JPH06273421A - Method for suppressing for activity of endotoxin - Google Patents

Abstract

PURPOSE:To provide the selective suppressing method for endotoxin (hereinafer ET), which can be performed by a simple operation by using reagent that can be obtained readily and the method and reagent for measuring the material having the ET activity contained in a sample other than ET (hereinafter described as ET-analogous material) by using the above described method. CONSTITUTION:In a sample containing endotoxin, a peptide derivative (or protein) having the property for suppressing the activity of ET in combination with ET and a surface-active agent are commonly present. This method has the above described feature. This is the method for suppressing the activity of ET and also the measuring method for the ET-analogous material contained in the sample, which is processed by the suppressing method.

Description

Detailed Description of the Invention

[0001]

The present invention relates to endotoxin (hereinafter referred to as
Abbreviated as ET. ), For example, a blood cell component solution of horseshoe crab (hereinafter abbreviated as AL solution), which activates an enzyme such as a protease contained in the solution (hereinafter simply referred to as an enzyme activation reaction). Yes.)
And a method of selectively suppressing the property of causing gelation reaction and the like (hereinafter abbreviated as ET activity), and a substance having ET activity contained in a sample treated by this method (hereinafter, And a method other than ET (hereinafter abbreviated as ET-related substance).

[0002]

BACKGROUND OF THE INVENTION ET is a lipopolysaccharide (LPS) existing in the outer wall of cell wall of Gram-negative bacteria, and is known as a strong pyrogenic substance. Therefore, the detection of ET in injectable drugs and the like is considered to be important, and the endotoxin test method is listed in the pharmacopoeias of the United States and Japan. Further, ET is considered to be a major cause of shock in Gram-negative bacterial infection, and in clinical diagnosis, measurement of endotoxin in plasma is diagnostic of Gram-negative bacterial infection, therapeutic effect and prognosis of Gram-negative bacterial infection. It is used for the determination of, and early diagnosis of endotoxin shock. The AL solution has the property of causing an enzyme (protease, etc.) activation reaction or gelation reaction by ET, which was used in the fields of medicine, pharmacy, and microbiology.
A simple and inexpensive ET detection method, for example, a method in which the degree of activation of an enzyme (protease, etc.) is measured by a colorimetric method, a so-called limulus test using a gelation reaction, etc. (hereinafter, these are collectively referred to as limulus test, etc. Is widely used.

However, the reagents used for the Limulus test and the like are ET related substances such as (1 → 3) -β-D.
-Because it also reacts with glucan and / or its derivative (hereinafter abbreviated as βG etc.) (Kakinuma et al., Bioche
m.Biophys.Res.Commun., vol.101,434-439 (1981), Morit
a et al., FEBS Lett., vol.129, 318-321 (1981)), when βG and the like coexisted with ET in the measurement sample, there was a problem that a positive error occurs in the measurement value. .

Further, βG and the like are interfering substances that give a positive error when measuring ET, but since they are also cell wall components of fungi such as yeasts and molds, βG and the like can be detected using an AL solution. If it is carried out, it is considered that it can be used for diagnosis of fungal infections, and therefore development of a measuring method thereof is also under consideration. However, since reagents currently used in the Limulus test and the like are naturally reacted with ET, it is said that ET in a sample affects the measured value when attempting to measure βG or the like using these reagents. There was a problem.

In order to solve such a problem, AL
Factors that cause the enzyme (protease, etc.) activation reaction or gelation reaction by reacting the solution with ET present in the AL solution by treating the solution with various chromatographies (hereinafter, ET
Abbreviated as susceptibility factor. ) Is removed (JP-A-59-2)
7828, JP-A-2-138193, JP-A-4-76459), or a peptide having an affinity for ET
A method of inactivating the ET susceptibility factor by adding it to a solution (JP-A-2-207098) or a method of inactivating an ET susceptibility factor by adding an antibody against the ET susceptibility factor to an AL solution (JP-A-4-52558). No. gazette) has been reported. However, since all of these methods are methods for treating the AL solution itself, ET widely existing in normal environments is used.
It has a drawback that there is a high risk that the AL solution will be contaminated during the treatment by β or βG. Furthermore, these methods require aseptic equipment and complicated aseptic operation for avoiding such contamination, and peptides having affinity for ET and antibodies for ET susceptibility factor are available. It is difficult and expensive, and as a result, it is an economically and technically problematic method.

As a method for inactivating ET by treating a measurement sample instead of an AL solution, a heat treatment method for the sample has been reported (Japanese Patent Laid-Open No. 2-141666). Although this method overcomes the drawbacks of the above-mentioned method, it has a problem that a long treatment time is required to sufficiently inactivate ET, and thus it cannot be said to be a preferable method.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and is a method for selectively suppressing ET activity which can be carried out by a simple operation using an easily available reagent, and
It is an object of the present invention to provide a method for measuring an ET analog in a sample using such a method and a reagent therefor.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a sample containing ET, a peptide derivative (or protein) having the property of binding to ET to suppress the activity of ET (hereinafter abbreviated as ET suppressing peptide), and a surfactant. And coexist with,
It is an invention of a method for suppressing ET activity.

The present invention further comprises reacting a sample treated by the above-mentioned inhibition method with an AL solution and measuring the activity of an enzyme activated by the resulting enzyme activation reaction, or gelation resulting therefrom. ET present in a sample characterized by measuring the degree of change in the turbidity of the reaction solution based on the reaction or the degree of gelation by an instrument or visually, ET
It is an invention of a method for measuring a related substance.

Furthermore, the present invention is an aqueous solution containing an ET inhibitory peptide and a surfactant, which does not react with the AL solution and does not inhibit or promote the reaction between the AL solution and the ET related substance. And a pretreatment liquid for suppressing ET activity.

Furthermore, the present invention provides an ET inhibitory peptide,
It is an invention of a reagent for measuring an ET related substance, which comprises a surfactant and an AL solution.

That is, the present inventors have used an AL solution,
In the course of earnest research to find out a specific assay method for AL solution activators such as ET and βG, a surfactant and an ET inhibitory peptide were allowed to coexist in a sample or a reaction solution at the time of assaying an AL solution activator. In this case, it was found that only the ET activity contained in these was suppressed, and other ET related substances such as βG could be specifically detected, and the present invention was completed.

