Method for obtaining information on target polypeptide and reagent kit

Abstract

To provide a method for acquiring information regarding a size of a target polypeptide more accurately, and a reagent kit.SOLUTION: An information acquisition method for target polypeptide includes the processes of: acquiring, by fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy, a diffusion time of a fluorescent labeled target polypeptide and diffusion times of a plurality of fluorescent labeled reference polypeptides; and acquiring information regarding a size of the fluorescent labeled target polypeptide based upon the diffusion times of the plurality of fluorescent labeled reference polypeptides, wherein information on respective sizes of the plurality of fluorescent labeled reference polypeptides is already known, and the plurality of reference polypeptides differ in size from one another.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for obtaining information on a target polypeptide and a reagent kit. [Background technology]

[0002] Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are known as analytical methods for obtaining information about the size of polypeptides. Non-Patent Document 1 describes structural changes due to protein inactivation using FCS. This document describes that the size of a sample protein is estimated by proportional calculation using the diffusion time obtained by FCS for a dye with a known diffusion coefficient and a sample protein. Non-Patent Document 2 describes that a standard sample containing dye-labeled polystyrene beads of different sizes is used to determine the diffusion time of polystyrene beads of each size by fluorescence correlation spectroscopy, and a calibration curve is created. This document describes that the diffusion time obtained by FCS for the target protein is applied to the calibration curve to calculate the size of the target protein. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Eilon Sherman, Biophysical Journal Volume 94, June 2008, 4819-4827, “Using Fluorescence Correlation Spectroscopy to Study Conformational Changes in Denatured Proteins” [Non-Patent Document 2] Tingjuan Gao, PROTEIN SCIENCE 2011 VOL 20:437-447, “Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy” Summary of the Invention [Problem to be solved by the invention]

[0004] The present inventors have found that conventional methods have large measurement errors and are problematic in terms of accuracy. An objective of the present invention is to provide a method and a reagent kit for obtaining information regarding the size of a target polypeptide more accurately. [Means for solving the problem]

[0005] One embodiment of the present invention relates to a method for acquiring information on a target polypeptide, the method for acquiring information on the target polypeptide comprising the steps of acquiring a diffusion time of a fluorescently labeled target polypeptide and a diffusion time of each of a plurality of fluorescently labeled reference polypeptides by fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy, and acquiring information on the size of the fluorescently labeled target polypeptide from the diffusion time of the fluorescently labeled target polypeptide using the diffusion times of the fluorescently labeled reference polypeptides as references, wherein information on the size of each of the fluorescently labeled reference polypeptides is known, and the sizes of the reference polypeptides are different from one another.

[0006] According to the method for obtaining protein information of this embodiment, compared to Non-Patent Documents 1 and 2, information regarding the size of the target polypeptide can be obtained more accurately. Certain embodiments of the present invention relate to a reagent kit.

[0007] One embodiment of the reagent kit relates to a reagent kit including a plurality of fluorescently labeled reference polypeptides, each of which has known size information and which are different in size from one another, and which is used in a method for obtaining information on a protein.

[0008] Another embodiment of the reagent kit relates to a reagent kit comprising a plurality of reference polypeptides and a fluorescently labeled antibody that binds to each of the plurality of reference polypeptides. The fluorescently labeled reference polypeptides each have known size information, and the plurality of reference polypeptides are different in size from each other. The reagent kit is used in a method for obtaining information on the target polypeptide.

[0009] According to the reagent kit of the above embodiment, compared to Non-Patent Documents 1 and 2, information regarding the size of the target polypeptide can be obtained more accurately. Effect of the Invention

[0010] More precisely, information regarding the size of the target polypeptide can be obtained. [Brief description of the drawings]

[0011] [Figure 1] Figure 1 shows an overview of the present invention, which is an example using FCCS. [Diagram 2] FIG. 2 shows the appearance of the reagent kit. [Diagram 3] FIG. 3 shows a schematic of the proportional conversion used in Comparative Example 1. [Figure 4]Figure 4(A) shows the results after measuring and normalizing the hydrodynamic radius in PBS. Figure 4(B) shows the results after measuring and normalizing the hydrodynamic radius in diluted plasma. Figure 4(C) shows the difference in the calibration curves when PBS is used as the aqueous solvent and when diluted samples are used. In Figure 4(C), the abbreviations stand for AP: aprotinin, L: lysozyme, CA: carbonic anhydrase, BS: bovine serum albumin, BA: β-amylase, AF: apoferritin, and T: thyroglobulin, respectively. Error bars indicate the standard deviation of triplicate measurements. [Diagram 5] FIG. 5(A) is a calibration curve created based on the diffusion time measured using diluted serum. FIG. 5(B) is a calibration curve created based on the diffusion time measured using diluted plasma 1. FIG. 5(C) is a calibration curve created based on the diffusion time measured using diluted plasma 2, which is different from diluted plasma 1. [Figure 6] Fig. 6(A) shows the difference between the predicted value and the theoretical value for β-Amylase obtained by the method of the present invention, and Fig. 6(B) shows the relationship between the absolute value of the difference between the predicted value and the theoretical value obtained for each reference polypeptide and the reference polypeptide. [Figure 7] Fig. 7(A) shows the difference between the predicted value and the theoretical value for β-Amylase obtained by the method of Comparative Example 1. Fig. 7(B) shows the relationship between the absolute value of the difference obtained for each reference polypeptide in each sample and the molecular size of the reference polypeptide. [Figure 8] Fig. 8(A) shows the cross-correlation G(τ) and lag time before normalization when recombinant VWF protein was measured by FCCS, and Fig. 8(B) shows the predicted value, theoretical value, and the difference between them for recombinant VWF protein calculated based on the method of the present invention. [Figure 9] Fig. 9(A) shows the cross-correlation G(τ) and lag time before normalization when egg white lysozyme was measured by FCCS. Fig. 9(B) shows the predicted value, theoretical value, and the difference between them for egg white lysozyme protein calculated based on the method of the present invention. [Figure 10]Fig. 10(A) shows the diffusion time (μs) and predicted hydrodynamic radius of each reference polypeptide when measured by FCCS using PBS as an aqueous solvent. Fig. 10(B) shows a calibration curve created based on Fig. 10(A). Fig. 10(C) shows the absolute value of the difference between the predicted value and the theoretical value obtained in Example 4, and the accuracy. Fig. 10(D) shows the absolute value of the difference between the predicted value and the theoretical value, and the accuracy obtained in Comparative Example 2. [Figure 11] Figure 11(A) shows the diffusion time (μs) and predicted hydrodynamic radius of each reference polypeptide measured by FCS using PBS as an aqueous solvent. Figure 11(B) shows the calibration curve prepared based on Figure 11(A). Figure 11(C) shows the calibration curve and regression equation obtained by the reference example. [Figure 12] 12(A) shows the results of Example 5, FIG. 12(B) shows the results of the Reference Example, and FIG. 12(C) shows the results of Comparative Example 3. [Figure 13] Figure 13(A) shows comparative data on the absolute value of the difference in β-Amylase, and Figure 13(B) shows comparative data on the absolute value of the difference in Thyroglobulin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 1. Method for obtaining information on target polypeptide 1-1.Explanation of terms Reference polypeptide is a polypeptide that is used as a reference for obtaining information about the size of target polypeptide, and the information about size is known.In addition, it is preferable to use multiple reference polypeptides that are different in size from each other.For example, two, three, four, five, six or seven kinds of reference polypeptides can be used.

[0013] The size of the reference polypeptide is appropriately selected according to the expected size of the target polypeptide. For example, the hydrodynamic radius of the reference polypeptide is 1 nm or more and 20 nm or less. The multiple reference polypeptides include at least a first reference polypeptide and a second reference polypeptide. The first reference polypeptide and the second reference polypeptide are different in size from each other. In one embodiment, the difference between the hydrodynamic radius of the first reference polypeptide and the hydrodynamic radius of the second reference polypeptide is at least 2 times. In another embodiment, the hydrodynamic radius of the first reference polypeptide is 1 nm or more and less than 5 nm, and the hydrodynamic radius of the second reference polypeptide is 5 nm or more and 10 nm or less.

