Comparison and Optimization of Quantification Methods for Shigella flexneri Serotype 6 O-antigen Containing Galacturonic Acid and Methyl-Pentose |
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Authors: | Maria Michelina Raso Oscar Vassallo Francesca Micoli Carlo Giannelli |
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Affiliation: | 1.GSK Vaccines Institute for Global Health (GVGH) S.r.l., Via Fiorentina 1, 53100 Siena, Italy; (M.M.R.); (O.V.); (F.M.);2.Merck, Via Einaudi 11, 00012 Guidonia, Italy |
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Abstract: | Shigella is a leading diarrheal cause of morbidity and mortality worldwide, especially in low- and middle-income countries and in children under five years of age. Increasing levels of antimicrobial resistance make vaccine development an even higher global health priority. S. flexneri serotype 6 is one of the targets of many multicomponent vaccines in development to ensure broad protection against Shigella. The O-antigen (OAg) is a key active ingredient and its content is a critical quality attribute for vaccine release in order to monitor their stability and to ensure appropriate immune response. Here, the optimization of two methods to quantify S. flexneri 6 OAg is reported together with the characterization of their performances. The optimized Dische colorimetric method allows a tenfold increment of the sensitivity with respect to the original method and is useful for fast analysis detecting selectively methyl-pentoses, as rhamnose in S. flexneri 6 OAg. Also, a more specific HPAEC-PAD method was developed, detecting the dimer galacturonic acid-galactosamine (GalA-GalN) coming from S. flexneri 6 OAg acid hydrolysis. These methods will facilitate characterization of S. flexneri 6 OAg based vaccines. The colorimetric method can be used for quantification of other polysaccharide containing methyl-pentoses, and the HPAEC-PAD could be extended to other polysaccharides containing uronic acids. |
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Keywords: | S. flexneri O-antigen HPAEC-PAD Dische colorimetric method quantification polysaccharide vaccine design of experiment galacturonic acid methyl-pentose |
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