Biomaterial Database

Optimalization of a chromogenic assay for endotoxin in blood. 1985

A Sturk, and K Joop, and J W ten Cate, and L L Thomas

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.

UI	MeSH Term	Description	Entries
D008033	Limulus Test	Sensitive method for detection of bacterial endotoxins and endotoxin-like substances that depends on the in vitro gelation of Limulus amebocyte lysate (LAL), prepared from the circulating blood (amebocytes) of the horseshoe crab, by the endotoxin or related compound. Used for detection of endotoxin in body fluids and parenteral pharmaceuticals.	Limulus Tests,Test, Limulus,Tests, Limulus
D008070	Lipopolysaccharides	Lipid-containing polysaccharides which are endotoxins and important group-specific antigens. They are often derived from the cell wall of gram-negative bacteria and induce immunoglobulin secretion. The lipopolysaccharide molecule consists of three parts: LIPID A, core polysaccharide, and O-specific chains (O ANTIGENS). When derived from Escherichia coli, lipopolysaccharides serve as polyclonal B-cell mitogens commonly used in laboratory immunology. (From Dorland, 28th ed)	Lipopolysaccharide,Lipoglycans
D002863	Chromogenic Compounds	Colorless, endogenous or exogenous pigment precursors that may be transformed by biological mechanisms into colored compounds; used in biochemical assays and in diagnosis as indicators, especially in the form of enzyme substrates. Synonym: chromogens (not to be confused with pigment-synthesizing bacteria also called chromogens).	Chromogenic Compound,Chromogenic Substrate,Chromogenic Substrates,Compound, Chromogenic,Compounds, Chromogenic,Substrate, Chromogenic,Substrates, Chromogenic
D004731	Endotoxins	Toxins closely associated with the living cytoplasm or cell wall of certain microorganisms, which do not readily diffuse into the culture medium, but are released upon lysis of the cells.	Endotoxin
D006493	Heparin	A highly acidic mucopolysaccharide formed of equal parts of sulfated D-glucosamine and D-glucuronic acid with sulfaminic bridges. The molecular weight ranges from six to twenty thousand. Heparin occurs in and is obtained from liver, lung, mast cells, etc., of vertebrates. Its function is unknown, but it is used to prevent blood clotting in vivo and vitro, in the form of many different salts.	Heparinic Acid,alpha-Heparin,Heparin Sodium,Liquaemin,Sodium Heparin,Unfractionated Heparin,Heparin, Sodium,Heparin, Unfractionated,alpha Heparin
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D006863	Hydrogen-Ion Concentration	The normality of a solution with respect to HYDROGEN ions; H+. It is related to acidity measurements in most cases by pH	pH,Concentration, Hydrogen-Ion,Concentrations, Hydrogen-Ion,Hydrogen Ion Concentration,Hydrogen-Ion Concentrations
D013696	Temperature	The property of objects that determines the direction of heat flow when they are placed in direct thermal contact. The temperature is the energy of microscopic motions (vibrational and translational) of the particles of atoms.	Temperatures