JP2016183966A - Method for measuring target substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of measuring a target substance specifically with high sensitivity.SOLUTION: Provided is a method for measuring a target substance, the method including: 1) cutting a fluorescent molecule labeled complex including the target substance, a first affinity molecule fixed to a solid phase via a cleavable linker, and a second affinity molecule labeled by a fluorescent molecule and fixed to a solid phase via a cleavable linker, specifically at a portion of the cleavable linker in a solution, and obtaining an ejected solution including the fluorescent molecule labeled complex that is ejected from the solid phase; 2) measuring a fluorescence signal with a confocal fluorescence detector in the ejected solution; 3) determining the presence or quantity of the fluorescent molecule labeled complex included in the ejected solution on the basis of the characteristic of a fluorescence signal specific to the fluorescent molecule labeled complex that is ejected from the solid phase; and 4) evaluating the presence or quantity of the target substance on the basis of the presence or quantity of the fluorescent molecule labeled complex included in the ejected solution.SELECTED DRAWING: None

Description

  The present invention relates to a method for measuring a target substance.

  As a method for measuring a target substance, an immunological method has been conventionally performed. In an immunological method, a target substance is typically measured by contacting the target substance with a labeled antibody to form a labeled complex, and then using the label in the formed labeled complex. . As such a method, a measurement method using a solid phase (eg, Non-Patent Document 1) and a measurement method using a homogeneous liquid phase system without using a solid phase (eg, Patent Document 1, Non-Patent Document 1). ) Etc. have been reported.

  Further, confocal fluorescence detection is used for highly sensitive measurement of a target substance by an immunological method (Patent Document 1, Non-Patent Documents 1 and 2).

JP-A-2005-345311

Todd et al. , Clin. Chem. , 2007, vol. 53, no. 11, pp. 1990-1995 Fujii et al. , Analytical Biochemistry, 2007, vol. 370, pp. 131-141

  The present inventors have not always been able to specifically measure a target substance by confocal fluorescence detection using a conventional immunological method, in other words, the measurement accuracy of the target substance is not sufficient. We found that there was a problem. For example, in such conventional fluorescence detection methods, not only specific signals but also non-specific signals are measured, but these signals are often indistinguishable, so there is room for improving the measurement accuracy of the target substance. It was.

  Specifically, in the method described in Non-Patent Document 1, a fluorescent molecule label complex containing a target substance (magnetic fine particle-streptavidin-biotin-labeled capture antibody-target substance-fluorescent molecule label) fixed to magnetic fine particles. By treating the detection antibody) with a denaturing agent (urea), each component of the fluorescent molecule-labeled complex is dissociated indiscriminately. The solution containing the released fluorescent molecule-labeled antibody is optically analyzed with a confocal detector to detect a fluorescent signal generated from the fluorescent molecule-labeled antibody. In this method, the target substance is measured on the assumption that the number of events of the detected fluorescent signal corresponds to the number of fluorescent molecule-labeled complexes containing the target substance (that is, the number of target substances).

  However, in this method, it is considered that a fluorescent molecule labeled antibody (non-specific substance) nonspecifically bound to the magnetic fine particles is also released from the magnetic fine particles by the treatment of the magnetic fine particles with a denaturant. Therefore, this method has a problem that a non-specific signal generated from a non-specific substance is measured. Further, in this method, it is discriminated whether the measured fluorescent signal is derived from the fluorescent molecule labeled antibody derived from the complex or the fluorescent molecular labeled antibody (non-specific substance) bound nonspecifically to the magnetic fine particles. There is a problem that it cannot be done.

  In the methods described in Patent Document 1 and Non-Patent Document 2, the target substance (prion protein) is measured in a homogeneous liquid phase system without using a solid phase such as magnetic fine particles. These methods measure target substances in confocal fluorescence detection using fluorescence correlation method (FCS) / fluorescence cross correlation method (FCCS), developed as a methodology that enables differential measurement of specific signals. Therefore, at first glance, it is likely that a specific signal generated from a fluorescent molecule labeled complex (specific complex) including a target substance and a fluorescent molecule labeled antibody can be specifically measured.

  However, the present inventors have found that these methods are not sufficient for specific measurement of specific signals, and still have problems.

  First, these methods have a problem (Problem I) that sufficient measurement accuracy cannot be obtained because a large amount of unreacted fluorescent molecule-labeled antibody remains in the liquid phase. When measuring an antigen by an immunological method, generally a larger amount of a capture antibody or a labeled antibody is used than the amount of an antigen as a target substance. Therefore, in a general immunological method, many unreacted fluorescent molecule-labeled antibodies remain in the liquid phase, and many nonspecific signals are detected. If it does so, the ratio of the nonspecific signal in the signal detected will become high, and calculation (fitting) precision will fall as a result. Specifically, in confocal fluorescence detection using FCS / FCCS, for example, what percentage of the detected signal is derived from a non-specific signal (NSS) and what percentage is derived from a specific signal (SS) (ie, NSS And SS ratio) are obtained as output. Then, in a homogeneous liquid phase system containing a large amount of free fluorescent molecules, when the fluorescent molecule labeling complex that generates SS is present in a very small amount compared to the free fluorescent molecule that generates NSS, the value (signal) for SS However, it is hidden within the variation in the value (background) of NSS, and the value of SS cannot be specifically recognized.

  Secondly, in these methods, there is a problem (Problem II) that the specific signal cannot be sufficiently identified when the characteristics (eg, molecular weight) of the non-specific complex are similar to those of the specific complex. Specifically, in these methods, the fluorescent molecule-labeled antibody may bind nonspecifically to contaminants or the like in the sample. Therefore, a nonspecific complex that does not contain a target substance due to such nonspecific binding can be obtained. When a non-specific complex is formed and the characteristics (eg, molecular weight) of the non-specific complex are similar to those of the specific complex, the specific signal generated from the specific complex is sufficiently different from the non-specific signal generated from the non-specific complex. It cannot be identified, and as a result, sufficient measurement accuracy cannot be obtained.

  These problems are also particularly a concern when the amount of target substance in the sample is relatively small and the amount of contaminants and the like in the sample is relatively large.

  Therefore, the first object of the present invention is to improve the measurement accuracy in highly sensitive measurement of a target substance by confocal fluorescence detection.

  In addition, in confocal fluorescence detection using a conventional immunological method, noise derived from a matrix component (fluorescent component) of a measurement sample, for example, mechanical noise derived from a measurement device such as vibration, an electric field from the outside, Due to electrical noise such as electron emission due to thermal fluctuations and induction noise from the ground, non-specific signals are detected even at zero concentration where the fluorescent label is not contained in the measurement sample, and the specific signal is accurate. It may be difficult to detect.

  In fact, Table 1 of Non-Patent Document 1 shows that the signal intensity is not zero even when the fluorescently labeled antibody (target substance) in the measurement sample has a zero concentration. Therefore, the method described in Non-Patent Document 1 has a problem that the detection accuracy of the specific signal is greatly affected and the detection accuracy is lowered particularly when the concentration of the target substance is low.

  Therefore, the second object of the present invention is to provide a fluorescent label detection system and a fluorescent label detection system that can improve detection accuracy by eliminating noise derived from a matrix component (component that emits fluorescence) of a measurement sample. It is to provide a fluorescent label detection method used.

(First purpose)
In a high-sensitivity measurement system based on confocal fluorescence detection, the analyte to be optically detected needs to be in the liquid phase, so that a fluorescent molecule-labeled complex containing the target substance is formed in the liquid phase, It is considered necessary to measure the signal generated from the fluorescent molecules in this complex. In addition, as described in Patent Document 1 and Non-Patent Document 2, it is considered that the specific signal generated from the target complex can be detected more specifically by confocal fluorescence detection using FCS / FCCS. However, in such a measurement system, as described above, it is assumed that sufficient measurement accuracy cannot still be obtained.

  First, as a result of intensive studies under these circumstances, the present inventors have not formed a fluorescent molecule-labeled complex containing a target substance in a liquid phase, but the complex once formed in a solid phase. It was conceived that the measurement accuracy can be improved in high-sensitivity measurement of a target substance by confocal fluorescence detection by specifically releasing the compound from the solid phase into the liquid phase while maintaining the binding state (specific release).

  Specifically, this methodology solves issues I and II as follows.

  First, the fluorescent molecule-labeled complex containing the target substance is not formed in the liquid phase but once in the solid phase, so that the solid phase is removed from the liquid phase system containing a large amount of free fluorescent molecules. It becomes possible to separate. As a result, the proportion of free fluorescent molecules that generate a non-specific signal, and thus the background value, can be reduced, and as a result, the recognition of specific signals can be improved. Therefore, the problem I can be solved by such a methodology.

  Secondly, by specifically releasing the fluorescent molecule-labeled complex containing the target substance formed in the solid phase from the solid phase into the liquid phase while maintaining the binding state, the specific signal can be distinguished. Can be improved. A fluorescent molecular label complex (specific complex) containing a target substance generally has a high molecular weight. As described above, when a non-specific complex containing a fluorescent molecule-labeled antibody and not containing a target substance is formed, and the molecular weight of the non-specific complex is similar to that of the specific complex, the non-specific complex is also It also has a high molecular weight similar to a specific complex. The specific complex once formed in the solid phase is specifically released from the solid phase into the liquid phase while maintaining its binding state (in other words, the non-specific complex non-specifically bound to the solid phase Next, in the liquid phase, the specific signal generated from the fluorescent molecule in the specific complex is measured with a confocal fluorescence detector, whereby the recognition of the specific signal can be improved. Therefore, the problem II can be solved by such a methodology.

Next, the present inventors have intensively studied in order to realize such an idea. As a result, the inventors have made it possible for the specific release to have a target substance, a first affinity molecule immobilized on a solid phase via a cleavable linker, and a second affinity molecule labeled with a fluorescent molecule. The present inventors have found that a fluorescent molecule labeling complex immobilized on a solid phase via a cleavable linker can be used as a solid phase system, and the present invention has been completed.
As far as the present inventors know, there is no known method for realizing high-sensitivity measurement of a target substance by using both solid phase and confocal fluorescence detection.

That is, the present invention is as follows.
[1] Method for measuring target substance, including:
1) Immobilized to a solid phase via a cleavable linker, including a target substance, a first affinity molecule immobilized on a solid phase via a cleavable linker, and a second affinity molecule labeled with a fluorescent molecule Cleaving the fluorescent molecule labeling complex specifically in the solution at the site of the cleavable linker to obtain a release solution comprising the fluorescent molecule labeling complex released from the solid phase;
2) measuring the fluorescence signal in the release solution with a confocal fluorescence detector;
3) determining the presence or amount of the fluorescent molecular label complex contained in the release solution based on the characteristics of the fluorescent signal specific to the fluorescent molecular label complex released from the solid phase; and 4) Assessing the presence or amount of the target substance based on the presence or amount of the fluorescent molecular label complex contained in the release solution.
[2] The method according to [1], wherein the cleavable linker is a covalent linker or an affinity linker.
[3] The method of [1] or [2], wherein the target substance is an antigen, and each of the first affinity molecule and the second affinity molecule is an antibody.
[4] The method according to [3], wherein the first affinity molecule is an antibody against a first epitope of an antigen, and the second affinity molecule is an antibody against a second epitope of an antigen.
[5] The method according to any one of [1] to [4], further comprising preparing a fluorescent molecule-labeled complex by contacting a sample containing a target substance with the first affinity molecule and the second affinity molecule. Either way.
[6] The method of [5], further comprising washing the solid phase during and / or after the preparation of the fluorescent molecule-labeled complex.
[7] The method according to any one of [1] to [6], wherein the step 1) is performed as follows:
1-1) A release solution containing a fluorescent molecule-labeled complex released from a solid phase by specifically cleaving the fluorescent molecule-labeled complex at a site of a cleavable linker in the solution, and a two-phase containing a solid phase Obtaining the system; and 1-2) removing the solid phase from the two-phase system to obtain a release solution containing the fluorescent molecule-labeled complex and no solid phase.
[8] The method according to any one of [1] to [7], wherein the step 2) is performed by a photon counting method.
[9] The method according to any one of [1] to [8], wherein the property is a molecular weight or a fluorescence property.
[10] The method according to any one of [1] to [9], wherein the step 3) is performed using a fluorescence correlation method or a fluorescence cross-correlation method.
[11] The method according to any one of [1] to [10], wherein the solid phase is a magnetic bead.
[12] The method according to any one of [1] to [11], which is a method for measuring a target substance including:
a1) A sample containing a target substance is brought into contact with a first affinity molecule immobilized on a solid phase via a cleavable linker and a second affinity molecule labeled with a fluorescent molecule in a liquid phase, and the target substance A first affinity molecule immobilized on a solid phase via a cleavable linker, and a fluorescent molecule labeling complex immobilized on the solid phase via a cleavable linker, comprising the second affinity molecule, and a liquid phase Obtaining a first two-phase system comprising:
a2) removing the liquid phase from the first two-phase system to obtain the fluorescent molecule-labeled complex;
a3) A release solution containing the fluorescent molecule labeling complex released from the solid phase by specifically cleaving the fluorescent molecule labeling complex at the site of the cleavable linker in the solution, and a second second phase containing the solid phase Obtaining a system;
a4) removing the solid phase from the second two-phase system to obtain a release solution containing a fluorescent molecule-labeled complex and no solid phase;
a5) measuring the fluorescence signal in the release solution with a confocal fluorescence detector;
a6) determining the presence or amount of the fluorescent molecule label complex contained in the release solution based on the characteristics of the fluorescent signal specific to the fluorescent molecule label complex released from the solid phase; and a7) Assessing the presence or amount of the target substance based on the presence or amount of the fluorescent molecular label complex contained in the release solution.

