JP2010187634A - METHOD FOR DETERMINING beta-GLUCAN CONCENTRATION AND CONCENTRATION DETERMINATION KIT - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for quickly and sensitively determining β-glucan of a level non-detectable by conventional β-glucan determination method without using a dedicated device. <P>SOLUTION: Concentration of β-glucan in a specimen is quantitatively determined by reacting an determination object specimen, a reagent containing a G factor which is activated by bonding with β-glucan and a luminescent synthetic substrate obtained by bonding a luminescent substrate to a peptide to liberate the luminescent substrate from the luminescent synthetic substrate, reacting the liberated luminescent substrate with a luminescent enzyme, determining generated light intensity and determining β-glucan concentration based on the result. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for measuring the concentration of β-glucan contained in a sample and a kit for measuring the concentration, and more particularly to a method for measuring the concentration of β-glucan using a bioluminescence reaction and a kit for measuring the concentration.

  β-glucan is a substance known to exist as a skeletal constituent of cell walls of yeast and mold, a major polysaccharide component of many basidiomycetous fruit bodies (mushrooms), and an elution component from membranes used for hemodialysis. is there. By measuring this, it is believed that early diagnosis of deep mycosis, which is a serious infectious disease, and detection of contamination of medical devices by fungi can be performed. In particular, fungi are eukaryotic as we are, and antifungal drugs (antifungal agents) cause some damage to the human body, so their side effects are much larger and cautious than antibacterial antibiotics. Administration is necessary. For this reason, it is very important to detect fungal infections at an extremely early stage, and it is possible to reduce the dose of antifungal drugs by treating them before the infection spreads, thereby significantly reducing damage. Become.

  Conventional β-glucan is measured using a serine protease cascade that is activated by β-glucan, which is a component of the fungus cell wall, from the blood cell extract (Amebosite lysate) of horseshoe crab. It has been. FIG. 1 shows the process of gelation reaction of the blood cell extract of horseshoe crab with β-glucan. A factor G pathway that specifically reacts with β-glucan exists in the blood cell extract of horseshoe crab. The “factor G pathway” is constituted by the following reaction cascade. First, β-glucan binds tightly to Factor G (Factor G) and activates Factor G. Next, the factor G activated by the binding of β-glucan activates a proclotting enzyme to generate a clotting enzyme. This clotting enzyme partially hydrolyzes its substrate, coagulogen. As a result, peptide C (peptide C) is released from coagulogen to produce coagulin, a coagulation protein. The coagulation action causes gelation (see Non-Patent Document 1).

  The β-glucan measurement method by the Limulus test is an application of a process in which the blood cell extract of horseshoe crab gels with β-glucan. As the Limulus test, methods such as a gelation overturning method (gelation method), a chromogenic synthetic substrate method (colorimetric method), and a turbidimetric time analysis method (turbidimetric method) are known because of differences in determination or measurement methods.

  In the “gelling overturning method”, a liquid containing a factor G-related substance is mixed with a sample to be examined in a test tube and reacted under a certain condition (for example, at 30 ° C. for 30 to 60 minutes), and then the test tube is turned over. Alternatively, it is a method of determining whether the sample remains liquid or solidified when tilted. In the former case, β-glucan is negative, and in the latter case, β-glucan is positive. This method does not require special equipment and is relatively easy to operate, but the measurement results are likely to change depending on the raw materials and production lots, and it is less objective than the optical method for human judgment. Because of the problems, it is usually only used simply.

  The “chromogenic synthetic substrate method” is a method for calculating the amount of β-glucan by colorimetrically determining the amount of the chromogenic group released by absorbance using a chromogenic synthetic peptide substrate as a substrate for the coagulation enzyme. As the chromogenic synthetic peptide substrate, a substrate simulating the amino acid sequence of the site where the coagulase hydrolyzes the natural substrate coagulogen is used. A color developing group such as paranitroaniline (pNA) is bonded to the site cleaved by the coagulation enzyme, and the color develops when the color developing group is released by cleavage with the enzyme. When the coloring group is paranitroaniline, the absorbance at 405 nm, which is the absorption wavelength of paranitroaniline, is measured over time. Further, when the chromophore is diazo-coupled with paranitroaniline, the absorbance at 545 nm is measured over time. Thereafter, the change in the amount of transmitted light with time is analyzed to measure the β-glucan concentration. The chromogenic substrate method has problems such as relatively expensive reagents and complicated operations, but is excellent in quantitativeness, sensitivity, and objectivity.

