JPH075176A - Method and apparatus for measuring endotoxin - Google Patents

Abstract

PURPOSE:To provide method and apparatus for measuring endotoxin in which the operability is enhanced while facilitating automation. CONSTITUTION:The method for measuring endotoxin comprises a step for introducing an adsorbent 12 into a column 4 through an opening 1 at the tip of the column having means 3 for passing a solution specimen and holding the endotoxin adsorbent, a step for introducing the solution specimen into the column 4 through an opening at the tip thereof and discharging the solution specimen through an opening 2 at the rear end thereof to bring into contact with the adsorbent, and a step for introducing a detection reagent into the column 4 through the opening 1 to cause reaction on the endotoxin adsorbed by the adsorbent 12. The measuring apparatus comprises the column 4, a vessel 6 containing the solution specimen or the like, a column sustaining means 7, and means 11 for applying negative pressure to the column. This method and apparatus allows convenient measurement of endotoxin and facilitate automation.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an improved method and apparatus for measuring endotoxin.

[0002]

2. Description of the Related Art Endotoxin, which is a type of pyrogen, is measured by a febrile test using rabbits, and a blood cell extract of Limulus polyphemus (Limulus Amebocyte Lysate; Limulus Amebocyte Lysate;
LAL] is being used (limulus method), but this limulus method is often affected by inhibition and promotion by substances coexisting in the measurement system, and thus pretreatment is often required. Examples of such pretreatment include dilution, heating, perchloric acid treatment, etc., but none of them can be said to be sufficient in terms of specificity, sensitivity, and quantitativeness when applied to endotoxin measurement. In order to solve such a problem, the present inventors have already developed a measuring method using an endotoxin adsorbent (Japanese Patent Laid-Open No. 2-193072).

In order to improve the operability of the method, an improved method of adsorbing endotoxin to an adsorbent and reacting the adsorbed endotoxin with a Limulus reagent in a centrifugal separator is also proposed. (JP-A-4-72
571).

[0004]

However, in order to accurately measure endotoxin by these methods,
During operation, it was necessary to pay close attention to prevent the contamination of endotoxin, reaction inhibitor, etc. from the outside. Further, in these methods, it is not always easy to automate the operation so that a large amount of sample can be processed simply and in a short time. In view of such circumstances, the present invention provides
It is an object of the present invention to provide an improved endotoxin measuring method and measuring apparatus with improved operability and which can be easily automated.

[0005]

The present invention provides a method for measuring endotoxin by reacting endotoxin adsorbed on an adsorbent having an ability to specifically adsorb endotoxin with a reagent for detecting endotoxin. An opening, a rear end opening, and the inside of the sample fluid are passed through the opening,
And, using a column having means for packing and holding the endotoxin adsorbent, (2) introducing the endotoxin adsorbent into the column from the opening of the tip of the column, and packing and holding the endotoxin adsorbent in the column (3) Then, the sample liquid is introduced into the column from the front end opening of the column, discharged from the rear end opening, and the sample and the adsorbent are brought into contact with each other to adsorb endotoxin to the adsorbent, and (4) further, the detection reagent The present invention provides a method for measuring endotoxin, which comprises a step of reacting an endotoxin adsorbed on an adsorbent. Usually, the above step (4) in this method is carried out by discharging the adsorbent adsorbing endotoxin into the detection reagent from the column tip opening or by introducing the detection reagent into the column from the column tip opening. You can When the step (4) is carried out by introducing the detection reagent into the column, the adsorbent and the detection reagent are discharged from the column tip opening to the outside of the column after the introduction of the reagent into the column, if necessary. May be added.

The present invention is also a measuring device for measuring endotoxin by reacting an endotoxin adsorbed on an adsorbent having an ability to specifically adsorb endotoxin with a reagent for detecting endotoxin, which comprises a tip opening and a rear end. A column having an opening and a means for allowing the sample liquid to pass therethrough and filling and holding the endotoxin adsorbent,
Maintain at least one container or column in which one end is open, at least the tip of the column can be inserted therein, and which can store the adsorbent suspension, sample liquid or detection reagent, inside the container Characterized by comprising a maintenance means and a negative pressure loading means in communication with the rear end of the column,
An endotoxin measuring device suitable for automation is also provided. Hereinafter, the present invention will be described in detail.

