Immunoassay method, accuracy management method, standard sample, and immunoassay reagent

Abstract

This immunoassay method comprises: a step for detecting a signal corresponding to the concentration or titer of an antigen contained in a biological sample; and a step for calculating the concentration or titer of the antigen in the biological sample from the signal by using a calibration curve indicating the correlation between the signal and the concentration or titer of the antigen. The calibration curve is created using a standard sample containing a fusion protein. The fusion protein has an amino acid sequence of at least a portion of the antigen and an amino acid sequence of at least a portion of the constant domain of immunoglobulin.

Description

Immunoassay method, quality control method, standard sample and immunoassay reagent

[0001] The present invention relates to an immunoassay method, a quality control method, a standard sample, and an immunoassay reagent. This application claims priority to Japanese Patent Application No. 2024-047613, filed on March 25, 2024, the contents of which are incorporated herein by reference.

[0002] To quantify the target components in an immunoassay in a biological sample, a calibration sample must be used. A calibration sample is a sample containing the target components and is used as an internal standard or a standard for concentration calibration (calibrator). To obtain accurate quantitative values ​​of the target components, a calibration sample that is stable over time and with temperature changes is required.

[0003] Such calibration samples include those in which the antigen to be measured itself is purified from a biological sample, or proteins expressed by genetic recombination. For example, Patent Document 1 discloses a calibration sample solution that is a liquid composition containing a pulmonary surfactant protein.

[0004] International Publication No. 2023 / 145916

[0005] When purifying antigens themselves from biological samples as proteins contained in calibration samples in immunoassays, the purification process can be complicated. Furthermore, when expressing proteins through genetic recombination, sufficient expression levels may not be obtained depending on the type of antigen.

[0006] Thus, there is room for further improvement in the standard samples used in immunoassays.

[0007] An object of the present invention is to provide an immunoassay method, a quality control method, and a standard sample and an immunoassay reagent to be used therein.

[0008] The present invention encompasses the following aspects: [1] An immunoassay method comprising the steps of: detecting a signal corresponding to the concentration or titer of an antigen contained in a biological sample; and calculating the concentration or titer of the antigen in the biological sample from the signal using a calibration curve showing the relationship between the concentration or titer of the antigen and the signal, wherein the calibration curve is prepared using a standard sample containing a fusion protein, and the fusion protein contains at least a partial amino acid sequence of the antigen and at least a partial amino acid sequence of an immunoglobulin constant region. [2] The immunoassay method according to [1], wherein at least a partial amino acid sequence of the immunoglobulin constant region is derived from a rabbit antibody. [3] The immunoassay method according to [1] or [2], wherein the antigen is a protein that exists as a multimer in vivo. [4] The immunoassay method according to [1] or [2], wherein the antigen is at least one of P1NP, procalcitonin, SP-D, and TARC. [5] The immunoassay method according to [4], wherein the antigen is P1NP. [6] A quality control method for antigen immunoassay, comprising the steps of: detecting a signal corresponding to the concentration or titer of a fusion protein in a standard sample containing a fusion protein at a predetermined concentration or titer; calculating the concentration or titer of the fusion protein from the detected signal; and evaluating the accuracy of the immunoassay by comparing the calculated concentration or titer with the predetermined concentration or titer, wherein the fusion protein contains at least a partial amino acid sequence of the antigen and at least a partial amino acid sequence of an immunoglobulin constant region. [7] The quality control method for antigen immunoassay according to [6], wherein at least a partial amino acid sequence of the immunoglobulin constant region is derived from a rabbit antibody. [8] The quality control method for antigen immunoassay according to [6] or [7], wherein the antigen is a protein that exists as a multimer in vivo. [9] The quality control method for antigen immunoassay according to [6] or [7], wherein the antigen is one of P1NP, PCT, SP-D, and TARC.

[10] The method for controlling the accuracy of immunoassays of antigens according to [9], wherein the antigen is P1NP.

[11] A standard sample used in the immunoassay method according to any one of [1] to [5] or the method for controlling the quality of immunoassay according to any one of [6] to

[10] , the standard sample containing the fusion protein of at least a part of the amino acid sequence of the antigen and at least a part of the amino acid sequence of the constant region of the immunoglobulin at a known concentration or a known titer.

[12] An immunoassay method comprising the steps of forming a complex between an antibody and the fusion protein in a solution and detecting a signal emitted from a labeling substance bound to the fusion protein, the fusion protein being a fusion protein of at least a part of the amino acid sequence of an antigen capable of binding to the antibody and at least a part of the amino acid sequence of the constant region of immunoglobulin.

[13] An immunoassay method comprising the steps of: obtaining a reaction system in which a biological sample possibly containing an antigen, an antibody capable of binding to the antigen, and a competitor capable of competitively binding to the antigen with the antibody and bound to a labeling substance, are present; and measuring a signal from the labeling substance contained in the reaction system, wherein the competitor is a fusion protein of at least a portion of the amino acid sequence of the antigen and at least a portion of the amino acid sequence of an immunoglobulin constant region.

[14] The immunoassay method according to

[13] , further comprising, between the steps of obtaining the reaction system and measuring the signal, a step of removing by washing the antigen and the competitor that are not bound to the antibody while leaving the antibody in the reaction system, thereby obtaining an assay system containing the antigen bound to the antibody and the competitor.

[15] The immunoassay method according to

[12] or

[13] , wherein at least a portion of the amino acid sequence of the immunoglobulin constant region is derived from a rabbit antibody.

[16] The immunoassay method according to any one of

[12] to

[15] , wherein the antigen is a protein that exists as a multimer in vivo.

