JPH0274864A - Fractional measurement - Google Patents

Abstract

PURPOSE:To judge whether a material which is incorporated in a specimen to be checked and generates gelling reaction is endotoxin or not by making the specimen to be checked react with Limulus blood-cell component extract, and reading a curve expressing the degree of optical change based on the gelling reaction after the elapse of specified time. CONSTITUTION:The frozen dry material of Limulus blood-cell component extract (AL solution) derived from Limulus of a Limulus genus is dissolved with distilled water. A specimen comprising endotoxin (ET) solution or glucan (GL) solution having a specified concentration is added into the solution (LAL solution) obtained by said method. After agitation, the change in amount of transmitted light during the specified elapsed time in said mixed liquid is measured at the maintained temperature of 37 deg.C. As a result, the trends in changes in the amounts of the transmitted lights are different depending on the case when the ET solution is used as the specimen and the case when the GL solution is used as the specimens. Whether the gelling material incorporated in the specimen is ET or not is judged based on the curves.

Description

Detailed Description of the Invention [Field of Application of the Invention] The present invention is directed to the detection of horseshoe crab blood cell components (Am) in a test sample.
oebocyte Lysate) extract (hereinafter referred to as A
L warm solution is abbreviated. ) and which causes a gelling reaction (hereinafter abbreviated as gelling substance).

[Background of the Invention Coendotoxin (hereinafter abbreviated as ET) is a ribopolysaccharide that mainly exists on the cell surface of Durum-negative bacteria, and is also known as a type of pyrogen. Therefore, the measurement of ETfi degree in a sample is
It is one of the important fields in the fields of pharmaceutical science and microbiology.

At present, this ET measurement method utilizes the phenomenon of activation and gelation by AL warm solution ET.
The so-called Limulus test is widely used because of its simplicity and low cost.

However, gelling substance 1 other than this AL hot solution ET
For example, it was found that it also reacts with carboxymethylated β-1,3-glucan [Kakinumaem a
l,,Biochem,Biophys,Re5ear
ch Communication, BΩ Engineering η-,
434-439 (1981)], this phenomenon is caused by a factor (hereinafter referred to as GL) that reacts with β-1,3-glucan (hereinafter abbreviated as GL) present in the AL solution and induces a coagulation reaction.
It is abbreviated as GL sensitivity factor. ) is GL or its derivative (
Hereinafter, it will be abbreviated as GLD. C. Iwanaga et al. 2 Japanese Journal of Bacteriology, Kagamiyama, 781-803 (1983)].

Therefore, most of the Limulus tests currently on the market react with not only ET but also GL or GLD to form a gel. It is difficult to determine whether there is one or a mixture of these, and its specificity has been a problem.

In order to solve these problems, a method has been reported to prepare a reagent specific for ET by removing GL-sensitive factors from a warm AL solution (Japanese Patent Application Laid-open No. 13516/1983, 59-27828), however, in all of the methods disclosed in these publications, treatment is carried out by AL hot solution gel transfer method or chromatography using a carrier bound with heparin or dextran sulfate, followed by solidification. The GL-sensitive factor is removed by separating into a fraction of the enzyme precursor, a fraction of the GL-sensitive factor, and a fraction of the factor that reacts with ET to cause a gelation reaction (hereinafter abbreviated as the ET-sensitive factor). This is a method that requires extremely complicated operations. Therefore, special equipment is required to perform this operation, and
In order to finally obtain a reagent specific to ET, each fraction had to be appropriately mixed again.

[Object of the Invention] The present invention has been made in view of the above-mentioned circumstances, and an object of the present invention is to provide a simple measuring method capable of separately measuring gelled substances in a test sample.

[Structure of the Invention] The present invention measures an optical change based on a gelation reaction that occurs when a test sample and an AT=solution are reacted, and measures an optical change based on a gelation reaction over a predetermined elapsed time. This is a method for determining whether or not a substance that causes a gelation reaction contained in a test sample is ET from the shape of a curve representing the degree of physical change, and the differential obtained by differentiating the curve. This method estimates the ET concentration and the GL or/and GLD concentration in the test sample from the maximum value of the coefficient and the elapsed time until the maximum value is reached.

