Life Technologies plans to launch in mid-2010 an "open kit" for its line of TaqMan Protein Expression Assays, which will enable researchers to design their own real-time qPCR-based proximity-ligation assays for protein analysis.
The launch of the flexible open kit follows a set of fixed TaqMan Protein Expression Assays for stem cell research that the company released in August.
David Ruff, principal scientist for genomic assays R&D at Life Technologies, disclosed the upcoming launch during a discussion of protein analysis applications for the TaqMan platform at last month's qPCR Symposium in Millbrae, Calif.
He said that the availability of the open kit, which will enable researchers to design their own protein expression assays, will bring qPCR-based protein-expression analysis "to the masses."
The kit is currently available for early-access customers, with a full product release scheduled for next year.
The TaqMan protein-expression line is based on proximity-ligation assay, or PLA, technology licensed from Olink Biosciences of Uppsala, Sweden. The assay relies on two biotinylated antibodies that are each conjugated to a different streptavidin-linked oligonucleotide — one 3' and one 5'. When the two conjugated antibodies bind to a target protein and are in close proximity, the oligonucleotides are ligated and serve as the template for real-time PCR amplification and quantification.
The company holds an exclusive license for the PLA technology for in vitro applications with amplification and readout by real-time PCR, a Life Technologies spokesperson told PCR Insider. The license is specific to the commercialization of reagents for research use only.
While the concept of PCR-based protein analysis is not new — immuno-PCR, which links a detection antibody to a DNA molecule that serves as a PCR template, was first described in 1992 — the use of the technology for protein analysis has not been broadly adopted.
Ruff noted in his talk last month that the PLA-based approach differs from immuno-PCR because its use of two antibodies rather than one reduces the amount of background noise.
"PLA requires two separate antibody-antigen binding events — this results in better selectivity and hence much lower background template for the PCR process," he explained via e-mail this week. "And because the PLA process uses two antibodies, protein-protein interactions can be detected and quantified."
The method requires about an hour and a half of hands-on time, Ruff said, with a total analysis time of around three and a half hours.
The current assay menu for stem cell research includes ready-made assays for hOCT3/4, hNANOG, hSOX2, and hLIN28, which are markers for pluripotency in stem cells, as well as for hICAM1 and hCSTB, which serve as controls.
Ruff said that the TaqMan protein assays are more sensitive than Western blots for measuring expression of these markers, with a limit of detection of between 10 and 35 cells per well as opposed to 10,000 to 30,000 cells per lane for Western blot. The qPCR-based approach is also much quicker, he noted, requiring several hours as opposed to up to two days for Western blot.
The upcoming open kit will include the streptavidin-linked oligos and customers can supply their own antibodies to create their own proximity probes, Ruff said.
In a comparison of the approach to an ELISA for measuring the expression of human TNF-R1, the TaqMan protein assay exhibited 2.5-fold greater sensitivity and 10-fold greater dynamic range than ELISA, Ruff said during his talk, though he noted this week that "this is not always the case."
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In general, he noted, when the company has compared antibodies developed as ELISA pairs to published standard ELISA data, "our assays often show equal or better sensitivity and up to 10 times greater dynamic range — all using 25 to 50 times less sample input for the assays, and using a homogeneous format, unlike typical assays that utilize washing steps."
Tina Settineri, director of the genomic assays product line at Life Technologies, said via e-mail that the cost of running the assays is "competitively priced, especially considering the very small sample input required." She did not elaborate.
'Sensitive and Robust'
Greg Shipley, director of the Quantitative Genomics Core Laboratory at the University of Texas Medical School at Houston, explained via e-mail that real-time qPCR shows promise for protein analysis because "it has the largest quantitative dynamic range of any technique and is thus a very sensitive and robust method."
Shipley added that researchers working with ELISAs "are thrilled with 4-log of linearity … so if you can apply a more robust method for detecting proteins, it would be very desirable."
He said that his lab currently runs MesoScale and AlphaLisa ELISA assays and would "welcome PLAs if they worked well because we are well set up to run real-time qPCR instrumentation."
Ruff said that so far, Life Technologies has validated more than 50 human targets in-house for the protein assay open kit and this number is "growing every month."
"Most of the original screening was performed with polyclonal antibodies directed against recombinant protein antigens," he explained. "We have also validated the process for some monoclonal antibody pairs. Also, we have found some polyclonal and monoclonal antibodies directed against peptide immunogens have worked well in our open kit process."
Ruff noted that a "good guide" for selecting antibodies to screen "is to use antibody pairs that perform satisfactorily in an ELISA type of immunoassay format."
Shipley noted that one potential limit to the capability of the qPCR-based PLA approach would be antibody quality. Quantitative protein assays "are only as good as the specificity of the antibodies used in the assay" — a fact that applies to PLA assays as well as ELISAs, he said.
He cautioned that "there are many commercial [antibodies] available out there, but not all of them live up to their advertised specificity." Shipley recommended that anyone developing an antibody-based assay should purchase multiple antibodies and find the ones that work best. "That would certainly be true for the proximity ligation assay as well."
Life Technologies said it foresees a broad range of applications for the qPCR-based PLA approach, including protein-activation studies, tumor characterization, protein localization experiments, quantification of cell surface-marker expression, analysis of secreted proteins, fixed-tissue studies, and of protein-protein–interaction analysis.
The company began exploring the PLA technology around four and a half years ago, Ruff said, in order to address a gap in its assay portfolio. While there was "good assay coverage" for genomic, DNA, RNA, and miRNA analysis, the company determined that assays for post-translational research were "missing," and made its initial investment in the PLA approach.
Ruff said he believes that the current TaqMan Protein Expression Assay portfolio is just "the tip of the iceberg" for the approach. "We envision that eventually not just protein expression, but all protein biology will be studied with this tool," he said. "A new horizon awaits qPCR platforms."