Document Detail

Biochemical characterization of a beta cell membrane fraction antigenic for autoreactive T cell clones.
MedLine Citation:
PMID:  10882061     Owner:  NLM     Status:  MEDLINE    
The two NOD-derived T cell clones, BDC-2.5 and BDC-6.9, are CD4+, Vbeta4+, islet-specific, and diabetogenic. These two T cell clones show different response patterns to whole islet cell antigen, but were found to respond to the same fraction isolated from beta granule membranes. The clones were used to follow the antigenic activity in the biochemical purification of a beta cell membrane detergent lysate subjected to HPLC anion exchange (IEX) and size exclusion chromatography (SEC). Antigenic activity could be retained after lysis in only one detergent (octyl-beta-glucoside) among several tested. In order to detect solubilized antigen, beta membrane proteins were covalently linked to microlatex beads prior to being added to T cell proliferation assays, a technique that eliminated detergent toxicity and resulted in increased assay sensitivity. To purify the antigen, membrane proteins were absorbed onto an anion exchange column and after elution using a salt gradient, activity for the clones was found in a fraction containing 0.15-0.2 M NaCl. Subsequent analysis of this material by size exclusion chromatography provided an apparent molecular weight of the antigen to be between 50 and 80 kDa. Further attempts to purify the protein by SDS-PAGE resulted in loss of antigenic activity. It is possible that the elusive nature of this protein is a clue to its importance as an autoantigen.
B Bergman; J L McManaman; K Haskins
Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Journal of autoimmunity     Volume:  14     ISSN:  0896-8411     ISO Abbreviation:  J. Autoimmun.     Publication Date:  2000 Jun 
Date Detail:
Created Date:  2000-10-17     Completed Date:  2000-10-17     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  8812164     Medline TA:  J Autoimmun     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  343-51     Citation Subset:  IM    
Barbara Davis Center for Childhood Diabetes and Department of Immunology, University of Colorado Health Sciences Center, Denver 80262, USA.
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MeSH Terms
Antigens, Surface / immunology*,  isolation & purification
Autoantigens / immunology*,  isolation & purification
Cell Membrane / chemistry,  immunology
Chromatography, Gel
Chromatography, Ion Exchange
Clone Cells / immunology
Diabetes Mellitus, Type 1 / immunology
Electrophoresis, Polyacrylamide Gel
Epitopes, T-Lymphocyte / immunology
Islets of Langerhans / chemistry,  immunology*
Membrane Proteins / immunology,  isolation & purification
Mice, Inbred NOD
Mice, SCID
T-Lymphocytes / immunology*
Grant Support
Reg. No./Substance:
0/Antigens, Surface; 0/Autoantigens; 0/Detergents; 0/Epitopes, T-Lymphocyte; 0/Membrane Proteins

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