cDNA Display Mediated Immuno-PCR (cD-IPCR): A Novel PCR-based Antigen Detection Method

Chathuni Jayathilake; Takuya Terai; Naoto Nemoto

doi:10.21769/BioProtoc.3457

cDNA Display Mediated Immuno-PCR (cD-IPCR): A Novel PCR-based Antigen Detection Method

Bio Protoc. 2019 Dec 20;9(24):e3457. doi: 10.21769/BioProtoc.3457.

Authors

Chathuni Jayathilake¹, Takuya Terai¹, Naoto Nemoto^{1

2}

Affiliations

¹ Graduate School of Science and Engineering, Saitama University, Sakura-ku, Saitama, Japan.
² Epsilon Molecular Engineering, Inc., 255 Shimo-Okubo, Sakura-ku, Saitama, Japan.

Abstract

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 in vitro transcription. A puromycin DNA linker is then ligated to the mRNA and then in vitro 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.

Keywords: Immuno-detection; Immuno-polymerase chain reaction; VHH; cDNA display; qPCR.