RU2625031C1 - Immunoenzymometric test system for animals anaerobic enterotoxemia serum diagnostics and postvaccinal immunity stress control - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: invention relates to the immunoenzymometric test system for the anaerobic enterotoxemia serum diagnostics of agricultural animals, caused by Clostridium perfringens (Cl. perfringens) bacteria, as well as to control the postvaccinal immunity stress. The presented immunoenzyme test system contains the specific antigens of strains Cl. perfringens No.28 (type A), No.1 (type B), No.392 (type C), No.213 (type D), obtained by sounding the bacterial cells at the ultrasonic disintegrator, then precipitating the endotoxin with the ammonium sulfate and the dialysis against the piped water. The antigens are the specific protein in the concentration of 8-10 mcg/cm³, adsorbed on the surface of polystyrene wells in the carbonate-bicarbonate buffer. Also, the set contains the control positive serum, obtained for the antigens of Clostridium perfringens of types A, B, C, D with activity in ELISA 1:6400-1:12800, the control negative serum, the antispecies conjugate, the potassium phosphate monosubstituted, the potassium phosphate dibasic, the sodium chloride, the chromogen (orthophenylenediamine), the hydrogen peroxide, the stop reagent and the panels for the enzyme immunoassay reaction establishing.

EFFECT: test system allows to provide diagnostic of animals anaerobic enterotoxemia and determine the postvaccination immunity stress.

2 tbl, 5 ex

Description

The invention relates to the field of veterinary microbiology and biotechnology and relates to an enzyme-linked immunosorbent assay system for the serological diagnosis of animal anaerobic enterotoxemia and control of post-vaccination immunity.

In the structure of the incidence of farm animals, the gastrointestinal diseases of young animals are of the greatest economic importance. In the etiology of these diseases, many domestic and foreign authors note the increasing importance of the bacteria Clostridium perfringens (Cl. Perfringens) and the toxic infectious disease caused by them - anaerobic enterotoxemia.

Anaerobic enterotoxemia is an acute disease of animals of various species (sheep, calves, piglets, fur animals, birds, etc.), characterized by general toxicosis of the body with signs of damage to the nervous system and gastrointestinal tract, resulting from intensive reproduction of various types of Cl in the intestines. perfringens and absorption of the resulting toxins (Kurilenko A.N. et al., 2006; Van Kruingen H.J. et al., 2009). It affects more often young young children.

The causative agent of the disease is Cl. perfringens, which are divided into six types according to the release of toxins: A, B, C, D, E and F, which differ from each other in the antigenic structure of the toxins they produce. In lambs, anaerobic enterotoxemia is caused by the causative agent of types B, D; in calves - types A, C, D; in piglets - mainly type C, less often others (Urguyev K.R., 1987; Salimov V.A. et al., 2002; Spiridonov G.N. et al., 2010; Ceci L. et al., 2006; Gurjar A. et al., 2010).

To date, the diagnosis of infectious enterotoxemia of animals is made on the basis of epizootological, clinical, pathological and anatomical data and laboratory test results (Methodological instructions for laboratory diagnosis of animal infectious enterotoxemia and anaerobic dysentery of lambs. Laboratory studies in veterinary medicine. Bacterial infections. Reference. Edited by B. I. . Antonova, 1986).

Laboratory research is carried out in two directions:

- detection of toxin in the contents of the small intestine;

- isolation of the pathogen culture from pathological material with subsequent verification of its toxicity.

To detect toxin, the filtrate of the intestinal extract is administered intravenously or intraperitoneally to two white mice at a dose of 0.5 ml. In the presence of a toxin, animals die within 12 hours. To determine the type of major lethal toxins Cl. perfringens put a neutralization reaction on white mice or rabbits. For this purpose, 1.0 ml of the intestinal extract extract filtrate is poured into five test tubes and 1.0 ml of diagnostic antitoxic Cl sera are added. perfringens diluted with sterile saline to 10AE in 1.0 ml: type A serum in the first tube, type C in the second, type D in the third, and type E in the fourth. 1.0 ml of saline is added to the fifth tube (the control). The tubes are kept at 37 ° C for 30 minutes. The mixture from each tube is administered intravenously or intraperitoneally in a dose of 0.5 ml to two white mice or intradermally in 0.2 ml of a guinea pig or rabbit. The result of the neutralization reaction is taken into account in the death of control white mice or the formation of necrosis in control guinea pigs (rabbits).

