Immunological analysis method and immunological analysis reagent kit

Abstract

The present invention addresses the problem of providing a feature for improving sensitivity in an immunological analysis method and accurately analyzing a detected substance.　The aforementioned problem can be solved by a method for immunological analysis of a detected substance, the method including bringing a biological sample that could contain the detected substance, and a conjugate of a mark substance and polyethylene-glycol-modified antibodies, into contact with each other.

Description

[Technical field]

[0001] The present invention relates to an immunological analysis method and an immunological analysis reagent kit for detecting an analyte. The present invention also relates to a method for reducing measurement errors in an immunological analysis method for detecting an analyte. The present invention also relates to a method for preparing a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex. [Background technology]

[0002] When analyzing the amount of a substance to be detected in a biological sample, immunological analysis methods that use antibodies against the substance to be detected are sometimes used. Among immunological analysis methods, methods that measure the substance to be detected in a sample by detecting luminescence or fluorescence allow simple and highly sensitive analysis, and can be automated using analytical devices such as immunoplate readers, so they are used in many fields, including clinical testing.

[0003] In the method of detecting luminescence or fluorescence, it is necessary to subtract the blank value to which no biological sample is added when obtaining the measured value of the amount of the detected substance. However, when a highly hydrophobic labeling substance is conjugated to an antibody, the labeled antibody may adhere to the carrier, making it impossible to accurately measure the amount of the detected substance. In addition, it has been observed that the labeled antibodies may aggregate with each other, making it impossible to accurately measure the amount of the detected substance. Patent Document 1 teaches a chemiluminescent immunoassay reagent characterized by comprising a conjugate of an antibody and a hydrophilic compound having a dissociable group or a polar group and a substituent capable of reacting with an amino group of the antibody, bound to a water-soluble polymer and a chemiluminescent substance. In Patent Document 1, when a labeling substance with low solubility under neutral pH conditions is used, a linker with a low isoelectric point is added to the antibody to lower the isoelectric point of the labeled antibody and increase the solubility under neutral pH conditions. However, in order to improve sensitivity, a technology for further improving sensitivity by using an antibody labeled with a larger number of labeling substances and accurately measuring the amount of the substance to be detected has been desired. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 58-77662 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a technique for improving the sensitivity in an immunological analysis method and accurately analyzing the amount and / or presence of a substance to be detected. [Means for solving the problem]

[0006] The present inventors have discovered that in an immunological analysis method for a substance to be detected, when a biological sample which may contain the substance to be detected, a carrier on which a capture antibody is immobilized, and a labeled antibody in which a labeling substance is conjugated to a polyethylene glycol-modified antibody are contacted, aggregation of the labeled antibody, nonspecific adsorption of the labeled antibody to the carrier, etc. can be prevented, and the substance to be detected can be accurately analyzed, thereby completing the present invention. Specifically, the present invention is as follows. <1> A biological sample possibly containing a substance to be detected is contacted with a conjugate of a polyethylene glycol-modified antibody and a labeling substance. A method for immunologically analyzing a substance to be detected, comprising: <2> The biological sample that may contain the substance to be detected is one or more selected from the group consisting of blood, plasma, serum, and urine. <1> The immunological analysis method according to claim 1. <3> The weight average molecular weight (MW) of the polyethylene glycol is 250 to 15,000. <1> or <2> The immunological analysis method according to claim 1. <4> The labeling substance is an electrochemiluminescent substance, and the immunological analysis method is an electrochemiluminescence method (ECL method); <1> ~ <3> 13. The immunological analysis method according to any one of the preceding claims. <5> The polyethylene glycol-modified antibody is a polyethylene glycol-modified IgM antibody or a polyethylene glycol-modified IgG antibody. <1> ~ <4> 13. The immunological analysis method according to any one of the preceding claims. <6> The number of molecules of the labeling substance bound to one antibody molecule is 10 to 200. <1> ~ <5> 13. The immunological analysis method according to any one of the preceding claims. <7> Polyethylene glycol-modified antibody and labeling substance conjugate A reagent kit for immunological analysis of a substance to be detected, comprising: <8> For use on one or more biological samples selected from the group consisting of blood, plasma, and serum, <7> A reagent kit for immunological analysis according to claim 1. <9> The weight average molecular weight (MW) of the polyethylene glycol is 250 to 15,000. <7> or <8> A reagent kit for immunological analysis according to claim 1. <10> The labeling substance is an electrochemiluminescent substance, and the immunological analysis is an electrochemiluminescence method (ECL method); <7> ~ <9> 13. The immunological analysis reagent kit according to claim 12, <11> The polyethylene glycol-modified antibody is a polyethylene glycol-modified IgM antibody or a polyethylene glycol-modified IgG antibody. <7> ~ <10> 13. The immunological analysis reagent kit according to claim 12, <12> The number of molecules of the labeling substance bound to one antibody molecule is 10 to 200. <7> ~ <11> 13. The immunological analysis reagent kit according to claim 12, <13> contacting a biological sample possibly containing a substance to be detected with a conjugate of a polyethylene glycol-modified antibody and a labeling substance; A method for reducing measurement errors in immunological analysis of a substance to be detected, comprising: <14> The biological sample that may contain the substance to be detected is one or more selected from the group consisting of blood, plasma, and serum. <13> The measurement error reduction method according to claim 1. <15> The weight average molecular weight (MW) of the polyethylene glycol is 250 to 15,000. <13> or <14> The measurement error reduction method according to claim 1. <16> A method for preparing a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex, comprising the steps of: (1) a first complex formation step of contacting an antibody with a ruthenium complex to form a first complex; (2) a conjugate formation step of contacting polyethylene glycol with the first complex to form a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex; (3) A free ruthenium complex removal step for removing free ruthenium complexes that are not bound to the antibodies. <17> The conjugate formation step (2) is carried out after the free ruthenium complex removal step (3). <16> A method for preparing a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex according to claim 1. The present invention also encompasses the following embodiments. (a) the antibody is a monoclonal antibody; <1> ~ <6> 13. The immunological analysis method according to any one of the preceding claims. (b) the antibody is a monoclonal antibody; <7> ~ <12> 13. The immunological analysis reagent kit according to claim 12, (c) the antibody is a monoclonal antibody; <13> ~ <15> 2. The method for reducing measurement errors according to claim 1 . (d) the antibody is a monoclonal antibody; <16> or <17> A method for preparing a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex according to claim 1. Effect of the Invention

