Supplementary Material for: Detection of Circulating Peanut Components in Serum after Ingestion

Introduction: Detection of specific food allergens brought into circulation following ingestion is complicated by the minute amounts present in serum. We therefore aimed to assess the utility of selective removal of IgE against peanut components using ImmunoCAP combined with the basophil histamine release (HR) assay (BHRA) to identify the absorbed allergen components after ingestion of peanut. Methods: Serum from six healthy individuals was drawn before and 1 h after ingestion of 100 g peanut. Serum from two peanut allergic patients was depleted for IgE against single allergen components by incubation with ImmunoCAPs coupled with either Ara h 1; 2; 3 or 6 before donor basophils were passively sensitized with the serum preparations. The sensitized cells were challenged with serum obtained from the six individuals before and after ingestion of peanut, and HR was measured after serum provocation. Results: Ara h 2 and Ara h 6 were detected in serum 1 h after ingestion in 6/6 individuals by negative selection. Depletion of specific IgE against Ara h 2 or Ara h 6 almost completely abolished the response to serum provocation, indicating a sequence homology between the two allergen components in serum. Ara h 1 was demonstrated in 5/6 sera and Ara h 3 in 1/6 sera. Conclusion: This study is a proof of concept showing that passive sensitization of basophils with sera depleted of component-specific IgE can be used to identify food allergen components present in serum after ingestion.

引言：食入食物后，过敏原可进入血液循环，但因其在血清中含量极微，给特异性检测带来了极大挑战。为此本研究旨在评估，采用免疫CAP检测系统（ImmunoCAP）联合嗜碱性粒细胞组胺释放试验（basophil histamine release assay, HR，简称BHRA）选择性清除针对花生过敏原组分的免疫球蛋白E（IgE），以此识别花生食入后血清中存在的吸收性过敏原组分的应用价值。

方法：招募6名健康受试者，分别在食入100g花生前及食入后1小时采集血清样本。针对2名花生过敏患者的血清，先将其分别与偶联了花生过敏原1（Ara h 1）、花生过敏原2（Ara h 2）、花生过敏原3（Ara h 3）或花生过敏原6（Ara h 6）的ImmunoCAP孵育，以清除针对单一过敏原组分的IgE；随后用上述处理后的血清制剂对供体嗜碱性粒细胞进行被动致敏。将致敏后的嗜碱性粒细胞分别用6名受试者食入花生前后的血清进行激发，并检测激发后的组胺释放水平。

结果：通过阴性筛选法，在全部6名受试者食入花生后1小时的血清中均检测到了Ara h 2与Ara h 6。清除针对Ara h 2或Ara h 6的特异性IgE后，血清激发的反应几乎完全被阻断，这表明血清中这两种过敏原组分存在序列同源性。5份血清样本中检测到了Ara h 1，1份血清样本中检测到了Ara h 3。

结论：本研究为概念验证性研究，结果表明，采用经组分特异性IgE清除处理的血清对嗜碱性粒细胞进行被动致敏，可用于识别食入后血清中存在的食物过敏原组分。