Serum LPS Quantification Using LAL Assay

Serum LPS concentrations were measured with a ToxinSensor Chromogenic Limulus Amebocyte Lysate (LAL) Endotoxin Assay Kit (GenScript), following the manufacturer’s instructions [83 (link)]. Briefly, to minimize inhibition or enhancement by contaminating proteins, the samples were diluted 10- to 50-fold with endotoxin-free water, adjusted to the recommended pH, and heated for 10 min at 70 °C. The lyophilized endotoxin standard was dissolved by adding 2 mL of LAL reagent water and mixed thoroughly for 15 min with a vortexer to obtain an endotoxin stock solution. LAL reagents were added to the serum and incubated at 37 °C for 45 min, and the absorbance was read at 545 nm. A spiked control at 0.45 EU/mL was included for each sample to check that no significant inhibition or activation occurred. The lower limit of detection (LLOD) was 0.01 EU mL⁻¹. The coefficient of variation (C.V.) equals 100 times the standard deviation of a group of values divided by the mean and is expressed as a percent. The C.V. absorbance was less than 10%.