A Monoclonal Antibody-Based Immunoradiometric Assay for h-LH

Abstract

An immunoradiometric assay (IRMA) for h-LH based upon an 125I-labelled mouse monoclonal antibody (MAb) to h-LH with an effective equilibrium constant of 5·8 × 109 l/mol is described. A total incubation time of 3 h at room temperature was required, separation by means of the sucrose layering procedure took a further 1 h and counting times were 1 min/tube. Using the first IRP for h-LH (prep. 68/40) as standard, the detection limit was 0·1 U/l serum and the within-assay CV for duplicate determinations was <10% over the range 1–280 U/l and <3% at 10–100 U/l. The epitope, with which the MAb reacted, shared structures, on the α- and β-subunits of LH since the assay responded to the intact hormone, but not to either of the subunits. Specificity was > 100 000:1 for h-LH vs. h-FSH and > 10 000:1 for h-LH vs. h-TSH. h-CG and h-LH were approximately equipotent. The results on 604 unselected samples were generally very similar to those found by RIA except at levels below 2 U/l for which the IRMA regularly gave lower results suggesting relative freedom from non-specific serum effects. The new assay, based upon potentially limitless supplies of a very stable reagent offers advantages of speed, sensitivity, range, and precision over conventional RIA. The specificity appears to be excellent. Although there are marginally more steps the total staff involvement is less than with conventional methods employing centrifugation.