fgfr3 cd333  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

doi: 10.3390/ijms22126240

Figure Lengend Snippet: ( A ) Following biotin-X-DHPE labeling on FGFR3-overexpressing cells (PDC #1), FGFR3 expression levels on the cell surface were analyzed by FACS analysis. Biotin-X-DHPE was detected using Streptavidin-FITC, and FGFR3 was detected using PE-direct conjugated antibody. ( B ) The optimal reaction temperature and time for labeling the living cells with biotin-X-DHPE were analyzed by FACS analysis. ( C ) Optimal reaction buffer analysis for attaching magnetic beads on cells. ( D ) The capture yield of live cells attached with magnetic beads was compared using both direct and indirect method through cell counting and microscopic observation. *** p < 0.001 paired T test. ( E ) Microscopic observation of live cells attached with magnetic beads (magnification 400×).

Article Snippet: After washing with 1% FBS (pH 7.4, PBS), the cells were stained with human FGFR3 PE-conjugated antibody (R&D systems, FAB766P, Minneapolis, MN, USA) and the FGFR3 expression level on the cell surface was analyzed by FACS analysis.

Techniques: Labeling, Expressing, Magnetic Beads, Cell Counting

Journal: International Journal of Molecular Sciences

Article Title: Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

doi: 10.3390/ijms22126240

Figure Lengend Snippet: ( A ) Phage pool binding to FGFR3-overexpressing cells in a flow cytometer; comparison of cell surface binding between amplified phage pools obtained using conventional bio-panning (blue histogram) only or with the introduction of cell panning (red histogram). Comparison of CDR-H3 sequence similarity of isolated antibodies through cell panning introduction. ( B ) Phylogenetic tree of similar sequences built on the basis of CDR-H3 (Kabat numbering) using CLUSTAL W multiple sequence alignment programs. Each phylogenetic tree was analyzed for 13 clones selected through conventional bio-panning only and 6 clones selected through introduction of cell panning. The A1D06, S2D05, S3A06, and S3B09 clones were grouped into “Clade A”. ( C ) Comparison of CDR-H3 loop structure alignment using POSA analysis (interactive multiple protein structure alignment). The CDR-H3 of the A1D06 (yellow), S2D05 (green), S3A06 (cyan), and S3B09 (magenta) clones of Clade A were aligned based on the VH region in the Fv modeling annotation. CDR-H3 loop structure and length are different for each clone (gray, framework).

Article Snippet: After washing with 1% FBS (pH 7.4, PBS), the cells were stained with human FGFR3 PE-conjugated antibody (R&D systems, FAB766P, Minneapolis, MN, USA) and the FGFR3 expression level on the cell surface was analyzed by FACS analysis.

Techniques: Binding Assay, Flow Cytometry, Comparison, Amplification, Sequencing, Isolation, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

doi: 10.3390/ijms22126240

Figure Lengend Snippet: Physicochemical property analysis of anti-FGFR3 antibodies derived from cell panning. ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified antibodies in non-reducing and reducing conditions. In the non-reducing condition, 150 kDa band indicates the whole IgG, and in the reducing condition, 25 kDa and 50 kDa indicate the light chain and heavy chain of the antibody, respectively. ( B ) The purity of the anti-FGFR3 antibodies were analyzed using size exclusion-high performance liquid chromatography (SEC-HPLC). All anti-FGFR3 antibodies showed more than 95% purity of monomer.

Article Snippet: After washing with 1% FBS (pH 7.4, PBS), the cells were stained with human FGFR3 PE-conjugated antibody (R&D systems, FAB766P, Minneapolis, MN, USA) and the FGFR3 expression level on the cell surface was analyzed by FACS analysis.

Techniques: Derivative Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, High Performance Liquid Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

doi: 10.3390/ijms22126240

Figure Lengend Snippet: Binding characterization analysis of anti-FGFR3 antibody derived following the introduction of semi-automated bio-panning. ( A ) The binding specificity of selected FGFR3 antibodies to FGFR3 isoforms and interspecies was evaluated using ELISA. ( B ) The binding ability of FGFR3 antibodies to FGFR3-overexpressing cells (PDC#1) and FGFR3 negative cells (PDC#2) was determined using flow cytometry.

Article Snippet: After washing with 1% FBS (pH 7.4, PBS), the cells were stained with human FGFR3 PE-conjugated antibody (R&D systems, FAB766P, Minneapolis, MN, USA) and the FGFR3 expression level on the cell surface was analyzed by FACS analysis.

Techniques: Binding Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

doi: 10.3390/ijms22126240

Figure Lengend Snippet: Binding affinities (KD) of selected clones to human FGFR3 isotypes and interspecies FGFR3.

Article Snippet: After washing with 1% FBS (pH 7.4, PBS), the cells were stained with human FGFR3 PE-conjugated antibody (R&D systems, FAB766P, Minneapolis, MN, USA) and the FGFR3 expression level on the cell surface was analyzed by FACS analysis.

Techniques: Binding Assay, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

doi: 10.3390/ijms22126240

Figure Lengend Snippet: Biological functional assay of anti-FGFR3 antibodies. Inhibitory effect of clone S3B09 (Clade A) and S3C04 selected from conventional bio-panning on proliferation of FGFR3-overexpressing cells (PDC #1) was evaluated. Cells were cultured in the presence of 10 ng/mL FGF1 plus 10 μg/mL heparin sulfate or with S3B09 or S3C04 antibodies. Relative cell growth was evaluated through ( A ) cell viability after 96 h incubation with antibodies. Data represent mean ± SD; ***, p < 0.001 using one-way ANOVA. ( B ) Cell surface FGFR3 degradation assay. Individual antibodies were treated on FGFR3-overexpressing cells (PDC #1) and incubated for 1 h at 37 °C, and FGFR3 expression on the cell surface was detected through FACS analysis. ( C ) Total FGFR3 degradation assay. After the FGFR3-overexpressing cells (PDC #1) were treated with the antibody, total FGFR3 contained in the cell lysate was detected through sandwich ELISA. Data represent mean ± SD; *, p < 0.03 using one-way ANOVA.

Article Snippet: After washing with 1% FBS (pH 7.4, PBS), the cells were stained with human FGFR3 PE-conjugated antibody (R&D systems, FAB766P, Minneapolis, MN, USA) and the FGFR3 expression level on the cell surface was analyzed by FACS analysis.

Techniques: Functional Assay, Cell Culture, Incubation, Degradation Assay, Expressing, Sandwich ELISA