胭脂红酸人工抗原合成及多克隆抗体的制备
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摘要
为了快速高效检测食品中胭脂红酸含量,本研究通过羟基二咪唑法制备了胭脂红酸人工抗原;通过免疫新西兰大耳白兔获得了胭脂红酸抗血清,经硫酸铵沉淀法分离纯化获得抗胭脂红酸多克隆抗体;利用棋盘滴定法检测了抗体效价,并探究了溶液pH值、离子强度和有机溶剂对ELISA反应的影响,优化出最佳ELISA反应条件;在最佳反应条件下,建立了胭脂红酸的间接竞争ELISA抑制曲线。紫外光谱扫描结果显示,免疫抗原(CA-BSA)和检测抗原(CA-OVA)均与载体蛋白成功偶联;经分离纯化获得的胭脂红酸多克隆抗体效价为1:16000;检测抗原最佳浓度为1μg/m L、ELISA反应最适缓冲液为pH7.4、含50mmol/L Na~+且无甲醇的磷酸盐缓冲液;间接竞争ELISA标准曲线的线性范围为7.8～1150ng/mL,检测限为1ng/mL,灵敏度IC_(50)值为95.2ng/mL。本研究为开发CA快速检测试剂盒对食品中胭脂红酸含量的大量快速测定奠定了基础。
In order to detect rapidly and efficiently the content of carminic acid(CA) in food, the artificial antigen of CA was prepared by hydroxydiimidazole method; A high-titer polyclonal antibody(Pab) against CA was obtained through immunizing New Zealand white rabbits to obtain antiserum(against CA) for further separation and purification by ammonium sulfate precipitation. The titer of the antibody was tested by checkerboard titration, and the effects of factors such as solution pH, ionic strength, and organic solvent were investigated to optimize the conditions of the enzyme-linked immunosorbent assay(ELISA) reaction. Under the optimal conditions, an indirect competitive ELISA inhibition curve for CA was established. Ultraviolet(UV) spectroscopy analysis showed that both the immunogenic antigen(CA-BSA) and coating antigen(CA-OVA) were successfully conjugated with carrier protein. The titer of the Anti-CA polyclonal antibody obtained after separation and purification was 1:16000; The optimal concentration of the detection antigen was 1 μg/m L, the optimum buffer for ELISA reaction was the phosphate buffer at pH 7.4 containing 50 mmol/L Na~+ but no methanol. The linear range of the indirect competitive ELISA standard curve was 7.8～1150 ng/mL, with the limit of detection(LOD) as 1 ng/mL, and the sensitivity IC50 was 95.2 ng/mL. This study has laid the foundation for the development of a rapid CA rapid test kit for the determination of CA in food.
In order to detect rapidly and efficiently the content of carminic acid(CA) in food, the artificial antigen of CA was prepared by hydroxydiimidazole method; A high-titer polyclonal antibody(Pab) against CA was obtained through immunizing New Zealand white rabbits to obtain antiserum(against CA) for further separation and purification by ammonium sulfate precipitation. The titer of the antibody was tested by checkerboard titration, and the effects of factors such as solution pH, ionic strength, and organic solvent were investigated to optimize the conditions of the enzyme-linked immunosorbent assay(ELISA) reaction. Under the optimal conditions, an indirect competitive ELISA inhibition curve for CA was established. Ultraviolet(UV) spectroscopy analysis showed that both the immunogenic antigen(CA-BSA) and coating antigen(CA-OVA) were successfully conjugated with carrier protein. The titer of the Anti-CA polyclonal antibody obtained after separation and purification was 1:16000; The optimal concentration of the detection antigen was 1 μg/m L, the optimum buffer for ELISA reaction was the phosphate buffer at pH 7.4 containing 50 mmol/L Na~+ but no methanol. The linear range of the indirect competitive ELISA standard curve was 7.8～1150 ng/mL, with the limit of detection(LOD) as 1 ng/mL, and the sensitivity IC50 was 95.2 ng/mL. This study has laid the foundation for the development of a rapid CA rapid test kit for the determination of CA in food.
