5 M ethanolamine, 0.5 M NaCl pH 8.3 and screening assay again 0.1 M AcONa, 0.5 M NaCl pH 4 were passed through the column (6 column volumes for each buffer). The column was stored in 0.05 M Na2HPO4, 0.1% NaN3 pH 7.0 at 4 °C. A syringe was used for all wash steps. Flow through and wash solutions were analysed by phenol sulphuric assay to calculate the amount of sugar linked to the resin. Blood containing anti-Salmonella antibodies was venesected from a healthy adult and left to clot at 22 °C for 4 h before separating by centrifugation at 4 °C and freezing in aliquots at − 80 °C. Ethical approval for the use

of human serum in this study was granted by the Life and Health Sciences Ethical Review Committee of the University of Birmingham. Informed written consent was obtained prior to venesection. Ammonium sulphate was added as a solid to 1 ml of human serum to give a final concentration of 0.5 g/ml and the mixture was placed on ice for 5 min. The serum was centrifuged at 4 °C, 3300 × g for

5 min and the supernatant discarded. The precipitate was washed twice with 1 ml 0.5 g/ml ammonium sulphate. The pellet was solubilized in 0.3 ml PBS and dialysed overnight against PBS at 4 °C. NHS HiTrap columns with activated OAg were equilibrated with PBS (6 column volumes) before applying the serum protein solution to the column and incubating overnight at 4 °C. Columns were then washed with PBS (6 column volumes), followed by 50 mM NaH2PO4, 500 mM NaCl pH 7.2 (6 column volumes). Bound antibodies were eluted in 5 column volumes of elution buffer, collecting fractions of 0.5 ml each. Protease Inhibitor Library nmr 0.1 M glycine, 0.1 M NaCl at pH 2.4, 2.6, 2.8 and 3.0; 20% ethanol; 4 M MgCl2 in 10 mM Tris base pH 7; 8 M urea; and 100 mM Tris base pH 9 were tested as elution buffers. Following elution with glycine buffers using a pH 2.4–3.0,

the pH was adjusted to 7.0 with 2 M Tris pH 9. Individual eluate fractions were analysed for protein content by measuring absorption at 280 nm. After elution, all eluates were dialysed overnight against PBS at 4 °C. Purified antibodies were stored at 4 °C. Retention of antibodies on columns was investigated by applying 1% SDS to columns and analysing SDS-eluates by SDS-PAGE. Columns were washed with PBS and stored in 0.05 M Na2HPO4, 0.1% NaN3 O-methylated flavonoid pH 7.0 at 4 °C. 96-well flat bottom plates (NUNC Maxisorp) were incubated with 100 μl per well of 5 μg/ml TLR-grade smooth LPS from S. Typhimurium (Alexis Biochemicals) or 15 μg/ml S. Typhimurium OAg, overnight at 4 °C. After coating, plates were washed with PBS 0.05% Tween and incubated with 200 μl blocking buffer (PBS 1% BSA) per well for 1 h at 37 °C. Plates were washed again with PBS 0.05% Tween and incubated for 1 h at 37 °C with 100 μl serial dilutions of antibody solution diluted with PBS 1% BSA 0.05% Tween.