Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Generation of multispecific DB-VHH constructs. Trastuzumab IgG1-K409R mAb (anti-HER2) C-termini are fused to VHHs directed against EGFR, IL6R or NKG2D via a GS-linker. CS06 IgG1-F405L mAb (anti-c-MET) is fused to the same VHH molecules in the same manner. After recombinant production and purification, parental IgG-VHHs are recombined pairwise by reduction and reoxidation. The matching K409R and F405L mutations drive the generation of heterodimeric multispecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Construct, Recombinant, Purification

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Binding affinities measured for each paratope using recombinant antigens

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant, Construct

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Biolayer interferometry analysis of simultaneous antigen binding of tri- and tetraspecific DB-VHHs. (a), (b) and (c) show exemplary sensorgrams for trispecific molecules and (d) a tetraspecific DB-VHH. The first association step represents binding of the DB-VHH (200 nM) via its CS06 paratope to biotinylated c-MET immobilized to streptavidin biosensors. Second (and third for (D)) association step is performed using an IL6R, EGFR or NKG2D recombinant protein (200 nM). The last association step is performed using HER2 (200 nM). Kinetic buffer (KB) controls were applied as negative controls for each association step.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Specific cell clustering due to simultaneous binding of the DB-VHHs to three different cancer cell lines. Flow cytometry cytograms represent the fluorescence signals of the different cell populations. (a) Cells without antibody construct. Upper left gate = HCC-1954 (HER2 +++ ) cells stained with DeepRed, lower left gate = MDA-MB-468 (EGFR +++ ) cells stained with CMRA, lower right gate = EBC-1 (c-MET ++ ) cells stained with CFSE, upper right gate = HCC-1954 + EBC-1 cell doublets. Events in all three fluorescence channels (cell triplets) were marked in red. Cells were incubated in the presence of 1 nM (b) bispecific DB, (c) and (d) trispecific DB-VHHs, (e) and (f) tetraspecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Flow Cytometry, Fluorescence, Construct, Staining, Incubation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: Simultaneous interaction of the DB-VHHs with HCC-1954 and EBC-1 target cells and binding of recombinant IL6R. Flow cytometry cytograms represent the fluorescence signals of the two cell populations and bound recombinant IL6R, detected with an anti-His6 detection antibody. (a) Cells without antibody construct. Upper left gate = HCC-1954 (HER2+++) cells stained with DeepRed, lower right gate = EBC-1 (c-MET++) cells stained with CFSE, upper right gate = HCC-1954 + EBC-1 cell doublets. Events in all three fluorescence channels (HCC-1954 + EBC-1 + bound recombinant IL6R-His Tag) were marked in blue. Cells were incubated in the presence of 10 nM (b) bispecific DB, (c) and (d) trispecific DB-VHHs, (e) and (f) tetraspecific DB-VHHs.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Binding Assay, Recombinant, Flow Cytometry, Fluorescence, Construct, Staining, Incubation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet: DB-VHHs elicit potent and specific NK cell-mediated target cell killing. Tumor cells were incubated with primary effector cells (NK cells) at a 1:5 ratio in the presence of antibody constructs in different concentrations. Error bars represent standard deviation of two biological replicates. Wildtype CS06 and trastuzumab with active Fc effector functioning were used as an ADCC reference (green). (a) and (b): c-MET-positive EBC-1 target cells, (c) and (d) HER2-overexpressing SK-BR-3 target cells. (a) and (c): NK cell cytotoxicity triggered by parental antibodies. (b) and (d): DB-VHHs serve as NK cell engager and mediate tumor cell killing.

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Incubation, Construct, Standard Deviation

Journal: mAbs

Article Title: Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities

doi: 10.1080/19420862.2021.2018960

Figure Lengend Snippet:

Article Snippet: The biosensors were then rinsed in kinetics buffer (KB; PBS + 0.1% Tween-20 + 1% bovine serum albumin (BSA)) for 45 s (baseline) and the corresponding antigen was associated to the biosensor for 300 s. Different antigen concentrations (1:2 dilution rows in KB) were tested in order to identify the dynamic range of the binding kinetics and the most suitable concentration ranges were determined as follows: HER2/ErbB2 ECD-His Tag (Sino Biological, Cat: 10004- H08H) – 0.78 nM to 50 nM, c-MET ECD-strepII-His Tag (in-house production) – 0.39 nM to 25 nM, human EGFR-His Tag (ACRO Biosystems, Cat: EGR-H5222) – 1.5 nM to 100 nM, human IL6R ECD-His Tag (Sino Biological, Cat: 10398-H08H) – 0.78 nM to 50 nM, and human NKG2D-His Tag (Sino Biological, Cat: 10575-H07B) – 0.78 nM to 50 nM.

