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Molecular & Cellular Proteomics 8:1688-1696, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Immunocapture and Identification of Cell Membrane Protein Antigenic Targets of Serum Autoantibodies^*,

Edward Littleton^,, Mathias Dreger^¶,,||, Jackie Palace^** and Angela Vincent ^,

From the Neurosciences Group, Weatherall Institute of Molecular Medicine and
**Department of Clinical Neurology, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom and
¶Proteomics Laboratory, Henry Wellcome Centre for Gene Function, South Parks Road, Oxford OX3 7BN, United Kingdom

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.

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