Anti-Human B7-H2 Recombinant Antibody (20B10) (CAT#: TAB-1141CL)

Figure 1 Specificity and reactivity of anti-ICOSL mAbs with the transfectants.

L929/mock, L929/B4 and L929/ICOSL cell lines were stained with the anti-ICOSL mAbs (20B10, 2B4 and 2D3) or IgG2a isotype control antibody (MOPC-173) (open black histograms) for 30 min followed by PE-goat anti-mouse IgG. Then, the cells were analyzed by flow cytometry.

Hu, X., Wu, J., An, J., Hu, Y., Shen, Y., Liu, C., & Zhang, X. (2016). Development of a novel monoclonal antibody to human inducible co-stimulator ligand (ICOSL): biological characteristics and application for enzyme-linked immunosorbent assay. International immunopharmacology, 36, 151-157.

Figure 2 Western blot analysis of the specific mouse anti-human ICOSL mAbs.

The commercial ICOSLIg protein was loaded for SDS-PAGE electrophoresis and transferred onto nitrocellulose membranes. The membranes were incubated with the anti-ICOSL mAbs followed by goat antimouse IgG-HRP. The protein bands were visualized using chemical reagents. 2D3 mAb was used as a positive control in this experiment.

Hu, X., Wu, J., An, J., Hu, Y., Shen, Y., Liu, C., & Zhang, X. (2016). Development of a novel monoclonal antibody to human inducible co-stimulator ligand (ICOSL): biological characteristics and application for enzyme-linked immunosorbent assay. International immunopharmacology, 36, 151-157.

Figure 3 Dot blot analysis of the specific mouse anti-human ICOSL mAbs.

Hu, X., Wu, J., An, J., Hu, Y., Shen, Y., Liu, C., & Zhang, X. (2016). Development of a novel monoclonal antibody to human inducible co-stimulator ligand (ICOSL): biological characteristics and application for enzyme-linked immunosorbent assay. International immunopharmacology, 36, 151-157.

Figure 4 Determination of the binding ability of the obtained mAbs to immobilized ICOSL-Ig antigen by indirect ELISA.

The recombinant ICOSL-Ig protein was serially diluted two-fold to different concentrations with carbonate buffer and coated on the ELISA plate. Then, the indirect ELISA was performed with the obtained mAbs (clones 20B10 and 2B4) and HRP-goat anti-mouse IgG. The absorbance of the wells was measured at 450 nm by a microplate reader.

Hu, X., Wu, J., An, J., Hu, Y., Shen, Y., Liu, C., & Zhang, X. (2016). Development of a novel monoclonal antibody to human inducible co-stimulator ligand (ICOSL): biological characteristics and application for enzyme-linked immunosorbent assay. International immunopharmacology, 36, 151-157.

Figure 5 Effect of the anti-ICOSL mAbs on ICOS/ICOSL interaction.

(A) L929/ICOSL cells were incubated with the indicated doses of different mAbs for 30 min at 4 °C. Then, the cells were reacted with ICOS-Ig fusion protein followed by PE goat anti-human IgG(Fc) (red lines) for 45 min at 4 °C. At the same time, L929/ICOSL cells were stained directly with ICOS-Ig protein followed by PE-goat anti-human IgG(Fc) as a positive control (blue lines) or human IgG as a negative control (red histograms). The fluorescence intensity of the stained cells was analyzed by flow cytometry. (B) In vitro stimulating of PBMC using 20B10 mAb increases the proliferation of CD3+ T cells. CFSE-labeled cells were incubated with a pool of CD3 antibody in the presence or absence of 20B10 mAb for 6 days and stained for the respective markers. (C) The supernatants from the 20B10 stimulating PBMC after 6 days were collected, and interleukin (IL)-17, IL-2, IL-4 and IFN-γ production in the presence of 20B10 compared with the control IgG group was measured by the CBA assay. The results are shown as the mean ± SD of triplicate wells. *P < 0.05, **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Hu, X., Wu, J., An, J., Hu, Y., Shen, Y., Liu, C., & Zhang, X. (2016). Development of a novel monoclonal antibody to human inducible co-stimulator ligand (ICOSL): biological characteristics and application for enzyme-linked immunosorbent assay. International immunopharmacology, 36, 151-157.

Figure 6 Establishment of the sICOSL ELISA.

(A and B) The correlated line of the OD450 nm absorbance and sICOSL concentration at different detection limits. The capture anti-ICOSL mAb (20B10) was at 2 μg/ml, the biotinylated anti-ICOSL mAb (bio-2B4) was at 0.5 μg/ml. The two-fold serial dilutions of the soluble ICOSL Ig protein starting from 100 ng/ml were detected by the ELISA. The absorbance was measured at 450 nm using a microplate reader. R2 represents the correlation coefficient. (C) The specificity of sICOSL ELISA. The two-fold serial dilutions of the sICOSL, sB7H4, sICOS and sPD-L1 proteins starting from 20 ng/ml were detected by the ELISA. The absorbance was measured at 450 nm using a microplate reader. These results are representative of three independent experiments.

Hu, X., Wu, J., An, J., Hu, Y., Shen, Y., Liu, C., & Zhang, X. (2016). Development of a novel monoclonal antibody to human inducible co-stimulator ligand (ICOSL): biological characteristics and application for enzyme-linked immunosorbent assay. International immunopharmacology, 36, 151-157.