White mice that received a mixture of toxin with homologous antitoxic serum remain alive, but in guinea pigs and rabbits necrosis does not develop.

If toxins of a certain type are detected in the intestinal contents, further work on the isolation of the culture is not carried out.

Isolation of the culture of the pathogen.

Inoculations from parenchymal organs are done on Wednesday Kitta-Tarozzi, MPB and MPA, from the contents of the small intestine - only on Wednesday Kitta-Tarozzi. Crops are incubated in a thermostat at 37-38 ° C for 20-24 hours. Special methods are used to clean the pathogen culture from concomitant microflora: the method of frequent reseeding in Kitta-Tarozzi medium; method of heating the culture in a water bath at 65 ° C for 10 minutes On Wednesday Kitta-Tarozzi Cl. perfringens gives rapid growth with a strong uniform turbidity of the broth and profuse gas. Gram-positive short thick sticks with chopped ends without spores are found in smears from cultures. On glucose-blood agar Cl. perfringens forms large, smooth, convex colonies with smooth edges, surrounded by one or two zones of strong opaque (incomplete) hemolysis. Colonies typical of the pathogen are discarded onto Kitta-Tarozzi medium. To determine the toxicity using an 8-16-hour culture grown on medium Kitta-Tarozzi. The culture is administered intravenously or intraperitoneally at a dose of 0.5 ml to white mice weighing 16-18 g.

If you suspect the presence of Cl. Type D and E perfringens are activated by adding 0.5% pancreatin or 0.25% trypsin at pH 8.0-8.2. To do this, the culture is alkalinized with a 10% sodium hydroxide solution, pancreatin or trypsin is added and kept at 37-38 ° С for 2 hours, shaking occasionally. After activation, the toxicity of type D and E crops increases significantly, and the toxicity of type C crops decreases dramatically. The toxicity of type B cultures can be maintained at the same level due to the activation of epsilon toxin.

The determination of the type of toxin is carried out in a neutralization reaction, as described above.

The diagnosis of infectious enterotoxemia of animals is considered established in the case of:

- detection of toxin in the filtrate of the contents of the small intestine by the biological method and determination of its type in the neutralization reaction with type-specific sera;

- isolation from the contents of the small intestine of a culture with properties characteristic of a given pathogen producing a toxin, the type of which is established in the neutralization reaction with type-specific sera.

The disadvantages of the laboratory method are:

- the complexity and duration of studies requiring diagnosis up to 8 working days (Laboratory studies in veterinary medicine. Bacterial infections. Handbook. Edited by B.I. Antonov, 1986);

- the high cost of the study (the high cost of nutrient media, reagents, the cost of the acquisition and maintenance of laboratory animals).

Currently, for the diagnosis of many infectious diseases, serological methods are used (RSK, RDP, RNGA, ELISA, etc.), which allow detecting sick animals by titers of specific antibodies in the blood serum, as well as determining the immunity in vaccinated animals.

An associative vaccine against anaerobic enterotoxemia and colibacillary diarrhea based on Cl antigens has been developed at the Federal State Budgetary Institution “FTsTRB-VNIVI”. perfringens of serotypes A, C, D and adhesive antigens of E. coli K99 and A20 (RF Patent No. 24282002, 09/10/2011). However, to date, the Russian Federation has not yet developed a method for determining specific antibodies to bacteria Cl. perfringens in the blood serum of animals, which makes it difficult to control the intensity of post-vaccination immunity.

The technical result to which the invention is directed is to obtain a test system with high specificity and sensitivity, which allows to simultaneously determine specific antibodies to 4 Cl serotypes. perfringens (A, B, C and D) and their tiers reliably diagnose anaerobic enterotoxemia among unvaccinated livestock and control the intensity of post-vaccination immunity in vaccinated animals.

