Journal: Antibodies

Article Title: Evaluating the Therapeutic Efficacy of an Anti-BAFF Receptor Antibody Using a Rheumatoid Arthritis Mouse Model

doi: 10.3390/antib14040090

Figure Lengend Snippet: Antibody-mediated dose-dependent inhibition of B220+/CD21+CD23+ B-cell proliferation analyzed by flow cytometry. ( A ) CD21+ CD23+ cells, ( B ) B cells. Splenocytes from C57BL/6 mice (1 × 10 6 cells/mL) were treated with or without the following: BAFF [7 ng/mL]. mIgG2a isotype control mAb (clone-c1.18.4), mIgG1 isotype control mAb (clone-MOPC-21), V3-1 mIgG2a mAb, V3-46s mIgG2a mAb, and V3-46s mIgG1 mAb were added in a dose-dependent manner, as indicated, with 3-fold dilutions starting at 3 µg/mL, and cultured for 72 h at 37 °C. Cells were then stained for B220, CD21, and CD23, and visualized on a CytoflexL4.

Article Snippet: Cells were then stained for CD45 (Biolegend, catalog BLG-103108), B220 (Miltenyi, catalog 130-110-846), CD21/CD35 (Miltenyi, catalog 130-111-732), CD23 (Miltenyi, catalog 130-118-761), and DAPI (Sigma, catalog D9542), and were visualized on a CytoflexL4 (Beckman Coulter) flow cytometer.

Techniques: Inhibition, Flow Cytometry, Control, Cell Culture, Staining

Journal: Journal of pharmacological sciences

Article Title: Establishment of enzyme-linked immunosorbent facilitated antigen binding as a biomarker assay for Japanese cedar pollen allergy immunotherapy.

doi: 10.1016/j.jphs.2019.07.001

Figure Lengend Snippet: Fig. 1. Formation of allergen-IgE-CD23 complexes depending on concentration of immobilized CD23. Indicator serum was incubated with JC pollen (1 mg/mL) before immune complex capture. Data was expressed as absorbance at 450 nm.

Article Snippet: The following antibodies and reagents were used: monomeric human recombinant soluble CD23 (sCD23, 123-FE-050; R&D Systems, MN); biotin-conjugated anti-human IgE antibody (555858, BD Biosciences, NJ); ExtrAvidin-Peroxidase (E2886-1ML, Sigma, MO); tetramethyl benzidine (T2885, Sigma, MO); Anti-CD23 monoclonal antibody (Ab16702, Abcam plc, Cambridge, UK); JC pollen allergen extract (200 mg of protein/mg of dry extract, Torii, Japan).

Techniques: Concentration Assay, Incubation

Journal: Journal of pharmacological sciences

Article Title: Establishment of enzyme-linked immunosorbent facilitated antigen binding as a biomarker assay for Japanese cedar pollen allergy immunotherapy.

doi: 10.1016/j.jphs.2019.07.001

Figure Lengend Snippet: Fig. 2. Effect of JC pollen allergen concentration on allergeneIgE binding to immobi- lized CD23. The indicator serum was applied to this experiment. Three independent experiments were performed. Data are expressed as mean ± SE (absorbance at 450 nm). Heavy dashed line: Optimal allergen: IgE ratio. Vertical dotted lines: IgE excess (1:3) or allergen excess (3:1). NS: Non-significant.

Article Snippet: The following antibodies and reagents were used: monomeric human recombinant soluble CD23 (sCD23, 123-FE-050; R&D Systems, MN); biotin-conjugated anti-human IgE antibody (555858, BD Biosciences, NJ); ExtrAvidin-Peroxidase (E2886-1ML, Sigma, MO); tetramethyl benzidine (T2885, Sigma, MO); Anti-CD23 monoclonal antibody (Ab16702, Abcam plc, Cambridge, UK); JC pollen allergen extract (200 mg of protein/mg of dry extract, Torii, Japan).

Techniques: Concentration Assay, Binding Assay

Journal: Journal of pharmacological sciences

Article Title: Establishment of enzyme-linked immunosorbent facilitated antigen binding as a biomarker assay for Japanese cedar pollen allergy immunotherapy.

doi: 10.1016/j.jphs.2019.07.001

Figure Lengend Snippet: Fig. 3. Assessment of assay dependency. Three experiments were performed for each test. Values represent mean ± SE. (a) CD23 dependency was investigated using the anti-CD23 antibody. Data are shown as percentage of inhibition binding which indicator serum without adding of anti-CD23 was normalized 100%. (b) IgE dependency was assessed using heat-denatured serum. Data was expressed as % of complex formation relative to un-heat denatured serum. NT; Non treated.

Article Snippet: The following antibodies and reagents were used: monomeric human recombinant soluble CD23 (sCD23, 123-FE-050; R&D Systems, MN); biotin-conjugated anti-human IgE antibody (555858, BD Biosciences, NJ); ExtrAvidin-Peroxidase (E2886-1ML, Sigma, MO); tetramethyl benzidine (T2885, Sigma, MO); Anti-CD23 monoclonal antibody (Ab16702, Abcam plc, Cambridge, UK); JC pollen allergen extract (200 mg of protein/mg of dry extract, Torii, Japan).

