JP3950470B2 - Limulus reaction-activating substance, method for inactivating this substance, and method for measuring this substance and endotoxin - Google Patents

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 a Limulus reaction false positive substance mixed in a biological preparation on the Limulus reaction. In other words, 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 present in a biologic, and to eliminate the influence of this causative agent from the Limulus reaction system. 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). The present inventors have found that the influence of this causative substance can be eliminated from the Limulus reaction system by coexisting a biologic with a surfactant .

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 group consisting of a vaccine preparation, a blood preparation and an antibiotic. The Limulus reaction activator according to (1) above, wherein a certain amount of a biologic selected from the above and a surfactant are allowed to coexist in a certain amount at a temperature exceeding the freezing point temperature of the surfactant to 50 ° C. or less. Use of a surfactant to inactivate in the biological product ; (3) selected from the group consisting of vaccine preparations, blood products and antibiotics by using the surfactant of (2) above And (4) a method for measuring the limulus reaction activator using the method for producing endotoxin according to (3).

  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 separated from various biological means (for example, blood products, vaccines, antibiotics, etc.) using various physical and chemical properties of the Limulus reaction activator of the present invention. Can be purified. 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 biological product is applied to the chromatography column, and endotoxin and β-D-glucan-free distilled water are eluted as a solvent. The flow rate of the solvent is not particularly limited, but is preferably about 0.5 ml / min.

An aqueous solution of polymyxin B sulfate was added to the fraction eluted under the conditions of (13) below to a final concentration of 0.5 mg / ml to completely suppress endotoxin activity, and this fraction was treated with endotoxin-specific limulus. The Limulus reaction-activating substance of the present invention can be obtained by measuring with a reagent and collecting the positive fraction. 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 properties described in (1) to (12 ) below . 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) that are not endotoxin.
(6) (1 → 3) is not a -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.
(13) TSKgel G4000SW and TSKgel G3000SW (any color problem size even 7 × 30 mm, volume 14.3 cm ³⁾ using a column ligated and to the flow rate of the solvent and 0.5 ml / min.

  Although the in vivo action and toxicity 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. 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-mentioned limulus reaction activator of the present invention is contained in a biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics , a false positive reaction will be induced in the limulus reagent. This is not preferable because the detection accuracy of a desired substance (for example, endotoxin) is lowered.

  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% (final concentration when contacted with a biological product selected from the group consisting of vaccine preparations, blood preparations and antibiotics ). W / V), preferably 0.01% to 0.5% (W / V), more preferably 0.05% to 0.3% (W / V), etc., but these are only examples However, it is not limited to these.

  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 an order and method for coexisting a surfactant or an aqueous solution of a surfactant with a biological preparation selected from the group consisting of a vaccine preparation, a blood preparation and an antibiotic containing a Limulus reaction activator, The surfactant is not particularly limited as long as it is present in the biological preparation so as to have a predetermined concentration without any modification or destruction. Vaccine formulations, methods coexist with biologics and surfactant selected from the group consisting of blood products and antibiotics is usually added and the biologics and surfactant are thoroughly mixed Is achieved. Further, vaccine formulations, the order in which coexist with biologics and surfactant selected from the group consisting of blood products and antibiotics in advance by adding a surfactant or the like in the Limulus reagent, the biological product Although the object of the invention can be achieved by carrying out the mixing and reaction simultaneously with it, it is more desirable in view of the effect that it is preliminarily mixed with the biological preparation before the Limulus reaction. The amount of the aqueous solution of the surfactant or the like to be mixed with a biological preparation selected from the group consisting of a vaccine preparation, a blood preparation and an antibiotic is 0.1% of the surfactant solution per volume of the biological preparation . Although it is preferable to carry out by 1-10 volume, it will not specifically limit if the surfactant of the predetermined density | concentration is added and the effective density | concentration is maintained in the said biological preparation .

  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.

  Also, 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, and 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 biological agent selected from the group consisting of vaccine preparations, blood preparations and antibiotics and the surfactant to coexist at a temperature above the freezing point temperature of the surfactant to 50 ° C. or less is particularly The endotoxin molecule and its associated micelles are not particularly limited as long as they are physicochemically stable and do not adsorb to the container, but it is usually several seconds to 5 minutes, and about 1 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 method for inactivating the Limulus reaction activator of the present invention described above is also used for inactivating the Limulus reaction activator of the present invention in a biological product selected from the group consisting of vaccine preparations, blood preparations and antibiotics. It can be applied as it is. Thus, the present invention provides a biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics and a surfactant at a temperature above the freezing point temperature of the surfactant to a temperature of 50 ° C. or less. The use of a surfactant to inactivate the Limulus reaction activator of the present invention, which coexists in Further, as will be described later, it is more preferable that an alkylamine coexists in addition to the surfactant in the biological preparation .

