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

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.