JP2547264B2 - Horseshoe crab amebosite lysate factor G activation inhibitor - Google Patents

Description

TECHNICAL FIELD The present invention relates to an agent that inhibits the activation of certain zymogens, and more specifically, to horseshoe crab amebosite.
Among the factors involved in the lysate coagulation system (1 → 3)
The present invention relates to a substance that inhibits activation of a (1 → 3) -β-D-glucan-sensitive factor, that is, factor G in a system in which coagulation is initiated by a reaction with -β-D-glucan, that is, a factor G activation inhibitor.

Background Art Horseshoe Crab Amebosite Lysate (Horseshoe
crab Amebocyte Lysate-hereinafter sometimes abbreviated as LAL), was discovered by Levin and Bang in 1964, in which a gram-negative bacterial toxin (endotoxin) caused immediate coagulation (gelation) [J.Levin and FBBang. (1964) Bu
LL.Johns Hopkins Hosp., 115 , 265-274], LAL has been widely used as a specific detection assay for endotoxin (bacterial endotoxin), a so-called “Limulus Test” reagent. The extant species of horseshoe crab are 3 genera and 4 species, Li
mulus polyphemus , Tachypleus tridentatus , T. gig
as and Carcinoscorpius rotundicauda are known, among which L. polyphemus , Japanese, Chinese T.
Commercialization of the "Rimulus test" reagent using amebosite lysate from tridentatus has been carried out. [For example, Progress in Clinical and Biological Research; v
olume 93, Endotoxins and their Detection with the L
imulus Amebocyte Lysate Test, Stanly W. Watson, Jack
Levin and Thomas J. Novitsky, Editors: Published in 1
982 by Alan R. Liss, Inc.page 7-24, Title: The Limulus
Test and Bacterial Endotoxins: Some Perspectives by
See J. Levin].

LAL was initially thought to react specifically with endotoxin only, but recent studies have revealed that LAL reacts with (1 → 3) -β-D-glucan as well as endotoxin. Like the mammalian blood coagulation system, the LAL coagulation system consists of a system of stepwise reactions of multiple coagulation factors. In addition to the system that initiates reaction by endotoxin (factor C activation system), (1 → 3 It has been confirmed that there is also a system (factor G activation system) that initiates reaction by) -β-D-glucan [T. Morita et al. (1981) FEBS LETTER
S, 129 , 318-321 and S. Iwanaga et al. (1986) J. Prote.
in Chem., 5 , 255-268]. Therefore, studies have been conducted to make the Limulus test as endotoxin-specific as possible. For example, T. Obayashi et al. (1985) Cli.
n.Chim.Acta, 149 , 55-65, the fraction of the coagulation factor of LAL-
A method for measuring endotoxin using a reagent from which factor G has been separated and removed by reconstitution has been proposed, and an endotoxin-specific measurement kit based on this method is sold by Seikagaku Corporation under the trade name of “endospecy”.

However, although the above-mentioned proposed assay has an extremely strong demand as a detection assay specific to endotoxin, separation and removal of factor G from horseshoe crab amebosite lysate, which is composed of multiple coagulation factors, can be performed using endotoxin or (( It is necessary to perform fractionation operation of each factor in the absence of 1 → 3) -β-D-glucan, and endotoxin or (1 → 3) -β-D for instruments, devices and drugs used for this purpose.
-Complicated operation for complete removal of glucan is required in advance, amebosite lysate is diluted with fractionation operation, and concentration operation is required if necessary, and activity of each factor is decreased with each operation. And loss of fractions,
Core Guilurogen (coagulation protein precursor) together with Factor G
Therefore, the measurement method obtained by reconstitution of other factors cannot be applied to the gelling method, nephelometry, nephelometry, etc. that utilizes the gelation phenomenon in the "Limulus test". However, it has a drawback that it is applicable only to the synthetic substrate method, which is accompanied by a limitation of use.

Among the LAL coagulation mechanisms, the present inventors have found that (1 → 3) -β
In the course of detailed research on the system (factor G activation system) in which the coagulation (gelation) reaction is initiated by the involvement of -D-glucan, it was thought that it was only involved in the activation of factor G until now ( 1 → 3) Among β-D-glucans,
A (1 → 3) -β-D-glucoside structural unit containing a structural portion in which a specific number of consecutively bonded units is
Surprisingly, they found that the inhibitory effect is completely opposite to the activation of factor G, and completed the present invention.

DISCLOSURE OF THE INVENTION Thus, according to the present invention, the following formula A poly- (2 → 370) -bonded (1 → 3) -β-D-glucoside structural unit (molecular weight: 162)
Provided is a horseshoe crab amebocyte lysate factor G activation inhibitor, which comprises a polyglycoside containing at least one (1 → 3) -β-D-glucoside structure portion as an active ingredient.

According to the present invention, there is also provided a method for inhibiting activation of factor G present in LAL, which comprises adding an effective amount of the above polyglycoside to horseshoe crab amebosite lysate (LAL).

Hereinafter, the present invention will be described in more detail. The polyglycoside used as an active ingredient in the inhibitor of the present invention has the following formula (1 → 3) -β-D-glucoside structural unit represented by 2 to 370, preferably 3 to 310, and more preferably 4 to 180 poly- (1 → 3) -β -D-Glucoside structure part [Hereinafter, this is referred to as "poly (1 →
3) "Glucoside structure part"] is contained in one molecule.

Thus, as long as the polyglycoside used in the present invention contains at least one poly (1 → 3) glucoside structure portion in one molecule, the structure of other portions of the polyglycoside molecule is not particularly limited. You can choose from a wide range. However, it is important that the other structural parts do not substantially interact with the endotoxin and factor C activation system. For example, the polyglycoside used in the present invention is a polyglycoside consisting essentially of only one poly (1 → 3) glucoside structural moiety, for example, the following formula: In the formula, n is 2-370, preferably 3-310, and more preferably 4-.
It is an integer of 180, and can be poly- (1 → 3) -β-D-glucoside represented by, or one poly (1 → 3) glucoside structure part has the following formula: One or more of the (1 → 4) -β-D-glucoside structural units shown by and / or the following formula One or more of the (1 → 6) -β-D-glucoside structural units represented by and / or the following formula In formulas (IV), (V) and (VI), at least one of R ₁ , R ₂ and R ₃ is a chemical group such as methyl group, hydroxymethyl group, carboxymethyl group, acetyl group, sulfuric acid group and phosphoric acid group. A group capable of forming a group and a salt that can be introduced into a group represent a residue selected from a metal salt, an ammonium salt and an organic amine salt, and the rest represent a hydrogen atom. -Glucoside structural unit 1
It may have a structure in which a sugar chain composed of three or more [this sugar chain may be bonded as a branched chain to the above-mentioned poly (1 → 3) glucoside structure portion] is bonded.

