Method for measuring endotoxin

Abstract

A method for rapidly and highly sensitively measuring endotoxin is provided. Endotoxin is measured using an endotoxin-measuring agent comprising the proteins (1) to (3) below, each of which is a recombinant protein obtainable by being expressed using insect cells as a host: (1) a factor C derived from Tachypleus tridentatus, which factor C does not have a His-tag sequence at the C-terminus; (2) a factor B of a horseshoe crab; and (3) a proclotting enzyme of a horseshoe crab.

What is claimed is: 
     
         1 . A method for measuring an endotoxin in a test sample, the method comprising steps (a) and (b) below:
 (a) a step of mixing an endotoxin-measuring agent with the test sample; and   (b) a step of measuring progress of a cascade reaction mediated by proteins (1) to (3) below:   wherein the endotoxin-measuring agent comprises the proteins (1) to (3) below, each of which is a recombinant protein:   (1) a horseshoe crab factor C or variant thereof having factor C activity, which factor C does not have His-tag sequence at the C-terminus;   (2) a horseshoe crab factor B or variant thereof having factor B activity; and   (3) a horseshoe crab proclotting enzyme or variant thereof having proclotting enzyme activity.   
     
     
         2 . The method according to  claim 1 , which further comprises a step of adding a substrate of the endotoxin-measuring agent to a reaction system for detection of progress of the cascade reaction, wherein the reaction system is the mixture of the endotoxin-measuring agent and the test sample. 
     
     
         3 . The method according to  claim 2 , which further comprises a step of calculating the endotoxin level in the test sample on the basis of reaction of said substrate. 
     
     
         4 . The method according to  claim 1 , wherein said horseshoe crab is  Tachypleus tridentatus.    
     
     
         5 . The method according to  claim 1 , wherein said factor C is the protein (A) or (B) below; said factor B is the protein (C) or (D) below; and said proclotting enzyme is the protein (E) or (F) below:
 (A) a protein comprising the amino acid sequence shown in SEQ ID NO:2;   (B) a protein comprising the amino acid sequence shown in SEQ ID NO:2 but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, which protein has factor C activity;   (C) a protein comprising the amino acid sequence shown in SEQ ID NO:4;   (D) a protein comprising the amino acid sequence shown in SEQ ID NO:4 but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, which protein has factor B activity;   (E) a protein comprising the amino acid sequence shown in SEQ ID NO:6; and   (F) a protein comprising the amino acid sequence shown in SEQ ID NO:6 but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, which protein has proclotting enzyme activity.