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

Ravi, G. (2015) Antibodies to ribitol and its phosphate: application in immunoassays for riboflavin and its coenzymes. PhD thesis, University of Mysore.

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Abstract

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 <1% cross reactivity with other water-soluble vitamins. Food samples were subjected to acid hydrolysis to obtain total riboflavin (TRF), and with methanol: methylene chloride (9:10, v/v) to obtain non-degraded individual flavins, and were used as the source of competing analyte in the immunoassay. Quantification of individual flavins (riboflavin and FMN) by icELISAs showed variation in their values compared to TRF, and were in good agreement with the HPLC and AOAC spectrofluorimetric methods. Further, spiking and recovery analysis a a vi of food sample using inter and intra-assays showed no significant matrix effects with good recovery, sensitivity, accuracy and precision. The present study also describes the use of antibodies to ribitol and ribitol-5-phosphate for the immunological detection of Haemophilus influenzae type b (Hib) bacteria by indirect immunofluorescence and its polyribosyl-ribitol-phosphate (PRP)-containing glycoconjugate vaccines (Hiberix™, Sii HibPro™ and Pentavac SD® containing PRP conjugated to tetanus toxoid) using icELISA.

Item Type: Thesis (PhD)
Uncontrolled Keywords: D-Ribitol, D-ribitol-5-phosphate, vitamin B2, riboflavin, antibodies
Subjects: 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 31 Sugar Chemistry
500 Natural Sciences and Mathematics > 07 Life Sciences > 03 Biochemistry & Molecular Biology > 17 Vitamin Biochemistry
Divisions: Dept. of Biochemistry
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 19 May 2016 06:59
Last Modified: 19 May 2016 06:59
URI: http://ir.cftri.com/id/eprint/12175

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