引文
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[10]Ruwona T B,Johnson V J,Hettick J M,et al.Production,characterization and utility of a panel of monoclonal antibodies for the detection of toluene diisocyanate haptenated proteins[J].Journal of Immunological Methods,2011,373(2):127-135
[11]Wang Z H,Mi T,Ross C B,et al.Hapten synthesis,monoclonal antibody production and development of a competitive indirect enzyme-linked immunosorbent assay for erythromycin in milk[J].Food Chemistry,2015,171(15):98-107
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[2]GB 2760,食品安全国家标准食品添加剂使用标准[S]GB 2760,National Standard for Food Safety Standard for Use of Food Additives[S]
[3]高敏,和玲,胡敏,等.胭脂红酸老化的紫外光谱法研究[J].湖南大学学报(自然科学版),2013,40(4):83-86GAO Min,HE Ling,HU Min,et al.Study on the aging of carminic acid by UV-vis spectrophotometry[J].Journal of Hunan University(Natural Sciences),2013,40(4):83-86
[4]郭蒙京,李堃,赵丹莹.食品中胭脂虫红的高效液相色谱测定法[J].职业与健康,2016,32(18):2508-2510GUO Meng-jing,LI Kun,ZHAO Dan-ying.Determination of carminic acid in food by HPLC[J].Occupation and Health,2016,32(18):2508-2510
[5]张正挺.酶联免疫检测技术测定牛奶中青霉素含量和试剂盒研制[D].杭州:浙江大学,2014ZHANG Zheng-ting.Determination of penicillin content in milk by ELISA and preparation of ELISA kits[D].Hangzhou:Zhejiang University,2014
[6]张岩蔚,金艳丹,栗慧,等.氨苄西林人工抗原的合成及鼠源多克隆抗体的制备[J].黑龙江畜牧兽医,2018,1:227-229ZHANG Yan-wei,JIN Yan-dan,SU Hui,et al.Synthesis of ampicillin artificial antigen and preparation mouse polyclonal antibodies[J].Heilongjiang Animal Science and Veterinary Medicine,2018,1:227-229
[7]杨凡凡.雌二醇人工抗原合成及其多克隆抗体的制备[J].江西畜牧兽医杂志,2017,3:4-7YANG Fan-fan.Synthesis of estradiol artificial antigen and preparation of its polyclonal antibodies[J].Jiangxi Journal of Animal Husbandry&Veterinary Medicine,2017,3:4-7
[8]Xing W.Development of a sensitive and group-specific polyclonal antibody-based enzyme-linked immunosorbent assay for detection of malachite green and leucomalachite green in water and fish samples[J].Sic.Food Agric.,2009,89:2165-2173
[9]Yang F,Zhu L,Meng W,et al.Novel artificial antigen synthesis for antibody production and development of an indirect competitive ELISA of cyanocobalamin[J].Anal Methods,2015,7:5275-5281
[10]Ruwona T B,Johnson V J,Hettick J M,et al.Production,characterization and utility of a panel of monoclonal antibodies for the detection of toluene diisocyanate haptenated proteins[J].Journal of Immunological Methods,2011,373(2):127-135
[11]Wang Z H,Mi T,Ross C B,et al.Hapten synthesis,monoclonal antibody production and development of a competitive indirect enzyme-linked immunosorbent assay for erythromycin in milk[J].Food Chemistry,2015,171(15):98-107
[12]王亚楠,王晓斐.牛琳琳,等.G族黄曲霉毒素半抗原分子设计、抗原合成及抗体特性[J].西北农业学报,2018,27(5):626-633WANG Ya-nan,WANG Xiao-pei,NIU Lin-lin,et al.Hapten molecular design and antigen synthesis and antibody characterization of G-group aflatoxins[J].Acta Agriculturae Boreali-occidentalis Sinica,2018,27(5):626-633