JP4471214B2 - Endotoxin measurement method and reagent kit for measurement - Google Patents

Description

  The present invention relates to a method for measuring endotoxin (hereinafter referred to as ET) and a reagent kit for measurement. The method for measuring ET of the present invention is an enzyme immunoassay (EIA) for ET, and in particular, a method using a luminescent substrate.

ET is a lipopolysaccharide polymer complex produced by Escherichia coli, cholera, Pseudomonas aeruginosa, Salmonella, Shigella, etc. and is a component of bacterial cells and is released by cell destruction. Its action is relatively weak in toxicity and has characteristics such as strong pyrogenicity, heat resistance, and destruction of tumor cells.
If a medical device that comes into direct contact with medicines or blood is contaminated with ET, even a very small amount can cause serious results, and the amount of ET contamination must be strictly controlled. The ET test method is also listed on the website. In addition, attempts have been made to quantify ET in blood for early diagnosis of sepsis due to Gram-negative bacterial infection, determination of therapeutic effect and prognosis of Gram-negative bacterial infection.
In this way, ET is interested in various fields, and various methods for measuring ET have been proposed.

Conventional methods for measuring ET include the rabbit fever test, chicken embryo lethal test, radioimmunoassay, gas mass spectrum, and Limulus test. Used in contamination tests and clinical tests.
In this ET measurement method using the Limulus test, when ET is added to a horseshoe crab blood cell extract (lysate), factor C (ET sensitivity factor) is activated, and subsequent chain enzyme reactions are sequentially triggered by different pathways. In this method, the activated clotting enzyme hydrolyzes the reaction substrate. In general, 1) The clotting enzyme finally activated by the C-factor reaction in ET and lysate changes the turbidity that occurs during the process of gelling by converting coagulogen to coagulin by its protease activity. Nephelometry, measured by optical analyzer, and 2) hydrolysis of coagulogen when coagulation enzyme finally activated by C-factor reaction in ET and lysate converts coagulogen to coagulin by its protease activity. A colorimetric method for measuring the absorbance of pNA released by the amidase activity of a coagulation enzyme, using a chromogenic synthetic substrate in which a chromogenic group p-nitroaniline (pNA) is bound to a synthetic peptide having a sequence similar to the amino acid sequence of the site. Are used.
However, since the conventional ET measurement using the Limulus test is a conventional method, there are problems such as variations in measurement values depending on the measurer and the necessity of a sensor for each measurement.

Furthermore, as an improvement of the Limulus test, an ET measurement reagent comprising β-1,3-glucanase (Patent Document 1), an ET measurement method using a turbidimetric time analysis method (Patent Document 2), or an ET adsorbent A measurement method used (Patent Document 3) and the like have also been proposed. In addition, polymyxin B (hereinafter referred to as PMX) is known as a substance that neutralizes ET activity, and methods for analyzing the interaction by SPR have been reported (Non-patent Documents 1 and 2). In addition, there is a proposal of a reagent comprising a carrier and a color-developing reagent in which a ligand (mainly antibiotic) having affinity for ET is covalently bonded, but the measurement sensitivity required for dialysate has not been achieved (Patent Document 4). .
JP-A-11-178599 JP-A-8-211063 JP-A-2-193072 JP-A-6-66806 FEBS Letters 445 (1999) 420-424 JBC 274 (42) 29624-29627 (1999)

  An object of the present invention is to provide a novel ET measurement method that replaces the Limulus test. In other words, by using the EIA method, which has heretofore been impossible as an ET measurement method, it is intended to provide an easier ET measurement method. The prior art does not disclose or suggest means for quickly and easily measuring ET from a specimen using the EIA method.

  When measuring the ET using the EIA method, the inventors immobilized a substance having the ability to specifically adsorb ET such as polymyxin B (hereinafter referred to as PMX) on a carrier, and then using the EIA method. The present invention was completed by finding that ET can be measured very easily and rapidly from a specimen using the above principle and the selected enzyme.

