Journal: Frontiers in Pharmacology

Article Title: Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

doi: 10.3389/fphar.2019.00847

Figure Lengend Snippet: EC 50 values in dose–response ELISA for clones identified through fishing.

Article Snippet: For blocking, Raji or CLL cells were incubated with a polyclonal antibody (rabbit anti-CD22 (Sino Biological, 11958-T26-50), goat anti-FCRL5 (Invitrogen, PA5-48003), and goat anti-ROR1 (R&D Systems, AF2000)), 100 µg/ml, 25 µl/well, or buffer, for 1 h at +4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay

Journal: Frontiers in Pharmacology

Article Title: Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

doi: 10.3389/fphar.2019.00847

Figure Lengend Snippet: Binding analysis of antibodies generated through fishing with recombinant CD22, ROR1, and FCRL5 proteins. (A) Dose–response ELISA of one representative antibody (human IgG1) per protein. Each antibody was analyzed for binding to the specific protein used for fishing (target protein) and a non-related protein carrying the same tag (non-target protein). Binding was detected using an HRP-labeled anti-human antibody and a luminescent substrate. (B) Flow cytometry analysis of one representative antibody (human IgG1 or scFv) per target showing the binding to target cells, that is, chronic lymphocytic leukemia (CLL) cells (anti-FCRL5) or Raji cells (anti-CD22 and anti-ROR1), with or without a prior blocking step with a commercial polyclonal antibody of the same specificity as the tested antibody. Binding was detected using an APC-labeled anti-human antibody (anti-CD22 and anti-ROR1) or an AF647-labeled anti-tag antibody (anti-FCRL5).

Article Snippet: For blocking, Raji or CLL cells were incubated with a polyclonal antibody (rabbit anti-CD22 (Sino Biological, 11958-T26-50), goat anti-FCRL5 (Invitrogen, PA5-48003), and goat anti-ROR1 (R&D Systems, AF2000)), 100 µg/ml, 25 µl/well, or buffer, for 1 h at +4°C.

Techniques: Binding Assay, Generated, Recombinant, Enzyme-linked Immunosorbent Assay, Protein Binding, Labeling, Flow Cytometry, Blocking Assay

Journal: Frontiers in Pharmacology

Article Title: Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

doi: 10.3389/fphar.2019.00847

Figure Lengend Snippet: Protein and gene expression levels in chronic lymphocytic leukemia (CLL) cells (target cells) and CD19− PBMC from healthy donors (non-target cells) for receptors successfully used in fishing.

Article Snippet: For blocking, Raji or CLL cells were incubated with a polyclonal antibody (rabbit anti-CD22 (Sino Biological, 11958-T26-50), goat anti-FCRL5 (Invitrogen, PA5-48003), and goat anti-ROR1 (R&D Systems, AF2000)), 100 µg/ml, 25 µl/well, or buffer, for 1 h at +4°C.

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

doi: 10.3389/fphar.2019.00847

Figure Lengend Snippet: Frequency, based on NGS analysis, of discovered clones in the different strategies including the input phage pool. Median frequency is shown with red bars. (A) Antibodies, previously discovered through direct screening with corresponding specificities, CD23 ( n = 107), CD72 ( n = 132), and CD200, CD21, CD32, or HLA-DR ( n = 34). (B) Antibodies, discovered through fishing, binding FCRL5, ROR1, or CD22 ( n = 17). For statistical analysis, Friedman’s test with Dunn’s multiple comparison was done using GraphPad Prism.

Article Snippet: For blocking, Raji or CLL cells were incubated with a polyclonal antibody (rabbit anti-CD22 (Sino Biological, 11958-T26-50), goat anti-FCRL5 (Invitrogen, PA5-48003), and goat anti-ROR1 (R&D Systems, AF2000)), 100 µg/ml, 25 µl/well, or buffer, for 1 h at +4°C.

Techniques: Clone Assay, Binding Assay

Journal: Free radical biology & medicine

Article Title: Extracellular vesicles released by ALL patients contain HNE-adducted proteins: Implications of collateral damage.

doi: 10.1016/j.freeradbiomed.2024.12.006

Figure Lengend Snippet: Fig. 3. Immunoblotting of protein markers in EV lysates. For all graphs in this figure, individual fold change was measured, followed by averaging the overall fold change across the different collection time points. Quantification of CD22 (A) and CD19 (B) as cell surface markers of leukemia. A significant increase of CD22 was detected in the EVs at consolidation day 15 compared to induction day 29 (p < 0.05). (C) A significant decrease in GFAP expression was observed in the EV lysates at consolidation days 1 and 8 compared to induction day 29 (p < 0.05). (D) An insignificant decline in the neuronal marker, NeuN, in the EVs was observed during consolidation phase compared to pre-treatment or induction day 29. (E) A statistically significant decrease in BDNF was observed in the EV lysates during both induction and consolidation phase collection points compared to pre-treatment (p < 0.05). *denotes p < 0.05 when compared to pre-treatment and # denotes p < 0.05 when compared to induction day 29. N.S. = No statistically significant difference.

