Supports, kits and methods for fluorescence polarization immunoassays

Abstract

Provided are a support for fluorescence polarization immunoassay, a kit, and a method thereof, the support including a reaction portion for fluorescence polarization immunoassay of a target substance in a sample, the reaction portion being loaded with an antibody having a binding ability with the target substance and a fluorescent labeling substance that labels the target substance with a fluorescent dye.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of Japanese Patent Application No. 2021-1841, filed on January 8, 2021, the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] This disclosure relates to a support for fluorescence polarization immunoassay loaded with antibodies and fluorescently labeled substances, a fluorescence polarization immunoassay kit, and fluorescence polarization immunoassay using the support for fluorescence polarization immunoassay. Background Technology

[0004] Fluorescence polarization immunoassay is known as an immunoassay method that uses fluorescence. It is known that the fluorescence polarization measured by fluorescence polarization immunoassay is proportional to the effective volume of the target substance. Unexamined Japanese Patent Application Publication No. H03-103765 ​​describes a fluorescence polarization immunoassay using a reagent in which an antibody (or antigen) is immobilized on a substance having a larger molecular weight than the antibody, and then the significant change in fluorescence polarization caused by a specific antigen-antibody reaction between the reagent and the fluorescently labeled antigen (or antibody) is utilized.

[0005] Another method uses fluorescence polarization analysis to measure high molecular weight substances (Japanese Patent No. 3255293). In an example of this method, pyrene butyrate is used as a fluorescent dye, and an anti-HDL polyclonal antibody is used as an antibody that specifically binds to the target substance to create a high-density lipoprotein (HDL) calibration curve.

[0006] This fluorescence polarization immunoassay can be performed using a multi-well plate with multiple wells. Unexamined Japanese Patent Application Publication No. 2005-521032 describes a module containing multiple detection domains, including a first detection domain with a first reagent and a second detection domain with a second reagent, wherein, with reduced interference between the luminescence emitted from the first detection domain and the luminescence emitted from the second detection domain, the first detection domain is capable of producing luminescence at least 10 times brighter than the second detection domain. This is preferably performed in a detection module with an integrated electrode having a reader device configured to induce luminescence and measure the induced luminescence (Summary of the Invention). According to Unexamined Japanese Patent Application Publication No. 2005-521032, various types of antibodies can be immobilized on the detection domains, and multiple test measurements can be performed using a module with multiple detection domains.

[0007] Another type of porous test plate has multiple wells containing: a binding surface on which the capture reagent is fixed; a drying reagent that can be restored, and the drying reagent is arranged on the surface of the wells in such a way that the drying reagent does not overlap with the binding surface (Japanese Patent Application Publication No. 2009-521686, Unexamined). Because the drying reagent does not overlap with the binding surface, even if an additional liquid reagent containing a test control is dispensed and dried, the test can be performed without physical contact between the dried test reagent and the dried test control.

[0008] Besides the high sensitivity and accuracy of the instruments and measurement systems used, general assays also require factors such as ease of operation and low reagent volume. Fluorescence polarization immunoassay is based on the principle of competitive binding immunoassay and uses two types of reagents: fluorescently labeled compounds, where molecules identical to the target molecule are labeled with a fluorescent substance; and antibodies that specifically bind to the target molecule. When using multi-well assays, multiple reagents need to be added to each well, which complicates the operation. Therefore, it is desirable to develop easy-to-use supports and reagent kits for fluorescence polarization immunoassay.

[0009] In fluorescence polarization immunoassay, the concentration of the target compound can be quantified within a range where there is a correlation between fluorescence polarization and the target substance concentration. If the concentration of the target substance in the sample solution exceeds this measurement range, the sample solution needs to be diluted and measured again. Therefore, when the measurement range is wide, the sample solution dilution process can be omitted.

[0010] In view of the above-mentioned current situation, this disclosure aims to provide a support for fluorescence polarization immunoassay and a fluorescence polarization immunoassay kit suitable for fluorescence polarization immunoassay, which has an increased measurement range and is easy to use.

[0011] This disclosure also aims to provide a method for fluorescence polarization immunoassay using a support for fluorescence polarization immunoassay. Summary of the Invention

[0012] As a result of a detailed investigation into fluorescence polarization immunoassay, this disclosure was made possible by the following findings: for example, it was discovered that when the reaction portion of the support is preloaded with an antibody capable of binding to the target substance and a fluorescent labeling substance that labels the target substance with a fluorescent dye, fluorescence polarization immunoassay can be performed simply by adding a sample solution containing the target substance.

[0013] In other words, this disclosure provides a support having a reaction portion for fluorescence polarization immunoassay of a target substance in a sample, wherein the reaction portion is loaded with an antibody capable of binding to the target substance and a fluorescent labeling substance for labeling the target substance with a fluorescent dye.

[0014] This disclosure also provides a support for fluorescence polarization immunoassay, characterized in that the reaction portion is loaded with at least one of fluorescent labeling substances or antibodies at different concentrations.

[0015] This disclosure also provides a support for fluorescence polarization immunoassay, characterized in that the reaction portion is loaded with antibodies that have different binding affinities for the target substance.

[0016] This disclosure also provides a support for fluorescence polarization immunoassay, characterized in that the reaction section is further loaded with a pH adjuster.

[0017] This disclosure also provides a support for fluorescence polarization immunoassay, characterized in that the reaction portion is a microfluidic channel.

[0018] This disclosure also provides a support for fluorescence polarization immunoassay, characterized in that some reaction portions are connected via a transport path.

