Generation of antibodies specific to d-mannitol, a unique haptenic allergen, using reductively aminated d-mannose–bovine serum albumin conjugate as the immunogen

Abstract

d-Mannitol is commonly used as a food additive and excipient due to its sweetness, non-toxicity, and low calorific value. However, several cases of hypersensitivity reactions to mannitol have been reported. Owing to its inert nature, mannitol cannot produce an immunological response. In order to explain the mechanism of immunogenicity of mannitol, a method to obtain mannitol epitopes on a carrier protein, which serves as an immunogen to generate antibodies against mannitol, is described. In the present investigation, d-mannitol-specific polyclonal antibodies were generated by immunizing rabbits with reductively aminated mannose–bovine serum albumin (BSA) (33 haptens/molecule) as the hapten–carrier conjugate. Anti-mannitol IgG antibodies were purified from the immune serum by hapten-affinity chromatography on a d-mannitol-keyhole limpet hemocyanin (KLH)-Sepharose CL-6B affinity matrix. The yield of mannitol-specific antibodies was ∼40 μg per mL of rabbit antiserum. The specificity of the purified antibodies towards d-mannitol was demonstrated by hapten-inhibition in enzyme-linked immunosorbent assay (ELISA). The affinity-purified antibodies were found to be very specific to d-mannitol showing less than 5% cross-reactivity with other sugars and sugar alcohols, with the exception of its epimer, sorbitol, which showed 8.8% cross-reactivity. Importantly, the antibodies showed <1% cross-reactivity with l-mannitol epitope, thus exhibiting configurational specificity. The inhibition studies provided evidence for the haptenic nature of mannitol and confirmed that the mannitoyl group is a single epitope. The reaction scheme utilized here for the generation of mannitol epitopes provides the basis for the immunogenicity of mannitol.