JP2004109147A - Pretreatment agent, pretreatment method, measurement method by pretreated sample, measuring kit, and determination method of sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a rapid measurement method having a high detection rate for β-glucan in a blood-derived sample. <P>SOLUTION: (1) This pretreatment agent contains an alkali metal hydroxide as a main constituent. (2) This pretreatment method is characterized by mixing the pretreatment agent described in (1) with a sample and heating them. (3) This measurement method is used for measuring (1-3)-β-D-glucan in the sample by using a limulus reaction and for detecting variation of a substrate by pretreating the sample by the method (2), and by mixing and reacting the pretreated sample with a limulus reagent. (4) This measuring kit comprises (A) the pretreatment agent and (B) the limulus reagent. (5) This determination method of the sample is used for quantitatively measuring the (1-3)-β-D-glucan by the measurement method to determine that the sample is affected by an infectious disease when the substance exceeds a certain amount. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to a pretreatment agent for a sample containing a reaction interfering factor with respect to a Limulus reaction, a pretreatment method thereof, a measurement method using a pretreated sample, a measurement kit used for the measurement method, and a method for determining a sample. In particular, the present invention relates to a method for measuring (1 → 3) -β-D-glucan in a biological sample, particularly a blood sample, containing a factor that interferes with the Limulus reaction using horseshoe crab amebosite lysate. The treatment of the blood-derived sample, which is particularly effective for diagnosing a fungal infection by measuring (1 → 3) -β-D-glucan in a blood-derived sample of a patient with a fungal infection with high accuracy, and It concerns the measurement method.

Conventionally, the measurement of a target substance in a biological sample using a biochemical reaction has been widely performed in connection with clinical diagnosis. For example, a typical biochemical diagnosis includes detection of fungi or gram-negative bacteria using a Limulus reaction. The Limulus reaction is a biochemical reaction using a Limulus reagent.

Horseshoe crab amebosite lysate (hereinafter sometimes simply referred to as “lysate”) contained in the Limulus reagent includes a cascade-type coagulation system (factor C system) that is activated by reacting with endotoxin and (1 → 3) Cascade-type coagulation system (Factor G system) which is activated by reacting with -β-D-glucan (hereinafter sometimes also referred to as “β-glucan”) (FIG. 1); A method of specifically measuring endotoxin using only the latter system and a method of specifically measuring β-glucan using only the latter system are known (Non-Patent Document 1). In addition, patients with fungal infections have increased β-glucan in the blood, and patients with gram-negative infections have increased endotoxin in the blood. It is also known that a fungal infection or a gram-negative bacterial infection can be diagnosed by measuring β-glucan or endotoxin in the blood, respectively.

Such a method for measuring β-glucan using a factor G system and endotoxin using a factor C system is very suitable for detection of trace amounts of the above substances in a biological sample due to extremely high detection sensitivity. Its effectiveness in the diagnosis of sexually transmitted fungal infections and gram-negative bacterial infections has been studied and confirmed, and it has begun to be used in clinical tests. By the way, when β-glucan and endotoxin in a biological sample, particularly blood, are measured using a cascade reaction of a lysate by a factor G system and a factor C system, the reaction utilizes the reaction of a serine protease in the lysate. to, various reaction interfering factors contained therein (e.g., thrombin or factor Xa to show the effect similar to clotting enzyme in the lysate, and false positives factor, alpha ₂ - plasmin inhibitor, alpha ₁ - antitrypsin In addition, antithrombin III strongly inhibits the reaction and becomes a false negative factor), and requires a pretreatment to inactivate or remove the antithrombin III. Conventionally, for this purpose, a blood sample is conventionally subjected to a specific treatment to prepare platelet-rich plasma (PRP), which is further heated at 37 ° C. by adding perchloric acid, and then the denatured precipitate is centrifuged. And then removing the supernatant, collecting the supernatant, neutralizing it with an alkali to obtain a test solution (Non-Patent Document 1), but the separation operation of the denatured precipitate is complicated, and all the operation steps are required. In many cases, there are problems such as a risk of contamination by substances that affect the reaction system during the operation.

By the way, all of the pretreatment methods described above for measuring endotoxin and β-glucan perform pretreatment in a test tube, and furthermore, a part of the sample thus pretreated is transferred to another test tube. It was performed by taking out and performing a Limulus reaction. Further, when the measurement is performed by the synthetic substrate method, an end-point method of converting the p-nitroaniline generated by cleavage of the substrate to a red dye by a diazotization reaction and measuring the absorbance after the Limulus reaction is generally used. Was used to. The end point method is a method that is usually complicated and requires a long measurement time, and a method that can process a large number of samples in a short time at a time is desired. If a microplate is used instead of a test tube, a large number of samples can be handled at the same time, but continuous automatic measurement is difficult with the endpoint method.

