(.fwdarw.)-.beta.- d-glucan binding protein, an antibody recogni

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...


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435 731, 435 732, 435 78, 435 793, 435 794, 435 795, 435 23, 435962, 530350, 5303882, 5303891, 5303911, 530396, 530413, 530857, G01N 3353, G01N 33579, C07K 14435, C07K 1618






This invention relates to a (1.fwdarw.3)-.beta.-D-glucan binding protein or variants thereof obtained from horseshoe crab amoebocytes and an antibody thereto.
Further, the present invention relates to a (1.fwdarw.3)-.beta.-D-glucan assay agent composed of said protein, a kit composed of said protein and said assay agent, a kit composed of said protein and said antibody, and the method for assaying (1.fwdarw.3)-.beta.-D-glucan using said protein.
In addition, this invention relates to a (1.fwdarw.3)-.beta.-D-glucan removing agent composed of the protein and a carrier bound to the protein and a method for removing (1.fwdarw.3)-.beta.-D-glucan using the removing agent.
Furthermore, this invention relates to a method for inhibiting activation of factor G which may exist in horseshoe crab amoebocyte lysate using the protein.
Still further, this invention relates to a method for assaying endotoxin using the protein.


In 1964, coagulation or gel formation of horseshoe crab, amoebocyte lysate (hereinafter may be abbreviated LAL), with a very small amount of an intracellular toxin of Gram negative bacteria (hereinafter abbreviated as endotoxin (Et) or lipopolysaccharide (LPS)) was discovered, and multiple factors, serine protease precursors, including Et (LPS) sensitive factor (factor C), participating in the gel formation have been found. This reaction is composed of a cascade mechanism which resembles to the coagulation system of blood of mammals, and similar mechanisms have also been reported in the other invertebrates
LAL has been known to react with a very small amount of (1.fwdarw.3)-.beta.-D-glucan (hereinafter abbreviated as .beta.-glucan) to cause gel formation in addition to Et, and a sensitive factor G which recognizes .beta.-glucan has been found. An induction of gel formation as well as Et by a quite different coagulation cascade route (factor G system) from a route by way of factor C (factor C system) has been elucidated. Further, .beta.-glucan is a constructive polysaccharide of fungal cell wall and this route is presumed to be closely related to a defense system of a living body as well as factor C system, Heretofore, .beta.-glucan binding protein such as blood coagulating factor G of horseshoe crab (FEBS Lett., 129, 318-321 (1981)), .beta.-glucan recognizing protein of silkworm (prophenol oxidase) (J. Biol. Chem., 263, 12056-12062 (1988)), .beta.-glucan receptor of human monocytes (J. Exp. Med., 173, 1511-1520 (1991)), an adjuvant receptor accompanied with localized opsonin formation (J. Immunol., 124, 3307-3315 (1985)), .beta.-glucan elicitor to plant cells (J. Cell Biol., 78, 627 (1978)), a glucan binding protein derived from Streptococcus sobrinus (Infect Immun., 60 (12) 5291-5293 (1992)) and .beta.-glucan specific lectin derived from great wax moth (Galleria mellonella L., (Matha V., 64, 35-42 (1990)) have been reported.


The inventors of the present invention have been investigating the gel formation factor in LAL and found a protein which specifically binds to .beta.-glucan to inhibit the activation of factor G, and isolated the protein. Furthermore, the inventors of the present invention found an antibody which selectively recognizes the protein.
Therefore, the inventors of the present invention investigated the characteristic features of these proteins and the antibody and found their uses.
The object of the present invention is to provide a novel protein which specifically binds to such .beta.-glucan and its antibody and to apply them for the assay of .beta.-glucan and endotoxin, removal of .beta.-glucan, and further for the treatment of fungal infections.
The present invention relates to a .beta.-glucan binding protein obtained from horseshoe crab amoebocytes, purified with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to give a single band, and having the following physicochemical properties. non-reducing conditions) and about 170 k dalton (SDS-PAGE under reducing conditions)

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Enghild et al., 1996 .alpha.-Macroglobulin from Limulus polyphemus exhibitsproteinase inhibitory activity and participates in a hemplytic system. Biochemistry 29: 10070-10080.


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