Detection method and detection reagent

Abstract

The objective is to provide a method for suppressing non-specific reactions using a highly versatile method without affecting specific reactions. [Solution] A method for detecting a target antigen in a sample using a specific antibody that specifically binds to the antigen, wherein a modified antibody is used in which part or all of the variable regions of the L chain and / or H chain of the specific antibody are modified, and the detection method is characterized by comprising the steps of: a) contacting the sample with the modified antibody, and b) contacting the sample with the specific antibody simultaneously with or after step a), thereby providing a method for suppressing nonspecific reactions with excellent versatility.

Description

Technical Field

[0001] The present invention relates to a detection method and a detection reagent using a specific antibody against an antigen in a sample. More specifically, in order to suppress non-specific reactions, a modified antibody of the specific antibody is used to a) contact the sample with the modified antibody, and b) simultaneously with or after the step a), contact the sample with the specific antibody, and the detection method and the detection reagent are characterized in that they include the above steps.

Background Art

[0002] As a measurement method in the field of diagnostic agents, an immunological measurement method for detecting a measurement target antigen present in a biological sample using a specific antibody can be mentioned. Here, in addition to the measurement target antigen, various substances are present in the biological sample, and substances that cause non-specific reactions, so-called non-specific factors, are often included. When non-specific factors are present in the sample, binding not based on specific reactions is promoted or specific immune reactions are hindered, which causes measurement errors. As non-specific factors, the presence of heterophilic antibodies and rheumatoid factor (RF) has been clarified, but there are also various other substances, and there are still many unclear points. By the way, as techniques for suppressing non-specific reactions in immunological measurement methods, for example, Patent Documents 1 to 4 are known. However, these methods cannot be applied to all measurement items in principle, and there are actually measurement items for which non-specific reactions cannot be suppressed.

[0003] Patent Document 1 discloses an immunoassay method that suppresses nonspecific reactions by performing an immunoassay in the presence of a monoclonal antibody-derived substance, in which the specific antibody activity of the monoclonal antibody used in the immunoassay method has been completely or substantially lost, but its nonspecific activity has been substantially retained. Specifically, the monoclonal antibody-derived substance is prepared by denaturing the antibody structure through methods such as sonication, organic solvent treatment, or acid / alkali treatment, thereby substantially eliminating the antibody's original antibody activity while substantially retaining its nonspecific activity.

[0004] Patent Document 2 discloses a method for detecting a target antigen in a sample using an insoluble carrier carrying a fragment antibody, in which a nonspecific reaction is suppressed by contacting the sample with a denatured fragment antibody beforehand or simultaneously. The denaturation is carried out using methods commonly used for protein denaturation, such as heating, acid, alkali, reducing agents, and chaotropic salts.

[0005] Furthermore, Patent Document 3 discloses a method for detecting analytes, particularly tumor markers, by reducing interference in immunoassays by adding a substance containing a peptide sequence derived from the framework region of a variable domain such as a detection antibody to the sample. The peptide sequence shown is a specific sequence located in framework region 1 and framework region 3 of the heavy chain of an antibody.

[0006] Furthermore, Patent Document 4 discloses a method for eliminating the influence of interfering substances by adding a large excess of an inhibitory substance to an immunoassay, which maintains specificity for the interfering substance but no longer reacts with the antigen to be analyzed, or has been altered to the point of a clear decrease in reaction. Methods for preparing the inhibitory substance include methods for selectively removing the antigen-binding structure from the reagent antibody by degrading the antibody using an enzyme, and methods for altering the antigen-binding structure of the reagent antibody by mutation during hybridoma culture. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Patent No. 2561134 [Patent Document 2] Patent No. 6431766 [Patent Document 3] Patent No. 4334065 [Patent Document 4] Japanese Patent Application Publication No. 1-28560 [Overview of the project] [Problems that the invention aims to solve]

[0008] However, in the method described in Patent Document 1, the monoclonal antibody-derived substance that functions as a non-specific reaction inhibitor is prepared by heat treatment, and it has been reported that Fab is substantially removed. In order to deactivate the reactivity of the antibody using this method, high temperature or prolonged heating is required, and it is extremely difficult to find the optimal conditions. When the present inventors attempted to prepare a non-specific reaction inhibitor by heat-treating a monoclonal antibody using the method described in the same document, they confirmed that a precipitate formed at the same time as the antibody's reactivity was deactivated, and that the non-specific reaction inhibitory effect could not be observed. Therefore, this method has the problem of lacking versatility.

[0009] Furthermore, the method described in Patent Document 2 is limited to cases where a fragment antibody is used as the detection antibody. The denatured fragment antibody described in Patent Document 2 requires the use of an antibody with a different recognition epitope from the detection fragment antibody, and is cumbersome because it requires the creation of multiple monoclonal antibodies and the identification of the recognition epitope of each antibody. Moreover, similar to Patent Document 1, it has the problem of lacking versatility because it requires optimal conditions to be set regarding the antibody denaturation method.

[0010] Furthermore, the method described in Patent Document 3 provides little information other than the use of a peptide containing specific sequences derived from the disclosed antibody framework region 1 and framework region 3, and is considered to be effective only when the cause of the non-specific reaction depends on those sequences. Therefore, it suffers from the same lack of versatility as Patent Documents 1 and 2.

[0011] Furthermore, while Patent Document 4 describes a method for separating the Fab region from the Fc region as a specific example of an enzyme-based antibody degradation method, the region separated and removed is large, and it is considered effective only in non-specific reactions dependent on the Fc region, and ineffective in non-specific reactions dependent on the variable region. In addition, because this method utilizes mutant antibodies obtained through mutations that may occur during the hybridoma culture process, it is very rare to obtain mutant antibodies with the expected mutations, resulting in a lack of versatility and reproducibility. The present invention aims to solve the above problems and to provide a method for effectively suppressing nonspecific reactions using a highly versatile method. [Means for solving the problem]

[0012] In order to solve the above problem, the inventors of the present invention investigated whether there was a method that could suppress nonspecific reactions using a highly versatile method. As a result, they came to the conclusion that if a substance that acts as a "decoy" for the specific antibody against the target antigen could be coexisted in the antigen-antibody reaction system, nonspecific reactions could be suppressed even more reliably. They succeeded in suppressing nonspecific reactions by performing an antigen-antibody reaction between the specific antibody and the target antigen in the presence of a modified antibody in which part or all of the variable region of the L chain and / or H chain of the specific antibody has been modified, and thus completed the present invention. That is, the present invention has the following configuration. <1> A method for detecting a target antigen in a sample using a specific antibody that specifically binds to the antigen, a) A step of bringing the sample into contact with the modified antibody, b) The step of contacting the sample with the specific antibody simultaneously with or after step a), The modified antibody is an antibody in which part or all of the variable region of the antibody light chain (hereinafter abbreviated as L chain) and / or antibody heavy chain (hereinafter abbreviated as H chain) of the specific antibody has been modified. The detection method characterized by the above. <2> The modified antibody is an antibody in which at least one of the six complementarity-determining regions (hereinafter abbreviated as CDRs) of the L chain and H chain of the specific antibody and / or a part or all of the region related to the three-dimensional structure of the CDR is modified, and at least one CDR different from the said CDR is not modified. <1> The detection method described above. <3> The reactivity of the modified antibody to the target antigen is 30% or less of the reactivity of the specific antibody to the target antigen. <1> or <2> The detection method described above. <4> The concentration ratio of the modified antibody to the specific antibody in the antigen-antibody reaction system is 1 to 29 times. <1> ~ <3> A detection method described in any of the following. <5> The modified antibody is either an intact antibody or a fragment antibody. <1> ~ <4> A detection method described in any of the following. <6> It comprises one or more of the aforementioned specific antibodies, and includes a modified antibody against at least one of the aforementioned specific antibodies. <1> ~ <5> A detection method described in any of the following. <7> The specific antibody comprises a first specific antibody and a second specific antibody, and the modified antibody comprises a modified antibody of at least one of the first specific antibody and the second specific antibody. <1> ~ <6> A detection method described in any of the following. <8> The specific antibody is either an intact antibody or a fragment antibody containing a binding site for the target antigen. <1> ~ <7> A detection method described in any of the following. <9> The detection method according to any one of <1> to <8>, wherein the detection method is a method based on a homogeneous method or a heterogeneous method. <10> The detection method according to any one of <1> to <9>, wherein at least one kind of the specific antibody is an antibody bound to an insoluble carrier or labeled with a labeling agent. <11> The detection method according to <10>, wherein the insoluble carrier is latex particles, gold colloid, magnetic particles, a well-shaped plate, or a porous membrane. <12> The detection method according to any one of <1> to <11>, wherein the step of bringing the target antigen in the sample into contact with the specific antibody is performed in a solution. <13> The detection method according to <12>, further comprising a step of optically detecting the degree of aggregation of latex particles in the solution. <14> The detection method according to <13>, wherein the step of optically detecting includes optically detecting the color development of a color developing agent that has reacted depending on the amount of the antigen-antibody complex in the solution, or the amount of the labeling agent. <15> A detection reagent for detecting a target antigen in a sample with a specific antibody that specifically binds to the antigen, the detection reagent comprising the following. (1) A specific antibody that specifically binds to the antigen (2) A modified antibody in which a part or all of the variable region of the L chain and / or H chain of the specific antibody is modified <16> The detection reagent according to <15>, wherein the modified antibody is an antibody in which at least one CDR and / or a part or all of the region related to the three-dimensional structure of the CDR among the six CDRs of the L chain and H chain of the specific antibody is modified, and at least one CDR different from the CDR is not modified. <17> The detection reagent according to <15> or <16>, wherein the reactivity of the modified antibody with respect to the target antigen is 30% or less with respect to the reactivity of the specific antibody with respect to the target antigen. <18> The modified antibody was prepared such that the concentration ratio of the modified antibody to the specific antibody in the antigen-antibody reaction system was 1 to 29 times. <15> ~ <17> A detection reagent as described in any of the following. <19> The modified antibody is either an intact antibody or a fragment antibody. <15> ~ <18> A detection reagent as described in any of the following. <20> It comprises one or more of the aforementioned specific antibodies, and includes a modified antibody against at least one of the aforementioned specific antibodies. <15> ~ <19> A detection reagent as described in any of the following. <21> The specific antibody comprises a first specific antibody and a second specific antibody, and the modified antibody comprises a modified antibody of at least one of the first specific antibody and the second specific antibody. <15> ~ <20> A detection reagent as described in any of the following. <22> The specific antibody is either an intact antibody or a fragment antibody containing a binding site for the target antigen. <15> ~ <21> A detection reagent as described in any of the following. <23> The detection reagent is a reagent based on a homogeneous method or a heterogeneous method. <15> ~ <22> A detection reagent as described in any of the following. <24> At least one of the aforementioned specific antibodies is bound to an insoluble carrier or is labeled with a labeling agent. <15> ~ <23> A detection reagent as described in any of the following. <25> The insoluble carrier is latex particles, gold colloid, magnetic particles, well-shaped plates, or a porous membrane. <24> The detection reagent described above. <26> A detection reagent kit comprising reagents (1) and (2) for detecting a target antigen in a sample using a specific antibody that specifically binds to the antigen, characterized in that at least one of reagents (1) or (2) contains a modified antibody in which part or all of the variable region of the L chain and / or H chain of the specific antibody has been modified. (1) Reagent containing buffer (2) Reagents containing specific antibodies <27> A method for suppressing non-specific reactions in a method for detecting a target antigen, characterized by using a combination of a specific antibody that specifically binds to the target antigen in a sample and a modified antibody in which part or all of the variable regions of the L chain and / or H chain of the specific antibody have been modified. <28> A non-specific reaction inhibitor used in conjunction with a specific antibody that specifically binds to a target antigen in a sample, the antibody comprising an antibody in which part or all of the variable region of the L chain and / or H chain of the specific antibody has been modified. <29> An antibody used in conjunction with a specific antibody that specifically binds to a target antigen in a sample, wherein part or all of the variable region of the light chain and / or heavy chain of the specific antibody has been modified. [Effects of the Invention]

