Res., 75: 229-235.

Methods for binding agents to monoclonal antibody molecules include forming a non-covalent or covalent bond. Since the non-covalent bonds are readily disrupted before the antibody complex reaches the target site, covalent bonds are preferred. For example, a carbodiimide bond may be formed between the carboxy groups of the pharmaceutical agent and the amino groups of the antibody molecule. Two functional agents such as dialdehydes or imidoesters can be used to link the amino group of the drug to the amino groups of the antibody molecule. Schiff base reactions may be used to couple the drug to the antibody molecule. This method requires the use of periodate to oxidize a drug or cytostatic agent that contains a glycol or hydroxyl group. An aldehyde is formed which then reacts with the antibody molecule. Attachment occurs through the formation of a Schiff base with the amino groups of the antibody molecule. Drugs with reactive sulfhydryl groups can additionally coupled to antibody molecules.

Detection and determination of soluble fibrin in vitro

As mentioned above, hemostatic mechanisms require a complex sequence of reactions by which fibrinogen is completely converted to fibrin by thrombin. The end result of these reactions is the formation of a thrombus (blood clot). The sequence of reactions can simply be illustrated by a three-step process:

Step 1 - Proteolysis: Fibrinogen (+ thrombin) -> fibrin monomers + fibrinopeptides A and B

Step 2 - Polymerization: Fibrin monomers <===> soluble fibrin polymers

Step 3 - Precipitation: Soluble fibrin polymer -> fibrin clot.

Fibrinogen consists of three pairs of non-identical polypeptide chains: Αα, Ββ and gamma. See L. Stryer, Biochemistry, 3rd Edition, p. 249, V.H. Freeman and Company, New York (1988). In the initial step in which fibrinogen is transformed into fibrin, designated as step 1 above, fibrinogen is cleaved by thrombin, whereby the amino-terminated ends of the two fibrinogen A and retazco give rise to fibrinopeptide A. The resulting monomer is DesAA fibrin monomer. As also shown in step 1, at the same time, but much slower thrombin cleaves fibrinopeptide B from the amino-terminated ends of the two fibrinogen β-chains as well. As a result of fibrinopeptide release, new terminal amino groups are exposed and exposed to β α and fibrin chains. In step 1, the resulting molecules are fibrinopeptide A, fibrinopeptide B, and fibrin monomers DesAA and DesAABB, indicated in the above scheme as fibrin monomers. See V. Nieuweizen, Blood Coaqulation and Fibrinolysis, 4:93-96 (1993). As shown in step 2, fibrin monomers then form non-covalent (non-crosslinked) and covalent (crosslinked) bonds to form soluble fibrin polymers. The soluble fibrin polymers then form a fibrin clot, as shown in step 3.

Soluble fibrin is defined as any kind of molecules, arising from fibrinogen or fibrin that can lead to the formation of fibrin polymers, or from a fibrin (ogen) in a derived species that has a molecular weight greater than that of natural fibrinogen while maintaining in the form of a solution in the blood. The non-crosslinked and crosslinked DesAABB fibrin polymers that were formed in the above-mentioned second step are two species of several types of soluble fibrin. In addition, the term soluble fibrin includes various other species, such as DesAA fibrin polymers, complexes formed by the interaction between fibrin monomers (either DesAA or DesAABB fibrin monomers) and fibrinogen X, Y, D and R degradation products (see Monoclonal Antibody, fibrin specificity) and also, for example, DesAA and DesAABB monomers complexed with fibrinogen, see Nieuwenhuizen, pages 93-94.

Soluble fibrin polymers are intermediate precursors of insoluble fibrin, i.e. a blood clot. The associated elevation in plasma levels of soluble fibrin polymers is considered to be an indication of endangerment or involvement of individuals with thrombosis (the formation of intracranial blood clots). Detection and determination of the amount of these polymers in the blood, particularly soluble DesAABB fibrin polymers, will therefore be useful as an indicator of early blood clot formation. See Bang et al., U.S. Patent No. 5,206,140.

Some soluble fibrin species have been previously detected or determined by a variety of methods, including, for example, the determination of fibrinopeptide A using an antibody to Aa and gamma epitopes exposed to the conversion of fibrinogen to fibrin, Nieuwehuizen, 94-96, and D-dimer assays, Marder et al. U.S. Patent No. 5,206,140. Other methods already used to determine the presence or amount of soluble fibrin include determining agglutination of fibrin-coated erythrocytes in the presence of soluble fibrin, gel chromatography, increasing plasminogen activation with plasminogen activator ΐ-PA, see Nieuwenhuizen. on page 94, gelling with ethanol or protamine sulphate, N-terminal analysis of plasma-purified fibrinogen residues, incorporation of labeled glycine-ethyl ester and agarose gel chromatography, see Bang and Chang on pages 111 to 118. However, neither of these methods allows specifically prove or specify the content of those two types of polymers soluble crosslinked and non-crosslinked soluble fibrin polymers.

In vitro determination of soluble crosslinked fibrin polymers and soluble uncrosslinked fibrin polymers.

The present invention is based on the finding that in vitro determination of the presence and determination of the amount of soluble crosslinked DesAABB fibrin polymers and soluble uncrosslinked DesAABB fibrin polymers can be performed. Said polymers comprise DesAABB fibrin polymers. (A) fibrinogen, (b) degradation

I fibrinogen products, (c) DesAA fibrin monomers, (d) DesAA fibrin polymers, (e) DesAABB fibrin monomers, (f) cross-linked fibrinogen (Factor XIIIa-treated fibrinogen) (g) DesAA fibrin monomer-fibrin-58 gene complex and (h) fibrin degradation products (types (a) to (h)). Fibrinogen decomposition products and fibrin decomposition products are those produced by the action of plasmin on fibrinogen or fibrin, as described in Determining cross-reactivity with fibrin (ogen) degradation products.

