Method for measuring analytes and measurement reagent using immune reaction

Abstract

The present invention addresses the problem of improving sensitivity when measuring an analyte in a sample through a sandwich immunoassay method.　In a sandwich immunoassay method using a first antibody and a second antibody, sensitivity can be remarkably improved by making one or both of the first antibody or the second antibody a mixture of: a monoclonal antibody that recognizes a linear epitope; and a monoclonal antibody that recognizes a higher-order epitope.

Description

Technical Field

[0001] The present invention relates to a method for measuring an analyte using an immune reaction and a measurement reagent. More specifically, in a sandwich immunoassay using a first antibody and a second antibody, the present invention relates to a method for measuring an analyte and a measurement reagent in which an antibody that recognizes a linear epitope and an antibody that recognizes a higher-order structure epitope are mixed and used as the first antibody and / or the second antibody.

Background Art

[0002] PIVKA-II refers to a protein that is part of prothrombin, the blood coagulation factor II, which lacks coagulation factor activity and is also known as abnormal prothrombin. Prothrombin is synthesized in the liver, and its production process requires the conversion of glutamic acid (Glu) residues to γ-carboxyglutamic acid (Gla) residues by vitamin K-dependent γ-glutamyl carboxylase. There are 10 Gla residues near the N-terminus of normal prothrombin, but in PIVKA-II, all or some of the 10 remain as Glu residues without being converted to Gla. PIVKA-II was initially found in the blood of patients with vitamin K deficiency or those receiving vitamin K antagonist medications. In recent years, due to its increased blood concentration associated with hepatocellular carcinoma, it has been measured as a tumor marker for hepatocellular carcinoma. PIVKA-II is an abbreviation of protein induced by Vitamin K absence or antagonists-II and is also called des-γ-carboxy prothrombin (DCP) (References: Weitz, I. C., and Liebman, H. A.; (1993) Hepatology 18, 990-997, Suzuki M, Shiraha H, Fujikawa T, Takaoka N, Ueda N, Nakanishi Y, Koike K, Takaki A, Shiratori Y.; J Biol Chem. 2005 Feb 25;280(8):6409-15, A. Nakao, A. Virji, Y. Iwaki, B. Carr, S. Iwatsuki, and E. Starzl; Hepatogastroenterology. 1991 October; 38(5): 450-453).

[0003] The Picolumi® PIVKA-II MONO Kit (Sekisui Medical Co., Ltd.) is commercially available as a reagent for specifically measuring PIVKA-II in samples. This product uses beads conjugated with anti-PIVKA-II mouse monoclonal antibody as the solid phase, and employs a sandwich electrochemiluminescence immunoassay (ECLIA) method using an anti-prothrombin mouse monoclonal antibody labeled with a ruthenium (Ru) complex that emits light upon electrochemical change. In the first reaction, the anti-PIVKA-II mouse monoclonal antibody-conjugated beads are reacted with the sample, resulting in binding of PIVKA-II in the sample to the anti-PIVKA-II mouse monoclonal antibody on the beads. In the second reaction, the beads are washed, and then ruthenium-labeled anti-prothrombin mouse monoclonal antibody is reacted with the PIVKA-II bound to the beads, resulting in a sandwich binding. Furthermore, after washing the beads, application of electrical energy to the electrode causes the ruthenium complex to emit light in response to the amount of ruthenium-labeled anti-prothrombin mouse monoclonal antibody bound to the beads via PIVKA-II. By measuring this luminescence, the amount of PIVKA-II in the sample can be accurately measured (see the Picolumi® PIVKA-II MONO Kit package insert, 5th edition). The Picolumi® PIVKA-II MONO Kit has a measurement range of 10 to 75,000 mAU / mL, featuring high sensitivity and a wide measurement range.

[0004] On the other hand, the upper reference limit for serum or plasma PIVKA-II in healthy adults is 28 mAU / mL, and the cutoff value calculated based on sensitivity and specificity in patients with hepatocellular carcinoma, liver cirrhosis, and chronic hepatitis is 40 mAU / mL. Therefore, this cutoff value is near the lower end of the wide measurement range of the Picolumi (registered trademark) PIVKA-II MONO Kit, and there is a need for the development of a more sensitive measurement method.

[0005] Japanese Patent Application Laid-Open No. 6-113830 discloses that by using a mixture of a plurality of monoclonal antibodies that specifically react with human hemoglobin bound to a carrier, a specific agglutination reaction of human hemoglobin can be easily and accurately performed, human hemoglobin in a biological sample can be specifically and highly sensitively measured, and it can be used for measuring hemoglobin in human feces and urine, and can be applied to the diagnosis of colorectal cancer, kidney diseases, etc.

