WO2022138460A1 - Dupan-2 antigen measurement reagent and measurement kit - Google Patents

Dupan-2 antigen measurement reagent and measurement kit Download PDF

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Publication number
WO2022138460A1
WO2022138460A1 PCT/JP2021/046591 JP2021046591W WO2022138460A1 WO 2022138460 A1 WO2022138460 A1 WO 2022138460A1 JP 2021046591 W JP2021046591 W JP 2021046591W WO 2022138460 A1 WO2022138460 A1 WO 2022138460A1
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Prior art keywords
dupan
antigen
antibody
binds
less
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PCT/JP2021/046591
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French (fr)
Japanese (ja)
Inventor
瞳 池田
仁 小野
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ミナリスメディカル株式会社
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Priority to JP2022571385A priority Critical patent/JPWO2022138460A1/ja
Publication of WO2022138460A1 publication Critical patent/WO2022138460A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a DUPAN-2 antigen measuring reagent and a measuring kit.
  • Monoclonal antibody DUPAN-2 is a monoclonal antibody produced using cultured human pancreatic cancer cells HPAF as an immunogen, and has been reported to react with a mutin-like protein (antigen) produced at a high rate from pancreatic cancer cells. (Non-Patent Documents 1 and 2). This antigen is called DUPAN-2 antigen and is used as a cancer marker for pancreatic cancer and biliary tract cancer.
  • Non-Patent Document 1 competition radioimmunoassay (Non-Patent Document 1) and Enzyme-Linked Immunosorbent Assay (Patent Document 1) have been reported.
  • An object of the present invention is to provide a reagent and a kit capable of measuring a DUPAN-2 antigen in a sample with high sensitivity and accuracy.
  • the present inventors have used a DUPAN-2 antigen measuring reagent containing an insoluble carrier and an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof to ascite water.
  • the measured values of the DUPAN-2 antigen derived from DUPAN-2 antigen and the DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF were examined, the measured values of DUPAN-2 antigen derived from ascites and the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF were examined. It has been found that the measuring reagent having a predetermined ratio of the DUPAN-2 antigen to the measured value tends to have high sensitivity, and the invention of the present disclosure has been completed.
  • a reagent for measuring the DUPAN-2 antigen in a sample comprises an insoluble carrier and an antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
  • (A) means the measured value of DUPAN-2 antigen derived from ascites
  • (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites.
  • the antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
  • a method for measuring the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from the cultured human pancreatic cancer cell HPAF is (1) The insoluble carrier and an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof are reacted with the DUPAN-2 antigen derived from ascites or the DUPAN-2 antigen derived from the human pancreatic cancer cultured cell HPAF. A step of producing an immune complex containing the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the DUPAN-2 antigen on the insoluble carrier; and (2) produced in step (1).
  • the process of measuring immune complexes The reagent according to [1], which is a method comprising.
  • the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen is the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
  • One of the two affinity substances is bound to the insoluble carrier, and the other of the two affinity substances is bound to the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen.
  • a measurement kit for the DUPAN-2 antigen in a sample comprises a first reagent comprising an aqueous medium and a second reagent comprising an insoluble carrier and an antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
  • (A) means the measured value of DUPAN-2 antigen derived from ascites
  • (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites.
  • the antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
  • a method for measuring DUPAN-2 antigen derived from ascites and DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF is (1) The insoluble carrier is reacted with the DUPAN-2 antigen derived from ascites or the DUPAN-2 antigen derived from the cultured human pancreatic cancer cell HPAF, and the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen is reacted with the insoluble.
  • the process of measuring the body The kit according to [7], which is a method including.
  • a measurement kit for DUPAN-2 antigen in a sample comprises an insoluble carrier, a first reagent containing a first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, and a second reagent that contains a second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
  • (A) means the measured value of DUPAN-2 antigen derived from ascites
  • (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites.
  • the antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
  • a measurement kit for the DUPAN-2 antigen in a sample Includes a first reagent containing an insoluble carrier, a second reagent containing a first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, and a third reagent that contains a second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
  • a kit that satisfies the following formula (X) when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the measurement kit.
  • (A) means the measured value of DUPAN-2 antigen derived from ascites
  • (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites.
  • the antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
  • a method for measuring DUPAN-2 antigen derived from ascites and DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF is (1) The insoluble carrier, the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and the DUPAN-2 to the DUPAN-2 antigen derived from ascites or the DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF.
  • the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, the DUPAN-2 antigen, and the DUPAN- 2 A step of generating an immune complex containing a second antibody or an antibody fragment thereof that binds to an antigen; and (2) a step of measuring the immune complex produced in step (1).
  • One of the two affinity substances is bound to the insoluble carrier, and the other of the two affinity substances is bound to the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen.
  • the concentration of DUPAN-2 antigen derived from ascites and the concentration of DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF are 40 U / mL to 400 U / mL, in any one of [7] to [13].
  • the embodiments described below show an example of a typical embodiment of the present disclosure, and the scope of the disclosure is not narrowly interpreted by this.
  • the numerical range indicated by using “-” indicates a range including the numerical values before and after "-" as the minimum value and the maximum value, respectively.
  • the amount of each component in the solution means the total amount of the plurality of substances present in the reagent when a plurality of substances corresponding to each component are present in the solution, unless otherwise specified.
  • “% (w / v)” means a percentage of mass (g) based on volume (100 mL).
  • the antibody and the DUPAN-2 antigen when expressed as “binding” or “reacting”, or the antibody “recognizes” the DUPAN-2 antigen, the meaning commonly used in the art of the present disclosure is used. Including, both are used synonymously.
  • Methods for confirming that the antibody and the DUPAN-2 antigen "bind” include an antigen-immobilized ELISA method, a competitive ELISA method, a sandwich ELISA method, a surface plasmon resonance method, and an immunochromatography method, which are well known to those skilled in the art. It can be performed by a method using a principle such as a quartz crystal microbalance method.
  • the reagent for measuring the DUPAN-2 antigen in sample includes an insoluble carrier and an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof.
  • the antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof may be a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof. ..
  • the reagent for measuring the DUPAN-2 antigen in the sample is an insoluble carrier, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a second antibody that binds to the DUPAN-2 antigen. Includes the antibody fragment.
  • the DUPAN-2 antigen in the present disclosure is the monoclonal antibody "DUPAN-2" (Metzgar et al., "Antigens of Human Pancreatic Adenocarcinoma Cells Defined by Murine Monoclonal Antibodies", CA A protein (glycoprotein), a sugar chain to which the monoclonal antibody "DUPAN-2” binds, or a peptide containing the sugar chain.
  • the DUPAN-2 antigen is a sialyl Lewis C sugar chain (Kawa et al., "Epitope Analysis of Span-1 and DUPAN-2 using Synthesized Glycoconjugate", which has been reported to bind to the monoclonal antibody "DUPAN-2".
  • -Fucopentaose II and Siallylact-N-Tetoraose ", Pancreas (1994), 9 (6): 692-697)
  • a protein having the sugar chain or a peptide having the sugar chain.
  • the DUPAN-2 antigen contained in the ascites of a human pancreatic cancer patient or the culture supernatant of human pancreatic cancer cultured cells HPAF can be used.
  • Human pancreatic cancer cultured cell HPAF can be obtained from ATCC (American Type Culture Collection) under the name of "HPAF-II".
  • the insoluble carrier in the present disclosure is particularly limited as long as it can bind an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof and can be used as a reagent for measuring the DUPAN-2 antigen in the sample in the present disclosure.
  • Examples include slide glasses, ELISA plates (microtiter plates), beads, latex particles, magnetic particles, filters, films and membranes.
  • Examples of the material of the insoluble carrier include those composed of an inorganic base material, a polysaccharide base material, a polymer base material, and the like, for example, glass, silicon, ceramic, cellulose, nitrocellulose, nylon, polycarbonate, polyethylene, polypurpyrene, polystyrene, and the like. Examples include polyethylene terephthalate and polyurethane.
  • the concentration of the insoluble carrier in the DUPAN-2 antigen measuring reagent in the sample of the present disclosure is 0.001 mg / mL or more, 0.01 mg / mL or more, 0.02 mg / mL or more, 0.03 mg / mL or more, 0. .04 mg / mL or more, 0.05 mg / mL or more, 0.06 mg / mL or more, 0.07 mg / mL or more, 0.08 mg / mL or more, 0.09 mg / mL or more, 0.1 mg / mL or more, 0.
  • the concentration of the insoluble carrier in the measurement reagent is 0.6 mg / mL or less, 0.7 mg / mL or less, 0.8 mg / mL or less, 0.9 mg / mL or less, 1 mg / mL or less, 2 mg / mL or less, It may be 3 mg / mL or less, 4 mg / mL or less, 5 mg / mL or less, 6 mg / mL or less, 7 mg / mL or less, 8 mg / mL or less, 9 mg / mL or less, 10 mg / mL or less.
  • the concentration of the insoluble carrier in the measurement reagent may be 0.01 mg / mL to 10 mg / mL, 0.03 mg / mL to 5 mg / mL, or 0.1 mg / mL to 2 mg / mL. It may be mL.
  • an arbitrary numerical value is selected from an example of an upper limit value and an example of a lower limit value for a certain concentration or weight, and a numerical range obtained by combining them is also considered to be exemplified in the present disclosure.
  • an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof (a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof) is referred to as an antibody thereof or an antibody fragment thereof.
  • an antibody or antibody fragment thereof that binds to the DUPAN-2 antigen is disclosed in WO 2019/189874.
  • DUPAN-2 antigen having the following sugar chain structure Reacts with NCC-ST-439 antigen having the following sugar chain structure It may be an antibody or an antibody fragment thereof that does not react with the NCC, and may be an antibody or an antibody fragment thereof having a reactivity with the NCC-ST-439 antigen of less than 10% of the reactivity with the DUPAN-2 antigen.
  • -An antibody or an antibody fragment thereof having a reactivity to the ST-439 antigen of less than 1% of the reactivity to the DUPAN-2 antigen may be used, and these antibodies may be rat antibodies.
  • the antibody that binds to the DUPAN-2 antigen may be a mouse antibody, a rat antibody, a rabbit antibody, a human antibody, a humanized antibody or a chimeric antibody, or may be an antibody derived from another species.
  • Antibodies that bind to the DUPAN-2 antigen can be any class of immunoglobulin molecules (eg, IgG, IgE, IgM, IgD and IgA), any subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). ) Can be.
  • Examples of the antibody that binds to the DUPAN-2 antigen include a monoclonal antibody, a polyclonal antibody, a dimer, a multimer, and the like, and a monoclonal antibody is preferable from the viewpoint of homogeneity and stability.
  • the antibody fragment refers to a part of an antibody that binds to the DUPAN-2 antigen and contains a variable domain of the antibody or at least an antigen-binding region.
  • the antibody fragments in the present disclosure include, for example, Fab, Fab', F (ab') 2 , Fv fragment, linear antibody, single-chain antibody (scFv), sc (Fv) 2 , Fab 3 , and domain antibody (dAb). , Diabody, Triabody, Tetrabody and Minibody.
  • An "Fv fragment” is the smallest antibody fragment that contains a complete antigen recognition region and antigen binding region.
  • the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen of the present disclosure may or may not be bound to an insoluble carrier.
  • the insoluble carrier include the above-mentioned insoluble carrier.
  • the method for binding an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen to an insoluble carrier include the binding method described below.
  • a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof and a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof are used. You may.
  • the first antibody or its antibody fragment that binds to the DUPAN-2 antigen and the second antibody or its antibody fragment that binds to the DUPAN-2 antigen have the same site of the DUPAN-2 antigen to which each antibody or its antibody fragment binds. However, they may be different. Further, the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof and the second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof may be the same antibody or different antibodies. good.
  • the antibody that binds to the DUPAN-2 antigen in the present disclosure can be obtained by a conventional method for producing an antibody using a DUPAN-2 antigen or a cancer cell that produces the DUPAN-2 antigen as an immunogen.
  • it can be obtained by producing a cell (hybridoma) in which an antibody-producing cell obtained by immunizing an animal with the immunogen and a myeloma cell is produced, and a monoclonal antibody produced by the hybridoma is obtained.
  • the monoclonal antibody that binds to the DUPAN-2 antigen As the monoclonal antibody that binds to the DUPAN-2 antigen, the above-mentioned monoclonal antibody "DUPAN-2" (Metzgar et al., "Antigens of Human Cancer Cellrecined by Murine Monoclonial ) Etc. can be mentioned. In the present disclosure, the monoclonal antibody DUPAN-2 is also referred to as "DUPAN-2 antibody”.
  • the concentration of the antibody that binds to the DUPAN-2 antigen or its antibody fragment and the concentration of the first antibody that binds to the DUPAN-2 antigen or its antibody fragment in the reagent for measuring the DUPAN-2 antigen in the sample of the present disclosure are as follows.
  • 6 ⁇ g / mL or more 0.7 ⁇ g / mL or more, 0.8 ⁇ g / mL or more, 0.9 ⁇ g / mL or more, 1 ⁇ g / mL or more, 2 ⁇ g / mL or more, 3 ⁇ g / mL or more, 4 ⁇ g / mL or more, 5 ⁇ g / mL or more , 6 ⁇ g / mL or higher, 7 ⁇ g / mL or higher, 7.5 ⁇ g / mL or higher, 8 ⁇ g / mL or higher, or 9 ⁇ g / mL or higher.
  • the concentration of the antibody or its antibody fragment that binds to the DUPAN-2 antigen and the concentration of the first antibody or its antibody fragment that binds to the DUPAN-2 antigen in the measurement reagent are 10 ⁇ g / mL or less and 11 ⁇ g / mL.
  • the concentration of the antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen and the concentration of the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen in the measurement reagent are 0.01 ⁇ g / mL to 100 ⁇ g / mL. It may be 0.1 ⁇ g / mL to 30 ⁇ g / mL, 6 ⁇ g / mL to 30 ⁇ g / mL, or 10 ⁇ g / mL to 20 ⁇ g / mL.
  • the antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, or the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof is bound to a microtiter plate and does not contain an aqueous medium and is in a dry state. If, the concentration of the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the amount of the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in one well of the microtiter plate is 0.001 ⁇ g or more.
  • the concentration of the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the amount of the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in one well of the microtiter plate are 0.6 ⁇ g or less.
  • ⁇ g or less 0.7 ⁇ g or less, 0.8 ⁇ g or less, 0.9 ⁇ g or less, 1 ⁇ g or less, 2 ⁇ g or less, 3 ⁇ g or less, 4 ⁇ g or less, 5 ⁇ g or less, 6 ⁇ g or less, 7 ⁇ g or less, 7.5 ⁇ g or less, 8 g / mL or less, 9 ⁇ g or less, 10 ⁇ g below, it may be 11 ⁇ g or less, 12 ⁇ g or less, 13 ⁇ g or less, 14 ⁇ g or less, 15 ⁇ g or less, 16 ⁇ g or less, 17 ⁇ g or less, 18 ⁇ g or less, 19 ⁇ g or less, 20 ⁇ g or less, 30 ⁇ g or less, 40 ⁇ g or less, 50 ⁇ g or less, or 100 ⁇ g or less.
  • the concentration of the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the amount of the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in one well of the microtiter plate are 0.01 ⁇ g to 30 ⁇ g. It may be 0.1 ⁇ g to 10 ⁇ g, 0.3 ⁇ g to 3 ⁇ g, 0.3 ⁇ g to 1 ⁇ g, or 0.5 ⁇ g to 1 ⁇ g.
  • the concentration of the second antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen in the DUPAN-2 antigen measuring reagent in the sample of the present disclosure is 0.001 ⁇ g / mL or more, 0.01 ⁇ g / mL or more, 0. 02 ⁇ g / mL or more, 0.03 ⁇ g / mL or more, 0.04 ⁇ g / mL or more, 0.05 ⁇ g / mL or more, 0.06 ⁇ g / mL or more, 0.07 ⁇ g / mL or more, 0.08 ⁇ g / mL or more, 0.09 ⁇ g It may be / mL or more, or 0.1 ⁇ g / mL or more.
  • the concentration of the second antibody or its antibody fragment that binds to the DUPAN-2 antigen in the measurement reagent is 0.2 ⁇ g / mL or less, 0.3 ⁇ g / mL or less, 0.4 ⁇ g / mL or less, 0.5 ⁇ g /.
  • mL or less 0.6 ⁇ g / mL or less, 0.7 ⁇ g / mL or less, 0.8 ⁇ g / mL or less, 0.9 ⁇ g / mL or less, 1 ⁇ g / mL or less, 2 ⁇ g / mL or less, 3 ⁇ g / mL or less, 4 ⁇ g / mL or less 5, 5 ⁇ g / mL or less, 6 ⁇ g / mL or less, 7 ⁇ g / mL or less, 7.5 ⁇ g / mL or less, 8 g / mL or less, 9 ⁇ g / mL or less, 10 ⁇ g / mL or less, 11 ⁇ g / mL or less, 12 ⁇ g / mL or less, 13 ⁇ g / mL or less, 14 ⁇ g / mL or less, 15 ⁇ g / mL or less, 16 ⁇ g
  • the concentration of the second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in the measurement reagent may be 0.01 ⁇ g / mL to 100 ⁇ g / mL, or 0.03 ⁇ g / mL to 30 ⁇ g / mL. It may be 0.1 ⁇ g / mL to 20 ⁇ g / mL.
  • the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen may or may not be bound to the insoluble carrier.
  • the reagent for measuring the DUPAN-2 antigen of the present disclosure can be used.
  • the insoluble carrier that can be used is not particularly limited, and examples thereof include binding by physical adsorption and binding by chemical bonding. Examples of the physical adsorption include electrostatic bonds, hydrogen bonds, hydrophobic bonds and the like. Examples of the chemical bond include a covalent bond and a coordinate bond.
  • the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen directly bind to the insoluble carrier by utilizing the above-mentioned physical adsorption and / or chemical bond. It may be allowed to bind or indirectly bind to an insoluble carrier.
  • an antibody that binds to the DUPAN-2 antigen or an antibody thereof by utilizing the specific binding of a set of affinity substances such as biotin and avidins (avidin, streptavidin, neutravidin, etc.) or the like.
  • Examples thereof include a method of binding a fragment and a first antibody that binds to a DUPAN-2 antigen or an antibody fragment thereof to an insoluble carrier, a method of binding to an insoluble carrier by covalent bonding via a linker, and the like.
  • an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, or a DUPAN-2 antigen is bound.
  • An insoluble carrier to which one antibody or an antibody fragment thereof is bound can be produced.
  • a method for binding an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen and a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen to an insoluble carrier is generated in a reaction solution for an antigen-antibody reaction described later.
  • an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen to which one of the pair of affinity substances (A) is bound, or a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen may be used.
  • An antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, or DUPAN-2 by reacting an insoluble carrier to which the other (a) of a set of affinity substances is bound in the reaction solution of the antigen-antibody reaction An insoluble carrier to which a first antibody that binds to an antigen or an antibody fragment thereof is bound can be produced in the reaction solution of an antigen-antibody reaction.
  • Examples of the combination of Aa include the following combinations and the like. ⁇ Combination of biotin and avidins (avidin, neutral avidin, streptavidin, etc.); ⁇ Combination of avidins (avidin, neutravidin, streptavidin, etc.) and biotin; -A combination of the Fc region of an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, or the Fc region of a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, and an antibody that binds to the Fc region.
  • the linker covalently binds both the functional group on the surface of the insoluble carrier and the functional group of the antibody that binds to the DUPAN-2 antigen or its antibody fragment, or the first antibody that binds to the DUPAN-2 antigen or its antibody fragment.
  • Examples thereof include molecules capable of reacting with a functional group contained in an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, or a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof. It is a molecule having a reaction active group and a second reaction active group capable of reacting with a functional group on the surface of the insoluble carrier at the same time, and the first reaction active group and the second reaction active group are different groups. Molecules are preferably used.
  • the functional group of the antibody that binds to the DUPAN-2 antigen or its antibody fragment, or the functional group of the first antibody that binds to the DUPAN-2 antigen or its antibody fragment, and the functional group held on the surface of the insoluble carrier are carboxyl.
