JPH07128337A - Measuring method for (1-3)-beta-d-glucan - Google Patents

Abstract

PURPOSE:To provide a method for measuring (1 3)-beta-D-glucan quickly and efficiently with a high detection rate while allowing use of a nonspecific limulus reagent even when a sample contains a reaction interference factor for limulus factor G system and/or endotoxin and the (1 3)-beta-D-glucan in the sample has triple spiral structure. CONSTITUTION:A sample containing (1 3)-beta-D-glucan principally having triple spiral structure and can contain metal ions is pretreated under strong alkaline conditions and under presence of chelate agent, as required, thus destroying or inactivating endotoxin or endotoxin and a reaction interfering factor G, which may be contained in the sample as a reaction interfering factor, before limulus reaction takes place using a limulus reagent.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to (1 → 3) -β-D-
The present invention relates to a method for measuring glucan (hereinafter, also referred to as "β-glucan"). In particular, it interferes with the endotoxin (hereinafter also referred to as “Et”) or Et and Limulus factor G and β-glucan involved in the Limulus factor C system reaction, which may be contained in the sample as a reaction-interfering factor. In a sample or Ringer's solution that may contain a reaction interfering factor (hereinafter also referred to as “factor G reaction interfering factor”)
The present invention relates to a method capable of measuring β-glucan in a sample such as a dialysate that may contain metal ions with high accuracy using a Limulus reagent.

[0002]

2. Description of the Related Art Horseshoe crab amebosite lysate (hereinafter also simply referred to as "lysate") contained in Limulus reagent includes a cascade type coagulation system (factor C system) and β-glucan that are activated in response to Et. A coagulation type coagulation system (factor G system) that is activated by reacting with is coexistent (FIG. 1), and the lysate reacts not only with β-glucan but also with Et. Et using only the former system
A method of specifically measuring β-using only the latter system
Methods for specifically measuring glucan are known (Obayashi T. et al., Clin. Chim. Acta, 149,
55-65 (1985)).

It is also known that patients with fungal infections have increased β-glucan in blood, and fungal infections can be diagnosed by measuring β-glucan in blood.
The β-glucan assay method using such a G factor system is
Since the detection sensitivity is very high, it is suitable for detection of a trace amount in a biological sample. Especially, its effectiveness in diagnosing deep-seated fungal infections has been examined and confirmed, and it has begun to be used for clinical tests.

The reaction of β-glucan with G factor involves not only the molecular weight but also the higher-order structure thereof. Those having a triple helix structure do not activate G factor, and when it takes a single helix structure, G factor is It is known to activate (Sait
o H. et al., Carbohydr. Res., 217, 181-190 (1991);
Tanaka S. et al., Carbohydr. Res., 218, 167-174 (19
91); Aketagawa J. et al., J. Biochem., 113,683-686 (19
93)). On the other hand, β-glucan often exists in the most stable form of triple helix structure in nature,
And often coexists with Et.

Therefore, in a usual Limulus reagent (non-specific Limulus reagent) using the whole lysate as a raw material,
Due to the existence of the factor G system and the factor C system, not only β-glucan but also Et reacted, and β-glucan could not be measured. In order to solve this problem, conventionally, the G factor component was fractionated and reconstituted from the lysate (eg, Obayashi T. et al., Supra), or the C factor was specifically inhibited, adsorbed, or removed. What to do (for example, WO9
1/19981 and WO92 / 16651), β-glucan in a sample as a β-glucan-specific Limulus reagent that does not substantially contain a factor C component or inhibits a factor C reaction and does not react with endotoxin. Was being measured.

In addition, Et is inactivated by pretreatment of the sample regardless of the means such as fractionation / reconstitution of factor G system, adsorption removal of factor C system or specific inhibition as described above. After that, a method for measuring β-glucan by allowing lysate to act has been proposed (Japanese Patent Laid-Open No. 2-141666). This method heats the sample liquid to be tested, but in reality, treatment at 100 ° C. for 2 hours is required.
Moreover, in this treatment, the β-glucan having a triple helix structure is
It converts little to the single helix structure and therefore cannot measure the total amount of β-glucan.

By the way, when β-glucan in a biological sample, particularly blood, is measured by utilizing the cascade reaction of the lysate factor G system, the reaction utilizes the reaction of serine protease in the lysate. Various factor G-type reaction-interfering factors contained therein (for example, thrombin and factor Xa have similar effects to coagulase in lysate, and thus become false positive factors, α ₂ -plasmin inhibitor, α ₁ -antitrypsin and Antithrombin
III strongly inhibits the factor G system reaction and becomes a false negative factor) and requires pretreatment to inactivate or remove it.

For this purpose, in the past, platelet-rich plasma (PRP) was prepared from a blood sample, perchloric acid was added thereto, and the mixture was heated at 37 ° C. and then the denatured precipitate was removed by centrifugation. Then, the supernatant was collected and neutralized with alkali to obtain a test solution (Obayashi T. et a
L., J. Lab. Clin. Med. 104, 321-3301 (1984)), but the separation operation of modified precipitates is complicated and many operation steps are required. There was a problem such as danger.

