Immunoassay method using site-specifically modified body of igg antibody with igg-binding peptide

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 loss of function. However, in conventional methods, denaturation or loss of function of the antibody immobilized on the material surface has occurred, and the antigen-binding ability has sometimes decreased or been lost.

[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 modified body of an IgG antibody using an IgG-binding peptide that can bind to IgG derived from rodents and has little denaturation or functional loss even when immobilized on the surface of a material.

Means for Solving the Problems

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

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

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

[0010] The present invention also relates to an immunochromatographic test strip used in the above immunoassay method, which includes a labeled antibody holding part that holds a labeled antibody and a detection region where a capture antibody is immobilized, and the labeled antibody or the capture antibody or both are a complex of an IgG-binding peptide to which an IgG and a functional ligand are bound.

[0011] The present invention further relates to a method for producing the above solid phase, which includes a step of covalently bonding or physically adsorbing an anchor protein to the solid phase, and a step of covalently bonding or physically adsorbing a complex of an 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 body of an IgG antibody using an IgG-binding peptide that can bind to IgG derived from rodents and has little denaturation or functional loss even when immobilized on the surface of a material.

Brief Description of the Drawings

[0013]

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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 engineering 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), mouse IgG (IgG1, IgG2a, IgG2b, IgG2c or IgG3).

[0017] Examples of the functional ligand include drugs, proteins, peptides, nucleic acids, enzymes, radiolabeled substances, fluorescent substances, and chemical cross-linkers (Cross-linker) having a functional group capable of covalently bonding to the surface of the detection substrate material. More specifically, the functional ligand may be biotin or an azide compound (azide). The functional ligand may bind directly to the IgG-binding peptide or may bind via a molecule such as PEG (polyethylene glycol).

[0018] The binding between the IgG-binding peptide and the functional ligand can be carried out by known methods, for example, the reaction between an azide group (-azide) and a DBCO (Dibenzocyclooctyne) group, the reaction between a maleimide group and a sulfhydryl group (-SH), etc.

[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 the IgG-binding peptide 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) for human and rodent IgG-Fc. The IgG-binding peptide may include a peptide selected from the peptides represented by SEQ ID NOs: 1 to 9, and may include a peptide in which one or more amino acids are substituted, deleted, or added in these peptides 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 are substituted, deleted, or added in these peptides and which has the ability to bind to the Fc region of IgG, to which a functional ligand or a functional group for adding a functional ligand is added. Note that SEQ ID NO: 1 is an amino acid sequence in which all Lys included in Z34C are substituted with Arg, and is denoted 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 denoted as εZ34C. SEQ ID NO: 3 is an amino acid sequence in which Gly is added to the N-terminus of αZ34C, and is denoted as α-1Z34C. SEQ ID NO: 4 is an amino acid sequence in which the 7th Arg of αZ34C is substituted with Lys. SEQ ID NO: 5 is an amino acid sequence in which the 7th Arg of εZ34C is substituted with Lys. SEQ ID NO: 6 is an amino acid sequence in which the 8th Arg of α-1Z34C is substituted with Lys. SEQ ID NO: 7 is an amino acid sequence in which the 5th Cys of αZ34C is substituted with Gln and the C-terminal Cys is removed. SEQ ID NO: 8 is an amino acid sequence in which the 5th Cys of αZ34C is substituted with Gln, the C-terminal Cys is removed, and Pro-Ser-Arg-Arg-Lys-Arg is added to the C-terminus, and is denoted as Z33-38.SEQ ID NO: 9 is an amino acid sequence in which the 5th Cys of αZ34C is replaced with Gln, the 7th and 28th Arg are replaced with Lys, the C-terminal Cys is removed, and Pro-Ser-Arg-Arg-Lys-Arg-Arg-Lys-Arg-Arg-Lys is added to the C-terminus, and is designated as Z33-5.

[0020] 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.

