RU2192012C2 - Method of assay of antibody raised to chicken reoviral tenosynovitis antigen - Google Patents

Abstract

FIELD: veterinary virology, biotechnology. SUBSTANCE: method involves preparing virus-containing material from the strain "VNIVIP-DEP" and the following purification of virus-containing material is carried out by method of molecular-sieve chromatography on macroporous glass with pore diameter corresponding to diameter of virus virion to be purified. The best result is obtained using macroporous glass modified with polyvinylpyrrolidone with pore diameter 1200

, not less. Then method involves adsorption of highly purified antigen in the protein concentration 5-10 mcg/0.1 cm³, not less. Antispecies immunoperoxidase conjugate raised against to chicken IgG is used as conjugate. In carrying out spectrophotometric recording results in assay of final titer of sample to be analyzed by a single dilution 1:100 the following equation is used: lg titer = 2.19 x (LgS/P) + 3.59 where S/P = (S-NKx)/(PKx + NKx); S means value of optical density of analyzed serum; P means value of optical density of positive control; NKx means an average value of optical density of negative control; PKx means an average value of optical density of positive control. Invention can be used for determination of specific antibodies raised to chicken tenosynovitis reovirus by method of immunoenzyme analysis. EFFECT: enhanced sensitivity and specificity of analysis. 4 cl, 2 tbl

Description

 The invention relates to veterinary virology and biotechnology and can be used to determine specific antibodies to chickens tenosynovitis reovirus by enzyme immunoassay.

 Tenosynovitis is a contagious reoviral disease of the bird, registered in all countries of the world, has a subacute, chronic or latent-persistent course. Widely distributed among chickens, turkeys, geese and synanthropic birds. Bird reoviruses are antigenically different from mammalian and human reoviruses. Serological and virological studies have established a widespread (90-95%) reovirus infection in chickens in the Russian Federation [1]. Therefore, the diagnosis of this disease based on epizootological, clinical and pathological data, laboratory results (isolation of the virus and its identification in the neutralization reaction, bioassay) and detection of specific antibodies in the blood serum of birds by enzyme-linked immunosorbent assay is extremely important.

 A known method of obtaining a vaccine against proventriculitis (disease of birds) [2]. The method comprises isolating proventriculitis reovirus from reovirus containing bird tissue affected by proventriculitis, inoculating a cell culture capable of supporting the growth and replication of these reoviruses, extracting the virus from cellular and extracellular components, and converting the reoviruses into a form suitable for administration to poultry.

 The disadvantage of this method of producing chicken reovirus antigen is a low concentration of viral protein, which does not allow to obtain a highly active antigen used in enzyme-linked immunosorbent assay (ELISA).

 There is also known a method of producing an immunoperoxidase conjugate for detecting Brucella antigens, comprising immunizing rabbits with a mixture of water-soluble Bg antigens. abortus 544, Vg. melitensis 565 and Br. suis 1330 taken in equal proportions, then the isolation of specific immunoglobulins from the immune serum and conjugation with horseradish peroxidase [3].

 However, the preparation of an immunoperoxidase conjugate by this method does not solve the problem of detecting specific antibodies to chicken reovirus tenosynovitis.

 The basis of the invention is the comprehensive task of creating a method for the determination of antibodies to the chickens rheovirusovirus antigen, which allows to increase the sensitivity and specificity of the analysis.

для вирусной гельхроматографии. Далее разведение полученного очищенного антигена теносиновита кур проводят фосфатно-буферным раствором до концентрации 5,0-10,0 мкг/0,1 см³, адсорбцию полученного разведенного антигена проводят на поверхности полистироловых планшет, затем на планшеты наносят исследуемые сыворотки крови кур, детекцию комплекса антиген-антитело осуществляют с использованием антивидового иммунопероксидазного конъюгата против IgG кур. Учет результатов реакции ИФА в исследуемых сыворотках крови проводят либо визуально путем сравнения цветового окрашивания содержимого лунок исследуемой сыворотки с интенсивностью окраски продукта пероксидазной реакции титруемого контроля, либо спектрофотометрически.The problem is solved in that according to the invention, the virus-containing material is obtained using the strain "VNIVIP-DEP", the purification of the obtained virus-containing material is carried out by viral gel chromatography on macroporous glass with a pore diameter corresponding to the diameter of the virus to be cleaned. The best result is obtained using macroporous glass modified with polyvinylpyrrolidone (PVP) with a pore diameter of at least 1200

