4. For calculating the amount of water to be added, consider the
real μg of sugar weighed taking account of purity and the
eventual hydration water.
5. For a 2-mL scale, use 4-mL Wheaton glass vials, using either a
boiling water bath or an oven; for a 2-L scale, use a heatjacketed glass round-bottom flask connected to a condenser;
for a 30-L scale, perform the hydrolysis directly in the bioreactor, maintaining the temperature constant at 100 Æ 0.5
C
through the double jacket and the closed loop thermostat
system.
6. Purification process described for 1 L of “post-hydrolysis
supernatant.”
7. During concentration and diafiltration steps, keep retentate
volume constant, maintaining input pressure (Pin) at
1.8–2.0 bar and transmembrane pressure (TMP) at
1.1–1.2 bar. Membranes with a different molecular weight
cut-off could be used according to the OAg size.
8. The formed precipitate during this step can be discarded, containing protein and nucleic acid impurities.
9. Further purify “post-pH 3” by cation-exchange chromatography, through a Sartobind S MA75 filter, if the residual protein
content after the precipitation at pH 3 is >3% (w/w respect to
total sugar content). Before proceeding with filtration, equilibrate the Sartobind S cartridge with 20 mM citrate, pH 3.
Load the OAg sample and collect it in the flow through.
Indicate the solution as “post-Sartobind S.”
10. Or to “post-Sartobind S.”
11. The formed precipitate during this step containing nucleic
acids can be discarded.
12. “Post hydrolysis supernatant” and “post-EtOH/CaCl 2 ” samples are analyzed after desalting against water on a HiTrap
desalting column, 5 mL, prepacked with Sephadex G-25
Superfine (GE Healthcare), to avoid interference from the
matrix in the colorimetric methods.
13. This is specific for S. paratyphi A OAg, related to its OAg chain
and core sugar composition. The same method can be applied
to S. typhimurium and S. enteritidis OAg.
14. For S. paratyphi A OAg KDO quantification should be in good
agreement with GlcNAc quantification by HPAEC-PAD confirming the presence of one α-ketoacid per OAg chain.
15. Alternatively, Speedvac can be used or the product can be
lyophilized.
16. If necessary, the solubilization can be carried out at 30
C or by
mixing overnight.
O-Antigen Purification and Conjugation
301
real μg of sugar weighed taking account of purity and the
eventual hydration water.
5. For a 2-mL scale, use 4-mL Wheaton glass vials, using either a
boiling water bath or an oven; for a 2-L scale, use a heatjacketed glass round-bottom flask connected to a condenser;
for a 30-L scale, perform the hydrolysis directly in the bioreactor, maintaining the temperature constant at 100 Æ 0.5
C
through the double jacket and the closed loop thermostat
system.
6. Purification process described for 1 L of “post-hydrolysis
supernatant.”
7. During concentration and diafiltration steps, keep retentate
volume constant, maintaining input pressure (Pin) at
1.8–2.0 bar and transmembrane pressure (TMP) at
1.1–1.2 bar. Membranes with a different molecular weight
cut-off could be used according to the OAg size.
8. The formed precipitate during this step can be discarded, containing protein and nucleic acid impurities.
9. Further purify “post-pH 3” by cation-exchange chromatography, through a Sartobind S MA75 filter, if the residual protein
content after the precipitation at pH 3 is >3% (w/w respect to
total sugar content). Before proceeding with filtration, equilibrate the Sartobind S cartridge with 20 mM citrate, pH 3.
Load the OAg sample and collect it in the flow through.
Indicate the solution as “post-Sartobind S.”
10. Or to “post-Sartobind S.”
11. The formed precipitate during this step containing nucleic
acids can be discarded.
12. “Post hydrolysis supernatant” and “post-EtOH/CaCl 2 ” samples are analyzed after desalting against water on a HiTrap
desalting column, 5 mL, prepacked with Sephadex G-25
Superfine (GE Healthcare), to avoid interference from the
matrix in the colorimetric methods.
13. This is specific for S. paratyphi A OAg, related to its OAg chain
and core sugar composition. The same method can be applied
to S. typhimurium and S. enteritidis OAg.
14. For S. paratyphi A OAg KDO quantification should be in good
agreement with GlcNAc quantification by HPAEC-PAD confirming the presence of one α-ketoacid per OAg chain.
15. Alternatively, Speedvac can be used or the product can be
lyophilized.
16. If necessary, the solubilization can be carried out at 30
C or by
mixing overnight.
O-Antigen Purification and Conjugation
301