(Second purpose)
The present inventors have also intensively investigated the elimination of noise derived from the matrix component (fluorescent component) of the measurement sample. As a result, by combining the pulse counting method and the analysis method such as FCS and FCCS, the above-described problem can be obtained. As a result, the present invention has been completed.

That is, the present invention provides the following [1 ′] to [8 ′].
[1 ′] a confocal detector that detects fluorescent photons generated in the measurement sample;
It is connected to the confocal detection unit, generates primary measurement data based on the pulse signal data generated by the confocal detection unit, calculates the presence ratio of the fluorescent label or the target substance containing the fluorescent label, A fluorescent label detection system including a fluorescent label or a calculation unit that determines the number of target substances including the fluorescent label based on the primary measurement data and the abundance ratio to form secondary measurement data.
[2 ′] The confocal detection unit is connected to the laser beam generation unit that generates and outputs a laser beam, and irradiates the measurement sample with the laser beam. The fluorescent label detection system according to [1 ′], including an optical processing unit that emits the fluorescent photons generated in step 1 and a detection unit that is connected to the optical processing unit and detects the fluorescent photons.
[3 ′] The detection unit is connected to the optical processing unit, the fluorescent photons emitted from the optical processing unit are incident, and the incident fluorescent photons are converted into an electrical signal based on the amount of light. [2 ′] including a sensor unit that outputs and an electrical signal processing unit that is connected to the sensor unit, amplifies the electrical signal output by the sensor unit, converts the electrical signal into an electrical pulse signal, and outputs the electrical pulse signal. The fluorescent label detection system according to 1.
[4 ′] The detection unit is connected to the electrical signal processing unit, the electrical pulse signal output from the electrical signal processing unit is input, and count data is formed based on the input electrical pulse signal, The fluorescent label detection system according to [3 ′], further including a counting unit that outputs the same.
[5 ′] The calculation unit records the pulse signal data output from the detection unit, re-counts the pulse signal data to calculate re-count data, compares the re-count data with a threshold value, The number of valid events exceeding the threshold is counted to generate primary measurement data, autocorrelation function data is generated based on the pulse signal data, and the autocorrelation function data is statistically processed to obtain a fluorescent label or a fluorescent label A calculation unit that calculates the abundance ratio of a target substance containing a fluorescent label based on the primary measurement data and the abundance ratio, or generates a secondary measurement data by determining the number of target substances containing the fluorescence label. The fluorescent label detection system according to any one of [2 ′] to [4 ′].
[6 ′] a step in which the confocal detection unit detects fluorescent photons generated in the measurement sample, and generates pulse signal data based on the number of detected fluorescent photons;
A calculation unit calculates a primary measurement value based on the pulse signal data and generates primary measurement data; and
The calculation unit calculates a fluorescent label based on the pulse signal data or a ratio of the target substance containing the fluorescent label; and
And a step of generating a secondary measurement data by determining the number of fluorescent labels or a target substance containing the fluorescent label based on the primary measurement data and the abundance ratio.
[7 ′] In the step of generating the primary measurement data, the calculation unit re-counts the pulse signal data with a predetermined binning width to generate re-count data, and the signal intensity value and threshold value of each event of the re-count data The method for detecting a fluorescent label according to [6 ′], further comprising a step of re-counting only events exceeding the threshold value to calculate a primary measurement value and generating primary measurement data.
[8 '] The step of calculating the existence ratio includes the step of calculating the autocorrelation function based on the pulse signal data by the calculation unit and generating autocorrelation function data, and the calculation unit calculating the autocorrelation function data. The method for detecting a fluorescent label according to [6 ′] or [7 ′], comprising a step of statistically processing and calculating a fluorescent label or a ratio of a target substance containing the fluorescent label.

Since the target substance can be specifically measured, the method of the present invention is useful for qualitative and quantitative measurement of the target substance. The method of the present invention can also detect a target substance with extremely high sensitivity even when the sample contains only an extremely small amount of the target substance.
Further, according to the present invention, there is provided a fluorescent label detection system capable of detecting and quantifying a fluorescent label more precisely and specifically even at an extremely low concentration, and a fluorescent label detection method using such a fluorescent label detection system. Can be provided.

FIG. 1 shows labeling with a target substance, a solid phase on which a first affinity molecule having a cleavable linker is immobilized (that is, a first affinity molecule immobilized on a solid phase via a cleavable linker), and a fluorescent molecule. It is a schematic diagram which shows an example (sandwich method) of the preparation of the fluorescent molecule label complex fixed to the solid phase via the cleavable linker by the contact of the made second affinity molecule. The washing of the solid phase is an optional step, but preferably the solid phase is washed. FIG. 2 is a schematic diagram showing the release of a fluorescent molecule labeling complex from a solid phase by specifically cleaving the fluorescent molecule labeling complex immobilized on the solid phase via a cleavable linker at the site of the cleavable linker. FIG. FIG. 3 is a diagram showing an outline of measuring a fluorescence signal generated from a fluorescent molecule in a fluorescent molecule-labeled complex released from a solid phase with a confocal fluorescence detector. FIG. 4 shows that a specific signal generated from a fluorescent molecule in a fluorescent molecule labeling complex is distinguished from a nonspecific signal based on characteristics specific to the fluorescent molecule labeling complex released from the solid phase in the release solution. FIG. 2 is a diagram showing an example of measurement with a confocal fluorescence detector [combination of single molecule pulse counting and fluorescence cross-correlation (FCS)]. FCS can determine the molecular weight by measuring the fluctuation (diffusion coefficient) of molecules in the confocal area, and can identify the abundance of specific signals and the abundance of non-specific signals with respect to fluorescent signals. FIG. 5 is a schematic functional block diagram illustrating a configuration example of the fluorescence detection system. FIG. 6 is a schematic functional block diagram illustrating a configuration example of the detection unit. FIG. 7 is a flowchart for explaining the fluorescence detection method. FIG. 8 is a graph showing an example of the recount data. FIG. 9 is a graph showing an example of autocorrelation function data. FIG. 10 is a diagram showing an example of recount data obtained by recounting the fluorescence signal data of the fluorescent molecule labeled complex cleaved through a sugar linker, measured by a confocal fluorescence detector. FIG. 11 is a diagram showing the relationship between the AFP concentration (ng / mL) and the abundance ratio (%) of the fluorescently labeled complex in the conventional method (Comparative Example 1). FIG. 12 is a diagram showing the relationship between AFP concentration (ng / mL) and secondary measurement data in the method of the present invention.

The present invention provides a method for measuring a target substance.
The method of the present invention includes:
1) Immobilized to a solid phase via a cleavable linker, including a target substance, a first affinity molecule immobilized on a solid phase via a cleavable linker, and a second affinity molecule labeled with a fluorescent molecule Cleaving the fluorescent molecule-labeled complex specifically at the site of the cleavable linker in the solution to obtain a release solution containing the fluorescent molecule-labeled complex released from the solid phase (cleavage reaction);
2) measuring the fluorescence signal in the release solution with a confocal fluorescence detector (counting);
3) determining the presence or amount of the fluorescent molecular label complex contained in the release solution based on the characteristics of the fluorescent signal specific to the fluorescent molecular label complex released from the solid phase (detection); and 4) Evaluating the presence or amount of the target substance based on the presence or amount of the fluorescent molecule-labeled complex contained in the release solution (evaluation).

1) Cleavage reaction In this step, a target substance, a first affinity molecule immobilized on a solid phase via a cleavable linker, and a second affinity molecule labeled with a fluorescent molecule are used via a cleavable linker. The fluorescent molecular label complex immobilized on the solid phase is specifically cleaved at the site of the cleavable linker in the solution. Thereby, the release solution containing the fluorescent molecule label complex released from the solid phase can be obtained.

  In the present invention, a cleavable linker refers to a molecule or molecular complex that can be cleaved specifically by a specific treatment according to the type. By specifically cleaving the cleavable linker, the release of a substance that is non-specifically bound to the solid phase (eg, a non-specific substance as described below) is suppressed, and the fluorescent molecule-labeled complex immobilized on the solid phase The body can be released into solution. By using the obtained release solution for the measurement, it is possible to reduce the background noise and improve the S / N (signal / noise) ratio. Examples of the cleavable linker include a covalent linker and an affinity linker.

  A covalent linker is a molecule that can be specifically cleaved through cleavage of a covalent bond by a specific treatment according to its type, and the fluorescent molecule-labeled complex is immobilized on the solid phase through the covalent bond. Used for. Examples of the treatment for specifically cleaving the covalent linker include a treatment with a cleaving agent (substance) such as an enzyme and a stimulus (non-substance).

  An affinity linker is a molecular complex that can be specifically cleaved (dissociated) through the dissociation of an affinity substance by a specific treatment according to its type, and a fluorescent molecular label complex through an affinity substance. Is used to fix to the solid phase. The treatment for specifically cleaving the affinity linker includes, for example, treatment with a competitor (eg, one of a pair of affinity substances and similar substances).

  The fluorescent molecule label complex includes a target substance, a first affinity molecule immobilized on a solid phase via a cleavable linker, and a second affinity molecule labeled with a fluorescent molecule.

  The target substance to be measured by the method of the present invention is not particularly limited as long as it is a substance that can be measured by the method of the present invention. For example, amino acid polymers (eg, oligopeptides, polypeptides, proteins), sugars (eg , Oligosaccharides, polysaccharides), nucleic acids (eg, DNA, RNA), and low molecular weight substances, and derivatives thereof. The target substance may also be a component of an immunological complex (eg, antigen or antibody). A preferred example of the target substance is an antigen. The method of the present invention can measure an antigen in a sample. Examples of such antigens include antigens that are allergenic agents, antigens derived from pathogenic microorganisms, and antigens that are biomarkers such as disease markers (tumor markers, heart disease markers, and kidney disease markers). Another preferred example of the target substance is an antibody. The method of the present invention can measure antibodies in a sample (eg, autoantibodies in blood).

The target substance can also be classified into a low molecular substance and a substance having a high molecular weight from the viewpoint of molecular weight.
The term “small substance” refers to a compound having a molecular weight of less than 1,500. The low molecular weight substance is a natural substance or a synthetic substance. The molecular weight of the low molecular weight substance may be less than 1,200, less than 1,000, less than 800, less than 700, less than 600, less than 500, less than 400, or less than 300. The molecular weight of the low molecular weight substance may also be 50 or more, 100 or more, 150 or more, or 200 or more. Examples of low molecular weight substances include vitamins (eg, vitamin D such as vitamin D2 and vitamin D3), hormones (eg, steroid hormones such as follicular hormone, thyroid hormone), amino acid compounds, ligands, lipids, fatty acids, opioids, nerves Examples include transmitters (eg, catecholamines), nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, and oligopeptides, or pharmaceuticals, toxicants, and metabolites.
The term “substance having a high molecular weight” refers to a compound having a molecular weight of 1,500 or more. A substance having a high molecular weight is, for example, 2,000 or more, 3,000 or more, 5,000 or more, 10,000 or more, 15,000 or more, 20,000 or more, 30,000 or more, or 50,000 or more. It may have a molecular weight. A substance having a high molecular weight may also have a molecular weight of, for example, less than 1,000,000, less than 500,000, or less than 300,000.

