RU2323743C2 - Method of determination of specific antibodies to virus of goose enteritis - Google Patents

Abstract

FIELD: medicine, veterinary.

SUBSTANCE: specific antibodies to virus of goose enteritis are determined by IEA where virus received following injection of epizootic strain "P-75" of enteritis virus, is used as an antigen, and macro porous glass with diameter not less than 700 is used for purification of virus-containing material, the goose Ig G specific immunoperoxidase conjugate is used as an anti-specific agent, and the IEA results are calculated by use of the formula: titer = antiIg[2.02(lg S/P)+3.51], where S is for optical density of tested serum; P is for optical density of the positive control.

EFFECT: method reduces the test time and economical costs.

2 tbl, 2 ex

Description

The invention relates to veterinary medicine, and in particular to methods for determining specific antibodies to enteric virus (parvovirus) of geese.

Goose viral enteritis (parvovirus infection, Keep disease) - Enteritis virosa anserculorum is a contagious parvovirus disease of young birds, characterized by a predominant lesion of the gastrointestinal tract, liver, heart, pancreas and other parenchymal organs and causing its high mortality (from 30 to 100% ) The causative agent of the disease is parvovirus geese. The disease is widespread in European countries (Germany, France, England, Hungary, Czech Republic, Slovakia, the Netherlands) and on other continents. Serological and virological studies have established the spread of parvovirus infection in geese in most of the studied farms in various zones of the Russian Federation. At the same time, economic damage reaches from 25 to 100 thousand rubles. per 1000 heads of birds (1). Therefore, the diagnosis of this disease based on epizootological, clinical, pathological and anatomical data, laboratory tests (isolation of the virus and its identification in the neutralization reaction, bioassay), as well as the detection of specific antibodies in the blood serum of birds by enzyme-linked immunosorbent assay is extremely important.

Currently, to determine antibodies to enteritis virus, as a rule, a precipitation reaction in an agar gel is used. However, this method does not allow quantitative analysis (2).

A known method for the diagnosis of viral enteritis by examining samples of blood serum of geese using the neutralization reaction, which consists in the fact that to the double dilutions of serum add an equal volume of virus-containing suspension containing 100 or 1000 TCD ₅₀ antigen. The mixture is incubated at room temperature for 1 hour and infect the cell culture. The titer of neutralizing antibodies is expressed in log ₂ (3).

The disadvantage of this method is its duration - (5-6 days), which is unacceptable in the midst of an epizootic, as well as an expensive, time-consuming method and is associated with the seasonal laying of geese.

A known method for determining antibodies to exotoxin of pertussis bacteria by enzyme-linked immunosorbent assay includes adsorption of exotoxin on the surface of polystyrene, introduction of the studied antibodies, conjugation of antispecies antibodies with a substrate enzyme, and before the adsorption of toxin the surface of the polystyrene is treated with partially denatured human ceruloplasmin (4).

However, this method does not allow the diagnosis of viral enteritis of geese.

Closest to the technical nature of the claimed method is a known method for determining antibodies to the antigen of rheovirus tenosynovitis chickens (5).

According to the method, the virus-containing material is obtained using the strain "VNIVIP-DEP", the purification of the obtained bio-materials is carried out by viral gel chromatography on macroporous glass with a pore diameter corresponding to the diameter of the virus to be purified, the purified tenosynovitis antigen is diluted with phosphate-buffered solution to a concentration of 5.0-10 0 mg / 0.1 cm ^3, the adsorption of diluted antigen is carried on the surface of the polystyrene plate, and then applied to the plates of serum chicken blood, detection of complex antigen ntitelo performed using immunoperoxidase conjugate antispecies IgG against chicken and take into account the results of the ELISA reaction.

The results of the ELISA of the studied blood serum are taken into account both visually by comparing the color staining of the contents of the wells of the test serum with the color intensity of the peroxidase reaction product of the titrated control, and using the mathematical formula.

However, this method also does not allow the diagnosis of enteritis of geese.

The problem to be solved within the framework of this invention is the creation of a method for determining antibodies to the enteritis virus of geese, which has high sensitivity and specificity, as well as allowing to reduce research time and reduce economic costs.