Furthermore, the present invention provides a method for measuring the ET concentration in a test sample by measuring an optical change based on a gelation reaction that occurs when a test sample is reacted with an AL hot solution. A method of measuring the ET concentration in a test sample from the maximum value of the differential coefficient obtained by differentiating a curve representing the degree of optical change due to gelation reaction over time,
and measuring the ET concentration in the test sample by this method,
On the other hand, by measuring the optical change based on the gelation reaction that occurs when the test sample and the AL hot solution are reacted in the presence of polymyxin B, it is possible to detect GL or/and G in the test sample.
This method measures the concentration of LD and tests the reliability of the ET concentration in the test sample obtained by the above method from the GL or/and GLD concentration. By measuring the optical changes based on the gelation reaction that occurs when the sample and the AL hot solution are reacted,
This is a method for measuring the concentration of GL and/or GLD in a test sample.

That is, the present inventors are in the process of researching a method that can easily separate and measure ET and GL or/and GLD in a test sample. The shape of the curve representing the degree of optical change due to the gelation reaction resulting from the reaction is different, and more specifically, the optical change due to the gelation reaction when GL or/and GLD is reacted with AL hot solution. The degree of E
It was found that the temperature was smaller at the initial stage of the reaction than when T was reacted with AL in a warm solution, and based on the shape of the curve representing the degree of optical change due to the gelation reaction over a predetermined elapsed time, it was found that The gelatinized substance is ET, GL
It has been found that it is possible to determine that it is GLD or/and GLD or a mixture thereof. Furthermore, the curve representing the degree of optical change over a predetermined elapsed time is differentiated to obtain a differential coefficient, and its maximum value (hereinafter abbreviated as Dmax) and the elapsed time until the maximum value is reached are calculated. (hereinafter abbreviated as TP), it has been found that the abundance ratio of gelling substances contained in the test sample, in other words, the abundance ratio of ET and GL or/and GLD can be estimated, and the present invention reached.

In addition, from this fact, the time required for the optical change to reach a certain value (hereinafter referred to as Tg When the so-called turbidimetric method is used to measure the ET concentration in the test sample from Although the reliability of the measured values is sufficiently recognized, it was once again confirmed that the reliability of the measured values decreases when the gelling substance is a mixture of ET and GL or/and GLD. Therefore, GL or/and GLD
As a result of repeated research on a method that can reliably measure ET even when ET is mixed in the test sample, we found that if Dmax is used as an index instead of Tg, GL and/or GLD can be measured.
It has been found that the ET concentration in a test sample containing a mixture of ET can be measured with high accuracy.

It has been found that depending on the concentration of GL and/or GLD, there is a range of ET concentrations that can be measured with high accuracy.

Therefore, as a result of extensive research into a method that can easily and accurately measure the GL and/or GLD concentration in a test sample, we found that when reacting a test sample with an AL warm solution, ET
When coexisting with polymyxin B (hereinafter abbreviated as PMB), which has a strong affinity for GL and/or GLD, PMB inhibits the reaction between ET and AL warm solution, but surprisingly It was found that the reaction of the warm solution was not inhibited.

Utilizing this phenomenon, the test sample and AL in the coexistence of PMH
By measuring the optical change based on the gelation reaction that occurs when a hot solution is reacted, GL or/and GL in the test sample can be detected.
It has been found that D can be measured easily and accurately. Furthermore, GL or/in the test sample obtained by this method
By using the GLD concentration and the GLD concentration, the E in the test sample obtained by the method of measuring ET using Dmax described above can be
It was discovered that the reliability of the measured value of T concentration can also be tested,
The present invention has now been completed.

The present invention may be carried out, for example, as follows.

That is, first, after mixing the test sample and the AL hot solution, the optical change of this solution over a predetermined elapsed time is measured, and the degree of optical change due to the gelation reaction at 11 fl at the predetermined elapsed time is determined. Create a curve representing . Next, by comparing this curve with a curve representing the degree of optical change based on the gelation reaction over a predetermined elapsed time, which was prepared in advance using a specimen containing only ET, GL, or/and GLD, the test sample can be determined. It is determined whether the gelling substance contained therein is ET. Further, the curve representing the degree of optical change due to the gelation reaction in a predetermined elapsed time is differentiated to obtain a differential coefficient, Dmax and Tp are obtained, and these are determined in advance by predetermined ET-ET and GL. or/and GLD
, or ET and GL or/and GLD contained in the test sample by applying it to a graph showing the correlation between Dmax and Tp measured using a sample containing GL or/and GLD.
Estimate the content of In addition, the Dm obtained for the test sample
The ET concentration in the test sample is measured by applying ax to a calibration curve of Dmax and ET concentration prepared in advance using a sample containing ET at a predetermined concentration. Furthermore.

