Analyte measurement method and measurement reagent using immunoreactions

Abstract

To improve sensitivity when measuring an analyte in a sample using the sandwich immunoassay.SOLUTION: In a sandwich immunoassay using a first antibody and a second antibody, sensitivity can be significantly improved by using a mixture of a monoclonal antibody that recognizes linear epitopes and a monoclonal antibody that recognizes conformational epitopes as either or both of the first antibody and the second antibody.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method and reagent for measuring an analyte using an immune reaction, and more specifically to a sandwich immunoassay using a first antibody and a second antibody, in which an antibody that recognizes a linear epitope and an antibody that recognizes a conformational epitope are mixed as the first antibody and / or the second antibody. [Background technology]

[0002] PIVKA-II, also known as abnormal prothrombin, is a type of blood coagulation factor II that lacks clotting factor activity. Prothrombin is synthesized in the liver, and its synthesis requires the conversion of glutamic acid (Glu) residues to gamma-carboxyglutamic acid (Gla) residues by vitamin K-dependent gamma glutamyl carboxylase. Normal prothrombin contains 10 Gla residues near the N-terminus, but in PIVKA-II, some or all of the 10 remain as Glu residues without being converted to Gla. PIVKA-II was initially identified in the blood of patients with vitamin K deficiency or receiving vitamin K antagonists. Recently, PIVKA-II has been measured as a tumor marker for hepatocellular carcinoma (HCC), as its blood concentration increases with the development of HCC. PIVKA-II stands for protein induced by Vitamin K absence or antagonists-II and is also called des-γ-carboxy prothrombin (DCP) (References: Weitz, IC, and Liebman, HA; (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 Laid-Open Publication No. 6-113830 discloses that by using a mixture of multiple monoclonal antibodies that react specifically with human hemoglobin bound to a carrier, a human hemoglobin specific agglutination reaction can be performed accurately and simply, and human hemoglobin in a biological sample can be measured specifically and with high sensitivity, and the method can be used to measure hemoglobin in human feces and urine, and can be applied to the diagnosis of colon cancer, kidney diseases, etc.

[0006] While the inventors were working hard to develop a highly sensitive method for measuring PIVKA-II, they noticed that in a sandwich immunoassay using a first antibody and a second antibody, when multiple labeled monoclonal antibodies are used, there are combinations that significantly improve sensitivity compared to when those multiple antibodies are used alone, and combinations that do not show any improvement in sensitivity.

[0007] The present inventors determined the epitopes of these antibodies and surprisingly found that the antibody combination that significantly improved sensitivity was a combination of a monoclonal antibody that recognizes a linear epitope and a monoclonal antibody that recognizes a conformational epitope, thereby completing the present invention. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Publication No. 6-113830 [Patent Document 2] Special Publication No. 5-43357 Summary of the Invention [Problem to be solved by the invention]

[0009] The problem to be solved by the present invention is to improve the sensitivity of sandwich immunoassays for measuring an analyte in a sample, particularly when the cutoff value for the analyte is near the lower limit of the measurement range of the assay. [Means for solving the problem]