[0007] According to the present invention, the variation in blank value can be reduced. More specifically, according to the present invention, even if the number of molecules of the labeling substance bound to one antibody molecule is increased, the variation in blank value, the increase in blank value due to non-specific adsorption of the labeled antibody to the carrier, and the aggregation of the labeled antibodies via the labeling substance can be prevented. Therefore, according to the present invention, the amount of the substance to be detected can be measured accurately with high sensitivity. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic diagram showing an example of a conjugate of a polyethylene glycol-modified antibody and a labeling substance used in the immunological analysis method of the present invention. [Diagram 2] FIG. 1 is a schematic diagram showing the procedure and principle of the ECL method, which is one embodiment of the immunological analysis method of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] (Substance to be detected) The substance to be detected in the immunological analysis method of the present invention is a substance that can be an antigen and can be measured by utilizing an antigen-antibody reaction. Examples of the substance to be detected in the immunological analysis method of the present invention include the following substances. Myocardial markers such as CK-MB, HFABP, troponin (troponin I, troponin T), and myoglobin; coagulation and fibrinolysis markers such as fibrin degradation products (e.g. D-dimer), soluble fibrin, TAT (thrombin-antithrombin complex), and PIC (plasmin-plasmin inhibitor complex); circulation-related markers such as oxidized LDL and BNP (brain natriuretic peptide); metabolism-related markers such as adiponectin; tumor markers such as CEA (carcinoembryonic antigen), AFP (alpha-fetoprotein), PSA (prostate-specific antigen), and mucin-type glycoprotein; inflammation-related markers such as CRP (C-reactive protein) and LRG (leucine-rich alpha glycoprotein); infection-related markers such as influenza, HIV (human immunodeficiency virus), HBV (hepatitis B virus), HCV (hepatitis C virus), toxoplasma, chlamydia, syphilis, Staphylococcus aureus, Escherichia coli, and procalcitonin.

[0010] (Biological samples) In the present invention, examples of biological samples that may contain a substance to be detected include mainly substances derived from living organisms (organisms) and extracts obtained by extracting a substance to be detected from them. Specific examples of substances derived from living organisms (organisms) include blood (whole blood), serum, plasma, lymph, urine, feces, ascites, pleural effusion, and cerebrospinal fluid. In the present invention, it is preferable to use one or more body fluids selected from the group consisting of blood (whole blood), serum, plasma, and urine as the biological sample. Examples of biological samples that may contain a substance to be detected include components separated and fractionated from the whole blood by means of centrifugation, filtration, purification, etc., components extracted with organic solvents, etc., components solubilized with surfactants, etc., components diluted with buffer solutions, etc., and components modified or altered by chemical reactions, etc.

[0011] (Immunological analysis method) The immunological analysis method of the present invention includes, but is not limited to, electrochemiluminescence immunoassay (ECL), enzyme-linked immunosorbent assay (ELISA), latex agglutination immunoassay (LTIA), chemiluminescence immunoassay, fluorescent antibody assay, etc. The immunological analysis method of the present invention is preferably electrochemiluminescence immunoassay (ECL). In addition, in this specification, "analysis" includes verification of the presence and / or quantification of the analyte. A conjugate of a polyethylene glycol-modified antibody and a labeling substance may be referred to as a PEG-modified labeled antibody.

[0012] Electrochemiluminescence immunoassay (ECL) refers to a method in which a labeling substance emits light by passing an electric current through it, and the amount of the substance to be detected is measured by detecting the amount of the light emitted. In electrochemiluminescence immunoassay (ECL), a ruthenium complex can be used as the labeling substance. An electrode is placed on a solid phase (such as a microplate) and radicals are generated on the electrode, which excites the ruthenium complex and causes it to emit light. The amount of light emitted by the ruthenium complex can then be detected. An electrochemiluminescence immunoassay (ECL) can be performed by using a first antibody as a solid-phase antibody and a second antibody that recognizes an epitope different from that of the first antibody as a detection antibody (labeled antibody). A polyethylene glycol-modified antibody (PEG-modified labeled antibody) bound to a labeling substance is used as the labeled antibody. When a plurality of identical epitopes exist on the substance to be detected, the same antibody can be used as the solid-phase antibody and the PEG-modified labeled antibody. The measurement principle when magnetic particles are used as the insoluble carrier particles and a ruthenium complex is used as the PEG-modified labeled substance is as follows. The following shows the measurement principle in one embodiment of the present invention, and does not limit the scope of the present invention in any way. 1. When a biological sample is brought into contact with magnetic particles on which antibodies have been immobilized, the substance to be detected in the biological sample binds to the immobilized antibody. 2. After that, when the PEG-modified labeled antibody is brought into contact, the PEG-modified labeled antibody binds to the substance to be detected that is bound to the magnetic particles. 3. After removing the unbound PEG-modified labeled antibody, the detector emits light according to the amount of PEG-modified labeled antibody bound to the target substance when a current is applied. By measuring the amount of light emitted, the amount of the target substance in the sample can be accurately measured.

[0013] Among immunological analysis methods, enzyme-linked immunosorbent assay (ELISA) using enzyme labels is also preferred because it allows simple and rapid measurement of targets. In the case of sandwich ELISA, an insoluble carrier on which a first antibody (solid-phase antibody) that recognizes the substance to be detected is immobilized, and a second antibody (labeled antibody) labeled with a labeling substance are used. As the labeled antibody, a polyethylene glycol-modified antibody (PEG-modified labeled antibody) to which a labeling substance is bound is used. When the first antibody used recognizes multiple epitopes on the substance to be detected, the first antibody can be used as both the solid-phase antibody and the labeled antibody. The insoluble carrier is preferably a plate (immunoplate). The solid-phase antibody immobilized on the insoluble carrier captures the analyte in the biological sample and forms an antibody-analyte complex on the insoluble carrier. The PEG-modified labeled antibody binds to the captured analyte and forms a sandwich with the antibody-analyte complex. The analyte in the sample can be measured by measuring the amount of the labeling substance by a method appropriate for the labeling substance. Specific methods such as the method of immobilizing the antibody on the insoluble carrier and the method of binding the antibody to the labeling substance can be any method known to those skilled in the art without any particular restrictions.