for viral gel chromatography. Then, dilution of the obtained purified chicken tenosynovitis antigen is carried out with a phosphate-buffered solution to a concentration of 5.0-10.0 μg / 0.1 cm ³ , adsorption of the obtained diluted antigen is carried out on the surface of polystyrene tablets, then the studied chicken blood serum is applied to the tablets, and the complex is detected the antigen-antibody is carried out using an anti-species anti-IgG chicken conjugate immunoperoxidase conjugate. The results of the ELISA reaction in the studied blood serums are taken into account either visually by comparing the color staining of the contents of the wells of the test serum with the color intensity of the product of the peroxidase reaction of the titrated control, or spectrophotometrically.

The final titer of the test blood serum is determined by one dilution of 1: 100, using the formula
Lg titer = 2.19 (Lg S / P) +3.59,
where S / P = (S-NK _x ) / (PK _x -NK _x ),
where S is the optical density of the investigated serum;
P is the optical density value of the positive control;
NK _x is the average optical density of the negative control;
PK _x is the average optical density of the positive control;
in this case, a serum sample at an S / P ratio below 0.2 is regarded as negative, and at an S / P ratio equal to or greater than 0.2, as positive.

The claimed properties of the method are determined primarily by using the strain "VNIVIP-DEPT", whose activity is 5.5-6.0 Ig EID ₅₀ . The strain is deposited in the collection of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines under the number "VNIVIP-DEP" [4].

The authors found that the optimal dose of reovirus antigen for adsorption on a polystyrene tablet is 5-10 μg / 0.1 cm ³ , since an increase above 10 μg / 0.1 cm ³ leads to multilayer sorption of antigen, thereby reducing the sensitivity of the reaction, and reducing it below 5 μg / 0.1 cm ³ does not lead to a monolayer on polystyrene, which also negatively affects the result.

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

 The method is as follows.

 Chicken tenosynovitis virus strain "VNIVIP-DEP" is stored in VGNKI of veterinary preparations and is issued at the request of a biological enterprise at least 1 time per year with a passport characterizing the properties of the strain.

 To obtain virus-containing material, the method of intermittent passages was used on developing chicken embryos of 9–11 days old, which were infected with the freshly attenuated vaccine strain “VNIVIP-DEP” of chicken reovirus tenosynovitis. The activity of chicken tenosynovitis embryonic virus was determined by titration on chicken embryos [5].

(ВГХ), модифицированном ПВП.The biological activity of the virus-containing fluid should not be lower than 5.5 Ig EID ₅₀ . The active and specific antigen of the chicken tenosynovitis virus is obtained by purification of the virus-containing embryonic fluid by molecular sieve chromatography on macroporous glass with a pore diameter of 1200

(VGH), modified 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 . Next, the purified virus preparation is diluted with 0.01 M phosphate-buffered saline with 0.15 M sodium chloride (FBI) pH 7.2-7.4 to a final concentration of 5-10 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 plate, since an increase in the concentration of antigen above 10 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 chicken tenosynovitis virus antigen is divided into two batches (parts), one of which is used to immunize chickens to obtain hyperimmune blood serum, and the second for adsorption in the wells of an ELISA plate.

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

 Then, the contents of the plate wells are removed by sharp shaking and washed three times with washing buffer of the following composition: phosphate-buffered saline (pH 7.2-7.4) 0.01 mol / L; sodium chloride 0.5 M, tween-20, tween-80 or triton X-100 0.1%, after which 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) ^o С, a household refrigerator, a pH meter type 121 or 340, a spectrophotometer of any design with a vertical beam light at a wavelength of 450 nm.

For research, 20-25 samples (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 are delivered to the laboratory.

Then proceed as follows: prepare immunospecific and nonspecific components and working solutions:
Component 1 - positive blood serum of chickens to tenosinovitis reovirus (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 (K.1. );
Component 2 - negative blood serum (negative control), not containing antibodies to chicken tenosynovitis reovirus, 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 3 - chicken tenosynovitis reovirus antigen, purified and adsorbed at 0.1 cm ³ in the wells of an ELISA plate - 2 tablets (K.3.);
Component 4 - antiviral immunoperoxidase conjugate against chicken IgG, lyophilized 0.2 or 1.0 cm ³ - 1 ampoule (vial), working dilution 1: 21 (K.4.);
Component 5 - concentrated diluent - 1.5 M potassium phosphate buffer, pH 7.2-7.4, liquid, 10 cm ³ - 1 bottle (K.5.);
Component 6 - diluted detergent Tween-20, Tween-80 or Triton X-100, liquid, 5 cm ³ - 1 bottle (K.6.);
Component 7 - 5-aminosalicylic acid, powder, 20 mg - 1 vial or 1 tube for microprobe (K.7.);
Component 8 - hydroperite, tablet in a standard package, 0.75 or 1.5 g - 1 pc. (K.8.).