[0019] This disclosure also provides a support for fluorescence polarization immunoassay, characterized in that the fluorescent dye is one or more selected from the group consisting of fluorescein, dansyl sulfonyl, pyrene, rhodamine, dialkylaminonaphthalene, dialkylaminonaphthalenesulfonyl, pseudoindole, and ruthenium.

[0020] This disclosure also provides a support for fluorescence polarization immunoassay, characterized in that the fluorescent dye has a fluorescence lifetime of 1-3,000 nanoseconds.

[0021] This disclosure also provides a fluorescence polarization immunoassay kit, which includes a support for fluorescence polarization immunoassay and a solvent for dissolving the target substance.

[0022] This disclosure also provides a method for fluorescence polarization immunoassay, characterized in that: a sample solution containing a target substance is added to a reaction section of a support for fluorescence polarization immunoassay; the target substance, antibody, and fluorescently labeled substance react in the reaction section; and fluorescence polarization immunoassay of the reaction section is performed at a temperature of 4-40°C.

[0023] It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory, and are not intended to limit this disclosure.

[0024] According to this disclosure, a support for fluorescence polarization immunoassay and the like is provided, wherein the reaction portion is loaded with an antibody capable of binding to a target substance and a fluorescent labeling substance for labeling the target substance using a fluorescent dye. Additionally, a method for fluorescence polarization immunoassay using the support for fluorescence polarization immunoassay is provided. Attached Figure Description

[0025] A more complete understanding of this application can be obtained by considering the following detailed description in conjunction with the accompanying drawings, wherein:

[0026] Figure 1 It is a diagram of a fluorescence polarization immunoassay support having circular reaction sections formed in three vertical and horizontal rows;

[0027] Figure 2 This is a schematic diagram of a fluorescent polarization immunoassay support, in which a series of reaction sections are connected by a linear transport path.

[0028] Figures 3A-3C It is used to describe Figure 2 A diagram illustrating the application of a fluorescence polarization immunoassay support;

[0029] Figure 4 This is a diagram of a fluorescence polarization immunoassay support with five microfluidic channels serving as reaction sections;

[0030] Figure 5 This is a diagram of a fluorescent polarization immunoassay support, in which the reaction sections are arranged in a 3×3 configuration, and each reaction section is loaded with different concentrations of fluorescent labeling material and different concentrations of antibody;

[0031] Figure 6 This is a graph showing the results of measuring fluorescence polarization relative to the concentration of the antibody by changing the concentration of the target substance;

[0032] Figure 7 This is a graph showing the results of measuring fluorescence polarization relative to the target material while keeping the fluorescently labeled material and the target material constant;

[0033] Figure 8 This is a graph showing the results of measuring fluorescence polarization relative to the concentration of a target substance using measurement antibodies with different binding constants;

[0034] Figure 9 This is a diagram of a fluorescent polarization immunoassay support, in which different fluorescently labeled substances and antibodies with different binding affinities for the target substance are loaded in a 3×3 reaction section;

[0035] Figure 10This is a diagram of a fluorescent polarization immunoassay support, in which a certain amount of fluorescently labeled material and measuring antibody are loaded in each of the 3×3 reaction sections, and different pH adjusters are further loaded in each row;

[0036] Figure 11 This is a diagram showing the pattern of the reaction section of the fluorescence polarization immunoassay support at different temperatures T1, T2, and T3;

[0037] Figure 12 The figure shows the results of Example 1, which illustrates the results of measuring fluorescence polarization relative to the concentration of the measurement antibody using measurement antibodies with different binding constants.

[0038] Figure 13 The figure shows the results of Example 1, which illustrates the results of measuring fluorescence polarization relative to the concentration of the target substance using measurement antibodies with different binding constants.

[0039] Figure 14 This is a graph showing the results of measuring fluorescence polarization relative to the concentration of a target substance using a support loaded with fluorescently labeled substances of different concentrations and a measuring antibody; and

[0040] Figure 15 This is a graph showing the results of measuring fluorescence polarization relative to the concentration of a target substance by changing the concentration of the fluorescently labeled substance. Detailed Implementation

[0041] This disclosure provides a support having a reaction section for fluorescence polarization immunoassay of a target substance in a sample, wherein the reaction section is loaded with an antibody capable of binding to the target substance and a fluorescent labeling substance in which the target substance is labeled with a fluorescent dye. Since the antibody and fluorescent labeling substance are pre-loaded in the reaction section, the concentration of the target substance can be measured by a simple operation of adding a sample containing a certain amount of the target substance and measuring the fluorescence polarization, without performing the addition of reaction reagents.

[0042] (1) Target material

[0043] The target substance refers to the compound or composition to be measured by the fluorescence polarization immunoassay support of this disclosure. A measurable target substance is a compound in which at least a portion can be used as an epitope for antibody preparation. Examples include: proteins, glycoproteins, peptides, polypeptides, oligonucleotides, polynucleotides, antibodies, antigens, haptens, hormones, drugs, enzymes, and receptors. Note that, for ease of interpretation, when the target substance is an antibody, it is described as a "test receptor antibody".

