Preparation,Identification And Preliminary Application Of Monoclonal Antibodies Against Liver-Specific Protein

Hepatocellular carcinoma (HCC) is the main type of primary carcinoma of the liver.The incidence ratio of HCC disease and death is high. So, it is important to research basicand clinic HCC and explore effective methods of diagnose and therapy. The technic ofmonoclonal antibody is the important tools in the research of modern life science. It hasindispensable affection in studying structure and function of gene and protein. Furthermore, itis widely applied in basic research, clinic and therapy of tumors. In this research, twomonoclonal antibodies were obtained against liver-specific protein LFIRE-l/HFREP-1 inorder to provide experimental researching tools for LFIRE-l/HFREP-1. The process andresults were as following: BALB/c mice were given three immunizations with LFIRE-l/HFREP-1 protein purifiedfrom prokaryotic expression system. The serum was separated to develop indirect ELISA.The fusion was performed 3 days after the fourth immunization with LFIRE-l/HFREP-1protein, polyethylene glycol (PEG) acting as the fusogen. The ratio of splenic lymphocytes toSP2/0 myeloma cells was 9:1. The fusion rate was 94.1%, the positive rate was 89% afterscreening with indirect ELISA. Two hybridomas producing anti-LFIRE-l/HFREP-1monoclonal antibodies, named LF307-1, LF560-4, respectively, were obtained bylimiting dilution after 3 times subcloning and Western blot detection. Large amounts ofmonoclonal antibodies were produced by production of ascites. Collecting all the fluidpurified by protein G-argarose, the characterization of antibodies were performed by SDS-PAGE, ELISA test. The results indicated: The molecular weight of monoclonal antibodywas 156.4KDa, the titer of ascitic Mabs reached 1:1 X 107. The Ig subclass of LF307-1,LF560-4 were IgG2a (K ) and IgG ( K ), respectively. Using monoclonal antibody, LFIRE-l/HFREP-1 protein was detected in normal humanliver tissue, HCC cell lines and other tumor cell lines. The results indicated: the expression ofLFIRE-l/HFREP-1 was selectively in normal human liver tissue, whereas the expressipnlevel was reduced or undetectable in HCC cell lines and other tumor cell lines. Then Mabdetected the expression of MFREP-1, which is ortholog gene of LFIRE-l/HFREP-1 , inmouse liver, spleen, stomach, kidney, lung and heart. The results indicated: the expression ofMFREP-1 protein was specifically in mouse liver tissue, but the expression level wasundetectable in mouse's other visceras. At last, the location of LFIRE-l/HFREP-1 protein inHepG2 was ascertained by cell immunoflourescence technic. The results indicated: LFIRE-l / H F R E P - 1 p r o t e i n i s l o c a t e d i n c y t o p l a s m o f H e p G 2 .Anti-liver-specific LFIRE-l/HFREP-1 protein Mab was prepared successfully and appliedpreliminarily, it provides a useful reagent and experimental means for further studying thefunction of LFIRE-l/HFREP-1. VICandidate: Liu Hailan Major: Basic Veterinary Science Supervisor: Yan Yunqin...