This problem was solved as a result of the creation of a method for the determination of specific antibodies to the geese enteritis virus (parvovirus) by means of ELISA, including the production of antigen as a result of the introduction of the geese enteritis virus epizootic strain P-75 and purification of the virus-containing material by molecular sieve chromatography macroporous glass modified with polyvinylpyrrolidone (PVP) with a pore diameter of at least 700 Å, and a specific conjugate is used as an anti-species immunoperoxidase conjugate to IgG of geese, and ELISA results are taken into account according to the formula: titer = antilg (2.02 (Ig S / P) +3.51), where

S is the optical density of the test serum;

P is the optical density value of the positive control.

The claimed properties of the method are primarily due to the use of strain "P-75", the infectious titer of which is 4.0-4.5 lg ELD ₅₀ in 0.3 cm ³ . The strain of the virus is deposited in the collection of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines under the number "P-75".

It was found that the optimal dose of geese enteritis virus antigen for adsorption on a polystyrene tablet is 5-8 μg / 0.1 cm ³ , since an increase above 8 μg / 0.1 cm ³ leads to multilayer sorption of antigen, thereby reducing the sensitivity of the reaction, and its decrease below 5 μg / 0.1 cm ³ does not lead to a monolayer on polystyrene, which also negatively affects the results.

A specific complex, an antigen-unlabeled antibody, is detected using an anti-species immunoperoxidase conjugate specific for goose IgG, as this is determined by its biological properties.

The method is as follows.

The enteritis virus of geese strain P-75 is stored in VGNKI of veterinary preparations and is issued at the request of the enterprise at least 1 time per year with a passport characterizing the properties of the strain.

To obtain a virus-containing material using generally accepted methods (3).

The infectious titer of the virus-containing fluid should not be lower than 4.0 lg ELD ₅₀ in 0.3 cm ³ . The active and specific geese enteritis virus antigen is obtained by purification of virus-containing embryonic fluid by molecular sieve chromatography on macroporous glass with a pore diameter of 700 Å (VHC) modified with PVP.

In a purified virus preparation, the amount of protein is determined by the Lowry method, and it usually ranges from 35 to 50 μg / cm ³ . Then the purified virus preparation is diluted with 0.01 M phosphate-buffered saline (PBS) with a content of 0.15 M sodium chloride, pH 7.2-7.4, to a final concentration of 5-8 μg / 0.1 cm ³ . This value is the optimal parameter for adsorption of the purified antigen of the virus in the wells of the tablet, since an increase in antigen concentration above 8 μg / 0.1 cm ³ and a decrease below 5 μg / 0.1 cm ³ reduces the sensitivity of the reaction.

After determining the activity and specificity, the purified virus antigen is divided into two batches (parts), one of which is used for immunization of goslings in order to obtain virus-specific blood serum, and the second for adsorption in the wells of an ELISA plate.

Solid-phase immunosorbent is obtained by sorption in the wells of an ELISA plate of 0.1 cm ³ diluted in the FBI to a final concentration of purified antigen for 16-18 hours at 2-4 ° C.

Then the contents of the plate wells are removed by sharp shaking and washed three times with a buffer of the following composition: 0.01 M potassium phosphate buffer containing 0.5 M sodium chloride with a final concentration of detergent (tween-20, tween-80 or triton X-100) 0, 1%, pH 7.2-7.4. After that, the wells of the tablet are dried by tapping on filter paper.

Enzyme-linked immunosorbent assay is performed using: single and eight-channel automatic pipettes with interchangeable tips of different volumes, a thermostat with a heating temperature (37.0 ± 0.5) ° С, a household refrigerator, a pH meter type 121 or 340, a spectrophotometer of any design with a vertical a light beam at a wavelength of 450 nm.

For research, 20-25 samples are delivered to the laboratory in a volume of 0.2-0.3 cm ^{3 of} blood serum of birds of different age groups from poultry farms where there is a suspicion of this disease.

Then prepare immunospecific and non-specific components and working solutions:

Component No. 1 - positive geese serum to enteritis virus (positive control), diluted 1: 100 with 0.15 M sodium chloride solution containing 1% fetal serum, lyophilized, 1.0 cm ³ - 1 ampoule or vial (KI);

Component No. 2 - negative blood serum of geese (negative control), diluted 1: 100 with 0.15 M sodium chloride solution containing 1% fetal serum, lyophilized, 1.0 cm ³ - 1 ampoule or vial (K.2.);

Component No. 3 - geese enteritis virus antigen, purified and adsorbed at 0.1 cm ³ in the wells of an ELISA plate - 2 tablets (K.3.);