  The first affinity molecule and the second affinity molecule are fluorescent molecules immobilized on a solid phase, including a target substance, a first affinity molecule immobilized on the solid phase, and a second affinity molecule labeled with a fluorescent molecule. The label complex is not particularly limited as long as it can be formed by affinity binding (eg, immunological reaction). Such affinity molecules include, for example, components of immunological complexes (eg, antibodies and antigens). For example, when the target substance is an antigen, an antibody is suitably used as the first affinity molecule and the second affinity molecule. On the other hand, when the target substance is an antibody, an antigen is suitably used as the first affinity molecule, and a desired molecule can be appropriately selected as the second affinity molecule.

Preferably, each of the first affinity molecule and the second affinity molecule is an antibody. In such a case, any antibody can be used as the first affinity molecule and the second affinity molecule, but these affinity molecules are preferably monoclonal antibodies. Each of the first antibody and the second affinity molecule may be any isotype of immunoglobulin (eg, IgG, IgM, IgA, IgD, IgE, IgY). The first antibody and the second antibody may also be full length antibodies. A full-length antibody refers to an antibody comprising a heavy chain and a light chain comprising a variable region and a constant region, respectively (eg, an antibody comprising two Fab portions and an Fc portion). The first antibody and the second antibody may also be antibody fragments derived from such full-length antibodies. The antibody fragment is a part of a full-length antibody, and examples thereof include constant region deleted antibodies (eg, F (ab ′) ₂ , Fab ′, Fab, Fv). The first antibody and the second antibody may also be modified antibodies such as a single chain antibody.

  Examples of the solid phase include a solid phase that can contain or mount a liquid phase (eg, a support such as a plate, a membrane, or a test tube, and a container such as a well plate, a microchannel, a glass capillary, a nanopillar, or a monolith column). And a solid phase that can be suspended or dispersed in a liquid phase (eg, a solid phase carrier such as beads). Examples of the solid phase material include glass, plastic, metal, and carbon. A non-magnetic material or a magnetic material can also be used as the solid phase material, but a magnetic material is preferred from the viewpoint of ease of operation. The solid phase is preferably a solid phase carrier, more preferably a magnetic solid phase carrier, and even more preferably a magnetic bead.

The first affinity molecule immobilized on the solid phase via a cleavable linker can be prepared by a known method.
When the cleavable linker is a covalent bondable linker, the first affinity molecule may be immobilized on the cleavable linker after the cleavable linker is immobilized on the solid phase, or the cleavable linker is bound to the first affinity molecule. Thereafter, the first affinity molecule may be immobilized on the solid phase via the cleavable linker. When the cleavable linker is an affinity linker, after fixing one of the pair of affinity substances to the solid phase and binding the other to the first affinity molecule, the first affinity molecule is determined by the affinity between the affinity substances. Can be immobilized on a solid phase. The fixing of the cleavable linker to the solid phase and the binding of the first affinity molecule and the cleavable linker may be any type of binding as long as a specific cleavage reaction of the cleavable linker can be appropriately performed. For example, it can be performed by physical adsorption, covalent bond, ionic bond, bond utilizing affinity, and combinations thereof. For example, covalent bonding can be achieved using a cross-linking agent. Examples of such cross-linking agents include maleimide reagent, carbodiimide reagent, glutaraldehyde, and cyanuryl chloride (peptide synthesis method, Maruzen Co., Ltd. (Showa 50); enzyme immunoassay, Kyoritsu Shuppan (1987) ); Protein Nucleic Acid Enzyme See the separate volume No. 31 (1987)). Examples of the maleimide reagent include N-succinimidyl maleimide acetic acid, N-succinimidyl 4-maleimidobutyric acid (GMBS), N-succinimidyl maleimide hexanoic acid, N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1- Carboxylic acid, N-sulfosuccinimidyl 4-maleimidomethyl-cyclohexane-1-carboxylic acid. Examples of the carbodiimide reagent include dicyclohexylcarbodiimide (DCC) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC). The carbodiimide reagent can achieve the fixation of the cleavable linker to the solid phase and the binding of the first affinity molecule and the cleavable linker by introducing a reactive group (eg, amide group, ester group).

  As the fluorescent molecule, a fluorescent molecule generally used as a fluorescent labeling reagent in this technical field can be used. For example, Alexa Fluor (registered trademark) compounds (eg, Alexa Fluor 532, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 633, Alexa Fluor 647), ATTO compounds (ATTO 488, ATTO 532, ATTOTO N6 Isothiocyanate), 6-FAM (6-carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein), HEX (hexachlorofluorescein), 6-JOE (6-carboxy-4 ′, 5′-dichloro-2 ′, 7'-dimethoxyfluorescein), ROX (carboxy-6-rhodamine), and TAMRA ((6-tetramethylrhodamine) N-5 (6) -carboxamide) hexanoate), and fluorescent proteins such as R-phycoerythrin (red fluorescent protein) and GFP (green fluorescent protein).

  The second affinity molecule labeled with a fluorescent molecule can be prepared by a known method that enables the antibody to be labeled with the fluorescent molecule. The binding between the second affinity molecule and the fluorescent molecule may be in any binding mode as long as the specific cleavage reaction of the cleavable linker can be appropriately performed, such as physical adsorption, covalent bond, ionic bond, It can be performed by binding utilizing affinity, a combination thereof, or the like. For example, it can be performed using a crosslinking agent as described above.

  Specifically, the preparation of the first affinity molecule immobilized on the solid phase and the preparation of the second affinity molecule labeled with a fluorescent molecule are described in JP-A-3-115862 and JP-A-10-197535. It may be performed by a method disclosed in a previously published report.

  The solution used in the cleavage reaction does not inhibit cleavage at the site of the cleavable linker and retains the fluorescent molecule-labeled complex (eg, target substance, first affinity molecule immobilized on the solid phase, and fluorescence). Any suitable aqueous solution may be used as long as it enables the maintenance of the affinity binding of the molecule-labeled second affinity molecule. Examples of such an aqueous solution include water (eg, distilled water, sterilized water, sterilized distilled water, pure water), and a buffer solution. Examples of the buffer include Tris-based buffers such as Tris-HCl and TE (Tris-EDTA) buffers, phosphate buffers, and carbonate buffers. The pH of the buffer is appropriately near neutral. Such pH is, for example, pH 5.0 to 9.0, preferably 5.5 to 8.5, more preferably 6.0 to 8.0, and even more preferably 6.5 to 7.5. The solution used in the cleavage reaction may contain a small amount of an organic solvent (eg, alcohol) and other components.

  The volume of the solution used in the cleavage reaction is generally, for example, 1-1000 μL, preferably 3-500 μL, more preferably 5-200 μL, and even more preferably 10-100 μL.

  The specific cleavage reaction can be performed by an appropriate treatment according to the type of the cleavable linker in the fluorescent molecule labeling complex (for example, see Tables 1 and 2). For example, when a fluorescent molecule-labeled complex is treated with a substance (eg, a cleaving agent and a competitor), the cleavage reaction is usually 4 to 45 ° C. (eg 10 to 42 ° C., preferably 15 to 40 ° C.) for 1 second. For about 6 hours (for example, 3 seconds to 1 hour, preferably 5 seconds to 10 minutes).

  In one embodiment, the first affinity molecule is an affinity molecule for the first target site of the target substance, and the second affinity molecule is an affinity molecule for the second target site of the target substance. In the method of the present invention, by using such a combination of the first affinity molecule and the second affinity molecule, a specific signal generated from the fluorescent molecule in the fluorescent molecule-labeled complex can be measured. Preferably, the first affinity molecule is an antibody against the first epitope of the antigen and the second affinity molecule is an antibody against the second epitope of the antigen.

  In another embodiment, the first affinity molecule is an affinity molecule for the target site of the target substance, and the second affinity molecule is an affinity molecule for the complex comprising the target substance and the first affinity molecule. is there. In the method of the present invention, by using such a combination of the first affinity molecule and the second affinity molecule, a specific signal generated from the fluorescent molecule in the fluorescent molecule-labeled complex can be measured. Preferably, the first affinity molecule is an antibody against an epitope of the antigen and the second affinity molecule is an antibody against a complex comprising the antigen and the first affinity molecule. Such a second affinity molecule can be prepared, for example, using the method described in WO2013 / 042426.

  The method of the present invention includes a target substance, a first affinity molecule immobilized on a solid phase via a cleavable linker, and a second affinity molecule labeled with a fluorescent molecule. The method may further comprise providing a fluorescent molecular label complex immobilized on the substrate. For example, the method of the present invention comprises contacting a sample containing a target substance with a first affinity molecule immobilized on a solid phase via a cleavable linker and a second affinity molecule labeled with a fluorescent molecule, It may further comprise preparing a fluorescent molecular label complex immobilized on a solid phase via a cleavable linker. Such preparation can be performed by a known method such as a sandwich method such as a one-step method, a delay one-step method, and a two-step method. The contact is performed by incubating the target substance, the first affinity molecule, and the second affinity molecule simultaneously or with a time difference. Incubation is usually performed at 4 to 45 ° C. (for example, 10 to 42 ° C., preferably 15 to 40 ° C.) for about 1 second to 6 hours (for example, 3 seconds to 1 hour, preferably 10 seconds to 10 minutes).

  The sample used in the method of the present invention is a sample containing the target substance as described above. The origin of the sample is not particularly limited, and may be a biological sample derived from a living organism, or an environmental sample. Examples of organisms from which biological samples are derived include animals such as mammals (eg, humans, monkeys, mice, rats, rabbits, cows, pigs, horses, goats, sheep), birds (eg, chickens), insects, and the like. , Microorganisms, plants, fungi, and fish, preferably mammals, and still more preferably humans. Biological samples also include, for example, blood related samples (eg, whole blood, serum, plasma), saliva, urine, milk, tissue and cell extracts, and mixtures thereof. Examples of the environmental sample include samples derived from soil, seawater, and fresh water.

  In the method of the present invention, the sample may be subjected to pretreatment prior to preparation of the fluorescent molecular label complex. Examples of such pretreatment include centrifugation, extraction, filtration, precipitation, heating, freezing, refrigeration, and stirring.

(Removal of non-specific substances in the liquid phase by washing)
Where a fluorescent molecular label complex is prepared, the method of the present invention preferably further comprises washing the solid phase during and / or after preparation of the fluorescent molecular label complex. Washing may take place at different stages during and / or after preparation, preferably at one or more (eg, 1-3) different stages during preparation and after preparation. The frequency | count of washing | cleaning in each step is 1-10 times, for example, Preferably it is 2-8 times, More preferably, it is 3-6 times. By such washing, non-specific substances and the like in a trace amount solution adhering to the solid phase can be excluded. The washing solution retains the fluorescent molecule-labeled complex immobilized on the solid phase (eg, affinity binding of the target substance, the first affinity molecule immobilized on the solid phase, and the second affinity molecule labeled with the fluorescent molecule). Is not particularly limited as long as it enables the maintenance of the above), and an appropriate aqueous solution can be used. Examples of such an aqueous solution include water (eg, distilled water, sterilized water, sterilized distilled water, pure water), and a buffer solution. Examples of the buffer include Tris-based buffers such as Tris-HCl and TE (Tris-EDTA) buffers, phosphate buffers, and carbonate buffers. The pH of the buffer is appropriately near neutral. Such pH is, for example, pH 5.0 to 9.0, preferably 5.5 to 8.5, more preferably 6.0 to 8.0, and even more preferably 6.5 to 7.5. The cleaning liquid may contain a small amount of an organic solvent (eg, alcohol) and other components.

Specifically, the preparation of the fluorescent molecular label complex may be performed as follows:
a1) A sample containing a target substance is brought into contact with a first affinity molecule immobilized on a solid phase via a cleavable linker and a second affinity molecule labeled with a fluorescent molecule in a liquid phase, and the target substance A fluorescent molecule-labeled complex immobilized on a solid phase via a cleavable linker, comprising: a first affinity molecule immobilized on a solid phase via a cleavable linker; and a second affinity molecule labeled with a fluorescent molecule Obtaining a two-phase system comprising a body (solid phase) and a liquid phase; and a2) removing the liquid phase from the two-phase system and immobilizing the fluorescent molecule labeled complex immobilized on the solid phase via a cleavable linker To obtain a body (solid phase).

a1) may be performed by the following steps:
a1-1) A sample containing a target substance is brought into contact with a first affinity molecule immobilized on a solid phase via a cleavable linker in a liquid phase, and the target substance and the solid substance via a cleavable linker are brought into contact with the solid phase. Obtaining a first two-phase system comprising an intermediate complex (solid phase) immobilized on a solid phase via a cleavable linker, comprising an immobilized first affinity molecule, and a liquid phase;
a1-2) removing the liquid phase from the first two-phase system to obtain an intermediate complex (solid phase) fixed to the solid phase via a cleavable linker; and a1-3) via a cleavable linker The intermediate complex (solid phase) immobilized on the solid phase is brought into contact with the second affinity molecule labeled with a fluorescent molecule in the liquid phase and immobilized on the solid phase via the target substance and a cleavable linker. A fluorescent molecule-labeled complex (solid phase) immobilized on a solid phase via a cleavable linker, comprising a first affinity molecule and a second affinity molecule labeled with a fluorescent molecule, and a liquid phase Obtain a second two-phase system.