  The “turbidimetric time analysis method” captures the increase in turbidity due to gelation as a change in the amount of transmitted light, and gels the time required for the ratio of transmitted light in the reaction solution to decrease to a certain threshold (usually around 90%). This is a method for calculating the β-glucan value using a standard curve created from the relationship between gelation time and β-glucan concentration. Quantitative and objectivity is very good, but a dedicated device is required for measurement.

Tatsushi Muta, Noriaki Seki, Yoshie Takaki, Ryuji Hashimoto, Toshio Oda, Atsufumi Iwanaga, Fuminori Tokunaga, and Sadaaki Iwanaga, J. Biol. Chem., 270: 892-897 (1995)

  However, various problems have been pointed out in the measurement of β-glucan even when using the Limulus test such as the chromogenic synthetic substrate method and the turbidimetric time analysis method, which are excellent in quantification and objectivity. For example, a high sensitivity measurement of 5 pg / ml or less requires a reaction time of about 2 hours, and the measurement of β-glucan of 0.2 pg / ml or less is used for diagnosis at the early stage of infection because the measurement result is unreliable. Can not do it. As described above, it is considered that there is still a problem in the measurement sensitivity of the present β-glucan limulus test.

  The present invention has been made in view of the above problems, and provides a method for quickly and highly sensitively measuring β-glucan at a level that could not be detected by a conventional β-glucan measurement method without using a dedicated device. With the goal.

The present invention includes the following inventions in order to solve the above problems.
[1] A method for measuring the concentration of β-glucan contained in a sample, comprising a sample, a reagent containing a factor G activated by binding to β-glucan, and a luminescent synthesis comprising a luminescent substrate bound to a peptide A luminescent substrate releasing step for reacting a substrate and releasing the luminescent substrate from the luminescent synthetic substrate; and a luminescence measuring step for measuring a luminescence amount by allowing a luminescent enzyme to act on the luminescent substrate released by the luminescent substrate releasing step; And a concentration quantification step of quantifying the β-glucan concentration in the sample based on the measurement value obtained by the luminescence amount measurement step.
[2] The luminescent synthetic substrate has a structure in which the bond between the luminescent substrate and the peptide is cleaved by the action of an activated G factor or coagulase generated by the activation of the G factor. The method for measuring the concentration of β-glucan according to [1] above.
[3] The β-glucan concentration measurement method according to [2], wherein the luminescent substrate is aminoluciferin and the luminescent enzyme is luciferase.
[4] The luciferase is derived from a beetle selected from the group consisting of North American fireflies, Genji fireflies, Heike fireflies, Japanese fireflies, Japanese fireflies, mad fireflies, skulls, light beetles, and railroad insects. β-glucan concentration measurement method.
[5] The luciferase is a mutant luciferase, and the amino acid sequence of the wild-type luciferase is modified so that the luminescence intensity of the mutant luciferase is increased as compared with the wild-type luciferase. [3] The method for measuring the concentration of β-glucan according to [4].
[6] The β-glucan concentration measurement method according to [5], wherein the mutant luciferase is one selected from the group consisting of the following (i) to (vi):
(i) A mutant firefly luciferase comprising an amino acid sequence of the wild-type North American firefly luciferase shown in SEQ ID NO: 10, wherein isoleucine at position 423 is replaced with leucine and aspartic acid at position 436 is replaced with glycine
(ii) A mutant firefly luciferase comprising an amino acid sequence of the wild-type North American firefly luciferase shown in SEQ ID NO: 10, wherein isoleucine at position 423 is replaced with leucine and leucine at position 530 is replaced with arginine
(iii) A mutant firefly luciferase comprising an amino acid sequence of the wild-type North American firefly luciferase shown in SEQ ID NO: 10, wherein aspartic acid at position 436 is replaced with glycine and leucine at position 530 is replaced with arginine
(iv) In the amino acid sequence of the wild-type North American firefly luciferase shown in SEQ ID NO: 10, leucine at position 423 was substituted with leucine, aspartic acid at position 436 was substituted with glycine, and leucine at position 530 was substituted with arginine. Variant firefly luciferase consisting of amino acid sequence
(v) In the amino acid sequence of wild-type North American firefly luciferase shown in SEQ ID NO: 10, isoleucine at position 47 is threonine, asparagine at position 50 is serine, methionine at position 59 is threonine, and threonine at position 252 is serine. Mutant firefly luciferase having an amino acid sequence in which leucine at position 423 is replaced with leucine and leucine at position 530 is substituted with arginine
(vi) The amino acid sequence of wild-type North American firefly luciferase shown in SEQ ID NO: 10 consists of an amino acid sequence in which asparagine at position 50 is replaced with serine, isoleucine at position 423 is replaced with leucine, and leucine at position 530 is replaced with arginine. Mutant firefly luciferase [7] The reagent containing G factor is a horseshoe crab blood cell extract component, and the protein concentration of the horseshoe crab blood cell extract component is 1.5 mg / mL to 3.5 mg / mL with respect to the total amount of the reaction system The method for measuring β-glucan concentration according to any one of [1] to [6] above.
[8] A kit for measuring β-glucan concentration, comprising the following (a), (b), and (c) as constituent components.
(a) a reagent containing factor G that is specifically bound to and activated by β-glucan
(b) Luminescence having a structure in which a bond between a luminescent substrate and a peptide is cleaved by the action of an activated factor G or coagulase produced by the activation of factor G by binding a luminescent substrate to the peptide Synthetic substrate
(c) Luminescent enzyme