Therefore, the present inventor previously proposed a method of treating with an alkali metal hydroxide and the like (Japanese Patent Application No. 5-100426) as a rapid pretreatment method which does not require a centrifugation operation. . However, in the previous application, removal of the Limulus reaction-interfering factor is the main focus, and in the case of measuring Et and β-glucan specifically, it is necessary to use a special lysate reagent specific to each substance. It was That is, it is not possible to find a condition that satisfies the inactivation of Et and the inactivation of the factor G system reaction-interfering factor at the same time while maintaining the advantages of the method. It was difficult to measure β-glucan using a specific Limulus reagent. In addition, if the β-glucan had a triple helix structure under such sample conditions, it was even more difficult to measure it sufficiently reproducibly when a non-specific Limulus reagent was used. on the other hand,
When β-glucan in a sample that may contain metal ions such as Ringer's solution, phosphate buffered saline, cell culture medium and dialysate in hemodialysis is measured using a Limulus reagent, β-glucan in these samples is measured. Has a triple-helical structure, the structure is stabilized and the factor G system reaction does not proceed sufficiently, so that there is a problem that the total amount of β-glucan cannot be accurately measured.

[0010]

DISCLOSURE OF THE INVENTION The present invention is intended to solve the above-mentioned conventional problems, in which a sample contains endotoxin and / or a factor G type reaction-interfering factor, and β-glucan in the sample is contained. The present invention provides a pretreatment method capable of using a non-specific Limulus reagent as a Limulus reagent even when it has a triple helix structure, and a method capable of rapidly and efficiently measuring β-glucan at an extremely high detection rate. Furthermore, according to the present invention, even when the sample contains a metal ion and β-glucan has a triple helix structure in the sample, the triple helix structure of β-glucan is G
Provided is a method capable of rapidly and efficiently measuring β-glucan with an extremely high detection rate by being converted into a structure having factor activation ability.

[0011]

According to the present invention, a sample containing (1 → 3) -β-D-glucan having a triple helix structure as a main component is pretreated under strongly alkaline conditions and used as a reaction interfering factor. The method is characterized by destroying or inactivating endotoxin alone or endotoxin and a factor G system reaction-interfering factor that may be contained in a sample, and then subjecting to a Limulus reaction by a Limulus reagent (1 → 3) -β
-D-glucan measuring method, the Limulus reagent is a non-specific Limulus reagent, the above-mentioned (1 → 3) -β-D-glucan measuring method, alkali metal hydroxide in a sample The method for measuring (1 → 3) -β-D-glucan described above or under the condition of being strongly alkaline under the condition that the alkali metal hydroxide concentration in the sample is 0.03 to 2.0 mol / L. Pretreatment is performed under the following, the measurement method described above, and the measurement method according to any one of the above, wherein the pretreatment is performed under a condition of 0 to 135 ° C., and the sample may contain a metal ion. The method for measuring (1 → 3) -β-D-glucan described above, in which a sample is pretreated in the presence of a chelating agent, mainly having a triple helix structure (1 →
3) A sample that may contain -β-D-glucan and a metal ion is pretreated in the presence of a chelating agent under strongly alkaline conditions and then subjected to a Limulus reaction with a Limulus reagent (1 → 3) ) Method for measuring -β-D-glucan, the pretreatment, after adding a chelating agent to the sample,
It is carried out under strong alkaline conditions or when the chelating agent is added to the sample under strong alkaline conditions (1.
→ 3) -β-D-glucan measurement method, Limulus reagent does not substantially contain factor C component of horseshoe crab hemolymph coagulation system, or factor C reaction is inhibited and does not react with endotoxin, (1 → 3) -β described above, which is specific to (1 → 3) -β-D-glucan.
-A method for measuring D-glucan.

The present invention will be specifically described below. The first aspect of the present invention is to treat a sample such as endotoxin, factor G system reaction-interfering factor, etc. by pretreating the sample under strongly alkaline conditions.
When measuring β-glucan by Limulus reagent, destroys or inactivates a reaction interfering factor that interferes with the reaction,
When β-glucan has a triple helix structure, it is solved to convert it into a structure that activates factor G, and then mixed with Limulus reagent to change synthetic peptide substrate or gelation of lysate by a known method. Alternatively, β-glucan is measured by detecting a change in turbidity accompanying it. The second aspect of the present invention pretreats the sample in the presence of a chelating agent under strong alkaline conditions to eliminate the influence of metal ions in the sample, which may contain metal ions such as Ringer's solution and dialysate, to remove β- The triple helix structure of glucan is converted into a structure capable of activating factor G, and then β-glucan is measured using Limulus reagent in the same manner as above.

