JPH0531744B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for measuring endotoxin in a test liquid. Endotoxin is a typical pyrogen, and when endotoxin-contaminated blood, transfusions, or injections are injected into a living body, it can cause severe side effects such as strong fever and shock. Are known. For this reason, in the manufacturing process of pharmaceuticals such as injection solutions, it has become essential to measure the amount of endotoxin in raw water and washing water to prevent contamination. In addition, endotoxin measurement has become widely used as a function test for ultrapure water production membranes or a water quality test for cleaning water used in semiconductor production. In recent years, as a method for measuring endotoxin, it has been discovered that Limulus amoebocyte lysate (limeshoe crab blood cell extract, hereafter abbreviated as LAL) reacts with endotoxin and turns into a gel, and a method for measuring endotoxin using this has been developed. has been done. This measurement method involves mixing the test liquid and LAL reagent in a test tube, leaving it at a constant temperature for a certain period of time, then tilting or overturning the reaction test tube and visually observing whether the sample has gelled. The positive (+) or negative (-) endotoxin in the test solution is determined semi-quantitatively by comparing the results of a similar reaction between an endotoxin sample of known concentration and the LAL reagent. However, this judgment method requires skill, and since the +/- judgment criteria depend on the subjectivity of the measurer, there are large individual differences in judgment, and the judgment limit is around 0.05 ng/ml, making it impossible to detect low-concentration endotoxins. There were some drawbacks. On the other hand, a measurement method has also been developed in which a chromogenic peptide derivative is used as a synthetic substrate, and the endotoxin concentration is determined by colorimetrically quantifying the degree of color development when the substrate is hydrolyzed by LAL and endotoxin in the sample. However, it has not yet been introduced into routine testing due to the narrow measurement range of several pg/ml to 100 pg/ml and the extremely complicated measurement operation. As a result of intensive research to solve the above-mentioned conventional drawbacks, the present inventors were able to detect gelation of LAL due to endotoxin in the test solution down to low concentrations using an objective standard. The present invention provides a method for quantifying endotoxin concentrations over a wide range from low to high concentrations with high precision and extremely ease. That is, the present invention irradiates a cube holding a sample solution containing an endotoxin gelling reagent and a test solution with light, and calculates the amount of light transmitted from the sample solution at time t, I(t), and the progress of the reaction. Find the ratio of the amount of transmitted light from the sample liquid to the initial value _I0 before it starts decreasing, and make sure that the ratio is 75% or more when viewed as I(t)/ _I0 .
The time when an arbitrarily set constant value in the range of 97% or less is reached is determined as the gelation time of the sample liquid, and the time from the mixing time of the sample liquid to the gelation determination time is the gelation of the sample liquid. This is an invention of a method for measuring endotoxin, which is characterized in that the gelation time is determined as time and used as an index when measuring endotoxin concentration. In the present invention, for example, the optical system shown in FIG. 1 can be used to measure the transmitted light of the sample. That is, the measurement cuvette 1 (usually 6 mm inner diameter)
Use items with an inner diameter of 12 mm. ) Sample solution 2 containing a mixture of test solution and LAL reagent
(Normally, 0.1ml of test solution and 0.1ml of LAL reagent, or
Consists of lyophilized LAL reagent and 0.2ml of test solution. ) is irradiated with a light beam from a light source 5 through an aperture 3. The light beam that has passed through the sample liquid 2 passes through an aperture 4 and is converted by a photoelectric detector 6 into an amount of electricity corresponding to the amount of transmitted light. Here, the light source 5 is, for example, a light emitting element such as an LED (light emitting diode) or a tungsten lamp.
