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

Abstract

The present application relates to an immunoassay method including a process of detecting a signal corresponding to the concentration or titer of an antigen contained in a biological sample, and a process of calculating the concentration or titer of the antigen in the biological sample from the signal using a calibration curve indicating the correlation between the concentration or titer of the antigen and the signal, the calibration curve being prepared using a standard sample containing a fusion protein including 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 an immunoglobulin.

Description

Technical Field

[0001] This invention relates to immunoassay methods, precision management methods, standard samples, and immunoassay reagents.

[0002] 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. Background Technology

[0003] When quantifying the target component for immunoassay in biological samples, calibration samples are required. Calibration samples are samples containing the target component used for purposes such as internal standards or concentration calibration references (calibrators). To obtain accurate quantitative values ​​of the target component, calibration samples must be stable over time and under temperature variations.

[0004] Such calibration samples contain the antigen itself of the analyte, purified from a biological sample, and proteins expressed through genetic recombination. For example, Patent Document 1 discloses a calibration sample solution as a liquid composition containing pulmonary surfactant protein.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: International Publication No. 2023 / 145916 Summary of the Invention

[0008] The problem the invention aims to solve

[0009] When proteins are contained in calibrating samples used in immunoassays, the purification process can sometimes become cumbersome when purifying the antigen itself from a biological sample. Furthermore, when proteins are expressed through gene recombination, sufficient expression levels may not be obtained depending on the type of antigen.

[0010] In this respect, there is still room for further improvement regarding the standard samples used in immunoassays.

[0011] The purpose of this invention is to provide immunoassay methods, precision management methods, and standard samples and immunoassay reagents for these methods.

[0012] Problem Solving Methods

[0013] The present invention includes the following methods.

[0014] [1] An immunoassay method comprising: a step of detecting a signal corresponding to the concentration or titer of an antigen contained in a biological sample; and a step of using a calibration curve representing the relationship between the concentration or titer of the antigen and the signal and calculating the concentration or titer of the antigen in the biological sample based on the signal, wherein the calibration curve is prepared using a standard sample containing a fusion protein, the fusion protein containing at least a portion of the amino acid sequence of the antigen and at least a portion of the amino acid sequence of a constant region of an immunoglobulin.

[0015] [2] According to the immunoassay method described in [1], at least a portion of the amino acid sequence of the constant region of the immunoglobulin is derived from rabbit antibody.

[0016] [3] According to the immunoassay method described in [1] or [2], wherein the antigen is a protein that exists in the body in the form of a polymer.

[0017] [4] According to the immunoassay method described in [1] or [2], wherein the antigen is at least one of P1NP, procalcitonin, SP-D and TARC.

[0018] [5] According to the immunoassay method described in [4], the antigen mentioned above is P1NP.

[0019] [6] A method for precision management of an antigen immunoassay, comprising: a step of detecting a signal corresponding to the concentration or titer of a fusion protein in a standard sample containing a pre-set concentration or titer; a step of calculating the concentration or titer of the fusion protein based on the detected signal; and a step of evaluating the precision of the immunoassay by comparing the calculated concentration or titer with the pre-set concentration or titer, 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 the constant region of an immunoglobulin.

[0020] [7] The method for precision management of the immunoassay of the antigen according to [6], wherein at least a portion of the amino acid sequence of the constant region of the immunoglobulin is derived from rabbit antibody.

[0021] [8] A method for managing the accuracy of an immunoassay of an antigen according to [6] or [7], wherein the antigen is a protein that exists in the body in the form of a polymer.

[0022] [9] The method for precision management of the immunoassay of the antigen according to [6] or [7], wherein the antigen is one of P1NP, PCT, SP-D and TARC.

[0023]

[10] The method for managing the accuracy of the immunoassay of the antigen described in [9], wherein the antigen is P1NP.

[0024]

[11] A standard sample, which is a standard sample for the immunoassay method described in any one of [1] to [5] or the precision management method for the immunoassay described in any one of [6] to

[10] , wherein the standard sample contains, at a known concentration or known titer, 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.

[0025]

[12] An immunoassay method comprising: forming a complex of an antibody and a fusion protein in a solution and detecting a signal emitted by 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 a constant region of an immunoglobulin.

[0026]

[13] An immunoassay method comprising: a step of obtaining a reaction system in which a biological sample, an antibody, and a competing substance bound to a labeling substance coexist, wherein the biological sample may contain an antigen, the antibody is capable of binding to the antigen, and the competing substance is capable of competitively binding to the antibody against the antigen; and a step of measuring a signal of the labeling substance contained in the reaction system, wherein the competing substance 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 the constant region of an immunoglobulin.

[0027]

[14] According to the immunoassay method described in

[13] , between the step of obtaining the above reaction system and the step of measuring the above signal, a step of obtaining a assay system is further included, in which the above antibody is retained in the above reaction system and the above antigen and the above competing substance that are not bound to the above antibody are removed by washing, thereby obtaining an assay system containing the above antigen and the above competing substance that are bound to the above antibody.