Therefore, measurement by a kinetic method (Patent Document 1) in which such a microplate can be used and a change in the substrate can be directly measured automatically is desired, but the reaction volume of the microplate is small, and the reaction solution is turbid. There is a problem that the measurement cannot be performed with high accuracy due to the influence of the above.

Obayashi @ T. et @ al. , Clin. Chim. Acta, (1985), 149, 55-65. JP-A-3-220456

The present invention is intended to solve the above-mentioned problems of the conventional pretreatment method, and includes a reaction interfering factor for a Limulus reaction, such as a blood interfering sample containing a reaction interfering factor for a G factor system in a Limulus reaction. The β-glucan in the blood-derived sample can be extremely reduced by removing or denaturing the factor by a simple treatment, employing a method that does not require the separation operation of the denatured precipitate, and adopting a method that does not cause turbidity during the measurement by the Limulus reaction. An object of the present invention is to provide a pretreatment agent, a pretreatment method, a measurement method, a measurement kit, and a method for determining a sample that can be measured quickly and efficiently at a high detection rate.

Means for Solving the Problems The present invention comprises the following means, which can solve the above-mentioned problems.
1) Before using a sample containing a reaction interfering factor to the Limulus reaction prior to the Limulus reaction when measuring (1 → 3) -β-D-glucan in the sample using the Limulus reaction What is claimed is: 1. A pre-treatment agent comprising an alkali metal hydroxide as a main component.
2) The pretreatment agent according to the above 1), wherein the pretreatment agent is stored as a plurality of solutions, and the solutions are mixed at the time of use.
3) When detecting (1 → 3) -β-D-glucan contained in a sample containing a reaction interfering factor to the Limulus reaction by using the reaction, the sample is treated prior to the Limulus reaction. In the pretreatment method, the pretreatment agent described in 1) or 2) above is mixed with a sample and heated.
4) The pretreatment method according to 3) above, wherein the sample is a blood-derived sample.
5) A method for measuring (1 → 3) -β-D-glucan in a sample using the Limulus reaction, wherein the sample is pretreated by the method described in 3) or 4) above, and A measurement method comprising mixing a sample with a Limulus reagent and reacting to detect a change in a substrate.
6) A measurement kit for measuring (1 → 3) -β-D-glucan, which comprises at least the following constituent reagents.
(A) The pretreatment agent according to 1) above.
(B) Limulus reagent obtained from horseshoe crab amebosite lysate as a raw material.
7) The measurement kit according to 6) above, wherein the Limulus reagent of (B) is a Limulus reagent that specifically reacts with (1 → 3) -β-D-glucan.
8) The kit for measurement according to 6) above, further comprising (C) below as a constituent reagent.
(C) A standard reagent containing a fixed amount of (1 → 3) -β-D-glucan.
9) (1 → 3) -β-D-glucan in a sample derived from a living body is quantified by the measuring method according to claim 5, and when the measured value of the substance exceeds a certain amount, the living body suffering from infectious disease is determined. A method for determining a sample, comprising determining that the sample is derived from the sample.
Hereinafter, the present invention will be described specifically.

The present invention can provide a pretreatment agent that can effectively perform a pretreatment of a biological sample such as blood to which various biochemical reactions are applied, and particularly, a plasma, serum, or the like containing a factor G-based reaction interfering factor. When measuring β-glucan in a sample derived from a living body, the reaction system of the reaction interfering factor can be measured by adopting a simple means of treating the sample with a pretreatment agent containing an alkali metal hydroxide as a main component. In addition to completely removing the influence on the test solution, the increase in turbidity of the test liquid after the treatment was able to be suppressed. In addition, since there is no need to separate and remove the modified precipitate after the treatment, the entire operation step can be shortened. Further, the measurement method of the present invention can automatically measure β-glucan in a biological sample in a microplate by a kinetic method, so that simple, quick, highly accurate and highly reproducible results can be obtained. By applying the measurement method of the present invention to a clinical test, it is possible to quickly and accurately diagnose a deep fungal infection, which is extremely difficult to diagnose with a normal test method.

