双抗体夹心法检测肺炎衣原体循环免疫复合物的方法学建立
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摘要
目的：建立一种敏感而且特异的双抗体夹心法检测血液中的循环免疫复合物(CIC)，并讨论血液中肺炎衣原体(Cpn)CIC与冠心病的关系。方法：本实验分为三个阶段。1．第一阶段为根据传统双抗体夹心间接法检测血清中的CpnCIC。在PVD酶标板上包被已知特异性羊抗人IgG单克隆抗体(CpnTW183IgG)，4℃过夜。接着加入经过7％聚乙烯乙二醇6000(PEG6000)沉淀处理的，从冠心病患者血清中提取的大分子蛋白质，与固相抗体反应。再加入特异性鼠抗人CpnTW183IgG，与大分子蛋白质中可能存在的CpnCIC中的CpnAg结合。然后加入辣根过氧化物酶(HRP-)标记的羊抗鼠IgG单克隆抗体。最后，通过底物显色反应检测患者CpnCIC阳性率。2．第二阶段根据第一阶段实验结果，建立新的简便敏感的双抗体夹心法。包被鼠抗人CpnTW183IgG，4℃，过夜。再加入经过7％聚乙烯乙二醇6000(PEG6000)沉淀处理的，从冠心病患者血清中提取的大分子蛋白质，与固相抗体反应。然后加入HRP-羊抗人IgG单克隆抗体孵育。最后，通过底物显色反应检测患者CpnCIC阳性率。3．第三阶段为补充实验。根据分子生物学检测细胞核粗提物中检测到极微量的DNA序列特异性结合蛋白的凝胶滞后实验而推导衍生出来的已知特异性抗体与待测抗原抗体复合物结合的凝胶滞后实验。本法采用非变性，非还原聚丙烯酰胺凝胶电泳技术。选取第二阶段实验确证为阳性的血清标本如上述方法处理，样品分为两份。一份与鼠抗人CpnTW183IgG于37℃孵育2小时，反应后的混合液与另一份样品加入8％的聚丙烯酰胺凝胶板中，40mA约4小时。胶板经过考马斯亮兰染色4小时，最后脱色4—5次。结果：1．传统间接双抗体夹心法检测待检标本，阴性对照，空白对照，均出现强阳性或假阳性。2．改进后的方法检测冠心病组CpnCIC的阳性率(63.8％)高于对照组CpnCIC的阳性率(40.0％)，经过统计学检验，差异具有显著性意义(x~2=7.78，P＜0.05)。3．凝胶滞后实验检测已知CpnCIC阳性标本出现滞后条带。结论：1．传统的双抗体夹心间接法在此不能特异地检测出血清中的CpnCIC。2．本实验建立的双抗体夹心法检测CpnCIC有较高的敏感性和特异性，可用于批量临床标本的检测；血清中中分子量CpnCIC的存在与冠心病之间有密切关系。3．本实验建立的新的滞后实验能够有效的检测出血清中的CpnCIC。
Objective: to establish a sensitive and special method of double mono-cloned antibody for examining the circular immune complex (CIC) in blood, and to further probe the relation between CIC of chlamydia pneumoniae (Cpn) and the coronary heart disease.
Methods: This experiment was consisted of three steps. 1. At the first step, we planned to detect the CpnCIC existing in the serum according to the classical indirect double mono-cloned method. Covering the special goat anti human IgG antibody in the plate under 4# for 12 hours. The samples were added to react with the above fixed IgG antibody, as followed the special mouse anti human CpnTW183IgG. The peroxidase labeled goat anti mouse IgG could combine with CpnlgG on CpnCIC if it existed. At last, the result was got through the reaction of substrate. 2. At the second step, according to the result of the first step, we tried to create a novel method. Covering the special mouse anti human CpnTW183IgG under 4癈 for 12 hours, after this step ,we added CpnCIC extracted from the blood of patients with coronary heart disease by 7%PEG6000. As the peroxidase labeled goat anti human IgG could combine with antibody in CIC, we can get the positive rate of experimental group through the reaction of substrate. 3. At the
third step, according to the Gel electrophoresis DNA-binding assay, we changed it partly to detect CpnCIC through the retarding belt which was caused by being combined with CpnAb under 37# for 2 hours. It was supposed that the belt of CpnCIC would lost or become lighter.
