Demonstration of GAD-65 as the main immunogenic isoform of glutamate decarboxylase in type 1 diabetes and determination of autoantibodies using a radioligand produced by eukaryotic expression.
Plasmids containing cDNA for the rat 67- and 65-kD isoforms of glutamate decarboxylase (GAD-67 and GAD-65) were expressed in COS-cells, and lysates of [35S]methionine-labeled cells were used for immunoprecipitations. Sera from 38 patients with type 1 (insulin-dependent) diabetes mellitus, which precipitated a 64-kD antigen from rat islets, reacted with recombinant GAD-65 in relation to their anti-64-kD titers. The eight strongest sera also precipitated recombinant GAD-67, suggesting that certain epitopes are common to both isoforms. Subsequently, [35S]methionine-labeled GAD-65 was purified from COS cell lysates and employed in a binding assay with 50 sera of patients with recent onset of type 1 diabetes mellitus. 38 sera (76%) precipitated labeled GAD-65 with titers that correlated with islet cell antibodies (ICA), determined in a standard immunofluorescence assay. 2 sera were GAD positive but ICA negative, 4 were positive only for ICA, and 6 were negative for both GAD and ICA, as were the sera of 20 controls. The data illustrate that antibodies against GAD-65 are present in a majority of patients with type 1 diabetes mellitus and that autoantibodies against other islet cell antigens also exist. The radioligand-binding assay, which is convenient and sensitive for detecting GAD antibodies, will facilitate the screening of individuals with autoimmune islet cell disease.
Velloso
L A
LA
Department of Internal Medicine, University Hospital, Uppsala, Sweden.
Kämpe
O
O
Hallberg
A
A
Christmanson
L
L
Betsholtz
C
C
Karlsson
F A
FA
D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D002460:Cell Line; D002648:Child; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000201:enzymology* / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005968:Glutamate Decarboxylase / Q000097:blood* / Q000235:genetics / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007515:Islets of Langerhans / Q000276:immunology; D007527:Isoenzymes / Q000097:blood* / Q000235:genetics / Q000276:immunology; D007668:Kidney; D008297:Male; D008875:Middle Aged; D008970:Molecular Weight; D051381:Rats; D012016:Reference Values; D014162:Transfection
D001323:Autoantibodies; D007527:Isoenzymes; C024211:islet cell antibody; D004247:DNA; D005968:Glutamate Decarboxylase