The “sample” targeted in the present invention is β
There is no particular limitation as long as it is a sample for measuring glucan, but it is particularly effective when it contains a reaction-interfering factor and when β-glucan has a triple helix structure. Here, "reaction-interfering factor" means E that initiates a C-factor reaction.
In addition to t, β-glucan is added at any stage of the stepwise enzymatic reaction (cascade reaction) of the factor G system of the lysate shown in FIG. Is a false-positive factor that reacts independently or a false-negative factor that inhibits the reaction. Since the factor G system reaction-interfering factor functions similarly in the factor C system, it is also referred to as a limulus reaction-interfering factor. In particular,
Aqueous solution containing β-glucan, drug, Ringer's solution,
In addition to dialysate, cell culture medium, buffer, physiological saline, etc., body fluids of patients with fungal infections that may contain fungal β-glucan (blood samples such as plasma, serum and whole blood, urine , Spinal fluid, ascites, etc.) are exemplified.

As the Limulus reagent, a non-specific Limulus reagent can be used in the above-mentioned first invention, but it is composed of a component specific to the factor G system reaction or the factor C system reaction is inhibited to react with Et. A β-glucan-specific Limulus reagent that does not can also be used. The use of the latter can pretreat mainly for the conversion of the triple helix structure of β-glucan and the removal of the factor G system reaction-interfering factor, so that the condition of Et inactivation can be neglected and the tolerance of the pretreatment condition is broadened. effective. In the above-mentioned second invention, since the destruction or inactivation of Et is not an essential requirement and the pretreatment is performed mainly for the conversion of the triple helix structure and the removal of the factor G system reaction-interfering factor, it is possible to include Et. It is preferable to use a β-glucan-specific Limulus reagent in the case of targeting a biological sample.

Here, the term "non-specific Limulus reagent" means
Prepared by a known method (for example, J. Biochem., 80 , 1011-1021 (1976)) from the hemolymph of Limulus polyphemus, Tachypleus tridentatatus, Tachypleus gigas, Carcinoscorpius rotundicauda, etc. It is meant to include the horseshoe crab amebosite lysate, and contains at least the G factor component and the C factor component of the hemolymph coagulation system involved in the G factor reaction and the C factor reaction of FIG. 1. However, it may contain a synthetic peptide substrate described below. Here, the term “metal ion” means that the triple helix structure of β-glucan is prevented from being converted into a structure having factor G activating ability, or the converted β-glucan returns to the triple helix structure. Is a metal ion that promotes the ionization of ions such as Mg ²⁺ , Ca ²⁺ and other alkaline earth metal ions and Mn.
Ions of transition elements such as ²⁺ , Fe ²⁺ , Fe ³⁺ , Al ³⁺ and Ga ³⁺ .

The non-specific Limulus reagent that can be used in the present invention is not limited to the production method, composition, etc., as long as it has the functions as described above. In the pretreatment method used in the present invention, it is an essential condition to make the sample strongly alkaline. However, when the sample further contains metal ions, the chelate is used when pretreating under strongly alkaline conditions. It is necessary to make the agent coexist. This is because the triple helix structure of β-glucan is converted into a structure capable of activating factor G by the strong alkaline treatment, which is a kind of metal ion, that is, an ion of alkaline earth metal such as Mg ²⁺ , Ca ^2+. Or Mn ²⁺ ,
This is because ionization of transition element ions such as Fe ²⁺ , Fe ³⁺ , Al ³⁺ , and Ga ³⁺ is released. The method of making the sample strongly alkaline is not particularly limited, but it is preferable to use an alkali metal hydroxide. Specific examples thereof include potassium hydroxide (KOH) and sodium hydroxide (NaOH).

When an alkali metal hydroxide is used, its concentration is usually 0.03 to 2.0 mol / L. As the chelating agent, a metal ion contained in the sample can be converted into a chelate compound, and the concentration of free metal ion can be made equal to or lower than the minimum concentration showing the above-mentioned inhibitory action, and is not particularly limited as long as it does not inhibit the Limulus reaction. No, but when processing samples containing unknown metal ions,
Those that produce a stable and strong chelate compound with many metal ions are preferable. Such chelating agents include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid,
Trans-1,2-diaminocyclohexane-N, N,
N ', N'-tetraacetic acid, diethylenetriamine-N, N,
N ′, N ″, N ″ -pentaacetic acid, hydroxyethylenediaminetriacetic acid, glycol etherdiaminetetraacetic acid, triethylenetetraminehexaacetic acid, diaminopropanoltetraacetic acid, diaminopropanetetraacetic acid, ethylenediaminetetrakis (methylenephosphonic acid), citric acid Examples thereof include acids and their alkali salts. The pretreatment of the sample can be basically carried out by adding a chelating agent to the sample and mixing the sample with a substance that can make the sample strongly alkaline (hereinafter also simply referred to as alkali). The order of addition when adding both the alkali and the chelating agent is
It does not matter as long as the chelating agent is not added after the addition of the alkali, but after adding the chelating agent to the sample, the addition of the alkali easily causes the structural conversion of β-glucan in the sample by adding the chelating agent at a low concentration (this Under strong alkaline conditions, metal ions tend to coordinate between β-glucan molecular chains rather than to chelating agents, which seems to prevent the conversion of triple helices into single-helix structures.) . After the addition of these pretreatment agents, if desired, they can be heated in a thermostat or in an autoclave, or by high frequency using a microwave oven or the like. The treatment temperature at this time is usually 0 to 135 ° C., particularly 30 to 60 ° C.
Is preferable, and the treatment time is 1 to 60 minutes, especially 5 to 3
The range of 0 minutes is preferred.