Results: 1. The results of all the samples at the first step were positive; 2. The positive rate of CpnCIC of the experimental group is higher than that of the control group (63.8%/40%), (x2=7.78, p<0.05). 3. The retarding belt abstracted from the mixture of CpnlgG and the positive sample which was certaintied by the second step appeared, and the belt of CpnCIC lost correspondingly.
Conclusions: 1. The classical method of double mono-cloned method could not be
used to detect CpnCIC in serum. 2. It was a sensitive and special method of the double mono-cloned antibody for detecting CpnCIC, which could be used for the samples in clinic, and there is a relation between CIC of chlamydia pneumoniae and the coronary heart disease. 3. The novel Gel shift assay could detect CpnCIC existing in the blood finely.
Objective: to establish a sensitive and special method of double mono-cloned antibody for examining the circular immune complex (CIC) in blood, and to further probe the relation between CIC of chlamydia pneumoniae (Cpn) and the coronary heart disease.
Methods: This experiment was consisted of three steps. 1. At the first step, we planned to detect the CpnCIC existing in the serum according to the classical indirect double mono-cloned method. Covering the special goat anti human IgG antibody in the plate under 4# for 12 hours. The samples were added to react with the above fixed IgG antibody, as followed the special mouse anti human CpnTW183IgG. The peroxidase labeled goat anti mouse IgG could combine with CpnlgG on CpnCIC if it existed. At last, the result was got through the reaction of substrate. 2. At the second step, according to the result of the first step, we tried to create a novel method. Covering the special mouse anti human CpnTW183IgG under 4癈 for 12 hours, after this step ,we added CpnCIC extracted from the blood of patients with coronary heart disease by 7%PEG6000. As the peroxidase labeled goat anti human IgG could combine with antibody in CIC, we can get the positive rate of experimental group through the reaction of substrate. 3. At the
third step, according to the Gel electrophoresis DNA-binding assay, we changed it partly to detect CpnCIC through the retarding belt which was caused by being combined with CpnAb under 37# for 2 hours. It was supposed that the belt of CpnCIC would lost or become lighter.
Results: 1. The results of all the samples at the first step were positive; 2. The positive rate of CpnCIC of the experimental group is higher than that of the control group (63.8%/40%), (x2=7.78, p<0.05). 3. The retarding belt abstracted from the mixture of CpnlgG and the positive sample which was certaintied by the second step appeared, and the belt of CpnCIC lost correspondingly.
Conclusions: 1. The classical method of double mono-cloned method could not be
used to detect CpnCIC in serum. 2. It was a sensitive and special method of the double mono-cloned antibody for detecting CpnCIC, which could be used for the samples in clinic, and there is a relation between CIC of chlamydia pneumoniae and the coronary heart disease. 3. The novel Gel shift assay could detect CpnCIC existing in the blood finely.
