CS260747B1 - Mouse lymphocyte hybrids producing antibody against human erythrocyte group antigen B - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against human erythrocyte group antigen B, stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation HEB-20. The monoclonal antibody of the hybridoma HEB-20 is suitable as part of a diagnostic panel also containing monoclonal antibodies HE-10, HE-14 and HEB-27 to clarify the pathological anatomical diagnosis of small and large intestinal tumors and lung tissue tumors.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line Sp 2/0 and a mouse spleen lymphoid cell producing an antibody against the human erythrocyte group antigen B.

Diagnostic sera against human erythrocyte group antigen B (abbreviated as anti-B diagnostic sera) are an essential component of sets of diagnostic sera used to determine the group membership 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, haematological 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 blood transfusion, maternal serological examinations and neonatal examinations.

The presence of ABO blood group antigen is not limited to erythrocyte membranes. In the form of glyboproteins, they are found, for example, in the secretions of glandular cells of the gastrointestinal, genital and respiratory tract. Antigenic carbohydrate structures are also part of the cell membranes of epithelial tissue, paying attention to changes in the expression of these antigens in connection with developmental, differentiation and especially tumor processes. A large number of immunoassays, chemical studies have shown that, for example, lung, stomach, prostate and bladder cancers have lost A and B isoantigens, which are normally present in healthy epithelial tissue.

Blood group antigens A and B are also present in mucosal epithelial cells (colon of human fetus. They remain in the proximal part after birth but disappear quickly in the distal part of the colon. Blood group antigens reappear in distally localized cancers - eg. in the esophagus and rectum - and become an important diagnostic feature of tumor development.

Anti-B diagnostic sera have so far been mostly prepared from human blood from selected donors that have previously been shown to have naturally high levels of anti-B agglutinins or from blood of voluntary donors intentionally immunized with saliva of the group specific substance B, resp. Substance B prepared from human saliva or animal material --- eg gastric mucosa of pigs or horses.

Substances used to immunize human volunteers need to be subjected to state control of sterility, harmlessness and efficacy. Since the concentration of the desired antibodies in the serum decreases after immunization with time, immunization should be repeated. Immunization of volunteers with isolated substances may be accompanied by adverse events that can be prevented by desensitization of immunized individuals. The method used for the preparation of anti-B diagnostic sera thus far places considerable demands on material, personnel, methodological and organizational support of the production process.

In addition to the desired anti-B antibodies, many other antibodies occur in the serum of each intentionally immunized and non-immunized donor, in a heterogeneous - always unique and unique composition - some of which directly affect the response of serum and crythrocytes and impair the diagnostic efficacy of the obtained serum. 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, since the inherent nature of the present preparation of sera implies the inability to obtain two production batches of the same characteristics. A disadvantage of the prior art method for the preparation of anti-B diagnostic sera is the consumption of large amounts of human blood.

Many of these problems of the prior art method of preparing diagnostic human anti-B sera and a number of difficulties resulting from the adverse properties of the current diagnostic sera can be overcome by introducing the preparation of murine anti-B monoclonal antibodies produced by lymphocyte hybridomas.

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

These are products of companies:

Serotex (England),

Immunotech (France),

Biotest-Diagnostics (NSR)

Diagnostics Pasteur (France),

Celtech (England).

Antibodies offered abroad are intended for serological examination of blood groups. None of these foreign companies mention the possibility of using the offered antibodies in immunohistochemistry.

This disadvantage is overcome by a hybridoma deposited in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská 1083 under the name IMG HEB-20, which is a source of monoclonal antibody against human erythrocyte group antigen B.

Said hybridoma was obtained by a method known in the literature (Fazekas de St. Groth, S., Scheidigger, O .: Production of monoclonal antibodies: Strategies and tactics, J. Immunol. Meth .: 35: 1, 1980; Galfré, G. Howe, SS, Milstein, C., Butcher, GW, Howard, JC: Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature, 2166: 550, 1977) And cells obtained from the spleen of F1 mice (BALB / c XB

10.A) immunized with Group B erythrocytes

The HEB-20 lymphocyte hybridoma produces a completely homogeneous antibody, a monoclonal antibody specifically reacting with human type erythrocytes B, AiB and A to B and which does not react with A, A and A erythrocytes. media suitable for animal cells or in vivo conditions in the peritoneal cavity of Fi mice (BALB / c X Β 10.A).

