JP7762913B2 - Immunoassay method using site-specifically modified IgG antibodies using IgG-binding peptides - Google Patents

Description

The present invention relates to an immunoassay method using site-specifically modified IgG antibodies using IgG-binding peptides.

In immunoassay methods, antibodies are often immobilized on the surface of a material via covalent bonds, typically obtained by physical adsorption or amine coupling. In this case, it is necessary to avoid denaturation or functional loss of the antibody immobilized on the surface. However, with conventional methods, denaturation or functional loss of the antibody immobilized on the surface can occur, resulting in a decrease or loss of antigen-binding ability.

Therefore, the CCAP method (chemical conjugation by affinity peptide) has been developed as a method for modifying human IgG with minimal loss of antigen binding ability (Non-patent document 1).

Kishimoto et al. , “Site-Specific Chemical Conjugation of Antibodies by Using Affinity Peptide for the Development of Therapeutic Antibody Format.”, Bioconjug. Chem. 2019, 30 (3), 698-702.

However, the conventional CCAP method was not suitable for IgG derived from rodents such as mice and rats, which are commonly used in in vitro diagnosis.

The present invention aims to provide an immunoassay method using site-specifically modified IgG antibodies with IgG-binding peptides that are capable of binding to rodent-derived IgG and that undergo minimal denaturation or loss of functionality when immobilized on a material surface.

The present invention relates to an immunoassay method using a complex of IgG and an IgG-binding peptide bound to a functional ligand.

The present invention also relates to an in vitro diagnostic reagent comprising a complex of IgG and an IgG-binding peptide bound to a functional ligand.

The present invention also relates to a solid phase on which a complex of IgG and an IgG-binding peptide bound to a functional ligand is immobilized.

The present invention also relates to an immunochromatographic test piece for use in the above-mentioned immunoassay method, which comprises a labeled antibody holding section that holds a labeled antibody and a detection area to which a capture antibody is immobilized, and the labeled antibody, the capture antibody, or both, are a complex of IgG and an IgG-binding peptide bound to a functional ligand.

The present invention further relates to a method for producing the above solid phase, which comprises the steps of covalently binding or physically adsorbing an anchor protein to the solid phase, and covalently binding or physically adsorbing a complex of IgG and an IgG-binding peptide to the anchor protein bound to the solid phase.

The present invention provides an immunoassay method using site-specifically modified IgG antibodies that use IgG-binding peptides, which are capable of binding to rodent-derived IgG and undergo minimal denaturation or loss of functionality even when immobilized on a material surface.

FIG. 1 is a diagram showing a model of site-specific covalent bond formation of a complex to the surface of a detection platform material via an anchor protein. FIG. 2 shows a model for the direct, site-specific covalent bond formation of a complex to the surface of a detection platform material without the intervention of an anchor protein. FIG. 3 shows a cross-linking model of IgG-Fc and Z34C. FIG. 4 shows the results of SDS-PAGE examining the reactivity of Z34C variants (αZ34C, εZ34C, and α-1Z34C) with mouse IgG1 or mouse IgG2a. FIG. 5 shows the results of SDS-PAGE examining the reactivity of Z34C variants (αZ34C, εZ34C, and α-1Z34C) with human IgG1. FIG. 6 shows the results of SDS-PAGE examining the reactivity of αZ34C with IgG derived from human, mouse, rabbit, or rat. FIG. 7 shows the results of SDS-PAGE examining the reactivity of the Z34C variant Z33-38biotin with human IgG1 or mouse IgG2a. FIG. 8 shows the results of SDS-PAGE used in Test Examples 4 and 5 to examine the reactivity of Z33-38biotin with mouse IgG1 or mouse IgG2a. FIG. 9 shows the results of examining the antigen reactivity of biotin-conjugated IgG obtained using the CCAP method and biotin-conjugated IgG obtained using the random amine coupling method when each antibody was applied to an ELISA system. FIG. 10 shows the results of examining the antigen reactivity of biotin-conjugated IgG obtained using the CCAP method and biotin-conjugated IgG obtained using the random amine coupling method when each antibody was applied to the RPLA method. FIG. 11 shows an image of actual latex agglutination when the RPLA reaction was performed. FIG. 12 shows the results of examining the antibody titers by ELISA for biotin-conjugated IgG obtained using the CCAP method and biotin-conjugated IgG obtained using the random amine coupling method. FIG. 13 shows the results of examining influenza antigen-specific agglutination reaction using particles on which anti-influenza antibodies were immobilized by site-specific covalent bonding using the CCAP method.

