Revelation of specificity of 64K autoantibodies in IDDM serums by high-resolution 2-D gel electrophoresis. Unambiguous identification of 64K target antigen

Baekkeskov, S. and Warnock, G. and Christie, M. and Rajotte, R.V. and Larsen, P.M. and Fey, S. (1989) Revelation of specificity of 64K autoantibodies in IDDM serums by high-resolution 2-D gel electrophoresis. Unambiguous identification of 64K target antigen. Diabetes, 38 (9). pp. 1133-1141. ISSN 0012-1797

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Item Type:Article
Item Status:Live Archive

Abstract

Antibodies in serum from newly diagnosed insulin-dependent (type I) diabetes mellitus (IDDM) patients and individuals experiencing early phases of β-cell destruction specifically immunoprecipitate a minor pancreatic islet cell membrane protein of 64,000 M(r) (64K). In this report, we demonstrate the use of two-dimensional (2-D) gel electrophoresis to umambiguously 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 α and β, 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 >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 analyses demonstrate that purification of membrane protein fractions from both human and rat islets before the immunoprecipitation efficiently removes background proteins and substantially increases the specificity of 64K autoantibody measurements by traditional methods.

Keywords:autoimmunity, controlled study, electrophoresis, human, human cell, insulin dependent diabetes mellitus, measurement, priority journal, Animal, Antibody Specificity, Autoantibodies, Diabetes Mellitus, Insulin-Dependent, Electrophoresis, Gel, Two-Dimensional, Electrophoresis, Polyacrylamide Gel, Insulin Antibodies, Islets of Langerhans, Molecular Weight, Precipitin Tests, Rats, Rats, Inbred Strains, Support, Non-U.S. Gov't
Subjects:A Medicine and Dentistry > A100 Pre-clinical Medicine
Divisions:College of Science > School of Life Sciences
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ID Code:18171
Deposited On:31 Jul 2015 15:00

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