Furthermore, the polyglycoside used in the present invention is 2
One or more of the above poly (1 → 3) glucoside structure moieties, together with other sugar chain structure moieties, have the following formula A ₁ -B ₁ -A ₂ -B ₂ -... Each of A ₁ , A ₂ , ... Is ₂ (1 → 3) -β-D-glucoside structural units represented by the formula (I).
~ 370, preferably 3 to 310, more preferably 4 to 18
Representing 0-linked poly- (1 → 3) -β-D-glucoside structural moieties, the number of units of formula (I) constituting each structural moiety of A ₁ , A ₂ , ... May be different,
And B ₁ , B ₂ , ... Represents the same or different sugar chain structure portions, respectively, and may have a structure linked as shown in.
Here, as the other sugar chain structure represented by B ₁ , B ₂ , ..., For example, the above formulas (II), (III),
A structural portion consisting of one or more blocks of the structural unit represented by (IV), (V) or (VI) can be mentioned.

Furthermore, the polyglycoside used in the present invention includes the poly (1 → 3) glucoside structure portion, the above B ₁ , B ₂ , and
The length in which 371 or more consecutive (1 → 3) -β-D-glucoside structural units represented by the above formula (I) are bound to another sugar chain structure represented by It may have a structure in which the poly- (1 → 3) -β-D-glucoside structure portion of the chain is linked.

Therefore, the polyglycoside used in the present invention is not particularly limited in its molecular weight as long as it contains at least one poly (1 → 3) glucoside structure portion in one molecule, As the solubility of the product decreases and the viscosity increases, handling becomes difficult, which may be disadvantageous in terms of obtaining a certain quality when preparing the endotoxin measurement kit, so the molecular weight is generally 500,00.
0 or less, preferably 500 to 240,000, more preferably 650
Those within the range of to 80,000 are convenient.

Further, the polyglycoside used in the present invention preferably consists essentially of one containing at least one poly (1 → 3) glucoside structure portion in the molecule, but it does not necessarily have to be so. , For example, a high molecular weight poly- (1 → 3) -β-D-glucoside in which 371 or more consecutive (1 → 3) -β-D-glucoside structural units represented by the above formula (I) are bound. Other polyglycoside containing a structural portion may be mixed. What is important is that the polyglycoside according to the present invention can be used as a factor G which is an initiation factor of the factor G activation system of LAL and a high molecular weight poly- (1 → 3) -β-D which is a factor G activator. -Binds faster and stronger than glucoside and inhibits activation to activated factor G, and the presence of such high molecular weight poly- (1 → 3) -β-D-glucoside has a substantial effect on its inhibitory action. Because there is no.

When a polyglycoside mixed with other components as described above is used as an inhibitor, the content of the polyglycoside according to the present invention in the inhibitor is not particularly limited, but if the content is too low, the factor G Since it is uneconomical to use a large amount of the polyglycoside to inhibit the activation of the above, it generally accounts for at least 5% by weight, preferably 10% by weight or more, more preferably 20% by weight or more. desirable.

In the present specification, the molecular weight of polyglycoside is
Using a standard substance of known molecular weight, perform gel permeation chromatography under the following conditions to create a standard curve, then perform the chromatography on the sample under the same conditions, and compare the results with the standard curve. It is the value obtained by.

Column: TSKgel G-PW _XL series (Tosoh Corporation) 7.8
× 300 mm Several types Mobile phase: 0.3 M NaOH Flow rate: 0.5 ml / min Sample solution concentration: 0.1-5 mg / ml Sample solution injection amount: 0.1 ml Column temperature: Room temperature Detection method: Measurement with differential refractometer (LKB) Or sugar quantification by phenol-sulfuric acid method Standard substances: TSK standard polyethylene oxide (Tosoh Corporation) and polyethylene glycol (Hanai Chemical Co., Ltd.) with weight average molecular weights of 1,000 to 860,000 are used.

The polyglycoside having the above-mentioned properties, which is used as a factor G activation inhibitor in the present invention, may be naturally derived, or synthesized or represented by the above formula (I) (1 → 3) A part of poly (1 → 3) -β-D-glucoside containing 3 or more -β-D-glucoside structural units may be chemically modified. Usually, naturally derived ones are easier to obtain.
Specific examples of such polyglycoside include those described below.

(1) (1 → 3) -β-D-g represented by the formula (I)
Substantially straight-chain consisting essentially of rucoside structural units
Polyglucosides: For example, Alcaligenes (Alcali
genes) (1 → 3) -β-D-gluca derived from bacteria
Flagella (Euglena) Derived paramylon; of higher plants
Kalo extracted from β-glucan of fiber tissue or sieve tube
The above (1 → 3) -β-D-glucan orLaminaria
(Kombus spp.),EiseniaDerived from brown algae such as
The high degree of polymerization contained in partial hydrolyzates such as laminarans
D-glucose polymer composed of (1 → 3) -β-bond;
Laminari dextrin (polymerization degree 10-20), Lamina
Li-oligosaccharide (having a degree of polymerization of 10 or less), etc.

(2) Poly- (1 → 3) -β- represented by the above formula (I)
A polyglycoside containing both the D-glucoside structural unit and the (1 → 6) -β-D-glucoside structural unit represented by the formula (III): for example, a) consisting of (1 → 3) -β-bond Glucose or glucose polymer in which one to several (1 → 6) -β-bonded chains are incorporated in the main chain, for example, laminarans derived from Eisenia brown algae.

b) The sugar chain of (1 → 3) -β-bond is further branched to the glucose or glucose polymer linked with the (1 → 3) -β-bond of the above a) by the (1 → 6) -β-bond. , And particularly those that may contain other sugar moieties in a part of the sugar chain, such as Lamina
ria Laminarans from brown algae, Ochromona
s , Phaeodactylum , Skeletonema , Biddulphia , Coscino
Chrysoraminarans derived from diatoms such as discus and Chaetoceros , and pakiman derived from Poria .

c) having more branches and having a dendritic structure,
For example, glucan derived from the cell wall of Phytophthora , and the like.

d) a glucose having a (1 → 6) -β-bond linked to a linear glucan composed of a (1 → 3) -β-bond, for example, branched at a ratio of 1 residue per 3 glucose unit residues. Schizophyll from Sclerotinia
um (Suehirotake), Schizophyllan, Sclerotium , Cort
Scleroglucans derived from icium , Stromatinia , etc.

In addition, the ratio of 2 residues per 5 glucose units of the linear glucan consisting of (1 → 3) -β-bond is (1 → 6).
Those in which glucose is bound by -β-bond, for example,
Lentinus from Lentinus , etc.

e) A linear glucan composed of (1 → 6) -β-bonds, in which a plurality of glucose chains are branched from the C-3 position of glucose by (1 → 3) -β-bonds, for example, Saccharomyce
β-glucan derived from the cell wall of s (baking yeast), and the like.

(3) (1 → 3) -β-D-glucoside structural unit represented by the formula (I) and (1 → 4) represented by the formula (II).
A polyglycoside containing both -β-D-glucoside structural units: for example, rhihenans derived from Cetraria , Usnea , Evernia and the like, β-glucan contained in barley endosperm and the like.

