Immunoassay of soluble fibrin polymers

 

(57) Abstract:

The invention relates to immunology. The method of determining in vitro the presence or amount of soluble cross crosslinked fibrin polymers Des and transversely not crosslinked fibrin polymers Des in the sample obtained from the mammal, includes contacting the specified sample with the antibody or immunoreactive fragment. The latter is not linked specifically a) fibrinogen, (C) degradation products of fibrinogen, C) with fibrin monomers Des, d) with fibrin polymers Des, e) with fibrin monomers Des, f) cross-cross-linked fibrinogen, g) with complex fibrin monomer Des - fibrinogen and h) with the breakdown products of fibrin. Methods for assessing the predisposition to thrombotic state, confirmation of the presence of thrombotic state and monitoring thrombotic condition in a mammal include determining the amount of soluble cross crosslinked fibrin polymers Des and soluble cross is not crosslinked fibrin polymers Des in the sample obtained from the mammal. The proposed sets to determine in vitro the presence or amount of soluble sample to determine predisposition to thrombotic condition in a mammal, to confirm the diagnosis of the presence of thrombotic state and for determining the stage of thrombotic condition in a mammal. The invention allows to reliably determine the predisposition to thrombotic condition in a mammal. 8 C. and 26 C.p. f-crystals, 12 tab., 8 Il.

1. The technical field

The invention relates to a method for producing monoclonal antibodies. In this way are vaccinated sterile animals. In the claimed invention is also the use of these monoclonal antibodies and polyclonal antibodies derived from a vaccinated sterile animals for clinical diagnostics in vitro and in vivo and in therapy. The aim of the invention is also a fibrin-specific monoclonal antibody.

2. Background of the invention

It is recognized that Kohler and Milstein developed a technique, which successfully led to the first hybridoma producing monoclonal antibodies (G. Kohler and C. Mistein, 1975, Nature 256, 495-497; 1976, Eur. J. Immun. , 6, 511-519). Merging cells producing antibodies (B-lymphocytes of the spleen), with myeloma cells (malignant cells of primary tumors of the bone marrow), they got a line of hybrid cells resulting from edinstvena as lymphocytes, and lines myeloma cells. As lymphocytes, hybridoma secretes one type of immunoglobulin; moreover, like the myeloma cells, hybridoma capable of unlimited cell division. The combination of these two properties provides clear advantages over conventional antisera.

The antisera obtained from vaccinated animals, are changing the composition of a mixture of polyclonal antibodies, which are not identical to reproduce. Monoclonal antibodies are highly specific for the immunoglobulin single type. One type of immunoglobulin secreted by hybridomas is specific to one and only one antigenic determinant, or epitope, antigen - complex molecule with many antigenic determinants. So, if the antigen is a protein, the antigenic determinant may be one of many peptide sequences (generally 6-7 amino acids; M. Z. Atassi. 1980. Molec. Cell. Biochem. 32, 21-43) in the protein molecule. Thus, the monoclonal antibodies produced against one antigen may differ from each other depending on determinants that triggers their education; however, for any hybridoma in vitro and in vivo and allows to obtain antibodies with extremely high concentration.

Monoclonal antibodies can be used as a probe to detect their antigen. Thus monoclonal antibodies used for the diagnosis in vitro, for example in radioimmune assays and enzyme immunosorbent assays (ELISA) for the diagnosis and in vivo, for example for imaging in vivo using the containing tag monoclonal antibodies. Monoclonal antibody can also be used as a carrier for drug delivery to such antigen antibodies.

However, before a monoclonal antibody can be used for these purposes, it is necessary to monoclonal antibody could be in contact with interest antigen, i.e., the target antigen. This selection procedure can be very tedious, because you may want to take a lot, for example, presumably several thousand, monoclonal antibodies will be identified before hybridoma producing an antibody able to bind with the target antigen. Therefore, it is necessary to develop a method of obtaining monoclonal antibodies, which exceeds the probability that hybridoma produces the antibody for the target antigen is lokalnych antibodies to the antigen, including:

a) immunization of sterile animal specified antigen to allow producing antibody to the cells to reproduce antibodies to a specific antigen

b) allocating at least part of the above mentioned producing antibody cells from the specified sterile animal,

c) obtaining hybridoma merge one of these producing antibody cells with immortalize cage, with the indicated hybridoma capable of producing monoclonal antibody to a specific antigen

d) growing the specified hybridoma, and

e) isolation of monoclonal antibodies produced by the specified hybridomas.

In the present invention claimed methods of using monoclonal antibodies and polyclonal antibodies obtained from sterile animal. The present invention also claimed the fibrin-specific monoclonal antibody and methods of using such monoclonal antibodies.

Brief description of drawings

Fig. 1(A) - determination of immunoreactivity mh1c with fibrin monomer DesAABB using affinity chromatography.

Fig. 1(B) is a control experiment to confirm the binding capacity of the matrix anorectoplasty unstitched fibrin polymers DesAABB and crosslinked fibrin polymers DesAABB in in vitro assays for soluble fibrin polymers DesAABB using mh1c.

Fig. 3 - formation of DesAA fibrin during processing of plasma fibrinogen by batroxobin.

Fig. 4 - immunoreactivity of soluble fibrin polymers DesAA in in vitro test system for soluble fibrin polymers DesAABB using mh1c.

Fig. 5 - the immunoreactivity of the antibodies mh1c with fibrinogen treated with activated factor XIII (factor XIIIa).

Fig. 6 - determining formation of soluble fibrin polymer DesAABB in conditions in virto using analysis of soluble fibrin mh1c.

Fig. 7 - demonstration purification of soluble fibrin from plasma using column sepharose-mh1c.

Fig. 8 - determination of soluble fibrin polymers DesAABB in the blood of patients with chest pain and installed myocardial infarction.

4. Detailed description of the invention

4.1. Sterile animals

The present invention relates to the use of sterile animals to produce monoclonal antibodies. Sterile animals were first obtained at the end of the 19th century and since then intensively used.

Sterile animal is gnotobiotes, which is free from ties with all possible forms of life, vkliuchvane or view, obtained by aseptic caesarean section or sterile removal of eggs, which are grown and which is constantly monitoring using sterile methods in terms of the insulator, and in which the composition of any associated fauna and flora, if it exists, is completely determined by the current methodology. (It should be noted that all mice contain latent leukemogenic virus, but because the mouse that buspirone except this leukemogenic virus should be considered as a sterile animal for the purposes of the present invention).

The essence amicrobic system is to create a barrier against the entry of undesirable microbial invaders. In addition to the physical barrier of plastic, metal, rubber and glass, for which the animal is placed, the system requires operational barriers for air filtration, sterilization of food and water, manipulation with gloves, which form an integral part of the barrier system. In addition, the supply of food in the insulator must be carried out under sterile conditions.

We believe that the present invention can be any sterile animal. The most common sterile animals are titelnoj, especially the mouse Balb/c mice.

4.2. The breeding, care and care sterile animals

There are many publications devoted to the breeding, care and care of sterile animals. For example. B. S. Wostmann, Ed., "Gnotobiotes: Standards and Guidelines for the Breeding, Care and Management of Laboratory Animals, National Research Council, National Academy of Sciences, Washington, D. C. 1970; M. E. Coates et al., "The Germfree Animal in Research, Academic Press, London, 1968; J. R. Pleasants, "Gnotobiotics", in: Handbook of Laboratory Animal Science", Vol. 1 (E. C. Melby et al., Eds.). CRC Press, Boca Raton, Fla., 117 (1974), which are given here for reference.

The following are excerpts from the publication of B. S. Wostmann, Ed. (1970) "Gnotobiotes: Standards and Guidelines for the Breeding, Care and Management of Laboratory Animals, National Research Council, National Academy of Sciences, Washington. D. C., which describes the removal, care and care for sterile rats and mice. It should be noted that such methods of breeding, care and maintenance similar to other animals.

External laboratory Environment

Premises, equipment and methods of cultivation should be prepared and implemented in such a way as to provide maximum control over the surrounding conditions and optimum comfort for the animals. Cages, feeders and waterers should be designed and constructed so that the animals felt Maxi.

Desirable floor plan to conduct intensive research in sterile conditions should include:

1) work area for Assembly I sterilization insulators,

2) the area to sustain animals in isolators, and

3) laboratory area to monitor the status geobiological environment.

In the floor plan may also be included office and cooking area.

Laboratory conditions for maintenance gnotobiology insulators shall meet the standards established for the construction of a conventional laboratory nurseries. The design must protect against the penetration of insects, and the walls and floor should be waterproof. The lighting must be uniform with the same cycles of light - dark time throughout the year. Ventilation must act quickly to remove any odors arising from chemical sterilization, and climatic conditions should be controlled as follows.

Temperature. The most appropriate temperature in the room where the animals constituting 21-27oC, may be, if necessary, modified so that the temperature in the insulator sleep for a person level, average of 40-60%. However, if for ventilation insulator used room air, the recommended relative humidity is 40-50%.

Ventilation. Change of air should be sufficient to quickly remove any odors arising from chemical sterilization. The recommended rate of air exchange in the room is from ten to fifteen volumes per hour. Staff must have the masks with fresh air to protect against dangerous concentrations of vapors of chemical agents.

Sterile equipment (see E. Sacquiet 1968, "Equipment design and management: General technique of maintaining germ-free animals, p. 1-22, in: M. E. Coates (Ed.) "The germ-free animals in research", Academic Press, London; P. C. Trexler 1968, "Equipment design and management: Transport of germ-free animals and current developments in equipment design, p. 23-35, in: M. E. Coates (Ed.) "The germ-free animals in research", Academic Press, London).

Complete elimination of germs from the Environment requires the establishment of an absolute barrier. Successful surgery depends on the constant maintenance of the integrity of the specified barrier. There are two main types of insulators made of metal and plastic. Some metal structures are developed in such a way that they are kept inside the action of water n the N. Y. Acad. Sci. 78:37). Other designs place inside a large autoclave to conduct a primary sterilization (see B. E. Gustafsson, 1959. "Lightweight stainless steel systems for rearing germ-free animals, Ann N. Y. Acad. Sci. 78:17).

The most popular currently received flexible film isolators (see P. C. Trexler and L. I. Reynolds 1957. "Flexible film appararus for rearing and use of germ-free animals, Appl Environ. 5:406). They are usually made of flexible multilayer films of vinyl polymers and subjected to chemical sterilization; however, they can easily be adapted for a specific purpose. Sterilizers another type, which is made from large nylon tubes, stacked with each other, can be sterilized in the autoclave. (See M. Lev 1962. "An autoclavable plastic unit for rearing animals under germ-free conditions". J. Appl. Bacteriol. 25:30). Also developed insulators made of organic glass and disposable flexible film blocks. Many of them are quite light, so they can be placed one above the other on a single rack, which saves space inside the room.

For insulators, sensitive to heat, use a special cylinder for sterilization of food and nutrition. The insulators shall be designed so that they had great site Phil is being aware of contamination", Lab. Anim. Care 13: 572). Alternatively, the cylinder can be provided to drain the felling connected with atmosphere that allows you to remove air and condensate in the sterilization process without the use of vacuum. (See N. E. Jaworski and C. E. Miller 1963. "Refinement of cylinder technique for supplying germ-free isolators", Lab. Anim. Care 13:591).

Sterilization

All equipment, food, litter, water and air used in the isolator must be absolutely sterile. Used this method and conditions are determined individually in each case.

The use of steam supplied under pressure, is the best known methods of sterilization. It is particularly suitable for porous substances that are resistant to the action of heat. Each zone, which can be expected to serve as a shelter microbes, should be exposed to direct contact with water vapor. The exposure time depends on the applied temperature. Recommend to the least accessible part of the downloadable components (middle package) was processed at least 15 minutes at a temperature of 121oC. higher temperatures and shorter processing times can be used after a thorough research on the completeness of sterilisation. The standard size of the UPA is the origin of water vapor would be consistent and predictable.

For sterilization supplied to the isolator air use dry heat (see M. Miyakawa 1959. "The Miyakawa remote-control germfree rearing unit", Ann. N. Y. Acad. Sci., 78:37; B. E. Gustafsson 1959. "Lightweight stainless steel systems for rearing germ-free animals, Ann. N. Y. Acad. Sci. 78:17).

Peracetic acid (CH3COOOH) is widely used in the case vulnerable to heat non-porous materials, especially flexible film boxes. This acid is used in the form of 2% solution with a wetting agent (detergent) (see P. C. Trexler and L. I. Reynolds 1957. "Flexible film apparatus for the rearing and use of hermfree animals" Appl. Environ. 5:406). In specific cases, can be applied to other chemical reagents, in particular hypochlorites, iodophore or Quaternary ammonium compound for filling liquid separators, in which is placed the newborn after the removing of the uterus, or mercury chloride for placing eggs in sterile conditions before hatching.

For sterilization nesmeshivayuschikhsya sensitive to the action of heat boxes you can use ethylene oxide. Sterilization time depends on temperature, humidity, pressure and concentration of ethylene oxide. The ethylene oxide may chemically interact with bedding and food components with the formation of toxic or undesirable compounds should be limited to manufactured gas mixtures containing not more than 20% of ethylene oxide.

For sterilization of the incoming air using filters made of fiberglass. They should function as absolute filters.

Not to pull the liquid to heat, can be applied membrane filters, provided that these membranes are absolute filters, such as filters, preventing particles with a diameter of more than 0.22 microns.

For sterilization write or other special components can be sources of gamma rays or electrons. Used doses ranging from 2.5 to 6106happy.

Internal environment

Temperature. The temperature inside the insulator depends on room temperature and must be maintained in the range of 22-26oC.

The humidity. In the event of an overload, insufficient ventilation, or under the influence of both these factors in isolation may condense moisture. The air entering the isolator must have a relative humidity less than 50 percent and preferably not lower than 40 percent.

The air supply. The multiplicity of the exchange of air in the isolator should be from 12 to 20 volumes per hour, and the excess pressure is 3-5 inches (8-13 cm) vodkajust system centralized air supply it is recommended to use an air compressor turbine type, because oil pumps tend to allocate aerosols of oil in the supply line of the air.

The insulator to the diffusion of air (see P. C. Trexler 1968. "Equipment design and management: Transport of germ-free animals and current developments in equipment design", p. 23-35. in: M. E. Coates (Ed.) "The germ-free animal in research, Academic Press, London) is insensitive to loss of ventilation in case of power failure. However, this type has the disadvantage of allowing a smaller ratio of air exchanges per hour and can not create protective excessive pressure, which could prevent contamination in case of small damages in the barrier.

Protective measures in cases of emergencies.

