RU2592232C1 - Method of producing diagnostic serum for detecting aleutian disease of mink virus - Google Patents

Abstract

FIELD: veterinary science; virology.

SUBSTANCE: disclosed method of producing serum for diagnosis of Aleutian disease of mink (ADM) comprises obtaining high-purity viral antigen by homogenisation organs infected with ADM followed by triple freezing/defrosting of homogenate, disintegration of cell homogenate with triple ultrasonic treatment, deposition of destructed cells with double-speed centrifugation, followed by deposition of immune complexes, seal residue low-speed centrifugation, by extraction of immune complexes (IC) with minimum amounts of TOE buffer by triple extraction in cold for 1-2 hours with subsequent low-speed centrifugation, deposition of IC by high-speed centrifugation of combined extracts at 30,000 rpm for 2.5 hours, extraction of IC from sludge with minimum amounts of TOE buffer by triple extraction in cold for 1-2 hours with subsequent low-speed centrifugation, dissociation of immune complexes in an acidic medium for 1 hour in cold, separation of ADM-F from antibodies by high-speed ultracentrifugation, dissolving residue in minimum amount of carbonate buffer, ferritin separation from viral particles on a column with sepharose 6B in a carbonate buffer. Further, method includes immunising rabbits with trace amounts of purified antigen in five steps to obtain serum with high titre of antibodies to ADM - 1/16384 virus.

EFFECT: obtaining diagnostic serum with high titre, making it possible to test ADM virus in various biological fluids.

4 cl, 2 ex, 4 dwg

Description

The invention relates to veterinary virology, in particular to the production of diagnostic preparations, in particular highly active sera during immunization of animal producers with various antigens, can be used to determine the Aleutian mink disease virus (VABN) in fur farms in order to protect animals from the disease, and also for laboratory research and production of diagnostic test kits VABN.

Aleutian Mink Disease (AB) is an infectious viral disease that causes major damage to fur farming. A feature of AB is the inability of the resulting antibodies to neutralize the virus. Vaccination only aggravates the pathological process, and the only method for controlling AB is timely diagnosis and culling of sick animals. The existing methods for diagnosing AB in the world are technological, but they have not very high sensitivity. In addition, highly sensitive methods are time-consuming, expensive, require a lot of time for their execution, which increases dramatically when examining a large number of animals.

The Aleutian mink disease caused by a pathogen from the genus Parvovirus is difficult to detect by the enzyme immunoassay due to the fact that the virions form a complex with the iron-containing protein ferritin (F) and antibodies to this virus. Ferritin and antibodies interfere with sorption of the virus on a styrene plate and reactions with immunoglobulins. Ferritin also makes it impossible to obtain a highly purified antigen to obtain antiserum. The complexity of determining the virus from biological fluids, as noted above, is associated with the formation of very resistant aggregates of VABN-F. Existing determination methods require preliminary isolation and purification of the virus in the laboratory using expensive complex equipment and the use of strong surface-active substances (surfactants).

A sensitive and at the same time high-tech method is required, which allows one to obtain an antiserum for conducting a mass examination of minks on fur farms.

The well-known "Method for serological diagnosis of Aleutian mink disease" (copyright certificate No. 1427651, A61K 39/00, 09/15/1990). In this method, an antigen containing an inactivated Aleutian mink disease virus is pre-concentrated, washed with buffer, re-concentrated by ultrafiltration and iodinated with an isotope using NaJ in the presence of chloramine T and sodium metabisulfate. After adding the indicated antigen to the test serum, this mixture is incubated for 1-1.5 hours, separated, the precipitate is centrifuged and its radioactivity is measured, which serves as a measure of the antibody content in the test serum.

The specified method is time consuming and expensive, because isotopes are used to produce the antigen and, in addition, the diagnostic antiserum has a low titer.

