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- 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, 1983-
(författare)
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Solid-phase Proximity Ligation Assays : High-performance and multiplex protein analyses
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein biomarkers circulating in blood hold the promise of improved diagnosis, prognosis and follow-up of treatment of disease via minimally invasive procedures. For the discovery and validation of such biomarkers, methods are needed that can facilitate parallel, highly specific and in-depth analysis of the blood proteome. The work presented in this thesis intends to develop and apply such assays, building on the concept of the proximity ligation assay (PLA). In paper I, I present an easy and non-expensive alternative for the conjugation of oligonucleotides to antibodies via biotin-streptavidin-biotin interaction. This approach can be used when large sets of antibodies and/or oligos need to be validated for their performance as probes in PLA reactions. In paper II, a solid-phase variant of PLA (SP-PLA) for the detection and quantification of proteins in blood is presented. SP-PLA exhibited an improved limit of detection compared to commercial ELISA assays by two orders of magnitude. In addition SP-PLA exhibited a broader dynamic range by at least one order of magnitude and required only 5 μl of sample, rendering the method very well suited for analyses of precious bio-banked material. Last but not least, SP-PLA was used to validate the diagnostic potential of GDF-15 as a biomarker for cardiovascular disease in a set of cardiovascular disease patients and healthy controls. Paper III discusses the development of a multiplex SP-PLA (MultiPLAy) for the simultaneous detection of 36 proteins in just 5 μl of sample. MultiPLAy exhibited an improved LOD when compared to state-of-the-art bead-based sandwich assays. Most importantly, we observed only a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that much higher levels of multiplexing will be possible. The assay was used to identify putative biomarkers in sample cohorts of colorectal cancer (CRC) and cardiovascular disease (CVD). Subsequent multivariate analysis revealed previously known diagnostic biomarkers. Furthermore, we successfully applied next-generation sequencing as a readout for the protein assays, allowing for the first time digital recording of protein profiles in blood. In paper IV, we investigated the suitability of prostasomes as blood biomarkers in patients with prostate cancer using a newly developed PLA assay (4PLA) that utilizes five binders for the detection of complex target molecules. The assay successfully detected significantly elevated levels of prostasomes in blood samples from prostate cancer patients prior to radical prostatectomy, compared to controls and men with benign biopsy results.
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