If the test sample and the AL warm solution are reacted together and the measurement is performed in the presence of PMB, the ET in the test sample will not cause the AL warm solution reaction, and the GL or /
and a gelation reaction triggered only by GLD occurs. Therefore, optical changes caused by this gelation reaction are measured, Tg, Dmax, etc. are determined, and the degree of GL and/or GLDII in the test sample is determined from these values. ET in the test sample previously determined based on this GL or/and GLD concentration.
Test the reliability of the concentration.

The AL hot solution that can be used in the method of the present invention is one that can be used for ordinary ET measurement and is GL or GL.
It can be used without particular limitation as long as it also reacts with D and causes a gelation reaction, but for example, A
CC(ASSOCIATES OF CAPE C0
Products prepared from commercially available freeze-dried AL warm solutions manufactured by D), Hemakem, MAB, Mallinckrodt, Teikoku Kinki Co., Ltd., etc., may be used, and Limulus spp. .

It is extracted from the blood cells of horseshoe crabs belonging to the genus Tachypleus or Carcinoscorpius.
It may be mentioned without particular limitation as long as it causes a gelation reaction by reaction with GL or GLD.

The operating method and reaction conditions for carrying out the present invention may be carried out in accordance with the nephelometric time analysis method, which is a well-known ET measurement method, and other reagents used may also be carried out according to the nephelometric time analysis method, which is a well-known ET measurement method. They may be selected and used as appropriate.

In the present invention, the pH at which optical changes are measured may be any pH that does not deactivate the factors that react with ET, GL, or GLD in the AL solution and cause a coagulation reaction. , usually in the range of 6 to 8 is preferably used. The temperature at the time of measurement may be any temperature that does not deactivate the factors that react with ET, GL, or GLD in the AL solution and cause a coagulation reaction.1 Usually, 0 to 40°C, more preferably 25 to 40°C. 40°C is used.

The method of the present invention can be carried out using, for example, a toxinometer ET-201.
(manufactured by Wako Pure Chemical Industries, Ltd.), LAL-5000 (ACC
It can be carried out using a specialized device for turbidimetric time analysis, such as a nephelometric analysis method (manufactured by Co., Ltd.), or it can be similarly carried out using a measuring device that uses other optical principles, such as a spectrophotometer.

In the method of measuring GL and/or GLD concentration of the present invention, the concentration of PMB used is not particularly limited as long as it can inhibit the gelation reaction of ET and AL warm solutions, but it is usually It is used so that the final concentration in the reaction solution during solution reaction is 100 ng/ml to 10 mg/ml.

GL and GLD that can be measured by the method of the present invention are not particularly limited as long as they are polysaccharides containing GL as a component, but include, for example, various bacteria (e.g. Alcaligenes genus, Lam1nari
a genus, Agrobacterium genus, etc.), yeasts (
For example, Saccharomyces genus), mushrooms
M (e.g. Shiitake, Suehirotake, Kawaratake, etc.)
Natural polysaccharides obtained from cell walls such as curdlan, pachyman, sclerotane, lentinan, etc.
Schizophyllan, Coriolan, etc., or storage polysaccharides of algae (e.g., brown algae, euglenoid, diatom, etc.), specifically, for example, laminaran, paramylon, etc., or conventional methods for these, e.g., Large Organic Chemistry Vol. , 7th edition, pp. 70-101, supervised by Munio Kotake, May 10, 1962, Breakfast Shoten;
A, E, C1arke et al., Phyt.

chemist, ry, soil, 175-188 (196
7); T, 5asaki et al., Europe.

J. Cancer, 15, 211-215 (1987)
Examples include derivatives obtained by introducing a carboxymethyl group, a carboxylethyl group, a methyl group, a hydroxyethyl group, a hydroxypropyl group, a sulfopropyl group, etc. according to the method described in et al.

Further, when GL and/or GLD are measured by the method of the present invention, the reactivity of the AL warm solution may differ depending on the origin of GL, the type and number of introduced substituents, etc. Therefore, by the method of the present invention, GL or/and GLD
When measuring GL or GLD, it is desirable to use a specific GL or GLD as a reference substance and to indicate the concentration of GL or/and GLD contained in the test sample as the concentration converted to the reference substance.

The present invention will be explained in more detail by reference examples, experimental examples, and examples below, but the present invention is not limited by these in any way.

