【作者】 谭三勤；

【导师】 陈方平；

【作者基本信息】 中南大学 ， 血液病学， 2010， 博士

【摘要】 免疫学分型在急性白血病(acute leukemia, AL)诊断中具有重要意义。目前在临床上,AL免疫分型主要是通过两种方法实现,一种是借助显微镜观察的人工计数法(免疫细胞化学或免疫荧光方法),另一种是使用流式细胞仪的流式细胞术(flow cytometry, FCM)。前者具有主观性较强、重复性较差、费时且每次只能检测一种抗原(即低通量)等缺陷；而FCM又因仪器与试剂昂贵难以在基层医院推广应用。因此,建立一种具有高通量、使用简便、灵敏度高、经济、准确、客观且易于临床推广的免疫分型新技术具有十分重要的意义。本研究将细胞酶联免疫分析(cell enzyme linked immunosorbent assay, cELISA)技术与电化学分析方法相结合,形成cELISA电化学分析新技术,该技术将cELISA中的酶催化显色反应改为酶催化银沉积反应,再通过电化学分析检测银沉积后电导率的大小来反应抗原量的多少。我们首先研究具有pA级超微电流检测灵敏度的多功能微小新型电化学免疫分析仪。其次,建立一种cELISA酶催化银沉积于插指电极阵列电化学免疫分析(简称为cELISA插指电化学)新方法,并探讨该仪器用于该方法对临床AL标本免疫分型的可行性。最后,探讨该仪器用于电化学阻抗法检测临床AL细胞内髓过氧化物酶(myeloperoxidase, MPO)活性的可行性。目的：研究适合于微型电化学传感器超微电流检测的多功能微小新型电化学分析仪。方法：首先,设计硬件电路,包括信号采集放大及转换器、滤波器和短路报警器等电路设计；其次,设计软件程序,包括微控制器程序、通讯软件和上位机软件设计。最后,采用线性扫描伏安法及电化学阻抗法对设计的新型电化学免疫分析仪的性能进行了测试。结果：新型电化学免疫分析仪检测到小于10pA量级的微电流；分析仪产生、显示和记录出阻抗波形图。结论：性能测试结果表明,新型电化学免疫分析仪满足微电流、多功能的设计要求,是一套具有应用价值的高集成度和高精度的微小仪器。目的：探讨新型电化学免疫分析仪用于本研究建立的cELISA插指电化学分析新方法对临床AL标本免疫分型的可行性。方法：在微孔板内,用多聚左旋赖氨酸(Poly-L-Lysine, P-L-L)包被白血病细胞,依次加入鼠抗人一抗及碱性磷酸酶(alkaline phosphatase, ALP)标记的马抗鼠二抗,再将银溶液和ALP底物加入到该微孔板内,待溶液中的银离子还原成银单质沉积到插指电极表面后,用新型电化学免疫分析仪测定电导率。首先建立cELISA插指电化学分析新方法,即对实验条件如多聚赖氨酸分子量的大小、抗体的浓度等进行优化及分析cELISA插指电化学分析新方法的特异性、灵敏度及其与FCM的相关性。然后,采用该方法与FCM分别测定72例相同的AL患者白细胞表面分化(cluster of differentiation, CD)抗原,并将这两种方法的检测结果进行比较。结果：最佳P-L-L分子量为7-15万道尔顿；最佳包被细胞数为1.6×105个；最佳一抗体浓度为20μg/ml;1／500为最适二抗浓度；可检测出300个左右的HL-60细胞；cELISA插指电化学分析新方法与FCM方法检测结果相关系数为0.93；经χ2计学分析,72例AL患者8种白细胞CD抗原检测结果与FCM分析结果无统计学差异(P=0.373)。结论：我们创建的cELISA插指电化学分析新方法对白血病细胞株及临床AL标本检测结果与FCM分析结果一致,这表明两种方法吻合程度较高,cELISA插指电化学分析新方法特异性好,灵敏度高,为临床白血病免疫分型提供一种新方法,新型电化学免疫分析仪可望成为临床白血病免疫分型的新仪器。目的：探讨新型电化学免疫分析仪用于电化学阻抗法检测AL髓过氧化物酶(myeloperoxidase, MPO)活性的可行性。方法：用Tris-HCL(pH 8.7)溶液裂解白血病细胞,将其滴加到金电极表面,再依次加入过氧化物酶(Peroxidase, POX)染液A和B,用新型电化学免疫分析仪测定催化反应前、后电极的电化学阻抗图,并计算出酶催化反应前、后阻抗实部半径差值。首先,分析电化学阻抗法的特异性；然后,分别用此方法和增强化学发光法分析25例相同的AL患者MPO活性,并将这两种方法的检测结果进行比较。结果：25例AL患者中,电化学阻抗法MPO活性阳性15例,阴性10例；增强化学发光法MPO阳性14例,MPO阴性11例,增强化学发光法检测MPO阳性的14例,电化学阻抗法检测也为阳性,增强化学发光法检测阴性的11例中,1例电化学阻抗法检测为阳性。经χ2计学分析,两种方法检测的阳性率无统计学差异(P=1)。结论：电化学阻抗法与增强化学发光法检测结果一致,这表明两种方法吻合程度好。电化学阻抗法特异性好,灵敏度高,为临床AL标本MPO活性检测提供了一种新方法。新型电化学免疫分析仪可用于该方法检测AL白血病细胞内MPO活性,是一台灵敏度高的仪器。本研究表明,新型电化学免疫分析仪是一台可检测pA量级微电流,支持多种电化学分析方法的多功能灵敏仪器。该仪器不仅可用来检测细胞表面抗原,还可用来检测细胞内MPO活性,且操作简单、携带方便、准确度高,具有临床推广价值和市场应用前景。更多还原

【Abstract】 Immunophenotyping of acute leukemia (AL) plays a very important role in the diagnosis of AL. At present, there are two methods of immunophenotyping of AL:one is immunocytochemistry or immunofluorescence method by means of microcsope and the other is immunofluorescence by means of flow cytometry (FCM). However, the former is subjectivity, poor reproducibility, time consuming,one kind of antigen being detected(ie, low flux) and et al. Because of expensive equipment and reagents, it is difficult to apply FCM in the primary hospitals.Therefore, it is essential to develop a rapid, simple, high-through-put, high sensitivity, economical and more objective new technology for immunophenotyping of AL.In our studies,a new method of cELISA electrochemical analysis was developed by combining cell enzyme-linked immunoassay technique with electrochemical methods,in which enzymatic silver deposition reaction took the place of