| 1. |
- Darmanis, Spyros, et al.
(författare)
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Multiplexed solid-phase proximity ligation assays: Highly specific and parallel protein measurements with DNA sequencing readout
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Identification and validation of protein biomarkers is a very important step towards the understanding of the underlying mechanisms of disease, early diagnosis and efficient patient treatment. To carry out this task, methods are needed that would allow us to mine the proteome with sufficient sensitivity and specificity in large sets of samples. We present herein the development of a Multiplexed Proximity Ligation Assay (MultiPLAy), to facilitate efficient protein profiling in a parallel, sensitive and specific manner. We showed that for the simultaneous analysis of 35 proteins MultiPLAy exhibited an improved sensitivity over conventional sandwich assays as well as a smaller susceptibility to background signal increase in the transition from singleplex to multiplex. We used MultiPLAy to identify putative biomarkers in two separate sample cohorts of colorectal cancer (CRC) and cardiovascular disease (CVD) and with the use a novel multivariate analysis approach were able to identify new, as well as already known diagnostic biomarkers. Furthermore we were able to combine MultiPLAy with the use of next-generation sequencing allowing for the first time digital recording of protein profiles in blood. We demonstrated good reproducibility of MultiPLAy coupled to next-generation sequencing, as well as a satisfactory correlation to standard real-time PCR readout. We conclude that MultiPLAy has great potential as a basis for highly multiplexed protein detection assays that can be utilized for the identification of large numbers of proteins or protein variants. This will allow extensive validation of protein expression patterns in biobanked samples and in prospective studies, and can provide a much-needed platform for efficient validation of diagnostic markers for clinical use.
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| 2. |
- Darmanis, Spyros, et al.
(författare)
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ProteinSeq : high-performance proteomic analyses by proximity ligation and next generation sequencing
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:9, s. e25583-
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Tidskriftsartikel (refereegranskat)abstract
- Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 μl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use.
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| 3. |
- Darmanis, Spyros, et al.
(författare)
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Sensitive plasma protein analysis by microparticle-based proximity ligation assays
- 2010
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 9:2, s. 327-335
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Tidskriftsartikel (refereegranskat)abstract
- 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 microparticle-based 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-of-the-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.
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