Processing method for soluble GPC3-containing samples in immunoassays of soluble GPC3

Abstract

The present invention provides a method and reagent useful for testing for soluble GPC3. More specifically, the present invention provides (1), (2), and (3). (1) A processing method for specimens in soluble GPC3 immunoassays, that includes mixing a soluble GPC3-containing specimen with a reducing agent. (2) A soluble GPC3 immunoassay method that includes (a) and (b). (a) Mixing the soluble GPC3-containing specimen with a reducing agent; and (b) using at least one type of antibody against soluble GPC3 and measuring the amount of soluble GPC3 in the mixed liquid obtained in (a). (3) A soluble GPC3 immunoassay method that includes (a) and (b). (a) A reducing agent; and (b) at least one antibody against soluble GPC3.

Description

Technical Field

[0004] , , , , , , ,

[0001] The present invention relates to a method for treating a soluble GPC3-containing specimen in an immunoassay of soluble glypican-3 (GPC3), etc.

Background Art

[0002] GPC3 is a protein having a molecular weight of about 65 kDa belonging to the glypican family, which is a proteoglycan having a heparan sulfate chain and is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor present at its C-terminus. GPC3 is cleaved between the arginine residue at position 358 and the serine residue at position 359 by an enzyme called Furin in the Golgi apparatus to generate an N-terminal fragment (about 40 kDa) and a C-terminal fragment containing a GPI anchor (about 30 kDa). However, it has been confirmed that such two fragments are usually linked by a disulfide bond. Therefore, GPC3 is considered to be bound to the cell membrane via a GPI anchor in the form of a full-length protein containing two fragments linked by a disulfide bond.

[0003] GPC3 is also a protein that shows specific expression in cancers such as liver cancer, etc., and thus, the development of a method useful for the examination of cancer patients targeting GPC3 has been explored. For example, in Patent Document 1, an examination method for cancer patients by measuring soluble GPC3 has been proposed. Also, in Patent Document 2, a measurement method for GPC3 using two different antibodies that bind to different epitopes present in the N-terminal region of GPC3 has been proposed.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

[0005] The object of the present invention is to provide a method and reagents useful for testing soluble GPC3. [Means for solving the problem]

[0006] As a result of diligent research, the inventors have found that in an immunoassay of soluble GPC3, treating the sample with a reducing agent can improve the accuracy of distinguishing between positive and negative samples. Furthermore, the inventors have found that in an immunoassay of soluble GPC3, treating the sample with both a reducing agent and a surfactant can improve the accuracy of distinguishing between positive and negative samples and reduce matrix effects, thus completing the present invention.

[0007] In other words, the present invention is as follows: [1] A method for processing a soluble GPC3-containing sample in an immunoassay for soluble GPC3, comprising mixing the soluble GPC3-containing sample with a reducing agent. [2] The method of [1], wherein the reducing agent is used at a final concentration of 0.05 to 1,000 mM. [3] The method of [1] or [2], further comprising mixing the soluble GPC3-containing sample with a surfactant. [4] The method of [3], wherein the surfactant is used at a final concentration of 0.005 to 10% by weight. [5] Any of the methods [1] to [4], wherein the immunoassay is a sandwich immunoassay using two or more different antibodies against two or more different epitopes in soluble GPC3. [6] Any of the methods [1] to [5], wherein the soluble GPC3-containing sample is a blood sample. [7] A soluble GPC3-containing sample is obtained from a subject suffering from cancer by any of the methods described in [1] to [6]. [8] The cancer is liver cancer, by the method in [7]. [9] Immunoassay method for soluble GPC3 comprising (a) and (b) below: (a) Mixing a soluble GPC3-containing sample with a reducing agent; and (b) The amount of soluble GPC3 in the mixture obtained in (a) is measured using one or more antibodies against soluble GPC3.

[10] The method of [9], further comprising mixing a soluble GPC3-containing sample with a surfactant.

[11] The method of [9] or

[10] , wherein the measurement is performed by a sandwich immunoassay using two or more different antibodies against two or more different epitopes in soluble GPC3.

[12] Immunoassay reagent for soluble GPC3 comprising (a) and (b) below: (a) Reducing agent; and (b) One or more antibodies against soluble GPC3.

[13] The immunoassay reagent of

[12] further comprising (c) a surfactant.

[14] The immunoassay reagent of

[12] or

[13] , wherein one or more antibodies against soluble GPC3 are two or more different antibodies against two or more different epitopes in soluble GPC3, and the immunoassay reagent is for sandwich immunoassay.

[15] Any of the immunoassay reagents from

[12] to

[14] , wherein the reagent is used for the diagnosis of cancer. [Effects of the Invention]

[0008] According to the present invention, the accuracy of distinguishing between positive and negative samples in an immunoassay of soluble GPC3 can be improved. Therefore, the present invention is useful in achieving high diagnostic sensitivity for specific conditions (e.g., diseases such as cancer) in an immunoassay of soluble GPC3. [Brief explanation of the drawing]

[0009] [Figure 1]Figure 1 shows the analysis of the recognition sites of antibodies A and B in antigen proteins by Western blotting (WB). Recombinant human GPC3 (rhGPC3), consisting of amino acid residues 1-559 of human GPC3, and the culture supernatant of human hepatocellular carcinoma cells (HepG2) with high GPC3 expression were used as antigen proteins. The amount of protein applied to each lane was 1.4 ng / lane and 7.2 ng / lane, respectively. [Modes for carrying out the invention]

[0010] This invention provides a method for processing samples in an immunoassay of soluble GPC3.

