33株抗心肌肌钙蛋白T(cTnT)的单克隆抗体制备、鉴定及应用
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摘要
旨在制备抗心肌肌钙蛋白T(cTnT)的单克隆抗体(m Ab),对单抗进行初步评价鉴定,并建立(cTnT)的化学发光定量检测试剂。首先利用外购的cTnT抗原免疫BALB/c小鼠,利用常规m Ab制备技术和间接ELISA法筛选m Ab,以表达和合成的cTnT片段对筛选到的m Ab进行表位鉴定。使用双抗体夹心ELISA方法筛选检测cTnT抗原的配对m Ab,并建立cTnT全自动化学发光定量检测试剂。使用220例临床标本评价该试剂与罗氏试剂的检测一致性,另外使用238例临床样本和784例体检人群样本评价该试剂的临床应用。我们成功筛选到33株稳定分泌抗cTnT抗体的杂交瘤细胞株,并对单抗的表位进行初步鉴定。我们筛选到能检测10 pg/m L cTnT抗原的配对m Ab E16H8和C8G11,并使用该配对研制出全自动化学发光定量试剂。该试剂与罗氏试剂相关系数r达到0.959 9,检测一致率95%,利用该试剂盒检测临床样本灵敏度为97.5%,特异性为99.15%,99%体检人群的cTnT浓度分布小于0.080 6 ng/m L,符合WHO对急性心肌梗死的定义标准。综上,初步建立了cTnT诊断优势表位单抗,并利用这些优势表位的单抗建立全自动管式化学发光定量检测试剂,与罗氏试剂检测结果符合率高。
The aim of this study is to prepare and characterize cardiac troponin T(cTnT) monoclonal antibodies(m Ab),and further develop a chemiluminescence quantitative detection assay for cTnT.BALB/c mice were immunized with recombinant cTnT antigen,and specific m Abs were prepared using conventional hybridoma technique and screened by indirect ELISA method.To identify the epitopes,several cTnT peptide fragments were synthesized or expressed by genetic engineering.A double antibody sandwich ELISA method was used to screen the m Ab pairs for cTnT detection,and the automatic chemiluminescence detection assay for cTnT was developed.In total 220 clinical specimens were used for system comparison between our assay and Roche cTnT assay;further performance characteristics was evaluated by testing 238 clinical samples and 784 physical examination samples.We successfully screened 33 strains of hybridoms against cTnT,and the m Abs' epitopes were identified.Mab E16H8 and C8G11 with a detection limit of 10 pg/m L cTnT antigen were selected to develop the full automatic chemiluminescence quantitative assay.The correlation coefficient of our reagent with Roche's was 0.959 9,with a coincidence rate of 95%.The assay presented a sensitivity of 97.5%,and a specificity of 99.15% in detection of clinical samples.The cTnT concentration was less than 0.080 6 ng/m L in 99% of general population,which agrees with the definition of WHO on patients with acute myocardial infarction(AMI).In summary,we developed monoclonal antibodies against predominant epitopes for diagnostics of cTnT,and an automatic tubular chemiluminescence quantitative detection assay was further developed,which presents a high coincidence rate with Roche's.
The aim of this study is to prepare and characterize cardiac troponin T(cTnT) monoclonal antibodies(m Ab),and further develop a chemiluminescence quantitative detection assay for cTnT.BALB/c mice were immunized with recombinant cTnT antigen,and specific m Abs were prepared using conventional hybridoma technique and screened by indirect ELISA method.To identify the epitopes,several cTnT peptide fragments were synthesized or expressed by genetic engineering.A double antibody sandwich ELISA method was used to screen the m Ab pairs for cTnT detection,and the automatic chemiluminescence detection assay for cTnT was developed.In total 220 clinical specimens were used for system comparison between our assay and Roche cTnT assay;further performance characteristics was evaluated by testing 238 clinical samples and 784 physical examination samples.We successfully screened 33 strains of hybridoms against cTnT,and the m Abs' epitopes were identified.Mab E16H8 and C8G11 with a detection limit of 10 pg/m L cTnT antigen were selected to develop the full automatic chemiluminescence quantitative assay.The correlation coefficient of our reagent with Roche's was 0.959 9,with a coincidence rate of 95%.The assay presented a sensitivity of 97.5%,and a specificity of 99.15% in detection of clinical samples.The cTnT concentration was less than 0.080 6 ng/m L in 99% of general population,which agrees with the definition of WHO on patients with acute myocardial infarction(AMI).In summary,we developed monoclonal antibodies against predominant epitopes for diagnostics of cTnT,and an automatic tubular chemiluminescence quantitative detection assay was further developed,which presents a high coincidence rate with Roche's.
