CS260746B1 - Mouse lymphocyte hybrid producing antibody against human erythrocyte group anigeau A - Google Patents

Abstract

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

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusion of a mouse mysoloma cell line P3-X63-Ag8.653 and a mouse splenic lymphoid cell producing an antibody against human erythrocyte group A antigen.

Diagnostic sera against human erythrocyte group antigen A (abbreviated as diagnostic anti-A sera) 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 a blood transfusion, serological examinations of parturient women and examinations of newborns.

The occurrence of antigens of the ABO blood group system is not limited to erythrocyte membranes. In the form of glycoproteins, they are found, for example, in the secretions of glandular cells of the gastrointestinal, genital and respiratory tracts. Antigenic carbohydrate structures are also part of the cell membranes of epithelial tissue, and attention is focused on changes in the expression of these antigens in connection with developmental, differentiation and especially tumor processes. A significant number of immunohistochemical studies have shown that, for example, in lung, gastric, prostate and bladder carcinomas, there is a loss of A and B isoantigens, which are normally present in healthy epithelial tissue.

Blood group antigens A and B are also present in the epithelial cells of the human fetal colon. After birth, they are retained in the proximal part, but they rapidly disappear in the distal part of the colon. Blood group antigens reappear in distally located carcinomas — e.g., in the sigmoid colon and rectum — and become an important diagnostic feature of tumor development.

Diagnostic anti-A sera are still prepared from human blood of selected donors, in whom natural high values of anti-A agglutinins have been previously demonstrated, or from the blood of volunteer donors intentionally immunized with saliva of secretors of group-specific substance A, or with substance A prepared from human saliva or animal material — — e.g. gastric mucosa of pigs or horses. Substances used for immunization of human volunteers 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-A sera therefore places considerable demands on the material, personnel, methodological and organizational support of the production process.

In addition to the desired anti-A antibodies, the serum of every intentionally immunized and non-immunized donor, whose organism is continuously exposed to various antigenic stimuli, contains many other antibodies in a heterogeneous — always unique and unrepeatable mixture 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 serums is difficult and always incomplete with the current method of preparation, since 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-A sera is the consumption of large amounts of human blood.

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

Such hybridomas producing antibodies have been prepared in a number of countries, including the Czechoslovak Socialist Republic, and in some countries they are already commercially available and are beginning to be used in clinical serological practice.

These include, for example, products from companies:

Orphan (England),

Immunoteclh (France,

Biotest-Diagnostics (NSR),

Diagnostics Pasteur (France),

Celts (England).

Hybridomas protected in the CSSR so far (HE-24 AO 229 996, HE-02 AO 228 859, HE-18 AO 229 996) produce antibodies unsuitable for use in immunohistochemistry. Antibodies offered abroad are intended for serological examination of blood groups. None of the listed foreign companies mention the possibility of using the offered antibodies in immunohistochemistry.

The above disadvantages are eliminated by the hybridoma stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská ul. 1083 under the designation IMG CZAS HE-10, which is a source of monoclonal antibody against human erythrocyte group A antigen.

The hybridoma was obtained by a method known from the literature (Fazekas de St. Groth, S., Scheidigger, O.: Production of monoclonal antibodies: Strategies and tactics, J. Immunol. Meth.: 35: 1, 1980; Galfré,

280746

G., Howe, S. S., Milstein, C., Bultcher, 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, resulting from the fusion of cells of the mouse myeloma line P3-X63-Ag-8.853 and cells obtained from the spleen of BALB/c mice, immunized with the saliva of a group-specific substance A secretor.

The lymphocytic hybridoma HE-10 produces a completely homogeneous antibody, the so-called monoclonal antibody, specifically reacting with human erythrocyte types Ai, AtB and A2 and which does not react with erythrocytes AzB, B and O. The hybridoma HE-10 can be cultivated in vitro in culture media suitable for animal cells or in vivo in the peritoneal cavity of BALB/c mice. The hybridoma HE-10 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 hybridoma HE-10 is specific for human erythrocyte group antigen A and is free of any ballast antibodies.