It will be understood that the above assay is particularly useful in the clinical diagnosis of conditions characterized by thrombosis.

The assay may be performed using a suitable sample of body fluids, but preferably is performed using a mammalian blood sample. Suitable mammals include, for example, rabbits, monkeys and humans, of which humans are most preferred.

Known or future developed means for the determination of soluble cross-linked DesAABB fibrin polymers and soluble non-cross-linked DesAABB fibrin polymers, compositions which do not capture species (a) to (h), may be used in the above assays. Upon detection, the sample is determined for the amount of soluble non-crosslinked DesAABB fibrin polymers and soluble DesAABB non-crosslinked polymers either by comparison with the control or using standard solutions containing known amounts of soluble cross-linked DesAABB fibrin polymers and soluble non-crosslinked DesAABB fibrin polymers. Standards do not include species (a) to (h).

It should be noted that the means of determination is any means by which the presence of the proven material in the sample can be determined. A preferred means of determination is an antibody to soluble crosslinked DesAABB fibrin polymer and soluble noncrosslinked DesAABB fibrin polymer. Said antibody does not cross-react with (a) fibrinogen (b) fibrinogen degradation products, (c) DesAA fibrin monomers, (d) DesAA fibrin polymers, (e) DesAABB fibrin monomers, (f) cross-linked

I by fibrinogen (Factor XHIa treated fibrinogen), (g) DesAA fibrin monomer fibrinogen complex and (h) fibrin degradation products. To determine the presence and to determine the amount of soluble non-crosslinked DesAABB fibrin polymer and soluble non-crosslinked DesAABB fibrin polymer in a sample, the antibody described in this paragraph can be used. Such antibodies include polyclonal and monoclonal antibodies, preferably monoclonal antibodies. The antibody can be derived from any species. Preferably, however, the antibody is of human, murine or rabbit origin. Such antibodies further include chimeric antibodies, single chain antibodies, and Fab fragments, wherein the antibodies are not restricted by the agents.

An example of said antibodies is the monoclonal antibody MH1, which is already described in the sections Determining cross-reactivity with fibrinogen to Determining cross-reactivity with uncrosslinked fibrin and non-crosslinked fibrin clots, and also in the section Hybridoma Deposition. The sections describe the characteristics of the method of binding said MH1 antibody and moreover, it does not bind to the fibrinogen-fibrin degradation products complex. This property of the MH1 antibody can enhance its efficacy as a means of determining the presence and determination of the amount of soluble crosslinked and soluble uncrosslinked DesAABB fibrin polymers.

It should be noted that two or more antibodies may be used in the same assay and assay. Antibodies of different specificity can then be used in combination to determine the presence and determination of the amount of soluble crosslinked and soluble uncrosslinked DesAABB fibrin polymers, for example in samples where neither antibody alone is able to complex with both kinds of polymers: soluble DesAABB crosslinked and soluble uncrosslinked DesAABB fibrin polymers, while a combination of two or more antibodies can form such complexes. Of course, none of the antibodies can cross-react with species (a) to (h).

It should be noted that it may be necessary to use species-specific antibodies when using antibodies.

To produce antibodies, various host animals can be immunized by injecting soluble cross-linked DesAABB fibrin polymers and soluble non-cross-linked DesAABB fibrin polymers as antigens. The host animals may be rabbits, mice, rats, and so on, but are not limited thereto. Said polymers can be selectively isolated using, for example, an MH1 antibody bound to a Sepharose-MH1 antibody affinity column. Polymers can be prepared as follows: first, soluble fibrin can be prepared in vitro by adding a low level of thrombin to the citrate plasma. After decreasing thrombin activity by adding a strong thrombin inhibitor such as hirudin, plasma samples are transferred to a Sepharose-MH1 column. This Sepharose-MH1 column can be prepared by binding MH1 antibody to cyanogen bromide activated Sepharose 4B (Pharmacia), as is conventional in the art. See Pharmacia Products, insert with methods for preparing an Sepharose 4B antibody column using pre-activated Sepharose 4B. Initially, soluble fibrin is transferred to a Sepharose-MH1 column in the presence of phosphate buffered saline (PBS). After binding to a Sepharose-MH1 column, soluble fibrin bound to the Sepharose-MH1 column can be eluted using sodium thiocyanate solution in the presence of PBS. After dialysis against PBS, the isolated soluble fibrin polymer can be stored at -70 ° C.

Various adjuvants may be used in conjunction with the isolated polymers to enhance the immune response, depending on the host species. The following adjuvants may be used, but are not limited thereto, including Freund's adjuvants (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluron polyols, polyanions, peptides, oil emulsions, ready suction hemocyanine , dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.

Monoclonal antibodies specific for soluble, cross-linked and soluble, non-cross-linked DesAABB fibrin polymers that do not cross-react with species (a) to (g) can be prepared using any technique that allows the production of antibody molecules in culture with a continuous cell line. These techniques include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (Nature, 1975, 256: 495-497), a much later hybridoma technique with human B cells (Kosbor et al., 1983, Immunology Toady 4:72) and the EBV-hybridoma technique (Cole et.al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pages 77-96). According to another embodiment of the invention, monoclonal antibodies can be prepared in sterile animals using current technology, see Materials and Procedures to Use of the Antigen-Free Animal System for the preparation of a fibrin-specific monoclonal antibody. An example of such an antibody preparation is the MH1 preparation described above in Preparation of Monoclonal Antibodies Using Antigen-Free Mice to Hybridoma Deposition.

