双抗夹心酶联免疫吸附法检测β-伴大豆球蛋白和大豆球蛋白
CSTR:
作者:
作者单位:

1.四川成都中农大现代农业产业研究院,四川 成都 611430;2.中国农业大学食品科学与营养 工程学院,北京 100083;3.河南轻工职业学院,河南 郑州 450011;4.华测检测认证集团 北京有限公司,北京 101111

作者简介:

肖昊蚺 男 研究生 研究方向为食品安全 E-mail:XHR20010415@126.com

通讯作者:

车会莲 女 教授 研究方向为食物过敏、免疫学 E-mail:chehuilian@cau.edu.cn

中图分类号:

R155

基金项目:

四川成都中农大现代农业产业研究院建设省市农业补助专项


Development of sandwich ELISA for detection of soybean allergens β-Conglycinin and glycinin
Author:
Affiliation:

1.China Agricultural University-Sichuan Advanced Agricaltural & Industrial Institute, Sichuan Chengdu 611430, China;2.College of Food Science and Nutrition Engineering, China Agricultural University, Beijing 100083, China;3.He’nan Light Industry Vocational College, He’nan Zhengzhou 450011, China;4.CTI Certification Group Beijing Co.Ltd, Beijing 101111, China

Fund Project:

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    目的 以大豆中主要过敏原β-伴大豆球蛋白(β-Conglycinin)和大豆球蛋白(glycinin)为目标,分别建立双抗体夹心酶联免疫吸附(sELISA)法。方法 使用单因素试验优化从脱脂大豆蛋白粉中分离富集β-Conglycinin和glycinin的方法,确定沉淀剂CaCl2和NaHSO3的添加量,分离时间和转速提高对分离效果的影响;使用棋盘法确定捕获抗体和检测抗体的最佳工作浓度,确定各步骤反应时间,确定目标蛋白稀释溶剂,建立ELISA检测方法并对其性能进行评价。结果 在离心分离β-Conglycinin和glycinin前添加10 mmol/L沉淀剂CaCl2和0.01 mmol/L还原剂NaHSO3,沉淀过夜并将离心转速提高至10 000 r/min,使β-Conglycinin和glycinin的提取率分别达到41.81%和54.06%,且蛋白条带分离清晰;β-Conglycinin双抗sELISA以0.5 μg/mL 3A9 mAb作为捕获抗体,以1∶4 000稀释2E2mAb-HRP作为检测抗体,抗原和检测抗体各孵育90 min,显色15 min,采用四参数拟合和线性拟合绘制标准曲线。该方法的检出限(LOD)为1.36 ng/mL,标准曲线在5~1 215 ng/mL的浓度下线性良好,在实际样品加标回收试验中回收率在96%~108%。批次内和批次间变异系数均<15%;glycinin的双抗sELISA以0.5 μg/mL的7D3 mAb作为捕获抗体,以1∶1 000稀释4G4mAb-HRP作为检测抗体,抗原和检测抗体各孵育120 min,显色15 min,采用线性拟合绘制标准曲线。该方法的LOD为28.75 ng/mL,标准曲线在31.25~8 000 ng/mL的浓度下线性良好,在实际样品加标回收试验中回收率在90.3%~107.2%。批次内和批次间变异系数均<15%,且两种检测方法均与其他过敏原无交叉反应,特异性良好。结论 建立的两种双抗sELISA方法灵敏、快速、准确、稳定性好,可用于食品中β-Conglycinin和glycinin的检测。

    Abstract:

    Objective In this study, we established sandwich enzyme-linked immunosorbent assay (sELISA) methods to detect major soybean allergens β-Conglycinin and glycinin.Methods A single-factor experiment was conducted to optimize the method for isolating and enriching β-Conglycinin and glycinin from defatted soy protein powder, determining the addition amounts of the precipitants CaCl2 and NaHSO3, and the effect of the separation time and speed on the separation. The checkerboard method was used to determine the optimal working concentrations of the capture and detection antibodies, the reaction time for each step, and the dilution solvent for the target protein. An ELISA detection method was established and its performance was evaluated.Results Before centrifugal separation of β-Conglycinin and glycinin, 10 mmol/L CaCl2 and 0.01 mmol/L NaHSO3 were added as precipitants and reducing agents, respectively. The precipitation was left overnight and the centrifugation speed was increased to 10 000 r/min, resulting in extraction rates of 41.81% and 54.06% for β-Conglycinin and glycinin, respectively, with clear protein bands. For the sELISA of β-Conglycinin, 0.5 μg/mL 3A9 mAb was used as the capture antibody, and 2E2mAb-HRP was diluted 1∶4000 as the detection antibody. The antigen and detection antibody were incubated for 90 min each, and the color development was for 15 min. A four-parameter fit and linear fit were used to draw the standard curve. The LOD of this method was 1.36 ng/mL, and the standard curve shows good linearity in the concentration range of 5-1 215 ng/mL. In the actual sample spiked recovery experiment, the recovery rate was between 96% and 108%, and the intra-batch and inter-batch coefficient of variation were both less than 15%. For the sELISA of glycinin, 0.5 μg/mL 7D3 mAb was used as the capture antibody, and 4G4mAb-HRP was diluted 1∶1 000 as the detection antibody. The antigen and detection antibody were incubated for 120 min each, and the color development was for 15 min. A linear fit was used to draw the standard curve. The LOD of this method was 28.75 ng/mL, and the standard curve shows good linearity in the concentration range of 31.25-8 000 ng/mL. In the actual sample spiked recovery experiment, the recovery rate was between 90.3% and 107.2%, and the intra-batch and inter-batch coefficient of variation were both less than 15%, both two detection methods had no cross-reaction with other allergens, and the specificity was good.Conclusion The sELISA methods established are sensitive, rapid, accurate, and stable, and can be used for the detection of β-Conglycinin and glycinin in food.

    参考文献
    相似文献
    引证文献
引用本文

肖昊蚺,汪洋翰,张浩,付永康,王倩男,车会莲.双抗夹心酶联免疫吸附法检测β-伴大豆球蛋白和大豆球蛋白[J].中国食品卫生杂志,2025,37(4):316-326.

复制
相关视频

分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2024-01-09
  • 最后修改日期:
  • 录用日期:
  • 在线发布日期: 2025-07-11
  • 出版日期:
文章二维码
严正声明
关闭