Two‐Site Immunoassay for Acetylcholinesterase in Brain, Nerve, and Muscle

Two‐site methods were developed for immunoassay of acetylcholinesterase (AChE; EC 3.1.1.7) in crude extracts of rat and human tissues. A radiometric assay for human AChE utilized a specific monoclonal AChE antibody adsorbed to polystyrene microtiter wells at alkaline pH. AChE bound strongly to this antibody after 24 h at 4°C. Bound enzyme was detected with an ¹²⁵I‐labeled antibody against a different AChE epitope. The assay signal was quasilinearly related to AChE concentration in purified and crude samples, with a detection threshold near 100 pg. Tetrameric and dimeric AChE behaved equivalently in the assay. Two‐site methods with a different pair of species‐selective antibodies worked equally well for immunoassay of rat AChE. Assays of the rat enzyme showed that immunoreactivity was lost as rapidly as enzyme activity during heating to 54°C. On the other hand, immunoreactivity was preserved despite loss of enzyme activity after exposure to anti cholinesterases or trypsin. A biotinylated second antibody detected by alkaline‐phosphatase‐conjugated avidin was used to develop an AChE enzyme‐linked immunosorbent assay (ELISA) with a sensitivity similar to that of the radio‐metric assay. Either the ELISA or the radiometric immunoassay may be useful whenever proteolysis or other mechanisms are suspected of dissociating enzyme activity and immunoreactivity. In denervated muscle and ligated peripheral nerve, application of the two‐site method showed closely parallel variations in immunoreactivity and enzyme activity.