CS261297B1 - A mouse lymphocyte hybridoma producing anti-human blood antibody B group antigen - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against human blood group antigen B, stored in the hybridoma collection of the Molecular Genetics Collection of the Czechoslovak Academy of Sciences under the designation IMG ČZAS HEB-29. The monoclonal antibody of the hybridoma HEB-29 can be used as a routine diagnostic agent for serological examinations of human blood group antigen B.

Description

The invention relates to a novel hybridoma, that is, a hybrid unicellular organism constructed by fusion of a cell of the mouse myeloma line Sp 2/0 and a mouse splenic lymphoid cell producing an antibody against human erythrocyte group B antigen.

Diagnostic sera against human erythrocyte group antigen B /abbreviated diagnostic sera anti-B/ are the basic component of diagnostic serum sets used to determine the group affiliation of human erythrocytes in the ABO system. Examination of group affiliation in the ABO system is the basic and most common serological examination in human, hematological and transfusion practice. It is performed, for example, as an essential part of a complete pre-transfusion examination, bedside blood group control at the beginning of transfusion, serological examination of parturient women and examination of newborns.

Diagnostic anti-B sera are still mostly prepared from human blood of selected donors, in whom natural high values of anti-B agglutinins have been previously demonstrated, or from the blood of voluntary donors intentionally immunized with saliva of secretors of group-specific substance B, or with substance B prepared from human saliva or animal material - e.g. gastric mucosa of pigs or horses. Substances used for immunization of human voluntary donors must be subjected to state control of sterility, harmlessness and efficacy. Since the concentration of desired antibodies in serum decreases with time after immunization, it is necessary to perform immunization repeatedly. Immunization of volunteers with isolated substances may be accompanied by adverse effects, which can only be prevented by desensitization of immunized individuals. The method used so far for preparing diagnostic anti-B sera therefore places considerable demands on the material, personnel, methodological and organizational support of the production process.

In addition to the desired anti-B antibodies, the serum of each intentionally immunized and non-immunized donor contains many other antibodies in a heterogeneous mixture - always unique and unrepeatable in composition - some of which directly affect the reaction of the serum with erythrocytes and degrade the diagnostic efficacy of the obtained serum. It is necessary to remove such antibodies from the serum. Standardization of individual batches of diagnostic serum is difficult and always incomplete with the current method of preparation, because the very nature of the current preparation of serums makes it impossible to obtain two production batches with identical properties. An unfavorable feature of the current method of preparation of diagnostic anti-B serums is the consumption of large amounts of human blood.

Many of the problems mentioned in the previously used procedure for preparing diagnostic human anti-B sera and a number of difficulties resulting from the unfavorable properties of current diagnostic sera can be eliminated by introducing the preparation of mouse monoclonal anti-B antibodies produced by lymphocyte hybridomas.

Similar antibody-producing hybridomas have been prepared in a number of countries, and in some of them they are already commercially available and are beginning to be used in clinical serological practice.

These include products from companies such as Serotec (England), Immunotech (France), Biotest-Diagnostics (NSR), Diagnostics Pasteur (France), Celtech (England). The antibodies offered abroad are intended for serological examination of blood groups. In Czechoslovakia, hybridomas HEB-20 and HEB-27 were prepared and are protected, producing antibodies against the blood group antigen B, - suitable only for use in immunohistochemistry and unsuitable for use in serology.

The mentioned deficiency is eliminated by the hybridoma stored in the hybridoma collection of the Molecular Genetics Collection of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská ul. 1 083 under the designation IMG HEB-29, which is the source of monoclonal antibodies against human erythrocyte group antigen B. The mentioned hybridoma was obtained by a method known from the professional literature (Fazekas de St. Groth, S., Scheidigger, 0.: Production of monoclonal antibodies: Strategies and tactics, J. Immunol. Meth.: 35: 1,

1980; Galfré, G., Howe, S. S., Milstein, C., Butoher, G. W., Howard. J. C.: Antibodies to major histocompatibility antigens produced by hybrid cell lines, Nature, 266: 550, 1977) by cloning a set of hybrid cells, created by fusion of cells of the mouse myeloma line Sp 2/0 and cells obtained from the spleen of F^ mice, /BALB/c χ B 10.A/, immunized with group B erythrocytes. .

