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 sulph one reactive groups and a chromogen, can elicit IgE-mediated occupational a sthma (OA) by haptenatian. Human serum albumin (HSA) is known as the most r eliable carrier protein for the vRD, the IgE epitopes of vRD-HSA are not we ll characterized. In this study we evaluated the epitope of vRD-HAS-specifi c 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), w ere haptenated to HSA, respectively. vRD-HSA was denatured by heat or merca ptoethanol treatment and the allergenicities of denatured and non-denatured vRD-HSA were compared by ELISA and IgE immunoblotting using the sera of si x vRD-OA patients. vRD-HSA-specific, Procion Red-MX-SB (pRD)-HSA-specific a nd VS-HAS-specific IgE were also measured with ELISA and the cross-reactivi ty 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 HS A, its allergenicity also disappeared or was markedly attenuated compared w ith the vRD-HSA in five of six sera. Mercaptoethanol treatment markedly aff ected 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 immunobl otting from denatured SDS PAGE did not show IgE affinity. Among six RD-OA p atients, five and four patients had pRD-HSA-specific and VS-HSA-specific Ig E, 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 th e chromogen and reactive groups of the vRD would contribute to the formatio n of IgE epitope. Our results also confirmed the heterogeneity of IgE epito pes in the RD-HSA complex.