The present invention is described in detail below. As used herein, "antibodies directly bind to the surface of a detection platform material" refers to a mode in which a covalent bond is formed between the surface of the detection platform material and the IgG-binding peptide. On the other hand, "physical adsorption" refers to a mode in which antibodies are adsorbed to the surface of a detection platform material through electrostatic interactions or hydrophobic bonds.

An immunoassay method according to one embodiment uses a complex of IgG and an IgG-binding peptide bound to a functional ligand. The immunoassay method according to this embodiment does not require genetic modification of antibody molecules through antibody engineering, and allows peptide/IgG binding under conditions that do not place a burden on the antibody, allowing for rapid reaction at room temperature.

The IgG may be mammalian IgG. For example, the IgG may be 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).

Functional ligands include drugs, proteins, peptides, nucleic acids, enzymes, radiolabeled substances, fluorescent substances, and chemical crosslinkers with functional groups that can be covalently bound to the surface of the detection base 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 may be bound via a molecule such as PEG (polyethylene glycol).

The IgG-binding peptide can be linked to a functional ligand 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).

In one embodiment, the IgG-binding peptide is a peptide derived from Protein A, including, 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 variants thereof. Z34C is derived from the B domain of Protein A and has been optimized using a phage library method. Z34C exhibits affinity (binding ability) particularly for human and rodent IgG-Fc. The IgG-binding peptide may also include a peptide selected from the peptides represented by SEQ ID NOs: 1 to 9, or a peptide in which one or more amino acids have been substituted, deleted, or added, and which has the ability to bind to the Fc region of IgG. The IgG-binding peptide may also include a peptide selected from the peptides represented by SEQ ID NOs: 1 to 9, or a peptide in which one or more amino acids have been substituted, deleted, or added, and which has 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. SEQ ID NO: 1 is an amino acid sequence in which all Lys in Z34C are substituted with Arg, and is designated as αZ34C. SEQ ID NO: 2 is an amino acid sequence in which Phe at the N-terminus of αZ34C is substituted with Lys, and is designated as εZ34C. SEQ ID NO: 3 is an amino acid sequence in which Gly is added to the N-terminus of αZ34C, and is designated as α-1Z34C. SEQ ID NO: 4 is an amino acid sequence in which the 7th Arg in αZ34C is substituted with Lys. SEQ ID NO: 5 is an amino acid sequence in which the 7th Arg in εZ34C is substituted with Lys. SEQ ID NO: 6 is an amino acid sequence in which the 8th Arg in α-1Z34C is substituted with Lys. SEQ ID NO: 7 is an amino acid sequence in which the 5th Cys in αZ34C is substituted with Gln and the C-terminal Cys is removed. SEQ ID NO: 8 is an amino acid sequence in which Cys at position 5 of αZ34C is substituted with Gln, the C-terminal Cys is deleted, and Pro-Ser-Arg-Arg-Lys-Arg is added to the C-terminus, and is designated as Z33-38. SEQ ID NO: 9 is an amino acid sequence in which Cys at position 5 of αZ34C is substituted with Gln, Arg at positions 7 and 28 is substituted with Lys, the C-terminal Cys is deleted, and Pro-Ser-Arg-Arg-Lys-Arg-Arg-Lys-Arg-Arg-Lys is added to the C-terminus, and is designated as Z33-5.

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

Z34C variants can be synthesized by known methods. Examples of synthesis methods include solid-phase synthesis methods such as Fmoc synthesis and Boc synthesis, and liquid-phase synthesis methods such as fragment condensation. However, from the standpoint of operational simplicity, solid-phase synthesis is preferred. When an IgG-binding peptide such as a Z34C variant is modified with a cross-linking agent, as described below, such an IgG-binding peptide can be produced by modifying the synthesized IgG-binding peptide with the cross-linking agent, or by performing peptide synthesis using amino acid residues modified with the cross-linking agent.

In one embodiment, the IgG-binding peptide is believed to be bound to the side chain of Lys248 in the Fc region of IgG, with the two linked via a cross-linker, e.g., DSG (Disuccinimidyl Glutarate). Other examples of the crosslinking agent include crosslinking agents containing preferably two or more succinimidyl groups, such as DSS (Disuccinimidyl suberate), crosslinking agents containing preferably two or more imide ester moieties, such as DMA (Dimethyl Adipimidate Dihydrochloride), DMP (Dimethyl Pimellimidate Dihydrochloride), and DMS (Dimethyl Suberimidate Dihydrochloride), crosslinking agents containing preferably two or more imide ester moieties, such as DTBP (Dimethyl 3,3'-dithio-bis(propionimidate) Dihydrochloride) and DSP (Dithiobis(succinimidyl Examples of crosslinking agents include crosslinkers having an S-S bond such as methylpropionate.