[0014] In immunoassays, a signal may be detected even in the absence of an antigen due to nonspecific adsorption of the labeled antibody to the magnetic particles (or the carrier to which the antibody is bound). For example, in the case of the ECL method, a complex of the antibody bound to the magnetic particles and the electrochemiluminescent substance-labeled antibody may be formed at a certain rate even in the absence of an antigen, and a certain amount of luminescence may be detected even in the absence of an antigen. In particular, when the labeling substance is a ruthenium complex or the like having a high degree of hydrophobicity, false detection is likely to occur due to nonspecific adsorption of the labeled antibody to the magnetic particles or carrier. In addition, compared with IgG antibodies, which are often used in general immunoassay systems, IgM antibodies are more likely to be detected even in the absence of an antigen due to nonspecific adsorption to the magnetic particles (or the carrier to which the antibody is bound) due to the high hydrophobicity of the IgM antibody itself. In order to avoid this, the hydrophilicity of the labeled antibody is increased by polyethylene glycol modification. This makes it possible to suppress nonspecific adsorption of the labeled antibody to the magnetic particles or carrier, suppress the blank value, and improve the reproducibility of the measured value when a solution having the same antigen concentration is measured. In addition, the detection sensitivity can be increased by increasing the amount of the labeling substance bound to each antibody molecule. However, when a labeling substance with high hydrophobicity is labeled to an antibody, the hydrophobicity of the labeled antibody increases as the amount of the labeling substance bound to each antibody molecule increases. That is, when the amount of the labeling substance bound to each antibody molecule increases, the detection sensitivity increases, but disadvantages such as an increase in the blank value and a deterioration in reproducibility may occur. By modifying the labeled antibody with polyethylene glycol, even if the amount of the highly hydrophobic labeling substance (e.g., ruthenium complex, etc.) bound to each antibody molecule is increased, the increase in the blank value and the deterioration in reproducibility can be suppressed, and therefore, the improvement of sensitivity and the suppression of the increase in the blank value and the deterioration in reproducibility can be realized.

[0015] The antibody used in the immunological analysis method of the present invention can be prepared by known methods, either monoclonal or polyclonal, but monoclonal antibodies are preferred. Monoclonal antibodies can be obtained, for example, by isolating spleen cells or lymph node cells, which are antibody-producing cells, from a non-human mammal immunized with a substance to be detected or a fragment thereof, fusing the cells with a myeloma-derived cell line having high proliferation ability to produce a hybridoma, and purifying the antibody produced by the hybridoma. Polyclonal antibodies can be obtained from the serum of an animal immunized with a substance to be detected or a fragment thereof. A fragment is a partial fragment of the substance to be detected, and an antibody that binds to the fragment recognizes the substance to be detected.

[0016] As the antibody, in addition to the whole antibody molecule, it is also possible to use an antibody fragment having antigen-antibody reaction activity, and in addition to those obtained through the immunization process of animals as described above, it is also possible to use those obtained using genetic recombination technology and chimeric antibodies. As the antibody fragment, functional fragments are preferred, and examples thereof include F(ab')2, Fab', scFv, etc., and these fragments can be produced by treating the antibody obtained as described above with a protease (e.g., pepsin, papain, etc.) or by cloning the DNA of the antibody and expressing it in a culture system using E. coli or yeast.

[0017] The antibody used in the immunological analysis method of the present invention is preferably an antibody that specifically reacts with the analyte. "Specifically reacts with the analyte" means that it reacts with the analyte but does not substantially react with other substances. The meaning of "does not substantially react" will be described later.

[0018] In this specification, "reacting" with a substance to be detected, "recognizing" a substance to be detected, and "binding" to a substance to be detected are used synonymously, but are not limited to these examples and should be interpreted in the broadest sense. Confirmation of whether an antibody "reacts" with an antigen (compound) can be performed by antigen immobilization ELISA, competitive ELISA, sandwich ELISA, etc., as well as by a method utilizing the principle of surface plasmon resonance (SPR method). The SPR method can be performed using an apparatus, sensor, and reagents commercially available under the name Biacore (registered trademark).

[0019] The term "not reacting" between an antibody used and a certain compound means that the antibody used in the present invention does not substantially react with a certain compound, and "not substantially reacting" means that, for example, when the antibody used in the present invention is immobilized and measured using Biacore (registered trademark) T100 or T200 based on the above SPR method, no enhancement in the reactivity of the antibody used in the present invention is observed. In detail, it means that there is no significant difference in the reactivity between the antibody and the compound compared to the reactivity of a control (no compound added). It goes without saying that "not substantially reacting" can be confirmed by methods or means well known to those skilled in the art other than the above SPR method.

[0020] In this specification, the term "carrier" refers to a substance on which an antibody or the like that specifically recognizes a substance to be detected is immobilized. Examples of the carrier include, but are not limited to, insoluble substances such as immunoplates, membranes, latex particles, and magnetic particles.

[0021] (Polyethylene glycol) The polyethylene glycol (sometimes simply referred to as PEG) used in the immunological analysis method of the present invention is not particularly limited as long as the effects of the present invention can be obtained, but the degree of polymerization is preferably 2 to 300, more preferably 2 to 200, even more preferably 2 to 150, even more preferably 2 to 100, even more preferably 2 to 50, even more preferably 2 to 20, even more preferably 2 to 10, and most preferably 4 to 10. The degree of polymerization means "n" in the structural formula "HO-(CH2-CH2-O)n-H" of PEG. In the case of branched PEG, the degree of polymerization means the number of all repeating units of (CH2-CH2-O). That is, when each branch of the branched PEG contains "(CH2-CH2-O)l", "(CH2-CH2-O)m", and "(CH2-CH2-O)n", the degree of polymerization means the total value of l+m+n. The PEG used in the immunological analysis method of the present invention has a weight average molecular weight (MW) of preferably 250 to 15,000, more preferably 250 to 10,000, even more preferably 250 to 5,000, even more preferably 250 to 2,000, and even more preferably 250 to 1,000. In this specification, when the term "2 to 200" is used, the values ​​at both ends are included, and in this case, the range includes 2 and 200. PEG may be either branched or linear, or may include both. The weight average molecular weight (MW) can be measured using size exclusion chromatography (SEC).