[0014] The multiple reference polypeptides may include a first reference polypeptide, a second reference polypeptide and a third reference polypeptide.The first reference polypeptide, the second reference polypeptide and the third reference polypeptide are each different in size.In one embodiment, the hydrodynamic radius of the first reference polypeptide is 1 nm or more and less than 3 nm, the hydrodynamic radius of the second reference polypeptide is 3 nm or more and less than 6 nm, and the hydrodynamic radius of the third reference polypeptide is 6 nm or more and less than 9 nm.

[0015] Specific examples of reference polypeptides that can be used include aprotinin (hydrodynamic radius 1.4 nm), Hen egg-white lysozyme (lysozyme) (hydrodynamic radius 1.9 nm), carbonic anhydrase (CA) (hydrodynamic radius 2.1 nm), bovine serum albumin (BSA) (hydrodynamic radius 3.5 nm), β-amylase (hydrodynamic radius 5.4 nm), apo-ferritin (hydrodynamic radius 6.4 nm), thyroglobulin (hydrodynamic radius 8.6 nm), etc. Among these, two or more types can be selected depending on the expected size of the target polypeptide.

[0016] The target polypeptide is a polypeptide to be measured. The target polypeptide may be a polypeptide derived from a specimen collected from a living body. Examples of specimens include whole blood, plasma, serum, cerebrospinal fluid, and urine. The target polypeptide may be a protein, a protein fragment, or a polypeptide aggregate. A polypeptide aggregate is also called a "polymer" or "multimer" and is formed by physical and / or chemical polymerization of multiple monomer molecules. Specific examples of aggregates include von Willebrand factor aggregates, amyloid beta aggregates, tau protein aggregates, serum-amyloid-A-protein aggregates, IgG-light chain aggregates, AapoAI aggregates, AapoAII aggregates, ATTR aggregates, DISC1 aggregates, FUS aggregates, IAPP aggregates, SOD1 aggregates, α-synuclein aggregates, TDP-43 aggregates, huntingtin aggregates, and lysozyme aggregates. Polypeptide aggregates may be used to determine the presence or absence of a disease. For example, von Willebrand factor exists as aggregates (von Willebrand factor multimers) in the blood of healthy individuals, but in acquired von Willebrand factor syndrome, these aggregates may be degraded. In other words, information on the size of von Willebrand factor can be an indicator of acquired von Willebrand factor syndrome. In addition, information on the size of amyloid beta aggregates, tau protein aggregates, etc. can be an indicator of neurological diseases such as Alzheimer's disease.

[0017] Fluorescence correlation spectroscopy (hereinafter sometimes referred to as "FCS") is a method for detecting a fluorescent dye of one color bound to one molecule. Fluorescence cross-correlation spectroscopy (hereinafter sometimes referred to as "FCCS") is a method for detecting fluorescent dyes of two colors bound to one molecule. Apparatuses for performing analysis by FCS or FCCS are known. For example, by combining an inverted microscope Zeiss AxioObserver (Carl Zeiss K.K.) with the company's Zen software, both FCS and FCCS analysis are possible. In addition, as an FCS analysis apparatus, for example, the FCS Compact series of fluorescence correlation spectroscopy apparatuses (Hamamatsu Photonics K.K.) can be mentioned. As an FCCS analysis apparatus, for example, the FCCS Compact series of fluorescence cross-correlation spectroscopy apparatuses (Hamamatsu Photonics K.K.) can be mentioned.

[0018] The diffusion time is generally represented by the symbol τ D etc., and can be expressed in units of, for example, microseconds (μs) or milliseconds.

[0019] Examples of size information obtained by FCS or FCCS include hydrodynamic radius, hydrodynamic diameter, and volume. Hydrodynamic radius and hydrodynamic diameter can be expressed in units of nanometers (nm). Volume can be expressed in units of cubic nanometers (nm 3 ) can be expressed in units such as

[0020] Since FCS uses a single color fluorescent dye, any fluorescent dye within the range that can be detected by the FCS analyzer can be used.

[0021] On the other hand, FCCS uses two kinds of fluorescent dyes and detects these fluorescent dyes almost simultaneously. For this reason, it is preferable that the fluorescent wavelengths of the two kinds of fluorescent dyes used are different from each other. For example, when the peak of the fluorescent wavelength of one of the two fluorescent dyes is in the range of 400 nm to 550 nm, it is preferable that the peak of the fluorescent wavelength of the other fluorescent dye is in the range of 600 nm to 800 nm. In terms of excitation wavelength, when the peak of the excitation wavelength of one of the two fluorescent dyes is in the range of 400 nm to 500 nm, it is preferable that the peak of the excitation wavelength of the other fluorescent dye is in the range of 530 nm to 700 nm. For example, when Alexa Fluor (trademark) 488, FITC, Cy3, Cy2, etc. is used as one fluorescent dye, Alexa Fluor (trademark) 647, rhodamine, Cy5, etc. can be used as the other fluorescent dye. When two kinds of fluorescent dyes with overlapping fluorescent spectra are used, the fluorescent signal may be detected, for example, through a band pass filter or the like.

[0022] The fluorescent dye with which the target polypeptide is labeled and the fluorescent dye with which the reference polypeptide is labeled are preferably the same.

[0023] A reference polypeptide or a target polypeptide can be labeled with a fluorescent dye via a chemical bond such as a covalent bond by a known method. Hereinafter, a fluorescent labeling method via a chemical bond is also referred to as a direct labeling method. For example, a fluorescent dye can be covalently labeled to a polypeptide using amino labeling, thiol labeling, etc. In amino labeling, an N-hydroxysuccinimide (NHS) ester, sulfodicholorphenol (SDP) ester, tetrafluorophenyl (TFP) ester, etc. of a fluorescent dye can be used to label the fluorescent dye by an amine coupling reaction between the ester and the amine of the polypeptide. In addition, in thiol labeling, a fluorescent dye can be labeled by a covalent bond between a maleimide derivative of a fluorescent dye and a thiol group of a polypeptide.

[0024] When labeling, the polypeptide and the fluorescent dye ester or fluorescent dye derivative are mixed in a predetermined reaction buffer. The mixing ratio of the polypeptide and the fluorescent dye ester or fluorescent dye derivative can be 2 to 50 moles of the fluorescent dye ester or fluorescent dye derivative per mole of the polypeptide. When two fluorescent dyes are used, for example, the ratio can be 2 to 50 moles of the first fluorescent dye ester or first fluorescent dye derivative and 2 to 50 moles of the second fluorescent dye ester or second fluorescent dye derivative per mole of the polypeptide. It is preferable to mix the first and second fluorescent dye esters or fluorescent dye derivatives so that the molar ratios are the same.

[0025] The labeling reaction can be carried out in the dark, for example, at 20° C. to 28° C. with stirring, for about 30 minutes to 2 hours.

[0026] The fluorescently labeled polypeptide can be purified by desalting the reaction mixture using a desalting / buffer exchange column, etc., and removing the unreacted fluorescent dye ester or fluorescent dye derivative. For example, HiTrap Desalting (GE Healthcare Cat. No. 29048684) can be used as a desalting / buffer exchange column.

