JP4761448B2 - Blood endotoxin measurement method - Google Patents

Description

  The present invention relates to a method for measuring endotoxin in blood.

  "Endotoxin (endotoxin)" is one of the components constituting the outer membrane of Gram-negative bacteria, and the main body of its activity is LPS (lipopolysaccharide: lipopolysaccharide).

  The presence of endotoxin in the living body is present as a part of the outer membrane on the surface layer of Gram-negative bacteria (0101) (0102) as shown in FIG. In general, after the death of Gram-negative bacilli, it is present in the bloodstream as free endotoxin (0103). Furthermore, some endotoxins are present in a detoxified state by binding to leukocyte-derived inactivating factors such as BIP and CAP, proteins such as anti-endotoxin protein HDL, and albumin (0104) (0105). Many of these free endotoxins are formed on monocytes (0108), which are a type of white blood cell, after forming a complex (0107) with LBP (LPS binding protein: LPS binding protein) (0106) in the blood. It binds to the cell surface antigen CD14 (0109). Subsequently, this CD14 associates with TLR (Toll-like receptor: Toll-like receptor) (0110). By this association, endotoxin binding information is transmitted to the nucleus (0112) via the intracellular signal transduction pathway (0111), and the expression of inflammatory cytokine genes such as TNFα (0113) and IL-6 is induced. Sex cytokines are produced. “Cytokine” is a collective term for humoral factors that are secreted extracellularly and exhibit physiological activity in a very small amount, and functions as a signal transduction factor (mediator) between cells in the formation and maintenance of biological functions. (Non-Patent Document 1)

  By the way, when endotoxin is present in a certain amount or more in the blood, excessive inflammatory cytokines are produced in monocytes, granulocytes and the like by stimulation of the endotoxin. As a result, symptoms such as fever, sepsis, septic shock, or multiple organ failure called endotoxemia are caused. The onset of endotoxemia correlates with the amount of endotoxin contained in the blood. Therefore, accurate measurement of endotoxin in blood is considered to be an extremely important process for elucidating the relationship between endotoxin and various pathological conditions and providing information for early diagnosis and treatment method development.

  Conventional endotoxin measurement methods apply the method of directly injecting the sample to be examined into the body of a rabbit and measuring the amount of endotoxin based on the rise in body temperature, or the phenomenon in which the blood crab blood cell extract is gelled by endotoxin. The Limulus test is known. The method of directly injecting into the rabbit has problems in terms of cost, time to obtain the result, poor sensitivity, etc., and the Limulus test is now the mainstream method for measuring endotoxin.

  FIG. 2 shows the process of gelation reaction using a blood cell extract of horseshoe crab with endotoxin. There is a factor C pathway that specifically reacts with endotoxin in the blood cell extract of horseshoe crab. The “factor C pathway” is composed of the following reaction cascade. First, endotoxin binds tightly to factor C (Factor C) and activates factor C (0201). Next, factor C activated by endotoxin binding activates factor B (0202). Subsequently, the activated factor B further activates a clotting enzyme to generate a clotting enzyme (0203). This clotting enzyme partially hydrolyzes its substrate, coagulogen. As a result, peptide C (peptide C) is released from coagulogen to produce coagulin, a coagulation protein. This coagulin coagulation action causes gelation (0204). (Non-Patent Document 2)

  In addition, in the blood cell extract of horseshoe crab, there is a factor G pathway that induces gelation of the blood cell extract in addition to the factor C pathway. In the reaction cascade of the factor G pathway, first, a glucan-like substance such as β-D-glucan (0205) binds to factor G (Factor G) to activate factor G (0206). Next, the activated factor G activates the procoagulase to generate a coagulation enzyme (0203). The subsequent steps are the same as the C factor pathway. By the way, this β-D-glucan is a plant polysaccharide and is also known as a component of fungal cell walls such as yeast and mold. Since yeasts, molds and the like are widely present in normal environments, glucan-like substances such as β-D-glucan and BGLA are likely to be mixed into the endotoxin test target sample. When a test target sample contaminated by the contamination is used, the Limulus test results in overestimation of the endotoxin amount in the test target sample. Therefore, when using a blood cell extract of horseshoe crab to perform the Limulus test, inactivation of the factor G pathway is usually used. Inactivation of the factor G pathway is achieved by performing a pretreatment that removes or inactivates factor G in the sample.

  The endotoxin measurement method by the Limulus test is an application of a process in which the blood cell extract of horseshoe crab gels with endotoxin. In the Limulus test, methods such as the gel overturning method (gelation method), the chromogenic synthetic substrate method (colorimetric method), and the turbidimetric time analysis method (turbidimetric method) shown in Fig. 3 are known because of differences in judgment or measurement methods. It has been. (Non-Patent Document 2)

  As shown in FIG. 3A, the “gelling overturning method” mixes a test object sample (0301) with a liquid (0302) containing a factor C-related substance in a test tube, and is subjected to certain conditions (for example, 30 to 30 ° C. at 30 to 30 ° C.). After the reaction (0303) for 60 minutes, the test tube is turned over (0304) or tilted to determine whether the sample remains liquid (0305) or solidified (0306). The former is endotoxin negative, and the latter is endotoxin positive. This method does not require special equipment and is relatively easy to operate, but the measurement results are likely to change depending on the raw materials and production lots, and it is less objective than the optical method for human judgment. Because of the problems, it is usually only used simply.