[0013] According to the present invention, in a method for detecting a target antigen in a sample using a specific antibody that specifically binds to the antigen, performing the antigen-antibody reaction in the presence of a modified antibody of the specific antibody broadly suppresses nonspecific reactions caused by s contained in the sample, making it possible to accurately measure the target antigen. [Brief explanation of the drawing]

[0014] [Figure 1] This document outlines the acquisition and sequence analysis of variable region genes from antibody-producing cells. After preparing total RNA from antibody-producing cells, full-length cDNA was synthesized using reverse transcription and template switching. Variable region gene fragments of the H and L chains were obtained by PCR using primers specific to the 5' end and constant region. After cloning, the variable region gene sequences were determined by sequencing the colony PCR amplification products. [Figure 2] Examples of analysis results for representative H-chain and L-chain variable region gene amplification fragments are shown. PCR amplification products of approximately 0.5 kb were detected by agarose electrophoresis. A Quick-Load Purple 1kb Plus DNA Ladder was used as a size marker. [Figure 3]The following outlines the method for determining the base sequence after subcloning. After cloning the antibody variable region gene fragment into a vector, monocloning was achieved by introducing the gene into E. coli and transforming it. Clones presumed to be antibody genes were selected by colony PCR using vector sequence-specific primers, and their base sequences were determined. [Figure 4] The results of agarose electrophoresis of representative colony PCR products are shown. Amplification products of approximately 0.9 kb (indicated by "←" in the figure) were detected for both the H and L chains, and after purification, the base sequences were determined. A Quick-Load Purple 1kb Plus DNA Ladder was used as a size marker. [Figure 5] The results of transient expression analysis of the 92204R antibody by immunoblotting are shown. The H and L chains were co-expressed in CHO-K1(F) cells, and the antibody in the culture supernatant was subjected to immunoblotting under non-reducing conditions and detected using anti-Rat IgG HRP. Mock: Culture supernatant from transgene-negative CHO-K1(F) cells cultured for the same period was used. [Figure 6] The results of the reactivity analysis of the transient expression 92204R antibody are shown. The reactivity of the transient expression antibody (culture supernatant) to the antigen was analyzed by solid-phase ELISA. Mock: Culture supernatant from CHO-K1(F) cells cultured for the same period was used. [Figure 7] The CDR sequences of mutant antibodies with various point mutations added for screening the variable regions involved in the reaction with the antigen are shown. To search for regions strongly involved in reactivity to the antigen, mutant antibodies were prepared in which the amino acids of CDR1(A), CDR2(B), and CDR3(C) of the predicted H chain variable regions were replaced with alanine (underlined in the figure). [Figure 8] This section shows the transient expression results of representative mutant antibodies by immunoblotting. The H and L chains were co-expressed in CHO-K1(F) cells, and the antibodies in the culture supernatant were subjected to immunoblotting under non-reducing conditions and detected using anti-Rat IgG HRP. Mock: Culture supernatant from transduction-negative CHO-K1(F) cells cultured for the same period was used. [Figure 9]This report presents the results of analyzing the reactivity of representative mutant antibodies to antigens using solid-phase ELISA, specifically the reactivity of transiently expressed mutant antibodies (culture supernatant) to antigens. WT: Transiently expressed wild-type antibody; Mock: Transduction-negative CHO-K1(F) cells cultured for the same period; PC: Positive control; Purified antibody derived from ascites fluid was used. [Figure 10] This figure shows the types of the heavy chains and light chains, and the modifications, for the heavy chain mutant antibody (H_105-107-A, H_Δ105-107) and the light chain mutant antibody (L_CDR3-A). Gene fragments of both antibodies were obtained by artificial gene synthesis and cloned into mammalian cell expression vectors. [Figure 11] The results of immunoblotting analysis of modified IgG antibodies are shown. Modified IgG antibodies in the culture supernatant of transiently expressed ExpiCHO cells were detected by anti-Rat IgG HRP. [Figure 12] This figure shows the purity analysis results of the purified antibody. After purification, the antibody was subjected to SDS-PAGE under reducing and non-reducing conditions, followed by CBB staining. Cont.(#IL-701): Purified 92204R antibody derived from ascites fluid. [Figure 13] The results of the isoelectric point analysis of the 92204R_H_Δ105-107 antibody and the 92204R_L_CDR3-A antibody are shown. [Figure 14] This figure shows the results of the reactivity analysis of the mutant antibodies of the present invention. Each mutant antibody was prepared to 100 μg / mL, and its reactivity to the antigen was evaluated by solid-phase ELISA. Blank: Wells without the antigen were used as negative controls. [Figure 15] This figure shows the results of verifying the effect of adding a CDR-modified antibody on specific reactions in the LTIA method. The horizontal axis represents the sIL-2R concentration (U / mL), and the vertical axis represents the sensitivity (mAbs.). [Figure 16] This figure shows the modifications made to the light chain CDR modified antibodies of the anti-BNP IgG monoclonal antibody 33236: 33236-(W51A), 33236-(K95A), and 33236-(CDR3-A). [Modes for carrying out the invention]

[0015] (Modified antibody) In the method of detecting a target antigen in a sample using a specific antibody that specifically binds to the antigen, the antibody coexisting to suppress non-specific reactions refers to a modified antibody in which part or all of the variable region of the L chain and / or H chain of the specific antibody has been modified (hereinafter, this may be referred to as the modified antibody of the present invention, or simply as the modified antibody). Modifying part or all of the variable region of the L chain and / or H chain of the specific antibody means that at least one amino acid in the variable region has been modified, and also includes modifying multiple amino acids or all of them. The term "modification" is used to mean any mutation, including substitution, deletion, or addition. The multiple amino acids refer to two or more amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. Furthermore, preferred ranges include 2 to 20, 2 to 15, 2 to 10, and 2 to 5.

[0016] Therefore, the modified antibodies of the present invention include antibodies in which part or all of the variable region of the L chain and / or H chain of the specific antibody is replaced with other amino acids, antibodies in which part or all of the variable region is deleted, or antibodies in which one or more amino acids are added to the variable region. Furthermore, the modified antibodies are modified antibodies in which part or all of the region (generally the CDR) that is particularly involved in the antigen-antibody reaction among the variable regions of the L chain and / or H chain of the specific antibody is modified, or modified antibodies in which part or all of the region related to the three-dimensional structure of the CDR is modified, and it is desirable that the modified region be as small as possible. Examples of regions related to the three-dimensional structure of the CDR include the neighboring region adjacent to the N-terminus and / or C-terminus of the amino acid sequence of each CDR, and the region that interacts with the three-dimensional structure of each CDR.

[0017] For example, a modified antibody is preferable in which at least one of the six CDRs in the variable regions of the L and H chains of the specific antibody is partially or entirely modified, and at least one CDR different from the aforementioned CDR remains completely unmodified. This is because, by leaving at least one CDR completely unmodified, non-specific reactions dependent on the sequence of that CDR can be suppressed, potentially allowing for broad suppression of non-specific reactions. Therefore, it is desirable to have as many unmodified CDRs as possible; at least one is sufficient, two or more are preferable, three or more are even preferable, four or more are even preferable, and five are most preferable. Furthermore, other regions of the modified antibody may be identical to or different from those of the specific antibody.

[0018] Another form of modified antibody is one in which the binding of the specific antibody to the target antigen is reduced, for example, by modifying the amino acids adjacent to the N-terminus and / or C-terminus of the amino acid sequence of each CDR, or by modifying the amino acids in the framework region involved in the three-dimensional structure of the CDR. Examples of amino acids adjacent to each CDR include 1 to 5 amino acids from the N-terminus and / or C-terminus of the amino acid sequence of each CDR.

[0019] The modified antibody of the present invention is also an antibody that has been modified to such an extent that, when reacting with a target antigen, it does not compete with and bind to a specific antibody (wild-type antibody) that does not contain amino acid modifications when the modified antibody is present in the reaction system. Whether the modification does not compete with the wild-type antibody can be specifically evaluated by its specificity for the target antigen. The modified antibody preferably has a specificity for the target antigen of 50% or less compared to the WT (wild-type) antibody, more preferably 40% or less, even more preferably 30% or less, even more preferably 20% or less, and most preferably 10% or less. Specificity can be analyzed, for example, by solid-phase ELISA as shown in the examples described later.

[0020] Examples of modified antibodies of the present invention include antibodies in which part or all of the variable region of the L chain and / or H chain of an antibody that binds to a target antigen described later is substituted with other amino acids. More specifically, examples include antibodies produced from an anti-sIL-2R antibody-producing hybridoma (accession number: NITE BP-02123) in which part or all of the variable region of the L chain and / or H chain of the antibody is substituted with other amino acids, and antibodies produced from an anti-BNP antibody-producing hybridoma (acceptance number: NITE ABP-03799) in which part or all of the variable region of the L chain and / or H chain of the antibody is substituted with other amino acids. Furthermore, the modified antibody may be any type of antibody, such as an intact antibody (full-length antibody) or a fragment antibody, as long as it contains at least a portion of the variable region of the specific antibody and specifically binds to the target antigen, thereby suppressing the non-specific reaction of the specific antibody.

[0021] Specifically, if the specific antibody is a full-length antibody, the modified antibody may be a full-length antibody or a fragment antibody in which the CDR of the specific antibody is included and the amino acids in the CDR and / or the region near the CDR have been modified. Also, if the specific antibody is a fragment antibody, the modified antibody may be a fragment antibody in which the amino acids in the CDR and / or the region near the CDR of the fragment antibody have been modified, or a full-length antibody containing the modified fragment antibody.

[0022] Whether the specific antibody is a full-length antibody or a fragment antibody, by using protease treatment or fragments derived from individual specific antibodies produced as genetically modified proteins, the sites causing non-specific reactions can be identified and used to select, design, and produce the desired modified antibody.

