Method for detection of gram-negative bacterial liposaccharides

Details Method for detection of gram-negative bacterial liposaccharides Method for detection of gram-negative bacterial liposaccharides

1993-02-05

1994-10-18

Scheiner, Toni R.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

435 732, 435 735, 435 737, 435 791, 435 792, 435 794, 435810, 435879, 435 23, 435 24, 436 71, 436501, 436518, 436815, 436821, G01N 33579, G01N 3353, G01N 33569

Patent

active

053567781

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The present invention relates to a method for the detection of bacterial endotoxin in a biological fluid or in a fluid intended for clinical or pharmaceutical use, using antibodies cross-reactive with a broad range of gram-negative bacteria to act as lipopolysaccharide capture agents. In particular, the method provides a sensitive assay that can be tailored to detect the endotoxin of selected gram-negative bacteria.


______________________________________ LIST OF ABBREVIATIONS ______________________________________ Haemophilus influenzae type b Hib lipooligosaccharide LOS polymyxin B PMB immunolimulus IML chromogenic Limulus CLAL amoebocyte assay Limulus amoebocyte lysate LAL lipopolysaccharide LPS monoclonal antibodies MAbs enzyme-linked ELISA immunosorbent assay outer membrane vesicles OMV sodium dodecyl sulfate SDS polyacrylamide gel PAGE electrophoresis pyrogen-free pf immunoglobulin G IgG phosphate buffered saline PBS Tween 20 Tw bovine serum albumin BSA cerebrospinal fluid CSF colony forming units CFU brain heart infusion broth BHIs supplemented with Levinthal base ______________________________________


DESCRIPTION OF THE RELATED ART

Septicemia is a potentially fatal clinical condition which is currently increasing in importance, possibly because of the longer survival of immunocompromised patients and greater use of invasive techniques in medicine (1,2). It has been estimated that the incidence of this disease has increased ten-fold during the last 20 years and that the number of cases annually is from 100,000 to 300,000 in the United States alone (3). From 20% to 40% of the patients with gram-negative bacterial septicemia have shock and, of these, approximately 75% will die (1). In children, Haemophilus influenzae type b (Hib) is responsible for about 40% of cases of septic shock (4). Pseudomonas aeruginosa bacteremia in neutropenic patients is a particularly virulent form of septicemia.
Specific laboratory diagnosis of gram-negative septicemia is usually performed by culturing blood samples. These methods, however, are relatively slow, requiring several hours to days to detect bacterial growth.
Endotoxin is considered to be a key element in the initiation of the inflammatory cascade during gram-negative bacterial infections (5). Therefore, quantitation of these molecules in blood samples of septic patients has been considered to be important. An easy and relatively sensitive way to detect endotoxin involves the Limulus amoebocyte lysate (LAL) assay (6). The LAL assay, however, has several problems which have limited its usefulness in the diagnosis of septicemia. This assay is sensitive to trace amounts of LPS contamination in laboratory fluids and reagents, which then cause false-positive reactions. Furthermore, plasma of patients has several nonspecific activators and inhibitors of the enzymes involved in the LAL reaction. Finally, the color and turbidity of normal plasma impedes the high sensitivity of a recent refinement of the LAL method known as the CLAL assay. The latter method measures color generated by the action of activated lysate enzymes on a synthetic chromogenic substrate. Because of these problems, the sensitivity and specificity of the LAL and CLAL assays are thought to be suboptimal for reliable clinical diagnosis.
The basic structure of lipopolysaccharide (LPS) involves three relatively well defined regions and is similar in all gram-negative bacteria. These regions are an O-specific side chain, the core oligosaccharide, and lipid A. The O-specific region is composed of repeating oligosaccharide units each having 2-6 saccharides. The core lies between the O-specific side chains and lipid A and is a branching oligosaccharide having representative sugars such as glucose, N-acetylglucosamine and galactose. In the core region proximal to Lipid A, heptose and keto-deoxyoctonate are commonly found. There is considerable structural variation among the gram-negative bacteria in the O-chain region,

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