More specifically, it has been well known that each of the surfactants and the ET inhibitory peptides represented by polymyxin has the property of inactivating the ET activity. It was necessary to add a large amount in order to completely deactivate the ET activity. Therefore, when the ET related substance in the sample is measured by the Limulus test or the like for the sample treated with any of these alone, there is an influence such as inhibition of the activation of the enzyme in the AL solution. However, there is a problem in that an accurate measured value cannot be obtained. However, the inventors of the present invention found that the combined use of these can completely deactivate the ET activity in the sample even when an amount within the range that does not affect the Limulus test or the like is added. It came to completion.

The surfactant used in the present invention does not inhibit or promote the activation reaction (enzyme activation reaction, gelation reaction, etc.) of the AL solution by the ET analogue, and Examples thereof are not particularly limited as long as they do not cause non-specific turbidity during the reaction, and specific examples thereof include polyoxyethylene alkyl such as polyoxyethylene cetyl ether and polyoxyethylene lauryl ether. Ethers such as polyoxyethylene octyl phenyl ether, polyoxyethylene nonyl phenyl ether, and other polyoxyethylene alkyl phenyl ethers such as polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostea rate Polyoxyethylene alkyl esters such as polyoxyethylene sorbitan trioleate,
For example, octanoyl-N-methylglucamide, nonanoyl-
Methylglucamide derivatives such as N-methylglucamide and decanoyl-N-methylglucamide, for example n-octyl-β-D-
Nonionic surfactants such as alkyl sugar derivatives such as glucosides, such as sodium dodecyl sulfate (SDS), laurylbenzenesulfonic acid, deoxycholic acid, cholic acid, tris (hydroxymethyl) aminomethane dodecylsulfate (Tris DS), etc. Anionic surfactants, such as octadecylamine acetate, tetradecylamine acetate, stearylamine acetate, laurylamine acetate, lauryldiethanolamine acetate, and other alkylamine salts, such as octadecyltrimethylammonium chloride, dodecyltrimethylammonium chloride, cetyl chloride Trimethylammonium, cetyltrimethylammonium bromide, allyltrimethylammonium methylsulfate, benzalkonium chloride, tetradecyldimethylbenzylammonium chloride, octadecyldichloride Chill benzyl ammonium,
Quaternary ammonium salts such as lauryldimethylbenzylammonium chloride, cationic surfactants such as alkylpyridinium salts such as laurylpyridinium chloride and stearylamidomethylpyridinium chloride, 3-[(3-colamidoamidopropyl) dimethylammonio] Amphoteric surfactants such as -1-propane sulfonate, 3-[(3-colamidoamidopropyl) dimethylammonio] -2-hydroxy-1-propane sulfonate, natural saponins (derived from soybeans), digitonin, etc. Although surfactants such as surfactants can be mentioned, nonionic surfactants or amphoteric surfactants are particularly preferred in view of the influence on the enzyme system and the like during the reaction.
These may be used alone or in any combination. The concentration of these used varies depending on the kind of the surfactant and the sample to be treated, but is a concentration at which the ET activity can be sufficiently suppressed when an ET inhibitor peptide coexists, and an ET analogue such as βG. There is no particular limitation as long as it is a concentration that neither promotes nor inhibits the reactivity with respect to the AL solution, but more specifically, ET
The concentration in the sample containing, or the concentration in the solution when the sample is reacted with the AL solution is usually 0.005 to 5 w / v%, preferably 0.05 to 2.5 w / v%, and more preferably 0.1 to 1 w. It is used by appropriately selecting from the range of / v%.

The ET inhibitory peptide or the like used in the present invention is not particularly limited as long as it is a peptide derivative (or protein) having the property of binding to ET and inhibiting the activity of ET. Those having strong basicity or having many hydrophobic sites, or having both of these properties are preferable. Examples of such peptide derivatives (or proteins) include polymyxins produced by Bacillus polymyxa and the like, for example, tachyplesin and polyphemsin derived from horseshoe crab blood cell components.
hemusin), anti-lipopolysaccharide factor (Anti-LPS Factor) and the like are preferable. In addition, tachyplesin (Tachyplesi
n), polyphemusin, anti-lipopolysaccharide factor (Anti-LPS Factor) and the like are generally difficult to obtain and expensive, and thus it is economically advantageous to use polymycin. As the polymyxin, for example, polymyxins A, B, C, D, E, K, M, P, etc., which are purified as a single component, or a mixture of some of these, The salt may be in the form of a salt, and may be any salt without limitation, but a sulfate of polymyxin B is preferable in view of easy availability. In addition, as this usage concentration,
Although it depends on the type of ET inhibitory peptide and the sample to be treated, it is a concentration that can sufficiently suppress ET activity when a surfactant coexists, and also promotes the reactivity of ET analogs such as βG with respect to the AL solution. There is no particular limitation as long as it is a concentration that does not inhibit, but more specifically, it is usually a concentration in a sample containing ET or a concentration in a solution when the sample is reacted with an AL solution. 0.00001 ~ 0.1
w / v%, preferably 0.0001 to 0.05 w / v%, more preferably
It is used by appropriately selecting from the range of 0.001 to 0.01 w / v%.

To carry out the suppressing method of the present invention, for example, the following may be carried out. That is, when the sample containing ET or when the sample is reacted with the AL solution, it is sufficient that the surfactant and the ET-suppressing peptide as described above are coexistent so as to have the concentrations as described above. When the ET related substance is measured for the sample once treated by the suppressing method of the present invention, the concentration of the surfactant and the ET suppressing peptide during the reaction with the AL solution is lower than the above-mentioned concentration. It goes without saying that it does not matter if it becomes.

When the suppression method of the present invention is used to suppress ET activity in a heatable sample, the ET activity can be suppressed more effectively by using heat treatment in combination. Especially when measuring ET related substances in samples containing inhibitory factors such as plasma and serum by the Limulus test, etc.
It is desirable to use heat treatment together as a pretreatment method for removing the influence of an inhibitory factor or the like. The heat treatment may be carried out either before or after the coexistence of the surfactant and the ET-suppressing peptide, but after the coexistence, the effect is often high, which is desirable.
The heating temperature in the heat treatment is not particularly limited, but is usually 60 to 220 ° C., preferably about 70 to 100 ° C. The heating time is not particularly limited, but is usually 3 to 60 minutes. It is preferably about 5 to 15 minutes.
The heating method may be any method as long as it can heat the sample under the heating conditions described above, and for example, a heating method using an incubator and a heating method using an autoclave are preferably mentioned.

