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Up to 50% of patients with stinging-insect allergy have double-positive RAST results to honeybee and yellow jacket (YJ) venom. True double sensitization and crossreactivity through venom hyaluronidases are considered main reasons for this multiple reactivity.We investigated the role of antibodies against cross-reactive carbohydrate determinants in venom double positivity.CAP inhibition experiments were performed with crude oilseed rape (OSR) and timothy grass pollen extracts and a neoglycoprotein construct displaying a MUXF glycan, as present in pineapple-stem bromelain (MUXF-BSA). CAP to OSR was used as a rough measure for carbohydrate-specific IgE in individual sera.CAP results to OSR pollen were positive in 2 of 14 single-positive honeybee venom sera, 2 of 16 single-positive YJ venom sera, and 33 (80.5%) of 41 double-positive sera (P.00001, chi(2) test). CAP inhibition was performed in 16 selected patients with a CAP class of 3 or higher to both venoms. In 9 of 11 patients with a highly positive CAP result to OSR (CAP score to OSRCAP score to second venom), pollen extracts, MUXF-BSA, or both were able to completely inhibit IgE binding to one of the venoms, whereas this was not the case in 5 patients with a negative or weakly positive CAP result to OSR (CAP score to OSRCAP score to second venom).The data suggest that carbohydrate-specific IgE is a major cause for the double positivity to honeybee and YJ venom seen in patients with Hymenoptera allergy. Because these antibodies may have low clinical relevance, they may severely impede the correct diagnosis of Hymenoptera venom allergy.

Background Yellow jacket hyaluronidase (YJ-HYA) is considered a major allergen in yellow jacket allergy. It shows 50% homology with the hyaluronidase from honeybee venom, Api m 2. Recently, IgE binding to YJ-HYA and cross-reactivity with Api m 2 has been shown to be due to cross-reactive carbohydrate determinants (CCDs). Objective We sought to quantify the importance of YJ-HYA in yellow jacket allergy and the cross-reactivity with Api m 2 by discriminating between carbohydrate and peptide epitopes. Methods IgE binding to Vespula species venom was studied by means of Western blotting in 136 patients with yellow jacket allergy (31 in vitro single positive to yellow jacket venom and 105 in vitro double-positive to yellow jacket-honeybee). Inhibition studies were carried out with MUXF-BSA (isolated bromelain glycopeptides linked to bovine serum albumin) and purified Api m 2. Results Among yellow jacket single-positive sera, only 1 of 31 bound with YJ-HYA, whereas this was the case in 87% of 105 double-positive sera. Of 83 patients in whom inhibitions were performed, 65% reacted with hyaluronidase through CCDs alone, 27% reacted with both CCDs and peptide epitopes, and 8% reacted only with the hyaluronidase peptide. The protein-specific reactivity with YJ-HYA was cross-inhibited by Api m 2 in 48% (14/29). Antigen 5 and phospholipase A 1 were each recognized by around 90% of sera from both groups, together identifying 97% of patients. Conclusion Hyaluronidase is a minor yellow jacket venom allergen, and only 10% to 15% of patients with yellow jacket allergy are estimated to have IgE against the hyaluronidase protein. Peptide-specific cross-reactivity with Api m 2 occurs in half of these sera. Component-resolved diagnosis with antigen 5 and phospholipase would detect virtually all patients with yellow jacket venom allergy.

Background Cross-reactive carbohydrate determinants (CCDs) are probably the most widely occurring IgE epitopes. Approximately one fifth of patients with allergy develop IgE antibodies against such glycans. However, they appear to be of low clinical significance. Objective We wanted to elucidate the reasons for this lack of clinical symptoms on contact with CCD allergens by determination of the binding affinities of patients' IgE and IgG antibodies. Methods IgE and IgG against CCDs were affinity-purified from sera of selected patients. The binding affinity to defined glyco-epitopes was measured by surface plasmon resonance. Results From a pool of CCD-positive sera, we isolated 0.1 and 25 μg CCD-specific IgE and IgG, respectively. The binding affinity of purified IgE antibodies to core α1,3-fucosylated glycans was in the 10 −10 mol/L range. The affinity was highest when both fucose and xylose were present, whereas xylosylation alone did not cause IgE binding. CCD-specific IgG exhibited a dissociation constant of approximately 10 −8 mol/L. IgG 4 amounted to only 20% of the CCD-specific IgG (as well as total IgG). Conclusion Low binding affinity of anti-CCD IgE cannot be the reason for the observed clinical insignificance of IgE against plant/insect glycan epitopes. Notably, the affinity of IgG to CCDs is higher than that to protein allergens, and it may therefore function as blocking antibody.

Rationale The 23 kDa thaumatin-like protein (TLP) from cherry was identified as a major allergen and designated Pru av 2. Its amino acid sequence contains a single potential N-glycosylation site. The glycosylation of Pru av 2 and its role in IgE-binding was investigated in this study. Methods Natural (n)Pru av 2 was purified from fresh cherries by ion exchange chromatography. An anti-horse radish peroxidase (HRP) antibody was used to detect glycosylation. 20 sera from cherry allergic patients from Italy were selected by high IgE-binding to TLP and were used to study IgE-binding to nPru av 2 and to carbohydrate moieties of HRP and Api g 5, a glycoprotein from celery. ELISA inhibition experiments were performed with 10 μg/ml HRP. Results 180 mg nPru av 2 were purified from 1.5 kg ripe cherries. The anti-HRP-antibody as well as IgE from all sera bound to nPru av 2. Three of 20 sera were strongly reactive with the glycoproteins HRP and Api g 5, two sera were weakly reactive with both proteins, and three sera were only positive to Api g 5. IgE-binding to nPru av 2 was inhibited by preincubation with HRP when using HRP positive sera but also with some of the sera laking reactivity in the HRP-ELISA. Conclusions The cherry TLP very likely contains simple N-glycans similar to those of HRP or Api g 5. The correct conformation and the glycosylation of Pru av 2 have an influence on its IgE-binding capacity.