Some of the polyglycosides mentioned above are available as commercial products, and they can be used as they are as the inhibitor of the present invention, but if necessary,
Fractions rich in polyglycoside containing (1 → 3) -β-D-glucoside structural unit represented by the formula (I) in the above-mentioned specific amount by partially decomposing saccharides and / or subjecting to fractionation treatment. It is also possible to prepare a portion and utilize it for the inhibitor of the present invention.

The partial decomposition and fractionation of the sugar chain can be carried out by a method known per se. For example, partial decomposition of sugar chains can be performed by acid or alkali, hydrolysis using β-glucanase, acetylation decomposition, sonication, or the like. Further, the molecular weight fractionation can be performed by a fractional precipitation method using an organic solvent such as alcohol, acetone, ether or the like, or a salt, or a fractionation using a molecular sieve or a molecular sieve membrane.

In addition, the polyglycoside as exemplified in the above (1) to (3), a part of the sugar chain is an alkyl group such as a methyl group,
It may be chemically modified with a hydroxyalkyl group such as a hydroxymethyl group, a carboxyalkyl group such as a carboxymethyl group, an acid group such as an acetyl group, a sulfuric acid group and a phosphoric acid group, and other groups. They can be prepared by introducing such a group by a method known per se [eg, (1) Ando, Terayama, Nishizawa, Yamakawa ed., Biochemical Research Method I, 284-303 (1967), Asakura Shoten,
(2) Whistler, RLed.; Methods in Carbohydrate Che
mistry III, 193-267, 271-331 (1964), Academic Pres
s etc.]. In particular, it has a molecular weight of about 6 which has a G factor activating effect.
More than 10,000 (1 → 3) -β-D-glucans have their poly- (1 → 3) -β-D-glucans due to partial chemical modification.
By setting the number of continuous bonds of the (1 → 3) -β-D-glucoside structure unit represented by the above formula (I) in the glucoside structure portion to 370 or less, it can be used as the inhibitor of the present invention.

The specific examples of the polyglycoside that can be preferably used in the present invention are as follows.

-Laminari oligosaccharide having a molecular weight of 342 to 1,638-Laminaridextrin having a molecular weight of 1,800 to 3,258- (1 → 3) -β-D-glucan having an average molecular weight of 2,000 to 60,000-Laminaran having an average molecular weight of 3,000 to 23,000-Average molecular weight 3,000 to 20,000 sclerotan, -Schizophyllan with an average molecular weight of 500,000 or less-Lentinan with an average molecular weight of 1,100,000 or less-Baker's yeast glucan water-soluble matter with an average molecular weight of 12,000 or less-Richhenan with an average molecular weight of 33,000 or less-Average molecular weight of 200,000 or less Barley β-glucan, for example, partial carboxymethylated (1 → 3) -β-D-glucan having an average molecular weight of 40,000 to 240,000 obtained by partial carboxymethylation of curdlan and salts thereof (substitution degree: 0.003 to 1.0),・ Partially carboxymethylated laminaran with an average molecular weight of 23,000 or less and its salts (substitution degree: 1.0 or less) ・ Average molecular weight of 80,000 or more Lower partial methylation (1 → 3) -β
-D-glucan (substitution degree: 0.003 to 1.0),-Partially sulfated laminaran having an average molecular weight of 23,000 or less and a salt thereof (substitution degree: 1.0 or less).

The polyglycoside according to the present invention described above has an action of strongly inhibiting the activation of factor G present in LAL, as demonstrated in the examples described later, and therefore, in the limulus test, factor G It can be used to specifically detect and measure endotoxin in a sample without being affected by the activation system. At the time of use, polyglycoside as a factor G activation inhibitor is usually used in the LIM used in the Limulus test in an amount greater than or equal to the amount necessary to completely block the activation of factor G in the LAL during measurement. Added or pre-added to LAL,
It can be added when preparing and extracting LAL.

Here, the amount of the inhibitor required to completely block the activation of factor G in LAL can be determined, for example, as follows.

L under a constant amount of LAL under ice-cooling under normal measurement conditions
Add a certain amount of a G factor activator that sufficiently activates AL (one that does not contain endotoxin, and one that does not contain a G factor activation inhibitor as much as possible), to which an inhibitor (does not contain endotoxin) is added. )) At different concentrations and react under the same conditions as when using normal LAL. Under this condition, the concentration of the inhibitor that inhibits LAL activation by 100% is determined.

Next, add the above-determined concentration of inhibitor to the above-mentioned fixed amount of LAL.
In addition, by changing the amount of the factor G activator,
Make sure that LAL is not activated at any concentration.

By the above operation, the amount (concentration) of the inhibitor necessary to completely inhibit the activation of factor G in a certain amount of LAL can be determined.

The amounts of the G factor activation inhibitors required to completely block the G factor activation of various commercially available lysates thus obtained are shown below.

As is clear from the required amount of the inhibitor shown in Table 1 above, the Limulus test reagent comprising LAL contains at least 50 ng, preferably 100 ng or more, more preferably 100 to 230 ng, most preferably the polyglycoside according to the present invention per 1 ml of LAL. Is suitable to be added within the range of 230 to 500 ng.

Hereinafter, a method for preparing the factor G activation inhibitor of the present invention, a mechanism of action, a Limulus test reagent using the inhibitor, a kit and the like will be described in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a molecular sieve (GPC) fractionation pattern of commercially available curdlan, and FIG. 2 is a re-chromatography fractionation pattern of Nos. 44 to 46 in FIG.

[Preparation Method of Factor G Activation Inhibitor of the Present Invention] The factor G activation inhibitor of the present invention can be prepared, for example, by the method shown in the following Preparation Example. In addition, (1
→ 3) Among β-D-glucans, those within the scope of the present invention can be used as they are.

Preparation Example 1: Preparation by molecular sieve chromatography fractionation from commercially available curdlan Curdlan (Wako Pure Chemical Industries, Reagent, Lot No. PEQ 908
0, Mn> 136,000, Mw / Mn> 2.76) 0.3g of 1g of sample No. 101
The solution was dissolved in NaOH at a concentration of 5 mg / ml, and 100 μl of each was subjected to gel permeation chromatography (hereinafter GPC) at room temperature under the following conditions. {Column: TSKgel G6000P
W _XL and G5000PW _XL (both 7.8 x 300 mm) are connected in series,
Mobile phase: 0.3 M NaOH, flow rate: 0.5 ml / min}. The eluted low molecular weight fraction (No.44-46) was collected and re-chromatographed to obtain 0.015 mg of a sample having a number average molecular weight of 3,050 and a polydispersity of 1.29 (Sample No.1). The GPC fractionation pattern is shown in the attached FIG. Further, the attached fractional pattern is shown in FIG.

This sample No. 1 was digested with β-1,3-glucanase (Zymolyce-100T, manufactured by Seikagaku Corporation), and the enzyme digestion solution was treated with GP.
C (column: TSKgel G4000PW _XL , G3000PW _XL , G2500PW _{XL in-} series; mobile phase: distilled water, flow rate: 0.6 ml / min) and analyzed for sugar composition (glucose 40%, laminaribiose)
30%, laminaritriose 20%, laminaritetraose 8%, laminaripentaose 2%, recovery rate 94%) were confirmed. From this, the sugar structure of this sample (No. 1) is (1 →
3) It can be seen that the β-polyglucoside contains a -β-D-glucoside structure portion.

