JP2006284606A - Limulus reaction activating substance, inactivation method and measuring method for the substance, and method of measuring limulus reaction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for specifying and isolating a novel causative agent of exhibiting a false positive in an endotoxin specific limulus reaction, which exists mainly in a biological preparation, and for removing an influence of the causative agent from the limulus reaction, and a method of measuring the causative agent. <P>SOLUTION: The false positive substance is removed from a limulus reaction system, by making a limulus measuring sample coexisting with a surfactant at a temperature ranging from a freezing point of the surfactant to 50 °C or less. The present invention provides a method of measuring the false positive substance itself, and endotoxin based on the limulus reaction, using the means for removing the false positive substance, or the like. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a limulus reaction that is a reaction that activates amebosite lysate, a blood cell component of horseshoe crab used for detection of endotoxin and the like, and particularly, in a limulus reaction specific to endotoxin, (1) initiated by the endotoxin. A non-endotoxin-like substance activated by reacting with factor C, which is the first factor of the system, (2) means for eliminating the influence of this non-endotoxin-like substance in the Limulus reaction, (3) endotoxin using the means for eliminating this influence And (4) a technical field related to the measurement means by the Limulus reaction of this non-endoxin-like substance. Furthermore, the present invention relates to (5) a technical field relating to a means for measuring a true endotoxin content in a biological preparation, in which the influence of the non-endotoxin-like substance is particularly problematic in the measurement of endotoxin using a Limulus reaction. Belonging to.

  Endotoxin is a lipopolysaccharide present in the outer cell wall membrane of Gram-negative bacteria, and is known as a strong pyrogen. In addition to fever, endotoxin induces various pathologies caused by bacterial infection, even in trace amounts, such as the release of inflammatory cytokines such as TNF and interleukin-1 (IL-1) associated with macrophage activation and the induction of endotoxin shock. It is known to wake up. For this reason, detection of endotoxins in injectable drugs and the like is important, and endotoxin test methods are listed in the pharmacopoeia and biopharmaceutical standards of the United States and Japan. Endotoxin is considered to be the main cause of shock in Gram-negative bacterial infections. For purposes such as diagnosis of Gram-negative bacterial infection, determination of its therapeutic effect and prognosis, and early diagnosis of endotoxin shock, Endotoxin measurements are being made.

  The Limulus test, which is an endotoxin test method, is a reaction in which endotoxin derived from Gram-negative bacteria activates the blood cell extract component (Amebosite lysate) of horseshoe crab (this reaction is called Limulus reaction, and the reagent used for it is called Limulus reagent The endotoxin detection method, more specifically, the gelation method, the turbidimetric method using the turbidity change of the gel as an index, and the colorimetric method using the color developed by hydrolysis of the chromogenic synthetic substrate as an index And is now broadly classified. In 1978, the US FDA approved the Limulus test method as an alternative method because of its high correlation with the rabbit fever test, and was used for safety testing of pharmaceuticals and medical devices including biological products such as vaccines and blood products. Since this test has been widely applied, it has been listed in Japanese Pharmacopoeia and Biopharmaceutical Standards as an alternative to the rabbit fever test in Japan.

  The horseshoe crab, amebocyte lysate (hereinafter sometimes simply referred to as lysate) contained in the Limulus reagent includes a cascade type coagulation system (factor C system) activated by reaction with endotoxin and (1 → 3) -β. -D-glucan (hereinafter also referred to as β-D-glucan) and a cascade type coagulation system (factor G system) activated by reaction coexist (see FIG. 1), the former A method for specifically measuring endotoxin using only this system and a method for specifically measuring β-D-glucan using only the latter system are known (Obayashi T. et al. , Clin. Chim. Acta, 149, 55-65 (1985), for endotoxin-specific measurements; WO 90/02951, USP 5,155,032, USP 5,179,006, WO 92/037 36, WO92 / 066381, JP-A-6-258326, JP-B-2-18080, β-D-glucan-specific measurement method; WO91 / 19981, WO92 / 16651), and Limulus reagent for specific measurement of each has already been released Has been. In particular, the biopharmaceutical standard in Japan stipulates that a Limulus reagent specific for endotoxin is used because β-D-glucan is often mixed in a sample for measuring Limulus.

  By the way, even though this endotoxin-specific Limulus reagent is used, there are substances that react in addition to endotoxin depending on the sample for measuring Limulus (in the present specification, a sample used for measurement using the Limulus reaction). Are known. That is, serine proteases such as trypsin, thrombin, factor Xa, etc. (the Limulus reaction uses the reaction of serine protease in lysate and is positive because it exhibits a similar action to the coagulation enzyme therein), chymotrypsin (factor C) "A substance that reacts in an endotoxin-specific limulus reaction other than endotoxin (endotoxin-specific limulus reaction false positive substance)" is known. Therefore, when measuring endotoxin in a sample for measuring Limulus containing these substances, that is, blood (whole blood, plasma, serum) with Limulus reagent, this Limulus reaction false-positive substance is used without impairing the endotoxin activity. Methods to eliminate are known.

For example, on the premise of heat treatment, a means for diluting a sample for measuring Limulus with an aqueous solution containing a surfactant and eliminating the influence of false positive substances in the sample for measuring Limulus is disclosed (Japanese Patent Laid-Open No. 6-1994). No. 118086: Patent Document 1). Further, means for treating a sample for measuring Limulus with an acid such as perchloric acid (PCA) and eliminating the influence of a Limulus reaction false positive substance in the sample for measuring Limulus (Japanese Patent Publication No. 63-55671: Patent Document) 2) or means for treating a sample for measuring Limulus with a hexadimethrin compound such as polybrene and an alkali metal hydroxide to eliminate the influence of Limulus reaction interfering factors in the sample for measuring Limulus (Japanese Patent Laid-Open No. Hei 6-70796) : Patent Document 3) and the like are known. Patent Documents 4 to 7 are presented in a notice of reasons for refusal of the parent application as citation examples of the parent application of the present application.
Japanese Patent Laid-Open No. 6-118086 Japanese Examined Patent Publication No. 63-55671 JP-A-6-70796 JP-A-4-16765 JP-A-6-273421 JP 7-239332 A JP-A-2-141666

  However, recently, a phenomenon that is positive in endotoxin-specific Limulus reaction has been observed mainly in biological products, although it does not show pyrogenicity in the Japanese Pharmacopoeia pyrogen test, Since the listed endotoxin test method (endotoxin-specific Limulus reaction) was used, it was possible that the safety of the drug could be evaluated improperly. The present inventors analyzed this phenomenon, and the causative substance exhibiting false positive in this endotoxin-specific Limulus reaction is different from the conventionally known Limulus reaction activator, and any of the above Limulus reaction false positive substances It has been found that it is difficult to eliminate this causative substance even by means of exclusion.

  Therefore, it has become a very important issue to provide a method for accurately measuring only endotoxin by eliminating the influence of the Limulus reaction false positive substance mixed in these preparations on the Limulus reaction. That is, the problem to be solved by the present invention is to identify and isolate a novel causative agent that exhibits a false positive in an endotoxin-specific limulus reaction mainly present in a biological product, and to further determine the influence of this causative agent on the Limulus reaction system. It is an object of the present invention to provide means for eliminating the above and a method for measuring the causative substance.

  As a result of intensive studies on the novel causative substance and its exclusion means, the present inventor established an isolating means for the causative substance, and surprisingly, under non-heating conditions (specific temperatures will be described later). It was found that the influence of this causative substance can be eliminated from the Limulus reaction system by coexisting a sample for measuring Limulus with a surfactant, or a surfactant and an alkylamine.

  That is, the present invention mainly includes (1) a Limulus reaction activator having specific properties produced by a specific production method (details will be described later); (2) a sample for measuring Limulus and a surfactant. Use of the surfactant for inactivating the Limulus reaction activator described in (3) above, preferably coexisting at a temperature exceeding the freezing point temperature of the surfactant and below 50 ° C. Use of the same surfactant in the presence of an amine; (4) an inactivating agent for the Limulus reaction activator described in (1) above, comprising the surfactant, a Limulus reagent comprising this inactivating agent, and A kit for measuring Limulus comprising an inactivating agent; (5) a sample for measuring Limulus in which the Limulus reaction activator described in (1) above is inactivated by a surfactant; (6) an end including a specific step; Providing and (7) Measurement method of limulus reaction-activating substance according the above (1) containing a specific process; Shin measurement methods.