The term "sample containing a reaction interfering factor with respect to the Limulus reaction" as a target in the pretreatment agent of the present invention refers to a sample that may contain the target substance measured by the Limulus reaction. It is included to the extent that it affects the Limulus reaction. The sample is typically a biological sample, especially a blood sample. The blood-derived sample is typically plasma or serum itself obtained by treating blood collected from mammals including humans by a known method, or proteases, protease inhibitors, blood-derived protein preparations, and the like. Or plasma or serum containing As used herein, the term “response interfering factor” refers to a factor that reacts independently of the Limulus reaction (false positive factor) or a factor that inhibits the reaction at any stage (false negative factor). And the factors contained therein.

血漿 In order to prepare plasma from blood, it is usually sufficient to add a blood coagulation inhibitor such as heparin to blood and centrifuge to precipitate blood cells. At that time, if the centrifugation is performed at a low rotation speed (for example, about 150 × g), platelet-rich plasma (PRP) containing a large amount of platelets is obtained, and when the centrifugation is performed at a high rotation speed (for example, about 1000 × g). Platelet poor plasma (PPP) is obtained. When the blood-derived sample to be measured in the present invention is plasma, it may be either PRP or PPP.

血清 Serum is obtained by removing blood cells and some blood coagulation factors from blood, and is usually prepared by leaving collected blood in a container and separating and removing the formed clot. By treating the blood-derived sample or the like with the pretreatment agent of the present invention, the influence of the reaction interfering factor on the Limulus reaction is removed.

In the present invention, the term "limulus reaction" refers to a G-factor-based component (a component containing at least factor G and a coagulase precursor) of a lysate (limulus amoebocyte lysate) obtained by extracting horseshoe crab amebosite (blood cell) with a hypotonic solution or the like. ) And β-glucan. "Limulus reagent" and "Limulus reagent obtained from horseshoe crab amebosite lysate" mean both reagents for measuring β-glucan by the Limulus reaction, and include Limulus polyphemus, Taxipulus tridentatus, and Taxipulus. Contains normal horseshoe crab amebosite lysate prepared from a horseshoe crab hemolymph such as Gigas and Carcinoscorpius rotundicauda by a known method (see, for example, J. @Biochem., 80, 1011-1021 (1976)). This is a reagent to which a peptide synthesis substrate described below is added as necessary. The term "Limulus reagent specifically reacting with β-glucan" refers to specifically inhibiting, adsorbing and removing factor C of the lysate (for example, WO91 / 19981, WO92 / 16651), or It is prepared by fractionation and reconstitution (Obayashi T. et al., Clin. Chim. Acta, 149, 55-65 (1985)). Therefore, the Limulus reagent for β-glucan was prepared so that the reaction system was specifically activated by β-glucan without being activated by endotoxin.

リ ム The Limulus reagent that can be used in the present invention may be any one having the above functions, and is not limited to a production method, a composition, or the like. In the Limulus reaction, as a reaction interfering factor when β-glucan is to be measured, a factor affecting a factor G-based reaction can be mentioned. Here, the “reaction interfering factor” is independent of β-glucan at any stage of the stepwise enzyme reaction (cascade reaction) of the lysate factor G system shown in FIG. 1 which is initiated by β-glucan. The false positive factor or false negative factor that reacts.

Hereinafter, the pretreatment agent of the present invention will be specifically described.
The present invention provides a pretreatment agent for measuring β-glucan containing alkali metal hydroxide as a main component (pretreatment agent B for measuring β-glucan).
The alkali metal hydroxide is a hydroxide of an alkali metal such as potassium, lithium, and sodium, and specifically includes potassium hydroxide (KOH), lithium hydroxide (LiOH), and sodium hydroxide (NaOH). is there. As the pretreatment agent, an aqueous solution containing one or more alkali metal hydroxides is usually used. Most preferred are KOH and NaOH.

Usually, it is preferable to adjust the concentration of the alkali metal hydroxide in the pretreatment agent so that the concentration of the alkali metal hydroxide in the test solution is 0.04 to 0.4 mol / l.
Other additives may be added to this pretreatment agent as needed. Examples of such additives include substances having an action of preventing β-glucan from being adsorbed to blood components to suppress the factor G cascade reaction, stabilizing the reaction system, and improving reproducibility. Can be For example, an alkali metal halide is preferably exemplified.
The pretreatment agent of the present invention may be stored as a plurality of solutions, and the solutions may be mixed at the time of use. If the alkali metal hydroxide and the alkaline earth metal halide and / or the nonionic surfactant are mixed and stored for a long time, there is an inconvenience such as precipitation. It is preferable to mix during pretreatment. In particular, it is preferable that KOH and CaCl ₂ and Triton X-100 be stored separately.

