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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.

The large enhancement in the rate of alkaline hydrolysis of p-nitrophenyl hexanoate in aqueous solutions of tetradecyltrimethylammonium bromide in the presence of bovine serum albumin was found. A maximum in the rate enhancement appeared in the vicinity of the critical micelle concentration of the surfactant. It was suggested that the rate enhancement was attributable to the formation of protein–surfactant complexes providing a new pseudo-phase for the reaction.