As a method of coexisting a surfactant and an ET-suppressing peptide in a sample containing ET or in reacting the sample with the AL solution, the activation of the AL solution by ET is started. Any method may be used as long as the above-mentioned concentration of the surfactant and the ET-suppressing peptide are added to the sample or the AL solution, and examples thereof include the following methods.

1) A sample containing ET was treated with a surfactant and ET.
A solution containing an appropriate amount of an inhibitory peptide (or a solution containing an appropriate amount of a surfactant and a solution containing an appropriate amount of an ET inhibitory peptide), which does not activate the AL solution and reacts the AL solution with an ET analog A method of appropriately diluting with a solution that neither inhibits nor promotes.

2) In the case of measuring an ET analogue, a method in which an appropriate amount of a surfactant and an ET-suppressing peptide are previously contained in an AL solution, and the AL solution and a measurement sample are appropriately mixed.

In the case of the method of 1) above, the dilution ratio of the sample is not particularly limited, but when the sample is plasma etc., if the dilution ratio is too low, the denaturation of plasma protein during heating will occur. Since the viscosity of the liquid diluted with plasma may increase, problems such as precipitation in the liquid may occur, and if the dilution ratio is too high, problems such as not being able to properly detect ET may occur. In such cases, usually 5
20 times, preferably about 8 to 12 times.

The solution containing the surfactant and the ET-inhibiting peptide used in the above method 1), or the solution containing each of them separately, is the surfactant or
And ET inhibitory peptide in an aqueous solution of appropriate concentration,
It is desirable to use after confirming that the AL solution is not activated and neither promotes nor inhibits the reaction such as Limulus test. The solution containing the surfactant and / or the ET suppressing peptide may be autoclaved at 121 ° C. for 20 minutes for sterilization. The concentration of the surfactant in the solution is not particularly limited as long as the final concentration after diluting the target sample is the concentration as described above, but usually 0.01 to 10 w / in consideration of operability. The range of v% is preferably 0.1 to 5 w / v%, more preferably 0.2 to 2 w / v%. The concentration of the ET-suppressing peptide in the solution is not particularly limited as long as the final concentration after diluting the target sample is in the above-mentioned concentration, but in consideration of operability, it is usually 0.00002- 0.2w / v%, preferably 0.0002-0.1w / v%, more preferably 0.002-0.0
The range of 2 w / v% is mentioned.

AL containing the ET-inhibiting peptide and the surfactant at a predetermined concentration used in the above method 2)
It is naturally possible to freeze-dry the solution once and store it in the form of a reagent, and then re-dissolve it in water containing no AL solution activating substance such as distilled water for injection before use.

According to the suppression method of the present invention, the ET activity in a sample can be completely deactivated by using a small amount of the ET suppression peptide and a surfactant together. It is a very economical method. Further, since the sample treated by the suppression method of the present invention has a low content of the ET-suppressing peptide and the surfactant, measurement of ET related substances using the limulus test etc. without considering the influence of these on the limulus test etc. It can be used as a sample for use.

Depending on the type (origin) of ET, for example, in the case of ET derived from E. coli O127: B8, a low concentration of E
It is possible to sufficiently suppress the ET activity only by the T suppressor peptide. However, when it is necessary to suppress ET activity in order to measure ET related substances in a sample in which it is not clear what kind of ET is contained, it is better to use the suppression method of the present invention. Needless to say, it is preferable.

The endotoxin activity suppressing method according to the present invention can be effectively used in the following cases, for example. That is, when the amount of β-glucan in a sample is measured by the Limulus test or the like, it can be used to prevent the endotoxin mixed in the sample from affecting the measurement result. Also, by adding LAL to the sample, LA
In the method of activating L and then adding a synthetic substrate to measure endotoxin or β-glucan in a sample, it can be used to eliminate endotoxin contamination of a reagent used such as a synthetic substrate. Further, for example, in experiments using cultured cells and the like, when the cultured cells have a property of being easily affected by endotoxin, they can be used to eliminate contamination of endotoxin of the reagent used.

In measuring the ET related substances in the sample treated by the suppression method of the present invention, the so-called Limulus test or the like, that is, Toxinometer ET-201 (manufactured by Wako Pure Chemical Industries, Ltd.), Toxinometer MT- 251 (manufactured by Wako Pure Chemical Industries, Ltd.), LAL-5000 [ACC (ASSOCIAT
ES OF CAPE COD), etc.] turbidimetric time analysis method using a dedicated device, or a synthetic substrate method that measures the protease activity that appears when the AL solution is activated using a synthetic substrate, the activity of AL It suffices to carry out by a conventional method using an AL solution such as a gelation inversion method in which a gel formed by crystallization is visually determined.

ET related substances according to the present invention include ET
There is no particular limitation as long as it is a substance other than the above and reacts with the AL solution to cause an enzyme activation reaction or a gelation reaction, and examples thereof include (1 → 3) -β-D-glucan and The derivative is mentioned as a typical thing. The (1 → 3) -β-D-glucan and its derivative are not particularly limited as long as they are polysaccharides containing (1 → 3) -β-D-glucan as a constituent component. However, for example, various types of bacteria (eg, Alca
ligenes genus, Agrobacterium genus, etc., yeasts (eg Sa
ccharomyces, Candida, Cryptococcus, Trichosp
Oron, Rhodotorula, etc.) Molds (Aspergillus, Mu)
Genus cor, Penicillium, Trichophyron, Sporothrix
Genus, Phialophora, etc.), actinomycetes (Actinomyces, No)
cardia, etc.), mushrooms (for example, shiitake, shirohirotake, kawaratake, etc.) and other natural polysaccharides obtained from cell walls, such as curdlan, pakiman, sclerotan, lentinan, schizophyllan, coriolan, or the like, or Storable polysaccharides of algae (for example, brown algae, euglena, diatoms, etc.), specifically, for example, laminaran, paramylon, etc., or to these, for example, large organic chemistry, vol. 19, 7th edition, 70 to 101. Page, Mitsuo Kotake, May 10, 1942, Asakura Shoten; AEClarke et al., Phytochemistry, vol.1,175-188
(1967); T. Sasaki et al., Europ. J. Cancer, vol.15, 211-215 (1
967) and the like, for example, derivatives obtained by introducing a sulfate group, a carboxymethyl group, a carboxyethyl group, a methyl group, a hydroxyethyl group, a hydroxypropyl group, a sulfopropyl group and the like are preferred. It