[17] The immunoassay method according to any one of

[12] to

[15] , wherein the antigen is one of P1NP, PCT, SP-D, and TARC.

[18] The immunoassay method according to

[17] , wherein the antigen is P1NP.

[19] An immunoassay reagent comprising the fusion protein, for use in the immunoassay method according to any one of

[12] and

[14] to

[18] .

[20] An immunoassay reagent for use in the immunoassay method according to any one of

[13] to

[18] , comprising the competitive substance.

[0009] According to the above aspects, it is possible to provide an immunoassay method, a quality control method, and a standard sample and an immunoassay reagent used therein.

[0010] Figure 1 is a graph showing the correlation between P1NP measurement values ​​calculated from a calibration curve prepared using a calibrator prepared using P1NP-RbFc and P1NP measurement values ​​obtained using the procollagen intact P1NP reagent. Figure 2 is a graph showing the correlation between P1NP measurement values ​​calculated from a calibration curve prepared using a calibrator prepared using P1NP in human serum and P1NP measurement values ​​obtained using the procollagen intact P1NP reagent.

[0011] Hereinafter, one embodiment of the present invention will be described with reference to the drawings. In this specification and claims, when a numerical range is expressed using "to", the numerical range includes the numerical values ​​on both sides of "to".

[0012] The biological sample referred to herein mainly refers to a body fluid derived from a living organism. Examples of biological samples include blood, serum, plasma, nasopharyngeal swabs, nasal swabs, saliva, sputum, and amniotic fluid, with serum and plasma being preferred. Subjects from which biological samples are collected include humans or animals (e.g., monkeys, dogs, or cats), preferably humans. The biological sample may be a sample directly from the subject, or may be a sample that has been subjected to a commonly performed treatment such as dilution or concentration. The person who collects and prepares the biological sample used in the present invention may be the same person as the person who performs the measurement method of the present invention, or may be a different person. Furthermore, the biological sample used in the measurement method of the present invention may be collected or prepared at the time of carrying out the measurement method of the present invention, or may be collected or prepared in advance and stored.

[0013] <Immunoassay method and standard sample> An immunoassay method in one aspect of the present invention includes the steps of: detecting a signal corresponding to the concentration or titer of an antigen contained in a biological sample; and calculating the concentration or titer of the antigen in the biological sample from the signal using a calibration curve showing the relationship between the concentration or titer of the antigen and the signal, wherein the calibration curve is prepared using a standard sample containing a fusion protein, and the fusion protein contains at least a portion of the amino acid sequence of the antigen and at least a portion of the amino acid sequence of an immunoglobulin constant region.

[0014] In one aspect of the present invention, a standard sample is used in an immunoassay method, and contains the fusion protein of at least a portion of the amino acid sequence of the antigen and at least a portion of the amino acid sequence of the constant region of the immunoglobulin at a known concentration or titer. The concentration of the fusion protein can be calculated using the BCA method, the Bradford method, the Lowry method, or an existing test reagent for measuring the antigen contained in the fusion protein. The titer of the fusion protein can be calculated by protease titer measurement, etc.

[0015] The standard sample can be used as a calibrator to prepare a calibration curve for a clinical diagnostic reagent, and can also be used as a control to be measured for quality control of the clinical diagnostic reagent.

[0016] The fusion protein contains an amino acid sequence identical to or a part of an antigen to be measured in an immunoassay, such as brain natriuretic peptide (BNP), C-reactive protein (CRP), D-dimer, heart-type fatty acid binding protein (H-FABP), Krebs von der Lungen Nr. 6 (KL-6), LDL receptor relative with 11 ligand-binding repeats (LR11), matrix metalloproteinase-3 (MMP-3), N-terminal pro-brain natriuretic peptide (NT-proBNP), type I procollagen-N-propeptide (P1NP), procalcitonin (PCT), protein induced by vitamin K absorption or antagonist-II (PIVKA-II), prostate-specific antigen (PSA), pulmonary surfactant protein D (SP-D), soluble interleukin-2 receptor (sIL-2R), Th2 chemokine (TARC), and cardiac troponin. The antigen may be at least one of P1NP, PCT, SP-D, and TARC. The antigen to be measured is preferably an oligopeptide consisting of about 30 to 1,000 amino acids or a complex thereof, and more preferably an oligopeptide consisting of about 50 to 700 amino acids or a complex thereof.

[0017] When a fusion protein contains an amino acid sequence identical to a portion of an antigen to be measured, the fusion protein preferably contains at least an amino acid sequence identical to an epitope of the antigen to be measured. When a fusion protein contains an amino acid sequence identical to a portion of an antigen to be measured, the "portion of the antigen to be measured" may be one portion or multiple portions. That is, the fusion protein may contain multiple amino acid sequences identical to the sequence of a portion of the antigen to be measured. For example, when the antigen to be measured is IL-2R, LR11, P1NP, or the like, the fusion protein preferably contains an amino acid sequence identical to a portion of the antigen to be measured. For example, the fusion protein may contain an amino acid sequence identical to the α1-1 sequence and an amino acid sequence identical to the α1-2 sequence of the α1 chain of P1NP.

[0018] The antigen to be measured may be a protein that exists as a multimer in vivo. The antigen to be measured may exist as a homomultimer or a heteromultimer in vivo. For example, the antigen to be measured may be IL-2R, P1NP, SP-D, etc.

