Calibrators, complexes, and methods for measuring IgA aggregates

Abstract

The present application provides a calibrator for use in a determination of IgA aggregates, and a determination method of IgA aggregates using the calibrator. The present application solves the problem by a calibrator containing IgA having a biotin group and an avidin.

Description

[Technical Field]

[0001] This invention relates to calibrators for obtaining the concentration of IgA lectins in a sample. This invention relates to complexes containing biotin-based IgA and avidin derivatives. This invention relates to a method for determining IgA lectins in a sample using lectins. [Background Technology]

[0002] Patent Document 1 describes a method for detecting IgA agglutinations in a sample using an enzyme-linked immunosorbent assay (ELISA) employing a lectin that specifically reacts with IgA agglutinations. Furthermore, Patent Document 1 describes that when comparing the detection results of IgA purified from the serum of patients with IgA nephropathy and healthy individuals, the patient-derived purified IgA showed a particularly strong reaction to the lectin.

[0003] [Existing Technical Documents]

[0004] [Patent Documents]

[0005] [Patent Document 1] Patent No. 6066220

[0006] [Summary of the Invention]

[0007] [The problem the invention aims to solve]

[0008] In the quantitative determination of test substances by immunoassays such as ELISA, calibrators are used. A calibrator generally refers to a reagent containing a standard substance corresponding to the test substance. In the field of immunoassays, standard substances are often isolated from biological samples or synthesized from the test substance itself. However, suitable calibrators for the determination of IgA agglutinations are unknown. Therefore, this invention aims to provide: a calibrator used in the determination of IgA agglutinations, a complex containing biotinylated IgA and avidin contained in the calibrator, and a method for determining IgA agglutinations using the calibrator.

[0009] [Methods used to solve problems]

[0010] This invention provides a calibrator containing biotin-based IgA and avidin for obtaining the concentration of IgA lectins in a sample. This invention also provides a complex containing biotin-based IgA and avidin for obtaining the concentration of IgA lectins in a sample. Furthermore, this invention provides a method for determining the concentration of IgA lectins in a sample using lectins, comprising using a calibrator containing biotin-based IgA and avidin to obtain the concentration of IgA lectins in the sample.

[0011] [The effects of the invention]

[0012] According to the present invention, a calibrator for use in the determination of IgA condensates, a complex containing IgA with a biotin group and an avidin, and a method for determining IgA condensates using the calibrator are provided.

[0013] [Brief explanation of the attached image]

[0014]

【 Figure 1 [IgA] is a schematic diagram of a complex of biotinylated IgA and avidin.

[0015]

【 Figure 2 [IgA] is a schematic diagram of a complex of IgA with a biotinylate, avidin, and a polypeptide with a biotinylate and a glycan that binds to lectins.

[0016]

Figure 3

[0017]

Figure 4

[0018]

Figure 5

Detailed Implementation Methods

[0019] This embodiment describes a method for determining IgA agglutination using lectins. IgA agglutination refers to polymeric IgA formed by the aggregation of IgA in the blood of patients with IgA nephropathy. Currently, it is difficult to separate and purify IgA agglutination from the blood of patients with IgA nephropathy. Therefore, it is impossible to use the IgA agglutination itself in calibrators. On the one hand, IgA monomers with normal glycan chains are readily available. However, such IgA monomers do not bind to lectins. Therefore, as mentioned above, suitable calibrators for the determination of IgA agglutination are unknown.

[0020] The method of this embodiment is characterized by obtaining the concentration of IgA condensates in the sample using a calibrator containing IgA with a biotin group and an avidin. "Biotin group" refers to the heterocyclic portion containing an imidazolyl lysine ring in the chemical structure of a biotin. In this specification, the term "biotin" includes biotin and its analogues. Examples of biotin analogues include, for example, desulfurized biotin and oxybiotin. In this specification, the term "avidin" includes avidin and its analogues. Examples of avidin analogues include, for example, streptavidin, neutral avidin (trademark), and tamavidin (registered trademark). Avidins generally exist as tetramers composed of four subunits. Hereinafter, as with "one molecule of streptavidin," one molecule of avidin refers to the tetramer form of avidin. One molecule of avidin can bind to four biotin groups.

[0021] IgA with a biotinylated group (hereinafter also referred to as "biotinylated IgA") can be obtained by combining an IgA monomer and a biotinylate using known methods such as cross-linking agents. Commercially available biotin-labeling reagents can also be used. The number of biotinylated groups in one molecule of biotinylated IgA is not particularly limited; it can be one or more. When the IgA monomer is of human origin, it can be either natural IgA or recombinant IgA. The isotype of IgA can also be either IgA1 or IgA2. The preferred isotype is IgA1.

[0022] In the calibrator used in the method of this embodiment, a complex of biotinylated IgA and avidin is formed by the combination of a biotinylated group and an avidin class. That is, the calibrator is a reagent containing a complex of biotinylated IgA and avidin. In this embodiment, this complex corresponds to a standard substance for IgA condensates. An example of the complex is shown below. Figure 1 In the diagram, "STA" represents streptavidin, and "B" represents biotinyl. Figure 1 In this example, one molecule of streptavidin binds to four molecules of biotinylated IgA, but the invention is not limited to this example. Figure 1 As shown, the biotinylated IgA and avidin-like complexes are pseudo-agglutination aggregates of IgA. Because this complex can bind to lectins used in the determination of IgA agglutination aggregates, the concentration of IgA agglutination aggregates in a sample can be obtained by using a calibrator containing this complex. Furthermore, in Figure 1 For convenience, only the biotinylated IgA group is shown in the image, but as mentioned above, biotinylated IgA may also have multiple biotinylated groups.

