[1]谭玉华,朱应竹,江 燚,等.时间分辨免疫荧光法检测结核感染T细胞释放γ-干扰素的方法建立及初步临床应用[J].现代检验医学杂志,2018,33(06):21-25.[doi:10.3969/j.issn.1671-7414.2018.06.006]
　TAN Yu-hua,ZHU Ying-zhu,JIANG Yi,et al.Establishment and Preliminary Clinical Application of Interferon Gamma Release Assays for T Cells Infected With Mycobacterium Tuberculosis Based on Time-Resolved Fluoroimmunoassay[J].Journal of Modern Laboratory Medicine,2018,33(06):21-25.[doi:10.3969/j.issn.1671-7414.2018.06.006]
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时间分辨免疫荧光法检测结核感染T细胞释放γ-干扰素的方法建立及初步临床应用()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第33卷
期数:
2018年06期
页码:
21-25
栏目:
论著
出版日期:
2018-12-24

文章信息/Info

Title:
Establishment and Preliminary Clinical Application of Interferon Gamma Release Assays for T Cells Infected With Mycobacterium Tuberculosis Based on Time-Resolved Fluoroimmunoassay
文章编号:
1671-7414(2018)06-021-05
作者:
谭玉华; 朱应竹; 江 燚; 陶佳俊; 吴能伟
广州市丰华生物工程有限公司体外诊断试剂研发中心,广州 510730
Author(s):
TAN Yu-hua; ZHU Ying-zhu; JIANG Yi; TAO Jia-jun; WUNeng-wei
R & D Center of IVD Reagents,Guangzhou Fenghua Bioengineering Co.,Ltd.,Guangzhou 510730,China
关键词:
结核分枝杆菌;  T细胞;  γ-干扰素释放试验;  时间分辨免疫荧光法;  性能评估
分类号:
R378.911; R392-33
DOI:
10.3969/j.issn.1671-7414.2018.06.006
文献标志码:
A
摘要:
目的 建立一种检测结核感染T细胞释放γ-干扰素(IFN-γ)的时间分辨免疫荧光法(TRFIA)及初步临床应用。方法 选择73例结核分枝杆菌阳性的肺结核患者,77例结核分枝杆菌阴性的肺结核患者,25例肺外结核患者,52例呼吸道疾病以外的非结核患者,70例非结核呼吸道疾病(含肺癌)患者作为研究对象。利用早期分泌抗原靶蛋白6(ESAT6)和培养滤液蛋白10(CFP-10)作为结核分枝杆菌特异性抗原,刺激患者全血标本中的结核分枝杆菌抗原特异性T细胞释放IFN-γ。植物血凝素L作为有丝分裂原,可非特异地刺激IFN-γ的产生。应用双抗体夹心TRFIA定量测定血浆中的IFN-γ浓度。评价TRFIA检测IFN-γ的精密度、检测低限、生物检测限、功能灵敏度、线性、准确度和特异度等分析性能指标,并与酶联免疫法(ELISA)进行比对试验研究。方法间结果差异比较采用χ2检验,P<0.05为差异具有统计学意义。结果 TRFIA检测IFN-γ的捕获抗体包被浓度为5.0 μg/ml,生物素标记抗体使用工作稀释度为1:1 800和铕标记物使用工作稀释度为1:500; 实验内变异系数(CV)<5%,实验间CV<10%; 检测低限0.69 pg/ml,生物检测限0.90 pg/ml,功能灵敏度1.80 pg/ml; 线性范围2～5 000 pg/ml; 检测国际生物标准参考品的偏倚不超过5%; 其他常见的细胞因子和常见内源性干扰物(胆红素、血红蛋白和三酰甘油)样本对该方法检测结果无明显影响。TRFIA检测297例临床样本结果判断与临床诊断的一致率可达85.19%; TRFIA与ELISA的结果具有高度一致性(κ=0.9 931),且TRFIA与ELISA的阳性检出率结果差异无统计学意义(χ2=0,P>0.05)。结论 TRFIA检测IFN-γ的精密度好,灵敏度高,线性范围宽,准确度好,特异度强,临床符合率高,能满足临床检测需要。
Abstract:
Objective To establish an interferon gamma(IFN-γ)release assays for T cells infected with mycobacterium tuberculosis basedon time-resolved fluoroimmunoassay(TRFIA),and evaluate its preliminary clinical application.Methods 73 patients with sputum smear-positive pulmonary tuberculosis,77 patients with sputum smear-negative pulmonarytuberculosis,25 patients with extrapulmonary tuberculosis,52 patients with non-tuberculosis and non-respiratory diseases,and 70 patients with respiratorydiseases(including lung cancer)and non-tuberculous were selected as the studysubjects.Early secreting antigen target protein 6(ESAT6)and culture filter protein 10(CFP-10)were used as mycobacterium specific antigen,which stimulate effector T cells of Mycobacterium tuberculosis antigen in patients peripheralblood.Phytohaemagglutinin L was mitogen and could stimulate the production ofIFN-γ without selection.The concentration of IFN-γ was quantitatively determined by double antibody sandwich TRFIA.The precision,low limit of detection,biologic limit of detection,functional sensitivity,linear range,accuracy,specificity,and other analytical performance indicators of TRFIA were evaluated,and RFIA was compared with enzyme-linked immunosorbent assay(ELISA).The χ2 test was used to test the difference between self-established method and the reference method of ELISA.If P value of χ2 test was less than0.05,the difference was statistically significant.Results The TRFIA capture IFN-γ antibody coating concentration was 5.0 μg/ml,the biotin-labeled IFN-γ antibody working dilution was 1:1 800,and the TRFIAeuropium marker working dilution was 1:500.The intra-assay and inter-assay coefficients of variation(CV)were less than 5% and 10% respectively,the low limit of detection was 0.69 pg/ml,the biologic limit of detection was 0.90pg/ml,the functional sensitivity was 1.80 pg/ml,the linear range was 2～5 000 pg/ml,the bias compared with international biological reference standard wasless than 5% and other cytokines and common endogenous interference(e.g.,bilirubin,hemoglobin and triglyceride)in the plasma did not interfere the detection results.The coincidence rate between the results of TRFIA and clinical diagnosis was 85.19%.The results of TRFIA and ELISA were highly consistent(κ=0.9 931),and the positive detection rate was not statistically significant(χ2=0,P>0.05).Conclusion The TRFIA methodologyhas good precision,high sensitivity,wide linear range,good accuracy,strongspecificity and high clinical compliance rate,which was valuable for clinical application.