Thus, a means for inactivating the aforementioned limulus reaction activator of the present invention is provided, whereby the limulus reaction activator of the present invention is directly inactivated by a surfactant , vaccine preparation, blood A biological product selected from the group consisting of a formulation and an antibiotic is provided, but this inactivation means can be used in various forms. For example, the Limulus reaction activation comprising a surfactant based on the premise that the biologic and the surfactant coexist at a temperature exceeding the freezing point temperature of the surfactant to a temperature of 50 ° C. or less. It is possible to apply to the form as a substance 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 allows a surfactant to coexist in a biological product selected from the group consisting of vaccine preparations, blood preparations and antibiotics at a temperature exceeding the freezing point temperature of the surfactant to a temperature of 50 ° C. or lower. And a method for eliminating the influence of the limulus reaction activator of the present invention on the limulus reaction from the biological preparation .

Furthermore, the present invention includes a biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics, wherein the Limulus reaction-activating substance of the present invention is inactivated by the surfactant.

In the present invention, “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 (eg, 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, endotoxin in a biological product selected from the group consisting of vaccine preparations, blood preparations and antibiotics comprising the following steps: A measurement method is provided.
(1) A biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics, and a surfactant at a temperature above the freezing point temperature of the surfactant to 50 ° C. or less. A first step of inactivating the Limulus reaction-activating substance of the present invention in the biological preparation by coexisting so that the final concentration of the agent is 0.005 to 0.8% (W / V);
(2) The second step of measuring endotoxin by causing the Limulus reaction-activating substance inactivated biological preparation obtained in the first step to undergo a Limulus reaction with a Limulus reagent.

In the first step, the biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics, and the surfactant at a temperature above the freezing point temperature of the surfactant to a temperature of 50 ° C. or less. It is more preferable to coexist .

  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 a biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics does not contain β-D-glucan, it is necessary to use an endotoxin-specific Limulus reagent. Rimulus reagent that detects both endotoxin and β-D-glucan 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 of the present invention and the method for measuring endotoxin of the present invention, a surfactant is added to a biological product selected from the group consisting of vaccine preparations, blood preparations and antibiotics. When coexisting at a temperature exceeding the freezing point temperature of 50 ° C. or less, it is preferable that alkylamine coexist at the same time.

There is no particular limitation on the method of coexisting a biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics , surfactants , and alkylamines. It is preferable to mix the liquid mixture so as to have a concentration of 1 and then directly mix the liquid mixture with the biological preparation . If the alkylamine is maintained at a predetermined concentration for the biological product , a method of coexisting the surfactant after coexisting the alkylamine, or coexisting the alkylamine after coexisting the surfactant There is no particular limitation such as a method or a method in which an alkylamine is previously present in the Limulus reagent.

For example, in an aqueous solution of a surfactant prepared to a predetermined concentration, the final concentration when alkylamine is brought into contact with a biological product selected from the group consisting of vaccine preparations, blood preparations and antibiotics is usually 0. The effect can be easily exerted simply by adding 0001% to 0.05% (W / V), preferably 0.002% to 0.01% (W / V). Thus, the effects of the present invention can be further enhanced.

  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 inactivates the Limulus reaction activator of the present invention present in a biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics , and specifically measures endotoxin only, that is, a pharmaceutical product. In addition, the present invention provides a method for appropriately performing only the limulus reaction activator of the present invention. 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 a biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics . 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, the biological preparation is treated by adding an acid or alkali to a biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics .

The concentration of these acids or alkalis in the biological product selected from the group consisting of vaccine preparations, blood products and antibiotics is not particularly limited, but the final concentration in the biological product is 0. 05M or more is preferable, and about 0.05 to 0.2M is more preferable.

Note that a biological product selected from the group consisting of endotoxin-inactivated vaccine preparations, blood preparations and antibiotics 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 a Limulus reagent in a biological product selected from the group consisting of a vaccine product, a blood product and an antibiotic using a Limulus reagent.
(2) Separately, a second step of measuring endotoxin in the biological preparation by the endotoxin measurement method 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 activator of the present invention in a biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics, comprising the following steps.
(1) A first step of treating a biological preparation selected from the group consisting of a vaccine preparation, a blood preparation and an antibiotic with an endotoxin inactivating substance, particularly an acid or an alkali.
(2) The endotoxin-inactivated biological product obtained in the first step is subjected to a Limulus reaction with a Limulus reagent, and the change in this reaction system is measured to measure the Limulus reaction-activating substance of the present invention. The second step. As described above, the endotoxin-inactivated biological preparation 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.

