Immunoassay method using site-specific modifications of IgG antibodies with IgG-binding peptides

Abstract

To provide an immunoassay method using a site-specifically modified body of an IgG antibody with an IgG-binding peptide, which is capable of binding to IgG derived from rodents and undergoes minimal denaturation or functional loss even upon immobilization onto a material surface.SOLUTION: The present invention pertains to an immunoassay method that uses a complex of IgG and an IgG-binding peptide that is bound to a functional ligand.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an immunoassay method using a site-specific modified form of an IgG antibody using an IgG-binding peptide.

Background Art

[0002] In immunoassay methods, antibodies are often immobilized on the surface of a material by covalent bonds obtained by physical adsorption or amine coupling. In this case, it is required that the antibody immobilized on the material surface does not undergo denaturation or functional loss. However, in conventional methods, denaturation or functional loss of the antibody immobilized on the material surface may occur, resulting in a decrease or loss of antigen-binding ability.

[0003] Therefore, as a method for modifying human IgG with less decrease in antigen-binding ability, the CCAP method (chemical conjugation by affinity peptide) has been developed (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, the conventional CCAP method is not applicable to IgG derived from rodents such as mice and rats, which are often used in in vitro diagnosis.

[0006] The present invention aims to provide an immunoassay method using a site-specific modification of an IgG antibody that utilizes an IgG-binding peptide, which can bind to rodent-derived IgG and exhibits minimal denaturation or functional loss even when immobilized on a material surface. [Means for solving the problem]

[0007] This invention relates to an immunoassay method using a complex of IgG and an IgG-binding peptide to which a functional ligand is bound.

[0008] The present invention also relates to an in vitro diagnostic agent comprising a complex of IgG and an IgG-binding peptide to which a functional ligand is bound.

[0009] The present invention also relates to a solid phase in which a complex of IgG and an IgG-binding peptide conjugated with a functional ligand is immobilized.

[0010] The present invention also relates to an immunochromatographic test piece for use in the above-mentioned immunoassay method, comprising a labeled antibody holding portion for holding a labeled antibody and a detection region on which a capture antibody is immobilized, wherein the labeled antibody, the capture antibody, or both are a complex of IgG and an IgG-binding peptide to which a functional ligand is bound.

[0011] The present invention further relates to a method for producing the solid phase, comprising the steps of covalently bonding or physically adsorbing an anchor protein to the solid phase, and covalently bonding or physically adsorbing a complex of IgG and an IgG-binding peptide to the anchor protein bonded to the solid phase. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide an immunoassay method using a site-specific modified IgG antibody that uses an IgG-binding peptide, which can bind to rodent-derived IgG and exhibits minimal denaturation or functional loss even when immobilized on a material surface. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 shows a model of site-specific covalent bond formation on the surface of the detection substrate material of the complex via anchor proteins. [Figure 2] Figure 2 shows a model of site-specific covalent bond formation on the surface of the detection substrate material of the complex, without the use of anchor proteins. [Figure 3] Figure 3 shows a crosslinking model between IgG-Fc and Z34C. [Figure 4] Figure 4 shows the results of SDS-PAGE, which investigated the reactivity of Z34C variants (αZ34C, εZ34C, and α-1Z34C) to mouse IgG1 or mouse IgG2a. [Figure 5] Figure 5 shows the results of SDS-PAGE, which investigated the reactivity of Z34C variants (αZ34C, εZ34C, and α-1Z34C) to human IgG1. [Figure 6] Figure 6 shows the results of SDS-PAGE, which examined the reactivity of αZ34C to IgG derived from humans, mice, rabbits, or rats. [Figure 7] Figure 7 shows the results of SDS-PAGE, which investigated the reactivity of the Z34C variant Z33-38biotin to human IgG1 or mouse IgG2a. [Figure 8] Figure 8 shows the results of SDS-PAGE, which examined the reactivity of Z33-38biotin to mouse IgG1 or mouse IgG2a, as used in Test Examples 4 and 5. [Figure 9] Figure 9 shows the results of investigating the antigenic reactivity of biotin-bound IgG obtained using the CCAP method and biotin-bound IgG obtained using the random amine coupling method when each antibody was applied to an ELISA system. [Figure 10] Figure 10 shows the results of investigating the antigenic reactivity of biotin-bound IgG obtained using the CCAP method and biotin-bound IgG obtained using the random amine coupling method when the antibodies were applied to the RPLA method. [Figure 11]Figure 11 shows the actual latex aggregation image when the RPLA reaction was performed. [Figure 12] Figure 12 shows the results of examining the antibody titers of biotinylated IgG obtained by the CCAP method and biotinylated IgG obtained by the random amine coupling method by ELISA. [Figure 13] Figure 13 shows the results of examining the influenza antigen-specific aggregation reaction using particles obtained by immobilizing an anti-influenza antibody by site-specific covalent bond formation by the CCAP method.

