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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 陈自敏等 /CK-MB 特异性单抗的制备方法及其应用 January 25, 2017, 33(1): Chin J Biotech, All rights reserved 生物技术与方法 141 CK-MB 陈自敏 1, 周国梁 1, 徐伟玲 2, 郑小红 2, 童勋章 2, 柯起沈 2, 宋浏伟 1, 葛胜祥 ,,,. CK-MB., 2017, 33(1): Chen ZM, Zhou GL,Xu WL, et al. Development and application of CK-MB specific monoclonal antibodies. Chin J Biotech, 2017, 33(1): 摘要 : 本研究拟建立肌酸激酶同工酶 MB (CK-MB) 特异性单克隆抗体 (mab) 的研制方法, 对抗 CK-MB 单抗进行评价分类及性质鉴定, 并初步建立 CK-MB 定量检测试剂以 CK-MB 抗原免疫 BALB/c 小鼠, 利用常规单抗制备技术, 使用间接和捕获 ELISA 差异筛选法筛选单抗利用肌酸激酶同工酶 (CK-MM/BB/MB) 抗原对所制备单抗的抗原识别表位进行鉴定, 另通过免疫印迹法及合成 CK-MM CK-BB 差异性的线性表位肽鉴定对所制备的单抗进行评价分类使用双抗体夹心 ELISA 方法筛选检测 CK-MB 抗原的配对 mab, 并初步建立 CK-MB 定量检测试剂使用 74 例临床标本初步评价该试剂与罗氏试剂的检测一致性最终, 我们成功筛选到 22 株稳定分泌抗 CK-MB 抗体的杂交瘤细胞株, 这些单抗可以分为线性偏构象的 CK-MB 和 CK-MM 或者 CK-BB 交叉的单抗以及与 CK-MB 特异反应的偏构象型单抗, 并使用偏构象型单抗研制出 CK-MB 定量检测试剂, 该试剂与罗氏试剂相关系数 r 达到综上所述, 本研究建立了研制 CK-MB 偏构象型特异性单抗的筛选方法, 通过对所筛选的单抗进行分析鉴定并建立了 CK-MB 定量检测试剂, 与罗氏试剂检测结果符合率高 : 肌酸激酶同工酶 MB (CK-MB), 急性心肌梗死 (AMI), 单克隆抗体, 构象型单抗, 捕获法 Received: May 31, 2016; Accepted: November 24, 2016 Supported by: Xiamen Science and Technology Program (No. 3502Z ). Corresponding author: Shengxiang Ge. Tel/Fax: ; sxge@xmu.edu.cn (No. 3502Z )

2 142 ISSN CN /Q Chin J Biotech January 25, 2017 Vol.33 No.1 Development and application of CK-MB specific monoclonal antibodies Zimin Chen 1, Guoliang Zhou 1, Weiling Xu 2, Xiaohong Zheng 2, Xunzhang Tong 2, Qishen Ke 2, Liuwei Song 1, and Shengxiang Ge 1 1 National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Sciences, Xiamen University, Xiamen , Fujian, China 2 Xiamen Innodx Biotech Co., Ltd., Xiamen , Fujian, China Abstract: The aim of this study is to develop creatine kinase isoenzyme MB (CK-MB) specific monoclonal antibodies (mab), and characterize the monoclonal antibody and further development of quantitative detection assay for CK-MB. The BALB/c mice were immunized with purchased CK-MB antigen, then monoclonal antibodies were prepared according to conventional hybridoma technique and screened by indirect and capture ELISA method. To identify the epitopes and evaluate the classification, purchased creatine kinase isoenzyme MB (CK-MM/BB/MB) antigen was used to identify the epitopes, with immunoblotting and synthetic CK-MM and CK-BB in different linear epitope. A double antibody sandwich ELISA was applied to screen the mab pairs for CK-MB detection, and the quantitative detection assay for CK-MB was developed. We used 74 cases of clinical specimens for comparison of our assay with Roche s CK-MB assay. We successfully developed 22 strains of hybridoms against CK-MB, these mabs can be divided into linear, partial conformational CK-MB, CK-MM or CK-BB cross monoclonal antibody and CK-MB specific reaction with partial conformational monoclonal antibody, and CK-MB quantitative detection assay was developed by using partial conformational monoclonal antibody. The correlation coefficient factor r of our reagent and Roche s was This study established a screening method for CK-MB partial conformational specific monoclonal antibody, and these monoclonal antibodies were analyzed and an established quantitative detection assay was developed. The new assay had a high concordance with Roche s. Keywords: creatine kinase isoenzyme MB, acute myocardial infarction (AMI), monoclonal antibodies, conformational monoclonal antibodies, capture method (AMI) [1] AMI (CK) (AST) (LDH) AMI MB (CK-MB) CK-MB MM (CK-MM) 94% 96% CK-MB 5% CK-MB CK-MB 6% [2-3] CK B M 43 kda M B 80% 3 CK-MM CK-BB CK-MB 3 3 [4] CK-M CK-B [5]