[0019] The fusion protein comprises at least a portion of the amino acid sequence of an immunoglobulin constant region. Examples of immunoglobulins include IgG, IgA, IgM, IgD, and IgE. Preferably, at least a portion of the amino acid sequence of the immunoglobulin constant region is at least a portion of the amino acid sequence of an IgG constant region. Preferably, at least a portion of the amino acid sequence of the immunoglobulin constant region is derived from a rabbit antibody. When the fusion protein comprises at least a portion of the amino acids of an immunoglobulin constant region, a sufficient amount of the fusion protein is expressed in the culture supernatant of transformed cells, even when expressed using a common plasmid.

[0020] The fusion protein of this embodiment preferably further comprises an affinity tag for protein purification. Examples of affinity tags include Histag, which is a sequence of 6 to 9 consecutive histidine residues, FLAG-tag, Spot-tag, C-tag, and Strep-tag.

[0021] The fusion protein of this embodiment may further contain a linker for the purpose of improving the reactivity between the fusion protein and the antibody. The length and sequence of the linker are appropriately selected taking into consideration the sequences of the antigen and the constant region of the immunoglobulin. For example, the linker may be 2 to 20 residues long. The linker may be positioned between the affinity tag and a portion of the antigen to be measured, or between a portion of the antigen to be measured and at least a portion of the constant region of the immunoglobulin. Furthermore, when the fusion protein contains an amino acid sequence identical to the amino acid sequence of multiple positions of a portion of the antigen to be measured, a linker may be positioned between the amino acids.

[0022] The fusion protein of this embodiment preferably comprises at least a portion of the amino acid sequence of an immunoglobulin constant region at the C-terminus or N-terminus of the amino acid sequence of the antigen to be measured. Inclusion of at least a portion of the amino acid sequence of an immunoglobulin constant region at the C-terminus or N-terminus of the amino acid sequence of the antigen to be measured is preferred because it allows the N-terminus or C-terminus of the antigen amino acid sequence to be exposed, and does not impair the reactivity between the antigen and the antibody when the antibody recognizes the N-terminus or C-terminus of the antigen.

[0023] The fusion protein of this embodiment can be obtained by introducing into cells an expression vector containing a signal sequence and a base sequence encoding the fusion protein and allowing the cells to express the fusion protein. The signal peptide translated from the signal sequence is degraded and removed during the process of extracellular secretion of the fusion protein. Because the fusion protein of this embodiment is expressed in large amounts extracellularly, it can be easily recovered from cell culture medium and is highly productive.

[0024] In the antigen immunoassay method of this embodiment, a calibration curve is used that is prepared by measuring the concentration or titer of the fusion protein contained in the above-mentioned standard sample. The calibration curve can be prepared by a general method. For example, it can be prepared by plotting the signal when measuring the standard sample or the signal change per unit time on the vertical axis and the concentration of the antigen contained in the standard sample on the horizontal axis. The calibration curve may be prepared by the person performing the immunoassay, or a calibration curve that has been prepared in advance may be used.

[0025] In this embodiment, a signal corresponding to the concentration or titer of an antigen to be measured contained in a biological sample is detected, and the concentration or titer of the antigen in the biological sample is calculated from the signal using a calibration curve showing the correlation between the antigen concentration or titer and the detected signal. For example, the signal may be transmitted light, scattered light, fluorescence, chemiluminescence, electrochemiluminescence, or coloration.

[0026] The immunoassay of this embodiment is not particularly limited, but examples thereof include LTIA (latex immunoturbidimetry), ELISA (enzyme-linked immunosorbent assay), electrochemiluminescence immunoassay, and immunochromatography.

[0027] (LTIA) LTIA is an immunoassay method that utilizes latex particle agglutination, which occurs when an antibody supported on the surface of the latex particle binds to an antigen to be measured. The latex particles are not particularly limited as long as they are latex particles commonly used in in vitro diagnostics. The concentration and average particle size of the latex particles during agglutination reaction measurement can be appropriately set depending on the sensitivity or performance. When using latex turbidimetric immunoassay as the immunoassay method of this embodiment, the measurement procedure and principle are as follows: (1) The sample is contacted with latex particles supporting antibodies. (2) The antigen in the sample forms a complex with at least two antibodies, causing the latex particles to agglutinate. (3) The sample is irradiated with near-infrared light (e.g., a wavelength of 600 nm), and the transmitted light or scattered light (i.e., the signal) is measured. The concentration or titer of the antigen is calculated based on the calibration curve created using the standard sample of this embodiment and the measured signal.

[0028] As the measuring device, a biochemical automatic analyzer (for example, Hitachi Automatic Analyzer 3500 manufactured by Hitachi Ltd.) can be used.

[0029] (ELISA) As used herein, ELISA refers to a method in which an antigen to be measured contained in a sample is captured using an antibody and then detected using an enzyme reaction. A plate (also called an immunoplate) is preferably used as the solid phase. HRP or ALP can be used as the label. When sandwich ELISA is used as the immunoassay method of this embodiment, the measurement procedure and principle are as follows: (1) A sample is contacted with a solid phase on which an antibody is immobilized, and the antigen in the sample is allowed to bind to the solid-phase antibody. (2) A labeled antibody, i.e., a labeled antibody, is added to the solid phase and reacted to form a solid-phase antibody-antigen-labeled antibody complex, i.e., a sandwich, in which the labeled antibody binds to the antigen. (3) After washing, the labeled substance is allowed to develop color, and the absorbance (i.e., signal) is measured. The concentration or titer of the antigen is calculated based on the calibration curve created using the standard sample of this embodiment and the measured signal.

