l
Column compartment: 30
C.
l
Autosampler compartment: 4
C.
l
Fluorimeter detector: excitation wavelength set at
345 nm; emission wavelength acquired at 480 nm
(photomultiplier gain level set at 1) (see Note 26).
l
Chromatographic conditions:
– Separation performed in isocratic condition: eluent 65% ACN and 35% water (mixture generated
by HPLC pump); after analyte peak elution, column cleaning step with 95% ACN and 5% water.
Detailed elution conditions are reported in
Table 7.
– Flow rate: 1 mL/min.
– Run time: 50 min.
– Sample injection volume: 100 μL.
(d) Calculate percentage of free NH 2 groups as molar ratio %
of free NH 2 groups (see Note 27) divided by total NH 2
groups introduced after derivatization with ADH (quantified by TNBS).
9. Total active ester group quantification by A260 [22].
(a) Solubilize OAg-ADH-SIDEA sample in water to a sugar
concentration of 5 mg/mL.
(b) Immediately after solubilization, add 250 μL of this solution to 500 μL of water and measure A260 (as blank).
(c) Add 250 μL of the OAg solution to 500 μL 0.1 M
NH4OH to release the N-hydroxy succinimide groups
and measure the absorption of the N-hydroxy succinimidate anion at 260 nm.
(d) Build the calibration curve with 20–200 nmol/mL NHydroxy succinimide.
(e) Percentage of derivatization with SIDEA is calculated as %
molar ratio of linked active ester groups (subtracting the
moles of free active ester groups quantified by RP-HPLC)
to total NH2 groups before derivatization measured by
TNBS. The ratio indicates the % in moles of NH2 groups
activated as esters.
10. Free SIDEA quantification by RP-HPLC.
(a) SIDEA Calibration curve.
l
In prelabeled Eppendorf tubes, prepare the dilutions
of the SIDEA 15 μM standard solution with Water/
ACN 50% and mix them by vortexing (Table 8).
O-Antigen Purification and Conjugation
295
Column compartment: 30
C.
l
Autosampler compartment: 4
C.
l
Fluorimeter detector: excitation wavelength set at
345 nm; emission wavelength acquired at 480 nm
(photomultiplier gain level set at 1) (see Note 26).
l
Chromatographic conditions:
– Separation performed in isocratic condition: eluent 65% ACN and 35% water (mixture generated
by HPLC pump); after analyte peak elution, column cleaning step with 95% ACN and 5% water.
Detailed elution conditions are reported in
Table 7.
– Flow rate: 1 mL/min.
– Run time: 50 min.
– Sample injection volume: 100 μL.
(d) Calculate percentage of free NH 2 groups as molar ratio %
of free NH 2 groups (see Note 27) divided by total NH 2
groups introduced after derivatization with ADH (quantified by TNBS).
9. Total active ester group quantification by A260 [22].
(a) Solubilize OAg-ADH-SIDEA sample in water to a sugar
concentration of 5 mg/mL.
(b) Immediately after solubilization, add 250 μL of this solution to 500 μL of water and measure A260 (as blank).
(c) Add 250 μL of the OAg solution to 500 μL 0.1 M
NH4OH to release the N-hydroxy succinimide groups
and measure the absorption of the N-hydroxy succinimidate anion at 260 nm.
(d) Build the calibration curve with 20–200 nmol/mL NHydroxy succinimide.
(e) Percentage of derivatization with SIDEA is calculated as %
molar ratio of linked active ester groups (subtracting the
moles of free active ester groups quantified by RP-HPLC)
to total NH2 groups before derivatization measured by
TNBS. The ratio indicates the % in moles of NH2 groups
activated as esters.
10. Free SIDEA quantification by RP-HPLC.
(a) SIDEA Calibration curve.
l
In prelabeled Eppendorf tubes, prepare the dilutions
of the SIDEA 15 μM standard solution with Water/
ACN 50% and mix them by vortexing (Table 8).
O-Antigen Purification and Conjugation
295