GPC3 measurement method and kit therefor

Abstract

Disclosed are: a GPC3 measurement method in which an increase in the count value of a negative sample is reduced; and an immunoassay kit. This GPC3 measurement method is a method for measuring GPC3 by means of immunoassay, the method including subjecting, to an antigen-antibody reaction, GPC3 contained in a liquid sample separated from a living body and a labeled anti-GPC3 antibody labeled with a labeling substance or an antigen-binding fragment thereof, and comprises bringing a solution containing a labeled anti-GPC3 antibody or an antigen-binding fragment thereof and a polyoxyethylene-polyoxypropylene block copolymer into contact with GPC3 in a sample.

Description

GPC3 measurement method and kit therefor

[0001] The present invention relates to a method for measuring GPC3 and a kit therefor.

[0002] Glypican-3 (GPC3) is a protein expressed during malignant transformation of hepatocytes and exists in an immobilized state on the cell membrane of hepatocytes. GPC3 is cleaved by furin between arginine 358 and serine 359 (furin cleavage site), and is separated into an N-terminal subunit extending from the N-terminus to arginine 358 and a C-terminal subunit extending from serine 359 to the C-terminus. The N-terminal and C-terminal subunits are linked by a single intramolecular disulfide bond.

[0003] Since GPC3 is also a protein that is specifically expressed in cancers such as hepatocellular carcinoma, efforts are being made to develop a method that targets GPC3 and is useful for testing cancer patients. For example, Patent Document 1 proposes a method for testing cancer patients by measuring GPC3. Furthermore, Patent Document 2 proposes an immunoassay method for GPC3 that uses two different antibodies that bind to different epitopes present in the N-terminal region of GPC3. Patent Document 3 proposes a method for measuring GPC3 in which a sample containing GPC3 is reduced.

[0004] International Publication No. WO 2004 / 038420 International Publication No. WO 2015 / 097928 International Publication No. WO 2022 / 154119

[0005] The present inventors have found that when measuring GPC3 in a liquid sample isolated from a living body by immunoassay, there is a problem that the count value of a liquid sample (negative sample) derived from a healthy subject increases. Therefore, an object of the present invention is to provide a method for measuring GPC3 and an immunoassay kit that reduce the increase in the count value of a negative sample.

[0006] As a result of extensive research, the present inventors have found that in a method for measuring GPC3 by immunoassay, the increase in the count value of negative samples can be reduced by adding a polyoxyethylene-polyoxypropylene block copolymer to a solution containing a labeled antibody, and have thus completed the present invention.

[0007] That is, the present invention provides the following: (1) A method for measuring GPC3 by immunoassay, comprising the step of causing an antigen-antibody reaction between GPC3 contained in a liquid sample separated from a living body and a labeled anti-GPC3 antibody or an antigen-binding fragment thereof that has been labeled with a labeling substance, the method comprising contacting GPC3 in the sample with a solution containing the labeled anti-GPC3 antibody or antigen-binding fragment thereof and a polyoxyethylene-polyoxypropylene block copolymer. (2) The method according to (1), wherein the immunoassay is a sandwich method. (3) The method according to (1) or (2), wherein the ethylene oxide content in the polyoxyethylene-polyoxypropylene block copolymer is 70% or more and 90% or less. (4) The method according to (3), wherein the number of moles of ethylene oxide added in the polyoxyethylene-polyoxypropylene block copolymer is 150 or more and 300 or less. (5) The method according to any one of (1) to (4), wherein the concentration of the polyoxyethylene-polyoxypropylene block copolymer in the solution containing the labeled anti-GPC3 antibody or antigen-binding fragment thereof and the polyoxyethylene-polyoxypropylene block copolymer is 0.01 w / v% to 6.0 w / v%. (6) The method according to any one of (1) to (5), wherein the labeling substance is an enzyme. (7) A kit for measuring GPC3, comprising a solution containing an anti-GPC3 antibody labeled with a labeling substance and a polyoxyethylene-polyoxypropylene block copolymer. (8) Use of a solution containing an anti-GPC3 antibody labeled with a labeling substance and a polyoxyethylene-polyoxypropylene block copolymer for measuring GPC3. (9) Use of a solution containing an anti-GPC3 antibody labeled with a labeling substance and a polyoxyethylene-polyoxypropylene block copolymer for producing a kit for measuring GPC3.

[0008] According to the present invention, it is possible to provide a method for measuring GPC3 and a kit for measuring the same, which suppresses an increase in the count value of negative samples.

[0009] The method of the present invention is a method for measuring GPC3 by mixing GPC3 contained in a liquid sample separated from a living body with a solution containing an anti-GPC3 antibody labeled with a labeling substance and a polyoxyethylene-polyoxypropylene block copolymer.

