title: Biochemical principle of Limulus test for detecting bacterial endotoxins.
abstract: A hemocyte lysate from horseshoe crab (Limulus) produced a gel, when exposed to Gram-negative bacterial endotoxins, lipopolysaccharides (LPS). This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1,3)-β-D-glucan-mediated coagulation pathway which also results in the formation of gel. Up to now, six protein components, designated coagulogen, proclotting enzyme, factor B, factor C, and factor G, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. The molecular structures of these proteins have also been elucidated. Moreover, the reconstitution experiments using the isolated clotting factors, factor C, factor B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin gel. Here, I will focus on the biochemical principle of Limulus test for detecting bacterial endotoxins, and its activation and regulation mechanism on the LPS-mediated coagulation cascade.
authors.affiliation: The Chemo-Sero-Therapeutic Research Institute, 1-6-1 Okubo, Kumamoto 812-8581, Japan . ; Research adviser.
authors.forename: Sadaaki
authors.initials: S
authors.lastname: Iwanaga
keywords: Limulus test; bacterial endotoxins; clotting factors; horseshoe crab (Tachypleus tridentatus); innate immunity; lipopolysacchalides (LPS)
references.citation: Curr Opin Microbiol. 2000 Feb;3(1):16-22
references.pmid: 10679426
references.citation: Ann N Y Acad Sci. 1994 Apr 15;712:102-16
references.pmid: 8192325
references.citation: Methods Enzymol. 1993;223:358-64
references.pmid: 8271965
references.citation: Immunobiology. 2006;211(4):237-49
references.pmid: 16697917
references.citation: Clin Chim Acta. 1985 Jun 30;149(1):55-65
references.pmid: 3896576
references.citation: Proc Natl Acad Sci U S A. 1999 Aug 31;96(18):10086-91
references.pmid: 10468566
references.citation: EMBO J. 1996 Dec 16;15(24):6789-97
references.pmid: 9003754
references.citation: J Biol Chem. 1996 Sep 27;271(39):23768-74
references.pmid: 8798603
references.citation: J Biol Chem. 2002 Apr 19;277(16):14281-7
references.pmid: 11830593
references.citation: J Biol Chem. 1994 Nov 25;269(47):29462-7
references.pmid: 7961928
references.citation: J Biol Chem. 2000 Nov 10;275(45):35297-301
references.pmid: 10948203
references.citation: J Biol Chem. 1995 Jan 13;270(2):892-7
references.pmid: 7822328
references.citation: Curr Opin Immunol. 1996 Feb;8(1):41-7
references.pmid: 8729445
references.citation: Trends Immunol. 2004 Jun;25(6):289-94
references.pmid: 15145318
references.citation: J Biochem Mol Biol. 2005 Mar 31;38(2):128-50
references.pmid: 15826490
references.citation: EMBO J. 1999 May 4;18(9):2313-22
references.pmid: 10228146
references.citation: EMBO J. 2005 Dec 21;24(24):4404-14
references.pmid: 16362048
references.citation: Curr Opin Immunol. 2002 Feb;14(1):87-95
references.pmid: 11790537
references.citation: J Biochem. 1998 Jan;123(1):1-15
references.pmid: 9504402
references.citation: Pigment Cell Res. 2002 Feb;15(1):2-9
references.pmid: 11837452
references.citation: Thromb Res. 1992 Oct 1;68(1):1-32
references.pmid: 1448796
references.citation: J Biol Chem. 2001 Jul 20;276(29):27166-70
references.pmid: 11375396
references.citation: J Biol Chem. 2007 Feb 9;282(6):3962-7
references.pmid: 17135239
references.citation: Proc Natl Acad Sci U S A. 2004 Jan 27;101(4):953-8
references.pmid: 14722355
references.citation: Dev Comp Immunol. 1999 Jun-Jul;23(4-5):391-400
references.pmid: 10426430
references.citation: Methods Enzymol. 1993;223:378-88
references.pmid: 7903786
references.citation: Adv Exp Med Biol. 2001;484:195-202
references.pmid: 11418985
references.citation: Front Biosci. 1998 Sep 01;3:D973-84
references.pmid: 9727083
references.citation: Prog Mol Subcell Biol. 1996;15:154-89
references.pmid: 8963461
references.citation: Dev Comp Immunol. 1999 Jun-Jul;23(4-5):291-301
references.pmid: 10426423
references.citation: Fish Shellfish Immunol. 2002 May;12(5):421-37
references.pmid: 12194453
references.citation: Bull Johns Hopkins Hosp. 1964 Sep;115:265-74
references.pmid: 14209047
references.citation: J Biol Chem. 2000 Sep 22;275(38):29264-7
references.pmid: 10880508
references.citation: Cell Mol Life Sci. 2004 Jun;61(11):1257-65
references.pmid: 15170505
references.citation: Clin Chim Acta. 1994 Apr;226(1):109-12
references.pmid: 8070129
references.citation: Thromb Haemost. 1993 Jul 1;70(1):48-55
references.pmid: 8236113
references.citation: Haemostasis. 1978;7(2-3):183-8
references.pmid: 658779
references.citation: Biol Chem. 1997 Mar-Apr;378(3-4):283-7
references.pmid: 9165082
references.citation: Immunol Rev. 2004 Apr;198:97-105
references.pmid: 15199957
references.citation: J Biol Chem. 2000 Jun 16;275(24):17929-32
references.pmid: 10770921
references.citation: Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):13519-24
references.pmid: 11707569
references.citation: Curr Opin Immunol. 1993 Feb;5(1):74-82
references.pmid: 8452677
references.citation: J Biol Chem. 2002 Oct 18;277(42):40084-90
references.pmid: 12189150