A novel immuno-PCR method using cDNA display.

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.