[0030] A secondary antibody can also be used in the sandwich ELISA method. By using a secondary antibody, the reaction can be amplified and detection sensitivity can be increased. When a secondary antibody is used, the following procedures (1) to (5) can be adopted: (1) A sample is added to a solid phase on which an antibody is immobilized, followed by incubation, and then the sample is removed and washed. (2) An antibody capable of binding to the antigen to be measured is added, followed by incubation and washing. (3) An enzyme-labeled secondary antibody is then added and incubated. (4) A substrate is added to develop color. (5) The developed color (i.e., the signal) is measured using a plate reader or the like. The concentration or titer of the antigen is calculated based on the calibration curve created using the standard sample of this embodiment and the measured signal value.

[0031] (Electrochemiluminescence Immunoassay) Electrochemiluminescence immunoassay refers to a method for measuring the amount of an antigen to be measured contained in a sample by passing an electric current through a labeling substance to cause it to emit light and then detecting the amount of light emitted (i.e., the signal). In electrochemiluminescence immunoassay, a ruthenium complex can be used as the labeling substance. An electrode is placed on a solid phase (such as a microplate) and radicals are generated on this electrode, which excites the ruthenium complex and causes it to emit light. The amount of light emitted by this ruthenium complex can then be detected.

[0032] The measurement procedure and principle when magnetic particles are used as the solid phase and a ruthenium complex is used as the labeling substance are as follows.

[0033] (1) A sample is contacted with magnetic particles on which a solid-phase antibody has been immobilized, and the antigen in the sample is allowed to bind to the solid-phase antibody. (2) After washing the magnetic particles, a labeled antibody is contacted with the magnetic particles, and the labeled antibody is allowed to bind to the antigen bound to the magnetic particles. (3) After washing the magnetic particles, an electric current is applied, and the amount of luminescence corresponding to the amount of labeled antibody bound to the antigen is measured, thereby measuring the amount of antigen in the biological sample.

[0034] (Immunochromatography) Immunochromatography is an immunoassay method that utilizes the property of labeled antibodies bound to antigens or the flow of labeled antibodies across a membrane. The general principle of immunochromatography for measuring antigens contained in a sample is as follows: An antibody against the antigen is immobilized on an insoluble membrane carrier, which serves as a chromatographic medium, to create a detection section, which serves as a stationary phase. A conjugate (a labeled substance sensitized with an antibody capable of binding to the antigen) is then used as the mobile phase. The analyte is specifically reacted with the conjugate, which serves as the mobile phase. Furthermore, in the detection section, which serves as a stationary phase, the analyte bound to the conjugate is specifically reacted with the antibody immobilized on the detection section. Signals such as coloration or fluorescence derived from the labeled substance bound to the detection section via the analyte are detected to determine whether the sample contains an antigen or the antigen concentration in the sample. Examples of labeled substances include colloidal gold particles, colloidal platinum particles, colored latex particles, and magnetic particles. Colloidal gold particles are preferred. Those skilled in the art can appropriately adjust the type and particle size of these labeled substances according to the desired sensitivity.

[0035] The measurement procedure and principle when immunochromatography is used as the immunoassay method of the present invention are as follows: (1) The sample is brought into contact with a sample supply unit for supplying the sample. (2) The antigen in the sample is brought into contact with a conjugate to form a first complex. The conjugate is a first monoclonal antibody bound to a labeled substance. (3) The first complex is brought into contact with a second monoclonal antibody immobilized on a detection unit to form a second complex. (4) The amount or presence of the antigen in the sample is measured by measuring the intensity of the signal derived from the labeled substance contained in the conjugate.

[0036] According to the immunoassay method of this embodiment, the standard sample of this embodiment is used, and therefore high measurement accuracy equivalent to that of a substance purified from a biological sample can be obtained.

[0037] Furthermore, since the standard sample of this embodiment contains the fusion protein containing at least a portion of the amino acid sequence of the constant region of immunoglobulin, a sufficient amount of the standard sample is expressed in the culture supernatant of transformed cells even when expressed using a general plasmid. Furthermore, the use of the standard sample of this embodiment provides high measurement accuracy equivalent to that of a standard sample produced using a biological sample.

[0038] <Immunoassay method and immunoassay reagent> In another aspect of this embodiment, the immunoassay method includes the steps of forming a complex between an antibody and a fusion protein in a solution and detecting a signal emitted from a labeling substance bound to the fusion protein, wherein the fusion protein is a fusion protein of at least a partial amino acid sequence of an antigen capable of binding to the antibody and at least a partial amino acid sequence of an immunoglobulin constant region. The labeling substance may be bound to the fusion protein in advance, or the labeling substance may be bound to the antibody in advance, and the fusion protein and the labeling substance may be bound via the antibody when the antibody and the fusion protein form a complex.

[0039] In yet another aspect of this embodiment, the immunoassay method includes the steps of obtaining a reaction system in which a biological sample that may contain an antigen, an antibody capable of binding to the antigen, and a competitor capable of binding to the antigen competitively with the antibody and to which a labeling substance is bound, and measuring a signal from the labeling substance contained in the measurement system, wherein the competitor is a fusion protein of at least a partial amino acid sequence of the antigen and at least a partial amino acid sequence of an immunoglobulin constant region. The immunoassay method may include, after the step of obtaining the reaction system, a step of removing the antigen and the competitor that are not bound to the antibody by washing, while leaving the antibody in the reaction system.

[0040] In this immunoassay method, an antibody capable of binding to the antigen to be measured is used. The above-mentioned fusion protein is capable of binding to this antibody and functions as a competitor to the antigen to be measured. The fusion protein of this embodiment may be bound to an enzyme that reacts with a substrate to emit a signal. The other components of the fusion protein are the same as those of the standard sample described in <Immunoassay method and standard sample>, so a description thereof will be omitted.

[0041] The immunoassay method of this embodiment is not particularly limited as long as it is a method that can detect a target substance using the competitive substance of this embodiment, and examples include competitive ELISA, LTIA, electrochemiluminescence immunoassay, and immunochromatography.

