Method for measuring neutrophil count using trace amounts of biological samples

Abstract

The present invention aims to develop a technique for measuring neutrophil counts using small amounts of blood, such as 1 μL or less, as obtained by fingertip puncture. [Solution] A method for measuring the number of neutrophils in a blood sample is provided, which includes measuring superoxide anions in the presence of bortezomib or ixazomib or a salt thereof.

Description

Technical Field

[0001] The present invention relates to a method for measuring the number of neutrophils in a trace amount of biological sample.

Background Art

[0002] Since the number of neutrophils in peripheral circulating blood varies sensitively according to diseases and drugs, it is an important test item in patients. The conventional technique for measuring neutrophils is based on white blood cell classification by flow cytometry, and generally requires about 2 mL of venous blood sampling. Clinical tests using venous blood sampling can examine not only the number of neutrophils but also the total blood cell count and biochemical items, and are versatile. However, it cannot be easily performed in infants and the elderly who have difficulty in venous blood sampling. Therefore, a method for measuring the number of neutrophils using a trace amount of blood is required.

[0003] As a test using a trace amount of blood, tests by fingertip puncture blood sampling have become popular in glucose measurement, hemoglobin measurement, and some biochemical tests. However, currently, there is no technique that can measure the number of neutrophils by fingertip puncture blood sampling.

[0004] On the other hand, since the production of reactive oxygen species due to excessive activation of neutrophils causes oxidative stress in the living body, it has been proposed to evaluate the oxidative stress state of the living body based on the evaluation of neutrophil activity (Patent Document 1, Patent Document 2). In Patent Document 1 and Patent Document 2, a method for adding a neutrophil stimulant to a biological sample, measuring the superoxide production activity, and evaluating the activity of neutrophils based on the obtained measurement results is described. However, these methods only evaluate the oxidative stress state of the living body simply by a decrease or increase in neutrophil activity, do not quantitatively measure the number of neutrophils in the sample, and further include a method for simultaneously measuring myeloperoxidase activity.

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] Patent No. 6285691 [Patent Document 2] Patent No. 7092565 [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to develop a technique for measuring neutrophil counts using minute biological samples of 1 μL or less, such as those obtained by fingertip blood sampling. [Means for solving the problem]

[0007] The inventors focused on the fact that neutrophils produce superoxide anions in response to stimuli such as bacterial-derived substances, and diligently researched whether it would be possible to measure the number of neutrophils based on their superoxide anion production ability. Surprisingly, they found that by measuring superoxide anions in a small amount of blood sample in the presence of bortezomib as a sensitizer for superoxide anion detection, the number of neutrophils in the small amount of blood sample could be measured quantitatively and rapidly using the measurement results as an indicator. Thus, the present invention was completed.

[0008] This invention provides a method for quantitatively measuring the number of neutrophils using a small amount of biological sample, such as a blood sample of 1 μL or less, with the potent superoxide production capacity of neutrophils as an indicator. For example, this application provides the following: [1] A method for measuring the number of neutrophils in a biological sample, comprising measuring superoxide anions in the presence of bortezomib or ixazomib or a salt thereof. [2] The method according to [1], wherein the biological sample is a blood sample. [3] The method according to [2], wherein the blood sample comprises more than 0.01 μL and less than 2 mL of blood. [4] The method according to [2], wherein the blood sample comprises more than 0.01 μL and 1 μL or less of blood. [5] The method according to any one of [2] to [4], wherein the blood sample is whole blood. [6] The method according to any one of [1] to [5], comprising measuring superoxide anions in a system using a chemiluminescent probe, a fluorescent probe, or a color-developing probe. [7] The method according to any one of [1] to [6], further comprising converting the measured amount of superoxide anion to the amount of xanthine oxidase based on a calibration curve obtained by a reaction system of xanthine oxidase and xanthine. [8] A kit for measuring the number of neutrophils in a biological sample, comprising bortezomib or ixazomib or a salt thereof. [9] The kit according to [8] further comprising a probe or capture agent and / or measuring device used in a detection method used for the measurement.

[10] The kit according to [9], wherein the probe is selected from the group consisting of chemiluminescent probes, fluorescent probes, and color-developing probes.

[11] The kit according to [9] or

[10] , wherein the measuring device is a portable luminometer. [Effects of the Invention]

[0009] The method of the present invention makes it possible to measure neutrophil counts in minute samples, and can be used, for example, by using minute blood samples obtained by fingertip puncture. Therefore, according to the present invention, the frequency of venous blood sampling for patient blood tests can be reduced, thereby reducing invasiveness for patients and the workload of medical personnel. Furthermore, since the method of the present invention does not require the isolation of neutrophils, rapid testing and diagnosis are possible, with results obtained within one hour from sample collection. [Brief explanation of the drawing]

