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

Publication date: Available online 12 May 2017
Source:Journal of Allergy and Clinical Immunology
Author(s): Wolfgang Hemmer, Friedrich Altmann, Friedrich Holzweber, Clemens Gruber, Felix Wantke, Stefan Wöhrl
RationaleCross-reactive carbohydrate determinants (CCDs) in plants and insect venoms are a common cause of irrelevant positive test results during in-vitro allergy diagnosis. We observed that some CCD-positive sera show non-specific IgE-binding even with CCD-free recombinant allergens when using the Phadia ImmunoCAP platform.ObjectiveWe investigated if cellulose used as allergen carrier in the ImmunoCAP harbors residual N-glycans causing non-specific background-binding in CCD-positive sera.MethodsIgE binding to six samples of blank ImmunoCAPs coupled with either streptavidin (SA-CAP 1-2) or non-allergenic maltose-binding protein (MBP-CAP 1-4) and to a panel of four recombinant allergens was compared in CCD-positive sera before and after inhibition with a CCD inhibitor (MUXF-HSA).ResultsOut of 52 CCD-positive sera (bromelain 1.01-59.6 kUa/L) tested on SA-CAP-1, 35 (67%) showed IgE-binding greater than 0.35 kUa/L (0.41-4.22 kUa/L). Among those with anti-CCD IgE levels >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 pre-incubation with the CCD inhibitor (n=15). Binding scores with SA-CAP-2 and MBP-CAPs 1-4 were generally lower but strongly correlated with SA-CAP-1 and bromelain. IgE reactivity of 10 CCD-positive sera (14.0-52.5 kUa/L) with the recombinant allergens rPhl p12, rFel d1, rAra h2, and rPru p3 was positive to at least one allergen in 8/10 (0.36-1.63 kUa/L) and borderline in 2/10 (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 2-3% of the reactivity seen with bromelain.ConclusionsCellulose used as a solid phase allergen carrier may contain varying amounts of CCDs sufficient to cause false-positive test results up to 2 kUa/L with non-glycosylated recombinant allergens in patients with high levels of anti-CCD IgE antibodies.

Teaser

Cellulose-based IgE assays may deliver false-positive test results of up to 2 kUa/L with recombinant allergens in strongly CCD-positive patient sera due to the presence of glycoproteins in the cellulose matrix.


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