  • Examples thereof include a group, an amino group, a glycidyl group, a sulfhydryl group, a hydroxyl group, an amide group, an imino group, an N-hydroxysuccinyl group, a maleimide group and the like.
  • Examples of the reactive active group in the linker include allyl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, maleimide, N-hydroxysuccinimide ester, pentafluorophenyl (PFP) ester, solarene, pyridyl disulfide, vinyl sulfone and the like. The group is mentioned.
  • the second antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen may or may not be bound to the insoluble carrier.
  • the method for binding the second antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen to the insoluble carrier is not particularly limited as long as it is an insoluble carrier that can be used as the measurement reagent for the DUPAN-2 antigen of the present disclosure.
  • the above-mentioned bonding method can be mentioned.
  • the first antibody that binds to the DUPAN-2 antigen or the antibody fragment thereof is bound to the insoluble carrier, and the second antibody that binds to the DUPAN-2 antigen.
  • the antibody or antibody fragment thereof may be bound to an insoluble carrier.
  • the insoluble carrier to which the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen binds and the insoluble carrier to which the second antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen binds are the same type of insoluble carrier. It may be an insoluble carrier of a different type.
  • the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof and the second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof may be bound on the same insoluble carrier.
  • an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen and a second antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen. And may be used, and the DUPAN-2 antigen can be measured, for example, by a method including the following steps.
  • the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, the DUPAN-2 antigen, and the DUPAN-2 antigen are reacted on the insoluble carrier by reacting the second antibody or the antibody fragment thereof.
  • the concentration of the DUPAN-2 antigen in the sample can also be determined by performing the following steps (3) and (4) after the step (2B).
  • the DUPAN-2 antigen derived from ascites refers to the DUPAN-2 antigen contained in the ascites of a pancreatic cancer patient.
  • the ascites of a pancreatic cancer patient may be used as it is, or phosphate buffered saline (10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7. 2.
  • Ascites diluted with PBS or the like may be used, or a DUPAN-2 antigen solution obtained by diluting DUPAN-2 antigen purified from ascites of a pancreatic cancer patient with PBS or the like may be used.
  • the concentration of the DUPAN-2 antigen derived from ascites is not particularly limited as long as it enables the measurement of the DUPAN-2 antigen, and may be 10 U / mL to 2000 U / mL, and may be 30 U / mL to 1600 U / mL. It may be 40 U / mL to 400 U / mL, and may be 40 U / mL to 350 U / mL.
  • the DUPAN-2 antigen concentration unit "U / mL” "when the serum of a specific pancreatic cancer patient is measured by a competitive radioimmunoassay method, the concentration of the DUPAN-2 antigen contained in the serum is 1000 U / mL.
  • the DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF is the DUPAN-2 antigen contained in human pancreatic cancer cultured cell HPAF, or the DUPAN-2 antigen contained in the culture supernatant obtained when human pancreatic cancer cultured cell HPAF is cultured.
  • the DUPAN-2 antigen is contained in the human pancreatic cancer cultured cell HPAF
  • the cell lysate of the human pancreatic cancer cultured cell HPAF may be used as it is, or the cell solubilized product diluted with PBS or the like may be used. May be good.
  • the culture supernatant When the DUPAN-2 antigen is contained in the culture supernatant, the culture supernatant may be used as it is, or the culture supernatant diluted with PBS or the like may be used.
  • the medium used for culturing human pancreatic cancer cultured cells HPAF include Medium Essential Medium Eagle (Eagle's minimum essential medium) containing 10% fetal bovine serum.
  • the concentration of DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF is not particularly limited as long as it enables measurement of DUPAN-2 antigen, and may be 10 U / mL to 2000 U / mL, and may be 30 U / mL. It may be up to 1600 U / mL, may be 40 U / mL to 400 U / mL, and may be 40 U / mL to 350 U / mL.
  • step (1B) an insoluble carrier, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and DUPAN- are added to the DUPAN-2 antigen derived from ascites or the DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF.
  • a second antibody that binds to two antigens or an antibody fragment thereof is reacted, and the first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, the DUPAN-2 antigen, and the DUPAN on the insoluble carrier.
  • This is a step of producing an immune complex containing a second antibody that binds to an antigen or an antibody fragment thereof.
  • step (1B) a DUPAN-2 antigen derived from ascites or a DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, an insoluble carrier, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and DUPAN-2.
  • the reaction with the second antibody or its antibody fragment that binds to the antigen binds to the first antibody or its antibody fragment that binds to the DUPAN-2 antigen, the DUPAN-2 antigen, and the DUPAN-2 antigen on an insoluble carrier.
  • an immune complex containing the second antibody or an antibody fragment thereof is produced, and for example, a DUPAN-2 antigen derived from ascites water or a DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, an insoluble carrier and the like.
  • the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof is reacted to generate an immune complex of the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof and the DUPAN-2 antigen on an insoluble carrier.
  • a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof may be reacted, or the DUPAN-2 antigen derived from ascites water or the DUPAN-2 antigen derived from the cultured human pancreatic cancer cell HPAF is used as an insoluble carrier.
  • the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof may be mixed and reacted with the second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof.
  • the order of adding the insoluble carrier, the first antibody that binds to the DUPAN-2 antigen or its antibody fragment, and the second antibody that binds to the DUPAN-2 antigen or its antibody fragment may be any order.
  • the cleaning solution used for cleaning the insoluble carrier is not particularly limited as long as it is a cleaning solution capable of measuring the DUPAN-2 antigen, and examples thereof include PBS and PBS containing a surfactant.
  • the surfactant include nonionic surfactants such as Tween (registered trademark) 20.
  • the first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen and the insoluble carrier may be previously bound before reacting with the DUPAN-2 antigen.
  • the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen can be bound to the insoluble carrier, for example, by the method described above.
  • the concentration of the insoluble carrier in the reaction solution is not particularly limited as long as it is a concentration capable of measuring the DUPAN-2 antigen, and is 0.001 mg / mL or more, 0.002 mg / mL or more, 0.003 mg / mL or more.
  • 0.004 mg / mL or more 0.005 mg / mL or more, 0.01 mg / mL or more, 0.02 mg / mL or more, 0.03 mg / mL or more, 0.04 mg / mL or more, 0.05 mg / mL or more, 0.06 mg / mL or more, 0.07 mg / mL or more, 0.08 mg / mL or more, 0.09 mg / mL or more, 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0 It may be 0.4 mg / mL or more, or 0.5 mg / mL or more.
  • the concentration of the insoluble carrier in the reaction solution is 0.6 mg / mL or less, 0.7 mg / mL or less, 0.8 mg / mL or less, 0.9 mg / mL or less, 1 mg / mL or less, 2 mg / mL or less. It may be 3 mg / mL or less, 4 mg / mL or less, 5 mg / mL or less, 6 mg / mL or less, 7 mg / mL or less, 8 mg / mL or less, 9 mg / mL or less, or 10 mg / mL or less.
  • the concentration of the insoluble carrier in the reaction solution may be 0.003 mg / mL to 10 mg / mL, 0.01 mg / mL to 3 mg / mL, or 0.05 mg / mL to 1 mg. It may be / mL.
  • the concentration of the first antibody or its antibody fragment that binds to the DUPAN-2 antigen in the reaction solution is 0.01 ⁇ g / mL or more, 0.02 ⁇ g / mL or more, 0.03 ⁇ g / mL or more, 0.04 ⁇ g / mL.
  • the concentration of the first antibody or its antibody fragment that binds to the DUPAN-2 antigen in the reaction solution is 10 ⁇ g / mL or less, 11 ⁇ g / mL or less, 12 ⁇ g / mL or less, 13 ⁇ g / mL or less, 14 ⁇ g / mL or less.
  • the concentration of the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in the reaction solution may be 0.01 ⁇ g / mL to 100 ⁇ g / mL, or 0.1 ⁇ g / mL to 30 ⁇ g / mL. It may be 6 ⁇ g / mL to 30 ⁇ g / mL, or 10 ⁇ g / mL to 20 ⁇ g / mL.
  • the concentration of the second antibody or its antibody fragment that binds to the DUPAN-2 antigen in the reaction solution is 0.001 ⁇ g / mL or more, 0.01 ⁇ g / mL or more, 0.02 ⁇ g / mL or more, 0.03 ⁇ g / mL. 0.04 ⁇ g / mL or more, 0.05 ⁇ g / mL or more, 0.06 ⁇ g / mL or more, 0.07 ⁇ g / mL or more, 0.08 ⁇ g / mL or more, 0.09 ⁇ g / mL or more, or 0.1 ⁇ g / mL That may be the above.
  • the concentration of the second antibody or its antibody fragment that binds to the DUPAN-2 antigen in the reaction solution is 0.2 ⁇ g / mL or less, 0.3 ⁇ g / mL or less, 0.4 ⁇ g / mL or less, 0.5 ⁇ g.
  • / ML or less 0.6 ⁇ g / mL or less, 0.7 ⁇ g / mL or less, 0.8 ⁇ g / mL or less, 0.9 ⁇ g / mL or less, 1 ⁇ g / mL or less, 2 ⁇ g / mL or less, 3 ⁇ g / mL or less, 4 ⁇ g / mL 5 ⁇ g / mL or less, 6 ⁇ g / mL or less, 7 ⁇ g / mL or less, 7.5 ⁇ g / mL or less, 8 g / mL or less, 9 ⁇ g / mL or less, 10 ⁇ g / mL or less, 11 ⁇ g / mL or less, 12 ⁇ g / mL or less, 13 ⁇ g / ML or less, 14 ⁇ g / mL or less, 15 ⁇ g / mL or less, 16 ⁇ g / m
  • the concentration of the second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in the reaction solution may be 0.01 ⁇ g / mL to 100 ⁇ g / mL, or 0.03 ⁇ g / mL to 30 ⁇ g / mL. It may be 0.1 ⁇ g / mL to 20 ⁇ g / mL.
  • the step (1B) is preferably performed in an aqueous medium.
  • the aqueous medium include deionized water, distilled water, a buffer solution and the like, and a buffer solution is preferable.
  • the buffer used for preparing the buffer include acetate buffer and Good's buffer. One type of buffer may be used alone, or two or more types may be used in combination.
  • salts include lithium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride and the like.
  • metal ion include sodium ion, magnesium ion, manganese ion, zinc ion and the like.
  • saccharides include mannitol and sorbitol.
  • protein include bovine serum albumin (hereinafter referred to as BSA) and the like.
  • surfactant include anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants and the like.
  • the reaction temperature in the step (1B) is not particularly limited as long as it is a temperature capable of measuring the DUPAN-2 antigen, for example, 0 to 50 ° C., preferably 4 to 45 ° C., and particularly 20 to 40 ° C. preferable.
  • the reaction time of the step (1B) is not particularly limited as long as it enables the measurement of the DUPAN-2 antigen, and is, for example, 5 minutes to 3 hours, preferably 10 minutes to 2 hours.
  • step (2B) the measured value of DUPAN-2 antigen in the sample is determined by measuring the immune complex produced in step (1B) using the following method.
  • the second antibody that binds to the DUPAN-2 antigen or the antibody that binds to the antibody fragment thereof (hereinafter referred to as the third antibody). ) Is bound to the labeled third antibody, the immune complex can be measured. That is, the immune complex is reacted with the labeled third antibody to form a complex 3 of the immune complex and the labeled third antibody, and the labeling of the complex 3 is measured by the method described below. Thereby, the amount of the immune complex produced in the step (1B) can be measured.
  • the third antibody include a second antibody that binds to the DUPAN-2 antigen or an antibody that binds to the Fc region of an antibody fragment thereof.
  • the reaction temperature of the reaction between the labeled third antibody and the immune complex is not particularly limited as long as it is a temperature that enables measurement of the DUPAN-2 antigen, and may be, for example, 0 to 50 ° C. 4 It may be ⁇ 45 ° C. and may be 20-40 ° C.
  • the reaction time of the reaction is not particularly limited as long as it enables the measurement of the DUPAN-2 antigen, and may be, for example, 1 minute to 4 hours, 105 minutes to 3 hours, or 10 minutes. It may be up to 2 hours.
  • the concentration of the labeled third antibody in the reaction solution is not particularly limited as long as it is a concentration capable of measuring the DUPAN-2 antigen, and may be, for example, 0.01 to 100 ⁇ g / mL, and may be 0.1 to 0.1. It may be 20 ⁇ g / mL.
  • Step (2B) can be performed by measuring the immune complex with a label.
  • Methods of binding the label to a second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen are known in the art of immunoassay.
  • the label can be attached to the antibody via one or several amino acids, or via a linker with one or several amino acids.
  • various kits for binding the label to the protein are commercially available.
  • Labels include enzymes, radioactive isotopes, fluorescent substances, luminescent substances, DNA, RNA, co-enzymes, or substances that specifically bind to co-enzymes (biotin, avidin), which are used in ordinary immunoassay methods.
  • tags substances that absorb in the ultraviolet to infrared regions, color-developing fine particles, fluorescent fine particles, metallic fine particles (gold colloidal particles, etc.), magnetic substances, substances that have properties as spin labeling agents, colored latex, and the like. Be done.
  • Examples of the enzyme include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase and the like.
  • Examples of the radioisotope include 3H , 14C , 35S , 32P , 125P , 131I and the like.
  • Examples of the fluorescent substance include FITC (fluorescein isothiocyanate) and RITC (rhodamine B-isothiocyanate).
  • Examples of the luminescent substance include acridinium and its derivatives, ruthenium complex compounds, loffin and the like.
  • the immune complex can be measured by detecting the signals resulting from these labels.
  • the method for measuring the signal generated from the label may be appropriately selected depending on the label to be used.
  • the label is a color-developing substance, that is, a substance that absorbs light of a certain wavelength
  • the label can be measured by measuring the absorbance using a spectrophotometer, a multi-well plate reader, or the like.
  • the label is a fluorescent substance
  • the label can be measured by measuring the fluorescence intensity using a fluorometer, a fluorescent multi-well plate reader, or the like.
  • the label is a luminescent substance
  • the label can be measured by measuring the luminescence intensity using a luminescence photometer, a luminescence multi-well plate reader, or the like.
  • the label when the label is a radioisotope, the label can be measured by measuring the radioactivity with a scintillation counter, a ⁇ -well counter, or the like.
  • the label when the label is an enzyme, the label can be measured by measuring the enzyme activity.
  • the label can be measured by reacting the substrate of an enzyme with the enzyme and measuring the produced substance.
  • the alkaline phosphatase activity can be measured by, for example, a luminescence method.
  • the method for measuring the alkaline phosphatase activity by the luminescence method include a method of reacting alkaline phosphatase with a substrate thereof and measuring the intensity of the generated luminescence with a luminescence photometer, a luminescence multi-well plate reader or the like.
  • Examples of the substrate for alkaline phosphatase include 3- (2'-spirodamantan) -4-methoxy-4- (3'-phosphoryloxy) phenyl-1,2-dioxetane / disodium salt (AMPPD), 2-chloro-.
  • divalent or higher metal ion particularly a divalent metal ion
  • the labeling enzyme is alkaline phosphatase.
  • divalent or higher metal ions include alkaline earth metal ions such as beryllium ion, magnesium ion, calcium ion, strontium ion and barium ion; titanium ion, chromium ion, manganese ion, iron ion, cobalt ion and nickel ion.
  • first transition element ions such as copper ion and zinc ion.
  • the reagent for measuring the DUPAN-2 antigen in the sample of the present disclosure is the following formula when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from the cultured human pancreatic cancer cell HPAF are measured according to the above method. X) is satisfied.
  • Such a DUPAN-2 antigen measuring reagent becomes a highly sensitive DUPAN-2 antigen measuring reagent. 0.8 ⁇ (B) / (A) ⁇ 1.2 (X) (In the formula, (A) means the measured value of DUPAN-2 antigen derived from ascites, and (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites.
  • the antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
  • the measured value of DUPAN-2 antigen derived from ascites (A) and the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF (B) may be a signal such as absorbance, luminescence intensity or fluorescence intensity described later. It may be the concentration of the above-mentioned DUPAN-2 antigen.
  • the (B) / (A) value may be 0.7 or more, 0.8 or more, and 0.9 or more. Further, the (B) / (A) value may be 1.1 or less and 1.2 or less. The above numerical values can be freely combined. For example, the (B) / (A) value may be 0.7 to 1.2, 0.8 to 1.2, or 0.9 to 1.2.
  • the reagent for measuring the DUPAN-2 antigen in the sample of the present disclosure can also take the form of a kit from the viewpoint of storage, transportation, distribution and the like.
  • a kit from the viewpoint of storage, transportation, distribution and the like.
  • preferred embodiments of the DUPAN-2 antigen measurement kit in the sample of the present disclosure will be illustrated.
  • an insoluble carrier an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a second antibody that binds to the DUPAN-2 antigen.
  • the antibody fragment thereof includes, for example, the above-mentioned insoluble carrier, an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a DUPAN-2 antigen.
  • Antibodies or antibody fragments thereof can be used.
  • a measurement kit comprising a first reagent comprising an aqueous medium; and a second reagent comprising an insoluble carrier and an antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
  • Measurement kit (2) A first reagent containing an insoluble carrier and a first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen; A measurement kit comprising a second reagent containing a second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
  • the measurement kit (2) and the measurement kit (3) may contain a reagent containing an aqueous medium.
  • aqueous medium in the measurement kit (1), the measurement kit (2), and the measurement kit (3) include the above-mentioned aqueous medium.
  • the concentration of the insoluble carrier in the second reagent of the measurement kit (1), the concentration of the insoluble carrier in the first reagent of the measurement kit (2), and the concentration of the insoluble carrier in the first reagent of the measurement kit (3) are , 0.001 mg / mL or more, 0.01 mg / mL or more, 0.02 mg / mL or more, 0.03 mg / mL or more, 0.04 mg / mL or more, 0.05 mg / mL or more, 0.06 mg / mL or more, 0.07 mg / mL or more, 0.08 mg / mL or more, 0.09 mg / mL or more, 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0.4 mg / mL or more, or It may be 0.5 mg / mL or more.
  • the concentration of the insoluble carrier in the second reagent of the measurement kit (1), the concentration of the insoluble carrier in the first reagent of the measurement kit (2), and the insoluble carrier in the first reagent of the measurement kit (3) is 0.6 mg / mL or less, 0.7 mg / mL or less, 0.8 mg / mL or less, 0.9 mg / mL or less, 1 mg / mL or less, 2 mg / mL or less, 3 mg / mL or less, 4 mg / mL or less.
  • It may be 5, 5 mg / mL or less, 6 mg / mL or less, 7 mg / mL or less, 8 mg / mL or less, 9 mg / mL or less, or 10 mg / mL or less.
  • the concentration of the insoluble carrier in the second reagent of the measurement kit (1), the concentration of the insoluble carrier in the first reagent of the measurement kit (2), and the insoluble carrier in the first reagent of the measurement kit (3) may be 0.01 mg / mL to 10 mg / mL, 0.03 mg / mL to 5 mg / mL, or 0.1 mg / mL to 2 mg / mL.
  • the concentration of the fragment and the concentration of the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (3) are 0.01 ⁇ g / mL or more, 0.02 ⁇ g / mL or more, and 0.
  • ⁇ g / mL or more 1 ⁇ g / mL or more, 0.2 ⁇ g / mL or more, 0.3 ⁇ g / mL or more, 0.4 ⁇ g / mL or more, 0.5 ⁇ g / mL or more, 0.6 ⁇ g / mL or more, 0.7 ⁇ g / mL or more, 0.8 ⁇ g It may be / mL or more, 0.9 ⁇ g / mL or more, 1 ⁇ g / mL or more, 2 ⁇ g / mL or more, 3 ⁇ g / mL or more, or 4 ⁇ g / mL or more.
  • the concentration of the antibody or antibody fragment thereof that binds to the DUPAN-2 antibody in the second reagent of the measurement kit (1), the first antibody that binds to the DUPAN-2 antigen in the first reagent of the measurement kit (2), or The concentration of the antibody fragment and the concentration of the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (3) are 5 ⁇ g / mL or less, 6 ⁇ g / mL or less, and 7 ⁇ g / mL.
  • It may be 0.1 ⁇ g / mL to 30 ⁇ g / mL, 6 ⁇ g / mL to 30 ⁇ g / mL, or 10 ⁇ g / mL to 20 ⁇ g / mL.
