| 1. |
- 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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| 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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| 3. |
- Tavoosidana, Gholamreza, et al.
(författare)
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Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer
- 2011
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:21, s. 8809-8814
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Tidskriftsartikel (refereegranskat)abstract
- Prostasomes are microvesicles (mean diameter, 150 nm) that are produced and secreted by normal and malignant prostate acinar cells. It has been hypothesized that invasive growth of malignant prostate cells may cause these microvesicles, normally released into seminal fluid, to appear in interstitial space and therewith into peripheral circulation. The suitability of prostasomes as blood biomarkers in patients with prostate cancer was tested by using an expanded variant of the proximity ligation assay (PLA). We developed an extremely sensitive and specific assay (4PLA) for detection of complex target structures such as microvesicles in which the target is first captured via an immobilized antibody and subsequently detected by using four other antibodies with attached DNA strands. The requirement for coincident binding by five antibodies to generate an amplifiable reporter results in both increased specificity and sensitivity. The assay successfully detected significantly elevated levels of prostasomes in blood samples from patients with prostate cancer before radical prostatectomy, compared with controls and men with benign biopsy results. The medians for prostasome levels in blood plasma of patients with prostate cancer were 2.5 to sevenfold higher compared with control samples in two independent studies, and the assay also distinguished patients with high and medium prostatectomy Gleason scores (8/9 and 7, respectively) from those with low score (<= 6), thus reflecting disease aggressiveness. This approach that enables detection of prostasomes in peripheral blood may be useful for early diagnosis and assessment of prognosis in organ-confined prostate cancer.
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| 4. |
- Wallin, Ulrik, et al.
(författare)
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Growth differentiation factor 15 : a prognostic marker for recurrence in colorectal cancer
- 2011
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Ingår i: British Journal of Cancer. - : Springer Science and Business Media LLC. - 0007-0920 .- 1532-1827. ; 104:10, s. 1619-1627
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Growth differentiation factor 15 (GDF15) belongs to the transforming growth factor beta superfamily and has been associated with activation of the p53 pathway in human cancer. The aim of this study was to assess the prognostic value of GDF15 in patients with colorectal cancer (CRC). METHODS: Immunohistochemistry and tissue microarrays were used to analyse GDF15 protein expression in 320 patients with CRC. In a subgroup of 60 patients, the level of GDF15 protein in plasma was also measured using a solid-phase proximity ligation assay. RESULTS: Patients with CRC with moderate to high intensity of GDF15 immunostaining had a higher recurrence rate compared with patients with no or low intensity in all stages (stages I-III) (HR, 3.9; 95% CI, 1.16-13.15) and in stage III (HR, 10.32; 95% CI, 1.15-92.51). Patients with high plasma levels of GDF15 had statistically shorter time to recurrence (P = 0.041) and reduced overall survival (P = 0.002). CONCLUSION: Growth differentiation factor 15 serves as a negative prognostic marker in CRC. High expression of GDF15 in tumour tissue and high plasma levels correlate with an increased risk of recurrence and reduced overall survival.
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