4 Notes
1. Dicarboxylic acid anhydrides can also be utilized in place of
disuccinimidyl esters. In this case, an additional step to transform the resulting carboxylic acid derivatives into activated
esters is necessary before the conjugation to carrier proteins.
2. The monosaccharide samples are used to prepare standard
stock solutions for obtaining the calibration curve in the analysis of carbohydrate loading of the conjugate. The monosaccharides used should be the same as those in the carbohydrate
antigen and in the same molar ratio equivalent. For example,
the Globo-H antigen is composed of one Fuc unit, three Gal
units, one GalNAc unit, and one Glc unit; thus the monosaccharide mixture should be made of Fuc, Gal, GalNAc, and Glc
in a molar ratio of 1:3:1:1 [15, 20].
3. Commercial zinc powder should be activated before use by
treatment with diluted HCl aq. solution (0.1 N), which is
followed by thorough washing with distilled water until the
washing becomes neutral.
4. Other tertiary amines, such as trimethyl and triethyl amines,
can be used to neutralize the reaction. However, the use of
primary and secondary amines as bases should be avoided
because these compounds can interfere with the acylation
reaction [7].
5. DMF is added as a cosolvent to help dissolve disuccinimidyl
dicarboxylic acid ester as it is not soluble in water or PBS.
6. N-Hydroxysuccinimide esters can be hydrolyzed slowly in
water. Thus, they should be stored as dry solids before being
added to reaction solutions and the activated intermediates
should be applied to the next step as soon as possible.
7. The product should be washed with ethyl acetate thoroughly to
remove any excessive dicarboxylic acid disuccinimidyl ester
because its presence can affect subsequent antigen conjugation
with carrier protein.
8. The antigen–protein conjugate elutes much earlier from the
size-exclusion column than the unconjugated oligosaccharide
antigen because of the large difference between their molecular
sizes.
9. A spectrophotometer in combination with a Pierce BCA protein assay kit can be used for the detection of proteins.
10. Staining of carbohydrates with 15% (v/v) H 2 SO 4 in EtOH:
Apply the eluted fractions onto a silica gel TLC plate, and then
dip the plate in the 15% (v/v) H 2 SO 4 in EtOH solution. Wipe
the back of the plate clean and char it on a hot plate until the
310
Brittany R. Smith and Zhongwu Guo
1. Dicarboxylic acid anhydrides can also be utilized in place of
disuccinimidyl esters. In this case, an additional step to transform the resulting carboxylic acid derivatives into activated
esters is necessary before the conjugation to carrier proteins.
2. The monosaccharide samples are used to prepare standard
stock solutions for obtaining the calibration curve in the analysis of carbohydrate loading of the conjugate. The monosaccharides used should be the same as those in the carbohydrate
antigen and in the same molar ratio equivalent. For example,
the Globo-H antigen is composed of one Fuc unit, three Gal
units, one GalNAc unit, and one Glc unit; thus the monosaccharide mixture should be made of Fuc, Gal, GalNAc, and Glc
in a molar ratio of 1:3:1:1 [15, 20].
3. Commercial zinc powder should be activated before use by
treatment with diluted HCl aq. solution (0.1 N), which is
followed by thorough washing with distilled water until the
washing becomes neutral.
4. Other tertiary amines, such as trimethyl and triethyl amines,
can be used to neutralize the reaction. However, the use of
primary and secondary amines as bases should be avoided
because these compounds can interfere with the acylation
reaction [7].
5. DMF is added as a cosolvent to help dissolve disuccinimidyl
dicarboxylic acid ester as it is not soluble in water or PBS.
6. N-Hydroxysuccinimide esters can be hydrolyzed slowly in
water. Thus, they should be stored as dry solids before being
added to reaction solutions and the activated intermediates
should be applied to the next step as soon as possible.
7. The product should be washed with ethyl acetate thoroughly to
remove any excessive dicarboxylic acid disuccinimidyl ester
because its presence can affect subsequent antigen conjugation
with carrier protein.
8. The antigen–protein conjugate elutes much earlier from the
size-exclusion column than the unconjugated oligosaccharide
antigen because of the large difference between their molecular
sizes.
9. A spectrophotometer in combination with a Pierce BCA protein assay kit can be used for the detection of proteins.
10. Staining of carbohydrates with 15% (v/v) H 2 SO 4 in EtOH:
Apply the eluted fractions onto a silica gel TLC plate, and then
dip the plate in the 15% (v/v) H 2 SO 4 in EtOH solution. Wipe
the back of the plate clean and char it on a hot plate until the
310
Brittany R. Smith and Zhongwu Guo