There is increasing interest in the role of
antibodies targeting specific
membrane proteins in neurological and other diseases. The target(s) of these pathogenic
antibodies is known in a few diseases, usually when candidate
cell surface proteins have been tested. Approaches for identifying new
antigens have mainly resulted in the identification of
antibodies to intracellular
proteins, which are often very useful as diagnostic markers for disease but unlikely to be directly involved in disease pathogenesis because they are not accessible to circulating
antibodies. To identify
cell surface antigens, we developed a "conformational membrane
antigen isolation and identification" strategy. First, a cell line is identified that reacts with patient sera but not with control sera. Second, intact cells are exposed to sera to allow the binding of presumptive
autoantibodies to their cell surface targets. After washing off non-bound serum components, the cells are lysed, and
immune complexes are precipitated. Third, the bound
surface antigen is identified by mass spectrometry. As a model system we used a muscle cell line, TE671, that endogenously expresses muscle-specific
tyrosine receptor kinase (
MuSK) and sera or plasmas from patients with a subtype of the
autoimmune disease myasthenia gravis in which patients have
autoantibodies against
MuSK.
MuSK was robustly detected as the only
membrane protein in immunoprecipitates from all three patient samples tested and not from the three
MuSK antibody-negative control samples processed in parallel. Of note, however, there were many intracellular
proteins found in the immunoprecipitates from both patients and controls, suggesting that these were nonspecifically immunoprecipitated from
cell extracts. The conformational membrane
antigen isolation and identification technique should be of value for the detection of highly relevant antigenic targets in the growing number of suspected antibody-mediated autoimmune disorders. The approach would also be very suitable for the analysis of human or experimental antitumor responses.