JP2009085880A - Endotoxin measuring vessel - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique which enables the shortening of the amount of time required for gelation by mixture of an LAL reagent and a specimen and quickly and easily detecting endotoxin or measuring the concentration of endotoxin. <P>SOLUTION: Inside of a set of a glass cuvette 3 and an aluminum cap 5 that have been sterilized by dry heat, a magnetic stirrer bar 7 that has also been sterilized by dry heat is put in advance, so that an endotoxin measuring kit 1 is prepared. Further, a boundary portion 10 between the aluminum cap 5 and the glass cuvette 3 is sealed by a shrink-wrap plastic 9. The LAL reagent and the specimen are introduced into the endotoxin measuring kit 1, and then the magnetic stirrer bar 7 is driven externally. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a container for measuring endotoxin for detecting endotoxin or measuring its concentration.

  If endotoxin present in the outer membrane of Gram-negative bacteria adheres to and mixes with injection drugs, surgical instruments, fluids, etc., as well as dialysate, dialysis machines and instruments in artificial dialysis, etc. There is a risk of causing an exothermic reaction and various immune reactions. Therefore, it is obliged to manage endotoxin so that it is not mixed into the above-mentioned medicines and instruments.

  Common methods for measuring the concentration of endotoxin include gelation and turbidimetry. In these methods, the endotoxin measurement reagent and the sample are mixed in a glass measurement cell in which endotoxin has been previously inactivated by dry heat sterilization or the like, or in a separate container. Is mixed and moved to a glass measuring cell. Thus, in the gelation method, endotoxin is detected depending on whether the mixed solution gels within a certain time. In the turbidimetric method, the endotoxin concentration in the sample is measured by measuring the change in turbidity of the admixture over time.

As described above, in the gelation method, it is possible to determine whether or not endotoxin exceeding the reference exists in the sample by confirming whether or not the mixed solution has gelled after a certain time. On the other hand, in the turbidimetric method, gelation is detected from the change in turbidity over time, but the time required for gelation becomes shorter as the concentration of endotoxin in the specimen becomes higher. The concentration can be measured.
JP 2004-061314 A JP-A-10-293129

  Here, when measuring the concentration of endotoxin by the turbidimetric method, as described above, a mixed solution of the endotoxin measuring reagent and the sample is generated in the glass measurement cell subjected to the dry heat sterilization treatment. Then, the glass measurement cell is sealed with a cap subjected to dry heat sterilization, and then left standing, and the gelation of the mixed solution is optically measured from the outside.

  As described above, the gelation of the mixed solution is measured while leaving the mixed solution of the endotoxin measurement reagent and the specimen standing, and the detection of endotoxin is often performed by the gelation method from the past. Based on the historical background that the turbidimetric method has been developed for detailed concentration measurements. That is, in the gelation method, the mixture of the endotoxin measurement reagent and the sample is allowed to stand, and after a certain period of time, the glass measurement cell containing the mixture is turned upside down, and the dripping of the mixture in the cell is observed. This is because the gelation of the mixed solution was determined.

  As described above, if the endotoxin concentration measurement is performed by leaving the mixed solution of the endotoxin measurement reagent and the sample to stand, the measurement itself takes a long time, and measurement of endotoxin at a low concentration may not be practical. there were.

On the other hand, after mixing the endotoxin measurement reagent and the sample, gelation time can be shortened by continuously stirring the mixture using a magnetic stirrer in a glass heat-treated cell. Can be considered. However, in this case, there is a possibility that the preparation and procedure of the measurement may be complicated, such that it is necessary to further sterilize the magnetic stirrer at the start of endotoxin measurement.

  The present invention has been devised in view of the above-mentioned problems, and its object is to shorten the time required for gelation of a mixture of an endotoxin measurement reagent and a sample in endotoxin detection or concentration measurement. It is possible to provide a technique capable of performing highly accurate detection or measurement with a simpler procedure.

  In order to achieve the above object, according to the present invention, a container for measuring endotoxin is formed by preliminarily storing a magnetic stir bar sterilized by dry heat in a set of a glass measurement cell and cap sterilized by dry heat. This is the biggest feature.

More specifically, an endotoxin measurement container formed by sealing a glass measurement cell that should contain a specimen with a heat-resistant lid,
The glass measuring cell and the lid are sterilized by dry heat,
The container has a magnetic stirrer sterilized by dry heat sterilization.