The well-known "Method for producing serum for the diagnosis of cattle anaplasmosis in the reaction of indirect immunofluorescence by hyperimmunization of rabbits" (RF patent No. 2368393, A61K 39/395, 09/27/2009). The method consists of administering antigen and a levamisole immunostimulant to rabbits. In this case, an antigen is used as an antigen, recovered by alternately freezing and thawing Vero cells grown in growth medium in a growth support medium. The MEM needle with the addition of embryonic serum, amino acids and antibiotics of the live culture Anaplasma sp. Omsk after eight-day incubation.

The known method has the following disadvantages: low titers of antibodies formed in the blood of rabbits and low activity of the obtained antisera.

Also known is the "Method for producing serum against mink viral enteritis" (RF patent No. 2049478, IPC A61K 39/23, priority from 04/16/1992). In the known method, hyperimmunization of animals is carried out with an antigen, followed by blood sampling and separation of the target product. As an antigen, a purified and concentrated strain of Virus enteritis lutreo larum "Springs" is used. Goats are used from animals, and hyperimmunization is carried out by administering 5-12 cm ^{3 of} antigen sequentially with an interval between injections of 7-10 days intravenously, intramuscularly, intravenously, subcutaneously with Freund's complete adjuvant, and then after 28-30 days intravenously.

In a known method, goats are used from animals, not rabbits, which requires large doses of antigen.

Also known is the “Method for the production of antiviral serum” (see Abstract of dissertation for the degree of candidate of biological sciences Martynenko MV “Immunochemical and molecular genetic properties of the Aleutian disease virus (ADV) circulating in the south of Primorsky Territory, and development of methods for its diagnosis ”Vladivostok 2006, p. 5-8). The method includes obtaining a purified virus preparation from mink organs with a positive response in serological tests for the presence of ADV (the name of the Far Eastern virus isolate ADV-DV) by differential centrifugation in a Tris buffer system, dissociation of immune complexes with HC1-glycerol buffer, elimination of ferritin impurity by chromatography on sepharose 6B and immunization of rabbits with a drug with complete Freund's adjuvant according to the scheme of M. Horvitz and M. Sharff in their own modification, precipitation of IgG from the blood of mink sulfato ammonium and purification on a column of Sephadex G-200. Conjugation of viral proteins and IgG were performed with ¹²⁵ I by chloramine T. iodate oxidation resulting rabbit polyclonal antiserum against ADV-DV, which contained antibodies to virion proteins have been reacted with LRM virus preparation. ADV-DV showed itself in this method of preparation of the middle immunogen (titer of specific antibodies, according to the RDD, 1: 512-1024).

The serum obtained by a known method is characterized by an insufficient degree of purification of antigens from impurities of ferritin and a low titer of antiserum obtained by immunization of rabbits.

The basis of the claimed technical solution is the task of obtaining diagnostic serum with a high titer for the determination of ABN viruses, which can be used to diagnose ABN in the field during a mass examination of minks.

The problem is solved in that in the method for producing serum for the diagnosis of ABN, including isolation and purification of the virus and immunization of animals, the antigen is obtained by homogenization of organs infected with ABN minks, followed by three-fold freezing / thawing of the homogenate, disintegration of it three times with ultrasonic treatment, sedimentation of undisturbed cells twice centrifugation, precipitation of immune complexes, sediment compaction by low-speed centrifugation, extraction mmun complexes with minimal amounts by triple extraction in the cold for 1-2 hours, followed by low-speed centrifugation, precipitation by IR high-speed centrifugation from the combined extracts, extraction of immune complexes from the precipitate with minimal amounts of TNE buffer by three times extraction in the cold for 1-2 hours, followed by low-speed centrifugation, dissociation of immune complexes in an acidic environment for 1 hour in the cold, separation of VABN-F from antibodies is high speed ultracentrifugation, dissolving the precipitate in a minimum amount of carbonate buffer, ferritin separation from virus particles on a Sepharose 6B in carbonate buffer system. For better purification, chromatography is performed twice. Spectrophotometry control is carried out in the ultraviolet wavelength range 220-310. After that, the estimated concentration of the virus is calculated according to the Kalkar formula:

K = 1.45 × E ₂₈₀ -0.74 × E ₂₆₀

Then, to obtain the maximum titer of antibodies to VABN, the following rabbit immunization schedule is carried out:

