A novel immuno-PCR method using cDNA display

Anal Biochem. 2019 Aug 1;578:1-6. doi: 10.1016/j.ab.2019.04.017. Epub 2019 Apr 25.

Abstract

Immuno-PCR (IPCR) provides sensitive and versatile detection of a variety of antigens by conjugating a PCR-amplifiable DNA reporter to a specific antibody or an aptamer. Several methodologies have been developed to prepare appropriate DNA-antibody conjugates, but in most cases, it remains difficult to label polypeptides with high site-specificity and fixed stoichiometry. To address this issue, we first demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA via puromycin at the single molecule level. Several other in vitro display technologies (e.g., ribosome display, mRNA display) have similar simple nucleic acid-peptide linkage. However, they should be unsuitable for diagnostic applications because of their lability against heat and RNase. The newly developed system here, termed cDNA display mediated immuno-PCR (cD-IPCR), proved to work in direct- and sandwich-type detection of target proteins. Detection of a target in serum was also possible, using a VHH (variable domain of the heavy chain of a heavy chain antibody) antibody as a binding molecule. Although further improvement on sensitivity and quantitativity is necessary before the method becomes useful, we believe this work demonstrated a potential of cD-IPCR as an alternative novel format of IPCR.

Keywords: Immuno-polymerase chain reaction; Nanobody; Variable domain of the heavy chain of a heavy chain antibody; cDNA display.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Complementary / chemistry*
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Polymerase Chain Reaction / methods
  • Protein Interaction Domains and Motifs
  • Single-Chain Antibodies / chemistry
  • Staphylococcal Protein A / chemistry*

Substances

  • DNA, Complementary
  • Single-Chain Antibodies
  • Staphylococcal Protein A