The AL solution that can be used in this case is not particularly limited as long as it can be used for ordinary ET measurement, and examples thereof include ACC, Hemachem and Whitaker. Bio Product, End Safe, Teikoku Organ Co., Ltd., Seikagaku Corporation
A lyophilized product of a commercially available lyophilized AL solution produced by
s) Extracted from blood cells of horseshoe crab belonging to the genus Tachypleus or the genus Carcinoscorpius. Activation of enzyme (protease etc.) and gelation by reaction with AL solution activator Any reaction may be used without limitation.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0031]

【Example】

Example 1. [Reagent] ・ ET solution E.coli O55: B5 LPS (manufactured by Difco) was weighed 10 mg and dissolved in 10 ml of distilled water for injection to prepare a 1 mg / ml solution, which was used as a stock solution and injected. The product was appropriately diluted with distilled water for use. -AL solution A freeze-dried AL solution derived from Limulus spp. (Hereinafter abbreviated as LAL; sold by Wako Pure Chemical Industries, Ltd., for 2 ml).
LAL dissolution buffer (HS) (manufactured by Wako Pure Chemical Industries, Ltd.)
The LAL solution obtained by dissolving in 1. was used.・ Surfactant aqueous solution Polyoxyethylene glycol pt-octyl phenyl ether (nonionic surfactant, Wako Pure Chemical Industries, Ltd.
121) was diluted with distilled water for injection to 10 w / v%.
What was autoclaved at ℃ for 20 minutes was used as a stock solution, and after appropriately diluting this with distilled water for injection, it was confirmed that the AL solution was not activated and that the measurement of βG etc. by the Limulus test etc. was not inhibited or promoted. Was used. -Polymyxin B (hereinafter abbreviated as PxB) aqueous solution PxB sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) with distilled water for injection
What was diluted to 1.0 w / v% was autoclaved at 121 ° C for 20 minutes to make a stock solution, and this was diluted appropriately with distilled water for injection (or 0.2 w / v% surfactant aqueous solution prepared above). After that, it was confirmed that the AL solution was not activated and that the measurement of βG and the like by the Limulus test or the like was not inhibited or promoted. [Procedure] 20 μl of an ET solution having a predetermined concentration was added to 2.0 ml of heparinized normal human plasma, and the obtained ET-added plasma 10
0 μl of 10-fold diluted with distilled water for injection, PxB aqueous solution of a predetermined concentration or 900 μl of a surfactant aqueous solution of a predetermined concentration was heat-treated at 80 ° C. for 5 minutes and immediately ice-cooled (end of ET). The concentration is 2 ng / ml, the final concentration of PxB is 0.0018-
0.009w / v%, the final concentration of surfactant is 0.09-0.45w / v
%. ). The ET concentration in the obtained diluted plasma was measured using a toxinometer MT-251 (manufactured by Wako Pure Chemical Industries, Ltd.).
It was measured as follows according to a conventional method. That is, 0.1 ml of L
Add 0.1 ml of the diluted plasma to the AL solution, stir and mix,
The time (hereinafter, abbreviated as Tg) until the amount of transmitted light of the above-mentioned mixed liquid decreased by 5% was measured while keeping the temperature at 37 ° C. The obtained Tg was applied to a calibration curve showing the relationship between the ET concentration and Tg, which was prepared in advance by performing the same operation using ET solutions having various concentrations as samples, and the ET concentration in the diluted plasma was determined. [Results] The obtained results are shown in FIGS. Incidentally, FIG.
Shows the results obtained for plasma diluted with a surfactant solution, and the gelation time (Tg) of the diluted heated plasma obtained for each surfactant concentration (at the time of heat treatment) on the horizontal axis is shown vertically. It is the connection of points plotted along the axis. Also,
FIG. 2 shows the results obtained for plasma diluted with PxB solution. The gelation time (Tg) of the diluted plasma obtained for each PxB concentration (at the time of heat treatment) on the horizontal axis is plotted on the vertical axis. The points plotted along are connected, and in the figure,-△
− Represents the results obtained using an aqueous solution containing only PxB,
-□-is the result obtained using an aqueous solution containing 0.2 w / v% of a surfactant (polyoxyethylene glycol pt-octylphenyl ether) together with PxB (surfactant concentration during heat treatment is 0.18 w / v%.) respectively. Also, FIG.
In (), Tg was 90 minutes or more, in other words, ET was not detected. As is clear from the results of FIG. 1, the Tg obtained for plasma diluted with distilled water for injection was 7.8 minutes, whereas when plasma was diluted with a 0.1-0.5% aqueous surfactant solution ( Surfactant concentration during heat treatment is 0.09 to 0.45w / v%), T
It was found that g was delayed from 16.3 to 33.7 minutes, and the ability of ET to activate AL solution (ET activity) was decreased (the shorter Tg, the ET
The activity is high. ). Moreover, as is clear from the result of FIG.
Tg obtained for plasma diluted with distilled water for injection
Was 7.8 minutes, while plasma had 0.002-0.01 w / v% P
It can be seen that when diluted with an xB aqueous solution (PxB concentration during heat treatment is 0.0018 to 0.009 w / v%), Tg is delayed to 24.2 to 32.9 minutes and ET activity is lowered. Furthermore, from the results of FIG. 2, when plasma was diluted with 0.002 to 0.01 w / v% PxB and an aqueous solution containing 0.2% surfactant (the surfactant concentration during the heat treatment was 0.18%, the PxB concentration was Is 0.0018 to 0.0
09w / v%. ), ET is no longer detected (Tg is 90 minutes or more). From the above results, it is clear that the ET activity of E. coli O55: B5 LPS added to plasma is completely inhibited by the coexistence of the surfactant and PxB.