3 陈自敏等 /CK-MB 特异性单抗的制备方法及其应用 143 CK-MM CK-BB CK-MB CK-MB 4 h h 3 4 d CK-MB (AMI) [6] CK-MB CK-MB CK-MB CK-MB CK-MB 1 材料与方法 1.1 材料 CK-MB CK-MM CK-BB Meridian ( VTI820 VTI830 VT810) SP2/0 (CCTCC) BALB/c PEG1500 DMSO 和 (HRP) Sigma RPMI 1640 Gibco PAA AMI 76 (AMI WHO ) 1.2 方法 BALB/c CK-MB (CFA) (Sigma St. Louis MO) (IFA) (Sigma St. Louis MO) 2 2 PBS 3 d [7] ) 3 CK-MM/BB/MB (20 mmol/l CB ph 9.6) HRP ( 20 mmol/l PBS (ph 7.4) ) 2) (GAM) 20 mmol/l PBS (ph 7.4) ( 20 mmol/l PBS ph 7.4) HRP 3 (CK-MM/BB/MB) ) mab 3 CK-MM/BB/MB (20 mmol/l CB ph 9.6) HRP 2) mab cjb@im.ac.cn

4 144 ISSN CN /Q Chin J Biotech January 25, 2017 Vol.33 No.1 GAM 3 HRP 3) 3 (CK-MM/ BB/MB) 5% (Blocking) 1 h 1 22 ( Primary antibody incubation 5 μg/ml) 1 h (20 mmol/l PBS (ph 7.4)+0.05% Tween20) IgG (GAM-AP) ( Secondary antibody incubation) 1 h ( ) 15 min 4) CK-MM CK-BB aa (CK-MM QKIEEIFKKAGHPFM CK-BB TQIETLFKSK DYEFM) 15 aa ng/ml mg EDC (10 mg EDC HCl 1 ml ) 80 μg 1 ml 1% (ph 7.4) PBS ( ) (ph 8.0) PBS (20 mmol/l PBS ph 7.4) PBS GPS GraphPad Prism 2 结果与分析 2.1 CK-MB 单克隆抗体的制备与鉴定 ELISA ELISA 22 CK-MB 2.2 单抗活性评价 ELISA ELISA ( 1 μg/ml) 3 ( ) Ⅰ CK-MM/BB/MB Ⅱ Ⅲ CK-MM/MB CK-BB/MB Ⅳ Ⅵ CK-MM/MB CK-BB/MB 2 Ⅴ CK-MB 2.3 免疫印迹法评价单抗性质 2 Ⅰ/Ⅱ/Ⅲ ELISA Ⅳ/Ⅴ/Ⅵ CK-MB

陈自敏等 /CK-MB 特异性单抗的制备方法及其应用 145 Ⅳ/Ⅴ/Ⅵ Ⅰ/Ⅱ/Ⅲ 2.

100 ng/ml Ⅰ/Ⅱ/Ⅲ CK-MM/BB 3 CK-MB 图 1 单抗 6

1 Mapping of evaluation for the activity of

5 陈自敏等 /CK-MB 特异性单抗的制备方法及其应用 145 Ⅳ/Ⅴ/Ⅵ Ⅰ/Ⅱ/Ⅲ 2.5 单抗配对筛选 22 HRP 3 4 CK-MM/CK-BB 500 ng/ml CK-MB 100 ng/ml Ⅰ/Ⅱ/Ⅲ CK-MM/BB 3 CK-MB 图 1 单抗 6 种活性评价方法图谱 Fig. 1 Mapping of evaluation for the activity of monoclonal antibodies using 6 methods. 图 2 免疫印迹法评价单抗性质图谱 Fig. 2 Evaluation of monoclonal antibodies with immune blotting method. 2.4 线性表位验证单抗性质 3 ( 10 μg/ml) Ⅰ/Ⅱ/Ⅲ CK-MM CK-BB aa 图 3 线性表位评价单抗性质图谱 Fig. 3 Mapping of linear epitope evaluation of monoclonal antibody properties cjb@im.ac.cn