  • the amount of the first antibody or antibody fragment thereof that binds to the antigen is 0.001 ⁇ g or more, 0.01 ⁇ g or more, 0.02 ⁇ g or more, 0.03 ⁇ g or more, 0.04 ⁇ g or more, 0.05 ⁇ g or more, 0.06 ⁇ g or more, It may be 0.07 ⁇ g or more, 0.08 ⁇ g or more, 0.09 ⁇ g or more, 0.1 ⁇ g or more, 0.2 ⁇ g or more, 0.3 ⁇ g or more, 0.4 ⁇ g or more, or 0.5 ⁇ g or more.
  • the amount of the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in one well of the microtiter plate is 0.6 ⁇ g or less, 0.7 ⁇ g or less, 0.8 ⁇ g or less, 0.9 ⁇ g or less, 1 ⁇ g. 2 ⁇ g or less, 3 ⁇ g or less, 4 ⁇ g or less, 5 ⁇ g or less, 6 ⁇ g or less, 7 ⁇ g or less, 7.5 ⁇ g or less, 8 g / mL or less, 9 ⁇ g or less, 10 ⁇ g or less, 11 ⁇ g or less, 12 ⁇ g or less, 13 ⁇ g or less, 14 ⁇ g or less, 15 ⁇ g or less.
  • 16 ⁇ g or less 16 ⁇ g or less, 17 ⁇ g or less, 18 ⁇ g or less, 19 ⁇ g or less, 20 ⁇ g or less, 30 ⁇ g or less, 40 ⁇ g or less, 50 ⁇ g or less, or 100 ⁇ g or less.
  • the amount of the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in one well of the microplate may be 0.01 ⁇ g to 30 ⁇ g, or 0.1 ⁇ g to 10 ⁇ g. It may be 0.3 ⁇ g to 3 ⁇ g, 0.3 ⁇ g to 1 ⁇ g, or 0.5 ⁇ g to 1 ⁇ g.
  • the concentration of the antibody or its antibody fragment is 0.001 ⁇ g / mL or more, 0.01 ⁇ g / mL or more, 0.02 ⁇ g / mL or more, 0.03 ⁇ g / mL or more, 0.04 ⁇ g / mL or more, 0.05 ⁇ g / mL or more.
  • the concentration of the second antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (2) and the DUPAN-2 antigen in the third reagent of the measurement kit (3) is not limited.
  • the concentration of the second antibody or its antibody fragment is 0.2 ⁇ g / mL or less, 0.3 ⁇ g / mL or less, 0.4 ⁇ g / mL or less, 0.5 ⁇ g / mL or less, 0.6 ⁇ g / mL or less, 0.7 ⁇ g / mL or less.
  • mL or less 0.8 ⁇ g / mL or less, 0.9 ⁇ g / mL or less, 1 ⁇ g / mL or less, 2 ⁇ g / mL or less, 3 ⁇ g / mL or less, 4 ⁇ g / mL or less, 5 ⁇ g / mL or less, 6 ⁇ g / mL or less, 7 ⁇ g / mL Below, 7.5 ⁇ g / mL or less, 8 g / mL or less, 9 ⁇ g / mL or less, 10 ⁇ g / mL or less, 11 ⁇ g / mL or less, 12 ⁇ g / mL or less, 13 ⁇ g / mL or less, 14 ⁇ g / mL or less, 15 ⁇ g / mL or less, 16 ⁇ g It may be / mL or less, 17 ⁇ g / mL or less, 18 ⁇ g
  • the concentration of the second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (2), and the DUPAN-2 antigen that binds to the DUPAN-2 antigen in the third reagent of the measurement kit (3) may be 0.01 ⁇ g / mL to 100 ⁇ g / mL, 0.03 ⁇ g / mL to 30 ⁇ g / mL, or 0.1 ⁇ g / mL to 20 ⁇ g / mL. May be.
  • the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the DUPAN-2 antigen measurement kit in the sample of the present disclosure, for example, the measurement is carried out by the above-mentioned method. can do.
  • an insoluble carrier an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, the first 3 Antigen, DUPAN-2 antigen derived from ascites, DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and as a label, for example, the above-mentioned insoluble carrier, an antibody that binds to DUPAN-2 antigen or an antibody fragment thereof, DUPAN-2.
  • First antibody or antibody fragment thereof that binds to the antigen, second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, third antibody, DUPAN-2 antigen derived from ascites, DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF , And signs can be used.
  • the insoluble carrier in the reaction solution is used.
  • Concentration of antibody or antibody fragment thereof that binds to DUPAN-2 antigen, first antibody or antibody fragment thereof that binds to DUPAN-2 antigen, second antibody or antibody fragment thereof that binds to DUPAN-2 antigen, and third antibody For example, the above-mentioned concentration can be mentioned.
  • the reaction temperature may be, for example.
  • the above-mentioned reaction temperature is mentioned, and examples of the said reaction time include the above-mentioned reaction time.
  • the DUPAN-2 antigen measurement kit in the sample of the present disclosure has the following formula when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF are measured according to the above method. X) is satisfied.
  • Such a DUPAN-2 antigen measurement kit is a highly sensitive DUPAN-2 antigen measurement kit. 0.8 ⁇ (B) / (A) ⁇ 1.2 (X) (In the formula, (A) means the measured value of DUPAN-2 antigen derived from ascites, and (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites.
  • the antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
  • the measured value of DUPAN-2 antigen derived from ascites (A) and the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF (B) may be a signal such as the above-mentioned absorbance, luminescence intensity or fluorescence intensity. It may be the concentration of the above-mentioned DUPAN-2 antigen.
  • the (B) / (A) value may be 0.7 or more, 0.8 or more, and 0.9 or more. Further, the (B) / (A) value may be 1.1 or less and 1.2 or less. The above numerical values can be freely combined. For example, the (B) / (A) value may be 0.7 to 1.2, 0.8 to 1.2, or 0.9 to 1.2.
  • the reagent and measurement kit for measuring the DUPAN-2 antigen in the sample can be used in the method for measuring the DUPAN-2 antigen in the sample.
  • Examples of the method for measuring the DUPAN-2 antigen in the sample include methods including the following (1) to (3).
  • a method including the following (1) and (2) can be mentioned.
  • (1) The DUPAN-2 antigen in the sample is reacted with an insoluble carrier, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof.
  • a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, a DUPAN-2 antigen, and a second antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen are placed.
  • a step of producing an immune complex containing; and (2) a step of measuring the immune complex produced in step (1).
  • Examples of the insoluble carrier, an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof include, for example.
  • Examples thereof include the above-mentioned insoluble carrier, an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, and a second antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen. Be done.
  • the DUPAN-2 antigen measuring reagent in the sample of the present disclosure and the DUPAN-2 antigen measuring kit in the sample of the present disclosure include the above-mentioned aqueous medium, salts, metal ions, saccharides, proteins, surfactants and polyethylene. A glycol derivative or the like may be contained.
  • aqueous medium examples include deionized water, distilled water, a buffer solution and the like, and a buffer solution is preferable.
  • examples of the buffer used for preparing the buffer include acetate buffer and Good's buffer. One type of buffer may be used alone, or two or more types may be used in combination.
  • the aqueous medium may contain the above-mentioned salts, metal ions, saccharides, proteins, surfactants and the like.
  • the polyethylene glycol derivative is not particularly limited as long as it is a polyethylene glycol derivative that enables the method for measuring the DUPAN-2 antigen of the present disclosure or can be used in the measuring reagent or measuring kit of the present disclosure.
  • examples thereof include polyethylene glycol, polyoxyethylene alkyl phenyl ether, polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, and polyoxyethylene polycyclic phenyl ether.
  • Polyoxyethylene alkyl phenyl ether and polyoxyethylene polycyclic phenyl ether are polyoxyethylene nonionic surfactants having a phenyl group.
  • polyethylene glycol examples include polyethylene glycol having a number average molecular weight of 100 to 25,000, preferably polyethylene glycol having a number average molecular weight of 200 to 20,000, and polyethylene glycol having a number average molecular weight of 6,000 to 20,000. Especially preferable.
  • Examples of the alkyl in the polyoxyethylene alkyl phenyl ether include an alkyl having 8 to 9 carbon atoms. Examples of the alkyl having 8 to 9 carbon atoms include octyl and nonyl.
  • Commercially available products of polyoxyethylene alkyl phenyl ether include, for example, Triton (registered trademark) X-100 (polyoxyethylene octylphenyl ether) (manufactured by Sigma Aldrich Japan GK) and Emargen (registered trademark) 810 (polyoxyethylene octylphenyl).
  • Emargen (registered trademark) 910 Emargen (registered trademark) 930 (above, polyoxyethylene nonylphenyl ether) (above, manufactured by Kao Co., Ltd.), Nikkor (registered trademark) OP-10 (polyoxyethylene octylphenyl ether) ), Nikkor® NP-10 (polyoxyethylene nonylphenyl ether) (above, manufactured by Nikko Chemicals Co., Ltd.), nonion HS-210, nonion HS-215, nonion HS-220 (above, polyoxyethylene octylphenyl).
  • Nonion NS-210 Nonion NS-215, Nonion NS-220 (above, polyoxyethylene nonylphenyl ether) (above, manufactured by Nichiyu Co., Ltd.), BLAUNON NK-810 (manufactured by Aoki Oil & Fat Industry Co., Ltd.), etc. Can be mentioned.
  • alkyl in the polyoxyethylene alkyl ether examples include alkyl having 8 to 24 carbon atoms, and among these, alkyl having 10 to 20 carbon atoms is preferable.
  • alkyl having 8 to 24 carbon atoms include octyl, isooctyl, nonadecylic, decyl, isodecyl, undecylic, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), and oleyl.
  • alkyl having 10 to 20 carbon atoms include decyl, isodecyl, undecylic, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl, nonadecylic, and icosyl. Can be mentioned.
  • polyoxyethylene alkyl ether Commercially available products of polyoxyethylene alkyl ether include, for example, Brij (registered trademark) 35 (polyoxyethylene lauryl ether), Brij (registered trademark) 99 (polyoxyethylene oleyl ether), and Brij (registered trademark) 56 (polyoxyethylene).
  • Cetyl ether Cetyl ether
  • Brij registered trademark
  • BC-23 polyoxyethylene cetyl ether
  • Nonion P-210, Nonion P-213 aboveve, polyoxyethylene cetyl ether
  • Nonion K-220 polyoxyethylene lauryl ether
  • Nonion E-205 Nonion E-215, Nonion E-230 (above, poly) Oxyethylene oleyl ether
  • Nonion S-215 Nonion S-220 (above, polyoxyethylene stearyl ether) (above, manufactured by Nichiyu Co., Ltd.)
  • Emargen registered trademark
  • 120 polyoxyethylene lauryl ether
  • Emargen aboveve) Registered trademark
  • 220 polyoxyethylene cetyl ether
  • Emargen registered trademark
  • Examples of the fatty acid in the polyoxyethylene sorbitan fatty acid ester include saturated or unsaturated fatty acids having 8 to 24 carbon atoms, and saturated or unsaturated fatty acids having 12 to 18 carbon atoms are preferable.
  • Saturated or unsaturated fatty acids having 8 to 24 carbon atoms include, for example, octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linolenic acid, linolenic acid, eicosatrienoic acid, arachidonic acid, and the like.
  • Examples thereof include icosanoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosanoic acid, docosahexaenoic acid, tetradocosanic acid, tetracosapentaenoic acid and the like.
  • saturated or unsaturated fatty acids having 12 to 18 carbon atoms include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid.
  • polyoxyethylene sorbitan fatty acid ester examples include polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, and polyoxyethylene sorbitan monopalmitate.
  • Commercially available products of polyoxyethylene sorbitan fatty acid ester include, for example, Tween (registered trademark) 20 (polyoxyethylene sorbitan monolaurate; manufactured by Sigma Aldrich Japan LLC) and Tween (registered trademark) 40 (polyoxyethylene sorbitan monopalmitate).
  • the polycyclic phenyl in the polyoxyethylene polycyclic phenyl ether is a phenyl group in which two or more groups having one aromatic ring (substituent) are substituted in the group, and a group having two or more aromatic rings in the group (substituent). Examples thereof include a phenyl group in which one or more substituents) are substituted. Examples of the group having one aromatic ring in the group include benzyl, 1- (phenyl) ethyl and the like. Examples of the group having two or more aromatic rings in the group include naphthyl and the like.
  • Newcol (registered trademark) 704 Newcol (registered trademark) 706, Newcol (registered trademark) 707, Newcol (registered trademark) 708, and Newcol (registered).
  • New Call (Registered Trademark) 709 New Call (Registered Trademark) 710, New Call (Registered Trademark) 711, New Call (Registered Trademark) 712, New Call (Registered Trademark) 714, New Call (Registered Trademark) 740, New Call (Registered Trademark) 610, New Call (registered trademark) 2607, New Call (registered trademark) 2609, New Call (registered trademark) 2614 (all manufactured by Nippon Embroidery Co., Ltd.) and the like can be mentioned.
  • sample The sample in the present disclosure is not particularly limited as long as it is a sample that may contain the DUPAN-2 antigen, and is not particularly limited. Examples thereof include any one or more biological samples selected from the group consisting of, preferably whole blood, plasma, serum, ascites and the like. Further, it may be a cell lysate of cultured human pancreatic cancer cells HPAF, or a culture supernatant obtained when cultured human pancreatic cancer cells HPAF are cultured. Further, it may be the DUPAN-2 antigen purified from the biological sample, the cell solubilized product or the culture supernatant.
  • DUPAN-2 antigen measurement kits 1 and 2 containing a DUPAN-2 antibody-binding microplate and a peroxidase (POD) -labeled DUPAN-2 antibody solution were prepared.
  • the measurement kit 1 had a DUPAN-2 antibody concentration of 10 ⁇ g / mL in the DUPAN-2 antibody solution
  • the measurement kit 2 had a DUPAN-2 antibody concentration of 20 ⁇ g / mL. Used for.
  • ⁇ DUPAN-2 antibody binding microplate> In each well of a 96-well polystyrene plate (manufactured by Thermo Fisher Scientific), use a binding buffer [0.01 mol / L phosphate buffer (pH 7.3) containing 0.14 mol / L sodium chloride]. 50 ⁇ L of the prepared DUPAN-2 antibody solution was added, and the mixture was incubated at 25 ° C. for 16 hours. The DUPAN-2 antibody solution is then removed and a phosphate buffer (pH 7.2) containing a blocking solution [1% (w / v) BSA, 0.05% (w / v) Tween® 20). ] To each well and wash, then 250 ⁇ L of the blocking solution was added to each well and incubated at 4 ° C. for 16 hours. Then, the blocking solution was removed to prepare a DUPAN-2 antibody-binding microplate.
  • a binding buffer [0.01 mol / L phosphate buffer (pH 7.3) containing 0.
  • ⁇ POD-labeled DUPAN-2 antibody solution> Using Peroxidase Labeling Kit-NH 2 (manufactured by Dojin Kagaku Kenkyusho Co., Ltd.), the DUPAN-2 antibody was labeled with POD according to the instruction manual of the kit to prepare a POD-labeled DUPAN-2 antibody. Using the obtained POD-labeled DUPAN-2 antibody, a POD-labeled DUPAN-2 antibody solution having the following composition was prepared.
  • a DUPAN-2 antibody-binding microplate was prepared with a DUPAN-2 antibody concentration of 5 ⁇ g / mL in the DUPAN-2 antibody solution.
  • a measurement kit A in which the DUPAN-2 antibody-binding microplate and the POD-labeled DUPAN-2 antibody solution prepared by the method described in Example 1 were combined was prepared as a comparative example.
  • DUPAN-2 antigen derived from human ascites is 40 U / mL in an antigen diluted solution [1% (w / v) BSA, 0.05 mol / L Tris buffer (pH 8.0) containing 0.15 mol / L sodium chloride]. , 100 U / mL, 350 U / mL to prepare human ascites-derived DUPAN-2 antigen solution.
  • the DUPAN-2 antigen derived from the culture supernatant of human pancreatic cancer cultured cell HPAF-II is diluted with the antigen diluted solution to 40 U / mL, 100 U / mL, 350 U / mL, and human pancreatic cancer cultured cell HPAF-II.
  • a DUPAN-2 antigen solution of origin was prepared.
  • sample diluent 100 ⁇ L of sample diluent was added to each well of the DUPAN-2 antibody-bound microplate. Then, 20 ⁇ L of a human ascites-derived DUPAN-2 antigen solution or a human pancreatic cancer cultured cell HPAF-II-derived DUPAN-2 antigen solution was added to each well and incubated at 25 ° C. for 1 hour (primary reaction). After the primary reaction, each well of the plate was subjected to a washing solution [0.05% (w / v) Tween® 20, 0.15 mol / L sodium chloride-containing 0.01 mol / L phosphate buffer (pH 7.
  • Kit 1 and Kit 2 were smaller than 1.3.
  • Kit 1 and Kit 2 showing such (B) / (A) values tend to have a larger amount of DUPAN-2 antibody bound than Comparative Example Kit A, and are measurement kits with high detection sensitivity. There was found.
  • Example 3 A DUPAN-2 antigen measurement kit 3 containing the following magnetic particle suspension, biotin-conjugated DUPAN-2 antibody solution, and alkaline phosphatase-labeled DUPAN-2 antibody solution was prepared.
  • Magnetic particle suspension As the magnetic particles, a commercially available magnetic particle [Dynabeads MyOne Streptavidin T1 (manufactured by Thermo Fisher Scientific Co., Ltd.)] to which streptavidin was bound was used to prepare a magnetic particle suspension having the following composition.
  • Biotin-bound DUPAN-2 antibody solution Using Biotin Labeling kit-NH 2 (manufactured by Dojin Kagaku Kenkyusho Co., Ltd.), a biotin-bound DUPAN-2 antibody in which biotin was bound to the DUPAN-2 antibody was prepared according to the instruction manual of the kit. Using the obtained biotin-bound DUPAN-2 antibody, a biotin-bound DUPAN-2 antibody solution having the following composition was prepared.
  • Alkaline Phosphatase Labeling kit-SH 2 (manufactured by Dojin Kagaku Kenkyusho Co., Ltd.) was used to prepare an alkaline phosphatase-labeled DUPAN-2 antibody in which alkaline phosphatase was bound to DUPAN-2 antibody according to the instruction manual of the kit.
  • an alkaline phosphatase-labeled DUPAN-2 antibody solution having the following composition was prepared.
  • Example 4 ⁇ Measurement method 2 of DUPAN-2 in the sample> The 350 U / mL ascites-derived DUPAN-2 antigen solution prepared in Example 2 and the 350 U / mL human pancreatic cancer cultured cell HPAF-II-derived DUPAN-2 antigen solution were used as measurement samples.
  • Kit 3 From the results in Table 2, the (B) / (A) values of Kit 3 were smaller than 1.3. Kit 3 showing such (B) / (A) values tended to have a larger amount of DUPAN-2 antibody bound than Comparative Example Kit A, and was found to be a measurement kit with high detection sensitivity. ..
  • the present disclosure provides a highly sensitive and accurate measurement reagent and measurement kit for DUPAN-2 antigen in a sample, which is effective for cancer diagnosis such as pancreatic cancer and biliary tract cancer.

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Abstract

The present application discloses a reagent for measuring DUPAN-2 antigen in a sample, the measurement reagent including an insoluble carrier and an antibody or antibody fragment that binds to DUPAN-2 antigen, and satisfying formula (X) when DUPAN-2 antigen derived from ascitic fluid and DUPAN-2 antigen derived from human pancreatic cancer cultured cells (HPAF) are each measured using the measurement reagent. (X): 0.8 ≤ (B)/(A) ≤ 1.2 (In the formula, (A) represents the measurement value for DUPAN-2 antigen derived from ascitic fluid, and (B) represents the measurement value for DUPAN-2 antigen derived from human pancreatic cancer cultured cells (HPAF), the antigen concentration ratio of DUPAN-2 antigen derived from ascitic fluid and DUPAN-2 antigen derived from human pancreatic cancer cultured cells (HPAF) being 1:1.)

