Labeled antibody, method for manufacturing same, immunological measurement reagent, and immunological measurement method

Abstract

A labeled antibody according to the present invention is obtained by using a linker to bind a full-length antibody and a labeling substance. The linker is formed by binding a first reactive group of a crosslinking agent to the full-length antibody and binding a second reactive group of the crosslinking agent to the labeling substance. The molecular weight of the labeling substance is 10,000 or greater, and the molecular weight of the crosslinking agent is 270 to 1350.

Description

Labeled Antibody, Method for Producing the Same, Immunological Measurement Reagent, and Immunological Measurement Method

[0001] The present invention relates to a labeled antibody, a method for producing the same, an immunological measurement reagent, and an immunological measurement method. This application claims priority to Japanese Patent Application No. 2024-166001 filed in Japan on September 25, 2024, the content of which is incorporated herein by reference.

[0002] Conventionally, biochemical measurement methods are known as means for detecting various components contained in human biological samples. Among biochemical measurement methods, immunological measurement methods are highly specific measurement methods because they utilize specific binding between proteins such as antigen-antibody reactions.

[0003] In immunological measurement methods, labeled antibodies may be used. A labeled antibody is an antibody labeled with a labeling substance. By detecting the signal from this labeling substance, the detection and quantification of the target component can be performed.

[0004] In immunological measurement methods, binding that does not occur based on the original specific reaction or the specific immune reaction may be hindered. These are called non-specific reactions, and the causative substances may be called non-specific factors. Examples of non-specific factors include rheumatoid factor (RF), bilirubin, and milk. When such non-specific reactions occur in biochemical measurements, measurement errors occur. Therefore, various methods have been studied for the purpose of suppressing non-specific reactions.

[0005] RF, which is a non-specific factor, is known to cause non-specific reactions by binding to the Fc region of an antibody. Therefore, antibodies used in immunological measurements are often fragments obtained by cleaving the Fc region from full-length antibodies using digestive enzymes.

[0006] Patent Document 1 discloses reacting a substance that causes a non-specific reaction contained in a sample with a non-specific reaction inhibitor, and then performing a specific immune reaction.

[0007] Patent Document 2 discloses a method for preparing labeled antibodies, which involves attaching a labeling substance to antibodies that have been fragmented by a reduction treatment.

[0008] International Publication No. 2013 / 002309 JP 4-291155 Publication

[0009] Patent Document 1 focuses on the presence of substances in the sample that react with carrier proteins bound to maleimide groups, thereby suppressing measurement errors. On the other hand, in immunological measurement methods, if an insoluble carrier is present, the insoluble carrier and the labeled antibody may react nonspecifically without involving the target substance, potentially leading to measurement errors.

[0010] As mentioned above, one known method for suppressing non-specific reactions is to cleave the Fc portion of a full-length antibody with a digestive enzyme. However, when attempting to fragment IgG2a and IgG2b subclass antibodies by digestion, mainly Fab / Fc is produced, and the F(ab')2 fraction, which has antibody activity, is not sufficiently obtained. It is known that further digestion of Fab / Fc leads to digestion of peptides with lower molecular weight than the F(ab')2 fraction. Patent document 2 discloses a method to solve this problem by treating the antibody with a reducing agent without digesting it with pepsin to obtain halves of the antibody with the sulfhydryl group released. However, setting the appropriate processing conditions is difficult because the appropriate conditions differ depending on the antibody.

[0011] Furthermore, even with antibodies other than IgG2a or IgG2b subclass antibodies, depending on the type of antibody, overdigestion by pepsin may occur, resulting in either no F(ab') fraction being obtained or, even if F(ab') fractions are obtained, a low yield.

[0012] The object of the present invention is to provide a labeled antibody containing a full-length antibody to which a labeling substance is conjugated via a linker, an immunological assay reagent containing the labeled antibody, and an immunological assay method.

[0013] The present invention encompasses the following embodiments: [1] A labeled antibody comprising a full-length antibody and a labeled substance linked by a linker, wherein the linker is formed by a first reactive group of a crosslinking agent being bound to the full-length antibody and a second reactive group of the crosslinking agent being bound to the labeled substance, wherein the molecular weight of the labeled substance is 10,000 or more, and the molecular weight of the crosslinking agent is 270 or more and 1350 or less. [2] The labeled antibody according to [1], wherein the full-length antibody is overdigested by treatment with a proteolytic enzyme. [3] The labeled antibody according to [1] or [2], wherein the full-length antibody is an IgG2 subclass antibody. [4] The labeled antibody according to any one of [1] to [3], wherein the labeled substance is an enzyme. [5] The labeled antibody according to any one of [1] to [4], wherein the first reactive group of the crosslinking agent that binds to the full-length antibody is a group having a maleimide skeleton. [6] The labeled antibody according to any one of [1] to [5], wherein the second reactive group of the crosslinking agent that binds to the labeling substance is an N-hydroxyester or a sulfo-N-hydroxyester. [7] The labeled antibody according to any one of [1] to [6], wherein the linker is an alkylene glycol unit. [8] The labeled antibody according to [7], wherein the alkylene glycol unit is an ethylene glycol unit. [9] The labeled antibody according to [7] or [8], wherein the number of alkylene glycol units contained in the linker is 2 or more and 12 or less.

[10] An immunoassay reagent comprising the labeled antibody according to any one of [1] to [9].

[11] An immunoassay method comprising the step of mixing a sample with the immunoassay reagent according to

[10] and measuring by chemiluminescence.

[12] A method for producing a labeled antibody, comprising the steps of: introducing a sulfhydryl group into a full-length antibody; reacting a second reactive group of a crosslinking agent with a labeling substance to bind the crosslinking agent and the labeling substance; and reacting the sulfhydryl group with a first reactive group of the crosslinking agent to bind the crosslinking agent and the full-length antibody, thereby obtaining a labeled antibody in which the full-length antibody and the labeling substance are bound via a linker formed by the crosslinking agent, wherein the molecular weight of the labeling substance is 10,000 or more, and the molecular weight of the crosslinking agent is 270 or more and 1350 or less.Another aspect of the present invention is that it encompasses the following embodiments:

[13] A labeled antibody comprising a full-length antibody and a labeled substance linked by a linker, wherein the linker is formed by a first reactive group of a crosslinking agent being bound to the full-length antibody and a second reactive group of the crosslinking agent being bound to the labeled substance, wherein the molecular weight of the labeled substance is 40,000 or more and 200,000 or less, and the molecular weight of the crosslinking agent is 270 or more and 1350 or less, as described in [1].