In the test solution pretreated by the method of the present invention, even if the β-glucan before the treatment has a triple helix structure which does not activate factor G, the structure rapidly activates factor G. The structure is retained even if the β-glucan before the treatment is not in the triple helix structure and already has the factor G activating ability.

That is, β-measured in the present invention
The glucan is not particularly limited and includes those existing in fungi and the like, and the molecular structure, higher order structure, origin, etc. are not particularly limited. Further, these β-glucans include those contained in the body fluid of patients with fungal infections. In the present invention β
-Conversion of the glucan to another structure of triple helix structure can be specifically confirmed by ¹³ C-NMR or the like.

The test liquid obtained by treating the sample with the above-described pretreatment agent of the present invention is then mixed with the Limulus reagent. The mixed liquid is adjusted to an optimum pH in order to complete the factor G reaction. It is preferable that the pH is usually adjusted to 7 to 9 with a conventionally known buffer solution. The false positive factor, false negative factor, and Et inactivated by the pretreatment of the present invention have been proved to have no regenerated activity from the examples described later. On the contrary, in the mixing of the relatively high-concentration basic (alkaline) test liquid treated with the pretreatment agent with the G factor-based component, the pretreatment agent causes each component in the G factor-based component to be mixed. It is considered that the buffering effect does not adversely affect the above.

In the reaction mixture, β-
To measure glucan, the amidase activity or protease activity of the clotting enzyme, which is generated by being activated by the factor G cascade reaction of the lysate of FIG. 1, as described above, may be measured by a known method. Here, the substrate may be either synthetic or natural, and is hydrolyzed by the clotting enzyme to be introduced into a product that can be easily detected, thereby causing a reaction mixture to undergo a change based on an enzymatic reaction. It is a substrate, and it is acceptable if this change can be measured qualitatively or quantitatively.

For example, the amidase activity can be measured by bringing the Limulus reagent containing a synthetic peptide substrate into contact with the pretreated test solution to carry out the reaction. As such a synthetic peptide substrate, a peptide that can be a substrate for the above clotting enzyme (eg, methoxycarbonyl-D-hexahydrotyrosyl-Gly-
Arg; Leu-Gly-Arg with N-terminal protection,
Ile-Glu-Ala-Arg and other peptides) C-terminal arginine carboxyl group has a chromophoric residue (eg, p-nitroaniline, p- (N, N-diethylamino) aniline, p- (N -Ethyl-N-β-
(Hydroxyethyl) aniline, etc.), a fluorescent residue (eg, 7-amino-4-methylcoumarin, etc.), a luminescent residue, or a synthetic peptide substrate in which ammonia or the like is substituted by an amide bond. That is, the amidase activity can be measured by measuring the reaction products (p-nitroaniline, ammonia, etc.) produced by the clotting enzyme acting on these synthetic substrates. Specifically, the method adopted for the measurement of Et can be exemplified. For example, a dye produced by the above-mentioned cascade reaction and, if necessary, conversion reaction of the product to another dye , A fluorescent substance, a luminescent substance, or ammonia, respectively.
6871, Japanese Examined Patent Publication No. 3-66319, etc.), a fluorometer, a luminescence measuring device, an electrode for ammonia detection (Japanese Patent Laid-Open No. 62-14).
8860) and the like to measure β-glucan.

On the other hand, to measure the protease activity of clotting enzyme, for example, coagulogen, which is an ordinary natural substrate contained in Limulus reagent, is acted on by clotting enzyme produced by the cascade reaction to form coagulin gel. The reaction to be performed is measured by, for example, an appropriate device (for example, a turbidity measuring device, a viscosity measuring device, etc.), or a method (e.g., Japanese Patent Publication No. 4-14310) adopted for the determination of Et judged by the naked eye is used can do.

The method for measuring β-glucan according to the present invention is useful for early diagnosis of fungal infections, especially deep-seated fungal infections that are extremely difficult to diagnose. That is, after treating a blood-derived sample collected from a patient with the pretreatment agent of the present invention, β-glucan is measured, and when β-glucan in the blood significantly exceeds a normal value, the patient is infected with a fungal infection. It can be determined that they are affected.

In the present invention, a desired measurement kit can be constructed by appropriately combining the respective types of the pretreatment agent and the Limulus reagent. The kit includes
A standard reagent containing a fixed amount of β-glucan, distilled water for blanking, a reaction reagent dissolving / reaction buffer, etc. can be added. As the buffer solution, a Good buffer solution (for example,
HEPES (N-2-hydroxyethylpiperazine-
N'-2-ethanesulfonic acid) buffer solution, etc., Tris-hydrochloric acid buffer solution, etc. can be exemplified.