In case of cessation of air for a period of not more than a few minutes (when the power or mechanical damage) should provide appropriate security measures for maintaining the air pressure inside the isolator. Failure is not secured emergency should support film insulators may eventually lead to suffocation of animals, but the immediate danger is that the animals can reach the film or gloves and damage them. This can be prevented by temporarily down air only emergency power source. Centralized air supply system must have a second turbine compressor to back air flow.

It is recommended to enter the graphical record of temperature and pressure. In a centralized system air supply must be installed audiovisual system alerts, which operate in the event of a pressure drop in the line due to power loss, and in case of mechanical damage. A similar alert system should indicate undesirable deviations in the temperature of the air. For a system of individual air supply is recommended for continuous graphic registration conditions inside the building.

Cells and located inside equipment

Equipment. A list of basic equipment for the laboratory may include cells with protective covers, water bottles, feeders, protective cloth gloves rubber gloves, additional sealing of the door or cap strip, long rubber coated tweezers, hemostatic clamps, scissors, towels, gauze swabs, containers with a capacity of two quarts for storage of tools, cans are coated with a capacity of four quarts for HDI shall be constructed of smooth, corrosion resistant material. They must be impermeable to liquids and is easy to undergo sterilization. Acceptable materials are plastics, stainless steel and glass. Metals plated with rust and they are not recommended, as contamination by trace metals may influence the results of the experiment.

The size of the cells is usually limited by the size of the boot opening. The minimum area required for one female mouse and straw bedding for her, is 50 square inches (970 sq. cm). In most cases, for one animal may need more space.

In table a presents the recommended space for one animal when placing mice and rats depending on the weight of the group.

Other recommendations

To ensure the integrity of the barrier system, it is recommended to test the PA for leaks using freon.

For each box should be your Protocol operations to record in chronological order all the events in Boxing since its Assembly and sterilization. Such records are usually stored in the form of a card file on metal stellarone, in particular replacement of gloves. On the same rack can be stored records of breeding offspring.

Due to limited available space within the insulator for food and litter, as well as for transportation of animals between insulators, United sterile aisles, recommend the use of paper and folding packages. Inside the prison, you cannot use the ether, as it may explode if static discharge. As volatile non-flammable anesthetic tools recommended fluoran (bromchlordifluormethane).

Food, litter and water

General recommendations

Need a complete formula for manufactured food, with all the ingredients and their contents, including preservatives, antioxidants and other additives. Must be clearly marked with date of manufacture. The manufacturer must ensure that food:

1) has a normal acceptable limits of natural hormonal activity;

2) does not contain supplements containing stimulants, hormones, antibiotics or other substances that create abnormal physiological conditions or in conflict with the experimental method is tons of pollutants from rodents or vermin;

5) free from all neprivrednih residues meat or fish products, which may contain pathogens.

Enrichment of food

The food sterile animals are increased requirements on the content of certain nutrients, to compensate for the loss of vitamins caused by heat treatment for sterilization (especially some of the B vitamins and vitamin A and D), and loss of nutritional value of proteins (reducing the number of available lysine, methionine, arginine and tryptophan). Food should also contain the necessary nutrients that normal animals produced by microbial synthesis in the gastrointestinal tract (see B. S. Reddy, B. S. Wostman and J. R. Pleasants 1968. "Nutritionally adequate diets for germ-free animals, p. 87-111, in: M. E. Coates (Ed. ) "The germ-free animal in research, Academic Press, London). An example of such a food is L-485 - inexpensive food that has been extensively tested (see T. F. Kellogg and B. S. Wostman 1969. "Rat and mouse stock diet L-485", Lab.Anim.Care) and is produced industrially (see Table B). To compensate for the losses in the qualitative composition of proteins, you should not increase the total protein content, and add specific amino acids. The increase in the total protein content in the diet will lead to greater consumption and selected the e can be placed in the isolator.

Sterilization of water vapor (see B. S. Reddy, B. S. Wostman and J. R. Pleasants 1968. "Nutritionally adequate diets for germ-free animals, p. 87-111, in: M. E. Coatcs (Ed.) "The germ-free animal in research, Academic Press, London).

The specific method will depend on the existing hand equipment. As a rule, an important role is played by three factors:

1. Preliminary vacuum but at least 20 inches (508 mm) of mercury promotes the penetration of water vapor inside the poor in the autoclave or the cylinder is connected to atmosphere. If the cylinder for feeding poor is not connected with the atmosphere, it is recommended that a vacuum of 28 inches (711,2 mm Hg or more.

2. The use of minimum phase sterilization, which guarantees total sterility, plus secure borders defined by the equipment and experience of the experimenter. The temperature measured in the inner parts of the food must reach at least 121oC. At this temperature, the actual phase sterilization must be at least 15 minutes. At higher temperatures sterilization sterilization time will be slightly shorter.

3. Vacuum after sterilization accelerates the cooling of food. This leads to the prevention of unwanted decomposition of nutrients. However, developed the

In the process of sterilizing food water vapor aim is to avoid as incomplete sterilization and unwanted destruction of nutrients, caused by excessive heating. Although losses of some nutrients, it is difficult to avoid, quite satisfactory results can be obtained by controlling the following factors:

(a) Technical parameters, such as temperature pre-sterilization and vacuum after sterilization, as well as the size of the tablets.

(b) the water Content in food. The increase in water content leads to a better preservation of vitamins after sterilization (see D. R. Zimmerman and B. S. Wostmann 1963. "Vitamin stability in diets for germ-tree animals", J. Nutr. 79:318). For solid foods recommended water content is up to 25 percent or may be such as not to degrade the quality of the food during storage. The change in water content in food should be accompanied by a new test of the speed with which food reaches the sterilization temperature.

Sterilization under the action of radiation.

(See B. S. Reddy, B. S. Wostman and J. R. Pleasants 1968. "Nutritionally adequate diets for germ-free animals, p. 87-111, in: M. E. Coates (Ed.) "The germ-free animal in research, Academic Press, London; F. J. Ley, J. Bleby, M. E. Coates and J. S. Paterson 1969. "Sterilization is zlecenia. Although in General it is believed that sterilization using radiation results in less destruction of nutrients, currently recommend that the food has passed the sterilization of water vapor.

Tests for sterility

To control the degree of sterilisation, which is achieved with the use of specific methods of sterilization, it is recommended to use the indicator paper with spores of Bacillus stearothermophilus. Strips of indicator paper put food inside. Periodically microbiological monitoring is subjected to the insulator and animals. The specified control is necessary for incidental impurities resulting from the destruction of barriers insulator or improper sterilization facility or its contents. The specified procedure is carried out as described by B. S. Wostmann, Ed., "Gnotobiotes: Standards and Guidelines for the Breeding, Care and Management of Laboratory Animals". National Research Council, National Academy of Sciences, Washington, D. C. 1970. pp. 28-39.

Assessment of the extent of the loss of nutrients in the sterilization process

As a useful check of the extent of the loss of vital nutrients, it is recommended to use the definition of the number of extracted thiamine as an indicator of the number of allocation thiamine, dobavlayet to a significant deterioration of the overall nutritional value of food. Using the proper equipment and proper precautions should be allocated 50% or more.

Storage of solid food

Because the experiments under sterile conditions usually is very expensive, you should take extra precautions and never use food that has largely lost its nutritional properties. It is recommended that (a) unsterilized food is always kept in the refrigerator and in any case not more than one month, and (b) the storage of sterilized food inside the insulator should be one week or less and not to exceed ten days.

Litter

The bedding should be changed at least once a week. It is recommended that the litter material can be easily sterilized and that he, too, was absorbed by animals. He should not be identified in the sterilization of toxic substances. It is recommended to use dust-free white pine chips (sawdust and shavings. Acceptable chips American basswood or poplar or crushed corn cobs. It is not recommended to apply products diatomaceous earth, cedar, resinous trees rocks and trees tn the question of the possible formation of harmful substances.

Water

Drinking water should be sterilized. It is treated in an autoclave in square packing containers, vessels Mason or tanks attached to the box. Inside each container should be left a small space.

Principles of getting gnotobiotic with caesarean section

Part of the key to any successful cesarean section is that pregnancy has completely reached its deadline. This is especially important for animals with short periods of pregnancy, when the fetal fetus can add 20% of their weight in the last 24 hours before delivery. The timing of mating undoubtedly useful, however, for animals, giving a large litter (rats and mice), it is useful to wait to before the surgery, the female gave the first offspring. For Guinea pigs the most satisfactory method is the selection of females for surgery by measuring the degree of stretching of the pubic bone (see B. P. Philihs, P. A. Wolfe and H. A. Gordon 1959. "Studies on the rearing guinea pig germ-free", Ann.N.Y.Acad.Sci 78: 183).

Received caesarean section babies should be transferred to amicrobic environment before they make their first breath of air. They can directly Valera or move in a sterile isolator through bactericidal trap by removing the uterus. Conventional surgical preparation females prior to surgery caesarean section involves removal of hair on the belly, the cleaning and disinfection of surgical procedure. Anesthesia is carried out preferably by displacement of the cervical vertebrae rat or mouse, although you can use anesthesia on the middle line of the belly or to use General anesthesia, without causing a significant weakening or death of the fetus. In the case of Guinea pigs operation usually carried out after the preliminary exposure to sedative agent and under local anesthesia.

Transfer cubs after a caesarean section through the membrane barrier requires special equipment for carrying out insulation. Surgical Boxing Range stainless steel (see J. A. Reyniers 1965. "Germ-free life methodology (gnotobiotics) and experimental nutrition, p. 458-466, in: Proc. 3rd Intertat. Congr. Biochem., Bruxelles) has a built-in horizontal metal wall that divides the upper and lower branch of Boxing. The partition has a round hole covered with a film of Mylar, preserving the integrity of the upper branch. Female, prepared, placed in the lower compartment so that its belly is pressed against the Mylar. All surgical instruments are placed in VSU of electrocautery or scalpel. For dissection of the skin is preferable to use semesterised blade electrocautery. The edges of the skin and Mylar are pressed to each other and unbend. On the belly put sterile surgical towel, covering the cut edges of the skin, and open the connective tissue before opening the abdominal cavity is treated with warm disinfectant (benzalkonium chloride 1:1000). You should be particularly careful not to damage the intestine. It may be useful to placing a pair of hemostatic clamps between the abdominal wall and internal organs. Then open the uterus and remove the pups, the shell of the fruit is removed, compress the umbilical cord and cut off. Calves gently wipe and massage to stimulate breathing. Their transfer to the Department for cultivation, where they feed the nurse or feed them manually. Another sheet of Mylar you can close the surgical opening and the procedure is repeated for 5 or 6 females without much risk to introduce contamination. Birth caesarean section can be carried out as using the operating plastic insulator or Boxing with gloves. The external surface of the insulator previously subjected to sterilization, and then it pressed belly galacticomm the barrier can be closed with sterile tape and surgical procedure is repeated for other pregnant females.

Birth excision of the uterus usually spend in plastic insulators (see H. Foster 1959. "A procedure for obtaining nucleus srock for a pathogen-free animal colony", Lab. Anim. Care 9:135). The uterus naked under aseptic conditions and compress directly in front of the neck. Isecheno the uterus in amicrobic compartment through the trap with a germicidal liquid. As soon as the uterus will be in prison, as soon as possible carry out delivery of babies, so that they do not inhaled separated at birth waters. Cubs usually wipe and stimulate them breath, and then compress the umbilical cord and cut it. Next cubs passed the nurse or feed manually.

Excision of the uterus can be successfully carried out for mice, rats, and pigs. However, for Guinea pigs, it is preferable dissection of the uterus, since most mortality occurs in the case from the moment of separation from the source of the mother's blood and before placing inside the insulator is held over two minutes.

The system of cultivation in gnotobiology colonies

Inbred breed

The usual system of cultivation in which it is possible mating brother x sister, also can be used to obtain gnotobiology colonies.

Ainbridge with the waiting siblings and cousins. Although this pairing in weinbrenner colonies try to avoid, we obtain the system of crossing does not reduce the level of inbreeding to the possible limit values. Acceptable any system with minimal inbreeding. (See D. S. Falconer 1967. "Genetic aspects of breeding methods", p. 72-96, in: "The UFAQ handbook on the care and management of laboratory animals, 3rd ed., E and S Lovingstone, Ltd., London; National Research Council, Institute for Laboratory Animal Resources. 1969. "A guide to genetic standards for laboratory animals, National Academy of Sciences, Washington, D. C.).

Comparison of conventional and gnotobiology colonies

For comparison it is desirable to include both conventional and geobiological colony, with their similar genetic Constitution can be maintained by introducing obtained caesarean section offspring of effective cultivated populations of normal colonies in geobiological colony. It's probably a matter of choice for those manufacturers that the greatest attention is paid to the cultivation magnetobiological rats or mice, however, this way has the disadvantage that prevents the establishment of microbiological origin, which could facilitate microbiological control. Ideally, the control could be implemented using offspring from specific crosses, which could slugishly be used as a family to grow geobiological colony. If this procedure is repeated after every second or third generation, the genetic Constitution of both colonies will be very similar, of course, provided that in each colony used the same system crossing.

Alternatively, the offspring grown from effective population geobiological colonies can be used to create or maintain a normal colony. Genetic sequence will be identical provided that uses the same technique.

Save the registration entries (see G. L. Wolff 1967. "Practical mating systems and record-keeping in dreeding colony", p. 97-113, in The UFAW handbook on the care and management of laboratory animals, 3rd ed., E and S Livingstone, Ltd., London).

Must be kept appropriate records of animals and maintenance of the facility, records of animals must determine the efficiency of the operation and the biological activity of animals. Records about the insulator must contain, in chronological order all the events associated with the insulator so as to help to find a gap in the barrier when the contamination.

4.3. No antigens animals

In a preferred embodiment of the present sobreamrica, water soluble food, which is subjected to ultrafiltration. It is believed that this food provides complete control over the consumption of animal nutrients and antigens. Such foods are usually completely prepared from individual ingredients known chemical nature, in particular amino acids, simple sugars, lipids, vitamins and minerals. In the present invention chemically defined food includes amino acids, simple sugars, lipids, vitamins and minerals and does not contain any other components with a molecular weight of about 10,000 daltons, thus, all the components are chemically specific food has a low molecular weight and are natural nutrients that exist in the animal organism, and therefore it is considered that such components do not cause immune responses. The above references relate to sterile animals that are grown with the use of chemically defined food as animals do not have antigens".

It is also desirable to use for litter filter paper, otherwise sterile animals can eat the litter, which will lead to immune responses. Suppose that p/P> Specific chemically defined food for these species should include such components in such proportions and quantities to meet the known requirements for nutritional value for these species. Below is the composition and preparation of the preferred chemically defined food for sterile mice.

