JP2007240397A - METHOD OF MEASURING (1->3)-beta-D-GLUCAN - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an amebocyte lysate (AL) reagent for specifically measuring (1→3)-β-D-glucan (βG), and to provide a method of specifically measuring the βG by using the same. <P>SOLUTION: The AL reagent for measuring the βG contains the AL and polymyxin or its salt, including the βG and/or a related material whose concentration is equal to or lower than the maximum value that does not induce enzyme reaction of a limulus AL. A kit using the AL reagent, a method for preparing the AL reagent and a method for specifically measuring the βG by using the AL reagent are also provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention reacts with a horseshoe crab blood cell component and (1 → 3) -β-D-glucan (hereinafter sometimes abbreviated as “βG”), and analyzes the reaction to perform βG-specificity. Related to the measurement method.

  βG is a substance known to exist as a skeletal constituent of yeast and mold cell walls, a major polysaccharide component of many basidiomycetous fruit bodies (mushrooms), and an elution component from membranes used for hemodialysis. . Although the biological activity of βG is not as clear as endotoxin (hereinafter abbreviated as ET), it is thought that by measuring this, early diagnosis of mycosis and contamination of medical devices by fungi can be detected. Yes.

  On the other hand, ET is a lipopolysaccharide (LPS) present in the outer cell wall of Gram-negative bacteria and is known as a strong pyrogenic substance.

  βG reacts with a solution (hereinafter abbreviated as AL solution) containing a horseshoe crab blood cell extract (Amebocyte Lysate, hereinafter abbreviated as AL) to activate an enzyme (such as a protease) or a gelation reaction. As a simple and inexpensive βG detection method using this in the fields of medicine, pharmacy and microbiology, for example, the degree of activation of an enzyme (protease etc.) is measured by a colorimetric method. A so-called Limulus test or the like utilizing a gelation reaction (hereinafter collectively referred to as a Limulus test or the like) is widely used.

  However, since the AL reagent used for the Limulus test reacts not only with βG but also with ET (Non-patent document 1, Non-patent document 2), when βG is to be measured using these, it depends on the ET in the sample. There was a problem that the specificity was affected by the measurement value.

  In order to solve such problems, factors that cause the activation reaction or gelation reaction of enzymes (proteases, etc.) by reacting the AL solution with various chromatography and reacting with ET present in the AL solution ( (Patent Document 1, Patent Document 2, Patent Document 3), a method of inactivating the ET sensitivity factor by adding a peptide having affinity for ET to the AL solution (Patent Document 4) ), A method of inactivating the ET sensitivity factor by adding an antibody against the ET sensitivity factor to the AL solution (Patent Document 5) and the like have been reported. However, since these methods are all methods for treating the AL solution itself, there is a drawback that there is a high risk that the AL solution is contaminated during the treatment by ET, βG, etc. widely present in normal environments. Have.

  In addition, these methods require aseptic equipment and complicated aseptic operation to avoid such contamination, and peptides having affinity for ET and antibodies against ET sensitivity factors are available. As is clear from the fact that it is difficult and expensive, it is a method that has many problems in terms of economy and technology.

In addition, dimethyl sulfoxide and polymyxin B were added in a method for quantifying the amount of ET or βG in a sample by measuring the concentration of coagulin peptide C produced by the reaction of LAL with ET or βG by ELISA. It has been reported that when ELISA is performed after reacting LAL with a sample, only reactivity to ET is inhibited (Non-patent Document 3).
In addition, as a method for inactivating ET by treating a measurement sample instead of an AL solution, a method of heating the sample has been reported (Patent Document 6). Although this method overcomes the disadvantages of the above method, it has a problem that a long processing time is required to sufficiently inactivate the ET, and is not a preferable method.

  Furthermore, the activity of ET can be obtained by heating the sample containing ET together with a surfactant and a peptide derivative (or protein) having a property of binding to ET and suppressing the activity of ET, such as polymyxin B. A method for specifically suppressing βG has also been reported (Patent Document 7). However, this method has a problem that the operation is complicated because the sample needs to be pretreated.

JP 59-27828 A Japanese Unexamined Patent Publication No. 2-138193 Japanese Patent Laid-Open No. 4-76459 JP-A-2-07098 Japanese Patent Laid-Open No. 4-52558 JP-A-2-141666 Japanese Patent No. 3106839 Kakinuma et al., Biochem. Biophys. Res. Commun., 1981, vol. 101, p.434-439 Morita et al., FEBS Lett., 1981, vol.129, p.318-321 Zhang et al., J. Clin. Microbiol., 1994, vol.32, No.6, p.1537-1541

  This invention is made | formed in view of the above situations, and makes it a subject to provide the AL reagent for measuring βG specifically, and the specific measurement method of βG using the same.

The present invention has been made for the purpose of solving the above problems, and has the following configuration.
(1) An AL reagent for βG measurement, comprising polymyxin or a salt thereof and AL, which contains βG or / and its related substances only at a concentration that does not cause an enzymatic reaction of AL.
(2) A method for preparing an AL reagent for βG measurement, characterized in that polymyxin or a salt thereof containing βG or / and its related substance only in a concentration that does not cause an enzymatic reaction of AL coexists with AL.
(3) A βG measurement kit comprising polymyxin or a salt thereof containing only βG or / and its related substance at a concentration that does not cause an enzymatic reaction of AL, and AL as components.
(4) A kit for measuring βG, which contains a reagent containing AL that contains polymyxin or a salt thereof that contains βG or / and its related substance only at a concentration that does not cause an enzymatic reaction of AL.
(5) A sample containing ET is reacted with an AL reagent containing polymyxin or a salt thereof containing βG or / and its related substance only at a concentration not causing an enzyme reaction of AL, and the resulting enzyme activation reaction Measuring the activity of the enzyme activated by the above, or measuring the degree of turbidity change and gelation state of the reaction solution based on the resulting gelation reaction by instrument or visual observation, A method for specifically measuring βG.
(6) A sample containing ET is reacted with AL in the presence of polymyxin or a salt thereof containing βG or / and its related substance only at a concentration that does not cause an enzyme reaction of AL, and the resulting enzyme activation reaction The activity of the activated enzyme is measured, or the degree of change in turbidity or the degree of gelation of the reaction solution based on the resulting gelation reaction is measured by instrument or visual observation. Specific measurement method.

  That is, the present inventors are able to suppress the activity of ET coexisting with a commercially available polymyxin or a salt thereof in a reaction system with a sample in the course of earnest research to find a specific method for measuring βG using an AL solution. If the AL reagent coexists with the AL reagent, the AL reagent is activated. However, when polymyxin or a salt thereof purified to a high purity coexists with the AL reagent, such activation of the AL reagent does not occur. Found that when this AL reagent was reacted with a sample containing βG and ET, AL did not react with ET and βG could be measured specifically. As a result of further research, it was determined that many of the commercially available polymyxins or their salts are contaminated with βG or / and its related substances. By purifying, βG or / and its related substance can be effectively removed without losing polymyxin or its salt, and βG or / and its related substance is below the concentration that does not cause the enzymatic reaction of AL. It has been found that polymyxin or its salt can be obtained without containing.

  If βG in a sample is measured using an AL reagent in which polymyxin or a salt thereof coexists, βG can be specifically measured without being affected by ET coexisting in the sample. The present invention has been completed.

  The present invention provides a novel βG measurement AL reagent, a method for preparing the same, and a βG-specific measurement method using the βG measurement AL reagent prepared by this method. If βG is measured in this way, the effect of ET present in the sample can be eliminated without performing pretreatment of the sample as in the prior art, and βG can be measured specifically.

  The βG measurement AL reagent of the present invention is “a βG measurement AL reagent comprising polymyxin or a salt thereof and AL that contain βG or / and its related substances only at a concentration that does not cause an enzymatic reaction of AL”. Is mentioned.

  The AL used in the present invention can be mentioned without particular limitation as long as it can be used for ordinary βG measurement. For example, the genus Limulus, Tachypleus, or Carcinosco There is no particular limitation as long as it is a lysate obtained from the blood cells of horseshoe crabs belonging to the genus Carcinoscorpius and the like, as long as it causes an enzyme (protease activation or gelation reaction by reaction with βG).

  In addition, for example, from ACC (Associates of Cape Cod), Hemakem, Cambrex Corp., Charles River Laboratories, Seikagaku Corporation, and Wako Pure Chemical Industries, Ltd. Of course, those prepared based on commercially available lyophilized products of AL solutions can also be used.