Mode for Carrying Out the Invention

[0014] Hereinafter, the present invention will be described in detail. In this specification, "the antibody binds directly to the surface of the detection substrate material" means a mode in which a covalent bond is formed between the surface of the detection substrate material and the IgG-binding peptide. On the other hand, "physical adsorption" means a mode in which the antibody is adsorbed on the surface of the detection substrate material by electrostatic interaction or hydrophobic bond.

[0015] The immunoassay method according to one embodiment uses a complex of IgG and an IgG-binding peptide to which a functional ligand is bound. By using the immunoassay method according to this embodiment, it is possible to bind peptide / IgG under conditions that do not require genetic modification of antibody-engineered antibody molecules, react rapidly at room temperature, and do not impose a burden on the antibody.

[0016] IgG can be mammalian IgG. IgG can be, for example, human IgG (IgG1, IgG2, IgG3 or IgG4), rabbit IgG, rat IgG (IgG1, IgG2a, IgG2b or IgG2c), or mouse IgG (IgG1, IgG2a, IgG2b, IgG2c or IgG3).

[0017] Examples of functional ligands include drugs, proteins, peptides, nucleic acids, enzymes, radiolabeled substances, fluorescent substances, and chemical crosslinkers having functional groups that can be covalently bonded to the surface of the detection substrate material. More specifically, the functional ligand may be biotin or an azide compound. The functional ligand may be directly bound to the IgG-binding peptide or bound via a molecule such as PEG (polyethylene glycol).

[0018] The binding of IgG-binding peptides to functional ligands can be carried out by known methods, such as the reaction of an azide group (-azide) with a DBCO (dibenzocyclooctyne) group, or the reaction of a maleimide group with a sulfhydryl group (-SH).

[0019] In one embodiment, the IgG-binding peptide is a peptide derived from protein A, and includes, for example, a partial peptide of the B domain or Z domain of protein A or a variant thereof. Examples of IgG-binding peptides include Z34C and its variants. Z34C is derived from the B-domain of protein A and has been optimized by the phage library method. Z34C exhibits particularly high affinity (binding ability) to human and rodent IgG-Fc. The IgG-binding peptide may also include peptides selected from the peptides represented by SEQ ID NOs: 1 to 9, and may include peptides in which one or more amino acids are substituted, deleted, or added, which have the ability to bind to the Fc region of IgG. Furthermore, the IgG-binding peptide may include peptides selected from the peptides represented by SEQ ID NOs: 1 to 9, or peptides in which one or more amino acids are substituted, deleted, or added, which have the ability to bind to the Fc region of IgG, to which a functional ligand or a functional group for attaching a functional ligand has been added. Sequence ID 1 is an amino acid sequence in which all Lys molecules in Z34C are replaced with Arg molecules, and is denoted as αZ34C. Sequence ID 2 is an amino acid sequence in which the Phe molecule at the N-terminus of αZ34C is replaced with Lys, and is denoted as εZ34C. Sequence ID 3 is an amino acid sequence in which Gly is added to the N-terminus of αZ34C, and is denoted as α-1Z34C. Sequence ID 4 is an amino acid sequence in which the 7th Arg molecule of αZ34C is replaced with Lys. Sequence ID 5 is an amino acid sequence in which the 7th Arg molecule of εZ34C is replaced with Lys. Sequence ID 6 is an amino acid sequence in which the 8th Arg molecule of α-1Z34C is replaced with Lys. Sequence ID 7 is an amino acid sequence in which the 5th Cys molecule of αZ34C is replaced with Gln, and the Cys molecule at the C-terminus is removed. Sequence ID 8 is an amino acid sequence obtained by substituting the 5th Cys of αZ34C with Gln, removing the C-terminal Cys, and adding Pro-Ser-Arg-Arg-Lys-Arg to the C-terminus, and is denoted as Z33-38.Sequence ID 9 is an amino acid sequence obtained by substituting the 5th Cys of αZ34C with Gln, substituting the 7th and 28th Arg with Lys, removing the C-terminal Cys, and adding Pro-Ser-Arg-Arg-Lys-Arg-Arg-Lys-Arg-Arg-Lys to the C-terminus, and is denoted as Z33-5.