[0023] A complex of biotinylated IgA and avidin can be obtained by mixing biotinylated IgA and avidin. The mixing conditions are not particularly limited as long as they do not denature the protein. For example, a solution of biotinylated IgA and a solution of avidin can be mixed and stirred or allowed to stand at 4°C to 40°C, preferably 15°C to 37°C.

[0024] In this embodiment, the complex preferably consists of one molecule of avidin and two, three, or four molecules of biotinylated IgA. Theoretically, when the biotinylated IgA has multiple biotinylate groups, the complex could become a giant molecule with multiple molecules of avidin and multiple molecules of biotinylated IgA linked together. However, the goal is to prevent such a giant molecule from forming substantially, or even if it does, to the extent that it has no impact on the method of this embodiment. The vast majority of complexes are complexes of one molecule of avidin and two, three, or four molecules of biotinylated IgA, particularly one molecule of avidin and four molecules of biotinylated IgA. This is considered to be because, due to steric hindrance, etc., the linked binding of biotinylated IgA and avidin does not occur.

[0025] In a further embodiment, the calibrator may also contain a polypeptide (hereinafter also referred to as "biotinylated carrier protein") having a sugar chain and a biotinylate group that binds to lectins. The term "polypeptide" refers to a substance composed of multiple amino acids linked by peptide bonds, and also includes proteins and their fragments. In this calibrator, a complex of biotinylated IgA, avidin, and biotinylated carrier protein is formed through the binding of the biotinylate group and avidin-like compounds. That is, this calibrator is a reagent containing a complex of biotinylated IgA, avidin-like compounds, and biotinylated carrier protein.

[0026] Biotinylated carrier proteins can be obtained by combining a polypeptide (hereinafter also referred to as "carrier protein") having a sugar chain that binds to lectins with biotin using known methods such as cross-linking agents. Commercially available biotinylated reagents can also be used. The number of biotin groups in one molecule of a biotinylated carrier protein is not particularly limited; it can be one or more.

[0027] The carrier protein is not particularly limited as long as it has a glycan that the lectin used in the method of this embodiment can bind to. When the lectin is known, the sequence or structure of the glycan that specifically binds to the lectin is well known. Examples of carrier proteins include M2BP (Mac-2 binding protein), MUC1 (Mucin 1, a cell surface-associated glycoprotein), and α2M (α-2-macroglobulin). These carrier proteins are particularly suitable for the method of this embodiment in which WFA (Wisteria floribunda lectin) is used as the lectin.

[0028] In this embodiment, the complex of biotinylated IgA, avidin, and biotinylated carrier protein corresponds to a standard substance for the IgA condensate. An example of this complex is shown in... Figure 2 .exist Figure 2 In this example, one molecule of streptavidin is bound to two molecules of biotinylated IgA and two molecules of biotinylated carrier protein, but the invention is not limited to this example. Figure 2 As shown, the complex of biotinylated IgA, avidin-like proteins, and biotinylated carrier proteins is a pseudo-agglutination body of IgA with glycan chains bound to lectins. Because this complex can bind to the lectins used in the determination of IgA agglutination bodies via the glycan chains of the biotinylated carrier proteins, the concentration of IgA agglutination bodies in the sample can be obtained by using a calibrator containing this complex. Furthermore, in Figure 2 For convenience, only the biotinylate group of one biotinylated IgA and only the biotinylate group of one biotinylated carrier protein are shown. However, as mentioned above, the biotinylated IgA may have multiple biotinylate groups, and the biotinylated carrier protein may have multiple biotinylate groups.

[0029] In this embodiment, the complex preferably consists of 1 molecule of an avidin, 1, 2, or 3 molecules of biotinylated IgA, and 1, 2, or 3 molecules of biotinylated carrier protein (however, the total number of molecules of biotinylated IgA and biotinylated carrier protein is 2 or more but less than 4). When the biotinylated IgA and / or biotinylated carrier protein have multiple biotinylate groups, theoretically, the complex could become a giant molecule with multiple molecules of avidin, multiple molecules of biotinylated IgA, and multiple molecules of biotinylated carrier protein linked together. However, as described above, such a giant molecule does not substantially occur, and even if it does, it does not affect the method of this embodiment to a certain extent.

[0030] A complex of biotinylated IgA, avidin derivatives, and a biotinylated carrier protein can be obtained by mixing these components. The mixing conditions are not particularly limited as long as they do not denature the protein. For example, a solution of biotinylated IgA, a solution of avidin derivatives, and a solution of the biotinylated carrier protein can be mixed and stirred or allowed to stand at 4°C to 40°C, preferably 15°C to 37°C. Preferably, the mixing order is such that the solutions of biotinylated IgA, avidin derivatives, and biotinylated carrier protein are mixed substantially simultaneously. Alternatively, the solutions of biotinylated IgA and biotinylated carrier protein can be mixed first, followed by mixing the resulting solution with the avidin derivative solution.

