wall and play a central role in the pathogenesis of endo-Endotoxin or lipopolysaccharide (LPS), a major cell toxemia and septic shock syndromes (1-3). Structursurface component of gram-negative bacteria, which ally, the bacterial LPS is composed of an O-antigenic could bind to different cell types when released into polysaccharide chain, oligosaccharide core region, and the bloodstream, plays a central role in the pathogenelipid A moiety (4). The lipid A is known as the endotoxic sis of septic shock syndromes. We have established a center of LPS molecule, affecting a wide range of cell biotinylation procedure for labeling purified LPS types such as monocytes, macrophages, neutrophils, molecules from Salmonella minnesota R595 and Escheplatelets, and vascular endothelium. The subsequent richia coli J5 bacteria. The biotin group was conjupathophysiological process involves fever, hypotension, gated to the bacterial LPS either by chemical oxidaintravascular coagulation, lactic acidosis, and deleterition of the LPS carbohydrate moiety (inner core ous inflammatory reactions that lead to multiorgan region), followed by reduction with biotin-LC-hydrafunction failure and shock (3, 5, 6). Localization of LPSzide (biotinamido hexanoyl hydrazide), or by photoacbinding sites and identification of membrane receptors tivatable cross-linking with biotin-LC-ASA [1-(4are important for developing antisepsis drugs and reazidosalicylamido -) -6 ( biotinamido ) -hexane ], which cently have gained keen interest (7). Labeled LPS liwas randomly attached to the carbohydrate and fatty gand having a functional stability remains an imacid (lipid A) groups of the LPS. Both labeled products portant tool for binding analysis and pharmacological retained biological activity (or endotoxicity) as eviassays in studies of endotoxin-receptor interactions.
denced by coagulation of the Limulus amoebocyte
The biochemical nature of bacterial endotoxin, which lysate. To determine its ability to bind avidin/streptahas both hydrophobic and hydrophilic properties and vidin which in turn could be conjugated with enzycarries both positively and negatively charged domatic and fluorescent probes, the biotinylated LPS was used in enzyme immunoassay, Western blot, and mains, has hampered its labeling procedures. Curflow cytometry. These assays were also used to analyze rently, the commonly used LPS probes are the radiolathe binding of LPS ligand to its counterreceptor(s) on beled ligand forms that are made either by metabolic whole cell surface, membrane fragments, and in deterincorporation of the entire LPS molecules with 14 C, 3 H, gent lysates from human endothelial and monocytic and 51 Cr isotopes or by iodination with Na 125 I usually cells. The described biotinylated LPS probes can be following tyramination treatment or photoaffinity applied in a wide variety of techniques in receptor biocross-linking with amino-group carrier (8-11). Neverchemistry, immunohistochemistry, and molecular cell theless, these radiolabeling procedures are laborious, biology.