Method for measuring endotoxin

Abstract

The present invention aims to provide a method for measuring endotoxin concentration of dialysate with higher accuracy using a calibration curve made of water, and a kit for the method. The present invention provides a method for measuring endotoxin as follows: mixing a prescribed amount of a test sample with a dry powder containing a solubilizer reagent to prepare a first reaction solution, then incubating the first reaction solution for a prescribed time, mixing the first reaction solution with a dry powder containing a luciferase bioluminescence reagent under light shielding to prepare a second reaction solution, measuring the amount of luminescence of the second reaction solution, and measuring the endotoxin concentration of the test sample, which is dialysate or water, from the amount of luminescence, the second reaction solution having a sodium ion concentration of 250 to 390 mM, a calcium ion concentration of 0.5 to 10 mM, a chloride ion concentration of 250 to 361.5 mM, and a Tris concentration of 20 to 30 mM.

Description

Technical Field

[0001] This invention relates to a method for determining endotoxins in water and dialysate using a solution reaction and bioluminescence. Background Technology

[0002] Endotoxins are lipopolysaccharides that make up the cell walls of Gram-negative bacteria and are highly potent toxins. Therefore, detecting endotoxin contamination in water, pharmaceuticals, food, and other beverages is crucial. In particular, since dialysate administered intravenously to dialysis patients is typically administered long-term, even very low concentrations of endotoxins can have serious consequences. Therefore, from a safety perspective, controlling endotoxin contamination in dialysate is extremely important.

[0003] Endotoxin detection is generally performed using a lysate reaction with a lysate reagent prepared from amybocyte lysate (a component of horseshoe crab blood cells). This lysate reagent contains factors C and B, as well as a pro-clotting enzyme, all derived from horseshoe crabs. When the lysate reagent is mixed into a sample containing endotoxin, factor C is activated by the endotoxin, factor B is activated by the active factor C, and the pro-clotting enzyme is activated by the active factor B to generate a clotting enzyme. The activity of this clotting enzyme is then used as an indicator to determine the amount of endotoxin.

[0004] Traditionally, coagulase activity has been determined using turbidimetry to measure the amount of insoluble coagulin gel produced by the hydrolysis of coagulogen; alternatively, a synthetic matrix, formed by the binding of chromogenic pigments to peptides via the digestion sites of the coagulase, is pre-added to the reaction system, followed by colorimetric determination of the amount of chromogenic pigment released from the synthetic matrix by the coagulase digestion. In recent years, a method using bioluminescence has been developed as a more sensitive and rapid method for determining coagulase activity (Non-Patent Literature 1). This method uses a bioluminescent matrix modified with peptides as the coagulase matrix, luciferase, and adenosine triphosphate (ATP). The bioluminescent matrix released by the coagulase digestion emits light via ATP and luciferase. Based on the amount of light emitted, the coagulase activity can be determined, thereby allowing the determination of the amount of endotoxin.

[0005] Firefly luciferase is widely used in luciferin-luciferase luminescence reaction. Wild-type firefly luciferase is affected by sodium chloride, and the enzyme reaction is inhibited in a high concentration of sodium chloride environment, and the luminescence intensity is reduced to about 20 to 50% compared to an aqueous solution not containing sodium chloride. Therefore, in the method described in Non-Patent Literature 1, when the endotoxin concentration of a dialysate having a high sodium chloride concentration of about 130 to 150 mM is measured, a salt-resistant luciferase is used (Patent Literature 1).

[0006] Prior art documents

[0007] Patent Literature

[0008] Patent Literature 1: Japanese Patent Application Publication No. 2017-225372

[0009] Non-Patent Literature

[0010] Non-Patent Literature 1: Oda, Yu, et al. "Endotoxin and Natural Immunity Research", 2019, Vol. 22, pp. 17-20. SUMMARY

[0011] Problems to be solved by the invention

[0012] By using a salt-resistant luciferase, although it is possible to measure the endotoxin concentration in a dialysate having a high sodium chloride concentration, the effect of inhibition by the dialysate component cannot be completely removed. Therefore, in a measurement method using bioluminescence, the amount of luminescence in a dialysate having an endotoxin concentration is smaller than that in water having the same endotoxin concentration. Therefore, when a calibration curve made of water is used, the endotoxin concentration in a dialysate is measured as a lower concentration than the actual endotoxin concentration. Thus, in order to more accurately measure the endotoxin concentration of a dialysate, it is necessary to use a calibration curve made of a dialysate, not water, but it is cumbersome to use two kinds of calibration curves.

[0013] Therefore, an object of the present application is to provide a method for more accurately measuring the endotoxin concentration of a dialysate using a calibration curve made of water, and a kit for the method.

[0014] Technical solution to the problem

[0015] In order to achieve the above object, the present inventors have conducted intensive studies, and as a result, have found that in a method for measuring endotoxin using a solubilized substance reaction and a bioluminescence method, by adjusting the salt concentration in the reaction solution at the time of luciferase reaction within a specific range, while sufficiently ensuring the detection sensitivity of endotoxin, the difference in the amount of luminescence when the test sample is water and when the test sample is a dialysate can be reduced, thereby completing the present application.

[0016] That is, the application of the present application is the following 1st to 13th aspects.

[0017] The first aspect of the present application is a method for measuring endotoxin, which is a method for measuring endotoxin, comprising: a lysate reaction step of mixing a predetermined amount of a test sample with a dry powder containing a lysate reagent to prepare a first reaction solution, and then incubating the first reaction solution for a predetermined time; a luciferase reaction step of mixing the first reaction solution after the lysate reaction step with a dry powder containing a luciferase bioluminescence reagent in the dark to prepare a second reaction solution, and measuring the amount of luminescence of the second reaction solution; and an endotoxin measurement step of measuring the endotoxin concentration of the test sample, which is dialysate or water, based on the amount of luminescence obtained in the luciferase reaction step, wherein the lysate reagent contains a C factor, a B factor, and a pre-activated coagulation enzyme, the luciferase bioluminescence reagent contains a luminescence synthetic substrate, a salt-resistant luciferase, and ATP, the luminescence synthetic substrate is a substance that releases a luminescent substrate by digestion with an activated C factor, an activated B factor, or a coagulation enzyme obtained by conversion of the pre-activated coagulation enzyme, and in the luciferase reaction step, the second reaction solution has a sodium ion concentration of 250 to 390 mM, a calcium ion concentration of 0.5 to 10 mM, a chloride ion concentration of 250 to 361.5 mM, and a Tris concentration of 20 to 30 mM.

[0018] The second aspect of the present application is the method for measuring endotoxin according to the first aspect, wherein in the luciferase reaction step, the second reaction solution has a pH of 7.7 to 8.5.

[0019] The third aspect of the present application is the method for measuring endotoxin according to the first aspect or the second aspect, wherein in the luciferase reaction step, the second reaction solution has a magnesium ion concentration of 10 to 20 mM and a carbonate ion concentration of 1 to 5 mM.

[0020] The fourth aspect of the present application is a method for measuring endotoxin, which is a method for measuring endotoxin, wherein a predetermined amount of a test sample is mixed with a lysate reagent and a dry powder containing a luciferase bioluminescence reagent to prepare a reaction solution, the reaction solution is incubated for a predetermined time in the dark, and then the endotoxin concentration of the test sample is measured based on the amount of luminescence of the reaction solution, the test sample is dialysate or water, the lysate reagent contains a C factor, a B factor, and a pre-activated coagulation enzyme, the luciferase bioluminescence reagent contains a luminescence synthetic substrate, a salt-resistant luciferase, and ATP, the luminescence synthetic substrate is a substance that releases a luminescent substrate by digestion with an activated C factor, an activated B factor, or a coagulation enzyme obtained by conversion of the pre-activated coagulation enzyme, and the reaction solution has a sodium ion concentration of 250 to 390 mM, a calcium ion concentration of 0.5 to 10 mM, a chloride ion concentration of 250 to 361.5 mM, and a Tris concentration of 20 to 30 mM.

[0021] The fifth aspect of the present application is the endotoxin measurement method according to the fourth aspect, wherein the pH of the reaction solution is 7.7 to 8.5.

[0022] The sixth aspect of the present application is the endotoxin measurement method according to the fourth aspect or the fifth aspect, wherein the magnesium ion concentration of the reaction solution is 10 to 20 mM and the carbonate ion concentration is 1 to 5 mM.

[0023] The seventh aspect of the present application is the endotoxin measurement method according to any one of the first aspect to the sixth aspect, wherein the lysate reagent is a component extracted from limulus hemocytes.

[0024] The eighth aspect of the present application is the endotoxin measurement method according to any one of the first aspect to the seventh aspect, wherein, in the endotoxin measurement step, the endotoxin concentration of the test sample is measured based on a calibration curve prepared using a dilution series of water containing endotoxins of known concentration and the amount of luminescence obtained in the luciferase reaction step.

[0025] The ninth aspect of the present application is a kit for endotoxin measurement, which is a kit for measuring the endotoxin concentration of dialysate or water, and contains: a first container containing a dry powder containing a lysate reagent; and a second container containing a dry powder containing a luciferase bioluminescence reagent, wherein a prescribed amount of dialysate or water is mixed in the first container to prepare a first reaction solution, and the total amount of the obtained first reaction solution is then mixed into the second container to obtain a second reaction solution, and the sodium ion concentration of the obtained second reaction solution is 250 to 390 mM, the calcium ion concentration is 0.5 to 10 mM, the chloride ion concentration is 250 to 361.5 mM, and the Tris concentration is 20 to 30 mM.

[0026] The tenth aspect of the present application is the kit for endotoxin measurement according to the ninth aspect, wherein the pH of the second reaction solution is 7.7 to 8.5, the magnesium ion concentration is 10 to 20 mM, and the carbonate ion concentration is 1 to 5 mM.

[0027] The eleventh aspect of the present application is a kit for endotoxin measurement, which is a kit for measuring the endotoxin concentration of dialysate or water, and contains a container containing a dry powder containing a lysate reagent and a luciferase bioluminescence reagent, wherein the sodium ion concentration of a reaction solution prepared by mixing a prescribed amount of dialysate or water in the container is 250 to 390 mM, the calcium ion concentration is 0.5 to 10 mM, the chloride ion concentration is 250 to 361.5 mM, and the Tris concentration is 20 to 30 mM.

