4. Purify the reaction mixture through desalting against water on
a G-25 column and designate the derivatized OAg as “OAgADH.”
5. Characterize “OAg-ADH” 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) TNBS colorimetric method and free ADH RP-HPLC
analysis for % activation with ADH linker.
l
Calculate selective activation of the terminus KDO as
moles of linked ADH per mole of KDO %, indicating
the % of activated OAg chains.
l
Correct total NH 2 groups by subtracting the number
of NH 2 groups eventually already present in underivatized OAg and the number of free ADH detected
by RP-HPLC.
Scheme 2 Conjugation scheme. OAg is derivatized at the terminus KDO with ADH followed by SIDEA linkage
and conjugation to carrier protein
O-Antigen Purification and Conjugation
279
a G-25 column and designate the derivatized OAg as “OAgADH.”
5. Characterize “OAg-ADH” 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) TNBS colorimetric method and free ADH RP-HPLC
analysis for % activation with ADH linker.
l
Calculate selective activation of the terminus KDO as
moles of linked ADH per mole of KDO %, indicating
the % of activated OAg chains.
l
Correct total NH 2 groups by subtracting the number
of NH 2 groups eventually already present in underivatized OAg and the number of free ADH detected
by RP-HPLC.
Scheme 2 Conjugation scheme. OAg is derivatized at the terminus KDO with ADH followed by SIDEA linkage
and conjugation to carrier protein
O-Antigen Purification and Conjugation
279