Fluorescence-polarized immunoassay

Abstract

To provide a fluorescence polarization immunoanalysis method that uses a fluorescent label material in which a single domain antibody is labeled by a fluorescence dye.SOLUTION: Provided is a fluorescence polarization immunoanalysis method for analyzing a measurement object material in a sample, said method including a binding step in which a fluorescent label material in which a single domain antibody having ability to bind to the measurement object material is labeled by a fluorescence dye is bound to the measurement object material included in the sample, and a measurement step in which a fluorescence polarization change of the fluorescent label material to which the measurement object material is bound is measured, the fluorescence polarization change of the fluorescent label material being measured by drawing a sigmoid curve having a saturation region on the lower and the upper sides of fluorescence polarization. Measurement can be made of a sample containing a measurement object material in low concentration, or a measurement object material having a high molecular weight.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure relates to fluorescence-polarized immunoassay using a fluorescently labeled substance in which a fluorescent dye is conjugated to a single-domain antibody. In the law To relate to. [Background technology]

[0002] One immunoassay method using fluorescence is fluorescence polarization immunoassay. As described in XQ Guo et al., Anal. Chem. 1998, 70, 632-637, the following relationship exists between the degree of fluorescence polarization and the volume of the substance being measured. (1 / P-1 / 3)=(1 / P0-1 / 3)(1+kTτ / ηV) P: degree of polarization, P0: degree of polarization without rotational diffusion, k: Boltzmann constant, η: viscosity of solution, T: absolute temperature, τ: fluorescence lifetime, V: molecular volume Patent Document 1 describes a fluorescence polarization immunoassay method that uses a reagent in which an antibody (or antigen) is immobilized on a substance with a larger molecular weight compared to the antibody, and utilizes the fact that a large change in fluorescence polarization occurs due to a specific antigen-antibody reaction between this reagent and a fluorescently labeled antigen (or antibody).

[0003] There is also a method for measuring high molecular weight substances using fluorescence polarization immunoassay (Patent Document 2). This method is characterized by using a fluorescently labeled protein to which a fluorescent dye with a fluorescence lifetime of 10 to 200 nanoseconds is covalently bound as the antibody that specifically binds to the substance to be measured. In the example, pyrenebutanoic acid, a long-lived fluorescent dye, is used as the fluorescent dye, and an HDL calibration curve is created using an anti-HDL polyclonal antibody as the antibody that specifically binds to the substance to be measured, and an LDL calibration curve is created using an anti-LDL polyclonal antibody as the antibody that specifically binds to the substance to be measured.

[0004] Furthermore, as a method for measuring high molecular weight substances, some methods use fluorescently labeled low molecular weight antibodies to amplify the change in molecular weight before and after binding of the fluorescently labeled substance to the substance to be measured (Patent Document 3). Patent Document 3 describes Fab fragments, Fab' fragments, and scFv antibodies (single chain antibodies) that include at least an antigen recognition site as low molecular weight antibodies. In the example, Fab-labeled compounds were prepared by reacting Fab fragments with FITC (Fluorescein isothiocyanate), and the fluorescence polarization degree was measured by adding human serum albumin at various concentrations to these compounds. As a result, the low concentration region was 2-3 × 10⁻⁶. -8 It should be stated that it is possible to measure down to approximately M (1-2 μg / mL).

[0005] Here, IgG antibodies, for example, are Y-shaped antibodies consisting of two heavy chains and two light chains, with each heavy chain and light chain possessing a variable region. Fab antibodies and scFv antibodies are composed of a portion of such antibodies and include variable regions of both the heavy chain and the light chain. In contrast, there are also heavy chain antibodies that do not contain a light chain and consist of two heavy chains linked in a Y-shape. Each heavy chain of a heavy chain antibody has a variable region. The variable region includes a framework region consisting of F1-F4 and a complementary determining region (CDR) consisting of CDR1-CDR3, and can specifically bind to antibodies via this variable region. Since the variable region exerts antigen-binding activity through the framework region and CDR, if these are considered a single domain (hereinafter referred to as a single domain), then a heavy chain antibody has two single domains. Heavy chain antibodies are contained in the serum of camelid animals, and each variable region of the heavy chain is called a VHH (Variable domain of a heavy chain antibody). Camel-derived VHH antibodies are single-domain antibodies. Patent document 4 describes a heat-stable VHH antibody in which specific amino acids of the VHH antibody are substituted with glycine or the like, for the purpose of heat stabilization of the VHH antibody. [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] Japanese Patent Application Publication No. 11-44688 [Patent Document 4] Japanese Patent Publication No. 2019-210267 [Overview of the project] [Problems that the invention aims to solve]

[0007] Immunofluorescence chromatography (MRI) involves observing the change in the degree of fluorescence polarization of a fluorescently labeled substance before and after binding to the target substance. Since the degree of fluorescence polarization depends on molecular weight, measuring high molecular weight substances using fluorescently labeled substances with large molecular weights is not easy. Patent document 2 describes the preparation and use of a fluorescently labeled substance using pyrenebutanoic acid, a long-lived fluorescent dye, to measure high molecular weight substances (approximately 500,000 or more), such as substances larger than viruses (approximately 20 nm or more as particles). However, long-lived fluorescent dyes are expensive, and there is a need for the development of an immunofluorescence chromatography method that can measure high molecular weight substances using highly versatile fluorescent dyes with fluorescence lifetimes of 10 nanoseconds or less, such as fluorescein.

[0008] Furthermore, Patent Document 3 describes using a small molecule antibody to produce 2-3 × 10 -8 It is stated that measurements are possible down to approximately M. However, in order to enable measurement with trace amounts of sample, the development of fluorescence polarization immunoassay, which can detect even lower concentrations, is desired.

[0009] Furthermore, while Patent Document 4 discloses a VHH antibody with high thermal stability, it only discloses the preparation of a variant and does not provide any examples of its use in fluorescence-polarized immunoassay. It also does not describe any single-domain antibodies other than VHH antibodies.