[0028] The endotoxin assay kit according to the 11th aspect of the present application, wherein the reaction solution has a pH of 7.7 to 8.5, a magnesium ion concentration of 10 to 20 mM, and a carbonate ion concentration of 1 to 5 mM.

[0029] The endotoxin assay kit according to any one of the 9th to 12th aspects of the present application, which is used for the endotoxin assay method according to any one of the 1st to 8th aspects of the present application.

[0030] Effects of the Invention

[0031] The endotoxin assay method according to the present application can more accurately measure the endotoxin concentration in dialysate even when a calibration curve prepared using water is used.

[0032] In addition, the endotoxin assay method can be more simply and easily performed by using the endotoxin assay kit according to the present application. DETAILED DESCRIPTION

[0033] The endotoxin assay method according to the present application is a method for measuring the endotoxin concentration in water or dialysate, and utilizes a lysate reaction and a bioluminescence method. In the bioluminescence, a photosynthetic substrate, luciferase, and ATP are used, and the photosynthetic substrate is a luminescent photosynthetic substrate in which a luminescent substrate is liberated by protease activity of active C factor, active B factor, or coagulase produced by the lysate reaction. In this method, when the test sample contains endotoxin, the C factor is activated by the endotoxin, the B factor is activated by the active C factor, and the pre-coagulase is converted into coagulase by the active B factor. The luminescent photosynthetic substrate is decomposed by the protease activity of the produced active C factor, active B factor, or coagulase. As a result, the liberated luminescent substrate (fluorescein or a derivative thereof) bioluminesces by the luciferase and ATP. The more the amount of endotoxin in the test sample, the more the amount of coagulase produced, and as a result, the amount of luminescence produced by the luciferase increases, and thus the S / N (Signal / Noise) is excellent compared to the turbidimetry in which the amount of transmitted light is observed.

[0034] Specifically, water or dialysate is used as the test sample, and the following lysate reaction step, luciferase reaction step, and endotoxin assay step are performed.

[0035] The lysate reaction step mixes a predetermined amount of the test sample with a dry powder containing a lysate reagent to prepare a first reaction solution, and then incubates the first reaction solution for a predetermined time.

[0036] a luciferase reaction step of mixing the first reaction solution with a dry powder containing a luciferase bioluminescent reagent to prepare a second reaction solution, and measuring the amount of luminescence of the second reaction solution, under light shielding, after the above-described lysate reaction step.

[0037] an endotoxin measurement step of measuring the endotoxin concentration of the test sample based on the amount of luminescence obtained in the above-described luciferase reaction step.

[0038] The lysate reagent used in the present application contains a C factor, a B factor, and a pre- coagulase. As the lysate reagent used in the present application, there is no particular limitation as long as it is a reagent generally used in the reaction. For example, a dry powder of a lysate reagent formed from components extracted from limulus hemocytes can be used as the dry powder containing a lysate reagent in the present application. The lysate reagent can be prepared from limulus hemocytes by a conventional method.

[0039] In the present application and the specification of the present application, "limulus" refers to, for example, limulus belonging to the genus Tachypleus such as Tachypleus tridentatus or Tachypleus gigas, limulus belonging to the genus Limulus such as Limulus polyphemus, and limulus belonging to the genus Carcinoscorpius such as Carcinoscorpius rotundicauda.

[0040] The C factor, the B factor, and the pre-coagulase contained in the lysate reagent used in the present application can also be recombinant proteins. As the "C factor composed of a recombinant protein", it can be a recombinant protein composed of the same amino acid sequence as a C factor purified from a wild limulus hemocyte extract (wild-type C factor), it can be a mutant protein (mutant) in which various mutations are introduced into the wild-type C factor, and it can be a variant in which other peptides or proteins are fused to the N terminus or the C terminus of the wild-type C factor or the mutant. Similarly, as the "B factor composed of a recombinant protein", it can be a recombinant protein composed of the same amino acid sequence as a B factor purified from a wild limulus hemocyte extract (wild-type B factor), it can be a mutant in which various mutations are introduced into the wild-type B factor, and it can be a variant in which other peptides or proteins are fused to the N terminus or the C terminus of the wild-type B factor or the mutant. As the "pre-coagulase composed of a recombinant protein", it can be a recombinant protein composed of the same amino acid sequence as a pre-coagulase purified from a wild limulus hemocyte extract (wild-type pre-coagulase), it can be a mutant in which various mutations are introduced into the wild-type pre-coagulase, and it can be a variant in which other peptides or proteins are fused to the N terminus or the C terminus of the wild-type pre-coagulase or the mutant.

[0041] As the mutant, a variant that does not impair the respective activities is acceptable. For example, if a known variant exists, it is acceptable as it is, and a variant obtained by reengineering the wild type is also acceptable. In the case of reengineering, for example, a variant obtained by designing the C factor, the B factor, or the prekallikrein activity so that the enzymatic activity is higher than that of the wild type is preferred. For example, an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added in the amino acid sequence of the wild type can be mentioned. Here, "in which one or several amino acids are deleted, inserted, substituted, or added" means that a polypeptide is mutated by a known polypeptide mutation method such as site-directed mutagenesis so that one or several amino acids (preferably 10 or less, more preferably 7 or less, and most preferably 5 or less) are deleted, inserted, substituted, or added. In addition, the sequence identity of the amino acid sequence of the wild type and the mutant is preferably 70% or more, more preferably 80% or more, further preferably 90% or more, and most preferably 95% or more.

[0042] Note that the sequence identity (homology) between the amino acid sequences is obtained by aligning the two amino acid sequences while inserting gaps in the portions corresponding to the insertions and deletions so that the corresponding amino acids are maximally identical, and thereby obtaining the proportion of the identical amino acids in the obtained alignment with respect to the entire amino acid sequences excluding the gaps. The sequence identity between the amino acid sequences can be obtained using various homology search software known in the art.

[0043] As the peptide or protein fused to the C factor, the B factor, and the prekallikrein in the wild type or the mutant, there is no particular limitation as long as the respective activities are not impaired. As the peptide and the like, for example, a tag such as a histidine tag, an HA (hemagglutinin) tag, a Myc tag, and a Flag tag, which are widely used for the expression and purification of recombinant proteins, can be mentioned.

[0044] The C factor, the B factor, and the prekallikrein composed of the recombinant proteins are obtained, for example, by introducing the genes encoding the respective proteins into cells as a host, allowing them to be expressed in the obtained transformants, and purifying them. As the cells as a host, Escherichia coli, yeast, insect cells, mammalian cells, and cell-free expression systems using extracts of these cells can be used. In addition, the production of the transformants by gene introduction, the culture of the obtained transformants, the expression of the recombinant proteins, and the purification of the recombinant proteins from the culture can be performed by conventional methods.

[0045] The genes encoding wild-type C factor, B factor, and pre- coagulase are known from the literature and databases (e.g., EMBL Nucleotide Sequence Database (http: / / www.ebi.ac.uk / embl / )). Thus, for example, the gene encoding B factor is described in J. Biol. Chem. 268, 21384-21388 (1993), the gene encoding C factor is described in J. Biol. Chem. 266, 6554-6561 (1991), the gene encoding pre-coagulase is described in the base sequence information described in International Publication No. 2008 / 004674, and the like, and can be amplified and cloned by PCR and the like. Furthermore, the genes encoding mutant C factor, B factor, and pre-coagulase can be produced by chemical synthesis based on the base sequence encoding a protein composed of the target amino acid sequence, or can be produced by appropriately modifying the respective wild-type genes. Furthermore, the genes encoding C factor, B factor, and pre-coagulase introduced into the transformant used in the present application are preferably genes in which the degenerate codons are modified to codons that are more frequently used in the host.

[0046] Furthermore, in the present application, a dry powder of a commercially available lysate reagent can also be used.

[0047] The ratio of the content of B factor to C factor (B factor / C factor) in the lysate reagent used in the present application is preferably 10 / 1 to 0.1 / 1, and more preferably 2 / 1 to 0.5 / 1, in terms of mass ratio. Furthermore, the ratio of the content of pre-coagulase to C factor (pre-coagulase / C factor) in the lysate reagent is preferably 10 / 1 to 0.1 / 1, and more preferably 2 / 1 to 0.5 / 1, in terms of mass ratio.

[0048] The lysate reagent used in the present application can appropriately contain other components, such as salts, pH adjusting agents, and the like, which are not derived from the extract of Limulus hemocytes.

[0049] The luciferase bioluminescence reagent used in the present application contains a light-emitting synthetic substrate, salt-resistant luciferase, and ATP. A dry powder of the luciferase bioluminescence reagent used in the present application is obtained by preparing a solution containing a light-emitting synthetic substrate, salt-resistant luciferase, and ATP, and drying the solution by freeze-drying or the like.

[0050] The light-emitting synthetic substrate used in the present application is a substance that releases a light-emitting substrate by digestion with active C factor, active B factor, or coagulase generated by the lysate reaction. As the light-emitting substrate, there is no particular limitation as long as it is a substance that bioluminesces by luciferase and ATP, and for example, firefly luciferin or a derivative thereof, and the like can be used. As the derivative of luciferin, for example, aminoluciferin can be mentioned.

[0051] As the luminescent synthetic substrate used in the present application, for example, a linker of a luminescent substrate (fluorescein or a derivative thereof) and a peptide can be given, and the linking site of the luminescent substrate and the peptide is an amino acid sequence which is cleaved by protease activity of at least any one of the activated C factor, the activated B factor, and the coagulation enzyme. Among them, a substance in which the amino group of the aminofluorescein forms an amide bond with the carboxyl group of the peptide, and the amide bond is cleaved by protease activity of at least any one of the activated C factor, the activated B factor, and the coagulation enzyme is preferable. The number of amino acid residues and the amino acid sequence of the peptide are not limited, and from the viewpoints of specificity, cost of synthesis, ease of handling, and the like, the number of amino acid residues is preferably 2 to 10. In addition, the kind of the peptide can be alone or a combination of two or more.

[0052] Specifically, as the peptide having the recognition sequence of the coagulation enzyme, 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 given. The N-terminal of the peptide can be protected by a protecting group. As the protecting group, any group which is generally used in the field can be used without limitation. Specifically, for example, N-succinyl, t-butoxycarbonyl, benzoyl, p-toluenesulfonyl, and the like can be given.

