New microassay for quantitation of endotoxin using Limulus amebocyte lysate combined with enzyme-linked immunosorbent assay

Abstract

A combined method of the Limulus amebocyte lysate (LAL) test and the enzyme-linked immunosorbent assay for quantitation of endotoxin was developed based on our observation that the antigenicity of coagulogen, a major protein in LAL, was lost when LAL reacted with endotoxin as shown by immunoblotting. Determination of the residual coagulogen by an enzyme-linked immunosorbent assay system with monoclonal antibody against coagulogen revealed that the loss of the antigenicity of coagulogen was proportional to the concentration of endotoxin. An inverse linear curve was established between the endotoxin concentration and absorbance. Standard curves of the LAL enzyme-linked immunosorbent assay with different detection limits (from 0.1 to 100 pg of the control standard endotoxin per ml) were obtained from one batch of commercial LAL by adjusting incubation time and dilution of LAL. The reaction curves of various endotoxins were parallel to one another, whereas the kinetics differed from that of (1-3)-beta-D-glucan. The LAL enzyme-linked immunosorbent assay is a highly reproducible microassay, using only 10 microliter of test sample and LAL reagent; because the color and turbidity of plasma samples do not interfere with the assay, it is well suited for quantitation of endotoxins in clinical specimens.