Example

In order to propagate HE-10 hybridoma cells in vitro, 1 X 10 ⁶ hybridoma cells were inoculated into a Legroux culture flask containing 20 ml of complete RPMI culture medium with fetal calf serum (10%) (Institute of Molecular Biology, Czech Academy of Sciences). After three days of hybridoma cell culture growth, the culture medium enriched with the monoclonal antibody HE-10 released into the medium by the hybridoma cells was obtained from the flask. The efficacy of the monoclonal antibody HE-10 was tested by successive two-fold dilution of the culture medium containing the monoclonal antibody in an isotonic sodium chloride solution in agglutination tubes according to the industry standard ON 84 3225. The culture medium agglutinated (according to ON 84 3225) erythrocytes A.1 at a dilution of 1:16, erythrocytes AiB at a dilution of 1:16, erythrocytes A2 at a dilution of 1:8. The culture medium containing the monoclonal antibody HE-10 did not react with erythrocytes A2B, B and O. Agglutination of erythrocytes Al and AlB by the antibody HE-10 was not inhibited by N-acetyl-D-galactosamine.

To test the reactivity of the HE-10 antibody on tissue sections, biopsy samples were taken from several sites of the colon, fixed in formaldehyde and embedded in paraffin. After deparaffinization of the sections, HE-10 antibody (diluted supernatant) was applied and its binding to target structures was monitored using a horseradish peroxidase-labeled swine anti-mouse immunoglobulin antibody (Institute of Serum and Vaccines, Prague).

The HE-10 antibody strongly stained the perinuclear region of crypt epithelial cells in normal tissue sections, with a maximum in the proximal colon and disappearance of positivity in the esophageal loop and rectum. A positive reaction was observed in the distal colon only in samples from tumor tissue (sigmoid adenocarcinoma).

It is significant that the HE-10 antibody also gives a positive reaction in the supranuclear region of some epithelial cells lying in the vicinity of tumor foci and classified by conventional histological approaches as completely normal cells. The picture obtained when using the HE-14 antibody is fundamentally different; the cells are stained diffusely and the positive reaction is only in tumor cells. The results of the comparison of the reactivity of individual MPs are summarized in the table.

Table

Monoclonal antibody reactivity in tissue sections ^{of 1} patient with blood group A

Monoclonal antibody Target antigen Colon Distal colon Adenocarcinoma of the distal colon HE-10 A ^X2 —. '+ ² HE-14 A '+ — HE-02 A — — — HE-18 A — ,— — ΗΕ-24 A — — — HEB-20 B — — —

Note:

¹ formalin fixation, paraffin embedding ² localization in cells in the Golgi apparatus area ,2 6 0 7

HE-10 hybridoma cells have an ultrastructural appearance typical of myeloma cells, where the predominant organelles are free and membrane-bound polyribosomes. Under in vitro conditions they grow as semi-suspension cultures. The basic culture media are RPMI or Eagle's essential medium with Hanks' salt mixture, supplemented with non-essential amino acids, L-glutamine (3 mM) and sodium pyruvate (1 mM) (medium referred to as H-MEMd, Molecular Genetics Department of the Czechoslovak Academy of Sciences).

For the cultivation of the HE-10 hybridoma, the media are supplemented with penicillin, streptomycin, 2-mercaptoethanol (5 X 10 ^-5 U), HEPES buffer (2 to 4 X 10 ^-3 U) and inactivated bovine serum for TK (Bioveta np, Ivanovice na Hané, 10%). To obtain the culture medium enriched with the HE-10 monoclonal antibody (supernatant of hybridoma cell culture) for diagnostic purposes, the inactive bovine serum in the culture medium is replaced with fetal calf serum.

Hybridoma HE-10 is cultured at 37°C. The median generation time is 18 hours, the modal chromosome number 20 months after construction (fusion) is 82 chromosomes. The monoclonal antibody produced is a mouse immunoglobulin of the IgM class, kappa.

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

Claims (1)

SUBJECT Mouse lymphocyte hybridoma IMG CZAS HE-10, producing a monoclonal antibody OF THE INVENTION against human erythrocyte group antigen A class IgM.