According to the present invention, human antibodies can be used or can be obtained using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci., 80: 2026-2030), or by transforming human B cells

Monoclonal

Liss, Inc., pages 77-96). Indeed, techniques developed to prepare chimeric antibodies can be used according to the invention (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81, 6851-6855; Neuberger et al., 1984, Nature, 312: 604 Takeda et al., 1985, Nature, 314: 452-454) by cleaving genes from an antibody molecule from a mouse of appropriate antigen specificity together with genes from a human antibody molecule with a suitable biological activity; such antibodies are within the scope of the invention.

According to the present invention, the technique described for the preparation of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to prepare specific single chain antibodies.

s

Another embodiment of the present invention utilizes the technique described for the construction of Fab expression libraries (Huse et al., 1989, Science, 246: 1275-1281). This will allow the rapid and easy identification of monoclonal Fab fragments requiring EBV virus in vitro Antibodies and Cancer (Cole et al., Therapy, Alan R.)

by their specificity for soluble crosslinked and soluble uncrosslinked DesAABB fibrin polymers.

Antibody fragments that contain specific binding sites to soluble cross-linked and soluble non-cross-linked DesAABB fibrin polymers can be prepared by known techniques. For example, fragments include, but are not limited to: F (ab) ₂ fragments, which can be produced by pepsin digestion of an antibody molecule, and Fab fragments, which can be produced by reducing the disulfide bridges of F (ab) ₂ fragments.

The antibodies of the invention can be used in any of the immunoanalytical systems known in the art, including, but not limited to, radioimmunoassay, enzyme immunoadsorbent analysis, sandwich analysis, precipitate reactions, gel diffusion immunodiffusion analysis, agglutination analysis, fluorescence immunoassay, ) immunoassay and immunoelectrophoresis analysis, as examples of some of the possible methods. U.S. Patent No. 4,629,783 and the patents cited therein also describe suitable analytical methods.

The antibodies can be used as baseline reagents in a variety of different immunoassays to determine the presence of soluble cross-linked and soluble non-cross-linked DesAABB fibrin polymers in a blood sample or other body fluids. In general, antibodies can be used in any type of immunoassay, both qualitative and quantitative. This includes double sandwich analysis as well as single-site sandwich immunoassay of non-competitive type as well as traditional competitive binding assays.

Sandwich analysis, or otherwise, a paired antibody assay is particularly preferred due to the ease of detection and the quantitative nature of the assay. There are a number of different modifications to this test. All these modifications are included in the present invention.

For example, in a typical procedure, a sandwich assay, the unlabeled antibody is immobilized on a solid substrate, for example, in a well of a culture plate, and the test sample is contacted with a binding molecule. After a suitable time in63 with control samples the amount of antigen. A variety of cubes of sufficient length to allow the formation of a binary antibody-antigen complex is added with a second antibody, which is labeled with a reporter molecule capable of generating a detectable signal. Incubation is continued, allowing sufficient time to bind to the antigen at a different site of the molecule and to form a ternary antibody-antigen labeled antibody complex. The unreacted fraction is removed by washing, the presence of the antigen is determined by monitoring a signal that can be quantified by comparison (standards) containing known sandwich assay adjustments involving a simultaneous assay in which the sample and antibody are simultaneously added to the binding antibody or reverse (reverse) sandwich assay. in which the labeled antibody and test sample are combined, incubated and added to the surface of the unlabeled bound antibody. These techniques are well known to those skilled in the art, and it will be appreciated that small adjustments to procedures are readily possible. As used herein, the term sandwich test is intended to include all modifications of the basic dual technique.

A more detailed example of a typical sandwich test. The first antibody is either covalently or passively bound to a solid support. The solid surface is usually glass or polymer, the most commonly used polymers are cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid carriers should be in the form of tubes, beads, discs or microplates, or any other surface shape suitable for execution. immunoassay. Binding procedures are well known in the art. Preparation for the assay is continued by washing the bound solid-antibody complex. An aliquot of the test body fluid is then added to the solid complex and incubated for a time sufficient to allow any soluble non-crosslinked DesAABB fibrin polymer and soluble cross-linked DesAABB fibrin polymer to bind to the antibody specific for the above proteins. A second antibody is then added to the solid complex and incubated for a time sufficient to allow the second antibody to bind to the primary antibody-antigen complex. The second antibody binds to a reporting molecule whose visible signal is used to indicate binding of the second antibody to any antigen of the sample. The term reporting molecule in this detailed description means a molecule which, by its chemical nature, provides an analytically detectable signal that allows the detection of an antigen-bound antibody. Detection must be quantifiable, at least by comparison with known samples, to allow determination of the amount of antigen in the sample. Said amount can be calculated absolutely or can be determined by comparison with standards (or series of standards) containing a known level of antigen content.