  The “chromogenic synthesis substrate method” is a method for calculating the amount of endotoxin by colorimetric determination of the amount of released chromogenic groups by absorbance, using a chromogenic synthetic peptide substrate as the substrate of the coagulation enzyme as shown in FIG. 3B. Yes (Non-Patent Document 4). As the chromogenic synthetic peptide substrate (0307), a substrate (0309) simulating the amino acid sequence of the site where the coagulase (0308) hydrolyzes the natural substrate coagulogen is used. A color developing group such as paranitroaniline (pNA) (0310) is bonded to the site cleaved by the coagulation enzyme, and the color develops when the color developing group is released by cleavage with the enzyme. When the coloring group is paranitroaniline, the absorbance at 405 nm, which is the maximum absorption wavelength of paranitroaniline, is measured over time. When the chromophore is diazo coupled with paranitroaniline (0311), the absorbance at 545 nm is measured over time. Thereafter, the endotoxin concentration is measured by computer analysis of the change in the amount of transmitted light with time. As the chromogenic synthetic substrate method, an endpoint synthetic substrate method and a kinetic synthetic substrate method are known, but at present, a kinetic synthetic substrate method with improved false positive reaction is mainly used. The chromogenic substrate method has problems such as relatively expensive reagents and complicated operation, but is extremely excellent in quantitativeness, sensitivity, and objectivity.

  As shown in FIG. 3C, the “turbidimetric time analysis method” captures an increase in turbidity due to gelation as a change in the amount of transmitted light, and the ratio of the amount of transmitted light in the reaction solution is constant (usually around 90%) (0312 This is a method for calculating the endotoxin value using a standard curve created from the relationship between the gelation time and the endotoxin concentration. Although a dedicated device is required, it is easy to operate and has excellent quantitative and objectivity.

  However, various problems have been pointed out when measuring endotoxins in blood even when using Limulus tests such as the chromogenic synthetic substrate method and turbidimetric time analysis method, which are excellent in quantification and objectivity. For example, there is a problem that clinical symptoms and results do not always correspond. Specifically, despite the clinical diagnosis of severe Gram-negative bacterial infection caused by infection of Gram-negative bacteria in the blood, the blood endotoxin concentration level by the Limulus test is positive. Applicable when not shown. The cause of this is thought to be a major problem in the preparation of samples for endotoxin measurement, such as the sensitivity of the Limulus test, the timing of blood collection from specimens, the method of separating and storing plasma, etc. It has been.

  Conventionally, plasma separated from whole blood has been used as a blood sample subjected to the Limulus test. This is primarily because red blood cells and their hemoglobin affect the results of the Limulus test. Plasma is suitable for the Limulus test because it is the main component after removing red blood cells and blood pigments present in large quantities in the blood. Second, there are many endotoxins in the blood that are free and bound to proteins in the blood, and endotoxins that are present on the surface of bacterial cells are also centrifuged during plasma preparation. This is because it can be left in the plasma by adjusting the conditions. Therefore, it has been considered that measuring the endotoxin level in plasma is sufficient for measuring the endotoxin level in blood.

However, the endotoxin in the blood is in a state where it is bound to the surface of the microbial cell, in a free state, bound to the protein in the blood, or bound to the surface of the leukocyte cell membrane or taken into the leukocyte cell. Existing. In addition, as shown in FIG. 1, most of the endotoxins bound to free endotoxin in blood and some proteins in blood are taken up into the white blood cells in a state of being bound to the membrane surface of white blood cells over time after infection. Transition to the specified state. In addition, leukocytes that have already bound endotoxin at the site of infection may migrate into the blood. Therefore, it is difficult to say that the amount of endotoxin in blood is accurately determined only by measuring the amount of endotoxin contained in plasma. If a certain period of time elapses after the infection until blood is collected, the endotoxin level in the state of being bound to the membrane surface of the white blood cell or taken into the white blood cell may be dominant. is there. In addition, pathological conditions such as symptoms such as septic shock are often caused as a result of induction of cytokine production by binding endotoxin to the membrane surface of white blood cells as described above. Thus, unless the amount of endotoxin bound to the membrane surface of leukocyte cells or endotoxin taken into leukocyte cells is taken into account, the relationship with the disease state cannot be clarified.
Japanese Patent Laid-Open No. 6-118086 JP 2004-117127 A Takeda Kiyoshi, Akira Yoshio, Infectious Diseases and Toll-like receptor. Latest Medicine, 2003; 58 (5); 123-128. Endo Shigetaka, Inada Shinya, Endotoxin and Pathology. Helus Publishing, 1995 http://www.bc-cytometry.com/reagent/productlist-Lysing.htm

  For the reasons described above, the present inventors have applied for a patent for a method for measuring endotoxin contained in white blood cells (Patent Document 2). According to this method, it is possible to measure endotoxin bound to the membrane surface of white blood cells, which has not been noticed in the past, and endotoxin taken into white blood cells. However, this method, on the contrary, excludes endotoxin contained in plasma, which was a conventional measurement target. As a result, if blood is collected at a short time after infection, the amount of endotoxin contained in the blood may be underestimated, and the amount of endotoxin in the blood can be comprehensively and accurately grasped. However, in terms of obtaining a result that clarifies the relationship between the amount of endotoxin in the blood and the pathological condition, an unsatisfactory problem remains.

  In view of the problems of the prior art, the present invention provides a method for separating and removing only red blood cells from whole blood, and measuring the amount of endotoxin contained in the blood for measurement obtained by the method of separating and removing. An object of the present invention is to provide a method for measuring blood endotoxin, which can comprehensively measure endotoxin contained in leukocytes and endotoxin contained in plasma and can improve the sensitivity of blood endotoxin measurement.

  According to the present invention, it is possible to more accurately grasp the amount of endotoxin in blood, which has been insufficient with conventional measurement methods. This makes it possible to clarify the relationship between the amount of endotoxin in the blood and the disease state. Such elucidation of the relationship between endotoxin and various pathological conditions can be expected to lead to early diagnosis of endotoxemia and provision of information for the creation of a therapeutic method.

