The Role of Anti-CCD Antibodies in Grape Allergy Diagnosis

Background: Allergens are mostly composed of glycoprotein structures. It is believed that glycan-specific antibodies may lead to false-positive reactions in immunoassays. In this study we investigated the glycosylation state of grape allergens as well as the presence of antibodies to cross-reactive carbohydrate determinants (anti-CCDs) in sera from grape-sensitive individuals. Methods: Grape extract proteins were electrotransferred onto PVDF membranes and their glycosylation states were analyzed by blotting methods. To assess the presence of anti-CCDs, natural and mildly deglycosylated proteins were immunoblotted with grape-allergic subjectschr('۳۹') sera. We also measured the IgE reactivity of each subject’s sera with other fruit extracts via an indirect ELISA. Results: Immunoblotting studies showed that mildly deglycosylated grape proteins had lower IgE-binding capacity than their intact natural counterparts, which could be due to the presence of anti-CCDs. Biotinylation studies confirmed that the glycosylation levels of the ۲۴, ۳۲, and ۶۰ kDa IgE-reactive proteins were higher than those of the ۳۸ and ۴۵ kDa ones. Lectin blotting showed that the ۲۴ and ۶۰ kDa bands were highly mannosylated, with the highest level of mannosylation on the ۲۴ kDa allergen. Conclusion: This study showed that some grape allergens are glycosylated and that anti-CCD antibodies may cause weakly false-positive results during assessment of IgE reactivity to grape allergens.