Preparation Example 2: Fractionation Based on Difference in Solubility of Curdlan in Water Commercially available curdlan (Sample No. 101) (50 g) was suspended in distilled water and fractionated by the operation shown in the following flow sheet.

Preparation Example 3: Preparation of curdlan water-insoluble sugar fraction by formic acid decomposition 45 g of sample No. 102 was subjected to the method of K. Ogawa et al. [Carbohydr.
s., 29 , 397-403 (1973)]. The operation details are shown in the flow sheet below.

Preparation Example 4-1: Re-fractionation of the water-soluble fraction of curdlan formic acid decomposition product by molecular sieve. Water-soluble fraction obtained in Preparation Example 3 shown above (Sample No. 3).
Dissolve 0.15 g in 30 ml of distilled water and GPC (column: TSK gelG3000P
W _XL x 2, G2500PW _XL x 1, mobile phase: distilled water, flow rate 0.5 ml /
min) was collected for each 0.5 ml, and re-chromatography was performed to obtain 6 kinds of samples (No. 11 to 16) having different molecular weights.

Preparation Example 4-2: Re-fractionation of water-insoluble fraction of curdlan formic acid decomposition product by molecular sieve 40 g of 0.2 g of the water-insoluble fraction (Sample No. 4) obtained in Preparation Example 3
Dissolve in 0.3 ml of 0.3 M NaOH solution, GPC (column: TSK gel G300
0PW _XL x 2, G2500PW _XL x 1, mobile phase: 0.3 M NaOH solution, flow rate 0.5 ml / min) was fractionated and rechromatographed in the same manner as in Preparation Example 4-1 above, and the eluent was 0.3 M HCl solution. Two types of samples with different molecular weights (No. 17 and 18)
I got

Preparation Example 5: Preparation of sample by sonication from curdlan water-insoluble fraction 1 g of sample No. 102 was suspended in about 100 ml of 5 mM NaOH solution,
Sonic generator under ice cooling, Sonicator ^TM (Otake Manufacturing, model
5202PZT, Tokyo) was used to reduce the molecular weight by sonication at 20 KHz, 80w for 12 minutes.

Using 5M NaOH as the treatment liquid to make a final 0.3M NaOH solution, perform chromatographic fractionation according to Preparation Example 4-2 above, and perform 8 types of samples with different molecular weights (No. 19 to 22 and 103 to
106) was obtained.

Preparation Example 6-1: Preparation of Inhibitor from Seaweed (I) The sample derived from arame ( Eisenia bicyclis ) is T. Usui.
Et al. Agric.Biol.Chem. 43 , 603-611 (1979) according to the method, after crushing 100 g of dried dried algae (Tokyo, Suita Shoten),
The low molecular weight soluble fraction is extracted and removed with 80% ethanol, and the laminaran fraction is extracted from the residue using a 2% CaCl ₂ aqueous solution. Then 95% ethanol was used for the extract to a final concentration of 75
% Solution, the resulting precipitates are collected by centrifugation and washed with ethanol to obtain a crude laminaran sample. The crude sample was redissolved in distilled water to remove acidic substances (alginic acid etc.) and pigments contaminated by anion exchanger (DEAE-Toyopearl),
Sample No. 25 was obtained from ethanol reprecipitation.

Preparation Example 6-2: Preparation of Inhibitor from Seaweed (II) JJConne is a sample derived from Laminaria japonica
ll et al., J. Chem. Soc., 3494 (1950), crushed 100 g of dried mackerel dried algal cells (Tokyo, Suita Shoten) to 0.09
Static extraction with M HCl solution for about 3 days, insoluble matter is filtered off,
The filtrate was allowed to stand for another day, the resulting small amount of precipitate was removed by centrifugation, and 3 times the volume of ethanol was added to the supernatant to give about 75%.
A solution was prepared, and the resulting precipitate was collected by centrifugation, washed with alcohol and dried to obtain a water-soluble laminaran fraction (Sample No. 27).

Preparation Example 7-1: Sample Sukurerotan from fungi prepared from the inhibitor (I) fungi Sclerotinia Libertiana is Kitahara et al,岐大agricultural Report 8, 100-105 (1957) defatted dry powder sclerotia of Sclerotinialibertiana according to the method of (3
The residue obtained by sufficiently extracting 0 g) with water was extracted with 7% NaOH solution, 10% CuSO ₄ solution was added to the extract to precipitate, and this was separated and washed with hydrochloric acid-acidic methanol to remove copper. , 80
% Of methanol to remove HCl, and washing and drying with methanol and ether were repeated 3 times to obtain 6 g of sample No. 28.

Preparation Example 7-2: Preparation of fungal-derived inhibitory substance (II) Fungus Schizophyllum commune : Samples derived from Suehirotake are commercially available Schizofiran (Kaken Pharmaceutical: trade name Sonifilan, drug: Lot No. J61040), K. Tabata et al., Carbohydr .
Res., 89 121-135 (1981), in accordance with the procedure of Preparation Example 5 described above, after sonication for 10 hours in an aqueous solution, three kinds of samples (No.
9, 30, 31) was obtained.

Preparation Example 7-3: Preparation of Inhibitor from Fungus (III) Yeast Saccharomyces cerevisiae : β from Baker's Yeast
-The glucan sample is a commercially available baker's yeast glucan (Sigma Lo
t No.56F-4027) 90 ml with distilled water 50 ml, stirred at room temperature for 2 hours and then centrifuged, and about 50 ml of the supernatant is concentrated under reduced pressure.
The insoluble matter was made up to 1 ml, and the insoluble matter was again removed by centrifugation to obtain 0.64 mg of a sample (No. 33) from the supernatant.

Preparation Example 8: Preparation of sample derived from barley β-glucan Commercial barley β-glucan (Sigma, Lot No.56F-065
2) was made into a 5 mg / ml solution with 0.3 M NaOH, and the above Preparation Example 4 was used.
-2, a β-glucan sample (No. 36) having a narrow molecular weight distribution was obtained by molecular sieve fractionation under alkaline conditions.

Further, the above-mentioned commercially available barley β-glucan was dissolved in hot water at a concentration of 5 mg / ml, and the supernatant obtained by centrifugation (3,500 rpm, 10 minutes) was distilled water as a mobile phase according to Preparation Example 4-1. 100 μl
GPC fractions were collected 50 times each and further re-fractionated under the same conditions to obtain two kinds of samples (Sample Nos. 37 and 38) having different molecular weights.

