JPH11178599A - Reagent for measuring endotoxin and/or peptide glucan - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a reagent for measuring an endotoxin for the diagnoses of gram-negative bacterium-infected diseases and measuring a peptide glucan for the safety tests of medicines, etc., and the microorganism tests of foods, etc., by including β-1,3-glucanase. SOLUTION: This reagent for measuring an endotoxin and/or a peptide glucan comprisesβ-1,3-glucanase, and a composition containing the inactivated factors of a body fluid-coagulating system cascade originating from an arthropod or a phenol oxidase precursor cascade and a substance reacting with the β-1,3- glucanase to activate the cascade. The reagent is useful for measuring the endotoxin to early diagnose septicemia infected with gram-negative bacteria, for measuring the peptide glucan existing in the cell walls of almost all procaryotes to test the safety of medicines, etc., test the presence of microorganisms in water, foods, etc., and diagnose infectious diseases, etc. The endotoxins are lipopolysaccharides existing in the outer cell wall membranes of gram-negative bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a treating agent and a treating method for a reagent for measuring endotoxin and / or peptidoglycan, and to a reagent for measuring endotoxin and / or peptidoglycan and a method for measuring peptidoglycan.

[0002]

2. Description of the Related Art Endotoxin (hereinafter abbreviated as ET) is a lipopolysaccharide present in the outer membrane of the cell wall of Gram-negative bacteria and has various biological activities such as pyrogenicity.
Therefore, medical devices that come into direct contact with drugs and blood are ET
ET contamination can cause severe results even in very small amounts, and these ET contamination levels must be strictly controlled, and ET test methods are listed in the United States and Japanese pharmacopeias. . Attempts have also been made to quantify ET in blood for the early diagnosis of sepsis due to Gram-negative bacterial infection, and the determination of the therapeutic effect and prognosis of Gram-negative bacterial infection.

On the other hand, peptidoglycan (hereinafter abbreviated as PG) is a glycoprotein containing N-acetylmuramic acid or N-glycolylmuramic acid and D-amino acids, and is a macrophage, B lymphocyte, T lymphocyte. Various effects on immune response cells, such as platelet destruction, promotion of fibroblast proliferation, promotion of bone resorption, activation of complement, enhancement or suppression of humoral immune response, enhancement of cellular immunity, cell endothelial system Irritation, transient leukopenia and subsequent leukocyte increase, enhanced interferon-inducing factor action, enhanced natural resistance, experimental induction of autoimmune diseases, induction of fever, increased sensitivity to ET toxicity, increased sleep or It has various biological activities such as suppression, epithelioid granuloma formation, induction of hemorrhagic necrotic inflammation at sites treated with Mycobacterium tuberculosis, acute and chronic toxicity. Also, PG is present in both Gram-positive and Gram-negative bacteria, and most prokaryotes have PG in their cell walls, except for archaea that have neither ET nor PG,
Since PG does not exist in eukaryotic cell components such as mammals, it is considered that bacteria exist where PG exists. Therefore, the measurement of PG is useful for micro-detection of microorganisms such as bacteria and blue-green algae that use this as a component of the cell wall, safety test of pharmaceuticals, microbial test of water and food, diagnosis of infectious disease Application to such applications is expected.

As a method for measuring these ET and PG, a method using a reagent containing an inactive factor of a body fluid coagulation cascade or a phenol oxidase precursor cascade (hereinafter abbreviated as proPO) derived from an arthropod body fluid. Is mentioned. For example, in the case of ET, a horseshoe crab blood cell component liquid such as a so-called Limulus test (hereinafter abbreviated as AL solution).
(Yin et al., Biochim. Biophys. Acta.
261 , 284-289 (1972)) and the like are used because of their simplicity and low cost. In the case of PG, a method using a reagent derived from insect body fluid (Japanese Patent Publication No. 7-11470)
No. 7) has attracted attention because of its simplicity and the like.

[0005] However, in this arthropod body fluid,
A substance that reacts with β-1,3-glucan to activate the cascade, for example, in an AL solution, reacts with β-1,3-glucan (hereinafter abbreviated as βG) to activate the enzyme. Substance causing gelling or gelling reaction, so-called Factor G
And a substance that reacts with ET to activate the enzyme or cause a gelling reaction, so-called Factor C, coexist (Morita, T. e.
etal., FEBS Lett. 129 , 318-321 (1981)) and a substance that binds to βG and activates the proPO cascade, a so-called βG recognition protein (hereinafter abbreviated as βGRP) in insect body fluid. ) And proPO by binding to peptidoglycan
Because a substance that activates the cascade coexists with a so-called PG recognition protein (Yoshida, H., Ochiai, M., and Ashida,
M. (1986) Biochem. Biophys. Res. Commun. 141 , 1177-
1184), the above method has a problem in that the reagent used reacts not only with ET or PG but also with βG, thereby impairing its specificity.

