Method of determination of specific antibodies to virus of goose enteritis

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

 

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

Viral enteritis of geese (parvovirus infection, disease Hold) - Enteritis virosa anserculorum - contagious parvovirus disease of young birds, characterized by a primary lesion of the gastrointestinal tract, liver, heart, pancreas and other parenchymatous organs and causing high mortality (30 to 100%). The disease is parvovirus geese. The disease is widely distributed in European countries (Germany, France, England, Hungary, the 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 investigated farms of different zones of the Russian Federation. The economic damage ranges from 25 to 100 thousand rubles per 1000 birds (1). Thus it is very important to diagnose this disease, based on epidemiological, clinical and anatomic data, results of laboratory tests (virus isolation and identification in the neutralization, bioassays), and detection of specific antibodies in the serum of birds enzyme-linked immunosorbent assay.

In the us Aasee time for the detection of antibodies to the virus enteritis use, as a rule, the reaction of precipitation in agar gel. However, this method does not allow quantitative analysis (2).

A known method for the diagnosis of viral enteritis by studies of blood serum samples geese using the neutralization, namely, that a twofold dilutions of serum add an equal volume of vaccinated suspension containing 100 or 1000 TCD50the antigen. The mixture is incubated at room temperature for 1 hour and infect the cell culture. The titer of neutralizing antibodies expressed in log2(3).

The disadvantage of this method is its duration (5-6 days), which is unacceptable in the midst of the epidemic, and how expensive, time-consuming and associated with the seasonality of egg-laying geese.

A known method of detecting antibodies to exotoxin pertussis bacteria by enzyme immunoassay, comprising the adsorption of exotoxin on the surface of the polystyrene, the introduction of an investigational antibody conjugate antivitamin antibody with the enzyme substrate, and before the adsorption of the toxin to the surface of polystyrene handle partially denatured ceruloplasmin person (4).

However, this method does not allow you to diagnose viral enteritis of geese.

The closest in technical essence to the claimed method is well-known methods for the detection of antibodies to the antigen reoviruses tenosynovitis chickens (5).

According to the method vaccinated material was obtained using strain "UNIVAP - DEPT", treatment received biosera carried out by viral gelchromotography on macroporous glass with a pore size corresponding to the diameter of the purified virus dilution of purified antigen tenosynovitis chickens spend a phosphate buffer solution to a concentration of 5.0-10.0 µg/0.1 cm3the adsorption of the diluted antigen is performed on the surface of polystyrene tablet, then the tablet put analyzed blood serum of chickens, the detection of complex antigen-antibody carried out using individualo immunoperoxidase conjugate IgG against chickens and take into account the results of the ELISA reaction.

Records of the results of the ELISA reaction studied serum spend as visually by comparing the colour content of the wells of the test serum with the color intensity of the product peroxidase reaction titrated control, and using a mathematical formula.

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

Problem to be solved in the framework of this invention is to provide a method for detection of antibodies to the virus enteritis of geese, with high sensitivity and specificity, as well as to reduce the time of the study and reduce the economy the economic costs.

This problem was solved in the create method of determination of specific antibodies to the virus enteritis of geese (parvovirus) by the IFA, including the production of antigen in the introduction into the organism of epizootic strain "P-75 virus enteritis of geese and cleaning vaccinated material by molecular-sieve chromatography on macroporous glass, a modified polyvinylpyrrolidone (PVP) with a pore diameter of not less than 700 Åand as individualo immunoperoxidase conjugate using conjugate specific for IgG geese, and the results of ELISA account by the formula: titer=antilg (2.02(Ig S/P)+3.51), where

S - the value of the optical density of the serum;

A p - value of the optical density of the positive control.

The claimed properties of the method are primarily caused by the use of strain P-75", the infectious titer of which is 4.0-4.5 lg EDS500.3 cm3. The virus strain deposited in the collection of the all-Russian state research Institute for control, standardisation and certification of veterinary preparations under the number "P-75".

Found that the optimal dose of antigen virus enteritis of geese for adsorption on polystyrene tablet is 5-8 µg/0.1 cm3because increasing it above 8 µg/0.1 cm3leads to megol Inoi sorption antigen, resulting in reduced sensitivity reaction, and the reduction is below 5 µg/0.1 cm3not result in monolayer polystyrene, which also affects the results.

