DD271709B1 - PROCESS FOR PREPARING MONOCLONAL ANTIBODY AGAINST PMSG - Google Patents

Description

Field of application of the invention

The invention relates to a method for the production of monoclonal antibodies which react specifically with PMSG. The monoclonal antibodies can be used for the quantitative determination of PMSG by an enzyme immunoassay in veterinary diagnostics, affinity chromatographic isolation and high purification of this hormone, as well as to control the sexual cycle of female farm animals.

Characterization of the known technical solutions

To value the value of PMSG preparations, only bioassays are used which are costly and time consuming, extremely poorly standardizable and ethically anfp.ntbsr. The alternative value assessment of PMSG by radio or enzyme immunological methods requires a degree of purity of the reagents which can be determined conventionally, ie. H. It is not possible to achieve this by chromatographic purification or by immunization of animals or only with unacceptably high expenditure. This is apparently primarily due to the detection of PMSG or components of the hormone, which are still antigenic but have no biological activity. By means of conventional antisera, it is not possible to differentiate between effective and inactive components of PMSG, nor is molecular differentiation possible. In addition, such conventional antisera generally have a number of other disadvantages, so antisera of different animals are not identical, on the contrary, there may be considerable differences in the content of antibodies, in quantitative and qualitative terms. These variations and the batch-dependency of the antisera do not allow a continuous standardization of the assays based on them. Monoclonal antibodies, which are secreted by permanently growing and producing hybridomas, are available in practically sufficient quantities and over a theoretically unlimited period of time, so that reproducible and standardizable immunoassays can be built on their basis.

It is already known how monoclonal antibodies are produced. The principle solution of hybridoma production was described by KÖHLER and MILSTEIN (Nature 256, [1975], p. 495).

There has hitherto been known a monoclonal antibody which reacts with PMSG (MOYAERT et al., Breeding Hygiene 20, 49-53, 1985). However, this monoclonal antibody is not accurately described in terms of its binding reactivity, but it mentions as a criterion a neutralizing (blocking) effect of PMSG binding in vitro in an enzyme immunoassay, and describes its effects on in vivo administration to cows.

Object of the invention The aim of the invention is to provide a process for the preparation of monoclonal antibodies which is easily controllable It allows the cost-effective production of larger quantities of specific antibodies against PMSG. The monoclonal antibodies are intended both for the quantitative determination of PMSG by means of a radio or Enzyme immunoassays as well as for affinity chromatography isolation and high purification after coupling to a

solid carrier are suitable.

By providing such monoclonal antibodies, the production control and the production of

highly purified, indication-specific PMSG preparations are significantly improved.

By injecting suitable monoclonal antibodies into female farm animals, control of the sexual cycle is to be improved

become.

Explanation of the essence of the invention The invention has the object of providing a traceable method for producing monoclonal antibodies against Specify PMSQ, in the course of which hybridomas are generated that are easy and inexpensive to handle and

their cultivation is technically and economically advantageously possible. Using these hybridomas, monoclonal antibodies are to be generated in subsequent process steps which react specifically with PMSG and are used for the quantitative determination, isolation and partial or complete blockage of the biological activity of the

PMSG are suitable. The object is achieved in that initially isolated by the known methods PMSG covalently on HMmocyanin

is coupled and this high molecular conjugate is used as Imtniinogen for immunization. Preferably, 6-8 week old female BALB / c mice are used for immunization. The hyperimmunization of the animals is induced by repeated administration of the immunogen.

From the spleens of such hyperimmunized mice, the lymphocytes are separated in a conventional manner. The B lymphocytes of this cell mixture are fused with murine myeloma cells. The appropriate mouse myeloma cells P3-X63 Ag 8,653 (KEARNEY et al., J. Immunol. 123, (19791, p. 1548) are translated into RPMM640 with 10% fetal calf serum (FCS).

drawn.

The fusion is triggered by polyethylene glycol or by Feldsprungtechnik. In a culture medium containing hypoxanthine, aminopterin and thymidine (UTTLEFIELD, Science 145, (1964), p.709),

the hybrid cells formed are separated from the unfused myeloma cells and according to the invention cell clones

which secrete antibodies of the desired specificity and other desired properties. The selection is made by dividing the cells immediately after the fusion into a plurality of separate 200 Ml cultures.

The supernatants of the cultures containing the growing hybrid cells are assayed for the presence of antibodies

which bind to solid phase adsorbed PMSG. For this purpose, the culture supernatants with a corresponding

Enzyme immunoassay tested. By testing with a suitable range of other substances, the specificity for

the PMSG detected.