[0006] The present inventors have been earnestly working on the development of a highly sensitive measurement method for PIVKA-II. In a sandwich immunoassay using a first antibody and a second antibody, when a plurality of labeled monoclonal antibodies are used, it was found that there are combinations in which the sensitivity is significantly improved and combinations in which no improvement in sensitivity is observed, as compared with the case where these plurality of antibodies are used alone.

[0007] When the present inventors determined the epitopes of these antibodies, surprisingly, it was found that the combination of antibodies with significantly improved sensitivity is a combination of a monoclonal antibody that recognizes a linear epitope and a monoclonal antibody that recognizes a higher-order structure epitope, and the present invention was completed.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0009] The problem to be solved by the present invention is to improve the sensitivity when measuring an analyte in a sample by a sandwich immunoassay. In another aspect, the problem to be solved by the present invention is to improve the sensitivity of measurement, particularly when the cut-off value of the analyte is located near the lower limit of the measurement range of the measurement method. [Means for Solving the Problems]

[0010] As a result of intensive studies to solve the above problems, the present inventors have found that in a sandwich immunoassay using a first antibody (single or plural; the same shall apply hereinafter) and a second antibody (single or plural; the same shall apply hereinafter), by using, as one or both of the first antibody and the second antibody, a mixture of a monoclonal antibody that recognizes a linear epitope and a monoclonal antibody that recognizes a higher-order structure epitope, the sensitivity can be significantly improved, and thus the present invention has been completed. That is, the present invention has the following configurations. [Embodiment 1] A sandwich immunoassay for measuring an analyte using a first antibody and a second antibody, wherein one or both of the first antibody and the second antibody is a mixture of a monoclonal antibody that recognizes a linear epitope and a monoclonal antibody that recognizes a higher-order structure epitope. [Embodiment 2] A sandwich immunoassay for measuring an analyte in a sample, comprising the following steps: Providing a sample; Contacting the sample with a first antibody to provide a first reactant; Optionally, recovering a first antibody-analyte complex contained in the first reactant; Contacting the first reactant or the first antibody-analyte complex with a second antibody to provide a second reactant; Recovering a first antibody-analyte-second antibody complex contained in the second reactant; and Measuring the analyte in the sample by measuring the signal of the label of the recovered first antibody-analyte-second antibody complex. Here, the first antibody and the second antibody are antibodies having different epitopes with respect to the analyte, one or both of the first antibody and the second antibody is a mixture of a monoclonal antibody that recognizes a linear epitope and a monoclonal antibody that recognizes a higher-order structure epitope. [Embodiment 3] The method according to Embodiment 1 or 2, wherein the first antibody is an immobilized antibody. [Embodiment 4] The method according to Embodiment 1 or 2, wherein the second antibody is an immobilized antibody. [Embodiment 5] The method according to any one of Embodiments 1 to 3, wherein the second antibody is a labeled antibody. [Embodiment 6] The method according to any one of Embodiments 1, 2, and 4, wherein the first antibody is a labeled antibody. [Embodiment 7] The method according to any one of Embodiments 1 to 6, wherein the analyte is PIVKA-II. [Embodiment 8] The monoclonal antibody that recognizes the linear epitope is an antibody that recognizes a peptide having the amino acid sequence ((E / γ)AF(E / γ)AL(E / γ)SSTATDVFWAKY) of the 26th to 44th amino acids of PIVKA-II, or a peptide having the amino acid sequence ((E / γ)AL(E / γ)SSTATDVFWAKY) of the 29th to 44th amino acids of PIVKA-II, and the method according to any one of Embodiments 1 to 7. [Embodiment 9] The monoclonal antibody that recognizes the higher-order structure epitope is an antibody that recognizes the higher-order structure of the amino acid sequence (SSTATDVFWAKYT) of the 33rd to 45th amino acids of PIVKA-II, or the amino acid sequence (TATDVFWAKYT) of the 35th to 45th amino acids, or the amino acid sequence (TATDVFWA) of the 35th to 42nd amino acids of PIVKA-II, and the method according to any one of Embodiments 1 to 8. [Embodiment 10] A reagent for a sandwich immunoassay for measuring an analyte, comprising a first antibody and a second antibody, wherein one or both of the first antibody and the second antibody is a mixture of a monoclonal antibody that recognizes a linear epitope and a monoclonal antibody that recognizes a higher-order structure epitope. [Embodiment 11] The method according to any one of Embodiments 1 to 9, or the reagent according to Embodiment 10, wherein the mixture is a mixture of two monoclonal antibodies derived from two hybridomas. [Embodiment 12] The method according to any one of Embodiments 1 to 9, or the reagent according to Embodiment 10, wherein the monoclonal antibody that recognizes the linear epitope is a monoclonal antibody derived from a first hybridoma, and the monoclonal antibody that recognizes the higher-order structure epitope is a monoclonal antibody derived from a second hybridoma. [Embodiment 13] The method according to any one of Embodiments 1 to 9, or the reagent according to Embodiment 10, wherein the mixture consists of two immunoglobulin molecules. [Embodiment 14] The method according to any one of Embodiments 1 to 9, or the reagent according to Embodiment 10, wherein the monoclonal antibody that recognizes the linear epitope consists of a first immunoglobulin molecule, and the monoclonal antibody that recognizes the higher-order structure epitope consists of a second immunoglobulin molecule. [Advantages of the Invention]