[0021] The variants of Z34C can be synthesized by known methods. Examples of the 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. From the viewpoint of ease of operation, the solid-phase synthesis method is preferred. When an IgG-binding peptide such as a variant of Z34C is modified with a crosslinking agent described later, such an IgG-binding peptide can also be produced by modifying the synthesized IgG-binding peptide with the crosslinking agent, or can be produced by performing peptide synthesis using an amino acid residue modified with the crosslinking agent.

[0022] In one embodiment, the IgG-binding peptide is considered to bind to the side chain of Lys248 in the Fc region of IgG, and the two are bound via a cross-linking agent, for example, DSG (Disuccinimidyl Glutarate). Other cross-linking agents include cross-linking agents preferably containing two or more succinimidyl groups such as DSS (Disuccinimidyl suberate), cross-linking agents preferably containing two or more imide ester moieties such as DMA (Dimethyl Adipimidate Dihydrochloride), DMP (Dimethyl Pimelimidate Dihydrochloride), DMS (Dimethyl Suberimidate Dihydrochloride), cross-linking agents having an SS bond such as DTBP (Dimethyl 3,3’-dithio-bis(propionimidate) Dihydrochloride), DSP (Dithiobis (succinimidyl propionate)), and the like.

[0023] Examples of immunoassay methods include 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), biolayer interferometry (BLI), surface plasmon resonance, and the like.

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

[0025] A solid phase according to an embodiment has immobilized thereon a complex of an IgG-binding peptide in which IgG and a functional ligand are bound.

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

[0027] The above complex may be immobilized via an anchor protein on the surface of a detection substrate material having a functional group that reacts with an amino group (Fig. 1). The anchor protein (Protein in Fig. 1) is not particularly limited, and can be streptavidin or the like. In view of the balance with non-specific reactions, BSA or HSA used as a blocking agent is suitable.

[0028] As shown in Fig. 1, a functional group that reacts with an amino group is reacted with the amino group in the anchor protein, or a functional group that reacts with a carboxy group is reacted with the carboxy group in the anchor protein, etc. Further, the amino group in the anchor protein is reacted with DBCO-NHS, or the -SH group in the anchor protein is reacted with DBCO-maleimide, so that -DBCO can be introduced into the anchor protein bound to the surface of the detection substrate material. Further, a click reaction can occur between the -DBCO introduced into this anchor protein and the -azide introduced into the IgG-binding peptide in the complex, and the complex can be indirectly immobilized on the surface of the detection substrate material. Note that the functional group to be introduced is not limited to -DBCO, and any functional group that can be used in the click reaction may be used. 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. may be used. In addition to this click reaction, reactions using -SH and -maleimide or bromoacetyl can also be used.

[0029] Examples of the functional group that reacts with an amino group include a carboxy group, a tosyl group, an epoxy group, an isocyanate group, an isothiocyanate group, an N-hydroxysuccinimide group (-NHS), a maleimide group, etc. As the detection substrate with a functional group that reacts with an amino group, an immobilizer (amino) (Thermo Fisher Scientific K.K. (former Nalge Nunc 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 reactive with the above amino group without an anchor protein (Figure 2). In this case, before immobilization, -SH or -azide is introduced into the IgG-binding peptide, and if necessary, a protecting group for -SH is also introduced. Deprotect if necessary, and react -SH or -azide with a functional group having an alkyne introduced by reacting it with a functional group reactive with the amino group on the surface of the detection substrate material (cyclooctyne, DBCO, BCN, etc.) to form a covalent bond, and the above complex can be immobilized on the surface of the detection substrate material.

[0031] Examples of the protecting group include SATA (N-Succinimidyl S-acetylthioacetate), SATP (N-Succinimidyl S-Acetylthiopropionate), etc.

[0032] Specific examples of the above complex immobilized on the surface of a detection substrate material with a functional group reactive with the amino group without an anchor protein include Figure 2(b) and (c).