引文
1. Linnanma k E, Leinonen M, Mattula K et al. Chlamydia Pneumoniae--specific circulating immune complex in patients with chronic coronary heart disease. Circulation, 1993, 87(4): 130
2. Saikku P, Leinonen M, Mattila K et al. Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infection. Lancet, 1988, 2: 983
3. Wimmer M L, Sandmann S R, Saikku P et al. Association of chlamydiae infection with cerebrovascular disease.sttoke, 1996,27(12): 207
4. Wimmer ML, Sandmann SR, Saikku P, et al. Association of chlamydiae infection with cerebrovascular disease. Stroke,1996,27(12): 2207
5. Blasi F, Denti F, Erba M, et al. Detection of Chlamydia pneumoniae but not Helicobacter pylori in atherosclerotic plaques of aortic aneurysms. J Clin Microbiol, 1996,34(11): 2766
6. Grayston JT, Kuo CC, Coulson AS, et al. Chlamydia pneumoniae (TWAR) in atherosclerosis of the carotid artery. Circulation, 1995,92(12): 3397
7. Saikku P, Leinonen M, Tenkanen L, Et al. Chronic chlamydia penumoniae infection as a risk factor for coronary heart disease in the Helsinki heart study. Ann Intern Med, 1992,116(3): 273
8. Kuo CC,Shor A,Campbell LA,et al.Demonstration of chlamydia penumoniae in atheroscleritie lesions of coronary arteries.J Infect Dis, 1993,167(4): 841
9. Grayston J T, Wang S P, History of chlamydia penumoniae (TWAR). In: Alleg L, Blasi F, eds. chlamydia penumoniae The lung and the heart. Milano: Springer, 1999,1
10. Saikku P, Leinonen M, Matilla K, et al. Serological evidence of an association of a novel chlamydia, TWAR, with chronic heart disease and acute myocardial infarction. Lancet, 1988,2: 983
11.. Valtonen VV. Rode of infections in atherosclerosis. Am Heart J, 1999,138: 431
12. Muhlestain JB, Hammond EH, Carlquist JF, et al. Increased incidence of chlamydia species within the coronary arteries of patients with symptomatic atherosclerotic versus other forms of cardiovascular disease. J Am Coll Cardiol, 1996,27: 1555
13. Puorakkainen M, Kuo CC, Shor A, et al. Serological response to Chlarnydia pneumoniae in adults with coronary artherial fatty streaks and fibrolipid plaque J Clin Microbiol, 1993,31: 2212
14. Kuo CC, Grayston JT, Goo YA, et al. Chlamydia penumoniae (TWAR) in coronary artery of young (15-35y) adults. Proc Natl Acad Sci USA, 1995,92: 6911
15. Wang SP. The microimmunofluorescence test for Chlamydia penumoniae infection: technoque and interpretation. J Infect Dis, 2000,181: s421
16. Saikku P, Leinonen M, Tenkanen L, et al. Chronic chlamydia penumoniae infection as a risk factor for coronary heart in the Helsinki heart study. Ann Intern Med, 1992,116: 273
17. Leinonen M, Laurila A, Laitinen K, et al. Immunology of Chlamydia penumoniae. In: Altegra L, Blasi F, eds. Chlamydia penumoniae infection. Berlin: Springer, 1995: 39
18. Campbell LA, O'Brien ER, Cappuccio AL, et al. Detection of Chlamydia penumoniae TWAR in human coronary atherectomy tissue. J Infect Dis, 1995, 172(2): 585
19. Gray GC, Rodier GR, Matrasmaslin VC, et al. Serologic evidence of respiratory and rickettsial infections among Somali refugees. Am J Trop Med Hyg, 1995,52(4): 349