[0011] According to the present invention, more accurate measurement of an analyte becomes possible. In addition, diagnosis or auxiliary diagnosis of a disease or clinical condition using the analyte as a marker can be performed with higher reliability. In particular, when the cut-off value of the analyte is located near the lower limit of the measurement range of an existing sandwich immunoassay using a first antibody and a second antibody, diagnosis or auxiliary diagnosis of a disease or clinical condition can be performed with higher reliability. [Brief Description of the Drawings]

[0012]

Figure 1

Mode for Carrying Out the Invention

[0013] [Definition]

[0014] First antibody / second antibody: In this specification, the term "first antibody" means one antibody of the sandwich structure (antibody-analyte-antibody complex) formed in the sandwich immunoassay, and the term "second antibody" means the other antibody that forms a complex with the first antibody via the analyte. That is, in the sandwich structure, the first antibody and the second antibody sandwich the analyte. In this specification, when referring to the "first antibody", in one aspect, it means the first monoclonal antibody. In this case, the "first antibody" is not a mixture of a plurality of monoclonal antibodies. However, in another aspect, the term "first antibody" means a set of the first monoclonal antibodies. In this case, the "first antibody" is a mixture of a plurality of monoclonal antibodies. The same applies to the term "second antibody". It will be apparent to those skilled in the art that in order to form the sandwich structure, the first antibody and the second antibody need to recognize different epitopes (not inhibit each other's binding). On the other hand, when the first antibody (or the second antibody) is a mixture of a plurality of monoclonal antibodies, the epitopes of these plurality of monoclonal antibodies do not necessarily need to be different, and they may be the same or partially overlapping. Sample: As used herein, the term "sample" means a biological sample from a mammal, preferably a human. The biological sample may be any sample in which prothrombin may be present (e.g., a sample derived from a tissue expressing prothrombin or a body fluid in which prothrombin is circulating), but blood, serum, plasma, or lymph fluid is preferred. Analyte: With respect to the analyte, there is no particular limitation as long as it is a molecule capable of utilizing an antigen-antibody reaction. Examples include PIVKA-II, prothrombin, CRP (C-reactive protein), Lp(a), MMP3 (matrix metalloproteinase 3), type IV collagen, PSA (prostate-specific antigen), BNP (brain natriuretic peptide), insulin, microalbumin, cystatin C, antiphospholipid antibody, anti-Treponema pallidum antibody, FDP (fibrin-fibrinogen degradation product), D-dimer, SF (soluble fibrin), TAT (thrombin-antithrombin III complex), factor XIII, pepsinogen I and II, etc. However, in principle of the present invention, low molecular weight compounds or short peptide molecules that cannot conceive of linear epitopes or higher-order structure epitopes cannot be used as analytes. PIVKA-II: As used herein, the term "PIVKA-II" means mammalian, preferably human PIVKA-II. Prothrombin: In this specification, both normal and abnormal prothrombin are collectively referred to as "prothrombin". Also, in this specification, "abnormal prothrombin" means PIVKA-II, and "normal prothrombin" means prothrombin other than PIVKA-II. Sandwich immunoassay: The term "sandwich immunoassay" used herein is used in a meaning well-known to those skilled in the art. More specifically, it means an immunoassay that enables specific measurement of an analyte by both a first antibody and a second antibody binding to the analyte to form a so-called "sandwich" structure. Examples of "sandwich immunoassays" known to those skilled in the art include electrochemiluminescence immunoassay (ECLIA), enzyme-linked immunosorbent assay (ELISA), latex turbidimetry immunoassay (LTIA), and lateral flow immunoassay. Linear epitope: As used herein, the term "linear epitope" refers to a site of an antigen that is recognized or bound by an antibody and is formed by the primary structure of a protein or polypeptide. In this specification, terms such as "linear" structure, "linear" structure, "primary" structure, and "1-degree" structure are used synonymously. Higher-order structure epitope: As used herein, the term "higher-order structure epitope" refers to a site of an antigen that is recognized or bound by an antibody, and means a site where an antibody that does not react with a linear peptide extracted from the primary structure of a protein reacts in an antigen having a native higher-order structure. In this specification, the term "conformation" is used synonymously with the term "higher order structure". Monoclonal antibody: As used herein, the term "monoclonal antibody (a / the monoclonal antibody)" refers to a collection of immunoglobulin molecules of a single molecular species having the same structure. When referring to "monoclonal antibody" in this specification, it may be the antibody itself or a fragment having antigen-binding activity such as a Fab fragment or an F(ab')2 fragment. The monoclonal antibody may be obtained by classical immunization of a non-human animal with an antigen, or by genetic recombination technology, gene immunization method, etc., regardless of its acquisition method. In addition, the antibody may be bound to a known labeling substance such as a ruthenium (Ru) complex, peroxidase, alkaline phosphatase, biotin, metal colloid, FITC, etc. Also, the "reagent containing an antibody" may contain an appropriate salt, buffer, preservative, surfactant, reducing agent, cryoprotective substance, etc. In the present invention, the use of two types of monoclonal antibodies is essential, but this is not intended to exclude the use of the third and subsequent types. Furthermore, the two types of monoclonal antibodies immobilized on carrier particles are not excluded from being formulated in a free state without being immobilized on carrier particles. Immobilization: In this specification, the term "immobilization" is used in the same meaning as "fixation" or "sensitization". Carrier particles: Examples of the carrier particles (insoluble carriers) used in the present invention include magnetic beads, organic polymer powders, inorganic substance powders, microorganisms, blood cells, and cell fragments. Examples of the above organic polymer powders include natural polymer powders such as insoluble agarose, cellulose, and insoluble dextran, and synthetic polymer powders such as polystyrene, styrene-styrene sulfonate copolymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylic acid ester copolymer, and vinyl acetate-acrylic acid ester copolymer. In particular, latex particles in which synthetic polymer powders are uniformly suspended are preferable. Examples of the above inorganic substance powders include metal pieces such as gold, titanium, iron, and nickel, and silica, alumina, and carbon powder. The average particle size of the above insoluble carrier is usually 0.05 to 10.0 μm. The particle sizes and materials of the two types of carrier particles carrying two types of monoclonal antibodies may be the same or different. Contacting: In the present specification, when the carrier particles are "contacted" with a biological sample, it means mixing them in any form of solid, aqueous solution, or suspension.