Various methods have been reported to solve such problems (JP-A-58-13516, JP-A-59-5959).
-27828, JP-B-7-114707, JP-B7-15474
Publication, Biochem. Biophys. Research Communication,
101 (2) , 434-439 (1981), etc.), but all of these methods remove factor G and βGRP present in the arthropod body fluid from the arthropod body fluid by affinity chromatography or the like. Since the method is a method, there is a problem that an extremely complicated operation is required and a problem that the measurement sensitivity of the obtained reagent may be significantly reduced.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and has a new ET or / and P
Processing agent for reagent for G measurement, processing method using the same, and E
It is an object of the present invention to provide a reagent for measurement specific to T and / or PG and a method for measuring PG.

[0008]

In order to achieve the above object, the present invention has the following constitution.

(1) A reagent for measuring ET and / or PG comprising β-1,3-glucanase.

(2) A reagent for measuring ET or / and PG, comprising an arthropod body fluid and β-1,3-glucanase.

(3) an arthropod-derived composition comprising an inactive factor of the body fluid coagulation cascade or the proPO cascade and a substance which reacts with β-1,3-glucan to activate the cascade; ET or / and PG measurement reagent comprising -1,3-glucanase.

(4) A reagent for ET measurement comprising a composition containing an inactive factor and a factor G of a body fluid coagulation cascade derived from horseshoe crab blood cells, and β-1,3-glucanase.

(5) a composition containing an inactive factor of the proPO cascade derived from insect body fluid and βGRP;
A reagent for measuring PG, comprising 1,3-glucanase.

(6) A treating agent for a reagent for measuring ET or / and PG, which comprises β-1,3-glucanase.

(7) The reagent for measuring ET or / and PG is
The above-mentioned (6), which is the measuring reagent containing an arthropod body fluid
The processing agent according to any one of the above.

(8) The reagent for measuring ET or / and PG is
Fluid coagulation cascade or pr from arthropods
The treatment according to the above (6), wherein the measurement reagent comprises a composition comprising an inactive factor of the oPO cascade and a substance which reacts with β-1,3-glucan to activate the cascade. Agent.

(9) The reagent for ET or / and PG measurement is
ET containing an inactive factor of the fluid coagulation cascade derived from a horseshoe crab blood cell component and a composition comprising factor G
The treatment agent according to the above (6), which is a measurement reagent.

(10) The treatment according to (6) above, wherein the reagent for measuring ET or / and PG is a reagent for measuring PG containing a composition containing an inactive factor of the proPO cascade derived from insect body fluid and βGRP. Agent.

(11) A method for treating a reagent for measuring ET or / and PG, which comprises treating with β-1,3-glucanase.

(12) The treatment method according to the above (11), wherein the reagent for measuring ET or / and PG is the measuring reagent containing an arthropod body fluid.

(13) The reagent for measuring ET or / and PG is an arthropod-derived inactive factor of the body fluid coagulation cascade or proPO cascade and β-1,3-
A substance that reacts with glucan to activate the cascade;
The processing method according to 1).

(14) The reagent according to the above (11), wherein the reagent for measuring ET or / and PG is a reagent for measuring ET containing a composition containing an inactive factor of a body coagulation cascade derived from a horseshoe crab blood cell component and factor G. The processing method described.

(15) The treatment according to the above (11), wherein the reagent for measuring ET or / and PG is a reagent for measuring PG containing a composition containing an inactive factor of the proPO cascade derived from insect body fluid and βGRP. Method.

(16) In the presence of β-1,3-glucanase, a sample is reacted with a reagent for measuring PG containing an inactive factor of the proPO cascade derived from insect body fluid and a composition containing βGRP. PG measurement method.

That is, the present inventors consider that ET and / or PG
As a result of intensive studies in search of a method that can easily prepare a reagent specific to, a β-1,3-glucanase can be converted from a body fluid coagulation system cascade or a proPO cascade (for example, from arthropod body fluids known per se). Hereinafter, it may be abbreviated as the cascade according to the present invention.)
A substance that reacts with G to activate the cascade, and a reagent for ET or / and PG measurement containing
A reagent capable of reacting with βG present in the reagent and activating the cascade according to the present invention can suppress the effect, and a reagent capable of specifically measuring ET or / and PG can be easily prepared. The inventors have found that the present invention can be obtained, and have completed the present invention.

