Preparation And Application Of A Polyclonal Antibody Against Human α-L-fucosidase From Primary Hepatocarcinoma Tissue

Part IAIM: To purify and characterizeα-L-fucosidase (AFU, EC3.2.1.51) from human hepatocellular carcinoma (HCC).MATERIALS AND METHODS: Ion exchange chromatography on CM-52 and ultrafiltration were used to separate AFU from crude extract of primary hepatocarcinoma tissue excised from operation. SDS-PAGE was performed to indicate the molecular weight of subunit ofα-L-fucosidase. Bradford method was used to detect protein concentration. 4-methylumbelliferyl-α-L-fucopyranoside (4-MUF) was used as a fluorescent substrate to quantify purified AFU activity in each step. Standard curve of known product of enzyme-substrate reaction was generated according to each specific optical density value of certain quantity of product.RESULTS: Human HCCα-L-fucosidase was purified 74–fold to apparent homogeneity with 15% yield. SDS-PAGE indicated the presence of one subunit of Mr 55000. Specific activity of AFU in pooled fraction by chromatography was 10085μkat/g.CONCLUSION: The purified fucosidase through Ion exchange chromatography can be used as an immunogen to generate specific antibody against human hepatocellular carcinoma. PartⅡOBJECTIVE: To prepare and purify a polyclonal antibody againstα-L-fucosidase from human liver cancer tissue and detect the localization of AFU in the tumorous tissue.METHODS: A polyclonal antibody was raised against purified AFU by immunizing the rabbit and obtained by anion exchange chromatography on DEAE-52 with stepwise elution after saturated ammonium sulfate fractionation. Working concentration of polyclonal antibody was measured using indirect ELISA. Expression of AFU in the malignant and adjacent liver tissue was observed by immuohistochemical staining using purified antiserum as a primary antibody.RESULTS: Titer and Working concentration of the polyclonal antibody measured by indirect ELISA were 1:32 000, 1: 4 000, respectively. Western-blot analysis indicated that the polyclonal antibody can recognize one protein band of Mr 55kD. Expression of AFU was observed in the cytoplasm and/or cell membrane of liver cancer tissue but not in that of adjacent tissue.CONCLUSION: The polyclonal antibody against AFU prepared in this experiment has high specificity and can be applied to the diagnosis of HCC by indirect ELISA.