[0010] As shown in the Examples, when measuring GPC3 in a liquid sample isolated from a living body by immunoassay, it was found that the count value of a liquid sample (negative sample) derived from a healthy subject increases when the measurement reagent is stored for a predetermined period. By including a polyoxyethylene-polyoxypropylene block copolymer in the solution containing the labeled anti-GPC3 antibody, the increase in the count value of the negative sample can be suppressed.

[0011] In the present invention, the GPC3 contained in the liquid sample separated from a living body is mainly soluble GPC3 secreted from GPC3-expressing cells. GPC3 derived from any subject can be used as the GPC3. Such subjects are preferably mammals, including primates such as humans, monkeys, and chimpanzees; rodents such as mice and rats, and dodonts such as rabbits; ungulates such as cattle, pigs, goats, horses, and sheep; and carnivores such as dogs, cats, and ferrets. GPC3 is widely conserved in animals, and its amino acid sequence is particularly highly conserved among mammals. From the perspective of clinical application, the subject is preferably human. Therefore, the GPC3 is preferably human GPC3.

[0012] The human GPC3 is preferably an N-terminal fragment generated by cleavage between the arginine residue at position 358 and the serine residue at position 359 in a 580-amino acid-containing human soluble GPC3 protein (accession number: P51654.1), or a soluble full-length GPC3 released by cleavage of the GPI anchor at the C-terminus of the human GPC3 protein. Examples of soluble full-length GPC3 include a GPC3 fragment in which the N-terminal fragment and the C-terminal fragment generated by cleavage between the arginine residue at position 358 and the serine residue at position 359 in the human GPC3 protein are linked to each other via a disulfide bond. More specifically, such human soluble GPC-3 is (a) an N-terminal fragment or variant thereof consisting of amino acid residues at positions 1 to 358 in the amino acid sequence of SEQ ID NO: 1, (b) a soluble full-length GPC-3 in which an N-terminal fragment or variant thereof consisting of amino acid residues at positions 1 to 358 in the amino acid sequence of SEQ ID NO: 1 and a soluble C-terminal fragment or variant thereof consisting of amino acid residues at positions 359 to 560 in the amino acid sequence of SEQ ID NO: 1 are linked via a disulfide bond, or (c) a variant thereof that may naturally occur between races and / or individuals. Such variants are (a) an N-terminal fragment or variant thereof, or (b) a soluble full-length GPC-3 or variant thereof, into which one or more amino acid residue mutations (e.g., substitution, insertion, deletion) that may naturally occur between races and / or individuals have been introduced. The number of amino acid residue mutations in such a mutant may be, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, and particularly preferably 1, 2, 3, 4, or 5.

[0013] In the present invention, the liquid sample separated from a living body may be any liquid sample. Examples of liquid samples include body fluids separated from a subject (e.g., blood, lymph, urine, milk, saliva, and tears), tissue extracts separated from a subject, lavage fluids collected from a subject (e.g., obtained by washing mucosal tissues such as bronchi), and liquids obtained from cell cultures derived from a subject, as well as liquid samples obtained by processing (e.g., fractionating) these. From the viewpoint of easily obtaining a liquid sample rich in GPC3, the liquid sample is preferably a blood specimen (e.g., whole blood, serum, plasma).

[0014] In the present invention, a negative sample refers to a liquid sample derived from a subject who is not affected by the target disease. A subject who is not affected by the target disease is, for example, a subject who is not affected by liver disease, preferably a subject who is not affected by liver cancer, more preferably a subject who is not affected by hepatocellular carcinoma. A negative GPC3 sample is, for example, a sample in which the GPC3 concentration is 100.0 pg / mL or less, preferably 95.0 pg / mL or less. The GPC3 concentration in the sample may be measured as the N-terminal subunit from the N-terminus to the 358th arginine, the C-terminal subunit from the 359th serine to the C-terminus, or GPC3 in which the N-terminal subunit and the C-terminal subunit are linked.

[0015] In the present invention, an anti-GPC3 antibody refers to an antibody that can specifically bind to GPC3. Examples of the antibody include polyclonal antibodies and monoclonal antibodies. From the viewpoint of reproducibly obtaining antibodies with uniform reaction specificity, the antibody is preferably a monoclonal antibody. The antibody can also be identified by its isotype. Examples of such isotypes include IgG, IgM, IgA, IgD, IgE, and IgY. Preferably, the antibody is IgG, IgM, or IgA, more preferably IgG or IgM, and even more preferably IgG. The antibody may also be a chimeric antibody, a humanized antibody, or a human antibody. The antibody may also be an antigen-binding fragment. The antigen-binding fragment may be any antibody fragment, or a low molecular weight compound to which a variable region fragment is linked, as long as it maintains the binding ability of the original antibody to the corresponding antigen (antigen-antibody reactivity). Specific examples include, but are not limited to, Fab, Fab', F(ab')2, scFv, and VHH antibodies. Furthermore, "specifically bind" means having the ability to specifically bind, and having the ability to undergo an antigen-antibody reaction. The term "recognize" is sometimes used interchangeably with "specifically bind."

