| Human leukocyte differentiation antigens (HLDA), the most important immune cell surface markers, are named as cluster of differentiation (CD) by the international workshop on leukocyte differentiation antigen. Platelet and I cell activation antigen I (PTA 1) is a novel HLDA and was designated as CD226 in the 7th HLDA. Initially this molecule was identified by mAb Leo Al and named T lineage-specific activation antigen I (TLiSA 1) since it was expressed on activated T cells in 1985. In 1989, the molecule was also found on platelet and was involved in platelet activation and aggregation, therefore it was renamed PTA 1. More experiments showed that CD226 was also expressed on NK and stimulated endotheliocytes and could be regulated by various cytokines. The human CD226 was cloned in 1996 and turned out to be a 67 kDa heavily glycosylated protein which belongs to iinmunoglobulin superfamily (IgSF) containing 2 lg-like domains of the V-set and encoded by a gene containing 7 exons on human chromosome I 8q22.3. The PTA 1 genes of gibbon and monkey were cloned in 1997 and the homology similarity of PTA 1 amino acids among human, gibbon and monkey is more than 93%. The development of NK and CTh could be inhibited when the Leo Al mAb was added at the beginning of mixed lymphocyte culture (MLC), and Leo Al mAb could up-regulate the NK cytotoxicily in redirected cytotoxicity assay (RCA). In addition, CD226 mediates cellular adhesion to other cells bearing an unidentified ligand and 2 cross-linking CD226 with antibodies causes cellular activation. Furthermore, some preliminary clinic research has revealed that the level of PTA 1 expression on lymphocytes was related to some virus infection diseases and autoimmune diseases. In this paper, we purified natural CD226 from platelets by affinity chromatography and prepared polyclonal antibody (PoAb) anti CD226 with the titer of 4 X I 0~ by immunizing the rabbit with the purified CD226 antigen. The MW of the purified natural CD226 is 67 kDa probed by CD226 PoAb in Western blot. Meanwhile Balb/c mice were immunized with purified natural CD226 antigen and another seven hybridomas named FMU 1-7 were obtained, which could secret mAbs against CD226. All seven mAbs reactivated well with purified natural CD226 and PTA lug fusion protein (but not with hIgG) in indirect ELISA as well as bound the COS7 transfected with the cDNA of CD226. The results of pairwise testing of epitope mapping analysed by biosensor showed that FMU1-7 together with the perviously reported PTA1 mAbs Leo Al and New El could recognize 7 different epitopes, which were Leo Al, New El, FMU3, FMU6 and FMU7 recognizing different epitopes respectively, while FMUI and FMU2 or FMU4 and FMU5 reacted with a same epitope, respectively. Moreover the Leo Al, New El, FMU1, and FMU2 epitopes or the FMU6 and FMU7 epitopes might be close to one another because the binding of one mAb to PTAI could interfere the binding of other mAbs in pairwise testing of epitope mapping assay. Finally the Leo Al (FMUI or FMU2) and FMU3 could be used in sandwich ELISA to detect the soluble CD226 (sCD226). A sandwich ELISA was established for detecting sCD226 either in culture supernatants of PBMC activated with TPA, PHA or CD3 mAb or in human serum. Kinetic investigation on sCD226 in cell culture supernatants and the membrane form of CD226 (mCD226) on activated PBMC showed that the time of sCD226 production was later than that of mCD226 expression, suggesting that the sCD226 may be derived f...
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