摘要
肿瘤标志物(Tumor markers,TM),在恶性肿瘤的辅助诊断、疗效监测、预后判断等方面具有非常重要的应用价值。因此,不断探索新的TM及其检测方法研究已经成为肿瘤防治的热点课题之一。流动注射分析(Flow iniection analysis,FIA)能在非平衡状态下高效率地完成试样的在线处理与测定,因而分析速度快、重现性好、易于自动化,已被广泛地应用于临床诊断、药物分析、环境和食品质量监控与处理等方面。电化学与化学发光免疫传感器(Electrochemical and chemiluminescent immunosensors)和时间分辨荧光免疫检测技术(Time-resolved fluoroimmunoassay,TRFIA)以其高灵敏度、高特异性、高准确度等优点而倍受关注。这些检测技术与分析方法的结合,为肿瘤标志物的高效检测奠定了基础。本文围绕肿瘤标志物的临床应用、流动注射-时间分辨荧光免疫分析新方法的建立和电化学与化学发光免疫传感器的研制等方面,开展了以下工作:
1.肿瘤标志物一胃泌素前体释放肽在肺癌诊断中的应用价值
ProGRP(Progastrin-releasing peptide)是脑肠激素的一种,是胃泌素释放肽(gastrin-releasing peptide,GRP)的前体。前人已建立检测ProGRP的酶免疫测定方法(Enzyme immunoassay,EIA)并用于小细胞肺癌(Small cell carcinoma,SCLC)的免疫珍断。本文利用该方法,对78例SCLC患者、15例肺良性疾病(肺结核、肺气肿、气胸)患者,15例健康人血清中ProGRP含量进行测定并对比分析,探讨了ProGRP在肺癌诊断中的应用价值。结果显示,SCLC患者血清ProGRP水平显著高于对照组(P<0.01);以大于46pg/ml为临界值,ProGRP对SCLC的阳性率高达64.6%;患者血清ProGRP与神经元特异性烯醇化酶(Neuron specific enolase,NSE)水平呈高度正相关,r=0.9,P<0.01。肺癌患者血清中ProGRP水平检测可望成为小细胞肺癌的辅助诊断指标。
The determination of serum tumor markers (TM) plays an important role in screening and auxiliary diagnosing a disease, monitoring therapy in the tumor prevention stage, as well as in the follow-up examination during therapy for the patients with certain tumor-associated diseases. The developments of new TM and novel immunoassay methods have been the focused object in tumor diagnosis. Flow injection analysis (FIA) has been applied to many fields such as clinical, pharmaceutical, environmental and food assays due to the shortened assay time, good reproducibility and easy automation for high sample throughput in non-equilibration state. The immunosensors based on electrochemical, chemiluminescent and time-resolved fluorescent detection methods have been interestingly researched due to the high sensitivity, characterization and accuracy. The combination of the different detection techniques and immunoassay methods has been the basis for the efficient determinations for the TM. This thesis focuses on the clinical application of TM, flow injection- time resolved fluorescence and the preparation of immunosensors based on the electrochemical and chemiluminescent detection.1. Detection and clinical significance of serum progastrin-releasing peptide in patients with lung cancerProgastrin-releasing peptide (ProGRP) is one kind of brain-gut hormone, and the precursor of gastrin-releasing peptide. The enzyme immunoassay method for ProGRP has been developed and applied in the diagnosis of small cell carcinoma. The levels of
serum progastrin-releasing peptide ProGRD in 78 patients with lung cancer were detected by using ELISA and compared with healthy controls and lung benign group to evaluate and probe into their clinical significance in distinctive diagnosis of lung cancer. The serum ProGRP levels in patients with small cell carcinoma (SCLC) were obviously higher than those of controls (P<0.01). Using 46 pg/ml as cutoff value the positive rate of ProGRP for SCLC was 64.6%. There was a proportional relation between ProGRP and NSE levels with a correlation coefficient of 0.9 (P<0.01). The detection of serum ProGRP was useful in auxiliary diagnosis of small cell lung cancer.2. Flow injection immunoassay for carcinoembryonic antigen combined with time-resolved fluorometric detectionA simple, sensitive and specific method has been developed for immunoassay of serum carcinoembryonic antigen (CEA) by combining the time-resolved fluorescence with high sensitivity and flow injection analysis with high flexibility. Based on a sandwich immunoassay format, a monoclonal antibody immobilized immunoaffinity column inserted in a flow system was used for immunoreactions. The Eu-labeled antibody and analyte antigen were injected into the immunoaffinity column respectively to form the sandwich immunocomplex. The enhancement solution that was used to cleave the Eu-labels from the immunocomplex was injected into the column after the above immunoreaction. Finally, the cleaved solution was collected and then detected by time-resolved fluorescence. Serum CEA could be detected in the linear range from 2.5 to 100 ng/ml with a correlation coefficient of 0.997 and the detection limit of 1.0 ng/ml. The whole assay time for one sample was 25 min. The coefficient variations for the concentrations of 2.0, 10 and 50 ng/ml were 3.5%, 1.0% and 7.65% for 10 times detection. Twenty-four human serum samples have been detected by this method were in good agreement with the results obtained by the electrochemiluminescence immunoassay (Elecsys(?)2010). This method realized the basic requirements for rapid, low-cost, sensitive and accurate assay and could be further developed for fast practical clinical detection of serum CEA levels.3. Chemiluminescent and electrochemical immunosensors for the tumor markersA chemiluminescent immunosensor for carbohydrate antigen 19-9 (CA19-9) based on the immobilization of CA19-9 on the cross-linked chitosan membrane was
developed. Based on a noncompetitive immunoassay format, after an off-line incubation of the analyte CA19-9 with ho
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