[0023] The modified antibody of the present invention can broadly suppress nonspecific reactions caused by the binding of nonspecific factors to specific antibodies by binding to nonspecific factors present in the sample instead of the specific antibody. Therefore, the modified antibody of the present invention functions as an active ingredient in a nonspecific reaction inhibitor. In other words, it can be said to be an antibody that acts as a "decoy" or "substitute" for the specific antibody against nonspecific factors. Antibodies with such effects are included in the modified antibody of the present invention. Previously, in order to measure target antigens with high sensitivity and stability, it was common practice to create antibodies with higher reactivity to the target antigen by modifying existing antibodies. However, in this invention, conversely, by utilizing specific antibodies that have been partially modified and have low reactivity to the antigen, it has become possible to suppress nonspecific reactions effectively and with high versatility. In other words, we have succeeded in providing a modified specific antibody, a method for suppressing nonspecific reactions using the modified antibody, and a detection reagent that utilizes the action of the modified antibody.

[0024] The outline of the creation of the modified antibody of the present invention is as follows. First, to obtain the genetic information of the specific antibody for the target antigen, the H / L chain gene is cloned, its base sequence is determined, the expected amino acid sequence is identified, and then the functional domain is analyzed. Next, the modified antibody of the present invention is searched for. Various mutations are introduced into the variable region of the H chain or L chain of the specific antibody using genetic recombination technology to make modifications. Among the variable regions of the H chain or L chain, the CDR region, the neighboring region adjacent to the N-terminus and / or C-terminus of the amino acid sequence of each CDR, or the region that interacts with the three-dimensional structure of each CDR is preferred.

[0025] The search for modified antibodies of the present invention can be carried out in detail by the following procedure. CDR-modified antibodies are created in which all amino acids in CDR1, CDR2, and CDR3 are replaced with alanine, and the CDRs that contribute to binding to the antigen are determined by comparing and evaluating their reactivity to the antigen with that of the wild type. These CDR-modified antibodies may also suppress nonspecific reactions. Since it is desirable that the modification sites be as small as possible, point mutation antibodies are created in which each amino acid in the CDR identified by the above method is mutated with alanine, and the important amino acid is determined by comparing and evaluating its reactivity with that of the wild type as described above. These point mutation antibodies may also suppress nonspecific reactions. Since modified antibodies with further reduced reactivity to the antigen may be required, modified antibodies are created in which the amino acids determined above are replaced with amino acids other than alanine, or multiple modified antibodies in which the area around that amino acid is replaced with alanine, or modified antibodies in which that amino acid is deleted, or modified antibodies in which the area around that amino acid is multiplely deleted, or modified antibodies in which new amino acids are added before and after that amino acid, and modified antibodies are selected by similarly comparing and evaluating their reactivity with that of the wild type.

[0026] The reduction in reactivity of the obtained modified antibody candidates to the target antigen is confirmed by the following method. First, modified antibodies are produced using a transient expression system in animal cells or other organisms. Next, modified antibodies that show a significant decrease in reactivity to the antigen compared to the wild type are selected using antigen-solid phase ELISA or similar methods. Cell-free systems or expression systems using E. coli or other organisms can also be used to produce these transient expression antibodies. Furthermore, modified antibodies that exhibit reactivity with non-specific substances in the sample equivalent to that of the wild type can be selected using antibody-solid-phase ELISA or other methods that utilize the selected modified antibodies. By following these steps, a modified antibody can be obtained that does not affect the reactivity of the specific antibody of the present invention with the target antigen, and that suppresses non-specific reactions.

[0027] In the detection method of the present invention, which includes the step of contacting an antigen in a sample with a specific antibody in the presence of the modified antibody, the specific antibody may be one type or two or more types, and the modified antibody may include a modified antibody against at least one of the specific antibodies. In another embodiment, the specific antibody includes two types, a first specific antibody and a second specific antibody, and the modified antibody includes a modified antibody of at least one of the first specific antibody and the second specific antibody. Furthermore, the specific antibody includes two types: a first specific antibody and a second specific antibody. The modified antibody may also consist of two or more modified antibodies of either the first or second specific antibody, or it may include two types: one modified antibody of the first specific antibody and one modified antibody of the second specific antibody. The modified antibody may be used individually, as a mixture of two or more types, or conjugated to an insoluble carrier.

[0028] In the present invention, the concentration of the modified antibody is sufficient to exert a desired non-specific reaction suppression effect without strongly affecting the antigen-antibody reaction, and can be appropriately set by those skilled in the art depending on the type of antigen and sample to be measured. The concentration of the modified antibody in the antigen-antibody reaction system varies depending on the reagent composition, but is not limited to 0.75 to 750 μg / mL, preferably 3.0 to 450 μg / mL, and more preferably 9.0 to 375 μg / mL. Furthermore, the concentration ratio of the modified antibody to the specific antibody in the antigen-antibody reaction system is not limited to 0.1 to 140 times, preferably 0.4 to 85 times, and more preferably 1.2 to 70 times. For example, a ratio of less than 30 times may be preferred. More specifically, it may be 1 to 29 times, 5 to 20 times, 10 to 15 times, or less than 10 times, such as 1 to 9 times, 2 to 7 times, 3 to 6 times, etc. The modified antibody of the present invention exhibits an inhibitory effect on non-specific reactions even at low concentration ratios relative to the specific antibody, thereby further reducing the influence of the specific antibody on the antigen-antibody reaction. Because the concentration of the modified antibody can be kept low, reagent costs can be reduced, and it is expected to have minimal impact on other components in the measurement system, making it usable regardless of the type of measurement principle.

[0029] Modified antibodies can be used individually, as a mixture of two or more modified antibodies, or as modified antibodies bound to an insoluble carrier. When using two or more modified antibodies in combination, the combined concentration of both antibodies must be within the specified concentration range. If the detection reagent of the present invention is to contain the modified antibody beforehand, it should be added to the detection reagent beforehand so that the concentration is within the reaction system described above. Furthermore, in another aspect of using the modified antibody of the present invention, a sample containing the target antigen can be pre-treated with the modified antibody to remove non-specific factors that may potentially bind to the specific antibody and cause a non-specific reaction. The specific antibody can then be reacted with the sample from this pre-treated sample.

[0030] (specific antibody) The specific antibody in this invention may be any antibody that is specific to the antigen to be measured, and antibodies such as polyclonal antibodies and monoclonal antibodies (including recombinant antibodies and functional fragments of each antibody) can be selected. Among these, monoclonal antibodies are preferably used. Furthermore, the specific antibody in this invention may be an intact antibody (full-length antibody) or a fragment antibody (functional fragment) that contains a binding site with the target antigen. In addition to those obtained through an immunization process on common animals (mice, rats, rabbits, goats, sheep, etc.), the specific antibody in this invention may also be an antibody (chimeric antibody, humanized antibody, or fully humanized antibody, etc.) in which the amino acid sequence of the immunogen (substance to be measured) has been changed to that of an animal species different from the animal immunized by genetic recombination technology, etc. Examples of fragment antibodies include F(ab')2, Fab', and single-chain antibodies (scFv), which are fragments having antigen-binding activity. These fragment antibodies can be produced by treating the antibody obtained as described above with a protease (e.g., pepsin or papain).

[0031] In the present invention, the specific antibody may be used in either a free or bound form, and the form is selected according to the measurement principle. For example, in enzyme-linked immunosorbent assay (ELISA), the specific antibody may be bound to a well plate or labeled with a labeling substance; in latex immunoturbidimetry (LTIA), it may be bound to latex particles; in immunochromatography, it may be bound to gold colloid particles or a porous membrane. Furthermore, in chemiluminescence, it may be bound to magnetic particles. When there are two or more specific antibodies, one may be bound to an insoluble carrier while the other is free (ELISA, chemiluminescence), or both may be bound to an insoluble carrier (LTIA, immunochromatography), and so on. Furthermore, when two types of specific antibodies bind to an insoluble carrier, there are cases where both are intact antibodies, where one is a fragment antibody, or where both are fragment antibodies.

[0032] (sample) Examples of samples containing the target antigen of the present invention include human or animal blood, serum, plasma, culture supernatant, urine, cerebrospinal fluid, saliva, sweat, ascites, or cell or tissue extracts. The samples in the present invention include not only the specimens themselves obtained from living organisms, but also specimens that have undergone pretreatment such as dilution and purification.

[0033] (Target antigen) In the present invention, various antigens contained in the sample can be used as target antigens for measurement. Examples of target antigens include proteins, peptides, amino acids, glycoproteins, lipids, carbohydrates, glycolipids, nucleic acids, and haptens, but there are no particular limitations as long as the substance is theoretically measurable. Examples include CRP (C-reactive protein), Lp(a), MMP3 (matrix metalloproteinase 3), antiphospholipid antibodies, type IV collagen, PSA, BNP (brain natriuretic peptide), sIL-2R (soluble interleukin-2 receptor), insulin, albumin, cystatin C, RF (rheumatoid factor), KL-6, procalcitonin, FDP, D-dimer, SF (soluble fibrin), TAT (thrombin-antithrombin III complex), PAI-1, as well as phenytoin, phenobarbital, carbamazepine, valproic acid, and theophylline.

[0034] Methods for introducing the modified antibody of the present invention into an antigen-antibody reaction system to bring the antigen in the sample into contact with the specific antibody include using it as one of the reagent components of a detection reagent, or adding it to a sample diluent or pretreatment solution. The term "within the antigen-antibody reaction system" refers to the liquid phase in which the sample and the liquid detection reagent are mixed and the immunoassay takes place, for example, when the detection reagent is a liquid reagent. For example, in the case of the LTIA method, the sample may be mixed with an LTIA detection reagent containing a modified antibody, or the sample may be mixed beforehand with a pretreatment solution containing a modified antibody and then mixed with the detection reagent. In the case of the ELISA method, the sample may be mixed with a pretreatment solution containing the modified antibody before being dropped onto a well plate, or the sample may be mixed with a solution containing the modified antibody and a specific antibody before being dropped onto a well plate. In addition, in the case of chemiluminescence reagents, the sample may be mixed with a pretreatment solution containing the modified antibody beforehand and then mixed with the detection reagent, or the detection reagent (for example, a solution containing a specific antibody, magnetic particles, etc.) may contain the modified antibody. Furthermore, when an immunoassay is performed on a solid phase, such as by immunochromatography, the antigen-antibody reaction system refers to the solid phase on which the immunoassay between the liquid sample and the specific antibody takes place. In this case, the sample may be pre-mixed with a pretreatment solution containing the modified antibody before being dropped onto the immunochromatographic test piece, or the modified antibody may be kept dry in the sample supply area and dissolved when the sample is dropped, causing the solid phase to develop and the modified antibody to be present in the reaction system.