[0010] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have discovered that in a sandwich immunoassay using a first antibody(ies) and a second antibody(ies), sensitivity can be significantly improved by using a mixture of a monoclonal antibody that recognizes a linear epitope and a monoclonal antibody that recognizes a conformational epitope as one or both of the first and second antibodies, and have thus completed the present invention. That is, the present invention has the following features. [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 are a mixture of monoclonal antibodies that recognize linear epitopes and monoclonal antibodies that recognize conformational epitopes. [Embodiment 2] A sandwich immunoassay method for determining an analyte in a sample, comprising the steps of: providing a sample; contacting the sample with a first antibody to provide a first reactant; optionally, recovering the first antibody-analyte complex contained in said first reaction; 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 reaction product; and measuring the signal from the label of the recovered first antibody-analyte-second antibody complex to measure the analyte in the sample; where: the first antibody and the second antibody are antibodies that have different epitopes against the analyte, One or both of the first and second antibodies is a mixture of monoclonal antibodies that recognize linear epitopes and monoclonal antibodies that recognize conformational epitopes. [Embodiment 3] 3. The method of claim 1 or 2, wherein the first antibody is an immobilized antibody. [Embodiment 4] 3. The method of claim 1 or 2, wherein the second antibody is a solid-phase antibody. [Embodiment 5] 4. The method according to any one of embodiments 1 to 3, wherein the second antibody is a labeled antibody. [Embodiment 6] 5. The method of any one of embodiments 1, 2, and 4, wherein the first antibody is a labeled antibody. [Embodiment 7] 7. The method of any of embodiments 1 to 6, wherein the analyte is PIVKA-II. [Embodiment 8] A method according to any one of embodiments 1 to 7, wherein the monoclonal antibody recognizing the linear epitope is an antibody recognizing a peptide having the sequence of amino acids 26 to 44 of PIVKA-II ((E / γ)AF(E / γ)AL(E / γ)SSTATDVFWAKY), or a peptide having the sequence of amino acids 29 to 44 of PIVKA-II ((E / γ)AL(E / γ)SSTATDVFWAKY). [Embodiment 9] The method according to any one of embodiments 1 to 8, wherein the monoclonal antibody recognizing the conformational epitope is an antibody recognizing the conformation of the amino acid sequence from the 33rd to the 45th positions of PIVKA-II (SSTATDVFWAKYT), or the amino acid sequence from the 35th to the 45th positions of PIVKA-II (TATDVFWAKYT), or the amino acid sequence from the 35th to the 42nd positions of PIVKA-II (TATDVFWA). [Embodiment 10] A sandwich immunoassay reagent 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 conformational 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 types of monoclonal antibodies derived from two types of hybridomas. [Embodiment 12] 11. The method according to any one of embodiments 1 to 9, or the reagent according to embodiment 10, wherein the monoclonal antibody recognizing the linear epitope is a monoclonal antibody derived from a first hybridoma, and the monoclonal antibody recognizing the conformational epitope is a monoclonal antibody derived from a second hybridoma. [Embodiment 13] 11. The method according to any one of embodiments 1 to 9, or the reagent according to embodiment 10, wherein the mixture consists of two types of immunoglobulin molecules. [Embodiment 14] 11. The method according to any one of embodiments 1 to 9, or the reagent according to embodiment 10, wherein the monoclonal antibody recognizing the linear epitope comprises a first immunoglobulin molecule, and the monoclonal antibody recognizing the conformational epitope comprises a second immunoglobulin molecule. [Effects of the Invention]

[0011] The present invention enables more accurate measurement of analytes. Furthermore, diagnosis or assistance in diagnosis of diseases or clinical conditions using the analytes as markers can be performed with higher reliability. In particular, when the cutoff value of the analyte is near the lower limit of the measurement range of existing sandwich immunoassays using a first antibody and a second antibody, diagnosis or assistance in diagnosis of diseases or clinical conditions can be performed with higher reliability. [Brief explanation of the drawings]

[0012] [Figure 1]The graph shows plots of the measured values ​​(counts) of standard antigen solutions of 10 mAU / mL, 100 mAU / mL, and 1000 mAU / mL, divided into two cases: (1) when only a monoclonal antibody recognizing a linear epitope or a monoclonal antibody recognizing a conformational epitope was used as the Ru-labeled anti-F1 antibody, and (2) when a mixture of a monoclonal antibody recognizing a linear epitope and a monoclonal antibody recognizing a conformational epitope was used as the Ru-labeled anti-F1 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0013] [Definition]