[0026]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 0.1 mL of Schizophyllan (trade name: Sonifilan, sold by Kaken Pharmaceutical Co., Ltd., 2 μg / mL aqueous solution) consisting of 100% triple helix structure and Et (E. coli 0111: B4)
Origin, sold by Difco, 80 ng / mL aqueous solution)
0.2 mL of various concentrations of NaOH aqueous solution (pretreatment agent) was added to 1 mL, mixed, and then heated at 60 ° C. for 10 minutes. This is diluted 1000 times with distilled water for injection, and Sonifiran 1
A solution of ng / mL and Et 0.02 ng / mL (concentration when Et is not destroyed) was prepared. These treated liquids were used as test liquids, and distilled water for injection was used as a blank.
And 50 μL of each was separately taken into the wells of a β-glucan-free microplate (Toxipet plate 96F, sold by Seikagaku Corporation).

Β-glucan and Et present in the test liquid
The amount of was determined by the following method. The Obayash
i, T. A β-glucan-specific chromogenic synthetic substrate method containing a factor G component prepared from horseshoe crab amebosite lysate and a chromogenic synthetic substrate (Boc-Leu-Gly-Arg-pNA (p-nitroanilide)) according to these methods Limulus reagent (hereinafter referred to as "G test") or non-specific Limulus reagent for chromogenic synthetic substrate method (toxicolor,
Seikagaku Kogyo Co., Ltd.) or the endotoxin-specific chromogenic synthetic substrate method Limulus reagent (Endospecie, Seikagaku Kogyo Co., Ltd.) is used to add 0.2 to 50 μL of the test solution.
Mol / L HEPES buffer (pH 7.6) dissolved in 50 μL of G test solution or 0.2 mol / L Tris
Add 50 μL of Toxicolor solution or Endospecy solution dissolved in -HCl buffer (pH 8.0), and add 3 at 37 ° C.
The reaction was carried out by heating for 0 minutes, and then 0.04% (weight / volume) sodium nitrite (1 mol / L hydrochloric acid solution) 50
μL, 0.3% (weight / volume) ammonium sulfamate 50 μL and 0.07% (weight / volume) N
-1-naphthylethylenediamine dihydrochloride (14% (volume / volume) N-methyl-2-pyrrolidone solution) 50 μL
Were sequentially added to carry out diazo coupling, and quantification was carried out by measuring the absorbance at 545 nm using 630 nm as a control wavelength by a microplate reader.

Further, the structure change of the triple helix β-glucan when the NaOH concentration of the pretreatment agent solution is variously changed to variously adjust the NaOH concentration of the test solution is described by Saito H. e.
Tal. (Carbohydr. Res., 74, 227-240 (1979)), and was measured by ¹³ C-NMR. Table 1 shows the ratio of the triple helix structure and the measured value (absorbance) of each test of the pretreated test liquid.

[0029]

[Table 1]

In Table 1, the NaOH concentration (mol / L)
Indicates the concentration in the test solution, and the absorbance is the value obtained by subtracting the blank value. Moreover, the case where the Et aqueous solution was replaced with distilled water or the sonifiran aqueous solution was replaced with distilled water was also shown as the test liquid. According to Table 1, in the G test, β-glucan was not detected at all when treated with only distilled water for injection, and the concentration of NaOH was 0.03 mol / L.
In the above, the value of absorbance is constant (Sample No. 1-1
And 1-4 to 10). This means that from the result of observing the structural change of β-glucan in the pretreated test liquid,
This corresponds to the fact that the absorbance increases as the triple helix structure of β-glucan is solved. Further, in the case of Toxicolor, treatment with only distilled water for injection reacts with Et but does not react with β-glucan at all (Sample Nos. 1-1 and 1-2). NaOH concentration
When 0.03 mol / L or more, the absorbance becomes constant and β
-Glucan can be detected (Sample No. 1-4
-10). In the case of endoscopy, treatment with only distilled water for injection reacts with Et and does not react with β-glucan at all (Sample Nos. 1-1 and 1-2). NaO
It can be seen that when the H concentration is 0.03 mol / L or more, the absorbance becomes 0 and the activity of Et is lost.

That is, in this embodiment, when the sample is an aqueous solution, when the NaOH concentration is around 0.03 mol / L or more,
Pretreatment at 0 ° C. for 10 minutes inactivates Et and unravels the triple helix structure of β-glucan to G
Since the G value is activated by converting it into a structure having factor activation ability, the absorbance value becomes constant, and the true value of β-glucan in the test solution is of course the same as with the β-glucan-specific Limulus reagent. It is clear that even if a non-specific Limulus reagent is used, it can be detected accurately and with high reliability and reproducibility.

Example 2 To 180 μL of PRP of a healthy person, lentinan consisting of 99% triple helix structure (sold by Ajinomoto Co., Inc., 200 ng / mL)
Aqueous solution) 10 μL and Et (Serratia marce
10 μL of scens, sold by Sigma, 8 ng / mL aqueous solution), and 800 μL of distilled water or KOH aqueous solution of various concentrations were added thereto, and the mixture was heated at 37 ° C. for 30 minutes. After cooling with ice, 50 μL of the treated mixture was placed in a test tube, and 50 μL of an aqueous HCl solution was added to neutralize the mixture.
100 μL of G test solution, Toxicolor solution or Endospecy solution was added to this and heated at 37 ° C. for 30 minutes to react, and then 0.04% sodium nitrite (0.48
(Mol / L hydrochloric acid solution), 0.3% ammonium sulfamate, and 0.07% N-1-naphthylethylenediamine dihydrochloride (0.5 mL each) were sequentially added to perform diazo coupling, and the absorbance was measured at 545 nm. When a precipitate was formed, it was centrifuged and the supernatant was measured. Table 2 shows the measured values (absorbance) of each test of the pretreated test liquid.

