| 1. |
- de Oliveira, Felipe Marques Souza, et al.
(författare)
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Detection of post-translational modifications using solid-phase proximity ligation assay.
- 2018
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Ingår i: New biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784. ; 45:October, s. 51-59
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Tidskriftsartikel (refereegranskat)abstract
- Post-translational modifications (PTMs) regulate protein activities to help orchestrate and fine-tune cellular processes. Dysregulation of PTMs is often related with disorders and malignancies, and may serve as a precise biomarker of disease. Developing sensitive tools to measure and monitor low-abundant PTMs in tissue lysates or serum will be instrumental for opening up new PTM-based diagnostic avenues. Here, we investigate the use of solid-phase proximity ligation assay (SP-PLA) for detection of different PTMs. The assay depends on the recognition of the target protein molecule and its modification by three affinity binders. Using antibodies and lectins, we applied the method for detection of glycosylated CD44 and E-Cadherin, and phosphorylated p53 and EGFR. The assay was found to have superior dynamic range and limit of detection compared to standard ELISAs. In summary, we have established the use of SP-PLA as an appropriate method for sensitive detection of PTMs in lysates and sera, which may provide a basis for future PTM-based diagnostic and prognostic biomarkers.
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| 2. |
- Löf, Liza, et al.
(författare)
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Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.
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| 3. |
- Siart, Benjamin, et al.
(författare)
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Measurements ofinflammation protein biomarkers in venous plasma, earlobe capillary plasma andin capillary plasma stored on filter paper
- 2017
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Ingår i: Cytokine. - 1043-4666 .- 1096-0023.
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Multiplex panels for protein biomarkers are increasingly used to analyze blood samples in various fields of clinical research. However, they are rarely used in studies performed outside of a clinical setting. This may in part be due to the relative invasiveness of drawing blood from the vein and because of problems associated with the separation and transport of plasma. Samples collected from the earlobe would be less invasive and could be collected more conveniently. Transportation and storage of such samples could be made easier by blotting plasma on filter paper as dried plasma spots (DPS). The objective of this study was to compare values of multiple protein biomarkers for inflammation measured from three different sources (1) venous plasma, (2) plasma derived from capillary blood from the earlobe and (3) from capillary plasma stored as DPS. Samples from twelve male individuals were assessed with a panel of 92 inflammation related proteins using multiplex proximity extension assay technology. Correlations between the three sample types varied greatly between analytes. In general, a high correlation was observed between capillary plasma and DPS, with 33 analytes showing a correlation value of ρ > 0.8 in the two sample types. At this level of correlation (ρ > 0.8) 14 analytes correlated between venous and capillary plasma in contrast to only six analytes in the comparison of venous blood with DPS. We conclude that collecting samples from the earlobe is a feasible and less invasive alternative to venipuncture, but that the comparability with measures obtained from venous samples varies greatly between proteins.
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| 4. |
- Siart, Benjamin, et al.
(författare)
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Protein measurements in venous plasma, earlobe capillary plasma and in plasma stored on filter paper
- 2019
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Ingår i: Analytical Biochemistry. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 0003-2697 .- 1096-0309. ; 566, s. 146-150
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Tidskriftsartikel (refereegranskat)abstract
- In this study, levels of inflammatory protein biomarkers in venous plasma, plasma derived from capillary blood from the earlobe, and capillary plasma stored as dried plasma spots (DPS) were compared. Samples from 12 male individuals were assessed with a panel of 92 inflammation-related proteins using multiplex proximity extension assay. Correlations between sample types varied greatly between analytes. A high correlation of rho > 0.8 was observed between capillary plasma and DPS for 32 analytes. At this level of correlation, 13 analytes correlated between venous and capillary plasma and 5 analytes in the comparison of venous blood with DPS.
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