  Examples of βG or / and related substances thereof (hereinafter sometimes abbreviated as “βGs”) according to the present invention are not particularly limited as long as they are polysaccharides containing βG as a component. However, for example, those having the property of causing an enzymatic reaction of AL can be mentioned. Specifically, for example, various bacteria (for example, Alcaligenes genus, Agrobacterium genus, etc.), yeasts (for example, Saccharomyces genus, Candida genus, Cryptococcus genus, Trichosporon genus, Rhodotorula genus, etc.) molds (Aspergillus genus, Mucor genus, Natural polysaccharides obtained from cell walls such as Penicillium genus, Trichophyton genus, Sporothrix genus, Phialophora genus), actinomycetes (Actinomyces genus, Nocardia genus etc.), mushrooms (eg Shiitake mushroom, Suhirotake mushroom, Kawaratake etc.), specifically For example, curdlan, pachyman, sclerotane, lentinan, schizophyllan, coriolan, etc., or storable polysaccharides of algae (eg, brown algae, euglena, diatoms, etc.), specifically laminaran, paramylon, etc. These include conventional methods, for example, Dai Organic Chemistry Vol. 19, 7th edition, pages 70 to 101, supervised by Fujio Kotake, May 10, 1967, Asakura Shoten; AE Clarke et al., Phytochemistry, vol.1, 175 -18 8 (1967); according to the method described in T. Sasaki et al., Europ. J. Cancer, vol. 15, 211-215 (1967), etc., for example, sulfate group, carboxymethyl group, carboxyethyl group, methyl group, hydroxyethyl And derivatives obtained by introducing a group, hydroxypropyl group, sulfopropyl group and the like.

  The concentration in the case of “the concentration of βG” used in the present invention is a β-glucan test Wako “β-glucan standard product” commercially available from Wako Pure Chemical Industries, Ltd. Lysate (lysate, especially abbreviated as LAL) obtained from blood cells of horseshoe crab belonging to the genus Limulus as AL, and having a property of increasing turbidity by reaction with βG It means the value (concentration) obtained by measurement.

  Examples of the polymyxin of the polymyxin according to the present invention or a salt thereof (hereinafter abbreviated as “polymyxins”) include polymyxin A, B, C, D, E, K, M, P, and the like. Examples include those produced by Bacillus polymyxa, but are not particularly limited. Further, it may be purified as a single component, may be a mixture of some of these, or may be in the form of a salt, and is not particularly limited. Examples of polymyxin salts include sulfates. Considering availability, polymyxin B sulfate is preferable.

  The concentration of the polymyxins of the present invention in the AL reagent for βG measurement according to the present invention is a concentration that can sufficiently suppress the activity of ET when reacted with a sample and, for example, the reaction of βG with an AL solution The concentration is not particularly limited as long as it does not promote or inhibit the sex. When the sample and the AL reagent are mixed and measured one-to-one, if the ET concentration at the time of the reaction is about 1 ng / mL, if the polymyxin of about 4 μg / mL is contained in the β reagent for measuring AL Measurement is possible without being affected by ET. However, in the field of clinical diagnosis, there are also samples containing ET at a concentration of about several tens of ng / mL. Further, the concentration varies depending on the type of βG measurement method, and when the turbidimetric time analysis method is performed, it is desirable to add polymyxins having a higher concentration than when the endpoint synthetic substrate method is performed. In the field of clinical diagnosis, in order to specifically measure βG, it is necessary to contain polymyxins at a concentration that can sufficiently suppress ET activity in any sample. Therefore, in order to be able to measure βG specifically by sufficiently suppressing the activity of ET regardless of what measurement method is used or when using a clinical sample containing any concentration of ET. The concentration of the polymyxins of the present invention is 0.02 to 80 mg / mL, preferably 0.2 to 80 mg / L, more preferably 0.4 to 20 mg / mL, more preferably 1 in the AL reagent for βG measurement. About 4 mg / mL.

  The polymyxins according to the present invention should not contain an amount of βG that affects the measurement. That is, it should contain βGs only at a concentration that does not cause the enzymatic reaction of AL (such polymyxins may be hereinafter referred to as “polymyxins of the present invention”).

  AL has different sensitivity to βGs depending on the preparation method and measurement method. In the present invention, it is important to prevent the enzymatic reaction of AL from being caused by βGs derived from polymyxins. Therefore, the concentration of βGs in polymyxins is such that when polymyxins are coexisted in the AL solution so as to have a required concentration, the concentration of βGs in the AL reagent is the final acceptable concentration (ie, It is required that the concentration be less than or equal to a concentration that does not cause activation of the AL solution. The concentration of βG that does not cause the enzymatic reaction of AL varies depending on the βG sensitivity of AL, but is about several pg / mL for general AL. For example, when measuring using LAL, the concentration of (1 → 3) -β-D-glucan in the LAL solution is 10 pg / mL or less, preferably 1 pg / mL or less, more preferably about 0.2 pg / mL. Is less than.

  Therefore, the content of βGs in the polymyxins according to the present invention is such that the concentration of βG in the AL solution when the polymyxins are added to the βG measurement AL solution so as to have the concentration as described above. Is 10 pg / mL or less. Therefore, when converted into the concentration of curdlan which is a linear (1 → 3) -β-D-glucan, it is 0.9 ng / mL or less, preferably 0.09 ng / mL or less, more preferably 0 0.018 ng / mL or less.

  Conventionally, in order to specifically measure βG in a sample, a method for specifically measuring βG in a sample by adding polymyxins to the sample and suppressing ET activity in the sample has been attempted. However, polymyxins alone cannot sufficiently suppress ET activity, or the background value thereof cannot be suppressed. As a result of diligent research, the present inventors have found that the polymyxin itself used may be contaminated with βGs, which is a cause of an increase in the background value when βG is measured. In addition, due to contamination of polymyxins by βGs, it was not possible to add polymyxins in an amount that could sufficiently suppress ET activity, but polymyxins alone could not sufficiently suppress ET activity. I also found out that it was the cause.

  Therefore, the polymyxins used in the present invention are polymyxins having a βGs content below the required amount as described above, or pretreated to remove βGs from the polymyxins so as to be a predetermined concentration or less. Need to be.

  As a method of removing βGs from a solution containing βGs, generally, for example, a membrane having a positive charge such as Zetapore membrane (trade name of Cuno Co., Ltd.), Ultipore (trade name of Nippon Pole Co., Ltd.), Bayodyne, etc. Nylon 66 filters such as Nippon Pole Co., Ltd., such as polytetrafluoroethylene filters such as PTFE membranes (manufactured by Nitto Denko Corporation), and methods using activated carbon are known. It has been.

  As a result of diligent research on a method of performing pretreatment for removing βGs from polymyxins, the present inventors have included polymyxins, particularly by treating a solution containing polymyxins with a positively charged membrane. The present inventors succeeded in effectively removing βGs from the solution and obtaining polymyxins (“polymyxins of the present invention”) containing βGs only at a concentration that does not cause the enzymatic reaction of AL. Moreover, according to this method, it discovered that there was little loss of polymyxins in the said solution.

  Examples of the positively charged membrane used for removing βGs from polymyxins include a nylon 66 membrane capable of removing fine particles, ET, and the like by an adsorption action of zeta potential. Actually, for example, a commercially available product such as a filter made of nylon 66 such as Zetapore membrane (trade name of Cuno Co., Ltd.), Ultipore (trade name of Nippon Pole Co., Ltd.), Bayodyne (trade name of Nihon Pole Co., Ltd.) may be used. The pore size of the positively charged film is not particularly limited, and examples thereof include 0.1 to 2 μm.

  As a matter of course, the cellulose-based membrane cannot be used for this purpose because there is a risk that the βG-like substance is eluted from the membrane.

  In the case where βGs are removed by passing through a membrane having a positive charge, for example, the polymyxins used are made into a solution having an appropriate concentration (for example, about 0.1 to 8 W / V%), and then the membrane is supported on the support. Set and filtered. The polymyxin solution may be subjected to filtration treatment under the above conditions after preparing a solution having a target concentration.

  The solvent of the solution containing polymyxins used in the treatment of the solution containing polymyxins with a positively charged membrane is free of ET and βG, and also inhibits the reaction between AL and βGs. Those which are not accelerated are desirable, and examples thereof include distilled water (for example, distilled water for injection free of ET and βG), buffer solution and the like. As the buffer constituting the buffer, all those usually used in this field can be used. Specifically, for example, phosphate, borate, acetate, Tris buffer, Good's buffer, etc. The concentration of use may be appropriately selected from the use concentration range in this field.

  Alternatively, a solution containing polymyxins may be treated once or several times with a positively charged membrane. In addition, it is optional to appropriately combine the processing methods using the conventional filters as described above. Moreover, you may use combining methods, such as ultrafiltration. In particular, it is desirable to perform a normal ultrafiltration treatment after treating a solution containing polymyxins with a membrane having a positive charge, and then treating the solution again with a membrane having a positive charge more effectively. ΒGs can be removed from solutions containing sucrose.

  The content of βGs in polymyxins should be below the concentration that does not cause the enzymatic reaction of AL when coexisting with AL. However, by treating polymyxins with this method, The βGs can be effectively removed with almost no loss of polymyxins.