Immunoassay methods include ELISA, immunochromatography, immuno-latex agglutination (LA), turbidimetric immunoassay (TIA), chemiluminescent immunoassay (CLIA), pulse immunoassay, time-resolved fluorescence resonance energy transfer (TR-FRET), quartz crystal microbalance (QCM), and BioLayer interferometry (BioLayer). Examples of such methods include a laser interferometry (BLI) method and a surface plasmon resonance method.

An in vitro diagnostic reagent according to one embodiment includes a complex of IgG and an IgG-binding peptide bound to a functional ligand. When included in an in vitro diagnostic reagent, the IgG-binding peptide may be modified with an enzyme, a radioactively labeled substance, a fluorescent substance, or the like. Use of the above immunoassay method can suppress a decrease in the antigen affinity of the antibody and enhance the immobilization orientation on the material surface. Therefore, when applied to an in vitro diagnostic reagent using an immunoassay method, for example, highly sensitive measurements are believed to be possible.

In one embodiment, the solid phase has immobilized thereon a complex of IgG and an IgG-binding peptide bound to a functional ligand.

The complex may be immobilized directly on the surface of a detection substrate material by physical adsorption, either without or via an anchor protein. Examples of detection substrates include latex particles (LTX), immunoassay plates made primarily of organic materials such as polypropylene or polystyrene, or glass.

The above complex may be immobilized via an anchor protein on the surface of a detection substrate material with functional groups that react with amino groups (Figure 1). The anchor protein (Protein in Figure 1) is not particularly limited, but can be streptavidin or the like. In terms of balancing non-specific reactions, BSA or HSA, which are used as blocking agents, are preferred.

As shown in Figure 1, functional groups reactive with amino groups can be reacted with amino groups in the anchor protein, or functional groups reactive with carboxy groups can be reacted with carboxy groups in the anchor protein. Furthermore, amino groups in the anchor protein can be reacted with DBCO-NHS, or -SH groups in the anchor protein can be reacted with DBCO-maleimide, thereby introducing -DBCO into the anchor protein bound to the surface of the detection platform material. Furthermore, a click reaction can occur between -DBCO introduced into the anchor protein and -azide introduced into the IgG-binding peptide in the complex, thereby indirectly immobilizing the complex to the surface of the detection platform material. The functional group introduced is not limited to -DBCO, and any functional group that can be used in a click reaction can be used. The functional group introduced can be, for example, a compound containing an alkyne, more specifically, cyclooctyne, BCN (bicyclo[6.1.0]nonyne), etc. In addition to this click reaction, a reaction using -SH and -maleimide or bromoacetyl can also be used.

Functional groups that react with amino groups include carboxyl groups, tosyl groups, epoxy groups, isocyanate groups, isothiocyanate groups, N-hydroxysuccinimide groups (-NHS), maleimide groups, etc. An example of a detection substrate with a functional group that reacts with amino groups is Immobilizer (Amino) (Thermo Fisher Scientific Co., Ltd. (formerly Nalge Nunc International Co., Ltd.)).

The above complex may be immobilized without an anchor protein on the surface of a detection platform material equipped with functional groups that react with the amino groups (Figure 2). In this case, -SH or -azide is introduced into the IgG-binding peptide before immobilization, and if necessary, a protecting group for -SH is also introduced. Deprotection is performed as necessary, and the -SH or -azide is reacted with a functional group on the surface of the detection platform material that reacts with amino groups, thereby forming a covalent bond with the introduced alkyne-containing functional group (cyclooctyne, DBCO, BCN, etc.), thereby immobilizing the above complex on the surface of the detection platform material.

Examples of protecting groups include SATA (N-Succinimidyl S-acetylthioacetate) and SATP (N-Succinimidyl S-acetylthiopropionate).

Figures 2(b) and (c) are specific examples of the above complex being immobilized on the surface of a detection base material with functional groups that react with amino groups without an anchor protein.