[14] A labeled antibody comprising a full-length antibody and a labeled substance linked by a linker, wherein the linker is formed by a first reactive group of a crosslinking agent being bound to the full-length antibody and a second reactive group of the crosslinking agent being bound to the labeled substance, wherein the molecular weight of the labeled substance is 75,000 or more and 200,000 or less, and the molecular weight of the crosslinking agent is 270 or more and 1350 or less, as described in

[13] .

[15] A labeled antibody comprising a full-length antibody and a labeling substance linked by a linker, wherein the linker is formed by a first reactive group of a crosslinking agent being bound to the full-length antibody and a second reactive group of the crosslinking agent being bound to the labeling substance, wherein the molecular weight of the labeling substance is 10,000 or more, and the molecular weight of the crosslinking agent is 350 or more and 750 or less, as described in [1].

[16] The labeled antibody according to any one of

[13] to

[15] , wherein the full-length antibody is overdigested by treatment with a proteolytic enzyme.

[17] The labeled antibody according to any one of

[13] to

[16] , wherein the full-length antibody is an IgG2 subclass antibody.

[18] The labeled antibody according to any one of

[13] to

[17] , wherein the labeling substance is an enzyme.

[19] The labeled antibody according to any one of

[13] to

[18] , wherein the first reactive group of the crosslinking agent that binds to the full-length antibody is a group having a maleimide skeleton.

[20] The labeled antibody according to any one of

[13] to

[19] , wherein the second reactive group of the crosslinking agent that binds to the labeling substance is an N-hydroxyester or a sulfo-N-hydroxyester.

[21] The labeled antibody according to any one of

[13] to

[20] , wherein the linker is an alkylene glycol unit.

[22] The labeled antibody according to

[21] , wherein the alkylene glycol unit is an ethylene glycol unit.

[23] The labeled antibody according to

[21] or

[22] , wherein the linker contains two or more but no more than twelve alkylene glycol units.

[24] An immunoassay reagent comprising the labeled antibody according to any one of

[13] to

[23] .

[25] An immunoassay method comprising the step of mixing a sample with the immunoassay reagent according to

[24] and measuring by chemiluminescence.

[26] A method for producing a labeled antibody according to

[12] , comprising the steps of: introducing a sulfhydryl group into a full-length antibody; reacting a second reactive group of a crosslinking agent with a labeling substance to bind the crosslinking agent and the labeling substance; and reacting the sulfhydryl group with a first reactive group of the crosslinking agent to bind the crosslinking agent and the full-length antibody, thereby obtaining a labeled antibody in which the full-length antibody and the labeling substance are bound via a linker formed by the crosslinking agent, wherein the molecular weight of the labeling substance is 75,000 or more and 200,000 or less, and the molecular weight of the crosslinking agent is 270 or more and 1350 or less.

[27] A method for producing a labeled antibody according to

[12] , comprising the steps of: introducing a sulfhydryl group into a full-length antibody; reacting a second reactive group of a crosslinking agent with a labeling substance to bind the crosslinking agent and the labeling substance; and reacting the sulfhydryl group with a first reactive group of the crosslinking agent to bind the crosslinking agent and the full-length antibody, thereby obtaining a labeled antibody in which the full-length antibody and the labeling substance are bound via a linker formed by the crosslinking agent, wherein the molecular weight of the labeling substance is 10,000 or more, and the molecular weight of the crosslinking agent is 350 or more and 750 or less.

[0014] According to the above embodiment, it is possible to provide a labeled antibody containing an unfragmented full-length antibody, an immunological assay reagent containing the labeled antibody, and an immunological assay method, in which the nonspecific reaction between the labeled antibody and an insoluble carrier (e.g., beads described later) without the presence of a target substance is suppressed.

[0015] Figure 1 is a schematic diagram showing a labeled antibody in one aspect of the present invention. Figure 2 is a schematic diagram showing a full-length antibody in one aspect of the present invention. Figure 3 is a schematic diagram showing an immunological measurement method in one aspect of the present invention.

[0016] The embodiments will be described in detail below with reference to the drawings.

[0017] In this invention, the terms "react" and "bind" are used synonymously to describe the reactivity between an antibody and an antigen.

[0018] In this invention, "target substance" and "antigen" are used synonymously.

[0019] When a numerical range is written as, for example, "1-10" or "1 to 10," it means the range from 1 to 10, including the lower limit of 1 and the upper limit of 10. Furthermore, the upper and lower limits of a numerical range can be combined in any way. In addition, the numerical ranges for each physical property, composition, and measurement process can be combined in any way.

[0020] <Labeled Antibody> The labeled antibody in this embodiment is a labeled antibody in which a full-length antibody and a labeling substance are linked by a linker. The linker is formed when the first reactive group of the crosslinking agent is bound to the full-length antibody, and the second reactive group of the crosslinking agent is bound to the labeling substance. The molecular weight of the labeling substance is 10,000 or more, and the molecular weight of the crosslinking agent is 270 or more and 1350 or less.

[0021] The labeled antibody of this embodiment will be described with reference to Figure 1. Figure 1 is a schematic diagram showing a labeled antibody in one aspect of this embodiment. The labeled antibody 1 consists of a full-length antibody 10 and a labeling substance 20 linked by a linker 30.

[0022] The labeled antibody 1 preferably has 1 to 5 molecules of labeling substance 20 bound to 1 molecule of full-length antibody 10, and more preferably 1 to 3 molecules of labeling substance 20 bound to 1 molecule of full-length antibody 10. The composition of the labeled antibody 1 can be estimated from the molecular weight of the labeled antibody 1 and the molecular weights of the full-length antibody 10 and the labeling substance 20, respectively. The molecular weights of the full-length antibody 10 and the labeling substance 20 can be determined by liquid chromatography analysis, using standard samples with known molecular weights as a reference.

[0023] Figure 2 is a schematic diagram showing the full-length antibody 10 of this embodiment. In this specification, the term "full-length antibody 10" is used in the same sense as the term "full-length antibody" used in the academic field of immunology. That is, the full-length antibody 10 means an antibody that includes a Fab region 101, an Fc region 102, and a hinge region 103 connecting them. The Fab region 101 includes an antigen-binding site 104, and a sulfhydryl group 105 is introduced into the Fc region 102. Therefore, antibodies that have undergone digestion by proteolytic enzymes or treatment with reducing agents do not qualify as full-length antibodies.

[0024] In this embodiment, the labeled antibody 1 does not require the preparation of an antibody fragment of F(ab')2 or F(ab'), and the full-length antibody 10 can be used. The class of the full-length antibody 10 is not particularly limited and may be any one class selected from the group consisting of IgG, IgM, IgA, IgD, and IgE, but the IgG class is preferred. Any subclass of the antibody class may be used as the antibody subclass, but an antibody of the IgG2a or IgG2b subclass is more preferred.

