Antigen selectivity characteristic of polyclonal antibodies against omega-conotoxin GVIA and N-type voltage-dependent calcium channels.

Abstract

The antibodies against omega-conotoxin GVIA (omega-CTX GVIA; N-type voltage-dependent calcium channel [VDCC] blocker) and B1Nt (N-terminal segment [residues 1-13] of BI alpha1 subunits of VDCCs) were prepared, and the selectivity for each antigen omega-CTX GVIA and B1Nt was investigated. For the antigen selectivity of anti-omega-CTX GVIA antibody against omega-CTX GVIA, ELISA, and immunoprecipitation were used. The reactions for ELISA and immunoprecipitation were observed except when antibody IgG purified by Protein A-Sepharose CL-4B from nonimmunized serum (purified NI-Ab) was used. The specific reactions were inhibited by 10 nM omega-CTX GVIA, but not by omega-CTX SVIB (N-type VDCC blocker), omega-CTX MVIIC (N- and P-type VDCC blocker), or omega-Aga IVA (P-type VDCC blocker). For the antigen selectivity of the anti-B1Nt antibody, analyses by ELISA, immunoprecipitation, and Western blotting were conducted. The reactions were observed except when NI-Ab was used. The ELISA and immunoprecipitation reactions were inhibited by the antigen peptide B1Nt, and the IC50 values were about 1.2 x 10(-8) and 1.3 x 10(-8) M, respectively. The bands of 210 and 190 kD by Western blotting of crude membranes from chick brain were also inhibited by 1 microM B1Nt. These results suggest that the antibodies prepared against omega-CTX GVIA and B1Nt in this work have high selectivity for their antigen. Therefore we assume that the antibodies against omega-CTX GVIA and B1Nt are useful tools for the analyses of the function and distribution of N-type VDCCs. The anti omega-CTX GVIA antibody must also be useful for the radioimmunoassay of omega-CTX GVIA.