To achieve the named technical result, an enzyme-linked immunosorbent assay system for serological diagnosis of anaerobic enterotoxemia in animals and for monitoring post-vaccination immunity tension contains specific antigens of Cl. perfringens No. 28 (type A), LD-1 (type B), No. 392 (type C), No. 213 (type D), obtained by washing them 3 times with physiological saline, mixing these bacteria in equal proportions of 5 billion m .to. in 1 cm ^{3 of} each serotype, followed by destruction of bacterial cells by ultrasound at a frequency of 20 MHz for 10-15 minutes at 4 ° C, further precipitation of endotoxin with ammonium sulfate at 40% saturation with pH 7.0 and dialysis against tap water, representing a specific protein at a concentration of 8-10 μg / cm ³ adsorbed on the surface of polystyrene wells in carbonate-bicarbonate buffer, a control positive serum obtained for Cl antigens. perfringens of serotypes A, B, C, D with an activity of 1: 6400-1: 12800, control negative serum, antispecific conjugate, monosubstituted potassium phosphate, disubstituted potassium phosphate, sodium chloride, detergent, chromogen (orthophenylenediamine), hydrogen peroxide, stop and panels for staging the reaction of enzyme immunoassay.

The proposed enzyme-linked immunosorbent assay is universal, has high specificity and sensitivity, does not require sophisticated equipment and is acceptable for analysis in bulk quantities. It can be used both for the diagnosis of anaerobic enterotoxemia of animals in stationary dysfunctional farms, and for determining the intensity of post-vaccination immunity and studying the immune status in vaccinated animals. The study time is only 4 hours, while currently existing laboratory tests - up to 8 days.

According to the “Instructions for use of the enzyme immunoassay system for the detection of specific antibodies to bacteria Cl. perfringens ”one set of the test system is designed to determine on one tablet the simultaneous analysis of 46 test samples of blood serum in one dilution and 2 control sera (in duplicate) or 10 test and 2 control samples of blood serum in eight dilutions.

The composition of the proposed ELISA test system includes:

1. Polystyrene 96-well microplate plates for enzyme-linked immunosorbent assay adsorbed by the bacterial antigen Cl. perfringens - 2 pcs.;

2. Control positive serum, lyophilized (K ⁺ ), titer in ELISA 1: 6400 - 1: 12800, volume 1 cm ³ - 1 amp., No. 1;

3. Control negative serum, lyophilized (K ^- ), volume 1 cm ³ , - 1 amp., No. 2;

4. Diagnostic antibodies against bovine IgG class immunoglobulins labeled with horseradish peroxidase (conjugate), volume 0.2 cm ³ - 1 amp. Number 3;

5. Phosphate-saline buffer solution (PBS), 20-fold concentrate, 20 cm ³ - 2 fl., No. 4;

6. Detergent (tween-80), 1.0 cm ³ - 1 bottle, No. 5;

7. Citrate-phosphate buffer solution (TSFBR), a volume of 10 cm ³ - 2 vials, No. 6;

8. Chromogen (orthophenylenediamine - OFD), 5 mg - 2 fl., No. 7;

9. Hydrogen peroxide (H ₂ O ₂ ) 3% solution, volume 10 cm ³ - 1 vial, No. 8;

10. 1 M sulfuric acid (stop reagent), volume 10 cm ³ - 2 vials, No. 9.

Example 1. Preparation of kit components.

Antigen Preparation In the inventive test system antigen Cl. perfringens is prepared as follows: get the biomass of bacteria Cl. perfringens of each serotype individually (A, B, C, D), release it from the remains of the nutrient medium by 3 times washing with saline. Suspensions of bacteria Cl. perfringens with a concentration of 20 billion m.k. in 1 cm ³ . Then a suspension of bacteria Cl. perfringens are mixed in equal proportions. It turns out a suspension of bacteria Cl. perfringens with a content of 1 cm ³ at 5 billion m. each serotype. The resulting suspension (20 billion cubic meters in 1 cm ³ ) is voiced on an ultrasonic disintegrator with a frequency of 20 MHz for 10-15 minutes at 4 ° C until the cells are completely destroyed. Then, endotoxin is precipitated with ammonium sulfate at 40% saturation, pH 7.0, which are released by dialysis against tap water.

Example 2. Preparation of control sera.

2.1. The manufacture of a control positive serum for Cl. perfringens is carried out by hyperimmunization of clinically healthy young cattle of 6-7 months of age. For this purpose, the corpuscular antigen and toxoids of the industrial bacteria strains Cl. perfringens of serotypes A (No. 28), B (LD-1), C (No. 392) and D (No. 213).