As the adsorbent having the ability to specifically adsorb endotoxin used in the present invention, endotoxin is specifically adsorbed, and by adsorption to the adsorbent,
The endotoxin is not particularly limited as long as it does not lose the reactivity with the reagent for detecting endotoxin, and for example, the adsorbent described in JP-A-2-193072 of the present inventors can be used. Above all, a water-insoluble carrier such as cellulose or agarose among the above is preferably used an adsorbent in which a nitrogen-containing heterocyclic compound selected from histidine, histamine and adenine is bonded via a spacer such as alkylenediamine. be able to. Further, regarding the shape of the adsorbent, in consideration of the ease of filling the column, the pressure loss, etc., the shape of the adsorbent is preferably a bead shape. Such an adsorbent may be commercially available, and Pyrosep (trade name of Tanabe Seiyaku Co., Ltd.) and the like are preferable.

The method of the present invention uses a column having a front end opening, a rear end opening, and a means for allowing a sample liquid to pass therethrough and for filling and holding an endotoxin adsorbent therein, using the above (2) to The step of packing the endotoxin adsorbent in (4) into the column, introducing the sample liquid into the column, adsorbing the endotoxin, and reacting the adsorbed endotoxin with a detection reagent to detect and measure endotoxin, Then, the reaction solution is discharged from the column and incubated to promote detection and measurement of endotoxin. The adsorbent may be brought into contact with the sample liquid in advance and then filled in the column, and the filling step and the adsorption step may be performed in one step.

As a means for filling and holding the endotoxin adsorbent provided inside the column to be used, for example, a filter having a property of allowing the sample liquid to pass but not allowing the adsorbent to pass, for example, glass wool or the like can be mentioned.

Thus, in carrying out the method of the present invention, first, in order to fill and retain the endotoxin adsorbent in the column, the endotoxin adsorbent suspension is introduced into the column through the opening of the tip of the column. Although not particularly limited, a suspension containing 1 to 50% by weight of an adsorbent in endotoxin-free water is usually used. In general,
A negative pressure is applied to the opening at the rear end of the column to introduce the endotoxin adsorbent suspension into the column and fill it. The negative pressure is usually applied by suction with a plunger such as a syringe, a vacuum pump, a tube pump, etc., and it is preferable to keep the column vertical in order to facilitate uniform filling and holding. .

Then, the negative pressure is further increased to introduce the sample liquid into the column through the opening at the tip of the column. The sample used for the measurement is a liquid such as a solution, and for example, an aqueous solution, particularly an aqueous solution is preferable. By introducing the sample liquid,
The endotoxin can be quantified accurately. The sample liquid is sucked by negative pressure, passes through the packed bed of the endotoxin adsorbent, and is discharged from the rear end opening of the column. During this period, the endotoxin in the sample liquid comes into contact with the adsorbent and is adsorbed by the adsorbent. Usually, after adsorbing endotoxin to the adsorbent, non-specific adsorbed substances are washed and removed with endotoxin-free water, buffer solution or the like. It should be noted that the adsorbent is brought into contact with the sample liquid in advance, and if necessary, the nonspecific adsorbate is washed and removed, and then the adsorbent having adsorbed endotoxin is packed in the column as described above, and then the above steps are performed. You may change.

In the step of reacting the detection reagent with the endotoxin adsorbed on the adsorbent, the adsorbent adsorbing endotoxin is discharged from the column tip opening into the detection reagent, or the negative pressure is increased to increase the tip opening. From the above, the detection reagent is introduced into the column, and the endotoxin and the detection reagent are brought into contact with each other while adsorbing the endotoxin to the adsorbent, and the reaction is carried out. The reaction and quantification when the detection reagent is introduced into the column can be performed while the adsorbent is still filled in the column, and the negative pressure load is removed after the detection reagent is introduced into the column. Alternatively, or conversely, pressure can be applied from the rear end opening of the column to discharge the adsorbent and the detection reagent from the front end opening of the column to an incubation container or the like.