  In a1), the contact is carried out by incubating the target substance, a first affinity molecule immobilized on the solid phase via a cleavable linker, and a second affinity molecule labeled with a fluorescent molecule. The contact conditions (eg, incubation conditions) are the same as those described above.

  In a1-1), the contact is performed by incubating the target substance and the first affinity molecule immobilized on the solid phase via a cleavable linker. The contact conditions (eg, incubation conditions) are the same as those described above.

  In a1-2), the non-specific substance derived from the sample and the non-specific substance derived from the liquid containing the first affinity molecule fixed to the solid phase via the cleavable linker are removed by removing the liquid phase. be able to. Further, after a1-2), it is preferable to wash the intermediate complex (solid phase) fixed to the solid phase via a cleavable linker. By such washing, non-specific substances in a trace amount solution adhering to the solid phase can be excluded. Cleaning conditions such as the cleaning liquid and the number of cleanings are the same as those described above.

  In a1-3), the contact is carried out by incubating an intermediate complex immobilized on the solid phase via a cleavable linker and a second affinity molecule labeled with a fluorescent molecule. The contact conditions (eg, incubation conditions) are the same as those described above.

  In a2), by removing the liquid phase, the non-specific substance derived from the sample, or the non-specific substance derived from the liquid containing the first affinity molecule immobilized on the solid phase via the cleavable linker (or the cleavable linker) The non-specific substance derived from the liquid containing the intermediate complex immobilized on the solid phase) and the non-specific substance derived from the liquid containing the second affinity molecule labeled with the fluorescent molecule can be excluded. Moreover, after a2), it is preferable to wash the fluorescent molecule label complex (solid phase) fixed to the solid phase via a cleavable linker. By such washing, non-specific substances in a trace amount solution adhering to the solid phase can be excluded. Cleaning conditions such as the cleaning liquid and the number of cleanings are the same as those described above.

(Removal of non-specifically bound substances on the solid phase)
In 1), a release solution containing the fluorescent molecule-labeled complex released from the solid phase is obtained by the cleavage reaction. Since the substance nonspecifically bound to the solid phase can maintain the state bound to the solid phase even after the cleavage reaction, it is possible to collect the release solution from the system after the cleavage reaction (in other words, after the cleavage reaction). By removing the solid phase from the system, substances that are non-specifically bound to the solid phase (eg, labeled molecules, second affinity molecules labeled with labeled molecules) can be eliminated. Some of the substances that are nonspecifically bound to the solid phase may be released from the solid phase to the liquid phase in the course of treatment. Incorporation of specifically bound substances into the release solution can be avoided.

Therefore, in order to avoid contamination of the release solution with the substance nonspecifically bound to the solid phase during the treatment, the step 1) is preferably performed as follows:
1-1) Immobilization to a solid phase via a cleavable linker comprising a target substance, a first affinity molecule immobilized on a solid phase via a cleavable linker, and a second affinity molecule labeled with a fluorescent molecule The resulting fluorescent molecule-labeled complex is specifically cleaved at the site of the cleavable linker in the solution to obtain a release solution containing the fluorescent molecule-labeled complex released from the solid phase and a two-phase system containing the solid phase And 1-2) the release solution containing the fluorescent molecule labeled complex released from the solid phase, and the solid phase is removed from the two-phase system including the solid phase, the fluorescent molecule labeled complex is included, and the solid phase To obtain a release solution free of

By the way, when a fluorescent molecule labeling complex is prepared, as described above in the steps a1-2) and a2), removal of a liquid phase that may contain a nonspecific substance derived from a sample or the like, and / or a nonspecific substance The non-specific substance in the liquid phase and the trace solution can be eliminated by washing the solid phase to which the trace solution that can contain the liquid is attached. Thus, the release solution has already been treated by the above-described process so as not to contain non-specific substances that are easily distributed in the liquid phase.
On the other hand, in the step of obtaining the release solution [particularly the steps of 1-1) and 1-2), it is possible to avoid contamination of the release solution with a substance nonspecifically bound to the solid phase. Therefore, the release solution has already been processed so as not to contain non-specific substances that are easily adsorbed to the solid phase by the above-described steps.
Therefore, according to the method of the present invention, both non-specific substances that are easily distributed in the liquid phase and non-specific substances that are easily adsorbed to the solid phase can be excluded. It can be reduced efficiently, and the S / N (signal / noise) ratio can be improved. That is, the method of the present invention realizes highly sensitive measurement of a target target substance by physically removing both a non-specific substance that is easily distributed in a liquid phase and a non-specific substance that is easily adsorbed to a solid phase. be able to.

2) Measuring the fluorescence signal with a confocal fluorescence detector (counting)
In this step, the fluorescence signal is measured with a confocal fluorescence detector in the release solution. Thereby, measurement data (pulse signal data) of the fluorescence signal based on the number of photons generated in the release solution is obtained.

  The step of measuring the fluorescence signal by the confocal fluorescence detector can be performed by a photon counting method in which the number of photons is counted based on an electric pulse signal obtained by binarizing the signal. This provides pulse signal data based on the number of photons generated in the release solution.

  The pulse signal data obtained by the photon counting method is data relating to a time-series electric pulse signal, and may be time-series electric pulse signal data, or the time-series electric pulse signal may be converted into a signal number per arbitrary time (count number / Count data counted as seconds, Hz) may also be used. This count data is data relating to the number of signals per predetermined unit time (photometric binning time), for example, and “number of counted fluorescent photons / second” or “Hz” is used as the unit.

3) Determination of the presence or amount of the fluorescent molecule label complex contained in the release solution In this step, based on the characteristics of the fluorescent signal specific to the fluorescent molecule label complex, the fluorescent molecule label complex contained in the release solution The presence or amount of is determined.

  The release solution described above contains not only a fluorescent molecule-labeled complex released from the solid phase that generates a target specific signal (SS), but also a small amount of a non-specific substance that generates a non-target nonspecific signal (NSS). May contain. Such non-specific substances include 1) a free fluorescent molecule, 2) a second affinity molecule labeled with a fluorescent molecule, 3) a second affinity molecule labeled with a fluorescent molecule, and a target substance. Examples include incomplete complexes that do not include one affinity molecule, and 4) complexes that do not include a target substance, including a second affinity molecule labeled with a fluorescent molecule. Examples of the complex that does not include a target substance and includes a second affinity molecule labeled with a fluorescent molecule include 4a) a second affinity molecule labeled with a fluorescent molecule, and a first affinity molecule, A non-specific complex containing no target substance, and 4b) a non-specific complex comprising a second affinity molecule labeled with a fluorescent molecule, and a contaminant (eg, a contaminant in a sample containing the target substance). Can be mentioned. Even if a release solution is obtained by performing the above-mentioned method (eg, the method of 1) above for the purpose of completely eliminating non-specific substances that generate NSS, only a small part of non-specific substances that generate NSS is obtained. Can be mixed into the release solution. Therefore, simply counting in this case can measure not only SS but also NSS, which may hinder accurate measurement of the target substance. On the other hand, characteristics such as molecular weight may be different between a fluorescent molecular label complex released from a solid phase that generates SS and a small amount of non-specific substance that generates NSS. Therefore, based on the characteristics of the fluorescent signal specific to the fluorescent molecule-labeled complex released from the solid phase, the presence or amount of the fluorescent molecule-labeled complex contained in the release solution can be determined to obtain a more accurate target substance. Measurement is possible.

  The presence or amount of the fluorescent molecular label complex can be determined by utilizing the characteristics specific to the fluorescent molecular label complex released from the solid phase. The characteristic specific to the fluorescent molecular label complex released from the solid phase is a characteristic that allows the fluorescent molecular label complex released from the solid phase to be distinguished from a non-specific substance that generates a non-specific signal. The fluorescent molecule-labeled complex released from the solid phase is a complex in which the target substance, the first affinity molecule cleaved by the cleavable linker, and the second affinity molecule labeled with the fluorescent molecule are bound to each other. Therefore, it is possible to use a characteristic that can evaluate such a binding state as an index. Therefore, specific characteristics of the fluorescent molecule labeling complex released from the solid phase that can be used in the present invention include, for example, molecular weight (molecular size) and fluorescent characteristics (eg, labeling with two or more fluorescent molecules). Resulting from a difference in the labeling mode of the complex, and due to a change in fluorescence intensity due to interaction).

  Specifically, the presence or amount of the fluorescent molecule labeling complex can be determined using, for example, fluorescence correlation spectroscopy (FCS) (Analytical Biochemistry, 2007, vol. 370, pp. 131-141). Chem., Phys., 4, 390-401, 1974; Biopolymers, 13, 1-27, 1974; Physical Rev. A, 10: 1938-19945, 1974; in Topics in Fluorescence Spectroscopy, 1, pp. 337-378. , Plenum Press, New York and London, 1991; R. Rigler, ES Elson (Eds.), Fluorescence Correlation Speciosos. opy.Theory and Applications, Springer, Berlin, 2001). According to FCS, SS can be obtained by analyzing the measured fluorescence signal based on the difference in molecular weight. FCS uses a laser confocal microscope system to capture the Brownian motion of a target molecule labeled with fluorescence in a minute region, and then analyzes the diffusion time from fluctuations in fluorescence intensity using an autocorrelation function. This is performed by measuring (molecular weight, number of molecules), and analysis by FCS that captures molecular fluctuations in such a minute region is effective in detecting intermolecular interactions with high sensitivity. Means.

  The determination of the presence or amount of fluorescent molecular labeling complexes can also be performed using fluorescence cross-correlation (FCCS) (Analytical Biochemistry, 2007, vol. 370, pp. 131-141; Biochemistry, Vol. 82). No. 12, pp. 1103-1116, 2010; Biophysics 48 (5), 290-293, 2008). When FCCS is performed, the first affinity molecule immobilized on the solid phase is used as the first affinity molecule immobilized on the solid phase, and the first affinity molecule is labeled with the fluorescent molecule. It is necessary that the wavelength of the fluorescent molecule used in the above is different from the wavelength of the fluorescent molecule used for labeling the second affinity molecule. In FCCS, these two types of fluorescence intensity fluctuations can be analyzed using a cross-correlation function, and the interaction between the two types of fluorescent molecules can be detected. According to FCCS using two kinds of fluorescent molecules, even if there is no difference in molecular weight (molecular size) between a fluorescent molecule labeling complex that generates SS and a non-specific substance that generates NSS, Depending on the characteristics, SS can be obtained by analyzing the measured fluorescence signal.

  Specifically, in this step, after calculating a correlation value (autocorrelation value or cross-correlation value) based on pulse signal data, correlation function data (autocorrelation function data or cross-correlation function data) is generated, and then correlation is performed. Statistically process (fitting) function data. Thereby, the number of molecules having a molecular weight corresponding to the fluorescent molecule labeling complex contained in the release solution and / or the fluorescent molecules constituting the fluorescent molecule labeling complex for all the fluorescent molecules contained in the release solution The existence ratio is calculated. Next, based on the calculated number of molecules or abundance ratio, the presence or amount of the fluorescent molecule label complex contained in the release solution is determined.

  In a preferred embodiment, step 2) in the method of the present invention may be performed by a combination of a photon counting method and a pulse counting method.

In the pulse counting method, the number of valid events is generated from pulse signal data. Specifically, first, the pulse signal data is recounted with a predetermined binning width to calculate recount data. Thereby, the fluorescence signal intensity value included in each binning width (event) is obtained.
Next, the signal intensity value of each event recounted with a predetermined binning width is compared with a preset threshold value, and only events whose signal intensity value exceeds the preset threshold value are counted as the number of valid events. .

  The predetermined binning width is preferably about 10 μsec to about 10 msec, more preferably about 100 μsec to about 1 msec.

  The threshold value is not particularly limited as long as an arbitrary signal / noise ratio is maintained. For example, a predetermined SD value of the average signal intensity value when a solution not containing a fluorescent label is measured can be set as the threshold value. The threshold value is preferably 1SD to 10SD, more preferably 3SD to 8SD.