  The present invention provides a method for easily and rapidly inactivating a non-endotoxin limulus reaction-activating substance having no pyrogenic activity mixed in a biological preparation, and measuring only endotoxin with a limulus reagent with high accuracy. According to the present invention, the amount of endotoxin in a biological product can be measured correctly, and thus the safety evaluation of the biological product can be performed more appropriately, leading to a great contribution to medicine. In addition, the activated substance may have a novel physiological action, and the possibility of creating a new pharmaceutical product is also recognized. Thus, the Limulus reaction activator according to the present invention, and the endotoxin and the method for measuring the activator have great medical significance.

A. About specific isolation means of Limulus reaction activator:
The Limulus reaction activator according to the present invention (hereinafter also referred to as the Limulus reaction activator of the present invention) is a novel causative substance that exhibits a false positive in the endotoxin-specific Limulus reaction, and is specifically isolated by the following means.

  That is, the Limulus reaction activator of the present invention is obtained from the sample for measuring Limulus [mainly biological preparations (for example, blood preparations, vaccines, antibiotics, etc .: these biological preparations will be described later)]. It can be purified by various separation means utilizing the physical and chemical properties of the Limulus reaction activator. Examples of the separation means include treatment with an ordinary protein precipitating agent, ultrafiltration, gel filtration, centrifugation, electrophoresis, gel permeation chromatography, ion exchange chromatography, affinity chromatography, reverse phase chromatography, hydrophobic chromatography. Graphy, dialysis, etc. Needless to say, these separation means can be used in appropriate combination.

  Among these separation means, gel permeation chromatography, particularly gel permeation chromatography using high performance liquid chromatography is preferred. Specifically, the sample for measuring Limulus is applied to the chromatography column and eluted with endotoxin and β-D-glucan-free distilled water as a solvent. The flow rate of the solvent is not particularly limited, but is preferably about 0.5 ml / min.

To the eluted fraction, an aqueous solution of polymyxin B sulfate was added to a final concentration of 0.5 mg / ml. Endotoxin activity was completely suppressed, and this fraction was measured with an endotoxin-specific Limulus reagent. By collecting the above, the limulus reaction activator of the present invention can be obtained. The Limulus reaction-activating substance of the present invention that can be produced in this manner is mainly present in biological preparations such as vaccines and albumin preparations, and has the following properties. Further, these properties will be described in detail mainly in examples described later.
(1) It has the ability to activate Limulus reaction.
(2) Reacts with endotoxin-specific Limulus reagent.
(3) Activate factor C derived from horseshoe crab amebocyte lysate.
(4) Exothermic substance test shows no exothermicity.
(5) Not endotoxin.
(6) Not (1 → 3) -β-D-glucan.
(7) No serine protease activity.
(8) Even if polymyxin B coexists with this substance, the ability to activate the Limulus reaction of this substance does not decrease.
(9) Even if colistin coexists with this substance, the ability to activate the Limulus reaction of this substance does not decrease.
(10) Even if this substance is treated with 0.2 M hydrochloric acid at 37 ° C. for 60 minutes, the ability to activate the Limulus reaction of this substance does not decrease.
(11) Even if this substance is treated with 0.2 M potassium hydroxide at 37 ° C. for 60 minutes, the ability to activate the Limulus reaction of this substance does not decrease.
(12) By treating this substance with polyoxyethylene hexadecyl ether, the ability to activate the Limulus reaction of this substance disappears.

  Although the in vivo action, toxicity, etc. of the Limulus reaction activator of the present invention are unknown, it enhances the toxicity of endotoxin as reported by TSST-1 (Staphylococcal toxic shock syndrome toxin-1) of staphylococcal exotoxin There is a possibility of having similar physiological action and toxicity as endotoxin such as synergistic action (H. Fujikawa et al., Infect Immun., 52, 134 (1986)) or production of inflammatory mediators. Therefore, the Limulus reaction-activating substance of the present invention isolated for the first time according to the present invention is extremely important for clarifying the action of the substance in vivo and in various pathological conditions. In addition, the measurement method of the Limulus reaction activator of the present invention provided by the present invention provides extremely useful information like the endotoxin measurement method.

  In addition, the limulus reaction-activating substance of the present invention is mainly present in biological preparations and the like, and that a relatively large amount of the substance is contained in biological preparations imported from outside Japan. Have been confirmed. Although the origin of the substance of the present invention is not clear, the possibility of contamination in the process of manufacturing a biological preparation cannot be denied. The method for measuring a Limulus reaction-activating substance of the present invention is also expected to be used for checking the contamination of the substance and the final product in the manufacturing process of the biological product. It is also expected to lead to the provision of practical preparations. The method for measuring the Limulus reaction-activating substance of the present invention will be described later.

B. About the means for inactivating the Limulus reaction-activating substance of the present invention (hereinafter also referred to as the method of deactivating the Limulus reaction-activating substance of the present invention):
When the above-described Limulus reaction-activating substance is contained in the Limulus measurement sample, a false positive reaction is induced in the Limulus reagent, thereby reducing the detection accuracy of a desired substance (for example, endotoxin). This is not preferable.

  As a means for inactivating the Limulus reaction activator of the present invention, a method in which the Limulus reaction activator and a surfactant coexist at a temperature from the temperature exceeding the freezing point of the surfactant to 50 ° C. or less is specifically mentioned. Can be mentioned. As the surfactant used in the present invention, as long as it does not impair the Limulus reactivity as a result of the dissociation action on endotoxin micelles, and does not activate the Limulus reaction and does not inhibit the active serine protease, it is positive. There are no particular restrictions on ionic surfactants, anionic surfactants, amphoteric surfactants, nonionic surfactants or naturally occurring surfactants, but nonionic surfactants with little direct action on endotoxins It is preferable to select. In addition, it is also possible to use these surfactants in combination as appropriate.

Of the nonionic surfactants, surfactants having a structure having polyoxyethylene in the hydrophilic portion (hereinafter also referred to as “polyoxyethylenes”) are preferable. Polyoxyethylenes include polyoxyethylene alkyl ethers (general formula, C _n H _{2n + 1} (OCH ₂ CH ₂ ) _x OH, usually abbreviated as C _n E _x ), alkyl chains, Examples include polyoxyethylene alkylphenyl ether (C _n ФE _x ) having a phenyl group between polyoxyethylene chains and acyl polyoxyethylene sorbitan (C _n sorbitan E _x ), which are respectively Brij (C _{n E x), Triton X (C} n ФE x), is called by Tween (C _n sorbitan E _x) that Nonproprietary name (trade name), widely used in many purposes such as solubilization of membrane proteins.

The polyoxyethylene chain of the polyoxyethylenes used in the present invention (“(OCH ₂ CH ₂ ) _x OH” in the above general formula, also abbreviated as “Ex”) is not particularly limited, but x = 2 to 2. A compound having an integer of 25, preferably an integer of x = 4 to 23, more preferably an integer of x = 7 to 13 is preferable. The number of carbon atoms of the alkyl group of the polyoxyethylenes used in the present invention (“C _n H _{2n + 1} ” in the above formula, also abbreviated as “Cn”) is not particularly limited, but n = 8 to A compound that is an integer of 18 is preferred.

  Examples of such polyoxyethylenes include polyoxyethylene dodecyl ether, polyoxyethylene hexadecyl ether (also referred to as polyoxyethylene cetyl ether), polyoxyethylene isooctyl phenyl ether, polyoxyethylene nonyl phenyl ether, polyoxyethylene. Examples include fatty acid esters and polyoxyethylene sorbitol esters. Among these, polyoxyethylene hexadecyl ether is very preferable. These surfactants are preferably used as aqueous solutions and have a certain micelle size.