  As a method for preparing the AL reagent for βG measurement of the present invention, the polymyxin of the present invention or a salt thereof may be coexistent with AL.

As a method of obtaining the β reagent for measuring βG measurement according to the present invention by allowing polymyxins to coexist in AL or a solution thereof, polymyxins as described above are allowed to coexist in AL or the solution so as to have a concentration as described above. It's enough. The specific method is not particularly limited as long as the polymyxins can be finally added to AL or a predetermined concentration thereof in the solution. For example, (1) a solution containing the polymyxins of the present invention is used. A method for dissolving a freeze-dried product of AL,
(2) A method of adding and dissolving an appropriate amount of the polymyxins of the present invention in an AL solution,
(3) A method of adding an appropriate amount of a solution containing the polymyxins of the present invention to an AL solution, and the like.

  In the methods (1) and (3) above, the concentration of polymyxins in the solution containing polymyxins when preparing the AL reagent for βG measurement is the final concentration when the target AL reagent for βG measurement is prepared. Is not particularly limited as long as the concentration is in the range as described above.

  In the above methods (2) and (3), the concentration of the AL solution when preparing the β reagent for measuring AL is the final concentration in the sample when the target reagent for measuring β G is prepared. There is no particular limitation as long as the concentration is suitable for use in βG measurement.

  Further, when βG is measured by a synthetic substrate method described later, the βG measurement AL reagent of the present invention further contains a synthetic substrate in addition to AL and a predetermined concentration of polymyxins.

  In addition, it is confirmed that the solution containing polymyxins used in the preparation method of the present invention and the solvent for preparing the AL solution are ET and βG free and do not inhibit or promote the reaction between AL and βG. Is desirable. For example, distilled water (for example, βG-free distilled water for injection), buffer solution and the like can be mentioned. As the buffer constituting the buffer, all those usually used in this field can be used. Specifically, for example, phosphate, borate, acetate, Tris buffer, Good's buffer, etc. The concentration of use may be appropriately selected from the use concentration range in this field.

  The AL reagent for βG measurement of the present invention obtained by the above method may remain in a solution state, but is once freeze-dried and stored as a reagent form, and then with water not containing ET and βG such as distilled water. It is of course possible to redissolve and use it for the measurement of βG.

  Among the above-mentioned βG measurement AL reagents or those freeze-dried, there may be, for example, phosphate buffers, Good's Buffers, etc., as long as they do not inhibit or promote the measurement of βG by the Limulus test method. Needless to say, a buffering agent or a chelating agent such as ethylenediaminetetraacetic acid (EDTA) may be contained.

  In addition, among the AL reagents for βG measurement or those freeze-dried, reaction accelerators, stabilizers, preservatives, and others used in this field inhibit the stability of coexisting reagents. However, it may contain those usually used in the target measurement as long as they do not inhibit or promote the measurement of βG. Also, the concentration may be used in a concentration range usually used in this field.

Moreover, as the βG measurement kit of the present invention,
(I) Polymyxins that contain βGs only at a concentration that does not cause the enzymatic reaction of AL, and those that contain AL as a constituent, or (ii) βGs that contain no more than a concentration that does not cause the enzymatic reaction of AL A reagent containing a polymyxin-containing AL as a constituent,
Is mentioned.

  Preferred embodiments and specific examples of each component are as described above.

  In order to carry out the βG measurement method of the present invention, for example, the following two methods can be mentioned.

  (1) A sample containing βG is reacted with the AL reagent for βG measurement of the present invention, and the activity of the enzyme activated by the resulting enzyme activation reaction is measured, or based on the resulting gelation reaction A method for specifically measuring βG, wherein the degree of turbidity change and the degree of gelation of the reaction solution are measured by an instrument or visual observation.

  (2) A sample containing βG is reacted with AL in the presence of polymyxins (β polymyxins of the present invention) containing βGs only at a concentration that does not cause an enzymatic reaction of AL, and the resulting enzyme activation The activity of the enzyme activated by the reaction is measured, or the degree of change in turbidity or the degree of gelation of the reaction solution based on the resulting gelation reaction is measured by instrument or visual observation. A method for specifically measuring βG.

  In order to carry out the above method (1), other than using the AL reagent for βG measurement of the present invention, the conventional measurement conditions (for example, reaction time, temperature at the time of measurement) can be determined by a known LAL method using AL. Measurement wavelength, etc.) may be measured by a measurement operation.

  In the method of (1) above, as a method of reacting a sample containing βG with an AL reagent for βG measurement of the present invention, for example, an AL reagent solution for βG measurement of the present invention (the polymyxins of the present invention are added at a predetermined concentration). A method of reacting the sample with the sample (eg, an AL reagent for βG measurement (an AL solution containing the polymyxins of the present invention at a predetermined concentration) once freeze-dried, and re-dissolved to react with the sample) Is mentioned.

  In the method of (2) above, when a sample containing βG is reacted with AL, polymyxins (β polymyxins of the present invention) containing βGs only at a concentration that does not cause an enzymatic reaction of AL are predetermined. The concentration should be present. Specifically, for example, a sample containing βG, AL or an AL solution, and the polymyxins of the present invention or a solution thereof are mixed, and then the LAL method using per se known AL, similar to the method of (1) above. For example, a method for measuring βG may be used.

  Specific examples of AL used in the method (2) are as described above.

  The concentration of polymyxins in the reaction solution when the βG measurement AL reagent is reacted with a sample containing βG is 0.01 to 40 mg / mL, preferably 0.1 to 40 mg / mL, more preferably It is 0.2-10 mg / mL, More preferably, it is 0.5-2 mg / mL.

  As the LAL method using AL known per se, for example, a sample is reacted with the AL reagent for βG measurement of the present invention, and the activity of the enzyme activated by the resulting enzyme activation reaction is measured, or the result is obtained. Conventional methods such as the so-called LAL method, which measures the degree of change in turbidity and the degree of gelation state of the reaction solution based on the gelation reaction with an instrument or visual observation and measures the ET based on the measurement results, are listed. It is done.

  For example, FDA guidelines (Guidelines on Validation of the Limulus Amoebocyte Lysate Test as an End-Product Endotoxin Test for Human and Animal Parenteral Drugs, Biologics and Medical Devices, Food and Drug Adm. (1987)) And the synthetic substrate method, turbidimetric time analysis method, gelation overturning method and the like described in the above.

  That is, the synthetic substrate method is a method for measuring the activity of an enzyme (such as protease) activated with the activation of AL using a synthetic substrate (Nakamura, S. et al., J. Biochem., 81 , 1567-1569 (1977)), and turbidimetric time analysis methods include, for example, Toxinometer MT-5500, Toxinometer ET-201 (manufactured by Wako Pure Chemical Industries, Ltd.), Toxinometer MT-251 ( After mixing βG and AL using a dedicated device such as Wako Pure Chemical Industries, Ltd.), LAL-5000 [ACC (Associates of Cape Cod)], microplate reader Tmax (Molecular Devices), etc. Method by measuring the time (gelation time) until the transmitted light changes by a certain rate (Oishi, H. et al., J. Parenter. Sci. Techn. 39, 194-200 (1985)) The gel overturning method is effective for the formation of gels formed by the activation of AL. (Cooper, JF et al., J. Lab. Clin. Med., 78, 138-148 (1971)), and a turbidimetric method for measuring the turbidity that accompanies coagulation (Bondar, RJL et al., Biomedical Applications of the Horseshoe Crab (ed. Cohan, E.), 435-451, Alan R. Liss, Inc. New York (1979)).

  Also, the reaction is started by mixing βG and AL, and the time required for the amount of change in absorbance based on the resulting gelation reaction to reach a certain value (arrival time) is obtained, and this value is calculated as the arrival time. This may be done by fitting to a calibration curve representing the relationship between the ET concentration and the ET concentration.

  More specifically, the βG measurement method according to the present invention is as follows.

a) Synthetic Substrate Method A substrate (synthetic substrate) in which a sample and the AL reagent for βG measurement of the present invention are mixed and then partially cleaved by the action of an enzyme activated to release a chromogenic substance After mixing in the presence of, the mixture is reacted for 25 hours at a temperature of 25 to 40 ° C. Thereafter, a reaction stop solution (for example, hydrochloric acid solution, acetic acid solution, etc.) is added to the reaction solution to stop the reaction, and the predetermined absorbance (or fluorescence intensity) of the obtained final solution is measured with an appropriate measuring instrument (for example, spectrophotometer). Measure using a meter, microplate reader, fluorimeter, etc.). The obtained measurement value is applied to a calibration curve representing the relationship between the βG concentration prepared in advance using a βG solution having a known concentration and the measurement value, and the βG concentration in the sample is obtained.

b) Turbidity Time Analysis Method First, the βG measurement AL reagent of the present invention and a sample containing βG are mixed, and the mixture is irradiated with light. Next, using a suitable measuring instrument (for example, a spectrophotometer, a microplate reader, etc.), for example, a change in transmittance, a change in absorbance, a variation in the transmitted light ratio Rt, and a logarithmic value of the transmitted light ratio Rt. The time required for the optical change such as fluctuation to reach a predetermined value after the start of light irradiation (gelation time, Tg) is measured. The obtained time is applied to a calibration curve representing the relationship between the gelation time and βG concentration prepared in advance using a βG solution having a known concentration, and the βG concentration in the sample is obtained.

c) Gelling overturning method In a 10 x 75 mm test tube, the sample and the AL reagent for βG measurement of the present invention are mixed, and then reacted for 60 minutes while being kept at 25 to 40 ° C. Then, this is turned over 180 degreeC and the presence or absence of gel formation is determined visually.