[0031] The complex contained in the calibrator can be in solid form (e.g., powder, crystals, lyophilized products, etc.) or liquid form (e.g., solution, suspension, emulsion, etc.). The solvent is not particularly limited as long as it is suitable for the preservation of proteins (especially antibodies), and examples include physiological saline and buffer solutions. The buffer solution is preferably a buffer with a pH near neutral (e.g., pH between 6 and 8). Examples of such buffer solutions include Good buffers for HEPES, MES, and PIPES, Tris-buffered saline (TBS), and phosphate-buffered saline (PBS).

[0032] Calibrators may also contain known additives. Examples of additives include protein stabilizers such as bovine serum albumin (BSA), preservatives such as sodium azide, and inorganic salts such as sodium chloride.

[0033] The calibrator can be in the form of a single reagent or in the form of a reagent group containing multiple reagents. As a reagent, the calibrator may be, for example, a container containing a complex of biotinylated IgA and avidin, or a container containing a complex of biotinylated IgA, avidin, and a biotinylated carrier protein.

[0034] When the calibrator is a reagent set, each reagent contained in the reagent set will be referred to as a "calibrator reagent" below. A reagent set may include multiple calibrator reagents, each containing a complex of biotinylated IgA and an avidin, or multiple calibrator reagents, each containing a complex of biotinylated IgA, an avidin, and a biotinylated carrier protein. In this reagent set, the concentrations of the complexes in the multiple calibrator reagents are different from each other. The reagent set may, for example, include multiple containers, each containing multiple calibrator reagents. The complexes are contained in the multiple containers at different concentrations.

[0035] The number of calibrator reagents in a reagent set is not particularly limited and can be selected from, for example, 2, 3, 4, 5, and 6. The concentration of the complex in each calibrator reagent is not particularly limited, as long as it allows for the creation of a calibration curve. For example, among multiple calibrator reagents, the calibrator reagent with the lowest complex concentration can contain the complex at a concentration between 1 μg / mL and 200 μg / mL, while the calibrator reagent with the highest complex concentration can contain the complex at a concentration between 2 and 1000 times that of the lowest.

[0036] In this embodiment, the concentration of IgA clumps in the sample is obtained by simultaneously measuring the IgA clumps in the sample using a lectin and the complex contained in the aforementioned calibrator. Next, the method for measuring IgA clumps in a sample using a lectin will be described.

[0037] In this embodiment, the sample is not particularly limited as long as it contains or is suspected of containing IgA agglutinants. Preferred samples are biological samples. Examples of biological samples include blood (whole blood), plasma, serum, urine, lymph, tissue fluid, cerebrospinal fluid, saliva, and other extracellular fluids. Among these, blood, plasma, serum, and urine are preferred. When the sample contains insoluble impurities such as cells, these impurities can be removed from the sample by known methods such as centrifugation and filtration. The sample can also be diluted with a suitable aqueous medium as needed. Such an aqueous medium is not particularly limited as long as it does not interfere with the determination described later; examples include water, physiological saline, and buffer solutions. The same applies to buffer solutions.

[0038] Lectin, also known as agglutinin, is a protein that specifically binds to a specified sugar chain. In this embodiment, the lectin is not particularly limited as long as it binds to IgA lectins. Such lectins are well known, and examples include WFA, HHL (Hippeastrum Hybrid lectin), GNA (Galanthus nivalis lectin), NPA (Narcissus pseudonarcissus lectin), SBA (Soybean lectin), VVA (Vicia villosa lectin), BPL (Bauhinia purpurea lectin), TJA (Trichosanthes japonica lectin)-II, PHA-L (Phaseolus vulgaris leukocyte lectin), and AOL (Aspergillus oryzae lectin). Among these, WFA is particularly preferred.

[0039] Naturally occurring WFA exists as a tetramer composed of four subunits. Monomeric or dimer WFA can be obtained from tetrameric WFA using known methods such as the use of reducing agents. In this specification, unless the number of subunits is specifically stated, the term "WFA" includes monomeric WFA, dimeric WFA, trimeric WFA, and tetrameric WFA. In this specification, when referring to WFA composed of a specified number of subunits, the number of subunits is explicitly stated, for example, as in "monomeric WFA," "dimeric WFA," "trimeric WFA," and "tetrameric WFA."

[0040] In this embodiment, the WFA can be a tetrameric WFA, or a monomeric WFA or a dimer WFA obtained from the tetrameric WFA. Among them, the dimer WFA is preferred from the perspective of its high reactivity with the IgA aggregate. A method for obtaining the dimer WFA from the tetrameric WFA using a crosslinking agent is described, for example, in U.S. Patent Application Publication No. 2016 / 0363586 (which is incorporated herein by reference).

[0041] Methods for determining IgA agglutination using lectins are well known, as described, for example, in Japanese Patent No. 6066220. As such a method, a method based on the principle of ELISA is preferred. Specifically, a method using lectins bound to IgA agglutinations can be cited as an alternative to the capture antibody or detection antibody in ELISA. The capture antibody is an antibody that specifically binds to the test substance and is used to capture the test substance on a solid phase by immobilizing itself on the solid phase. The detection antibody is an antibody that specifically binds to the test substance and has a labeled substance to provide a signal detectable by that labeled substance. The detection antibody is preferably not immobilized on a solid phase. In the assay, the immune complex transfer method described in Japanese Patent Application Publication No. 1-254868 can also be used.

[0042] ELISA methods can be any of the following: sandwich method, competitive binding method, direct method, indirect method, etc. In a preferred embodiment, IgA agglutination is measured by a sandwich ELISA method using a lectin and a capture body (hereinafter also referred to as "labeled capture body") containing a labeled substance that specifically binds to IgA agglutination. In this case, the lectin is equivalent to the capture antibody in the sandwich ELISA method, and the labeled capture body is equivalent to the detection antibody in the sandwich ELISA method.

