[Quantitative endotoxin determination. Automated kinetic Limulus amebocyte lysate microtiter test with measurement of sample-related interferences]

Abstract

A turbidometric, automated limulus amoebocyte lysate (LAL) microtiter test has been developed based on the evaluation of the LAL-endotoxin reaction kinetics. The maximal increase in optical density of each reaction mixture within 1 min is recorded. With this method an endotoxin standard curve is achieved which is linear over a concentration range of six decades. With presently available LAL methods sample-related inhibition or enhancement of the LAL endotoxin reaction may be overlooked and lead to false results. The quality of interfering factors can be characterized with our methods by spiking serial dilutions of the sample with constant endotoxin concentrations. The additional introduction of an internal standardization in our system allows the determination of endotoxin with simultaneous detection of quality and quantity of sample-induced interference. This procedure is based on a mathematic model which describes interference-caused alterations of the reaction revealed by addition of endotoxin in increasing concentrations. In comparison to the LAL tube test and the turbidometric determination at a given time the advantages of the developed method are demonstrated using three different samples (gelatin solution, adenine-HCl solution and a concentrate of coagulation factors (PPSB)). These are paradigmaticly selected because and enhancement of the LAL endotoxin reaction.