Examples

[0015] [Experimental materials and methods] <Monoclonal antibody (anti-PIVKA-II antibody): MU-3 antibody> (1) Preparation method As the anti-PIVKA-II monoclonal antibody (MU-3 antibody), an antibody prepared by the method described in Example 1 of Japanese Patent Publication No. 5-43357 was used.

[0016] Hereinafter, the antibody preparation method described in Example 1 of Japanese Patent Publication No. 5-43357 is partially omitted and cited. To 100 mg / mL of BaSO4 and BaCO3 were added to the plasma of B warfarin users respectively, and stirred for 120 minutes to adsorb and remove normal prothrombin. Next, it was added to DE-52 cellulose for ion exchange, and then passed through an affinity column using a monoclonal antibody against the common part of both normal prothrombin and PIVKA-II, eluted with 4M guanidine hydrochloride, dialyzed, concentrated, and PIVKA-II was purified. The obtained PIVKA-II (50 μg) was administered intraperitoneally to BALB / C mice (female, 4 weeks old) together with the same volume of Freund's complete adjuvant. Two weeks later, PIVKA-II (15 μg) was administered intravenously through the tail. Three days later, spleen cells were removed and fused with the tumor cell line P3U1. Cell fusion was performed by the method of Watanabe et al. using polyethylene glycol 4000. Next, three rounds of cloning were performed by the limiting dilution method using a 96-well microplate. For the assay for cloning, the carboxylated human prothrombin in the above A and finally native PIVKA-II were used. Each of the cell lines of antibody-producing hybridomas established by cloning was labeled with identification symbols such as..., MU-3,.... From the cell line MU-3, a monoclonal anti-PIVKA-II antibody was obtained by a conventional method.

