fcγr‐specific mabs  (STEMCELL Technologies Inc)

Journal: EMBO Molecular Medicine

Article Title: Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel

doi: 10.15252/emmm.202114182

Figure Lengend Snippet: BW5147 reporter cells stably expressing human FcγR‐ζ chain chimeras or BW5147 parental cells (grey/dashed) were stained with FcγR specific conjugated mAbs as indicated and measured for surface expression of FcγRs via flow cytometry. Bar graphs show means of three independent experiments performed in technical duplicates. Error bars = SD. FCS coating of an ELISA microtiter plate allows for suspension of subsequently added IgG. Plate bound IgG was quantified via ELISA. Bar graph shows means of one experiment performed in technical triplicates. Error bars = SD. Immobilized IC, immobilized IgG and IgG opsonized cells represent qualitatively similar ligands for FcγRs. Response curves of human FcγRs activated by opsonized cells (293T cells stably expressing CD20 + Rituximab [Rtx]), immobilized IC (rec. soluble CD20 + Rtx) and immobilized IgG (Rtx). sICs formed using monovalent antigen (rec. soluble CD20 + Rtx) do not activate human FcγRs. X‐Axis shows sample concentration determined by antibody molarity. Y‐Axis shows FcγR activation determined by reporter cell mouse IL‐2 production (OD 450 nm). Two independent experiments performed in technical duplicates. Error bars = SD. Schematic of used assay setups. BW5147 reporter cells expressing chimeric human FcγR receptors express endogenous CD69 or secrete mouse IL‐2 in response to FcγR activation by clustered IgG. sICs are generated using mAbs and multivalent antigens. sIC suspension requires pre‐blocking of an ELISA plate using PBS supplemented with 10% FCS (FCS coat, grey‐dashed).

Article Snippet: FcγR‐specific mAbs were obtained from Stem Cell technologies (CD16: clone 3G8; CD32: IV.3).

Techniques: Stable Transfection, Expressing, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Concentration Assay, Activation Assay, Generated, Blocking Assay

Journal: EMBO Molecular Medicine

Article Title: Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel

doi: 10.15252/emmm.202114182

Figure Lengend Snippet: Ultra‐pure antigen (Ag, TNF‐α) mixed with therapy‐grade mAb (infliximab, Ifx) was used to generate sICs. X‐Axis: concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC (expressed as mAb molarity) at a mAb:Ag ratio of 1:2. Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs. Immobilized IgG (Rtx) or immobilized FcγR‐specific mAbs served as positive controls. Two independent experiments performed in technical duplicates. Error bars = SD. Error bars smaller than symbols are not shown. Left panel: IL‐2 quantification 16 h after reporter cell activation. Background (blank) was subtracted (dashed line). IL‐2 was measured via anti‐IL‐2 ELISA (A 450nm ) and IL‐2 concentrations were calculated from an IL‐2 standard. Right panel: Reporter cell CD69 expression 4 h post trigger was measured using flow cytometry. Mean florescence intensity (MFI) were normalized to untreated cells (ctrl.) and are presented as fold‐change increase.

Article Snippet: FcγR‐specific mAbs were obtained from Stem Cell technologies (CD16: clone 3G8; CD32: IV.3).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: Detection and functional resolution of soluble immune complexes by an FcγR reporter cell panel

doi: 10.15252/emmm.202114182

Figure Lengend Snippet: Two different multivalent ultra‐pure antigens (Ag) mixed with respective therapy‐grade mAbs were used to generate sICs as indicated for each set of graphs (top to bottom). IC pairs: mepolizumab (Mpz) and rhIL‐5; bevacizumab (Bvz) and rhVEGFA. X‐Axis: concentrations of stimulant expressed as molarity of either mAb or Ag monomer and IC (expressed as mAb molarity) at a mAb:Ag ratio of 1:2. Soluble antigen or soluble antibody alone served as negative controls and were not sufficient to activate human FcγRs. FcγR responses were normalized to immobilized rituximab (Rtx) at 1 µg/well (set to 1) and a medium control (set to 0). Two independent experiments performed in technical duplicates. Error bars = SD. Error bars smaller than symbols are not shown.

Article Snippet: FcγR‐specific mAbs were obtained from Stem Cell technologies (CD16: clone 3G8; CD32: IV.3).

Techniques: