Method for processing a sample containing soluble gpc3 in an immunoassay for soluble gpc3

Abstract

The present application provides a method and reagent useful for examination of soluble GPC3. More specifically, the present application provides the following (1), (2) and (3). (1) A method for processing a sample in an immunoassay of soluble GPC3, comprising mixing a sample containing soluble GPC3 with a reducing agent. (2) An immunoassay method of soluble GPC3, comprising the following (a) and (b): (a) mixing a sample containing soluble GPC3 with a reducing agent; (b) measuring the amount of soluble GPC3 in the mixture obtained in (a) using one or more antibodies against soluble GPC3. (3) An immunoassay reagent for soluble GPC3, comprising the following (a) and (b): (a) a reducing agent; (b) one or more antibodies against soluble GPC3.

Description

Technical Field

[0001] This invention relates to a method for processing soluble GPC3-containing samples in the immunoassay of soluble phosphatidylinositol proteoglycan-3 (GPC3). Background Technology

[0002] GPC3 is a protein with a molecular weight of approximately 65 kDa, belonging to the phosphatidylinositol proteoglycan family of proteoglycans containing a heparan sulfate chain, and bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor located at its C-terminus. GPC3 is cleaved between an arginine residue at position 358 and a serine residue at position 359 by an enzyme called furin in the Golgi apparatus, resulting in an N-terminal fragment (approximately 40 kDa) and a C-terminal fragment containing the GPI anchor (approximately 30 kDa). However, it has been confirmed that these two fragments are usually linked by a disulfide bond. Therefore, it is believed that GPC3 binds to the cell membrane via the GPI anchor in its full-length protein form, containing the two fragments linked by disulfide bonds.

[0003] Furthermore, since GPC3 is a protein specifically expressed in cancers such as liver cancer, methods useful for testing cancer patients targeting GPC3 are being explored. For example, Patent Document 1 proposes a method for testing cancer patients based on the assay of soluble GPC3. Additionally, Patent Document 2 proposes a method for measuring GPC3 using two different antibodies that bind to different epitopes present in the N-terminal region of GPC3.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: International Publication No. 2004 / 038420

[0007] Patent Document 2: International Publication No. 2015 / 097928 Summary of the Invention

[0008] The technical problem that the invention aims to solve

[0009] The technical problem of this invention is to provide a method and reagent useful for the examination of soluble GPC3.

[0010] Technical means to solve technical problems

[0011] The inventors conducted in-depth research and found that, in the immunoassay of soluble GPC3, treating the sample with a reducing agent can improve the accuracy of distinguishing between positive and negative samples. The inventors also found that, in the 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 the matrix effect, thus completing this invention.

[0012] That is, the present invention is as follows.

[0013] [1] A method for processing soluble GPC3-containing samples in an immunoassay of soluble GPC3, comprising: mixing the soluble GPC3-containing sample with a reducing agent.

[0014] [2] According to the method of [1], the reducing agent is used at a final concentration of 0.05 to 1000 mM.

[0015] [3] The method according to [1] or [2] further comprises: mixing the soluble GPC3 sample with a surfactant.

[0016] [4] The method according to [3], wherein the surfactant is used at a final concentration of 0.005 to 10% by weight.

[0017] [5] The method according to any one of [1] to [4], wherein the immunoassay is a sandwich immunoassay using two or more different antibodies targeting two or more different epitopes in soluble GPC3.

[0018] [6] The method according to any one of [1] to [5], wherein the soluble GPC3 sample is a blood sample.

[0019] [7] The method according to any one of [1] to [6], wherein the soluble GPC3 sample is obtained from the cancerous subject.

[0020] [8] According to the method described in [7], the cancer is liver cancer.

[0021] [9] An immunoassay method for soluble GPC3, comprising the following (a) and (b):

[0022] (a) Mixing the sample containing soluble GPC3 with a reducing agent;

[0023] (b) The amount of soluble GPC3 in the mixture obtained in (a) was determined using one or more antibodies against soluble GPC3.

[0024]

[10] The method according to [9] further comprises: mixing the soluble GPC3 sample with a surfactant.

[0025]

[11] The method according to [9] or

[10] , wherein the determination is performed by using sandwich immunoassay with two or more different antibodies targeting two or more different epitopes in soluble GP C3.

[0026]

[12] An immunoassay reagent for soluble GPC3, comprising the following (a) and (b):

[0027] (a) Reducing agent;

[0028] (b) One or more antibodies against soluble GPC3.

[0029]

[13] The immunoassay reagent according to

[12] further comprises: (c) a surfactant.

[0030]

[14] The immunoassay reagent according to

[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 used for sandwich immunoassay.

[0031]

[15] An immunoassay reagent according to any one of

[12] to

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

[0032] Invention Effects

[0033] According to the present invention, the accuracy of distinguishing positive and negative samples in the immunoassay of soluble GPC3 can be improved. Therefore, the present invention is useful for achieving high diagnostic sensitivity for specific conditions (such as diseases like cancer) in the immunoassay of soluble GPC3. Attached Figure Description

[0034] [ Figure 1 ] Figure 1 This is a diagram illustrating the resolution of recognition sites for antibody A and antibody B in antigen proteins based on Western blot (WB). Recombinant human GPC3 (rhGPC3) containing amino acid residues 1–559 of human GPC3 and culture supernatants from human hepatocellular carcinoma cells HepG2 with high GPC3 expression were used as antigen proteins. The amounts of protein added to the lanes were 1.4 ng / lane and 7.2 ng / lane, respectively. Detailed Implementation

[0035] This invention provides a method for processing samples in the immunoassay of soluble GPC3.

