HAL Allergy, J. H. Oortweg, Leiden, The Netherlands; Department of Experimental Immunology, Amsterdam University Medical Centers, Amsterdam, The Netherlands.
HAL Allergy, J. H. Oortweg, Leiden, The Netherlands.
J Allergy Clin Immunol. 2023 Aug;152(2):436-444.e6. doi: 10.1016/j.jaci.2023.03.025. Epub 2023 Apr 5.
Surprisingly, IgE cross-reactivity between the major peanut allergens Ara h 1, 2, and 3 has been reported despite very low sequence identities.
We investigated the unexpected cross-reactivity between peanut major allergens.
Cross-contamination of purified natural Ara h 1, 2, 3, and 6 was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot test, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and sandwich enzyme-linked immunosorbent assay (ELISA). IgE cross-reactivity was studied with sera of peanut-allergic patients (n = 43) by ELISA and ImmunoCAP inhibition using both intact natural and recombinant allergens and synthetic peptides representing postulated Ara h 1 and Ara h 2 cross-reactive epitopes.
Both purified nAra h 1 and nAra h 3 were demonstrated to contain small but significant amounts of Ara h 2 and Ara h 6 (<1%) by sandwich ELISA, SDS-PAGE/Western blot analysis, and LC-MS/MS. IgE cross-inhibition between both 2S albumins and Ara h 1 and Ara h 3 was only observed when using natural purified allergens, not recombinant allergens or synthetic peptides. Apparent cross-reactivity was lost when purified nAra h 1 was pretreated under reducing conditions, suggesting that Ara h 2 and Ara h 6 contaminations may be covalently bound to Ara h 1 via disulfide interactions.
True cross-reactivity of both peanut 2S albumins with Ara h 1 and Ara h 3 could not be demonstrated. Instead, cross-contamination with small quantities was shown to be sufficient to cause significant cross-inhibition that can be misinterpreted as molecular cross-reactivity. Diagnostic tests using purified nAra h 1 and nAra h 3 can overestimate their importance as major allergens as a result of the presence of contaminating 2S albumins, making recombinant Ara h 1 and Ara h 3 a preferred alternative.
尽管主要花生过敏原 Ara h 1、2 和 3 的序列同一性非常低，但已有报道称它们之间存在 IgE 交叉反应，这令人惊讶。
我们调查了花生主要过敏原之间意想不到的交叉反应。
通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、Western blot 试验、液相色谱-串联质谱（LC-MS/MS）和夹心酶联免疫吸附试验（ELISA）评估纯化天然 Ara h 1、2、3 和 6 的交叉污染。使用花生过敏患者（n=43）的血清通过 ELISA 和免疫 CAP 抑制研究 IgE 交叉反应，使用完整天然和重组过敏原以及代表推测的 Ara h 1 和 Ara h 2 交叉反应表位的合成肽进行抑制。
夹心 ELISA、SDS-PAGE/Western blot 分析和 LC-MS/MS 均表明，纯化的 nAra h 1 和 nAra h 3 均含有少量但显著量的 Ara h 2 和 Ara h 6（<1%）。只有使用天然纯化过敏原时，才能观察到 2S 白蛋白与 Ara h 1 和 Ara h 3 之间的 IgE 交叉抑制，而不是重组过敏原或合成肽。当纯化的 nAra h 1 在还原条件下预处理时，明显的交叉反应消失，这表明 Ara h 2 和 Ara h 6 的污染可能通过二硫键相互作用与 Ara h 1 共价结合。
未能证明花生 2S 白蛋白与 Ara h 1 和 Ara h 3 之间存在真正的交叉反应。相反，少量的交叉污染足以导致显著的交叉抑制，这可能被误解为分子交叉反应。由于存在污染的 2S 白蛋白，使用纯化的 nAra h 1 和 nAra h 3 的诊断测试可能会高估它们作为主要过敏原的重要性，因此重组 Ara h 1 和 Ara h 3 是更好的选择。