[0033]

[Table 2]

In Table 2, KOH concentration (mol / L)
Indicates the concentration in the test solution, and the absorbance is the value obtained by subtracting the blank value. As a blank, distilled water was used instead of PRP, and distilled water for injection was used as a pretreatment agent. In addition, the case where the Et aqueous solution was replaced with distilled water for injection and / or the lentinan aqueous solution was replaced with distilled water as the test liquid was also shown.

According to Table 2, since there is a Limulus reaction-interfering factor in PRP, β-glucan and Et were hardly detected by the treatment with only distilled water for injection (N
o. 2-1 to 4), it is found that this factor is inactivated by heating in KOH of 0.06 mol / L or more at 37 ° C. for 30 minutes (Sample No. 2-6). ~ 1
2). Further, Et decreases in activity with an increase in the concentration of treated KOH, and is completely inactivated by the treatment of 0.7 mol / L or more (Sample No. 2-10 to 12). on the other hand,
Lentinan having a 99% triple-helix structure changes its triple-helix and exhibits high reactivity when treated with 0.06 mol / L or more of KOH. That is, the KO is 0.7 mol / L or more when plasma treated with Et and β-glucan is treated.
Pretreatment with aqueous H solution at 37 ° C for 30 minutes will result in Et and β-
It is found that even in a sample in which glucan coexists, only β-glucan can be accurately detected by a normal Limulus reagent (non-specific Limulus reagent) that reacts with both without using G test that is β-glucan-specific reagent. (Sample No.
2-10-12).

When clinical samples were actually treated with the pretreatment agent according to Example 2 and measured with toxicocolor, only the patients with deep-seated mycosis showed high values. Example 3 0.2 mL of an aqueous solution containing metal ions shown in Table 3 to which 10% schizophyllan (trade name Sonifiran) having a triple helix structure was added
And 0.125 mol / L EDTA4Na salt solution 0.025
After adding mL and standing for 5 minutes under ice cooling, 0.025 mL of 3 mol / L sodium hydroxide solution is added (sequential addition) or 1.5 mol / L containing 0.20 mol / L EDTA4Na salt solution L sodium hydroxide 0.05mL
Was added (simultaneous addition), allowed to stand at room temperature for 10 minutes, then diluted 10-fold with distilled water for injection, and 0.05 mL of the diluted solution was used as a test solution using the G test as in Example 1. The reactivity was investigated. The results are shown in Table 3. In the table, 100% of the case where the sodium ion solution containing no metal ion was treated with a sodium hydroxide solution containing no EDTA4Na salt (0.3 mol / L at the time of treatment), and instead of the EDTA4Na solution and the sodium hydroxide solution, The relative activity was 0% when using distilled water for injection. In order to investigate the effect of adding EDTA4Na salt when measuring a sample containing metal ions, EDTA4N was added in the above sequential addition.
An experiment was also performed using an equal amount of distilled water for injection instead of the salt solution a. An experiment was also conducted in which the addition order of the above-mentioned sequential addition was reversed.

[0037]

[Table 3] Table 3 ――――――――――――――――――――――――――――――――――― Metal Ion Aqueous Solution Addition of EDTA4Na and NaOH Relative activity Presence (+/-) and addition method (%) EDTA4Na NaOH addition method (A) (B) ―――――――――――――――――――――――――――― ――――――――― Distilled water for injection (control example) − − 0 Distilled water for injection (control example) − + 100 10 mM MgCl ₂ − + 2.6 10 mM MgCl ₂ + + Sequential (A → B) 124.0 10 mM MgCl ₂ + + Sequential (B → A) 0.6 10 mM CaCl ₂ − + 2.8 10 mM CaCl ₂ + + Simultaneous 105.9 0.5 mM FeCl ₂ − + 2.0 0.5 mM FeCl ₂ + + Sequential (A → B) 98.8 0.5 mM FeCl ₃ − + 0 0.5 mM FeCl ₃ + + Simultaneous 95.6 0.05 mM MnSO ₄ − + 4.0 4.0 mM MnSO ₄ + + Sequential (A → B) ) 105.0 Ringer's solution (containing 2.24 mM CaCl ₂ ) + + Simultaneously 113.2 For artificial kidney Dialysate- + 3.8 Dialysis fluid for artificial kidney + + Simultaneous 97.5 (including 1.5 mM CaCl ₂ , 0.5 mM MgCl ₂ ) PBS (+) + + Sequential (A → B) 107.4 (including 0.901 mM CaCl) ₂ , 0.492mM MgSO ₄ ) Hank's solution + + Sequential (A → B) 105.2 (including 1.26mM CaCl ₂ , 0.812mM MgSO ₄ ) ―――――――――――――――――― ―――――――――――――――――

From the results shown in Table 3, the presence of Mg ²⁺ or Ca ²⁺ in the aqueous solution of schizophyllan having a triple helix structure does not increase the G test reactivity even if treated only with alkali, but the chelating agent is added with alkali. If added at the same time as or before the addition, the G-test reactivity of Schizophyllan increases,
It was found that the detection and quantification can be performed with high sensitivity. An experiment was conducted using potassium hydroxide instead of sodium hydroxide in the above experiment, and almost the same results as above were obtained.

