Microsoft Word 王伟华校正.doc

Size: px

Start display at page:

Download "Microsoft Word 王伟华校正.doc"

秩铁阎
7 years ago
Views:

1 5 6 Vol. 5 No Journal of Food Safety and Quality Jun., 2014 icelisa 王伟华 1, * 田木星 1, 韩占江 2 (1. /, ; 2. /, ) 摘要 : 目的, icelisa( ) 方法, CPF-H1 CPF-H2,,, icelisa, 结果 ;, ; ; icelisa, : PBST, 1:1000, 1:1000, 40 min, 30 min,, IC ng/ml, IC 20 IC ng/ml, LOD ng/ml 结论, 关键词 : ; ; icelisa; Preparation of chlorprifos monoclonal antibody and optimization of icelisa detection method WANG Wei-Hua 1*, TIAN Mu-Xing 1, HAN Zhan-Jiang 2 (1. Xinjiang Production & Construction Corps Key Laboratory of Deep Processing of Agricultural Products in South Xinjiang, College of Life Science, Tarim University, Alar , China; 2. Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, College of Plant Science, Tarim University, Alar , China) ABSTRACT: Objective To prepare monoclonal antibodies of chlorpyrifos, and establish and optimize ice- LISA (indirect competitive enzyme-linked immunosorbent assay) detection methods. Methods Hapten CPF-H1 and CPF-H2 were prepared based on chlorpyrifos, artificial antigen were prepared through conjugated protein, and then specific monoclonal antibodies were obtained through immunization, cellfusion and screening. icelisa of chlorpyrifosis was established, and its analysis conditions were optimized. Results Mass spectrometry results indicated that the chlorpyrifos hapten was successfully prepared; hapten conjugated proteins, UV wavelength scanning verified the successful preparation of artificial antigen; animals were immunized, cells were fusioned and monoclonal antibodies were prepared; icelisa detection method was established, the optimal reaction conditions were optimized to be: PBST was used as the standard diluent, the optimal coating 基金项目 : (2012BB012) ( ) Fund: Supported by the Doctor Fundation of Xinjiang Production and Construction Crops(2012BB012); and National College Students' Innovation and Entrepreneurship Training Program( ) * 通讯作者 :,,, wangweihua6688@163.com *Corresponding author: WANG Wei-Hua, Ph.D., Associate Professor, College of Life Science, Tarim University, Alar , China. wangweihua6688@163.com

2 antigen dilution was 1:1000, the optimal antibody dilution was 1:1000, the optimal reaction time of antibody was 40 min, the optimal HRP-IgG response time was 30 min, the standard curve of chlorpyrifos was prepared, the chlorpyrifos antibody IC 50 was ng/ml, the linear range of IC 20 ~IC 80 was ng/ml, and the LOD was ng/ml. Conclusion The paper laid theoretical foundation for chlorpyrifos rapid detection, which is of great significance for the protection of people's health. KEY WORDS: chlorprifos; monoclonal antibody; icelisa; optimization 1 引言 (chlorpyrifos, CPF) [1], [2],,, 14 [3],,,, [4],,,, - -,, [5], (ELISA),, [6-7] [8] IgG1,, 1%; [9], icelisa, 40~420 ng/ml, 30 ng/ml,, ;, icelisa,, icelisa 2 材料与方法 2.1 实验试剂 4- ; (99.5%) ; (98.5%) ; (BSA) (OVA) (DCC) N- (NHS) Freund PEG4000( Sigma ); HRP IgG ( Southern Biotech ); RPMI1640 HAT HT( Gibco ); ( ); 18.2 mω, SP2/0 ( ); Balb/c (6 8, g, ) 2.2 实验仪器 imark ( Biorad ); LCQ DECA XP - ( Thermo Finnigan ); / ( Kyoto ); 6K-15 ( Sigma ); ( Biotech ); XSB-1A ( ); ( ) 2.3 实验方法半抗原 CPF-H1 的合成 3- NaOH, 100 ml,, ; 50 ml, 2 h,, NaCl