The β-1,3-glucanase used in the present invention has an action of hydrolyzing β-1,3-glucan to produce oligosaccharide or glucose, and reacts with βG. The substance having the action of activating the cascade according to the present invention may be any substance having the property of suppressing the action, and is not particularly limited. Also,
The origin is not particularly limited. For example, those derived from microorganisms such as bacteria, filamentous fungi, and fungi, for example, barley,
Examples thereof include those derived from plants such as soybeans and rice, and those derived from animals such as sea urchins. Above all, Arthrobact
er sp., Rhizoctonia sp., Trichoderma sp., Aspergil
Preferred are those derived from lus sp. Among them, the optimum temperature is usually 5 to 80 ° C, preferably 20 to 40 ° C, and the optimum pH is usually 4 to 9, preferably 6 to
8 and has a stable pH of usually 3 to 10, preferably 4
Which is present in the range of from 1 to 9, and has a molecular weight of usually from 1 to 9.
Those with 500,000, preferably 2-100,000 are particularly preferred.

The concentration of the β-1,3-glucanase used in the present invention may be a reagent for measuring ET or / and PG, for example, a clotting cascade or a proPO cascade derived from an arthropod body fluid. Any concentration may be used as long as it is a concentration present in a reagent for measuring ET or / and PG containing an active factor and reacting with βG to activate the cascade, which can suppress the action of activating the cascade. Not done. Specifically, the concentration in the reagent for measuring ET or / and PG slightly varies depending on the type of β-1,3-glucanase to be used.
l, preferably 2-1000u / ml, more preferably 10-500u
/ Ml in an appropriate range. More specifically, for example, the factor G in the reagent for measuring ET or / and PG
For an amount of 50 μg / ml or less, preferably 1 μg / ml or less,
Usually, it is appropriately selected from the range of 0.2 to 2000 u / ml, preferably 2 to 1000 u / ml, more preferably 10 to 500 u / ml, and the amount of βGRP in the reagent for measuring ET or / and PG is 50 μm.
g / ml or less, preferably 1 μg / ml or less.
It is appropriately selected from the range of 2 to 2000 u / ml, preferably 2 to 1000 u / ml, more preferably 10 to 500 u / ml. Further, the β-1,3-glucanase according to the present invention may be used alone or in an appropriate combination of two or more. Incidentally, the measurement of the factor G amount is carried out, for example, by the following method (Muta, T. et al., J. Biol. Chem. 270 , 892-897 (1.
995)). That is, 0.01 ml of sample and 0.1 M
Tris-HCL buffer (0.5 mg / ml bovine serum albumin and 125 ng
/ Ml curdlan, pH8.0) 0.19ml, 37 ℃
And react for 20 minutes. Next, 2 mM Boc was added to the reaction solution.
-Glu (OBzl) -Gly-Arg-MCA (t-butyloxycarbonyl-γ-benz
yl-L-glutamyl-glycyl-L-arginine-4-methyl-coumaryl-
Add and mix 0.01ml of 7-amide) and react at 37 ℃ for 30 minutes. After the reaction, 0.79 ml of 0.6 M acetic acid is added to the reaction solution to stop the reaction. Next, the amount of free AMC (7-amino-4-methyl-coumarin) present in the reaction solution is measured with a fluorescence spectrophotometer. The obtained measured value is compared with a previously obtained measured value of the purified factor G having a known concentration, the activity value of the activated factor G is calculated, and the amount of the factor G in the sample can be obtained. The amount of βGRP is measured, for example, by the following method (Ochiai, M., Ashid
a, M. (1988) J. Biol. Chem. 263 , 12056-12062). That is, in a centrifuge tube, 1 ml of the sample and 0.
Β-GRP contained in the sample is adsorbed to the curdlanger by mixing with 0.2 ml of 3% curdlanger. Next, the centrifugal tube is subjected to a centrifugal separation treatment to collect curdlan gel. The recovered curdlan gel is mixed with distilled water, 1
M sodium chloride aqueous solution, 8M urea aqueous solution, and distilled water were sequentially washed, and then the distilled water was added to the curdlan gel.
50 μl and SDS-electrophoresis solubilization buffer [1% SD
S, 82 mM Tris-HCL containing 1% β-mercaptoethanol, 30% glycerol and 0.01% bromophenol blue
(PH 8.8)], add and mix, and heat-treat at 100 ° C for 5 minutes. After centrifugation again, a part of the supernatant is taken out and subjected to SDS-electrophoresis (acrylamide concentration 12%, under reducing conditions). After the electrophoresis, the electrophoresis gel is stained with Coomassie brilliant blue, and the staining intensity of the βGRP band is measured with a densitometer. SDS-electrophoresis is similarly performed on the purified βGRP, and a calibration curve for the amount of βGRP and the staining intensity is prepared. Using the calibration curve, the amount of βGRP in the sample can be determined from the staining intensity of βGRP in the sample measured with a densitometer.