  According to the β-glucan concentration measurement method according to the present invention, the bioluminescence reaction is applied to the β-glucan concentration measurement, and the β-glucan concentration is quantified based on the amount of luminescence generated. The level of β-glucan that did not exist can be measured easily and quickly without using a dedicated measuring device.

It is a figure which shows the process of the gelatinization reaction of the blood cell extract of horseshoe crab by (beta) glucan. It is a figure which shows an example of the density | concentration measuring method of the beta glucan based on this invention. 2 is a graph plotting the emission intensity at each sample concentration in Example 1. FIG. It is the graph which plotted the light absorbency in each sample concentration of a comparative example.

1. β Glucan Concentration Measurement Method First, the mechanism of the β glucan concentration measurement method according to the present invention will be described with reference to FIGS. 1 and 2. FIG. 1 is a diagram showing the process of gelation reaction of a blood crab blood cell extract (LAL) using β-glucan, and FIG. 2 is a diagram showing an example of a method for measuring β-glucan concentration according to the present invention. It is. As shown in FIG. 1, β-glucan binds tightly to factor G in the blood cell extract of horseshoe crab and activates factor G, and activated factor G (active factor G) activates procoagulase. And clotting enzymes are produced. The coagulation enzyme hydrolyzes partially using coagulogen as a substrate to produce coagulin, a coagulation protein.

  As shown in FIG. 2, when a sample containing β-glucan is brought into contact with a blood cell extract of horseshoe crab, the Limulus reaction system is activated by β-glucan in the sample. Therefore, when “benzoyl-Leu-Arg-Arg-aminoluciferin” is added to the reaction system as a substrate for the coagulation enzyme, the bond between Arg and aminoluciferin is cleaved by the coagulation enzyme generated by the activation of the Limulus reaction system. Aminoluciferin, a luminescent substrate, is released. Light is generated by allowing luciferase (a luminescent enzyme) to act on the aminoluciferin. The luminescence reaction of luciferin / luciferase is shown in the following reaction formula (I).

  The β-glucan concentration measurement method according to the present invention is a method for measuring the amount of light generated by the above-described mechanism and quantifying the β-glucan concentration in the sample based on the obtained measurement value (amount of luminescence). .

Next, the method for measuring β-glucan concentration according to the present invention will be described in detail. The concentration measurement method for β-glucan according to the present invention includes:
(1) A sample, a reagent containing factor G activated by binding to β-glucan, and a luminescent synthetic substrate formed by binding a luminescent substrate to a peptide are reacted to release the luminescent substrate from the luminescent synthetic substrate. Luminescent substrate releasing step (2) Luminescent enzyme measurement is performed by causing a luminescent enzyme to act on the luminescent substrate released in the luminescent substrate releasing step, and measuring the amount of luminescence. (3) In the sample based on the measurement value obtained by the luminescent amount measuring step Any method may be used as long as it includes a concentration quantification step of quantifying the β-glucan concentration. The β-glucan that is the measurement target of the present invention may be any β-glucan that can activate factor G by binding to factor G in the blood cell extract of horseshoe crab, and preferably (1 → 3) -β-D-glucan. It is.

(1) Luminescent substrate release step The luminescent substrate release step comprises a sample, a reagent containing factor G activated by binding to β-glucan (hereinafter referred to as “G-factor-containing reagent”), and a peptide. This is a step of reacting with a luminescent synthetic substrate formed by binding to release the luminescent substrate from the luminescent synthetic substrate.

〔sample〕
The sample is not particularly limited, and any sample that may contain β-glucan can be suitably used as the sample. Among them, clinical samples such as blood (plasma), body fluid, urine and the like are suitable as samples for diagnosis of deep mycosis. In addition, a sample in which contamination with fungi (molds or the like) is a problem can be suitably used as a sample. Specific examples include pharmaceuticals such as injections, infusion solutions, and dialysates, collected materials from medical devices and medical devices, collected materials from clean rooms, and the like. In clinical samples, when inactivation treatment of interfering factors (inhibitors and enhancement factors) of the Limulus reaction system is necessary, a known inactivation treatment method (for example, perchloric acid treatment method (PCA method) or New PCA method). Etc.) is preferably pretreated.

[Factor containing G factor]
As the factor G-containing reagent, horseshoe crab blood cell extract components conventionally used in the Limulus test can be suitably used. For example, it is obtained from blood cells of horseshoe crab belonging to the genus Limulus, Tachypleus, or Carcinoscorpius, and can produce a coagulation enzyme by reaction with β-glucan There is no particular limitation. Therefore, a commercially available Limulus reagent (LAL reagent) for β-glucan measurement or a Limulus reagent (LAL reagent) attached to a β-glucan measurement kit can be suitably used.

  It is also possible to use a recombinant G factor derived from a recombinant gene synthesized on the basis of a part or all of the gene for G factor of horseshoe crab. Recombinant factor G can be obtained by preparing an expression vector into which a horseshoe crab factor G gene is inserted according to a known genetic engineering technique, introducing it into a suitable host cell, expressing the recombinant protein, and purifying it. .

  When a horseshoe crab blood cell extract component (for example, a commercially available Limulus reagent for β-glucan measurement) conventionally used for the Limulus test is used as the G-factor-containing reagent, the active G-factor is reacted with a sample containing β-glucan. And clotting enzymes are produced. These are all known to be proteins having protease activity. Therefore, in this case, as the luminescent synthetic substrate, those having an active factor G recognition sequence and those having a coagulation enzyme recognition sequence can be used. However, since the β-glucan concentration measurement method according to the present invention uses the protease activity of either active G factor or coagulation enzyme as an index, one kind of chromogenic synthetic substrate corresponding to the enzyme used as the index is used. .

  On the other hand, when recombinant factor G is used as the G-factor-containing reagent, there is no precoagulation enzyme in the reagent, so that the active recombinant factor G is produced by the reaction with the sample containing β-glucan. Only. Accordingly, in this case, a substrate having an active factor G recognition sequence is used as the luminescent synthetic substrate.

  When a horseshoe crab blood cell extract component (for example, a commercially available Limulus reagent for β-glucan measurement) conventionally used in the Limulus test is used as the Factor G-containing reagent, the amount used is about 40% to about 80% of the conventional amount. % Is preferred. It is clear that the conventional use amount (100%) hinders the luminescence reaction and the luminescence intensity decreases, and that high luminescence intensity is obtained when using about 40% to about 80% of the conventional use amount. Because it became. About 40% to about 80% of the conventional use amount corresponds to a range of 1.5 mg / mL to 3.5 mg / mL when converted to the protein concentration of horseshoe crab blood cell extract component (LAL) with respect to the total amount of the measurement system. . More preferably, it is 2.0 mg / mL to 3.3 mg / mL. Since high-sensitivity measurement is possible with an amount smaller than the conventional use amount, the β-glucan concentration measurement method according to the present invention is very useful industrially.

[Luminescent synthetic substrate]
The luminescent synthetic substrate is formed by binding a luminescent substrate to a peptide, and any substrate can be used as long as the luminescent substrate is liberated from the luminescent synthetic substrate by the reaction of the sample, the G-factor-containing reagent and the luminescent synthetic substrate. Such a luminescent synthetic substrate preferably has a structure in which the bond between the luminescent substrate and the peptide is cleaved by the action of activated factor G or coagulase generated by activating factor G. In the present specification, the “luminescent substrate” means a substance that emits light as a reaction substrate by bioluminescence. Luciferin can be preferably used as the luminescent substrate. Of these, aminoluciferin represented by the following formula (II) is preferable. This is because the amino group of aminoluciferin can form an amide bond with the carboxyl group of an adjacent amino acid.

  The peptide that binds to aminoluciferin may be any peptide having an amino acid sequence in which the amide bond with aminoluciferin at the C-terminus of the peptide is cleaved by the active factor G or the protease activity of the clotting enzyme. The number of amino acid residues and the amino acid sequence are not limited as long as this condition is satisfied, but the number of amino acid residues is preferably 2 to 10 from the viewpoint of specificity, synthesis cost, ease of handling, and the like.

  Specifically, examples of the peptide having a coagulation enzyme recognition sequence include Gly-Val-Ile-Gly-Arg- (SEQ ID NO: 1), Val-Leu-Gly-Arg- (SEQ ID NO: 2), Leu-Arg- Arg- (SEQ ID NO: 3), Ile-Glu-Gly-Arg- (SEQ ID NO: 4), Leu-Gly-Arg- (SEQ ID NO: 5), Val-Ser-Gly-Arg- (SEQ ID NO: 6), Val- Gly-Arg- (SEQ ID NO: 7) and the like can be mentioned. Moreover, as a peptide which has the recognition sequence of an active factor G, Thr-Leu-Ser-Arg-Gln-Arg-Arg- (sequence number 8), Thr-Thr-Thr-Thr-Arg- (sequence number 9) Etc.