Description

ＤＵＰＡＮ－２抗原の測定試薬及び測定キットDUPAN-2 antigen measurement reagent and measurement kit
　本発明は、ＤＵＰＡＮ－２抗原の測定試薬及び測定キットに関する。 The present invention relates to a DUPAN-2 antigen measuring reagent and a measuring kit.
　モノクローナル抗体ＤＵＰＡＮ－２は、ヒト膵癌培養細胞ＨＰＡＦを免疫原として用いて作製されたモノクローナル抗体であり、膵癌細胞から高率に産生されるムチン様タンパク質（抗原）に反応することが報告されている（非特許文献１、２）。この抗原は、ＤＵＰＡＮ－２抗原と呼ばれており、膵癌及び胆道系癌などの癌マーカーとして用いられている。 Monoclonal antibody DUPAN-2 is a monoclonal antibody produced using cultured human pancreatic cancer cells HPAF as an immunogen, and has been reported to react with a mutin-like protein (antigen) produced at a high rate from pancreatic cancer cells. (Non-Patent Documents 1 and 2). This antigen is called DUPAN-2 antigen and is used as a cancer marker for pancreatic cancer and biliary tract cancer.
　ＤＵＰＡＮ－２抗原の測定方法としては、ｃｏｍｐｅｔｉｔｉｏｎ　ｒａｄｉｏｉｍｍｕｎｏａｓｓａｙ（非特許文献１）及びＥｎｚｙｍｅ－Ｌｉｎｋｅｄ　ＩｍｍｕｎｏＳｏｒｂｅｎｔ　Ａｓｓａｙ（特許文献１）が報告されている。 As a method for measuring the DUPAN-2 antigen, competition radioimmunoassay (Non-Patent Document 1) and Enzyme-Linked Immunosorbent Assay (Patent Document 1) have been reported.
国際公開第２０１９／１８９８７４号パンフレットInternational Publication No. 2019/189874 Pamphlet
　ＤＵＰＡＮ－２抗原は、膵癌及び胆道系癌などの癌マーカーとして用いられているため、より感度が高く、正確な測定ができる測定試薬及び測定キットが必要とされていた。
　本発明の課題は、試料中のＤＵＰＡＮ－２抗原を高感度で且つ正確に測定できる試薬及びキットを提供することにある。 Since the DUPAN-2 antigen is used as a cancer marker for pancreatic cancer and biliary tract cancer, a measuring reagent and a measuring kit capable of more sensitive and accurate measurement have been required.
An object of the present invention is to provide a reagent and a kit capable of measuring a DUPAN-2 antigen in a sample with high sensitivity and accuracy.
　本発明者らは、上記課題を解決すべく鋭意検討を行った結果、不溶性担体と、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片とを含むＤＵＰＡＮ－２抗原の測定試薬を用いて、腹水由来のＤＵＰＡＮ－２抗原及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定した際の測定値を調べたところ、腹水由来のＤＵＰＡＮ－２抗原の測定値と、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値との割合が所定の値である測定試薬が、感度が高い傾向にあるということを見出し、本開示内容の発明を完成するに至った。 As a result of diligent studies to solve the above problems, the present inventors have used a DUPAN-2 antigen measuring reagent containing an insoluble carrier and an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof to ascite water. When the measured values of the DUPAN-2 antigen derived from DUPAN-2 antigen and the DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF were examined, the measured values of DUPAN-2 antigen derived from ascites and the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF were examined. It has been found that the measuring reagent having a predetermined ratio of the DUPAN-2 antigen to the measured value tends to have high sensitivity, and the invention of the present disclosure has been completed.
　すなわち、本開示は一態様において以下を提供する。
［１］試料中のＤＵＰＡＮ－２抗原の測定試薬であって、
　不溶性担体と、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、を含み、
　前記測定試薬を用いて、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定した場合に、下記式（Ｘ）を満たす、試薬。
　０．８≦（Ｂ）／（Ａ）≦１．２　（Ｘ）
（式中、（Ａ）は、腹水由来のＤＵＰＡＮ－２抗原の測定値、（Ｂ）は、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値を意味し、ここで、腹水由来のＤＵＰＡＮ－２抗原とヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原との抗原濃度比は、１：１である。）
［２］前記腹水由来のＤＵＰＡＮ－２抗原及び前記ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定する方法が、
（１）前記腹水由来のＤＵＰＡＮ－２抗原又は前記ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原に、前記不溶性担体と、前記ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、を反応させ、
　前記不溶性担体上に、前記ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原と、を含む免疫複合体を生成させる工程；及び
（２）工程（１）で生成された免疫複合体を測定する工程、
を含む方法である、［１］に記載の試薬。
［３］前記ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片が、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、及び、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片である、［１］又は［２］に記載の試薬。
［４］前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片が前記不溶性担体に結合している、［３］に記載の試薬。
［５］前記不溶性担体には２つの親和性物質のうちの片方が結合しており、前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片には２つの親和性物質のうちのもう一方が結合している、［３］又は［４］に記載の試薬。
［６］前記腹水由来のＤＵＰＡＮ－２抗原の濃度、及び前記ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の濃度が４０Ｕ／ｍＬ～４００Ｕ／ｍＬである、［１］～［５］のいずれか１に記載の試薬。 That is, the present disclosure provides the following in one aspect.
[1] A reagent for measuring the DUPAN-2 antigen in a sample.
It comprises an insoluble carrier and an antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
A reagent that satisfies the following formula (X) when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the measurement reagent.
0.8 ≤ (B) / (A) ≤ 1.2 (X)
(In the formula, (A) means the measured value of DUPAN-2 antigen derived from ascites, and (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites. The antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
[2] A method for measuring the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from the cultured human pancreatic cancer cell HPAF is
(1) The insoluble carrier and an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof are reacted with the DUPAN-2 antigen derived from ascites or the DUPAN-2 antigen derived from the human pancreatic cancer cultured cell HPAF.
A step of producing an immune complex containing the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the DUPAN-2 antigen on the insoluble carrier; and (2) produced in step (1). The process of measuring immune complexes,
The reagent according to [1], which is a method comprising.
[3] The antibody or antibody fragment thereof that binds to the DUPAN-2 antigen is the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen. The reagent according to [1] or [2].
[4] The reagent according to [3], wherein the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof is bound to the insoluble carrier.
[5] One of the two affinity substances is bound to the insoluble carrier, and the other of the two affinity substances is bound to the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen. The reagent according to [3] or [4] to which is bound.
[6] Any of [1] to [5], wherein the concentration of the DUPAN-2 antigen derived from the ascites and the concentration of the DUPAN-2 antigen derived from the human pancreatic cancer cultured cell HPAF are 40 U / mL to 400 U / mL. The reagent according to 1.
［７］試料中のＤＵＰＡＮ－２抗原の測定キットであって、
　水性媒体を含む第１試薬、並びに、不溶性担体、及びＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片を含む第２試薬を含み、
　前記測定キットを用いて、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定した場合に、下記式（Ｘ）を満たす、キット。
　０．８≦（Ｂ）／（Ａ）≦１．２　（Ｘ）
（式中、（Ａ）は、腹水由来のＤＵＰＡＮ－２抗原の測定値、（Ｂ）は、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値を意味し、ここで、腹水由来のＤＵＰＡＮ－２抗原とヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原との抗原濃度比は、１：１である。）
［８］腹水由来のＤＵＰＡＮ－２抗原及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定する方法が、
（１）腹水由来のＤＵＰＡＮ－２抗原又はヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原に、前記不溶性担体と、前記ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、を反応させ、前記不溶性担体上に、前記ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原と、を含む免疫複合体を生成させる工程；及び
（２）工程（１）で生成された免疫複合体を測定する工程、
を含む方法である、［７］に記載のキット。 [7] A measurement kit for the DUPAN-2 antigen in a sample.
It comprises a first reagent comprising an aqueous medium and a second reagent comprising an insoluble carrier and an antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
A kit that satisfies the following formula (X) when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the measurement kit.
0.8 ≤ (B) / (A) ≤ 1.2 (X)
(In the formula, (A) means the measured value of DUPAN-2 antigen derived from ascites, and (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites. The antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
[8] A method for measuring DUPAN-2 antigen derived from ascites and DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF is
(1) The insoluble carrier is reacted with the DUPAN-2 antigen derived from ascites or the DUPAN-2 antigen derived from the cultured human pancreatic cancer cell HPAF, and the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen is reacted with the insoluble. A step of producing an immune complex containing the DUPAN-2 antigen or an antibody fragment thereof and the DUPAN-2 antigen on a carrier; and (2) the immune complex produced in step (1). The process of measuring the body,
The kit according to [7], which is a method including.
［９］試料中のＤＵＰＡＮ－２抗原の測定キットであって、
　不溶性担体、及びＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片を含む第１試薬、並びに、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片を含む第２試薬を含み、
　前記測定キットを用いて、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定した場合に、下記式（Ｘ）を満たす、キット。
　０．８≦（Ｂ）／（Ａ）≦１．２　（Ｘ）
（式中、（Ａ）は、腹水由来のＤＵＰＡＮ－２抗原の測定値、（Ｂ）は、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値を意味し、ここで、腹水由来のＤＵＰＡＮ－２抗原とヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原との抗原濃度比は、１：１である。） [9] A measurement kit for DUPAN-2 antigen in a sample.
It comprises an insoluble carrier, a first reagent containing a first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, and a second reagent that contains a second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
A kit that satisfies the following formula (X) when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the measurement kit.
0.8 ≤ (B) / (A) ≤ 1.2 (X)
(In the formula, (A) means the measured value of DUPAN-2 antigen derived from ascites, and (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites. The antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
［１０］試料中のＤＵＰＡＮ－２抗原の測定キットであって、
　不溶性担体を含む第１試薬、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片を含む第２試薬、及びＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片を含む第３試薬を含み、
　前記測定キットを用いて、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定した場合に、下記式（Ｘ）を満たす、キット。
　０．８≦（Ｂ）／（Ａ）≦１．２　（Ｘ）
（式中、（Ａ）は、腹水由来のＤＵＰＡＮ－２抗原の測定値、（Ｂ）は、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値を意味し、ここで、腹水由来のＤＵＰＡＮ－２抗原とヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原との抗原濃度比は、１：１である。）
［１１］腹水由来のＤＵＰＡＮ－２抗原及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定する方法が、
（１）腹水由来のＤＵＰＡＮ－２抗原又はヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原に、前記不溶性担体と、前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片と、を反応させ、　前記不溶性担体上に、前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原と、前記ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片と、を含む免疫複合体を生成させる工程；及び
（２）工程（１）で生成された免疫複合体を測定する工程、
を含む方法である、［９］又は［１０］に記載のキット。
［１２］前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片が前記不溶性担体に結合している、［９］又は［１１］に記載のキット。
［１３］前記不溶性担体には２つの親和性物質のうちの片方が結合しており、前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片には２つの親和性物質のうちのもう一方が結合している、［９］～［１１］のいずれか１に記載のキット。
［１４］腹水由来のＤＵＰＡＮ－２抗原の濃度、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の濃度が４０Ｕ／ｍＬ～４００Ｕ／ｍＬである、［７］～［１３］のいずれか１に記載のキット。 [10] A measurement kit for the DUPAN-2 antigen in a sample.
Includes a first reagent containing an insoluble carrier, a second reagent containing a first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, and a third reagent that contains a second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen. ,
A kit that satisfies the following formula (X) when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the measurement kit.
0.8 ≤ (B) / (A) ≤ 1.2 (X)
(In the formula, (A) means the measured value of DUPAN-2 antigen derived from ascites, and (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites. The antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
[11] A method for measuring DUPAN-2 antigen derived from ascites and DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF is
(1) The insoluble carrier, the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and the DUPAN-2 to the DUPAN-2 antigen derived from ascites or the DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF. The first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, the DUPAN-2 antigen, and the DUPAN- 2 A step of generating an immune complex containing a second antibody or an antibody fragment thereof that binds to an antigen; and (2) a step of measuring the immune complex produced in step (1).
The kit according to [9] or [10], which is a method comprising the above.
[12] The kit according to [9] or [11], wherein the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof is bound to the insoluble carrier.
[13] One of the two affinity substances is bound to the insoluble carrier, and the other of the two affinity substances is bound to the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen. The kit according to any one of [9] to [11] to which is bound.
[14] The concentration of DUPAN-2 antigen derived from ascites and the concentration of DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF are 40 U / mL to 400 U / mL, in any one of [7] to [13]. The kit described.
　本開示によれば、試料中のＤＵＰＡＮ－２抗原を高感度で且つ正確に測定できる試薬及びキットを提供することができる。 According to the present disclosure, it is possible to provide a reagent and a kit capable of measuring the DUPAN-2 antigen in a sample with high sensitivity and accuracy.
　以下、本開示を実施するための形態ついて詳細に説明する。なお、以下に説明する実施形態は、本開示の代表的な実施形態の一例を示したものであり、これにより開示の範囲が狭く解釈されることはない。
　なお、「～」を用いて示された数値範囲は、「～」の前後に記載される数値をそれぞれ最小値及び最大値として含む範囲を示す。また、溶液中の各成分の量は、溶液中に各成分に該当する物質が複数存在する場合、特に断らない限り、試薬中に存在する当該複数の物質の合計量を意味する。
　本開示において、「％（ｗ／ｖ）」とは、体積（１００ｍＬ）を基準とする質量（ｇ）の百分率を意味する。 Hereinafter, the mode for carrying out the present disclosure will be described in detail. It should be noted that the embodiments described below show an example of a typical embodiment of the present disclosure, and the scope of the disclosure is not narrowly interpreted by this.
The numerical range indicated by using "-" indicates a range including the numerical values before and after "-" as the minimum value and the maximum value, respectively. Further, the amount of each component in the solution means the total amount of the plurality of substances present in the reagent when a plurality of substances corresponding to each component are present in the solution, unless otherwise specified.
In the present disclosure, "% (w / v)" means a percentage of mass (g) based on volume (100 mL).
　本開示において、抗体とＤＵＰＡＮ－２抗原とが「結合する」、「反応する」、あるいは抗体がＤＵＰＡＮ－２抗原を「認識する」と表現する場合、本開示の分野で通常使用される意味を含み、いずれも同義で用いる。抗体とＤＵＰＡＮ－２抗原とが「結合する」ことを確認する方法としては、当業者に周知の抗原固相化ＥＬＩＳＡ法、競合ＥＬＩＳＡ法、サンドイッチＥＬＩＳＡ法、表面プラズモン共鳴法、免疫クロマトグラフィー法、水晶振動子マイクロバランス法等の原理を利用した方法などにより行うことができる。 In the present disclosure, when the antibody and the DUPAN-2 antigen are expressed as "binding" or "reacting", or the antibody "recognizes" the DUPAN-2 antigen, the meaning commonly used in the art of the present disclosure is used. Including, both are used synonymously. Methods for confirming that the antibody and the DUPAN-2 antigen "bind" include an antigen-immobilized ELISA method, a competitive ELISA method, a sandwich ELISA method, a surface plasmon resonance method, and an immunochromatography method, which are well known to those skilled in the art. It can be performed by a method using a principle such as a quartz crystal microbalance method.
「試料中のＤＵＰＡＮ－２抗原の測定試薬」
　試料中のＤＵＰＡＮ－２抗原の測定試薬は、不溶性担体と、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片とを含む。ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片は、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、及び、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片であってもよい。すなわち、一実施形態において、試料中のＤＵＰＡＮ－２抗原の測定試薬は、不溶性担体と、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片とを含む。 "Reagent for measuring DUPAN-2 antigen in sample"
The reagent for measuring the DUPAN-2 antigen in the sample includes an insoluble carrier and an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof. The antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof may be a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof. .. That is, in one embodiment, the reagent for measuring the DUPAN-2 antigen in the sample is an insoluble carrier, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a second antibody that binds to the DUPAN-2 antigen. Includes the antibody fragment.
（ＤＵＰＡＮ－２抗原）
　本開示におけるＤＵＰＡＮ－２抗原は、モノクローナル抗体「ＤＵＰＡＮ－２」（Ｍｅｔｚｇａｒら、“Ａｎｔｉｇｅｎｓ　ｏｆ　Ｈｕｍａｎ　Ｐａｎｃｒｅａｔｉｃ　Ａｄｅｎｏｃａｒｃｉｎｏｍａ　Ｃｅｌｌｓ　Ｄｅｆｉｎｅｄ　ｂｙ　Ｍｕｒｉｎｅ　Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ”、ＣＡＮＣＥＲ　ＲＥＳＥＡＲＣＨ（１９８２），４２：６０１－６０８）が結合するムチン様タンパク質（糖タンパク質）、モノクローナル抗体「ＤＵＰＡＮ－２」が結合する糖鎖、又は当該糖鎖を含むペプチドをいう。具体的には、ＤＵＰＡＮ－２抗原は、モノクローナル抗体「ＤＵＰＡＮ－２」が結合すると報告されているシアリルルイスＣ糖鎖（Ｋａｗａら、“Ｅｐｉｔｏｐｅ　Ａｎａｌｙｓｉｓ　ｏｆ　Ｓｐａｎ－１　ａｎｄ　ＤＵＰＡＮ－２　ｕｓｉｎｇ　Ｓｙｎｔｈｅｓｉｚｅｄ　Ｇｌｙｃｏｃｏｎｊｕｇａｔｅｓ　Ｓｉａｌｙｌｌａｃｔ－Ｎ－Ｆｕｃｏｐｅｎｔａｏｓｅ　ＩＩ　ａｎｄ　Ｓｉａｌｙｌｌａｃｔ－Ｎ－Ｔｅｔｒａｏｓｅ”、Ｐａｎｃｒｅａｓ（１９９４），９（６）：６９２－６９７）、当該糖鎖を有するタンパク質、又は当該糖鎖を有するペプチドをいう。ＤＵＰＡＮ－２抗原は、ヒト膵癌患者の腹水、又はヒト膵癌培養細胞ＨＰＡＦの培養上清に含まれるＤＵＰＡＮ－２抗原を使用することができる。ヒト膵癌培養細胞ＨＰＡＦは、「ＨＰＡＦ－ＩＩ」という名称でＡＴＣＣ（Ａｍｅｒｉｃａｎ　Ｔｙｐｅ　Ｃｕｌｔｕｒｅ　Ｃｏｌｌｅｃｔｉｏｎ）から入手することができる。 (DUPAN-2 antigen)
The DUPAN-2 antigen in the present disclosure is the monoclonal antibody "DUPAN-2" (Metzgar et al., "Antigens of Human Pancreatic Adenocarcinoma Cells Defined by Murine Monoclonal Antibodies", CA A protein (glycoprotein), a sugar chain to which the monoclonal antibody "DUPAN-2" binds, or a peptide containing the sugar chain. Specifically, the DUPAN-2 antigen is a sialyl Lewis C sugar chain (Kawa et al., "Epitope Analysis of Span-1 and DUPAN-2 using Synthesized Glycoconjugate", which has been reported to bind to the monoclonal antibody "DUPAN-2". -Fucopentaose II and Siallylact-N-Tetoraose ", Pancreas (1994), 9 (6): 692-697), a protein having the sugar chain, or a peptide having the sugar chain. As the DUPAN-2 antigen, the DUPAN-2 antigen contained in the ascites of a human pancreatic cancer patient or the culture supernatant of human pancreatic cancer cultured cells HPAF can be used. Human pancreatic cancer cultured cell HPAF can be obtained from ATCC (American Type Culture Collection) under the name of "HPAF-II".
（不溶性担体）
　本開示における不溶性担体は、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片を結合することができ、本開示における試料中のＤＵＰＡＮ－２抗原の測定試薬に使用可能な不溶性担体であれば特に制限はなく、例えば、スライドグラス、ＥＬＩＳＡプレート（マイクロタイタープレート）、ビーズ、ラテックス粒子、磁性粒子、フィルター、フィルム及びメンブレンが挙げられる。不溶性担体の材料としては、無機基材、多糖類基材、高分子基材等からなるものが挙げられ、例えばガラス、シリコン、セラミック、セルロース、ニトロセルロース、ナイロン、ポリカーボネート、ポリエチレン、ポリプルピレン、ポリスチレン、ポリエチレンテレフタレート及びポリウレタンが挙げられる。 (Insoluble carrier)
The insoluble carrier in the present disclosure is particularly limited as long as it can bind an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof and can be used as a reagent for measuring the DUPAN-2 antigen in the sample in the present disclosure. Examples include slide glasses, ELISA plates (microtiter plates), beads, latex particles, magnetic particles, filters, films and membranes. Examples of the material of the insoluble carrier include those composed of an inorganic base material, a polysaccharide base material, a polymer base material, and the like, for example, glass, silicon, ceramic, cellulose, nitrocellulose, nylon, polycarbonate, polyethylene, polypurpyrene, polystyrene, and the like. Examples include polyethylene terephthalate and polyurethane.