The most commonly used intelligence molecules for this type of immunoassay are either enzymes or fluorophores. When using enzyme immunoassay, the enzyme is coupled to a second antibody, often using glutaraldehyde or periodate. It is well known to those skilled in the art that many kinds of different conjugation techniques are known to attach the enzyme to the antibody. Commonly used enzymes include, among others, horseradish peroxidase, glucose oxidase, β-galactosidase, and alkaline phosphatase. Substrates used with specific enzymes are generally selected to provide a detectable color change upon hydrolysis with the corresponding enzymes. For example, p-nitrophenyl phosphate is suitable for use with an alkaline phosphatase conjugate; For the peroxidase conjugate, 1,2-phenylenediamine or toluidine is usually used. Fluorogenic substrates that provide a fluorescent product instead of a chromogenic substrate, as described above, may also be utilized. In all cases, an enzyme-labeled antibody is added to bind to the complex. The excess reagent is removed by washing. A solution containing the appropriate substrate is then added to the triple antibody-antigen-labeled antibody complex. The substrate reacts with the enzyme bound to the second antibody to produce a visually detectable qualitative signal which can be further quantified, usually spectrophotometrically, to give an indication of the amount of antigen present in the serum sample.

Optionally, fluorescent compounds such as fluorescein or rhodamine may chemically bind to the antibodies (without altering their ability to bind). Activated by light illumination of a certain wavelength, the fluorochrome labeled antibody absorbs light energy, induces a energized state of the molecule, followed by emission of light with a characteristic wavelength that is greater than the original wavelength. The emission appears as a characteristic color, visually detectable by an optical microscope. Similar to enzyme immunoassay (EIA), a fluorescently labeled antibody may bind to the first antigen complex. After removal of the excess reagent by washing, the remaining three-fold complex is exposed to light of the appropriate wavelength and fluorescence is observed to indicate the presence of the antigen. Both methods, immunofluorescence analysis and EIA, are well known in the art and are particularly preferred. However, other reporting molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules, may also be used. It will be apparent to those skilled in the art how the process can be adapted to suit the specific application desired.

Optionally, a test sample, either mammalian blood or body fluid, containing soluble non-crosslinked fibrin DesAABB polymer and soluble cross-linked DesAABB fibrin polymer, can be used for a one-site immunoassay in which the sample is captured on a solid substrate, either covalently or non-covalently. The unlabeled antibody is contacted with a sample bound to a solid substrate. After a suitably long incubation period, such that the time is sufficient to allow the formation of an antibody-antigen binary complex, a second antibody, labeled with a news molecule capable of generating a detectable signal, is added. Incubation is continued for a suitable time to allow the formation of a triple antigen-antibody-labeled antibody complex. In a one-site immunoassay, the second antibody may be a general antibody (i.e., a zenogenic antibody to an immunoglobulin, particularly anti- (IgM and IgG) bound to a reporting molecule) capable of binding an antibody that is specific to soluble crosslinked fibrin polymer and soluble uncrosslinked fibrin polymer. .

Detection and determination of the amount of soluble, non-crosslinked DesAABB fibrin polymers and insoluble cross-linked desAABB fibrin polymers in vitro are particularly useful where detection and assay are performed using patient plasma to obtain an indication of an emerging or preexisting thrombotic event due to or occurring thrombosis. . See, for example, V. Nieuwenhuizen, p. 94, Bang and Chang, pages 109-122 and Marder et al., U.S. Patent No. 5,206,140. Such events include, for example, deep vein thrombosis (DVT), a condition that results from a blood clot in the deep veins of the legs; pulmonary embolism (PE) that occurs when a thrombus (blood clot) detaches from the deep veins and embolizes into the pulmonary vascular bundle; diffuse intracranial coagulation that results from the continuous activation of a blood coagulation cascade (for example in bacterial infections); myocardial infarction (MI), which results from a thrombus occluded on the arteries that supply blood to the heart muscle; defeat and intracardiac thrombi formed as a result of articular fibrillation. By using the assay to detect and determine soluble non-cross-linked DesAABB fibrin polymers and soluble cross-linked DesAABB fibrin polymers in combination with observing other patient symptoms, a type of thrombotic event can be diagnosed. The patient's symptoms are those commonly used in the clinical diagnosis of occurring or existing thrombotic events. Said detection and determination of soluble non-cross-linked DesAABB fibrin polymers and soluble cross-linked DesAABB fibrin polymers are particularly useful as a means for differential diagnosis of chest pain patients due to onset MI from patients having chest pain due to other conditions.

Examples of materials and methods

protein

Albumin and bovine serum thrombin were obtained from ICN (Costa Mesa, CA, USA). Hirudin, horseradish peroxidase and dihydrochloride o-phenylenediamine spstrate tablets were supplied by Sigma Chemical Co. (St. Louis, MO, USA). Human fibrinogen (purity L) was purchased from Helena Laboratories (Beaumont, TX, USA). Fibrinogen was further purified by ammonium sulfate precipitation as reported by Holm et al., Thromb. Res., 37,165-176 (1985). The anti-fibrin monoclonal antibody MH1 used in the assay system was prepared as described in the Monoclonal antibody, fibrin specificities section above. The 45J antifibrinogen monoclonal antibody was prepared as described in the Monoclonal Antibody, fibrin specificity section herein.

DesAABB fibrin monomer was prepared by dissolving uncrosslinked fibrin polymer in a solution of 50 mM sodium acetate buffer (pH 5.3) containing sodium bromide.

Soluble cross-linked DesAABB polymers were prepared by incubating fibrinogen or citrate plasma with low thrombin (0.025 units / ml) for 5-7 minutes. The reaction mixture also contained Factor XIIa. The reaction was stopped by adding hirudin to the reaction mixture, resulting in a concentration of 10 ATU / ml).

Uncrosslinked soluble DesAABB fibrin polymers were prepared by incubating citrate plasma or fibrinogen with a thrombin solution containing EDTA (12 mM, final concentration) for 7 to 8 minutes at 37 ° C. The reaction was stopped by adding excess hirudin to the reaction mixture (final concentration 10 ATU / ml).