  According to this, the glass measurement cell sterilized by dry heat sterilization is sealed with the lid sterilized by dry heat sterilization, and a magnetic stirrer previously sterilized by dry heat is housed therein for endotoxin measurement. Can provide a container. In this endotoxin measurement container, at the time of detection or concentration measurement of endotoxin, the lid of the container is removed, the endotoxin measurement reagent and the sample are introduced into the cell, and then sealed again with the lid. Samples for endotoxin detection as well as concentration measurements can be made. Moreover, initial contamination with endotoxin can be prevented more reliably, and turbidity can be measured by gelation while continuously stirring the endotoxin measurement reagent and the specimen. Therefore, it is possible to shorten the time required for endotoxin detection and concentration measurement, and to perform highly accurate detection or concentration measurement by a simpler procedure.

  Further, in the container for measuring endotoxin of the present invention, a sealing reinforcing member that reinforces the sealing property of the container by covering at least the boundary portion between the glass measuring cell and the lid on the outer surface of the container. You may make it have further.

  If it does so, it can prevent more reliably that bacteria etc. penetrate | invade into the inside of a container from the boundary part between a glass-made measurement cell and a heat-resistant lid | cover later.

  Further, the endotoxin measurement container of the present invention may further include a lyophilized LAL reagent inside the container.

  That is, by storing in advance a dry heat sterilized magnetic stirrer and a freeze-dried LAL reagent as an endotoxin measuring reagent in a set of dry heat sterilized glass measuring cell and heat resistant lid, endotoxin A container for measurement is prepared. Then, endotoxin detection and concentration measurement can be performed by a very simple procedure of introducing the specimen into the container.

In this case, the container further includes a sealing reinforcing member that reinforces the sealing property of the container by covering at least the boundary between the glass measuring cell and the heat-resistant lid on the outer surface of the container. As a result, it is possible to more reliably prevent moisture from entering the container. Then, it is possible to better maintain the storage state of the lyophilized LAL reagent in the container.

  The means for solving the above-described problems of the present invention can be used in combination as much as possible.

  In the present invention, in the detection or concentration measurement of endotoxin, the time required for gelation of the mixed solution of the endotoxin measurement reagent and the sample can be shortened, and more accurate detection or concentration can be performed with a simpler procedure. Measurements can be made.

  The best mode for carrying out the present invention will be exemplarily described in detail below with reference to the drawings.

  It is known that a horseshoe crab blood extract (LAL: Limulus amoebocyte lysate) gels when it reacts with endotoxin (ribopolysaccharide), which is a major component of the outer membrane of Gram-negative bacteria.

  Since this LAL gels by reacting with endotoxin with very high sensitivity, an extremely small amount of endotoxin can be detected. Therefore, the LAL method using this LAL as a measurement reagent is widely used as a means for detecting endotoxin.

  Specifically, in the LAL method, a LAL reagent and a specimen containing endotoxin are mixed. As a result, the reaction of the LAL reagent and endotoxin produces fine gel particles in the mixed solution, and the mixed solution gels. A method of optically measuring the presence of the gel particles using light scattering is called a light scattering particle measurement method. A method of optically measuring the turbidity of the mixed solution due to the formation of gel particles or gelation of the mixed solution by the light transmittance is referred to as a turbidimetric method.

  Next, specific operations when endotoxin detection or concentration measurement is performed by the LAL method will be described with reference to FIG. Conventionally, when endotoxin detection or concentration measurement is performed by the LAL method, a glass cuvette 3 and an aluminum cap 5 sterilized by dry heat as shown in FIG. 4A are used. Then, as shown in FIG. 4B, a mixed solution of the LAL reagent and the sample mixed in a separate container is put into a glass cuvette 3 and further sealed with the above-described dry heat sterilized aluminum cap 5. Then, as shown in FIG. 4C, the mixture was allowed to stand to wait for gelation of the mixed solution, and endotoxin was detected or the concentration was measured by optically detecting the occurrence of gelation of the mixed solution.

  Here, the glass cuvette 3 and the aluminum cap 5 are preliminarily sterilized by dry heat because the LAL reacts with a very small amount of endotoxin as described above. This is because when the sample is initially contaminated, endotoxin detection or concentration measurement cannot be accurately performed on the specimen.

  However, in the conventional detection method and measurement method as described above, since the reaction proceeds by leaving the mixture of the LAL reagent and the specimen stationary, the time required for the reaction is prolonged and the measurement time is prolonged. It was.

In addition, it is conceivable to continuously stir the mixture from the outside using a magnetic stirrer bar (magnetic stirrer) to increase the reaction rate. In this case, it is necessary to sterilize the magnetic stirrer bar separately. Therefore, it has been difficult to efficiently detect endotoxin or measure the concentration in a short time.