Component No. 4 - anti-species immunoperoxidase conjugate against IgG geese, lyophilized, 0.2 or 1.0 cm ³ - 1 ampoule (bottle), working dilution 1:21 (K.4.);

Component No. 5 - buffer solution concentrate, 5 cm ³ - 1 bottle (K.5.);

Component No. 6 - diluted detergent Tween-20, Tween-80 or Triton X-100, 5 cm ³ - 1 bottle (K.6.);

Component No. 7 - 5-aminosalicylic acid, powder, 20 mg - 1 vial or 1 tube for microprobe (K.7.);

Component No. 8 - hydroperite, tablet, 0.75 or 1.5 g - 1 pc. (K.8.);

Component No. 9 - 1% NaOH solution, colorless transparent liquid, 15 cm ³ - 1 bottle (K.9.);

Component No. 10 - sodium chloride, crystalline powder, 22 g - 1 bottle (K.10.).

Preparation of working solutions is as follows.

Solution No. 1 - 0.01 M potassium phosphate buffer containing 0.5 M sodium chloride with a final concentration of detergent (Tween-20, Tween-80 or Triton X-100) 0.1%, pH 7.2-7, four. For its preparation, take bottles with a concentrate of a buffer solution (K.5.) And sodium chloride (K.10.), The contents are dissolved in 750 cm ^{3 of} distilled water, and the pH is checked. Then diluted detergent (K.6.) Is added.

The solution is used to dilute the positive, negative, studied samples of geese blood serum, antiviral conjugate and washing the wells of the plates. Store no more than 15 days in a household refrigerator at 4-8 ° C.

Solution No. 2 - the contents of the ampoule (vial) with positive blood serum of geese (K.1.) Are dissolved in 1.0 cm ^{3 of} solution No. 1, a 1: 100 dilution of positive serum is obtained. Prepare before use. Storage in the freezer of a domestic refrigerator is allowed for up to 15 days.

Solution No. 3 - the contents of the ampoule (vial) with negative geese blood serum (K.2.) Are dissolved with 1.0 cm ^{3 of} solution No. 1, a 1: 100 dilution of negative serum is obtained. Prepare before use. Storage in the freezer of a household refrigerator is allowed for up to 15 days.

Solution No. 4 - the contents of the ampoule with the antispecific conjugate (K.4.) Are dissolved in 21 cm ^{3 of} solution No. 1. Prepare before use. Storage in the freezer of a domestic refrigerator is allowed for up to 15 days.

Solution No. 5 - a tablet of hydroperite 0.75 or 1.5 g (K.8.) Is dissolved in 22.5 or 45 cm ^{3 of} distilled water. The solution is stored in a dark place at 4-8 ° C for no more than 10 days.

Solution No. 6 - substrate indicator mixture. 5-aminosalicylic acid (K.7.) Is dissolved in 25 cm ^{3 of} hot distilled water (70 ± 2) ° С with constant stirring on a magnetic stirrer, then cooled to room temperature and adjusted to pH 6.0 ± 0.1 using 1% NaOH solution (K.9.), Then add 0.1 cm ³ solution No. 5. The solution is prepared immediately before use, do not store.

The studied sera are diluted 1: 100 with solution No. 1. To this end, 0.01 cm ³ (10 μl) of avian blood serum is added to 1.0 cm ^{3 of} solution No. 1. Then, 0.1 cm ^{3 of} positive blood serum (solution No. 2) is introduced into three wells (A1, B1, C1) of the vertical row of the tablet (solution No. 2) and 0.1 cm ^{3 of} negative serum are introduced into the next three wells (D1, E1, F1) blood (solution No. 3). 0.1 cm ^{3 of} solution No. 1 is added to wells G1, H1, which serve to control the conjugate and the substrate-indicator mixture.

In the remaining wells of rows B2-12 ... H2-12, 0.1 cm ^{3 of} solution No. 1 are added, and in the wells of the horizontal row A2-12 0.2 cm ^{3, the} studied blood serum samples are tested and they are calibrated in vertical rows, starting from a dilution of 1: 100 to 1: 12800. For this purpose, 0.1 cm ^{3 of} diluted test blood serums are taken from A2-12 wells and transferred to wells of B2-12 of the corresponding row, pipetted and double dilution is carried out by transfer of 0.1 cm ³ into the next hole of the vertical row of the tablet, removing from the last holes H2-12 at 0.1 cm ³ .

