RU2461008C2 - Elisa test system for serum diagnosis of cattle parvoviral infection and level detection of vaccine-induced antibodies - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: test system contains: specific antigen of type I cattle parvovirus from the strain Parvo 32459-DEP in the concentration of 3-5 mcg/cm³ adsorbed on the well surface of polystyrene plates; parvovirus (positive and negative) reference serums diluted 1:400 - 1:1600; anti-species conjugate; potassium dihydrogen phosphate; dipotassium phosphate; sodium chloride; chromogen (orthophenylenediamine); hydrogen peroxide; stop solution and a panel for ELISA reaction.

EFFECT: test system enables detecting antibodies in sampled blood serums once diluted, evaluating antibodies to type one cattle parvovirus in the infected, recovered and vaccinated animals in four dilutions of blood serum.

6 tbl, 1 dwg

Description

The invention relates to veterinary virology and biotechnology, in particular the creation of an ELISA test system for establishing the immune status of livestock in one dilution and quantifying antibodies in four dilutions of blood serum to cattle parvovirus of the first serotype in patients who have been ill and vaccinated animals.

Currently, scientists from around the world, based on the results of extensive studies, have created a complete picture of the pathogenic potentials of numerous viruses secreted from sick animals and their significance in the etiopathogenesis of respiratory and intestinal diseases of newborn animals and the pathology of reproductive organs of adult livestock associated with early embryonic mortality abortion and stillbirth. In this regard, among them, along with herpesvirus type I (RTI), viral diarrhea virus - diseases of the mucous membranes, respiratory syncytial virus, adenoviruses of the 1st and 2nd subgroups and reoviruses, bovine parvoviruses poorly studied in the Russian Federation that are capable of selectively replicate in dividing cells, such as intestinal cells and embryonic cells.

The etiological agent of parvovirus infection in cattle is a small, DNA-containing virus belonging to the genus Bocavirus of the family Parvoviridae, causing a disease with a variety of clinical manifestations, causing significant economic damage to livestock in many countries.

The first isolation of parvovirus was reported in 1959 by F.R.Abinanti and M.S. Warfield, who isolated it from the feces of sick calves with the symptom complex of diarrhea and named it because of their hemadsorbing properties, as HADEN-virus (HemADsorbingENterovirus). Since then, this strain of the virus has been considered a reference for parvoviruses of cattle of the first serotype.

Parvoviruses isolated from cattle in the USA, Europe and North America did not differ antigenically, turned out to be serologically related to the prototype strain “Haden” and were classified as type I cattle parvovirus.

Strain C-134, isolated in Japan, was serologically different from the Haden virus and was assigned to the 2nd type of cattle parvovirus (Inaba et al., 1973).

In our country, bovine parvovirus (strain B-2) was first isolated in 1972 from the nasal mucus of a sick calf with respiratory syndrome, which differed from the HADEN reference strain in antigenic structure and a number of biological properties. In particular, this virus did not have hemagglutinating activity in the native state. Therefore, it was assigned to the third type of cattle parvovirus (Zudilina Z.F., Mikhailov P.N., Zhidkov S.A., 1976; Kryukov N.N., Tretyakova I.A., Zudilina Z.F. and et al., 1988, B. Orlyankin et al., 1985, Storz I. et al., 1973).

Parvovirus infection manifests itself in young cattle, mainly in the form of damage to the digestive system: diarrhea develops 24-48 hours after infection (Spahn, GJ, et al., Characteristics of hemadsorbing enteric (HADEN) virus / Canad. J. Microbiol. - 1966. - V.12. - P.653-660 .; Durham PJK et al., Epidemiological studies of parvovirus infection in calves on endeinically infected properties / J. Res. Vet. Sci. - 1985. - V.38. - No. 2. - P.234-240; Sandals, WCD Prevalence and seroepidemiology of bovine parvovirus / MSc Thesis, University of Guelph, 1989. - P.432-437).

Due to the fact that parvoviruses multiply in actively dividing cells, the digestive tract plays a major role in the reproduction of the virus due to the constant renewal of mucosal cells. There is evidence in the literature that, in calves, a parvovirus infection may be accompanied first by a respiratory syndrome and then by an upset digestive tract (Yurov K.P. et al. 1994).

It is also known that parvoviruses are able to penetrate the placental barrier and can cause serious problems in the pathology of reproductive function in cows, in which abortion and embryonic mortality are observed (Yurov K.P. et al. 1994; Bachman R.A., 1971; Storz J . et. al., 1978; Bodine et. al., 1981; Athayde de Costa, Dreonde Albuquerque, 1986).

These viruses are stable in the environment, and can be stored indoors for 4-6 months. The leading mechanism of transmission of parvovirus infection is fecal-oral and aerogenic. However, a possible transplacental transmission of infection cannot be ruled out.

According to foreign researchers, laboratory diagnosis of parvovirus infection in cattle is carried out by isolating the virus in a cell culture, detecting the viral antigen directly in samples of pathological material (intestines, feces, aborted fetus) by immunofluorescence in sections or imprints of the intestinal mucosa. The results should be confirmed by the detection of an increase in virus-neutralizing antibodies or antihemagglutinins during the convalescence period compared with blood sera taken in the acute stage of the disease (Tijssen R., 1990; David Sandals W.C. et. Al., 1994 and others).

However, to date, in the Russian Federation, it seems to us that the features of the clinical manifestation and spread of parvovirus infection in cattle have not been sufficiently disclosed, the etiological role of parvovirus in respiratory and intestinal diseases of young animals and impaired reproductive organs of adult livestock has been underestimated for many years. Issues such as the epizootology of the disease, the features of post-infectious immunity, the tasks associated with the development of methods and means of laboratory diagnostics and specific prophylaxis remained without proper attention.