試 料 The treatment of a sample using the above pretreatment agent can be basically performed by adding and mixing the sample and heating. Then, if desired, the mixture can be heated while being subjected to stirring or vibration. The processing temperature at this time is appropriately selected depending on the type of the sample, and is usually preferably in the range of 25 to 70 ° C., particularly preferably 37 to 56 ° C., and the processing time is preferably in the range of 5 to 40 minutes, particularly 5 to 20 minutes. .

A method for measuring β-glucan in a sample using the Limulus reaction is to mix a sample treated with the pretreatment agent of the present invention with a Limulus reagent obtained from a horseshoe crab, amebosite lysate, and to react with the mixture. This can be done by detecting a change.
The pretreated sample can be directly subjected to the Limulus reaction without centrifugation or neutralization. In the measurement method using the Limulus reaction, a Limulus reagent is added to a pretreated sample, the mixture is reacted at about 37 ° C. and pH 7 to 9 for an appropriate time, and the change in the substrate is measured by a reaction measuring means corresponding to the substrate. This is performed by calculating the content of β-glucan in a sample from a calibration curve prepared using a standard reagent in advance.

In addition, it is necessary to mix and react the sample treated with the pretreatment agent B for β-glucan measurement of the present invention with a Limulus reagent containing at least a factor G-based component that reacts with β-glucan. It is very preferable to select a substance that specifically reacts with only β-glucan. Examples of such a Limulus reagent include those containing a factor G component and a factor C component removed or inhibited.

Therefore, the reaction mixture for measuring β-glucan is preferably adjusted to the vicinity of the optimal pH of the factor G system, and usually adjusted to a pH of 7 to 9 by a conventionally known buffer as desired. . The false positive factors and false negative factors inactivated by the pretreatment according to the present invention have been found to have no activity regenerated from the examples described later. Conversely, when mixing the test solution containing a relatively high concentration of a basic substance treated with the pretreatment agent with the Limulus reagent, the pretreatment agent reacts with each of the components in the factor G system. It is considered that the buffering action does not adversely affect the water content.

In the reaction mixture, the β-glucan in the test solution was measured by the amidase activity on the substrate of the clotting enzyme generated by the activation of the lysate of FIG. 1 by the factor G cascade reaction as described above. Alternatively, the protease activity may be measured by a known method. Here, the substrate may be any of synthetic and natural ones, and is hydrolyzed by the clotting enzyme to lead to an easily detectable product, which causes a change based on an enzymatic reaction in the reaction mixture. It is only required that the substance be a substrate and that this change can be measured qualitatively or quantitatively.

For example, an amidase activity can be measured by bringing a reaction system containing a lysate for β-glucan measurement and a peptide synthesis substrate into contact with a test solution to carry out a reaction. As such a peptide synthesis substrate, a peptide that can be a substrate of the above-mentioned clotting enzyme (for example, methoxycarbonyl-D-hexahydrotyrosyl-Gly-Arg; Leu-Gly-Arg, Ile-Glu having an N-terminal protected). -A chromogenic residue (for example, p-nitroaniline, p- (N, N-diethylamino) aniline, p- (N-ethyl-) at the carboxyl group of arginine at the C-terminus of a peptide having a sequence such as -Ala-Arg or the like. N- [beta] -hydroxyethyl) aniline, etc.), a fluorescent residue (e.g., 7-aminomethylcoumarin, etc.), a luminescent residue, or a peptide synthesis 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.) generated by the action of the clotting enzyme on these synthetic substrates. Specifically, a reaction (a cascade reaction and, if necessary, other dyes of the product, etc.) is carried out in the presence of the peptide synthesis substrate in a reaction system containing the pretreated test liquid and a factor G-based component of the lysate. And a dye, a fluorescent substance, a luminescent substance, or ammonia generated by the reaction, respectively, into a spectrophotometer (JP-B-63-26871, JP-B-3-66319, etc.), a fluorometer, a chemiluminescence measuring device. And an ammonia detection electrode (Japanese Patent Application Laid-Open No. 62-148860).

Particularly, the pretreatment agent of the present invention is effectively used for measurement in which pretreatment is performed with the pretreatment agent in a microplate, and then a Limulus reaction is performed. In particular, since it can be suitably used for measurement by simultaneous two-wavelength photometry in the kinetic method, quick and accurate measurement of a desired substance is possible.