Conjugates MH1 and 45J with horseradish peroxidase

(Taken from: Vilson and Nakane, Immunofluorescence and

Related Staining Techniques, Knapp, et al. (eds.), pp. 215-224, Elsevier / North Holland Biomedical Press, Amsterdam (1978)).

Horseradish peroxidase (HRP) (RZ> 3) (10 mg) was dissolved in 1 ml of distilled water. Freshly prepared 0.1 M sodium periodate solution (0.4 mL) was added and the mixture was vigorously stirred for 20 minutes. Using a Centricon 10 set microconcentrator, the solution was then transferred to sodium acetate buffer (1 mM, pH

4.4) at 4 ° C.

The pH of the solution was increased to 9.5 by adding 40 μΐ of 0.2 M sodium carbonate and bicarbonate buffer (pH 9.5). Immediately after increasing the pH, the antibody MH1 (20 mg) was added in 2.0 ml of 0.01 M sodium carbonate-bicarbonate buffer (pH

9.5). The solution was stirred gently at room temperature for 2 hours. 0.2 ml of freshly prepared sodium borohydride solution (4 mg / ml) was then added. The mixture was allowed to stand at 4 ° C for two hours.

Finally, the HRP antibody conjugate was dialyzed against 5x4 liters of PBS at 2-8 ° C.

The conjugate was stored in a brown container at 2-8 ° C.

Blood samples

Blood samples were collected with the consent of healthy volunteers and patients from intensive care units with symptoms of MI, such as severe chest pain. Blood samples were placed in sodium citrate using Vacutainers (Becton Dickinson). The plasma was separated by centrifugation at 2400 rpm for 15 minutes. Plasma was either used immediately or frozen at -70 ° C.

Determination of soluble fibrin by ELISA

Soluble fibrin was determined in fresh or frozen plasma samples using the sandwich type enzyme immunoadsorbent system. The bound antibody in the present invention was the antifibrin antibody MH1. The detection (or labeled) antibody was an HRP conjugate of the same antibody. Alternatively, the 45J anti-fibrinogen antibody conjugate was used as the detection antibody.

Polyvinyl chloride 96-well microtiter plates were coated with monoclonal antibody MH1 by incubating 100 μ puf of Mab solution (at 50μ§ / πι1) at 4 ° C in a buffer coating (sodium borate, pH 8.5) for 12-16 hours. Wash the wells to remove unbound antibody from the plates. Wash three times with PBS-Tween. The MH1 coated wells were overlaid with BSA by incubation with 200 μΐ of a solution of BSA (1%) in PBS (PBS-BSA) for 1 hour at 37 ° C. Unbound BSA was removed after inverting the plates by gently drying them with a paper towel. Citrate samples (50 μΐ) containing soluble fibrin were incubated in wells occupied with MH1-BSA. Incubation was performed at 37 ° C for 30 minutes. Unbound substances were removed by rotating the culture plates and gently drying with a paper towel. The wells were then washed three times with PBS-Tween. The bound soluble fibrin is first incubated with 100 μΐ of PBS-BSA solution of MH1-HRP (or HRP conjugate with antifibrin (ogen) antibody 45J) for 30 minutes at 37 ° C. Unbound conjugate was removed by inverting the plates and washing the wells with PBS-Tween three times. Bound conjugate was detected by the addition of 100 μΐ solution of O-phenylenediamine dihydrochloride at room temperature for 10 minutes. The substrate solution was prepared by dissolving the O-phenylenediamine dihydrochloride tablet in a sodium citrate solution containing H ₂ O ₂ .

The color development was stopped after 10 minutes by the addition of 25 μ 25 of 1 M sulfuric acid solution. At 490 nm, the absorbance of the solutions in each well was determined using a Thermomax microtiter plate reader (Molecular Device, Menlo Park, CA, USA).

detected the conjugate conglomerate

Preparation of affinity chromatography columns

Sepharose-MH1 columns were prepared as follows:

1 g of freeze-dried and cyanogen bromide activated Sepharose 4 B (Pharmacia) was weighed and suspended in 1 mM HCl solution. The precipitated gel was washed for 15 minutes with 1 mM HCl solution on a sintered glass filter.

In the binding buffer (0.1 M NaHCO 3 solution, pH 8.3, containing 0.5 M NaCl), the MH1 antibody (20 mg) was dissolved and carefully mixed with the precipitated gel. Stirring was performed in a sealed vessel for 2 hours at room temperature or at 4 ° C overnight.

After mixing, the gel was washed with binding buffer to remove excess antibody. Unoccupied active sites were occupied with 0.1 M Tris HCl, pH 8.0 (or 1 M ethanolamine solution, pH 9.0) for 2 hours at room temperature. Then, the bound antibody gel was washed three times with 0.1 M acetate buffer (pH 4.0, containing 0.5

M NaCl), followed by Tris buffer (0.1 M solution, pH 8, containing 0.5 M NaCl). The bound antibody was stored at 4 ° C in sodium azide solution (1% solution).

Sepharose-DesAABB fibrin monomer columns were prepared as follows: DesAABB fibrin monomers bound to Sepharose 4B, activated with cyanogen bromide in the presence of 1 M NaBr in 0.1 M borate buffer (pH 8.2), in the same manner as used to prepare a Sepharose column -MHl.