- the first immunization with 200 μg of antigen + Freund's complete adjuvant (USA) was carried out subcutaneously at 9-12 points;

- the second immunization (7 days after the first) - 200 μg of antigen + Freund's complete adjuvant (USA) intramuscularly and subcutaneously at 6-8 points;

- the third immunization (7 days after the second) - 300 μg of antigen + Freund's complete adjuvant (USA) intramuscularly and subcutaneously at 6-8 points;

- the fourth immunization (14 days after the third) - 200 μg of antigen + Freund's complete adjuvant (USA) intramuscularly and subcutaneously at 6-8 points;

- fifth immunization (7 days after the fourth) - 200 μg of antigen intravenously.

The antibody titer was 1/16384.

In addition, 50 mM Tris-HC1, 0.15 M NaC1, pH 7.2 are used as a buffer system, 1% Triton X-100, 0.4 mM phenylmethylsulfonyl fluoride, 1% sodium deoxycholate (Sigma USA) are used as detergents 10 mM Tris-HC1, 0.1 M NaC1, 1 mM EDTA-Na, 0.05% NP-40 (Sigma USA), pH 7.4 are used as a TNE buffer.

In the claimed method, the common features for him and for his prototype are:

- obtaining purified antigen;

- immunization of animals with antigen.

A comparative analysis of the proposed technical solution and prototype shows that the first has, in contrast to the prototype, the following essential features:

- a modified method for producing a highly purified virus preparation from a complex complex with iron-containing ferritin protein and antibodies to the Aleutian mink disease virus;

- An improved five-stage immunization schedule for rabbits to obtain the maximum titer of antibodies to VABN.

The set of essential features of the claimed technical solution has a causal relationship with the achieved technical result - obtaining diagnostic serum with a high titer. Based on the foregoing, we can conclude that the claimed technical solution is new, has a technical level and is suitable for industrial use.

The invention is illustrated by drawings, where in FIG. 1 shows the titer of rabbit antiserum for VABN in an indirect ELISA, FIG. 2 - antigenic relationships of 2 strains of VABN, in FIG. 3 - the effect of surfactants on the complex VABN-F in determining the virus, in FIG. 4 - titration curve of the drug VABN-F after exposure to surfactants. In the drawings, the following notation:

FIG. one

Antiserum to VABN 1 from rabbit No. 1

Antiserum to VABN 2 from rabbit No. 2

Control - normal rabbit antiserum

1/1000 Anti-Species Conjugate Dilution

The antigen concentration is 5 μg / ml.

FIG. 2.

VABN cleaning. preparation 1 - virus isolate from Moscow region

VABN cleaning. preparation 2 - isolate from a local fur farm

Initial virus concentration of 50 μg / ml

1/1000 Anti-Species Conjugate Dilution

FIG. 3.

VABN cleaning. preparation 2 - isolate from a local fur farm

VABN-F - a complex of virus with ferritin

VABN-F + X100 60 min. - VABN-F complex treated with Triton X100 for 60 min.

Control - healthy mink blood plasma diluted 100 times

Initial virus concentration of 50 μg / ml

Dilution of the antispecific conjugate 1/1000.

FIG. four.

VABN-F - a complex of virus with ferritin

Control - healthy mink blood plasma diluted 100 times.

