Patent ID: 9040254
Filing Date: 2015-05-26
Classification: C07K,C12N,C12Q,G01N,Y02P

Abstract:
1. A method of detecting an endotoxin in a sample, comprising the steps of (a) to (c): (a) mixing the components of (1)-(3), the sample, and the substrate, to produce a detection mixture, wherein no magnesium salts are added to said mixture: (1) a recombinant pro-clotting enzyme, wherein said recombinant pro-clotting enzyme is produced by a method comprising: growing an insect cell harboring a baculovirus, wherein said baculovirus harbors a DNA fragment encoding a protein comprising the amino acid sequence of SEQ ID NO: 4; and collecting pro-clotting enzyme from said grown insect cell; (2) recombinant factor C, wherein the recombinant factor C has the same sequence as factor C produced by a horseshoe crab selected from the group consisting of (3) recombinant factor B, wherein the recombinant factor B has the same sequence as factor B produced by a horseshoe crab selected from the group consisting of (b) quantitatively or qualitatively detecting an enzymatic activity in said mixture, said enzymatic activity having been induced through conversion of said recombinant pro-clotting enzyme to a clotting enzyme, and wherein said detection uses the color of a synthetic chromogenic substrate as a signal, said synthetic chromogenic substrate being Boc-Leu-Gly-Arg-pNA, and (c) detecting endotoxin in the sample by using said detected enzymatic activity as an index of the existing amount or the existence of endotoxin in the sample.