[0011] In this invention, soluble GPC3 refers to soluble GPC3 secreted from GPC3-expressing cells. As soluble GPC3, soluble GPC3 derived from any subject can be used. Examples of such subjects include mammals (e.g., primates such as humans and monkeys; rodents such as mice, rats, and rabbits; ungulates such as cattle, pigs, goats, horses, and sheep; carnivores such as dogs and cats) and birds (e.g., chickens). Preferably, the subject is a mammal such as a human. GPC3 is widely conserved in animals, and its amino acid sequence is particularly highly conserved among mammals. From the viewpoint of clinical application, the subject is preferably a human. Therefore, soluble GPC3 is preferably human soluble GPC3.

[0012] Preferably, human soluble GPC3 is either the N-terminal fragment generated by cleavage between the arginine residue at position 358 and the serine residue at position 359 in the human GPC3 protein (accession number: P51654.1), which consists of 580 amino acid residues, or soluble full-length GPC3 released by cleavage of the GPI anchor located 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 generated by cleavage between the arginine residue at position 358 and the serine residue at position 359 in the human GPC3 protein and the C-terminal fragment are linked to each other via a disulfide bond. More specifically, such human soluble GPC3s are (a) an N-terminal fragment or variant consisting of amino acid residues at positions 1-358 of the amino acid sequence of SEQ ID NO: 1, (b) a soluble full-length GPC3 formed by disulfide bonds linking the N-terminal fragment or variant consisting of amino acid residues at positions 1-358 of the amino acid sequence of SEQ ID NO: 1 with a soluble C-terminal fragment or variant consisting of amino acid residues at positions 359-560 of the amino acid sequence of SEQ ID NO: 1, or (c) naturally occurring variants thereof between races and / or individuals. Such variants are formed by introducing one or more naturally occurring amino acid residue mutations (e.g., substitutions, insertions, deletions) between races and / or individuals into (a) the N-terminal fragment or its variant, or (b) the soluble full-length GPC3 or its variant. The number of amino acid residue mutations in such mutants 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 this invention, an immunoassay refers to an immunoassay of soluble GPC3 using one or more antibodies against soluble GPC3. Examples of antibodies include polyclonal antibodies and monoclonal antibodies. Preferably, the antibody is a monoclonal antibody. The antibody can also be specified by 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 a full-length antibody or a fragment thereof, containing a heavy chain and a light chain, each containing a variable region and a constant region, respectively. Examples of antibody fragments include F(ab')2, Fab', Fab, and Fv. The antibody may also be a single-chain antibody (scFv) or a VHH antibody.

[0014] The antibody against soluble GPC3 used in the present invention is not particularly limited as long as it is one or more, and may be one, two, three, four, or five. When two or more antibodies against soluble GPC3 are used in the present invention, such two or more antibodies can recognize the same or different epitopes. Preferably, such two or more antibodies may recognize different epitopes. Since various epitopes available in the immunoassay of soluble GPC3 (e.g., two or more different epitopes available in a sandwich assay) and antibodies against such epitopes are known (see, for example, International Publication No. 2015 / 097928, International Publication No. 2004 / 038420, International Publication No. 2004 / 022739), such known epitopes and antibodies against them may be used in the present invention. In addition, since antibodies against soluble GPC3 are commercially available, commercially available antibodies can also be used in the present invention. From the viewpoint of a simple immunoassay or the like, the use of one or two antibodies against soluble GPC3 is preferable. The antibody against soluble GPC3 used in the present invention is preferably an antibody having the ability to bind to a region in the N-terminal fragment of GPC3, and more preferably an antibody having the ability to specifically bind to a region in the N-terminal fragment of GPC3.

[0015] The immunoassay can be performed by any immunoassay using one or more antibodies against soluble GPC3. Examples of such immunoassays include chemiluminescent immunoassay (CLIA) [e.g., chemiluminescent enzyme immunoassay (CLEIA)], immunoturbidimetry (TIA), enzyme immunoassay (EIA) (e.g., direct ELISA, indirect ELISA, competitive ELISA, and sandwich ELISA), radioimmunoassay (RIA), latex agglutination reaction method, fluorescence immunoassay (FIA), and immunochromatography method, Western blotting, and immunostaining.

[0016] The immunoassay can also be performed by any immunoassay using two or more antibodies against soluble GPC3, including a labeled antibody and a solid-phase antibody against soluble GPC3. The labeled antibody is an antibody labeled with a labeling substance or an antibody that is labeled with a labeling substance in the process of the immunoassay. Examples of the labeling substance include a fluorescent substance, a luminescent substance, a dye, and an enzyme. The solid-phase antibody is an antibody fixed to a solid phase or an antibody that is fixed to a solid phase in the process of the immunoassay. Examples of the solid phase include particles (e.g., microparticles, nanoparticles, microbeads, nanobeads, microspheres, nanospheres), supports (e.g., membranes), and substrates (e.g., plates). The solid phase may be a solid phase having magnetism (e.g., magnetic particles).