  According to the pulse counting method, a low level signal can be eliminated. Further, since it is not affected by variations in signal height, signal measurement at a high S / N ratio can be realized in a low signal range. In particular, when a very low concentration of fluorescently labeled protein is measured by the confocal fluorescence detection method, the level of the signal varies depending on the time that the fluorescently labeled molecule passes through the measurement volume of several fL level. In other words, even when the same molecule passes through the measurement volume, the amount of signal varies depending on the time (path) that the molecule passes through the measurement volume. In principle, the method of counting the number is not quantitative. In such a case, the pulse counting method for counting the number of effective events exceeding the threshold value is very effective regardless of the signal level.

In another preferred embodiment, the above steps 2) and 3) in the method of the present invention can be performed by a combination of a photon counting method and a correlation method (eg, FCS, FCCS). In this case, steps 2) and 3) may each be performed as follows:
2 ') Measure the fluorescence signal with a confocal fluorescence detector by the photon counting method to obtain pulse signal data;
3a ′) generating correlation function data (eg, autocorrelation function data, cross-correlation function data) based on the pulse signal data; and 3b ′) a fluorescent molecular label complex contained in the release solution based on the correlation function data. To determine the presence or amount of.

In still another preferred embodiment, the above steps 2) and 3) in the method of the present invention can be performed by calculating the number of molecules based on a combination of a photon counting method and a correlation method (eg, FCS, FCCS). . In this case, steps 2) and 3) may each be performed as follows:
2-1) Measure the fluorescence signal with a confocal fluorescence detector by the photon counting method to obtain pulse signal data;
3-1a) generating correlation function data (eg, autocorrelation function data, cross-correlation function data) based on pulse signal data;
3-1b) calculating the number of molecules having a molecular weight corresponding to the fluorescent molecular label complex released from the solid phase based on the correlation function data; and 3-1c) calculating the number of molecules in the release solution from the calculated number of molecules. Determining the presence or amount of a fluorescent molecular labeling complex contained in

In still another preferred embodiment, the above steps 2) and 3) in the method of the present invention can be performed by calculating the abundance ratio based on a combination of a photon counting method and a correlation method (eg, FCS, FCCS). In this case, each of steps 2) and 3) may be performed as follows.
2-2) Measure the fluorescence signal with a confocal fluorescence detector by the photon counting method to obtain pulse signal data;
3-2a) generating correlation function data (eg, autocorrelation function data, cross-correlation function data) based on the pulse signal data;
3-2b) Based on the correlation function data, calculating the abundance ratio of the fluorescent molecule-labeled complex released from the solid phase with respect to all fluorescent molecules emitting fluorescent signals;
3-2c) Based on the abundance ratio of the fluorescent molecule label complex released from the solid phase, the pulse signal data is corrected, and the number of pulse signals (number of photons) corresponding to the number of fluorescent molecule label complexes released from the solid phase )
3-2d) From the number of pulse signals (number of photons), determine the presence or amount of the fluorescent molecule-labeled complex contained in the release solution.

  In still another embodiment, the above steps 2) and 3) in the method of the present invention can be performed by a combination of a photon counting method, a pulse counting method, and a correlation method (eg, FCS, FCCS).

If a combination of photon counting method, pulse counting method and correlation method (eg, FCS, FCCS) is utilized, steps 2) and 3) may be performed as follows:
2-3) Measure the fluorescence signal with a confocal fluorescence detector by the photon counting method to obtain pulse signal data;
3-3a) generating correlation function data (eg, autocorrelation function data, cross-correlation function data) based on pulse signal data;
3-3b) calculating the abundance ratio of the fluorescent molecule-labeled complex released from the solid phase with respect to all fluorescent molecules emitting fluorescent signals based on the correlation function data;
3-3c) generating the number of valid events (primary measurement data) from the pulse signal data by the pulse counting method;
3-3d) correcting the number of effective events (primary measurement data) based on the ratio to obtain the number of events (secondary measurement data) corresponding to the number of fluorescent molecule-labeled complexes released from the solid phase; 3-3e) Determining the presence or amount of the fluorescent molecule-labeled complex contained in the release solution from the number of events (secondary measurement data) corresponding to the number of fluorescent molecule-labeled complexes released from the solid phase.

  The above steps 2) and 3) and the above specific steps 2-1) to 2-3), 3-1a) to 3-1c), 3-2a) to 3-2d), 3-3a) to 3-3e For example, the method of Example 2 can be referred to. In addition, since at least a part of these steps are known, a previously reported method (e.g., Japanese Patent Application Laid-Open No. 2005-345311; Analytical Biochemistry, 2007, vol. 370, pp. 131-141; Journal of Immunological Methods 260, 117-124, 2002; Biochemistry, Vol. 82, No. 12, pp. 1103-1116, 2010; Biophysics 48 (5), 290-293, 2008). The correction may be performed, for example, by multiplying the pulse signal data or the number of effective events (primary measurement data) by the abundance ratio of the fluorescent molecule label complex released from the solid phase.

  In the present invention, NSS can be removed by optical analysis as shown in the above steps 2) and 3), so that background noise caused by non-specific substances that generate NSS can be reduced. N (signal / noise) ratio can be improved. That is, in the present invention, highly sensitive measurement of the target substance can be realized by optically removing NSS in the optical analysis.

4) Evaluation In this step, the presence or amount of the target substance is evaluated based on the presence or amount of the fluorescent molecule label complex contained in the release solution. For example, when the presence of the fluorescent molecule-labeled complex is confirmed in all or part of the release solution, it can be evaluated that the target substance is present. Further, the amount of the target substance can be evaluated based on the amount of the fluorescent molecule label complex in the whole or a part of the release solution. For example, when the value obtained in 3-2c) or 3-3d) is the number of pulse signals (number of photons) or the number of events, the concentration and number of molecules of the target substance can be evaluated using a separately prepared calibration curve. it can. For example, when the number of molecules of the target substance is calculated by fitting in 3-1b), the number of molecules of the target substance may be evaluated from the obtained number of complexes without creating a calibration curve.

  The above steps 2) to 4) are, for example, FCS unit (C9413, manufactured by Hamamatsu Photonics), FCCS unit (C10874-01, manufactured by Hamamatsu Photonics), confocal spectrum imager (LSM880, Carl Zeiss Microscopy Co., Ltd.) Company), a confocal microscope (Leica TCS SP8 SMD FCS, Leica Microsystems), MF20 (manufactured by Olympus Corporation).

  As far as the present inventors know, there has been no report on a method for measuring a target substance by combining non-specific substance removal (physical removal) and non-specific signal removal (optical removal). The methods of the present invention include physical removal (eg, removal of non-specific material by washing and / or removal of the solid phase) and optical removal (eg, differential measurement of specific signals with a confocal fluorescence detector, and / or Under the concept of realizing both non-specific signal removal by pulse counting), background noise caused by both non-specific substance and non-specific signal can be reduced, and S / N (signal / noise) ) Ratio can be improved. In the conventional method using the fluorescence correlation method, since many unreacted fluorescent molecule-labeled antibodies remain in the liquid phase, the calculation (fitting) accuracy is lowered, and sufficient measurement accuracy cannot be obtained. In particular, when there is only a very small amount of the target substance in the sample, the detection of the target substance is extremely difficult and practically impossible. Further, in the conventional method using only the confocal fluorescence detection method, a nonspecific signal cannot be removed from the fluorescence signal, so that sufficient measurement sensitivity cannot be obtained. On the other hand, in the method of the present invention, non-specific signals can be sufficiently reduced by performing physical removal before optical removal, which greatly improves the calculation (fitting) accuracy in optical removal. can do. Therefore, the method of the present invention is useful in that the target substance can be detected with extremely high sensitivity even when the sample contains only an extremely small amount of the target substance.

  As described above, the main embodiments of the method of the present invention have been described by assuming steps 1) to 4) as essential, but in certain embodiments, the method of the present invention can also omit step 1). . In this case, the method of the present invention can be carried out by steps 2) to 4) (and specific forms thereof). The “fluorescent molecule label complex released from the solid phase” used in steps 2) to 4) is used. Terms relating to fluorescent molecule labeling complexes such as “containing release solution”, “fluorescent molecular label complex released from solid phase”, and “fluorescent molecular label complex contained in release solution” are simply “fluorescent molecular label complex” It can be read as “body”. Moreover, such a method of the present invention can be applied not only to the fluorescent molecule label complex but also to the fluorescent molecule itself. Therefore, the “release solution containing the fluorescent molecule label complex released from the solid phase”, “the fluorescent molecule label complex released from the solid phase”, and “included in the release solution” used in steps 2) to 4) A term relating to a fluorescent molecule labeling complex such as “fluorescent molecule labeling complex” may simply be read as “fluorescent molecule”. The same applies to the following.

  In a specific embodiment, when the steps 2) and 3) in the method of the present invention are performed by a combination of a photon counting method, a pulse counting method, and a correlation method, the steps (2) and (3) The detection may be performed by a fluorescence detection system or a fluorescence detection method using the fluorescence detection system.

Examples of such systems and methods include the following.
<1> A confocal detection unit that detects fluorescent photons generated in a measurement sample, and is connected to the confocal detection unit, and generates primary measurement data based on pulse signal data generated by the confocal detection unit A calculation unit that calculates the abundance ratio of the fluorescent molecule-labeled complex, determines the number of fluorescent molecule-labeled complexes based on the primary measurement data and the abundance ratio, and forms secondary measurement data. Detection system.
<2> The confocal detection unit is connected to a laser light generation unit that generates and outputs laser light, and the laser light generation unit, and irradiates the measurement sample with the laser light. The fluorescence detection system according to <1>, comprising: an optical processing unit that emits the generated fluorescent photons; and a detection unit that is connected to the optical processing unit and detects the fluorescent photons.
<3> The detection unit is connected to the optical processing unit, the fluorescent photons emitted from the optical processing unit are incident, and the incident fluorescent photons are converted into electrical signals based on the light amount. <2> fluorescent light comprising: a sensor unit that outputs the signal; and an electric signal processing unit that is connected to the sensor unit, amplifies the electric signal output from the sensor unit, converts the signal into an electric pulse signal, and outputs the signal. Detection system.
<4> The detection unit is connected to the electrical signal processing unit, the electrical pulse signal output from the electrical signal processing unit is input, and count data is formed based on the input electrical pulse signal, The fluorescence detection system according to <3>, further including a counting unit that outputs.
<5> The calculation unit records the pulse signal data output from the detection unit, re-counts the pulse signal data to calculate re-count data, and compares the re-count data with a threshold value. To generate primary measurement data, and generate correlation function data based on the pulse signal data, and statistically process the correlation function data to determine the abundance ratio of the fluorescent molecule-labeled complex. Fluorescence detection according to any one of <1> to <4>, which is an arithmetic unit that calculates and determines the number of fluorescent molecule-labeled complexes based on the primary measurement data and the abundance ratio to generate secondary measurement data system.
<6> A step in which the confocal detection unit detects fluorescent photons generated in the measurement sample and generates pulse signal data based on the number of the detected fluorescent photons, and a calculation unit adds the pulse signal data to the pulse signal data. On the basis of calculating a primary measurement value, generating primary measurement data, the calculation unit calculating the abundance ratio of the fluorescent molecule label complex based on the pulse signal data, and the calculation unit, Determining the number of fluorescent molecule-labeled complexes based on the primary measurement data and the abundance ratio and generating secondary measurement data.
<7> In the step of generating the primary measurement data, the calculation unit re-counts the pulse signal data with a predetermined binning width to generate re-count data, and determines the signal intensity value and threshold value of each event of the re-count data. In contrast, the fluorescence detection method according to <6>, including a step of re-counting only events exceeding the threshold value to calculate a primary measurement value and generating primary measurement data.
<8> The step of calculating the existence ratio includes the step of calculating the correlation function based on the pulse signal data by the calculation unit and generating correlation function data, and the calculation unit statistically calculating the correlation function data. Processing to calculate the abundance ratio of the fluorescent molecule-labeled complex, and the fluorescence detection method according to <6> or <7>.
Hereinafter, such a system and method will be described in detail.

<Fluorescence detection system>
The fluorescence detection system is connected to a confocal detection unit that detects fluorescent photons generated in a measurement sample, and the confocal detection unit, and primary measurement data is obtained based on pulse signal data generated by the confocal detection unit. And a calculation unit that calculates the abundance ratio of the fluorescent molecule labeling complex, determines the number of the fluorescent molecule labeling complex based on the primary measurement data and the abundance ratio, and obtains the secondary measurement data.

