This research proposal involves the characterization of autoantibodies with reactivity for the muscle receptor for acetylcholine (AChR) responsible for the neuromuscular disorder, myasthenia gravis (MG). Past observations indicate a frequent disparity between the level of circulating anti-AChR antibodies produced in MG patients and the severity of resulting disease symptoms. Therefore, the objective of this investigation is to demonstrate distinctions among the individual species of anti-AChR antibodies produced by individual patients that might allow the categorization of antibodies into subsets associated with 1) particular patterns of antigen binding fine-specificity (including reactivity with the main immunogenic region (MIR) and the intriguing crossreactivities noted in the past (i.e., between AChR and myosin]), and 2) with high or low disease-causing potential. The strategy to be used is to combine determinations of antigen binding fine-specificity with the analysis and purification of antibodies based on their isoelectric point (i.e., preparative isoelectric focusing). In this way, relationships may be revealed between particular patterns of reactivity with the AChR and """"""""clonotypic"""""""" B cell products (i.e., anti-AChR antibodies). This should allow a different perspective on such relationships than have past studies of the polyclonal antibodies found in unfractionated MG sera. Accordingly, this project will address specific aims based on several criteria of patient antibody binding and purification described below. Additionally, relationships will be sought between particular sub sets of anti body and their binding specificity, with their ability to influence the expression of AChR and their disease-causing potential.
SPECIFIC AIM 1. Determine the reactivity profile and potential immunopathological relationships involving -clonotypic species of anti- AChR antibodies. Analytical IEF will be used to assess the reactivity and crossreactivity profiles of anti-AChR antibodies with respect to the MIR- associated peptide and myosin, as well as for associations with the 11E10 idiotype. Preparative IEF (pIEF) will be used to purify clonotypically restricted anti-AChR antibodies for tests of binding specificity, receptor crosslinking/down-modulating activities, and for tests of disease-causing potential via passive antibody transfer studies into immunologically naive rats.
SPECIFIC AIM 2. Determine serological and potential immunopathological relationships involving clonotypic species of anti-AChR antibodies with specificity for an MlR-associated synthetic peptide. Antibodies are to be studied that have been affinity-purified by adsorption to and elution from Sepharose columns to which the alpha61-76 MlR-associated peptide has been attached. Evaluations of these antigen-specific antibodies will be similar to those described for Specific Aim 1.
SPECIFIC AIM 3. Determine serological and potential immunopathological relationships involving clonotypic species of anti-AChR antibodies that lack specificity for an MIR-associated synthetic peptide. In order to further clarify the serological and immunopathological importance of antibodies with reactivity to the MIR, patient Ig that has been depleted of reactivity with the alpha61-76 MlR-associated peptide will be examined. Evaluations of these antigen-specific antibodies will be similar to those described for Specific Aim 1.