[0033] The above complex can be produced by a step of mixing an IgG-binding peptide to which an IgG and a functional ligand are bound. The mixing conditions are not particularly limited as long as a cross-linking 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 at room temperature in an appropriate buffer. When a cross-linking agent is bound to the IgG-binding peptide to which the functional ligand is bound, an appropriate amount of a catalyst for promoting the cross-linking reaction may be added and mixed if necessary.

[0034] In order to enhance the binding affinity between the IgG-binding peptide conjugated with the functional ligand and IgG, the reaction conditions may be adjusted. For example, Protein A, from which the Z34C peptide is derived, is known to increase its binding affinity with human IgG3 under conditions where the pH is 8 or higher, and is also known to increase its binding affinity with mouse IgG1 by increasing the salt concentration such as NaCl. Referring to such findings, the conditions for the cross-linking reaction of the present invention can be set.

[0035] The mixing step of the IgG-binding peptide conjugated with the functional ligand 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-binding peptide conjugated with the functional ligand and IgG can be IgG:peptide = 1:1 to 20.

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

[0038] After the above mixing step, if necessary, a step of separating impurities such as the IgG-binding peptide conjugated with the unreacted functional ligand, IgG, and reagents from the obtained mixture and purifying the complex may be further carried out. The purification step can be performed by known methods such as chromatography including gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, HPLC, etc.

[0039] The immunochromatographic test strip according to one embodiment is used in the above immunoassay method, and includes a labeled antibody holding part that holds a labeled antibody and a detection region where a capture antibody is immobilized, and the labeled antibody or the capture antibody is a complex of IgG and an IgG-binding peptide conjugated with a functional ligand.

[0040] The method for manufacturing a solid phase according to one embodiment includes a step of covalently bonding or physically adsorbing an anchor protein to the solid phase, and a step of covalently bonding, physically adsorbing, or specifically binding a complex of IgG and an IgG-binding peptide to the anchor protein bound to the solid phase.

Examples

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

[0042] <Preparation of Monoclonal Antibody, Antigen, and Peptide> Trastuzumab was purchased for research use from iRxMedicine. As the mouse IgG1 control antibody, mouse IgG1, κ isotype control purchased from Crown Bioscience, Inc., or mouse IgG1 isotype control purchased from the Institute of Medical Biology was used. As the mouse IgG2 control antibody, InVivoMAb anti-human / rat HER2 purchased from BioCell Technology, LLC was used. The anti-CA19-9 H7D1 antibody and the anti-CA19-9 G6C8 antibody were purchased from BBI Solutions. The 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 by Eurofins.

[0043] <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 is amidated. After removing the protecting groups, the peptides were purified using reverse-phase HPLC.

[0044] <Formation of intramolecular disulfide bond> The following steps were 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 reverse-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 evaporated off and lyophilized for 20 hours. The lyophilized product was dissolved in DMSO to a concentration of 10 mM.

[0045] <DSG binding> The following steps were 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 solution of disuccinimidyl glutarate (DSG) dissolved in acetonitrile were mixed so that the 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. The fractions separated using an Inert-Sustain C18 reverse-phase column (5 μm, 7.6×250 mm) were collected, and the desired fractions were lyophilized for 20 hours.

[0046] <Binding of biotin and DSG to Z33-38azide> The following procedure was performed on Z33-38azide to obtain Z33-38biotin with biotin 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 such that the molar ratio of peptide:DBCO-PEG4-Biotin:DSG was 1:1:20, and pyridine was added to a final concentration of 5%. Thereafter, the mixture was incubated at 50 °C for 2 hours.

[0047] <Peptide Structure Modeling> The model structure of the complex of 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] <Binding of Three Types of Z34C Variants (αZ34C, εZ34C, and α-1Z34C), Z33-38biotin, or Z33-5azide to 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) and the peptide reagent diluted to 10 mM in DMSO were mixed such that the molar ratio of IgG:peptide was 1:5. The mixture was incubated at each reaction temperature (25 °C, 37 °C, 50 °C) for 1 hour or overnight (about 16 hours). The results of the structural simulation of the binding of the three types of Z34C variants (αZ34C, εZ34C, and α-1Z34C) to IgG are shown in Figure 3.