[0010] In addition, in order to enable measurement with a small amount of sample, measuring instruments equipped with microchannels have been developed. A microchannel is a device having a channel of about several tens to several hundreds of micrometers fabricated using a semiconductor process. A chemical reaction system using this microchannel has advantages such as a significant reduction in reaction time and a stable chemical reaction due to excellent uniformity of temperature and concentration compared to ordinary bulk-sized chemical reactions. The device used in fluorescence polarization immunoassay needs to be composed of members that do not affect the fluorescence polarization degree, and polydimethylsiloxane (PDMS) is often used. PDMS has transparency comparable to quartz glass and low autofluorescence, so it is suitable for analyses utilizing fluorescence reactions. In addition, since it is liquid and has a low viscosity, microfabrication on the submicron to micron order is possible. However, fluorescence polarization immunoassay utilizes an antigen-antibody reaction. When a fluorescent labeling substance, a measurement target substance, and their conjugate adhere to PDMS, accurate measurement cannot be performed.

[0011] In view of the above situation, an object of the present disclosure is to provide a fluorescence polarization immunoassay using a fluorescent labeling substance in which a fluorescent dye is bound to a single domain antibody.

[0012] Another object of the present disclosure is to provide a fluorescence polarization immunoassay that can also be measured with a measuring instrument having a microchannel.

Means for Solving the Problems

[0014] As a result of detailed examination of fluorescence polarization immunoassay by the present inventors, it has been found that a single domain antibody can quantify a measurement target of a high molecular weight of 150 kDa even when bound to a fluorescent dye having a fluorescence lifetime of 4 nanoseconds, that the fluorescent labeling substance thus obtained is excellent in the sensitivity of fluorescence polarization degree and can be measured even at a low concentration, and moreover, the conjugate of the fluorescent labeling substance and the measurement target substance does not adhere to the microchannel, and thus the present disclosure has been completed.

[0017] In other wordsThe present disclosure relates to a fluorescence polarization immunoassay for analyzing a substance to be measured in a sample, a binding step of binding a fluorescent labeling substance in which a fluorescent dye is bound to the N-terminus of a single-domain antibody to the substance to be measured contained in the sample, and a measurement step of supplying the sample containing the fluorescent labeling substance or the fluorescent labeling substance to which the substance to be measured is bound to a microchannel formed of PDMS and measuring a change in the fluorescence polarization degree of the fluorescent labeling substance to which the substance to be measured is bound, and provides a fluorescence polarization immunoassay including the same.

Effects of the Invention

[0019] According to the present disclosure, a fluorescence polarization immunoassay using a fluorescent labeling substance in which a fluorescent dye is bound to a single-domain antibody is provided ru.

Brief Description of the Drawings

[0020] [Figure 1] It is a diagram explaining the relationship between the mass of the substance to be measured, the fluorescence polarization degree, and the fluorescence lifetime of the fluorescent dye. [Figure 2] It is a diagram showing the results of a standard curve of rabbit IgG by fluorescence polarization degree measurement in Example 1 and the results of a standard curve of rabbit IgG by fluorescence polarization degree measurement in Comparative Example 1. [Figure 3] It is a diagram showing the results of a standard curve of Her2 using a fluorescent labeling substance in which Alexa Fluor 488 is bound to the SH group of an antibody in Example 2. [Figure 4] It is a diagram showing the results of a standard curve of Her2 using a fluorescent labeling substance in which Alexa Fluor 488 is bound to the amino group of an antibody in Example 3. [Figure 5] It is a diagram showing a schematic configuration of a fluorescence polarization degree measurement device used in Example 4. [Figure 6] It is a diagram explaining a microchannel within the effective viewing field for observing a sample light-emitting section. [Figure 7] It is a diagram showing a fluorescence polarization degree measurement image of the microchannel used in Example 4. [Figure 8]This figure shows the standard curve for rabbit IgG measured by fluorescence polarization in Example 4, and the fluorescence polarization results for the measured samples. [Figure 9] This figure shows the results of the standard curve for rabbit IgG obtained by fluorescence polarization measurement in Example 5. [Figure 10] This figure shows the results of the standard curve for rabbit IgG obtained by fluorescence polarization measurement in Comparative Example 2. [Modes for carrying out the invention]

[0021] The first aspect of this disclosure is a fluorescence-polarized immunoassay method for analyzing a target substance in a sample, A binding step in which a fluorescently labeled substance, which is a single-domain antibody having the ability to bind to the substance to be measured and is labeled with a fluorescent dye, is bound to the substance to be measured contained in the sample, and This is a fluorescence polarization immunoassay method that includes a measurement step of measuring the change in the degree of fluorescence polarization of a fluorescently labeled substance to which the target substance is bound. By using a single-domain antibody, the detection sensitivity is improved, and it is possible to measure even samples containing the target substance at low concentrations or high molecular weight target substances.

[0022] Heavy chain antibodies, which consist solely of heavy chains, are known to be produced in the bodies of camelid animals such as Bactrian camels, dromedary camels, llamas, alpacas, vicuñas, and guanacos, as well as cartilaginous fish such as sharks and rays. The variable region of heavy chain antibodies derived from camelid animals is called a VHH antibody, and the variable region of heavy chain antibodies derived from cartilaginous fish is called a vNAR (new antigen receptor) antibody. Each variable region of a heavy chain antibody is a single-domain antibody that exhibits antigen-binding activity through a framework region and a CDR, respectively. In this disclosure, "single-domain antibody" means an antibody composed of one variable region. Therefore, VHH antibodies and vNAR antibodies can be used as single-domain antibodies.

[0023] The single-domain antibodies used in this disclosure are limited to those that have the ability to bind to a specific target substance. At least one part of the target substance must be an epitope, and the antibody must have the ability to recognize and bind to this epitope.

[0024] Single-domain antibodies with such binding ability can be prepared by preparing a heavy-chain antibody that uses the target substance as an antigen and then cleaving a portion of it. For example, a heavy-chain antibody-producing animal can be immunized with the target substance as an antigen, and a heavy-chain antibody that binds to the antigen can be selected from the B cells of the immunized animal. Variable regions of VHH antibodies or vNAR antibodies obtained by cleaving heavy-chain antibodies with enzymes or other means can be used as single-domain antibodies. Furthermore, single-domain antibodies are not limited to isolates from heavy-chain antibodies. They may also be genetically engineered to have specific binding ability to a particular substance by referring to the DNA sequences of conventionally known VHH antibodies or vNAR antibodies, or by using antibody libraries. In addition, some amino acids in the single-domain antibody prepared in this way may be substituted with other amino acid residues for purposes such as improving heat resistance, chemical resistance, pressure resistance, or other purposes, as long as the binding ability to the target substance is not impaired. Furthermore, a single variable region may be extracted from conventionally known Fab antibodies or scFv antibodies and used as a single-domain antibody.