[0025] Furthermore, the full-length antibody used in this embodiment is preferably a full-length antibody from which it is difficult to obtain an F(ab')2 antibody fragment by a digestion reaction using proteolytic enzymes.

[0026] The full-length antibody of this embodiment is used without proteolytic enzyme treatment or reducing agent treatment. Therefore, a type of full-length antibody that is prone to overdigestion by proteolytic enzyme treatment or reducing agent treatment can be used as the full-length antibody of this embodiment. Examples of full-length antibodies prone to overdigestion include those in which digestion fragments with a molecular weight smaller than F(ab')2 appear when digested with a proteolytic enzyme. Pepsin is preferred as the proteolytic enzyme. In other words, the full-length antibody of this embodiment is preferably a full-length antibody that is prone to overdigestion by pepsin. The conditions for digesting the full-length antibody with a proteolytic enzyme can be general conditions that maintain the activity of the digestive enzyme. In the case of pepsin, examples include 37°C, pH 3, and an enzyme reaction time of 60 minutes. Methods for confirming whether digestion fragments with a molecular weight smaller than F(ab')2 appear include, for example, gel filtration chromatography, ion exchange chromatography, and known protein separation methods such as SDS-PAGE. Furthermore, the content of digested fragments can be determined by known methods such as ultraviolet absorbance, BCA method, BRADFORD method, LOWRY method, and comparison of the intensity of stained bands on SDS-PAGE. Note that if digestion does not proceed as intended and over-digestion occurs, numerous disordered digested fragments will be generated. When a sample containing such digested fragments is separated by gel filtration chromatography, a broad peak consisting of two or more peaks, or three or more overlapping peaks, may be detected in the range of 12.4 to 124 kDa.

[0027] Conventional labeled antibodies often use the F(ab') fraction. The F(ab') fraction is obtained by reducing the disulfide bonds of the antibody's heavy chain, and therefore sulfhydryl groups are present in a free state. The antibody and the labeling substance can be bound via these free sulfhydryl groups. On the other hand, since the full-length antibody 10 does not have free sulfhydryl groups, it is necessary to introduce sulfhydryl groups in order to bind the labeling substance 20 to the full-length antibody 10. The introduction of sulfhydryl groups 105 into the full-length antibody 10 can be carried out using a sulfhydryl group introduction reagent. As the sulfhydryl group introduction reagent, one or more reagents selected from the group consisting of succinimidyl acetylthioacetate (SATA) reagent, succinimidyl acetylthiopropionate (SATP) reagent, N-succinimidyl S-acetyl (thiotetraethylene glycol), and 2-iminothiolane hydrochloride are preferred.

[0028] The molecular weight of the labeling substance 20 is 10,000 or more, preferably 20,000 or more. If the molecular weight of the labeling substance 20 is above the lower limit, it is possible to prevent nonspecific factors from binding to the Fc region of the full-length antibody. The molecular weight of the labeling substance 20 is 10,000 or more, preferably 10,000 to 200,000, more preferably 40,000 to 200,000, and even more preferably 75,000 to 200,000.

[0029] There are no particular limitations on the types of labeling substances 20, and examples include enzymes, fluorescent substances, chemiluminescent substances, avidin, gold colloid particles, and colored latex.

[0030] Some crosslinking agents, whose diagrams are not shown, contain a primary reactive group, a secondary reactive group, and an intermediate structural component (also called a spacer arm) connecting the primary and secondary reactive groups within a single molecule.

[0031] The lower limit of the molecular weight of the crosslinking agent is 270 or more, preferably 300 or more, and more preferably 350 or more. The upper limit of the molecular weight of the crosslinking agent is 1350 or less, preferably 900 or less, more preferably 800 or less, even more preferably 750 or less, particularly preferably 700 or less, and most preferably 650 or less. The molecular weight of the crosslinking agent is 270 or more and 1350 or less, preferably 300 or more and 900 or less, more preferably 300 or more and 800 or less, even more preferably 300 or more and 700 or less, particularly preferably 350 or more and 750 or less, and particularly preferably 300 or more and 650 or less. When the molecular weight of the crosslinking agent is below the upper limit, the gap between the full-length antibody 10 and the labeling substance 20 becomes smaller, and the binding of non-specific factors contained in the biological sample to the Fc region of the full-length antibody 10 can be suppressed. When the molecular weight of the crosslinking agent is above the lower limit, sufficient measurement sensitivity can be obtained. As a result, even with labeled antibody 1 containing unfragmented full-length antibody 10, it is possible to suppress nonspecific reactions between labeled antibody 1 and substances that cause nonspecific reactions without the involvement of a target substance. The molecular weight of the crosslinking agent can be calculated from the types and number of elements that make up the crosslinking agent, using the atomic weights of the elements that constitute the crosslinking agent. Alternatively, it can be determined by mass spectrometry based on standard samples with known molecular weights.

[0032] Examples of crosslinking agents include MBS Crosslinker (m-Maleimidobenzol-N-hydroxysuccinimide ester) represented by chemical formula (1), e.g., manufactured by ProteoChem, product number: c1114; Sulfo-MBS Crosslinker (m-Maleimidobenzol-N-hydroxysulfosuccinimide ester) represented by chemical formula (2), e.g., manufactured by ProteoChem, product number: c1115; and SMCC represented by chemical formula (3). Crosslinker (Succinimimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, e.g., manufactured by ProteoChem, product number: c1108), represented by chemical formula (4) Sulfo-SMCC Crosslinker (Sulfosuccinimimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate, e.g., manufactured by ProteoChem, product number: c1109), represented by chemical formula (5) EMCS Crosslinker (N-(e-Maleimidocaproyloxy)succinimilide Examples include ester (e.g., manufactured by ProteoChem, product number: c1123), and those having a structure derived from NHS-PEG-maleimide represented by chemical formula (6).

[0033] In NHS-PEG-maleimide, represented by chemical formula (6), n in the structure indicates the number of ethylene glycol units. An example of a crosslinking agent containing two ethylene glycol units is NHS-PEG2-maleimide (e.g., manufactured by Thermo Fisher Scientific, catalog number: 22102). An example of a crosslinking agent containing six ethylene glycol units is NHS-PEG6-maleimide (e.g., manufactured by Thermo Fisher Scientific, catalog number: 22105).

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] As the first reactive group that binds to the full-length antibody 10 of the cross-linking agent, there is no particular limitation as long as it can bind to the sulfhydryl group 105, and examples include groups having a maleimide skeleton (for example, those in which the hydrogen atom of the imide group of the maleimide group has been removed), pyridyldithiol, and iodoacetic acid.