[0017] The immunoassay can also be performed in any manner. Examples of such a manner include a direct method, an indirect method, a competitive method, and a sandwich method. Preferably, the immunoassay may be performed by a sandwich immunoassay using two or more different antibodies against two or more different epitopes of soluble GPC3. In such a sandwich immunoassay, as two or more different antibodies against two or more different epitopes of soluble GPC3, two or more antibodies including a labeled antibody and a solid-phase antibody against soluble GPC3 are used. From the viewpoint of a simple immunoassay or the like, the immunoassay may be performed by a sandwich immunoassay using two antibodies (a labeled antibody and a solid-phase antibody) against two different epitopes in soluble GPC3. The two antibodies (a labeled antibody and a solid-phase antibody) against two different epitopes of soluble GPC3 may be two antibodies against different epitopes of the N-terminal fragment or two antibodies against different epitopes of the C-terminal fragment. Alternatively, such two antibodies may be a combination of one antibody against an epitope of the N-terminal fragment and one antibody against an epitope of the C-terminal fragment. Preferably, such two antibodies are two antibodies against different epitopes of the N-terminal fragment.

[0018] The method for processing a soluble GPC3-containing sample according to the present invention includes mixing the soluble GPC3-containing sample with a reducing agent. This produces a mixture of the soluble GPC3-containing sample and the reducing agent.

[0019] A soluble GPC3-containing sample is any sample containing the soluble GPC3 described above. Examples of soluble GPC3-containing samples include liquid samples obtained from a subject (e.g., blood, lymph, urine, breast milk, saliva, tears), tissue extracts obtained from a subject, washing solutions that can be recovered from a subject (e.g., obtained by washing mucosal tissue such as bronchi), samples obtained from cell cultures derived from a subject, and samples containing recombinant soluble GPC3 (e.g., soluble GPC3 preparations), as well as liquid samples obtained by processing (e.g., fractionation) these samples. From the viewpoint of easily obtaining samples rich in soluble GPC3, the soluble GPC3-containing sample is preferably a blood sample (e.g., whole blood, serum, plasma).

[0020] In certain embodiments, the soluble GPC3-containing sample may be obtained from a subject in a specific condition. Such subjects include, for example, subjects suffering from a specific disease and subjects potentially suffering from a specific disease. Examples of specific diseases include cancer (e.g., liver cancer, prostate cancer, malignant melanoma) and liver diseases (e.g., hepatitis, cirrhosis) (see, for example, International Publication No. 2004 / 038420; International Publication No. 2007 / 081790; International Publication No. 2005 / 039380; Detection of glypican-3-specific CTLs in chronic hepatitis and liver cirrhosis, Oncology Reports 22, pp. 149-54, 2009).

[0021] When mixing a sample containing soluble GPC3 with a reducing agent, any reducing agent can be used. Examples of such reducing agents include 2-(dimethylamino)ethanethiol (DEAET), tris(2-carboxyethyl)phosphine (TCEP), 2-mercaptoethylamine, 2-mercaptoethanol, dithiothreitol, thioglycerol, sodium sulfite, and borohydrides, as well as their salts. Examples of salts include metal salts (e.g., monovalent metal salts such as sodium salts and potassium salts, and divalent metal salts such as calcium salts and magnesium salts), inorganic salts (e.g., halide salts such as fluoride, chloride, bromide, and iodide, and ammonium salts), organic salts (e.g., alkyl-substituted ammonium salts), and acid addition salts (e.g., salts with inorganic acids such as sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, and phosphoric acid, and salts with organic acids such as acetic acid, oxalic acid, lactic acid, citric acid, trifluoromethanesulfonic acid, and trifluoroacetic acid). From the viewpoint of using a reducing agent that is highly stable in solution, DEAET, TCEP, or salts thereof are preferred.

[0022] The reducing agent can be used at a final concentration that reduces the detection signal intensity (e.g., count) from negative samples. The final concentration is the concentration at the time of mixing. Therefore, the final concentration can also be expressed as the concentration in the mixture produced by mixing. Such a final concentration is, for example, 0.05 to 1,000 mM, preferably 0.5 to 500 mM, more preferably 1 to 300 mM, and even more preferably 3 to 200 mM.

[0023] The mixing of a soluble GPC3-containing sample and a reducing agent may include further mixing of the soluble GPC3-containing sample with a surfactant. In such a case, a mixture of the soluble GPC3-containing sample, the reducing agent, and the surfactant is produced.