The corpuscular antigen is a formalin-inactivated bacteria Cl. perfringens containing 2.5 billion m.k. each serotype in 1 cm ³ . To obtain the toxoid bacteria Cl. perfringens is grown in a liquid nutrient medium for 7 hours, then the toxin is inactivated with formalin at a temperature of 37 ° C for 10 days. The toxoid is then purified by centrifugation and filtration of the culture medium through bacterial filters.

Hyperimmunization of animals is carried out 4 times with an interval of 14 days. In this case, the animals are administered simultaneously corpuscular antigen subcutaneously and toxoid - intravenously. To prevent anaphylactic shock, starting from the second cycle of immunization, animals are injected with 2 cm ^{3 of} antigen subcutaneously 30-40 minutes before the main dose of antigen. The hyperimmunization scheme of producing bulls is presented in table 1.

20 days after the last administration of the antigen in animals from the jugular vein, a blood sample is taken to determine the titer of specific antibodies. Production blood sampling is permitted in the presence of antibodies in the blood serum of Cl-producing bulls. perfringens in titers 1: 6400-1: 12800 in ELISA and provided that the total body temperature of the animals does not exceed 39.5 ° C, as well as after preliminary exposure of the animals to a hungry diet for 12 hours with unlimited watering.

2.2. Preparation of negative control serum.

A control negative serum is obtained from young cattle of 6-7 months of age from a clostridiosis-free farm after keeping them in quarantine for 2 months. Pre-serum from animals examined in ELISA. In clinically healthy non-immunized animals that do not contain specific antibodies to the bacteria Cl. perfringens, conduct sterile blood sampling from the jugular vein at the rate of 600 cm ^{3 of} blood per 100 kg of animal weight. Blood is incubated for 2 hours at a temperature of 20-22 ° C, then circled and defended at a temperature of 4 ° C. The settled serum is aspirated into vials under sterile conditions, packaged in 1 cm ³ ampoules and lyophilized.

Example 3. Standardization of the basic conditions for research, the claimed test system ELISA.

3.1. Determination of optimal antigen dilution.

The concentration of antigen for adsorption on a polystyrene 96-well plate is selected. In this case, the intensity of the enzyme immunoassay is evaluated at the following concentrations of antigen in solution: 2, 2.5, 5, 8, 10 μg / cm ³ . As test samples, “positive” and “negative” control samples of animal blood serum are used. The concentration of antigen sorption was selected by parallel testing of various dilutions of the conjugate. The most acceptable indicator of the titer of blood serum of animals was revealed when diluting the conjugate 1: 2500 and the concentration of sorbed antigen 5-8 μg / cm ³ . Thus, this dilution of the conjugate, at an initial concentration of sorbed antigen of 5-8 μg / cm ³ was considered optimal and used for further work.

3.2. Selection of the optimal exposure time for antigen adsorption.

For this purpose, immobilization modes were tested for 6, 18, 24 hours at a temperature of 4 ° C. The antigen adsorption process was evaluated by the intensity of the reaction with control hyperimmune positive and negative sera in serial dilutions. The criterion was to achieve the maximum OD value. The minimum optical density was observed during incubation at a temperature of 4 ° C for 18 and 24 hours. As a result of the studies, it was found that it is effective to use the antigen immobilization regime for 18 hours at a temperature of 4 ° C.

3.3. Determination of the optimal exposure time of the studied sera.

The process of formation of stable immune complexes in the ELISA test system is a complex process and requires a certain time, for the establishment of which the reaction intensity was evaluated in 3 repetitions depending on the time of incubation of specific and negative sera in serial dilutions at a temperature of 37 ° C. In this case, the conjugate was used in a working dilution of 1: 2500. It was found that the optical density of the studied sera in the range from 15 to 60 minutes increased, and then stabilized. The observed effect was recorded for sera with different levels of specific activity. On this basis, a 60-minute exposure to sera was considered sufficient to achieve a maximum and stable response.

3.4. Determination of the positive-negative threshold of the ELISA test system.

To take into account and interpret the results obtained by the ELISA test system, a positive-negative threshold of the test system was determined, which is in the range of <15% -> 22%. In the future, all samples with a Ksv value less than the positive-negative threshold were considered negative, and samples with a Ksv value equal to or higher than this indicator were considered positive.

Example 4. Illustration of the application of the ELISA test system.