  Moreover, you may measure using the commercially available kit for ET measurement by LAL method.

  Further, the LAL method can be used without particular limitation as long as it is a commonly used method.

Other reagents used in these known measurement methods include, for example, 3,4-dihydroxyphenylalanine (dopa), enzyme substrates to be measured such as synthetic substrates, conjugate enzymes, coenzymes, etc. Buffers, color formers, activators such as divalent metal ions (Ca ²⁺ , Mg ^2+, etc.), stabilizers, surfactants, etc. And reagents used in the above.

  The reaction pH in these known measurement methods varies depending on the measurement method, the type of enzyme to be measured, and the like, but is usually pH 4 to 11, preferably pH 6 to 9. In addition, a buffer may be used to maintain the reaction pH, and the type and concentration used are not particularly limited as long as they do not affect the reaction. For example, phosphate, boric acid Salt, acetate, Tris buffer, Good's buffer, and the like.

  The reaction temperature and reaction time are not particularly limited as long as the reaction proceeds at the temperature and time. The reaction temperature is usually 0 to 50 ° C., preferably 4 to 30 ° C., and the reaction time is usually 1 It is suitably selected from the range of seconds to 20 hours, preferably 10 seconds to 2 hours.

  Examples of the sample according to the present invention include, but are not limited to, clinical specimens such as plasma, serum, urine, lymph, cerebrospinal fluid, pleural effusion, and ascites, pharmaceuticals, medical devices, foods, and the like.

  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. Β-glucan removal from polymyxin sulfate B solution-1
(Preparation of each reagent)
(1) Polymyxin B (hereinafter abbreviated as “PxB”) aqueous solution Polymyxin B sulfate (manufactured by Wako Pure Chemical Industries, Ltd., biochemical reagent) is added to endotoxin-free distilled water for injection (Otsuka Pharmaceutical Co., Ltd.). The product dissolved in the product was prepared to obtain 200 mL of a 1 W / V% PxB aqueous solution.
(2) βG standard solution A solution obtained by dissolving 10 mg of curdlan (manufactured by Wako Pure Chemical Industries, Ltd.) in 10 mL of 0.1N sodium hydroxide solution was used as a βG stock solution. In use, the βG stock solution was diluted with distilled water for injection to obtain a βG standard solution.
The curdlan used was confirmed to correspond to curdlan 1 ng = (1 → 3) -β-D-glucan 11 pg by measurement using LAL.
(3) AL solution AL freeze-dried product (shoe crab blood cell extract HS-J, sold by Wako Pure Chemical Industries, Ltd., gelation sensitivity: 0.03 EU / mL, for 5.2 mL) What was melt | dissolved in 2 mL was prepared, and it was set as AL solution.

[Preparation of AL reagent]
AL solution is 12.5 V / V%, t-butyloxycarbonyl-Thr-Gly-Arg-p-nitroaniline (Boc-Thr-Gly-Arg-pNA) is 0.4 mM, and MgSO ₄ is 100 mM. A solution dissolved in 100 mM Tris-HCl pH 8.0 was prepared and used as an AL reagent.

[Filtration material]
(1) PVDF (polyvinylidene difluoride) membrane Milex VV (manufactured by Millipore, syringe pressure filter unit, pore size 0.1 μm, φ33 mm, filter material / PVDF, γ-ray sterilized).
(2) Ultrafiltration membrane Biomax 10 (Biomax 10, trade name of Millipore, PBGC ultrafiltration disk, NMWL10,000, φ47 mm, filter material, polyethersulfone).
(3) Positively charged membrane Zetapore membrane (Zetapore disk, trade name of Cuno Co., Ltd., pore diameter 0.65 μm, φ47 mm).

[Rough purification of PxB aqueous solution]
200 mL of a 1 W / V% PxB aqueous solution was filtered under pressure with Milex VV to obtain a filtrate. The obtained filtrate was ultrafiltered with Biomax 10 under pressure. The resulting filtrate was further filtered with suction through a zetapore membrane. 2 mL of the filtrate obtained for each filtration treatment was sampled, and the PxB concentration and βG concentration in each filtrate were measured.

[Measurement of PxB concentration]
Absorbance at 256 nm of each filtrate was measured using a spectrophotometer UVmini-1240 (manufactured by Shimadzu Corporation).
The PxB concentration in each sampled filtrate was calculated based on the value obtained by measuring the absorbance at 256 nm using the aqueous solution with the PxB concentration in the 1 W / V% PxB aqueous solution being 10 mg / mL.

[Measurement of βG concentration]
The βG concentration in each filtrate was measured by the endpoint synthetic substrate method according to the following method.
First, 50 μL of filtrate was added to a microplate (Costar 3595, manufactured by Corning), and then 50 μL of AL reagent was added. After reacting at 37 ° C. for 30 min, 0.04% NaNO ₂ (0.9 M HCl) 50 μL, 0.3 W / V% ammonium amidosulfate solution 50 μL, 0.07 W / V% N-1-naphthylethylenediamine dihydrochloride 50 μL And mixed.
Thereafter, the absorbance at 545 nm to 630 nm was measured using Spectramax 250 (manufactured by Molecular Devices).
Furthermore, a βG solution having a known concentration was prepared using a βG standard solution, and the same measurement was performed using the βG solution. A calibration curve representing the relationship between the βG concentration and the absorbance was prepared. Based on this calibration curve, the concentration of βG in each obtained filtrate was calculated.

〔result〕
Table 1 shows the PxB concentration measurement results and βG concentration measurement results in the 1 W / V% PXB aqueous solution and each filtrate.
The βG concentration is expressed as βG content per gram of PxB.

  As is apparent from the results in Table 1, the filtration treatment with a 0.1 μm PVDF membrane could remove only a small amount of βGs mixed in PxB. Even by filtration using an ultrafiltration membrane of NMWL 10,000, only a small amount of βGs in PxB could be removed.

  On the other hand, when filtration is performed with a 0.65 μm zetapore membrane (a membrane having a positive charge), the βG concentration in PxB is reduced to 1/100 or less. That is, after filtration with a 0.1 μm pore size PVDF membrane and an NMWL 10,000 ultrafiltration membrane, the βGs in PxB were filtered with a zeta pore membrane with a pore size of 0.65 μm. It can be seen that it has been removed efficiently. On the other hand, since the PxB concentration in the solution has not changed at all by the filtration treatment with the zetapore membrane, it can be seen that there is no adsorption of PxB to the zetapore membrane.

  From the above, it is clear that treatment with a positively charged film is effective in removing βGs.

Example 2 Β-glucan removal in PxB solution-2
(Preparation of reagents)
(1) PxB aqueous solution prepared by dissolving polymyxin sulfate B (manufactured by B company, Japanese pharmacopoeia polymyxin sulfate B) in distilled water for endotoxin-free injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), 1 W / V% 200 mL of PxB aqueous solution was obtained.
(2) βG standard solution The same as that used in Example 1.
(3) AL solution The same as that used in Example 1.

[Preparation of AL reagent]
In the same manner as in Example 1, an AL reagent was prepared.

[Filtration material]
(1) For activated carbon chromatograph (manufactured by Wako Pure Chemical Industries, Ltd.).
(2) Positively charged membrane Zetapore membrane (Zetapore disk, trade name of Cuno Co., Ltd., pore diameter 0.2 μm, φ47 mm).
(3) Ultrafiltration membrane Biomax 5 (Biomax 5, trade name of Millipore, PBCC ultrafiltration disk, 5,000 NMWL, φ47 mm, filter material, polyethersulfone).

[Rough purification of PxB aqueous solution]
2 g of activated carbon was placed in a 500 mL Erlenmeyer flask and sterilized by dry heat at 260 ° C. for 3 hours. Subsequently, 200 mL of 1 W / V% PxB aqueous solution was added and stirred overnight. Centrifugation (3,000 rpm × 10 min) was performed, and the supernatant was collected to remove activated carbon. The obtained supernatant was subjected to suction filtration with a zetapore membrane, and the filtrate was further ultrafiltered with Biomax 5 under pressure. 2 mL of the filtrate obtained for each filtration treatment was sampled, and PxB concentration and βG concentration in each filtrate were measured in the same manner as in Example 1.

