化学发光酶免疫分析检测全血γ-干扰素诊断结核杆菌感染方法的建立及应用
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摘要
结核病是一种由结核分枝杆菌引起的慢性传染病,全身组织器官几乎都有感染的可能,临床以肺结核最为常见。结核分枝杆菌感染人体后,主要是免疫系统在发挥作用,免疫力强时,机体处于感染状态,不发病,但当机体免疫力下降时,就可能发展为结核病。目前筛查结核杆菌感染的方法主要是结核菌素试验(Tuberculin skin test,TST),它使用结核杆菌纯蛋白衍化物(Puified protein derivative, PPD)作为抗原,而PPD皮肤试验的抗原是一种混合抗原,它包含卡介苗和其它非结核分枝杆菌的共同抗原,此试验的特异性较差,卡介苗接种者也呈阳性反应,不能有效地区分结核感染和非结核分枝杆菌感染以及卡介苗接种。
CFP-10/ESAT-6是结核杆菌特异性融合抗原,可特异地刺激结核杆菌感染者外周血单个核淋巴细胞分泌高水平的γ-干扰素,而未感染者γ-干扰素的水平很低。用化学发光酶免疫分析对全血γ-干扰素进行检测,可以定量地检测pg/ml(10-12g/ml)水平的γ-干扰素,为筛查及诊断结核杆菌感染提供了一种稳定、简单、快速、特异性强、灵敏度很高的检测方法,弥补了PPD试验的不足。
本文对阳性刺激物(植物血凝素PHA)的刺激浓度0、10、20、40、80μg/ml, CFP-10/ESAT-6融合抗原的刺激浓度0、10、20、40、80μg/ml,培养条件有无二氧化碳,培养时间18-24h和48h,底物作用时间2、5、8min等进行了比较试验研究,并对方法的稳定性进行了重复试验研究,用受试者工作曲线(ROC曲线)确定临界值,最终建立的试验方法为：PHA和CFP-10/ESAT-6融合抗原终浓度均为20μg／ml,培养时有无二氧化碳都可以,培养时间为18～24小时,底物作用时间为5分钟,临界值为379.5pg/ml(10-12g/ml)。
以传统的PPD皮肤试验为对照,用新建立的全血γ-干扰素方法对393名2009年新入伍的战士进行结核杆菌感染的筛查,结果显示PPD试验的阳性率为46.6%,全血γ-干扰素的阳性率为21.6%,两种方法的一致率为60.8%。210例PPD阴性战士中,28例全血γ-干扰素阳性,阳性率为13.3%；183例PPD阳性战士中,57例全血γ-干扰素阳性,阳性率为31.1%。
总之,在CFP-10/ESAT-6特异性融合蛋白的刺激下利用化学发光酶免疫分析法检测全血γ-干扰素可以提高结核杆菌感染的敏感性和特异性,有望成为检测结核感染的新方法。
Tuberculosis is a chronic disease caused by mycobacterium tuberculosis, which may infect almost all body tissues and organs, but pulmonary tuberculosis is the most common in clinic. Once human bodies are infected by mycobacterium tuberculosis, immune system will play a great role in defence:with strong immunity, bodies won't fall for TB even in the infectious status, but when immunity becomes weaker, they might develop into TB. As a main present method of screening TB, tuberculin skin test, (TST), puified protein derivative (PPD) is it's antigen, PPD test has weak specifidity becasue of its hybrid antigens, which contain BCG and other non-TB antigens. Since the test for BCG inoculators is positive, it can't effectively distinguish TB from non-TB and BCG inoculation.
CFP-10/ESAT-6, a tuberculosis bacili specific fusion antigen, can specifically stimulate peripheral blood mononuclear lymphocyte of the infected and make them secretey-IFN, but the y-IFN level of the non-infected is very low. Using chemiluminescence enzyme immunoassay to test whole bloody-IFN can quantitatively detect y-IFN at pg/ml (10-12 g/ml) level and it provides a test method of screening and diagnosing TB, which is characterized by stability, simplicity, rapidity, strong specificity and high sensitivity and makes up for the weakness of PPD test.
After comparatively testing stimulation concentration of positive stimulus (plant hemagglutinin PHA) of 0,10,20,40 and 80ug/ml, that of CFP-10/ESAT-6 fusion antigen of 0,10,20,40 and 80ug/ml, culturing conditions:existence and non-existence of carbon dioxide during culturing period, training time for 18-24 hours and 48 hours respectively, and substrate duration for 2,5,8 minutes, etc., and determining critical value by subjects'working curve (ROC curve), the article finally established a stable and effective test method, whose stability was also examined repeatedly:the final concentration of PHA and CFP-10/ESAT-6 fusion antigen was 20 ug/ml, it didn't matter that there was with or without carbon dioxide druing culturing periods, training time was 18-24 hours, substrate duration was 5 minutes and critical value was 379.5 pg/ml(10-12 g/ml).
