Support body for fluorescent polarization immunoassay, kit for fluorescent polarization immunoassay, and method for fluorescent polarization immunoassay

Abstract

To provide a supporting body having a plurality of reaction parts for performing fluorescent polarization immunoassay to measure a target substance in a sample.SOLUTION: A fluorescent polarization immunoassay supporting body includes: a reaction part for carrying an antibody and a fluorescent-labeling substance. A first fluorescent polarization immunoassay supporting body includes a reaction part for serving as a microchannel. A second fluorescent polarization immunoassay supporting body includes a reaction part for serving as a recess, with some of the reaction parts being connected through a communication passage.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a support for fluorescence polarization immunoassay on which an antibody and a fluorescent labeling substance are carried, a kit for fluorescence polarization immunoassay, and a fluorescence polarization immunoassay method using the support for fluorescence polarization immunoassay. [Background technology]

[0002] Fluorescence polarization immunoassay is an immunoassay method that uses fluorescence. It is known that the degree of fluorescence polarization measured by fluorescence polarization immunoassay is proportional to the effective volume of the substance to be measured. Patent Document 1 describes a fluorescence polarization immunoassay that uses a reagent in which an antibody (or antigen) is immobilized on a substance with a larger molecular weight than the antibody, and that utilizes the fact that a large change in the degree of fluorescence polarization occurs due to a specific antigen-antibody reaction between this reagent and the fluorescently labeled antigen (or antibody).

[0003] There is also a method for measuring high molecular weight substances using fluorescence polarization immunoassay (Patent Document 2). In the examples, pyrenebutanoic acid is used as the fluorescent dye, and an anti-HDL polyclonal antibody is used as the antibody that specifically binds to the substance to be measured, to create a calibration curve for HDL.

[0004] Such fluorescence polarization immunoassays can be performed using multiwell plates containing multiple wells. Patent Document 3 describes a module containing multiple assay domains, including a first assay domain containing a first reagent and a second assay domain containing a second reagent, where the first assay domain is capable of generating luminescence at least 10 times brighter than the second assay domain, and where interference between the luminescence emitted from the first assay domain and the luminescence emitted from the second assay domain is reduced. Preferably, the assay is performed in an assay module having an integrated electrode and a reader device configured to induce electrode-induced luminescence and measure the induced luminescence (abstract). Patent Document 3 states that multiple types of antibodies can be immobilized in a single assay domain, and multiple test measurements can be performed using a module with multiple assay domains.

[0005] There is also a multi-well assay plate in which multiple wells contain a binding surface for immobilizing a capture reagent and a reconstitutable dry reagent, and the dry reagent is arranged on the surface of the well so that it does not overlap with the binding surface (Patent Document 4). Because the dry reagent does not overlap with the binding surface, even if an additional liquid reagent containing an assay control is dispensed and dried, the assay can be performed without the dried detection reagent and the dried assay control coming into physical contact with each other. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 3-103765 [Patent Document 2] Patent No. 3255293 [Patent Document 3] Special Publication No. 2005-521032 [Patent Document 4] Special Publication No. 2009-521686 Summary of the Invention [Problem to be solved by the invention]

[0007] In general assays, in addition to high sensitivity and accuracy of the equipment and measurement system used, factors such as easy operation and low reagent volume are required. Fluorescence polarization immunoassay is based on the principle of competitive binding immunoassay and uses two types of reagents: a fluorescently labeled compound in which the same molecule as the target molecule is labeled with a fluorescent substance, and an antibody that specifically binds to the target molecule. When using multi-well plates, multiple reagents must be added to each well, making the operation complicated. Therefore, the development of a support and kit for fluorescence polarization immunoassay that are easy to operate is desired.

[0008] Furthermore, in fluorescence polarization immunoassay, the concentration of a target compound can be quantified within a range where the relationship between the concentration of the target compound and the degree of fluorescence polarization is consistent. If the concentration of the target compound contained in the sample solution falls outside this measurement range, the sample solution must be diluted and remeasured. Therefore, if the measurement range is wide, the dilution step of the sample solution can be omitted.

[0009] In view of the above-described current situation, an object of the present disclosure is to provide a support for fluorescence polarization immunoassay that has a wide measurement range and is easy to use, and is suitable for fluorescence polarization immunoassay, and a kit for fluorescence polarization immunoassay.

[0010] Another object of the present disclosure is to provide a fluorescence polarization immunoassay method using a support for fluorescence polarization immunoassay. [Means for solving the problem]

[0011] As a result of detailed investigation into fluorescence polarization immunoassay, the present inventors have discovered that if an antibody capable of binding to the substance to be measured and a fluorescently labeled substance in which the substance to be measured is labeled with a fluorescent dye are carried in the reaction zone of a support, fluorescence polarization immunoassay can be performed simply by adding a sample solution containing the substance to be measured, and have completed the present disclosure.