[0043] Labeled traps can be obtained by directly or indirectly binding a labeling substance to a trap that specifically binds to IgA foci. For example, cross-linking agents, commercially available labeling kits, etc., can be used to bind the IgA foci-specific trap to the labeling substance. Alternatively, a labeled secondary antibody targeting the IgA foci-specific trap can be used. A labeled secondary antibody is an antibody to which a labeling substance is bound, specifically to an antibody that specifically binds to the IgA foci-specific trap.

[0044] The trapping body that specifically binds to IgA thallus is not particularly limited as long as it is a substance that can bind to a site in the IgA thallus that is different from the site where the lectin binds. Examples of such trapping bodies include, for example, antibodies (anti-IgA antibodies) or aptamers that specifically bind to IgA, and antigens that are recognized by IgA in the IgA thallus.

[0045] Labeling substances can be signal-generating substances or substances that catalyze the reaction of other substances to produce a detectable signal. Examples of signal-generating substances include fluorescent substances and radioactive isotopes. Examples of substances that catalyze the reaction of other substances to produce a detectable signal include enzymes. Examples of enzymes include alkaline phosphatase (ALP), peroxidase, β-galactosidase, glucose oxidase, tyrosinase, acid phosphatase, and luciferase. Examples of fluorescent substances include fluorescein isothiocyanate (FITC), rhodamine, Alexa Fluor (registered trademark), anthocyanin dyes, and fluorescent proteins such as GFP. Examples of radioactive isotopes include… 125 I, 14 C 32 P, etc. Among them, enzymes are preferred, especially ALP.

[0046] The following example illustrates the determination of the sample and calibrator using a sandwich ELISA method. In this example, the IgA condensates in the sample are measured first, followed by the determination of the complexes in the calibrator; however, the invention is not limited to this example. The IgA condensates in the sample may also be measured after the complexes in the calibrator are measured. Alternatively, the IgA condensates in the sample and the complexes in the calibrator may be measured substantially simultaneously.

[0047] First, the IgA agglutination mass is measured using a sample, agglutinin, and a labeled capture body. Specifically, as follows: An immune complex containing the IgA agglutination mass from the sample, agglutinin, and a labeled capture body is formed on a solid phase. This immune complex can be formed by mixing the sample, agglutinin, and labeled capture body. Furthermore, by contacting the aforementioned immune complex with a solid phase in which agglutinin can be immobilized, the aforementioned immune complex can be formed on the solid phase. In this embodiment, a solid phase in which agglutinin has been pre-immobilized is preferably used. That is, by mixing the solid phase in which the agglutinin is immobilized, the sample, and the labeled capture body, the aforementioned immune complex can be formed on the solid phase.

[0048] The method of fixing lectin to a solid phase is not particularly limited. For example, lectin and solid phase can be directly bonded, or lectin and solid phase can be indirectly bonded via other substances. Examples of direct bonding include physical adsorption. Examples of indirect bonding include, for example, bonding via a combination of biotin and avidin. In this case, by pre-modifying the lectin with a biotin and pre-binding the solid phase with avidin, the lectin and solid phase can be indirectly bonded via the combination of biotin and avidin.

[0049] The raw materials for the solid phase are not particularly limited and can be selected from, for example, organic polymers, inorganic compounds, and biological polymers. Examples of organic polymers include latex, polystyrene, and polypropylene. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, and ferrite, etc.), silicon oxide, alumina, and glass. Examples of biological polymers include insoluble agarose, insoluble dextran, gelatin, and cellulose. Two or more of these can also be used in combination. The shape of the solid phase is not particularly limited and can include, for example, particles, microplates, microtubes, membranes, and test tubes. Among these, microplates and particles (especially magnetic particles) are preferred.

[0050] Furthermore, the IgA agglutination count can be obtained by detecting the immune complex formed on the solid phase using methods known in the art. For example, when using an anti-IgA antibody bound to a labeled substance as a labeled trap, the IgA agglutination count in the sample can be obtained by detecting the signal generated by the labeled substance. The same method can be used to obtain the IgA agglutination count in the sample when using a labeled second antibody.

[0051] In this specification, "detection signal" includes qualitative detection of the presence or absence of a signal, quantitative detection of signal strength, and semi-quantitative detection of signal strength. Semi-quantitative detection refers to representing signal strength in stages, such as "no signal," "weak," "medium," and "strong." In this embodiment, quantitative detection of signal strength is preferred.

[0052] The substrate for an enzyme can be appropriately selected from known substrates corresponding to the type of enzyme. For example, when using alkaline phosphatase as an enzyme, substrates that can be cited include chemiluminescent substrates such as CDP-Star (4-chloro-3-(methoxyspiro[1,2-dioxane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate 2 sodium), CSPD (3-(4-methoxyspiro[1,2-dioxane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate 2 sodium), chromogenic substrates such as 5-bromo-4-chloro-3-indole phosphate (BCIP), 5-bromo-6-chloro-indole phosphate 2 sodium, and p-nitrophenyl phosphate. In addition, when peroxidase is used as an enzyme, examples of substrates include chemiluminescent substrates such as LUMINOR and its derivatives, and chromogenic substrates such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate ammonium) (ABTS), 1,2-phenylene diamine (OPD), and 3,3',5,5'-tetramethylbenzidine (TMB).

