[1]朱业培,王玮,吕青骎,等.动物过敏原牛血清白蛋白间接竞争ELISA检测方法的建立[J].南京农业大学学报,2016,39(2):305-311.[doi:10.7685/jnau.20150814]
 ZHU Yepei,WANG Wei,L Qingqin,et al.Development of an indirect competitive ELISA for detection of animal allergen bovine serum albumin[J].Journal of Nanjing Agricultural University,2016,39(2):305-311.[doi:10.7685/jnau.20150814]
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动物过敏原牛血清白蛋白间接竞争ELISA检测方法的建立()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
39卷
期数:
2016年2期
页码:
305-311
栏目:
出版日期:
2016-03-18

文章信息/Info

Title:
Development of an indirect competitive ELISA for detection of animal allergen bovine serum albumin
作者:
朱业培 王玮 吕青骎 钱行 徐幸莲 周光宏
南京农业大学国家肉品质量安全控制工程技术研究中心/农业部畜产品加工重点实验室/食品安全与营养协同创新中心, 江苏 南京 210095
Author(s):
ZHU Yepei WANG Wei L Qingqin QIAN Hang XU Xinglian ZHOU Guanghong
National Center of Meat Quality and Safety Control/Key Laboratory of Animal Products Processing, Ministry of Agriculture/Synergetic Innovation Center of Food Safety and Nutrition, Nanjing Agricultural University, Nanjing 210095, China
关键词:
牛血清白蛋白动物过敏原单克隆抗体间接竞争ELISA检测
Keywords:
bovine serum albuminanimal allergenmonoclonal antibodyIC-ELISAdetection
分类号:
TS207.3
DOI:
10.7685/jnau.20150814
摘要:
[目的] 制备鼠抗牛血清白蛋白(bovine serum albumin,BSA)单克隆抗体,在此基础上建立可用于定量检测动物过敏原BSA含量的间接竞争酶联免疫检测方法(IC-ELISA)。[方法] 采用BSA纯品为抗原免疫BALB/c小鼠,通过杂交瘤细胞融合技术制备抗BSA单克隆抗体,并对其特异性进行鉴定;在此基础上,建立BSA间接竞争ELISA检测方法,并对其分析条件进行优化。[结果] 采用自行制备的特异性抗BSA单克隆抗体,建立间接竞争ELISA检测方法,优化得到最佳反应条件:抗原最佳包被浓度为0.25 μg·mL-1,单抗最佳工作浓度为1:32 000,酶标二抗最佳工作浓度为1:10 000,最低检测限为6.71 ng·mL-1,线性检测范围为3.593 8~460 ng·mL-1。线性回归直线方程为y=-47.448 9x+135.866 3,相关系数R2=0.997 3,P<0.000 1。[结论] 该方法特异性强、灵敏度高、可靠性好,适用于食品中过敏原BSA的现场快速检测。
Abstract:
[Objectives] An indirect competitive enzyme-linked immunosorbent assay(indirect competitive enzyme-linked immunosorbent assay,IC-ELISA)was developed for the quantitative detection of bovine serum albumin(BSA)using a self-prepared mouse anti-BSA monoclonal antibody. [Methods] BSA standard protein was immunized to BALB/c mice to prepare the specific monoclonal antibody against BSA by hybridoma technology. An IC-ELISA method for BSA based on monoclonal antibodies was developed,and its analysis conditions were optimized. [Results] The IC-ELISA method was established using specific monoclonal antibodies. The optimal concentrations of the coating antigen,monoclonal antibody against BSA and HRP conjugated goat anti-mouse IgG were 0.25 μg·mL-1,1:32 000 and 1:10 000,respectively. The limited detection was 6.71 ng·mL-1 in a linear range of 3.593 8-460 ng·mL-1. The regression equation of standard curve was y=-47.448 9x+135.866 3(the correlation coefficient R2=0.997 3,P<0.000 1). [Conclusions] The IC-ELISA method constructed in the paper has high specificity,good sensitivity and reliability,and can be used for rapid detection of animal allergen BSA in food.

参考文献/References:

[1] El-Agamy E I. The challenge of cow milk protein allergy[J]. Small Ruminant Research,2007,68(1):64-72.
[2] 陈颖. 食物过敏与食品过敏原[J]. 食品安全质量检测学报,2012,3(4):233-234. Chen Y. Food allergy and food allergens[J]. Journal of Food Safety and Quality,2012,3(4):233-234(in Chinese with English abstract).
[3] 王玮,朱业培,范巧君,等. 高静压降低食物致敏性的研究进展[J]. 南京农业大学学报,2015,38(4):525-531. DOI:10.7685/j.issn.1000-2030.2015.04.001. Wang W,Zhu Y P,Fan Q J,et al. Research progress of reducing food allergenicity by high hydrostatic pressure[J]. Journal of Nanjing Agricultural University,2015,38(4):525-531(in Chinese with English abstract).
[4] Wang W,Han J X,Wu Y J,et al. Simultaneous detection of eight food allergens using optical thin-film biosensor chips[J]. Journal of Agricultural and Food Chemistry,2011,59(13):6889-6894.
[5] Pourpak Z,Motsafaie A,Hasan Z,et al. A laboratory method for purification of major cow’s milk allergens[J]. Journal of Immunoassay and Immunochemistry,2004,25(4):385-397.
[6] Hochwallner H,Schulmeister U,Swoboda I,et al. Cow’s milk allergy:from allergens to new forms of diagnosis,therapy and prevention[J]. Methods,2014,66(1):22-33.
[7] Wal J M. Cow’s milk allergens[J]. Allergy,1998,53(11):1013-1022.
[8] Fiocchi A,Restani P,Riva E. Beef allergy in children[J]. Nutrition,2000,16(6):454-457.
[9] 郭玲,刘爱国,胡志和. 牛血浆中主要蛋白质的研究进展[J]. 食品科学,2009,30(21):489-492. Guo L,Liu A G,Hu Z H. Research advances in main proteins in bovine blood plasma[J]. Food Science,2009,30(21):489-492(in Chinese with English abstract).
[10] Wang H Y,Li G,Wu Y J,et al. Development of an indirect competitive immunoassay for walnut protein component in food[J]. Food Chemistry,2014,147:106-110.
[11] Ecker C,Ertl A,Pulverer W,et al. Validation and comparison of a sandwich ELISA,two competitive ELISAs and a real-time PCR method for the detection of lupine in food[J]. Food Chemistry,2013,14:407-418.
[12] 王玮,韩建勋,袁飞,等. 多重PCR同时检测常见8种食物过敏原[J]. 中国食品学报,2011,11(6):152-157. Wang W,Han J X,Yuan F,et al. Detection of eight food allergens with multiplex PCR[J]. Journal of Chinese Institute of Food Science and Technology,2011,11(6):152-157(in Chinese with English abstract).
[13] Eischeid A C. Development and evaluation of a real-time PCR assay for detection of lobster,a crustacean shellfish allergen[J]. Food Control,2016,59:393-399.
[14] 李慧,陈敏,李赫,等. 反相高效液相色谱法测定乳清蛋白中的α-乳白蛋白和β-乳球蛋白[J]. 色谱,2007,25(10):116-117. Li H,Chen M,Li H,et al. Determination of α-lactalbumin and β-lactoglobulin in whey protein by reversed phase high performance liquid chromatography[J]. Chinese Journal of Chromatography,2007,25(10):116-117(in Chinese with English abstract).
[15] Monaci L,Pilolli R,de Angelis E,et al. Multi-allergen detection in food by micro high-performance liquid chromatography coupled to a dual cell linear ion trap mass spectrometry[J]. Journal of Chromatography A,2014,1358:136-144.
[16] 王守法,阚春月,许学书. 酶联免疫吸附试验在食品检测中的应用[J]. 食品科学,2009,30(23):489-492. Wang S F,Kan C Y,Xu X S. Application of ELISA method in food detection[J]. Food Science,2009,30(23):489-492(in Chinese with English abstract).
[17] Khamehchian S,Madani R,Golchinfar F,et al. Development of a sandwich enzyme-linked immunosorbent assay(ELISA)for determining of bovine serum albumin(BSA)in trivalent measles-mumprubella(MMR)vaccines[J]. Human Vaccines,2008,4(5):375-378.
[18] 张加利,蔡芳,高强,等. 牛血清白蛋白单克隆抗体的筛选与双抗体夹心ELISA 检测方法的建立[J]. 中国生物制品学杂志,2008,21(11):999-1001. Zhang J L,Cai F,Gao Q,et al. Screening of monoclonal antibody against bovine serum albumin and development of double antibody sandwich ELISA for BSA[J]. Chinese Journal of Biologicals,2008,21(11):999-1001(in Chinese with English abstract).
[19] 陈贺,匡渤海,刘志刚,等. 口虾蛄主要过敏原TM的单抗制备和免疫学特性鉴定[J]. 免疫学杂志,2010,26(11):991-995. Chen H,Kuang B H,Liu Z G,et al. Preparation and immunological properties identification of monoclonal antibodies against Oratosquilla oratoria de Haan TM allergen[J]. Immunological Journal,2010,26(11):991-995(in Chinese with English abstract).
[20] 王海燕,袁飞,吴亚君,等. 食品中过敏原胡桃蛋白间接竞争ELISA检测方法研究[J]. 中国食品学报,2010,10(5):217-222. Wang H Y,Yuan F,Wu Y J,et al. Detection of allergen walnut protein in food by a indirect competitive enzyme-linked immunosorbent assay(ELISA)[J]. Journal of Chinese Institute of Food Science and Technology,2010,10(5):217-222(in Chinese with English abstract).
[21] Kanny G,de Hauteclocque C,Moneret-Vautrin D A. Food anaphylaxis to bovine serum albumin[J]. Journal of Allergy and Clinical Immunology,1998,101(1):137-139.
[22] 郑义成,华萍,杨安树,等. 食物中过敏原检测技术研究进展[J]. 食品科学,2010,31(21):417-421. Zheng Y C,Hua P,Yang A S,et al. Research advance in detection technologies for allergen in food[J]. Food Science,2010,31(21):417-421(in Chinese with English abstract).

相似文献/References:

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 WANG Wei,TENG Shuang,ZHU Yepei,et al.Development of a surface plasmon resonance sensor for detection of animal allergen bovine serum albumin[J].Journal of Nanjing Agricultural University,2017,(2):739.[doi:10.7685/jnau.201612009]

备注/Memo

备注/Memo:
收稿日期:2015-8-13。
基金项目:国家自然科学基金项目(31501395);江苏省自然科学基金项目(BK20140722)
作者简介:朱业培,硕士研究生。
通信作者:王玮,讲师,主要从事食品质量安全控制研究,E-mail:wangwei821220@njau.edu.cn。
更新日期/Last Update: 1900-01-01