[0027] In another embodiment, a fluorescent dye can be labeled via a probe by a known method using a fluorescently labeled probe for a reference polypeptide or a target polypeptide. Hereinafter, the fluorescent labeling method via a probe is also referred to as an indirect labeling method. Herein, the probe is not limited as long as it can specifically bind to each reference polypeptide or target polypeptide. In this specification, specifically binding means that it may include a non-specific reaction that does not adversely affect the measurement. Probes include antibodies, lectins, aptamers, and the like. The probe is preferably an antibody. As the antibody, any of polyclonal antibodies, monoclonal antibodies, and fragments thereof (e.g., Fab, F(ab'), F(ab)2, and the like) can be used. The class and subclass of immunoglobulin are not particularly limited. The antibody may be one screened from an antibody library, and may be a chimeric antibody, scFv, single domain antibody (sdAb), and the like. The antibody may also include a VHH (variable domain of heavy chain of heavy chain antibody) domain of a heavy chain antibody derived from a camelid, a VNAR (variable new antigen receptor) domain derived from a cartilaginous fish (e.g., shark) heavy chain antibody IgNAR (new antigen receptor), etc. The antibody used as the fluorescently labeled probe is preferably Fab, VHH, VNAR, or scFv, which has a small molecular size itself.

[0028] In FCS, each reference polypeptide, or probe that binds to a target polypeptide, can be labeled with a fluorescent dye of the same fluorescent wavelength, preferably the same fluorescent dye.

[0029] In FCCS, two types of fluorescent dyes with different fluorescent wavelengths as described in 1-1 above can be bound to a polypeptide to fluorescently label the reference polypeptide so that it contains two types of fluorescent dyes. In this case, it is preferable to bind a first probe containing one of the two types of fluorescent dyes and a second probe containing the other fluorescent dye to the polypeptide. Similarly, the target polypeptide can be fluorescently labeled by binding two types of fluorescent dyes with different fluorescent wavelengths to two different probes so that the target polypeptide contains two types of fluorescent dyes. In this case, it is preferable that the two types of fluorescent dyes labeled to the reference polypeptide are the same as the fluorescent dyes labeled to the target polypeptide.

[0030] The regions in a polypeptide recognized by the first probe and the second probe may be different or the same, but are preferably different. When the probes are antibodies, the epitope of the first antibody containing the first fluorescent dye and the epitope of the second antibody containing the second fluorescent dye are preferably different sites in a single polypeptide.

[0031] The fluorescently labeled probe can be mixed and bound to each reference polypeptide or target polypeptide in a buffer such as PBS, in a sample, or in a diluted sample. The diluted sample can be a sample diluted about 1.5 to 10 times with PBS, physiological saline, or the like. The reaction time is, for example, about 3 to 7 minutes at room temperature (about 23°C to 27°C). The fluorescently labeled antibody can be added at a protein concentration of, for example, about 50 nM to 150 nM. By this reaction, each fluorescently labeled reference polypeptide or fluorescently labeled target polypeptide can be prepared.

[0032] 1-2. Method for obtaining information on target polypeptide An embodiment of the present invention relates to a method for acquiring information on a target polypeptide (hereinafter, may be simply referred to as "information acquisition method"). The information acquisition method includes a first step of acquiring the diffusion time of a fluorescently labeled target polypeptide and the diffusion times of multiple fluorescently labeled reference polypeptides by FCS or FCCS, and a second step of acquiring information on the size of the fluorescently labeled target polypeptide based on the diffusion times of the multiple fluorescently labeled reference polypeptides.

[0033] (1) First step: Diffusion time acquisition step In the first step, the diffusion time of the fluorescently labeled reference polypeptide is obtained from each standard sample. The standard sample can be prepared using a fluorescently labeled reference polypeptide by direct labeling or a fluorescently labeled reference polypeptide by indirect labeling. The standard sample includes a first standard sample containing a fluorescently labeled first reference polypeptide and a second standard sample containing a fluorescently labeled second reference polypeptide. Thus, the diffusion time obtained from each standard sample includes the diffusion time of the fluorescently labeled first reference polypeptide and the diffusion time of the fluorescently labeled second reference polypeptide. When the standard sample further includes a third standard sample containing a fluorescently labeled third reference polypeptide, the diffusion time obtained from the standard sample includes at least the diffusion time of the fluorescently labeled first reference polypeptide, the diffusion time of the fluorescently labeled second reference polypeptide, and the diffusion time of the fluorescently labeled third reference polypeptide.

[0034] When each reference polypeptide is fluorescently labeled by a direct labeling method, in one embodiment, the reference polypeptide is reacted with a fluorescent dye in an aqueous solvent, and the reaction solution can be used as a standard sample. In another embodiment, the fluorescently labeled reference polypeptide is mixed with an aqueous solvent, and the mixture can be used as a standard sample. A standard sample is prepared for each reference polypeptide. The "aqueous solvent" in this specification is not particularly limited as long as it is a liquid solvent. Examples of the aqueous solvent include water, a buffer solution, and physiological saline. In addition, a liquid specimen collected from a living body may be used as the "aqueous solvent". The specimen may contain insoluble components such as cells, and examples of the specimen include whole blood, plasma, serum, cerebrospinal fluid, and urine. In addition, the specimen may be pretreated, such as removing insoluble components or diluting, by a known method as necessary.

[0035] When the reference polypeptide is fluorescently labeled by the indirect labeling method, in one embodiment, the reference polypeptide is reacted with a fluorescent probe in an aqueous solvent. In this embodiment, the reaction solution can be used as the standard sample. In another embodiment, the fluorescently labeled reference polypeptide is mixed with an aqueous solvent, and the mixture can be used as the standard sample.

[0036] Furthermore, in the first step, the diffusion time of the fluorescently labeled target polypeptide is obtained from the measurement sample.

[0037] A measurement sample can be prepared using a target polypeptide fluorescently labeled by a direct labeling method or a target polypeptide fluorescently labeled by an indirect labeling method. When a target polypeptide is fluorescently labeled by a direct labeling method, in one embodiment, the target polypeptide is reacted with a fluorescent dye in an aqueous solvent, and the reaction solution can be used as a measurement sample.

[0038] When the target polypeptide is fluorescently labeled by the indirect labeling method, in one embodiment, the target polypeptide is reacted with a fluorescent probe in an aqueous solvent. In this embodiment, the reaction solution can be used as the measurement sample. In another embodiment, the fluorescently labeled target polypeptide is mixed with an aqueous solvent, and the mixture can be used as the measurement sample.

[0039] The viscosity of the aqueous solvent affects the diffusion time of molecules in the liquid. Therefore, preferably, the viscosity of the aqueous solvent containing the fluorescently labeled reference polypeptide is comparable to that of the aqueous solvent containing the fluorescently labeled target polypeptide. More preferably, the aqueous solvent containing the fluorescently labeled reference polypeptide is the same as the aqueous solvent containing the fluorescently labeled target polypeptide. When the measurement sample contains the fluorescently labeled target polypeptide in an aqueous solvent containing impurities such as a specimen collected from a living body, it is preferable to prepare the standard sample by mixing each fluorescently labeled reference polypeptide with the same aqueous solvent as the aqueous solvent used for the measurement sample. In this case, in a more preferred embodiment, the aqueous solvent such as a specimen collected from a living body is divided into multiple portions, one of which is added with a fluorescently labeled probe that labels the target polypeptide, and the other is added with a fluorescently labeled reference polypeptide. This allows the measurement sample containing the fluorescently labeled target polypeptide and the standard sample containing the fluorescently labeled reference polypeptide to be prepared. More specifically, the specimen containing the target polypeptide collected from a living body is divided into a first aliquot, a second aliquot, and a third aliquot. A measurement sample containing a fluorescently labeled target polypeptide can be prepared by mixing the first aliquot with a fluorescently labeled probe that binds to the target polypeptide. A first standard sample can be prepared by mixing the second aliquot with a fluorescently labeled first reference polypeptide. A second standard sample can be prepared by mixing the third aliquot with a fluorescently labeled second reference polypeptide.

[0040] The viscosity of the measurement sample containing each fluorescently labeled reference polypeptide or fluorescently labeled target polypeptide is preferably about 0.800 to 30.00 mPa.s at 23° C. In addition, the amount of the fluorescent dye contained in the peptide solution is preferably adjusted to about 0.1 nM to 1000 nM.

