Concentration estimation kit and concentration estimation method
The concentration estimation kit and method enhance the range of measurable antigen concentrations in FPIA by derivatizing antigens with different rates and measuring polarization changes, addressing the limitations of existing FPIA methods.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- TIANMA JAPAN LTD
- Filing Date
- 2022-06-01
- Publication Date
- 2026-06-30
Smart Images
Figure 0007882691000004 
Figure 0007882691000005 
Figure 0007882691000006
Abstract
Description
[Technical Field]
[0001] This disclosure relates to a concentration estimation kit and a concentration estimation method. [Background technology]
[0002] One immunoassay method using fluorescence is fluorescence polarization immunoassay (FPIA). The degree of fluorescence polarization measured by FPIA is proportional to the effective volume of the substance being measured. Patent document 1 describes an FPIA that utilizes the fact that the degree of fluorescence polarization changes due to a specific antigen-antibody reaction between a reagent in which an antibody is immobilized on a substance with a larger molecular weight than the antibody and a fluorescently labeled antigen.
[0003] FPIA uses antibodies that specifically bind to the target substance as an antigen. To obtain such antibodies, the target substance must possess immunogenicity, which is the activity that induces antibody production. When using a hapten as the target substance, which binds to antibodies but does not exhibit immunogenicity on its own due to its small molecular weight, the hapten must be conjugated to immunogenicity in order to obtain the antibody. For example, Patent Document 2 discloses an antigen-antibody reaction assay for quantifying antipyrine, using an antibody prepared with an antipyrine derivative, which is antipyrine conjugated to serum albumin, as the antigen. [Prior art documents] [Patent Documents]
[0004] [Patent Document 1] Japanese Patent Application Publication No. 3-103765 [Patent Document 2] Japanese Unexamined Patent Publication No. 51-104029 [Overview of the project] [Problems that the invention aims to solve]
[0005] FPIA detects changes in fluorescence polarization in response to antigen-antibody binding, but the range of concentrations of antigens that bind to antibodies, i.e., the target substance, is limited. Therefore, the range of target substance concentrations that can be estimated from the fluorescence polarization measurement value based on the calibration curve in FPIA depends on the affinity between the target substance and the antibody. If there are no multiple antibodies with different affinities to the target substance, the range of estimated target substance concentrations becomes limited.
[0006] This disclosure is made in view of the above circumstances and aims to provide a concentration estimation kit and concentration estimation method that can expand the range of concentrations of the target substance that can be estimated. [Means for solving the problem]
[0007] The concentration estimation kit relating to the first aspect of this disclosure is Derivatize at least a portion of the antigens contained in the mixed sample. do , amount of substance but each other Different multiple invitation Conductorizing reagent, An antibody against the aforementioned antigen, The antigen modified with a dye, It is equipped with.
[0008] The concentration estimation method relating to the second aspect of this disclosure is: A sample preparation step involves mixing a sample containing an antigen with a derivatization reagent that derivatizes at least a portion of the antigen contained in the sample, thereby obtaining a plurality of samples with different derivatization rates, which is the ratio of the derivatized antigen to the antigen contained in the sample. A mixing step involves mixing each of the aforementioned samples with an antibody against the antigen and the antigen modified with a dye to obtain a plurality of target solutions. A measurement step of measuring the degree of polarization of each of the aforementioned solutions to be measured, Includes. [Effects of the Invention]
[0009] According to the present disclosure, the range of the concentration of the measurable substance that can be estimated can be extended.
Brief Description of the Drawings
[0010] [Figure 1] It is a diagram showing the ratio of the antigen bound to the antibody against the antigen when the derivatization rate and the concentration of the antibody are changed. [Figure 2] It is a diagram showing the polarization degree calculated from the derivatization rate. [Figure 3] It is a diagram schematically showing a multi-well plate having an antibody, a tracer which is an antigen modified with a dye, and a derivatization reagent having different amounts of substance in each row. [Figure 4] It is a diagram showing the configuration of the fluorescence polarization measurement device according to the embodiment. [Figure 5] It is a diagram showing the microchannel within the effective field of view. [Figure 6] It is a diagram showing the polarization degree with respect to the histamine concentration of samples with different amounts of acylation reagent added.