[0012] That is, the present disclosure provides a support having a plurality of reaction sites for performing fluorescence polarization immunoassay on a substance to be measured in a sample, the support comprising: The present invention provides the following support for fluorescence polarization immunoassay, characterized in that the reaction zone carries an antibody capable of binding to the substance to be measured and a fluorescently labeled substance in which the substance to be measured is labeled with a fluorescent dye.

[0013] The present disclosure provides a first support for fluorescence polarization immunoassay, characterized in that the reaction section is a microchannel.

[0014] The present disclosure also provides a second support for fluorescence polarization immunoassay, characterized in that the reaction areas are recesses, and some of the reaction areas are connected by a communication path.

[0015] The present disclosure also provides the support for fluorescence polarization immunoassay, wherein the fluorescent dye is one or more selected from the group consisting of fluorescein, dansyl, pyrene, rhodamine, dialkylaminonaphthalene, dialkylaminonaphthalenesulfonyl, indolenine, and ruthenium.

[0016] The present disclosure also provides the support for fluorescence polarization immunoassay, wherein the fluorescent dye has a fluorescence lifetime of 1 to 3,000 nanoseconds.

[0017] The present disclosure also provides a fluorescence polarization immunoassay kit comprising the support for fluorescence polarization immunoassay and a solvent for dissolving the substance to be measured.

[0018] The present disclosure also provides a method for detecting a fluorescence polarization immunoassay by adding a sample solution containing the substance to be measured to a reaction zone of the support for fluorescence polarization immunoassay; reacting the target substance, the antibody, and the fluorescently labeled substance in the reaction section; Next, a fluorescence polarization immunoassay is performed on the reaction zone at a temperature of 4 to 40°C. [Effects of the Invention]

[0019] According to the present disclosure, there is provided a support for fluorescence polarization immunoassay, in which a reaction part carries an antibody capable of binding to a substance to be measured and a fluorescently labeled substance obtained by labeling the substance to be measured with a fluorescent dye. There is also provided a fluorescence polarization immunoassay method using the support for fluorescence polarization immunoassay. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram of a support for fluorescence polarization immunoassay on which circular reaction sites are formed in three rows and three columns. [Figure 2]FIG. 1 is a diagram of a support for fluorescence polarization immunoassay in which reaction sections are connected in series via linear communication paths. [Figure 3] FIG. 3 is a diagram illustrating a method for using the support for fluorescence polarization immunoassay shown in FIG. [Figure 4] FIG. 1 is a diagram of a support for fluorescence polarization immunoassay with five microchannels as reaction sections. [Figure 5] FIG. 1 is a diagram of a support for fluorescence polarization immunoassay in which reaction sites are arranged in a 3×3 array, and each reaction site carries a different concentration of fluorescently labeled substance and a different concentration of measurement antibody. [Figure 6] FIG. 10 shows the results of measuring the degree of fluorescence polarization versus the concentration of the antibody to be measured, with the concentration of the substance to be measured being changed. [Figure 7] FIG. 10 is a diagram showing the results of measuring the degree of fluorescence polarization for a substance to be measured, with the fluorescently labeled substance and the substance to be measured kept constant. [Figure 8] FIG. 10 shows the results of measuring the fluorescence polarization degree versus the concentration of a substance to be measured using measurement antibodies with different binding constants. [Figure 9] FIG. 1 is a diagram of a support for fluorescence polarization immunoassay in which different fluorescently labeled substances and antibodies with different binding affinities to the substance to be measured are carried in 3×3 reaction zones. [Figure 10] This is a diagram of a support for fluorescence polarization immunoassay, in which a fixed amount of a fluorescently labeled substance and a measurement antibody are carried in 3 x 3 reaction zones, and a different pH adjuster is carried in each row. [Figure 11] FIG. 1 is a diagram illustrating an embodiment in which the reaction zone of a support for fluorescence polarization immunoassay is measured at different temperatures T1, T2, and T3. [Figure 12] FIG. 1 shows the results of Example 1, and shows the results of measuring the fluorescence polarization degree versus the concentration of measurement antibodies using measurement antibodies with different binding constants. [Figure 13] FIG. 1 shows the results of Example 1, measuring the degree of fluorescence polarization versus the concentration of a substance to be measured using antibodies for measuring different binding constants. [Figure 14]FIG. 10 shows the results of measuring the degree of fluorescence polarization versus the concentration of the substance to be measured using a support carrying a fluorescently labeled substance and an antibody for measurement at different concentrations. [Figure 15] FIG. 10 is a diagram showing the results of measuring the degree of fluorescence polarization relative to the concentration of the substance to be measured, with the concentration of the fluorescent labeling substance being changed. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present disclosure relates to a support having a plurality of reaction zones for performing fluorescence polarization immunoassay on a substance to be measured in a sample, characterized in that the reaction zones support an antibody capable of binding to the substance to be measured and a fluorescently labeled substance obtained by labeling the substance to be measured with a fluorescent dye. Because the antibody and the fluorescently labeled substance are supported in advance in the reaction zones, adding a sample containing a fixed amount of the substance to be measured and measuring the degree of fluorescence polarization eliminates the need to add a reaction reagent, and allows concentration measurement to be performed with a simple procedure.