Hereinafter, embodiments will be described with reference to the drawings. Each drawing only schematically shows the shape, size, and arrangement of the components to the extent that the invention can be understood. The present invention is not limited to the following description, and each component can be appropriately changed without departing from the gist of the present invention. In the following description, the same components are denoted by the same reference numerals, and overlapping descriptions may be omitted.

  A configuration example of the fluorescence detection system will be described with reference to FIG. FIG. 5 is a schematic functional block diagram illustrating a configuration example of the fluorescence detection system 10.

  As shown in FIG. 5, the fluorescence detection system 10 includes a confocal detection unit 12 and a calculation unit 14 connected to the confocal detection unit 12. Hereinafter, each part is demonstrated concretely.

(Confocal detection unit)
The confocal detection unit 12 has a function capable of irradiating a measurement sample with laser light, detecting a fluorescent photon (fluorescence signal) generated from a fluorescent label in the measurement sample, and outputting the detected fluorescence signal as an electric pulse signal. Part.

  In this configuration example, the confocal detection unit 12 includes a laser light generation unit 20 connected to the calculation unit 14 via an electric communication line, an optical processing unit 30 optically connected to the laser light generation unit 20, A detection unit 40 that is optically connected to the optical processing unit 30 and connected to the calculation unit 14 by an electric communication line is provided. The laser light generation unit 20 may be configured not to be connected to the calculation unit 14 and not controlled by the calculation unit 14.

  Here, “optically connected” means connected so that optical data, an optical signal, and the like can be exchanged directly or through an optical waveguide such as an optical fiber. Further, “connected by a telecommunication line” means that it is connected so that data, control signals, etc. can be exchanged via a wired or wireless information line using a medium such as electricity or light. ing.

  The laser beam generator 20 is a functional unit that can generate and output a laser beam. The intensity or the like of the laser light output from the laser light generation unit 20 can be optimized by control by the calculation unit 14, control by another configuration, or autonomous control.

  As described above, when the laser light generation unit 20 is not controlled by the calculation unit 14, the laser light can be controlled by turning on / off a power switch provided in the laser light generation unit 20 itself or opening / closing a mechanical shutter or the like. The laser beam can also be controlled by an optical filter or the like.

  As the laser beam generator 20, any conventionally known and suitable laser oscillator capable of generating a laser beam having a wavelength suitable for the fluorescent label to be used can be used.

The optical processing unit 30 is a functional unit that can irradiate the measurement sample with the laser light output from the laser light generation unit 20 and cause the fluorescence photons generated by the fluorescent label in the measurement sample to enter the detection unit 40. The optical processing unit 30 includes a conventionally known arbitrary suitable optical element, and is configured to be able to construct a confocal system. Specifically, the optical processing unit 30 includes, for example, one or two or more lenses including an objective lens arranged to face the measurement sample, and one or two or more mirrors (dichroic mirrors) arranged on the optical path. Any suitable optical elements such as pinholes may be provided in any suitable arrangement corresponding to the desired design. The optical processing unit 30 may include a mechanical shutter, an optical filter, and the like, and can control the characteristics of the laser beam output from the laser beam generation unit by opening and closing the mechanical shutter. The characteristics of the laser beam can be controlled.

The detection unit 40 is a functional unit that can detect a fluorescent photon generated by a fluorescent label in a measurement sample, which is generated by irradiation with laser light, and can change the photon into a time-series electric pulse signal. This is a functional unit that receives fluorescent photons from the unit 30, detects the incident fluorescent photons, changes them to time-series electric pulse signals, and outputs pulse signal data to the calculation unit 14.
The pulse signal data output from the detection unit 40 may be time-series electric pulse signal data, or count data obtained by counting time-series electric pulse signals as the number of signals per arbitrary time (count number / second, Hz). It may be.

A configuration example of the detection unit 40 will be described with reference to FIG. FIG. 6 is a schematic functional block diagram illustrating a configuration example of the detection unit 40.

As shown in FIG. 6, the detection unit 40 includes a sensor unit 42 optically connected to the optical processing unit 30, an electrical signal processing unit 44 connected to the sensor unit 42 via an electrical communication line, A counting unit 46 connected to the electrical signal processing unit 44 via an electrical communication line and connected to the arithmetic unit 14 via an electrical communication line is provided.
In the later-described process, when electric pulse signal data is used as pulse signal data, the counting unit 46 can be omitted.

  The sensor unit 42, the electric signal processing unit 44, and the counting unit 46 may be integrally configured.

  The sensor unit 42 is a functional unit that receives the fluorescence photons generated by the fluorescent label from the optical processing unit 30, and converts the incident fluorescence photons into an electrical signal (photoelectric) based on the light amount and outputs the electrical signal. is there. As the sensor unit 42, any conventionally known suitable photomultiplier tube (PMT), photon counting head, avalanche photodiode (APD), superconducting single photon detector (SSPD), or the like can be used.

The electric signal processing unit 44 is a functional unit that can generate and output an electric pulse signal by amplifying the electric signal output from the sensor unit 42 and performing predetermined signal processing. The electric signal processing unit 44 includes a signal processing circuit such as a conventionally known arbitrary suitable amplifier circuit (a device including an amplifier circuit) and a conventionally known arbitrary suitable wave height discrimination circuit (a device including a wave height discrimination circuit).

  The counting unit 46 is a functional unit that can generate count data and output the count data. More specifically, the counting unit 46 receives the electric pulse signal output from the confocal detection unit 12, that is, the electric signal processing unit 44, and counts the number of signals per arbitrary time (counting) based on the input electric pulse signal. It is a functional unit capable of forming count data by counting (number / second, Hz) and outputting it. As the counting unit 46, any conventionally known suitable frequency counter circuit (an apparatus including a frequency counter circuit) can be used. The operation method of the counting unit 46 is not particularly limited. Examples of the operation method include a gate method and a reciprocal method. As the operation method, it is preferable to adopt a reciprocal method in consideration of the light amount, resolution, and the like when immunological measurement is assumed, for example.

(Calculation unit)
The calculation unit 14 is a functional unit that can control the operation of functional units directly or indirectly connected to the outside such as the detection unit 40 (electric signal processing unit 44, counting unit 46). Specifically, the calculation unit 14 records the pulse signal data output from the detection unit 40, reads the pulse signal data, re-counts the acquired pulse signal data with a predetermined binning width, and calculates re-count data. The recount data and the threshold value are compared, the number of recount data events exceeding the threshold value (the number of valid events) is counted to calculate the primary measurement value, and the primary measurement data is generated or the pulse signal data is Correlation function (eg, autocorrelation function, cross-correlation function) is calculated based on this, and correlation function data is generated, or the correlation function data is statistically processed to calculate the abundance ratio of the fluorescent molecule-labeled complex. It has a function of determining the number of fluorescent molecule-labeled complexes based on primary measurement data, abundance ratio, etc., and generating secondary measurement data (details will be described later).

  The calculation unit 14 includes, for example, a microprocessor (CPU), a graphic processor (GPU), an external function unit, an interface such as a serial connection and a parallel connection for exchanging data with the device, input data, and formed data A computer hardware resource including a storage device for recording, an input interface such as a keyboard, a display device that can visually display data, a printer that can output data recorded on a paper medium, etc., or the above function units It can be realized by dedicated hardware resources that can perform the corresponding function.

The fluorescence detection system 10 includes, for example, an FCS unit (C9413 manufactured by Hamamatsu Photonics Co., Ltd.), a confocal spectrum imager (LSM880 manufactured by Carl Zeiss Microscopy Co., Ltd.), a confocal microscope (Leica TCS SP8 SMD FCS manufactured by Leica Microsystems), It can be configured using a molecular fluorescence analysis system (MF20 manufactured by Olympus Corporation).
In addition, the above-described functional units described above may be combined as appropriate and configured integrally.

<Fluorescence detection method>
The fluorescence detection method is a fluorescence detection method using the above-described fluorescence detection system, in which the confocal detection unit detects the fluorescence photons generated in the measurement sample, and the confocal detection unit detects the detected fluorescence photons. And generating a primary measurement value (number of valid events) based on the pulse signal data, and generating a primary measurement data. A step of calculating the abundance ratio of the fluorescent molecule labeling complex based on the calculation unit, and a step of generating the secondary measurement data by determining the number of the fluorescent molecule labeling complex based on the primary measurement data and the abundance ratio. Including.

  The fluorescence detection method will be described with reference to FIG. FIG. 7 is a flowchart for explaining the fluorescence detection method.

  In carrying out the fluorescence detection method, for example, a previously reported method (eg, Japanese Patent Application Laid-Open No. 2005-345311; Analytical Biochemistry, 2007, vol. 370, pp. 131-141; Journal of Immunological Methods 260, 117-124, 2002; Biochemistry, Vol. 82, No. 12, pp. 1103 to 1116, 2010; Biophysics 48 (5), 290 to 293, 2008).

  As shown in FIG. 7, the fluorescence detection method preferably includes a step (measurement sample preparation step) (S1) in which a measurement sample is prepared.

  In the fluorescence detection method, a liquid phase measurement sample is used. The measurement sample is preferably the release solution.

The fluorescence detection method can more effectively eliminate the influence of measurement noise. Such noise includes noise from the measurement sample (eg, buffer solution, component, pH, temperature), mechanical noise from the measurement equipment (eg, vibration, material of the measurement sample injection container), and electrical noise. (For example, electric field from the outside, electron emission due to thermal fluctuation, induction noise from the ground, after pulse from the detector itself) and the like. Examples of a buffer that can be a noise source include Tris buffer (eg, Tris-HCl, Tris-EDTA), phosphate buffer, hydrochloric acid-potassium chloride buffer, glycine-hydrochloric acid buffer, citrate buffer, acetic acid. Buffer, citrate
Examples include phosphate buffer, glycine-sodium hydroxide buffer, carbonate-bicarbonate buffer, borate buffer, and tartaric acid buffer. Examples of components that can be noise sources include components that can be used in immunoassays such as ELISA, and components that can be mixed into measurement samples such as biological components. Accordingly, the release solution used in the present invention (eg, the release solution used in the above step 1) may be under such conditions, and may contain, for example, such a buffer or component. .

[Confocal detection unit detects fluorescent photons generated in the measurement sample and generates pulse signal data based on the number of fluorescent photons]
In this step, the confocal detection unit 12 detects fluorescent photons generated in the measurement sample, generates pulse signal data based on the number of detected fluorescent photons, and further calculates the obtained pulse signal data. This is a step (S2) of inputting to the part.

  This step is performed by a photon counting method in which the signal is binarized and the number of fluorescent photons is counted according to the number of pulses.

  Specifically, first, the laser light generation unit 20 of the confocal detection unit 12 to which the control signal from the calculation unit 14 is input generates laser light, and the generated laser light is incident on the optical processing unit 30. Next, the optical processing unit 30 irradiates the measurement sample with the introduced laser light. The laser light applied to the measurement sample causes fluorescent photons to be emitted by the fluorescent label in the measurement sample. Next, the optical processing unit 30 separates the emitted fluorescent photons and laser light, emits only the separated fluorescent photons, and enters the detection unit 40. The fluorescent photons incident in this way are detected by the detection unit 40.

  Next, the detection unit 40 of the confocal detection unit 12 generates pulse signal data that is a digital signal based on the detected fluorescent photons, and inputs the pulse signal data to the calculation unit 14. The computing unit 14 once records the acquired pulse signal data.

  Here, the pulse signal data is data relating to time-series electric pulse signals, and may be time-series electric pulse signal data, or the number of signals per arbitrary time (count number / second, Hz). The counted data may be counted as

  When counting data is used as the pulse signal data, for example, the counting unit 46 generates the counting data by counting the number of fluorescent photons based on the electric pulse signal input from the electric signal processing unit 44.

  This counted data is data relating to the number of signals per predetermined unit time (photometric binning time), for example, and “number of counted fluorescent photons / second”, “Hz” is used as the unit.

  Next, the counting unit 46 inputs the count data to the calculation unit 14.

  The measurement frequency in the photon counting method is preferably set to a certain time resolution or higher in consideration of processing performed later by FCS, FCCS, etc., for example, 5 kHz or higher, preferably higher than 80 kHz. .

  In this way, pulse signal data by the photon counting method is acquired.

[Processing unit for calculating primary measurement value based on pulse signal data and generating primary measurement data]
In this step, the calculation unit 14 calculates a primary measurement value based on the pulse signal data, and generates primary measurement data. Specifically, this step is performed by, for example, a pulse counting method. This will be specifically described below.

