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

被引:34
作者
Woo, W
LaGasse, JM
Zhou, Z
Patel, R
Palmer, JP
Campus, H
Hagopian, WA [1 ]
机构
[1] Pacific NW Res Inst, Seattle, WA 98122 USA
[2] Univ Washington, Dept Med, Seattle, WA 98195 USA
[3] Puget Sound Vet Hlth Care, Seattle, WA 98108 USA
关键词
prediction; autoimmunity; islet cell antibodies; GAD autoantibodies; IA2/ICA512; autoantibodies; insulin autoantibodies;
D O I
10.1016/S0022-1759(00)00259-3
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Prediction of Type 1 diabetes for study of preventive therapies requires screening the general population, where 85% of new cases occur. Even with HLA-based prescreening, nearly 20% of all children will need multiple serum autoantibody 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 membrane-bound Protein A. Each microtiter plate contained a standard negative control serum, and low-, medium-, and high-level positive control sera. All sera were evaluated in triplicate. This readily allowed quality control criteria both for triplicates of individual sera and for each 96-well plate. Inter-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-met-Phogrin, I-125-insulin, and could be used for simultaneous screening of reactivity to both GAD65 and ICA512/IA2 in the same and well. Diagnostic accuracy compared favorably with microcentrifuge tube-based Protein A-agarose GAD65 and IA2 autoantibody radiobinding assays and with acid-charcoal-polyethylene glycol (PEG) based competitive insulin autoantibody assays. In the case of I-125-insulin, comparing signal in the absence versus presence of cold 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 less than all other commonly used formats, and should be useful for population screening. (C) 2000 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:91 / 103
页数:13
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