[0053] The luminescent synthetic substrate used in the present application can be commercially available or synthesized. As the commercially available luminescent synthetic substrate, for example, "Proteasome-Glo Assay Systems" (benzoyl-Leu-Arg-Arg-aminofluorescein) which is commercially available from Promega Corporation can be given. As the method of synthesis, for example, the method described in Japanese Patent Application Laid-Open No. 2005-530485 (International Publication No. WO 2003 / 066611) can be given. TM Assay Systems" accompanying the commercially available luminescent synthetic substrate (benzoyl-Leu-Arg-Arg-aminofluorescein) which is commercially available from Promega Corporation. As the method of synthesis, for example, the method described in Japanese Patent Application Laid-Open No. 2005-530485 (International Publication No. WO 2003 / 066611) can be given.

[0054] The salt-resistant luciferase used in the present application is a mutant luciferase which is less susceptible to the inhibition of luminescence caused by sodium chloride, as compared with the wild-type luciferase. The salt-resistant luciferase is a mutant luciferase in which a mutation is introduced into the wild-type luciferase, and the luminescence intensity in a 0.9 mass% sodium chloride solution is 50% or more of the luminescence intensity in a solution not containing sodium chloride. The residual activity of luciferase activity of the salt-resistant luciferase used in the present application is preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, particularly preferably 80% or more, and most preferably 90% or more.

[0055] Note that the "residual activity (%) of luciferase" is the relative value of the luminescence intensity in a 0.9 mass% sodium chloride solution, when the luminescence intensity in a solution not containing sodium chloride is taken as 100% ([luminescence intensity in a 0.9 mass% sodium chloride solution] / [luminescence intensity in a solution not containing sodium chloride] x 100) (%). Note that the "solution not containing sodium chloride" in the luciferin-luciferase luminescence reaction is a reaction solution prepared without mixing sodium chloride. The "solution not containing sodium chloride" and the "0.9 mass% sodium chloride solution" for the calculation of the residual activity are identical in composition except for sodium chloride, and the reaction conditions such as the reaction temperature and the reaction time are also identical.

[0056] The salt-resistant luciferase used in the present application is preferably a mutant of the wild-type luciferase derived from a beetle, and particularly preferably a mutant beetle luciferase having at least one mutation selected from one or more of the following (a), (b), (c), and (d) in the amino acid sequence encoding the wild-type beetle luciferase (Patent Document 1).

[0057] (a) the amino acid corresponding to the valine at position 288 in the amino acid sequence of the wild-type North American firefly luciferase is mutated to isoleucine, leucine, or phenylalanine.

[0058] (b) the amino acid corresponding to the leucine at position 376 in the amino acid sequence of the wild-type North American firefly luciferase is mutated to proline.

[0059] (c) the amino acid corresponding to the glutamic acid at position 455 in the amino acid sequence of the wild-type North American firefly luciferase is mutated to valine, alanine, serine, leucine, isoleucine, or phenylalanine.

[0060] (d) the amino acid corresponding to the glutamic acid at position 488 in the amino acid sequence of the wild-type North American firefly luciferase is mutated to valine, alanine, serine, leucine, isoleucine, or phenylalanine.

[0061] In the present application and the specification of the present application, "wild-type beetle luciferase" refers to, for example, Photinus pyralis luciferase (SEQ ID NO: 8), Luciola lateral is luciferase (SEQ ID NO: 9), Luciola cruciata luciferase (SEQ ID NO: 10), Luciola minorelica luciferase, Lampyris noctiluca luciferase, Pyrophorus plagiophthalamus luciferase (SEQ ID NO: 11), and the like. Note that the amino acid sequences of various wild-type beetle luciferases can be searched for in databases (for example, EMBL-EBI Database (http: / / www.ebi.ac.uk / queries / )).

[0062] When the wild-type beetle luciferase is not Photinus pyralis luciferase, the "amino acid corresponding to the Xth amino acid in the amino acid sequence of Photinus pyralis luciferase" in the amino acid sequence of the wild-type beetle luciferase refers to the amino acid located at a position corresponding to the "Xth amino acid in the amino acid sequence of Photinus pyralis luciferase" when the amino acid sequences of the wild-type beetle luciferase and Photinus pyralis luciferase are aligned in such a manner that the homology (sequence identity) is maximized, using amino acid sequence homology analysis software (for example, "Micro Genie", manufactured by Beckman Coulter), or the like.

[0063] Specifically, the amino acid corresponding to the valine at position 288 in the amino acid sequence of wild-type Photinus pyralis luciferase corresponds to the valine at position 290 in the amino acid sequence of wild-type Luciola cruciata luciferase, the valine at position 290 in the amino acid sequence of wild-type Luciola iturni luciferase, the valine at position 285 in the amino acid sequence of wild-type Luciola luctuosa luciferase, respectively. The leucine at position 376 in the amino acid sequence of wild-type Photinus pyralis luciferase corresponds to the leucine at position 378 in the amino acid sequence of wild-type Luciola cruciata luciferase, the leucine at position 378 in the amino acid sequence of wild-type Luciola iturni luciferase, respectively. The glutamic acid at position 455 in the amino acid sequence of wild-type Photinus pyralis luciferase corresponds to the glutamic acid at position 457 in the amino acid sequence of wild-type Luciola cruciata luciferase, the glutamic acid at position 457 in the amino acid sequence of wild-type Luciola iturni luciferase, the glutamic acid at position 452 in the amino acid sequence of wild-type Luciola luctuosa luciferase, respectively. The glutamic acid at position 488 in the amino acid sequence of wild-type Photinus pyralis luciferase corresponds to the glutamic acid at position 490 in the amino acid sequence of wild-type Luciola cruciata luciferase, the glutamic acid at position 490 in the amino acid sequence of wild-type Luciola iturni luciferase, the glutamic acid at position 489 in the amino acid sequence of wild-type Luciola luctuosa luciferase, respectively.

[0064] When the salt-resistant luciferase used in the present application has the mutation of (a) described above, as the mutation, the amino acid corresponding to the valine at position 288 in the amino acid sequence of wild-type Photinus pyralis luciferase is preferably mutated to isoleucine or leucine, more preferably mutated to isoleucine.

[0065] When the salt-resistant luciferase used in the present application has the mutation of (c) described above, as the mutation, the amino acid corresponding to the glutamic acid at position 455 in the amino acid sequence of wild-type Photinus pyralis luciferase is preferably mutated to valine, leucine, isoleucine or alanine, more preferably mutated to valine.

[0066] When the salt-resistant luciferase used in the present application has the mutation of (d) described above, as the mutation, the amino acid corresponding to the glutamic acid at position 488 in the amino acid sequence of wild-type Photinus pyralis luciferase is preferably mutated to valine, leucine, isoleucine or alanine, more preferably mutated to valine.

[0067] The salt-resistant luciferase used in the present application can have only one of the mutations of (a), (b), (c), and (d) above, or two or more of them in combination. Among them, since the luminescent intensity produced by the luciferin-luciferase luminescence reaction in a 0.9 mass% sodium chloride solution is greater than that of the wild-type beetle luciferase, in the mutations of (a), (b), (c), and (d) above, a mutant beetle luciferase having only the mutation of (a) above, a mutant beetle luciferase having only the mutation of (b) above, a mutant beetle luciferase having only the mutation of (d) above, a mutant beetle luciferase having the mutations of (a) and (b) above, a mutant beetle luciferase having the mutations of (a) and (d) above, or a mutant beetle luciferase having the mutations of (a) and (b) and (d) above is preferred, and further since the residual activity is also high, in the mutations of (a), (b), (c), and (d) above, a mutant beetle luciferase having only the mutation of (a) above, a mutant beetle luciferase having only the mutation of (d) above, a mutant beetle luciferase having the mutations of (a) and (b) above, a mutant beetle luciferase having the mutations of (a) and (d) above, or a mutant beetle luciferase having the mutations of (a) and (b) and (d) above is more preferred.

[0068] As the salt-resistant luciferase used in the present application, for example, a mutant beetle luciferase in which the amino acid corresponding to the valine at position 288 in the amino acid sequence of wild-type North American firefly luciferase is substituted with isoleucine, a mutant beetle luciferase in which the amino acid corresponding to the leucine at position 376 in the amino acid sequence of wild-type North American firefly luciferase is substituted with proline, a mutant beetle luciferase in which the amino acid corresponding to the glutamic acid at position 455 in the amino acid sequence of wild-type North American firefly luciferase is substituted with valine, a mutant beetle luciferase in which the amino acid corresponding to the glutamic acid at position 488 in the amino acid sequence of wild-type North American firefly luciferase is substituted with valine, a mutant beetle luciferase in which the amino acid corresponding to the valine at position 288 in the amino acid sequence of wild-type North American firefly luciferase is substituted with isoleucine and the amino acid corresponding to the leucine at position 376 is substituted with proline, a mutant beetle luciferase in which the amino acid corresponding to the valine at position 288 in the amino acid sequence of wild-type North American firefly luciferase is substituted with isoleucine and the amino acid corresponding to the glutamic acid at position 488 is substituted with valine, a mutant beetle luciferase in which the amino acid corresponding to the valine at position 288 in the amino acid sequence of wild-type North American firefly luciferase is substituted with isoleucine, the amino acid corresponding to the leucine at position 376 is substituted with proline, and the amino acid corresponding to the glutamic acid at position 488 is substituted with valine, and the like can be mentioned.

[0069] Note that the mutant beetle luciferase having at least one mutation selected from one or more of the above (a), (b), (c), and (d) in the amino acid sequence of the wild-type beetle luciferase can be synthesized by the method described in Patent Literature 1.

[0070] The amount of the light-emitting synthetic substrate in the dried powder of the luciferase bioluminescent reagent used in the present application is preferably an amount in which the concentration of the light-emitting synthetic substrate in the second reaction solution prepared in the luciferase reaction step is 0.1 to 100 μM, and more preferably an amount in which the concentration of the light-emitting synthetic substrate in the second reaction solution prepared in the luciferase reaction step is 1 to 20 μM. If the concentration of the light-emitting synthetic substrate is equal to or higher than the lower limit value described above, sufficient detection sensitivity can be obtained, whereas if the concentration of the light-emitting synthetic substrate is equal to or lower than the upper limit value described above, material costs can be suppressed.