That is, this invention consists of the following.
1. Polymyxin B (hereinafter PMX) a solid obtained by immobilized via a spacer to the gold thin film phase, is contacted with the dialysate, captures endotoxin in hemodialysis solution (ET) to the solid phase was captured ET the enzyme-labeled substance directly or indirectly bound, contacting the luminescent substrate guide the luminescence reaction, method of measuring ET that by measuring the light emission amount after amplified by a photomultiplier tube against.
2. A solid phase obtained by immobilizing PMX on a gold thin film via a spacer is obtained by reacting aminoethanethiol with a gold thin film, then reacting with disuccinimidyl suberic acid, and then reacting with PMX. 2. The method for measuring ET according to item 1 above, which is a phase.
3 . 3. The method for measuring ET according to 1 or 2 above, wherein the enzyme is alkaline phosphatase or peroxidase.
4. 4. The method for measuring ET according to any one of items 1 to 3 , wherein the luminescent substrate is a 1,2-dioxetane compound.
5 . 5. The method for measuring ET according to any one of items 1 to 4 , wherein ET is Lipopolysaccharides, endotoxin, lipopolysaccharide, pyrogen, or pyrogen.
6 . 6. The method for measuring ET according to any one of 1 to 5 above, wherein an enzyme labeling substance having reactivity with the captured ET is brought into contact and then reacted with a luminescent substrate .
7 . A substance reactive with the captured ET (first substance) is brought into contact and reacted, and then an enzyme labeling substance (second substance) that reacts specifically with the first substance is brought into contact and reacted. The method for measuring ET according to any one of 1 to 5 above , wherein a luminescent substrate is contacted .
8 . 8. A method for determining ET contamination by the method for measuring ET according to any one of 1 to 7 above.

  The ET measurement method of the present invention uses the principle of the EIA method in which PMX or the like that specifically binds to ET is immobilized on a carrier. If the specimen contains ET, the PMX or the like on the carrier ET is captured, and this is used to measure ET by measuring enzyme activity using sandwich technique of EIA method. The present inventors have found for the first time that the ET measurement method using the present invention is simple and that the EIA method, which has heretofore been impossible, can be applied to the ET measurement method.

  The basic reagent used in the method for measuring ET of the present invention is prepared as a solid phase by immobilizing a substance having the ability to specifically adsorb ET such as PMX on a carrier. The substance having the ability to specifically bind to ET is preferably PMX or anti-ET antibody, but is not limited thereto as long as it has the ability to specifically bind to ET. For example, a nitrogen-containing heterocyclic compound selected from histidine, histamine and adenine can also be used. PMX is commercially available and can be used as it is or after purification. As the anti-ET antibody, a polyclonal antibody or a monoclonal antibody can be appropriately prepared by a known technique, but these are also commercially available because they are also commercially available. For example, there are Nanotools Antikorpertechnik (Germany) for anti-ET antibodies, QED Bioscience, Inc. (USA), and Biogenesis LTD. (UK) for labeled anti-ET antibodies. ET is generally called by various names, but LPS, endotoxins, lipopolysaccharides, pyrogens, pyrogens, etc. are targeted, and these are administered to animals together with an appropriate adjuvant in order to produce antibodies. It can be prepared or further prepared by hybridoma preparation techniques.

  As the carrier, a known plastic, glass, paper, resin or the like can be widely used in the EIA method, but a metal thin film, particularly a gold thin film is more preferable. The shape of the container is not particularly limited, such as a tube shape, a petri dish shape, a microtube or a membrane shape known per se.

  The carrier and the substance having the ability to specifically bind to ET are bound directly or via a spacer. Examples of the spacer include a monolayer film made of a linear alkyl compound having a thiol group or dithiol group at one end and a functional group capable of binding to a substance capable of specifically binding to ET at the other end. However, it is not limited to this. It is preferable that the substance having the ability to specifically bind to ET is a 10 to 600 μg / mL dimethyl sulfoxide (DMSO) solution, which is contacted for 2 to 12 hours to be immobilized. Further, a blocking process or the like may be performed.

  The solid phase, which is the basic reagent for ET measurement of the present invention, may be provided as an immobilized substance having the ability to specifically bind to the carrier and ET in advance. It is also possible to provide a kit containing a reagent containing at least a carrier and a substance capable of specifically binding to ET.