[0049] <sds-page> 450 μL of 4× Laemmli Sample Buffer (Bio-Rad Laboratories, Inc.) and 50 μL of DTT solution (1 M DTT, 1 mM EDTA) were mixed to prepare 4× reducing sample buffer. The sample solution was mixed with the 4× reducing sample buffer and incubated at 95 °C for 10 minutes. The obtained sample was applied to an SDS-PAGE gel (Mini Protean TGX precast Gels Any kD; Bio-Rad Laboratories, Inc.) at 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> Streptavidin was coated on an ELISA plate (Nunc-Immuno Module plate Maxisorp; Thermo Fisher Scientific, Inc.) at 300 ng / well and incubated at 4 °C overnight. After washing the plate, 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 at 4 °C overnight. Biotinylated antibody was added at 150 ng / well and incubated at 37 °C for 1 hour. After washing the plate, human CA19-9 or human IgE was added at each concentration and incubated at 37 °C for 1 hour. After washing the plate, HRP-labeled antibody was added at an amount of 30 ng / well and incubated at 37 °C for 1 hour. After washing the plate, TMB substrate was added and incubated at 25 °C for 30 minutes. After adding the same amount of 0.3 M H2SO4 as the TMB substrate, the absorbance at 450 nm and 630 nm was measured. Biotinylated antibody (-NHS) was prepared using Biotin Labeling kit-NH2 (Dojindo Laboratories). HRP-labeled antibody was prepared using Peroxidase Labeling kit-NH2 (Dojindo Laboratories).

[0051] <Preparation of Antibody-Bound Beads for RPLA (Reverse Passive Latex Agglutination Reaction)> 1 mL of PBS was mixed with 60 μL of a 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 stirred, followed by rotation 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 again 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 at a 1:1 ratio and added to a V-bottom 96-well microplate (25 μL / well). 25 μL of the antibody-bound bead slurry was added to each well. After mixing for 1 minute on 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 to find the condition with the highest modification rate. The modification rate was evaluated by SDS-PAGE (Figure 4).

[0054] According to Figure 4, it can be seen that the pH of the reaction solution has a great influence on the modification efficiency. Furthermore, αZ34C had reactivity above a certain level in both mouse IgG1 (Figure 4(a)) and mouse IgG2a (Figures 4(b)(c)), which are often used in immunoassay methods. Furthermore, the modification of these three types of Z34C variants to human IgG1 was also confirmed (Figure 5).

[0055] According to this reaction, theoretically monovalent or divalent modification occurs (Figure 4(d)). The actual modification rate for 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 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 has a tendency to have a higher yield of synthetic peptide compared to the three types of Z34C variants (αZ34C, εZ34C, and α-1Z34C), and is easier and cheaper to synthesize.

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

[0059] The biotinylated antibody was added to a plate coated with streptavidin, and sandwich ELISA measurements were performed. The biotinylated antibody was prepared by the random amine coupling method (-NHS) or the CCAP method (-CCAP). As a result of measuring by changing the concentration of the IgE antigen, a significant difference was observed between the random amine coupling method and the CCAP method, and the CCAP method was more sensitive than the random amine coupling method (Figure 9). This is presumably because, firstly, the antibody modified by the CCAP method has less reduction in antibody reactivity to the antigen than the antibody modified by the random amine coupling method. Secondly, it is presumably because the CCAP method has a good orientation of the capture antibody to the plate and a large number of antigen-binding sites capable of binding to the antigen.

[0060] (Test Example 5) Using an antibody modified with biotin by the random amine coupling method or an antibody modified with biotin by the CCAP method, the antigen detection ability in RPLA was compared. The biotin-labeled antibody was adsorbed onto streptavidin-coated beads, and antigens at each concentration were prepared and allowed to act on the antibody-bound beads. In the systems using CA19-9 or IgE as antigens, the test was carried out with n = 3.

