摘要
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。
To optimize an indirect sandwich ELISA kit for serum MUC1 detection,the rabbits and rats were immunized by subcutaneous injection of purified recombinant MUC1-GST and MUC1-MBP fusion protein that have been successfully constructed in our laboratory respectively.We harvested and purified the anti-MUC1 serum.In the indirect sandwich ELISA kit,rabbit anti-human MUC1 antibodies and rat anti-MUC1 antibodies were used as coating antibodies and detecting antibodies,respectively.The sensitivity of the indirect ELISA kit was up to 0.2 ng/ml.The indirect ELISA was used to detect the levels of MUC1 protein in the peripheral blood serum of 40 cases of breast cancer patients,18 cases of breast benign disease patients,and 120 healthy subjects.After ROC curve analysis,The cut off point of breast cancer and breast benign disease patients is 2.75 ng/ml,while the cut off point of breast disease and healthy subjects is 1.86 ng/ml.testing results also showed that the positive detection rate of breast cancer patients and the benign breast disease patients was 97.5% and 66.7%,and the specificity of healthy subjects was 96.7%.The indirect ELISA kit was significantly higher than the CA15-3 kit in the accuracy of diagnosis of breast cancer.All the results supports that a sensitive and specific anti-MUC1 polyclonal antibody-sandwich ELISA is established and expected to be developed as a conventional ELISA kit in clinical diagnosis.Especially,the method can be applied to breast cancer mass screening and early diagnosis.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2012年第5期436-440,共5页
Immunological Journal
基金
国家自然科学的基金(30972782)
吉林省科技厅计划支撑重点项目(20080931)