[0041] In one embodiment, the binding mode between the target polypeptide and the fluorescent dye is the same as the binding mode between the reference polypeptide and the fluorescent dye. For example, when the fluorescent dye is bound to the target polypeptide by direct labeling, the fluorescent dye may be bound to the reference polypeptide by direct labeling. When the fluorescent dye is bound to the target polypeptide by indirect labeling, the fluorescent dye may be bound to the reference polypeptide by indirect labeling. In this case, the type of fluorescently labeled probe used for indirect labeling of the target polypeptide and the fluorescent dye is preferably the same as the type of fluorescently labeled probe used for indirect labeling of the reference polypeptide and the fluorescent dye. As a specific example, the target polypeptide is labeled with a fluorescently labeled antibody that specifically binds to the target polypeptide, and the reference polypeptide is labeled with a fluorescently labeled antibody that specifically binds to the reference polypeptide.

[0042] In another embodiment, the binding mode between the target polypeptide and the fluorescent dye is different from the binding mode between the reference polypeptide and the fluorescent dye. The reference polypeptide does not necessarily have to have the same binding mode, as long as it can serve as a reference for the target polypeptide with respect to information regarding the size of the polypeptide. For example, when the fluorescent dye is bound to the target polypeptide by direct labeling, the fluorescent dye may be bound to the reference polypeptide by indirect labeling. When the fluorescent dye is bound to the target polypeptide by indirect labeling, the fluorescent dye may be bound to the reference polypeptide by direct labeling. As a specific example, the target polypeptide is labeled with a fluorescently labeled antibody that specifically binds to the target polypeptide, and the reference polypeptide is labeled with a fluorescent dye via a covalent bond.

[0043] In obtaining the diffusion times described below, the diffusion times of the fluorescently labeled target polypeptide in the measurement sample, the diffusion time of the fluorescently labeled first reference polypeptide in the first standard sample, and the diffusion time of the fluorescently labeled second reference polypeptide in the second standard sample can each be obtained.

[0044] Each sample is individually dispensed in about 30 μl into, for example, a 384-well glass-bottom plate (Sigma-Aldrich, M4437-16EA) and subjected to measurement. The standard sample and the measurement sample are preferably measured at about the same temperature, for example, about 23° C. to 25° C.

[0045] There are no limitations on the laser used for the measurement, as long as it can excite the fluorescent dye contained in each sample. The laser is focused to about the 1fL region, and the fluorescent signal emitted when the fluorescently labeled polypeptide in the sample passes through the confocal region is detected. In FCS, one laser is used to excite the fluorescent dye, and the fluorescent signal is acquired over time. In FCCS, two lasers are used to excite the fluorescent dye, and the fluorescent signal is acquired over time. The raw data of the detected fluorescent signal is a data group represented by the signal intensity and the measurement time. The processing of this raw data differs between FCS and FCCS, so the two are explained separately below.

[0046] The raw data may be optionally pre-processed to remove measurements that exhibit baseline drift or bursts (fluorescence signal more than three-fold higher than the mean fluorescence intensity).

[0047] (i)FCS The raw data of single-wavelength photon counts is acquired using the FCS analysis device described above. Autocorrelation analysis is performed on the raw data to obtain the raw curve of the autocorrelation function. An appropriate fitting model is performed on the autocorrelation function curve to obtain the correlation curve drawn by the correlation function G(τ) and lag time(τ).

[0048] When the measurement sample and the standard sample are prepared using a specimen collected from a living body, fitting by the autocorrelation function can be performed with a two-component model. The first component used in the two-component model can be, for example, an unreacted fluorescently labeled probe. The second component can be each complex of each polypeptide and its corresponding fluorescently labeled probe. For example, on 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 probe. Next, on the same software, a two-component model is selected, the diffusion time of the first component is fixed to the diffusion time of the first component measured earlier, and the diffusion time of the second component is measured.

[0049] (ii)FCCS The raw photon count data of two wavelengths is obtained by the above-mentioned FCCS analysis device. A cross-correlation analysis is performed on the raw data to obtain a raw curve of the cross-correlation function. An appropriate fitting model is performed on the cross-correlation function curve to obtain a correlation curve in which, for example, the X-axis is represented by lag time (τ) and the Y-axis is represented by the cross-correlation function G(τ). Specific functions are shown in Eq.1A, Eq.1B, Eq.2, and Eq.3 in the examples.

[0050] In FCCS, the correlation function can be fitted with a one-component model. One component is each complex between each polypeptide and its corresponding fluorescently labeled probe. Select the one-component model on software such as Zen software and measure the diffusion time of each complex. FCCS uses two types of fluorescent dyes for one polypeptide, allowing for more accurate analysis.

[0051] Here, the value of G(τ) when τ=0 is the value of the origin, and the diffusion time indicates the lag time τ when the value of the origin becomes 1 / 2. Specifically, when the G(τ) value is 0, that is, when G(0) is a, the diffusion time is intended to be the lag time τ where G(τ)=a×1 / 2. More specifically, when the value at the time of G(τ) is converted to 1 in the correlation function G(τ), the diffusion time is the lag time τ where G(τ)=0.5. Therefore, the correlation function G(τ) may be standardized so that the maximum value is "1" and the minimum value is "0". When the correlation function G(τ) is standardized, the combination of the maximum value and the minimum value can be set appropriately. For example, the combination may be the maximum value of "2" and the minimum value of "1", or the combination may be the maximum value of "100" and the minimum value of "0".

[0052] (2) The second step: information acquisition step The diffusion time corresponding to the predetermined G(τ) value of the reference polypeptide contained in each standard sample obtained in the first step is used as a reference to obtain information on the size of the target polypeptide from the diffusion time of the target polypeptide. When the diffusion time of the fluorescently labeled first reference polypeptide and the diffusion time of the fluorescently labeled second reference polypeptide are obtained, the diffusion time is used as a reference to obtain information on the size of the target polypeptide. When the diffusion time of the fluorescently labeled first reference polypeptide, the diffusion time of the fluorescently labeled second reference polypeptide, and the diffusion time of the fluorescently labeled third reference polypeptide are obtained, the diffusion time is used as a reference to obtain information on the size of the target polypeptide.

[0053] Preferably, a regression equation is obtained based on the diffusion time corresponding to a predetermined G(τ) value of the reference polypeptide contained in each standard sample and the previously known size of each reference polypeptide to create a calibration curve. The diffusion time corresponding to the G(τ) value of the target polypeptide that is the same as the predetermined G(τ) value of the reference polypeptide is obtained. The obtained diffusion time of the target polypeptide is applied to the calibration curve to obtain information regarding the size of the target polypeptide. The size of the target polypeptide obtained here is the size of the target polypeptide that is not fluorescently labeled.

[0054] (3) Example of embodiment The principle of the method of this embodiment will be described using a specific example shown in Fig. 1. The example shown in Fig. 1 is merely one example, and the present invention is not limited to this example. Fig. 1 shows an information acquisition method using fluorescence cross-correlation spectroscopy.

[0055] For multiple reference polypeptides labeled with fluorescent dyes in step A, the diffusion time (also called lag time) τ is obtained by FCCS. Molecules in a solution are always moving freely due to Brownian motion, etc. FCS and FCCS use a confocal laser to detect a fluorescent signal emitted when a molecule in a solution passes through a confocal region. The raw data of the detected fluorescent signal is expressed as signal intensity and measurement time. In FCCS, this is fitted with a cross-correlation function, and a correlation curve is obtained in which the X-axis is the diffusion time (τ) and the Y-axis is the correlation function G(τ). In the case of FCS, the raw data of the detected fluorescent signal is fitted with an autocorrelation function to obtain a curve of the correlation function G(τ). Here, the correlation function G(τ) shown in step A is a correlation curve after normalization.

[0056] In step B, a calibration curve is prepared. Since the size of the reference polypeptide is known in advance, a regression equation can be obtained from the diffusion time corresponding to a certain G(τ) value for each reference polypeptide and the size (here, the hydrodynamic radius) of each reference polypeptide to prepare a calibration curve.

