A novel high-throughput method for accurate, rapid, and economical measurement of multiple Type 1 diabetes autoantibodies

Prediction of Type 1 diabetes for study of preventive therapies requires sc reening the general population, where 85% of new cases occur. Even with HLA -based prescreening, nearly 20% of all children will need multiple serum au toantibody testings. High-throughput. economical, and accurate methods are therefore essential. We have developed such a radiobinding method, using 96 -well microtiter plates and a novel immune complex capture method via membr ane-bound Protein A. Each microtiter plate contained a standard negative co ntrol serum, and low-, medium-, and high-level positive control sera. All s era were evaluated in triplicate. This readily allowed quality control crit eria both for triplicates of individual sera and for each 96-well plate. In ter-assay coefficients of variation (CVs) were all less than or equal to 16 %, while intra-assay CVs were all less than or equal to 10%. The assay was found to be sensitive (to detect autoantibodies in patients) and specific ( low reactivity in thousands of healthy volunteers). The format worked well using diverse antigens such as S-35-met-GAD65, S-35-met-ICA512/IA2, S-35-me t-Phogrin, I-125-insulin, and could be used for simultaneous screening of r eactivity to both GAD65 and ICA512/IA2 in the same and well. Diagnostic acc uracy compared favorably with microcentrifuge tube-based Protein A-agarose GAD65 and IA2 autoantibody radiobinding assays and with acid-charcoal-polye thylene glycol (PEG) based competitive insulin autoantibody assays. In the case of I-125-insulin, comparing signal in the absence versus presence of c old insulin competitor was not necessary. Total serum volumes required were only 6 mul for GAD and ICA512, and only 15 mul for IAA. The method costs l ess than all other commonly used formats, and should be useful for populati on screening. (C) 2000 Elsevier Science B.V. All rights reserved.