Abstract
Background: A new ELISA for 65 kDa isoform of glutamic acid decarboxylase autoantibodies (GAD 65 Abs), which depends on GAD 65 Ab acting divalently and forming a bridge between immobilized GAD 65 and liquid-phase GAD 65–biotin, is described. Methods: Sera (25 μl) were incubated in GAD 65-coated ELISA plate wells followed by washing and incubation with GAD 65–biotin. After a further wash step, GAD 65–biotin bound was quantitated by addition of streptavidin peroxidase followed by tetramethylbenzidine. Assay calibration was with WHO reference preparation 97/550. Results: Using a cut-off for positivity of 5 units/ml, sera from 0.7% (2/300) healthy blood donors (HBDs), 100% (39/39) selected type 1 diabetes mellitus (DM) patients, 1.6% (1/62) type 2 diabetes mellitus patients and 3% (4/119) autoimmune disease controls were GAD 65 Ab positive in the ELISA. Levels of positivity in an immunoprecipitation assay (IPA) based on 125I-labelled GAD 65 (cut-off 25 units/ml) were 1%, 82%, 0% and 3%, respectively. ELISA inter-assay coefficients of variation ( n=12) were 7.3%, 3.8%, 7.2% and 6.3% at 5.4, 40.8, 137 and 382 units/ml, respectively. Conclusions: The ELISA we have described has sensitivity and specificity at least as high as current radioactive assays. It has good precision and handling making it suitable for routine use.
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