Fig 2: Detection of Ara h 2-beads, diluted 1:10, in an artificial peptide library of scrambled Ara h 2 beads. Beads were incubated with serum from two different peanut-allergic patients, and IgE-binding was detected with fluorophore-labeled anti-IgE antibodies. Phase contrast images are shown on the left, corresponding fluorescence images of the appropriate channel on the right side of the panel. (A) Beads incubated with serum from patient 1, diluted to approximately 90 ng Ara h 2-specific IgE/ml; IgE-binding detected with phycoerythrin-labeled monoclonal anti-IgE antibody; (B) Beads incubated with serum from patient 1, diluted to approximately 23 ng Ara h 2-specific IgE/ml; IgE-binding detected with DyLight488-labeled polyclonal anti-IgE antibody; (C) Beads incubated with total serum from patient 2, diluted to approximately 33 ng Ara h 2-specific IgE/ml; IgE-binding detected with phycoerythrin-labeled monoclonal anti-IgE antibody; (D) Beads incubated only with phycoerythrin-labeled monoclonal anti-IgE antibody; (E) Beads incubated with IgG-depleted serum from patient 2, diluted to approximately 33 ng Ara h 2-specific IgE/ml; IgE-binding detected with phycoerythrin-labeled monoclonal anti-IgE antibody. Quantitation of fluorescence intensities can be found in the Supplementary Figure 3.

Fig 3: Sporadic cross-reactivity of anti-IgE antibodies with bead-bound random peptides in an OBOC-library. (A) DyLight550-labeled, polyclonal anti-IgE antibody; (B) phycoerythrin-labeled, monoclonal anti-IgE antibody. Phase contrast images are shown on the left, corresponding fluorescence images on the right side of the panel.

Fig 4: Identification of specific epitope-carrying beads in a pre-adsorbed OBOC library. Specific c-myc- (A,B) or Ara h 2- (C) beads were mixed 1:1,000 with an OBOC library. Beads were incubated with a FITC-labeled monoclonal anti-IgE antibody to block cross-reactive sequences and subsequently screened with anti-c-myc IgE (A,B) or serum from a peanut-allergic patient (C) followed by detection of IgE-binding with a phycoerythrin-labeled monoclonal anti-IgE antibody. Phase contrast images are shown on the left, corresponding fluorescence images of the green and the red channel are in the middle and on the right side of the panel. “True positive” beads carrying the specific epitope sequence are recognizable by strong fluorescence only in the red channel (arrows) and only slight autofluorescence in the green channel; pre-adsorbed “cross-reactive” beads show a strong fluorescence with corona in the green channel (open arrowheads), and no signal in the red channel. A number of fluorescent beads cannot be clearly assigned to either “positive” or “cross-reactive,” exhibiting similar fluorescence intensities and/or coronas in both, the red and the green channel (closed arrowheads).

Fig 5: Sorting of OBOC beads after pre-adsorption with the phycoerythrin (PE)-labeled anti-IgE antibody. Representative dot plots with full gating of (A) the total pre-adsorbed OBOC library showing the anti-IgE cross-reactive beads in the upper PE+ gate and the non-cross-reactive beads in the lower PE- gate, and (B) re-analysis of the pre-cleaned non-cross-reactive bead fraction (PE-) after sorting.