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RAb bound, demonstrating the specificity of CLL69C -derived rAbs to
(A) Title Loaded From File Nucleotide and deduced amino acid sequence comparison on the UL10 Fab IgHV. coli extract expressing UL10 Fab; lane 3, purified UL10 Fab. (B) Binding pattern of CLL69C homologous UL10 Fab to OSE. Chemiluminescent ELISA for the binding of UL10 Fab (1 mg/ml) to each from the plated antigens (5 mg/mL) for 1.5 hour at RT. The information are expressed as RLU/100 ms and represent values from three independent experiments (imply six SD), every value determined in triplicate. doi:ten.1371/journal.pone.0065203.gPLOS A single | www.plosone.orgClonal Choice of CLL B Cells by MAA EpitopesFigure six. Binding of CLL69 Abs to OSE on apoptotic cells and atherosclerotic lesions. (A) Flow cytometry evaluation of CLL69 rAb binding to apoptotic cells. Representative flow cytometry contour plots of apoptotic murine thymocytes and histogram plots for the binding of rAb to apoptotic cells. Murine thymocytes have been induced to undergo apoptosis by incubation with ten ng/ml PMA for 16 hours. Apoptotic cells had been stained with rAbs CLL69 A , isotype VH3-21, or no major Ab, followed by detection with a FITC-conjugated anti-human IgG1 and staining with PE-labeled Annexin-V and 7-AAD to determine the viable and apoptotic cells. The contour plot (upper left panel) identifies the viable cells (Q3, PE-Annexin-V2/7-AAD2), earlyPLOS 1 | www.plosone.orgClonal Choice of CLL B Cells by MAA Epitopesapoptotic (Q4, Annexin-V+/7-AAD2) and late apoptotic cells (Q2, Annexin-V+/7-AAD+). Histogram panels Q3 (bottom left), Q4 (bottom Title Loaded From File suitable) and Q2 (upper suitable) represent rAb staining of viable cells, early apoptotic cells, and late apoptotic cells, respectively. Relative cell fluorescence of rAbs CLL69 A and IGHV3-21 manage rAb are indicted by colored lines, and staining with the secondary Ab alone is shown in grey.RAb bound, demonstrating the specificity of CLL69C -derived rAbs towards the MAA-LDL adducts (Fig. 6C).PLOS 1 | www.plosone.orgClonal Choice of CLL B Cells by MAA EpitopesFigure four. Sequence evaluation and comparison of the IgHV of CLL69C towards the very homologous UL10 Fab. (A) Nucleotide and deduced amino acid sequence comparison of your UL10 Fab IgHV. Comparison was made with all the closest CLL69C IgHV gene sequence. Dots indicate homology and dashes indicate no sequence at that position. (B) Amino acid sequence alignment of your IgHV of CLL69C with all the UL10 Fab isolated from an umbilical-cord phage-display Fab library. Complementarity determining regions (CDR) are labeled above the amino acid sequence and indicated in bold. doi:ten.1371/journal.pone.0065203.gCLL69 Abs Recognized OSE in Atherosclerotic LesionsAs we and other people [18] have shown that MAA epitopes are enriched in atherosclerotic lesions, we examined if CLL69C rAb could immunostain such lesions. CLL69C rAb strongly immunostained atherosclerotic lesions from human carotid endarterectomy(CEA) specimens (Fig. 6D) or WHHL rabbit aortas (Figure S2). In specific, the necrotic cores with the lesions had been strongly stained with CLL69C rAb, but not handle Ab (Figure S2), equivalent to observations obtained in our lab with all the MAA-specific NAb LRO4 (data not shown).Figure five. Expression and purification of active UL10 Fab Ab. (A) The UL10 Fab was developed in E. coli and purified by affinity chromatography from soluble fraction of E.
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