[0057] Step C is a step of determining the diffusion time of the fluorescently labeled target polypeptide. The diffusion time can be determined in the same manner as the diffusion time of the reference polypeptide.

[0058] Steps A and C correspond to steps of acquiring the diffusion time of a fluorescently labeled target polypeptide and the diffusion time of multiple fluorescently labeled reference polypeptides. The order of steps A and C is not limited. Step A may be performed before step C, step C may be performed before step A, or steps A and C may be performed simultaneously.

[0059] Step D is a step of obtaining information about the size of the target polypeptide from the calibration curve prepared in step B and the diffusion time of the target polypeptide obtained in step C. The diffusion time of the target polypeptide corresponding to the G(τ) value when the calibration curve was prepared is applied to the calibration curve to obtain information about the molecular size of the target polypeptide.

[0060] 2. Reagent Kit An embodiment of the present invention relates to a reagent kit. The reagent kit includes a plurality of fluorescently labeled reference polypeptides as described in 1-2 above. This reagent kit can be used as a calibrator for obtaining information about the size of a target polypeptide. The fluorescently labeled reference polypeptides are preferably contained in separate containers. The reagent kit can be provided to a user as a kit as illustrated in FIG. 2. The reagent kit 50 includes an outer box 55, a first container 51 for storing a fluorescently labeled first reference polypeptide, a second container 52 for storing a fluorescently labeled second reference polypeptide, a third container 53 for storing a fluorescently labeled third reference polypeptide, and an attachment 54 for the reagent kit. The attachment 54 can include a method for handling the reagent kit, storage conditions, expiration date, and the like. A container containing an aqueous solvent for dilution may be packed in the outer box 55.

[0061] Alternatively, the reagent kit may be provided to the user as a reagent kit containing a plurality of reference polypeptides and fluorescently labeled antibodies that bind to each of the plurality of reference polypeptides.

[0062] The fluorescently labeled antibody that binds to each of the multiple reference polypeptides may include a first fluorescently labeled antibody and a second fluorescently labeled antibody that bind to one reference polypeptide. Preferably, the first fluorescently labeled antibody is labeled with a fluorescent dye whose fluorescence wavelength is different from that of the fluorescent dye labeled to the second fluorescently labeled antibody. In addition, it is preferable that the epitope of the first fluorescently labeled antibody is different from that of the second fluorescently labeled antibody. EXAMPLES

[0063] The present invention will be described in more detail below using examples. However, the present invention is not limited to the examples.

[0064] I. Method 1. Reference Polypeptide As reference polypeptides, Aprotinin (Sigma-Aldrich, A3886-1VL, hydrodynamic radius 1.4 nm); Hen egg-white lysozyme (lysozyme) (Sigma-Aldrich, L4919-500MG, hydrodynamic radius 1.9 nm); Carbonic anhydrase (CA) (Sigma-Aldrich, C7025-1VL, hydrodynamic radius 2.1 bovine serum albumin (BSA) (Sigma-Aldrich, A8531-1VL, hydrodynamic radius 3.5 nm); β-amylase (Sigma-Aldrich, A8781-1VL, hydrodynamic radius 5.4 nm), Apo-ferritin (Sigma-Aldrich, A3660-1VL, hydrodynamic radius 6.4 nm); nm), Thyroglobulin (Sigma-Aldrich, T1001-100MG, A hydrodynamic radius of 8.6 nm was used.

[0065] 2. Fluorescent labeling of polypeptides (1) Direct labeling of polypeptides with fluorescent dyes The amines of the reference and target polypeptides were labeled with fluorescent dyes by amine coupling reaction using the N-hydroxy succinimidyl (NHS) ester of Alexa Fluor™ 647 (Invitrogen, Cat. No. A20006) and / or Alexa Fluor™ 488 (Invitrogen, Cat. No. A20000). Each polypeptide measured by FCS was labeled with one of the above fluorescent dyes. Each polypeptide measured by FCCS was labeled with two of the above fluorescent dyes. The amine coupling reaction was carried out by mixing the fluorescent dye and the polypeptide in PBS and stirring at 400 rpm at room temperature in the dark for 1 hour.

[0066] In FCS, the mixing ratios of polypeptide and NHS ester of fluorescent dye were, in molar ratio, Aprotinin: fluorescent dye = 1:2, CA: fluorescent dye = 1:2, lysozyme: fluorescent dye = 1:2, BSA: fluorescent dye = 1:5, β-amylase: fluorescent dye = 1:20, Apo-ferritin: fluorescent dye = 1:20, Thyroglobulin: fluorescent dye = 1:50. When two fluorescent dyes were used in FCCS, they were mixed so that the molar ratio of the first and second fluorescent dyes was the same, for example, Aprotinin: first fluorescent dye: second fluorescent dye = 1:2:2.

[0067] After the amine coupling reaction, the reaction mixture was desalted using a HiTrap Desalting column (GE Healthcare Cat. No. 29048684) to remove unreacted fluorescent dye, and the desalted reaction mixture was further subjected to ultra-high performance liquid chromatography (UHPLC) using a TSKgel G3000SWXL column (TOSOH Cat. No. 08541) to analyze the quality of the fluorescently labeled polypeptide.

[0068] The quality of the fluorescently labeled polypeptide was evaluated by obtaining a fluorescence spectrum in the region from 230 nm to 700 nm and observing the peaks in the 280 nm region originating from the protein and in the fluorescence wavelength region of each fluorescent dye.

[0069] (2) Indirect labeling of polypeptides with fluorescent dyes Indirect labeling has been used in assays to detect target polypeptides in samples containing multiple components, such as serum or plasma.

[0070] The target polypeptide was indirectly fluorescently labeled by reacting each polypeptide contained in the sample with a labeled antibody bound to Alexa Fluor(TM) 647 or Alexa Fluor(TM) 488 against the target polypeptide. Each polypeptide to be measured by FCCS was labeled with both Alexa Fluor(TM) 647-labeled antibody and Alexa Fluor(TM) 488-labeled antibody.

[0071] The serum or plasma was added with the same amount of fluorescently labeled antibody solution as the specimen. After adding the fluorescently labeled antibody, the mixture was mixed and incubated in the dark at room temperature for 5 minutes. Then, the mixture was centrifuged at 13,000 rpm for 5 minutes, and the supernatant was used as the measurement sample.

[0072] 3. Measurement (1) Measurement of fluorescently labeled reference polypeptide When PBS was used as the aqueous solvent, each fluorescently labeled reference polypeptide, which was directly labeled with a fluorescent dye, was added to PBS at 100 nM to prepare a standard sample for preparing a calibration curve. When a diluted sample was used as the aqueous solvent, each fluorescently labeled reference polypeptide was added to serum or plasma at 100 nM to prepare a standard sample for preparing a calibration curve.

[0073] Each standard sample was added in an amount of 30 μl to a 384-well glass-bottom plate (Sigma-Aldrich, M4437-16EA) that had been previously blocked with blocking reagent N101 (NOF, 71S410050011) and subjected to measurement. Measurements were performed using a Zeiss AxioObserver Confocor3 setup equipped with an LSM710 laser module. Alexa Fluor™ 488 was excited with a 488 nm laser. Alexa Fluor™ 647 was excited with a 633 nm laser. The 488 nm and 633 nm lasers were output at 8 mW and 1.2 mW, respectively. Ten measurements were performed for each well, with one measurement lasting 20 seconds. In other words, the total measurement time for each well was 200 seconds. The correlation function was calculated based on these ten measurements. The temperature during measurement was 23°C. Zen software from Zeiss was used for FCS analysis and FCCS analysis.

[0074] (2) Fitting of each function and estimation of polypeptide size The raw data were preprocessed using Zen software to remove measurements that showed baseline drift or bursts (fluorescence signals >3-fold higher than the mean fluorescence intensity). The cross-correlation curves used in FCCS and the autocorrelation curves used in FCS were fitted using one- or two-component fitting (3D, or 3D+3D, or T×3D, or T×{3D+3D}) based on a 3D diffusion model.