Specific complex - antigen-its antibody detected using individualo immunoperoxidase conjugate specific for IgG geese, because it is determined by its biological properties.

The method is as follows.

Virus enteritis of geese strain P-75 is stored in VGNKI veterinary preparations and issued at the request of the company not less than 1 time per year with a passport describing the properties of the strain.

To get vaccinated material using conventional techniques (3).

Infectious titers of vaccinated liquid should not be below 4.0 lg EDS500.3 cm3. Active and specific antigen of the virus enteritis of geese is obtained by purification vaccinated embryonic fluid by molecular-sieve chromatography on macroporous glass with a pore diameter of 700 Å (GSH), a modified PVP.

In a purified preparation of the virus protein is determined by the method of Lowry, and it usually ranges from 35 to 50 mcg/cm3. Then the purified preparation of virus diluted in 0.01 M phosphate buffer solution (FBI) containing 0.15 M sodium chloride, pH 7,2-7,4, to ConECs the th concentration 5-8 µg/0.1 cm 3. This value is the optimal parameter for the adsorption of purified virus antigen in the wells, because the increase in the concentration of the antigen more than 8 µg/0.1 cm3and reduce it below 5 µg/0.1 cm3reduces the sensitivity of the reaction.

After determination of activity and specificity of the purified virus antigen divided into two parties (part), one of which is used for immunization of goslings with the aim of obtaining virousspecificakih serum, and the second adsorption in the wells for ELISA.

Solid state immunosorbent get through sorption in the wells for ELISA 0.1 cm3divorced FBI to final concentration of the purified antigen for 16-18 hours at 2-4°C.

Then the contents of the wells to remove sharp shaking and washed three times with buffer of the following composition: 0.01 M potassium phosphate buffer containing 0.5 M sodium chloride to a final concentration of detergent (tween-20, tween-80 or Triton X-100) of 0.1%, pH of 7.2 to 7.4. Then wells dried by tapping the filter paper.

For setting immunoassay use: one - and eight-channel automatic pipette with interchangeable tips of different sizes, thermostat with temperature (37,0±0,5)°With, a household refrigerator, a pH meter type 121 or 340, spectrafor the meter of any structure with a vertical beam of light at a wavelength of 450 nm.

For research in the laboratory take 20-25 samples in a volume of 0.2-0.3 cm3blood sera of birds of different age groups from poultry farms where there is suspicion for this disease.

Then prepare immunospecificity and nonspecific components and working solutions:

Component No. 1 - positive serum geese to enteritis virus (positive control), diluted 1:100 0.15 M solution of sodium chloride with 1% fetal serum, lyophilized, 1.0 cm3- 1 ampoule or vial (K.I.);

Component No. 2 - negative serum geese (negative control)diluted 1:100 0.15 M solution of sodium chloride with 1% fetal serum, lyophilized, 1.0 cm3- 1 ampoule or vial (K.2.);

Component # 3 - antigen of the virus enteritis of geese, purified and adsorbed 0.1 cm3in the wells for ELISA - 2 tablet (K.3.);

Component No. 4 - individuai immunoperoxidase conjugate IgG against geese, dried, 0.2 or 1.0 cm3- 1 vial (bottle), working dilution 1:21 (K.4.);

Component # 5 - concentrate buffer solution, 5 cm3- 1 bottle (C.);

Component No. 6 - diluted detergent tween-20, tween-80 or Triton X-100, 5 cm3- 1 bottle (C.);

Component No. 7 - 5-aminosalicylic acid, powder, 20 mg - 1 bottle or 1 tube microprobe (C);

Component No. 8 - hydroponic, tablet of 0.75 or 1.5 g - 1 piece (office 8.);

Component No. 9 - 1% NaOH solution, colorless transparent liquid, 15 cm3- 1 bottle (C.);

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

Preparation of working solutions is as follows.

Solution No. 1 - 0.01 M potassium phosphate buffer containing 0.5 M sodium chloride to a final concentration of detergent (tween-20, tween-80 or Triton X-100) of 0.1%, pH of 7.2 to 7.4. To make it, take the vials with buffer solution (C.) and sodium chloride (C.), the contents are dissolved in 750 cm3distilled water, check the pH. Then add the diluted detergent (C.).

