Optimalization of a chromogenic assay for endotoxin in blood

Early detection of Gram-negative septicemia or endotoxemia may become feasible with sensitive and reliable endotoxin (LPS) measurements. We recently published an assay for LPS in blood, based upon the LPS dependent activation of Limulus amebocyte lysate (LAL) and the subsequent chromogenic measurement of the activated enzyme(s). Inhibitors and activated clotting factors potentially interfering in the assay were removed by dilution and heating. In the present study we describe the further improvement of the assay. Optimal conditions include: blood anticoagulated with 30 I.U./ml heparin; dilution of the platelet-rich plasma (PRP) in water; 5 min. heating at 75 degrees C; 15 mM Mg2+, 1.5 mM Ca2+, 125 mM Na+, 50 mM TRIS, pH 8.5 in the LAL activation step; substrate step without extra addition of Ca2+, Mg2+, or Na+, but in the presence of 50 mM TRIS at pH = 9.5. Under those optimal conditions less than 10 pg LPS per ml blood (PRP) can easily be detected. Prospective clinical trials are presently envisaged to investigate the clinical usefulness of this extremely sensitive LPS assay.