[0075] The fitting function G(τ) for obtaining a three-dimensional one-component correlation curve (3D) is expressed by the following Eq. 1A. The fitting function G(τ) for obtaining a three-dimensional two-component correlation curve (3D+3D) is expressed by the following Eq. 1B. Both functions indicate the translational diffusion process. When calculating the translational diffusion time from the cross-correlation curve, a fitting model such as Eq. 1A or Eq. 1B was used.

[0076]

number

number

[0077] In Eq.1A, N represents the amplitude (number of molecules). τ D indicates the diffusion time (μs). τ indicates the lag time. ω (structure factor) is the ratio of the horizontal (XY) width to the vertical (Z) axis width of the laser focal volume (w z / w xy In Eq.1B, Q is the quantum yield.

[0078] Structure factors were assigned at the start of each day based on known diffusion times obtained from a dye mixture of 100 nM Alexa Fluor™ 488 and 10 nM Alexa Fluor™ 647 used as a reference.

[0079] To calculate the triplet state transition time (T), a fitting model was used as shown in Eq. 2 below. Eq. 2 below shows the triplet state transition.

[0080]

number

[0081] For the autocorrelation function, a fitting model was used, including both the translational diffusion time and the triplet state transition time of the dye (T×3D, or T×{3D+3D}), as shown in Eq. 3 below.

[0082]

number

[0083] (i) FCS analysis using samples FCS was performed by fitting the autocorrelation function with a two-component model. The first component was the unreacted fluorescently labeled antibody. The second component was each immune complex between each reference polypeptide and its corresponding fluorescently labeled antibody. First, a one-component model was selected on Zen software, and the diffusion time of the first component was measured for each fluorescently labeled antibody in a separate independent experimental system that did not contain the antigen (Tp 488 or Tp 647 Next, select the two-component model on Zen software and calculate the diffusion time of the first component by the diffusion time of the first component (Tp 488 or Tp 647 The diffusion time of the second component was measured. A regression equation between the diffusion time and the hydrodynamic radius (nm) of each reference polypeptide was obtained, and a calibration curve was created.

[0084] The diffusion time of the target polypeptide was also determined in the same manner as for the reference polypeptide, and the hydrodynamic radius was calculated from the calibration curve.

[0085] (ii) FCCS analysis using diluted samples FCCS performed correlation function fitting using a one-component model. One component is each immune complex between each polypeptide and its corresponding fluorescently labeled antibody. The one-component model was selected on Zen software, and the diffusion time of each immune complex was measured. A regression equation between the obtained diffusion time and the hydrodynamic radius (nm) of each reference polypeptide was obtained, and a calibration curve was created. The diffusion time of the target polypeptide was also determined in the same manner as for the reference polypeptide, and the hydrodynamic radius was calculated from the calibration curve.

[0086] II. Comparative Example 1 As Comparative Example 1, the proportional conversion described in Non-Patent Document 1 was used. An outline of the proportional conversion is shown in FIG. 3. In the proportional conversion, first, the hydrodynamic radius R HC Alexa Fluor™ 488 (hydrodynamic radius R HC = 0.7 nm) and the diffusion time of Alexa Fluor™ 647 (hydrodynamic radius R HC =0.8 nm) was measured by FCS (step 1 in FIG. 3). The temperature during measurement was 23°C. Next, the autocorrelation function of the standard was obtained and the diffusion time of the fluorescent dye was corrected (step 2 in FIG. 3). Next, the diffusion time of the fluorescently labeled target polypeptide was measured by FCS, and the diffusion time of the fluorescent dye τ DC (μs) and the diffusion time τ of the fluorescently labeled target polypeptide D (μs) and the hydrodynamic radius R as standard HC from the hydrodynamic radius R of the fluorescently labeled target polypeptide H asked for.

[0087] III. Example 1 According to the method described in I. above, PBS or diluted samples were used as the aqueous solvent, and aprotinin, lysozyme, CA, BSA, β-amylase, apo-ferritin, and thyroglobulin were used as reference polypeptides to measure the diffusion time by FCCS and create a calibration curve. In addition, one serum sample and two plasma samples from different subjects were used as samples to create a calibration curve.

[0088] Figure 4 shows the difference in diffusion time (lag time (s)) between the case where PBS was used as an aqueous solvent and the case where diluted plasma was used. Figure 4(A) shows the results after the hydrodynamic radius was measured in PBS and normalized. Figure 4(B) shows the results after the hydrodynamic radius was measured in diluted plasma and normalized. In Figure 4(B), the correlation curve shifted to the right compared to Figure 4(A). Figure 4(C) shows the difference in the calibration curve between the case where PBS was used as an aqueous solvent and the case where diluted plasma was used. The slope of the calibration curve was the same for the case where PBS was used as an aqueous solvent and the case where diluted plasma was used, but the intercept was lower for PBS. This indicates that the molecular migration speed is slower in diluted plasma, which has a higher viscosity than PBS.

[0089] Figure 5 shows the calibration curves obtained using three different types of specimens. Figure 5(A) is a calibration curve created based on the diffusion time measured using diluted serum. Figure 5(B) is a calibration curve created based on the diffusion time measured using diluted plasma 1. Figure 5(C) is a calibration curve created based on the diffusion time measured using diluted plasma 2, which is different from diluted plasma 1. Compared to plasma, serum had a shorter diffusion time. It was also shown that there were differences in diffusion time depending on the specimen, even with regard to plasma.

[0090] From these results, it was considered preferable to perform the measurement of the reference polypeptide in a solvent environment equivalent to that of the target polypeptide in order to predict the size of the target polypeptide and to prepare a calibration curve.

[0091] In addition, each of the reference polypeptides was assumed to be a target polypeptide, and the predicted value of the hydrodynamic radius of each reference polypeptide was calculated based on the calibration curve obtained in the measurement system using the diluted sample of the FCCS of the present invention. The samples used were serum, plasma 1, or plasma 2. The molecular size described as a gel filtration chromatography marker was then used as the theoretical value of the hydrodynamic radius to determine the difference between the predicted value and the theoretical value, and the absolute value of the difference. Figure 6(A) shows the difference value calculated for β-Amylase. Figure 6(B) shows the relationship between the absolute value of the difference calculated for each reference polypeptide in each sample and the hydrodynamic radius of the reference polypeptide.

[0092] Further, the predicted values ​​of the reference polypeptides were calculated in the same manner according to the method of Comparative Example 1, and compared with the theoretical values ​​of each reference polypeptide. The results are shown in FIG. 7. FIG. 7(A) shows the difference between the predicted value and the theoretical value calculated for β-Amylase. FIG. 7(B) shows the relationship between the absolute value of the difference calculated for each reference polypeptide in each sample and the molecular size of the reference polypeptide. In the method described in Comparative Example 1, even when the fluorescent dyes Alexa Fluor (trademark) 488 and Alexa Fluor (trademark) 647 were used as standards, the difference between the predicted value and the theoretical value was large. In addition, the difference between the predicted value and the theoretical value was large regardless of which of the three types of samples was used. In particular, it was shown that the difference between the predicted value and the theoretical value increased as the hydrodynamic radius of the reference peptide increased.

[0093] These results show that the information gathering method of the present invention has higher prediction accuracy than the comparative example.

[0094] IV. Example 2 To prove that the information acquisition method of the present invention is capable of predicting the hydrodynamic radius of large polypeptides, the molecular size of von Willebrand factor (VWF; molecular weight approximately 250 kDa) was predicted according to the information acquisition method of the present invention using FCCS.

[0095] A positive sample was prepared by adding recombinant VWF protein (Merck, 681300-100UGCN) to plasma to a final concentration of 25 nM. A negative sample was prepared by adding the same amount of buffer as the recombinant VWF protein solution to plasma without adding recombinant VWF protein. The positive and negative samples were treated in the same manner as described below.