The photoelectric detector 6 may be a light receiving element such as a photodiode or a photocell. The amount of transmitted light I(t) detected by the photoelectric detector 6 shows a change over time as shown in FIG. 2 with respect to the reaction time t after mixing the test liquid and the LAL reagent. That is, there is a stage a in which the amount of transmitted light at the beginning of the reaction shows an almost constant initial value _I0 , then a stage b where the amount of transmitted light I(t) rapidly decreases, and finally a value where I(t) shows almost no fluctuation. Step c, the sample gels through the above three steps. The inventors,
This stage b is a state in which gelation is rapidly progressing within the sample, and the sample liquid that has reached stage c is a state in which the gelation reaction is determined to be positive by conventional visual measurement. Furthermore, it was experimentally discovered that the time required from the start of the reaction to reach stage b and stage c is correlated with the endotoxin concentration in the test solution, and it has good accuracy over a wide measurement range and is suitable for routine testing. We have completed the endotoxin measurement method of the present invention, which can be applied to. In order to correct the difference in the amount of transmitted light due to the turbidity and coloring that the sample liquid originally has, the present invention uses the amount of transmitted light I(t) and the initial value I of the amount of transmitted light before it starts to decrease due to the progress of the reaction. It is characterized by measuring the ratio R(t) with respect to ₀ . That is, R(t)=[I(t)/I ₀ ]×100
The ratio R(t) determined by [%] exhibits similar behavior to I(t) as shown in FIG. (t) is 75%
Set the threshold value Rth within the range of 97% or less, and R(t)
The time when Rth is reached is determined as the time when the sample solution gels. Further, the reaction time from the time of mixing the sample solution to the time of gelation determination is detected as the gelation time T _G and used for quantitative determination. Note that Rf indicates R(t) at stage c, where I(t) is a constant value with almost no fluctuation. The initial value I ₀ of the amount of transmitted light in the present invention is as follows:
Whether it is the maximum value of the amount of transmitted light I(t) or the average value of the amount of transmitted light I(t) within a certain time in the initial stage, the amount of transmitted light I(t _s ) at any point t _s within a certain time in the initial stage However, it may also be the minimum value of the amount of transmitted light I(t) within a certain period of time in the initial stage. FIG. 4 is a plot of the relationship between endotoxin concentration and R(t) using the elapsed time from the time of mixing the sample liquid as a parameter. The reagents and measurement methods used are as follows. LAL reagent: Associates of Cape Cod, Inc. Visual gelation fallover method using FDA reference Sensitivity: 0.05 ng/ml Endotoxin: Escherichia coli UKT-B
Purified from strain (Osaka University Institute of Technology). Content verified by FDA reference. Measurement method: The above LAL reagent and endotoxin were added to distilled water for injection (Otsuka Pharmaceutical Co., Ltd.).
Dissolve 0.1 ml of each in diameter
Stir and mix in a 10 mm test tube and measure the change in the amount of transmitted light. In the figure, A, B, C, D, E, and F each indicate elapsed time 5.
Indicates minutes, 10 minutes, 20 minutes, 35 minutes, 60 minutes, and 120 minutes. From FIG. 4, it can be seen that at all concentrations measured, R(t) decreases with gelation, and the higher the concentration of the sample, the earlier the decrease begins. Furthermore, it is clear from this figure that the final value Rf, at which R(t) shows almost no decrease over time, does not show any significant difference in response to changes in concentration and is not suitable as an indicator of the amount of endotoxin. It is. On the other hand, Rth is the value before R(t) reaches Rf (for example, in FIG. 4, a broken line is drawn with R(t) = 95% as Rth).
By detecting that Rth has been reached, gelation of the sample solution can be determined over a wide range of concentrations _.
It can be seen that a wide range of endotoxin concentrations can be quantified by measuring as . The time it takes for R(t) to reach Rf depends on the sensitivity of the LAL reagent used,
Since it differs depending on the shape of the cube, etc., in the present invention, in consideration of these points, the value of R(t) is determined to ensure gelation over a wide concentration range between R(o) and Rf. It was decided to set the threshold value Rth for gelling detection in the detectable range of 75% or more and 97% or less. If Rth is smaller than 75%, e.g.
