Rapid and simple immunoassays for measurement of human chorionic gonadotropin using monoclonal antibodies

Two monoclonal antibodies, designated Gab-35 and Gab-144, were used in competitive radioimmunoassay (RIA) and solid-phase sandwich enzyme immunoassay. By SDS-polyacrylamide gel electrophoresis and protein blot enzyme immunobinding assay, Gab-35 and Gab-144 were shown to react specifically to the alpha-subunit and beta-subunit of human chorionic gonadotropin (hCG), respectively. The immunoglobulin class and subclass of these two monoclonal antibodies were found to be IgG1 by the Ouchterlony double gel immunodiffusion test. The beta-subunit specific monoclonal antibody of Gab-144 was used in competitive RIA for determination of serum hCG in the range of 5-500 mIU/ml. The assay results correlated well with the monoclonal antibody-based RIA system and commercial RIA kits using polyclonal antisera. Moreover, the present competitive RIA system showed a shorter incubation time (1 hour 20 minutes) and a shorter total assay time (1 hour 40 minutes), as compared to those of the commercial RIA kit. A solid-phase sandwich enzyme immunoassay was developed using a combination of the Gab-35 antibody-coated microtiter plate and the beta-subunit specific antibody of Gab-144 conjugated with horseradish peroxidase (HRP). The assay range was 20-200 mIU/ml, and the incubation time was only 30 minutes. In addition, the solid-phase sandwich enzyme immunoassay developed in the present study could also be used as a qualitative hCG assay with a cutoff point of 50 mIU/ml as an early pregnancy test at or just a few days before the missed period.