  In addition, the solvent of the aqueous solution of these surfactants may be a buffer solution. The buffer is preferably a buffer adjusted to around the optimum pH of the factor C system (specifically, about pH 7 to 9). For example, Good buffer [for example, HEPES (N-2-hydroxyethyl) Piperazine-N′-2-ethanesulfonic acid buffer), collamine chloride buffer, BES buffer, MOPS buffer, TES buffer, HEPPS buffer (N-2-hydroxyethylpiperazine-N′-3-propane) Sulfonic acid), Tricine buffer, glycinamide buffer, Bicine buffer, TAPS buffer, etc.], Tris-hydrochloric acid buffer, and the like.

  The amount of the surfactant coexisting with the limulus reaction activator of the present invention can be appropriately changed depending on the amount of the present limulus reaction activator present and the type and amount of the coexisting surfactant, and is particularly limited. Not. The specific surfactant concentration is usually 0.005% to 0.8% (W / V), preferably 0.01% to 0.5% as the final concentration when contacting the sample for measuring Limulus. % (W / V), more preferably 0.05% to 0.3% (W / V) and the like are exemplified, but these are merely examples and are not limited thereto.

  Furthermore, a desired aqueous surfactant solution can be prepared by subjecting the aqueous surfactant solution to endotoxin-free means such as activated carbon treatment, membrane filter treatment, and autoclave treatment.

  As a sequence and method for coexisting a surfactant or an aqueous solution of a surfactant with a sample for measuring Limulus containing a Limulus reaction activator, the surfactant is not modified or destroyed at all. There is no particular limitation as long as it is present in the sample for measuring Limulus so as to have a predetermined concentration. The method for allowing the sample for measuring Limulus and the surfactant to coexist is usually achieved by adding the sample for measuring Limulus and the surfactant and mixing them well. In addition, the order in which the sample for measuring Limulus and the surfactant coexist can achieve the object of the invention by adding a surfactant or the like in the Limulus reagent and mixing and reacting with the sample for measuring Limulus simultaneously. However, it is more desirable from the viewpoint of the effect that it is preliminarily mixed with the sample for measuring Limulus before the Limulus reaction. The amount of the aqueous solution of the surfactant or the like to be mixed with the Limulus measurement sample is preferably 0.1 to 10 volumes of the surfactant solution per 1 volume of the Limulus measurement sample. There is no particular limitation as long as a predetermined concentration of surfactant or the like is added and the effective concentration is maintained.

  Furthermore, in order to inactivate the Limulus reaction activator of the present invention, it is necessary to coexist the above-mentioned surfactant at a temperature exceeding the freezing point of this surfactant to 50 ° C. or less. At a temperature below the freezing point of the surfactant, the solution in which the surfactant and the Limulus reaction activator coexist is frozen, making it difficult to smoothly carry out the desired inactivation reaction. If it exceeds, the substance to be measured such as endotoxin is inactivated, and its accurate measurement becomes difficult, which is not preferable.

  Moreover, the Limulus reaction activity of the present invention is preferably carried out at a temperature of 1 ° C. or more and 50 ° C. or less, more preferably 4 ° C. or more and 50 ° C. or less, further preferably 4 ° C. or more and 45 ° C. or less, particularly preferably 15 ° C. or more and 40 ° C. or less. The chemical can be contacted with a surfactant. This is a point that is greatly different from the prior art (Japanese Patent Laid-Open No. 6-118086) in which a surfactant is used and the false positive substance of the Limulus reaction is inactivated by positive heating as in the present invention.

  The time required for the sample for measuring Limulus and the surfactant to coexist at a temperature exceeding the freezing point temperature of the surfactant to 50 ° C. or less is particularly that the endotoxin molecule and its associated micelles are physicochemically stable, Although it is not particularly limited as long as it does not adsorb to the liquid, it usually takes several seconds to 5 minutes, and about 1 minute to 2 minutes is sufficient. Thus, the purpose of the inactivating action of the surfactant used in the present invention on the Limulus reaction-activating substance can be achieved in a very short time.

  The above-described method for inactivating the Limulus reaction-activating substance of the present invention can be directly applied to the inactivation of the Limulus reaction-activating substance of the present invention in the sample for measuring Limulus. The present invention inactivates the Limulus reaction-activating substance of the present invention, in which the sample for measuring Limulus and the surfactant coexist at a temperature exceeding the freezing point temperature of the surfactant to 50 ° C. or less. Use of a surfactant. As will be described later, since it is more preferable that a sample for measuring Limulus coexists with an alkylamine in addition to a surfactant, the present invention includes a sample for measuring Limulus, a surfactant and an alkylamine. Also included is the use of a surfactant to inactivate the Limulus reaction activator of the present invention that coexists at a temperature above the freezing point temperature of the activator up to 50 ° C.

  Thus, means for inactivating the above-described Limulus reaction-activating substance of the present invention is provided, whereby a Limulus-measuring sample in which the Limulus reaction-activating substance of the present invention is inactivated by a surfactant is directly provided. Although provided, this inactivation means can be used in various forms. For example, a Limulus reaction activator comprising a surfactant based on the premise that a sample for measuring Limulus and a surfactant coexist at a temperature exceeding the freezing point temperature of the surfactant to 50 ° C. or less. It is possible to apply to the form as an inactivating agent.

  It is also possible to apply to the form of a Limulus reagent comprising this inactivating agent, a Limulus measuring kit comprising this Limulus reagent, or a Limulus measuring kit comprising an inactivating agent and a Limulus reagent. It is. In the present specification, “Limulus measurement” means measurement using a Limulus reaction, and “Limulus measurement kit” means a kit containing at least a Limulus reagent used for measurement using a Limulus reaction. . In addition to the above-described Limulus reagent, any component reagent can be added to the Limulus measurement kit as necessary. Examples of such reagents include, but are not limited to, blank test distilled water, reaction reagent dissolution / reaction buffer, and the like.

  Thus, the present invention provides an inactivating agent for the Limulus reaction-activating substance of the present invention comprising a surfactant, a Limulus reagent comprising the inactivating agent, and a Limulus measurement comprising the Limulus reagent and the inactivating agent. A kit is also provided. The Limulus reagent is preferably an endotoxin-specific Limulus reagent. Furthermore, the present invention is characterized in that a surfactant is allowed to coexist in a sample for measuring Limulus at a temperature exceeding the freezing point temperature of the surfactant to a temperature of 50 ° C. or less, and the Limulus reaction of the Limulus reaction activator of the present invention is characterized in that Also included is a method for eliminating the influence on the sample for measuring Limulus.

  Further, the present invention includes a sample for measuring Limulus in which the Limulus reaction-activating substance of the present invention is inactivated by a surfactant in this way. The limulus measurement sample to be inactivated by the limulus reaction activator described herein is not particularly limited as long as endotoxin or the like is to be detected using the limulus reaction. Biologically-derived limulus measurement samples in which a large amount of reaction activator is observed are preferred, and biological preparations are particularly suitable targets.

  This “biological product” is a concept that excludes blood fractions containing whole blood, that is, whole blood, plasma, serum and the like. Specifically, vaccine preparations (generally prepared for the purpose of increasing the blood antibody titer against the target virus and reducing the morbidity by inoculating the vaccine) Inactivated vaccine (for example, influenza vaccine (influenza HA vaccine), Japanese encephalitis vaccine, etc.) or attenuated obtained by degrading and inactivating highly purified virus particles after growing in a target host such as or in the brain And the like. ], Blood products (usually starting with plasma, varying the conditions such as pH, ionic strength, ethanol concentration, etc., and using a fractional precipitation method (Cohn fractionation method, etc.) based on protein solubility, And human plasma proteins such as human serum albumin prepared], or antibiotics.

  In addition, said vaccine takes the form as a colorless and transparent or slightly cloudy odorless liquid goods which added stabilizers, such as gelatin normally. There is also a freeze-dried preparation, which is a powdery solid with a slightly yellowish white taste, and is usually used in a liquid form by adding a prescribed amount of distilled water.

