Preparation And Application Of Functional Monoclonal Antibody Against Human B7-1 Molecule

Human B7-1 (CD80), a 44-54KD type I transmembrane glycoprotein, belongs to immunoglobulin superfamily (IgSF). B7-1 is expressed on many types of cells including dendritic cells, macrophagocytes and actived B cells. B7 can ligand its receptor CD28/CTLA-4, delivering necessary signals promoting T cell activation, proliferation and functional commitment, which are the most radical costimulatory signals. For these reasons, enhancing costimulatory signals is available for immunotherapy of cancer, while, blocking the signals result in the immune tolerance specific to T cells and contributes to treatment of autoimmune disease, hypersensitivity and allogenetic graft rejection. Later research showed that the concentrations of soluble B7-1 (sB7-1) in patient serums are significantly higher than those in healthy volunteers, but the significance is unknown so far. Therefore, the preparation of anti-B7-1 mAbs and detection of sB7-l have significanttheoretic and clinical value.1. Preparation of monoclonal antibody against B7-1After pretreatment with mitomycin, Daudi, a human malignant B lymphoma cell line, which highly expressing B7-1 molecule, was used to immunize BALB/c mice. According to the hybridoma technique, the immunized mice spleencytes were fused with mouse myeloma cells (SP2/0). L929-B7-1 was used as positive screening cell line. Through repeated cloning and screening, one hybridoma (10D9) was eventually obtained. The hybridoma grew well after long-term culturing and storage in liquid nitrogen. Fast-strip analysis showed that subclass of 10D9 was IgG1. BALB/c mice were primed with pristane. Ascites was induced by intraperitoneal injection of well-grown hybridoma and the output is average to 5ml each mouse. Affinity chromatography was used to purify mAb, and the concentration of protein is 1.5~2.0mg/ml. Indirect immunofluorescence assay suggested the engagement of purified B7-1 mAb to cells was 0.2-1 μg/l×10~6 cells. The results of Western blotting showed that the strap was specific. Competition experiment indicated that 10D9 recognized a different antigen epitope from that of commercial mAb 2D10.