Preparation Of Mouse Anti-human PD-1 Monoclonal Antibody And Characterization Of Human Soluble PD-1

The most intensively characterized CMs are those of the B7/CD28 family. The PD-1 receptor, which belongs to CD28 family, is a 55KDa type I transmembrane protein of Ig superfamily with an extracellular IgV-like domain. The domain plays an important role in binding to ligands. Ligation PD-1 with its two ligands, PD-L1 and PD-L2, triggers inhibitory signals to inhibit T and B cell activation and production of cytokines. PD-L and PD-1 interaction involves in maintenance of peripheral tolerance. In addition, the PD-1 expression plays an important role in the occurrence and development of a series of diseases. Studies on mouse models showed that PD-1 receptor was a promising target in the intervention of human malignant cancers, autoimmune disease and viral diseases.In our study, a stable human PD-1 transfected cell line L929/PD-1 was used as an antigen to immunize BALB/c mice. By means of the cell fusion technique, multiple cell subcloning and repeated screening with L929/PD-1 as target cells while L929/mock was used as the negative control, the hybridomas specifically secreting mouse anti-human PD-1 monoclonal antibody were generated, named as 6E2 and 10B5. For additional analysis, western blotting was performed with PD-1 membrane protein extracted from L929/PD-1 cell line. Both of the two mAbs could identify a protein with approximately 55 kDa, importantly both mAbs also could recognize new epitopes of cell surface PD-1 membrane molecule.The sandwich ELISA was prepared by utilizing two anti-hPD-1 monoclonal antibodies 6E2 and 10B5 as capture and detecting antibody respectively. Then its stability, precision and specificity were analyzed. The detection range of the ELISA permits a reliable assay of sPD-1 from 1.56 up to 100ng/ml, and with high stability, veracity and specificity.The target gene encoding full length human PD-1(PD-1) was cloned by RT-PCR, then two fragments of PD-1Δex3 gene were amplified and assembled by TP-PCR, PD-1Δex3 gene was obtained. Then the two genes PD-1 andΔPD-1 were inserted into the eukaryotic expressing vector pIRES2-EGFP respectively to construct the recombinant vectors. Then the recombinants were transfected into 293T cells with Lipofect2000 Reagent. Flow cytometry and Western blot revealed that 293T cells transfected with vector pIRES2-EGFP/PD-1 could express PD-1 protein on the cell surface but no soluble PD-1 in the supernatant of transfected cells, on the contrary, 293T cells transfected with vector pIRES2-EGFP/ΔPD-1 could express soluble PD-1 in the culture supernatant but no membrane PD-1. Indirect immunofluorescence assay indicated theΔPD-1 protein could bind to the two ligands of PD-1 on the cells surface.In conclusion, two monoclonal antibodies for human PD-1 have been generated. Base on the two mAbs, a ELISA assay for the detection of soluble PD-1 with high stability, veracity and specificity was developed. Furthermore, we have successfully cloned PD-1Δex3 gene and constructed the recombinant eukaryotic expressing vector. the PD-1Δex3 gene could encode a soluble form of PD-1 protein.ΔPD-1 protein remainded the biological activity as PD-1. This study provides the initial material for further study of PD-1Δex3 in PD-1/PD-L signaling pathway.