Multiplexed Proximity Ligation Assays to Profile Putative Plasma Biomarkers Relevant to Pancreatic and Ovarian Cancer

Simon, Fabrice; Horecka, Joe; Brustugun, Odd Terje; Schlingemann, Joerg; Koong, Albert C.; Tibshirani, Rob; Davis, Ronald W.

doi:10.1373/clinchem.2007.093195

Cited by 87 publications

(66 citation statements)

References 29 publications

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“…In this way, we hope to use conformation-switching aptamers with a high signal-to-noise ratio, and to amplify the small signals that occur in the presence of even small amounts of protein analytes. By utilizing different probes for different conformation-switching aptamers it should be straightforward to adapt these assays to a multiplex format, as has recently also been done for the proximity ligation assay [49].…”

Section: Discussionmentioning

confidence: 99%

Real-time PCR detection of protein analytes with conformation-switching aptamers

Yang

Ellington

2008

Analytical Biochemistry

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We have developed a novel method that utilizes conformation-switching aptamers for real-time PCR analysis of protein analytes. The aptamers have been designed so that they assume one secondary structure in the absence of a protein analyte, and a different secondary structure in the presence of a protein such as thrombin or PDGF. The protein-bound structure in turn assembles a ligation junction for the addition of a real-time PCR primer. Protein concentrations could be specifically detected into the picomolar range, even in the presence of cell lysates. The method has advantages relative to both immunoPCR (since no signal is produced by background binding) and to the proximity ligation assay (PLA; since only one epitope on a protein surface must be bound, rather than two).

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Section: Discussionmentioning

confidence: 99%

Real-time PCR detection of protein analytes with conformation-switching aptamers

Yang

Ellington

2008

Analytical Biochemistry

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“…The assay has also been performed on solid supports by immobilizing antibodies directly on the walls of PCR tubes (2) or by immobilizing biotinylated antibodies on the surface of streptavidin-coated tubes (3). The PLA technique has been implemented for a wide variety of applications, including to visualize proteins in situ (4), to reveal infectious agents (3) and protein-DNA interactions (5), and for biomarker detection in both singleplex (6,7) and multiplex (9,10).…”

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confidence: 99%

Sensitive Plasma Protein Analysis by Microparticle-based Proximity Ligation Assays

Darmanis

Nong

Hammond

et al. 2010

Molecular & Cellular Proteomics

114

132

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Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticlebased solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-ofthe-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples. Molecular & Cellular Proteomics 9:327-335, 2010.Analyses of the plasma proteome, its protein content, their modifications, and interactions, hold great promise to improve detection, classification, and prognostication of pathological conditions such as cancer (1). The attraction of serum or plasma biomarkers lies in their potential to reveal disease processes throughout the body and to guide selection of therapy and follow-up using minimally invasive blood sampling.This optimism is tempered by the molecular complexity of plasma and the fact that the abundance of known plasma proteins varies over at least 12 orders of magnitude (1), posing great challenges for immunoassays used to investigate the plasma proteome. Thus, new assay formats are needed that can offer improved sensitivity and specificity over a broad dynamic range with good precision to assess new protein biomarkers for analysis in plasma, serum, or whole blood.The proximity ligation assay (PLA), 1 first described by Fredriksson et al. (2) in 2002, is an immunoassay for detection of protein molecules via DNA ligation and amplification, offering high specificity and sensitivity. In PLA, pairs of affinity probes directed against the same target molecule are modified by attaching short single-stranded DNA m...

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“…The Stanford group was one of the first groups to develop and apply PLA in a biomarker discovery effort for cancer patients. Fredriksson et al [33] used 4 multiplex panels with each for detecting 6 or 7 markers, to compare 18 pancreatic cancer patients with 19 matched healthy controls and 20 ovarian cancer patients with 20 matched healthy controls. CA19-9 and CA125 were the most significant biomarkers to differentiate patients from controls for the two cancers, respectively.…”

Section: Pla For Cancer Patientsmentioning

confidence: 99%

Biomarker Studies on Radiotherapy to Hepatocellular Carcinoma

et al. 2013

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Radiotherapy (RT) has been gradually integrated into the multimodality treatment for hepatocellular carcinoma (HCC). The various patterns of failure in HCC patients undergoing RT drive the need of effective biomarkers to guide treatment decisions. Limited numbers of biomarkers have been investigated in HCC, with even fewer of them for patients treated by RT. Serum or plasma biomarkers measured by enzyme-linked immunosorbent assay were the most common practice. Of particular interest are those biomarkers that are detectable early in the course of radiotherapy which correlated with ultimate clinical outcome. Functional magnetic resonance imaging (MRI) is increasingly used to evaluate the imaging pattern indicative of disease control following RT. Positron emission tomography shows that pre-RT standard uptake values associate with various types of recurrence after treatment. Proximity ligation assay (PLA) is evolving with the unique features of dual-probe identification, ligation and amplification to allow the small volume of serum/plasma samples for evaluating multiple biomarkers. We demonstrate the screening work of biomarkers by PLA with pre- and post-RT serum samples from HCC patients undergoing RT. Efforts are being made to search for the potential biomarkers for HCC patients treated by RT.

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Multiplexed Proximity Ligation Assays to Profile Putative Plasma Biomarkers Relevant to Pancreatic and Ovarian Cancer

Abstract: BACKGROUND: Sensitive methods are needed for biomarker discovery and validation. We tested one promising technology, multiplex proximity ligation assay (PLA), in a pilot study profiling plasma biomarkers in pancreatic and ovarian cancer.

Cited by 87 publications

References 29 publications

Real-time PCR detection of protein analytes with conformation-switching aptamers

Real-time PCR detection of protein analytes with conformation-switching aptamers

Sensitive Plasma Protein Analysis by Microparticle-based Proximity Ligation Assays

Biomarker Studies on Radiotherapy to Hepatocellular Carcinoma

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