The above complex can be produced by mixing IgG with an IgG-binding peptide bound to a functional ligand. The mixing conditions are not particularly limited as long as they are conditions that allow a cross-linking reaction between the IgG and the IgG-binding peptide bound to a functional ligand. For example, the reaction can be carried out by mixing the IgG-binding peptide bound to a functional ligand with IgG in an appropriate buffer at room temperature. If a cross-linking agent is bound to the IgG-binding peptide bound to a functional ligand, mixing may be carried out with the addition of an appropriate amount of a catalyst that promotes the cross-linking reaction, as needed.

Reaction conditions may be adjusted to enhance the binding affinity between IgG and the IgG-binding peptide bound to a functional ligand. For example, Protein A, from which the Z34C peptide is derived, is known to have increased binding affinity with human IgG3 at pH 8 or higher, and its binding affinity with mouse IgG1 is known to increase with increasing salt concentrations such as NaCl. The conditions for the crosslinking reaction of the present invention can be set with reference to these findings.

The mixing process of the IgG-binding peptide bound to the functional ligand with the 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.

The mixing ratio (molar ratio) of the IgG-binding peptide bound to a functional ligand to IgG can be IgG:peptide = 1:1-20.

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

After the above mixing step, if necessary, a step of separating impurities, such as unreacted IgG-binding peptides bound to functional ligands, IgG, and reagents, from the resulting mixture to purify the complex may be carried out. The purification step can be carried out by known methods, such as chromatography, such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse-phase column chromatography, and HPLC.

In one embodiment, the immunochromatographic test piece is used in the above-mentioned immunoassay method and comprises a labeled antibody holding section that holds a labeled antibody and a detection area in which a capture antibody is immobilized, and the labeled antibody or capture antibody is a complex of IgG and an IgG-binding peptide bound to a functional ligand.

In one embodiment, a method for producing a solid phase comprises the steps of covalently binding or physically adsorbing an anchor protein to a solid phase, and covalently binding, physically adsorbing, or specifically binding a complex of IgG and an IgG-binding peptide to the anchor protein bound to the solid phase.

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.

<Preparation of monoclonal antibodies, antigens, and peptides>
Trastuzumab was purchased for research use from iRxMedicine. Mouse IgG1 control antibodies were mouse IgG1, κ isotype control purchased from Crown Bioscience, Inc., or mouse IgG1 isotype control purchased from Medical and Biological Laboratories. Mouse IgG2 control antibodies were InVivoMAb anti-human/rat HER2 purchased from BioCell Technology, LLC. 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 standard Fmoc solid phase synthesis methods by Eurofins.

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

<Intramolecular disulfide bond formation>
The following procedure was performed for the three Z34C variants (αZ34C, εZ34C, and α-1Z34C). Equal volumes of a 10 mM peptide solution dissolved in DMSO and 0.2 M Tris-HCl (pH 8.3) were mixed 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 reverse-phase column (Waters) equilibrated with 0.1% TFA solution. After washing with 0.1% TFA solution, the peptide was eluted with 60% acetonitrile containing 0.1% TFA. The acetonitrile was removed by evaporation, and the column was lyophilized for 20 hours. The lyophilized product was dissolved in DMSO to a concentration of 10 mM.

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

<Binding of biotin and DSG to Z33-38azide>
The following steps were performed on Z33-38azide to obtain Z33-38biotin, in which biotin was introduced into Z33-38azide. 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 at 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.

<Peptide structure modeling>
A model structure of the complex between Fc and the bound 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).

<Binding of three types of Z34C variants (αZ34C, εZ34C, and α-1Z34C), Z33-38biotin, or Z33-5azide to IgG>
1 μM IgG diluted in 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 at a molar ratio of 1:5 IgG:peptide. The mixture was incubated at various reaction temperatures (25°C, 37°C, 50°C) for 1 hour or overnight (approximately 16 hours). The structural simulation results of the binding of three types of Z34C variants (αZ34C, εZ34C, and α-1Z34C) with IgG are shown in Figure 3.

