Assay of Endotoxin in Human Plasma Using Immobilized Histidine, Limulus Amoebocyte Lysate and Chromogenic Substrate

Abstract

Summary:The Limulus amoeboeyte 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 fllter unit, a chromogenic Limulus amoeboeyte 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 amoeboeyte lysate-interfering substances are removed, and the Limulus amoeboeyte lysate test. Preheating of plasma samples (40-fold dilution with distilled water, at 100 °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 frorii plasma by the immobilized histidine method was almost l. Moreover, by increasing the sample volume and extending the Limulus amoeboeyte lysate reaction time, the sensitivity could be increased. By using the immobilized histidine method, 50-200 units/1 of endotoxin in plasma samples can be accurately assayed. The method was used for the determination of plasma endotoxin in rabbits.

Detection of lipopolysaccharide in hemoglobin‐vesicles by Limulus amebocyte lysate test with kinetic–turbidimetric gel clotting analysis and pretreatment of surfactant

“…Considering this background information, we aimed to find the optimal condition for the pretreatment of HbVs using a surfactant to release LPS28,29 with a minimal interference effect for the subsequent kinetic–turbidimetric LAL assay using a Toxinometer®. For a better detection limit, we tested the histidine‐immobilized agarose gel that effectively adsorbs LPS to concentrate the trace amount of LPS and to eliminate the solutes that interfere with the LAL assay 30–33…”