[0053] When the labeled substance is a radioactive isotope, the radiation as a signal can be measured using a known device such as a scintillation counter. When the labeled substance is a fluorescent substance, the fluorescence as a signal can be measured using a known device such as a fluorescence microplate reader. The excitation wavelength and fluorescence wavelength can be appropriately determined according to the type of fluorescent substance used.

[0054] The signal detection result can be used as a measurement value for the IgA coagulant. For example, when quantitatively detecting signal intensity, the measured value of the signal intensity itself or the value obtained from that measurement can be used as a measurement value for the IgA coagulant. Examples of values ​​obtained from the measured signal intensity include, for example, the value obtained by subtracting the measured value of a negative control sample or the background value from that measured value. A suitable negative control sample can be selected, for example, a sample obtained from a healthy individual.

[0055] Next, the determination value of the complex in the calibrator is obtained using the calibrator, lectin, and labeled capture body. "The complex in the calibrator" refers to a complex of biotinylated IgA and avidin, or a complex of biotinylated IgA, avidin, and a biotinylated carrier protein. Specifically, as follows: An immune complex containing the complex in the calibrator, lectin, and labeled capture body is formed on a solid phase. This immune complex can be formed by mixing the calibrator, lectin, and labeled capture body. Furthermore, by contacting the aforementioned immune complex with a solid phase capable of immobilizing lectin, the aforementioned immune complex can be formed on the solid phase. In this embodiment, a solid phase pre-immobilized with lectin is preferably used. That is, by mixing the solid phase immobilized with lectin, the calibrator, and the labeled capture body, the aforementioned immune complex can be formed on the solid phase. Details of the solid phase are as described above.

[0056] Furthermore, the measured value of the complex in the calibrator can be obtained by detecting the immune complex formed on the solid phase using methods known in the art. For example, when using an anti-IgA antibody bound to a labeled substance as a labeled capture body, the measured value of the complex in the calibrator can be obtained by detecting the signal generated by the labeled substance. The measured value of the complex in the calibrator can also be obtained similarly when using a labeled second antibody. The details of signal detection are as described above.

[0057] In this embodiment, it is preferable to measure the concentrations of multiple calibrators that differ from each other. By obtaining the measured values ​​from multiple calibrators, a calibration curve, as described later, can be generated.

[0058] In this embodiment, it is preferable to determine the IgA agglutinin and calibrator in the sample using a sandwich ELISA method that uses a lectin immobilized on a solid phase and an anti-IgA antibody labeled with a labeled substance. When the solid phase is magnetic particles, the determination can also be performed using commercially available devices such as the HISCL series (Sysmex Corporation).

[0059] In this embodiment, Bound / Free (BF) separation, which removes unreacted free components that do not form immune complexes, can also be performed between the formation and detection of immune complexes. Unreacted free components refer to components that do not constitute immune complexes. Examples include lectins and labeled capture bodies that do not bind to IgA agglutinins. The method of BF separation is not particularly limited. When the solid phase is particles, BF separation can be performed by centrifuging to recover only the solid phase containing the captured immune complexes. When the solid phase is a container such as a microplate or microtube, BF separation can be performed by removing the liquid containing unreacted free components. Furthermore, when the solid phase is magnetic particles, BF separation can be performed by removing the liquid containing unreacted free components from the nozzle while the magnetic particles are magnetically confined by a magnet; this is preferred from an automation perspective. After removing the unreacted free components, the solid phase containing the captured immune complexes can also be washed with a suitable aqueous medium such as PBS.

[0060] In this embodiment, the concentration of IgA phagocytokines in the sample is obtained from the measured value of IgA phagocytokines in the sample and the measured value of the complex in the calibrator. The concentration of IgA phagocytokines in the sample is not only a commonly used concept of concentration (e.g., mass ratio, volume ratio, mole fraction, mass per unit volume, number of moles per unit volume, etc.), but also includes a value that serves as an indicator of concentration. Examples of values ​​that serve as indicators of concentration include, for example, numerical values ​​(grades) that semi-quantitatively indicate the concentration of IgA phagocytokines in the sample.

[0061] Since the concentration of the complex in the calibrator is known, the concentration of the IgA phagocytogen in the sample can be calculated from the measured value of the IgA phagocytogen based on the measured value of the complex. In a preferred embodiment, a calibration curve is constructed from the measured values ​​of the complex, and the concentration of the IgA phagocytogen in the sample is obtained based on the calibration curve and the measured values ​​of the IgA phagocytogen. The calibration curve can be constructed, for example, by plotting the measured values ​​of the complex obtained from multiple calibrators on an XY plane with the concentration of the complex in the calibrator on the X-axis and the measured values ​​(e.g., signal intensity) on the Y-axis, and obtaining a straight line or curve using known methods such as the least squares method. By applying the measured values ​​of the IgA phagocytogen to the calibration curve, the concentration of the IgA phagocytogen in the sample can be obtained.

[0062] Currently, IgA nephropathy is diagnosed solely by renal biopsy. However, as mentioned above, it is known that patients with IgA nephropathy have increased IgA agglutination in their blood and urine. Therefore, the concentration of IgA agglutination obtained by the method of this embodiment can also be used in determining whether a subject has IgA nephropathy. For example, the present invention provides a method to assist in the determination of IgA nephropathy. This method includes a step of measuring the amount of IgA agglutination in a sample obtained from a subject using a lectin, and a step of obtaining the concentration of IgA agglutination in the sample using a calibrator containing biotin-based IgA and avidin, wherein a concentration of IgA agglutination above a specified threshold indicates a high risk of the subject having IgA nephropathy.

