DEVELOPMENT AND VALIDATION OF A COMPETITIVE ENZYME-IMMUNOASSAY FOR CHUM SALMON PROLACTIN - A COMPARISON TO RADIOIMMUNOASSAY

An enzyme immunoassay (EIA), based on a competitive assay system, for the measurement of prolactin (PRL) in the pituitary of salmonid fishes and of hormone released in medium from incubated pituitary was developed using a rabbit antiserum to chum salmon PRL (PRL, a combination of PRL I and PRL II). Chum salmon PRL was coupled to horseradish peroxidase (HRP). The incubation procedure for the antigen-antibody reaction was analogous to that in the radioimmunoassay (RIA) for PRL. The antibody-bound HRP-PRL was separated by a double antibody method. The enzyme activity in the precipitate was followed by a colorimetric method, in which 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and o-phenylenediamine were used as substrates. PRL, PRL I, and PRL II showed exactly the same competitive curves in the EIA system. PRL (127-158) showed the highest cross-reactivity among the fragments of PRL examined. Low cross-reactivity was seen with other hormones isolated from chum salmon pituitary. The displacement curves for pituitary extracts from several salmonids, including chum salmon, coho salmon, and rainbow trout, were parallel to that of the PRL standard, whereas those from the carp and tilapia showed negligible cross-reactivity. A parallel displacement curve to the PRL standard was also seen with incubation medium of the pars distalis of the chum salmon pituitary. Plasma from chum salmon, coho salmon, and rainbow trout gave nonspecific HRP activity in the EIA. The values of PRL-EIA were significantly correlated (y = 0.99x + 1.06, r = 0.942, P < 0.05, n = 24) with those obtained in PRL-RIA. The ED50 of PRL-EIA was 11.5 ± 0.86 ng (n = 10). The interassay coefficient was 23.7% at ED50 (n = 10). Intraassay coefficients of variation were 12.8% at 5 ng and 10.6% at 20 ng (n = 10). © 1990.