[0044] The receptor antibody being tested can be a monoclonal antibody, a multispecific antibody, a bifunctional antibody, a human antibody, a humanized antibody, an antibody derived from birds such as chickens, mammals such as humans and cattle, non-primates such as camels, and other animals, a recombinant antibody, a chimeric antibody, a single-chain Fv (“scFv”), a single-chain antibody, a single-domain antibody, a Fab fragment, an F(ab') fragment, an F(ab')2 fragment, a disulfide-linked Fv (“sdFv”), and an anti-idiotype antibody (“anti-Id”), a two-domain antibody, a bivariate antibody, or the like. When the target substance is classified according to its source or characteristics, substances such as biological substances, pharmaceutical substances, viral substances, and bacterial substances can also be measured. Biological substances include various components produced by organisms within an organism (such as immunoglobulins), various components excreted from an organism, and the organism itself, where organisms include plants and animals. Pharmaceutical substances include agricultural chemicals and the like, but are not limited to medicines prescribed for humans or animals.

[0045] (2) Measure antibody

[0046] An antibody loaded in the reaction portion of a support for fluorescence polarization immunoassay is an antibody capable of binding to a target substance. The antibody is required to have binding ability to recognize and bind at least a portion of the target substance as an epitope. If such an antibody is commercially available, a commercially available product can be used. If such an antibody is not commercially available, it can be generated by using an immunogen, wherein the immunogen carrier material binds to the target compound via an amide bond or other groups. The immunogen carrier material can be selected from conventionally known carrier materials. The immunogen carrier material can be any of the following immunogenic proteins: polypeptides, carbohydrates, polysaccharides, lipopolysaccharides, nucleic acids, and the like. The immunogen carrier material is preferably a protein or polypeptide, more preferably bovine serum albumin (BSA), keyhole cyanin (KLH), and thyroglobulin. Such immunogens can be used for the preparation of polyclonal and monoclonal antibodies using well-known methods. Typically, the immunogen, preferably a mixture of immunogen and adjuvant, is injected into one or more different sites in a host animal such as a rabbit, goat, guinea pig, or horse. Additional injections are made at the same or different sites at regular or irregular intervals. The titer is determined appropriately to obtain the desired antibody. Antibodies can be collected by collecting blood from a host animal.

[0047] Note that, to distinguish it from "receptor antibody," an antibody loaded in the reaction portion used for measurement and possessing the ability to bind to the target substance is called a "measurement antibody." Measurement antibodies include monoclonal antibodies, multispecific antibodies, bifunctional antibodies, human antibodies, humanized antibodies, antibodies derived from birds such as chickens, mammals such as humans and cattle, non-primates such as camels, and other animals, recombinant antibodies, chimeric antibodies, single-chain Fv ("scFv"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fv ("sdFv"), and anti-idiotype antibodies ("anti-Id"), dual-domain antibodies, bivariate antibodies, and the like. This is because the antibody is sufficient to bind to at least one epitope of the target compound. Additionally, some amino acids can be replaced with other amino acid residues in the prepared measurement antibody in this way to improve heat resistance, chemical resistance, pressure resistance, or other purposes without compromising the binding ability to the target substance.

[0048] (3) Fluorescent labeling substances

[0049] The fluorescently labeled substance loaded in the reaction section is a compound obtained by labeling the target substance with a fluorescent dye.

[0050] A fluorescent dye is a dye that emits fluorescence. Each fluorescent dye has its own fluorescence lifetime. In this disclosure, depending on the molecular weight of the target substance, fluorescent dyes with fluorescence lifetimes of 1-10 nanoseconds, fluorescent dyes with fluorescence lifetimes exceeding 10-200 nanoseconds, and fluorescent dyes with fluorescence lifetimes exceeding 200-3,000 nanoseconds can be appropriately selected and used. Examples of fluorescent dyes with fluorescence lifetimes of 1-10 nanoseconds include: pseudoindole; fluorescein compounds such as chlorotriazinylaminofluorescein, 4'-aminomethylfluorescein, 5-aminomethylfluorescein, 6-aminomethylfluorescein, 6-carboxyfluorescein, 5-carboxyfluorescein, 5- and 6-aminofluorescein, thiourea-fluorescein, and methoxytriazinylaminofluorescein; rhodamine derivatives such as rhodamine B, rhodamine 6G, and rhodamine 6GP; and the Alexa Fluor series of dyes, which are registered trademarks and product names, such as Alexa Fluor 488, BODIPY series, DY series, ATTO series, DY Light series, Oyster series, HiLyte Fluor series, Pacific Blue, Marina Blue, Acridine, Edans, Courmarin, 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. Examples of fluorescent dyes with fluorescence lifetimes exceeding 10–200 nanoseconds include: naphthalene ring derivatives such as dialkylaminonaphthalenesulfonyl, and pyrene derivatives such as N-(1-pyrene)maleimide, aminopyrene, pyrenebutyric acid, and alkynylpyrene. Furthermore, examples of fluorescent dyes with fluorescence lifetimes exceeding 200–3,000 nanoseconds include: metal complexes such as platinum, rhenium, ruthenium, osmium, and europium.

[0051] The target substance can be labeled with a fluorescent dye, for example, by covalently binding the fluorescent dye to the target substance, or by binding the fluorescent dye to the target substance via a suitable linking group such as oligoethylene glycol or an alkyl chain. The fluorescent dye has functional groups capable of binding to carboxyl, amino, hydroxyl, thiol, phenyl, or similar groups. The target substance can be a protein or other substance capable of covalently binding to the above functional groups. The corresponding functional groups of the fluorescent dye and the target substance can react under conditions known to those skilled in the art to produce a fluorescently labeled substance. After the reaction is complete, unreacted fluorescent dye can be removed by conventional methods. Note that the number of molecular bonds of the fluorescent dye introduced into the fluorescently labeled substance can be arbitrarily chosen. Preferably, one or more fluorescent dye molecules bind to a single target substance molecule, and more preferably, two to five fluorescent dye molecules bind to a single target substance molecule. In this disclosure, the fluorescently labeled substance in which the target substance is labeled with a fluorescent dye is simply referred to as a "fluorescently labeled substance".

