DD277916A1 - METHOD FOR THE PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST FOLLICLE STIMULATING HORMONE (FSH) - Google Patents

Abstract

The invention relates to a method for obtaining monoclonal antibodies against follicle-stimulating hormone (FSH), which can be used in medical diagnostics. The process according to the invention is characterized in that initially in a manner known per se, spleen cells of Balb / c mice which have been immunized with purified FSH are fused with X63-Ag8.653 myeloma cells in the cell culture which selects antibody-producing hybrid cell clones , propagated and used according to the invention for the production of monoclonal antibodies by cultivation or after transplantation. The hybrid cell clones used for the antibody production according to the invention were deposited in the ZIM Deposit Center for Cell Lines in the Central Institute of Molecular Biology under the following numbers: Name of the cloneRegister number B33-AE10 ZIM-0332 B33-BC12 ZIM-0333 B33-AF5 ZIM-0334 B33-AD7 ZIM- 0335 B33-BH8 ZIM-0336

Description

NamedßsKlons Restriernummer ß 33-AE10 ZIM-0332 B33 BC12 ZIM-0333 B 33-Αι "5 ZIM-0334 B 33-AD 7 ZIM-0335 B33 BH8 ZIM-0336

The monoclonal antibodies to FSH synthesized by the deposited hybrid cell clones B33-AE10, B33-BC12 and B33-AF5 react exclusively with FSH and not with LH, TSH, HCG or β-HCG, as shown in the table below.

Reaction of the produced antibodies

Clone with 125 I-labeled FSH HCG β-HCG LH TSH

B33-AE10 + + + - -

B 33-BC12

B 33-AF 5

B 33-AO 7

B33-BH8 ++ "++ - ++ + +

The antibodies produced by clones B33-AD7 and B33-BH8 show cross-reactions, but are also suitable for constructing test sets in combination with specific antibodies in 2-side binding assays.

The immunoglobulin class or subclass of the antibodies produced by the hybrid cell clones is shown in the next table.

Nama of the clone immunoglobulin class or subclass

the antibody produced

B33-AE10 IgM

B 33-BC12 IgGI

B 33-AF 5 'IgGI

B33-AD7 IgG2a

B33-BH8 LGG3

All hybrid cell clones have good growth properties and show stable antibody production over several months in cell culture without the need for recloning. They can be very well transplanted in the form of ascites to obtain large quantities of antibody. The antibodies are detectable in the ascites fluid at a dilution of more than 1: 10OOCOO; All clones are consequently one of the strong producing hybridomas. The invention enables the production of highly specific and highly reactive antibodies against FSH and, consequently, the development of effective test kits that can be used in conjunction with LH test kits to detect ovulation and thus the optimal timing of a concept. The invention will be explained in more detail below using an exemplary embodiment.

embodiment

1. Balb / c mice were immunized with purified FSH and the spleen cells of these animals were used to fuse with X63-Ag8.653 myeloma cells. The fusion, selection with selection medium, cloning, etc. was carried out according to the standard method of G. Köhler and C.Milstein, Nature 256 (1975), 495 and L.Hudson and F.C. Hay Practical Immunology 2.Aufl. Oxford 1980.

2. The selection of antibody-producing hybrid cells was carried out with a solid-phase radioimmunoassay according to established method.! Micheel et al., J. Immunol., Msth. 46 [1981], 41). For this purpose, anti-mouse immuno-myobulin was adsorbed on the PVC tablet portfolio, followed by incubation with the culture supernatants of the hybrid cell colonies and then with 125 I-labeled FSH.

3. Speification testing of the synthesized antibodies was performed with the same solid phase radioimmunoassay using 125 I-labeled FSH, HCG, β-HCG, LH and TSH in the last incubation step.

According to these tests, the monoclonal antibodies synthesized by the hybrid cell clones B33-AE10, B33-BC12 and B33-AF5 react exclusively with FSH and not with HCG, β-HCG, LH and TSH, whereas those by clones B33-AO7 and B33-BH8 antibodies reacted with FSH, HCG, LH and TSH.

4. The subclass of antibodies was determined by a solid phase radioimmunoassay (B. Micheel et al., Biomed.Biochim.Acta 42 [19831, 13] and a hemagglutination inhibition assay using sub-mass specific rabbit anti-mouse immunoglobulin.

According to this, the antibodies produced by clones selected from d ^: e belong to the following classes or subclasses: IgM (B33-AE10), IgG1 (B33-BC12 and B33-AF5), IgG2a (B33-AD7), IgG3 (B33-BH8).