[0039]

INDUSTRIAL APPLICABILITY The present invention provides a β-glucan mainly composed of a triple helix structure, as well as a β-glucan in a sample containing Et and / or factor G system reaction-interfering factor (limulus reaction-interfering factor).
When measuring glucan, the simple effect of treating the sample with a strongly alkaline pretreatment agent is employed to completely eliminate the influence of the Et and / or G factor reaction-interfering factors on the reaction system. And a β-glucan that has a triple helix structure that does not activate factor G can be converted into a structure capable of activating factor G, and thus is non-specific that also has reactivity with Et. The Limulus reagent can be used to accurately quantify β-glucan. Further, the present invention, when measuring β-glucan in a sample containing a metal ion in addition to β-glucan mainly having a triple helix structure, the sample is treated with a strong alkaline pretreatment agent in the presence of a chelating agent. By treatment, it is possible to prevent the metal ion from inhibiting the conversion of the triple helix structure of β-glucan into a structure having factor G activating ability by strong alkaline treatment. It is possible to accurately quantify β-glucan in a sample which may contain metal ions such as. Furthermore, when β-glucan in a biological sample is measured using the measuring method of the present invention, highly accurate and highly reproducible results can be obtained. By applying the method for measuring β-glucan of the present invention to a clinical test, a deep-seated fungal infection, which is extremely difficult to diagnose by an ordinary test method, can be quickly and accurately diagnosed.

[Brief description of drawings]

Figure 1: Horseshoe crab, amebosite lysate (1
→ 3) The reaction mechanism of the cascade reaction by -β-D-glucan and endotoxin is shown.

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[Procedure amendment]

[Submission date] December 8, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 8

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0001

[Correction method] Change

[Correction content]

[0001]

BACKGROUND OF THE INVENTION The present invention relates to (1 → 3) -β-D-
The present invention relates to a method for measuring glucan (hereinafter, also referred to as "β-glucan"). In particular, endotoxin (hereinafter also referred to as “Et”) involved in the Limulus factor C system reaction, which may be contained in the sample as a reaction-interfering factor, alone or a specific reaction between Et and Limulus factor G and β-glucan is described. The β-glucan in a sample that may contain both reaction-interfering factors (hereinafter also referred to as “G factor-based reaction-interfering factors”) that interfere with each other or in a sample that may contain metal ions such as Ringer's solution and dialysate is tested using a Limulus reagent. The present invention relates to a method that can be used to perform measurement with high accuracy.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction content]

[0011]

According to the present invention, a sample containing (1 → 3) -β-D-glucan having a triple helix structure as a main component is pretreated under strongly alkaline conditions and used as a reaction interfering factor. The method is characterized by destroying or inactivating endotoxin alone or endotoxin and a factor G system reaction-interfering factor that may be contained in a sample, and then subjecting to a Limulus reaction by a Limulus reagent (1 → 3) -β
-D-glucan measuring method, the Limulus reagent is a non-specific Limulus reagent, the above-mentioned (1 → 3) -β-D-glucan measuring method, alkali metal hydroxide in a sample The method for measuring (1 → 3) -β-D-glucan described above or under the condition of being strongly alkaline under the condition that the alkali metal hydroxide concentration in the sample is 0.03 to 2.0 mol / L. Pretreatment is performed under the following, the measurement method described above, and the measurement method according to any one of the above, wherein the pretreatment is performed under a condition of 0 to 135 ° C., and the sample may contain a metal ion. The method for measuring (1 → 3) -β-D-glucan described above, in which a sample is pretreated in the presence of a chelating agent, mainly having a triple helix structure (1 →
3) A sample that may contain -β-D-glucan and a metal ion is pretreated in the presence of a chelating agent under strongly alkaline conditions and then subjected to a Limulus reaction with a Limulus reagent (1 → 3) ) Method for measuring -β-D-glucan, the pretreatment, after adding a chelating agent to the sample,
Method of measuring (1 → 3) -β-D- glucan of the claimed to strongly alkaline conditions at the time of adding or carried out in strongly alkaline conditions, or a chelating agent to the sample, the Limulus reagent, Limulus blood It does not substantially contain the factor C component of the lymphoid coagulation system, or the factor C system reaction is inhibited, and it does not react with endotoxin, and (1 → 3) -β-D-
It is specific to glucan (1 →
3) A method for measuring -β-D-glucan.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0036

[Correction method] Change

[Correction content]

