ImmunoCAP cellulose displays cross-reactive carbohydrate determinant (CCD) epitopes and can cause false-positive test results in patients with high anti-CCD IgE antibody levels

Abstract

Cross-reactive carbohydrate determinants (CCDs) in plants and insect venoms are a common cause of irrelevant positive test results during invitro allergy diagnosis. We observed that some CCD-positive sera show nonspecific IgE binding even with CCD-free recombinant allergens when using the Phadia ImmunoCAP platform. We investigated whether cellulose used as an allergen carrier in ImmunoCAP harbors residual N-glycans, causing nonspecific background binding in CCD-positive sera. IgE binding to 6 samples of blank ImmunoCAPs coupled to either streptavidin (SA-CAP-1 or 2) or nonallergenic maltose-binding protein (MBP; MBP-CAP-1 to 4) and binding to a panel of 4 recombinant allergens were compared in CCD-positive sera before and after inhibition with a CCD inhibitor (MUXF3-human serum albumin). Of 52 CCD-positive sera (bromelain, 1.01-59.6 kilounits of antigen per liter [kUA/L]) tested on SA-CAP-1, 35 (67%) showed IgE binding of greater than 0.35 kUA/L (0.41-4.22 kUA/L). Among those with anti-CCD IgE levels of greater than 7.0 kUA/L, 90% (26/29) were positive. IgE binding to SA-CAP-1 correlated with IgE binding to bromelain (r=0.68) and was completely abolished by serum preincubation with the CCD inhibitor (n=15). Binding scores with SA-CAP-2 and MBP-CAP-1 to MBP-CAP-4 were generally lower but strongly correlated with those of SA-CAP-1 and bromelain. IgE reactivity of 10 CCD-positive sera (14.0-52.5 kUA/L) with the recombinant allergens rPhl p 12, rFel d 1, rAra h 2, and rPru p 3 was positive to at least 1 allergen in 8 of 10 (0.36-1.63 kUA/L) and borderline in 2 of 10 (0.21-0.25 kUA/L). Binding correlated with antibody binding to bromelain (r=0.61) and to all blank ImmunoCAPs (r>0.90) and could be completely blocked by the CCD inhibitor. Overall, mean background binding to cellulose CCDs corresponded to 2% to 3% of the reactivity seen with bromelain. Cellulose used as a solid-phase allergen carrier can contain varying amounts of CCDs sufficient to cause false-positive test results up to 2 kUA/L with nonglycosylated recombinant allergens in patients with high levels of anti-CCD IgE antibodies.