DD259635A1 - PROCESS FOR PREPARING MONOCLONAL ANTIBODY AGAINST HUMAN INTERLEUKIN-2 - Google Patents

Abstract

By means of the method according to the invention, monoclonal antibodies to human interleukin-2 are produced with high affinity constant and high specificity. Maize spleen cells immunized with human interleukin-2 are fused with mouse myeloma cells. The hybridoma cells are selected and cloned using immunochemical screening methods. With regard to their reactivity with interleukin-2, the properties of the monoclonal antibodies obtained are determined. The production of the antibodies by the hybridoma clones is carried out either in vitro with biotechnological methods of cell culture or in vivo in ascites form in mice. The specific hybridomas were deposited in the ZI for Molecular Biology (List 1987) under the numbers 0173 to 0192. Areas of application are medicine and immunology.

Description

embodiment

1. Preparation of hybridomas from the spleen cells of Balb / c mice immunized with recombinant and natural IL-2 in various ways using the patent DD 235027A1 and X 63-Ag 8,653 and P ₃ O ₁ myeloma cells, respectively. y Fusion, cultivation, cloning, etc. modified according to the original work KÖHLER, G. u. CMILSTEIN, Nature 1975, 256, 435-437.

2. The selection of antibody-producing hybridomas was carried out according to DD 221884 A1.

3. The properties of the monoclonal antibodies obtained are further demonstrated by examples.

table

depository Marked gungsnr. voltage 173 Zl-Hl A4C8 174 Zl-Hl A4G8 175 Zl St. 176 Zl-Hl A6 177 Zl-Hl A7 178 ZI-HI11 179 Z1 K13H8 180 ZI-K13E4 181 CI 1.12. 182 CI 2.10. 183 Zl- 9.2. 184 ZI-K15F7 185 ZI-K14H9 186 Z1 K14A1 187 ZI-K15B10 188 ZI-K15F12 ⁺ 189 ZI-HIA3A5 190 Zl-Hl A3B7 191 Zl-Hl A3B1 192 Zl-Hl A4H7

Reactivity with human IL-2 recomb. Naturally

with natural mouse IL-2

Antibody class

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

iggi

iggi

n.d.

n.d.

n.d.

Ig2b

iggi

iggi

Ig2b

Ig2b

n.d.

n.d.

n.d.

n.d.

Affinity constant 9 · 10 ⁹ l / mol

Claims (1)

A method of producing monoclonal antibodies against human IL-2 by immunization of Balb / c mice with human interleukin-2, fusion of their spleen cells with illyloma cells, selection of the obtained hybridomas, selection of the specific hybridomas and their culture or transplantation to mice in the Ascitesform, characterized in that for culturing or transplantation the hybridomas (ZIM List 87) 0173 to 0192 are used. Field of application of the invention The invention relates to a method for producing monoclonal antibodies to human interleukin-2. Fields of application are medical diagnostics and immunology. Characteristic of the known technical solutions Human interleukin-2 is of essential importance for the activation, the course and effect of an immunological reaction. It is becoming increasingly important for the elucidation of immunoregulatory relationships and as an immunotherapeutic agent. This development goes hand in hand with the necessity of determining interleukin-2 (IL-2) in an increasing number of samples in both IL-2 production and in diagnosis and therapy. This calls for the replacement of the z. Z. Mostly practiced, very time consuming and complicated biological detection system by a simple and fast test system. The use of monoclonal IL-2 antibodies in a quanitative IL-2 assay, e.g. B. in a sandwich ELISA system, is the method of choice. To date, there have been 4 groups internationally that have produced monoclonal antibodies (mAbs) to human IL-2 (KASmith et al., J. Immunol., 131, 1808-1815 [1983], MACousin et al., Lymphokines Res , 319-329 [1985], E.Brandt et al., Immunobiol. 172, 33-53 [1986] and A. Altmann et al., PNAS 81, [1984]). Almost all groups have received only one or very few mAbs and the quality of the antibodies obtained and their applicability to various immunological methods are very different. Object of the invention The invention aims to produce the largest possible group of mAbs against human IL-2 with appropriate affinity constants and high specificity. For example, the antibodies are expected to be useful for the development of rational, quantitative IL-2 assay systems that can be used in IL-2 production, medical diagnostics, and immunological research, as well as for the production of effective immunosorbents for immunoaffinity chromatographic purification IL-2. The most favorable combination results from the specific task. Explanation of the essence of the invention The inventive method for the production of monoclonal antibodies with different properties] against human IL-2 is characterized in that first in a conventional manner spleen cells of Balb / c mice, which - because of the task to obtain as many different mAK - in various combinations with purified human recombinant or natural IL-2, both in soluble form and RP-18 bound (Patent KÖRNER, I., DETTMER, R., KOPP, J., DD 235027 A1) and applied by different routes (both subcutaneously and intraperitoneally, directly into the spleen or as an in vitro immunization), fused with either X 63-Ag 8.653 or P ₃ O myeloma cells in cell culture with the addition of PEG 4000 and seeded the obtained hybridomas in microtest plates , The antibody-producing hybridomas are detected by a solid phase ELISA assay system, performed as follows: Highly purified antigen (either recombinant or naturally human IL-2 or natural mouse IL-2) is adsorbed on nitrocellulose or microplates followed by incubation with the culture supernatant of the hybridomas and then with peroxidase-labeled affinity chromatographically purified anti-mouse immunoglobulin antibodies. The ELISA-reactive hybridomas are cloned, further propagated and optionally used according to the invention after storage in liquid nitrogen for the production of the MAb by culturing or transplantation. From the recovered ascites, the MAb are purified by hydroxyapatite and their properties determined in various test systems. The hybridomas used for antibody production according to the invention were deposited at the Central Institute of Molecular Biology (list 1987) under the following numbers:
0173 to 0192 (see table) The invention enables the production of many mAbs with different properties against human IL-2. Such a range of antibodies has not yet been available. All mAbs specifically recognize human IL-2 and do not react with mouse IL-2. Most react with both recombinant and natural IL-2, but some only with natural IL-2. The mAbs belong to the immunoglobulin classes IgM, IgG 1 and IgG2b, have partial neutralizing activity and different affinity (highest affinity 9 χ 10 ⁹ l / mol), most react with IL-2 after SDS gel electrophoresis and subsequent blotting on nitrocellulose as well as in various immunological tests.