电化学发光免疫分析方法及其在医学中的应用研究
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摘要
电化学发光免疫分析(ECLIA)方法作为一种新型的标记免疫学方法，具有灵敏度高、选择性好的优点，已经越来越受到人们的关注。国外有几个研究组在从事这种方法的开发和应用，并有公司生产了一套完整的仪器，但售价昂贵。而国内对电化学发光免疫分析的研究少见报道，所以本论文在充分调研国际、国内有关电化学发光分析研究、电化学发光免疫分析研究的历史和进展的前提下，在如何提高电化学发光效率和发光检测的灵敏度方面做了一定的改进，作为主要贡献者参与研制了一套高灵敏度的电化学发光免疫分析仪器。并用自制的仪器检测了人血清p53抗体的浓度，得到了初步但非常有意义的结果，证实了其用于临床诊断的可行性。
本工作首先参与研制了一台高灵敏度的电化学发光免疫分析仪。采用单光子计数模块作为光子探测器，能够灵敏的检测光学信号；并在样品池的工作电极与探测器的光电阴极之间用光纤束进行导光，避免工作电极下的磁铁对探测器的影响。
电极形状对电化学发光效率影响很大。对不同形状电极的电化学发光效率进行比较，设计了圆片、四孔圆片和螺旋丝状三种不同形状的电极，对其电化学发光图像和电化学发光强度进行检测，结果发现电化学发光集中在电极的边缘，圆片电极检测Ru(bpy)_3Cl_2·6H_2O的极限是1fmol／L，而四孔圆片电极检测的极限可以达到1amol／L，提高了3个数量级。由此得出结论，对于一定形状的电极，如果能做成筛网状的，也就是最大限度的增加边缘的长度，同时也是提高了比表面积，就可以达到最大的发光效率。检测Ru(bpy)_3Cl_2·6H_2O的线性范围可达到7个数量级，浓度从0.1pmol／L到1μmol／L，为定量检测提供了较宽的动态检测范围。
电化学发光免疫分析实验中用到的发光标记探针——三联吡啶钉的复合物Ru(bpy)_3~(2+)—NHS ester由本研究组自行合成。通过分析其红外光谱确定了该物质，又通
中文摘要
过其紫外一可见吸收光谱确定了其产率为75．2％。
把电化学发光免疫分析应用于肿瘤早期诊断，本文设计了一个新的肿瘤早期诊断
的方法，并从实验上证明了其可行性。用磁免疫电化学发光（IM－ECL）分析方法检测
稀释于正常人血清中的极低浓度的P53抗体，并且做出标准曲线，然后定量分析了临
床诊断过的癌症病人血清中的P53抗体的浓度。结果显示，用本文设计的IMECL检测
分析方法检测肺癌血清的P53抗体阳性率为28．6％，鼻咽癌血清的P53抗体阳性率
非常低，为 8．33％，都与前人的报道相一致。分析结果发现，随着肺癌临床分期的升
高，p53抗体浓度增加。
总之，电化学发光免疫分析方法是一种简单、快速、灵敏度高的标记免疫学方法。
该方法可以定性或定量的分析临床样品，为临床检测肿瘤标记物一P53抗体等提供了
一种新的分析方法。
Electrochemiluminescence immunoassay (ECLJA) is a new tag immunology method. It has been won the attention because of the advantages of high sensitivity and better selectivity. Some foreign teams had been at the work on the exploiture and application of ECLIA. And some companies had made the apparatus but with high price. Few teams in our contrary have been studied in the field of ECLIA. So we investigated the status and progress of the electrochemiluminescence (ECL) and ECLIA. Then some studies in improving the ECL efficiency and making a high-sensitive ECLIA device was done. At last, we used the analyzer to detect the concentration of p53 antibodies in human sera. The results are primary but significative, suggesting that ECLIA is a feasible method for clinical diagnosis.
Firstly, a high-sensitive ECLIA device was improved. In the device, a single photon multiplier rube was used to detect the weak photon signal. And a quartz fiber bundle was used between the working electrode and PMT to avoid the influence of magnet under the working electrode. Thus, make a high efficiency coupling of photons; the detection efficiency was obviously improved.