[Measurement of PxB concentration]
The same operation as in Example 1 was performed.

[Measurement of βG concentration]
The same operation as in Example 1 was performed.

〔result〕
Table 2 shows the PxB concentration measurement results and βG concentration measurement results in the 1 W / V% PXB aqueous solution and each filtrate.
The βG concentration is expressed as βG content per gram of PxB.

  As is apparent from the results in Table 2, it was found that the PxB product from a manufacturer different from Example 1 was contaminated with βGs as a result of examining the contamination with βGs.

  Moreover, in the method of processing PxB aqueous solution using the conventional activated carbon, since PxB will adsorb | suck to activated carbon, it turns out that the loss of PxB is large. Furthermore, although the βG concentration in the solution was lowered by the activated carbon treatment, the removal was insufficient.

  On the other hand, it was found that when filtration was performed with a zetapore membrane (a membrane having a positive charge) and an ultrafiltration membrane, there was almost no adsorption of PxB to the membrane, and there was almost no loss of PxB due to the filtration treatment. .

  Further, it can be understood that βGs in the PxB aqueous solution, which could not be removed by the activated carbon treatment, can be almost completely removed by combining the membrane having a positive charge and the filtration treatment by the ultrafiltration membrane.

Example 3 Effect of PxB on the reaction between AL and βG or AL and ET (endpoint synthetic substrate method)
(Preparation of reagents)
(1) PxB aqueous solution The same as that used in Example 2.
(2) PxB sample solution The supernatant or filtrate obtained by treating with activated carbon sampled in each treatment step of Example 2 was used.
(3) βG sample A solution obtained by dissolving 10 mg of curdlan (manufactured by Wako Pure Chemical Industries, Ltd.) in 10 mL of 0.1N sodium hydroxide solution was used as a βG stock solution. This was diluted with distilled water for injection to prepare an 11 pg / mL βG sample.
(4) Endotoxin sample (hereinafter referred to as “ET sample”)
A purified lipopolysaccharide derived from Escherichia coli 0111: B4 (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved and diluted with distilled water for injection to prepare a 2 ng / mL ET sample.
(5) AL solution Same as that used in Example 1.

[Preparation of AL reagent]
AL solution 12.5V / V%, Boc-Thr -Gly-Arg-pNA is 0.4 mM, so that MgSO ₄ is 100 mM, and PxB solution, the PxB reagent or distilled water for injection, PxB concentration 400ng / ML to 1.2 mg / mL or 0 solubilized in 100 mM Tris-HCl pH 8.0 was prepared and used as the AL reagent.

[Reactivity test of AL reagent for βG and ET]
The reactivity of the AL reagent containing PxB against βG and ET was measured by the endpoint synthetic substrate method according to the following method.

First, after adding 50 μL of 11 pg / mL βG sample or 2 ng / mL ET sample to a microplate (Costar 3595, manufactured by Corning), 50 μL of AL reagent was added. After reacting at 37 ° C. for 30 min, 50 μL of 0.04% NaNO ₂ (0.9 M HCl), 0.3 W / V% ammonium amidosulfate 50 μL, 0.07 W / V% N-1-naphthylethylenediamine dihydrochloride 50 μL Added and mixed.

  The final concentration of βG upon reaction with the AL reagent when 11 pg / mL βG sample is added is 5.5 pg / mL. In addition, when a 2 ng / mL ET sample is added, the final concentration of ET upon reaction with the AL reagent is 1 ng / mL ET.

  Thereafter, the absorbance at 545 nm to 630 nm of each solution was measured using Spectramax 250 (manufactured by Molecular Devices).

  As a control, instead of βG sample or ET sample, ET and βG-free distilled water for injection were used in the same manner, and the absorbance at 545 nm to 630 nm was measured by the endpoint synthetic substrate method.

〔result〕
The results are shown in FIGS. FIG. 1 shows a 1 W / V% PxB aqueous solution before the filtration treatment, FIG. 2 shows a supernatant obtained by treating activated carbon with a 1 W / V% PxB aqueous solution (PxB solution), and FIG. FIG. 4 is obtained by using the filtrate (PxB solution) obtained by further ultrafiltration of the filtrate under pressure with Biomax 5 as the PxB reagent, respectively. The results are shown respectively.

  1 to 4,-●-indicates the results obtained when the βG sample is used, and-▲-indicates the results obtained when the ET sample is used. Moreover,-♦-shows the result when distilled water for injection is used (control).

  The PxB concentration on the horizontal axis in FIGS. 1 to 4 indicates the PxB concentration in the reaction solution when the AL reagent is reacted with the ET sample, βG sample, or control (distilled water for injection). Therefore, the concentration of PxB in the used AL reagent is twice the concentration of PxB on the horizontal axis.

  As is apparent from the results of FIG. 1, when an AL reagent prepared using an untreated PxB aqueous solution (before filtration) was reacted with an ET sample (the ET content during the reaction was 1 ng) / ML), the absorbance was hardly measured when the concentration of PxB at the time of reaction was 2 μg (2 × 10 −6 g) / mL. Therefore, if the ET concentration at the time of reaction was about 1 ng / mL, In the presence of this concentration of PxB, the reaction between AL and ET was suppressed.

  However, as the amount of PxB added to the AL reagent is further increased (as the PxB concentration during the reaction is increased), the absorbance increases again. In addition, when the reactivity between the control (distilled water for injection) and the AL reagent was tested, the absorbance increased (-♦-), and the sample containing neither ET nor βG was reacted with the AL reagent. Nevertheless, since the AL reagent has reacted, it can be seen that the background of measurement increases as the concentration of PxB increases (FIG. 1).

This is because βGs coexist in the PxB aqueous solution, and as the concentration of PxB in the AL reagent increases, the concentration of βGs in PxB also increases, and the βGs react with AL. It is believed that there is. For example, from Table 2, the βG concentration in a 1 W / V% PxB aqueous solution is 70,000 pg / g PxB. Then, when the PxB concentration in the AL reagent is 4 μg [the PxB concentration during the reaction is 2 μg (2 × 10 ⁻⁶ g) / mL], the amount of βG derived from the PxB solution in the AL reagent is 0.28 pg / mL ( The reaction was 0.14 pg / mL), and the AL reagent used this time did not react at this concentration, but the PxB concentration in the AL reagent was 12 μg / mL (6 μg / mL during the reaction). mL), that is, when the βG concentration derived from the PxB solution in the AL reagent is 0.84 pg / mL (0.42 pg / mL during the reaction), the reaction with the AL reagent has occurred and it is determined as a false positive. It turns out that there is a possibility.

For example, some samples used for clinical diagnosis contain ET at a concentration of about several tens of ng / mL. In order to sufficiently suppress the ET activity even in clinical samples where it is unknown how much ET is contained, a higher concentration of PxB is allowed to coexist in the AL reagent. It is desirable to keep it. For that purpose, it is desirable to contain PxB in the AL reagent at least 200 μg / mL or more (100 μg / mL or more at the time of reaction), preferably 400 μg / mL or more (200 μg / mL or more at the time of reaction). However, as apparent from FIG. 1, an AL reagent containing 400 μg / mL of PxB was prepared using an untreated PxB aqueous solution, and the reactivity of the AL reagent with ET was tested (FIG. 1). 1,-▲-) (PxB concentration during the reaction was 200 μg (2 × 10 ⁻⁴ g) / mL), and the activity of ET could not be suppressed. In addition, when the sample containing βG is measured (FIG. 1,-●-), it can be seen that the absorbance has increased. That is, it is found that it is difficult to specifically measure βG in a sample when using an AL reagent containing 400 μg / mL PxB that has not been subjected to βG removal treatment.

  On the other hand, when FIGS. 1 to 4 are compared, when an AL reagent prepared using a PxB solution that has been subjected to activated carbon treatment, then zetapore membrane filtration treatment, and further ultrafiltration treatment, high concentration PxB is used as the AL reagent. Even when added, it can be seen that the reactivity increase between AL and ET, and between AL and control (distilled water for injection) is gradually suppressed at each treatment stage. This is because the βGs coexisting in the PxB were effectively removed as the PxB aqueous solution was sequentially treated. Therefore, the βGs coexisting in the AL reagent prepared using this treatment solution were removed. This is probably because the amount decreased as a result, and the increase in the background value of the measurement was suppressed.