A method of obtaining a diagnostic serum is implemented as follows. Getting the maximum titer of antibodies. For this, it is necessary to obtain a viral preparation of the highest degree of purification. The antigen is obtained by homogenization of organs of mink infected with ABN (for example, spleen, liver, kidneys) in a buffer system containing detergents in a ratio of 1 / 3-1 / 5 of the volume (in a mortar with the addition of quartz sand and then in the homogenizer), followed by three times freezing / thawing of the homogenate, disintegration of the cell homogenate by three-fold ultrasonic treatment at a frequency of 22 kHz / s on an UZDN-2 apparatus (for 20 seconds with an interval of several seconds), deposition of intact cells by a double low-speed centrifuge (clarification of the homogenate (at 6000-8000 rpm for 20 min), precipitation of immune complexes (IR) with 10% polyethylene glycol (PEG-6000) or 25% ammonium sulfate for 4 hours or night, compaction of the precipitate by low-speed centrifugation (12000 rpm for 30 min), extraction of immune complexes with minimal amounts (1/10 of the initial volume) of TNE buffer by three times extraction in the cold for 1-2 hours, followed by low-speed centrifugation at 12000 rpm for 20 minutes, High Speed Centrifuge IR Deposition by extracting from the combined extracts at 30,000 rpm for 2.5 hours, extracting the immune complexes from the precipitate with minimal amounts of TNE buffer by triple extraction in the cold for 1-2 hours, followed by low-speed centrifugation at 12,000 rpm for 20 minutes, dissociation of immune complexes in acidic medium (pH 2.4) of HC1-glycine buffer in the ratio of 1V extract to 2V buffer for 1 hour in the cold, separation of VABN-F from antibodies by high-speed ultracentrifugation (30000 rpm for 2.5 hours) following by dissolving the precipitate in a minimum amount (not more than 2 ml) of a pH 9.6 carbonate buffer and separating ferritin from viral particles on a 6B Sepharose column in a carbonate buffer system, then chromatography is carried out twice, spectrophotometry is carried out in the ultraviolet wavelength range 220 -310, and the calculation of the estimated concentration of the virus is carried out according to the Kalkar formula:

K = 1.45 × E ₂₈₀ -0.74 × E ₂₆₀ ,

rabbits are used as animals for immunization, and the immunization itself is carried out in five stages:

- the first immunization with 200 μg of antigen + Freund's complete adjuvant (USA) was carried out subcutaneously at 9-12 points;

- the second immunization (7 days after the first) - 200 μg of antigen + Freund's complete adjuvant (USA) intramuscularly and subcutaneously at 6-8 points;

- the third immunization (7 days after the second) - 300 μg of antigen + Freund's complete adjuvant (USA) intramuscularly and subcutaneously at 6-8 points;

- the fourth immunization (14 days after the third) - 200 μg of antigen + Freund's complete adjuvant (USA) intramuscularly and subcutaneously at 6-8 points;

- fifth immunization (7 days after the fourth) - 200 μg of antigen intravenously.

To confirm the technical result — obtaining high titer serum — the obtained diagnostic serum was tested. Testing of serum was carried out with a purified preparation of 2 strains of VABN (local and from Moscow region) (Fig. 2). It can be seen from the drawing that a heterologous strain can also be determined with an antiserum to a local strain of VABN.

Example No. 1 of the implementation of serum testing.

20 μl of diluted biological fluid (blood plasma, urine, saliva) is added to the wells of the polystyrene plate and 0.1 ml of a 10% solution of X-100 triton in bicarbonate buffer (0.05 M, pH 9.6) is added to destroy the VABN- complex F. Then a standard dilution is carried out and the plate is incubated. Wells are washed three times with PBS buffer (0.01 M, pH 7.5) with the addition of Tween-20 (0.05%).

The conjugate and antiserum were applied in 0.01 M phosphate buffer (pH 7.5) supplemented with 0.1% bovine serum albumin (Serva). At a certain dilution (Fig. 3), ferritin together with triton is washed and does not interfere with the determination of the virus. The substrate (orthophenylenediamine 0.05%) is applied in phosphate-citrate buffer (0.05 M, pH 5.0) with 0.003% hydrogen peroxide. The enzymatic reaction was stopped after 30 minutes. 4 H sulfuric acid and measured the optical density at a wavelength of 492 nm.

Example No. 2 implementation of the testing of serum.

1 ml of diluted antiserum is added to the wells of the polystyrene plate and incubated to adsorb antibodies to the plate. The tablet is washed and biological material (mink blood) pretreated with Triton X-100 diluted from 2000 to 20,000 times is added to the wells (Fig. 4). The tablet is incubated. Further processing is carried out as in example 1.