When a known ET or / and PG measuring reagent is treated with the above-mentioned β-1,3-glucanase, factor G, βGRP, etc. present in the ET or / and PG measuring reagent can be obtained. The substance that reacts with βG to activate the cascade according to the present invention can be activated by βG to suppress the effect of activating the cascade, and ET or / and PG can be specifically measured. The reagent can be easily prepared.

The ET known per se used in the present invention
And / or and / or PG measurement reagents include ET and / or P
Blood clotting cascade or p activated by G
roPO Cascade (Muta, T., and Iwanaga, S., Pro
gress in Molecular and Subcellular Biology. 15 , 15
4-189 (1996), Ashida, M., and Yamazaki, HI Molt
ing and Metamorphosis, 239-265, Japan Sci. Soc. Pr
ess (1990), Johansson, MW, and Soderhall, K., P.
arasitol. Today. 5 , 171-176 (1989)) and a substance that reacts with βG to activate the cascade and reacts with ET and PG present in the cell wall of the microorganism. It is not particularly limited as long as it has. Also, the origin is not particularly limited, for example, insects,
Preferable examples include arthropod body fluids such as horseshoe crabs and crustaceans. Insects are not particularly limited but are preferably as large as possible and have established breeding methods. And beetles such as Orthoptera and Scutellaria beetles. Among them, for example, Bombyx
Bombyx mori belonging to the genus Bombyx mori such as mori and Bombyx mori mandarina are particularly preferred. The horseshoe crab is not particularly limited, and examples thereof include horseshoe crab belonging to the genus Limulus, the genus Tachypleus, the genus Carcinoscorpius, and the like. As beetles, for example, genus Astacus, Cambarus
Crayfish such as genus, Procambarus, and Cambaroides are preferred. In addition, the reagent for ET or / and PG measurement containing the body fluid of the arthropod as described above includes a body fluid itself, a blood cell component thereof,
Those containing hemolymph, etc., or those containing factors or cascades that react with ET or PG purified therefrom are included.

The ET known per se used in the present invention
Among the reagents for measuring ET, among the reagents for measuring ET, those obtained from the blood cell component (Lysate) of horseshoe crab as described above, the body fluid coagulation cascade (Muta, T., and Iwanaga, S. , Progress in Molecular
and Subcellular Biology. 15 , 154-189 (1996)) which have not been activated (inactive factor) and which have the effect of reacting with βG to activate the cascade. A reagent containing a substance (Factor G) and having a property of causing the activation of an enzyme (protease or the like) or a gelling reaction or a property of increasing turbidity by a reaction with ET or βG, a so-called AL solution is particularly preferable. It is preferably used. In addition, as the AL solution,
A substance (including a so-called synthetic substrate) that is a substrate of an enzyme that is activated as a result of reacting with G or ET and that produces an appropriate dye or the like (a fluorescent or luminescent substance) by the action of the enzyme. Additions can also be used.

Among the known ET and / or PG measurement reagents used in the present invention, the reagents for measuring PG include those obtained from the body fluids of insects as described above, and the proPO cascade. [Ashida and Yam
azaki, Molting and Metamorphosis, 239-265, Japan S
ci. Soc. Press (1990)] and those in which activation has not occurred (inactive factor) or a substance that reacts with βG to activate the cascade (βGR
A reagent containing P) and having the property of activating proPO by reacting with βG or PG present in the cell wall of the microorganism, a so-called PPO reagent, is particularly preferably used. In addition, PPO
The reagent is a substrate of an enzyme which is usually activated as a result of the reaction with βG or PG, and produces a suitable dye or the like (fluorescent or luminescent) by the action of the enzyme (so-called synthesis). And the like).

