Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia; School of Biological Sciences, University of Sydney, Sydney, Australia.
School of Biological Sciences, University of Sydney, Sydney, Australia.
Food Chem. 2017 Apr 15;221:335-344. doi: 10.1016/j.foodchem.2016.10.063. Epub 2016 Oct 14.
The clinical importance of peanut (Arachis hypogaea) allergies demands standardized allergen extraction protocols. We determined the effectiveness of common extraction conditions (20 buffers, defatting reagents, extraction time/temperatures, processing, extraction repeats) on crude protein and Ara h 1 and 2 yields. Despite similar 1D-gel profiles, defatting with n-hexane resulted in significantly higher yields of crude protein, Ara h 1, and Ara h 2 than with diethyl ether. The yields were affected by the composition and pH of the extraction buffers and other conditions, but crude protein yield did not always correlate with Ara h 1 and 2 yields. Denaturants, reducing agents, acidic buffers, and thermal processing of peanuts perturbed allergen quantification in ELISAs, probably via exposure of additional epitopes. Allergen detection in 2D-Western blots with PBS resulted in greater sensitivity than with TBS or Tris. We recommend that allergen extraction conditions be selected based on the research question being investigated.
花生（Arachis hypogaea）过敏的临床重要性要求标准化的过敏原提取方案。我们确定了常见提取条件（20 种缓冲液、脱脂试剂、提取时间/温度、处理、提取重复）对粗蛋白和 Ara h 1 和 2 产量的影响。尽管 1D 凝胶图谱相似，但正己烷脱脂比二乙醚脱脂产生的粗蛋白、Ara h 1 和 Ara h 2 产量显著更高。产量受提取缓冲液的组成和 pH 值以及其他条件的影响，但粗蛋白产量并不总是与 Ara h 1 和 2 产量相关。变性剂、还原剂、酸性缓冲液和花生的热加工会通过暴露额外的表位来干扰 ELISA 中的过敏原定量，可能会导致更大的影响。使用 PBS 在 2D-Western 印迹中检测过敏原比使用 TBS 或 Tris 更敏感。我们建议根据正在研究的研究问题选择过敏原提取条件。
