Fluorescence labeled anti ige antibodies

The serum samples were analyzed for IgE reactivity to microarrayed allergen molecules by using the MeDALL chip, which is based on the ISAC microarray platform (Phadia Multiplexing; Thermo Fisher Scientific, Uppsala, Sweden) but differs from the commercially available ISAC regarding outlay and the number of allergens. The technical details and features of the MeDALL chip together with the cutoff of 0.3 ISAC standardized units for IgE detection (ISU-E) or greater are described in detail by Lupinek et al.22 (link) IgE reactivity profiles and levels were measured for PR-10 proteins (Bet v 1, Mal d 1, Aln g 1, Cor a 1.04, Ara h 8, Pru p 1, Api g 1, Act d 8, and Gly m 4). A level of 0.3 ISU-E or greater was considered positive. Briefly, aliquots of 35 μL of serum were incubated on the microarray, and after 120 minutes of incubation at room temperature, slides were washed, and fluorescence-labeled anti-IgE antibodies (Thermo Fisher) were added and incubated for 30 minutes. Chips were then washed, dried, and analyzed with a Laser Scan Confocal microarray reader (LuxScan 10K/A; Capital-Bio, Beijing, China). The results were evaluated by using Phadia Microarray Image Analysis (MIA) software and are reported in ISU-E.22 (link)

Serum samples were initially tested with ImmunoCAP (Thermo Fisher AB, Uppsala, Sweden) for allergen-specific IgE antibodies to cat (e1) and dog (e5) extract. The results were expressed in kilounits of allergen per liter, and a positive test result was defined as 0.35 kU_A/L or greater. An IgE antibody level of greater than 100 kU_A/L was given the value of 101 kU_A/L in statistical evaluations.
IgE reactivity to the cat allergen molecules Fel d 1, 2, and 4, as well as the dog allergen molecules Can f 1, 2, 3, 5, and 6, were analyzed with an allergen chip based on ISAC technology (Thermo Fisher) developed in the MeDALL FP7-funded research program.22 (link) In brief, serum aliquots of 35 μL were incubated on the microarray for 120 minutes at room temperature, and slides were washed and incubated with fluorescence-labeled anti-IgE antibodies (Thermo Fisher) for 30 minutes. Chips were then washed, dried, and analyzed with a Laser Scan Confocal microarray reader (LuxScan 10K/A; Capital-Bio, Beijing, China). The results were evaluated with Phadia Microarray Image Analysis software and are reported in ISAC standardized units. The cutoff was set at 0.3 ISAC standardized units for IgE detection (ISU-E).