引文
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[11]Swaanenburg JCJM,Klaase JM,De Jongste MJL,et al.Troponin I,troponin T,CKMB-activity and CKMB-mass as markers for the detection of myocardial contusion in patients who experienced blunt trauma.Clin Chim Acta,1998,272(2):171–181.
[12]Giannitsis E,Müller-Bardorff M,Kurowski V,et al.Independent prognostic value of cardiac troponin T in patients with confirmed pulmonary embolism.Circulation,2000,102(2):211–217.
[13]Herman EH,Lipshultz SE,Rifai N,et al.Use of cardiac troponin T levels as an indicator of doxorubicin-induced cardiotoxicity.Cancer Res,1998,58(2):195–197.
[14]Harlow E,Lane D.Monoclonal antibodies//Harlow E,Lane D,Eds.Antibodies:A Laboratory Mannual.New York:Cold Spring Harbor Laboratory Press,1998:139–312.
[15]Liu PG,Li GQ,Chen YX,et al.Chemiluminescent immunoassay for high-sensitivity C-reactive protein.Chin J Biotech,2010,26(8):1150-1156(in Chinese).刘平果,李国强,陈毅歆,等.一种定量检测人血清高敏C反应蛋白的化学发光免疫方法.生物工程学报,2010,26(8):1150–1156.
[16]Liu YN,Li XY,Li ZH,et al.Preparation and identification of monoclonal antibodies against infectious bursal disease virus(IBDV)VP4.Chin J Biotech,2014,30(12):1660–1668(in Chinese).柳亚楠,李夏莹,李忠华,等.传染性法氏囊病病毒VP4蛋白单克隆抗体的制备及鉴定.生物工程学报,2014,30(11):1660–1668.
[17]Tijssen P,Kurstak E.Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays.Anal Biochem,1984,136(2):451–457.
[18]Guo YL,Chen YX,Huang DD,et al.Preparation and selection of the monoclonal antibody used for kits to detect the p24 antigen of HIV-1.Chin J Zoon,2007,23(6):527–531(in Chinese).郭永利,陈毅歆,黄德党,等.适于HIV-1 p24抗原检测试剂的单抗制备与筛选.中国人兽共患病学报,2007,23(6):527–531.
[19]Chen LY,Lin HJ,Weng ZX,et al.Development of detection kit for T cell infected with Mycobacterium tuberculosis based on full-automatic chemiluminescence immune analyzer.J Mol Diagn Ther,2015,7(5):296–301(in Chinese).陈鹭颖,林海军,翁祖星,等.基于全自动管式化学发光免疫检测系统的人结核感染T细胞检测方法的建立.分子诊断与治疗杂志,2015,7(5):296–301.
[20]Qi JL,Shao J,Peng K,et al.Preparation of monoclonal antibodies against neutrophil gelatinase-associated lipocalin(NGAL)and development of an antibody-based chemiluminescence immune quantification assay.Chin J Biochem Drugs,2015,35(4):5–9(in Chinese).齐家龙,劭佳,彭宽,等.中性粒细胞明胶酶相关脂质运载蛋白的单克隆抗体制备及化学发光免疫定量检测试剂研究.中国生化药物杂志,2015,35(4):5–9.
