SU1751202A1 - Strain of hybridous cultured mammalian cells mus musculus l , producing monoclonal antibodies to 146 s-component of foot-and-mouth virus a@@@ (aglie) - Google Patents

Abstract

Uses Biotechnologists, Virologists, Immunologists Summary of the Invention: The strain was obtained by fusing mouse myeloma cells of the PA line with cells of the spleen of the BAIB / C mouse line, previously immunized with the 146 S component of the culture virus AV (Alia}. On media for mammalian cell cultures

Description

The invention relates to biotechnology, in particular, hybridoma technology, and represents a new strain of hybrid cells producing monoclonal antibodies (Math) to cell virus and ascitic fluids Ab (Alia) used in virology and immunology for scientific research. and to obtain diagnostic therapeutic and prophylactic anti-schur drugs.

The closest to the proposed are the strains of the hybrid cultured Musmusculus L cells, producing MAb to the schur virus, As vaccine, As Parma, As Westerwald (AsW), and As Ispani -86

Mat to the 146S component of shchura A.5 virus vaccine studied in ELISA RIA and reactions

netralization (PH) in cell culture and mice. It has been established that the Mabs identified in ELISA and RIA are not active in RSK, however, some of them had virus-neutralizing properties. In ELISA, MAb are active against a homologous virus and did not react with heterologous (From and Ci) antigens. The obtained MAbs are useful in studying serological relatedness among various virus strains in epizootological studies and for controlling the quality of reagents in vaccine production.

The study of the Mat to the Schur As Parma virus was performed in a competitive analysis with 146S and 12 S particles. Besides,. The 146S antigen was treated with trypsin and chemotrypsin.

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Working with the Mat obtained for the AsW virus established a wide range of antibody specificity for variants of the type A virus, but they differed in activity depending on the strain of the Mat for the AgW virus in the PH, were inactive for the Ot and C viruses, but neutralized isolates containing antigen As (3).

The properties of mAbs obtained on the 146S antigen of the schur As Ispani -86 virus were studied in immunochemical and serological reactions. Mats are highly active and specific in RIA and inactive in PH.

The conducted research allowed us to divide the obtained MAbs into 5 groups, which reacted differently with the components of the schur virus, including after treatment with enzymes

MAbs with different spectrum of characteristics can be used to make a panel of antibodies. However, MAbs obtained for the Ad Pzpma, AsW, As Ispani -86 virus are not identical to antibodies produced by As (Alia), and differ in activity in immunological reactions. x PH and protection reactions (RE)

The Asp (Asie) virus is a manufacturing agent for the manufacture of diagnostic products and vaccines in Eastern European countries. The Mat to the 146S component of the Schur As virus (Allie) is not commercially available in the USSR and abroad.

The aim of the invention is to obtain a strain of hybrid cultured animal cells Musmusculus L, producing a Mat to the 146S component of the As virus (Alia), highly active in ELISA and PH, having high specific activity in ELISA and PH against As (Alia) and A and not active with heterologous strains of the A22, Ai2, Ayu, Och, d. Azi -1

The goal has been achieved by obtaining a new strain of hybrid cultured animal cells Musmusculus L, SCC (P) 476D, which produces monoclonal antibodies to the 1465 component of the schur As virus (Allie).

Monoclonal antibodies obtained from the proposed strain are homogenous. The titer of these antibodies is constant, the specificity is high. Hybrid cells can be grown in desired volumes.

The obtained MAbs against the schur As virus (Allie) reacted in ELISA with the 146S antigen of the As virus (Alli) and A, which indicates its high antigenic relationship and high specificity. Similar results were obtained in PH and RH in suckling mice.

The proposed strain under the author's name No. 7 of GPNA A-5-89 was deposited in the All-Union Cell Culture Collection (Leningrad) under registration number VSKK (P) 476D.

The proposed strain is obtained as follows.