[0010] [Figure 1A]The results of L-012 luminescence measurements using mouse blood (whole blood, cardiac blood collection) are shown. In the figure, "Bort" represents bortezomib (10 μM), "PMA" represents the neutrophil stimulant phorbol 12-myristate 13-acetate (200 nM), and "DMSO" represents dimethyl sulfoxide, the solvent used as a control for bortezomib or PMA. The Y axis shows the total luminescence (total RLU) measured 30 minutes after PMA stimulation, and the X axis shows the blood volume (μL). [Figure 1B] This figure shows the time-series measurement results of L-012 luminescence using 0.1 μL of mouse blood (whole blood, cardiac blood). In the figure, "Bort" represents bortezomib, and "buffer" represents the control (i.e., no blood added) using Krebs-HEPES buffer instead of blood sample. The Y axis represents luminescence (RLU), and the X axis represents measurement time (minutes). [Figure 1C] This figure shows the results of L-012 luminescence measurements using mouse blood (cardiac blood collection) after red blood cell lysis (RBC lysis). In the figure, "Bort" refers to bortezomib. The Y axis shows the total RLU measured over 30 minutes, and the X axis shows the blood volume (μL). [Figure 1D] The results of L-012 luminescence measurements using mouse blood (whole blood, cardiac blood) stored at room temperature for 1 to 24 hours are shown. In the figure, "Bort" refers to bortezomib. The Y axis shows the total luminescence (total RLU) measured over 30 minutes, and the X axis shows the blood volume (μL). [Figure 2A] The results of L-012 luminescence measurements using 0.1 μL of mouse blood (whole blood, tail vein blood collection) are shown. In the figure, "Heart" refers to the whole blood sample collected from the heart (control), and "Tail 1st," "Tail 2nd," and "Tail 3rd" refer to the whole blood samples obtained from the first, second, and third blood collections from the tail vein, respectively. The Y-axis shows the total luminescence (total RLU) measured over 30 minutes, the X-axis shows the blood sample (containing 0.1 μL of blood), and "0" indicates a control using Krebs-HEPES buffer instead of a blood sample. [Figure 2B]This figure shows the results of time-series luminescence measurements of L-012 (with bortezomib added and PMA stimulated) using 0.1 μL of mouse blood (whole blood, tail vein collection). In the figure, "Heart" represents the whole blood sample collected from the heart (control), "Tail 1st," "Tail 2nd," and "Tail 3rd" represent the whole blood samples obtained from the first, second, and third collections from the tail vein, respectively, and "buffer" represents the control using Krebs-HEPES buffer instead of blood samples. The Y axis represents luminescence (RLU), and the X axis represents measurement time (minutes). [Figure 3A] This graph shows the correlation between the number of isolated mouse neutrophils and the amount of L-012 luminescence (with bortezomib and PMA stimulation). The Y-axis represents the total luminescence (total RLU) measured over 30 minutes, and the X-axis represents the number of neutrophils. The graph also shows the linear regression line, the regression equation (y = 1665.1 × -149641), and the R² value (0.9266). [Figure 3B] This graph plots the signal-to-noise ratio (S / N), obtained by dividing the L-012 luminescence (Signal) shown in Figure 3A by the buffer control (Noise), on the Y-axis. The graph also shows the linear regression line, the regression equation (y = 0.0703 × -6.7643), and the R² value (0.7902). [Figure 3C] This graph shows the correlation between the number of isolated mouse neutrophils (small amounts) and the L-012 luminescence (with PMA stimulation). The Y-axis represents the total luminescence (total RLU) measured over 30 minutes, and the X-axis represents the number of neutrophils. "Bort" indicates measurements taken in the presence of bortezomib, and "DMSO" indicates measurements taken using DMSO instead of bortezomib. The graph also shows the linear regression line and regression equation (Bort: y = 1804.1 × +2921.5; DMSO: y = 270.21 × +4627.3) and R2 value (Bort: 0.9266; DMSO: 0.999). [Figure 3D]This graph shows the correlation between the number of isolated mouse cells (in small quantities) and the L-012 luminescence (with bortezomib added and PMA stimulation). The Y-axis represents the total luminescence (total RLU) measured over 30 minutes, and the X-axis represents the number of each cell type. The graph also shows the linear regression line, the regression equation (neutrophils: y = 1804.1 × +2921.5; monocytes: y = 228.49 × +7434.1), and the R2 value (neutrophils: 0.9266). [Figure 4A] This graph shows the correlation between neutrophil count and L-012 (bortezomib-added, PMA-stimulated) luminescence in neutropenic and neutropenic model mice. The Y-axis represents the total RLU measured over 30 minutes in 0.1 μL of blood collected from the tail vein, and the X-axis represents the neutrophil count in 0.1 μL of blood calculated by flow cytometry using blood collected from the heart of the same mouse. The graph shows the linear regression line, the regression equation (y = 1640.8 × +16994), and the R² value (0.7221). In the figure, "sal(day7)" represents a control mouse 7 days after administration of physiological saline, "sal(day1)" represents a control mouse 1 day after administration of physiological saline, "5-FU(day7)" represents a neutropenic model mouse 7 days after administration of the anticancer drug 5-FU, and "G-CSF(day1)" represents a sample from a neutropenic model mouse 1 day after administration of recombinant G-CSF. [Figure 4B] Figure 4A shows a graph plotting the S / N ratio (L-012 luminescence (Signal) divided by the buffer control (Noise) on the Y axis. The graph shows a linear regression line, the regression equation (y = 0.1209 × +0.9739), and the R² value (0.7224). In the figure, "sal(day7)" represents a control mouse 7 days after administration of physiological saline, "sal(day1)" represents a control mouse 1 day after administration of physiological saline, "5-FU(day7)" represents a neutropenic model mouse 7 days after administration of the anticancer drug 5-FU, and "G-CSF(day1)" represents a sample from a neutropenic model mouse 1 day after administration of recombinant G-CSF. [Figure 5]Shows the measurement results of L-012 luminescence in neutrophils isolated from neutrophil-increased model mice and control mice. The Y-axis shows the total luminescence (total RLU) measured for 30 minutes, and the X-axis shows the neutrophil-increased model mouse group (G-CSF) and the control mouse group (saline). [Figure 6A] Shows a calibration curve of the luminescence amount of L-012 (with bortezomib added) by xanthine oxidase. The Y-axis shows the total luminescence (total RLU) measured for 30 minutes, and the X-axis shows the concentration of xanthine oxidase. In the graph, a linear regression line, the regression equation (y = 453823x - 47648), and the R2 value (0.9383) are shown. In the figure, "XO" represents xanthine oxidase. [Figure 6B] Shows a graph indicating the correlation between the total luminescence for 30 minutes corrected by the xanthine oxidase activity value and the number of neutrophils. The Y-axis shows the concentration of xanthine oxidase corresponding to the total luminescence of each sample obtained from the xanthine oxidase calibration curve in Figure 6A. The X-axis shows the number of Ly6G-positive neutrophils in 0.1 μL of the blood sample. In the figure, "sal(day1)" represents control mice 1 day after administration of saline, "G-CSF(day1)" represents neutrophil-increased model mice 1 day after administration of recombinant G-CSF, and "5-FU(day7)" represents samples derived from neutrophil-decreased model mice 7 days after administration of the anticancer drug 5-FU. [Figure 7A] Is a graph showing the correlation between the number of neutrophils and the luminescence amount of L-012 (with bortezomib added and PMA stimulation) in human blood samples. The Y-axis shows the total luminescence (total RLU) measured for 30 minutes in 0.1 μL collected from humans, and the X-axis shows the number of neutrophils in 0.1 μL of blood calculated using an automated blood cell analyzer or blood image analysis using blood collected from the same human. In the graph, a linear regression line, the regression equation (y = 1680.5x + 15763), and the R2 value (0.8853) are shown. [Figure 7B]This graph shows the correlation between the total luminescence over 30 minutes, corrected for xanthine oxidase activity, and the number of neutrophils. The Y-axis represents the xanthine oxidase concentration corresponding to the total luminescence of each sample, as determined from the xanthine oxidase calibration curve. The X-axis represents the number of neutrophils in 0.1 μL of blood, calculated using an automated hematology analyzer or blood smear analysis. The graph also shows the linear regression line, the regression equation (y=0.002x-0.2459), and the R² value (0.9182). [Modes for carrying out the invention]