The composition and preparation of chemically defined food L489E14Se

Ingredient: Amount, grams per 100 g of ingredient

To 192 ml of water Milli-Q at a temperature of 70oC add the following components:

Leucine - 1,9

Phenylalanine - 0,74

Isoleucine - 1,08

Methionine - 1,06

Tryptophan - 0,37

Valine - 1,23

Aspargine - 0,91

Hydrochloride arginine - 0,81

Threonine - 0,74

Lysine hydrochloride - 1,77

Hydrochloride histidine - 0,74

The solution is cooled to 45oC and add to it the following components:

Glycine - 0,59

Proline is 1.48

Serine - 1,33

Alanine - 0,59

Monosodium glutamate is 3.40

The hydrochloride of the ethyl ester of L-tyrosine - 0,62

Gluconate iron (II) - 0,05

Salts 35D (1) - 0,105

Sodium Selenite (2) - 0,074

The solution is cooled to 5oC and add to it the following components:

Glycyrrhizinate calcium - 5,22

G 680 mg KI) - 0,086

The mixture 111E5 vitamins B (3) - 0,09

Vitamin B12 (4) - 1,20 mg

Chloride choline - 0,31

Potassium acetate - 1,85

To 108 ml oxen Milli-Q at a temperature of 70oC add:

D-dextrose anhydrous - 71,28

The solution is cooled to 5oC, mixed together and subjected to ultrafiltration.

(1) the Composition of salts salt 35D mixture given in Table 3.

(2) Add additional selenate sodium contained in the mixture of salts 35D.

(3) the Composition of the mixture 111E5 vitamins B are presented in Table 4.

(4) Add in addition to vitamin B-12 found in the composition of the mixture 111E5 of B vitamins (see below).

The specified composition fed mice or rats ad libitum.

The composition of lipid Supplement LADEK 69E6

Ingredient: Amount per daily dose (*) an adult animal (0385 ml)

Purified salt triglycerides (1) - 0.33 G

Retinilpalmitat - 6,34 mg (11,7 I. E.)

Cholecalciferol - 0,0288 mg (1,15 I. E.)

2-Ambo-alpha-tocopherol - 3,3 mg

Acetate 2-ambo-alpha-tocopherol - 6.6 mg

Phylloquinone - 72 mg

(*) Lactating mouse get a double dose for a normal animal.

(1) Includes: 12% palmitate, 1.5% stearate, 24% oleate, 55% of linoleate, 8% linole the R. Pleasants et al., "Germ-free Research: Microflora Control and its Application to the Biomedical Sciences, B. S. Wostmann. Ed., p. 87, Liss, New York (1985); B. S. Wostmann et al., J. Nutr. , 112, 552 (1982); and J. R. Pleasants et al., J. Nutr., 100, 498 (1970) described here for reference.

4.4. Obtaining monoclonal antibodies

Sterile animal is then used to produce monoclonal antibodies. Amicrobic the system can be used to obtain monoclonal antibodies to any antigen that can produce an animal in a non-sterile condition. A sample list of the antigens described in U.S. patent 3935074. We believe that the sterile animal allows you to get a much greater response immune reaction to antigen. So, you can increase the possibility that B-lymphocyte that produces the antibody is able to bind to specific epitope of the antigen. This is the main advantage of the present invention. Further, we believe that amicrobic system is particularly useful for generating high-affinity antibodies to those antigens that have many epitopes.

Sterile animal can be subjected to vaccination with standard methods. However, it is preferable to expose the sterile animal vaccination at least three times with perezia before performing the merge. We believe that the increased level of vaccination may be necessary, since it was observed that after two vaccinations, as is usually practiced, the greater part still are B-lymphocytes secreting IgM, but not B-lymphocytes secreting IgG.

4.5. Somatic cells

Somatic cells sterile animal, potentially capable of producing the antibody and, in particular, B-lymphocytes, suitable for fusion with B-cell myeloma line. Those producing the antibody cells, which are under the division of lymphoblast, mainly merge easier. Somatic cells can be obtained from lymph nodes, spleen and peripheral blood system premirovany sterile animals, and the choice of lymphatic cells largely depends on their determined in practice the usefulness of a particular system merge. But usually I prefer somatic cells obtained from the spleen, once defeating sterile animals premirovanii or hyperventilatie, you can use them as a source of lymphocytes producing antibodies. Lymphocytes of mice give a large percentage of stable fusions with myeloma mouse lines that opinionmeter sterile animal depends on the choice of antigen, because it is important that sterile animal among their B-lymphocytes were B-lymphocyte capable of producing antibody against this antigen.

4.6. Immortalize cells

From limfozitah tumors specialized myeloma cell lines for use in obtaining a hybrid (G. Kohler and C. Milstein, 1976, Eur. J. Immunol. 6:511-519; M. Schulman et al., 1978, Nature 276:269-270). Cell lines developed at least three reasons. The first reason is that it is necessary to facilitate the selection of fused myeloma cells. This is usually carried out using myeloma with deficiency of the enzyme that makes them incapable of growing in certain selective media which promote the growth of hybridomas. The second reason stems from the potential limfozitah tumor cells to produce their own antibodies. The purpose of using monoclonal techniques are eliminated to obtain immortalized fused hybrid cell lines that produce one need specific antibody in genetic control of somatic cells, which is a component of hybridoma. To prevent the production of the antibodies hybridomas tumor cells, use Milan cnie line with the deficit mechanism of secretion of antibodies. The third reason for the choice of such cell lines is that they are suitable and efficient to merge.

For the production of fused cell hybrids can be used multiple myeloma cell lines, including NS-1, X63-A8, NIS-Ag4/1, MPC11-45.6TG1, X63-Ag8.653, Sp2/0-Agf14, FO and S194/5XX O. Bu.1., which are all derived from mice, and 210-.RCY3.Ag1.2.3, which receive from rats (G. L. Hammerling, U. Hammerling and J. F. Kearnly, eds., 1981, "Monoclonal antibodies and hybridomas", in: J. L. Turk, eds. "Research Monographs in Immunology" Vol. 3, Elsevier/North Holland Biomedical Press, New York).

4.7. The merger

Methods for generating hybrids of spleen cells or cells of lymph nodes, producing antigens, and immortalized cells usually consist in mixing somatic cells with immortalizing cells in proportions that can vary from about 20:1 to about 1:1, in the presence of an agent or agents (chemical, viral or electric), which accelerate the fusion of cell membranes. It is often preferable to those same bodies of animal would serve as a source of somatic and immortalized cells used in the fusion. Methods merge describe Kohler and Milstein (1975, Nature 256:495-497; 1976. Eur. J. Immunol. , 6: 511-519), Gefter et al. (1977. Somatic Cell Genet. 3:231-236) and Kozbor et al. , 1983, Immunology Today 4, 72. Area and polyethylene glycol.

You can also use a recently developed technique EBV-transformation (Cole et al., 1985. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

4.8. Selection of clones and antibody

The probability of obtaining a viable hybrids on the mail merge procedure is usually very low and is approximately 110-6110-8. Due to the low probability of obtaining a viable hybrids, it is important to have at hand a tool for the selection of fused cell hybrids from the remaining Nikitich cells, in particular Nikitich myeloma cells. Also needed a way to determine the right producing a hybrid antibody amongst the obtained fused cell hybrids.

Usually merged cells grown in a selective medium, such as medium HAT, which contains gipoksantin, aminopterin and thymidine. Wednesday HAT promotes the growth of hybrid cells and inhibits growth Nikitich myeloma cells, which under normal conditions would continue to share continuously. Aminopterin blocks de novo synthesis of purine and pyrimidine by suppressing receiving tetrahydrofolate. Adding thymidine allows you to bypass the blocking of the synthesis of pyrimidine and gipoksantin introduced into the environment in order inhibin are mutants, have no gipoksantin phosphoribosyl-transferase (HPRT), but because they can't use the way of recycling. In a viable hybrids genetic information for the production of this enzyme provides a B-lymphocyte.

Since B-cells have a limited life cycle in culture (approximately two weeks), the only cells that can grow in the environment of the HAT, are hybrids derived from myeloma cells and spleen cells.

To facilitate selection of the antibodies secreted by the hybrids, and to prevent individual hybrids developed other, the mixture is fused myeloma and B-lymphocytes dilute environment HAT and grow in the cells of the tablet for micrometrology. After two or three weeks, when the clones are visible in the microscope, the liquid above the sediment in the individual cells containing hybrid clones analyzed for production of specific antibodies.

The analysis should be sensitive, simple and fast. Analytical methods include radioimmunoassays, enzyme immunosorbent assays, assays for cytotoxicity and trombotsitnoy analysis.

4.9. Growth of cells and producera is lnyh cell lines producing antibodies, each cell line germinated in one of two standard methods. Sample hybridoma injected into histocompatible animal of the same class, which is used to produce somatic and myeloma cells when performing a merge. In subjected to injection, the animal develops a tumor secreting specific monoclonal antibody produced by fused cell hybrid. Circulating in the animal body fluids such as serum and ascitic fluid can be pumped out through the puncture and to obtain monoclonal antibodies in high concentration. Alternatively, the individual cell lines can be grown in laboratory vessels with culture in vitro. Environment with a culture containing high concentrations only specific monoclonal antibodies can be distinguished by decantation, filtration or centrifugation.

4.10. The use of monoclonal antibodies

Monoclonal antibodies obtained by the method of the present invention can be used in any known techniques or methods that will appear in the future and employ monoclonal antibody.

Monoclonal antibodies used - titulo. Such analyses are usually heterogeneous or homogeneous. In homogeneous immunoassays immunological reaction usually involves the specific antibody containing the label defined in the analysis of the substance and analyzed OPSEC. The signal from the tag, directly or indirectly modified in the binding of an antibody to containing the label defined in the analysis substance. As an immunological response, and determining its duration is carried out in homogeneous solution. Used immunological labels include free radicals, fluorescent dyes, enzymes, bacteriophages, jointly acting enzymes, etc. the Main advantage of a homogeneous immunoassay is that the specific antibody need not be separated from containing the label defined in the analysis of substances.

In heterogeneous immunoassay as reagents analyzed using sample-specific antibody and a means to obtain a signal that can be detected. The sample is usually placed on a substrate, such as a tablet or a glass slide, and he is in contact with the antibody in the liquid phase. Then the substrate is separated from the liquid phase and either the phase on the substrate or the liquid phase of research, the al indicates the presence in the sample determined in the analysis of substances. Means for receiving the detected signal include the use of radioactive labels, fluorescent substances, enzymes, etc. are Examples of heterogeneous immunoassays are the radioimmunoassay analysis, immunofluorescence methods, ELISA tests, etc.

For a more detailed discussion of the above techniques of immunoassays, see "Enzyme-Immunoassays" Edward D. Maggio, CRC Press, Inc., Boca Raton, Fla., 1980. Cm. the U.S. Patents numbers 3690834; 3791932; 3817837; 3850578; 3853987; 3867517; 3901654; 3935074; 3984533; 3996345 and 4098876, and this list is far from complete.

Another important use of monoclonal antibodies is in vivo imaging and therapy. Monoclonal antibodies may contain a label, radioactive compounds, such as radioactive iodine, and be given to a patient intravenously. The antibody may also contain a magnetic label. For the precise location of the antigen it is possible to apply the method of nuclear magnetic resonance. Lokalizovan antibodies the antigen, the antigen can be detected using the methods of emission tomography and radionuclide scanning, thus determining the location of the antigen.

For example, the purified monoclonal antibody is suspended in a suitable noice, with the proper dosage and administered intravenously, for example, by continuous intravenous infusion over several hours, as specified in the monograph by Miller et al. "Hybridomas in Cancer Diagnosis and Therapy" (1982), which is reproduced here for reference.

Monoclonal antibodies but the present invention can be used for therapeutic purposes. Antibodies with relevant biological properties, directly may be useful as therapeutic agents alternatively, antibodies can be contacted with the toxin with the formation of immunotoxin or radioactive substance or drug education radiopharmaceutical or a pharmaceutical preparation. Methods of obtaining immunotoxins or radiopharmaceuticals antibodies are well known (see, for example, Cancer Treatment Reports (1984) 68:317-328).

In addition, we believe that the polyclonal antibodies obtained from sterile animals, can also be used in immunoassays and to give the best results compared to polyclonal antibodies obtained from normal animals.

Polyclonal antibodies from sterile animals can be obtained by using sterile animals, ka the animals using conventional methods, in particular, by separating the serum of the animal.

Fibrin-specific monoclonal antibody

5.1. Prerequisites

The mechanism of bleeding is a complex physiological response of the body in the repair of broken blood vessels. Hemostasis is achieved by joint cooperative action of the walls of damaged blood vessel, platelets and clotting system. The role of coagulating system is to create a three-dimensional network of fibrin, which is stabilized and secured the tube of platelets, which is formed on subendothelial the structure of the damaged vessel. The formation of insoluble fibrin matrix from circulating in the blood fibrinogen is the result of a complex sequence of transformations, the culmination of which is the explosive generation of thrombin in the right place. Coagulation is a process of complication of the structure, including the chain of enzymatic reactions, in which proenzymes (factors of blood clotting) sequentially activated and transformed into reactive enzymes. The process of polymerization of fibrin in clots controlled razlichnye components of fibrinolytic system, such as tissue plasminogen activator (t-PA) and its fast-acting inhibitor (PAI).

In accordance with the hypothesis about the mechanism stop bleeding, expressed in the publication T. Astrup, Blood 11, 781-806 (1956), there is an equilibrium between the formation of fibrin (coagulation) and the dissolution of fibrin (fibrinolysis). In the normal or healthy condition of these functions is strictly balanced. However, once the process of stopping bleeding weakened, coagulation and fibrinolysis pathologically expressed in terms of thrombosis and bleeding, respectively. Pathological manifestations of pathological thrombosis or thrombolytic diseases are extremely varied and include the consumption coagulopathy, thrombosis of the deep veins, arterial and venous thrombosis. Thromboembolism and thrombolytic complications of other diseases of blood vessels (in particular, atherosclerosis) can lead to blockage of the main artery that leads to ischemia of the organ and the accompanying life-threatening conditions, such as stroke (stroke), myocardial infarction, etc.

Fibrinolytic process is the transformation of the inactive proferment, plasminogen, proteolytic protein, plasmin, through the actions of agents who naten, however, it is known that in the process of formation of the fibrin plasminogen attached to where it can be brought into an active state by plasminogen activators, in particular t-PA. According to this scheme, the formation of plasmin occurs inside of a blood clot, where it is protected from deactivating the effects of the main inhibitor of plasmin - A2-antiplasmin.

Under the action of plasmin fibrinogen and fibrin break up to form the project of destruction. Fibrin is degraded to fragments X and Y, and at the further action of plasmin - fragments D and E. Fibrin in the form of unstitched D-dimer is decomposed into fragments X, Y, D, and E, and cross-linked fibrin is decomposed into complex D-D/E, dimer Y, dimer Y-D and oligomer X.

