Der p 2 isoallergens have different allergenicity, and quantification with 2-site ELISA using monoclonal antibodies is influenced by the isoallergens.

BACKGROUND: Der p 2 isoallergens have been reported and the possibility of different allergenicity has also been suggested. In addition, the quantification with 2-site ELISA may be affected by the isoallergens. OBJECTIVES: Two different recombinant Der p 2 (rDer p 2) isoallergens were compared in terms of human IgE responses and the reliability of quantification of them with two-site ELISA kits which use monoclonal antibodies (mAbs) as capture and detection of Der p 2. METHODS: Seven different Der p 2 cDNA from the cultured Dermatophagoides pteronyssinus (DP) were cloned and polymorphism in nine amino acid residues was found. Two different recombinant isoallergens (rDer p 2A and rDer p 2B) were expressed and compared to their human IgE immune responses by ELISA and the ELISA inhibition test with 23 sera of DP-allergic patients. The reliability of quantification of two different available 2-site ELISA kits, which used mAbs for capture and detection of Der p 2, was evaluated. RESULTS: The ELISA optical density of rDer p 2B-specific IgE (sIgE) was higher than that of rDer p 2A (P < 0.001). The ELISA inhibition curve of rDer p 2B sIgE in pool I sera (n = 5; high sIgE both to rDer p 2A and rDer p 2B) did not show any differences in the 50% inhibition concentration and maximum inhibitory percentage of rDer p 2A and rDer p 2B sIgE. However, with pool II sera (n = 5; markedly higher sIgE to rDer p 2B than rDer p 2A), the 50% inhibitory concentrations (10 microg/mL vs. 40 ng/mL) and maximum inhibitory percentage (61% vs. 99%) of rDer p 2B sIgE with the two recombinant isoallergens were quite different. rDer p 2B could be quantified with two different 2-site ELISA kits, but rDer p 2A was detected by only one kit. CONCLUSION: We conclude that isoallergens of Der p 2 may have different IgE immune responses. Quantification of Der p 2 with 2-site ELISA kits that adopted mAbs, might be affected by the prevalent form of the isoallergens in reservoir dust.

Heterogeneity of IgE epitopes of vinyl sulphone reactive dye: human serum albumin that react with IgE.

BACKGROUND: Vinyl sulphone reactive dye (vRD), which consists of vinyl sulphone reactive groups and a chromogen, can elicit IgE-mediated occupational asthma (OA) by haptenation. Human serum albumin (HSA) is known as the most reliable carrier protein for the vRD, the IgE epitopes of vRD-HSA are not well characterized. In this study we evaluated the epitope of vRD-HAS-specific IgE. METHODS: Two vRD (Remazole Black-GR and Remazole Orange-3R), Procion Red-MX-5B, which has a dichlorotriazine reactive group, and vinyl sulphone (VS), were haptenated to HSA, respectively. vRD-HSA was denatured by heat or mercaptoethanol treatment and the allergenicities of denatured and non-denatured vRD-HSA were compared by ELISA and IgE immunoblotting using the sera of six vRD-OA patients. vRD-HSA-specific, Procion Red-MX-5B (pRD)-HSA-specific and VS-HAS-specific IgE were also measured with ELISA and the cross-reactivity between them was evaluated with ELISA inhibition. RESULTS: Denaturation of vRD-HSA by heat affected its allergenicity markedly in five of six sera of RD-OA. When vRD was conjugated to the pre-heated HSA, its allergenicity also disappeared or was markedly attenuated compared with the vRD-HSA in five of six sera. Mercaptoethanol treatment markedly affected the allergenicity of the RD-HSA in all six RD-OA sera. Immunoblotting from non-denatured PAGE showed strong IgE affinity to vRD-HSA but immunoblotting from denatured SDS PAGE did not show IgE affinity. Among six RD-OA patients, five and four patients had pRD-HSA-specific and VS-HSA-specific IgE, respectively. However, the vRD-HSA-specific IgE was neither inhibited by pRD-HSA nor VS-HSA CONCLUSION: We considered that the conformational structure of HSA would be critical for the IgE epitopes during the haptenation process and both of the chromogen and reactive groups of the vRD would contribute to the formation of IgE epitope. Our results also confirmed the heterogeneity of IgE epitopes in the RD-HSA complex.