Techniques: Variant Assay, Hydrophobic Interaction Chromatography, Mutagenesis, Molecular Weight, High Performance Liquid Chromatography

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: CLCF1 binds recombinant and serum ApoE. ( A ) Recombinant CLCF1 (200 ng) and ApoE2, 3 or 4 (250 ng) were incubated alone or in combination for 16 h. The samples were subjected to immunoprecipitation (IP) with anti-CLCF1 and protein G agarose. Immunoprecipitated proteins were analyzed by WB using mAbs specific for ApoE (upper panel) or CLCF1 (lower panel). Lanes “CLC 2 ng” and “ApoE3 20 ng”, show results of recombinant proteins directly subjected to SDS-PAGE and WB blot analysis. ( B ) CLCF1 and ApoE form a complex when co-expressed in HEK-293 cells. The cell culture medium from stable transfectants expressing the indicated proteins in combination with CRLF1 or from cells transfected with empty expression vector (lane D5) was subjected to immunoaffinity chromatography with rat-anti-human ApoE and anti-rat Ig Agarose. The eluates were analysed by WB using anti-ApoE or anti-CLC mAbs (upper panels). Supernatants of the stable transfectants were analyzed for the presence of ApoE or CLCF1 by WB (lower panels). ( C and D ) CLCF1 binds mouse and human serum ApoE. ( C ) Mouse serum samples isolated from WT or ApoE −/− mice were diluted 1:20, mixed with biotinylated mouse CLCF1 and the complexes immunoprecipitated with rabbit anti-mouse ApoE and anti-rabbit Ig agarose. The eluates were analysed for ApoE or CLCF1 by WB using anti-mouse ApoE or HRP-labelled streptavidin. The last two lanes show the signals obtained with 40 ng of biotinylated mouse CLCF1 or ApoE included as control. ( D ) Human serum samples were diluted 1:20, mixed with biotinylated human CLCF1 and the complexes immunoprecipitated with rat anti-ApoE and anti-rat Ig agarose. The eluates were analyzed for ApoE or CLCF1 by WB using anti-ApoE or HRP-labelled streptavidin. The last two lanes show the signals obtained with 40 ng of biotinylated human CLCF1 or ApoE included as control. Blots’ images where cropped to show relevant areas.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: Recombinant, Incubation, Immunoprecipitation, SDS Page, Cell Culture, Expressing, Transfection, Plasmid Preparation, Chromatography, Isolation

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: AlphaLISA proximity assay shows CLCF1-ApoE complex formation that is increased by the lipid DMPC. ( A ) ApoE2, 3 or 4 were mixed with CLCF1 (10 nM) in PBS 0.1% BSA for 1 h with IgG rat anti-ApoE (3 nM) and anti-IgG rat acceptor beads (10 μg/mL). Streptavidin donor beads (40 μg/mL) were further added for 1 h and 615 nm fluorescence signal assessed. ( B ) The indicated ApoE isotypes (30 nM) were mixed at 37 °C with increasing amount of DMPC for 1 h. ApoE-DMPC mix were further incubated with CLCF1 and subjected to AlpahLISA proximity assay. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: Proximity Assay, Fluorescence, Incubation

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: ApoE3 increases CLCF1-induced CNTFR activation. ( A ) Ba/F3 transfected with CNTFRα, gp130 and LIFRβ cDNA were successively incubated with mouse biotinylated CLCF1 (1 μg/ml) alone or in the presence of ApoE3 (10 mg/ml) and PE-labelled streptavidin and fluorescence was assessed by flow cytometry. Vehicle: cells incubated with PE-streptavidin alone. ( B ) Ba/F3-CNTFR were stimulated with CLCF1 (100 ng/ml) or ApoE3 (10 mg/ml) alone or in combination for 15 min. STAT3 tyrosine phosphorylation was assessed by intracellular staining with anti-pSTAT3 mAb and flow cytometry. Vehicle: cells incubated with PE-streptavidin alone. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: Activation Assay, Transfection, Incubation, Fluorescence, Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: CLCF1 co-purifies or co-elutes with serum lipoprotein complexes. ( A ) WT mouse, ( B ) human or ( C ) ApoE −/− mouse sera were mixed with corresponding biotinylated CLCF1. The mixtures were fractionated by FPLC using a Superose 6 HR 10/ column in order to obtain 40 fractions. The fractions were analyzed for ApoE or CLCF1 by WB using anti-ApoE or HRP-labelled streptavidin. The right lanes show the signals obtained with 40 ng of ApoE or biotinylated CLCF1 included as control. Blots’ images where cropped to show relevant areas.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques:

