期刊
CLINICAL CHEMISTRY
卷 61, 期 4, 页码 627-635出版社
OXFORD UNIV PRESS INC
DOI: 10.1373/clinchem.2014.232728
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BACKGROUND: Small molecules classified as haptens are generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. We created a method for developing sandwich immunoassays to measure haptens on the basis of antimetatype antibodies. METHODS: We generated antimetatype monoclonal antibodies against a hapten-antibody immunocomplex using an ex vivo antibody development system, the Autonomously Diversifying Library (ADLib) system. We selected 2 haptens, estradiol (E2) and 25-hydroxyvitamin D [25(OH) D], as analytes. Sandwich immunoassays for these 2 haptens were developed by use of a 96-well microtiter plate and a fully automated chemiluminescence analyzer, and the performances of these immunoassays were investigated. RESULTS: The developed assays exhibited sensitivity high enough to detect target haptens in serum samples. The limit of detection of the ELISA for E-2 was 3.13 pg/mL, and that of the fully automated chemiluminescent enzyme immunoassay (CLEIA) system was 2.1 ng/mL for 25(OH) D. The cross-reactivity with immunoreactive derivatives was effectively improved compared with the competitive assay. The CVs for the sandwich ELISA for E2 were 4.2%-12.6% (intraassay) and 6.2%-21.8% (total imprecision). The CVs for the sandwich CLEIA for 25(OH) D were 1.0%-2.3% (intraassay) and 1.9%-3.5% (total imprecision). In particular, the sandwich CLEIA for 25(OH) D showed correlations of r = 0.99 with bothLC-MS/MSand a commercially available I-125 RIA. CONCLUSIONS: Our method represents a potentially simple and practical approach for routine assays of haptens, including vitamins, hormones, drugs, and toxins. (C) 2015 American Association for Clinical Chemistry
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