RU2782275C1 - Method for diagnosis of classical swine fever by indirect immunohistochemical analysis based on monoclonal antibodies - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: invention relates to the field of veterinary virology and can be used in research institutions and veterinary laboratories for the diagnosis of classical swine fever in histological sections of susceptible organs by detecting the virus antigen on the surface and in the cytoplasm of infected cells during immunohistochemical examination based on monoclonal antibodies. The technical problem is the need to develop an effective and affordable method for diagnosing classical swine fever to detect the virus antigen in pig tissues and organs, simply feasible in histological laboratories without the use of expensive consumables, expanding the arsenal of methods for diagnosing classical swine fever. A method for the diagnosis of classical swine fever virus by indirect immunohistochemical analysis based on the use of monoclonal antibodies is proposed, in which pig organs are examined for the presence of virus antigen using monoclonal mouse antibodies of clone 3c3, specific to the recombinant protein E2 (rE2) of the virus, and secondary donkey antibodies against mouse antibodies labeled with horseradish peroxidase, followed by specific staining and the identification of red-brown conglomerates as a positive result.

EFFECT: determination of the antigen of the classical swine fever virus in susceptible organ cells, the expansion of the arsenal of methods for diagnosing classical swine fever.

1 cl, 4 dwg

Description

The invention relates to the field of veterinary virology and can be used in research institutions and veterinary laboratories for the diagnosis of classical swine fever in histological sections of susceptible organs by detecting the virus antigen on the surface and in the cytoplasm of infected cells during immunohistochemical studies based on monoclonal antibodies.

State of the art

For the diagnosis of CSF, a variety of laboratory methods are used to isolate the virus, detect its antigen or nucleotide sequence. Among the studies aimed at achieving this goal, virus isolation in cell cultures, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), fluorescent in situ hybridization and immunohistochemical study (IGHI) are used [Stafford V.V., Streltsova Ya.B. . Immunohistochemistry and in situ hybridization in the diagnosis of classical swine fever. Veterinary and nutrition. No. 6. - 2018. - P. 14-16., Internet site of the World organization for animal health (OIE) "Manual of Diagnostic Tests and Vaccines for Terrestrial Animals" (Chapter 3.9.3) [Electronic resource]. - Access mode: https://www.oie.int/en/what-we-do/standards/codes-and-manuals/terrestrial-manual-online-access/].

Foreign experts, for IHCA of organs and tissues from CSF-infected pigs, despite a large number of modifications of the method, adhere to the protocol published in the World Organization for Animal Health (OIE) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Chapter 3.9.3). The methodology is as follows. As pathological material, tonsils, spleen, lymph nodes, etc. are taken from pigs, which are fixed with cold and stored at a temperature of -70°C. After defrosting, the areas necessary for the study are excised from the organs, and native histological preparations are made using a cryotome. Histological sections prepared by this method can be used 20 minutes after drying, or additionally fixed with acetone, which makes it possible to store slides with sections without damage for 2-3 years at a temperature of -70°C. Dried freshly prepared sections of organs are fixed in acetone for 10 minutes at a temperature of -20°C, after which the sections are re-dried in an air stream. Next, a working dilution of monoclonal antibodies specific to the pathogen antigen is prepared with the addition of phosphate-buffered saline (PBS) pH 7.6, 0.01% Tween 80 and 5% horse serum. Before applying the antibody working solution, the sections are rinsed in PBS, then excess liquid is removed, the antibody solution is applied, and the sections are incubated in a humid chamber for 30 minutes at 37°C. After incubation, the sections are washed in PBS 6 times for 10 seconds each at room temperature. Washed sections are stained with freshly prepared chromogen substrate solution for 15 minutes at room temperature. To prepare a solution, 1 ml of a mixture of 0.4% 3-amino-9-ethylcarbazole and N,N-dimethylformamide is added to 19 ml of 0.05 M sodium acetate (pH 5.0), activation of the solution is carried out by adding 10 μl of a 30% peroxide solution hydrogen. After the sections are rinsed in 0.05M sodium acetate pH 5.0, then in distilled water and sealed with mounting medium. This protocol is used when performing a direct IHCA method with horseradish peroxidase labeled antibodies. If an indirect reaction method is used, then after incubation of the sections with primary antibodies specific to the antigen of the virus (without a label), they are rinsed and then secondary antibodies labeled with horseradish peroxidase are applied. By adding this step, the analysis time is increased, but its specificity is greatly increased. The remaining stages of IHCA do not differ in execution both in the direct and in the indirect version of the study [Internet site World organization for animal health (OIE) "Manual of Diagnostic Tests and Vaccines for Terrestrial Animals" (Chapter 3.9.3) [Electronic resource] . - Access Mode: https://www.oie.int/en/what-we-do/standards/codes-and-manuals/terrestrial-manual-online-access/1. We took this development as a prototype.