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: Biotinylated LDL and VLDL bind CLCF1. ( A ) Interaction between CLCF1 and lipoproteins was assessed using ligand blotting assays: 0.1–0.2 μg of hCLC, or 1 μg of BSA were subjected to non-reducing SDS-PAGE. Proteins were revealed by Coomassie blue staining (left section) or electro-transferred to nitrocellulose and incubated successively with 30 μg/ml biotinylated VLDL, LDL or HDL and HRP-labeled neutravidin (middle 3 panels). Signal was revealed by chemiluminescence. Image in the far right section shows results of incubation with HRP-labeled neutravidin alone. Lane hCLC biot; 40 ng of biotinylated CLC was used as positive control for neutravidin detection. Blots’ images where cropped to show relevant areas. ( B ) The formation of LDL and VLDL complexes can be detected using an AlphaLISA proximity assay. Biotinylated hCLCF1 was mixed with LDL, HDL and VLDL covalently coupled to acceptor beads (10 μg/mL). Streptavidin donor beads (40 μg/mL) were further added for 1 h and 615 nm fluorescence signal assessed. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: SDS Page, Staining, Incubation, Labeling, Positive Control, Proximity Assay, Fluorescence

Journal: Scientific Reports

Article Title: Effect of human very low-density lipoproteins on cardiotrophin-like cytokine factor 1 (CLCF1) activity

doi: 10.1038/s41598-018-22400-y

Figure Lengend Snippet: VLDL increases CLCF1 binding to CNTFR-expressing cells. Ba/F3 transfected with CNTFRα, gp130 and LIFRβ cDNA, IMR32 neuroblastoma or 3T3-L1 pre-adipocytes were successively incubated with species-matched biotinylated CLCF1 (1 μg/ml) alone or in the presence of LDL, HDL or VLDL (all at 100 μg/ml). Cells were then incubated with PE-conjugated streptavidin and fluorescence was assessed by flow cytometry. Vehicle: cells incubated with PE-streptavidin alone. Histograms indicate the mean fluorescence intensity of 3 independent samples ± SD.

Article Snippet: Eluates were analyzed by WB using HRP-streptavidin (GE-Healthcare Life Science, Cedarlane, Burlington, ON) or rabbit anti-mouse ApoE Ab (Abcam Inc, Cedarlane) followed by HRP-labelled TrueBlot™ anti-rabbit Ab (Rockland Immunochemicals Inc).

Techniques: Binding Assay, Expressing, Transfection, Incubation, Fluorescence, Flow Cytometry

Journal: Scientific Reports

Article Title: B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

doi: 10.1038/s41598-024-67912-y

Figure Lengend Snippet: Activated Cγ1 CD63-emGFP B cells undergoing Cre-mediated recombination in vitro express emGFP. ( a ) Schematic for Cγ1 CD63-emGFP reporter strain design. Transgenic mice expressing Cre recombinase driven by transcription of the Ig γ1 constant region gene segment (Cγ1) was crossed with a silenced reporter mouse, resulting in CD63-emerald GFP expression driven by the CAG promoter. ( b ) Percentage of emGFP + Cγ1 CD63-emGFP and Cγ1 Cre control B cells following stimulation with IL-4 or LPS + IL-4 for 3 days. Gates indicate GFP low/- B cells, GFP + B cells and GFP + B220 low/− B cells. ( c ) Percentage of cell surface expression of CD69, PNA and CD138 in GFP low/− , GFP + and GFP + B220 low/− B cells at day 3, for the Cγ1 CD63-emGFP reporter mice in panel ( b ). Gates were set on negative control samples (dotted black histograms). ( d ) Frequencies of IgG1 + B cells in B cells gated on GFP expression at days 3 and 7, for the reporter mice in ( b ). ( e ) Concentration of IgG1 in the culture medium of B cells from the reporter mice in ( b ). All data are expressed as mean ± SEM. Results shown are representative of at least three independent experiments. P = **0.01 and ***0.001, with unpaired, two-tailed t -test.

Article Snippet: Cγ1 CD63-emGFP B6 mice were subcutaneously immunized with influenza A virus PR8/34 recombinant HA protein (10 μg/mouse, Sino Biological, Wayne, PA) in the adjuvant Addavax on days 0, 14, and 21.

