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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。
OBJECTIVE: To prepare and identify monoclonal antibody (mAb) of matrix metalloproteinase-2 (MMP-2) for detection in human ovarian cancer and xenografted tumors in nude mice. Methods: The artificial antigens MMP-2-BSA and MMP-2-KLH were prepared by glutaraldehyde method. The mice were immunized with the standard hybridoma technique. The monoclonal cell lines were subcloned to prepare ascites. Purification of mAbs. Antibody was identified by ELISA and Western blot. By immunohistochemistry and commercial multi-resistance comparison, combined with CA125 self-made antibodies used in clinical human ovarian cancer and xenograft tumor detection. Results: The artificial antigens MMP-2-BSA and MMP-2-KLH were successfully synthesized. Three hybridoma cell lines stably secreting anti-MMP-2 (3G10, 4D12 and 1E8) were obtained. The antibody subtypes were both IgG1. Among them, 3G10 is the most reactive. The titer of purified antibody was 1: 1 × 106 by indirect ELISA. The self-made mAb was applied to the detection of human ovarian cancer tissue sections and nude mice model, and the difference was statistically significant (P <0.01) between ovarian cancer tissues and normal tissues. The positive rate of combined detection of CA125 and MMP-2 in ovarian cancer patients was higher than that in single test (P <0.01). The staining of self-made mAb was similar to the commercial antibody, and the detection rate was higher than that of commercial antibody (P <0.01). Conclusion: The anti-MMP-2 mAb associated with ovarian cancer was successfully prepared by using synthetic peptides as immunogens.