[0020] In the peptides represented by SEQ ID NOs: 1 to 9, the number of amino acid residues that are substituted, added, or deleted can be, for example, 1 to 10, 1 to 5, 1 to 3, or 1 to 2.

[0021] Modified Z34C can be synthesized by known methods. Examples of synthesis methods include solid-phase synthesis methods such as the Fmoc synthesis method and the Boc synthesis method, and liquid-phase synthesis methods such as the fragment condensation method. However, from the viewpoint of ease of operation, solid-phase synthesis methods are preferred. When IgG-binding peptides such as modified Z34C are modified with a crosslinking agent, such IgG-binding peptides can be produced by modifying the synthesized IgG-binding peptide with the crosslinking agent, or by performing peptide synthesis using amino acid residues modified with the crosslinking agent.

[0022] In one embodiment, the IgG-binding peptide is thought to be bound to the side chain of Lys248 in the Fc region of IgG, and the two are linked via a crosslinking agent, such as DSG (Disuccinimidyl Glutarate). Other examples of the above crosslinking agent include crosslinking agents that preferably contain two or more succinimidyl groups, such as DSS (Disuccinimidyl suberate); crosslinking agents that preferably contain two or more imide ester portions, such as DMA (Dimethyl Adipimidate Dihydrochloride), DMP (Dimethyl Pimelimidate Dihydrochloride), and DMS (Dimethyl Suberimidate Dihydrochloride); and crosslinking agents having SS bonds, such as DTBP (Dimethyl 3,3'-dithio-bis(propionimidate) Dihydrochloride) and DSP (Dithiobis (succinimidyl propionate)).

[0023] Immunoassay methods include ELISA, immunochromatography, latex agglutination (LA), turbidimetric immunoassay (TIA), chemiluminescent immunoassay (CLIA), pulsed immunoassay, time-resolved fluorescence resonance energy transfer (TR-FRET), quartz crystal microbalance (QCM), biolayer interferometry (BLI), and surface plasmon resonance (SPL) assays.

[0024] An in vitro diagnostic agent according to one embodiment includes a complex of IgG and an IgG-binding peptide to which a functional ligand is bound. When included in the in vitro diagnostic agent, the IgG-binding peptide may be modified with an enzyme, a radiolabeled substance, a fluorescent substance, etc. By using the above immunoassay method, it is possible to suppress the decrease in antigen affinity of the antibody and enhance the immobilization orientation of the material surface. Therefore, it is thought that if applied to an in vitro diagnostic agent using an immunoassay method, for example, highly sensitive measurement will be possible.

[0025] In one embodiment, the solid phase has a complex of IgG and an IgG-binding peptide to which a functional ligand is bound immobilized.

[0026] The above-mentioned composite may be immobilized directly on the surface of the detection substrate material by physical adsorption, either without an anchor protein or via an anchor protein. Examples of detection substrates include latex particles (Ltx), organic materials such as polypropylene and polystyrene, or immunoassay plates (plates) mainly composed of glass.

[0027] The above complex may be immobilized on the surface of a detection substrate material equipped with a functional group that reacts with an amino group, via an anchor protein (Figure 1). The anchor protein (Protein in Figure 1) is not particularly limited, but can be streptavidin, etc., and BSA or HSA, which are used as blocking agents, are preferred in consideration of the balance with non-specific reactions.

[0028] As shown in Figure 1, -DBCO can be introduced into an anchor protein bound to the surface of a detection substrate material by reacting a functional group that reacts with an amino group with an amino group in the anchor protein, or by reacting a functional group that reacts with a carboxyl group with a carboxyl group in the anchor protein, or by further reacting the amino group in the anchor protein with DBCO-NHS, or the -SH group in the anchor protein with DBCO-maleimide. Furthermore, a click reaction can be generated between the -DBCO introduced into this anchor protein and the -azide introduced into the IgG-binding peptide in the complex, thereby indirectly immobilizing the complex on the surface of the detection substrate material. Note that the functional group to be introduced is not limited to -DBCO, but any functional group that can be used in a click reaction is acceptable. The functional group to be introduced may be, for example, a compound having an alkyne, and more specifically, cyclooctyne, BCN (bicyclo[6.1.0]nonyne), etc. In addition to this click reaction, reactions using -SH and -maleimide or bromoacetyl can also be used.

[0029] Functional groups that react with amino groups include carboxyl groups, tosyl groups, epoxy groups, isocyanate groups, isothiocyanate groups, N-hydroxysuccinimide groups (-NHS), and maleimide groups. For detection substrates with functional groups that react with amino groups, the Immobilizer (Amino) (Thermo Fisher Scientific K.K. (formerly Nalgenunk International K.K.)) can be used.