[0025] The single-domain antibodies used in this disclosure are labeled with a fluorescent dye and used as fluorescent labeling substances.

[0026] In this disclosure, "fluorescence" means light emission produced by irradiating with light that excites electrons. "Fluorescent dye" means a dye that emits fluorescence. Since phosphorescence, like fluorescence, involves atoms absorbing energy and becoming excited, dyes that emit phosphorescence are also included in the definition of fluorescent dyes in this disclosure. If a fluorescent dye emits phosphorescence, the degree of fluorescence polarization based on phosphorescence may be measured instead of fluorescence.

[0027] Fluorescent dyes that can be used in this disclosure include fluorescein compounds such as chlorotriazinylaminofluorescein, 4'-aminomethylfluorescein, 5-aminomethylfluorescein, 6-aminomethylfluorescein, 6-carboxyfluorescein, 5-carboxyfluorescein, 5 and 6-aminofluorescein, thioureafluorescein, and methoxytriazinylaminofluorescein; nitrobenzoxadiazole derivatives such as nitrobenzoxadiazole chloride; indorenine; dansyl derivatives such as dansyl; naphthalene derivatives such as dialkylaminonaphthalene and dialkylaminonaphthalenesulfonyl; pyrene derivatives such as N-(1-pyrenyl)maleimide, aminopyrene, pyrenbutanoic acid, and alkynylpyrene; metal complexes such as platinum, rhenium, ruthenium, osmium, and europium; rhodamine derivatives such as rhodamine B, rhodamine 6G, and rhodamine 6GP; and registered trademarks or trade names such as Alexa Fluor 488 and other Alexa Fluor series, BODIPY series, and DY series. Examples include the ATTO series, Dy Light series, Oyster series, HiLyte Fluor series, Pacific Blue, Marina Blue, Acridine, Edans, Coumarin, DANSYL, FAN, Oregon Green, Rhodamine Green-X, NBD-X, TET, JOE, 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. Ruthenium emits phosphorescence, and its fluorescence lifetime is 2,700 nanoseconds.

[0028] Figure 1 schematically shows the relationship between the fluorescence lifetime of a fluorescent dye, the degree of fluorescence polarization, and the molecular weight. This figure was created by referring to "Use of a Long-Lifetime Re(I) Complex in Fluorescence Polarization Immunoassays of High-Molecular-Weight Analytes", Analytical Chemistry, 1998, Vol.70, p.632. In Figure 1, the horizontal axis represents molecular weight, and the vertical axis represents the degree of fluorescence polarization. Figure 1 shows that the total mass range measurable by the degree of fluorescence polarization differs depending on the fluorescence lifetime of the fluorescent dye, and that the target substance can be quantified within a predetermined range of fluorescence polarization (approximately 0.05 to 0.35 in Figure 1). For example, if a fluorescent dye with a fluorescence lifetime of 4 nanoseconds is used, 1 × 10⁻⁶ 3 ~1 × 10 5 In the (Da) region, if a fluorescent dye with a fluorescence lifetime of 100 nanoseconds is used, then 1 × 10 4 ~1 × 10 7 In the (Da) region, if a fluorescent dye with a fluorescence lifetime of 2,700 nanoseconds is used, then 1 × 10 6 ~1 × 10 8 The fluorescence polarization changes significantly in the (Da) region. The fluorescence polarization is resolved by rotational diffusion of the fluorescent labeling substance to which the target substance is bound between excitation and fluorescence emission. By using a longer-lived fluorescent dye, the change in fluorescence polarization can be measured in the higher molecular weight region. For example, when a fluorescent dye with a fluorescence lifetime of 4 nanoseconds is bound to a 150 kDa IgG antibody and used as a fluorescent labeling substance, the fluorescent labeling substance already has a fluorescence polarization of 0.37, so even if it binds to a high molecular weight target substance, the fluorescence polarization hardly changes. In the example of Patent Document 2, by using a long-lived dye such as a pyrene derivative with the IgG antibody, the fluorescence polarization of the fluorescent labeling substance alone is reduced, and high molecular weight substances such as C-reactive protein (CRP; molecular weight 120,000), high-density lipoprotein (HDL; molecular weight approximately 400,000), and low-density lipoprotein (LDL; molecular weight 3,000,000) are measured.

[0029] However, in the present disclosure, a fluorescent dye having a fluorescence lifetime of 4 to 3,000 nanoseconds can be used regardless of the molecular weight of the substance to be measured. However, it may be appropriately selected within the above fluorescence lifetime range according to the molecular weight of the substance to be measured, measurement conditions such as excitation wavelength, and autofluorescence of the measurement sample. For example, when the mass of the substance to be measured is about 15,000 to 2×10 5 Da, a conventional fluorescent dye having a fluorescence lifetime of about 4 nanoseconds is used, and when the mass of the substance to be measured is about 2×10 5 ~10 8 Da, a fluorescent dye having a fluorescence lifetime of about 100 nanoseconds may be used. As shown in the examples described later, in the present disclosure, a fluorescent dye having a short fluorescence lifetime such as Alexa Fluor 488 can be used to quantify a high molecular weight substance to be measured of about 150 kDa. Moreover, the lower limit of quantification is calculated to be 0.45 nM, enabling detection at low concentrations. Thus, the reason why a highly sensitive and high molecular weight substance to be measured can be measured using a fluorescent dye such as Alexa Fluor 488 with high versatility is not clear, but it is presumably due to the synergistic action of the single domain antibody having a low molecular weight, the ability to bind a fluorescent dye near the antigen recognition region, and the reduction in the fluctuation of the binding between the fluorescent labeled substance and the substance to be measured, resulting in an increase in the sensitivity of fluorescence polarization.