When Rth = 70%, gelation cannot be detected,
If a calibration curve cannot be obtained, or if the concentration range in which a calibration relationship can be obtained is, for example, endotoxin concentration 0.1~
It is very narrow at 10ng/ml. On the other hand, Rth to 97%
If the value is larger than , the fluctuation in R(t) due to vibration (convection) or movement of air bubbles in the sample solution immediately after mixing the LAL reagent and test solution may be mistakenly judged as gelation. There is a possibility that it will be stored away. Therefore, a noise margin of 3% is required to prevent false detection due to such noise. That is, in the present invention, it is an essential component to set Rth to a constant value in the range of 75% or more and 97% or less, more preferably 80% or more and 95% or less. Figure 5 compares the results of determining gelation using the method of the present invention with Rth = 95% for the same test solution, and the results of determining gelation using the conventional visual gelation inversion method. be. More specifically, the shorter the gelation time obtained by the method of the present invention, the higher the endotoxin concentration in the sample.
(Tm = 25 minutes in Figure 5) If shorter, positive;
If the gelation time is longer than Tm, it is considered negative, and FIG. 5 shows a correlation diagram with the determination result by the conventional visual gelation overturning method. Note that the reagents and measurement method used in the method of the present invention are as described in Section 4.
It is the same as that in the figure. As is clear from FIG. 5, the determination results of both methods are in good agreement. Since the conventional gelation inversion method only makes a binary judgment of positive (+) or negative (-) on the gelation state of the sample solution after 60 minutes, the judgment according to the present invention can also be made within a certain measurement time Tm. Automatic judgment positive (AMJ+)
Alternatively, a binary judgment of automatic negative judgment (AMJ-) was performed. In the conventional method, only a sample liquid in which R(t) reaches Rf can be determined as positive (+), whereas according to the present invention, a positive determination can be made at an early stage before R is reached. That is, in this example, Tm=25 minutes, and the same determination result could be obtained in less than half the measurement time of the conventional method.
Moreover, no skill is required for the determination, and objective results without individual differences can be obtained. or,
By extending the Tm, it is also possible to detect gelation at low concentrations, which was impossible to determine using conventional methods. The present invention also enables highly accurate quantitation over a wide measurement range that was impossible with conventional measurement ranges, and the detection sensitivity and measurement range vary depending on the sensitivity of the LAL reagent used, but any In this case, the sensitivity is much higher than that obtained by the conventional visual gelation inversion method, and the dynamic range is much wider than that of the colorimetric method using a synthetic substrate. As described above, the present invention provides an objective, sensitive, and rapid method for measuring endotoxin by measuring the amount of light transmitted through a sample liquid using a unique configuration. The present invention provides an excellent method for measuring endotoxin that can achieve high-accuracy quantification over a wide measurement range with simple operations, which is impossible to achieve with conventional methods, and thus makes an extremely large contribution to this industry. The effects of the present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples in any way, and may be applied to various specific examples within the scope of the present invention. It goes without saying that it is possible. Example 1 Endotoxin concentration was measured under the following measurement conditions. LAL reagent: Visual gelation inversion method using FDA reference manufactured by Associates of Cape Cod. Sensitivity: 0.05 ng/ml Endotoxin Purified from Escherichia coli UKT-B strain (Osaka University Institute of Technology). Content verified by FDA reference. Measurement method: The above LAL reagent and endotoxin were added to distilled water for injection (Otsuka Pharmaceutical Co., Ltd.).