[0011] Neutrophils are known to migrate to foreign substances such as fungi and bacteria that invade the body, phagocytosing and killing them. Neutrophils highly express the superoxide-producing enzyme NOX2 / NADPH oxidase, and their bactericidal action is exerted by the production of reactive oxygen species such as superoxide anions through NOX2 activation induced by bacterial-derived substances. This invention includes determining the number of neutrophils based on the superoxide anion production capacity of neutrophils, that is, using the measurement results of NOX2-dependent superoxide anion production as an indicator.

[0012] In this application, superoxide anion is a type of reactive oxygen species, O2 - This refers to chemical substances that contain [the specified substance].

[0013] Superoxide anions are extremely reactive and have a short lifetime, making direct measurement difficult. Therefore, indirect measurement methods are commonly used, employing probes that rapidly react with superoxide anions to produce stable and quantifiable reaction products, or scavenging agents that rapidly capture superoxide anions. Examples of such superoxide anion measurement methods include chemiluminescence, fluorescence, spectrophotometrics, and electron spin resonance (ESR). In chemiluminescence, superoxide anions are measured by quantifying the amount of light emitted by the reaction between a chemiluminescent probe and superoxide anions. In fluorescence, superoxide anions are measured by measuring the fluorescence produced by the reaction between a fluorescent probe and superoxide anions, or by quantifying the specific reaction product between the probe and superoxide anions using liquid chromatography-mass spectrometry. In spectrophotometrics, a color change is induced by the reaction between a color-developing probe and superoxide anions, and superoxide anions are measured by measuring this change in absorbance. In the ESR method, superoxide anions are captured by a spin trapping agent, and the superoxide anions are measured by measuring the characteristic ESR spectrum produced by this capture.

[0014] Furthermore, in recent years, sensitizers have been developed that enhance the detection sensitivity of superoxide anions in indirect measurement methods. In this invention, by using a sensitizer for superoxide anion detection (Patent No. 7371941) containing the peptide boronic acid compound bortezomib or ixazomib, we have discovered the remarkable effect of being able to detect superoxide anions produced by neutrophils contained in trace amounts of biological samples of less than 1 μL with high sensitivity, thereby enabling the quantitative measurement of the number of neutrophils in the sample.

[0015] Accordingly, in one aspect of the present invention, a method for measuring the number of neutrophils in a biological sample is provided, comprising measuring superoxide anions in the presence of bortezomib or ixazomib or a salt thereof (hereinafter also referred to as "the method of the present invention").

[0016] Bortezomib or ixazomib used in the method of the present invention may be in the form of a salt, and this includes, for example, inorganic salts such as hydrochloride, sulfate, phosphate, or hydrobromide, or acid addition salts such as organic salts such as acetate, fumarate, maleate, oxalate, citrate, methanesulfonate, benzenesulfonate, or toluenesulfonate, or base addition salts such as ammonium salts, alkali metal salts (e.g., lithium salts, sodium salts, potassium salts), alkaline earth metal salts (e.g., calcium salts, magnesium salts), or other metal salts such as iron salts and zinc salts.

[0017] Bortezomib or ixazomib, or a salt thereof, may be provided as a composition (also called a sensitizer) containing bortezomib or ixazomib, or a salt thereof. The composition may be bortezomib or ixazomib, or a salt thereof, or may contain additional components other than bortezomib or ixazomib, or a salt thereof. The additional components are not particularly limited as long as they do not interfere with the action of bortezomib or ixazomib, or a salt thereof, but examples include solvents such as water, stabilizers, buffers, etc.

