title: Recognition of flavin mononucleotide, Haemophilus influenzae type b and its capsular polysaccharide vaccines by antibodies specific to D-ribitol-5-phosphate.
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 × 10(8) 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 D-ribitol 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.
authors.affiliation: Department of Biochemistry and Nutrition, CSIR-Central Food Technological Research Institute (CFTRI), 'Chaluvamba Vilas', KRS Road, Mysore, 570020, Karnataka State, India.
authors.forename: G
authors.initials: G
authors.lastname: Ravi
authors.forename: Yeldur P
authors.initials: YP
authors.lastname: Venkatesh
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D016667:Bacterial Capsules / Q000276:immunology*; D002846:Chromatography, Affinity; D003429:Cross Reactions / Q000276:immunology; D005486:Flavin Mononucleotide / Q000276:immunology*; D005182:Flavin-Adenine Dinucleotide / Q000276:immunology; D018073:Haemophilus Vaccines / Q000276:immunology*; D019741:Haemophilus influenzae type b / Q000276:immunology*; D006241:Haptens / Q000276:immunology; D007106:Immune Sera; D007150:Immunohistochemistry; D008297:Male; D010428:Pentosephosphates / Q000276:immunology*; D011135:Polysaccharides, Bacterial / Q000276:immunology*; D011817:Rabbits; D012255:Ribitol / Q000276:immunology; D012256:Riboflavin / Q000276:immunology; D012267:Ribosemonophosphates / Q000737:chemistry / Q000378:metabolism
chemical_list: D000907:Antibodies, Bacterial; D018073:Haemophilus Vaccines; C044237:Haemophilus influenzae type b polysaccharide vaccine; D006241:Haptens; D007106:Immune Sera; D010428:Pentosephosphates; D011135:Polysaccharides, Bacterial; D012267:Ribosemonophosphates; D005182:Flavin-Adenine Dinucleotide; C041025:ribitol-5-phosphate; D012255:Ribitol; C031626:ribose-5-phosphate; D005486:Flavin Mononucleotide; D012256:Riboflavin
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