Modes for Carrying Out the Invention
[0011] Embodiments according to the present disclosure will be described with reference to the drawings. Note that the present disclosure is not limited by the following embodiments and drawings. In the following embodiments, expressions such as "having", "including", or "containing" also include the meaning of "consisting of" or "composed of".
[0012] The concentration estimation kit according to the present embodiment is a kit for FPIA that estimates the concentration of an antigen, which is a substance to be measured in a sample, by utilizing the specific binding of the antigen by an antibody. For convenience of explanation, hereinafter, the antigen in the sample will be referred to as the "substance to be measured". The sample is not particularly limited as long as it is an object to be inspected or analyzed. For example, the sample is a cell, tissue, cell culture supernatant, cell extract, tissue extract, body fluid such as blood, saliva, urine, and lymph fluid obtained from a human or non-human animal, and biological samples such as nasal or nasopharyngeal aspirates, beverages, foods, and cleaning liquids of objects.
[0013] The concentration estimation kit according to this embodiment comprises a derivatization reagent, an antibody against the substance to be measured, and a dye-modified antigen. The derivatization reagent derivatizes at least a portion of the substance to be measured contained in the mixed sample. Derivatization here means adding substituents to the substance to be measured using not only hydrogen atoms constituting the substance to be measured, but also functional groups such as hydroxyl groups, amino groups, carboxyl groups, mercapto groups, carbonyl groups, and thiol groups. Examples of derivatization include silylation, acylation, esterification, and oximation. Derivatization may also be carried out by adding substituents to the substance to be measured using a known crosslinking agent. The substituent is any substituent that can be substituted on atoms constituting the substance to be measured by a known method. Examples of substituents are acyl groups and alkyl groups, and acyl groups are preferred. When adding acyl groups to the substance to be measured, an acylation reagent (acylation agent) that substitutes hydrogen atoms such as hydroxyl groups, amino groups, and mercapto groups of the substance to be measured with acyl groups (RCO-) can be used as the derivatization reagent. For example, acylation reagents include acid chlorides, acid anhydrides, ketones, and carboxylic acids. Specific examples of acylation reagents include trifluoroacetic anhydride, imidazole trifluoroacetate, and 4-chlorobutyryl chloride. Silylation reagents include hexamethyldisilazane (HMDS) and N-trimethylsilylimidazole. Esterification reagents include acid-alcohols, N,N-dimethylformamide, dimethylacetal, on-column methylating agents, and diazomethane. Oximation reagents include pentafluorobenzyl and hydroxyamine hydrochloride. Crosslinking agents include aldehydes and ketones; for example, glutaraldehyde is a crosslinking agent.
[0014] Derivatization reagents are used to obtain multiple samples with different derivatization rates, which is the ratio of the derivatized target substance to the target substance contained in the sample. The derivatization rate depends on the amount of derivatization reagent (amount of substance) relative to the amount of target substance contained in the sample. If the derivatization reagent is in the form of multiple solutions with different amounts of derivatization reagent, then when estimating the concentration of the target substance in the same sample, multiple samples with different derivatization rates can be obtained by mixing each of the same volume samples with multiple solutions containing different amounts of derivatization reagent. For example, multiple solutions with different amounts of derivatization reagent may consist of a solution with a minimum amount M and multiple solutions with different amounts of derivatization reagent, where the amount of derivatization reagent is a positive real multiple of M. The derivatization reagent may be a solution containing multiple derivatization reagents of the same concentration but different volumes, or a solution containing multiple derivatization reagents of different concentrations but the same volume.