[0028]

[15] According to the immunoassay method described in

[12] or

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

[0029]

[16] An immunoassay method according to any one of

[12] to

[15] , wherein the antigen is a protein that exists in the body in the form of a polymer.

[0030]

[17] The immunoassay method according to any one of

[12] to

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

[0031]

[18] According to the immunoassay method described in

[17] , wherein the antigen is P1NP.

[0032]

[19] An immunoassay reagent comprising the fusion protein, wherein the immunoassay reagent is used in any of the immunoassay methods described in

[12] and

[14] to

[18] .

[0033]

[20] An immunoassay reagent comprising the above-mentioned competing substance, wherein the immunoassay reagent is used in any of the immunoassay methods described in

[13] to

[18] .

[0034] The effects of the invention

[0035] Based on the above methods, it is possible to provide immunoassay methods, precision management methods, and standard samples and immunoassay reagents for these methods. Attached Figure Description

[0036] Figure 1 This is a graph showing the correlation between P1NP measurements calculated using a calibration curve and P1NP measurements based on pre-reagent collagen Intact P1NP, the calibration curve being prepared using a calibrator made with P1NP-RbFc.

[0037] Figure 2 This is a graph showing the correlation between P1NP measurements calculated using a calibration curve and P1NP measurements based on pre-reagent collagen Intact P1NP, the calibration curve being prepared using a calibrator made from P1NP in human serum. Detailed Implementation

[0038] Hereinafter, one aspect of the present invention will be described with reference to the accompanying drawings.

[0039] In this specification and claims, when “~” is used to indicate a numerical range, the numerical range includes the values ​​on both sides of “~”.

[0040] The biological samples used in this specification can primarily be bodily fluids derived from biological organisms. Examples of biological samples include blood, serum, plasma, nasopharyngeal swabs, nasal swabs, saliva, sputum, and amniotic fluid, with serum and plasma being preferred. The subjects from which biological samples are collected include humans or animals (e.g., monkeys, dogs, or cats), with humans being preferred. The biological sample can be the sample itself derived from the subject, or it can be a sample that has undergone conventional dilution or concentration treatments. It should be noted that the person collecting and preparing the biological samples used in this invention can be the same person as the person performing the assay method of this invention, or a different person. Furthermore, the biological samples used in the assay method of this invention can be samples collected or prepared during the implementation of the assay method of this invention, or samples that have been collected or prepared and stored in advance.

[0041] <Immunoassay Methods and Standard Samples>

[0042] An immunoassay method according to one aspect of the present invention includes: a step of detecting a signal corresponding to the concentration or titer of an antigen contained in a biological sample; and a step of using a calibration curve representing the correlation between the concentration or titer of the antigen and the signal, and calculating the concentration or titer of the antigen in the biological sample based on the signal, wherein the calibration curve is prepared using a standard sample containing a fusion protein, the fusion protein containing at least a portion of the amino acid sequence of the antigen and at least a portion of the amino acid sequence of a constant region of an immunoglobulin.

[0043] In one embodiment of the present invention, the standard sample is a standard sample for an immunoassay method, comprising the fusion protein containing 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. The concentration of the fusion protein can be calculated using the BCA method, the Bradford method, the Lowry method, and existing diagnostic reagents for the determination of the antigen contained in the fusion protein. The titer of the fusion protein can be calculated using protease titer assays, etc.

[0044] Standard samples can be used as calibrators for preparing calibration curves for clinical laboratory reagents. Additionally, standard samples can be used as controls for measurements taken in the precision management of clinical laboratory reagents.

[0045] Fusion proteins contain the same amino acid sequence as the antigen used in the immunoassay method, or a portion thereof. Examples of antigens used as assay targets include 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-terminal propeptide (P1NP), procalcitonin (PCT), protein induced by vitamin K absence 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 can be at least one of P1NP, PCT, SP-D, and TARC. The antigen of the test subject is preferably an oligopeptide or a complex thereof composed of about 30 to 1000 amino acids, more preferably an oligopeptide or a complex thereof composed of about 50 to 700 amino acids.

[0046] When the fusion protein contains an amino acid sequence identical to a portion of the antigen of the target antigen, the fusion protein preferably contains at least an amino acid sequence identical to an epitope of the target antigen. This "portion of the target antigen" can be one location or multiple locations. That is, the fusion protein can contain multiple amino acid sequences identical to portions of the target antigen. For example, when the target antigen is IL-2R, LR11, or P1NP, the fusion protein preferably contains an amino acid sequence identical to a portion of the target antigen. For example, the fusion protein can contain an amino acid sequence identical to the α1-1 sequence in the α1 chain of P1NP and an amino acid sequence identical to the α1-2 sequence.

[0047] The antigens used for testing can be proteins that exist as polymers in the body. These antigens can exist as homopolymers or heteropolymers in the body. For example, antigens used for testing can be IL-2R, P1NP, and SP-D.

