2a11  (Bio-Rad)

Journal: The Journal of Biological Chemistry

Article Title: Epitope-specific antibody fragments block aggregation of AGelD187N, an aberrant peptide in gelsolin amyloidosis

doi: 10.1016/j.jbc.2024.107507

Figure Lengend Snippet: Schematic overview of the phage display strategy leading to Fabs 1 to 23. A , the initial scFv phage pool was panned separately against all antigens until significant enrichment was observed, as determined by the number of colonies on output plates and phage immunoreactivity. B , VL-VH regions of selected phage pools were extracted and subcloned into an expression vector for soluble scFv. Single scFv clones were expressed in E. coli , screened, and characterized. C , selected clones were converted to Fabs, expressed in CHO cells, and characterized. D , alternatively, the VL and VH regions from selected scFv outputs were extracted as pools and recombined with human CL and CH1 domains to randomly generate VL/VH-shuffled Fab genes. These genes were subcloned back into the phagemid. Three rounds of panning were performed to select high-affinity binders. E , variable regions of selected phage pools were extracted and subcloned into an expression vector for expression in E. coli and screening of soluble, monoclonal Fabs. F , selected clones were expressed in CHO cells and characterized. CHO, Chinese hamster ovary; Fab, antigen-binding fragment; scFv, single-chain variable fragment; VH, antibody heavy chain variable; VL, antibody light chain variable domain.

Article Snippet: Fabs were processed as described above for scFv, with the exception that Fabs were cloned into the pAK400 screening vector and detected with Eu-labeled anti-human IgG CH1 antibody 2A11 (Hytest).

Techniques: Expressing, Plasmid Preparation, Clone Assay, Binding Assay

Journal: PLoS ONE

Article Title: Candida albicans Induces Selective Development of Macrophages and Monocyte Derived Dendritic Cells by a TLR2 Dependent Signalling

doi: 10.1371/journal.pone.0024761

Figure Lengend Snippet: (A) Fluorescence mean intensity of TLR2, TLR4 and Dectin-1 staining with the specific monoclonal antibodies (number on the right) and their respective isotype control (number on the left) measured by flow cytometry, in Lin − progenitor cells. (B) Identification of SP population. Hoeschst Red and Hoeschst Blue parameters were examined allowing the SP population to be identified and gated; the percentage of SP cells measured in the total nucleated viable cells is indicated. The SP cells were then displayed for their Sca-1 versus c-Kit plot: the gate indicates the percentage of double positive population. (C) Fluorescence mean intensity of TLR2, TLR4 and Dectin-1 staining with the specific monoclonal antibodies (number on the right) and their respective isotype control (number on the left) measured by flow cytometry, in SP gated cells. The results shown represent the data from one representative experiment of two.

Article Snippet: FITC-labeled anti-Dectin-1 (clone 2A11) monoclonal was purchased from AbD Serotec (Oxford, UK).

Techniques: Fluorescence, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Candida albicans Induces Selective Development of Macrophages and Monocyte Derived Dendritic Cells by a TLR2 Dependent Signalling

doi: 10.1371/journal.pone.0024761

Figure Lengend Snippet: Lin − progenitor cells (400,000 cells/well in 0.5 ml) from C57BL/6, MyD88 −/− , TLR2 −/− , TLR4 −/− and Dectin-1 −/− mice were cultured in the presence of SCF, FL and IL-7 with medium alone (Medium), Pam 2 CSK 4 (0.125 µg/ml), Curdlan (100 µg/ml) or inactivated yeasts of C. albicans ATCC 26555 [120 µg (dry weight) of cells/ml] for 7 days. (A) and (C) Cells were labelled with anti-CD11b and anti-CD11c antibodies and analyzed by flow cytometry. Cells were gated as population A (CD11b − CD11c + , violet gate), population B (CD11b + CD11c + , green gate), population C (CD11b + CD11c − , red gate) and population D (CD11b high CD11c + , blue gate). (B) Expression of mPDCA-1, Ly6C, CD8α, CD4 and F4/80 were analyzed in the gated populations; the percentage of positive cells is indicated. (D) The expression of c-Kit was analyzed by flow cytometry and indicated as % of positive cells, whereas the total number of cells was determined microscopically. (E) Expression of F4/80 was analyzed in CD11b high CD11c + and CD11b high CD11c low populations (blue gates). All cell populations analyzed are F4/80 positive but differ in the mean fluorescence intensity, as indicated Data represent means ± SD, from one representative experiment of three. * P <0.05, ** P <0.01 with respect to cells incubated with medium alone (Medium).

Article Snippet: FITC-labeled anti-Dectin-1 (clone 2A11) monoclonal was purchased from AbD Serotec (Oxford, UK).

Techniques: Cell Culture, Flow Cytometry, Expressing, Fluorescence, Incubation