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

Sandwich ELISA
Streptavidin was coated onto an ELISA plate (Nunc-Immuno Module plate Maxisorp; Thermo Fisher Scientific, Inc.) at 300 ng/well and incubated overnight at 4°C. After washing, the plate was added with plate blocking solution (100 mM Tris-HCl, pH 7.6, containing 100 mM NaCl, 0.05% Tween 20, and 0.5% BSA) and incubated overnight at 4°C. Biotinylated antibodies were added at 150 ng/well and incubated at 37°C for 1 hour. After washing, human CA19-9 or human IgE was added at various concentrations and incubated at 37°C for 1 hour. After washing the plate, HRP-labeled antibody was added at 30 ng/well and incubated for 1 hour at 37° C. After washing the plate, TMB substrate was added and incubated for 30 minutes at 25° C. After adding an equal amount _of 0.3 M _H2SO4 to the TMB substrate, absorbance was measured at 450 nm and 630 nm.
Biotinylated antibodies (-NHS) were prepared using Biotin Labeling kit-NH2 (Dojindo Laboratories). HRP-labeled antibodies were prepared using Peroxidase Labeling kit-NH2 (Dojindo Laboratories).

<Preparation of antibody-bound beads for RPLA (reverse passive latex agglutination reaction)>
1 mL of PBS was mixed with 60 μL of streptavidin-coated bead (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 loosened, followed by rotation at 25°C for 2 hours. After centrifugation, the supernatant was removed, and 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, and 1 mL of blocking solution was added to suspend the beads, followed by centrifugation to remove the supernatant and washing (performed three 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.

<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 at a 1:1 ratio and added to a V-bottom 96-well microplate (25 μL/well). 25 μL of antibody-bound bead slurry was added to each well. After mixing on a shaker for 1 minute, the plate was incubated overnight at 25°C and the aggregation state was observed.

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

Figure 4 shows that the pH of the reaction solution has a significant effect on modification efficiency. Furthermore, αZ34C exhibited a certain level of reactivity with both mouse IgG1 (Figure 4(a)) and mouse IgG2a (Figures 4(b) and (c)), which are often used in immunoassay methods. Furthermore, modification of human IgG1 with these three types of Z34C variants was also confirmed (Figure 5).

This reaction theoretically results in monovalent or divalent modification (Figure 4(d)). The actual modification rate for the H chain was often around 50% (Figures 4 and 5).

(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).

(Test Example 3)
The binding of Z33-38biotin, obtained by introducing biotin into Z33-38azide by 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 produce a higher synthetic peptide yield than the three Z34C variants (αZ34C, εZ34C, and α-1Z34C), and is easier and less expensive to synthesize.

(Test Example 4)
Sandwich ELISA was performed using a modified antibody in which Z33-38 biotin was covalently bound to mouse IgG using the CCAP method, and human CA19-9 and human IgE were used as antigens.

Biotinylated antibodies were added to streptavidin-coated plates and subjected to sandwich ELISA. Biotinylated antibodies were prepared using either the random amine coupling method (-NHS) or the CCAP method (-CCAP). Measurements using varying concentrations of IgE antigen revealed significant differences between the random amine coupling and CCAP methods, with the CCAP method demonstrating higher sensitivity than the random amine coupling method (Figure 9). This is likely due, first, to the fact that antibodies modified using the CCAP method exhibit less reduction in their reactivity toward the antigen than antibodies modified using the random amine coupling method. Second, the CCAP method provides favorable orientation of the capture antibody relative to the plate, resulting in a greater number of antigen-binding sites available for binding to the antigen.

(Test Example 5)
Antigen detection ability in RPLA was compared using antibodies modified with biotin using the random amine coupling method or the CCAP method. Biotin-labeled antibodies were adsorbed onto streptavidin-coated beads, and various concentrations of antigen were prepared and reacted with the antibody-conjugated beads. For systems using CA19-9 or IgE as the antigen, tests were performed with n=3.

In each system using CA19-9 as the antigen, an increase in score depending 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 coupled with a control antibody were used, no agglutination reaction would normally occur, resulting in a score of (-). However, in the system using the random amine coupling method, a nonspecific agglutination reaction occurred. 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 the lysine residues on the antibody surface, exposing the hydrophobic portions. On the other hand, no such nonspecific reaction was observed in the system using the CCAP method (Figure 10(a)).

In systems using IgE as an antigen, an increase in score dependent on antigen concentration was observed only in systems using the CCAP method. When the random amine coupling method was used, no bead agglutination reaction was observed at any concentration (Figure 10(b)). Figure 11 shows an image of actual agglutination when the RPLA reaction was performed.

(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 the amine coupling method according to the manufacturer's instructions. Binding kinetics was analyzed in single-cycle kinetics mode using five concentrations of IgG diluted in analysis 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). 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 analysis 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.

