WO2025205534A1 - Immunoassay method, accuracy management method, standard sample, and immunoassay reagent - Google Patents

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

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.

To quantify the components to be measured by immunoassay in biological samples, a calibration sample must be used. Calibration samples are samples containing the components to be measured and are used as internal standards or concentration calibration standards (calibrators). To obtain accurate quantitative values for the components to be measured, a calibration sample that is stable over time and with temperature changes is required.

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

International Publication No. 2023/145916

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, depending on the type of antigen, sufficient expression levels may not be obtained.

As such, there is room for further improvement in the standard samples used in immunoassays.

The object of the present invention is to provide an immunoassay method, a quality control method, and standard samples and immunoassay reagents for use therein.

The present invention includes the following aspects.
[1] 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 a constant region of an immunoglobulin.
[2] The immunoassay method according to [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 [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 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 evaluating the quality of the immunoassay by comparing the calculated concentration or titer with the predetermined 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 an immunoglobulin constant region.
[7] The method for controlling the accuracy of immunoassays of antigens according to [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 [6] or [7], wherein the antigen is a protein that exists as a multimer in vivo.
[9] The method for quality control of immunoassay of an antigen 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 quality control method for immunoassay according to any one of [6] to [10], the standard sample comprising 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 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.
[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 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 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 [12] or [13], wherein at least a part of the amino acid sequence of the constant region of the immunoglobulin 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 containing the fusion protein, which is used 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.

The above aspects provide an immunoassay method, a quality control method, and standard samples and immunoassay reagents used therein.

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

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".

As used herein, biological samples primarily refer to bodily fluids derived from living organisms. 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 and animals (e.g., monkeys, dogs, or cats), preferably humans. Biological samples may be the sample itself from the subject, or may be samples that have been collected and subjected to conventional treatments such as dilution or concentration. The person who collects and prepares the biological sample used in the present invention may be the same 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 the measurement method of the present invention is performed, or may be collected or prepared in advance and stored.

<Immunoassay method and standard sample>
In one aspect of the present invention, the immunoassay method 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.

In one aspect of the present invention, the standard sample is a standard sample 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 known titer. The concentration of the fusion protein can be calculated using the BCA method, Bradford method, Lowry method, or existing test reagents for measuring the antigen contained in the fusion protein. The titer of the fusion protein can be calculated by protease titer measurement, etc.

Standard samples can be used as calibrators to create calibration curves for clinical diagnostic reagents. Standard samples can also be used as controls measured for quality control of clinical diagnostic reagents.

The fusion protein contains an amino acid sequence identical to the antigen to be measured in the immunoassay, or an amino acid sequence identical to a portion of the antigen. Antigens to be measured include brain natriuretic peptide (BNP), C-reactive protein (CRP), D-dimer, heart-type fatty acid-binding protein (H-FABP), and 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 abundance 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 approximately 30 to 1,000 amino acids or a complex thereof, and more preferably an oligopeptide consisting of approximately 50 to 700 amino acids or a complex thereof.

When the fusion protein contains an amino acid sequence identical to a portion of the antigen to be measured, it is preferable that the fusion protein contains at least an amino acid sequence identical to an epitope of the antigen to be measured. When the fusion protein contains an amino acid sequence identical to a portion of the antigen to be measured, the "portion of the antigen to be measured" may be one location or multiple locations. In other words, 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, etc., it is preferable that the fusion protein 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 the α1-2 sequence of the α1 chain of P1NP.

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.

The fusion protein contains 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 contains 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.

The fusion protein of this embodiment preferably further includes 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.

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 that is identical to the amino acid sequence of multiple positions in a portion of the antigen to be measured, a linker may be positioned between the amino acids.

The fusion protein of this embodiment preferably includes 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. Including 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 preferable 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.

The fusion protein of this embodiment is 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 secreting the fusion protein outside the cells. Because the fusion protein of this embodiment is expressed in large amounts outside the cells, it is easy to recover from the cell culture medium and has high productivity.

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 standard sample. The calibration curve can be prepared using a conventional method. For example, it can be prepared by plotting the signal when measuring the standard sample or the amount of 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.

