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Biochemical principle of Limulus test for detecting bacterial endotoxins.

Iwanaga S - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2007)

Bottom Line: This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins.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.

View Article: PubMed Central - PubMed

Affiliation: The Chemo-Sero-Therapeutic Research Institute, 1-6-1 Okubo, Kumamoto 812-8581, Japan . ; Research adviser.

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.

No MeSH data available.


Related in: MedlinePlus

A light (A and B) and electron (C) micrographs of horseshoe crab (T. tridentatus) hemocytes/amebocytes, and major defense molecules (C) that have been identified in large and small cell granules.9)
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f1-83_110: A light (A and B) and electron (C) micrographs of horseshoe crab (T. tridentatus) hemocytes/amebocytes, and major defense molecules (C) that have been identified in large and small cell granules.9)

Mentions: The hemolymph is collected by inserting a needle into a joint between the cephalothorax and abdominal region. Fifty to 150 ml of the hemolymph per individual is drawn under sterilized condition, and the hemocytes/amebocytes are collected by centrifuging pooled hemolymph at 2,500 rpm for 10 min. The hemocytes thus prepared are lysed and the lysate is used for endotoxin assay. On the basis of cell morphology, the hemocyte is an oval, and plate shaped structure, 15∼20 μm in its longest dimension.16), 18)Fig. 1 shows a light and an electron micrographs of the hemocytes separated from Japanese horseshoe crab, Tachypleus(T) tridentatus. The cell contains numerous dense granules classed into two major types: large(L) and small(S) granules.9) The former is larger (up to 1.5 μm in diameter) and less dense than the latter (< 0.6 μm in diameter). The L-granules contain more than 25 proteins, the majority of which have molecular masses between 8 and 123 kDa.17), 19), 20) In contrast, the S-granules contain at least 6 proteins with molecular masses of less than 30 kDa, in addition to several antimicrobial peptides and their analogues, such as tachyplesins, tachystatins, tachycitins and big defensins, all of which show antimicrobial activities against Gram-negative and -positive bacteria and fungi.21)–27)


Biochemical principle of Limulus test for detecting bacterial endotoxins.

Iwanaga S - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2007)

A light (A and B) and electron (C) micrographs of horseshoe crab (T. tridentatus) hemocytes/amebocytes, and major defense molecules (C) that have been identified in large and small cell granules.9)
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756735&req=5

f1-83_110: A light (A and B) and electron (C) micrographs of horseshoe crab (T. tridentatus) hemocytes/amebocytes, and major defense molecules (C) that have been identified in large and small cell granules.9)
Mentions: The hemolymph is collected by inserting a needle into a joint between the cephalothorax and abdominal region. Fifty to 150 ml of the hemolymph per individual is drawn under sterilized condition, and the hemocytes/amebocytes are collected by centrifuging pooled hemolymph at 2,500 rpm for 10 min. The hemocytes thus prepared are lysed and the lysate is used for endotoxin assay. On the basis of cell morphology, the hemocyte is an oval, and plate shaped structure, 15∼20 μm in its longest dimension.16), 18)Fig. 1 shows a light and an electron micrographs of the hemocytes separated from Japanese horseshoe crab, Tachypleus(T) tridentatus. The cell contains numerous dense granules classed into two major types: large(L) and small(S) granules.9) The former is larger (up to 1.5 μm in diameter) and less dense than the latter (< 0.6 μm in diameter). The L-granules contain more than 25 proteins, the majority of which have molecular masses between 8 and 123 kDa.17), 19), 20) In contrast, the S-granules contain at least 6 proteins with molecular masses of less than 30 kDa, in addition to several antimicrobial peptides and their analogues, such as tachyplesins, tachystatins, tachycitins and big defensins, all of which show antimicrobial activities against Gram-negative and -positive bacteria and fungi.21)–27)

Bottom Line: This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins.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.

View Article: PubMed Central - PubMed

Affiliation: The Chemo-Sero-Therapeutic Research Institute, 1-6-1 Okubo, Kumamoto 812-8581, Japan . ; Research adviser.

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.

No MeSH data available.


Related in: MedlinePlus