[0024] Examples of surfactants include nonionic surfactants, amphoteric surfactants, anionic surfactants, and cationic surfactants, as well as their salts. The salts are the same as those described above. Examples of nonionic surfactants include polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkylphenyl ethers, and polyoxyethylene alkyl ethers. Examples of polyoxyethylene sorbitan fatty acid esters include polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Tween 40), polyoxyethylene sorbitan monostearate (Tween 60), and polyoxyethylene sorbitan monooleate (Tween 80). Examples of polyoxyethylene alkylphenyl ethers include polyoxyethylene (10) octylphenyl ether (Triton X-100), polyoxyethylene (8) octylphenyl ether (Triton X-114), polyoxyethylene (30) octylphenyl ether (Triton X-305), and polyoxyethylene (40) octylphenyl ether (Triton X-405). Examples of polyoxyethylene alkyl ethers include polyoxyethylene (23) lauryl ether (Brij 35) and polyoxyethylene (20) cetyl ether (Brij 58). Preferred examples of nonionic surfactants include polyoxyethylene sorbitan monolaurate (Tween 20) and polyoxyethylene (10) octylphenyl ether (Triton X-100). Examples of amphoteric surfactants include sulfobetaine-type surfactants.Examples of sulfobetaine-type surfactants include 3-[(3-collamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[3-collamidopropyl]dimethylammonio)-2-hydroxypropanesulfonate (CHAPSO), N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate. Examples of anionic surfactants include sodium dodecyl sulfate (SDS), sodium N-lauroyl sarcosinate (NLS) lithium dodecyl sulfate, sodium dodecylbenzenesulfonate, and deoxycholate. Examples of cationic surfactants include decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, hexadecyltrimethylammonium chloride, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and hexadecyltrimethylammonium bromide. Preferably, the surfactant is a nonionic surfactant, an amphoteric surfactant, or a salt thereof.

[0025] The surfactant can be used at a final concentration that can enhance the reducing effect of the reducing agent in reducing the detection signal intensity from negative samples and / or reduce the matrix effect of the samples. The final concentration is the concentration at the time of mixing. Therefore, the final concentration can also be expressed as the concentration in the mixture produced by mixing. Such a final concentration is, for example, 0.005 to 10% by weight, preferably 0.01 to 9% by weight, more preferably 0.02 to 7.5% by weight, even more preferably 0.05 to 6% by weight, and particularly preferably 0.05 to 5% by weight.

[0026] Preferably, the reducing agent and the surfactant can be used in a ratio that enhances their effect in reducing the detection signal intensity from negative samples and / or reducing the matrix effect of the samples. Such a ratio can be defined by the concentration range of the surfactant per 1 mM of reducing agent. The concentration range of the surfactant per 1 mM of reducing agent is, for example, 0.000025 to 3% by weight, preferably 0.00005 to 0.1% by weight, more preferably 0.0005 to 0.01% by weight, and even more preferably 0.005 to 0.05% by weight.

[0027] When a sample containing soluble GPC3 is mixed with both a reducing agent and a surfactant, the mixing can be performed simultaneously or separately. If the mixing is performed simultaneously, the sample containing soluble GPC3 can be mixed with a mixture of the reducing agent and the surfactant. If the mixing is performed separately, the sample containing soluble GPC3 may be mixed first with the reducing agent and then with the surfactant, or first with the surfactant and then with the reducing agent.

[0028] Reducing agents and / or surfactants can be used dissolved in aqueous solutions. Examples of such aqueous solutions include water (e.g., distilled water, sterile water, sterile distilled water, pure water) and buffer solutions. Examples of buffer solutions include phosphate buffer, MES buffer, citrate buffer, Tris buffer, carbonate buffer, HPEPS buffer, and MOPS buffer. The pH of the buffer solution varies depending on factors such as the type and concentration of the reducing agent, but may be 1.0 to 9.0 (preferably 4.0 to 6.0) from the viewpoint of improving the effect of the reducing agent. From the viewpoint of stably exhibiting the effect of the reducing agent within a desired pH range, the aqueous solution is preferably a buffer solution. The aqueous solution may contain other components such as organic solvents (e.g., alcohol). The ratio of the volume to be mixed between the soluble GPC3-containing sample and the aqueous solution containing the reducing agent and / or surfactant (soluble GPC3-containing sample: aqueous solution containing the reducing agent and / or surfactant) is, for example, 10:1 to 1:10, preferably 5:1 to 1:5, and more preferably 2:1 to 1:2.

[0029] The mixing is carried out under conditions sufficient for treating the sample with the reducing agent alone or with both the reducing agent and the surfactant. Such temperature conditions are, for example, 15 to 60°C, preferably 20 to 50°C, and more preferably 25 to 45°C. The mixing time is, for example, 30 seconds or less. From the viewpoint of rapid processing, the mixing time is preferably 20 seconds or less, and more preferably 15 seconds or less. By mixing under such conditions, the detection signal intensity from negative samples can be reduced.

[0030] The method of the present invention may further include incubating the mixture after mixing. The incubation time varies depending on factors such as the type and concentration of the reducing agent, whether or not a surfactant is used in combination, the type and concentration of the surfactant, the mixing time, the desired degree of reduction in the detection signal intensity, and, if an antibody-based measurement (immunoassay) is performed, the time required for that measurement. For example, it is 120 minutes or less, preferably 60 minutes or less, and more preferably 30 minutes or less. From the viewpoint of rapid processing, the incubation time may be even more preferably 20 minutes or less, and particularly preferably 10 minutes or less, or 5 minutes or less. The incubation temperature is the same as the temperature conditions in the mixing described above.

