Preparation Of Monoclonal Antibody Against Macrobrachium Nipponense Molt-inhibiting Hormone

Molting in crustaceans is inhibited by the molt-inhibiting hormone(MIH), a neuropeptide member of the CHH family. Besides MIH, CHH family neuropeptides include the crustacean hyperglycemic hormone(CHH), gonad-inhibiting hormone(GIH) and mandibular organ-inhibiting hormone(MOIH). These peptides have common amino acid compositions and organizations. All members of this family generally have 70~78 amino acids residue.In this study, the recombinant MIH is highly expressed in the E.coli prokaryotic expression system, and it existed in a form of inclusion body. By denaturation, renaturation and washing, the inclusion body was purified. The recombinant protein was used as immunogen to prepare the monoclonal antibody in BALB/C mice. The high quality of the antibody was successfully identified by ELISA. PEG(Mr=4000) was used to induce cell fusion of SP2/0 and immunized mice’s spleen cells, and get hybridoma cells.After fusion, 18 wells of all the wells with appearance of hybridoma showed strongly positive. After further subclone, 1 hybridoma cell line that had stable nature and high titer was screened, and the titre of its supernatant reached 1:204800. This hybridoma cell was elected to make ascites, and we collect ascites to purify monoclnal antibody, whose titre reached 1:204800. The concentration of monoclonal antibody is 0.65mg/mL.The monoclonal antibody provided technical support for mechanism investigation of the MIH. It also provided an effective tool for measuring the concentration of MIH in blood lymphocytes.