[0022] (1) Measurement target substance The term "analyte" refers to a compound or composition to be measured using the support for fluorescence polarization immunoassay of the present disclosure. Measurable analytes are compounds for which antibodies can be prepared, with at least a portion of the compound serving as an epitope. Examples include proteins, glycoproteins, peptides, polypeptides, oligonucleotides, polynucleotides, antibodies, antigens, haptens, hormones, drugs, enzymes, and receptors. For ease of explanation, when the analyte is an antibody, it will be referred to as "analyte antibody."

[0023] Analyte antibodies may include monoclonal antibodies, multispecific antibodies, bifunctional antibodies, human antibodies, humanized antibodies, antibodies derived from birds such as chickens, mammals such as humans and cows, non-primates such as camels, and other animals, recombinant antibodies, chimeric antibodies, single-chain Fvs ("scFvs"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs ("sdFvs"), as well as anti-idiotype ("anti-Id") antibodies, dual-domain antibodies, and dual-variable domain antibodies. Categorizing the substances to be measured based on their origin and properties allows for the measurement of biological substances, pharmaceuticals, viruses, bacteria, and the like. Biological substances include various components produced by living organisms, such as immunoglobulins, various components excreted outside the body, and the living organisms themselves. Living organisms include plants and animals. Pharmaceuticals are not limited to those administered to humans or animals, but also include pesticides and the like.

[0024] (2) Antibody for measurement The antibody supported on the reaction zone of the support for fluorescence polarization immunoassay is an antibody capable of binding to the analyte. It must have the ability to recognize and bind to at least a portion of the analyte as an epitope. If such an antibody is commercially available, it can be used. If not, it can be produced using an immunogen in which an immunogenic carrier substance is bound to the analyte via an acid amide bond or other group. The immunogenic carrier substance can be selected from conventionally known substances. It may be any immunogenic protein, polypeptide, carbohydrate, polysaccharide, lipopolysaccharide, nucleic acid, etc. Proteins or polypeptides are preferred, and bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), and thyroglobulin are more preferred. Such immunogens can be used to prepare polyclonal or monoclonal antibodies using well-known methods. Typically, an immunogen, preferably a mixture of the immunogen and adjuvant, is injected into one or more sites of a host animal such as a rabbit, goat, mouse, guinea pig, or horse. Further injections may be given at the same or different sites at regular or irregular intervals. The titer may be assessed as appropriate to obtain the desired antibody. The antibody may be recovered by, for example, drawing blood from the host animal.

[0025] To distinguish it from an "analyte antibody," an antibody capable of binding to a substance to be measured and supported in a reaction zone for measurement is referred to as an "antibody for measurement." Measurement antibodies in the present disclosure include monoclonal antibodies, multispecific antibodies, bifunctional antibodies, human antibodies, humanized antibodies, antibodies derived from birds such as chickens, mammals such as humans and cows, non-primates such as camels, and other animals, recombinant antibodies, chimeric antibodies, single-chain Fvs ("scFvs"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs ("sdFvs"), as well as anti-idiotype ("anti-Id") antibodies, dual-domain antibodies, dual-variable domain antibodies, and the like. This is because it is sufficient for an antibody to bind to at least one epitope of the compound to be measured. Furthermore, some of the amino acids in the measurement antibody prepared in this manner may be substituted with other amino acid residues for the purpose of improving heat resistance, chemical resistance, pressure resistance, or other purposes, as long as the binding ability to the substance to be measured is not impaired.

[0026] (3) Fluorescent labeling substances The fluorescently labeled substance carried in the reaction section is a compound in which the substance to be measured is labeled with a fluorescent dye.

