# US 9,040,254 B2 · Methods for detecting an endotoxin using a horseshoe crab clotting enzyme > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,040,254 B2 - Document ID: US9040254B2 - Status: Granted - Kind: B2 - Application number: 13/942,293 - Filed: 2013-07-15 - Published: 2013-11-21 - Granted: 2015-05-26 - Earliest claimed: 2006-07-07 - Assignees: Seikagaku Corporation - Applicants: Seikagaku Corporation - Inventors: Hiroshi Tamura; Shoji Takahashi - Web page: https://patentgrep.com/patent/US9040254B2 ## CPC - C12Q 1/56: involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen - C07K 14/43509: {from crustaceans} - C12N 9/6402: {from non-mammals} - C12N 9/6408: {Serine endopeptidases (3.4.21)} - G01N 33/579: involving limulus lysate - C12N 2799/026: where the vector is derived from a baculovirus - Y02P 20/582: Recycling of unreacted starting or intermediate materials - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS ## Abstract Objects of the present invention are to provide a DNA fragment encoding a limulus-derived pro-clotting enzyme, a virus harboring the DNA fragment, a cell harboring the virus, a method of producing the pro-clotting enzyme by use of the cell, and means for assaying an endotoxin or (1→3)-β-D-glucan employing the enzyme, wherein these elements are capable of producing an endotoxin or (1→3)-β-D-glucan assay reagent of satisfactory quality, steadily, at low cost, and on a large scale. In the present invention, for example, a DNA fragment encoding a protein having an amino acid sequence defined by SEQ ID NO: 4 is selected as a nucleic acid fragment encoding a limulus-derived pro-clotting enzyme, and the corresponding recombinant pro-clotting enzyme. Use of the enzyme can provide a high-sensitivity method and kit for detecting (1→3)-β-D-glucan and an endotoxin, utilizing a cascade reaction system in a horseshoe crab lysate. ## Claims ### Claim 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 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. ### Claim 2 (depends on 1) The method according to claim 1, wherein said DNA fragment comprises the nucleotide sequence of nucleotides 1 to 1143 of SEQ ID NO: 3. ### Claim 3 (depends on 1) The method according to claim 1, wherein the baculovirus is nuclear polyhedrosis virus. ### Claim 4 (depends on 1) The method according to claim 1, wherein calcium chloride is not added to said mixture. ### Claim 5 (depends on 1) The method according to claim 1, wherein, in production of the recombinant factor B, MOI (Multiplicity of Infection) is about 0.1 and the period of culture at 28° C. is about 96 hours when the insect cell is infected with the baculovirus DNA fragment to which the gene coding factor B is introduced and culture supernatant of the baculovirus-infected insect cell is obtained.