[0017] As described in the above citation, the screening of the anti-PIVKA-II antibody produced by the hybridoma obtained by immunizing with PIVKA-II was carried out through assays for cloning of antibody-producing hybridomas using carboxylated human prothrombin and finally native PIVKA-II.

[0018] In addition, the epitope site of the MU-3 antibody has been identified and confirmed to be the "decarboxypeptide site at amino acid positions 13 to 23 of prothrombin" through a detailed study of the binding ability using various lengths of peptide fragments synthesized based on the Gla region amino acid sequence of PIVKA-II (Japanese Patent Laid-Open No. 7-20127). Therefore, in addition to the native PIVKA-II and decarboxylated human prothrombin described in Japanese Patent Publication No. 5-43357, various lengths of peptide fragments synthesized based on the Gla region amino acid sequence of PIVKA-II described in Japanese Patent Laid-Open No. 7-20127, etc., can be appropriately combined to compare and confirm the binding ability of the antibody to PIVKA-II and substances other than PIVKA-II, thereby screening for anti-PIVKA-II antibodies. Thus, new anti-PIVKA-II antibodies other than the MU-3 antibody can also be obtained. This specification includes the entire descriptions of Japanese Patent Publication No. 5-43357 (Japanese Patent Publication No. 5-43357) and Japanese Patent Laid-Open No. 7-20127 by reference.

[0019] Furthermore, anti-PIVKA-II antibodies are also known other than MU-3. For example, the 2G4 antibody described in Japanese Patent Laid-Open No. 9-43237, the antibodies described in Japanese Patent Publication No. 60-60557, and the antibodies described in Japanese Patent Laid-Open No. 7-313186 are also considered to be usable as the anti-PIVKA-II antibody of the present invention.

