title: Screening of single-stranded DNA (ssDNA) aptamers against a zearalenone monoclonal antibody and development of a ssDNA-based enzyme-linked oligonucleotide assay for determination of zearalenone in corn.
abstract: A hypotoxic immunosorbent assay for the detection of zearalenone (ZEN) was developed, by identifying a single-stranded DNA (ssDNA) aptamer with high specificity and affinity for a ZEN monoclonal antibody (mAb-ZEN). ssDNA aptamers, which could mimic ZEN epitopes, were identified using the modified systematic evolution of ligands by an exponential enrichment (SELEX) technique. The purified mAb-ZEN was coated on microtiter plates as a target recognized by the random oligonucleotide ssDNA library. The binding affinity between the aptamers and mAb-ZEN during each round was measured by the biotin–streptavidin–horseradish peroxidase system. During 15 rounds of screening, an increasing binding affinity was observed. The enriched ssDNA library binding to mAb-ZEN with high affinity was cloned, sequenced, and analyzed. One aptamer (number 46), which displays the highest affinity and specificity for the mAb-ZEN, was used to establish an indirect competition enzyme-linked oligonucleotide assay (ELONA) to measure the ZEN concentration in corn. Under optimal conditions, the regression equation for quantification of ZEN was y = −0.0778x + 0.713 (R2 = 0.9981). The detection limit and IC50 were 0.01 and 0.2 ng/mL, respectively, with a working range of 0.03–2.5 ng/mL. The recovery rates of the spiked samples in the ELONA ranged from 95 to 105%. Aptamers, which can mimic many types of low-weight analytes in agricultural products, could serve as surrogates for the development of hypotoxic, environmentally friendly immunological detection methods.
authors.forename: Yuan-Kai
authors.initials: YK
authors.lastname: Wang
authors.forename: Qi
authors.initials: Q
authors.lastname: Zou
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Sun
authors.forename: Heng-an
authors.initials: HA
authors.lastname: Wang
authors.forename: Xingmin
authors.initials: X
authors.lastname: Sun
authors.forename: Zhi-Fei
authors.initials: ZF
authors.lastname: Chen
authors.forename: Ya-Xian
authors.initials: YX
authors.lastname: Yan
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D052157:Aptamers, Nucleotide / Q000737:chemistry; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry; D007124:Immunoenzyme Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D052156:SELEX Aptamer Technique / Q000379:methods; D012639:Seeds / Q000737:chemistry; D012680:Sensitivity and Specificity; D003313:Zea mays / Q000737:chemistry* / Q000235:genetics; D015025:Zearalenone / Q000032:analysis* / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D015025:Zearalenone