Optimization of Radioligand Assays for Islet Autoantibodies / 中国医师杂志

ABSTRACT

Objective To optimize the radioligand assays for glutamic acid decarboxylase antibody (GAD-Ab), tyrosine phosphatase antibody (IA-2A) and insulin autoantibody (IAA) and to facilitate their application in clinical practice. Methods The radioligand assays established in our laboratory were optimized in several aspects, including the mode and time of antigen-antibody incubation, pH value of TBST buffer, times of washing, and type of centrifuge. The sensitivity and specificity of the optimized assays were calculated by the Diabetes Antibody Standardization Program (DASP, 2003). And the results were compared to those measured with domestic radioimmune assay kits. Results The optimal condition for radiligand assay were as followed incubating the antigens of GAD-Ab and IA-2A with serum by rotating slowly for 24 hours and IAA for 72 hours respectively; washing the precipitation complex of GAD-Ab and IA-2A with TBST buffer (pH value 7 2～7 4) 4 times and IAA 5 times respectively; and applying the horizontal type of centrifuge. The results from DASP demonstrated that sensitivity and specificity of the optimized assays were 82% and 98% respectively for GAD-Ab, and 64% and 100% for IA-2A. The proportion of consistency between our results for 82 IAA gradient index of optimized assay and those measured with domestic radioimmune kits was 89%. The extensive analysis showed that the consistency reached 100% for those with IAA index less than 0 04 or more than 1 00, and the inconsistency was for the bordline positive ones (index 0 04～1 0). Conclusion The optimized radioligand assays for islet autoantibodies are high sensitivity and specificity, and are applicable in clinical practice.