　本開示の、試料中のＤＵＰＡＮ－２抗原の測定試薬における不溶性担体の濃度は、０．００１ｍｇ／ｍＬ以上、０．０１ｍｇ／ｍＬ以上、０．０２ｍｇ／ｍＬ以上、０．０３ｍｇ／ｍＬ以上、０．０４ｍｇ／ｍＬ以上、０．０５ｍｇ／ｍＬ以上、０．０６ｍｇ／ｍＬ以上、０．０７ｍｇ／ｍＬ以上、０．０８ｍｇ／ｍＬ以上、０．０９ｍｇ／ｍＬ以上、０．１ｍｇ／ｍＬ以上、０．２ｍｇ／ｍＬ以上、０．３ｍｇ／ｍＬ以上、０．４ｍｇ／ｍＬ以上、０．５ｍｇ／ｍＬ以上であってよい。また、前記測定試薬における不溶性担体の濃度は、０．６ｍｇ／ｍＬ以下、０．７ｍｇ／ｍＬ以下、０．８ｍｇ／ｍＬ以下、０．９ｍｇ／ｍＬ以下、１ｍｇ／ｍＬ以下、２ｍｇ／ｍＬ以下、３ｍｇ／ｍＬ以下、４ｍｇ／ｍＬ以下、５ｍｇ／ｍＬ以下、６ｍｇ／ｍＬ以下、７ｍｇ／ｍＬ以下、８ｍｇ／ｍＬ以下、９ｍｇ／ｍＬ以下、１０ｍｇ／ｍＬ以下であってよい。 The concentration of the insoluble carrier in the DUPAN-2 antigen measuring reagent in the sample of the present disclosure is 0.001 mg / mL or more, 0.01 mg / mL or more, 0.02 mg / mL or more, 0.03 mg / mL or more, 0. .04 mg / mL or more, 0.05 mg / mL or more, 0.06 mg / mL or more, 0.07 mg / mL or more, 0.08 mg / mL or more, 0.09 mg / mL or more, 0.1 mg / mL or more, 0. It may be 2 mg / mL or more, 0.3 mg / mL or more, 0.4 mg / mL or more, and 0.5 mg / mL or more. The concentration of the insoluble carrier in the measurement reagent is 0.6 mg / mL or less, 0.7 mg / mL or less, 0.8 mg / mL or less, 0.9 mg / mL or less, 1 mg / mL or less, 2 mg / mL or less, It may be 3 mg / mL or less, 4 mg / mL or less, 5 mg / mL or less, 6 mg / mL or less, 7 mg / mL or less, 8 mg / mL or less, 9 mg / mL or less, 10 mg / mL or less.
　前記の数値は自由に組み合わせることができる。例えば、前記測定試薬における不溶性担体の濃度は、０．０１ｍｇ／ｍＬ～１０ｍｇ／ｍＬであってもよく、０．０３ｍｇ／ｍＬ～５ｍｇ／ｍＬであってもよく、０．１ｍｇ／ｍＬ～２ｍｇ／ｍＬであってもよい。
　本開示では、ある濃度又は重量について、上限値の例と下限値の例とからそれぞれ任意の数値を選択し、それらを組み合わせて得られる数値範囲も、本開示に例示されているものとみなす。 The above numerical values can be freely combined. For example, the concentration of the insoluble carrier in the measurement reagent may be 0.01 mg / mL to 10 mg / mL, 0.03 mg / mL to 5 mg / mL, or 0.1 mg / mL to 2 mg / mL. It may be mL.
In the present disclosure, an arbitrary numerical value is selected from an example of an upper limit value and an example of a lower limit value for a certain concentration or weight, and a numerical range obtained by combining them is also considered to be exemplified in the present disclosure.
（ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片）
　本開示において、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片（ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片）とは、ＤＵＰＡＮ－２抗原のシアリルルイスＣを認識し、結合することができる抗体をいう。
　一実施形態において、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片は、国際公開第２０１９／１８９８７４号パンフレットに開示される、
下記糖鎖構造を有するＤＵＰＡＮ－２抗原
Figure JPOXMLDOC01-appb-C000001
 と反応し、かつ、
下記糖鎖構造を有するＮＣＣ－ＳＴ－４３９抗原
Figure JPOXMLDOC01-appb-C000002
 と反応しない抗体又はその抗体断片であってよく、上記ＮＣＣ－ＳＴ－４３９抗原に対する反応性が上記ＤＵＰＡＮ－２抗原に対する反応性の１０％未満である抗体又はその抗体断片であってよく、上記ＮＣＣ－ＳＴ－４３９抗原に対する反応性が上記ＤＵＰＡＮ－２抗原に対する反応性の１％未満である抗体又はその抗体断片であってよく、また、これらの抗体はラット抗体であってよい。 (Antibody that binds to DUPAN-2 antigen or an antibody fragment thereof)
In the present disclosure, an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof (a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof) is referred to as an antibody thereof or an antibody fragment thereof. An antibody capable of recognizing and binding to the DUPAN-2 antigen, sialyl Lewis C.
In one embodiment, an antibody or antibody fragment thereof that binds to the DUPAN-2 antigen is disclosed in WO 2019/189874.
DUPAN-2 antigen having the following sugar chain structure
Figure JPOXMLDOC01-appb-C000001
 Reacts with
NCC-ST-439 antigen having the following sugar chain structure
Figure JPOXMLDOC01-appb-C000002
 It may be an antibody or an antibody fragment thereof that does not react with the NCC, and may be an antibody or an antibody fragment thereof having a reactivity with the NCC-ST-439 antigen of less than 10% of the reactivity with the DUPAN-2 antigen. -An antibody or an antibody fragment thereof having a reactivity to the ST-439 antigen of less than 1% of the reactivity to the DUPAN-2 antigen may be used, and these antibodies may be rat antibodies.
　ＤＵＰＡＮ－２抗原に結合する抗体は、マウス抗体、ラット抗体、ウサギ抗体、ヒト抗体、ヒト化抗体又はキメラ抗体であってよく、又は他の種由来抗体であってもよい。また、ＤＵＰＡＮ－２抗原に結合する抗体は、免疫グロブリン分子の任意のクラス（例えば、ＩｇＧ、ＩｇＥ、ＩｇＭ、ＩｇＤ及びＩｇＡ）、任意のサブクラス（例えば、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４、ＩｇＡ１及びＩｇＡ２）であり得る。 The antibody that binds to the DUPAN-2 antigen may be a mouse antibody, a rat antibody, a rabbit antibody, a human antibody, a humanized antibody or a chimeric antibody, or may be an antibody derived from another species. Antibodies that bind to the DUPAN-2 antigen can be any class of immunoglobulin molecules (eg, IgG, IgE, IgM, IgD and IgA), any subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). ) Can be.
　ＤＵＰＡＮ－２抗原に結合する抗体は、モノクローナル抗体、ポリクローナル抗体、二量体、多量体等が挙げられ、均質性及び安定性の観点からはモノクローナル抗体が好ましい。 Examples of the antibody that binds to the DUPAN-2 antigen include a monoclonal antibody, a polyclonal antibody, a dimer, a multimer, and the like, and a monoclonal antibody is preferable from the viewpoint of homogeneity and stability.
　本開示において、抗体断片とは、前記ＤＵＰＡＮ－２抗原に結合する抗体の一部であって、抗体の可変ドメインを含むか、少なくとも抗原結合領域を含むものを言う。本開示における抗体断片としては、例えばＦａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、Ｆｖ断片、線状抗体、一本鎖抗体（ｓｃＦｖ）、ｓｃ（Ｆｖ）２、Ｆａｂ３、ドメイン抗体（ｄＡｂ）、ダイアボディ、トリアボディ、テトラボディ及びミニボディが挙げられる。「Ｆｖ断片」は最小の抗体断片であり、完全な抗原認識領域と抗原結合領域を含む。 In the present disclosure, the antibody fragment refers to a part of an antibody that binds to the DUPAN-2 antigen and contains a variable domain of the antibody or at least an antigen-binding region. The antibody fragments in the present disclosure include, for example, Fab, Fab', F (ab') 2 , Fv fragment, linear antibody, single-chain antibody (scFv), sc (Fv) 2 , Fab 3 , and domain antibody (dAb). , Diabody, Triabody, Tetrabody and Minibody. An "Fv fragment" is the smallest antibody fragment that contains a complete antigen recognition region and antigen binding region.
　本開示のＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片は、不溶性担体に結合していてもよいし、結合していなくてもよい。不溶性担体としては、例えば前述の不溶性担体が挙げられる。ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片の、不溶性担体への結合方法としては、例えば後述の結合方法が挙げられる。 The antibody or antibody fragment thereof that binds to the DUPAN-2 antigen of the present disclosure may or may not be bound to an insoluble carrier. Examples of the insoluble carrier include the above-mentioned insoluble carrier. Examples of the method for binding an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen to an insoluble carrier include the binding method described below.
　本開示のＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片として、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片とを用いてもよい。ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、及びＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片は、それぞれの抗体又はその抗体断片が結合するＤＵＰＡＮ－２抗原の部位は同じであっても、異なっていてもよい。また、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片とは同一の抗体であってもよいし、異なる抗体であってもよい。 As the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen of the present disclosure, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof and a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof are used. You may. The first antibody or its antibody fragment that binds to the DUPAN-2 antigen and the second antibody or its antibody fragment that binds to the DUPAN-2 antigen have the same site of the DUPAN-2 antigen to which each antibody or its antibody fragment binds. However, they may be different. Further, the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof and the second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof may be the same antibody or different antibodies. good.
　本開示におけるＤＵＰＡＮ－２抗原に結合する抗体は、ＤＵＰＡＮ－２抗原、又はＤＵＰＡＮ－２抗原を産生する癌細胞を免疫原として用いて、通常の抗体の作製方法により取得することができる。例えば、前記免疫原を動物に免疫することで得られる抗体産生細胞と、ミエローマ細胞とを融合した細胞（ハイブリドーマ）を作製し、前記ハイブリドーマが産生するモノクローナル抗体を得ることにより取得することができる。ＤＵＰＡＮ－２抗原に結合するモノクローナル抗体としては、前述のモノクローナル抗体「ＤＵＰＡＮ－２」（Ｍｅｔｚｇａｒら、“Ａｎｔｉｇｅｎｓ　ｏｆ　Ｈｕｍａｎ　Ｐａｎｃｒｅａｔｉｃ　Ａｄｅｎｏｃａｒｃｉｎｏｍａ　Ｃｅｌｌｓ　Ｄｅｆｉｎｅｄ　ｂｙ　Ｍｕｒｉｎｅ　Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ”、ＣＡＮＣＥＲ　ＲＥＳＥＡＲＣＨ（１９８２），４２：６０１－６０８）等が挙げられる。なお、本開示において、当該モノクローナル抗体ＤＵＰＡＮ－２を「ＤＵＰＡＮ－２抗体」とも記載する。 The antibody that binds to the DUPAN-2 antigen in the present disclosure can be obtained by a conventional method for producing an antibody using a DUPAN-2 antigen or a cancer cell that produces the DUPAN-2 antigen as an immunogen. For example, it can be obtained by producing a cell (hybridoma) in which an antibody-producing cell obtained by immunizing an animal with the immunogen and a myeloma cell is produced, and a monoclonal antibody produced by the hybridoma is obtained. As the monoclonal antibody that binds to the DUPAN-2 antigen, the above-mentioned monoclonal antibody "DUPAN-2" (Metzgar et al., "Antigens of Human Cancer Cellrecined by Murine Monoclonial ) Etc. can be mentioned. In the present disclosure, the monoclonal antibody DUPAN-2 is also referred to as "DUPAN-2 antibody".
　本開示の試料中のＤＵＰＡＮ－２抗原の測定試薬における、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片の濃度、及び、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の濃度は、０．０１μｇ／ｍＬ以上、０．０２μｇ／ｍＬ以上、０．０３μｇ／ｍＬ以上、０．０４μｇ／ｍＬ以上、０．０５μｇ／ｍＬ以上、０．０６μｇ／ｍＬ以上、０．０７μｇ／ｍＬ以上、０．０８μｇ／ｍＬ以上、０．０９μｇ／ｍＬ以上、０．１μｇ／ｍＬ以上、０．２μｇ／ｍＬ以上、０．３μｇ／ｍＬ以上、０．４μｇ／ｍＬ以上、０．５μｇ／ｍＬ以上、０．６μｇ／ｍＬ以上、０．７μｇ／ｍＬ以上、０．８μｇ／ｍＬ以上、０．９μｇ／ｍＬ以上、１μｇ／ｍＬ以上、２μｇ／ｍＬ以上、３μｇ／ｍＬ以上、４μｇ／ｍＬ以上、５μｇ／ｍＬ以上、６μｇ／ｍＬ以上、７μｇ／ｍＬ以上、７．５μｇ／ｍＬ以上、８μｇ／ｍＬ以上、又は９μｇ／ｍＬ以上であってよい。また、前記測定試薬における、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片の濃度、及び、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の濃度は、１０μｇ／ｍＬ以下、１１μｇ／ｍＬ以下、１２μｇ／ｍＬ以下、１３μｇ／ｍＬ以下、１４μｇ／ｍＬ以下、１５μｇ／ｍＬ以下、１６μｇ／ｍＬ以下、１７μｇ／ｍＬ以下、１８μｇ／ｍＬ以下、１９μｇ／ｍＬ以下、２０μｇ／ｍＬ以下、３０μｇ／ｍＬ以下、４０μｇ／ｍＬ以下、５０μｇ／ｍＬ以下、又は１００μｇ／ｍＬ以下であってよい。 The concentration of the antibody that binds to the DUPAN-2 antigen or its antibody fragment and the concentration of the first antibody that binds to the DUPAN-2 antigen or its antibody fragment in the reagent for measuring the DUPAN-2 antigen in the sample of the present disclosure are as follows. 0.01 μg / mL or more, 0.02 μg / mL or more, 0.03 μg / mL or more, 0.04 μg / mL or more, 0.05 μg / mL or more, 0.06 μg / mL or more, 0.07 μg / mL or more, 0 .08 μg / mL or more, 0.09 μg / mL or more, 0.1 μg / mL or more, 0.2 μg / mL or more, 0.3 μg / mL or more, 0.4 μg / mL or more, 0.5 μg / mL or more, 0. 6 μg / mL or more, 0.7 μg / mL or more, 0.8 μg / mL or more, 0.9 μg / mL or more, 1 μg / mL or more, 2 μg / mL or more, 3 μg / mL or more, 4 μg / mL or more, 5 μg / mL or more , 6 μg / mL or higher, 7 μg / mL or higher, 7.5 μg / mL or higher, 8 μg / mL or higher, or 9 μg / mL or higher. The concentration of the antibody or its antibody fragment that binds to the DUPAN-2 antigen and the concentration of the first antibody or its antibody fragment that binds to the DUPAN-2 antigen in the measurement reagent are 10 μg / mL or less and 11 μg / mL. 12 μg / mL or less, 13 μg / mL or less, 14 μg / mL or less, 15 μg / mL or less, 16 μg / mL or less, 17 μg / mL or less, 18 μg / mL or less, 19 μg / mL or less, 20 μg / mL or less, 30 μg / mL Hereinafter, it may be 40 μg / mL or less, 50 μg / mL or less, or 100 μg / mL or less.
　前記の数値は自由に組み合わせることができる。例えば、前記測定試薬における、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片の濃度、及びＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の濃度は、０．０１μｇ／ｍＬ～１００μｇ／ｍＬであってもよく、０．１μｇ／ｍＬ～３０μｇ／ｍＬであってもよく、６μｇ／ｍＬ～３０μｇ／ｍＬであってもよく、１０μｇ／ｍＬ～２０μｇ／ｍＬであってもよい。 The above values can be freely combined. For example, the concentration of the antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen and the concentration of the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen in the measurement reagent are 0.01 μg / mL to 100 μg / mL. It may be 0.1 μg / mL to 30 μg / mL, 6 μg / mL to 30 μg / mL, or 10 μg / mL to 20 μg / mL.
　ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片、又は、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片がマイクロタイタープレートに結合しており、且つ、水性媒体を含んでおらず乾燥状態である場合、マイクロタイタープレートの１ウェルにおける、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片の濃度、及びＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の量は、０．００１μｇ以上、０．０１μｇ以上、０．０２μｇ以上、０．０３μｇ以上、０．０４μｇ以上、０．０５μｇ以上、０．０６μｇ以上、０．０７μｇ以上、０．０８μｇ以上、０．０９μｇ以上、０．１μｇ以上、０．２μｇ以上、０．３μｇ以上、０．４μｇ以上、又は０．５μｇ以上であってよい。また、前記マイクロタイタープレートの１ウェルにおける、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片の濃度、及びＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の量は、０．６μｇ以下、０．７μｇ以下、０．８μｇ以下、０．９μｇ以下、１μｇ以下、２μｇ以下、３μｇ以下、４μｇ以下、５μｇ以下、６μｇ以下、７μｇ以下、７．５μｇ以下、８ｇ／ｍＬ以下、９μｇ以下、１０μｇ以下、１１μｇ以下、１２μｇ以下、１３μｇ以下、１４μｇ以下、１５μｇ以下、１６μｇ以下、１７μｇ以下、１８μｇ以下、１９μｇ以下、２０μｇ以下、３０μｇ以下、４０μｇ以下、５０μｇ以下、又は１００μｇ以下であってよい。 The antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, or the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof is bound to a microtiter plate and does not contain an aqueous medium and is in a dry state. If, the concentration of the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the amount of the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in one well of the microtiter plate is 0.001 μg or more. , 0.01 μg or more, 0.02 μg or more, 0.03 μg or more, 0.04 μg or more, 0.05 μg or more, 0.06 μg or more, 0.07 μg or more, 0.08 μg or more, 0.09 μg or more, 0.1 μg or more , 0.2 μg or more, 0.3 μg or more, 0.4 μg or more, or 0.5 μg or more. The concentration of the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the amount of the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in one well of the microtiter plate are 0.6 μg or less. 0.7 μg or less, 0.8 μg or less, 0.9 μg or less, 1 μg or less, 2 μg or less, 3 μg or less, 4 μg or less, 5 μg or less, 6 μg or less, 7 μg or less, 7.5 μg or less, 8 g / mL or less, 9 μg or less, 10 μg Below, it may be 11 μg or less, 12 μg or less, 13 μg or less, 14 μg or less, 15 μg or less, 16 μg or less, 17 μg or less, 18 μg or less, 19 μg or less, 20 μg or less, 30 μg or less, 40 μg or less, 50 μg or less, or 100 μg or less.
　前記の数値は自由に組み合わせることができる。例えば、前記マイクロタイタープレートの１ウェルにおける、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片の濃度、及びＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の量は、０．０１μｇ～３０μｇであってもよく、０．１μｇ～１０μｇであってもよく、０．３μｇ～３μｇであってもよく、０．３μｇ～１μｇであってもよく、０．５μｇ～１μｇであってもよい。 The above values can be freely combined. For example, the concentration of the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the amount of the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in one well of the microtiter plate are 0.01 μg to 30 μg. It may be 0.1 μg to 10 μg, 0.3 μg to 3 μg, 0.3 μg to 1 μg, or 0.5 μg to 1 μg.
