Preparation Of Mouse Anti-human PD-l2 Monoclonal Antibody And Development Of Sandwich ELISA Systems For Evaluating Human Soluble PD-l2

Costimulatory molecules play an important role in immune regulation, PD-1(CD279) mediated negative signals can lead to T cell apoptosis . As a second ligand of PD-1 , PD-L2(CD273) inhibit T cell activation and secretion of IFN-γ. Being one of the B7 superfamily, the structure of PD-L2 contains IgV, IgC-like area, transmembrane domain and a short cytoplasmic tail. Recent studies have shown, PD-L2 is closely related with schistosomiasis, tuberculosis and allergic asthma. PD-L2 also plays an important role in the allogeneic reaction and autoimmune diseases. Studies also show that PD-L2 expressing on the surface of dendritic cells interact with PD-1 on the T cells ,can inhibit the biological function of T cells.The absence of PD-L2 led to abnormal PD-1/PD-L2 pathway, and trigger the body to be suffered autoimmune disease.Mounting data demonstrated that many costimulatory molecules assume two forms of expression. Except membrane-bound forms, soluble forms have been found for several members of B7 family including OX40L,CD40L,B7-H3 and PD-1. The soluble protein is either shed from the membrane or produced by the special mRNA. Many soluble proteins are valuable for clinical diagnosis. However, the functions and biological significance of sPD-L2 remain unknown.In this study, we intend to generate and characterize monoclonal antibodies against human PD-L2. Then the mAbs were used to develope sandwich enzyme-linked immunosorbent assay (ELISA) systems for the detection and quantification of sPD-L2.Part 1 Generation and characterization of monoclonal antibodies against human PD-L2Objective : To prepare functional monoclonal antibodies against human PD-L2 molecule and analysis of their biological characteristics. Method : A stable human PD-L2 transfected cell line L929/PD-L2 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-L2 as target cells while L929/mock was used as the negative control, the hybridoma specifically secreting mouse anti-human PD-L2 monoclonal antibody was generated. Then its biological characterization was investigated by Western bloting, rapid murine Ig subclass typing method, indirect immunofluorescene, mutual competitive inhibition test.Results: After multiple fusion technique and repeated screening, One mouse anti-human PD-L2 monoclonal antibody was generated successfully, the monoclonal antibody (named as 8F2) could bind to human PD-L2 specially, and it recognized a new epitope of PD-L2. Furthermore, we use FCM to analyze the expression patterns of PD-L2 on human different cell lines.The datas indicate that PD-L2 express in THP-1, SHI-1, U937 monocytes and other sources of tumor cell lines, as well as increased expression in mature dendritic cells and activated T cells.Conclusion: One mouse anti-human PD-L2 monoclonal antibody was generated successfully, which recognize different epitopes. The PD-L2 monoclonal antibody provide the initial material for further study of the role of human PD-1/PD-L2 signaling pathway in the biological role of immune response.PartПDevelopment of sandwich ELISA systems for evaluating human soluble PD-L2Objective : To establish specific ELISA systems with high sensitivity to detect human soluble PD-L2 proteins respectively. Then detect The level of sPD-L2 of patients with asthma by the sPD-L2 ELISA .Method : Mouse anti-human PD-L2 mAb (8F2), used as coating antibody, was precoated in the ELISA plate with the CBS. Another biotin-anti-PD-L2 mAb (10D6) was used as detecting antibody to recognize the 8F2-bound soluble PD-L2 protein. Then the Streptavidin-HRP was added to the reaction system. Finally, TMB substrate was added for colorable reaction. The absorbance was measured by the microplate reader.Results: The results showed that the ELISA system for detecting human sPD-L2 was established successfully. It could be used to detect human sPD-L2 protein specially without any cross-reaction with other proteins. Standard curve of the system displayed favorable linear correlation when the concentration of sPD-L2 was 1.56-100ng/ml. The ELISA system had favorable stability, precision and specificity. The result showed that levels of sPD-L2 were significantly increased in patients with asthma serum.Conclusion: Two ELISA systems with high specificity and sensitivity provide useful methods to detect human sPD-L2. And levels of sPD-L2 were significantly increased in serum of patients with asthma.In conclusion, one monoclonal antibodies for human PD-L2 have been generated. Base on the mAbs, a ELISA assay for the detection of soluble PD-L2 with high stability, veracity and specificity was developed.