C. Endotoxin measurement means by using the inactivation means of the Limulus reaction activator of the present invention (hereinafter also referred to as the present endotoxin measurement method):
By using such a means for inactivating the limulus reaction-activating substance of the present invention, a method for measuring endotoxin in a sample for measuring limulus comprising the following steps is provided.
(1) A sample for measuring Limulus and a surfactant are allowed to coexist at a temperature exceeding the freezing point temperature of the surfactant to a temperature of 50 ° C. or less, and the Limulus reaction-activating substance of the present invention in the sample for measuring Limulus is not used. The first step to be activated;
(2) A second step of measuring endotoxin by causing the Limulus reaction-activated sample obtained in the first step to undergo a Limulus reaction using a Limulus reagent.

  In the first step, it is more preferable that the sample for measuring Limulus, the surfactant and the alkylamine coexist at a temperature exceeding the freezing point temperature of the surfactant to 50 ° C. or less. The technical significance of adding this alkylamine will be described later. The sample for measuring Limulus is preferably a biological product. Preferred biologics are described above.

  In addition, this invention endotoxin measuring method can be implemented more simply by using the Limulus reagent containing the inactivation agent of the above-mentioned Limulus reaction activator containing the surfactant. In addition, the Limulus reagent that can be used in the endotoxin measurement method of the present invention is not particularly limited as long as it can detect endotoxin, but the Limulus reaction activator of the present invention is false positive in the endotoxin detection system. An endotoxin-specific Limulus reagent is preferred.

  However, when it is clear that the sample for measuring Limulus does not contain β-D-glucan, it is not always necessary to use an endotoxin-specific Limulus reagent, and both endotoxin and β-D-glucan are detected. Limulus reagents can also be used.

  As long as the Limulus reagent can detect endotoxin, in addition to the synthetic substrate method (endpoint assay, kinetic assay), ordinary gelation and turbidimetric methods (endpoint assay, kinetic assay), etc. There is no particular limitation. Various commercially available Limulus reagents include Toxicolor System LS-6, LS-20, LS-200, Endspecy ES-6, Endspecy ES-200, Pyrodic, Pregel, Pregel-S, Pregel-M, Pyrotel Multitest, Pyro. Tel Single Test, Pyroteru T (sold by Seikagaku Corporation), Limulus J Test Wako, Limulus HS-J Test Wako, Limulus J Single Test Wako, Limulus HS-J Single Test Wako, Limulus F Test Wako, Limulus HS-F Test Wako, Limulus F Single Test Wako, Limulus HS-F Single Test Wako, Limulus ES-II Test Wako, Limulus ES-II Single Test Wako, Limulus ES-III Test Wako, Limulus ES-J Test Wako Pure Drug Sales), Pyrogent, Pyrogent Multi Test, Pyrogent Single Test, QCL-1000, Kinetic QCL System (manufactured by BioWittaker, Daiichi Chemical Sales), Cotest Endotoxin (Chromogenic AB Sales) ), Endchrome, Endchrome M (Charles River Laboratories End Safe Sales) Pyrosate (Hemakem Sales), Pyrochrome (Cape Cod Sales).

  Among these commercially available Limulus reagents, there are two types specific to endotoxin: Endospecies and Limulus ES test. Further, without being limited to these commercially available Limulus reagents, any one that activates a series of factor C-based enzyme systems (coagulation systems) by reaction with endotoxin can be used as Takipreus tridentatsu, Takipreus gigas. It is also possible to prepare and use a lysate by a known method from Tachypleus rotan dikauda (Asian horseshoe crab) and Limulus polyphemus (North American) horseshoe crab blood cells.

  Specifically, lysates can be prepared from the hemolymph of these horseshoe crabs by, for example, the method described in J. Biochem., 80, 1011-1021 (1976). “Endotoxin-specific Limulus reagent” specifically inhibits, adsorbs or removes factor G of the lysate (for example, WO 90/02951, USP 5,155,032, USP 5,179,006, WO 92/03736, WO 92/3736). 06381, Japanese Patent Application No. 5-61464, or fractionation and reconstitution of C-factor components (eg, Japanese Patent Publication No. 2-18080, Japanese Patent Publication No. 3-18080, Obayashi T. etal., Clin. Chim. Acta, 149 55-65 (1985)).

  It can also be prepared from factor C, a peptide synthesis substrate, buffer solution, divalent metal salt, etc. described later, or factor C, factor B, a peptide synthesis substrate, buffer solution, divalent metal salt, etc., described below. As a peptide synthesis substrate, a peptide that can be a substrate of active factor C (for example, a peptide consisting of a sequence such as Val-Pro-Arg, Leu-Gly-Arg, Ile-Glu-Ala-Arg, etc., in which the N-terminus is protected, A chromogenic residue (eg, p-nitroaniline, p- (N, N-diethylamino) aniline, p- (N) is added to the carboxyl group of arginine at the C-terminal of methoxycarbonyl-D-hexahydrotyrosyl-Gly-Arg). -Ethyl-N-β-hydroxyethyl) aniline, etc.), fluorescent residues (for example, 7-aminomethylcoumarin, etc.), luminescent residues, ammonia, etc. are substituted with amide bonds. . That is, endotoxin can be measured by measuring amidase activity by measuring reaction products (p-nitroaniline, ammonia, etc.) produced by the action of active factor C on these synthetic substrates. . Specifically, the above-mentioned peptide synthesis substrate is allowed to coexist in a reaction system containing a test solution and a factor C, buffer solution, divalent metal salt, etc. (conversion reaction of the product to other dyes, if necessary) A dye, a fluorescent substance, a luminescent substance or ammonia produced by the reaction, respectively, a spectrophotometer (Japanese Examined Patent Publication No. Sho 63-26871, Japanese Examined Patent Publication No. 3-66319, etc.), a fluorophotometer, a chemiluminescence measuring device, an ammonia detection electrode (Japanese Patent Laid-Open No. Sho 62-148860) can be used, and a method that has already been adopted for the measurement of endotoxin can be used.

  In addition, by appropriately adding alkylamine to the detection system, the association state and dispersion state of endotoxin can be maintained satisfactorily, and the difference in solubility and reactivity depending on the bacterial species can be minimized as much as possible. It was first discovered by the inventors that some data could be obtained. That is, in the method for inactivating the limulus reaction activator or the method for measuring endotoxin of the present invention, a surfactant is allowed to coexist in a sample for measuring limulus at a temperature above the freezing point temperature of the surfactant at a temperature of 50 ° C. or lower. At this time, it is preferable to coexist alkylamine at the same time.

  There are no particular limitations on the method of coexisting the sample for measuring Limulus, a surfactant and an alkylamine, but the alkylamine is mixed with a surfactant in advance to a predetermined concentration, and the mixture is used for Limulus measurement. A method of directly mixing the sample is preferred. If alkylamine is maintained at a predetermined concentration relative to the sample for measuring Limulus, a method of coexisting alkylamine and coexisting surfactant or a method of coexisting surfactant and coexisting alkylamine Alternatively, there is no particular limitation such as a method in which an alkylamine is previously present in the Limulus reagent.

  For example, in an aqueous solution of a surfactant prepared at a predetermined concentration, alkylamine is usually 0.0001% or more and 0.05% (W / V) or less in a final concentration when contacting the sample for measuring Limulus. Preferably, the effect can be easily exerted only by adding 0.002% or more and 0.01% (W / V) or less, and the effect of the present invention can be further enhanced. It was.

  In addition, when the content of the alkylamine is less than 0.001% (W / V), it is difficult to exert the desired effect, and when the content exceeds 0.05% (W / V), it is contained. An increase in the effect corresponding to the increase in the amount does not appear, which is not preferable.

  The alkylamine may have a substituent and is not particularly limited as long as it is soluble in a normal polar solvent and water and is not easily decomposed, but preferably methylmethanamine, ethylethanamine, etc. Secondary amines, and more preferably tertiary amines such as dimethylamine and diethylethanamine. Specifically, N-ethylethanamine (diethylamine), 2,2′-iminodiethanol, bis (2-hydroxyethyl) amine (diethanolamine), N, N-dimethylmethanamine (trimethylamine), N, N-diethyl Examples include ethanamine (triethylamine) and tris (2-hydroxyethyl) amine (triethanolamine).

