Ultrasensitive two-site immunometric assay of human lutropin by time-resolved fluorometry

Abstract

Abstract A rapid, sensitive two-site immunometric assay for human lutropin (LH), with two monoclonal antibodies directed against the beta subunit, has been developed with time-resolved fluorescence as the detection principle. The cross-reactivities with human thyrotropin and human follitropin are negligible and that with human choriogonadotropin is 1% in the standard two-step protocol. Free beta subunits of LH are also detected. Besides the standard procedure (45 + 15 min), more rapid alternative protocols (15 + 15 min or 30 min in one step) have also been evaluated. The minimum detectable dose per well is approximately 1 amol (10(-18) mol), corresponding to less than 0.01 int. unit/L, for a sample volume of 25 microL and less than 0.001 int. unit/L for 200-microL samples. The intra- and interassay CV is less than 6% for LH concentrations from 0.2 to 250 int. units/L. With 200-microL sample volumes, intra-assay variation is less than 6% at 0.04 int. unit/L and interassay variation is 7% at 0.05 int. unit/L. The very high sensitivity and reproducibility of the presented method enables assay of low prepubertal and of suppressed LH concentrations, and represents a clear improvement over currently available immunoassays.