Preparation Example 9: Partial carboxymethylation (1 → 3) -β-D-
Preparation of glucan (degree of substitution DS = 0.63) The water-insoluble matter of curdlan obtained according to Preparation Example 2 was treated with AEClar.
ke and BAStone: Phytochemistry 1 , 175〜188 (196
Carboxymethylated according to the method of 2). That is, 1
00g curdlan water insoluble matter under nitrogen flow at 0 ℃ 1 5M
A solution prepared by dissolving 236 g of monochloroacetic acid in 200 ml of water was added dropwise while stirring in a NaOH solution, and after the addition, the mixture was stirred at 60 to 65 ° C for 2 hours. The resulting gel was vortexed in 2.5 volumes of ethanol, pulverized and filtered. 70%
After thoroughly washing with ethanol, washed with ethanol, ether and ether, and dried. This product was dissolved in 7 liters of water, neutralized with 1M acetic acid, 40 g of activated carbon was added, and the mixture was stirred at room temperature for 1 hour and filtered. The filtrate was concentrated under reduced pressure to 1 to add 3 volumes of ethanol to form a precipitate, which was washed with ethanol and ether and dried over concentrated sulfuric acid under reduced pressure to obtain 113.85 g.

The obtained curdlan partial carboxymethylation (1 →
3) -β-D-Glucan was prepared by DFDurso's Uranyl Nitrate Method Methods in Carbobydrate Chem.VIII, 127-129 (198).
0) The degree of etherification (degree of substitution: Degr
The ee of Substitution (DS) was 0.63. This means that 0.63 out of 3 replaceable hydroxyl groups per glucose residue forming a sugar chain were replaced.

Obtained partial carboxymethylation (1 → 3) -β-D
-Dissolve 25 mg of glucan in 5 ml of 0.1 M ammonium acetate aqueous solution, GPC (column: Toyopearl HW65F, 5 x 100 cm;
Mobile phase: 0.1 M ammonium acetate solution; Flow rate: 5.8 ml / mi
was fractionated collected by n), GPC (column with another column: TSKgelG6000PW _XL + G5000PW using _XL + G3000PW _XL in series; mobile phase: aqueous 0.1M ammonium acetate; flow rate: 0.6 ml /
re-fractionation of sample No.41 with narrow molecular weight distribution
(Mn = 231,000) was obtained.

In addition, partial carboxymethylation (1 → 3) -β-D-
Dissolve 0.3 g of glucan in 30 ml of distilled water and sonicate (9 kH
z, 180-130W, 1 hour, using a sound generator, Kubota Seisakusho, Insonator Model 201M) to lower the molecular weight, and after adding 0.5 ml of 1 M ammonium acetate aqueous solution to 4.5 ml of the mixture and mixing, GPC fraction collection and GPC re-fractionation collection were performed in the same manner as the procedure for obtaining .41 to obtain two types of samples (No. 39 and 40) having different molecular weights.

Preparation Example 10: Partial carboxymethylation with a degree of substitution of 1.2 (1 →
3) Preparation of -β-D-glucan 10 g of carboxymethylated (1 → 3) -β-D-glucan having a degree of substitution (DS) of 0.63 obtained according to Preparation Example 9 under nitrogen stream at 0 ° C was added to 25 ml. Add to 10.5M NaOH to make a paste,
While stirring well, add monochloroacetic acid aqueous solution (10 g /
12 ml), heated to 60 ° C., stirred for 4 hours, cooled, added 30 ml of 2M HCl, and then poured into 200 ml of hydrochloric acid-acidified ethanol (40 ml HCl / ethanol), and the resulting precipitate was collected to obtain 70% ethanol. After being washed with, washed with ethanol and ether, and dried under reduced pressure to obtain a sample No.107 standard product.

The degree of substitution was measured by the same method as for the partial carboxymethylated (1 → 3) -β-D-glucan with DS = 0.63 shown in Preparation Example 9, and as a result, it was DS = 1.20.

Preparation Example 11: Preparation of partially carboxymethylated laminaran The partially carboxymethylated laminaran is Laminaria dig.
Using itata laminaran (Lot No. 77F-3885, Sigma), as in the partial carboxymethylation method of Preparation Example 9, AE
Clarke and BAStone:. Phytochem 1, 175 was prepared according to the method described in (1962), to obtain a preparation of the sample No.42 (DS = 0.06).

Preparation Example 12: Preparation of partially methylated (1 → 3) -β-D-glucan 3.0 g of a curdlan water-insoluble substance obtained according to Preparation Example 2 was added to M.Sa.
According to the method of mec, Kolloid-Beihefte 51 , 369 (1940),
Suspend in 80 ml of water and under a nitrogen stream, saturated aqueous caustic soda solution 1.
35 ml was added and completely dissolved, 60 g of dimethylsulfate was gradually added thereto at 4 ° C., and after about 1 hour, the reaction solution was added dropwise to acetone, and the resulting precipitate was collected, washed thoroughly with acetone and concentrated. After drying under reduced pressure on sulfuric acid, the title preparation (Sample No. 43, DS
0.13) was obtained.

Preparation Example 13: Preparation of partially-sulfated laminaran The laminaran derived from Laminaria digitata was subjected to sulfuric esterification using pyridine-3 sulfur oxide complex (Wako Pure Chemical Industries, Ltd., Lot No. PPL8823) in pyridine as follows.

Fully dried Laminaria digitata- derived laminaran (Sigma, Lot No.77F-3885) 0.5 g is dissolved in 50 ml of dehydrated pyridine, 1 g of pyridine-3 sulfur oxide complex is added, and reacted at 60 ° C. for 1 hour, Add 100 ml of water, cool, N
It is neutralized with aOH, dialyzed against water using a dialysis membrane (Spectropore 1,000 cuts) that has been thoroughly washed with an alkaline aqueous solution to remove glucan in advance, then concentrated and added with 2 volumes of acetone to add sugar components. Was precipitated, washed with acetone, and dried under reduced pressure on concentrated sulfuric acid to obtain 0.38 g of the title preparation (sample No.44, DS = 0.14).

In addition, the substitution degree of the methyl group and the sulfate group of each of the preparations shown in Preparation Examples 12 to 13 was measured and calculated according to the method of the following literature.

Ochiai, Tsuda, Sakamoto; Organic quantitative analysis method (minor volume), Nanzandou (1956); Whistler, RLed., Methods in Carbohydrate Chemist
ry III, p229 to 235, 277 to 280 (1964), Academic Press [Commercial Sample] The following commercially available sample was used for measurement as it was or after being solubilized with an alkali and neutralized.

Glucose: (Wako Pure Chemicals, JIS Special Grade Reagent): Sample No.108 Laminarioligosaccharide: (Seikagaku Corporation, Pure Reagent): Sample Nos. 5-10 Laminaran Eisenia araborea Origin: (Hanai Chemistry, Reagent): Sample No.23 〃 E. araborea origin: (Tokyo Kasei, reagent):
Sample No.24 〃 Laminaria digitata origin: (Sigma reagent): Sample No.26 lentinan Lentinus edodes (Shiitake) origin: (Yamanouchi Pharmaceutical, Pharmaceutical Lot No.CKC7): Sample No.32 Richenan Cetraria islandica origin: (Sigma) ,
Reagent): Sample No.34 from Usnea barbata : (Sigma, Reagent): Sample No.35 Examples 1-44 The following table shows the measurement results of the molecular weight of each of the above samples, the factor G activation inhibitory potency, etc. 2 shows.

