Preparation Of Mouse Anti-human PD-1 Monoclonal Antibody And Identification Of Its Biological Function

PD-1,a type I transmembrane glycoprotein with 288 amino acids,belonged to the immunoglobulin superfamily.PD-1 expressed on activated T cells,B cells,NK cells,DC cells and so on.PD-1 played the negative immuno-regulatory function,to be maintaining immune homeostasis by interacting with its ligand PD-L1 or PD-L2.PD-1/PD-Ls immunoregulatory dysfunction participated the pathological mechanism of autoimmune diseases and tumors.In order to provide research tools and experimental basis for the study of PD-1/PD-Ls pathway,in this study,we prepared a mouse anti-human PD-1 monoclonal antibody,and identified its biological function.Part? Construction of human PD-1 gene transfected cell lineObjective: Constructing the human PD-1 gene transfected cell line,for the preparation of PD-1 monoclonal antibody.Methods: The recombinant retrovirus expression vector p EGZ-term-R/PD-1 was transfected into 293 T cells by liposome transfection method.Virus supernatant was collected and infected with L929 cells.After selection by G418,flow cytometry and Western Blot were used to identify the expression of PD-1 molecule in this cell line.Results: A gene-transfected cell line L929/PD-1 stably expressing human PD-1 molecule was obtained.Conclusion: Successfully constructed a gene-transfected cell line which stably expressing human PD-1 molecule,provided an immunogen for the preparation of PD-1 monoclonal antibody.Part? Preparation and identification of mouse anti-human PD-1 monoclonal antibodyObjective: Preparation of PD-1 monoclonal antibody provided a research tool for the study of the PD-1/PD-Ls pathway.Methods: The BALB/c mice were immunized with stably expressing human PD-1 gene transfected cell line L929/PD-1.Using conventional B-lymphoma hybridoma technology,a hybridoma cell strain named 15D9,which stably produced mouse anti-human PD-1 monoclonal antibody was obtained by HAT culture screening and indirect immunofluorescence analysis.Rapid qualitative test strips were used to identify the class and type of the monoclonal antibody.Using flow cytometry to detect competitive binding sites and antibody titers and the identification of PD-1 molecule by 15D9 was confirmed by immunocytochemical method.Results:A hybridoma cell line stably produced mouse anti-human PD-1 monoclonal antibody was obtained and named as 15D9.The heavy chain of 15D9 was Ig G1,and the light chain was ? chain.The competitive binding assay results showed that 15D9 m Ab and 6E1 m Ab recognized similar sites.Antibody titer results showed that 0.1 ?g/test could be used for indirect labeling flow cytometry,1 ?g/test could be used for direct labeling flow cytometry.And the immunocytochemical method result showed that 15D9 could specifically recognize PD-1 molecule.Conclusion: A hybridoma cell strain 15D9 stably produced mouse anti-human PD-1 monoclonal antibody was successfully obtained.15D9 could be used for immunofluorescence labeling,flow cytometry,and immunohistochemistry experiments.Part? Identification of blocking function of mouse anti-human PD-1 monoclonal antibodyObjective: By observing the role of monoclonal antibody 15D9 in the activation of PBMC cells,the biological function of monoclonal antibody 15D9 was initially identified.Methods: Activated CD3 monoclonal antibody was coated and combined with activated CD28 monoclonal antibody to activate PBMC.The expression of PD-1 and PD-L1 was detected at 24 h,48 h,and 72 h.After the addition of 15D9 m Ab,the morphological changes of PBMC were observed and changes in the number of PBMC were determined by cell counting.And the expression levels of CD69 and CD25,were measured at 24 h,48 h,and 72 h by flow cytometry.ELISA was used to detect the secretion of IL-2 and IFN-? at 96 h.Results: Activated CD3 monoclonal antibody combined with activated CD28 monoclonal antibody could activate PBMC,and PBMC cells expressed PD-1 and PD-L1 molecules.After 15D9 m Ab was added,compared with the CD3+CD28+Ig G group,the morphology of PBMC of the CD3+CD28+15D9 group changed more significantly: the colony diameter increased,the number of colonies increased.And the expression of PBMC activation molecules CD69 and CD25 increased.What is more,the secretion of IFN-? was higher.Conclusion: Activated CD3 monoclonal antibody combined with activated CD28 monoclonal antibody could activate PBMC,and the activation of PBMC was increased after the addition of 15D9 m Ab,indicating that 15D9 was a monoclonal antibody with blocking function.