Compared with traditional PPD skin test, we screened infection due mycobacterium tuberculosis for 393 new recurits in 2009 by using the newly established method of whole bloodγ-IFN. The results showed that positive rate of whole bloodγ-IFN was 21.6%, PPD skin test 46.6%, and consistent rate of both was 60.8%. Among 210 PPD negative cases,28 were whole blood y-IFN positive, positive rate was 13.3%. while among 183 PPD positive cases,57were whole bloodγ-IFN positive. positive rate was 31.1%.
In words, it can improve mycobacterium tuberculosis'infectious sensitivity and specificity to use chemical luminescence enzyme immunoassay to test whole bloodγ-IFN under the stimulation of CFP-10/ESAT-6specific fusion antigen; therefore, the method is expected to be a new diagnostic method of screening and examining TB infection.
CFP-10/ESAT-6是结核杆菌特异性融合抗原,可特异地刺激结核杆菌感染者外周血单个核淋巴细胞分泌高水平的γ-干扰素,而未感染者γ-干扰素的水平很低。用化学发光酶免疫分析对全血γ-干扰素进行检测,可以定量地检测pg/ml(10-12g/ml)水平的γ-干扰素,为筛查及诊断结核杆菌感染提供了一种稳定、简单、快速、特异性强、灵敏度很高的检测方法,弥补了PPD试验的不足。
本文对阳性刺激物(植物血凝素PHA)的刺激浓度0、10、20、40、80μg/ml, CFP-10/ESAT-6融合抗原的刺激浓度0、10、20、40、80μg/ml,培养条件有无二氧化碳,培养时间18-24h和48h,底物作用时间2、5、8min等进行了比较试验研究,并对方法的稳定性进行了重复试验研究,用受试者工作曲线(ROC曲线)确定临界值,最终建立的试验方法为：PHA和CFP-10/ESAT-6融合抗原终浓度均为20μg／ml,培养时有无二氧化碳都可以,培养时间为18～24小时,底物作用时间为5分钟,临界值为379.5pg/ml(10-12g/ml)。
以传统的PPD皮肤试验为对照,用新建立的全血γ-干扰素方法对393名2009年新入伍的战士进行结核杆菌感染的筛查,结果显示PPD试验的阳性率为46.6%,全血γ-干扰素的阳性率为21.6%,两种方法的一致率为60.8%。210例PPD阴性战士中,28例全血γ-干扰素阳性,阳性率为13.3%；183例PPD阳性战士中,57例全血γ-干扰素阳性,阳性率为31.1%。
总之,在CFP-10/ESAT-6特异性融合蛋白的刺激下利用化学发光酶免疫分析法检测全血γ-干扰素可以提高结核杆菌感染的敏感性和特异性,有望成为检测结核感染的新方法。
Tuberculosis is a chronic disease caused by mycobacterium tuberculosis, which may infect almost all body tissues and organs, but pulmonary tuberculosis is the most common in clinic. Once human bodies are infected by mycobacterium tuberculosis, immune system will play a great role in defence:with strong immunity, bodies won't fall for TB even in the infectious status, but when immunity becomes weaker, they might develop into TB. As a main present method of screening TB, tuberculin skin test, (TST), puified protein derivative (PPD) is it's antigen, PPD test has weak specifidity becasue of its hybrid antigens, which contain BCG and other non-TB antigens. Since the test for BCG inoculators is positive, it can't effectively distinguish TB from non-TB and BCG inoculation.
CFP-10/ESAT-6, a tuberculosis bacili specific fusion antigen, can specifically stimulate peripheral blood mononuclear lymphocyte of the infected and make them secretey-IFN, but the y-IFN level of the non-infected is very low. Using chemiluminescence enzyme immunoassay to test whole bloody-IFN can quantitatively detect y-IFN at pg/ml (10-12 g/ml) level and it provides a test method of screening and diagnosing TB, which is characterized by stability, simplicity, rapidity, strong specificity and high sensitivity and makes up for the weakness of PPD test.