The ET measurement method of the present invention measures ET by directly or indirectly binding an enzyme labeling substance to the captured ET and measuring the enzyme activity. The reaction conceptual diagram is shown in FIG. 3 based on the example.
When an enzyme labeling substance is directly bound to the captured ET, a substance obtained by modifying the enzyme labeling substance to a substance having the ability to specifically adsorb ET such as the PMX or anti-ET antibody can be used. . As the enzyme, a system widely used in the EIA method can be used, particularly preferably alkaline phosphatase or peroxidase (POD), more preferably alkaline phosphatase (ALP). When the enzyme-labeled substance is directly bound to the captured ET, for example, when an anti-ET antibody is selected, the added amount of the whole system after addition to the solid phase (surface area of about 10 to 50 mm ² ) is selected. The concentration is preferably about 0.01-300 μg / ml, preferably about 1-100 μg / ml.

On the other hand, when an enzyme-labeled substance (second substance) is indirectly bound to the captured ET, it reacts with the ET to be measured as a substance interposed between the captured ET and the second substance. A substance (first substance) with As the first substance, PMX or anti-ET antibody can be used as well as the substance having the ability to specifically adsorb ET, but anti-ET antibody is preferable. The amount of the first substance added when the second substance is indirectly bound is, for example, when an anti-ET antibody is selected for the substance, and the entire system after addition to the solid phase (surface area of about 10 to 50 mm ² ). It is preferable to add so that the concentration of is about 10-1000 ng / ml, preferably about 50-500 ng / ml.

Furthermore, an enzyme labeling substance (second substance) that specifically reacts with the first substance is used, and ET is detected using this enzyme reaction system.
For example, when an anti-ET antibody is selected as the first substance, the second substance is an antibody against the anti-ET antibody, and a polyclonal antibody or a monoclonal antibody can be appropriately prepared by a known technique, but these are also commercially available. They are readily available and widely available anti-IgG antibodies can be used. Enzymatic labeling can be carried out using the means commonly used for labeling antibodies such as enzymes (Implementation of clinical tests, Immunoassay Kanehara Publishing). The amount of the second substance added when the second substance is indirectly bound is, for example, when an anti-IgG antibody is selected as the substance, and the entire system after addition to the solid phase (surface area of about 10 to 50 mm ² ). Is preferably added so that the concentration of is about 0.01 to 300 μg / ml, preferably about 1 to 100 μg / ml.

The immobilized enzyme-labeled substance is washed to remove unnecessary enzyme-labeled compounds, then brought into contact with a substrate for the enzyme to induce an enzyme reaction, and ET is measured by measuring the enzyme activity. The enzyme reaction is carried out under the optimum conditions for each enzyme. The amount of the substrate is 2-20 μg, preferably 3-10 μg, to 2 μg of the enzyme-labeled substance added.
The substrate liberates a marker substance such as a dye, fluorescence or luminescence by an enzymatic reaction, and measures it optically, physically, or chemically. The optimum method is a method using a chemiluminescence reaction. The substrate used in the luminescence method is, for example, an adamantyl-1,2-dioxetane derivative (Fujirebio: trade name Lumipulse) which is a substrate for ALP, an oxidizing agent under alkaline conditions (H ₂ O ₂ , HClO, O ₂ , MnO ₄ ⁻ , I ₂ etc.) Luminol (5-amino-2,3-dihydro-1,4 phthalazine ion) derivative (ortho: trade name Bistro ECi) (Fe, Co, Mn, Cu, etc.) Illustrative examples include N-methylacridinium derivatives (chiron: trade name ACS: 180) as an oxidation reaction substrate, but are not limited to these. The offending system is widely available.
Luminescence occurs when, for example, an excited state occurs due to contact between the enzyme and the substrate, and when this transitions to the ground state, luminescence occurs (see the lower part of FIG. 3). This light emission amount is converted as an ET amount. Luminescence can be measured with higher sensitivity by using a photomultiplier tube. This system amplifies light in a vacuum tube. When light entering through the entrance window 1 strikes the photocathode 2 coated with a photosensitizer, electrons jump out. The electrons are accelerated by the electric field generated by the collector electrode 3 and led to the first electron multiplier 41 to be collided. Due to the collision, a plurality of electrons are knocked out from the electrode (secondary electron emission effect), which is accelerated and collides with the second electron multiplier 42. Thereafter, this process is repeated in the electron multiplier 4 to reach the final dynode 6, and by the final stage, the amplification factor of each electrode is multiplied by the number of electrodes, resulting in a multiplication of 1 to 10 million times. Effective measurement becomes possible. The measurement can be performed by a commercially available photo counter (manufactured by Hamamatsu Photonics, IT Research, etc.). FIG. 4 shows a conceptual diagram of the photomultiplier tube.

