Joerger R D, Truby T M, Hendrickson E R, Young R M, Ebersole R C
Central Research & Engineering Department, E. I. DuPont Co., Wilmington, DE 19880-0173, USA.
Clin Chem. 1995 Sep;41(9):1371-7.
We demonstrate immuno-polymerase chain reaction (PCR) assays for two clinical analytes--human thyroid-stimulating hormone and chorionic gonadotropin (hTSH, hCG)--using DNA-labeled antibodies and PCR for amplification of assay response. DNA-antibody conjugates were synthesized by using heterobifunctional cross-linker chemistries to covalently attach single- or double-stranded DNA labels through amine or sulfhydryl groups on the analyte antibodies. These approaches yielded molecular chimeras possessing both analyte-specific antibody binding and nucleic acid amplification functionalities. Dose-response relationships were demonstrated for immuno-PCR assays of both analytes in a microtiter plate-based, two-antibody sandwich assay format. Detection limits for hTSH (1 x 10(-19) mol, < 1.4 mIU/L) and hCG (5 x 10(-18) mol, 0.025 IU/L) exceeded those of conventional enzyme immunoassays by 2-3 orders of magnitude. We also evaluated various DNA design factors influencing label amplification and assay performance, such as primer sequence, strand number, and DNA length. Our findings, in concert with previous reports, suggest that this hybrid technology could provide the basis for a new generation of ultra-sensitive immunoassays offering multianalyte capabilities.
我们展示了针对两种临床分析物——人促甲状腺激素和绒毛膜促性腺激素（hTSH、hCG）的免疫聚合酶链反应（PCR）分析方法，该方法使用DNA标记抗体并通过PCR扩增分析反应。通过使用异双功能交联剂化学方法，使单链或双链DNA标记通过分析物抗体上的胺基或巯基共价连接，从而合成DNA-抗体偶联物。这些方法产生了兼具分析物特异性抗体结合和核酸扩增功能的分子嵌合体。在基于微量滴定板的双抗体夹心分析形式中，展示了两种分析物免疫PCR分析的剂量反应关系。hTSH（1×10⁻¹⁹摩尔，<1.4 mIU/L）和hCG（5×10⁻¹⁸摩尔，0.025 IU/L）的检测限比传统酶免疫分析高出2 - 3个数量级。我们还评估了影响标记扩增和分析性能的各种DNA设计因素，如引物序列、链数和DNA长度。我们的研究结果与先前的报告一致，表明这种杂交技术可为新一代提供多分析物能力的超灵敏免疫分析提供基础。