[0031] The present invention also provides an immunoassay method for soluble GPC3, comprising (a) and (b) below: (a) Mixing a soluble GPC3-containing sample with a reducing agent; and (b) The amount of soluble GPC3 in the mixture obtained in (a) is measured using one or more antibodies against soluble GPC3.

[0032] The definitions, examples, and preferred examples of the various elements in the immunoassay method for soluble GPC3 are the same as those described in the method for processing soluble GPC3-containing specimens according to the present invention.

[0033] Step (a) can be carried out in the same manner as the method for processing a soluble GPC3-containing sample according to the present invention. Step (b) can be carried out by the immunoassay described above. Steps (a) and (b) can be carried out in parallel or separately. For example, when steps (a) and (b) are carried out in parallel, the processing of the soluble GPC3-containing sample with the reducing agent (and surfactant) and the antigen-antibody reaction can be carried out simultaneously by simultaneously mixing the soluble GPC3-containing sample, the reducing agent (and surfactant), and one or more antibodies against soluble GPC3. However, in order to sufficiently reduce the detection signal intensity (e.g., count) from negative samples, it is preferable to thoroughly process the soluble GPC3-containing sample with the reducing agent (and surfactant), and then measure the amount of soluble GPC3 by an antigen-antibody reaction using one or more antibodies against soluble GPC3. Furthermore, if the antibody used in the present invention contains a disulfide bond, if the antibody and the reducing agent are kept together for a long time, the reducing agent will destroy the antibody by cleaving the disulfide bond in the antibody, which may affect the measurement accuracy of the immunoassay. Therefore, if the antibody used in the present invention contains a disulfide bond, it is preferable that steps (a) and (b) be carried out separately. Of course, if the antibody used in the present invention does not contain a disulfide bond (e.g., a single-chain antibody), it is also preferable that steps (a) and (b) be carried out in parallel.

[0034] The present invention further provides an immunoassay reagent for soluble GPC3 comprising (a) and (b) below: (a) Reducing agent; and (b) One or more antibodies against soluble GPC3.

[0035] The reagent of the present invention may further contain (c) a surfactant.

[0036] The definitions, examples, and preferred examples of the reducing agents, antibodies, and surfactants of components (a) to (c) are the same as those described in the method for processing soluble GPC3-containing samples according to the present invention.

[0037] In certain embodiments, the reagent of the present invention may be a reagent in which one or more antibodies against soluble GPC3 are two or more different antibodies against two or more different epitopes in soluble GPC3, and the reagent is for sandwich immunoassay. Preferably, such a reagent includes, as the two or more antibodies, a labeled antibody and / or a solid-phase antibody against soluble GPC3. Alternatively, if such a reagent does not include a labeled antibody labeled with a labeling substance and / or a solid-phase antibody immobilized on a solid phase, it may include a labeling substance and / or a solid phase. Examples of labeling substances include fluorescent substances, luminescent substances, dyes, and enzymes. If the labeling substance is an enzyme, such a reagent may include an enzyme substrate (e.g., a substrate that generates a detection signal, or a substrate that is converted by the enzyme to a product that generates a detection signal, or a substrate in a reaction that can be coupled with other enzymatic reactions that utilize a substrate that generates a detection signal, or a substrate that is converted by the enzyme to a product that generates a detection signal). The solid phase is as described above.

[0038] The reagents of the present invention can be used to determine specific conditions (e.g., disease diagnosis). These specific conditions (e.g., diseases) are the same as those described above. Preferably, the reagents of the present invention can be used to diagnose cancers such as liver cancer and prostate cancer. [Examples]

[0039] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0040] Reference Example 1: Confirmation of reactivity between glypican-3 (GPC3) and antibody Western blotting using recombinant human GPC3 (rhGPC3) (R&D Systems) antigen consisting of amino acid residues 1-559 of GPC3, or the culture supernatant of human hepatocellular carcinoma cells (HepG2) that highly express GPC3, was used to analyze the recognition sites of anti-GPC3 antibody A and anti-GPC3 antibody B (both monoclonal IgG antibodies).

[0041] SDS-PAGE sample buffer was added to the rhGPC3 and HepG2 culture supernatants, and the rhGPC3 and HepG2 culture supernatants were applied to 5-20% polyacrylamide gels (Superset Ace 5-20%, WAKO) to achieve concentrations of 1.4 ng protein / lane and 7.2 ng protein / lane, respectively. After electrophoresis (30 mA, 60 min), the proteins were transferred to blotting membranes (Imobilon ISEQ, Merck Millipore) using a Transblot® SD semi-dry electrophoresis transfer cell (Bio-Rad) (15 V, 60 min). The blotting membranes were lightly washed with TBS-T and then shaken at room temperature for 1 hour in blocking solution (0.5% ECL Block (GE Healthcare Life Sciences), 0.5% BSA, TBS-T). After washing twice with TBS-T, the blotting membrane was shaken overnight at 4°C in a solution containing anti-GPC3 antibody A and anti-GPC3 antibody B, each diluted to 1 μg / mL with the reaction solution (blocking solution diluted 2-fold with TBS-T). After washing four times with TBS-T, the blotting membrane was shaken for 1 hour in a solution containing POD-labeled anti-mouse F(ab')2 (Jackson), diluted 20,000-fold with the reaction solution, and then washed with TBS-T. The colorimetric reaction was performed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), and development was performed using a chemiluminescence imaging system (FUSION SYSTEM, Bilbar-Rumat). The results of the Western blotting analysis are shown in Figure 1. As a result, both anti-GPC3 antibody A and anti-GPC3 antibody B reacted to a 40 kDa band corresponding to the N-terminal fragment of GPC3 (the fragment N-terminal to amino acid residue 358) (Figure 1). This indicates that anti-GPC3 antibody A and anti-GPC3 antibody B recognize the N-terminal region of GPC3. Therefore, it was confirmed that anti-GPC3 antibody A and anti-GPC3 antibody B recognize soluble GPC3.