[0052] (4) Support components

[0053] As for the material used for the support of the fluorescence polarization immunoassay of this disclosure, for example, polypropylene, polyethylene, polymethylpentene, ethylene-tetracyclododecene copolymer, polyacetal, acrylonitrile-butadiene-styrene resin, hydroxybenzoic acid polyester, polyetherimide, methacrylic acid resin, polyethylene terephthalate, polybutylene terephthalate, polycyclohexyl terephthalate, polyethylene naphthalate, polyacrylonitrile, polystyrene, polyamide, polycarbonate, polyvinyl alcohol, polylactic acid, or other resins, glass, quartz, or the like can be used. As for the support, various plates with multiple grooves conventionally referred to as microplates, multi-well plates, microplates, immunoassay plates, and the like can be used for various immunoassays, such as radioimmunoassay, enzyme immunoassay, and fluorescence immunoassay using fluorophores.

[0054] (5) Reaction section

[0055] The grooves formed in the support can be used as reaction portions of the support for the fluorescence polarization immunoassay of this disclosure. The shape of the reaction portions is not particularly limited and can be semi-circular, cylindrical (flat-bottomed), disc-shaped, hemispherical (U-shaped bottom), or similar. Similarly, the number of reaction portions is not particularly limited, and at least two or more reaction portions can be present in the support. The number of reaction portions is preferably 6-1,000, and more preferably 10-100. Note that the size and shape of the support are not particularly limited and can be appropriately selected depending on the fluorescence polarization measuring instrument. Furthermore, the arrangement of the reaction portions in the support is not particularly limited. If the reaction portions of the support are arranged vertically and horizontally, the fluorescence polarization of a large number of samples in small doses can be efficiently measured using a microplate reader for detecting and measuring absorption, fluorescence, and luminescence. Note that the volume of the reaction portions is 0.01-1 ml, preferably 0.1-0.4 ml, so that the reaction portions can be loaded with measuring antibodies and fluorescently labeled substances, and can receive a certain amount of sample solution containing the target substance.

[0056] Figure 1 An example of support 1 for fluorescence polarization immunoassay is shown. Figure 1 The support 1 is shown, in which circular grooves are formed in three vertical and horizontal rows to form the reaction section 3. Measurement antibodies and fluorescently labeled substances are loaded in the reaction section 3.

[0057] Multiple reaction portions formed in the support for fluorescence polarization immunoassay can be connected to each other via a transport path. Figure 2 The diagram illustrates a configuration where three square reaction sections 3 are connected in series via a linear transport path 5. Figure 2 In the example, branch path 7 originates from transmission path 5. Figure 3A As shown, multiple reaction portions 3, loaded with fluorescently labeled material 9 and measuring antibody 11, are connected to a transport path 5 via branch paths 7 leading to the respective reaction portions 3. The transport path 5 can be used as a sample solution injection path. Figure 3B As shown, when sample solution 13 is injected from the left end of conveying path 5, all three reaction sections 3 can be filled with sample solution 13 in a single operation. Figure 3C As shown, after the sample solution 13 is filled, instead of the sample solution 13, it can be removed from the transport path 5 by feeding a sealant 15 (such as air, nitrogen, or other gas that does not affect fluorescence polarization measurement). Instead of air or other gases, a liquid such as silicone resin, fluorinated inert liquid, or similar liquid that does not affect fluorescence polarization measurement can be fed as the sealant 15.

[0058] In addition, polydimethylsiloxane (PDMS) microfluidic channels can be used as the reaction portion of a support for fluorescence polarization immunoassay. Figure 4 The support 1, which has five microfluidic channels as the reaction section 3, is shown.

[0059] (6) Loading

[0060] The reaction section of the support for fluorescence polarization immunoassay is loaded with measuring antibodies and fluorescently labeled substances. The term "loaded" as used herein refers to a state in which the measuring antibodies and fluorescently labeled substances are bound in such a way that when a sample solution containing the target substance is added to the reaction section, the measuring antibodies and fluorescently labeled substances can be released from the surface of the reaction section into the solution. Therefore, cases where measuring antibodies and such substances are covalently bonded to the reaction section are excluded. Additionally, cases where the reaction section has undergone plasma treatment or other surface treatments to enhance the binding force of the measuring antibodies or fluorescently labeled substances so that they cannot be released from the surface of the reaction section even upon addition of the sample solution are also excluded.

[0061] There are no particular limitations on the method of loading the measuring antibody or fluorescent labeling substance into the reaction section. For example, a solution containing dissolved or dispersed measuring antibody and a solution containing dissolved or dispersed fluorescent labeling substance can be separately added dropwise to the reaction section and dried by freeze-drying, vacuum drying, heat drying, low-temperature drying, or the like, thereby loading the measuring antibody and fluorescent labeling substance into the reaction section. A mixed solution of measuring antibody and fluorescent labeling substance dissolved or dispersed in a specific ratio can be prepared in advance, added dropwise to the reaction section as described above, and loaded by freeze-drying or the like.