[0071] The amount of the salt-resistant luciferase in the dried powder of the luciferase bioluminescent reagent used in the present application is preferably an amount in which the concentration of the salt-resistant luciferase in the second reaction solution prepared in the luciferase reaction step is 1 ng / μL to 10 μg / μL, and more preferably an amount in which the concentration of the salt-resistant luciferase in the second reaction solution prepared in the luciferase reaction step is 10 ng / μL to 100 ng / μL. If the concentration of the salt-resistant luciferase is equal to or higher than the lower limit value described above, sufficient detection sensitivity can be obtained, whereas if the concentration of the salt-resistant luciferase is equal to or lower than the upper limit value described above, material costs can be suppressed.

[0072] The amount of the ATP in the dried powder of the luciferase bioluminescent reagent used in the present application is preferably an amount in which the concentration of the ATP in the second reaction solution prepared in the luciferase reaction step is 10 -7 M to 10 -3 M, and more preferably an amount in which the concentration of the ATP in the second reaction solution prepared in the luciferase reaction step is 10 -6 M to 10 -4 M. If the concentration of the ATP is equal to or higher than the lower limit value described above, ATP deficiency in the bioluminescent reaction can be prevented, whereas if the concentration of the ATP is equal to or lower than the upper limit value described above, material costs can be suppressed and hindrance of the bioluminescent reaction due to excess ATP can be prevented.

[0073] In the present application, first, a prescribed amount of a test sample (water or dialysate) is mixed with a dry powder containing a solubilizer reagent to prepare a first reaction solution, and then the first reaction solution is incubated for a prescribed time (solubilizer reaction step). After the test sample and the dry powder containing the solubilizer reagent are mixed, the resulting first reaction solution is preferably stirred. The amount of the test sample mixed with the dry powder containing the solubilizer reagent is not particularly limited, and can be, for example, 10 μL to 1 mL, preferably 100 to 800 μL, and more preferably 100 to 500 μL. The temperature and time for incubating the first reaction solution are not particularly limited as long as they are a temperature and time at which a solubilizer reaction can be performed, and can be, for example, 20 to 40°C for 5 to 40 minutes, and preferably 37°C for 10 to 30 minutes.

[0074] Next, the first reaction solution is mixed with a dry powder containing a luciferase bioluminescence reagent under light shielding to prepare a second reaction solution, and the amount of luminescence of the second reaction solution is measured (luciferase reaction step). After the first reaction solution is mixed with the dry powder containing the luciferase bioluminescence reagent, the resulting second reaction solution is preferably stirred. The reaction temperature of the bioluminescence reaction, that is, the temperature before the amount of luminescence of the prepared second reaction solution is measured, is not particularly limited as long as it is a temperature at which a bioluminescence reaction can be performed, and can be, for example, room temperature to 40°C, and preferably 20 to 40°C. Furthermore, the reaction time of the bioluminescence reaction, that is, the time from when the second reaction solution is prepared until the amount of luminescence of the reaction solution is measured, is not particularly limited, and can be, for example, 0 seconds to 2 minutes, and preferably 0 to 30 seconds. Note that a reaction time of 0 seconds means that the amount of luminescence is measured immediately after the first reaction solution is mixed with the dry powder containing the luciferase bioluminescence reagent.

[0075] The amount of luminescence of the second reaction solution can be detected, for example, by a photomultiplier (PMT). The measurement time of the amount of luminescence is not particularly limited, and can be, for example, 10 to 60 seconds. In addition to this, a commercially available bioluminescence measuring device can be used to measure the amount of luminescence of the second reaction solution. As the bioluminescence measuring device, for example, "Lumitester (registered trademark) C1000" (manufactured by Wako Pure Chemical Industries, Ltd.), and the like can be given.

[0076] The solubilizer reaction step and the luciferase reaction step in the present application can be measured, for example, by using a sample liquid analysis system described in Japanese Patent No. 5979318, and a commercially available endotoxin meter "Luminutes (registered trademark) -ET" (manufactured by Toa DKK Corporation).

[0077] Finally, the endotoxin concentration of the test sample is determined from the amount of luminescence obtained in the luciferase reaction step (endotoxin determination step). Specifically, for a dilution series of an endotoxin standard of known concentration, the amount of luminescence is similarly determined, and a calibration curve showing the relationship between the endotoxin concentration and the amount of luminescence is prepared. From this calibration curve and the amount of luminescence obtained for the test sample in the luciferase reaction step, the endotoxin concentration of the test sample is calculated. The amount of luminescence obtained in the luciferase reaction depends on the amount of active C factor, active B factor, or coagulase produced by the lysate reaction and the amount of luminescence substrate (luciferin or its derivative) produced in the luciferase reaction. Therefore, for the dilution series of the endotoxin standard used in the preparation of the calibration curve and the test sample for which the endotoxin concentration is calculated using the prepared calibration curve, it is preferable that the amount of the test sample used for the reaction, the temperature and time of incubation of the first reaction solution, and the temperature and time of incubation of the second reaction solution are determined under the same conditions.

[0078] Even the salt-resistant luciferase is affected by the concentration of sodium chloride in the reaction solution. That is, the higher the concentration of sodium chloride in the second reaction solution, the lower the amount of luminescence produced in the luciferase reaction. Since the second reaction solution contains sodium chloride from the test sample, the concentration of sodium chloride in the second reaction solution differs when the test sample is water and when the test sample is dialysate. Therefore, even if the endotoxin concentrations are the same, the amount of luminescence obtained when dialysate is used as the test sample is less than the amount of luminescence obtained when water is used as the test sample.

[0079] In the present application, in either case where the test sample is water and where the test sample is dialysate, the concentration of each ion and the Tris concentration of the second reaction solution are adjusted to be within a certain range, thereby suppressing the difference in the amount of luminescence of the second reaction solution when the test sample is water and when the test sample is dialysate. Therefore, in the present application, the endotoxin concentration of dialysate can be determined with high precision using a calibration curve prepared using a dilution series of an endotoxin standard of known concentration dissolved in water.

[0080] In the present application, in either case where the test sample is water and where the test sample is dialysate, the concentration of sodium ions in the second reaction solution is adjusted to be 250 to 390 mM, the concentration of calcium ions is adjusted to be 0.5 to 10 mM, the concentration of chloride ions is adjusted to be 250 to 361.5 mM, and the Tris concentration is adjusted to be 20 to 30 mM. For example, when the concentration of sodium chloride is 250 to 390 mM, the difference in the amount of luminescence due to the difference in the concentration of sodium chloride is small. Therefore, if the concentration of sodium chloride of the second reaction solution prepared when the test sample is water and the concentration of sodium chloride of the second reaction solution prepared when the test sample is dialysate are both within the range of 250 to 390 mM, the endotoxin concentration in dialysate can be determined with high precision even if a calibration curve prepared using water is used. The same applies to calcium chloride and Tris.

[0081] In the present application, in either case where the test sample is water and where the test sample is dialysate, the magnesium ion concentration of the second reaction solution is preferably adjusted to 10 to 20 mM, and the carbonate ion concentration is preferably adjusted to 1 to 5 mM. If the magnesium ion concentration of the second reaction solution prepared when the test sample is water and the magnesium ion concentration of the second reaction solution prepared when the test sample is dialysate are both within the range of 10 to 20 mM, the difference in the amount of luminescence due to the difference in the concentration of magnesium ions can be further reduced. The same applies to the carbonate ion, and if the carbonate ion concentration of the second reaction solution prepared when the test sample is water and the carbonate ion concentration of the second reaction solution prepared when the test sample is dialysate are both within the range of 1 to 5 mM, the difference in the amount of luminescence due to the difference in the concentration of carbonate ions can be further reduced.

[0082] In the present application, in either case where the test sample is water and where the test sample is dialysate, the pH of the second reaction solution is preferably adjusted to 7.7 to 8.5. If the pH is 7.7 to 8.5, the amount of luminescence is greater even if the concentration of endotoxins is the same, and the detection sensitivity of endotoxins can be further improved.

[0083] The concentrations of the respective ions and the Tris concentration, and the pH of the second reaction solution can be adjusted by adjusting the concentrations of the respective ions and the Tris concentration in the dry powder of the lysate reagent used in the lysate reaction step and the dry powder of the luciferase bioluminescent reagent used in the luciferase reaction step. For example, in the case where the concentrations of sodium salt, calcium salt, magnesium salt, chloride salt, carbonate salt, and Tris contained in the dry powder of the lysate reagent and the dry powder of the luciferase bioluminescent reagent are measured, and the first reaction solution and the second reaction solution are prepared using a prescribed amount of water, the amount that is insufficient for the concentrations of the respective ions and the Tris concentration of the second reaction solution to be above the lower limit value of the prescribed concentration range is investigated. This insufficient amount is added to either one of the dry powder of the lysate reagent and the dry powder of the luciferase bioluminescent reagent. Alternatively, the addition can be performed by dividing the two, the dry powder of the lysate reagent and the dry powder of the luciferase bioluminescent reagent. At this time, in the case where the first reaction solution and the second reaction solution are prepared using a prescribed amount of dialysate, attention is paid so that the concentrations of the respective ions and the Tris concentration of the second reaction solution do not exceed the upper limit value of the prescribed concentration range. For example, by investigating the concentrations of the respective ions of the dialysate as the test sample in advance, the amounts of the respective salts contained in the dry powder of the lysate reagent and the dry powder of the luciferase bioluminescent reagent can be easily adjusted so that the concentrations of the respective ions of the second reaction solution are within the prescribed range regardless of whether water or dialysate is used.

[0084] For example, the sodium ion concentration of the dialysate is usually 100 to 140 mM. Therefore, the content of sodium salt contained in the dry powder of the solubilizing agent-containing reagent and the dry powder of the luciferase bioluminescent reagent is adjusted so that the concentration of sodium ions other than those from the test sample in the second reaction solution is 250 to 390 mM. Similarly, since the chloride ion concentration of the dialysate is usually 100 to 140 mM, the content of chloride salt contained in the dry powder of the solubilizing agent-containing reagent and the dry powder of the luciferase bioluminescent reagent is adjusted so that the concentration of chloride ions other than those from the test sample in the second reaction solution is 250 to 361.5 mM.

[0085] In addition, the dry powder of the solubilizing agent-containing reagent used in the solubilizing reaction step and the dry powder of the luciferase bioluminescent reagent used in the luciferase reaction step can be mixed in advance to form a single agent, and the series of steps of the solubilizing reaction step and the luciferase reaction step can be performed under light shielding. Specifically, first, a prescribed amount of the test sample is mixed with the solubilizing agent and the dry powder of the luciferase bioluminescent reagent (hereinafter referred to as "single-agentized dry powder") to prepare a reaction solution under light shielding. Next, the obtained reaction solution is incubated for a prescribed time under light shielding, and the solubilizing reaction and the luciferase reaction are performed at the same time. Then, the endotoxin concentration of the test sample is measured from the luminescence amount of the reaction solution.