In particular, as is apparent from FIG. 4, the reactivity of the AL reagent prepared using the PxB solution subjected to all treatments of activated carbon treatment → zetapore membrane filtration → ultrafiltration and ET was tested. Even when the PxB concentration of ET was 2 mg / mL [PxB concentration during reaction was 1 mg (1 × 10 ⁻³ g) / mL] or more, the activity of ET could be suppressed and no increase in background was observed. . From Table 2, the βG concentration in the ultrafiltration filtrate is <55 pg / g PxB, so the PxB concentration in the AL reagent is 400 μg / mL [the PxB concentration during the reaction is 200 μg (2 × 10 ⁻⁴ g) / mL]. In this case, the amount of βG derived from the PxB solution is <0.022 pg / mL (<0.011 pg / mL during the reaction), and the PxB concentration in the AL reagent is 2 mg / mL [the PxB concentration during the reaction is 1 mg (1 In the case of × 10 ⁻³ g) / mL], the amount of βG derived from the PxB solution is <0.11 pg / mL (<0.055 pg / mL during the reaction). That is, when an AL reagent containing PxB was prepared using a PxB solution that had been subjected to all treatments of activated carbon treatment → zetapore membrane filtration → ultrafiltration, the amount of βGs derived from the PxB solution was small. It is presumed that PxB at a high concentration of 2 mg / mL can coexist with AL.

  Moreover, about the AL reagent prepared using the PxB solution which performed all the processes of activated carbon treatment-> zetapore membrane filtration-> ultrafiltration, the result (reagent blind value) with the distilled water for injection was tested (figure) 4,-◆-), even if the PxB concentration in the AL reagent becomes 2 mg / mL (PxB concentration at the time of measurement is 1 mg / mL), the absorbance is almost 0, and the background value of the measurement increases. It was able to suppress enough. That is, by purifying PxB in advance, it can be seen that there is no risk of activation of AL by βGs in the PxB solution even when high concentration of PxB is added.

  Further, as apparent from FIG. 1, the AL reagent prepared using an untreated PxB aqueous solution also increased its reactivity with βG as the concentration of PxB in the reagent was increased ( FIG. 1,-●-). This is because the reactivity of βG and the AL reagent apparently increased because the concentration of βGs contained in the AL reagent increased. When this AL reagent was used, the βG concentration was increased with high accuracy. It is suggested that it is difficult to measure. However, the AL reagent prepared by sequentially purifying the PxB aqueous solution suppresses not only the reactivity with ET but also the increase in reactivity unrelated to the βG concentration as the PxB aqueous solution is processed. You can see that In particular, in the case of FIG. 4, as a result of testing the reactivity between the sample containing βG and the AL reagent (FIG. 4,-●-), the absorbance became constant even when the PxB concentration in AL increased. From this, it can be seen that the reactivity between AL and βG is not affected by the PxB concentration.

  From the above results, it can be seen that by allowing PxB not containing βG to coexist in AL, only ET activity coexisting in the sample to be measured can be suppressed and βG can be specifically measured.

Example 4 Reactivity test for ET and βG of AL reagent prepared using PxB from which βG contamination has been removed (endpoint synthetic substrate method)
(Preparation of reagents)
(1) PxB test solution The ultrafiltration filtrate obtained in Example 2 (activated carbon treatment → zetapore membrane filtration → ultrafiltration treatment) was used.
(2) βG sample A solution obtained by dissolving 10 mg of curdlan (manufactured by Wako Pure Chemical Industries, Ltd.) in 10 mL of 0.1N sodium hydroxide solution was used as a βG stock solution. This was diluted with distilled water for injection to prepare βG samples of 0.0093 pg / mL to 92 pg / mL.
(3) ET sample
Purified lipopolysaccharide derived from Escherichia coli 0111: B4 (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved and diluted with distilled water for injection to prepare 0.06 pg / mL to 200 ng / mL ET samples.
(4) AL solution The same as that used in Example 1.

[Preparation of AL Reagent of the Present Invention]
AL solution is 12.5 V / V%, Boc-Thr-Gly-Arg-pNA is 0.4 mM, MgSO ₄ is 100 mM, and 100 mM Tris-HCl using PxB reagent so that the PxB concentration is 2 mg / mL. What was dissolved in pH 8.0 was prepared, and the AL reagent of the present invention containing 2 mg / mL PxB was obtained. From Table 2, since the βG concentration in the ultrafiltration treated filtrate is <55 pg / gPxB, the βG concentration in this AL reagent is <0.11 pg / mL.

[Preparation of AL reagent not containing PxB]
An AL reagent containing no PxB was prepared using the same reagent as the AL reagent of the present invention except that distilled water for injection was used instead of the PxB reagent solution.

[Reactivity test of AL reagent for βG and ET]
The reactivity of the AL reagent containing PxB against βG and ET was measured by the endpoint synthetic substrate method according to the following method.

First, after adding 50 μL of βG sample, ET sample, or distilled water for injection (control) to a microplate (Costar 3595, manufactured by Corning), 50 μL of the AL reagent of the present invention was added and reacted at 37 ° C. for 30 minutes. . At this time, the PxB concentration in the reaction solution is 1 mg / ml. After the reaction time, 50 μL of 0.04% NaNO ₂ (0.9 M HCl), 0.3 W / V% ammonium amidosulfate 50 μL, 0.07 W / V% N-1-naphthylethylenediamine dihydrochloride 50 μL was added and mixed. .

  Thereafter, the absorbance at 545 nm to 630 nm was measured using Spectramax 250 (manufactured by Molecular Devices).

  Separately, reactivity tests on βG samples and ET samples were similarly performed using an AL reagent containing no PxB instead of the AL reagent of the present invention.

[result]
The results are shown in FIGS. FIG. 5 shows the test result of the reactivity of the AL reagent to ET (ET sensitivity), and FIG. 6 shows the test result of the reactivity of the AL reagent to βG (βG sensitivity). 5 and 6,-●-is in the presence of 1 mg / mL PxB (when the AL reagent of the present invention containing 2 mg / mL PxB is used), and-◆-is in the absence of PxB ( The results of (when using an AL reagent not containing PxB) are shown.

  The ET concentration and βG concentration on the horizontal axis in FIGS. 5 and 6 indicate the ET concentration and βG concentration in the reaction solution at the time of reaction with the AL reagent, respectively.

As is apparent from FIG. 5, in the absence of PxB (-♦-), AL reacts with ET in a concentration dependent manner. On the other hand, in the presence of 1 mg / mL PxB (when the AL reagent of the present invention containing 2 mg / mL PxB is used (-●-)), an ET concentration of 100 ng (10 ^-7 g / mL) exists. Even so, there is no reaction with ET.

  On the other hand, as apparent from FIG. 6, the reactivity (sensitivity) of AL to βG is equal regardless of whether or not PxB coexists, and PxB according to the present invention affects the reaction between AL and βG. It turns out not to give.

  From the above, it can be seen that if βG is measured by the endpoint synthetic substrate method using the AL reagent of the present invention, ET activity in the sample can be suppressed and βG can be specifically measured.

  In addition, when polymyxin treated by the method of the present invention is contained in the AL reagent, it can contain PxB at a high concentration of 2 mg / mL. It can be seen that it is possible to provide an AL reagent specific for βG that can suppress ET activity.

Example 5 FIG. Reactivity test for ET and βG of AL reagent prepared using PxB with βG contamination removed (turbidimetric time analysis method)
(Preparation of reagents)
(1) PxB sample solution The ultrafiltration filtrate obtained in Example 2 (activated carbon treatment → zetapore membrane filtration → ultrafiltration treatment) was used.
(2) βG Sample The same βG stock solution used in Example 1 was diluted with distilled water for injection to prepare βG samples of 0.092 pg / mL to 2750 pg / mL.
(3) ET sample The same ET sample as used in Example 3 was diluted with distilled water for injection to prepare 0.2 pg / mL to 200 ng / mL ET sample.
(4) AL solution The same as that used in Example 1.

[Preparation of AL Reagent of the Present Invention]
AL freeze-dried product (Hymen Crab Blood Cell Extract HS-J, sold by Wako Pure Chemical Industries, Ltd., gelation sensitivity: 0.03 EU / mL, for 5.2 mL) with 5.2 mL of 2 mg / mL PxB solution The AL reagent of the present invention was obtained that dissolved and contained 2 mg / mL PxB. From Table 2, since the βG concentration in the ultrafiltration treated filtrate is <55 pg / mL, the βG concentration in this AL reagent is <0.11 pg / mL.

[Preparation of AL reagent not containing PxB]
Dissolve AL lyophilized product (Horb Crab Blood Cell Extract HS-J, sold by Wako Pure Chemical Industries, Ltd., gel sensitivity: 0.03 EU / mL, for 5.2 mL) with 5.2 mL of distilled water for injection. An AL reagent containing no PxB was obtained.

[Reactivity test of AL reagent for βG sample and ET sample]
The reactivity of the AL reagent containing PxB against βG and ET was measured by a turbidimetric time analysis method according to the following method.