[0042] Reference Example 2: Preparation of anti-GPC3 antibody-immobilized particles Anti-GPC3 antibody A was added to magnetic particles in 10 mM MES buffer (pH 5.0) to obtain a suspension containing 0.2 mg / mL anti-GPC3 antibody A and 0.01 g / mL magnetic particles. This suspension was incubated at 5°C for 1 hour with gentle stirring to immobilize the anti-GPC3 antibody A onto the magnetic particles. Subsequently, the magnetic particles were collected with a magnet and washed with a washing solution (50 mM Tris buffer, 150 mM NaCl, 2.0% BSA, pH 7.2) to obtain anti-GPC3 antibody A immobilized particles. For measurement, the anti-GPC3 antibody A immobilized particles were suspended in a particle diluent (50 mM Tris buffer, 1 mM EDTA2Na, 0.1% NaN3, 2.0% BSA, pH 7.2).

[0043] Reference Example 3: Preparation of Alkaline Phosphatase-Labeled Anti-GPC3 Antibody Desalted alkaline phosphatase (ALP) and N-(4-maleimidobutyryloxy)-succinimide (GMBS) (final concentration 0.3 mg / mL) were mixed and allowed to stand at 30°C for 1 hour to maleimideize the ALP. Next, the Fab'-modified anti-GPC3 antibody B and the maleimide-modified ALP were mixed in a 1:1 molar ratio in a coupling reaction solution (100 mM phosphate buffer, 1 mM EDTA 2Na, pH 6.3) and reacted at 25°C for 1 hour. Using column chromatography on a Superdex200 10 / 300 (GE Healthcare), the major peak was isolated and purified with a purification buffer (50 mM MES buffer, 150 mM NaCl, 0.1% NaN3, pH 8.0) at a flow rate of 0.5 mL / min to obtain ALP-labeled anti-GPC3 antibody B. For the measurement, ALP-labeled anti-GPC3 antibody B was suspended in a labeling diluent (50 mM MES buffer, 150 mM NaCl, 0.3 mM ZnCl2, 1 mM MgCl2, 0.1% NaN3, 2.0% BSA, pH 6.8).

[0044] Reference Example 4: Measurement of Soluble GPC3 20 μL of pretreatment solution was dispensed into the reaction vessel, followed by 20 μL of the sample. The mixture of pretreatment solution and sample was incubated at 37°C for 6.5 minutes. Then, 50 μL of anti-GPC3 antibody A immobilized particles were dispensed into the reaction vessel, and the mixture was stirred. The mixture was incubated at 37°C for 8 minutes, followed by B / F separation and washing. 50 μL of ALP-labeled anti-GPC3 antibody B was dispensed into the reaction vessel, and the mixture was stirred. The mixture was incubated at 37°C for 8 minutes, followed by B / F separation and washing. Subsequently, 200 μL of Lumipulse substrate solution containing the chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane-2-sodium salt (AMPPD) was dispensed into the reaction vessel, and the mixture was stirred. The mixture was then incubated at 37°C for 4 minutes, and the luminescence was measured using a luminometer. The actual measurements were performed using a fully automated chemiluminescent enzyme immunoassay system (Lumipulse L2400 (Fujirebio Corporation)).

[0045] Example 1: Sample pretreatment with reducing agent DEAET in the measurement of soluble GPC3 Serum samples from healthy individuals (negative samples) and serum samples from liver cancer patients positive for α-fetoprotein (AFP) (Trina) (positive samples) were used as samples for evaluating soluble GPC3. A phosphate buffer containing 6.25 mM to 600 mM 2-(dimethylamino)ethanethiol hydrochloride (DEAET) (10 mM phosphate buffer, 6.25-600 mM DEAET, pH 6.0) was used as a pretreatment solution. A 10 mM phosphate buffer (pH 6.0) was used as a control solution. Negative samples, positive samples, and buffer samples (10 mM phosphate buffer) were each mixed with the pretreatment solution in a 1:1 volume ratio and reacted at 37°C for 6.5 minutes (pretreatment applied). Similarly, negative samples, positive samples, and buffer samples were each mixed with the control solution and reacted (no pretreatment).

[0046] For each sample, soluble GPC3 was measured using the measurement method described in Reference Example 4. The counts for each sample are shown in Table 1. In addition, the (1) reduction rate (%), (2) difference between positive and negative counts (%), and (3) matrix difference (%) calculated from the counts using the following formulas are also shown in Table 1.