Known reagents for detecting endotoxin include known reagents such as Limulus reagent (including Limulus amebocyte lysate and a chromogenic substrate having a chromophore or a fluorophore), a labeled antibody against endotoxin, etc. Can be used in any condition. For example, the Limulus reagent is, as disclosed in JP-A-4-72571, 25 to 40 ° C., usually 10 to 37 ° C.
The reaction is carried out by incubating for 120 minutes, and after the reaction is stopped, absorbance measurement, fluorescence measurement and the like are performed. In any case, a known method can be adopted for the quantification. For example, it is possible to measure the absorbance and the fluorescence intensity using a measuring instrument and quantify using a calibration curve prepared in advance. Alternatively, it can be quantified visually by comparing the intensity of color development with those of various standard samples.

The measuring device of the present invention comprises a column having a front end opening, a rear end opening, and means for allowing a sample liquid to pass through and filling and holding the endotoxin adsorbent therein.
Maintain at least one container or column in which one end is open, at least the tip of the column can be inserted therein, and which can store the adsorbent suspension, sample liquid or detection reagent, inside the container It consists of a maintaining means and a negative pressure loading means communicating with the rear end of the column, one specific example of which is shown in the schematic diagram of the attached FIG.

In the specific example of the measuring apparatus of the present invention shown in FIG. 1, as a means for allowing a sample liquid to pass through the inside of the front end opening 1, the rear end opening 2, and filling and holding an endotoxin adsorbent. A column 4 having a glass wool filter 3 that allows the sample liquid to pass through but does not allow the adsorbent to pass through is provided. The column 4 is not particularly limited, but usually has an inner diameter of 1 to 2.5 mm and a length of 50.
A small diameter column of about 150 mm is preferable. The material is not particularly limited as long as it does not hinder the measurement by the Limulus reaction, but glass, metal, synthetic resin or the like can be used.

Further, in the apparatus, one end 5 is opened, at least the tip of the column can be inserted therein, and a container 5 capable of containing an adsorbent suspension, a sample liquid or a detection reagent.
have. The container may be made of glass, metal or synthetic resin, and in FIG. 1, the container is a test tube, but not only the test tube but also a cuvette, a cell or the like may be used. The same container may be used for the adsorbent suspension, the sample liquid and the detection reagent, as well as for the above-mentioned incubations, or each storage item, for each use, may have a separate identical or different shape. A plurality of containers may be provided as a set.

Further, the apparatus has a maintaining means 7 for maintaining the column 4 in a storage state inside the container. The maintaining means 7 is a lid for sealing the container opening 5 in order to effectively fill the adsorbent into the column by negative pressure and to introduce the sample liquid and the detection reagent, and a ventilation hole (suction / exhaust hole). 8
And the column 4 penetrates the lid,
Keep the column contained within the container. In FIG. 1, for example, a plug of silicon rubber that fits into the container opening 5 and through which the column penetrates, so that the column 4 is inside the container 6 and the end opening 1 of the column is downward. It is kept vertical. The air holes 8 are filled with glass wool 9 for preventing contamination from the outside.

The rear end opening 2 of the column 4 communicates with the negative pressure load means 11 via a connecting pipe 10. As a result, negative pressure can be applied to the column to fill the adsorbent into the column and introduce the sample liquid and the detection reagent. In FIG. 1, the negative pressure loading means 11 is a syringe, but other negative pressure generating sources such as a plunger, a vacuum pump, a tube pump, etc. can be adopted. The material of the connecting tube 10 is not particularly limited as long as it does not interfere with the endotoxin measurement, and specific examples thereof include glass, synthetic resin, and metal.