C The selected clones producing anti-FSH antibodies were frozen in liquid nitrogen in a known manner and used in cell culture and in ascites form mice to obtain antibodies (RH Kennet, TJ McKearn and KB Bechtol, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analysis, New York and London 1981). All clones obtained in cell culture showed stable antibody production over several weeks without the need for recloning. After transplantation to mice, they showed good production stability (loss of anti-body production after more than 5 transplant passages). Because antibodies (especially those of the

Clones B33-AE10 and B33-BC12 produced) could still be detected at dilutions of ascitic fluids of more than 1: 1000000, the most important selected clones belong to the »strongly producing hybridomas. The seloktierton hybrid cell clones have been deposited in the ZIM cell site depository at the Central Institute of Molecular Biology under the following numbers:

NamedesKlons Registration number B33-AE10 ZIM-0332 B33BC12 ZIM-0333 B 33 AF 5 ZIM-0334 B33 AD7 ZIM-0335 B 33-BH 8 ZIM-0336

Claims (2)

Method of producing monoclonal antibodies against follicle stimulating hormone (FSH) by immunizing Balb / c mice with FSH, fusion of their spleen cells with myeloma cells, selection of antibody-producing hybrid cells and propagation of hybrid cell clones by culturing or transplantation to mice in the ascitic form, characterized the clones B33-AE10, B33-BC12, B33-AF5, B33-AD7 or B33-BH8 are used for the cultivation or transplantation. Field of application of the invention , The invention relates to a method for producing monoclonal antibodies of different reaction specificity against the follicle stimulating hormone (FSH).
Field of application of the invention is medical diagnostics. Characteristic of the known state of the art Follicle stimulating hormone (FSH) i = t, like the closely related hormones human chorionic gonadotropin (HCG), luteinizing hormone (LH) and thyroid stimulating hormone (TSH), a glycoprotein composed of two subunits, an α chain and a β chain is. While the α-chain is almost identical in these hormones, the β-chains, in addition to a considerable amino acid sequence homology, also have a number of specific segments. FSH appears in the serum and urine of women in increased concentration in the middle of the normal menstrual cycle. FSH promotes hollicle growth and maturation and is therefore an indicator of the maturity of Graaf's follicle. The measurement of the FSH concentration in body fluids can also provide information on primary disturbances in the production of this gonadotropic hormone and on the hypothalamic-pituitary-gonadal function. For the proof and. Quantitative detection of follicle stimulating hormone (FSH) requires specific antibodies that can distinguish FSH from closely related hormones in a specific immunoassay. For the construction of 2-site binding assays are e.g. B. Two monoclonal antibodies (MoAK) against two different epitopes needed, of which one of the antibodies must be specific. Since the specific epitopes of the gonadotropins are evidently in close spatial proximity, for such assays the combination of a specific with a cross-reacting antibody that reacts with a distant epitope is most favorable. Especially for the selection of the most effective combinations that give the most sensitive tests, often very few antibodies are suitable. The production of specific antibodies is made considerably more difficult by the amino acid sequence homologies of the related hormones and the associated cross-reactivity during immunization. A sign of these difficulties is the fact that so far only very few FSH-specific monoclonal antibodies have been described in an international context (Patent DE 3507849A1, 1986; H. Hojo and R. J. Ryan, Endocrinol 117 [1985], 2428). For the insufficient number of previous antibodies speaks the z.Z. small selection of commercial test kits (Serono). An important prerequisite for all immunological detection and purification methods is the presence of stable and well-producing hybrid cell lines. For the hybrid cell clones described so far, no data are available for stability and productivity. Object of the invention The invention aims to produce stable clones which produce monoclonal antibodies to FSH of strong binding ability in good yields. The antibodies are expected to be useful for establishing a specific assay system for the qualitative and quantitative detection of FSH and to discriminate the closely related HCG, LH and TSH hormones, as well as other immunological assays (e.g., immunohistochemical methods) and purification methods. Explanation of the essence of the invention The inventive method for the production of monoclonal antibodies to FSH is characterized in that first in a conventional manner spleen cells of Balb / c mice, dia were immunized with purified FSH, with X 63-Ar 8.6Ü3 myeloma cells in the cell culture with addition of PEG 1500, which sows fusion mixture into microtest plates, selects hybrid cells with a selection medium and determines the antibody-producing hybrids with a solid-phase immunoassay. The hybrid cells which produce specific antibodies against FSH are selected, propagated and optionally after storage in liquid nitrogen, according to the invention for the production of monoclonal antibodies by cultivation or after transplantation to mice in the form of ascites used. The hybrid cell clones used for antibody production according to the invention were deposited in the ZIM deposit facility for cell inks at the Central Institute of Molecular Biology under the following numbers: