CS275909B6 - Murine lymphocyte hybridoma img czas he-193 producing antibody against human group antigen a - Google Patents

Abstract

The lymphocyte hybridoma produces pro human blood group antigen A, and is deposited in the collection of hybridomas Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague under the designation IMG CZAS HE-193. Alone HE-193 hybridoma monoclonal antibody it is suitable for medical use practice as a group diagnostician antigen A in group investigations of human erythrocytes in the system ABO

Description

The present invention relates to a novel hybridoma, i.e. a hybrid unicellular 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 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 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 in whom naturally high levels of anti-A agglutinins have been demonstrated or from the blood of voluntary donors deliberately immunized with saliva of group-specific substance A, or substance A prepared from human saliva or animal 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, many other antibodies are present in the serum of each intentionally immunized and non-immunized donor, whose organism is continuously exposed to various anigenic stimuli, in heterogeneous - always unique and unrepeatable - mixtures, some of which directly affect the serum reaction with erythrocytes. and 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 with the current method of preparation, because the very 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 aforementioned problems of the previously used method for 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 the Czechoslovak Socialist Republic, 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 patent applications and copyright protection. These are hybridomas HE-O2 (Czechoslovak author's certificate No. 228 859), HE-18 (Czechoslovak author's certificate No. 229 996) and HE-24 (Czechoslovak author's certificate No. 229 995), which produce monoclonal ones. antibodies useful in medical practice for the safe differentiation of A ^ B erythrocytes from AjB erythrocytes ^{and the} hybridomas HE-10 (Czech author's certificate No. 260 746) and HE-14 (Czech author's certificate No. 260 748), producing monoclonal antibodies useful for detection of group antigen A on tissue sections in immunohistochemical diagnosis of tumor diseases of epithelial tissues - for example colonic mucosa. From the point of view of wide use in medical practice, the most important among monoclonal antibodies against human group antigen A are those antibodies which react with erythrocytes A ₂ B in hemagglutination tests used for typing group membership in ABO system and meet specificity, efficiency and avidity conditions of industry standard. ON 86 4511 of the Ministry of Health of the Czechoslovak Republic for anti-A diagnostics. In the Czechoslovak Republic, the subject of the invention has so far become the only lymphocyte hybridoma producing a monoclonal antibody of the above-mentioned properties - the HE-195 hybridoma. Due to the fundamental importance of anti-A monoclonal diagnostics, compliant with the industry standard ON 86 4511, in medical practice and to the fact that the needs of large-scale cultivation of hybridomas, hybridomas to the same extent, it is desirable to ensure the successful production of anti-A diagnostics by several hybridomas producing monoclonal antibodies of the required properties.

Another hybridoma producing monoclonal antibodies against human group antigen A, which meets the requirements of industry standard ON 86 4511 for anti-A diagnostics, is a lymphocyte hybridoma, 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-193 .

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 S. C., 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, J. Immunol. . 35: 1, 1980) by cloning a set of hybrid cells, cells resulting from fusion of a mouse myeloma cell line Sp2 / O-Ag 14, and cells obtained from the spleen of Balb / c mice immunized glycolipids prepared from human erythrocytes of blood group a ₂ <

The HE-193 lymphocyte hybridoma produces a monoclonal antibody against group antigen A, which reacts with human type erythrocytes, A 1 E, A ₂ , A ₂ B and does not react with erythrocytes B and O. The HE-193 hybridoma can be cultured under in-situ conditions. in vitro in culture media suitable for animal (mammalian) cells or in vivo in the peritoneal cavity of BALB / c mice. The HE-193 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-193 hybridoma is specific for human blood group A antigens and is free of any ballast antibodies.

Example:

In order to propagate HE-193 hybridoma cells in vitro, 1.5 x 10 6 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 growing the hybridoma cell culture, a culture medium enriched in monoclonal antibody HE-193 released into the medium by the hybridoma cells was obtained from the flask. The efficacy of the HE-193 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-193 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-193 according to ON 86 4500

Blood cells group Number of examinations Average titer Minimum acceptable titre according to ON 86 4511 ^A 1 23 256 + 256 + ^A 2 5 256 + 128 + AjB 5 256 + 256 + a ₂ b 5 32 ++ 32 + ^A 3 . - not tested V 3 · + / the standard does not require

diagnostic serum anti-A SEVAC achieves a 4 + titer with A ^ blood cells.

The set of examinations, the results of which are shown in Table 1, shows that the culture medium containing the monoclonal antibody HE-193 meets the conditions of the branch standard MZ ČSR ON 86 4511 for group diagnostic serum anti-A.

The avidity of the HE-193 monoclonal antibody 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 the monoclonal antibody and determining the reaction time according to Article 9 of the common industry standard ON 86 4500. HE-193 with typical erythrocytes and a comparison with the maximum acceptable reaction times according to the industry standard ON 86 4511 are given in Table 2.

TABLE 2

Results of avidity examination of culture medium containing monoclonal antibody HE-193 according to ON 86 4500

Blood cells group X / Reaction time Maximum acceptable time ^xx ^ ^A 1 15 s 15 s ^A 2 17 s 30 s ^A 1 ^B 15 s 30 s ^A 2 ^B 15 s 45 s

x / average value from 3 measurements.

^χχ / according to industry standard ON 86 4511

From the results given in table no. 2 shows that the culture medium containing the monoclonal antibody HE-193 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 monoclonal antibody HE-193 did not react with type erythrocytes B (7 examinations) and O (13 examinations).

HE-193 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 pyruvate and fetal calf serum. The hybridoma is cultured at 37 ° C. The median generation time is 16.2 hours and the modal number of chromosomes was 14 months after the construction of the 83 chromosome hybridoma. The monoclonal antibody produced is a murine IgM immunoglobulin.

The monoclonal antibody produced by the murine lymphocyte hybridoma HE-193 can be used in medical practice as a diagnostician of blood group A antigen.

Claims (1)

PATENT CLAIMS Mouse lymphocyte hybridoma IMG CZAS HE-193, producing an IgM class monoclonal antibody against human blood group A antigen.