[0041] As the second reactive group that binds to the labeling substance 20 of the cross-linking agent, there is no particular limitation as long as it can bind to the labeling substance 20, and examples include N-hydroxy esters and groups having a succinimide skeleton (for example, those in which the hydrogen atom of the hydroxyl group of N-hydroxysulfosuccinimide has been removed or those in which the hydroxyl group has been substituted with an ester group).

[0042] The linker 30 is formed from the first reactive group after binding to the full-length antibody 10 and the second reactive group after binding to the labeling substance 20 among the cross-linking agents. Further, the intermediate structure portion may be included between the first reactive group and the second reactive group. There is no particular limitation on the type of the linker 30, and examples include a linker containing an alkylene glycol unit and a linker containing a cyclohexane structure.

[0043] The alkylene glycol unit is a divalent linking group represented by the chemical formula "-(R1-O)n-". R1 represents an alkylene group, and there is no particular limitation on the number of carbon atoms. For example, 1 to 4 are preferable, and 2 to 3 are more preferable. Within these preferable ranges, the length of the linker 30 becomes suitable, and the influence of the non-specific reaction substance 60 during the use of the labeled antibody 1 can be further reduced. Also, the cross-linking reaction between the labeling substance 20 and the full-length antibody 10 becomes easier.

[0044] In the alkylene glycol unit “-(R1 - O)n-”, n represents the number of repetitions of the alkylene glycol unit. It is preferably 2 or more and 12 or less, more preferably 2 or more and 8 or less, and even more preferably 2 or more and 6 or less.

[0045] When both ends of the alkylene glycol unit are respectively bonded to the first reactive group and the second reactive group, the alkylene glycol unit forms a form that constitutes all of the intermediate structure portion of the crosslinking agent.

[0046] <Method for producing labeled antibody> The method for producing the labeled antibody of this embodiment includes a step of introducing a sulfhydryl group into a full-length antibody, a step of binding a second reactive group of a crosslinking agent to a labeling substance, and a step of binding the sulfhydryl group to the first reactive group of the crosslinking agent to obtain a labeled antibody 1. The molecular weight of the labeling substance is 100,000 or more, and the molecular weight of the crosslinking agent is 270 or more and 1350 or less. The preferred range of the molecular weight of the crosslinking agent is the same as the above-mentioned range.

[0047] In the production method of this embodiment, first, a sulfhydryl group 105 is introduced into the full-length antibody 10. When introducing the sulfhydryl group 105 into the full-length antibody 10, there is no particular limitation on the molar ratio of the full-length antibody 10 to the sulfhydryl group-introducing reagent, but it is preferable to use an excessive amount of the sulfhydryl group-introducing reagent. The molar ratio represented by the full-length antibody 10: sulfhydryl group-introducing reagent is preferably 1:2 to 1:20, more preferably 1:2 to 1:10, and even more preferably 1:2.5 to 1:5.

[0048] Next, the second reactive group of the crosslinking agent is bound to the labeling substance 20. When binding the labeling substance 20 to the crosslinking agent, there is no particular limitation on the molar ratio of the labeling substance 20 to the crosslinking agent. For example, the molar ratio represented by the labeling substance 20: crosslinking agent is preferably 1:2 to 1:15, more preferably 1:2.5 to 1:10, and even more preferably 1:2.5 to 1:7.5.

[0049] Next, the sulfhydryl group 105 of the full-length antibody 10 is bound to the first reactive group of the crosslinking agent to which the labeling substance 20 is bound. There are no particular limitations on the molar ratio of the full-length antibody 10 to the crosslinking agent to which the labeling substance 20 is bound when the full-length antibody 10 and the crosslinking agent to which the labeling substance 20 is bound are bound, but it is more preferable to use an excess amount of the crosslinking agent to which the labeling substance 20 is bound.

[0050] In the above description, the crosslinking agent and the labeling substance 20 are bound together first, and then the full-length antibody 10 is bound to them. However, the present invention is not limited to this. The crosslinking agent and the full-length antibody 10 may be bound together first, and then the labeling substance 20 may be bound together, or the crosslinking agent, the full-length antibody 10, and the labeling substance 20 may be bound together simultaneously.

[0051] As a method for reacting the full-length antibody 10, the labeling substance 20, and the crosslinking agent, physical adsorption, glutaraldehyde method, maleimide method, pyridyl disulfide method, or periodic acid method, etc., which are available to those skilled in the art, can be used. When an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP) is used as the labeling substance, the enzyme activity can be measured using the enzyme's specific substrate. For example, if the enzyme is HRP, O-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine (TMB) can be used. In the case of ALP, the enzyme activity can be measured using p-nitrophenyl phosphate. When biotin is used as the labeling substance, the first antibody can be labeled with biotin and reacted with avidin or streptavidin labeled with an enzyme, dye, or fluorescent label (preferably HRP).

[0052] By the above steps, a labeled antibody 1 according to one embodiment of the present invention is obtained.

[0053] <Immunological assay reagent> An immunoassay reagent is a reagent used to detect the immunological binding of a biochemical target substance generated in a biological sample to a substance that has a specific affinity for that target substance. The immunoassay reagent in this embodiment includes labeled antibody 1.

[0054] The immunoassay reagent may have any configuration that includes the labeled antibody 1 in one embodiment of the present invention. For example, one configuration may include a first reagent containing a diluent of the biological sample, a second reagent containing antibody-conjugated beads, and a third reagent containing the labeled antibody 1. Furthermore, it may also contain other components such as a washing solution.

[0055] The immunoassay reagent of this embodiment contains a labeled antibody 1 in which a full-length antibody 10 and a labeled substance 20 are bound with a crosslinking agent having a molecular weight of 270 to 1350. Therefore, nonspecific reactions between the full-length antibody 10 and substances in the biological sample that cause nonspecific reactions can be suppressed. As a result, the nonspecific reaction of the labeled antibody 1 containing the full-length antibody 10 to the antibody-conjugated beads 3 without the target substance 2 can be suppressed.

[0056] <Immunological Measurement Method> The immunological measurement method according to this embodiment comprises the steps of mixing a biological sample with the above-described immunological measurement reagent and measuring by chemiluminescence. Specifically, the mixing of the biological sample and the immunological measurement reagent may consist of a first contact step of bringing the biological sample into contact with antibody-conjugated beads, and a second contact step of bringing the antigen-antibody-conjugated beads obtained in the first contact step into contact with the labeled antibody of the present invention.