[Example] Reference example 1. Preparation of carboxymethylated curdlan Curdlan (Wako Pure Chemical Industries, Ltd.) Toluene 54 to aog
0ml and 80ml of ethanol were added thereto, and 61g of a 50% aqueous sodium hydroxide solution was added dropwise thereto, then heated to 50°C and stirred for 1 hour. In addition, monochloric acid fi 35
g to 100ml of a mixed solvent of toluene:ethanol=9:1
1 was added to the mixture, and the mixture was further stirred at 50°C for 1 hour. After repeating the above operation twice, adding an aqueous sodium hydroxide solution and a monochloroacetic acid solution to the reaction solution,
Cool and store overnight. Add this to 90% methanol 1
1, filter the resulting precipitate and dry it to form 142
A crude crystal of g was obtained. The obtained crude crystals were dissolved in 1420 ml of distilled water, and the pH of this solution was adjusted to 8 using dilute hydrochloric acid.

12.781 methanol was added dropwise to this while stirring,
The resulting precipitate was collected by filtration, washed with 500 ml of 90% methanol, and dried to obtain the desired carboxymethylated curdlan (
Hereinafter, it will be abbreviated as CMGL. ) was obtained.

Experimental Example 1゜(Reagent) ・ET solution E. coli reference endotoxin (manufactured by Wako Pure Chemical Industries, Ltd., lipopolysaccharide derived from E. coli UKT-8 strain, 1
Per Vial FDA Reference Endotoxin E
Contains an amount equivalent to 500 ng as C-2, for 5 ml. ) was appropriately diluted with distilled water for injection.

・GL solution Add 5 mg of ET-free curdlan (manufactured by Wako Pure Chemical Industries, Ltd.) to a ET-free 50 mM NaOH aqueous solution.
After dissolving the solution to a volume of 1.0 ml, the solution was appropriately diluted with distilled water for injection and used.

・AL warm solution Freeze-dried AL warm solution derived from horseshoe crabs of the genus Mullus (
Hereinafter, it will be abbreviated as LAL. Manufactured by Wako Pure Chemical Industries, Ltd., gelling sensitivity: 0.03 El/m for 1.5 ml. ) in 5 ml of distilled water for injection was used.

(sample) An ET solution or GL solution with a predetermined concentration was used as a specimen.

(Operating method) Using a toxin meter ET-201 (manufactured by Wako Pure Chemical Industries, Ltd.), the following procedure was carried out according to a conventional method.

0.1 ml of the sample was added to 0.1 ml of the LAL solution, and after stirring, the mixture was kept at 37° C. and the change in the amount of transmitted light over a predetermined elapsed time of the mixture was measured.

(Results) The results are shown in Figures 1 and 2. In addition, Figures 1 and 2
In the figure, the change in the transmitted light amount is expressed as the transmitted light amount ratio (%) (L
Relative value of the amount of transmitted light in a predetermined time, when the amount of transmitted light immediately after mixing the AL solution and the sample is taken as 100%. ).

As is clear from this result, the tendency of the change in the amount of transmitted light over time is different when the ET solution is used as the sample and when the GL solution is used as the sample. From this, from the shape of the curve representing the degree of change in the amount of transmitted light over a predetermined elapsed time, it is clear that the gelling substance contained in the sample is
It was suggested that it is possible to determine whether it is T or not.

Further, the curve obtained here representing the degree of change in the amount of transmitted light for each specimen over a predetermined elapsed time was differentiated to obtain a differential coefficient, and Dmax and TP as shown in FIG. 3 were obtained.

In Fig. 3, a represents a curve representing the degree of change in the amount of transmitted light over a predetermined elapsed time, b represents a differential curve obtained by differentiating a, and Th represents the ratio of the amount of transmitted light. Indicates a certain value. A graph showing the correlation between the obtained Dmax and Tp is shown in the fourth rgI. In addition, in the v14 diagram, O
. indicates the results obtained using the GL solution as the sample, and . indicates the results obtained using the ET solution as the sample.

As is clear from the ρ results, in the correlation between Dmax and TP, there is a difference between samples containing ET and samples containing GL.
It can be seen that different linear relationships are obtained for each.

Experimental example 2゜ (reagent) The same one as in Experimental Example 1 was used.

(Sample) A solution containing ET, GL, or ET and GL at a predetermined concentration was used as a specimen.

(Operation method) The same procedure as in Experimental Example 1 was carried out.

(Results) Tables 1-1 and 1-2 show Dmax and T obtained for each sample.
The actual measured values of p are shown in Fig. 5, and the D w obtained for each sample is shown in Fig. 5.
A graph showing the correlation between ax and Tp is shown. In addition, No. S
In the figure, . indicates the results obtained with the sample containing only ET, O indicates the results obtained with the sample containing only GL, and Δ indicates the results obtained with the sample containing ET and GL. show.

