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.