Studies of markers of thrombotic diseases was conducted recently with the use of polyclonal antibodies in radioimmunoassays and analyses the type of latex agglutination. It has been shown (P. J. Gaffney, Ann. X. Y. Acad. Sci., 408, 407-423 (1983)), these tests are quite unreliable. More specific and sensitive immunoassays such as enzyme-linked immunosorbent assay) using monoclonal antibodies become common practice in clinical laboratories. The limiting factor for these diagnostic metazocine highly specific antibodies for any potential impairment indicator hemostasis is constrained by low levels of indicators, and antigenic relatedness of a particular token with its predecessor, which is usually found in plasma at much greater concentrations. Examples are the formation of complexes between enzymes and their inhibitors, in particular thrombin - antithrombin III, plasmin - A2-doesn? t, t-PA - PAI-1. The number of new antigenic sites that occur due to the formation of these complexes is very small and makes it difficult to obtain immunological probes such as monoclonal antibodies).

Similarly, the main problem associated with the susceptibility of monoclonal antibodies to fibrin, is a structural and conformational similarity between fibrin and its physiological predecessor fibrinogen. Assessment, conservation covalent structure during the conversion of fibrinogen to fibrin exceeds 98% (E. F. Plow et al., 1982, Semin. Thromb. Haemostas, 8, 36), but because only a small percentage of epitopes in the molecule of fibrin actually are neoantigens (and unique to fibrin). In most of these approaches used to generate antibodies of fibrin, the main attention is given to vaccination of animals soluble fragments of fibrin and synthesis of peptides that mimic the treated nevant the stas., 50, 591-94 (1983); B. Kurdryk et al., Mol. Immunol., 21, 89-94 (1984)). Suppose, however, that the binding site of these antibodies is stored in the dissolution of fibrin, and therefore these antibodies can also contact the degradation products of fibrin.

The present invention enables a completely different approach and use the original antigen fibrin in combination with enhanced immunological activity does not contain the antigen of the animal with the aim of producing a fibrin-specific monoclonal antibodies. In accordance with the present invention, the fibrin-specific monoclonal antibody binds to fibrin but not fibrinogen degradation products of fibrinogen or breakdown products of fibrin.

5.2. Materials and methods

5.2.1. Animals

Sterile mouse BALB/cAnN get from amicrobic colony of the University of Wisconsin. Animals transported in amicrobic conditions. Not containing antigen colony creating, moving pregnant sterile mice receiving food L-485 from natural ingredients (see J. P. Pleasants et al., J. Netr, 116, 1949-1964 (1986)) does not contain antigens insulator, in which the mice immediately begin to give chemically certain foods. Their offspring that never poluchennuyu food and is designated as the first generation, no antigens. Not containing antigens of mice pair and contain pairs up until the females will appear obvious signs of pregnancy; after this, the female is separated, so that the female could obtain your complete diet lipids. Offspring tokaut from breast milk at the age of 24 days.

5.2,2. Home

Not containing antigens manufacturers are placed in pairs in one half of the standard cells for mice, made of polycarbonate, with dimensions 28x17,8x12,7 see the Bottom cut out and replaced with a false bottom made of stainless steel mesh. The longitudinal partition stainless steel is bolted to the ends of the plastic cells, acting far above and beyond to keep in place the wire stainless steel cover. This cover with a cavity, which is typically inserted into the cage, turned around so that above the false bottom came extra space. On the top of the lid weld stainless steel hinges of the appropriate size so that they can be mounted bottle capacity 60 ml On the sides of the longitudinal partition at half the distance from the false bottom to the top of the cell munology in Aging" (M. M. Kay, T. Makinodan, eds. ), pp. 257-297, CRC Press, Boca Raton, F1, (1981)). Bottles with food made from glass brown. As bottles with food and water bottles have plastic covers which are drilled holes. Bottle fill out and turn in their loops. Lipid additive measure daily Cup of stainless steel. Under each cage was placed a tray for collection of waste.

Filter paper, which serves as a litter, and how edible fiber, is obessolennuyu Whatman filter paper No. 41, which is bought in the form of scraps ("Sargent Welch"). For litter paper cut into strips. Uncut pieces of square paper forms used for cleaning of the insulator. The paper is treated in an autoclave for 25 minutes at a temperature of 12oC or irradiated with dose of 4.5 Mrad in plastic bags. All mice in one corner of the cage lay a sufficient amount of paper. Paper replaced when it becomes wet, yellow or dirty.

Cells are placed inside a flexible insulator of Trexler size 1,37x0,60 m (P. C. Trexler, Lab.Anim.Care, 13, 572-581)) and apply the standard geobiological laboratory (see B. S. Wostman Ed., "Gnotobiotics Standards and Guideliner for the Breeding, Care and Management of L is raspisanie 12 hours dark and light time.

To the upper part of the insulator paste the tube of Tigana with a diameter of 2.5 cm and a length of 7.55 cm, which is at the top and bottom closed vinyl covers. It serves as a channel for sterile filtration of food, water and oil.

5.2.3. Food

Table 1 lists the composition of food and the order of dissolution of ingredients in subjected to ultrafiltration water Milli-Q ("Millipore", Massachusetts). Amino acids and dextrose receive from the company "Sigma"; they correspond to the purity of the drugs used for growing crops. Vitamins also receive from the company "Sigma", except net retinilpalmitat, who kindly provided by Hoffman-La Roche, Inc." (Natli, new Jersey). The purity of the other reagents certified Fisher or has a similar quality. Completely soluble in water, food filtered cold through an ultra-filter Amicon Diaflo TC3 using three Pm10 membranes with a diameter of 150 mm ("Amicon").

Value cutoff membrane ultrafilter is 10,000 daltons. The apparatus ultrafilter after Assembly is sterilized before use by passing through it to 0.15% sodium hypochlorite solution followed by thorough rinsing. Subjected to ultrafiltration food to the COI is ATOR using a 0.2 μm filter of nylon ("MSI"), placed in the holder, each filter of the autoclave, which is a tube insertion type with tube 6 made of neoprene. To this end the top of the vinyl tube in the tube of Tigana is removed and the inside of the tube serves sterilizing solution 2% peracetic acid containing 0.1% alkylarylsulfonate. The tube of the filter holder is placed instead of the top tube. After 20 minutes, remove the bottom plug (inside the prison) and food or water filter inside the insulator under pressure 20 psig (137,9 kPa) of nitrogen.

The lipid composition of the additive shown in Table 2. Soybean triglycerides are a drug derived from those of the methyl esters, which, after vacuum distillation in the temperature range give esters from palmitate to linolenate. The resulting esters are then subjected to interesterification with glycerin and get a mixture of triglycerides ("NuChek Prep, Elysian, Minnesota). Before filtering the mixture into the insulator to the glycerides add soluble in oil vitamins while it is heated to a temperature of 50oC, and then filtered in the insulator is similar to the method specified for filtering water-soluble portions of food.

The consumption of lipids appreciate in the 0.375 ml/size offspring also increases in comparison with animals grown in normal conditions. Lactating females receive a double portion of lipid supplements.

Note to Table 1:

(1) the Composition of salts salt 35D mixture given in Table 3.

(2) Add additional selenate sodium contained in the mixture of salts 35D.

(3) the Composition of the mixture 111E5 vitamins B are presented in Table 4.

(4) Add in addition to vitamin B-12 found in the composition of the mixture 111E5 of B vitamins (see below).

Water is subjected to ultrafiltration water Milli-Q, and it is filtered in the insulator as well as food.

5.2.4. Microbiological monitoring

Not containing antigens system test for the presence of microbiological contaminants according to the method described in B. S. Wostman, Ed. (1970), "Gnotobiotes: Standards and Guidelines for the Breeding, Care and Management of Laboratory Animals, National Research Council, National Academy of Sciences, Washington, D. C. in brief, tampons soaked in food and water inside the insulator, take a fresh fecal smears from mice and waste that have accumulated under each cage. Take swabs also from the walls of the prison, in particular around the boot holes. Every time I go on a two stroke. One set is subjected to the direct study under the microscope for the presence of Bakov. Before install the negativity of the culture allow people to go three weeks.

Microbiological tests are conducted approximately every two weeks, or a few days after placing the insulator new animals.

5.3. Obtaining monoclonal antibodies from not containing antigens of mice

The above does not contain antigens of mice used as donor lymphocytes in obtaining monoclonal antibodies. The solutions of antigens prepared under sterile conditions in a fume hood with laminar flow.

When vaccination is not having the antigens of the mouse used the following methodology. Antigen (25-50 μg) dissolved in sterile physiological saline (100 μl) and emuleret with an equal volume of complete adjuvant's adjuvant. Before receiving the emulsion in solution add interferon (1000 units). For all vaccinations use sterile syringes and needles. Syringes are transferred into not containing antigens insulator through the loading hole and there they are sterilized, Spryskova 2% solution of peracetic acid. Have injections activator, using the same amount of antigen, replacing the full beta-blockers on incomplete beta-blockers. Spend three activaction days before the date of the merger. All vaccination spend vnutribruchinno. Mice removed from the facility on the day of the merger and instantly put to death, stifling their carbon dioxide. Remove the spleen and merge splenic macrophages with myeloma cells of the mouse (NS1), using standard hybridoma technology.

5.4. The use of the system does not contain antigens of the animal to obtain a fibrin-specific monoclonal antibodies

Not containing antigens used to generate a fibrin-specific monoclonal antibodies. The antibody is highly specific and does not recognize fibrinogen degradation products, fibrin or degradation products of fibrinogen. Hybridoma cell line is produced by the fusion of macrophages from spleen containing antigens in BALB/c mice vaccinated with fibrin person, and myeloma NS1 cells.

5.4.1. The schedule of vaccinations

Three do not contain antigens female mice aged eight weeks Vaccinium 33 micrograms of the drug fibrin person. The drug is a frozen sample of crushed fibrin, which is obtained as follows.

The human fibrinogen is transformed into fibrin by using thrombin and factor XIIIa.tion crushed fibrin is dispersed in physiological saline solution and get a clear solution of cross-linked fibrin XL-Fn with a final concentration of 1 mg/ml For vaccination of animal use 33 μl of the antigen of fibrin. The volume of antigen solution is brought to 100 μl with sterile physiological saline and emuleret with complete adjuvant's adjuvant, as described in the previous section. Activating vaccination are at intervals of three weeks, using the same concentration of antigen in incomplete Freund's adjuvant. The last activation is conducted for 4 days prior to fusion. Use the same concentration of antigen and adjuvant replaced with physiological saline.

5.4.2. Detection and determination of antibodies

Qualitative and quantitative determination of antibodies is carried out using an enzyme immunosorbent assay (ELISA). ELISA tests conducted using fibrin person immobilized on poly (vinyl chloride) tablet with 96 cells ("Costar"). Covered with fibrin cells for analysis prepared by incubation in the incubator 100 μl of a solution of fibrinogen ("Kabi", the cleanliness class L) (50 μg/ml of the borate/saline buffer) over night at a temperature of 4oC. Unbound fibrin is removed by washing, phosphate buffered physiological saline solution containing 0.05% Tween 80. Febrile solution of thrombin (10 units of the National Institute of health, USA 1 ml), containing 2 mm of calcium chloride for 1 h at a temperature of 37oC. Standard calibration curves for antigen build using the drug antibody, which is homogeneous according to electrophoresis in a polyacrylamide gel-dodecylsulfonate sodium (SDS-PAGE).

To prevent nonspecific binding covered with fibrin tablets thermostatic in 1% solution of bovine serum albumin in buffered physiological salt solution with a pH of 4. Then add the solution containing the antibody (100 µl), and incubated in an incubator at a temperature of 37oC for 90 minutes. At the end of each stage in the procedure, the cells are thoroughly washed with phosphate buffered saline solution containing Tween 80. Bound peroxidase antibody determined by adding diluted 1000 times the antibody rabbit - anti-mouse associated with alkalinebattery (Sigma) diluted in buffered saline containing 1% bovine serum albumin, pH 8.0.

5.4.3. Getting a hybrid - fusion

Mice killed by suffocation in carbon dioxide and immediately carry out the removal of the spleen. The spleen cells of vaccinated mice merge, using as srnet in T-flask for 1 week. After this period, cells are placed on the tablets 5x96 cells, of which 93 show growth. Of these 19 cells show positive reaction to the antigen of fibrin. One of these clones, F492D8 (later renamed mh1c), produces an antibody that recognizes the antigen of fibrin, but does not cross-react with fibrinogen. This clone, mh1c, three powerhaul re-cloning by limited dilution triple phase for cloning, which is carried out for a single cell to cell. After the stabilization of cell lines it is transferred into serum-free medium, and cell line producing antibody with a concentration of about 7.5 mg/l

5.4.4. Purification of monoclonal antibodies

Before cleaning (portion 4 l) of liquid precipitation over tissue culture centrifuged to remove cellular residue and filtered through 0.8 mm nylon membrane to remove any remnants of the original materials. The liquid above hybridomas concentrate at a temperature of 4oC to a volume of 500 ml using a membrane type YM ("Amicon") as ultrafiltration system with a spiral winding, which cuts molecules with a molecular weight of 30000. Replacement buffer 20 mm solution of 2-(N-Mohali the driver. After further concentration to a final volume of 100 ml of the antibody solution before proceeding with clean filtered through a nylon membrane 0,451 microns. A concentrated solution of antibodies purified by liquid chromatography on a liquid chromatograph high resolution firm "Waters" using ABx column size of 7.75 mm 10 cm ("J. T. Baker, Philippsburg castles, new Jersey). Column offset buffer A and place the sample (100 ml) with a feed rate of 1.0 ml/min After extensive washing with buffer A elute antibody from the column in a gradient of buffer A to 100% buffer B (1 M sodium acetate pH 7) with a feed rate of 1 ml/min Collected fractions (2 ml) and those that contain monoclonal antibody (according to enzyme immunosorbent analysis) are combined and subjected to dialysis against phosphate buffered physiological saline solution (20 mm phosphate, 150 mm sodium chloride, a pH of 7.4) and stored at minus 20oC with a concentration of > 1 mg/ml ABx Column recovered by washing for 5 minutes and 100% buffer B, followed by equilibration wash buffer A in a quantity of 15 volumes of the column.

5.5. Determination of specificity in relation to the fibrin

Pervin what about from the microplate, covered with fibrin, and tablets, coated with fibrinogen. Acceptable are only producing antibody cell lines, which do not allow cross-reacts with fibrinogen.

Further evidence of specificity in relation to the fibrin is done using competitive analysis with fibrinogen in solution, thus confirming that the antibody does not recognize fibrinogen in solution.

