RU1791453C - Strain of hybridous cultured cells mus musculus l used for preparation of monoclonal antibodies to membrane-bound and soluble forms of human h-antigen - Google Patents

Abstract

Usage: biotechnology, biomedical research and forensic examination. SUMMARY OF THE INVENTION: Preparation of a strain of hybrid cultured Mus musculus L cells producing monoclonal antibodies (MABs) of increased sensitivity to human H antigen. The strain is obtained by hybridization of mouse splenocytes (TWO / 2xBa lb / c) FI with myeloma cells RZ-KhbZ-Ad.8.653. MCA are class M Ig. Their titer in the culture fluid, as determined in the Micropanel agglutination reaction with group erythrocytes Oh, it is 1: 256-1: 512, in ascites it is 1: 256000. The resulting MCAs are capable of interacting with both membrane bound and soluble forms of human H antigen. 2 tab. . ate with

Description

The invention relates to biotechnology and can be used for the administration of H-antigen on the surface of human red blood cells and its soluble form in the saliva of excretory individuals.

Strain H-86/44 is known which produces monoclonal antibodies (MABs) to membrane-bound and soluble forms of human H-antigen.

However, these antibodies have insufficient specificity and sensitivity.

An object of the invention is to provide a hydridoma producing MAB to membrane-bound and soluble forms of human H antigen with increased sensitivity.

The strain is obtained as follows.

Female mice TWO / 2 x BaL B / cF1 age 6 weeks. they are immunized with five times washed group O erythrocytes obtained from several donors. The first immunization is carried out with a 50% suspension of red blood cells in saline and Freund's complete complete adjuvant (0.3 ml intraperitoneally and 0.2 ml subcutaneously). A second immunization was carried out after 2 weeks. intravenously with a 3% suspension of erythrocytes п о 0.25 ml / mouse. In 2 days after booster immunization, splenocytes from 5 immune mice hybridize with P3-X63 Ad8.653 mouse myeloma cells, scattered at a concentration of 0.45 x 10 cells / ml the day before.

Before hybridization, cell suspensions are washed three times in serum-free medium.

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Fusion is performed in 50 ml conical tubes in the presence of 40% polyethylene glycol (molar mass 3000-3700) in DMEM medium With 15% DMCO for 1 min (300 x 106 splenocytes and 150 x 106 myelo cells we).

Then, 5 ml of serum-free DMEM medium was added to the cell suspension over 4 minutes, and then the volume of the suspension was adjusted to 50 ml with medium. DMEM with 2% embryo, rionic calf serum (ETS). The cell suspension is centrifuged for 8 min at 800 rpm, the pellet is resuspended in complete nutrient medium at a concentration of 1 x 10 6 cells / ml and the cells are placed in 96-cell flat-bottom plates in a volume of 0.2 ml (2 x 105 cells) per cell.

Hybrid cells were cultured in complete DMEM nutrient medium supplemented with antibiotics, 4 mM glutamine, 20 mM Hepes buffer, 1 mM sodium pyruvate, 5 x M 2-mercaptoethanol and 10 - 20% ETS.

Clones were selected on GAT medium (2% 50-fold concentrate for 7 days after fusion). In 1 week when half the volume of the medium is changed, instead of GAT, 2% of a 50-fold concentrate of HT (medium containing guanine and thymidine) is added.

Monoclocal antibody (MAB) screening was performed on days 11-18. after fusion by agglutination and enzyme immunoassay. As a result of testing, a hybridoma H-89/8 was selected, producing MAB to the H-antigen on the surface of human red blood cells and its soluble form. The hybridoma was cloned twice, clonirvanic efficiency of 92%.

Strain H - 89/9 is characterized by the following features.

Cultural properties.