Role of skin prick test and serological measurement of specific IgE in the diagnosis of occupational asthma resulting from exposure to vinyl sulphone reactive dyes.

OBJECTIVES: Some patients with occupational asthma resulting from exposure to reactive dyes have skin reactivity to the causative dyes and specific IgE to reactive dyes have been found in these patients. However, the usefulness of skin prick tests (SPTs) and serological measurement of specific IgE in screening, diagnosis, and monitoring the occupational asthma resulting from exposure to reactive dyes have not yet been assessed. In this study, the clinical validation of SPTs and measurement of specific IgE to vinyl sulphone reactive dyes by enzyme linked immunosorbent assay (ELISA) was evaluated. METHODS: 42 Patients with occupational asthma from reactive dyes (true positive group) were enrolled. In these the causative reactive dye was confirmed by bronchial challenge test. 93 Asymptomatic factory workers with negative challenge to the reactive dye (true negative group) and 16 unexposed controls with negative challenge to the reactive dye were also enrolled. Skin prick tests were done with 10 mg/ml reactive dye in 0.4% phenol/0.9% saline. IgE specific to reactive dye conjugated to human serum albumin (HSA) was measured with enzyme linked immunosorbent assays (ELISAs). RESULTS: None of the unexposed controls had a positive response to SPTs. The sensitivity (76.2% v 53.7%), specificity (91.4% v 86.0%), positive predictive value (80.0% v 62.9%), and negative predictive value (89.5% v 80.8%) of SPTs were higher than those of ELISAs. The mean weal size of reaction to reactive dye was weakly correlated with the ELISA optical density of IgE to reactive dye conjugate in patients with occupational asthma from reactive dyes (n=41, r=0.337, p<0.05). In four patients with occupational asthma from reactive dyes and eight control subjects exposed to reactive dye, IgE specific to reactive dye conjugated to HSA was detected with ELISA even though they showed negative skin reactivity. Six patients completely avoided the reactive dye for a mean (SD) 27.8 (10.3) months, IgE specific to reactive dyes decreased in all six patients (p<0.05) during this time. CONCLUSIONS: Both SPTs and detection of IgE specific to reactive dye in serum samples could be valuable for screening, diagnosis, and monitoring occupational asthma resulting from exposure to reactive dyes. These two tests would complement each other.

Identification and characterization of the major allergens of buckwheat.

BACKGROUND: Buckwheat (BW) has been recognized as a common food allergen in Korea, Japan, and other countries. Until now, serologic findings of BW food-allergic patients and its major allergenic components have not been clarified. In this study, we analyzed the serologic findings of BW food allergy and characterized its major allergenic components. METHODS: Nineteen BW-allergic subjects with symptoms after BW ingestion and 15 asymptomatic control subjects with positive skin prick test to BW were recruited. BW-specific IgE was measured with the Pharmacia CAP kit. Allergenic components of BW were analyzed by IgE immunoblotting, periodate oxidation, two-dimensonal PAGE, and sequencing of N-terminal amino acids. RESULTS: From the BW-allergic patients and asymptomatic controls, the sensitivity (100%), specificity (53%), and negative (100%) and positive predictive values (73%) of Pharmacia CAP specific IgE for diagnosis were estimated. The prevalence of IgE binding to 24-kDa (pI 8.3), 16-kDa (pI 5.6), and 9-kDa (pI 5.0/ 6.0) allergens was higher than 50% in BW-allergic and asymptomatic subjects. However, the specific IgE to split 19-kDa (pI 6.5/7.0) allergens were more specifically found in BW-allergic patients than in asymptomatic subjects (78% vs 7%). N-terminal amino-acid sequences of 19-kDa and 16-kDa allergens showed moderate and weak homology to the 19-kDa globulin protein of rice and alpha-amylase/trypsin inhibitor of millet, respectively. The N-terminus of the 9-kDa isoallergens were not different from each other and were identified as the reported trypsin inhibitors of BW. Attenuation of the IgE binding to the 9-kDa allergen was found with periodate oxidation. CONCLUSIONS: The allergens of 24, 19, 16, and 9 kDa are strong candidates to be major allergens, and the 19-kDa allergen was relatively specific for BW-allergic patients. Moreover, measurement of BW-specific IgE and the features of immunoblotting should be very useful tools in the diagnosis of BW allergy.