Mice of the BALB / C line at the age of 1.5– 2 months were injected with 146S component of the cultured scurvy virus As (Alie) in an amount of 100 μg / mouse intraperitoneally in a 1: 1 ratio with complete Freund-adjuvant. After 21 days, mice were hyper-immunized intraperitoneally without mice. adjuvant with the same antigen at a dose of 100 µg / mouse

After 3 days after the reintroduction of antigen, the serum of the mice in the ELISA was tested for the presence of specific antibodies. Donors with an antibody titer in the serum of at least 103 were selected for the test.

For hybridization in immunized mice, the spleen was sterilely taken and resuspended in 50 ml of DM EM culture medium without serum. The PAI myeloma cells in the logarithmic growth phase were washed in DMEM medium without serum mixed with immune cells of the spleen at a ratio of 1 1 and precipitated by centrifugation at 1000 rev. A min. 50% solution of polyethylene glycol (PEG) with mol 4000, containing 5% dimethyl sulfoxide (DMZO) was added dropwise to the precipitate within a minute. Cells were left for 3 minutes with PEG. Then 5 ml of DMEM medium was added without serum and left for contact for 3 minutes. The resulting cell suspension was diluted with DMEM containing 20% fetal serum and hypoxanthine-aminopterin-thymidine (GAT) in an amount of 13.6 mg / l, 0.176 mg / L and 3.78 mg / L, respectively, in working concentrations. Cells were applied to 96- and 24-well plates from Flow Laboratories (UK) 0.2-1 in the amount of 100-20 thousand / well, respectively. After 10 days, the supernatants of the hybrid clones were tested for the presence of antibodies in the ELISA. Positive clones made up 5-7% of the total number of hybrid cells. These clones were subcloned twice by the limiting dilution method. The selected stable clone with the highest antibody titer was designated No. 7 GPNA5-89 and deposited into the All-Union Cell Culture Collection (Leningrad) under registration number VSKK (P) 476D

The resulting strain has the following morphological and cultural characteristics

Morphological features

The cells are round, of various sizes. The nuclei occupy most of the cell,

Caryology culture.

The karyotype corresponds to the species (mouse). The modal class is 84 (16%), 86 (10%). 88 (16%) chromosomes. Modal class for the parent myeloma line PAI-64 chromosome.

Cultural signs

The strain grows on media for mammalian cultures 1MDM, DMEM, RPMH640 with the addition of 10-20% fetal bovine serum and 50 µg / ml gentamicin. When hybrid cells are selected, aminopterin 0.176 mg / l, hypoxanthine 13.6 mg / l, thymidine 3.78 mg / l are added. The optimal growth concentration of cells is 150000 cells / ml. The optimum pH of the medium is 6.8-7.4, the cells are incubated in a humid atmosphere with 5% CO2 at 37 ° C. When seeding, single cells form round-shaped colonies with smooth edges, then the colonies merge to form a solid monolayer,

At a planting dose of 150 thousand cells / ml, a continuous monolayer is formed on the 2-3rd day. Cells have good adhesion and are easily removed from the walls of the dish by shaking without using trypsin or versa. Cell viability is maintained by regular transfers to fresh nutrient medium. The frequency of reseeding 2-3 times a week. The multiplicity of sieving 1F2 1.5 Growth type -stationary suspension When administered hybridomas intraperitoneally to BALB / C mice, ascites or solid tumors are formed. Before the introduction of cells in 5-20 days, 0.5 ml of Angarsk oil (TU 38-101-84-81) is intraperitoneally injected into the animals. The antibody titer in ascitiche fluids in ELISA is 10 -10J. 3-4 ml of ascites are obtained from one mouse. Ascites occurs after 14–20 days.

Cell preservation.

Hybridomas were frozen for 20–25 passages of 4–10 million cells in a vial (volume 1.0–1.5 ml) in a cryoprotective medium containing 50% culture medium, 43% fetal serum and 7% DMSO. Cells in a cryoprotective environment are frozen on a programmatic freezer and stored at - 196 ° C in liquid nitrogen. Ottaipioi rapid in a water bath at 38 ° С

The number of viable cells after thawing is 60-90%. Once the medium becomes liquid, the cells are transferred to a conical plastic beaker with 50 ml of cold nutrient medium and precipitated by centrifugation at 1000 rpm.