  Therefore, in this example, a kit for measuring endotoxin is prepared by combining a glass cuvette, an aluminum cap and a magnetic stirrer bar that have been sterilized by dry heat in advance, so that endotoxin measurement can be performed more efficiently. I was able to.

  FIG. 1 shows an endotoxin measurement kit 1 in this example and a method for producing the kit.

  As shown in FIG. 1 (a), the endotoxin measurement kit 1 in this example houses a magnetic stirrer bar 7 in a glass cuvette 3 and is further sealed with an aluminum cap 5. Then, dry heat sterilization is performed at 250 ° C. for 2 hours. Therefore, in the state of FIG. 1B, the sterilization process inside the endotoxin measurement kit 1 is completed, and it is unlikely that the endotoxin is contaminated.

  In this embodiment, in the state of FIG. 1B, the heat shrink film 9 is further covered so as to cover the boundary portion 10 between the glass cuvette 3 and the aluminum cap 5, and then the heat shrink film 9 sufficiently shrinks. The endotoxin measurement kit 1 is heated to the temperature at which Thereby, the heat shrink film 9 seals the aluminum cap 5 and the boundary portion 10 as shown in FIG. In this state, the final endotoxin measurement kit 1 is completed. By doing so, it is possible to more reliably prevent bacteria and the like from entering the endotoxin measurement kit 1 after completion.

  When endotoxin is measured using this endotoxin measurement kit 1, first, the heat shrink film 9 is peeled off, the aluminum cap 5 is removed, and the LAL reagent and the sample are introduced therein. Thereafter, the aluminum cap 5 is sealed, and in this state, the magnetic stirrer bar 7 is driven from the outside to continue the stirring of the mixed solution while optically generating gel particles in the mixed solution or gelling the mixed solution. taking measurement. If it does so, generation | occurrence | production of the gel particle in a liquid mixture or gelatinization of a liquid mixture can be accelerated | stimulated by simple operation, and the time required for a detection or a density | concentration measurement can be shortened.

  As described above, when endotoxin is measured using the endotoxin measurement kit 1 in the present embodiment, the heat shrink film 9 and the aluminum cap 5 of the endotoxin measurement kit 1 are removed, and the glass cuvette 3 is attached to the LAL. The preparation for endotoxin detection or concentration measurement is completed simply by introducing the mixed solution of the reagent and the specimen and closing the aluminum cap 5 again. In addition, by driving the magnetic stir bar 7 from the outside, the LAL reagent and the sample can be reacted while continuing to stir the admixture, and endotoxin measurement can be performed in a shorter period and more efficiently. It becomes possible. Further, since the endotoxin measurement kit 1 is surely subjected to the dry heat sterilization treatment, it is possible to more reliably prevent initial contamination with endotoxin.

  In the above description, in FIG. 1A, the glass cuvette 3, the aluminum cap 5 and the magnetic stirrer bar 7 are combined and then subjected to the dry heat sterilization treatment. The cap 5 and the magnetic stirrer bar 7 may be separately subjected to a dry heat sterilization treatment, and then combined with each other.

In the above embodiment, the endotoxin measurement kit 1 is expected to be stored for a long time, and the aluminum cap 5 and the boundary portion 10 are sealed with the heat shrink film 9, but this is only an endotoxin measurement kit. This is to further improve the reliability of 1. This is not always necessary when the storage period of the endotoxin measurement kit 1 is short.

  Here, the glass cuvette 3 corresponds to a glass measuring cell in this embodiment. The aluminum cap 5 corresponds to a heat resistant lid. The heat shrink film 9 corresponds to a sealing reinforcement member. Further, the endotoxin measurement kit 1 corresponds to a container for endotoxin measurement. The material of the heat-resistant lid is not necessarily aluminum, and may be other metals such as iron and titanium, or ceramic. Furthermore, the opening of the glass cuvette 3 may be sealed with aluminum foil or the like. Moreover, as a sealing reinforcement member, materials other than a heat shrink film may be used, for example, a paraffin film may be used.

  Next, a second embodiment of the present invention will be described. In this example, an example will be described in which a previously lyophilized LAL reagent is enclosed in the endotoxin measurement kit in Example 1.

  FIG. 2 shows an endotoxin measurement kit 11 in this example and a method for producing the kit. In this embodiment, first, as shown in FIG. 2A, dry heat sterilization is performed on each of the glass cuvette 3, the aluminum cap 5, and the magnetic stirrer bar 7. Then, the magnetic stirrer bar 7 is stored in the glass cuvette 3.