Competitive analysis, confirming the specificity of antibodies against fibrin, spend the same way as previously described for enzyme immunosorbent analysis, pre-soaking in the incubator antibody together with fibrinogen in solution. In short, the liquid above the sediment of hybridoma kept in the incubator at a temperature of 37oC for 30 minutes with a solution of fibrinogen in physiological concentrations (4 mg/ml) containing bovine serum albumin (10 mg/ml) to prevent unspecific binding of the antigen to the fibrinogen. A solution of fibrinogen/antibody is then transferred to the microtiter wells are coated with fibrin. The inhibitor of fibrin add GlyProArgPro to prevent the polymerization of fibrin under the influence of the residual thrombin on the walls of the cell. C is authorized antigen. In all experiments to determine the specificity of antibodies mh1c against fibrin as a control connections use a second antibody 45J. 45J gives excellent reaction with fibrin and fibrinogen.

5.5.1. Definition of cross-reaction with fibrinogen

5.5.1.1. Immobilized fibrin and fibrinogen

Cell line mh1c produces a mouse monoclonal antibody, which gives cross-react with fibrin, immobilized on the surface of the polyvinyl chloride tablet for micrometrology (table 5). In the same analysis, the antibody does not recognize immobilized on tablets fibrinogen. As shown by the data presented in Table 5, the immunoreactivity increases sharply as soon as the fibrinogen is converted into fibrin by the action of thrombin, which clearly indicates the impact or the formation of neoepitope in the molecule of fibrin. At the same time, the control antibody, 35J, explicitly recognizes the epitope, which is stored in the conversion of fibrinogen to fibrin.

5.5.1.2. Competitive analysis with fibrin and fibrinogen

The specificity of the antibodies, antibody mh1c, in relation to the fibrin further demonstrates competitive analysis, in which the liquid over hybridomas perawat in the incubator with a solution of fibrinogen (final concentration 4 mg/ml). Because in this competitive analysis using a high concentration of fibrinogen (x500 against the antibody concentration), then added to the mixture of bovine serum albumin (final concentration 10 mg/ml). To prevent polymerization of fibrin by the action of the residual thrombin coated fibrin cell type peptide GlyProArgPro. The results of this analysis show that the antibody mh1c does not recognize fibrinogen in solution (table 6).

If we assume that each cell microplate associated 400 ng of fibrin, the content of fibrinogen in this competitive analysis 1000 times the amount of bound antigen fibrin. In addition, it corresponds to 400-fold excess of the content of antibodies in the liquid above the sediment fabrics.

5.2.2. Determination of ability to cross-interaction with degradation products of fibrin (fibrinogen)

The degradation products of fibrinogen get soaking fibrinogen with plasmin in the incubator with the temperature of the 37oC from 10 minutes to 3 hours. At the right time fibrinogenesis interrupt the addition of the drug (100 units inhibitor of kallikrein in milliliter) and 20 mm Epsilon-aminocaproic acid.

the cited U.S. 1 ml) to a solution of fibrinogen (5 mg/ml in physiological saline solution with Tris buffer (pH 7,4, 50 mm Tris HCl, 150 mm NaCl) containing 10 mm calcium chloride, plasminogen (0.25 mg/ml) and urokinase (50 m E./ml). The mixture was kept in an incubator at a temperature of 37oC and fermentation of fibrin interrupt through various time intervals, as described previously for the decay products of fibrinogen.

To measure the ability of antibodies to cross-react with degradation products and fibrin degradation products fibrinogen corresponding decay products are placed in the tablets for microteriofauna and spend enzyme immunosorbent analysis in the usual manner. As indicated in Table 7, the antibody mh1c is not involved in the cross-interaction with any degradation products of fibrinogen, formed under the influence of fibrin. As indicated in Table 8, the antibody does not enter into cross-interaction with degradation products XL-fibrin. A similar pattern is observed when the antibody investigate on the subject of cross-reaction by the method of Western blotting.

We can conclude that the antibody recognizes an epitope of the original molecules of fibrin, which is missing or is not formed in the molecule precursor, fibrinogen. Probably, as can be seen from Table 8, the epitope is destroyed by fermentation stitched fibre analnoe antibody that can be characterized as follows: in accordance with the present invention, the fibrin-specific monoclonal antibody is a monoclonal antibody that:

1. In a competitive analysis to determine the ability to cross-reaction with fibrin and fibrinogen, as indicated previously, monoclonal antibody has approximately less than 75%, preferentially approximately less than 10% of the ability to cross-react with fibrinogen, when the amount of fibrinogen in 1000 times the amount of fibrin.

2. In a competitive analysis to determine the ability to cross-reaction with crosslinked fibrin and degradation products of fibrin, as indicated previously, the reactivity of monoclonal antibodies against fibrin subjected to enzymatic effects of plasmin for about 3 hours, is approximately less than 50%, mainly approximately less than 40% of the reactivity of monoclonal antibodies against fibrin at time zero.

3. In a competitive analysis to determine the ability to cross-react with fibrinogen and degradation products of fibrinogen, as umentation the effects of plasmin for approximately 4 h, does not exceed the reactivity of monoclonal antibodies against fibrinogen at time zero.

Antibody mh1c further characterized by its affinity for fibrin. The affinity determined by the analysis of Scatchard (Frankel et al., Molecular Immunology, 16, 101-106 (1979)) using antibody mh1c containing the tag125I. the value of the dissociation constants KDis 6,710-10M. the affinity of about 5000 times greater than the affinity of t-PA to fibrin.

Analysis by the method of Western blotting was also shown that the antibody mh1c shall not cross-react with A, B or gamma chains of fibrinogen. Using the same method it was found that the antibody mh1c shall not cross-react treated with thrombin (A, B, or gamma chains of fibrinogen. (Processing of fibrinogen by thrombin leads to the release of fibrinopeptide A and fibrinopeptide B of the A-chain and B-chain, respectively, with the formation of the alpha-chain and beta-chain of fibrin).

Further, the results in Table 7 show that the control antibody, antibody 45J, binds to fibrinogen and shall not cross-react with the products of decomposition of fibrinogen. Thus, such a monoclonal gaining. Fibrinogen-specific monoclonal antibody can be used in any immunoassay that can be used for determination of fibrinogen in plasma in vitro. Antibody 45J differs in that, as has been discovered, the epitope 45J is located on the alpha-chain of fibrinogen in the area around 206 amino acids to about 424 amino acids, and most likely in the area around 207 amino acids to about 231 amino acids. In accordance with the present invention, the fibrin-specific monoclonal antibody is an antibody in which the analysis is by definition the ability to engage in cross-react with fibrinogen and degradation products of fibrinogen, as indicated earlier, demonstrates the reactivity towards fibrinogen subjected to enzymatic effects of plasmin for about 40 minutes, which is approximately 50% of the reactivity of monoclonal antibodies against fibrinogen at time zero.

Hybridoma 45J receive conventional methods using conventional Balb/c mice, while mice Vaccinium fibrin. However, the fibrinogen can also be used as anti unstitched fibrin

Using the method of enzyme immunosorbent analysis it was shown that the antibody mh1c enters into cross-react not only with crosslinked fibrin (XLFn), but unstitched fibrin (NONXLFn). Enzyme immunosorbent analysis carried out as follows:

1. Tablet for micrometrology with 96 cells cover 100 μl of a solution of fibrinogen (50 mg/ml in borate buffered saline solution with pH 8.5) overnight at a temperature of 4oC.

2. Cross-linked fibrin get coated with fibrinogen cells by incubation in the incubator for 1 h at a temperature of 37oC together with a solution of thrombin (10 units of the National Institute of health, USA 1 ml) in physiological salt solution with Tris buffer (pH of 7.4, 50 mM Tris HCl, 150 mM NaCl) containing 2 mm calcium chloride and 10 mm cysteine.

3. Unstitched fibrin get coated with fibrinogen cells maturing in the incubator for 1 h at a temperature of 37oC together with a solution of thrombin (10 units of the National Institute of health, USA 1 ml) in phosphate buffer (pH 6,1) containing add with the final concentration of 0.0125 M

4. Bound antibody is detected by keeping in the incubator with artemisinin conjugate alkalinity. the functions are controlled by an automatic device at a wavelength of 405 nm.

The results of the analysis are shown in Table 9 and from them it is possible to conclude that the ability of antibodies to cross-reaction with crosslinked fibrin stronger than the ability to cross-reaction with unstitched fibrin. The simplest explanation may lie in the fact that covalent cross-linking, the existing crosslinked structure, serves to lock or freeze the conformation recognized by the antibody. In unstitched samples this conformation, although it is formed is not stable covalent bonds of the polymer.

It was also shown that the antibody enters into cross-react with both stitched and unstitched clots obtained in vitro. Cross-linked fibrin get, soaking in the incubator solution of fibrinogen (in physiological saline solution with Tris buffer (50 mm, pH 7,4) containing 2 mm calcium chloride and 10 mm cysteine) with thrombin (10 units of the National Institute of health, USA 1 ml) for 3 h at a temperature of 37oC. Unstitched fibrin get soaking in the incubator solution of fibrinogen (3 mg/ml) in phosphate buffer (pH 6,1) containing add with the final concentration of 0.0125 M, with thrombin (10 units of the National Institute of health, USA 1 ml) for 3 h at a temperature of 37
antibody mh1c tagged with125I. In different periods of time take a small aliquot and the amount of bound antibody is determined in the plasma using the count of gamma radiation.

Table 10 shows data on the binding of an antibody as unstitched and stitched by clots.

5.6. The depositing of hybridoma

Mh1c and 45J deposited in the American type culture collection (ATCC) on 9 June 1988 and received a registration number HB 9739 and HB 9740, respectively. Address the American type culture collection - 12301 Parklawn Drive, Rockville, MD, 20852. Antibody mh1c is an antibody IgG1 with Kappa light-chain, and it was shown that the mh1c enters into cross-react not only with human fibrin, and fibrin rabbit.

The present invention is not limited by the amount of the deposited hybridomas, and they only serve as an example of hybridomas that produce the fibrin-specific monoclonal antibody and fibrinogen-specific monoclonal antibody, as described previously. Any functionally equivalent cell line included in the scope of claims of the present invention. The term "functionally equivalent" means that the antibody is capable of competing with the antibody mh1c Egan, attached antibody 45J, respectively. Hereinafter, the term covers a fibrin-specific monoclonal antibody and fibrinogen-specific monoclonal antibodies, as described previously, which are connected to the epitope different from the epitope to which is attached antibody mh1c, or to the epitope to which is attached antibody 45J, respectively.

5.7. The use of fibrin-specific monoclonal antibodies for diagnosis in vivo and in therapy

5.7.1. Localization of the disease clot/vessel

Fibrin-specific monoclonal antibodies of the present invention is able to note fibrin clots or aggregation of fibrin in vivo. Therefore they can be used to localize the possible damage of tissues or blood vessels in humans and to monitor vascular diseases. Fibrin-specific monoclonal antibody is particularly preferred for use in these purposes, as specified monoclonal antibody is not connected with fibrinogen degradation products and fibrin degradation products fibrinogen, thereby reducing background interaction and allowing more precisely localize the fibrin clot or aggregation of fibrin.

For at methods liquid chromatography high resolution. Purification of monoclonal antibodies to assign a person may also include the purification by precipitation with ammonium sulfate or sodium sulfate followed by dialysis against physiological saline solution and sterilized by filtration. As an alternative for the purification of monoclonal antibodies can be used methods immunoaffinity chromatography.

Purified monoclonal antibodies can be marked with radioactive compounds, such as isotopes123I125I131I99mTc,111In, and assign to the patient intravenously. The antibody may also contain the label of the magnetic sample. The exact location of the clot can then be determined by the method of nuclear magnetic resonance. After localization of antibodies in the clot or the Assembly of fibrin can discover a method emission tomography and scanning radioactive nuclei, thereby establishing the location, for example, blood clot or tumor covered with thrombin.

To illustrate: purified monoclonal antibody is suspended in a suitable carrier, in particular a physiological saline solution containing or not containing albumin human, with the appropriate dosage, and appoint patiene several hours.

5.7.2. Treatment of vascular diseases conjugate monoclonal antibodies

Monoclonal antibodies of the present invention can be used in conjunction with a wide range of pharmaceutical agents, such as cytotoxic agents and thrombolytic agents, including t-PA, urokinase, streptokinase and other protease capable of leasing of fibrin. This application is especially preferred because the fibrin-specific monoclonal antibodies of the present invention allows a very efficient use of these reagents, because none of these reagents will not be lost due to binding with fibrinogen degradation products, fibrin or degradation products of fibrinogen. Various reviews on this issue, see Bale et al. , 1980, Cancer Research, 40: 2965-297; Ghose and Blair, 1978, J. Natl. Cancer Inst. , 61(3): 657-676; Gregoriadis, 1977, Nature, 265:407-411; Gregoriadis, 1980 Pharmac. Ther., 10:103-108; Trouet et al., 1980, Recent Results Cancer Res., 75:229-235.

Methods that are used for binding of these agents with a monoclonal antibody can include education as non-covalent and covalent bonds. Since non-covalent communication can break down faster than the complex of the antibody reaches the target, then the covalent bond more predpochtite which may be formed by carbodiimide linkages. Bifunctional agents such as dialdehyde or imidiately, can be used to link the amino groups of drugs with the amino group of the antibody molecules. To associate drugs with a molecule antibodies can be used for the formation of Schiff bases. This method involves the oxidation of cytotoxic drugs or products containing glycolic acid or hydroxyl group by the action of periodate with the formation of aldehyde, which then reacts with a molecule of antibody. Accession shall be effected by forming a Schiff base with the amino group of the antibody molecules. In addition, medications containing reactive sulfhydryl group, is associated with antibody molecules.

6. Detecting and determining the concentration of soluble fibrin in vitro

6.1. Prerequisites

As indicated earlier (see Section 5.1.), the mechanism of bleeding involves a complex sequence of reactions, in which fibrinogen is fully converted to fibrin by the action of thrombin. The end result of these reactions is the formation of a thrombus (blood clot). The sequence of reactions in a simplified form can be represented in the form of a three-stage process.


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Fibrinogen is composed of three pairs of nonidentical polypeptide chains: A, B, and (see L. Stryer, "Biochemistry", 3rd Edition, p. 249, W. H. Freeman and Company, New York (1988)). At the initial stage, when fibrinogen is converted into fibrin, which is shown above in Stage 1, fibrinogen is cleaved by the action of thrombin, releasing fibrinopeptide A from having the amino group of the endings of the two A-chains of fibrinogen. The resulting monomer is a monomer DesAA fibrin. As shown in Stage 1, at the same time, but more slowly, thrombin also cleaves fibrinopeptide B has the amino group of the endings of the two B-chains of fibrinogen. Result in the release of fibrinopeptides on-chain and a-chain of fibrin new aminobenzene areas. As indicated above, the molecules formed in Stage 1 are fibrinopeptide a, fibrinopeptide B and the monomer DesAA fibrin, fibrin monomer DesAABB shown in the diagram as "monomers of fibrin (see W. Nieuwenhuizen, Blood Coagulation and Fibrinolysis, 4:93-96 (1993)). As described above in Stage 2, the monomers of fibrin next form as non-covalent (unstitched) and covalent (crosslinked) polymers with the formation of soluble fibrin polymers. As shown in Stage 3, soluble fibrin polymers then form a clot fibrinogen, which can lead to the formation of the fibrin polymer, or any molecular fragment derived from fibrin (fibrinogen), molecular weight which is greater than the molecular weight of native fibrinogen, which is contained in dissolved form in the blood.

