Fractionation of antibodies to acetylcholine receptor according to antigenic specificity

Abstract

Acetylcholine receptor (AChR) is a major autoantigen involved in the neuromuscular disease myasthenia gravis (MG). Injection of experimental animals with purified AChR from electric fish leads to the elicitation of an immune response to this immunogen accompanied by a neuromuscular weakness, designated experimental autoimmune myasthenia gravis (EAMG) (reviewed [l-3]). Antibodies to AChR were shown to block the physiological activity of AChR [4-61 and to inhibit the binding of ar-bungarotoxin to the receptor [7]. However, it is still not known which parts or immunogenic determinants in the AChR molecule are responsible for its myasthenic activity and whether or not these overlap with sites involved in the physiological function of the receptor. Detailed immunochemical analysis of AChR should shed light on these questions. We have demonstrated that a denatured preparation of AChR (RCM-AChR, i.e., reduced and carboxymethylated AChR) cross reacts partially with antiAChR antibodies by reacting with antibodies directed against only part of the antigenic determinants present in the intact receptor [8]. Moreover, RCM-AChR by itself elicits an immune response which is not associated with any myasthenic symptoms, and is capable of specifically suppressing EAMG [9]. Analysis of the specificity of the immune response elicited by AChR and RCM-AChR led us to propose that the denaturation of AChR destroys some structural antigenic determinants which are important for the induction of EAMG, and which may be located close to the toxin binding site [8,9]. AChR elicits antibodies against both myasthenic determinants in the AChR molecule which are involved in the disease and other antigenic determinants which are not involved in the disease. RCM-AChR cross reacts only with antibodies of the latter specificity and by itself elicits an immune response only to determinants other than the myasthenic ones. Here we describe the fractionation of two antibody populations different in their immunological and physiological specificity from rabbit anti-AChR serum. The fractionation was achieved by using an immunoadsorbent of RCM-AChR bound to Sepharose. Both the adsorbed and unadsorbed subpopulations of antibodies bind to iritact AChR, whereas only the subpopulation which does not bind to RCM-AChR blocks the in vitro binding of cw-bungarotoxin to AChR.