[0014] First antibody / second antibody: As used herein, the term "first antibody" refers to one of the antibodies in the sandwich structure (antibody-analyte-antibody complex) formed in a sandwich immunoassay, and the term "second antibody" refers to 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. As used herein, the term "first antibody" refers, in one embodiment, to a first monoclonal antibody. In this case, the "first antibody" is not a mixture of multiple monoclonal antibodies. However, in another embodiment, the term "first antibody" refers to a set of first monoclonal antibodies. In this case, the "first antibody" is a mixture of multiple monoclonal antibodies. The same applies to the term "second antibody." It will be clear to those skilled in the art that, in order to form the sandwich structure, the first and second antibodies must recognize different epitopes (i.e., they do not inhibit each other's binding). On the other hand, when the first antibody (or second antibody) is a mixture of multiple monoclonal antibodies, the epitopes of these multiple monoclonal antibodies do not necessarily have to be different, and may be the same or may partially overlap. Sample: As used herein, the term "sample" refers to a mammalian, preferably human, biological sample. The biological sample may be any sample in which prothrombin may be present (e.g., a sample derived from a tissue that expresses prothrombin or a body fluid in which prothrombin circulates), but blood, serum, plasma, or lymph is preferred. Analyte: The substance to be measured is not particularly limited as long as it is a molecule that can utilize an antigen-antibody reaction, and 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 antibodies, anti-Treponema pallidum antibodies, FDP (fibrin-fibrinogen degradation products), D-dimer, SF (soluble fibrin), TAT (thrombin-antithrombin III complex), factor XIII, and pepsinogen I and II. However, due to the principles of the present invention, low-molecular-weight compounds and short peptide molecules that do not have linear or conformational epitopes cannot be used as analytes. PIVKA-II: When "PIVKA-II" is referred to herein, it refers to mammalian, preferably human, PIVKA-II. Prothrombin: In this specification, both normal and abnormal prothrombin are collectively referred to as "prothrombin." Furthermore, in this specification, "abnormal prothrombin" refers to PIVKA-II, and "normal prothrombin" refers to prothrombin other than PIVKA-II. Sandwich immunoassay: As used herein, the term "sandwich immunoassay" is used in the sense known to those skilled in the art. More specifically, it refers to an immunoassay in which both a first antibody and a second antibody bind to an analyte to form a so-called "sandwich" structure, thereby enabling specific measurement of the analyte. Examples of "sandwich immunoassays" known to those skilled in the art include electrochemiluminescence immunoassay (ECLIA), enzyme-linked immunosorbent assay (ELISA), latex turbidimetric immunoassay (LTIA), and lateral flow immunoassay. Linear epitope: As used herein, the term "linear epitope" refers to a site on an antigen that is recognized or bound by an antibody and is formed by the primary structure of a protein or polypeptide. As used herein, the terms "linear," "linear," "primary," "primary" and "primary" structure are used interchangeably. Conformational epitope: As used herein, the term "conformational epitope" refers to a site on an antigen that is recognized or bound by an antibody, and that reacts with an antibody that does not react with a linear peptide derived from the primary structure of a protein, in an antigen that has a native higher order structure. As used herein, the term "conformation" is used synonymously with the term "higher order structure." Monoclonal antibody: As used herein, the term "monoclonal antibody" refers to a group of immunoglobulin molecules of a single molecular species with the same structure. The term "monoclonal antibody" as used herein refers to not only the antibody itself but also a fragment thereof, such as a Fab fragment or F(ab')2 fragment, that has antigen-binding activity. The monoclonal antibody may be obtained by any method, including classical methods such as immunization of non-human animals with an antigen, as well as methods such as genetic recombination and genetic immunization. The antibody may be conjugated to a known labeling substance, such as a ruthenium (Ru) complex, peroxidase, alkaline phosphatase, biotin, metal colloid, or FITC. The "antibody-containing reagent" may also contain appropriate salts, buffers, preservatives, surfactants, reducing agents, cryoprotectants, etc. Although the present invention requires the use of two types of monoclonal antibodies, this does not preclude the use of three or more types. Furthermore, the two types of monoclonal antibodies that are used by immobilizing them on carrier particles may also be formulated in a free state without being immobilized on carrier particles. Immobilization: As used herein, the term "immobilization" is used synonymously with "immobilization" or "sensitization." Carrier particles: Examples of carrier particles (insoluble carriers) used in the present invention include magnetic beads, organic polymer powders, inorganic powders, microorganisms, blood cells, and cell fragments. Examples of the organic polymer powder 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-acrylate copolymer, and vinyl acetate-acrylate copolymer. Latex particles, which are uniformly suspended synthetic polymer powders, are particularly preferred. Examples of the inorganic powder include metal pieces such as gold, titanium, iron, and nickel, silica, alumina, and carbon powder. The insoluble carrier usually has an average particle size of 0.05 to 10.0 μm. The particle sizes and materials of the two types of carrier particles that carry the two types of monoclonal antibodies may be the same or different. Contacting: As used herein, "contacting" carrier particles with a biological sample means mixing them together in the form of a solid, an aqueous solution, or a suspension. [Example]

