A new solid-phase radioimmunoassay to detect anti-GAD65 autoantibodies.
This paper describes a simple, rapid, routine method to detect anti-GAD65 autoantibodies by a solid-phase radioimmunoassay using human recombinant GAD65 coated microwells and 125I-protein A to reveal antibody binding. Both recombinant and radiolabelled proteins are commercially available. This new method was validated by investigating the presence of GAD65 autoantibodies in two different studies (A and B); the first including subjects originating from our own case histories (group A sera), the second made up of recoded subjects and standards sent to our lab by the Second International GAD Antibody Workshop organizers (group B sera). In study A we tested sera from 52 normal subjects, 25 newly diagnosed type 1 diabetics and 3 stiff man syndrome (SMS) subjects detecting GAD65 autoantibodies in 72% of IDDM and 100% of SMS patients. In study B we tested (in blind fashion) 89 recoded sample sera or standards that were part of the larger group used in the Second International GAD Antibody Workshop, finding GAD65 autoantibodies in 3.3% of healthy control subjects (1/30), 60% of IDDM patients (18/30), 100% of ICA + nondiabetic subjects (3/3) but in none of 4 nondiabetic patients with Graves disease. Comparing our solid-phase RIA results with those published for the same sera from the Second International GAD Antibody Workshop we obtained for our method a sensitivity of 85.7%, a specificity of 93.9% and a consistency of 100%. These result indicate that our assay, which is based on commercially available reagents, should be a useful tool for the detection of GAD65 autoantibodies in large scale studies.
Tiberti
C
C
Department of Endocrinology, La Sapienza University, Rome, Italy.
Falorni
A
A
Torresi
P
P
Vecci
E
E
Anastasi
E
E
Dotta
F
F
Di Mario
U
U
D000293:Adolescent; D000328:Adult; D001323:Autoantibodies / Q000032:analysis*; D002648:Child; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis / Q000276:immunology*; D005260:Female; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007527:Isoenzymes / Q000276:immunology; D008297:Male; D008875:Middle Aged; D011863:Radioimmunoassay / Q000379:methods*; D011994:Recombinant Proteins / Q000276:immunology; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000276:immunology; D016750:Stiff-Person Syndrome / Q000276:immunology
D001323:Autoantibodies; D007527:Isoenzymes; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D005968:Glutamate Decarboxylase