l
Read each tube UV ABS, using as blank for the spectrophotometer the 0 μg/mL standard.
l
Calculate a linear regression between concentration
and ABS for the standards and calculate on it the total
sugar concentration of the samples.
2. HPAEC-PAD for neutral sugars quantification: Rha, Gal, Glc
and Man, sugars of the OAg chain repeating unit, and GlcNAc,
present as a unique sugar in the core region, are estimated by
HPAEC-PAD after acid hydrolysis of unconjugated or conjugated OAg samples to release the monosaccharides. Commercial monomer sugars are used for building the calibration
curves (see Note 22).
Paratose (Par), the other monosaccharide present in
S. paratyphi A OAg chain, could not be determined by this
method as no commercially available standard exists, therefore
the presence and amount of Par is determined by
1
H NMR.
(a) Calibration curve standard solutions.
l
In 2 mL screw cap vials, prepare in duplicate the dilutions
starting from the 11.25 μg/mL neutral sugar standard mix,
as indicated in Table 2:
(b) Sample preparation.
l
Dilute the sample with water to a suitable concentration to
fit the calibration curve.
l
Prepare three (in triplicate) 2 mL screw cap vials containing
450 μL of the diluted sample.
(c) Sample/standard treatment.
Table 3
Dilutions for preparing the GlcNAc calibration curve for HPAEC-PAD analysis
μg/mL (GlcNAc)
11.25 μg/mL
standards solution (μL)
Water (μL)
0
0
450
0.5
20
430
1.0
40
410
2.5
100
350
5.0
200
250
7.5
300
150
10
400
50
284
Francesca Micoli et al.
Read each tube UV ABS, using as blank for the spectrophotometer the 0 μg/mL standard.
l
Calculate a linear regression between concentration
and ABS for the standards and calculate on it the total
sugar concentration of the samples.
2. HPAEC-PAD for neutral sugars quantification: Rha, Gal, Glc
and Man, sugars of the OAg chain repeating unit, and GlcNAc,
present as a unique sugar in the core region, are estimated by
HPAEC-PAD after acid hydrolysis of unconjugated or conjugated OAg samples to release the monosaccharides. Commercial monomer sugars are used for building the calibration
curves (see Note 22).
Paratose (Par), the other monosaccharide present in
S. paratyphi A OAg chain, could not be determined by this
method as no commercially available standard exists, therefore
the presence and amount of Par is determined by
1
H NMR.
(a) Calibration curve standard solutions.
l
In 2 mL screw cap vials, prepare in duplicate the dilutions
starting from the 11.25 μg/mL neutral sugar standard mix,
as indicated in Table 2:
(b) Sample preparation.
l
Dilute the sample with water to a suitable concentration to
fit the calibration curve.
l
Prepare three (in triplicate) 2 mL screw cap vials containing
450 μL of the diluted sample.
(c) Sample/standard treatment.
Table 3
Dilutions for preparing the GlcNAc calibration curve for HPAEC-PAD analysis
μg/mL (GlcNAc)
11.25 μg/mL
standards solution (μL)
Water (μL)
0
0
450
0.5
20
430
1.0
40
410
2.5
100
350
5.0
200
250
7.5
300
150
10
400
50
284
Francesca Micoli et al.