[0016] Anti-GPC3 monoclonal or polyclonal antibodies can be produced by conventional methods using GPC3 (preferably soluble GPC3 protein or a peptide thereof) as an immunogen. For example, in the case of polyclonal antibodies, the antibodies of the present invention can be obtained by immunizing an animal with the immunogen and producing antibodies from the antiserum by conventional methods. Alternatively, in the case of monoclonal antibodies, the antibodies of the present invention can be obtained by the following method. First, antibody-producing cells, such as spleen cells, from an animal immunized with the immunogen are fused with tumor cells, such as myeloma cells, using a fusing agent such as polyethylene glycol to produce hybridomas. The hybridomas are then selected using a selective medium such as HAT medium, monoclonalized by an appropriate method such as limiting dilution, and cultured. The culture supernatant is then analyzed by an appropriate immunoassay such as enzyme immunoassay, and clones producing the desired anti-GPC3 antibody are selected to obtain the antibodies of the present invention. These monoclonal antibodies can be produced by known methods, such as those of Köhler and Milstein (Nature 256, 495-497 (1975)) and Scherrer (Nature 285, 446-450 (1980)). The production of antigen-binding fragments of antibodies is also known. For example, Fab and F(ab')2 can be obtained by treating antibodies with protease enzymes such as papain and pepsin, as is well known. Methods for producing scFv (single chain fragment of variable region, single-chain antibody) and low molecular weight compounds are also well known, and they can be produced according to known methods. Furthermore, anti-GPC3 antibodies and antigen-binding fragments thereof are commercially available, so commercially available products can also be used. In the following description prior to the examples, unless otherwise clear from the context, the term "antibody" means "antibody or antigen-binding fragment thereof."

[0017] In the present invention, examples of the labeling substance include enzymes, fluorescent proteins, fluorescent substances, luminescent substances, radioactive substances, and dyes. The labeling substance is preferably an enzyme. Examples of the enzyme include alkaline phosphatase (ALP), peroxidase (POD (e.g., HRP)), luciferase, and β-galactosidase. Examples of the fluorescent protein include green fluorescent protein and red fluorescent protein. Examples of the fluorescent substance include fluorescein, fluorescein isothiocyanate, and rhodamine. Examples of the luminescent substance include acridinium derivatives. Examples of the radioactive substance include ３ H. １４ C. ３２ P. ３５ S. １２５ Examples of radioactive elements include I.

[0018] In the present invention, a labeled anti-GPC3 antibody can be produced by any conventionally known method or a method based thereon, and the labeling substance and the anti-GPC3 antibody can be bound directly or indirectly.

[0019] Examples of direct binding methods include adding an active group to a labeling substance, or using a labeling substance having such an active group and binding the antibody via a covalent bond via the active group. When preparing an enzyme-labeled anti-GPC3 antibody, for example, an enzyme into which an active ester group has been introduced can be mixed with an anti-GPC3 antibody to prepare an enzyme-labeled anti-GPC3 antibody. Alternatively, an enzyme-labeled anti-GPC3 antibody can be prepared by reacting an enzyme into which a maleimide group has been introduced with a reduced anti-GPC3 antibody.

[0020] As a method for indirect immobilization, for example, the labeling substance and the anti-GPC3 antibody can be indirectly bound via a pair of affinity substances. Examples of the pair of affinity substances include a combination of biotin and avidin (or streptavidin). For example, by binding biotin to the anti-GPC3 antibody and avidin (or streptavidin) to the labeling substance, and then mixing the biotinylated antibody with the avidinated labeling substance, the antibody and the labeling substance can be indirectly bound.

[0021] In the present invention, the polyoxyethylene-polyoxypropylene block copolymer is a triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene, and HO(C ２ H ４ O) a-(C ３ H ６ O) b-(C ２ H ４ O)cH (Formula 1) (wherein a, b, and c represent any integer). Polyoxyethylene: (C ２ H ４ O) a or (C ２ H ４ O)c is an abbreviation for EO, ​​POE, etc., and is abbreviation for polyoxypropylene: (C ３ H ６ O)b may be represented by abbreviations such as PO, POP, PPG, etc., and similar notations may be used in this specification. The polyoxyethylene polyoxypropylene block copolymer is, for example, a poloxamer, and commercially available products can be used. There are several ways to represent the components of polyoxyethylene polyoxypropylene block copolymers, and although identity and similarity can be confirmed between each notation, the numerical values ​​between each notation may not completely match even for commercially available products with the same name. However, these are specific to polymerizable polymers and would be naturally understood by those skilled in the art.

