Centre for Biotechnology, Maharshi Dayanand University, Rohtak, Haryana, India,
Institute of Synthetic Biology (iSynBio), Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
Int J Nanomedicine. 2018 Dec 12;13:8523-8535. doi: 10.2147/IJN.S181052. eCollection 2018.
Immuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of antigen.
GNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of -specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR.
Transmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 10-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6.
Unlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis.
免疫聚合酶链反应（I-PCR）是一种超灵敏的方法，它将 ELISA 的多功能性与 PCR 的指数扩增能力相结合。检测抗体与报告 DNA 的偶联是 I-PCR 的关键步骤。金纳米粒子（GNPs）和磁性珠（MBs）相对容易与抗体和 DNA 结合。因此，我们设计了基于 MB 偶联的 GNP 的 I-PCR（MB-GNP-I-PCR）检测方法用于检测抗原。
通过化学还原和种子介导合成法合成 GNPs。通过将 GNPs 与检测抗体和报告 DNA 偶联来制备功能化的 GNPs，并对其进行了表征。通过 MB-GNP-I-PCR 测定了特异性纯化的早期分泌抗原靶 6（ESAT-6）（Rv3875）的检测限。
透射电子显微镜显示，20nm 和60nm 大小的 GNPs 呈球形且略有多分散性。抗体与 GNPs 的偶联通过吸收最大值从 524nm 向 534nm 的移动来指示，这通过透射电子显微镜得到了证实。ELISA 的显色反应和 PCR 的 76bp 产物的存在进一步验证了检测抗体和信号 DNA 与功能化 GNPs 的偶联。此外，MB 上捕获抗体的附着通过磁酶联免疫吸附试验得到了证实。通过 MB-GNP-I-PCR 测定，纯化 ESAT-6 的检测限为 10fg/mL，比类似的 ELISA 低 10 倍。值得注意的是，在健康个体的唾液样本中加入纯化的 ESAT-6 后，未观察到样品基质效应。
与传统的 I-PCR（固相）不同，MB-GNP-I-PCR（液相）相对简单，背景信号较低，可进一步用于结核病的临床诊断。
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