The measuring apparatus of the present invention may be provided with a plurality of sets of the above-mentioned column, column maintaining means and container,
This allows multiple samples to be measured simultaneously. Further, it is automated by a known method such as filling the adsorbent into the column, introducing the sample liquid and the detection reagent, further discharging the adsorbent and the detection reagent from the column, and computer control programmed with the measurement cycle. It is also possible.

Next, a specific example of the measuring method of the present invention using the measuring apparatus of the present invention shown in FIG. 1 will be described. The column 4 is filled with the endotoxin adsorbent 12 by negative pressure such as suction force. Then, the sample liquid is introduced into the column 4, and the endotoxin in the sample is adsorbed by the adsorbent 12. This adsorption operation is performed, for example, by introducing the sample aqueous solution into a column filled with an adsorbent and allowing it to pass through. The conditions can be appropriately selected according to the individual sample solution, but normally, the sample solution 1
Using 3 to 100 mg of adsorbent per ml, 4 to 40 ° C
At pH 4 to 8, preferably 5 to 7, and ionic strength of 0.2 or less, endotoxin is specifically and efficiently adsorbed.
By passing the sample aqueous solution through the column at a flow rate of about 2 ml / min or less under the conditions, the endotoxin in the sample aqueous solution is almost completely adsorbed on the adsorbent and is concentrated on the adsorbent. Further, the measurement sensitivity can be increased by increasing the amount of the sample liquid passed through the column.
As mentioned above, the packing of the adsorbent suspension into this column is
The sample liquid and the adsorbent may be mixed in a container such as a test tube in advance, stirred and brought into contact with each other, and then carried out.

If necessary, after the adsorption operation, the non-specifically adsorbed substances are washed and removed with pyrodiene-free water and a buffer solution. Then, an endotoxin detection reagent is introduced into the column and brought into contact with the adsorbent. Further, the adsorbent and the endotoxin detection reagent are discharged into the container 6, reacted under appropriate conditions, the absorbance of the reaction solution and the like are measured, and the endotoxin concentration in the sample is calculated.

In the present invention, a commercially available automatic dispensing device,
The measurement operation can be automated by appropriately combining and using an incubator for the Limulus reaction, a plate reader for reading the absorbance of the reaction solution, and the like.
That is, for example, a cylindrical column having an appropriate inner diameter can be attached to the probe part of an automatic dispensing device such as RSP5051 manufactured by Tecan Co., Ltd. to make a measuring device of the present invention, and the operation of each step described above is automatically performed. Can be carried out.

Further, by carrying out the above operation in a clean bench, it is possible to prevent endotoxin contamination from the outside of the operation system, and it is possible to perform accurate measurement.

[0024]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. Example 1 (Study of calibration curve) A calibration curve was examined as follows using the measuring apparatus shown in FIG. An endotoxin aqueous solution (concentration: 0.00125 and 0.0025 endotoxin unit (EU)) was prepared by adding a predetermined amount of endotoxin (EC-5) to an endotoxin-free phosphate buffer (pH 6.0, μ = 0.08). / Ml) 4 ml was prepared. Endotoxin adsorbent (Pyrosep; trade name manufactured by Tanabe Seiyaku) 1
20 mg of suspension (suspended in 1 ml of endotoxin-free water) was filled in a glass column (inner diameter: 1.68 mm, length 130 mm) connected to a suction syringe through a connecting pipe, and the adsorbent 4 ml of either the endotoxin aqueous solution or the phosphate buffer solution was sucked into the packed column to pass the solution. Then, the column is washed with 1 ml of a 20 mM sodium chloride aqueous solution to remove nonspecific adsorbates, and then Limulus reagent [QCL-100
0 (trade name of Bio Whittaker)
0.2 ml of a mixed solution of the Limulus amebocyte lysate solution and the synthetic substrate solution for color development. The mixture of the adsorbent and Limulus reagent was discharged into a test tube and incubated at 37 ° C. for 1 hour, then 25%
The reaction was stopped by adding 0.1 ml of an aqueous acetic acid solution. The reaction mixture was centrifuged to remove the adsorbent, and the supernatant was transferred to a 96-well microplate and then 405n with a plate reader.
The absorbance at m was measured. As a result of measurement as described above, a good calibration curve was obtained as shown in FIG.