  First, 100 μL of the AL reagent of the present invention was added to a reaction test tube. Then, 100 μL of ET sample, βG sample, or distilled water for control (control) was added. Immediately, it was set in a toxinometer ET301 and turbidimetric time analysis was performed. The PxB concentration in the reaction solution at the time of analysis is 1 mg / mL.

  Separately, the turbidimetric time analysis was conducted in the same manner using an AL reagent not containing PxB instead of the AL reagent of the present invention.

[result]
The results are shown in FIGS.
FIG. 7 shows the test results of the reactivity of the AL reagent to ET (ET sensitivity), and FIG. 8 shows the test results of the reactivity of the AL reagent to βG (βG sensitivity). 7 and 8,-●-is in the presence of 1 mg / mL PxB (when the AL reagent of the present invention containing 2 mg / mL PxB is used), and-♦-is in the absence of PxB (PxB The results of (when using an AL reagent not containing) are shown.

The ET concentration and βG concentration on the horizontal axis in FIGS. 7 and 8 indicate the ET concentration and βG concentration in the reaction solution during the reaction with the AL reagent, respectively.
In the turbidimetric time analysis method, a shorter turbidimetric time indicates that an AL reaction occurs.

As is clear from FIG. 7, in the absence of PxB (-♦-), AL reacts with ET at an ET concentration of 1 pg (1 × 10 ⁻¹² g) / mL or more in a concentration dependent manner. In contrast, in the presence of 1 mg / mL PxB (when using the AL reagent of the present invention containing 2 mg / mL PxB,-●-), the ET concentration is 30 ng (3 × 10 ⁻⁸ g) / mL or more. Only reacting. That is, the reactivity of the AL reagent of the present invention containing PxB with respect to ET is approximately 1 / 30,000 of the reactivity of the AL reagent not containing PxB with respect to ET.

  On the other hand, as apparent from FIG. 8, the reactivity (sensitivity) of AL to βG is the same regardless of whether or not PxB coexists, and PxB affects the reaction between AL and βG by the turbidimetric time analysis method. It turns out not to give.

  From the above, it can be seen that when βG is measured by turbidimetric time analysis using the AL reagent of the present invention, ET activity in the sample can be suppressed and βG can be specifically measured.

Comparative Example 1 Reactivity test 1 for ET and βG of an AL reagent prepared using PxB without removing βG contamination (endpoint synthetic substrate method)
(Preparation of reagents)
(1) PxB test solution The PVDF membrane filtration filtrate obtained in Example 1 (βGs contamination was not removed) was used.
(2) βG sample A solution obtained by dissolving 10 mg of curdlan (manufactured by Wako Pure Chemical Industries, Ltd.) in 10 mL of 0.1N sodium hydroxide solution was used as a βG stock solution. This was diluted with distilled water for injection to prepare βG samples of 0.0093 pg / mL to 92 pg / mL.
(3) ET sample
Purified lipopolysaccharide derived from Escherichia coli 0111: B4 (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved and diluted with distilled water for injection to prepare 0.06 pg / mL to 200 ng / mL ET samples.
(4) AL solution The same as that used in Example 1.

[Preparation of AL reagent containing PxB]
AL solution was 12.5 V / V%, Boc-Thr-Gly-Arg-pNA was 0.4 mM, MgSO ₄ was 100 mM, and 100 mM of PxB was dissolved using PxB reagent so that the PxB concentration was 6 μg / mL. Tris-HCl pH 8.0 was prepared to obtain an AL reagent containing PxB. From Table 1, since the βG concentration in the PVDF membrane filtration filtrate is 53,900 pg / g PxB, the βG concentration in this AL reagent is 0.3234 pg / mL.

[Preparation of AL reagent not containing PxB]
An AL reagent containing no PxB was prepared using the same reagent as the AL reagent of the present invention except that distilled water for injection was used instead of the PxB reagent solution.

[Reactivity test of AL reagent for βG and ET]
The reactivity of the AL reagent containing PxB against βG and ET was measured by the endpoint synthetic substrate method according to the following method.

First, after adding 50 μL of βG sample, ET sample, or distilled water for injection (control) to a microplate (Costar 3595, manufactured by Corning), 50 μL of an AL reagent containing PxB is added and reacted at 37 ° C. for 30 minutes. It was. The PxB concentration in the reaction solution at this time is 3 μg / mL. After the reaction time, 50 μL of 0.04% NaNO ₂ (0.9 M HCl), 50 μL of 0.3 W / V% ammonium amidosulfate solution, and 0.07 W / V% N-1-naphthylethylenediamine dihydrochloride 50 μL were added and mixed. did.

  Thereafter, the absorbance at 545 nm to 630 nm was measured using Spectramax 250 (manufactured by Molecular Devices).

  Separately, the reactivity test with respect to the βG sample and the ET sample was similarly performed using an AL reagent not containing PxB instead of the AL reagent containing PxB.

〔result〕
The results are shown in FIGS. FIG. 9 shows the test results of the reactivity of the AL reagent to ET (ET sensitivity), and FIG. 10 shows the test results of the reactivity of the AL reagent to βG (βG sensitivity). In FIGS. 9 and 10, − ● − is in the presence of 3 μg / mL PxB (when an AL reagent containing 6 μg / mL PxB is used), and − ◆ − is in the absence of PxB (PxB The results of the tests performed in the case of using an AL reagent that does not contain are shown respectively.

  The ET concentration and βG concentration on the horizontal axis in FIGS. 9 and 10 indicate the ET concentration and βG concentration in the reaction solution at the time of reaction with the AL reagent, respectively.

As is clear from FIG. 9, AL reacts with ET in an ET concentration-dependent manner in the absence of PxB (-♦-). In contrast, in the presence of 3 μg / mL PxB (− ● −), the reaction between AL and ET is suppressed as compared with the case where PxB is not present, but the concentration of ET is 1 ng (1 × 10 ⁻ It can be seen that AL is reacting with ET at ⁹ g) / mL. Therefore, when the reactivity with E was tested using an AL reagent prepared using a PxB test solution filtered with a PVDF membrane, in the presence of 3 μg / mL PxB (the βG concentration derived from the PxB solution during the reaction) Is 0.1617 pg / mL), the amount of PxB added is insufficient, and it is clear that the reaction between AL and ET is not sufficiently suppressed. In the presence of PxB (− ● −), the absorbance in the presence of PxB is higher than that in the absence of PxB in the ET concentration range of 0.3 ng (3 × 10 ⁻¹⁰ g) / mL or less. You can see that Even when the ET concentration is 0, the absorbance is higher in the presence of 3 μg / mL of PxB than in the absence of PxB. This is considered to be due to the reaction of βGs mixed in PxB with AL, indicating that the background value is high.

Further, as is clear from FIG. 10, when the βG concentration becomes lower than 0.1 pg (10 ⁻¹ pg) / mL, the relationship between the βG concentration and the absorbance is not linear, and such low concentration βG is quantified. It turns out to be difficult.

As is clear from the above, when PxB that does not sufficiently remove βGs mixed in PxB coexists in the AL reagent at 6 μg / mL (PxB at the time of reaction is 3 μg / mL), βGs derived from the PxB solution are measured. Measurement of βG below 0.1 pg (10 ⁻¹ pg) / mL becomes impossible. Further, even in the presence of 3 μg / mL PxB, even 2 × 10 ⁻¹⁰ g / mL ET activity cannot be suppressed. Considering these facts, when PxB that does not sufficiently remove βGs is used, an amount of PxB that can sufficiently suppress the activity cannot coexist in the AL reagent. As a natural result, it is understood that when such an AL reagent is used, the reaction between AL and ET cannot be sufficiently suppressed, and βG-specific measurement cannot be performed.

Comparative Example 2 Reactivity test 2 for the ET and βG of the AL reagent prepared using PxB without removing βG contamination (endpoint synthetic substrate method)
(Preparation of reagents)
(1) PxB test solution The PVDF membrane filtration filtrate obtained in Example 1 (βGs contamination was not removed) was used.
(2) βG sample A solution obtained by dissolving 10 mg of curdlan (manufactured by Wako Pure Chemical Industries, Ltd.) in 10 mL of 0.1N sodium hydroxide solution was used as a βG stock solution. This was diluted with distilled water for injection to prepare βG samples of 0.0093 pg / mL to 92 pg / mL.
(3) ET sample
Purified lipopolysaccharide derived from Escherichia coli 0111: B4 (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved and diluted with distilled water for injection to prepare 0.2 pg / mL to 200 ng / mL ET samples.
(4) AL solution The same as that used in Example 1.

[Preparation of AL reagent containing PxB]
AL solution was 12.5 V / V%, Boc-Thr-Gly-Arg-pNA was 0.4 mM, MgSO ₄ was 100 mM, and 100 mM of PxB was dissolved using PxB reagent so that the PxB concentration was 200 μg / mL. Tris-HCl pH 8.0 was prepared to obtain an AL reagent containing 200 μg / mL PxB. From Table 1, the βG concentration in the PVDF membrane filtration filtrate is 53,900 pg / g PxB, and the βG concentration in this AL reagent is 10.78 pg / mL.