  The specimen for ET measurement of the present invention is not particularly limited as long as it is an object that may contaminate ET. It can be widely applied to biological samples such as blood and urine, culture solution, hemodialysis solution and its waste solution, water for injection, pharmaceuticals, pure water and the like. The ET measurement in the lipid component requires pretreatment with a surfactant or the like in advance, and any of them can be suitably measured as long as the specimen is liquefied. The ET to be measured can detect LPS, endotoxin, lipopolysaccharide, pyrogen, pyrogen and the like as essentially equivalents.

The mixing ratio at the time of contact between the sample and the solid phase is, for example, that the sample 50-5000 μl is contacted with the solid phase (surface area of about 10 to 50 mm ² ) for 2-60 minutes, preferably 1000-3000 μl is contacted over 6-40 minutes. Do that.

  In the measurement according to the present invention, the increase in ET in the specimen can provide a calibration curve as shown in FIGS. 1 and 2, and it has been confirmed that the measurement method of the present invention is extremely useful for quantitative analysis of ET. It was.

  Thus, the measurement method of the present invention can be applied to the measurement of specimens that are widely contaminated with ET, and in particular, ET contamination determination methods, etc. (culture solution, hemodialysis solution and its waste solution, water for injection, pharmaceuticals, pure water, etc.) It is particularly useful for the ET contamination determination method of hemodialysis fluid. Its measurement sensitivity enables measurement at a level of about 1 EU / l, which could not be achieved before.

  Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples. However, the present invention is not limited to these, and the ability to specifically adsorb ET when measuring ET using an EIA system. As long as a substance having s is immobilized on a carrier and ET is measured using a luminescence method, all are included in the technical idea of the present invention.

Example 1 Preparation of Solid Phase for ET Measurement Reaction A gold thin film was washed with a piranha solution in a clean bench, and then ultrasonically washed with ethanol. Then, the ethanol solution of 1mM aminoethanethiol, immersed gold thin film (room temperature, 12 hours), washed with ethanol, 1mM di succinic Imijirusuberin acid (hereinafter DSS) / dimethyl sulfoxide (hereinafter DMSO) solution was immersed for 2 hours, Washed with DMSO. Next, it was immersed in a 10 to 1000 μg / mL polymyxin B (hereinafter PMX) / DMSO solution for 2 hours and washed with DMSO and ET-free water. Finally, after immersing a diluted solution of Block Ace (Dainippon Pharmaceutical Co., Ltd.) for 2 hours, PBS containing ET-free 0.05% polyoxyethylene sorbitan fatty acid ester (137 mM sodium chloride, 8.1 mM disodium hydrogenphosphate 12 hydrate, A solid phase for ET reaction was prepared by washing with 1.47 mM potassium dihydrogen phosphate and 2.68 mM potassium chloride (hereinafter PBST).