As a method for obtaining a blood cell component from horseshoe crab as described above, for example, blood obtained by collecting blood from the head and thorax and abdomen of horseshoe crab is allowed to stand in NaCl containing N-ethylmaleimide for a while, Blood cells (Amoebocyt
e) collecting a blood cell component from the blood cell (J.
Levin, FB Bang., Bull. Johns Hopkins Hosp., 11
5 , 265-274 (1964)). Also, for example, A
CC (Associates of Cape Cod), Bio Whittaker bioproducts, Inc., Charles River Endosafe, In
c.), a lyophilized product of an AL solution commercially available from Seikagaku Corporation, Wako Pure Chemical Industries, Ltd., etc. can also be used.

When the body fluid is obtained from the insect as described above, the body fluid may be hemolymph obtained from a body cavity.
mph) is the easiest to obtain and is more common. For example, after collecting insects on ice and stopping the movement, the sugarcane factor (glucose,
Sucrose or a sugarcane factor itself containing impurities (a polymer substance composed of an amino acid or the like) or, for example, (p
-Amidinophenyl) methanesulfonyl fluoride (p
-APMSF), phenylmethanesulfonyl fluoride (PMSF), diisopropylfluorophosphoric acid (DF
P), p-nitrophenyl-p'-guanidinobenzoic acid (NPGB), dihydroxychloromethylcoumarin (D
CC) or the like, a physiological saline solution containing a serine protease inhibitor or the like is injected into the body cavity, and then left to stand for a while to collect hemolymph from the body cavity. Addition and collection method (Ashida, M., Inse
ct Biochem., 11 , 57-65, 1981, JP-A-1-14266) and the like. The hemolymph obtained in this manner is subjected to centrifugation to remove blood cells, and then dialyzed to obtain plasma usable as the PPO reagent according to the present invention. As such a reagent, a commercially available reagent (for example, SLP ^™ reagent set, manufactured by Wako Pure Chemical Industries, Ltd.) may be used.

The method of treating a known ET or / and PG measuring reagent with the β-1,3-glucanase according to the present invention includes,
Any method can be used as long as it can cause -1,3-glucanase to coexist, and is not particularly limited.
β-1,3-glucanase itself may be directly added to and mixed with the reagent for measuring ET or / and PG,
-Glucanase is contained in an appropriate solvent to form a solution, which is mixed with the reagent for measuring ET or / and PG, etc., so that β-
For example, a method of adding and mixing 1,3-glucanase to coexist is used.

In the present invention, the solvent containing β-1,3-glucanase does not activate the blood coagulation cascade or the proPO cascade, and
-Is not particularly limited as long as it does not inhibit the property of glucanase, which activates the cascade by reacting with βG, to inhibit the activating action, for example, distilled water,
Buffers and the like can be mentioned. As the buffer constituting the buffer, all those usually used in this field can be used, and specifically, for example, phosphate, borate, acetate,
Tris buffer, Good's buffer and the like can be mentioned, and the use concentration may be appropriately selected from the use concentration range in this field.

The treating agent of the reagent for measuring ET or / and PG of the present invention contains β-1,3-glucanase as described above, and preferred embodiments and specific examples of the constituent elements are described above. As expected.

The reagent for measuring ET or / and PG comprising the β-1,3-glucanase of the present invention is a known reagent for E
T or / and PG measurement reagent, for example, AL solution, PP
ET and / or P comprising arthropod body fluids such as O reagent
It can be easily obtained by coexisting the aforementioned β-1,3-glucanase with the reagent for G measurement in the above-mentioned concentration range. By using the thus obtained reagent for measuring ET or / and PG of the present invention and measuring ET or / and PG in the reagent by a method known per se,
ET and / or PG can be specifically measured without being affected by coexisting βG.

The measuring method known per se is not particularly limited as long as it uses a reagent for measuring ET and / or PG known per se as described above. For example, as a measurement method using an AL solution, for example, after mixing a sample and an AL solution, the mixture is incubated at an appropriate temperature for a certain period of time, and a gel inversion method (Cooper, J .;
F. et al., J. Lab.clin.Med., 78 , 138-148 (197
1)), a turbidimetric method (Bond
ar, RJL et al., Biomedical Applications of th
e Horseshoe Crab (ed. Cohan, E.), 435-451, Alan R.
Liss, Inc. New York (1979)), a turbidimetric time analysis method that measures the time required for the turbidity produced by coagulation to reach a constant value (Oishi, H. et al., J. Parenter. Sci. Techn.
39 , 194-200 (1985)), an enzyme activated by the reaction between a component of an AL solution and ET (eg, a protease or the like)
Substrate method (Nakamu
ra, S. et al., J. Biochem., 81 , 1567-1569 (1977))
And the like.