In view of this situation, in order to identify the prevalence of parvovirus infection among cattle in the Middle Volga region, in 2006-2009, we carried out seromonitoring for this infection by examining 1117 animal serum samples from the hemagglutination inhibition test (RTGA) from 35 farms of the Republic of Tatarstan, Bashkortostan, the Chuvash Republic and the Nizhny Novgorod Region. The work was carried out according to the “Instructions for the use of a set of drugs for serodiagnosis of parvovirus infection in cattle in the inhibition of hemagglutination inhibition (RTGA)”, approved by the management of the Federal State Institution “FTsTRB-VNIVI”. In this case, the parvovirus strain “Parvo 32459” belonging to the first serotype was used as antigen. The research results are shown in table 1.

Table 1 The results of serological studies of cattle in several regions of the Middle Volga Region Number of households Number of samples Of which are seropositive RTGA Qty % Republic of Tatarstan 24 901 229 25,4 Chuvash Republic 2 87 21 24 Republic of Bashkortostan four 80 8 10 Nizhny Novgorod Region 5 49 16 32.6 Total 35 1117 403 36.1

From the data of table 1 it follows that from 1117 samples of blood serum of cattle specific antibodies to parvovirus of cattle are detected in rtga in 403 (36.1%) animals. Moreover, antibody titers reached significant values: 1: 160-1: 1280. Therefore, a significant number of animals were found to be seropositive to the bovine parvovirus antigen. The results indicate the circulation of this pathogen among the examined livestock.

Considering the polyetiologicity of ubiquitously recorded respiratory-intestinal viral infections of calves and the pathologies of the reproductive organs of adult livestock caused by them, applicants for the grant of this patent of the Russian Federation created the “Associated inactivated emulsion vaccine against parvovirus, reovirus, herpesvirus type I infections and viral diarrhea - mucous membranes of large horn "With high antigenic and immunogenic activity.

At present, the veterinary service of the Russian Federation does not have modern methods and laboratory diagnostics, including kits for serological monitoring of infectious diseases such as parvovirus and reovirus infections in cattle that are poorly studied in the Russian Federation, which can cause various forms of pathology in this animal species and most often in the form of mixed infections.

Currently, the ELISA method is the main test for the determination of antibodies in the diagnosis of many viral infections, not only to establish the immune status, but also to quantify the specific antibodies contained in the blood serum of animals.

Domestic researchers (Yurov K.P., Shcherbakova G.B. 1993; Yurov K.P. et al. 1994, Yurov K.P., Tretyakova I.A., Vecherkina A.S. - Parvovirus infection of cattle. / Veterinary medicine, 1994, No. 5, pp. 26-27) research was carried out on the creation of enzyme-linked immunosorbent assay systems designed to indicate antigen and detect antibodies to bovine parvovirus of the 3rd serotype. However, to date, not a single enzyme immunoassay system has acquired its completed form and has not been introduced into veterinary practice.

Also known is the ELISA test system (an abstract for the academic degree of candidate of biological sciences Shcherbakova TB "Immunoassay diagnosis of parvovirus infection in cattle" // Moscow, 1993. - p.21 "). However, in the indicated scientific work, which is closest to the proposed one, only the principal possibility of determining the antigen of parvovirus of cattle of the 3rd type (strain B-2) in the excretions of experimentally infected calves is presented using the sandwich ELISA method. In this abstract, the antigen for ELISA is made from a strain of the virus "B-2", referred to the third serotype. The authors performed the serological identification of the isolate using monospecific sheep sera obtained for the Haden and B-2 strains in the HH and RTGA reactions. As shown by the results of cross-reactions, that these strains in the tests used do not have antigenic affinity (Kryukov N.N., Zudilina et al., 1988).

Therefore, it can be assumed that the obtained antigen from strain B-2 has lower specificity and activity against parvovirus infection, caused mainly by the I-serotype virus, as evidenced by the high seropositivity of the livestock in the Middle Volga region, in which over the past 5 -6 years, more than 30 thousand heads of pedigree highly productive cattle were imported from countries of Western Europe, North America and Australia.

In addition, bovine parvovirus isolates registered in most countries, except those isolated in the former USSR and Japan, are serologically identical to the prototype strain Haden and belong to the first type of parvovirus (K. Yurov et al., 1994. - Parvovirus infection of cattle / Veterinary medicine, No. 5, 1994, pp. 26-27).

One of the characteristic properties of bovine parvovirus is its ability to agglutinate red blood cells. The difference in hemagglutinating activity of parvovirus strains can serve as a genetic sign and taken into account when identifying them. For example, the HADEN virus, according to Abinanti, Warned, agglutinates red blood cells of guinea pigs and humans. Strain Andrewes No. 32459, isolated from the feces of a fallen 3-month-old calf with signs of enteritis, was identical to the HADEN virus in the neutralization reaction and in the RTGA (Kryukov N.N. et al., 1988).

In all foreign countries, retrospective diagnosis of parvovirus infection in cattle is carried out in rtga, preferably using the antigen of the reference strain of parvovirus type I

Most isolated strains are serologically related to the HADEN strain. They belong to the parvovirus of cattle of the 1st type (Athayde de Costa, Dreon de Albuquerque - Rev. Cent. Cienc. Rurais, 1986, 3; Bates RC, Storz J., Reed - J. infect. Dis., 1972, 126; Dirham Johnson - Veter. Microbiol., 1985, 10; Huck, Woods, Orr - Vet. Rec., 1975, 96; Inaba et al. - Jap. J. Microbiol., 1973, 17; Inaba et al - Arch . ges. Virusforsch., 1973, 42; Inaba et al. - Jap. J. Vet. Sci., 1971, 33; Spahn, Mohanty, Hetrick - Cnad. J. Microbiol., 1966, 12).