6 146 ISSN CN /Q Chin J Biotech January 25, 2017 Vol.33 No.1 图 4 Fig. 4 抗体正交配对评价 Evaluation of the cross reaction with antibodies. Ⅳ/Ⅴ/Ⅵ CK-MB Ⅵ CK-BB CK-MB 4 Roche Ⅳ

7 陈自敏等 /CK-MB 特异性单抗的制备方法及其应用 147 (E19B12)-Ⅵ (D22G9) Ⅴ (B14H8)-Ⅵ (D22G9) Ⅴ (B14H8)-Ⅳ (E19B12) Ⅵ (D22G9)-Ⅳ (E19B12) Ⅵ (D22G9)-Ⅴ (B14H8) 5 Ⅵ (D22G9)-Ⅳ (E19B12) 2.6 单抗在管式化学发光平台原料的应用 CK-MB Roche CK-MB 74 (r 2 = P< ) 6 图 5 Fig. 5 比较不同抗体配对评价 (A) CK-MB 抗原和 (B) 样本 Comparison of different pairs of antibodies in detection of CK-MB antigen (A) and samples (B). 图 6 Fig. 6 与同类试剂比较 Comparison with similar assay. 3 讨论 CK CK-MB CK-MB 20% [8] ctni CK-MB AMI [9-19] CK-MB CK-MM/BB/MB cjb@im.ac.cn

8 148 ISSN CN /Q Chin J Biotech January 25, 2017 Vol.33 No Ⅰ CK-MM/BB/MB Ⅱ Ⅲ CK-MM/MB CK-BB/MB Ⅳ Ⅵ CK-MM/MB CK-BB/MB Ⅴ CK-MB CK-MM CK-BB Ⅰ/Ⅱ/ Ⅲ Ⅳ/Ⅴ/Ⅵ 3 3 CK-MB D22G9 E19B12 CK-MB r 2 = P< CK-MB CK-MB CK-BB CK-MM CK-MB M B MB CK-MB CK-MB [20-22] CK-MB CK-MB B M CK-MM CK-BB CK-MB CK-MB [23-25] CK-MB CK-MB REFERENCES [1] Wang LL. Classification and clinical application of myocardial markers. Int J Lab Med, 2004, 25(5):

9 陈自敏等 /CK-MB 特异性单抗的制备方法及其应用 (in Chinese)..., 2004, 25(5): [2] Tucker JF, Collins RA, Anderson AJ, et al. Value of serial myoglobin levels in the early diagnosis of patients admitted for acute myocardial infarction. Ann Emerg Med, 1994, 24(4): [3] Lin XJ, Gong XD. Application of chemiluminescence detection of ctni/myo/ck-mb in coronary heart disease. Chin J Healthy Birth Child Care, 2014, 20(4): (in Chinese).,. ctni/myo/ CK-MB., 2014, 20(4): [4] Zhao X, Han B, Yang ZH. Electrophoretic separation and fluorescence scanning of creatine kinase isoforms. J Clin Lab Sci, 1999, 17(3): (in Chinese).,,.., 1999, 17(3): [5] Tai DF, Ho YF, Wu CH, et al. Artificial-epitope mapping for CK-MB assay. Analyst, 2011, 136(11): [6] Sachdeva A, Paul B. Newer cardiac biomarkers in myocardial necrosis. J Assoc Physicians India, 2014, 62(10): [7] Harlow E, Lane D. Antibodies//Harlow E, Lane D, Eds. Antibodies: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press, 1998: [8] Wang F, Li J, Lin Y, et al. To discuss the values of CK,CK-MB in early diagnosis of acute myocardial infarction. Chin J Mod Med, 2010, 12(8): (in Chinese).,,,. CK CK-MB., 2010, 12(8): [9] Zhang H, Dong M, Zhong HQ. Clinical value of joint detection of Mb, CK-Mb and ctni inearly diagnosis of acute myocardial infarction. Chin J Lab Med, 2010, 20(6): (in Chinese).,,. Mb CK-MB ctni AMI., 2010, 20(6): [10] Wang HM. The application value of the detection of myocardial markers in the rapid diagnosis of acute myocardial infarction. The Medical Forum, 2015, 19(23): (in Chinese)..., 2015, 19(23): [11] Hu ZJ, Zhou YQ, Zhang HB, et al. Clinical value of monitoring serum cardiac biomarkers in pulmonary thromboembolism-induced myocardial injury. J First Mil Med Univ, 2008, 28(10): (in Chinese).,,,.., 2008, 28(10): [12] Wang GH. Diagnostic value of troponin T, troponin I and creatine kinase isoenzyme in acute myocaridal infarction. Clin Pharm, 2015, 8(12): (in Chinese).. T Ⅰ., 2015, 8(12): [13] Dohi T, Maehara A, Brener SJ, et al. Utility of peak creatine kinase-mb measurements in predicting myocardial infarct size, left ventricular dysfunction, and outcome after first anterior wall acute myocardial infarction (from the INFUSE-AMI trial). Am J Cardiol, 2015, 115(5): [14] Wang QH, Clinical significance of hs-crp, ctni, Myo and CK-MB detection in serum of patients with acute myocardial infarction. Lab Med, 2012, 9(25): (in Chinese).. hs-crp ctni Myo CK-MB., 2012, 9(25): [15] Brandt DR, Gates RC, Eng KK, et al. Quantifying the MB isoenzyme of creatine kinase with the Abbott "IMx" immunoassay analyzer. Clin Chem, 1990, 36(2): [16] Panteghini M, Bonora R, Pagani F. A two-tube immunochemical method for determination of cjb@im.ac.cn

