Tsk gel 3000 pwxl column

Vi, freeze dried as the sodium salt, was solubilized in water and H₂O₂ was added to give a final concentration of 2.5 mg/mL Vi and 5% (wt/v) H₂O₂ in water. The mixture was heated at 80±0.5°C for 2h. The mixture was then injected into a Hiscreen Capto Q [GE Healthcare] column (4.7 mL of resin loading up to 100 mg of fragmented Vi mixture) equilibrated with buffer A and populations of different average size were separated using a gradient step method. NaH₂PO₄ 20 mM pH 7.2 and NaH₂PO₄ 20 mM NaCl 1M pH 7.2 were used as buffer A and B respectively. Pools at average size Vi of 8.6 and 43 kDa were eluted at 25 and 37% of buffer B respectively. Each pool was desalted on a Sephadex G-25 column [GE Healthcare] equilibrated with water. The average size of the fragmented Vi pools was determined by HPLC-SEC equipped with a TSK gel 3000 PW_XL column and a TSK gel PW_XL guard column (Tosoh Bioscience). Dextrans (5, 25, 50, 80, 150 kDa) were used as standards (Sigma Aldrich). The mobile phase was 0.1 M NaCl, 0.1 M NaH₂PO₄, 5% CH₃CN, pH 7.2, at the flow rate of 0.5 mL/min (isocratic method for 30 min). HPAEC-PAD was used to measure Vi content [10 (link), 21 (link)]. ¹H NMR was used to verify Vi identity and confirm O-acetylation levels were >60% [10 (link), 21 (link)].

S. flexneri 2a and 1b GMMA were produced and purified as previously described [31 (link)]. GMMA total protein content was estimated by bicinchoninic acid assay (BCA) using BSA as a reference. The total OAg amount and sugar composition were determined by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), after performing acid hydrolysis directly on GMMA. In particular, the OAg amount was quantified based on the detection of rhamnose, as previously described [33 (link),34 (link)]. The OAg extracted was characterized by high performance liquid chromatography–size exclusion chromatography (HPLC-SEC) (TSK gel 3000 PWXL column with TSK gel PWXL guard column equilibrated in 0.1 NaCl, 0.1 NaH₂PO₄, 5% CH₃CN, Tosoh Bioscience, Tokyo, Japan) with differential refractive index (dRI) detection, using dextrans as the standards to estimate the molecular size distribution as previously described [31 (link),33 (link)]. OAg structures were confirmed by ¹H-NMR analysis measured with a Bruker AvanceIII 400 spectrometer at 400 MHz and 323 K.

S. sonnei, S. flexneri 2a, and S. Typhimurium GMMA were produced and purified as previously described [6 (link)]. The total protein content was estimated by bicinchoninic acid assay (BCA) using BSA as a reference. The total OAg amount and sugar composition were determined by HPAEC–PAD analysis, after performing acid hydrolysis directly on GMMA. In particular, the OAg amount was quantified based on the detection of repeating unit-specific monosaccharides (2-aminouronic acid for S. sonnei; rhamnose for S. flexneri 2a and S. Typhimurium), as previously described [34 (link),35 (link),36 (link)]. Purity and particle sizes were established by HPLC–SEC MALS, using TSK gel G6000PW + G4000PW columns (Tosoh Bioscience, Tokio, Japan) in series equilibrated in Phosphate Buffer Saline (PBS) [37 (link)]. The OAg extracted was characterized by HPLC–SEC (TSK gel 3000 PWXL column with TSK gel PWXL guard column equilibrated in 0.1 NaCl, 0.1 NaH₂PO₄, 5% CH₃CN, Tosoh Bioscience, Tokio, Japan) with differential refractive index (dRI) detection using dextrans as the standards to estimate the molecular size distribution, as previously described [34 (link),35 (link)]. The ability of each GMMA to stimulate IL-6 release from human PBMC was evaluated by MAT [38 (link)].