[0020] <Monoclonal antibody (anti-prothrombin F1 region antibody): 242202 antibody, 242203 antibody, 242205 antibody, 242206 antibody> (1) Preparation method i) Preparation of hybridoma Purified silkworm-expressed PIVKA-II (manufactured by Ozeki Co., Ltd.) or synthetic peptide (amino acid sequence positions 29-47) (SEQ ID NO: 1) and Freund's complete adjuvant (manufactured by GIBCO) were mixed and emulsified one-to-one to form an emulsion, and administered subcutaneously to 8-week-old female BALB / c mice (manufactured by Charles River Laboratories Japan) at a dose of 20 μg / 100 μL five times at 2-week intervals. Three days after the final immunization, the spleen was removed. Spleen cells obtained from the removed spleen and myeloma cells SP2 / O-Ag14 were mixed at a ratio of 10 to 1, and cell fusion was performed in the presence of 50 wt% polyethylene glycol 1540 (manufactured by Wako Pure Chemical Industries, Ltd.). The fused cells were suspended in HAT medium to a concentration of 2.5×10 6 cells / mL and dispensed 0.2 mL each into a 96-well culture plate (manufactured by CORNING). This was cultured at 37°C in a 5% CO2 incubator. Approximately 2 weeks later, the culture supernatant of the wells in which hybridomas had grown was evaluated according to the ELISA method shown below, and hybridomas producing antibodies reactive to PIVKA-II were selected. Specifically, first, PIVKA-II prepared by the method of Bajah et al. (the method described in S. Paul Bajah, Paul A. Price, and William A. Russell, Decarboxylation of γ-Carboxyglutamic Acid Residues in Human Prothrombin, Journal of Biological Chemistry 257(7):3726, 1982) was immobilized at 1 μg / mL on a 96-well plate for ELISA (manufactured by NUNC). After reacting each culture supernatant with this, a peroxidase-labeled goat anti-mouse IgG antibody (jackson Immuno Research) was reacted, and then OPD coloring solution was added to cause coloring. After adding the stop solution to stop the coloring, it was measured with a microplate reader (Abs. 492 nm), and hybridoma strains that react with PIVKA-II were selected. From among the selected hybridoma strains, hybridomas 242202, 242203, 242205, and 242206 that give good results when constructing a sandwich immunoassay system in combination with the MU-3 antibody were obtained. Hybridomas 242202, 242203, and 242205 were obtained from mice immunized with PIVKA-II, and hybridoma 242206 was obtained from mice immunized with a synthetic peptide. ii) Preparation of monoclonal antibody From hybridomas 242202, 242203, 242205, and 242206, 242202 monoclonal antibody (202 antibody), 242203 monoclonal antibody (203 antibody), 242205 monoclonal antibody (205 antibody), and 242206 monoclonal antibody (206 antibody) were prepared by the following methods, respectively. The hybridomas were suspended in a serum-free medium (ESCLONE SF-B) at 1×10 5After seeding in a flask at 4 mL and culturing up to 1 L, a culture solution containing the antibody was obtained. The culture solution was centrifuged to obtain the supernatant. The supernatant was mixed with an equal volume of adsorption buffer (3 mol / L NaCl - 1.5 mol / L Glycine-NaOH, pH 8.5) and then filtered. This filtrate was passed through a Protein A column (HiTrap rProteinA FF, manufactured by GE Healthcare Japan) equilibrated with the adsorption buffer to adsorb the antibody to the column. Then, it was eluted with 0.1 mol / L citrate buffer (pH 3.0) to purify the monoclonal antibody. (2) Determination of Monoclonal Antibody Epitope i) Analysis with Linear Peptides By preliminary analysis, it was revealed that all of the antibodies produced by hybridomas 242202, 242203, 242205, and 242206 bind to the Fragment 1 (F1) region of PIVKA-II. To identify the epitopes of these antibodies, the antibodies were screened by their reactivity with peptides corresponding to the amino acid sequence from the 26th to the 47th amino acids of PIVKA-II. This region is the region from the C-terminal side of the Gla domain in the F1 region of PIVKA-II to just upstream of the thrombin cleavage site (position 51↓position 52). The region after the 33rd amino acid of PIVKA-II has an amino acid sequence common to the corresponding region of prothrombin. For the screening, peptides L1 - L7 (SEQ ID NOs: 2 - 8) purchased from Toray Industries, Inc. were used. The N-terminus of each peptide was biotinylated. The peptides were dissolved in PBS to a concentration of 10 nM. DynabeadsTM M-270 Streptavidin was suspended in these peptide solutions to prepare a 10 mg / mL solution. After standing for 10 minutes, it was washed three times with the following Ru diluent (1) to obtain peptide solid-phase magnetic beads, and the beads were diluted to 1 mg / mL to obtain a peptide solid-phase magnetic bead solution. To 1 mL of anti-mouse IgG Fab antibody (prepared at 1 mg / mL), 68 μL (10 mg / mL) of a Ru complex compound of succinimidyl group-modified ruthenium tris(dipyridyl) was added, and the reaction was carried out with stirring at room temperature for 30 minutes. 50 μL of 2 mol / L glycine was added to stop the reaction, and the reaction was further carried out with stirring at room temperature for 10 minutes. Finally, the sample was flowed through Sephadex G-25 (equilibrated with 10 mmol / L phosphate buffer), and the protein fraction with Ru binding was collected to prepare a Ru-labeled anti-mouse IgG Fab antibody (hereinafter sometimes abbreviated as "Ru-labeled anti-mouse antibody"). The obtained Ru-labeled anti-mouse IgG Fab antibody was diluted to a final concentration of 2 μg / mL with Ru diluent (1) at the time of use. The composition of Ru diluent (1) is shown below. Composition of Ru diluent (1): 50 mM HEPES, 1 mM EDTA-4Na, 0.05% NaN3, 1% BSA, 0.1% Tween20, pH 7.8 Using the prepared peptide solid-phase magnetic bead solution, Ru-labeled anti-mouse antibody solution, and ECLIA automatic measuring device "Picolumi III" (manufactured by Sekisui Medical Co., Ltd.), the epitope of the obtained monoclonal antibody was analyzed by electrochemiluminescence immunoassay. To 100 μL of Ru diluent (1), 20 μL of each monoclonal antibody solution (0.5 μg / mL) and 12.5 μL of peptide solid-phase magnetic beads were added and reacted for 9 minutes. After adding 200 μL of Ru-labeled anti-mouse antibody solution diluted to 0.2 mg / mL with Ru diluent (1) and reacting for 9 minutes, the magnetic particles were washed and measured. A count value less than 2000 was recorded as -, 2000 or more and less than 10000 as +-, 10000 or more and less than 20000 as +, 20000 or more and less than 30000 as ++, and 30000 or more as +++. The analysis results were summarized in Table 1 below.

