2.3 Conjugation of
Mono-activated Ester
of Oligosaccharide
Antigen to Carrier
Protein
1. Dram vial.
2. Magnetic stir bar.
3. Stir plate.
4. Bio-gel A 0.5 m gel.
5. Separation column.
6. 0.1 M PBS.
7. Test tubes (13 Â 100 mm).
8. Dialysis tube or bag (with molecular cut of 5–10 kDa).
9. Bicinchoninic acid (BCA).
10. 15% (v/v) H 2 SO 4 in EtOH.
11. Freeze dryer.
2.4 Analysis
of Carbohydrate
Loading
of the Resultant
Glycoconjugate
1. Commercial monosaccharides appropriate for the oligosaccharide antigen (see Note 2).
2. Dry test tubes (10 mL).
3. Cuvettes (1 mL).
4. Microliter pipette and tips.
5. UV-Vis spectrometer.
6. 96% H 2 SO 4 (concentrated).
7. 4% phenol (aq. solution).
8. Blank carrier protein.
3 Methods
3.1 Reduction
of the Azido Group
in Oligosaccharide
Antigen
1. Dissolve the azido derivative of oligosaccharide antigen
(50 μmol) in distilled water (3.0 mL).
2. Add zinc powder (164 mg, 2.5 mmol) (see Note 3) to the
above solution.
3. Add acetic acid (29 μL).
4. Stir the reaction mixture at ambient temperature for 24 h.
5. Add N,N-diisopropylethylamine (DIPEA) (see Note 4) to neutralize the reaction.
Scheme 1 Oligosaccharide antigen conjugation to carrier proteins via dicarboxylic acid linkers
Oligosaccharide-Protein Conjugation
307