[0015] The concentration estimation kit may include a de-derivatization reagent used to obtain a sample with a derivatization rate of 0. If the concentration estimation kit includes a solution containing multiple derivatization reagents of the same concentration but different volumes, the concentration estimation kit may also include a de-derivatization reagent that shares the same solvent as the solution containing the derivatization reagent and does not contain the derivatization reagent. If the concentration estimation kit includes a solution containing multiple derivatization reagents of different concentrations but the same volume, the concentration estimation kit may also include a de-derivatization reagent that shares the same solvent as the solution containing the derivatization reagent, has the same volume, and does not contain the derivatization reagent.
[0016] The antibody is not limited insofar as it specifically binds to the substance to be measured, and may include, for example, monoclonal antibodies, multispecific antibodies, bifunctional antibodies, human antibodies, humanized antibodies, antibodies derived from birds such as chickens, non-primates such as camels, mammals other than humans, and other animals, recombinant antibodies, chimeric antibodies, single-chain Fv, single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fv, anti-idiotype antibodies, bidomain antibodies, and bivariable domain antibodies.
[0017] The substance to be measured is not particularly limited, as long as its affinity for the antibody can be altered by derivatization with a derivatization reagent. Considering the efficiency of derivatization, the substance to be measured is preferably a low molecular weight substance rather than a high molecular weight substance such as a protein. Preferably, the substance to be measured is a hapten. A hapten is a substance that binds to an antibody but does not exhibit immunogenicity, which is the activity that induces antibody production, on its own due to its small molecular weight. Examples of haptens include histamine, γ-aminobutyric acid (GABA), dopamine, thyroid hormones, and steroid hormones. More specifically, thyroid hormones include triiodothyronine, thyroxine, and 3,5-diiodo-L-thyronine. Steroid hormones include estrone, estradiol, estriol, progesterone, cortisol, testosterone, and dehydroepiandrosterone sulfate. Haptens may also be low molecular weight peptide hormones, catecholamines, columbar enzyme vitamins, drugs, antibiotics, and their metabolites.
[0018] Haptens become immunogenic complete antigens by binding to immunogenic substances such as proteins. Examples of immunogenic substances include immunogenic proteins, polypeptides, carbohydrates, polysaccharides, lipopolysaccharides, and nucleic acids. The immunogenic substance is preferably a protein or polypeptide, with bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), and thyroglobulin being examples of immunogenic substances.
[0019] When the substance to be measured is a hapten, preferably the antibody is an antibody that uses a hapten derivative, in which an immunogenic substance is bound to the hapten via a linker, as its immunogen. The linker is an atomic group introduced between the immunogenic substance and the hapten. Examples of linkers include amides, disulfides, thioethers, hydrazones, hydrazides, imines, oximes, urea, thiourea, amidines, amines, and sulfonamides.
[0020] Antibodies using hapten derivatives ((hapten)-(linker)-(immunogenic substance)) as immunogens can be obtained by known methods. Typically, antibody acquisition involves injecting the immunogen into host animals such as rabbits, goats, mice, guinea pigs, or horses. Preferably, a mixture of the immunogen and an adjuvant is injected to enhance immunogenicity. Further injections of the immunogen may be administered at regular or irregular intervals to the same or different sites on the host animal. Antibody titers can be evaluated as appropriate, and antibodies that specifically bind to the hapten derivative can be recovered by blood collection from the host animal or other means.
[0021] When the substance to be measured is a hapten and an antibody using a hapten derivative as an immunogen is used, preferably the derivatization reagent imparts the same structure to the hapten in the sample as at least a part of the linker. For example, if the linker contains an acyl group, a derivatization reagent that imparts an acyl group to the hapten should be used.