[0018] The amount of bortezomib or ixazomib, or a salt thereof, used in the method of the present invention is not particularly limited and can be appropriately determined by those skilled in the art. For example, the concentration of bortezomib or ixazomib in the reaction solution for measuring superoxide anions is not limited, but for example, about 3 to 100 μM, preferably about 5 to 50 μM, and more preferably about 10 to 30 μM. Depending on the amount used, the detection sensitivity of superoxide anions is increased by at least about 2 to 10 times compared to when they are not used, for example, by at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, or at least 9 times, and preferably by 10 times or more.

[0019] The method of the present invention employs an indirect method for measuring superoxide anions, and the measurement of said superoxide anions may be based, for example, on the detection of chemiluminescence, fluorescence, absorbance, or ESR spectra. Furthermore, the method of the present invention employs a probe or capture agent for the indirect method for measuring superoxide anions. Examples of said probes include chemiluminescent probes, fluorescent probes, color-developing probes, etc. Examples of said capture agents include spin traps. More specifically, but not limited to, examples of chemiluminescent probes include L-012 (8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione), lucigenin, luminol, isoluminol, methyl cypridina luciferin analog (MCLA), and Diogenes (manufactured by National Diagnostics). Examples of fluorescent probes include, but are not limited to, dihydroethidium (DHE, also known as hydroethidine), H2DCFDA (also known as DCFDA, 2',7'-dichlorofluorescein diacetate), H2DCFDA derivatives, dihydrorhodamine 123, and CellROX® (manufactured by Thermo Fisher Scientific). Examples of chromogenic probes include, but are not limited to, tetrazolium sulfonate salts (e.g., WST-1), cytochrome c, and tetrazolium nitroblue salt (NBT). Examples of spin trapping agents include, but are not limited to, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In the method of the present invention, a probe or scavenger suitable for the measurement method of the superoxide anion used may be used.

[0020] The biological sample is an animal-derived sample, preferably a mammal-derived sample. Mammals are not limited to humans, monkeys, dogs, mice, rats, etc., and more preferably, human-derived samples are used. The biological sample may be any biological sample containing neutrophils. Samples with a low neutrophil content may also be used. Examples include blood, saliva, and gingival crevicular fluid. As the biological sample, a sample collected from a living organism may be used directly (as is, untreated), or it may be used after one of the following treatments. Such treatments include, for example, dilution of the sample collected from a living organism, and concentration or isolation of neutrophils in the sample. For dilution, for example, physiological saline or buffer solution may be used. For concentration or isolation, for example, flow cytometry may be used. Because the method of the present invention has high detection sensitivity for superoxide anions, concentration or isolation of neutrophils in the biological sample is not required. Therefore, preferably, an untreated biological sample or a diluted biological sample is used.

[0021] The biological sample is preferably a blood sample. Because the method of the present invention has high detection sensitivity for superoxide anions, the neutrophil content in the sample used in the method of the present invention may be low. Therefore, the blood sample may be whole blood, or it may be a sample containing blood that has undergone red blood cell lysis, neutrophil concentration, or neutrophil isolation. The blood sample is preferably a sample containing whole blood. Since whole blood can be used in the method of this application, it is particularly useful in clinical settings.

[0022] The amount of biological sample used in the method of the present invention may be any amount and is not limited. Because the method of the present invention has high detection sensitivity for superoxide anions, the amount of biological sample used in the method of the present invention may be minute. For example, in the case of a blood sample, the amount of blood used may be, for example, less than 2 mL, 1 mL or less, 50 μL or less, 30 μL or less, 10 μL or less, 5 μL or less, 3 μL or less, 2 μL, 1 μL or less, 0.5 μL or less, 0.3 μL or less, or 0.1 μL or less, and preferably more than 0.01 μL, and may be less than 2 mL, 1 mL or less, 50 μL or less, 30 μL or less, 10 μL or less, 5 μL or less, 3 μL or less, 2 μL, 1 μL or less, 0.5 μL or less, 0.3 μL or less, or 0.1 μL or less. Blood samples can also be obtained by fingertip puncture, in which case the amount of blood sample is, for example, 1 μL or less, preferably 0.5 μL or less, more preferably 0.3 μL or less, even more preferably 0.1 μL or less, or even more preferably more than 0.01 μL and 1 μL or less, more than 0.01 μL and 0.5 μL or less, more than 0.01 μL and 0.3 μL or less, or more than 0.01 μL and 0.1 μL or less.

[0023] The method of the present invention includes adding bortezomib or ixazomib, or a salt thereof, to the superoxide anion detection system when measuring superoxide anions in a sample. For example, bortezomib or ixazomib, or a salt thereof, may be added to the sample, incubated as appropriate if necessary, and then a superoxide anion detection probe or capture agent may be added to the sample, incubated as appropriate if necessary, and then the superoxide anions may be measured. Alternatively, for example, bortezomib or ixazomib, or a salt thereof, and a superoxide anion detection probe or capture agent may be added to the sample simultaneously, incubated as appropriate if necessary, and then the superoxide anions may be measured.