Unstitched and stitched polymers DesAABB fibrin formed in Stage 2, represent two of several derivatives of soluble fibrin, and also are two fragments of soluble fibrin polymer. In addition, the term soluble fibrin includes other compounds, for example polymers of DesAA fibrin, the complexes formed by the interaction of fibrin monomers (monomers DesAA fibrin and DesAABB), and the degradation products of fibrinogen X, Y, D, and U (see section 5.1 above. for descriptions of these decay products), as well as, for example, monomers DesAA and DesAABB in the form of a complex with fibrinogen (see Nieuwenhuizen pp. 93-94).

Soluble fibrin polymers are the immediate precursors of insoluble fibrin, i.e., clot, and thus, the content of soluble polymers in the plasma, it is believed, increases in patients prone to thrombosis or at risk of thrombosis (a blood clot inside the vessel). Detection and determination of the content to the as indicating the beginning of the formation of a blood clot (see Bang and Chang, pp. 119-121; Nieuwenhuizen, p. 94; Marder et al. , U.S. patent 5206140).

Some fragments of soluble fibrin previously found and determined their content, using various methods, including, for example, the determination of fibrinopeptide A determination with the use of antibodies to epitopes A and formed during the conversion of fibrinogen to fibrin, Nieuwenhuizen, 94-96, and the determination of D-dimers. Marder et al., U.S. patent 5206140. Other methods used for the detection or for the determination of soluble fibrin include determining the amount of agglutination of erythrocytes coated with fibrin in the presence of soluble fibrin, exclusion gel chromatography, strengthening plasminogen activity by plasminogen activator t-PA (see Nieuwenhuizen, p. 94), gelation under the action of ethanol or Protamine sulfate. N-terminal analysis of fibrinogen fractions isolated from plasma, the inclusion of the ethyl ester of glycine containing the tag14C, and gel chromatography on agarose (see Bang and Chang, p. 111-118). None of these tests will not allow to detect and to determine the specific content of soluble cross-linked polymers and soluble unstitched fibrin polymers.

6.2. The analysis in plonetheme the invention is based on the opening, that it is possible to analyze in vitro with the aim to detect and determine the content rate of the soluble unstitched fibrin polymers DesAABB composed of fibrin monomers DesAABB, analysis system, which are not detected (a) fibrinogen, (b) the degradation products of fibrinogen, (c) fibrin monomers DesAA, (d) fibrin polymers DesAA, (e) fibrin monomers DesAABB, (f) cross-linked fibrinogen (fibrinogen treated with factor XIIIa), (g) complexes fibrin monomer DesAA - fibrin and (h) the breakdown products of fibrin ("fragments (a)-(h)"). The degradation products of fibrinogen and degradation products of fibrin occur when the enzymatic effect of plasmin on fibrinogen or fibrin, as described earlier in section 5.5.2.

We believe that the above analysis is particularly useful for the clinical diagnosis of conditions defined by thrombosis.

The analysis is performed using any suitable sample of the fluid circulating inside the body, but it is preferably carried out using the blood of mammals. Suitable mammals include, for example, rabbits, monkeys and people, thus people are preferred.

When conducting this analysis in vitro can be applied EOW and soluble DesAABB unstitched fibrin polymers DesAABB, however, these methods cannot detect fragments (a)-(h). Upon discovery, the content of soluble crosslinked fibrin polymers and soluble DesAABB unstitched fibrin polymers DesAABB in the sample can be determined by comparing with a control sample or using a standard with a known content of soluble crosslinked fibrin polymers and soluble DesAABB unstitched fibrin polymers DesAABB, while these standards do not contain fragments of (a)-(h).

It should be noted that by definition is any means by which it is possible to determine the presence in the sample of interest connections. The preferred means of determining is antibody against soluble crosslinked fibrin polymer and soluble DesAABB unstitched fibrin polymer DesAABB, this antibody does not enter into cross-react with (a) fibrinogen, (b) degradation products of fibrinogen, (c) fibrin monomers DesAA, (d) fibrin polymers DesAA, (e) monomers DesAABB, (f) crosslinked fibrinogen (g) complex fibrin monomer DesAA - fibrin and (h) the breakdown products of fibrin. The specified antibody can be used for detection and determination of the content in the sample soluble stitched the polyclonal or monoclonal antibodies, mainly monoclonal antibodies. The antibody may be derived from any species. However, antibodies are predominantly of human or murine origin, or get them from rabbits. Further, such antibodies include, but not limited to, chimeric antibodies, antibody single-chain fragments of the monoclonal antibodies.

An example of such antibodies is a monoclonal antibody mh1c, which is described above in sections 5.5.1-5.5.3 and section 5.6. We believe that the mh1c, besides the fact that it possesses the above characteristics does not attach well to the complex fibrin - degradation product of fibrin. This property mh1c can increase its effectiveness as a means of detection and determination of the soluble crosslinked and soluble unstitched fibrin polymers DesAABB.

It should also be noted that as a means of detection can be used two or more antibodies. So, for the detection and determination of the content in the sample soluble crosslinked fibrin polymers and soluble DesAABB unstitched fibrin polymers DesAABB can use a combination of antibodies with different specificity, when none of the antibodies does not in itself capable obrazovym and polymers DesAABB, and two or more antibodies can form these complexes. Of course, none of the antibodies may not engage in cross-react with fragments of (a)-(h).

It should also be noted that when using antibodies, it may be necessary to apply the antibody is specific to this form.

For the production of antibodies, various host animals Vaccinium by injection of soluble crosslinked fibrin polymers and soluble DesAABB unstitched fibrin polymers DesAABB as antigen, and these include including, but not limited to, rabbits, mice, rats, etc., These polymers can be selectively allocate using, for example, antibodies mh1c bound on column separate for affinity chromatography antigen mh1c. These polymers can be obtained as follows: soluble fibrin can be obtained in vitro by the addition of small amounts of thrombin to plasma containing citrate buffer. After the suppression of the activity of thrombin by adding a potential inhibitor of thrombin, such as hirudin, plasma samples can be placed on a column of sepharose-NH1. This column NH1 can be obtained by linking antibodies NH1 with SEFI "Pharmacia" about getting speakers antibody-sepharose 4B, using a pre-activated sepharose 4B. In column sepharose-mh1c first place fibrin in the presence of phosphate buffered physiological saline. After the tie in column sepharose-mh1c in the presence of phosphate buffered physiological salt solution associated to sepharose-mh1c soluble fibrin can be blueraven with a solution of sodium thiocyanate. After dialysis against phosphate buffered physiological salt solution selected soluble fibrin polymer can be stored at a temperature of minus 70oC.

In combination with the isolated polymers in order to enhance the immunological response, depending on the host species can use different adjuvants, which include, but not limited to, beta-blockers (complete and incomplete), mineral gels such as aluminum hydroxide, surface active compounds, such as lysolecithin, plutonomies polyols, polyanion, peptides, oil emulsions, hemocyanin, dinitrophenol, and potentially suitable adjuvants, such as BCG (Bacillus Calmette-Gerina) and corynebacterium parvum.

Monoclonal antibodies specific for soluble crosslinked and soluble unstitched fibrin polymers DesAABB, prby methods, which allow you to produce molecules antibodies continuous cell lines in culture. They include, but not limited to, hybridoma methods, which were first described by Kohler and Milstein (Nature, 1975, 256: 495-497), later developed methods of producing hybrid B-cells (Kosbor et al., 1983, Immunology Today 4: 72) and methods of EBV-hybridoma (Cole et al., 1985, Monoclonal antibodies and Cancer Therapy, Alan R. Liss, Inc., p. 77-96). In yet another embodiment of the present invention, monoclonal antibodies can be produced in sterile animals, using the above technology (see sections 5.2-5.4). An example of the production of such antibodies is getting mh1c, which is described earlier in sections 5.3-5.6.

In accordance with the present invention, human antibodies can be used and can be obtained by using a hybrid of man (Cote et al. , 1983, Proc.Natl.Acad.Sci., 80: 2026-2030) or by transforming B-human cells exposed to virus EBV in vitro (Cole et al., 1985, in: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77-96). In fact, in accordance with the present invention can be used in methods designed to obtain a "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81: 6851-6855; Neuberger et al., 1984, Nature, 312: 604-608; Takeda et al., 1985, Nature, 314: 452-454) by playingmohegan antigen person, with relevant biological activity; these antibodies are included in the scope of claims of the present invention.

In accordance with the present invention methods are described for obtaining single-chain antibodies (U.S. patent 4946778), can be used to obtain specific single-chain antibodies.

In yet another embodiment of the present invention use a technique developed for constructing libraries of expression of monoclonal antibodies Huse et al., 1989, Science, 246:1275-1281) to quickly and easily identify fragments of monoclonal antibodies with desired specificity with respect to soluble crosslinked and soluble unstitched fibrin polymers DesAAABB.

Antibody fragments that have specific binding sites for soluble crosslinked and soluble unstitched fibrin polymers DesAAABB, can be formed by known methods. For example, these fragments include, but not limited to, fragments F(ab')2that can be produced by fermentation of antibody molecules under the action of pepsin, and fragments of monoclonal antibodies that can be generated when restoring disul the change in well-known from the field of engineering systems, immunoassay, which include, but not limited to, radioimmunoassays, enzyme immunosorbent assays, "sandwich" assays, reactions of precipitin, immunodiffusion analyses in the gel agglutination assays, fluorescent immunoassays, immunoassays, protein A and immunoelectrophoretic analyses. Suitable methods of analysis are also presented in U.S. patent 4629783.

Antibodies can be used as key reagents in a number of different immunoassays to detect the presence of soluble crosslinked and soluble unstitched fibrin polymers DesAAABB in samples of blood or other fluid circulating in the body. In General, antibodies can be used in any type of immunoassays, both qualitative and quantitative. They include dvuhsimovyiy sandwich analysis, and single-site immunoassay non-competitive types, as well as traditional analyses by the method of competitive binding.

Especially preferred because of the ease of detection and due to the fact that he is quantitative - sandwich analysis, or the analysis with the two antibodies, which has many varieties, and they are all covered by the present invention.

Example: is the surrounding area cell tablet for micrometrology, and the analyzed sample is introduced into contact with the bound molecule. After the required incubation period, i.e. the time interval required for the formation of a binary complex of antibody-antigen, add the second antibody containing the tag molecule-reporter, able to give a signal that can be registered, and the incubation continued for a time sufficient for the antigen was able to join on another site and formed a ternary complex of antibody-antigen - containing tag antibody. Any unreacted substance wash, and the presence of the antigen set by observing the signal, which can be quantified by comparing with the control samples (standards), containing a known amount of antigen. Variations of the direct sandwich analysis are continuous analysis, in which both the sample and the antibody at the same time add to the related antibody, or reverse sandwich analysis, which contains the label of the antibody and the test sample is first mixed, incubated in the incubator and add to not containing the label to the antibody on the surface of the carrier. These techniques are well known to experts in the art, the Liz covers all variations of the basic dvuhslotovoj methods.

As a more specific example, in a typical direct sandwich the analysis of the primary antibody covalently or passively attached to the solid carrier. Solid carrier is typically glass or a polymer, the most commonly used polymers is cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. A solid carrier can be in the form of tubes, beads, discs or microplasmin or have other surface suitable for conducting immunoassay. The binding process is well known from the technical field. After binding the complex of solid phase antibody wash and prepare for the test sample. Then an aliquot of the liquid to be tested is added to the specified solid phase complex and incubated in the incubator for a time sufficient to bind any present soluble unstitched fibrin polymer and soluble DesAABB crosslinked fibrin polymer with any antibody specific for the indicated proteins. To solid phase complex then add the second antibody, and optionally kept in the incubator for a time sufficient for binding to primary education primary solid phase to the use for indicating the binding of the second antibody to the antigen of the sample. Under the "molecule-reporter" in the present description means the molecule, which by their chemical nature allows to obtain a stable signal to be detected by analytical methods, i.e., to detect the complex antigen-associated antigen. Detection must be at least relatively, quantitative, so that you can determine the amount of antigen in the sample, can be calculated in absolute units or as to compare with the standard (or a series of standards containing a known normal level of antigen content.

The most commonly used molecules reporters in analyses of this type are either enzymes or fluorophores. In the case of enzyme immunoassay enzyme linked to the second antibody, often using glutaraldehyde or periodate. Obviously, there are many different methods of binding, which are well known to specialists in this field of technology. Commonly used enzymes include, among others, horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase. The substrates that will be used with the specific enzymes are usually chosen by a noticeable color change when affected by conjugates exude alkaline-phosphatase; for peroxidase conjugates typically use 1,2-phenylenediamine or toluidine. You can also apply fluorogenic substrates that yield fluorescent products, not listed above chromogenic substrates. In any case, the antibody labeled with an enzyme, is added to the first complex of the antibody and allowed to form a complex, and the excess reagent and then wash. Then to the triple complex of antibody-antigen - containing tag antibody add a solution containing a suitable substrate. The substrate interacts with the enzyme linked to the second antibody, giving a qualitative visual signal, which can then be quantified, usually spectrophotometrically, thereby estimating the amount of antigen, which is present in the serum sample.

Otherwise, fluorescent compounds such as fluorescein or rhodamine, may be chemically bonded to antibodies without altering their binding capacity. When activated under the action of light with a specific wavelength antibody labeled with fluorochrome, absorbs light energy transferring molecule in an excited state, followed by emission of a characteristic of light with a longer wavelength. Radiation has a characteristic color, which is labeled antibody provide an opportunity to communicate with the first antigen complex. After washing out unreacted reagent obtained ternary complex is exposed to light with an appropriate wavelength, and the observed fluorescence indicates the presence of antigen. Both immunofluorescence and enzyme immunoassays are well designed and are most preferred for use in the present invention. However, you can apply other molecules reporters, such as radioisotopes, chemiluminescent or bioluminescent molecule. For professionals it is obvious how to modify the technique to suit the respective requirements.

