CS276354B6 - mouse lymfphocytar hybridom img-czas he-195 producing antitoxin against human group antigene - Google Patents

Abstract

The mouse lymphocyte hybridoma produces an antibody against human blood group antigen And, it is stored in the Institute's hybridoma collection Molecular Genetics of the Czechoslovak Academy of Sciences under the designation IMG HE-195. Monoclonal alone the hybridoma antibody HE-195 is suitable for use in medical practice as a diagnostician group A antigen in the investigation group affiliation erythrocytes in the ABO system.

Description

The invention relates to a novel hybridoma, i.e. a hybrid single-celled organism, constructed by the fusion of a mouse myeloma line Sp2 / O-Ag 14 and a mouse spleen lymphoid cell producing an antibody against a human blood group A antigen.

Diagnostic sera against human blood group A antigen (abbreviated as anti-A diagnostic sera) are an essential component of diagnostic files used to determine the group affiliation of human erythrocytes in the ΛΒΟ 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 monitoring at the beginning of a blood transfusion, serological examinations for post-transfusion events, prenatal examinations of mothers and examinations of newborns.

Anti-A diagnostic sera have so far been prepared from the human blood of selected donors who have been shown to have naturally high levels of anti-A agglutinins or from the blood of voluntary donors deliberately immunized with group-specific substance A or material - for example, gastric mucosa of pigs or horses. Substances used to immunize human volunteers must be subjected to state control for sterility, antigenic efficacy and safety. Because serum concentrations of the desired antibodies decrease over time after immunization, immunizations must be performed repeatedly. Immunization of volunteers with isolated substances may be accompanied by adverse events that can only be prevented by desensitization of the immunized individuals. Measures to prevent the spread of AIDS are a serious problem in the immunization of volunteers. It is obvious that the hitherto used method of preparation of anti-A diagnostics places considerable demands on the material, personnel, methodological and organizational provision of the production process.

In addition to the desired antibodies against group antigen A, the serum of every intentionally immunized and non-immunized donor, whose body is constantly exposed to a variety of antigenic stimuli, contains a heterogeneous - always unique and unrepeatable mixture of many other antibodies, some directly affecting serum erythrocytes. degrade the diagnostic efficacy of the serum obtained. Such antibodies must be removed from the serum. The standardization of individual batches of diagnostic sera is difficult and always incomplete in the current method of preparation, because the inherent nature of the current preparation of sera results in the impossibility of obtaining two production batches of the same properties. An unfavorable feature of the current method of preparing diagnostic anti-A sera is the consumption of large amounts of human blood. ·

Many of the above problems of the current method of preparing diagnostic anti-A human sera can be overcome by introducing the preparation of anti-A monoclonal antibodies produced by lymphocyte hybridomas.

Hybridomas producing monoclonal antibodies against human group antigen A have already been prepared in a number of countries. Anti-A monoclonal antibodies commercially available, for example, from Celtech (UK), Serotec (UK), Biotest-Diagnostics (NSR) and Diagnostics Pasteur (France), are gradually being introduced into clinical practice as serological typing diagnostics of group antigen A.

Also in Czechoslovakia, a number of lymphocyte hybridomas producing monoclonal antibodies against human group antigen A have been prepared. Some of them have already been the subject of the application and copyright protection. These are hybridomas HE-02 (Czech author's certificate No. 228 859), HE-18 (Czech author's certificate No. 229 996) and HE-24 (Czech author's certificate No. 229 995), producing monoclonal antibodies useful in medical practice for the safe differentiation of A- ^ B erythrocytes from AjB erythrocytes and HE-10 and HE-14 hybridomas, producing monoclonal antibodies useful for the detection of group A antigen on tissue sections in the immunohistochemical diagnosis of epithelial cancers

CS 276 354 B6 tissues - such as colonic mucosa. None of the monoclonal antibodies against group antigen A produced by the hybridomas react under the conditions of the assays used to type group membership in the AgO system with AgB erythrocytes and therefore cannot be used as anti-A diagnostics according to industry standard.

The above-mentioned disadvantages of previously protected hybridomas are eliminated by a lymphocyte hybrid, stored in the collection of hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague. 4, Vídeňská 1083 under the designation IMG CZAS HE-195, which is a source of a monoclonal antibody against human blood group A antigen meeting the requirements of the industry standard ON 86 4511 for anti-A diagnostics.