Resuspend in 5 ml of DMEM with 20% fetal serum and GAT and plated on a 50 cm 3 glass mattress. Cells are incubated at 37 ° C. When the colonies of cells 5 fill 50-70% of the area of the mattress, a sample of the culture fluid is taken to determine the titer of antibodies to the scur virus using ELISA. The highest antibody titer is marked with complete monolayer.

0 The culture fluid can be used as a diagnostic drug, however, to obtain a higher antibody titer, up to 10 hybrid cells at a dose of 1-10 min / 0.5 ml are administered intravenously to BALB / C mice, primed with intra-syrupin Angarsk oil.

14-30 days after the appearance of ascites or solid tumors in mice, a liquid is collected (3-4 ml), the cells precipitate

0 by centrifugation at 1000 rpm for 10 minutes. The supernatant contains Mat.

The pellet containing the hybrid cells is cultured or reintroduced into myocardium. To increase the specificity of the MAb, they are purified and pure immunoglobulins are isolated. An immunoglobulin fraction is obtained by precipitating twice with 10% PEG (a) m, m. 6000 (to antibodies

0 add an equal volume of 20% PEG and centrifuge at 3500 rpm for 30 minutes. The pellet is resuspended in a reduced amount of phosphate buffer solution (PBS). The solution was dialyzed against FBI for 24 hours at 4 ° C. The culture fluid contains 10-20 µg / ml, and ascites - 20-30 mg / ml of specific immunoglobulins,

Contaminant line information. No bacteria were detected, no fungi were detected, mycoplasmas were not detected.

Example. FMD antigen As (All) is identified in the ELISA using the Mat. To this end, MAbs, diluted in 0.02 molar carbonate bicarbonate buffer pH 9.5, in an amount of 200 µl per well at a concentration of 3-100 ng / ml are added to a well of polystyrene plates for serological reactions and incubated for 2- 14 h at a temperature of 200 4 ° C. The antibody solution is removed and the PBS wells are washed 3-5 times. Then, 200 μl each of the test solution is administered to the FBI with 10% fetal serum each time (the antigen is prepared by tenfold dilutions in the same

5 solution): Wells with antigen and antibodies are incubated for 2 hours at 37 ° C and washed 3-5 times in PBS. Then the conjugate (monoclonal antibodies labeled with horseradish peroxidase) is introduced into the wells at the working dilution. The conjugate is diluted in PBS with 10% fetal bovine serum to a concentration of 1-5 µg / ml per IgC and incubated for 1-2 hours at room temperature. Then the wells are washed 345 times with PBS and the substrate containing orthophenylene diamine 0.4 mg / ml in 0.1 M citrate-phosphate buffer, pH 5.0 and 0.01% H202 is added to them. After incubation at room temperature for 1-5 minutes, the reaction was recorded by determining the optical density of the solution on an Fltertebe Multlscan automatic 8-channel photometer from Fow Laboratories (United Kingdom). The reaction can be observed visually by the intensity of the color brown.

It has been established that the strain of hybrid cells is stable both in cultural and morphological characteristics, and in the titer of specific antibodies in ELISA, PH and RE.

The activity and specificity of the MAb were determined in ELISA, PH, RH, RSK and RDSK with homo- and heterologous strains of the schur virus.

The data for determining the specific activity of ascitic fluid containing the mAb obtained from strain No. VSKK (P) 476D is presented in the table

The data show that the tested MAb in ELISA have a high specific activity towards the homologous (As) and closely related (Ay) antigen and are not at all active against viruses

A22, Oi No. 194, Ci No. 564, and Azi -1. Similar results were obtained in PH. Mat to the As virus does not possess complement-binding activity in RSK and RDSK.

The resulting Mat can be used for

detecting antigens of shchura virus in ELISA, PH, P3, as well as for studying their antigenic structure.

Claims (1)

The claims of the strain of the hybrid cultured animal cells Musmusculus L. SCC (P) 476D, producing monoclonal antibodies to the 1465 component of Schur As (Allier)  n / a - inactive.