[0027] A fluorescent dye is a dye that emits fluorescence. Each fluorescent dye has its own unique fluorescence lifetime. In the present disclosure, 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 3,000 nanoseconds can be appropriately selected and used depending on the molecular weight of the substance to be measured. For example, fluorescent dyes with a fluorescence lifetime of 1 to 10 nanoseconds include fluorescein compounds such as indolenine, chlorotriazinylaminofluorescein, 4'-aminomethylfluorescein, 5-aminomethylfluorescein, 6-aminomethylfluorescein, 6-carboxyfluorescein, 5-carboxyfluorescein, 5 and 6-aminofluorescein, thioureafluorescein, and methoxytriazinylaminofluorescein; rhodamine derivatives such as rhodamine B, rhodamine 6G, and rhodamine 6GP; and trademarks such as the Alexa Fluor series, including Alexa Fluor 488, the BODIPY series, the DY series, the ATTO series, the Dy Light series, the Oyster series, the HiLyte Fluor series, Pacific Blue, Marina Blue, Acridine, Edans, Coumarin, DANSYL, FAN, Oregon Green, Rhodamine Green-X, NBD-X, TET, JOE, and Yakima. 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 are examples of fluorescent dyes with a fluorescence lifetime of more than 10 nanoseconds to 200 nanoseconds. These include naphthalene derivatives such as dialkylaminonaphthalenesulfonyl, and pyrene derivatives such as N-(1-pyrenyl)maleimide, aminopyrene, pyrenebutanoic acid, and alkynylpyrene. Furthermore, metal complexes of platinum, rhenium, ruthenium, osmium, and europium are examples of fluorescent dyes with a fluorescence lifetime of more than 200 nanoseconds to 3,000 nanoseconds.

[0028] Labeling a target substance with a fluorescent dye can be achieved by, for example, covalently bonding the fluorescent dye to the target substance or by bonding via a suitable linker such as oligoethylene glycol or an alkyl chain. The fluorescent dye has a functional group capable of bonding to a carboxyl group, an amino group, a hydroxyl group, a thiol group, a phenyl group, or the like. The target substance may be a protein or the like, and can be covalently bonded to the above functional group. A fluorescently labeled substance can be produced by reacting the functional groups of the fluorescent dye and the target substance under conditions well known to those skilled in the art. After the reaction is complete, unreacted fluorescent dye can be removed by a conventional method. The number of fluorescent dye molecules bonded to the fluorescently labeled substance can be freely selected. Preferably, there is one or more molecules, and more preferably two to five molecules, per molecule of the target substance. In the present disclosure, a fluorescently labeled substance in which a target substance is labeled with a fluorescent dye is also referred to simply as a "fluorescently labeled substance."

[0029] (4) Support Materials that can be used for the support for fluorescence polarization immunoassays of the present disclosure include, for example, resins such as polypropylene, polyethylene, polymethylpentene, ethylene-tetracyclododecene copolymer, polyacetal, acrylonitrile-butadiene-styrene resin, hydroxybenzoic acid polyester, polyetherimide, methacrylic resin, polyethylene terephthalate, polybutadiene terephthalate, polycyclohexylene dimethylene terephthalate, polyethylene naphthalate, polyacrylonitrile, polystyrene, polyamide, polycarbonate, polyvinyl alcohol, and polylactic acid, as well as glass and quartz. Supports that can be used include various plates with multiple recesses, such as microplates, multiwell plates, microwell plates, and immunoplates, which have been conventionally used for various immunoassays such as radioimmunoassays, enzyme immunoassays, and fluorescent immunoassays using fluorescent substances.

[0030] (5) Reaction section The recesses formed on the support can be used as reaction zones in the support for fluorescence polarization immunoassay of the present disclosure. The shape of the reaction zones is not particularly limited, and may be semicircular dish-like, cylindrical (flat-bottom), disc-like, hemispherical (U-bottom), or the like. The number of reaction zones is also not particularly limited, as long as at least two are present on the support. The number is preferably 6 to 1,000, more preferably 10 to 100. The size and shape of the support are not particularly limited and can be selected appropriately depending on the fluorescence polarization measurement device. The arrangement of the reaction zones on the support is also not particularly limited. A support in which the reaction zones are aligned vertically and horizontally allows efficient fluorescence polarization measurement of a small amount and a large number of samples using a microplate reader for detecting and measuring absorbance, fluorescence, and luminescence. The volume of the reaction zones is sufficient to support the antibody to be measured and the fluorescently labeled substance, and to accommodate a certain amount of sample solution containing the substance to be measured. It is 0.01 to 1 ml, preferably 0.1 to 0.4 ml.

[0031] An example of a support 1 for fluorescence polarization immunoassay is shown in Figure 1. In Figure 1, circular recesses are formed in three rows and three columns on the support 1 as reaction areas 3. The reaction areas 3 carry an antibody to be measured and a fluorescently labeled substance.

[0032] Multiple reaction sites formed on a support for fluorescence polarization immunoassay may be interconnected via a communication channel. Figure 2 shows an embodiment in which three rectangular reaction sites 3 are serially connected via a linear communication channel 5. In Figure 2, a branch channel 7 is formed in the communication channel 5. As shown in Figure 3A, multiple reaction sites 3 carrying fluorescently labeled substances 9 and assay antibodies 11 are connected to the communication channel 5 via the branch channels 7 leading to each reaction site 3. The communication channel 5 can be used as a sample solution injection channel. As shown in Figure 3B, by injecting sample solution 13 into the left end of the communication channel 5, three reaction sites 3 can be filled with the sample solution 13 in one operation. As shown in Figure 3C, after filling the reaction sites 3 with sample solution 13, a sealant 15 such as air, nitrogen gas, or other gas that does not affect fluorescence polarization measurement can be blown in place of the sample solution 13 to remove the sample solution 13 from the communication channel 5. Instead of a gas such as air, a solution that does not affect fluorescence polarization measurement, such as silicone or a fluorine-based inert liquid, can be used as the sealant 15.

