Affinity purification of antibodies specific for 1,4-dihydropyridine Ca2+ channel blockers

High-affinity antibodies specific for the 1,4-dihydropyridine Ca2+ channel blockers have been produced in sheep and affinity purified using a dihydropyridine-Sepharose affinity column. Dihydropyridine-Sepharose affinity matrix was synthesized by reaction of aminohexyl-Sepharose with an affinity analogue of nifedipine, dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridine-dicarbo xylate. Residual amine groups were then blocked by carbodiimide-catalyzed acetylation. [3H]Nitrendipine-binding activity in serum was specifically absorbed by the dihydropyridine-Sepharose affinity column. The bound antibody was eluted with diethylamine (pH 11.5) in 10% dioxane or with a low-affinity dihydropyridine ligand (diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate), pH 7.4. Thirty-six milligrams of highly pure IgG antibody, as demonstrated by sodium dodecyl sulfate-gel electrophoresis, was isolated from 50 ml hyperimmune sheep serum. The affinity-purified anti-dihydropyridine antibodies have been shown to have high affinity (Kd approximately 0.1 nM) and specificity for the 1,4-dihydropyridine Ca2+ channel blockers and, therefore, exhibit dihydropyridine-binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+ channel blockers. Immunoblot staining of an azidopine-bovine serum albumin conjugate with affinity-purified antidihydropyridine antibodies demonstrated that the anti-dihydropyridine antibodies recognize the 1,4-dihydropyridine Ca2+ channel blockers when covalently coupled to protein and, therefore, should be useful in the identification and purification of receptors covalently labelled with 1,4-dihydropyridine Ca2+ channel blockers.