[0022] For example, examples of polyoxyethylene-polyoxypropylene block copolymers include poloxamer 388, poloxamer 407, poloxamer 188, poloxamer 217, poloxamer 237, poloxamer 238, poloxamer 288, and poloxamer 108. Commercially available polyoxyethylene-polyoxypropylene block copolymers include, for example, Pluronic F108 (average molecular weight 14,600, EO content about 80%, number of EO units (a+c) 264), Pluronic F127 (average molecular weight 12,600, EO content about 70%, number of EO units (a+c) 202), Pluronic F68 (average molecular weight 8,400, EO content about 80%, number of EO units (a+c) 160), Pluronic F77 (average molecular weight 6,600, EO content about 70%, number of EO units (a+c) 104), Pluronic F87 (average molecular weight 7,700, EO content about 70%, number of EO units (a+c) 128), Pluronic Examples of such compounds include compounds from the PLURONIC (registered trademark) series, such as Pluronic F88 (average molecular weight 11,400, EO content about 80%, number of EO units (a+c) 207), Pluronic F98 (average molecular weight 13,000, EO content about 80%, number of EO units (a+c) 236), and Pluronic F38 (average molecular weight 4,700, EO content about 80%, number of EO units (a+c) 85).

[0023] The ethylene oxide content (EO content, w / w %) in the polyoxyethylene-polyoxypropylene block copolymer is 60% or more and 95% or less, preferably 70% or more and 90% or less.

[0024] The number average molecular weight of the polyoxyethylene-polyoxypropylene block copolymer is, for example, 4,000 to 20,000, preferably 8,000 to 17,000, and more preferably 9,000 to 15,000.

[0025] The average number of moles of ethylene oxide added (the number of EO units (a+c)) in the polyoxyethylene-polyoxypropylene block copolymer is 100 or more and 300 or less, preferably 150 or more and 300 or less, and more preferably 200 or more and 300 or less.

[0026] The polyoxyethylene-polyoxypropylene block copolymer is preferably poloxamer 388, poloxamer 407, or poloxamer 188, and more preferably poloxamer 388 or poloxamer 407.

[0027] In the present invention, the concentration of the polyoxyethylene-polyoxypropylene block copolymer in the solution containing the labeled anti-GPC3 antibody and the polyoxyethylene-polyoxypropylene block copolymer is not particularly limited, as long as it is a concentration that can suppress an increase in the count value of negative samples when measuring GPC3. Such a concentration is, for example, 0.001 to 10.0 w / v%, preferably 0.01 to 6.0 w / v%, more preferably 0.01 to 4.0 w / v%, and even more preferably 0.25 to 4.0 w / v%.

[0028] In the present invention, the concentration of the labeled anti-GPC3 antibody in the solution containing the labeled anti-GPC3 antibody and the polyoxyethylene-polyoxypropylene block copolymer is not particularly limited as long as it is a concentration at which GPC3 can be measured. Such a concentration is, for example, 0.75 to 3.0 μg / mL, preferably 1.2 to 1.8 μg / mL.

[0029] In the present invention, the liquid serving as the base of the solution containing the labeled anti-GPC3 antibody and polyoxyethylene-polyoxypropylene block copolymer is not particularly limited, as long as it is an aqueous liquid. For example, water (e.g., distilled water, sterilized water, sterilized distilled water, pure water) and buffer solutions can be used. Examples of buffer solutions include phosphate buffer, MES buffer, citrate buffer, Tris buffer, carbonate buffer, HEPES buffer, and MOPS buffer. The pH of the buffer solution may be 5.0 to 9.0 (preferably 6.0 to 8.0) as long as it can stably retain the labeling substance and anti-GPC3 antibody. The solution is preferably a buffer solution.

[0030] In the present invention, the solution containing the labeled anti-GPC3 antibody and the polyoxyethylene-polyoxypropylene block copolymer may further contain a water-soluble polymer such as BSA, a sugar such as sucrose, or a surfactant such as a nonionic surfactant, a cationic surfactant, an anionic surfactant, or an amphoteric surfactant. As the surfactant, a nonionic surfactant is preferred.

[0031] The method for measuring GPC3 of the present invention is a method for measuring GPC3 by immunoassay using one or more antibodies against GPC3.

[0032] In the present invention, the immunoassay method is preferably any sandwich immunoassay method using two or more antibodies against GPC3, including a solution containing a labeled anti-GPC3 antibody (labeled antibody) and an anti-GPC3 antibody (solid-phase antibody) for capturing GPC3 on a solid phase. Sandwich immunoassay methods are well known in the art and can be performed according to well-known sandwich immunoassay methods. In addition, in the present invention, the term "measurement" includes detection, quantification, and semi-quantification.