[0086] The solubilizing agent or the luciferase bioluminescent reagent used can be the same as described above. Furthermore, the content of each component of the solubilizing agent in the single-agentized dry powder is preferably adjusted so that the concentration in the reaction solution obtained by mixing a prescribed amount of the test sample is equivalent to the concentration in the first reaction solution described above. Similarly, the content of each component of the luciferase bioluminescent reagent in the single-agentized dry powder is preferably adjusted so that the concentration in the reaction solution obtained by mixing a prescribed amount of the test sample is equivalent to the concentration in the second reaction solution described above. Specifically, the sodium ion concentration, the calcium ion concentration, the chloride ion concentration, the Tris concentration, the pH, the magnesium ion concentration, and the carbonate ion concentration of the reaction solution in which a prescribed amount of the test sample and the single-agentized dry powder are mixed are preferably 250 to 390 mM, 0.5 to 10 mM, 250 to 361.5 mM, 20 to 30 mM, 7.7 to 8.5, 10 to 20 mM, and 1 to 5 mM, respectively, and more preferably 250 to 390 mM, 0.5 to 10 mM, 250 to 361.5 mM, 20 to 30 mM, 7.7 to 8.5, 10 to 20 mM, and 1 to 5 mM, respectively.

[0087] By making a kit of the various reagents used in the endotoxin assay method of the present application, the method can be more simply carried out. As a kit for assaying the endotoxin concentration of dialysate or water, for example, it is preferable to contain: a first container containing a dry powder of a lysate reagent; and a second container containing a dry powder of a luciferase bioluminescence reagent. Further, instead of the first and second containers, it is preferable to contain: a container containing a single-dose dry powder. Also, it is preferable to contain written materials or the like describing the operation manual for carrying out the endotoxin assay method of the present application using the kit.

[0088] In the kit, the concentrations of the salts and Tris contained in the dry powder of the lysate reagent and the dry powder of the luciferase bioluminescence reagent are preferably adjusted so that the sodium ion concentration is 250 to 390 mM, the calcium ion concentration is 0.5 to 10 mM, the chloride ion concentration is 250 to 361.5 mM, and the Tris concentration is 20 to 30 mM in the second reaction solution obtained by mixing the entire amount of the first reaction solution prepared by mixing a prescribed amount of dialysate or water in the first container into the second container. Further, it is more preferable to adjust so that the pH of the second reaction solution is 7.7 to 8.5, the magnesium ion concentration is 10 to 20 mM, and the carbonate ion concentration is 1 to 5 mM. Likewise, the concentrations of the salts and Tris contained in the single-dose dry powder are preferably adjusted so that the sodium ion concentration is 250 to 390 mM, the calcium ion concentration is 0.5 to 10 mM, the chloride ion concentration is 250 to 361.5 mM, and the Tris concentration is 20 to 30 mM in the reaction solution prepared by mixing a prescribed amount of dialysate or water in the container containing the single-dose dry powder. Further, it is more preferable to adjust so that the pH of the second reaction solution is 7.7 to 8.5, the magnesium ion concentration is 10 to 20 mM, and the carbonate ion concentration is 1 to 5 mM.

[0089] Example

[0090] Hereinafter, the present application will be further explained by examples, but the present application is not limited to the following examples.

[0091] [Example 1]

[0092] Endotoxin in water was assayed using a lysate reaction and a bioluminescence reaction, and the effect of the sodium concentration in the reaction solution was investigated.

[0093] The endotoxin used was endotoxin derived from E. coli 0113:H10 (manufactured by Fuji Photo Film Co., Ltd. and Wako Pure Chemical Industries, Ltd.). Further, unless otherwise noted, water was pyrogen-free water not containing endotoxin. In this example, as the test sample, an endotoxin aqueous solution in which endotoxin was dissolved in pyrogen-free water so that the endotoxin activity value would be 0.01 EU / mL was used, and as the blank (control test sample), pyrogen-free water was used.

[0094] The endotoxin assay used an endotoxin meter "Luminutes (registered trademark) -ET" (manufactured by Toa DKK Corporation). 200 μL of the test sample was added to a container containing a dry powder of a lysate reagent and stirred, and then the container was fixed to a prescribed position of the endotoxin meter, and a lysate reaction was performed by incubation at 37°C for 20 minutes. A dry powder of a luciferase bioluminescence reagent containing a luciferase was added to the reaction solution after the end of the reaction, and a bioluminescence reaction was induced. The bioluminescence was performed at 37°C as with the lysate reaction. The amount of luminescence generated during a period of 40 seconds from the start of the bioluminescence reaction was detected by a PMT and measured.

[0095] As the lysate reagent, a dry powder of an extracted component of limulus hemocytes (lysate reagent) was used, which was a dry powder of a lysate reagent (manufactured by Toa DKK Corporation) attached to the endotoxin meter as a special kit.

[0096] In the luciferase bioluminescence reagent, as the salt-resistant luciferase, a mutant (V288I+E488V) of the firefly luciferase of North America was used (Patent Document 1). Further, as the luminescence synthetic substrate, benzoyl-Leu-Gly-Arg-amino luciferin (manufactured by AAT Bioquest Corporation) was used.

[0097] As the dry powder of the luciferase bioluminescence reagent containing a luciferase, a dry powder obtained by freeze-drying 30 μL of a solution (pH 8.0) containing 0.25 μg / μL of a salt-resistant luciferase, 5 mM of ATP, 93 mM of magnesium acetate, and 0.1 M of a luminescence synthetic substrate was used. As the dry powder of the lysate reagent containing a lysate reagent, a dry powder obtained by freeze-drying 30 μL of a solution (pH 8.0) containing 167 mM of Tris-Cl and 0, 233, 466, 1000, 1333, or 1666 mM of sodium chloride was used. Since the luciferase bioluminescence reagent contains substantially no salt or Tris, in the case where the test sample is water, the sodium ion concentration, the calcium ion concentration, the magnesium ion concentration, the chloride ion concentration, the carbonate ion concentration, and the Tris concentration of the reaction solution of the bioluminescence reaction are derived from the dry powder of the lysate reagent containing a lysate reagent.

[0098] The luminescence after subtracting the measured value of the luminescence of the blank from the measured value of the luminescence of each test zone was taken as the luminescence of each test zone. The relative luminescence (%) of each test zone was calculated by taking the luminescence of the test zone using the luciferase bioluminescence reagent having a sodium chloride concentration of 0 mM (the test zone having a sodium chloride concentration of 0 mM in the reaction solution of the bioluminescence reaction) as 100%. The calculation results are shown in Table 1.

[0099] Table 1

[0100] Sodium chloride concentration [mM] in the reaction solution of the bioluminescence reaction Relative luminescence amount [%] 100 100.0 135 62.1 170 60.2 250 48.7 320 44.6 390 42.1

[0101] As shown in Table 1, when the concentration of sodium chloride in the reaction solution of the bioluminescence reaction is increased, there is a tendency for the relative light emission amount to decrease, but when the concentration of sodium chloride in the reaction solution is 250 to 390 mM, the relative light emission amounts are substantially equal, and it is understood that if the concentration is within this range, the difference in light emission amount due to the difference in the concentration of sodium chloride can be sufficiently reduced. For example, in the case where the dry powder containing the solubilizer reagent and the dry powder containing the luciferase bioluminescence reagent are prepared so that the concentration of sodium chloride from outside the test sample in the reaction solution of the bioluminescence reaction is 150 mM, the concentration of sodium chloride in the reaction solution of the bioluminescence reaction is 150 mM when the test sample is water, and the concentration of sodium chloride in the reaction solution of the bioluminescence reaction is 250 to 390 mM when the test sample is dialysate (the concentration of sodium chloride is 100 to 140 mM). It is understood that the concentration of sodium chloride in the reaction solution of the bioluminescence reaction is within the range of 250 to 390 mM when the test sample is water or dialysate, and the difference in light emission amount between the two is small, and even when the dialysate is the test sample, the calibration curve prepared using water can be used to determine the concentration of endotoxin.

[0102] Example 2

[0103] As the dry powder containing the solubilizer reagent, a dry powder obtained by freezing and drying 30 μL of a solution (pH 8.0) containing 167 mM of Tris-Cl, 0 or 1000 mM of sodium chloride, and 0, 3.3, 6.6, 13.3, 33.3, 66.6, 133, or 200 mM of calcium chloride was used, and otherwise, in the same manner as in Example 1, the concentration of endotoxin in a test sample containing 0.01 EU / mL of endotoxin was determined using the solubilizer reaction and the bioluminescence reaction. The results of the determination are shown in Table 2.

[0104] Table 2

[0105]

[0106] As shown in Table 2, as the concentration of calcium chloride in the reaction solution of the bioluminescence reaction becomes higher, there is a tendency for the relative light emission amount to decrease, and this tendency is more significant as the concentration of sodium chloride in the reaction solution of the bioluminescence reaction becomes higher. When the concentration of calcium chloride in the reaction solution of the bioluminescence reaction is 0.5 to 10 mM regardless of the concentration of sodium chloride, the difference in the light emission amount due to the difference in the concentration of calcium chloride is small. Based on this, when the dry powder containing the solubilizer reagent and the dry powder containing the luciferase bioluminescence reagent are prepared in such a manner that the concentration of calcium chloride from outside the test sample in the reaction solution of the bioluminescence reaction becomes 0.5 to 10 mM, it is known that, regardless of whether the test sample is water or dialysate, the concentration of calcium chloride in the reaction solution of the bioluminescence reaction is within the range of 0.5 to 10 mM, and the difference in the light emission amount between the two is small, and even when the dialysate is the test sample, the calibration curve prepared using water can be used to more accurately measure the endotoxin concentration.

[0107] Example 3

[0108] As the dry powder containing the solubilizer reagent, a dry powder obtained by preparing a solution containing 167 mM of Tris-Cl, 150 mM of sodium chloride, and 13.3 mM of calcium carbonate (pH 7.0, 7.5, 8.0, 8.5, or 9.0) and freeze-drying 30 μL of the solution was used, and otherwise, similarly to Example 1, the endotoxin concentration of a test sample containing 0.01 EU / mL of endotoxin was measured using the solubilization reaction and the bioluminescence reaction. The measurement results are shown in Table 3.

