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1 5 3 Vol. 5 o Journal of Food Safety and Quality Mar., 2014 李桂敏, 宁保安, 白家磊, 赵 * 丽, 赵翠娇, 高志贤 (,, ) 摘要 : 目的 (diazepam, DZP), 方法 1- -(3- ) [1-ethyl-3-(3-dimethyllaminopropyl) carbodi imide hydrochloride, EDC H], Balb/C, 1:16000 SP2/0,,, -, (surface plasmon resonance, SPR) 结果, Balb/C 7 1:16000,, (dissociation constants, KDs) mol/l, ELISA, IC 50 =10.8 ng/ml, 0.45~ ng/ml 结论, 关键词 : ; ; ; Preparation of anti-diazepam monoclonal antibody and establishment of its enzyme-linked immunosorbent assay method LI Gui-Min, IG Bao-An, BAI Jia-Lei, ZHA Li, ZHA Cui-Jiao, GA Zhi-Xian * (Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Health and Environmental Medicine, Academy of Military Medical Sciences, Tianjin , China) ABSTRACT: bjectives To establish a rapid and effective method for the detection of diazepam (DZP), we prepared anti-dzp monoclonal antibody and identified the characteristics of the antibody. Methods The immunogen and coating antigen were synthesized by using the method of EDC and the mice were immunized with the immunogen until the titer reach 1: To obtain the hybridoma, splenocytes from immunized mice were fused with SP2/0 under the condition of PEG4000. Then, hybridoma clones were screened by indirect complete enzyme-linked immunosorbent assay (ELISA) and the positive cell lines were subclone three times using the method of limit dilution assay. In order to prepare ascites monoclonal antibody, the positive cell lines were injected into the Balb/C mice which have been injected into paraffin oil seven days ago. The ascites mo- 基金项目 : (2011 BAK10B02-13) Fund: Supported by ational Science and Technology Support Project (2011 BAK10B02-13) * 通讯作者 :,,, gaozhx@163.com *Corresponding author: GA Zhi-Xian, Professor, Institute of Health and Environmental Medicine, o.1 Dali Road, Tianjin , China. gaozhx@163.com

2 3, : 957 noclonal antibody was purified by bitterness-ammonium sulfate precipitation and the characteristics were determined by a method based on ELISA. Results The immunogen and coating antigen were synthesized successfully and the titer reach 1:16000 after seven times immunization. The results showed that the limit of detection (LD) was 0.45 ng/ml and IC 50 =10.8 ng/ml. The dissociation constants (KDs) of the monoclonal antibody by surface plasmon resonance (SPR) was mol/l and the cross-reaction was not obvious except temazepam (35.4%). Conclusion The anti-dzp monoclonal antibody was prepared successfully which was very meaningful for the rapid detection of DZP. KEY WRDS: diazepam; hybridoma technology; monoclonal antibody; enzyme-linked immunosorbent assay 1 引言 (diazepam, DZP),,,,,,,,,, [1] [2] [3-4],,, (enzyme-linked immunosorbent assay, ELISA) [5],, 2 材料与方法 2.1 主要材料与试剂 (carbonate buffer solution, CBS); (phosphate buffered saline, PBS); (tetramethyl benzidine, TMB ); - (-hydroxysuccinmide HS, Sigma ); 1- -(3- ) [1-ethyl-3-(3-dimethylaminoproply) carbodi imide hydrochloride, EDC, Sigma ]; (bovine serum albumin, BSA, Sigma ) (ovalbumin, VA, Sigma ) / ( Sigma ); (, ); (UC, ); (, ); ( ) 2.2 主要仪器设备 MK3 (Thermo, ); C 2 (Thermo, ); ( ); ( ); (lympus DP11, ), (surface plasmon resonance, SPR)( Autolab SPR ESPRIT) 2.3 实验动物 SPF Balb/C, 6~8, 20 g/,, : SCXK-( ) 方法半抗原的改造, : DZP 3 -CH, 1(DZP1), 1 2 : DZP 3, -CH, 2(DZP2) 完全抗原的制备 EDC, DZP1 DZP2 BSA, DZP2 VA

