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Recognition of riboflavin and the capsular polysaccharide of Haemophilus influenzae type b by antibodies generated to the haptenic epitope D-ribitol.

Ravi, G. and Venkatesh, Y. P. (2014) Recognition of riboflavin and the capsular polysaccharide of Haemophilus influenzae type b by antibodies generated to the haptenic epitope D-ribitol. Glycoconjugate Journal, 31. pp. 247-258.

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Abstract

D-Ribitol, a five–carbon sugar alcohol, is an important metabolite in the pentose phosphate pathway; it is an integral part of riboflavin (vitamin B2) and cell wall polysaccharides in most Gram-positive and a few Gram-negative bacteria. Antibodies specific to D-ribitol were generated in New Zealand white rabbits by using reductively aminated Dribose- BSA conjugate as the immunogen. MALDI-TOF and amino group analyses of ribitol-BSA conjugate following 120 h reaction showed ~27–30 mol of ribitol conjugated per mole BSA. The presence of sugar alcohol in the conjugates was also confirmed by an increase in molecular mass and a positive periodic acid–Schiff staining in SDS-PAGE. Caprylic acid precipitation of rabbit serum followed by hapten affinity chromatography on ribitol–KLH–Sepharose CL-6B resulted in pure ribitol–specific antibodies (~45–50 μg/mL). The affinity constant of ribitol antibodies was found to be 2.9× 107 M−1 by non-competitive ELISA. Ribitol antibodies showed 100 % specificity towards ribitol, ~800 % cross– reactivity towards riboflavin, 10–15 % cross–reactivity with sorbitol, xylitol andmannitol, and 5–7%cross–reactivity with L-arabinitol and meso-erythritol. The specificity of antibody to ribitol was further confirmed by its low cross-reactivity (0.4 %) with lumichrome. Antibodies to D-ribitol recognized the purified capsular polysaccharide of Haemophilus influenzae type b, which could be specifically inhibited by ribitol. In conclusion, antibodies specific to D-ribitol have been generated and characterized, which have potential applications in the detection of free riboflavin and ribitol in biological samples, as well as identification of cell-surface macromolecules containing ribitol.

Item Type: Article
Uncontrolled Keywords: Haemophilus influenzae type b . Immunogenicity . Polyribosyl ribitol phosphate . Reductive amination . Ribitol–specific antibodies . Riboflavin
Subjects: 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 28 Polysaccharide Chemistry
Divisions: Dept. of Biochemistry
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 25 Apr 2014 09:56
Last Modified: 25 Apr 2014 09:56
URI: http://ir.cftri.com/id/eprint/11465

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