[0033] In a sandwich immunoassay (two-step sandwich immunoassay), for example, an anti-GPC3 antibody immobilized on a solid phase or an anti-GPC3 antibody (solid-phase antibody) that can be immobilized on a solid phase in a reaction step is first mixed with GPC3 in a liquid sample, and an antigen-antibody reaction between the solid-phase antibody and GPC3 occurs, forming an immune complex between GPC3 and the solid-phase antibody (primary reaction). If the solid-phase antibody is an anti-GPC3 antibody that can be immobilized on a solid phase, the solid-phase antibody may be immobilized on the solid phase after or simultaneously with the primary reaction. Then, B / F separation is performed. Next, the GPC3 bound to the solid-phase antibody is mixed with a solution containing a labeled anti-GPC3 antibody (labeled antibody), and an antigen-antibody reaction between GPC3 and the labeled antibody occurs, forming an immune complex between the solid-phase antibody, GPC3, and the labeled antibody (secondary reaction). Next, B / F separation is performed, and the GPC3 in the liquid sample can be measured by measuring the signal derived from the label of the labeled antibody bound to the GPC3 captured on the solid phase (an immune complex of the solid-phase antibody, GPC3, and labeled antibody). Washing may be performed after B / F separation. Furthermore, the signal derived from the label of the immune complex of the solid-phase antibody, GPC3, and labeled antibody may be measured while the immune complex is still formed, or the portion containing the label may be dissociated from the immune complex, and then the signal derived from the dissociated label may be measured.

[0034] Alternatively, first, GPC3 in a liquid sample is mixed with a solution containing a labeled antibody to cause an antigen-antibody reaction between GPC3 and the labeled antibody, forming an immune complex between GPC3 and the labeled antibody (primary reaction), followed by an antigen-antibody reaction between a solid-phase antibody and GPC3 to form an immune complex between the solid-phase antibody, GPC3, and the labeled antibody (secondary reaction), followed by B / F separation. Next, GPC3 in the liquid sample can be measured by measuring the signal derived from the labeling substance of the labeled antibody bound to the GPC3 bound to the solid phase. Washing may be performed after B / F separation.

[0035] It is also possible to simultaneously react GPC3 in a liquid sample, a solid-phase antibody (anti-GPC3 antibody), and a labeled anti-GPC3 antibody (one-step sandwich immunoassay).

[0036] The primary reaction and secondary reaction can be carried out under any suitable conditions, as long as they allow an antigen-antibody reaction to occur and an immune complex to be formed. For example, the primary reaction and secondary reaction can be carried out at 4 to 45°C, preferably 20 to 37°C, at a pH of about 5.0 to 9.0, preferably 6.0 to 8.0, for about 1 minute to 12 hours, preferably 3 minutes to 1 hour.

[0037] In the present invention, immunoassays can be classified into, for example, chemiluminescent enzyme immunoassays (CLEIA), chemiluminescent immunoassays (CLIA), enzyme immunoassays (EIA), radioimmunoassays (RIA), and fluorescent immunoassays (FIA), depending on the type of label used. Chemiluminescent enzyme immunoassays are immunoassays that use an enzyme as a label and a substrate that generates a chemiluminescent compound (e.g., AMPPD when alkaline phosphatase is used as the enzyme) as a substrate. Enzyme immunoassays are immunoassays that use an enzyme as a label (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase, etc.). The substrate for each enzyme is a compound that can be quantified by absorbance measurement or the like. For example, in the case of peroxidase, 1,2-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine (TMB) is used; in the case of alkaline phosphatase, p-nitrophenyl phosphate (pNPP) is used; in the case of β-galactosidase, MG: 4-methylumbelliferyl galactoside or NG: nitrophenyl galactoside is used; and in the case of luciferase, luciferin is used. Radioimmunoassay (RIA) is a method that uses a radioactive substance as a label. Fluorescent immunoassay (FIA) is a method that uses a fluorescent substance or fluorescent protein as a label. Immunoassays using these labels are well known in the art and are described, for example, in US8039223B and US20150309016A1.

[0038] In the above-described immunoassay methods, the solid phase can be, for example, particles (e.g., sepharose beads, agarose beads, magnetic particles), supports (e.g., membranes), or containers (e.g., plates such as plastic plates, tubes, microchannels), but from the viewpoint of automation and shortening the time required, magnetic particles are preferred. The antibody can be immobilized on the solid phase either directly or indirectly, and any conventional method can be used. For example, immobilization can be achieved by physical adsorption, covalent bonding, methods using affinity substances (e.g., biotin, streptavidin), or ionic bonding.

[0039] The present invention also provides a measurement kit for measuring GPC3 in a liquid sample using the above-described measurement method of the present invention.

[0040] The measurement kit of the present invention comprises a solution containing the above-mentioned labeled anti-GPC3 antibody and a polyoxyethylene-polyoxypropylene block copolymer.

[0041] The assay kit of the present invention may be a sandwich immunoassay assay kit and may further comprise a solid-phase antibody. Preferred conditions for the labeling substance, anti-GPC3 antibody, polyoxyethylene-polyoxypropylene block copolymer, solution, assay method, GPC3, etc. are the same as those described above.