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 treating each biological product under the conditions shown in the table below, they 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 and the like has a function of directly activating endotoxin-sensitive 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 biological product .
・ 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 Limulus reaction activity of the present invention 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 that have similar actions, such as Triton N-101 and Tergitol, in addition to Brij56, and aqueous solutions of these surfactants coexist in biological products as appropriate. It was found that, by making it possible to eliminate the influence (false positive) of the Limulus reaction activator contained in the biological preparation on the Limulus reaction, 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) prepared 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 a surfactant aqueous solution prepared at an appropriate concentration is used, nonspecific endotoxin reaction does not remain, and conditions for specifically measuring endotoxin alone should be set. Is possible. In other words, the endotoxin recovery rate is good and the non-specific endotoxin activity (Limulus reaction activation after mixing with the biologic product is good only by mixing the surfactant aqueous solution and the biologic formulation of appropriate concentration respectively. It can be seen that the reactivity of the substance can be eliminated and only endotoxin in the biological product can be specifically measured. From Table 3, it is clear that IHA can achieve the above objectives with Brij 56, JEC with Tergitol, Triton N-101 and Brij 56, and HSA with the appropriate concentration of Brij56 aqueous solution. 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 activator mixed in the biological product can be inactivated and only endotoxin can be specifically measured.

[Example 5] Effect of temperature at Brij mixing and standing time at each temperature after mixing in endotoxin measurement in various biological preparations
Brij 56 aqueous solution was added to 25 μl of influenza HA vaccine (IHA-LotB) so that it would be 0.125% (W / V) at the time of mixing, and mixed at 4-80 ° C. 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 keep the endotoxin recovery rate and to suppress the limulus activity to the maximum by simply adding an aqueous solution of the surfactant to the biological preparation 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 rather not stably maintained, and it has been impossible to accurately measure endotoxin in biological preparations .

[Example 6] Effect of polymyxin B, EDTA-4Na and Brij 56 on endotoxin measurement in various biological preparations
A total of 6 lots of 25 μl containing the same biological products as in Example 3 were used, 25 μl of polymyxin B sulfate (Sigma, 2 mg / ml) aqueous solution 25 μl or 10 mM EDTA-4Na aqueous solution 25 μl or 0.25% (W / V) 25 μl of Brij 56 aqueous solution was mixed, 50 μl of endospecy was added, and the endotoxin concentration (EU / ml) was determined in the same manner as described in Example 1. 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 biological product 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 biological product 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 apparent from FIGS. 5-A and B (similar tests performed in different lots), the sum of each of the biologics 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 activator and specifically measure endotoxin by coexisting an aqueous solution of Brij56 with a biological preparation so as to have a predetermined concentration. I understand.

[Example 7] Dilution dose response of various biological preparations
An equal amount of 0.25% (W / V) Brij 56 aqueous solution was added to the same biological preparation as in Example 3, and then 25 μl of a diluted solution prepared in distilled water or 0.125% Brij 56 aqueous solution to × 2 to × 32 In addition, take 25 μl of the diluted solution prepared in × 2 to × 32 with distilled water in advance on the Toxipet Plate 96F, add 25 μl each of distilled water and 0.25% (W / V) Brij 56 aqueous solution, and stir well. 50 μl 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 measured value obtained by adding 25 μl of 0.25% (W / V) Brij 56 aqueous solution to 25 μl of biological product , at each dilution ratio Plotted. As shown in FIGS. 6A to 6C, when a biological product previously diluted with distilled water and an aqueous solution of Brij 56 were added, stable relative activity was obtained at each dilution rate, whereas biological When the aqueous solution of Brij 56 was added to the preparation and then diluted with distilled water, the measured value tended to increase as the dilution rate increased. In contrast, when an aqueous solution of Brij 56 is added to a biological product and then diluted with an aqueous solution of 0.125% (W / V) Brij 56 instead of distilled water, the biological product 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 activator has been inactivated by Brij 56, it is diluted by adding distilled water (ie, the concentration of Brij 56 is diluted in the biological product ). In some cases, a reversible recovery phenomenon is observed, but inactivation with Brij 56 and the biological product diluted with Brij 56 to the same concentration as this concentration, such recovery phenomenon is completely It is not recognized, and 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 biological product . 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 the influence of the limulus reaction activator of the present invention is eliminated and only true endotoxin in a biological product is accurately measured with a limulus reagent, a constant concentration of the specific surfactant is always present. It is necessary to keep it. 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] Endotoxin measurement in biological preparations using various Limulus reagents
Equal amounts of 0.3% (W / V) Brij 30 aqueous solution are added to various biological preparations used in Example 3, and Endospecies or various Limulus reagents are added to determine the endotoxin concentration (EU / ml) in the biological preparations. Measurement was performed 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 biological preparation , the endotoxin concentration in the biological preparation was measured using various Limulus reagents. It can be seen that only endotoxin can be specifically measured only by allowing Brij30 to coexist in a biologic 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. 8-A and B. 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 became clear that endotoxins in biological products can be measured more accurately.

