Figure 1.
Detection of Api m 12 and Ves v 6 in A. mellifera and V. vulgaris venom.
A SDS-PAGE and Coomassie blue staining of the high molecular weight fraction of honeybee venom. The arrow indicates the 200 kDa band that was subjected to MS/MS-based sequencing. B IgE Immunoreactivity of pooled sera from YJV-sensitized patients with the venom of V. vulgaris in Western Blot (AlaBlot™). The arrow indicates the reactive 200 kDa protein band.
Figure 2.
Alignment of Api m 12 and Ves v 6.
Alignment of A. mellifera vitellogenin Api m 12 (Genbank accession NP_001011578) and V. vulgaris vitellogenin Ves v 6 (Genbank accession AER70365) reveals an identity of 40% on protein level. Asterisks, colons, and periods indicate fully conserved, strongly similar, and weakly similar residues, respectively. Peptides identified by mass spectrometry are underlined, signal sequences are italicized, and putative N-glycosylation sites are highlighted in grey.
Figure 3.
Domain architecture of Api m 12, Ves v 6 and other vitellogenins.
Comparison of the domain architecture of Api m 12 and Ves v 6 with that of the vitellogenins from the hymenoptera species Bombus ignitus (Genbank accession ACM46019) and Nasonia vitripennis (Genbank accession XP_001607388) as well as with that of vitellogenin allergens of the mites Dermatophagoides pteronyssinus (Der p 14, Genbank accession AAM21322) and Euroglyphus maynei (Eur m 14, Genbank accession AAF14270), the fish Oncorhynchus mykiss (Onc m Vg, Genbank accession CAA63421), and Gallus gallus (Gal d 6, Genbank accession AAA49139). DUF, domain of unknown function; VWD, von Willebrand factor type D domain.
Figure 4.
Recombinant expression and immunoreactivity of Api m 12 and Ves v 6.
A, B SDS-PAGE and immunoblot analyses of Api m 12 and Ves v 6 recombinantly produced in Sf9 insect cells visualized by Coomassie blue staining, monoclonal anti-V5 epitope antibody and Galanthus nivalis agglutinin (GNA), recognizing terminal mannose 1,2-, 1,3-, and 1,6-linked to mannose. C, D Immunoreactivity of recombinant Api m 12 and Ves v 6 in ELISA using the monoclonal anti-V5 epitope antibody and polyclonal anti-HRP antiserum specific for α1,3-fucose residues, the underlying principle of hymenoptera venom cross-reactive carbohydrate determinant (CCD) reactivity.
Figure 5.
IgE immunoreactivity of individual patient sera with recombinant Api m 12 and Ves v 6.
A IgE reactivity of individual sera from HBV-sensitized patients with Api m 12 in ELISA. B IgE reactivity of individual sera from YJV-sensitized patients with Ves v 6 in ELISA. C IgE reactivity of exemplary sera from patients that show a monosensitization to either HBV or YJV in intradermal skin test with Api m 12 and Ves v 6 in ELISA. Sera 1 and 2 correspond to sera 3 and 23 in figure 5B and serum 3 to serum 41 in figure 5A. The lower end functional cut-off of the ELISAs is represented as solid line.