4.1. Preparation of biological material.

For analysis, blood serum of cattle is used. If the analysis is carried out within 24 hours after selection, samples of the biomaterial are stored at a temperature of 4 ° C. During longer storage, the samples are frozen at a temperature of minus 20 ° C. Before the study, frozen samples are quickly (for 5-10 minutes) heated in a water bath at a temperature of 37 ° C. In case of precipitation, the samples must be clarified by centrifugation for 10 min at 2000 g. Repeated freezing and thawing of samples is not allowed.

4.2. Preparation of working solutions and reagents.

Before work, all reagents can withstand 30 minutes at room temperature.

4.2.1. Enzyme-linked immunosorbent assay plate with Cl. perfringens is ready to eat.

4.2.2. The working solution for diluting the conjugate is 0.01 M phosphate-buffered saline (PBS). The contents of the bottle No. 4 was adjusted with freshly prepared distilled water to a volume of 400 cm ³ . It is allowed to store no more than 1 month at a temperature of 4 ± 2 ° C.

4.2.3. Working solution for washing tablets (PBS / t). To 500 cm ³ phosphate-buffered saline (PBS) add 0.25 cm ³ tween-80 (bottle No. 5) and mix until completely dissolved.

4.2.4. Serum of cattle positive (No. 1) and negative (No. 2) are dissolved with distilled water to 1 cm ³ .

4.2.5. The contents of the ampoule with the conjugate (No. 3) are dissolved in phosphate-buffered saline that does not contain tween-80 (PBS) and working dilution.

4.2.6. The contents of the ampoule with hydrogen peroxide (No. 8) are dissolved in distilled water in the volume indicated on the label of the ampoule.

4.2.7. The substrate-indicator mixture is prepared immediately before use in the reaction as follows: the contents of bottle No. 7 (chromogen) are dissolved in 10 cm ^{3 of} citrate-phosphate buffer solution (bottle No. 6) and 0.17 cm ^{3 of} hydrogen peroxide is added.

4.2.8. Stop reagent - ready for use.

4.3. Analysis.

4.3.1. In all wells of the plate with sorbed antigens, 0.1 cm ³ of PBS / t buffer solution is added. Control (negative and positive) and test sera are diluted 40 times with FSBR / t buffer solution and added to the horizontal wells of the plate in a volume of 0.1 cm ³ . At the same time, control negative serum is added to well A1, control positive to well A2 and test sera to wells A3 to A12 and double dilutions from 1:80 to 1: 10240 are prepared. The tablet is closed with a lid and incubated at 37 ± 0.5 ° C for 1 h.

4.3.2. Washing the tablet. The tablet is washed 5 times with PBS / t buffer solution (filling the wells to the top), in each case completely removing the liquid by tapping the inverted tablet on filter paper.

4.3.3. The introduction of immunoperoxidase conjugate. 0.1 cm ³ immunoperoxidase conjugate in working dilution was added to each well. Incubated for 1 h at 37 ± 0.5 ° C.

4.3.4. Washing the tablet.

4.3.5. The introduction of a substrate-indicator mixture. 0.1 cm ^{3 of} substrate-indicator mixture is added to all wells of the plate. The tablet is covered with a lid and incubated in the dark for 20 minutes at room temperature.

4.3.6. Stop the reaction. Without draining the contents, 0.05 cm ³ stop reagent (1 M sulfuric acid solution) is added to all wells of the plate.

4.3.7. Evaluation and interpretation of reaction results.

ELISA results are taken into account after stopping the reaction according to the readings of the optical density of the substrate-indicator mixture on a spectrophotometer at a wavelength of 492 nm (OD ₄₉₂ ). The reaction is considered positive if the optical density of the substrate-indicator mixture in the wells in which the positive specific serum and the test sera were previously incubated is 2 or more times the optical density in the wells in which the negative (control) serum was previously incubated.

Example 5. Testing the ELISA test system in a production environment.

In a production test of the effectiveness of the kit, blood samples of cattle were used, which were delivered from cattle-breeding farms of the Republic of Tatarstan that were epizootically different in clostridioses. At the same time, blood serum was studied from sick, unvaccinated and healthy, vaccinated animals from clostridiosis-poor households, as well as from intact animals from successful farms. The results of the study of blood serum are presented in table 2. The table shows that the components of the kit have a high specificity in ELISA. They allow the determination of specific antibodies to bacteria Cl. perfringens in 98.1% of healthy vaccinated animals and 93.0% of animals with anaerobic enterotoxemia.

