Conjugation of CPS and TetHc

CPS and TetH_c conjugation was performed as detailed previously [16] (link), [17] (link). Briefly, purified CPS was solubilised at 5 mg/mL in PBS (Sigma) and sodium meta-periodate (Alpha Aesar) was added to a final 30 mM concentration. The mixture was gently stirred at room temperature for 40 min and afterwards, excess oxidising agent was removed by dialysis (GeBAflex-tube maxi). To the dialysed polysaccharide solution, TetH_c protein was added to a final concentration of 5 mg/mL in PBS (Sigma). Sodium cyanoborohydride (Sigma: 1 M NaBH₃CN in 10 mM NaOH) was then added at 10 μL per mL of conjugation mixture and then stirred at room temperature for 4 days. Following this, a further 10 μL of sodium borohydride (Sigma: 1 M NaBH₄ in 10 mM NaOH) was added to each mL of conjugation mixture and stirring was continued at room temperature for 40 min. The conjugate reactions were then dialysed against distilled water (GeBAflex-tube maxi) and analysed by SDS-PAGE (12%, Expedeon) and western blot using anti-CPS monoclonal antibody DSTL189. The resulting products were then re-dissolved in ultrapure water and stored at −20 °C.