食品中脱氧雪腐镰刀菌烯醇胶体金免疫快速检测技术研究
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摘要
脱氧雪腐镰刀菌烯醇(DON)是单端孢霉烯族毒素之一,广泛存在于谷物和饲料中,严重危害人类和动物的健康。目前的检测方法操作繁琐,大多需要专门的仪器进行配套检测,无法满足快速、简便、现场检测的要求。本文以多克隆抗体和胶体金合成标记技术为基础,根据免疫层析的原理,建立了食品中DON胶体金免疫快速检测方法。
通过DCC法合成DON完全抗原,DON与BSA的初始摩尔比为50:1,得到DON完全抗原中DON与BSA的摩尔比为9.7:1。通过红外光谱扫描以及ELISA实验验证了DON-BSA的偶联成功。将DON-BSA免疫新西兰大白兔,得到抗体的最高效价为12800,抑制率IC_(50)为10μg/mL,与T-2毒素、NIV的交叉反应率分别为9.1%,4.9%。
采用柠檬酸三钠还原法成功制备了粒径为10 nm和20 nm的胶体金,并在紫外图谱上进行了表征。胶体金标记抗体的最适pH为9.0,最适抗体加入量为0.066 mg/mL、0.072 mg/mL。胶体金标记抗血清得到免疫胶体金探针,胶体金、抗体溶液、免疫探针复合物做紫外扫描,280 nm附近有蛋白特征峰出现,与胶体金复合后,原出现在520 nm处的胶体金的特征峰,峰值增高,并平移至540 nm附近,证明大分子蛋白抗体与胶体金偶联成功。
标记前后的抗体蛋白红外光谱扫描的结果表明,标记前后抗体蛋白的特征基团结构没有发生明显的变化。对抗体探针进行荧光光谱扫描分析,胶体金对抗体蛋白的内源荧光有猝灭作用,并且随着胶体金浓度的增加,抗体的荧光强度呈现有规律的降低,发射光的位置不变。
以胶体金标记多克隆抗体作为探针,DON-BSA作为竞争抗原,构建了胶体金免疫检测体系(GICA)。确定最佳包被抗原浓度为10μg/mL,缓冲液体系:10%甲醇+0.05%吐温20的pH 7.4 PBS溶液,胶体金的最佳粒径20 nm左右。GICA目测检出限为1 mg/kg,检测时间为10~15 min。方法的重复性良好。方法的稳定性良好,试纸条室温保存3个月,4℃保存6个月。对自然污染的样品进行ELISA和GICA检测,结果证明两者有良好的一致性。
Deoxynivalenol(DON), as one fungi toxin belonging to the group of trichothecenes, is widely distributed in cereal and feedstuffs, and its rapid detection is important for food safety. However most of the currently used methods are time-consuming and also need expensive instruments, which cannot satisfy the requirement for a rapid, convenient, and on-site detection of the presence of DON in food samples.Thus,an immunochromatographic strip system using colloidal gold-labeled polyclonal antibody(PcAb) specific to DON as the maker was developed for rapid detection of DON in foods.
The full-antigen of DON was first produced by conjugating DON to bovine serum albumin (BSA) according to the method of DCC. A molar ration of 9.7:1 between DON and BSA was abtained in the conjugation reaction from an initial molar ratio of 50:1.After the successful conjugation of BSA to DON was verified by ELISA and IR spectra, the PcAb to DON-BSA was then produced by immunizing rabbits. The results of characterizing PcAb showed a highest titer of 12800 with an IC50 of 10μg/mL, and its cross reactivity with T-2 toxin and NIV was 9.1% and 4.9% respectively.
Colloidal gold were prepared through reducing HAuCl4·3H2O by sodium citrate, and it was then characterized by UV-spectra. The optimum pH for antibody labelling with colloidal gold (also called gold labeled probe) was 9.0, and the concentration of antibody was 0.066mg/mL,0.072 mg/mL. The probe colloidal gold particles, and antibody were analyzed by UV-spectra, and the results showed that the conjugation was successful.
Combination of PcAb with colloidal gold particles was also characterized by IR spectra and fluorescence spectroscopy. The experimental resluts showed that the structure of PcAb was not changed, but its fluorescence can be quenched by the colloidal gold, and its power was strengthened as the concentration increased. However no changes of the emission spectrum profile was found .
A rapid, simple and sensitive colloidal gold labeled immunochromatographic test(GICA) for detection of DON in foods was established. The optimum concentration of envelope antigen was 10μg/mL. A buffer system composed of PBS (pH 7.4)+10%methanol+0.05%Tween20 was used to detect the DON. The optimum particle diameter of colloidal gold was found to be 20nm, and the detection process could be finished within 15min, which is reproducible and stable. With visual observation, the lowest limit was found to be 1mg/L corresponding to 1mg/Kg DON in samples. The shelf-life of immunostrips was three months at room temperature and six months at 4℃.Comparing with commercial ELISA kit using naturally contaminated food samples, consistent result was obtained using the developed GICA method.