Techniques: In Vitro, Transgenic Assay, Expressing, Control, Negative Control, Concentration Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

doi: 10.1038/s41598-024-67912-y

Figure Lengend Snippet: In vivo generation of Cγ1 CD63-emGFP GC B cells and progeny IgG1 + memory B cells and plasma cells express emGFP after immunization with NP-KLH. ( a ) Flow cytometry gating strategy of GC B cells, IgG1 + SWM B cells, and plasma cells that express emGFP in the spleens of Cγ1 Cre and Cγ1 CD63-emGFP mice immunized with NP-KLH for 14 days. ( b ) Percentages of emGFP + cells within each gated B cell subset are shown. ( c ) Numbers of emGFP + B cells in spleens for the reporter mice shown in panel ( b ). ( d ) Immunofluorescence confocal microscopy of emGFP + B cells in the lymph nodes of mice immunized with NP-KLH for 14 days. ( e ) Serum total and NP-specific IgG1 antibody levels in naïve Cγ1 Cre and Cγ1 CD63-emGFP mice and after immunization with NP-KLH for 14 days. Data are expressed as mean ± SEM. Results shown in panels ( a – c ) and ( e ) are representative of three independent experiments and in panel d from > 20 images of two independent experiments. P = *0.05, **0.01, ***0.001 and ****0.0001, with unpaired, two-tailed t -test.

Article Snippet: Cγ1 CD63-emGFP B6 mice were subcutaneously immunized with influenza A virus PR8/34 recombinant HA protein (10 μg/mouse, Sino Biological, Wayne, PA) in the adjuvant Addavax on days 0, 14, and 21.

Techniques: In Vivo, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Two Tailed Test

Journal: Scientific Reports

Article Title: B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

doi: 10.1038/s41598-024-67912-y

Figure Lengend Snippet: Activated Cγ1 CD63-emGFP B cells undergoing Cre-mediated recombination in vitro produce EVs that express emGFP, tetraspanins, and IgG. ( a ) Nanoparticle tracking analysis of total and GFP + EVs isolated from cell culture medium from Cγ1 CD63-emGFP B cells stimulated with LPS + IL-4 for 7 days. ( b ) EVs were visualized by cryoelectron microscopy. Images are representative of three independent EV preparations. ( c , d ) Lysates from EVs isolated from cell culture medium from Cγ1 CD63-emGFP B cells stimulated with IL-4, LPS, and LPS + IL-4 for 7 days were probed by Western blot for the presence of CD9, CD81, CD63, GFP, and IgG under non-reducing conditions, and IgG under reducing conditions (10 μg/lane). Under reducing conditions, IgG bands from EV samples 1 and 2 are juxtaposed from the same blot as indicated by vertical line, from full length blots shown in Supplementary Fig. b. Results show two EV preparations and are representative of at least six independent experiments. ( e ) ImageStream analysis of individual emGFP + EVs isolated from the cell culture medium of Cγ1 CD63-emGFP B cells stimulated with LPS + IL-4 and stained for surface IgG or CD16/32 (Alexa Fluor 647), CD64 (PE), and binding of exogenous mouse IgG (DyLight 405). ( f ) ImageStream analysis of peritoneal B cells stained for surface B220 (FITC) and binding of exogenous mouse IgG. ( g ) ImageStream analysis of peritoneal macrophages stained for surface CD11b (FITC) and binding of exogenous mouse IgG in the absence (left panel) or presence of 2.4G2 Fc block (right panel). The results shown are representative of three independent experiments.

Article Snippet: Cγ1 CD63-emGFP B6 mice were subcutaneously immunized with influenza A virus PR8/34 recombinant HA protein (10 μg/mouse, Sino Biological, Wayne, PA) in the adjuvant Addavax on days 0, 14, and 21.

Techniques: In Vitro, Isolation, Cell Culture, Cryo-Electron Microscopy, Western Blot, Staining, Binding Assay, Blocking Assay

Journal: Scientific Reports

Article Title: B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

doi: 10.1038/s41598-024-67912-y

Figure Lengend Snippet: Circulating EVs produced from Cγ1 CD63-emGFP B cells bind specific antigen and express GFP and EV tetraspanin markers after immunization with NP-KLH. ( a ) ELISA measurements of NP-specific total (NP32-BSA) and high-affinity (NP4-BSA) IgG antibody levels in whole serum (left two panels) and EVs isolated from serum (right two panels) from Cγ1 CD63-emGFP mice unimmunized or immunized with NP-KLH for 14 days. Sample dilutions shown are 1:1000–1:30,000 for serum and 3.3 × 10 7 –3.7 × 10 6 particles/ml for EVs. ( b ) ELISA measurements for the presence of both CD63 and CD9 and GFP from NP-specific EVs, for the reporter mice in panel ( a ). Data are expressed as mean ± SEM. The results shown are representative of three independent experiments.