[0109] Table 3

[0110] pH of the reaction solution of the bioluminescence reaction Relative luminescence amount [%] 7.0 55 7.5 100 8.0 224 8.5 209 9.0 98

[0111] As shown in Table 3, the bioluminescence reaction is affected by the pH of the reaction solution. In particular, in the test zone in which the pH of the reaction solution of the bioluminescence reaction is 8.0 to 8.5, the relative light emission amount is very high, and the detection sensitivity of endotoxin is high.

[0112] Example 4

[0113] As the test sample, water containing 0.01 EU / mL of endotoxin or dialysate used in Example 1 was used. As the dry powder containing the solubilizer reagent, a dry powder obtained by preparing a solution containing 150 mM of sodium chloride and 13.3 mM of calcium carbonate and a solution containing 133, 166, 200, 266, 333, or 466 mM of Tris-Cl (pH 8.0) and freeze-drying 30 μL of the solution was used, and otherwise, similarly to Example 1, the endotoxin concentration of the test sample was measured using the solubilization reaction and the bioluminescence reaction.

[0114] As the dialysate, "Sublood (registered trademark) hemofiltration supplement BSG" (manufactured by Fusoban Pharmaceutical Co., Ltd.) was used. The sodium ion concentration of this dialysate was 140.0 mM (140.0 mEq / L), the potassium ion concentration was 2.0 mM (2.0 mEq / L), the calcium ion concentration was 3.5 mM (3.5 mEq / L), the magnesium ion concentration was 1.0 mM (1.0 mEq / L), the chloride ion concentration was 111.5 mM (111.5 mEq / L), and the carbonate ion concentration was 35 mM (70 mEq / L). The measurement results are shown in Table 4.

[0115] Table 4

[0116]

[0117] As shown in Table 4, the bioluminescence reaction was affected by the Tris concentration of the reaction solution. In particular, when the test sample was water, the effect of the Tris concentration was stronger than when the test sample was the dialysate, and the difference in the amount of luminescence when the test sample was water and when the test sample was the dialysate became larger. If the Tris concentration of the reaction solution of the bioluminescence reaction was in the range of 20 to 30 mM, the difference in the amount of luminescence when the test sample was water and when the test sample was the dialysate was small, and it was found that even when the dialysate was the test sample, the calibration curve prepared using water could be used to measure the endotoxin concentration.

[0118] Example 5

[0119] The amount of endotoxin in water and the dialysate was measured using a dry powder in which the solubilizer reagent and the luciferase bioluminescence reagent were single-dosed. As the test sample, a solution in which 0, 0.001, 0.003, 0.005, or 0.01 EU / mL of endotoxin was dissolved in water or the dialysate was used. The water and the dialysate used were the water and the dialysate used in Example 4.

[0120] As the single-dosed dry powder, a dry powder obtained by freezing and drying 30 μL of a solution in which a solubilizer reagent (0.38 μg / μL), 0.25 μg / μL of salt-resistant luciferase, 5 mM of ATP, and 0.1 M of a luminescence synthesis substrate were dissolved in Tris buffer (25 mM, pH 8.0) together with an appropriate amount of sodium chloride, sodium bicarbonate, and calcium chloride was used. The solubilizer reagent, the salt-resistant luciferase, and the luminescence synthesis substrate were the same as those used in Example 1.

[0121] The endotoxin was measured using an endotoxin meter "Luminutes (registered trademark)-ET" (manufactured by Toa DKK Inc.) in the same manner as in Example 1. 200 μL of the test sample was added to a container containing a single-dose dry powder and stirred, and then the container was fixed to a prescribed position of the endotoxin meter, and a lysate reaction was performed by incubation at 37°C for 20 minutes in the dark. The amount of luminescence generated during 40 seconds from the end of the reaction was detected and measured by PMT.

[0122] The concentrations of the components of the reaction solution at the time of luminescence measurement when the test sample was water and when the test sample was dialysate are shown in Table 5. Further, the results of measurement of the luminescence of the reaction solution of each test sample are shown in Table 6. The luminescence of each reaction solution was measured in a manner corrected so that the sample luminescence when the endotoxin concentration of the test sample was water was 0 EU / mL was 0.

[0123] Table 5

[0124]

[0125] Table 6

[0126]