On the territory of the Russian Federation, for the diagnosis of classical swine fever, a bioassay, polymerase chain reaction, immunofluorescence reaction, neutralization reaction and enzyme immunoassay are used [Stafford V.V., Streltsova Ya.B. Immunohistochemistry and in situ hybridization in the diagnosis of classical swine fever. Veterinary and nutrition. No. 6. - 2018. - P. 14-16., Guidelines for the laboratory diagnosis of classical swine fever // Approved. Ministry of Agriculture and Food of Russia 30.12.1996 N 13-4-2 / 809.].

The technical problem is the need to develop an effective and affordable method for diagnosing classical swine fever, for detecting the antigen of the virus in tissues and organs of pigs, which is simply feasible in histological laboratories without the use of expensive consumables, expanding the arsenal of methods for diagnosing classical swine fever.

Disclosure of the essence of the invention

The technical result is the determination of the antigen of the classical swine fever virus in susceptible organ cells, expanding the arsenal of methods for diagnosing classical swine fever.

This method allows you to determine the localization of the antigen in the body, as well as to compare the presence of the antigen with histopathological changes, thereby confirming the pathological effect of the virus on infected tissues and organs. The use of this method is important for pathomorphological studies and contributes to accurate differential diagnosis, the study of the pathogenesis of the immune response in a sick animal.

The proposed method is as follows.

To perform IHCA on native histological sections from pig organs, primary mouse monoclonal antibodies of clone 3c3 specific to the recombinant protein E2 (rE2) of the classical swine fever virus and secondary donkey antibodies against mouse antibodies labeled with horseradish peroxidase are used. Primary mouse antibodies specific to the antigen of the virus were obtained at the Institute of Virology. DI. Ivanovsky Federal State Budgetary Institution "National Research Center for Epidemiology and Microbiology" N.F. Gamaleya" of the Ministry of Health of Russia. Donkey secondary antibodies against mouse antibodies manufactured by SIGMA (USA). Obtaining and characterization of monoclonal antibodies (mAbs).

BALB/c female mice were used for immunization. The recombinant protein E2 (rE2) of the Shi-Men strain of HCSF, obtained in SF-21 cell culture with baculovirus as a vector, was taken as antigens. Mice were immunized with purified rE2 HCSF with Freund's adjuvant (Difco, USA) 4 times with an interval of 2 weeks at a concentration of 50 μg/mouse. After that, splenocytes were obtained from immunized mice, which were hybridized with a transplantable Sp2/0 mouse myeloma cell line that does not produce its own immunoglobulins. PEG/DMSO Solution (Sigma, USA) was used for fusion. Hybridization was carried out according to the method of G. Kohler. After 10-12 days, cells grown in 96-well plates were tested by indirect ELISA. Positive hybridoma cells were cloned.

