A newly established ELISA showing the effect of the androgenic gland on secondary-vitellogenic-specific protein in the hemolymph of the crayfish Cherax qundricarinatus

A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to m onitor the onset of secondary vitellogenesis in Cherax quadricarinatus fema les and in intersex individuals (having both male and female reproductive s ystems) after removal of the androgenic gland (AG). As a prerequisite for t he assay, the 106-kDa polypeptide was separated from newly laid C. quadrica rinatus eggs by SDS-PAGE, and anti-106-kDa antibody was raised in rabbit. T he specificity of the anti-106-kDa polypeptide for proteins specific for th e hemolymph of secondary-vitellogenic females was confirmed by double immun odiffusion and immunoblot cross-reactivity tests. A characteristic standard ELISA curve, using egg high-density lipoprotein (HDL), showed linearity be tween 16 and 500 ng (r = 0.953) and was sensitive for amounts as low as 8 n g. The inter- and intraassay coefficients of variance were 14.8 and 7.2%, r espectively. Only traces of egg HDL equivalents were detected in the hemoly mph of male and primary-vitellogenic females (11 to 110 mu g/ml), confirmin g the specificity of the assay, whereas high levels of such a protein (8-35 mg/ml) were detected in the hemolymph of secondary-vitellogenic females. R emoval of the AG from intersex individuals leads to a significant increase in the concentration of vitellogenic-specific protein in the hemolymph (up to 2 mg/ml). Moreover, a significantly lower concentration was found in fem ales subjected to AG transplant (79.3 mu g/ml). The ELISA thus provided an accurate and sensitive tool to investigate the influence of the AG on the e xpression of a vitellogenic-specific protein in female and intersex C. quad ricarinatus, confirming the central role of this gland in tuning sexual pla sticity in this species. (C) 1999 Academic Press.