17. In some cases, the total NH 2 groups also include phosphoethanolamine groups on the OAg.
18. Weigh a known amount of lyophilized OAg-ADH-SIDEA,
solubilize it in water and carry out all the characterization
using this solution.
19. Alternatively, other purification methods can be used (e.g.,
TFF, hydrophobic interaction chromatography [HIC]) [24].
20. Alternative methods can be used for free OAg quantification,
for example, solid phase extraction (SPE) using a C4 cartridge
(Vydac Bioselect) followed by HPAEC-PAD for quantification.
21. This step is critical for the color development. The sulfuric acid
must be added quickly (automatic pipette with high drop
speed) keeping the tip end about 2–3 cm above the sample
solution. Moreover, the dropped volume must reach the sample/standard solution without coming into contact with the
glass tube. In this way the sulfuric acid hydration is fast and
highly exothermic and generates the heat necessary to drive the
reaction. The color development fails if part of sulfuric acid
mixed in the tube forms a two-phase system: In this case, the
sample/standard tube must be discarded.
22. Note that this method can be applied on OAg containing these
monosaccharides, such as S. paratyphi A, S. typhimurium, and
S. enteritidis OAg. For any other polysaccharide, appropriate
calibration curves and conditions of hydrolysis need to be
identified and optimized.
23. Use an electronic positive displacement pipette as the acid can
ruin a Gilson pipette.
24. Perform system equilibration with elution buffer before starting sample analysis. This equilibration step lasts 70 min if the
column system must be equilibrated with a different eluent;
60 min or more are also needed to warm up the PDA lamps
before the analysis. This step can be skipped if the system
(HPLC and columns) is already equilibrated with the buffer
and lamps are on. After the last chromatographic analysis, store
the column in 0.02% NaN 3 .
25. For example, for S. paratyphi A (or S. typhimurium and S.
enteritidis) OAg, the H-6 of Rha or Paratose (or Abequose
and Tyvelose, respectively) can be used for comparison with
the acetate ester signal.
26. Fluorescence detector is set with excitation wavelength at
345 nm and emission wavelength at 480 nm to only see the
emission of the diderivatized PBA-ADH-PBA and not the
reactant PBA.
27. Amino group concentration is twice the ADH concentration.
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Francesca Micoli et al.
18. Weigh a known amount of lyophilized OAg-ADH-SIDEA,
solubilize it in water and carry out all the characterization
using this solution.
19. Alternatively, other purification methods can be used (e.g.,
TFF, hydrophobic interaction chromatography [HIC]) [24].
20. Alternative methods can be used for free OAg quantification,
for example, solid phase extraction (SPE) using a C4 cartridge
(Vydac Bioselect) followed by HPAEC-PAD for quantification.
21. This step is critical for the color development. The sulfuric acid
must be added quickly (automatic pipette with high drop
speed) keeping the tip end about 2–3 cm above the sample
solution. Moreover, the dropped volume must reach the sample/standard solution without coming into contact with the
glass tube. In this way the sulfuric acid hydration is fast and
highly exothermic and generates the heat necessary to drive the
reaction. The color development fails if part of sulfuric acid
mixed in the tube forms a two-phase system: In this case, the
sample/standard tube must be discarded.
22. Note that this method can be applied on OAg containing these
monosaccharides, such as S. paratyphi A, S. typhimurium, and
S. enteritidis OAg. For any other polysaccharide, appropriate
calibration curves and conditions of hydrolysis need to be
identified and optimized.
23. Use an electronic positive displacement pipette as the acid can
ruin a Gilson pipette.
24. Perform system equilibration with elution buffer before starting sample analysis. This equilibration step lasts 70 min if the
column system must be equilibrated with a different eluent;
60 min or more are also needed to warm up the PDA lamps
before the analysis. This step can be skipped if the system
(HPLC and columns) is already equilibrated with the buffer
and lamps are on. After the last chromatographic analysis, store
the column in 0.02% NaN 3 .
25. For example, for S. paratyphi A (or S. typhimurium and S.
enteritidis) OAg, the H-6 of Rha or Paratose (or Abequose
and Tyvelose, respectively) can be used for comparison with
the acetate ester signal.
26. Fluorescence detector is set with excitation wavelength at
345 nm and emission wavelength at 480 nm to only see the
emission of the diderivatized PBA-ADH-PBA and not the
reactant PBA.
27. Amino group concentration is twice the ADH concentration.
302
Francesca Micoli et al.