To study the problems of ECL efficiency and detection efficiency, three kinds of platinum electrodes with different geometry, such as round disk, four-hole round disk and spiral thread, were designed. The relation of the ECL intensity with the geometry of these electrodes has been investigated. The optical character of these electrodes was directly studied by observation of ECL image and measurement of the ECL intensity. The results showed that the ECL not only concentrated on the edge of the electrode but also concentrated on the edge of holes, which contain in the electrode. The detection limit was improved to 10-18 mol/L
Ru(bpy)3C12 6H2O on four-hole round disk electrode, three times higher than that on round disk electrode. So we concluded, given an identical surface area, when the length of the edge is added, the surface area of per gram is increased; the ECL efficiency can be improved. Results showed that the dynamic linear range extended over five orders, from 10-13 mol/L to 10-8 mol/L.
In the experiments of ECLIA, the ECL probe-Ru(bpy)32+-NHS ester was chemosynthesized by ourselves. From the studies of the infrared spectroscopy and ultraviolet-visible absorption spectra, we calculated the yield was 75.2%.
Furthermore, we applied the ECLIA in tumor early diagnosis. We designed a new method to detect p53 antibodies in human sera. The results suggested that ECLIA is a feasible method for clinical diagnosis. We used ECLIA to detect an extremely low concentration p53 antibodies introduced in normal human sera. The calibrated assay was then used to quantitatively evaluate the presence of p53 antibodies in cancerous human sera that was determined with conventional diagnosis. The positive rate of p53 antibodies was 28.6% in lung carcinoma and 8.33% in nasopharyngeal carcinoma, respectively. P53 antibody concentration in the carcerous human sera was quantified from the calibration curve. To lung carcinoma, a trend was found that higher the p53 antibody concentration in the serum likely linked to a higher stage of the cancer.
In conclusion, results of the study suggested that ECLIA is a simple, rapid, sensitive method. This method could provide either qualitative or quantitative results for analysis clinical samples. It is a feasible method for rapid and sensitive detection of p53 antibodies in human serum. ECLIA, thus, may provide a new means for quantitatively evaluated the production of p53 antibodies.
本工作首先参与研制了一台高灵敏度的电化学发光免疫分析仪。采用单光子计数模块作为光子探测器，能够灵敏的检测光学信号；并在样品池的工作电极与探测器的光电阴极之间用光纤束进行导光，避免工作电极下的磁铁对探测器的影响。
电极形状对电化学发光效率影响很大。对不同形状电极的电化学发光效率进行比较，设计了圆片、四孔圆片和螺旋丝状三种不同形状的电极，对其电化学发光图像和电化学发光强度进行检测，结果发现电化学发光集中在电极的边缘，圆片电极检测Ru(bpy)_3Cl_2·6H_2O的极限是1fmol／L，而四孔圆片电极检测的极限可以达到1amol／L，提高了3个数量级。由此得出结论，对于一定形状的电极，如果能做成筛网状的，也就是最大限度的增加边缘的长度，同时也是提高了比表面积，就可以达到最大的发光效率。检测Ru(bpy)_3Cl_2·6H_2O的线性范围可达到7个数量级，浓度从0.1pmol／L到1μmol／L，为定量检测提供了较宽的动态检测范围。
电化学发光免疫分析实验中用到的发光标记探针——三联吡啶钉的复合物Ru(bpy)_3~(2+)—NHS ester由本研究组自行合成。通过分析其红外光谱确定了该物质，又通
中文摘要
过其紫外一可见吸收光谱确定了其产率为75．2％。
把电化学发光免疫分析应用于肿瘤早期诊断，本文设计了一个新的肿瘤早期诊断
的方法，并从实验上证明了其可行性。用磁免疫电化学发光（IM－ECL）分析方法检测
稀释于正常人血清中的极低浓度的P53抗体，并且做出标准曲线，然后定量分析了临
床诊断过的癌症病人血清中的P53抗体的浓度。结果显示，用本文设计的IMECL检测
分析方法检测肺癌血清的P53抗体阳性率为28．6％，鼻咽癌血清的P53抗体阳性率
非常低，为 8．33％，都与前人的报道相一致。分析结果发现，随着肺癌临床分期的升
高，p53抗体浓度增加。
总之，电化学发光免疫分析方法是一种简单、快速、灵敏度高的标记免疫学方法。
该方法可以定性或定量的分析临床样品，为临床检测肿瘤标记物一P53抗体等提供了
一种新的分析方法。
Electrochemiluminescence immunoassay (ECLJA) is a new tag immunology method. It has been won the attention because of the advantages of high sensitivity and better selectivity. Some foreign teams had been at the work on the exploiture and application of ECLIA. And some companies had made the apparatus but with high price. Few teams in our contrary have been studied in the field of ECLIA. So we investigated the status and progress of the electrochemiluminescence (ECL) and ECLIA. Then some studies in improving the ECL efficiency and making a high-sensitive ECLIA device was done. At last, we used the analyzer to detect the concentration of p53 antibodies in human sera. The results are primary but significative, suggesting that ECLIA is a feasible method for clinical diagnosis.
