Antigen Selectivity Characteristic of Polyclonal Antibodies Against Omega-Conotoxin GVIA and N-Type Voltage-Dependent Calcium Channels

Abstract

The antibodies against omega-conotoxin GVIA (ω-CTX GVIA; N-type voltage-dependent calcium channel [VDCC] blocker) and B₁Nt (N-terminal segment [residues 1–13] of BI α₁ subunits of VDCCs) were prepared, and the selectivity for each antigen ω-CTX GVIA and B1Nt was investigated. For the antigen selectivity of anti–ω-CTX GVIA antibody against ω-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 ω-CTX GVIA, but not by ω-CTX SVIB (N-type VDCC blocker), ω-CTX MVIIC (N- and P-type VDCC blocker), or ω-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 × 1028 and 1.3 × 1028 M, respectively. The bands of 210 and 190 kD by Western blotting of crude membranes from chick brain were also inhibited by 1 μM B1Nt. These results suggest that the antibodies prepared against ω-CTX GVIA and B1Nt in this work have high selectivity for their antigen. Therefore we assume that the antibodies against ω-CTX GVIA and B1Nt are useful tools for the analyses of the function and distribution of N-type VDCCs. The anti ω-CTX GVIA antibody must also be useful for the radioimmunoassay of ω-CTX GVIA.