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AssigneeHiroshima University
InventorsAkio KURODA, Kenichi NODA
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The present invention provides a method comprising allowing a reaction of a sample, a reagent containing Factor C, which can be activated by binding with endotoxin, and a synthetic luminescent substrate comprising a luminescent substrate bound to a peptide, for release of the luminescent substrate from the synthetic luminescent substrate, allowing a luminescent enzyme to act on the luminescent substrate released in the luminescent substrate release step, for measurement of the luminescence intensity, and quantifying the level of endotoxin in the sample based on a measured value obtained in the luminescence measuring step, the method enabling endotoxin to be simply and quickly measured at a level that cannot be detected in conventional methods for endotoxin measurement, without use of any dedicated measuring device.
1-7. (canceled)
A kit for measurement of endotoxin level, comprising, as components, a reagent containing Factor C, which can be activated by a specific binding with endotoxin, a synthetic luminescent substrate comprising a luminescent substrate bound to a peptide, and a luminescent enzyme.
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Chemiluminescence; Bioluminescence: bioluminescence
Measuring or testing processes involving enzymes, nucleic acids or microorganisms ; Compositions therefor; Processes of preparing such…
For microorganisms, e.g. protozoa, bacteria, viruses: bacteria
Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing:…
Term end not estimated
The present invention relates to a method and a kit for measurement of endotoxin level in a sample, and in particular relates to a method and a kit for measurement of endotoxin level using a bioluminescent reaction.
“Endotoxin” is a component of the cell wall in the outer membrane of gram-negative bacteria, and its activity is mainly attributed to LPS (lipopolysaccharide). In the living body, endotoxin exists as a part of the outer membrane in the surface layer of gram negative bacteria. Generally, after death of gram-negative bacteria, endotoxin is liberated and is present in a free form in blood.
When more than a certain level of endotoxin is present in blood, the endotoxin stimulates monocytes, granulocytes, etc., resulting in excessive production of inflammatory cytokines. Consequently, so called endotoxinemia accompanied by symptoms such as fever, sepsis, septic shock, multiple organ failure, etc. is induced. For this reason, detection of endotoxin in pharmaceuticals for injection, etc. is crucial, and thus the bacterial endotoxin test is prescribed by the Japanese, U.S. and European pharmacopeias. From the aspect of clinical diagnosis, precise measurement of blood endotoxin level is considered crucial for early diagnosis and therapeutic effect evaluation.
FIG. 1 shows a gelation process of horseshoe crab amebocyte lysate triggered by endotoxin.
FIG. 2 shows an exemplary method for measurement of endotoxin level according to the present invention.
FIG. 3 is a graph of luminescence intensity plotted against the sample concentration in Example 1.
Firstly, the mechanism of the method for measurement of endotoxin level will be illustrated by reference to FIGS. 1 and 2. FIG. 1 shows a gelation process of horseshoe crab amebocyte lysate triggered by endotoxin, and FIG. 2 shows an exemplary method for measurement of endotoxin level according to the present invention.
As shown in FIG. 1, endotoxin firmly binds with Factor C and thereby activates the Factor C, and then activated Factor C (active Factor C) activates Factor B. Next, activated Factor B (active Factor B) activates a proclotting enzyme and thereby a clotting enzyme is produced. The clotting enzyme partially hydrolyzes a substrate called coagulogen, and thereby a clotting protein called coagulin is produced.
As shown in FIG. 2, when an endotoxin-containing sample has been brought into contact with horseshoe crab amebocyte lysate, endotoxin in the sample triggers activation of the limulus clotting cascade. Then, by addition of “benzoyl-Leu-Arg-Arg-aminoluciferin” as a substrate of the clotting enzyme to the reaction system, the clotting enzyme produced by the activation of the limulus clotting cascade, cleaves the bond of Arg and aminoluciferin, resulting in release of a luminescent substrate aminoluciferin. Luciferase (luminescent enzyme) is allowed to act on the aminoluciferin, resulting in light emission. The luciferin/luciferase luminescent reaction is shown in the following reaction formula (I).