[0036] In this invention, soluble GPC3 refers to soluble GPC3 secreted by GPC3-expressing cells. Soluble GPC3 can be derived from any subject. 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). Human mammals are preferred as subjects. GPC3 is widely conserved in animals, especially among mammals, where its amino acid sequence is highly conserved. From a clinical point of view, humans are preferred as subjects. Therefore, human soluble GPC3 is preferred.

[0037] Preferably, the human soluble GPC3 is an N-terminal fragment generated by cleaving between the arginine residue at position 358 and the serine residue at position 359 in the human GPC3 protein (accession number: P51654.1), which contains 580 amino acid residues, or a soluble full-length GPC3 fragment freed by cleaving the GPI anchor present at the C-terminus of the human GPC3 protein. Examples of soluble full-length GPC3 fragments include those formed by linking the N-terminal fragment (generated by cleaving between the arginine residue at position 358 and the serine residue at position 359) with a C-terminal fragment via a disulfide bond. More specifically, such human soluble GPC3 is: (a) an N-terminal fragment or a variant thereof comprising amino acid residues 1–358 of the amino acid sequence of SEQ ID NO. 1; (b) a soluble full-length GPC3 formed by linking an N-terminal fragment or a variant thereof comprising amino acid residues 1–358 of the amino acid sequence of SEQ ID NO. 1 with a soluble C-terminal fragment or a variant thereof comprising amino acid residues 359–560 of the amino acid sequence of SEQ ID NO. 1 via disulfide bonds; or (c) a mutant thereof that is naturally occurring among people and / or individuals. Such a mutant is a mutant that introduces a mutation (e.g., substitution, insertion, deletion) of one or more amino acid residues that is naturally occurring among people and / or individuals into (a) the N-terminal fragment or a variant thereof, or (b) the soluble full-length GPC3 or a variant thereof. The number of mutations in such mutant amino acid residues can 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.

[0038] In this invention, immunoassay refers to the immunoassay of soluble GPC3 using one or more antibodies against soluble GPC3. Examples of antibodies include polyclonal antibodies and monoclonal antibodies. Monoclonal antibodies are preferred. Antibodies can also be determined by isotype. Examples of such isotypes include IgG, IgM, IgA, IgD, IgE, and IgY. Preferred antibodies are IgG, IgM, or IgA, more preferably IgG or IgM, and even more preferably IgG. Antibodies can also be chimeric antibodies, humanized antibodies, or human antibodies. Furthermore, antibodies can be full-length antibodies or fragments thereof comprising heavy and light chains containing variable and constant regions, respectively. Examples of antibody fragments include F(a b')2, Fab', Fab, and Fv. Additionally, antibodies can also be single-chain antibodies (scFv) or VHH antibodies.

[0039] The use of one or more antibodies against soluble GPC3 in this invention is not particularly limited; one, two, three, four, or five types can be used. When two or more antibodies against soluble GPC3 are used in this invention, these two or more antibodies can recognize the same or different epitopes. Preferably, these two or more antibodies can recognize different epitopes. Various epitopes and antibodies are known as epitopes available in immunoassays of soluble GPC3 (e.g., two or more different epitopes available in sandwich assays) and antibodies against those epitopes (e.g., see International Publication Nos. 2015 / 097928, 2004 / 038420, and 2004 / 022739). Therefore, such known epitopes and antibodies against those epitopes can also be used in this invention. Furthermore, antibodies against soluble GPC3 are commercially available; therefore, commercially available antibodies can also be used in this invention. From the viewpoint of simple immunoassay, it is preferable to use one or two antibodies against soluble GPC3. The antibody against soluble GPC3 used in this invention is preferably an antibody capable of binding to a region in the N-terminal fragment of GPC3, and more preferably an antibody capable of specifically binding to a region in the N-terminal fragment of GPC3.

[0040] Immunoassays can be performed using any immunoassay employing one or more antibodies against soluble GPC3. Examples of such immunoassays include chemiluminescent immunoassay (CLIA) [e.g., chemiluminescent enzyme immunoassay (CLEIA)], immunoturbidimetric assay (TIA), enzyme immunoassay (EIA) (e.g., direct ELISA, indirect ELISA, competitive ELISA, and sandwich ELISA), radioimmunoassay (RIA), latex agglutination assay, fluorescence immunoassay (FIA), immunochromatography, Western blotting, and immunostaining.