[0063] In a further embodiment, the method for assisting in the determination of IgA nephropathy may also include: a step of obtaining a measurement value of IgA globulins in the sample using a sample obtained from the subject, a lectin, and a trap containing a labeled substance that specifically binds to IgA globulins; a step of obtaining a measurement value of a complex in the calibrator using a calibrator, the lectin, and the trap; a step of obtaining the concentration of IgA globulins in the sample from the measurement value of the IgA globulins and the measurement value of the complex; and a method in which a concentration of IgA globulins above a specified threshold indicates that the subject has a high risk of IgA nephropathy.

[0064] The specified threshold is not particularly limited and can be set appropriately. For example, biological samples are collected from both a healthy group and an IgA nephropathy patient group, and the concentration of IgA coagulants in the samples is obtained using the method of this embodiment. The value that most accurately distinguishes the healthy group from the patient group is determined from the obtained IgA coagulant concentration data, and this value is set as the threshold. Sensitivity, specificity, median positive rate, median negative rate, etc., may also be considered when setting the threshold.

[0065] A further embodiment of the present invention is a calibrator containing biotinylated IgA and avidin for obtaining the concentration of IgA condensates in a sample. The calibrator of this embodiment may also further contain a biotinylated carrier protein. The calibrator of this embodiment is suitably used in the method described above. Details of the calibrator are the same as those described for the method of this embodiment.

[0066] In one embodiment, the calibrator is a reagent containing a complex of biotinylated IgA and avidin. In a further embodiment, the calibrator is a reagent containing a complex of biotinylated IgA, avidin, and a biotinylated carrier protein. The details of these complexes are the same as those described for the method of this embodiment. The reagent may also be in the form of a container holding the aforementioned complex. This container may also be bundled into a box and provided to the user. An accompanying document may also be included in the box. The accompanying document may also describe the composition, usage, and storage methods of the calibrator of this embodiment.

[0067] An example of a calibrator is shown in Figure 3 .exist Figure 3 In this document, 10 represents the calibrator of this embodiment, 11 represents the first container that contains a complex of biotinylated IgA and avidin, or a complex of biotinylated IgA, avidin and biotinylated carrier protein, 12 represents the packaging box, and 13 represents the accompanying documents. Figure 3 The present invention is not limited to displaying calibrators in the form of a single reagent. Calibrators may also be in the form of a reagent group containing multiple calibrator reagents. Details of the calibrators as a reagent group are the same as those described for the method of this embodiment.

[0068] In a further embodiment, the calibrator is a reagent group comprising a reagent containing biotinylated IgA and a reagent containing an avidin. A complex of biotinylated IgA and an avidin can be obtained by mixing the reagent containing biotinylated IgA and the reagent containing an avidin. The details of the mixing conditions are the same as those described for the method of this embodiment. An example of this calibrator is shown in... Figure 4 .exist Figure 4 In this document, 20 represents the calibrator of this embodiment, 21 represents the first container containing biotinylated IgA, 22 represents the second container containing avidin, 23 represents the packaging box, and 24 represents the accompanying documents.

[0069] In a further embodiment, the calibrator is a reagent group comprising a reagent containing biotinylated IgA, a reagent containing avidin, and a reagent containing a biotinylated carrier protein. A complex of biotinylated IgA, avidin, and biotinylated carrier protein can be obtained by mixing the reagent containing biotinylated IgA, the reagent containing avidin, and the reagent containing a biotinylated carrier protein. The details of the mixing conditions are the same as those described for the method of this embodiment. An example of this calibrator is shown in… Figure 5 .exist Figure 5 In this document, 30 represents the calibrator of this embodiment, 31 represents the first container containing biotinylated IgA, 32 represents the second container containing avidin, 33 represents the third container containing biotinylated carrier protein, 34 represents the packaging box, and 35 represents the accompanying documents.

[0070] A further embodiment of the present invention is a complex containing biotinylated IgA and avidin for obtaining the concentration of IgA condensates in a sample. The complex of this embodiment may be a complex of biotinylated IgA and avidin, or a complex of biotinylated IgA, avidin, and a biotinylated carrier protein. The complex of this embodiment is suitably used in the method of this embodiment described above. The details of the complex of this embodiment are the same as those described for the method of this embodiment.

[0071] A further embodiment of the present invention is the use of biotin-based IgA and avidin derivatives in the manufacture of calibrators for obtaining the concentration of IgA condensates in a sample. In this embodiment, a calibrator containing a complex of biotin-based IgA and avidin derivatives is obtained by mixing biotin-based IgA and avidin derivatives.

[0072] A further embodiment of the present invention is the use of biotinylated IgA, avidin derivatives, lectin-bound sugar chains, and biotinylated polypeptides to manufacture a calibrator for obtaining the concentration of IgA lectins in a sample. In this embodiment, a calibrator containing a complex of biotinylated IgA, avidin derivatives, and a biotinylated carrier protein is obtained by mixing biotinylated IgA, avidin derivatives, and a biotinylated carrier protein.

[0073] The present invention will now be described in detail with reference to embodiments, but the present invention is not limited to these embodiments.

