Human monoclonal IgG antibodies derived from a patient allergic to birch pollen as tools to study the in situ localization of the major birch pollen allergen,

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Human monoclonal IgG antibodies derived from a patient allergic to birch pollen as tools to study the in situ localization of the major birch pollen allergen, Bet v 1, by immunogold electron microscopy Monika Grote, PhDa, Petra Wiedemann, MScb, Serge Lebecque, MDc, Rudolf Valenta, MDb Journal of Allergy and Clinical Immunology Volume 101, Issue 1, Pages 60-66 (January 1998) DOI: 10.1016/S0091-6749(98)70194-0 Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 1 Detection of nitrocellulose-blotted natural birch pollen extract (Betula verrucosa) and recombinant Bet v 1 (r Bet v 1) with human monoclonal antibodies BAB1, BAB2, BAB4, and isotype controls (IgG1, IgG2, IgG4). Journal of Allergy and Clinical Immunology 1998 101, 60-66DOI: (10.1016/S0091-6749(98)70194-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 2 Survey of pollen grain showing part of pollen wall, peripheral cytoplasm, and nucleus after mapping of Bet v 1 with human monoclonal antibody BAB4 and goat anti-human IgG/gold. Gold particles are distributed in cytoplasm and nucleus, whereas there is no labeling in pollen wall (magnification × 40,000; bar: 0.25 μm). GP, Gold particle; ST, starch; L, lipid; M, mitochondrion; N, nucleus; W, wall. Journal of Allergy and Clinical Immunology 1998 101, 60-66DOI: (10.1016/S0091-6749(98)70194-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 3 Part of pollen grain showing wall and cytoplasm after probing with human monoclonal antibody BAB1 followed by goat anti-human IgG/gold. Gold markers predominantly associate with electron-dense matrix between various cell organelles like mitochondria, dictyosomes, endoplasmic reticulum, and vesicles (magnification × 46,000; bar: 0.25 μm). E, Exine; ER, endoplasmic reticulum; GP, gold particle; I, intine; L, lipid; M, mitochondrion; ST, starch. Journal of Allergy and Clinical Immunology 1998 101, 60-66DOI: (10.1016/S0091-6749(98)70194-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 4 Detail of pollen grain cytoplasm after probing with human monoclonal antibody BAB2 followed by goat anti-human IgG/gold. Most gold particles are localized between numerous cisternae and vesicles (magnification × 54,000; bar: 0.25 μm). Inset, Detail of birch pollen cytoplasm after anhydrous-liquid fixation in p-formaldehyde/glycerol. Well-preserved ribosomes line endoplasmic membranes (magnification × 56,000; bar: 0.125). D, dictyosome; ER, endoplasmic reticulum; GP, gold particle; Ri, ribosome. Journal of Allergy and Clinical Immunology 1998 101, 60-66DOI: (10.1016/S0091-6749(98)70194-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 5 Control experiment for BAB2. Section was labeled with normal human IgG (subclass 4, kappa) followed by goat anti-human IgG/gold. Detail from cytoplasm shows very few nonspecifically adsorbed gold particles (magnification × 54,000; bar: 0.25 μm). ER, Endoplasmic reticulum; GP, gold particle; L, lipid; M, mitochondrion. Journal of Allergy and Clinical Immunology 1998 101, 60-66DOI: (10.1016/S0091-6749(98)70194-0) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 6 Multiple sequence alignment of Bet v 12 with Bet v 1 homologous proteins and allergens. Amino-acid sequence of Bet v 1 was aligned by hand with homologous proteins according to their database entry. Sources of proteins and sequence identities with Bet v 1 (MPAV_BETVE) are as follows: MPAG_ALNGL: major alder allergen, Aln g 1 (79.6%); MPA1_CARBE: major hornbeam allergen, Car b 1 (72.8%); MPAA_CORAV: major hazel allergen, Cor a 1 (71%); PR1_PHAVU: PR protein from Phaseolus vulgaris (43.8%); SAM2_SOYBN: PR protein from soybean (46.3%); L1R18R: PR protein from Lupinus luteus (45.1%); VUPR42: PR protein from Vigna unguiculata (43.2%); MAL1_MALDO: major apple allergen, Mal d 1 (53.1%); DRR1_PEA: PR protein from pea (51.2%); PRS1_SOLTU: PR protein from potato (43.2%); PR11_PETCR: PR protein from parsley (39.5%); MPAG_APIGR: major celery allergen, Api g 1 (38.9%); PR1_ASPOF: PR protein from asparagus (32.7%). Identical amino acids are indicated by dashes and blanks represent gaps introduced for maximal fit. Journal of Allergy and Clinical Immunology 1998 101, 60-66DOI: (10.1016/S0091-6749(98)70194-0) Copyright © 1998 Mosby, Inc. Terms and Conditions