A SENSITIVITY MULTIANALYTE IMMUNOASSAY USING COVALENT DNA-LABELED ANTIBODIES AND POLYMERASE CHAIN-REACTION
A SENSITIVITY MULTIANALYTE IMMUNOASSAY USING COVALENT DNA-LABELED ANTIBODIES AND POLYMERASE CHAIN-REACTION
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DOI：
10.1093/nar/23.3.522
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发表时间：
1995-02-11
影响因子：
14.9
通讯作者：
EBERSOLE, RC
中科院分区：
文献类型：
--
作者：
HENDRICKSON, ER;TRUBY, TMH;EBERSOLE, RC
使用DNA标记抗体和聚合酶链反应（PCR）扩增检测响应，证明了同时检测三种分析物（hTSH、hCG和β-Gal）的多分析物免疫测定法。通过将独特设计的DNA寡核苷酸与每种分析物特异性单克隆抗体共价偶联来制备标记抗体。每个DNA寡核苷酸标记含有相同的引物序列，以促进通过单个引物对的共扩增。使用双抗体夹心测定形式和三种DNA标记抗体的混合物进行测定。证明了每种分析物的剂量-反应关系。检测分析物的灵敏度超过传统的酶免疫测定约三个数量级。hTSH、β-Gal和hCG的检测限分别为1 × 10 ~（-19）、1 × 10 ~（-17）和1 × 10 ~（-17）mol。考虑到PCR提供的巨大扩增和现有的基于大小或序列差异区分DNA的能力，使用DNA标记的抗体可以提供以比现有抗原检测系统更高的灵敏度同时检测许多分析物的基础。这些发现与以前的报告一致表明，这种混合技术可以提供新一代的超灵敏多分析物免疫测定。
A multianalyte immunoassay for simultaneous detection of three analytes (hTSH, hCG and beta-Gal) has been demonstrated using DNA-labeled antibodies and polymerase chain reaction (PCR) for amplification of assay response. The labeled antibodies were prepared by covalently coupling uniquely designed DNA oligonucleotides to each of the analyte-specific monoclonal antibodies. Each of the DNA oligonucleotide labels contained the same primer sequences to facilitate coamplification by a single primer pair. Assays were performed using a two-antibody sandwich assay format and a mixture of the three DNA-labeled antibodies. Dose-response relationships for each analyte were demonstrated. Analytes were detected at sensitivities exceeding those of conventional enzyme immunoassays by approximately three orders of magnitude. Detection limits for hTSH, beta-Gal and hCG were respectively 1 x 10(-19), 1 x 10(-17) and 1 x 10(-17) mol. Given the enormous amplification afforded by PCR and the existing capability to differentiate DNA based on size or sequence differences, the use of DNA-labeled antibodies could provide the basis for the simultaneous detection of many analytes at sensitivities greater than those of existing antigen detection systems. These findings in concert with previous reports suggest this hybrid technology could provide a new generation of ultra-sensitive multianalyte immunoassays.