  The best mode for carrying out the present invention will be described below. Note that the present invention is not limited to these embodiments, and can be implemented in various modes without departing from the scope of the invention. Note that it is assumed that all of the instruments, reagents, water, and the like used in the present invention are endotoxin-free or that contain an extremely small amount of endotoxin that does not affect the endotoxin measurement results.

  The first embodiment will mainly describe claims 1, 2, 3, and the like.

  The second embodiment will mainly describe claims 4, 6, 7, 8, 9, 10 and the like.

  The third embodiment will mainly describe claims 4, 5, 7, 8, and the like.

  << Embodiment 1 >>

<Embodiment 1: Overview>
The first embodiment will be described. In the present embodiment, component blood obtained by removing only red blood cells is extracted from the collected whole blood, and blood for measurement is prepared by destroying white blood cells in the component blood under conditions where endotoxin in the component blood is not destroyed. A blood pretreatment method for measuring blood endotoxin.

  <Embodiment 1: Configuration>

  FIG. 4 schematically illustrates the first embodiment. This embodiment includes a whole blood collection step (S0401), a component blood extraction step (S0402), and a measurement blood pretreatment step (S0403).

  “Whole blood collection step” (S0401) is a step of collecting whole blood.

  “Whole blood” in the present invention includes all substances contained in collected blood.

  The term “collecting whole blood” in the present invention refers to collecting whole blood directly from a living body or indirectly from what is once stored after being collected from the living body. For example, in the case of direct collection, the case of collecting blood by inserting an injection needle into a blood vessel of a living body corresponds. The blood vessel that pierces the injection needle may be a vein or an artery.

  The number and timing of collecting whole blood in the whole blood collecting step are not particularly limited as long as they are from the same individual. For example, whole blood collected only once from the same individual, whole blood collected in multiple times at almost the same time, or whole blood collected in multiple times over time It may be.

  In collecting the whole blood, means for preventing blood from coagulating may be taken. For example, the inside of a syringe used for blood collection may be coated in advance with a substance that prevents or inhibits blood coagulation such as heparin.

  The blood after blood collection is preferably subjected to anticoagulation treatment such as adding an appropriate amount of a substance that prevents or inhibits coagulation. For example, heparin may be added at a concentration that does not affect the Limulus test. In this case, the final concentration of heparin is preferably 10 to 100 units / mL.

  The whole blood to be collected can be only the blood of a human or a non-human mammal.

  The “component blood extraction step” (S0402) is a step of extracting component blood obtained by removing only red blood cells from whole blood.

  In the present invention, “erythrocytes” mean red blood cells and hemoglobin (hemoglobin). “Leukocytes” mean lymphocytes, neutrophils, eosinophils, basophils, monocytes, and macrophages. Unless otherwise noted in the present invention, platelets are also included in leukocytes.

  The “component blood” in the present invention refers to a residual component obtained by removing red blood cells from whole blood. That is, blood cell components other than red blood cells such as leukocytes and other blood components such as plasma are included. In addition, substances other than blood components such as bacteria contained in blood and their remains, free endotoxin, and endotoxin bound to blood protein are also applicable.

  The method for separating red blood cells from whole blood may be a method in which collected whole blood is separated from other components by centrifugation, or the whole blood is mixed with a specific gravity solution and then left to stand for a certain period of time. May be separated from the other components by separation by centrifugation or by centrifugation, or may be separated according to the attached protocol using a reagent such as a commercially available blood cell separation agent.

  The specific gravity means a solution that is smaller than the specific gravity of erythrocytes and larger than the specific gravity of leukocytes when mixed with blood. For example, dextran and ficoll solutions are applicable. The amount of specific gravity added is preferably about 1/10 of the blood volume used for the measurement of endotoxin, but is not limited to this range. In addition, when blood and a specific gravity liquid are mixed and allowed to stand for separation, it may be left for about 30 to 60 minutes, but the standing time is not limited as long as it is sufficiently separated visually. The temperature for separating red blood cells from whole blood may be room temperature to 37 ° C, but is preferably around 20 ° C.

  In any method, layers other than the red blood cell layer are collected. Usually, red blood cells migrate to the lowermost layer, and in that case, all of the upper layer above the lowermost layer is collected. After adding and preparing water so that the volume of the collected upper layer sample is equal to the original whole blood, mix well to homogenize the distribution of cells in the liquid. A buffer solution such as Tris buffer may be used instead of water. This is a test sample. By this operation, for example, the endotoxin concentration per mL of whole blood can be expressed in the same unit as the endotoxin amount per mL of plasma expressed in the conventional measurement of plasma endotoxin concentration. In the present invention, the amount of endotoxin per mL of the test sample is expressed as the amount of endotoxin per mL of component blood.

