Hyperglycemic hormone in fresh water prawn Macrobrachium rosenbergii: Purification from eyestalk nervous tissue and quantification by ELISA in hemolymph following various stresses

Macrobrachium rosenbergii is a fresh water prawn and one of the most abundantly cultured species in India. The purpose of this study was to purify and to develop and validate a sensitive enzyme-linked immunosorbent assay specific for M. rosenbergii hyperglycemic hormone (Mar-CHH). Mar-CHH purified by high performance liquid chromatography revealed that among five immunoreactive peptide fractions four exhibited hyperglycemic activity. The polypeptide with greater hyperglycemic activity was used to raise antibodies in rabbit and an ELISA was developed for this peptide. The detection limit of the assay was 0.4 fmol of peptide and the working range extended to 40 fmol. Recovery of purified CHH was 89.5 ± 5.43; intra-assay variation was 5.9% and inter-assay variation was 9.4%. We used ELISA to analyze the circulatory levels of CHH after various environmental stresses. Significant increase in plasma CHH was observed during exposure to high temperature and heavy metal (cadmium chloride) stresses. Concentrations of hemolymph glucose also increased concomitantly with increases in CHH. Development of the CHH-ELISA allowed rapid and convenient analysis of plasma samples to evaluate exposure to different stresses.