RU2614256C2 - Method of detecting of cell culture contamination with viruses by immunoperoxidase method - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to biochemistry. Method of detecting contamination with viruses of cell culture by immunoperoxidase method is described, involving reaction of antigen with labelled antibodies in clean cell culture and accounting of reaction results by color density of formed complex under microscope. Wherein reaction uses tablets with analyzed cell culture, contaminated with diarrhoea virus, and after adding into tablet wells of 0.10–0.12 ml of specific homologous serum diluted by 1:64 – 1:128 it is incubated, washed, then 0.10–0.12 ml of anti-specific immunoenzymometric conjugate is introduced, consisting of peroxidase marked antibodies against globulins of cattle blood serum, it is incubated, washed, substrate mixture is introduced consisting of 5-aminosalicylic acid and hydrogen peroxide, and 30–40 minutes later results are accounted under light microscope by formation of brown color in virus-containing cells. Invention improves control system of cell lines latent contamination with viruses used in laboratory practice and biological industry.

EFFECT: invention allows to prevent biological contamination and is extremely important stage in production of vaccines and diagnostic preparations; this method can be used for certification at absence of viruses in again coming cell cultures; invention is practically feasible, cheap method of monitoring of cell cultures for content of virus and may be recommended for extensive use both in scientific research and practical virological laboratories, and in production in making diagnostic preparations and vaccines.

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Description

The present invention relates to the field of veterinary virology, in particular to veterinary biotechnology, namely the detection of artifacts (viruses) in cell culture by the “indirect” immunoperoxidase method.

The contamination of cell cultures with viruses is a problem both for scientific research in virology and biotechnology, and for the production of various biological products (diagnosticums and vaccines).

The source of infection of cell cultures can be media, reagents, serum, laboratory personnel, etc. Diagnostics made from contaminated raw materials can give false research results. In laboratory practice, cases of contamination of transplanted cell cultures by the diarrhea virus of non-cytopathogenic biotype are known.

A known method for detecting contamination of cell cultures by the virus of cattle diarrhea in the polymerase chain reaction (PCR) (1).

There is also a method of detecting contamination of a cell culture by the diarrhea virus in an immunofluorescence reaction, for which a cell monolayer is washed with a phosphate-buffered solution of pH 7.2 and fixed with acetone. Cells are incubated with immunoglobulins of mice against VD-BS labeled with FITC taken in working dilution for 45 min at 37 ° C. After that, the cells are washed with phosphate-buffered saline 3-4 times, after which they are examined under a luminescent microscope. VD-BS virus is judged by yellow-green fluorescence in cells (2).

The research objective was to develop a specific, sensitive, equipment-free method for detecting contamination of cell cultures by the cattle diarrhea virus.

The essence of the proposed method is as follows.

Wells of a plate with a grown monolayer of cell culture are washed 3-4 times with phosphate-buffered saline (pH 7.2-7.4), fixed with acetone, washed with buffer, specific serum is applied to the monolayer in a working dilution of 1:64 - 1: 128 in volume 0.10-0.12 ml, incubated 1.5-2 hours at a temperature of 36-37 ° C, washed, applied, 0.10-0.12 ml of anti-enzyme immunoassay conjugate consisting of horseradish peroxidase-labeled antibodies to immunoglobulins of specific blood serum cattle, incubated under the same conditions, washed and after adding substrate see B 0.10-0.12 ml carried out keeping the reaction results after 30-40 minutes for the virus under a light microscope. The method can also be carried out on slides.

Example 1

At the first stage of the reaction, the wells of the plate were washed from the growth medium with phosphate buffer pH 7.2, and fixed with acetone. Then, a 0.1 ml hyperimmune serum to a diarrhea virus in titer 1:64 was applied to the cell monolayer with an automatic pipette, incubated in an incubator for 1.5 hours at 36 ° C, then washed from unbound antibodies and introduced 0.10-0.12 ml anti-enzyme immunoassay conjugate consisting of anti-species immunoglobulins labeled with horseradish peroxidase. The plate was incubated in a thermostat at 36 ° C for 1.5 h and again washed with phosphate-buffered saline. Then, 0.1 ml of a substrate mixture was applied, consisting of a solution of 5-aminosalilicylic acid and hydrogen peroxide. The tablet was viewed under a light microscope. The result was taken into account after 30 min from the appearance of brown to light brown color in the cell cytoplasm of the resulting reaction product.