[0041] Immunoassays can also be performed using any immunoassay containing two or more antibodies against soluble GPC3, including labeled antibodies against soluble GPC3 and solid-phase antibodies. Labeled antibodies are antibodies labeled with a labeling substance or antibodies labeled with a labeling substance during the immunoassay step. Examples of labeling substances include fluorescent substances, luminescent substances, dyes, and enzymes. Solid-phase antibodies are antibodies immobilized on a solid phase or antibodies immobilized on a solid phase during the immunoassay step. Examples of solid phases include particles (e.g., microparticles, nanoparticles, microbeads, nanospheres, nanospheres), supports (e.g., membranes), and substrates (e.g., plates). The solid phase can also be a magnetic solid phase (e.g., magnetic particles).

[0042] Immunoassays can be performed in any manner. Examples of such methods include direct methods, indirect methods, competitive methods, and sandwich methods. Preferably, the immunoassay can be performed using a sandwich immunoassay employing two or more different antibodies targeting two or more different epitopes of soluble GPC3. In such a sandwich immunoassay, two or more antibodies, comprising a labeled antibody and a solid-phase antibody, are used as the two or more different antibodies targeting two or more different epitopes of soluble GPC3. From the viewpoint of simplified immunoassays, the immunoassay can be performed using a sandwich immunoassay employing two antibodies (labeled antibody and solid-phase antibody) targeting two different epitopes of soluble GPC3. The two antibodies (labeled antibody and solid-phase antibody) targeting two different epitopes of soluble GPC3 can be two antibodies targeting different epitopes of the N-terminal fragment or two antibodies targeting different epitopes of the C-terminal fragment. Alternatively, such two antibodies could be a combination of one antibody targeting an epitope on the N-terminal fragment and one antibody targeting an epitope on the C-terminal fragment. Preferably, such two antibodies are two antibodies targeting different epitopes on the N-terminal fragment.

[0043] The method for processing soluble GPC3 samples according to the present invention includes mixing the soluble GPC3 sample with a reducing agent. This generates a mixture containing the soluble GPC3 sample and the reducing agent.

[0044] A soluble GPC3-containing sample is any sample containing the soluble GPC3. 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., substances obtained from washing mucous tissues such as bronchi); samples obtained from cell cultures derived from a subject; and samples containing recombinant soluble GPC3 (e.g., soluble GPC3 standards), as well as liquid samples obtained by processing (e.g., grading) these samples. From the viewpoint of easily obtaining a sample rich in soluble GPC3, soluble GPC3-containing samples are preferably blood samples (e.g., whole blood, serum, plasma).

[0045] In certain embodiments, the soluble GPC3-containing sample may also be obtained from a sample in a specific state. Examples of such samples include those with a specific disease and those potentially suffering from a specific disease. Examples of specific diseases include cancers (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; Determination of glypican-3-specific CTLs in chronic hepatitis and liver cirrhosis, Oncology Reports 22, pp. 149-54, 2009).

[0046] When mixing a soluble GPC3 sample 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 and potassium salts, and divalent metal salts such as calcium and magnesium salts), inorganic salts (e.g., halide salts such as fluorides, chlorides, bromides, and iodides, 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 or TCEP or their salts are preferred.

[0047] The reducing agent can be used at a final concentration capable of reducing the intensity (e.g., counting) of the detection signal derived from a negative sample. The final concentration refers to the concentration at the time of mixing. Therefore, the final concentration can also be expressed as the concentration in the mixture generated by mixing. Such a final concentration is, for example, 0.05–1000 mM, preferably 0.5–500 mM, more preferably 1–300 mM, and even more preferably 3–200 mM.

[0048] In the mixture containing soluble GPC3 sample and reducing agent, further mixing of the soluble GPC3 sample with a surfactant may also be included. In this case, a mixture containing soluble GPC3 sample, reducing agent, and surfactant is generated.

[0049] Examples of surfactants include nonionic surfactants, amphoteric surfactants, anionic surfactants, and cationic surfactants, as well as their salts. Salts are the same as 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 (Tween20), polyoxyethylene sorbitan monopalmitate (Tween40), polyoxyethylene sorbitan monostearate (Tween60), and polyoxyethylene sorbitan monooleate (Tween80). 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-100). Examples of polyoxyethylene alkyl ethers include polyoxyethylene (23) lauryl ether (Brij35) and polyoxyethylene (20) cetyl ether (Brij58). Examples of preferred nonionic surfactants include polyoxyethylene sorbitan monolaurate (Tween20) and polyoxyethylene (10) octylphenyl ether (Triton X-100). Examples of amphoteric surfactants include, for example, sulfobetaine-type surfactants. Examples of sulfobetaine-type surfactants include 3-[(3-cholesterol aminopropyl)dimethylamino]-1-propanesulfonic acid (CHAPS), 3-[3-cholesterol aminopropyl]dimethylamino)-2-hydroxypropanesulfonic acid (CHAPSO), N-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate, N-tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate, and N-hexadecyl-N,N-dimethyl-3-ammonium-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. The preferred surfactant is a nonionic surfactant, an amphoteric surfactant, or a salt thereof.

[0050] Surfactants can be used at a final concentration that enhances the effect of reducing agents that lower the intensity of detection signals derived from negative samples and / or reduces the matrix effect of the sample. The final concentration refers to the concentration at the time of mixing. Therefore, the final concentration can also be expressed as the concentration in the mixture generated by mixing. Such a final concentration is, for example, 0.005–10% by weight, preferably 0.01–9% by weight, more preferably 0.02–7.5% by weight, even more preferably 0.05–6% by weight, and particularly preferably 0.05–5% by weight.

