检测外周血MUC1蛋白双抗体夹心ELISA方法的建立及初步应用

Establishment and preliminary application of indirect sandwich ELISA for MUC1 protein detection in peripheral blood

目的为提高外周血MUC1蛋白检测的敏感度,建立了抗MUC1多克隆抗体的夹心ELISA试剂盒,并对其进行了初步测试。方法首先成功构建-表达并纯化了MUC1-GST和MUC1-MBP融和蛋白;通过免疫家兔和大鼠,获得抗MUC1血清,经饱和硫酸铵沉淀、Protein A/G纯化及抗GST和MBP抗体吸收的进一步纯化获得纯的家兔抗人及大鼠抗人MUC1多克隆抗体;通过凝血酶溶解MUC1-GST获得MUC1标准品。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MUC1抗体作为检测抗体的双抗体夹心试剂盒,敏感度可达到0.2 ng/ml。结果对32例乳腺癌患者和20例健康受试者外周血血清中MUC1蛋白水平的检测结果显示,乳腺癌患者的阳性检出率达到53.1%(17例/32例),而健康对照组检出率为0。结论本研究建立的抗MUC1多克隆抗体双夹心试剂盒有望应用于临床诊断。

To establish and apply an indirect sandwich ELISA kit for MUC1 protein detection in peripheral blood.The MUC1-GST and MUC1-MBP fusion protein were expressed and purified,and then anti-MUC1 serum was obtained by immunization of rabbits and rats. In order to further purify rabbit and rat anti-human MUC1 polyclonal antibody,the serum was purified using saturated ammonium sulfate, protein A/G,and anti-GST and anti-MBP antibody absorption.The standard MUC1 peptide was prepared with MUC1-GST dissolved by thrombin.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 sandwich ELISA kit was up to 0.2 ng/ml.It was detected that the levels of MUC1 protein in the peripheral blood serum of 32 cases of breast cancer patients and 20 healthy subjects,which means the positive detection rate of breast cancer patients was 53.1%（17 cases / 32 cases）,while 0 in healthy control group.All the results support that the established anti-MUC1 polyclonal antibody indirect sandwich ELISA kit is sensitive and expected to be used in clinical diagnosis.