抗人肝癌组织α-L-岩藻糖苷酶抗体的制备及运用

Preparation and application of a polyclonal antibody against human ɑ-L-fucosidase from primary hepatocarcinoma tissues

目的:制备和纯化肝癌特异性α-L-岩藻糖苷酶(α-L-fucosidase,AFU)多克隆抗体及初步研究其在肝癌诊断中的临床作用。方法:用自行提取和纯化的人肝癌组织AFU免疫家兔,以间接ELISA检测抗血清效价和工作浓度。抗血清经阴离子交换法纯化后采用Western-blot鉴定纯化的AFU和免疫组化方法定位AFU在肝癌组织中的分布情况。结果:抗血清的效价和工作浓度经间接ELISA检测分别为1∶32000和1∶4000。抗血清经过饱和硫酸铵沉淀,阴离子交换树脂DEAE-52纯化后可在第115收集管前后出现一明显洗脱峰。Western-blot显示纯化的多克隆抗体可特异性地识别相对分子质量为55×103的AFU。免疫组化表明,AFU多表达于肝癌组织的细胞质和(或)胞膜而不表达于癌旁组织。结论:本实验制备的抗人肝癌组织AFU多克隆抗体可用于检测肝癌组织和血清中AFU的表达水平。

OBJECTIVE： To prepare and purify a polyclonal antibody against α-L-fucosidase （AFU） from human liver cancer tissue and detect the localization of AFU in the tumorous tissues. 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. The working concentration of polyclonal antibody was measured by u- sing indirect ELISA. Expression of AFU in the malignant and adjacent liver tissues was observed by the immuohistochemical staining using purified antiserum as a primary antibody. RESULTS： The 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 55× 10^3. Expression of AFU was observed in the cytoplasm and/or cell membrane of liver cancer tissues but not in that of adjacent tissues. 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.