[0042] The competitive ELISA method can employ the following steps (1) to (5): (1) A reaction system is obtained in which a sample and the competitor of this embodiment are present on a solid phase on which an antibody is immobilized, and then the reaction system is incubated. (2) The antigen and the competitor that are not bound to the antibody are removed by washing, while the antibody remains in the reaction system, to obtain a measurement system containing the antigen and the competitor that are bound to the antibody. (3) A substrate is added to develop color. (4) The color development (i.e., the signal) of the labeling substance contained in the measurement system is measured using a plate reader or the like. (5) The concentration or titer of the antigen is calculated based on the calibration curve and the measured signal value.

[0043] The antigen to be measured is not particularly limited, but examples include IL-2R, LR11, NT-proBNP, P1NP, PCT, PSA, SP-D, and TARC.

[0044] By using an immunoassay reagent containing the fusion protein of this embodiment in an immunoassay method, the antigen that is a component of the reagent can be produced stably and easily.

[0045] <Method for controlling accuracy of immunoassay of antigen> The method for controlling accuracy of immunoassay of antigen of the present embodiment is a method for controlling accuracy of immunoassay of antigen, comprising the steps of: detecting a signal corresponding to the concentration or titer of a fusion protein of a predetermined concentration or titer for a standard sample containing the fusion protein; calculating the concentration or titer of the fusion protein from the detected signal; and comparing the calculated concentration or titer with the predetermined concentration or titer to evaluate the accuracy of the immunoassay, wherein the fusion protein contains at least a portion of the amino acid sequence of the antigen and at least a portion of the amino acid sequence of an immunoglobulin constant region.

[0046] In the quality control method for antigen immunoassay, the standard sample is used as a control for quality control of clinical diagnostic reagents. The composition of the fusion protein contained in the standard sample is the same as that described in <Immunoassay method and standard sample>, so its description is omitted.

[0047] One aspect of the quality control method for antigen immunoassays of this embodiment is a method for controlling the measurement accuracy of clinical diagnostic reagents and clinical testing devices. Specifically, a standard sample is prepared to contain a fusion protein at a predetermined concentration or titer. Then, a standard sample of known concentration and titer and a clinical diagnostic reagent are used to detect a signal corresponding to the concentration or titer of the fusion protein. The concentration or titer of the fusion protein is calculated from the detected signal. The calculated concentration or titer is compared with the previously set concentration or titer to evaluate the accuracy of the immunoassay and control whether the clinical diagnostic reagent, clinical testing device, etc. can perform accurate measurements.

[0048] The immunoassay to which the quality control method of this embodiment can be applied is not particularly limited as long as it is a method that can be quality controlled using the standard sample of this embodiment, and examples include LTIA, ELISA, electrochemiluminescence immunoassay, and immunochromatography.

[0049] The antigens to be measured in the quality control method of this embodiment are not particularly limited, but examples include P1NP, PCT, SP-D, and TARC.

[0050] According to the method for controlling the accuracy of antigen immunoassay of this embodiment, the standard sample of this embodiment is used, so that it is possible to perform control with high accuracy equivalent to that of a substance purified from a biological sample.

[0051] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the examples described below.

[0052] <Plasmids> Plasmids used for expressing fusion proteins were prepared by inserting into pcDNA3.1(-) Mammalian Expression Vector (ThermoFisher SCIENTIFIC) a signal sequence (SEQ ID NO: 1) shown in Table 1, the entire or partial sequence of an antigen, Histag, rabbit antibody Fc (RbFc), and a base sequence encoding an appropriate linker (Linker-Histag-1 (SEQ ID NO: 2) or Linker-Histag-2 (SEQ ID NO: 3), and Linker-RbFc sequence (SEQ ID NO: 4)). Plasmids for expressing fusion antigens of NT-proBNP (SEQ ID NO: 7), PCT (SEQ ID NO: 9), PSA (SEQ ID NO: 10), SP-D (SEQ ID NO: 11), and TARC (SEQ ID NO: 12) contained base sequences encoding the entire antigen sequences. The nucleotide sequence (SEQ ID NO: 5) encoding the extracellular domain sequence was used for the plasmid used to express the fusion antigen of IL-2R. The nucleotide sequence (SEQ ID NO: 6) encoding a partial sequence of the antigen was used for the plasmid used to express the fusion antigen of LR11. The nucleotide sequence (SEQ ID NO: 8) encoding a peptide in which two amino acid sequences in the α1 chain are linked by a linker was used for the plasmid used to express the fusion antigen of P1NP. The respective nucleotide sequences are shown in Tables 1 to 4.

[0053]

[0054]

[0055]

[0056]

[0057] Table 5 shows the combination of base sequences inserted into each fusion protein expression plasmid.

[0058]

[0059] The plasmids used for the comparative experiments were a plasmid containing a nucleotide sequence (Linker-HSA (SEQ ID NO: 13)) encoding HSA instead of the rabbit antibody Fc shown in Table 6, and a plasmid not containing a sequence encoding rabbit antibody Fc. Table 7 shows the combinations of nucleotide sequences inserted into each fusion protein expression plasmid or Histag-binding protein expression plasmid.