[0042] The GPC3 assay kit of the present invention may further contain other reagents that are generally contained in assay kits, such as an appropriate substrate solution depending on the labeling substance used, and a washing solution.

[0043] The present invention will be specifically described below based on examples. However, the present invention is not limited to the following examples. In the following, "%" indicates weight / volume percentage (w / v %: g / 100 mL) unless otherwise specified.

[0044] Reference Example 1 Preparation of anti-GPC3 antibody A immobilized particle solution Anti-GPC3 antibody A, which recognizes the N-terminal subunit of GPC3, was added to magnetic particles in 10 mM MES buffer (pH 5.0), and the mixture was incubated at 25°C for 1 hour with gentle stirring. After the reaction, the magnetic particles were collected with a magnet and washed with a washing solution to obtain anti-GPC3 antibody A immobilized particles. During measurement, the anti-GPC3 antibody A immobilized particles were diluted with a particle diluent (50 mM Tris buffer, 1 mM EDTA2Na, 0.1% NaN ３ The antibody was suspended in a final concentration of 0.05% in a 2.0% BSA solution (pH 7.2) to prepare a solution of immobilized anti-GPC3 antibody A particles.

[0045] Reference Example 2 Preparation of alkaline phosphatase-labeled anti-GPC3 antibody Anti-GPC3 antibody B, which recognizes the N-terminal subunit of GPC3, was labeled with alkaline phosphatase (ALP) according to a standard method, and purified by column chromatography using Superdex 200 16 / 600 (trade name, manufactured by GE) in a purification buffer (100 mM MES buffer, 150 mM NaCl, 0.1% NaN ３ The main peak was isolated and purified at a flow rate of 1.0 mL / min in a 1000 ml / min aqueous solution of 100% ethanol (pH 6.8) to obtain ALP-labeled anti-GPC3 antibody B.

[0046] ALP-labeled anti-GPC3 antibody C was prepared in the same manner as above, except that anti-GPC3 antibody C, which recognizes the C-terminal subunit of GPC3, was used instead of anti-GPC3 antibody B. This was designated as ALP-labeled anti-GPC3 antibody C.

[0047] Example 1 Preparation of a solution containing alkaline phosphatase-labeled anti-GPC3 antibody As a control, ALP-labeled anti-GPC3 antibody B was diluted with a labeled antibody diluent (50 mM MES buffer, 150 mM NaCl, 0.3 mM ZnCl) containing no nonionic polymer. ２ , 1 mM MgCl ２, 0.1% Proclin 300, 2.0% BSA, pH 6.8) to prepare ALP-labeled anti-GPC3 antibody solution B (condition 1). Furthermore, each antibody was suspended in a labeled antibody diluent containing Pluronic F108, polyvinyl alcohol (PVA), or polyvinylpyrrolidone (PVP) at a final concentration of 1.00% (final labeled antibody concentration: 1.5 μg / mL) to prepare ALP-labeled anti-GPC3 antibody solution B (conditions 2 to 4).

[0048] ALP-labeled anti-GPC3 antibody C solution was prepared in the same manner as above, except that ALP-labeled anti-GPC3 antibody C was used instead of ALP-labeled anti-GPC3 antibody B (conditions 5 to 8).

[0049] Measurement of Negative Serum Samples Two negative serum samples (hereinafter referred to as negative serum samples) (purchased from ProMedDX) derived from healthy individuals were used as measurement subjects. Both had a GPC3 concentration of 95.0 pg / mL or less.

[0050] Negative serum samples were evaluated using two types of measurement systems: one was a measurement system combining anti-GPC3 antibody A immobilized particles with ALP-labeled anti-GPC3 antibody B (A-B measurement system), and the other was a measurement system combining anti-GPC3 antibody A immobilized particles with ALP-labeled anti-GPC3 antibody C (A-C measurement system).

[0051] The method for measuring the A-B measurement system using a two-step sandwich immunoassay (2-step mode) is as follows. 50 μL of anti-GPC3 antibody A solid-phase particle solution and 20 μL of sample were dispensed into a cuvette and mixed. This was then incubated at 37°C for 8 minutes, the particles in the cuvette were collected with a magnet, and the inside of the cuvette was washed with a cleaning solution (0.05% Tween (registered trademark) 20 / PBS). 50 μL of ALP-labeled anti-GPC3 antibody B solution was dispensed into the cuvette, stirred, and then incubated at 37°C for 8 minutes, the particles in the cuvette were collected with a magnet, and the inside of the cuvette was washed with a cleaning solution. Then, 200 μL of Lumipulse® substrate solution (Fujirebio) containing the chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) was dispensed into the cuvette, stirred, and incubated at 37°C for 4 minutes, after which the amount of luminescence (counts) was measured using a luminometer. Measurements were performed using a fully automated chemiluminescent enzyme immunoassay system (Lumipulse L2400 (Fujirebio)).