enzyme-catalyzed color reaction,and the number of antigen was detected by the number of deposition silver. First,we developed a small new electrochemical immunoassay analyzer whose current detection limit was pA level.Secondly, a new methods of cELISA enzymatic silver deposition in the interdigitated electrode array (referred as cELISA interpolation electrochemical) was developed, the feasibility of the new type of electrochemical immunoassay analyzer applied in immunophenotyping of AL was explored. Finally, the feasibility of the new type of electrochemical immunoassay analyzer applied in detecting of MPO in AL with the electrochemical impedance method was explored.Objective:To develope a multi-function small new type of electrochemical immune analyser suitable for detecting of micro-current in micro-electrochemical sensor.Methods:First, the hardware circuitry designed included the circuitry of signal acquisition and conversion amplification, filters and short-circuit alarm, et al. Secondly, software programs designed was made up of micro-controller program, communications software and PC software. Finally, the performance of new electrochemical immunoassay analyzer was tested using linear sweep voltammetry (LSV) and electrochemical impedance impedance method. Results:The current of less than 10pA was detected by the new type of electrochemical immunologic analyser; a impedance waveforms was displayed and recorded by it. Conclusion:The new electrochemical immune analyzer has been developed, its performance meets the design requirements of multi-functional, high sensitivity and small-type.Objective:To explore the feasibility of the new type of electrochemical immune analyser applied in immunophenotyping of AL patients by cELISA interdigitated electrochemistry method established in this work. Methods:Leukemic cells were coated with Poly-L-Lysine (P-L-L) in the wells of microplate,after primary antibody of mouse against human and second antiobody of horse against mouse labled with alkaline phosphatase (ALP) was added one by one,the solution containing silver and ALP substrate was added, after the silver deposition on the surface of interdigitated electrode, the electrical conductance was measured with the new electrochemical immunologic analyzer.First,a new method of cELISA interdigitated electrode electrochemistry was established, which included optimizing the parameters such as the molecular size of P-L-L used, the concentration of antibodies,et al and analyzing the specificity and sensitivity of the cELISA interdigitated electrode electrochemistry method. Subsequently, the expression of 8 cluster of differentiation (CD) antigens on the surface of leukemic cells in 72 AL patients were determined by the method and FCM, and their results compared with each other. Results:The optimal molecular weight of poly-L-lysine was 7-15 million Dalton; The optimal number of cells coated on a microplate was 1.6×105, The optimal concentration of the primary antibody was 20μg/ml, and the optimal concentration of the second antibody was a ratio of 1:500 dilution; The sensitivity of the method of the cELISA interdigitated electrochemistry was about 300 of HL-60 