[0048] The fusion protein contains at least a portion of the amino acid sequence of the constant region of an immunoglobulin. Examples of immunoglobulins include IgG, IgA, IgM, IgD, and IgE. The amino acid sequence of at least a portion of the constant region of the immunoglobulin is preferably a portion of the amino acid sequence of the constant region of IgG. The amino acid sequence of at least a portion of the constant region of the immunoglobulin is preferably derived from rabbit antibodies. When the fusion protein contains at least a portion of the amino acids of the constant region of an immunoglobulin, a sufficient amount of the fusion protein will be expressed in the culture supernatant of transformed cells, even when expressed using a conventional plasmid.

[0049] The fusion protein of this embodiment preferably further includes an affinity tag for protein purification. Examples of affinity tags include Histag, FLAG-Tag, Spot-tag, C-Tag, and Strep-tag, which are composed of 6 to 9 consecutive histidine residues.

[0050] The fusion protein of this embodiment may further include a linker to improve the reactivity of the fusion protein with the antibody. The length and sequence of the linker can be appropriately selected considering the sequences of the constant regions of the antigen and immunoglobulin. For example, the length of the linker may be 2 to 20 residues. The linker may be located between the affinity tag and a portion of the antigen of the target, or between a portion of the antigen of the target and at least a portion of the constant region of the immunoglobulin. In addition, if the fusion protein contains an amino acid sequence that is identical to the amino acid sequence at multiple positions of a portion of the antigen of the target, a linker may be arranged between the aforementioned amino acids.

[0051] The fusion protein of this embodiment preferably contains at least a portion of the constant region of an immunoglobulin on the C-terminus or N-terminus side of the amino acid sequence of the antigen to be measured. When the amino acid sequence of the antigen to be measured contains at least a portion of the constant region of an immunoglobulin on the C-terminus or N-terminus side, the N-terminus or C-terminus of the antigen's amino acid sequence can be exposed, and the reactivity of the antigen and antibody will not be impaired when the antibody recognizes the N-terminus or C-terminus of the antigen, which is therefore preferred.

[0052] The fusion protein of this embodiment can be obtained by introducing an expression vector containing a signal sequence and a base sequence encoding the fusion protein into cells, and then expressing the fusion protein in the cells. The signal peptide translated from the signal sequence is broken down and removed during the secretion of the fusion protein extracellularly. Because the fusion protein of this embodiment is expressed in large quantities extracellularly, it is easy to recover from the cell culture medium and has high productivity.

[0053] In the immunoassay method for the antigen of this embodiment, a calibration curve is used, which is prepared by measuring the concentration or titer of the fusion protein contained in the standard sample. The calibration curve can be prepared using conventional methods. For example, it can be prepared by plotting the signal measured with respect to 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 can be a curve prepared by the person performing the immunoassay, or a pre-prepared calibration curve can be used.

[0054] In this embodiment, a signal corresponding to the concentration or titer of the antigen contained in the biological sample as the analyte is detected. A calibration curve representing the correlation between the antigen concentration or titer and the detected signal is used to calculate the concentration or titer of the antigen in the biological sample based on the signal. Examples of signals include transmitted light, scattered light, fluorescence, chemiluminescence, electrochemiluminescence, or coloring.

[0055] The immunoassay method used in this embodiment is not particularly limited, and examples include LTIA (latex immunoturbidimetric assay), ELISA (enzyme-linked immunosorbent assay), electrochemiluminescence immunoassay, and immunochromatography.

[0056] (LTIA)

[0057] LTIA is an immunoassay method that utilizes the aggregation of latex particles caused by the binding of antibodies supported on the surface of latex particles to the antigens used as the analyte. There are no particular restrictions on the latex particles used in in vitro diagnostic reagents. The concentration of latex particles and the average particle size during the aggregation reaction assay can be appropriately set according to sensitivity or performance requirements. The assay procedure and principle when using latex immunoturbidimetry as the immunoassay method in this embodiment are described below.

[0058] (1) Make the latex particles carrying the antibody come into contact with the sample.

[0059] (2) The antigen in the sample forms a complex with at least two antibodies, and the latex particles aggregate.

[0060] (3) Irradiate the sample with near-infrared light (e.g., wavelength 600 nm) and measure the transmitted or scattered light (i.e., the signal). Calculate the concentration or titer of the antigen based on the calibration curve prepared using the standard sample of this embodiment and the measured signal value.

[0061] As the measuring device, an automated biochemical analyzer can be used (e.g., Hitachi Automated Analyzer 3500).

[0062] (ELISA)

[0063] In this specification, ELISA refers to a method that uses antibodies to capture antigens contained in a sample, which are then detected by an enzyme reaction. The solid phase is preferably a plate (also called an immunoassay plate). HRP or ALP can be used as a label. The assay procedure and principle when using a sandwich ELISA as the immunoassay method of this embodiment are described below.

[0064] (1) Contact the sample with the solid phase immobilized with antibody so that the antigen in the sample binds to the antibody in the solid phase.

[0065] (2) Add labeled antibodies to the solid phase to make it react and form a solid phase antibody-antigen-labeled antibody complex, i.e., a sandwich, in which the labeled antibody and the antigen are bound.