[0030] The above complex may be immobilized on the surface of a detection substrate material with a functional group that reacts with the above amino group, without an anchor protein (Figure 2). In this case, before immobilization, -SH or -azide is introduced to the IgG-binding peptide, and if necessary, a protecting group of -SH is also introduced. If necessary, it is deprotected, and the -SH or -azide is reacted with a functional group having an alkyne (cyclooctin, DBCO, BCN, etc.) that reacts with the amino group on the surface of the detection substrate material to form a covalent bond, thereby immobilizing the above complex on the surface of the detection substrate material.

[0031] Examples of protecting groups include SATA (N-Succinimidyl S-acetylthioacetate) and SATP (N-Succinimidyl S-Acetylthiopropionate).

[0032] Figures 2(b) and (c) show specific examples of the above complex being immobilized without an anchor protein on the surface of a detection substrate material that has a functional group that reacts with an amino group.

[0033] The above complex can be produced by mixing IgG with an IgG-binding peptide to which a functional ligand is bound. The mixing conditions are not particularly limited as long as they are conditions under which a crosslinking reaction occurs between the IgG-binding peptide to which the functional ligand is bound and IgG. For example, the reaction can be carried out by mixing the IgG-binding peptide to which the functional ligand is bound and IgG in a suitable buffer at room temperature. If a crosslinking agent is bound to the IgG-binding peptide to which the functional ligand is bound, an appropriate amount of catalyst to promote the crosslinking reaction may be added as needed before mixing.

[0034] The reaction conditions may be adjusted to enhance the binding affinity between the functional ligand-bound IgG-binding peptide and IgG. For example, it is known that protein A, from which the Z34C peptide is derived, exhibits increased binding affinity to human IgG3 at a pH of 8 or higher, and that increasing the salt concentration, such as NaCl, increases its binding affinity to mouse IgG1. The conditions for the crosslinking reaction of the present invention can be set by referring to such findings.

[0035] The mixing step of the IgG-binding peptide to which the functional ligand is bound and IgG can be carried out under conditions of pH 4.5 to 8.5, more preferably under conditions of pH 5.0 to 8.0, and even more preferably under conditions of pH 6.0 to 7.7.

[0036] The mixing ratio (molar ratio) of the IgG-bound peptide with the functional ligand to IgG can be IgG:peptide = 1:1 to 20.

[0037] The mixing time (reaction time) between the functional ligand-bound IgG-binding peptide and IgG can be, for example, overnight, 30 minutes to 20 hours, 1 minute to 5 hours, 10 minutes to 2 hours, or 15 minutes to 1 hour.

[0038] After the mixing step described above, if necessary, a further step may be taken to separate impurities from the resulting mixture, such as unreacted functional ligand-bound IgG-binding peptides, IgG, and reagents, and to purify the complex. The purification step can be carried out by known methods, such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reversed-phase column chromatography, HPLC, and other chromatography methods.

[0039] An immunochromatographic test piece according to one embodiment is used in the above immunoassay method and comprises a labeled antibody holding portion that holds a labeled antibody and a detection region on which a capture antibody is immobilized, wherein the labeled antibody or capture antibody is a complex of IgG and an IgG-binding peptide to which a functional ligand is bound.

[0040] A method for producing a solid phase according to one embodiment comprises the steps of covalently bonding or physically adsorbing an anchor protein onto a solid phase, and covalently bonding, physically adsorbing, or specifically binding a complex of IgG and an IgG-binding peptide to the anchor protein bonded to the solid phase. [Examples]

[0041] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples.

[0042] <Preparation of monoclonal antibodies, antigens, and peptides> Trastuzumab was purchased for research purposes from iRxMedicine. Mouse IgG1 control antibodies were purchased from either Crown Bioscience, Inc. (mouse IgG1, κ isotype control) or the Medical & Biological Laboratories (MEB). Mouse IgG2 control antibodies were purchased from BioCell Technology, LLC (InVivoMAb anti-human / rat HER2). Anti-CA19-9H7D1 and anti-CA19-9G6C8 antibodies were purchased from BBI Solutions. Anti-IgE antibody [5D4] was purchased from abcam. Human CA19-9 was purchased from BBI Solutions. Human IgE was purchased from abcam. Streptavidin was purchased from Prospec-Tany Technogene, Ltd. Streptavidin-HRP (horseradish peroxidase streptavidin) was purchased from Vector lab, Inc. All peptides were prepared by the standard Fmoc solid-phase synthesis method using Eurofins.