Article Snippet: The primary antibodies and their dilutions used were: flotillin-1 (Flot-1) (1:20) from Bioss (catalog #: BS7798R), HSC70 (1:20) from Santa Cruz (catalog #: sc-7298), CD63 (1:10) from Santa Cruz (catalog #: sc-5275), ApoA1 (1:50) from Cell Signaling (catalog #: 3350), CD22 (1:50) from ProteinTech (catalog #: 66103-1-Ig) CD19 (1:10) from Abcam (catalog #: ab227019), GFAP (1:20) from Aviva Systems (catalog #: OAEB01041), BDNF (1:50) from Abcam (catalog #: ab10505), and NeuN from Novus (catalog #: NBP192716).

Techniques: Western Blot, Expressing, Marker

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD22 , REA340 , 50 , 130-124-223 , FITC , Miltenyi Biotec.

Techniques: Imaging

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: IgG membrane tail does not alter tyrosine phosphorylation of CD22 or ERK phosphorylation. Splenocytes from IgM and IgMG transgenic mice were stimulated with 50 μg/ml anti-IgM F(ab′) 2 . Total cellular proteins (A) or immunoprecipitated CD22 (B) were fractionated by SDS-PAGE and Western blotted with antiphosphotyrosine antibody (A and B, top) or anti-CD22 antibody (B, bottom). The ratios of phosphorylated CD22 to total CD22 are indicated. (C and D) Mean fluorescence intensity (MFI) of permeabilized B220 + CD21 medium CD23 + follicular B cells stained by flow cytometry for phosphorylated ERK either (C) at the indicated times after stimulation with 50 μg/ml anti-IgM F(ab′) 2 or (D) in unstimulated (−) versus stimulated (+) cells after 2 min in the presence of MEK inhibitors PD98059, U0126, or DMSO as diluent controls. Data are representative of two experiments.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Phospho-proteomics, Transgenic Assay, Immunoprecipitation, SDS Page, Western Blot, Fluorescence, Staining, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: IgG membrane tail evokes an augmented Ca 2+ response independent of CD22. RBC-depleted splenocytes from mice of the indicated genotypes were labeled with 1 μM Indo-1 for 30 min at 37°C. (A and B) The cells were counterstained with antibodies against B220 and CD21 for the final 10 min of Indo-1 loading. The stained cells were at first acquired for 30 s and then stimulated with the indicated concentrations of anti-IgM F(ab′) 2 antibody (A, 5 μg/ml; and B, 0.5 μg/ml). Lines show the mean Indo-1 ratio in B220 + CD21 medium cells. Data from one out of three independent experiments are shown. (C and D) Indo-1–loaded cells stained with antibody against B220 were simulated with the indicated concentrations of HEL conjugated to PE (HEL-PE; C, 5 μg/ml; and D, 0.5 μg/ml), and the Indo-1 ratio was measured on gated HEL + B220 + cells.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Labeling, Staining

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: IgG membrane tail increases antibody production independent of CD22. 5 × 10 5 HEL-binding splenic B cells from IgMG or IgM transgenic donors of the indicated CD22 genotypes were adoptively transferred into nonirradiated C57BL/6 mice, and the recipient mice were immunized with HEL in CFA. The concentration of anti-HEL IgM a antibody 10 d after immunization was measured in the serum of individual recipient mice (circles) by ELISA. Data are representative of three separate experiments. Significant differences were determined by the Mann-Whitney test. *, P < 0.05; **, P < 0.01.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Membrane, Binding Assay, Transgenic Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Enhancement and suppression of signaling by the conserved tail of IgG memory–type B cell antigen receptors

doi: 10.1084/jem.20061923

Figure Lengend Snippet: CD22 deficiency suppresses IgM but not IgMG marginal zone B cell differentiation. Splenocytes were stained with HEL and combinations of antibodies against CD21/CD23/HyHEL9/B220 (A and B) or CD21/CD1d/HyHEL9/B220 (C and D). (A and C) The displayed profiles are gated on B220 + HEL-binding cells, and the percentage of B220 + HEL-binding cells in each window is shown. (B and D) Percentages of HEL-binding B cells in the marginal zone subset in individual mice (circles). Significant differences, as determined by the Student's t test, are indicated.

Article Snippet: After stripping, the membranes were stained with goat anti–mouse Siglec-2 (CD22) antibody (R&D Systems), followed by horseradish peroxidase–conjugated anti–goat antibody (Jackson ImmunoResearch Laboratories).

Techniques: Cell Differentiation, Staining, Binding Assay