[0022] (Polyethylene glycol modified antibody) As used herein, the term "polyethylene glycol-modified antibody" refers to an antibody that is conjugated to PEG by contacting the antibody with PEG and causing a chemical reaction with the PEG. As long as the effects of the present invention can be obtained, the conjugation may occur at any position on the antibody, and may occur, for example, by reaction between an amino group and PEG. It is preferable to use PEG having an NHS group at one end to react with the amino group of the antibody and conjugate the antibody. The antibody used in the immunological analysis method of the present invention is a polyethylene glycol-modified antibody, preferably a polyethylene glycol-modified IgM antibody or a polyethylene glycol-modified IgG antibody, and more preferably a polyethylene glycol-modified IgM antibody. Compared with an IgG antibody, an IgM antibody is more hydrophobic, and becomes even more hydrophobic when labeled with a labeling substance having a strong hydrophobicity. By modifying an IgM antibody with polyethylene glycol, aggregation between IgM antibodies due to strong hydrophobicity can be effectively prevented. The modification of an antibody with polyethylene glycol may be performed immediately before analyzing a substance to be detected, or an antibody prepared in advance may be used. By using a polyethylene glycol-modified antibody, it is possible to prevent a complex of an antibody and a labeling substance from being adsorbed to a carrier or the like, or the complex from being aggregated.

[0023] (Conjugate of polyethylene glycol-modified antibody and labeling substance) The conjugate of a polyethylene glycol-modified antibody and a labeling substance that can be used in the immunological analysis method of the present invention, i.e., a PEG-modified labeled antibody, is not limited as long as the effects of the present invention can be obtained, but is preferably a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex. The conjugate of a polyethylene glycol-modified antibody and a ruthenium complex that can be used in the immunological analysis method of the present invention is not limited as long as the effects of the present invention can be obtained, but can be prepared, for example, by a method including the following steps (1) to (3). (1) a first complex formation step of contacting an antibody with a ruthenium complex to form a first complex; (2) a conjugate formation step of contacting polyethylene glycol with the first complex to form a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex; (3) A free ruthenium complex removal step for removing free ruthenium complexes that are not bound to the antibodies. In the preparation method, in order to reduce the blank value, it is preferable that the conjugate formation step (2) is performed after the free ruthenium complex removal step (3). Even when other labeling substances are used, the conjugates can be prepared in a similar manner.

[0024] In the (1) first complex formation step, the mixing ratio of the ruthenium complex to the antibody, based on the number of molecules of the ruthenium complex and the antibody, is preferably 1:1 to 1:200, more preferably 1:5 to 1:200, even more preferably 1:10 to 1:200, even more preferably 1:20 to 1:200, and even more preferably 1:50 to 1:150. In the conjugate formation step (2), the mixing ratio of the first conjugate to polyethylene glycol can be 1:1 to 1:1000, 1:1 to 1:500, 1:5 to 1:500, 1:10 to 1:500, or 1:30 to 1:300 based on the molecular number of the first conjugate and polyethylene glycol.

[0025] The more the number of ruthenium complexes bound to one antibody molecule, the higher the detection sensitivity. On the other hand, in this case, if the labeling substance is hydrophobic, the amount of the target substance may not be accurately measured due to the adsorption of the complex of the antibody and the labeling substance to a carrier or the like, or the complex aggregates. However, even in such cases, the target substance can be analyzed with high sensitivity and accuracy by using a polyethylene glycol-modified antibody.

[0026] In the conjugate of the present invention of the polyethylene glycol-modified antibody and the labeling substance, the number of molecules of the labeling substance, preferably the ruthenium complex, bound per antibody molecule is preferably 1 to 200, more preferably 10 to 200, even more preferably 15 to 200, and still more preferably 20 to 100. The labeling substance may be bound to the antibody directly or via a linker or the like.

[0027] (labeled substance) As used herein, the term "labeling substance" refers to a substance that directly or indirectly binds to a substance to be detected and generates a signal for detecting the substance to be detected. The signal derived from the labeling substance may be measured according to a known method, for example, by measuring absorbance or intensity of reflected light. The signal may be confirmed visually or by using a specific measuring device. Examples of labeling substances for preparing labeled antibodies include enzymes, fluorescent substances, electrochemiluminescent substances, biotin, avidin, radioisotopes, gold colloid particles, and colored latex. Those skilled in the art can appropriately select an immunological analysis method depending on the antibody and labeling substance used. Electrochemiluminescent substances are substances that emit light when excited by electrical energy, and include ruthenium complexes and osmium complexes. Ruthenium complexes include ruthenium pyridine complexes. It is preferable to use electrochemiluminescence immunoassay (ECL) as the immunological analysis method, and to use an electrochemiluminescent substance, particularly a ruthenium complex, as the labeling substance. Electrochemiluminescent substances, particularly ruthenium complexes, are highly hydrophobic. Therefore, the effect of the immunological analysis method of the present invention can be greatly enjoyed in the ECL method. The immunological analysis method of the present invention can include a step of measuring a signal derived from a labeling substance. The step can include subtracting a blank value.

[0028] (Reduction of measurement error) It is known that in immunological measurement methods, positive or negative measurement errors in measured values ​​occur due to an increase in the blank value as described above or adhesion of the labeled antibody to the carrier, etc. In this specification, "reduction of measurement error" refers to bringing the positive or negative measurement error of the measured value closer to the original measured value (true value).

[0029] (Immunological analysis reagent kit) The reagent kit for immunological analysis of a substance to be detected of the present invention may be any kit containing a reagent containing a polyethylene glycol-modified antibody and a conjugate of a labeling substance (PEG-modified labeled antibody). In a reagent containing a PEG-modified labeled antibody (hereinafter sometimes referred to as a labeled reagent), the PEG-modified labeled antibody may have a concentration of 0.01 to 1000 μg / mL, 0.05 to 200 μg / mL, 0.1 to 200 μg / mL, 0.1 to 100 μg / mL, or 0.1 to 20 μg / mL in the labeled reagent. In the labeled reagent, the PEG-modified labeled antibody is preferably contained in a form that can be adjusted to 0.1 to 100 μg / mL, particularly 0.1 to 20 μg / mL in a mixed state of all reagents and a sample to be added to a measurement system.

