| Immune response is a complicated and strictly regulated process, which involves a variety of immune cells and molecules (membrane-anchored and soluble). Costimulatory molecules, a series of membrane-anchored ones, perform an essential function due to their regulative expression, interaction and signal transduction during the process, and one of them is B7/CD28 interaction.Human B7-1(CD80), a 44-54KD type I transmembrane glycoprotein, which is encoded by a gene located at 2q33 and regarded as radical costimulatory molecules, belongs to immunoglobulin superfamily (IGSF). CD80 is expressed on many types of cells including actived B, T, dendritic cells and monocyte actived by interferon Y , and binds its receptor, CD28/CTLA-4, which mediates necessary signals promoting T cell activation, proliferation and function commitment. For this reason, enhancing costimulatory signals is available for immunotherapy of cancer; paradoxically, blocking them results in the immune tolerance specific to T cells and contributes to treatment of autoimmune disease, hypersensitivity and allogenetic graft rejection. Therefore, the research of anti-CD80 mAbs and investigating of their regulatory functions on B7/CD28 signal transduction may have significant theoretic and clinical value.In this study, a multiple myeloma (MM) cell line transfected with human B7-1 gene XG7-B7, was used to immunize Balb/c mice. The immunized spleencytes were fused with mouse myeloma cells (SP2/0) by using polyethylene glycol (PEG), and then they were cultured in HAT selection medium. With L-B7cells, a murine fibroblast cell line transfected with human B7-1 gene, the hybridomas secreting specific mAbs were screened by immunoflurescence assay, repeatedly cloning and screening, and four hybridoma cell lines (1F11, 3H8, 6H2 and 7B10) continuously and steadily secreting specific anti-human B7-1 (CD80) were obtained. These hybridomas grew well after long-term culturing in vitro and stored in liquid nitrogen.These mAbs were produced by in-vivo procedures in mouse peritoneal cavity and the ascitic titers was all over 1:1000 dilution by flow cytometry assay, in which every 5X 105 cells required 0.25-2.00 microgram purified mAb. Fast-strip method analyses displayed that these mAbs belong to mouse IgGl (1F11, 6H2 and 7B10) or IgM (3H8) subclass, respectively.To further elucidate the recognition ability of these prepared mAbs against B7-1 molecule, the phenotype analysis was utilized by flow cytometry assay. The result indicated that these mAbs could recognize both B7-1 molecule expressed on B7-1 transfected cells (i.e. XG7-B7, L-B7) and on the lymphoma Raji cell line as well as on B cells immortalized by Epstein-Barr virus (EBV), DC, PBTC and tonsil lymphocytes, but not on negative contols, i.e. XG7 and L cells. These results suggested that the antigen-antibody binding in this particular circumstance to cells had high specificity and affinity.In the competition test with PE-conjugated standard CDSOmAb (Immunotech), 1F11, 6H2 and 7B10 could not completely block the binding of standard mAb to B7-1 molecule on XG7-B7 cells. It suggested that these mAbs recognized the different epitope from that of standard mAb. In the interest of distinguishing the different epitope between these mAbs (1F11, 6H2 and 7B10), an additional matching competition test was bestowed by using of these mAbs labeled with isotope 125I to analyze the recognizing epitopes. The results demonstrated that IF 11 recognized the different epitope from these of 6H2 and 7B10; the epitope recognized by 6H2 and 7B10 was identical or similar. This wasthe precondition to establish sCDSO ELISA kit and we successfully established preliminary ELISA methods for measuring sCDSO on the basis of identifying their different epitopes. Thus, these mAbs might have a potential value in the diagnosis for related diseases.The neutralizing activity of these mAbs was determined by 3H-TdR incorporation. The results showed that these three mAbs (1F11, 6H2, 7B10, except 7B10) could block the proliferation of re...
|
PDF Full Text Request