  Next, in that state, in FIG. 2B, a necessary amount of the LAL reagent is further introduced into the glass cuvette 3 and freeze-dried. And after that, it seals with the aluminum cap 5 in FIG.2 (c). The subsequent FIG. 2D and FIG. 2E are the same as those in FIG. 1B and FIG. 1C, and are omitted here.

  In the endotoxin measurement kit 11 in the present embodiment, the procedure for detecting endotoxin or measuring the concentration can be greatly simplified. That is, the heat shrink film 9 of the endotoxin measurement kit 11 is peeled off, the aluminum cap 5 is removed, only the specimen is introduced into the glass cuvette 3, and the aluminum cap 5 is closed again. The liquid can be generated in a glass cuvette 3. If the magnetic stirrer bar 7 is driven from the outside, the LAL reagent and the specimen can be reacted while continuing to stir the mixture. Therefore, according to this example, it becomes possible to more efficiently detect endotoxin or measure the concentration. Moreover, since the initial contamination by the endotoxin of the measurement kit 11 itself can be more reliably prevented, improvement in measurement accuracy can be achieved.

  Next, a third embodiment of the present invention will be described. In this example, instead of sealing the aluminum cap and the boundary portion with the heat shrink film in Example 2, before sealing the glass cuvette 3 with the aluminum cap, the sealing performance is enhanced with a heat-resistant rubber plug. An example will be described.

  FIG. 3 shows an endotoxin measurement kit 21 in this example and a method for producing the kit. In this embodiment, first, as shown in FIG. 3A, in addition to the glass cuvette 3, the aluminum cap 5, and the magnetic stirrer bar 7, the rubber plug 8 is also subjected to dry heat sterilization. Then, the magnetic stirrer bar 7 is stored in the glass cuvette 3.

Next, in that state, in FIG. 3B, as in FIG. 2B, the required amount of LAL reagent is further introduced into the glass cuvette 3 and freeze-dried. Then, after that, in FIG. 3C, air is removed from the glass cuvette 3 and the rubber stopper 8 is attached. In FIG. 3D after that, the aluminum cap 5 is further sealed from above the rubber stopper 8. Thereby, the endotoxin measurement kit 21 is completed as shown in FIG.

  Also in the endotoxin measurement kit 21 in the present embodiment, the procedure at the time of endotoxin detection or concentration measurement can be greatly simplified. That is, the aluminum cap 5 and the rubber plug 8 of the endotoxin measurement kit 21 are removed, the required amount of sample is introduced into the glass cuvette 3, and the aluminum cap 5 is closed again. Can be produced in the glass cuvette 3. And if the magnetic stirrer bar 7 is driven from the outside, the LAL reagent and the specimen can be reacted while continuing to stir the mixture.

  In this embodiment, after the LAL reagent is freeze-dried, the air is removed and the rubber stopper 8 is used to maintain the endotoxin measurement kit 21 in a negative pressure state even after completion. Accordingly, moisture absorption of the LAL reagent can be prevented more reliably and bacteria and the like can be prevented from entering. As a result, according to the present embodiment, the deterioration due to moisture absorption of the LAL reagent and the initial contamination of the measurement kit 21 itself with endotoxin can be more reliably prevented, so that further improvement in measurement accuracy can be achieved.

  In this embodiment, the rubber plug corresponds to a sealing reinforcement member. In the present invention, after the endotoxin measurement kit 21 is completed, the sealing property may be further enhanced by a heat shrink film or the like.

It is a figure for demonstrating the kit for an endotoxin measurement in Example 1 of this invention. It is a figure for demonstrating the kit for an endotoxin measurement in Example 2 of this invention. It is a figure for demonstrating the kit for an endotoxin measurement in Example 3 of this invention. It is a figure for demonstrating the method of the conventional endotoxin measurement.

Explanation of symbols

1, 11, 21 ... Endotoxin measurement kit 3 ... Glass cuvette 5 ... Aluminum cap 7 ... Magnetic stirrer bar 8 ... Rubber stopper 9 ... Heat-shrinkable film 10 ...・ Boundary part

Claims (3)

A container for measuring endotoxin formed by sealing a glass measuring cell that should contain a specimen with a heat-resistant lid,
The glass measuring cell and the lid are sterilized by dry heat,
A container for measuring endotoxin, comprising a magnetic stirrer sterilized by dry heat inside the container.   The sealing reinforcing member which reinforces the sealing property of the container by covering at least the boundary portion between the glass measuring cell and the lid on the outer surface of the container. Container for endotoxin measurement.   The container for measuring endotoxin according to claim 1 or 2, further comprising a lyophilized LAL reagent inside the container.

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