D. Means for measuring the Limulus reaction-activating substance of the present invention (hereinafter also referred to as the method of measuring the Limulus reaction-activating substance of the present invention):
The present invention provides a method for inactivating the limulus reaction activator of the present invention present in a biologic and the like and specifically measuring only endotoxin, that is, a method for more appropriately performing a pharmaceutical safety test. At the same time, the present invention provides a method for specifically measuring only the Limulus reaction-activating substance. Specifically, the total amount of the Limulus reaction-activating substance and endotoxin of the present invention (total amount of the substance that reacts with the Limulus reagent) is measured using a normal Limulus test, and the value of only endotoxin obtained by various means is measured. Calculate by subtracting.

  In addition, endotoxin is inactivated only by using a substance (also referred to as endotoxin inactivating substance) having a property to specifically inactivate only endotoxin, such as polymyxin such as acid, alkali or polymyxin B and colistin. A method for measuring the limulus reaction activator of the present invention by a normal limulus test is also included in the present invention.

  Of these endotoxin inactivating substances, polymyxins such as polymyxin B and colistin may not always accurately reflect the measured value of this substance. That is, a substance that suppresses the endotoxin neutralizing action of polymyxins may coexist in the sample for measuring Limulus. On the other hand, when processing with an acid or an alkali, there is no such a problem and it can mention as a preferable endotoxin processing method.

  Here, the acid to be used is not particularly limited, but strong acids such as hydrochloric acid, sulfuric acid, and nitric acid are preferable. Moreover, although an alkali is not specifically limited, Strong alkalis, such as sodium hydroxide and potassium hydroxide, are preferable. Usually, an acid or alkali is added to a sample for measuring Limulus, and the sample for measuring Limulus is processed by coexisting it.

  The concentration of these acids or alkalis in the sample for measuring Limulus is not particularly limited, but the final concentration in the sample for measuring Limulus is preferably 0.05M or more, more preferably about 0.05 to 0.2M. .

  Note that the endotoxin-inactivated limulus measurement sample must be neutralized before measurement with the limulus reagent. After neutralization, it is preferable to neutralize so that the pH is about 6-9.

Specifically, the present invention is a method for measuring a Limulus reaction-activating substance of the present invention comprising the following steps.
(1) A first step of measuring a substance that reacts with the Limulus reagent in the Limulus measurement sample using the Limulus reagent.
(2) Separately, a second step of measuring endotoxin in the sample for measuring Limulus by the method for measuring endotoxin of the present invention.
(3) Using the difference between the measured value of the substance reacting with the Limulus reagent obtained in the first step and the measured value of endotoxin obtained in the second step, the amount of the Limulus reaction-activating substance of the present invention is calculated. 3rd process to do.

  Here, the Limulus reagent requires that the Limulus reagent used in the first step and the Limulus reagent used in the second step are the same type of reagents. That is, when an endotoxin-specific limulus reagent is used in the first step, it is necessary to use an endotoxin-specific limulus reagent also in the second step. The Limulus reagent that can be used is the same as the Limulus reagent that can be used in the above-described endotoxin measurement method of the present invention, and is preferably an endotoxin-specific Limulus reagent.

The present invention also includes a method for measuring the Limulus reaction-activating substance of the present invention in a sample for measuring Limulus, comprising the following steps.
(1) A first step of treating a sample for measuring Limulus with an endotoxin inactivating substance, particularly an acid or an alkali.
(2) The limulus reaction-activating substance of the present invention is measured by measuring the change in this reaction system by causing the limulus reaction using the limulus reagent for the endotoxin-inactivated limulus measurement sample obtained in the first step. The second step. As described above, the endotoxin-inactivated limulus measurement sample obtained in the first step needs to be neutralized before measurement with the limulus reagent. The Limulus reagent used here is preferably an endotoxin-specific Limulus reagent.

E. Means for measuring the amount of true endotoxin in biological products (method for measuring endotoxin in biological products of the present invention):
The method for measuring endotoxin in a biologic according to the present invention enables measurement of the true endotoxin content in a biologic by a Limulus reaction, which has been found to be impossible by a conventionally known method. The method of the present invention includes a series of methods for inactivating the limulus reaction-activating substance of the present invention contained in a biological preparation, and then measuring endotoxin with a limulus reagent.

  The inactivation of the Limulus reaction activator of the present invention in the biological preparation is carried out by bringing the biological preparation and the surfactant to be used for the Limulus reagent measurement to a temperature not lower than the freezing point temperature of the surfactant and not higher than 50 ° C. In the presence of a surfactant. In addition, as described above, it is preferable that an alkylamine coexist in addition to the surfactant in the biological preparation to be used in the Limulus reagent, that is, the biological preparation, surfactant and alkylamine to be used in the Limulus reagent measurement. It is more preferable to use a surfactant and an alkylamine which are allowed to coexist at a temperature exceeding the freezing point temperature of the surfactant to a temperature of 50 ° C. or less.

  Further, the present invention is characterized in that the surfactant is allowed to coexist at a temperature exceeding the freezing point temperature of the surfactant to a temperature of 50 ° C. or less to inactivate the Limulus reaction activator according to claim 1. In addition, a pretreatment method of a biological product to be subjected to Limulus reagent measurement is also included. Furthermore, the present invention provides a biological preparation for use in measuring a Limulus reagent, wherein the Limulus reaction activator according to claim 1 is inactivated by a surfactant obtained by this pretreatment method. Is also included.

The inactivation of the Limulus reaction false positive substance in such a biological product provides a method for measuring endotoxin in the biological product comprising the following steps.
(1) Limulus reaction activation according to claim 1, wherein the biological preparation and the surfactant coexist at a temperature above the freezing point temperature of the surfactant to a temperature of 50 ° C. or less. A first step of inactivating the substance;
(2) A second step of measuring endotoxin by causing the biological preparation obtained in the first step to undergo a Limulus reaction with a Limulus reagent and measuring changes in this reaction system.

  In the first step, it is more preferable that the sample for measuring Limulus, the surfactant and the alkylamine coexist at a temperature exceeding the freezing point temperature of the surfactant at 50 ° C. or less. The Limulus reagent that can be used is the same as the Limulus reagent that can be used in the above-described endotoxin measurement method of the present invention, and more preferably an endotoxin-specific Limulus reagent.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
[Production Example] Production of the Limulus Reaction Activating Substance of the Present Invention Influenza HA vaccine (IHA-LotA) and Japanese encephalitis vaccine (JEC-LotA) 50 μl were added to TSKgel G4000SW and TSKgel G3000SW (both manufactured by Tosoh, each column size of 7 × Applied to gel permeation chromatography using high-performance liquid chromatography coupled with 30 mm, volume 14.3 cm ³ ), 0.5 ml / min using distilled water from which endotoxin and β-D-glucan have been removed as a solvent. Elution was performed at a flow rate, and fractions were each 1 ml. The elution pattern when using the influenza HA vaccine is shown in FIG. To each eluted fraction, an aqueous solution of polymyxin B sulfate was added to a final concentration of 0.5 mg / ml, and the endotoxin activity was completely suppressed, followed by endospecy [endotoxin-specific Limulus reagent, sold by Seikagaku Corporation. ), The Limulus reaction activity was measured, and the positive fraction (the fraction having activity) was collected to obtain the Limulus reaction activator of the present invention.