  An example of a specific method of the component blood extraction step is shown in FIG. 5 and will be described below. First, 1 mL (0501) of whole blood that has been subjected to anticoagulation treatment by adding heparin is placed in a centrifuge tube, and centrifuged at room temperature using a swing rotor at a centrifugal acceleration of 1400 × g for 5 to 10 minutes. If the component blood can be separated, the conditions for centrifugation are not limited to these conditions. For example, when separation is not good due to blood, the centrifugal force may be increased, the centrifugation time may be extended, or an angle rotor may be used instead of a swing rotor. After centrifugation, buffy coat (0503) layered on the upper part of red blood cell layer (0502) located at the lowest layer of the centrifuge tube and plasma (0504) on the upper layer of the buffy coat are used with a syringe or the like. Collect each separately. The “buffy coat” is mainly composed of white blood cells and platelets. Next, about 1 mL of water is added to the collected buffy coat and mixed gently, and the mixture is allowed to stand for about 30 seconds to hemolyze the erythrocytes mixed during buffy coat collection with low osmotic pressure (0505). At this time, the amount of water to be added is not particularly limited as long as only red blood cells are hemolyzed by the final osmotic pressure and white blood cells are not destroyed. Further, a hemolytic agent based on ammonium chloride or a commercially available hemolytic agent may be used (Non-patent Document 3). Subsequently, the buffy coat that has been subjected to the hemolysis treatment is again put into the centrifuge tube, and centrifuged at room temperature using a swing rotor at a centrifugal acceleration of 1400 × g for 5 to 10 minutes. The centrifugation conditions are not limited to the above conditions as long as the leukocyte fraction can be separated. After centrifugation, the precipitate in the centrifuge tube is collected as a leukocyte fraction (0506) using a syringe or the like. The upper layer (0507) containing hemoglobin eluted in the sample by hemolysis is discarded. The white blood cell fraction is a fraction containing white blood cells, platelets and the like without containing red blood cells. The leukocyte fraction is gently added to the collected plasma and mixed. Finally, water or the like is added to adjust the total volume to the original 1 mL as component blood (0508). In this way, the amount of endotoxin present in the original whole blood sample can be accurately calculated by supplementing the original amount with water with the amount reduced by the removal of red blood cells and the like.

  “Measurement blood pretreatment step” (S0403) prepares measurement blood containing destroyed leukocytes by destroying white blood cells under the condition that endotoxin in the component blood extracted in the component blood extraction step is not destroyed. It is a step to do.

  In the present invention, “measurement blood” refers to component blood that has been subjected to a predetermined treatment before endotoxin measurement in order to accurately and highly sensitively measure endotoxin in the component blood. The predetermined treatment refers to destruction of white blood cells under conditions where endotoxin is not destroyed.

  As a method for destroying white blood cells, a method using a dilution high temperature heating method, a method using an ultrasonic destruction method, a method using a physical disruption method, a method using a freeze-thaw method, etc. are usually used. If it is a method which can fully destroy, it will not be restricted to these methods.

  The “dilution high temperature heating method” is a method of destroying white blood cells by adding water to the above component blood and diluting it 3 to 10 times and heating it in the range of 70 to 100 ° C. for about 5 to 10 minutes. The heating method is not limited as long as the component blood is sufficiently heated at the temperature of the previous period. For example, the container may be heated in a thermostat. The “constant temperature bath” may be a water bath or a heat block. Moreover, an air incubator may be used. Further, at this time, in the turbidimetric time analysis method, a surfactant such as Triton X-100 may be added at a concentration that does not decrease the endotoxin activity (final concentration 0.02%). The sample may be cooled by ice cooling or the like after heating.

  The “ultrasonic destruction method” is a method in which by applying ultrasonic waves to the component blood, the cell membrane of white blood cells is destroyed by ultrasonic vibration, and the cell contents are eluted. The ultrasonic treatment may be performed using an apparatus such as an ultrasonic cell crusher (sonicator). The intensity of the ultrasonic wave in this method can destroy the cell membrane of white blood cells, but may be within a range that does not destroy endotoxin, and may be appropriately adjusted depending on the amount of component blood, the oscillation output of the ultrasonic wave, and the transmission time.

  The “physical disruption method” is a method for destroying white blood cells by applying a physical external force to the component blood. For the physical crushing treatment, an apparatus such as a homogenizer may be used. The strength of the external force in the method may be within a range that can destroy the cell membrane of white blood cells but does not destroy endotoxin.

  The “freezing and thawing method” is a method for destroying white blood cells in the component blood by performing an operation of rapidly freezing the component blood in liquid nitrogen or dry ice and then thawing it with warm water or the like. It is based on the principle of breaking the cell membrane by volume expansion due to crystallization of intracellular water by rapid freezing and subsequent crystal melting. The operation of freezing and thawing may be repeated a plurality of times until the white blood cells in the component blood are sufficiently destroyed.

  An example of a specific method by the dilution high temperature heating method of the blood pretreatment step for measurement is shown in FIG. 6 and will be described below. First, 0.1 mL is taken from 1 mL of component blood obtained in the component blood extraction step and transferred to a 1.5 mL tube (0601). After adding 0.9 mL of water to the tube and diluting it 10 times (0602), the tube is set in a heat block (0603) that is capped and pre-filled with water, and heated at 70 ° C. for 10 minutes. . Immediately after heating, transfer in ice to cool (0604). Let the sample after cooling be the blood for a measurement (0605).

<Embodiment 1: Effect>
By the blood pretreatment method according to the present embodiment, it is possible to provide a blood sample for measuring endotoxin capable of measuring endotoxin contained in blood comprehensively and accurately without being affected by red blood cells.

  << Embodiment 2 >>

<Embodiment 2: Overview>
A second embodiment will be described. In this embodiment, in order to measure endotoxin contained in component blood obtained by removing only red blood cells from whole blood, blood for measuring endotoxin in measurement blood obtained in the measurement blood pretreatment step of the first embodiment. Endotoxin measurement method. In particular, after removing or inactivating the Limulus test interference factor from the blood for measurement, the endotoxin in the blood for measurement is measured using a C-factor-related substance that is a substance containing C factor.

  <Embodiment 2: Configuration>

  FIG. 7 schematically illustrates the third embodiment. Of the present embodiment, the whole blood collection step (S0701), the component blood extraction step (S0702), and the measurement blood pretreatment step (S0703) are the same as in the first embodiment. This embodiment has an interference factor elimination step (S0704) and an endotoxin measurement step (S0705) following the measurement blood pretreatment step.

  Since the whole blood collection step (S0701), the component blood extraction step (S0702), the measurement blood pretreatment step (S0703) and the first embodiment are the same as those in the first embodiment, description thereof will be omitted.

  The “interference factor elimination step” (S0704) is a step of preparing component blood in which the Limulus test interference factor contained in the component blood containing the destroyed leukocytes obtained in the blood pretreatment step for measurement is removed or inactivated. .