[0035] (Detection method) The present invention relates to an immunological detection method for detecting a target antigen in a sample using a specific antibody, wherein the immune reaction is carried out in the presence of a modified antibody. Immunological detection methods are further broadly classified into homogeneous and heterogeneous methods. The homogeneous method is a measurement method that specifically detects the reaction proceeding by the target substance in a mixed solution (reaction solution) of the sample and reagent without performing B / F (bound / unbound) separation. The heterogeneous method is a measurement method in which excess components that did not participate in the measurement reaction are washed and removed by a B / F separation operation, and then the main reaction is allowed to proceed and detected. The heterogeneous method has the drawback of being time-consuming due to the washing step, but it has the advantage of being relatively less susceptible to the influence of non-specific reactants. In contrast, the homogeneous method does not involve a washing step, and therefore tends to be more susceptible to the influence of non-specific reactions. However, it is simpler, requires fewer steps, and takes less time to measure, making it a widely sought-after method in the field of clinical diagnosis.

[0036] Homogeneous methods include immunoaggregation (TIA) and immunochromatography (lateral flow and flow-through). TIA is a method for qualitatively or quantitatively detecting analytes (target substances) in a sample based on the degree of aggregation of immune complexes formed by the cross-linking action of analytes by specific antibodies such as antibodies. Among these, LTIA, which uses latex particles as an insoluble carrier to amplify the agglutination signal, is suitable for optical detection and is easy to automate, making it a highly versatile measurement method applied to various test items. Examples of heterogeneous methods include ELISA using well plates and chemiluminescence methods.

[0037] While the present invention can utilize any of the above immunological detection methods, the homogeneous method, which is relatively susceptible to the effects of nonspecific reactions, is expected to more effectively utilize the benefits of the present invention. In this specification, the term "detection" is used in its broadest sense, including proof of the existence of an object and / or quantitative determination, and also includes the concept of "measurement," which means quantitative determination.

[0038] (Detection reagent) The detection reaction of the present invention is carried out using a detection reagent, which contains not only a specific antibody against the target antigen, which is the main reaction component, but also a modified version of the specific antibody. Furthermore, some reagents also contain an insoluble carrier. Furthermore, the detection reagent may contain components to buffer and adjust the pH, ionic strength, and osmotic pressure of the sample, such as buffer solutions of acetic acid, citric acid, phosphoric acid, Tris, glycine, boric acid, carbonic acid, and Good's, or their sodium, potassium, and calcium salts. It may also contain polymers such as polyvinylpyrrolidone and phospholipid polymers as components to enhance aggregation formation. In addition, it may contain one or a combination of commonly used components such as proteins, amino acids, sugars, metal salts, surfactants, reducing substances, and chaotropic substances as components to control aggregation formation.

[0039] (Insoluble carrier) The insoluble carrier used in the present invention can be any material capable of carrying an antibody specific to the desired target component, and known particles can be used; in the case of particles, they are specifically referred to as insoluble carrier particles. Preferably, latex particles made of polymer materials such as polystyrene can be used, but depending on the method of carrying a binding partner specific to the target antigen, inorganic particles such as metal colloids, silica, and carbon can also be used as insoluble carrier particles in the present invention. The size of the insoluble carrier particles can be appropriately selected from the range of 0.04 to 1 μm, taking into consideration the optical measurement method used (e.g., turbidimetry for measuring transmitted light, or turbidimetry for measuring scattered light), in order to obtain the desired measurement sensitivity and measurement range. While an average particle diameter of 0.1 to 0.4 μm is commonly used in optical measurements using automated analyzers, the size is not limited to this range. The average particle size of the immunoassay particles can be confirmed using a particle size analyzer or transmission electron microscope. The concentration of the immunoassay particles in the reagent can be appropriately selected from a range such as 0.0001 mg / mL to 10 mg / mL, depending on the particle size of the immunoassay particles used and the overall design of the measurement system.

[0040] When latex particles are used as the insoluble carrier particles of the present invention, there are no particular limitations on the synthetic polymer constituting the latex particles, but examples include polystyrene, vinylnaphthalene, styrene-styrene sulfonate copolymer, methacrylic acid polymer, acrylic acid polymer, itaconic acid polymer, and styrene-hydrophilic carboxymonomer copolymer: for example, styrene-methacrylic acid copolymer, styrene-acrylic acid copolymer, styrene-itaconic acid copolymer, etc. An immunoaggregation method that uses latex particles as insoluble carrier particles is specifically called latex immunoturbidimetric analysis (LTIA, described later).

[0041] Specific antibodies against the target antigen can be attached to latex particles by known methods such as physical adsorption, chemical binding, or a combination thereof. It is preferable that the specific antibodies carried by the latex particles are of multiple types in order to form a sandwich. However, if the target antigen is a polyvalent antigen, one type of specific antibody may suffice. For example, if the specific antibody is a monoclonal antibody, it is common to use a combination of multiple monoclonal antibodies with different recognition sites. Furthermore, if it is necessary to perform treatment to suppress spontaneous aggregation of latex particles or nonspecific reactions, the latex particles may be treated by known methods, such as contacting and coating the surface with proteins such as bovine serum albumin (BSA), casein, gelatin, ovalbumin or its salts, surfactants, or skim milk powder, to perform a blocking treatment (masking treatment) of the carrier. The nonspecific reaction inhibitor containing the modified antibody of the present invention is expected to be effective even against nonspecific reactions that cannot be suppressed even after such general nonspecific reaction suppression treatments.

[0042] (Latex immunoturbidimetry) This document describes latex immunoturbidimetric analysis (LTIA), one of the immunological measurement methods of the present invention. Methods for measuring target substances using LTIA can be broadly classified into two categories. The first method involves reacting latex particles to which a specific antibody against the target substance is bound with the target substance to form a sandwich-type immune complex, and measuring the target substance based on the degree of aggregation of the latex particles accompanying the formation of the immune complex. The second method involves adding proteins or the like, which contain multiple target substances or their analogues (including fragments thereof), to a reagent. These proteins compete with the target substances in the sample, thereby inhibiting the formation of immune complexes between the target substances in the reagent and latex particles to which specific antibodies against the target substances are bound. The target substance (antigen) is then measured based on the degree of inhibition of aggregation of the latex particles resulting from the inhibition of immune complex formation. Specifically, the following steps are examples. (1) A step of bringing the sample into contact with the modified antibody in the liquid phase. (2) A step in which latex particles conjugated with a specific antibody are added to the liquid phase simultaneously with or after step (1) and brought into contact with the specific antibody. (3) A step after step (2) to measure the aggregation reaction between the substance to be measured and the latex particles. Here, step (3) means "a step in which the agglutination reaction between the target antigen and specific antibody-bound latex particles is measured either during or after step (2), without going through a washing and separation step."

[0043] LTIA allows for the measurement of a test substance by optically or electrochemically observing the degree of aggregation that occurs. Optical observation methods include measuring scattered light intensity, absorbance, or transmitted light intensity using optical instruments (endpoint method, rate method, etc.). The absorbance and other measurements obtained from measuring the sample are compared with the absorbance and other measurements obtained from measuring a standard substance (a sample in which the concentration of the target substance is known) to calculate the concentration (quantitative value) of the target substance contained in the sample. The measurement of absorbance and other measurements of transmitted or scattered light may be performed using one wavelength or two wavelengths (difference or ratio between two wavelengths). The measurement wavelength is generally selected from 500 nm to 900 nm.

[0044] The detection of the target antigen in the sample of the present invention may be performed manually or using a measuring device. The measuring device may be a general-purpose automated analyzer or a dedicated measuring device (dedicated machine). Furthermore, it is preferable to carry out this measurement by a method consisting of multiple operating steps, such as a two-step method (two-reagent method).

[0045] (Reagent configuration) The detection reagent of the present invention is composed of a single test solution or a plurality of test solutions of two or more test solutions. Examples of the plurality of test solutions include a test solution composed of a buffer solution for the purpose of adjusting the target antigen to a concentration suitable for measurement, adjusting the environment of the antigen-antibody reaction, etc., and a test solution containing specific antibody-binding particles. The modified antibody of the specific antibody of the present invention can exert an effect of suppressing non-specific reactions derived from the sample in the mixed state at the time of detection, and may be included in any or all of the constituent reagents as long as it does not affect the stability of the constituent reagents. In the case of a detection reagent using latex particles as an insoluble carrier, a detection reagent can be exemplified that includes a first reagent containing a buffer solution and a second reagent containing latex, and contains a modified antibody in either one or both of them. Further, the modified antibody may be contained in the specimen pretreatment solution. The concentration of the modified antibody in each constituent reagent may be included as long as it can be adjusted to the concentration in the antigen-antibody reaction system in the mixed state of the reagent and the sample at the time of measurement, and varies depending on the composition of each detection reagent.

[0046] The above has described the detection / measurement method and reagent composition mainly centered on LTIA, but other detection / measurement methods for suppressing non-specific reactions in the antigen-antibody reaction system using the modified antibody of the present invention will also be described. <ELISA method> The ELISA method is a method that utilizes various combinations of antigen-antibody reactions, and finally incorporates an enzyme-labeled antigen or antibody into the reaction system to detect enzyme activity. For the detection of enzyme activity, a substrate whose absorption spectrum changes by the reaction is used, and examples include the direct method, indirect method, sandwich method, competitive method, etc. depending on the combination of antigen-antibody reactions. The reagents in the case where the detection method of the present invention is the sandwich ELISA method will be exemplified. (a) An insoluble carrier bound with a specific antibody that reacts with the substance to be measured (b) A specific antibody labeled with a labeling substance and reacting with the target antigen As the insoluble carrier in (a), a well-shaped plate is preferable, and the labeling substance can be appropriately selected and used. The specific antibody bound to the insoluble carrier captures the target antigen in the solution containing the sample and forms a complex on the insoluble carrier. The specific antibody labeled with the labeling substance of (b) binds to the captured target substance and forms a sandwich with the aforementioned complex. The amount of the target substance in the sample can be measured by measuring the amount of the labeling substance using a method appropriate to the labeling substance. Specific methods such as the method for binding the specific antibody to the insoluble carrier and the method for binding the specific antibody to the labeling substance can be any method well known to those skilled in the art without particular limitation. In this ELISA method, the modified antibody of the present invention can be present in the reaction system by, for example, adding it to a sample diluent or pretreatment solution, or by adding it to the solution in which the antigen-antibody reaction is carried out.

[0047] <Immunochromatography Method> The reagent configuration (test piece configuration) when the principle of the detection method of the present invention is immunochromatography will be described below. Immunochromatographic specimen; When a specific antibody is used, the test piece is a sheet-like insoluble carrier such as a porous membrane, and in the direction of the spread of the solution containing the sample, it has the following features: 1. a sample supply site, 2. a site for holding the labeled specific antibody (labeled antibody holding site), and 3. a site for binding the specific antibody to capture the complex formed by the labeled antibody and the specific antibody (capture antibody site). In immunochromatography, when a predetermined amount of a sample containing the target antigen, which includes at least the test piece described above, is added to the sample supply site, the sample enters the label retention site by capillary action, and the target antigen and the labeled specific antibody bind to form a complex. When this complex expands the membrane and enters the capture antibody site, it is captured by the specific antibody (capture antibody) bound to the membrane, forming a complex of capture-specific antibody-target antibody-labeled specific antibody. The target substance can then be detected by detecting the label using any method (for example, by observing the agglutination image in the case of a visible label such as gold colloid, or by a color reaction caused by adding a substrate in the case of an enzyme). In this immunochromatography method, the modified antibody of the present invention can be present in the reaction system, for example, by adding it to a sample diluent or pretreatment solution, or by incorporating it into the sample supply site or label retention site of a test piece and keeping it dry.