[0030] The reagent kit for immunological analysis of a substance to be detected of the present invention preferably further includes a reagent (hereinafter, sometimes referred to as a capture reagent) containing a carrier to which an antibody (hereinafter, sometimes referred to as a "capture antibody") against the substance to be detected that is not labeled with a labeling substance is bound. The carrier to which the capture antibody is bound may have a concentration of 0.01 to 1000 mg / mL, 0.05 to 200 mg / mL, 0.1 to 200 mg / mL, 0.1 to 100 mg / mL, or 0.1 to 2 mg / mL in the capture reagent. In the capture reagent, the carrier to which the capture antibody is bound is preferably contained in a form that can be adjusted to 0.1 to 10 mg / mL, particularly 0.1 to 2 mg / mL, in a mixed state of all reagents and a sample to be added to a measurement system.

[0031] The reagent kit for immunological analysis of a substance to be detected of the present invention may also contain other test reagents such as a standard antigen substance and an antigen sample for quality control, a specimen diluent, and / or instructions for use.

[0032] In the reagent kit for immunological analysis of a substance to be detected of the present invention, the PEG-modified labeled antibody and the capture antibody may recognize different epitopes, and when the PEG-modified labeled antibody recognizes multiple epitopes on an antigen, the same antibody (i.e., recognizing the same epitope) may be used as the PEG-modified labeled antibody and the capture antibody.

[0033] When the ECL method is used, the reagent kit for immunological analysis of an analyte of the present invention can contain the following (A) and (B). (A) a labeling reagent comprising a conjugate of a polyethylene glycol-modified antibody and an electrochemiluminescent substance (e.g., a ruthenium complex, etc.); and (B) A solid phase on which a capture antibody that binds to the substance to be detected is immobilized. For example, in a kit using magnetic particles as a solid phase, a biological sample is added to the magnetic particles on which a capture antibody that binds to the substance to be detected is immobilized, and the biological sample is then reacted, and the sample is removed and washed. Next, a conjugate of a polyethylene glycol-modified antibody and an electrochemiluminescent substance (e.g., a ruthenium complex, etc.) is added and reacted. After washing the magnetic particles, electrical energy is applied to cause light emission, and the amount of light emitted by the labeling substance is measured, allowing the substance to be analyzed.

[0034] When using the sandwich ELISA method, the reagent kit for immunological analysis of a substance to be detected of the present invention can contain the following (A) and (B). (A) Labeling reagent containing PEG-modified labeled antibody (B) Insoluble support onto which a capture antibody is immobilized. In such a kit, a biological sample is first added to an insoluble carrier on which a capture antibody that binds to the substance to be detected is immobilized, followed by incubation, removal of the sample, and washing. Next, a labeling reagent is added, followed by incubation, and a substrate is added to cause color development. The substance to be detected can be analyzed by measuring the color development using a plate reader or the like.

[0035] The present invention will now be described in detail with reference to examples, but these are not intended to limit the scope of the present invention. Unless otherwise specified, % means mass %. In addition, the "Ru-Ab mixing ratio" in the table indicates the ratio of the number of ruthenium complex molecules when the number of antibody molecules is taken as 1. EXAMPLES

[0036] [Preparation Example 1] Preparation of the antibody used The antibodies used were anti-Dupan-2 antibody and anti-procalcitonin antibody. The anti-Dupan-2 antibody is an IgM antibody, and the anti-procalcitonin antibody is an IgG antibody. Both antibodies are monoclonal. The anti-Dupan-2 antibody was prepared by culturing clone No. S19201R and purifying the culture supernatant containing the anti-Dupan-2 antibody by anion exchange and gel filtration chromatography. The antibody was dialyzed against 150 mM potassium phosphate / 150 mM NaCl (pH 7.8) and then used. The antibody was stored in a refrigerator until use. Anti-procalcitonin antibodies were produced by transplanting clone No. S02211 and S02203R cells into BALB / c mice and forming ascites. Ascites containing each antibody was purified from the S02211 clone by anion exchange chromatography. Ascites from the S02203R clone was purified by protein G chromatography. Each purified antibody was dialyzed against 150 mM potassium phosphate / 150 mM NaCL (pH 7.8) and stored in a refrigerator until use.

[0037] [Preparation Example 2-1] Binding of ruthenium complex to anti-Dupan-2 antibody 1.73 mg / mL of purified antibody (anti-Dupan-2 antibody, dissolved in 150 mM potassium phosphate / 150 mM NaCL (pH 7.8)) was placed in a 2.0 mL Eppendorf tube. 10 mg / mL of ruthenium complex (dissolved in DMSO) was placed in the Eppendorf tube so that the ratio of the number of antibody molecules to the number of ruthenium complex molecules was 1:5, 1:20, 1:50, or 1:100, respectively. The mixture was then stirred and mixed at room temperature for 30 minutes. Ruthenium(II)Tris(bipyridyl)-NHS Ester was used as the ruthenium complex. In order to block the NHS group (N-hydroxysuccinimide group) of the free ruthenium complex with an excess of the amino group of glycine, 2M glycine (dissolved in distilled water) was added to the reaction solution so that the final concentration of glycine was 100mM. The mixture was then mixed and stirred at room temperature for 20 minutes. The reaction solution was applied to a Sephadex G25 column (gel volume: 20 mL) equilibrated with 10 mM potassium phosphate / 150 mM NaCl (pH 6.0) to remove free ruthenium complexes. The absorbance of the eluate at A280 nm was measured, and fractions showing absorption at A280 nm, the absorption wavelength of protein, were collected. The protein concentration of the collected fractions was quantified using a Micro BCA Protein Assay Kit (Thermo Fisher), and the number of antibody molecules was calculated from the quantitative value. The absorbance at A455 nm, which is the absorption of ruthenium complexes, was also measured, and the number of ruthenium complex molecules in the collected fractions was calculated from the A455 nm absorbance. From the calculated number of antibody molecules and the number of ruthenium complex molecules, the number of ruthenium complex molecules labeled per antibody molecule was calculated for the ruthenium-labeled antibody in the collected fraction (Table 1). The prepared ruthenium-labeled antibody was stored in a refrigerator until use.

[0038] [Table 1]

[0039] [Preparation Example 2-2] Binding of ruthenium complex to anti-procalcitonin antibody The procedure was the same as for anti-Dupan-2 antibody, except that the antibody purified from ascites using the S02211 clone was used as the labeled antibody, and the ratio of the number of antibody molecules to the number of ruthenium complex molecules was mixed at a ratio of 1:20. The number of ruthenium complex molecules bound to one antibody molecule was 10.