The tablet is gently shaken to mix the contents, close the lid and transfer to a thermostat (37.0 ± 0.5) ° C for 30 minutes. Then the wells of the tablet are freed from the contents by shaking, washed three times with solution No. 1 in a volume of 0.2 cm ³ and dried by tapping the inverted tablet on filter paper.

Prepare solution No. 4 and add 0.1 cm ³ to all wells of the plate, then place in a thermostat (37.0 ± 0.5 ° C) for 30 minutes. After incubation, the conjugate solution is removed, the wells are washed three times with solution No. 1 and dried by tapping an inverted plate on filter paper.

Then prepare the substrate-indicator mixture and contribute to all wells of the tablet 0.1 cm ³ . The tablet is left at room temperature in a dark place for 10-15 minutes.

Identification of specific antibodies in the blood serum of geese by an enzyme immunoassay can be carried out without the test serum being tested. In this case, 0.1 cm ^{3 of} diluted 1: 100 positive blood serum (positive control) is added to three wells (A1, B1, C1) of the vertical row of the tablet (0 control), and 0 are added to the next three wells (D1, E1, F1) , 1 cm ³ diluted 1: 100 negative serum (negative control). 0.1 cm ^{3 of} solution No. 1 is added to wells G1, H1, which serve to control the conjugate and substrate-indicator mixture, and 0.2 cm ^{3 of} positive serum are added to wells A2 and they are grafted in a vertical row A2-H2. In the remaining wells of rows A3-12 ... H3-12 make 0.1 cm ^{3 of the} studied samples of blood serum of geese at a dilution of 1: 100, using one well for each sample. The remaining stages of the reaction are carried out similarly.

When spectrophotometric accounting, the reaction is stopped by adding to all wells of the tablets 50 μl (0.05 cm ³ ) of 1% NaOH solution (K.9.).

Analysis results are taken into account after 10-15 minutes visually - according to the intensity of staining the contents of the wells of the tablet or instrumentally - using a spectrophotometer with a vertical beam of light at a wavelength of 450 nm.

With the visual method of accounting, the results are initially evaluated according to the control wells of the tablet: the contents of the wells with positive serum (positive control) should be intensely colored in brown. The contents of the wells with negative serum (negative control) and the control of the conjugate should be colorless or have a pale straw color. In the absence of the need for rigorous quantitative determination of the content of specific antibodies in the studied blood serum samples, the results of ELISA are carried out visually by comparing the color staining of the contents of the wells of the test serum with the color intensity of the peroxidase reaction product of the titrated positive control. The color intensity of the peroxidase reaction product is proportional to the content of antibodies to the geese enteritis virus.

The leveling of samples of the tested blood serum makes it possible to quantify the concentration of specific antibodies. For the titer of serum, its highest dilution is taken, in which the difference in color staining is still quite clearly visible in comparison with negative blood serum.

Spectrophotometric recording of ELISA results allows you to quantify the content of antibodies in the studied samples by measuring the optical density (OD) while recording the reaction results from each well of the tablet on a special paper tape or sheet of paper. The average OD value of the negative controls should not be higher than 0.150. The difference between the average value of the OD of the positive control and the average value of the OD of the negative control (P-N) is valid in the range of 0.340-0.780. If it goes beyond these values, the results are considered unreliable and the samples are re-examined. The presence or absence of antibodies to the geese enteritis virus is established by comparing the optical density of the test serum (S) with a positive control (P). A serum sample with an S / P ratio below 0.18 should be considered negative. If the S / P ratio is equal to or greater than 0.18, then the test sample should be regarded as positive. When determining the final titer of the test sample by one dilution of 1: 100, use the formula Titer = antilg [2.02 (lgs / p) +3.51], where

S is the optical density of the test serum; P is the optical density value of the positive control.

If in the blood serum of birds of different age groups unvaccinated against VEG, specific antibodies are found in titers of 1: 100 and higher in more than 50% of the studied samples, it can be considered that the economy is permanently unfavorable for parvovirus infection.

To confirm the diagnosis, virological studies are carried out to isolate the virus from pathological material (heart, kidneys, spleen, liver, pieces of the duodenum 12).

Evaluation of the effectiveness of the vaccine is the detection of specific antibodies in the blood serum of the vaccinated geese in titers no lower than 1: 400 in 80% of the vaccinated bird.

Example 1

A comparative analysis of the results of a study of blood serum samples of geese was carried out using the neutralization reaction (pH) and using the proposed method.

