Method for obtaining information on von Willebrand factor, method for preparing measurement samples, and reagent kit.

Abstract

To provide means for reducing an affection of VWF concentration, in measurement of a von Willebrand factor (VWF) by a fluorescence correlation spectroscopy (FCS) using a fluorescent label capture body or a fluorescence cross-correlation spectroscopy (FCCS).SOLUTION: There is provided a method for performing modification processing of a VWF by urea, then performing fluorescence labeling of the modified VWF by using a capture body including a fluorescent substance and coupling to the modified VWF, then acquiring information on a size of the VWF which has been subjected to the fluorescence labeling by FCS or FCCS. In the method, when acquiring the information by the FCS, the capture body is a polyclonal antibody or a plurality of monoclonal antibodies or aptamers which couples to a different epitope, and when acquiring information by the FCCS, out of two kinds of capture bodies, the capture body including a first fluorescent substance is the polyclonal antibody or the plurality of monoclonal antibodies or aptamers which couples to a different epitope.SELECTED DRAWING: Figure 14

Description

[Technical Field]

[0001] This invention relates to a method for obtaining information on the von Willebrand factor (VWF). This invention also relates to a method for preparing a sample for measurement. This invention also relates to a reagent kit used in a method for obtaining information on VWF. [Background technology]

[0002] VWF is a high molecular weight plasma glycoprotein that plays a crucial role in primary hemostasis. Specifically, VWF binds to the subendothelial tissue of damaged blood vessels at the bleeding site, where it promotes platelet adhesion and platelet thrombus formation. While VWF itself is a protein of approximately 250 kDa, it polymerizes and exists in the blood as multimers of various sizes (approximately 500 kDa to 15,000 kDa). High molecular weight multimers of VWF have higher platelet adhesion activity than low molecular weight multimers and are therefore important for primary hemostasis.

[0003] Von Willebrand disease (VWD) is a congenital coagulation disorder characterized by impaired primary hemostasis and a bleeding tendency due to quantitative reduction, complete absence, or qualitative abnormalities of von Willebrand fiber (VWF). VWD is diagnosed by classifying disease types based on the amount, activity, and multimer composition of VWF. The multimer composition of VWF is traditionally analyzed using a combination of SDS-gel electrophoresis and Western blotting with anti-VWF antibodies. This analysis separates VWF multimers according to their molecular weight, allowing for the determination of the proportions of high-molecular-weight, medium-molecular-weight, and low-molecular-weight multimers.

[0004] In recent years, attempts have been made to measure the molecular size of VWF using fluorescence imaging techniques called fluorescence correlation spectroscopy (FCS) or fluorescence cross-correlation spectroscopy (FCCS). FCS measures the fluctuations in the fluorescence signal (e.g., the change in fluorescence intensity over time) caused by molecules labeled with one fluorescent substance moving in and out of a minute observation area formed by the laser of a confocal optical system due to Brownian motion. FCCS measures molecules labeled with two fluorescent substances. In FCS, the obtained fluctuations are analyzed using an autocorrelation function, while in FCCS, the simultaneity of the fluctuations of the two fluorescent substances is analyzed using a cross-correlation function. FCS and FCCS can provide information such as the number and size of molecules. For example, Non-Patent Literature 1 describes the measurement of VWF in the plasma of healthy individuals and VWD patients using FCS with a fluorescently labeled anti-VWF antibody, and a comparison of the measured values. Furthermore, Patent Document 1 describes how the molecular size of recombinant VWF was measured by FCCS using two types of fluorescently labeled anti-VWF antibodies. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2021-173705 [Non-patent literature]

[0006] [Non-Patent Document 1] Torres R. et al., Clin.Chem., vol.58, pp.1010-1018, 2012 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] In antigen measurement using FCS and FCCS with fluorescently labeled antibodies, the antigen concentration in the sample may affect the results. This is due to the dissociation of the fluorescently labeled antibody and the antigen. When an antibody with a predetermined dissociation constant is used at a constant final concentration, the number of complexes formed by the antibody and the antigen depends on the antigen concentration in the sample. Therefore, the lower the antigen concentration in the sample, the greater the effect of dissociation between the fluorescently labeled antibody and the antigen. Non-patent document 1 also describes that the diffusion time obtained by FCS changed depending on the VWF concentration in the sample. Diffusion time is the average time required for a fluorescently labeled molecule to pass through the observation area and is a parameter representing molecular size. Smaller molecules pass through the observation area more quickly than larger molecules, resulting in shorter diffusion times. In other words, if the number of complexes decreases due to dissociation between the fluorescently labeled antibody and the antigen in the sample, the diffusion time value for that sample will decrease. Therefore, for samples with low antigen concentrations, it is unclear whether the molecular size information obtained by FCS or FCCS reflects the actual molecular size of the antigen.

[0008] On the other hand, when measuring the fluorescent substance itself or beads to which the fluorescent substance is covalently bonded (fluorescent beads) using FCS, it is known that even if the concentrations of the fluorescent substance or fluorescent beads differ between the measurement samples, the molecular size measurement results obtained from each measurement sample hardly change (see, for example, Gendron PO. et al., J.Fluoresc., vol.18, pp.1093-1101, 2008 and Yamamoto J. et al., Opt.Exp., vol.27, pp.14835-14841, 2019). Furthermore, the present inventors also confirmed that when VWF, which is covalently bonded to two types of fluorescent substances, was measured using FCCS, the diffusion time for each measurement sample remained almost constant even if the concentrations of VWF differed between the measurement samples.

[0009] However, in order to measure VWF in biological samples collected from subjects using FCS or FCCS, indirect labeling of VWF with a fluorescently labeled capture agent is necessary. On the other hand, the concentration of VWF in biological samples collected from subjects may differ from subject to subject. Therefore, the present inventors aim to provide a means to reduce the influence of VWF concentration in the measurement of VWF using FCS and FCCS with a fluorescently labeled capture agent. [Means for solving the problem]

[0010] The present invention provides a method for obtaining information on VWF, comprising the steps of: denaturing VWF contained in a biological sample with urea; fluorescently labeling the denatured VWF using a capture body containing a fluorescent substance and binding to the denatured VWF; and obtaining information on the size of the fluorescently labeled VWF using FCS or FCCS, wherein when information is obtained by FCS, the capture body comprises a polyclonal antibody or a plurality of monoclonal antibodies or aptamers that bind to different epitopes; and when information is obtained by FCCS, the capture body comprises a capture body containing a first fluorescent substance and binding to the denatured VWF, and a capture body containing a second fluorescent substance and binding to the denatured VWF, wherein the second fluorescent substance is a fluorescent substance having a fluorescence emission maximum in a wavelength range different from that of the first fluorescent substance, and the capture body containing the first fluorescent substance and binding to the denatured VWF is a polyclonal antibody containing the first fluorescent substance, or a plurality of monoclonal antibodies or aptamers, each containing the first fluorescent substance and binding to different epitopes.

[0011] The present invention provides a method for preparing a sample for measurement using FCS or FCCS, comprising the steps of: denaturing VWF contained in a biological sample with urea; and fluorescently labeling the denatured VWF using a capture body containing a fluorescent substance and binding to the denatured VWF, wherein when the VWF fluorescently labeled with the capture body is used for measurement by FCS, the capture body comprises a polyclonal antibody or a plurality of monoclonal antibodies or aptamers that bind to different epitopes; and when the VWF fluorescently labeled with the capture body is used for measurement by FCCS, the capture body comprises a capture body containing a first fluorescent substance and binding to the denatured VWF, and a capture body containing a second fluorescent substance and binding to the denatured VWF, wherein the second fluorescent substance is a fluorescent substance having a fluorescence emission maximum in a wavelength range different from that of the first fluorescent substance, and the capture body containing the first fluorescent substance and binding to the denatured VWF is a polyclonal antibody containing the first fluorescent substance, or a plurality of monoclonal antibodies or aptamers each containing the first fluorescent substance and binding to different epitopes.

[0012] The present invention provides a reagent kit for use in the above method, comprising urea and a capture agent containing a fluorescent substance and binding to VWF denatured with urea, wherein the capture agent comprises a polyclonal antibody or a plurality of monoclonal antibodies or aptamers that bind to different epitopes.

[0013] The present invention provides a reagent kit for use in the above-mentioned method by FCCS, comprising urea, a capture agent containing a first fluorescent substance and binding to VWF denatured with urea, and a capture agent containing a second fluorescent substance and binding to VWF denatured with urea, wherein the second fluorescent substance is a fluorescent substance having maximum absorption in a wavelength range different from that of the first fluorescent substance, and the capture agent containing the first fluorescent substance and binding to VWF denatured with urea is a polyclonal antibody containing the first fluorescent substance, or a plurality of monoclonal antibodies or aptamers, each containing the first fluorescent substance and binding to different epitopes. [Effects of the Invention]