[0040] [Preparation Example 3-1] Modification of ruthenium-labeled antibody with PEG As PEGs with one end NHS-modified, MS(PEG)4 Methyl-PEG-NHS-Ester Regent (Thermo Fisher, MW: about 330) with a degree of polymerization of 4, MS(PEG)24 Methyl-PEG-NHS-Ester Regent (Thermo Fisher, MW: about 1210) with a degree of polymerization of 24, and Acrylate-PEG7500-NHS (Merck, average Mn: about 7500) with a degree of polymerization of about 170 were used. Each PEG was dissolved in distilled water to 0.1 mM immediately before use. Each ruthenium-labeled antibody prepared in Preparation Example 2-1 to Preparation Example 2-2 was diluted 2-fold with 150 mM potassium phosphate / 150 mM NaCL (pH 7.8) to make the solution pH weakly alkaline, and the 2-fold diluted ruthenium-labeled antibody molecule and various PEG molecules were mixed in any ratio and stirred at room temperature for 16 hours. In order to block the NHS group (N-hydroxysuccinimide group) of the free ruthenium complex with an excess of the amino group of glycine, 2M glycine (dissolved in distilled water) was added to the stirred solution in an amount to make the final concentration of glycine 100mM, and the mixture was stirred at room temperature for 20 minutes. The prepared PEG-modified ruthenium-labeled antibody was stored in a refrigerator until use. Details of the prepared PEG-modified ruthenium-labeled antibodies are shown in Table 2 below.

[0041] [Table 2]

[0042] [Preparation Example 3-2] Binding of ruthenium complex to antibody and PEG modification of ruthenium-labeled antibody To examine the effect of removing the free ruthenium complex after PEG modification of the antibody, a PEG-modified ruthenium-labeled antibody was prepared as follows. 1.73 mg / mL of purified antibody (anti-Dupan-2 purified IgM antibody, dissolved in 150 mM potassium phosphate / 150 mM NaCL (pH 7.8)) was poured into 2.0 mL Eppendorf tubes (total of 3 tubes) in amounts of 1.84 mg, 0.92 mg, and 1.5 mg. 4.8 μL (for 1.84 mg of antibody), 6.0 μL (for 0.92 mg of antibody), and 19.6 μL (for 1.5 mg of antibody) of 10 mg / mL ruthenium complex (M.W1057, dissolved in DMSO) were poured therein so that the ratio of antibody molecule number:ruthenium complex molecule number = 1:20, 1:50, and 1:100, respectively, and the mixture was stirred at room temperature for 30 minutes. Next, MS(PEG)4 Methyl-PEG-NHS-Ester Reagent (Thermo Fisher), which is a four-molecule PEG with one end NHS-modified, was dissolved in 150 mM potassium phosphate / 150 mM NaCL (pH 7.8) immediately before use to make the concentration 1 mM, mixed so that the ratio of the number of ruthenium-labeled antibody molecules to the number of PEG molecules was 1:30, and stirred at room temperature for 1 hour. In order to block the NHS groups of the free ruthenium complex and free PEG with an excess of the amino group of glycine, 2 M glycine (dissolved in distilled water) was added to the reaction solution in an amount to make the final concentration of glycine 100 mM, and the mixture was mixed and stirred at room temperature for 20 minutes. The reaction solution was applied to a Sephadex G25 column (gel volume: 20 mL) equilibrated with 10 mM potassium phosphate / 150 mM NaCl (pH 6.0) to remove free ruthenium complex and free PEG. The absorbance of the eluate at A280 nm was measured, and fractions showing absorption at A280 nm, the absorption wavelength of protein, were collected. The protein concentration of the collected fractions was quantified using a Micro BCA Protein Assay Kit (Thermo Fisher), and the number of antibody molecules was calculated from the quantitative value. The absorbance at A455 nm, which is the absorption of ruthenium complexes, was also measured, and the number of ruthenium molecules in the collected fractions was calculated from the A455 nm absorbance. The number of ruthenium complex molecules labeled per antibody molecule was calculated from the calculated number of antibody molecules and the number of ruthenium complex molecules for the ruthenium-labeled antibody in the collected fraction (Table 3). The prepared ruthenium-labeled antibody was stored in a refrigerator until use.

[0043] [Table 3]

[0044] Details of the prepared PEG-modified ruthenium-labeled antibodies are shown in Table 4 below.

[0045] [Table 4]

[0046] [Preparation Example 4] Preparation of antibody-bound magnetic particles 1 mL of 30 mg / mL magnetic particles with a diameter of 3 μm having an epoxy group on the surface was taken, and the magnetic particles were collected using a magnet to remove the solvent. In order to wash the magnetic particles, 1 mL of 150 mM potassium phosphate / 150 mM NaCL (pH 7.8) was mixed with the magnetic particles and stirred, and then the magnetic particles were collected again using a magnet to remove the solvent. 1 mL of 0.6 mg / mL of each purified antibody (anti-Dupan-2 antibody, anti-procalcitonin antibody dissolved in 150 mM potassium phosphate / 150 mMNaCL (pH 7.8)) was added to the magnetic particles from which the solvent had been removed, and the antibodies were bound to the magnetic particles by stirring with an end-over-end stirrer at 25°C for 17 hours. The antibody-bound magnetic particles were washed and stored in a refrigerator until use.

[0047] [Example 1-1] Analysis of Dupan-2 using anti-Dupan-2 antibody (1-1) Preparation of ruthenium-labeled antibody solution Each of the PEG-unmodified ruthenium-labeled anti-Dupan-2 antibody and antibodies No. 2 to 11 prepared in Preparation Example 3-1 was diluted to 5 μg / mL and used in the measurement.

[0048] (1-2) Preparation of antibody-bound magnetic particle solution The antibody-bound magnetic particles prepared in Preparation Example 4 were diluted to 2 mg / mL and used in the measurement.

[0049] (1-3) Measuring equipment Measurements were performed using an electrochemiluminescence immunoassay device, Picolumi III (manufactured by Sekisui Medical Co., Ltd.).