[0015] [Experimental Materials and Methods] <Monoclonal antibody (anti-PIVKA-II antibody): MU-3 antibody> (1) Creation method The anti-PIVKA-II monoclonal antibody (MU-3 antibody) used was an antibody produced by the method described in Example 1 of Japanese Patent Publication No. 5-43357.

[0016] The antibody production method described in Example 1 of Japanese Patent Publication No. 5-43357 will be quoted below with some omissions. "B. Plasma from a warfarin patient was treated with 100 mg / mL BaSO4 and 100 mg / mL BaCO3. The mixture was stirred for 120 minutes to remove normal prothrombin. The mixture was then loaded onto DE-52 cellulose for ion exchange, applied to an affinity column using monoclonal antibodies against both normal prothrombin and PIVKA-II. The column was eluted with 4 M guanidine hydrochloride, dialyzed, and concentrated to purify PIVKA-II. The resulting PIVKA-II (50 μg) was administered intraperitoneally to BALB / C mice (female, 4 weeks old) with an equal volume of Freund's complete adjuvant. Two weeks later, PIVKA-II (15 μg) was administered intraperitoneally via the tail vein. Three days later, splenocytes were removed and fused with the tumor cell line P3U1. Cell fusion was performed using polyethylene glycol 4000 according to the method of Watanabe et al. Three clones were then cloned by limiting dilution in a 96-well microplate. The assays for cloning used decarboxylated human prothrombin (A) and ultimately native PIVKA-II. The antibody-producing hybridoma cell lines established by cloning were given the identification symbols ..., MU-3, and .... Monoclonal anti-PIVKA-II antibodies were obtained from cell line MU-3 by standard methods.

[0017] As described in the above reference, screening of anti-PIVKA-II antibodies produced by hybridomas obtained by immunization with PIVKA-II was carried out through assays for cloning antibody-producing hybridomas using decarboxylated human prothrombin and ultimately native PIVKA-II.

[0018] Furthermore, detailed binding studies using peptide fragments of various lengths synthesized based on the amino acid sequence of the Gla region of PIVKA-II have identified and confirmed that the epitope site of the MU-3 antibody is the "decarboxylated peptide site at positions 13 to 23 of the prothrombin amino acid sequence" (JP 7-20127 A). Therefore, in addition to native PIVKA-II and decarboxylated human prothrombin described in JP 5-43357 A, peptide fragments of various lengths synthesized based on the amino acid sequence of the Gla region of PIVKA-II described in JP 7-20127 A can be appropriately combined to compare and confirm the binding ability of antibodies to PIVKA-II and substances other than PIVKA-II. Screening for anti-PIVKA-II antibodies can also be performed to obtain new anti-PIVKA-II antibodies other than the MU-3 antibody. The entire disclosures of JP 5-43357 A (JP 5-43357 A) and JP 7-20127 A are incorporated herein by reference.

