Monoclonal antibodies against the alpha-bungarotoxin-binding protein of chick optic lobe

The nicotinic acetylcholine (ACh) receptor probe alpha-bungarotoxin (alpha-Butx) binds with high affinity to a membrane protein of the vertebrate central nervous system. To characterize further this putative neuronal ACh receptor, we have prepared monoclonal antibodies (mAbs) against the alpha-Butx-binding protein of chick optic lobe. Mice were immunized with affinity-purified protein preparations which were estimated to be 10 to 20% pure. Spleen cells from an immunized mouse were fused with the mouse myeloma cell line X63-Ag 8.653. From this fusion, 14 stable hybridoma lines were isolated which produce mAbs against the chick neuronal alpha-Butx-binding protein. Most of the antibodies inhibited alpha-Butx-binding to membrane fractions and/or detergent extracts of chick optic lobe. Some of the mAbs cross-reacted with the alpha-Butx-binding protein of the rat pheochromocytoma cell line PC12. However, none of the mAbs bound to a significant extent to the nicotinic ACh receptor of chick skeletal muscle or of Torpedo californica electric organ. All antibodies specifically isolated a polypeptide of Mr = 57,000 (+/- 2,000) from radiolabeled neuronal protein preparations. The present data show that these mAbs constitute useful tools for the further molecular and functional characterization of the neuronal alpha-Butx-binding protein.