[0066] (3) After cleaning, the labeled substance is made to develop color and the absorbance (i.e., signal) is measured. Based on the calibration curve prepared using the standard sample of this embodiment and the measured signal value, the concentration or titer of the antigen is calculated.

[0067] Secondary antibodies can also be used in sandwich ELISA. By using secondary antibodies, the reaction is amplified, which can improve the detection sensitivity. When using secondary antibodies, the following steps (1) to (5) can be taken.

[0068] (1) After adding the sample to the solid phase immobilized with antibody, incubate it, remove the sample and clean it.

[0069] (2) Add antibodies that can bind to the antigens used for testing, and then incubate and wash.

[0070] (3) Further add enzyme-labeled secondary antibody and incubate.

[0071] (4) Add a substrate to make it develop color.

[0072] (5) Measure the color development (i.e., signal) using an enzyme-linked immunosorbent assay (ELISA) reader or similar instrument. Calculate the concentration or titer of the antigen based on the calibration curve prepared using the standard sample of this embodiment and the measured signal value.

[0073] (Electrochemiluminescence immunoassay)

[0074] Electrochemiluminescence immunoassay (ECIA) is a method that determines the amount of antigen contained in a sample by detecting the emission of light from a labeled substance through an electric current, and then detecting the amount of light emitted (i.e., the signal). In EIA, ruthenium complexes can be used as the labeled substance. Electrodes are placed on a solid phase (such as a microplate), and free radicals are generated on the electrodes, thereby exciting the ruthenium complex and causing it to emit light. The amount of light emitted by the ruthenium complex can then be detected.

[0075] The determination procedure and principle when using magnetic particles as the solid phase and ruthenium complex as the labeling substance are described below.

[0076] (1) Make the magnetic particles immobilized with solid phase antibody come into contact with the sample, so that the antigen in the sample binds to the solid phase antibody.

[0077] (2) After cleaning the magnetic particles, bring them into contact with the labeled antibody so that the labeled antibody binds to the antigen bound to the magnetic particles.

[0078] (3) After cleaning the magnetic particles, an electric current is applied, and the amount of light emitted is measured by measuring the amount of labeled antibody that has bound to the antigen, thereby determining the amount of antigen in the biological sample.

[0079] (Immunochromatography)

[0080] 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 determining antigens contained in a sample is as follows.

[0081] Antibodies against antigens are immobilized on an insoluble membrane carrier, which serves as the chromatographic medium, to create a detection unit that acts as the stationary phase. A conjugate (a labeled substance sensitized by an antibody capable of binding to the antigen) is then used as the mobile phase. The analyte reacts specifically with the conjugate in the mobile phase, and then, in the detection unit, which is the stationary phase, the analyte bound to the conjugate reacts specifically with the antibody immobilized in the detection unit. The presence of antigen in the sample, or the concentration of antigen in the sample, is determined by detecting signals such as coloring or fluorescence from the labeled substance bound to the detection unit by the analyte. Examples of labeled substances include gold colloidal particles, platinum colloidal particles, colored latex particles, and magnetic particles. Gold colloidal particles are preferred. The type and particle size of these labeled substances can be appropriately adjusted by those skilled in the art according to the desired sensitivity.

[0082] The assay steps and principles when using immunochromatography as the immunoassay method of the present invention are as follows.

[0083] (1) Make the sample come into contact with the sample supply section used to supply the sample.

[0084] (2) The antigen in the sample comes into contact with the conjugate to form the first complex. The conjugate is formed by the first monoclonal antibody binding to the labeled substance.

[0085] (3) The first complex is brought into contact with the second monoclonal antibody immobilized in the detection section to form a second complex.

[0086] (4) The amount of antigen in the sample or its presence can be determined by measuring the signal intensity of the marker substance contained in the conjugate.

[0087] According to the immunoassay method of this embodiment, since the standard sample of this embodiment is used, the same high assay accuracy as that of substances purified from biological samples can be obtained.

[0088] Furthermore, according to the standard sample of this embodiment, since the fusion protein contains at least a portion of the amino acid sequence of the constant region of immunoglobulins, a sufficient amount of the standard sample will be expressed in the culture supernatant of transformed cells even when expressed using conventional plasmids. Additionally, when using the standard sample of this embodiment, the same high measurement accuracy as that obtained with standard samples manufactured using biological samples can be obtained.

[0089] <Immunoassay Methods and Reagents>

[0090] As another aspect of this embodiment, the immunoassay method includes the steps of forming a complex of an antibody and a fusion protein in solution and detecting a signal emitted by a labeling substance bound to the fusion protein, wherein the fusion protein is a fusion protein comprising 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 a constant region of an immunoglobulin. The labeling substance may be pre-bound to the fusion protein, or the labeling substance may be pre-bound to the antibody, and the fusion protein may bind to the labeling substance via the antibody when the antibody and fusion protein form a complex.

