2.4. Limulus amoebocyte lysate (LAL) assay of endotoxin content

The amount of LPS (endotoxin) present in each antigen preparation was determined using the Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher, catalog no. 88282). Briefly, 50 ​μl of each antigen sample was dispensed into a preheated (37 ​°C) 96-well plate, followed by 50 ​μl of LAL reagent. The plate was incubated at 37 ​°C for 10 ​min. One hundred microliters of chromogenic substrate were added and incubation was continued for 10 ​min. One hundred microliters of stop solution was added and sample absorbance was read at 405 ​nm. The concentration of endotoxin was extrapolated using a standard curve generated by purified E. coli (serotype 011: B4) LPS. The range of detection was 0.1–1 EU/ml (EU ​= ​endotoxin units).