3 6, : icelisa 1711 ph 11, 3 [10],, CPF-H 半抗原 CPF-H2 的合成 1, ml 2 h, 20 ml 0 3,,, O-, O-,, 10 min,, 2 h, O- -O O- -O-4- -,, 0, 0 2 ml 180 mg NaOH 220 mg 4-,, ph 3, 3,, (O- -O-4- ), CPF-H 人工抗原的合成 DMF, DCC NHS, 4, A ; BSA OVA PBS, B ;, A B, 4 12 h, 4 PBS 3 d, 3 1 mg/ml 0.5 ml, -20 [11] CPF-H1-BSA CPF-H2-OVA, nm BSA OVA BSA OVA 单克隆抗体的制备 (1) : ;,, ELISA ; 1:1000, [12] 1 CPF-H1 Fig. 1 Synthetic route of chlorpyrifos hapten CPF-H1 2 CPF-H2 Fig. 2 Synthetic route of chlorpyrifos hapten CPF-H2

4 (2) :,, (3) : 3~4 ; 2~3, ; 5~7, HT HAT 3~4, 6~7,,,, Balb/c icelisa 方法的建立及优化, B/B 0 B/B 0,, -, 0 B 0, B, : % 1 B 抑制率 ( ) = - B 100% 0 Originlab 7.5 ELISA S, A max IC 50 A max /IC 50 icelisa CPF-H1-BSA CPF-H2-OVA 37,, CPF-H2-OVA, (1:500, 1:1000, 1:1500, 1:2000) 37 ( ), 1:5000, PBST ( PBS PBST), ng/ml, icelisa CPF-H2-OVA 1:1000, 37 (12 h), (1:500, 1:1000, 1:1500, 1:2000), 1:5000, PBST CPF-H2-OVA 1:1000, 37 (12 h), 1:1000, 1:5000, icelisa min, 30 min, PBST CPF-H2-OVA 1:1000, 37 (12 h), 1:1000, 1:5000, icelisa 40 min, min, PBST icelisa,, IC 50 (LOD) (IC 20 -IC 80 ) 3 结果与分析 3.1 半抗原 CPF-H1 的合成与鉴定 CPF-H1, 3, ESI-MS(-)m/z: 209[M-H] - 3 CPF-H1, CPF-H1 3.2 半抗原 CPF-H2 的合成与鉴定 CPF-H2, 4, APCI-MS (negative) m/z: [2M-H] - 4 CPF-H2, CPF-H2 3.3 人工抗原的合成及鉴定 5 6 :, CPF-H1 CPF-H2 BSA CPF-H2-OVA CPF-H1-BSA,,,,

5 6, : icelisa 1713 Fig. 3 3 The mass spectra of CPF-H1 Fig. 4 4 CPF-H2 The mass spectra of CPF-H2 5 CPF-H1-BSA Fig. 5 The UV wavelength scanning spectra of CPF-H1-BSA 6 CPF-H2-OVA Fig. 6 The UV wavelength scanning spectra of CPF-H2-OVA

6 单克隆抗体的制备 d, 84%, ELISA, 33%, icelisa, 3, 1 AS4- R1,,, 3.5 icelisa 方法的建立及优化包被原的确定 CPF-H1-BSA, IC ng/ml, CPF-H2-OVA, IC ng/ml,, CPF-H2-OVA icelisa Fig. 7 7 The inhibition curves when coated with two different coating antigens 包被原浓度的确定 8-a 8-b,,, 1000 icelisa, A 450 nm, 1: 药物稀释液的优化 ph,,, -, [13], icelisa, A max ; PBS PBST A max, PBS PBST, IC 50 A max /IC 50, -20 icelisa, PBST 抗体稀释倍数的确定 CPF-H2-OVA 1:1000, 37 (12 h), (1:500, 1:1000, 1:1500, 1:2000), 1:5000, PBST a 10-b, 500, IC 50,, A max, 1: 竞争反应时间优化的确定, 30 min, 40 min IC 50 A max /IC 50, 40 min Fig. 8 8 Optimization of coating antigen dilution factors

7 6, : icelisa Fig. 9 Optimization of drug diluent Fig Optimization of antibody dilution factors Fig Optimization of reaction time of antibody 优化二抗反应时间的确定, IC 50, 30 min IC 50 A max /IC 50,, 30 min - : CPF-H2-OVA, PBST ; 1:1000; 1:1000;

