A universal immuno-PCR platform for comparative and ultrasensitive quantification of dual affinity-tagged proteins in complex matrices
Journal Publication ResearchOnline@JCUAbstract
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.
Journal
Analyst
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N/A
Volume
137
ISBN/ISSN
0003-2654
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Issue
22
Pages Count
4
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Publisher
Royal Society of Chemistry
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EISSN
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DOI
10.1039/c2an35857c