Techniques: Inhibition, Binding Assay

Journal: Journal of pharmacological sciences

Article Title: Establishment of enzyme-linked immunosorbent facilitated antigen binding as a biomarker assay for Japanese cedar pollen allergy immunotherapy.

doi: 10.1016/j.jphs.2019.07.001

Figure Lengend Snippet: Fig. 4. Inhibitory activity of allergeneIgE binding to CD23 in patients with JC pollen SCIT. Data are expressed as median values with 1st and 3rd quartiles (n ¼ 11). Sta- tistical significance was detected by using the KruskaleWallis test followed by the Dunn post hoc test. **p < 0.01 and ***p < 0.001, NS: Non-significant. Control: indicator serum diluted with medium. Allergic group: Sera from JC pollen-sensitized subjects diluted with indicator serum. SCIT group: Sera from JC pollen AIT patients diluted with indicator serum.

Article Snippet: The following antibodies and reagents were used: monomeric human recombinant soluble CD23 (sCD23, 123-FE-050; R&D Systems, MN); biotin-conjugated anti-human IgE antibody (555858, BD Biosciences, NJ); ExtrAvidin-Peroxidase (E2886-1ML, Sigma, MO); tetramethyl benzidine (T2885, Sigma, MO); Anti-CD23 monoclonal antibody (Ab16702, Abcam plc, Cambridge, UK); JC pollen allergen extract (200 mg of protein/mg of dry extract, Torii, Japan).

Techniques: Activity Assay, Binding Assay, Control

Journal: bioRxiv

Article Title: The dynamics of hinge flexibility in receptor bound immunoglobulin E revealed by electron microscopy

doi: 10.1101/2023.02.17.528943

Figure Lengend Snippet: A) Representation of the interplay and functional effects of IgE bound to its receptors FcεRI on basophils and mast cells (left) and CD23 on antigen presenting cells (right). B) Schematic outline of the IgE molecule with domain names. The IgE Fc moiety is formed by the Cε2-Cε4 domains in the two heavy chains (green and blue), whereas the Fab is formed by the heavy chain domains VH and Cε1 together with the light chain (light blue and light green) VL and CL domains. The Fc moiety can interconvert between a symmetric straight conformation (top) and a bent conformation (bottom). C) Two orientations of the consensus cryo EM map of the IgE-Fc ε RI α complex contoured at 16 s. Density around the receptor and the IgE Fc is grey and pink, respectively, whereas density at around the distal Fab (dFab) is green and density around the proximal Fab (pFab) is blue. D) As in panel C, but displaying a local resolution filtered map contoured at 6 s to provide a better impression of the Fab arms. At the proximal Fab, the cleft between the variable the constant domains are visible. E) Map from the focused refinement centered on IgE-Fc and Fc ε RI α contoured at 20 s. F-I) Examples of map quality from the focused refinement in C ε 2 (F), C ε 3 (G), C ε 4 (H), and Fc ε RI α (I). The map is contoured at 12s. In panels F-H, carbon atoms in one IgE heavy chain are colored blue, while carbons in the other heavy chain are green. In panel I, carbon atoms in the receptor α -subunit are colored grey, notice the clear density for the glycan linked to Asn42.

Article Snippet: The preincubated immunocomplexes were transferred to overnight coated and BSA-blocked recombinant CD23 (625 ng/ 96 well in PBS (R&D systems 123-FE-050)).

Techniques: Functional Assay, Cryo-EM Sample Prep, Glycoproteomics

Journal: bioRxiv

Article Title: The dynamics of hinge flexibility in receptor bound immunoglobulin E revealed by electron microscopy

doi: 10.1101/2023.02.17.528943

Figure Lengend Snippet: A) Schematic drawing of the two glycans recognized by the HMM5 IgE. The lower affinity antigen MMXF3 is found on HRP, the higher affinity antigen MUXF3 on pineapple stem bromelain. B) SDS-PAGE analysis of HMM5 IgE and HMM5L IgE under non-reducing and reducing conditions after size exclusion chromatography. The band around 170 kDa represents the fully assembled antibody, whereas the band at 60 kDa under reducing corresponds to the ε-chain. The light chains are not visible under these conditions. C) The identity of IgE was further confirmed by immunoblot using an anti-human IgE antibody labeled with alkaline phosphatase for detection. D) Binding and cross-linking of the high-affinity receptor FcεRI was assessed by detection of β-hexosaminidase release from RBL-S38X cells upon passive loading with IgE followed by exposure to the antigens. Both IgE variants elicit β-hexosaminidase release, but HMM5L is less active with the lower affinity MMXF3 antigen. E) The ability of IgE to form immune complexes and the subsequent binding to immobilized CD23 was analyzed in ELIFAB assays. A clear reduction of complex binding to CD23 for HMM5L compared to HMM5, in particular for the lower affinity antigen, suggests a more efficient complexation or sterically different complex architecture.

Article Snippet: The preincubated immunocomplexes were transferred to overnight coated and BSA-blocked recombinant CD23 (625 ng/ 96 well in PBS (R&D systems 123-FE-050)).

Techniques: SDS Page, Size-exclusion Chromatography, Western Blot, Labeling, Binding Assay