[0048] <Chemiluminescence> The reagent configuration for the detection method of the present invention, where the principle is chemiluminescence, will be described below. The chemiluminescence method involves contacting magnetic particles bound to an antigen or antibody with the target antigen and a labeled antibody to form a complex. After separating the complex from the unreacted labeled antibody, a luminescent reagent is added, and the amount of light emitted is measured. When an enzyme is used for labeling, it is called chemiluminescent enzyme immunoassay (CLEIA method). When a metal complex such as a ruthenium pyridine complex is used for labeling and the luminescence intensity is measured by an electrochemical reaction, it is called electrochemiluminescence immunoassay (ECLIA method). When a chemiluminescent substance is used for labeling, it is called chemiluminescence immunoassay (CLIA method). Examples of reagents used when the detection method of the present invention is the CLEIA method are given below. (a) Magnetic particles to which a specific antibody that reacts with the target antigen is bound. (b) Enzyme-labeled specific antibodies that react with the target antigen. (c) Luminescent reagent Specific antibodies bound to magnetic particles capture target antigens in a solution containing the sample and form a complex. Specific antibodies labeled with an enzyme-labeled substance bind to the captured target antigen, forming a sandwich with the aforementioned complex. By reacting the enzyme-labeled substance with a luminescent reagent and measuring the amount of light emitted, the target antigen in the sample can be measured. In this CLEIA method, the modified antibody of the present invention can be present in the reaction system by, for example, adding it to a sample diluent or pretreatment solution, or by adding it to the solution in which the antigen-antibody reaction is carried out.

[0049] (Methods for suppressing nonspecific reactions / Nonspecific reaction inhibitors) Certain components in biological samples can often cause aggregation of immunoassay particles bound to specific antibodies against the target antigen that should not occur (positive measurement error), or conversely, prevent aggregation that should occur (negative measurement error). These are called nonspecific reactions and are known to cause various measurement errors. In the present invention, suppressing nonspecific reactions means acting (passively) on factors (nonspecific factors) in a sample that cause the above-mentioned nonspecific reactions, and acting as a decoy for specific antibodies to suppress the influence of reactions other than the target specific reaction on detection. In the present invention, nonspecific reactions can be suppressed by performing an immune reaction in the presence of a modified antibody of the specific antibody against the target antigen. Therefore, the modified antibody of the present invention is also an active ingredient of the nonspecific reaction inhibitor.

[0050] (Reagent Kit) The reagent kit of the present invention is characterized by comprising at least a specific antibody of the target antigen and a modified version of said specific antibody in its kit composition. The kit composition may include reagents related to immunological detection, as well as a sample diluent, a sample extract, etc., and the modified antibody of the present invention may be included in one or more of these. Typically, the reagents include (1) and (2), and at least one of reagents (1) or (2) contains a modified antibody in which part or all of the variable region of the L chain and / or H chain of the specific antibody has been altered. (1) Reagent containing buffer (2) Reagents containing specific antibodies Examples of reagents containing specific antibodies include reagents in which the specific antibody is bound to an insoluble carrier such as latex. In addition to the above, the kit also includes an instruction manual and sample collection tools (such as a collection pipette, syringe, cotton swab, and filter).

[0051] (Other reagent components) The detection reagent of the present invention may contain polymers such as polyethylene glycol, polyvinylpyrrolidone, and phospholipid polymers as components that enhance the aggregation of insoluble carrier particles. Furthermore, as components that control aggregation, the reagent may contain one or a combination of commonly used components such as proteins, amino acids, sugars, metal salts, surfactants, reducing substances, and chaotropic substances. [Examples]

[0052] The present invention will be described in detail below with reference to examples of nonspecific reaction inhibitors containing the modified antibody of the present invention, detection methods utilizing the same, detection reagents, and methods for suppressing nonspecific reactions, but the present invention is not limited thereto.

[0053] [Test Example 1] Preparation of CDR-modified antibody 1. Analysis of antibody variable region gene sequences We analyzed the antibody variable region gene sequences using general molecular and cellular biological techniques (Figure 1). Specifically, RNA was extracted from approximately 1 million cultured anti-sIL-2R antibody-producing hybridomas (accession number: NITE BP-02123), and full-length cDNA was obtained by reverse transcription and template switching. Using this cDNA as a template, the genes in the variable regions of the H and L chains were amplified by RT-PCR (Figure 2). The amplified sequences were inserted into a vector, and E. coli were transformed. The transformed E. coli were cultured, and approximately 12 colonies were picked. The cloned sequences were amplified by colony PCR (Figures 3 and 4). After purifying these PCR amplification products, sequence information was obtained by direct sequencing, and those without translation frame fragmentation were analyzed. Finally, the gene sequences of the H and L chains of the anti-sIL-2R monoclonal antibody (92204R antibody) produced from anti-sIL-2R antibody-producing hybridomas were determined by estimating the 5' signal sequence from a public database and combining it with known constant region sequences.

[0054] 2. Equivalence analysis of recombinant antibodies The obtained H and L chain genes were transiently expressed in mammalian cells, and their reactivity was analyzed to confirm whether they were equivalent to the hybridoma-derived 92204R antibody. Specifically, the H and L chain genes were cloned into a mammalian cell expression vector by artificial gene synthesis, and IgG was transiently expressed in the culture supernatant by introducing the genes into CHO cells (Figure 5). This culture supernatant was collected, and its reactivity to the antigen was analyzed by solid-phase ELISA, confirming that it had reactivity equivalent to the purified antibody (Figure 6). Therefore, it was confirmed that the obtained H chain and L chain genes are genes that constitute the hybridoma-derived antibody. For solid-phase ELISA, 50 ng of hIL-2Ra (R&D Systems, Cat.# 2232A025 / CF) was used as the antigen.

[0055] 3. Screening of modified antibodies We obtained mutant antibody genes by synthesizing gene sequences in which the complementarity determining region (CDR) of the antibody H chain, estimated from a public database, was replaced with alanine. We then incorporated these mutant antibodies into a mammalian cell expression vector to prepare mutant antibody expression vectors (Figure 7A, B, C). Next, the antibody was transiently expressed in CHO cells, as described above, and IgG was detected in the culture supernatant (Figure 8). Furthermore, the reactivity to the antigen was analyzed by solid-phase ELISA. As a result, a mutant with significantly reduced reactivity was found in the point mutation antibody of the H chain CDR3 (Figure 9). Therefore, it was inferred that the amino acids around position 106 in CDR3 are mainly involved in the antigen recognition and binding of the 92204R antibody.

[0056] 4. Production of modified antibodies The results described above suggest that amino acids around position 106 within CDR3 are strongly involved in the antigen recognition and binding of the 92204R antibody. Therefore, as candidate modified antibodies of the present invention, a multiple mutant antibody (105-107-A) and a deletion antibody (Δ105-107) containing amino acids before and after position 106 were prepared. An antibody in which all amino acids of the L-chain CDR3 were replaced with alanine was also prepared (Figure 10), and transient expression and reactivity analysis were performed. (Although transient expression was attempted for an antibody in which all amino acids of the H-chain CDR3 were replaced with alanine after preparing the expression vector, IgG conversion could not be confirmed, so it was not used in subsequent experiments.) As before, the reactivity to the antigen was analyzed by solid-phase ELISA (Figure 11). The results showed a significant decrease in reactivity for Δ105-107 and L_CDR3-A, so these modified antibodies were used in subsequent experiments. First, these modified antibodies were expressed using the ExpiCHO cell expression system (TheromoFisher), and IgG in the culture supernatant was confirmed by immunoblotting. Since a sufficient amount of IgG, including wild-type antibodies, was confirmed, these antibodies were purified using a Protein G column. Analysis of the purity of the purified antibodies by CBB (Coomassie Brilliant Blue) staining confirmed that it was equivalent to known purified antibodies (Figure 12), so the isoelectric point was further analyzed. Isoelectric focusing electrophoresis revealed that H_Δ105-107 had isoelectric points equivalent to the wild type, while L_CDR3-A had slightly higher isoelectric points (both in the pH range of 6.0-6.9). Therefore, it was clear that there was no significant change in isoelectric point due to the modification (Figure 13). Finally, the reactivity of the purified antibodies was confirmed by solid-phase ELISA. Antibody solutions were prepared by diluting various purified antibodies 10-fold, 100-fold, 1,000-fold, ..., and 10,000,000-fold from 1 μg / mL, and their reactivity was analyzed by solid-phase ELISA. As a result, the positive control (PC) 92204R (#IL-107), which is a purified antibody derived from ascites fluid, and the antibody 92204R produced by genetic recombination technology of the same antibody showed equivalent antigen reactivity. However, at a concentration of 100 ng / mL, the modified antibody showed approximately 23% of the reactivity of the positive control (PC) for H_D105-107 and approximately 5% of the reactivity of the PC (equivalent to Blank) for L_CDR3-A (Figure 14). In this study, the positive control, which is an unmodified antibody, was a purified antibody derived from ascites fluid. Therefore, the decrease in reactivity of the modified antibody compared to the positive control was shown to be due to the modification of the amino acids themselves, rather than a decrease in antibody reactivity due to transient expression. Based on the above, it was concluded that the modified antibody produced has the potential to suppress non-specific reactions in sample measurement.

[0057] [Test Example 2] Verification of the effect of adding a CDR-modified antibody on inhibiting nonspecific aggregation of antibody-conjugated latex (LTIA method) This paper describes a test in which the degree of aggregation between components in a sample and one type of antibody-conjugated latex was evaluated using a buffer solution to which the modified CDR antibody and the unmodified antibody of the present invention were added. Since the antigen sIL-2R is not a polyvalent antigen with multiple identical epitopes within its molecule, agglutination cannot be formed when sIL-2R in the sample reacts with only one type of antibody-conjugated latex, and therefore the absorbance does not change. Accordingly, the change in absorbance observed in the evaluation method shown in this test example can be considered to be due to nonspecific aggregation of nonspecific factors in the sample with the antibody-conjugated latex.

[0058] 1. Measurement reagents (1) Preparation of the first reagent [Comparative Example 1] The first reagent was prepared according to the method described in Japanese Patent Publication No. 2017-181377. [Comparative Example 2] 92204R-(WT) antibody (unmodified antibody) was added to the first reagent of Comparative Example 1 to a concentration of 100 μg / mL. [Example 1] 92204R-(Δ105-107) modified antibody was added to the first reagent of Comparative Example 1 to a concentration of 100 μg / mL. [Example 2] The 92204R-(CDR3-A) modified antibody was added to the first reagent of Comparative Example 1 to a concentration of 100 μg / mL.

