A universal immuno-PCR platform for comparative and ultrasensitive quantification of dual affinity-tagged proteins in complex matrices

Askin, Samuel P., and Schaeffer, Patrick M. (2012) A universal immuno-PCR platform for comparative and ultrasensitive quantification of dual affinity-tagged proteins in complex matrices. Analyst, 137 (22). pp. 5193-5196.

[img] PDF (Published Version) - Published Version
Restricted to Repository staff only

View at Publisher Website: http://dx.doi.org/10.1039/c2an35857c
 
6
1


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.

Item ID: 24159
Item Type: Article (Refereed Research - C1)
ISSN: 0003-2654
Funders: National Health and Medical Research Council (NHMRC), Queensland Tropical Health Alliance, Smart Futures Fund
Date Deposited: 05 Dec 2012 05:27
FoR Codes: 06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060101 Analytical Biochemistry @ 100%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 100%
Downloads: Total: 1
More Statistics

Actions (Repository Staff Only)

Item Control Page Item Control Page