As a control, negative serum was used instead of hyperimmune serum.

To control the reaction under the same mode, a non-contaminated diarrhea virus cell culture was treated.

7 experiments were conducted on the development of a method for detecting contamination of cell culture by cattle diarrhea virus by immunoperoxidase method with a positive result.

In control preparations, staining was absent.

Example 2

The reaction was carried out analogously to example 1.

At the first stage of the reaction, the wells of the plate were washed from the growth medium with phosphate buffer pH 7.2, and fixed with acetone. Then, 0.1 ml hyperimmune serum to the diarrhea virus in titer 1: 128 was applied to the cell monolayer with an automatic pipette, incubated in a thermostat for 2 h at 37 ° C, then washed from unbound antibodies and an anti-type immunosorbent conjugate consisting of anti-type immunoglobulins labeled horseradish peroxidase. The plate was incubated in a thermostat at 37 ° C for 2 h and again washed with phosphate-buffered saline. Then, 0.1 ml of a substrate mixture consisting of a solution of 5-aminosalicylic acid and hydrogen peroxide was applied. The tablet was viewed under a light microscope. The result was taken into account after 40 min from the appearance of brown to light brown color in the cell cytoplasm of the resulting reaction product. The control is similar to example 1.

5 experiments were carried out to identify the contamination of cell culture with cattle diarrhea virus by immunoperoxidase method with a positive result.

In control preparations, staining was absent.

The proposed method is sensitive and specific, in laboratory conditions reveals non-cytopathogenic strains of the diarrhea virus. It is simple in execution, not laborious, does not require special equipment.

The proposed method, using a set of specific sera for various pathogens, can significantly reduce the time to detect contamination of cell culture.

Improving the control system aimed at preventing biological pollution is an extremely important stage in the production of biological preparations (vaccines, diagnostic kits, etc.).

The proposed method for detecting the contamination of cell cultures with cattle diarrhea virus by immunoperoxidase method was tested with a positive result in 2014 in the Virology Laboratory of FSBIU VIEV named after Ya.R. Kovalenko.

In connection with the foregoing, the problem of improving the control system for covert virus contamination of cell lines used in laboratory practice and the biological industry is aimed at preventing biological contamination and is an extremely important stage in the production of vaccines and diagnostics. This method can be used for certification for the absence of viruses in newly introduced cell cultures.

The method is almost easily feasible for monitoring cell cultures for the content of viruses in them and is recommended for widespread use both in research and practical virological laboratories, and in production in the manufacture of diagnosticums and vaccines.

Information sources

1. Newsletter, “Cell Culture”. M .: VIEV, 2011, no. 27, pp. 80-88.

2. Russian Veterinary Journal. "Farm animals", Moscow: 2013, 1, p. 15-18.

Claims (1)

A method for detecting viral contamination of cell culture by the immunoperoxidase method, including the interaction of antigen with labeled antibodies in an uninfected cell culture and taking into account the results of the reaction by the intensity of staining of the resulting complex under a microscope, using plates with the analyzed cell culture contaminated with diarrhea virus in the reaction and after wells of a specific homologous serum tablet of 0.10-0.12 ml at a dilution of 1:64 - 1: 128 are incubated, washed, then 0.10-0.12 ml of anti a specific enzyme immunoassay conjugate consisting of horseradish peroxidase-labeled antibodies against bovine serum globulins is incubated, washed, a substrate mixture consisting of 5-aminosalicylic acid and hydrogen peroxide is introduced, and after 30-40 minutes the results are counted under a light microscope to form brown staining in virus-containing cells.