In this embodiment, a signal corresponding to the concentration or titer of the antigen to be measured contained in the biological sample is detected, and a calibration curve showing 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 from the signal. Examples of signals include transmitted light, scattered light, fluorescence, chemiluminescence, electrochemiluminescence, or coloration.

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

(LTIA)
LTIA is an immunoassay method that utilizes the agglutination of latex particles, which occurs when an antibody supported on the surface of the latex particles binds to an antigen to be measured. There are no particular limitations on the latex particles, as long as they are latex particles commonly used in in vitro diagnostic reagents. The concentration of latex particles during agglutination reaction measurement, the average particle size of the latex particles, and other parameters can be appropriately set depending on sensitivity or performance. When using latex immunoturbidimetry as the immunoassay method of this embodiment, the measurement procedure and principle are as follows.
(1) The sample is brought into contact with latex particles carrying 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., wavelength 600 nm) and the transmitted light or scattered light (i.e., signal) is measured. The concentration or titer of the antigen is calculated based on the calibration curve prepared using the standard sample of this embodiment and the measured signal.

A biochemical automatic analyzer (e.g., Hitachi Automated Analyzer 3500, manufactured by Hitachi) can be used as the measuring device.

(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. The solid phase is preferably a plate (also called an immunoplate). HRP or ALP can be used as a 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 brought into contact with a solid phase on which an antibody has been immobilized, and the antigen in the sample is allowed to bind to the solid-phase antibody.
(2) 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 labeling 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 prepared using the standard sample of this embodiment and the measured signal value.

In the sandwich ELISA method, a secondary antibody can also be used. By using a secondary antibody, the reaction is amplified, and the detection sensitivity can be increased. When a secondary antibody is used, the following steps (1) to (5) can be adopted.
(1) A sample is added to a solid phase on which an antibody has been immobilized, followed by incubation, followed by removal of the sample and washing.
(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) Add a substrate to develop color.
(5) The color development (i.e., the signal) is measured using a plate reader, etc. The concentration or titer of the antigen is calculated based on the calibration curve prepared using the standard sample of this embodiment and the measured signal value.

(Electrochemiluminescence Immunoassay)
Electrochemiluminescence immunoassay is a method for measuring the amount of an antigen to be measured 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 the electrode, exciting the ruthenium complex to emit light. The amount of light emitted by the ruthenium complex can then be detected.

The measurement procedure and principle when using magnetic particles as the solid phase and a ruthenium complex as the labeling substance are as follows:

(1) A sample is brought into contact with magnetic particles on which solid-phase antibodies are immobilized, and the antigen in the sample is allowed to bind to the solid-phase antibodies.
(2) After washing the magnetic particles, the magnetic particles are contacted with a labeled antibody, 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 passed through them, and the amount of luminescence corresponding to the amount of labeled antibody bound to the antigen is measured, thereby determining the amount of antigen in the biological sample.

(Immunochromatography)
Immunochromatography is an immunoassay method that utilizes the properties 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 an antigen is immobilized on an insoluble membrane carrier, which serves as a chromatographic medium, to create a detection section serving 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, serving as the mobile phase, and then, in the detection section serving as the 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 gold colloid particles, platinum colloid particles, colored latex particles, and magnetic particles. Gold colloid 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.

When immunochromatography is employed as the immunoassay method of the present invention, the measurement procedure and principle are as follows.
(1) The sample is brought into contact with a sample supplying section for supplying the sample.
(2) The antigen in the sample is brought into contact with the conjugate to form a first complex, which is a first monoclonal antibody bound to a labeling substance.
(3) The first complex is contacted with a second monoclonal antibody immobilized on a detection section to form a second complex.
(4) The amount or presence of the antigen in the sample is determined by measuring the intensity of the signal derived from the labeling substance contained in the conjugate.

The immunoassay method of this embodiment uses the standard sample of this embodiment, thereby achieving high measurement accuracy equivalent to that of substances purified from biological samples.

Furthermore, according to the standard sample of this embodiment, because the fusion protein contains at least a portion of the amino acid sequence of the immunoglobulin constant region, a sufficient amount of the standard sample is expressed in the culture supernatant of transformed cells even when expressed using a general plasmid. Furthermore, when the standard sample of this embodiment is used, high measurement accuracy equivalent to that of standard samples produced using biological samples can be obtained.