[0033] Furthermore, the PDMS microchannels can be used as the reaction zones of a support for fluorescence polarization immunoassay. Figure 4 shows a support 1 with five microchannels as reaction zones 3.

[0034] (6) Support The reaction zone of the support for fluorescence polarization immunoassay supports a measurement antibody and a fluorescently labeled substance. In the present disclosure, "supported" refers to a state in which the measurement antibody and the fluorescently labeled substance are bound to each other to such an extent that they can be released from the surface of the reaction zone into the solution when a sample solution containing the substance to be measured is added to the reaction zone. Therefore, this does not include a reaction zone in which the measurement antibody or the like is immobilized by a covalent bond. It also does not include a reaction zone in which plasma treatment or other surface treatment has been performed to increase the binding strength of the measurement antibody or the fluorescently labeled substance, so that the measurement antibody or the fluorescently labeled substance cannot be released from the surface of the reaction zone even when a sample solution is added.

[0035] The method for loading the measurement antibody or fluorescently labeled substance into the reaction section is not particularly limited. For example, a solution in which the measurement antibody is dissolved or dispersed, or a solution in which the fluorescently labeled substance is dissolved or dispersed, can be dropped into the reaction section, and then dried by freeze-drying, vacuum drying, hot drying, low-temperature drying, or the like, to load them into the reaction section. A mixed solution in which the measurement antibody and the fluorescently labeled substance are dissolved or dispersed in a specific ratio can be prepared in advance, and this can be dropped into the reaction section, and then loaded by freeze-drying or the like in the same manner as above.

[0036] (7) Fluorescence polarization immunoassay Fluorescence polarization immunoassay utilizes the competitive reaction of substances and the change in polarization that occurs with changes in the molecular weight of the competing substance. When a fluorescent dye in a liquid maintains a steady state in an excited state, it emits polarized fluorescence in the same plane. However, if it rotates due to Brownian motion during the excited state, it emits fluorescence in a different plane from the excitation plane, resulting in the loss of fluorescence polarization. Fluorescence polarization indicates the degree of rotation of a fluorescent molecule between excitation and emission of fluorescence. Low-molecular-weight molecules rotate more vigorously due to Brownian motion in solution, resulting in a low degree of polarization, while large-molecular-weight molecules exhibit weaker Brownian motion, resulting in a higher degree of polarization. For example, in a solution containing analyte A, antibody B, which specifically binds to analyte A, and fluorescent-labeled substance C, which is analyte A labeled with a fluorescent dye, analyte A, antibody B, and fluorescent-labeled substance C compete with each other in the solution. Therefore, a high concentration of analyte A increases the amount of binding between analyte A and antibody B, while increasing the amount of free fluorescent-labeled substance C that does not bind to antibody B. If there is a difference in mass between fluorescently labeled substance C and the complex of antibody B and fluorescently labeled substance C, the concentration of target substance A can be measured using the change in polarization as an index.

[0037] (8) Loading amount As described above, fluorescence polarization immunoassay utilizes a competitive reaction between the analyte, the measurement antibody, and the fluorescently labeled substance. Therefore, differences in the amount of the fluorescently labeled substance or the measurement antibody carried by the measurement antibody change the degree of fluorescence polarization, and also change the measurable range of the analyte. In the fluorescence polarization immunoassay support of the present disclosure, the multiple reaction zones may each carry different amounts of the fluorescently labeled substance or the measurement antibody. The amounts carried by such supports with different amounts can be easily changed by changing the amount of solution of the fluorescently labeled substance or the measurement antibody dripped onto the reaction zone, or by changing the concentration of the dripped solution.

[0038] As one such example, Figure 5 shows a schematic diagram of a support 1 having reaction zones 3 arranged in a 3x3 pattern, each reaction zone 3 carrying a different concentration of fluorescently labeled substance 9 and a different concentration of measurement antibody 11. While Figure 5 shows a configuration in which the amounts of both the fluorescently labeled substance 9 and the measurement antibody 11 carried are varied, the amount of either one carried may also be varied. This can be selected appropriately depending on the properties of the substance to be measured and other factors.

[0039] For example, if the loading amount of all fluorescently labeled substances in m1 rows of reaction sections arranged in an n × m array is defined as Dm1 and the loading amount of fluorescently labeled substances in m2 rows is defined as Dm2, then a calibration curve can be created and a sample solution can be measured simultaneously under the conditions of loading amounts of fluorescently labeled substances Dm1 and Dm2. Even if the concentration of the substance to be measured in the sample solution is unknown, measurement can be performed using either calibration curve, expanding the measurement range and reducing the number of dilution steps for the sample solution.