[0047] calculation formula (1) Reduction rate (%) = 100 - (Count of "with pretreatment" for each DEAET concentration / Count of "without pretreatment") × 100 (2) Difference in the number of positive and negative samples (%) = (average count of positive samples) / (average count of negative samples) × 100 (3) Matrix difference (%) = (count of buffer sample) / (average count of negative sample) × 100

[0048] The reduction rate (%) is an indicator for evaluating the effect of sample preparation on individual samples. A larger reduction rate for negative samples and a smaller reduction rate for positive samples widens the difference between the measured values ​​of negative and positive samples, making them more distinguishable and thus enabling higher diagnostic sensitivity. Therefore, a larger reduction rate for negative samples and a smaller reduction rate for positive samples are useful as sample preparation conditions for soluble GPC3 measurement systems.

[0049] The difference in positive and negative counts (%) is an index used to evaluate the difference in counts between positive and negative samples after pretreatment. A larger difference in positive and negative counts (%) indicates higher diagnostic sensitivity, making it useful for measuring soluble GPC3.

[0050] The matrix difference (%) is an index used to evaluate the difference in counts between buffered samples and negative samples. In immunoassays using samples, the immune response can be affected by substances contained in the sample (matrix effect). The closer the matrix difference (%) is to 100%, the greater the reduction in the matrix effect. A reduced matrix effect is useful for measuring soluble GPC3 because it enables the output of true values ​​that are independent of the sample type.

[0051] [Table 1]

[0052] As shown in Table 1, when negative samples were pretreated with DEAET, the count of negative samples decreased at all concentrations examined (6.25 mM to 600 mM), and the rate of decrease was very large. On the other hand, when positive samples were pretreated with DEAET, the count of positive samples did not decrease, or if it did decrease, the rate of decrease was smaller compared to negative samples. In particular, when DEAET was added at concentrations of 50 mM to 400 mM, the difference (%) between positive and negative counts was very large. This indicates that sample pretreatment with the reducing agent DEAET can improve the accuracy of distinguishing between positive and negative samples in the measurement of soluble GPC3. Furthermore, when negative samples were pretreated with DEAET, the matrix difference approached 100%, and the matrix effect was reduced. Therefore, it was confirmed that sample pretreatment with the reducing agent DEAET is useful for the measurement of soluble GPC3.

[0053] Example 2: Sample pretreatment with reducing agent 2MEA in the measurement of soluble GPC3 As a pretreatment solution, a phosphate buffer containing 6.25 mM to 100 mM 2-mercaptoethylamine hydrochloride (2MEA) (10 mM phosphate buffer, 6.25-100 mM 2MEA, pH 6.0) was used. As a control solution, a 10 mM phosphate buffer (pH 6.0) was used. Negative samples, positive samples, and buffer samples were each mixed with the pretreatment solution in a 1:1 volume ratio and reacted at 37°C for 6.5 minutes (pretreatment applied). Similarly, negative samples, positive samples, and buffer samples were each mixed with the control solution and reacted (no pretreatment applied).

[0054] For each sample, soluble GPC3 was measured using the measurement method described in Reference Example 4. The counts for each sample are shown in Table 2. In addition, as in Example 1, the reduction rate (%), the difference between positive and negative counts (%), and the matrix difference (%) were calculated.

[0055] [Table 2]

[0056] As shown in Table 2, when negative samples were pretreated with 2MEA, the count of negative samples decreased at all concentrations examined (6.25 mM to 100 mM), and the rate of decrease was very large. On the other hand, when positive samples were pretreated with 2MEA, the count of positive samples did not decrease, or if it did decrease, the rate of decrease was smaller compared to negative samples. This indicates that sample pretreatment with the reducing agent 2MEA can improve the accuracy of distinguishing between positive and negative samples in the measurement of soluble GPC3. Furthermore, when negative samples were pretreated with 2MEA, the matrix difference approached 100%, and the matrix effect was reduced. Therefore, it was confirmed that sample pretreatment with the reducing agent 2MEA is useful for the measurement of soluble GPC3.

[0057] Example 3: Sample pretreatment with reducing agent TCEP in the measurement of soluble GPC3 As a pretreatment solution, a phosphate buffer containing 6.25 mM to 100 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (10 mM phosphate buffer, 6.25-100 mM TCEP, pH 6.0) was used. As a control solution, a 10 mM phosphate buffer (pH 6.0) was used. Negative samples, positive samples, and buffer samples were each mixed with the pretreatment solution in a 1:1 volume ratio and reacted at 37°C for 6.5 minutes (pretreatment applied). Similarly, negative samples, positive samples, and buffer samples were each mixed with the control solution and reacted (no pretreatment applied).

[0058] For each sample, soluble GPC3 was measured using the measurement method described in Reference Example 4. The counts for each sample are shown in Table 3. In addition, as in Example 1, the reduction rate (%), the difference between positive and negative counts (%), and the matrix difference (%) were calculated.