[0051] Preferably, the reducing agent and surfactant can be used in a ratio that enhances the effect in reducing the intensity of the detection signal derived from the negative sample and / or reducing the matrix effect of the sample. Such a ratio can be given 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.

[0052] When mixing soluble GPC3 sample with both a reducing agent and a surfactant, the mixing can be performed simultaneously or separately. When mixing simultaneously, the mixture containing the soluble GPC3 sample, reducing agent, and surfactant can be combined. When mixing separately, the soluble GPC3 sample can be mixed first with the reducing agent, then with the surfactant, or vice versa.

[0053] The reducing agent and / or surfactant can be dissolved in an aqueous solution for use. 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, and can be 1.0 to 9.0 (preferably 4.0 to 6.0) from the viewpoint of maximizing the effect of the reducing agent. From the viewpoint of ensuring the stable effect of the reducing agent within the desired pH range, the aqueous solution is preferably a buffer solution. The aqueous solution may contain other components such as organic solvents (e.g., alcohols). The volume ratio of the soluble GPC3 sample to the aqueous solution containing the reducing agent and / or surfactant (soluble GPC3 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.

[0054] The mixing is carried out under sufficient conditions of either using a reducing agent alone or by treating the sample with both a reducing agent and a surfactant. Such temperature conditions are, for example, 15–60°C, preferably 20–50°C, and more preferably 25–45°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 intensity of the detection signal originating from negative samples can be reduced.

[0055] The method of the present invention may include further incubation of the mixture after mixing. The incubation time varies depending on factors such as the type and concentration of the reducing agent, the presence or absence of a surfactant, the type and concentration of the surfactant, the mixing time, the desired reduction in the detection signal intensity, and the time required for assays using antibodies (immunoassays), and is, for example, 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 further preferably 20 minutes or less, particularly preferably 10 minutes or less or 5 minutes or less. The incubation temperature is the same as the temperature conditions in the mixing process.

[0056] The present invention also provides an immunoassay method for soluble GPC3, comprising the following (a) and (b):

[0057] (a) Mixing the sample containing soluble GPC3 with a reducing agent;

[0058] (b) The amount of soluble GPC3 in the mixture obtained in (a) was determined using one or more antibodies against soluble GPC3.

[0059] The definitions, examples, and preferred embodiments of various elements in the immunoassay method for soluble GPC3 are the same as those described in the processing method for soluble GPC3-containing samples of the present invention.

[0060] Step (a) can be performed in the same manner as the method for processing soluble GPC3-containing samples of the present invention. Step (b) can be performed by the immunoassay. Steps (a) and (b) can be performed simultaneously or separately. For example, when steps (a) and (b) are performed simultaneously, by simultaneously mixing the soluble GPC3-containing sample, the reducing agent (and surfactant), and one or more antibodies against soluble GPC3, the processing of the soluble GPC3-containing sample using the reducing agent (and surfactant) and the antigen-antibody reaction can be performed simultaneously. However, in order to sufficiently reduce the detection signal intensity (e.g., counting) derived from negative samples, it is preferable to sufficiently process the soluble GPC3-containing sample using the reducing agent (and surfactant), and then determine the amount of soluble GPC3 by using an antigen-antibody reaction using one or more antibodies against soluble GPC3. Furthermore, in the case where the antibody used in the present invention contains disulfide bonds, if the antibody and the reducing agent coexist for a long time, the reducing agent will destroy the antibody by cleaving the disulfide bonds in the antibody, thus affecting the accuracy of the immunoassay. Therefore, when the antibody used in this invention contains disulfide bonds, steps (a) and (b) are preferably performed separately. Of course, when the antibody used in this invention does not contain disulfide bonds (e.g., a single-chain antibody), steps (a) and (b) are preferably performed simultaneously.

[0061] The present invention also provides an immunoassay reagent for soluble GPC3, comprising the following (a) and (b):

[0062] (a) Reducing agent;

[0063] (b) One or more antibodies against soluble GPC3.

[0064] The reagents of the present invention may also include: (c) surfactants.

[0065] The definitions, examples, and preferred examples of the reducing agents, antibodies, and surfactants constituting components (a) to (c) are the same as those described in the processing method for soluble GPC3 specimens of the present invention.

[0066] In a specific embodiment, the reagent of the present invention may be one or more antibodies against soluble GPC3, or 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 comprises a labeled antibody against soluble GPC3 and / or a solid-phase antibody as the two or more antibodies. Alternatively, such a reagent may contain a labeled substance and / or a solid phase without containing a labeled antibody labeled with a labeling substance and / or a solid-phase antibody immobilized on a solid phase. Examples of labeling substances include fluorescent substances, luminescent substances, pigments, and enzymes. When the labeling substance is an enzyme, such a reagent may contain an enzyme substrate (e.g., a substrate that generates a detection signal, or a substrate that is converted by an enzyme into a product that generates a detection signal, or a substrate in a reaction conjugated with another enzyme that utilizes a substrate that generates a detection signal or a substrate that is converted by an enzyme into a product that generates a detection signal). The solid phase is the same as described above.