Therefore, the current task is to create an enzyme-linked immunosorbent assay system designed for serological diagnosis of parvovirus infection in cattle based on the type I parvovirus antigen, its testing in laboratory and industrial conditions in comparison with similar methods.

This goal is achieved in that it contains a specific antigen of parvovirus cattle type I from the strain "Parvo 32459-DEPT", which is a polypeptide complex at a concentration of 3-5 μg / ml adsorbed on the surface of polystyrene wells, parvovirus (positive) and normal ( negative) control serum at a dilution of 1: 400-1: 1600 in incubation buffer, antispecific conjugate, monosubstituted potassium phosphate, disubstituted potassium phosphate, sodium chloride, chromogen (orthophenylenediamine), cross there is hydrogen, a stop reagent and panels for staging the enzyme immunoassay. In addition, the detection of antibodies is carried out in one dilution, and the determination of the level of antibodies in four dilutions, while the criterion for differentiation of positive and negative results is the positive-negative threshold, which is in the range of <15-≥25%.

The composition of the proposed ELISA test system includes:

1. Tablets for enzyme immunoassay with the adsorbed antigen of parvovirus type I cattle - 5 pcs.

2. Control positive serum, (K ⁺ ), 0.5 cm ³ - 2 amp. No. 1.

3. Control negative serum, (K ^- ), 1 cm ³ - 2 fl. No. 2.

4. 10-fold concentrate of phosphate-buffered solution with Tween-20 for washing tablets (FBI-T), 100 cm ³ - 2 fl. Number 3.

5. The buffer for dilution of the conjugate (DBK), 10 cm ³ - 1 fl. Number 4.

6. Diagnostic antibodies against immunoglobulins G (IgG) of cattle labeled with horseradish peroxidase (conjugate), 0.2 cm ³ - 1 amp. No. 5.

7. Phosphate-citrate buffer (FCB), 10 cm ³ - 5 fl. No. 6.

8. Chromogen, orthophenylenediamine (CRF), 4 mg - 5 fl. Number 7.

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

10. 0.1 M Sulfuric acid (Stop reagent), 50 cm ³ - 1 vial. No. 9.

The technical result from the use of the present invention is to expand the arsenal of modern methods and tools used to diagnose viral infections and increase the specificity of the claimed ELISA test system, designed to detect post-infectious and post-vaccination antibodies of animals due to the use of a specific sorbing viral antigen obtained from strain " Parvo 32459-DEP "parvovirus of the first type.

1. The strain "Parvo 32459-DEL" parvovirus cattle was obtained in 1979 by Professor Gaffarov Kh.Z. from the CVL of the UK Ministry of Agriculture and accepted by the applicant as a production one, is identical in RVI and RTGA with the reference strain “Haden” type I (Kryukov N.N. et al., 1988), isolated in 1959 in the USA from a fallen calf with signs of enteritis (Abinanti FR, Warfield MS, 1961), has high biological, antigenic and immunogenic activity, retains its native immunobiological properties after inactivation, in its native form and after inactivation, is highly productive in sensitive biological cultivation systems, recognized as fit for the production of highly sensitive and specific diagnostic tools.

Cattle parvovirus is a DNA virus. Virions are non-shell particles of cubic symmetry with a diameter of 20-23 nm. The “Parvo 32459-DEP” strain of cattle parvovirus type I is resistant to changes in pH from 3 to 8, ether, chloroform and 1% trypsin, and heating at 70 ° C for 2 hours reduces the infectious activity by 100 times , thermostable at 37 and 56 ° C, agglutinates erythrocytes of guinea pig and human 0-group. In vivo, cattle are susceptible to the virus. Parvovirus is pathogenic for calves, it penetrates the tissues of various parts of the intestine and adjacent lymph nodes, and is also able to cross the placental barrier, causing death of the embryo, causing abortions in cows, and for a long time to persist in the cow's body.

An effective reproductive system of the Parvo 32459-DEPT strain of parvovirus type I is a primary trypsinized culture of cow embryonic kidney cells (PEC). Parvovirus is active in hemagglutination reactions (RGA), inhibition of hemagglutination inhibition (RTGA), in the neutralization reaction (PH), and enzyme-linked immunosorbent assay (ELISA). The virus titer in RGA with 1% guinea pig erythrocytes is 1: 512-1: 1024, in PH 10 ^4.5 -10 ^-5.5 TCD _{50 / ml} .

The strain "Parvo 32459-DEP" of parvovirus type I cattle was deposited in the laboratory of the Museum of Microorganism Strains of Especially Dangerous Diseases (SAR) of Federal State Institution FTSTRB-VNIVI on December 9, 2009, registration number 2. The storage location of the strain was determined to be the Laboratory of the Museum of Microorganism Strains of SOB Federal State Institution “FTsTRB-VNIVI”, 420075, Kazan, Scientific Town-2.

In the manufacture of an ELISA test system designed for serological diagnosis of parvovirus infection in cattle and the quantitative determination of antibodies, the “Parvo 32459-DEPT” strain I of cattle parvovirus is used in the blood sera of infected, ill and vaccinated animals, it is reproduced in the primary trypsinized culture of cow embryonic kidney cells (PEK) before virus accumulation in titer 1: 1024 GAE, the cell mass is precipitated by centrifugation, destroyed by ultrasound, followed by centrifugation by carbonation to relieve cell debris, inactivate with a 0.1% solution of 1,2-aminoethylaziridine. Subsequent purification of the antigen is carried out by ultracentrifugation through 20% sucrose. To perform indirect solid-phase ELISA, the purified parvovirus antigen of the first serotype Parvo 32459-DEPT is immobilized on the surface of the polystyrene plate wells and diluted samples of the tested blood sera and control positive and negative sera are introduced into the wells, incubated for 2 hours at 37 ° C, after which is added peroxidase antiviral conjugate at a dilution of 1: 5000, which is also incubated at 37 ° C for 1 hour. At the last stage of the reaction, a substrate solution containing orthophenyldiamine (RPD) and 3% hydrogen peroxide is added. The reaction is read after 5-15 minutes on a Bio-Rad Model 680 spectrophotometer at a wavelength of 490 nm.