[0057] Figure 3 is a schematic diagram illustrating the mixing of a biological sample and an immunoassay reagent in an immunoassay method according to one embodiment of the present invention. In this embodiment, the biological sample contains a nonspecific reactant 60 that can react nonspecifically with the antibody.

[0058] The biological sample is not particularly limited as long as it is a sample derived from a living organism, but liquid samples such as body fluids are preferred, at least one selected from the group consisting of blood, plasma, and serum is more preferred, and any one selected from the group consisting of blood, plasma, and serum is even more preferred. The living organism is preferably a human or mammal (for example, a mouse, guinea pig, rat, monkey, dog, cat, hamster, horse, cow, and pig), and more preferably a human. The biological sample may be collected or prepared at the time of carrying out the present invention, or it may be collected or prepared in advance and stored.

[0059] Examples of nonspecific reactants 60 that are contained in a biological sample and can react nonspecifically with the antibody include rheumatoid factor (RF), bilirubin, and lipemia. In one embodiment of the present invention, the labeled antibody 1 has a short linker 30, which can suppress the reaction of nonspecific reactants 60 to the full-length antibody 10. One reason for this is that the labeling substance 20 is already bound to the full-length antibody 10 via the linker 30. It is thought that the short length of the linker 30 narrows the space between the labeling substance 20 and the full-length antibody 10, preventing the nonspecific reactants 60 from coming into contact with the surface of the full-length antibody 10.

[0060] The antibody-conjugated bead 3 is a carrier bead 41 on which an antigen-conjugated antibody 42 is immobilized. The carrier bead 41 is not particularly limited as long as it is possible to immobilize the antigen-conjugated antibody 42 that can bind to the target substance 2 on the bead surface, and examples include magnetic beads, Sepharose beads, and agarose beads.

[0061] The mixing of the biological sample and the immunoassay reagent begins with a first contact step. In this first contact step, the biological sample is brought into contact with the antibody-conjugated beads 3. This causes the target substance 2 in the biological sample to bind to the antigen-conjugated antibody 42 on the antibody-conjugated beads 3, thereby obtaining antigen-antibody-conjugated beads. Next, unbound substances in the supernatant of the biological sample containing the antigen-antibody-conjugated beads are removed, and the antigen-antibody-conjugated beads are washed. After that, the antigen-antibody-conjugated beads are diluted with a solvent.

[0062] There are no particular limitations on the temperature and reaction time when contacting the antibody-conjugated beads 3 with the biological sample. The temperature is preferably 0 to 50°C, more preferably 10 to 40°C, and even more preferably 20 to 40°C. The reaction time is preferably 0.5 to 60 mins, and more preferably 1 to 40 mins.

[0063] In the second contact step, the antigen-antibody conjugated beads obtained in the first contact step are brought into contact with the labeled antibody 1. This causes the target substance 2, which is bound to the antibody conjugated beads 3, to bind to the labeled antibody 1. The resulting complex of labeled antibody 1, target substance 2, and antibody conjugated beads 3 may hereafter be referred to as the "measurement sample," which is X in Figure 3. Next, the supernatant of the solution containing the measurement sample X is removed, and the unbound substances are washed away. After that, the measurement sample is diluted with a solvent.

[0064] There are no particular limitations on the temperature and reaction time when contacting the antigen-antibody conjugated beads with the labeled antibody 1. The temperature is preferably 0 to 50°C, more preferably 10 to 40°C, and even more preferably 20 to 40°C. The reaction time is preferably 0.5 to 60 mins, and more preferably 1 to 40 mins.

[0065] There are no particular limitations on the solvent used to dilute the antigen-antibody binding beads and the measurement sample, but it is preferable that it contains a buffer solution.

[0066] In the above description, a second contact step is performed in which the labeled antibody 1 is brought into contact with the biological sample after the first contact step in which the biological sample is brought into contact with the antibody-conjugated beads 3. However, the present invention is not limited to this. The first contact step may be performed after the second contact step. Specifically, the biological sample may be brought into contact with the labeled antibody 1, and then into contact with the antibody-conjugated beads 3. Furthermore, the first and second contact steps may be performed simultaneously. Specifically, the biological sample, the labeled antibody 1, and the antibody-conjugated beads 3 may be brought into contact at the same time.

[0067] In the embodiment shown in Figure 3, the antigen-binding antibody 42 is immobilized on the carrier beads 41, and then the target substance 2 is attached to the beads by a direct method, but the present invention is not limited to this. For example, an indirect method may be applied in which the antigen-binding antibody 42 and the target substance 2 are attached, and then the carrier beads 41 are attached.

[0068] The sample X obtained by mixing a biological sample with an immunoassay reagent is measured by chemiluminescence.

[0069] Next, we will explain the measurement process using chemiluminescence. Specific examples of chemiluminescence measurement methods include chemiluminescent enzyme immunoassay (CLEIA), enzyme immunoassay (ELISA), and fluorescence enzyme immunoassay (FLEIA).

[0070] The CLEIA method is a method in which an antigen is reacted with an immobilized antibody, then an enzyme-labeled antibody is reacted with the antigen, and a chemiluminescent substrate is added to measure the luminescence intensity. When the immunological measurement method in this embodiment is the CLEIA method, an insoluble carrier 3 such as carrier beads 41 can be used, which includes a full-length antibody 10 that is a first antibody that binds to the target substance 2, a linker 30, an enzyme (e.g., alkaline phostapase, etc.) as the labeling substance 20, and an antigen-binding antibody 42 that binds to the target substance 2.

[0071] Furthermore, the labeled antibody 1 and the insoluble carrier 3 may each be contained in the reaction buffer. In addition, as the chemiluminescent substrate, an enzyme substrate solution (for example, a solution containing 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2'-(5-chlorotricyclo[3.3.1.13.7]decane])-4-yl]-1-phenyl phosphate disodium (CDP-Star®)) can be used.

[0072] In the CLEIA method, a biological sample is brought into contact with antibody-conjugated beads 3, then labeled antibody 1 is brought into contact with the antigen-antibody-conjugated beads, and an enzyme substrate is added to the resulting sample X to be measured and reacted. Subsequently, the amount of luminescence is measured to analyze the target substance. In one configuration, after reacting the antibody-conjugated beads 3 with the biological sample, unbound substances can be removed and the antigen-antibody-conjugated beads can be washed.

[0073] ELISA is a method for detecting antigens or antibodies, which are substances to be measured, contained in a biological sample, by capturing them using antibodies or antigens against the target substance and then detecting them using an enzymatic reaction. When the immunological measurement method in this embodiment is ELISA, a labeled antibody 1 containing a full-length antibody 10 that binds to the target substance 2, a linker 30, and an enzyme (such as HRP or ALP) as a labeling substance 20, and a carrier bead 41 on which an antigen-binding antibody 42 that binds to the target substance 2 is immobilized, can be used as antibody-binding beads 3.