Example 2 ET Measurement Method As a preliminary preparation, a photon counter (manufactured by IT Research) used for measurement is installed in a dark room, and the shield box surrounding the photon counter is further covered with a dark screen, and light from a personal computer for measurement, etc. Anything that emits was placed outside the darkroom.
Next, 3 mL of the measurement sample was brought into contact with the reaction solid phase prepared in Example 1 in a clean bench at room temperature for 30 minutes, washed with PBST, and then 1 mL of 1 μg / mL anti-ET IgG antibody solution was added. Contacted for 30 minutes and washed with PBST. Next, 1 mL of a 100-fold diluted solution of anti-Mouse IgG alkaline phosphatase conjugate (manufactured by Sigma) was contacted for 30 minutes, and then washed thoroughly with PBST to give 3- (2′-spiroadamantyl) -4-methoxy. After contacting with 50 μL of -4- (m-phosphoryloxyphenyl) -1,2 dioxetane (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter referred to as AMPPD), measurement was performed with a photon counter placed in a dark room. The sample was placed approximately 2 cm away from the light-receiving surface of the photon counter, and measurement was started 80 seconds after the gold substrate and AMPPD were brought into contact with each other.
The measurement results are shown as calibration curves in FIGS. Here, 0 seconds in the calibration curve means 80 seconds after the AMPPD is contacted. As a result, a useful calibration curve in a straight line was achieved. In addition, the conceptual diagram of the measuring method and luminescent reaction was shown in FIG. In the figure, PMB is PMX, anti-ET is anti-ET IgG antibody, secondary antibody / reverse Y-ALP is anti-Mouse IgG alkaline phosphatase conjugate, ALP is alkaline phosphatase, and specific examples of luminescent substrate are 3 -(2'-Spiroadamantyl) -4-methoxy-4- (m-phosphoryloxyphenyl) -1,2 dioxetane, Light means luminescence generated by the reaction of the enzyme substrate.

  In the measurement system according to the present invention, an increase in ET in a specimen can provide a linear calibration curve, and the measurement method of the present invention is extremely useful for quantitative analysis of ET. Thus, the measurement method of the present invention can be applied to the measurement of specimens that are widely contaminated with ET, and in particular, a method for diagnosing bacterial infection, a method for determining ET contamination (culture solution, hemodialysis solution and its waste solution, water for injection, etc. , Pharmaceuticals, pure water, etc.). It is particularly effective for ET contamination judgment method in hemodialysis fluid.

FIG. 5 is a relationship diagram when time is plotted on the horizontal axis and photons are plotted on the vertical axis when 0, 1, 10, 50, 250 EU / L ET samples are measured in Example 2. FIG. 5 is a relationship diagram when the horizontal axis represents ET concentration and the vertical axis represents photons when 0, 1, 10, 50, 250 EU / L ET samples were measured in Example 2. It is a reaction conceptual diagram. It is a photomultiplier tube conceptual diagram.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Incident window 2 ... Photoelectric surface 3 ... Focusing electrode 4 ... Electron multiplication part 41 ... 1st electron multiplication part 42 ... 2nd electron multiplication part 5 ... Anode 6 ... Final dynode

Claims (8)

Polymyxin B (hereinafter PMX) a solid obtained by immobilized via a spacer to the gold thin film phase, is contacted with the dialysate, captures endotoxin in hemodialysis solution (ET) to the solid phase was captured ET the enzyme-labeled substance directly or indirectly bound, contacting the luminescent substrate guide the luminescence reaction, method of measuring ET that by measuring the light emission amount after amplified by a photomultiplier tube against. A solid phase obtained by immobilizing PMX on a gold thin film via a spacer is obtained by reacting aminoethanethiol with a gold thin film, then reacting with disuccinimidyl suberic acid, and then reacting with PMX. The method for measuring ET according to claim 1, which is a phase. The method for measuring ET according to claim 1 or 2 , wherein the enzyme is alkaline phosphatase or peroxidase. The method for measuring ET according to any one of claims 1 to 3 , wherein the luminescent substrate is a 1,2-dioxetane compound. ET is, Lipopolysaccharides, endotoxin, lipopolysaccharide, pyrogens, or the measurement method of ET according to any one of claims 1-4 is pyrogen. The method for measuring ET according to any one of claims 1 to 5, wherein an enzyme labeling substance having reactivity with the captured ET is brought into contact and then reacted with a luminescent substrate . A substance reactive with the captured ET (first substance) is brought into contact and reacted, and then an enzyme labeling substance (second substance) that reacts specifically with the first substance is brought into contact and reacted. , contacting a luminescent substrate, method of measuring ET according to any one of claims 1-5. Method for determining ET contamination by the measuring method of the ET according to any one of claims 1-7.