CSP27 or CSP9 was initially concentrated using an Amicon ultra-15 centrifugal filter unit (10 kD molecular weight cut-off, MWCO). Briefly, the filter was washed by spinning 10 mL of MQ at 4000 g for 20 minutes at 4 °C then discarding the flow through and any retained water. 2 mg of CSP27 or 1 mg CSP9 was added to the membrane and made up to 10 mL in 3% NaHCO3, then spun at 4000 g for 60 minutes at 4 °C. The flow-through was discarded and the retained protein made up to 10 mL in 3% NaHCO3, then respun at 4000 g for 60 minutes at 4 °C. The flow-through was discarded and the retained protein was made up to final volume of 1 mL per mg of original starting material in 3% NaHCO+. This was then transferred into a pre-soaked 3.5 kD MWCO dialysis tubing and dialysed overnight, then for 4 hours in 1 L 3% NaHCO3 at 4 °C.

Next, NP-ε-Aminocaproyl-Osu (NP-CAP-Osu) was dissolved in DMF to a final concentration of 10 mg/mL. We determined empirically that a 2:1 ratio of NP:CSP in the final conjugated product required a 20:1 ratio of NP:CSP at the conjugation step. For other NP:CSP ratios (6:1 and 10:1) this 10-fold increase during conjugation was also used; i.e., 60:1 and 100:1, respectively. The dialysed CSP and required NP-CAP-Osu were combined in sterile 2 mL tubes, covered with foil to protect from light, then rotated for 4 hours at RT to conjugate. The conjugated products were then dialysed in 1 L 3% NaHCO3 at 4 °C for 5 hours, overnight then 4 hours before dialysis in 1 L PBS for 4 hours and overnight. Conjugated products were stored at 4 °C with 0.1% sodium azide to prevent bacterial growth.

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