[0127] As shown in Table 6, the samples having the same endotoxin concentration had the same luminescence whether the test sample was water or dialysate. From these results, it was found that by using the single-dose dry powder, the endotoxin concentration in dialysate can be measured with high precision using a calibration curve prepared using water. SEQUENCE LISTING <110> Toa DKK Inc. <120> Method for measuring endotoxin <130> J32459A1 <160> 11 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Consensus Sequence <400> 1 Gly Val Ile Gly Arg 1 5 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Coagulase recognition sequence <400> 2 Val Leu Gly Arg 1 <210> 3 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Coagulase recognition sequence <400> 3 Leu Arg Arg 1 <210> 4 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Coagulase recognition sequence <400> 4 Ile Glu Gly Arg 1 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Coagulase recognition sequence <400> 5 Leu Gly Arg 1 <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Coagulase recognition sequence <400> 6 Val Ser Gly Arg 1 <210> 7 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Coagulase recognition sequence <400> 7 Val Gly Arg 1 <210> 8 <211> 550 <212> PRT <213> Photinus pyralis <220> <223> Luciferase <400> 1 Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro 1 5 10 15 Leu Glu Asp Gly Thr Ala Gly Glu Gin Leu His Lys Ala Met Lys Arg 20 25 30 Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu 35 40 45 Val Asn Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala 50 55 60 Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val 65 70 75 80 Cys Ser Glu Asn Ser Leu Gin Phe Phe Met Pro Val Leu Gly Ala Leu 85 90 95 Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg 100 105 110 Glu Leu Leu Asn Ser Met Asn Ile Ser Gin Pro Thr Val Val Phe Val 115 120 125 Ser Lys Lys Gly Leu Gin Lys lie Leu Asn Val Gin Lys Lys Leu Pro 130 135 140 Ile lie Gin Lys lie lie lie Met Asp Ser Lys Thr Asp Tyr Gin Gly 145 150 155 160 Phe Gin Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe 165 170 175 Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr lie 180 185 190 Ala Leu lie Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val 195 200 205 Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp 210 215 220 Pro lie Phe Gly Asn Gin lie lie Pro Asp Thr Ala lie Leu Ser Val 225 230 235 240 Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu 245 250 255 Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu 260 265 270 Phe Leu Arg Ser Leu Gin Asp Tyr Lys lie Gin Ser Ala Leu Leu Val 275 280 285 Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr 290 295 300 Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser 305 310 315 320 Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile 325 330 335 Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr 340 345 350 Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe 355 360 365 Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val 370 375 380 Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly 385 390 395 400 Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly 405 410 415 Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His Phe 420 425 430 Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Tyr Gln 435 440 445 Val Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro Asn Ile 450 455 460 Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu 465 470 475 480 Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys 485 490 495 Glu Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu 500 505 510 Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly 515 520 525 Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys 530 535 540 Gly Gly Lys Ser Lys Leu 545 550 <210> 9 <211> 548 <212> PRT <213> Genji firefly [[ID=3�]]<220> <223> Luciferase <400> 2 Met Glu Asn Met Glu Asn Asp Glu Asn Ile Val Tyr Gly Pro Glu Pro 1 5 10 15 Phe Tyr Pro Ile Glu Glu Gly Ser Ala Gly Ala Gln Leu Arg Lys Tyr 20 25 30 Met Asp Arg Tyr Ala Lys Leu Gly Ala Ile Ala Phe Thr Asn Ala Leu 35 40 45 Thr Gly Val Asp Tyr Thr Tyr Ala Glu Tyr Leu Glu Lys Ser Cys Cys 50 55 60 Leu Gly Glu Ala Leu Lys Asn Tyr Gly Leu Val Val Asp Gly Arg Ile 65 70 75 80 Ala Leu Cys Ser Glu Asn Cys Glu Glu Phe Phe Ile Pro Val Leu Ala 85 90 95 Gly Leu Phe Ile Gly Val Gly Val Ala Pro Thr Asn Glu Ile Tyr Thr 100 105 110 Leu Arg Glu Leu Val His Ser Leu Gly Ile Ser Lys Pro Thr Ile Val 115 120 125 Phe Ser Ser Lys Lys Gly Leu Asp Lys Val Ile Thr Val Gln Lys Thr 130 135 140 Val Thr Ala Ile Lys Thr Ile Val Ile Leu Asp Ser Lys Val Asp Tyr 145 150 155 160 Arg Gly Tyr Gln Ser Met Asp Asn Phe Ile Lys Lys Asn Thr Pro Pro 165 170 175 Gly Phe Lys Gly Ser Ser Phe Lys Thr Val Glu Val Asn Arg Lys Glu 180 185 190 Gln Val Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys 195 200 205 Gly Val Gln Leu Thr His Glu Asn Ala Val Thr Arg Phe Ser His Ala 210 215 220 Arg Asp Pro Ile Tyr Gly Asn Gln Val Ser Pro Gly Thr Ala Ile Leu 225 230 235 240 Thr Val Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly 245 250 255 Tyr Leu Thr Cys Gly Phe Arg Ile Val Met Leu Thr Lys Phe Asp Glu 260 265 270 Glu Thr Phe Leu Lys Thr Leu Gln Asp Tyr Lys Cys Ser Ser Val Ile 275 280 285 Leu Val Pro Thr Leu Phe Ala Ile Leu Asn Arg Ser Glu Leu Leu Asp 290 295 300 Lys Tyr Asp Leu Ser Asn Leu Val Glu Ile Ala Ser Gly Gly Ala Pro 305 310 315 320 Leu Ser Lys Glu Ile Gly Glu Ala Val Ala Arg Arg Phe Asn Leu Pro 325 330 335 Gly Val Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Ile 340 345 350 Ile Thr Pro Glu Gly Asp Asp Lys Pro Gly Ala Ser Gly Lys Val Val 355 360 365 Pro Leu Phe Lys Ala Lys Val Ile Asp Leu Asp Thr Lys Lys Thr Leu 370 375 380 Gly Pro Asn Arg Arg Gly Glu Val Cys Val Lys Gly Pro Met Leu Met 385 390 395 400 Lys Gly Tyr Val Asp Asn Pro Glu Ala Thr Arg Glu Ile Ile Asp Glu 405 410 415 Glu Gly Trp Leu His Thr Gly Asp Ile Gly Tyr Tyr Asp Glu Glu Lys 420 425 430 His Phe Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly 435 440 445 Tyr Gln Val Pro Pro Ala Glu Leu Glu Ser Val Leu Leu Gln His Pro 450 455 460 Asn Ile Phe Asp Ala Gly Val Ala Gly Val Pro Asp Pro Ile Ala Gly 465 470 475 480 Glu Leu Pro Gly Ala Val Val Val Leu Glu Lys Gly Lys Ser Met Thr 485 490 495 Glu Lys Glu Val Met Asp Tyr Val Ala Ser Gln Val Ser Asn Ala Lys 500 505 510 Arg Leu Arg Gly Gly Val Arg Phe Val Asp Glu Val Pro Lys Gly Leu 515 520 525[[ID=⑦]] Thr Gly Lys Ile Asp Gly Lys Ala Ile Arg Glu Ile Leu Lys Lys Pro 530 535 540 Val Ala Lys Met 545 [[ID=十五]] <210> 10 <211> 548 <212> PRT <213> Genji Firefly <220> <223> Luciferase <400> 3 Met Glu Asn Met Glu Asn Asp Glu Asn Ile Val Val Gly Pro Lys Pro 1 5 10 15 Phe Tyr Pro Ile Glu Glu Gly Ser Ala Gly Thr Gln Leu Arg Lys Tyr<00005②5>20 25 30 Met Glu Arg Tyr Ala Lys Leu Gly Ala Ile Ala Phe Thr Asn Ala Val 35 40 45 Thr Gly Val Asp Tyr Ser Tyr Ala Glu Tyr Leu Glu Lys Ser Cys Cys 50 ⑤5 60 Leu Gly Lys Ala Leu Gln Asn Tyr Gly Leu Val Val Asp Gly Arg Ile 65 70 75 80 <00005③3>It should be noted that there may be some inaccuracies in the translation due to the lack of clear context for some terms. For example, "源氏萤" is directly translated as "Genji Firefly", which may not be the most accurate translation without more background information. Also, the numbering in the original text seems a bit inconsistent, and the translated text tries to follow it as closely as possible while maintaining readability. If possible, it would be beneficial to have more information about the specific content and purpose of these sequences for a more precise translation.Ala Leu Cys Ser Glu Asn Cys Glu Glu Phe Phe Ile Pro Val Ile Ala 85 90 95 Gly Leu Phe Ile Gly Val Gly Val Ala Pro Thr Asn Glu Ile Tyr Thr 100 105 110 Leu Arg Glu Leu Val His Ser Leu Gly Ile Ser Lys Pro Thr Ile Val 115 120 125 Phe Ser Ser Lys Lys Gly Leu Asp Lys Val Ile Thr Val Gln Lys Thr 130 135 140 Val Thr Thr Ile Lys Thr Ile Val Ile Leu Asp Ser Lys Val Asp Tyr 145 150 155 160 Arg Gly Tyr Gln Cys Leu Asp Thr Phe Ile Lys Arg Asn Thr Pro Pro 165 170 175 Gly Phe Gln Ala Ser Ser Phe Lys Thr Val Glu Val Asp Arg Lys Glu 180 185 190 Gln Val Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys 195 200 205 Gly Val Gln Leu Thr His Glu Asn Thr Val Thr Arg Phe Ser His Ala 210 215 220 Arg Asp Pro Ile Tyr Gly Asn Gln Val Ser Pro Gly Thr Ala Val Leu 225 230 235 240 Thr Val Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly 245 250 255 Tyr Leu Ile Cys Gly Phe Arg Val Val Met Leu Thr Lys Phe Asp Glu 260 265 270 Glu Thr Phe Leu Lys Thr Leu Gln Asp Tyr Lys Cys Thr Ser Val Ile 275 280 285 Leu Val Pro Thr Leu Phe Ala Ile Leu Asn Lys Ser Glu Leu Leu Asn 290 295 300 Lys Tyr Asp Leu Ser Asn Leu Val Glu Ile Ala Ser Gly Gly Ala Pro 305 310 315 320 Leu Ser Lys Glu Val Gly Glu Ala Val Ala Arg Arg Phe Asn Leu Pro 325 330 335 Gly Val Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Ile 340 345 350 Ile Thr Pro Glu Gly Asp Asp Lys Pro Gly Ala Ser Gly Lys Val Val 355 360 365 Pro Leu Phe Lys Ala Lys Val Ile Asp Leu Asp Thr Lys Lys Ser Leu 370 375 380 Gly Pro Asn Arg Arg Gly Glu Val Cys Val Lys Gly Pro Met Leu Met 385 390 395 400 Lys Gly Tyr Val Asn Asn Pro Glu Ala Thr Lys Glu Leu Ile Asp Glu 405 410 415 Glu Gly Trp Leu His Thr Gly Asp Ile Gly Tyr Tyr Asp Glu Glu Lys 420 425 430 His Phe Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly 435 440 445 Tyr Gln Val Pro Pro Ala Glu Leu Glu Ser Val Leu Leu Gln His Pro 450 455 460 Ser Ile Phe Asp Ala Gly Val Ala Gly Val Pro Asp Pro Val Ala Gly 465 470 475 480 Glu Leu Pro Gly Ala Val Val Val Leu Glu Ser Gly Lys Asn Met Thr 485 490 495 Glu Lys Glu Val Met Asp Tyr Val Ala Ser Gln Val Ser Asn Ala Lys 500 505 510 Arg Leu Arg Gly Gly Val Arg Phe Val Asp Glu Val Pro Lys Gly Leu 515 520 525 Thr Gly Lys Ile Asp Gly Arg Ala Ile Arg Glu Ile Leu Lys Lys Pro 530 535 540 Val Ala Lys Met 545 <210> 11 <211> 543 <212> PRT <213> Firefly beetle <220> <223> Luciferase <400> 4 Met Met Lys Arg Glu Lys Asn Val Val Tyr Gly Pro Glu Pro Lys His 1 5 10 15 Pro Leu Gly Asn Phe Thr Ala Gly Glu Met Leu Tyr Asn Ala Leu His 20 25 30 Lys His Ser His Ile Pro Gln Ala Ile Leu Asp Val Met Gly Asn Glu 35 40 45 Ser Leu Ser Tyr Gln Glu Phe Phe Asp Thr Thr Val Lys Leu Gly Gln 50 55 60 Ser Leu Gln Asn Cys Gly Tyr Lys Met Asn Asp Val Val Ser Ile Cys 65 70 75 80 Ala Glu Asn Asn Lys Arg Phe Phe Ile Pro Ile Ile Ser Ala Trp Tyr 85 90 95 Ile Gly Met Val Val Ala Pro Val Asn Glu Asp Tyr Ile Pro Asp Glu 100 105 110 Leu Cys Lys Val Thr Gly Ile Ser Lys Pro Ile Leu Val Phe Thr Thr 115 120 125 Arg Lys Ile Leu Pro Lys Val Leu Glu Val Lys Asp Arg Thr Asn Tyr 130 135 140 Ile Lys Arg Ile Ile Ile Leu Asp Ser Glu Glu Asn Leu Leu Gly Cys 145 150 155 160 Glu Ser Leu His Asn Phe Met Ser Arg Tyr Ser Asp Asn Asn Leu Gln 165 170 175 Thr Phe Lys Pro Leu His Tyr Asp Pro Val Asp Gln Val Ala Ala Ile 180 185 190 Leu Cys Ser Ser Gly Thr Thr Gly Leu Pro Lys Gly Val Met Gln Thr 195 200 205 His Arg Asn Ile Cys Val Arg Leu Thr His Ala Ser Asp Pro Arg Val 210 215 220 Gly Thr Gln Leu Ile Pro Gly Val Ser Val Leu Ala Tyr Leu Pro Phe 225 230 235 240 Phe His Ala Phe Gly Phe Ser Ile Asn Leu Gly Tyr Phe Met Val Gly 245 250 255 Leu Arg Val Val Met Leu Arg Arg Phe Asn Gln Glu Val Phe Leu Lys 260 265 270 Ala Ile Gln Asp Tyr Glu Val Arg Ser Val Ile Asn Val Pro Ser Thr 275 280 285 Ile Leu Phe Leu Ser Lys Ser Pro Leu Val Asp Lys Tyr Asp Leu Ser 290 295 300 Thr Leu Ala Glu Leu Cys Cys Gly Ala Ala Pro Leu Ala Lys Glu Val 305 310 315 320 Ala Glu Ile Ala Val Lys Arg Leu Asn Leu Pro Gly Ile Arg Cys Gly 325 330 335 Tyr Gly Leu Thr Glu Ser Thr Ser Ala Asn Ile His Thr Leu His Asn 340 345 350 Glu Phe Lys Ser Gly Ser Leu Gly Lys Val Thr Pro Tyr Met Ala Ala 355 360 365 Lys Ile Ile Asp Arg Asn Thr Gly Glu Ala Leu Gly Pro Asn Gln Val 370 375 380 Gly Glu Leu Cys Ile Trp Gly Pro Met Val Thr Lys Gly Tyr Val Asn 385 390 395 400 Asn Pro Gln Ala Thr Lys Glu Ala Ile Asp Asp Asp Gly Trp Leu His 405 410 415 Ser Gly Asp Phe Gly Tyr Tyr Asp Glu Asp Glu Tyr Phe Tyr Ile Val 420 425 430 Asp Arg Tyr Lys Glu Leu Ile Lys Tyr Lys Gly Tyr Gln Val Ala Pro 435 440 445 Val Glu Leu Glu Glu lie Leu Leu Gin His Pro Gly lie Arg Asp Val 450 455 460 Ala Val Val Gly lie Pro Asp lie Glu Ala Gly Glu Leu Pro Ala Gly 465 470 475 480 Phe Val Val Lys Gin Pro Gly Ala Gin Leu Thr Ala Lys Glu Val Tyr 485 490 495 Asp Phe Leu Ala Gin Arg Val Ser His Ser Lys Tyr Leu Arg Gly Gly 500 505 510 Val Arg Phe Val Asp Ser lie Pro Arg Asn Val Thr Gly Lys lie Ser 515 520 525 Arg Lys Glu Leu Arg Glu Ala Leu Met Glu Lys Ala Ser Lys Leu 530 535 540

