Development of glutamic acid decarboxylase 65 (GAD65) autoantibody assay using biotin-GAD65 fusion protein.
We evaluated a biotin-glutamic acid decarboxylase 65 (GAD65)-based enzyme-linked immunosorbent assay (B-ELISA) to detect GAD65 autoantibodies (GAD65Ab) in 78 sera from individuals with newly diagnosed type 1 diabetes. The GAD65Ab index of patients with type 1 diabetes (mean value of GAD65Ab index of 1.891) was significantly higher than those in 50 sera from healthy control group (mean value of 0.068). The intra- and inter-assay coefficients of variation (CV) were calculated to be 1.042 and 10.703%, respectively. The specificity of the B-GAD65 ELISA was comparable to the standard radioimmunoassay (RIA) which is routinely used in the laboratory. We describe the optimal conditions of the binding kinetics from each assay-step for the detection of GAD65Ab using the WHO standard serum 97/550 as a model autoantibody serum. We concluded that incubation times of 15, 90, and 90 min for step 1 (pre-incubation of Biotin14-GAD65 with serum), step 2 (binding the Ab/Ag complex to NeutrAvidin plate), and step 3 (incubation with HRPO-anti-human IgG), respectively, along with human serum dilutions of 1:50, would provide an optimal assay signal within a relatively short timeframe.
Luo
Dong
D
Department of Medicine, University of Washington, P.O. Box 357710, HSB K-165, 1959 Pacific Street N.E., Seattle, WA 98195-7710, USA.
Rogers
Chad N
CN
Steed
Jordan T
JT
Gilliam
Lisa K
LK
Hampe
Christiane S
CS
Lernmark
Ake
A
D001324:Autoantigens / Q000097:blood / Q000276:immunology; D015415:Biomarkers / Q000097:blood*; D001710:Biotin / Q000276:immunology*; D001774:Blood Chemical Analysis / Q000379:methods; D003922:Diabetes Mellitus, Type 1 / Q000097:blood* / Q000175:diagnosis / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005968:Glutamate Decarboxylase / Q000097:blood* / Q000276:immunology*; D006801:Humans; D011993:Recombinant Fusion Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001324:Autoantigens; D015415:Biomarkers; D011993:Recombinant Fusion Proteins; D001710:Biotin; D005968:Glutamate Decarboxylase