[0062] (7) Fluorescence polarization immunoassay

[0063] Fluorescence polarization immunoassay utilizes the competitive reactions of substances and the polarization changes caused by variations in the molecular weight of competing substances. When a fluorescent dye in a liquid remains stable in its excited state, it emits polarized fluorescence in the same plane. However, when the fluorescent dye rotates in the excited state due to Brownian motion, it emits fluorescence in a plane different from the excitation plane, thus eliminating fluorescence polarization. Fluorescence polarization indicates the degree of rotation of the fluorescent molecule between the time of molecule excitation and the time of fluorescence emission. Low molecular weight molecules rotate vigorously in solution due to Brownian motion, resulting in lower polarization, while high molecular weight molecules have weaker Brownian motion, resulting in enhanced polarization. For example, in a solution containing a mixture of target substance A, antibody B with specific binding ability to target substance A, and fluorescent labeling substance C in which target substance A is labeled with a fluorescent dye, target substance A, antibody B, and fluorescent labeling substance C compete for a reaction in solution. Therefore, a high concentration of target substance A increases the amount of binding between target substance A and antibody B, and also increases the amount of free fluorescent labeling substance C that does not bind to antibody B. If there is a difference between the mass of fluorescently labeled substance C and the mass of the conjugate of antibody B and fluorescently labeled substance C, the change in polarization can be used as an indicator to measure the concentration of target substance A.

[0064] (8) Load capacity

[0065] As described above, since fluorescence polarization immunoassay utilizes a competitive reaction between the target substance, the measuring antibody, and the fluorescently labeled substance, different loading amounts of the fluorescently labeled substance or the measuring antibody will alter the fluorescence polarization and thus the measurable range of the target substance. In the support used for the fluorescence polarization immunoassay of this disclosure, multiple reaction portions can have different loading amounts of fluorescently labeled substance and measuring antibody for their respective reaction portions. Using such a support with different loading amounts, the loading amounts can be easily changed by altering the solution volume of the fluorescently labeled substance and measuring antibody to be added to the reaction portion, or by altering the concentration of these solutions.

[0066] As an example, Figure 5 A schematic diagram illustrates a configuration in which reaction portions 3 are arranged in a 3×3 configuration within a carrier 1, and each reaction portion 3 is loaded with different concentrations of fluorescently labeled material 9 and different concentrations of measuring antibody 11. Note that, although Figure 5 The diagram illustrates a pattern where the loading amounts of both fluorescently labeled substance 9 and measuring antibody 11 are varied, but the loading amount of only one of the fluorescently labeled substance 9 and measuring antibody 11 can be varied. The loading amounts can be appropriately selected based on the target substance and such characteristics.

[0067] For example, when the loading of all fluorescently labeled substances in horizontal row m1 of the reaction section arranged vertically (n × m) is Dm1, and the loading of fluorescently labeled substances in horizontal row m2 is Dm2, the creation of calibration curves and the measurement of sample solutions can be performed simultaneously under the condition that the loading of fluorescently labeled substances is Dm1 and Dm2. Even when the concentration of the target substance in the sample solution is unknown, one of the calibration curves can be used to measure the concentration, which increases the width of the measurement range and, in turn, reduces the dilution process of the sample solution.

[0068] (9) Antibodies with different binding affinities

[0069] The measuring antibody can be an antibody with different binding affinities for the target substance. By using antibodies with different binding affinities, the measurement range can be broadened.

[0070] If the target substance is an antigen, then fluorescence polarization immunoassay can be considered as an antigen-antibody reaction. The equilibrium formula for binding in the antigen-antibody reaction can be expressed as: Ka = ([AgAb]) / ([Ag][Ab]) and B / F = ([AgAb]) / [Ag], where Ag: antigen concentration, Ab: antibody concentration, Ka: binding constant, B: binding concentration, and F: free concentration.

[0071] If the initial input concentrations of Ag and Ab are p and q, respectively, then [Ag] = p - [AgAb], [Ab] = q - [AgAb], and (B / F) 2 +(B / F)(1+Kap-Kaq)-Kaq=0.

[0072] When the concentration of the target substance is defined as x and x = [Ag], and the concentration of the fluorescently labeled substance is defined as p and p is replaced by p → p + x, then the ternary notation (B / F) is used. 2 +(B / F)(1+Kap+Kax-Kaq)-Kaq=0 can be expressed as B / F(Ka,p,x,q). Assuming (B / F)=R, the above equation can be transformed into R.

[0073]

[0074] When the fluorescence polarization of the fluorescently labeled substance is Fh when bound and Fl when ionized, the fluorescence polarization value can be expressed as the variable in the following equation (1):

[0075] [Formula 1]

[0076]

[0077] Figure 6The results of Equation (1) above are shown when the concentration of the fluorescently labeled substance and the binding constant of the measuring antibody are the same, by changing the concentration of the measuring antibody. Under the conditions shown in Table 1 with three types of concentrations of the target substance, the binding constant Ka = 1 × 10⁻⁶. 10 M -1 Fh = 0.3, Fl = 0.07. As shown in curves a, b, and c, fluorescence polarization decreases with increasing target substance concentration.