(Test Example 7)
The titers of the biotinylated antibodies used in the sandwich ELISA system and the RPLA method were confirmed by ELISA using antigen-immobilized plates. For both the anti-CA19-9 G6C8 antibody and the anti-CA19-9 H7D1 antibody, the random amine coupling method (-NHS) showed little difference from the CCAP method. The anti-IgE antibody titer was significantly higher with the CCAP method than with the random amine coupling method (Figure 12).

(Test Example 8)
Using the CCAP method, an IgG-binding peptide (Z33-5azide) was conjugated to an anti-influenza virus antibody to obtain a conjugate. The carboxyl groups of latex particles bearing carboxyl groups on their surfaces were converted to -DBCO by amine coupling. The conjugate and the -DBCO-introduced latex particles were mixed, and covalent bonds were formed via a click reaction. After blocking with casein, a latex particle suspension was obtained by ultrasonic dispersion. The resulting latex particle suspension (FluA: 0.010% or FluB: 0.016%) was mixed with equal amounts of influenza antigen solution (type A: 800 pfu/mL or type B: 3300 pfu/mL), and the scattered light signal, which increased with the progression 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 demonstrates that the latex agglutination method functions in a system using the CCAP method.

The difference in sensitivity of immunoassay systems due to differences in modification methods is thought to be primarily due to the following two factors. First, the effect on affinity to the antigen, and second, the effect on binding orientation on the material surface. These results demonstrate that, as seen with the anti-IgE 5D4 antibody, differences in IgG modification methods result in differences in affinity to the antigen. However, in some cases, such as with the anti-CA19-9 G6C8 antibody and anti-CA19-9 H7D1 antibody, there was no significant difference in affinity to the antigen (Figure 12). Furthermore, the random amine coupling method does not allow for selective modification. 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 antigen binding ability is thought to be limited by reduced orientation. On the other hand, in the case of biotin-modified antibodies using the CCAP method, biotin is modified site-specifically, allowing for uniform binding orientation on the material surface. Therefore, it is thought that a larger number of molecules can maintain their binding ability to the antigen. In particular, in the RPLA method, it is thought that the number of beads that bind to the antigen and undergo agglutination reaction is limited from the viewpoint of steric hindrance (Figure 10(c)).

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 immunoassay measurements. It is believed that the immunoassay method of the present invention can also be used to achieve highly sensitive measurements of antibodies from other hosts, such as human IgG and rabbit IgG, similar to rodent IgG.

Claims (37)

