THE DEVELOPMENT OF AN ENZYMEIMMUNOMETRIC ASSAY FOR LH AND THE EFFECTSOF THE METHODS ON THE IMMUNOREACTIVITY OF THE CONJUGATES

Using an affinity purified sheep anti-human luteinizing hormon IgG-hor seradish peroxidase conjugate (sheep anti-hLH IgG-HRP) and rabbit anti -human luteinizing hormone antiserum (rabbit anti-hLH) directed agains t different antigenic determinants, a solid-phase ''sandwich'' enzyme immunometric assay (EIMA) for human luteinizing hormone (hLH) was deve loped. The assay was validated and compared with a liquid phase ''two site'' immunoradiometric assay (IRMA) for hLH which uses same two anti bodies. The sheep anti-hLH IgG, which had been affinity purified by el uting at PH 3,5 from a hLH-sepharose 4 B column, was labelled with I-1 25. The IRMA is based on simultaneous addition of two antibodies to st andards and samples. After overnight incubation, separation was achiev ed by addition of Sheep anti-Rabbit Fc (SARFc) antiserum. In EIMA; par tially denaturated (at PH 2.5) Sheep anti-Rabbit FcIgG (SARFcIgG) coat ed polystyrene tubes or microtitre plates were employed as solid-phase second antibody. The substrate was N, N'-o-phenylene diamine (2 mg/ml ) and H2O2 (0,02 %). Two methods, modified NaIO4 and 4-(N-maleimido me thyl)-cyclohexane-1-carboxylic acid N-hydroxy succinimide ester (SMCC) , were employed in the preparation of sheep anti-hLH IgG-HRP conjugate . The immunoreactivity and peroxidase activity of conjugate prepared w ith NaIO4 method was impared to various extends. Both EIMA and IRMA ha d good specificity, were not susceptible to interference from serum co mponents and exhibited very low non-specific binding. The values deter mined by EIMA were independent of the serum volume employed. Standard added to serum samples was accurately determined and the results obtai ned from the analysis of serum samples correlated closely with those o btained by IRMA.