6 3 Vol. 6 o. 3 2015 3 Journal of Food Safety and Quality Mar., 2015 * 熊波, 吴胜泽, 苏焕斌, 张燕, 刘辉 (, 528300) 摘要 : 目的 (sildenafil, SD) 方法 (SDH) (SDH-BSA),, SD, 结果 SD, 3.8 10 5, 0.02~3.8 ng/ml,, 结论 SD, SD 关键词 : ; ; Preparation and specificity analysis of sildenafil polyclonal antibody XIG Bo, WU Sheng-Ze, SU Huan-Bin, ZHAG Yan, LIU Hui * (Guangdong Provincial Product Quality Supervision and Inspection Institute, Shunde 528300, China) ABSTRACT: bjective To prepare high sensitivity and specificity polyclonal antibody against sildenafil (SD). Methods Sildenafil hapten (SDH) and Sildenafil antigen (SDH-BSA) were prepared by artificial methods. Then, through immunized rabbits the SD polyclonal antibody was prepared. Results After immunity, the polyclonal antibody against SD was generated. The titer of the antibody was 3.8 10 5. The linear detection of the polyclonal antibody was ranged from 0.02 ng/ml to 3.8 ng/ml. The polyclonal antibody had a high cross reaction rate to vardenafil, but had a low response rate to hongdenafil and calais. Conclusion SD polyclonal antibody had a high specificity and sensitivity, and it could be applied to the development of SD rapid detection kit. KEY WRDS: sildenafil; polyclonal antibody; ELISA 1 引言 (sildenafil, SD) FDA1998 (erectile dysfunction, ED), 5, cgmp GMP, cgmp,,, ED [1-3], [4],,,, 基金项目 : (2013B090600059) Fund: Supported by Guangdong Province Science and Technology Plan Project (2013B090600059) * 通讯作者 :,, E-mail: lh403@163.com *Corresponding author: LIU Hui, Engineer, Guangdong Provincial Product Quality Supervision and Inspection Institute, Shunde 528300, China. E-mail: lh403@163.com
3, : 1023,, SD, - [5-7],, SD, SD, SD 2 材料与方法 2.1 材料与试剂 2,, 3 kg, ; Freund, - (DMF) - (HS) (DCC) (BSA) (VA) 3, 3, 5, 5 - (TMB) Sigma ; Alfa Aesar ; 2.2 仪器与设备 HP1100 (, Agilent); Multiskan MK3 (, Thermo ); Wellwash MK2 (, Thermo ) 2.3 实验方法 2.3.1 SD 半抗原的合成 4.75 g (4.75 g, 10 mmol), 30 ml DMF, 520 mg 60 1 h, 2.34 g, 15 min, 60 16 h, DMF, 20 ml,,,,,, 6 ml, 3 ml (TFA), 2 h, TLC,, SDH, 1 2.3.2 人工抗原制备与鉴定 [8] SDH-BSA, SDH-VA 0.1 mmol 0.5 ml DMF, 0.2 mmol DCC 0.2 mmol HS,, (4000 r/min, 5 min),, BSA VA( BSA VA 60:1)pH 9.6, 4 h,,, 2, 0.8%, -20 1 SDH Fig. 1 The synthesis of SDH hapten
1024 6 200~400 nm,, (BSA VA) [9] [10] 2.3.3 多克隆抗体的制备 3 kg, 0.5mg 2 ml 2 ml 3,,, ELISA, 10 d, (4000 r/min, 5 min), -20 2.3.4 抗体特异性分析 SD, SD 3 结果与分析 3.1 SD 半抗原的鉴定 - (LC-MS), 2 3, 589.2 [M+H] +, SDH 533.4[M+H] +, 3.2 SD 抗原的制备及鉴定 4 SDH BSA VA SDH-BSA SDH-VA,, SDH-BSA 276~277 nm, D, 245 nm, D, 300~320 nm ; SDH-VA 276~277 nm, D, 248 nm, D, SDH BSA VA, SDH BSA VA,,, SDH 3.3 抗体效价的测定及其标准曲线的绘制 icelisa,,, A 450nm 1.0 [11], (D) 1.0, 0.25 μg/ml, 3.8 10 5 Fig. 2 2 SDH The mass spectrogram of SDH intermediate
3, : 1025 Fig. 3 3 SDH The mass spectrogram of SDH hapten Fig. 4 4 SDH UV spectrum of SDH artificial antigens and haptens 5 ELISA Fig. 5 The icelisa standard curve of SD, riginlab 7.5 ELISA S, SDH-VA, ELISA 5, IC 50 0.16 ng/ml, (IC 20 ~IC 80 ) 0.02~3.8 ng/ml 3.4 SD 多克隆抗体特异性分析 icelisa, icelisa, SD, SD
1026 6 Table 1 表 1 SD 与结构类似物或功能类似物的交叉反应率结果 (n=3) Cross reactivity of polyclonal antibody with analogous compound of SD (n=3) IC 50 (ng/ml) S H 0.16 100 S H 0.22 72.7 CH3 H 3550.44 <0.01 H 1392.62 <0.01, 50%, [12] 100 IC50 (SD) CR(%) IC (cross - reactant) 50 cgmp PDE-5 PcAb-SDH-BSA, (72.7%), (<0.01%) SD, SD, SD, SD,, 4 讨论, [13], [14], [15] SD, SD, SD, SD, 0.01%, 72.7%,, SD 参考文献 [1] Behr-Roussel D, udot A, Caisey S, et al. Daily treatment with sildenafil reverses endothelial dysfunction and oxidative stress in an animal model of insulin resistance [J]. European urology, 2008, 53(6): 1272 1281. [2] Fleshner, Harvey M, Adomat H, et al. Evidence for contamination of herbal erectile dysfunction products with phosphodiesterase type-5 inhibitors [J]. Urology, 2005, 174(2): 636. [3],,. [J]., 1999, 21(6): 493 496. Wang J, Ren J, Wang ZG. Mode of Action of Sildenafil [J] Acta Acad Med Sinicae, 1999, 21(6): 493 496. [4] McCulley TJ, Luu JK, Marmor MF, et al. Effects of sildenafil citrate (Viagra) on choroidal congestion [J]. phthalmologica, 2002, 216 (6): 455 458. [5],. [J]., 2008, 28(8): 673 674. Pang XX, Lv HC. Fast detection method of sildenafil in Chinese patent drug and traditional Chinese medicine health care products
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