Supplementary Material for: Do <b><i>Plantago lanceolata</i></b> Skin Prick Test-Positive Patients Display IgE to Genuine Plantain Pollen Allergens? Investigation of Pollen Allergic Patients from the North-East of France

Background: English plantain (Plantago lanceolata) is an important weed pollen allergen source triggering allergic symptoms during summer. To elucidate genuine versus cross-reactive sensitization, we investigated IgE reactivity patterns and inhibition capacities of plantain-sensitized patients. Methods: Sera of 35 rhinoconjunctivitis patients from the north-east of France with positive skin prick tests (SPT) to Plantago lanceolata pollen were tested with clinically relevant allergen sources using ELISA, ImmunoCAP, and immunoblot inhibition. Results: The patients were multisensitized with additional reactivity to grass (94.3%), ash (74.3%), birch (71.4%), and mugwort (55.2%) pollen in SPT. Sensitization prevalence to allergen molecules was 34.3% (Pla l 1), 94.3% (Phl p 1/5), 60.0% (Ole e 1), 65.7% (Bet v 1), 37.1% (profilin), and 40.0% (CCD). In immunoblot, IgE reactivity to plantain pollen was inhibited with relevant pollen extracts and purified rPla l 1. Two sera did not reveal any IgE cross-reactivity, while reactivity to plantain was efficiently inhibited by grass pollen in the sera of 10 patients. The sera from 17 different patients could be inhibited by grass, birch, or ash pollen to varying degrees. Thus, only 37.1% of our patients demonstrated true plantain pollen sensitization, while 62.9% were solely positive due to IgE cross-reactive molecules from other clinically relevant pollen. Conclusions: Plantain pollen-sensitized patients are multi-reactors demonstrating varying and complex IgE-reactivity profiles. In vivo and in vitro tests using extracts are typically blurred due to the presence of homologous allergens or CCD in grass, birch, or ash pollen. So far, Pla l 1 represents the only indicative marker allergen for the diagnosis of genuine plantain pollen sensitization.背景：长叶车前草（Plantago lanceolata）是一类重要的杂草花粉变应原来源，可在夏季引发过敏性症状。为阐明原发性致敏与交叉反应性致敏的差异，本研究针对车前草致敏患者的IgE反应模式及抑制能力展开了探究。 方法：收集法国东北部35例对长叶车前草花粉皮肤点刺试验（skin prick tests, SPT）结果呈阳性的变应性鼻结膜炎患者血清，采用酶联免疫吸附试验（Enzyme-Linked Immunosorbent Assay, ELISA）、ImmunoCAP检测及免疫印迹抑制实验，针对临床相关变应原进行检测。 结果：本研究纳入的患者均存在多重致敏现象，皮肤点刺试验显示其对禾本科花粉（94.3%）、白蜡树花粉（74.3%）、桦树花粉（71.4%）及艾蒿花粉（55.2%）亦存在反应性。针对变应原分子的致敏阳性率分别为：Pla l 1（34.3%）、Phl p 1/5（94.3%）、Ole e 1（60.0%）、Bet v 1（65.7%）、profilin（37.1%）及交叉反应性碳水化合物决定簇（cross-reactive carbohydrate determinants, CCD，40.0%）。免疫印迹实验显示，长叶车前草花粉的IgE反应可被相关花粉提取物及纯化重组Pla l 1所抑制。2份血清未检测到任何IgE交叉反应性，而10份患者血清中，针对长叶车前草的反应可被禾本科花粉有效抑制。另有17份患者血清的反应可被禾本科、桦树或白蜡树花粉不同程度地抑制。综上，仅37.1%的患者表现为真正的长叶车前草花粉致敏，剩余62.9%的阳性结果仅源于其他临床相关花粉来源的IgE交叉反应性分子。 结论：长叶车前草花粉致敏患者多为多重反应者，其IgE反应谱呈现多样化且复杂的特征。由于禾本科、桦树或白蜡树花粉中存在同源变应原或交叉反应性碳水化合物决定簇，基于提取物的体内及体外检测结果通常易出现混淆。截至目前，Pla l 1仍是诊断真正长叶车前草花粉致敏的唯一特异性标记变应原。