Development And Preliminary Application Of A Double MAb Sandwich ELISA To Detect Soluble CD226 And The Study On Production Mechanism Of Soluble CD2

Postgraduate Ning Shuang-feiSupervisor Jin Bo-quanAssistant Tutor Jia WeiDepartment of Immunology, Fourth Military Medical University, Xi'an ChinaApril 2002Many members of human leukocyte differentiation antigens (LDA) have been shown to generate their soluble counterparts. These soluble forms in human body fluids may play an important role under physiological or pathological conditions, such as serving as antagonists, agonists, carrier proteins or markers of disease activity. Some soluble forms of cytokines and adhesion molecules may be used as therapeutic or diagnostic agents.CD226, also was named platelet and T cell activation antigen 1 (PTA1) and DNAX accessory molecule-1 (DNAM-1), was found in 1985 through an monoclonal antibody (mAb) LeoAl, which was produced by immunizing the Balb/c mice with the activated T lymphocytes from mixed lymphocytes culture. CD226 expresses on activated T lymphocytes, NK cells, and megakaryocyte/platelet lineage and may be involved in T cell activation and differentiation, cytotoxicity inhibition of NK. and LAK cells, platelet activation and aggregation as well as signal transduction. Recently, the soluble counterpart of CD226 has been found in human serum by sandwich ELISA using rabbit polyclonal sera paired with monoclonal antibody. The serum soluble CD226(sCD226) levels in patients with tumor and graft rejection reaction are higher than that in healthy people. In this study, wedeveloped a double monoclonal antibodies (mAb) sandwich ELISA for detection of sCD226 and investigated the production mechanism of sCD226.Firstly, we developed a sandwich ELISA with two mAb LeoAl and FMU3 recognizing different epitopes of CD226 extracellular region. The ELISA system was optimized with the detection limitation of HOng/L when ABTS used as substrate. The very low or negative absorbance obtained from ELISA by using human IgG and RPMI 1640 complete medium instead of PTAl/IgG and the result of blocking test showed that the system is specific. The coefficients of variant (CV) of intra-assay (3.4%~4.3%) and inter-assay (11.8%~15.6%) indicated that the ELISA is reproducible. Sera from healthy adults and some patients then were detected by this system. Statistical analysis showed that the serum level in patients with rheumatoid arthritis (RA) was significantly higher than that in normal control subjects. However, the serum levels of sCD226 in some individuals are lower than the detection limitation and cannot be quantified.To improve the sensitivity, we employ TMB as substrate and re-develop the ELISA system. As a result, the detection limitation of the new system comes to 27ng/L. the results of cross-reaction test and blocking test proved its specificity. Its CVs of intra-assay and inter-assay are between 4.0 %~4.5 % and 11.3 %~18.4 %, respectively. Applying it to detect the sera from healthy adults and some patients, we found that the sera levels of sCD226 in patients with rheumatoid arthritis (RA), psoriasis as well as hemorrhagic fever with renal syndrome (HFRS) are significantly higher than that in normal control adults. Particularly the serum sCD226 level from patients with psoriasis may be refered to as a clinic marker.According to the cDNA of CD226, we designed two primers before PBMC was isolated and the RT-PCR was performed. The result showed there was no alternative splicing form in PBMC or maybe the express level of CD226 mRNA is too low to detectable. But sCD226 in the supernatants of PBMC can be detected. We also transfected the plasmid pEF-BOS-CD226 containing the whole open reading frame of CD226 cDNA into COS? cells and cultured. The supernatants and the cells werecollected in various times and analyzed by ELISA and FCM, respectively. The results showed that not only can the transfected COS? cells express membrane form of CD226, but also produce soluble form of CD226 into the supernatants. Thus we concluded that the production of sCD226 depends mainly on the proteolytic cleavage. Although the mechanism o...