Figure 1.
Detection of allergen conjugation on micro-beads using mouse monoclonal and rabbit polyclonal antibodies.
Panel A: Each antibody preparation was tested on the respective allergen-coupled micro-bead. The other two micro-beads were not in the tube. NP: Not performed in this singlepex testing. * Values are expressed as Median Fluorescence Intensity [MFI]. Upper right corner: an example of blank fluorescence reading of the three conjugated micro-beads without serum. Panel B: Antibodies selected on the basis of the findings reported in panel A were tested on the three allergen-coupled micro-beads, all found in the same testing tube. * Values are expressed as Median Fluorescence Intensity [MFI]. Upper right corner: an example of micro-bead fluorescence scatter plot and micro-bead clusters.
Figure 2.
Detection of allergen conjugation on micro-beads using human pooled sera containing specific IgE.
Selected pooled human sera with IgE specific for the three candidate allergens were tested to verify that allergen coupling was acceptable also for human immunoglobulin determination and to estimate interference among different allergen-conjugated micro-beads. Panel A: Each serum pool was tested on the respective allergen-coupled micro-bead. The other two micro-beads were not in the tube. NP: Not performed in this singlepex testing. ° Values are expressed as kU/l. * Values are expressed as Median Fluorescence Intensity [MFI]. Upper right corner: an example of blank fluorescence reading of conjugated micro-beads without serum. Panel B: Serum pools were tested on the three allergen-coupled micro-beads present in the same tube. ° Values are expressed as kU/l. * Values are expressed as Median Fluorescence Intensity [MFI]. Upper right corner: an example of micro-bead fluorescence clusters.
Figure 3.
Testing human sera containing specific IgE on allergen conjugated micro-beads.
Three human individual serum samples (49812, 49141, 33020), each of them known to have specific IgE to one allergen and not to the others were tested on the three micro-beads present in the same tube. Serum 31851 was from a subject recognizing nDer s 1 and nPen m 1, but not nPru p 3. A non allergic donor was used for control purposes. # Serum sample numbers; ° Values are expressed as kU/l; * Values are expressed as Median Fluorescence Intensity [MFI]. Upper right corner: an example of micro-bead fluorescence clusters.
Figure 4.
ABA versus ISAC correlation results on 137 serum samples selected on the basis of nDer s 1, nPen m 1, and nPru p 3 mutually exclusive IgE positivity are reported.
Panel A: All 411 IgE values, obtained by testing the three allergens; Panel B: 137 IgE results obtained on nDer s 1 allergen; Panel C: 137 IgE results obtained on nPen m 1 allergen; Panel D: 137 IgE results obtained on nPru p 3 allergen. For graphical visualization needs on log scales, zero values for ABA were set at 10 MFI on the X axis, and at 0.01 kU/l for ISAC values on the Y axis. The Spearman r correlation coefficient, the χ2 and the Fisher's exact tests were used where applicable.
Figure 5.
ABA versus ISAC IgE correlation results on 1,519 serum samples selected on the basis of any of the allergen specificities reported in Table S1, using the two micro systems.
Letter flag (A) in figure 5 indicates it as part of the results shown also in figures 6, 7, and 8. Consecutive letters are used on purpose to recall result type continuity across the four figures. Allergen nature, being either natural or recombinant, matched for both tests. Vertical dashed lines represent the arbitrary ABA negative cut off value. Horizontal dashed lines mark the value range where ISAC IgE determinations are not always reproducible (unpublished data). For graphical visualization needs on log scales, zero values for ABA were set at 10 MFI on the X axis, and at 0.01 kU/l for ISAC values on the Y axis. The Spearman r correlation coefficient was calculated and the χ2 test was used for statistical purposes. Statistical results are reported below the graph.
Figure 6.
ABA versus ISAC correlation results on serum samples selected on the basis of the allergen specificities reported in each panel and listed in Table S1.
Letter flags, namely B, C, D, E, F, G, in figure 6 indicate them as parts of the results shown also in figures 5, 7, and 8. Consecutive letters are used on purpose to recall result type continuity across the four figures. Allergen nature, being either natural or recombinant, matched for both tests. Vertical dashed lines represent the arbitrary ABA negative cut off value. Horizontal dashed lines mark the value range where ISAC IgE determinations are not always reproducible (unpublished data). For graphical visualization needs on log scales, zero value for ABA was set at 10 MFI on the X axis, and at 0.01 kU/l for ISAC value on the Y axis. The Spearman r correlation coefficient was calculated and the Fisher's exact test was used for statistical purposes. Statistical results are reported below each graph.
Figure 7.
ABA versus ISAC correlation results on serum samples selected on the basis of the allergen specificities reported in each panel and listed in Table S1.
Letter flags, namely H, I, J, K, L, M, in figure 7 indicate them as parts of the results shown also in figures 5, 6, and 8. Consecutive letters are used on purpose to recall result type continuity across the four figures. Allergen nature, being either natural or recombinant, matched for both tests. Vertical dashed lines represent the arbitrary ABA negative cut off value. Horizontal dashed lines mark the value range where ISAC IgE determinations are not always reproducible (unpublished data). For graphical visualization needs on log scales, zero value for ABA was set at 10 MFI on the X axis, and at 0.01 kU/l for ISAC value on the Y axis. The Spearman r correlation coefficient was calculated and the Fisher's exact test was used for statistical purposes. Statistical results are reported below each graph.
Figure 8.
ABA versus ISAC correlation results on serum samples selected on the basis of the allergen specificities reported in each panel and listed in Table S1.
Letter flags, namely N, O, P, Q, in figure 8 indicate them as parts of the results shown also in figures 5, 6, and 7. Consecutive letters are used on purpose to recall result type continuity across the four figures. Allergen nature, being either natural or recombinant, matched for both tests. Vertical dashed lines represent the arbitrary ABA negative cut off value. Horizontal dashed lines mark the value range where ISAC IgE determinations are not always reproducible (unpublished data). For graphical visualization needs on log scales, zero value for ABA was set at 10 MFI on the X axis, and at 0.01 kU/l for ISAC value on the Y axis. The Spearman r correlation coefficient was calculated and the Fisher's exact test was used for statistical purposes. Statistical results are reported below each graph.
Figure 9.
Selected examples of Allergen micro-Beads Array (ABA) IgE testing report.
Eleven different micro-beads were tested in the same tube in each run for each sample. Examples were selected on purpose among the samples with high total IgE. All IgE positive and negative ABA results matched the ISAC results. Numbers in the upper left corner indicate the Table S1 row numbers and patients' ID. In each panel: the upper left graph shows clustered micro-beads by their dimension; the upper right: scatter plots of each fluorescent bead; the lower left: fluorescence intensity and event counts; the lower right: summary table with median fluorescence values. Samples reported in each of the six panels had the following total IgE values: Panel A = 9,730 IU/l; Panel B = 1,351 IU/l; Panel C = 1,220 IU/l; Panel D = 1,931 IU/l; Panel E = 20,900 IU/l; Panel F = 23,540 IU/l.