When comparing with the prototype method, the following advantages of the obtained diagnostic serum were revealed:

- the ability to test the virus of Aleutian mink disease in various biological fluids;

- carry out testing without preliminary expensive purification of the virus in the laboratory;

- reduce the cost of analysis.

The use of the invention in veterinary virology will allow the mass production of highly active serum to detect the Aleutian mink disease virus.

Claims (4)

1. A method of obtaining serum for the diagnosis of Aleutian mink disease (ABN), comprising obtaining a purified virus and immunizing animals with an antigen, characterized in that the antigen is obtained by homogenizing organs of infected mink ABN, for example, a spleen, liver, kidney, in a buffer system containing detergents in the ratio of 1 / 3-1 / 5 of the volume, in a mortar with the addition of quartz sand and then in the homogenizer, followed by three freezing / thawing of the homogenate, disintegration of the cell homogenate three times ultra by sound processing at a frequency of 22 kHz / s on an UZDN-2 apparatus, for 20 s with an interval of several s, sedimentation of undamaged cells by double low-speed centrifugation - clarification of the homogenate at 6000-8000 rpm for 20 min, deposition of immune complexes (IR) 10% polyethylene glycol (PEG-6000) or 25% ammonium sulfate for four hours or a night, compaction of the precipitate by low-speed centrifugation - 12,000 rpm for 30 minutes, extraction of immune complexes with minimal amounts, 1/10 of the initial volume, TNE buffer p threefold extraction in the cold for 1-2 hours, followed by low-speed centrifugation at 12,000 rpm for 20 minutes, precipitation by IR high-speed centrifugation from the combined extracts at 30,000 rpm for 2.5 hours, and the extraction of immune complexes from the sediment with minimal amounts of TNE buffer by triple extraction in the cold for 1-2 hours, followed by low-speed centrifugation at 12000 rpm for 20 min, dissociation of immune complexes in an acidic environment, pH 2.4, Hcl-glycine buffer Era in the ratio of 1V extract to 2V buffer for 1 hour in the cold, separation of VABN-F from antibodies by high-speed ultracentrifugation - 30000 rpm for 2.5 hours, followed by dissolution of the precipitate in a minimum amount, not more than 2 ml, of carbonate buffer pH 9.6 and separation of ferritin from the viral particles on a 6B Sepharose column in a carbonate buffer system, followed by chromatography twice, with control being carried out by spectrophotometry in the ultraviolet in the wavelength range 220-310, and the estimated concentration of virus sa is carried out according to the formula Kalkara:
K = 1.45 × E ₂₈₅ -0.74 × E ₂₆₀ ,
rabbits are used as animals for immunization, and the immunization itself is carried out in five stages:
- the first immunization with 200 μg of antigen + Freund's complete adjuvant (USA) was carried out subcutaneously at 9-12 points;
- the second immunization, 7 days after the first, - 200 μg of antigen + Freund's complete adjuvant (USA) intramuscularly and subcutaneously at 6-8 points;
- the third immunization, 7 days after the second, - 300 μg of antigen + Freund's complete adjuvant (USA) intramuscularly and subcutaneously at 6-8 points;
- the fourth immunization, 14 days after the third, - 200 μg of antigen + Freund's complete adjuvant (USA) intramuscularly and subcutaneously at 6-8 points;
- the fifth immunization, 7 days after the fourth, - 200 μg of the antigen intravenously, while the antibody titer was 1/16384. 2. A method for producing serum for the diagnosis of ABN according to claim 1, characterized in that 50 mM Tris-Hcl, 0.15 M NaCl, pH 7.2 are used as a buffer system. 3. A method for producing serum for the diagnosis of ABN according to claim 1, characterized in that 1% Triton X-100, 0.4 mM phenylmethylsulfonyl fluoride, 1% sodium deoxycholate (Sigma USA) are used as detergents. 4. A method for producing serum for the diagnosis of ABN according to claim 1, characterized in that 10 mM Tris-Hcl, 0.1 M NaCl, 1 mM EDTA-Na, 0.05% NP-40 are used as the TNE buffer (Sigma USA ), pH 7.4.

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