[0040] (9) Antibodies with different binding affinities The measurement antibodies may have different binding affinities to the substance to be measured. The measurement range can also be widened by using antibodies with different binding affinities.

[0041] Fluorescence polarization immunoassay can be thought of as an antigen-antibody reaction, where the substance to be measured is an antigen. The equilibrium equation for binding in an antigen-antibody reaction can be expressed as Ka = ([AgAb]) / ([Ag][Ab]), B / F = ([AgAb]) / [Ag], where Ag is the antigen concentration, Ab is the antibody concentration, Ka is the binding constant, B is the bound concentration, and F is the free concentration.

[0042] If the initial concentrations of Ag and Ab are p and q, then [Ag] = p - [AgAb], [Ab] = q - [AgAb], and (B / F) 2 +(B / F)(1+Kap-Kaq)-Kaq=0.

[0043] Let the concentration of the substance to be measured be x, x = [Ag], and the concentration of the fluorescently labeled substance be p, and p → p + x, then the ternary system is expressed as (B / F) 2 +(B / F)(1+Kap+Kax-Kaq)-Kaq=0, and can be expressed as B / F(Ka,p,x,q). If (B / F)=R, the above equation becomes R(Ka,p,x,q)=-(1+Kap+Kax+(-Ka)q) / 2+√((1+Kap+Kax+(-Ka)q) 2 +4Kaq) / 2.

[0044] If the fluorescence polarization index of a fluorescently labeled substance is Fh when bound and Fl when free, the value of the fluorescence polarization index can be expressed by the variables in the following formula (1).

[0045]

number

[0046] The concentration of the fluorescent label and the binding constant of the antibody used for measurement are the same, and the binding constant Ka is 1 x 10 10 M -1 Figure 6 shows the results of plotting the above formula (1) by varying the concentration of the antibody used for measurement under the conditions shown in Table 1, where Fh = 0.3, Fl = 0.07, and the three concentrations of the substance to be measured. As shown by curves a, b, and c, the degree of fluorescence polarization decreases as the concentration of the substance to be measured increases.

[0047] [Table 1]

[0048] On the other hand, the concentration of the substance to be measured is kept constant (q = 4 × 10 -10 M), and the concentration of the fluorescent labeling substance was also kept constant (concentration p = 1 × 10 -10 The results of measuring the fluorescence polarization degree versus the antibody concentration for measurement are shown in Figure 7. In Figure 7, the white area surrounded by the dashed line shows the 1 × 10 -10 M~1×10 -8 It can be seen that there is a certain correlation between the measurement antibody and the degree of fluorescence polarization within the range of M, and this range is the measurement range for the substance to be measured. This result coincides with the fact that the measurement range width expands when the amount of measurement antibody carried in the reaction zone is changed.

[0049] Next, as shown in Table 2, antibodies with different binding constants Ka were used for measurement, and the concentrations of the antibodies and fluorescent labeling substances were varied to create fluorescence polarization curves in the same manner as above. The results are shown in Figure 8. The measurable range differs depending on the curve, and curve d shows a range of 1 × 10 -11 M~1×10 -10 The range of M is the measurable range, and for curve e, it is 1×10 -10 M~1×10 -9 The range of M is 1 × 10 for curve f. -9 M~1×10 -8 The range of M is the measurable range of the substance to be measured. In the present disclosure, by loading antibodies with different binding affinities for the substance to be measured onto the reaction section and by loading different concentrations of fluorescently labeled substances and measurement antibodies onto the reaction section, it is possible to obtain the measurement range width, for example, within the range indicated by the dashed line in Figure 8.

[0050] [Table 2]

[0051] 9 shows a schematic diagram of a support 1 having a 3 × 3 reaction zone 3, on which a fluorescently labeled substance 9 and antibodies (measurement antibodies) 11 with different binding affinities for the substance to be measured are supported. Antibodies with different binding affinities for the substance to be measured can be prepared, for example, by inoculating an immunogen, in which an immunogenic carrier substance such as a polysaccharide is bound to the compound to be measured, into one or more different sites of a rabbit or other host animal to obtain antibodies, and then evaluating the titers as appropriate and collecting antibodies with different titers.

[0052] (10) pH adjuster The reaction zone may further contain a pH adjuster. The binding affinity between the analyte and the antibody for measurement varies depending on the pH, ensuring a wide measurement range. Examples of such pH adjusters include glycine-NaOH (pKa 9.60), Tris-HCl (pKa 8.20), Tricine-HCl (pKa 8.15), HEPES-NaOH (pKa 7.55), NaH2PO4-Na2HPO4 (pKa 7.22), MOPS-NaOH (pKa 7.20), MES-NaOH (pKa 6.15), acetate-NaOH (pKa 4.80), glycine-NaOH (pKa 2.34), and GTA buffer. To load the pH adjuster into the reaction zone, prepare a solution of the adjuster, dropwise add it to the reaction zone, and then dry it in the same manner as loading the fluorescently labeled substance and antibody for measurement. The pH adjuster can be selected appropriately depending on the properties of the substance to be measured, the fluorescent labeling substance, and the measurement antibody. Figure 10 shows a schematic diagram of a 3x3 array of reaction sites, each carrying a fixed amount of fluorescent labeling substance and measurement antibody, and each row carrying a different pH adjuster.

