CS260748B1 - Mouse lymphocyte hybridoma producing antibody against human erythrocyte group antigen A - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against human erythrocyte group antigen A, stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation HE-14. The monoclonal antibody of the hybridoma HE-14 is suitable as part of a diagnostic panel also containing monoclonal antibodies HE-10, 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 fusing a mouse myeloma cell line P3-X63 to Ag8.653 and a mouse spleen lymphoid cell producing an antibody against the human erythrocyte group antigen A.

Diagnostic sera against human erythrocyte group antigen A (abbreviated diagnostic seraanti-A) 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 start of blood transfusion, maternal serological examinations and neonatal examinations.

The presence of ABO blood group antigens is not limited to erythrocyte membranes. In the form of glycoproteins, they are found, for example, in secretions of the glandular, gastrointestinal, genital and respiratory tract cells. 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 immunohistochemical 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 human fetal lining epilelial 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-A 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-A agglutinins or from blood of voluntary donors deliberately immunized with saliva of the group specific substance A, resp. Substance A 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 antibody in serum decreases after immunization with time, immunization must be repeated. Immunization of volunteers with isolated substances may be accompanied by adverse effects.

In addition to the desired anti-A antibodies, the serum of each intentionally immunized and non-immunized donor occurs in a · heterogeneous - always unique and unique composition - many other antibodies, some of which directly affect the reaction of serum with erythrocytes 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. An unfavorable feature of the current method of preparing diagnostic sera anti-A consumption of large amounts of human blood.

Many of the problems of the prior art method of preparing diagnostic human anti-A 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-A monoclonal antibodies produced by lymphocyte hybridomas.

Such antibody-producing hybridomas have been prepared in a number of countries and in the CSSR, and in some of them are commercially available and are beginning to be used in clinical serological practice. These are products of companies

Serotec (England),

Immunotech (France),

Biotest-Diagnostics (NSR)

Diagnostics Pasteur (France)

Celtech (England).

Hybridomas protected in Czechoslovakia (HE-24 AO 229 996, HE-02 AO 228 859) produce antibodies unsuitable for use in immunohistochemistry. 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 overcome by a hybridoma deposited in the collection of hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská ul. 1083 under the designation IMG CZAS HE-14, which is a source of monoclonal antibody against human erythrocyte group antigen A.

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, 1985; Galfré, G. Howe, SS, Milstein, C., Butcher, GW, Howard, JC: 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 murine myeloma cell line P3- X63-Ag8.653 and cells obtained from the spleen of BALB / c mice immunized with saliva of the secretor of group specific substance A.

The lymphocyte hybridoma HE-14 produces a completely homogeneous antibody, called. monoclonal antibody specifically reacting with human type erythrocytes A1, AiB, and A2 and which does not react with erythrocytes A to B, B and O. HE-14 hybridoma can be cultured under in vitro conditions in animal cell culture media or in vivo in peritoneal cavity of BALB / c mice. The HE-14 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 requiring additional antigen. The monoclonal antibody produced by HE-14 is specific for human erythrocyte group antigen A and is free of any ballast antibodies.

Example

In order to propagate HE-14 hybridoma cells in vitro, 1 X 10® hybridoma cells were introduced into a Legroux culture flask containing 20 ml of RPMI culture medium (IMG CSAV). After three days of growth of the hybridoma cell culture, a culture medium supplemented with monoclonal antibody HE-14 released into the handle by hybridoma cells was recovered from the flask. The efficacy of the HE-14 monoclonal antibody was tested by successive two-fold dilutions of the culture medium containing the monoclonal antibody in isotonic sodium chloride medium in agglutination tubes according to the industry standard T a b at 1 k a

ON 84 3225. The culture medium agglutinated to γ- (according to ON 84 3225) erythrocytes Ai at 1: 32 dilution, erythrocytes AiB at 1: 16 dilution, erythrocytes Only at 1: 2 dilution. With erythrocytes ToB, B and O culture medium containing monoclonal antibody HE-14 did not respond.

To test the reactivity of HE-14 on tissue sections, bio -iptic samples were taken from several colon sites, fixed in formaldehyde and embedded in paraffin. After dewaxing of the sections, HE-14 antibody (diluted supernatant) was applied and its binding to target structures was monitored with a horseradish peroxidase labeled porcine anti-mouse immunoglobulin antibody (Institute of Serums and Vaccines, Prague). normal tissues diffuse crypt epithelial cells with a maximum in the prcximal part of the large intestine and disappearance of the positivity in the area of the esophageal loop and rectum. The image obtained using HE-10 was fundamentally different, only supranuclenar cell areas were stained, and a positive reaction was also present in epithelial cells adjacent to the tumor, and a strong positive reaction was also observed with erythrocytes present. are summarized in the table.

Reactivity of monoclonal antibodies in tissue sections ¹

Monoclonal antibody Target antigen of patients with blood type A

Colon Distal colon Adenocarcinoma of the distal colon

HE-14

HE-10

HE-02

HE-18

HE-24

HEB-20 B! + - 1! + ² - + ²

Note:

¹ fixation with formol, embedding in paraffin ² localization in cells only in the area of Golgi apparatus

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HE-14 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free and membrane-bound polyribosomes. They grow in the form of semi-suspension cultures under in vitro conditions. The basic culture media are RPMI or Eagle's minimal essential medium with Hanks salt mixture, supplemented with non-essential amino acids, L-glutamine (3mM) and sodium pyruvate (1mMj medium referred to as H-MEMd, The Institute of Molecular Genetics of the Czechoslovak Academy of Sciences). Media for HE-14 hybridomas is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (5 X ΙΟ ^-5 M), HEPES buffer (2 - 4 X 10 ^-3 M) and inactivated bovine serum for BP (Bloveta np, Ivanovice in Haná, 101%). In order to obtain culture medium enriched for the monoclonal antibody HE-14 (culture supernatant of hyhridoma cells), the culture medium is replaced with inactivated bovine serum with inactivated fetal calf serum.

The HE-14 hybridoma is cultured at 37 ° C. The mean generation time is 17 hours, the modal number of chromosomes 40 months after construction (fusion) is 70 chromosomes. The monoclonal antibody produced is a mouse immunoglobulin of the IgM class, kappa.

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

Claims (1)

SUBJECT IMG CZAS HE-14 mouse lymphocyte hybridoma producing monoclonal antibody YNALEZU against human erythrocytic group A antigen of the IgM class.