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

[0020] <Monoclonal antibodies (anti-prothrombin F1 domain antibodies): 242202 antibody, 242203 antibody, 242205 antibody, 242206 antibody> (1) Creation method i) Preparation of hybridomas Purified silkworm-expressed PIVKA-II (Ozeki Co., Ltd.) or synthetic peptide (amino acid sequence of positions 29-47) (SEQ ID NO: 1) was mixed 1:1 with Freund's complete adjuvant (GIBCO) to form an emulsion, which was administered subcutaneously at a dose of 20 μg / 100 μL to 8-week-old female BALB / C mice (Charles River Japan) five times at 2-week intervals. Three days after the final immunization, spleens were removed. Spleen cells obtained from the removed spleens were mixed with myeloma cells SP2 / O-Ag14 at a 10:1 ratio and fused in the presence of 50% by weight polyethylene glycol 1540 (Wako Pure Chemical Industries, Ltd.). The fused cells consisted of 2.5 × 10 spleen cells. 6 The cells were suspended in HAT medium at a concentration of 1000 / mL and dispensed in 0.2 mL aliquots into 96-well culture plates (Corning). These were cultured at 37°C in a 5% CO2 incubator. After approximately two weeks, the culture supernatants from wells in which hybridomas had grown were evaluated by the following ELISA method, and hybridomas producing antibodies reactive to PIVKA-II were selected. Specifically, PIVKA-II (prepared by the method of Bajah et al. [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 ELISA plate (NUNC). Each culture supernatant was reacted with a peroxidase-labeled goat anti-mouse IgG antibody (Jackson Immuno Research), followed by the addition of OPD color development solution to develop color. The color development was stopped by the addition of stop solution, and the plate was read using a microplate reader (Abs. 492 nm) to select hybridoma lines that reacted with PIVKA-II. From the selected hybridoma lines, hybridomas 242202, 242203, 242205, and 242206 were selected, which gave good results when combined with the MU-3 antibody to construct a sandwich immunoassay system. Hybridomas 242202, 242203, and 242205 were obtained from mice immunized with PIVKA-II, and hybridoma 242206 was obtained from a mouse immunized with a synthetic peptide. ii) Preparation of monoclonal antibodies Monoclonal antibodies 242202 (antibody 202), 242203 (antibody 203), 242205 (antibody 205), and 242206 (antibody 206) were prepared from hybridomas 242202, 242203, 242205, and 242206, respectively, by the methods described below. Hybridomas were grown in serum-free medium (Esculon SF-B) at 1 × 10 5The cells were seeded into a flask at 4 mL / ml and cultured until the volume reached 1 L, yielding a culture medium containing the antibody. The culture medium 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. The filtrate was passed through a Protein A column (HiTrap rProteinA FF, GE Healthcare Japan) equilibrated with the adsorption buffer, allowing the antibody to adsorb to the column. The monoclonal antibody was then purified by elution with 0.1 mol / L citrate buffer (pH 3.0). (2) Determination of monoclonal antibody epitopes i) Analysis with linear peptides Preliminary analysis revealed that the antibodies produced by hybridomas 242202, 242203, 242205, and 242206 all bind to the Fragment 1 (F1) region of PIVKA-II. To identify the epitopes of these antibodies, the antibodies were screened for reactivity with a peptide corresponding to the sequence from amino acids 26 to 47 of PIVKA-II. This region extends from the C-terminus of the Gla domain in the F1 region of PIVKA-II to immediately upstream of the thrombin cleavage site (positions 51 to 52). The region of PIVKA-II from amino acid 33 onward shares an amino acid sequence with the corresponding region of prothrombin. Peptides L1 to L7 (SEQ ID NOS: 2 to 8) purchased from Toray Industries, Inc. were used for screening. The N-terminus of each peptide was biotinylated. The peptides were dissolved in PBS to a concentration of 10 nM. Dynabeads™ M-270 Streptavidin was suspended in these peptide solutions to prepare a 10 mg / mL solution. After standing for 10 minutes, the solution was washed three times with the Ru dilution solution (1) described below to obtain peptide-immobilized magnetic beads. The beads were then diluted to 1 mg / mL to prepare a peptide-immobilized magnetic bead solution. To 1 mL of anti-mouse IgG Fab antibody (prepared at 1 mg / mL), 68 μL (10 mg / mL) of succinimide-modified ruthenium tri-dipyridyl complex was added and incubated at room temperature for 30 minutes with stirring. The