[0030] Fluorescently labeled substances can be prepared by labeling a single-domain antibody with a fluorescent dye. Fluorescent dyes generally have functional groups such as amino groups, carboxyl groups, halogens, and nitro groups introduced into them. Since single-domain antibodies are polypeptides, the reaction between the single-domain antibody and the fluorescent dye can be carried out according to conditions well known to those skilled in the art. For example, the functional groups of the fluorescent dye can be activated and mixed with the single-domain antibody, and the reaction can be carried out at 4-65°C for several hours to form a covalent bond. Unreacted fluorescent dye can be purified by conventional methods after the reaction is complete. Single-domain antibodies have excellent heat resistance, so reactions at high temperatures are possible, and fluorescently labeled substances can be prepared under various reaction conditions. When single-domain antibodies are genetically engineered, amino acid residues having amino groups, carboxyl groups, thiol groups, etc. that can react with the fluorescent dye can be introduced at the desired position for fluorescent dye binding, and a fluorescent dye having the corresponding functional group can be reacted with it. This allows the fluorescent dye to be attached near the variable region, at the N-terminus, C-terminus, the -NH2 group derived from arginine, asparagine, glutamine, or lysine, the -SH group derived from cysteine, or at any other arbitrary position on the single-domain antibody. Alternatively, the single-domain antibody and the fluorescent dye may be linked via any linker.

[0031] The number of fluorescent dye molecules bound to a single-domain antibody molecule can be arbitrarily selected. Preferably, it is one or more molecules per single-domain antibody molecule, and more preferably two to five molecules. The average mass of the single-domain antibody is 12 to 15 kDa, and binding five or more molecules may impair its ability to bind to the target substance.

[0032] The substances that can be measured in this disclosure are not particularly limited, as long as it is possible to prepare a single-domain antibody that uses at least a part of the substance as an epitope. For example, a preferred mass is 1.5 × 10⁻⁶. 3 ~1 × 10 8 Da, comfortable 1×10 5 ~1 × 10 8The measurement is of Da. Preferably, the stalk diameter is 1 nm to 10 μm, and more preferably 3 nm to 10 μm. Measurement is possible within this range. Furthermore, by classifying the substances to be measured according to their origin and characteristics, it is also possible to measure biological substances, pharmaceuticals, viruses, or bacteria. Biological substances include various components produced within the body of an organism, various components excreted outside the body, and the organism itself; organisms include both plants and animals. Pharmaceuticals are not limited to pharmaceuticals administered to humans or animals, but also include pesticides, etc.

[0033] Biological substances include hormones, which are physiologically active substances synthesized and secreted by endocrine organs such as the hypothalamus, pituitary gland, thyroid gland, parathyroid gland, adrenal gland, pancreas, and reproductive organs; metabolites such as nucleic acids, uric acid, purines, C-reactive protein (CRP), apolipoproteins, HDL, LDL, and glycated hemoglobin; shellfish toxins and bacterial toxins such as mycotoxins, aflatoxin B1, and botulinum toxin A; plant-derived alkaloids such as morphine, atropine, quinine, and cocaine; and bacteria themselves, such as E. coli, streptococci, bacilli, salmonella, and Pseudomonas aeruginosa. Pharmaceuticals include antibiotics such as chloramphenicol and cyclosporine, and pesticides such as fungicides, antifungal agents, insecticides, herbicides, rodenticides, and plant growth regulators used to improve agricultural efficiency. Viruses are extremely small infectious structures that replicate themselves by utilizing the cells of other organisms. Measurable viruses include influenza virus, coronavirus, hepatitis B virus, hepatitis A virus, hepatitis C virus, and HIV.

[0034] In the fluorescence polarization immunoassay method disclosed herein, the degree of fluorescence polarization of a fluorescently labeled substance to which the target substance is bound is measured. Depending on the characteristics of the target substance contained in the sample, the sample solution is appropriately diluted with pure water or other diluent, and impurities are removed as necessary to prepare the sample solution. The fluorescently labeled substance is mixed with this sample solution to bind the target substance and the fluorescently labeled substance. Then, the degree of fluorescence polarization of the compound of the target substance and the fluorescently labeled substance is measured. Any polarization measuring device can be used to measure the degree of fluorescence polarization. The measurement should be performed at a temperature range of 4 to 40°C, preferably at a constant temperature within the above range, as long as the target substance does not denature. To quantify the target substance, a calibration curve can be created by performing the same procedure as above using a solution containing the target substance at a known concentration, and this can be compared with the measured value of the sample solution.

[0035] The same procedure applies when analyzing microorganisms such as bacteria as the target substance. A single-domain antibody that specifically binds to a portion of the bacteria as an epitope is prepared beforehand, and a fluorescently labeled substance is prepared by binding this single-domain antibody to a fluorescent dye. The fluorescently labeled substance is added to the sample solution to bind the bacteria in the sample solution to the fluorescently labeled substance. Then, the change in the degree of fluorescence polarization of the fluorescently labeled substance to which the bacteria are bound is measured. The bacterial quantity can be quantified by comparing the measured value of the sample solution with a calibration curve prepared using a solution containing the target substance at a known concentration. The same procedure applies when measuring viruses instead of bacteria.

[0036] In this disclosure, there are no limitations on the apparatus as long as it can measure the degree of fluorescence polarization. On the other hand, by using a measuring device composed of microfluidic channels, highly sensitive measurements can be performed using minute amounts of sample. Microfluidic channels used in fluorescence polarization measurement methods must be composed of materials that do not affect the degree of fluorescence polarization, and PDMS is often used. However, it has been found that when a fluorescently labeled substance is used, conjugated products of the fluorescently labeled substance and the substance to be measured adhere to the PDMS. In contrast, when a fluorescently labeled substance is used, which is obtained by reacting a single-domain antibody with a fluorescent dye, conjugated products of the fluorescently labeled substance and the substance to be measured do not adhere to the microfluidic channels formed with PDMS, enabling rapid and accurate measurement. A single-domain antibody has one variable region, but a Fab antibody contains four variable regions, and correspondingly its volume is 3 to 4 times larger. It is presumed that these differences in volume and structure result in differences in adhesion ability to PDMS.