[0022] The antigen included in the concentration estimation kit according to this embodiment is modified with a dye and functions as a tracer in the immunoassay. Hereinafter, the dye-modified antigen included in the concentration estimation kit will also be referred to as the "tracer". As the dye, a fluorescent dye that emits fluorescence is preferred. Each fluorescent dye has a fluorescence lifetime. Depending on the molecular weight of the substance to be measured, a fluorescent dye with a fluorescence lifetime of 1 to 10 nanoseconds, a fluorescent dye with a fluorescence lifetime of more than 10 nanoseconds to 200 nanoseconds, or a fluorescent dye with a fluorescence lifetime of more than 200 nanoseconds to 3000 nanoseconds may be appropriately selected. For example, fluorescent dyes with a fluorescence lifetime of 1 to 10 nanoseconds include fluorescein compounds such as indorenine, chlorotriazinylaminofluorescein, 4'-aminomethylfluorescein, 5-aminomethylfluorescein, 6-aminomethylfluorescein, 6-carboxyfluorescein, 5-carboxyfluorescein, 5-aminofluorescein, 6-aminofluorescein, thioureafluorescein, and methoxytriazinylaminofluorescein; rhodamine derivatives such as rhodamine B, rhodamine 6G, and rhodamine 6GP; and registered trademarks or trade names such as Alexa Fluor 488 and other Alexa Fluor series, BODIPY series, DY series, ATTO series, Dy Light series, Oyster series, HiLyte Fluor series, Pacific Blue, Marina Blue, Acridine, Edans, Coumarin, DANSYL, FAN, Oregon Green, Rhodamine Green-X, NBD-X, TET, JOE, and Yakima. Examples include Yellow, VIC, HEX, R6G, Cy3, TAMRA, Rhodamine Red-X, Redmond Red, ROX, Cal Red, Texas Red, LC Red 640, Cy5, Cy5.5, and LC Red 705. Fluorescent dyes with fluorescence lifetimes of over 10 nanoseconds to 200 nanoseconds include naphthalene derivatives such as dialkylaminonaphthalenesulfonyl, and pyrene derivatives such as N-(1-pyrenyl)maleimide, aminopyrene, pyrenebutanoic acid, and alkynylpyrene. Fluorescent dyes with fluorescence lifetimes of over 200 nanoseconds to 3000 nanoseconds include metal complexes such as platinum, rhenium, ruthenium, osmium, and europium.
[0023] To modify an antigen with a dye, for example, the dye and the antigen can be directly covalently bonded, or they can be bonded via a linker such as an oligoethylene glycol and an alkyl chain. When the antigen is a hapten and an antibody using a hapten derivative as an immunogen is used, preferably the dye is bonded to the hapten via a structure that is at least the same as a part of the linker interposed between the hapten and the immunogenic substance in the hapten derivative. For example, if the linker interposed between the hapten and the immunogenic substance in the hapten derivative contains an acyl group, it is preferable that the linker interposed between the dye and the antigen also contains an acyl group.
[0024] The dye has functional groups that can bind to carboxyl groups, amino groups, hydroxyl groups, thiol groups, and phenyl groups of the antigen. By reacting the functional groups of the dye and the antigen under known conditions, the antigen can be labeled with the dye. The number of dye molecules used to modify one antigen molecule can be arbitrarily selected. Preferably, there is one or more dye molecules per antigen molecule, and it may be two to five molecules.
[0025] Next, the concentration estimation method according to this embodiment will be described using the above-mentioned concentration estimation kit as an example. The concentration estimation method includes a sample preparation step, a mixing step, and a measurement step. In the sample preparation step, a sample containing the substance to be measured is mixed with the above-mentioned derivatization reagent to obtain a plurality of samples with different derivatization rates. These plurality of samples may be obtained by mixing each of the solutions containing multiple derivatization reagents with the same concentration but different volumes with the sample, or by mixing each of the solutions containing multiple derivatization reagents with different concentrations but the same volume with the sample. For example, the same amount of sample can be dispensed into each well of a multi-well plate, and each solution can be added to each well.
[0026] In the mixing step, each sample is mixed with the antibody and tracer to obtain multiple target solutions. If using the multi-well plate described above, the antibody and tracer should be added to each well containing each sample.