  First, the calculation unit 14 re-counts the pulse signal data with a predetermined binning width to generate re-count data (S3).

  Specifically, the calculation unit 14 reads the recorded pulse signal data, re-counts the pulse signal data with a predetermined binning width, and calculates re-count data. Thereby, the fluorescence signal intensity value included in each binning width (event) is obtained. The “predetermined binning width” is preferably about 10 μsec to about 10 msec, more preferably about 100 μsec to about 1 msec.

  Next, the computing unit 14 compares the signal intensity value of each event of the recount data with a threshold value (S4).

  Specifically, the calculation unit 14 compares the signal intensity value of each event recounted with a predetermined binning width with a threshold value calculated from measurement data of a zero concentration sample, a preset threshold value, or the like.

  Next, the calculation unit 14 counts only events that exceed the threshold, calculates a primary measurement value (number of valid events), and generates primary measurement data (S5 to S9). Hereinafter, these steps will be described in detail.

First, the calculation unit 14 determines whether or not the signal intensity value of each event of the recount data exceeds a preset threshold value (S5). The calculation unit 14 performs re-counting by the pulse counting method that counts the corresponding event as the number of valid events only when the signal intensity value of each event exceeds the threshold (Yes in S5) (S6). When the signal strength value of the event is equal to or less than the threshold value (No in S5), the calculation unit 14 does not count the event (not counting) (S7). The computing unit 14 determines whether or not the processing of S6 or S7 has been completed for all events (S8). S6 or S7 for all events
When the process is completed (Yes in S8), the calculation unit 14 calculates primary measurement values based on the counted number of events (number of valid events), generates primary measurement data (S9), and further performs primary measurement. Record data. If the process of S6 or S7 has not been completed for all events (No in S8), the process returns to S5 and is repeated until the process is completed.

An example of the recount data is shown in FIG. FIG. 8 shows that a solution containing a fluorescent label Alexa Fluor 532 is irradiated with an excitation laser having an excitation wavelength of 532 nm and an excitation light intensity of 1 mW, and the generated fluorescence photons are acquired as an electric pulse signal at 80 kHz for 3 seconds, and a binning width of 1 ms. The re-count data re-counted in FIG. In FIG. 8, fluorescence signal intensity values included in each of about 2000 events are obtained. For example, in FIG. 8, when the threshold is set to 6SD, which is an average signal intensity value when a solution containing no fluorescent label is measured, the number of effective signals is 118.
Here, the threshold value is not particularly limited as long as an arbitrary signal / noise ratio is maintained. As the threshold value, for example, a predetermined multiple value of the value of the average signal intensity value (SD) when a solution containing no fluorescent label is measured can be used. The threshold value can be preferably 1SD to 10SD, more preferably 3SD to 8SD.

[Step of calculating the abundance ratio of the fluorescent molecule labeling complex based on the pulse signal data by the arithmetic unit]
In this step, the calculation unit 14 calculates the abundance ratio of the fluorescent molecule label complex based on the pulse signal data. Here, the abundance ratio is a parameter for obtaining secondary measurement data relating to the number of fluorescent molecule-labeled complexes.

  Examples of data for calculating the abundance ratio used for obtaining the secondary measurement data include autocorrelation values calculated by FCS and data related to cross-correlation values calculated by FCCS.

  As a preferred example of the process in which the calculation unit 14 calculates the existence ratio, the calculation unit 14 calculates a correlation function based on the pulse signal data, generates correlation function data, and the calculation unit 14 statistically calculates the correlation function data. The process of calculating the abundance ratio of the fluorescent molecule-labeled complex after processing in detail will be specifically described as an example.

First, the calculating part 14 calculates a correlation function based on pulse signal data, and produces | generates correlation function data (S10).
Specifically, this step is preferably performed by FCS. Hereinafter, the example which implements this process by FCS is demonstrated. The calculation of the autocorrelation function by FCS is not particularly limited as long as it is a known method. For example, it can be performed by a multiple tau method.
Hereinafter, the calculation process of the autocorrelation function by the multiple tau method will be specifically described.

  First, the calculation unit 14 reads the recorded pulse signal data, calculates an autocorrelation function based on the pulse signal data and the following equation (1), generates autocorrelation function data, and further autocorrelation. Record function data.

  In formula (1), the sign “<>” represents the harmonic mean, I represents the fluorescence intensity signal, δI (t) represents the fluctuation component of the fluorescence intensity signal, and δ (t + τ) is after τ (time). Represents the fluctuation component of the fluorescence intensity signal.

FIG. 9 shows an example of autocorrelation function data (data indicating the relationship between the autocorrelation value G (t) and time) calculated by the multiple tau method.
Specifically, FIG. 9 shows a result of calculating an autocorrelation function in a minimum tau of 200 μs from pulse signal data photon-counted at 80 kHz using a molecule obtained by fluorescently labeling a protein with Alexa Fluor 532 as a sample.

  Next, the calculation unit 14 statistically processes the autocorrelation function data to calculate the abundance ratio of the fluorescent molecule label complex (S11).

  Specifically, the calculation unit 14 reads the recorded autocorrelation function data and statistically processes (fitting) the data using, for example, the following equation (2).

In Equation (2), T represents the ratio of the triplet state, τ _trip represents the translational diffusion time of the triplet state, τ _i represents the translational diffusion time of the i component (Index), and f _i represents i It represents the ratio of the components, N represents the number of molecules, M represents the number of molecular species, and s represents the ratio of the major axis radius to the minor axis radius of the observation region.

In this way, T, τ _trip , τ _i , f _i , N, M, and s (referred to as calculation parameters) are calculated, and the fluorescent molecule labeled complex among the total number of fluorescent photons (number of events) counted. The abundance ratio of the body can be calculated. And the calculating part 14 records the calculated numerical value (existence ratio).

  The fitting in this step can be performed by, for example, the Levenberg Marcadol method.

[Processing section for obtaining secondary measurement data by determining the number of fluorescent molecule-labeled complexes based on primary measurement data and abundance ratio]
This step is a step in which the calculation unit 14 determines the number of fluorescent molecule-labeled complexes based on the primary measurement data and the abundance ratio to obtain secondary measurement data (S12).

  Specifically, the calculation unit 14 determines the number (concentration) of the fluorescent molecule-labeled complex using the primary measurement data (primary measurement value) and the abundance ratio calculated by the statistical processing. The abundance ratio used at this time may be obtained by any of the above methods, or a combination thereof.

The computing unit 14 determines the number (concentration) of the target fluorescent molecule label complex from the primary measurement data and the abundance ratio, and records the obtained determination value (secondary measurement data). The secondary measurement data can be obtained based on the following calculation formula, for example.
Calculation formula: Primary measurement data (primary measurement value) x abundance ratio = secondary measurement data (decision value)

  Through the above steps, the number (concentration) of the fluorescent molecule-labeled complex to be measured can be accurately determined.

  In the fluorescence detection method described above, the example of performing the steps (S3 to S9) of obtaining primary measurement data after the step of obtaining pulse measurement data (S2) has been described. However, the step of obtaining pulse measurement data (S2) Next, the step (S10 and S11) of obtaining the existence ratio based on the pulse measurement data may be performed, and then the steps (S3 to S9) of obtaining the primary measurement data may be performed.

  According to such a fluorescence detection method, by combining the photon counting method and the pulse counting method, it is not affected by the variation in the height of the pulse signal. Therefore, even in a low level pulse signal region, it has a high S / N ratio. Signal measurement can be performed, and only low-level and non-specific pulse signals can be excluded with high accuracy. Furthermore, since the correlation method is combined, noise derived from matrix components of the measurement sample, mechanical noise, electrical noise As a result of suppressing background noise caused by static noise, etc., fluorescent labels (target substances containing fluorescent labels) can be detected and quantified more precisely and accurately even at extremely low concentrations. .

The present invention also relates to a program for causing a computer to execute the fluorescence detection method described above.
Specifically, such a program is a program for causing the calculation unit 14, which is a computer hardware resource (computer), to execute, for example, the following steps.
(1) The step of causing the confocal detection unit 12 to detect fluorescent photons generated in the measurement sample (2) The computer causes the confocal detection unit 12 to output pulse signal data based on the detected fluorescent photons. Step (3) of generating and acquiring Step (4) in which the computer re-counts the acquired pulse signal data with a predetermined binning width to generate and count re-counting data. A step of comparing the signal intensity value of an event with a threshold value, re-counting only events exceeding the threshold value to calculate a primary measurement value (number of valid events), and generating primary measurement data (5) Step of calculating autocorrelation function based on data and generating autocorrelation function data (6) The computer statistically processes the autocorrelation function data, Step of calculating the existence ratio of the labeled complex (7) computer, determining the number of fluorescent molecules labeled complex on the basis of the primary measurement data and proportions

(Example)
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples.

(material)
1) Preparation of dextran-crosslinked anti-α-fetoprotein (AFP) monoclonal antibody-fixed magnetic beads 167 μL of 0.1 M water-soluble carbodiimide (EDC) was added to 5 mg of carboxylated magnetic beads (Life Technologies) and stirred at room temperature for 30 minutes. did. After washing with purified water, 167 µL of a 50 mM MES pH 5.5 solution of aminated dextran was added, incubated at room temperature for 30 minutes, and then washed with purified water to obtain aminated dextran magnetic beads. After adding 320 μL of 1 mg / mL GMBS (N-succinimidyl-4-maleimidobutyric acid) ethanol solution to 4 mg of aminated dextran magnetic beads and reacting for 1 hour, the beads were washed with phosphate buffer and 200 μL of 0.4 mg / mL. After adding an anti-AFP monoclonal antibody Fab ′ solution and reacting for 2 hours, the beads were washed to obtain dextran-crosslinked anti-AFP monoclonal antibody-immobilized magnetic beads.

2) Preparation of anti-AFP monoclonal antibody Fab ′ Using a PD-10 column (GE Healthcare Bioscience), the anti-AFP monoclonal antibody solution was replaced with citrate buffer (pH 3.5), and pepsin was used as the antibody 1 / 100 (w / w) amount was added and left at 37 ° C. for 1 hour. This solution was purified using a Superdex 200 column (GE Hercare Bioscience) to obtain F (ab ′) ₂ . After adding 1/20 (v / v) of 0.2M 2-mercaptoethylamine to this F (ab ′) ₂ and leaving it at 37 ° C. for 3 hours, 2-mercaptoethylamine was removed with a PD-10 column. Fab ′ fragment was obtained.
10 equivalents of Alexa Fluor 532 C5-maleimide (Life Technologies) was added to the anti-AFP monoclonal antibody Fab ′ and left at 4 ° C. overnight, then unreacted dye was removed with a PD-10 column, and Alexa Fluor 532 was removed. A labeled anti-AFP monoclonal antibody Fab ′ was obtained. The anti-AFP monoclonal antibody used when labeling Alexa Fluor 532 was an antibody different from the anti-AFP monoclonal antibody immobilized on the magnetic beads.

3) Production of biotin-antibiotin antibody cross-linked anti-AFP monoclonal antibody-immobilized magnetic beads 3a) Production of anti-biotin antibody-immobilized magnetic beads Anti-biotin antibody is added to the magnetic beads and allowed to react at room temperature for 1 hour with stirring. The magnetic beads are washed three times by magnetic attraction to obtain anti-biotin antibody-immobilized magnetic beads.

3b) Preparation of biotin-labeled anti-AFP monoclonal antibody An NHS-biotin solution is added to the anti-AFP monoclonal antibody solution, and the resulting solution is allowed to stand at room temperature for 60 minutes. A biotin-labeled anti-AFP monoclonal antibody is obtained by desalting and purification (removal of unreacted biotin) using a PD-10 column.

3c) Affinity reaction between anti-biotin antibody-immobilized magnetic beads and biotin-labeled anti-AFP monoclonal antibody To 250 μL of anti-biotin antibody-immobilized magnetic beads solution, 250 μL of biotin-labeled anti-AFP monoclonal antibody solution was added and stirred. Leave at 30 ° C. for 30 minutes.
Thereby, biotin-anti-biotin antibody cross-linked anti-AFP monoclonal antibody fixed magnetic beads are obtained by affinity reaction.
The magnetic beads are washed three times by magnetic attraction to obtain a biotin-antibiotin antibody cross-linked anti-AFP monoclonal antibody immobilized magnetic bead solution.