Example 2 (Recovery of endotoxin from plasma) Endotoxin-free water (1.96 ml) in a test tube
And plasma (0.04 ml) were mixed by stirring and heat-treated in a boiling water bath for 10 minutes (control solution). In addition, endotoxin-free water 1.96 ml and endotoxin-containing plasma 0.9.
After mixing with 04 ml (EC-5, concentration: 0.1 EU / ml), the same heat treatment was performed. The control solution and the endotoxin-containing plasma solution were treated in the same manner as in Example 1, the amount of endotoxin adsorbed on the adsorbent was determined using the calibration curve in FIG. 2, and the recovery rate was calculated. As a result, it was found that 90% or more of the added endotoxin was recovered.

Example 3 (Measurement of Endotoxin in Injection, Recovery of Endotoxin from Injection) 30% Glucose Injection to 0.5, 1.0 and 2.0 Endotoxin Units (EU) / ml As a result, an endotoxin aqueous solution was added to prepare a sample solution. Endotoxin-free endotoxin adsorbent (PYROSEP; trade name of Tanabe Seiyaku) 0.15 mg suspended in phosphate buffer (pH 6.0, μ = 0.02) 4 ml suspension
To the above, 0.04 ml of the above sample solution was added and stirred. Using a glass column (internal diameter: 1.68 mm, length: 130 mm) connected to a suction syringe via a connecting pipe, the adsorbent suspension was sucked to remove the pyrosep in the suspension. The column was packed and the liquid was continuously passed. Then, the column was washed by suctioning 1 ml of a 20 mM sodium chloride aqueous solution to remove nonspecific adsorbates, and then Limulus reagent [QCL-1000
0.075 ml of a mixed solution of Limulus amebocyte lysate solution (trade name of Bio Whittaker) and synthetic substrate solution for color development] was introduced into the column. The column into which the Limulus reagent had been introduced was incubated at 37 ° C for 40 minutes to develop the color. Separately, an endotoxin aqueous solution was added to endotoxin-free water at a concentration of 0.5, 1.0, 2.0 EU / ml to prepare a standard solution. This standard solution was treated in the same manner as the above sample solution, and the degree of color development of the sample and the standard solution after incubation was compared. As a result, the color development of the standard solution was identifiable at each concentration, and the color development of the sample solution was similar to that of the standard solution of the corresponding concentration. Therefore, it was found that the approximate concentration of endotoxin in the injection can be easily determined by using this simple method.

[0027]

INDUSTRIAL APPLICABILITY The measuring method using the device of the present invention can measure endotoxin much more easily than the conventional method, and the measuring device of the present invention can be used with existing automatic dispensing devices, incubators and plates. The operation can be automated by combining with a reader or the like. Also, by installing the measurement system including these devices in a clean bench,
Since it is possible to make the system almost completely closed, the method and apparatus of the present invention have the excellent features that the endotoxin contamination from the outside can be prevented and the endotoxin can be accurately measured.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing one specific example of a measuring apparatus of the present invention.

FIG. 2 is a graph showing a calibration curve obtained in Example 1.

[Explanation of symbols]

 1 Column Top Opening 2 Column Rear End Opening 3 Adsorbent Packing and Holding Means 4 Column 5 Container Opening 6 Container 7 Column Maintaining Means 8 Intake / Exhaust Holes 9 Filter-10 Connecting Pipe 11 Negative Pressure Loading Means 12 Adsorbent

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Makoto Masuda 2-2-1 Sumiregaoka, Takarazuka-shi, Hyogo Prefecture

Claims (23)

