原发性肝癌肿瘤标志物化学发光酶免疫分析方法及应用研究
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摘要
原发性肝癌早期症状不明显,以致于确诊时多发展为癌症中、晚期,因此肝癌患者的死亡率较高,预后效果差。对抗肝癌的最有效手段是早期诊断,进行早期治疗。肿瘤标志物的检测在癌症的早期诊断中起着重要的作用；化学发光以灵敏、快速、无污染等特征,在生物分析方面得到越来越多的关注,并在临床检验领域得到了广泛的应用。本论文采用化学发光酶免疫分析方法,对肝癌肿瘤标志物的检测展开一系列研究,并对其临床检测效果进行了评价。主要内容如下：
1,综述了原发性肝癌的早期诊断方法,肝癌肿瘤标志物检测方法研究进展以及化学发光免疫分析在临床检验方面的应用。总结了建立免疫分析方法需要解决的主要的问题,并对其应用进行了展望。
2,为了提高原发性肝癌肿瘤标志物甲胎蛋白(AFP)的检测范围和灵敏度,在本研究中采用经过表面修饰的磁性微粒子作为固相载体,与固相抗体通过共价键特异结合,实现了AFP的宽范围检测。具体的工作有：
以碱性磷酸酶(ALP)为标记酶的化学发光酶免疫分析方法检测血清AFP含量。异硫氰酸荧光素(FITC)标记的AFP单克隆抗体、AFP样本和ALP标记的AFP抗体三者的免疫反应在均相中完成,形成三明治夹心复合物。再加入FITC抗体包被的磁颗粒用于免疫复合物与液相的分离,有效缩短了免疫分析时间,极大提高了线性检测范围(5.7-1300 ng-mL-1).
采用辣根过氧化物酶(HRP)为标记酶的化学发光免疫分析方法检测血清中AFP含量,有效降低了背景值和提高了检测灵敏度。在此工作中,磁颗粒包被固相一抗直接参与免疫反应,与AFP样本和HRP标记的AFP抗体三者形成夹心免疫复合物,实现了一步免疫分析。另外,磁颗粒和聚苯乙烯包被管两种固相载体的化学发光免疫分析方法进行了对比。结果显示,磁颗粒作为固相载体,有效缩短了总分析时间,提高了免疫试剂的利用率和线性检测范围(1.5-2000 ng-mL-1)。
3,AFP在原发性肝癌的早期诊断中存在阳性检出率不高的问题,因此寻找更灵敏、特异的标志物在原发性肝癌早期诊断中具有重要的临床价值。磷脂酰基醇蛋白聚糖-3(Glypican-3, GPC3)在肝癌患者的肝脏组织中显著表达,是潜在的肝癌肿瘤标志物,但多采用取样复杂的免疫组织学检测GPC3。在本部分工作中,实现了GPC3血清免疫学检测,并考察其在临床诊断中的应用价值。具体的工作如下：
125I标记GPC3蛋白与待测样本竞争结合羊抗人GPC3多克隆抗体,实现了血样中N端和C端GPC3的同时测定,并着重考察GPC3的血清水平在正常肝脏、乙肝、丙肝、肝硬化和原发性肝癌患者中的差异。结果显示,GPC3在原发性肝癌患者血清中的含量明显高于其它良性肝脏疾病患者和正常肝脏人群。并且与AFP和AFP/CA19-9联合检测相比,GPC3诊断灵敏度和特异性较高。
为了缩短分析时间,避免放射性污染以及提高分析灵敏度,在以上工作基础上建立了磁颗粒化学发光酶免疫分析方法检测血清GPC3,并考察了该方法用于诊断原发性肝癌的灵敏度与特异性,以及肝癌患者采取肝动脉化疗栓塞术(transarterial chemoembolization, TACE)后血清GPC3水平的变化。临床检测数据显示,GPC3在原发性肝癌早期诊断和预后监测中均有重要的临床参考价值；另外,在本工作中还着重分析了纳米磁颗粒与微米磁颗粒在免疫反应中的影响。结果显示,纳米磁颗粒在免疫分析中可更有效的保持生物分子的活性,提高免疫试剂利用率,缩短免疫分析时间。
4,建立了基于微流控芯片的肝癌肿瘤标志物的同时化学发光免疫分析方法。通过微通道表面的三维修饰,提高了抗体包被效果,增加了抗体包被量,设计了用于同时检测肝癌肿瘤标志物AFP和铁蛋白(Ferritin)微通道。该方法的特异性高,不存在交叉反应,AFP的检测范围可达到0.5-500 ng·mL-1, Ferri的检测范围可达到0.65-800 ng·mL-1。
5,在微流控芯片平台上实现了人外周血中肝癌细胞的快速化学发光检测。本工作结合免疫细胞化学的特异性、化学发光检测的灵敏性和微流控芯片平台的快速性,实现了肝癌细胞的定量检测,为肝癌的早期诊断和预后监测提供了更加灵敏、特异的检测平台。
Hepatocellular carcinoma (HCC) in its early stage is asymptomatic, and consequently the diagnosis of HCC is often delayed. Thus, the mortality rate is still high for pateints with liver cancers because of poor prognosis. An early diagnosis is the key to an effective treatment of HCC and tumor markers play an important role in early clinical diagnosis of cancers. Chemiluminescence has attracted great attentions in terms of sensitivity and small footprint in bioananlytical field, and has been applied in clinical diagnosis with a great market share. In this work, several chemiluminescence enzyme immunoassay(CLEIA) methods have been developed for the detection of HCC tumor markers as well as their applications in clinics. The major works are as follows:
1. The diagnostic methods for HCC and their development are reviewed. The application of chemiluminescence immunoassay for detection of tumor markers are introduced in detail. Then, we summarized whatever key works should be concerned and have a overlook about the application of CLEIA.
