Record Details

Antibodies to ribitol and its phosphate: application in immunoassays for riboflavin and its coenzymes

IR@CSIR-CFTRI

View Archive Info
 
 
Field Value
 
Relation http://ir.cftri.com/12175/
 
Title Antibodies to ribitol and its phosphate: application in immunoassays for riboflavin and its coenzymes
 
Creator Ravi, G.
 
Subject 31 Sugar Chemistry
17 Vitamin Biochemistry
 
Description D-Ribitol and D-ribitol-5-phosphate are non-immunogenic, five-carbon sugar molecules; they are important metabolites in the pentose phosphate pathway and are an integral part of bacterial cell wall polysaccharides, and also constituent components of water-soluble vitamin B2 (riboflavin), and its coenzyme flavin mononucleotide (FMN; riboflavin-5-phosphate), respectively. The main focus of the present study was to generate antibodies to ribitol and ribitol-5-phosphate by utilizing hapten-carrier conjugates containing respective epitopes and to utilize these rabbit IgG antibodies to develop immunoassays to detect and quantitate riboflavin and FMN, respectively, in some selected foods.
Antibodies specific to D-ribitol was generated using reductively aminated D-ribose-BSA conjugate (~30 moles of ribitol/mole of BSA) as the immunogen in rabbits. Caprylic acid precipitation of immune serum to remove serum albumin followed by hapten affinity chromatography on ribitol–KLH–Sepharose CL-6B resulted in pure ribitol–specific antibodies (~45–50 μg/mL) with an affinity constant of 2.9 × 107 M−1 and showing 100% specificity towards ribitol, ~800% cross–reactivity towards riboflavin, and low cross-reactivity with various sugars, sugar alcohols and riboflavin derivatives in indirect-competitive ELISA (icELISA).
a
a v
Similarly, Antibodies specific to D-ribitol-5-phosphate were generated using reductively aminated D-ribose-5-phosphate-BSA conjugate (~32 moles of ribitol-5-phosphate/mole of BSA) as the immunogen in rabbits. Antibodies specific to ribitol-5-phosphate were purified by caprylic acid precipitation of immune serum to remove serum albumin followed by hapten affinity chromatography on ribitol-5-phosphate–KLH–Sepharose CL-6B (yield: ~60–65 μg/mL) with an affinity constant of 7.1 × 108 M−1 and showing 100% specificity towards ribitol-5-phosphate, ~230% cross–reactivity towards FMN, and low cross-reactivity with various sugars, sugar alcohols, riboflavin and FAD in icELISA.
For the development of ELISAs for quantifying riboflavin and FMN in foods, inhibition curves were prepared using icELISA by competition between affinity-purified hapten-specific anti-ribitol and anti-ribitol-5-phosphate antibodies with riboflavin (LOD = 0.02 ng/mL) and FMN (LOD = 0.24 ng/mL), respectively, which showed linear range between 0.1 and 100 ng of competitors and
 
Contributor Venkatesh, Y. P.
 
Date 2015
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Language en
 
Rights —
 
Identifier http://ir.cftri.com/12175/1/Ravi.pdf
Ravi, G. (2015) Antibodies to ribitol and its phosphate: application in immunoassays for riboflavin and its coenzymes. PhD thesis, University of Mysore.