　本開示の、試料中のＤＵＰＡＮ－２抗原の測定試薬における、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の濃度は、０．００１μｇ／ｍＬ以上、０．０１μｇ／ｍＬ以上、０．０２μｇ／ｍＬ以上、０．０３μｇ／ｍＬ以上、０．０４μｇ／ｍＬ以上、０．０５μｇ／ｍＬ以上、０．０６μｇ／ｍＬ以上、０．０７μｇ／ｍＬ以上、０．０８μｇ／ｍＬ以上、０．０９μｇ／ｍＬ以上、又は０．１μｇ／ｍＬ以上であってよい。また、前記測定試薬における、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の濃度は、０．２μｇ／ｍＬ以下、０．３μｇ／ｍＬ以下、０．４μｇ／ｍＬ以下、０．５μｇ／ｍＬ以下、０．６μｇ／ｍＬ以下、０．７μｇ／ｍＬ以下、０．８μｇ／ｍＬ以下、０．９μｇ／ｍＬ以下、１μｇ／ｍＬ以下、２μｇ／ｍＬ以下、３μｇ／ｍＬ以下、４μｇ／ｍＬ以下、５μｇ／ｍＬ以下、６μｇ／ｍＬ以下、７μｇ／ｍＬ以下、７．５μｇ／ｍＬ以下、８ｇ／ｍＬ以下、９μｇ／ｍＬ以下、１０μｇ／ｍＬ以下、１１μｇ／ｍＬ以下、１２μｇ／ｍＬ以下、１３μｇ／ｍＬ以下、１４μｇ／ｍＬ以下、１５μｇ／ｍＬ以下、１６μｇ／ｍＬ以下、１７μｇ／ｍＬ以下、１８μｇ／ｍＬ以下、１９μｇ／ｍＬ以下、２０μｇ／ｍＬ以下、３０μｇ／ｍＬ以下、４０μｇ／ｍＬ以下、５０μｇ／ｍＬ以下、又は１００μｇ／ｍＬ以下であってよい。 The concentration of the second antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen in the DUPAN-2 antigen measuring reagent in the sample of the present disclosure is 0.001 μg / mL or more, 0.01 μg / mL or more, 0. 02 μg / mL or more, 0.03 μg / mL or more, 0.04 μg / mL or more, 0.05 μg / mL or more, 0.06 μg / mL or more, 0.07 μg / mL or more, 0.08 μg / mL or more, 0.09 μg It may be / mL or more, or 0.1 μg / mL or more. The concentration of the second antibody or its antibody fragment that binds to the DUPAN-2 antigen in the measurement reagent is 0.2 μg / mL or less, 0.3 μg / mL or less, 0.4 μg / mL or less, 0.5 μg /. mL or less, 0.6 μg / mL or less, 0.7 μg / mL or less, 0.8 μg / mL or less, 0.9 μg / mL or less, 1 μg / mL or less, 2 μg / mL or less, 3 μg / mL or less, 4 μg / mL or less 5, 5 μg / mL or less, 6 μg / mL or less, 7 μg / mL or less, 7.5 μg / mL or less, 8 g / mL or less, 9 μg / mL or less, 10 μg / mL or less, 11 μg / mL or less, 12 μg / mL or less, 13 μg / mL or less, 14 μg / mL or less, 15 μg / mL or less, 16 μg / mL or less, 17 μg / mL or less, 18 μg / mL or less, 19 μg / mL or less, 20 μg / mL or less, 30 μg / mL or less, 40 μg / mL or less, 50 μg / It may be mL or less, or 100 μg / mL or less.
　前記の数値は自由に組み合わせることができる。例えば、前記測定試薬におけるＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の濃度は、０．０１μｇ／ｍＬ～１００μｇ／ｍＬであってもよく、０．０３μｇ／ｍＬ～３０μｇ／ｍＬであってもよく、０．１μｇ／ｍＬ～２０μｇ／ｍＬであってもよい。 The above values can be freely combined. For example, the concentration of the second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in the measurement reagent may be 0.01 μg / mL to 100 μg / mL, or 0.03 μg / mL to 30 μg / mL. It may be 0.1 μg / mL to 20 μg / mL.
　本開示の、試料中のＤＵＰＡＮ－２抗原の測定試薬において、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片は不溶性担体に結合していてもよいし、結合していなくてもよい。 In the reagent for measuring the DUPAN-2 antigen in the sample of the present disclosure, the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen may or may not be bound to the insoluble carrier.
　ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片、及びＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の、不溶性担体への結合方法としては、本開示のＤＵＰＡＮ－２抗原の測定試薬に使用可能な不溶性担体であれば特に制限はなく、物理吸着による結合、化学結合による結合等が挙げられる。物理吸着としては、静電的結合、水素結合、疎水結合等が挙げられる。化学結合としては、共有結合、配位結合等が挙げられる。 As a method for binding an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof to an insoluble carrier, the reagent for measuring the DUPAN-2 antigen of the present disclosure can be used. The insoluble carrier that can be used is not particularly limited, and examples thereof include binding by physical adsorption and binding by chemical bonding. Examples of the physical adsorption include electrostatic bonds, hydrogen bonds, hydrophobic bonds and the like. Examples of the chemical bond include a covalent bond and a coordinate bond.
　ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片、及びＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片は、前述の物理吸着及び／又は化学結合を利用して、直接、不溶性担体に結合させてもよいし、間接的に不溶性担体に結合させてもよい。間接的な結合方法としては、ビオチンとアビジン類（アビジン、ストレプトアビジン、ニュートラアビジン等）等の一組の親和性物質の特異的結合を利用して、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片、及びＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片を不溶性担体に結合する方法、リンカーを介した共有結合により不溶性担体に結合する方法等が挙げられる。 The antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen directly bind to the insoluble carrier by utilizing the above-mentioned physical adsorption and / or chemical bond. It may be allowed to bind or indirectly bind to an insoluble carrier. As an indirect binding method, an antibody that binds to the DUPAN-2 antigen or an antibody thereof by utilizing the specific binding of a set of affinity substances such as biotin and avidins (avidin, streptavidin, neutravidin, etc.) or the like. Examples thereof include a method of binding a fragment and a first antibody that binds to a DUPAN-2 antigen or an antibody fragment thereof to an insoluble carrier, a method of binding to an insoluble carrier by covalent bonding via a linker, and the like.
　一組の親和性物質を用いる場合、一組の親和性物質の一方（Ａ）が結合したＤＵＰＡＮ－２抗原に結合する抗体若しくはその抗体断片、又はＤＵＰＡＮ－２抗原に結合する第１抗体若しくはその抗体断片と、一組の親和性物質の他方（ａ）が結合した不溶性担体とを、結合させることにより、ＤＵＰＡＮ－２抗原に結合する抗体若しくはその抗体断片、又はＤＵＰＡＮ－２抗原に結合する第１抗体若しくはその抗体断片が結合した不溶性担体を生成させることができる。 When a set of affinity substances is used, an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen to which one of the sets of affinity substances (A) is bound, or a first antibody or a first antibody thereof that binds to the DUPAN-2 antigen. By binding an antibody fragment and an insoluble carrier to which the other (a) of a set of affinity substances is bound, an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, or a DUPAN-2 antigen is bound. An insoluble carrier to which one antibody or an antibody fragment thereof is bound can be produced.
　ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片、及びＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の、不溶性担体への結合方法は、後述する抗原抗体反応の反応液中で生成されてもよく、この場合、一組の親和性物質の一方（Ａ）が結合したＤＵＰＡＮ－２抗原に結合する抗体若しくはその抗体断片、又はＤＵＰＡＮ－２抗原に結合する第１抗体若しくはその抗体断片と、一組の親和性物質の他方（ａ）が結合した不溶性担体とを、抗原抗体反応の反応液中で反応させることにより、ＤＵＰＡＮ－２抗原に結合する抗体若しくはその抗体断片、又はＤＵＰＡＮ－２抗原に結合する第１抗体若しくはその抗体断片が結合した不溶性担体を、抗原抗体反応の反応液中で生成させることができる。 A method for binding an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen and a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen to an insoluble carrier is generated in a reaction solution for an antigen-antibody reaction described later. In this case, an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen to which one of the pair of affinity substances (A) is bound, or a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen may be used. , An antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, or DUPAN-2 by reacting an insoluble carrier to which the other (a) of a set of affinity substances is bound in the reaction solution of the antigen-antibody reaction. An insoluble carrier to which a first antibody that binds to an antigen or an antibody fragment thereof is bound can be produced in the reaction solution of an antigen-antibody reaction.
　Ａ－ａの組み合わせとしては、以下の組み合わせ等が挙げられる。
・ビオチンとアビジン類（アビジン、ニュートラアビジン、ストレプトアビジン等）との組み合わせ；
・アビジン類（アビジン、ニュートラアビジン、ストレプトアビジン等）とビオチンとの組み合わせ；
・ＤＵＰＡＮ－２抗原に結合する抗体若しくはその抗体断片のＦｃ領域、又はＤＵＰＡＮ－２抗原に結合する第１抗体若しくはその抗体断片のＦｃ領域と、Ｆｃ領域と結合する抗体との組み合わせ。 Examples of the combination of Aa include the following combinations and the like.
・ Combination of biotin and avidins (avidin, neutral avidin, streptavidin, etc.);
・ Combination of avidins (avidin, neutravidin, streptavidin, etc.) and biotin;
-A combination of the Fc region of an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, or the Fc region of a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, and an antibody that binds to the Fc region.
　リンカーとしては、不溶性担体表面の官能基と、ＤＵＰＡＮ－２抗原に結合する抗体若しくはその抗体断片、又はＤＵＰＡＮ－２抗原に結合する第１抗体若しくはその抗体断片が有する官能基との両者を共有結合できる分子等が挙げられ、例えば、ＤＵＰＡＮ－２抗原に結合する抗体若しくはその抗体断片、又はＤＵＰＡＮ－２抗原に結合する第１抗体若しくはその抗体断片が有する官能基と反応することができる第１の反応活性基と、不溶性担体表面の官能基と反応することができる第２の反応活性基とを同時に持つ分子であって、第１の反応活性基と第２の反応活性基が異なる基である分子が好ましく用いられる。ＤＵＰＡＮ－２抗原に結合する抗体若しくはその抗体断片、又はＤＵＰＡＮ－２抗原に結合する第１抗体若しくはその抗体断片が有する官能基、及び不溶性担体がその表面に保持している官能基としては、カルボキシル基、アミノ基、グリシジル基、スルフヒドリル基、水酸基、アミド基、イミノ基、Ｎ－ヒドロキシサクシニル基、マレイミド基等が挙げられる。リンカーにおける反応活性基としては、アリルアジド、カルボジイミド、ヒドラジド、アルデヒド、ヒドロキシメチルホスフィン、イミドエステル、イソシアネート、マレイミド、Ｎ－ヒドロキシスクシンイミドエステル、ペンタフルオロフェニル（ＰＦＰ）エステル、ソラレン、ピリジルジスルフィド、ビニルスルホン等の基が挙げられる。 The linker covalently binds both the functional group on the surface of the insoluble carrier and the functional group of the antibody that binds to the DUPAN-2 antigen or its antibody fragment, or the first antibody that binds to the DUPAN-2 antigen or its antibody fragment. Examples thereof include molecules capable of reacting with a functional group contained in an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, or a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof. It is a molecule having a reaction active group and a second reaction active group capable of reacting with a functional group on the surface of the insoluble carrier at the same time, and the first reaction active group and the second reaction active group are different groups. Molecules are preferably used. The functional group of the antibody that binds to the DUPAN-2 antigen or its antibody fragment, or the functional group of the first antibody that binds to the DUPAN-2 antigen or its antibody fragment, and the functional group held on the surface of the insoluble carrier are carboxyl. Examples thereof include a group, an amino group, a glycidyl group, a sulfhydryl group, a hydroxyl group, an amide group, an imino group, an N-hydroxysuccinyl group, a maleimide group and the like. Examples of the reactive active group in the linker include allyl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, maleimide, N-hydroxysuccinimide ester, pentafluorophenyl (PFP) ester, solarene, pyridyl disulfide, vinyl sulfone and the like. The group is mentioned.
　本開示の、試料中のＤＵＰＡＮ－２抗原の測定試薬において、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片は不溶性担体に結合していてもよいし、結合していなくてもよい。ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の、不溶性担体への結合方法としては、本開示のＤＵＰＡＮ－２抗原の測定試薬に使用可能な不溶性担体であれば特に制限はなく、例えば前述の結合方法が挙がられる。
　本開示の、試料中のＤＵＰＡＮ－２抗原の測定試薬において、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片が不溶性担体に結合しており、かつ、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片が不溶性担体に結合していてもよい。ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片が結合する不溶性担体と、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片が結合する不溶性担体とは、同じ種類の不溶性担体であってもよいし、異なる種類の不溶性担体であってもよい。また、同一の不溶性担体上に、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片とが結合していてもよい。 In the reagent for measuring the DUPAN-2 antigen in the sample of the present disclosure, the second antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen may or may not be bound to the insoluble carrier. The method for binding the second antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen to the insoluble carrier is not particularly limited as long as it is an insoluble carrier that can be used as the measurement reagent for the DUPAN-2 antigen of the present disclosure. The above-mentioned bonding method can be mentioned.
In the reagent for measuring the DUPAN-2 antigen in the sample of the present disclosure, the first antibody that binds to the DUPAN-2 antigen or the antibody fragment thereof is bound to the insoluble carrier, and the second antibody that binds to the DUPAN-2 antigen. The antibody or antibody fragment thereof may be bound to an insoluble carrier. The insoluble carrier to which the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen binds and the insoluble carrier to which the second antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen binds are the same type of insoluble carrier. It may be an insoluble carrier of a different type. Further, the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof and the second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof may be bound on the same insoluble carrier.
（腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定方法）
　本開示の、試料中のＤＵＰＡＮ－２抗原の測定試薬を用いて、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定する場合、例えば以下の工程を含む方法により測定することができる。
（１Ａ）腹水由来のＤＵＰＡＮ－２抗原、又はヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原に、不溶性担体と、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、を反応させ、前記不溶性担体上に、前記ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原と、を含む免疫複合体を生成させる工程；及び
（２Ａ）工程（１Ａ）で生成された免疫複合体を測定する工程。 (Measurement method of DUPAN-2 antigen derived from ascites and DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF)
When the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the DUPAN-2 antigen measuring reagent in the sample of the present disclosure, for example, the following steps are included. It can be measured by the method.
(1A) The insoluble carrier is reacted with the DUPAN-2 antigen derived from ascites or the DUPAN-2 antigen derived from the cultured human pancreatic cancer cell HPAF with an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and the insoluble carrier is described. Above, a step of producing an immune complex comprising the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the DUPAN-2 antigen; and (2A) the immune complex produced in step (1A). The process of measuring.
　前述の測定方法において、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片として、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片とを用いてもよく、例えば以下の工程を含む方法により、ＤＵＰＡＮ－２抗原を測定することができる。
（１Ｂ）腹水由来のＤＵＰＡＮ－２抗原、又はヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原に、不溶性担体と、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片と、を反応させ、前記不溶性担体上に、前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原と、前記ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片と、を含む免疫複合体を生成させる工程；及び
（２Ｂ）工程（１Ｂ）で生成された免疫複合体を測定する工程。 In the above-mentioned measurement method, as an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen and a second antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen. And may be used, and the DUPAN-2 antigen can be measured, for example, by a method including the following steps.
(1B) To DUPAN-2 antigen derived from ascites or DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF, an insoluble carrier, a first antibody that binds to DUPAN-2 antigen or an antibody fragment thereof, and DUPAN-2 antigen. The first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, the DUPAN-2 antigen, and the DUPAN-2 antigen are reacted on the insoluble carrier by reacting the second antibody or the antibody fragment thereof. A step of producing an immune complex containing a second antibody or an antibody fragment thereof that binds to; and a step of measuring the immune complex produced in step (2B) (1B).
　さらに、工程（２Ｂ）の後に、以下の工程（３）及び工程（４）を行うことにより、試料中のＤＵＰＡＮ－２抗原の濃度を決定することもできる。
（３）試料として、既知濃度のＤＵＰＡＮ－２抗原を用いて前記工程（１Ｂ）から（２Ｂ）を行い、ＤＵＰＡＮ－２抗原濃度と測定値との関係を表す検量線を作成する工程；
（４）工程（３）で作成された検量線と、工程（２Ｂ）の測定により得られた測定値とから、試料中のＤＵＰＡＮ－２抗原の濃度を決定する工程。 Furthermore, the concentration of the DUPAN-2 antigen in the sample can also be determined by performing the following steps (3) and (4) after the step (2B).
(3) A step of performing steps (1B) to (2B) using a known concentration of DUPAN-2 antigen as a sample to create a calibration curve showing the relationship between the DUPAN-2 antigen concentration and the measured value;
(4) A step of determining the concentration of the DUPAN-2 antigen in the sample from the calibration curve prepared in the step (3) and the measured value obtained by the measurement in the step (2B).
　腹水由来のＤＵＰＡＮ－２抗原とは、膵癌患者の腹水に含まれるＤＵＰＡＮ－２抗原をいう。腹水由来のＤＵＰＡＮ－２抗原として、膵癌患者の腹水をそのまま用いてもよいし、リン酸緩衝化生理食塩水（０．１５ｍｏｌ／Ｌの塩化ナトリウムを含有する１０ｍｍｏｌ／Ｌリン酸緩衝液、ｐＨ７．２、以下、ＰＢＳと記す）等で希釈された腹水を用いてもよいし、膵癌患者の腹水から精製したＤＵＰＡＮ－２抗原をＰＢＳ等で希釈したＤＵＰＡＮ－２抗原溶液を用いてもよい。 The DUPAN-2 antigen derived from ascites refers to the DUPAN-2 antigen contained in the ascites of a pancreatic cancer patient. As the DUPAN-2 antigen derived from ascites, the ascites of a pancreatic cancer patient may be used as it is, or phosphate buffered saline (10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7. 2. Ascites diluted with PBS or the like may be used, or a DUPAN-2 antigen solution obtained by diluting DUPAN-2 antigen purified from ascites of a pancreatic cancer patient with PBS or the like may be used.
　腹水由来のＤＵＰＡＮ－２抗原の濃度としては、ＤＵＰＡＮ－２抗原の測定を可能とする濃度であれば特に限定はなく、１０Ｕ／ｍＬ～２０００Ｕ／ｍＬであってよく、３０Ｕ／ｍＬ～１６００Ｕ／ｍＬであってよく、４０Ｕ／ｍＬ～４００Ｕ／ｍＬであってよく、４０Ｕ／ｍＬ～３５０Ｕ／ｍＬであってよい。なお、ＤＵＰＡＮ－２抗原の濃度単位「Ｕ／ｍＬ」について、「特定の膵癌患者の血清を競合ラジオイムノアッセイ法により測定した場合の、当該血清に含まれるＤＵＰＡＮ－２抗原の濃度を１０００Ｕ／ｍＬする」と定義される（Ｍｅｔｚｇａｒら、“Ｄｅｔｅｃｔｉｏｎ　ｏｆ　ａ　ｐａｎｃｒｅａｔｉｃ　ｃａｎｃｅｒ－ａｓｓｏｃｉａｔｅｄ　ａｎｔｉｇｅｎ　（ＤＵ－ＰＡＮ－２　ａｎｔｉｇｅｎ）　ｉｎ　ｓｅｒｕｍ　ａｎｄ　ａｓｃｉｔｅｓ　ｏｆ　ｐａｔｉｅｎｔｓ　ｗｉｔｈ　ａｄｅｎｏｃａｒｃｉｎｏｍａ”、Ｐｒｏｃｅｅｄｉｎｇｓ　ｏｆ　ｔｈｅ　Ｎａｔｉｏｎａｌ　Ａｃａｄｅｍｙ　ｏｆ　Ｓｃｉｅｎｃｅｓ　ｏｆ　ｔｈｅ　Ｕｎｉｔｅｄ　Ｓｔａｔｅｓ　ｏｆ　Ａｍｅｒｉｃａ、１９８４年，Ｖｏｌ．８１，ｐｐ．５２４２－５２４６）。 The concentration of the DUPAN-2 antigen derived from ascites is not particularly limited as long as it enables the measurement of the DUPAN-2 antigen, and may be 10 U / mL to 2000 U / mL, and may be 30 U / mL to 1600 U / mL. It may be 40 U / mL to 400 U / mL, and may be 40 U / mL to 350 U / mL. Regarding the DUPAN-2 antigen concentration unit "U / mL", "when the serum of a specific pancreatic cancer patient is measured by a competitive radioimmunoassay method, the concentration of the DUPAN-2 antigen contained in the serum is 1000 U / mL. 」と定義される（Ｍｅｔｚｇａｒら、“Ｄｅｔｅｃｔｉｏｎ　ｏｆ　ａ　ｐａｎｃｒｅａｔｉｃ　ｃａｎｃｅｒ－ａｓｓｏｃｉａｔｅｄ　ａｎｔｉｇｅｎ　（ＤＵ－ＰＡＮ－２　ａｎｔｉｇｅｎ）　ｉｎ　ｓｅｒｕｍ　ａｎｄ　ａｓｃｉｔｅｓ　ｏｆ　ｐａｔｉｅｎｔｓ　ｗｉｔｈ　ａｄｅｎｏｃａｒｃｉｎｏｍａ”、Ｐｒｏｃｅｅｄｉｎｇｓ　ｏｆ　ｔｈｅ　Ｎａｔｉｏｎａｌ　Ａｃａｄｅｍｙ　ｏｆ　Ｓｃｉｅｎｃｅｓ　ｏｆ　ｔｈｅ　Ｕｎｉｔｅｄ　Ｓｔａｔｅｓ　ｏｆ　Ａｍｅｒｉｃａ、 1984, Vol. 81, pp. 5242-5246).
　ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原とは、ヒト膵癌培養細胞ＨＰＡＦに含まれるＤＵＰＡＮ－２抗原、又はヒト膵癌培養細胞ＨＰＡＦを培養した際に得られる培養上清に含まれるＤＵＰＡＮ－２抗原をいう。ＤＵＰＡＮ－２抗原がヒト膵癌培養細胞ＨＰＡＦに含まれる場合、ヒト膵癌培養細胞ＨＰＡＦの細胞可溶化物（ｃｅｌｌ　ｌｙｓａｔｅ）をそのまま用いてもよいし、ＰＢＳ等で希釈された細胞可溶化物を用いてもよい。ＤＵＰＡＮ－２抗原が前記培養上清に含まれる場合、前記培養上清をそのまま用いてもよいし、ＰＢＳ等で希釈された培養上清を用いてもよい。ヒト膵癌培養細胞ＨＰＡＦの培養に用いられる培地としては、１０％ウシ胎仔血清を含むＭｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ　Ｅａｇｌｅ（イーグル最少必須培地）等が挙げられる。 The DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF is the DUPAN-2 antigen contained in human pancreatic cancer cultured cell HPAF, or the DUPAN-2 antigen contained in the culture supernatant obtained when human pancreatic cancer cultured cell HPAF is cultured. To say. When the DUPAN-2 antigen is contained in the human pancreatic cancer cultured cell HPAF, the cell lysate of the human pancreatic cancer cultured cell HPAF may be used as it is, or the cell solubilized product diluted with PBS or the like may be used. May be good. When the DUPAN-2 antigen is contained in the culture supernatant, the culture supernatant may be used as it is, or the culture supernatant diluted with PBS or the like may be used. Examples of the medium used for culturing human pancreatic cancer cultured cells HPAF include Medium Essential Medium Eagle (Eagle's minimum essential medium) containing 10% fetal bovine serum.
　ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の濃度としては、ＤＵＰＡＮ－２抗原の測定を可能とする濃度であれば特に限定はなく、１０Ｕ／ｍＬ～２０００Ｕ／ｍＬであってよく、３０Ｕ／ｍＬ～１６００Ｕ／ｍＬであってよく、４０Ｕ／ｍＬ～４００Ｕ／ｍＬであってよく、４０Ｕ／ｍＬ～３５０Ｕ／ｍＬであってよい。 The concentration of DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF is not particularly limited as long as it enables measurement of DUPAN-2 antigen, and may be 10 U / mL to 2000 U / mL, and may be 30 U / mL. It may be up to 1600 U / mL, may be 40 U / mL to 400 U / mL, and may be 40 U / mL to 350 U / mL.
＜工程（１Ｂ）＞
　工程（１Ｂ）は、腹水由来のＤＵＰＡＮ－２抗原、又はヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原に、不溶性担体と、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片と、を反応させ、前記不溶性担体上に、前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原と、前記ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片と、を含む免疫複合体を生成させる工程である。 <Process (1B)>
In step (1B), an insoluble carrier, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and DUPAN- are added to the DUPAN-2 antigen derived from ascites or the DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF. A second antibody that binds to two antigens or an antibody fragment thereof is reacted, and the first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, the DUPAN-2 antigen, and the DUPAN on the insoluble carrier. -2 This is a step of producing an immune complex containing a second antibody that binds to an antigen or an antibody fragment thereof.
　工程（１Ｂ）において、腹水由来のＤＵＰＡＮ－２抗原又はヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原と、不溶性担体と、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片との反応は、不溶性担体上に、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、ＤＵＰＡＮ－２抗原と、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片とを含む免疫複合体が生成する限りは特に制限はなく、例えば、腹水由来のＤＵＰＡＮ－２抗原又はヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原に、不溶性担体と、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片とを反応させて、不溶性担体上にＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片とＤＵＰＡＮ－２抗原との免疫複合体を生成させた後に、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片を反応させてもよいし、腹水由来のＤＵＰＡＮ－２抗原又はヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原に、不溶性担体と、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片とを混合して反応させてもよい。
　この場合、不溶性担体、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、及びＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片を添加する順番は、いかなる順番であってもよい。 In step (1B), a DUPAN-2 antigen derived from ascites or a DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, an insoluble carrier, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and DUPAN-2. The reaction with the second antibody or its antibody fragment that binds to the antigen binds to the first antibody or its antibody fragment that binds to the DUPAN-2 antigen, the DUPAN-2 antigen, and the DUPAN-2 antigen on an insoluble carrier. There is no particular limitation as long as an immune complex containing the second antibody or an antibody fragment thereof is produced, and for example, a DUPAN-2 antigen derived from ascites water or a DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, an insoluble carrier and the like. The first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof is reacted to generate an immune complex of the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof and the DUPAN-2 antigen on an insoluble carrier. After the reaction, a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof may be reacted, or the DUPAN-2 antigen derived from ascites water or the DUPAN-2 antigen derived from the cultured human pancreatic cancer cell HPAF is used as an insoluble carrier. , The first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof may be mixed and reacted with the second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof.
In this case, the order of adding the insoluble carrier, the first antibody that binds to the DUPAN-2 antigen or its antibody fragment, and the second antibody that binds to the DUPAN-2 antigen or its antibody fragment may be any order.
　不溶性担体上に、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片とＤＵＰＡＮ－２抗原との免疫複合体を生成させた後に、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片を反応させる場合には、当該免疫複合体を生成させた後に洗浄工程を設けることもできる。不溶性担体の洗浄の際に使用する洗浄液としては、ＤＵＰＡＮ－２抗原の測定を可能とする洗浄液であれば特に制限はなく、例えばＰＢＳ、界面活性剤を含有するＰＢＳ等が挙げられる。界面活性剤としては、例えばＴｗｅｅｎ（登録商標）　２０等の非イオン性界面活性剤が挙げられる。 On an insoluble carrier, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof and an immune complex of the DUPAN-2 antigen are generated, and then a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof is formed. In the case of reaction, a washing step can be provided after the immune complex is generated. The cleaning solution used for cleaning the insoluble carrier is not particularly limited as long as it is a cleaning solution capable of measuring the DUPAN-2 antigen, and examples thereof include PBS and PBS containing a surfactant. Examples of the surfactant include nonionic surfactants such as Tween (registered trademark) 20.
　工程（１Ｂ）において、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と不溶性担体とは、ＤＵＰＡＮ－２抗原と反応させる前に予め、結合させておいてもよい。ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の不溶性担体への結合は、例えば前述の方法で結合することができる。 In step (1B), the first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen and the insoluble carrier may be previously bound before reacting with the DUPAN-2 antigen. The first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen can be bound to the insoluble carrier, for example, by the method described above.
　前記反応溶液中の不溶性担体の濃度は、ＤＵＰＡＮ－２抗原の測定を可能とする濃度であれば特に限定はなく、０．００１ｍｇ／ｍＬ以上、０．００２ｍｇ／ｍＬ以上、０．００３ｍｇ／ｍＬ以上、０．００４ｍｇ／ｍＬ以上、０．００５ｍｇ／ｍＬ以上、０．０１ｍｇ／ｍＬ以上、０．０２ｍｇ／ｍＬ以上、０．０３ｍｇ／ｍＬ以上、０．０４ｍｇ／ｍＬ以上、０．０５ｍｇ／ｍＬ以上、０．０６ｍｇ／ｍＬ以上、０．０７ｍｇ／ｍＬ以上、０．０８ｍｇ／ｍＬ以上、０．０９ｍｇ／ｍＬ以上、０．１ｍｇ／ｍＬ以上、０．２ｍｇ／ｍＬ以上、０．３ｍｇ／ｍＬ以上、０．４ｍｇ／ｍＬ以上、又は０．５ｍｇ／ｍＬ以上であってよい。また、前記反応溶液中における不溶性担体の濃度は、０．６ｍｇ／ｍＬ以下、０．７ｍｇ／ｍＬ以下、０．８ｍｇ／ｍＬ以下、０．９ｍｇ／ｍＬ以下、１ｍｇ／ｍＬ以下、２ｍｇ／ｍＬ以下、３ｍｇ／ｍＬ以下、４ｍｇ／ｍＬ以下、５ｍｇ／ｍＬ以下、６ｍｇ／ｍＬ以下、７ｍｇ／ｍＬ以下、８ｍｇ／ｍＬ以下、９ｍｇ／ｍＬ以下、又は１０ｍｇ／ｍＬ以下であってよい。 The concentration of the insoluble carrier in the reaction solution is not particularly limited as long as it is a concentration capable of measuring the DUPAN-2 antigen, and is 0.001 mg / mL or more, 0.002 mg / mL or more, 0.003 mg / mL or more. , 0.004 mg / mL or more, 0.005 mg / mL or more, 0.01 mg / mL or more, 0.02 mg / mL or more, 0.03 mg / mL or more, 0.04 mg / mL or more, 0.05 mg / mL or more, 0.06 mg / mL or more, 0.07 mg / mL or more, 0.08 mg / mL or more, 0.09 mg / mL or more, 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0 It may be 0.4 mg / mL or more, or 0.5 mg / mL or more. The concentration of the insoluble carrier in the reaction solution is 0.6 mg / mL or less, 0.7 mg / mL or less, 0.8 mg / mL or less, 0.9 mg / mL or less, 1 mg / mL or less, 2 mg / mL or less. It may be 3 mg / mL or less, 4 mg / mL or less, 5 mg / mL or less, 6 mg / mL or less, 7 mg / mL or less, 8 mg / mL or less, 9 mg / mL or less, or 10 mg / mL or less.
　前記の数値は自由に組み合わせることができる。例えば、前記反応溶液中の不溶性担体の濃度は、０．００３ｍｇ／ｍＬ～１０ｍｇ／ｍＬであってもよく、０．０１ｍｇ／ｍＬ～３ｍｇ／ｍＬであってもよく、０．０５ｍｇ／ｍＬ～１ｍｇ／ｍＬであってもよい。 The above values can be freely combined. For example, the concentration of the insoluble carrier in the reaction solution may be 0.003 mg / mL to 10 mg / mL, 0.01 mg / mL to 3 mg / mL, or 0.05 mg / mL to 1 mg. It may be / mL.
　前記反応溶液中の、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の濃度は、０．０１μｇ／ｍＬ以上、０．０２μｇ／ｍＬ以上、０．０３μｇ／ｍＬ以上、０．０４μｇ／ｍＬ以上、０．０５μｇ／ｍＬ以上、０．０６μｇ／ｍＬ以上、０．０７μｇ／ｍＬ以上、０．０８μｇ／ｍＬ以上、０．０９μｇ／ｍＬ以上、０．１μｇ／ｍＬ以上、０．２μｇ／ｍＬ以上、０．３μｇ／ｍＬ以上、０．４μｇ／ｍＬ以上、０．５μｇ／ｍＬ以上、０．６μｇ／ｍＬ以上、０．７μｇ／ｍＬ以上、０．８μｇ／ｍＬ以上、０．９μｇ／ｍＬ以上、１μｇ／ｍＬ以上、２μｇ／ｍＬ以上、３μｇ／ｍＬ以上、４μｇ／ｍＬ以上、５μｇ／ｍＬ以上、６μｇ／ｍＬ以上、７μｇ／ｍＬ以上、７．５μｇ／ｍＬ以上、８ｇ／ｍＬ以上、又は９μｇ／ｍＬ以上であってよい。また、前記反応溶液中の、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の濃度は、１０μｇ／ｍＬ以下、１１μｇ／ｍＬ以下、１２μｇ／ｍＬ以下、１３μｇ／ｍＬ以下、１４μｇ／ｍＬ以下、１５μｇ／ｍＬ以下、１６μｇ／ｍＬ以下、１７μｇ／ｍＬ以下、１８μｇ／ｍＬ以下、１９μｇ／ｍＬ以下、２０μｇ／ｍＬ以下、３０μｇ／ｍＬ以下、４０μｇ／ｍＬ以下、５０μｇ／ｍＬ以下、又は１００μｇ／ｍＬ以下であってよい。 The concentration of the first antibody or its antibody fragment that binds to the DUPAN-2 antigen in the reaction solution is 0.01 μg / mL or more, 0.02 μg / mL or more, 0.03 μg / mL or more, 0.04 μg / mL. Above, 0.05 μg / mL or more, 0.06 μg / mL or more, 0.07 μg / mL or more, 0.08 μg / mL or more, 0.09 μg / mL or more, 0.1 μg / mL or more, 0.2 μg / mL or more , 0.3 μg / mL or more, 0.4 μg / mL or more, 0.5 μg / mL or more, 0.6 μg / mL or more, 0.7 μg / mL or more, 0.8 μg / mL or more, 0.9 μg / mL or more, 1 μg / mL or more, 2 μg / mL or more, 3 μg / mL or more, 4 μg / mL or more, 5 μg / mL or more, 6 μg / mL or more, 7 μg / mL or more, 7.5 μg / mL or more, 8 g / mL or more, or 9 μg / mL It may be mL or more. The concentration of the first antibody or its antibody fragment that binds to the DUPAN-2 antigen in the reaction solution is 10 μg / mL or less, 11 μg / mL or less, 12 μg / mL or less, 13 μg / mL or less, 14 μg / mL or less. , 15 μg / mL or less, 16 μg / mL or less, 17 μg / mL or less, 18 μg / mL or less, 19 μg / mL or less, 20 μg / mL or less, 30 μg / mL or less, 40 μg / mL or less, 50 μg / mL or less, or 100 μg / mL It may be as follows.
　前記の数値は自由に組み合わせることができる。例えば、前記反応溶液中の、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の濃度は、０．０１μｇ／ｍＬ～１００μｇ／ｍＬであってもよく、０．１μｇ／ｍＬ～３０μｇ／ｍＬであってもよく、６μｇ／ｍＬ～３０μｇ／ｍＬであってもよく、１０μｇ／ｍＬ～２０μｇ／ｍＬであってもよい。 The above values can be freely combined. For example, the concentration of the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in the reaction solution may be 0.01 μg / mL to 100 μg / mL, or 0.1 μg / mL to 30 μg / mL. It may be 6 μg / mL to 30 μg / mL, or 10 μg / mL to 20 μg / mL.
　前記反応溶液中の、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の濃度は、０．００１μｇ／ｍＬ以上、０．０１μｇ／ｍＬ以上、０．０２μｇ／ｍＬ以上、０．０３μｇ／ｍＬ以上、０．０４μｇ／ｍＬ以上、０．０５μｇ／ｍＬ以上、０．０６μｇ／ｍＬ以上、０．０７μｇ／ｍＬ以上、０．０８μｇ／ｍＬ以上、０．０９μｇ／ｍＬ以上、又は０．１μｇ／ｍＬ以上であってよい。また、前記反応溶液中の、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の濃度は、０．２μｇ／ｍＬ以下、０．３μｇ／ｍＬ以下、０．４μｇ／ｍＬ以下、０．５μｇ／ｍＬ以下、０．６μｇ／ｍＬ以下、０．７μｇ／ｍＬ以下、０．８μｇ／ｍＬ以下、０．９μｇ／ｍＬ以下、１μｇ／ｍＬ以下、２μｇ／ｍＬ以下、３μｇ／ｍＬ以下、４μｇ／ｍＬ以下、５μｇ／ｍＬ以下、６μｇ／ｍＬ以下、７μｇ／ｍＬ以下、７．５μｇ／ｍＬ以下、８ｇ／ｍＬ以下、９μｇ／ｍＬ以下、１０μｇ／ｍＬ以下、１１μｇ／ｍＬ以下、１２μｇ／ｍＬ以下、１３μｇ／ｍＬ以下、１４μｇ／ｍＬ以下、１５μｇ／ｍＬ以下、１６μｇ／ｍＬ以下、１７μｇ／ｍＬ以下、１８μｇ／ｍＬ以下、１９μｇ／ｍＬ以下、２０μｇ／ｍＬ以下、３０μｇ／ｍＬ以下、４０μｇ／ｍＬ以下、５０μｇ／ｍＬ以下、又は１００μｇ／ｍＬ以下であってよい。 The concentration of the second antibody or its antibody fragment that binds to the DUPAN-2 antigen in the reaction solution is 0.001 μg / mL or more, 0.01 μg / mL or more, 0.02 μg / mL or more, 0.03 μg / mL. 0.04 μg / mL or more, 0.05 μg / mL or more, 0.06 μg / mL or more, 0.07 μg / mL or more, 0.08 μg / mL or more, 0.09 μg / mL or more, or 0.1 μg / mL That may be the above. The concentration of the second antibody or its antibody fragment that binds to the DUPAN-2 antigen in the reaction solution is 0.2 μg / mL or less, 0.3 μg / mL or less, 0.4 μg / mL or less, 0.5 μg. / ML or less, 0.6 μg / mL or less, 0.7 μg / mL or less, 0.8 μg / mL or less, 0.9 μg / mL or less, 1 μg / mL or less, 2 μg / mL or less, 3 μg / mL or less, 4 μg / mL 5 μg / mL or less, 6 μg / mL or less, 7 μg / mL or less, 7.5 μg / mL or less, 8 g / mL or less, 9 μg / mL or less, 10 μg / mL or less, 11 μg / mL or less, 12 μg / mL or less, 13 μg / ML or less, 14 μg / mL or less, 15 μg / mL or less, 16 μg / mL or less, 17 μg / mL or less, 18 μg / mL or less, 19 μg / mL or less, 20 μg / mL or less, 30 μg / mL or less, 40 μg / mL or less, 50 μg It may be less than / mL or less than 100 μg / mL.
　前記の数値は自由に組み合わせることができる。例えば、前記反応溶液中の、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の濃度は、０．０１μｇ／ｍＬ～１００μｇ／ｍＬであってもよく、０．０３μｇ／ｍＬ～３０μｇ／ｍＬであってもよく、０．１μｇ／ｍＬ～２０μｇ／ｍＬであってもよい。 The above values can be freely combined. For example, the concentration of the second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in the reaction solution may be 0.01 μg / mL to 100 μg / mL, or 0.03 μg / mL to 30 μg / mL. It may be 0.1 μg / mL to 20 μg / mL.
　工程（１Ｂ）は水性媒体中で行うことが好ましい。水性媒体としては、脱イオン水、蒸留水、緩衝液等が挙げられ、緩衝液が好ましい。緩衝液の調製に使用される緩衝剤としては、酢酸緩衝液、グッド緩衝剤等が挙げられる。緩衝剤は、１種を単独で使用してもよいし、２種以上を併用してもよい。 The step (1B) is preferably performed in an aqueous medium. Examples of the aqueous medium include deionized water, distilled water, a buffer solution and the like, and a buffer solution is preferable. Examples of the buffer used for preparing the buffer include acetate buffer and Good's buffer. One type of buffer may be used alone, or two or more types may be used in combination.