[21]Feng HZ,Biesiadecki BJ,Yu ZB,et al.Restricted N-terminal truncation of cardiac troponin T:a novel mechanism for functional adaptation to energetic crisis.J Physiol,2008,586(14):3537–3550.
[22]Huang CH,Yuan Q,Chen PJ,et al.Influence of mutations in hepatitis B virus surface protein on viral antigenicity and phenotype in occult HBV strains from blood donors.J Hepatol,2012,57(4):720–729.
[23]Müller-Bardorff M,Hallermayer K,Schr?der A,et al.Improved troponin T ELISA specific for cardiac troponin T isoform:assay development and analytical validation.Clin Chem,1997,43(3):458–466.
[24]Gámiz-Gracia L,Garc??a-Campa?a AM,Soto-Chinchilla JJ,et al.Analysis of pesticides by chemiluminescence detection in the liquid phase.Tr AC Trends Analyt Chem,2005,24(11):927–942.
[25]Arakawa H,Maeda M,Tsuji A.Enzyme immunoassay of cortisol by chemiluminescence reaction of luminol-peroxidase.Bunseki Kagaku,1977,26(5):322–327.
[2]Thygesen K.Alpert JS.White HD on behalf of the joint ESC/ACCF/AHA/WHF task force for redefinition of myocardial infarction.JACC,2007,50:2173–2195.
[3]Huang JF,Zhang XF,Chen PJ.Values of D-dimer,c Tn T and myocardial enzymogram detections in the diagnosis of acute myocardial infarction.Int J Lab Med,2015,36(10):1325–1326,1329(in Chinese).黄建锋,张小锋,陈丕绩.D-二聚体、c Tn T及心肌酶谱检测在急性心肌梗死诊断中的价值.国际检验医学杂志,2015,36(10):1325–1326,1329.
[4]Hu XB,Xiang XJ,Guo XC,et al.Significance investigation of high-sensitivity cardiac troponin T for viral myocarditis in children.Labor Med,2014,29(4):307–311(in Chinese).胡孝彬,向小节,郭晓聪,等.高敏肌钙蛋白T对儿童病毒性心肌炎诊断价值探讨.检验医学,2014,29(4):307–311.
[5]Lin HM,Sun QK,Hu LY.Clinical significance of NT-pro BNP,c Tn T,hs-CRP mesurements in patients with chronic heart failure.Int J Lab Med,2013,34(15):1965–1966,1968(in Chinese).林慧铭,孙全昆,胡礼仪.慢性心衰患者血NT-pro BNP、c Tn T、hs-CRP检测的临床意义.国际检验医学杂志,2013,34(15):1965–1966,1968.
[6]Kitamura M,Hata N,Takayama T,et al.High-sensitivity cardiac troponin T for earlier diagnosis of acute myocardial infarction in patients with initially negative troponin T test-comparison between cardiac markers.J Cardiol,2013,62(6):336–342.
[7]Roberts A.Biomarkers:hs-c Tn T measurement to rule out MI.Nat Rev Cardiol,2014,11:311.
[8]Heeschen C,Hamm CW,Goldmann BU,et al.for PRISM study investigators.Troponin concentrations for stratification of patients with acute coronary syndromes in relation to therapeutic efficacy of tirofiban.Lancet,1999,354:1757–1762.
[9]Masson S,Pirelli S,Pulitano G,et al.Circulating markers of myocyte injury predict first recurrence of atrial fibrillation.New Orleans:AHA,2008,11:8–12.
[10]Lauer B,Niederau C,Kühl U,et al.Cardiac troponin T in patients with clinically suspected myocarditis.JACC,1997,30(5):1354–1359.
[11]Swaanenburg JCJM,Klaase JM,De Jongste MJL,et al.Troponin I,troponin T,CKMB-activity and CKMB-mass as markers for the detection of myocardial contusion in patients who experienced blunt trauma.Clin Chim Acta,1998,272(2):171–181.