On the other hand, in the measurement of the protease activity of the clotting enzyme, for example, a clotting enzyme generated by a cascade reaction acts on a limulus reagent for measuring β-glucan containing a coagulogen which is a natural substrate of the clotting enzyme. The coagulin gel formation reaction is measured by, for example, a suitable instrument (for example, a turbidity measuring device, a viscosity measuring device, or the like) or a method adopted for the measurement of endotoxin which is visually determined (Japanese Patent Publication No. 4-14310). Etc.) can be used. As the Limulus reagent used in the above reaction, a reagent (for β-glucan) specifically inhibiting or adsorbing and removing factor C of the lysate is preferably used. These Limulus reagents usually contain coagulogen, but may of course be added separately.

The method for measuring β-glucan according to the present invention is useful for early diagnosis of fungal infections, particularly deep fungal infections, which are extremely difficult to diagnose. In order to diagnose a fungal infection, after treating a blood-derived sample collected from a patient suspected of having a fungal infection with the pretreatment agent of the present invention, β-glucan is measured, and β-glucan in the blood is determined. When the amount exceeds a certain amount (normal value), it can be determined that the patient has a fungal infection.

In the present invention, a desired measurement kit can be constituted by combining the pretreatment agent of the present invention with a Limulus reagent for β-glucan measurement.

キ ッ ト The kit of the present invention can optionally add other optional constituent reagents. Examples of such a reagent include a standard reagent containing a certain amount of β-glucan, distilled water for blank test, and a buffer for dissolving and reacting the reaction reagent. Examples of the buffer include Good buffer (for example, HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer) and Tris-HCl buffer.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
Example 1-1: Recovery test of β-glucan addition to PRP (KOH concentration of pretreatment agent)
2 ml of blood of a healthy person collected by adding 5 units of heparin per 1 ml of blood was centrifuged at 150 × g for 10 minutes to obtain platelet-rich plasma (PRP).

To 190 μl of this PRP sample, 0.01M hydroxylation of a β-glucan preparation (pakiman; prepared according to the method of Saito et al., Agric. Biol. Chem., 32, 1261-1269 (1968)) derived from Bacillus bacterium (Poria @ cocos). Add 10 μl of an aqueous sodium (NaOH) solution (1.0 ng / ml), mix well, and transfer 5 μl to a β-glucan-free microplate (Toxipet plate 96F, trade name, manufactured by Seikagaku Corporation). , 20 μl of an aqueous potassium hydroxide (KOH) solution [pretreatment agent] selected within the range of 0 to 1.0 mol / l (KOH concentration in the test solution becomes 0 to 0.8 mol / l) ) And kept at 37 ° C. for 10 minutes to obtain a test solution.

量 The amount of β-glucan present in the test solution was determined by the following method. Obayashi, T .; et @ al. (Clin. Chim. Acta, 149, 55-65 (1985)). Factor G-based components prepared from horseshoe crab amebosite lysate and a chromogenic synthetic substrate (Boc-Leu-Gly-Arg-pNA (p-nitro). Anilide)) and a freeze-dried product of a color-developing synthetic substrate method reagent for β-glucan measurement (hereinafter referred to as “G test”) containing 0.2 mol / l Tris-HCl buffer (pH 8.0) in 25 μl of the test solution. 50 μl of the G test solution dissolved in 0) and 50 μl of distilled water are added, and the mixture is heated at 37 ° C. for 30 minutes to react, and then 0.04% (weight / volume) sodium nitrite (1 mol / l hydrochloric acid solution) 50 μl, 50 μl of 0.3% (weight / volume) ammonium sulfamate and 0.07% (weight / volume) N-1-naphthylethylenediamine dihydrochloride (1 % (Weight / volume) N- methyl-2-pyrrolidone solution) were successively diazo coupling by adding 50 [mu] l, it was quantified by measuring the absorbance at 545nm and 630nm as a reference wavelength in a microplate reader (545-630Nm).

Table 1 shows the recovery of β-glucan addition and recovery when the KOH concentration in the test solution was variously adjusted by variously changing the KOH concentration of the pretreatment agent solution, and the β-glucan-free PRP pretreated under the same conditions. The results are shown together with the measurement results (absorbance).

In Table 1, the KOH concentration (mol / l) indicates the concentration in the test solution when PRP was treated, and the absorbance of the test solution without β-glucan was the same as that of the pretreated PRP without β-glucan. The absorbance measured at 545-630 nm is shown. The recovery rate of β-glucan addition is shown as a percentage of the recovery rate of the test liquid (β-glucan addition pre-treated PRP) to which β-glucan was added, assuming that the measured value of the control was 100%. The control uses physiological saline for injection in place of PRP, uses distilled water for injection in place of the pretreatment agent, and uses the values measured with and without β-glucan as a reference.