[0091] As another aspect of this embodiment, the immunoassay method includes: a step of obtaining a reaction system in which a biological sample, an antibody, and a competing substance bound to a labeled substance coexist, wherein the biological sample may contain an antigen, the antibody is capable of binding to the antigen, and the competing substance is capable of competitively binding to the antibody against the antigen; and a step of measuring the signal of the labeled substance contained in the reaction system, wherein the competing substance 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 the constant region of an immunoglobulin. The immunoassay method may, after obtaining the reaction system, include a step of retaining the antibody in the reaction system and removing the antigen and the competing substance that are not bound to the antibody by washing.

[0092] In this immunoassay method, an antibody capable of binding to the antigen being measured is used. The aforementioned fusion protein can bind to this antibody, acting as a competing substance relative to the analyte. The fusion protein of this embodiment may bind to an enzyme that emits a signal upon reaction with a substrate. The other components of the fusion protein are the same as those of the standard sample described in <Immunoassay Method and Standard Sample>, therefore their description is omitted.

[0093] The immunoassay method described in this embodiment is not particularly limited as long as it is a method that can detect the target substance using the competing substance described in this embodiment. Examples include competitive methods such as ELISA, LTIA, electrochemiluminescence immunoassay, and immunochromatography.

[0094] The competitive ELISA method can be performed using the following steps (1) to (5).

[0095] (1) After obtaining a reaction system in which the sample and the competing substance of this embodiment coexist with the solid phase immobilized with the antibody, incubation is performed.

[0096] (2) By leaving the antibody in the above reaction system and removing the antigen and the competing substance that have not bound to the antibody by washing, an assay system containing the antigen and the competing substance that have bound to the antibody is obtained.

[0097] (3) Add a substrate to make it develop color.

[0098] (4) Use an enzyme-linked immunosorbent assay (ELISA) reader or similar device to measure the color development (i.e., signal) of the labeling substance contained in the assay system.

[0099] (5) The concentration or titer of the antigen is calculated based on the calibration curve and the measured value of the signal.

[0100] There are no particular limitations on the antigens that can be used for testing; examples include IL-2R, LR11, NT-proBNP, P1NP, PCT, PSA, SP-D, and TARC.

[0101] By using an immunoassay reagent containing the fusion protein of this embodiment in an immunoassay method, antigens that are components of the reagent can be produced stably and easily.

[0102] <Precision Management Methods for Antigen Immunoassay>

[0103] The method for managing the accuracy of an antigen immunoassay according to this embodiment includes: a step of detecting a signal corresponding to the concentration or titer of a fusion protein in a standard sample containing a pre-set concentration or titer; a step of calculating the concentration or titer of the fusion protein based on the detected signal; and a step of evaluating the accuracy of the immunoassay by comparing the calculated concentration or titer with the pre-set concentration or titer, 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 the constant region of an immunoglobulin.

[0104] In the precision management methods for antigen immunoassays, standard samples are used as controls for precision management of clinical laboratory reagents. The composition of the fusion protein contained in the standard samples is the same as that described in <Immunoassay Methods and Standard Samples>, therefore its description is omitted.

[0105] As one method for managing the accuracy of the antigen immunoassay according to this embodiment, a method for managing the assay accuracy of clinical laboratory reagents and clinical laboratory apparatus can be cited. Specifically, a standard sample is prepared to contain a fusion protein at a predetermined concentration or titer. Then, a signal corresponding to the concentration or titer of the fusion protein is detected using the standard sample with known concentration and titer and the clinical laboratory reagent. The concentration or titer of the fusion protein is calculated based on the detected signal. The accuracy of the immunoassay is evaluated by comparing the calculated concentration or titer with the predetermined concentration or titer, thereby managing whether the clinical laboratory reagents and clinical laboratory apparatus can perform assays with good accuracy.

[0106] The accuracy management method of this embodiment is not particularly limited to any type of immunoassay that can be applied to. Any method that can use the standard sample of this embodiment for accuracy management is acceptable, such as LTIA, ELISA, electrochemiluminescence immunoassay, and immunochromatography.

[0107] In the accuracy management method of this embodiment, the antigen used as the test object is not particularly limited, and examples include P1NP, PCT, SP-D and TARC.

[0108] According to the method for precision management of antigen immunoassay according to this embodiment, since the standard sample of this embodiment is used, it is possible to perform management with the same high precision as substances purified from biological samples.

[0109] Example

[0110] The present invention will be described in more detail below through embodiments, but the present invention is not limited to the embodiments described below.

[0111] <plasmid>

[0112] As plasmids for expressing fusion proteins, plasmids were used that inserted the signal sequence shown in Table 1 (SEQ ID NO. 1), the full or partial sequence of the antigen, Histag, rabbit antibody Fc (RbFc), and appropriate linker base sequences (Linker-Histag-1 (SEQ ID NO. 2), Linker-Histag-2 (SEQ ID NO. 3), and Linker-RbFc (SEQ ID NO. 4), respectively) into pcDNA3.1(-)Mammalian Expression Vector (ThermoFisher SCIENTIFIC). Plasmids used 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) used base sequences encoding the full sequence of the antigen. The plasmid used for expressing the IL-2R fusion antigen used a base sequence encoding the extracellular domain (SEQ ID NO. 5). The plasmid used for LR11 fusion antigen expression uses a base sequence encoding a partial sequence of the antigen (SEQ ID NO. 6). The plasmid used for P1NP fusion antigen expression uses a base sequence encoding a peptide consisting of two amino acid sequences linked by a linker in the α1 chain (SEQ ID NO. 8). The base sequences are shown in Tables 1-4.