Table 1-1 Table 1-2 As is clear from Table 1-1, Table 1-2 and Figure 5, ET
In samples in which the GL concentration was varied while keeping the concentration constant, there was a tendency for the position of the plotted point to move in parallel to the left as the GL concentration in the sample increased; It can be seen that when the concentration is changed, the position of the plotted point tends to move toward the upper left as the ET concentration in the sample increases. From this, we have determined in advance that Dmax and Tp at each ET or GL concentration for samples containing only ET and samples containing only GL.
It was suggested that the ET concentration and 0L211 degrees in an unknown sample can be estimated by measuring the above, creating a graph showing the respective correlations, and determining the Dmax and TP of the unknown sample.

In addition, from this result, it was found that D[1lax is mainly proportional to the ET concentration in the sample and is hardly affected by coexisting GL, suggesting that the ET concentration in the sample can be measured with high accuracy from Dmax. Ta.

Therefore, FIG. 6 shows the results of plotting the relationship between Dmax and ET concentration of each sample obtained here.

Here, * indicates the results obtained with the sample containing only ET, and ○ indicates the results obtained with the specified ET and 0.1 ng/ml GL.
The results obtained from the sample containing
The results obtained with the sample containing 0 g/ml of GL,
△ indicates the results obtained with the sample containing the specified ET and 10 ng/ml of GL;
The results obtained with samples containing ml of GL are shown.

As is clear from Figure 6, the difference between the actual measured value and true value of ET for each sample obtained based on Dmax becomes larger as the concentration of ET in the sample is lower and the concentration of coexisting GL is higher. I understand. In other words, there is a range of ET concentrations that can be measured by the measurement method using Dmax depending on the concentration of coexisting GL. In other words, the reliability of the ET concentration obtained by this measurement method depends on the coexisting GL concentration. It turns out that it will be done.

Example 1゜ (reagent) ・ET solution The same one as in Experimental Example 1 was used.

- CMGL solution The CMGL obtained in Reference Example 1 was dissolved in distilled water for injection, and then appropriately diluted and used.

・AL solution The same one as in Experimental Example 1 was used.

(Sample) ET solution of 0.250 EU/ml and CM of specified concentration
A sample was prepared by mixing the sample with the GL solution at a ratio of 1:1.

(Procedure) Using a toxinometer ET-201 (manufactured by Wako Pure Chemical Industries, Ltd.), the following procedure was carried out according to a conventional method.

0.1 ml of the sample was added to 0.1 ml of the LAL solution, and after stirring, the mixture was kept at 37° C. and the change in the amount of transmitted light over a predetermined elapsed time of the mixture was measured.

Analyze this data to determine Dmax and T for each sample.
g was calculated. These values were obtained in advance using a solution containing ET at a predetermined concentration as a sample. Apply external pressure to the calibration curve of ET concentration and Dmax, or ET211 degrees and Tg, and calculate the ET of each sample.
The actual measured value was obtained.

(result) The measurement results are shown in Table 2.

Table 2 As is clear from these results, in measuring the ET concentration in a solution in which CMGL coexists, the measurement accuracy is higher when Dmax is used as a measurement index than Tg.

However, even when using Dmax as an index,
When the concentration of CMGL in the sample increases, the effect is that E
The actual value of T is higher than the theoretical value. Therefore, in order to actually perform accurate measurements, it is necessary to
It can be seen that it is necessary to measure the GL concentration in parallel.

Experimental example 3. Effect on measured values when PMB coexists PMB specifically binds to ET, and the reaction of the AL warm solution,
That is, it is already known that it inhibits the gelation reaction. However, it is currently unknown whether PMB inhibits the reaction of GL or/and GLD with AL warm solution. Therefore, when PMB coexists, CM
We investigated the progress of the reaction between GL and AL hot solutions.

(reagent) The same material as in Example 1 was used.

(Sample) 0.125 El/ml ET solution and 1 ng/ml
CMGL solution, and PMB (Wako Pure Chemical Industries, Ltd.) respectively.
The sample was prepared by adding 0.2 mg/ml of sulfate (manufactured by Co., Ltd.) to the sample.

(Operation method) Toxinometer ET-201 (Wako Pure Chemical Industries, Ltd.)
The following procedure was carried out using a conventional method.