Example 2. [Reagent] The same AL solution, surfactant aqueous solution and PxB aqueous solution as in Example 1 were used.・ ET solution E.coli O111: B4 LPS (manufactured by Difco) was weighed 10 mg and dissolved in 10 ml of distilled water for injection to prepare a 1 mg / ml solution to prepare a stock solution, which was distilled water for injection. It was used by appropriately diluting with. [Procedure] ET-added plasma obtained by adding 12 μl of ET solution of a predetermined concentration to 0.6 ml of normal human plasma treated with heparin
0 μl of distilled water for injection, 0.04 w / v% surfactant aqueous solution, 0.2 w / v% surfactant aqueous solution or 0.01 w / v% PxB
Aqueous solution [containing 0.2 w / v% surfactant (polyoxyethylene glycol pt-octyl phenyl ether). ]
A 10-fold dilution with 900 μl was heat-treated at 80 ° C for 5 minutes and immediately cooled with ice (final concentration of ET was 1.96 ng / m 2.
l. ). The ET concentration in the obtained diluted plasma was determined by the same operation method as in Example 1. [Results] The results obtained are shown in Table 1.

[0033]

[Table 1]

As is clear from the results shown in Table 1, the Tg obtained for plasma diluted with distilled water for injection was 10.0 minutes, while the plasma was diluted with 0.2 w / v% surfactant aqueous solution. In some cases (concentration of surfactant during heat treatment is 0.18
w / v%. ), Tg is delayed to 40.4 minutes and ET activity is lowered. In addition, 0.01 w / v% PxB aqueous solution (0.
Contains 2w / v% surfactant. ) (PxB concentration during heat treatment is 0.009w / v%, surfactant concentration is 0.18w / v%), ET is no longer detected (Tg is 90).
It will be more than a minute). Incidentally, when plasma was diluted with a 0.04% aqueous solution of a surfactant, Tg was smaller (ET activity was higher) than that obtained with plasma diluted with distilled water for injection. It is present in plasma due to the addition of detergent, ET and AL
It is speculated that this is because the influence of the inhibitor on the reaction with the solution was reduced. As is clear from the above results, the ET activity of E. coli O111: B4 LPS added to plasma is completely inhibited by the coexistence of the surfactant and PxB.

Example 3. [Reagent] The same AL solution, surfactant aqueous solution and PxB aqueous solution as in Example 1 were used.・ ET solution E.coli O127: B8 LPS (manufactured by Difco) was weighed at 10 mg and dissolved in 10 ml of distilled water for injection to prepare a 1 mg / ml solution to prepare a stock solution, which was distilled water for injection. It was used by appropriately diluting with. [Procedure] 14 μl of ET solution with a predetermined concentration was added to 0.7 ml of heparin-treated normal human plasma, and the resulting ET-added plasma 10
0 μl of distilled water for injection, 0.04 w / v% surfactant aqueous solution, 0.2 w / v% surfactant aqueous solution, 0.01 w / v% PxB aqueous solution or 0.2 w / v% surfactant ( Polyoxyethylene glycol p-t-octylphenyl ether) was diluted 10 times with 900 μl of a 0.01 w / v% PxB aqueous solution containing polyoxyethylene glycol (p-t-octylphenyl ether), which was heat-treated at 80 ° C. for 5 minutes and immediately cooled with ice (E
The final concentration of T was 1.96 ng / ml. ). The ET concentration in the obtained diluted plasma was determined by the same operation method as in Example 1. [Results] The results obtained are shown in Table 2.

[0036]

[Table 2]

As is clear from the results in Table 2, the Tg obtained for the plasma diluted with distilled water for injection was 10.6 minutes, while the plasma was diluted with a 0.2 w / v% aqueous surfactant solution. In some cases (concentration of surfactant during heat treatment is 0.18
w / v%. ), Tg was delayed to 28.6 minutes and ET activity decreased. In addition, plasma is 0.01 w / v% PxB aqueous solution or
When diluted with 0.01 w / v% PxB aqueous solution containing 0.2 w / v% surfactant (PxB concentration during heat treatment is
0.009w / v%, surfactant concentration 0.18w / v%. ), ET is no longer detected (Tg is 90 minutes or more).
Incidentally, when plasma was diluted with a 0.04% aqueous solution of a surfactant, Tg was smaller (ET activity was higher) than that obtained with plasma diluted with distilled water for injection. It is speculated that this is because the addition of the surfactant reduced the influence of the inhibitor on the reaction between ET and AL solution present in plasma. As is clear from the above results, E. coli O127: B8 LPS added to plasma was used.
It can be seen that the ET activity of PxB is completely inhibited by 0.009 w / v% of PxB, or the coexistence of a surfactant and PxB.

Example 4. [Reagent] The same AL solution, surfactant aqueous solution and PxB aqueous solution as in Example 1 were used.・ ET solution E.coli O128: B12 LPS (manufactured by Difco) was weighed 10 mg and dissolved in 10 ml of distilled water for injection to prepare a 1 mg / ml solution, which was used as a stock solution. This was distilled water for injection. It was used by appropriately diluting with. [Procedure] 13 μl of ET solution having a predetermined concentration was added to 1.3 ml of heparinized normal human plasma, and the obtained ET-added plasma 10
0 μl of distilled water for injection, a predetermined concentration of surfactant aqueous solution, a predetermined concentration of PxB aqueous solution or 0.1 w / v% or 0.2 w /
v% surfactant (polyoxyethylene glycol p-
Tx-octyl phenyl ether) containing Px at a specified concentration
A 10-fold dilution with 900 μl of B aqueous solution was heat-treated at 80 ° C for 5 minutes and immediately ice-cooled (final concentration of ET was 2 ng.
/ ml, the final concentration of PxB is 0.00009-0.009w / v%, and the final concentration of the surfactant is 0.09-0.9w / v%. ). The ET concentration in the obtained diluted plasma was determined by the same operation method as in Example 1. [Results] The obtained results are shown in FIGS. In addition, FIG.
The results obtained for plasma diluted with a surfactant solution are shown, and the gelation time (Tg) of diluted heated plasma obtained for each surfactant concentration (at the time of heat treatment) on the horizontal axis is shown on the vertical axis. It is the connection of the points plotted along. FIG. 4 shows P
showing the results obtained for plasma diluted with xB solution,
The gelation time (Tg) of the diluted heated plasma obtained for each PxB concentration (at the time of heat treatment) on the horizontal axis is plotted along the vertical axis. FIG. 5 shows the results obtained for plasma diluted with an aqueous solution containing a surfactant and PxB. The gelation time of the diluted plasma obtained for each PxB concentration (at the time of heat treatment) on the horizontal axis. It is a combination of points plotted by plotting (Tg) along the vertical axis. In the figure, −
□-is the result obtained by using an aqueous solution containing 0.1 w / v% of the surfactant (surfactant concentration at the time of heat treatment is 0.09 w / v%.), -X- is 0.2 w / v Using an aqueous solution containing 10% of surfactant (concentration of surfactant during heat treatment is 0.18 w / v
%. The results obtained are shown below. Further, (○) in FIG. 4 and (□) and (×) in FIG. 5 are T
It means that g was 90 minutes or more, in other words, ET was not detected. As is clear from the results of FIGS. 3 to 5, PxB was allowed to coexist with 0.009 w / v%, or the surfactant and P
It is understood that ET activity in plasma can be completely inhibited when coexisting with xB. In addition, if the suppression method of the present invention is used to suppress ET activity, the amount of PxB used can be reduced (1/100 of the case of PxB alone).
Quantity), in other words, the method of the present invention uses conventional Px
It is also found that the method is economically superior to the method using B alone.