Nature of soluble fibrin, investigated by in vitro analysis of soluble fibrin polymers using MHI to detect soluble fibrin

The fibrin specificity assay has already stated that the MH1 antibody does not recognize fibrinogen, which is a precursor of fibrin polymer in plasma. It has also been mentioned in the said section that the antibody used recognizes both fibrin polymers, both crosslinked and uncrosslinked fibrin polymer. In addition, it has been shown in this section that the breakdown of fibrin by plasmin (or any process that leads to the breakdown of the fibrin polymer structure) causes a decrease in the immune recognition of fibrin by said antibody. Accordingly, the MH1 antibody does not recognize any of the known plasmin-formed, breakdown products of cross-linked fibrin.

Evidence that the MH1 antibody only recognizes the polymeric structures of DesAABB fibrin and does not recognize monomeric desAABB fibrin units was obtained by affinity chromatography. Briefly, 0.5 mg of MH1 antibody was loaded onto a 5 milliliter Sepharose-desAABB fibrin monomer column. The column was washed with buffer (0.1 M tris buffered saline (TBS), pH 8.5). No significant binding of the antibody to the column was found. Figure 1A shows how the antibody elutes when passing through the column. When the column was eluted with a 6 M guanidine hydrochloride solution, it was released and a small amount of bound protein was obtained. Protein was detected by monitoring all fractions at 280 nm. The antibody was determined by assaying the immunoreactivity of the protein by an ELISA method with a solid adsorbent using fibrin coated microtiter wells. See section Proof of Presence and Antibody Detection for a description of the ELISA method used. The column bound protein is due to the presence of a small amount of contaminating soluble DesAABB fibrin polymer retained on the DesAABB fibrin monomer column. This impurity originates from the starting material used in the preparation of the DesAABB fibrin monomer column. To demonstrate that the Sepharose-DesAABB fibrin monomer column is capable of binding antibody, in a control experiment, a fibrinogen specific monoclonal antibody (Mab) was passed through the column. It can be seen from Figure 1B that elution of 45J requires column-bound control Mab, 45J antifibrinogen antibody, and guanidine hydrochloride (6 M) treatment.

The above evidence that MH1 recognizes both cross-linked and non-cross-linked soluble DesAABB fibrin polymers was obtained by an experiment in which soluble DesAABB fibrin polymers were formed by the action of thrombin on citrated plasma in the presence and absence of EDTA (EDTA prevents Factor XIIla fibrin from crosslinking DesAABB). Said cross-linked and non-cross-linked DesAABB fibrin polymers were prepared as described in the Proteins section and their respective immunoreactivities were determined using ELISA analysis as described in the Determination of soluble fibrin by ELISA. As shown in Figure 2, the assay system used found no significant difference in immunoreactivity between crosslinked (B) and uncrosslinked (C) fibrils by the new polymers.

Evidence that the MH1 antibody does not recognize soluble desAA fibrin polymer was obtained by an experiment in which the DesAA fibrin polymer was prepared from fibrinogen or plasma by treatment with the snake venom-derived batroxobin enzyme from Bothrops atrox, which selectively cleaves fibrinopeptide A from the fibrinogen molecule. Batroxobin does not cleave fibrinopeptide B (FPB), unlike thrombin, which cleaves both FPA and FPB. The removal of FPA results in the polymerization of the desAA fibrin monomer unit and the formation of the DesAA fibrin clot. Figure 3 shows clot formation by measuring the absorbance of the reaction mixture at 340 nm when a fibrinogen sample was incubated with a batroxobin solution. Incubation was carried out for 15 minutes at room temperature. Absorbance gradually increased over time as the length and concentration of soluble DesAA fibrin polymers increased until a precipitate finally formed. In an MH1 assay for recognition of desAA fibrin, the desAA fibrin polymer was prepared in vitro by incubating a fibrinogen sample with batroxobin (final concentration of 0.5 units / ml) at 37 ° C. An aliquot was removed from the batroxobin-treated sample after seven minutes and assayed by the ELISA method described in Determining Soluble Fibrin by ELISA. No reactivity was observed in this sample (sample C), as shown in Figure 4. From this experiment, it can further be concluded that the assay system does not recognize the fibrinogen complex with DesAA fibrin, which is another source of soluble fibrin. The reaction mixture obtained in this experiment would clearly contain such soluble fibrin elements, since batroxobin produces desAA fibrin monomers, which can interact unhindered with other desAA monomers, or with digestion unaffected by fibrinogen molecules.

Since no reaction was shown after the addition of batroxobin by the assay system, it can be concluded that these fibrinogen-desAA fibrin units are not detected in the assay. Sample B, which is a positive control, consisted of DesAABB fibrin polymers produced by processing a plasma sample with thrombin and hirudin. Untreated fib73 rhinogen - sample A - was selected as a negative control.

The soluble fibrin complex may also be formed by the effect of Factor XIIla by crosslinking the original fibrinogen molecules to fibrinogen dimer molecules (Kanaide et al., J. Lab. Clin.Med., 86: 574-579 (1975)). In this experiment, fibrinogen in the wells of microtiter plates was first treated with thrombin activated with Factor XIIla for 1.5 hours at 37 ° C. After discontinuing the remaining thrombin activity, the MH1 antibody was incubated in wells treated with FXIIIa and Anti-mouse alkaline phosphatase conjugate was used to detect bound MH1 The bound mouse antibody was detected by addition of alkaline phosphatase substrate The bound MH1 level in the cells was determined by reading the absorbance at 490 nm The result of the sample, designated Fg + FXIIIa in Figure 5, there was no MH1 binding in the wells, indicating that As a positive control, fibrinogen wells were treated with thrombin to give desAABB fibrin polymers, which were tested for immunoreactivity with the MH1 antibody - in Figure 5, this is designated as Fg + Thrombin.