reaction was stopped by adding 50 μL of 2 mol / L glycine, and the incubation continued at room temperature for an additional 10 minutes with stirring. Finally, the sample was loaded onto Sephadex G-25 (equilibrated with 10 mmol / L phosphate buffer), and the Ru-bound protein fraction was collected to prepare Ru-labeled anti-mouse IgG Fab antibody (hereafter referred to as "Ru-labeled anti-mouse antibody"). The resulting Ru-labeled anti-mouse IgG Fab antibody was diluted with Ru diluent (1) to a final concentration of 2 μg / mL before use. The composition of Ru diluent (1) is shown below. Composition of Ru dilution solution (1): 50 mM HEPES, 1 mM EDTA-4Na, 0.05% NaN3, 1% BSA, 0.1% Tween20, pH 7.8 The epitopes of the obtained monoclonal antibodies were analyzed by electrochemiluminescence immunoassay using the prepared peptide solid-phase magnetic bead solution, Ru-labeled anti-mouse antibody solution, and the ECLIA automatic measurement device "Picolumi III" (manufactured by Sekisui Medical Co., Ltd.). 20 μL of each monoclonal antibody solution (0.5 μg / mL) and 12.5 μL of peptide solid-phase magnetic beads were added to 100 μL of Ru Dilution Solution (1) and allowed to react for 9 minutes. 200 μL of Ru-labeled anti-mouse antibody solution diluted to 0.2 mg / mL with Ru Dilution Solution (1) was added and reacted for 9 minutes. After that, the magnetic particles were washed and measured. Count values ​​less than 2000 were rated as -, 2000 to 10,000 as +-, 10,000 to 20,000 as +, 20,000 to 30,000 as ++, and 30,000 or more as +++. The analysis results are summarized in Table 1 below. [Table 1] The analysis results confirmed that antibody 205 reacts with the peptide sequence at positions 26-44 of PIVKA-II, and antibody 206 reacts with the peptide sequence at positions 29-44 of PIVKA-II. On the other hand, no strong binding of antibodies 202 and 203 to peptides L1 to L7 in Table 1 was confirmed. ii) Analysis using peptides with higher-order structure Epitope analysis of antibodies 202 and 203 was performed using PEPPERMAP (registered trademark) Conformational Epitope Mapping service from PEPPERPRINT. We synthesized 480 peptides consisting of consecutive 7, 10, and 13 amino acids, each offset by one amino acid from the N-terminus of the PIVKA-II fragment 1 region, by adding the GSGSGSG sequence to the N- and C-termini. Each peptide was equipped with a cysteine ​​at the C-terminus and a suitable functional group reactive with cysteine ​​at the N-terminus. The synthesized peptides were cyclized by forming a thioester bond between the cysteine ​​and the functional group to enhance their conformation, and then printed on a microarray. The microarray was blocked with Rockland blocking buffer MB-070 for 30 minutes. Antibodies 202 and 203, prepared at 1, 10, and 100 μg / mL in incubation buffer (Rockland blocking buffer MB-070 diluted 1:10 with PBS, 0.005% Tween 20, pH 7.4), were incubated for 16 hours at 4°C with shaking at 140 rpm. After washing twice for 10 seconds with PBS, 0.005% Tween 20, pH 7.4, the cells were incubated with Goat anti-mouse IgG (H+L) DyLight680 diluted 5000-fold in incubation buffer at room temperature for 45 minutes, and then analyzed using a LI-COR Odyssey Imaging System with an offset of 0.65 mm and a resolution of 21 μm. As a result, the 202 antibody most strongly bound to the cyclic peptide SSTATDVFWAKYT (peptide number 40), and the 203 antibody most strongly bound to the cyclic peptide STATDVFWAKYTA (peptide number 41). Therefore, it was shown that the 202 antibody and the 203 antibody recognize the higher-order structural epitope at positions 35-45 of PIVKA-II. More specifically, it was shown that the 202 antibody is an antibody that recognizes the higher-order structural epitope at positions 35-44 of PIVKA-II, and the 203 antibody is an antibody that recognizes the higher-order structural epitope at positions 36-45 of PIVKA-II / prothrombin. Furthermore, it was shown that both antibodies may recognize the motif of TATDVFWA at positions 35-42. This region forms a loop and an α-helix separate from the loop corresponding to the sequence (positions 13-23) recognized by the MU-3 antibody of PIVKA-II in the crystal structure of prothrombin (PDB ID: 6BJR (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=6BJR)). Therefore, it is suggested that the 202 antibody and the 203 antibody recognize the higher-order structural epitope of the loop and / or α-helix formed by the amino acids at positions 33-45 located on the C-terminal side of the Gla domain (positions 8-45) of PIVKA-II.