The solution is used for cultivation of positive, negative, studied blood serum samples geese, individualo conjugate and wash holes tablets. Store no more than 15 days in refrigerator at 4-8°C.

Solution No. 2 - the contents of the ampoules (vials) with positive serum geese (K.1.) dissolved in 1.0 cm3solution No. 1, receive the breeding of positive sera 1:100. Prepare before use. Be stored in the freezer household refrigerator for up to 15 days.

Solution No. 3 - the contents of the ampoules (vials) with a negative serum geese (K.2.) dissolved with 1.0 cm3the solution is the 1, get breeding negative serum 1:100. Prepare before use. Be stored in the freezer household refrigerator for up to 15 days.

Solution No. 4 - contents of the ampoule with antivitamin conjugate (K.4.) dissolved in 21 cm3solution No. 1. Prepare before use. Be stored in the freezer household refrigerator for up to 15 days.

Solution No. 5 - pill hydropenia of 0.75 or 1.5 g (office 8.) dissolve 22.5 or 45 cm3of distilled water. Solution store protected from light place at 4-8°With not more than 10 days.

Solution No. 6 - substrate-indicator mixture. 5-aminosalicylic acid (C.) dissolved in 25 cm3hot distilled water (70±2)°with constant stirring on a magnetic stirrer, then cooled to room temperature and adjusted pH to 6.0±0,1 using 1% NaOH solution (C.), then add 0.1 cm3solution No. 5. The solution was prepared immediately before use, do not store.

Studied syvorotki diluted 1:100 solution No. 1. With this objective 1.0 cm3solution No. 1 added 0.01 cm3(10 µl) of serum birds. Then in three wells (A1, B1, C1) vertical row of the tablet make a 0.1 cm3positive serum (solution No. 2) and in the following three wells (D1, E1, F1) contribute to 0.1 cm3negative serum (solution No. 3). the wells G1, H1 contribute to 0.1 cm3solution No. 1, serving to control conjugate and substrate-indicator mixture.

In the remaining wells of rows B2-12...H2-12 contribute to 0.1 cm3solution No. 1, and in the wells of a horizontal row A2-12 0.2 cm3the studied samples of blood sera and spend restframe vertical rows, starting from a dilution of 1:100 to 1:12800. With this purpose from the wells A2-12 select 0.1 cm3divorced investigated serum and transferred to wells B2-12 relevant number will pipeinput and conduct a two-fold dilution by transferring 0.1 cm3once the hole of the vertical row of the plate, removing from the last holes H2-12 to 0.1 cm3.

The tablet gently shaken to mix the contents, close the lid and transferred to thermostat (37,0±0,5)°With 30 minutes Then wells exempt from the contents by shaking, washed three times with solution No. 1 in the amount of 0.2 cm3and dried by tapping the inverted tablet on filter paper.

Prepare a solution # 4 and make all wells at 0.1 cm3then placed in a thermostat (37,0±0,5° (C) at 30 minutes After incubation, the conjugate solution is removed, the wells washed three times with solution No. 1 and dried by tapping the inverted tablet on filter paper.

Then prepare substrate in katarou mixture and make into all wells of 0.1 cm 3. The tablet is left at room temperature in the dark for 10-15 minutes

Detection of specific antibodies in the blood serum of geese by ELISA can be performed without rascaroli of the tested sera. In this case, three wells (A1, B1, C1) vertical row of the tablet make a 0.1 cm3diluted 1:100 positive serum (positive control), but in the next three wells (D1, E1, F1) contribute to 0.1 cm3diluted 1:100 negative serum (negative control). In wells G1, H1 contribute to 0.1 cm3solution No. 1, serving to control conjugate and substrate-indicator mixture, as in wells A2 0.2 cm3positive sera and spend restframe vertical row A2-H2. In the remaining wells series A3-12...H3-12 contribute to 0.1 cm3the studied samples of blood sera geese in a dilution of 1:100, using one well for each sample. The remaining stages of the formulation of the reaction carried out in the same way.