Determination of the course of formation of soluble DESAABB fibrin polymer

Soluble DesAABB fibrin polymers were prepared in vitro by adding a low level of thrombin (0.025 NIH units / ml) to the citrate plasma sample. The sample was incubated at 37 ° C. Aliquots were taken from the sample at 1 minute intervals. Thrombin activity was always discontinued in the collected fraction by the addition of an effective thrombin inhibitor, hirudin (final concentration 2 ATU / ml). The amount of soluble DesAABB fibrin polymer in each aliquot was determined by ELISA as described in Determining soluble fibrin by ELISA.

After the addition of thrombin to plasma after an initial induction period of 4 to 5 minutes, the amount of soluble fibrin polymer increases as shown in Figure 6 by the absorbance at 490 nm over time. The level of soluble fibrin polymers peaked at approximately 7.5 minutes after the addition of thrombin to plasma. The sudden decrease in soluble fibrin levels observed after 9 to 10 minutes coincides with gelation and formation of insoluble fibrin polymers (clot formation).

Purification of soluble fibrin polymers from plasma by affinity chromatography on sepharose - MH1

The plasma sample was incubated with thrombin (0.025 units / ml) for 7 minutes at 37 ° C. Thrombin activity was quenched by addition of excess hirudin. The reaction mixture was passed through a column. The ELISA method described in the Determination of Soluble Fibrin by ELISA method evaluated the immunoreactivity with MH1 from the starting material, the passed liquid (unbound material), the bound protein (purified protein), which eluted using NaSCN and dialyzed against PBS. Immunoreactivities of thrombin-treated plasma upon discontinuation of thrombin activity by hirudin (starting material - in the figure: Starting material), bound protein after elution from the column and dialysis (eluate - in the figure: Elution) and unbound material (unbound - in figure: Nonbound) The starting material and bound protein after dialysis were immunoreactive with MH1, while unbound material did not have immunoreactivity.

In vitro determination and determination of soluble crosslinked and soluble uncrosslinked fibrin polymers for MI diagnosis

36 different plasma samples were tested. It was determined

The level of soluble cross-linked and soluble non-cross-linked DesAABB fibrin polymer using the ELISA described in the Determination of Soluble Fibrin by Method

ELISA with MH1 as anchor antibody. After collection, plasma samples were mixed with anticoagulant and frozen. Thawed samples were tested for soluble crosslinked and soluble noncrosslinked DesAABB fibrin polymer. Ten of the 36 samples were obtained from laboratory staff who were in excellent health and who did not experience chest pain even after fatigue. All other samples were taken from patients at the local public hospital. All patients in the emergency rooms had clinical symptoms (chest pain) indicating a myocardial infarction (MI); 15 patients were confirmed to have MI; all other 11 patients were confirmed not to be at risk for MI. The latter group of patients was diagnosed with various clinical conditions including angina, anxiety or pulmonary edema. Blood samples were taken from patients immediately upon admission to the room for sudden cases. In most cases, the duration of symptoms was reported. In this study, all patients reported that chest pain occurred 1 to 3 hours before admission to the hospital. As shown in Figure 8, in all cases, patients with confirmed MI had higher levels of soluble fibrin DesAABB polymers than control healthy patients (3 to 30 fold difference). In patients who experienced chest pain but without MI, the level of soluble DesAABB fibrin polymers was not significantly different from healthy patients. (The levels of soluble crosslinked and soluble uncrosslinked DesAABB fibrin polymers are shown as: Level of soluble fibrin in ELISA units (x 1000) [in the figure: Level of Soluble Fibrin Elisa Units (x 1000)].

It follows that the sensitivity of the test system was 100% (for n = 36) and that the specificity was also 100% (for n = 36), that is, there was neither one negative nor one positive false case. Therefore, it can be argued that for myocardial infarction this assay system has a high level of diagnostic accuracy.

Kit for in vitro detection of cross-linked soluble fibrin polymers and soluble non-cross-linked fibrin polymers

It is understood that the invention is not limited to the use of a monoclonal antibody in an assay. However, if such an antibody is used in connection with a kit described below, the kits comprise a set of standards, a first antibody (i.e., a binding antibody such as MH1) that can be immobilized on a solid surface and a second antibody labeled with a signal source, as described above. Such kits contain standards in the form of known amounts of soluble crosslinked DesAABB fibrin polymer and soluble noncrosslinked DesAABB fibrin polymer. Said standards can be prepared by isolating soluble crosslinked and soluble uncrosslinked DesAABB fibrin polymers using an MH1-Sepharose affinity column as described above. The kits may also contain specific buffers, release agents, and control samples. The kits may contain aids and chemicals for processing the samples to be evaluated.

All publications and patents cited above are incorporated herein by reference.