An immunoassay method using a complex of IgG and an IgG-binding peptide bound to a functional ligand, wherein the IgG and the IgG-binding peptide are covalently bound via disuccinimidyl glutarate, disuccinimidyl suberate, dimethyl adipimidate dihydrochloride, dimethyl pimelimidate dihydrochloride, dimethyl suberimidate dihydrochloride, dimethyl 3,3'-dithio-bis(propionimidate) dihydrochloride, or dithiobis(succinimidyl propionate), the IgG-binding peptide is bound to the side chain of Lys248 in the Fc region of the IgG, the functional ligand is selected from the group consisting of a functional group capable of being covalently bound to the surface of a detection platform material, a chemical crosslinker having such a functional group, and biotin, and the complex is immobilized on the surface of the detection platform material. The method of claim 1, which is a two-site immunoassay using two types of antibodies, a first antibody and a second antibody, wherein the first antibody or the second antibody is IgG. The method of claim 1, which is a multi-site immunoassay using a first antibody, a second antibody, and a third antibody or more antibodies, wherein the first antibody, the second antibody, or the third antibody is IgG. The method according to claim 2 or 3, wherein the first antibody is the IgG and is used as a capture antibody in a state where it is bound to the surface of a detection platform material via the functional ligand . The method of claim 2 or 3, wherein the first antibody is an antibody other than the IgG and is used as a tracer antibody without being directly bound to the surface of the detection base material. The method according to claim 2 or 3, wherein both the first antibody and the second antibody are IgG and are used as capture antibodies in a state where they are bound to the surface of a detection base material via the functional ligand . The method of claim 3, wherein both the first antibody and the second antibody are antibodies other than IgG and are used as tracer antibodies without being directly bound to the surface of the detection base material. The method according to claim 2 or 3, wherein the first antibody is the IgG and is used as a capture antibody bound to the surface of the detection base material via the functional ligand , and the second antibody is an antibody other than the IgG and is used as a tracer antibody that does not directly bind to the surface of the detection base material. The method according to claim 3, wherein the first antibody, the second antibody, the third antibody, or all of the more antibodies are IgG and are used as capture antibodies in a state where they are bound to the surface of a detection base material via the functional ligand . The method according to claim 3, wherein any one of the first antibody, the second antibody, the third antibody or more antibodies is the IgG and is used as a capture antibody in a state where it is bound to the surface of the detection base material via the functional ligand , and the other antibody is an antibody other than the IgG and is used as a tracer antibody in a state where it is not directly bound to the surface of the detection base material. The method according to claim 3, wherein any one of the first antibody, the second antibody, the third antibody or more antibodies is an antibody other than the IgG and is used as a tracer antibody in a state where it is not directly bound to the surface of the detection base material, and the other antibody is the IgG and is used as a capture antibody in a state where it is bound to the surface of the detection base material via the functional ligand . The method of any one of claims 1, 4, and 5, wherein the immunoassay method is selected from the group consisting of ELISA, immunochromatography, immuno-latex agglutination (LA), quartz crystal microbalance (QCM), biolayer interferometry (BLI), and surface plasmon resonance. The immunoassay method includes ELISA, immunochromatography, immunolatex agglutination (LA), turbidimetric immunoassay (TIA), chemiluminescent immunoassay (CLIA), pulse immunoassay, time-resolved fluorescence resonance energy transfer (TR-FRET), quartz crystal microbalance (QCM), and biolayer interferometry (BioLayer Interferometry). The method according to any one of claims 1, 2, and 4 to 8, wherein the method is selected from the group consisting of a BLI method and a surface plasmon resonance method. The immunoassay method includes ELISA, immunochromatography, immunolatex agglutination (LA), turbidimetric immunoassay (TIA), chemiluminescent immunoassay (CLIA), pulse immunoassay, time-resolved fluorescence resonance energy transfer (TR-FRET), quartz crystal microbalance (QCM), and biolayer interferometry (BioLayer Interferometry). The method according to any one of claims 1, 3, and 4 to 11, wherein the method is selected from the group consisting of a BLI method and a surface plasmon resonance method. The method according to any one of claims 1 to 7, 10, 11, 13, and 14, wherein the immunoassay method is a sandwich ELISA method, the functional ligand of the complex is biotin, and the complex is bound via the biotin to a solid phase coated with avidin or streptavidin. The method according to any one of claims 1 to 14, wherein the immunoassay method is an immunochromatography method. The method according to any one of claims 1 to 14, wherein the immunoassay method is a latex agglutination method. The method according to any one of claims 1 to 14, wherein the immunoassay method is a latex agglutination method, the functional ligand of the complex is biotin, and the complex is bound via the biotin to a latex surface coated with avidin or streptavidin. The method of any one of claims 1, 4, 5, and 12, wherein the immunoassay method is surface plasmon resonance and the complex is immobilized on a sensor chip. The method according to any one of claims 1 to 19, wherein the IgG-binding peptide is a peptide having the amino acid sequence of any one of SEQ ID NOs: 1 to 9, or a peptide in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence of any one of SEQ ID NOs: 1 to 9, and which has the ability to bind to the Fc region of IgG. An in vitro diagnostic agent comprising a complex of IgG and an IgG-binding peptide bound to a functional ligand, wherein the IgG and the IgG-binding peptide are covalently bonded via disuccinimidyl glutarate, disuccinimidyl