[0067] The reagent of the present invention can be used to determine specific conditions (e.g., the diagnosis of a disease). The specific conditions (e.g., a disease) are the same as described above. Preferably, the reagent of the present invention can be used for the diagnosis of cancers such as liver cancer and prostate cancer.

[0068] Example

[0069] The present invention will now be described in more detail using examples; however, the present invention is not limited to these examples.

[0070] Reference Example 1: Confirmation of the reactivity of phosphatidylinositol proteoglycan-3 (GPC3) with antibodies

[0071] The recognition sites of anti-GPC3 antibody A and anti-GPC3 antibody B (both monoclonal IgG antibodies) were analyzed by using Western blotting of the recombinant human GPC3 (rhGPC3) (R&D Systems) antigen composed of amino acid residues 1 to 559 of GPC3, or the culture supernatant of human hepatocellular carcinoma cells HepG2 with high GPC3 expression.

[0072] Add SDS-PAGE sample buffer to the culture supernatants of rhGPC3 and HepG2 to prepare 1.4 ng protein / lane and 7.2 ng protein / lane, respectively, and apply the supernatants to a 5-20% polyacrylamide gel (Supersep Ace 5-20%, WAKO). After electrophoresis (30 mA, 60 min), transfer the proteins to a blot membrane (IMMOBILON ISEQ, MERCKMILLIPORE) using a TRANS-BLOT (registered trademark) SD semi-dry electrophoresis transfer unit (Bio-Rad) (15 V, 60 min). After gently washing the blot membrane with TBS-T, shake in blocking buffer (0.5% ECL Block (GE Healthcare Life Sciences), 0.5% BSA, TBS-T) at room temperature for 1 hour. After washing twice with TB ST, the blot membrane was incubated overnight at 4°C with solutions containing anti-GPC3 antibody A and anti-GPC3 antibody B diluted to 1 μg / mL with reaction solution (blocking solution diluted 2-fold with TBS-T). After washing four times with TBS-T, the blot membrane was incubated for 1 hour with solutions containing POD-labeled anti-mouse F(ab')2 (Jackson) diluted 20,000-fold with reaction solution, followed by washing with TBS-T. Colorimetric reactions were performed using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and developed using a Chemiluminescence Imaging System (FUSIONSYSTEM, Vilber Lourmat). The resolution results of the protein blot are shown below. Figure 1 .

[0073] As a result, both anti-GPC3 antibody A and anti-GPC3 antibody B reacted with a 40 kDa band corresponding to the N-terminal fragment of GPC3 (the fragment located to the N-terminus of 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 is confirmed that anti-GPC3 antibody A and anti-GPC3 antibody B recognize soluble GPC3.

[0074] Reference Example 2: Preparation of solidified anti-GPC3 antibody particles

[0075] 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. The suspension was incubated at 5°C for 1 hour with slow stirring to immobilize anti-GPC3 antibody A onto the magnetic particles. The magnetic particles were then magnetized using a magnet and washed with washing buffer (50 mM Tris buffer, 150 mM NaCl, 2.0% BSA, pH 7.2) to obtain immobilized anti-GPC3 antibody A particles. In the assay, the immobilized anti-GPC3 antibody A particles were suspended in particle dilution buffer (50 mM Tris buffer, 1 mM EDTA-2Na, 0.1% NaN3, 2.0% BSA, pH 7.2).

[0076] Reference Example 3: Preparation of alkaline phosphatase-labeled anti-GPC3 antibody

[0077] Desalted alkaline phosphatase (ALP) was mixed with N-(4-maleimidebutyryl)-succinimide (GMBS) (final concentration 0.3 mg / mL) and incubated at 30°C for 1 hour to maleimide ALP. Next, in a coupling reaction buffer (100 mM phosphate buffer, 1 mM EDTA-2Na, pH 6.3), Fab'-labeled anti-GPC3 antibody B was mixed with maleimide ALP at a 1:1 molar ratio and reacted at 25°C for 1 hour. Purification was performed using a Superdex 200 10 / 300 (GE Healthcare) column chromatography, with the major peak separated at a flow rate of 0.5 mL / min using purification buffer (50 mM MES buffer, 150 mM NaCl, 0.1% NaN3, pH 8.0) to obtain ALP-labeled anti-GPC3 antibody B. In the assay, ALP-labeled anti-GPC3 antibody B was suspended in the label dilution buffer (50 mM MES buffer, 150 mM NaCl, 0.3 mM ZnCl2, 1 mM MgCl2, 0.1% NaN3, 2.0% BSA, pH 6.8).

[0078] Reference Example 4: Determination of soluble GPC3

[0079] 20 μL of pretreatment solution was aliquoted 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 was aliquoted 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 aliquoted 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. Then, 200 μL of the LUMIPULSE matrix solution containing 3-(2'-spiroadamantane)-4-methoxy-4-(3”-phosphonooxy)phenyl-1,2-dioxane·2-sodium salt (AMPPD) as the chemiluminescent matrix 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 intensity was measured using a luminometer. The actual measurement was performed using a fully automated chemiluminescent enzyme immunoassay system (LUMIPULSE L2400 (FUJIREBIO)).