[Example 1] Comparison of physical properties and properties of Limulus reaction activator and endotoxin mixed in vaccine preparation Table 1 shows the above production from influenza HA vaccine (IHA-LotA) and Japanese encephalitis vaccine (JEC-LotA). The physical properties of the Limulus reaction-activating substance of the present invention produced in the examples were compared with various purified endotoxins ( E. coli 0111: B4, E.coli 055: B5, E.coli UKT-B, Salmonella minnesota R595 , S. marcescens , S. typhimurium , Westphal type endotoxin prepared from S. abrtus equi ). After processing each sample for measuring limulus under the conditions shown in the table below, these were treated with endotoxin-specific limulus reagent (Endospecy, Seikagaku) and endotoxin and (1 → 3) -β-D-glucan-free. Change in absorbance [A405nm-492nm (control wavelength)] per minute by reacting in microplate radar (Well Reader SK601, Seikagaku) for 30 minutes using a microtiter plate (Toxipet Plate 96F, Seikagaku) The endotoxin concentration was automatically calculated from the rate (mAbs / min), and the remaining activity (%) of the limulus reaction activator and endotoxin was determined when the untreated control was taken as 100%. From Table 1, the Limulus reaction activator mixed in influenza HA vaccine, Japanese encephalitis vaccine, etc., has a function of directly activating endotoxin sensitivity factor (factor C) present in horseshoe crab blood cells, non-serine protease (C No ability to activate factors and clotting enzyme precursors, and no direct hydrolytic ability to synthetic substrates) and non- (1 → 3) -β-D-glucan (no ability to activate factor G) It was revealed that these molecules have physical properties similar to those of endotoxin that forms polymeric amphiphilic micelles. The activated substance was found not to be adsorbed on a glass test tube even when left at a low temperature for a long time, and has the property of stably maintaining the Limulus activity.

Note) ・ Pyrogenicity: Carry out in accordance with the rabbit fever test described in the Japanese Pharmacopoeia, and positive if the value obtained by subtracting the starting body temperature from the maximum body temperature (rectal temperature) after 3 hours is> 0.55 ° C.
Polymyxin B: An aqueous solution of the same sulfate (Sigma, used: 0.5 mg / ml)
・ Acid: 0.2M hydrochloric acid, treated at 37 ℃, 60 minutes ・ Alkali: 0.2M potassium hydroxide, treated at 37 ℃, 60 minutes ・ Thermal stability: treated at 100 ℃, 60 minutes ・ Solution stability: 0.02M phosphate buffer (PH 7.0), left at 4 ° C. for 10 days. Synthetic substrate: Boc-Leu-Gly-Arg-pNA hydrochloride (during reaction: 3.0 mM)
-Factor C activation ability: Using purified factor C (prepared by the method of Nakamura T. et al., Eur. J. Biochem. 154 , 511-521 (1986)), Measured by amidase activity.
Inhibition by anti-factor C antibody: Measurement was started after adding 1 volume of a 250-fold diluted solution of mouse monoclonal antibody (2C12) to factor C to 1 volume of Limulus measurement sample.
・ Activity of factor G: Coagulation enzyme of active factor G using purified factor G (prepared by the method of Obayashi T. et al., Clin. Chi, Acta, 149, 55-65 (1985)) Precursor activation ability was measured by the amidase activity of the synthetic substrate.
Coagulase precursor activation ability: Like factor G, the ability of the coagulase precursor purified by the method of Obayashi et al. To activate coagulase is measured by the amidase activity of the synthetic substrate.
· Metal _{Ion: NaCl, KCl, MgCl 2,} CaCl 2 aqueous solution (processing time: 0~1.5M)
・ Endotoxin adsorbent: END-X-B15 (adsorbent with endotoxin neutralizing factor in horseshoe crab blood cells as a ligand, manufactured by Cape Cod, sold by Seikagaku Corporation), Pyrosep A & C (adsorbent with histidine as a ligand, manufactured by Tanabe Seiyaku) , Wako Pure Chemical Industries, Ltd.), collecting the unadsorbed fractions, measuring endotoxin with endospecies, and calculating the adsorption rate (%) on the carrier.
*: See Example 2

[Example 2] Effect of various surfactants on Limulus reaction activator and endotoxin The present Limulus reaction activity obtained from influenza HA vaccine (IHA-LotA) or Japanese encephalitis vaccine (JEC-LotA) produced in the above production example Take 25 μl of chemical substance and E. coli 0111: B4 endotoxin (Westphal type, Et-B4, 2EU / ml) on Toxipet Plate 96F, and use polyoxyethylene or other nonionic surfactant (trade names Tween, Triton) , Brij et al., Sigma, Aldrich, Wako Pure Chemical, and Dojindo Laboratories) 25 μl of aqueous solution and mixed well, followed by addition of 50 μl of endospecy and reaction at 37 ° C. for 30 minutes. The residual activity (%) was calculated using distilled water as a control. From Table 2, it can be seen that when a surfactant having an appropriate concentration is added, there is a large difference in the residual activity depending on the surfactant. For example, by mixing with Brij56, it is clear that the Limulus reaction activator mixed in IHA can be completely inactivated while maintaining the endotoxin activity well. As is clear from Table 2, there are other surfactants having similar actions such as Triton N-101 and Tergitol in addition to Brij56, and an aqueous solution of such a surfactant coexists in the sample for measuring Limulus as appropriate. It was found that, by making it possible to eliminate the influence (false positive) on the Limulus reaction of the Limulus reaction activator mixed in the Limulus measurement sample, only endotoxin can be specifically measured.

[Example 3] Effect of various surfactants on endotoxin measurement in various biological preparations Biological preparations containing high concentrations of Limulus reaction activator [influenza HA vaccine (IHA-LotB, undiluted solution), Japanese encephalitis vaccine (JEC-LotB, undiluted solution) and human serum albumin (HSA-LotY1) adjusted to 2.5% (W / V)] were dispensed into microplates, respectively, and Tergitol, Triton N-101 and Brij 56 (all Sigma After mixing, add 50 μl of Endospecy, and add endotoxin concentration according to the method shown in Example 1. The residual ratio of the Limulus activity (%) was calculated as to how much the value remained compared to the value when no addition (distilled water) was used as a control. In addition, endotoxin (Et-B4) with a known concentration (1.2EU / ml) is added to various biological products that contain almost no Limulus reaction activator, and the recovery rate (%) is calculated with distilled water as 100%. did. As can be seen from Table 3, if an aqueous solution of a surfactant prepared to an appropriate concentration is used, non-specific endotoxin reaction does not remain, and conditions under which only endotoxin can be specifically measured should be set. Is possible. That is, by mixing a surfactant aqueous solution and a sample for measuring Limulus at an appropriate concentration, the endotoxin recovery rate is good and non-specific endotoxin activity (Limulus reaction activation after mixing with the sample for Limulus measurement). It can be seen that the reactivity of the substance can be eliminated, and only endotoxin in the sample for measuring Limulus can be specifically measured. From Table 3, it is clear that IHA can achieve the above objectives using Brij 56, JEC, Tergitol, Triton N-101 and Brij 56, and HSA using an aqueous solution of appropriate concentration of Brij56. is there.

[Example 4] Influence of Brij56 concentration on endotoxin measurement in various biological preparations Prepared to be 0-0.5% (W / V) when mixed in 25 μl of various biological preparations used in Example 3 After adding 25 μl of Brij 56 aqueous solution, 50 μl of Endospecy was added, and the endotoxin concentration (EU / ml) was measured by the method described in Example 1. At the same time, the endotoxin addition recovery rate (%) at the concentration of each Brij 56 aqueous solution was measured. The results are summarized in FIG. As the concentration of the Brij 56 aqueous solution increases, the limulus activity (non-specific endotoxin concentration) and the endotoxin addition recovery rate decrease significantly. Therefore, the concentration of Brij 56 capable of suppressing the nonspecific endotoxin (Limulus reaction activator) activity to the maximum while appropriately maintaining the addition recovery rate of endotoxin is appropriately selected. In this selected concentration range, it is clear that the Limulus reaction-activating substance mixed in the sample for measuring Limulus can be inactivated and only endotoxin can be specifically measured.

[Example 5] Influence of Brij temperature during endotoxin measurement in various biological preparations and standing time at each temperature after mixing 0.125% (W / V) when mixed with 25 μl of influenza HA vaccine (IHA-LotB) Brij 56 aqueous solution was added so that it might become, and it mixed at 4-80 degreeC for 1 minute. After mixing separately, the mixture was further allowed to stand at the same temperature for 1 to 20 minutes. The relative values of the endotoxin activities (remaining limulus activity when distilled water was used as a control (100%)) and the endotoxin addition recovery rate (%) were calculated. FIG. 4 shows the results when stirring was performed for 1 minute at each temperature. From FIG. 4, it is possible to maintain the endotoxin recovery rate and to suppress the limulus activity to the maximum by simply adding an aqueous solution of the surfactant to the sample for measuring Limulus and stirring at 4 to 50 ° C. for 1 minute. (At 4 to 40 ° C., the limulus activity remaining rate and the endotoxin addition recovery rate were not particularly affected even when left for 5 minutes.) Furthermore, under the heating conditions of 60 ° C. or higher, endotoxin activity is not maintained stably, and it has been impossible to accurately measure endotoxin in a sample for measuring Limulus.