[12]Giannitsis E,Müller-Bardorff M,Kurowski V,et al.Independent prognostic value of cardiac troponin T in patients with confirmed pulmonary embolism.Circulation,2000,102(2):211–217.
[13]Herman EH,Lipshultz SE,Rifai N,et al.Use of cardiac troponin T levels as an indicator of doxorubicin-induced cardiotoxicity.Cancer Res,1998,58(2):195–197.
[14]Harlow E,Lane D.Monoclonal antibodies//Harlow E,Lane D,Eds.Antibodies:A Laboratory Mannual.New York:Cold Spring Harbor Laboratory Press,1998:139–312.
[15]Liu PG,Li GQ,Chen YX,et al.Chemiluminescent immunoassay for high-sensitivity C-reactive protein.Chin J Biotech,2010,26(8):1150-1156(in Chinese).刘平果,李国强,陈毅歆,等.一种定量检测人血清高敏C反应蛋白的化学发光免疫方法.生物工程学报,2010,26(8):1150–1156.
[16]Liu YN,Li XY,Li ZH,et al.Preparation and identification of monoclonal antibodies against infectious bursal disease virus(IBDV)VP4.Chin J Biotech,2014,30(12):1660–1668(in Chinese).柳亚楠,李夏莹,李忠华,等.传染性法氏囊病病毒VP4蛋白单克隆抗体的制备及鉴定.生物工程学报,2014,30(11):1660–1668.
[17]Tijssen P,Kurstak E.Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassays.Anal Biochem,1984,136(2):451–457.
[18]Guo YL,Chen YX,Huang DD,et al.Preparation and selection of the monoclonal antibody used for kits to detect the p24 antigen of HIV-1.Chin J Zoon,2007,23(6):527–531(in Chinese).郭永利,陈毅歆,黄德党,等.适于HIV-1 p24抗原检测试剂的单抗制备与筛选.中国人兽共患病学报,2007,23(6):527–531.
[19]Chen LY,Lin HJ,Weng ZX,et al.Development of detection kit for T cell infected with Mycobacterium tuberculosis based on full-automatic chemiluminescence immune analyzer.J Mol Diagn Ther,2015,7(5):296–301(in Chinese).陈鹭颖,林海军,翁祖星,等.基于全自动管式化学发光免疫检测系统的人结核感染T细胞检测方法的建立.分子诊断与治疗杂志,2015,7(5):296–301.
[20]Qi JL,Shao J,Peng K,et al.Preparation of monoclonal antibodies against neutrophil gelatinase-associated lipocalin(NGAL)and development of an antibody-based chemiluminescence immune quantification assay.Chin J Biochem Drugs,2015,35(4):5–9(in Chinese).齐家龙,劭佳,彭宽,等.中性粒细胞明胶酶相关脂质运载蛋白的单克隆抗体制备及化学发光免疫定量检测试剂研究.中国生化药物杂志,2015,35(4):5–9.
[21]Feng HZ,Biesiadecki BJ,Yu ZB,et al.Restricted N-terminal truncation of cardiac troponin T:a novel mechanism for functional adaptation to energetic crisis.J Physiol,2008,586(14):3537–3550.
[22]Huang CH,Yuan Q,Chen PJ,et al.Influence of mutations in hepatitis B virus surface protein on viral antigenicity and phenotype in occult HBV strains from blood donors.J Hepatol,2012,57(4):720–729.
[23]Müller-Bardorff M,Hallermayer K,Schr?der A,et al.Improved troponin T ELISA specific for cardiac troponin T isoform:assay development and analytical validation.Clin Chem,1997,43(3):458–466.
[24]Gámiz-Gracia L,Garc??a-Campa?a AM,Soto-Chinchilla JJ,et al.Analysis of pesticides by chemiluminescence detection in the liquid phase.Tr AC Trends Analyt Chem,2005,24(11):927–942.
[25]Arakawa H,Maeda M,Tsuji A.Enzyme immunoassay of cortisol by chemiluminescence reaction of luminol-peroxidase.Bunseki Kagaku,1977,26(5):322–327.
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