  In the present invention, the “limulus test interference factor” includes α2-plasmin inhibitor, antithrombin III, α1-antitrypsin, etc., which are inhibitors of limulus test contained in plasma, and factor Xa, thrombin, trypsin, etc., which are enhancement factors of limulus test. Applicable. In order to measure endotoxin in blood, it is necessary to remove or inactivate the Limulus test interfering factor by treating the endotoxin measurement sample in advance. The removal or inactivation of the Limulus test interference factor varies somewhat depending on the Limulus test measurement method. Hereinafter, general removal or inactivation methods in the case of the chromogenic substrate method and the turbidimetric time analysis method will be described respectively.

  In the chromogenic synthetic substrate method, the Limulus test interference factor is removed or inactivated mainly by a PCA method, a New PCA method, or the like. However, the method is not limited to these methods as long as the chromogenic substrate method is not affected and the Limulus test interference factor can be removed or inactivated.

  The “PCA method” is a method performed by the following procedure. First, PCA (perchloric acid) is added to the target sample. Next, the precipitate of the denatured product generated by the addition of PCA is removed by centrifugation. Subsequently, NaOH is added to the supernatant for alkali treatment and then used for measurement.

  A specific example of the PCA method is shown below. Add 0.32M PCA to 0.1 mL of component blood, react at 37 ° C. for 20 minutes, and then centrifuge at 800 × g for 15 minutes. The supernatant is collected, 50 μL of 0.18 M NaOH is added, and then mixed well for measurement.

  The “New PCA method” is a method in which a small amount of NaOH is added before the target sample is treated with PCA, and the precipitate generated by the PCA treatment is solubilized with NaOH for measurement.

  A specific example of the New PCA method is shown below. 0.1 mL of 0.18M NaOH is added to 0.1 mL of the component blood, mixed, and allowed to react at 37 ° C. for 5 minutes. Then, 0.32M PCA is added and reacted again at 37 ° C. for 10 minutes. Subsequently, 0.2 mL of 0.18M NaOH is added to the resulting precipitate and dissolved sufficiently. Take 50 μL of sample, dispense into a separate container, add 50 μL of 0.2 M Tris-HCl buffer (pH 8.0), mix well, and use for measurement.

  In the turbidimetric time analysis method, the Limulus test interference factor is removed or inactivated mainly by a dilution heating method. However, the method is not limited to this as long as it does not affect the turbidimetric time analysis method and can remove or inactivate the Limulus test interference factor.

  The “dilution heating method” is a method in which water or a buffer solution is added to a target sample to dilute it, and then an interference factor is inactivated by heating for measurement. The dilution rate is diluted 3 to 10 times, the temperature is in the range of 70 to 100 ° C., and the Limulus test interference factor is destroyed by heating for about 5 to 10 minutes. At this time, you may dilute with water which added 0.02% of surfactant, such as Triton X-100. The heating method is not limited as long as the component blood is sufficiently heated at the above temperature. For example, the container may be heated in a thermostat. The thermostatic bath may be a water bath or a heat block. Moreover, an air incubator may be used. The sample may be ice-cooled after heating.

  The dilution heating method can be considered to conform to the dilution high temperature heating method of the blood pretreatment step for measurement. Therefore, when measuring endotoxin by the turbidimetric time analysis method, the interference factor exclusion step may be omitted.

  The “endotoxin measurement step” (S0705) is a step of measuring endotoxin contained in the measurement blood obtained in the interference factor exclusion step using a factor C-related substance, which is a substance containing factor C.

  In the present embodiment, the “factor C-related substance” corresponds to a substance that contains factor C and participates in the factor C pathway.

  The factor C-related substance may be prepared from a blood cell extract of horseshoe crab. The thing prepared from the blood cell extract of a horseshoe crab corresponds to what further processed to the blood cell extract of a horseshoe crab. For example, the G factor in the blood cell extract of horseshoe crab that reacts with β-D-glucan is removed or the G factor is subjected to processing such as inactivation.

  “A method for measuring endotoxin contained in blood for measurement using a factor C-related substance, which is a substance containing factor C,” uses a mixed solution in which blood for measurement and a factor C-related substance are mixed. Is a method for measuring endotoxin contained in

  As a method for measuring endotoxin, for example, a method for measuring the amount of light transmitted through the mixed solution may be used. The method for measuring the amount of transmitted light may be a measurement of a change in the amount of transmitted light over time or a change rate of the amount of transmitted light. In the case of measuring the change in the amount of transmitted light over time, for example, the amount of endotoxin is determined by measuring the amount of light transmitted immediately after mixing the C-factor-related substance and then detecting the change from the amount of transmitted light immediately after that. You may make it measure. In the case of measuring the rate of change in the amount of transmitted light, it may be one using the chromogenic synthetic substrate method described in the background art. For example, the amount of endotoxin is measured by measuring the amount of light transmitted immediately after mixing the factor C and measuring the time required for the transmitted light amount ratio of the mixture to change to a certain value (for example, around 8%). You may do it. In this case, a standard curve created based on the rate of change obtained from a standard endotoxin whose concentration is known in advance may be used. The method may use the turbidimetric time analysis method described in the background art.

  In the endotoxin measurement step, each of the blood collected in multiple times over time from the same individual in the whole blood collection step is collected after the component blood extraction step, the interference factor elimination step, and the measurement blood pretreatment step. Including performing comparative measurements on an hourly basis. As a result, it is possible to grasp a change in the amount of endotoxin in the blood over time after infection, and an accurate endotoxemia diagnosis can be performed.

<Embodiment 2: Effect>
The blood endotoxin measurement according to the present embodiment provides an endotoxin measurement blood sample capable of comprehensively and accurately measuring endotoxin contained in blood without being affected by red blood cells using a factor C-related substance. be able to.