Diagnostic parameters and quality indicators of the developed ELISA test system for serological diagnosis of parvovirus infection in cattle meet the requirements of the OIE standards (Guidelines for the diagnosis and production of vaccines, OIE publication, 2004, pp.21-29).

In the process of creating a diagnostic test system, the conditions for conducting ELISA in one dilution (1: 400) were optimized to establish the immune status of the livestock and quantify antibodies in four dilutions (1: 800, 1: 1600, 1: 3200, 1: 6400) of sera blood to the parvovirus of cattle of the first serotype of infected, ill and vaccinated animals.

In the study of blood serum in one dilution, a criterion for the differentiation of positive and negative ELISA results by calculating the binding coefficient (Ksv.): If the value of Ksv. less than 15%, the sample is considered negative if the value of Ksv. more than 25%, the sample is considered positive.

In order to obtain reliable results, acceptable values of the optical density of control sera were established: in the range from 0.5 to 0.709 for a positive control and from 0.05 to 0.08 for a negative control.

The main indicators of the quality of the enzyme immunoassay system are sensitivity and specificity. These parameters are determined based on the correlation of the results of studies of blood serum in rtga and RVN. The diagnostic sensitivity of the test system with respect to RVN is 96.07%, the diagnostic specificity is 93.82%, and the coincidence of the results is 95.1%.

In the study of blood serum in ELISA and rtga, obtained from obviously negative and positive animals, the coincidence of the results is 100%.

One set of test system is designed for simultaneous analysis of 46 test blood serum samples in one dilution (in duplicate) and 2 control sera (in duplicate) or 20 test blood serum samples (in four dilutions) and 2 control sera on one tablet (in four dilutions). One test system is designed to study 230 samples of blood serum in one dilution and 100 samples in four dilutions.

The set is stored at a temperature of 2 to 8 ° C and a relative humidity of not more than 80%. Freezing of kit reagents is not allowed.

The shelf life of the kit is 12 months from the date of manufacture.

In the inventive ELISA test system designed to detect and quantify antibodies to cattle parvovirus, a specific antigen is prepared using the Parvo 32459-DEP strain of cattle parvovirus adapted to a primary trypsinized cell culture of a cow embryo kidney (PEC) .

For infection with parvovirus, mattresses with a complete monolayer of PEC cells are selected, growth medium is removed, 5 cm ^{3 of} viral material is introduced into each mattress. Incubate in an incubator at 37 ° C for one hour. After that, without removing the viral material, a supporting medium is introduced in a volume of 150-200 cm and the culture is placed in a thermostat at 37 ° C. The characteristic cytopathogenic effect of parvovirus on PEC cell culture (granularity, rounding and cell destruction) is controlled by microscopy of mats within 72-96 hours after infection, frozen three times at minus 20 ° C, then 5 cm ³ samples are taken from each mattress for inoculation on sterility and titer determination in RGA.

Concentration is carried out by sedimentation of the cell mass by centrifugation (3000 rpm for 15 min). Infected cells are destroyed on a Bandelin GM 3100 ultrasonic disintegrator at a frequency of 20 kHz ± 500 Hz impulsively 6 times for 30 seconds, followed by centrifugation (5000 rpm for 30 minutes) to free them from ballast proteins. Inactivate the virus with a 0.1% solution of 1,2-aminoethylaziridine. Subsequent purification of the antigen isolated from the cells containing the virus is carried out by ultracentrifugation at 30,000 g on a “pad” of 20% sucrose. The pellet was resuspended in carbonate-bicarbonate buffer (pH 9.6–9.8) and used as parvovirus antigen to sensitize the polystyrene plate wells. The degree of purification and concentration of the antigen is estimated by the hemagglutinating activity determined in the RGA, and by the optical density of the viral antigen protein (Table 2) on a LOMO SF-46 spectrophotometer at wavelengths of 235 and 280 nm, adjusted for dilution in a cuvette according to the following formula :

According to the formula:

where - OP ₂₃₅ ; OP ₂₈₀ - optical density at a wavelength of 235 and 280 nm, respectively;

- 30 - dilution of the test sample in the cuvette (1:30).

table 2 The activity of cattle parvovirus concentrated by ultracentrifugation on a “pillow” of 20% sucrose (n = 3; P <0.05) No. p / p Test material Activity in the RSA, 1 / n Protein concentration, mg / ml one Raw viral feed 2048 1.2 ± 0.03 2 After sonication 8192 1.1 ± 0.02 3 After inactivation 2048 1.0 ± 0.02 four After concentration 16384 0.9 ± 0.03

According to the results of chess titration of antigen and control sera, the optimal working concentration of parvovirus antigen is established in ELISA, which in this case is 3-5 μg / ml.

2. The production of a control positive type-specific serum against Parvo 32459-DEPT strain of parvovirus type I is carried out by hyperimmunization of clinical healthy young cattle of 6-8 months of age, concentrated, purified and inactivated virus suspension with hemagglutinating activity of at least 1: 1280- 1: 2560.

Before immunization of animals, the blood and serum are taken from the jugular vein and checked for the presence of antihemagglutinins for cattle parvovirus. In the absence of specific antibodies in them, animals are considered suitable for hyperimmunization.