に よ According to Table 1, β-glucan was not detected at all in the treatment with distilled water for injection alone, and it was found that the recovery rate was significantly increased as the concentration of KOH in the test solution was increased. When the concentration of KOH exceeds 0.6 mol / l or lower than 0.03 mol / l, the recovery of β-glucan decreases, but the concentration of about 0.04-0.4 mol / l. Can eliminate the effects of false positive factors and inhibitors (false negative factors) of factor G-based reactions, and reproduce the true value of β-glucan in PRP accurately and with high reliability It is clear that it is possible to detect sexually well.

That is, when the absorbance of the pretreated PRP without β-glucan was the same as that of the control without β-glucan, it indicates that the factor G reaction false positive factor in PRP was completely denatured, In addition, when the recovery rate of β-glucan in the example (β-glucan pre-treated PRP) measured by adding β-glucan is 100%, a factor G-based reaction inhibitory factor (false negative factor) in PRP Is completely denatured. Therefore, the conditions under which these reaction interfering factors can be simultaneously denatured and inactivated, that is, the measured values of the pre-treated PRP without β-glucan are almost the same as those of the control, and the β-glucan of the pre-treated PRP without β-glucan added The conditions under which the addition recovery rate is almost 100% are ideal.

From the results in Table 1, it was shown that when the KOH concentration of the test solution was 0.04 to 0.4 mol / l, the above conditions were satisfied.
Example 1-2: Recovery test of β-glucan addition to PRP (NaOH concentration of pretreatment agent)
10 μl of a 0.01 M NaOH aqueous solution (1.0 ng / ml) of the same β-glucan preparation (Pakiman) as in Example 1-1 was added to 190 μl of a PRP sample prepared by the same method as in Example 1-1 (test solution). The NaOH concentration in the solution is 0 to 0.8 mol / l). After mixing well, 5 μl of the mixture is placed on a toxicpet plate 96F, and an aqueous NaOH solution selected within the range of 0 to 0.8 mol / l [ Pretreatment agent] was added, and the mixture was heated at 37 ° C. for 10 minutes and used as a test solution.

量 The amount of β-glucan present in the test solution was measured in the same manner as in Example 1-1 using the G test. Table 2 shows the recovery rate of β-glucan addition when the NaOH concentration was variously changed, together with the measurement result (absorbance) of β-glucan-free PRP pretreated under the same conditions.

In Table 2, the NaOH concentration (mol / l) indicates the concentration in the test solution when PRP was treated, and the absorbance of the test solution without β-glucan was the same as that of the pre-treated PRP without β-glucan. The absorbance measured at 545-630 nm is shown, and the recovery rate of β-glucan added is the recovery rate of the test liquid to which β-glucan was added (PRP treated with β-glucan added), and the measured value of the control was 100%. As a percentage. The control uses physiological saline for injection in place of PRP, uses distilled water for injection in place of the pretreatment agent, and uses the values measured with and without β-glucan as a reference.

According to Table 2, it was found that β-glucan was not detected at all in the treatment with only distilled water for injection, and the recovery rate was significantly increased as the concentration of NaOH in the test solution was increased. From the results in Table 2, as in the case where the KOH aqueous solution of Example 1-1 was used as the pretreatment agent, the NaOH aqueous solution such that the NaOH concentration in the test solution was 0.04 to 0.4 mol / l. It was found that when was used as a pretreatment agent, the effects of false positive factors and false negative factors in PRP could be eliminated.
Example 1-3: Recovery test of β-glucan added to serum (treatment time)
3 ml of blood of a healthy person collected without adding an anticoagulant was allowed to stand at 4 ° C. for 1 hour, and then centrifuged at 1,000 × g for 10 minutes to obtain serum.
After adding 10 μl of an aqueous solution containing 10 pg of the same β-glucan preparation (Pakiman) as in Example 1-1 to 190 μl of this serum sample, 5 μl thereof was placed on a toxicpet plate 96F, and a 0.1 mol / l KOH aqueous solution was added. [Pretreatment agent] 20 μl was added, and the mixture was heated and maintained at 37 ° C. for a predetermined time, and used as a test liquid.

Then, β-glucan added to the test solution was measured by the same means as in Example 1-1.
The measurement results for each heating time are shown in Table 3 as in the case of Example 1-1.

According to Table 3, when the heating time is 0, that is, when the test solution is used immediately after the addition of the pretreatment agent, the recovery of β-glucan is very low. On the other hand, by using the one after a heating time of 5 minutes to 40 minutes, β-glucan added to serum could be quantitatively detected. That is, when pretreated under such conditions, false positive factors and inhibitors can be denatured, and the true value of β-glucan in serum can be detected accurately and with high reliability with good reproducibility. It is clear that this is possible.