[0037] An example of a fluorescence polarization measurement device having such microchannels is the fluorescence polarization measurement device used in Example 4 described later. A fluorescent labeling substance, a sample, or a fluorescent labeling substance bound to the substance to be measured is supplied to the microchannel, and the fluorescence polarization is measured. It is preferable to form a sample emission portion in the microchannel where the fluorescent labeling substance emits fluorescence upon irradiation with excitation light, and then measure the fluorescence polarization. With a microchannel, multiple channels can be formed within the effective field of view of the optical observation area for measuring fluorescence polarization, allowing for simultaneous measurement and image analysis of multiple samples. For example, even if the effective field of view of the optical observation area is approximately 3 mmφ, nine channels can be formed by setting the channel pitch to approximately 300 μm. The channel width and the space between channels can be arbitrarily set relative to this channel pitch. For example, by setting the channel width and the space between channels to equal intervals, the channel width can be set to 150 μm and the space between channels to 150 μm. Since the measurement sensitivity improves with greater channel depth, the channel depth can be set to 900 μm, for example. Furthermore, the microchannel material may be blackened to improve measurement sensitivity. The above is just one example. Regarding ease of manufacturing, there is a close relationship between the channel depth and channel width dimensions. For example, if the channel depth is 300 μm, the channel width can be 200 μm or more. A larger channel width leads to improved light extraction efficiency, regardless of the optical system configuration, and has the advantage of improving measurement uniformity.

[0038] According to the fluorescence polarization measurement method of this disclosure, the concentration of the target substance contained in the sample can be measured in the range of 100 pM to 10 μM, more preferably 1 to 1,000 nM, regardless of whether or not a microfluidic channel is used. Furthermore, according to the fluorescence polarization measurement method of this disclosure, a mass of 1.5 × 10⁻⁶ can be measured. 3 ~1 × 10 8 Da, comfortable 1×10 5 ~1 × 10 8The concentration of the target substance in Da can be quantified. Specifically, for a target substance with a mass of approximately 150 kDa, the concentration can be measured in the range of 0.4 to 10,000 nM. Furthermore, as shown in the examples described later, by using a fluorescently labeled substance in which a single-domain antibody is labeled with a fluorescent dye with a fluorescence lifetime of 1 to 10 nanoseconds, the range of variation in fluorescence polarization can be expanded compared to using Fab antibodies, even when quantifying high molecular weight target substances such as IgG.

[0039] The second aspect of this disclosure is a fluorescently labeled substance obtained by conjugating a fluorescent dye to a single-domain antibody. A single-domain antibody, as described above, means an antibody composed of a single variable region. VHH antibodies and vNAR antibodies can be used as single-domain antibodies. Conventionally known Fab antibodies and scFv antibodies may be degraded to isolate a single variable region and used as a single-domain antibody. Furthermore, conventionally known VHH antibodies and vNAR antibodies may be referenced in relation to their DNA sequences, or may be manufactured genetically using antibody libraries, etc., or modified as appropriate.

[0040] A fluorescent dye is a dye that emits fluorescence. It is preferable that the fluorescent dye has a functional group that can bind to carboxyl groups, amino groups, hydroxyl groups, thiol groups, phenyl groups, etc. This is because single-domain antibodies often have carboxyl groups, amino groups, hydroxyl groups, thiol groups, and phenyl groups, and if the fluorescent dye has a functional group that can bind to these groups, it facilitates the formation of fluorescently labeled substances. Furthermore, each fluorescent dye has its own unique fluorescence lifetime. In this disclosure, depending on the intended use of the fluorescent labeling material, fluorescent dyes with fluorescence lifetimes of 1 to 10 nanoseconds, fluorescent dyes with fluorescence lifetimes of more than 10 nanoseconds to 200 nanoseconds, and fluorescent dyes with fluorescence lifetimes of more than 200 nanoseconds to 3,000 nanoseconds can be appropriately selected and used. Fluorescent dyes with fluorescence lifetimes of 1 to 10 nanoseconds include fluorescein compounds such as indorenine, 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 registered trademarks or trade names such as Alexa Fluor 488 and other Alexa Fluor series, BODIPY series, DY series, ATTO series, Dy Light series, Oyster series, HiLyte Fluor series, Pacific Blue, Marina Blue, Acridine, Edans, Coumarin, DANSYL, FAN, Oregon Green, Rhodamine Green-X, NBD-X, TET, JOE, and Yakima. Yellow, VIC, HEX, R6G, Cy3, TAMRA, Rhodamine Red-X, Redmond Red, ROX, Cal Red, Texas Red, LC Red 640, Cy5, Cy5.5, LC Red 705.

[0041] Fluorescent dyes with fluorescence lifetimes ranging from over 10 nanoseconds to 200 nanoseconds include naphthalene derivatives such as dialkylaminonaphthalenesulfonyl, and pyrene derivatives such as N-(1-pyrenyl)maleimide, aminopyrene, pyrenebutanoic acid, and alkynylpyrene. Examples of fluorescent dyes with fluorescence lifetimes ranging from over 200 nanoseconds to 3,000 nanoseconds include metal complexes such as platinum, rhenium, ruthenium, osmium, and europium.

[0042] The fluorescent labeling material of this disclosure may be a single-domain antibody conjugated with a fluorescent dye having a fluorescence lifetime of 1 to 10 nanoseconds, a single-domain antibody conjugated with a fluorescent dye having a fluorescence lifetime of more than 10 nanoseconds to 200 nanoseconds, or a single-domain antibody conjugated with a fluorescent dye having a fluorescence lifetime of more than 200 nanoseconds to 3,000 nanoseconds. Using this fluorescent labeling material, a mass of 1.5 × 10⁻⁶ 3 ~1 × 10 8 Da, comfortable 1×10 5 ~1 × 10 8 The concentration of the target substance Da can be measured by fluorescence-polarized immunoassay.