[0060]

[0061]

[0062] <Expression of fusion proteins> For expression of fusion proteins, an ExpiCHO Expression System Kit (manufactured by Thermo Fisher Scientific, product number: A29133) was used, and the fusion proteins of interest were expressed according to the product protocol. More specifically, Expi CHO cells were cultured in a dedicated medium at a density of 3 to 4 × 10 ６ The following day, the plasmid DNA adjusted to 1 μg / μL was mixed with OptiPro medium, and then further mixed with a separately prepared mixture of OptiPro medium and ExpiFectanube CHO reagent, and the mixture was allowed to stand at room temperature for 1 to 5 minutes. The resulting mixture was diluted to 7 to 10 × 10 ６ The solution was added to ExpiCHO cells seeded at 1000 cells / mL and cultured. The next day, ExpiCHO Enhancer and ExpiCHO FeED were added to the cells in the amounts indicated in the protocol, and the cells were recultured. After 8 days, the supernatant was collected and used for each experiment.

[0063] Obtaining Antigen-Binding Monoclonal Antibodies Antibodies reactive with each antigen were prepared according to the method described in International Publication No. 2023 / 182353. BALB / c mice were immunized with each antigen, and spleen cells, iliac lymph node cells, and inguinal lymph node cells were collected from mice in which an increase in antibody titer in the blood was confirmed. The collected cells were fused with SP2 / 0 myeloma cells by electrofusion. The fused cells were cultured in a 96-well plate, and the culture supernatant was collected. Cell lines producing antibodies reactive with each antigen were selected by antigen solid-phase ELISA using each antigen as an immunogen. Antibody-containing ascites was collected from mice administered with the selected antibody cell lines, and the obtained ascites was purified using a protein A column or protein G column to obtain monoclonal antibodies reactive with each antigen. Specifically, the following antibodies were obtained: anti-IL-2R antibody abIL-2R, anti-LR11 antibody abLR11, anti-NT-proBNP antibody abNT-proBNP, anti-P1NP antibodies abP1NP-1 and abP1NP-2, anti-PCT antibody abPCT, anti-PSA antibody abPSA, anti-SP-D antibody abSP-D, and anti-TARC antibody abTARC. Each monoclonal antibody was biotinylated using Biotin Labeling Kit-NH2 (Dojindo Laboratories) according to a standard method, to obtain each biotinylated monoclonal antibody.

[0064] <Measurement of fusion protein expression level> The reactivity of the fusion protein in the culture supernatant with antibodies reactive with each antigen was evaluated. More specifically, 50 μL of antibodies reactive with each antigen diluted to 5 μg / mL with PBS was dispensed into each well of a 96-well ELISA plate and allowed to stand overnight at 4°C. The anti-P1NP antibody used was abP1NP-1. After washing each well three times with PBS containing 0.05% Tween 20 (hereinafter referred to as washing solution), 100 μL of PBS containing 1% BSA and 0.05% Tween 20 (hereinafter referred to as blocking solution) was dispensed into each well and allowed to stand overnight at 4°C. After removing the blocking solution from each well, 50 μL of the culture supernatant of each fusion protein diluted 5-fold with blocking solution was dispensed into each well and allowed to stand at room temperature for 1 hour. After washing each well three times with washing buffer, 50 μL of biotinylated monoclonal antibody solution reactive with each antigen, adjusted to 1 μg / mL with blocking buffer, was dispensed into each well and allowed to stand at room temperature for 1 hour. After washing each well three times with washing buffer, 50 μL of Streptavidin, Horseradish Peroxidase Conjugate (Thermo Fisher Scientific) adjusted to 0.2 μg / mL with blocking buffer was dispensed into each well and allowed to stand at room temperature for 30 minutes. After washing each well three times with washing buffer, 50 μL of OPD color development solution was dispensed into each well and allowed to stand at room temperature for 10 minutes. 50 μL of stop solution was dispensed into each well, and the absorbance at a wavelength of 492 nm was measured using a plate reader. The absorbance measurement results are shown in Table 8. In Table 8, "antigen" refers to the antigen contained in the fusion protein, and "fused protein or peptide" refers to the protein or peptide contained in the fusion protein. For example, the numerical value of the intersection point between IL-2R and RbFc, 3.934, indicates the absorbance value when a fusion protein containing IL-2R and RbFc is reacted with an antibody.

[0065]

[0066] When each antigen was expressed as a fusion protein with RbFc, a high signal derived from the reaction between the fusion protein in the culture supernatant and antibodies reactive with each antigen was confirmed. It was suggested that the RbFc fusion protein had a high expression level in the culture supernatant and good reactivity with antibodies reactive with each antigen. When LR11, P1NP, PCT, SP-D, and TARC were expressed as fusion proteins with HSA, the signal derived from the reaction between the fusion protein in the culture supernatant and antibodies reactive with each antigen was lower than in the case of the RbFc fusion protein. It is considered that the HSA fusion protein has a lower expression level in the culture supernatant or a lower reactivity with antibodies reactive with each antigen than the RbFc fusion protein. When PCT, PSA, or TARC was expressed in conjunction with Histag, the signal derived from the reaction between the fusion protein in the culture supernatant and antibodies reactive with each antigen was lower than in the case of the RbFc fusion protein. It is thought that the Histag-binding protein is expressed in lower amounts in the culture supernatant than the RbFc fusion protein, or that it has lower reactivity with antibodies that react with each antigen. From the above, it was found that various antigens can be stably expressed by fusing RbFc to antigens and expressing them.

[0067] <Confirmation of multimer formation of fusion protein> To confirm whether the fusion protein expressed by pDNA-SP-D-RbFc forms a multimer, it was examined whether the fusion protein could be detected by sandwich ELISA using the same antibody.