[0074] In the ELISA method, for example, a biological sample is brought into contact with antibody-conjugated beads 3 and incubated. Then, unbound substances are removed and the antigen-antibody-conjugated beads are washed. Next, labeled antibody 1 is added and incubated, and a substrate for the enzyme is added to induce color development. By measuring the color development, the target substance 2 can be analyzed.

[0075] The measurement principle of the FLEIA method is the same as that of the ELISA method, except that a fluorescent substrate is added instead of an enzyme substrate and the fluorescence intensity is measured.

[0076] The immunological measurement method of this embodiment uses a labeled antibody 1 in which a full-length antibody 10 and a labeled substance 20 are conjugated with a crosslinking agent having a molecular weight of 270 to 1350. Therefore, nonspecific reactions between the full-length antibody 10 and nonspecific reactive substances 60 contained in the biological sample can be suppressed. As a result, the nonspecific reaction of the labeled antibody 1 containing the full-length antibody 10 to the antibody-conjugated beads 3 without the target substance 2 can be suppressed.

[0077] The present invention will be described in more detail below with reference to experimental examples, but the present invention is not limited to the experimental examples described later.

[0078] ALP (manufactured by Roche, catalog number: 03535452103, molecular weight: 111,000) was used as the labeling substance 20. The crosslinking agents used are NHS-PEG2-maleimide (manufactured by Thermo Fisher Scientific, catalog number: 22102, molecular weight: 425.39), NHS-PEG6-maleimide (manufactured by Thermo Fisher Scientific, catalog number: 22105, molecular weight: 601.6), NHS-PEG24-maleimide (manufactured by Thermo Fisher Scientific, catalog number: 22114, molecular weight: 1394.55), and N-β-maleimidopropyl-oxysuccinimide ester (BMPS, Thermo Fisher A reagent from Scientific, Inc. (Catalog No. 22298, Molecular Weight: 266.21) was used. SATA (Thermo Fisher Scientific, Inc., Catalog No. 26102) was used as the sulfhydryl group introduction reagent. A NAP10 column (Cytiva, Inc., Catalog No. 17085401) was used as the desalting column. A Superdex 200 Increase 10 / 300 GL (Cytiva, Inc., Catalog No. 28990944) was used as the gel filtration chromatography column. Interference Check RF Plus (Sysmex, Inc., Catalog No. 79181) was used as the nonspecific factor.

[0079] <Experimental Example 1> [Preparation of Antibody-Conjugated Beads 3] Antibody-conjugated beads 3 were prepared according to the method described below. 150 mg of magnetic particles (3 w / v%, average particle size 3 μm, resin core particles coated with a magnetic layer and having epoxy groups on the surface) manufactured by Sekisui Medical Co., Ltd. were separated, and the solvent was removed by magnetizing the magnetic particles using a magnet. Next, 5 mL of borate buffer (0.3 mol / L borate, pH 8.5) was added to the magnetic particles and mixed and stirred to wash the magnetic particles. After that, the solvent was removed again by magnetizing using a magnet. 5 mL of MU-3 antibody (manufactured by Sekisui Medical Co., Ltd., prepared based on international publication WO2021 / 107105), which had been prepared to 1 mg / mL in borate buffer, was added to the magnetic particles and rotated and stirred at 25°C for 72 hours using a rotary rotor. Subsequently, the magnetic particles were washed three times with PBS-1 (140 mM K2HPO4, 10 mM KH2PO4, 150 mM NaCl, pH 7.8). After antibody immobilization, the particles were collected and washed, and then blocked using 5 mL of several solutions containing BSA, etc. This was used as the antibody-conjugated bead stock solution 3. Note that MU-3 antibody is another name for anti-PIVKA-II monoclonal antibody.

[0080] [Preparation of Labeled Antibody 1] Labeled antibody 1 was prepared according to the method described below. The anti-PIVKA-II monoclonal antibody was prepared by Sekisui Medical Co., Ltd. with reference to International Publication WO2021 / 107105.

[0081] 1 mg of full-length antibody 10 (anti-PIVKA-II monoclonal antibody) and 2.3 μL of SATA (54.5 mM), a sulfhydryl group introduction reagent, were mixed and stirred by rotation at 25°C for 30 minutes. 320 μL of hydroxylamine solution (1 mM Hydroxlamine, 25 μM 2Na (EDTA-2Na), PBS (5 mM KH2PO4, 15 mM Na2HPO4, 150 mM NaCl), pH 7.5) was added to the full-length antibody 10-SATA reaction mixture and stirred by rotation at 25°C for 120 minutes. The resulting solution was replaced using a NAP10 column to obtain the full-length antibody 10-SATA solution. PBS-1 buffer was used for the replacement solution.

[0082] Next, maleimide was introduced into ALP using the crosslinking agent NHS-PEG2-maleimide. After maleimide introduction, unreacted crosslinking agent was removed using a NAP10 column to obtain the ALP solution. PBS-1 buffer was used as the replacement solution.

[0083] The full-length antibody 10-SATA solution obtained above was mixed with the ALP solution and stirred overnight at 25°C. The mixture was then purified by gel filtration using Superdex® 200 Increase 10 / 300 GL to obtain labeled antibody 1 (hereinafter referred to as labeled antibody 1 [ALP-PEG2]). In addition, labeled antibody 1 was prepared by the same method except that NHS-PEG6-maleimide or NHS-PEG24-maleimide was used as the crosslinking agent (hereinafter referred to as labeled antibody 1 [ALP-PEG6] or labeled antibody 1 [ALP-PEG24]).

[0084] [Preparation of Measurement Samples] 1 mL of human serum was added to an Interference Check RF Plus RF vial to prepare RF 500 IU / mL. 1 mL of human serum was added to an Interference Check RF Plus blank vial to prepare RF 0 IU / mL. RF 500 IU / mL and RF 0 IU / mL were mixed in a 1:1 ratio to prepare RF 250 IU / mL. RF 250 IU / mL and RF 0 IU / mL were mixed in a 1:1 ratio to prepare RF 125 IU / mL. Note that the RF vial contains a known amount of RF, while the blank vial does not contain RF. The serum used contained the PIVKA-II antigen to be detected by the labeled antibody obtained above. The IU / mL in the names of the measurement samples prepared above indicates the concentration of RF contained in the sample.

[0085] [Preparation of Measurement Reagent] (R1 Reagent) The R1 reagent was prepared with the following composition: 50 mM HEPES (pH 7.8) 0.1% BSA 500 μg / mL Mouse-derived antibody

[0086] (R2 Reagent) The R2 reagent was prepared with the following composition: 25 mM Tris-HCl (pH 7.8), 0.5% BSA, 0.9 mg / mL antibody-conjugated beads (stock solution).