[0052] The method for measuring the AC measurement system in 2-step mode was carried out in the same manner as above, except that the ALP-labeled anti-GPC3 antibody solution C was used instead of the ALP-labeled anti-GPC3 antibody solution B.

[0053] Calculation of the Rate of Fluctuation (%) of Count Values ​​of Negative Serum Samples Due to Incubation The anti-GPC3 antibody A solid-phase particle solution was divided into two containers, and each was stored at 4°C and 37°C for three days. Similarly, the four ALP-labeled anti-GPC3 antibody B conditions (conditions 1 to 4) and the four ALP-labeled anti-GPC3 antibody C conditions (conditions 5 to 8) were each divided into two containers, and each was stored at 4°C and 37°C for three days. The anti-GPC3 antibody A solid-phase particle solution stored at 4°C was combined with the ALP-labeled anti-GPC3 antibody B solution stored at 4°C (conditions 1 to 4) and measured in the 2-step mode described above. The anti-GPC3 antibody A solid-phase particle solution stored at 37°C was combined with the ALP-labeled anti-GPC3 antibody B solution stored at 37°C (conditions 1 to 4) and measured in the 2-step mode described above (A-B). A solution of immobilized anti-GPC3 antibody A particles stored at 4°C was combined with an ALP-labeled anti-GPC3 antibody C solution stored at 4°C (conditions 5 to 8) and measured in a 2-step mode, and a solution of immobilized anti-GPC3 antibody A particles stored at 37°C was combined with an ALP-labeled anti-GPC3 antibody C solution stored at 37°C (conditions 5 to 8) and measured in a 2-step mode (A-C).

[0054] The fluctuation rate (%) of the count value of the negative serum specimen was calculated using (count value at 37°C) / (count value at 4°C)×100 (Equation 2).

[0055] The fluctuation rates (%) of the count values ​​measured using the A-B measurement system and the count values ​​of the negative serum samples are shown in Table 1, and the fluctuation rates (%) of the count values ​​measured using the A-C measurement system and the count values ​​of the negative serum samples are shown in Table 2.

[0056]

[0057]

[0058] The rate of variation (%) of count values ​​of negative serum samples is an index for evaluating the influence on measurement when the measurement reagent is kept warm for a predetermined period of time. The closer the rate of variation is to 100%, the more robust the reagent is, i.e., less affected by temperature.

[0059] Table 1 shows the results measured using the A-B assay system. When a label dilution solution containing no nonionic polymer was used, the count values ​​of negative serum samples significantly increased when stored at 37°C compared to when stored at 4°C (Table 1, condition 1). The variability of the count values ​​of negative serum samples was over 400%. This indicates that the temperature at which the assay reagent is stored affects the measurement of negative serum samples containing small amounts of GPC3. On the other hand, when a label dilution solution containing Pluronic F108 was used (Table 1, condition 2), the variability of negative serum samples was approximately 140%, demonstrating that the influence of temperature could be reduced. Furthermore, when a label dilution solution containing other nonionic polymers, PVA or PVP, was used (Table 1, conditions 3 and 4), the variability was approximately 350-400%, demonstrating that the condition using a label dilution solution containing Pluronic F108 best reduced the influence of temperature. Similarly, when a positive sample containing 100.0 pg / mL or more of GPC3 was measured under the same conditions as Condition 1, the fluctuation rate of the count value of the positive sample was within 100 to 110% (data not shown).

[0060] Table 2 shows the results of measurements using the AC measurement system. The results obtained using the AC measurement system were similar to those obtained using the AB measurement system.

[0061] From the above, it was suggested that, regardless of the type of anti-GPC3 antibody, when a label dilution solution containing Pluronic F108 is used, it becomes a highly robust reagent that is not affected by temperature.

[0062] Example 2 Preparation of a solution containing alkaline phosphatase-labeled anti-GPC3 antibody As a control, ALP-labeled anti-GPC3 antibody B was suspended in a labeled antibody diluent containing no nonionic polymer (final concentration of labeled antibody: 1.5 μg / mL) to prepare an ALP-labeled anti-GPC3 antibody B solution (condition 9).

[0063] To investigate the effective concentration of Pluronic F108, ALP-labeled anti-GPC3 antibody B was suspended in labeled antibody diluents containing Pluronic F108 at final concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1.0, 2.0, 4.0%, and 6.0% (final labeled antibody concentration: 1.5 μg / mL) (Conditions 10 to 18). To examine other Pluronic polymers, ALP-labeled anti-GPC3 antibody B was suspended in labeled antibody dilutions containing Pluronic F68 at final concentrations of 0.05, 0.25, and 1.00% (final labeled antibody concentration: 1.5 μg / mL) (conditions 19 to 21), and ALP-labeled anti-GPC3 antibody B was suspended in labeled antibody dilutions containing Pluronic F127 at final concentrations of 0.05, 0.25, and 1.00% (final labeled antibody concentration: 1.5 μg / mL) (conditions 22 to 24), to prepare ALP-labeled anti-GPC3 antibody B solutions.