150 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech January 25, 2017 Vol.33 No.1 CK-MB isoenzyme in serum evaluated. Clin Chem, 1990, 36(3): 550 553. [17] Plran U, Kohn DW, Uretsky LS, et al.

10 150 ISSN CN /Q Chin J Biotech January 25, 2017 Vol.33 No.1 CK-MB isoenzyme in serum evaluated. Clin Chem, 1990, 36(3): [17] Plran U, Kohn DW, Uretsky LS, et al. Immunochemiluminometric assay of creatine kinase MB with a monoclonal antibody to the MB isoenzyme. Clin Chem, 1987, 33(9): [18] Christenson RH, Vaidya H, Landt Y, et al. Standardization of creatine kinase-mb (CK-MB) mass assays: the use of recombinant CK-MB as a reference material. Clin Chem, 1999, 45: [19] Brener SJ, Lytle BW, Schneider JP, et al. Association between CK-MB elevation after percutaneous or surgical revascularization and three-year mortality. J Amer Coll Cardiol, 2002, 40(11): [20] Sadoh WE, Eregie CO, Nwaneri DU, et al. The diagnostic value of both troponin T and creatinine kinase isoenzyme (CK-MB) in detecting combined renal and myocardial injuries in asphyxiated infants. PLoS ONE, 2014, 9(3): e [21] Venta R, Cecchini BG, Geijo SA, et al. Serum creatinine kinase MB after acute myocardial infarction in a patient with IgA-CK-BB complex. Clin Chem,1994, 40(1): [22] Zeng LL, Jiang CX, Zhao YH, et al. Clinical application and diagnostic cutoff value analysis of cardiac markers in the diagnosis of acute myocaridial infarction. Lab Med Clin, 2013, 18: ,,,.., 2013, 18: [23] Friedman DL, Kesterson R, Puleo P, et al. Recombinant creatine kinase proteins and proposed standards for creatine kinase isoenzyme and subform assays. Clin Chem, 1993, 39(8): [24] Gao YS, Zhao TJ, Chen Z, et al. Isoenzyme-specific thermostability of human cytosolic creatine kinase. Int J Biol Macromol, 2010, 47(1): [25] Sunahara Y, Uchida K, Tanaka T, et al. Production of recombinant human creatine kinase (r-hck) isozymes by tandem repeat expression of M and B genes and characterization of r-hck-mb. Clin Chem, 2001, 47(3): ( 本文责编郝丽芳 )

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<453A5CCAB3C6B7BFC6D1A73131C4EA3134C6DA5C3134C6DA5C3032B7D6CEF6> 227 ELISA 刘沙洲 1,2, 桑小雪 1, 欧阳华学 3, 雷绍荣 3, 白林含 1, * (1. 四川大学生命科学学院, 四川成都 610065;2. 成都市食品药品检测中心, 四川成都 610045; 3. 四川省农业科学院分析测试中心, 四川成都 610066) 目的 : 比较