In solution PLA, a proximity ligation assay, for biomarker research and diagnostics

Protein detection in solution

The PLA™ can be configured in many formats depending on your particular analytical need. In situ PLA using the Duolink reagents is suitable for detecting proteins in a localized manner in fixed cells and tissues immobilized on glass slides. Proteins present in a solution, for example derived from a cell lysate or a serum/plasma sample, can also be detected by PLA and in order to distinguish it from in situ PLA we refer to as in solution PLA. This assay can provide enhanced sensitivity, specificity, lower sample consumption and multiplexing capability in comparison to other techniques.

Principle of in solution PLA

 

Schematic illustration of homogeneous in solution PLA showing a target protein bound by two proximity probes (antibodies linked to ligatable DNA-strands). 

In solution PLA employs two primary antibodies linked to DNA strands which upon simultaneous and proximal binding to the target protein enables the ligation of the two different DNA strands. Once ligated, they form a PCR amplicon typically detected with real-time quantitative PCR. This protein-to-DNA conversion provides a number of attractive features. The amplification ability of a DNA reporter drives sensitivity and lowers sample consumption, while also supporting multiplexing capabilities. The assay can be performed either as a convenient homogeneous assay without any washing steps or on a solid phase where the target analyte is first captured prior to binding the PLA probes.

Unique features provided by in solution PLA

The attractive features of the assay are the low sample consumption, high sensitivity, and multiplexing capability. As described in the publications listed below, the assay can detect down to low femto molar concentrations of target proteins in just 1 μL samples with a 5-log linear range even when performed in multiplex.

The figure below shows detection of the Vascular Endothelial Growth Factor (VEGF) in 1 uL samples by PLA performed with real-time PCR readout of the ligation products. This highly sensitive detection (<0.2 pg/mL or 0.01 pM) is performed in minute sample amounts and is especially applicable to biomarker studies in limited sample amounts.

Contract service

Olink Bioscience offers this research tool on a fee for service basis within the biomarker research field. Large numbers of samples can be profiled for many putative protein biomarkers indicative of disease states, outcomes, etc., in minute and precious biobanked sample collections. For further information on the service offered, please contact Simon Fredriksson (simon.fredriksson@olink.com).

PLA for regulated diagnostics

Strategic partnerships are under development for the use of PLA within regulated diagnostics.

EU FP-7 sponsored biomarker program, PROACTIVE

Olink Bioscience is currently coordinating an EU funded biomarker research effort in to colorectal cancer detection using high throughput multiplexed in solution PLA. This large scale biomarker effort comprises partners from academia, biotech and diagnostics industry around Europe. For more information, please visit the PROACTIVE website

Commercial partner in the life science research market

In 2007 Olink Bioscience entered into a exclusive license and collaboration agreement with Applied Biosystems (now a part of Life Technologies). Under the agreement, Applied Biosystems will co-develop the PLA technology for specific applications in the life science research market that include biomarker validation and characterization of complex biological processes. For more information, please read the press release

Further information on in solution PLA can be found in the following publications.

Multiplex in solution PLA for cancer biomarker research

Fredriksson S, Dixon W, Ji H, Koong AC, Mindrinos M, Davis RW: Multiplexed protein detection by proximity ligation for cancer biomarker validation. Nat Methods 2007, 4(4):327-329.

Fredriksson S, Horecka J, Brustugun OT, Schlingemann J, Koong AC, Tibshirani R, Davis RW. Multiplexed proximity ligation assays to profile putative plasma biomarkers relevant to pancreatic and ovarian cancer. Clin Chem. 2008 Mar;54(3):582-9. Epub 2008 Jan 2.

DNA-protein interactions

Gustafsdottir SM, Schlingemann J, Rada-Iglesias A, Schallmeiner E, Kamali-Moghaddam M, Wadelius C, Landegren U: In vitro analysis of DNA-protein interactions by proximity ligation. Proc Natl Acad Sci U S A 2007, 104(9):3067-3072.

Sensitive pathogen detection

Gustafsdottir SM, Nordengrahn A, Fredriksson S, Wallgren P, Rivera E, Schallmeiner E, Merza M, Landegren U: Detection of individual microbial pathogens by proximity ligation Clin Chem 2006, 52(6):1152-1160

First papers on in solution PLA for protein detection

Gullberg, M, Gustafsdottir, S M, Schallmeiner, E, Jarvius, J, Bjarnegard, M, Betsholtz, C, Landegren, U & Fredriksson, S, Cytokine detection by antibody-based proximity ligation Proc Natl Acad Sci U S A, 2004, 101, 8420-4

Fredriksson, S, Gullberg, M, Jarvius, J, Olsson, C, Pietras, K, Gustafsdottir, S M, Ostman, A & Landegren, U, Protein detection using proximity-dependent DNA ligation assays Nat Biotechnol, 2002, 20, 473-7

In solution PLA for interaction inhibition screening

Gustafsdottir SM, Wennström S, Fredriksson S, Schallmeiner E, Hamilton AD, Sebti SM, Landegren U. Use of proximity ligation to screen for inhibitors of interactions between vascular endothelial growth factor A and its receptors. Clin Chem. 2008 Jul;54(7):1218-25. Epub 2008 May 22.