Cultivation medium: DMEM supplemented with 3 mm L glutamine, 1 mM sodium pyruvate, 20 mM Hepes buffer, 5 x 10 M 2 mercaptoethanol, 10% ETS or deer serum and antibiotics. The frequency of passivation is 1: 2-1: 3 after 2-3 days. the density of cells in a liquid medium is 3-4 x 10 cells / m. Cells are grown at 37 ° C, 6% CO2 in air and 95-100% humidity. When cultivated in vivo, BAL B / s mice intraperitoneally for 7-30 days. prior to the introduction of the hybrid cells, pristan (0.25 ml) was administered. Hybrid cells are injected in an amount of 2-3 x 10 / mouse, ascites tumors form in 12-16 days.

Morphological signs.

Cells weakly attached to the substrate with the usual hybrid morphology.

Karyological signs. Marker chromosomes:

1 subtelocetric and metacentric. The model number of chromosomes is 92 (86-94).

Characteristic of a useful product: MCAs belong to Ig M. They reveal H-antigen on the surface of human erythrocytes, as well as its soluble form in the saliva of 0 excretory individuals.

Strain productivity. The titer of MCA in the culture fluid is 1: 256-1: 512, in ascites 1: 256 x 103.

The stability of the productive properties is maintained for 15 passages.

Contamination control. Bacteria, fungi and mycoplasma were not detected. Virus testing has not been conducted.

Freeze mode. Cryoprotective 0 Wednesday:

10% DMSO, 40% of any serum, growth medium.

Cells incubated during the day at - 70 ° C. then transferred to liquid nitrogen. 5 Cell viability after thawing is 60-65%.

The use of strain H-89/8 is illustrated by the following examples.

(Example 1. The serological properties of the supernatant containing MAB are evaluated for erythrocytes of various blood groups of the ABO system, which are characterized by a decrease in the content of H - antigen in the series 0-B-A1-A1B. Double 5 dilutions of MAB for 50 μl of phosphate-buffered saline or 0.9% NaCI is added to the wells of a 96-well round-bottom plate and mixed with an equal volume of 2% suspension of red blood cells obtained from one donor.

After 30-60 minutes of incubation at room temperature, the antibody titer is determined. The last dilution giving a visible agglutination reaction of the nation is taken as the MCA titer. The research results are presented in table 1.

The results shown in the table indicate that the titer of the obtained MCAs in the agglutination reaction with erythrocytes of group A1B is more than 100 times lower than with erythrocytes of group O. This proves the difference between antibodies H - 89/8 and known (H - 86/44), as well as their ability to interact with the membrane-bound form of 5 N - antigen, practically not discriminating against red blood cells of various blood groups of the ABO system,

PRI me R 2. Samples of the culture medium containing the MCA, diluted 2 times with phosphate-buffered saline (pH 7.4) with the addition of 0.3% Tween-20. On the nitrocellulose membrane, the saliva of individuals isolating various blood groups of the ABO system is diluted 100 times with Tris-glycine buffer. Pieces of the membrane are placed in the wells of a 96-cell flat-bottom plate and incubated for 1.5 hours at room temperature on a rotary shaker with 100 ml MCA samples. Then the membrane is washed in 200 μl of phosphate-buffered saline with 0.3% Tween-20 3 times for 10 minutes.

The membrane is then incubated for 1.5 hours at room temperature with 500-fold diluted antiglobulin serum labeled with peroxidase. After. incubation, the membrane is washed 3 more times for 10 min 200 μl of phosphate-saline buffer with Tween-20.

Peroxidase activity is detected in a solution of the substrate (4 - chloro - 1 - naphthol)

for 30 min at room temperature. The research results are presented in table.2.

The results presented in table 2 indicate that the activity of the obtained antibodies H - 89/8 in the enzyme immunoassay is maximum with the antigen contained in the saliva of individuals - blood group O isolators.

Thus, these antibodies are capable of detecting the H antigen in soluble form and can be used as a valuable reagent in the forensic examination of material evidence.

Claims (1)

SUMMARY OF THE INVENTION Mus musculus L. BCKK (N) hybrid cultured cell strain No. 461 used to produce monoclonal antibodies to membrane-bound and soluble forms of human H antigen. Table 1 table 2