[0061] In each system using CA19-9 as an antigen, an increase in score depending on the antigen concentration was observed (Figure 10(a)). The system using the CCAP method showed a higher score even at a lower antigen concentration, and the sensitivity was higher than that of the system using the random amine coupling method. Also, when using beads bound with a control antibody, no agglutination reaction should occur and the score should be (-), but a non-specific agglutination reaction occurred in the system using the random amine coupling method. This is presumably because in the system using the random amine coupling method, the structure of the antibody is not maintained due to the random coupling reaction occurring at the lysine residues on the antibody surface, and the hydrophobic part is exposed. On the other hand, such a non-specific reaction was not observed in the system using the CCAP method (Figure 10(a)).

[0062] In the system using IgE as an antigen, an increase in the score dependent on the antigen concentration was confirmed only in the system using the CCAP method. When the random amine coupling method was used, no agglutination reaction of the beads was observed at any concentration (Fig. 10(b)). Fig. 11 shows the actual agglutination image when the RPLA reaction was carried out.

[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 the amine coupling method according to the manufacturer's instructions. The binding kinetics were analyzed in single-cycle kinetics mode using five concentrations of IgG diluted in the assay 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 measurement of the binding process was carried out by adding each concentration of IgG for 2 minutes (flow rate 30 μL / min). The measurement of the dissociation process was carried out by flowing the assay buffer for 30 minutes (flow rate 30 μL / min). The diluted 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 the biotinylated antibodies used in the sandwich ELISA system and the RPLA method were confirmed by the ELISA method using plates immobilized with the antigen. For both the anti-CA19-9 G6C8 antibody and the anti-CA19-9 H7D1 antibody, there was not much difference between the random amine coupling method (-NHS) and the CCAP method. Regarding the anti-IgE antibody titer, the CCAP method was significantly higher than the random amine coupling method (Fig. 12).

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

[0068] The difference in the sensitivity of the immunoassay system due to such differences in the modification methods is considered to be mainly caused by the following two points. The first is the effect on the affinity for the antigen, and the second is the effect on the binding orientation to the material surface. From the above results, it can be seen that, as in the case of the anti-IgE 5D4 antibody, a difference in the affinity for the antigen occurs due to the difference in the modification method of IgG. However, in some cases, there was no significant difference in the affinity for the antigen, such as the anti-CA19-9 G6C8 antibody and the anti-CA19-9 H7D1 antibody (Figure 12). Also, in the random amine coupling method, the site to be modified cannot be selected. Therefore, when binding to the material surface such as a streptavidin-coated plate or streptavidin-coated beads via biotin modified by the random amine coupling method, it is difficult to make the orientation of the antibody uniform. The number of antibody molecules maintaining the binding ability to the antigen is considered to be limited by the decrease in orientation. On the other hand, in the case of the biotin-modified antibody by the CCAP method, since biotin is modified site-specifically, the binding orientation on the material surface can be made uniform. Therefore, it is considered that more molecules can maintain the binding ability to the antigen. In particular, in the RPLA method, it is considered that the number of beads leading to the aggregation reaction by binding to the antigen is limited from the viewpoint of steric hindrance (Figure 10(c)).

[0069] As described above, by using the immunoassay method of the present invention, the affinity of the antibody for rodent IgG can be maintained and the binding orientation can be improved, and as a result, highly sensitive measurement in the immunoassay method can be realized. It is considered that highly sensitive measurement can be realized for antibodies of other hosts such as human IgG and rabbit IgG in the same manner as rodent IgG by using the immunoassay method of the present invention.

Claims

1. An immunoassay method using a complex of IgG and an IgG-binding peptide bound to a functional ligand.

2. The method of claim 1, wherein the functional ligand is selected from the group consisting of a drug, a protein, a peptide, a nucleic acid, an enzyme, a radioactive label, a fluorescent substance, a functional group capable of being covalently bound to the surface of a detection base material, a chemical crosslinker having the functional group, and biotin.

3. The method according to claim 1 or 2, which is a one-site immunoassay using only the first antibody.

4. The method according to claim 1 or 2, which is a two-site immunoassay using two types of antibodies, a first antibody and a second antibody.