The molecular weights in the table are represented by the number average molecular weight (Mn) defined by the following equation determined by GPC, and the molecular weight distribution is represented by the polydispersity (Mw / Mn) defined by the following equation.

Polydispersity = Mw / Mn However, Hi represents the i-th peak height (sample concentration) when the chromatogram is divided into equal parts in time, and Mi represents the i-th molecular weight.

The factor G activation inhibitory titer was measured by [Activity titer measuring method for factor G activation inhibitor] shown below and shown as a unit per mg.

[Method for measuring activity titer of factor G activation inhibitor (hereinafter sometimes abbreviated as GI)] 200 μl of the reaction mixture contains the following. (1)
Specimen (Note 1) GI sample or pure water 50 μl [G factor activator (abbreviated as GA, Note 2)] 10 pg added or not added (2) Horseshoe crab lysate coagulation enzyme precursor fraction (A ₂₈₀ = 2.5) (Note 3) 30 μl (3) Horseshoe crab lysate factor G fraction (A ₂₈₀ = 0.9) (Note 3) 20 μl (4) Tris-HCl buffer (pH 8.0) 20 μmole (5) MgCl _{2 20} μmole (6) Boc-Leu- Gly-Arg-pNA (t-butoxycarbonyl-L-leucyl-glycyl-L-arginine-p-nitroanilide) 0.13 μmole After incubating the reaction solution at 37 ° C. for 30 minutes, release of pNA (paranitroaniline) 0.5 ml each of 0.04% sodium nitrite (0.48M HCl solution), 0.3% ammonium sulfamate and 0.07% N-1-naphthylethylenediamine dihydrochloride were added sequentially, and the color was converted by diazo coupling, and the absorbance at 545 nm was measured. (A ₅₄₅ )
Measure as quantity.

 GI activity is calculated by the following formula.

Under these conditions, the amount of GI that completely inhibits the activation of factor G by GA is 100 units.

(Note 1) Of the samples, those that are insoluble in water should be dissolved in 0.3M NaOH and then neutralized by adding an equal volume of 0.3M HCl.

(Note 2) GPC fraction purified sample of curdlan sonicated product prepared in Preparation Example 5 (Table-2, No. 106, molecular weight 216,00)
0).

(Note 3) Reference [T. Obayashi et al., Clin. Chim. Acta, 14
9 , 55-65 (1985)] according to Japanese horseshoe crab T. trident
Prepared from atus .

 The following is clear from the contents of Table 2 above.

(A) A commercially available curdlan known as a factor G activator (sample No. 101) contains a water-soluble low-molecular weight factor G activation inhibitor (samples Nos. 1 and 2).

(B) A polyglycoside in which the number of linked (1 → 3) -β-D-glucoside structural units of the polyglycoside composed of the (1 → 3) -β-D-glucoside structural portion is in the range of 2 to 370. Shows the G factor activation inhibitory action of the present invention.

(C) A fraction having a molecular weight of 60,000 or less obtained by preparing various high molecular weight β-glucan fractions (Sample No. 102) showing no inhibitory activity by various molecular weight reduction procedures has a factor G activation inhibitory potency. It was expressed (Sample Nos. 3, 4, 11 to 22).

(D) Barley in which a poly- (1 → 3) -β-D-glucoside structure portion having a degree of polymerization of 10 or less and a poly- (1 → 4) -β-D-glucoside structure portion are linked in a block shape β-glucan [Ballance et al., Carbohyd. Res., 61 , 107-118 (197
8)]] (Preparation Example 8, Sample Nos. 36, 37, 38) all show activity equivalent to laminaritetraose or laminaripentaose, and even if the whole glucan has a molecular weight of 60,000 or less, It can be used as an inhibitor of the present invention.

(E) Among the partially carboxymethylated (1 → 3) -β-D-glucans obtained in Preparation Example 10, the average degree of substitution is 1.
In the samples of 0 or more (Sample No.107 DS = 1.2), the factor G activation inhibitory effect disappeared, and the sample showing high factor G activation inhibitory titer mentioned as sample No.26 was partially carboxymethyl. When the chain length of the (1 → 3) -β-D-glucoside moiety was shortened by the chemical reaction (Preparation Example 11) (Sample No. 42) and by the modification with the sulfate ester of Preparation Example 13 (1 → 3) -β-D −
It was also confirmed that shortening the glucoside structure portion also reduces the factor G activation inhibitory effect.

(F) A (1 → 3) -β-glucan having a degree of polymerization of the (1 → 3) -β-D-glucoside structure portion of 370 or more and a molecular weight of 60,000 or more and exhibiting a G factor activating effect. Also, when the structure defined by the present invention is obtained by partial methylation (Preparation Example 12, Sample No. 43) and partial carboxymethylation (Preparation Example 9, Sample No. 41), a factor G activation inhibitory effect It has been confirmed that

[Mode of action of the inhibitor of the present invention on factor G] The factor G activation system of the horseshoe crab blood coagulation system is T.Mo.
It is shown as the chart below as reported by rita et al., FEBS Letters, 129 , 318-321 (1981).

The following experiment was conducted for the purpose of clarifying which part of the coagulation system the inhibitor of the present invention inhibits.

In a reaction mixture of 200 μl, 5 μg of a factor G activation inhibitor (laminariheptaose, abbreviated as sample No. 10, I)
g, Factor G activator (curdlan sonicated GPC fraction fraction, abbreviated as sample number 106, A) 3 pg, reference [T. Obayashi
et al., Clin. Chim. Acta, 149 , 55-65 (1985)], 20 μl of the factor G fraction (abbreviated as G), 30 μl of coagulase precursor fraction (abbreviated as P), and Tris- HCl buffer (pH8.0) 20 μmole, MgCl _{2 20} μmole, synthetic substrate Boc-Leu
-Gly-Arg-pNA (abbreviated as S) 0.13 μmole is included.

By changing the order of addition of each component and the heating conditions, the degree of inhibition of activation of the factor G activation system was determined in each experiment (1 to
In 5), the results of measurement with no inhibitor added as a control are shown below.

As is clear from Experiments 1, 2, and 3, when the inhibitor (I) of the present invention was added to the G factor (precursor), the G factor activating substance (A) Activation is 100% inhibited.

On the other hand, as is clear from Experiments 4 and 5, factor G is (A)
Once activated, the active factor G is not inhibited even if the inhibitor (I) is allowed to coexist.

Therefore, the inhibitor (I) of the present invention is a factor G (precursor)
It can be said that it is a substance that acts only on.

As long as the inhibitor (I) of the present invention is present in an amount that inhibits the activation of factor G present in LAL by 100%, no matter how large the amount of activator (A) is, Activation does not occur. However, when a large amount of the activating substance (A) is present in the presence of a small amount of the inhibitor (I) that cannot inhibit the G factor 100%, the G factor not inhibited by the inhibitor (I) is activated. It was also confirmed that it was activated by the activated substance (A).