Alternatively, the test sample or the blood of a mammal or other circulating in the body fluid containing soluble unstitched fibrin polymer DesAABB or soluble crosslinked fibrin polymer DesAABB can be used in the single-site immunoassay, where he covalently or ecovalence is attached to a solid carrier. Not containing the label antibody comes into contact with the sample, bound on a solid medium. After the desired incubation period in a period of time sufficient for formation of a binary complex of antibody-antigen, add the second antibody, Lieut within a reasonable amount of time, necessary for the formation of a ternary complex of antigen-antibody - containing tag antibody. The second antibody for single-site immunoassay may be an antibody of General type (i.e., xenogenous antibody for immunoglobulin, in particular anti-(IgM or IgG) associated with the molecule-reporter), which are capable of binding an antibody specific for soluble crosslinked fibrin polymer and soluble unstitched fibrin polymer.

Detection and determination of soluble unstitched fibrin polymer DesAABB or soluble crosslinked fibrin polymer DesAABB in vitro is particularly useful in the case when the detection and identification is done using the plasma of the patient, as an indication of emerging or existing thrombotic state, while the specified condition possibly caused or arising thrombosis (see, in particular, W. Nieuwenhuizen, p. 94; Bang and Chang, p. 109-122; Marder et al., U.S. patent 5206140).

Such States include, for example, thrombosis of the deep veins, a condition that occurs as a result of blood clots in the deep veins of the legs, pulmonary embolism, which occurs when a thrombus (blood clot) is shifted from the deep veins of their systematic activation of the cascade of blood coagulation (in particular, in the result of a bacterial infection); myocardial infarction, which occurs as a result of blockage by a blood clot in the coronary arteries that supply blood to the heart muscle; the shock and intracardiac thrombi resulting from atrial fibrillation. The type of thrombotic state can be set through the use of analysis for the detection and determination of the content of soluble unstitched fibrin polymers DesAABB or soluble crosslinked fibrin polymers DesAABB in combination with the observation of other symptoms of the patient. These symptoms are symptoms that are commonly used in clinical diagnosis occurring thrombotic state. For example, this detection and determination of soluble fibrin polymers DesAABB particularly useful as a tool to distinguish patients with chest pain due to myocardial infarction patients with chest pain due to other causes.

6.3. Examples materials and methods

6.3.1. Proteins

Bovine serum albumin and thrombin buy the company ICN (Costa Mesa, California). Hirudin, horseradish peroxidase and the substrate dihydrochloride o-phenylenediamine in the form of tablets buy es" (beaumont, Texas). Fibrinogen additionally purified by precipitation with ammonium sulfate, as described in the publication Holm et al. , Thromb. Res., 37:165-176 (1985). Antifibrin monoclonal antibody, mh1c, which is used in the analytical system, receive, as described earlier in section 5. Antifibrogenic monoclonal antibody 45J, receive, as described earlier in section 5.

Fibrin monomer DesAABB get, dissolving unstitched fibrin polymer in 50 mm buffer solution of sodium acetate (pH of 5.3) containing sodium bromide.

Stitched soluble fibrin polymers DesAABB get soaking in the incubator fibrinogen or plasma containing citrate buffer, together with a small amount of thrombin (of 0.025 units/ml) for 7-8 minutes. The reaction mixture also contains a factor XIIIa. The reaction is interrupted, adding to the reaction mixture hirudin with a final concentration of 10 Atu/ml).

Unstitched soluble fibrin polymers DesAABB get soaking in the incubator plasma containing citrate buffer or fibrinogen with thrombin solution containing add (final concentration 12 mm) for 7-8 minutes at a temperature of 37oC. the Reaction is interrupted, adding to the reaction mixture an excess of hirudin (final concentration 10 the Wilson and Nakane, Immunofluorescence and Related Staining Techniques, Knapp et al. (eds.), p. 215-224, Elsevier/North Holland Biomedical Press, Amsterdam (1978))

Peroxidase from horseradish (RZ > 3) (10 mg) dissolved in 1 ml of distilled water. Add a freshly prepared solution of 0.1 M periodate sodium (0.4 ml) and gently stirred for 20 minutes. Then the resulting solution is transferred into a buffer solution of sodium acetate (1% mm, pH 4,4) at a temperature of 4oC using microconcentrators Method 10.

Raise the pH to 9.5 by adding 40 μl of 0.2 M buffer solution of carbonate and bicarbonate (pH 9,5).

Then immediately add the antibody mh1c (20 mg) in 2.0 ml of 0.01 M buffer solution of carbonate and bicarbonate (pH 9,5). The resulting solution was gently stirred at room temperature for 2 h, and then add 0.2 ml of freshly prepared solution of sodium borohydride (4 mg/ml). Leave the mixture for 2 hours at a temperature of 4oC.

Finally, the conjugate of horseradish peroxidase with the antibody is subjected to dialysis against 5x4 litres of phosphate buffered physiological salt solution at a temperature of 2-8oC.

The conjugate was stored in a container of brown at the temperature of 2-8oC.

6.3.3. Blood samples

The blood samples received what symptoms of myocardial infarction, in particular, with severe pain in the chest. Blood samples are taken in a solution of sodium citrate with vacutainers ("Becton Dickinson). Plasma was separated by centrifugation at 2400 rpm for 15 minutes. Plasma or used immediately or stored frozen at minus 70oC.

6.3.4. The procedure of enzyme immunosorbent analysis for the determination of soluble fibrin

Soluble fibrin found in fresh or frozen plasma samples using system enzyme immunosorbent analysis of sandwich type. As the immobilized antibody in accordance with the present invention using antifibrin antibody mh1c. As an identifying (or labeled) antibody using peroxidase conjugate of horseradish with the same antibody. Alternatively, as labeled antibodies can be used conjugate of peroxidase from horseradish antifibrin antibody 45J.

Tablets for micrometrology PVC with 96 cells ("Costar" Cambridge, Massachusetts) cover a monoclonal antibody by incubating 100 μl of a solution of monoclonal antibodies mh1c (with a concentration of 50 μg/ml) used in dirigida washing tablets phosphate buffered saline, containing Tween 80. Antibody-coated mh1c cells treated with bovine serum albumin by incubation in an incubator with 200 μl of a solution of bovine serum albumin (BSA) (1%) in phosphate buffered saline for 1 h at a temperature of 37oC. Unbound BSA removed, turning the cell on a paper towel and gently tapping. Samples in citrate buffer (50 μl) containing soluble fibrin, kept in the incubator in the cells covered mh1c-BSA for 30 min at 37 toC. Unbound material is removed, turning the tablet and gently tapping. Then cells are washed three times with phosphate buffered saline solution containing Tween 80. Associated soluble fibrin identify, placing in the first cell 100 μl of conjugate solution mh1c - peroxidase from horseradish (or conjugate of peroxidase from horseradish and antifibrogenic antibody 45J) in phosphate buffered saline solution containing bovine serum albumin, and soaking in the incubator for 30 minutes at a temperature of 37oC. Unbound conjugate is removed, turning the cells and washing them three times with phosphate buffered saline solution containing Tween 80 and room temperature. The substrate solution is prepared by dissolving tablet dihydrochloride o-phenylenediamine solution of sodium citrate, hydrogen peroxide.

The colorimetric reaction is interrupted after 10 minutes by adding 25 μl of a solution of sulfuric acid (1 M). The absorption of the solution in each cell is determined on the length of 490 nm using a reader Thermomax ("Molecular Devices, Menlo Park, California).

6.3.5. Preparation of columns for carrying out affinity chromatography

Column sepharose mh1c obtained as follows.

Weigh 1 g sepharose 4B (Pharmacia"), activated dried by freezing the bromine cyan and suspended in 1 mm hydrochloric acid. The swollen gel is washed for 15 minutes in 1 mm hydrochloric acid solution on a glass funnel with filter bottom.

Antibody NH1 (20 mg) is dissolved in the buffer for binding (0.1 M sodium bicarbonate solution with a pH of 8.3, containing 0.5 M sodium chloride) and gently mixed with the swollen gel is supplied with a plug in the vessel for 2 h at room temperature or over night at a temperature of 4oC.

After mixing, the gel is washed with buffer to associate with in order to remove excess antibodies. Unlocked active space lock is the atur. The gel with immobilized antibody, washed three times with 0.1 M acetate buffer solution (pH of 4.0, containing 0.5 M sodium chloride), and then with Tris buffer (0.1 M with pH 8.0, containing 0.5 M sodium chloride). Linked antibody is stored at a temperature of 4oC in a solution of sodium azide (0.05 per cent).

Column sepharose - fibrin monomer DesAABB obtained as follows: DesAABB fibrin associated with activated bromine cyan-separate 4B in the presence of 1 M sodium bromide in 0.1 M borate buffer (pH 8,2) according to the method described previously to obtain the columns of sepharose - MN.

6.3.6. Defining properties form soluble fibrin recognized during the analysis of in vitro conditions for soluble fibrin polymers, using mh1c for the detection of soluble fibrin

It was shown (section 5.5.), the antibody mh1c does not recognize the fibrinogen contained in the plasma, the precursor of fibrin polymer. Previously it was also shown (section 5.5.), the antibody recognizes both stitched and unstitched fibrin polymer. Further, earlier in section 5.5. it was shown that the degradation of fibrin by the action of plasmin (or other process which leads to the destruction of the structure of the fibrin polymer) causes the specified antibodies loss of pic is of the collapse of cross-linked fibrin, formed under the action of plasmin.

Evidence that antibody mh1c recognizes only the polymer structure DesAABB fibrin and does not recognize the Monomeric form of DesAABB fibrin, produced by the method of affinity chromatography. Briefly, 0.5 mg of antibody mh1c is passed through a column of sepharose - fibrin monomer DesAABB. The column was washed with equilibrating buffer (physiological salt solution containing 0.1 M Tris, pH 8.5). Visible binding antibodies in the column is not observed. As shown in Fig. 1A, the antibody immediately eluted from the column. When the column elute 6 M solution of guanidine hydrochloride, then allocate a small amount of bound protein. Squirrels find spending control all factions at a wavelength of 280 nm. The antibody detects, analyzing the immunoreactivity of the protein by methods of solid-phase enzyme immunosorbent analysis using cells for micrometrology covered with fibrin. Description of the used enzyme immunosorbent analysis above in section 5.4.2. Binding of the protein in the column is the result of small amounts of impurities soluble fibrin polymer DesAABB joined the fibrin monomer DesAABB in the column. This impurity present is Elista, that column sepharose - fibrin monomer DesAABB capable of binding the antibody through the column in the control experiment, miss fibrinogen-specific monoclonal antibody. As shown in Fig. 1B, column connects the control monoclonal antibody, antifibrogenic antibody 45J, and for elution of antibody 45J you must use guanidine hydrochloride (6 M).

The proof that the above antibody mh1c recognizes both stitched and unstitched soluble fibrin polymers DesAABB, obtained from the experiment in which soluble fibrin polymers DesAABB generate by adding thrombin to the plasma samples containing citrate buffer in the presence or absence of add (add prevents linking of fibrin polymers DesAABB under the action of factor XIIIa). Both stitched and unstitched soluble fibrin polymers DesAABB receive, as described in section 6.3.1, and their relative immunoreactivity determined by the method of enzyme immunosorbent analysis, as described in section 6.3.4. As follows from Fig. 2, in this analytical system is no significant difference between the immunoreactivity stitched (B) and seamless (C) fibrin polymers.

Evidence tor the the enzyme batroxobin from snake venom from Bothrops atrox, which selectively cleaves fibrinopeptide A molecule of fibrin. Batroxobin does not break fibrinopeptide B in contrast to thrombin, which cleaves as fibrinopeptide A, and fibrinopeptide B. Remove fibrinopeptide A leads to the polymerization of fibrin monomer DesAA to the formation of a fibrin clot DesAA. In Fig. 3 depicts the formation of a clot, which is determined by the absorption of the reaction mixture at a wavelength of 340 nm, when the solution batroxobin kept in the incubator for 15 minutes at room temperature with a sample of fibrinogen. Absorption slowly increases over time, as increasing the length and concentration of soluble fibrin polymer DesAA, until, finally, does not form a clot. In the experiment in which test your ability mh1c to recognize DesAA fibrin, soluble fibrin polymer DesAA receive in vitro by incubation in an incubator sample of fibrinogen with batroxobin (with a final concentration of 0.5 units/ml) at a temperature of 37oC. From a sample treated with batroxobin, after 7 minutes of taking the sample and analyzed by the method of enzyme immunoassay in accordance with section 6.3.4. This sample (sample C) has no activity, as VI is n-DesAA fibrin, which is a source of soluble fibrin. Obviously, the reaction mixture obtained in this experiment will contain these forms of soluble fibrin", because batroxobin produces fibrin monomers DesAA who are free to interact with other fibrin monomers DesAA or not subjected to fermentation molecules of fibrinogen. Because after adding batroxobin in this analytical system response is not observed, we can conclude that the analysis does not detect these forms of fibrinogen-fibrin DesAA. Sample B, which is a positive control sample consists of fibrin polymers DesAABB formed during the processing of a sample of plasma thrombin and hirudin. As a negative control sample add sample A.

A complex of soluble fibrin may also be formed due to cross-linking of the molecules of native fibrinogen by the action of factor XIIIa with the formation of the dimer of fibrin (Kanaide et al., J. Lab. Clin. Med., 86: 574-579 (1975)). Fibrinogen treated with factor XIIIa, is not detected by the analytical system. In this experiment, the microplate coated with fibrinogen, initially treated with factor XIIIa, activetable, antibody mh1c kept in the incubator in the treated factor XIIIa cells and associated mh1c is determined using an anti-mouse alkaline phosphatase conjugate. Linked antibody anti-mouse find adding the substrate of alkaline phosphatase. The number mh1c associated in cells, determined by optical density on the length of 490 nm. In Fig. 5 the sample, denoted by Fg + FXIIIa, shows that the mh1c contacted in cells, indicating that the antibody does not recognize the structure of cross-linked fibrin. As a positive control, coated with fibrinogen cells treated with thrombin to obtain fibrin polymers DesAABB and examined the immunoreactivity of the antibody mh1c (Fig. 5, the sample is denoted by Fg + thrombin).

6.3.7. Study of the formation of soluble fibrin polymer DesAABB in vitro

Soluble fibrin polymers DesAABB receive in vitro by the addition of small amounts of thrombin (0,025 units of the American Institute of health 1 ml) to the sample plasma in citrate buffer. The sample is incubated in the incubator at a temperature of 37oC. With 1 minute interval from sample taking aliquots and activity of thrombin to inhibit the addition of hirudin, a potential inhibitor of thrombin (final Koke enzyme immunosorbent analysis, see 6.3.4.