Dissolve 0.1 ml of each in diameter
Stir and mix in a 10 mm test tube and measure the change in the amount of transmitted light. FIG. 6 shows the calibration relationship between gelation time and endotoxin concentration in this example, where Rth was set to 95%. As is clear from FIG. 6, in this example, a good calibration relationship was obtained over a very wide range from 0.0005 ng/ml to 100 g/ml. This is 100 times more sensitive than the detection sensitivity obtained by the visual gelation inversion method using the same reagents, and more than 1000 times more dynamic than the measurement range of the colorimetric method using a synthetic substrate. In this example, when Rth=70%, gelation cannot be detected and a calibration curve cannot be obtained. Example 2 Under the same measurement conditions as in Example 1, the gelation time was measured with 16 repetitions for six sample solutions having different endotoxin concentrations within the range of Example 1. Table 1 shows the gelation time T _G and the reproducibility of the concentration value converted using the calibration curve shown in FIG. As is clear from Table 1, very good results were obtained using this method, with the CV value of T _G being within 4.5% and the CV value of concentration being within 15%. Performing the above-mentioned highly accurate quantitative measurement over such a wide range of endotoxin concentrations was not possible before the disclosure of the technology according to the present invention, but with the method of the present invention, it has been possible for the first time. This can now be achieved using a simple measurement procedure similar to visual gelation and inversion.

【table】

[Table] Example 3 Endotoxin concentration was measured under the following measurement conditions. LAL reagent: Visual gelation inversion method using FDA reference manufactured by Associates of Cape Cod Sensitivity: 0.01 ng/ml Endotoxin: Same as Example 1 Measurement method: Same as Example 1 In this experiment, Rth was set to 95% in Figure 7. The calibration relationship between gelation time and endotoxin concentration in an example is shown. As is clear from FIG. 7, in this example, the endotoxin concentration ranged from 0.0001 ng/ml to 100 g/ml.
A calibration relationship was observed up to ml. This is higher than the detection sensitivity obtained by the visual gelation inversion method using the same reagents.
It is 100 times more sensitive and has a dynamic measurement range that is approximately 10,000 times greater than that of colorimetric methods using synthetic substrates. In this example as well, when Rth = 70%, gelation cannot be detected and a calibration curve cannot be obtained. Example 4 Endotoxin concentration was measured under the following measurement conditions. LAL reagent: Visual gelation inversion method using FDA reference manufactured by Associates of Cape Cod Sensitivity: 0.1 ng/ml Endotoxin: Same as Example 1 Measurement method: The above endotoxin was dissolved in distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) as the test liquid. Add freeze-dried LAL reagent and 0.2 ml of test solution to a 12 mm diameter test tube, stir and dissolve, and measure the change in the amount of transmitted light. FIG. 8 shows the calibration relationship between gelation time and endotoxin concentration in this example where Rth was set to 85%. As is clear from FIG. 8, in this example, the endotoxin concentration ranged from 0.02 ng/ml to 100 ng/ml.
A calibration relationship was observed in the range up to ml. This has a detection sensitivity five times higher than that of the visual gelation inversion method using the same reagent, and a dynamic range of measurement that is about 50 times that of the colorimetric method using a synthetic substrate. In this example, when Rth=70%, the concentration range in which a calibration relationship can be obtained is extremely narrowed to an endotoxin concentration of 0.1 to 10 ng/ml. Experimental Example 1 We investigated the relationship between Rth and reaction time when the production lots of endotoxin gelling reagents were different. (Endotoxin gelation reagent) LAL-A: manufactured by Associates of Cape Cod,
Visual gelation tipping method sensitivity with FDA reference
0.025ng/ml. LAL-B: Manufactured by Associates of Cape Cod,
(The lot is different from LAL-A.) Visual gelation fallover method sensitivity according to FDA reference is 0.025 ng/ml. LAL-C: Manufactured by Haemachem, visual gelation fall-over method sensitivity 0.025 ng/ml based on FDA reference. (Endotoxin) Same as Example 1 (Test solution) The above endotoxin was mixed with distilled water for injection (Otsuka Pharmaceutical
Co., Ltd.) to achieve an endotoxin concentration of 2 ng/
ml and 0.01 ng/ml solutions were prepared and used as test solutions. (Procedure) 0.1 ml of a predetermined endotoxin gelation reagent and 0.1 ml of a predetermined test liquid were stirred and mixed in a test tube with a diameter of 10 mm, and changes in the amount of transmitted light were measured. (Results) The obtained results are shown in Figure 9. The explanation of each symbol in FIG. 9 is as shown in Table 2.

