Journal: Antibody Therapeutics

Article Title: Discovery of candidate antibodies and antigens for acute myeloid leukemia therapy by combining whole-cell phage display selection and CRISPR-Cas9 library screening

doi: 10.1093/abt/tbag043

Figure Lengend Snippet: Characterization of mAb_11 using recombinant PTPRG protein. (A) Flow cytometry histograms of the binding of mAb_11 in IgG format in U937 cells in the presence of competing recombinant PTPRG protein (hPTPRG-N-hFc) (left panel) or rabbit anti-human PTPRG pAbs (rbAb; right panel), each at two different concentrations. Blue; cells treated with 2 μg/ml IgG_11 and 20 μg/ml of hPTPRG-N or rbAb. Green; cells treated with 2 μg/ml IgG_11 and 2 μg/ml of hPTPRG-N or rbAb. Red; cells treated with 2 μg/ml IgG_11 only. The results of U937 cells treated with secondary antibody only (second only, black), and U937 cells unstained (gray) are also shown. (B) SPR sensorgrams showing mAb_11 in Fab format bound the N-terminal structured domains of human PTPRG (hPTPRG-N-hFc) (K D = 4.73 × 10 −9 M) (left panel), the extracellular domain of human PTPRG (hPTPRG-ECD-hFc (K D = 4.23 × 10 −9 M) (middle panel), and the extracellular domain of mouse PTPRG (mPTPRG-ECD-hFc) (K D = 3.31 × 10 −9 M) (right panel).

Article Snippet: After Fab staining for 30 min on ice followed by washing with 150 μL flow cytometry buffer (PBS supplemented with 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide), cells were then stained with 25 μL of a 100-fold dilution of PE-conjugated goat anti-human IgG, F(ab′) 2 fragment-specific antibody (Jackson ImmunoResearch no. 109-116-097) in flow cytometry buffer.

Techniques: Recombinant, Flow Cytometry, Binding Assay

Journal: Antibody Therapeutics

Article Title: Discovery of candidate antibodies and antigens for acute myeloid leukemia therapy by combining whole-cell phage display selection and CRISPR-Cas9 library screening

doi: 10.1093/abt/tbag043

Figure Lengend Snippet: mAbs in BLUE category bound to Nectin-2. (A) Flow cytometry histograms of the binding of Fabs in the BLUE category (Fab_484, Fab_277, Fab_2430) to U937 cells. Binding of Fab in a different category (Fab_2810) was also measured as a control. Blue; cells transduced with two NECTIN2 gRNAs (Top1 and zf gRNAs). Cyan; cells transduced with NECTIN2 zf gRNA. Green; cells transduced with NECTIN2 Top1 gRNA. Red; parental U937 cells. Gray; unstained U937. (B) Epitope binning studies by flow cytometry. Binding of Alexa 647 labeled Fab_2282 to U937 cells in competition with unlabeled Fab_2430, Fab_277, Fab_484, and Fab_2282 itself. Binding of Alexa 647 labeled Fab_2282 with unlabeled Fab in a different category (Fab_2810) was also measured as a control. Binding of Alexa 647 labeled Fab_2282 with no unlabeled competitor (Fab 2282-647) is shown as a positive control. (C–E) Flow cytometry histograms of the binding of Fab_2282 (C), Fab_484 (D), and Fab_277 (E) to U937 cells, treated with hNectin-2-hFc [left panel of each of (C–E)] or treated with rabbit anti-human Nectin-2 pAbs [right panel or each of (C–E); rbAb], each at two different concentrations. Green; cells treated with 50 μg/ml of hNectin-2-hFc or rbAb. Blue; cells treated with 10 μg/ml hNectin-2-hFc or rbAb. Red; cells treated with 5 μg/ml each Fab in BLUE category only. Through (B–E), the results of U937 cells treated with secondary antibody only (second only, black), and unstained U937 cells (gray) are shown as negative controls.

Article Snippet: After Fab staining for 30 min on ice followed by washing with 150 μL flow cytometry buffer (PBS supplemented with 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide), cells were then stained with 25 μL of a 100-fold dilution of PE-conjugated goat anti-human IgG, F(ab′) 2 fragment-specific antibody (Jackson ImmunoResearch no. 109-116-097) in flow cytometry buffer.

Techniques: Flow Cytometry, Binding Assay, Control, Transduction, Epitope Binning, Labeling, Positive Control