Sera were tested for antibodies to goose enteritis virus in pH. The neutralization test is a classic test to detect specific antibodies for viral diseases, including parvovirus infection of geese. The results of a comparative study of the sensitivity of ELISA and the neutralization reaction in the detection of specific antibodies to enteritis virus are presented in table 1.

Table 1
The results of a comparative study of serological reactions Serum Activity No. p / p Test serum of geese Virus neutralization titer, log ₂ The claimed invention one Positive serum to the geese enteritis virus from 09.04. 9.5 1: 3800 2 Positive serum to the geese enteritis virus from 05.05. 10.0 1: 4570 3 Positive serum to the geese enteritis virus (positive control) from 03.05 9.0 1: 3235 four Positive serum to enteritis virus from daily goslings obtained from vaccinated geese 5.8 1: 370 5 Positive serum to enteritis virus from vaccinated geese of the parent herd 8.5 1: 2820 6 Negative serum (negative control) from 03.05 0.75 -

The results of the studies showed that the proposed method has high sensitivity and specificity and allows you to identify specific antibodies to the enteritis virus of geese in blood serum. Between the two compared methods, the correlation coefficient was 1.0 (p <0.05). The time of the study using the claimed invention was 3 hours, and in the case of pH - 5-6 days.

Example 2

Tests were conducted on the activity and specificity of the proposed method in an encrypted experience.

The following were submitted for the test:

1. Positive serum of geese (positive control) from 03.05.

2. Negative blood serum of geese (negative control) from 03.05.

3. Blood serum of goslings-convalescents - 10 samples.

4. Blood serum of geese vaccinated against viral enteritis - 20 samples.

5. Blood serum of geese from farms safe for viral enteritis - 25 samples.

6. Specific (heterologous) geese serum to hepatitis viruses and reovirus - 2 samples each.

For research, 6 geese blood serum samples were encrypted. The studied sera were diluted with the FBI 1: 100 and introduced into the wells of a plate with adsorbed purified geese enteritis virus antigen in a volume of 0.1 cm ³ , 2 wells per serum. An enzyme-linked immunosorbent reaction was carried out in a similar manner as previously indicated. The test results are presented in table.2.

table 2
Identification of specific antibodies in the blood serum of geese in an encrypted experiment Cipher Test sera Result one Positive for geese enteritis virus (positive control from 03.05) + 2 Negative geese serum (negative control from 03.05) - 3 Goslings-convalescents blood serum + four Serum of geese vaccinated against viral enteritis + 5 Serum of geese from farms safe for viral enteritis - 6 Positive geese serum to hepatitis virus - 7 Positive geese serum to reovirus - Note: "+" - the presence of antibodies to the enteritis virus of geese in ELISA; "-" - absence of antibodies in ELISA.

The results of the encrypted experiment showed that ELISA, when detecting specific antibodies to the enteric virus of geese in the blood serum of birds, has high sensitivity and specificity.

Thus, the claimed method can be used for the qualitative and quantitative detection of specific antibodies to enteritis virus in the blood serum of geese during serological monitoring of poultry head, as well as to determine the level of post-vaccination immunity against viral enteritis (parvovirus infection) of geese.

Information sources

1. Patent RU No. 21118539, A61K 39/15, G01N 33/535.

2. Gouf R.E., Applikation of the agar gel preeipiting and virus neutralization test to the serological stady of goose parvovirus. Avian Palhology, 13, 501-509.

3. Syurin V.L. "Guide to Veterinary Virology." M., 1966.

4. Patent SU No. 1475331, 1999.

5. Patent RU No. 2192012, A61K 39/15, G01N 33/535, 10.27.2002.

Claims (1)

The method of determining specific antibodies to the geese enteritis virus, including the production of antigen and purification of the virus by gel chromatography on macroporous glass, its adsorption on the surface of polystyrene plates, ELISA, which consists of introducing control and test blood serum and detecting the antigen-antibody complex using an antiviral immunoperoxidase conjugate the presence of a substrate-indicator mixture, characterized in that the virus obtained by introducing into the body is used as an antigen epizootic enteritis virus strain P-75, when cleaning virus-containing material, macroporous glass with a pore diameter of at least 700 A is used, an anti-species immunoperoxidase conjugate using goose-specific Ig G is used, and ELISA results are taken into account by the formula: titer = antilg [2, 02 (logS / P) +3.51], where S is the optical density of the studied serum; P is the optical density value of the positive control.