Article Snippet: Cγ1 CD63-emGFP B6 mice were subcutaneously immunized with influenza A virus PR8/34 recombinant HA protein (10 μg/mouse, Sino Biological, Wayne, PA) in the adjuvant Addavax on days 0, 14, and 21.

Techniques: Produced, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Scientific Reports

Article Title: B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

doi: 10.1038/s41598-024-67912-y

Figure Lengend Snippet: HA-specific EVs neutralize influenza infection. ( a ) Experimental strategy for testing HA-specific EVs in influenza infection in vivo. ( b ) Four groups of mice ( n = 6 mice per group) were intranasally infected with PR8 virus only (Vehicle), PR8 virus mixed with high dose of EVs isolated from serum from Cγ1 CD63-emGFP mice immunized with influenza A hemagglutinin (HA) protein for 21 days (HA-high), PR8 virus mixed with low dose of EVs from the same mice immunized with HA (HA-low), or PR8 virus mixed with high dose of EVs isolated from serum from Cγ1 CD63-emGFP mice immunized with NP-KLH for 21 days. Host survival and body weight change following PR8 challenge were monitored. Data are expressed as mean ± SEM. The results shown are representative of two independent experiments. P = **0.001 between the HA-high and HA-low body weight change, calculated by unpaired, two-tailed t -test. P = * < 0.05 for survival in HA-high and HA-low groups as compared with NP-high and Vehicle groups, calculated by log-rank test. ( c ) Analysis of EV samples from serum from Cγ1 CD63-emGFP mice immunized with HA protein by size exclusion chromatography collected in 0.5 ml fractions. The number of particles per fraction was measured by nanoparticle tracking analysis and the amount of soluble IgG per fraction was measured by ELISA. Results shown are representative of two independent experiments.

Article Snippet: Cγ1 CD63-emGFP B6 mice were subcutaneously immunized with influenza A virus PR8/34 recombinant HA protein (10 μg/mouse, Sino Biological, Wayne, PA) in the adjuvant Addavax on days 0, 14, and 21.

Techniques: Infection, In Vivo, Virus, Isolation, Two Tailed Test, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: B cells secrete functional antigen-specific IgG antibodies on extracellular vesicles

doi: 10.1038/s41598-024-67912-y

Figure Lengend Snippet: EVs derived from spontaneous germinal center B cell responses in Cγ1 CD63-emGFP Nba2 lupus-prone mice express surface IgG with self-reactivity. ( a ) Schematic for generating the Cγ1 CD63-emGFP reporter strain on the Nba2 lupus-prone congenic background. ( b ) Flow cytometry gating strategy of GC B cells, IgG1 + SWM B cells, and plasma cells that express emGFP in the spleens of 7-mo-old Cγ1 Cre Nba2 and Cγ1 CD63-emGFP Nba2 mice. ( c ) Percentages of emGFP + cells within each gated B cell subset are shown ( n = 9 mice per group). ( d ) Numbers of emGFP + B cells in spleens for the reporter mice shown in panel ( c ). ( e ) Immunofluorescence confocal microscopy of emGFP + B cells in the spleens of 7-mo-old Cγ1 CD63-emGFP Nba2 mice. ( f ) ELISA measurements of antinuclear IgG antibody levels in whole serum and EVs isolated from serum from Cγ1 CD63-emGFP Nba2 mice and Cγ1 CD63-emGFP B6 controls, for the reporter mice in panel ( c ). Sample dilutions shown are 1:500–1:3000 for serum and 6.6 × 10 8 –7.4 × 10 7 particles/ml for EVs. ( g , h ) ELISA measurements for the presence of antinuclear binding EVs that express either CD9 and CD81, CD63 and CD9, or CD63 and CD81, and emGFP from EVs, for the reporter mice in panel ( c ). Data are expressed as mean ± SEM. The results shown in panels ( b – d ) and ( f – h ) are representative of three independent experiments, and in panel e from two independent experiments. P = ***0.001 and ****0.0001, with unpaired, two-tailed t -test.