After comparatively testing stimulation concentration of positive stimulus (plant hemagglutinin PHA) of 0,10,20,40 and 80ug/ml, that of CFP-10/ESAT-6 fusion antigen of 0,10,20,40 and 80ug/ml, culturing conditions:existence and non-existence of carbon dioxide during culturing period, training time for 18-24 hours and 48 hours respectively, and substrate duration for 2,5,8 minutes, etc., and determining critical value by subjects'working curve (ROC curve), the article finally established a stable and effective test method, whose stability was also examined repeatedly:the final concentration of PHA and CFP-10/ESAT-6 fusion antigen was 20 ug/ml, it didn't matter that there was with or without carbon dioxide druing culturing periods, training time was 18-24 hours, substrate duration was 5 minutes and critical value was 379.5 pg/ml(10-12 g/ml).
Compared with traditional PPD skin test, we screened infection due mycobacterium tuberculosis for 393 new recurits in 2009 by using the newly established method of whole bloodγ-IFN. The results showed that positive rate of whole bloodγ-IFN was 21.6%, PPD skin test 46.6%, and consistent rate of both was 60.8%. Among 210 PPD negative cases,28 were whole blood y-IFN positive, positive rate was 13.3%. while among 183 PPD positive cases,57were whole bloodγ-IFN positive. positive rate was 31.1%.
In words, it can improve mycobacterium tuberculosis'infectious sensitivity and specificity to use chemical luminescence enzyme immunoassay to test whole bloodγ-IFN under the stimulation of CFP-10/ESAT-6specific fusion antigen; therefore, the method is expected to be a new diagnostic method of screening and examining TB infection.
引文
[1]World Health Organization.Gobal tuberculosis control:Surveillance, planning,financing.WHO Report 2005.Geneva,2005,349.
[2]WorldHealthorgernization,Http//www.who.int/gtb/publications/globrep01/,2001.
[3]端木宏谨.第四次全国结核病流行病学抽样调查报告[J].中华结核呼吸杂志,2002,25(1)3-7.
[4]Madhukar Pai,Lee W Riley,and John M Colford Jr Interferon-yassays in the immunodiagnosis of tuberculosis:a systematic review.Infections Diseases Vol 4 December,2004,761-773.
[5]Alton B,Farris,M D,John A.B randa,M.D.QuantiFERON-TB Gold Assay for TuberculosisInfection.ClinicalMicrobiologyNewsletter,2007,29:17129-135.
[6]Sorensen AL,Nagai S,Hou'en Qet al.Purification and characterization of a loq molecular mass T-cell antigen secreted by Mycobacterium tuberculosis[J].Ifect Immune 1995,63:1710-1717.
[7]吴雪琼、张俊仙、史迎昌,等.结核分支杆菌ESAT6蛋白的表达、纯化及抗原性研究[J].中国现代医学杂志2001,11(9)：14-17.
[8]蒋玉凤,钟森,史小玲,等.结核分枝杆菌ESAT-6真核表达质粒的构建及蛋白表达的鉴定[J].中国人兽共患病杂志,2003,19(5)：63-69.
[9]陈全,骆旭山,蒋英,等.结核分枝杆菌1hp基因原核表达载体的构建和表达[J].细胞与分子免疫学杂志,2003,19(4)：332-334.
[10]陈全,骆旭山,蒋英,等.结核分枝杆菌CFP10-ESAT6融合蛋白的制备[J].中华微生物学和免疫学杂志[J],2002,22(6)：648.
[11]李娟,吴雪琼,张俊仙,等。结核分支杆菌CFP10-ESAT6融合蛋白在大肠杆菌中的高效表达[J]。中国防痨杂志2004,26(4)：204-208.
[12]MUNKME,ORMEIM,SAXENARK.Use of ESAT-6 and CFP-10 antigens for diagnosis of extrapulmonary tuberculosis[J].Infect Dis,2001,183:175-176.
[13]都伟欣,陈保文,徐苗,等结核分支杆菌ESAT-6/CFP-10融合蛋白对不同分支杆菌致敏动物的鉴别[J].中国生物制品学杂志,2009,22,(8)：807-814.
[14]周永碧.CFP10-ESAT6融合蛋白为抗原的ELISA法鉴别MTB和BCG致敏鼠的实验研究[J].中华当代医学,2006,4(1)：8-9.
[15]Ravn P,Munk ME,Andersen AB,Lundgren B,Lundgren JD,Nielsen LN,Kok-Jensen A,Andersen P,Weldingh Kprospective evaluation of a whole-blood test using Mycobacterium tuberculosis-soecific antigens ESAT-6 and CFP-10 for diagnosis of active tuberculosis[J]. Clin Diagn Lab Immunol,2005,12:491-496.
[16]Dogra S,Narang P,Mendiratta DK,Chaturvedi P,Reingold AL,Colford Jr JM,et al Comparison of a whole blood interferon-gamma assay with tuberculin skin testing for the detection of tuberculosis infection in hospitalized children in rural India [J].Infect 2007,54(3):261-216.
