Development and characterization of a new, highly specific antibody to the human chorionic gonadotropin-beta fragment. In addition to high concentrations of hCG, pregnancy urine contains even higher concentrations of a fragment of the hCG beta-subunit. This biologically inactive material complicates immunological measurement of hCG, since it cross-reacts with many polyclonal and monoclonal antibodies to the hCG beta-subunit that are employed for assays of hCG in urine. Although we and others have developed antibodies to this fragment, specific measurement of the fragment in the presence of free hCG beta has remained difficult due to intrinsic cross-reactivity of these antibodies with the intact hCG beta. Rather than attempt to increase specificity by assay optimization, we developed a new, highly specific monoclonal antibody, designated B210, which cross-reacts less than 0.1% with the free hCG beta-subunit in both liquid and solid phase immunoassay formats. We have used this new monoclonal antibody in immunoradiometric assays to measure specifically the hCG beta fragment in urine throughout pregnancy as well as in the sera of two individuals with cancers producing the hCG beta-subunit. We discovered that the hCG beta fragment can bind three monoclonal antibodies simultaneously, indicating that although the epitope for antibody B210 is a new determinant exposed on the hCG beta fragment and not on intact hCG or on free hCG beta-subunit, the hCG beta fragment retains at least two other hCG beta-related epitopes intact, i.e. those that bind monoclonal antibodies B108 and B201. 