The lymphocytic hybridoma HEB-29 produces a completely homogeneous antibody, the so-called monoclonal antibody, specifically reacting with human erythrocyte types B, A^B and A ₂ B, which does not react with erythrocytes A^, A ₂ and 0. The HEB-29 hybridoma can be cultivated in vitro in culture media suitable for animal cells or in vivo in the peritoneal cavity of /BALB/c χ B ÍO.A/F^ mice. The HEB-29 hybridoma can be stored for a long time in cans stored in liquid nitrogen. Antibody production can be started after thawing the cell can, without the need for additional antigen. The monoclonal antibody produced by the HEB-29 hybridoma is specific for human erythrocyte group antigen B and is free of any ballast antibodies.

The monoclonal antibody HEB-29 forms the basis of diagnostics, which fully complies with all requirements of the current industry standard for diagnostic anti-B sera /i.e. avidity of 15 seconds with whole blood under bedside control conditions, minimum titer in the tube hemagglutination test of 256 and efficiency in the examination of agglutinogen B by slide, plate and tube methods/. The growth and production properties of the hybridoma are very good and, when compared to foreign antibodies, are the basis for economically advantageous use of the HEB-29 antibody as the basis of commercially prepared and sold test diagnostics.

Example

In order to propagate HEB-29 hybridoma cells in vitro, 1x10® hybridoma cells were inoculated into a Legroux culture flask containing 20 ml of complete RPMI culture medium with fetal calf serum Π8!. After three days of hybridoma cell culture growth, culture medium enriched with the monoclonal antibody HEB-29 released into the medium by the hybridoma cells was obtained from the flask.

The efficacy of the monoclonal antibody HEB-29 was tested by successive two-fold dilution of a 5-fold concentrated culture medium containing the monoclonal antibody in an isotonic sodium chloride solution in agglutination tubes according to the industry standard ON 84 3225. The concentrated culture medium agglutinated + /according to ON 84 3225/ erythrocytes Β at a dilution of 1:256. The concentrated culture medium containing the monoclonal antibody HEB-29 did not react with erythrocytes Ap A ₂ and 0.

The avidity of the monoclonal antibody HEB-29 was tested with a 10% suspension of B or A^B erythrocytes washed once and suspended in isotonic sodium chloride solution on a clean glass slide according to the industry standard ON 86 4511. The onset of agglutination of B erythrocytes from different donors occurred in 5 to 14 seconds, and the onset of agglutination of A^B erythrocytes from different donors occurred in 8 to 15 seconds.

Table

Comparison of the efficacy and avidity of the monoclonal antibody HEB-29 with the reactivity of monoclonal antibodies anti-B produced by hybridomas so far registered in the Czechoslovak Socialist Republic as inventions

Antibody B Erythrocytes A _x B titer/time ^A 1 and ₂ 0 HEB-29 256+/5-14S 256+/8-15s - - - HEB-20 32+/300S 16+/300S - - - HEB-27 16+/45-150s 16+/55-200S - - - diagnostic serum 256+/7-15S 256+/9-25S

anti-B

SEVAC

HEB-29 hybridoma cells have an ultrastructural image of typical myeloma cells, where the predominant organelles are free and membrane-bound polyribosomes. Under in vitro conditions they grow in the form of semi-suspension cultures. The basic culture media are RPMI or Eagle's minimal essential medium with Hanks' salt mixture, supplemented with non-essential amino acids, L-glutamine /3mM/ and sodium pyruvate /1mM/ /medium referred to as H-MEMd, Molecular Genetics Department of the Czech Academy of Sciences/. The media for the cultivation of HEB-29 hybridomas are supplemented with penicillin, streptomycin, — 5 -3

2-mercaptoethanol /5x10 M/, HEPES buffer /2 to 4x10 M/ and inactivated bovine serum for TK /Bioveta n.p., Ivanovice na Hané, 10%/. To obtain a culture medium enriched with monoclonal antibody HEB-29 /supernatant of hybridoma cell culture/ for diagnostic purposes, inactivated bovine serum is replaced in the culture medium with fetal calf serum /5 to 10%/.

Hybridoma HEB-29 is cultured at 37 °C. The median generation time is 15.5 hours, the modal chromosome number 40 months after construction /fusion/ is 84 chromosomes. The antibody produced is a mouse immunoglobulin of the IgM class, kappa.

The monoclonal antibody produced by the murine lymphocyte hybridoma HEB-29 can be used in medical practice for routine serological examinations of human blood group antigen B.

Claims (1)

object of the invention IMG CZAS HEB-29 mouse lymphocyte hybridoma producing an IgM class monoclonal antibody against human erythrocyte group antigen B. Severography, n, p., MOST Price 2,40 Kčs