0.1 ml of the sample was added to 0.1 ml of the LAL solution, and after stirring, the mixture was kept at 37° C. and the change in the amount of transmitted light over a predetermined elapsed time of the mixture was measured.

(Results) The results obtained using the ET solution as the sample are shown in Figure 7.
The results obtained using the L solution as the sample are shown in Figure 8.6
In each figure, (1) shows the case where a sample to which PMB was not added was used, and (2) shows the case where a sample to which P'MB was added was used.

As is clear from the results in Figures 7 and 8, ET and AL
It can be seen that the warm solution reaction is greatly affected by the addition of PMB, whereas CMGL is not affected by the addition of PMB. That is, in the presence of PMB, the sample is
It was suggested that it is possible to selectively measure GL and/or GLD in a specimen by reacting with a L-warm solution and measuring the change in the amount of transmitted light of the mixture over a predetermined elapsed time.

Experimental Example 4゜From the results of Experimental Example 3, GL in the specimen or /
It was suggested that only GLD and GLD could be selectively measured. Therefore, we investigated the effect on the calibration curve for CMGL when PMB, ET, or PMB and ET coexist.

(reagent) The same material as in Example 1 was used.

(Sample) CMGL solution with a predetermined concentration, PMB in each CMGL solution
(sulfate) 0.2 o+g/+1 and ET 0.1
The sample was added at a concentration of 25 EU/ml.

(Procedure) Using a toxinometer ET-201 (manufactured by Wako Pure Chemical Industries, Ltd.), the following procedure was carried out according to a conventional method.

Add 0.1ml of the sample to the LAI solution of 0.1+++1, stir it, keep it warm at 37℃, measure the change in the amount of transmitted light of the above mixture over a predetermined elapsed time, and calculate the ratio of the amount of transmitted light for each sample. The time Tg5 required for a 5% decrease was determined.

(Results) The results are shown in Figure 9. In the figure, -・- indicates a sample containing only CMGL, -〇- indicates a sample containing CMGL and ET,
-Δ- indicates the results when samples containing CMGL, PMB, and ET were used, respectively.

As is clear from this result, when using a sample containing CMGL and ET, as the CMGL concentration decreases,
The Tg5 of each sample is determined by the calibration curve between Tg5 and CMGL concentration obtained from a sample containing only CMGL (hereinafter referred to as CMGL
Abbreviated as calibration curve. ), but CMGL, PM
When using samples containing B and ET, the Tg of each sample
5 matched the CMGL standard curve.

From the above results, it was found that GL and/or GLD of each specimen can be quantified by adding PMB to the specimen.

Example 2゜ (test sample) A commercially available cupric ammonia oxide regenerated cellulose membrane hollow fiber filter (effective filtration area 150 c+n2) was washed with 50 ml of distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.), and the resulting washing solution was used as the test sample.

(Specimen) The stock solution of the test sample, the 10-fold dilution of this with distilled water for injection, and the ET added to 0.25 EU/ml or PMB 0.2 mg/ml, respectively. was used as the specimen.

(LAL solution) The same one as in Experimental Example 1 was used.

(Procedure) Using a toxinometer ET-201 (manufactured by Wako Pure Chemical Industries, Ltd.), the following procedure was carried out according to a conventional method.

Add 0.1 ml of the sample to 0.1 ml of L A L solution, stir, and then measure the change in the amount of transmitted light of the above mixture over a predetermined elapsed time while keeping it warm at 37°C. Based on this data, Dm
Ax and TP were determined.

(Results) The correlation between the obtained Dmax and TP is shown in FIG. In the figure, Mu indicates the result obtained using the test sample stock solution as the sample, Δ indicates the result obtained using the test sample diluted 10 times, and Kaku and Kuchi each indicate the result obtained using PMB at 0.2 rag. /m
The results obtained with the sample prepared by adding 0.25 EU/ml of ET are shown as the results obtained with the sample prepared by adding 0.25 EU/ml of ET, respectively. In addition, ○ in the figure indicates the results obtained with the GL warm solution (GL containing curdlan) at a predetermined concentration used in Experimental Example 1, and the straight line in the figure connects these results.

As is clear from Fig. 10, the D max -T obtained using the test sample and its 10-fold dilution as the test sample.
When plotting p, the Dma obtained from the GL warm solution
Plotted on a straight line showing the correlation between x and Tp, the substance contained in the test sample is GL or/and GL D
It was inferred that.