[0096] Alexa Fluor™ 488-labeled 2F2A9 antibody (BD Biosciences, 555849) and Alexa Fluor™ 647-labeled NMC4 Fab were added to each sample at a final concentration of 100 nM, and incubated for 5 minutes at room temperature in the dark. NMC4 Fab was prepared by genetic recombination from the amino acid sequence of a known NMC4 Fab. FCCS was measured according to the method described in 3 above.

[0097] The results are shown in Figure 8. Figure 8(A) shows the cross-correlation G(τ) and lag time before normalization. Figure 8(B) shows the predicted value, theoretical value, and the difference between them for the recombinant VWF protein calculated based on the information acquisition method of the present invention.

[0098] The theoretical hydrodynamic radius of recombinant VWF protein is 16.25 nm when not reacted with antibodies, which is a relatively large protein. Even for such a large protein, the difference between the predicted value and the measured value was only 2.3 nm, demonstrating that accurate prediction is possible.

[0099] V. Example 3 In order to prove that the hydrodynamic radius of small polypeptides can be predicted by the information acquisition method of the present invention, the hydrodynamic radius of egg white lysozyme (molecular weight approximately 14 kDa; hydrodynamic radius 3 nm) was predicted according to the information acquisition method of the present invention using FCCS.

[0100] A positive sample was prepared by adding egg white lysozyme protein (Sigma-Aldrich, L4919-500MG) to plasma to a final concentration of 100 nM. A negative sample was prepared by adding the same amount of buffer as the egg white lysozyme protein solution to plasma without adding egg white lysozyme protein. The positive and negative samples were treated in the same manner as described below.

[0101] Alexa Fluor™ 488-labeled cAB-Lys2 VHH and Alexa Fluor™ 647-labeled D2L24 VHH were added to each sample at a final concentration of 100 nM, and incubated for 5 minutes at room temperature in the dark. The cAB-Lys2 VHH and D2L24 VHH were prepared by genetic recombination from the known amino acid sequences of cAB-Lys2 VHH and D2L24 VHH, respectively. FCCS was measured according to the method described in 3 above.

[0102] The results are shown in Figure 9. Figure 9(A) shows the cross-correlation G(τ) and lag time before normalization. Figure 9(B) shows the predicted value, theoretical value, and the difference between them for egg white lysozyme protein calculated based on the information acquisition method of the present invention.

[0103] The theoretical hydrodynamic radius of egg white lysozyme protein is 3 nm when not reacted with an antibody, which is a relatively small protein. Even for such a small protein, the difference between the predicted value and the measured value was only 0.5 nm, demonstrating that accurate prediction is possible.

[0104] VI. Example 4 and Comparative Example 2 In a measurement system using PBS as the aqueous solvent, the prediction accuracy was evaluated when the information acquisition method of the present invention was carried out by FCCS.

[0105] In Example 4, the FCCS measurement system using PBS as the aqueous solvent described in 3 above was used, and a calibration curve was created using the reference polypeptide double-labeled with the above-mentioned two-color fluorescent dyes. Each reference polypeptide was assumed to be a target polypeptide, and the hydrodynamic radius of each reference polypeptide was obtained from the calibration curve. Figure 10(A) shows the diffusion time (μs) and predicted value of the hydrodynamic radius of each reference polypeptide in FCCS. Also, Figure 10(B) shows the calibration curve created based on Figure 10(A). Figure 10(C) shows the absolute value of the difference between the predicted value and the theoretical value obtained in Example 4, and the accuracy. The accuracy (%) was calculated by subtracting the % error from 100%. The % error was calculated by the formula 100 × [(absolute value of difference)÷(theoretical value)].

[0106] In Comparative Example 2, the diffusion time measured by FCCS, τ DC The hydrodynamic radius was predicted by the proportional conversion described in Non-Patent Document 1 in the same manner as in Comparative Example 1. However, for the standard, BSA bound to Alexa Fluor (trademark) 647-labeled anti-BSA antibody and Alexa Fluor (trademark) 488-labeled anti-BSA antibody instead of fluorescent dye was used so that it could be measured by FCCS. In addition, PBS was used as the aqueous solvent for the measurement system. The hydrodynamic radius R of dual fluorescent-labeled BSA HC is 3.5 nm, and the measured diffusion time τ DC is 230 μs. Each of the dual fluorescently labeled reference polypeptides was assumed to be the target polypeptide, and the hydrodynamic radius of each of the reference polypeptides was determined according to the method of Comparative Example 1. FIG. 10(D) shows the absolute value of the difference between the predicted value and the theoretical value determined by Comparative Example 2, and the accuracy. The definitions of the terms are the same as those in FIG. 10(C).

[0107] In Comparative Example 2, since BSA was used as the standard, the absolute value of the difference was 0 and the prediction accuracy was 100%, but in all other respects Example 4 of the present invention had higher accuracy.

[0108] VII. Example 5, Reference Example and Comparative Example 3 In a measurement system using PBS as the aqueous solvent, the prediction accuracy was evaluated when the information acquisition method of the present invention was carried out by FCS.

[0109] In Example 5, the FCS measurement system using PBS as the aqueous solvent described in 3 above was used, and a calibration curve was created using the reference polypeptides labeled with the two fluorescent dyes described above. Each reference polypeptide was assumed to be a target polypeptide, and the hydrodynamic radius of each reference polypeptide was determined from the calibration curve. The calibration curve in FIG. 11(A) shows the diffusion time (μs) and predicted hydrodynamic radius of each reference polypeptide in FCS. FIG. 11(B) shows the calibration curve created based on FIG. 11(A).

[0110] The reference example is an example of measurement using the method described in Non-Patent Document 2, which uses fluorescent beads as a calibrator. As shown in Fig. 11(C), the diffusion time (msec) of fluorescent beads of various particle sizes was measured by FCS, a regression equation was created based on the diffusion time and particle size, and the diffusion time of the fluorescently labeled target polypeptide measured by FCS was applied to this regression equation. The hydrodynamic radius was calculated assuming that each reference polypeptide reacted with an Alexa Fluor (trademark) 647-labeled antibody was the target polypeptide.

[0111] Comparative Example 3 is an example in which the hydrodynamic radius was determined by the same method as in Comparative Example 1 described in II. Alexa Fluor (trademark) 647 dye was used as a standard. In addition, PBS was used as the aqueous solvent in the measurement system. The hydrodynamic radius R of Alexa Fluor (trademark) 647 dye HC is 0.8 nm, and the measured diffusion time τ DC The time required for the hydrodynamic radius of each of the fluorescently labeled reference polypeptides was calculated according to the method of Comparative Example 1, assuming that each of the fluorescently labeled reference polypeptides was the target polypeptide.

[0112] Fig. 12(A) shows the results of Example 5, Fig. 12(B) shows the results of Reference Example, and Fig. 12(C) shows the results of Comparative Example 3. Example 5 showed good accuracy in the range of the reference polypeptide used. Reference Example and Comparative Example 3 showed poorer accuracy in the range of large hydrodynamic radii than Example 5, and showed a tendency for the hydrodynamic radii to appear smaller than the actual values.

[0113] Figure 13(A) shows comparative data on the absolute value of the difference in β-Amylase. Figure 13(B) shows comparative data on the absolute value of the difference in Thyroglobulin. Example 5 showed smaller absolute values ​​of the difference than Reference Example and Comparative Example 3 for both polypeptides.

[0114] Abnormal proteins may have a larger hydrodynamic radius than normal proteins. In such cases, the methods described in Non-Patent Documents 1 and 2 may estimate the hydrodynamic radius of the abnormal protein to be smaller than the actual value, which may lead to a risk of false negatives.

