Supplementary Material for: Localization of Four Allergens in <b><i>Artemisia</i></b> Pollen by Immunofluorescent Antibodies

<b><i>Background:</i></b> <i>Artemisia</i> pollens have a high potential to induce allergic symptoms. Seven allergen components have been identified, but only Art v 7 has been localized in the pollen grain. This study aimed to localize the allergens in the pollen grains of 4 <i>Artemisia</i> spp. <b><i>Methods:</i></b> Pollen extracts from 2 Chinese <i>Artemisia</i> spp., <i>A. argyi</i> and <i>A. annua</i>, were used to immunize BALB/c mice. Recombinant Art v 1 and Art v 3 allergens were used to select specific monoclonal antibodies (mAbs). Three mAbs were used to purify the natural allergens and were then analyzed by mass spectrometry. As reported previously, polyclonal antibodies were obtained from rabbits immunized with 3 synthesized peptides of Art an 7. Using conventional histology procedures with pollens from 4 <i>Artemisia</i> spp. (<i>A. argyi</i>, <i>A.</i> <i>annua</i>, <i>A. capilaris</i>, and <i>A. sieversiana</i>), allergen images were observed and recorded by fluorescence and confocal laser microscopy. <b><i>Results:</i></b> We obtained 2 specific mAbs against Art v 1, 1 against Art v 2, and 4 against Art v 3 homologs. The Art v 1 and Art v 3 homologs were mainly located on the pollen walls, and the Art v 7 homologous protein was localized intracellularly around nuclei. The location of the Art v 2 homologous protein varied across species, being intracellular around nuclei for <i>A. annua</i> and <i>A.</i> <i>argyi</i>, and in both the pollen wall and around nuclei for <i>A</i>. <i>capilaris</i> and <i>A.</i> <i>sieversiana</i>. <b><i>Conclusions:</i></b> Four mugwort allergens were localized in the pollen, and the major Art v 1 and Art v 3 allergens were located mainly in the pollen wall.**<i>背景：</i>** 蒿属（*Artemisia*）花粉具有极高的致敏潜能，目前已鉴定出7种变应原组分，但仅Art v 7已在花粉粒中完成定位。本研究旨在对4种蒿属（*Artemisia*）植物花粉粒中的变应原进行定位。 **<i>方法：</i>** 采用2种中国产蒿属植物——艾蒿（*A. argyi*）与黄花蒿（*A. annua*）的花粉提取物免疫BALB/c小鼠；以重组Art v 1及Art v 3变应原筛选特异性单克隆抗体（monoclonal antibody, mAb）。筛选获得3株单克隆抗体，用于纯化天然变应原，随后通过质谱（mass spectrometry）进行分析。如既往研究所述，以Art an 7的3条合成肽段免疫家兔，制备得到多克隆抗体。采用常规组织学流程，对4种蒿属植物（*A. argyi*、*A. annua*、*A. capilaris*及*A. sieversiana*）的花粉进行处理，通过荧光显微镜（fluorescence microscopy）与共聚焦激光扫描显微镜（confocal laser scanning microscopy）观察并记录变应原定位图像。 **<i>结果：</i>** 本研究成功获得2株针对Art v 1的特异性单克隆抗体、1株针对Art v 2的特异性单克隆抗体，以及4株针对Art v 3同源蛋白的特异性单克隆抗体。Art v 1与Art v 3同源蛋白主要定位于花粉壁；Art v 7同源蛋白定位于细胞核周围的胞内区域。Art v 2同源蛋白的定位因物种而异：在*A. annua*与*A. argyi*中，其定位于细胞核周围的胞内区域；而在*A. capilaris*与*A. sieversiana*中，其同时分布于花粉壁与细胞核周围区域。 **<i>结论：</i>** 本研究完成了4种蒿属变应原在花粉中的定位，主要变应原Art v 1与Art v 3主要定位于花粉壁。