[0064] <<Measurement of Negative Serum Samples (Step 2)>> Two negative serum samples similar to those in Example 1 were used as measurement subjects.

[0065] Calculation of the Percentage Fluctuation (%) of Count Values ​​of Negative Serum Samples Due to Incubation An anti-GPC3 antibody A solid-phase particle solution prepared by the same method as described in Example 1 was divided into three containers and stored at 4°C, 25°C, and 37°C for three days. The ALP-labeled anti-GPC3 antibody B solution under Condition 11 above was divided into three containers and stored at 4°C, 25°C, and 37°C for three days. The ALP-labeled anti-GPC3 antibody B solutions under Conditions 12 to 26 above, which used different labeled antibody dilutions, were each divided into two containers and stored at 4°C and 37°C for three days. The anti-GPC3 antibody A solid-phase particle solution stored at 4°C, 25°C, and 37°C and the ALP-labeled anti-GPC3 antibody B solution stored at 4°C, 25°C, and 37°C were combined at the same temperatures and measured in 2-step mode (Condition 9). Anti-GPC3 antibody A immobilized particle solution stored at 4°C and 37°C and ALP-labeled anti-GPC3 antibody B solution stored at 4°C and 37°C were combined at the same temperature and measured in 2-step mode (conditions 10 to 24).

[0066] The count values ​​and the rate of variation (%) of the count values ​​of the negative serum samples were calculated in the same manner as in Example 1. The results are shown in Table 3.

[0067]

[0068]

[0069] When a labeled antibody diluent containing 0.01% to 6.0% Pluronic F108 was used, the variation rate of negative serum samples was lower than when a labeled antibody diluent containing no nonionic polymer was used, demonstrating the effectiveness of reducing the effects of temperature (Table 3, conditions 10 to 18). Furthermore, when a labeled antibody diluent containing 0.25% to 4.0% Pluronic F108 was used, the variation rate of negative serum samples was less than 200%, demonstrating the effectiveness of reducing the effects of temperature (Table 3, conditions 13 to 17).

[0070] When a labeled antibody diluent containing 0.25% to 1.00% Pluronic F68 was used, the fluctuation rate of negative serum samples was lower than when a labeled antibody diluent containing no nonionic polymer was used, demonstrating the effectiveness of reducing the effects of temperature (Table 3, conditions 19 to 21).

[0071] When a labeled antibody diluent containing 0.05% to 1.00% Pluronic F127 was used, the fluctuation rate of negative serum samples was lower than when a labeled antibody diluent containing no nonionic polymer was used, demonstrating the effectiveness of reducing the effects of temperature (Table 3, conditions 22 to 24). The conditions containing a labeled antibody diluent containing Pluronic F127 showed the same effect as the conditions containing a labeled antibody diluent containing Pluronic F108.

Claims

1. A method for measuring GPC3 by immunoassay, comprising a step of causing an antigen-antibody reaction between GPC3 contained in a liquid sample separated from a living body and a labeled anti-GPC3 antibody or an antigen-binding fragment thereof that has been labeled with a labeling substance, the method comprising contacting the GPC3 in the sample with a solution containing the labeled anti-GPC3 antibody or an antigen-binding fragment thereof and a polyoxyethylene-polyoxypropylene block copolymer.

2. The method of claim 1, wherein said immunoassay is a sandwich method.

3. The method according to claim 1 or 2, wherein the ethylene oxide content in said polyoxyethylene-polyoxypropylene block copolymer is 70% or more and 90% or less.

4. The method according to claim 3, wherein the number of moles of ethylene oxide added in said polyoxyethylene-polyoxypropylene block copolymer is 150 or more and 300 or less.

5. The method according to claim 1 or 2, wherein the concentration of the polyoxyethylene-polyoxypropylene block copolymer in the solution containing the labeled anti-GPC3 antibody or antigen-binding fragment thereof and the polyoxyethylene-polyoxypropylene block copolymer is 0.01 w / v % to 6.0 w / v %.

6. The method according to claim 1 or 2, wherein the labeling substance is an enzyme.

7. A kit for measuring GPC3, comprising an anti-GPC3 antibody labeled with a labeling substance and a solution containing a polyoxyethylene-polyoxypropylene block copolymer.

8. Use of a solution containing an anti-GPC3 antibody labeled with a labeling substance and a polyoxyethylene-polyoxypropylene block copolymer for measuring GPC3.

9. Use of a solution containing an anti-GPC3 antibody labeled with a labeling substance and a polyoxyethylene-polyoxypropylene block copolymer for the manufacture of a kit for measuring GPC3.