Preparation of working solutions:
A solution of 1 - 0.01 M potassium phosphate buffer containing a 0.5 M solution of sodium chloride with a 0.1% final concentration of detergent (Tween-20, Tween-80 or Triton X-100), pH 7.2-7, 4.

To prepare it, it is necessary to dissolve the contents of the bottle with concentrated diluent (K.5.), The contents of the bottle with diluted detergent (K.6.) And 44.0 g of sodium chloride in 1500 cm ^{3 of} distilled water, check the pH. If necessary, adjust the pH to 0.1n. KOH or HCI.

The solution is used to dilute positive, negative test samples of chicken blood serum, an anti-species conjugate, and washing the wells of the plates. Store no more than 15 days in a household refrigerator at (4-8) ^o C.

Solution 2 - the contents of the ampoule (vial) with chicken positive blood serum (K. 1.) are dissolved in 1.0 cm ^{3 of} solution 1, a positive serum dilution of 1: 100 is obtained, prepared before use. Storage in the freezer of a household refrigerator is allowed for up to 15 days.

Solution 3 - the contents of the ampoule (vial) with negative chicken blood serum (K. 2.) is dissolved with 1.0 cm ^{3 of} solution 1, a negative serum dilution of 1: 100 is obtained, prepared before use.

 Storage in the freezer of a household refrigerator is allowed for up to 15 days.

Solution 4 - the contents of the ampoule with the anti-conjugate conjugate (K.4.) Are dissolved in 21 cm ^{3 of} solution 1. Prepare before use. Storage in the freezer of a household refrigerator is allowed for up to 15 days.

Solution 5 - 1% NaOH solution. Prepared by dissolving 1 g of sodium hydroxide in 99 cm ³ distilled water. Store no more than 15 days at room temperature 20 ^o C.

Solution 6 - a tablet of hydroperite 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 ^o C for no more than 10 days.

Solution 7 — the contents of the vial with 5-aminosalicylic acid (K.7.) Are dissolved in 25 cm ^{3 of} hot distilled water (70 ± 2) ^o С with constant stirring on a magnetic stirrer, then cooled to room temperature and adjusted to pH 6.0 ± 0.1 using solution 5. The solution is prepared immediately before use, do not store.

Solution 8 - to prepare a substrate-indicator mixture, add 0.1 cm ^{3 of} solution 6 to 3.3 cm ^{3 of} cold (4-8) ^o With distilled water, then 18 cm ^{3 of} solution is added to 2 cm ^{3 of the} resulting solution 7. The solution is prepared immediately before use, do not store.

Statement of the reaction:
Samples of the studied sera are diluted 1: 100 with solution 1. For this purpose, 0.01 cm ³ (10 μl) of blood serum of birds is added to 1.0 cm ^{3 of} solution 1. Then, 0.1 cm ^{3 of} diluted negative blood serum (solution 3) is added to three wells (A1, B1, C1) of the vertical row of the tablet (solution 3) and 0.1 cm ^{3 of} diluted 1 are added to the next three wells (D1, E1, F1) : 100 positive blood serum (solution 2). 0.1 cm ^{3 of} solution 1 are added to wells G1, HI, which serve to control the conjugate and the substrate-indicator mixture.

0.1 cm ^{3 of} solution 1 are added to the remaining wells of rows B2-12 ... H2-12, and 0.2 cm ^{3 of the} studied blood serum samples are added to the wells of the horizontal row A2-12 and the test is carried out in vertical rows, starting from dilutions 1: 100 to 1: 12800. For this purpose, 0.1 cm ^{3 of} diluted test blood sera are taken from wells A2-12 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 well of the vertical row of the tablet, removing 0.1 cm ³ from the last holes of H2-12.

The tablet is gently shaken to mix the contents, close the lid and transfer to a thermostat (37.0 ± 0.5 ^o C) for 30 minutes Then the wells of the tablet are freed from the contents by shaking, washed three times with solution 1 and dried by tapping on filter paper.