[0027] In the measurement step, the degree of polarization of each solution to be measured is measured. FPIA utilizes the change in degree of polarization associated with the change in molecular weight of the tracer when the tracer binds to the antibody to form a tracer-antibody complex. When a dye in solution maintains a steady state in the excited state, it emits polarized fluorescence in the same plane. However, when it rotates due to Brownian motion during the excited state, it emits fluorescence in a plane different from the excitation plane, thus eliminating the fluorescence polarization. The degree of fluorescence polarization indicates the degree to which the fluorescent molecule rotates between excitation and fluorescence emission. Molecules with small molecular weights rotate vigorously in solution due to Brownian motion, resulting in a low degree of polarization, while molecules with large molecular weights exhibit weaker Brownian motion, resulting in a higher degree of polarization. For example, in a solution containing a target substance A, an antibody B that specifically binds to target substance A, and a tracer C labeled with a fluorescent dye, target substance A and tracer C compete for antibody B in the solution. Therefore, if the concentration of target substance A is high, the amount of target substance A bound to antibody B increases (the amount of tracer C bound to antibody B decreases), and the amount of free tracer C not bound to antibody B increases. On the other hand, if the concentration of the substance A to be measured is low, the amount of substance A bound to antibody B decreases (the amount of tracer C bound to antibody B increases), and the amount of free tracer C not bound to antibody B decreases. If there is a difference between the mass of free tracer C and the mass of the complex formed when tracer C binds to antibody B, the concentration of substance A to be measured can be measured using the change in polarization as an indicator.
[0028] In FPIA, the change in molecular weight due to the binding of the tracer to the target substance is measured as a temporal change in molecular orientation. Any polarimeter can be used to measure the degree of polarization. The degree of polarization should be measured at a predetermined time after the reaction is complete. To quantify the target substance, a calibration curve can be created by performing the same procedure as above using a solution containing the target substance at a known concentration, and this can be compared with the measured value of the sample.
[0029] In the concentration estimation kit and concentration estimation method according to this embodiment, the polarization degree of each target solution prepared from multiple samples with different derivatization rates is measured to estimate the concentration of the target substance in the sample. If the target substance after derivatization is Ag_A, the target substance that has not been derivatized is Ag_B, the antibody is Ab, the complex of Ag_A and antibody is Ag_A-Ab, and the complex of Ag_B and antibody is Ag_B-Ab, then the binding constant K between Ag_A and Ab is... A and the coupling constant K between Ag_B and Ab B This is shown as follows:
[0030]
number
[0031] If we denote the ratio of bound to free Ag_A and the ratio of bound to free Ag_B as BF_A and BF_B, respectively, then the following applies.
[0032]
number
[0033] Then, the amounts of Ag_A (M), Ag_B (M), and Ab (M) added are p A , p B If we let and q be the given values, then BF_A can be found from the following cubic equation.
[0034]
number
[0035] Based on a cubic equation, when the derivatization rate and antibody concentration were changed, the ratio of antigen bound to the antibody to the total amount of antigen (derivatized and underivatized antigen) was estimated, and as shown in Figure 1, it was shown that changing the derivatization rate changes the affinity of the antigen to the antibody (K A :2E+9M -1 , K B :2E+6M-1 and total antigen amount (p A +p B ): 1E-8 M).
[0036] In FPIA, the polarization degree of the tracer is measured. The binding constant between the tracer and the antibody is assumed to be the same as the binding constant K A of the derivatized antigen. The B / F ratio of the tracer is equal to the B / F ratio (BF_A) of the derivatized antigen. Let the polarization degree when the tracer binds to the antibody be Fh and the polarization degree when the tracer is free be Fl. Then, the polarization degree P of the tracer in the FPIA system is represented by P = (Fh × BF_A + Fl) / (1 + BF_A). K A , K B , antibody concentration, tracer concentration, Fh, and Fl are set to 2E+9 M -1 , 2E+6 M -1 , 1E-7 M, 1E-8 M, 300 mP, and 100 mP, respectively. The polarization degree calculated from the derivatization rate is shown in Fig. 2. According to Fig. 2, it was shown that when the derivatization rate is different, the range of antigen concentration at which the polarization degree changes also changes.