[0043] The binding of the fluorescent dye to the single-domain antibody is preferably by covalent bonding. A fluorescently labeled substance can be produced by reacting the aforementioned functional groups of the fluorescent dye with the single-domain antibody under conditions well known to those skilled in the art. For example, the functional groups of the fluorescent dye can be activated, mixed with the single-domain antibody, and reacted at a temperature of 4-65°C for several hours to form a covalent bond. After the reaction is complete, the unreacted fluorescent dye can be removed by conventional methods. Alternatively, when genetically engineering a single-domain antibody, an amino acid residue having an amino group, carboxyl group, thiol group, etc., that can react with the fluorescent dye can be introduced at the desired position for fluorescent dye binding, and a fluorescent dye having the corresponding functional group can be reacted. This allows the fluorescent dye to be bound near the variable region, at the N-terminus, C-terminus, the -NH2 group derived from arginine, asparagine, glutamine, or lysine, the -SH group derived from cysteine, or any other arbitrary position on the single-domain antibody. Furthermore, the single-domain antibody and the fluorescent dye may be linked via a linker. Such linkers include oligoethylene glycol and alkyl chains. As shown in the examples described below, a fluorescently labeled substance in which a fluorescent dye is conjugated to the N-terminus corresponding to the vicinity of the variable region of a single-domain antibody exhibits excellent sensitivity to fluorescence change.

[0044] The fluorescently labeled substances of this disclosure can be used in fluorescence polarization immunoassay, sandwich immunoassay, and immunostaining.

[0045] Single-domain antibodies have a lower molecular weight than low-molecular-weight antibodies such as Fab antibodies and scFv antibodies. They readily revert to their natural structure even under denaturing conditions such as denaturing solutions of denaturing agents like guanidine hydrochloride and urea, as well as high temperature and high pressure, and exhibit excellent heat resistance, pressure resistance, and chemical resistance. In particular, they have excellent heat resistance; when returned to room temperature from high-temperature conditions of 90°C, they exhibit the same antigen-binding activity as before heat treatment. Their resistance to temperature changes is advantageous during distribution and storage. Furthermore, they have high solubility in aqueous solvents and excellent stability against surfactants, making them easy to handle when genetically engineering single-domain antibodies that can specifically bind to target substances. When used in fluorescence polarization immunoassay, they allow for the measurement of high molecular weight target substances using highly versatile fluorescent dyes with fluorescence lifetimes of 1 to 10 nanoseconds, and offer advantages such as the ability to measure at low concentrations, thus reducing the amount of sample required. [Examples]

[0046] The present disclosure will now be explained in detail with reference to examples, but these examples are not intended to limit the present disclosure in any way.

[0047] (Example 1) (1) To quantify rabbit IgG, rabbit IgG (manufactured by Sigma-Aldrich) was dissolved in phosphate-buffered saline (PBS) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare nine levels of rabbit IgG solutions: 3,230 nM, 1,080 nM, 360 nM, 120 nM, 40 nM, 13 nM, 4.4 nM, 1.5 nM, and 0.49 nM. (2) Anti-Rabbit IgG Alpaca-mono, recombinant VHH Alexa Fluor 488 modified (VHH) (Chromotek, mass 15 kDa) was used as a fluorescently labeled substance, which is a single-domain antibody capable of binding to the target substance (rabbit IgG) and labeled with a fluorescent dye. This was diluted 100-fold with PBS to prepare a fluorescently labeled substance solution. (3) Fetal bovine serum (FBS) (manufactured by Biowest) was diluted 10-fold with PBS to obtain an FBS solution. (4) 16 μL of the fluorescent labeling solution (containing 5 μg / mL of VHH), 60 μL of FBS solution, 60 μL of rabbit IgG solution at 9 levels, and 464 μL of PBS were mixed to prepare 600 μL of nine standard curve preparation samples. This preparation resulted in an antibody concentration of 10 nM. After preparing each sample, the fluorescence polarization was measured after standing at room temperature in the dark for 2 hours. (5) A spectrofluorometer F7100 (manufactured by Hitachi High-Tech Science) was used as the fluorescence polarization measurement device, and measurements were taken in fluorescence polarization mode. The excitation wavelength was 490 nm, the detection wavelength was 510-540 nm, the scan speed was 60 nm / min, the initial waiting time was 0 s, the fluorescence-side slit was 10 nm, the excitation-side slit was 10 nm, and the response was 0.002 s. 150 μL of a standard curve preparation sample containing 0.049 nM rabbit IgG was placed in the cell and the G value was measured. The standard curve preparation sample was placed in a quartz cell and the fluorescence polarization was measured. All data were measured three times. The results are shown in Figure 2 as VHH antibody (15 kDa).

[0048] (Comparative Example 1) The procedure was the same as in Example 1, except that Anti-Rabbit IgG Alexa-labeled Fab fragment (Fab) (Jackson ImmunoResearch, mass 50 kDa) was used instead of Anti-Rabbit IgG Alpaca-mono, recombinant VHH Alexa Fluor 488 modification (VHH). The results are shown in Figure 2 as Fab antibody (50 kDa).

[0049] As shown in Figure 2, the fluorescence polarization degree of Comparative Example 1, which used Fab antibody, fluctuated within the range of 0.108 to 0.138, with a fluctuation range of 0.03. On the other hand, the fluorescence polarization degree of Example 1, which used VHH antibody, fluctuated within the range of 0.082 to 0.13, with a fluctuation range of 0.048. Using VHH antibody resulted in a 1.6 times wider fluctuation range compared to using Fab antibody.

[0050] Furthermore, sigmoid curves were created for the results of Example 1 and Comparative Example 1 under the conditions shown in Table 1, and the lower and upper limits of quantification were calculated. [Table 1] As a result, the lower limit of quantification for Comparative Example 1, which used the Fab antibody, was 5.4 nM and the upper limit of quantification was 18 nM, while the lower limit of quantification for Example 1, which used the VHH antibody, was calculated to be 0.45 nM and the upper limit of quantification was 41 nM. It was found that measurement is possible even at a low concentration of 0.45 nM.