2. In order to improve the linear detection range and the sensitivity for the detection of AFP, magnetic particles with surface modification are employed, which can covently binding antibodies. The contents are briefly summarized as bellow,
CLEIA was developed by alkaline phosphatase (ALP) labeling AFP antibodies for the detection of AFP. A sanwich immo-complex was formed by immunoreaction between FITC labled AFP monoclonal antibodies, AFP samples and ALP labled AFP antibodies. Magnetic micro-particles coated with anti-FITC antibodies were used as solid phase to separate immuno-complex from the liquid system. The linear detection range was enlarged to 1300 ng-mL-1 with a shorter immunoassay time compared with conventional CLEIA based on microplates.
In order to decrease background interference and increase sensitivity, CLEIA with horseradish peroxidase labeling AFP antibodies was proposed for the detection of AFP. A one-step immunoassay was performed by reaction between magnetic micro-particles coated with AFP antibodies, AFP samples and HRP labled AFP antibodies to form sanwich immo-complex. Additionaly, polystyrene StartubesTM were used as solid phase for AFP antibodies coating in parallel with magnetic micro-particles. Our results indicated that the magnetic micro-particles based CLEIA showed better performance with less consumption of immunoreagents, larger linear detection range (2000 ng-mL-1) and shorter immunoassay time by comparing with StartubesTM based CLEIA.
3. AFP used in early diagnosis of HCC show low diagnosis positive rate, therefor more sensitive and specific tumor markers should be exlored in combination with or instead of AFP for the diagnosis of HCC. Glypican-3 (GPC3) highly expressed in liver tissues of HCC pateins, is a promising tumor marker for HCC, and its detection was often reported by molecular biochemistry. A serological immunoassay method was proposed in our work for detection of GPC3, and its application in clinical diagnosis was studied.
Firstly, N-terminal and C-terminal GPC3 circulated in blood were simultaneously determinaed by competitive radioimmunoassay between 125I labeled GPC3, GPC3 samples and goat anti-human GPC3 polyclonal antibodies. The levels of GPC3 were evaluated in normal liver, hepatitis B, hepatitis C, hepatocirrhosis and HCC by our proposed method. We got that the levels of GPC3 were obviously higher in pateints with HCC than other benigh liver diseases. Addtionaly, the diagnostic sensitivity and specificity of GPC3 were also much higher compared with AFP and AFP/CA19-9 combination detection.
Sencondly, in order to save assay time, avoid radio-pollution and increase sensitivity, magnetic nano-particles based CLEIA was proposed for detection of serological GPC3, and the performance of the method in terms of sensitivity and specificity were evaluated. The GPC3 levels in HCC after transarterial chemoembolization(TACE)treatment indicated that GPC3 was an effective tumor marker for prognosis of HCC patients. Additionally, the influence of nano-particles in immunoassay was compared with micro-particles. The results revealed that nano-particles based CLEIA exhibted better performance in terms of higher bioactivity, less consumption of immunoreagents and shorter immunoassay time than micro-particles based CLEIA.
4. Simultaneous detection of HCC tumor markers was developed by CLEIA on microfluidics device. Glass substrate was processed with three-dimensional modification to improve antibodies coating efficiency. AFP and Ferri were simultaneously determined on microfluidic chip with high performance. No cross-reaction occurred and high specificity was obtained. The linear detection range for AFP and Ferri were 500 ng-mL-1 and 800 ng-mL-1, respectively.
