Characterization of monoclonal and polyclonal antibodies to human choline acetyltransferase and epitope analysis

Choline acetyltransferase (ChAT) was partially purified from human placenta and brain. In order to raise monoclonal antibodies, Balb/c mice were immunized with a preparation from placenta or with a mixture of eight synthetic peptides that were deduced from the primary structures of porcine and human ChAT. Polyclonal antibodies were raised in rabbits against five synthetic peptides deduced from the amino acid sequence of human ChAT. The monoclonal and polyclonal antibodies were characterized by their ability to recognize ChAT in Various immunoassays: immunoblot, enzyme-linked immunosorbent assay (ELISA), two-side ELISA and immunohistochemistry. With one exception all monoclonal antibodies recognized ChAT on immunoblots, some were particularly sensitive; one bound active ChAT in ELISA when used as capture reagent; most antibodies recognized immobilized ChAT in ELISA. Two monoclonal antibodies out of nine gave particularly excellent results in staining cholinergic neurons and fibers on sections from rat and primate brain. With the help of nine synthetic peptides it was possible to evaluate two major binding sites for the monoclonal antibodies on the ChAT molecule, comprising amino acids 167 - 189 and 57 - 76, respectively.