<Immunoassay method and immunoassay reagent>
In another aspect of this embodiment, an 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.

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 competitively to the antigen with respect to the antibody and having a labeling substance bound thereto are present, and measuring the signal of the labeling substance contained in the assay 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. After the step of obtaining the reaction system, the immunoassay method may include the 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.

In this immunoassay method, an antibody capable of binding to the antigen to be measured is used. The fusion protein described above 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.

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, but examples include competitive ELISA, LTIA, electrochemiluminescence immunoassay, and immunochromatography.

The competitive ELISA method can employ the following steps (1) to (5).
(1) A reaction system is obtained in which a sample and the competitive substance 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 leaving the antibody in the reaction system, thereby obtaining a measurement system containing the antigen and the competitor that are bound to the antibody.
(3) Add a substrate to develop color.
(4) The color (i.e., signal) of the labeling substance contained in the measurement system is measured using a plate reader or the like.
(5) Calculate the antigen concentration or titer based on the calibration curve and the measured signal value.

The antigens to be measured are not particularly limited, but examples include IL-2R, LR11, NT-proBNP, P1NP, PCT, PSA, SP-D, and TARC.

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.

<Accuracy control method for antigen immunoassay>
The quality control method for antigen immunoassay of this embodiment is 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 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.

In the quality control method for antigen immunoassays, standard samples are used as controls 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 methods and standard samples>, so its explanation is omitted here.

One aspect of the method for controlling the accuracy of 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 the fusion protein at a predetermined concentration or titer. A standard sample of known concentration and titer and the clinical diagnostic reagent are then 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 reagents, clinical testing devices, etc. can perform accurate measurements.

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.

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.

The quality control method for antigen immunoassays of this embodiment uses the standard sample of this embodiment, allowing for control with the same high level of accuracy as that achieved with substances purified from biological samples.

The present invention will be explained in more detail below using examples, but the present invention is not limited to the examples described below.

<Plasmid>
The plasmid used to express the fusion protein was a pcDNA3.1(-) Mammalian Expression Vector (ThermoFisher SCIENTIFIC) containing the signal sequence (SEQ ID NO: 1) shown in Table 1, the entire or partial sequence of the antigen, Histag, rabbit antibody Fc (RbFc), and a nucleotide 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)). The nucleotide sequences encoding the entire antigen sequences were used for the plasmids used to express the fusion antigens 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). 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.

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

The plasmids used for the comparison experiment were a plasmid containing a base 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 base sequences inserted into each fusion protein expression plasmid or Histag-binding protein expression plasmid.

 

<Expression of fusion protein>
Fusion proteins were expressed using the ExpiCHO Expression System Kit (Thermo Fisher Scientific, product number: A29133) according to the product protocol. More specifically, Expi CHO cells were seeded at 3-4 x ¹⁰ cells/mL in dedicated medium and cultured. The next day, Plasmid DNA prepared at 1 μg/μL was mixed with OptiPro medium, and further mixed with a separately prepared mixture of OptiPro medium and ExpiFectanubeCHO reagent, and allowed to stand at room temperature for 1-5 minutes. The resulting mixture was added to Expi CHO cells seeded at 7-10 x ¹⁰ 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 re-cultured. After 8 days, the supernatants were collected and used for each experiment.

<Obtaining antigen-binding monoclonal antibodies>
Antibodies reactive with each antigen were produced according to the method described in WO 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 myeloma cells SP2/0 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 the selected antibody cell lines, and the resulting 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.

<Measurement of expression level of fusion protein>
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, were 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.