3 (DZP1) Fig. 1 Synthetic map of half antigen 1 (DZP1) 2 2(DZP2) Fig. 2 Synthetic map of half antigen 2 (DZP2) 动物免疫方案 6~8 Balb/C, 2 3, 3, 3, 2,, 阳性杂交瘤细胞的筛选 [6-7],,, 3, C 7 B 7 C 腹水的抽取及抗体的纯化 : 8 Balb/C, ml/, ~ /, [4], - SDS-PAGE 间接竞争 ELISA 标准曲线的绘制 1 μg/ml DZP2-VA (CBS), 100 μl, 4 1%VA (PBS), PBST 3 ( 260 μl, 3 min ), 50 μl μl, 37 2 h PBST 3 1:3000, 37 1 h 100 μl TMB, 5~7 min, 2 mol/l H 2 S 4 (50 μl/ ), 450 nm 630 nm 抗体亲和力的测定 SPR 1 mg/ml,, KDs 交叉反应率的测定 6 ( ) ( ),

第3期 CR(%)=(DZP 的 IC50 值/DZP 的结构类似物的 3 结 IC50 值) 100% 2.4.9 959 李桂敏, 等: 地西泮单克隆抗体的制备及其酶联免疫吸附检测方法的建立加标回收实验准确称取 15 份猪肉匀浆式样, 每个加标量 5 份, 果完全抗原的鉴定 3.1 由质谱鉴定结果可知 MWDZP1-BSA=69185.

4 第3期 CR(%)=(DZP 的 IC50 值/DZP 的结构类似物的 3 结 IC50 值) 100% 李桂敏, 等: 地西泮单克隆抗体的制备及其酶联免疫吸附检测方法的建立加标回收实验准确称取 15 份猪肉匀浆式样, 每个加标量 5 份, 果完全抗原的鉴定 3.1 由质谱鉴定结果可知 MWDZP1-BSA= , 每份 5 g 按照事先确定的加标量加入地西泮小分子 MWBSA= , 而 MWDZP1=384, 根据计算公式, 以后, 加入 10 g 的无水硫酸钠和 20 ml 乙腈, 涡旋振可得荡 10 min, 4000 r/min 离心 10 min, 重复上述操作一 MWDZP2-VA= , 次取上清加入 10 ml 乙腈饱和的正己烷洗涤至澄 MWDZP2=370, 同理计算可得 DZP2 与 VA 的偶联比清取澄清液体 50 氮气吹干, 最后用 2 ml 的无水为 3:1, 详见图 3 甲醇复溶取复溶物过 0.22 μm 的滤膜, 用于 ELISA 3.2 方法检测 DZP1 与 BSA 的偶联比约为 7:1; MWVA= , 而免疫后效价结果从图中可以看出随着免疫次数的增加, 血清效 Table 1 表 1 小鼠免疫方案 The mice immune scheme 免疫剂量免疫原注射方式天数(d) 50 µg (300 µl) 完全抗原+弗氏完全佐剂 µg (300 µl) 完全抗原+弗氏不完全佐剂 µg (300 µl) 完全抗原+弗氏不完全佐剂 µg (300 µl) 完全抗原+弗氏不完全佐剂 µg (300 µl) 完全抗原+弗氏不完全佐剂 µg(300 µl) 完全抗原 51 Fig. 3 图 3 地西泮完全抗原 DZP1-BSA(A)和 DZP2-VA(B)的质谱图 Mass spectra of diazepam complete antigen DZP1-BSA (A) and DZP2-VA (B)

960 食品安全质量检测学报价得到明显的上升, 且用 DZP1-BSA 作为免疫原免疫时效价的上升速度明显快于用 DZP2-BSA 作为免疫原时的效价上升速度, 用 DZP1-BSA 作为免疫原时, 第 7 次免疫后效价达到 1:16000, 可进行细胞融合, 详见图 4 3.

4 取包被原和纯化后抗体进行棋盘滴定, 确定最佳的包被原浓度为 1 µg/ml, 最佳的抗体稀释倍数为 1 000 倍按照最佳的包被原浓度和抗体稀释倍数, 测定地西泮单克隆抗体的标准曲线经 Logistic 曲线拟合可以得到方程为 Y=92.651-90.6546/[1+(X/0.0093) 0.7733 ], R2=0.99469, 由此算出 IC50=10.

6 图 6 地西泮抗体间接竞争 ELISA 标准曲线 ELISA standard curve of diazepam antibody indirect competitive 3.5 抗体亲和力的测定地西泮单克隆抗体的亲和力测定结果如图 7 所示, 用数据采集软件 Autolab ESPRIT Data Acquisition 4.