Firstly, a high-sensitive ECLIA device was improved. In the device, a single photon multiplier rube was used to detect the weak photon signal. And a quartz fiber bundle was used between the working electrode and PMT to avoid the influence of magnet under the working electrode. Thus, make a high efficiency coupling of photons; the detection efficiency was obviously improved.
To study the problems of ECL efficiency and detection efficiency, three kinds of platinum electrodes with different geometry, such as round disk, four-hole round disk and spiral thread, were designed. The relation of the ECL intensity with the geometry of these electrodes has been investigated. The optical character of these electrodes was directly studied by observation of ECL image and measurement of the ECL intensity. The results showed that the ECL not only concentrated on the edge of the electrode but also concentrated on the edge of holes, which contain in the electrode. The detection limit was improved to 10-18 mol/L
Ru(bpy)3C12 6H2O on four-hole round disk electrode, three times higher than that on round disk electrode. So we concluded, given an identical surface area, when the length of the edge is added, the surface area of per gram is increased; the ECL efficiency can be improved. Results showed that the dynamic linear range extended over five orders, from 10-13 mol/L to 10-8 mol/L.
In the experiments of ECLIA, the ECL probe-Ru(bpy)32+-NHS ester was chemosynthesized by ourselves. From the studies of the infrared spectroscopy and ultraviolet-visible absorption spectra, we calculated the yield was 75.2%.
Furthermore, we applied the ECLIA in tumor early diagnosis. We designed a new method to detect p53 antibodies in human sera. The results suggested that ECLIA is a feasible method for clinical diagnosis. We used ECLIA to detect an extremely low concentration p53 antibodies introduced in normal human sera. The calibrated assay was then used to quantitatively evaluate the presence of p53 antibodies in cancerous human sera that was determined with conventional diagnosis. The positive rate of p53 antibodies was 28.6% in lung carcinoma and 8.33% in nasopharyngeal carcinoma, respectively. P53 antibody concentration in the carcerous human sera was quantified from the calibration curve. To lung carcinoma, a trend was found that higher the p53 antibody concentration in the serum likely linked to a higher stage of the cancer.
In conclusion, results of the study suggested that ECLIA is a simple, rapid, sensitive method. This method could provide either qualitative or quantitative results for analysis clinical samples. It is a feasible method for rapid and sensitive detection of p53 antibodies in human serum. ECLIA, thus, may provide a new means for quantitatively evaluated the production of p53 antibodies.