When clinical samples were actually treated with the pretreatment agent according to Example 2 and measured with toxicocolor, only the patients with deep-seated mycosis showed high values. Example 3 Schizophyllan (trade name Sonifiran) having a 100% triple helix structure was added to an aqueous solution containing metal ions shown in Table 3.
0.2 mL added to give 1.0 ng / mL
And 0.125 mol / L EDTA4Na salt solution 0.025
After adding mL and standing for 5 minutes under ice cooling, 0.025 mL of 3 mol / L sodium hydroxide solution is added (sequential addition) or 1.5 mol / L containing 0.20 mol / L EDTA4Na salt solution L sodium hydroxide 0.05mL
Was added (simultaneous addition), allowed to stand at room temperature for 10 minutes, then diluted 10-fold with distilled water for injection, and 0.05 mL of the diluted solution was used as a test solution using the G test as in Example 1. The reactivity was investigated. The results are shown in Table 3. In the table, 100% of the case where the sodium ion solution containing no metal ion was treated with a sodium hydroxide solution containing no EDTA4Na salt (0.3 mol / L at the time of treatment), and instead of the EDTA4Na solution and the sodium hydroxide solution, The relative activity was 0% when using distilled water for injection. In order to investigate the effect of adding EDTA4Na salt when measuring a sample containing metal ions, EDTA4N was added in the above sequential addition.
An experiment was also performed using an equal amount of distilled water for injection instead of the salt solution a. An experiment was also conducted in which the addition order of the above-mentioned sequential addition was reversed.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0039

[Correction method] Change

[Correction content]

[0039]

INDUSTRIAL APPLICABILITY The present invention provides a β-glucan mainly composed of a triple helix structure, as well as a β-glucan in a sample containing Et and / or factor G system reaction-interfering factor (limulus reaction-interfering factor).
When measuring glucan, the simple effect of treating the sample with a strongly alkaline pretreatment agent is employed to completely eliminate the influence of the Et and / or G factor reaction-interfering factors on the reaction system. And a β-glucan that has a triple helix structure that does not activate factor G can be converted into a structure capable of activating factor G, and thus is non-specific that also has reactivity with Et. The Limulus reagent can be used to accurately quantify β-glucan. Further, the present invention, when measuring β-glucan in a sample containing a metal ion in addition to β-glucan mainly having a triple helix structure, the sample is treated with a strong alkaline pretreatment agent in the presence of a chelating agent. by processing, that the triple helix structure of β- glucan by strong alkali treatment is converted into a structure having a factor G activating ability, it is possible to prevent the phenomenon of metal ions to inhibit, dialysis solution, Ringer's solution It is possible to accurately quantify β-glucan in a sample which may contain metal ions such as. Furthermore, when β-glucan in a biological sample is measured using the measuring method of the present invention, highly accurate and highly reproducible results can be obtained. By applying the method for measuring β-glucan of the present invention to a clinical test, a deep-seated fungal infection, which is extremely difficult to diagnose by an ordinary test method, can be quickly and accurately diagnosed.

Claims (9)

[Claims] 1. Mainly having a triple helix structure (1 → 3)
A sample that may contain -β-D-glucan is pretreated under strongly alkaline conditions to destroy endotoxin alone or endotoxin and a factor G system reaction-interfering factor which may be contained in the sample as a reaction-interfering factor. A method for measuring (1 → 3) -β-D-glucan, which comprises subjecting to a Limulus reaction with a Limulus reagent after inactivation. 2. The (1 → 3) -β- of claim 1, wherein the Limulus reagent is a non-specific Limulus reagent.
A method for measuring D-glucan. 3. The method for measuring (1 → 3) -β-D-glucan according to claim 1 or 2, wherein an alkali metal hydroxide is present in the sample and the sample is subjected to strong alkaline conditions. 4. The (1 → 3) -β-D-glucan according to claim 3, which is pretreated under the condition that the concentration of the alkali metal hydroxide in the sample is 0.03 to 2.0 mol / L. Measuring method. 5. The (1 → 3) -β-D according to any one of claims 1 to 4, wherein the pretreatment is carried out under a condition of 0 to 135 ° C.
-A method for measuring glucan. 6. The method for measuring (1 → 3) -β-D-glucan according to claim 1, wherein the sample can contain a metal ion and is pretreated in the presence of a chelating agent. 7. A triple helix structure is mainly used (1 → 3).
A sample that may contain -β-D-glucan and metal ions,
A method for measuring (1 → 3) -β-D-glucan, which comprises subjecting to preliminary treatment in the presence of a chelating agent under strongly alkaline conditions and then subjecting it to a Limulus reaction with a Limulus reagent. 8. The method according to claim 1, wherein the pretreatment is performed under strong alkaline conditions after adding the chelating agent to the sample or under strong alkaline condition when adding the chelating agent to the sample. 3) A method for measuring -β-D-glucan. 9. The Limulus reagent is substantially free of Factor C component of the horseshoe crab hemolymph coagulation system, or C
Factor system reaction is inhibited, it does not react with endotoxin,
Specific to (1 → 3) -β-D-glucan,
The method for measuring (1 → 3) -β-D-glucan according to claim 8.