[0087] (R3 Reagent) The R3 reagent base solution was prepared with the following composition: 50 mM MES (pH 6.4) 0.5% BSA

[0088] R3-[ALP-PEG2] was prepared by adding 1 μg / mL labeled antibody 1 [ALP-PEG2] as labeled antibody 1 to the above R3 reagent base solution. R3-[ALP-PEG6] was prepared by adding 1 μg / mL labeled antibody 1 [ALP-PEG6] as labeled antibody 1 to the above R3 reagent base solution. R3-[ALP-PEG24] was prepared by adding 1 μg / mL labeled antibody 1 [ALP-PEG24] as labeled antibody 1 to the above R3 reagent base solution.

[0089] [Measurement Method] A fully automated clinical laboratory system, STACIA (manufactured by PHC Corporation, hereinafter referred to as "the device"), was used for measurement. The measurement samples (RF0 IU / mL, RF125 IU / mL, RF250 IU / mL, RF500 IU / mL) were dispensed into Hitachi sample cups and set in the device. Reagents R1, R2, and R3 were dispensed into reagent bottles and set in the designated positions on the device, after which the measurement was performed automatically with the desired parameters.

[0090] In the measurement, first, 20 μL of the sample was mixed with 100 μL of reagent R1 and incubated at 37°C for 3.5 minutes. Then, 25 μL of reagent R2 was added and incubated at 37°C for 2.7 minutes (first contact step).

[0091] Next, the sample was washed with BF washing solution, and then 100 μL of R3 reagent was added and incubated at 37°C for 4.4 minutes (second contact step).

[0092] Next, a second BF wash was performed to obtain sample X. Subsequently, substrate solution was added to sample X, incubated for 2.7 minutes, and then the luminescence was measured. PIVKA-II antigen (company-formulated product, not for sale) was prepared and measured at various concentrations, and the concentration of the sample was quantified from the created calibration curve.

[0093] [Example 1] The measurement samples (RF0 IU / mL, RF125 IU / mL, RF250 IU / mL, RF500 IU / mL) were measured using the measurement method described above. The R3 reagent used was R3-[ALP-PEG2].

[0094] [Example 2] The same procedure as in Example 1 was followed, except that the R3 reagent was replaced with R3-[ALP-PEG6].

[0095] [Comparative Example 1] The procedure was carried out in the same manner as in Example 1, except that the R3 reagent was R3-[ALP-PEG24].

[0096] [Results] Each sample was measured three times, and the average value was calculated. Furthermore, the relative values ​​(%) of RF125IU / mL, RF250IU / mL, or RF500IU / mL relative to the average value of RF0IU / mL were calculated. If the relative value was between 80% and 120%, it was determined that there was no interference by RF. The results are shown in Table 1.

[0097]

[0098] The relative values ​​for Examples 1 and 2 ranged from 93.3% to 116.7%, indicating no interference by RF and suggesting that the nonspecific reaction between labeled antibody 1 and nonspecific factor 60 was suppressed. When a sample containing a high concentration of the nonspecific factor RF, "RF 500 IU / mL," was measured, the relative values ​​for Examples 1 and 2 were 120% or less, indicating suppression of the nonspecific reaction. In contrast, the relative value for Comparative Example 1 exceeded 120%, indicating interference by the nonspecific reaction. This demonstrates that the labeled antibody of the present invention can suppress the nonspecific reaction.

[0099] <Experimental Example 2> [Preparation of Antibody-Conjugated Beads 3] Antibody-conjugated beads 3 were prepared according to the method described below. 150 mg of magnetic particles (average particle diameter 3 μm, resin core particles coated with a magnetic layer and having epoxy groups on the surface) manufactured by Sekisui Medical Co., Ltd. were separated, and the solvent was removed by magnetizing the magnetic particles using a magnet. Next, 5 mL of borate buffer (0.3 mol / L borate, pH 8.5) was added to the magnetic particles and mixed and stirred to wash the magnetic particles. After that, the solvent was removed again by magnetizing using a magnet. 5 mL of MU-3 antibody (manufactured by Sekisui Medical Co., Ltd., prepared based on international publication WO2021 / 107105), which had been prepared to 1 mg / mL in borate buffer, was added to the magnetic particles and rotated and stirred at 25°C for 72 hours using a rotary rotor. Subsequently, the magnetic particles were washed three times with PBS-1 (140 mM K2HPO4, 10 mM KH2PO4, 150 mM NaCl, pH 7.8). After antibody immobilization, the particles were collected and washed, and then blocked using 5 mL of several solutions containing BSA, etc. This was used as the antibody-conjugated bead stock solution 3. Note that MU-3 antibody is another name for anti-PIVKA-II monoclonal antibody.

[0100] [Preparation of Labeled Antibody 1] Labeled antibody 1 was prepared according to the method described below. The anti-PIVKA-II monoclonal antibody was prepared by Sekisui Medical Co., Ltd. with reference to International Publication WO2021 / 107105.

[0101] 1 mg of full-length antibody 10 (anti-PIVKA-II monoclonal antibody) was mixed with 2.3 μL of 54.5 mM SATA and stirred by rotation at 25°C for 30 minutes. 320 μL of hydroxylamine solution (1 mM Hydroxlamine, 25 μM 2Na (EDTA-2Na), PBS (5 mM KH2PO4, 15 mM Na2HPO4, 150 mM NaCl), pH 7.5) was added to the full-length antibody 10-SATA reaction mixture and stirred by rotation at 25°C for 120 minutes. The reaction solution was replaced using a NAP10 column to obtain the full-length antibody 10-SATA solution. PBS-1 buffer was used as the replacement solution.

[0102] Next, maleimide was introduced into ALP using NHS-PEG2-maleimide as the crosslinking agent. After introducing maleimide, unreacted crosslinking agent was removed using a NAP10 column to obtain ALP solution. PBS-1 buffer was used as the replacement solution. The full-length antibody 10-SATA solution and the ALP solution obtained above were mixed and rotated and stirred overnight at 25°C. Subsequently, the mixture was purified by gel filtration using Superdex® 200 Increase 10 / 300 GL to obtain labeled antibody 1 (hereinafter referred to as labeled antibody 1 [ALP-PEG2]). Labeled antibody 1 was also prepared by the same method except that NHS-PEG6-maleimide or BMPS was used as the crosslinking agent (hereinafter referred to as labeled antibody 1 [ALP-PEG6] or labeled antibody 1 [ALP-BMPS]).

