Production of antipeptide antibodies for detection of crustacean molt-inhibiting hormone

In crustaceans, the synthesis of steroid molting hormones, ecdysteroids, is negatively regulated by a neuropeptide molt-inhibiting hormone (MIH). Amino acid sequence data place MIH in the crustacean hyperglycemic hormone (CHH) family. As part of our studies of the control of growth and molting in blue crabs (Callinectes sapidus), we have generated antipeptide antibodies for detection of MIH. Using GCG (University of Wisconsin) sequence analysis software programs, two regions of the blue crab MIH peptide were selected for antibody production. These fragments were selected using multiple alignment and peptide structure programs that predicted regions of MIH having high surface probability, flexibility and antigenicity, but which were not highly conserved between MIH and other members of the CHH family. The two 10-mer peptides were commercially synthesized and used to generate polyclonal antisera in rabbits. The binding properties of the antisera were characterized by immunocytochemistry (ICC) and Western blot. Thin section ICC showed specific immunoreactivity in the X-organ/sinus gland complex of eyestalk neural ganglia with both antisera. Western blot analysis revealed that both antisera also bound to proteins of the predicted size in blue crab sinus gland extracts and cell lysates containing recombinant MIH protein. We anticipate that these antipeptide antibodies will be useful for developing an immunoassay for quantification of MIH in hemolymph. Supported by MS/AL Sea Grant and NSF.