[0024] The superoxide anion detection system described above is further modified by adding a neutrophil stimulant. By adding a neutrophil stimulant, superoxide anion production is detected when neutrophil NOX2 activity, which is normally in a static state, is maximally activated. This allows for a stable correlation between superoxide anion production and neutrophil count (e.g., unaffected by the state of neutrophils at the time of sample collection or storage), enabling quantitative measurement of neutrophil count. The neutrophil stimulant can be any substance that activates neutrophil function (e.g., migration, phagocytosis). Substances known as neutrophil stimulants in this field may be used, for example, but are not limited to, phorbol 12-myristate 13-acetate (PMA), formylmethionylleucylphenylalanine (fMLP), opsonized zymon (OZ), etc. One or more neutrophil stimulants may be used. The amount of neutrophil stimulant used is not particularly limited and can be appropriately determined by those skilled in the art depending on the type of neutrophil stimulant used. For example, in the case of PMA, the concentration in the reaction solution for measuring superoxide anion could be, for example, 0.2 μM, although this is not limited to PMA.

[0025] In this application, as shown in the examples described below, it was revealed that the amount of superoxide anion produced by neutrophils is proportional to the number of neutrophils. Therefore, the number of neutrophils in a sample can be determined based on the measurement results of the amount of superoxide anion produced in the sample. For example, by pre-determining the amount of superoxide anion produced at a known number of neutrophils, the number of neutrophils in a sample can be determined simply by measuring the amount of superoxide anion produced in the sample.

[0026] However, since superoxide anions are extremely unstable substances, it is difficult to create a calibration curve using superoxide anions themselves. Therefore, a xanthine oxidase-xanthine reaction system, which is a cell-free superoxide anion production system, is used to measure the amount of superoxide anions produced at known amounts of xanthine oxidase in the presence of an excess amount of xanthine and create a calibration curve. Xanthine oxidase is an enzyme that has superoxide anion production activity and produces superoxide anions using xanthine as a substrate. Furthermore, the amount of superoxide anions produced at known neutrophil counts is measured, and the correlation between the amount of superoxide anions produced (converted to the amount of xanthine oxidase) and the neutrophil count is determined based on the above calibration curve. Thus, by pre-determining the correlation between superoxide anion production (converted to xanthine oxidase levels) and neutrophil count, it is possible to determine the neutrophil count in a sample simply by measuring the superoxide anion production in the sample and converting the measured value to xanthine oxidase levels based on the calibration curve described above. Furthermore, it has been found that the measured value of superoxide anion can be affected by the measurement environment, such as the measuring equipment used. Therefore, it is preferable to create the calibration curve in parallel with or simultaneously with the measurement of superoxide anion production in the sample each time.

[0027] Accordingly, a further embodiment of the present invention provides a method that includes converting the amount of superoxide anion produced in a sample measured by the method of the present invention into the amount of xanthine oxidase based on a calibration curve obtained by a reaction system of xanthine oxidase and xanthine. Furthermore, in a preferred embodiment, a method is provided that includes determining the number of neutrophils by converting the amount of superoxide anion produced in a sample measured by the method of the present invention into the amount of xanthine oxidase based on a calibration curve created using a xanthine oxidase-xanthine reaction system measured in parallel for each experiment.

[0028] In yet another aspect of the present invention, a kit comprising bortezomib or ixazomib, or a salt thereof, for use in a method for measuring neutrophil count according to the present invention is provided. The kit may further include a probe suitable for the detection method used, e.g., a chemiluminescent probe, a fluorescent probe or a color-developing probe, or a capture agent, e.g., a spin trap agent, and / or a measuring device. The measuring device may preferably include a portable measuring device, in particular a small measuring device. Examples of the measuring device, but not limited to, include luminometers, preferably portable luminometers.

[0029] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0030] Example 1: Measurement of superoxide anions using mouse blood samples obtained by cardiac blood collection. Samples (10 μL) containing 10 μL, 1 μL, 0.1 μL, or 0.01 μL of blood were prepared using whole blood collected from the hearts of normal mice treated with EDTA (ethylenediaminetetraacetic acid) as an anticoagulant, either undiluted or diluted with Krebs-HEPES buffer (pH 7.4). Specifically, the 10 μL sample contained the undiluted blood as collected, the 1 μL sample used 10 μL of a 10-fold dilution of 1 μL of blood, the 0.1 μL sample used 10 μL of a 100-fold dilution of 1 μL of blood, and the 0.01 μL sample used 10 μL of a 1000-fold dilution of 1 μL of blood. Each blood sample was treated with PMA (200 nM) as a neutrophil stimulant, L-012 (Fujifilm Wako Pure Chemical Industries, Ltd.) (0.1 mM) as a chemiluminescent probe for superoxide anion detection, and bortezomib (10 μM) as a sensitizer. The L-012 emission was measured for 30 minutes at 37°C using a plate reader (Infinite® 200PRO, TECAN). As a control, an equal amount of dimethyl sulfoxide (DMSO), the solvent, was added instead of bortezomib and / or PMA, and the L-012 emission was measured similarly. Another control was performed using Krebs-HEPES buffer (pH 7.4) instead of blood samples. The results are shown in Figures 1A and 1B.

[0031] Furthermore, the above mouse whole blood samples (1 μL, 0.1 μL) to which EDTA had been added were subjected to erythrocyte lysis with ammonium chloride hemolysant, and the L-012 luminescence levels were measured for 30 minutes in the same manner as described above. The results are shown in Figure 1C.

[0032] Furthermore, the above mouse whole blood samples (1 μL, 0.1 μL) to which EDTA had been added were left to stand at room temperature for 1 to 24 hours, and then the L-012 luminescence levels were measured for 30 minutes in the same manner as described above. The results are shown in Figure 1D.

[0033] As is clear from Figures 1A and 1B, it was found that superoxide anions can be sufficiently detected with only 0.1 μL of blood sample using the sensitizer bortezomib. In time-course measurements of 0.1 μL blood sample, luminescence exceeding that of the control (buffer) was observed with the addition of bortezomib (Figure 1B).