[0074]

Example

[0075]

Example 1

[0076] 【1. Preparation of reagents for determination】

[0077] (1.1) Sample dilution buffer (reagent 1)

[0078] As a sample dilution buffer, prepare 10 mM HEPES (pH 7.5) containing 1% BSA.

[0079] (1.2) Magnetic particles immobilized by WFA (Reagent 2)

[0080] WFA lectin (VECTOR Laboratories) was added to 20 mM PBS (pH 7.5) to obtain a WFA-containing solution (WFA concentration 2.5 mg / mL). 5-(N-succinimideoxycarbonyl)pentyl D-biotinamide (Biotin-AC5-Osu, Dojin Chemical Research Institute Co., Ltd.) was added to this WFA-containing solution as a biotinylated crosslinking agent at a WFA / crosslinking agent molar ratio of 1 / 100. The resulting solution was incubated at 25°C for 90 minutes to allow the WFA and crosslinking agent to react. The resulting reaction product was purified by HPLC to obtain biotinylated dimer WFA. In HPLC, a gel filtration column (TSK gel G3000SWXL, Higashi Thor Co., Ltd.) was used as the separation column, and PBS (pH 6.5) was used as the dissolution solvent. HISCL R2 reagent (Sysmex Co., Ltd.) was used as the suspension containing streptavidin-bound magnetic particles. Biotinylated dimer WFA and HISCL R2 reagent were mixed to a concentration of 20 μg / mL of biotinylated dimer WFA and reacted. The resulting reaction product was washed three times with 100 mM MES buffer (pH 6.5) to obtain magnetic particles immobilized with WFA dimer (hereinafter referred to as "WFA immobilized particles").

[0081] (1.3) Solution containing ALP-labeled anti-IgA antibody (Reagent 3)

[0082] As the anti-IgA antibody, an anti-human IgA antibody (SouthernBiotech) was used. This anti-IgA antibody was combined with bovine small intestine-derived ALP (ORIENTAL Yeast Co., Ltd.) using a maleimide cross-linking agent, and the resulting solution containing the ALP-labeled anti-IgA antibody was purified by affinity chromatography.

[0083] (1.4) Buffer solution (reagent 4) and matrix solution (reagent 5) for assay

[0084] HISCL R4 reagent (Sysmex Corporation) was used as the assay buffer. HISCL R5 reagent (Sysmex Corporation) containing CDP-Star (trademark) (Applied Biosystems) as the chemiluminescent substrate for ALP was used as the matrix solution.

[0085] 2. Modulation of calibrators

[0086] (2.1) Modulation of biotinylated IgA

[0087] Human IgA (Native Human IgA protein, Abcam) was used as the IgA monomer. Human IgA was added to 20 mM PBS (pH 7.5) to obtain an IgA solution. Biotin-AC5-Osu (Dojin Chemical Research Institute Co., Ltd.) was added to this IgA solution at a molar ratio of IgA / crosslinking agent of 1 / 100. The resulting solution was incubated at 25°C for 90 minutes to obtain biotinylated IgA.

[0088] (2.2) Modulation of peptides with glycans and biotin groups that bind to lectins

[0089] M2BP (Sysmex), MUC1 (Sysmex), and α2M (Sigma-Aldrich) were used as carrier proteins. Each carrier protein was added to 20 mM PBS (pH 7.5) to obtain a carrier protein solution. Biotin-AC5-Osu (Dojin Chemical Research Institute Co., Ltd.) was added to this solution at a carrier protein / crosslinking agent molar ratio of 1 / 100. The resulting solution was incubated at 25°C for 90 minutes to obtain the biotinylated carrier protein.

[0090] (2.3) Modulation of the biotinylated IgA and streptavidin complex

[0091] A solution containing biotinylated IgA and a solution containing streptavidin (Streptavidin SQ, Roche Diagnostics Co., Ltd.) were mixed and allowed to stand at room temperature for 1 hour to obtain a complex of biotinylated IgA and streptavidin (hereinafter also referred to as "IgA-Bio-STA-Bio-IgA").

[0092] (2.4) Modulation of the complex of biotinylated IgA, streptavidin and biotinylated carrier protein

[0093] Solutions of each biotinylated carrier protein were mixed with a solution containing biotinylated IgA. The resulting mixture was then mixed with a solution containing streptavidin (Streptavidin SQ, Roche Diagnostics, Inc.), and allowed to stand at room temperature for 1 hour to obtain a complex of biotinylated IgA, biotinylated carrier protein, and streptavidin (0.2 mg / mL). Hereinafter, the complex containing M2BP will be referred to as "M2BP-Bio-STA-Bio-IgA", the complex containing MUC1 will be referred to as "MUC1-Bio-STA-Bio-IgA", and the complex containing α2M will be referred to as "A2M-Bio-STA-Bio-IgA".