Example 5. [Reagent] The same AL solution, surfactant aqueous solution and PxB aqueous solution as in Example 1 were used. -ET solution Salmonella typhimurium LPS (manufactured by Difco) was weighed in an amount of 10 mg, dissolved in 10 ml of distilled water for injection to prepare a 1 mg / ml solution, which was appropriately diluted with distilled water for injection. I used one. [Procedure] 10 μl of ET solution of a predetermined concentration was added to 1.0 ml of heparinized normal human plasma, and the resulting ET-added plasma 10
0 μl of distilled water for injection, 0.2 w / v% surfactant aqueous solution,
What was diluted 10-fold with 900 μl of 0.01 w / v% PxB aqueous solution containing 0.01 w / v% PxB aqueous solution or 0.2 w / v% surfactant (polyoxyethylene glycol pt-octylphenyl ether), After heat treatment at 80 ° C for 5 minutes, the mixture was immediately ice-cooled (final concentration of ET is 2 ng / ml). The ET concentration in the obtained diluted plasma was determined by the same operation method as in Example 1. [Results] The results obtained are shown in Table 3.

[0040]

[Table 3]

As is clear from the results in Table 3, the ET activity of Salmonella typhimurium LPS added to plasma is completely inhibited by the coexistence of the surfactant and PxB.

Example 6. [Reagent] The same AL solution, surfactant aqueous solution and PxB aqueous solution as in Example 1 were used.・ Β-Glucan aqueous solution A linear (1 → 3) -β-D-glucan derivative, carboxymethylated curdlan (manufactured by Wako Pure Chemical Industries, Ltd.)
Weigh 0 mg, dissolve in 10 ml distilled water for injection, and dissolve to 10 mg / ml.
A solution was prepared to prepare a stock solution, which was appropriately diluted with distilled water for injection and used. [Procedure] Add 100 μl of 50 ng / ml β-glucan aqueous solution to 0.2
w / v% surfactant solution, 0.01 w / v% PxB solution or
0.01 w / v% PxB aqueous solution containing 0.2 w / v% polyoxyethylene glycol pt-octylphenyl ether 90
Diluted 10 times with 0 μl to prepare β-glucan-added solution (β-glucan
The final concentration of glucan is 5 ng / ml. ). The β-glucan concentration in the solution was measured as follows using a toxinometer MT-251 (manufactured by Wako Pure Chemical Industries, Ltd.) according to a conventional method. After adding 0.1 ml of the above diluted solution to 0.1 ml of LAL solution and stirring and mixing, the amount of transmitted light of the above mixture was 5% while keeping the temperature at 37 ℃.
The time until the decrease (hereinafter abbreviated as Tg) was measured. The obtained Tg value was prepared by performing similar operations in advance using various concentrations of β-glucan aqueous solutions as samples.
Fit to a calibration curve showing the relationship between glucan concentration and Tg,
The β-glucan concentration was determined, and the β-glucan recovery rate was calculated based on this result. [Results] The results obtained are shown in Table 4.

[0043]

[Table 4]

As is clear from the results shown in Table 4, the recovery of β-glucan was about 90 to 110 regardless of which diluted solution was used.
% Was good. Therefore, it can be seen that neither diluted solution inhibits or promotes the reaction between the AL solution and β-glucan.

Example 7. [Reagent] The same AL solution, surfactant aqueous solution and PxB aqueous solution as in Example 1 were used.・ ET solution E.coli O26: B6 LPS (manufactured by Difco) was weighed 10 mg and dissolved in 10 ml of distilled water for injection to prepare a 1 mg / ml solution, which was used as a stock solution. It was used by appropriately diluting with.・ Aqueous surfactant solution Sodium deoxycholate (anionic surfactant, manufactured by Wako Pure Chemical Industries, Ltd.), Emulgen 709 (nonionic surfactant, polyoxyethylene higher alcohol ether, trade name of Kao Corporation), Amphitol 20N (amphoteric surfactant,
Various surfactant aqueous solutions were prepared in the same manner as in Example 1 except that Kao Corporation's trade name, main component: lauryldimethylamine oxide) was used as the surfactant. [Procedure] 17 μl of ET solution with a predetermined concentration was added to 1.7 ml of heparinized normal human plasma, and the obtained ET-added plasma 10
0 μl of distilled water for injection, 0.1 w / v% sodium deoxycholate aqueous solution, 0.2 w / v% Emulgen 709 aqueous solution, 0.4 w
/ v% Amphitol 20N aqueous solution, 0.01 w / v% PxB aqueous solution, 0.01 w / v% PxB containing 0.1 w / v% sodium deoxycholate aqueous solution, 0.01 w / v% PxB containing 0.2 w / v%
Emulgen 709 aqueous solution or 0.4 containing 0.01 w / v% PxB
A 10-fold dilution with 900 μl of a w / v% amphitrol 20N aqueous solution was heat-treated at 80 ° C. for 5 minutes and immediately cooled with ice (final concentration of ET was 4 ng / ml). The ET concentration in the obtained diluted plasma was determined by the same operation method as in Example 1. [Results] The results obtained are shown in Table 5.