[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-glycidoxy octyltrimethoxysilane 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 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 phosphate buffer containing 1% BSA was added, and blocking was carried out with stirring at room temperature for one day and night to prepare a solid-phase magnetic bead solution of MU-3 monoclonal antibody (hereinafter, may be 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 to 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 further stirred 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-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 Figure 1, when antibodies recognizing linear epitopes (antibodies 202 and 203) and antibodies recognizing conformational epitopes (antibodies 205 and 206) were used in combination (shown in shaded areas), a significant increase in the measurement value (count number) of 10 mAU / mL was observed.

[0027] Without being bound by any particular theory, it is thought that the F1 domain of PIVKA-II (which has the same primary sequence as the F1 domain of prothrombin) is partially denatured in the reaction mixture, losing its native conformation. It is thought that combining an antibody that recognizes a linear epitope with an antibody that recognizes a conformational epitope may enable complete capture of PIVKA-II contained in the reaction mixture. [Industrial Applicability]

[0028] The method and reagent for measuring an analyte using an immune reaction according to the present invention can be used for diagnosing or assisting in diagnosing hepatocellular carcinoma, etc. Furthermore, the present invention can improve the sensitivity of existing methods and reagents for measuring an analyte using an immune reaction.

Claims

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 are a mixture of monoclonal antibodies that recognize linear epitopes and monoclonal antibodies that recognize conformational epitopes.

2. A sandwich immunoassay method for determining an analyte in a sample, comprising the steps of: providing a sample; contacting the sample with a first antibody to provide a first reactant; optionally, recovering the first antibody-analyte complex contained in said first reaction; 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 reaction product; and measuring the signal from the label of the recovered first antibody-analyte-second antibody complex to measure the analyte in the sample; where: the first antibody and the second antibody are antibodies that have different epitopes against the analyte, One or both of the first and second antibodies is a mixture of monoclonal antibodies that recognize linear epitopes and monoclonal antibodies that recognize conformational epitopes.

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

6. A method according to any one of claims 1 to 5, wherein the monoclonal antibody recognizing the linear epitope is an antibody recognizing a peptide having the sequence of amino acids 26 to 44 of PIVKA-II ((E / γ)AF(E / γ)AL(E / γ)SSTATDVFWAKY), or a peptide having the sequence of amino acids 29 to 44 of PIVKA-II ((E / γ)AL(E / γ)SSTATDVFWAKY).

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

8. A sandwich immunoassay reagent 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 conformational epitope.

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