Said hybridoma was obtained in a manner known from the literature (Kohler G., Milstein C .: Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256: 495, 1975; Galfré G., Howe SC, Milstein C., Butcher GW, Howard JC: Antibodies to major histocompatibility antigen produced by hybrid cell lines, Nature 266: 550, 1977; Fazekas de St. Groth S., Scheidigger D .: Production of monoclonal antibodies: Strategy and tactics, 0. Immunol Meth. 35: 1 , 1980. (by cloning a set of hybrid cells resulting from a fusion of cells of the mouse myeloma line Sp2 / 0-Ag 14 and cells obtained from the spleen of BALB / c mice immunized with cells of the human epidermoid carcinoma line A 431.

The HE-195 lymphocyte hybridoma produces a monoclonal antibody against group antigen A, which reacts with human type erythrocytes Aβ ^ B, A ₂ , A ₂ 8 a and does not react with erythrocytes 8 and 0. The hybridoma HE-195 can be cultured in vitro. in culture media suitable for animal (mammalian) cells or in vivo in the peritoneal cavity of a BALB / c mouse strain. The HE-195 hybridoma can be stored for a long time in cans stored in liquid nitrogen. Antibody production can be initiated after thawing the cell can without the need for additional antigen to immunize. The monoclonal antibody produced by the HE-195 hybridoma is specific for human blood group A antigens and is free of any ballast antibodies.

Example

To propagate HE-195 hybridoma cells in vitro, 1.5 x 10 10 hybridoma cells were placed in a Legroux culture flask containing 20 ml of RPMI culture medium containing 10% fetal calf serum. After three days of growth of the hybridoma cell culture, a culture medium enriched in monoclonal antibody He-195 released into the medium by the hybridoma cells was obtained from the flask. The efficacy of the HE-195 monoclonal antibody was tested by macrotitration in agglutination tubes by successive two-fold dilutions of the culture medium containing the monoclonal antibody with isotonic sodium chloride solution according to common industry standard ON 86 4500. Results of HE-195 antibody efficacy test industry standards ON 86 4511 are listed in Table 1. '

Table 1

Results of serological examination of culture medium containing monoclonal antibody HE-195 according to ON 86 4500

Blood cells group . Number of examinations Average titer Minimum acceptable titre according to ON 86 4511 ^A 1 30 256 ++ 256 + a ₂ 14 256 + 128 + Α _χ Β a ₂ b ^A 3 6 256 ++ 256 + 11 2 64 + 8 + ^h ) 32 + the standard does not require

CS 276 354 B6

x) diagnostic serum anti-A SEVAC achieves a titer of 4 + with blood cells Aj.

The set of examinations, the results of which are shown in Table 1, shows that the culture medium containing the monoclonal antibody HE-195 meets the conditions of the branch standard MZ ČSR ON 86 4511 for group diagnostic serum anti-A.

The avidity of monoclonal antibody HE-195 was tested on a slide by mixing a 10% suspension of type blood cells in isotonic. sodium chloride solution with the same amount of culture medium containing monoclonal antibody and determination of reaction time according to Article 9 of the common industry standard ON 86 4500. Example of results of HE-195 antibody avidity test with type erythrocytes and comparison with maximum acceptable reaction times according to industry standard ON 86 4511 are listed in Table 2.

Table 2

Results of avidity examination of culture medium containing monoclonal antibody HE-195 according to ON 86 4500

Group cells' Reaction time ^x ^ Maximum acceptable time ^{* xx} )

Α _χ 5.3 s 15 s

A2 5.0 s 30 s

Α _χ Β 5.2 s 30 s'

A2B 8.3 s 45 s

x) average value from 3 measurements xx) according to Industry Standard ON 86 4511

The results shown in Table 2 show that the culture medium containing the monoclonal antibody HE-195 meets the requirements of the branch standard MZ ČSR ON 86 4511 for the avidity of the group diagnostic serum anti-A. .

Culture medium containing HE-195 monoclonal antibody did not react with type erythrocytes B (36 examinations) and 0 (41 examinations).

HE-195 hybridoma cells have a morphological picture of typical murine myeloma cells.

In vitro, they grow in the form of semi-suspension cultures. The basic culture medium is RPMI 1640 supplemented according to the usual recipe with penicillin, streptomycin, gentamycin, HEPES buffer, 2-mercaptoethanol, L-glutamine, sodium urea and fatal calf serum. The hybridoma is cultured at 37 ° C. The median generation time is 16.5 hours and the modal number of chromosomes was 14 months after the construction of the hybridoma of 81 chromosomes. The monoclonal antibody produced is a murine IgM immunoglobulin.

The monoclonal antibody produced by the murine HE-195 lymphocyte hybridoma can be used in medical practice as a diagnostician of blood group A antigen.

Claims (1)

PATENT CLAIMS. IMG-CZAS HE-195 mouse lymphocyte hybridoma producing an IgM class monoclonal antibody against human blood group A antigen.