[0050] (1-4) Measurement The magnetic particle liquid and the ruthenium-labeled antibody liquid or PEG-modified ruthenium-labeled antibody in the designated container were set in the designated place of the measurement device. 60 μL of the reaction solution was dispensed into the reaction container designated by the measurement device, and then 30 μL of the measurement sample was added. The reaction container containing the measurement sample was set in the designated place of the measurement device, and the measurement preparation was completed. When the measurement with the device was started, 25 μL of the magnetic particle liquid was added to the reaction container containing the measurement sample, and an antigen-antibody reaction was carried out at 28 ° C for about 5 minutes (first reaction). Then, BF separation was carried out with the BF separation solution designated by the device. Next, 100 μL of the ruthenium-labeled antibody or PEG-modified ruthenium-labeled antibody was added, and an antigen-antibody reaction was carried out at 28 ° C for about 3 minutes (second reaction). Then, BF separation was carried out with the BF separation solution designated by the device, and unreacted ruthenium-labeled antibody or PEG-modified ruthenium-labeled antibody was removed. The antigen-antibody sandwich complex produced by the reaction was subjected to electrical current in a solution of the luminescent substrate (tripropylamine) specified by the device at a predetermined location in the device, causing it to chemiluminesce (ECL luminescence).

[0051] The intensity of ECL emission varies depending on the number of antigen-antibody sandwich complexes (magnetic particle-bound antibody - antigenic substance in the sample - ruthenium-labeled antibody (or PEG-modified ruthenium-labeled antibody)) produced by the reaction, and the antigen concentration can be quantified from the difference in ECL emission intensity according to the antigen concentration in the measurement sample. The results are shown in Table 5 below.

[0052] [Table 5]

[0053] As a result, in the measurement system using PEG-unmodified ruthenium-labeled anti-Dupan-2 antibody, the blank reproducibility (variability: CV) significantly worsened and the blank value increased as the number of ruthenium molecules bound to the antibody increased. The only antibody with a CV of 10% or less was the ruthenium-labeled antibody with 5 ruthenium molecules per antibody molecule. On the other hand, the measurement system using various PEG-modified ruthenium-labeled anti-Dupan-2 antibodies showed a significant improvement in the reproducibility. The CV was also suppressed to 10% or less for the ruthenium-labeled antibody (antibody Nos. 8-10) with 67 ruthenium molecules per antibody molecule. The blank ECL emission intensity was also suppressed compared to the PEG-unmodified labeled antibody. There was no clear difference in the blank reproducibility or ECL emission intensity depending on the degree of polymerization of the PEG to be bound.

[0054] [Example 1-2] Test results to confirm the degree of effectiveness depending on the PEG binding method We confirmed whether differences in the performance of PEG-modified ruthenium-labeled anti-Dupan-2 antibody were observed due to differences in PEG binding methods. Using PEG-modified ruthenium-labeled anti-Dupan-2 antibody prepared by the method of Preparation Example 3-1 or Preparation Example 3-2, blank measurements were performed with N=10 to confirm the simultaneous reproducibility. In both PEG binding methods, PEG binding to the antibody was performed with a mixture ratio of ruthenium-labeled antibody molecules to PEG molecules of 1:30. The results are shown in Table 6.

[0055] [Table 6]

[0056] As a result, the PEG-modified ruthenium-labeled anti-Dupan-2 antibody prepared by either binding method showed an improvement in the blank reproducibility (variability: CV) compared to the unPEG-modified ruthenium-labeled anti-Dupan-2 antibody. However, the PEG-modified ruthenium-labeled anti-Dupan-2 antibody prepared by preparation method 3-1 showed a greater improvement in the reproducibility (variability: CV) compared to preparation method 3-2.

[0057] [Example 2-1] Confirmation test of the effect of different PEG mixing ratios on ruthenium-labeled anti-Dupan-2 antibody It was confirmed whether the performance of the PEG-modified ruthenium-labeled anti-Dupan-2 antibody differed depending on the mixing ratio of PEG to the antibody. The mixing ratio of PEG was adjusted so that the ratio of the ruthenium-labeled antibody to the number of PEG molecules was 1:30 or 1:300. Antibodies No. 2 and 3 prepared in Preparation Example 3-1 were used for the study. Using each antibody, a blank (N=10) and a Dupan-2 antigen (STD in Table 7: 0.05, 0.1, 0.5, 1, 2 μg / mL) prepared using a synthetic glycan that mimicked the Dupan-2 glycan sequence were measured, and the simultaneous reproducibility and detection sensitivity in each measurement system were confirmed. The results are shown in Table 7.

[0058] [Table 7]

[0059] As a result, in the measurement systems using either PEG-modified ruthenium-labeled anti-Dupan-2 antibody, the blank N=10 simultaneous reproducibility was improved (CV 3.7% and 7.7%) compared to the measurement system using unPEG-modified ruthenium-labeled anti-Dupan-2 antibody (CV 19.1%). On the other hand, when comparing the ECL emission intensity of the STD, the PEG-modified ruthenium-labeled anti-Dupan-2 antibody prepared with a ruthenium-labeled antibody and PEG mixing ratio of 1:30 gave a stronger ECL emission intensity than the one with a mixing ratio of 1:300.

[0060] [Example 2-2] Sensitivity test results of the Dupan-2 measurement system using PEG-modified ruthenium-labeled anti-Dupan-2 antibody For Dupan-2 measurement systems using various PEG-modified ruthenium-labeled anti-Dupan-2 antibodies and Dupan-2 measurement systems using PEG-unmodified ruthenium-labeled anti-Dupan-2 antibodies, measurements were performed on serum samples from four pancreatic cancer patients and Dupan-2 antigens (STD in Tables 8 and 9: 0.025, 0.05, 0.1 μg / mL) prepared using synthetic glycans that mimic the Dupan-2 glycan sequence, and the detection sensitivity of each measurement system was confirmed. The results are shown in Tables 8 and 9.