Comparative Example 1: Measuring method using a homogeneous liquid phase system (conventional method)
125 μL of 0.686 nM Alexa Fluor 532 labeled anti-AFP monoclonal antibody Fab ′ was added to 125 μL of 876 nM anti-AFP monoclonal antibody and stirred. Furthermore, after adding 20 μL of known AFP solution and stirring, and incubating at room temperature for 1 hour, the fluorescence signal was measured with a confocal fluorescence detector in the same manner as in Example 2 below. Using the correlation function, the abundance ratio of the fluorescently labeled complex containing the anti-AFP monoclonal antibody, AFP, and Alexa Fluor 532 labeled anti-AFP monoclonal antibody Fab ′ was determined.

Example 1 Preparation of Release Solution Containing Fluorescent Molecular Label Complex Released from Solid Phase 1-1) Preparation of Release Solution Containing Fluorescent Molecular Label Complex Released from Magnetic Beads (1)
I) Preparation of fluorescent molecule-labeled complex immobilized on magnetic beads via a cleavable linker (dextran) To 20 μL of AFP solution with a known concentration was added to 250 μL of dextran-crosslinked anti-AFP monoclonal antibody-immobilized magnetic beads, and the mixture was incubated at 37 ° C. Incubation was for 10 minutes. The magnetic beads were washed three times, 250 μL of 5 nM Alexa Fluor 532 labeled anti-AFP monoclonal antibody Fab ′ was added and incubated at 37 ° C. for 10 minutes. Thereby, the fluorescent molecule-labeled complex, that is, the anti-AFP monoclonal antibody Fab ′ (first affinity molecule) -AFP (target substance) -Alexa Fluor 532 labeled anti-AFP monoclonal antibody Fab ′ (second affinity molecule) is converted into dextran. It was fixed to the magnetic beads via.

II) Specific cleavage of the fluorescent molecule-labeled complex at the site of the cleavable linker (dextran) The magnetic beads bound with the fluorescent molecule-labeled complex were washed 5 times, 200 μL of 5 U / mL dextranase was added, and 37 ° C. After incubating for 10 minutes, the complex released by magnetic attraction was separated from the magnetic beads, and the fluorescence signal of the resulting fluorescent molecule labeled complex solution was measured with a confocal fluorescence detector.

1-2) Preparation of release solution containing fluorescent molecule-labeled complex released from magnetic beads (2)
I) Preparation of fluorescent molecule-labeled complex immobilized on magnetic beads via a cleavable linker (affinity linker) The antibody is used as the first affinity molecule immobilized on the solid phase via a cleavable linker, and the Alexa Fluor 532 labeled anti-AFP monoclonal antibody is used as the second affinity molecule labeled with a fluorescent molecule. The epitopes of the first affinity molecule and the second affinity molecule are different from each other, and both the first affinity molecule and the second affinity molecule can simultaneously bind to one molecule of AFP.
Add 250 μL of biotin-anti-biotin antibody cross-linked anti-AFP monoclonal antibody (first affinity molecule) immobilized magnetic bead solution to 250 μL of AFP standard solution 10 μL and stir, and leave the resulting solution at 37 ° C. for 10 minutes. Put. By immunological reaction, a complex (magnetic bead-anti-biotin antibody-biotin-first affinity molecule-AFP) immobilized on magnetic beads via biotin and anti-biotin antibody is obtained.
After washing magnetic beads (complex is fixed) three times by magnetic attraction, all washing liquid is removed.
250 μL of Alexa Fluor 532 labeled anti-AFP monoclonal antibody (second affinity molecule) solution is added to the washed magnetic beads (complex is fixed) and stirred, and the resulting solution is allowed to stand at 37 ° C. for 10 minutes. Fluorescent molecule labeled complex immobilized on magnetic beads via biotin and anti-biotin antibody by immunological reaction (magnetic beads-anti-biotin antibody-biotin-first affinity molecule-AFP-second affinity molecule-fluorescence Molecule) is obtained.
After washing the magnetic beads (fluorescent molecule-labeled complex) 6 times by magnetic attraction, all the washing solution is removed.

II) Specific cleavage of the fluorescent molecule-labeled complex at the site of the cleavable linker (affinity linker) 250 μL of biotin solution was added to the obtained magnetic beads (fluorescent molecule-labeled complex) and stirred, and the resulting solution Let stand at room temperature for 30 minutes. Biotin in the biotin solution competes with biotin in the fluorescent molecular label complex. Fluorescent molecules released from magnetic beads after the fluorescent molecule-labeled complex immobilized on magnetic beads via biotin and anti-biotin antibodies is specifically cleaved at the anti-biotin antibody-biotin site with biotin in the biotin solution Obtain 250 μL of the release solution containing the labeled complex (biotin-first affinity molecule-AFP-second affinity molecule-fluorescent molecule).

Example 2: Measurement and analysis of fluorescence signal with confocal fluorescence detector 2-1) Measurement of fluorescence signal with confocal fluorescence detector The total amount of measurement samples obtained in Comparative Example 1 and Example 1 or A part of the sample was dispensed into a blocking agent (Nunc cover glass chamber 155411, manufactured by Thermo Scientific) with a blocking agent (N101 manufactured by NOF Corporation), and then the fluorescence signal of the measurement sample was measured with a confocal fluorescence detector. did.

The measurement conditions were as follows.
Excitation laser: Coherent Japan Co., Ltd. Sapphire 532FP, excitation wavelength 532 nm, excitation light intensity 1 mW (sample position)
Objective lens: Olympus Corporation UAPON 40XW340
Sensor: Hamamatsu Photonics Co., Ltd. H7421-40, photometric binning time: 50 ns measurement time: 3.2 seconds × 10 loops were measured once, and 10 measurements were performed for each sample.

The obtained pulse signal data with a photometric binning time of 50 ns was integrated every 2 × 10 ^4, thereby calculating recount data with a binning width of 1 msec. One pulse of the recount data is regarded as one event, and the number of events in which the fluorescence signal intensity included in each event exceeds a threshold is counted (pulse counting), thereby obtaining primary measurement data. The threshold was set as an average value of fluorescence signal intensity included in each event when measuring the buffer solution + standard deviation × 6.
FIG. 10 shows one of the recount data when measuring 80 ng / mL AFP. The threshold value at this time was 4.

2-2) Analysis of pulse signal data The measured pulse signal data was analyzed by the fluorescence correlation method.
First, the autocorrelation value of the multiple tau calculation method was calculated from the obtained pulse signal data using the formula (1) to generate autocorrelation function data. FIG. 9 shows autocorrelation function data when measuring 200 ng / mL of AFP.

  Next, using the formula (2), Levenberg-Marcadol method fitting was performed from the obtained autocorrelation function data, and the abundance ratio of the target fluorescent molecule-labeled complex with respect to all fluorescent molecules emitting fluorescent signals was calculated.

Finally, using the primary measurement value (primary measurement data) and the abundance ratio, the number (concentration) of the fluorescent label or the target substance containing the fluorescent label as the secondary measurement value (secondary measurement data) Decided on the basis.
Calculation formula: primary measurement value x existence ratio = secondary measurement value

(Experimental results related to the first object of the present invention)
The results of Comparative Example 1 (conventional method) are shown in Table 3 and FIG. The measurement sensitivity is “the concentration at which the average value of measurement data when measuring a zero-concentration sample + two standard deviations (2SD) and the average value of measurement value data when measuring a sample of known concentration−2SD do not overlap. ”, The measurement sensitivity of Comparative Example 1 is between AFP concentrations of 200 and 2,000 ng / mL.
The results of Example 1-1 and Example 2 of the present invention are shown in Table 4 and FIG. When the measurement sensitivity was obtained in the same manner as in Comparative Example 1, the measurement sensitivity was between 10 and 80 ng / mL of the AFP concentration, and the present invention was more sensitive than the conventional method.
In the conventional method, a large amount of unreacted fluorescent molecule label remains in the liquid phase. Therefore, when the amount of the fluorescent molecule complex is small compared to the unreacted fluorescent molecule label, both signals are Although it cannot be sufficiently discriminated, in the present invention, after the unreacted fluorescent molecule label is washed and removed, the released fluorescent molecule label complex is measured, so even if the amount of the complex is smaller , Indicating that both signals can be distinguished.

(Experimental results related to the second object of the present invention)
Even when the target substance is at zero concentration, it is common for nonspecific signals to be detected due to the effects of matrix-derived noise such as the buffer solution of the measurement sample, mechanical noise, and environmental noise. Known to. Also in the example of the present invention, the average number of effective events of 82.6 is measured as the primary measurement data of the AFP zero concentration (Table 4).
However, in the present invention, the influence of noise or the like can be reduced by taking into account the abundance ratio of the fluorescent molecule complex calculated at the same time. In the examples, since the abundance ratio of the fluorescent molecule complex at zero AFP concentration was calculated to be zero, the secondary measurement data was also zero, and more specific measurement was realized (Table 4).

  The method of the present invention is useful for measurement of a target substance (that is, immunoassay) and is expected to be applied in diagnosis and the like.

DESCRIPTION OF SYMBOLS 10 Fluorescence detection system 12 Confocal detection part 14 Calculation part 20 Laser light generation part 30 Optical processing part 40 Detection part 42 Sensor part 44 Electric signal processing part 46 Counting part

Claims (12)

Method for measuring target substance, including:
1) Immobilized to a solid phase via a cleavable linker, including a target substance, a first affinity molecule immobilized on a solid phase via a cleavable linker, and a second affinity molecule labeled with a fluorescent molecule Cleaving the fluorescent molecule labeling complex specifically in the solution at the site of the cleavable linker to obtain a release solution comprising the fluorescent molecule labeling complex released from the solid phase;
2) measuring the fluorescence signal in the release solution with a confocal fluorescence detector;
3) determining the presence or amount of the fluorescent molecular label complex contained in the release solution based on the characteristics of the fluorescent signal specific to the fluorescent molecular label complex released from the solid phase; and 4) Assessing the presence or amount of the target substance based on the presence or amount of the fluorescent molecular label complex contained in the release solution.   The method of claim 1, wherein the cleavable linker is a covalent linker or an affinity linker.   The method according to claim 1 or 2, wherein the target substance is an antigen, and each of the first affinity molecule and the second affinity molecule is an antibody.   4. The method of claim 3, wherein the first affinity molecule is an antibody against a first epitope of an antigen and the second affinity molecule is an antibody against a second epitope of an antigen.   The sample containing a target substance is further contacted with the first affinity molecule and the second affinity molecule to further prepare the fluorescent molecule-labeled complex. the method of.   6. The method of claim 5, further comprising washing the solid phase during and / or after preparation of the fluorescent molecular label complex. The method according to any one of claims 1 to 6, wherein step 1) is carried out as follows:
1-1) A release solution containing a fluorescent molecule-labeled complex released from a solid phase by specifically cleaving the fluorescent molecule-labeled complex at a site of a cleavable linker in the solution, and a two-phase containing a solid phase Obtaining the system; and 1-2) removing the solid phase from the two-phase system to obtain a release solution containing the fluorescent molecule-labeled complex and no solid phase.   The method according to claim 1, wherein the step 2) is performed by a photon counting method.   The method according to claim 1, wherein the property is a molecular weight or a fluorescence property.   The method according to claim 1, wherein the step 3) is performed using a fluorescence correlation method or a fluorescence cross-correlation method.   The method according to claim 1, wherein the solid phase is a magnetic bead. The method according to any one of claims 1 to 11, which is a method for measuring a target substance comprising:
a1) A sample containing a target substance is brought into contact with a first affinity molecule immobilized on a solid phase via a cleavable linker and a second affinity molecule labeled with a fluorescent molecule in a liquid phase, and the target substance A first affinity molecule immobilized on a solid phase via a cleavable linker, and a fluorescent molecule labeling complex immobilized on the solid phase via a cleavable linker, comprising the second affinity molecule, and a liquid phase Obtaining a first two-phase system comprising:
a2) removing the liquid phase from the first two-phase system to obtain the fluorescent molecule-labeled complex;
a3) A release solution containing the fluorescent molecule labeling complex released from the solid phase by specifically cleaving the fluorescent molecule labeling complex at the site of the cleavable linker in the solution, and a second second phase containing the solid phase Obtaining a system;
a4) removing the solid phase from the second two-phase system to obtain a release solution containing a fluorescent molecule-labeled complex and no solid phase;
a5) measuring the fluorescence signal in the release solution with a confocal fluorescence detector;
a6) determining the presence or amount of the fluorescent molecule label complex contained in the release solution based on the characteristics of the fluorescent signal specific to the fluorescent molecule label complex released from the solid phase; and a7) Assessing the presence or amount of the target substance based on the presence or amount of the fluorescent molecular label complex contained in the release solution.

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