6. Dry the “OAg-ADH” through rotating evaporator (see Note
15).
7. Solubilize dried OAg-ADH in water/DMSO 1:9 (v/v) at a
concentration of 50 mg/mL. When the polysaccharide is
completely solubilized, add triethylamine (TEA) (molar ratio
TEA/total NH2 groups ¼ 5) and then SIDEA (molar ratio
SIDEA/total NH2 groups ¼ 12) (see Note 17). Perform this
operation within a chemical hood.
8. Mix the solution at RT for 3 h.
9. Purify the reaction mixture through precipitation with
100 mM citrate buffer pH 3 and EtOH.
(a) Add to the reaction mixture 2Â reaction mixture volume
of 100 mM citrate pH 3 and mix at 4
C for 30 min.
Under these conditions, unreacted SIDEA precipitates
and is discarded after centrifugation (12,000 RCF at
4
C for 30 min).
(b) Add to the supernatant absolute EtOH to have 80%
EtOH final concentration (v/v). The derivatized OAg
precipitates and is recovered after centrifugation (12,000
RCF at 4
C for 30 min).
(c) Wash the pellet after centrifugation twice with 1.5
volumes of 100% EtOH with respect to the initial reaction
mixture volume.
11. Lyophilize the pellet and indicate it as “OAg-ADH-SIDEA.”
12. Characterize “OAg-ADH-SIDEA” (see Note 18) by:
(a) Phenol sulfuric acid assay for quantifying total sugar content and HPAEC-PAD analysis for sugar composition and
content.
(b) HPLC-SEC for molecular size distribution.
(c) Total active esters group quantification by A260,
RP-HPLC for free SIDEA quantification.
l
Calculate % of derivatization with SIDEA as molar
ratio of linked active ester groups (subtracting the
moles of free active ester groups quantified by
RP-HPLC) to total NH 2 groups before derivatization
measured by TNBS. The ratio indicates the % in moles
of NH 2 groups activated with this reaction.
3.2.2 Conjugation
to CRM 197 and Conjugate
Purification
1. Solubilize “OAg-ADH-SIDEA” in phosphate buffer pH 7.2
and add CRM197 to give a protein concentration of 20 mg/
mL and a molar ratio of active ester groups to CRM197 of
30 to 1.
2. Mix the reaction at RT for 2 h.
280
Francesca Micoli et al.
15).
7. Solubilize dried OAg-ADH in water/DMSO 1:9 (v/v) at a
concentration of 50 mg/mL. When the polysaccharide is
completely solubilized, add triethylamine (TEA) (molar ratio
TEA/total NH2 groups ¼ 5) and then SIDEA (molar ratio
SIDEA/total NH2 groups ¼ 12) (see Note 17). Perform this
operation within a chemical hood.
8. Mix the solution at RT for 3 h.
9. Purify the reaction mixture through precipitation with
100 mM citrate buffer pH 3 and EtOH.
(a) Add to the reaction mixture 2Â reaction mixture volume
of 100 mM citrate pH 3 and mix at 4
C for 30 min.
Under these conditions, unreacted SIDEA precipitates
and is discarded after centrifugation (12,000 RCF at
4
C for 30 min).
(b) Add to the supernatant absolute EtOH to have 80%
EtOH final concentration (v/v). The derivatized OAg
precipitates and is recovered after centrifugation (12,000
RCF at 4
C for 30 min).
(c) Wash the pellet after centrifugation twice with 1.5
volumes of 100% EtOH with respect to the initial reaction
mixture volume.
11. Lyophilize the pellet and indicate it as “OAg-ADH-SIDEA.”
12. Characterize “OAg-ADH-SIDEA” (see Note 18) by:
(a) Phenol sulfuric acid assay for quantifying total sugar content and HPAEC-PAD analysis for sugar composition and
content.
(b) HPLC-SEC for molecular size distribution.
(c) Total active esters group quantification by A260,
RP-HPLC for free SIDEA quantification.
l
Calculate % of derivatization with SIDEA as molar
ratio of linked active ester groups (subtracting the
moles of free active ester groups quantified by
RP-HPLC) to total NH 2 groups before derivatization
measured by TNBS. The ratio indicates the % in moles
of NH 2 groups activated with this reaction.
3.2.2 Conjugation
to CRM 197 and Conjugate
Purification
1. Solubilize “OAg-ADH-SIDEA” in phosphate buffer pH 7.2
and add CRM197 to give a protein concentration of 20 mg/
mL and a molar ratio of active ester groups to CRM197 of
30 to 1.
2. Mix the reaction at RT for 2 h.
280
Francesca Micoli et al.