通过DCC法合成DON完全抗原,DON与BSA的初始摩尔比为50:1,得到DON完全抗原中DON与BSA的摩尔比为9.7:1。通过红外光谱扫描以及ELISA实验验证了DON-BSA的偶联成功。将DON-BSA免疫新西兰大白兔,得到抗体的最高效价为12800,抑制率IC_(50)为10μg/mL,与T-2毒素、NIV的交叉反应率分别为9.1%,4.9%。
采用柠檬酸三钠还原法成功制备了粒径为10 nm和20 nm的胶体金,并在紫外图谱上进行了表征。胶体金标记抗体的最适pH为9.0,最适抗体加入量为0.066 mg/mL、0.072 mg/mL。胶体金标记抗血清得到免疫胶体金探针,胶体金、抗体溶液、免疫探针复合物做紫外扫描,280 nm附近有蛋白特征峰出现,与胶体金复合后,原出现在520 nm处的胶体金的特征峰,峰值增高,并平移至540 nm附近,证明大分子蛋白抗体与胶体金偶联成功。
标记前后的抗体蛋白红外光谱扫描的结果表明,标记前后抗体蛋白的特征基团结构没有发生明显的变化。对抗体探针进行荧光光谱扫描分析,胶体金对抗体蛋白的内源荧光有猝灭作用,并且随着胶体金浓度的增加,抗体的荧光强度呈现有规律的降低,发射光的位置不变。
以胶体金标记多克隆抗体作为探针,DON-BSA作为竞争抗原,构建了胶体金免疫检测体系(GICA)。确定最佳包被抗原浓度为10μg/mL,缓冲液体系:10%甲醇+0.05%吐温20的pH 7.4 PBS溶液,胶体金的最佳粒径20 nm左右。GICA目测检出限为1 mg/kg,检测时间为10~15 min。方法的重复性良好。方法的稳定性良好,试纸条室温保存3个月,4℃保存6个月。对自然污染的样品进行ELISA和GICA检测,结果证明两者有良好的一致性。
Deoxynivalenol(DON), as one fungi toxin belonging to the group of trichothecenes, is widely distributed in cereal and feedstuffs, and its rapid detection is important for food safety. However most of the currently used methods are time-consuming and also need expensive instruments, which cannot satisfy the requirement for a rapid, convenient, and on-site detection of the presence of DON in food samples.Thus,an immunochromatographic strip system using colloidal gold-labeled polyclonal antibody(PcAb) specific to DON as the maker was developed for rapid detection of DON in foods.
The full-antigen of DON was first produced by conjugating DON to bovine serum albumin (BSA) according to the method of DCC. A molar ration of 9.7:1 between DON and BSA was abtained in the conjugation reaction from an initial molar ratio of 50:1.After the successful conjugation of BSA to DON was verified by ELISA and IR spectra, the PcAb to DON-BSA was then produced by immunizing rabbits. The results of characterizing PcAb showed a highest titer of 12800 with an IC50 of 10μg/mL, and its cross reactivity with T-2 toxin and NIV was 9.1% and 4.9% respectively.
Colloidal gold were prepared through reducing HAuCl4·3H2O by sodium citrate, and it was then characterized by UV-spectra. The optimum pH for antibody labelling with colloidal gold (also called gold labeled probe) was 9.0, and the concentration of antibody was 0.066mg/mL,0.072 mg/mL. The probe colloidal gold particles, and antibody were analyzed by UV-spectra, and the results showed that the conjugation was successful.
Combination of PcAb with colloidal gold particles was also characterized by IR spectra and fluorescence spectroscopy. The experimental resluts showed that the structure of PcAb was not changed, but its fluorescence can be quenched by the colloidal gold, and its power was strengthened as the concentration increased. However no changes of the emission spectrum profile was found .
A rapid, simple and sensitive colloidal gold labeled immunochromatographic test(GICA) for detection of DON in foods was established. The optimum concentration of envelope antigen was 10μg/mL. A buffer system composed of PBS (pH 7.4)+10%methanol+0.05%Tween20 was used to detect the DON. The optimum particle diameter of colloidal gold was found to be 20nm, and the detection process could be finished within 15min, which is reproducible and stable. With visual observation, the lowest limit was found to be 1mg/L corresponding to 1mg/Kg DON in samples. The shelf-life of immunostrips was three months at room temperature and six months at 4℃.Comparing with commercial ELISA kit using naturally contaminated food samples, consistent result was obtained using the developed GICA method.
引文
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