20. Vercellotti G. Infectious agents that play a role in atherosclerosis and vasculopathies. What are they? What do we about them? Can J Cardiol, 1999,15: 13B
21. Kuo CC, Jackson LA, Campbell LA, et al. Chlamydia penumoniae (TWAR).Clin Microbiol Rev, 1995,8(4): 451
22. Kauppinen MT, Herva E, Kujala P, et al. The etiology of community acquired pneumonia among hospitalized patients during a Chlamydia penumoniae epidemic in Finland. J Infect Dis, 1995,172(5): 1330
23. Gencay M, Dereli D Ertem El. Prevalence of Chlamydia penumoniae specific antibodies in different clinical situations and healthy subjects in Izmir, Turkey. Eur J Epidemiol, 1998,14(5): 505
24. Miyashita N, Kubota Y, Nakajima M, et al. Chlamydia pneumoniae and exacerbations of asthma in adults. Ann Allergy Asthma Immunol, 1998, 80 (5): 405
25．王卫群，袁洁，王岫南，余竹元．家兔的肺炎衣原体感染和动脉粥样硬化模型．上海医科大学学报，2000，27(4)：292
26．袁洁，余竹元，黄士通．冠状动脉粥样硬化性疾病患者肺炎衣原体抗体检测．中国人兽病共患杂志，1999，15(1)：12
27．操乐杰，王明丽，吴向辉等．痰培养及血清学联合检测肺炎衣原体急性呼吸道感染．中华结核和呼吸杂志，1997，20：95
28．倪安平，王辉，董平等．肺炎，支气管炎和急性呼吸道感染患者肺炎衣原体感染的研究．中华内科杂志，1995，34(6)：388
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30．王汉斌，史寅奎，孙成文等．Research on the Location ofthe thrombin receptor in the atherosclerotic cardiovascular tissues．免疫学杂志，1998，14(2)：71
31．罗显荣，宁波，王琳等．冠心病患者血清肺炎衣原体特异性抗体的检测．中华动脉硬化杂志，2000，8(3)：260
32．孙蓉，王晓玲．部分冠状动脉硬化症患者肺炎衣原体感染的血清学调查．预防医学文献信息，2001，7(5)：495
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34．吴向辉，操乐杰，张小玲等．两种免疫荧光方法检测肺炎衣原体抗体的比较．上海医学检验杂志，2002，17(4)：221
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2. Saikku P, Leinonen M, Mattila K et al. Serological evidence of an association of a novel Chlamydia, TWAR, with chronic coronary heart disease and acute myocardial infection. Lancet, 1988, 2: 983
3. Wimmer M L, Sandmann S R, Saikku P et al. Association of chlamydiae infection with cerebrovascular disease.sttoke, 1996,27(12): 207
4. Wimmer ML, Sandmann SR, Saikku P, et al. Association of chlamydiae infection with cerebrovascular disease. Stroke,1996,27(12): 2207
5. Blasi F, Denti F, Erba M, et al. Detection of Chlamydia pneumoniae but not Helicobacter pylori in atherosclerotic plaques of aortic aneurysms. J Clin Microbiol, 1996,34(11): 2766
6. Grayston JT, Kuo CC, Coulson AS, et al. Chlamydia pneumoniae (TWAR) in atherosclerosis of the carotid artery. Circulation, 1995,92(12): 3397
7. Saikku P, Leinonen M, Tenkanen L, Et al. Chronic chlamydia penumoniae infection as a risk factor for coronary heart disease in the Helsinki heart study. Ann Intern Med, 1992,116(3): 273
8. Kuo CC,Shor A,Campbell LA,et al.Demonstration of chlamydia penumoniae in atheroscleritie lesions of coronary arteries.J Infect Dis, 1993,167(4): 841
9. Grayston J T, Wang S P, History of chlamydia penumoniae (TWAR). In: Alleg L, Blasi F, eds. chlamydia penumoniae The lung and the heart. Milano: Springer, 1999,1
10. Saikku P, Leinonen M, Matilla K, et al. Serological evidence of an association of a novel chlamydia, TWAR, with chronic heart disease and acute myocardial infarction. Lancet, 1988,2: 983
11.. Valtonen VV. Rode of infections in atherosclerosis. Am Heart J, 1999,138: 431