[0059] [Table 3]

[0060] As shown in Table 3, when negative samples were pretreated with TCEP, the count of negative samples decreased at all concentrations examined (6.25 mM to 100 mM), and the rate of decrease was very large. On the other hand, when positive samples were pretreated with TCEP, the count of positive samples did not decrease, or if it did decrease, the rate of decrease was smaller compared to that of negative samples. This indicates that sample pretreatment with the reducing agent TCEP can improve the accuracy of distinguishing between positive and negative samples in the measurement of soluble GPC3. Furthermore, when negative samples were pretreated with TCEP, the matrix difference approached 100%, and the matrix effect was reduced. Therefore, it was confirmed that sample pretreatment with the reducing agent TCEP is useful for the measurement of soluble GPC3.

[0061] (Summary of Examples 1-3) Considering the results of Examples 1-3, it is believed that sample pretreatment with a reducing agent can improve the accuracy of distinguishing between positive and negative samples in the measurement of soluble GPC3, regardless of the type and structure of the reducing agent. Furthermore, it is believed that pretreatment of negative samples with a reducing agent can achieve true value output that is independent of the sample type by reducing matrix effects. Therefore, sample pretreatment with a reducing agent is useful for the measurement of soluble GPC3.

[0062] Example 4: Sample pretreatment using a combination of reducing agent and surfactant in the measurement of soluble GPC3 As pretreatment solutions, phosphate buffers (pH 6.0) from Test Examples 2-11 shown in Table 4A were used. The phosphate buffers from Test Examples 2-11 contained DEAET as a reducing agent and 3-[(3-colamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or polyoxyethylene sorbitan monolaurate (Tween20) as surfactants. As a control solution, 10 mM phosphate buffer (pH 6.0) from Test Example 1 shown in Table 4A was used. Negative samples, positive samples, and buffer samples were each mixed with the pretreatment solution in a volume ratio of 1:1 and reacted at 37°C for 6.5 minutes (with pretreatment). Similarly, negative samples, positive samples, and buffer samples were each mixed with the control solution and reacted (without pretreatment).

[0063] For each sample, soluble GPC3 was measured using the measurement method described in Reference Example 4. The counts for each sample are shown in Table 4B. In addition, as in Example 1, the reduction rate (%), the difference between positive and negative counts (%), and the matrix difference (%) were calculated.

[0064] [Table 4]

[0065] As shown in Table 4, when negative samples were pretreated with a combination of reducing agent and surfactant, the count of negative samples decreased at all surfactant concentrations examined (0.1–10% by weight), and the rate of decrease was very large. On the other hand, when positive samples were pretreated with a combination of reducing agent and surfactant, the rate of decrease in the count of positive samples was smaller than that of negative samples. In particular, when surfactants were added at predetermined concentrations (0.1–2.5% by weight CHAPS and 0.1–10% by weight Tween20), the difference (%) between positive and negative counts was very large. Furthermore, when negative samples were pretreated with a combination of reducing agent and surfactant, the matrix difference approached 100%, further reducing the matrix effect. This indicates that sample pretreatment with a combination of reducing agent and surfactant can improve the accuracy of distinguishing between positive and negative samples in the measurement of soluble GPC3, and can reduce the matrix effect of the samples. Therefore, it was confirmed that sample pretreatment with a combination of reducing agent and surfactant is useful for the measurement of soluble GPC3.

Claims

1. A method for processing a soluble GPC3-containing sample in an immunoassay of soluble GPC3, comprising mixing the soluble GPC3-containing sample with a reducing agent, A method in which the immunoassay is a sandwich immunoassay using two or more different antibodies against two or more different epitopes in soluble GPC3.

2. The method according to claim 1, wherein the reducing agent is used at a final concentration of 0.05 to 1,000 mM.

3. The method according to claim 1 or 2, further comprising mixing a soluble GPC3-containing sample with a surfactant.

4. The method according to claim 3, wherein the surfactant is used at a final concentration of 0.005 to 10% by weight.

5. The method according to claim 3 or 4, wherein the surfactant is a nonionic surfactant, an amphoteric surfactant, or a salt thereof.

6. The method according to any one of claims 1 to 5, wherein the soluble GPC3-containing sample is a blood sample.

7. The method according to any one of claims 1 to 6, wherein a soluble GPC3-containing specimen is obtained from a subject suffering from cancer.

8. The method according to claim 7, wherein the cancer is liver cancer.

9. An immunoassay method for soluble GPC3, comprising (a) and (b) below: (a) Mixing a soluble GPC3-containing sample with a reducing agent; and (b) The amount of soluble GPC3 in the mixture obtained in (a) is measured by a sandwich immunoassay using two or more different antibodies against two or more different epitopes in the soluble GPC3.

10. The method according to claim 9, further comprising mixing a soluble GPC3-containing sample with a surfactant.

11. The method according to claim 10, wherein the surfactant is a nonionic surfactant, an amphoteric surfactant, or a salt thereof.

12. A sandwich immunoassay reagent for soluble GPC3 comprising (a) and (b) below: (a) Reducing agent; and (b) Two or more different antibodies against two or more different epitopes in soluble GPC3.

13. The sandwich immunoassay reagent according to claim 12, further comprising (c) a surfactant.

14. The sandwich immunoassay reagent according to claim 13, wherein the surfactant is a nonionic surfactant, an amphoteric surfactant, or a salt thereof.

15. The sandwich immunoassay reagent according to any one of claims 12 to 14, wherein the reagent is for the diagnosis of cancer.

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