[0014] According to the present invention, in the measurement of VWF by FCS and FCCS using a fluorescently labeled capture body, the influence of the concentration of VWF can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] [Figure 1A] It is a figure which shows an example of the reagent kit of this embodiment which can be used for the measurement of VWF by FCS. [Figure 1B] It is a figure which shows an example of the reagent kit of this embodiment which can be used for the measurement of VWF by FCCS. [Figure 2A] It is a graph which shows the diffusion time by FCCS of the measurement sample containing VWF to which Alexa Fluor (registered trademark) 488 and Alexa Fluor (registered trademark) 647 are covalently bonded at various concentrations. [Figure 2B] It is a graph which shows the diffusion time by FCCS of the measurement sample containing VWF to which the monoclonal antibody labeled with Alexa Fluor (registered trademark) 488 and the monoclonal antibody labeled with Alexa Fluor (registered trademark) 647 are bound at various concentrations. [Figure 3A] It is a graph which shows the diffusion time by FCS of the measurement sample containing VWF which is non-denatured or denatured with urea at different concentrations, and the VWF is indirectly labeled with the monoclonal antibody labeled with Alexa Fluor (registered trademark) 488. [Figure 3B] It is a graph which shows the diffusion time by FCS of the measurement sample containing VWF which is non-denatured or denatured with urea at different concentrations, and the VWF is indirectly labeled with the monoclonal antibody labeled with Alexa Fluor (registered trademark) 647. [Figure 4] It is a graph which shows the diffusion time by FCS of the measurement sample containing VWF which is denatured with urea at different concentrations, and the VWF is indirectly labeled with the polyclonal antibody labeled with Alexa Fluor (registered trademark) 488. [Figure 5]This graph shows the diffusion time by FCS for measurement samples containing urea-denatured VWF at different concentrations, in which the VWF is indirectly labeled with two monoclonal antibodies labeled with Alexa Fluor® 488. [Figure 6] This graph shows the diffusion time by FCCS for measurement samples containing undenatured or urea-denatured VWF at different concentrations, with the VWF indirectly labeled using monoclonal antibodies labeled with Alexa Fluor® 488 and Alexa Fluor® 647. [Figure 7] This graph shows the diffusion time by FCCS for measurement samples containing urea-denatured VWF at different concentrations, in which the VWF is indirectly labeled with a polyclonal antibody labeled with Alexa Fluor® 488 and a monoclonal antibody labeled with Alexa Fluor® 647. [Figure 8] This graph shows the diffusion time by FCCS for measurement samples containing urea-denatured VWF at different concentrations, with the VWF indirectly labeled using two monoclonal antibodies labeled with Alexa Fluor® 488 and a monoclonal antibody labeled with Alexa Fluor® 647. [Figure 9] This graph shows the diffusion time by FCCS for measurement samples containing VWF denatured with various concentrations of urea, and indirectly labeled with polyclonal antibodies labeled with Alexa Fluor® 488 and Alexa Fluor® 647. [Figure 10] This graph shows the diffusion time by FCCS for measurement samples containing urea-denatured VWF at different concentrations, and the VWF indirectly labeled with reagents containing polyclonal antibodies labeled with Alexa Fluor® 488 and polyclonal antibodies labeled with Alexa Fluor® 647 at various concentration ratios. [Figure 11]This figure shows the results of analyzing unmixed or mixed standard human plasma using SDS-gel electrophoresis and Western blotting, and an example of their densitometry analysis. [Figure 12A] This graph shows the diffusion time by FCS for measurement samples that have different proportions of high molecular weight fraction (LMW index), contain urea-denatured VWF, and indirectly label the VWF with a polyclonal antibody labeled with Alexa Fluor® 488. [Figure 12B] This graph shows the diffusion time by FCS for measurement samples containing VWF that has a different LMW index and has been denatured with urea, and in which the VWF is indirectly labeled with a polyclonal antibody labeled with Alexa Fluor® 647. [Figure 12C] This graph shows the diffusion time by FCCS for measurement samples containing VWF that has different LMW indices and has been denatured with urea, and indirectly labeled with polyclonal antibodies labeled with Alexa Fluor® 488 and polyclonal antibodies labeled with Alexa Fluor® 647. [Figure 13A] This graph shows the diffusion time by FCS for measurement samples containing VWF at different concentrations, which is prepared from unmixed or mixed standard human plasma and denatured with urea, and indirectly labeled with a monoclonal antibody labeled with Alexa Fluor® 488. [Figure 13B] This graph shows the diffusion time by FCS for measurement samples containing VWF at different concentrations, which is prepared from unmixed or mixed standard human plasma and denatured with urea, and indirectly labeled with a monoclonal antibody labeled with Alexa Fluor® 647. [Figure 13C]This graph shows the diffusion time by FCCS for measurement samples containing VWF at different concentrations, which is prepared from unmixed or mixed standard human plasma and denatured with urea, and indirectly labeled with monoclonal antibodies labeled with Alexa Fluor® 488 and Alexa Fluor® 647. [Figure 14] This graph shows the diffusion time by FCS for measurement samples containing VWF at different concentrations, which is prepared from unmixed or mixed standard human plasma and denatured with urea, and indirectly labeled with a polyclonal antibody labeled with Alexa Fluor® 488. [Figure 15] This graph shows the diffusion time by FCCS for measurement samples containing VWF at different concentrations, which is prepared from unmixed or mixed standard human plasma and denatured with urea, and indirectly labeled with polyclonal antibodies labeled with Alexa Fluor® 488 and monoclonal antibodies labeled with Alexa Fluor® 647. [Modes for carrying out the invention]

[0016] In the method for obtaining VWF information of this embodiment (hereinafter also referred to as "the information acquisition method of this embodiment"), first, the VWF contained in the biological sample is denatured with urea. In this specification, the terms "von Willebrand factor" and "VWF" include monomers and multimers of any molecular size. A VWF multimer is formed from multiple monomeric VWF. A VWF multimer only needs to contain multiple monomeric VWF, and may also contain other formed elements (e.g., platelets). In a VWF multimer, the monomeric VWF do not need to be strongly bound to each other by covalent bonds or the like. Aggregates formed by looser bonds between multiple monomeric VWF are also included in the multimer. Preferably, the VWF is VWF expressed in the human body and contained in a biological sample taken from a human body.

[0017] A biological sample may be a specimen collected from a subject and containing VWF, or a sample prepared from such a specimen. Examples of biological samples include whole blood, plasma, and serum. When whole blood or plasma is used, an anticoagulant may be added. The type of anticoagulant is not particularly limited and examples include sodium citrate, potassium ethylenediaminetetraacetic acid (EDTA) salt, sodium EDTA salt, and heparin salt. Among these, sodium citrate is preferred. If necessary, the biological sample may be diluted with a suitable aqueous solvent. Examples of such aqueous solvents include water, physiological saline, and buffer solutions. Examples of buffer solutions include phosphate-buffered saline (PBS), Tris-HCl, and Good's buffer.

[0018] Denaturation of VWF in biological samples with urea can be performed, for example, by mixing urea with the biological sample. The urea added to the biological sample may be in solution or solid form. For ease of handling, it is preferable to use a urea solution. The solvent for the urea solution is not particularly limited as long as it can dissolve the urea, but the aqueous solvent described above is preferred. The concentration of the urea solution is not particularly limited, for example, it may be between 1M and 8M. Note that "M" is the unit of molar concentration, and is expressed as "mol / L" or "mol / dm³". 3 It is also written as "[...]". Denaturation of VWF in biological samples with urea is performed with the aim of increasing the number of traps that bind to each VWF molecule. It is thought that the action of urea changes the higher-order structure of VWF as a protein, and the epitopes to which traps bind are exposed more than in the undenatured case.

[0019] The denaturation treatment of VWF with urea is preferably carried out in the presence of urea at a concentration of 0.5 M to 1.75 M. In the denaturation treatment, the lower limit of the urea concentration in the mixture of the biological sample and urea may be, for example, 0.5 M or higher, 0.6 M or higher, 0.7 M or higher, 0.8 M or higher, or 0.9 M or higher. The upper limit of the urea concentration in the mixture of the biological sample and urea may be, for example, 1.75 M or lower, 1.7 M or lower, 1.6 M or lower, 1.5 M or lower, 1.4 M or lower, 1.3 M or lower, 1.2 M or lower, or 1.1 M or lower.

[0020] The denaturation treatment can be carried out, for example, by mixing a biological sample with urea and then incubating the resulting mixture at a temperature of 20°C to 45°C, preferably 30°C to 40°C. The incubation time may be, for example, 5 minutes to 240 minutes, preferably 10 minutes to 120 minutes.

[0021] Next, the denatured VWF is fluorescently labeled using a capture agent containing a fluorescent substance and binding to the denatured VWF (hereinafter also referred to as the "fluorescently labeled capture agent"). The fluorescently labeled capture agent can be obtained by binding a capture agent that binds to the denatured VWF with a fluorescent substance. Examples of capture agents include polyclonal antibodies, multiple monoclonal antibodies that bind to different epitopes, multiple aptamers that bind to different epitopes, and combinations thereof. It is believed that by including polyclonal antibodies, multiple monoclonal antibodies that bind to different epitopes, multiple aptamers that bind to different epitopes, and combinations thereof, the capture agent can bind to multiple epitopes on one VWF molecule. This can increase the number of capture agents that bind to one VWF molecule. The multiple monoclonal antibodies that bind to different epitopes include at least a first monoclonal antibody that binds to a first epitope of the VWF and a second monoclonal antibody that binds to a second epitope of the VWF. Multiple aptamers that bind to different epitopes include at least a first aptamer that binds to a first epitope of VWF and a second aptamer that binds to a second epitope of VWF.

[0022] In this specification, the term "antibody" includes full-length antibodies and their fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fd', Fv, light chains, variable regions of heavy chain antibodies derived from camelids (VHH), variable regions of heavy chain antibodies derived from cartilaginous fish (VNAR), reduced IgG (rIgG), and single-chain antibodies (scFv). The origin of the antibody is not particularly limited and may be derived from any animal, such as mammals like mice, rats, hamsters, rabbits, goats, horses, and camels, or cartilaginous fish like sharks. The antibody isotype may be IgG, IgM, IgE, or IgA, but is preferably IgG. The antibody that binds to denatured VWF may be a commercially available anti-VWF antibody or an antibody produced by a method known in the art. The aptamer may be a peptide aptamer or a nucleic acid aptamer. Aptamers can be prepared by known methods such as the SELEX method.

[0023] Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, Cy2®, Cy3®, Cy5®, and the Alexa Fluor® series, as well as fluorescent proteins such as green fluorescent proteins (GFP, EGFP, etc.), yellow fluorescent proteins (YFP, EYFP, etc.), blue fluorescent proteins (BFP, CFP, ECFP, etc.), and red fluorescent proteins (dsRed, mCherry, etc.). Among these, fluorescent dyes are preferred.

