Assay of endotoxin in human plasma using immobilized histidine, Limulus amoebocyte lysate and chromogenic substrate

Abstract

The Limulus amoebocyte lysate test for endotoxin is inhibited or enhanced by many substances. It is particularly difficult to determine endotoxin in plasma. In order to overcome this problem, we have modified the specific endotoxin assay method by using a membrane filter unit, a chromogenic Limulus amoebocyte lysate reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This immobilized histidine method consists of the endotoxin adsorption step on immobilized histidine, the separation step, in which Limulus amoebocyte lysate-interfering substances are removed, and the Limulus amoebocyte lysate test. Preheating of plasma samples (40-fold dilution with distilled water, at 100 degrees C for 7.5 min) was necessary, and it was necessary to dilute the sample more than 100-fold for the adsorption step. Under these conditions, the fraction of endotoxin recovered from plasma by the immobilized histidine method was almost 1. Moreover, by increasing the sample volume and extending the Limulus amoebocyte lysate reaction time, the sensitivity could be increased. By using the immobilized histidine method, 50-200 units/l of endotoxin in plasma samples can be accurately assayed. The method was used for the determination of plasma endotoxin in rabbits.