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Immunological Techniques: Monoclonal Antibodies Immunologische Analysenverfahren: Monoklonale Antiktrper B 22 Enzyme-Immunoassay for Intact hCG Using Monoclonal Antibodies

A. M. G. Bosch and W. J. H. M. Stevens

Organon Scientific Development Group, P.O. Box 20, NL-5340 BH, Oss, The Netherlands

Table 1. Effect of combinations of MCAs and pH of test medium on the sensitivity of the hCG EIA. The separate MCAs were tested in a competitive EIA [1]. Sensitivities were read at blank + 3 SD (sandwich EIA) or 80 % binding (competitive EIA)

Combina- Sensitivity (IU/1) Combina- Sensitivity (IU/1) tion A tion B

Buffer Serum Buffer Serum

Enzymimmunoassay fdr intaktes hCG unter Verwendung monoklonaler Antiktrper

Introduction. Immunoassays for hCG in serum are widely used for early detection of pregnancy. The majority of assays based on polyclonal antisera suffer from lack of specificity due to a significant cross-reaction with LH. In order to ensure a high assay specificity together with a constant quality of reagents, we used monoclonal antibodies (MCAs) for development of a sandwich enzyme-immunoassay (EIA) for hCG.

Methods. Preparation of MCAs including immunisation, cell fusion, screening of supernatants and characterization of hy- bridomas was performed as described earlier [1].

For development of the sandwich EIA MCAs directed against a determinant on the fl-subunit ofhCG (anti-t) were used for coating of microtitration strip plates [1]. The enzyme label used consisted of MCAs directed against a determinant on hCGe (anti-e) labelled with horse radish peroxidase according to the SPDP method [2]. The test procedure was as follows: 50 gl of test fluid (serum sample, standard or control) and 50gl of enzyme- labelled antibodies were added to antibody coated strip plates and incubated for 2h at room temperature. After washing (3 times) with distilled water, 100pl of substrate solution (tetramethylbenzidine + urea hydrogen peroxide) was added. After 30 min incubation at room temperature the reaction was stopped with 100 ~1 of 2 mol/1 sulphuric acid and the absorption measured at 450 nm.

Results. Various combinations of MCAs were screened for sensitivity for hCG and LH in the sandwich EIA. The highest sensitivity and specificity were obtained with combinations of solid phase anti-fl and enzyme-labelled anti-e. It is obvious from the results listed in Table 1, that the use of two different MCAs greatly improved the assay sensitivity when compared with that of the individual MCAs. Another increase in sensitivity, also shown in Table 1, could be obtained by varying the pH of the test medium. This is, however, strongly dependent on the MCA combination used.

With test combination B a detection limit (blank + 3 SD) of 2 IU hCG/1 serum was obtained (standard range: 5 - 3 0 I U hCG/1), while 200 IU LH/1 serum gave no reaction. This was tested with crude urinary hCG and LH preparations. The assay proved to be specific for intact hCG as virtually no cross- reactivity was found towards the subunits. The specificity of the

Effect combination vs. s e p a r a ~ M C A s

143A a 293A a + 117C 2.1 10.3 + 116C b - 1.8

143A 3,100 - 293A 430 - 117C 400 - 116C 1,450 -

Combina- Sensitivity (IU/1) Combina- Sensitivity (IU/1) tion A tion B

Buffer Serum Buffer Serum

Effect pH of test medium

pH 7.4 7.7 44.0 pH 7.4 - 1.8 6.9 4.8 34.8 6.9 - 1.6 6.4 3.1 24.0 6.4 - 2.2 5.9 2.3 10.3 5.9 - 1.9 5.4 1.8 12.2 5.4 - 4.6 4.9 1.9 13.8 4.9 - 25

" Solid phase b Enzyme label

assay was also checked by testing a large number of male and female sera (premenopausal, midcycle, postmenopausal). 4 out of the 477 samples tested gave a positive result (> 5 IU hCG/1) due to early pregnancy, the others were negative.

The assay recovery ranged between 98 and 107 %, the within- assay and from day-to-day coefficients of variation were < 10 and < 15 % respectively (tested at levels of 7.5 and 25 IU hCG/1 serum).

Conclusions. The high sensitivity and specificity makes the assay a useful tool for surveillance of very early pregnancies as well as for monitoring of tumour patients.

References

1. Bosch AMG, Stevens WJHM, Schuurs AHWM, Sch6nherr OT, Roelofs HWM (1981) In: Peeters H (ed) Protides of the biological fluids, vol 29, Pergamon Press, Oxford, pp 8 3 7 - 846

2. Carlsson J, Drevin H, Axen R (1978) Biochem J 173:723

Offprint requests to: A. M. G. Bosch Fresenius Z Anal Chem (1984) 317:725

�9 Springer-Verlag 1984

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