[0059] (2) Preparation of the second reagent containing one type of antibody-conjugated latex. 92204R antibody-conjugated latex was prepared according to the method of Reagent 2(ii) in Test Example 1 described in Japanese Patent Application Publication No. 2017-181377. Reagent 2 was prepared by adjusting the absorbance at a wavelength of 600 nm to 2.5 Abs. / mL in 5 mM MOPS buffer (pH 7.0).

[0060] 2. Sample We used samples in which the absorbance did not change with only one type of antibody-conjugated latex (92204R antibody-conjugated latex) (control samples: samples 1-5) and samples that exhibited a nonspecific reaction and whose absorbance changed with only 92204R antibody-conjugated latex (nonspecific agglutination samples: samples 6-9).

[0061] 3. Measurement Procedure The first and second reagents were combined, and the degree of non-specific agglutination of each sample and the 92204R antibody-conjugated latex alone was evaluated using a Hitachi 7180 automated analyzer. Specifically, 120 μL of the first reagent was added to 5.6 μL of the sample and incubated at 37°C for 5 minutes, then 40 μL of the second reagent was added and mixed. The change in absorbance associated with agglutination formation was then measured over a 5-minute period at a primary wavelength of 570 nm and a secondary wavelength of 800 nm.

[0062] 4.Measurement results The measurement results are shown in Table 1.

[0063] [Table 1]

[0064] 5. Discussion The effects of the present invention were considered based on the results of Comparative Examples 1 and 2 and Examples 1 and 2. (1) Based on the results of Comparative Example 1, in samples 1 to 5, the change in absorbance with 92204R antibody-conjugated latex alone was 2.0 mAbs. or less, and no agglutination was observed. On the other hand, in samples 6 to 9, the change in absorbance with 92204R antibody-conjugated latex alone was 2.0 mAbs. or more, and non-specific agglutination was observed. (2) When the first reagent containing 92204R-(WT) was used, the change in absorbance of the 92204R antibody-conjugated latex decreased significantly in samples 6-9 (below 2.0 mAbs.), and nonspecific agglutination was suppressed. No difference in the degree of agglutination was observed in control samples 1-5. This indicates that the nonspecific factors contained in samples 6-9 acted as a decoy by reacting with the 92204R-(WT) antibody added to the first reagent, suppressing nonspecific agglutination between the nonspecific factors and the 92204R antibody-conjugated latex after the addition of the second reagent (Comparative Example 2). (3) When the first reagent containing the 92204R-(Δ105-107) modified antibody was used, the change in absorbance of the 92204R antibody-conjugated latex decreased significantly (to 2.0 mAbs. or less) in samples 6-9, similar to Comparative Example 2, and non-specific agglutination was suppressed. No difference in the degree of agglutination was observed in control samples 1-5 (Example 1). (4) When the first reagent containing the modified 92204R-(CDR3-A) antibody was used, in samples 6-9, the change in absorbance of the 92204R antibody-conjugated latex decreased significantly (to 2.0 mAbs. or less), similar to Comparative Example 2 and Example 1, and nonspecific agglutination was suppressed. No difference in the degree of agglutination was observed in control samples 1-5 (Example 2). (5) Based on the above, in an immunoassay method, by including the CDR-modified antibody of the present invention in the antigen-antibody reaction system, it is possible to suppress nonspecific aggregation of antibody-sensitized latex caused by nonspecific factors in the sample by having the antibody act as a decoy.

[0065] [Test Example 3] Verification of the effect of adding a modified CDR antibody on specific reactions (LTIA method) The concentration of sIL-2R in the sample was measured using an LTIA reagent to which the modified antibody of the present invention was added. 1. Measurement reagents (1) Reagent 1 [Comparative Example 3] The first reagent shown in Comparative Example 1 was used. [Comparative Example 4] The first reagent shown in Comparative Example 2 was used. [Example 3] The first reagent shown in Example 1 was used. [Example 4] The first reagent shown in Example 2 was used. (2) Second reagent The second reagent, prepared according to the method of Test Example 1 described in Japanese Patent Publication No. 2017-181377, was used. The specific antibody concentration in this reagent was 20-30 μg / mL.

[0066] 2. Sample The following samples were used for measurement as samples with known concentrations. (1) Physiological saline solution (Otsuka Saline Solution) (2) IL-2R calibrator N (Sekisui Medical Co., Ltd.) prepared to the concentrations shown in Table 2.

[0067] 3. Measurement Procedure The first and second reagents were combined, and the above-mentioned samples of known concentration (physiological saline, IL-2R calibrator N) were measured using a Hitachi 7180 automated analyzer. Specifically, 120 μL of the first reagent was added to 5.6 μL of the sample and incubated at 37°C for 5 minutes, then 40 μL of the second reagent was added and stirred. The change in absorbance due to aggregation formation was then measured over a 5-minute period at a primary wavelength of 570 nm and a secondary wavelength of 800 nm.

[0068] 4.Measurement results The measurement results are shown in Table 2 and Figure 15.

[0069] [Table 2]

[0070] 5. Discussion (1) In Comparative Example 3, which did not contain antibody, a concentration-dependent increase in absorbance was observed, whereas in Comparative Example 4, which used the first reagent to which 92204R-(WT) antibody was added, no concentration-dependent increase in absorbance was observed. This indicates that the addition of 92204R-(WT) antibody inhibits the specific reaction between sIL-2R in the sample and antibody-conjugated latex. (2) In Example 3, which used the first reagent to which the 92204R-(Δ105-107) modified antibody was added, a concentration-dependent increase in absorbance was observed, similar to Comparative Example 3, and the degree of increase was also equivalent. Therefore, it was shown that the 92204R-(Δ105-107) modified antibody does not affect the specific reaction between sIL-2R and antibody-conjugated latex. (3) In Example 4, which used the first reagent to which the 92204R-(CDR3-A) modified antibody was added, a concentration-dependent increase in absorbance of sIL-2R was observed, similar to Comparative Example 3 and Example 3, and the degree of increase was also similar. Therefore, it was shown that the 92204R-(CDR3-A) modified antibody does not affect the specific reaction between sIL-2R and antibody-conjugated latex. (4) Based on the above, by modifying the CDR amino acid sequence of the specific antibody, we were able to obtain a non-specific reaction inhibitor that acts as a decoy and does not affect the specific reaction in the immunoassay method.

[0071] [Test Example 4] Measurement of sIL-2R (LTIA method) The concentration of sIL-2R in the sample was measured using an LTIA reagent to which the modified antibody of the present invention was added.

[0072] 1. Measurement reagents [Comparative Example 5] (Measurement of sIL-2R concentration by CLEIA method) Lumipulse Presto® IL-2R (Fujirebio Inc.) was used. The CLEIA method involves a B / F separation operation and a washing step. Therefore, it was used as a comparative example because it is a measurement method that is less affected by non-specific reactions originating from the sample. [Comparative Example 6] (Measurement of sIL-2R concentration by LTIA method) The first reagent and the second reagent were prepared according to the method described in Japanese Patent Publication No. 2017-181377. [Example 5] (Measurement of sIL-2R concentration by LTIA method with added 92204R-(Δ105-107) modified antibody) The reagents were prepared in the same manner as in Comparative Example 6, except that 92204R-(Δ105-107) modified antibody was added to the first reagent to a concentration of 100 μg / mL or 500 μg / mL. [Example 6] (Measurement of sIL-2R concentration by LTIA method with added 92204R-(CDR3-A) modified antibody) The reagents were prepared in the same manner as in Comparative Example 6, except that 92204R-(CDR3-A) modified antibody was added to the first reagent to a concentration of 100 μg / mL or 500 μg / mL.

[0073] 2. Sample We used samples that showed values ​​close to those measured by chemiluminescent enzyme immunoassay (CLEIA method) (Comparative Example 5) (control samples: sample numbers 1-5), and samples that showed nonspecific reactions and deviated significantly from the measurements of Comparative Example 5 (deviant samples: sample numbers 13-16).

[0074] 3. Measurement Procedure (1) Comparative Example 5 was measured using Lumipulse (registered trademark) - L2400 (Fujirebio Inc.) in accordance with the instructions in the reagent package insert. (2) In Comparative Example 6, Example 5, and Example 6, the sIL-2R concentration in the sample was measured using a Hitachi 7180 automatic analyzer by combining the first and second reagents. Specifically, 120 μL of the first reagent was added to 5.6 μL of the sample and incubated at 37°C for 5 minutes, then 40 μL of the second reagent was added and stirred. The change in absorbance due to aggregation formation was then measured over a 5-minute period at a primary wavelength of 570 nm and a secondary wavelength of 800 nm. The amount of absorbance change was then applied to a calibration curve obtained by measuring standard substances of known concentration, and the measured value was calculated.

[0075] 4.Measurement results The measurement results are shown in Table 3.

[0076] [Table 3]

[0077] 5. Results and Discussion (1) In control samples (sample numbers 1-5) where the measurement values ​​obtained by the CLEIA method (Comparative Example 5) and the measurement values ​​obtained by the LTIA method without antibody addition (Comparative Example 6) were equivalent, the measurement values ​​obtained by the LTIA method with the modified antibody of the present invention added (Examples 5 and 6) were also equivalent to those of Comparative Examples 5 and 6. Therefore, it was found that the addition of the modified antibody of the present invention does not affect the measurement values ​​of samples that do not exhibit a nonspecific reaction. (2) For sample number 13, the measured value using the CLEIA method (Comparative Example 5) was 331 U / mL, while the measured value using the LTIA method without antibody (Comparative Example 6) was 835 U / mL, showing a significant discrepancy in the measured values. On the other hand, the measured values ​​using the LTIA method with 100 μg / mL of the modified antibody of the present invention added (Examples 5 and 6) were 652 or 696 U / mL, showing a tendency to approach Comparative Example 5. Furthermore, the measured values ​​using the LTIA method with 500 μg / mL of the modified antibody added (Examples 5 and 6) were 451 or 487 U / mL, showing a tendency to approach the measured value using CLEIA (Comparative Example 5). Similar results were obtained for samples 14 to 16. (3) As described above, in the LTIA method, which is a homogeneous immunoassay method, the presence of the modified antibody of the present invention in the reaction system made it possible to suppress nonspecific reactions originating from the sample.

[0078] [Test Example 5] Measurement of sIL-2R by ELISA method The sIL-2R concentration in the sample was measured using an ELISA reagent to which the modified antibody of the present invention was added.