[0061] [Table 8]

[0062] [Table 9]

[0063] As a result, the measurement system using PEG-modified ruthenium-labeled antibodies (12, 22, and 67 ruthenium molecules / antibody) was able to label more ruthenium to the antibody while suppressing the CV of the simultaneous reproducibility to 10% or less, and was able to obtain a stronger emission intensity than the measurement system using ruthenium-labeled antibodies (5 ruthenium molecules / antibody) with a blank simultaneous reproducibility of CV of 10% or less. In addition, the ECL emission intensity increased as the number of ruthenium-labeled molecules to the antibody increased. In the relationship between the difference in the type of PEG to be bound and the ECL emission intensity, the lower the degree of polymerization of the labeled PEG (shorter PEG), the higher the ECL emission intensity of the sample. In the measurement system using PEG4-modified ruthenium-labeled antibodies, which obtained the strongest emission intensity in this test, the emission intensity was 13 times higher on average for serum samples from pancreatic cancer patients and 29 times higher for synthetic glycans than the measurement system using unmodified ruthenium-labeled antibodies.

[0064] [Example 3] Analysis of procalcitonin (3-1) Preparation of ruthenium-labeled antibody solution The ruthenium-labeled anti-procalcitonin antibody and the PEG-modified ruthenium-labeled anti-procalcitonin antibody prepared in Preparation Examples 1, 2-2, and 3-1 were diluted to 20 μg / mL and used in the measurement.

[0065] (3-2) Preparation of antibody-bound magnetic particle solution The antibody-bound magnetic particles prepared in Preparation Example 4 were diluted to 1 mg / mL and used in the measurement.

[0066] (3-3) Measuring equipment Measurements were performed using an electrochemiluminescence immunoassay device, Picolumi III (manufactured by Sekisui Medical Co., Ltd.).

[0067] (3-4) Measurement The magnetic particle liquid and the ruthenium-labeled antibody liquid or the PEG-modified ruthenium-labeled antibody in the container designated for the measurement device were set in the designated place of the measurement device. The reaction solution was dispensed into the reaction container designated for the measurement device, and then 20 μL of the measurement sample (physiological saline as a blank) was added. The reaction container containing the measurement sample was set in the designated place of the measurement device, and the measurement preparation was completed. When the measurement with the device was started, 50 μL of the magnetic particle liquid was added to the reaction container containing the measurement sample, and the antigen-antibody reaction was carried out at 28 ° C for about 5 minutes (first reaction). Next, 50 μL of the ruthenium-labeled antibody liquid or the PEG-modified ruthenium-labeled antibody was added, and the antigen-antibody reaction was carried out at 28 ° C for about 3 minutes (second reaction). After that, BF separation was carried out with the BF separation solution designated for the device, and the unreacted ruthenium-labeled antibody liquid or the PEG-modified ruthenium-labeled antibody was removed. The antigen-antibody sandwich complex generated by the reaction was subjected to electricity in the luminescent substrate (tripropylamine) solution designated for the device at the designated place of the device, and chemiluminescence (ECL luminescence) was caused. The results are shown in Table 10.

[0068] [Table 10]

[0069] With the PEG-unmodified ruthenium-labeled anti-procalcitonin antibody, a certain percentage of abnormally high values ​​(jumps) in ECL luminescence were observed, which were thought to be due to non-specific binding of the ruthenium-labeled antibody to the antibody-bound magnetic particles, but this was suppressed when the PEG-modified ruthenium-labeled anti-procalcitonin antibody was used (data not shown). As a result, the PEG-modified ruthenium-labeled anti-procalcitonin antibody improved the intra-sample reproducibility (variability: CV) (14%) compared to the PEG-unmodified ruthenium-labeled anti-procalcitonin antibody. [Industrial Applicability]

[0070] An object of the present invention is to provide a technique for improving the sensitivity in an immunological analysis method and for accurately analyzing a substance to be detected.

Claims

1. A method for immunological analysis of a target substance, comprising contacting a biological sample possibly containing the target substance with a conjugate of a polyethylene glycol-modified antibody and a labeling substance, wherein the labeling substance is an electrochemiluminescent substance, and the immunological analysis method is an electrochemiluminescence method (ECL method).

2. 2. The immunological analysis method according to claim 1, wherein the biological sample possibly containing the target substance is one or more selected from the group consisting of blood, plasma, serum and urine.

3. 3. The immunological analysis method according to claim 1, wherein the weight-average molecular weight (MW) of the polyethylene glycol is 250 to 15,000.

4. 4. The immunological analysis method according to claim 1, wherein the polyethylene glycol-modified antibody is a polyethylene glycol-modified IgM antibody or a polyethylene glycol-modified IgG antibody.

5. 5. The immunological analysis method according to claim 1, wherein the number of molecules of the labeling substance bound to one antibody molecule is 10 to 200.

6. A reagent kit for immunological analysis of a substance to be detected, comprising a conjugate of a polyethylene glycol-modified antibody and a labeling substance, wherein the labeling substance is an electrochemiluminescent substance, and the immunological analysis is an electrochemiluminescence method (ECL method).

7. 7. The immunological analysis reagent kit of claim 6, for use with one or more biological samples selected from the group consisting of blood, plasma, and serum.

8. 8. The reagent kit for immunological analysis according to claim 6, wherein the weight average molecular weight (MW) of the polyethylene glycol is 250 to 15,000.

9. 9. The reagent kit for immunological analysis according to claim 6, wherein the polyethylene glycol-modified antibody is a polyethylene glycol-modified IgM antibody or a polyethylene glycol-modified IgG antibody.

10. 10. The reagent kit for immunological analysis according to claim 6, wherein the number of molecules of the labeling substance bound to one antibody molecule is 10 to 200.

11. A method for reducing measurement errors in immunological analysis of a target substance, comprising contacting a biological sample possibly containing the target substance with a conjugate of a polyethylene glycol-modified antibody and a labeling substance, wherein the labeling substance is an electrochemiluminescent substance and the immunological analysis is an electrochemiluminescence method (ECL method).

12. 12. The method for reducing measurement errors according to claim 11, wherein the biological sample that may contain the target substance is one or more selected from the group consisting of blood, plasma, and serum.

13. The method for reducing measurement errors according to claim 11 or 12, wherein the weight average molecular weight (MW) of the polyethylene glycol is 250 to 15,000.

14. A method for preparing a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex, comprising the steps of: (1) a first complex formation step of contacting an antibody with a ruthenium complex to form a first complex; (2) a conjugate formation step of contacting polyethylene glycol with the first conjugate to form a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex; (3) A free ruthenium complex removal step for removing free ruthenium complexes that are not bound to antibodies.

15. The method for preparing a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex described in claim 14, wherein the conjugate formation step (2) is carried out after the free ruthenium complex removal step (3).

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