To obtain immune sera, a viral antigen is deposited in Freund's incomplete adjuvant, which is prepared by thoroughly mixing 15 cm ^{3 of} liquid paraffin and 5 ml of dehydrated lanolin. The resulting mixture is subjected to fractional sterilization in a water bath for 3 consecutive days for 60 minutes. A mixture of antigen with adjuvant (in equal volume) is heated in a water bath to 37 ° C, mix thoroughly and injected subcutaneously in a dose of 2 cm ³ . Subsequently, hyperimmunization of animals is carried out by triple intravenous administration of a culture virus suspension, purified from cells, in increasing doses of 10 cm ³ , 20 cm ³ and 30 cm ^3, respectively, with an interval between administrations of 14-16 days.

In order to prevent an anaphylactic reaction, starting from the second cycle of immunization, the animal is injected with 2 cm ^{3 of the} virus subcutaneously 40 minutes before the main dose of the virus.

14-16 days after the last virus introduction, blood samples are taken from the jugular vein in animals, serum is obtained and a hemagglutination inhibition test is performed.

Production bleeding is allowed in the presence of antibodies in the blood serum of a producer to cattle parvovirus not lower than 1: 1280 in RTGA or 1: 256 in pH. The first blood collection from the producers in volume should not exceed 0.6 liters of blood per 100 kg of animal weight, and in the subsequent - 1.6 liters per 100 kg of weight.

Blood from the jugular vein into sterile cylinders moistened with physiological saline is placed in a thermal room for 30-40 minutes at a temperature of + 37-38 ° C, after which the serum is circled and the cylinders are left for 18 hours at a temperature of + 4-6 ° C to settle serum. Then the serum is taken, merthiolate is added in a ratio of 1: 10000, the titer of antibodies in rtga, PBH and ELISA with a homologous antigen is determined.

Serum with an antihemagglutinating titer of at least 1: 1280 is packaged in 0.5 ml ampoules and lyophilized.

Control (negative) sera are obtained from the blood of clinically healthy non-immunized animals that do not contain specific antibodies to parovovirus of the first serotype when tested in RTGA and ELISA, they are poured into 0.5 ml ampoules and lyophilized.

3. Buffer preparation

Adsorption 0.01 M carbonate-bicarbonate buffer solution (KBB pH 9.6) is intended for dissolution and sorption of antigen in the well of tablets: 10.6 g of Na ₂ CO _{3 are} dissolved in 500 cm ³ dist. water, 8.4 g of NaHCO _{3 are} dissolved in 500 cm ³ dist. water, the resulting solutions are mixed with 16 cm ³ 0.2 M Na ₂ CO ₃ and 34 cm ³ 0.2 M NaHCO ₃ and get a 0.2 M KBB mother liquor. 5 cm ³ are taken from the mother liquor and another 95 cm ³ dist. water. This solution is used in the reaction (0.01 M KBB pH 9.6).

Phosphate-buffered solution with tween is intended for the dilution of control, test sera and antiviral conjugate, as well as for panel washing (PBS / t pH 7.2-7.4): 10 g of Na ₂ HPO ₄ × 12 H ₂ O and 1 are mixed g KH ₂ PO ₄ in 1 liter dist. water. Then, to 900 cm ^{3 of a} 0.85% NaCl solution, add 180 cm ^{3 of the} main FBI solution, dissolve 0.5 g of Tween-20.

Phosphate-citrate buffer solution pH 4.9 (FCB): to 12.15 cm ³ 0.01 M solution lim. acids add 12.85 cm ^{3 of a} 0.2 M solution of Na ₂ HPO ₄ × 12 H ₂ O and 25 cm ³ dist. water.

The substrate solution is prepared no earlier than 10 minutes before entering into the wells of the tablet. For this, 4 mg of OPD is dissolved in 10 cm ^{3 of} FCB and 0.14 cm ^{3 of} 3% H ₂ O _{2 is} added. The solution should be clear.

The solution that stops the enzymatic reaction is 0.1 M H ₂ SO ₄ . For this, 9.8 ml of concentrated H ₂ SO _{4 is} diluted to 1 liter with distilled water.

4. Standardization of the basic conditions for conducting research developed by the ELISA test system.

The effect of the concentration of antigens of parvovirus cattle on the results of ELISA is investigated at a content of 30, 15, 5, 3, μg / ml. The results of the studies showed that the maximum value of the coefficient of specificity is observed during adsorption of parvovirus cattle antigen at a concentration of 3-5 μg / ml. The use of its other concentrations leads to a decrease in the specificity of the reaction.

The effect of temperature and time factors on the sensitization of antigen into the wells of an immunological plate is determined at +4 and + 37 ° C with a duration of contact of the antigen with a solid phase of 18 and 3 hours, respectively. The research results are shown in the drawing.

When developing enzyme immunoassays in which a single dilution of the test serum is used for the study, it is important to select the conditions for sorption of the immune serum on a sensitized antigen plate. For this, control sera in dilutions from 1: 100 to 1: 12800 are introduced into the wells of the plate immobilized by antigen. The conjugate is used in a working dilution of 1: 5000, which was determined in preliminary experiments by the method of "chess" titration. The results are presented in table 3.

Table 3 Selection of sorption conditions for control sera on a parvovirus antigen-immobilized plate (n = 3; P <0.05) Duration of antigen-antibody interaction, h Antibody Titers 1 / n KSP 0.5 1600 2.71 ± 0.03 one 6400 2.80 ± 0.02 2 12800 3.53 ± 0.07 3 3200 2.60 ± 0.02

The optimal concentration of antigen and working dilutions of the components of the enzyme-linked immunosorbent assay system is considered to be their final values, which ensure that in wells with a positive control serum OD ₄₉₀ > 0.650, and in wells with negative serum, ≤0.150. From the drawing and table 3 it is seen that the following reaction parameters meet these conditions: antigen concentration - 3-5 μg / ml, dilutions of controls - 1: 400 and working dilution of the conjugate 1: 5000. The optimal time for antigen adsorption was its 18-hour incubation at + 4 ± 1 ° C, for serums this time is 2 hours at + 37 ± 1 ° C.