  << Embodiment 3 >>

  <Embodiment 3: Overview>

The third embodiment will be described.
In this embodiment, in order to measure endotoxin contained in component blood obtained by removing only red blood cells from whole blood, blood for measuring endotoxin in measurement blood obtained in the measurement blood pretreatment step of the first embodiment. Endotoxin measurement method. In particular, it is characterized by measuring endotoxin in blood for measurement using a factor C-related substance, which is a substance containing factor C, without removing or inactivating the Limulus test interference factor from the blood for measurement.

  <Embodiment 3: Configuration>

  FIG. 8 schematically illustrates the third embodiment. In this embodiment, the whole blood collection step (S0801), the component blood extraction step (S0802), and the measurement blood pretreatment step (S0803) are the same as those in the first embodiment. The present embodiment further includes an endotoxin measurement step (S0804) following the blood pretreatment step for measurement.

  In the present embodiment, the whole blood collection step (S0801), the component blood extraction step (S0802), and the measurement blood pretreatment step (S0803) are the same as those in the first embodiment, and thus description thereof is omitted. Further, since the endotoxin measurement step (S0804) is based on the endotoxin measurement step (S0705) of the second embodiment, the characteristic points of this embodiment will be described below.

  The “endotoxin measurement step” (S0804) of the present embodiment is a step of measuring endotoxin contained in the measurement blood obtained in the measurement blood pretreatment step using factor C.

  The factor C may be generated from a blood cell extract of horseshoe crab. The thing produced from the blood cell extract of horseshoe crab corresponds to the thing processed from the blood cell extract of horseshoe crab. For example, only factor C may be separated from the blood cell extract of horseshoe crab by using an affinity column or the like.

  The factor C may be based on characteristics of factor C, which is a blood component of horseshoe crab. "Based on the characteristics of factor C" means a recombinant gene-derived protein (recombinant protein) synthesized based on part or all of the gene sequence of horseshoe crab factor C, such as recombinant factor C Applicable. The protein synthesis system for synthesizing the recombinant protein is not limited as long as the recombinant protein as the final product has the amino acid sequence encoded by the gene that is the base of the recombinant protein. For example, any of bacterial systems, insect cell systems, mammalian cell systems, and cell-free systems may be used.

  Further, the factor C may be similar to factor C which is a blood cell extract of horseshoe crab. “Similar to factor C” includes orthologs of horseshoe crab C-factors generated from species other than horseshoe crab, recombinant proteins biosynthesized based on part or all of the ortholog gene sequence, and the like . The protein synthesis system for synthesizing the recombinant protein is not limited as long as the recombinant protein as the final product has the amino acid sequence encoded by the gene that is the base of the recombinant protein. For example, any of bacterial systems, insect cell systems, mammalian cell systems, and cell-free systems may be used.

  “Method for measuring endotoxin contained in blood for measurement using factor C” is a method for measuring endotoxin contained in blood for measurement using a mixture of blood for measurement and a factor C-related substance. is there.

  The “method for measuring endotoxin” corresponds to a method for detecting active factor C activated by binding to endotoxin. For example, by adding a substrate that generates fluorescence by the action of active factor C, the generated fluorescence is received by an appropriate device, and the emitted intensity is quantitatively measured by a computer to measure endotoxin. May be. Alternatively, the amount of endotoxin may be measured by colorimetric determination of the amount of chromogenic groups released by adding a chromogenic substrate that develops color by the action of active factor C. Furthermore, the measurement may be carried out using a commercially available kit such as PyoGene (CAMBREX, USA) according to the attached protocol.

  The endotoxin measurement step is a comparative measurement at each collection time after the component blood extraction step and the measurement blood pretreatment step for each blood sampled in multiple times over time from the same individual in the whole blood collection step Including performing. As a result, it is possible to grasp a change in the amount of endotoxin in the blood over time after infection, and it is possible to accurately diagnose endotoxemia.

<Embodiment 3: Effect>
The blood endotoxin measurement according to the present embodiment provides a blood sample for endotoxin measurement capable of comprehensively and accurately measuring endotoxin contained in blood without being affected by red blood cells by activating factor C. can do.

Examples of the present invention are shown below. The following examples illustrate specific aspects of the present invention and are not intended to limit the scope of the invention. Further, regarding the numerical values such as temperature, amount, time, etc. used in this embodiment, some experimental errors and deviations may be considered. Note that it is assumed that the instruments, reagents, water, and the like used in this example are all endotoxin-free or that contain a very small amount of endotoxin that does not affect the endotoxin measurement results.
<< Example 1 >>

  Endotoxin addition recovery experiment

  <Purpose>

  Many endotoxins bound to free endotoxin in the blood and some proteins in the blood move to the state where they are bound to the membrane surface of leukocytes or taken into the leukocytes over time after infection. Make sure you do. In addition, it is confirmed that the measurement of endotoxin contained in the component blood excluding red blood cells can more accurately measure the amount of endotoxin in the blood than the plasma fraction or leukocyte fraction.

  <Method>

  First, 10 mL of whole blood was collected from the vein of a healthy person (male, 33 years old), and then heparin was added at 10 to 100 units / mL for anticoagulation treatment. To the whole blood subjected to the anticoagulation treatment, 120 pg / mL of E. coli O111: B4 derived endotoxin (LPS) was added and heated under conditions in a humidified 37 ° C. 5% CO 2 incubator. After addition of endotoxin, 1 mL each was taken from the blood 10 minutes, 30 minutes and 2 hours later.

