[...]... Rietschel, E T., Kusumoto, S., Shiba, T., and Brade, H (1987) Immunogenicity and antigenicity of natural and synthetic Escherichia coli lipid A Prog Clin Biol Res 231, 75–97 7 Brade, L., Brandenburg, K., Kuhn, H.-M., Kusumoto, S., Macher, I., Rietschel, E T., and Brade, H (1987) The immunogenicity and antigenicity of lipid A are influenced by its physicochemical state and environment Infect Immun 55, 2636–2644... enterica, Escherichia coli, and Shigella In addition to being cross-reactive among the different strains in vitro, the antibody is cross-protective in vitro and in vivo in endotoxin and infection models (8,9) It is thus evident that LPS and lipid A serology provide many clinical and experimental applications Reliable LPS serology requires defined, preferably monoclonal, antibodies and structurally characterized... Chemistry and Safety (Keeler, R F., Mandava, N B., and Tu, A T., eds.), Alaken, Inc., Fort Collins, CO, pp 301–315 Endotoxin Preparation from Gram-Negative Bacteria 25 5 Hitchcock, P J., Leive, L., Maleka, P J., Rietschel, E Th., Strittmatter, W., and Morrison, D C (1986) A review of lipopolysaccharide nomenclature: past, present and future J Bacteriol 166, 699–705 6 Galanos, C., Luderitz, D., and Westphal,... Ramsay, G., Schreier, M H., McClelland, D B L., and Rietschel, E T (1993) A broadly cross-protective monoclonal antibody binding to Escherichia coli and Salmonella lipopolysaccharides Infect Immun 61, 3863–3872 9 Bailat, S., Heumann, D., Le Roy, D., Baumgartner, J D., Rietschel, E T., Glauser, M P., and Di Padova, F (1997) Similarities and disparities between core-specific and O-side-chain-specific antilipopolysaccharide... particularly surprising, therefore, that there are now numerous methods and modifications of methods, that have been published in the scientific literature describing various approaches that have been employed for the extraction and purification of endotoxin from bacteria It would be beyond the scope of this chapter to describe in detail all of these various methods Therefore, we shall provide only a brief historical... isolating and purifying endotoxins It would be of value at the outset to begin with a definition of exactly what is meant in this chapter when referring to the terms “endotoxin” and “lipopolysaccharide” (LPS) Although these two terms are often used interchangeably, it is important to note that they are both functionally and biochemically distinct From: Methods in Molecular Medicine, Vol 36: Septic Shock... Seydel, U., Brandenburg, K., Ulmer, A J., Mattern, T., Heine, H., Schletter, J., Loppnow, H., Schönbeck, U., Flad, H.-D., Hauschildt, S., Schade, U F., Di Padova, F., Kusumoto, S., and Schumann, R R (1996) Bacterial endotoxin: chemical constitution, biological recognition, host response, and immunological detoxification, in Current Topics in Microbiology and Immunology, Pathology of Septic Shock (Rietschel,... LPS, in phenol; the solubiliity of LPS in an aqueous environment (water); the total miscibility of phenol and water at elevated temperatures about 68°; and the relative ease by which phenol and water can be separated upon cooling and centrifugation In general, this method is relatively uncomplicated and can be carried out even by investigators who are not generally accustomed to doing chemical extraction... equal volumes of phenol and water When cooled to 5–10°C, the mixture resolves into three phases, an upper water layer (containing the LPS), a phenol layer, and at the interface between the two a variably sized layer of material that is both 20 Shnyra, Luchi, and Morrison water and phenol insoluble Extraction of the LPS into the upper water layer, that is then simply removed and subsequently manipulated,... of this protein This is From: Methods in Molecular Medicine, Vol 36: Septic Shock Edited by: T J Evans © Humana Press Inc., Totowa, NJ 37 38 Weiss particularly true if PMN-rich inflammatory exudates can be induced in experimental animals as this provides a highly enriched and abundant starting material for extraction and isolation of BPI Accordingly, in this chapter, methods for isolation of BPI from . blood handling and performing the assay must be powder-free, because the powder contains endotoxin and can contaminate the assay. 2. The chromogenic LAL reagent kit with endotoxin standard and diazotization reagents. only. 2.4. Endotoxin Standard 1. The reference standard endotoxin (RSE) is made from Escherichia coli 0113 and known as EC-6. Other endotoxin standards are related to RSE and their potency documented. Pyrochrome Buffer, endotoxin standard and the diazo- coupling reagents. Other manufactures supply the chromogenic LAL reagent suitable for the assay and an endotoxin standard. If not purchased, the