[Example 10] Measurement of endotoxin by parallel line determination in various biological preparations
Diluted water dilution series (4 points) of a total of 3 lots of various biologics (IHA-LotD, JEC-F, HSA-Y2), each in 25 μl, Brij mixed solution [0.25% (W / V) Brij56 + Add 0.010% (W / V) triethylamine], stir, add 50 μl of endospecy attached to the kit, and react with Well Reader SK601 at 37 ° C. for 30 minutes. Change rate of absorbance per minute (mAbs405-492 nm / min) The analysis was performed with dedicated parallel line quantitative software (EG301, Seikagaku Corporation), and the endotoxin concentration (EU / ml) was calculated using the Japanese Pharmacopoeia standard endotoxin ( E.coli UKT-B). As shown in FIG. 9, good parallel lines and reproducibility were obtained for any biological product , and this kit was able to specifically and accurately quantify endotoxin in the biological product. .

[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 0.2 M 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 (10)

In the gel permeation chromatography using the high performance liquid chromatography using distilled water from which endotoxin and (1 → 3) -β-D-glucan have been removed as a solvent, the biological product is subjected to gel permeation chromatography. When eluted, the Limulus reaction-activating substance present in the elution fraction between 1/3 to 4/5 of the column volume and having 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;
(13) A column in which TSKgel G4000SW and TSKgel G3000SW (each column size is 7 × 30 mm, volume 14.3 cm ³ ) is used, and the flow rate of the solvent is 0.5 ml / min. A biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics, and a surfactant used for measuring Limulus , and a surfactant at a temperature above the freezing point temperature of the surfactant at a temperature of 50 ° C. or lower. Use of the surfactant for inactivating the Limulus reaction activator according to claim 1, wherein the surfactant is allowed to coexist so that the final concentration of the surfactant is 0.005 to 0.8% (W / V). . The use of a surfactant according to claim 2, 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 surfactant according to 3. A method for measuring endotoxin in a biologic comprising the following steps:
(1) A biological preparation selected from the group consisting of vaccine preparations, blood preparations and antibiotics, and a surfactant at a temperature above the freezing point temperature of the surfactant to a temperature of 50 ° C. or less. The 1st which makes the final concentration of an active agent coexist so that it may become 0.005-0.8% (W / V), and inactivates the Limulus reaction activator of Claim 1 in this biological preparation Process;
(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. The method for measuring endotoxin according to claim 5 , wherein the surfactant is a nonionic surfactant. Nonionic surfactant is polyoxyethylene alkyl ethers, one selected from the group consisting of polyoxyethylene alkyl phenyl ether and acyl polyoxyethylene sorbitan or more non-ionic surfactants, claim 6. The method for measuring endotoxin according to 6 . Limulus reagent is an endotoxin-specific Limulus reagent, method of measuring endotoxin according to any one of claims 5-7. The method for measuring a Limulus reaction activator according to claim 1 in a biological product selected from the group consisting of a vaccine product, a blood product and an antibiotic, comprising the following steps:
(1) a first step of measuring a substance that reacts with a reagent in a biological preparation selected from the group consisting of a vaccine preparation, a blood preparation and an antibiotic using a Limulus reagent;
(2) A second step of measuring endotoxin in the same biological preparation as the first step using the endotoxin measurement method according to any one of claims 5 to 8 ;
(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 is used in claim 1 in the biological preparation. A third step of calculating the amount of the limulus reaction activator described. The method for measuring a Limulus reaction-activating substance according to claim 9 , wherein the Limulus reagent is an endotoxin-specific Limulus reagent.