Supplementary Material for: Involvement of Can f 5 in a Case of Human Seminal Plasma Allergy

<b><i>Background:</i></b> The existence of IgE binding to dog dander extract without IgE antibodies against the described dog allergens (Can f 1, 2, 3 and 4) implies the presence of other dog allergens yet to be identified. Recently, an IgE-binding protein was isolated from dog urine and identified as prostatic kallikrein; it has been named Can f 5. Cross-reactivity between a dog dander allergen and human prostate-specific antigen (PSA) has been described. The aim of this study was to identify the dog dander allergen that presents cross-reactivity with PSA and demonstrate its clinical relevance in our patient with human seminal plasma allergy. <b><i>Methods:</i></b> SDS-PAGE immunoblotting and inhibition tests were performed. Mass spectrometry was carried out to identify the protein involved in the allergy reactions. <b><i>Results:</i></b> SDS-PAGE immunoblotting-inhibition with an IgE-binding protein from dog prostatic secretion showed total IgE binding inhibition to a 28-kDa IgE-reactive band identified as PSA. The electroeluted protein from dog prostatic secretion was identified by mass spectrometry as Can f 5. IgE immunoblotting of human seminal plasma incubated with the serum of the patient revealed two IgE-binding bands (28 and 32.7 kDa). Both SDS-PAGE immunoblotting inhibition assays, with human seminal plasma or purified PSA in solid phase, showed complete IgE binding inhibition when the serum of the anaphylactic patient was preincubated with dog dander extract or recombinant Can f 5. <b><i>Conclusions:</i></b> The dog dander allergen that shows cross-reactivity with human PSA has been characterized and turns out to be the recently described Can f 5. We demonstrated the clinical relevance of this cross-reactivity in a patient.

<b><i>研究背景：</i></b> 当血清中存在可结合犬皮屑提取物的免疫球蛋白E（Immunoglobulin E, IgE），却未检测到针对已报道犬过敏原（Can f 1、Can f 2、Can f 3及Can f 4）的特异性IgE抗体时，提示存在尚未被鉴定的其他犬源性过敏原。近期有研究从犬尿液中分离得到一种可结合IgE的蛋白，经鉴定为前列腺激肽释放酶，将其命名为Can f 5。已有研究报道犬皮屑过敏原与人前列腺特异性抗原（Prostate-Specific Antigen, PSA）存在交叉反应。本研究旨在鉴定可与PSA发生交叉反应的犬皮屑过敏原，并验证其在该例人类精浆过敏患者中的临床相关性。 <b><i>研究方法：</i></b> 本研究采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳免疫印迹（SDS-PAGE immunoblotting）及抑制实验开展分析，并通过质谱分析（mass spectrometry）鉴定参与过敏反应的靶蛋白。 <b><i>研究结果：</i></b> 采用犬前列腺分泌液中IgE结合蛋白开展的SDS-PAGE免疫印迹抑制实验显示，该蛋白可完全抑制IgE结合一条分子量为28 kDa的IgE反应条带，该条带经鉴定为PSA。对犬前列腺分泌液中经电洗脱获得的蛋白进行质谱分析，确认其为Can f 5。采用患者血清对人类精浆进行IgE免疫印迹检测，可观察到两条IgE结合条带（分子量分别为28 kDa与32.7 kDa）。采用固相化的人类精浆或纯化PSA开展SDS-PAGE免疫印迹抑制实验，结果显示：当该过敏性休克患者的血清预先与犬皮屑提取物或重组Can f 5孵育后，IgE结合反应被完全抑制。 <b><i>研究结论：</i></b> 本研究鉴定出可与人PSA发生交叉反应的犬皮屑过敏原为新近报道的Can f 5，并证实该交叉反应在该例患者中具有临床相关性。