To obtain ascitic fluid with MAb, female BALB/c mice treated with pristane (Sigma, USA) were intraperitoneally injected with 5-10 million hybrid cells. MAbs obtained from ascitic fluid were purified by protein precipitation with caprylic acid (Sigma, USA). The concentration of antibodies after purification was measured on a spectrophotometer at a wavelength of 280 nm. The assessment of the immunological activity of MAb was carried out using indirect ELISA. [Kostina L.V., Nepoklonov E.A., Zaberezhny A.D. et al. Obtaining and characterization of monoclonal antibodies to the E2 protein (GP 55) of the classical swine fever virus. Questions of virology. No. 5 T 53. - 2008. - S. 36-40].

Performing IHCI with mouse mAbs obtained against classical swine fever virus antigen:

Tonsils and spleen are taken from dead animals as pathological material for determining the localization of the classical swine fever virus antigen. Native pathological material is cooled for 6 hours in a refrigerator at a temperature of 4°C. Transportation of samples is carried out in a cooler bag with ice packs that maintain a temperature of +2-4°C during the entire transportation stage. Then, for long-term storage, the material is placed in a low-temperature chamber and stored at a temperature of -70°C. Samples are thawed immediately before testing. Further, the necessary samples are excised from the selected organs, 1-2 cm in length and width and 0.5 cm in thickness. The excised parts of the organs are fixed on the tables for cryotomy, filling the area between the table and the tissue with a special cryogel. Tables with objects are placed in the cryotome chamber at -34°C for 40-60 minutes. Slices 5 µm thick are cut from the finished block and glued onto polarized glasses. Dried glass sections are immersed in a mixture of isopropyl alcohol 30 ml and saline 70 ml for 60 minutes at a temperature of +4°C. The treated sections are dried in a stream of air for 40 minutes. The dried sections are rinsed in 3 portions of phosphate-buffered saline (PBS) pH 7.4 for 5 minutes each at room temperature. Next, to remove endogenous peroxidase, 3% hydrogen peroxide solution 300 μl is applied to the sections for 40 minutes at room temperature. Then repeat the rinsing of sections in 3 portions of PBS pH 7.4 for 5 minutes each at room temperature. Then prepare a blocking solution of 5% non-fat dry milk in PBS pH 7.4 at the rate of 200 µl per glass. The solution on sections is incubated in a thermostat for 40 minutes at a temperature of 37°C. After incubation, the sections are washed in 3 portions of PBS pH 7.4 for 5 minutes each at room temperature. Parallel to the process of rinsing the sections, a working solution of primary antibodies (AT) specific to the classical swine fever virus antigen is prepared. For this, PBS pH 7.4 was added to AT. The concentration of antibodies is selected empirically depending on their sensitivity. The dilution of AT is from 1:200 to 1:400. Next, 200 μl of primary antibody solution is applied to the sections, moist chamber conditions are created and placed in a thermostat for 80 minutes at a temperature of 37°C. After, the sections are washed in 3 portions of PBS pH 7.4 for 5 minutes each at room temperature. Simultaneously with the process of washing the sections, a working solution of secondary antibodies labeled with horseradish peroxidase is prepared. AT solution is made in PBS pH 7.4 with the addition of 5% skimmed milk powder to prevent non-specific binding of AT. The dilution of secondary AT is 1:200. 200 μl of secondary AT solution is applied to the sections, moist chamber conditions are created and placed in a thermostat at a temperature of 37°C for 60 minutes. Next, the sections are washed in 3 portions of PBS pH 7.4 for 5 minutes each at room temperature. Then, a working solution of the substrate is prepared in Na-phosphate buffer for specific staining of the peroxidase label. The working solution is prepared immediately before use and applied in an amount of 200 μl to one slide, after which the slides are placed in a humid chamber in a thermostat at 37°C for 30 minutes.

Chromogen stock solution: 0.02 g of 3-amino-9-ethylcarbazole was dissolved in 3 ml of dimethylformamide. Work must be done with gloves and in a fume hood, as the solution has carcinogenic properties (stored at +4°C).

