[Preparation and identification of a monoclonal antibody against human PD-L1].

ABSTRACT

OBJECTIVE: To prepare mouse anti-human PD-L1 monoclonal antibodies (mAbs) and identify their biological characteristics. METHODS: BALB/c mice were immunized with recombinant GST-PDL1 protein,and the strain of hybridoma cell secreting anti-PD-L1 mAb was obtained. The specificity of anti-PD-L1 mAb was checked and evaluated with ELISA and Western blot. Its titer, immunoglobulin subtype were also measured. The combining capacity of anti-PD-L1 mAb with PD-L1 on MDA-MB-231 cells, which had been induced with IFN-gamma for 72 hours, was identified through immunohistochemistry and flow cytometry. The peripheral blood mononuclear cell was labeled with CFSE, and the effect of anti-PD-L1 mAb on the activation and proliferation of the lymphocyte induced with antigens was evaluated by flow cytometry analysis. RESULTS: A strain of hybridoma cell secreting anti-PD-L1 mAb was successfully obtained and identified belong to IgG1 subtype. Its titers in cultural supernatant and ascetic fluid were 16400 and 1102400, respectively, by ELISA. The purified mAbs showed good affinity and specificity against GST-PDL1 protein in Western blot, also could block the PD-1/PD-L1 pathway and promote the proliferation of lymphocyte induced with antigens. CONCLUSION: Hyperactivity mouse anti-human PD-L1 hybridoma cell lines and their secreted monoclonal antibodies have been successfully obtained and identified.