[0034] In 10 μL and 1 μL whole blood samples, PMA-dependent L-012 luminescence was observed. However, the luminescence levels were lower than those of the control sample (0 μL blood sample, using buffer instead of blood), suggesting that the presence of a large number of red blood cells introduced into the assay inhibited the luminescence (Figure 1A, "DMSO-PMA"). In both cases, the addition of bortezomib increased the luminescence (Figure 1A, "DMSO-PMA" and "Bort-PMA" in 10 μL and 1 μL blood samples). On the other hand, no inhibition of luminescence by red blood cells was observed in 0.1 μL and 0.01 μL blood samples (Figure 1A). Furthermore, PMA-dependent luminescence was detected only in the presence of bortezomib in the 0.1 μL blood sample, but not in the 0.01 μL blood sample (Figure 1A). This suggests that a 0.01 μL blood sample is insufficient to produce enough neutrophils for the assay.

[0035] To eliminate the effect of erythrocyte-mediated inhibition of luminescence, L-012 luminescence was measured using blood samples treated with erythrocyte lysis. A significant increase in luminescence was observed compared to whole blood samples (Figure 1A) (Figure 1C). Therefore, it was shown that erythrocyte-mediated inhibition of luminescence occurs when using whole blood samples, and that this effect can be eliminated by subjecting the blood sample to erythrocyte lysis. On the other hand, no PMA-dependent enhancement of luminescence was observed in erythrocyte-lysis treated blood samples (Figure 1C, especially in the 0.1 μL RBC lysis blood sample), suggesting that neutrophils were activated by erythrocyte lysis.

[0036] In storage experiments of whole blood samples at room temperature, approximately 90% of the activity was maintained after 3 hours of standing at room temperature compared to after 1 hour (Figure 1D). On the other hand, it was almost completely inactivated after 24 hours (Figure 1D). Therefore, it was found that it is preferable to use blood collected within 3 hours.

[0037] Example 2: Measurement of superoxide anions using mouse blood samples collected from the tail vein. A sample (10 μL) containing 0.1 μL of whole blood collected from the tail vein of normal mice treated with EDTA as an anticoagulant was added. PMA (200 nM) was added as a neutrophil stimulant, L-012 (0.1 mM) as a chemiluminescent probe for superoxide anion detection, and bortezomib (10 μM) as a sensitizer. The L-012 emission level was measured using a plate reader for 30 minutes under 37°C conditions. The blood sample was collected three times by repeating tail vein puncture three times from the same mouse. As a control, the L-012 emission level was similarly measured using whole blood collected from the heart. As another control, Krebs-HEPES buffer (pH 7.4) was used instead of blood samples for the same measurement. All blood samples were prepared in the same manner as described in Example 1. The results are shown in Figures 2A and 2B.

[0038] As is clear from Figures 2A and 2B, higher L-012 luminescence was observed in blood samples collected via tail vein compared to those collected from the heart. This difference in luminescence depending on the collection site is thought to be due to differences in the number of blood cells depending on the collection site. In other words, since the cell density is higher in peripheral sites, the number of blood cells is higher in samples from the peripheral site for the same volume, and therefore the amount of L-012 luminescence is also higher. Furthermore, a tendency for L-012 luminescence to decrease was observed when tail vein puncture was repeated in the same individual.

[0039] Example 3: Correlation between cell number and L-012 luminescence in isolated mouse neutrophils Whole blood was collected from the hearts of normal mice. Neutrophils were isolated and counted using a magnetic bead method with an antibody against Ly6G, a neutrophil-specific surface antigen. The samples were then diluted appropriately with Krebs-HEPES buffer (pH 7.4) to prepare blood samples (10 μL) containing neutrophils of 10,000, 1,000, and 100 cells. PMA (200 nM) was added to each blood sample as a neutrophil stimulant, L-012 (0.1 mM) as a chemiluminescent probe for superoxide anion detection, and bortezomib (10 μM) as a sensitizer. The amount of L-012 emission was measured in the same manner as in Example 1. Figure 3A shows graphs plotting the amount of L-012 emission at neutrophil counts of 10,000, 1,000, and 100. Furthermore, as a control, the amount of L-012 emission was similarly measured using Krebs-HEPES buffer (pH 7.4) instead of blood samples. The signal-to-noise ratio (S / N ratio) was calculated by dividing the L-012 emission level (Signal) of each blood sample by the L-012 emission level (Noise) of the buffer control. Figure 3B shows a graph plotting the S / N ratio on the Y axis.

[0040] Furthermore, to investigate the correlation between small cell counts and L-012 luminescence, blood samples (10 μL) containing 3, 10, 30, and 100 Ly6G-positive neutrophils were prepared. To each blood sample, PMA (200 nM) was added as a neutrophil stimulant, L-012 (0.1 mM) as a chemiluminescent probe for superoxide anion detection, and bortezomib (10 μM) as a sensitizer. L-012 luminescence was measured in the same manner as in Examples 1 and 2. Additionally, as a control, DMSO was added instead of bortezomib, and L-012 luminescence was measured similarly. The results are shown in Figure 3C.

[0041] Furthermore, in addition to the Ly6G-positive neutrophils mentioned above, CD115-positive monocytes were isolated and counted from whole blood samples of normal mice. The remaining cells in the whole blood samples (including lymphocytes, hereinafter referred to as "other cells") were also counted. Blood samples (10 μL) containing 3, 10, 30, and 100 CD115-positive monocytes and other cells were prepared, respectively. PMA (200 nM), L-012 (0.1 mM), and bortezomib (10 μM) were added to each blood sample, and the L-012 luminescence was measured in the same manner as in Examples 1 and 2. The results are shown in Figure 3D.