Claims (26)

An in vitro method for detecting the presence or determination of soluble cross-linked DesAABB fibrin polymers and soluble non-cross-linked DesAABB fibrin polymers in mammalian samples, comprising contacting said samples with an antibody or immunoreactive fragment thereof that specifically binds to soluble cross-linked DesAABB fibrin polymer and soluble non-cross-linked DesAABB fibrin polymer to form an immunocomplex thereof whose antibody or immunoreactive fragment thereof does not specifically bind to: (a) fibrinogen, (b) fibrinogen degradation products, (c) DesAA fibrin monomer, (d) a polymer, (e) a DesAABB fibrin monomer, (f) a cross-linked fibrinogen, (g) a DesAA fibrin monomer-fibrinogen complex, and (h) a fibrin degradation product, and detects the presence or amount of the immunocomplex. The method of claim 1, wherein the antibody specifically binds to an epitope recognized by a monoclonal antibody designated MH1 produced by the hybridoma ATCC HB 9739. The method of claim 2, wherein said antibody is a monoclonal antibody, designated MH1, produced by the hybridoma ATCC HB 9739. The method of claim 1, wherein the mammal sample is a human sample. The method of claim 4, wherein the human sample is a body fluid. and The method of claim 5, wherein the body fluid is blood. The method of claim 1, wherein said immunocomplex is detected or quantified using a secondary antibody or immunoreactive fragment thereof that specifically binds to said immunocomplex, wherein said secondary antibody or immunoreactive fragment thereof is labeled with a news molecule that provides a detectable signal, which is measured and which correlates with the presence or amount of said immunocomplex. The method of claim 7, wherein the secondary antibody is an antibody that binds to an epitope recognized by the monoclonal antibody, designated 45J, produced by the hybridoma ATCC HB 9740. The method of claim 8, wherein the secondary antibody is a 45 J monoclonal antibody produced by the hybridoma ATCC HB 9740. A method of evaluating a predisposition to a thrombotic event, supporting the diagnosis of a thrombotic event, or monitoring a thrombotic event in a mammal, comprising determining the amount of soluble cross-linked DesAABB fibrin polymers and soluble non-cross-linked DesAABB fibrin polymers in the sample comprising said mammal sample; Or an immunoreactive fragment thereof to form an immunocomplex of which the antibody, or immunoreactive fragment thereof, does not specifically bind to: (a) fibrinogen, (b) fibrinogen degradation products, (c) DesAA fibrin monomer, (d) DesAA fibrin polymer, (e) DesAABB fibrin monomer, (f) cross-linked fibrinogen, (g) a DesAA fibrin monomer-fibrinogen complex, and (h) a fibrin degradation product to form an immunocomplex therewith, comprising detecting a proportion of said immunocomplex and comparing said proportion with a control sample, it was possible to evaluate the predisposition to a thrombotic event, or the development of a thrombotic event, or to monitor the thrombotic event of a mammal. The method of claim 10, wherein said antibody specifically binds to an epitope recognized by a monoclonal antibody, designated MH1, produced by the hybridoma ATCC HB 9739. The method of claim 11, wherein said antibody is a monoclonal antibody, designated MH1, produced by the hybridoma ATCC HB 9739. The method of claim 10, wherein the thrombotic event is selected from the group consisting of myocardial infarction, deep vein thrombosis, pulmonary embolism, widespread intracranial coagulation, defeat, and intracardiac thrombus resulting from cardiac fibrillation. The method of claim 10, wherein the mammal sample is a human sample. 15. The method of claim 14, wherein the human sample is a body fluid. The method of claim 15, wherein the body fluid is blood. 17. The method of claim 10, wherein the amount of the immunocomplex is determined using a secondary antibody or immunoreactive fragment thereof that specifically binds to an immunocomplex in which the secondary antibody or immunoreactive fragment thereof is labeled with a reporter molecule that provides a detectable signal that is measured and which is correlated with the amount of said immunocomplex. I The method of claim 17, wherein the secondary antibody is an antibody that binds to an epitope recognized by the monoclonal antibody designated 45J produced by the hybridoma ATCC HB 9740. The method of claim 18, wherein the secondary antibody is a 45J monoclonal antibody produced by the ATCC 9740 hybridoma. A kit for in vitro detection of the presence or determination of soluble crosslinked DesAABB fibrin polymers and soluble noncrosslinked DesAABB fibrin polymers in a mammalian sample, the kit comprising: (a) measured amounts of standards comprising said soluble crosslinked DesAABB fibrin and soluble uncrosslinked DesAABB fibrin polymers; (b) means for forming an immunocomplex with said soluble cross-linked DesAABB fibrin polymers and said soluble uncrosslinked DesAABB fibrin polymers, said means comprising an antibody or immunoreactive fragment thereof, which antibody or immunoreactive fragment does not specifically bind to: (i) fibrinogen, (ii) (iii) DesAA fibrin monomer; (iv) DesAA fibrin polymer; (v) DesAABB fibrin monomer; (vi) crosslinked fibrinogen; (vii) DesAA fibrin monomer fibrinogen complex; and (viii) fibrin breakdown product; and (c) means for determining the amount of said immunocomplex. 21. The kit of claim 20, wherein the means for forming an immunocomplex with said soluble crosslinked DesAABB fibrin polymers and said soluble uncrosslinked DesAABB fibrin polymers is an antibody, or immunoreactive fragment thereof, that specifically binds to an epitope recognized by a monoclonal antibody, designated MH1, produced by the hybridoma ATCC HB 9739. / 22. The kit of claim 21, wherein the antibody is a monoclonal antibody, designated MH1, produced by the hybridoma ATCC HB 9739. The kit of claim 20, wherein the means for determining the proportion of said immunocomplex is a secondary antibody, or an immunoreactive fragment thereof, labeled with a reporting molecule that provides a detectable signal that is measured and correlates with the amount of said immunocomplex. The kit of claim 23, wherein the secondary antibody specifically binds to an epitope recognized by a monoclonal antibody, designated 45J, produced by the hybridoma ATCC HB 9740. The kit of claim 24, wherein the secondary antibody is a 45J monoclonal antibody produced by the hybridoma ATCC HB 9740. The kit of claim 20, further comprising a control sample useful for determining a predisposition to a thrombotic event, a thrombotic event, or during a thrombotic event of a mammal. 1/9 absorbance (280nm)