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目的优化双抗体间接夹心ELISA试剂盒,并探讨其在乳腺癌患者中检测MUC1黏蛋白水平的应用价值。方法用基因重组MUC1-GST和MUC1-MBP融和蛋白免疫家兔和大鼠,获得抗MUC1血清,并对其纯化,获得纯化的家兔抗人及大鼠抗人MUC1多克隆抗体;经不同的筛选确立了以家兔抗人MUC1抗体作为包被抗体、大鼠抗人MUC1抗体作为检测抗体的双抗体间接夹心试剂盒,敏感度可达到0.2 ng/ml。结果应用建立的试剂盒对40例乳腺癌,18例乳腺良性疾病和120健康对照者血清中MUC1蛋白水平的进行检测,检测结果绘制ROC曲线,分析得出以2.75 ng/ml为乳腺癌患者与乳腺良性疾病患者的临界值,以1.86 ng/ml为乳腺疾病与正常人为临界值,检测结果表明本研究对乳腺癌诊断的阳性率高达97.5%,乳腺良性疾病的阳性率为66.7%,正常人特异性为96.7%。对于乳腺癌同一病例样本用酶联免疫法CA15-3诊断试剂盒进行对比检测,其检出率为3.33%,特异度为100%。绘制ROC曲线对比显示,本研究所建立的双抗体夹心ELISA方法对乳腺癌诊断的准确度明显高于CA15-3试剂盒。结论本研究成功建立了特异性强,灵敏度良好的双抗体间接夹心ELISA试剂盒,有望开发为临床辅助诊断的常规试剂盒,尤其有望应用于乳腺癌的大规模筛查及早期诊断。
Objective To optimize the double antibody indirect sandwich ELISA kit and to explore its value in the detection of MUC1 mucin in breast cancer patients. Methods Rabbit and rat were immunized with gene fusion MUC1-GST and MUC1-MBP fusion protein to obtain anti-MUC1 serum and purified to obtain purified rabbit anti-human and rat anti-human MUC1 polyclonal antibody. After different The rabbit anti-human MUC1 antibody as coating antibody and rat anti-human MUC1 antibody as the detection antibody were used to establish the indirect antibody sandwich ELISA kit with a sensitivity of 0.2 ng / ml. Results The serum levels of MUC1 protein in 40 cases of breast cancer, 18 cases of benign breast diseases and 120 healthy controls were detected by using the established kit. The ROC curve was drawn and the results showed that with 2.75 ng / ml for breast cancer patients and The critical value of benign breast disease patients, the threshold value of 1.86 ng / ml for breast disease and normal people, the test results show that the diagnosis of breast cancer in this study as high as 97.5%, breast benign disease was 66.7%, normal The specificity is 96.7%. The same case of breast cancer ELISA kit CA15-3 for comparative testing, the detection rate was 3.33%, the specificity of 100%. Draw ROC curve comparison shows that the double antibody sandwich ELISA established in this study has a significantly higher diagnostic accuracy for breast cancer than CA15-3 kit. Conclusion This study successfully established a double-antibody indirect sandwich ELISA kit with strong specificity and good sensitivity. It is expected to be developed as a routine kit for clinical assistant diagnosis, especially for large-scale screening and early diagnosis of breast cancer.