SU1560549A1 - Strain of hybrid cultivated cells of animals mus musculus used for obtaining monoclonal antibodies to igm of cattle - Google Patents

Abstract

This invention relates to hybridoma technology and can be used in veterinary medicine. The purpose of the invention is to increase the specificity of monoclonal antibodies (monAT). The strain of hybrid cells of mice was obtained by fusion of myeloma cells RZH63-AG8. 653 with splenocytes of BALB / C mice immunized with cattle JG M, and deposited under the number of VSKK (P) N 166D. The strain produces monat specifically reacting with bovine JG M, cross-reactivity with JGG and JGA is not detected. MonAT is detected in a diffusion precipitation reaction (RDP) and in an enzyme immunoassay (ELISA) test. The titer of specific antibodies in the culture fluid is in the RDP 1: 8-1: 16, in the ELISA 0.5 ^. 10 ³ -1 ^. 10 ³ . The strain was transplanted into BALB / C mice with primed prism in the form of ascitic tumors, the titer of specific antibodies in which is in the RDP 1: 160-1: 640, in the ELISA - 0.5 ^. 10 ⁶ - 1 ^. 10 ⁶ . MonAT produced by the strain is used for the immunochemical detection of JGM and the preparation of an enzyme immunoassay for the diagnosis of specific JGM. 1 tab.

Description

sheep, cattle IgM (IgM) and IgG KPC.

Reklonirovanie carried out in semi-liquid agate - 1%, as well as the method of limiting dilutions on the plates. As a result, a clone producing monoclonal antibodies (monAT), which form a precipitation ring with sheep serum and are positive for IgH KPC and sheep in the immuno-enzyme test, was selected and propagated; no cross-reactions with IgG were observed

A positive clone of cells was successively propagated in 96-24 and 12 well plates, and then in culture mattresses (100 ml).

The obtained strain is designated as MIM, RS.Ts-205 is deposited under VSKK (P) No. 166D number and is characterized by the following features.

Cultural features. Standard culture conditions are on DAYS or RPM-1640 medium with 10% fetal bovine serum or horse serum, 1 mM sodium pyruvate, 2% glutamine, 50 µg / ml gentamycin. Stationary suspension. Cells are transplanted in 2-5 days with the replacement of at least 50% growth medium sieving ratio 1: 3 - 1: 5. Seeding dose (0.3-0.5) 10y cells / ml.

Cultivation in the animal,

When administered intraperitoneally, the strain in the amount of 10–10 cells per mouse in 3AL3 / C mice causes the formation of ascites.

Characteristics of a useful product. The strain produces mouse IgG monat class specific for IgM. of cattle, does not react with IgA and G, Produces activity, In the culture fluid monAT accumulates in titers of 1: 500-1: 1000 (IAF) or 1: 3-1: 16 ( RDP is the diffusion precipitation reaction),

3 ascites fluid titers in ELISA 0.5 - ()), in RDP 1: 160-1: 640, Contamination, Nikoplasms, bacteria, mold fungi and yeast are not detected.

Cryopreservation. Freezing medium: 45% Eagle's medium, 45% fetal serum K, RS, 10% DMSO, Freezing mode to -30 ° C at a rate of 1 deg / min (up to -30 ° C at a rate of 2 deg / min, then - liquid nitrogen. Thawed at 40 ° C, center

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fugiruyut at 1000 rpm for 10 min, resuspended in the growth medium to a concentration of 0.5 x 10e cells / ml.

The use of the strain and monat produced is illustrated by the following examples.

Example 1. Clone cells are passaged on Dulbecco's medium with 10% serum of a cow embryo, 1 mI of sodium pyruvate in an atmosphere of 5% CC2.

The titer of specific antibodies in the culture fluid in the RDP is 1: 8 - 1:16, in the enzyme immunoassay test 1: 500 - 1: 1000.

Example 2. The clone cells were introduced into the abdominal cavity of BALB / c mice, previously for 7 days, primed with 0.5 ml of pristane, 2 cells each in 0.5 ml of phosphate-saline solution.

On day 16, ascitesic fluid was obtained, the titer of specific antibodies in it was 1: 600 - 1: 640 in the RDP, and 1U106 in the ELISA (1: 500) x10e.

Example 3 A hybridoma liquid containing specific anti-IgM antibodies with a titer in the RDP of 1: 8 is used to determine IgM in cattle blood serum using the Mancini method. 1 ml of culture liquid was used to prepare 10 ml of 1.2% agar (per glass) for 48 serum samples by radial immunodiffusion. In the presence of IgM in serum samples, a clear precipitation ring is formed around the wells after 2 days, the diameter of which increases and compares with a standard sample to determine the amount of IgM.

Example 4 MonAT preparations obtained from ascites and culture liquid by salting out ammonium sulfate at 50% saturation were conjugated by the periodate method to horseradish peroxidase in a 1: 1 ratio (w / w). The working dilution of the conjugate in ELISA using cattle IgM (U μg / well) as an antigen is 1: 1000. The reaction to cattle IgG and IgA in the ELISA test is at the level of the control with non-specific protein.

Characteristics of MIM RSC-20 class produced antibodies, their specificity and activity in serological tests are presented in the table.

Example 5. 100 µl of immunoglobulin preparation IgM, IgG, IgA in phosphate buffer at pH 7.2 with a protein content of 500 µg / ml are added to the wells of polystyrene plates and at the same time another solution of JSA concentration. Adsorption is carried out at 37 ° C for 2 hours. After 3 times liquid washing with 0.05% Tween-20 on a phosphate-saline buffer, 1000 µl of hybridoma liquid is added to each row of wells, incubated 18 h at 4 ° C. After 6 times washing with detergent solution, 100 ml of anti-mouse peroxidase conjugate are added to the wells at optimal dilution. Incubate for 2 hours at 37 ° C, thoroughly wash the boards with a washing solution (6 times) and add 100 µl per well of the substrate mixture — 0.08% solution of 5-aminosalicylic acid with 0.005%, pK5.8. After 30 min, a visual account of the reaction for the formation of a brown color is made. Staining is observed only in a series of wells with adsorbed cattle IgM before a dilution of 1: 320-1: 640.

The use of a new hybridoma strain will make it possible to obtain monoclonal antibodies of high specificity with precipitating properties.

Claims (1)

Invention Formula The strain of hybrid cultured animal cells Mus musculus VSKK (P) No. 166 D used to produce monoclonal antibodies to bovine IgM. Igg Compiled by G.Smirnova. L. Veselovska Editor. Tehred L. Oliynyk - Corrector S. Shekmar Order 951 Circulation 498 VNIIPI State Committee for Inventions and Discoveries at the State Committee on Science and Technology of the USSR 113035, Moscow, Zh-35, Raushsk nab. 4/5 0.2-2.0- 1: 160- 1: 2- 0.5-1: 3200, 35.0 1: 640 1:32 2-10- 1: 2560 Subscription