[0080] Example 1: Pretreatment of samples using the reducing agent DEAET in the determination of soluble GPC3

[0081] Serum samples from healthy individuals (negative samples) and serum samples from α-cell protein (AFP)-positive hepatocellular carcinoma patients (Trina Biotechnology) (positive samples) were used as soluble GPC3 evaluation samples. As a pretreatment solution, phosphate buffer (10 mM phosphate buffer, 6.25–600 mM DEAET, pH 6.0) containing 6.25 mM–600 mM 2-(dimethylamino)ethanethiol hydrochloride (DEAET) was used. As a control solution, 10 mM phosphate buffer (pH 6.0) was used. Negative samples, positive samples, and buffer samples (10 mM phosphate buffer) were mixed with the pretreatment solution at a 1:1 volume ratio and reacted at 37°C for 6.5 minutes (with pretreatment). Similarly, negative samples, positive samples, and buffer samples were mixed with the control solution and reacted (without pretreatment).

[0082] For each specimen, soluble GPC3 was determined using the assay method described in Example 4. The counts for each specimen are shown in Table 1. In addition, (1) the reduction rate (%), (2) the difference between positive and negative counts (%), and (3) the matrix difference (%), calculated from the counts using the following formulas, are shown in Table 1.

[0083] Calculation formula

[0084] (1) Reduction rate (%) = 100 - (Count of DEAET concentrations with pretreatment / Count of DEAET concentrations without pretreatment) × 100

[0085] (2) Difference in counts between positive and negative samples (%) = (average count of positive samples) / (average count of negative samples) × 100

[0086] (3) Matrix difference (%) = (count of buffer samples) / (average count of negative samples) × 100

[0087] The reduction rate (%) is an indicator used to evaluate the impact of pretreatment on individual samples. A higher reduction rate for negative samples and a lower reduction rate for positive samples results in a greater difference between the measured values ​​of negative and positive samples, further distinguishing them and thus achieving higher diagnostic sensitivity. Therefore, a higher reduction rate for negative samples and a lower reduction rate for positive samples are more useful as pretreatment conditions for soluble GPC3 assay systems.

[0088] The percentage difference between positive and negative counts is an indicator used to evaluate the difference in counts between pretreated positive and negative samples. A larger percentage difference between positive and negative counts indicates higher diagnostic sensitivity, and is therefore useful in soluble GPC3 assay systems.

[0089] Matrix difference (%) is an indicator used to evaluate the difference in counts between buffer samples and negative samples. In immunoassays using samples, the immune response can sometimes be affected by substances contained in the sample (matrix effect). The closer the matrix difference (%) is to 100%, the greater the reduction in matrix effect. Reduced matrix effect allows for the output of true values ​​independent of sample type, and is therefore useful in systems for the determination of soluble GPC3.

[0090] [Table 1]

[0091]

[0092] As shown in Table 1, when negative samples were pretreated with DEAET, the counts of negative samples decreased significantly at all studied concentrations (6.25 mM to 600 mM). On the other hand, when positive samples were pretreated with DEAET, the counts of positive samples did not decrease, or if they did, the decrease was smaller than that of negative samples. Particularly when DEAET was added at concentrations of 50 mM to 400 mM, the difference in counts (%) between positive and negative samples was very large. This indicates that pretreatment of samples using the reducing agent DEAET can improve the discrimination accuracy between positive and negative samples in the determination of soluble GPC3. Furthermore, when negative samples were pretreated with DEAET, the matrix difference was close to 100%, indicating a reduction in the matrix effect. Therefore, pretreatment of samples using the reducing agent DEAET has been confirmed as useful for the determination of soluble GPC3.

[0093] Example 2: Pretreatment of samples using reducing agent 2MEA in the determination of soluble GPC3

[0094] As a pretreatment solution, a phosphate buffer containing 6.25 mM–100 mM 2-mercaptoethylamine hydrochloride (2 MEA) was used (10 mM phosphate buffer, 6.25–100 mM 2 MEA, pH 6.0). As a control solution, 10 mM phosphate buffer (pH 6.0) was used. Negative, positive, and buffer samples were mixed with the pretreatment solution at a 1:1 volume ratio and reacted at 37°C for 6.5 minutes (with pretreatment). Similarly, negative, positive, and buffer samples were mixed with the control solution and reacted (without pretreatment).

[0095] For each specimen, the soluble GPC3 was determined using the assay method described in Example 4. The counts for each specimen are shown in Table 2. Furthermore, the reduction rate (%), the difference between positive and negative counts (%), and the matrix difference (%) were calculated in the same manner as in Example 1.

[0096] [Table 2]

[0097]

[0098] As shown in Table 2, when negative samples were pretreated with 2MEA, the counts of negative samples decreased significantly at all studied concentrations (6.25 mM–100 mM). On the other hand, when positive samples were pretreated with 2MEA, the counts of positive samples did not decrease, or if they did, the decrease was smaller than that of negative samples. This indicates that pretreatment of samples using the reducing agent 2MEA can improve the discrimination accuracy between positive and negative samples in the determination of soluble GPC3. Furthermore, when negative samples were pretreated with 2MEA, the matrix difference was close to 100%, indicating a reduction in the matrix effect. Therefore, pretreatment of samples using the reducing agent 2MEA has been confirmed as useful for the determination of soluble GPC3.