Furthermore, the Dwax and Tp obtained from the sample to which PMB was added were in good agreement with the measurement results for the sample to which no PMB was added.
It was inferred that it was only GLD or/and GLD. In addition, when the Dmax obtained here was extrapolated to a previously prepared calibration curve of GL (curdlan) 1 degree and Dmax, it was found that the GL and/or GLD concentration in the test sample was It was calculated to be about 110 g/ml.

In addition, the Dmax obtained from the sample to which ET was added is
By extrapolating to the calibration curve of ET concentration and Dmax created in advance and calculating each ET concentration, the ET concentration in the sample prepared based on the test sample stock solution was 0.384 Eυ
The ET concentration in the sample prepared based on the test sample diluted 10 times with 0.214 EU/ml was 0.214 EU/ml, which was almost the same as the added ET concentration.

[Effects of the Invention] As described above, the present invention provides a method for easily determining whether or not a substance that reacts with an AL hot solution in a sample to cause a gelation reaction is ET, or This method provides a method for estimating or measuring the concentration of ET and GL and/or GLD, and also a method for testing the reliability of the obtained measured value of ET concentration, which was difficult to distinguish and quantify using conventional methods. This invention has a remarkable effect in that it is possible to easily and accurately discriminate and quantify ET and GL and/or GLD coexisting in a sample, and it is a great invention that contributes to this industry.

[Brief explanation of the drawing]

FIG. 1 was obtained in Experimental Example 1. Change in the amount of transmitted light of a reaction solution over a predetermined elapsed time when a specimen containing endotoxin (hereinafter abbreviated as ET) is reacted with a horseshoe crab blood cell component extract (hereinafter abbreviated as AL warm solution) The results are shown for the transmitted light amount ratio (%) obtained for each elapsed time (minutes) on the horizontal axis for each sample (when the transmitted light amount immediately after mixing with the AL warm solution sample is taken as 100%). The relative value of the amount of transmitted light over a predetermined elapsed time.) is plotted along the vertical axis and the points are connected. Further, Th indicates a specific value of the transmitted light amount ratio. Figure 2 shows the predetermined dosage of the reaction solution obtained in Experimental Example 1 when the sample containing β-1,3-glucan (hereinafter abbreviated as GL) was reacted with the AL warm solution. Shows the results of measuring changes in the amount of transmitted light over time, and shows the ratio (%) of the amount of transmitted light obtained for each elapsed time (minutes) on the horizontal axis for each specimen.
It connects the points where the values of are plotted along the vertical axis. Further, Th indicates a specific value of the transmitted light amount ratio. FIG. 3 shows Dmax and Tp used in the present invention.
In the figure, a is the result of measuring the change in the ratio of transmitted light over a predetermined elapsed time of the reaction solution when a sample containing ET was reacted with the AL solution (curve ), which connects the points obtained by plotting the transmitted light amount ratio values obtained for each elapsed time on the horizontal axis along the vertical axis, and b
shows the curve of the differential coefficient obtained by differentiating the curve. The value of the differential coefficient obtained for each elapsed time on the horizontal axis is plotted along the vertical axis, and the points are connected. Also,
T h indicates a specific value of the transmitted light amount ratio. Figure 4 shows the correlation between Dmax and Tp obtained from samples containing predetermined ET or GL obtained in Experimental Example 1, and for each TP (minutes) on the horizontal axis. It connects the points obtained by plotting Dmax along the vertical axis. In the figure, * indicates the measurement results obtained with the sample containing ET, and O indicates the results obtained with the sample containing GL. FIG. 5 shows the predetermined ET, G obtained in Experimental Example 2.
Dma obtained from samples containing L, or ET and GL
It shows the correlation between x and TP, and each T on the horizontal axis
The obtained Dmax is plotted along the vertical axis against p (min). In the figure, * indicates the measurement results obtained with the sample containing ET, O indicates the results obtained with the sample containing GL, and Δ indicates the results obtained with the sample containing ET and GL. FIG. 6 shows the predetermined ET or E obtained in Experimental Example 2.
Dmax and ET concentration (El/m
1. ) indicates the correlation between the sample containing only ET and O indicates the correlation between the predetermined ET and 0.
．． △ indicates the results obtained with the sample containing 1 ng/ml GL, △ indicates the predetermined ET and 10B.
The results obtained with a sample containing GL/ml are shown, and the results obtained with a sample containing a given ET and 100 ng/ml of 6-7 are shown. FIG. 7 shows that when reacting the sample containing ET obtained in Experimental Example 3 with the AL warm solution, polymyxin B CJ
Under EA, it is abbreviated as PMB. ) is added and not added, the results of measuring the change in the amount of transmitted light over a predetermined elapsed time of the reaction solution are shown, and the ratio of the amount of transmitted light obtained for each elapsed time (minutes) on the horizontal axis ( %) values plotted along the vertical axis. In the figure, (1) is PM
(2) shows the case where B is not added, and (2) shows the case where PMB is added. Further, Th indicates a specific value of the transmitted light amount ratio. Figure 8 shows the cases in which PMB was added and not when reacting the sample containing carboxymethylated GL (hereinafter abbreviated as CMGL) with the AL warm solution obtained in Experimental Example 3. It shows the results of measuring the change in the amount of transmitted light of the reaction solution over a predetermined elapsed time, and the value of the ratio of transmitted light amount (%) obtained for each elapsed time (minutes) on the horizontal axis is plotted along the vertical axis. The plotted points are connected. In the figure, (1) shows the case where PMB is not added, and (2) shows the case where PMB is added. Further, Th indicates a specific value of the transmitted light amount ratio. Figure 9 shows CMGL, CM obtained in Experimental Example 4.
Time required for the transmitted light amount ratio of each reaction solution to decrease by 5% Tg5 and CM obtained as a result of reacting a sample containing GL and ET, or CMGI, ET, and PMB with an AL warm solution
The relationship with GL211 degrees is shown, and each CMGL concentration (
ng/ml) versus Tg5 (min) plotted along the vertical axis. In the figure, −・−
indicates a sample containing only CMGL, -〇- indicates a sample containing CMGL and ET
-Δ- indicates the results when using a sample containing CMGL, ET, and PMB. FIG. 10 shows Dmax and Tp obtained in Example 2.
This is a diagram showing the correlation with (min), where MU is the result of the test sample stock solution, Δ is the result of the 10-fold diluted test sample, and ■ and 0.2 B/ml of PMB are added to each. The results obtained with the samples prepared by
．． The results obtained with samples prepared by adding 25 EU/ml are shown. In addition, ○ in the figure indicates the results obtained with the GL warm solution (GL containing curdlan) at a predetermined concentration used in Experimental Example 1, and the straight line in the figure connects these results. Patent applicant: Wako Pure Chemical Industries, Ltd. December 4, 1989