Article Snippet: Cγ1 CD63-emGFP B6 mice were subcutaneously immunized with influenza A virus PR8/34 recombinant HA protein (10 μg/mouse, Sino Biological, Wayne, PA) in the adjuvant Addavax on days 0, 14, and 21.

Techniques: Derivative Assay, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Isolation, Binding Assay, Two Tailed Test

Journal: Gut Microbes

Article Title: Microbial metabolite Enterobactin impairs mitochondrial respiration and alleviates colitis

doi: 10.1080/19490976.2026.2662638

Figure Lengend Snippet: Monomeric form of Ent can disseminate into systemic circulation. Serum was collected from male germ-free (GF) rats and GF rats co-housed (GFC) with conventional mice for 10 d (8 weeks old, n = 5–6) at Taconic Facility. Serum metabolites were extracted using standard procedures for targeted metabolomics profiling. The extracted samples were analyzed using HPLC coupled to Agilent 6495 QQQ mass spectrometry (LC–MS). Ent was quantified as its monomeric form 2, 3-dihydroxy benzoic acid (2, 3-DHBA). The data were normalized with internal standards and log2-transformed on a per-sample basis. (A) Ent spectra (B) Fold change in serum 2, 3-DHBA. Note: Trace amounts of 2, 3-DHBA in GF animal sera are likely to be from dead bacteria in the diet. (C) Fecal Lcn2. Results are expressed as mean ± SEM. *** p < 0.001.

Article Snippet: Mouse recombinant Lcn2 was purchased from Sino Biological Inc. (50060-MNAH).

Techniques: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transformation Assay, Bacteria

Journal: Gut Microbes

Article Title: Microbial metabolite Enterobactin impairs mitochondrial respiration and alleviates colitis

doi: 10.1080/19490976.2026.2662638

Figure Lengend Snippet: Ent exacerbates mitochondrial dysfunction in Lcn2 −/− BMDMs and is rescued by rec-Lcn2. BMDMs were isolated from wild-type (WT) and Lcn2-deficient (Lcn2 −/− ) mice to assess mitochondrial function using a Seahorse XF Analyzer. (A and B) Oxygen consumption rate (OCR) was measured (C) BMDMs were treated with Ent alone or in combination with ferric iron (1:1 ratio) for 24 h to assess the impact on mitochondrial respiration. (D) Quantification of key mitochondrial parameters revealed that Ent treatment significantly reduced basal respiration, ATP production, maximal respiration, proton leak, and spare respiratory capacity in Lcn2KO BMDMs (E) Mito-respiration was measured with Ent, rec-Lcn2 or both. (F) Cotreatment with rec-Lcn2 on basal respiration, maximal respiration, ATP production, and spare respiratory capacity indicating OCR. (G) Iron-chelating activity was assessed in the supernatants using the chrome azurol S (CAS) liquid assay. Control reactions included Ent alone, and rec-Lcn2 alone. (H) Mitochondrial reactive oxygen species (ROS) were measured using MitoSOX™ Red staining followed by flow cytometry analysis. Data represent mean ± SEM. Statistical significance was determined using unpaired Student’s t -test or one-way ANOVA where appropriate (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: Mouse recombinant Lcn2 was purchased from Sino Biological Inc. (50060-MNAH).

Techniques: Isolation, Activity Assay, Control, Staining, Flow Cytometry

Journal: Gut Microbes

Article Title: Microbial metabolite Enterobactin impairs mitochondrial respiration and alleviates colitis

doi: 10.1080/19490976.2026.2662638

Figure Lengend Snippet: Ent impairs mitochondrial respiration in model intestinal epithelia. Caco-2, intestinal epithelial cells (IEC), were treated with iron-free Ent (25 μM) or iron-bound Ent-Fe³⁺ (25 μM; 1:1 ratio) or Fe +3 alone and assessed mitochondrial function. (A) Mitochondrial respiration was evaluated using the Seahorse XF Analyzer by measuring oxygen consumption rate (OCR). (B) Basal and maximal respiration, ATP-linked respiration, proton leak and spare respiratory capacity (C) Mito-respiration was measured with Ent, rec-Lcn2, or both. (D) IEC were treated with 25 µM Ent for 24 h and total cellular proteins were analyzed by immunoblotting using antibodies against key subunits of the mitochondrial oxidative phosphorylation (OXPHOS) complexes I–V of electron transport chain. Densitometry scanning of complex II (E) Immunoblots of Hif1α, p -AMPK, total AMPK, and loading control β-actin (F and G) IEC were treated with 25 µM enterobactin for 24 h, and mitochondrial ROS levels were assessed using MitoSOX™ Red dye followed by flow cytometry analysis. Data represent mean ± SEM from at least three independent experiments. Statistical significance was determined using one-way ANOVA where appropriate (* p < 0.05 and ** p < 0.01).

