Revelation of Specificity of 64K Autoantibodies in IDDM Serums by High-Resolution 2-D Gel Electrophoresis: Unambiguous Identification of 64K Target Antigen

作者：Steinunn Bækkeskov, Garth L. Warnock, Michael R. Christie, Ray V. Rajotte, Peter Mose Larsen, Stephen J. Fey · 发表于：Diabetes · 年份：1989 · DOI：10.2337/diab.38.9.1133 · 被引用次数：65 · 研究领域：Pancreatic function and diabetes、Diabetes and associated disorders、Diabetes Management and Research

Antibodies in serums from newly diagnosed insulin-dependent (type I) diabetes mellitus (IDDM) patients and individuals experiencing early phases of beta-cell destruction specifically immunoprecipitate a minor pancreatic islet cell membrane protein of 64,000 Mr (64K). In this report, we demonstrate the use of two-dimensional (2-D) gel electrophoresis to unambiguously identify the 64K antigen. By nonequilibrium pH-gradient gel electrophoresis in the first dimension and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second dimension, the 64K protein separates into two components, designated alpha and beta, that differ in size but display identical charge heterogeneity. The high resolution of the 2-D method efficiently separates the 64K components from background proteins in immunoprecipitates from crude detergent lysates of islets. The background proteins were identified as major cellular proteins carried nonspecifically through the immunoprecipitation procedure. The high affinity and specificity of the 64K autoantibodies were demonstrated by the exclusive and greater than 1000-fold purification of this minor protein by immunoprecipitation with IDDM serums. The 2-D analyses did not reveal additional proteins specifically immunoprecipitated by IDDM serums, suggesting that the 64K protein is the only protein antigen specifically and consistently recognized by IDDM autoantibodies in the relatively stringent conditions of immunoprecipitation. Moreover, the 2-D ana...