When spectrophotometric account for the reaction to stop making all wells tablets 50 ál of 0.05 cm3) 1% NaOH solution (K.9.).

The results of the analysis carried out in 10-15 min visually - intensity staining of the contents of the wells or instrumental - with the help of a spectrophotometer with a vertical beam of light at a wavelength of 450 nm.

In the visual method, taking into consideration the original evaluation test wells: the contents of the wells with positive serum (positive control) should be intensively stained in brown color. The contents of the wells with negative serum (negative control) and the control conjugate should be colorless or have a pale straw colour. In the absence of the need for strict quantitative determination of specific antibodies in samples of blood sera accounting ELISA results visually, by comparing the colour content of the wells of the test serum with the color intensity of the product peroxidase reaction titrated positive control. The intensity of the colour of the product of the peroxidase reaction proportional to the amount of antibodies to the virus enteritis of geese.

Rostirolla samples of the tested serum samples allows to quantify the concentration of specific antibodies. The titer of serum taken its highest dilution, which is still quite clearly see the difference in colour compared with the negative serum.

Spectrophotometric analysis ELISA allows to quantify the content of antibodies in the test sample by measuring the values of optical density (OD) with simultaneous recording of the results of the reactions with each of the wells on special paper tape or sheet of paper. The average OD value of the negative controls must not be higher 0,150. Differences in the indicators between the mean OD of the positive control and the average OD value of the negative control (P-N) is valid in the range 0,340-0,780. If it is outside these values, the results are considered unreliable and the sample was examined again. The presence or absence of antibodies to the virus enteritis of geese is established by comparing the values of optical density of test serum (S) with the positive control (P). Sample serum with S/P-ratio below 0,18 should be regarded as negative. If S/P-ratio is equal to or greater than 0,18, then the sample should be considered positive. In determining the final titer of the investigated samples at the same dilution 1:100, use the following formula : Titer=antilg [2.02(lgs/p)+3,51], where

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

When detected in the blood sera of birds of different age groups, unvaccinated against VEG, specific antibodies at titers of 1:100 and higher in more than 50% of the investigated samples suggest the economy permanently affected by parvovirus infection.

To confirm the diagnosis of conduct virological studies on the excretion of virus from pathological material (heart, kidneys, spleen, liver, pieces of 12 duodenal ulcer).

Assessment of the effectiveness of the vaccine is detected in the blood sera of vaccinated geese specific antibodies at a titer of not less than 1:400 at 80% vaccinio the Anna birds.

Example 1.

The comparative analysis of results of blood serum samples geese using the neutralization (pH) and using the proposed method.

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

Table 1

The results of a comparative study of serological reactions
Activity of sera
№ p/pThe investigated serum geeseThe titer of neutralizing antibodies log2The claimed invention
1Positive serum for virus enteritis of geese from 09.04.9.51:3800
2Positive serum for virus enteritis of geese from 05.05.10.01:4570
3Positive serum for virus enteritis of geese (put the capacity control) from 03.05 9.01:3235
4Positive serum for virus enteritis from daily goslings collected from vaccinated geese5.81:370
5Positive serum to enteritis virus from vaccinated geese parent stock8.51:2820
6Negative serum (negative control) from 03.050.75-

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

Example 2.

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

On the test were presented:

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

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

3. Serum goslings-patients - 10 samples.

4. Serum geese vaccinated against viral ENT the Rita - 20 samples.

5. Serum geese from farms with a good viral enteritis - 25 samples.

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

Research was encrypted 6 blood serum samples geese. Test serum was diluted FBI 1:100 and added into wells with adsorbed purified antigen of the virus enteritis of geese in the amount of 0.1 cm32 wells for each serum. Enzyme reaction was performed similarly as described previously. The test results presented in table 2.

Table 2

Detection of specific antibodies in the blood serum of geese in the encrypted experience
CipherTest serumResult
1Positive to the virus enteritis of geese (positive control from 03.05)+
2Negative serum geese (negative control from 03.05)-
3Serum goslings-patients+
4Serum geese vaccinated against viral enteritis+
5Serum geese from farms, blahop the meadow on viral enteritis -
6Positive serum geese to hepatitis-
7Positive serum geese to reovirus-
Note: "+" - the presence of antibodies to the virus enteritis of geese in IFA;
"-" - absence of antibodies in ELISA.