[0043] <Peptide synthesis> Synthetic peptides (αZ34C, εZ34C, α-1Z34C, Z33-38azide, and Z33-5azide) were synthesized using the Fmoc solid-phase method. The C-terminus of all peptides was amidated. After removing the protecting group, the peptides were purified using reverse-phase HPLC.

[0044] <Formation of intramolecular disulfide bond> The following procedure was performed on three types of Z34C variants (αZ34C, εZ34C, and α-1Z34C). A 10 mM peptide solution dissolved in DMSO and 0.2 M Tris-HCl (pH 8.3) were mixed in equal amounts and incubated at room temperature for 2 hours. The reaction mixture (1.6 mL) was acidified by adding trifluoroacetic acid (TFA) and passed through a Sep-Pak tC18 reversed-phase column (Waters) equilibrated with a 0.1% TFA solution. After washing with a 0.1% TFA solution, the peptide was eluted with a 60% acetonitrile solution containing 0.1% TFA. Acetonitrile was removed by evaporation and freeze-dried for 20 hours. The freeze-dried product was dissolved in DMSO to a concentration of 10 mM.

[0045] <DSG binding> The following procedure was performed on three types of Z34C variants (αZ34C, εZ34C, and α-1Z34C). A 10 mM oxidized peptide solution dissolved in DMSO and a 500 mM disuccinimidyl glutarate (DSG) solution dissolved in acetonitrile were mixed at a molar ratio of peptide:DSG = 1:30, and pyridine was added to a final concentration of 0.5%. Then, the mixture was incubated at 50 °C for 3 hours. Fractions separated using an Inert-Sustain C18 reversed-phase column (5 μm, 7.6 × 250 mm) were collected, and the target fraction was freeze-dried for 20 hours.

[0046] <Binding of biotin and DSG to Z33-38azide> The following procedure was performed on Z33-38azide to obtain Z33-38biotin, which is Z33-38azide into which biotin has been introduced. 40 mM Z33-38azide dissolved in DMSO, 100 mM DBCO-PEG4-Biotin (Thermo Fisher Scientific, Inc.) dissolved in DMSO, and 500 mM DSG dissolved in acetonitrile were mixed in a molar ratio of peptide:DBCO-PEG4-Biotin:DSG = 1:1:20, and pyridine was added to a final concentration of 5%. The mixture was then incubated at 50°C for 2 hours.

[0047] <Peptide structure modeling> A model structure of the complex between Fc and the binding peptide was constructed using the software MOE (Molecular Operating Environment, CCG) based on the crystal structure of human IgG-Fc and the peptide (1OQO.pdb).

[0048] <Conjugation of three Z34C variants (αZ34C, εZ34C, and α-1Z34C), Z33-38biotin, or Z33-5azide with IgG> 1 μM IgG diluted with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.4) or acetate buffer (100 mM, pH 5.5) was mixed with a peptide reagent diluted to 10 mM with DMSO, in a molar ratio of IgG:peptide = 1:5. The mixtures were incubated at each reaction temperature (25°C, 37°C, 50°C) for 1 hour or overnight (approximately 16 hours). Figure 3 shows the structural simulation results of the binding of three Z34C variants (αZ34C, εZ34C, and α-1Z34C) to IgG.

[0049] <sds-page> A 4× reducing sample buffer was prepared by mixing 450 μL of 4× Laemmli Sample Buffer (Bio-Rad Laboratories, Inc.) with 50 μL of DTT solution (1M DTT, 1 mM EDTA). The sample solution was mixed with the 4× reducing sample buffer and incubated at 95°C for 10 minutes. The resulting sample was applied to an SDS-PAGE gel (Mini Protean TGX precast Gels Any kD; Bio-Rad Laboratories, Inc.) at a concentration of 1-2 μg / well, and electrophoresis was performed. The gel after electrophoresis was stained with Bio-safe Comassie G-250 Stain (Bio-Rad Laboratories, Inc.).