[0046]

[Table 5]

As is clear from the results shown in Table 5, the ET activity of E. coli O26: B6 LPS added to plasma was found to be higher than that of surfactant and P.
It can be seen that coexistence with xB completely inhibits.

Example 8. [Reagent] The same AL solution, surfactant aqueous solution and PxB aqueous solution as in Example 1 were used.・ Surfactant aqueous solution Polyoxyethylene glycol pt-octyl phenyl ether (nonionic surfactant, Wako Pure Chemical Industries, Ltd.
Manufactured), Emulgen 709 (nonionic surfactant, polyoxyethylene higher alcohol ether, trade name of Kao Corporation), Amphitol 20N (amphoteric surfactant, trade name of Kao Corporation, main component: lauryl dimethylamine oxide) To
Various surfactant aqueous solutions were prepared in the same manner as in Example 1, except that the surfactant was used as the surfactant.・ ET solution Salmonella typhosa O901 LPS (manufactured by Difco)
Was dissolved in 10 ml of distilled water for injection to prepare a 1 mg / ml solution, which was used as a stock solution, which was appropriately diluted with distilled water for injection. [Procedure] 10 μl of ET solution of a predetermined concentration was added to 1.0 ml of heparinized normal human plasma, and the resulting ET-added plasma 10
0 μl of distilled water for injection, 0.2 w / containing 0.01 w / v% PxB
0.2% w / v containing v% polyoxyethylene glycol pt-octylphenyl ether aqueous solution, 0.01 w / v% PxB
% Emulgen 709 aqueous solution or 0.01 w / v% PxB 0.
A 10-fold dilution with 900 μl of a 4 w / v% amphitrol 20N aqueous solution was heat-treated at 80 ° C. for 5 minutes and immediately ice-cooled (final concentration of ET was 4 ng / ml). The ET concentration in the obtained diluted plasma was determined by the same operation method as in Example 1. [Results] The results obtained are shown in Table 6.

[0049]

[Table 6]

As is clear from the results shown in Table 6, the ET activity of Salmonella typhosa O901 LPS added to plasma is completely inhibited by the coexistence of the surfactant and PxB.

Example 9. [Reagent] The same AL solution, surfactant aqueous solution and PxB aqueous solution as in Example 1 were used.・ Β-Glucan aqueous solution 108 mg of curdlan (manufactured by Wako Pure Chemical Industries, Ltd.), which is a linear (1 → 3) -β-D-glucan, was weighed, and distilled water for injection was used.
After adding 10 ml, it was dissolved by adding 800 μl of 1N sodium hydroxide to prepare a 10 mg / ml solution.
A stock solution was prepared by diluting 10 times with distilled water for injection to 1 mg / ml, and this was diluted appropriately with distilled water for injection and used. [Procedure] 0.01 μl of 100 μl of 10 ng / ml β-glucan aqueous solution
1% w / v% PxB with 900 μl of a surfactant solution of the specified concentration
Diluted 0 times to make β-glucan-added solution (final concentration of β-glucan is 1 ng / ml. Also, P in β-glucan-added solution
The final concentration of xB is 0.009w / v%, and the final concentration of surfactant is 0.9-
4.5w / v%. ). The β-glucan recovery rate from each of the various β-glucan-added solutions obtained was determined by the same operation method as in Example 6. [Results] The obtained results are shown in FIG. 6 shows the concentration of each surfactant on the horizontal axis (concentration in the β-glucan-added solution. 0.0
Includes 09w / v% PxB. ) Is obtained by connecting the points plotted along the vertical axis with the value of the β-glucan recovery rate. As is clear from the results in FIG. 6, the β-glucan recovery rate was a constant value of about 90% even when the surfactant concentration was increased. From this, it is judged that the surfactant (polyoxyethylene glycol pt-octylphenyl ether) does not inhibit or promote the reaction between the AL solution and β-glucan within this concentration range.

Example 10. [Reagent] ET solution, AL solution, aqueous surfactant solution and P
The same xB aqueous solution as in Example 1 was used. [Procedure] 100 μl of an ET solution having a predetermined concentration was added with distilled water 0.2
w / v% surfactant (polyoxyethylene glycol p
-t-octyl phenyl ether) aqueous solution, 0.002-0.01w
/ v% PxB aqueous solution or 0.002-0.01w / v% PxB
1 with 900 μl of 0.2w / v% surfactant (polyoxyethylene glycol pt-octylphenyl ether) solution
Diluted 0 times to make ET addition solution (final concentration of ET is 2 ng / m
l. The concentration of PxB in the ET-added solution is 0.0018-0.0.
09w / v%, surfactant concentration 0.18w / v%). ET obtained
The ET concentration in the added solution was determined by the same operation method as in Example 1. [Results] The obtained results are shown in FIG. 7. 7. In FIG. 7, the gelation time (Tg) of the PtB concentration in the ET-added solution on the horizontal axis is plotted along the vertical axis. In the figure,-△-is the result obtained for the ET additive solution prepared using various concentrations of PxB aqueous solution, and-□-is the ET prepared using PxB aqueous solution containing 0.2 w / v% of surfactant. The results obtained for the additive solutions are shown respectively. As is apparent from the results of FIG. 7, it is found that the ET activity of E. coli O55: B5 LPS in the ET-added solution is completely inhibited by the coexistence of the surfactant and PxB.

Example 11. [Reagent] The same AL solution, surfactant aqueous solution and PxB aqueous solution as in Example 1 were used, and the same ET solution as in Example 8 was used. Also, the β-glucan solution is
The same as in Example 9 was used. [Procedure] 4 μg of heparinized normal human plasma 1.0 ml
10 μl / ml ET solution and 200 ng / ml β-glucan solution 10
ET and β-glucan-added plasma obtained by adding
100 μl of distilled water for injection, 0.2 w / v% surfactant (polyethylene glycol pt-octylphenyl ether) aqueous solution, 0.01 w / v% PxB aqueous solution or 0.01 w / v% P
xw-containing 0.2 w / v% surfactant (polyethylene glycol pt-octylphenyl ether) aqueous solution 900 μ
What was diluted 10 times with 1 and heat-treated at 80 ° C for 5 minutes was immediately cooled with ice (final concentration of ET was 3.92 ng / ml, and final concentration of β-glucan was 196 pg / ml). Β in the obtained diluted plasma
-The glucan concentration was determined by the same operation method as in Example 6. In addition, use the same plasma used above with distilled water for injection.
The β-glucan concentration was determined by performing the same procedure as above for the mixture containing 10 μl and 10 μl of 20 ng / ml β-glucan solution. Furthermore, based on these results, β-
The recovery rate of glucan was calculated. [Results] The results obtained are shown in Table 7.