12. Muhlestain JB, Hammond EH, Carlquist JF, et al. Increased incidence of chlamydia species within the coronary arteries of patients with symptomatic atherosclerotic versus other forms of cardiovascular disease. J Am Coll Cardiol, 1996,27: 1555
13. Puorakkainen M, Kuo CC, Shor A, et al. Serological response to Chlarnydia pneumoniae in adults with coronary artherial fatty streaks and fibrolipid plaque J Clin Microbiol, 1993,31: 2212
14. Kuo CC, Grayston JT, Goo YA, et al. Chlamydia penumoniae (TWAR) in coronary artery of young (15-35y) adults. Proc Natl Acad Sci USA, 1995,92: 6911
15. Wang SP. The microimmunofluorescence test for Chlamydia penumoniae infection: technoque and interpretation. J Infect Dis, 2000,181: s421
16. Saikku P, Leinonen M, Tenkanen L, et al. Chronic chlamydia penumoniae infection as a risk factor for coronary heart in the Helsinki heart study. Ann Intern Med, 1992,116: 273
17. Leinonen M, Laurila A, Laitinen K, et al. Immunology of Chlamydia penumoniae. In: Altegra L, Blasi F, eds. Chlamydia penumoniae infection. Berlin: Springer, 1995: 39
18. Campbell LA, O'Brien ER, Cappuccio AL, et al. Detection of Chlamydia penumoniae TWAR in human coronary atherectomy tissue. J Infect Dis, 1995, 172(2): 585
19. Gray GC, Rodier GR, Matrasmaslin VC, et al. Serologic evidence of respiratory and rickettsial infections among Somali refugees. Am J Trop Med Hyg, 1995,52(4): 349
20. Vercellotti G. Infectious agents that play a role in atherosclerosis and vasculopathies. What are they? What do we about them? Can J Cardiol, 1999,15: 13B
21. Kuo CC, Jackson LA, Campbell LA, et al. Chlamydia penumoniae (TWAR).Clin Microbiol Rev, 1995,8(4): 451
22. Kauppinen MT, Herva E, Kujala P, et al. The etiology of community acquired pneumonia among hospitalized patients during a Chlamydia penumoniae epidemic in Finland. J Infect Dis, 1995,172(5): 1330
23. Gencay M, Dereli D Ertem El. Prevalence of Chlamydia penumoniae specific antibodies in different clinical situations and healthy subjects in Izmir, Turkey. Eur J Epidemiol, 1998,14(5): 505
24. Miyashita N, Kubota Y, Nakajima M, et al. Chlamydia pneumoniae and exacerbations of asthma in adults. Ann Allergy Asthma Immunol, 1998, 80 (5): 405
25．王卫群，袁洁，王岫南，余竹元．家兔的肺炎衣原体感染和动脉粥样硬化模型．上海医科大学学报，2000，27(4)：292
26．袁洁，余竹元，黄士通．冠状动脉粥样硬化性疾病患者肺炎衣原体抗体检测．中国人兽病共患杂志，1999，15(1)：12
27．操乐杰，王明丽，吴向辉等．痰培养及血清学联合检测肺炎衣原体急性呼吸道感染．中华结核和呼吸杂志，1997，20：95
28．倪安平，王辉，董平等．肺炎，支气管炎和急性呼吸道感染患者肺炎衣原体感染的研究．中华内科杂志，1995，34(6)：388
29．冯根宝，鲁刚，朱清淤等．动脉粥样硬化症与肺炎衣原体感染：91例尸检结果报告，金陵医院学报，1999，12(2)：96
30．王汉斌，史寅奎，孙成文等．Research on the Location ofthe thrombin receptor in the atherosclerotic cardiovascular tissues．免疫学杂志，1998，14(2)：71
31．罗显荣，宁波，王琳等．冠心病患者血清肺炎衣原体特异性抗体的检测．中华动脉硬化杂志，2000，8(3)：260
32．孙蓉，王晓玲．部分冠状动脉硬化症患者肺炎衣原体感染的血清学调查．预防医学文献信息，2001，7(5)：495
33．王卫群，范卫，桂律等．检测人动脉瘤组织中肺炎衣原体特异性抗原的临床意义．中国临床医学，2001，8(2)：136
34．吴向辉，操乐杰，张小玲等．两种免疫荧光方法检测肺炎衣原体抗体的比较．上海医学检验杂志，2002，17(4)：221
35．吴向辉，操乐杰，张小玲等．间接免疫荧光法检测肺炎衣原体抗体的临床意义．上海免疫学杂志，2002，22(1)：51
36．葛长江，郭新贵．急性冠脉综合征病人肺炎衣原体抗体滴度的临床价值．免疫学杂志，2002，18(3)：218
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