[0113]

[0114]

[0115]

[0116]

[0117] Table 5 shows the combinations of base sequences inserted into the plasmids for expressing each fusion protein.

[0118]

[0119] As plasmids used in comparative experiments, plasmids containing the base sequence encoding HSA instead of rabbit antibody Fc (Linker-HSA (SEQ ID NO. 13)) shown in Table 6, and plasmids not containing the sequence encoding rabbit antibody Fc were used. Table 7 shows the combinations of base sequences inserted into each plasmid for fusion protein expression or Histag-binding protein expression.

[0120]

[0121]

[0122] <Expression of fusion proteins>

[0123] The fusion protein was expressed using the ExpiCHO Expression System Kit (Thermo Fisher Scientific, product number: A29133) according to the product operating protocol. More specifically, ExpiCHO cells were expressed in a dedicated culture medium at 3–4 × 10⁻⁶ cells / day. 6 Cells / mL were inoculated and cultured. The next day, Plasmid DNA prepared at 1 μg / μL was mixed with OptiPro medium, and then further mixed with a separately prepared mixture of OptiPro medium and ExpiFectanubeCHO reagent. The mixture was allowed to stand at room temperature for 1–5 minutes. The resulting mixture was then added to a concentration of 7–10 × 10⁻⁶ cells / mL. 6 Expi CHO cells were seeded at a concentration of cells / mL and cultured. The following day, ExpiCHO Enhancer and ExpiCHO FeED were added to the cells according to the instructions in the operating procedure, and the cells were cultured again. The supernatant was collected after 8 days and used for various experiments.

[0124] <Obtaining Antigen-Binding Monoclonal Antibodies>

[0125] Antibodies responsive to 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 recovered from mice whose antibody titers in the blood were confirmed to be elevated. The recovered cells were fused with myeloma cells SP2 / 0 using an electrofusion method. The fused cells were cultured in 96-well plates, and the culture supernatant was recovered. Cell lines producing antibodies responsive to each antigen were selected using antigen-based solid-phase ELISA with each antigen as an immunogen. Ascites fluid containing antibodies was collected from mice administered the selected antibody cell lines, and the ascites fluid was purified using a protein A column or a protein G column to obtain monoclonal antibodies responsive to each antigen. Specifically, abIL-2R (anti-IL-2R antibody), abLR11 (anti-LR11 antibody), abNT-proBNP (anti-NT-proBNP antibody), abP1NP-1 and abP1NP-2 (anti-P1NP antibodies), abPCT (anti-PCT antibody), abPSA (anti-PSA antibody), abSP-D (anti-SP-D antibody), and abTARC (anti-TARC antibody) were obtained. Each monoclonal antibody was biotinylated using the Biotin Labeling Kit-NH2 (Dongjin Chemical Research Institute) according to standard methods, resulting in biotinylated monoclonal antibodies.

[0126] <Determination of expression levels of fusion proteins>

[0127] The reactivity of the fusion proteins in the culture supernatant with antibodies reacting to each antigen was evaluated. More specifically, 50 μL of antibodies reacting to each antigen, diluted to 5 μg / mL with PBS, were aliquoted into each well of a 96-well ELISA plate and incubated 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 buffer), 100 μL of PBS containing 1% BSA and 0.05% Tween 20 (hereinafter referred to as blocking buffer) was aliquoted into each well and incubated overnight at 4°C. After removing the blocking buffer, 50 μL of the culture supernatant of each fusion protein, diluted 5-fold with the blocking buffer, was aliquoted into each well and incubated for 1 hour at room temperature. After washing each well three times with washing buffer, 50 μL of biotinylated monoclonal antibody solution (adjusted to 1 μg / mL with blocking buffer, reacting with each antigen) was added to each well and incubated 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 added to each well and incubated at room temperature for 30 minutes. After washing each well three times with washing buffer, 50 μL of OPD chromogenic solution was added to each well and incubated at room temperature for 10 minutes. 50 μL of stop solution was added to each well, and the absorbance was measured at 492 nm using a microplate reader. The absorbance 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 value of 3.934 at the intersection of IL-2R and RbFc represents the absorbance value when the fusion protein containing IL-2R and RbFc is reacted with the antibody.

