不同免疫表位抗Mam-A单克隆抗体用于原位乳腺癌诊断的实验研究

摘要

目的 基于表位特异性抗体免疫组化染色探讨乳腺珠蛋白A(mammaglobin A,Mam-A)表达或免疫活性在乳腺组织与乳腺癌组织之间的差异,并探讨这种差异用于区分正常乳腺组织与原位乳腺癌的可行性与诊断效能.方法 筛选针对Mam-A不同免疫表位的单克隆抗体,分别用于正常乳腺组织与乳腺癌组织的免疫组化染色;以平均光密度值作为指标,通过计算机辅助的光密度值识别定量分析免疫组化染色特征,筛选在两种组织之间染色特征差异较为显著的表位特异性抗体,进一步对筛选的表位特异性抗体用于原位乳腺癌诊断的可行性与效能进行评价.结果 筛选获得4株抗Mam-A表位特异性单抗mAb1152、mAb11617、mAb995与mAb656.分别对正常乳腺组织与原位乳腺癌组织的连续切片在相同条件下进行免疫组化染色显示,mAb1152、mAb11617与mAb995平均染色光密度值在两种组织之间差异较为显著;ROC曲线分析显示,基于mAb1 152、mAb11617与mAb995的曲线下面积均＞0.5,其中mAb11617优于mAb1152和mAb995;在各自最佳工作点下,mAb11617诊断效能最优,特异性90％,灵敏度59.62％;进一步分析显示,mAb11617与mAb995联合用于原位乳腺癌诊断的灵敏度达81.48％,特异性达90％,具有明显的诊断意义.结论 Mam-A蛋白表达或免疫活性在乳腺组织与乳腺癌组织之间存在明显差异,这种差异可通过表位特异性的抗体染色与计算机辅助技术进行一定程度识别,具有重要诊断价值.%Objective To investigate the difference between mammary gland tissues and breast cancer tissues.Methods Monoclonal antibodies against Mam-A immunized epitopes were screened for immunohistochemical staining of normal breast tissues and breast cancer tissues.The average optical density was used as an index to identify the quantitative data by computer-aided technology to screen epitope-specific antibodies with significant difference in staining characteristics between two types of tissues.Furthermore the feasibility and effectiveness of breast cancer diagnosis were evaluated.Results Four anti-Mam-A epitope-specific monoclonal antibodies,mAb1152,mAb11617,mAb995 and mAb656,were obtained.Immunohistochemical staining showed that the average density of mAb1152,mAb11617 and mAb995 was significantly different between the two types of tissues.The difference was significant between normal breast tissues and breast cancer tissues under the same conditions.The results showed that mAb11617 was better than mAb1152 and mAb995.At the best working point,mAb11617 was the best,the specificity was 90％ and the sensitivity was 59.62％.Further analysis showed that the sensitivity of mAb11617 combined with mAb995 in the diagnosis of in situ breast cancer was 81.48％ and the specificity was 90％,which was of great diagnostic significance.Conclusion There is significant difference between breast tissues and breast cancer tissues in Mam-A protein immunological activity or expression.This difference,which can be recognized by the specific antibody staining and computer aided technology,is of important diagnostic value.