The encrypted results experience has shown that ELISA in the detection of specific antibodies to the virus enteritis of geese in the blood serum of birds has a high sensitivity and specificity.

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

Sources of information

1. Patent RU No. 2118539, A61K 39/15, G01N 33/535.

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

3. Syurin V.L. "guidelines for veterinary Virology". M., 1966.

4. Patent SU # 1475331, 1999.

5. Patent RU NO. 2192012, AK 39/15, G01N 33/535, 27.10.2002.

The method of determination of specific antibodies to the virus enteritis of geese, including the production of the Academy of Sciences of the Egan and purification of the virus by gelchromotography on macroporous glass, its adsorption on the surface of polystyrene tablet, conducting ELISA, which consists in making the control and test serum samples and detection of complex antigen-antibody using individualo immunoperoxidase conjugate in the presence of substrate-indicator mixture, characterized in that the quality of the antigen used, the virus resulting from the introduction into the organism of epizootic strain of the virus enteritis "L-75", when cleaning vaccinated material used macroporous glass with a pore diameter of not less than 700 And, as individualo immunoperoxidase conjugate using specific Ig G geese, and the results of ELISA account by the formula: titer=antilg[2,02(lgS/P)+3,51], where S is the value of the optical density of the serum; P - value of the optical density of the positive control.



 

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1 tbl, 3 ex

FIELD: molecular biology, biotechnology, gene engineering, in particular diagnosis of atypical pneumonias.

SUBSTANCE: invention relates to DNA based on identified protein antigen epitope SARS-CoV. Particularly disclosed are nucleotide sequences of synthetic genes encoding recombinant protein fragments containing coronavirus protein antigen determinants SARS-CoV, represented in SEQ ID NO:1 - SEQ ID NO:8; as well as SARS-CoV virus protein fragments encoded by DNA sequences with amino acid sequences represented in SEQ ID NO:10 - SEQ ID NO:17. Said fragments are isolated by using preliminary obtained fusion protein by attachment to its N-terminal end GST-S-transferase protein with sequence represented in SEQ ID NO:9. Fusion proteins are useful in manufacturing of preparations for diagnosis of acute respiratory virus SARS-CoV.

EFFECT: new diagnostic agents for diagnosis of atypical pneumonias.

2 cl

FIELD: microbiology and immunology, in particular immunodiagnosis.

SUBSTANCE: atypical strain of melioidose Burkholderia pseudomallei-111-6-1 with altered phenotype defected with respect to synthesis of 8 antigen and acting as immunosuppressor is used as antigen for animal immunization. Immune serum is obtained after 2 immunization cycles of animal-producer with titer in gel immunodiffusion reaction not less than 1:128.

EFFECT: immune serum with increased specific activity.

2 tbl, 2 ex

The invention relates to the field of veterinary medicine and can be used for diagnosis of the causative agent of brucellosis in an L-shape

The invention relates to the field of medicine, namely biotechnology, diagnostic products, and can be used in the manufacture of medical immunobiological preparations intended for the detection of specific antibodies and antigens in biological substrates

The invention relates to the field of immunology, and can be used in the diagnosis of diseases associated with N. pylori using reaction coagglutination (RCA)

FIELD: veterinary science, virology, biotechnology.

SUBSTANCE: the suggested vaccine contains the suspension of avirulent and purified antigenic material out of the strain N 101 of cattle rotavirus (Research Institute of Animal Protection) obtained in mono-layer culture cells MDBK or SPEV at accumulation degree being 106 viral particles/ml, not less and activity in IEA being 5.0 log2, not less, and target additives: aluminum hydroxide and saponin in efficient ratios. The strain is deposited in collection of FGU VGNKI under registration number - a culture strain N 101 Research Institute of Animal Protection-DEP. The suggested vaccine is highly immunogenic, safe and areactogenic, it is capable to protect cattle stock against epizootic agent of rotaviral infection circulating in Russian territory.

EFFECT: higher efficiency.

14 cl, 9 ex, 10 tbl

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