As a result, A. Kakinuma et al., Biochem. Biophys.
Res.Commun., 101, pointed out 434~439 (1981), (1 →
3) -β-D-glucan derivative and T. Morita et al.
him.Biol.Res., 189 , 53-64 (1985)
It was possible to analyze the existence of the maximum activating concentration in the horseshoe crab factor G activating ability by glucan.

Example 45: Comparison of endotoxin-specific measurement ability of kits with and without addition of factor G activation inhibitor Substance comparison of endotoxin specificity with and without addition of the factor G activation inhibitor of the present invention to LAL-Test Was performed using various samples by the following operations.

The LAL-Test product used is Toxicolor Test ^™ (colorimetric method, manufactured by Seikagaku Corporation), which has the following structure.

Perchloric acid, sodium hydroxide, buffer, lysate + color-developing synthetic substrate, endotoxin-free distilled water,
Standard endotoxin, hydrochloric acid, sodium nitrite, ammonium sulfamate, N- (1-naphthyl) ethylenediamine dihydrochloride.

Of the above kit composition, a reaction solution group (A-kit) in which the LAL reaction main agent was dissolved using a solution of Sample No. 13 as a factor G activation inhibitor in a concentration of 5 μg / ml in a buffer solution, and As a reaction solution group (B-kit) in which a buffer solution containing no inhibitory substance was dissolved, the reactivity of both A and B kits for various samples was compared and shown in Table 3 below.

Specimens Nos. 1 to 5 are dissolved in the solvent as they are according to the measurement manual of the Toxicolor test, and 0.1 ml thereof is used as a sample for the reaction. Specimens No. 6 (ar) are T.Ob.
ayashi's method [J.Lad.Clin.Med., 104 , 321-330 (198
4)], a plasma sample was pretreated using the above-mentioned kit configuration, and 0.1 ml thereof was used as a sample for the reaction.

Regarding the reactivity of both A and B kits, each sample was added to the reaction solution prepared by using the kit composition, reacted at 37 ° C for 30 minutes, and the resulting pNA was developed with a coupling reagent of ~, and the absorbance at 545 nm Shown by value.

The maximum reactivity of this kit composition was ΔA ₅₄₅ = 1.5.

As can be seen from Table-3, both the A-kit and the conventional B-kit containing the factor G activation inhibitor of the present invention,
Although the results showed the same reactivity with endotoxin sample (No. 1), the curdlan water-insoluble fraction (sample No. 2), which is a factor G activator, was extremely high in the B-kit. Was exhibited. On the other hand, although the sample did not show any reactivity in the A-kit, when it was combined with endotoxin (sample No. 3), it showed the same reactivity as the endotoxin of sample No. 1.

Cellulose dialysis membrane washing solution known as Limulus test positive substance (non-endotoxinic) [FCPearson et al., Artif. Organs, 8 , 291-298 (198
4)] was used as a sample (No.4, No.5), the results are similar to those of the above-mentioned curdlan water-insoluble fraction and / or endotoxin addition in both A-kit and B-kit. It has been shown.

From the above results, the factor G activation inhibitor of the present invention was tested for LA
It can be seen that the combined use with L enables endotoxin-specific measurement.

Furthermore, by comparing the A-kit of the present invention with the conventional B-kit of the clinical blood sample that was not clearly determined by the conventional LAL-Test as to whether it was true endotoxemia, the result of bacterial culture was In the samples (No. 6; b to g) in which the presence of Gram-negative bacteria was confirmed, both A and B kits showed high reactivity, while (1 → 3) -β-D
-In the sample (the same: h to l) in which the presence of a fungus known to have glucan in the cell wall of the cell is confirmed, A
-No reactivity in the kit was shown, high reactivity was shown in the B-kit. Also, in the hemodialysis patient (chronic renal failure) specimen (m: r), which is not considered to be endotoxemia based on clinical symptoms, reactivity with A-kit was not observed, and abnormally high value with B-kit was observed. Is derived from dialysis membrane (1 → 3)
It was predicted to be due to -β-D-glucan.

By the combination of LAL-Test and the factor G activation inhibitor of the present invention, it is possible to measure a clinical specimen suspected of being an infectious disease or sepsis in which presence or absence of endotoxin such as the above-mentioned specimen is unclear. In addition to the advantage of being able to accurately discriminate negative bacterial infections (endotoxemia), since fungal infections can be discriminated, the selection, treatment, and treatment of an appropriate therapeutic agent for the infectious bacteria by early determination of the infectious bacterial type Providing a kit containing the inhibitor of the present invention, which enables the analysis of effects, can be expected to make a great contribution to advances in medicine such as diagnosis and medical treatment.

As a reference example, a preparation example of a kit for endotoxin-specific detection and measurement using a combination of the factor G activation inhibitor of the present invention and horseshoe crab amebocyte lysate is shown below.

Reference Example 1: Method for making an endotoxin-specific measurement kit by adding a factor G activation inhibitor to a commercial or existing Limulus test reagent at the time of endotoxin measurement 1-1. Limulus test reagent (lyophilized product) is as usual ,
A method for achieving the object by dissolving in a designated solution (distilled water or buffer) and adding the factor G activation inhibitor of the present invention to the sample simultaneously or separately (the order of addition does not matter) : For example, 0.1 ml of distilled water is added to "Pregel S" (freeze-dried product; gelling method Limulus test product; Seikagaku Corporation) to dissolve the inhibitor (GP of curdlan formic acid decomposition product GP
C fraction Fraction 4; Table 2, No. 14) aqueous solution (1.2 μg / ml) 0.01 m
l (120 μg / ml LAL) and 0.1 ml of the sample are added, and the mixture is gently shaken and left to stand for 60 minutes at 37 ° C. When heated, it reacts only with endotoxin to form a gel.

1-2. A method in which the inhibitor is dissolved in advance in a limulus test reagent solution and the limulus test reagent is dissolved using the solution to achieve the object: For example, "Cotest endotoxin" (synthetic substrate method limulus test product; kerbyvitrum). 1), 1 vial of LAL (freeze-dried product) is dissolved with 1.4 ml of distilled water in which 0.7 μg of the inhibitor (Laminaran; No. 26 in Table 2) is previously dissolved. 0.1 ml (500 ng / ml L
0.1 ml of the sample is added to AL) and heated at 37 ° C for 10 minutes, and 0.2 ml of buffer containing synthetic substrate (S-2423) is added and heated at 37 ° C for 3 minutes, it reacts only with endotoxin and becomes yellow. Present. For quantification, add 200 μl of 50% acetic acid solution and
Measure absorbance at 05 nm.

Reference Example 2: Method of preparing endotoxin-specific limulus test reagent kit by adding factor G activation inhibitor to LAL in advance 2-1. Commercially available LAL (so-called gelling method limulus test reagent) has the inhibitor in advance. A method that achieves the purpose by adding: Measured by a turbidimetric time analysis method using, for example, "Limulus HS-Test Wako" (freeze-dried product; gelling method Limulus test product, Wako Pure Chemical Industries, Ltd.) In case of LAL (lyophilized product), 1 vial of LAL (lyophilized product) was previously added to the inhibitor (curdlan formic acid degradation product GPC fraction 4;
o.14) Dissolve with 0.5 ml of distilled water in 5 ml. That 0.
Dispense 1 ml (100 ng / ml LAL) into a reaction test tube, add 0.1 ml of the sample, and gently shake, and use the turbidimetric time analyzer "Toxinometer ET-201" (Wako Pure Chemical Industries) Set the dedicated analysis module (37 ℃) at the specified photometric position and turn on the start switch. Only endotoxin reacts and the gel time is displayed.