[17]Harari A,Vallelian F,Meylan PR,Pantaleo G Functional heterogeneity of memory CD4T cell responses in different conditions of antigen exposure and persistence[J].Immunol,2005,174:1035-45.
[18]Detection of mycobacteriu tuberculosis infection in United States Navy Recruits using the tuberculin skin test or whole-blood interferon-γ release assays[J].Clin Infect Dis,2007,45(7):826-836.
[19]ANDERSEN P,DOHERTY TM,PAIM,et al.The prognosis of latent tuberculosis can disease be predicted? [J].Trends MolMed,2007,13(5):175-182.
[2]WorldHealthorgernization,Http//www.who.int/gtb/publications/globrep01/,2001.
[3]端木宏谨.第四次全国结核病流行病学抽样调查报告[J].中华结核呼吸杂志,2002,25(1)3-7.
[4]Madhukar Pai,Lee W Riley,and John M Colford Jr Interferon-yassays in the immunodiagnosis of tuberculosis:a systematic review.Infections Diseases Vol 4 December,2004,761-773.
[5]Alton B,Farris,M D,John A.B randa,M.D.QuantiFERON-TB Gold Assay for TuberculosisInfection.ClinicalMicrobiologyNewsletter,2007,29:17129-135.
[6]Sorensen AL,Nagai S,Hou'en Qet al.Purification and characterization of a loq molecular mass T-cell antigen secreted by Mycobacterium tuberculosis[J].Ifect Immune 1995,63:1710-1717.
[7]吴雪琼、张俊仙、史迎昌,等.结核分支杆菌ESAT6蛋白的表达、纯化及抗原性研究[J].中国现代医学杂志2001,11(9)：14-17.
[8]蒋玉凤,钟森,史小玲,等.结核分枝杆菌ESAT-6真核表达质粒的构建及蛋白表达的鉴定[J].中国人兽共患病杂志,2003,19(5)：63-69.
[9]陈全,骆旭山,蒋英,等.结核分枝杆菌1hp基因原核表达载体的构建和表达[J].细胞与分子免疫学杂志,2003,19(4)：332-334.
[10]陈全,骆旭山,蒋英,等.结核分枝杆菌CFP10-ESAT6融合蛋白的制备[J].中华微生物学和免疫学杂志[J],2002,22(6)：648.
[11]李娟,吴雪琼,张俊仙,等。结核分支杆菌CFP10-ESAT6融合蛋白在大肠杆菌中的高效表达[J]。中国防痨杂志2004,26(4)：204-208.
[12]MUNKME,ORMEIM,SAXENARK.Use of ESAT-6 and CFP-10 antigens for diagnosis of extrapulmonary tuberculosis[J].Infect Dis,2001,183:175-176.
[13]都伟欣,陈保文,徐苗,等结核分支杆菌ESAT-6/CFP-10融合蛋白对不同分支杆菌致敏动物的鉴别[J].中国生物制品学杂志,2009,22,(8)：807-814.
[14]周永碧.CFP10-ESAT6融合蛋白为抗原的ELISA法鉴别MTB和BCG致敏鼠的实验研究[J].中华当代医学,2006,4(1)：8-9.
[15]Ravn P,Munk ME,Andersen AB,Lundgren B,Lundgren JD,Nielsen LN,Kok-Jensen A,Andersen P,Weldingh Kprospective evaluation of a whole-blood test using Mycobacterium tuberculosis-soecific antigens ESAT-6 and CFP-10 for diagnosis of active tuberculosis[J]. Clin Diagn Lab Immunol,2005,12:491-496.
[16]Dogra S,Narang P,Mendiratta DK,Chaturvedi P,Reingold AL,Colford Jr JM,et al Comparison of a whole blood interferon-gamma assay with tuberculin skin testing for the detection of tuberculosis infection in hospitalized children in rural India [J].Infect 2007,54(3):261-216.
[17]Harari A,Vallelian F,Meylan PR,Pantaleo G Functional heterogeneity of memory CD4T cell responses in different conditions of antigen exposure and persistence[J].Immunol,2005,174:1035-45.
[18]Detection of mycobacteriu tuberculosis infection in United States Navy Recruits using the tuberculin skin test or whole-blood interferon-γ release assays[J].Clin Infect Dis,2007,45(7):826-836.
[19]ANDERSEN P,DOHERTY TM,PAIM,et al.The prognosis of latent tuberculosis can disease be predicted? [J].Trends MolMed,2007,13(5):175-182.