[0128]

[0129] When all antigens were expressed as fusion proteins with RbFc, high signal intensity was observed in the reactions of the fusion proteins derived from the culture supernatant with antibodies responding to each antigen. This indicates that the expression level of RbFc fusion proteins in the culture supernatant is high, and their reactivity with antibodies responding to each antigen is also good. When LR11, P1NP, PCT, SP-D, and TARC were expressed as fusion proteins with HSA, the reaction signal intensity of the fusion proteins derived from the culture supernatant with antibodies responding to each antigen was lower compared to the RbFc fusion protein expression. This suggests that the expression level of HSA fusion proteins in the culture supernatant is lower than that of RbFc fusion proteins, or that their reactivity with antibodies responding to each antigen is lower. When PCT, PSA, and TARC were expressed by binding to Histag, the reaction signal intensity of the fusion proteins derived from the culture supernatant with antibodies responding to each antigen was lower compared to the RbFc fusion protein expression. It can be assumed that the expression level of Histag-binding proteins in the culture supernatant is lower than that of RbFc fusion proteins, or that their reactivity with antibodies reacting to various antigens is lower. In summary, by fusing antigens with RbFc, multiple antigens can be stably expressed.

[0130] <Confirmation of multimer formation of fused proteins>

[0131] To confirm whether the fusion protein expressed by pDNA-SP-D-RbFc forms a multimer, an investigation was conducted into whether the fusion protein could be detected by a sandwich ELISA using the same antibody.

[0132] Add 50 μL of abSP-D diluted to 5 μg / mL with PBS to each well of a 96-well ELISA plate and incubate overnight at 4°C. Wash each well three times with washing buffer, then add 100 μL of blocking buffer to each well and incubate overnight at 4°C. After removing the blocking buffer, add 50 μL of culture supernatant containing SP-D-RbFc diluted 5–78–125 times with blocking buffer to each well and incubate for 1 hour at room temperature. Wash each well three times with washing buffer, then add 50 μL of biotinylated abSP-D solution adjusted to 1 μg / mL with blocking buffer to each well and incubate for 1 hour at room temperature. 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 added to each well, and the solution was incubated at room temperature for 30 minutes. After washing each well three times with washing buffer, 50 μL of OPD chromogenic solution was added to each well, and the solution was incubated at room temperature for 10 minutes. Finally, 50 μL of stop solution was added to each well, and the absorbance was measured at 492 nm using a microplate reader.

[0133] As a comparative experiment, the same procedure was followed to confirm whether culture supernatants containing SP-D-HSA or SP-D-Histag could be detected by sandwich ELISA. The results are shown in Table 9.

[0134]

[0135] SP-D-RbFc in the culture supernatant, like SP-D-Histag, can be detected by sandwich ELISA using the same antibody. That is, SP-D-RbFc and SP-D-Histag form polymers in the same way. On the other hand, while SP-D-has can be detected by sandwich ELISA using the same antibody at low dilutions, the correlation between antigen concentration and detection signal is poor.

[0136] <Calibration Performance>

[0137] 1. Preparation of LTIA reagent for P1NP detection

[0138] 1-1. Preparation of anti-P1NP antibody sensitized latex

[0139] Antibody solutions of abP1NP-1 and abP1NP-2 were prepared using 40 mM glycine buffer (hereinafter referred to as sensitization buffer) to achieve an absorbance of 0.5 at 280 nm. 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 the 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 the 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).

[0140] 1-2. Preparation of LTIA reagent

[0141] The first reagent and the second reagent were prepared as described below.

[0142] First reagent: 100mM HEPES pH 7.0

[0143] 500mM NaCl

[0144] 0.5% BSA

[0145] Second reagent: abP1NP-1 sensitized latex (absorbance at 600nm is 3.0)

[0146] abP1NP-2 sensitizing latex (absorbance at 600nm is 3.0)

[0147] 5mM MOPS pH7.0

[0148] 1-3. Preparation of calibrators

[0149] Four concentrations of random human serum containing P1NP were used. Collagen Intact P1NP (manufactured by Yamasa Soy Sauce Co., Ltd.) was used before use. The P1NP concentration in each sample was determined according to the method described in the accompanying instructions, and the concentration of P1NP in each antigen solution was calculated. Samples S1-S4, with determined P1NP concentrations, were used as calibrators 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.

[0150] 1-4. Determination based on LTIA reagent

[0151] The first and second reagents were placed in the reagent compartment, and the determination was performed using a Hitachi Automated Analyzer 3500 under the following conditions.

[0152] (Measurement conditions)

[0153] Analysis method: Two-point endpoint method

[0154] Metering point: 19~34

[0155] Measurement wavelength: 570 / 800nm

[0156] Sample volume: 5 μL

[0157] First reagent: 120 μL

[0158] Second reagent: 40 μL

[0159] 1-5. Preparation of Calibration Curves

[0160] The results of the measurements in items 1-4 were performed using calibrator 1 and physiological saline (P1NP concentration: 0 ng / mL) as samples. The result was plotted with the change in absorbance per unit time during the measurement as the vertical axis and the P1NP concentration of calibrator 1 as the horizontal axis to create calibration curve 1.

[0161] 1-6. Preparation of the test sample

[0162] 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.

[0163] 2. Confirmation of whether the fusion protein can be diluted and recovered.

[0164] Samples prepared by serially diluting calibrator 1's S5, P1NP-RbFc, and P1NP-HSA solutions with physiological saline were used as test samples for determinations 1-4. The P1NP concentration in each solution was calculated using calibration curve 1. The recovery rate of each protein was obtained by dividing the measured values ​​by the theoretical values ​​calculated based on the dilution ratio. The results are shown in Table 10.