As a measuring method using a PPO reagent, for example, a sample and a PPO reagent are mixed and reacted, and for example, N-α-benzoyl-L-
Arginine ethyl esterase (BAEEas
e), prophenol oxidase activating enzyme (PPA)
E), the activity of an enzyme such as phenol oxidase (PO) is measured according to a measurement method known per se, and a concentration of P
A method of calculating the amount of PG from a calibration curve prepared by the same operation using a standard solution of G (M. Tsuchiya et al. FEMS
Immunology and Medical Microbiology 15 (1996) 129-
134, JP-A-7-184690, etc.) After mixing the PPO reagent with the sample by utilizing the phenomenon that the time when proPO is activated to become PO depends on the PG concentration in the sample,
Method to measure the time required for the amount of reaction products obtained by the method to reach a certain value (M. Tsuchiya et al. FEMS Immunology a
nd Medical Microbiology 15 (1996) 129-134) and the like.

Other reagents used in these known methods include, for example, 3,4-dihydroxyphenylalanine (dopa), substrates of enzymes to be measured such as synthetic substrates, conjugate enzymes, coenzymes and the like. If you do, buffer,
Color formers, for example, divalent metal ions (Ca ²⁺ , Mg ^2+, etc.)
Examples of the method for measuring the target enzyme activity, such as activators, stabilizers, and surfactants, include reagents used in a method known per se. The reaction pH in these known measuring methods varies depending on the measuring method and the type of enzyme to be measured, but is usually pH 4 to 11, preferably pH 6
-9. In order to maintain the reaction pH, a buffer may be used. If the buffer does not affect the reaction, the type and concentration used are not particularly limited. For example, phosphate, boric acid, etc. Salt, acetate, Tris buffer, Good's buffer and the like. The reaction temperature and the reaction time are not particularly limited as long as the reaction proceeds at a temperature and time, and the reaction temperature is usually from 0 to 50.
° C, preferably 4 to 30 ° C, and the reaction time is appropriately selected from the range of usually 1 second to 20 hours, preferably 10 seconds to 2 hours. Further, among the measurement methods known per se as described above,
When a PPO reagent is used, a divalent metal ion in the range of 0.001 to 1000 mM, preferably 5 to 100 mM, such as C
It is desirable that a ²⁺ , Mg ^{2+ and the} like exist.

ET and / or PG by using the reagent of the present invention
There is no particular limitation on the sample from which the presence or absence of microorganisms in the sample can be detected, but it is not particularly limited, for example, washing water for semiconductors, for example, body fluids such as blood, plasma, serum, cerebrospinal fluid, urine, Examples include washing water after washing tap water, factory waste liquid, food, beverages, medical instruments, and the like.

ET and / or PG by using the reagent of the present invention
Can be measured, for example, as follows. That is,
For example, when ET is measured using an AL solution, the above-mentioned sample is mixed with the reagent for measuring ET containing β-1,3-glucanase according to the present invention, and the mixture is reacted under certain reaction conditions. (For example, 0 to 50 ° C, preferably 4 to 30 ° C)
Using a toxinometer (manufactured by Wako Pure Chemical Industries, Ltd.), the gelation time is measured until the transmitted light amount ratio, which decreases with the gelation of the reaction solution, reaches a preset threshold. . The ET content in the sample can be calculated from a calibration curve showing the relationship between the ET concentration and the gelation time, obtained in the same manner using a sample containing ET of a known concentration in advance.

When PG is measured using, for example, a PPO reagent, the above-described sample is mixed with the β according to the present invention.
PG measurement reagent containing -1,3-glucanase is mixed and reacted under a certain reaction condition (for example, at 0 to 50 ° C, preferably 4 to 30 ° C for 1 second to 20 hours, preferably 10 seconds ~
2 hours), for example, a commercially available microplate reader
Using a Thermo-Max (manufactured by Molecular Devices), a toxinometer (manufactured by Wako Pure Chemical Industries, Ltd.) or the like, the reaction time until the absorbance of the reaction solution reaches a preset threshold is measured. The measured value of the obtained reaction time is compared with a known concentration of P
PG obtained in the same manner using a sample containing G
From the calibration curve showing the relationship between the concentration and the reaction time, P
The G content can be calculated.

The method for measuring PG according to the present invention is based on a method known per se using the PPO reagent as described above, except that the β-1,3-glucanase according to the present invention is present in the above-mentioned concentration range. The other reagents to be used may be appropriately selected according to the above-described known measurement methods. That is, the PG in the sample is specifically measured by measuring the sample as described above in the presence of β-1,3-glucanase according to the above-described measurement method using a known PPO reagent. Can be.