[0037] According to the concentration estimation kit according to this embodiment, by measuring the polarization degrees of a plurality of samples containing measurement target substances with different derivatization rates, the affinity between the measurement target substance and the antibody can be changed. Since the measurable concentration range in FPIA depends on the affinity between the measurement target substance and the antibody, the range of the concentration of the measurement target substance that can be estimated can be expanded.
[0038] Note that the concentration estimation kit may include a multi-well plate, and different amounts of the derivatization reagent may be immobilized in each well of the multi-well plate. The derivatization reagent can be immobilized in the well by a known method. For example, a solution containing the derivatization reagent may be added to the well, and the solvent may be removed by drying or the like.
[0039] The multi-well plate described above is schematically illustrated in Figure 3. This multi-well plate has wells arranged in a 3x3 grid. Antibody, tracer, and derivatization reagent are immobilized in each well. The amount of immobilized antibody and tracer is the same in all wells. On the other hand, the amount of immobilized derivatization reagent differs from row to row, with the amounts of derivatization reagent immobilized in the first, second, and third row wells being M1, M2, and M3, respectively.
[0040] Using column A as an example, by adding the same volume of sample to each of the three wells in column A, the target substance in the sample can be derivatized according to the amount of derivatization reagent. By using this multi-well plate, multiple samples with different derivatization rates can be easily obtained. This multi-well plate also has columns B and C, which, like column A, have different amounts of derivatization reagent, making it useful for testing different samples or duplicates and triplicates. Note that the number of wells in the plate may be more than nine, and the number of rows and columns can be set appropriately according to the number of wells. [Examples]
[0041] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.
[0042] Histamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was modified with 5 / 6-TAMRA (Rhodamine) to obtain a histamine tracer. The histamine tracer was dissolved in pure water to prepare a 2 nM solution. Antihistamine antibody (manufactured by Progen Biotechnik) was diluted in phosphate buffer (PBS) to prepare a 21 nM solution. Histamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in pure water to prepare a 33 mg / mL solution. 100 μL of the obtained histamine solutions were divided into four test tubes, and the acylation reagent contained in the RIDA Screen Histamine ELISA Kit (manufactured by R-Biopharm) was added to each test tube. The amounts of acylation reagent added were 25 μL (Sample 1), 10 μL (Sample 2), and 5 μL (Sample 3), and no acylation reagent was added to one test tube (Sample 4).
[0043] Samples 1-4 were diluted 10-fold with pure water, and this process was repeated eight times to obtain nine different levels of samples for each of the nine levels. 25 μL of each of the nine obtained levels of samples, 25 μL of histamine tracer solution, and 25 μL of antibody solution were mixed and allowed to stand at room temperature in the dark for 60 minutes. The fluorescence polarization of the mixed solution was then measured.
[0044] For measuring the degree of fluorescence polarization, a fluorescence polarization measuring device having nine microchannels was used. The configuration of the fluorescence polarization measuring device 10 used is shown in Figure 4. The fluorescence polarization measuring device 10 comprises an LED light source unit 1, an excitation filter 2, an objective lens 3, a sample emission unit 4, a dichroic filter 5, a fluorescence filter 6, a digital imaging element (CMOS or CCD) 7, an imaging lens 8, and a liquid crystal element 9. Excitation light from the LED light source unit 1 with a central wavelength of 565 nm is irradiated onto the sample in the sample emission unit 4 via the excitation filter 2 and the objective lens 3. The fluorescence emitted by the sample is transmitted through the dichroic filter 5 and the fluorescence filter 6, and the transmitted light is acquired by the digital imaging element 7. By applying a voltage to the liquid crystal element 9 placed between the fluorescence filter 6 and the imaging lens 8 and modulating the voltage, the polarization direction of the transmitted fluorescence can be modulated. The modulation frequency and the acquisition frequency of the digital imaging element 7 are synchronized to acquire and calculate an image, and the degree of polarization P is extracted as a two-dimensional image.