5. Capturing and detecting liver cancer cells circulating in peripheral blood were performed on a microfluidic chip by chemiluminescence. In order to realize early diagnosis and prognosis of HCC by counting liver cancer cells, immunocytochemistry, chemiluminescence and microfluidic chip were integrated together in this work with advantages of high specificity, sensitivity and time-saving.
1,综述了原发性肝癌的早期诊断方法,肝癌肿瘤标志物检测方法研究进展以及化学发光免疫分析在临床检验方面的应用。总结了建立免疫分析方法需要解决的主要的问题,并对其应用进行了展望。
2,为了提高原发性肝癌肿瘤标志物甲胎蛋白(AFP)的检测范围和灵敏度,在本研究中采用经过表面修饰的磁性微粒子作为固相载体,与固相抗体通过共价键特异结合,实现了AFP的宽范围检测。具体的工作有：
以碱性磷酸酶(ALP)为标记酶的化学发光酶免疫分析方法检测血清AFP含量。异硫氰酸荧光素(FITC)标记的AFP单克隆抗体、AFP样本和ALP标记的AFP抗体三者的免疫反应在均相中完成,形成三明治夹心复合物。再加入FITC抗体包被的磁颗粒用于免疫复合物与液相的分离,有效缩短了免疫分析时间,极大提高了线性检测范围(5.7-1300 ng-mL-1).
采用辣根过氧化物酶(HRP)为标记酶的化学发光免疫分析方法检测血清中AFP含量,有效降低了背景值和提高了检测灵敏度。在此工作中,磁颗粒包被固相一抗直接参与免疫反应,与AFP样本和HRP标记的AFP抗体三者形成夹心免疫复合物,实现了一步免疫分析。另外,磁颗粒和聚苯乙烯包被管两种固相载体的化学发光免疫分析方法进行了对比。结果显示,磁颗粒作为固相载体,有效缩短了总分析时间,提高了免疫试剂的利用率和线性检测范围(1.5-2000 ng-mL-1)。
3,AFP在原发性肝癌的早期诊断中存在阳性检出率不高的问题,因此寻找更灵敏、特异的标志物在原发性肝癌早期诊断中具有重要的临床价值。磷脂酰基醇蛋白聚糖-3(Glypican-3, GPC3)在肝癌患者的肝脏组织中显著表达,是潜在的肝癌肿瘤标志物,但多采用取样复杂的免疫组织学检测GPC3。在本部分工作中,实现了GPC3血清免疫学检测,并考察其在临床诊断中的应用价值。具体的工作如下：
125I标记GPC3蛋白与待测样本竞争结合羊抗人GPC3多克隆抗体,实现了血样中N端和C端GPC3的同时测定,并着重考察GPC3的血清水平在正常肝脏、乙肝、丙肝、肝硬化和原发性肝癌患者中的差异。结果显示,GPC3在原发性肝癌患者血清中的含量明显高于其它良性肝脏疾病患者和正常肝脏人群。并且与AFP和AFP/CA19-9联合检测相比,GPC3诊断灵敏度和特异性较高。
为了缩短分析时间,避免放射性污染以及提高分析灵敏度,在以上工作基础上建立了磁颗粒化学发光酶免疫分析方法检测血清GPC3,并考察了该方法用于诊断原发性肝癌的灵敏度与特异性,以及肝癌患者采取肝动脉化疗栓塞术(transarterial chemoembolization, TACE)后血清GPC3水平的变化。临床检测数据显示,GPC3在原发性肝癌早期诊断和预后监测中均有重要的临床参考价值；另外,在本工作中还着重分析了纳米磁颗粒与微米磁颗粒在免疫反应中的影响。结果显示,纳米磁颗粒在免疫分析中可更有效的保持生物分子的活性,提高免疫试剂利用率,缩短免疫分析时间。
4,建立了基于微流控芯片的肝癌肿瘤标志物的同时化学发光免疫分析方法。通过微通道表面的三维修饰,提高了抗体包被效果,增加了抗体包被量,设计了用于同时检测肝癌肿瘤标志物AFP和铁蛋白(Ferritin)微通道。该方法的特异性高,不存在交叉反应,AFP的检测范围可达到0.5-500 ng·mL-1, Ferri的检测范围可达到0.65-800 ng·mL-1。
5,在微流控芯片平台上实现了人外周血中肝癌细胞的快速化学发光检测。本工作结合免疫细胞化学的特异性、化学发光检测的灵敏性和微流控芯片平台的快速性,实现了肝癌细胞的定量检测,为肝癌的早期诊断和预后监测提供了更加灵敏、特异的检测平台。
Hepatocellular carcinoma (HCC) in its early stage is asymptomatic, and consequently the diagnosis of HCC is often delayed. Thus, the mortality rate is still high for pateints with liver cancers because of poor prognosis. An early diagnosis is the key to an effective treatment of HCC and tumor markers play an important role in early clinical diagnosis of cancers. Chemiluminescence has attracted great attentions in terms of sensitivity and small footprint in bioananlytical field, and has been applied in clinical diagnosis with a great market share. In this work, several chemiluminescence enzyme immunoassay(CLEIA) methods have been developed for the detection of HCC tumor markers as well as their applications in clinics. The major works are as follows:
1. The diagnostic methods for HCC and their development are reviewed. The application of chemiluminescence immunoassay for detection of tumor markers are introduced in detail. Then, we summarized whatever key works should be concerned and have a overlook about the application of CLEIA.