Further, in the PG measurement method of the present invention,
When reacting a PPO reagent with a sample, the β
It is sufficient that -1,3-glucanase coexists in the above concentration range, and the method for coexistence is not particularly limited. Specifically, for example, a method in which the β-1,3-glucanase according to the present invention is contained in the PPO reagent as described above and the sample is mixed with the β-1,3-glucanase according to the present invention, for example, as described above, Examples include a method of diluting a sample with a solution such as a buffer containing 3-glucanase, and mixing the diluted sample with the PPO reagent as described above.

Hereinafter, the present invention will be described in more detail with reference to Reference Examples, Examples and Comparative Examples, but the present invention is not limited thereto. In addition, distilled water and water for injection (made by Otsuka Pharmaceutical Co., Ltd.) used in the following examples.
Is a commercially available SLP ^™ reagent set (Wako Pure Chemical Industries, Ltd.)
) Were used to confirm that βG, PG, and / or ET were not detected.

[0047]

〔reagent〕

(1) PPO reagent; Ashida. M., (1981) Insect Bioche
m., 11 , 57-65, a fraction of the silkworm body fluid obtained was used as a PPO reagent (βGRP amount: 1 μg / ml).
Less than). The amount of βGRP in the PPO reagent was determined by Ochiai,
M., Ashida, M. (1988) J. Biol. Chem. 263, 12056-120
Measured according to the method of 62. (2) Dopa-calcium solution; 0.1 mM MOPS buffer (pH 6.5), 6 mM dopa and 24 mM calcium chloride
And then filtered using a Zetapore membrane (manufactured by Cuno) to obtain a dopa-calcium solution. (3) β-1,3-glucanase solution; Tunicase R
70 (tunicase R 70, manufactured by Daiwa Kasei Co., Ltd., optimal temperature: 35
° C, optimum pH: 7.0-8.0, stable pH: 4.0-9.0, molecular weight: 5
5,000) was dissolved in a 0.1 M Tris-HCl buffer (pH 7.5) to a concentration of 10 mg / ml, and filtered using a zetapore membrane to obtain a β-1,3-glucanase solution. (4) PG standard solution; Micrococcus luteus (Microc
occus luteus) and 1 g of the dried cells were used according to the method of Schleifer, KH, and Kandlar, O., Bacterio.
l. Rev., 36 , 407-477 (1972)) to obtain 95 mg of trypsin-treated cell wall (PG). 20 mg of the obtained trypsin-treated cell wall (PG) was added to 20 ml of water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), and dispersed by ultrasonication to obtain a PG stock solution. In use, the PG standard solution was diluted with distilled water to prepare a dilution series from 1 ng / ml to 1 μg / ml, which was used as a PG standard solution. (5) βG standard solution; curdlan (Wako Pure Chemical Industries, Ltd.)
10G in 10 ml of 0.25N sodium hydroxide solution was used as a βG stock solution. In use, the βG
The standard solution was diluted with distilled water to prepare a dilution series from 10 pg / ml to 10 μg / ml, which was used as a βG standard solution.

[Measurement equipment] Microplate reader Th
The measurement was performed using ermo-Max (manufactured by Molecular Devices) under the following conditions. Temperature: 30 ° C. Time: 1 hour 40 minutes Onset OD: 0.01 Measurement wavelength: 650 nm The data was analyzed using Soft Max version 2.32.

[Operation] In a glass test tube previously sterilized by dry heat at 250 ° C. for 2 hours, 575 μl of PPO reagent,
375 μl of a calcium solution and 175 μl of a β-1,3-glucanase solution were dispensed and mixed to prepare a reaction reagent solution. Then
At a predetermined concentration of β
The G standard solution or the PG standard solution having a predetermined concentration was dispensed at 50 μl each, and then the reaction test solution was dispensed at 50 μl each, and the measurement was carried out using a microplate reader under the above conditions. As a control, instead of the β-1,3-glucan solution, a reaction solution prepared using 175 μl of distilled water (without β-1,3-glucanase) was used.
The measurement was performed as described above.

[Results] Table 1 shows the onset time (time (sec) until the onset OD = 0.01) obtained by using the PG standard solution, and the onset time obtained by using the βG standard solution. Table 2 shows the obtained onset times (Onset Time: time (seconds) until the onset OD = 0.01).

[0051]

[Table 1] * Nd in the table indicates that the onset OD was not reached within the measurement time.

[0052]

[Table 2] * Nd in the table indicates that the onset OD was not reached within the measurement time.

