Enzyme linked immunosorbent assay (ELISA) for grouper, Epinephelus coioides vitellogenin receptor: Technical paper no.1/1998
1998
Vichai Vatanakul (Department of Fisheries, Bangkok (Thialand). Songkhla National Inst. of Coastal Aquaculture)
Enzyme-linked immunosorbent assay (ELISA) was developed to study the vitellogenin binding site from solubilized grouper (Epinephelus coioides) oocyte membrane. Vitellogenin produced by estrogen stimulation of grouper was purified by anion-exchange chromatography on DEAE-Sephacel and gel filtration (Sephadex G-150) and identified by incorporation of 3H-leucine in vivo. The binding assay involved an adsorbtion of solubilized membrances in wells of microtitration plates. Scatchard analysis after competition experiments in different conditions (pH, time and temperature) revealed only single class of binding sites. The magnitude of the specific binding was dependent on the amount of oocyte membranes and was saturable. The binding show both tissue and protein specificity. The affinity (KD) of these receptors increase from the previtellogenic to vitellogenic stage.
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