[0078] [Table 1]

[0079] curve Ka Fluorescently labeled substance (p) Target substance (x) a <![CDATA[1×10 10 M -1 ]]> <![CDATA[1×10 -10 M]]> 0M b <![CDATA[1×10 10 M -1 ]]> <![CDATA[1×10 -10 M]]> <![CDATA[3×10 -10 M]]> c <![CDATA[1×10 10 M -1 ]]> <![CDATA[1×10 -10 M]]> <![CDATA[1×10 -9 M]]>

[0080] on the other hand, Figure 7 This shows that when the concentration of the target substance is constant (q = 4 × 10⁻⁶), the concentration of the target substance is constant. -10 M) and the concentration of the fluorescently labeled substance is also constant (concentration p = 1 × 10⁻⁶). -10 When M), the fluorescence polarization result is measured relative to the antibody concentration. Figure 7 This indicates that at 1×10 -10 M to 1×10 -8 Within the range of M, there is a correlation between antibody measurement and fluorescence polarization, which is outlined by a dashed line, and this range represents the measurement range of the target substance. This result is consistent with the fact that the width of the measurement range increases as the amount of antibody loaded in the reaction section changes.

[0081] Next, Figure 8 The results of creating fluorescence polarization curves in the same manner as described above are shown in Table 2, using measurement antibodies with different binding constants Ka and varying the concentrations of the measurement antibodies and the fluorescently labeled substance. The measurable range varies depending on the curve, where the measurable range of the target substance is 1 × 10⁻⁶ of curve d. -11 M to 1×10 -10 The range of M, the 1×10 of curve e -10 M to 1×10 -9 The range of M, the 1×10 of curve f -9 M to 1×10 -8 The range of M. In this disclosure, antibodies with different binding affinities to the target substance are loaded into the reaction section, and the concentration of the fluorescently labeled substance loaded in the reaction section is varied and the antibody concentration is measured, for example... Figure 8 The area outlined by the dashed line can be defined as the measurement range width.

[0082] [Table 2]

[0083] curve Ka Fluorescently labeled substance (p) Target substance (x) d <![CDATA[1×10 11 M -1 ]]> <![CDATA[1×10 -11 M]]> <![CDATA[1×10 -11 M]]> e <![CDATA[1×10 10 M -1 ]]> <![CDATA[1×10 -10 M]]> <![CDATA[1×10 -10 M]]> f <![CDATA[1×10 9 M -1 ]]> <![CDATA[1×10 -9 M]]> <![CDATA[1×10 -9 M]]>

[0084] Figure 9 The schematic diagram illustrates a support configuration in which a fluorescently labeled substance 9 and antibodies (measuring antibodies) 11 with different binding affinities to the target substance are loaded in a carrier 1 having a 3×3 reaction portion 3. Note, for example, antibodies with different binding affinities to the target substance can be prepared by inoculating an immunogen (which is the test compound bound to an immunogen carrier material such as a polysaccharide) into one or more different sites of a host animal (such as a rabbit or other host animal), appropriately determining the titer, and collecting antibodies with different titers.

[0085] (10) pH adjuster

[0086] pH adjusters can be further loaded into the reaction section. Since the binding affinity between the target substance and the measuring antibody changes depending on pH, an increased measurement range can be ensured. Examples of such pH adjusters include: glycine-NaOH (pKa 9.60), Tris-HCl (pKa 8.20), trimethylglycine-HCl (pKa 8.15), HEPES-NaOH (pKa 7.55), NaH2PO4-Na2HPO4 (pKa 7.22), MOPS-NaOH (pKa 7.20), MES-NaOH (pKa 6.15), acetic acid-NaOH (pKa 4.80), glycine-NaOH (pKa 2.34), and GTA buffer. The pH adjuster is loaded into the reaction section by preparing a solution of such a pH adjuster, adding the solution dropwise to the reaction section, and drying the solution in the same manner as loading fluorescently labeled substances or measuring antibodies. Note that the pH adjuster can be appropriately selected based on the characteristics of the target substance, the fluorescently labeled substance, and the measurement antibody. Figure 10 A schematic diagram illustrates a pattern in which a certain amount of fluorescently labeled material and measuring antibody are loaded into reaction sections arranged in a 3×3 pattern, and a different pH adjuster is further loaded for each row.

[0087] (11) Fluorescence polarization immunoassay kit

[0088] The support used for the fluorescence polarization immunoassay of this disclosure can be used as a fluorescence polarization immunoassay kit, which further includes a solvent for dissolving the target substance.

[0089] Examples of solvents used to dissolve the target substance include: water, such as pure water; alcohols, such as methanol, ethanol and butanol; ketones, such as acetone, diethyl ketone and methyl pentyl ketone; alkanes, such as hexane and heptane; ethers, such as diethyl ether; dimethyl sulfoxide, acetonitrile, chloroform and mixtures thereof.

[0090] (12) Measurement methods

[0091] Using the support for the fluorescence polarization immunoassay of this disclosure, the target substances contained in the sample solution can be analyzed as follows.

[0092] First, a sample solution is prepared by dissolving or dispersing the target substance in pure water or other solvents used to dissolve the target substance, and removing any foreign substances present in the solution by filtration or other means if necessary. A certain amount of sample solution is added to each reaction section of the support for fluorescence polarization immunoassay. In this way, the measuring antibody and fluorescently labeled substance loaded in the reaction section react with the target substance contained in the sample solution. Because this reaction is based on an antigen-antibody reaction, it is rapid and reproducible, and the reaction section contains conjugates of the target substance and the measuring antibody, conjugates of the fluorescently labeled substance and the measuring antibody, and so on. When the concentration of the target substance in the sample solution is high, the amount of binding between the target substance and the measuring antibody increases, and the amount of free fluorescently labeled substance not bound to the measuring antibody increases. In fluorescence polarization immunoassay, the change in molecular weight attributable to the binding between the fluorescently labeled substance and the target substance is measured as a change in molecular orientation over time. When there is a difference between the mass of the fluorescently labeled substance and the mass of the conjugate of the measuring antibody and the fluorescently labeled substance, the concentration of the target substance can be measured using the change in polarization as an indicator. The results of fluorescence polarization measurements can be obtained using any polarization measuring instrument. At a predetermined time after the reaction is complete, fluorescence polarization is measured. The measurement should be performed at a constant temperature within the range of 4-40°C, preferably 10-40°C, within which the target substance will not denature. A calibration curve can be pre-established by operating in the same manner as described above, using a solution containing a known concentration of the target substance and comparing the measured values ​​with those of the sample solution, thereby enabling the quantification of the target substance.