[Example 6] Effect of polymyxin B, EDTA-4Na and Brij 56 on endotoxin measurement in various biological products Polymyxin B was used in a total of 6 lots of 25 µl containing the same biological product as in Example 3. Sulfate (sold by Sigma, 2 mg / ml) aqueous solution 25 μl or 10 mM EDTA-4Na aqueous solution 25 μl or 0.25% (W / V) Brij 56 aqueous solution 25 μl, Endospecie 50 μl is added and described in Example 1 Similar to the method, the endotoxin concentration (EU / ml) was determined. Further, the control (total amount of endotoxin and Limulus reaction activator) to which none of polymyxin B, EDTA-4Na and Brij 56 was added was 1, and the ratio of the activity to the control under each condition was calculated. Only the endotoxin contained in the sample for measuring Limulus is specifically inactivated by the addition of polymyxin B or EDTA-4Na aqueous solution of the prescribed concentration, and only the Limulus reaction activator contained in the sample for measuring Limulus by adding Brij 56 Assuming that is specifically inactivated, the sum of the ratio of the value obtained with each addition to the control should equal 1 which is the total amount of endotoxin and limulus reaction activator. As is clear from FIGS. 5A and B (similar tests performed in different lots), the sum of each biological product is 1 ± 0.05 (CV = 5%, n = 3). ) Proved that the assumption was correct. From this, it is possible to inactivate only the Limulus reaction-activating substance and specifically measure endotoxin by coexisting an aqueous solution of Brij56 with a sample for measuring Limulus so as to have a predetermined concentration. I understand.

[Example 7] Dilution dose responsiveness of various biological products To a biological product similar to Example 3, an equal amount of 0.25% (W / V) Brij 56 aqueous solution was added, and then distilled water or 0.125% Take 25 μl of the diluted solution prepared in Brij 56 aqueous solution to × 2 to × 32 and 25 μl of the diluted solution prepared in advance to × 2 to × 32 with distilled water to the Toxipet Plate 96F, and add distilled water and 0.25% (W / V ) After adding 25 μl of an aqueous solution of Brij 56, the mixture was stirred well, 50 μl of endospecies was added, and the endotoxin concentration was measured by the method described in Example 1. Relative activity (%) obtained by dividing each value by the true endotoxin value, that is, the measurement value obtained by adding 25 μl of 0.25% (W / V) Brij 56 aqueous solution to 25 μl of Limulus measurement sample at each dilution ratio Plotted. As shown in FIGS. 6A to 6C, the sample for limulus measurement previously diluted with distilled water was added with an aqueous solution of Brij 56, while stable relative activity was obtained at each dilution rate. A sample diluted with distilled water after adding an aqueous solution of Brij 56 to the sample showed a tendency that the measured value increased as the dilution rate increased. On the other hand, after adding an aqueous solution of Brij 56 to a sample for measuring Limulus and then diluting with an aqueous solution of 0.125% (W / V) Brij 56 instead of distilled water, the sample for measuring Limulus previously diluted with distilled water As in the case of adding an aqueous solution of Brij 56, stable relative activity was exhibited and a good dilution dose response was observed. Therefore, once the Limulus reaction-activating substance that has been inactivated by Brij 56 is diluted by adding distilled water (that is, the concentration of Brij 56 is diluted from the sample for measurement), Some reversible recovery phenomena are observed, but this recovery phenomenon is completely observed when the sample for Limulus measurement is diluted with Brij 56 to inactivate with Brij 56 and the same concentration as that. It can be seen that all dilutions show a constant endotoxin value.

  This is a point that is greatly different from the properties of known Limulus reaction false positive substances and the like, and the inactivation method of Limulus reaction false positive substances produced mainly using blood as a sample for measuring Limulus. That is, it is known that once these false positive substances are inactivated by the inactivation method, the Limulus activity is no longer recovered at all after any treatment. The limulus reaction activator of the present invention is capable of suppressing its limulus reaction activation ability only in the presence of a specific concentration of the above-mentioned specific surfactant. Therefore, when eliminating the influence of the Limulus reaction activator of the present invention and accurately measuring only the true endotoxin in the Limulus measurement sample with the Limulus reagent, a certain concentration of the specific surfactant is always present together. It is necessary to keep. This point is greatly different from the endotokin measurement method related to the inactivation method of the conventional Limulus reaction false positive substance and the like.

[Example 8] Measurement of endotoxins in biological preparations using various Limulus reagents To the various biological preparations used in Example 3, an equal amount of 0.3% (W / V) Brij 30 aqueous solution was added to Endospecies or various Limulus reagents. And the endotoxin concentration (EU / ml) in the sample for measuring Limulus was measured using standard endotoxin ET-B4. The results are shown in Table 4.

  From Table 4, after adding an aqueous solution of Brij 30 to the sample for measuring Limulus, the endotoxin concentration in the sample for measuring Limulus was measured using various Limulus reagents. It can be seen that only endotoxin can be specifically measured only by allowing Brij30 to coexist in the sample for measuring Limulus, even if the measurement method and measurement reagent are different.

[Example 9] Effect of alkylamine on endotoxin reactivity Mainly various S-type and R-type endotoxins ( E.coli O111: B4 3 types, E.coli UKT-B, E .coli 0113, Salmonella minnesota R595, S.minnesota R5 (Rc), S.typhosa, S.enteritidis) each water dilution series (n = 9) aqueous solution of Brij to 25 [mu] l 56 (0.25% aqueous solution) 25 [mu] l added and the mixture Thereafter, 50 μl of endospecy was added, endotoxin concentration (EU / ml) was determined by the method shown in Examples, and plotted on a logarithmic axis. As a result, the titers differed depending on the type of endotoxin. However, when this heterogeneity was previously added to an aqueous solution of Brij 56 so as to be 0.005% (W / V), FIG. As shown in -A and B, the results showed good focusing (multiple correlation = 0.844). In addition, as a result of confirming dose dependency by adding various endotoxins to influenza HA vaccine (IHA-LotM) containing almost no endotoxin, only Brij 56 was allowed to coexist as shown in FIGS. It can be seen that better focusing (multiple correlation = 0.850) is shown when triethylamine is added than when adding triethylamine. Therefore, by adding a predetermined concentration of triethylamine to an aqueous solution of Brij 56, the dispersion state of endotoxin can be maintained well, and the difference in solubility and reactivity depending on the bacterial species can be minimized. It was revealed that endotoxin in the Limulus measurement sample can be measured more accurately.

[Example 10] Endotoxin measurement by parallel line quantification method in various biological products Diluted water dilution series (4 points) of 3 lots of various biological products (IHA-LotD, JEC-F, HSA-Y2) 25 μl each, 25 μl of Brij mixed solution [0.25% (W / V) Brij56 + 0.010% (W / V) triethylamine] was added and stirred, 50 μl of Endspecy attached to the kit was added, and 37 ° C. with well reader SK601. The reaction was performed for 30 minutes, and the rate of change in absorbance per minute (mAbs405-492nm / min) was analyzed with a dedicated parallel line quantitative software (EG301, Seikagaku Kogyo Co., Ltd.), and the Japanese Pharmacopoeia Standard Endotoxin ( E.coli UKT-B ) Was used to calculate the endotoxin concentration (EU / ml). As shown in FIG. 9, good parallel lines and reproducibility were obtained for any sample for measuring Limulus, and this kit was able to specifically and accurately quantify endotoxin in biological preparations. .

