Development of Vitellogenin-ELISA, an In Vivo Bioassay, and Identification of Two Vitellogenesis-Inhibiting Hormones of the Tiger Shrimp Penaeus monodon

Abstract:

A sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of vitellogenin in the hemolymph of Penaeus monodon is reported. Lipovitellin from the mature ovary was purified using hydroxylapatite column chromatography and used as the standard protein, which was serially diluted from 3336 to 6.51 μg and gave a linear plot. Sensitivity results showed ELISA was insensitive to samples that did not contain vitellogenin or lipovitellin. Specificity results showed the degree of the discrimination of the assay between positive samples (having vitellogenin or lipovitellin) and negative samples (devoid of vitellogenin or lipovitellin). Reproducibility studies showed that the intraassay coefficient of variation was 5.1% (n= 11) and the interassay coefficient of variation was 5.15% (n= 13). Separation of X-organ sinus gland peptides using a reversed phase column gave a total of 37 fractions. Two fractions were found to reduce the hemolymph vitellogenin concentration in a time-dependent manner and could be identified as vitellogenesis-inhibiting hormones I and II.