1. A novel immuno-PCR method using cDNA display
- Author
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Chathuni Jayathilake, Hiroki Anzai, Naoto Nemoto, Takeru Suzuki, and Takuya Terai
- Subjects
DNA, Complementary ,RNase P ,Aptamer ,Biophysics ,Enzyme-Linked Immunosorbent Assay ,Polymerase Chain Reaction ,01 natural sciences ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Complementary DNA ,mRNA display ,Protein Interaction Domains and Motifs ,Staphylococcal Protein A ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Heavy-chain antibody ,biology ,010401 analytical chemistry ,Cell Biology ,0104 chemical sciences ,chemistry ,Puromycin ,Ribosome display ,biology.protein ,Nucleic acid ,Single-Chain Antibodies - 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.
- Published
- 2019