Generation of an antibody specific to erythritol, a non-immunogenic food additive

Abstract

Erythritol, a simple sugar alcohol, is widely used as a food and drug additive owing to its chemical inertness, sweetness and non-toxicity. Adverse reactions to erythritol are rare and only three cases of allergic reactions to foods containing erythritol have been reported. Being inert, erythritol cannot produce an immunological response. In order to explain the mechanism of immunogenicity of erythritol, a method to obtain erythritol epitopes on a carrier protein, which can serve as an immunogen to develop antibodies against erythritol, is described. D-Erythrose was conjugated to bovine serum albumin at pH 8 by reductive amination. The reduction product of the Schiff base of D-erythrose–bovine serum albumin conjugate creates erythritoyl groups. Rabbits immunized with erythritol–bovine serum albumin conjugate (29 haptens/molecule) showed good antibody response (detection of 1 µg antigen, erythritol–keyhole limpet haemocyanin conjugate possessing 50% modified amino groups, at 1 : 50 000 dilution). Anti-erythritol immunoglobulin-G antibodies were purified from the immune serum using hapten-affinity chromatography on an erythritol-keyhole limpet haemocyanin-Sepharose CL-6B affinity matrix. The yield of erythritol-specific antibody was approximately 40 µg ml⁻¹ of rabbit antiserum. Enzyme-linked immunobsorbant assay inhibition studies using sugars, sugar alcohols and L-lysine showed minimal cross-reactivity (approximately 4%) when compared with erythritol; only dithioerythritol showed a cross-reactivity of approximately 33%. D-Threitol and L-threitol (isomers of erythritol) had cross-reactivities of 15 and 11%, respectively. The inhibition studies confirmed the haptenic nature of erythritol and indicated that the erythritoyl group is a single epitope. The reaction scheme outlined here for the generation of erythritol epitopes appears to provide a basis for the immunogenicity of erythritol.

Keywords:

• Allergen
• erythritol
• erythritol-bovine serum albumin
• erythritol-specific antibody
• food additive
• hapten
• reductive amination
• sugar alcohol
• sweetener

Acknowledgements

The authors thank Dr V. Prakash, Director, CFTRI, Mysore, for his support, keen interest and constant encouragement in this work. The research work was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi (Institutional Project No. MLP-1407 to Y. P. V.), and by a junior research fellowship to K. S.