Solution 4 is prepared and 0.1 cm ^{3 is} added to all wells of the plate, then placed in a thermostat (37.0 ± 0.5 ^° C) for 30 minutes. After incubation, the conjugate solution is removed, the wells are washed three times and dried by tapping on filter paper.

Then solutions 5, 6, 7 are prepared. 0.1 cm ^{3 of} solution 8 — the substrate-indicator mixture — is added to all wells of the plate and left at room temperature in a dark place for 15-20 minutes.

 Detection of specific antibodies in the blood serum of chickens by the enzyme immunoassay can also be carried out without testing the test sera.

In this case, 0.1 cm ^{3 of} diluted 1: 100 negative blood serum is added to three wells (A1, B1, C1) of the vertical row of the tablet (negative control), and 0 is added to the next three wells (D1, E1, F1) , 1 cm ³ diluted 1: 100 positive serum (positive control). 0.1 cm ^{3 of} solution 1 are added to wells G1, H1, which serve to control the conjugate and the substrate-indicator mixture. 0.1 cm ^{3 of} solution 1 is added to wells B2 ... H2, and 0.2 cm ^{3 of} positive serum is added to wells A2 and a trituration is carried out along the vertical row A2-H2. In the remaining wells of rows A3-12 ... H3-12 make 0.1 cm ^{3 of the} investigated samples of blood serum of chickens at a dilution of 1: 100, using one well for each sample. The remaining stages of the reaction are carried out similarly.

 Analysis results are taken into account after 15-20 minutes visually - according to the intensity of staining the contents of the wells of the tablet or instrumental - 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.

 If there is no need for strict 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 product of the peroxidase reaction of the titrated positive control. The intensity of color staining of the product of peroxidase reaction is proportional to the content of antibodies to chick tenosynovitis reovirus.

 The leveling of samples of the tested blood serum makes it possible to quantify the concentration of specific antibodies. The highest dilution is taken as the serum titer, at which the difference in color staining is still quite clearly visible compared 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 with simultaneous recording of the reaction results from each well of the tablet on a special paper tape or sheet of paper. The difference between the average value of the positive control and the average value of the negative control should be more than 0.10. The presence or absence of antibodies against reovirus 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 0.2 should be considered negative. If the S / P ratio is equal to or greater than 0.2, 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
Lg titer = 2.19 (Lg S / P) +3.59,
where S / P = (S-NK _x ) / (PK _x -NK _x ),
where S is the optical density of the investigated serum;
P is the optical density value of the positive control;
NK _x is the average optical density of the negative control;
PK _x is the average optical density of the positive control;
in this case, a serum sample at an S / P ratio below 0.2 is considered negative, and at an S / P ratio equal to or greater than 0.2, it is positive.

 Detection in blood serum of birds of different age groups unvaccinated against reovirus tenosinovitis, specific antibodies in titers of 1: 100 and higher in more than 50% of the studied samples, gives the basis for the diagnosis of this disease. In this case, take into account the epizootic situation in the poultry industry, the presence of clinical and pathological signs of the disease.

 For the final diagnosis of this disease, virological studies are carried out to isolate the virus from pathological material (affected joints, kidneys, spleen, liver).

 Evaluation of the effectiveness of post-vaccination immunity is the detection of specific antibodies in the blood serum of birds in titers of not less than 1: 400 in 80% of the vaccinated bird.

 The implementation of the claimed method is described in the examples.

 Example 1. To confirm the high sensitivity and specificity of the proposed method, a comparative analysis of the results of the study of samples of blood serum of chickens using the neutralization reaction and the proposed method.

 The sera studied by the proposed method are selected so that they are all checked for the presence of antibodies to the chicken tenosynovitis virus in the neutralization reaction (PH). The neutralization test is a classic test for the diagnosis of viral diseases, including the diagnosis of chicken reovirus tenosynovitis.

 The results of a comparative study of the sensitivity of the proposed method and the neutralization reaction in identifying specific antibodies to chicken tenosynovitis virus are presented in Table 1.

 The results of the study showed that the proposed method has high sensitivity and specificity and allows the detection of specific antibodies to chicken tenosienovitis virus in blood serum.

 No correlation was found between the two compared tests. The neutralization reaction reveals only virus-neutralizing antibodies, while the proposed method reveals a whole complex of antibodies: complement-binding, virus-neutralizing, precipitating, etc.