[0024] When obtaining information on the size of fluorescently labeled VWF using FCS, a capture body labeled with a single fluorescent substance is used. The single fluorescent substance may be a single fluorescent substance or a combination of multiple fluorescent substances that emit fluorescence at approximately the same wavelength. Examples of such combinations include FITC (fluorescence wavelength 522 nm) and Alexa Fluor® 488 (fluorescence wavelength 519 nm), rhodamine (fluorescence wavelength 570 nm) and Cy3® (fluorescence wavelength 570 nm), and Cy5® (fluorescence wavelength 667 nm) and Alexa Fluor® 647 (fluorescence wavelength 665 nm).

[0025] When acquiring the above information by FCCS, two types of traps labeled with two different fluorescent substances can be used. That is, the traps used in FCCS include a trap containing the first fluorescent substance and binding to denatured VWF (hereinafter also referred to as the "first fluorescently labeled trap") and a trap containing the second fluorescent substance and binding to denatured VWF (hereinafter also referred to as the "second fluorescently labeled trap"). Since FCCS detects two colors of fluorescence substantially simultaneously, the second fluorescent substance is a fluorescent substance that has a fluorescence emission maximum in a different wavelength range than the first fluorescent substance. The fluorescence emission maximum is the wavelength at which the fluorescence emitted by the excited fluorescent substance has the greatest intensity, and is also called the maximum fluorescence wavelength. For example, the fluorescence emission maximum of the second fluorescent substance may be in a wavelength range that is 30 nm or more, preferably 40 nm or more, away from the fluorescence emission maximum of the first fluorescent substance. The fluorescence emission maximum of a fluorescent substance can be easily determined by analyzing the fluorescence spectrum of the fluorescent substance using a known spectrofluorometer. More preferably, the second fluorescent substance is a substance that emits fluorescence of a different color from that of the first fluorescent substance. The color of fluorescence emitted by each fluorescent substance is not particularly limited, but for example, the first fluorescent substance may be a substance that emits green fluorescence (wavelength approximately 500 nm to approximately 550 nm), and the second fluorescent substance may be a substance that emits yellow fluorescence (wavelength approximately 580 nm to approximately 600 nm) or red fluorescence (wavelength approximately 610 nm to approximately 780 nm).

[0026] When obtaining the above information using FCCS, specifically, if at least one of FITC, Cy2(registered trademark), and Alexa Fluor(registered trademark) 488 is used as the first fluorescent substance, at least one of rhodamine, Cy3(registered trademark), Cy5(registered trademark), and Alexa Fluor(registered trademark) 647 can be used as the second fluorescent substance. The first and second fluorescent substances may each be a single fluorescent substance or a combination of multiple fluorescent substances that emit fluorescence at approximately the same wavelength.

[0027] When obtaining the above information by FCCS, it is preferable that at least one of the first fluorescently labeled capture body and the second fluorescently labeled capture body is a polyclonal antibody, multiple monoclonal antibodies that bind to different epitopes, multiple aptamers that bind to different epitopes, or a combination thereof. For example, the first fluorescently labeled capture body may be a polyclonal antibody containing a first fluorescent substance, or multiple monoclonal antibodies or aptamers each containing a first fluorescent substance and binding to different epitopes, and / or the second fluorescently labeled capture body may be a polyclonal antibody containing a second fluorescent substance, or multiple monoclonal antibodies or aptamers each containing a second fluorescent substance and binding to different epitopes. When either the first fluorescently labeled capture body or the second fluorescently labeled capture body is a polyclonal antibody, or multiple monoclonal antibodies or aptamers that bind to different epitopes, the other may be a single type of monoclonal antibody.

[0028] Labeling of a capture agent with a fluorescent substance can be performed by covalently bonding the fluorescent substance and the capture agent using known methods such as amine coupling or maleimide methods. The capture agent may also be fluorescently labeled using commercially available labeling kits or crosslinkers. Fluorescent substances with reactive groups suitable for labeling are also commercially available. For example, fluorescent substances having N-hydroxysuccinimide (NHS) ester, sulfodichlorophenol (SDP) ester, or tetrafluorophenyl (TFP) ester as reactive groups can label the capture agent by an amine coupling reaction between the ester and the amino group of the capture agent. Similarly, fluorescent substances having a maleimide group as a reactive group can label the capture agent by a reaction between the maleimide group and the sulfhydryl group of the capture agent. If the capture agent is a monoclonal antibody and the fluorescent substance is a fluorescent protein, the capture agent containing the fluorescent substance may be a fusion protein of the capture agent and the fluorescent substance. This can be produced by genetic engineering techniques known in the art.

[0029] When obtaining the above information by FCS, the fluorescent labeling of denatured VWF with a fluorescently labeled capture agent can be performed by mixing a mixture of the biological sample and urea with the fluorescently labeled capture agent. The fluorescently labeled capture agent is preferably in solution form. When obtaining the above information by FCCS, the fluorescent labeling of denatured VWF with a fluorescently labeled capture agent can be performed by mixing a mixture of the biological sample and urea with a first fluorescently labeled capture agent and a second fluorescently labeled capture agent. The mixture obtained by this fluorescent labeling process, containing the biological sample, urea, and fluorescently labeled capture agent, will hereinafter also be referred to as the "measurement sample".

[0030] Fluorescent labeling of denatured VWF is preferably carried out in the presence of urea at a concentration of 0.2 M to 1 M. When the fluorescent labeling capture agent is a solution, the amount of fluorescent labeling capture agent added to the mixture of the biological sample and urea is preferably such that the urea concentration in the measurement sample is between 0.2 M and 1 M. Although the denaturing effect of urea in the measurement sample extends to the fluorescent labeling capture agent, if the urea concentration in the measurement sample is within the above range, the effect of FCS and FCCS on the measurement is considered to be substantially negligible.

[0031] The concentration of the fluorescently labeled capture agent in the measurement sample is not particularly limited. For example, if the fluorescently labeled capture agent is an antibody, the fluorescently labeled capture agent can be added to the mixture of the biological sample and urea so that the final concentration of the antibody in the measurement sample is 1 nM or more and 100 nM or less, preferably 5 nM or more and 75 nM or less, and more preferably 10 nM or more and 50 nM or less. If the first and second fluorescently labeled capture agents are antibodies, they should be added so that the final concentration of each antibody in the measurement sample falls within the above range.

[0032] Fluorescent labeling can be performed, for example, by mixing a mixture of a biological sample and urea with a fluorescent labeling capture agent, and then incubating the resulting sample at a temperature of 20°C to 40°C, preferably 25°C to 37°C. The incubation time may be, for example, 1 minute to 120 minutes, preferably 3 minutes to 60 minutes.

[0033] In another embodiment, the denaturation of VWF with urea and the fluorescent labeling of VWF with a fluorescently labeled capture agent may be performed substantially simultaneously. In this case, the biological sample, urea, and fluorescently labeled antibody are mixed to prepare the measurement sample. These may be mixed substantially simultaneously. Alternatively, the biological sample and urea may be mixed first, followed by the addition of the fluorescently labeled antibody to prepare the measurement sample. The concentration of urea in the measurement sample is preferably 0.5 M to 1 M. In a measurement sample containing urea at such a concentration, denaturation of VWF with urea and fluorescent labeling of VWF with a fluorescently labeled antibody can be performed. The denaturation and fluorescent labeling can be performed, for example, by mixing the biological sample, urea, and fluorescently labeled capture agent, and then incubating the resulting measurement sample at a temperature of 20°C to 45°C, preferably 30°C to 40°C. The incubation time may be, for example, 10 minutes to 120 minutes, preferably 15 minutes to 60 minutes.

[0034] After the modified VWF is fluorescently labeled, information regarding the size of the fluorescently labeled VWF is obtained by FCS or FCCS. The information regarding the size of the fluorescently labeled VWF is not particularly limited, but examples include the diffusion time, hydrodynamic radius, hydrodynamic diameter, and volume of the fluorescently labeled VWF. The hydrodynamic radius, hydrodynamic diameter, and volume can be calculated from the diffusion time. The hydrodynamic radius and hydrodynamic diameter are usually expressed in nanometers (nm). The volume is usually expressed in cubic nanometers (nm). 3 It is expressed in units of ). Among these, it is preferable to obtain the diffusion time of fluorescently labeled VWF. As mentioned above, the diffusion time obtained by FCS or FCCS changes depending on the size of the fluorescently labeled molecule, so it is information that reflects the size of the molecule.

[0035] Diffusion time is the average time required for fluorescently labeled VWF in a sample to pass through the observation area formed by the laser of a confocal optical system. Diffusion time can also be described as the residence time (the time the fluorescently labeled VWF diffuses and remains in the observation area). Diffusion time is generally represented by the symbol "τ".D It is expressed as "fs" and the units used are microseconds (μs) or milliseconds (ms). The measurement and acquisition of diffusion time by FCS and FCCS are well known, and FCS and FCCS are described in various publications such as Eigen M. and Rigler R., Proc.Natl.Acad.Sci.USA. vol.91, pp.5740-5747, 1994; Bacia K. et al., Nat.Methods, vol.3, pp.83-89, 2006; and Bacia K. and Schwille P., Nat.Protoc., vol.2, pp.2842-2856, 2007.

[0036] The measurement of fluorescent molecules by FCS or FCCS can be performed using an instrument capable of forming an observation area with a confocal optical system laser and measuring the fluorescence signal generated from fluorescent molecules entering and exiting this observation area. Such instruments are well known and include, for example, confocal laser microscopes and fluorescence correlation spectrometers. Specifically, commercially available instruments include the Carl Zeiss LSM710 confocal laser microscope (Zeiss), FCS Compact (Hamamatsu Photonics K.K.), and FCS-101 (Toyobo Co., Ltd.). These commercially available instruments are equipped with analysis software such as Zen software (Zeiss), which allows for the analysis of detected fluorescence signals using autocorrelation and cross-correlation functions.