[0094] 【3. Determination of calibrators (1)】

[0095] Using reagents 1 through 5 described above, and with the HISCL 5000 (Sysmex Co., Ltd.) fully automated immunoassay apparatus as calibrators, the individual values ​​of M2BP-Bio-STA-Bio-IgA, MUC1-Bio-STA-Bio-IgA, and A2M-Bio-STA-Bio-IgA were measured. The measurement procedure using the HISCL-5000 is as follows: After mixing the calibrator (30 μL) and reagent 1 (100 μL), reagent 2 (30 μL) was added. The magnetic particles in the resulting mixture were magnetically collected, and the supernatant was removed. The magnetic particles were then washed with HISCL washing buffer (300 μL). After removing the supernatant, reagent 3 (100 μL) was added to the magnetic particles and mixed. The magnetic particles in the resulting mixture were magnetically collected, and the supernatant was removed. The magnetic particles were then washed with HISCL washing buffer (300 μL). After removing the supernatant, reagent 4 (50 μL) and reagent 5 (100 μL) were added to the magnetic particles to measure the chemiluminescence intensity. As controls, streptavidin (STA) and each biotinylated carrier protein (M2BP-Bio, MUC1-Bio, and A2M-Bio) were measured similarly. The measured values ​​(signal counts) are shown in Table 1.

[0096] Table 1

[0097] Calibration material count STA 531 M2BP-Bio 532 MUC1-Bio 542 A2M-Bio 1,279 M2BP-Bio-STA-Bio-IgA 144,375 MUC1-Bio-STA-Bio-IgA 201,568 A2M-Bio-STA-Bio-IgA 659,882

[0098] As shown in Table 1, significantly high signal counts were obtained when measuring M2BP-Bio-STA-Bio-IgA, MUC1-Bio-STA-Bio-IgA, and A2M-Bio-STA-Bio-IgA. This indicates that M2BP-Bio-STA-Bio-IgA, MUC1-Bio-STA-Bio-IgA, and A2M-Bio-STA-Bio-IgA can be used as calibrators for immunoassays using lectins and anti-IgA antibodies.

[0099] 【4. Determination of calibrators (2)】

[0100] M2BP-Bio-STA-Bio-IgA, MUC1-Bio-STA-Bio-IgA, A2M-Bio-STA-Bio-IgA, and IgA-Bio-STA-Bio-IgA were used as calibrators. Measurements were performed in the same manner as described above, except that each calibrator was diluted 5-, 25-, and 125-fold with 20 mM PBS (pH 7.5). The measured values ​​(signal counts) are shown in Table 2.

[0101] Table 2

[0102]

[0103] As shown in Table 2, the measured values ​​vary with the dilution ratio regardless of the calibrator used. This indicates that M2BP-Bio-STA-Bio-IgA, MUC1-Bio-STA-Bio-IgA, and A2M-Bio-STA-Bio-IgA can be used as calibrators for quantifying the concentration of IgA agglutinates in immunoassays using lectins and anti-IgA antibodies.

[0104] [Explanation of Symbols]

[0105] 10, 20, 30: Calibrators

[0106] 11, 21, 31: Container 1

[0107] 12, 23, 34: Bundled boxes

[0108] 13, 24, 35: Accompanying documents

[0109] 22, 32: Second container

[0110] 33: The 3rd container

Claims

1. A calibrator for obtaining a concentration of IgA aggregates in a sample, comprising: IgA having a biotin group, an avidin, and a polypeptide having a sugar chain bound to a lectin and a biotin group, wherein both the IgA and the polypeptide are bound to the avidin through binding of the biotin group and the avidin.

2. The calibrator according to claim 1, which is a reagent set comprising: a reagent containing the IgA having a biotin group, a reagent containing the avidin, and a reagent containing the polypeptide.

3. The calibrator according to claim 1, which is a reagent containing a complex of the IgA having a biotin group, the avidin, and the polypeptide.

4. A complex for obtaining a concentration of IgA aggregates in a sample, comprising: IgA having a biotin group, an avidin, and a polypeptide having a sugar chain bound to a lectin and a biotin group, wherein both the IgA and the polypeptide are bound to the avidin through binding of the biotin group and the avidin.

5. A method for measuring IgA aggregates in a sample using a lectin, comprising obtaining a concentration of IgA aggregates in the sample using a calibrator, wherein the calibrator comprises: IgA having a biotin group, an avidin, and a polypeptide having a sugar chain bound to a lectin and a biotin group, wherein both the IgA and the polypeptide are bound to the avidin through binding of the biotin group and the avidin.

6. The method according to claim 5, wherein the calibrator is a reagent containing a complex of: the IgA having a biotin group, the avidin, and the polypeptide.

7. The method according to claim 6, wherein the calibrator is a reagent set comprising a plurality of reagents containing the complex, and wherein concentrations of the complex in the plurality of reagents are different from each other.

8. The method according to claim 5, wherein the polypeptide contains all or a part of at least one glycoprotein selected from the group consisting of M2BP, MUC1, and α2M.

9. The method according to claim 6 or 7, comprising: a step of obtaining a measured value of the IgA aggregates using the sample, the lectin, and a capturer containing a labeling substance and specifically binding to the IgA aggregates, a step of obtaining a measured value of the complex using the calibrator, the lectin, and the capturer, and a step of obtaining a concentration of IgA aggregates in the sample from the measured value of the IgA aggregates and the measured value of the complex.

10. The method according to claim 9, wherein in the step of obtaining a concentration of IgA aggregates, a calibration curve is made from the measured value of the complex, and a concentration of IgA aggregates in the sample is obtained based on the calibration curve and the measured value of the IgA aggregates.

11. The method according to claim 5, wherein the lectin is immobilized on a solid phase.

12. The method according to claim 5, wherein the IgA is IgAl. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 13. The method of claim 5, wherein the lectin is at least one lectin selected from the group consisting of WFA, HHL, GNA, NPA, SBA, VVA, BPL, TJA-II, PHA-L, and AOL.

14. The method of claim 5, wherein the lectin is WFA.

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