Table 1

[0021] <Preparation of <MU-3 monoclonal antibody-immobilized magnetic bead solution>> 2.0 g of Micropearl EX-003 (particle size 3.01 μm, CV 3.1%, manufactured by Sekisui Chemical Co., Ltd.) as resin particles was ultrasonically dispersed in 40.0 g of ion-exchanged water to obtain a core particle dispersion liquid. Subsequently, while stirring under ultrasonic irradiation, 4.0 mL of magnetic fluid EMG707 (manufactured by Ferrotec Corporation) was added, and further ultrasonically dispersed for 30 minutes. The obtained dispersion liquid was filtered and washed with ion-exchanged water to remove the excess magnetic fluid, thereby obtaining magnetic-responsive beads. After ultrasonic dispersion of 1.0 g of the above magnetic-responsive beads in 400 g of ethanol, 10 mL of 28% aqueous ammonia solution (manufactured by Nacalai Tesque, Inc.), 1.0 g of tetraethyl orthosilicate, and 3.0 g of 8-glycidoxyoctyltrimethoxysilane were added, and ultrasonic dispersion was performed for 3 hours. After filtering the obtained dispersion, dispersion in ion-exchanged water and centrifugation were repeated three times to obtain magnetic-responsive beads EP having epoxy groups on the surface. After ultrasonic dispersion of the magnetic-responsive beads EP in PBS to a concentration of 3.0% by weight, 0.5 mL was aliquoted into a test tube. After trapping with a magnet and discarding the supernatant, 1 mL of MU-3 monoclonal antibody (1.07 mg / mL, 150 mM phosphate buffer, pH 7.8) was added to the magnetic beads, and the reaction was carried out with stirring at 25°C for 72 hours. After washing the magnetic beads, 2 mL of 1% BSA-containing phosphate buffer was added, and blocking was carried out with stirring at room temperature for one day and night to prepare a MU-3 monoclonal antibody-immobilized magnetic bead solution (hereinafter sometimes abbreviated as "MU-3 antibody beads"). At the time of use, the amount of magnetic beads was diluted to 1 mg / mL with a bead diluent and used. The composition of the bead diluent is shown below. Composition of bead diluent: 0.025 mol / L Tris buffer (pH 7.8), 0.01 mol / L NaCl, 0.025% Tween 20, 0.09% NaN3, 0.5 mM EDTA-2Na, 1% sucrose, 0.5% bovine serum albumin

[0022] <Preparation of <Ru>-labeled anti-prothrombin F1 region monoclonal antibody solution> To 1 mL of anti-prothrombin F1 region monoclonal antibody (prepared at 1 mg / mL), 68 μL (10 mg / mL) of a Ru complex compound of succinimidyl group-modified ruthenium tris-dipyridyl was added, and the reaction was carried out with stirring at room temperature for 30 minutes. 50 μL of 2 mol / L glycine was added to stop the reaction, and the mixture was further stirred at room temperature for 10 minutes. Finally, the sample was passed through Sephadex G-25 (equilibrated with 10 mmol / L phosphate buffer), and the protein fraction with Ru binding was collected to prepare a Ru-labeled anti-prothrombin F1 region monoclonal antibody (hereinafter sometimes abbreviated as "Ru-labeled anti-F1 antibody"). The obtained Ru-labeled anti-F1 antibody was diluted to a final concentration of 2 μg / mL with Ru diluent (2) at the time of use. The composition of Ru diluent (2) is shown below. Composition of Ru diluent (2): 0.05 mol / L HEPES buffer (pH 7.8), 0.09% NaN3, 0.2 mg / mL mouse IgG, 0.003 M EDTA-4Na, 0.1% Tween 20

[0023] <Measurement of PIVKA-II> PIVKA-II measurements were performed using the ECLIA Picolumi III automated analyzer. Standard antigen solutions containing PIVKA-II standard antigen at 0 mAU / mL, 10 mAU / mL, 100 mAU / mL, and 1000 mAU / mL were prepared. The required number of reaction tubes were prepared, and 100 μL of the reaction solution was poured into each reaction tube. 20 μL of the standard antigen solution was poured into each of two reaction tubes. For the first reaction, 25 μL of MU-3 monoclonal antibody-immobilized magnetic bead solution was poured, and the reaction was carried out at a temperature of 30 ± 2°C for 5 minutes. The reaction solution was stirred for several seconds at regular intervals during the reaction. A magnet was brought close to the reaction tube to collect the beads on the wall of the reaction tube, and the solution in the reaction tube was then aspirated and removed. For the washing step, 350 μL of Picolumi BF washing solution (manufactured by Sekisui Medical Co., Ltd.) was poured into the reaction tube and stirred. A magnet was brought close to the reaction tube to collect the beads on the wall of the reaction tube, and the liquid in the reaction tube was then aspirated and removed. This washing step was repeated once more. Next, for the second step, 100 μL of Ru-labeled anti-F1 antibody solution was poured into the reaction tube, and the reaction was carried out at a temperature of 30 ± 2°C for 3 minutes. The reaction solution was stirred for several seconds at regular intervals during the reaction. For the washing step, 350 μL of BF washing solution was poured into the reaction tube and stirred. A magnet was brought close to the reaction tube to collect the beads on the wall of the reaction tube, and the liquid in the reaction tube was then aspirated and removed. This washing step was repeated once more. Finally, 300 μL of luminescent electrolyte (manufactured by Sekisui Medical Co., Ltd.) was poured into the reaction tube, and the beads were introduced to a flow cell electrode to measure the amount of luminescence.