[0093] On the other hand, for example, when a calibration curve is created at a temperature of 10°C and the fluorescence polarization of a target substance contained in a sample solution is measured, there may be situations where the measurement results obtained using the calibration curve may be outside the measurement range depending on the concentration of the target substance. In this case, the measurement temperature can be changed to 40°C, and the measurement can be performed at a predetermined time after the reaction. Since the binding constant varies with temperature, a wider measurement range can be ensured by changing the temperature. Figure 11 The schematic diagram illustrates the mode in which the reaction portion is measured at temperatures T1, T2, and T3.

[0094] In this disclosure, the instrument is not limited, as long as fluorescence polarization can be measured. As described above, when using a support for fluorescence polarization immunoassay, wherein the reaction portion is configured as a microfluidic channel, highly sensitive measurements can be performed with a small amount of sample by using a measuring instrument capable of measuring the microfluidic channel.

[0095] (Example)

[0096] The present disclosure will now be described in detail with reference to embodiments, although these embodiments should not be construed as limiting the present disclosure in any way.

[0097] (Example 1)

[0098] Using the binding constant Ka = 2 × 10 6 M -1 Measurement of antibody A and binding constant Ka = 3 × 10 8 M -1 Measurement antibody B. Prepare measurement antibody A diluted to 5 × 10⁻⁶. -7 M, 3×10 -7 M, 1×10 -7 M, 6×10 -8 M, 3×10 -8 M, 2×10 -8 M, 8×10 -9 M, 4×10 -9 M and 2×10 -9 The solution of M, and the antibody B diluted to 6 × 10⁻⁶. -7 M, 3×10 -7 M, 1×10 -7 M, 8×10 -8 M, 4×10 -8 M, 2×10 -8 M, 1×10 -8 M, 5×10 -9 M and 2×10 -9 Solution M. Mix the respective concentrations of measurement antibody A or measurement antibody B with a solution containing 1×10⁻⁶... -8 The solution of the fluorescently labeled substance M was reacted, and the fluorescence polarization was measured. The results are shown in... Figure 12 Different fluorescence polarization curves can be obtained by using measurement antibodies with different binding constants.

[0099] Next, as shown in Table 3, the fluorescently labeled material was loaded into each reaction section to achieve a concentration of 4.5 × 10⁻⁶. -9 M, and will measure antibody A (binding constant Ka = 2 × 10⁻⁶). 6 M -1 ) is loaded into reaction section g, making the concentration 5 × 10-7 M, and will measure antibody B (Ka = 3 × 10) 8 M -1 The concentration was increased to 1 × 10⁻⁶ when the mixture was loaded into reaction section h. -8 M, thus preparing a support. Using this support, certain amounts of the target substance 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, 10,000 ng / ml, and 100,000 ng / ml, were added to reaction portions g and h, and fluorescence polarization was measured. The results are shown in... Figure 13 In the middle. For example Figure 13 As shown, on curve g loaded with antibody A, the concentration of the target substance at the lower limit of quantitation is 16 ng / ml, and the concentration of the target substance at the upper limit of quantitation is 2.7 × 10⁻⁶. 3 On the other hand, on curve h loaded with the measurement of antibody B, the concentration of the target substance at the lower limit of quantitation was 4.1 ng / ml, and the concentration of the target substance at the upper limit of quantitation was 1.5 × 10⁻⁶ ng / ml. 3 ng / ml. By loading measurement antibodies with different binding constants into the support, it is possible to measure the target substance within different measurement ranges.

[0100] [Table 3]

[0101] Reaction section Ka Fluorescent labeling substances Measuring antibodies g <![CDATA[2×10 6 M -1 ]]> <![CDATA[4.5×10 -9 M]]> <![CDATA[5×10 -7 M]]> h <![CDATA[3×10 8 M -1 ]]> <![CDATA[4.5×10 -9 M]]> <![CDATA[1×10 -8 M]]>

[0102] (Example 2)

[0103] The fluorescently labeled substance and the measuring antibody (where Ka = 1 × 10⁻⁶) are used. 10 M -1 The sample solutions were loaded into reaction sections i, j, and k at the concentrations shown in Table 4, and then a certain amount of sample solution containing different concentrations of the target substance was added to these reaction sections. Fluorescence polarization was calculated according to the following equation. Note that Fh = 0.3 and Fl = 0.07 were defined. The results are shown in... Figure 14 middle.

[0104] [Formula 2]

[0105]

[0106] [Table 4]

[0107] Reaction section Fluorescent labeling substances Measuring antibodies i <![CDATA[1×10 -11 M]]> <![CDATA[1×10 -10 M]]> j <![CDATA[2×10 -10 M]]> <![CDATA[2×10 -10 M]]> k <![CDATA[1×10 -9 M]]> <![CDATA[6×10 -10 M]]>

[0108] Based on the fluorescently labeled material and the measured antibody loading, curves i, j, and k were obtained. It was found that each measurement range was different, and that... Figure 14Within the area outlined by the dashed line, it is possible to perform measurements without diluting the sample solution.