Further, based on the results in Table 1, β-1,3
-Glucanase-free reaction solution and β-1,3-glucanase-containing reaction solution were used.
When a calibration curve representing the relationship between G and the onset time was created, a good calibration curve was obtained. By applying the data of Table 2 to these calibration curves (coordinate axes are logarithmic), based on the onset time at each concentration of the βG standard solution, the PG conversion value of βG when each reaction reagent was used was calculated. I asked. Table 3 shows the results.

[0054]

[Table 3]

As is apparent from the results in Table 3, β-1,
When 3-glucanase is contained in the PPO reagent,
Has almost no effect on the measurement of PG,
It can be seen that the effect of activating the O cascade can be suppressed. In other words, it can be seen that a PG-specific PPO reagent can be easily obtained by adding β-1,3-glucanase.

[0056]

The present invention provides a novel reagent for treating ET or / and PG, a treatment method using the same, and a simple reagent for measuring ET and / or PG and a method for measuring PG. And has a remarkable effect in that a measurement reagent specific to ET or / and PG can be easily obtained.

Claims (16)

[Claims] 1. A reagent for measuring endotoxin and / or peptidoglycan comprising β-1,3-glucanase. 2. A reagent for measuring endotoxin and / or peptidoglycan comprising an arthropod body fluid and β-1,3-glucanase. 3. A composition comprising an arthropod-derived inactive factor of a humor coagulation system cascade or a phenol oxidase precursor cascade and a substance which reacts with β-1,3-glucan to activate the cascade. And β-1,3-
A reagent for measuring endotoxin and / or peptidoglycan, comprising glucanase. 4. A reagent for measuring endotoxin, comprising a composition comprising an inactive factor and a factor G of a body fluid coagulation cascade derived from a horseshoe crab blood cell, and β-1,3-glucanase. 5. A reagent for measuring peptidoglycan, comprising a composition comprising an inactive factor of a phenol oxidase precursor cascade derived from an insect body fluid and a β-glucan recognition protein, and β-1,3-glucanase. 6. A treatment agent for a reagent for measuring endotoxin and / or peptidoglycan, which comprises β-1,3-glucanase. 7. The treatment agent according to claim 6, wherein the reagent for measuring endotoxin and / or peptidoglycan is a reagent containing arthropod body fluid. 8. A reagent for measuring endotoxin and / or peptidoglycan reacts with an inactive factor of an arthropod-derived fluid coagulation cascade or phenol oxidase precursor cascade and β-1,3-glucan. The treatment agent according to claim 6, which is a reagent for measurement containing a composition comprising: a substance that activates a cascade. 9. The method according to claim 6, wherein the reagent for measuring endotoxin and / or peptidoglycan is a reagent for measuring endotoxin comprising a composition containing an inactive factor of a body coagulation cascade derived from a horseshoe crab blood cell component and factor G. Processing agent. 10. The reagent for measuring endotoxin and / or peptidoglycan is a reagent for measuring peptidoglycan comprising a composition containing an inactive factor of a phenol oxidase precursor cascade derived from insect body fluid and a β-glucan recognition protein. Item 7. The treatment agent according to Item 6. 11. A method for treating a reagent for measuring endotoxin and / or peptidoglycan, which comprises treating with a β-1,3-glucanase. 12. The processing method according to claim 11, wherein the reagent for measuring endotoxin and / or peptidoglycan is the reagent for measurement containing an arthropod body fluid. 13. A reagent for measuring endotoxin and / or peptidoglycan reacts with an arthropod-derived inactive factor of a humor coagulation system cascade or a phenol oxidase precursor cascade and β-1,3-glucan to react with the β-1,3-glucan. The treatment method according to claim 11, wherein the measurement reagent comprises a composition comprising: a substance that activates a cascade. 14. The endotoxin measuring reagent according to claim 11, wherein the reagent for measuring endotoxin and / or peptidoglycan is a reagent for measuring endotoxin containing a composition containing an inactive factor of a body fluid coagulation cascade derived from a horseshoe crab blood cell component and factor G. Processing method. 15. The reagent for measuring endotoxin and / or peptidoglycan is a reagent for measuring peptidoglycan containing a composition containing an inactive factor of a phenol oxidase precursor cascade derived from insect body fluid and a β-glucan recognition protein. Item 12. The processing method according to Item 11. 16. In the presence of β-1,3-glucanase,
A method for measuring peptidoglycan, comprising reacting an inactive factor of a phenol oxidase precursor cascade derived from an insect body fluid and a peptidoglycan measurement reagent containing a composition containing a β-glucan recognition protein with a sample.