8 Fig Optimization of reaction time of HRP-IgG 40 min; 30 min ic ELISA 检测方法的标准曲线,,, IC ng/ml, IC 20 IC ng/ml, LOD ng/ml,,,,,,,,, ELISA, μg/ml, 0.05mg/L [15], 参考文献 13 Fig. 13 Indirect competition standard curve of chlorpyrifos 4 讨论,,,, [14],,,, [1],,,. [J]., 2004, 37(3): Wu G, HuangYL, Zhu GN, et al. Study on Immunochemical Assays of Organophosphorus Insecticide Chlorpyrifos [J]. Sci Agric Sinica, 2004, 37(3): [2],,. [J]., 2008, 31(2): Liu ZS, Huang AH, Xiao MS. Current Situation of Study Application Chlorpyrifos and It, s Industrial Development Prospects[J]. Word Pestic, 31(2): [3] Cink JH, Cots JR. Effect of concentration temperature, and soil moisture on the degradation of chlorpyrifos in an Urban lowa soil [J]. Pestic in Urban Enviromengts Fate and Significance, 1993, [4],,,.

9 6, : icelisa 1717 [J]., 2012, 4(5): 5 9. Su GY, Zhang XW, Wei S, et al. Review of Ecotoxicological and Risk Assessment for chlorpyrifos[j]. Environ Monitor Forewarning, 2012, 4(5): 5 9. [5] Brun EM, Garcés-García M, Puchades R, et al. Highly sensitive enzyme-linked immunosorbent assay for chlorpyrifos. Application to olive oil analysis[j]. J Agric Food Chem, 2005, 53(24): [6],. [J]., 2014, 30(4): Zhu H, Ji MS. Ecent Advances in Rapid Detection Technology of Pesticide Residue[J]. Chin Agric Sci Bull, 2014, 30(4): [7] Manclus JJ, Primo J, Montoya A. Development of enzyme-linked immunosorbentassays for the insecticide Chlorpyrifos. 1. monoclonal antibody production and immunoassay design[j]. J Agric Food Chem, 1996, 44: [8],,,. ELISA [J]., 2006, 34(2): Wang X, Liu Y, Liu R, et al. Preparation of Monoclonal Antibody against Chlorprifos and Development of ELISA for Detection of Chlorprifos Residues[J]. J Environ Occup Med, 2006, 34(2): [9],,,. [J]., 2003, 3: Yan CR, Liu JX, Yu XY, et al. Monoclonal Antibody Preparation and Its Property Analysis for Fipronil[J]. Jiangsu J Agric Sci, 2003, 3: [10],,,. [J]., 2008, 7(5): Liu B, Wei SH, Li XH, et al. Synthesis of the Antigens and Preparation of Polyclonal Antibody for Chlorpyrifos[J]. Mod Agrochem, 2008, 7(5): [11],,,. [J]., 2006, 29(3): Lian L, Gao ZX, Liu M, et al. Synthesis of the Artficial Antigen For Chlorpyrifos Detection [J]. J Prew Med Chin PLA, 2006, 29(3): [12],,,. [J]., 2006, 32(1): Jin RY, Gui WJ, Wu JQ, et al. Study on the Monoclonal Antibody to Chlorpyrifos[J]. J Zhejiang Univ, 2006, 32(1): [13],,,. [J]., 2013, 4(1): Li JJ, Hou CJ, Huo DQ, et al. Recent advance on fast detection methods and techniques for organophosphorus pesticide residues [J]. J Food Safe Qual, 2013, 4(1): [14],,,. [J]., 2008, 30(2): Feng CL, Zhang MR, Chen JF, et al. Synthesis and characterization of Artificial Antigen for the Organophosphorus Chlorpyvifos [J].Word Sci-Tech R&D, 2013, 4(1): [15],,,. - [J]., 2006, 12(1): Wan ZK, He J, Kang CA, et al. Application and Update Progresses of GC-MS in Determination of Pesticide Residues[J]. Anal Test Technol Instrum, 2006, 12(1): ( 责任编辑 : 赵静 ) 作者简介,,, wangweihua6688@163.com

<453A5CCAB3C6B7BFC6D1A73131C4EA3134C6DA5C3134C6DA5C3032B7D6CEF6>

<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