[0050] <Sandwich-ELISA> ELISA plates (Nunc-Immuno Module plate Maxisorp; Thermo Fisher Scientific, Inc.) were coated with streptavidin to a concentration of 300 ng / well and incubated overnight at 4°C. After washing the plates, plate blocking solution (100 mM Tris-HCl, pH 7.6, containing 100 mM NaCl, 0.05% Tween 20, and 0.5% BSA) was added, and the plates were incubated overnight at 4°C. Biotinylated antibody was added at 150 ng / well and incubated at 37°C for 1 hour. After washing the plates, human CA19-9 or human IgE was added at their respective concentrations and incubated at 37°C for 1 hour. After washing the plates, HRP-labeled antibody was added at a concentration of 30 ng / well and incubated at 37°C for 1 hour. After washing the plates, TMB substrate was added and incubated at 25°C for 30 minutes. After adding an equal amount of 0.3M H2SO4 to the TMB substrate, the absorbance at 450 nm and 630 nm was measured. Biotinylated antibodies (-NHS) were prepared using the Biotin Labeling kit-NH2 (Dojin Chemical Laboratories). HRP-labeled antibodies were prepared using the Peroxidase Labeling Kit-NH2 (Dojin Chemical Laboratories).

[0051] <Preparation of Antibody-Conjugated Beads for RPLA (Reverse Passive Latex Agglutination Reaction)> 1 mL of PBS was mixed with 60 μL of streptavidin-coated beads (Streptavidin Coated Microspheres 1.0 μm; Polysciences, Inc.) slurry and centrifuged. The supernatant was removed, and 600 μL of biotinylated antibody (20 μg / mL) diluted with PBS was added and stirred, and then rotated at 25 °C for 2 hours. After centrifugation, the supernatant was removed, 600 μL of blocking solution (PBS containing 0.5% BSA, pH 7.4) was added, and the mixture was rotated at 25 °C for 1 hour. After centrifugation, the supernatant was removed, 1 mL of blocking solution was added and suspended, then centrifuged to remove the supernatant, and washing was performed (performed 3 times in total). 2.1 mL of storage solution (PBS containing 0.5% BSA and 0.08% NaN3, pH 7.4) was added, suspended, and then sonicated.

[0052] <RPLA Reaction> Antigen (human CA19-9 or IgE) diluted with blocking solution (PBS containing 0.5% BSA, pH 7.4) and dilution buffer (PBS containing 0.1% BSA, pH 7.4) was mixed 1:1 and added to a V-bottom 96-well microplate (25 μL / well). 25 μL of antibody-conjugated bead slurry was added to each well. After mixing for 1 minute with a shaker, it was incubated overnight at 25 °C, and the agglutination state was observed.

[0053] (Test Example 1) Three types of Z34C variants (αZ34C, εZ34C, and α-1Z34C) were prepared and confirmed to react with mouse IgG1 and mouse IgG2a. Furthermore, the pH, reaction time, reaction molar ratio, and reaction temperature of the reaction solution were tested under several conditions, and the conditions with the highest modification rate were found. The modification rate was evaluated by SDS-PAGE (Figure 4).

[0054] As shown in Figure 4, the pH of the reaction solution significantly affects the modification efficiency. Furthermore, αZ34C exhibited a certain level of reactivity with both mouse IgG1 (Figure 4(a)) and mouse IgG2a (Figure 4(b)(c)), which are frequently used in immunoassays. In addition, modification of human IgG1 by these three Z34C variants was also confirmed (Figure 5).

[0055] This reaction theoretically results in monovalent or divalent modification (Figure 4(d)). In reality, the modification rate to the H chain was often around 50% (Figures 4 and 5).

[0056] (Test Example 2) αZ34C was prepared and confirmed to react with human IgG1, mouse IgG1, mouse IgG2a, mouse IgG2b, mouse IgG3, rabbit IgG, rat IgG1, rat IgG2a, rat IgG2b, and rat IgG2c. The modification rate was evaluated by SDS-PAGE (Figure 6).

[0057] (Test Example 3) The binding of Z33-38biotin, obtained by introducing biotin into Z33-38azide via a click reaction, to human IgG1 and mouse IgG2a was confirmed by SDS-PAGE. The results for human IgG1 are shown in Figure 7(b), and the results for mouse IgG2a are shown in Figure 7(c). Z33-38 tends to have a higher yield of synthesized peptides compared to the three Z34C variants (αZ34C, εZ34C, and α-1Z34C), and is easier and cheaper to synthesize.

[0058] (Test example 4) Sandwich ELISA was performed using modified antibodies in which Z33-38 biotin was covalently bound to mouse IgG using the CCAP method. Human CA19-9 and human IgE were used as antigens.

[0059] Biotinylated antibodies were added to streptavidin-coated plates, and sandwich ELISA measurements were performed. Biotinylated antibodies were prepared using either the random amine coupling method (-NHS) or the CCAP method (-CCAP). When measured with varying IgE antigen concentrations, a significant difference was observed between the random amine coupling method and the CCAP method, with the CCAP method being more sensitive than the random amine coupling method (Figure 9). This is likely because, firstly, antibodies modified by the CCAP method exhibit less reduction in reactivity to the antigen than antibodies modified by the random amine coupling method. Secondly, the CCAP method allows for better orientation of the captured antibody on the plate, resulting in a larger number of antigen-binding sites that can bind to the antigen.