The working solution is prepared in 0.02 M Na-phosphate buffer, pH 5.0 -5 ml, with the addition of 300 μl of stock solution and 1 μl of 3% hydrogen peroxide solution (shelf life 12 hours).

Next, 300 μl of working solution of chromogen is applied to the sections, conditions of a humid chamber are created and left for 20-40 minutes at room temperature. Then the sections are washed in 3 portions of PBS pH 7.4 for 5 minutes each at room temperature. After that, for differential staining of the structural elements of cells and tissues, Mayer's hematoxylin is applied to the sections for 5-10 minutes at room temperature. Stained sections are rinsed in distilled water, a mounting glycerin medium is applied and covered with a coverslip.

This method is applicable for indirect immunohistochemical analysis of native cryosections of tonsils, spleen, lymph nodes and other organs using mouse monoclonal antibodies of clone 3c3 to the classical swine fever virus antigen, as well as donkey secondary antibodies against mouse antibodies labeled with horseradish peroxidase. Detection of the virus antigen occurs on the surface and in the cytoplasm of susceptible cells of infected organs by the formation of antigen-antibody conglomerates and staining of the horseradish peroxidase label in red-brown color, which is clearly visible in the microscope both in preparations without additional staining of the structural elements of the tissue, and in preparations whose nuclei were stained with Mayer's hematoxylin. When using the proposed method, cell nuclei stained blue are visualized, and a positive reaction to the presence of the classical swine fever virus antigen is recorded as red-brown chromogen conglomerates on the surface and in the cytoplasm of susceptible cells (Fig. 1-2).

The proposed method has high specificity and sensitivity to the antigen of the classical swine fever virus and allows you to accurately detect the localization of the antigen of the virus, as well as to differentially diagnose CSF from other pathogens of swine infections with a similar clinical picture.

The Department of Pathomorphology of the Federal State Budgetary Scientific Institution Federal Scientific Center of the VIEV RAS has successfully tested the above method for diagnosing classical swine fever in scientific and practical activities. According to the test results, this research method can be recommended for use in research institutes and veterinary laboratories.

The implementation of the invention is illustrated by the following example:

Example #1

From 20 dead pigs, fresh material was selected for research (tonsils and spleen), since it was not possible to immediately deliver the material to the laboratory, it was cooled in a household refrigerator for 6 hours, then it was placed in a freezer at a temperature of -20 degrees for complete freezing. After freezing, the material was sent to the laboratory in a thermal bag with ice packs. In the laboratory, the material was thawed, samples 2 cm by 2 cm in size were excised from each organ, frozen on cryotomy tables in the cryostat chamber, and 2 sections were cut into glass from each organ. Sections 5 microns thick were placed on poly-L-lysine slides and dried. Dried glass sections were immersed in a mixture of isopropyl alcohol 30 ml and saline 70 ml for 60 minutes at a temperature of +4°C. The treated sections were dried in a stream of air for 40 minutes. The dried sections were rinsed in 3 portions of phosphate-buffered saline (PBS) pH 7.4 for 5 minutes each at room temperature. Next, to remove endogenous peroxidase, 3% hydrogen peroxide solution (300 µl) was applied to the sections for 40 minutes at room temperature. After, the sections were rinsed in 3 portions of PBS pH 7.4 for 5 minutes each at room temperature. Then a blocking solution was prepared with 5% non-fat dry milk in PBS pH 7.4 at the rate of 200 μl per glass. Sections with the solution were incubated in a thermostat for 40 minutes at a temperature of 37°C. After incubation, the sections were washed in 3 portions of PBS pH 7.4 for 5 minutes each at room temperature.