When each antigen was expressed as a fusion protein with RbFc, a high signal was observed due to the reaction between the fusion protein in the culture supernatant and antibodies reactive with each antigen. The RbFc fusion protein was highly expressed in the culture supernatant, suggesting 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 due to the reaction between the fusion protein in the culture supernatant and antibodies reactive with each antigen was lower than that of the RbFc fusion protein. It is thought that the HSA fusion protein is either expressed in lower amounts in the culture supernatant or has lower reactivity with antibodies reactive with each antigen than the RbFc fusion protein. When PCT, PSA, and TARC were expressed in conjunction with Histag, the signal due to the reaction between the fusion protein in the culture supernatant and antibodies reactive with each antigen was lower than that of the RbFc fusion protein. It is thought that Histag-binding proteins are expressed in lower amounts in the culture supernatant than RbFc fusion proteins, or have lower reactivity with antibodies that react with each antigen. From the above, it was found that a variety of antigens can be stably expressed by fusing RbFc to antigens and expressing them.

<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.

AbSP-D diluted to 5 μg/mL in PBS was dispensed at 50 μL per well of a 96-well ELISA plate and left 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 left 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 left 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 in blocking solution was dispensed into each well and left 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 a wavelength of 492 nm was measured using a plate reader.

In a control experiment, we also confirmed whether culture supernatant 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.

SP-D-RbFc in the culture supernatant could be detected by sandwich ELISA using the same antibody, just like SP-D-Histag. This indicates that SP-D-RbFc forms multimers, just 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, there was a poor correlation between the antigen concentration and the detection signal.

<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).

1-2. Preparation of LTIA Reagents 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)
5mM MOPS pH7.0

1-3. Preparation of Calibrators Four concentrations of human serum containing P1NP were used. Each sample was measured using intact procollagen P1NP (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.

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.

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

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.

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.

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.

P1NP-RbFc had a good recovery rate, similar to P1NP in serum. The recovery rate of P1NP-HSA decreased as the dilution ratio increased.

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.

Figure 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. Figure 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 calibrator antigen and 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.

The above aspects provide an immunoassay method, a quality control method, and standard samples and immunoassay reagents used therein.

Claims (20)

detecting a signal corresponding to the concentration or titer of an antigen contained in a biological sample;
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;
the calibration curve is prepared using a standard sample containing the fusion protein;
The immunoassay method, wherein 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 an immunoglobulin constant region. 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. The immunoassay method according to claim 1, wherein the antigen is a protein that exists as a multimer in vivo. The immunoassay method according to claim 1, wherein the antigen is at least one of P1NP, PCT, SP-D, and TARC. The immunoassay method according to claim 4, wherein the antigen is P1NP. A method for controlling the quality of an antigen immunoassay, comprising:
detecting a signal corresponding to the concentration or titer of a fusion protein in a standard sample containing the fusion protein at a predetermined concentration or titer;
calculating the concentration or titer of the fusion protein from the detected signal;
comparing the calculated concentration or titer with the predetermined concentration or titer to evaluate the accuracy of the immunoassay,
A method for controlling the accuracy of immunoassays of antigens, wherein 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 an immunoglobulin constant region. The method for controlling the accuracy of antigen immunoassays 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. The method for controlling the accuracy of antigen immunoassays according to claim 6, wherein the antigen is a protein that exists as a multimer in vivo. The method for quality control of antigen immunoassays according to claim 6, wherein the antigen is one of P1NP, PCT, SP-D, and TARC. The method for quality control of antigen immunoassays according to claim 6, wherein the antigen is P1NP. A standard sample used in the immunoassay method described in any one of claims 1 to 5 or the immunoassay quality control method described in any one of claims 6 to 10, the standard sample containing 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. 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,
The immunoassay method, wherein the fusion protein is a fusion protein of at least a portion of an amino acid sequence of an antigen capable of binding to the antibody and at least a portion of an amino acid sequence of a constant region of an immunoglobulin. 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 in a competitive manner with respect to the antibody and having a labeling substance bound thereto are present;
measuring a signal of the labeling substance contained in the reaction system,
An immunoassay method, 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. 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 competitor 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 competitor that are bound to the antibody. 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. The immunoassay method according to claim 12 or 13, wherein the antigen is a protein that exists as a multimer in vivo. The immunoassay method according to claim 12 or 13, wherein the antigen is one of P1NP, PCT, SP-D, and TARC. The immunoassay method according to claim 12 or 13, wherein the antigen is P1NP. An immunoassay reagent containing the fusion protein, for use in the immunoassay method described in claim 12. An immunoassay reagent containing the competitor, for use in the immunoassay method described in claim 13.