cells. The relative coefficient for the results obtained by the cELISA interdigitated electrochemistry method and FCM analysis was 0.93. The results of 8 CD antigens expression in 72 AL patients, which obtained by cELISA interdigitated electrochemistry method had no significant difference from those of FCM byχ2 test (P=0.373). Conclusion:The results of testing leukemia cell lines and clinical specimens by cELISA interdigitated electrochemical analysis which we created were consistent with those of the FCM analysis, this indicates that there is high consistency in two methods. CELISA interdigitated electrochemical analysis is specificity, high sensitivity and a new way of immunophenotyping of AL in clinic. The new type of electrochemical immune analyser can be applied in immunophenotyping of AL by cELISA interdigitated electrochemistry method.Objective:To explore the feasibility of the new type of electrochemical immunologic analyser applied in the detection of MPO activity in AL patients by electrochemical impedance spectroscopy method. Methods:Cells were lysis with the solution of Tris-HCL (pH 8.7), and added slowly on the surface of gold electrode, POX staining solution of A and B were added respectively. The conductivity before and after catalysis was measured with the new electrochemical immunologic analyzer. Firstly, the specificity of electrochemical impedance spectroscopy method was analyzed with HL-60 cell and Jurket cell. Subsequently, the activity of MPO in AL patients was analyzed by the electrochemical impedance spectroscopy method, and whose results compared with those obtained from the enhanced chemiluminescent assay Results:15 of 25 AL patients were MPO-positive, and the remaining 10 of AL patients were MPO-negative detected by the electrochemical impedance spectroscopy method. For the 14 MPO positive AL patients detected by the enhanced chemiluminescent assay, MPO positive were also obtained by the electrochemical impedance spectroscopy in the 14 AL patients;for the 11 MPO negative AL patients detected by the enhanced chemiluminescent assay,one case was detected as positive by the electrochemical impedance spectroscopy. Results obtained with the new method has no significant difference from the enhanced chemiluminescent assay byχ2 test (P=1).Conclusion:The results of MPO activity in 25 tested AL patients by electrochemical impedance spectroscopy are consistent with those of the enhanced chemiluminescent assay, which indicates that there is high consistency in the two methods. The electrochemical impedance spectroscopy is a specificity, high sensitivity method,which provides a new method for detecting the activity of MPO in AL patients. The new type of electrochemical immune analyser can be applied in detecting the activity of MPO in AL patients by the electrochemical impedance spectroscopy.This study showed that the new type of electrochemical immunoassay analyzer is a multifunction apparatus,which could detect micro-current such as pA level.This instrument could be used not only to detect cell surface antigens, but also to detect intracellular MPO activity. It is simple, portable,and has clinical value and market prospect.更多还原