Quantification of the endotoxin-neutralizing capacity of serum and plasma

A new procedure for quantifying the endotoxin-neutralizing capacity (ENC) of plasma or serum is described. Serially diluted samples were preincubated with endotoxin (lipopolysaccharide; LPS) and the sample dilution producing 50% inhibition of Tachypleus amebocyte lysate activation was measured by a Limulus peptide C enzyme-linked immunosorbent assay. The assay was not subject to interference from plasma or serum at a 500-fold dilution. The ENC of fresh sera from 120 healthy human donors, determined with Salmonella abortus LPS, had a median value of 7.7 kEU/ml (95% confidence limits 3-24 kEU/ml). Values for heparinized fresh plasma were close to those for the corresponding sera. Serum ENC varied greatly with different types of LPS. Neutralization of LPS by serum was rapid, heat-labile, and fully reversed by acidification. Addition of 2 mM EDTA to the serum diluent or pretreatment of LPS with 0.5% deoxycholate enhanced the ENC of serum about 25-fold or 10-fold respectively. The neutralization of LPS by polymyxin B or lysozyme could be demonstrated by the ENC assay, while that by human serum albumin, fibronectin or anti-LPS immunoglobulins was only detected in the presence of 2 mM EDTA. The kinetic changes of LPS and ENC during rabbit endotoxemia were also determined. The ENC assay may be used to study the significance of plasma ENC in Gram-negative infections and to identify the components contributing to plasma ENC.