Next, the extracted whole blood was transferred to two 1.5 mL tapered (conical) centrifuge tubes at 1 mL each time the sample was collected, and each fraction was prepared by performing the following operation on each sample.
(1) One tapered centrifuge tube was centrifuged for 5 minutes at 20 ° C. with a centrifugal acceleration of 1400 × g using a swing rotor. Only the uppermost plasma layer was collected using a syringe and transferred to a new 1.5 mL tube, and then water was added to adjust the total to 1 mL. After gently mixing, this was used as a plasma fraction.
(2) Collect the buffy coat of the test tube excluding the plasma using a syringe, transfer to a new 1.5 mL centrifuge tube, add 1 mL of water and mix gently to perform hemolysis, and then swing again -Centrifugation was performed at room temperature using a rotor at a centrifugal acceleration of 1400 xg for 5 minutes. After centrifugation, the precipitate was collected with a syringe and transferred to a 1.5 mL tube, and water was added to adjust the total to 1 mL. After gently mixing, this was used as the leukocyte fraction.
(3) The remaining one tapered centrifuge tube was centrifuged at a centrifugal acceleration of 1400 × g for 5 minutes at room temperature using a swing rotor. Only the uppermost plasma layer was collected using a syringe and transferred to a new 1.5 mL tube. In addition, only the buffy coat was collected using a syringe, transferred to another new 1.5 mL centrifuge tube, 1000 μL of water was added and gently mixed to perform hemolysis, and then again using a swing rotor at room temperature. Was centrifuged for 90 seconds at a centrifugal acceleration of 1400 × g. After centrifugation, the precipitate was collected with a syringe, and the precipitate was transferred to a 1.5 mL tube to which the plasma fraction was transferred. Water was added to adjust the total to 1 mL. After mixing gently, this was used as a component blood fraction.

Subsequently, blood pretreatment for measurement was performed on each of the fractions prepared in the above (1) to (3) by a dilution high temperature heating method. That is, 100 μL of each sample and 900 μL of water were added to each 1.5 mL tube, mixed, then heat-treated at 70 ° C. for 10 minutes using a heat block, cooled on ice, and used for measurement. Blood.

  As a standard solution of endotoxin, a commercially available endotoxin standard solution (E. coli O111: B4-derived endotoxin) was diluted with an endotoxin standard solution to prepare 1 mL of 1 / 10-fold diluted standard solution, and this was used as a standard solution. For details, the protocol attached to the endotoxin standard set (Wako Pure Chemical Industries, Ltd.) was followed.

  Next, 200 μL each was taken from the measurement blood and the standard solution endotoxin, transferred to a toxinometer dedicated reaction tube containing 200 μL of Limulus reagent, and then mixed for several seconds with a vortex mixer. . These were used as measurement standard samples and measurement samples. As the “Limulus reagent”, endotoxin-single test Wako (Wako Pure Chemical Industries, Ltd.), a reagent for turbidimetric time analysis, was used. For detailed handling, the attached protocol was followed.

  Finally, the measurement standard sample and the measurement sample were set in Toxinometer MT-358 (Wako Pure Chemical Industries, Ltd.), and the endotoxin concentration in the sample was measured by a turbidimetric time analysis method. The measured values of the obtained fractions were plotted for each whole blood collection time and graphed.

  <Result>

The results of Example 1 are shown in Table 1 and described below. Endotoxin added to the blood of healthy persons was detected as 74.5 pg / mL from the plasma fraction 10 minutes after the addition, and about 70% of the added endotoxin amount was detected. The amount of endotoxin in the plasma fraction thereafter decreased with time, and became about 1/3 or less after 120 minutes. On the other hand, the amount of endotoxin in the leukocyte fraction was only 15.0 pg / mL after 10 minutes of addition, but increased thereafter, and showed a high value of 37.4 pg / mL after 30 minutes of addition. After 120 minutes, the value was higher than the amount of endotoxin in the plasma fraction. In contrast, the amount of endotoxin contained in the component blood fraction was as high as 94.9 pg / mL 10 minutes after the addition. In addition, a high value of 66.3 pg / mL was maintained even 120 minutes after the addition.

  <Discussion>

  From the results of Example 1, endotoxin added to the blood is mostly present in the plasma fraction after addition, that is, after infection, but it decreases with time and conversely increases in the leukocyte fraction. It was shown that. This is because the endotoxin that was present in the plasma after the addition in the state of the cell surface, in the free state, or in a state bound to the protein in the blood was bound to the membrane surface of the white blood cell over time or the white blood cell It is suggested that the state has been transferred to the state. In other words, the state and localization of endotoxin in the blood may change depending on the time after infection.

  The measurement of endotoxin contained in the component blood fraction can correct the amount of endotoxin that is considered to have shifted from the plasma fraction to the leukocyte fraction, compared to the amount of endotoxin only in plasma that has been used in the past. is there. As a result, it was shown that the amount of endotoxin in the blood can be accurately measured regardless of the passage of time after the addition.

<< Example 2 >>
Relationship between clinical pathology and endotoxin content in each fraction

<Purpose>
Confirm that clinical symptoms and blood endotoxin concentration results are consistent with endotoxin measurements using component blood.

  <Method>

The experiment was conducted on the following two cases diagnosed as clinically severe Gram-negative bacterial infection.
(1) Specimen Example 1 collected 5 mL of blood from a vein 1 hour after the onset of septic shock from a 67-year-old female patient who had colon perforation due to idiopathic sigmoid colon necrosis.
(2) Specimen Example 2 collected 5 mL of blood from a 56-year-old female patient who had ovarian abscess 25 hours after the onset of septic shock.

  The method after blood collection is the same as that in Example 1 except that endotoxin was measured immediately after blood collection, and thus the description thereof is omitted.