2. In order to improve the linear detection range and the sensitivity for the detection of AFP, magnetic particles with surface modification are employed, which can covently binding antibodies. The contents are briefly summarized as bellow,
CLEIA was developed by alkaline phosphatase (ALP) labeling AFP antibodies for the detection of AFP. A sanwich immo-complex was formed by immunoreaction between FITC labled AFP monoclonal antibodies, AFP samples and ALP labled AFP antibodies. Magnetic micro-particles coated with anti-FITC antibodies were used as solid phase to separate immuno-complex from the liquid system. The linear detection range was enlarged to 1300 ng-mL-1 with a shorter immunoassay time compared with conventional CLEIA based on microplates.
In order to decrease background interference and increase sensitivity, CLEIA with horseradish peroxidase labeling AFP antibodies was proposed for the detection of AFP. A one-step immunoassay was performed by reaction between magnetic micro-particles coated with AFP antibodies, AFP samples and HRP labled AFP antibodies to form sanwich immo-complex. Additionaly, polystyrene StartubesTM were used as solid phase for AFP antibodies coating in parallel with magnetic micro-particles. Our results indicated that the magnetic micro-particles based CLEIA showed better performance with less consumption of immunoreagents, larger linear detection range (2000 ng-mL-1) and shorter immunoassay time by comparing with StartubesTM based CLEIA.
3. AFP used in early diagnosis of HCC show low diagnosis positive rate, therefor more sensitive and specific tumor markers should be exlored in combination with or instead of AFP for the diagnosis of HCC. Glypican-3 (GPC3) highly expressed in liver tissues of HCC pateins, is a promising tumor marker for HCC, and its detection was often reported by molecular biochemistry. A serological immunoassay method was proposed in our work for detection of GPC3, and its application in clinical diagnosis was studied.
Firstly, N-terminal and C-terminal GPC3 circulated in blood were simultaneously determinaed by competitive radioimmunoassay between 125I labeled GPC3, GPC3 samples and goat anti-human GPC3 polyclonal antibodies. The levels of GPC3 were evaluated in normal liver, hepatitis B, hepatitis C, hepatocirrhosis and HCC by our proposed method. We got that the levels of GPC3 were obviously higher in pateints with HCC than other benigh liver diseases. Addtionaly, the diagnostic sensitivity and specificity of GPC3 were also much higher compared with AFP and AFP/CA19-9 combination detection.
Sencondly, in order to save assay time, avoid radio-pollution and increase sensitivity, magnetic nano-particles based CLEIA was proposed for detection of serological GPC3, and the performance of the method in terms of sensitivity and specificity were evaluated. The GPC3 levels in HCC after transarterial chemoembolization(TACE)treatment indicated that GPC3 was an effective tumor marker for prognosis of HCC patients. Additionally, the influence of nano-particles in immunoassay was compared with micro-particles. The results revealed that nano-particles based CLEIA exhibted better performance in terms of higher bioactivity, less consumption of immunoreagents and shorter immunoassay time than micro-particles based CLEIA.
4. Simultaneous detection of HCC tumor markers was developed by CLEIA on microfluidics device. Glass substrate was processed with three-dimensional modification to improve antibodies coating efficiency. AFP and Ferri were simultaneously determined on microfluidic chip with high performance. No cross-reaction occurred and high specificity was obtained. The linear detection range for AFP and Ferri were 500 ng-mL-1 and 800 ng-mL-1, respectively.
5. Capturing and detecting liver cancer cells circulating in peripheral blood were performed on a microfluidic chip by chemiluminescence. In order to realize early diagnosis and prognosis of HCC by counting liver cancer cells, immunocytochemistry, chemiluminescence and microfluidic chip were integrated together in this work with advantages of high specificity, sensitivity and time-saving.
引文
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