[Example 11] Measurement of limulus reaction activator of the present invention in various biological preparations Add 50 µl of various biological preparations described in Example 8 to an equal volume of 0.2M NaOH and warm at 37 ° C for 1 hour. Thereafter, 50 μl of 0.2M HCl was added for neutralization. 50 μl of the endospecies was added to 50 μl, and the mixture was reacted at 37 ° C. for 30 minutes (A), and the amount of the limulus reaction activator of the present invention was measured. On the other hand, the total amount of endotoxin and the Limulus reaction activator of the present invention measured using distilled water instead of NaOH (C), and after adding 0.25% Brij 5625 μl to 25 μl of this preparation, add Endospecy 50 μl, and measure the endotoxin amount ( B) is also shown.

  As shown in Table 5, in each of the IHA, JEC, and HSA preparations, only the Limulus reaction activator of the present invention mixed in the preparation is specifically measured only by adding NaOH and heating at 37 ° C. It turns out that it is possible. In addition, the value obtained by adding the amount of the limulus activating substance of the present invention obtained by this method to the measured value of the endospecies obtained by adding an aqueous solution of Brij 56 to this preparation is the endotoxin and the limulus reaction activity of the present invention. It was clear that the amount of the Limulus reaction-activating substance of the present invention can be accurately measured by this method because the value was almost equal to the total amount of the activating substance.

It is the figure which showed the cascade mechanism of the horseshoe crab coagulation system. It is drawing which showed the elution pattern of the Limulus reaction activation substance of this invention in the influenza vaccine by a high performance liquid chromatography. 2 is a drawing showing the effect of various concentrations of surfactants on the measurement of endotoxins in biological products. It is the figure which showed the influence of the temperature and time after addition of surfactant in the measurement of endotoxin in biological preparations. It is drawing which showed the influence which it has on endotoxin measurement of polymyxin B, EDTA-4Na, and surfactant. It is drawing which showed dilution dose response. It is drawing which showed the influence (distilled water system) of the surfactant triethylamine on various endotoxin reactivity. It is the figure which showed the influence (biological formulation system) of surfactant and trimethylamine on various endotoxin reactivity. It is the figure which showed the endotoxin measurement in the biological preparation by a parallel-line quantitative method.

Claims (27)

When the biological product was eluted by gel permeation chromatography using high performance liquid chromatography using distilled water from which endotoxin and (1 → 3) -β-D-glucan were removed as a solvent, the biological volume was reduced to 1 Limulus reaction activator which is present in the elution fraction between 3/3 and 4/5 and has the following properties (1) to (12):
(1) has the ability to activate Limulus reaction;
(2) reacts with endotoxin-specific Limulus reagent;
(3) Activate factor C derived from horseshoe crab amebocyte lysate;
(4) Exothermic substance test shows no exothermicity;
(5) not endotoxin;
(6) not (1 → 3) -β-D-glucan;
(7) has no serine protease activity;
(8) Even if polymyxin B coexists with this substance, the ability to activate the Limulus reaction of this substance does not decrease;
(9) Even if colistin coexists with this substance, the ability to activate the Limulus reaction of this substance does not decrease;
(10) Even if this substance is treated with 0.2 M hydrochloric acid at 37 ° C. for 60 minutes, the Limulus reaction activation ability of this substance does not decrease;
(11) Even if this substance is treated with 0.2M potassium hydroxide at 37 ° C. for 60 minutes, the ability to activate the Limulus reaction of this substance does not decrease;
(12) By treating this substance with polyoxyethylene hexadecyl ether, the ability to activate the Limulus reaction of this substance disappears. The surfactant for inactivating the Limulus reaction activator according to claim 1, wherein the sample for measuring Limulus and the surfactant coexist at a temperature exceeding the freezing point temperature of the surfactant to 50 ° C or less. use. The use of a surfactant according to claim 2, wherein the surfactant is a nonionic surfactant. 4. The nonionic surfactant is one or more nonionic surfactants selected from the group consisting of polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether and acyl polyoxyethylene sorbitan. Use of the described surfactants. The inactivating agent for a Limulus reaction activator according to claim 1, comprising a surfactant. The inactivating agent for a Limulus reaction activator according to claim 5, wherein the surfactant is a nonionic surfactant. The nonionic surfactant is one or more nonionic surfactants selected from the group consisting of polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether and acyl polyoxyethylene sorbitan. Deactivators of the described Limulus reaction activator. A Limulus reagent comprising the Limulus reaction activator inactivating agent according to any one of Claims 5 to 7 in an effective amount for inactivating the Limulus reaction activator. The Limulus reagent according to claim 8, wherein the Limulus reagent is an endotoxin-specific Limulus reagent. A kit for measuring Limulus comprising an inactivating agent for a Limulus reaction-activating substance according to any one of Claims 5 to 7. The limulus measurement kit according to claim 10, wherein the limulus reagent contained in the limulus measurement kit is an endotoxin-specific limulus reagent. A sample for measuring Limulus, wherein the Limulus reaction-activating substance according to claim 1 is inactivated by a surfactant. The sample for measuring Limulus according to claim 12, wherein the surfactant is a nonionic surfactant. The nonionic surfactant is one or more nonionic surfactants selected from the group consisting of polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether and acyl polyoxyethylene sorbitan. The sample for limulus measurement as described. A method for measuring endotoxin in a sample for measuring Limulus comprising the following steps: (1) A sample for measuring Limulus and a surfactant are allowed to coexist at a temperature above the freezing point temperature of the surfactant at a temperature of 50 ° C. or less. A first step of inactivating the Limulus reaction-activating substance according to claim 1 in the Limulus measurement sample;
(2) A second step of measuring endotoxin by causing the Limulus reaction-activated sample obtained in the first step to undergo a Limulus reaction using a Limulus reagent. The method for measuring a Limulus reaction-activating substance according to claim 1 in a sample for measuring Limulus, comprising the following steps:
(1) a first step of measuring a substance that reacts with a reagent in a sample for measuring Limulus using a Limulus reagent;
(2) A second step of measuring endotoxin in the same sample for measuring Limulus as in the first step using the endotoxin measurement method according to claim 15;
(3) The difference between the measured value of the substance that reacts with the Limulus reagent obtained in the first step and the measured value of endotoxin obtained in the second step, in the sample for measuring Limulus according to claim 1 A third step of calculating the amount of the Limulus reaction activator. The method for measuring a Limulus reaction-activating substance according to claim 16, wherein the Limulus reagent is an endotoxin-specific Limulus reagent. The method for measuring a Limulus reaction-activating substance according to claim 1 in a sample for measuring Limulus, comprising the following steps:
(1) a first step of inactivating endotoxin in a sample for measuring Limulus by treating the sample for measuring Limulus with an endotoxin inactivating substance;
(2) The second step of measuring the limulus reaction-activating substance according to claim 1 by subjecting the endotoxin-inactivated limulus measurement sample obtained in the first step to a limulus reaction using a limulus reagent. The method for measuring a Limulus reaction-activating substance according to claim 18, wherein the endotoxin inactivating substance is an acid or an alkali. The method for measuring a Limulus reaction-activating substance according to Claim 23 or Claim 24, wherein the Limulus reagent is an endotoxin-specific Limulus reagent. The inactivation of the Limulus reaction activator according to claim 1, wherein the biological agent and the surfactant used for the measurement of Limulus are allowed to coexist at a temperature above the freezing point temperature of the surfactant to a temperature of 50 ° C or lower. Use of surfactants. The use of a surfactant according to claim 21, wherein the surfactant is a nonionic surfactant. The nonionic surfactant is one or more nonionic surfactants selected from the group consisting of polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, and acyl polyoxyethylene sorbitan. Use of the described surfactants. A biological preparation for use in limulus measurement, wherein the limulus reaction activator according to claim 1 is inactivated by a surfactant. 25. The biologic of claim 24, wherein the surfactant is a nonionic surfactant. 26. The nonionic surfactant is one or more nonionic surfactants selected from the group consisting of polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, and acyl polyoxyethylene sorbitan. The biologic described. A method for measuring endotoxin in a biologic comprising the following steps:
(1) The biological agent and the surfactant coexist at a temperature above the freezing point temperature of the surfactant to a temperature of 50 ° C. or less, and the Limulus reaction activator according to claim 1 in the biological agent A first step of inactivating
(2) A second step of measuring endotoxin by causing the biological preparation obtained in the first step to undergo a Limulus reaction with a Limulus reagent.