Detection of GAD-Ab index in diabetic patients using ~(35) S labeled recombinant human GAD _(65) antigen

Objective To establish a novel method for measuring glutamic acid decarboxylase autoantibodies(GAD Ab). Methods Recombinant human GAD 65 was used as the antigen, in vitro transcribed and translated 35 S GAD 65 as the tracer, a self designed rotating incubation apparatus as the incubator, protein A sepharose as the precipitator, and the liquid scintillation counter was used to measure radioactive count value to detect GAD Ab. The positive cut off point of GAD Ab index was determined as 0.05 by the 99.5% percentile in 109 healthy individuals. GAD Ab levels were determined in 43 type 1 and 226 type 2 diabetic patients. Results The optimized working conditions included SJ1515 35 S methionine for in vitro transcription and translation, 20～30 r/min setup of rotating incubation apparatus, test temperature 4～25 ℃, freshly prepared buffer of pH 7.2～7.4, and horizontal rotor centrifuge. The new method was better than original one, with intra assay CV of 4.9%～8 3% and inter assay CV of 7.1%～10.8%,specificity of 98.2%. The results were comparable with the figures issued by an international standardized laboratory (concordance was 98.3%, Kappa value 0.971). The positive rate of GAD Ab was 58.1%(25 of 43)in type 1 and 10.2%(23 of 226)in type 2 diabetes patients, but only 1 8% (2 of 109) in healthy individuals. Conclusion The new assay for GAD Ab is a highly sensitive, accurate, specific and reproducible method for clinical use.