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.

Toward Multiplexing Detection of Wound Healing Biomarkers on Porous Silicon Resonant Microcavities

“…This result confirms that the fluorescence‐based sensing approach by far exceeds the performance of the reflectance‐based sensor which gave a LOD in the order of 10 −8 m . Moreover, the fluorescence‐based pSiRM sensor outperforms the quantitative immuno‐PCR assay for SrtA in terms of LOD (10 −14 m vs 10 −12 m ) and analysis time (30 min vs 4 h including the PCR step), demonstrating its potential as a point‐of‐care diagnostic tool.…”

Defining the prototypical DNA replication fork trap in bacteria

“…The systematic analyses of individual Ter sites both in vitro and in vivo with respect to their affinity and kinetics for Tus, ability of forming a TT-lock structure as well as their position and orientation within the bacterial genome have provided a wealth of information as to how this seemingly simple protein-DNA interaction impedes replication forks. In fact, Tus-Ter has become one of the best-understood protein-DNA complexes, leading to the development of a variety of biotechnological applications (54)(55)(56)(57). Yet, we are only just starting to understand the modus operandi of Tus in vivo.…”

Tus- Ter -Lock Immuno-PCR Assays for The Sensitive Detection of Tropomyosin-Specific Ige Antibodies

“…There can be a trade-off between background and sensitivity when it comes to the number of wash steps performed in an immuno-PCR, as there is with ELISA. The number of washes in a TT-lock qIPCR has previously been optimised for different assay methods [40][41][42] and so formed the basis of washing protocols here. At 3 SD above the negative sample the positive cut-off value is 0.9 ∆Ct (mixture format).…”

“…Here we report the development of two new quantitative immuno-PCR (qIPCR) assays building on the TT-lock qIPCR platform [40][41][42]. We have engineered two new detection devices for the sensitive detection of TM-specific antibodies: TM tagged with the haemagglutinin A epitope (TM-HA) and Tus-TM ( Figure 1).…”