DD224770A1 - METHOD FOR THE PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST CARCINOEMBRYONAL ANTIGEN (CEA) - Google Patents

Abstract

The invention relates to a process for the production of monoclonal antibodies to various antigenic determinants of carcinoembryonic antigen (CEA). The aim of the invention is the production of antibodies of high specificity and strong binding ability in good yields. The method according to the invention is characterized in that spleen cells of Balb / c mice immunized with purified CEA are fused with X63-Ag8.653 myeloma cells in the cell culture, which selects antibody-producing hybridomas, their specificity with antibody inhibition, competition and determined immunohistological tests and selects the specific hybridomas and weitervermehrt in a known manner. Field of application of the invention is medical diagnostics.

Description

Dr. FIEBACH, Helmar dr. KARSTEH, Uwe dr. MICHEL, Burkhard dr. ZOTTER, Stefan GROSSMAMr, Heinz LOTSSITZER, Arndt

Process for the preparation of inonoclonal antibodies against carcinoembryonic antigen

(CEA)

Field of application of the invention

The invention relates to a process for the production of monoclonal antibodies against various antigenic determinants of carcinoembryonic antigen (CEA). Field of application of the invention is medical diagnostics.

Characteristics of the known technical solutions CEA is a glycoprotein which occurs in embryonic development and can point to malignant diseases of the gastrointestinal tract in the adult stage (eg colon carcinomas). Monoclonal antibodies to CEA have been known since 1980 (eg, ACOLLA et al., In Monoclonal Antibodies and T-Cell Hybridomas, Eds. IJ Hämmerling, U. Hämmerling and JP Kearney, Elsevier, Amsterdam, 1981, GRUUERT et al., Oncodev. Biol.Med., 1982, 3j1 ¹ ^ ¹ HEDHT et al., Mol. Immunol., 1982, 16, 1641).

These antibodies were produced by the known techniques of cell fusion and selection of antibody-producing hybridomas. The antibodies were i.a. used for the detection of CEA in the serum of tumor patients with the aid of immunoassays (BUCHEGGER et al., Immunol.lett 1982, 5, 85, HEDIlT et al., Proc.lTat.Acad.Sci., 1983, 80, 3470). The American company Hybritech commercializes a monoclonal anti-CEA antibody.

However, this antibody still shows a considerable response with so-called cross-reacting antigens, e.g. in lung and spleen, and is therefore not absolutely CEA specific.

Object of the invention

The invention aims to produce monoclonal antibodies of high specificity and strong binding ability in good yields. These antibodies should be suitable for the construction of a test system.

presentation; the essence of the invention

The process according to the invention for the production of monoclonal antibodies against various antigenic determinants of GEA is characterized in that spleen cells of Balb / c mice which have been immunized with purified CEA are fused with X63-Ag8.653 myeloma cells in the cell culture and the resulting hybridomas are fused in microtest plates sows. The antibody-producing hybridomas are detected with a pest phase immunoassay which is carried out as follows: anti-mouse immunoglobulin is bound to PVC, after which incubation is carried out with the culture supernatant of the hybridoma.

125

bridome and then with J-labeled CEA. The reactive hybridomas are cloned, their antigen specificity is determined according to the invention with an antibody inhibition test and a competition radioimmunoassay. The specific hybridomas are selected, further propagated, stored in liquid nitrogen and optionally transplanted for the production of monoclonal antibodies to mice in the ascitic form. An essential prerequisite for carrying out the invention is the antibody inhibition test for selecting the hybridomas which produce antibodies against certain antigenic determinants of CEA. It is carried out as follows: anti-mouse immunoglobulin is bound to the solid phase followed by incubation with monoclonal antibody which reacts with a particular antigenic determinant. It is then treated with normal mouse serum and then incubated with "\ T-labeled CEA in admixture with hybridoma culture supernatant

Phase bound, the monoclonal antibody in the mixture reacts against the same determinant as the antibody on the solid phase.

Another essential requirement for carrying out the method according to the invention is the determination of the specificity of the monoclonal antibodies by competition test and by immunohistological examinations. In the competition

125 test, the monoclonal antibody is incubated with J-labeled CEA and tissue extracts containing CEA or CEA-like glycoproteins. Subsequently, the antibody (with the bound antigen) is precipitated by addition of rabbit anti-mouse Ig-covered fixed staphylococci (strain Cowan I). The proportion of bound radioactivity in the different samples indicates the CEA specificity of the antibody or its cross-reactivity with CEA-like substances.

The same indications can be drawn from the immunohistology, tissue sections of different origin are incubated with the monoclonal antibodies and then with pluorescein or peroxidase-labeled anti-mouse Ig.

The invention enables the production of specific antibodies against CEA and thus the development of effective test kits for tumor diagnosis.

The invention will be explained in more detail below using an exemplary embodiment:

Embodiment;

1) Preparation of hybridomas from Balb / c mouse spleen cells and X63-Ag8.653 myeloma cells.

Fusion of spleen and plasmocytoma cells was carried out according to standard techniques (KÖHLER and MILSTEIN: Nature 256, 495, 1975, HUDSOLT and HERY: Practical Immunology, 2nd ed., Oxford 1930).

The myeloma line X63-Ag8.653 was prepared by KEARNEY et al., J. Immunol. 1979, 123, 1548. Cloning and further processing of the hybridomas was carried out according to modified techniques (KARSTEN et al., In preparation).

2) The selection of antibody-producing hybridomas was carried out using a pest phase radioimmunoassay according to MICHBEL et al., J. Immunol. Meth. 1981, J ^ S, 41.

3) Antibody Inhibition Assay, Competition Test, and Immunohistologist were performed on the culture supernatants of the various hybridomas having anti-CEA specificity.

Obtained monoclonal antibodies against various antigenic determinants of CEA

Antigen- Default- affinity Ig other deterministic antibody (1 / mol) class own minante · companies a ZIK-A42-B / B11 4 x 10 ⁸ IgG 1 various ZIK-A42-A / C1 2 χ 10 ⁸ IgG 1 Antigenic specificities, part ZIK-A42-B / B11C ' 1 x 10 ¹⁰ 211 CJlT holranyi "} * wise low ZIK-A42-A / G5a not tested U ti Yes. ctill 1 υ IgG1 Cross-reaction with KTormal ZIK-A42-B / B11b I! IgG2b g ewe be (B / B11a, B / B11C, A / C1) b ZIK-A42-B / A2a 2 χ 10 ⁹ IgG2b moderate ZIK-A42-A / G5b Not IgG2b Cross-reactivity with tested 17ormalgev; ebe (but schwa cher than the Hybritech- Antibody) C ZIK-A42-A / G6 Not IgG1 Kreuzreak tested tion with non-Cel relatives molecules d ZIK-A42-B / A2b 3 x 10 ⁹ IgG2b ' similar to antibody with b-spec fität

4) Preparation of the antibodies

The antibodies were obtained from the culture fluid of the cultured or the ascites fluid of the transplanted hybridomas according to customary methods (KEMETT, McKEAELT and BECHTOL: Monoclonal Antibodies Hybridomas: A New Dimension in Biological Analyzes Plenum Press, Hew York and London, 1981).

Claims (1)

invention claim Process for the preparation of monoclonal antibodies
against carcinoembryonic antigen (CEA), characterized, that one has spleen cells from Balb / c mice with
purified CSA, fused to X63-Ag8.653 myeloma cells in cell culture, which selects antibody-producing hybridomas, theirs
Determine specificity with antibody inhibition, competition and immunohistological assays which select specific hybridomas against diagnostically relevant determinants and cultured in known manner or transplanted to ascites form mice.