[0037] In measurements using a confocal laser microscope with FCS or FCCS, for example, the sample to be measured is dispensed into a 384-well glass-bottom plate, and the fluorescence signal is detected by irradiating it with a laser. The temperature of the sample to be measured during measurement is preferably around 23°C to 25°C. The wavelength of the laser is not particularly limited as long as it can excite the fluorescent substance in the sample to be measured. In the measurement, the laser irradiated from the light source of the instrument is focused to form a measurement area with a volume of, for example, 0.1 to 1 fL (femtoliter). The instrument then detects the fluorescence signal emitted when the fluorescently labeled VWF passes through the measurement area. In FCS, one type of laser is used to excite the fluorescent substance, and the fluorescence signal generated from the excited fluorescent substance is continuously acquired. In FCCS, two types of lasers with different wavelengths are used to excite a first and a second fluorescent substance, and the fluorescence signals generated from each of the excited first and second fluorescent substances are continuously acquired. The measurement time can be set as appropriate by a person skilled in the art, and can be set to, for example, 10 seconds or more. The measurement may be performed multiple times for a single sample to be measured. The raw data of the detected fluorescence signal is a set of data represented by fluorescence intensity (Hz) and measurement time (s). The raw data may be processed to remove measurements that show baseline drift or bursts (fluorescence signals more than three times higher than the average fluorescence intensity).

[0038] FCS measurements acquire raw data from single-wavelength photon measurements. Autocorrelation analysis is performed on the raw data to obtain the autocorrelation function curve. An appropriate fitting model is used to fit the autocorrelation function curve to obtain a correlation curve drawn by the correlation function G(τ) and the correlation time τ (also called lag time). FCS allows fitting using a two-component model with the autocorrelation function. The first component used in the two-component model can be, for example, an unreacted fluorescently labeled trap. The second component can be a composite of denatured VWF and the fluorescently labeled trap. For example, using analysis software such as Zen software, a one-component model is selected, and the diffusion time of the first component is measured for each fluorescently labeled trap. Next, using the same software, a two-component model is selected, and the diffusion time of the first component is fixed to the diffusion time of the first component measured in the previous one-component model, and the diffusion time of the second component is measured.

[0039] In FCCS measurements, raw photon measurement data is acquired for each of the two wavelengths. Cross-correlation analysis is performed on the raw data to obtain a cross-correlation function curve. Furthermore, autocorrelation analysis may be performed on the raw data for each wavelength to obtain autocorrelation function curves for the first and second fluorescent substances, respectively. An appropriate fitting model is used to fit the cross-correlation function curve to obtain a correlation curve drawn by the correlation function G(τ) and correlation time τ. FCCS allows fitting using a one-component model with the cross-correlation function. The one component can be a complex of denatured VWF and a fluorescently labeled trapper. For example, a one-component model is selected on analysis software such as Zen software, and the diffusion time of the complex is measured. In FCCS, two types of fluorescently labeled trappers are used for VWF, enabling more accurate analysis.

[0040] The following shows specific functions. All of the following functions are known (see, for example, Krichevsky O. and Bonnet G., Rep.Prog.Phys. vol.65, no.2, 251, 2002). The fitting function G(τ) for obtaining a one-component three-dimensional correlation curve is represented by Eq. 1A below. The fitting function G(τ) for obtaining a two-component three-dimensional correlation curve is represented by Eq. 1B below. Both of these functions represent a translational diffusion process. When calculating the translational diffusion time from the mutual correlation curve, a fitting model can be used as in Eq. 1A or Eq. 1B below.

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[0042]

Number

[0043] In Eq. 1A, N represents the number of molecules of the component in the measurement region (in the formula, it is the average number of molecules). τ D represents the diffusion time (μs). τ represents the correlation time. ω is the ratio of the radius (w xy ) of the laser focus volume (observation region) to half the length of the vertical axis (w z ) (w z / w xy ). In Eq. 1B, N1 represents the number of molecules of the first component in the measurement region, and N2 represents the number of molecules of the second component in the measurement region (in the formula, they are the average number of molecules). Q1 represents the quantum yield of the first component, and Q2 represents the quantum yield of the second component. τ D1 represents the diffusion time (μs) of the first component, and τ D2 represents the diffusion time (μs) of the second component. ω is preferably assigned at the start of measurement based on the known diffusion time obtained from a mixture of 100 nM Alexa Fluor® 488 and 10 nM Alexa Fluor® 647 used as a reference.

[0044] The number of molecules in a component is obtained from the value of G(τ) when τ=0. The value of G(τ) when τ=0 is taken as the origin value, and the correlation time τ when the origin value becomes 1 / 2 represents the diffusion time. Specifically, when G(τ) when the correlation time τ is 0, i.e., G(0), is a, the diffusion time is intended to be the correlation time τ where G(τ) = a × 1 / 2. More specifically, when the value of G(0) in the correlation function G(τ) is converted to 1, the diffusion time is the correlation time τ where G(τ) = 0.5. Therefore, the correlation function G(τ) may be normalized so that the maximum value is "1" and the minimum value is "0". When normalizing the correlation function G(τ), the combination of the maximum and minimum values ​​can be set as appropriate. For example, the combination may be that the maximum value is "2" and the minimum value is "1", or the combination may be that the maximum value is "100" and the minimum value is "0".

[0045] To calculate the triplet state transition time, a fitted model may be used, as shown in Eq. 2 below. Eq. 2 shows the triplet state transition. In Eq. 2, T represents the triplet state amplitude. τ T This represents the triplet state decay time of the fluorescent dye fluorophore.

[0046]

number

[0047] For the autocorrelation function, a fitting model may be used, as shown in Eq. 3 below, which includes both the translational diffusion time and the triplet state transition time of the fluorescent dye (T×3D or T×{3D+3D}). In Eq. 3, i represents the number of independent components.

[0048]

number

[0049] Diffusion time is also a parameter that changes depending on the size of the fluorescently labeled molecule. Information regarding the size of a fluorescently labeled VWF may be obtained based on the diffusion time of the VWF. The value obtained based on the diffusion time of a fluorescently labeled VWF may be a value calculated using the diffusion time. For example, the diffusion time (τ D ) and the radius of the observation area (w xy The diffusion coefficient (D) is obtained from ) D = w xy 2 / 4τ D It is calculated from the following formula. And, assuming that the fluorescently labeled molecule is a spherical particle, the hydrodynamic radius (R) of the molecule is... H ) is R H = k B It is calculated from the formula T / 6πηD. Here, k B θ is the Boltzmann constant, T is the temperature (K), and η is the viscosity of the sample being measured (Pa·s). From these equations and the diffusion time of the fluorescently labeled VWF, the hydrodynamic radius can be obtained as information about the size of the VWF.

[0050] The value obtained based on the diffusion time of fluorescently labeled VWF may be the molecular size obtained by applying the diffusion time of the VWF to a calibration curve created from the diffusion times of VWF with known molecular sizes. The molecular size does not need to be a quantitative value such as molecular weight or volume, but may be a relative value. Here, it is known that VWF is sheared by blood flow and becomes fragments with a small molecular size. As shown in Example 4 below, for example, by stirring normal human plasma, multiple measurement samples containing VWF with different ratios of high molecular weight VWF multimers can be prepared depending on the stirring time. The ratio of high molecular weight VWF multimers can be obtained, for example, by examining the composition of VWF multimers in the stirred and unstirred samples using the SDS-gel electrophoresis method and Western blotting method using anti-VWF antibodies as described above. Specifically, the ratio of the amount of high molecular weight VWF multimers in the stirred sample to the amount of high molecular weight VWF multimers in the unstirred sample is calculated by analyzing the Western blot image. The analysis and calculation of ratios of such VWF multimers are publicly known, for example, as described in Boender J. et al., Hemasphere, 5(3):e542, 2021.

[0051] In a preferred embodiment, for several reference samples with known ratios of polymer VWF multimers, the same denaturation treatment with urea, fluorescent labeling with a fluorescently labeled trapper, and diffusion time acquisition using FCS or FCCS are performed as with biological samples. A calibration curve is then created by plotting the diffusion time against the ratio of polymer VWF multimers. The diffusion time for VWF in the biological sample is then acquired and applied to the calibration curve to obtain the ratio of polymer VWF multimers for the biological sample as information regarding the size of VWF.

[0052] The advantage of the information acquisition method of this embodiment, as described above, is that it can obtain information on the size of VWF while reducing the influence of the VWF concentration in the biological sample or measurement sample. A further advantage is that the time required to obtain results with the information acquisition method of this embodiment is shorter than that of the conventional method, which is a combination of SDS-gel electrophoresis and Western blotting. With SDS-gel electrophoresis and Western blotting, it usually takes one or two days to obtain information on the size of VWF. However, with the information acquisition method of this embodiment, results can be obtained in about 30 minutes to 1 hour.

[0053] A further embodiment of the present invention relates to a method for preparing a measurement sample used in measurements by FCS or FCCS (hereinafter also referred to as "the preparation method of this embodiment"). According to the preparation method of this embodiment, a measurement sample suitable for obtaining the diffusion time of fluorescently labeled VWF can be prepared from a biological sample containing VWF. Specifically, first, the VWF contained in the biological sample is denatured with urea. The details of this urea denaturation treatment are the same as those described in the information acquisition method of this embodiment.

[0054] Next, the denatured VWF is fluorescently labeled using a fluorescently labeled capture agent. Details of the fluorescently labeled capture agent and the fluorescent label are the same as those described in the information acquisition method of this embodiment. The types of capture agents are the same as in the information acquisition method of this embodiment, and include polyclonal antibodies, multiple monoclonal antibodies that bind to different epitopes, multiple aptamers that bind to different epitopes, and combinations thereof.