  The N-terminus of the peptide may be protected with a protecting group. Any protecting group that can be used in this field can be used without limitation. Specifically, for example, an N-succinyl group, a tert-butoxycarbonyl group, a benzoyl group, a p-toluenesulfonyl group and the like can be mentioned.

The luminescent synthetic substrate used in the present invention can be synthesized, for example, by referring to the methods described in Example 6 and Example 7 of JP 2005-530485 (International Publication Number: WO2003 / 066661). Also, a luminescent synthetic substrate (benzoyl-Leu-Arg-Arg-aminoluciferin) attached to “Proteasome-Glo ^™ Assay Systems” commercially available from Promega can be used. When free aminoluciferin is contained in the luminescent synthetic substrate, it is preferable to remove it beforehand. Background luminescence can be suppressed by removing free aminoluciferin from the luminescent synthetic substrate. As a method for removing free aminoluciferin, for example, 0.8 mM coenzyme A, 1.5 mM ATP, 250 μg / ml firefly in 20 mM Tricine, 8 mM Mg ²⁺ , 0.13 mM EDTA buffer (pH 7.8) can be used. A method of mixing with a solution containing luciferase and 90 mM DTT and incubating at room temperature (25 ° C.) for 1 to 6 hours can be mentioned.

[Reaction procedure (method)]
The reaction procedure (method) of the sample, the G-factor-containing reagent, and the luminescent synthetic substrate is compatible with the conditions under which the luminescent substrate (for example, aminoluciferin) is released from the luminescent synthetic substrate by the reaction of these three members. There is no particular limitation. For example, a sample and a G-factor-containing reagent are mixed well and incubated at 37 ° C. for about 5 minutes to 60 minutes, and then a luminescent synthetic substrate is added and mixed well, followed by incubation at 37 ° C. for about 1 minute to 30 minutes. Can be mentioned.

(2) Luminescence measurement process The luminescence measurement process is a process in which the luminescence enzyme is allowed to act on the luminescence substrate released in the preceding luminescence substrate release process to measure the luminescence quantity.

[Luminescent enzyme]
The luminescent enzyme used in the present invention is not particularly limited as long as it catalyzes the bioluminescence of the luminescent substrate released from the luminescent synthetic substrate and generates light. When the luminescent substrate is luciferin, luciferase is preferably used as the luminescent enzyme. When the luminescent substrate is aminoluciferin represented by the above formula (II), it is preferable to use beetle-derived luciferase as the luminescent enzyme. As the beetle-derived luciferase, beetle-derived luciferases such as North American fireflies, Genji fireflies, Heike fireflies, Japanese fireflies, Japanese fireflies, mad fireflies, bots fireflies, light beetles and railroad worms can be suitably used. The amino acid sequences of these beetle-derived luciferases and the base sequences of the genes encoding them are shown in the known database (for example, EMBL Nucleotide Sequence Database (http://www.ebi.ac. uk / embl /)). The luciferase may be a natural protein or a recombinant protein. Luciferases that can be used in the present invention are commercially available from various companies as reagents.

  The luciferase is not limited to those having a wild type amino acid sequence, and may be a mutant luciferase having an amino acid sequence different from the wild type amino acid sequence as long as it has a function of catalyzing bioluminescence of a luminescent substrate. . Examples of the amino acid sequence different from the wild type amino acid sequence include an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the wild type amino acid sequence. Here, “one or several amino acids have been deleted, inserted, substituted or added” means deletion, insertion, substitution or addition by a known mutant polypeptide production method such as site-directed mutagenesis. It is intended that as many amino acids as possible (preferably 10 or less, more preferably 7 or less, most preferably 5 or less) are deleted, inserted, substituted or added.