[0079] 1.Measurement method [Comparative Example 7] (Measurement of sIL-2R concentration by CLEIA method) The procedure was carried out in the same manner as in Comparative Example 5. [Comparative Example 8] (Measurement of sIL-2R concentration by ELISA method) An anti-sIL-2R monoclonal antibody (clone number 92204R) was diluted to 10 μg / mL in 20 mM phosphate buffer (pH 7.2; hereafter referred to as PBS) containing 150 mM sodium chloride. 100 μL of this solution was dispensed into each well of a 96-well microplate and allowed to stand at room temperature for 1 hour. Each of the aforementioned wells was washed three times with 400 μL of PBS containing 0.05% Tween® 20 (hereinafter referred to as PBST), then 200 μL of PBST containing 1% bovine serum albumin (hereinafter referred to as BSA-PBST) was added, and the mixture was blocked at room temperature for 1 hour. This was used as the ELISA plate. After washing each well of the ELISA plate three times with 400 μL of PBST, 100 μL of the sample, diluted 20-fold with sample diluent (BSA-PBST with HBR-1 (SCANTIBODIES LABORATORY, INC.) added to 100 μg / mL), was added to each well and allowed to stand at room temperature for 1 hour. After washing each well three times with 400 μL of PBST, 100 μL of biotin-labeled anti-sIL-2R monoclonal antibody (clone number 92212), diluted to 1.0 μg / mL with BSA-PBST, was dispensed into each well and allowed to stand at room temperature for 1 hour. After washing each well three times with 400 μL of PBST, 100 μL of HRP-labeled streptavidin (Thermo Fishier Scientific), diluted to 0.2 μg / mL with BSA-PBST, was dispensed into each well and allowed to stand at room temperature for 30 minutes. After washing each well three times with 400 μL of PBST, 50 μL of citrate buffer (pH 5.0) containing 0.2% orthophenylenediamine and 0.02% hydrogen peroxide was added, and after standing at room temperature for 10 minutes, 100 μL of 4.5N sulfuric acid was added to stop the enzymatic reaction, and the absorbance at a wavelength of 492 nm was measured. A sample with a known concentration was used as a calibrator, and the sIL-2R value in the test sample was calculated. [Example 7] Measurement of sIL-2R concentration by ELISA method with added 92204R-(Δ105-107) modified antibody. The measurement was performed using the same method as in Comparative Example 8, except that 92204R-(Δ105-107) modified antibody was added to the sample diluent shown in Comparative Example 8 to a concentration of 100 μg / mL. [Example 8] (Measurement of sIL-2R concentration by ELISA method with added 92204R-(CDR3-A) modified antibody) The measurement was performed using the same method as in Comparative Example 8, except that 92204R-(CDR3-A) modified antibody was added to the sample diluent shown in Comparative Example 8 to a concentration of 100 μg / mL.

[0080] 2. Sample We used samples (control samples: sample numbers 17-19) that showed values ​​close to those measured by chemiluminescent enzyme immunoassay (CLEIA method) (Comparative Example 7), and samples that showed nonspecific reactions and deviated significantly from the measured values ​​of Comparative Example 7 (deviant sample: sample number 20).

[0081] 3.Measurement results The measurement results are shown in Table 4.

[0082] [Table 4]

[0083] 4. Results and Discussion (1) In control samples (sample numbers 17-19) where the measurement values ​​obtained by the CLEIA method (Comparative Example 7) and the measurement values ​​obtained by the ELISA method without antibody addition (Comparative Example 8) were equivalent, the measurement values ​​obtained by the ELISA method with the modified antibody of the present invention added (Examples 7 and 8) were also equivalent to those of Comparative Examples 7 and 8. Therefore, it was found that the addition of the modified antibody of the present invention does not have a significant effect on the measurement values ​​of samples that do not exhibit a nonspecific reaction. (2) For sample number 20, the measurement value obtained by the CLEIA method (Comparative Example 7) was 353 U / mL, while the measurement value obtained by the ELISA method without antibody (Comparative Example 8) was 756 U / mL, showing a significant discrepancy in the measured values. In contrast, the measurement values ​​obtained by the ELISA method with 100 μg / mL of the modified antibody of the present invention added (Examples 7 and 8) were 484 and 279 U / mL, respectively, both showing a tendency to approach that of Comparative Example 7. (3) As described above, in the ELISA method, which is a heterogeneous immunoassay method, the presence of the modified antibody of the present invention in the reaction system suppressed nonspecific reactions originating from the sample. Furthermore, it was found that this method of suppressing nonspecific reactions using the modified antibody is effective not only in the homogeneous measurement systems of Examples 1 to 6, but also in the heterogeneous measurement systems of Examples 7 and 8.

[0084] [Test Example 6] Verification of the effect of adding a CDR-modified antibody on inhibiting nonspecific aggregation of antibody-bound latex (LTIA method 2) This paper describes a test in which the degree of aggregation between components in a sample and one type of antibody-conjugated latex was evaluated using buffer solutions to which the modified CDR antibody and the unmodified antibody of the present invention were added, respectively. Since the antigen, brain natriuretic peptide (BNP), is not a polyvalent antigen with multiple identical epitopes within its molecule, a single type of antibody-conjugated latex cannot normally form agglutinations when reacting with BNP in the sample, and therefore the absorbance does not change. Accordingly, the change in absorbance observed in the evaluation method shown in this example can be considered to be due to nonspecific aggregation of nonspecific factors in the sample with the antibody-conjugated latex.

[0085] 1. Production of CDR-modified antibodies of anti-BNP monoclonal antibodies Three types of light chain CDR-modified antibodies, 33236-(W51A), 33236-(K95A), and 33236-(CDR3-A) were prepared based on the anti-BNP IgG monoclonal antibody 33236 produced by the hybridoma (NITE ABP-03799) using the same method as described in Test Example 1 above (Figure 16). All of these antibodies showed a significant decrease in reactivity to the antigen, BNP. 2. Measurement reagents (1) Preparation of the first reagent [Comparative Example 9] The first reagent shown below was prepared. 100mM Bicine buffer (pH8.0) 200 mM KCl 0.5% BSA [Comparative Example 10] 33236-(WT) antibody (unmodified antibody) was added to the first reagent of Comparative Example 9 to a concentration of 100 μg / mL. [Example 9] Modified antibody 33236-(W51A) was added to the first reagent of Comparative Example 9 to a concentration of 100 μg / mL. [Example 10] Modified antibody 33236-(K95A) was added to the first reagent of Comparative Example 9 to a concentration of 100 μg / mL. [Example 11] Modified antibody 33236-(CDR3-A) was added to the first reagent of Comparative Example 9 to a concentration of 100 μg / mL.

[0086] (2) Preparation of fragment antibodies Fragment antibody 33236F(ab')2 was prepared from anti-BNP IgG monoclonal antibody 33236 produced by hybridoma (NITE ABP-03799) according to a standard method.

[0087] (3) Preparation of the second reagent containing one type of antibody-conjugated latex. The 33236F(ab')2 antibody-conjugated latex was prepared as follows: An equal volume of 0.6 mg / mL of 33236F(ab')2 antibody solution was added to a 1% polystyrene latex solution with an average particle size of 270 nm (manufactured by Sekisui Medical Co., Ltd.), and the mixture was stirred at 4°C for 1 hour. Then, an equal volume of 2% BSA solution was added, and the mixture was stirred at 4°C for 1 hour to prepare the 33236F(ab')2 antibody-conjugated latex solution. The solution was adjusted with 5 mM MOPS buffer (pH 7.0) to obtain an absorbance of 5.25 Abs. / mL at a wavelength of 600 nm, and this was prepared as the second reagent.

[0088] 3. Sample A sample exhibiting a nonspecific reaction, in which the absorbance changed with only one type of antibody-conjugated latex (33236F(ab')2 antibody-conjugated latex) (nonspecific agglutination sample: sample 21), was used.

[0089] 4. Measurement Procedure The degree of nonspecific agglutination of each sample and the 33236F(ab')2 antibody-conjugated latex alone was evaluated using a Hitachi 7180 automated analyzer by combining the first and second reagents. Specifically, 150 μL of the first reagent was added to 12 μL of the sample and incubated at 37°C for 5 minutes, after which 50 μL of the second reagent was added and mixed. The change in absorbance associated with agglutination formation was then measured over a 5-minute period at a primary wavelength of 570 nm and a secondary wavelength of 800 nm.

[0090] 5.Measurement results The measurement results are shown in Table 5.

[0091] [Table 5]

[0092] 6. Discussion The effects of the present invention were considered based on the results of Comparative Examples 9-10 and Examples 9-11. (1) In Comparative Example 9, which did not contain antibody, the absorbance change of the 33236F(ab')2 antibody-conjugated latex alone was 2.0 mAbs. or more, indicating non-specific agglutination. (2) In Comparative Example 10, which used the first reagent to which 33236-(WT) was added, the change in absorbance of the 33236F(ab')2 antibody-conjugated latex decreased significantly (to 2.0 mAbs. or less), and nonspecific agglutination was suppressed. This indicates that the nonspecific factors contained in the sample acted as a decoy by reacting with the 33236-(WT) antibody added to the first reagent, suppressing nonspecific agglutination between the nonspecific factors and the 33236F(ab')2 antibody-conjugated latex after the addition of the second reagent. (3) In Example 9, which used the first reagent to which the 33236-(W51A) modified antibody was added, the change in absorbance of the 33236F(ab')2 antibody-conjugated latex decreased significantly (to 2.0 mAbs. or less), similar to Comparative Example 10, and nonspecific aggregation was suppressed. (4) In Example 10, which used the first reagent to which the 33236-(K95A) modified antibody was added, the change in absorbance of the 33236F(ab')2 antibody-conjugated latex decreased significantly (to 2.0 mAbs. or less), similar to Comparative Example 10, and nonspecific aggregation was suppressed. (5) In Example 11, which used the first reagent to which the 33236-(CDR3-A) modified antibody was added, the change in absorbance of the 33236F(ab')2 antibody-conjugated latex decreased significantly (to 2.0 mAbs. or less), similar to Comparative Example 10, and nonspecific aggregation was suppressed. (6) From the above, it was found that even with a different measurement target than in Test Example 2, by having the CDR-modified antibody of the present invention present in the antigen-antibody reaction system, it can act as a decoy and suppress nonspecific aggregation of antibody-sensitized latex caused by nonspecific factors in the sample.

[0093] [Test Example 7] Verification of the effect of adding a modified CDR antibody on specific reactions (LTIA method 2) The concentration of BNP in the sample was measured using an LTIA reagent to which the modified antibody of the present invention was added. 1. Measurement reagents (1) Reagent 1 [Comparative Example 11] The first reagent shown in Comparative Example 9 was used. [Comparative Example 12] The first reagent shown in Comparative Example 10 was used. [Example 12] The first reagent shown in Example 9 was used. [Example 13] The first reagent shown in Example 10 was used. [Example 14] The first reagent shown in Example 11 was used. (2) Second reagent The second reagent of Nanopia® BNP-A (Sekisui Medical Co., Ltd.) was used.

