Quantification of endotoxin inhibition in serum and plasma using a turbidimetric LAL assay

A modified Limulus amebocyte lysate (LAL) test was developed which quantifies the inhibition associated with lipopolysaccharide (LPS) in serum and plasma. The assay utilized the LR50 value to determine relative inhibition. The LR50, measured in ng/ml, represented the concentration of endotoxin needed to elicit a turbidimetric response equal to 50% of the maximum above the control (no added endotoxin). The LR50 concept was used to compare plasma and serum from a normal donor population. Plasma LR50 values ranged from 115 to 5000 ng/ml, while serum samples ranged from 48 to 615 ng/ml. Endotoxin in saline measured in a similar manner yielded values averaging 0.07 ng/ml (LAL sensitivity). Various means to remove inhibition were also tested by this method. Heating, dilution, and chloroform extraction of serum were all found to be efficient in removing inhibition. Although no direct attempt was made to elucidate the biochemical nature of the inhibitor(s) present in serum and plasma, the results presented were consistent with other studies and indicate the involvement of a heat labile component(s). The LR50 may provide a tool for the elaboration of these components. It also was suggested that LR50 may be the best in vitro indicator of the overall ability of serum or plasma to neutralize endotoxin.