Hybridoma HEB-20 can be stored for a long time in cans stored in liquid nitrogen. Antibody production can be initiated after thawing the cell can without requiring an additional antigen. The monoclonal antibody produced by the HEB-20 hybridoma is specific for the human erythrocyte group B antigen and is free of any ballast antibodies.

Example

To multiply HEB-20 hybridoma cells under in vitro conditions, 1 X 10 ⁶ hybridoma cells were introduced into a Legroux culture flask containing 20 ml of complete RPMI culture medium with fatal calf serum (10%). culture medium enriched for monoclonal antibody HEB-20 released into the medium by hybridoma cells The efficacy of monoclonal antibody HEB-20 was tested by successive two-fold dilutions of culture medium containing monoclonal antibody in isotonic sodium chloride medium in agglutination tubes according to industry standard ON 84 3225. agglutinated to (according to ON 84 3225) erythrocytes B at a 1: 32 dilution, erythrocytes AiB at a 1:16 dilution, erythrocytes A to B at a dilution of 1: 16.

To test the reactivity of HEB-20 on tissue sections, biopsy samples were taken from several colon sites, fixed in formaldehyde, and embedded in paraffin. After dewaxing of the sections, HEB-20 antibody (undiluted supernatant j) was applied and its binding to the target structures was monitored with horseradish peroxidase labeled anti-mouse immunoglobulin antibody (Institute of Serums and Vaccines, Prague).

The HEB-20 antibody stained diffuse mucinous crypt cells on normal tissue sections with a maximum in the proximal part of the colon and disappearance of positivity in the area of the sigmoid loop and rectum. Positive reaction was seen in the distal colon only in tumor tissue samples (adenocarcinoma sigmoidea). A strong positive reaction was also observed with erythrocytes present. The results of comparing the reactivity of the individual monoclonal antibodies are summarized in the table.

Table

Reactivity of monoclonal antibodies in tissue sections of ¹ blood group B patients

Monoclonal antibody Target antigen

Colon Distal Adenocarclum colon distal colon

HEB-20 B

HEB-27'B

HE-10

HE-14

Note:

¹ fixation with formol, embedded in paraffin

In similarly collected and processed lung tissue samples, a positive response, unlike the HEB-27 antibody, was observed only in the basal layer of the bronchial epithelium and in the secretion on the bronchial surface and in the lumens of a portion of the bronchial glands. The HEB-20 antibody also labeled erythrocytes and blood vessel endothelium present.

HEB-20 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is 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 minimum essential medium with Hank's salt mixture supplemented with non-essential amino acids, L-glutamine (3 mM) and sodium pyruvate (ImM) medium referred to as H-MEMd (Institute of Molecular Genetics of the Czechoslovak Academy of Sciences).

Media for HEB-20 hybridoma supplemented with penicillin, streptomycin, 2-mercaptoethanol (5 X 10 ~ ⁵ M), HEPES buffer (2-4 X 10 ^-3 M) and inactivated bovine serum for BP (Bioveta np, Iva260747 novice na Hané, 10 ( ¹⁰ / o). In order to obtain culture medium enriched for the monoclonal antibody HEB-120 (hybridoma cell culture supernatant) for diagnostic purposes, inactivated bovine serum is replaced with fetal calf serum in the culture medium.

The HEB-20 hybridoma is cultured at 37 ° C. The mean generation time is 17.0 hours, the modal number of chromosomes 40 months after construction (fusion) is 86 ohromosomes. The monoclonal antibody produced is a mouse immunoglobulin of the IgM class, kappa.

The monoclonal antibody produced by the murine lymphocyte hybridoma HEB-20 can be used in medical practice as part of a diagnostic panel containing also monoclonal antibodies HE-10, HE-14 and HEB-27 to refine the pathological anatomical diagnosis of small and large bowel cancer and lung tissue cancer. .

Claims (1)

Mouse lymphocyte hybridoma IMG CZAS HEB-20, producing the monoclonal antibody VYNALEZA against the human erythrocyte group B antigen of the IgM class.