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- Friedman, Mikaela, et al.
(författare)
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Engineering and characterization of a bispecific HER2 x EGFR-binding affibody molecule
- 2009
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 54, s. 121-131
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Tidskriftsartikel (refereegranskat)abstract
- HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G(4)S)(3) [(Gly(4)-Ser)(3)]-encoding gene fragment. The encoded 30 kDa affibody construct (Z(HER2))(2)-(G(4)S)(3)-(Z(EGFR))(2), with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.
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- Grönwall, Caroline, et al.
(författare)
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Selection and characterization of Affibody ligands binding to Alzheimer amyloid beta peptides
- 2007
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 128:1, s. 162-183
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Tidskriftsartikel (refereegranskat)abstract
- Affibody (Affibody) ligands specific for human amyloid beta (Abeta) peptides (40 or 42 amino acid residues in size), involved in the progress of Alzheimer's disease, were selected by phage display technology from a combinatorial protein library based on the 58-amino acid residue staphylococcal protein A-derived Z domain. Post-selection screening of 384 randomly picked clones, out of which 192 clones were subjected to DNA sequencing and clustering, resulted in the identification of 16 Affibody variants that were produced and affinity purified for ranking of their binding properties. The two most promising Affibody variants were shown to selectively and efficiently bind to Abeta peptides, but not to the control proteins. These two Affibody ligands were in dimeric form (to gain avidity effects) coupled to affinity resins for evaluation as affinity devices for capture of Abeta peptides from human plasma and serum. It was found that both ligands could efficiently capture Abeta that were spiked (100 microgml(-1)) to plasma and serum samples. A ligand multimerization problem that would yield suboptimal affinity resins, caused by a cysteine residue present at the binding surface of the Affibody ligands, could be circumvented by the generation of second-generation Affibody ligands (having cysteine to serine substitutions). In an epitope mapping effort, the preferred binding site of selected Affibody ligands was mapped to amino acids 30-36 of Abeta, which fortunately would indicate that the Affibody molecules should not bind the amyloid precursor protein (APP). In addition, a significant effort was made to analyze which form of Abeta (monomer, dimer or higher aggregates) that was most efficiently captured by the selected Affibody ligand. By using Western blotting and a dot blot assay in combination with size exclusion chromatography, it could be concluded that selected Affibody ligands predominantly bound a non-aggregated form of analyzed Abeta peptide, which we speculate to be dimeric Abeta. In conclusion, we have successfully selected Affibody ligands that efficiently capture Abeta peptides from human plasma and serum. The potential therapeutic use of these optimized ligands for extracorporeal capture of Abeta peptides in order to slow down or reduce amyloid plaque formation, is discussed.
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- Lindström, Sara, et al.
(författare)
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A microwell array device with integrated microfluidic components for enhanced single-cell analysis
- 2009
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 30:24, s. 4166-4171
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Tidskriftsartikel (refereegranskat)abstract
- Increasing awareness of the importance of cell heterogeneity in many biological and medical contexts is prompting increasing interest in systems that allow single-cell analysis rather than conventional bulk analysis (which provides average values for variables of interest from large numbers of cells). Recently, we presented a microwell chip for long-term, high-throughput single-cell analysis. The chip has proved to be useful for purposes such as screening individual cancer and stem cells for protein/gene markers. However, liquids in the wells can only be added or changed by manually rinsing the chip, or parts of it. This procedure has several well-known drawbacks - including risks of cross-contamination, large dead volumes and laboriousness - but there have been few previous attempts to integrate liquid rinsing/switching channels in "ready-to-use" systems for single-cell analysis. Here we present a microwell system designed (using flow simulations) for single-cell analysis with integrated microfluidic components (microchannels, magnetically driven micropumps and reservoirs) for supplying the cell culture wells with reagents, or rinsing, thus facilitating controlled, directed liquid handling. It can be used totally independently, since tubing is not essential. The practical utility of the integrated system has been demonstrated by culturing endothelial cells in the microwells, and successfully applying live-cell Calcein AM staining. Systems such as this combining high-density microwell chips with microfluidic components have great potential in numerous screening applications, such as exploring the important, but frequently undetected, heterogeneity in drug responses among individual cells.
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- Lindström, Sara, et al.
(författare)
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A microwell chip for parallel culture and analysis of stem cells
- 2009
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Ingår i: Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences. - : Chemical and Biological Microsystems Society. - 9780979806421 ; , s. 1309-1311
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Konferensbidrag (refereegranskat)abstract
- With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)-differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to maintain the cells as stem/progenitor cells over time was shown, as was isolation and clonal analysis.
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- Lindström, Sara, et al.
(författare)
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Comprehensive genetic evaluation of common E-cadherin sequence variants and prostate cancer risk : strong confirmation of functional promoter SNP
- 2005
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Ingår i: Human Genetics. - Umea Univ, Dept Radiat Sci, S-90187 Umea, Sweden. Karolinska Inst, Dept Med Epidemiol & Biostat, Stockholm, Sweden. Karolinska Inst, Ctr Genom & Bioinformat, Stockholm, Sweden. Univ Leicester, Dept Genet, Leicester LE1 7RH, Leics, England. Wake Forest Univ, Sch Med, Ctr Human Genom, Winston Salem, NC USA. Johns Hopkins Med Inst, Dept Urol, Baltimore, MD USA. CLINTECH, Karolinska Inst, Ctr Oncol, Stockholm, Sweden. : SPRINGER. - 0340-6717 .- 1432-1203. ; 118:3-4, s. 339-347
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Tidskriftsartikel (refereegranskat)abstract
- The E-cadherin gene (CDH1) has been proposed as a prostate cancer (PC) susceptibility gene in several studies. Aberrant protein expression has been related to prognosis and progression in PC. In addition, a functional promoter SNP (rsl6260) has been found to associate with PC risk. We performed a comprehensive genetic analysis of CDH1 by using the method of haplotype tagged SNPs in a large Swedish population-based case-control study consisting of 801 controls and 1,636 cases. In addition, Swedish PC families comprising a total of 157 cases sampled for DNA were analyzed for selected SNPs. Seven SNPs, including the promoter SNP rsl6260, that captured over 96% of CDH1 haplotype variation were selected as haplotype tagging SNPs and analyzed for associated PC risk. We observed significant confirmation of rsl6260 (P=0.003) for cases with a positive family history of PC (FH+) both in an independent case-control population and in PC families. In addition, a common haplotype (HapB, 25%) including the variant allele of rsl6260 was associated (P=0.004) with PC risk among FH+ cases. The promoter SNP rsl6260 as well as HapB were significantly transmitted to affected offspring in PC families. We report strong confirmation of the association between PC risk in FH+ cases and a functional CDH1 promoter SNP in an independent population. In conjunction with the biological importance of CDH1 our findings encourage further evaluation of genetic variation in CDH1 in relation to PC etiology. Due to the difficulties in replication of genetic association studies. this finding is unusual and novel.
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