[0068] 50 μL of abSP-D diluted to 5 μg / mL with PBS was dispensed into each well of a 96-well ELISA plate and allowed to stand overnight at 4°C. After washing each well three times with washing solution, 100 μL of blocking solution was dispensed into each well and allowed to stand overnight at 4°C. After removing the blocking solution from each well, 50 μL of culture supernatant containing SP-D-RbFc diluted 5-78125 times with blocking solution was dispensed into each well and allowed to stand at room temperature for 1 hour. After washing each well three times with washing solution, 50 μL of biotinylated abSP-D solution adjusted to 1 μg / mL with blocking solution was dispensed into each well and allowed to stand at room temperature for 1 hour. After washing each well three times with washing solution, 50 μL of Streptavidin, Horseradish Peroxidase Conjugate (Thermo Fisher Scientific) adjusted to 0.2 μg / mL with blocking solution was dispensed into each well and allowed to stand at room temperature for 30 minutes. After washing each well three times with washing solution, 50 μL of OPD color development solution was dispensed into each well and allowed to stand at room temperature for 10 minutes. 50 μL of stop solution was dispensed into each well, and the absorbance at 492 nm was measured using a plate reader.

[0069] In a control experiment, it was confirmed whether culture supernatants containing SP-D-HSA or SP-D-Histag could be detected by sandwich ELISA using the same procedure. The results are shown in Table 9.

[0070]

[0071] SP-D-RbFc in the culture supernatant could be detected by sandwich ELISA using the same antibody as SP-D-Histag. In other words, it was found that SP-D-RbFc forms multimers like SP-D-Histag. On the other hand, although SP-D-HSA could be detected by sandwich ELISA using the same antibody at low dilutions, the correlation between the antigen concentration and the detection signal was poor.

[0072] <Calibration Performance> 1. Preparation of LTIA Reagent for P1NP Detection 1-1. Preparation of Anti-P1NP Antibody-Sensitized Latex Antibody solutions of abP1NP-1 and abP1NP-2 were prepared in 40 mM glycine buffer (hereinafter referred to as sensitization buffer) so that the absorbance at 280 nm was 0.5. 1 mL of a 1% polystyrene latex solution (manufactured by Sekisui Medical Co., Ltd.) with an average particle size of 0.27 μm suspended in sensitization buffer was mixed with 1 mL of each antibody solution and stirred at 4°C for 2 hours. 1 mL of a 0.5% BSA solution prepared with sensitization buffer was added to the resulting latex solution, and the mixture was stirred at 4°C for 1 hour. The resulting antibody-sensitized latex solution was dialyzed against 5 mM MOPS solution (pH 7.0).

[0073] 1-2. Preparation of LTIA Reagent The first and second reagents were prepared as follows. First reagent: 100 mM HEPES pH 7.0, 500 mM NaCl, 0.5% BSA Second reagent: abP1NP-1-sensitized latex (absorbance at 600 nm: 3.0), abP1NP-2-sensitized latex (absorbance at 600 nm: 3.0), 5 mM MOPS pH 7.0

[0074] 1-3. Preparation of Calibrators Four concentrations of human serum containing P1NP were used. Each sample was measured using intact procollagen P1NP (manufactured by Yamasa Shoyu Co., Ltd.) according to the method described in the package insert, and the P1NP concentration in each antigen solution was calculated. S1 to S4, in which the P1NP concentration was determined, were designated as Calibrator 1. The P1NP concentrations in each antigen solution were S1: 29.6 ng / mL, S2: 65.4 ng / mL, S3: 130.7 ng / mL, and S4: 260.2 ng / mL.

[0075] 1-4. Measurement using LTIA reagent The first and second reagents were placed in a reagent cabinet, and measurements were performed using a Hitachi automatic analyzer 3500 under the following conditions.

[0076] (Measurement conditions) Analysis method: 2-point end Photometric point: 19-34 Measurement wavelength: 570 / 800 nm Sample volume: 5 μL First reagent: 120 μL Second reagent: 40 μL

[0077] 1-5. Preparation of calibration curve Calibrator 1 and physiological saline (P1NP concentration: 0 ng / mL) were used as samples to perform the measurements described in 1-4. Calibration curve 1 was prepared by plotting the change in absorbance per unit time during measurement on the vertical axis and the P1NP concentration of calibrator 1 on the horizontal axis.

[0078] 1-6. Preparation of measurement samples The culture supernatant containing P1NP-RbFc was purified by Ni-NTA affinity chromatography to obtain a P1NP-RbFc solution. The culture supernatant containing P1NP-HSA was purified by Ni-NTA affinity chromatography to obtain a P1NP-HSA solution.

[0079] 2. Confirmation of Recovery of Diluted Fusion Protein Calibrator 1 S5, P1NP-RbFc solution, and P1NP-HSA solution were each serially diluted with physiological saline and used as samples for measurements 1-4. The P1NP concentration in each solution was calculated using calibration curve 1. The measured value was divided by the theoretical value calculated based on the dilution ratio to determine the recovery rate of each protein. The results are shown in Table 10.

[0080]

[0081] The recovery rate of P1NP-RbFc was as good as that of P1NP in serum. The recovery rate of P1NP-HSA decreased as the dilution rate increased.

[0082] 3. Calibration using standard samples containing fusion protein P1NP-RbFc was appropriately diluted with saline to prepare S1' to S4'. Measurements were performed using S1' to S4' as samples according to the method described in 1-4, and the P1NP values ​​in S1' to S4' were calculated using calibration curve 1. S1' to S4' for which the P1NP values ​​were determined were designated as calibrator 2. The P1NP values ​​in each antigen solution were S1': 29.0 ng / mL, S2': 59.7 ng / mL, S3': 123.6 ng / mL, and S4': 253.8 ng / mL. Calibration curve 2 was created by plotting the change in absorbance per unit time during measurements of saline and calibrator 2 on the vertical axis and the P1NP concentration of calibrator 2 on the horizontal axis. 17 random human serum samples containing P1NP were used as samples according to the method described in 1-4. The P1NP concentration in the sample was calculated using calibrator 1 and calibrator 2. The correlation between the calculated measurement value and the measurement value when the same sample was measured using intact procollagen P1NP was confirmed.

