摘要
目的:制备和鉴定基质金属蛋白酶-2(MMP-2)单克隆抗体(mAb),检测其在人卵巢癌组织和裸鼠移植瘤中的表达。方法:利用戊二醛法制备人工抗原MMP-2-BSA和MMP-2-KLH,并免疫小鼠,采用标准杂交瘤技术,亚克隆筛选出单克隆细胞株,制备腹水,采用辛酸-硫酸铵法纯化mAb。以ELISA、Western blot法对抗体进行鉴定。通过免疫组织化学法与商品化多抗进行对比,并联合CA125将自制抗体应用于临床人卵巢癌组织和裸鼠移植瘤的检测。结果:人工抗原MMP-2-BSA和MMP-2-KLH合成成功。获取3株稳定分泌抗MMP-2的杂交瘤细胞株(3G10、4D12、1E8),抗体亚型均为IgG1。其中3G10反应性最强。经间接ELISA法检测纯化后抗体的效价达1∶1×106。自制mAb应用于人卵巢癌组织切片和裸鼠模型检测,卵巢癌组织与正常组织之间表达差异有统计学意义(P<0.01)。卵巢癌患者CA125和MMP-2联合检测阳性率较单一检测高(P<0.01)。自制mAb的染色特性与商品化抗体相似,且检出率较商品化多抗高(P<0.01)。结论:采用人工合成多肽作为免疫原成功制备了与卵巢癌相关的特异性抗MMP-2 mAb。
AIM： To prepare and characterize monoclonal antibodies against matrix metalloproteinase-2 （MMP-2）, check its expression in the tissues of human ovarian cancer and transplanted tumors in nude mice. METHODS： MMP-2 were linked to the carrier protein bovine serumalbumin （BSA） and keyhole limpet hemocyanin （KLH） using glutar- aldehyde method to obtain MMP-2-BSA and MMP-2-KLH, respectively. The anti-MMP-2 monoclonal antibody was obtained through hybridoma technique, We established the cell strains secreting mAb by hybridoma technique and pre- pared the mAb by induction of ascites in vivo. The prepared mAb was purified by salting out with ammonium sulfate and identified by ELISA and Western blotting. We compared the mAb and commercial polyclonal antibody by immunohisto- chemistry and detected the expressions of MMP-2 and CA125 in ovarian cancer issues and transplanted tumor. RESULTS： The artificial antigen and 3 hybridoma cell lines secreting monoclonal antibodies （mAb） against MMP-2 were obtained. The subclasses of mAb were all IgGl. The titer of peritoneal exudates was 1：1 × 106. The expressions of MMP-2 and CA125 in transplanted tumor and ovarian cancer tissues were all high. The positive expression rate of MMP-2 checked using generated antibody was 71.2% （57/80）in ovarian cancer tissues and 16.67% （5/30） in normal tissues, with significant difference between them （P 〈 0.01）. In early stage, the positive rate of MMP-2 and CAt25 combined detection was higher than that of CA125 detection alone （P 〈 0.01 ）. The mAb was suitable for detecting the expression of MMP-2 in human tissues and gave results consistent with commercial polyclonal antibody. The mAb was more specific than commercial mAb （P 〈 0.01）. CONCLUSION： The anti-human MMP-2 mAb is successfully prepared, which may serve as a valuable tool in the functional studies of ovarian cancer.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2012年第11期1177-1181,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家"重大新药创制"科技重大专项(2008ZX09203-003)
安徽省教育厅自然科学基金(KJ2011B155)
淮南联合大学校级科研项目(CYB1002)
关键词
基质金属蛋白酶-2
单克隆抗体
卵巢癌
裸鼠
联合检测
matrix metalloproteinase-2
monoclonal anti-body
ovarian cancer
nude mouse
com-bined detection