【table】

[Table] From the results in Figure 9, it is clear that the graph showing the relationship between R(t) and reaction time (minutes) differs depending on the lot of endotoxin gelling reagent, and that the measurement using LAL-A's endotoxin gelling reagent In this case, Rth can only be set within the range of 97% to 85%, but when using LAL-B or LAL-C endotoxin gelling reagents, Rth can be set within the range of approximately 97% to 75%. It can be seen that endotoxin can be measured even when Rth is set to .

[Brief explanation of the drawing]

Fig. 1 is a diagram of the principle of the optical system for measuring the amount of light transmitted through the sample liquid, Fig. 2 is a diagram showing the temporal change characteristics of the amount of light transmitted through the sample liquid I(t), and Fig. 3 is a diagram showing the amount of light transmitted through the sample liquid I(t). , Figure 4 is a diagram showing the temporal change characteristics of the ratio R(t) between the initial value I ₀ of the amount of transmitted light before it starts to decrease due to the progress of the reaction, and Figure 4 is a diagram showing the temporal change characteristics of the endotoxin concentration and R(t).
FIG. 5 is a correlation diagram between the gelation determination results according to the present invention and the determination results according to the conventional method (visual gelation overturning method);
FIG. 6, FIG. 7, and FIG. 8 are calibration relationship diagrams between endotoxin concentration and gelation time in Example 1, Example 3, and Example 4, respectively. Figure 9 shows
2 is a graph showing the relationship between R(t) and reaction time when endotoxin is reacted with various lots of endotoxin gelling reagents obtained in Experimental Example 1. 1... measurement cuvette, 2... sample solution, 3,
4...Aperture, 5...Light source, 6...Photoelectric detector.

Claims (1)

[Claims] 1. A cube holding a sample solution containing a mixture of an endotoxin gelling reagent and a test solution is irradiated with a light beam, and the amount of transmitted light I(t) from the sample solution at time t and the progress of the reaction are determined. Find the ratio of the amount of transmitted light from the sample liquid to the initial value _I0 before it starts to decrease, and select an arbitrary value in which the ratio is in the range of 75% or more and 97% or less when viewed as I(t)/ _I0 . The time when a certain value set in is reached is determined as the gelation time of the sample liquid, and the time from the mixing time of the sample liquid to the gelation determination time is determined as the gelation time of the sample liquid, and the gelation time is determined as the gelation time of the sample liquid. 1. A method for measuring endotoxin, characterized in that it is used as an index when measuring endotoxin concentration. 2. Measurement of endotoxin according to claim 1, in which the endotoxin concentration in the test liquid is determined by applying the gelation time to a calibration curve showing the relationship between the endotoxin concentration determined in advance and the gelation time. Method. 3. The method for measuring endotoxin according to claim 1 or 2, wherein the initial value I ₀ of the amount of transmitted light is the maximum value of the amount of transmitted light I(t). 4. The method for measuring endotoxin according to claim 1 or 2, wherein the initial value I ₀ of the amount of transmitted light is an average value of the amount of transmitted light I(t) within a certain period of time in the initial stage. 5. The endotoxin according to claim 1 or 2, wherein the initial value I ₀ of the amount of transmitted light is the amount of transmitted light I (t _s ) at any specific time t _s within a certain period of time in the initial stage How to measure. 6. The method for measuring endotoxin according to claim 1 or 2, wherein the initial value I ₀ of the amount of transmitted light is the minimum value of the amount of transmitted light I(t) within a certain period of time in the initial stage. 7. The method for measuring endotoxin according to any one of claims 1 to 6, wherein the cuvette for holding the sample liquid is a cylindrical test tube with an inner diameter in the range of 6 mm to 12 mm. 8 The sample solution is 0.1ml of endotoxin gelation reagent.
8. The method for measuring endotoxin according to any one of claims 1 to 7, comprising 0.1 ml of the test solution and a lyophilized endotoxin gelling reagent and 0.2 ml of the test solution.