Among the antibody-producing hybridomas are then in a second selection step those hybridomas

selected whose Antikörpe ^r also are capable, upon binding to a solid phase, for example on plastic surfaces with anti-

Mouse immunoglobulin have been coated to isolate PMSG from a mixture specifically, with high efficiency. The Testing whether PMSG was isolated from the test mixture is carried out by bioassay. In a further selection step, it is tested whether the monoclonal antibodies also the biological effect of PMSG

can block.

The hybridomas obtained by such selection are recloned and highly productive subclones are selected which are the

secrete the desired monoclonal antibody stably in high concentration. The resulting hybridomas received the

Designation H-BL-FMSG / 1 to 3. The preparation of the monoclonal antibody BL-PMSG / 1 to 3 is based on a method which refers to the in vitro or

/ n-v / Vo culture of hybridomas H-BL-PMSG / 1 to 3.

The hybridomas H-BL-PMSG / 1 to 3 are cultivated at the Life Sciences Section of Karl Marx University Leipzig

are cryopreserved and are available to interested parties. The monoclonal antibodies BL-PMSG / 1 to 3 have the

Registration numbers ZIM0272, ZlM0271 and ZIM 0270 were obtained from the Central Institute for Molecular Biology of the AdW of the GDR. The monoclonal antibody BL-PMSG / 1 has the following valuable and surprising properties: It binds solid phase-bound and dissolved PMSG specifically, while also binding PMSG in surface-bound form

and thus suitable for affinity-chromatographic isolation of the PMSG.

It binds to an epitope (A) of PMSG, which is essential for the biological action of PMSG. BL-PMSG / 1 blocks the / n-v / Vo effect of PMSG. In contrast, the monoclonal antibodies BL-PMSG / 2 and BL-PMSG / 3 bind to epitopes of PMSG different from the PMSG PMSG / 1 epitope (A epitope) differ. Both antibodies hinder the binding of PMSG to the monoclonal antibody BL-PMSG / 1 not. They can thus be used as detection reagents for PMSG which has been isolated by BL-PMSG / 1 in an enzyme or Radioimmunoassay be used advantageously. BL-PMSG / 2 and BL-PMSG / 3 react in turn with independent epitopes of PMSG, so that they are at the same time Application as a detection antibody, an increase in sensitivity or in alternative use, the differential diagnosis and Allow separation. embodiment Testing the binding of monoclonal antibodies to various substances

1. Polystyrene microtiter plates are coated with various hormones and serum proteins. For this purpose, the wells of the plate with 0.25 ml of the antigens (1 to 50 mg / l), in carbonate buffer (0.1 mol / l, pH 9.6) dissolved, filled and for 2 h at room temperature or 16h at + 4 ° C incubated. The cavities are then filtered off with suction and washed three times with each * 0.3 ml PBS (0.15 mol / l NaCl, 0.01 mol / l phosphate, pH 7.4).

2. The antibody-containing solutions (cell culture supernatant or ascites) are filled in different dilutions in the wells of the coated plate (each ΙΟΟμΙ) and kept for 2h at room temperature, then aspirated and washed three times with 0.3ml PBS / 0.05% Tween 80 each ,

3. Addition of ΙΟΟ-μΙ-POD-labeled anti-mouse immunoglobulin antibodies. After a reaction of 2 h at room temperature, the cavities are filtered off with suction and washed as indicated under 2.

4. Incubation with 0.2 ml of substrate-chromogen mixture consisting of H ₂ O ₂ and o-phenyldiamine, stopping the reaction after a sufficient development time (15-45 min) with 100 μM H ₂ SO ₄ (5 mol / l) and measuring the extinction at 492nm.

Claims (2)

1. A method for the production of monoclonal antibodies against various epitopes of PMSG (Pregnant Mare's Serum Gonatropln) by immunization of BALB / c mice with PMSG, fusion of the spleen derived B-lymphocytes with murine myeloma cells, selection of hybridomas, monoclonal antibodies which specifically react with epitopes of the PMSG, characterized in that a covalently coupled PMSG carrier conjugate is used as the immunogen; first, that the selection is by a monoclonal antibody of particular suitability as a detection reagent for solid-phase-bound PMSG-recognizing assay; in a second selection step, hybridomas are selected whose monoclonal antibodies in solid-phase-bound form specifically and effectively isolate PMSG from a mixture; in a third selection step hybridomas are selected for monoclonal antibodies which inhibit the hormone action in a PMSG-specific bioassay; it is demonstrated in a fourth selection step that the selected monoclonal antibodies recognize different epitopes and do not interfere with each other; the hybridomas H-BL-PMSG / 1 (ZIM 0272), H-BL-PMSG / 2 (ZIM 0271) and H-BL-PMSG / 3 (ZIM 0270) were selected and used to obtain the monoclonal antibody BL-PMSG / 1 -3 are used. 2. The method according to claim 1, characterized in that a covalent PMSG-hemocyanin conjugate is used to immunize the BALB / c mice.