Single-step, chromogenic Limulus amebocyte lysate assay for endotoxin

Abstract

A new reagent for the chromogenic Limulus amebocyte lysate (LAL) assay is described. LAL was formulated for optimal performance in either an endpoint procedure or a kinetic procedure with the chromogenic substrate, buffer, and LAL components colyophilized as a single reagent. The kinetic chromogenic method required an incubating microplate reader coupled to a computer for collection and analysis of data. The kinetic method had a longer incubation time than the endpoint method and spanned a range of over 3 orders of magnitude compared with the 1-order-of-magnitude range of the endpoint assay. The kinetic method was less subject to operator error, since readings were continuous and automatic. The endpoint test was more operator intensive, requiring both addition of acetic acid to stop the reaction and transfer of the sample to the reading device. A single-step chromogenic reagent was also prepared without lyophilization by mixing reconstituted gel clot LAL with a buffer and a chromogenic substrate. The reagent prepared in this manner performed as well as the colyophilized agent.