Kühne Yvonne, Reese Gerald, Ballmer-Weber Barbara K, Niggemann Bodo, Hanschmann Kay-Martin, Vieths Stefan, Holzhauser Thomas
Division of Allergology, Paul-Ehrlich-Institut, Langen, Germany.
Int Arch Allergy Immunol. 2015;166(3):213-24. doi: 10.1159/000381344. Epub 2015 Apr 24.
The identification of B-cell epitopes of food allergens can possibly lead to novel diagnostic tools and therapeutic reagents for food allergy. We sought to develop a flexible, low-tech, cost-effective and reproducible multipeptide microarray for the research environment to enable large-scale screening of IgE epitopes of food allergens.
Overlapping peptides (15-mer, 4 amino acid offset) covering the primary sequence of either peanut allergen Ara h 1 or all 3 subunits of the soybean allergen Gly m 5 were simultaneously synthesized in-house on a porous cellulose matrix. Identical peptide microarrays created with up to 384 duplicate peptide-cellulose microspots each were investigated for specificity and sensitivity in IgE immunodetection and in direct experimental comparison to the formerly established SPOT™ membrane technique.
The in-house microarray identified with 98% reproducibility the same IgE-binding peptides as the SPOT™ membrane technique. Additional IgE-binding peptides were identified using the microarray. While the sensitivity was increased between 2- and 20-fold, the amount of human serum required was reduced by at least two thirds over the SPOT™ membrane technique using the microarray. After subtraction of the potential background, we did not observe non-specific binding to the presented peptides on microarray.
The novel peptide microarray allows simple and cost-effective screening for potential epitopes of large allergenic legume seed storage proteins, and it could be adapted for other food allergens as well, to study allergenic epitopes at the individual subject level in large paediatric and adult study groups of food allergic subjects.
确定食物过敏原的B细胞表位可能会带来用于食物过敏的新型诊断工具和治疗试剂。我们试图开发一种灵活、低技术含量、具有成本效益且可重复的多肽微阵列，用于研究环境，以实现对食物过敏原IgE表位的大规模筛选。
在多孔纤维素基质上内部同时合成覆盖花生过敏原Ara h 1的一级序列或大豆过敏原Gly m 5的所有3个亚基的重叠肽（15肽，氨基酸偏移4个）。对每个最多有384个重复肽 - 纤维素微点的相同肽微阵列进行IgE免疫检测的特异性和敏感性研究，并与先前建立的SPOT™膜技术进行直接实验比较。
内部微阵列与SPOT™膜技术一样，以98%的重现性鉴定出相同的IgE结合肽。使用微阵列还鉴定出了其他IgE结合肽。虽然敏感性提高了2至20倍，但与使用SPOT™膜技术相比，使用微阵列所需的人血清量减少了至少三分之二。扣除潜在背景后，我们未观察到微阵列上呈现的肽有非特异性结合。
这种新型肽微阵列能够简单且经济高效地筛选大型致敏豆类种子储存蛋白的潜在表位，并且它也可适用于其他食物过敏原，以便在食物过敏受试者的大型儿科和成人研究组中，在个体受试者水平上研究过敏表位。
