Production And Characterization Of Anti-human Erythrocyte Acetylcholinesterase Monoclonal Antibodies

Acetylcholinesterase is the serine hydrolase that terminates nerve impulse transmission in cholinergic synapses by hydrolysis of the neurotransmitter acetylcholine. The most marked character of Acetylcholinesterase molecular structure is that there is a 2nm deep and narrow gorge on the surface. Active site lies at the bottom of the gorge and consists of a catalytic triad (Ser200, His440 and Glu327), the anionic site (Trp84, Gly199 and Tyr330) lies within the gorge, and the peripheral anionic site lies at the rim of the gorge (Try70, Tyr121, Trp279 and Asp72).Anti-idiotypic antibody (Ab2) is induced by the variable region of an antibody molecule (Ab1), having four categories: Ab2a, Ab2β, Ab2r and Ab2e. Ab2β carries idiotpic structures that are complementary to the antigen combining site of Ab1 and represents the image of the antigenic epitope recognized by Ab1. In accordance with the idiotypic network theory, if a monoclonal antibody against the active center of an native enzmy was usded as antigen to produce an anti-idiotypic antibody (Ab2β), the anti-idiotypic antibody (Ab2β) would display internal-image properties of the enzmy active center: having the similar structure and catalytic activity of the native enzmy.In this experiment, monoclonal antibodies were raised according to standard protocols. BALB/C mice were immuned with human erythrocyte AChE. Spleen lymphocytes were fused with SP2/0-Ag14. Monoclonal antibodies against AChE were screened by ELISA using AChE as antigen ,and monoclonal anti-AChE idiotypic antibodies were screened by ELISA using rabbit anti-AChE IgG as antigen. Six monoclonal antibodies against AChE were achieved, but no monoclonal anti-idiotypic antibody was achieved. Ascites fluid was purified by Caprylc acid - Ammonium sulfate protocol and was identified by SDS-PAGE. Effects of monoclonal antibodies on AChE activity demonstrated that McAb 12C3 and 13F6 couldinhibit AChE activity, inhibition ratio is 35.24+4.39% for 12C3 and 41.96+5.23% for 13F6. Effects of different AChE inhibitors on the binding of monoclonal antibodies to AChE indicated that none of the monoclonal antibodies could be inhibited by both tetramethylammonium (anionic site inhibitor) and pyridostigmine (active site inhibitor).So the determinants of McAb 12C3 and 13F6 didn't lie in the active center.