[0053] (11) Fluorescence polarization immunoassay kit The support for fluorescence polarization immunoassay of the present disclosure can be used as a fluorescence polarization immunoassay kit that further contains a solvent for dissolving the substance to be measured. Examples of solvents for dissolving the substance to be measured include water such as pure water; alcohols such as methanol, ethanol, and butanol; ketones such as acetone, diethyl ketone, and methyl amyl ketone; alkanes such as hexane and heptane; ethers such as diethyl ether; and mixed solvents of these, such as methyl sulfoxide, acetonitrile, and chloroform.

[0054] (12)Measurement method Using the support for fluorescence polarization immunoassay of the present disclosure, an analyte contained in a sample solution can be analyzed as follows. First, the analyte is dissolved or dispersed in pure water or another solvent suitable for dissolving the analyte. If necessary, impurities are removed from this solution by filtration or other means to prepare a sample solution. A fixed amount of this solution is then added to the reaction zone of the fluorescence polarization immunoassay support. The analyte antibody and fluorescently labeled substance supported in the reaction zone react with the analyte contained in the sample solution. Because the reaction is based on an antigen-antibody reaction, it is rapid and highly reproducible. The reaction zone contains a conjugate between the analyte and the analyte antibody, as well as a conjugate between the fluorescently labeled substance and the analyte antibody. A high concentration of the analyte in the sample solution increases the amount of binding between the analyte and the analyte antibody, resulting in an increase in the amount of free fluorescently labeled substance that does not bind to the analyte antibody. In fluorescence polarization immunoassay, the change in molecular weight associated with binding between the fluorescently labeled substance and the analyte is measured as a temporal change in molecular orientation. If there is a difference in mass between the fluorescently labeled substance and the conjugate between the analyte antibody and the fluorescently labeled substance, the concentration of the analyte can be determined using the change in polarization as an indicator. The degree of fluorescence polarization can be measured using any polarization measurement device. The degree of fluorescence polarization is measured at a predetermined time after the reaction is completed. The measurement can be performed at a temperature within a range that does not denature the substance to be measured, and is carried out at a constant temperature within a range of 4 to 40°C, preferably 10 to 40°C. To quantify the substance to be measured, a calibration curve can be prepared in advance using a solution containing the substance to be measured at a known concentration in the same manner as above, and compared with the measured value of the sample solution.

[0055] On the other hand, for example, if a calibration curve is created at a temperature of 10°C and the fluorescence polarization of the analyte contained in the sample solution is measured, the concentration of the analyte may not fall within the measurement range of this calibration curve. In this case, the measurement temperature may be changed to 40°C and measurement may be performed at a predetermined time after the reaction. Because the binding constant changes with temperature, changing the temperature can ensure a wide measurement range. Figure 11 shows a schematic diagram of an embodiment in which the reaction zone is measured at temperatures T1, T2, and T3.

[0056] In the present disclosure, there are no limitations on the device as long as it can measure the degree of fluorescence polarization. As described above, when using a support for fluorescence polarization immunoassay in which the reaction section is configured with a microchannel, a measurement device capable of measuring the microchannel can be used to perform highly sensitive measurements using a small amount of sample.

[0057] (Embodiment) The present disclosure will now be described in detail with reference to embodiments, but these embodiments do not limit the present disclosure in any way.

[0058] (Embodiment 1) Coupling constant Ka = 2 × 10 6 M -1 Antibody A for measurement, and binding constant Ka = 3 × 10 8 M -1 Antibody B for measurement was used. Antibody A for measurement was used at 5 × 10 -7 M, 3 x 10 -7 M, 1 x 10 -7 M, 6 x 10 -8 M, 3 x 10 -8 M, 2 x 10 -8 M, 8 x 10 -9 M, 4 x 10 -9 M, 2 x 10 -9 M, 6 × 10 antibody B for measurement -7 M, 3 x 10 -7 M, 1 x 10 -7 M, 8 x 10 -8 M, 4 x 10 -8 M, 2 x 10 -8 M, 1 x 10 -8 M, 5 x 10 -9 M, 2 x 10 -9A solution diluted to 1×10 M was prepared. -8 The M solution was reacted with various concentrations of measurement antibody A or measurement antibody B, and the fluorescence polarization was measured. The results are shown in Figure 12. Different fluorescence polarization curves were obtained by using measurement antibodies with different binding constants.