[0094] 2. Sample The following samples were used for measurement as samples with known concentrations. (1) Nanopia BNP BNP Calibrator (Sekisui Medical Co., Ltd.)

[0095] 3. Measurement Procedure The first and second reagents were combined, and the above-mentioned sample with known concentration (Nanopia BNP BNP calibrator) was measured using a Hitachi 7180 automated analyzer. Specifically, 150 μL of the first reagent was added to 12 μL of the sample and incubated at 37°C for 5 minutes, then 50 μL of the second reagent was added and stirred. The change in absorbance due to aggregation formation was then measured over a 5-minute period at a primary wavelength of 570 nm and a secondary wavelength of 800 nm.

[0096] 4.Measurement results The measurement results are shown in Table 6.

[0097] [Table 6]

[0098] 5. Discussion (1) In Comparative Example 12, which used the first reagent to which 33236-(WT) antibody was added, the absorbance was clearly lower than the BNP concentration-dependent absorbance measurement observed in Comparative Example 11, which did not contain antibody. This indicates that the addition of 33236-(WT) antibody inhibits the specific reaction between BNP in the sample and antibody-conjugated latex. (2) In Example 12, which used the first reagent to which the 33236-(W51A) modified antibody was added, a BNP concentration-dependent increase in absorbance was observed, similar to Comparative Example 11, and the degree of increase was also equivalent. Therefore, it was shown that the 33236-(W51A) modified antibody does not affect the specific reaction between BNP and antibody-conjugated latex. (3) In Example 13, which used the first reagent to which the 33236-(K95A) modified antibody was added, a BNP concentration-dependent increase in absorbance was observed, similar to Comparative Example 11 and Example 12, and the degree of increase was also similar. Therefore, it was shown that the 33236-(K95A) modified antibody does not affect the specific reaction between BNP and antibody-conjugated latex. (4) In Example 14, which used the first reagent to which the 33236-(CDR3-A) modified antibody was added, a BNP concentration-dependent increase in absorbance was observed, similar to Comparative Examples 11 and Examples 12 and 13, and the degree of increase was also similar. Therefore, it was shown that the 33236-(CDR3-A) modified antibody does not affect the specific reaction between BNP and antibody-conjugated latex. (5) Based on the above, even with a different target of measurement than in Test Example 3, by modifying the CDR amino acid sequence of the specific antibody, we were able to obtain a non-specific reaction inhibitor that does not affect the specific reaction in the immunoassay method and acts as a decoy. [Industrial applicability]

[0099] According to the present invention, in a method for detecting a target antigen in a sample using a specific antibody that specifically binds to the antigen, the following steps are performed using a modified antibody of the specific antibody. a) Steps to bring the sample into contact with the modified antibody. b) A step in which the sample is brought into contact with the specific antibody, either simultaneously with or after step a). The detection method, characterized by including the modified antibody, makes it possible to suppress non-specific reactions contained in the sample and perform accurate measurements. Furthermore, the modified antibody of the present invention is effective even at low concentration ratios to the specific antibody, and it is possible to further reduce the impact on the antigen-antibody reaction. Therefore, a detection reagent containing the modified antibody that can suppress non-specific reactions, and a non-specific reaction inhibitor with the modified antibody as an active ingredient can also be provided. [Accession Number]

[0100] [Reference to deposited biological materials] (1) Hybridomas that produce antibody number 92204R (i) The name and address of the depositary institution that deposited the biological material. National Institute of Technology and Evaluation (NITE) Patent Microorganism Depository Center Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818) Date of deposit of biological material with the depositary in Roy September 25, 2015 The depositary number assigned by the depositary in Hai to the deposit NITE BP-02123 (2) Hybridomas that produce antibody number 33236 (i) The name and address of the depositary institution that deposited the biological material. National Institute of Technology and Evaluation (NITE) Patent Microorganism Depository Center Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818) Date of deposit of biological material with the depositary in Roy December 16, 2022 The depositary number assigned by the depositary in Hai to the deposit NITE BP-03799 (Receipt number is NITE ABP-03799)

Claims

1. A method for detecting a target antigen in a sample using a specific antibody that specifically binds to the antigen, a) A step of bringing the sample into contact with the modified antibody, b) A step of contacting the sample with the specific antibody, either simultaneously with or after step a), A detection method characterized in that the modified antibody is modified by a mutation in which 1 to 20 amino acids are substituted or deleted, or 1 to 20 amino acids are added, in the variable region of the antibody light chain (hereinafter abbreviated as L chain) and / or antibody heavy chain (hereinafter abbreviated as H chain) of the specific antibody, and the modified antibody has a lower reactivity to the target antigen than the specific antibody.

2. The detection method according to claim 1, wherein the modified antibody is a modified antibody in which 2 to 20 amino acids in the variable region of the L chain and / or H chain of the specific antibody have been modified.

3. The detection method according to claim 1, wherein the reactivity of the modified antibody to the target antigen is 50% or less of the reactivity of the specific antibody to the target antigen.

4. The detection method according to claim 1, wherein the concentration ratio of the modified antibody to the specific antibody in the antigen-antibody reaction system is 1 to 29 times.

5. The detection method according to claim 1, wherein the modified antibody is an intact antibody or a fragment antibody.

6. The detection method according to claim 1, comprising one or more of the aforementioned specific antibodies, and further comprising a modified antibody against at least one of the aforementioned specific antibodies.

7. The detection method according to claim 1, wherein the specific antibody comprises a first specific antibody and a second specific antibody, and the modified antibody comprises a modified antibody of at least one of the first specific antibody and the second specific antibody.

8. The detection method according to claim 1, wherein the specific antibody is an intact antibody or a fragment antibody containing a binding site with the target antigen.

9. The detection method according to claim 1, wherein the detection method is based on a homogeneous method or a heterogeneous method.

10. The detection method according to claim 1, wherein at least one of the specific antibodies is bound to an insoluble carrier or is labeled with a labeling agent.

11. The detection method according to claim 10, wherein the insoluble carrier is latex particles, gold colloid, magnetic particles, well-shaped plates, or a porous membrane.

12. The detection method according to claim 1, wherein the step of contacting a target antigen in a sample with a specific antibody is performed in a solution.

13. Furthermore, the process includes a step of optically detecting the degree of aggregation of latex particles in the solution. The specific antibody is bound to the latex particle, The detection method according to claim 12, wherein agglutination occurs when the target antigen binds to the specific antibody.

14. The detection method according to claim 13, wherein the optical detection step includes a step of optically detecting the color development of a chromogenic agent that has reacted in a way that depends on the amount of antigen-antibody complex in the solution, or the amount of a label.

15. A detection reagent for detecting a target antigen in a sample using a specific antibody that specifically binds to the antigen, comprising the following: (1) A specific antibody that specifically binds to the antigen. (2) A modified antibody in which the variable region of the L chain and / or H chain of the specific antibody is modified by a mutation in which 1 to 20 amino acids are substituted or deleted, or 1 to 20 amino acids are added, and the reactivity to the target antigen is lower than that of the specific antibody.

16. The detection reagent according to claim 15, wherein the modified antibody is a modified antibody in which 2 to 20 amino acids in the variable region of the L chain and / or H chain of the specific antibody have been modified.

17. The detection reagent according to claim 15, wherein the reactivity of the modified antibody to the target antigen is 50% or less compared to the reactivity of the specific antibody to the target antigen.

18. The detection reagent according to claim 15, which is prepared such that the concentration ratio of the modified antibody to the specific antibody in the antigen-antibody reaction system is 1 to 29 times.

19. The detection reagent according to claim 15, wherein the modified antibody is an intact antibody or a fragment antibody.

20. The detection reagent according to claim 15, comprising one or more of the aforementioned specific antibodies, and further comprising a modified antibody against at least one of the aforementioned specific antibodies.

21. The detection reagent according to claim 15, wherein the specific antibody comprises a first specific antibody and a second specific antibody, and the modified antibody comprises a modified antibody of at least one of the first specific antibody and the second specific antibody.

22. The detection reagent according to claim 15, wherein the specific antibody is an intact antibody or a fragment antibody containing a binding site with the target antigen.

23. The detection reagent according to claim 15, wherein the detection reagent is a reagent based on a homogeneous method or a heterogeneous method.

24. The detection reagent according to claim 15, wherein at least one of the specific antibodies is bound to an insoluble carrier or is labeled with a labeling agent.

25. The detection reagent according to claim 24, wherein the insoluble carrier is latex particles, gold colloid, magnetic particles, a well-shaped plate, or a porous membrane.

26. A detection reagent kit for detecting a target antigen in a sample using a specific antibody that specifically binds to the antigen, comprising reagents (1) and (2), characterized in that at least one of reagents (1) or (2) contains a modified antibody which is modified by a mutation in which 1 to 20 amino acids are substituted or deleted, or 1 to 20 amino acids are added, in the variable region of the L chain and / or H chain of the specific antibody, and which has a lower reactivity to the target antigen than the specific antibody. (1) Reagents containing buffer (2) Reagents containing specific antibodies

27. A detection reagent kit for detecting a target antigen in a sample using a specific antibody that specifically binds to the antigen, comprising reagents (1) and (2), characterized in that (1) includes a modified antibody which is modified by a mutation in which 1 to 20 amino acids are substituted or deleted, or 1 to 20 amino acids are added, in the variable region of the L chain and / or H chain of the specific antibody, and which has a lower reactivity to the target antigen than the specific antibody. (1) Reagents containing buffer (2) Reagents containing specific antibodies

28. A method for suppressing non-specific reactions in a method for detecting a target antigen, characterized by using in combination a specific antibody that specifically binds to the target antigen in a sample, and a modified antibody that is modified by a mutation in which 1 to 20 amino acids are substituted or deleted, or 1 to 20 amino acids are added, in the variable region of the L chain and / or H chain of the specific antibody, and whose reactivity to the target antigen is lower than that of the specific antibody.

29. A non-specific reaction inhibitor, which is used together with a specific antibody that specifically binds to a target antigen in a sample, and whose active ingredient is an antibody that is modified by a mutation in which 1 to 20 amino acids are substituted or deleted, or 1 to 20 amino acids are added, in the variable region of the L chain and / or H chain of the specific antibody, and whose reactivity to the target antigen is lower than that of the specific antibody.

30. An antibody used in conjunction with a specific antibody that specifically binds to a target antigen in a sample, wherein the specific antibody is modified by a mutation in which 1 to 20 amino acids are substituted or deleted, or 1 to 20 amino acids are added, in the variable region of the L chain and / or H chain, and its reactivity to the target antigen is lower than that of the specific antibody.

31. The antibody according to claim 30, wherein the reactivity of the modified antibody to the target antigen is 50% or less compared to the reactivity of the specific antibody to the target antigen.

32. The antibody according to claim 30, characterized in that it inhibits the binding of the specific antibody, which specifically binds to the target antigen in the sample, to a non-specific factor.

33. The antibody according to claim 30, characterized in that it binds to a non-specific factor for the specific antibody that specifically binds to the target antigen in the sample.

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