Quantifying Soluble CD155 Levels by ELISA and CBA

Human sCD155 concentrations in the culture supernatants of HeLa (2.0 × 10⁴ cells/100 µl/well) 24 h after the start of culture were measured by ELISA, as described (Iguchi-Manaka et al., 2016 (link)). For measurement of mouse sCD155, we established a cytokine bead array (CBA) assay. Anti-CD155 mAb (TX56) was conjugated to BD CBA Functional Beads (BD Bioscience) in accordance with the manufacturer’s protocol. 2 μl of the TX56 beads was incubated with a 50-µl sample of sera, culture supernatants, or tumor extraction at room temperature or on ice for 2 h, washed with the wash buffer (BD Bioscience), and then incubated with 0.5 µg/ml of rabbit anti-mouse CD155 polyclonal antibody generated in our laboratory in 50 µl of the Assay Diluent (BD Bioscience) at room temperature for 1 h. The TX56 beads were then washed with the wash buffer, incubated with 1 µl of PE-conjugated donkey anti-rabbit polyclonal IgG (BioLegend) in 50 µl of the Assay Diluent at room temperature for 1 h, washed again with the wash buffer, and analyzed by flow cytometry. The culture supernatants of sCD155/BL6 and mock/BL6 (2.0 × 10⁴ cells/100 µl medium/well) obtained 24 h after the start of culture, sera of peripheral blood of mice injected with sCD155/BL6 or mock/BL6, and sera from the pulmonary vein of tumor-colonized lungs after clamping were subjected to the CBA assay.