Example 1-4: Recovery test of β-glucan added to PPP (pretreatment temperature)
2 ml of blood of a healthy person collected by adding 5 units of heparin per 1 ml of blood was centrifuged at 1,000 × g for 10 minutes to obtain platelet poor plasma (PPP). 0.01 μl / l NaOH containing 190 μl of this PPP sample and 40 pg of β-glucan (curdlan; Wako Pure Chemical Industries, Ltd.) derived from Alcaligenes faecalis var. Myxogenes IFO 13140. After adding 10 μl of the aqueous solution, 5 μl of the solution was placed on a toxicpet plate 96F, 20 μl of a 0.1 mol / l KOH solution was added, and the mixture was heated and maintained at a predetermined temperature for 10 minutes to obtain a test solution.

Then, β-glucan added to the test solution was measured by the same means as in Example 1-1. The measurement results for each heating temperature are shown in Table 4 as in the case of Example 1-1.

According to Table 4, when the treatment temperature was 4 ° C, the recovery of β-glucan was low, but when the treatment was carried out at a temperature of 25 ° C or higher, β-glucan added to PPP could be quantitatively detected. did it.
Example 1-5: Measurement of PRP sample of fungal infection patient Blood was collected from a patient suspected of having a fungal infection in the same manner as in Example 1 to prepare a PRP sample. 5 µl of the solution was placed in each well of the toxicpet plate 96F, and 20 µl of a 0.1 mol / l NaOH aqueous solution [pretreatment agent] was added. The mixture was heated and maintained at 37 ° C for 10 minutes to obtain a test solution. Thereafter, it was reacted with the G test by the same means as in Example 1-1, and the absorbance was measured. Using the same known amount of curdlan as the standard reagent as in Examples 1-4, the results of converting the β-glucan content in the test solution from the calibration curve separately prepared are shown in Table 5.

As shown in Table 5, a high concentration of β-glucan was detected in all cases (No. 1 to No. 6) (β-glucan content of healthy human: 0.2 ± 0.3 pg / ml). In three cases (No. 1 to No. 3), Candida albicans, Candida tropicalis and Cryptococcus neoformans were respectively detected in blood cultures, and Candida albicans, Candida tropicalis and Cryptococcus neoformans were respectively detected. In one case (No. 4), blood culture was negative, but Aspergillus fumigatus was detected by histopathological examination by dissection after death. The remaining two cases (No. 5 and No. 6) were negative in blood culture despite strongly suspected fungal infection from clinical symptoms, progress, drug sensitivity, etc., but administration of an antifungal agent (Miconazole). As a result, clinically remarkable improvement was observed, and it is considered that the patient was infected with the fungus.

Therefore, by treating β-glucan after treating PRP of a fungal infection patient with the pretreatment agent of the present invention, fungal infections, especially deep fungal infections, which are extremely difficult to diagnose by ordinary test methods, can be measured. The diagnosis could be made quickly and accurately.
Example 1-6: Measurement of Serum Specimen of Fungal Infection Patient Blood was collected from a patient suspected of having a fungal infection by the same method as in Example 1-3 to prepare a serum specimen. 5 µl of the solution was placed in each well of the toxicpet plate 96F, and 20 µl of a 0.1 mol / l KOH aqueous solution [pretreatment agent] was further added thereto. Thereafter, it was reacted with the G test by the same means as in Example 1-1, and the absorbance was measured. Table 6 shows the results obtained by using the same known amount of curdlan as in Example 1-4 as a standard reagent and converting the β-glucan content in the test solution from a calibration curve prepared separately.

As shown in Table 6, in all cases (No. 1 to No. 5), a high concentration of β-glucan was detected (β-glucan content of healthy person: 0.2 ± 0.2 pg / ml), of which In two cases (No. 1 and No. 2), Candida guilliermodi and Candida krusei were detected in the blood culture, and one case (No. 3) was the blood culture. Was negative, but Aspergillus fumigatus was detected by histopathological examination by anatomy after death. The remaining two cases (No. 4 and No. 5) were negative in blood culture despite strongly suspected fungal infection based on clinical symptoms, progress, drug sensitivity, etc., but the antifungal agents (amphotericin B, Fluconazole) showed a marked clinical improvement, suggesting that the patient was infected with a fungal infection.
Therefore, by treating β-glucan after treating the serum of a patient with a fungal infection with the pretreatment agent of the present invention, fungal infections, especially deep fungal infections that are extremely difficult to diagnose with ordinary testing methods, can be obtained. The diagnosis could be made quickly and accurately.