[Preparation of AL reagent not containing PxB]
An AL reagent containing no PxB was prepared using the same reagent as the AL reagent of the present invention except that distilled water for injection was used instead of the PxB reagent solution.

[Reactivity test of AL reagent for βG and ET]
The reactivity of the AL reagent containing PxB against βG and ET was measured by the endpoint synthetic substrate method according to the following method.

First, after adding 50 μL of βG sample, ET sample, or distilled water for injection (control) to a microplate (Costar 3595, manufactured by Corning), 50 μL of an AL reagent containing PxB is added and reacted at 37 ° C. for 30 minutes. It was. The PxB concentration in the reaction solution at this time is 100 μg / mL. After the reaction time, 50 μL of 0.04% NaNO ₂ (0.9 M HCl), 0.3 W / V% ammonium amidosulfate 50 μL, 0.07 W / V% N-1-naphthylethylenediamine dihydrochloride 50 μL was added and mixed. .

  Thereafter, the absorbance at 545 nm to 630 nm was measured using Spectramax 250 (manufactured by Molecular Devices).

  Separately, instead of the AL reagent containing PxB, the reactivity test for βG sample and ET sample was similarly conducted using AL reagent not containing PxB.

〔result〕
The results are shown in FIG. 11 and FIG. FIG. 11 shows the test results of the reactivity of the AL reagent to ET (ET sensitivity), and FIG. 12 shows the test results of the reactivity of the AL reagent to βG (βG sensitivity). 11 and 12,-●-is in the coexistence of 100 μg / mL PxB (when an AL reagent containing 200 μg / mL PxB is used), and-♦-is in the absence of PxB (does not contain PxB). The results of the test conducted in the case of using the AL reagent are shown respectively.

  The ET concentration and βG concentration on the horizontal axis in FIGS. 11 and 12 indicate the ET concentration and βG concentration in the reaction solution during the reaction with the AL reagent, respectively.

  As is apparent from FIG. 11, AL reacts with ET in a concentration-dependent manner in the absence of PxB (-♦-). In contrast, in the presence of 100 μg / mL PxB (− ● −), it can be seen that AL reacts regardless of the ET concentration.

  As is clear from FIG. 12, in the presence of PxB (-●-), AL reacts even when βG is not present (even when the concentration of βG is 0 pg / mL).

  As mentioned above, in order to prepare an AL reagent for βG measurement that can be used for clinical diagnosis performed on various samples with various ET concentrations, at least 200 μg / mL PxB coexists in the AL. It is desirable to keep it. However, if the removal of βGs mixed in PxB is not sufficient, βGs in PxB react with AL, so it is clear that βG measurement using AL cannot be performed.

The result of having tested the reactivity with respect to ET and (beta) G of the AL reagent prepared in Example 3 using the 1 W / V% PxB aqueous solution before the filtration process is shown. The result of having tested the reactivity with respect to ET and (beta) G of the AL reagent prepared using the supernatant (PxB solution) obtained by carrying out the activated carbon process of the 1 W / V% PxB aqueous solution obtained in Example 3 is shown. For the ET and βG of the AL reagent prepared in Example 3 using the filtrate (PxB solution) obtained by further subjecting the supernatant obtained by the activated carbon treatment to suction filtration using a zetapore membrane. The result of having tested the reactivity is shown. The ET and ET of the AL reagent prepared in Example 3 using the filtrate obtained by ultrafiltration of the filtrate obtained by suction filtration using a zetapore membrane with Biomax 5 under pressure. The result of having tested the reactivity with respect to (beta) G is shown. The result of having tested the reactivity with respect to ET of the AL reagent prepared using the ultrafiltration process filtrate of PxB aqueous solution obtained in Example 4 is shown. The result of having tested the reactivity with respect to (beta) G of the AL reagent prepared using the ultrafiltration process filtrate of PxB aqueous solution obtained in Example 4 is shown. The result of having tested the reactivity with respect to ET of the AL reagent prepared using the ultrafiltration process filtrate of PxB aqueous solution obtained in Example 5 is shown. The result of having tested the reactivity with respect to (beta) G of the AL reagent prepared using the ultrafiltration process filtrate of PxB aqueous solution obtained in Example 5 is shown. The result of having tested the reactivity with respect to ET of the AL reagent prepared using the PVDF membrane filtration various path fluid (PxB solution) obtained in the comparative example 1 is shown. The result of having tested the reactivity with respect to (beta) G of the AL reagent prepared in PVDF membrane filtration various path fluid (PxB solution) obtained in the comparative example 1 is shown. The result of having tested the reactivity with respect to ET of the AL reagent prepared in the comparative example 2 using the PVDF membrane filtration process filtrate (PxB solution) is shown. The result of having tested the reactivity with respect to (beta) G of AL reagent prepared using the PVDF membrane filtration process filtrate (PxB solution) obtained in the comparative example 2 is shown.

Explanation of symbols

  1 to 4,-●-indicates a βG sample,-▲-indicates an ET sample, and-♦-indicates a result when distilled water for injection (control) is used.

  5 and 6,-●-is in the presence of 1 mg / mL PxB (when the AL reagent of the present invention containing 2 mg / mL PxB is used), and-♦-is in the absence of PxB (PxB The results of (when using an AL reagent not containing) are shown.

  7 and 8,-●-is in the presence of 1 mg / mL PxB (when using an AL reagent containing 2 mg / mL PxB), and-♦-is in the absence of PxB (an AL reagent not containing PxB). The results are shown respectively.

  9 and 10,-●-is in the presence of 3 μg / mL PxB (when an AL reagent containing 6 μg / mL PxB is used), and-♦-is in the absence of PxB (an AL reagent not containing PxB). The results are shown respectively.

11 and 12,-●-is in the coexistence of 100 μg / mL PxB (when an AL reagent containing 200 μg / mL PxB is used), and-♦-is in the absence of PxB (an AL reagent not containing PxB). The results are shown respectively.

Claims (12)

Polymyxin or its salt and AL, which contain only (1 → 3) -β-D-glucan or / and its related substances below the concentration that does not cause an enzyme reaction of the horseshoe crab blood cell extract (hereinafter abbreviated as AL) An AL reagent for measuring (1 → 3) -β-D-glucan, comprising: The AL reagent according to claim 1, wherein the concentration of polymyxin or a salt thereof is 0.2 to 80 mg / mL. The AL reagent according to claim 1, wherein the polymyxin or a salt thereof is obtained by treating polymyxin or a salt thereof with a positively charged membrane. The AL reagent according to claim 1, wherein (1 → 3) -β-D-glucan or / and its related substance is (1 → 3) -β-D-glucan. The AL reagent according to claim 1, wherein the polymyxin or a salt thereof is polymyxin B or a salt thereof. The AL reagent according to claim 5, wherein the salt of polymyxin B is polymyxin B sulfate. It is characterized by coexisting with AL or polymyxin or a salt thereof containing only (1 → 3) -β-D-glucan or / and its related substance at a concentration not causing an enzymatic reaction of AL (1 → 3) ) Preparation of AL reagent for measuring β-D-glucan. The preparation method according to claim 1, wherein 0.2 to 80 mg / mL polymyxin or a salt thereof coexists in the (1 → 3) -β-D-glucan measurement AL reagent. Comprising polymyxin or a salt thereof containing only (1 → 3) -β-D-glucan or / and its related substances, and AL as constituents, which do not cause an enzyme reaction of AL (1 → 3) -β-D-glucan measurement kit. Containing a reagent that contains polymyxin or a salt thereof, which contains polymyxin or a salt thereof, containing only (1 → 3) -β-D-glucan or / and its related substances at a concentration that does not cause an enzymatic reaction of AL; (1 → 3) -β-D-glucan measurement kit. A sample containing endotoxin is reacted with an AL reagent containing polymyxin or a salt thereof containing only (1 → 3) -β-D-glucan or / and its related substance at a concentration not causing an enzymatic reaction of AL, Measure the activity of the enzyme activated by the resulting enzyme activation reaction, or measure the degree of turbidity change and gelation state of the reaction solution based on the resulting gelation reaction by instrument or visual observation A method for specifically measuring (1 → 3) -β-D-glucan. A sample containing endotoxin is reacted with AL in the presence of polymyxin or a salt thereof containing only (1 → 3) -β-D-glucan or / and its related substance at a concentration that does not cause an enzymatic reaction of AL. Measure the activity of the enzyme activated by the resulting enzyme activation reaction, or measure the degree of turbidity change and gelation state of the reaction solution based on the resulting gelation reaction by instrument or visual observation. A method for specifically measuring (1 → 3) -β-D-glucan.

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