Recognition of flavin mononucleotide, Haemophilus influenzae type b and its capsular polysaccharide vaccines by antibodies specific to D-ribitol-5-phosphate

Ravi, G. and Venkatesh, Y. P. (2014) Recognition of flavin mononucleotide, Haemophilus influenzae type b and its capsular polysaccharide vaccines by antibodies specific to D-ribitol-5-phosphate. Glycoconjugate Journal, 31. pp. 573-585.

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Abstract

D-Ribitol-5-phosphate (Rbt-5-P) is an important metabolite in the pentose phosphate pathway and an integral part of bacterial cell wall polysaccharides, specifically as polyribosyl ribitol phosphate (PRP) in Haemophilus influenzae type b (Hib). The major objective of this study was to investigate whether an antibody specific to Rbt-5-P can recognize the PRP of Hib. D-Ribose-5-phosphate was reacted with proteins in the presence of sodium cyanoborohydride to obtain Rbt-5-P epitopes; 120 h reaction resulted in conjugation of ~30 and ~17 moles of Rbt-5-P/mole of BSA and OVA, respectively, based on decrease in amino groups, MALDI–TOF analyses, an increase in apparent molecular weight (SDS–PAGE) and glycoprotein staining. Immunization of rabbits with Rbt-5-P–BSA conjugate generated antibodies to Rbt-5-P as demonstrated by dot immunoblot and non-competitive ELISA. Homogeneous Rbt-5-P-specific antibody was purified from Rbt-5-P–BSA antiserum subjected to caprylic acid precipitation followed by hapten–affinity chromatography; its affinity constant is 7.1×108 M−1. Rbt-5- P antibody showed 100 % specificity to Rbt-5-P, ~230 %, 10 % and 3.4 % cross-reactivity to FMN, riboflavin and FAD, respectively; the antibody showed ~4 % cross-reactivity to Dribitol and <3 % to other sugars/sugar alcohols. Rbt-5-P– specific antibody recognized Hib conjugate vaccines containing PRP which was inhibited specifically by Rbt-5-P, and also detected Hib cell-surface capsular polysaccharides by immunofluorescence. In conclusion, Rbt-5-P–protein conjugate used as an immunogen elicited antibodies binding to an epitope also present in PRP and Hib bacteria. Rbt-5-P–specific antibody has potential applications in the detection and quantification of free/bound Rbt-5-P and FMN as well as immunological recognition of Hib bacteria and its capsular polysaccharide.

Item Type:	Article
Uncontrolled Keywords:	Capsular polysaccharide . Conjugate vaccine . Haemophilus influenzaetypeb .Immunogenicity .Polyribosyl ribitol phosphate . Ribitol-5-phosphate–specific antibodies
Subjects:	500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 11 Antibiotics Chemistry
Divisions:	Dept. of Biochemistry
Depositing User:	Food Sci. & Technol. Information Services
Date Deposited:	19 Jan 2012 04:58
Last Modified:	29 Nov 2016 10:47
URI:	http://ir.cftri.res.in/id/eprint/10515

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