[0024] [Results and Discussion] PIVKA-II was measured using the following Ru-labeled anti-F1 antibodies: antibody 202 only, antibody 203 only, antibody 205 only, antibody 206 only, a mixture of antibodies 202 and 203, a mixture of antibodies 202 and 205, a mixture of antibodies 202 and 206, a mixture of antibodies 203 and 205, a mixture of antibodies 203 and 206, and a mixture of antibodies 205 and 206. The results are shown in Table 2 and Figure 1.

[0025] [Table 2]

[0026] As is clear from Table 2 and FIG. 1, when antibodies that recognize linear epitopes (antibody 202, antibody 203) and antibodies that recognize higher-order structure epitopes (antibody 205, antibody 206) were used in combination (indicated by hatching), a significant increase in the measured value (count number) of 10 mAU / mL was observed.

[0027] Although not bound by any particular theory, it is expected that in the reaction solution, a part of the F1 region of PIVKA-II (which has the same primary sequence as the F1 region of prothrombin) is denatured and has lost its native higher-order structure. By combining an antibody that recognizes a linear epitope and an antibody that recognizes a higher-order structure epitope, there is a possibility that PIVKA-II contained in the reaction solution can be captured without leakage.

Industrial Applicability

[0028] The analyte measurement method and measurement reagent using the immune reaction according to the present invention can be used for the diagnosis or auxiliary diagnosis of hepatocellular carcinoma and the like. Further, according to the present invention, the sensitivity of the existing analyte measurement method and measurement reagent using an immune reaction can be improved.

Claims

1. A sandwich immunoassay for measuring an analyte using a first antibody and a second antibody, wherein the first antibody and the second antibody form a sandwich structure via the analyte, and at least one of the first antibody and the second antibody is a mixture of a monoclonal antibody that recognizes a linear epitope and a monoclonal antibody that recognizes a higher-order structure epitope.

2. A sandwich immunoassay for measuring an analyte in a sample, comprising the following steps: Contacting the sample with a first antibody to provide a first reactant; Optionally, recovering the first antibody-analyte complex contained in the first reactant; Contacting the first reactant or the first antibody-analyte complex with a second antibody to provide a second reactant; Recovering the first antibody-analyte-second antibody complex contained in the second reactant; And Measuring the analyte in the sample by measuring the signal of the label of the recovered first antibody-analyte-second antibody complex; Here, The first antibody and the second antibody are antibodies with different epitopes for the analyte, And at least one of the first antibody and the second antibody is a mixture of a monoclonal antibody that recognizes a linear epitope and a monoclonal antibody that recognizes a higher-order structure epitope.

3. The method according to claim 1 or 2, wherein the first antibody is a solid-phase antibody.

4. The method according to any one of claims 1 to 3, wherein the second antibody is a labeled antibody.

5. The method according to any one of claims 1 to 4, wherein the analyte is PIVKA-II.

6. The monoclonal antibody that recognizes the linear epitope is an antibody that recognizes a peptide having the amino acid sequence ((E / γ)AF(E / γ)AL(E / γ)SSTA TDVF WAKY) from the 26th to the 44th amino acids of PIVKA-II, or a peptide having the amino acid sequence ((E / γ)AL(E / γ)SSTA TDVF WAKY) from the 29th to the 44th amino acids of PIVKA-II. The method according to any one of claims 1 to 5.

7. The method according to any one of claims 1 to 6, wherein the monoclonal antibody that recognizes the higher-order structure epitope is an antibody that recognizes the higher-order structure of the amino acid sequence from the 33rd to the 45th (SSTATDVFWAKYT), or the amino acid sequence from the 35th to the 45th (TATDVFWAKYT), or the amino acid sequence from the 35th to the 42nd (TATDVFWA) of PIVKA-II.

8. A reagent for a sandwich immunoassay for measuring an analyte, comprising a first antibody and a second antibody, wherein at least one of the first antibody and the second antibody that forms a sandwich structure via the analyte is a mixture of a monoclonal antibody that recognizes a linear epitope and a monoclonal antibody that recognizes a higher-order structure epitope.

Images