2-2. Method to achieve the objective by adding the inhibitor to the LAL prepared in advance before adding the sample: For example, in the case of measuring the nephelometric method using LAL,
1M Tris-HCl-1M MgCl in which 0.5 μg of the inhibitor (Laminaran; No. 26 in Table 2) was dissolved in 0.1 ml of lysate extracted from blood cells of horseshoe crab ( Tachypleus tridentatus ) using a hypotonic buffer. ₂ buffer solution (pH8.0) solution 0.01m
Add l (50ng / ml LAL). Add 0.1 ml of sample to this and heat at 37 ℃. Only endotoxin reacts and becomes cloudy. It can be quantified by measuring the absorbance at 660 nm over time.

Reference Example 3: A method for producing an endotoxin-specific limulus test reagent raw material lysate by adding a factor G activation inhibitor during extraction and preparation of LAL. For example, horseshoe crab ( Tachypleus tridentatus ,
T. gigas , Limulus polyphemus , Carcinoscorpius r
otundicauda (which may be any)
Blood cells (about 20 g) were obtained by centrifugation, and 2.0 mg / l of the inhibitor (partially carboxymethylated laminaran;
o.42) distilled water or 0.02M Tris-HCl buffer (pH 8.0)
After adding 100 ml of the inhibitor solution dissolved in a hypotonic buffer solution such as the above, after homogenizing with a Waring blender,
Fractionation into supernatant and precipitate by centrifugation (8,000 rpm; 30 minutes; 4 ° C). Repeat this operation once more, and
Obtain 150 ml as LAL. When a limulus test reagent (gelation method, turbidimetric method, turbidimetric time analysis method, synthetic substrate method) is prepared by using a fixed amount of this LAL instead of the LAL extracted by the conventional method, it is specific to endotoxin only. A reacting endotoxin-specific Limulus test reagent can be produced.

A synthetic substrate method for producing an endotoxin-specific Limulus test reagent includes, for example, US Pat. No. 4,495,29.
No. 4 (Method for determining bacterial endoto
xin and kit therefor)
R-Ile-Glu-Ala-Arg-pNA, methoxycarbonyl-D-hexahydro
A highly sensitive reagent can be produced by using a substrate such as tyrosyl-Gly-Arg-pNA / AcOH. Here, R is an acetyl group, α
-N-benzoyl group, α-N-carbobenzoxy group, N-
It represents a tert-butoxycarbonyl group, a p-toluenesulfonyl group, and other amino acid N-terminal protecting groups.

As an example, 0.04 ml of lysate was added with 1.5 μg of MgCl ₂ and a synthetic substrate (N-tert-butoxycarbonyl-Leu-Gly-
Arg-p-nitroanilide) (4.0 μg) and freeze-dried. For this freeze-dried product
When 0.1 ml of 0.2 M Tris-HCl buffer (pH 8.0) and 0.1 ml of sample are added and heated at 37 ° C for 30 minutes, only endotoxin reacts and a yellow color appears. For the gelation method, nephelometry, and nephelometry, the LAL reagent was used in 0.1 ml of lysate for MgCl 2.
It can be produced by adding 10.0 μg of ₂ and freeze-drying. This reagent was used in Reference Examples 1-1, 2-1, and 2-.
When measured in the same manner as in 2, it reacts only with endotoxin.

Industrial Applicability As described above, the factor G activation inhibitor of the present invention is
Endotoxin-specific LAL in combination with LAL
-Test is provided, which is especially useful when measuring infectious diseases in which the presence or absence of endotoxin is not clear, and clinical samples suspected of sepsis, and can accurately discriminate true Gram-negative bacterial infection (endotoxemia) In addition to the advantages, since fungal infections can be distinguished by using conventional LAL-Test together, selection of appropriate therapeutic agent for the infectious bacteria by early determination of infectious bacteria type, treatment, and its therapeutic effect. Providing a kit that enables analysis and that contains the inhibitor of the present invention can be expected to make a great contribution to advances in medicine such as diagnosis and medical treatment.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroshi Tamura 3-1293-10, Tateno, Higashiyamato-shi, Tokyo 2-408, Greentown 2-408 (72) Inventor, Ariyasu Shibata 5-3-9, Nakano, Nakano-ku, Tokyo (56) ) References BIOCHMICAL AND BI OPHYSICAL RESEARCH COMMUNICATIONS, Vol. 101, No. 2, (1981), p. 434 −439

Claims (7)

(57) [Claims] 1. A formula The poly- (1 → 3) -β-D in which 2 to 370 (1 → 3) -β-D-glucoside structural units represented by
A) a laminari-oligosaccharide having a molecular weight of 342 to 1,638, b) a laminaridextrin having a molecular weight of 1,800 to 3,258, and c) an average molecular weight of 2,000 to 60,000 (1 → 3) -β-D-
A horseshoe crab amebocyte lysate factor G activation inhibitor, which comprises glucan and d) a polyglycoside selected from the group consisting of laminaran having an average molecular weight of 3,000 to 23,000 as an active ingredient. 2. A polyglycoside is an inhibitor sample for a factor G activator, a horseshoe crab amebosite lysate coagulation precursor fraction, a horseshoe crab amebosite lysate factor G fraction, a buffer, magnesium chloride and a chromogenic synthetic substrate. A polyglycoside having a factor G activation inhibitory potency of at least 6,310,000 units / mg, which is measured by quantifying the chromophores released during the heating for a certain period of time after the addition. Inhibitor. 3. The inhibitor according to claim 1, wherein the polyglycoside is a partially degraded product of curdlan or a fractionated product of the partially degraded curdlan. 4. A polyglycoside is a partially decomposed product or a fractionated product of a curdlan, and contains a factor G activator, a horseshoe crab amebosite lysate coagulation enzyme precursor fraction, a horseshoe crab amebosite lysate. After the G factor fraction, buffer, magnesium chloride and chromogenic synthetic substrate were added to the inhibitor sample, the G factor activation inhibitory titer measured by quantifying the chromogenic group released during heating for a certain period of time An inhibitor according to claim 1 which is at least 6,310,000 units / mg of polyglycoside. 5. The activation of factor G which may be present in the horseshoe crab amebosite lysate, which comprises adding an effective amount of the polyglycoside according to claim 1 to the horseshoe crab amebosite lysate. How to block. 6. An endotoxin-specific limulus test reagent containing an effective amount of the polyglycoside according to claim 1. 7. A Limulus test reagent specific to endotoxin, which contains the polyglycoside according to claim 1 in an amount of at least 50 ng per 1 ml of horseshoe crab amebosite lysate.