CS260749B1 - Lymphocyte mice with hyliride producing antibody against human erythrocyte group B antigen - Google Patents

Abstract

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

Description

The present 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 lymphold cell producing an antibody against 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, blood group and bed control at the beginning of a blood transfusion, serological examinations of maternal women, and neonatal examinations.

The presence of the ABO blood-group system antigen is not limited to srytrocytes. of the membrane. In the form of glycoproteins, they are found, for example, in 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, differential, and especially tumor processes. A large number of immunohistochemical studies have shown that, for example, the lung, stomach, prostate and bladder cancers lose the A and B isoantigens that are normally present in healthy epithelial tissue.

Blood group antigens A and B are also present in human fetal mucosal epithelial cells. After birth they remain in the proximal part but disappear quickly in the distal part of the colon. Blood group antigens reappear in distally localized carcinomas - for example, in the S-loop 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 volunteer donated blood deliberately immunized with the saliva of the group specific substance B, or substance B prepared from human saliva; animal material such as stomach 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 only 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, some of which directly affect the serum-erythrocyte response and degrade the diagnostic efficacy of the obtained serum, occur in the serum of each intentionally immunized and non-immunized donor, heterogeneous - always unique and unique in composition. 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. An unfavorable feature of the current method of preparing diagnostic anti-B sera is the consumption of large amounts of human blood.

Many of the problems of the prior art method of preparing diagnostic human anti-B sera and a number of difficulties resulting from the adverse properties of 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 serological practice.

These are products of companies:

Serotec (England),

Immunotech (France),

Biotest-Diagnostics (NSR),

Pasteur Diagnostics (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.

These disadvantages are eliminated by a new hybridoma deposited in the collection of hyhridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská street 1083 ipod named IMG HEB-27, which is a source of monoclonal antibody against human erythrocyte group antigen B.

The 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, J., C .: Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature, 266: 550, 1977] by cloning a set of hybrid cells resulting from fusion of mouse myeloma cells line S _; p 2/0 and cells obtained from the spleen of Fi mice (BALB / c XB 10.A) immunized with Group B erythrocytes

The HEB-27 lymphocyte hybridoma produces a completely homogeneous antibody, the monoclonal antibody, specifically reacting with human type B, AiB-and A2B erythrocytes, and which does not react with A1, A and O erythrocytes. in culture media suitable for animal cells or in vivo in the peritoneal cavity of Fi mice (BALB / c X Β 10.A). Hybridoma HEB-27 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 additional antigen. The monoclonal antibody produced by HEB-27 is specific for human erythrocyte group B antigen and is free of any ballast antibodies.

Example

To multiply HEB-27 hybridoma cells in vitro, 1 X 10 ® hybridoma cells were introduced into a Legroux culture flask containing 20 ml of complete culture medium RPMI with fetal calf serum (10%). After three days of growth of the hybridoma cell culture, culture medium supplemented with the monoclonal antibody HEB-27 released into the medium by the hybridoma cells was recovered from the flask. The efficacy of the HEB-27 monoclonal antibody was tested by successive two-fold dilutions of monocin antibody containing culture medium in isotonic saline in agglutination tubes according to industry standard ON 84 3225. The culture medium agglutinated to + (according to ON 84 3225) erythrocytes B in dilution 1: 16, erythrocytes AiB at 1: 16 dilution, erythrocytes A to B at 1: 8 dilution.

To test the reactivity of HEB-27 on tissue sections, biopsy samples were taken from several colon sites, fixed in formaldehyde and embedded in paraffin. After the sections were dewaxed, antibody-HEB-27 (diluted supernatant) was applied and its binding to the target structures was monitored with horseradish peroxidase labeled porcine anti-mouse immunoglobulin antibody (Institute of Sera and Vaccines, Prague). HEB-27 strongly stained sections of normal tissue of diffuse mucinous crypt cells with a maximum in the proximal part of the large intestine and the disappearance of positivity in the ace-loop region and rectum. A positive reaction was seen in the distal colon area 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

HEB-27

HEB-20 B

HE-10

HE-14

Colon Distal Adenocarcitrator nom distal colon i - i +

Note:

¹ fixation with formol, embedded in paraffin

In the form of collected and processed lung tissue samples, a positive reaction, unlike the HEB-20 antibody, was found in all layers of the bronchial epithelium, in the secretion and in most bronchial gland cells. The HEB-27 antibody also labeled the present eytrocytes.

HEB-27 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free and membrane-bound polyribosones. They grow in half-suspension cultures under in vitro conditions. The basic culture media are RPMI or Eagle-essential medium with Hanks salt mixture supplemented with non-essential amino acids. L-glutamine (3mM) and sodium pyruvate (1mM) medium referred to as -H-MEM-d, (Institute of Molecular Genetics of the Czechoslovak Academy of Sciences). Media for HEB-27 hybridoma supplemented with penicillin, streptomycin, 2-mercaptoethanol (5 X ΙΟ ⁵ M), HEPES buffer (2-4 X 10 ³ M) and inactivated bovine serum for BP (Bioveta np, Ivanovice in Haná, 10%). To obtain culture medium enriched for monoclonal antibody HEB-27 (hybridoma cell culture supernatant) for diagnostic purposes, inactivated bovine serum is replaced with fetal calf serum in the culture medium.

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The HEB-27 hybridoma is cultured at 37 ° C. ^{: The} mean generation time is 16.4 hours; the molar number of chromosomes 40 months after construction (fusion) is 81 chromosomes. The monoclonal antibody produced is a mouse immunoglobulin of the IgM class, kappa.

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

Claims (1)

Mouse lymphocyte hybridoma IMG CZAS HEB-27, producing a monoclonal antibody OF THE INVENTION against the human erythrocytic group B antigen of the IgM class. Severography, n. Race 7, Most Price 2,40 Kčs