Claims

1. A method for obtaining information on a target polypeptide in a sample collected from a living body, comprising the steps of: Obtaining the diffusion time of a fluorescently labeled target polypeptide in the sample and the diffusion time of each of a plurality of fluorescently labeled reference polypeptides by fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy; obtaining information about the size of the fluorescently labeled target polypeptide from the diffusion time of the fluorescently labeled target polypeptide, using the diffusion times of the fluorescently labeled reference polypeptides as a reference; Including, The plurality of reference polypeptides are different kinds of proteins, and the size information of each of the plurality of fluorescently labeled reference polypeptides is known; the plurality of reference polypeptides are different in size from one another; In the fluorescently labeled target polypeptide, a fluorescent dye is bound to the target polypeptide via an antibody, the fluorescently labeled reference polypeptide, wherein a fluorescent dye is bound to the reference polypeptide via an antibody; The sample is a sample collected from a living body. A method for obtaining information on a target polypeptide.

2. The method for acquiring information on a target polypeptide according to claim 1, wherein the size is at least one selected from hydrodynamic radius, hydrodynamic diameter, and volume.

3. The method for obtaining information on a target polypeptide according to claim 1 or 2, wherein the hydrodynamic radius of the reference polypeptide is 1 nm or more and 20 nm or less.

4. the plurality of fluorescently labeled reference polypeptides includes at least a first fluorescently labeled reference polypeptide and a second reference polypeptide having the same fluorescent label as the first fluorescent label; the fluorescently labeled first reference polypeptide and the fluorescently labeled second reference polypeptide are different sizes from each other; In the diffusion time acquisition step, a diffusion time of the fluorescently labeled first reference polypeptide and a diffusion time of the fluorescently labeled second reference polypeptide are acquired; In the information acquiring step, information regarding the size of the fluorescently labeled target polypeptide is acquired from the diffusion time of the fluorescently labeled target polypeptide with reference to the diffusion time of the fluorescently labeled first reference polypeptide and the diffusion time of the fluorescently labeled second reference polypeptide. A method for acquiring information on a target polypeptide according to any one of claims 1 to 3.

5. the hydrodynamic radius of the first reference polypeptide is greater than or equal to 1 nm and less than 5 nm; The method for obtaining information on a target polypeptide according to claim 4, wherein the hydrodynamic radius of the second reference polypeptide is 5 nm or more and 10 nm or less.

6. the hydrodynamic radius of the first reference polypeptide is at least twice the hydrodynamic radius of the second reference polypeptide; A method for acquiring information on a target polypeptide according to any one of claims 1 to 5.

7. the plurality of fluorescently labeled reference polypeptides comprises at least a fluorescently labeled first reference polypeptide, a fluorescently labeled second reference polypeptide, and a fluorescently labeled third reference polypeptide, all of which are the same fluorescent label; the fluorescently labeled first reference polypeptide, the fluorescently labeled second reference polypeptide, and the fluorescently labeled third reference polypeptide are each a different size; In the diffusion time acquisition step, a diffusion time of the fluorescently labeled first reference polypeptide, a diffusion time of the fluorescently labeled second reference polypeptide, and a diffusion time of the fluorescently labeled third reference polypeptide are acquired; In the information acquiring step, information on the size of the fluorescently labeled target polypeptide is acquired from the diffusion time of the fluorescently labeled target polypeptide with reference to the diffusion time of the fluorescently labeled first reference polypeptide, the diffusion time of the fluorescently labeled second reference polypeptide, and the diffusion time of the fluorescently labeled third reference polypeptide. A method for acquiring information on a target polypeptide according to any one of claims 1 to 3.

8. the hydrodynamic radius of the first reference polypeptide is greater than or equal to 1 nm and less than 3 nm; the hydrodynamic radius of the second reference polypeptide is greater than or equal to 3 nm and less than 6 nm; the hydrodynamic radius of the third reference polypeptide is greater than or equal to 6 nm and less than 9 nm; A method for obtaining information on a target polypeptide according to claim 7.

9. the fluorescently labeled target polypeptide, wherein the fluorescent dye is chemically bound to the target polypeptide; In the fluorescently labeled reference polypeptide, the fluorescent dye is chemically bound to the reference polypeptide. A method for acquiring information on a target polypeptide according to any one of claims 1 to 8.

10. The method for obtaining information on a target polypeptide according to claim 9 , wherein the chemical bond is a covalent bond.

11. preparing the fluorescently labeled target polypeptide by reacting the target polypeptide with a fluorescently labeled antibody that recognizes the target polypeptide, prior to the step of acquiring the diffusion time; A method for acquiring information on a target polypeptide according to any one of claims 1 to 10, comprising:

12. In the information acquiring step, a calibration curve is created from the diffusion times of the plurality of fluorescently labeled reference polypeptides, and information regarding the size of the fluorescently labeled target polypeptide is acquired based on the calibration curve. A method for obtaining information on a target polypeptide according to any one of claims 1 to 11.

13. Before the diffusion time acquisition step, Dividing a sample containing the target polypeptide collected from a living body into a first aliquot, a second aliquot, and a third aliquot; Fluorescently labeling the target polypeptide contained in the first aliquot to prepare the fluorescently labeled target polypeptide; mixing said second aliquot with a fluorescently labeled first reference polypeptide; mixing said third aliquot with a fluorescently labeled second reference polypeptide; Includes In the diffusion time acquisition step, a diffusion time of a fluorescently labeled target polypeptide in a first aliquot, a diffusion time of a fluorescently labeled first reference polypeptide in a second aliquot, and a diffusion time of a fluorescently labeled second reference polypeptide in a third aliquot are acquired. A method for obtaining information on a target polypeptide according to any one of claims 1 to 12.

14. The method for obtaining information on a target polypeptide according to any one of claims 1 to 13, wherein the target polypeptide is derived from a specimen collected from a living body, and the specimen is whole blood, plasma, serum, cerebrospinal fluid, or urine.

15. The diffusion time acquisition step is carried out by fluorescence cross-correlation spectroscopy; The method for acquiring information on a target polypeptide according to any one of claims 1 to 14, wherein the fluorescently labeled target polypeptide contains two types of fluorescent dyes having different fluorescent wavelengths, and each of the plurality of fluorescently labeled reference polypeptides contains the two types of fluorescent dyes.

16. the fluorescently labeled target polypeptide is a complex of the target polypeptide, a first antibody containing a first fluorescent dye, and a second antibody containing a second fluorescent dye having a fluorescent wavelength different from that of the first fluorescent dye; A method for obtaining information on a target polypeptide according to claim 15.

17. The information acquisition method according to any one of claims 1 to 16, further comprising the step of adjusting the fluorescently labeled target polypeptide by mixing the sample containing the target polypeptide with an antibody that is labeled with the fluorescent dye and specifically binds to the target polypeptide, prior to the step of acquiring the diffusion time.

18. A reagent kit comprising a plurality of fluorescently labeled reference polypeptides, The plurality of reference polypeptides are different kinds of proteins, and the size information of each of the plurality of fluorescently labeled reference polypeptides is known; the plurality of reference polypeptides are different in size from one another; A reagent kit for use in the method according to any one of claims 1 to 17.

19. The reagent kit of claim 18, wherein the multiple fluorescently labeled reference polypeptides are each contained in a separate container.

20. A reagent kit comprising a plurality of reference polypeptides and a fluorescently labeled antibody that binds to each of the plurality of reference polypeptides, The plurality of reference polypeptides each have known size information; the plurality of reference polypeptides are different in size from one another; A reagent kit for use in the method for obtaining information on a target polypeptide according to any one of claims 1 to 17.

21. the fluorescently labeled antibodies that bind to each of the plurality of reference polypeptides include a first fluorescently labeled antibody and a second fluorescently labeled antibody that bind to one of the reference polypeptides; the first fluorescent-labeled antibody is labeled with a fluorescent dye having a fluorescent wavelength different from that of the fluorescent dye labeled with the second fluorescent-labeled antibody; the epitope of the first fluorescently labeled antibody is different from the epitope of the second fluorescently labeled antibody; The reagent kit according to claim 20.

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