[0103] [Preparation of Measurement Samples] PIVKA-II antigen (company-formulated product, not for sale) was added to a calibrator solution (50 mM Tris, 150 mM NaCl, 3% BSA) at the following concentrations to prepare the measurement samples. PIVKA-II Sample 1: No antigen added PIVKA-II Sample 2: Equivalent to 10 mAU / mL

[0104] [Preparation of Measurement Reagent] (R1 Reagent) The R1 reagent was prepared with the following composition: 50 mM HEPES (pH 7.8) 0.1% BSA 500 μg / mL Mouse-derived antibody

[0105] (R2 Reagent) The R2 reagent was prepared with the following composition: 25 mM Tris-HCl (pH 7.8), 0.5% BSA, 0.9 mg / mL antibody-conjugated beads (stock solution).

[0106] (R3 Reagent) The R3 reagent base solution was prepared with the following composition: 50 mM MES (pH 6.4) 0.5% BSA

[0107] R3-[ALP-PEG2] was prepared by adding 1 μg / mL labeled antibody 1 [ALP-PEG2] as labeled antibody 1 to the above R3 reagent base solution. R3-[ALP-PEG6] was prepared by adding 1 μg / mL labeled antibody 1 [ALP-PEG6] as labeled antibody 1 to the above R3 reagent base solution. R3-[ALP-BMPS] was prepared by adding 1 μg / mL labeled antibody 1 [ALP-PEG24] as labeled antibody 1 to the above R3 reagent base solution.

[0108] [Measurement Method] A fully automated clinical laboratory system STACIA (manufactured by PHC Corporation, hereinafter referred to as "the device") was used for measurement. The sample to be measured was dispensed into a Hitachi sample cup and set in the device. Reagents R1, R2, and R3 were dispensed into reagent bottles and set in the designated positions in the device, and then the measurement was performed automatically with arbitrary parameters. In the measurement, first, 100 μL of reagent R1 was mixed with 20 μL of the sample to be measured and incubated at 37°C for 3.5 minutes. Then, 25 μL of reagent R2 was added and incubated at 37°C for 2.7 minutes (first contact step). After that, washing was performed with BF washing solution, followed by the addition of 100 μL of reagent R3 and incubated at 37°C for 4.4 minutes (second contact step). After that, a second BF washing was performed to obtain sample X. Subsequently, substrate solution was added to sample X, incubated for 2.7 minutes, and then the amount of luminescence was measured.

[0109] [Example 3] The sample was measured using the measurement method described above. The R3 reagent used was R3-[ALP-PEG2].

[0110] [Example 4] The same procedure as in Example 3 was followed, except that the R3 reagent was R3-[ALP-PEG6].

[0111] [Comparative Example 2] The procedure was carried out in the same manner as in Example 3, except that the R3 reagent was R3-[ALP-BMPS].

[0112] [Results] Each sample was measured three times, and the average measurement count was calculated. Here, the measurement count indicates the intensity of the signal obtained; a higher measurement count indicates that a large amount of labeled antibody is bound to the antibody-conjugated beads. The results are shown in Table 2.

[0113]

[0114] When PIVKA-II sample 1, which did not contain the target substance PIVKA-II antigen, was measured, the average count values ​​for Examples 3 and 4 were 396 and 364, respectively. The average count value for Comparative Example 2 was 1237, which was higher than that of Examples 3 and 4. Furthermore, when PIVKA-II sample 2, which contained 10 mAU / mL of the target substance PIVKA-II antigen, was measured, the average count values ​​for Examples 3 and 4 were 785 and 696, respectively, while the average count value for Comparative Example 2 was 1442. These experimental examples demonstrate that using the labeled antibody of the present invention results in a lower count for samples that do not contain the target substance, and a difference in sensitivity can be obtained compared to samples that contain the target substance.

[0115] According to the present invention, it is possible to provide a labeled antibody containing an unfragmented full-length antibody in which the nonspecific reaction between the insoluble carrier and the labeled antibody without the involvement of a target substance is suppressed, as well as an immunological assay reagent and an immunological assay method containing the labeled antibody.

[0116] X...Measurement sample, 1...Labeled antibody, 2...Target substance, 3...Antibody-conjugated beads, 10...Full-length antibody, 20...Labeled substance, 30...Linker, 60...Non-specific adsorbent, 101...Fab region, 102...Fc region, 103...Hinge region, 104...Antigen-binding site, 105...Sulfhydryl group, 41...Carrier beads, 42...Antigen-conjugated antibody

Claims

1. A labeled antibody comprising a full-length antibody and a labeled substance linked by a linker, wherein the linker is formed by a first reactive group of a crosslinking agent being bound to the full-length antibody and a second reactive group of the crosslinking agent being bound to the labeled substance, the molecular weight of the labeled substance being 10,000 or more, and the molecular weight of the crosslinking agent being 270 or more and 1350 or less.

2. The labeled antibody according to claim 1, wherein the full-length antibody is overdigested by treatment with a proteolytic enzyme.

3. The labeled antibody according to claim 1, wherein the full-length antibody is an IgG2 subclass antibody.

4. The labeled antibody according to claim 1, wherein the labeling substance is an enzyme.

5. The labeled antibody according to claim 1, wherein the first reactive group of the crosslinking agent that binds to the full-length antibody is a group having a maleimide skeleton.

6. The labeled antibody according to claim 1, wherein the second reactive group of the crosslinking agent that binds to the labeled substance comprises an N-hydroxyester or a sulfo-N-hydroxyester.

7. The labeled antibody according to claim 1, wherein the linker comprises an alkylene glycol unit.

8. The labeled antibody according to claim 7, wherein the alkylene glycol unit is an ethylene glycol unit.

9. The labeled antibody according to claim 7, wherein the number of alkylene glycol units contained in the linker is 2 or more and 12 or less.

10. An immunoassay reagent comprising a labeled antibody according to any one of claims 1 to 9.

11. An immunoassay method comprising the step of mixing a sample with the immunoassay reagent described in claim 10 and measuring by chemiluminescence.

12. A method for producing a labeled antibody, comprising the steps of: introducing a sulfhydryl group into a full-length antibody; reacting a second reactive group of a crosslinking agent with a labeling substance to bind the crosslinking agent and the labeling substance; and reacting the sulfhydryl group with a first reactive group of the crosslinking agent to bind the crosslinking agent and the full-length antibody, thereby obtaining a labeled antibody in which the full-length antibody and the labeling substance are bound via a linker formed by the crosslinking agent, wherein the molecular weight of the labeling substance is 10,000 or more, and the molecular weight of the crosslinking agent is 270 or more and 1350 or less.

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