Claims

1. A method for determining endotoxins, comprising the following steps: The solubility reaction step involves mixing a specified amount of the test sample with a dry powder containing the solubility reagent to prepare a first reaction solution, and then incubating the first reaction solution for a specified time. The luciferase reaction step involves, after the solubilization reaction step, mixing the first reaction solution with a dry powder containing a luciferase bioluminescent reagent under light-protected conditions to prepare a second reaction solution, and measuring the luminescence amount of the second reaction solution. as well as The endotoxin assay step determines the endotoxin concentration of the test sample based on the amount of light emitted during the luciferase reaction step. The test sample is either dialysis fluid or water. The dissolved reagent contains factor C, factor B, and precoagulant enzyme. The luciferase bioluminescent reagent contains a luminescent synthesis matrix, salt-resistant luciferase, and ATP. The luminescent synthetic matrix is ​​a substance that releases the luminescent matrix through digestion by active C factor, active B factor, or a coagulating enzyme obtained from the conversion of the pre-coagulating enzyme. In the luciferase reaction step, the sodium ion concentration of the second reaction solution is 250-390 mM, the calcium ion concentration is 0.5-10 mM, the chloride ion concentration is 250-361.5 mM, and the Tris concentration is 20-30 mM. The salt-resistant luciferase is a mutant beetle luciferase derived from the wild-type luciferase of beetles, wherein the amino acid sequence encoding the wild-type beetle luciferase contains at least one mutation selected from (a), (b), (c), and (d) below: (a) The amino acid corresponding to valine at position 288 in the amino acid sequence of wild-type North American firefly luciferase is mutated to isoleucine, leucine or phenylalanine. (b) The amino acid corresponding to leucine at position 376 in the amino acid sequence of wild-type North American firefly luciferase is mutated to proline. (c) The amino acid corresponding to glutamic acid at position 455 in the amino acid sequence of wild-type North American firefly luciferase is mutated to valine, alanine, serine, leucine, isoleucine or phenylalanine. (d) The amino acid corresponding to glutamic acid at position 488 in the amino acid sequence of wild-type North American firefly luciferase is mutated to valine, alanine, serine, leucine, isoleucine, or phenylalanine.

2. The method for determining endotoxins according to claim 1, wherein, In the luciferase reaction step, the pH of the second reaction solution is 7.7 to 8.

5.

3. The method for determining endotoxins according to claim 1 or 2, wherein, In the luciferase reaction step, the magnesium ion concentration of the second reaction solution is 10-20 mM, and the carbonate ion concentration is 1-5 mM.

4. The method for determining endotoxins according to claim 1 or 2, wherein, The dissolving agent is a component extracted from horseshoe crab blood cells.

5. The method for determining endotoxins according to claim 3, wherein, The dissolving agent is a component extracted from horseshoe crab blood cells.

6. The method for determining endotoxins according to claim 1 or 2, wherein, In the endotoxin determination step, the endotoxin concentration of the test sample is determined based on a calibration curve prepared using a dilution series of water containing known concentrations of endotoxin and the luminescence amount obtained in the luciferase reaction step.

7. The method for determining endotoxins according to claim 3, wherein, In the endotoxin determination step, the endotoxin concentration of the test sample is determined based on a calibration curve prepared using a dilution series of water containing known concentrations of endotoxin and the luminescence amount obtained in the luciferase reaction step.

8. A method for determining endotoxins, wherein, A reaction solution is prepared by mixing a specified amount of the test sample with a dissolved reagent and a dry powder containing a luciferase bioluminescent reagent. The reaction solution was incubated in the dark for a specified time, and then the endotoxin concentration of the test sample was determined based on the amount of light emitted by the reaction solution. The test sample is either dialysis fluid or water. The dissolved reagent contains factor C, factor B, and precoagulant enzyme. The luciferase bioluminescent reagent contains a luminescent synthesis matrix, salt-resistant luciferase, and ATP. The luminescent synthetic matrix is ​​a substance that releases the luminescent matrix through digestion by active C factor, active B factor, or a coagulating enzyme obtained from the conversion of the pre-coagulating enzyme. The reaction solution has a sodium ion concentration of 250-390 mM, a calcium ion concentration of 0.5-10 mM, a chloride ion concentration of 250-361.5 mM, and a Tris concentration of 20-30 mM. The salt-resistant luciferase is a mutant beetle luciferase derived from the wild-type luciferase of beetles, wherein the amino acid sequence encoding the wild-type beetle luciferase contains at least one mutation selected from (a), (b), (c), and (d) below: (a) The amino acid corresponding to valine at position 288 in the amino acid sequence of wild-type North American firefly luciferase is mutated to isoleucine, leucine or phenylalanine. (b) The amino acid corresponding to leucine at position 376 in the amino acid sequence of wild-type North American firefly luciferase is mutated to proline. (c) The amino acid corresponding to glutamic acid at position 455 in the amino acid sequence of wild-type North American firefly luciferase is mutated to valine, alanine, serine, leucine, isoleucine or phenylalanine. (d) The amino acid corresponding to glutamic acid at position 488 in the amino acid sequence of wild-type North American firefly luciferase is mutated to valine, alanine, serine, leucine, isoleucine, or phenylalanine.

9. The method for determining endotoxin according to claim 8, wherein, The pH of the reaction solution is 7.7~8.

5.

10. The method for determining endotoxins according to claim 8 or 9, wherein, The magnesium ion concentration of the reaction solution is 10-20 mM, and the carbonate ion concentration is 1-5 mM.

11. The method for determining endotoxins according to claim 8 or 9, wherein, The dissolving agent is a component extracted from horseshoe crab blood cells.

12. The method for determining endotoxin according to claim 10, wherein, The dissolving agent is a component extracted from horseshoe crab blood cells.

13. The method for determining endotoxins according to claim 8 or 9, wherein, In the endotoxin determination step, the endotoxin concentration of the test sample is determined based on a calibration curve prepared using a dilution series of water containing known concentrations of endotoxin and the luminescence amount obtained in the luciferase reaction step.

14. The method for determining endotoxin according to claim 10, wherein, In the endotoxin determination step, the endotoxin concentration of the test sample is determined based on a calibration curve prepared using a dilution series of water containing known concentrations of endotoxin and the luminescence amount obtained in the luciferase reaction step.

15. The method for determining endotoxin according to claim 11, wherein, In the endotoxin determination step, the endotoxin concentration of the test sample is determined based on a calibration curve prepared using a dilution series of water containing known concentrations of endotoxin and the luminescence amount obtained in the luciferase reaction step.

16. The method for determining endotoxin according to claim 12, wherein, In the endotoxin determination step, the endotoxin concentration of the test sample is determined based on a calibration curve prepared using a dilution series of water containing known concentrations of endotoxin and the luminescence amount obtained in the luciferase reaction step.

17. An endotoxin assay kit for determining the endotoxin concentration in dialysate or water, comprising: The first container contains a dry powder containing a dissolved reagent; The second container contains a dry powder containing a luciferase bioluminescent reagent, wherein the luciferase bioluminescent reagent contains a luminescent synthesis matrix, salt-resistant luciferase, and ATP; A first reaction solution is prepared by mixing a specified amount of dialysis fluid or water in the first container. The total amount of the first reaction solution is then mixed into the second container. The resulting second reaction solution has a sodium ion concentration of 250-390 mM, a calcium ion concentration of 0.5-10 mM, a chloride ion concentration of 250-361.5 mM, and a Tris concentration of 20-30 mM. in, The salt-resistant luciferase is a mutant beetle luciferase derived from the wild-type luciferase of beetles, wherein the amino acid sequence encoding the wild-type beetle luciferase contains at least one mutation selected from (a), (b), (c), and (d) below: (a) The amino acid corresponding to valine at position 288 in the amino acid sequence of wild-type North American firefly luciferase is mutated to isoleucine, leucine or phenylalanine. (b) The amino acid corresponding to leucine at position 376 in the amino acid sequence of wild-type North American firefly luciferase is mutated to proline. (c) The amino acid corresponding to glutamic acid at position 455 in the amino acid sequence of wild-type North American firefly luciferase is mutated to valine, alanine, serine, leucine, isoleucine or phenylalanine. (d) The amino acid corresponding to glutamic acid at position 488 in the amino acid sequence of wild-type North American firefly luciferase is mutated to valine, alanine, serine, leucine, isoleucine, or phenylalanine.

18. The endotoxin assay kit according to claim 17, wherein, The second reaction solution has a pH of 7.7-8.5, a magnesium ion concentration of 10-20 mM, and a carbonate ion concentration of 1-5 mM.

19. The endotoxin assay kit according to claim 17 or 18, used for implementing the endotoxin assay method according to any one of claims 1 to 16.

20. An endotoxin assay kit, which is used to determine the endotoxin concentration in dialysate or water, wherein, The kit contains a container with dried powder containing a solvent reagent and a luciferase bioluminescent reagent, the luciferase bioluminescent reagent containing a luminescent synthesis matrix, salt-resistant luciferase, and ATP; The reaction solution prepared by mixing a specified amount of dialysis fluid or water in the container has a sodium ion concentration of 250-390 mM, a calcium ion concentration of 0.5-10 mM, a chloride ion concentration of 250-361.5 mM, and a Tris concentration of 20-30 mM. The salt-resistant luciferase is a mutant beetle luciferase derived from the wild-type luciferase, wherein the amino acid sequence encoding the wild-type beetle luciferase contains at least one mutation selected from (a), (b), (c), and (d) below: (a) The amino acid corresponding to valine at position 288 in the amino acid sequence of wild-type North American firefly luciferase is mutated to isoleucine, leucine or phenylalanine. (b) The amino acid corresponding to leucine at position 376 in the amino acid sequence of wild-type North American firefly luciferase is mutated to proline. (c) The amino acid corresponding to glutamic acid at position 455 in the amino acid sequence of wild-type North American firefly luciferase is mutated to valine, alanine, serine, leucine, isoleucine or phenylalanine. (d) The amino acid corresponding to glutamic acid at position 488 in the amino acid sequence of wild-type North American firefly luciferase is mutated to valine, alanine, serine, leucine, isoleucine, or phenylalanine.

21. The endotoxin assay kit according to claim 20, wherein, The reaction solution has a pH of 7.7-8.5, a magnesium ion concentration of 10-20 mM, and a carbonate ion concentration of 1-5 mM.

22. The endotoxin assay kit according to claim 20 or 21, used for implementing the endotoxin assay method according to any one of claims 1 to 16.

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