[0109] Next, using a binding constant Ka = 3 × 10 8 M -1 Antibodies were used to prepare reaction portions m and n as shown in Table 5. Sample solutions containing different concentrations of the target substance were added to these reaction portions, and fluorescence polarization was measured. The results are shown in... Figure 15 middle.

[0110] [Table 5]

[0111] Reaction section Fluorescent labeling substances Measuring antibodies m <![CDATA[4.5×10 -9 M]]> <![CDATA[1×10 -8 M]]> n <![CDATA[9×10 -10 M]]> <![CDATA[1×10 -8 M]]>

[0112] like Figure 15 As shown, the concentration at the lower limit of quantitation on curve m was 4.1 ng / ml, and the concentration at the upper limit of quantitation on curve m was 1.5 × 10⁻⁶. 3 ng / ml. On the other hand, the concentration of the target substance at the lower limit of quantitation on curve m is 2 ng / ml, and the concentration at the upper limit of quantitation is 2.4 × 10⁻⁶ ng / ml. 2 ng / ml. By loading fluorescently labeled substances of different concentrations into the support, the target substance can be measured within different measurement ranges.

[0113] Some exemplary embodiments have been described for illustrative purposes. While specific embodiments have been presented in the foregoing discussion, those skilled in the art will recognize that changes in form and detail may be made without departing from the broader spirit and scope of the invention. Therefore, the specification and drawings are to be regarded as illustrative rather than restrictive. Consequently, this detailed description should not be construed in a restrictive sense, and the scope of the invention is defined only by the scope of the included claims and the full scope of their authorized equivalents.

Claims

1. A support for fluorescence polarization immunoassay of a target substance in a sample, the support comprising: a plurality of grooves loaded with an antibody having a binding ability to a target substance and a fluorescent labeling substance that labels the target substance with a fluorescent dye, wherein the support comprises a plurality of types of grooves resulting from a difference in a loaded amount of the antibody and / or a difference in a loaded amount of the fluorescent labeling substance, wherein the support comprises a plurality of groups each comprising a plurality of grooves of the same kind loaded with the same substance and having the same loaded amount, and wherein the antibody and the fluorescent labeling substance are bound to a surface of the grooves in such a manner that the antibody and the fluorescent labeling substance are capable of being released from the surface of the grooves into a sample solution containing the target substance when the sample solution is added to the grooves.

2. A support for fluorescence polarization immunoassay of a target substance in a sample, the support comprising: a plurality of grooves loaded with an antibody having a binding ability to a target substance and a fluorescent labeling substance that labels the target substance with a fluorescent dye, wherein the support comprises a plurality of types of grooves resulting from a difference in a binding affinity of the antibody to the target substance, wherein the support comprises a plurality of groups each comprising a plurality of grooves of the same kind loaded with the same substance and having the same loaded amount, and wherein the antibody and the fluorescent labeling substance are bound to a surface of the grooves in such a manner that the antibody and the fluorescent labeling substance are capable of being released from the surface of the grooves into a sample solution containing the target substance when the sample solution is added to the grooves.

3. A support for fluorescence polarization immunoassay of a target substance in a sample, the support comprising: a plurality of grooves loaded with an antibody having a binding ability to a target substance and a fluorescent labeling substance that labels the target substance with a fluorescent dye, wherein at least one of the plurality of grooves is further loaded with a pH adjuster, and the support comprises a plurality of types of grooves resulting from a presence or absence of a pH adjuster and / or a difference in a type of pH adjuster, wherein the support comprises a plurality of groups each comprising a plurality of grooves of the same kind loaded with the same substance and having the same loaded amount, and wherein the antibody and the fluorescent labeling substance are bound to a surface of the grooves in such a manner that the antibody and the fluorescent labeling substance are capable of being released from the surface of the grooves into a sample solution containing the target substance when the sample solution is added to the grooves.

4. The support for fluorescence polarization immunoassay of a target substance in a sample according to any one of claims 1 to 3, wherein, The grooves are microfluidic channels.

5. The support for fluorescence polarization immunoassay of a target substance in a sample according to any one of claims 1 to 3, wherein, Some of the grooves are connected by a transfer path.

6. The support for fluorescence polarization immunoassay of a target substance in a sample according to any one of claims 1 to 3, wherein, The fluorescent dye is one or more selected from the group consisting of fluorescein, dansyl, pyrene, rhodamine, dialkylaminonaphthalene, dialkylaminonaphthalenesulfonyl, pseudoinode, and ruthenium.

7. The support for fluorescence polarization immunoassay of a target substance in a sample according to any one of claims 1 to 3, wherein, The fluorescent dye has a fluorescent lifetime of 1-3,000 nanoseconds.

8. A fluorescence polarization immunoassay kit, comprising: the support for fluorescence polarization immunoassay of a target substance in a sample according to any one of claims 1 to 3; and a solvent for dissolving the target substance.

9. A method for fluorescence polarization immunoassay, the method comprising: adding a sample solution containing a target substance to the recess of the support for fluorescence polarization immunoassay of a target substance in a sample according to any one of claims 1 to 3; causing the target substance, the antibody, and the fluorescently labeled substance to react in the recess; and performing fluorescence polarization immunoassay of the recess at a temperature of 4-40°C.

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