[0051] (Example 2) (1) Anti-Her2 Alpaca, monochronal, recombinant VHH (QVQ; 1 mg / mL) was used as a single-domain antibody capable of binding to the target substance Her2 (ErbB2 / Her2 Fc chimeric recombinant protein, manufactured by R&D Systems). (2) 20 μL of this antibody was mixed with 2.75 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) aqueous solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and left to stand in the dark at 37°C for 2 hours. 6 equivalents of Alexa Fluor 488 maleimide (manufactured by Thermo Scientific) were added to this mixture and left to stand in the dark at room temperature for 2 hours. Subsequently, the mixture was purified twice using a Zeba column (7 kDa) to produce a fluorescently labeled substance. In this fluorescently labeled substance, Alexa Fluor 488 is bound to the SH group of the antibody. (3) The substance to be measured, Her2, was diluted 50-fold with phosphate-buffered saline (PBS) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). This diluted solution was further diluted 5-fold sequentially with PBS to prepare Her2 solutions at six different concentrations: 410 nM, 82 nM, 16 nM, 3.3 nM, 0.66 nM, and 0.13 nM. (4) 92.5 μL of the fluorescent labeling solution was diluted with PBS to a concentration of 44 nM. This solution was mixed with 200 μL of Her2 solution of each concentration, 5 μL of fetal bovine serum (FBS) (Biowest), and 402.5 μL of PBS to obtain 500 μL of six standard curve preparation samples. This preparation resulted in a VHH concentration of 8.2 nM in the samples. Additionally, 80 μL of the fluorescent labeling solution was mixed with 2 μL of fetal bovine serum (FBS) (Biowest), and 161 μL of PBS to obtain a total of 200 μL of Her2-free control sample. After each sample preparation, the fluorescence polarization was measured after standing at room temperature in the dark for 2 hours. (5) A Tecan Infinite 200PRO F Plex was used as the fluorescence polarization measurement device. The excitation wavelength was 490 nm, and the detection wavelength was 510-540 nm. The results are shown in Figure 3. We were able to confirm an increase in fluorescence polarization degree in proportion to the Her2 concentration in the range of 0.66 to 1.6 nM.

[0052] (Example 3) (1) Anti-Her2 Alpaca, monochronal, recombinant VHH (QVQ; 1 mg / mL) was used as a single-domain antibody capable of binding to the target substance Her2 (ErbB2 / Her2 Fc chimeric recombinant protein, R&D Systems). 20 μL of this antibody and 5 equivalents of Alexa Fluor 488 SDP ester (Thermo Scientific) were mixed at pH 8.5 and stirred in the dark at room temperature for 1 hour. Subsequently, the mixture was purified twice using a Zeba column (7 kDa) to produce a fluorescently labeled substance. In this fluorescently labeled substance, the fluorescent dye Alexa Fluor 488 is bound to the -NH2 group of the antibody. (2) The procedure was carried out in the same manner as in Example 2, except that the fluorescent labeling substance obtained in (1) above was used, and the degree of fluorescence polarization was measured. The results are shown in Figure 4. When a VHH antibody with Alexa Fluor 488 bound to the -NH2 group was used, an increase in the degree of fluorescence polarization corresponding to the concentration was observed in the range of Her2 concentration from 0.66 to 8.2 nM. In addition, the degree of fluorescence polarization fluctuated between 0.158 and 0.187, and the degree of fluorescence polarization corresponding to the concentration was confirmed with better sensitivity than when bound to the -SH group in Example 2.

[0053] (Example 4) (1) To quantify rabbit IgG, seven levels of rabbit IgG solutions were prepared using rabbit IgG (Sigma-Aldrich) and phosphate-buffered saline (PBS) (Fujifilm Wako Pure Chemical Industries, Ltd.): 1,080 nM, 360 nM, 120 nM, 40 nM, 13 nM, 4.4 nM, and 0.15 nM. (2) Anti-Rabbit IgG Alpaca-mono, recombinant VHH Alexa Fluor 488 modified (VHH) (Chromotek, mass 15 kDa) was used as a fluorescently labeled substance, which is a single-domain antibody capable of binding to the target substance (rabbit IgG) and labeled with a fluorescent dye. This was diluted 100-fold with PBS to prepare a fluorescently labeled substance solution. (3) Bovine serum albumin (BSA) (manufactured by abcam) was dissolved in PBS to obtain a 1% BSA solution. (4) Fetal bovine serum (FBS) (Biowest) was diluted 10-fold with PBS to obtain an FBS solution. (5) 8 μL of the fluorescent labeling solution (containing 5 μg / mL of VHH), 30 μL of BSA solution, 30 μL of rabbit IgG solution of each concentration, and 232 μL of PBS were mixed to obtain 300 μL of each of the 7 levels of samples for creating standard curves. (6) Separately, 30 μL of rabbit IgG solution containing 13 nM and 120 nM rabbit IgG was prepared, and a sample for measurement was prepared by mixing 8 μL of the fluorescent labeling substance solution (containing 10 nM VHH), 30 μL of FBS solution, 30 μL of rabbit IgG solution, and 232 μL of PBS. (7) Using a fluorescence polarization analyzer having nine microchannels, the fluorescence polarization of the standard curve preparation sample and the measurement sample was measured with an excitation wavelength of 470±5nm and a detection wavelength of 520±5nm. The samples were prepared by injecting the seven levels of standard curve preparation samples described in (4) into seven of the nine microchannels, and the two levels of measurement samples described in (5) into the remaining two microchannels, and measuring them simultaneously. A schematic configuration of the fluorescence polarization analyzer used is shown in Figure 5. The apparatus 10 mainly consists of an LED light source unit 1, an excitation filter 2, a fluorescence filter 3, a dichroic filter 4, an objective lens 5, an imaging lens 6, a liquid crystal element 7 and a digital imaging element (CMOS or CCD camera) 8, and a sample emission unit 9. Excitation light from an LED light source unit 1 with a central wavelength of 470 nm is irradiated onto a sample in the sample emission unit 9 via an excitation filter 2 and an objective lens 5. The fluorescence emitted by the sample is transmitted through a dichroic filter 4 and a fluorescence filter 3, and the transmitted light is acquired by a CMOS camera 8. By applying a voltage to a liquid crystal element 7 placed between the fluorescence filter 3 and the imaging lens 6 and modulating the voltage, the polarization direction of the transmitted fluorescence can be modulated. The modulation frequency and the acquisition frequency of the CMOS camera 8 are synchronized to acquire and process an image, and the polarization degree P is calculated as a two-dimensional image. The effective field of view of the optical observation portion of the sample emission unit 9 of this apparatus 10 is approximately 3 mm in diameter. Nine channels are used to perform simultaneous measurement of the standard curve and the sample to be measured. As shown in Figure 6, within the effective field of view of φ3 mm shown as a circle, the channel width 11 and the space between channels 12 are equally spaced, with the channel width 150 μm and the space between channels 150 μm. The channel depth is 900 μm. By forming multiple microchannels within the sample emission unit 9, multiple samples can be measured simultaneously. Figure 7 shows the fluorescence polarization measurement image of the microchannel used in Example 4. (8) The results are shown in Figure 8. In Figure 8, the black circles represent the standard curve, and the black squares represent the measurement results for samples containing 1,3 nM and 12 nM of IgG. When using VHH antibody, measurements could be performed even with a measuring instrument equipped with a microfluidic channel.

