cDNA展示介导的免疫PCR（cD-IPCR）：一种基于PCR的新型抗原检测方法。
cDNA Display Mediated Immuno-PCR (cD-IPCR): A Novel PCR-based Antigen Detection Method.
作者信息
Jayathilake Chathuni, Terai Takuya, Nemoto Naoto
机构信息
Graduate School of Science and Engineering, Saitama University, Sakura-ku, Saitama, Japan.
Epsilon Molecular Engineering, Inc., 255 Shimo-Okubo, Sakura-ku, Saitama, Japan.
出版信息
Bio Protoc. 2019 Dec 20;9(24):e3457. doi: 10.21769/BioProtoc.3457.
Immuno-PCR (IPCR) is a powerful method in antigen detection where a PCR-amplifiable DNA reporter is conjugated to a specific antibody or an aptamer for the target molecule. In the development and application of IPCR, successful conjugation of a protein (an antibody) with a reporter DNA becomes challenging. To address this issue, we recently demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level. The cDNA display molecule for IPCR is generated first by transcribing the DNA that encodes the detection antibody into an mRNA by transcription. A puromycin DNA linker is then ligated to the mRNA and then translation and reverse-transcription are performed to generate the cDNA display molecule. The molecule is then directly used in antigen detection and subsequent qPCR. This method can be applied to detect various antigens in biological samples, if sequences of their single-domain antibodies (VHHs) or peptide aptamers are known.
免疫聚合酶链反应（IPCR）是抗原检测中的一种强大方法，其中可通过聚合酶链反应（PCR）扩增的DNA报告分子与针对目标分子的特异性抗体或适体结合。在免疫聚合酶链反应的开发和应用中，将蛋白质（抗体）与报告DNA成功结合变得具有挑战性。为了解决这个问题，我们最近证明了基于cDNA展示的免疫聚合酶链反应的可行性，即在单分子水平上多肽与其编码cDNA的1:1共价复合物。用于免疫聚合酶链反应的cDNA展示分子首先通过转录将编码检测抗体的DNA转录成mRNA产生。然后将嘌呤霉素DNA接头连接到mRNA上，接着进行翻译和逆转录以产生cDNA展示分子。然后将该分子直接用于抗原检测和随后的定量聚合酶链反应（qPCR）。如果已知其单域抗体（VHH）或肽适体的序列，该方法可应用于检测生物样品中的各种抗原。
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