Comparison and Optimization of Quantification Methods for Shigella flexneri Serotype 6 O-antigen Containing Galacturonic Acid and Methyl-Pentose

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.

Regulation on Pathway Metabolic Fluxes to Enhance Colanic Acid Production in Escherichia coli

“…The quantification of CA in broth was based on a modified Dische colorimetric method used to quantify CA indirectly by detecting Lfucose. 19 Briefly, 100 μL of aqueous samples was slowly mixed with 450 μL of 87% sulfuric acid and heated for 15 min. After the mixture was cooled to room temperature, the absorbance at 396 and 427 nm was measured to give A 1 .…”