5. 3. The method of claim 1 or 2, which is a multi-site immunoassay using a first antibody and a second antibody and a third antibody or more antibodies.

6. The method according to any one of claims 1 to 3, wherein the immunoassay method is selected from the group consisting of an ELISA method, an immunochromatography method, an immuno-latex agglutination (LA) method, a quartz crystal microbalance (QCM) method, a BioLayer Interferometry (BLI) method, and a surface plasmon resonance method.

7. The immunoassay method includes an ELISA method, an immunochromatography method, an immunolatex agglutination (LA) method, a turbidimetric immunoassay (TIA) method, a chemiluminescent immunoassay (CLIA) method, a pulsed immunoassay method, a time-resolved fluorescence resonance energy transfer (TR-FRET) method, a quartz crystal microbalance (QCM) method, a biolayer interferometry (BioLayer Interferometry (BIOLA) method, a fluoroscopy (FFT ... The method according to any one of claims 1, 2 and 4, wherein the method is selected from the group consisting of a BLI method and a surface plasmon resonance method.

8. The immunoassay method includes an ELISA method, an immunochromatography method, an immunolatex agglutination (LA) method, a turbidimetric immunoassay (TIA) method, a chemiluminescent immunoassay (CLIA) method, a pulsed immunoassay method, a time-resolved fluorescence resonance energy transfer (TR-FRET) method, a quartz crystal microbalance (QCM) method, a biolayer interferometry (BioLayer Interferometry (BIOLA) method, a fluoroscopy (FFT ... The method according to any one of claims 1, 2 and 5, wherein the method is selected from the group consisting of a BLI method and a surface plasmon resonance method.

9. The method according to any one of claims 3 to 8, wherein the first antibody is used as a capture antibody bound directly to the surface of the detection base material, by physical adsorption, without an anchor protein, or bound via an anchor protein.

10. The method according to any one of claims 3 to 8, wherein the first antibody is used as a tracer antibody without being directly bound to the surface of a detection base material.

11. The method according to any one of claims 4, 5, 7 and 8, wherein both the first antibody and the second antibody are used as capture antibodies bound directly to the surface of the detection base material, by physical adsorption, without an anchor protein or via an anchor protein.

12. 9. The method according to any one of claims 4, 5, 7 and 8, wherein both the first antibody and the second antibody are used as tracer antibodies without being directly bound to the surface of a detection base material.

13. The method according to any one of claims 4, 5, 7 and 8, wherein the first antibody is used as a capture antibody bound directly to the surface of the detection base material by physical adsorption, without an anchor protein or via an anchor protein, and the second antibody is used as a tracer antibody not bound directly to the surface of the detection base material.

14. The method according to claim 5 or 8, wherein the first antibody, the second antibody, the third antibody or all of the more antibodies are used as capture antibodies bound directly to the surface of the detection base material, by physical adsorption, without an anchor protein or via an anchor protein.

15. The method according to claim 5 or 8, wherein the first antibody, the second antibody, the third antibody or all of the more antibodies are used as tracer antibodies without being directly bound to the surface of the detection base material.

16. The method according to claim 5 or 8, wherein any one of the first antibody, the second antibody, the third antibody or more antibodies is used as a capture antibody in a state bound directly to the surface of the detection base material by physical adsorption, without an anchor protein or via an anchor protein, and the other antibodies are used as tracer antibodies in a state not bound directly to the surface of the detection base material.

17. The method according to claim 5 or 8, wherein any one of the first antibody, the second antibody, the third antibody or more antibodies is used as a tracer antibody without being directly bound to the surface of the detection base material, and the other antibodies are used as capture antibodies bound directly to the surface of the detection base material by physical adsorption, without an anchor protein or via an anchor protein.

18. The method according to any one of claims 1, 2, 4, 5, 7 to 13, 16 and 17, wherein the immunoassay method is a sandwich ELISA method, 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.