[Claims] 1. A method for measuring endotoxin by reacting an endotoxin adsorbed on an adsorbent having an ability to specifically adsorb endotoxin with a reagent for detecting endotoxin, comprising: (1) a front end opening and a rear end opening; A column having means for passing a sample liquid therein and for filling and holding the endotoxin adsorbent therein is used. (2) The endotoxin adsorbent is introduced into the column from the tip opening of the column, (3) Then, the sample liquid is introduced into the column from the front end opening of the column, discharged from the rear end opening, and the sample and the adsorbent are brought into contact with each other to adsorb the endotoxin to the adsorbent. (4) Furthermore, the step of reacting the above-mentioned detection reagent with endotoxin adsorbed on an adsorbent, characterized in that Relaxin measurement method. 2. The endotoxin measurement method according to claim 1, wherein the means for filling and holding the adsorbent is a filter which is provided at an intermediate portion or a rear end of the column and which allows the sample liquid to pass but does not pass the adsorbent. . 3. The method for measuring endotoxin according to claim 1, wherein the steps (2) to (3) are performed by applying a negative pressure from the rear end opening of the column. 4. The measuring method according to claim 1, wherein the step (4) is performed by introducing a detection reagent into the column by applying a negative pressure load from the column rear end opening. 5. The method for measuring endotoxin according to claim 3, wherein the negative pressure loading is performed by suction from the rear end opening of the column. 6. The endotoxin measurement method according to claim 1, further comprising the step of discharging the adsorbent and the detection reagent from the column tip opening to the outside of the column and incubating. 7. The method for measuring endotoxin according to claim 6, wherein the discharging is performed by releasing the negative pressure or by applying pressure from the rear end opening of the column. 8. The method for measuring endotoxin according to claim 1 or 6, wherein the column is maintained vertically with its tip facing down. 9. The method for measuring endotoxin according to claim 1, wherein a predetermined amount of the sample liquid is introduced into the column to quantify the endotoxin. 10. The method according to claim 1, wherein the measurement is performed continuously.
The endotoxin measurement method described. 11. The method for measuring endotoxin according to claim 1, wherein the detection reagent is a Limulus reagent. 12. The endotoxin assay method according to claim 1, wherein the endotoxin adsorbent is brought into contact with the sample solution in advance and introduced into the column, and the steps (2) and (3) are performed in one step. 13. A measuring device for measuring endotoxin by reacting an endotoxin adsorbed on an adsorbent having an ability to specifically adsorb endotoxin with a reagent for detecting endotoxin, comprising: a front end opening, a rear end opening, Pass the sample liquid inside,
A column having means for filling and holding the endotoxin adsorbent, one end of which is open, at least the tip of the column can be inserted therein, and an adsorbent suspension, a sample liquid, or a detection reagent can be stored. An endotoxin measuring device comprising: at least one container; a maintaining means for maintaining the column in the container in a stored state; and a negative pressure loading means communicating with a rear end of the column. 14. The filter for filling and holding the adsorbent is a filter which is provided at an intermediate portion or a rear end of the column and which allows a sample liquid to pass but does not pass an adsorbent.
The endotoxin measurement device described. 15. The endotoxin measurement device according to claim 13, wherein the storage state maintaining means of the column is a lid for sealing the container opening, has a vent hole, and the column penetrates the lid. 16. The endotoxin measurement device according to claim 13, wherein the lid is a stopper that fits into the container opening. 17. The endotoxin measurement device according to claim 13, wherein the negative pressure loading means is a suction means. 18. The endotoxin measuring device according to claim 13, wherein the negative pressure applying means is a plunger. 19. The column has an inner diameter of 1 to 2.5 mm and a length of 5
The endotoxin measurement device according to claim 13, which is a small-diameter column of 0 to 150 mm. 20. The endotoxin measurement device according to claim 13, wherein the column is maintained vertically with its tip facing down. 21. The endotoxin measurement device according to claim 13, comprising a plurality of containers for adsorbent suspension and / or sample liquid, detection reagent and incubation as one set. 22. The endotoxin measurement device according to claim 13, comprising a plurality of sets of columns, column maintenance means, and containers. 23. The endotoxin measurement device according to claim 13, which is for automatic measurement.