Among the mutant luciferases, it is preferable to use a mutant luciferase modified so that the luminescence intensity is increased. This is because an extremely small amount of β-glucan can be measured with high sensitivity. Examples of the mutant luciferase modified so as to increase the emission intensity include the following mutant luciferases.
(i) In the amino acid sequence of wild type North American firefly luciferase (SEQ ID NO: 10), isoleucine (Ile) at position 423 is replaced with leucine (Leu), and aspartic acid (Asp) at position 436 is replaced with glycine (Gly). Mutant firefly luciferase consisting of different amino acid sequences
(ii) In the amino acid sequence of wild-type North American firefly luciferase (SEQ ID NO: 10), isoleucine (Ile) at position 423 was replaced with leucine (Leu), and leucine (Leu) at position 530 was replaced with arginine (Arg). Variant firefly luciferase consisting of amino acid sequence
(iii) In the amino acid sequence of wild type North American firefly luciferase (SEQ ID NO: 10), aspartic acid (Asp) at position 436 is replaced with glycine (Gly), and leucine (Leu) at position 530 is replaced with arginine (Arg). Mutant firefly luciferase consisting of different amino acid sequences
(iv) In the amino acid sequence of wild-type North American firefly luciferase (SEQ ID NO: 10), isoleucine (Ile) at position 423 is replaced with leucine (Leu), and aspartic acid (Asp) at position 436 is replaced with glycine (Gly). Mutant firefly luciferase having an amino acid sequence in which leucine (Leu) at position 530 is replaced with arginine (Arg)
(v) In the amino acid sequence of wild-type North American firefly luciferase (SEQ ID NO: 10), isoleucine (Ile) at position 423 is replaced with leucine (Leu), and leucine (Leu) at position 530 is replaced with arginine (Arg); Isoleucine (Ile) at position 47 is threonine (Thr), Asparagine (Asn) at position 50 is serine (Ser), methionine (Met) at position 59 is threonine (Thr), and threonine (Thr) at position 252 is Mutant firefly luciferase consisting of an amino acid sequence substituted with serine (Ser)
(vi) A mutation comprising an amino acid sequence in which asparagine at position 50 is replaced by serine, isoleucine at position 423 is replaced by leucine, and leucine at position 530 is replaced by arginine in the amino acid sequence of wild-type North American firefly luciferase (SEQ ID NO: 10). Type firefly luciferase

  The luminescence intensity of each of the mutant firefly luciferases (i) to (v) is (i) 18 times, (ii) 18 times, (iii) 8 compared to the luminescence intensity of North American firefly luciferase (wild type), respectively. (Iv) 20 times and (v) 21 times (see JP 2007-97577 A). Note that the mutant luciferase modified to increase the emission intensity is not limited to the above (i) to (vi). A mutant luciferase modified so as to increase the emission intensity by appropriately selecting the position of the mutation and the amino acid to be substituted can be found and used. In particular, in the amino acid sequence of SEQ ID NO: 10, a mutation modified to increase luminescence intensity by combining amino acid substitutions at positions 47, 50, 59, 252, 423, 436, and 530 Type luciferase can be obtained.

  Mutant firefly luciferase is expressed as a recombinant protein by inserting a mutant firefly luciferase gene obtained by modifying a wild-type firefly luciferase gene into an expression vector by a known method, and introducing it into an appropriate host cell. And can be obtained by purification. The gene can be modified by methods well known to those skilled in the art, such as site-directed mutagenesis, random mutagenesis, and organic synthesis. The base sequence of the North American firefly luciferase gene (cDNA) is registered in a database (for example, EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/)) as an accession number: M15077. . Moreover, the mutant firefly luciferase described in (i) to (v) above can be produced by referring to the examples of JP-A-2007-97577.

  In addition, those skilled in the art also mutated Genji fireflies, Heike fireflies, Japanese fireflies, Japanese fireflies, light beetles, and railway insects by known methods based on the nucleotide sequences of these luciferase genes registered in known databases (see Table 1). A type luciferase can be easily obtained. Furthermore, by referring to the substituted amino acid of the mutant firefly luciferase derived from North American fireflies described in (i) to (vi) above, by replacing the amino acid at the equivalent position in the luciferase derived from other beetles, the person skilled in the art A mutant luciferase with increased luminescence intensity can be easily obtained.

[Reaction and measurement procedure (method)]
A luminescent enzyme (for example, luciferase) is added to the reaction liquid obtained in the preceding luminescent substrate releasing step, that is, the reaction liquid containing the released luminescent substrate (for example, aminoluciferin). As shown in the above reaction formula (I), ATP and divalent metal ions are required for the luciferin / luciferase luminescence reaction. For example, luciferase is dissolved in a buffer solution containing ATP and magnesium ions. It is preferable to add a luciferase solution. Specifically, for example, there is a method in which the reaction is performed at room temperature (25 ° C.) and the luminescence value is measured from 2 seconds to 10 seconds after the luciferase solution is added.

  A commercially available luminometer (luminescence measuring device) can be used for the measurement of the amount of luminescence. The manufacturer and performance are not particularly limited, but it is preferable that the apparatus can measure the relative light quantity measurement value in the range of 0 to 10 million. Specifically, specifications such as Kikkoman's Lumitester C1000 and Perkin Elmer's ARVO Light are suitable. The measurement may be performed according to the instruction manual of the device to be used.