suberate, dimethyl adipimidate dihydrochloride, dimethyl pimelimidate dihydrochloride, dimethyl suberimidate dihydrochloride, dimethyl 3,3'-dithio-bis(propionimidate) dihydrochloride, or dithiobis(succinimidyl propionate), the IgG-binding peptide is bound to the side chain of Lys248 in the Fc region of the IgG, the functional ligand is selected from the group consisting of a functional group capable of being covalently bonded to the surface of a detection platform material, a chemical crosslinker having such a functional group, and biotin, and the complex is immobilized on the surface of the detection platform material. The in vitro diagnostic reagent according to claim 21, wherein the IgG-binding peptide is a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 9, or an amino acid sequence of any one of SEQ ID NOs: 1 to 9 in which one or more amino acids have been substituted, deleted, or added, and which has the ability to bind to the Fc region of IgG. An immunochromatographic test piece for use in the method of any one of claims 1 to 14, comprising a labeled antibody retaining section that retains a labeled antibody and a detection area to which a capture antibody is immobilized, wherein the labeled antibody or the capture antibody, or both, are a complex of IgG and an IgG-binding peptide bound to a functional ligand, the IgG and the IgG-binding peptide are covalently bonded via disuccinimidyl glutarate, disuccinimidyl suberate, dimethyl adipimidate dihydrochloride, dimethyl pimelimidate dihydrochloride, dimethyl suberimidate dihydrochloride, dimethyl 3,3'-dithio-bis(propionimidate) dihydrochloride, or dithiobis(succinimidyl propionate), the IgG-binding peptide is bound to the side chain of Lys248 in the Fc region of the IgG, and the functional ligand is selected from the group consisting of a functional group capable of being covalently bonded to the surface of a detection base material, a chemical crosslinker having the functional group, and biotin. The immunochromatographic test piece according to claim 23, wherein the IgG-binding peptide is a peptide comprising an amino acid sequence of any one of SEQ ID NOS: 1 to 9, or an amino acid sequence of any one of SEQ ID NOS: 1 to 9 in which one or more amino acids have been substituted, deleted, or added, and which has the ability to bind to the Fc region of IgG. A solid phase on which a complex of IgG and an IgG-binding peptide bound to a functional ligand is immobilized, the IgG and the IgG-binding peptide being covalently bonded via disuccinimidyl glutarate, disuccinimidyl suberate, dimethyl adipimidate dihydrochloride, dimethyl pimelimidate dihydrochloride, dimethyl suberimidate dihydrochloride, dimethyl 3,3'-dithio-bis(propionimidate) dihydrochloride, or dithiobis(succinimidyl propionate), the IgG-binding peptide being bound to the side chain of Lys248 in the Fc region of the IgG, and the functional ligand being selected from the group consisting of a functional group capable of being covalently bonded to the surface of a detection base material, a chemical crosslinker having the functional group, and biotin. The solid phase described in claim 25, wherein the functional ligand of the complex is biotin, and the complex is bound to a solid phase coated with avidin or streptavidin via the biotin. The solid phase described in claim 25, wherein the functional ligand of the complex is a functional group capable of covalently binding to the surface of the detection platform material, or a chemical cross-linker having such a functional group, and the complex is covalently bound to the surface of the detection platform material without the intervention of an anchor protein. A solid phase comprising latex particles and particles made of a polymeric material on which a complex of IgG and an IgG-binding peptide bound to a functional ligand is immobilized, wherein the IgG and the IgG-binding peptide are covalently bonded via disuccinimidyl glutarate, disuccinimidyl suberate, dimethyl adipimidate dihydrochloride, dimethyl pimelimidate dihydrochloride, dimethyl suberimidate dihydrochloride, dimethyl 3,3'-dithio-bis(propionimidate) dihydrochloride, or dithiobis(succinimidyl propionate), the IgG-binding peptide is bound to the side chain of Lys248 in the Fc region of the IgG, and the functional ligand is selected from the group consisting of a functional group capable of covalently binding to the surface of a detection base material, a chemical crosslinker having such a functional group, and biotin. The solid phase described in claim 28, wherein the functional ligand of the complex is biotin, and the complex is bound to latex coated with avidin or streptavidin via the biotin. The solid phase of claim 28, wherein the functional ligand of the complex is a functional group capable of covalently binding to the surface of a latex material, or a chemical crosslinker having such a functional group, and the complex is covalently bound to the latex surface without the intervention of an anchor protein. The solid phase described in any one of claims 26 to 30, wherein the solid phase immobilizes an IgG-binding peptide complex via a functional group that reacts with an amino group. The solid phase described in any one of claims 26 to 30, wherein the solid phase immobilizes an IgG-binding peptide complex via a functional group that reacts with a succinimide group. The solid phase described in any one of claims 26 to 30, wherein the solid phase immobilizes an IgG-binding peptide complex via a functional group that reacts with a thiol group. The solid phase described in any one of claims 26 to 30, wherein the solid phase immobilizes an IgG-binding peptide complex via a functional group that reacts with a maleimide group. The solid phase described in any one of claims 26 to 30, wherein the solid phase immobilizes an IgG-binding peptide complex via a functional group that reacts with a carboxy group. The solid phase described in any one of claims 26 to 30, wherein the solid phase immobilizes an IgG-binding peptide complex via a functional group bonded by a click reaction. The solid phase according to any one of claims 26 to 36, wherein the IgG-binding peptide is a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 9, or an amino acid sequence of any one of SEQ ID NOs: 1 to 9 in which one or more amino acids have been substituted, deleted, or added, and which has the ability to bind to the Fc region of IgG.