[0099] Example 3: Pretreatment of Specimens Using Reducing Agent TCEP in the Determination of Soluble GPC3

[0100] As a pretreatment solution, a phosphate buffer containing 6.25 mM–100 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) was used (10 mM phosphate buffer, 6.25–100 mM TCEP, pH 6.0). As a control solution, 10 mM phosphate buffer (pH 6.0) was used. Negative, positive, and buffer samples were mixed with the pretreatment solution at a 1:1 volume ratio and reacted at 37°C for 6.5 minutes (with pretreatment). Similarly, negative, positive, and buffer samples were mixed with the control solution and reacted (without pretreatment).

[0101] For each sample, the soluble GPC3 was determined using the assay method described in Example 4. The counts for each sample are shown in Table 3. Furthermore, the reduction rate (%), the difference between positive and negative counts (%), and the matrix difference (%) were calculated in the same manner as in Example 1.

[0102] [Table 3]

[0103]

[0104] As shown in Table 3, when negative samples were pretreated with TCEP, the counts of negative samples decreased significantly at all studied concentrations (6.25 mM–100 mM). On the other hand, when positive samples were pretreated with TCEP, the counts of positive samples did not decrease, or if they did, the decrease was smaller than that of negative samples. This indicates that pretreatment of samples using the reducing agent TCEP can improve the discrimination accuracy between positive and negative samples in the determination of soluble GPC3. Furthermore, when negative samples were pretreated with TCEP, the matrix difference was close to 100%, indicating a reduction in the matrix effect. Therefore, pretreatment of samples using the reducing agent TCEP has been confirmed as useful for the determination of soluble GPC3.

[0105] (Summary of Examples 1-3)

[0106] Considering the results of Examples 1-3, it is believed that pretreatment of samples using a reducing agent is independent of the type and structure of the reducing agent and can improve the discrimination accuracy between positive and negative samples in the determination of soluble GPC3. Furthermore, it is believed that pretreatment of negative samples using a reducing agent can achieve true value output independent of sample type by reducing matrix effects. Therefore, pretreatment of samples using a reducing agent is useful for the determination of soluble GPC3.

[0107] Example 4: Pretreatment of Specimens Using a Combination of Reducing Agent and Surfactant in the Determination of Soluble GPC3

[0108] As a pretreatment solution, the phosphate buffer (pH 6.0) of Test Examples 2–11 shown in Table 4A was used. The phosphate buffer of Test Examples 2–11 contained DEAET as a reducing agent and 3-[(3-cholesterol aminopropyl)dimethylamino]-1-propanesulfonic acid (CHAPS) or polyoxyethylene sorbitan monolaurate (Tween 20) as a surfactant. As a control solution, the 10 mM phosphate buffer (pH 6.0) of Test Example 1 shown in Table 4A was used. Negative, positive, and buffer samples were mixed with the pretreatment solution at a volume ratio of 1:1 and reacted at 37°C for 6.5 minutes (with pretreatment). Similarly, negative, positive, and buffer samples were mixed with the control solution and reacted (without pretreatment).

[0109] For each specimen, the soluble GPC3 was determined using the assay method described in Example 4. The counts for each specimen are shown in Table 4B. Furthermore, the reduction rate (%), the difference between positive and negative counts (%), and the matrix difference (%) were calculated in the same manner as in Example 1.

[0110] [Table 4]

[0111]