5 960 食品安全质量检测学报价得到明显的上升, 且用 DZP1-BSA 作为免疫原免疫时效价的上升速度明显快于用 DZP2-BSA 作为免疫原时的效价上升速度, 用 DZP1-BSA 作为免疫原时, 第 7 次免疫后效价达到 1:16000, 可进行细胞融合, 详见图抗体纯化效果的鉴定利用 SDS-PAGE 对纯化效果进行鉴定纯化后杂蛋白被有效去除, 经过 BCA 蛋白定量得抗体浓度第5卷地西泮单克隆抗体标准曲线的绘制 3.4 取包被原和纯化后抗体进行棋盘滴定, 确定最佳的包被原浓度为 1 µg/ml, 最佳的抗体稀释倍数为倍按照最佳的包被原浓度和抗体稀释倍数, 测定地西泮单克隆抗体的标准曲线经 Logistic 曲线拟合可以得到方程为 Y= /[1+(X/0.0093) ], R2= , 由此算出 IC50=10.8 ng/ml, 检测范围为 0.45~ ng/ml, 详见图 6 为 5 mg/ml, 抗体亚型鉴定为 IgG1 型, 详见图 5 Fig. 4 图 4 免疫后效价变化情况 Immune after effect price changes Fig. 6 图 6 地西泮抗体间接竞争 ELISA 标准曲线 ELISA standard curve of diazepam antibody indirect competitive 3.5 抗体亲和力的测定地西泮单克隆抗体的亲和力测定结果如图 7 所示, 用数据采集软件 Autolab ESPRIT Data Acquisition 4.3 和数据分析软件 Kinetic Evaluation 5.0, 经过数据拟合最终得到抗体的解离常数 KDs= mol/L 表明抗体有较好的亲和力 3.6 单克隆抗体与结构类似物的交叉反应率交叉反应率结果如表 2 所示(其中 - 代表无交叉反应率), 交叉反应率最高的是替马西泮, 而与其他结构类似物基本上没有明显的交叉反应替马西泮与地西泮相比, 只是在仲胺上甲基取代了 H, 说明本抗体能够对地西泮中两个苯环(且其中一个氯代)进行图 5 地西泮单克隆抗体纯化效果的 SDS-PAGE 鉴定结果 Fig. 5 Result of effect of SDS-PAGE appraisal for diazepam monoclonal antibody purification A: 纯化前; B: 纯化后; M: Marker 特异性识别 3.7 加标回收实验结果实验中利用猪肉进行加标回收实验, 分别对每

6 3, : 961 Fig. 7 7 Gradient map of diazepam diluted with the antigen antibody Table 2 表 2 地西泮单抗与其结构类似物的交叉反应率结果 Results of diazepam single and its structural analogs cross reaction resistance ( ) H 100 H 3 C CH 3 F H 冸 H H H 35.4

7 962 5 ( ) 续表 2 H H 3.79 H 表 3 猪肉样品加标回收结果 (n=5) Table 3 Results of pork sample standard addition recovery (n=5) (ng) (ng) (%) , 78.33%~96.11% 4 结论与讨论,,,,, 1(DZP1) 2(DZP2 ) 2 3, 3 DZP1 DZP2 BSA DZP1-BSA DZP2-BSA, DZP1-BSA,, DZP1-BSA, [8,9], DZP2 VA DZP2-VA DZP1-BSA 1:16000,, [10], - [11], [12] Ka, Ka, ; KD S, mol/l, 10-6 ~10-8 mol/l,, KDs= mol/l, ELISA, 0.45 ng/ml~862 ng/ml, IC 50 =10.8 ng/ml [13,14] [15], ~96.11 参考文献 [1] Liu J, ing B, Liu M, et al. Construction of ribosome display library based on lipocalin scaffold and screening anticalins with specificity for estradiol [J]. Analyst, 2012, 137(10): [2],,,. [J]., 2007, 23(11): Li S, Lv J, Hu G, et al. Preparation and purification of polyclonal antibodies against LPS of E.coli [J]. Chin Agric Sci Bull, 2007, 23(11): [3],,,. [J]., 2009, 25 (4): Li XL, Li XF, ing BA, et al. Preparation of clenbuterol monoc-

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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