RU2352357C1 - Method of producing universal inactivated blutang virus antigen for serodiagnosis - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: method includes infecting the transplantable cell culture, cultivating the infected culture, settling the cells and cell detritus by centrifugation, preparing the cell lysate and freezing-defrosting. Herewith cell lysate is twice exposed to 12 mcm ultrasound within 1 minute every 1 min. After-centrifugation residual debris are refilled with distilled water, exposed to the same ultrasound. Supernatant fluids containing antigen are combined. Then antigen is double-activated with β-propiolactone in ratio 1:1000 at first at 37° constantly stirring, then β-propiolactone is introduced again in the same ratio, with exposition 18 hours and at temperature +4°C.

EFFECT: produced inactivated blutang antigen has high specificity and activity (tbl) and can be used for serodiagnosis in CFT, diffusion precipitation and EIA reactions.

1 tbl, 3 ex

Description

The invention relates to the field of veterinary medicine, namely to serodiagnosis of bluetong. According to the OIE, the bluetongue virus is classified as a particularly dangerous animal disease of List A. According to the classification of the State Committee for Epidemiological Surveillance, the bluetongue pathogen belongs to the 2nd group of pathogenicity for humans.

In the 20th century, bluetong caused a huge economic damage to sheep breeding in many countries of the world, more than all other infectious diseases of sheep combined. Especially the problem of bluetong became relevant for Russia after the epizootic of the disease in the Republic of Buryatia in 1993. Considering that in recent years, in countries with close economic contacts with Russia (Turkey, Bulgaria, Yugoslavia, Holland, etc.), extensive epizootics caused by the bluetongue virus have been noted, one of the most reliable ways of intravital detection of this pathogen is serodiagnosis, based on the determination the presence of specific antibodies in blood serum during their interaction with the bluetongue antigen in complement binding reactions (CSCs), diffusion precipitation reactions (RDPs), and immuno-enzyme production analysis (ELISA).

Previously, antigen was prepared from the brain tissue of hamsters and mice for the production of CSCs (Clark D. and Casals J., 1958, B.H. Syurin, 1966). Subsequently, a method was developed for the production of cultural antigen (A.A. Strizhakov, 1997). The PSHA cell culture was infected with bluetongue virus and cultured for 60-62 h. Then, the culture fluid was drained, and the infected cells were frozen, thawed twice, centrifuged at 3000 rpm for 30 min, and the supernatant was used as antigen.

The main disadvantage of such an antigen is that it contains an infectious virus and it can be used only when obtaining permission to work with microorganisms of the 2nd group of pathogenicity and requires special protective equipment, equipment and special care.

Therefore, the aim of the invention is to obtain an inactivated universal antigen for serodiagnosis, not inferior in antigenic activity and suitable for use in any diagnostic laboratory.

This goal is achieved by the fact that the method of obtaining a universal inactivated antigen of the bluetong virus includes infection of a transplanted cell culture, cultivation of an infected culture, sedimentation of cells and cell debris by centrifugation, preparation of a cell lysate, freezing-thawing, while the cell lysate is treated 2 times with ultrasound at 12 μm per for 1 min with an interval of 1 min, and the debris residue remaining after centrifugation is re-poured with distilled water, treated with ultrasound in t In the same mode, the supernatants are combined with the antigen, then the antigen is twice inactivated with β-propiolactone in a ratio of 1: 1000, while at first at 37 ° with constant stirring, then β-propiolactone is again added in the same ratio at an exposure of 18 hours + 4 ° FROM.

The proposed method for the preparation of antigen is characterized in that:

1) for greater destruction of cells using ultrasound treatment, which allows to increase the yield of viral antigen associated with debris of cells;

2) re-processing of cell debris remaining in the sediment after centrifugation, allows to increase by 2 times the yield of the resulting antigen using the same amount of viral raw material;

3) the use of the inactivant β-propiolactone allows you to get inactivated environmentally friendly antigen, which can be widely used in veterinary practice for serodiagnosis of bluetongue.

Examples of specific performance.

Example 1. A suspension culture of PSGK-60 cells in an amount of 10 l with a cell concentration of 0.7-1.0 million cells / ml was infected with bluetongue virus with a multiplicity of infection of 0,01 TCD ₅₀ / cell. Supporting medium - 0.25% FGM with 2% serum of cattle (cattle). The virus was cultured at 37 ° C, maintaining the pH of the medium in the range of 7.0-7.4 for 40-60 hours until 30% dead cells appeared in the suspension. After this, the cells were besieged by centrifugation at 4000 rpm for 30 minutes and a lysate of infected cells was prepared. For this purpose, distilled water up to 100 ml was added to the precipitate, resuspended and frozen at minus 20 ° С. After thawing, they were sonicated 2 times in 12 μm for 1 minute at intervals of 1 min and again the suspension with cell debris was frozen and thawed, then centrifuged at 4000 rpm for 30 min. The supernatant containing bluetongue antigen was removed, and the same amount of distilled water was added to the precipitate, resuspended, and the same operations were carried out with it. The resulting secondary clarified lysate was combined with the first and inactivated with β-propiolactone. To this end, 0.2 ml of β-propiolactone was added to 200 ml of the combined supernatant of lysed cells containing bluetongue virus antigen heated to 37 ° C and kept at this temperature for 2 hours with constant stirring. Then again introduced β-propiolactone in the same amount and kept for 18 hours at + 4 ° C. The completeness of antigen inactivation was checked in 3 consecutive passages in a sensitive PSGK cell culture. The resulting antigen did not contain residual infectivity, had high specificity and activity in the reactions of CSC (1: 32-1: 64), RDP (1: 2-1: 4), ELISA (1: 729-1: 2187, table).

