Solid-phase sandwich enzyme immunoassays of human chorionic gonadotropin using monoclonal antibodies

Abstract

A two-site sandwich enzyme immunoassay for human chrionic gonadotropin (hCG) employing monoclonal antibodies directed against β- and α-subunits is described. Monoclonal anti-β-hCG antibody was used for coating microtitration plates and monoclonal anti-α-hCG antibody labelled with 1 of the 3 enzymes namely horseradish peroxidase, alkaline phosphatase or β-galactosidase was used as tracer. The assay is able to detect up to 1 ng hCG/ml. No significant difference was observed with respect to sensitivity and range of assay with the 3 enzymes. The assay can be performed as a 'two-step' assay or reduced to a 'one-step' procedure with a linear relationship between absorbance and hormone concentration up to 31.25 ng hCG/ml. Beyond these concentrations an inflection of the dose curve was observed. This can, however, be avoided by increasing the concentration of antibody-enzyme conjugate. A higher sensitivity enabling detection up to 0.25 ng hCG/ml was attained in the sandwich enzyme immunoassay with the use of biotin-avidin interface. The hCG values obtained on 47 human urine samples either by the 'one-step' or 'two-step' procedure were similar with a correlation coefficient of 0.996. Results obtained by 'two-step' sandwich enzyme immunoassay on 22 human urine samples correlated well (r=0.968) with the values obtained by radioimmunoassay.