Article Snippet: Mouse recombinant Lcn2 was purchased from Sino Biological Inc. (50060-MNAH).

Techniques: Western Blot, Phospho-proteomics, Control, Flow Cytometry

Journal: Gut Microbes

Article Title: Microbial metabolite Enterobactin impairs mitochondrial respiration and alleviates colitis

doi: 10.1080/19490976.2026.2662638

Figure Lengend Snippet: Therapeutic oral administration of 2, 3-DHBA is effective in protecting against DSS-induced pathology than 2, 5-DHBA. Eight-weeks-old male C57BL/6J mice (WT, n = 3–5) were administered with 2% DSS in drinking water for 7 d, confirmed that mice were rectally bleeding. DSS water was replaced with regular water. Control mice were given regular water. After the cessation of DSS (i.e., during recovery phase), mice were administered with either 2, 3-dihydroxybenzoic acid (2, 3-DHBA) or 2, 5-dihydroxybenzoic acid (2, 5-DHBA: 500 µg/mouse, orally) or PBS for 7 d. Feces and blood were collected for the isolation of hemolysis-free serum. Intestinal barrier function was assessed via FITC-dextran administered 4 h before euthanasia. (A) Schematic representation of the experiment (B) Percent body weight loss/gain; (C) Gross colon images (D) Colon length (E) % Spleen weight (F) % Cecum weight (G) Serum fluorescence (H) Serum Lcn2 (I) Fecal Lcn2 (J) Serum KC (K) Colonic MPO. Data are presented as mean ± SEM from at least three independent experiments or biological replicates. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Mouse recombinant Lcn2 was purchased from Sino Biological Inc. (50060-MNAH).

Techniques: Control, Isolation, Fluorescence

Journal: Journal of innate immunity

Article Title: The Olfactomedin-4-Defined Human Neutrophil Subsets Differ in Proteomic Profile in Healthy Individuals and Patients with Septic Shock.

doi: 10.1159/000527649

Figure Lengend Snippet: Fig. 2. Differential proteomic profiles of Olfm4-H and Olfm4-L neutrophil subsets in healthy blood donors. Olfm4-H and Olfm4- L neutrophils isolated from healthy blood donors (n = 8) were sort- ed by FACS and analysed by LC-MS/MS. a Volcano plot showing median log2 abundance ratios between the identified proteins in Olfm4-H and Olfm4-L neutrophils and their p values. b Left box- and-whisker plot showing proteins with a statistically significant positive median log2 abundance ratio, indicating increased abun- dance in the Olfm4-H neutrophils. Right box-and-whisker plot showing proteins with a statistically significant negative median log2 abundance ratio, indicating increased abundance in the Olfm4-L neutrophils. c Neutrophils from healthy blood donors (n = 7) were isolated and stained for proteins of interest and analysed by live imaging flow cytometry. Box-and-whisker plot comparing median fluorescence intensity of the immunofluorescently stained proteins Rab3d, S100A7, Rab3a, and CXCR1 between Olfm4-H and Olfm4-L neutrophils. Wilcoxon signed rank tests were used to test for statistical significance. Common contaminants and his- tones are not shown.

Article Snippet: To quantify proteins of interest as identified by proteomics, AlexaFluor D ow nloaded from http://karger.com /jin/article-pdf/15/1/351/4124694/000527649.pdf by guest on 13 January 2025 Lundquist/Andersson/Chew/Das/ Turkina/Welin J Innate Immun 2023;15:351–364354 DOI: 10.1159/000527649 488-conjugated mouse anti-human Rab3d (sc-398727, Santa Cruz Biotechnology, Dallas, TX, USA) at 10 μg/mL, AlexaFluor 488-conjugated mouse anti-human S100A7 (NBP2-24911AF488, Novus Biologicals, Abingdon UK) at 31.5 μg/mL, AlexaFluor 488-conjugated mouse anti-human Rab3a (sc-365069, Santa Cruz Biotechnology) at 10 μg/mL or fluorescein isothiocyanate-conjugated mouse anti-human CXCR1 (320605, BioLegend) at 5 μg/mL were included in the primary antibody step.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Whisker Assay, Staining, Imaging, Flow Cytometry, Fluorescence

Journal: Immunity

Article Title: PD-L1:CD80 Cis -Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

doi: 10.1016/j.immuni.2019.11.003

Figure Lengend Snippet: (A) A Jurkat-Raji co-culture assay analyzing how PD-L1 interferes with CTLA-4-mediated CD80 depletion. Cartoons on the left depict the co-cultured cells. On the immediate right are representative flow-cytometry histograms of CD80 expression (anti-CD80 allophycocyanin) on Raji cells before (0 h) and after co-culture (0.5 h). Further on the right are representative confocal images for the Jurkat-Raji conjugate (scale bars, 10 μm). Rightmost is a bar graph showing CD80 MFI of Raji at 0.5 h, normalized to CD80 MFI at 0 h (mean ± SEM, n = 4).