[0054] (Example 5) (1) To quantify rabbit IgG, rabbit IgG (manufactured by Sigma-Aldrich) was dissolved in phosphate-buffered saline (PBS) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare nine levels of rabbit IgG solutions: 3,280 nM, 1,080 nM, 360 nM, 120 nM, 40 nM, 13 nM, 4.4 nM, 1.5 nM, and 0.49 nM. (2) As a fluorescently labeled substance obtained by labeling a single-domain antibody capable of binding to the target substance (rabbit IgG) with a fluorescent dye, Anti-Rabbit IgG Alpaca-mono, recombinant VHH Alexa Fluor 488 modified (VHH) (Chromotek, mass 15 kDa) was diluted 1000 times in PBS to prepare a fluorescently labeled substance solution. (3) 2.7 μL of the fluorescent labeling solution (containing 0.5 μg / mL of VHH), 10 μL of rabbit IgG solution of each concentration, and 87.3 μL of PBS were mixed to obtain 100 μL of each of the 9 levels of samples for creating standard curves. (4) Using a fluorescence polarization analyzer with nine microchannels, the fluorescence polarization of the sample for standard curve creation was measured with an excitation wavelength of 470±5nm and a detection wavelength of 520±5nm. The sample was prepared by injecting the nine levels of the standard curve creation sample described in (4) into each of the nine microchannels and measuring them simultaneously. The fluorescence polarization analyzer used was the same as that used in Example 4. The measurement results for Example 5 are shown in Figure 9.

[0055] (Comparative Example 2) The procedure was the same as in Example 5, except that Anti-Rabbit IgG Alexa-labeled Fab fragment (Fab) (Jackson ImmunoResearch, mass 50 kDa) was used instead of Anti-Rabbit IgG Alpaca-mono, recombinant VHH Alexa Fluor 488 modification (VHH). The measurement results for Comparative Example 2 are shown in Figure 10.

[0056] (result) As shown in Figure 9 of Example 5, the amount of IgG was easily quantified using VHH antibody with a measuring device having a microfluidic channel. On the other hand, as shown in Figure 10, when Fab antibody was used with a measuring device having a microfluidic channel, the increase in fluorescence polarization degree corresponding to the concentration observed in Example 1 was not observed, making it difficult to quantify the amount of IgG. This is presumed to be because Fab antibody has a larger molecular weight than VHH antibody, making it more easily adsorbed to the microfluidic channel wall. When an antibody like Fab antibody is adsorbed, rotational diffusion of the antibody molecule is suppressed, so it shows a high fluorescence polarization value even when it is not reacting with the antigen. Therefore, with a measuring device having a microfluidic channel, the result of the fluorescence polarization value increasing with the antigen concentration as in Example 1 was not obtained, and it is thought that the measurement of the antigen became difficult. In contrast, when an antibody that does not easily adsorb to the channel, such as VHH antibody, is used, the fluorescence polarization value increases as the antigen concentration increases even with a measuring device having a microfluidic channel, and measurement with high measurement sensitivity became possible, similar to Example 1. [Explanation of Symbols]

[0057] 1: LED light source unit, 2: Excitation filter, 3: Fluorescence filter, 4: Dichroic filter, 5: Objective lens, 6: Imaging lens, 7: Liquid crystal element, 8: CMOS camera, 9: Sample light emission unit, 10: Apparatus

Claims

1. A fluorescence polarization immunoassay method for analyzing target substances in a sample, A binding step in which a fluorescently labeled substance, which is a single-domain antibody having the ability to bind to the substance to be measured and is labeled with a fluorescent dye, is bound to the substance to be measured contained in the sample, and The measurement step includes measuring the change in the degree of fluorescence polarization of the fluorescently labeled substance to which the substance to be measured is bound, The fluorescently labeled substance, the sample, or the sample containing the fluorescently labeled substance to which the substance to be measured is bound is supplied to a microchannel formed by PDMS, and fluorescence polarization analysis is performed to simultaneously measure multiple samples in multiple microchannels. A fluorescence polarization immunoassay method that measures the change in the degree of fluorescence polarization of the fluorescently labeled substance by drawing a sigmoid curve having saturation regions at the lower and upper limits of the degree of fluorescence polarization.

2. A fluorescence polarization immunoassay method for analyzing target substances in a sample, A conjugation step in which a fluorescently labeled substance, in which a fluorescent dye is conjugated to the N-terminus of a single-domain antibody, is conjugated to the substance to be measured contained in the sample, and A fluorescence polarization immunoassay method comprising a measurement step of supplying the fluorescently labeled substance, or the sample containing the fluorescently labeled substance to which the substance to be measured is bound, to a microchannel formed by PDMS, and measuring the change in the degree of fluorescence polarization of the fluorescently labeled substance to which the substance to be measured is bound.

3. The fluorescence polarization immunoassay method according to claim 2, wherein the measurement step involves supplying a plurality of the samples to a plurality of the microchannels and simultaneously measuring the change in the degree of fluorescence polarization of the fluorescently labeled substance to which the substance to be measured is bound in the plurality of the samples.

4. The fluorescence polarization immunoassay method according to claim 2, comprising a measurement step of using a calibration curve based on the change in fluorescence polarization degree to measure the concentration of the substance to be measured contained in the sample in the range of 100 pM to 10 μM.

Images