[0042] As is clear from Figures 3A and 3B, a correlation was shown between the number of neutrophils in a blood sample and the amount of L-012 luminescence (i.e., superoxide anion production). Furthermore, as is clear from Figures 3C and 3D, a correlation was also shown between L-012 luminescence and neutrophils even with small cell counts.

[0043] Example 4: Correlation between neutrophil count and L-012 luminescence in neutropenic and neutropenic model mice. As a neutropenia model, mice 7 days after a single dose of the anticancer drug 5-FU (fluorouracil) at 100 mg / kg were used. As a neutrophilia model, mice 1 day after a single dose of recombinant G-CSF (granulocyte colony-stimulating factor) at 125 μg / kg or 250 μg / kg were used. There was no significant difference in neutrophilia between the 125 μg / kg and 250 μg / kg G-CSF doses. As a control, mice 1 or 7 days after administration of physiological saline were used. Blood was collected from the tail vein of each mouse and diluted with Krebs-HEPES buffer (pH 7.4) to prepare a blood sample (10 μL) containing 0.1 μL of the collected blood. Each blood sample was treated with PMA (200 nM) as a neutrophil stimulant, L-012 (0.1 mM) as a chemiluminescent probe for superoxide anion detection, and bortezomib (10 μM) as a sensitizer. The L-012 emission level was measured in the same manner as in Example 1. Blood was also collected from the hearts of each mouse, and the number of Ly6G-positive neutrophils was measured by flow cytometry. Figure 4A shows a graph plotting the L-012 emission level on the Y-axis and the number of neutrophils on the X-axis for each individual. The total number of plots in the graph corresponds to the total number of mice used in the experiment. Furthermore, as a control, the L-012 emission level was measured similarly using Krebs-HEPES buffer (pH 7.4) instead of blood samples. The S / N ratio was calculated by dividing the L-012 emission level (Signal) of each blood sample by the L-012 emission level (Noise) of the buffer control. Figure 4B shows a graph plotting the S / N ratio on the Y-axis. Furthermore, while flow cytometry requires at least 100 μL of blood, the technical limitations of collecting such a large volume of blood from the tail vein of a small animal like a mouse make it difficult. Therefore, the X-axis plotted the measurements of blood collected from the heart. As mentioned above, blood collected from the peripheral site (tail vein) has a higher cell density than blood collected from the heart. Based on known literature regarding the difference in cell density between the periphery and the heart, the number of Ly6G-positive neutrophils in peripheral blood is thought to be approximately three times higher than the number of Ly6G-positive neutrophils in cardiac blood.

[0044] As is clear from Figures 4A and 4B, a correlation was shown between the number of neutrophils in the blood sample and the amount of L-012 luminescence (i.e., superoxide anion production) in both model mice.

[0045] Example 5: L-012 luminescence of isolated neutrophils in a neutrophil-proliferating mouse model. Whole blood was collected from the hearts of neutrophilia model or control mice, and Ly6G-positive neutrophils and Ly6G-negative cells were isolated and counted using magnetic beads. Blood samples (10 μL) containing 10,000 Ly6G-positive neutrophils and 10,000 Ly6G-negative cells, respectively, were prepared, and PMA (200 nM), L-012 (0.1 mM), and bortezomib (10 μM) were added. The L-012 luminescence level was measured in the same manner as in Example 1. Neutrophilia model mice were used, which were mice 1 day after a single dose of recombinant G-CSF 125 μg / kg or 250 μg / kg. As a control, mice 1 day after administration of physiological saline were used. The results are shown in Figure 5.

[0046] As is clear from Figure 5, no difference was observed in the amount of L-012 luminescence in isolated neutrophils between the control mouse group and the neutrophil-reducing model mouse group. Therefore, it was found that even with an increase in neutrophils, the amount of superoxide anion produced per neutrophil unit does not change. In other words, it was shown that the change in L-012 luminescence reflects the increase or decrease in the number of neutrophils.

[0047] Example 6: Correction of luminescence using xanthine oxidase calibration curve Because superoxide anions are extremely unstable substances, it is difficult to create a calibration curve using superoxide anions themselves. Therefore, a calibration curve for the luminescence measurement system of this invention was created using xanthine oxidase, a cell-free superoxide anion production system. In order to strictly compare the measured values ​​of blood samples on different experimental days, the calibration curve was created each time in parallel with the measurement of blood samples. Specifically, xanthine oxidase (X4376, Sigma-A) at concentrations of 0.05 mU / mL, 0.5 mU / mL, and 1 mU / mL was added in the presence of bortemizob (10 μM), along with L-012 (0.1 mM) and substrate xanthine (245-00011, Fujifilm Wako Pure Chemical Industries) (0.2 mM), and the L-012 luminescence was measured for 30 minutes at 37°C using a plate reader (Infinite® 200PRO, TECAN). A calibration curve for xanthine oxidase was created by plotting the total RLU (total relative luminescence unit) of L-012 over 30 minutes on the Y axis and the xanthine oxidase concentration on the X axis (Figure 6A). The calibration curve equation obtained was y = 453823x - 47648.