Claims (7)

[Claims] (1) Test sample and horseshoe crab blood cell component extract (hereinafter referred to as A
It is abbreviated as L solution. ) is measured, and from the shape of the curve representing the extent of the optical change due to the gelation reaction over a predetermined elapsed time, it is determined whether the sample contains A method for determining whether a substance that causes a gelation reaction is endotoxin (hereinafter abbreviated as ET). (2) By using as an index the maximum value of the differential coefficient obtained by differentiating the curve representing the degree of optical change due to the gelation reaction over a predetermined elapsed time and the elapsed time until reaching that maximum value. The discrimination method according to claim 1, wherein the discrimination method is performed. (3) The substance other than ET that causes the gelation reaction contained in the test sample is β-1,3-glucan (hereinafter, GL
It is abbreviated as ) or/and a derivative thereof (hereinafter abbreviated as GLD), the discrimination method according to claim 1 or 2. (4) A method for measuring the ET concentration and GL or/and GLD concentration in a test sample by measuring the optical change based on the gelation reaction that occurs when the test sample and AL solution are reacted. Based on the maximum value of the differential coefficient obtained by differentiating the curve representing the degree of optical change due to the gelation reaction over a predetermined elapsed time and the elapsed time until the maximum value is reached, the A method for estimating the ET concentration and GL or/and GLD concentration of. (5) In the method of measuring the ET concentration in a test sample by measuring the optical change based on the gelation reaction that occurs when the test sample and the AL solution are reacted, A method for measuring the ET concentration in a test sample from the maximum value of the differential coefficient obtained by differentiating a curve representing the degree of optical change due to a gelation reaction. (6) By measuring the optical change based on the gelation reaction that occurs when the test sample and AL solution are reacted in the presence of polymyxin B, GL or/and G
The concentration of LD is measured, and the concentration of GL or/in the obtained test sample is measured.
A method for testing the reliability of the ET concentration in a test sample obtained by the method according to claim 5 from the concentration of ET and GLD. (7) By measuring the optical change based on the gelation reaction that occurs when the test sample and AL solution are reacted in the presence of polymyxin B, GL or/and G
A method of measuring the concentration of LD.