DD271572A1 - METHOD FOR THE QUANTITATIVE DETERMINATION OF PREGNANT MARE'S SERUM GONADOTROPHINE (PMSG) - Google Patents

Abstract

The invention relates to a method for the quantitative determination of Pregnant Mares Serum Gonadotrophin (PMSG) by means of a two-side immunoassay (sandwich principle) using only monoclonal antibodies as immunological reagents. The object is achieved by using the monoclonal antibody BL-PMSG / 1 as a capture antibody and the enzyme-labeled monoclonal antibodies BL-PMSG / 2, BL-PMSG / 3 or BL-PH (a) 24 individually or in combination. Fields of application are veterinary medical diagnostics and production control.

Description

an enzyme, ζ. B. Meerrettlch peroxydase (POD), via the known methods, ζ. B, via glutaraldehyde, via perlodataktlvierter

POD and the like. The cleaning of the Antlkorper-EnzynvKonJugates can by means of conventional methods, ι. B. by Qelchromatographle

or rPLC.

After incubation with the enzyme-labeled antibody (s), rinsing is carried out to remove it

bound Detektlonsantlkörper.

In another embodiment, the antibody or enzyme marker antibody is immediately labeled with the PMSG standard and the Test samples Plpetted into Reactive Vessels and incubated together. This is followed by removal of unbound Components by washing. The enzyme activity bound to the solid phase via the formed complex is then gomessen, whereby the for dao

selected marker on the usual subetrate chromogen Gemlsche be used. According to the sandwich *

Principle the more enzyme activity the solid phase gebundon, the more PMSG contained in the sample. The Concentration determination in the sample is made by comparison with PMSQ standards. The monoclonal antibodies used, BL-PMSG / 1 to 3 and BL-PH (a) 24, are in the ZIM of the AdW of the GDR under the numbers

Registered 0272,0271,0270 and 0146. The hybridomas are at the section Life Sciences of the Karl Marx Unlvereltat Leipzig In

Culture or cryokonsei are four and are accessible. exemplary Determination of the concentration of F.tiSG in mare serum

1. Polystyrene microtiter plates are coated with the monoclonal antibody BL-PMSG / 1. For this, the wells of the plate were filled with 0.25 ml of a solution of the antibody (50 mg / l), dissolved in carbonate buffer (0.1 mg / l, pH 9.6) and kept at room temperature for 2 h or + 4 ° C. for 16 h incubated. The cavities are then filtered off with suction and washed three times with 0.3 ml PBS (Q, 15 mol / l NaCl, 0.01 mol / l phosphate, pH 7.4).

2. Per 100μι PMSG'Standard (dilution series) or control sera or the test samples are placed in the wells for coated plate and incubated for 2 h at room temperature, then aspirated and washed three times with 0.3ml PBS / 0.05% Tweon 80 gewaschon.

3. Addition of 100 μM POD-labeled monoclonal antibody BL-PMSG / 3, prepared by coupling periodate-activated horseradish peroxidase with purified monoclonal antibody BL-PMSG / 3 (NAKANE, PK and J. AKAWAO: Histochem Cytochome 22 (19741, 1084) After incubation for 2 h at room temperature, the cavities are filtered off with suction and washed as indicated under 2.

4. Incubation with 0.2 ml substrate-chromogen mixture, consisting of H ₂ Oj and o-phenyldiamine, stopping the reaction after sufficient development time (15-45m! N) with 100μΙ HjSO ₄ (5mol / l) and measuring the absorbance at 492nm .

Claims (1)

-1-271672 claim: Method for the quantitative determination of pregnane. Mare's Scum üonado'rophin w "-d and when PanganlikÖrper serves means of two rare-immunoassay, characterized in that the assay using only \ monoclonal antibodies is performed as immunological reagents, wherein the monoclonal antibody BL-PMSG / 1 bound to a solid phase while the enzyme-labeled anti-monoclonal antibodies BL-PMSU / 2 and 3 as well as BL-PH (a) 24 are used, either in isolation or in combination, as detection antibodies. The monokional antibodies have the registration numbers 0272,0271,0270 and 014b in the ZIM of the AdW of the GDR. Field of application of the invention The invention relates to a method for the quantitative determination of the PMSQ by means of a Zwöloelten Immunoassay. Such a method permits the simple and rapid determination of the hormone during the production of and in veterinary diagnostics. Characteristic of the known technical solutions In the present case, bioassays are used for the quantitative determination of the PMSG. These bioassays are time consuming and KiU a Rilhe flawed. The quantitative BeJtIm nunp dia PMSO means of radio · or enzymlmmunologlscher methods has, in practice, inter alia, by not bowän., That could not be ensured by the previously used conventional antisera produced by immunization of experimental animals, that the measured values with correlate to biological activities. This is apparently primarily due to the detection of PMSQ or of components of the hormone which are still antigenic but have no biological activity. By means of conventional antisera, it is not possible to distinguish effective and inactive components of the PMSQ so that assays based on them do not give reliable values. In addition, such conventional antisera generally have a number of 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-dependent nature of the antisera do not allow for consistent standardization of assays based on them. Monoclonal antibodies, which are secreted by permanently growing and producing hybrids, are available in practically sufficient quantities and for 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 258, (1975), p. 495). However, no monoclonal antibodies to PMSG have been produced and used in a two-site immunoassay. Object of the invention The object of the invention is to develop a reliable reproducible values analytical method that allows a sensitive, specific, time-saving and cost-effective determination of PMSG. Explanation of the essence of the invention The invention has for its object to develop an analytical method based on the principle of the two-sided immunoassay for the quantitative determination of PMSG using only monoclonal antibodies as immunological reagents. According to the invention the object is achieved in that the monoclonal antibody BL-PMSG / 1 is bound to a solid phase. The monoclonal antibody is thereby used as a purified immunoglobulin fraction from ascites or cell culture supernatant. The coupling to the solid phase takes place either by adsorption or by covalent coupling. After removal of excess antibody by several washes, non-specific binding sites of the solid phase are blocked by loading with an inert protein. Subsequently, the incubation is carried out with a dilution series of a PMSG standard as well as with the test samples and corresponding controls. After this incubation, the samples are aspirated and the vessels with the fixed antibody, washed again. Thereafter, the PMSG bound to the monoclonal antibody is quantified by enzyme-labeled monoclonal antibodies reactive with an A-epitope of PMSG other than that recognized by BL-PMSG / 1. The monoclonal antibodies BL-PMSG / 2, BL-PMSG / 3 and BL-PH (a) 24, individually or in combination, can be used as the doping antibody. The Darstelluna the enzyme-linked antibodies by coupling the complete monoclonal antibody with