[0055] When preparing a sample for measurement by FCS, a capture body labeled with one color fluorescent substance is used. When preparing a sample for measurement by FCCS, a capture body labeled with each of two colors of fluorescent substances is used. That is, a first fluorescently labeled capture body and a second fluorescently labeled capture body are used as the fluorescently labeled capture bodies. As described above, it is preferable that the second fluorescent substance in the second fluorescently labeled capture body is a fluorescent substance that has a fluorescence emission maximum in a different wavelength range than the first fluorescent substance in the first fluorescently labeled capture body. Details of the first and second fluorescently labeled capture bodies and each fluorescent substance are the same as those described in the information acquisition method of this embodiment.

[0056] Further embodiments of the present invention relate to a reagent kit used in the information acquisition method and preparation method of this embodiment (hereinafter also referred to as the "reagent kit of this embodiment"). The reagent kit of this embodiment includes a first reagent containing urea and a second reagent containing a fluorescently labeled capture agent. Details of the fluorescently labeled capture agent are the same as those described in the information acquisition method of this embodiment.

[0057] The urea in the first reagent may be a solid or a solution. In a preferred embodiment, the first reagent preferably contains a urea solution. The solvent can be selected from the aqueous solvents described in the description of the information acquisition method in this embodiment. The concentration of urea in the first reagent is not particularly limited. For example, the concentration of urea in the first reagent may be such that the final concentration of urea in the mixture of the biological sample and the first reagent is 0.5 M or more and 1.75 M or less. Specifically, the concentration of urea in the first reagent may be 1 M or more and 8 M or less, preferably 1.5 M or more and 6 M or less, and more preferably 2 M or more and 4 M or less.

[0058] In the information acquisition method of this embodiment, when information regarding the size of VWF is acquired by FCS, or in the preparation method of this embodiment, when a measurement sample is prepared for measurement by FCS, the second reagent includes a capture body labeled with a single fluorescent substance. In the information acquisition method of this embodiment, when information regarding the size of VWF is acquired by FCCS, or in the preparation method of this embodiment, when a measurement sample is prepared for measurement by FCCS, the second reagent includes a first fluorescently labeled capture body and a second fluorescently labeled capture body. Alternatively, the first fluorescently labeled capture body and the second fluorescently labeled capture body may each be contained in separate reagents. In this case, the reagent kit of this embodiment includes a first reagent containing urea, a second reagent containing a first fluorescently labeled capture body, and a third reagent containing a second fluorescently labeled capture body. Details of the first and second fluorescently labeled capture bodies and each fluorescent substance are the same as those described in the information acquisition method of this embodiment.

[0059] The fluorescently labeled capture agent in the second reagent may be a solid (e.g., powder, crystal, lyophilized product, etc.) or a liquid (e.g., solution, suspension, emulsion, etc.). In a preferred embodiment, the second reagent preferably contains a solution of the fluorescently labeled capture agent. The solvent can be selected from the aqueous solvents described in the description of the information acquisition method in this embodiment. If necessary, stabilizers such as bovine serum albumin (BSA) and casein may be added to the aqueous medium. The concentration of the fluorescently labeled capture agent in the second reagent is not particularly limited. If the fluorescently labeled capture agent is an antibody, the concentration of the fluorescently labeled capture agent in the second reagent should be such that the final concentration of the antibody in the measurement sample is, for example, 1 nM or more and 100 nM or less. Specifically, the concentration of the fluorescently labeled capture agent in the second reagent may be 2 nM or more and 200 nM or less, preferably 5 nM or more and 150 nM or less, more preferably 10 nM or more and 100 nM or less. If the first and second fluorescently labeled capture agents are antibodies, the concentrations of each antibody in the reagent should be within the above range.

[0060] The reagent kit of this embodiment may be provided to the user in a box containing containers for each reagent. An accompanying document may be included in the box. The accompanying document may describe the composition of each reagent, how to use each reagent, how to store each reagent, etc. An example of the reagent kit of this embodiment is shown in Figure 1A. Referring to Figure 1A, 11 shows the reagent kit of this embodiment, 12 shows the first container containing the first reagent containing urea, 13 shows the second container containing the second reagent containing a fluorescently labeled trap, 14 shows the packaging box, and 15 shows the accompanying document. The second container 13 may contain the second reagent containing the first fluorescently labeled trap and the second fluorescently labeled trap.

[0061] Figure 1B shows an example of a reagent kit of this embodiment in which the first fluorescently labeled capture agent and the second fluorescently labeled capture agent are contained in the second reagent and the third reagent, respectively. Referring to Figure 1B, 21 shows the reagent kit of this embodiment, 22 shows the first container containing the first reagent containing urea, 23 shows the second container containing the second reagent containing the first fluorescently labeled capture agent, 24 shows the second container containing the third reagent containing the second fluorescently labeled capture agent, 25 shows the packaging box, and 26 shows the accompanying documentation.

[0062] Further embodiments relate to the use of a capture agent comprising urea and a fluorescent substance that binds to VWF denatured with urea. These substances are used in the manufacture of reagent kits for obtaining information on the size of VWF by FCS or for preparing a sample for measurement. The capture agent comprises a polyclonal antibody or multiple monoclonal antibodies or aptamers that bind to different epitopes. Details of the urea denaturation, fluorescent substance and capture agent are the same as those described in the information acquisition method of this embodiment.

[0063] Further embodiments relate to the use of a capture agent comprising urea, a first fluorescent substance, and a urea-denatured VWF, and a second fluorescent substance and a urea-denatured VWF. These substances are used in the manufacture of reagent kits for obtaining information on VWF size by FCCS or for preparing measurement samples. The capture agent comprising the first fluorescent substance and a urea-denatured VWF comprises a polyclonal antibody, or a plurality of monoclonal antibodies or aptamers that bind to different epitopes. Details of the urea denaturation, each fluorescent substance, and the capture agent are the same as those described in the information acquisition method of this embodiment.

[0064] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0065] Reference example When multiple samples containing VWF at different concentrations were measured by FCCS, we investigated whether there were differences in the diffusion time of VWF obtained from each sample. In this reference example, we compared the measurement of VWF directly labeled with a fluorescent dye with the measurement of VWF indirectly labeled with a fluorescently labeled antibody.

[0066] (1) Biological samples Human plasma-derived VWF protein (Merck) was labeled with Alexa Fluor® 488 and Alexa Fluor® 647 (Thermo Fisher Scientific) by amine coupling to obtain VWF covalently bonded to the two fluorescent dyes (hereinafter referred to as "488-VWF-647"). 488-VWF-647 was added to 1% (w / v) BSA-containing PBS (pH 7.4) (hereinafter referred to as "1% BSA-PBS") to a VWF concentration of 25 nM to prepare a 488-VWF-647 solution. Additionally, human plasma-derived VWF protein was added to 1% BSA-PBS to a concentration of 25 nM to prepare a VWF solution.

[0067] (2) Reagents containing fluorescently labeled antibodies NMC4 Fab and 2F2A9 antibody (BD Biosciences) were used as monoclonal antibodies that bind to VWF. These antibodies bind to different epitopes. NMC4 Fab was prepared using a known recombinant DNA method based on its publicly available amino acid sequence. Specifically, NMC4 light and heavy chains were first expressed in Expi293 cells, and then the culture supernatant was collected. The NMC4 Fab in this culture supernatant was purified by gel filtration and concentrated using a centrifugal concentrator. The obtained NMC4 Fab was labeled with Alexa Fluor® 647 by amine coupling to obtain Alexa Fluor 647-labeled NMC4 Fab (hereinafter referred to as "NMC4-647"). The 2F2A9 antibody was labeled with Alexa Fluor® 488 by the maleimide method, and the unbound fluorescent dye was removed by desalting column to obtain the Alexa Fluor 488-labeled 2F2A9 antibody (hereinafter referred to as "2F2A9-488"). Each antibody was added to 1% BSA-PBS to prepare Reagent 1 containing 2F2A9-488 (50 nM) and NMC4-647 (25 nM).

[0068] (3) Preparation of the sample (3.1) Sample containing 488-VWF-647 A 2-fold diluted sample was prepared by mixing 488-VWF-647 solution (50 μL) with 1% BSA-PBS (50 μL). A 10-fold diluted sample was prepared by mixing 488-VWF-647 solution (10 μL) with 1% BSA-PBS (90 μL). A 20-fold diluted sample was prepared by mixing 488-VWF-647 solution (5 μL) with 1% BSA-PBS (95 μL).

[0069] (3.2) Measurement samples containing VWF A 2-fold diluted sample was prepared by mixing VWF solution (50 μL) and reagent 1 (50 μL) and incubating at 37°C for 5 minutes. A 10-fold diluted sample was prepared by mixing VWF solution (10 μL), 1% BSA-PBS (40 μL), and reagent 1 (50 μL) and incubating at 37°C for 5 minutes. A 20-fold diluted sample was prepared by mixing VWF solution (5 μL), 1% BSA-PBS (45 μL), and reagent 1 (50 μL) and incubating at room temperature in the dark for 5 minutes.

[0070] (4) Measurement and analysis Each sample (30 μL) was added to a 384-well glass-bottom plate (Sigma-Aldrich) and measured using a Carl Zeiss LSM710 confocal laser microscope (Zeiss). Measurements were performed triplicately for each sample. Alexa Fluor® 488 was excited with a 488 nm laser, and Alexa Fluor® 647 was excited with a 639 nm laser. The output power of the 488 nm and 639 nm lasers was 1–10 μW. For each well, fluorescence intensity (kHz) was measured continuously with a measurement time of 10–15 seconds. 10–15 measurements were performed for each well. The mean correlation function of these 10–15 measurements was fitted. Analysis was performed using Zen software (Zeiss). First, the acquired raw data was processed to remove measurements showing baseline drift or bursts (fluorescence signals more than three times higher than the average fluorescence intensity). The cross-correlation curves used in FCCS were fitted using a one-component three-dimensional translational diffusion (3D) model. In fitting the cross-correlation function with the one-component model, the one component was the immunocomplex between the first and second fluorescently labeled antibodies and VWF. The one-component model was selected in Zen software. Then, for each sample, the diffusion time of the immunocomplex was obtained based on the signals of the first and second fluorescent substances.

