title: A universal immuno-PCR platform for comparative and ultrasensitive quantification of dual affinity-tagged proteins in complex matrices.
abstract: Protein detection in complex biological fluids and matrices has become a widely diversified field utilizing a number of different technologies. The quantification of target proteins in complex media such as serum remains a challenge for most technologies such as mass spectrometry, ELISA and western blot. Quantitative Immuno-PCR has been heavily used for antigen detection in immunoassays, but minimally so for quantifying affinity-tagged proteins expressed or circulating in complex matrices--despite its high sensitivity and robustness--because it suffers from detrimental background effects arising from its extreme detection power. We report the development of a universal qIPCR-based platform for the reproducible detection of dual affinity-tagged protein analytes in crude complex matrices such as serum and cell culture media or lysates. The system uses a couple of high-affinity antibodies against two affinity tags (GFP and HA) for the detection of dual-tagged proteins. The dual-tagged analyte is immuno-captured by one of its tags, while the second tag is bound by a detection device consisting of a new kind of self-assembled antibody-DNA conjugate. The new qIPCR platform enabled picomolar quantification of dual-tagged sortase in crude serum in 4 h including the PCR step.
authors.affiliation: Comparative Genomics Centre, School of Pharmacy and Molecular Sciences, James Cook University, DB21, James Cook Drive, Townsville, QLD 4811, Australia.
authors.forename: Samuel P
authors.initials: SP
authors.lastname: Askin
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Schaeffer
mesh_terms: D000345:Affinity Labels / Q000737:chemistry; D019881:Aminoacyltransferases / Q000097:blood; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D001426:Bacterial Proteins / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000097:blood; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D007074:Immunoglobulin G / Q000097:blood; D037102:Lectins / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000032:analysis*
chemical_list: D000345:Affinity Labels; D000906:Antibodies; D001426:Bacterial Proteins; D007074:Immunoglobulin G; D037102:Lectins; D011506:Proteins; C114923:hemagglutinin A, Porphyromonas gingivalis; D049452:Green Fluorescent Proteins; D004247:DNA; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases