Highly-sensitive and specific enzyme-linked immunosorbent assays for GAD65 autoantibodies using a thioredoxin-GAD65 fusion antigen.
Autoantibodies against glutamic acid decarboxylase (GAD65) are present in the sera of most patients with recently diagnosed insulin-dependent diabetes mellitus (IDDM). These antibodies appear years before the clinical symptoms, and they are considered to be early markers of the disease. To detect GAD65 autoantibodies (GADA), we developed new enzyme-linked immunosorbent assays (ELISA) with a fusion protein thioredoxin-GAD65 (Trx-GAD65) produced in E. coli as the antigen. These assays were compared with the reference radiobinding assay (RBA). Since most GADA are directed against native epitopes, and adsorption of GAD65 to plastic may cause disruption of its native conformation, the new assays rely on the following immobilization procedures: (a) capture ELISA (c-ELISA) with Trx-GAD65 (protocol A) or biotin-Trx-GAD (protocol B) indirectly immobilized by a non-adsorptive process; (b) ELISA with antigen-antibody preincubation in solution (p-ELISA) in which GADA were reacted first with Trx-GAD65 (protocol C) or biotin-Trx-GAD (protocol D) and the free antigen was determined by conventional ELISA. The results obtained with 42 newly diagnosed IDDM patients and 30 normal individuals were as follows: RBA had 79% sensitivity (percentage of IDDM patients detected) and 97% specificity (100% minus the percentage of false positives). c-ELISA showed low sensitivity (36 and 50%, respectively for protocols A and B), and high specificity (100 and 97%, respectively). p-ELISA were highly-sensitive (74 and 79%, respectively) and specific (97 and 93% for protocols C and D, respectively). Thus, protocols C and D had a performance similar to the reference method. The results reported here provide the basis for simple, highly-sensitive, specific, and widely-applicable tests for GADA that eliminate many of the drawbacks of the radioactive methods.
Papouchado
M L
ML
Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
Valdez
S N
SN
Ermácora
M R
MR
Gañan
S
S
Poskus
E
E
D000293:Adolescent; D001323:Autoantibodies / Q000276:immunology*; D001327:Autoimmune Diseases / Q000175:diagnosis* / Q000276:immunology; D001710:Biotin / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D005968:Glutamate Decarboxylase / Q000276:immunology*; D006801:Humans; D007223:Infant; D008297:Male; D011863:Radioimmunoassay / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D012680:Sensitivity and Specificity; D013879:Thioredoxins / Q000276:immunology
D001323:Autoantibodies; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D001710:Biotin; D005968:Glutamate Decarboxylase