[0071] (5) Results Figure 2A shows the diffusion time of each sample containing 488-VWF-647. Figure 2B shows the diffusion time of each sample containing VWF. As shown in Figure 2A, the diffusion time obtained from the sample containing 488-VWF-647 was almost constant regardless of the dilution ratio of the biological sample. This indicates that when VWF was directly labeled with a fluorescent dye, the molecular size of VWF obtained by FCCS was independent of the VWF concentration. On the other hand, as shown in Figure 2B, the diffusion time obtained from the sample containing VWF decreased as the dilution ratio of the biological sample increased. This indicates that when VWF was indirectly labeled with a fluorescently labeled antibody, the molecular size of VWF obtained by FCCS was dependent on the VWF concentration.

[0072] Regarding the above results, the inventors considered that the cause was insufficient exposure of the epitopes in undenatured VWF and a small number of fluorescently labeled antibodies bound to VWF. Insufficient exposure of the epitopes can affect the number of antibodies bound to VWF. Furthermore, a small number of antibodies bound to VWF can significantly affect the molecular size due to antibody dissociation from VWF. The inventors hypothesized that the lower the concentration of VWF in the measurement sample, the higher the frequency of antibody dissociation from VWF, thus decreasing the average size of the antibody-VWF complex. The inventors considered that in order to reduce the effect of VWF concentration, i.e., the effect of dissociation between VWF and antibodies, it is necessary to increase the number of antibodies bound per VWF molecule. The inventors considered denaturing VWF with a protein denaturant and changing the type of fluorescently labeled antibody.

[0073] Example 1 We investigated whether denaturation treatment of VWF and modification of the fluorescently labeled antibody could reduce the effect of VWF concentration.

[0074] (1) Biological samples Standard human plasma for coagulation testing (Sysmex Corporation) was used.

[0075] (2) Reagents (2.1) Denaturants A 3M urea solution was prepared by dissolving urea (9 g, Fujifilm Wako Pure Chemical Industries, Ltd.) in 1% BSA-PBS to make a 50 mL solution.

[0076] (2.2) Fluorescent substances As the first fluorescent agent, Alexa Fluor® 488 (Thermo Fisher Scientific) was used. As the second fluorescent agent, Alexa Fluor® 647 (Thermo Fisher Scientific) was used. Hereafter, the antibody labeled with Alexa Fluor® 488 will also be referred to as the "first fluorescently labeled antibody," and the antibody labeled with Alexa Fluor® 647 will also be referred to as the "second fluorescently labeled antibody."

[0077] (2.3) Reagents containing fluorescently labeled antibodies (i) Preparation of fluorescently labeled monoclonal antibodies In addition to the NMC4 Fab and 2F2A9 antibodies used in the above reference example, VWF635 antibody (Novus Biologicals) and SPM577 antibody (Novus Biologicals) were used as monoclonal antibodies that conjugate to VWF. The VWF635 antibody and SPM577 antibody conjugate to different epitopes. NMC4-647 and 2F2A9-488 were prepared from NMC4 Fab and 2F2A9 antibodies, respectively, in the same manner as in the above reference example. The VWF635 antibody and SPM577 antibody were each labeled with Alexa Fluor® 488 by amine coupling to obtain Alexa Fluor 488-labeled VWF635 antibody (hereinafter referred to as "VWF-488") and Alexa Fluor 488-labeled SPM577 antibody (hereinafter referred to as "SPM-488").

[0078] (ii) Preparation of fluorescently labeled polyclonal antibodies Polyclonal anti VWF IgG (Dako A / S) was used as the polyclonal antibody that binds to VWF. This polyclonal antibody was fragmented by pepsin digestion and purified by gel filtration to obtain Polyclonal anti VWF F(ab')2. The obtained F(ab')2 was labeled with Alexa Fluor® 488 or Alexa Fluor® 647 by amine coupling to obtain Alexa Fluor 488-labeled Polyclonal anti VWF F(ab')2 (hereinafter referred to as "DakoF-488") and Alexa Fluor 647-labeled Polyclonal anti VWF F(ab')2 (hereinafter referred to as "DakoF-647").

[0079] (iii) Preparation of reagents containing fluorescently labeled antibodies Reagents 1-3 were prepared by combining the fluorescently labeled antibodies described above. Reagent 1 was the same reagent as in the reference example above, namely containing 2F2A9-488 (50 nM) and NMC4-647 (25 nM). Reagent 2 was the same reagent as in the reference example above, namely containing DakoF-488 (50 nM) and NMC4-647 (25 nM). Reagent 3 was the same reagent as containing VWF-488 (25 nM), SPM-488 (25 nM), and NMC4-647 (25 nM). The composition of each reagent is shown in Table 1. In the table, "488-labeled antibody" and "647-labeled antibody" refer to antibodies labeled with Alexa Fluor® 488 and Alexa Fluor® 647, respectively. In the table, the concentrations of each antibody indicate the final concentrations in the reagent. 1% BSA-PBS was used as the solvent for reagents 1-3.

[0080] [Table 1]

[0081] (3) Preparation of the sample Multiple measurement samples with different VWF concentrations were prepared by diluting the biological sample. A 2x diluted measurement sample was prepared as follows: The biological sample (17 μL) was mixed with 1% BSA-PBS (17 μL) to dilute it 2x. 3M urea solution (17 μL) or 1% BSA-PBS (17 μL) was added to the diluted biological sample and incubated at 37°C for 15 minutes. At this time, the final urea concentration in the sample to which urea solution was added was 1 M. Then, reagent 1 (50 μL), reagent 2 (50 μL), or reagent 3 (50 μL) was added and incubated in the dark at room temperature for 5 minutes to obtain the measurement sample. A 10x diluted measurement sample was prepared in the same manner as the 2x diluted measurement sample, except that it was a mixture of biological sample (3.4 μL) and 1% BSA-PBS (30.6 μL). The final urea concentration in the sample to which urea solution was added was 0.495 M.

[0082] (4) Measurement and analysis Each sample (30 μL) was added to a 384-well glass-bottom plate (Sigma-Aldrich) and measured using a Carl Zeiss LSM710 confocal laser microscope (Zeiss). Alexa Fluor® 488 was excited with a 488 nm laser, and Alexa Fluor® 647 was excited with a 639 nm laser. The output power of the 488 nm and 639 nm lasers was 1–10 μW. For each well, fluorescence intensity (kHz) was continuously measured with a measurement time of 10–15 seconds. Measurements were performed 10–15 times. The mean correlation function of these 10–15 measurements was fitted. Analysis was performed using Zen software (Zeiss). First, the acquired raw data was processed to remove measurements showing baseline drift or bursts (fluorescence signals more than 3 times higher than the average fluorescence intensity). The cross-correlation curve used for FCCS was fitted using a one-component three-dimensional translational-diffusion model (3D). Furthermore, the autocorrelation curves used in FCS were fitted using a one-component or two-component three-dimensional translational diffusion + triplet state model (T×3D or T×{3D+3D}).

[0083] (i) Obtaining diffusion time using FCS In fitting the autocorrelation function using a one-component model, the one component was the immunocomplex of the first or second fluorescently labeled antibody with VWF. A one-component model was selected in Zen software. For each sample, the diffusion time of the immunocomplex was obtained based on the signal of the first or second fluorescent substance.

[0084] In fitting the autocorrelation function in the two-component model, the first component was the unreacted first or second fluorescently labeled antibody. The second component was the immunoconjugate of the first or second fluorescently labeled antibody with VWF. First, a one-component model was selected in Zen software. Then, each fluorescently labeled antibody was added to a sample without VWF (a mixture of 1% BSA-PBS (34 μL) and 3M urea solution (17 μL)), and the diffusion time of the first component was obtained (Tp 488 or Tp 647 Next, a two-component model was selected in Zen software. The diffusion time of the first component was the diffusion time of the first component measured earlier (Tp 488 or Tp 647 The sample was fixed in place. For each sample, the diffusion time of the second component was obtained based on the signal of the first or second fluorescent substance.

[0085] (ii) Obtaining diffusion time using FCCS In fitting the cross-correlation function using a one-component model, the one component was the immune complex between the first and second fluorescently labeled antibodies and VWF. A one-component model was selected in Zen software. For each sample, the diffusion time of the immune complex was obtained based on the signals of the first and second fluorescent substances.

[0086] (5) Results The diffusion times obtained by FCS or FCCS for each sample are shown in Figures 3A, 3B, and 4-8. In the figures, "PBS" represents the sample to which 1% BSA-PBS was added instead of 3M urea solution. "1M urea" represents the sample to which 3M urea solution was added. 1M was the final concentration of urea when 3M urea solution was added to the diluted biological sample. Table 2 shows the correspondence between these figures and the reagents used, measurement wavelengths, detected fluorescently labeled antibodies, and measurement methods.

[0087] [Table 2]

[0088] Figures 3A and 3B show the diffusion times obtained by FCS (measurement wavelength 488 nm or 647 nm) using Reagent 1. Reagent 1 contains two types of fluorescently labeled antibodies, 2F2A9-488 and NMC4-647, but the measurement wavelength was either 488 nm or 647 nm, so it was essentially the same as FCS using one type of fluorescently labeled antibody. As shown in Figures 3A and 3B, the diffusion time of the 10-fold diluted sample was shorter than that of the 2-fold diluted sample, regardless of whether or not urea denaturation treatment was performed. Reagent 1 was the same reagent used in the reference example above. This suggests that urea denaturation treatment alone cannot reduce the influence of the VWF concentration in the sample on the FCS measurement results.

[0089] Figures 4 and 5 show the diffusion times obtained by FCS (measurement wavelength 488 nm) using reagent 2 or reagent 3. Reagent 2 contains a fluorescently labeled antibody (DakoF-488) derived from a polyclonal antibody, and reagent 3 contains fluorescently labeled antibodies (VWF-488 and SPM-488) derived from two types of monoclonal antibodies that bind to different epitopes. Therefore, it was expected that the number of antibodies bound per VWF molecule would increase compared to when reagent 1 was used. As shown in Figures 4 and 5, when denaturation treatment with urea was performed, there was almost no difference in diffusion time between the 2-fold diluted measurement sample and the 10-fold diluted measurement sample. These results suggest that the influence of the VWF concentration in the measurement sample can be reduced by denaturing VWF in the biological sample with urea and by labeling polyclonal antibodies or two or more monoclonal antibodies that bind to different epitopes with a single fluorescent substance and using them in FCS. The denaturing effect of urea extends not only to VWF but also to fluorescently labeled antibodies. However, the results above suggest that a final urea concentration of approximately 0.5 M in the sample does not affect the measurement.

[0090] Figure 6 shows the diffusion times obtained by FCCS (measurement wavelengths 488 nm and 647 nm) using reagent 1. As shown in Figure 6, when urea denaturation treatment was not performed, the diffusion time of the 10-fold diluted sample was shorter than that of the 2-fold diluted sample. When denaturation treatment was performed, the difference in diffusion times between samples with different dilution ratios tended to be smaller compared to when no denaturation treatment was performed. These results suggest that urea denaturation treatment alone is not sufficient to reduce the influence of VWF concentration in the sample on the FCCS measurement results.

[0091] Figures 7 and 8 show the diffusion times obtained by FCCS using reagent 2 or reagent 3. As shown in Figures 7 and 8, when denatured with urea, there was almost no difference in diffusion time between the 2-fold diluted sample and the 10-fold diluted sample. Two types of fluorescent substances are used in FCCS. These results suggest that using one of the two fluorescent substances to fluorescently label a polyclonal antibody or two or more monoclonal antibodies that bind to different epitopes in FCCS is useful in reducing the influence of VWF concentration. It was also suggested that denaturing VWF with urea is useful. The above results also suggest that a final urea concentration of approximately 0.5 M in the sample does not affect the measurement.

[0092] Example 2 The concentration of urea used to denature VWF in biological samples was investigated. In Example 2, diffusion time was obtained by FCCS using a combination of polyclonal antibodies labeled with two different fluorescent substances.

[0093] (1) Biological samples and reagents As the biological sample, the same standard human plasma for coagulation testing as in Example 1 was used. As the denaturing agent, the same 3M urea solution as in Example 1 was used. In addition, urea (4.5 g or 15.75 g, Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 1% BSA-PBS to make 50 mL of solution to prepare 1.5M and 5.25M urea solutions. As the fluorescently labeled antibodies, DakoF-488 and DakoF-647 prepared in Example 1 were used. Reagent 4, containing these fluorescently labeled antibodies, was prepared, containing DakoF-488 (50 nM) and DakoF-647 (25 nM). 1% BSA-PBS was used as the solvent for Reagent 4.

[0094] (2) Preparation of the sample Multiple measurement samples with different concentrations of VWF and urea were prepared. A 2x diluted measurement sample was prepared as follows: The biological sample (17 μL) was mixed with 1% BSA-PBS (17 μL) to dilute it 2x. 1.5 M, 3 M, or 5.25 M urea solution (17 μL) was added to the diluted biological sample and incubated at 37°C for 15 minutes. At this time, the final urea concentration was 0.5 M, 1 M, or 1.75 M. Then, reagent 4 (50 μL) was added and incubated in the dark at room temperature for 5 minutes to obtain the measurement sample. A 10x diluted measurement sample was prepared in the same manner as the 2x diluted sample, except that it was a mixture of biological sample (3.4 μL) and 1% BSA-PBS (30.6 μL). The final urea concentration in the measurement sample was 0.224 M, 0.495 M, or 1.085 M.

[0095] (3) Measurement and analysis Diffusion time was obtained by FCCS using an LSM710 confocal laser microscope (Zeiss) and Zen software (Zeiss) in the same manner as in Example 1. The results are shown in Figure 9. The urea concentration in the figure is the final urea concentration when a 3M urea solution was added to the diluted biological sample. Table 3 shows the correspondence between Figure 9 and the reagents used, measurement wavelength, detected fluorescently labeled antibody, and measurement method.

[0096] [Table 3]

[0097] (4) Results As shown in Figure 9, no significant difference in diffusion time was observed between the 2-fold diluted and 10-fold diluted samples when denaturation treatment was performed with urea at concentrations of 0.5 M, 1 M, and 1.75 M. This result suggests that denaturation treatment of biological samples can be performed in the presence of urea at concentrations between 0.5 M and 1.75 M. Furthermore, it was suggested that using a combination of polyclonal antibodies labeled with two different fluorescent substances in FCCS is useful in reducing the influence of VWF concentration.

[0098] Example 3 We investigated the mixing ratio of polyclonal antibodies labeled with two different fluorescent substances.

[0099] (1) Biological samples and reagents The same standard human plasma and 3M urea solution for coagulation testing used in Example 1 were used as the biological sample and denaturing agent. DakoF-488 and DakoF-647, prepared in Example 1, were used as the fluorescently labeled antibodies. Reagents 5-9 were prepared containing these fluorescently labeled antibodies. The composition of each reagent is shown in Table 4. In the table, the concentration of each antibody indicates the final concentration in the reagent. 1% BSA-PBS was used as the solvent for reagents 5-9.

[0100] [Table 4]

[0101] (2) Preparation of the sample A 2x diluted sample was prepared as follows: The biological sample (17 μL) was mixed with 1% BSA-PBS (17 μL) to dilute the biological sample 2-fold. 3M urea solution (17 μL) was added to the diluted biological sample and incubated at 37°C for 15 minutes. At this time, the final concentration of urea was 1 M. Then, 50 μL of reagent 5, reagent 6, reagent 7, reagent 8, or reagent 9 was added and incubated in the dark at room temperature for 5 minutes to obtain the sample. A 10x diluted sample was prepared in the same manner as the 2x diluted sample, except that the biological sample (3.4 μL) was mixed with 1% BSA-PBS (30.6 μL). The final concentration of urea in the sample was 0.495 M.

[0102] (3) Measurement and analysis Diffusion time was obtained by FCCS using an LSM710 confocal laser microscope (Zeiss) and Zen software (Zeiss) in the same manner as in Example 1. The results are shown in Figure 10. The molar ratio of the fluorescently labeled antibody in the figure is the ratio of the final concentration of DakoF-488 to the final concentration of DakoF-647 in each reagent. Table 5 shows the correspondence between Figure 10 and the reagents used, measurement wavelength, detected fluorescently labeled antibody, and measurement method.

[0103] [Table 5]

[0104] (4) Results As shown in Figure 10, regardless of the molar ratio of DakoF-488 to DakoF-647 in the reagent, no significant difference in diffusion time was observed between the 2-fold diluted sample and the 10-fold diluted sample. This result suggests that the ratio of the concentration of the polyclonal antibody containing the first fluorescent substance to the concentration of the polyclonal antibody containing the second fluorescent substance in the reagent should be between 0.5 and 4 in molar ratio.

[0105] Example 4 In Example 4, standard human plasma for coagulation testing was stirred to prepare multiple measurement samples containing VWF of different molecular sizes. Each measurement sample was then measured using FCS or FCCS to investigate whether diffusion times corresponding to molecular size could be obtained.

[0106] (1) Biological samples Standard human plasma for coagulation testing (Sysmex Corporation) was used as the standard plasma sample. The standard plasma sample was divided into four aliquots. Three of the four aliquots were stirred in a vortex mixer for 10 minutes, 60 minutes, or 120 minutes, after which EDTA was added to a final concentration of 10 μg / mL. Analysis of the molecular size of VWF contained in each sample was commissioned to SRI Corporation. In this analysis, the composition of VWF multimers was examined by SDS-agarose gel electrophoresis and Western blotting using anti-VWF antibody. Figure 11 shows images of the Western blots for each sample. In the figure, "standard" refers to the standard plasma sample. As shown in Figure 11, it was shown that the molecular size of VWF decreased with increasing stirring time. That is, stirring prepared plasma samples containing VWF with different molecular sizes. In these Western blot images, the VWF bands were classified into four fractions according to their molecular size: Large, Medium, Small, and Smallest. Using ImageJ software (provided by NIH), band concentrations in images of each plasma sample were analyzed using densitometry. For each plasma sample, the ratio (L%) of the band concentration of the Large fraction to the total band concentration of all fractions was calculated. Using the L% values ​​for the standard plasma sample and each agitated sample, the LMW index (%) was calculated according to the following formula. For the analysis of VWF multimers based on Western blot images, refer to Boender J. et al., Hemasphere, 5(3):e542, 2021.

[0107] LMW index = [(L% value of stirred sample) / (L% value of standard plasma sample)] × 100

[0108] Based on the LMW index calculated for each sample, the standard plasma sample will be referred to as "100% SHP," a plasma sample stirred for 10 minutes as "75% SHP," a plasma sample stirred for 60 minutes as "50% SHP," and a plasma sample stirred for 120 minutes as "25% SHP."

[0109] (2) Reagents The same 3M urea solution as in Example 1 was used as the denaturing agent. Reagent 4, the same reagent as in Example 2, was used as the reagent containing the fluorescently labeled antibody.

[0110] (3) Preparation of the sample Diluted samples were prepared 10-fold as follows: 100% SHP (4 μL), 75% SHP (4 μL), 50% SHP (4 μL), or 25% SHP (4 μL) were mixed with 1% BSA-PBS (36 μL) to dilute each sample 10-fold. 3M urea solution (20 μL) was added to each diluted sample and incubated at 37°C for 15 minutes. At this time, the final urea concentration was 1 M. Then, reagent 4 (40 μL) was added and incubated in the dark at room temperature for 5 minutes to obtain the measurement sample. The final urea concentration in the measurement sample was 0.6 M.

[0111] (4) Measurement and analysis Diffusion time was acquired by FCS or FCCS using an LSM710 confocal laser microscope (Zeiss) and Zen software (Zeiss) in the same manner as in Example 1. Graphs plotting the diffusion time of each sample are shown in Figures 12A-C. Table 6 shows the correspondence between these figures, the reagents used, the measurement wavelength, the detected fluorescently labeled antibody, and the measurement method.

[0112] [Table 6]

[0113] (5) Results As shown in Figures 12A-C, in all measurements, the diffusion time increased as the LMW index increased. Furthermore, as shown in Figures 12A-C, the coefficient of determination was 0.9 or higher in all regression equations. These results suggest that the diffusion time corresponding to the molecular size of VWF can be obtained by measuring a sample containing urea-denatured VWF using FCS or FCCS with a fluorescently labeled polyclonal antibody.

[0114] Example 5 Dilution samples from 100% SHP and 50% SHP of Example 4, diluted 2 to 32 times, were measured by FCS and FCCS to investigate whether the effect of VWF concentration was reduced by urea denaturation treatment and the use of fluorescently labeled polyclonal antibodies.

[0115] (1) Biological samples and reagents As biological samples, the same 100% SHP and 50% SHP as in Example 4 were used. As a denaturing agent, the same 3M urea solution as in Example 1 was used. As reagents containing fluorescently labeled antibodies, the same reagents 1 and 2 as in Example 1 were used.

[0116] (2) Preparation of the sample A plasma sample with a 2-fold dilution was prepared by mixing 100% SHP (40 μL) and 1% BSA-PBS (40 μL). 40 μL was taken from the diluted plasma sample, and this sample (40 μL) was mixed with 1% BSA-PBS (40 μL) to prepare a plasma sample with a 4-fold dilution. The same procedure was repeated to prepare plasma samples with 8-fold, 16-fold, and 32-fold dilutions. The same dilution procedure was also performed on 50% SHP to prepare plasma samples with 2-fold, 4-fold, 8-fold, 16-fold, and 32-fold dilutions. 3M urea solution (17 μL) was added to each plasma sample (34 μL) obtained by dilution, and the mixture was incubated at 37°C for 15 minutes. At this time, the final urea concentration was 1 M. Then, reagent 1 (40 μL) or reagent 2 (40 μL) was added, and the mixture was incubated in the dark at room temperature for 5 minutes to obtain 2x, 4x, 8x, 16x, and 32x diluted samples. The final concentration of urea in the samples was 0.56 M.

[0117] (3) Measurement and analysis Diffusion time was acquired by FCS or FCCS using an LSM710 confocal laser microscope (Zeiss) and Zen software (Zeiss) in the same manner as in Example 1. Graphs plotting the diffusion time of each sample are shown in Figures 13A-C, 14, and 15. Table 7 shows the correspondence between these figures, the reagents used, the measurement wavelength, the detected fluorescently labeled antibody, and the measurement method.

[0118] [Table 7]

[0119] (4) Results As shown in Figures 13A-C, in FCS and FCCS using Reagent 1, the diffusion time decreased as the dilution ratio increased. This indicates that the low VWF concentration in the sample affected the FCS and FCCS measurements. Furthermore, as the dilution ratio increased, the difference in diffusion time between the sample derived from 100% SHP and the sample derived from 50% SHP decreased. This indicates that the size resolution of FCS and FCCS decreased as the VWF concentration in the sample decreased. Therefore, it is suggested that the molecular size information obtained from FCS and FCCS using a fluorescently labeled monoclonal antibody such as Reagent 1 may not accurately reflect the molecular size of VWF.

[0120] As shown in Figure 14, in FCS using reagent 2, there was almost no decrease in diffusion time even at high dilution ratios. As shown in Figure 15, in FCCS using reagent 2, the decrease in diffusion time due to increasing dilution ratio was significantly reduced. Furthermore, as shown in Figures 14 and 15, the difference in diffusion time between the measurement sample derived from 100% SHP and the measurement sample derived from 50% SHP was maintained at all dilution ratios. Therefore, it was suggested that the molecular size information obtained by FCS and FCCS using a fluorescently labeled polyclonal antibody such as reagent 2 accurately reflects the molecular size of VWF. [Explanation of Symbols]

[0121] 11, 21: Reagent kit 12, 22: 1st container 13, 23: 2nd container 14, 25: Packaging box 15, 26: Attached document 24: Third container

Claims

1. The process involves denaturing von Willebrand factor (VWF) contained in a biological sample with urea, A step of fluorescently labeling the modified VWF using a capture body containing a fluorescent substance and binding to the modified VWF, A step of obtaining information regarding the size of the fluorescently labeled VWF by fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy, Includes, When the aforementioned information is obtained by fluorescence correlation spectroscopy, the capture body includes a polyclonal antibody, or a plurality of monoclonal antibodies or aptamers that bind to different epitopes. When the aforementioned information is obtained by fluorescence cross-correlation spectroscopy, the capture body includes a capture body containing a first fluorescent substance and binding to the modified VWF, and a capture body containing a second fluorescent substance and binding to the modified VWF, The second fluorescent substance is a fluorescent substance having a fluorescence emission maximum in a different wavelength range than the first fluorescent substance. A method for obtaining information on von Willebrand factor, wherein the capture body containing the first fluorescent substance and binding to the denatured VWF is a polyclonal antibody containing the first fluorescent substance, or a plurality of monoclonal antibodies or aptamers, each containing the first fluorescent substance and binding to different epitopes.

2. The method according to claim 1, wherein the step of obtaining the information includes obtaining the diffusion time of the fluorescently labeled VWF by fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy.

3. The method according to claim 2, wherein the information is the diffusion time, or a value obtained based on the diffusion time.

4. The method according to claim 1, wherein, when the information is obtained by fluorescence cross-correlation spectroscopy, the capture body comprising the second fluorescent substance and bound to the denatured VWF is a polyclonal antibody comprising the second fluorescent substance, or a plurality of monoclonal antibodies or aptamers, each comprising the second fluorescent substance and bound to different epitopes.

5. The method according to claim 1, wherein the modification process is carried out in the presence of urea at a concentration of 0.5 M or more and 1.75 M or less.

6. The method according to claim 1, wherein the fluorescent labeling step is performed in the presence of urea at a concentration of 0.2 M or more and 1 M or less.

7. A method for preparing a sample for use in fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy, The process involves denaturing von Willebrand factor (VWF) contained in a biological sample with urea, A step of fluorescently labeling the modified VWF using a capture body containing a fluorescent substance and binding to the modified VWF, Includes, When VWF fluorescently labeled with the aforementioned capture agent is used for measurement by fluorescence correlation spectroscopy, the capture agent comprises a polyclonal antibody, or a plurality of monoclonal antibodies or aptamers that bind to different epitopes. When VWF fluorescently labeled with the aforementioned capture body is used for measurement by fluorescence cross-correlation spectroscopy, the capture body includes a capture body containing a first fluorescent substance and binding to the modified VWF, and a capture body containing a second fluorescent substance and binding to the modified VWF, The second fluorescent substance is a fluorescent substance having a fluorescence emission maximum in a different wavelength range than the first fluorescent substance. The capture body containing the first fluorescent substance and binding to the denatured VWF is a polyclonal antibody containing the first fluorescent substance, or a plurality of monoclonal antibodies or aptamers, each containing the first fluorescent substance and binding to different epitopes. Method for preparing the measurement sample.

8. The method according to claim 7, wherein the capture body containing the second fluorescent substance and binding to the denatured VWF is a polyclonal antibody containing the second fluorescent substance, or a plurality of monoclonal antibodies or aptamers, each containing the second fluorescent substance and binding to different epitopes.

9. The method according to claim 7, wherein the modification step is carried out in the presence of urea at a concentration of 0.5 M or more and 1.75 M or less.

10. The method according to claim 7, wherein the fluorescent labeling step is performed in the presence of urea at a concentration of 0.2 M or more and 1 M or less.

11. A reagent kit for use in the method of any one of claims 1 to 10, comprising urea and a capture agent containing a fluorescent substance and binding to von Willebrand factor (VWF) denatured with the urea, wherein the capture agent comprises a polyclonal antibody or a plurality of monoclonal antibodies or aptamers that bind to different epitopes.

12. The urea is contained in the urea reagent solution. The reagent kit according to claim 11, wherein the urea concentration in the urea reagent solution is 1 M or more and 8 M or less.

13. A reagent kit for use in the method of any one of claims 1 to 10 by fluorescence cross-correlation spectroscopy, comprising urea, a capture agent containing a first fluorescent substance and binding to von Willebrand factor (VWF) denatured with the urea, and a capture agent containing a second fluorescent substance and binding to VWF denatured with the urea, wherein the second fluorescent substance is a fluorescent substance having maximum absorption in a wavelength range different from that of the first fluorescent substance, and the capture agent containing the first fluorescent substance and binding to VWF denatured with the urea is a polyclonal antibody containing the first fluorescent substance, or a plurality of monoclonal antibodies or aptamers, each containing the first fluorescent substance and binding to different epitopes.

14. The reagent kit according to claim 13, wherein the capture body containing the second fluorescent substance and binding to VWF denatured with urea is a polyclonal antibody containing the second fluorescent substance, or a plurality of monoclonal antibodies or aptamers, each containing the second fluorescent substance and binding to different epitopes.

15. The urea is contained in the urea reagent solution. The reagent kit according to claim 13, wherein the urea concentration in the urea reagent solution is 1 M or more and 8 M or less.

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