Methods for detecting an endotoxin using a horseshoe crab clotting enzyme

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 Tachypleus tridentatus, Limulus polyphemus, Tachypleus gigas, and Tachypleus rotundicauda;
• and
• (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 Tachypleus tridentatus, Limulus polyphemus, Tachypleus gigas, and Tachypleus rotundicauda,
• (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.