[0054]

[Table 7]

As is clear from the results in Table 7, the effect of ET coexisting in the sample (the effect of giving a positive error to the measured value) was observed only when the surfactant and PxB were coexistent at the time of β-glucan measurement. It is understood that the measurement of β-glucan can be performed without receiving it.

[0056]

As is clear from the above description, the present invention provides a method for effectively suppressing ET activity in a sample, and the method of the present invention has been carried out conventionally. The ET activity can be suppressed more simply and surely than the method, and the ET related substance can be measured without being affected by ET, which contributes to the industry. It is a great invention.

[0057]

[Brief description of drawings]

FIG. 1 shows endotoxin in plasma (hereinafter abbreviated as ET) obtained in Example 1 (E. coli O55: B5 LP).
S) A graph showing the influence of the surfactant concentration in the measurement.

FIG. 2 shows ET (E.
5 is a graph showing the effect of polymyxin B (hereinafter abbreviated as PxB) concentration in measuring coli O55: B5 LPS).

FIG. 3 shows ET (E.
5 is a graph showing the influence of surfactant concentration in the measurement of coli O128: B12 LPS).

FIG. 4 ET (E.
5 is a graph showing the influence of PxB concentration in coli O128: B12 LPS) measurement.

FIG. 5 is a graph showing the effect of PxB concentration on the ET (E. coli O128: B12 LPS) measurement in plasma obtained in Example 4 in the presence of a surfactant.

6 is a graph showing β in the presence of PxB obtained in Example 9. FIG.
-A graph showing the effect of surfactant concentration on glucan measurement.

FIG. 7: ET (E. coli O5 obtained in Example 10)
5: B5 LPS) is a graph showing the influence of PxB concentration in the measurement of an aqueous solution containing 5: B5 LPS.

[Explanation of symbols]

In Fig. 2,-△-is the result obtained by using an aqueous solution containing only PxB, and-□-is 0.2 w / v% of the surfactant (polyoxyethylene glycol pt-octylphenyl) together with PxB. The results obtained using an aqueous solution containing ether) (the surfactant concentration during heat treatment is 0.18 w / v%) are shown. Further, (□) indicates that Tg was 90 minutes or more, in other words, ET was not detected.
In FIG. 4, (◯) indicates that Tg was 90 minutes or more, in other words, ET was not detected.
In FIG. 5,-□-is an aqueous solution containing 0.1 w / v% of surfactant (surfactant concentration at the time of heat treatment is 0.09).
w / v%) The obtained results were obtained by using an aqueous solution containing -0.2-0.2% surfactant (0.18w / v% surfactant concentration during heat treatment). The results are shown respectively. Also, (x)
And (□) indicate that Tg was 90 minutes or more, in other words, ET was not detected. In Fig. 7,-△-is the result obtained for the ET-added solution prepared using various concentrations of PxB aqueous solution, and-□-is 0.2 w / v.
E prepared using PxB aqueous solution containing 10% of surfactant
The results obtained for the T-added solution are shown respectively.

Claims (17)

[Claims] 1. A peptide derivative (or protein) having the property of binding endotoxin to suppress endotoxin activity (hereinafter abbreviated as endotoxin-inhibiting peptide) and a surfactant coexist in a sample containing endotoxin. A method for suppressing the activity of endotoxin, which comprises: 2. The suppression method according to claim 1, which further comprises heat treatment. 3. The sample is heat-treated after the endotoxin-suppressing peptide and the surfactant coexist.
Or the suppression method described in 2. 4. The concentration of the surfactant is 0.005 to 5.0 w / v%, and the concentration of the endotoxin-inhibiting peptide is 0.00001 to
The suppression method according to claim 1, wherein the suppression method is 0.1 w / v%. 5. The suppression method according to claim 2, wherein the heat treatment is heating at 60 to 220 ° C. for 3 to 60 minutes. 6. The method for suppressing according to claim 1, wherein the endotoxin-suppressing peptide is polymyxin. 7. The method for suppressing according to claim 1, wherein the surfactant is a nonionic surfactant or an amphoteric surfactant. 8. A sample treated by the method according to claim 1 is reacted with a horseshoe crab blood cell component solution (hereinafter abbreviated as AL solution), and the enzyme activated by the resulting enzyme activation reaction is reacted. Present in a sample, characterized by measuring the activity or measuring the degree of turbidity change or gelation state of the reaction solution based on the resulting gelation reaction with an instrument or visually. A method for measuring a substance having a property of reacting with a solution to cause an enzyme activation reaction or a gelation reaction, other than endotoxin (hereinafter abbreviated as endotoxin-related substance). 9. An endotoxin analogue is (1 → 3) -β.
-D-glucan or / and a derivative thereof.
Measurement method described in. 10. An aqueous solution containing an endotoxin-suppressing peptide and a surfactant, which does not react with an AL solution and does not inhibit or promote the reaction between the AL solution and an endotoxin-related substance. Pretreatment liquid for activity suppression of. 11. A surfactant concentration of 0.01 to 10.0 w / v% and an endotoxin-inhibiting peptide concentration of 0.00002 to
The pretreatment liquid according to claim 10, which has a concentration of 0.2 w / v%. 12. The pretreatment liquid according to claim 10, wherein the endotoxin-suppressing peptide is polymyxin. 13. The pretreatment liquid according to claim 10, wherein the surfactant is a nonionic surfactant or an amphoteric surfactant. 14. A reagent for measuring an endotoxin-related substance, which comprises an endotoxin-suppressing peptide, a surfactant, and an AL solution. 15. The measuring reagent according to claim 14, wherein the endotoxin-suppressing peptide is polymyxin. 16. The measuring reagent according to claim 14 or 15, wherein the surfactant is a nonionic surfactant or an amphoteric surfactant. 17. An endotoxin analogue is (1 → 3)-
The measuring reagent according to any one of claims 14 to 16, which is β-D-glucan or / and a derivative thereof.