(3) Concentration determination step The concentration determination step is a step of quantifying the β-glucan concentration in the sample based on the measurement value obtained in the light emission amount measurement step. Using a β-glucan solution with a known concentration, a calibration curve representing the relationship between the β-glucan concentration and the luminescence value at that concentration was created in advance, and the measurement sample obtained by the above luminescence measurement step was measured on this calibration curve. By applying the value, the β-glucan concentration in the sample can be determined.

2. Kit for Measuring β-Glucan Concentration The kit for measuring the concentration of β-glucan according to the present invention only needs to contain the following (a), (b), and (c) as constituent components.
(a) a reagent containing factor G that is specifically bound to and activated by β-glucan
(b) Luminescence having a structure in which a bond between a luminescent substrate and a peptide is cleaved by the action of an activated factor G or coagulase produced by the activation of factor G by binding a luminescent substrate to the peptide Synthetic substrate
(c) Luminescent enzyme The details of the above (a), (b), and (c) have already been described, and the description thereof is omitted here. The kit of the present invention may contain components other than the above (a), (b) and (c) as components. The specific kit configuration other than these is not particularly limited, and other necessary reagents, instruments, etc. may be appropriately selected to form the kit configuration. By using the kit according to the present invention, the β-glucan concentration measuring method according to the present invention can be carried out simply and quickly.

  As used herein, a “kit” is intended to be a package with a container (eg, bottle, plate, tube, dish, etc.) containing a specific material. Preferably, an instruction manual for using the material is provided. The instructions for use may be written or printed on paper or other media, or may be affixed to electronic media such as magnetic tape, computer readable discs or CD-ROMs.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these. The instruments, reagents, water, and the like used in the following examples were β-glucan-free or those containing an extremely small amount of β-glucan that did not affect the β-glucan measurement results.

[Example 1: Measurement of β-glucan by bioluminescence method]
<Method>
The Limulus reagent (LAL), β-glucan standard solution, and purified water were those attached to Glucatell (Associates of Cape Cod, Inc. (ACC)), an in vitro diagnostic agent for deep fungal infection. Benzoyl-Leu-Arg-Arg-aminoluciferin attached to Proteasome-Glo ^™ Assay Systems (Promega) was used as the luminescent synthetic substrate. Luciferase is a mutant firefly comprising an amino acid sequence in which asparagine at position 50 is substituted with serine, isoleucine at position 423 is replaced with leucine, and leucine at position 530 is replaced with arginine in the amino acid sequence of North American firefly luciferase (SEQ ID NO: 10). Luciferase was used. The mutant luciferase is produced by the present inventors in accordance with the method described in Examples of Japanese Patent Application Laid-Open No. 2007-97577.

  First, a β-glucan standard solution was diluted with purified water to prepare 6 steps in the range of 0.1 to 40 pg / mL, and this was used as a sample (see Table 1). Further, purified water not containing β-glucan was used as a blank. Subsequently, 50 μL each was taken out from each sample and blank, transferred to a reaction tube containing 50 μL Limulus reagent separately, and then mixed for several seconds with a vortex mixer. After mixing, the mixture was accurately heated with a 37 ± 1.0 ° C. incubator for 10 minutes. For detailed handling, the protocol attached to Glucatell was followed.

Next, 50 μL of 150 μM luminescent synthetic substrate (benzoyl-Leu-Arg-Arg-aminoluciferin) dissolved in 100 mM Tris-Cl (pH 8.0) containing 1 mM MgCl ₂ was added, and an incubator at 37 ± 1.0 ° C. For 5 minutes. After heating, the reaction solution was transferred to a bioluminescence measurement test tube (Lumitube, Kikkoman Corporation) and then dissolved in 100 mM Tris-Cl (pH 8.0) containing 10 ⁻⁵ M ATP and 1 mM MgCl _2. 50 μL was added and set in Lumitester C1000 (Kikkoman Corporation), and the amount of light emitted from the sample was measured. In addition, the density | concentration with respect to the reaction liquid whole quantity of the Limulus reagent used in the present Example was about 2.0 mg / mL. This amount of Limulus reagent corresponds to about 50% of the conventional amount used.

  As a comparative example, the same sample was measured by a kinetic colorimetric method using a chromogenic substrate attached to Glucatell instead of the luminescent synthetic substrate. The operating method followed the protocol attached to Glucatell.