[0048] On the other hand, using the same method as described in Example 4, samples (10 μL) containing 0.1 μL of blood collected from the tail vein of neutropenic mice 7 days after a single dose of the anticancer drug 5-FU (100 mg / kg), neutropenic mice 1 day after a single dose of recombinant G-CSF (125 μg / kg), and control mice 1 day after administration of physiological saline were prepared. PMA (200 nM), L-012 (0.1 mM), and bortezomib (10 μM) were added, and the L-012 luminescence was measured. The number of Ly6G-positive neutrophils in each blood sample was measured by flow cytometry. The total L-012 luminescence over 30 minutes in each sample was corrected using the xanthine oxidase activity value based on the xanthine oxidase calibration curve described above. Specifically, the total luminescence of each blood sample was converted to the corresponding xanthine oxidase concentration (x) based on the formula obtained from the xanthine oxidase calibration curve described above. Furthermore, the 30-minute total luminescence converted to xanthine oxidase concentration was plotted on the Y-axis, and the number of neutrophils in the blood sample was plotted on the X-axis to create a graph showing the correlation between the total luminescence converted to xanthine oxidase concentration and the number of neutrophils (Figure 6B). The experiment was conducted on two separate days, and each experiment was corrected using the xanthine oxidase calibration curve created for each experiment. Thus, a linear regression line (Figure 6B) showing the correlation between the 30-minute total luminescence corrected for xanthine oxidase activity and the number of neutrophils, the regression equation y = 0.0028x + 0.1494, and R 2 A value of 0.7428 was obtained.

[0049] As described above, by creating a xanthine oxidase calibration curve for each experiment and correcting the luminescence data obtained from sample measurements, universality and stability can be ensured in the measured values. Specifically, by measuring the amount of superoxide anion produced in the sample (e.g., the amount of luminescence measured by a chemiluminescent probe) and simultaneously creating a calibration curve for luminescence using xanthine oxidase, the measured value of superoxide anion in the sample can be converted to the amount of xanthine oxidase. Based on a graph showing the correlation between the total luminescence converted to the amount of xanthine oxidase and the number of neutrophils (e.g., the graph in Figure 6B), the number of neutrophils in the sample can be determined. Using this method, for example, data measured on different experimental days or using different instruments can be evaluated or compared simultaneously.

[0050] Example 7: Correlation between neutrophil count and L-012 luminescence in human blood samples Human blood samples (N=127) left over from blood tests during clinical practice were used to prepare 10 μL blood samples containing 0.1 μL of blood, diluted with Krebs-HEPES buffer (pH 7.4). PMA (200 nM) was added as a neutrophil stimulant, L-012 (0.1 mM) as a chemiluminescent probe for superoxide anion detection, and bortezomib (10 μM) as a sensitizer. The L-012 luminescence was measured using the same method as in Example 1. Neutrophil counts were measured at the Central Laboratory of Wakayama Medical University Hospital (using an automated blood cell analyzer or blood smear analysis) and are based on electronic medical record data. Figure 7A shows a graph plotting the total L-012 luminescence over 30 minutes for each individual on the Y-axis and the neutrophil count on the X-axis. The total number of plots in the graph corresponds to the total number of human blood samples used in the experiment. (Wakayama Medical University Ethics Review Committee Approval Number 4238, Prospective observational study on superoxide production and neutrophil count in trace amounts of blood)

[0051] A calibration curve for the luminescence measurement system of the present invention was created using xanthine oxidase in the same manner as in Example 6, and the total L-012 luminescence amount for 30 minutes in each sample was corrected by the xanthine oxidase activity value based on the xanthine oxidase calibration curve. Figure 7B shows a graph illustrating the correlation between the total luminescence amount converted to xanthine oxidase concentration and the number of neutrophils, with the total luminescence amount converted to xanthine oxidase concentration plotted on the Y axis and the number of neutrophils in the blood sample plotted on the X axis.

[0052] As is clear from Figures 7A and 7B, a correlation was shown between the number of neutrophils in human blood samples and the amount of L-012 luminescence (i.e., superoxide anion production). [Industrial applicability]

[0053] The neutrophil count measurement method of the present invention can be utilized in medical settings. For example, it is useful for identifying bacterial infections characterized by an increase in neutrophil count, identifying drug-induced neutropenia requiring regular monitoring of neutrophil counts (e.g., identifying agranulocytosis caused by anticancer drugs, clozapine, ticlopidine, sulfasalazine, etc.), diagnosing bacterial infections and prescribing appropriate antibiotics during fever, rapidly diagnosing neutrophil counts in infants and the elderly for whom venous blood sampling is difficult, minimally invasive diagnosis of chronic granulomatous disease, and rapidly diagnosing neutrophil counts in general hospitals and clinics that do not have clinical laboratories.

Claims

1. A method for measuring the number of neutrophils in a biological sample, comprising measuring superoxide anions in the presence of bortezomib or ixazomib or a salt thereof.

2. The method according to claim 1, wherein the biological sample is a blood sample.

3. The method according to claim 2, wherein the blood sample contains more than 0.01 μL and less than 2 mL of blood.

4. The method according to claim 2, wherein the blood sample contains more than 0.01 μL and 1 μL or less of blood.

5. The method according to claim 2, wherein the blood sample is whole blood.

6. The method according to any one of claims 1 to 5, comprising measuring superoxide anions in a system using a chemiluminescent probe, a fluorescent probe, or a color-developing probe.

7. The method according to claim 1, further comprising converting the measured value of superoxide anion to the amount of xanthine oxidase based on a calibration curve obtained by a reaction system of xanthine and xanthine.

8. A kit for measuring the number of neutrophils in a biological sample, comprising bortezomib or ixazomib, or a salt thereof.

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