[0060] (Test Example 5) Antigen detection capabilities in RPLA were compared using antibodies modified with biotin using random amine coupling or CCAP. Biotin-labeled antibodies were adsorbed onto streptavidin-coated beads, and antigens of various concentrations were prepared and treated with antibody-conjugated beads. For systems using CA19-9 or IgE as antigens, tests were conducted with n=3.

[0061] In each system using CA19-9 as the antigen, an increase in score dependent on the antigen concentration was observed (Figure 10(a)). The system using the CCAP method showed higher scores even at lower antigen concentrations, and its sensitivity was higher than that of the system using the random amine coupling method. Furthermore, when beads conjugated with the control antibody were used, although agglutination should not have occurred and the score should have been (-), a nonspecific agglutination reaction occurred in the system using the random amine coupling method. This is thought to be because, in the system using the random amine coupling method, the antibody structure was not maintained due to the random coupling reaction occurring at lysine residues on the antibody surface, and the hydrophobic portion was exposed. On the other hand, no such nonspecific reaction was observed in the system using the CCAP method (Figure 10(a)).

[0062] In systems using IgE as the antigen, an antigen concentration-dependent increase in score was observed only in the system using the CCAP method. When using the random amine coupling method, no bead aggregation reaction was observed at any concentration (Figure 10(b)). Figure 11 shows the actual aggregation pattern when the RPLA reaction was performed.

[0063] (Test Example 6) Surface plasmon resonance (SPR) measurements were performed at 25°C using a Biacore X100 instrument (GE Healthcare). Human IgE was immobilized on a CM5 sensor chip (GE Healthcare) by amine coupling according to the manufacturer's instructions. Binding dynamics were analyzed using five concentrations of IgG diluted in analytical buffer (120 mM NaCl, 7.1 mM Na2PO4, 2.36 mM KCl, 1.29 mM KH2PO4, 118 mM Tris, 0.05% Tween 20, pH 8.0) using single-cycle kinetics mode. The binding process was measured by adding each concentration of IgG for 2 minutes (flow rate 30 μL / min). The dissociation process was measured by flowing the analytical buffer for 30 minutes (flow rate 30 μL / min). The dilution concentrations of IgG are shown in Table 1, and the results are shown in Table 2.

[0064] [Table 1]

[0065] [Table 2]

[0066] (Test Example 7) The titers of biotinylated antibodies used in sandwich ELISA and RPLA methods were confirmed by ELISA using plates immobilized with the antigen. For both anti-CA19-9 G6C8 antibody and anti-CA19-9 H7D1 antibody, the random amine coupling method (-NHS) did not differ significantly from the CCAP method. However, for anti-IgE antibody titers, the CCAP method was significantly higher than the random amine coupling method (Figure 12).

[0067] (Test Example 8) An IgG-binding peptide (Z33-5azide) was conjugated to an anti-influenza virus antibody using the CCAP method to obtain a complex. The carboxyl groups on the surface of latex particles were converted to -DBCO by amine coupling. The above complex and the latex particles into which -DBCO was introduced were mixed, and covalent bonds were formed by a click reaction. After blocking with casein, a latex particle suspension was obtained by sonication. The obtained latex particle suspension (FluA: 0.010% or FluB: 0.016%) and influenza antigen solution (Type A: 800 pfu / mL or Type B: 3300 pfu / mL) were mixed in equal volumes, and the scattered light signal, which increased with the progress of the agglutination reaction, was measured using a nephelometer. As is clear from the results shown in Figure 13, an antigen-dependent agglutination signal was obtained. This indicates that the latex agglutination method functions in the system using the CCAP method.