[0165]

[0166] P1NP-RbFc showed good recovery rates similar to those of P1NP in serum. The recovery rate of P1NP-HSA decreased with increasing dilution factor.

[0167] 3. Calibration was performed using a standard sample containing the fusion protein.

[0168] P1NP-RbFc was appropriately diluted with physiological saline to prepare S1'~S4'. S1'~S4' were used as samples and measured using method 1-4. The P1NP values ​​in S1'~S4' were calculated using calibration curve 1. S1'~S4' with determined P1NP values ​​were used as calibrators 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 constructed by plotting the change in absorbance per unit time during the measurements with physiological saline and calibrator 2 on the ordinate and the P1NP concentration of calibrator 2 on the abscissa. Any human serum sample containing P1NP was used as a sample for the measurements in section 1-4. The P1NP concentration in the sample was calculated using calibrator 1 and calibrator 2. The correlation between the calculated values ​​and the values ​​obtained when the same specimens were measured using procollagen Intact P1NP was confirmed.

[0169] Figure 1 This is a graph showing the correlation between P1NP measurements calculated using calibration curve 2 (vertical axis) and P1NP measurements based on procollagen IntactP1NP (horizontal axis), which was prepared using calibrator 2 prepared using P1NP-RbFc. The slope of the regression line is 1.03, and the correlation coefficient is 0.967. Figure 2 This is a graph showing the correlation between P1NP measurements calculated using calibration curve 1 (vertical axis) and P1NP measurements based on procollagen Intact P1NP (horizontal axis), which was prepared using calibrator 1 prepared using human serum. The slope of the regression line is 1.0674, and the correlation coefficient is 0.964. P1NP measurements using P1NP-RbFc as the calibrator antigen and P1NP measurements using P1NP from human serum as the calibrator both show good correlation with the measurements based on procollagen Intact P1NP. That is, the calibrator prepared using P1NP-RbFc can be considered to have equivalent calibration performance to P1NP from human serum.

[0170] Industrial applicability

[0171] Based on the above methods, it is possible to provide immunoassay methods, precision management methods, and standard samples and immunoassay reagents for these methods.

Claims

1. An immunoassay method, comprising: A process for detecting a signal corresponding to the concentration or titer of an antigen contained in a biological sample; and The procedure of calculating the concentration or titer of the antigen in the biological sample based on the signal using a calibration curve representing the relationship between the concentration or titer of the antigen and the signal. The calibration curve was prepared using a standard sample containing the fusion protein. The fusion protein comprises 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.

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 rabbit antibodies.

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

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 precision management of an antigen immunoassay, comprising: The process of detecting a signal corresponding to the concentration or titer of a fusion protein in a standard sample containing a pre-set concentration or titer of the fusion protein; The process of calculating the concentration or titer of the fusion protein based on the detected signal; and The process of evaluating the accuracy of the immunoassay by comparing the calculated concentration or titer with the preset concentration or titer. The fusion protein comprises 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.

7. The method for accuracy management of antigen immunoassay according to claim 6, wherein, At least a portion of the amino acid sequence of the constant region of the immunoglobulin is derived from rabbit antibodies.

8. The method for accuracy management of antigen immunoassay according to claim 6, wherein, The antigen is a protein that exists in the body as a polymer.

9. The method for accuracy management of antigen immunoassay according to claim 6, wherein, The antigen is one of P1NP, PCT, SP-D, and TARC.

10. The method for accuracy management of antigen immunoassay according to claim 6, wherein, The antigen is P1NP.

11. A standard sample, which is a standard sample used in the immunoassay method according to any one of claims 1 to 5, or in the precision management method of the immunoassay according to any one of claims 6 to 10. in, The standard sample contains, at a known concentration or known titer, a fusion protein comprising 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.

12. An immunoassay method, comprising: The process of forming a complex of antibody and fusion protein in solution and detecting the signal emitted by the labeling substance bound to the fusion protein. The fusion protein is a fusion protein consisting 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 the constant region of an immunoglobulin.

13. An immunoassay method, comprising: A process for obtaining a reaction system in which a biological sample, an antibody, and a competing substance bound to a labeled substance coexist, wherein the biological sample may contain an antigen, the antibody is capable of binding to the antigen, and the competing substance is capable of competitively binding to the antibody against the antigen. and The step of measuring the signal of the labeled substance contained in the reaction system. The competing substance is a fusion protein consisting 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 an immunoglobulin.

14. The immunoassay method according to claim 13, wherein, Between the step of obtaining the reaction system and the step of measuring the signal, a further step of obtaining a measurement system is included, in which the antibody is retained in the reaction system and the antigen and the competing substance that are not bound to the antibody are removed by washing, thereby obtaining a measurement system containing the antigen and the competing substance that are 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 rabbit antibodies.

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

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 comprising the said fusion protein, The immunoassay reagent is used in the immunoassay method according to claim 12.

20. An immunoassay reagent comprising the competing substance, The immunoassay reagent is used in the immunoassay method according to claim 13.

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