Determination of the positive-negative threshold of the ELISA test system and interpretation of the results.

To account for and interpret the results obtained by the ELISA test system, it is necessary to determine the positive-negative threshold of the test system. For this purpose, the patterns of the distribution of OD ₄₉₀ values and the binding coefficient (Ksv.) Of virus-negative and positive sera are determined, i.e. threshold value (“cut off” or positive-negative threshold - PUP), delimiting the positive and negative reactions.

In the first experiment, 56 cow sera were used that did not contain virus-neutralizing antibodies to parvovirus antigen and 48 animal blood serum immunized with the associated vaccine against parvo-, reovirus, herpesvirus type I infections and viral diarrhea, an inactivated, emulsion cattle mucous membrane disease, with different levels of specific antibodies in RVN. Negative and positive sera were examined in four dilutions of 1: 100, 1: 200, 1: 400; 1: 800 each blood serum taken in a specific dilution was tested in three parallel wells.

Before taking into account the reaction according to the formula below, the average OD value of _{490 was} calculated for each test sample. The relative content of virus-specific antibodies in positive samples was determined by: a) antibody titer, i.e. the final dilution of the test sample, which is 2.1 times higher than the OD _{490 of the} negative control, and b) the binding coefficient (Ksv.), reflecting the content of antibodies in the test sample in relation to the content of antibodies in the positive control. Mathematically, this is expressed by the following formula:

where - OD ₄₉₀ IP _cf , OD ₄₉₀ K- and OD ₄₉₀ K + are the arithmetic mean values of optical density in the wells with the test sample, negative and positive controls, respectively.

Studies have shown that in order to obtain reliable results in the developed test system, test samples of cattle blood serum must be investigated in a dilution similar to controls, i.e. 1: 400.

The results of the experiments indicate that when the OD value _{490 of the} positive and negative controls is 0.58-0.869 and 0.05-0.068, respectively, the OD value of _{490 of} all obviously negative samples was within the range of 0.04-0.071, while the values of Kw. ranged from -2 to 15. In obviously positive blood sera, the value of OD ₄₉₀ exceeded 0.53, and the values of Ksv. were from 25 to 66.

When studying the dependence of the value of Ksv. from the titer of virus-specific antibodies in positive samples, a positive correlation was established between the two indicators (g = 0.69, P <0.05).

Further, extensive studies were conducted to determine the patterns of distribution of the values of Ksv. negative (n = 105) and positive (n = 123) according to RVN sera. The ELISA and accounting for the results were carried out similarly to the previous experiment.

An analysis of the results of the experiments showed the following: in most cases (93.3%) the value of Ksv. negative samples did not exceed 15. Of these, Ksv. <0 was in 52.4% of sera, in 44.8% of cases this indicator ranged from 0 to 15. Only in 3 samples (2.8%) did Ksv. was in the range of 15-32. In the majority of virus-specific blood sera (95.1%), the Ksv value. amounted to more than 25. At the same time, in 73.2% of the samples Ksv. it was more than 60, in 13.0% of cases it ranged from 40 to 60 and in 8.1% it was in the range of 25-40. In 7 samples (5.7%) the value of Ksv. was below 25%.

Thus, the analyzed sera having Ksv. less than 15 do not contain virus-specific antibodies. In virus-specific sera in most cases (98%), the OD value of ₄₉₀ exceeded the same value for the negative control by 2.1 times, and Ksv. was more than 25. Based on this, the PNP or “cut off” value for Ksv., which is a criterion for the differentiation of positive and negative samples and in which the minimum number of false positive and false negative results, is in the range of <15-≥25%. In the future, all tests, the value of Ksv. for which there was less PNP, were considered negative, and samples with a value of Ksv. equal to or greater than this indicator - positive.

Ideally, diagnostic test systems should be both sensitive and specific. In practice, the solution to these problems is quite complicated.

To confirm the reliability of diagnostically significant indicators, we carried out a number of statistical studies to determine the working dilution of serum, which would ensure maximum sensitivity and specificity of the developed test system. The research results are shown in table 4.

Table 4 Working dilution of the studied sera, providing maximum sensitivity and specificity of the ELISA test system Dilution of serum 1 / n Sensitivity% Specificity,% one hundred 97.4 83,2 200 97.8 86.0 400 96.07 93.8 800 87.3 94.2 1600 79.2 one hundred 3200 75.3 one hundred

From table 4 it follows that the maximum values of sensitivity (96.07%) and specificity (93.8%) of the test are achieved if serums in the dilution of 1: 400 are used in the reaction, this determined the further use of sera in this dilution.

Tests of diagnostic characteristics of the ELISA test system: specificity, sensitivity and reproducibility.

The main indicators of the quality of the enzyme immunoassay system are sensitivity and specificity. To obtain reliable results when testing sera from one dilution, the allowable optical density values of the control sera were determined. By studying 17 replicates of samples of positive and negative control sera at a dilution of 1: 400, we established the optimal values of the optical density of control sera: in the range from 0.5 to 0.709 for a positive control and from 0.05 to 0.08 for a negative control (Table. 5).

Table 5 Permissible OD values of control sera Indicators Positive Control (Pc) Negative Control (Nc) Difference Between Controls (Pc-Nc) Mean standard deviation ± m 0.620 ± 0.15 0.059 ± 0.01 0.570 ± 0.108 Confidence interval 0.557-0.746 0,053-0,068 0.517-0.720

If the optical density of the control sera is beyond the permissible values given above, then the reaction results are considered doubtful, and the samples should be re-examined.

The sensitivity and specificity of the test system was determined based on the correlation of the results of studies of blood serum in rtga and RVN (table 6).