[0112] As shown in Table 4, when negative samples were pretreated with a combination of reducing agent and surfactant, the count of negative samples decreased significantly at all surfactant concentrations studied (0.1–10 wt%). On the other hand, when positive samples were pretreated with a combination of reducing agent and surfactant, the decrease in the count of positive samples was smaller than that of negative samples. Particularly when surfactants were added at given concentrations (0.1–2.5 wt% CHAPS and 0.1–10 wt% Tween20), the difference in counts (%) between positive and negative samples was very large. Furthermore, when negative samples were pretreated with a combination of reducing agent and surfactant, the matrix difference was closer to 100%, further reducing the matrix effect. This indicates that pretreatment of samples using a combination of reducing agent and surfactant can improve the discrimination accuracy between positive and negative samples in the determination of soluble GPC3 and can reduce the matrix effect of the samples. Therefore, the use of pretreatment of samples using a combination of reducing agent and surfactant is confirmed to be useful for the determination of soluble GPC3. sequence list <110> Fuji Xerbio Co., Ltd. <120> Processing methods for soluble GPC3-containing samples in immunoassay of soluble GPC3 <130> RB-R033399 <150> JP2021-005934 <151> 2021-01-18 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 580 <212> PRT <213> Homo sapiens <400> 1 Met Ala Gly Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu 1 5 10 15 Ser Leu Asp Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp 20 25 30 Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly 35 40 45 Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val 50 55 60 Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys 65 70 75 80 Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala 85 90 95 Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln 100 105 110 Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala 115 120 125 Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe 130 135 140 Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp 145 150 155 160 Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro 165 170 175 Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu 180 185 190 Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe 195 200 205 Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln 210 215 220 Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile 225 230 235 240 Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu 245 250 255 Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys 260 265 270 Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly 275 280 285 Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu 290 295 300 Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu 305 310 315 320 Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys 325 330 335 Asn Ala Gly Lys Leu Thr Thr Thr Ile Gly Lys Leu Cys Ala His Ser 340 345 350 Gln Gln Arg Gln Tyr Arg Ser Ala Tyr Tyr Pro Glu Asp Leu Phe Ile 355 360 365 Asp Lys Lys Val Leu Lys Val Ala His Val Glu His Glu Glu Thr Leu 370 375 380 Ser Ser Arg Arg Arg Glu Leu Ile Gln Lys Leu Lys Ser Phe Ile Ser 385 390 395 400 Phe Tyr Ser Ala Leu Pro Gly Tyr Ile Cys Ser His Ser Pro Val Ala 405 410 415 Glu Asn Asp Thr Leu Cys Trp Asn Gly Gln Glu Leu Val Glu Arg Tyr 420 425 430 Ser Gln Lys Ala Ala Arg Asn Gly Met Lys Asn Gln Phe Asn Leu His 435 440 445 Glu Leu Lys Met Lys Gly Pro Glu Pro Val Val Ser Gln Ile Ile Asp 450 455 460 Lys Leu Lys His Ile Asn Gln Leu Leu Arg Thr Met Ser Met Pro Lys 465 470 475 480 Gly Arg Val Leu Asp Lys Asn Leu Asp Glu Glu Gly Phe Glu Ser Gly 485 490 495 Asp Cys Gly Asp Asp Glu Asp Glu Cys Ile Gly Gly Ser Gly Asp Gly 500 505 510 Met Ile Lys Val Lys Asn Gln Leu Arg Phe Leu Ala Glu Leu Ala Tyr 515 520 525 Asp Leu Asp Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr Pro 530 535 540 Lys Asp Asn Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His Ser 545 550 555 560 Pro Leu Lys Leu Leu Thr Ser Met Ala Ile Ser Val Val Cys Phe Phe 565 570 575 Phe Leu Val His 580

Claims

1. A method for processing soluble GPC3-containing samples in an immunoassay of soluble GPC3, comprising: The sample containing soluble GPC3 was mixed with a reducing agent. The reducing agent is: 2-(dimethylamino)ethanethiol or its salt, Tris(2-carboxyethyl)phosphine or its salts, 2-Mercaptoethylamine or its salts, 2-Mercaptoethanol, Dithiothreitol, Thioglycerin, Sodium sulfite, or borohydrides, Immunoassay is a sandwich immunoassay using two or more different antibodies targeting two or more different epitopes in the N-terminal fragment of soluble GPC3.

2. The method according to claim 1, wherein, The reducing agent is used at a final concentration of 0.05~1000mM.

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

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

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

6. The method according to claim 1 or 2, wherein, The sample containing soluble GPC3 is a blood sample.

7. The method according to claim 1 or 2, wherein, The soluble GPC3 sample was obtained from the cancerous specimen.

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

9. Use of the reducing agent in the manufacture of a processing agent containing soluble GPC3 sample for a sandwich immunoassay using two or more different antibodies targeting two or more different epitopes in the N-terminal fragment of soluble GPC3. in, The reducing agent is: 2-(dimethylamino)ethanethiol or its salt, Tris(2-carboxyethyl)phosphine or its salts, 2-Mercaptoethylamine or its salts, 2-Mercaptoethanol, Dithiothreitol, Thioglycerin, Sodium sulfite, or Boronhydrides.

10. Use of reducing agents and surfactants in the manufacture of processing agents containing soluble GPC3 samples for sandwich immunoassays using two or more different antibodies targeting two or more different epitopes in the N-terminal fragment of soluble GPC3. in, The reducing agent is: 2-(dimethylamino)ethanethiol or its salt, Tris(2-carboxyethyl)phosphine or its salts, 2-Mercaptoethylamine or its salts, 2-Mercaptoethanol, Dithiothreitol, Thioglycerin, Sodium sulfite, or Boronhydrides.

11. The use according to claim 10, wherein, The surfactant is an amphoteric surfactant or its salt, or a nonionic surfactant.

12. A sandwich immunoassay reagent for soluble GPC3, comprising the following (a) and (b): (a) A reducing agent, wherein the reducing agent is: 2-(dimethylamino)ethanethiol or its salt, Tris(2-carboxyethyl)phosphine or its salts, 2-Mercaptoethylamine or its salts, 2-Mercaptoethanol, Dithiothreitol, Thioglycerin, Sodium sulfite, or borohydrides; (b) Two or more different antibodies targeting two or more different epitopes in the N-terminal fragment of soluble GPC3.

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

14. The sandwich immunoassay reagent according to claim 12 or 13, wherein, The surfactant is an amphoteric surfactant or its salt, or a nonionic surfactant.

15. The immunoassay reagent according to claim 12 or 13, wherein, The reagent is used for cancer diagnosis.

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