[0059] Next, as shown in Table 3, a fluorescent labeling substance was added to each reaction section at a concentration of 4.5 × 10 -9 M, and the reaction section g was loaded with antibody A (binding constant Ka = 2 × 10 6 M -1 ) at a concentration of 5 x 10 -7 Add antibody B (Ka = 3 × 10) to reaction section h so that the total volume becomes M. 8 M -1 ) at a concentration of 1 x 10 -8 A support was prepared with the support loaded so that the concentration was M. Using this support, fixed amounts of the analyte at different concentrations, 0.001 ng / ml, 0.01 ng / ml, 0.1 ng / ml, 1 ng / ml, 10 ng / ml, 100 ng / ml, 1000 ng / ml, 10000 ng / ml, and 100000 ng / ml, were added to reaction zones g and h, respectively, and the degree of fluorescence polarization was measured. The results are shown in Figure 13. As shown in Figure 13, the lower limit of quantitation of the analyte for curve g, which loaded measurement antibody A, was 16 ng / ml, and the upper limit of quantitation was 2.7 x 10 3 On the other hand, the lower limit of quantification of the target substance for curve h, which carried antibody B for measurement, was 4.1 ng / ml, and the upper limit of quantification was 1.5 × 10 3 By supporting measurement antibodies with different binding constants on the support, it was possible to measure the target substance in different measurement ranges.

[0060] [Table 3]

[0061] (Embodiment 2) The fluorescently labeled substance and Ka = 1 × 10 were added to reaction zones i, j, and k at the concentrations shown in Table 4. 10 M -1The fluorescence polarization index was calculated based on the following formula when a fixed amount of sample solution containing different concentrations of the substance to be measured was added to the reaction zone. Fh = 0.3, Fl = 0.07. The results are shown in Figure 14.

[0062]

number

[0063] [Table 4]

[0064] Curves i, j, and k were obtained depending on the amount of fluorescent labeling substance and measurement antibody carried. Each curve had a different measurement range, and it was found that within the range enclosed by the dashed line in Figure 14, measurement was possible without diluting the test solution.

[0065] Then, the coupling constant Ka = 3 × 10 8 M -1 Using the antibody for measurement, reaction zones m and n shown in Table 5 were prepared. A fixed amount of sample solution containing different concentrations of the substance to be measured was added to these reaction zones, and the degree of fluorescence polarization was measured. The results are shown in Figure 15.

[0066] [Table 5]

[0067] As shown in Figure 15, the lower limit of quantification for curve m is 4.1 ng / ml, and the upper limit of quantification is 1.5 × 10 3 On the other hand, the lower limit of quantification of the target substance on curve m was 2 ng / ml, and the upper limit of quantification was 2.4 × 10 2 By carrying different concentrations of fluorescently labeled substances on the support, it is possible to measure the target substance in different measurement ranges. [Explanation of symbols]

[0068] 1···Support for fluorescence polarization immunoassay 3. Reaction section, 5...Communication path, 7...branch, 9. Fluorescent labeling substance, 11...antibody for measurement, 13... Sample solution, 15. Sealant

Claims

1. A support having a plurality of reaction sites for performing fluorescence polarization immunoassay on a substance to be measured in a sample, the reaction section is a microchannel, A support for fluorescence polarization immunoassay, characterized in that the reaction zone carries an antibody capable of binding to the substance to be measured and a fluorescently labeled substance in which the substance to be measured is labeled with a fluorescent dye.

2. A support having a plurality of reaction sites for performing fluorescence polarization immunoassay on a substance to be measured in a sample, the reaction section is a recess, Some of the reaction sections are connected by a communication passage, A support for fluorescence polarization immunoassay, characterized in that the reaction zone carries an antibody capable of binding to the substance to be measured and a fluorescently labeled substance in which the substance to be measured is labeled with a fluorescent dye.

3. 3. The support for fluorescence polarization immunoassay according to claim 1, wherein the fluorescent dye is one or more selected from the group consisting of fluorescein, dansyl, pyrene, rhodamine, dialkylaminonaphthalene, dialkylaminonaphthalenesulfonyl, indolenine, and ruthenium.

4. 3. The support for fluorescence polarization immunoassay according to claim 1, wherein the fluorescent dye has a fluorescence lifetime of 1 to 3,000 nanoseconds.

5. 5. A fluorescence polarization immunoassay kit comprising the support for fluorescence polarization immunoassay according to claim 1 and a solvent for dissolving the substance to be measured.

6. a sample solution containing the substance to be measured is added to the reaction zone of the support for fluorescence polarization immunoassay according to any one of claims 1 to 4; reacting the target substance, the antibody, and the fluorescently labeled substance in the reaction section; Then, the reaction zone is subjected to a fluorescence polarization immunoassay at a temperature of 4 to 40°C.

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