 Example 2. Tests were conducted on the activity and specificity of the proposed method in an encrypted experience.

For the test were presented 3 laboratory experimental series of immunospecific components for the detection of antibodies to chicken tenosynovitis virus by the proposed method, manufactured:
series 1 - 02.02.00
series 2 - 03.03.00
series 3 - 05.05.00
- a set for the diagnosis of SSYA-76 IFM, manufactured by VNIVIP (TU 10-07-146-89), manufactured on 06.99;
- a kit for detecting antibodies to the chicken infectious bronchitis virus enzyme immunoassay method, manufactured by VNIVIP (TU 10.07.080-93), manufactured on 04.2000;
- hyperimmune serums specific to the tenosynovitis virus of chickens from different age groups - 15 samples;
- positive serum to the chicken tenosynovitis virus from experimentally infected birds with various strains of the virus;
- field blood serum of chickens from birds of different ages - 60 samples;
- specific (heterologous) blood serum of chickens to viruses: bird flu, infectious laryngotracheitis. Marek's disease, infectious bursal disease. PMV-2 - 3 samples each;
- negative blood serum of chickens to the tenosynovitis virus - 10 samples.

For research, 10 chicken serum samples were encrypted. The test sera were diluted 1: 100 with the PBS and added to the wells of the plate with adsorbed purified reovirus 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.

 The results of the encrypted experience showed that the proposed method for detecting specific antibodies to tenosinovitis reovirus in chicken serum has high sensitivity and specificity.

 Thus, the claimed method for determining antibodies to the antigen of rheovirus tenosinovitis chickens has high sensitivity, specificity and effectiveness in detecting specific antibodies in the blood serum of chickens. The method can be used for the qualitative and quantitative detection of specific antibodies to the chicken tenosynovitis virus in blood serum and can be used for serological monitoring during epizootological examination of the poultry head and for determining the post-vaccination immunity against chicken tenosynovitis.

Sources of information
1. Trefilov B.B., Nikitina N.V. Reovirus infection of birds. - St. Petersburg, Information leaflet N 2-96, VNIVIP, 1996.

 2. EP 0101348 A2. Publ. 02.22.84, IPM 21, topics. issue 13, 1984.

 3. SU 1475331 A1 (Volgograd Research Anti-Plague Institute). Publ. 05/20/99.

 4. RU 2158304 (All-Russian Scientific Research Veterinary Institute of Poultry). Publ. 10.27.2000 30.

 5. Syurin V.L. Guide to Veterinary Virology. - M., 1966.

Claims (4)

1. The method of determining antibodies to the antigen of rheovirus tenosynovitis chickens, characterized in that the virus-containing material is obtained using the strain "VNIVIP-DEP", the purification of the obtained virus-containing material is carried out by viral gel chromatography on macroporous glass with a pore diameter corresponding to the diameter of the virus to be cleaned, then dilution purified antigen obtained tenosynovitis chickens performed with phosphate buffered saline to a concentration of 5.0-10.0 mg / 0.1 ml of a ^3, resulting diluted antigen adsorption etc. lead on the surface of the polystyrene plate, and then applied to the plates of serum chicken blood, detection of antigen-antibody complex is carried out using immunopereksidaznogo conjugate antispecies lg G against chickens and take into account the results of the ELISA reaction.  2. The method according to claim 1, characterized in that the results of the ELISA in the studied blood serum are taken into account visually by comparing the color staining of the contents of the wells of the test serum with the color intensity of the product of the titrated control peroxidase reaction. 3. The method according to claim 1, characterized in that the consideration of the results of the ELISA reaction in the test blood serum is carried out spectrophotometrically and the final titer of the test blood serum is determined by one dilution of 1: 100 using the formula
lg titer = 2.19 • (LgS / P) +3.59,
where S / P = (S-NKx) / (PKx-NKx), where S is the optical density of the studied serum;
P is the optical density value of the positive control;
NKx is the average optical density of the negative control;
PKx is the average optical density of the positive control,
in this case, a serum sample at a S / P ratio below 0.2 is regarded as negative, and at S / P a ratio equal to or greater than 0.2 is considered positive. 4. The method according to claim 1, characterized in that the virus-containing material is obtained by molecular sieve chromatography on macroporous glass modified with polyvinylpyrrolidone, with a pore diameter of at least 1200

for viral gel chromatography.

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