As shown in Fig. 6, the amount of soluble fibrin polymer, which is evident by the absorption at 490 nm, increases dramatically after the initial induction period of 4-5 minutes after the addition of thrombin to plasma. The peak of soluble fibrin polymer is observed in approximately 7.5 minutes after the addition of thrombin to plasma. Gentle reduction in the content of soluble fibrin observed after 9-10 minutes, coincides with the gelation and the formation of insoluble fibrin polymers (clot formation).

6.3.8. The allocation of soluble fibrin polymers from plasma by the method of affinity chromatography on a column of sepharose - mh1c

The plasma sample is kept in the incubator with thrombin (of 0.025 units/ml) for 7 minutes at a temperature of 37oC. the Activity of thrombin suppressed by adding an excess of hirudin. The reaction mixture was passed through a column of sepharose - mh1c. Source material and run (unbound material) through associated protein (purified protein), protein elute with sodium thiocyanate (3) and subjected to dialysis against phosphate buffered saline, and analyzed for immunoreactivity using lamina plasma after the suppression of the activity of the hirudin (source material), bound protein after separation from the column and dialysis (elution) and the unbound material (unbound material) shown in Fig. 7. Source material and associated protein after dialysis has immunoreactivity in relation to the mh1c, while the unbound material it is not.

6.3.9. Detection and determination in vitro, soluble crosslinked and soluble unstitched fibrin polymers for the diagnosis of myocardial infarction

36 different plasma samples examined on the content of soluble crosslinked and soluble unstitched fibrin polymers DesAABB using enzyme immunosorbent analysis described earlier in the section 6.3.4 using mh1c as immobilized antibodies. After selection, the plasma samples are transferred into the anticoagulant and freeze. After thawing the samples examined on the content of soluble crosslinked and soluble unstitched fibrin polymers DesAABB. Ten of these 36 samples taken from healthy people. Control samples collected from volunteers from the laboratory staff, who have good health and who do not have chest pain even after physical exertion. All other samples taken from patients from emergency departments p is ohms (chest pain), assuming the presence of myocardial infarction; in 15 of them the diagnosis of myocardial infarction was confirmed; for the remaining 11 patients it was found that they do not suffer from myocardial infarction. For the latter group were diagnosed with various clinical conditions, including angina pectoris, fear, edema of the lungs. Blood samples taken from patients immediately after admission in the ICU. In most cases, was conducted registration data about the positive symptoms. In this study, all patients felt pain in the chest for 1-3 h before admission. As shown in Fig. 8, in all cases, patients with a confirmed diagnosis of myocardial infarction the content of soluble fibrin polymers DesAABB considerably higher than their content in healthy control group (3-30 times). Patients experiencing pain in the chest, in which myocardial infarction is missing, the content of soluble fibrin polymers DesAABB slightly different from the values observed for the healthy control group. (The content of soluble crosslinked and soluble unstitched fibrin polymers DesAABB shown as the Content of soluble fibrin in units of enzyme immunosorbent and the specificity is 100% (n = 36), ie, there are no erroneous positive and false negative results. Hence we can conclude that the analytical system has a high level of accuracy in the diagnosis of myocardial infarction.

6.4. Kits for detecting in vitro soluble crosslinked and soluble unstitched fibrin polymers

It should be understood that the present invention is not limited to the use of monoclonal antibodies in the analysis. However, the use of these antibodies in the composition set forth such kits contain a number of standards, the first antibody (i.e., an immobilized antibody, e.g., mh1c) and second antibody containing the label of the signal generator, as already discussed earlier. These kits contain standards in the form of a known quantity of soluble crosslinked and soluble unstitched fibrin polymers DesAABB. Such standards can be obtained by extraction of soluble crosslinked and soluble unstitched fibrin polymers DesAABB using affinity column sepharose - mh1c, as indicated previously. The kits may also include specific buffer mixture, agents for the separation and control samples. Kits can include Azania above publications and patents are cited for reference.

1. The method of determining in vitro the presence or amount of soluble cross crosslinked fibrin polymers DesAABB and transversely not crosslinked fibrin polymers DesAABB in the sample obtained from the mammal, comprising contacting a specified sample with the antibody or immunoreactive fragment that specifically binds to soluble crosslinked fibrin polymers and soluble DesAABB unstitched fibrin polymers DesAABB with the formation of the immunocomplex, the antibody or immunoreactive fragment does not bind specifically (a) fibrinogen, (b) degradation products of fibrinogen, C) with fibrin monomers DesAA d) c fibrin polymers DesAA, e) with fibrin monomers DesAABB, f) cross-cross-linked fibrinogen, g) with complex fibrin monomer DesAA - fibrin and h) with the breakdown products of fibrin and determining the presence or amount of immunocomplex.

2. The method according to p. 1, wherein the antibody specifically binds to the epitope recognized by monoclonal antibody denoted MN and produced by hybridomas ADS HB 9739.

3. The method according to p. 2, wherein the antibody is a monoclonal antibody indicated MN and produced by g the EC, taken from the man.

5. The method according to p. 4, in which the sample is taken from the man, is the fluid of the body.

6. The method according to p. 5, in which the liquid body is blood.

7. The method according to p. 1, wherein immunocomplex detect or quantitatively determined using secondary antibodies or immunoreactive fragment that specifically binds with the specified immunocomplexes, but such secondary antibody or immunoreactive fragment contains a label in the form of molecules of the reporter, which produces the recorded signal, which is measured and which correlates with the presence or amount specified immunocomplex.

8. The method according to p. 7, wherein the secondary antibody is an antibody which binds to the epitope recognized by monoclonal antibody denoted 45J and produced by hybridomas ADS HB 9740.

9. The method according to p. 8 in which the secondary antibody is a monoclonal antibody indicated 45J and produced by hybridomas ADS HB 9740.

10. Method for assessing the predisposition to thrombotic condition in a mammal by determining the amount of soluble cross sshi is om the specified mammal, includes contacting the specified sample with the antibody or immunoreactive fragment to form with them complex, with subsequent determination of the number of formed complex, the comparison of this quantity with the reference sample and evaluation on the comparison of the susceptibility of a mammal to thrombotic state, and the antibody or immunoreactive fragment does not bind specifically (a) fibrinogen, (b) degradation products of fibrinogen, C) with fibrin monomers DesAA, d) with fibrin polymers DesAA, e) with fibrin monomers DesAABB, f) cross-cross-linked fibrinogen, g) with complex fibrin monomer DesAA - fibrin and h) with the breakdown products of fibrin.

11. The method according to p. 10, wherein the antibody specifically binds to the epitope recognized by monoclonal antibody denoted MN and produced by hybridomas ADS HB 9739.

12. The method according to p. 11, wherein the antibody is a monoclonal antibody indicated MN and produced by hybridomas ADS HB 9739.

13. The method according to p. 10, wherein the thrombotic condition selected from the group consisting of myocardial infarction, deep vein thrombosis, pulmonary embolism is a result of atrial fibrillation.

14. The method according to p. 10, wherein the sample taken from the mammal is a sample taken from the man.

15. The method according to p. 14, wherein the sample taken from the man, is the fluid of the body.

16. The method according to p. 15, in which the liquid body is blood.

17. The method according to p. 10, wherein the amount of complex determined using secondary antibodies or immunoreactive fragment that specifically binds with the specified immunocomplexes, but such secondary antibody or immunoreactive fragment contains a label in the form of molecules of the reporter, which produces the recorded signal, which is measured and which correlates with the amount specified immunocomplex.

18. The method according to p. 17, wherein the secondary antibody is an antibody that specifically binds to the epitope recognized by monoclonal antibody denoted 45J and produced by hybridomas ADS HB 9740.

19. The method according to p. 18 in which the secondary antibody is a monoclonal antibody indicated 45J and produced by hybridomas ADS HB 9740.

20. Set to determine in vitro the presence or quantity of rest the BB in the sample, obtained from a mammal, comprising (a) measured in the number of standards containing these soluble cross crosslinked fibrin polymers DesAABB and these soluble cross is not crosslinked fibrin polymers DesAABB; (b) the means for the formation of immunocomplex with the specified soluble cross crosslinked fibrin polymers DesAABB and these soluble cross is not crosslinked fibrin polymers DesAABB, these funds include the antibody or immunoreactive fragment, and an antibody or immunoreactive fragment does not bind specifically (a) fibrinogen, (b) degradation products of fibrinogen, C) with fibrin monomers DesAA, d) with fibrin polymers DesAA, e) with fibrin monomers DesAABB, f) cross-cross-linked fibrinogen, g) with complex fibrin monomer DesAA - fibrin and h) with the breakdown products of fibrin; (C) means for determining the amount specified immunocomplex.

21. Set on p. 20, in which the means for formation of a complex with the specified soluble cross crosslinked fibrin polymers DesAABB and these soluble cross is not crosslinked fibrin polymers DesAABB is an antibody or its immunoreacted Chemin MN and produced by hybridomas ADS HB 9739.

22. Set on p. 21, in which the antibody is a monoclonal antibody indicated MN and produced by hybridomas ADS HB 9739.

23. Set on p. 20, in which the means for determining the quantity of the specified complex is a secondary antibody or immunoreactive fragment containing the label in the form of molecules of the reporter, which produces the recorded signal, which is measured and which correlates with the presence or amount specified immunocomplex.

24. Set on p. 23, in which the secondary antibody specifically binds to the epitope recognized by monoclonal antibody denoted 45J and produced by hybridomas ADS HB 9740.

25. Set on p. 24, in which the secondary antibody is a monoclonal antibody indicated 45J and produced by hybridomas ADS HB 9740.

26. The way to confirm the diagnosis of the presence of a thrombotic condition in a mammal by determining the amount of soluble cross crosslinked fibrin polymers and soluble DesAABB cross is not crosslinked fibrin polymers DesAABB in the sample obtained from the specified mammal, comprising contacting a specified sample with the antibody or immunologica complex, by comparing this number with the control sample and evaluation by comparing the results of the diagnosis of thrombotic condition in a specified mammal, the antibody or immunoreactive fragment does not bind specifically (a) fibrinogen, (b) degradation products of fibrinogen, C) with fibrin monomers DesAA, d) with fibrin polymers DesAA, e) with fibrin monomers DesAABB, f) cross-cross-linked fibrinogen, g) with complex fibrin monomer DesAA - fibrin and h) with the breakdown products of fibrin and confirmed by comparing the results of diagnosis of thrombotic state.

27. The method according to p. 26, wherein the sample obtained from the mammal is a sample obtained from the individual.

28. The method according to p. 26, wherein the amount of complex determined using secondary antibodies or immunoreactive fragment that specifically attaches to the specified immunocomplex, but such secondary antibody or immunoreactive fragment contains a label in the form of molecules of the reporter, which produces the recorded signal, which is measured and which correlates with the amount specified immunocomplex.

29. Method of monitoring trobot is polimerov and soluble DesAABB cross is not crosslinked fibrin polymers DesAABB in the sample, obtained from the specified mammal, comprising contacting a specified sample with the antibody or immunoreactive fragment to form with them complex, with subsequent determination of the number of formed complex, the comparison of this quantity with the reference sample and evaluation of the results of the comparison for the purpose of monitoring thrombotic condition in a specified mammal, the antibody or immunoreactive fragment does not bind specifically (a) fibrinogen, (b) degradation products of fibrinogen, C) with fibrin monomers DesAA, d) with fibrin polymers DesAA, e) with fibrin monomers DesAABB, f) cross-cross-linked fibrinogen, g) with complex fibrin monomer DesAABB - fibrin and h) with the breakdown products of fibrin and determining the results of the comparison stage thrombotic state.

30. The method according to p. 29, wherein the sample obtained from the mammal is a sample obtained from the individual.

31. The method according to p. 29, wherein the amount of complex determined using secondary antibodies or immunoreactive fragment that specifically attaches to the specified immunocomplex; but such storemy signal, which is measured and which correlates with the amount specified immunocomplex.

32. The set is suitable for determining predisposition to thrombotic condition in a mammal comprising determining in vitro the presence or amount of soluble cross crosslinked fibrin polymers and soluble DesAABB cross is not crosslinked fibrin polymers DesAABB in the sample obtained from the mammal, comprising (a) measured in the number of standards containing these soluble cross crosslinked fibrin polymers DesAABB and these soluble cross unstitched fibrin polymers DesAABB; (b) the means for the formation of immunocomplex with the specified soluble cross crosslinked fibrin polymers DesAABB and these soluble cross is not crosslinked fibrin polymers DesAABB; these funds include the antibody or immunoreactive fragment, and an antibody or immunoreactive fragment does not bind specifically (a) fibrinogen, (b) degradation products of fibrinogen, C) with fibrin monomers DesAA, d) with fibrin polymers DesAA, e) with fibrin monomers DesAABB, f) cross-cross-linked fibrinogen, g) with complex fibrin monomial is of the complex; and (d) control sample, can be used to identify predisposition to thrombotic state.

33. The set is suitable for confirmation of the diagnosis of the presence of thrombotic condition in a mammal comprising determining in vitro the presence and quantity of soluble cross crosslinked fibrin polymers and soluble DesAABB cross is not crosslinked fibrin polymers DesAABB in the sample obtained from the specified mammal, comprising (a) measured in the number of standards containing these soluble cross crosslinked fibrin polymers DesAABB and these soluble cross is not crosslinked fibrin polymers DesAABB; (b) the means for the formation of immunocomplex with the specified soluble cross crosslinked fibrin polymers DesAABB and these soluble cross is not crosslinked fibrin polymers DesAABB; these funds include the antibody or immunoreactive fragment, and an antibody or immunoreactive fragment does not bind specifically (a) fibrinogen, (b) degradation products of fibrinogen, C) with fibrin monomers DesAA, d) with fibrin polymers DesAA, e) with fibrin monomers DesAABB f) cross-cross-linked fibrinogen, g) with complexo the specified immunocomplex; and (d) control sample, suitable for confirmation of the diagnosis of the presence of thrombotic state.

34. The set is suitable for determining the stage of thrombotic condition in a mammal comprising determining in vitro the presence or amount of soluble cross crosslinked fibrin polymers and soluble DesAABB cross is not crosslinked fibrin polymers DesAABB in the sample obtained from the mammal, comprising: (a) metered standards containing these soluble cross crosslinked fibrin polymers DesAABB and these soluble cross unstitched fibrin polymers DesAABB; (b) the means for the formation of immunocomplex with the specified soluble cross crosslinked fibrin polymers DesAABB and these soluble cross unstitched fibrin polymers DesAABB; these funds include the antibody or immunoreactive fragment, and an antibody or immunoreactive fragment does not bind specifically (a) fibrinogen, (b) degradation products of fibrinogen, C) with fibrin monomers DesAA, d) with fibrin polymers DesAA, e) with fibrin monomers DesAABB, f) cross-cross-linked fibrinogen, g) with complex fibrin monomial is complex; and (d) control sample, suitable for determining the stage of thrombotic state.

 

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