IMMUNOLOGICAL CROSS-REACTIVITY BETWEEN RAINBOW-TROUT GTH-I AND GTH-IIAND THEIR ALPHA-SUBUNIT AND BETA-SUBUNIT - APPLICATION TO THE DEVELOPMENT OF SPECIFIC RADIOIMMUNOASSAYS

Immunological cross-reactivities between rainbow trout GTH I and GTH I I and their alpha and beta subunits have been studied using highly pur ified rainbow trout gonadotropins and subunits and antibodies raised a gainst beta subunits. From these observations radioimmunoassays have b een developed for rainbow trout GTH I and GTH II. The GTH II RIA was h ighly specific and cross-reacted only with GTH II and its beta 1 and b eta 2 subunits, with beta 2 being less potent than pi in competing GTH II binding. There was no cross-reactivity with GTH I. Its sensitivity varied between 0.1 and 0.2 ng/ml, allowing GTH II measurement early i n the reproductive cycle. Variations between and within assays were le ss than 10%. There was a lack of specificity of GTH I RIA (44% crossre activity with GTH II, when using labelled native GTH I). Reasons for t his lack of sensitivity were studied. It cannot be attributed to beta subunits (less than 1.2% cross-reactivity). However, the cross-reactiv ity of a subunits was very important. This suggests that the presence of free a subunits in the medium can be responsible for the lack of sp ecificity. Labelling native GTH I resulted in conformational change in molecular weight and dissociation of the hormone into subunits, where as iodination did not induce GTH II dissociation. This dissociation ca n be avoided by labelling the stable form of GTH I. Using this radio-t racer, the specificity and the sensitivity of the assay were greatly i mproved (GTH II cross-reactivity was decreased to 3.7%, mean sensitivi ty 0.87 +/- 0.072 ng/ml). The sensitivity of the assay diminished with ageing of labelled GTH I. The assay variation was 4.6% within an assa y and 9.8% between assays. The use of labelled beta GTH I still increa ses the specificity (2.3% GTH II cross-reactivity), but with a 2.4-fol d loss of sensitivity. In both GTH I and GTH II RIA plasma and spiked plasma with purified GTHs gave displacement curves parallel to standar d. These assays were used to study pituitary responsiveness to a GnRH analogue in female rainbow trout prior to oocyte maturation. The effec ts of GnRH on GTH II secretion were confirmed. The peptide did not sig nificantly stimulate GTH I secretion, (C) 1998 Academic Press.