Preparation And Functional Characterization Of Anti-human Monoclonal Antibody Against Recombinant Extracellular Domain Of HAb18G/CD147

HAb18G, a hepatoma associated antigen, is a heavily glycosylated membrane protein. The cDNA sequence of HAbl8G has been cloned by hepatoma monoclonal antibody HAb18 screening from the human hepatoma cDNA library in our lab. The amino acid sequence of HAbl8G is identical to that of CD 147 molecule, a member of immunuglobulin super family (IgSF). CD 147 was also named as extracellular matrix metalloproteinase inducer (EMMPRIN), Basigin, Nureothelin, and so on. Previous studies have demonstrated that HAbl8G/CD147 stimulates fibroblast cells to produce elevated levels of several matrix metalloproteinases(MMPs) which degrade basement membrane and extracellular matrix. Furthermore, HAbl8G/CD147 is also a potential adhering molecule. It can paticipate in the adherence of cell-cell, cell-matrix through forming protein complex with α ₃ β 1, α 6 β ₁, which belong to family intergrin. The exertion of the function mostly depend on the expression and glycosylation of its extracellular domain. Therefore, HAb18G/CD147 extracellular domain(HAb18Ged) is the important functionalfragment, and was used as an immunogen in this experiment. Our study aimed to establish hybridoma cells which stably secrete anti-HAbl8Gedmonoclonal antibody and obtained the purified antibodies. The second purpose of the study is to identify the character and specificity. The thirdpurpose is to detect theirs biological functions in vivo and in vitro.The experiment consists of three parts. Firstly, Balb/c mice were immunized with HAb18Ged. Hybridoma cells were screened by cell fusion and subcloning approach. The monoclonal antibodies in the ascites were purified by ion exchange chromatography. The light chain and heavy chain gene of McAbs were amplified by RT-PCR. Then the sequences were analyzed. Secondly, character and specificity of the McAbs were identified by using indirect ELISA, Sigma ImmunoType?Kit, fluorescence-activated cell sorting analysis (FACs) and immunohistochemistry. Thirdly, the biological functions of the McAbs were investigated by employing the following methods respectively: gelatin zymography, collagenase type I zymography, matrigel-boyden degradation chamber method, chemotaxis migration, adhesion assay and angiogenesis experiment.Two hybridoma cells HAb18Gedomabl, HAb18Gedomab2 stably secreting anti-HAb18Ged monoclonal antibody were obtained. The purities of the McAbs were higher than 90%. The light chain and heavy chain gene of HAbl8Gedomab were amplified successfully. The titer of McAbs in ascites fluid were 1:10⁶,1:10⁵ respectively. The McAbs both belong to IgG1 subclass. HAb18Gedomabl showed high specificity and affinity to the antigen of FHCC-98 cell membrane and the tissue of hepatocellular carcinoma. The results of the biological function analysis suggested that the McAbs enhanced...