引文
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[2] Yalow R S, Berson S A. J. Clin. Incest., 1960, 39:1157
[3] Kohler G, Milstein C. Nature, 1975, 256:495
[4] Weber S G.. Comprehensive Analytical Chemistry. New York: Elservier Science Publishing Company Inc, 1992:91
[5] 吴健民．临床化学自动化免疫分析．北京：科学出版社，2000：52
[6] 李振甲．时间分辨荧光分析技术与应用．北京：科学出版社，1995：21
[7] Engvall E, Pearlmann P. Immunochemistry, 1971, 8:871
[8] 王自正．现代医学标记免疫学．北京：人民军医出版社，2000：17
[9] Blackburn G F, Shah H P, Kenten J H. Clin. Chem., 1991, 37/9:1534-1539
[10] Leland J K, Powell M J. J. Electrochem. Soc., 1990,137/10:3127～3131
[11] Nicbolas R, Hoyle N R, Eckert B. Clin. Chem., 1996, 42/9:1576-1578
[12] Tokel N E, Bard A J. J. Am. Chem. Soc., 1972, 94:2862～2863
[13] Bard AJ. Pure. Appl. Chem., 1992,64:185
[14] Zhang X, Bard A J. J. Phys. Chem., 1988,92:5566～5569
[15] Abruna H D, Bard A J. J. Am. Chem. Soc., 1982,104:2641～2642
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[21] Talahashi K, Suzuki N, Tanaka M. Clin. Chem., 1997, 43(6): S171
[22] Obenauer-Kutner LJ, Jacobs SJ, Kolz K. J. Immyno. Meth., 1997, 206:25
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[24] Ebert C, Uhl W. Clin.chem., 1997, 43(6): S232
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[30] Motmas K, Raus J,, Vandevyver C. J. Immuno. Meth., 1996, 190:107
[31] O'Conell C D, Juhasz A, Kuo C, Reeder D J, Hoon D S. Clin. Chem., 1998, 44(6): 1161
[1] 魏亚东、邢达、何永红．光电子·激光，2002，13(10)：1077—1080．
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[2] Yalow R S, Berson S A. J. Clin. Incest., 1960, 39:1157
[3] Kohler G, Milstein C. Nature, 1975, 256:495
[4] Weber S G.. Comprehensive Analytical Chemistry. New York: Elservier Science Publishing Company Inc, 1992:91
[5] 吴健民．临床化学自动化免疫分析．北京：科学出版社，2000：52
[6] 李振甲．时间分辨荧光分析技术与应用．北京：科学出版社，1995：21
[7] Engvall E, Pearlmann P. Immunochemistry, 1971, 8:871
[8] 王自正．现代医学标记免疫学．北京：人民军医出版社，2000：17
[9] Blackburn G F, Shah H P, Kenten J H. Clin. Chem., 1991, 37/9:1534-1539
[10] Leland J K, Powell M J. J. Electrochem. Soc., 1990,137/10:3127～3131
[11] Nicbolas R, Hoyle N R, Eckert B. Clin. Chem., 1996, 42/9:1576-1578
[12] Tokel N E, Bard A J. J. Am. Chem. Soc., 1972, 94:2862～2863
[13] Bard AJ. Pure. Appl. Chem., 1992,64:185
[14] Zhang X, Bard A J. J. Phys. Chem., 1988,92:5566～5569
[15] Abruna H D, Bard A J. J. Am. Chem. Soc., 1982,104:2641～2642
[16] 张津辉．军事医学科学院学位论文．北京，1997
[17] 张津辉，蒋中华，解放军医学情报，10(2)，94-97(1996)
[18] 蒋中华、张津辉，生物分子固定化技术及应用，化学工业出版社，1998：19
[19] 尹伯元，王仁芝，李振甲等．标记免疫学．北京：原子能出版社，1998：55
[20] Welch S L, Wright T, Permanente K. Clin. Chem., 1997, 43(6): S169
[21] Talahashi K, Suzuki N, Tanaka M. Clin. Chem., 1997, 43(6): S171
[22] Obenauer-Kutner LJ, Jacobs SJ, Kolz K. J. Immyno. Meth., 1997, 206:25
[23] Sapin R, Gasser F, Herbomez M. Clin. Chem., 1997, 43(3): 545
[24] Ebert C, Uhl W. Clin.chem., 1997, 43(6): S232
[25] Massey R J, Powell M I. PCT. Int. Appl. Wo., 1987, 87/06706
[26] Bard A J, Whitesides G M. US Patent, 1994, 5310687
[27] Yu H. J. Immuno. Methods, 1996, 192:63～71
[28] Yu H, Bruno J G. Appl. Environ. Microbio., 1996,62:587～592
[29] Yu H, Bruno J G, Chem T C. J. Immuno. Meth., 1996, 10:239
[30] Motmas K, Raus J,, Vandevyver C. J. Immuno. Meth., 1996, 190:107
[31] O'Conell C D, Juhasz A, Kuo C, Reeder D J, Hoon D S. Clin. Chem., 1998, 44(6): 1161
[1] 魏亚东、邢达、何永红．光电子·激光，2002，13(10)：1077—1080．
[1] James S. Symanski, Stanley Bruckenstein. J. Elecrochem Soc., 1988, 135:1985
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[3] Attila Szabo. J. Phys. Chem., 1987, 91:3108
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