Parallel to the process of rinsing the sections, a working solution of primary monoclonal antibodies (AT) of mouse clone 3c3 specific to the recombinant E2 protein (rE2), specific to the classical swine fever virus antigen, was prepared. For this, PBS pH 7.4 was added to AT. The concentration of antibodies was selected empirically depending on their sensitivity. In our case, the dilution of AT ranged from 1:200 to 1:400. Next, 200 μl of a solution of primary antibodies were applied to the sections, the conditions of a humid chamber were created, and they were placed in a thermostat for 80 minutes at a temperature of 37°C. After, the sections were washed in 3 portions of PBS pH 7.4 for 5 minutes each at room temperature. Simultaneously with the process of washing the sections, a working solution of secondary donkey antibodies, specific to primary ones, labeled with horseradish peroxidase, was prepared. The AT solution was made in PBS pH 7.4 with the addition of 5% skimmed milk powder to prevent non-specific binding of AT. The dilution of secondary AT was 1:200. The sections were covered with 200 μl of secondary AT solution, the conditions of a humid chamber were again created, and placed in a thermostat at a temperature of 37°C for 60 minutes. Next, the sections were washed in 3 portions of PBS pH 7.4 for 5 minutes each at room temperature. Then, a working solution of the substrate was prepared in Na-phosphate buffer for staining the peroxidase label. The working solution was prepared immediately before use and applied in an amount of 200 μl to one slide, after which the slides were placed in a humid chamber in a thermostat at 37°C for 30 minutes.

Chromogen stock solution: 0.02 g of 3-amino-9-ethylcarbazole was dissolved in 3 ml of dimethylformamide. The work was performed with gloves and in a fume hood, since the solution has carcinogenic properties (it is stored at a temperature of +4°C).

The working solution was prepared in 0.02 M Na-phosphate buffer, pH 5.0-5 ml, with the addition of 300 μl of stock solution and 1 μl of 3% hydrogen peroxide solution.

Next, 300 μl of the working solution of chromogen was applied to the sections, the conditions of a humid chamber were created, and left for 20-40 minutes at room temperature. Then the sections were washed in 3 portions of PBS pH 7.4 for 5 minutes each at room temperature. Afterwards, for differential staining of the structural elements of cells and tissues, Mayer's hematoxylin was applied to the sections for 10 minutes at room temperature. Stained sections were rinsed in distilled water, a mounting glycerol medium was applied, and covered with a coverslip.

As a result of the study of the material, it was revealed that 18 pigs were infected with classical swine fever.

A positive reaction to the antigen of the classical swine fever virus is shown in figures 1 and 2, where the staining of the chromogen in red-brown color is clearly visualized on the surface and cytoplasm of susceptible cells. Upon receipt of a negative test result for the presence of the antigen of the virus, there is no red-brown staining of the cells, which is clearly shown in figures 3 and 4.

Brief description of the drawings

Fig. 1 - Spleen. A positive IHCA result with the formation of chromogen conglomerates on the surface and in the cytoplasm of susceptible cells. Stained with Mayer's hematoxylin. SW. 630x.

Fig. 2 - Tonsil. A positive IHCA result with the formation of chromogen conglomerates on the surface and in the cytoplasm of susceptible cells. Stained with Mayer's hematoxylin. SW. 630x.

Fig. 3 - Spleen. Negative IHCA result, no chromogen conglomerates. Stained with Mayer's hematoxylin. At v. 630x.

Fig. 4 - Tonsil. Negative IHCA result, no chromogen conglomerates. Stained with Mayer's hematoxylin. At v. 630x.

Claims (1)

A method for diagnosing classical swine fever by indirect immunohistochemical analysis based on monoclonal antibodies, which includes performing an indirect immunohistochemical study using primary virus-specific monoclonal antibodies and secondary ones with a peroxidase label specific to primary antibodies, characterized in that the object of the study is native cryotomy sections from organs of pigs, mouse monoclonal antibodies of clone 3c3 to the recombinant protein E2 (rE2) of the classical swine fever virus, specific to the antigen of the virus, are used as primary antibodies, and donkey antibodies specific to primary and labeled with horseradish peroxidase are used as secondary antibodies, followed by staining marks and the formation of red-brown conglomerates on the surface and in the cytoplasm of susceptible cells.

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