磁性免疫层析试纸条联合定量检测NSE、CEA的应用研究

发布时间：2019-05-13 17:09

【摘要】：研究背景:原发性支气管肺癌严重危害人类健康,其发病率和死亡率,居全球癌症首位。因75%肺癌患者确诊时已到了晚期肺癌,直接导致了肺癌的5年生存率仅为15.6%。其中小细胞肺癌恶性程度高,易早期发生转移和播散,预后极差。但小细胞肺癌在早期对放疗、化疗很敏感。目前,许多血清肿瘤标记物已被用于肺癌筛查、诊断及预后判断的指标。神经元特异性烯醇化酶(Neuron-specific enolase,NSE)是小细胞肺癌最敏感最特异性的肿瘤指标。癌胚抗原(carcino-embryonic antigen,CEA)是被用于多系统肿瘤的诊断及预后判断。其中,约有30%～70%的肺癌患者CEA水平明显升高。对人血清中NSE和CEA进行联合检测有助提高肺癌的筛查阳性率及早期诊断的准确性。血清NSE及CEA的传统检测方法有酶联免疫吸附试验和电化学发光免疫检测法,该两种方法具有较高的灵敏度,但需要大型检测设备,复杂的检测步骤及专业的操作人员;这些使其无法应用在大规模人群中进行肺癌筛查。免疫层析测定法(Immunochromatographicassay,ICA)是近年发展起来的一种新型快速免疫检测技术。ICA通常以胶体金、纳米银粒子以及荧光材料等作标记物,在层析检测过程,免疫探针与待测物反应形成免疫复合物,被对应的捕获抗体捕获并富集于硝化纤维膜上,以检测区上信号条带的显色与否、颜色深度来定性分析或通过检测反射光强度来定量分析。近年来,利用磁性纳米粒子作为免疫探针来检测磁信号作定量分析成为免疫层析检测技术的研究热点。由于待测样品没有磁背景干扰及磁信号检测不受光漂白等影响,与传统光学免疫层析技术相比,磁性层析技术的信噪比更高,检测信号更稳定。结合配套的磁信号检测设备,不仅可以提供定量检测的精确值,还可以提高检测的灵敏度。研究方法:采用EDC碳二亚胺法分别制备NSE和CEA免疫探针;利用制备的免疫探针组装对应的磁性免疫层析联检试纸条,并优化制备条件;通过检测不同梯度浓度的NSE、CEA标准抗原,结合配套磁信号检测设备来进行定量分析;通过检测临床血清样本,评估该方法的敏感性、特异性和准确性。研究结果:该免疫层析试纸条可用于快速定量联合检测NSE和CEA,且具有特异性好、灵敏度高的优点(NSE的检测下限为0.094ng/mL,CEA检测下限为0.045ng.mL)。对130份临床血清进行验证,证实该磁性试纸条可用于未经预处理的血清检测。其检测结果与商业电化学发光法试剂盒的结果一致性良好(n= 30,R20.990,P0.001);加标回收试验证实该试纸条具有高的检测精确度。研究结论成功建立了基于磁性纳米粒子的免疫层析试纸条,实现对NSE和CEA的快速联合定量检测,且检测灵敏度高,特异性好,精确度高;可作为小型医院,社区门诊和家庭医生实现肺癌快速筛查的有效辅助手段。
[Abstract]:Background: primary bronchogenic cancer is seriously harmful to human health, and its incidence and mortality are among the highest in the world. Because 75% of patients with lung cancer had advanced lung cancer at the time of diagnosis, the 5-year survival rate of lung cancer directly caused by lung cancer was only 15.6%. Among them, small cell lung cancer is highly malignant, prone to early metastasis and dissemination, and the prognosis is very poor. But small cell lung cancer is sensitive to radiotherapy and chemotherapy at an early stage. At present, many serum tumor markers have been used for lung cancer screening, diagnosis and prognosis. Neuron-specific enolase (Neuron-specific enolase,NSE) is the most sensitive and specific tumor index for small cell lung cancer. Carcinoembryonic antigen (carcino-embryonic antigen,CEA) is used in the diagnosis and prognosis of multisystem tumors. Among them, the level of CEA was significantly increased in about 30% of lung cancer patients. The combined detection of NSE and CEA in human serum is helpful to improve the positive rate of lung cancer screening and the accuracy of early diagnosis. The traditional detection methods of serum NSE and CEA are enzyme-linked immunosorbent assay (Elisa) and electrochemiluminescence immunoassay (ECLIA). These two methods have high sensitivity, but need large detection equipment, complex detection steps and professional operators. These prevent it from being used for lung cancer screening in large populations. Immunochromatography (Immunochromatographicassay,ICA) is a new rapid immunodetection technique developed in recent years. ICA usually uses colloidal gold, silver nanoparticles and fluorescent materials as markers. The immune probe reacts with the object to form an immune complex, which is captured and enriched on the nitrocellulose membrane by the corresponding capture antibody to detect whether the signal band is colored or not in the area. Color depth for qualitative analysis or quantitative analysis by measuring the intensity of reflected light. In recent years, the use of magnetic nanoparticles as immune probe to detect magnetic signals for quantitative analysis has become a research focus of immune chromatography technology. Because there is no magnetic background interference and magnetic signal detection is not affected by photobleaching, compared with the traditional optical immunochromatography, the signal-to-noise ratio (SNR) of magnetic tomography is higher and the detection signal is more stable. Combined with the supporting magnetic signal detection equipment, it can not only provide the accurate value of quantitative detection, but also improve the sensitivity of detection. Methods: NSE and CEA immune probes were prepared by EDC carbodiimide method, and the corresponding magnetic immunochromatographic test strips were assembled with the prepared immune probes, and the preparation conditions were optimized. The sensitivity, specificity and accuracy of the method were evaluated by detecting NSE,CEA standard antigens with different gradient concentrations and combining with supporting magnetic signal detection equipment, and the sensitivity, specificity and accuracy of the method were evaluated by detecting clinical serum samples. The results showed that the immunochromatographic strip could be used for rapid and quantitative combined detection of NSE and CEA, and had the advantages of good specificity and high sensitivity (the detection limit of NSE was 0.094 ng / mL, and the detection limit of 0.045ng.mL was 0.094 ng / mL). 130 clinical serum samples were verified, and it was confirmed that the magnetic test strip could be used for unpretreated serum detection. The results of the test were in good agreement with those of the commercial electrochemical luminous kit (n = 30, R20.990, P0.001), and the standard recovery test confirmed that the test strip had high detection accuracy. Conclusion the immunochromatographic strip based on magnetic nanoparticles was successfully established to realize the rapid joint quantitative detection of NSE and CEA, and the detection sensitivity, specificity and accuracy were high. It can be used as an effective auxiliary means for rapid screening of lung cancer in small hospitals, community outpatients and family doctors.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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