Article Snippet: This home-made SNAP–CTLA-4–His 6 and human CD80–His, purchased from Sino Biological, were subjected to gel filtration chromatography, and monomeric fractions of each collected and used in the SPR assays.

Techniques: Co-culture Assay, Cell Culture, Flow Cytometry, Expressing, Co-Culture Assay

Journal: Immunity

Article Title: PD-L1:CD80 Cis -Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways

doi: 10.1016/j.immuni.2019.11.003

Figure Lengend Snippet: (A) Representative flow-cytometry histograms of CTLA-4-huFc staining of the indicated types of Raji cells. Bound CTLA-4-huFc was labeled by AF647 anti-human IgG Fc, the MFI of which was plotted against (CTLA-4-huFc). Shown in gray are Raji (CD80+CD86−) cells stained by isolated huFc domain. Means ± SEM, n ≥ 3.

Article Snippet: This home-made SNAP–CTLA-4–His 6 and human CD80–His, purchased from Sino Biological, were subjected to gel filtration chromatography, and monomeric fractions of each collected and used in the SPR assays.

Techniques: Flow Cytometry, Staining, Labeling, Isolation

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: One microgram per milliliter of each human (FGFR3-IIIb and -IIIc) (R&D systems, 1264-FR-050, Minneapolis, MN, USA), mouse (R&D systems, 710-MF-050, Minneapolis, MN, USA), and cynomolgus FGFR3 (Sino Biological, 90313-C02H, Beijing, China) protein was coated on 96-well EIA/RIA plates (Costar, #3590, Corning, NY, USA) at 4 °C for over-night, respectively.

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: One microgram per milliliter of each human (FGFR3-IIIb and -IIIc) (R&D systems, 1264-FR-050, Minneapolis, MN, USA), mouse (R&D systems, 710-MF-050, Minneapolis, MN, USA), and cynomolgus FGFR3 (Sino Biological, 90313-C02H, Beijing, China) protein was coated on 96-well EIA/RIA plates (Costar, #3590, Corning, NY, USA) at 4 °C for over-night, respectively.

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: One microgram per milliliter of each human (FGFR3-IIIb and -IIIc) (R&D systems, 1264-FR-050, Minneapolis, MN, USA), mouse (R&D systems, 710-MF-050, Minneapolis, MN, USA), and cynomolgus FGFR3 (Sino Biological, 90313-C02H, Beijing, China) protein was coated on 96-well EIA/RIA plates (Costar, #3590, Corning, NY, USA) at 4 °C for over-night, respectively.

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: One microgram per milliliter of each human (FGFR3-IIIb and -IIIc) (R&D systems, 1264-FR-050, Minneapolis, MN, USA), mouse (R&D systems, 710-MF-050, Minneapolis, MN, USA), and cynomolgus FGFR3 (Sino Biological, 90313-C02H, Beijing, China) protein was coated on 96-well EIA/RIA plates (Costar, #3590, Corning, NY, USA) at 4 °C for over-night, respectively.

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: One microgram per milliliter of each human (FGFR3-IIIb and -IIIc) (R&D systems, 1264-FR-050, Minneapolis, MN, USA), mouse (R&D systems, 710-MF-050, Minneapolis, MN, USA), and cynomolgus FGFR3 (Sino Biological, 90313-C02H, Beijing, China) protein was coated on 96-well EIA/RIA plates (Costar, #3590, Corning, NY, USA) at 4 °C for over-night, respectively.

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: One microgram per milliliter of each human (FGFR3-IIIb and -IIIc) (R&D systems, 1264-FR-050, Minneapolis, MN, USA), mouse (R&D systems, 710-MF-050, Minneapolis, MN, USA), and cynomolgus FGFR3 (Sino Biological, 90313-C02H, Beijing, China) protein was coated on 96-well EIA/RIA plates (Costar, #3590, Corning, NY, USA) at 4 °C for over-night, respectively.

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