磁性免疫層析試紙條聯(lián)合定量檢測NSE、CEA的應(yīng)用研究
發(fā)布時(shí)間：2019-05-13 17:09
【摘要】：研究背景:原發(fā)性支氣管肺癌嚴(yán)重危害人類健康,其發(fā)病率和死亡率,居全球癌癥首位。因75%肺癌患者確診時(shí)已到了晚期肺癌,直接導(dǎo)致了肺癌的5年生存率僅為15.6%。其中小細(xì)胞肺癌惡性程度高,易早期發(fā)生轉(zhuǎn)移和播散,預(yù)后極差。但小細(xì)胞肺癌在早期對放療、化療很敏感。目前,許多血清腫瘤標(biāo)記物已被用于肺癌篩查、診斷及預(yù)后判斷的指標(biāo)。神經(jīng)元特異性烯醇化酶(Neuron-specific enolase,NSE)是小細(xì)胞肺癌最敏感最特異性的腫瘤指標(biāo)。癌胚抗原(carcino-embryonic antigen,CEA)是被用于多系統(tǒng)腫瘤的診斷及預(yù)后判斷。其中,約有30%～70%的肺癌患者CEA水平明顯升高。對人血清中NSE和CEA進(jìn)行聯(lián)合檢測有助提高肺癌的篩查陽性率及早期診斷的準(zhǔn)確性。血清NSE及CEA的傳統(tǒng)檢測方法有酶聯(lián)免疫吸附試驗(yàn)和電化學(xué)發(fā)光免疫檢測法,該兩種方法具有較高的靈敏度,但需要大型檢測設(shè)備,復(fù)雜的檢測步驟及專業(yè)的操作人員;這些使其無法應(yīng)用在大規(guī)模人群中進(jìn)行肺癌篩查。免疫層析測定法(Immunochromatographicassay,ICA)是近年發(fā)展起來的一種新型快速免疫檢測技術(shù)。ICA通常以膠體金、納米銀粒子以及熒光材料等作標(biāo)記物,在層析檢測過程,免疫探針與待測物反應(yīng)形成免疫復(fù)合物,被對應(yīng)的捕獲抗體捕獲并富集于硝化纖維膜上,以檢測區(qū)上信號(hào)條帶的顯色與否、顏色深度來定性分析或通過檢測反射光強(qiáng)度來定量分析。近年來,利用磁性納米粒子作為免疫探針來檢測磁信號(hào)作定量分析成為免疫層析檢測技術(shù)的研究熱點(diǎn)。由于待測樣品沒有磁背景干擾及磁信號(hào)檢測不受光漂白等影響,與傳統(tǒng)光學(xué)免疫層析技術(shù)相比,磁性層析技術(shù)的信噪比更高,檢測信號(hào)更穩(wěn)定。結(jié)合配套的磁信號(hào)檢測設(shè)備,不僅可以提供定量檢測的精確值,還可以提高檢測的靈敏度。研究方法:采用EDC碳二亞胺法分別制備NSE和CEA免疫探針;利用制備的免疫探針組裝對應(yīng)的磁性免疫層析聯(lián)檢試紙條,并優(yōu)化制備條件;通過檢測不同梯度濃度的NSE、CEA標(biāo)準(zhǔn)抗原,結(jié)合配套磁信號(hào)檢測設(shè)備來進(jìn)行定量分析;通過檢測臨床血清樣本,評估該方法的敏感性、特異性和準(zhǔn)確性。研究結(jié)果:該免疫層析試紙條可用于快速定量聯(lián)合檢測NSE和CEA,且具有特異性好、靈敏度高的優(yōu)點(diǎn)(NSE的檢測下限為0.094ng/mL,CEA檢測下限為0.045ng.mL)。對130份臨床血清進(jìn)行驗(yàn)證,證實(shí)該磁性試紙條可用于未經(jīng)預(yù)處理的血清檢測。其檢測結(jié)果與商業(yè)電化學(xué)發(fā)光法試劑盒的結(jié)果一致性良好(n= 30,R20.990,P0.001);加標(biāo)回收試驗(yàn)證實(shí)該試紙條具有高的檢測精確度。研究結(jié)論成功建立了基于磁性納米粒子的免疫層析試紙條,實(shí)現(xiàn)對NSE和CEA的快速聯(lián)合定量檢測,且檢測靈敏度高,特異性好,精確度高;可作為小型醫(yī)院,社區(qū)門診和家庭醫(yī)生實(shí)現(xiàn)肺癌快速篩查的有效輔助手段。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2
本文編號(hào)：2476054
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2
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