Table 6 Comparative evaluation of results in ELISA and pH IFA results RVN result positive negative positive 196 10 negative 8 152 TOTAL 204 162

The results shown in table 6 indicate that the diagnostic sensitivity of the developed test system with respect to RVN was 196: 204 × 100% = 96.07%; diagnostic specificity 152: 162 × 100% = 93.82%; the coincidence of the results of the two methods 348: 366 × 100% = 95.1%.

At the same time, in the study of blood serum in ELISA and RTGA obtained from obviously negative (clinically healthy control animals from a well-off economy for this disease, n = 90) and obviously positive (experimental animals vaccinated with “Associated vaccine against parvo-, reovirus, herpesvirus type I infections and viral diarrhea - diseases of the mucous membranes of cattle inactivated emulsion ”, n = 90) cows, 100% coincidence of results was observed. In the first case, all research results were negative, in the second - positive.

The reproducibility of the ELISA test system was determined in 5 replicates, both within the same tablet (ELISA kit), and between different ELISA sets of the same series and sets of different series. The value of the coefficient of variation of the values of Ksv. in the study of positive samples (n = 10) by the ELISA test system of different series did not exceed 7.91%, and in the study of negative (n = 12) - 6.72%, which corresponds to the recommended parameters and does not exceed 10%. In this case, the correlation coefficient of the results in the study of positive samples was 0.9, and in the study of negative samples - 0.92.

5. Illustration of the application of the ELISA test system.

5.1. Preparation of biological material for research.

For analysis, blood serum of cattle is used. If the analysis is carried out within 24 hours after sampling, the samples of the biomaterial are stored at a temperature of + 4 ° C. For longer storage, the samples are frozen at -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 of red blood cells or protein, the samples must be clarified by centrifugation for 10 min at 2000 g. Repeated freezing and thawing of samples is not allowed. Do not use pooled sera from different animals. Hemolyzed and sprouted serums are not subject to research.

5.2. Preparation of working solutions.

Before starting work, all reagents can withstand at least 30 minutes at room temperature and mix thoroughly.

Working solution of the buffer for washing tablets FBI-T. The contents of the bottle No. 3 diluted 10 times with fresh distilled water. Example: 900 cm ^{3 of} distilled water are added to 100 cm ^{3 of} concentrate. If it is necessary to use part of the components, it should be borne in mind that approximately 400 cm ^{3 of} working solution is required to process one die. The working solution is stable at 4 ° C for 3 days.

Working chromogensubstrate solution. The solution is prepared immediately before use. The solution of chromogen (OFD - bottle No. 7) is diluted in phosphate-citrate buffer (FCB, bottle No. 6), hydrogen peroxide (bottle No. 8) is added and mixed. Example: to obtain 10 cm ^{3 of a} working chromogensubstrate solution, 4 mg of RPD is dissolved in 10 cm ^{3 of} phosphate-citrate buffer and 0.14 cm ^{3 of a} 3% hydrogen peroxide solution is added (flask. No. 8).

Conjugate Dilution Buffer (DBK), No. 4 - ready for use.

Control positive serum (K +), No. 1 - dissolve the contents of the vial in 0.5 cm ³ FBI-T.

Control negative serum (K ^- ) and 2 - dissolve the contents of the vial in 1 cm ³ FBI-T.

Conjugate solution (Conjugate), No. 5 - before use, dilute the required amount 5,000 times, for example, add 50 cm ³ DBK to 0.01 cm ^{3 of the} conjugate.

Stop reagent (1 M Sulfuric acid), No. 9 - ready for use.

10. All initial kit components are stable until the expiration date. Components remaining after partial use should be kept tightly closed in their original packaging. Do not pour into other dishes! Do not mix components from sets of different series!

11. Conducting an enzyme immunoassay.

Enzyme-linked immunosorbent assay (ELISA) is carried out in an indirect solid-phase version using the components included in the ELISA test system for serological diagnosis of parvovirus infection in cattle and to determine the level of post-vaccination antibodies.

Before starting work, the tablet is washed twice with a working solution of FBI-T in a volume of 0.15 cm ³ .

In a single dilution study, the test and control sera are used in a dilution of 1: 400 phosphate-buffered solution with tween (FBI-T) No. 4. For example, 0.01 cm ^{3 of} test serum is added to 3.99 cm ^{3 of} FBI-T.

0.1 cm ³ K ⁺ is added to wells E12-F12 at a working dilution of 1: 400.

0.1 cm ³ K ^is added to wells G12-H12 ^- at a working dilution of 1: 400.

In the remaining wells of the tablet, 0.1 cm ^{3 of the} test sample (AI) of the serum is diluted at a dilution of 1: 400 (two wells for each serum sample) according to the following scheme:

one 2 3 four 5 6 7 8 9 10 eleven 12 A IP1 IP5 B IP1 IP5 C IP2 IP6 D IP2 IP6 E IP3 etc. K + F IP3 K + G IP4 TO- H IP4 TO-

When tested in 4 dilutions, the test and control sera were used at a dilution of 1: 800. For example, 0.01 cm ^{3 of} test serum is added to 7.99 cm ^{3 of} FBI-T.

Previously, 0.1 cm ^{3 of} FBI-T is added to all wells of the plate.

0.1 cm ³ K ⁺ is added to well A11 in a working dilution of 1: 800.

0.1 cm ³ K ^is added to well A12 in a working dilution of 1: 800.

The remaining wells of the vertical row of the tablet (A1-A10) contribute 0.1 cm ^{3 of the} test serum at a dilution of 1: 800 and sequential double dilutions in four wells are carried out according to the following scheme:

one 2 3 four 5 6 7 8 9 10 eleven 12 A IP1 IP2 IP10 K + 1 K-1 B IP1.2 IP2.2 IP10.2 K + 2 K-2 C IP1.3 IP2.3 IP10.3 K + 3 K-3 D IP 1.4 IP2.4 IP10.4 K + 4 K-4 E IP11 etc. IP20 K + 1 K-1 F IP11.2 IP20.2 K + 2 K-2 G IP11.3 IP20.3 K + 3 K-3 H IP11.4 IP20.4 K + 4 K-4

- Cover the plate with a lid and incubate for 2 hours at 37 ° C.