  <Result>

  The results of Example 2 are shown in Table 1 and described below. In this example, the cut-off value of the endotoxin concentration in plasma was set to 1.1 pg / mL, and a value higher than that was diagnosed as positive for endotoxemia. In addition, the cut-off value of the endotoxin concentration of leukocytes and component blood was provisionally equivalent to the plasma endotoxin concentration.

“ET” in the table means endotoxin. “+” Means positive endotoxemia and “−” means negative endotoxemia.
(1) In Specimen Example 1, since 4.9 pg / mL endotoxin was detected in plasma, it was diagnosed as positive for endotoxemia. That is, in the case of the sample, the measurement of endotoxin in plasma, which is usually performed, results in the clinical symptom agreeing with the result of blood endotoxin concentration. On the other hand, endotoxin in the leukocyte fraction is 0.8 pg / mL, so that endotoxemia is negative. That is, in the case of the sample, the method for measuring endotoxin contained in the white blood cells previously invented by the present inventor (Patent Document 2) results in the clinical symptoms and the blood endotoxin concentration being inconsistent.

  (2) In sample example 2, since only 0.7 pg / mL endotoxin was detected in plasma, it was diagnosed as negative for endotoxemia. That is, in the case of the sample, the measurement of plasma endotoxin results in the clinical symptom and the blood endotoxin concentration being inconsistent. On the other hand, the endotoxin in the leukocyte fraction is 3.5 pg / mL, which is diagnosed as positive for endotoxemia. In other words, in the case of the sample, the method for measuring endotoxin contained in the white blood cells (Patent Document 2) results in a match between the clinical symptoms and the blood endotoxin concentration.

However, in the endotoxin measurement in the component blood obtained by removing only red blood cells from whole blood according to the present invention, 6.5 pg / mL and 9.7 pg / mL endotoxin were detected in Sample 1 and Sample 2, respectively. Therefore, in any case, the patient is diagnosed as positive for endotoxemia, and the clinical symptom and the result of blood endotoxin concentration are in agreement.

  <Discussion>

  As shown in Example 1, the state and localization of endotoxin in the blood are considered to change depending on the elapsed time after infection. That is, the localization of endotoxin in the collected blood varies depending on the time from blood collection after infection. Therefore, only the endotoxin measurement in the plasma or the endotoxin measurement in the leukocyte fraction is performed in spite of the fact that both the sample examples 1 and 2 are actually severe Gram-negative bacterial infections due to endotoxemia as in this example. Then, it may be diagnosed as endotoxemia negative. On the other hand, in the endotoxin measurement in the component blood obtained by removing only erythrocytes from the whole blood according to the present invention, the endotoxin blood sensitivity was improved from the results of diagnosing positive endotoxemia in any specimen. It was confirmed that it was clearly superior to endotoxin measurement using only plasma or only leukocyte fraction in terms of clinically accurate diagnosis of the disease.

The diagnostic sensitivity of endotoxemia is improved by the present invention, and early diagnosis becomes possible. It is also possible to clarify the relationship between clinical symptoms and endotoxemia. Alternatively, it can be expected to lead to the provision of information on the idea of treatment.

Presence of endotoxin in vivo Process of gelation reaction of horseshoe crab blood cell extract by endotoxin Conceptual diagram for explaining each method of Limulus test Flow chart of Embodiment 1 Conceptual diagram for explaining component blood extraction Conceptual diagram for explaining the dilution high-temperature heating method Flow chart of embodiment 2 Flow chart of Embodiment 3

Explanation of symbols

0501: Whole blood (1 ml)
0502: Red blood cell layer 0503: Buffy coat 0504: Plasma 0505: Hemolysis treatment 0506: Leukocyte fraction 0507: Upper layer containing hemolyzed hemoglobin 0508: Component blood

Claims (10)

Pretreatment for measuring endotoxin in the blood,
A whole blood collection step for collecting whole blood;
A component blood extraction step for extracting component blood obtained by removing only red blood cells from whole blood;
A blood pretreatment step for measurement for preparing a component blood containing destroyed white blood cells under the condition that the endotoxin in the component blood extracted in the component blood extraction step is not destroyed,
A blood pretreatment method comprising: The blood pretreatment method according to claim 1, wherein the whole blood is blood of a human or a non-human vertebrate. The blood pretreatment method according to claim 1 or 2, wherein the method of destroying leukocytes in the blood pretreatment step for measurement is a dilution high temperature heating method.   The blood endotoxin measuring method which measures the endotoxin in the blood for a measurement obtained at the blood pretreatment step for a measurement of the blood pretreatment method as described in any one of Claim 1 to 3. Following the blood pretreatment step for measurement of the blood pretreatment method,
The blood endotoxin measurement method according to claim 4, further comprising an endotoxin measurement step of measuring endotoxin in the measurement blood obtained in the measurement blood pretreatment step using factor C. Following the blood pretreatment step for measurement of the blood pretreatment method,
An interference factor exclusion step of preparing component blood from which the Limulus test interference factor contained in the component blood containing the destroyed leukocytes obtained in the blood pretreatment step for measurement is removed or inactivated;
An endotoxin measurement step of measuring endotoxin contained in the component blood obtained in the interference factor exclusion step using a factor C-related substance, which is a substance containing factor C;
The blood endotoxin measuring method according to claim 4, comprising: The blood endotoxin measurement method according to claim 5 or 6, wherein the endotoxin measurement step is performed on blood collected from the same individual in a plurality of times over time, and includes comparative measurement for each collection time. The blood endotoxin measurement method according to any one of claims 5 to 7, wherein the factor C-related substance is generated from a blood cell extract of horseshoe crab. The method for measuring blood endotoxin according to any one of claims 5 to 7, wherein the factor C is based on characteristics of factor C which is a blood component of horseshoe crab. The method for measuring blood endotoxin according to any one of claims 5 to 7, wherein the factor C is similar to factor C which is a blood component of horseshoe crab.

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