Immunochemical and functional characterization of an agonist-like monoclonal antibody against the M2 acetylcholine receptor

Monoclonal antibodies were raised against a peptide corresponding to the second extracellular loop of the M2 acetylcholine receptor. One of the monoclonal antibodies, B8ES, was selected for further characterization on the basis of its high yield, its isotype (IgG,,), its dissociation kinetics and its agonist like activity. The epitope recognized by B8E5 corresponded to the N-terminal part of the second extracellular loop of the receptor (V-R-T-V-E-) as determined by competition immunoassays and epitope scanning. The K, of B8E5 for the target peptide was assessed by surface plasmon resonance (SPR) to be 6.5 X 10(7) M-l by equilibrium and 3,7x10(7) M-l by kinetic analysis, B8E5 recognized the M2 acetylcholine receptor on rat cardiac tissue. It only recognized the non-reduced receptor in immunoblots. The antibody had no effect on antagonist binding but decreased the affinity for the agonist carbachol, B8E5 decreased the beating frequency of neonatal rat cardiomyocytes. The effect was specific since it was blocked by the target peptide and the antagonist atropine. The EC,, of the antibody corresponded to the K, measured by surface plasmon resonance. The physiological effect of the antibody did not lead to desensitization. The Fab fragments had no physiological effect;; subsequent addition of anti-mouse IgG however restored the physiological effect, These results confirm that the N-terminal of the second extracellular loop is a functional target for antibodies against the M2 acetylcholine receptor, They suggest that the functional epitope is only accessible in the non-reduced receptor. The antibodies act through a functional dimerization of the receptor.