ASSAY OF ENDOTOXIN IN HUMAN PLASMA USING IMMOBILIZED HISTIDINE, LIMULUS AMEBOCYTE LYSATE AND CHROMOGENIC SUBSTRATE

The Limulus amoebocyte lysate test for endotoxin is inhibited or enhan ced by many substances. It is particularly difficult to determine endo toxin in plasma. In order to overcome this problem, we have modified t he specific endotoxin assay method by using a membrane filter unit, a chromogenic Limulus amoebocyte lysate reagent, and immobilized histidi ne (which is a specific adsorbent for endotoxins). This immobilized hi stidine method consists of the endotoxin adsorption step on immobilize d histidine, the separation step, in which Limulus amoebocyte lysate-i nterfering substances are removed, and the Limulus amoebocyte lysate t est. Preheating of plasma samples (40-fold dilution with distilled wat er, 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 reactio n time, the sensitivity could be increased. By using the immobilized h istidine method, 50-200 units/l of endotoxin in plasma samples can be accurately assayed. The method was used for the determination of plasm a endotoxin in rabbits.