参考文献/References:

[1] 李 亮,唐神结,杜 建,等.结核病防控年度回顾及展望(2016年)[J].中华结核和呼吸杂志,2017,40(1):11-15.
Li L,Tang SJ,Du J,et al.Annual review and outlook for tuberculosis prevention and control(2016)[J].Chin J Tubere Respir Dis,2017,40(1):11-15.
[2] 中华人民共和国国家卫生和计划生育委员会疾病预防控制局.2016年全国法定传染病疫情概况[EB/OL].http://www.nhfpc.gov.cn/jkj/s3578/201702/38ca5990f8a54ddf9ca6308fec406157.shtmL,2017-02-23.
National Health and Family Planning Commission of the People's Republic of China.Overview of the national legal infectious disease epidemic in 2016[EB/OL].(2017-02-23).http://www.nhfpc.gov.cn/jkj/s3578/201702/38ca5990f8a54ddf9ca6308fec406157.shtml.
[3] 尹青琴,申阿东.不同国家γ干扰素释放试验临床应用指南的概述[J].国际呼吸杂志,2011,31(21):1663-1666.
Yin QQ,Shen AD.Outline of interferon gamma release assays clinicalapplication guidelines in different countries[J].International Journal of Respiration,2011,31(21):1663-1666.
[4] 中华医学会结核病学分会.肺结核诊断和治疗指南[J].中国实用乡村医生杂志,2013,20(2):7-11.
Chinese Medical Association Tuberculosis Branch.Tu-berculosis diagnosis and treatment guidelines[J].Chinese Practical Journal of Rural Doctor,2013,20(2):7-11.
[5] 中华人民共和国国家质量监督检验检疫总局.SN/T 3312-2012 结核分枝杆菌γ-干扰素体外检测方法[S].北京:中国标准出版社,2013:1-8.
General Administration of Quality Supervision,Inspe-ction and Quarantine of the People's Republic of China.SN/T 3312-2012 In vitro detection for mycobacterium tuberculosis-interferon-γ[S].Beijing:China Standard Press,2013:1-8.
[6] 谭玉华,孙宝清.总免疫球蛋白E时间分辨荧光免疫分析法的建立与评价[J].重庆医学,2014,43(33):4490-4493,4497.
Tan YH,Sun BQ.Establishment and evaluation of time resolved fluoroimmuno assay for quantification of total immuno globulin E[J].Chongqing Medicine,2014,43(33):4490-4493,4497.
[7] 谭玉华,孙宝清.TRFIA法检测总免疫球蛋白E的分析灵敏度及可报告范围的建立[J].山东医药,2013,53(46):21-24.
Tan YH,Sun BQ.Establishment of analysis sensitivity and reportablerange of total immunoglobulin E detected by time-resolved fluoroimmunoassay[J].Shandong Medical Journal,2013,53(46):21-24.
[8] 张培莉,刘义庆,邵 婧,等.结核感染T 淋巴细胞IFN-γ释放实验临床检测分析[J].现代检验医学杂志,2017,32(1):22-25,29.
Zhang PL,Liu YQ,Shao J,et al.Interferon gamma release assays from T lymphocytes in patients with tuberculosis infection[J].J Mod Lab Med,2017,32(1):22-25,29.
[9] 居金宏.结核分枝杆菌检测技术研究进展[J].微生物前沿,2014,9(3):64-69.
Ju JH.Research progress in detection technology of MycobacteriumTuberculosis[J].Advances in Microbiology,2014,9(3):64-69.
[10] World Health Organization.Use of tuberculosis interferon-gammarelease assays(IGRAs)in low- and middle-income countries:policy statement[S].WHO Library Cataloguing-in-Publication Data,2011:1-70.
[11] 中华人民共和国国家卫生和计划生育委员会.WS288-2017肺结核诊断[S].北京:中国标准出版社,2018:1-24.
National Health and Family Planning Commission of the People's Republic of China.WS288-2017 Diagnosis for pulmonary tuberculosis[S].Beijing:China Standard Press,2018:1-24.
[12] 蒋 俊.结核分枝杆菌感染T细胞斑点试验的质量控制与质量保证[J].中国防痨杂志,2014,36(5):380-384.
Jiang J.Quality control and quality assurance of T-SPOT.TB[J].Chin JAntituberc,2014,36(5):380-384.

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备注/Memo

备注/Memo:
作者简介:谭玉华(1980-),男,医学硕士,临床医学检验技师,医疗器械工程师,二级企业培训师,主要从事医疗器械(体外诊断试剂)的研发、应用、质量管理和医学检验工作,E-mail:tanywhy@aliyun.com。
更新日期/Last Update: 2018-11-30