- The plate was washed 3 times with buffer FBI-T (0.15 ^cm3) and the solution was removed by shaking, followed by tapping the filter paper to completely remove solution residues from the walls and bottom of the wells.

- 0.1 cm ³ of the conjugate working solution is added to each well.

- The tablet is closed with a lid and incubated for 1 h at 37 ° C.

- The tablet is washed 3 times with FBI-T buffer.

- Contribute 0.1 cm ^{3 of the} working chromogen substrate solution to each well.

- The tablet is incubated in the dark for 5-15 minutes at room temperature.

- Stop the reaction by adding 0.1 cm ³ stop reagent (1 M H ₂ SO ₄ ).

- After stopping the reaction, the optical density of the substrate mixture is measured on a spectrophotometer with a vertical beam at a wavelength of 490 nm (OD ₄₉₀ ).

12. Accounting and interpretation of the results.

When spectrophotometric accounting of the results of studies of blood serum in one dilution produce the following calculations:

- Calculate the arithmetic mean of the optical density (OD ₄₉₀ ) for samples K + (OD ₄₉₀ K + _sr );

- Calculate the arithmetic mean of the optical density (OD ₄₉₀ ) for samples K- (OD ₄₉₀ K- _cf );

- Calculate the arithmetic mean of the optical density (OD ₄₉₀ ) for each test sample SP (OP ₄₉₀ SP _avg );

- Calculate the binding coefficient (Ksv.) Conjugate serum antibodies according to the formula (II).

The sample was considered negative if the value of Ksv. less than 15%.

If the value of Ksv. More than 25% of the sample is considered positive.

The revealed results of serum studies in this range (from ≥15% to <25%) are considered doubtful, and the analysis should be repeated.

The detection of positive blood serum samples in one dilution during an epizootological examination of animals that have not previously been vaccinated against parvovirus infection in cattle indicates circulation in the herd of the pathogen.

Example 7. The analysis of the results in the study of sera in four dilutions calculate the specificity coefficient, which is equal to the ratio of the optical density of the reaction product in wells with a control positive (OD ₄₉₀ K +) or test serum (OD ₄₉₀ PI) to the optical density of the control negative serum (OD ₄₉₀ K-). The reaction is considered positive if the specificity coefficient is not lower than 2.1, and negative if below 2.1.

Example 7.1 Interpretation of Results

The identification of the growth dynamics of specific antibodies in paired serum samples, in the study of samples in four dilutions for the purpose of serological diagnosis, allows the diagnosis of parvovirus infection in the studied population.

7.2. The results of titration of sera in four dilutions determine the intensity of immunity in vaccinated animals 2-3 weeks after the second vaccination by dividing the number of samples with an antibody titer of 1: 1600 and higher to the total number of investigated sera and expressed as a percentage.

Animals are considered immune to parvovirus infection with an immunity intensity of 80 percent or more, i.e. if in 80 and more percent of blood serum samples the antibody titer reaches a value of 1: 1600 and above.

Thus, the proposed test system allows for large-scale studies of poorly studied parvovirus infection in cattle in the Russian Federation. The enzyme immunoassay system created by us meets modern requirements, and its introduction into the practice of the veterinary laboratories of the Russian Federation meets the needs of high-resolution immunological testing.

Bibliography

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9. Huck R.A. Isolation of a bovine parvovirus in the United Kingdom / R.A. Huck, D.W. Woods, J.P. Orr // J. Vet. Rec. - 1975 .-- V.96. - P.155-156.

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Claims (2)

1. ELISA test system for serological diagnosis of parvovirus infection in cattle and determine the level of post-vaccination antibodies, which is an indirect variant of solid-phase ELISA containing a specific antigen of parvovirus of cattle type I strain "Parvo 32459-DEPT" adapted for primary trypsinized culture cells of cow embryo kidneys (PEC) before virus accumulation in titer 1: 1024 GAE, inactivated by 0.1% solution of 1,2-aminoethylaziridine obtained by sedimentation of cell mass and destruction ltrazvukom followed by centrifugation to release from the cellular debris, concentrated and purified by ultracentrifugation through a layer of 20% sucrose, which is a polypeptide complex in a concentration of 5.3 g / cm ³ adsorbed on the surface of polystyrene wells plate parvovirus (positive) and the normal (negative ) control cattle serum at a dilution of 1: 400-1: 1600 in incubation buffer, antispecific conjugate, monosubstituted potassium phosphate, two potassium phosphate substituted, sodium chloride, chromogen (ortho-phenylenediamine), hydrogen peroxide, stop reagent and panels for setting the reaction of enzyme-linked immunosorbent assay, which allows epizootiological examination of animals not previously vaccinated against parvovirus infection in cattle, to detect antibodies in blood serum samples in one dilution (1: 400), based on the criterion of differentiation of positive and negative research results (positive-negative threshold), which is derived by calculating the coefficient I conjugate with serum antibodies and is in the range of <15-≥25%, characterized in that serological diagnosis is carried out by detecting the growth dynamics of specific antibodies in paired samples of blood serum, examining samples in 4 dilutions (1: 800, 1: 1600, 1: 3200, 1: 6400). 2. The ELISA test system for serological diagnosis of parvovirus infection in cattle and determining the level of post-vaccination antibodies according to claim 1, characterized in that the level of post-vaccination antibodies is determined by the results of a study of blood serum samples of vaccinated animals in 4 dilutions 2-3 weeks after the second vaccination by dividing the number of samples with an antibody titer of 1: 400-1: 800 and higher to the total number of investigated sera and the result is expressed as a percentage.

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