Preparation Of Anti-human B7-H4Monoclonal Antibody And Development Of An ELISA Kit For SB7-H4

B7-H4(B7x, B7S1) is a recently identified B7family member that negativelyregulates T cell immunity by the inhibition of T cell proliferation, cytokine production,and cell cycle progression. The genomic DNA of human B7-H4is mapped onchromosome1comprised of six exons and five introns spanning66kb, and encodes aprotein of282amino acids belonging to the immunoglobulin superfamily. B7-H4mRNA is widely distributed in lymphoid and non-lymphoid tissues, butimmunohistochemistry showed that B7-H4protein is only weakly expressed in somenormal tissues. In recent years, many studies have shown that B7-H4was highlyexpression in a majority of human cancers, such as ovarian cancer, breast cancer, lungcancer and others, suggesting that B7-H4may play a role in protecting tumor cells fromimmune surveillance. In addition, B7-H4also play an important role in maintainingimmune tolerance. Therefore, B7-H4pathway provide a new treatment ideas forcancer, autoimmune diseases, transplant rejection and other diseases. Moreover, B7-H4is expected to become a potential target for treatment of autoimmune diseases andtumors. Preparation of specific mouse anti-human B7-H4monoclonal antibodies anddevelopment of specific, sensitive, stable human soluble B7-H4enzyme assay kitprovide not only a useful tool for further studying B7-H4’s biological function incancers but also a new means for diagnosis and treatments of patients with cancers.PartⅠ The preparation of anti-human B7-H4monoclonal antibodies andcharacterization of their biological functionsObjective: To prepare anti-B7-H4monoclonal antibodies (mAbs) and characterizetheir biological functions.Methods: The L929/B7-H4transgenic cells were used to immunize BALB/c mice.The spleen B cells of the mice were fused with murine myeloma cells (SP2/0) andpositive clones were screened by FCM. The fast-strip analysis was used to investigate murine Ig subclass. The biological functions of mAbs were identified by Western blot,Dot blot, competitive inhibition test and T cell proliferation inhibition blocking test.Results: Three hybridoma cell lines steadily secreting specific anti-human B7-H4was obtained, named5C8,5G3and8F10. Fast-strip method analysis displayed thatmAb5C8was mouse IgG2a and mAbs5G3,8F10were mouse IgG1. Western blot andDot-blot showed that5C8,5G3and8F10could specifically bind to human B7-H4protein. Competitive inhibition test evidenced that (1) three mAbs bound differentepitopes on B7-H4from commercial antibody H74;(2) compared with other two mAbs(1F10and2B2) against B7-H4our lab have prepared, three mAbs bound differentepitopes on B7-H4from1F10, mAbs5C8and5G3recognized closing binding siteswith2B2, mAb8F10bound different epitope on B7-H4from2B2;(3) competingbetween each other, mAb5C8bound different epitopes on B7-H4from5G3, mAb8F10recognized closing binding site with5C8and5G3. Finally, all the mAbs5C8,5G3and8F10could block the inhibitory effects of B7-H4on T cell proliferation.Conclusion: Three hybridomas secreting anti-human B7-H4mAbs were generated.These mAbs provide useful tool for further studying B7-H4’s biological function incancer.Part Ⅱ Development of an ELISA kit for human soluble B7-H4(sB7-H4) andits primary applicationObjective: To establish human sB7-H4ELISA kit.Methods: On the basis of three mAbs (5C8,5G3and8F10) we generated in thisstudy and other two mAbs (1F10and2B2) prepared in our lab, we used1F10as coatingantibody and biotinylated8F10as detecting antibody. An ELISA kit for human sB7-H4was developed and applied for the examination of serum levels of sB7-H4in peripheralblood from normal volunteers.Results: An ELISA kit for human sB7-H4was established successfully with3.13ng/mL of lower limitation measurement. The retrievable rate is92.98%~107.4%and coefficient of variance (CV) is <±2.82after the kit was stored at4℃for one month,suggesting that the kit was of sensitivity, stability and accuracy. Using this kit, theserum level of sB7-H4in peripheral blood from normal volunteers was detected40.00±11.93ng/mL. Conclusion: A sensitive and specific ELISA kit for detecting human sB7-H4isestablished.In summary, three hybridomas secreting anti-hunan B7-H4monoclonal antibodieshave been obtained and an ELISA kit for detecting hunman sB7-H4established. ThesemAbs and ELISA kit provide useful tool for further studying B7-H4’s biological function.