Example 2. A suspension culture of BHK-21/13 cells in an amount of 12 L with a cell concentration of 0.7-1.0 million cells / ml was infected with bluetongue virus with a multiplicity of infection of 0, 001 TCD ₅₀ / cell. Maintenance medium - 0.25% FGM with 2% cattle serum. The virus was cultured in a 30 L bioreactor at 37 ° C, maintaining the pH of the medium in the range of 7.0-7.4 for 45-55 hours until 40% dead cells appeared in the suspension. Then the cells were besieged by centrifugation at 4000 rpm for 30 minutes and a lysate of infected cells was prepared. For this purpose, distilled water up to 120 ml was added to the precipitate, resuspended and frozen at minus 20 ° С. After thawing, they were sonicated 2 times in 12 μm for 1 minute at intervals of 1 min and again the suspension with cell debris was frozen and thawed, then centrifuged at 4000 rpm for 30 min. The supernatant containing bluetongue antigen was removed, and the same amount of distilled water was added to the precipitate, resuspended, and the same operations were carried out with it. The resulting secondary clarified lysate was combined with the first and inactivated with β-propiolactone. For this purpose, 0.24 ml of p-propiolactone was added to 240 ml of the combined supernatant of lysed cells containing bluetongue virus antigen heated to 37 ° C and kept at this temperature for 2 hours with constant stirring. Then again introduced β-propiolactone in the same amount and kept for 18 hours at + 4 ° C. The completeness of antigen inactivation was checked in 3 consecutive passages in an selective PSGK cell culture. The resulting antigen did not contain residual infectivity, had high specificity and activity in the reactions of CSC (1: 64-1: 128), RDP (1: 2-1: 4), ELISA (1: 2187-1: 6561, table).

Example 3. A monolayer cell culture CV-1, grown in a circular monolayer in 20 culture vessels with a capacity of 3 l, a filling volume of 0.5 l was infected with bluetongue virus with a multiplicity of infection of 0.004 TCD ₅₀ / cl. Supporting medium - Needle (MEM) with 2% cattle. The virus was cultured at 37 ° C and 8-12 rpm, maintaining the pH of the medium in the range of 7.0-7.4 for 60 hours until the development of 30-35% of the virus's CPD. Then the culture medium was drained, the monolayer of cells was frozen, followed by thawing, and after that, a lysate with destroyed infected cells was collected from all the bottles. Then distilled water up to 100 ml was added, resuspended and sonicated 2 times in 12 μm for 1 minute at intervals of 1 minute and again the suspension with cell debris was frozen and thawed, then centrifuged at 4000 rpm for 30 minutes. The supernatant containing bluetongue antigen was removed, and the same amount of distilled water was added to the precipitate, resuspended, and the same operations were carried out with it. The resulting secondary clarified lysate was combined with the first and inactivated with β-propiolactone. To this end, 0.2 ml of β-propiolactone was added to 200 ml of the combined supernatant of lysed cells containing bluetongue virus antigen heated to 37 ° C and kept at this temperature for 2 hours with constant stirring. Then, β-propiolactone was again added in the same amount and held for 18 h at + 4 ° C. The completeness of antigen inactivation was checked in 3 consecutive passages in an selective PSGK cell culture. The resulting antigen did not contain residual infectivity, had high specificity and activity in the reactions of CSC (1: 64-1: 128), RDP (1: 2-1: 4), ELISA (1: 2187-1: 6561, table).

Table The biological activity of the culture inactivated antigen No. p / p Cell culture Completeness of inactivation at passage 3 in cell culture Antigen activity in reactions RDP RSK IFA one PSGK-60 - * 1: 2-1: 4 1: 32-1: 64 1: 729-1: 2187 VNK-21 - 1: 2-1: 4 1: 64-1: 128 1: 2187-1: 6561 CV-1 - 1: 2-1: 4 1: 64-1: 128 1: 2187-1: 6561 Note: * - - no infectivity detected

Claims (1)

A method of obtaining a universal inactivated bluetongue virus antigen, including infection of a transplanted cell culture, culturing an infected culture, sedimentation of cells and cell debris by centrifugation, preparation of a cell lysate, freeze-thaw, characterized in that the cell lysate is treated 2 times with 12 μm ultrasound for 1 min with an interval of 1 min, and the debris sediments remaining after centrifugation are re-filled with distilled water, treated with ultrasound in the same mode, pumped full-time antigen-containing liquids are combined, then the antigen is inactivated twice with β-propiolactone in a ratio of 1: 1000, while at first at 37 ° with constant stirring, then β-propiolactone is again added in the same ratio at an exposure of 18 hours and a temperature of + 4 ° FROM.