Development of antibody-based assays for omega-conotoxin MVIIA.
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 titer is 1:8192. Thus, the assays based on this antibody are useful for the conotoxin analysis.
Wang
Feng
F
Department of Biochemistry, Zhejiang University Medical School, Hangzhou 310006, China.
Chen
Gao
G
Zhu
Yongping
Y
Guo
Zujian
Z
Zheng
Shu
S
Zhan
Jinbiao
J
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000235:genetics / Q000276:immunology; D008297:Male; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013879:Thioredoxins / Q000276:immunology; D020960:omega-Conotoxins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
D000906:Antibodies; D011993:Recombinant Fusion Proteins; D020960:omega-Conotoxins; D013879:Thioredoxins; C078452:ziconotide; D005982:Glutathione Transferase