Development of antibody-based assays for omega-conotoxin MVIIA

Journal of Biochemical and Biophysical Methods
Feng WangJinbiao Zhan

Abstract

Omega-conotoxin MVIIA (CTX MVIIA) is a specific peptide blocker of the N-type voltage-sensitive calcium channel in neurons. The synthetic version of CTX MVIIA, Ziconotide, has been recently approved by FDA for management of severe and chronic pains. Currently, the chemical synthetic CTX MVIIA has been analyzed by RP-HPLC, and there are no chemical or immunological assays available for determination of the peptide. In this article, we report a novel method for preparation of polyclonal antibody against CTX MVIIA, and the antibody-based assays for the analysis of CTX MVIIA. The DNA sequences encoding the conotoxin were chemically synthesized and then cloned into the expression vector pGEX-2T. The GST fusion protein of CTX MVIIA was expressed in E. coli BL21 (DE3) with induction of IPTG. The purified fusion protein was used to immunize the male rabbits with standard protocols. The produced antiserum was purified through anion-exchange chromatography. Another thioredoxin (Trx) fusion protein of CTX MVIIA was employed to cross-examine the antibody against the conotoxin. Our Western blot and ELISA results show that the polyclonal antibody was capable of binding the conotoxin parts of both GST and Trx fusion proteins, and the antibody...Continue Reading

References

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Citations

Nov 26, 2010·Proceedings of the National Academy of Sciences of the United States of America·Sheung Kwan LamRandy Schekman
Aug 30, 2014·Monoclonal Antibodies in Immunodiagnosis and Immunotherapy·Yanling YangZhenhong Zhuang
Jan 9, 2007·Neuroscience Letters·Shuang WangZhong Chen
Jul 3, 2013·Toxicon : Official Journal of the International Society on Toxinology·Bingmiao GaoSulan Luo

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