[0045] The effective field of view of the optical observation portion of the sample emission unit 4 of the fluorescence polarization measurement device 10 is approximately 3 mm in diameter. As shown in Figure 5, within the circular effective field of view of φ3 mm, the channel width 11 and the space between channel widths 12 are provided at equal intervals, with a channel width of 200 μm and a space between channel widths of 100 μm. The depth of the channel is 900 μm. By forming multiple microchannels within the sample emission unit 4, multiple samples can be measured simultaneously. The excitation wavelength was set to 546 ± 11 nm, and the detection wavelength to 590 ± 16.5 nm. Mixed solutions prepared from nine different levels of samples were injected into each of the nine microchannels and measured simultaneously.
[0046] (result) Figure 6 shows the degree of polarization as a function of antigen (histamine) concentration for samples 1-4, which have different amounts of acylation reagent added. The range of antigen concentrations at which the degree of polarization changes changed depending on the amount of acylation reagent added. This demonstrates that the range of histamine concentrations that can be estimated can be expanded by using multiple samples with different derivatization rates.
[0047] The embodiments described above are for illustrative purposes only and do not limit the scope of the present invention. That is, the scope of the present invention is defined not by the embodiments, but by the claims. Various modifications made within the scope of the claims and equivalent inventive meaning are considered to be within the scope of the present invention. [Explanation of symbols]
[0048] 1 LED light source unit, 2 excitation filter, 3 objective lens, 4 sample emission unit, 5 dichroic filter, 6 fluorescence filter, 7 digital imaging element, 8 imaging lens, 9 liquid crystal element, 10 fluorescence polarization degree measuring device, 11 flow path width, 12 space between flow paths
Claims
1. Multiple derivatization reagents, each in different amounts, derivatize at least a portion of the antigens contained in the mixed sample. An antibody against the aforementioned antigen, The antigen modified with a dye, A concentration estimation kit equipped with the following features.
2. The antigen is a hapten, The antibody is an antibody that uses a hapten derivative in which an immunogenic substance is bound to the hapten via a linker as an immunogen, The derivatization reagent imparts the same structure as at least a part of the linker to the hapten in the sample, The dye is bonded to the hapten via the same structure as at least a portion of the linker. The concentration estimation kit according to claim 1.
3. The aforementioned hapten is, Histamine, The concentration estimation kit according to claim 2.
4. The derivatization reagent is These are multiple solutions containing the derivatizing reagent at the same concentration but with different volumes. A concentration estimation kit according to any one of claims 1 to 3.
5. The derivatization reagent is Multiple solutions containing the derivatizing reagents at different concentrations but with the same volume, A concentration estimation kit according to any one of claims 1 to 3.
6. A sample preparation step involves mixing a sample containing an antigen with a derivatization reagent that derivatizes at least a portion of the antigen contained in the sample, thereby obtaining a plurality of samples with different derivatization rates, which is the ratio of the derivatized antigen to the antigen contained in the sample. A mixing step involves mixing each of the aforementioned samples with an antibody against the antigen and the antigen modified with a dye to obtain a plurality of target solutions. A measurement step of measuring the degree of polarization of each of the aforementioned solutions to be measured, A concentration estimation method, including the following.
7. The antigen is a hapten, The antibody is an antibody that uses a hapten derivative in which an immunogenic substance is bound to the hapten via a linker as an immunogen, The derivatization reagent imparts the same structure as at least a part of the linker to the hapten in the sample, The dye is bonded to the hapten via the same structure as at least a portion of the linker. The concentration estimation method according to claim 6.
8. The aforementioned hapten is, Histamine, The concentration estimation method according to claim 7.
9. In the sample preparation step, Multiple samples are obtained by mixing each of the multiple solutions containing the derivatizing reagent at the same concentration but with different volumes, with the sample. The concentration estimation method according to any one of claims 6 to 8.
10. In the sample preparation step, Multiple samples are obtained by mixing each of the multiple solutions containing the derivatizing reagent at different concentrations but with the same volume, with the sample. The concentration estimation method according to any one of claims 6 to 8.