<Result>
The results are shown in Table 1. Moreover, the graph which plotted the emitted light intensity in each sample density | concentration of Example 1 was shown in FIG. 3, and the graph which plotted the light absorbency in each sample density | concentration of a comparative example was shown in FIG. As is clear from Table 1, FIG. 3 and FIG. 4, the kinetic colorimetric method using a conventional chromogenic substrate is too low for β-glucan of less than 5 pg / mL to be quantitatively evaluated. Met. On the other hand, it was shown that the method of measuring luminescence intensity using a luminescent synthetic substrate can be measured even at a concentration of 0.1 pg / mL or less.

  The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  The present invention can be used in the medical field, in particular, in fields such as clinical examination and pharmaceutical production.

Claims (8)

A method for measuring the concentration of β-glucan contained in a sample,
Luminescence that liberates the luminescent substrate from the luminescent synthetic substrate by reacting the sample, a reagent containing factor G activated by the binding of β-glucan, and the luminescent synthetic substrate formed by binding the luminescent substrate to the peptide A substrate release step;
A luminescence measuring step in which a luminescent enzyme is allowed to act on the luminescent substrate released in the luminescent substrate releasing step and the amount of luminescence is measured; and
A concentration determination step of determining the concentration of β-glucan in the sample based on the measurement value obtained by the light emission amount measurement step.   The luminescent synthetic substrate has a structure in which the bond between the luminescent substrate and the peptide is cleaved by the action of an activated factor G or a coagulase produced by activating the factor G. The β glucan concentration measurement method according to claim 1.   The method for measuring the concentration of β-glucan according to claim 2, wherein the luminescent substrate is aminoluciferin, and the luminescent enzyme is luciferase.   The concentration of β-glucan according to claim 3, wherein the luciferase is derived from a beetle selected from the group consisting of North American fireflies, Genji fireflies, Heike fireflies, Japanese fireflies, Japanese fireflies, Japanese fireflies, Obata fireflies, light beetles and railroad insects. Measuring method.   The luciferase is a mutant luciferase, and the amino acid sequence of the wild-type luciferase is modified so that the luminescence intensity of the mutant luciferase is increased as compared with the wild-type luciferase. A method for measuring the concentration of β-glucan as described in 1. above. 6. The method for measuring β-glucan concentration according to claim 5, wherein the mutant luciferase is one selected from the group consisting of the following (i) to (vi).
(i) A mutant firefly luciferase comprising an amino acid sequence of the wild-type North American firefly luciferase shown in SEQ ID NO: 10, wherein isoleucine at position 423 is replaced with leucine and aspartic acid at position 436 is replaced with glycine
(ii) A mutant firefly luciferase comprising an amino acid sequence of the wild-type North American firefly luciferase shown in SEQ ID NO: 10, wherein isoleucine at position 423 is replaced with leucine and leucine at position 530 is replaced with arginine
(iii) A mutant firefly luciferase comprising an amino acid sequence of the wild-type North American firefly luciferase shown in SEQ ID NO: 10, wherein aspartic acid at position 436 is replaced with glycine and leucine at position 530 is replaced with arginine
(iv) In the amino acid sequence of the wild-type North American firefly luciferase shown in SEQ ID NO: 10, leucine at position 423 was substituted with leucine, aspartic acid at position 436 was substituted with glycine, and leucine at position 530 was substituted with arginine. Variant firefly luciferase consisting of amino acid sequence
(v) In the amino acid sequence of wild-type North American firefly luciferase shown in SEQ ID NO: 10, isoleucine at position 47 is threonine, asparagine at position 50 is serine, methionine at position 59 is threonine, and threonine at position 252 is serine. Mutant firefly luciferase having an amino acid sequence in which leucine at position 423 is replaced with leucine and leucine at position 530 is substituted with arginine
(vi) The amino acid sequence of wild-type North American firefly luciferase shown in SEQ ID NO: 10 consists of an amino acid sequence in which asparagine at position 50 is replaced with serine, isoleucine at position 423 is replaced with leucine, and leucine at position 530 is replaced with arginine. Mutant firefly luciferase   The reagent containing the factor G is a horseshoe crab blood cell extract component, and the protein concentration of the horseshoe crab blood cell extract component with respect to the total amount of the reaction system is 1.5 mg / mL to 3.5 mg / mL. The method for measuring the concentration of β-glucan according to any one of -6. A kit for measuring the concentration of β-glucan comprising the following (a), (b), and (c) as constituent components.
(a) a reagent containing factor G that is specifically bound to and activated by β-glucan
(b) Luminescence having a structure in which a bond between a luminescent substrate and a peptide is cleaved by the action of an activated factor G or coagulase produced by the activation of factor G by binding a luminescent substrate to the peptide Synthetic substrate
(c) Luminescent enzyme