At the same time, it was found that the components of the kit do not react with blood sera obtained from intact animals from farms that are safe for anaerobic enterotoxemia.

Thus, the proposed test system allows titers of specific antibodies in the blood serum to identify sick animals among unvaccinated livestock, as well as to determine the intensity of immunity in vaccinated animals.

Information sources

1. Kurilenko A.N. Bacterial and viral diseases of young farm animals / A.N. Kurylenko, V.L. Krupelnik, N.V. Pimenov. - M .: KolosS, 2006 .-- 296 p.

2. Guidelines for laboratory diagnosis of infectious enterotoxemia in animals and anaerobic dysentery of lambs. Laboratory research in veterinary medicine. Bacterial infections. Directory. / Ed. B.I. Antonova, 1986.

3. RF patent. Vaccine associated against anaerobic enterotoxemia and escherichia diarrhea of calves / G.N. Spiridonov, A.A. Ivanov, H.N. Macaev, M.T. Khuramshina, A.G. Spiridonov, E.R. Galiullina; Applicant and patent holder of the Federal Center for Toxicological, Radiation and Biological Safety. - No. 2428202, publ. 09/10/2011, Bull. Number 25.

4. Salimov V.A. Some features of the pathological diagnosis of anaerobic enterotoxemia of calves caused by Cl. perfringens type A / B.A. Salimov, N.P. Salimova // Actual problems of diseases of young animals in modern conditions: mat. scientific - prakt. conf. - Voronezh, 2002 .-- S. 527-528.

5. Spiridonov G.N. Infectious enterotoxemia of young farm animals in the Middle Volga and Ural regions. / G.N. Spiridonov, A.F. Makhmutov, M.T. Khuramshina, A.G. Spiridonov // Actual problems of veterinary medicine of Siberia: mat. scientific - prakt. conf. - Krasnoobsk, 2010 .-- S. 134-139.

6. Urguyev K.R. Clostridiosis of animals / K.R. Urguyev. - M .: Rosselkhozizdat, 1987.- 183 p.

7. Ceci L. Haemorrhagic bowel syndrome in dairy cattle: possible role of Clostridium perfringens type A in the disease complex / L. Ceci, P. Paradies, M. Sasanelly, D. de Caprariis, F. Guarda // J. Vet. Med. - 2006 .-- 53 - P. 518-523.

8. Gurjar A. Characterization of Toxin Plasmids in Clostridium perfringens type C isolates / A. Gurjar, J. Li, B.A. McClane // Infection and Immunity. - 2010 .-- 78 - 11 - P. 4860-4869.

9. Van Kruiningen H.J. Clostridial abomasal disease in Connecticut dairy calves / H.J. Van Kruiningen, C.A. Nyaoke, I.F. Sidor, J.J. Fabis, L.S. Hinckley, K.A. Lindell // CVJ. - 2009. - 50 - P. 857-860.

Claims (1)

Enzyme-linked immunosorbent assay system for the serological diagnosis of animal anaerobic enterotoxemia and control of post-vaccination immunity, characterized in that it contains specific antigens of Clostridium perfringens strains No. 28 (type A), LD-1 (type B), No. 392 (type C), No. 213 (type D) obtained by washing them 3 times with physiological saline by mixing these bacteria in equal proportions of 5 billion c.c. in 1 cm ^{3 of} each serotype, followed by destruction of bacterial cells by ultrasound at a frequency of 20 MHz for 10-15 minutes at 4 ° C, further precipitation of endotoxin with ammonium sulfate at 40% saturation with pH 7.0 and dialysis against tap water, representing a specific protein at a concentration of 8-10 μg / cm ³ adsorbed on the surface of polystyrene wells in carbonate-bicarbonate buffer, a control positive serum obtained for Cl antigens. perfringens of serotypes A, B, C, D with an activity of 1: 6400-1: 12800, control negative serum, antispecific conjugate, monosubstituted potassium phosphate, disubstituted potassium phosphate, sodium chloride, detergent, chromogen (orthophenylenediamine), hydrogen peroxide, stop and panels for staging the reaction of enzyme immunoassay.