[0068] The differences in sensitivity of immunoassay systems due to these modification methods are thought to be mainly due to the following two points: firstly, the effect on affinity to the antigen, and secondly, the effect on binding orientation to the material surface. From these results, it can be seen that differences in IgG modification methods result in differences in affinity to the antigen, as seen in the anti-IgE 5D4 antibody. However, in some cases, there was no significant difference in affinity to antigens such as anti-CA19-9 G6C8 antibody and anti-CA19-9 H7D1 antibody (Figure 12). Furthermore, with the random amine coupling method, it is not possible to select the modification site. Therefore, when binding to material surfaces such as streptavidin-coated plates or streptavidin-coated beads via biotin modified by the random amine coupling method, it is difficult to achieve uniform antibody orientation. The number of antibody molecules that maintain binding ability to the antigen is thought to be limited by the decrease in orientation. On the other hand, in the case of biotin-modified antibodies by the CCAP method, biotin is modified site-specifically, so the binding orientation to the material surface can be made uniform. Therefore, it is thought that more molecules can maintain binding ability to the antigen. In particular, with the RPLA method, the number of beads that bind to the antigen and lead to the agglutination reaction is thought to be limited from the perspective of steric hindrance (Figure 10(c)).

[0069] As described above, by using the immunoassay method of the present invention, it is possible to maintain the affinity of antibodies to rodent IgG and improve binding orientation, thereby achieving highly sensitive measurement in immunoassays. It is believed that the immunoassay method of the present invention can also be used to achieve highly sensitive measurement of antibodies from other hosts, such as human IgG and rabbit IgG, similar to rodent IgG.

Claims

1. A solid phase to which a complex of IgG and an IgG-binding peptide is bound, wherein the IgG-binding peptide is covalently bonded to an anchor protein, the anchor protein is covalently bonded to the surface of the solid phase, and the IgG and the IgG-binding peptide are covalently bonded via disuccinimidyl glutarate, disuccinimidyl sverate, dimethyl adipimide dihydrochloride, dimethyl pimerimide dihydrochloride, dimethyl sverimide dihydrochloride, dimethyl 3,3'-dithio-bis(propionimidate) dihydrochloride, or dithiobis(succinimidylpropionate), and the IgG-binding peptide is a peptide containing the amino acid sequence of any of SEQ ID NOs: 1 to 9, or a peptide in which one or more amino acids are substituted, deleted, or added in the amino acid sequence of any of SEQ ID NOs: 1 to 9, and which has the ability to bind to the Fc region of IgG.

2. The solid phase according to claim 1, wherein the IgG-binding peptide and the anchor protein are covalently bonded via a functional group that reacts with an amino group, and the IgG-binding peptide complex is immobilized on the solid phase.

3. The solid phase according to claim 1, wherein the IgG-binding peptide and the anchor protein are covalently bonded via a functional group that reacts with a succinimide group, and the IgG-binding peptide complex is immobilized on the solid phase.

4. The solid phase according to claim 1, wherein the IgG-binding peptide and the anchor protein are covalently bonded via a functional group that reacts with a thiol group, and the IgG-binding peptide complex is immobilized on the solid phase.

5. The solid phase according to claim 1, wherein the IgG-binding peptide and the anchor protein are covalently bonded via a functional group that reacts with a maleimide group, and the IgG-binding peptide complex is immobilized on the solid phase.

6. The solid phase according to claim 1, wherein the IgG-binding peptide and the anchor protein are covalently bonded via a functional group that reacts with a carboxyl group, and the IgG-binding peptide complex is immobilized on the solid phase.

7. The solid phase according to claim 1, wherein the IgG-binding peptide and the anchor protein are covalently bonded via a functional group formed by a click reaction, and the IgG-binding peptide complex is immobilized on the solid phase.

8. A solid phase to which a complex of IgG and an IgG-binding peptide is bound, wherein the IgG-binding peptide is physically adsorbed to an anchor protein, and the anchor protein is physically adsorbed to the surface of the solid phase, and the IgG and the IgG-binding peptide are covalently bonded via disuccinimidyl glutarate, disuccinimidyl sverate, dimethyl adipimide dihydrochloride, dimethyl pimerimide dihydrochloride, dimethyl sverimide dihydrochloride, dimethyl 3,3'-dithio-bis(propionimidate) dihydrochloride, or dithiobis(succinimidylpropionate), and the IgG-binding peptide is a peptide containing the amino acid sequence of any of SEQ ID NOs: 1 to 9, or a peptide in which one or more amino acids are substituted, deleted, or added in the amino acid sequence of any of SEQ ID NOs: 1 to 9, and which has the ability to bind to the Fc region of IgG.

9. A method for producing a solid phase according to any one of claims 1 to 7, comprising the steps of covalently bonding an anchor protein to a solid phase and covalently bonding a complex of IgG and an IgG-binding peptide to the anchor protein bonded to the solid phase.

10. A method for producing a solid phase according to claim 8, comprising the steps of physically adsorbing an anchor protein onto a solid phase and physically adsorbing a complex of IgG and an IgG-binding peptide onto the anchor protein bound to the solid phase.

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