6. Filter the reaction mixture through a Celite pad and wash the
pad with water.
7. Combine the filtrates and concentrate them in vacuo.
8. Use the resultant crude amine product in the next reaction
without further purification.
3.2 Coupling
of Amino Derivative
of Oligosaccharide
Antigen
with Disuccinimidyl
Ester of Dicarboxylic
Acid Linker
1. Dissolve the above-obtained amino derivative of oligosaccharide antigen (50 μmol) in a 4:1 mixture of N,N-dimethylformamide (DMF) and 0.1 M PBS (0.5 mL) (see Note 5).
2. Add the disuccinimidyl ester of a dicarboxylic acid, such as
disuccinimidyl glutarate (DSG) (245 mg, 750 μmol) (see
Note 6).
3. Stir the reaction mixture at ambient temperature for 4 h.
4. Remove solvents in vacuo.
5. Wash the resultant mono-activated ester with ethyl acetate ten
times (10 Â 1.0 mL) (see Note 7).
6. Dry the solid product under high vacuum.
7. Use the mono-activated ester in the next step without further
purification (see Note 6).
3.3 Conjugation
of Mono-activated
Ester
of Oligosaccharide
Antigen to Carrier
Protein
1. Dissolve the carrier protein (33 μmol) and the above-obtained
mono-activated ester of antigen (50 μmol) in 0.1 M PBS
(0.5 mL) (see Note 6).
2. Stir the solution at ambient temperature for 3 days.
3. Apply the solution to a Bio-gel A 0.5 column (1 Â 15 cm) and
elute the column with 0.1 M PBS to separate the conjugate
from the unconjugated oligosaccharide (see Note 8).
4. Combine fractions that contain both the protein indicated by
the BCA assay (see Note 9) and the carbohydrate indicated by
staining with 15% (v/v) H 2 SO 4 in EtOH (see Note 10).
5. Dialyze the combined fractions against distilled water for
2 days.
6. Lyophilize the solution to afford the conjugate as a white solid.
3.4 Analysis
of Carbohydrate
Loading
of the Resultant
Glycoconjugate
3.4.1 Colorimetric
Method
Preparation of calibration curve
1. Mix stoichiometric amounts of monosaccharides contained in
the oligosaccharide antigen (see Note 2).
2. Use this mixture to prepare a standard stock solution (1.0 mg/
mL) in distilled water.
3. Transfer the standard stock solution to ten different test tubes
in 5.0 μL increments, ranging from 5.0 to 50.0 μL.
4. Add 500 μL of 4% phenol (aq. solution) and 2.5 mL of 96%
H 2 SO 4 in each test tube (see Note 11).
308
Brittany R. Smith and Zhongwu Guo
pad with water.
7. Combine the filtrates and concentrate them in vacuo.
8. Use the resultant crude amine product in the next reaction
without further purification.
3.2 Coupling
of Amino Derivative
of Oligosaccharide
Antigen
with Disuccinimidyl
Ester of Dicarboxylic
Acid Linker
1. Dissolve the above-obtained amino derivative of oligosaccharide antigen (50 μmol) in a 4:1 mixture of N,N-dimethylformamide (DMF) and 0.1 M PBS (0.5 mL) (see Note 5).
2. Add the disuccinimidyl ester of a dicarboxylic acid, such as
disuccinimidyl glutarate (DSG) (245 mg, 750 μmol) (see
Note 6).
3. Stir the reaction mixture at ambient temperature for 4 h.
4. Remove solvents in vacuo.
5. Wash the resultant mono-activated ester with ethyl acetate ten
times (10 Â 1.0 mL) (see Note 7).
6. Dry the solid product under high vacuum.
7. Use the mono-activated ester in the next step without further
purification (see Note 6).
3.3 Conjugation
of Mono-activated
Ester
of Oligosaccharide
Antigen to Carrier
Protein
1. Dissolve the carrier protein (33 μmol) and the above-obtained
mono-activated ester of antigen (50 μmol) in 0.1 M PBS
(0.5 mL) (see Note 6).
2. Stir the solution at ambient temperature for 3 days.
3. Apply the solution to a Bio-gel A 0.5 column (1 Â 15 cm) and
elute the column with 0.1 M PBS to separate the conjugate
from the unconjugated oligosaccharide (see Note 8).
4. Combine fractions that contain both the protein indicated by
the BCA assay (see Note 9) and the carbohydrate indicated by
staining with 15% (v/v) H 2 SO 4 in EtOH (see Note 10).
5. Dialyze the combined fractions against distilled water for
2 days.
6. Lyophilize the solution to afford the conjugate as a white solid.
3.4 Analysis
of Carbohydrate
Loading
of the Resultant
Glycoconjugate
3.4.1 Colorimetric
Method
Preparation of calibration curve
1. Mix stoichiometric amounts of monosaccharides contained in
the oligosaccharide antigen (see Note 2).
2. Use this mixture to prepare a standard stock solution (1.0 mg/
mL) in distilled water.
3. Transfer the standard stock solution to ten different test tubes
in 5.0 μL increments, ranging from 5.0 to 50.0 μL.
4. Add 500 μL of 4% phenol (aq. solution) and 2.5 mL of 96%
H 2 SO 4 in each test tube (see Note 11).
308
Brittany R. Smith and Zhongwu Guo