ImmunoCAP ISAC assay

For the evaluation of allergen-specific IgE antibodies in blood, the ImmunoCAP ISAC (Immuno-Solid phase Allergy Chip) assay was used, which is a semi-quantitative molecular diagnostic test that reports results in ISAC standard units (ISU), indicating allergen-specific IgE levels; the operating range is 0.3-100 ISU. The specific ISAC microarray chip that was used was developed through the European Union-funded project Mechanisms for the Development of Allergies; (MeDALL) (33), which can measure IgE antibodies to 176 allergen components (34), using only 20 µl of serum. A sample consisting of 5 ml of venous blood was collected from each patient in a red-top blood collection tube (without anticoagulant or preservative). After collection of the whole blood, it was allowed to clot by leaving it undisturbed at room temperature for 30 min, and then the clot was removed by centrifugation at 1,500 x g for 10 min at room temperature. The resulting supernatant was immediately apportioned into 0.5 ml aliquots and stored at -70˚C until further processing. Allergen-specific IgE was measured in sera from ragweed-allergic patients using a fluoroenzyme immunoassay auto-analyser (Thermo Fisher Scientific Inc., Phadia AB), according to the manufacturer's guidelines. Briefly, 20 µl of serum was added to each microarray and incubated at room temperature for 120 min. The samples were washed and then incubated for 30 min with 20 µl of fluorescence-labeled antihuman IgE antibodies. Unbound antibodies were removed by washing and the fluorescence of the processed ISAC slides was measured in a GenePix4000B microarray scanner (Molecular Devices). Image analysis was performed using a microarray image analyzer software (MIA, Thermo Fisher Scientific Inc., Phadia AB). Fluorescence measurements were compared with a calibration curve and expressed as ISU. Established cut-off values were used to interpret the results: Values <0.3 ISU were considered negative (undetectable or very low) with regards to sensitization to a specific allergen, and values of ≥0.3 ISU were distributed into three classes, as follows: For ISU values ≥0.3 to <1, class 1 (low); for ISU values ≥1 to <15, class 2 (moderate to high); and for ISU values ≥15, class 3 (very high). IgE class according to ISU was also correlated with the intensity of the response to ragweed pollen extract in the SPT. The chip included only the established major allergen from ragweed pollen, Amb a 1, and not other ragweed pollen allergens, nor ragweed pollen extract. The sensitivity and specificity of ImmunoCAP ISAC were compared against the SPT, which was considered the standard method for allergy diagnosis, as it was the method used for patient selection.