Example 1-7: β-Glucan Measurement Gelation Method (Nephelometry) Kit A β-glucan measurement gelation method (nephelometry) kit (for 50 samples) was prepared, comprising the following components.
(A) Pretreatment agent 1.0ml
0.1 mol / l NaOH aqueous solution (B) Factor G-based reaction reagent (lyophilized product) Appropriate amount 15% (W /%) in commercially available lysate derived from Limulus polyphemus (Limulus HSII-Test Wako, sold by Wako Pure Chemical Industries, Ltd.) V) Add dextran (molecular weight 70,000), centrifuge at 3,500 rpm for 10 minutes, mix with a porous cellulose gel (Cellulofine GC-200m, manufactured by Seikagaku Corporation), and filter with a glass filter. And the filtrate is freeze-dried.
(C) Buffer for dissolution / reaction of factor G reaction reagent 5.0 ml
0.1 mol / l Tris-HCl buffer (pH 8.0)
(D) Standard β-glucan reagent (lyophilized product) Appropriate amount Commercially available curdlan (see Example 1-4)
(E) 1.0 ml of distilled water (β-glucan free) for dissolving a standard β-glucan reagent
(F) 1.0 ml of distilled water (β-glucan free) for blank test

Example 1-8: Chromogenic synthetic substrate method kit for β-glucan measurement A chromogenic synthetic substrate method kit for β-glucan measurement (for 100 samples) was prepared, comprising the following components.
(A) Pretreatment agent 2.0ml
0.1 mol / l KOH aqueous solution (B) Factor G-based reaction reagent (lyophilized product) Appropriate amount 15% (W / V) dextran (molecular weight 40,000) is added to lysate derived from Takipreus tridentatas at 3,500 rpm After centrifugation for 10 minutes, the mixture was passed through a nylon membrane filter having a pore size of 0.20 μm (Nalgen syringe filter, diameter: 25 mm, manufactured by Narge), and the passing solution was mixed with MS (0.8 M magnesium chloride and 6 mM Boc-Leu-Gly-Arg). -PNA) and freeze-dried.
(C) Buffer for dissolution / reaction of factor G reaction reagent 5.0 ml
0.2 mol / l Tris-HCl buffer (pH 8.0)
(D) Standard β-glucan reagent (freeze-dried product) Appropriate amount β-glucan preparation (pakiman) derived from Bacillus cerevisiae (see Example 1-1)
(E) 2.0 ml of distilled water (β-glucan free) for dissolving a standard β-glucan reagent
(F) Distilled water for blank test (β-glucan free) 2.0ml

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

Claims (9)

A pretreatment agent used to treat a sample containing a reaction interfering factor to the Limulus reaction prior to the Limulus reaction when measuring (1 → 3) -β-D-glucan in the sample using the Limulus reaction A pretreatment agent comprising an alkali metal hydroxide as a main component. The pretreatment agent according to claim 1, wherein the pretreatment agent is stored as a plurality of solutions, and the solutions are mixed at the time of use. Pretreatment for treating a sample prior to the Limulus reaction when detecting (1 → 3) -β-D-glucan contained in a sample containing a reaction interfering factor to the Limulus reaction using the reaction A pretreatment method comprising mixing the sample with the pretreatment agent according to claim 1 or 2, and heating the mixture. The pretreatment method according to claim 3, wherein the sample is a blood-derived sample. A method for measuring (1 → 3) -β-D-glucan in a sample using the Limulus reaction, wherein the sample is pretreated by the method according to claim 3 or 4, and the sample after the treatment is subjected to Limulus. A measuring method characterized by detecting a change in a substrate by mixing and reacting with a reagent. A measurement kit for measuring (1 → 3) -β-D-glucan, comprising at least the following constituent reagents:
(A) The pretreatment agent according to claim 1.
(B) Limulus reagent obtained from horseshoe crab amebosite lysate as a raw material. 7. The measurement kit according to claim 6, wherein the Limulus reagent of (B) is a Limulus reagent that specifically reacts with (1 → 3) -β-D-glucan. The measurement kit according to claim 6, further comprising (C) below as a constituent reagent.
(C) A standard reagent containing a fixed amount of (1 → 3) -β-D-glucan. (1 → 3) -β-D-glucan in a sample derived from a living body is quantified by the measuring method according to claim 5, and when the measured value of the substance exceeds a certain amount, it is derived from a living body suffering from an infectious disease. A method for determining a sample, comprising determining that the sample is a sample.

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