2.7. Limulus Amebocyte Lysate Test

To assay endotoxin levels, serum samples were collected aseptically, then diluted 1:5 with the Limulus Amebocyte Lysate (LAL) reagent solution in a nonpyrogenic tube by means of a commercially available Pyrochrome Limulus Amebocyte Lysate Kit (Associates of Cape Cod, Falmouth, MA, USA). E. coli O113:H10 served as the standard; a standard series ranging from 0.04 to 1.28 EU/mL was used to evaluate the concentration of endotoxin. Standards, samples, and a negative control were heated for 15 min at 75 °C, then added into wells of a 96-well plate with 50 μL of pyrochrome already present in each well, mixed for 30 s, and incubated at 37 °C for 25 min. To stop the reaction, 25 μL of 50% acetic acid was added. Optical density (OD) was determined at 405 nm on a Multiskan™ GO ELISA reader (Thermo Scientific, Waltham, MA, USA). Calculations were performed in endotoxin testing with concentrations expressed in EU/mL.