19. The method according to any one of claims 1 to 14, 16 and 17, wherein the immunoassay method is an immunochromatography method.

20. The method according to any one of claims 1 to 14, 16 and 17, wherein the immunoassay method is a latex agglutination method.

21. The method according to any one of claims 1 to 14, 16 and 17, 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.

22. The method according to any one of claims 1 to 3, 6, 9 and 10, wherein the immunoassay method is a surface plasmon resonance method, and the complex is immobilized on a sensor chip.

23. The method according to any one of claims 1 to 22, 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.

24. An in vitro diagnostic agent comprising a complex of IgG and an IgG-binding peptide bound to a functional ligand.

25. The in vitro diagnostic agent according to claim 24, 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.

26. An immunochromatographic test piece for use in the method according to any one of claims 1 to 17, comprising a labeled antibody retaining section that retains a labeled antibody and a detection area to which a capture antibody is fixed, 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.

27. The immunochromatographic test piece according to claim 26, 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.

28. A solid phase on which a complex of IgG and an IgG-binding peptide bound to a functional ligand is immobilized.

29. 29. The solid phase of claim 28, wherein the functional ligand of the complex is biotin and the complex is bound via the biotin to an avidin or streptavidin coated solid phase.

30. The solid phase described in claim 28, wherein the functional ligand of the complex is a functional group capable of covalently binding to the surface of a detection base material, or a chemical cross-linking agent having the functional group, and the complex is covalently bound to the surface of the detection base material without the intervention of an anchor protein.

31. A solid phase comprising latex particles on which a complex of IgG and an IgG-binding peptide bound to a functional ligand is immobilized, and particles made of a polymeric material.

32. 32. The solid phase of claim 31 , wherein the functional ligand of the complex is biotin and the complex is bound via the biotin to latex coated with avidin or streptavidin.

33. 32. The solid phase of claim 31 , wherein the functional ligand of the complex is a functional group capable of covalently binding to a surface of a latex material, a chemical cross-linker having said functional group, and the complex is covalently bound to the latex surface without the intervention of an anchor protein.

34. A solid phase having bound thereto a complex of IgG and an IgG-binding peptide, said IgG-binding peptide being covalently bound to an anchor protein, said anchor protein being covalently bound to a surface of the solid phase.

35. The solid phase according to any one of claims 28 to 34, wherein said solid phase is for immobilization of IgG-binding peptide complexes via functional groups that react with amino groups.

36. The solid phase according to any one of claims 28 to 34, wherein said solid phase is for immobilization of IgG-binding peptide complexes via functional groups that react with succinimide groups.

37. The solid phase according to any one of claims 28 to 34, wherein said solid phase is for immobilization of IgG-binding peptide complexes via functional groups that react with thiol groups.

38. The solid phase according to any one of claims 28 to 34, wherein said solid phase is for immobilization of an IgG-binding peptide complex via a functional group that reacts with a maleimide group.

39. The solid phase according to any one of claims 28 to 34, wherein said solid phase is for immobilization of IgG-binding peptide complexes via functional groups that react with carboxy groups.

40. The solid phase according to any one of claims 28 to 34, wherein the solid phase is for immobilization of an IgG-binding peptide complex via a functional group bonded by a click reaction.

41. A solid phase having a complex of IgG and an IgG-binding peptide bound thereto, the IgG-binding peptide being physically adsorbed to an anchor protein, the anchor protein being physically adsorbed to a surface of the solid phase.

42. The solid phase according to any one of claims 28 to 41, 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.

43. A method for producing a solid phase according to any one of claims 34 to 40, comprising the steps of covalently binding an anchor protein to the solid phase, and covalently binding a complex of IgG and an IgG-binding peptide to the anchor protein bound to the solid phase.

44. A method for producing a solid phase as described in claim 41, comprising the steps of physically adsorbing an anchor protein to the solid phase and physically adsorbing a complex of IgG and an IgG-binding peptide to the anchor protein bound to the solid phase.

45. The method according to claim 43 or 44, 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.

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