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A sensitive non-isotopic assay for GAD65 autoantibodies
Helen Brooking1, Rossitza Ananieva-Jordanova, Clare Arnold
1FIRS Laboratories, RSR Limited, Parc Ty Glas, Llanishen, Cardiff, South Wales, CF14 5DU, UK.
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 microl) 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.