[0083] FIG. 1 is a graph showing the correlation between P1NP measurement values ​​(vertical axis) calculated using calibration curve 2 prepared using calibrator 2 prepared using P1NP-RbFc, and P1NP measurement values ​​(horizontal axis) using intact procollagen P1NP. The slope of the regression line was 1.03, and the correlation coefficient was 0.967. FIG. 2 is a graph showing the correlation between P1NP measurement values ​​(vertical axis) calculated using calibration curve 1 prepared using calibrator 1 prepared using human serum, and P1NP measurement values ​​(horizontal axis) using intact procollagen P1NP. The slope of the regression line was 1.0674, and the correlation coefficient was 0.964. The P1NP measurement values ​​when P1NP-RbFc was used as the antigen for the calibrator and the P1NP measurement values ​​when P1NP in human serum was used as the calibrator both showed good correlation with the measurement values ​​using intact procollagen P1NP. In other words, it can be said that the calibrator prepared using P1NP-RbFc has calibration performance equivalent to that of P1NP in human serum.

[0084] According to the above aspects, it is possible to provide an immunoassay method, a quality control method, and a standard sample and an immunoassay reagent used therein.

Claims

1. An immunoassay method comprising: detecting a signal corresponding to the concentration or titer of an antigen contained in a biological sample; and calculating the concentration or titer of the antigen in the biological sample from the signal using a calibration curve showing the relationship between the concentration or titer of the antigen and the signal, wherein the calibration curve is prepared using a standard sample containing a fusion protein, and the fusion protein contains at least a portion of the amino acid sequence of the antigen and at least a portion of the amino acid sequence of an immunoglobulin constant region.

2. The immunoassay method according to claim 1, wherein at least a portion of the amino acid sequence of the constant region of the immunoglobulin is derived from a rabbit antibody.

3. The immunoassay method according to claim 1, wherein the antigen is a protein that exists as a multimer in vivo.

4. The immunoassay method according to claim 1, wherein the antigen is at least one of P1NP, PCT, SP-D, and TARC.

5. The immunoassay method according to claim 4, wherein the antigen is P1NP.

6. A method for quality control of immunoassay of an antigen, comprising the steps of: detecting a signal corresponding to the concentration or titer of a fusion protein in a standard sample containing a fusion protein at a predetermined concentration or titer; calculating the concentration or titer of the fusion protein from the detected signal; and comparing the calculated concentration or titer with the predetermined concentration or titer to evaluate the quality of the immunoassay, wherein the fusion protein contains at least a portion of the amino acid sequence of the antigen and at least a portion of the amino acid sequence of an immunoglobulin constant region.

7. The method for controlling the accuracy of immunoassays of antigens according to claim 6, wherein at least a portion of the amino acid sequence of the constant region of the immunoglobulin is derived from a rabbit antibody.

8. The method for controlling the accuracy of immunoassays of antigens according to claim 6, wherein the antigen is a protein that exists as a multimer in vivo.

9. The method for quality control of antigen immunoassays according to claim 6, wherein the antigen is one of P1NP, PCT, SP-D, and TARC.

10. The method for controlling the accuracy of antigen immunoassays according to claim 6, wherein the antigen is P1NP.

11. A standard sample used in the immunoassay method according to any one of claims 1 to 5 or the method for controlling the quality of immunoassay according to any one of claims 6 to 10, the standard sample comprising the fusion protein of at least a portion of the amino acid sequence of the antigen and at least a portion of the amino acid sequence of the constant region of the immunoglobulin at a known concentration or known titer.

12. An immunoassay method comprising the steps of forming a complex between an antibody and a fusion protein in a solution and detecting a signal emitted from a labeling substance bound to the fusion protein, wherein the fusion protein is a fusion protein of at least a portion of the amino acid sequence of an antigen capable of binding to the antibody and at least a portion of the amino acid sequence of an immunoglobulin constant region.

13. An immunoassay method comprising the steps of: obtaining a reaction system in which a biological sample that may contain an antigen, an antibody capable of binding to the antigen, and a competitor capable of binding to the antigen competitively with the antibody and having a labeling substance bound thereto; and measuring the signal of the labeling substance contained in the reaction system, wherein the competitor is a fusion protein of at least a portion of the amino acid sequence of the antigen and at least a portion of the amino acid sequence of an immunoglobulin constant region.

14. The immunoassay method according to claim 13, further comprising, between the step of obtaining the reaction system and the step of measuring the signal, a step of removing by washing the antigen and the competing substance that are not bound to the antibody while leaving the antibody in the reaction system, thereby obtaining an assay system containing the antigen and the competing substance bound to the antibody.

15. The immunoassay method according to claim 12 or 13, wherein at least a portion of the amino acid sequence of the constant region of the immunoglobulin is derived from a rabbit antibody.

16. The immunoassay method according to claim 12 or 13, wherein the antigen is a protein that exists as a multimer in vivo.

17. The immunoassay method according to claim 12 or 13, wherein the antigen is one of P1NP, PCT, SP-D, and TARC.

18. The immunoassay method according to claim 12 or 13, wherein the antigen is P1NP.

19. An immunoassay reagent containing the fusion protein, for use in the immunoassay method according to claim 12.

20. An immunoassay reagent containing the competitive substance, for use in the immunoassay method according to claim 13.

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