5 6 Vol. 5 No. 6 2014 6 Journal of Food Safety and Quality Jun., 2014 icelisa 王伟华 1, * 田木星 1, 韩占江 2 (1. /, 843300; 2. /, 843300) 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 50 73.25 ng/ml, IC 20 IC 80 32.52 260 ng/ml, LOD 19.34 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 843300, 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 843300, 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) (2013107570010) Fund: Supported by the Doctor Fundation of Xinjiang Production and Construction Crops(2012BB012); and National College Students' Innovation and Entrepreneurship Training Program(2013107570010) * 通讯作者 :,,, E-mail: wangweihua6688@163.com *Corresponding author: WANG Wei-Hua, Ph.D., Associate Professor, College of Life Science, Tarim University, Alar 843300, China. E-mail: wangweihua6688@163.com
1710 5 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 73.25 ng/ml, the linear range of IC 20 ~IC 80 was 32.5 260 ng/ml, and the LOD was 19.34 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, 15 25 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 2.3.1 半抗原 CPF-H1 的合成 3- NaOH, 100 ml,, ; 50 ml, 2 h,, NaCl
6, : icelisa 1711 ph 11, 3 [10],, CPF-H1 2.3.2 半抗原 CPF-H2 的合成 1, 5 0 30 ml 2 h, 20 ml 0 3,,, O-, -20 2 O-,, 10 min,, 2 h, 100 200 O- -O-4- - 3 O- -O-4- -,, 0, 0 2 ml 180 mg NaOH 220 mg 4-,, ph 3, 3,, 100 200 4-(O- -O-4- ), CPF-H2 2.3.3 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, 200 400 nm BSA OVA BSA OVA 2.3.4 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
1712 5 (2) :,, (3) : 3~4 ; 2~3, ; 5~7, HT HAT 3~4, 6~7,,,, Balb/c 2.3.5 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 2.3.5.1 CPF-H1-BSA CPF-H2-OVA 37,, 2.3.5.2 CPF-H2-OVA, (1:500, 1:1000, 1:1500, 1:2000) 37 ( ), 1:5000, PBST 2.3.5.3 ( PBS PBST), 0 15.6 31.25 62.5 125 250 500 1000 ng/ml, icelisa 2.3.5.4 CPF-H2-OVA 1:1000, 37 (12 h), (1:500, 1:1000, 1:1500, 1:2000), 1:5000, PBST 2.3.5.5 CPF-H2-OVA 1:1000, 37 (12 h), 1:1000, 1:5000, icelisa 20 30 40 min, 30 min, PBST 2.3.5.6 CPF-H2-OVA 1:1000, 37 (12 h), 1:1000, 1:5000, icelisa 40 min, 10 20 30 40 min, PBST 2.3.5.7 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: 694.8 [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,,,,
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
1714 5 3.4 单克隆抗体的制备 2.3.4 10 d, 84%, ELISA, 33%, icelisa, 3, 1 AS4- R1,,, 3.5 icelisa 方法的建立及优化 3.5.1 包被原的确定 CPF-H1-BSA, IC 50 251.69 ng/ml, CPF-H2-OVA, IC 50 156.5 ng/ml,, CPF-H2-OVA icelisa Fig. 7 7 The inhibition curves when coated with two different coating antigens 3.5.2 包被原浓度的确定 8-a 8-b,,, 1000 icelisa, A 450 nm, 1:1000 3.5.3 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 3.5.4 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 10 10-a 10-b, 500, IC 50,, A max, 1:1000 3.5.5 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
6, : icelisa 1715 9 Fig. 9 Optimization of drug diluent Fig. 10 10 Optimization of antibody dilution factors Fig. 11 11 Optimization of reaction time of antibody 3.5.6 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;
1716 5 Fig. 12 12 Optimization of reaction time of HRP-IgG 40 min; 30 min 3.5.7 ic ELISA 检测方法的标准曲线,,, IC 50 73.25 ng/ml, IC 20 IC 80 32.52 260 ng/ml, LOD 19.34 ng/ml,,,,,,,,, ELISA, 0.019 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): 382 386. Wu G, HuangYL, Zhu GN, et al. Study on Immunochemical Assays of Organophosphorus Insecticide Chlorpyrifos [J]. Sci Agric Sinica, 2004, 37(3): 382 386. [2],,. [J]., 2008, 31(2): 59 61. Liu ZS, Huang AH, Xiao MS. Current Situation of Study Application Chlorpyrifos and It, s Industrial Development Prospects[J]. Word Pestic, 31(2): 59 61. [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, 62 69. [4],,,.
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