　また、工程（１Ｂ）において、塩類、金属イオン、糖類、蛋白質、界面活性剤等が含有されてもよい。塩類としては、塩化リチウム、塩化ナトリウム、塩化カリウム、塩化カルシウム、塩化マグネシウム等が挙げられる。金属イオンとしては、ナトリウムイオン、マグネシウムイオン、マンガンイオン、亜鉛イオン等が挙げられる。糖類としては、マンニトール、ソルビトール等が挙げられる。蛋白質としては、ウシ血清アルブミン（以下、ＢＳＡと記す）等が挙げられる。界面活性剤としては、陰イオン性界面活性剤、陽イオン性界面活性剤、両イオン性界面活性剤、非イオン性界面活性剤等が挙げられる。 Further, in the step (1B), salts, metal ions, sugars, proteins, surfactants and the like may be contained. Examples of salts include lithium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride and the like. Examples of the metal ion include sodium ion, magnesium ion, manganese ion, zinc ion and the like. Examples of saccharides include mannitol and sorbitol. Examples of the protein include bovine serum albumin (hereinafter referred to as BSA) and the like. Examples of the surfactant include anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants and the like.
　工程（１Ｂ）の反応温度は、ＤＵＰＡＮ－２抗原の測定を可能とする温度であれば特に制限はなく、例えば、０～５０℃であり、４～４５℃が好ましく、２０～４０℃が特に好ましい。また、工程（１Ｂ）の反応時間は、ＤＵＰＡＮ－２抗原の測定を可能とする時間であれば特に限定はなく、例えば、５分間～３時間であり、１０分間～２時間が好ましい。 The reaction temperature in the step (1B) is not particularly limited as long as it is a temperature capable of measuring the DUPAN-2 antigen, for example, 0 to 50 ° C., preferably 4 to 45 ° C., and particularly 20 to 40 ° C. preferable. The reaction time of the step (1B) is not particularly limited as long as it enables the measurement of the DUPAN-2 antigen, and is, for example, 5 minutes to 3 hours, preferably 10 minutes to 2 hours.
　ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片が予め結合した不溶性担体を用いる場合であっても、前述の反応液中の濃度、反応温度及び反応時間で用いることができる。 Even when a first antibody that binds to the DUPAN-2 antigen or an insoluble carrier to which the antibody fragment thereof is bound in advance is used, it can be used at the above-mentioned concentration in the reaction solution, reaction temperature and reaction time.
＜工程（２Ｂ）＞
　工程（２Ｂ）において、工程（１Ｂ）で生成した免疫複合体を以下の方法を用いて測定することにより、試料中のＤＵＰＡＮ－２抗原の測定値が決定される。 <Process (2B)>
In step (2B), the measured value of DUPAN-2 antigen in the sample is determined by measuring the immune complex produced in step (1B) using the following method.
［ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片に標識が結合していない場合］　ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片に結合する抗体（以下、第３抗体と記す）に標識が結合した標識化第３抗体を用いて、前記免疫複合体を測定することができる。すなわち、前記免疫複合体に前記標識化第３抗体を反応させ、前記免疫複合体と、標識化第３抗体との複合体３を形成させ、当該複合体３の標識を後述の方法により測定することにより、工程（１Ｂ）で生成した免疫複合体の量を測定することができる。第３抗体としては、例えばＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片のＦｃ領域に結合する抗体等が挙げられる [When the label is not bound to the second antibody that binds to the DUPAN-2 antigen or its antibody fragment] The second antibody that binds to the DUPAN-2 antigen or the antibody that binds to the antibody fragment thereof (hereinafter referred to as the third antibody). ) Is bound to the labeled third antibody, the immune complex can be measured. That is, the immune complex is reacted with the labeled third antibody to form a complex 3 of the immune complex and the labeled third antibody, and the labeling of the complex 3 is measured by the method described below. Thereby, the amount of the immune complex produced in the step (1B) can be measured. Examples of the third antibody include a second antibody that binds to the DUPAN-2 antigen or an antibody that binds to the Fc region of an antibody fragment thereof.
　標識化第３抗体と、前記免疫複合体との反応の反応温度は、ＤＵＰＡＮ－２抗原の測定を可能とする温度であれば特に制限はなく、例えば、０～５０℃であってよく、４～４５℃であってよく、２０～４０℃であってよい。当該反応の反応時間は、ＤＵＰＡＮ－２抗原の測定を可能とする時間であれば特に制限はなく、例えば、１分間～４時間であってよく、１０５分間～３時間であってよく、１０分間～２時間であってよい。標識化第３抗体の反応溶液中の濃度は、ＤＵＰＡＮ－２抗原の測定を可能とする濃度であれば特に制限はなく、例えば、０．０１～１００μｇ／ｍＬであってよく、０．１～２０μｇ／ｍＬであってよい。 The reaction temperature of the reaction between the labeled third antibody and the immune complex is not particularly limited as long as it is a temperature that enables measurement of the DUPAN-2 antigen, and may be, for example, 0 to 50 ° C. 4 It may be ~ 45 ° C. and may be 20-40 ° C. The reaction time of the reaction is not particularly limited as long as it enables the measurement of the DUPAN-2 antigen, and may be, for example, 1 minute to 4 hours, 105 minutes to 3 hours, or 10 minutes. It may be up to 2 hours. The concentration of the labeled third antibody in the reaction solution is not particularly limited as long as it is a concentration capable of measuring the DUPAN-2 antigen, and may be, for example, 0.01 to 100 μg / mL, and may be 0.1 to 0.1. It may be 20 μg / mL.
［ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片に標識が結合している場合］
　工程（２Ｂ）は、標識を用いて、前記免疫複合体を測定することにより行うことができる。
　標識をＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片に結合させる方法は、免疫測定の技術分野において公知である。例えば、１個若しくは数個のアミノ酸を介して、又は、１個又は数個のアミノ酸とリンカーを介して、抗体に標識を結合させることができる。また、標識をタンパク質に結合させるキットも各種市販されている。 [When the label is bound to the second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen]
Step (2B) can be performed by measuring the immune complex with a label.
Methods of binding the label to a second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen are known in the art of immunoassay. For example, the label can be attached to the antibody via one or several amino acids, or via a linker with one or several amino acids. In addition, various kits for binding the label to the protein are commercially available.
　標識としては、通常の免疫測定方法において用いられる、酵素類、放射性同位元素、蛍光性物質、発光性物質、ＤＮＡ、ＲＮＡ、補酵素又は補酵素と特異的に結合するもの（ビオチン、アビジン）、タグ、紫外部～赤外部に吸収を有する物質、発色性微粒子、蛍光性微粒子、金属性微粒子（金コロイド粒子等）、磁性物質、スピンラベル化剤としての性質を有する物質、着色ラテックス等が挙げられる。 Labels include enzymes, radioactive isotopes, fluorescent substances, luminescent substances, DNA, RNA, co-enzymes, or substances that specifically bind to co-enzymes (biotin, avidin), which are used in ordinary immunoassay methods. Examples include tags, substances that absorb in the ultraviolet to infrared regions, color-developing fine particles, fluorescent fine particles, metallic fine particles (gold colloidal particles, etc.), magnetic substances, substances that have properties as spin labeling agents, colored latex, and the like. Be done.
　酵素としては、アルカリホスファターゼ、ペルオキシダーゼ、ガラクトシダーゼ、グルクロニダーゼ、ルシフェラーゼ等が挙げられる。放射性同位元素としては、３Ｈ、１４Ｃ、３５Ｓ、３２Ｐ、１２５Ｐ、１３１Ｉ等が挙げられる。蛍光性物質としては、ＦＩＴＣ（フルオレッセイン　イソチオシアナート）、ＲＩＴＣ（ローダミンＢ－イソチオシアナート）等が挙げられる。発光性物質としては、アクリジニウム及びその誘導体、ルテニウム錯体化合物、ロフィン等が挙げられる。 Examples of the enzyme include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase and the like. Examples of the radioisotope include 3H , 14C , 35S , 32P , 125P , 131I and the like. Examples of the fluorescent substance include FITC (fluorescein isothiocyanate) and RITC (rhodamine B-isothiocyanate). Examples of the luminescent substance include acridinium and its derivatives, ruthenium complex compounds, loffin and the like.
　これらの標識から生じるシグナルを検出することにより、前記免疫複合体を測定することができる。
　前記標識から生じるシグナルの測定方法は、用いる標識により適宜選択すればよい。
　標識が発色物質、すなわちある波長の光を吸収する物質の場合には、分光光度計及びマルチウェルプレートリーダー等を用いて吸光度を測定することにより、標識を測定することができる。
　標識が蛍光物質の場合には、蛍光光度計及び蛍光マルチウェルプレートリーダー等を用いて蛍光強度を測定することにより、標識を測定することができる。
　標識が発光物質の場合には、発光光度計及び発光マルチウェルプレートリーダー等を用いて、発光強度を測定することにより、標識を測定することができる。
　標識が放射性同位元素である場合、放射活性をシンチレーションカウンター、γ－ウェルカウンター等により、放射活性を測定することにより、標識を測定することができる。
　標識が酵素である場合には、酵素活性を測定することにより、標識を測定することができる。例えば酵素の基質を当該酵素と反応させ、生成した物質を測定することにより、標識を測定することができる。 The immune complex can be measured by detecting the signals resulting from these labels.
The method for measuring the signal generated from the label may be appropriately selected depending on the label to be used.
When the label is a color-developing substance, that is, a substance that absorbs light of a certain wavelength, the label can be measured by measuring the absorbance using a spectrophotometer, a multi-well plate reader, or the like.
When the label is a fluorescent substance, the label can be measured by measuring the fluorescence intensity using a fluorometer, a fluorescent multi-well plate reader, or the like.
When the label is a luminescent substance, the label can be measured by measuring the luminescence intensity using a luminescence photometer, a luminescence multi-well plate reader, or the like.
When the label is a radioisotope, the label can be measured by measuring the radioactivity with a scintillation counter, a γ-well counter, or the like.
When the label is an enzyme, the label can be measured by measuring the enzyme activity. For example, the label can be measured by reacting the substrate of an enzyme with the enzyme and measuring the produced substance.
　酵素がアルカリホスファターゼである場合には、例えば発光法等によりアルカリホスファターゼ活性を測定することができる。発光法によりアルカリホスファターゼ活性を測定する方法としては、例えばアルカリホスファターゼとその基質とを反応させ、生成した発光の強度を発光光度計及び発光マルチウェルプレートリーダー等で測定する方法等が挙げられる。 When the enzyme is alkaline phosphatase, the alkaline phosphatase activity can be measured by, for example, a luminescence method. Examples of the method for measuring the alkaline phosphatase activity by the luminescence method include a method of reacting alkaline phosphatase with a substrate thereof and measuring the intensity of the generated luminescence with a luminescence photometer, a luminescence multi-well plate reader or the like.
　アルカリホスファターゼの基質としては、例えば３－（２’－スピロアダマンタン）－４－メトキシ－４－（３’－ホスホリルオキシ）フェニル－１，２－ジオキセタン・二ナトリウム塩（ＡＭＰＰＤ）、２－クロロ－５－｛４－メトキシスピロ［１，２－ジオキセタン－３，２’－（５’－クロロ）トリシクロ［３．３．１．１３．７］デカン］－４－イル｝フェニルホスフェート・二ナトリウム塩（ＣＤＰ－ＳｔａｒＴＭ）、３－［４’－メトキシ－５－クロロスピロ［トリシクロ［３．３．１．１３，７］デカン－２，３’－［１，２］ジオキセタン］－４’－イル］フェニルホスフェート・二ナトリウム塩（ＣＳＰＤＴＭ）、９－［（フェニルオキシ）（ホスホリルオキシ）メチリデン］－１０－メチルアクリダン・二ナトリウム塩、９－［（４－クロロフェニルチオ）（ホスホリルオキシ）メチリデン］－１０－メチルアクリダン・二ナトリウム塩（ＬｕｍｉｇｅｎＴＭ　ＡＰＳ－５）等が挙げられる。 Examples of the substrate for alkaline phosphatase include 3- (2'-spirodamantan) -4-methoxy-4- (3'-phosphoryloxy) phenyl-1,2-dioxetane / disodium salt (AMPPD), 2-chloro-. 5- {4-Methylspiro [1,2-dioxetane-3,2'-(5'-chloro) tricyclo [ 3.3.1.13.7 ] decane] -4-yl} phenylphosphate disodium Salt (CDP-Star TM ), 3- [4'-methoxy-5-chlorospiro [tricyclo [3.3.1.1 3,7 ] decane-2,3'-[1,2] dioxetane] -4' -Il] phenyl phosphate disodium salt (CSPD TM ), 9-[(phenyloxy) (phosphoryloxy) methylidene] -10-methylacridan disodium salt, 9-[(4-chlorophenylthio) (phosphoryloxy) ) Methylidene] -10-methylacridan disodium salt (Lumigen TM APS-5) and the like.
　二価以上の金属イオン、特に二価の金属イオンは、アルカリホスファターゼの活性を上昇させることができるため、標識酵素がアルカリホスファターゼである場合に、使用することが好ましい。二価以上の金属イオンとして、例えば、ベリリウムイオン、マグネシウムイオン、カルシウムイオン、ストロンチウムイオン、バリウムイオン等のアルカリ土類金属イオン；チタンイオン、クロムイオン、マンガンイオン、鉄イオン、コバルトイオン、ニッケルイオン、銅イオン、亜鉛イオン等の第一遷移元素イオン等が挙げられる。 Since a divalent or higher metal ion, particularly a divalent metal ion, can increase the activity of alkaline phosphatase, it is preferable to use it when the labeling enzyme is alkaline phosphatase. Examples of divalent or higher metal ions include alkaline earth metal ions such as beryllium ion, magnesium ion, calcium ion, strontium ion and barium ion; titanium ion, chromium ion, manganese ion, iron ion, cobalt ion and nickel ion. Examples thereof include first transition element ions such as copper ion and zinc ion.
　本開示の、試料中のＤＵＰＡＮ－２抗原の測定試薬は、前記方法に従い、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定した場合に、下記式（Ｘ）を満たす。このようなＤＵＰＡＮ－２抗原の測定試薬は、高感度なＤＵＰＡＮ－２抗原の測定試薬となる。
　０．８≦（Ｂ）／（Ａ）≦１．２　（Ｘ）
（式中、（Ａ）は、腹水由来のＤＵＰＡＮ－２抗原の測定値、（Ｂ）は、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値を意味し、ここで、腹水由来のＤＵＰＡＮ－２抗原とヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原との抗原濃度比は、１：１である。） The reagent for measuring the DUPAN-2 antigen in the sample of the present disclosure is the following formula when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from the cultured human pancreatic cancer cell HPAF are measured according to the above method. X) is satisfied. Such a DUPAN-2 antigen measuring reagent becomes a highly sensitive DUPAN-2 antigen measuring reagent.
0.8 ≤ (B) / (A) ≤ 1.2 (X)
(In the formula, (A) means the measured value of DUPAN-2 antigen derived from ascites, and (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites. The antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
　腹水由来のＤＵＰＡＮ－２抗原の測定値（Ａ）及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値（Ｂ）は、後述の吸光度、発光強度又は蛍光強度のようなシグナルであってもよく、前述のＤＵＰＡＮ－２抗原の濃度であってもよい。 The measured value of DUPAN-2 antigen derived from ascites (A) and the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF (B) may be a signal such as absorbance, luminescence intensity or fluorescence intensity described later. It may be the concentration of the above-mentioned DUPAN-2 antigen.
　（Ｂ）／（Ａ）値は０．７以上、０．８以上、０．９以上であってよい。また、（Ｂ）／（Ａ）値は１．１以下、１．２以下であってよい。前記の数値は自由に組み合わせることができる。例えば、（Ｂ）／（Ａ）値は０．７～１．２であってもよく、０．８～１．２であってもよく、０．９～１．２であってもよい。 The (B) / (A) value may be 0.7 or more, 0.8 or more, and 0.9 or more. Further, the (B) / (A) value may be 1.1 or less and 1.2 or less. The above numerical values can be freely combined. For example, the (B) / (A) value may be 0.7 to 1.2, 0.8 to 1.2, or 0.9 to 1.2.
「試料中のＤＵＰＡＮ－２抗原の測定キット」
　本開示の試料中のＤＵＰＡＮ－２抗原の測定試薬は、保存、運搬、流通等の観点からキットの形態を取ることもできる。
　以下、本開示の、試料中のＤＵＰＡＮ－２抗原の測定キットの好適な実施形態を例示する。なお、下記の実施形態において、不溶性担体、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、及びＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片としては、例えば前述の不溶性担体、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、及びＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片を用いることができる。 "Measurement kit for DUPAN-2 antigen in sample"
The reagent for measuring the DUPAN-2 antigen in the sample of the present disclosure can also take the form of a kit from the viewpoint of storage, transportation, distribution and the like.
Hereinafter, preferred embodiments of the DUPAN-2 antigen measurement kit in the sample of the present disclosure will be illustrated. In the following embodiments, an insoluble carrier, an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a second antibody that binds to the DUPAN-2 antigen. Alternatively, the antibody fragment thereof includes, for example, the above-mentioned insoluble carrier, an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a DUPAN-2 antigen. 2 Antibodies or antibody fragments thereof can be used.
［測定キット（１）］
　水性媒体を含む第１試薬；並びに
　不溶性担体、及びＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片を含む第２試薬
を含む、測定キット。 [Measurement kit (1)]
A measurement kit comprising a first reagent comprising an aqueous medium; and a second reagent comprising an insoluble carrier and an antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
［測定キット（２）］
　不溶性担体、及びＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片を含む第１試薬；並びに、
　ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片を含む第２試薬
を含む、測定キット。 [Measurement kit (2)]
A first reagent containing an insoluble carrier and a first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen;
A measurement kit comprising a second reagent containing a second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
［測定キット（３）］
　不溶性担体を含む第１試薬；
　ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片を含む第２試薬；及び、
　ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片を含む第３試薬
を含む、測定キット。 [Measurement kit (3)]
First reagent containing an insoluble carrier;
A second reagent containing a first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen; and
A measurement kit comprising a third reagent containing a second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
　前記測定キット（２）及び前記測定キット（３）は、水性媒体を含む試薬を含んでいてもよい。前記測定キット（１）、前記測定キット（２）及び前記測定キット（３）における水性媒体としては、例えば、前述の水性媒体が挙げられる。 The measurement kit (2) and the measurement kit (3) may contain a reagent containing an aqueous medium. Examples of the aqueous medium in the measurement kit (1), the measurement kit (2), and the measurement kit (3) include the above-mentioned aqueous medium.
　前記測定キット（１）の第２試薬における不溶性担体の濃度、前記測定キット（２）の第１試薬における不溶性担体の濃度、及び、前記測定キット（３）の第１試薬における不溶性担体の濃度は、０．００１ｍｇ／ｍＬ以上、０．０１ｍｇ／ｍＬ以上、０．０２ｍｇ／ｍＬ以上、０．０３ｍｇ／ｍＬ以上、０．０４ｍｇ／ｍＬ以上、０．０５ｍｇ／ｍＬ以上、０．０６ｍｇ／ｍＬ以上、０．０７ｍｇ／ｍＬ以上、０．０８ｍｇ／ｍＬ以上、０．０９ｍｇ／ｍＬ以上、０．１ｍｇ／ｍＬ以上、０．２ｍｇ／ｍＬ以上、０．３ｍｇ／ｍＬ以上、０．４ｍｇ／ｍＬ以上、又は０．５ｍｇ／ｍＬ以上であってよい。また、前記測定キット（１）の第２試薬における不溶性担体の濃度、前記測定キット（２）の第１試薬における不溶性担体の濃度、及び、前記測定キット（３）の第１試薬における不溶性担体の濃度は、０．６ｍｇ／ｍＬ以下、０．７ｍｇ／ｍＬ以下、０．８ｍｇ／ｍＬ以下、０．９ｍｇ／ｍＬ以下、１ｍｇ／ｍＬ以下、２ｍｇ／ｍＬ以下、３ｍｇ／ｍＬ以下、４ｍｇ／ｍＬ以下、５ｍｇ／ｍＬ以下、６ｍｇ／ｍＬ以下、７ｍｇ／ｍＬ以下、８ｍｇ／ｍＬ以下、９ｍｇ／ｍＬ以下、又は１０ｍｇ／ｍＬ以下であってよい。 The concentration of the insoluble carrier in the second reagent of the measurement kit (1), the concentration of the insoluble carrier in the first reagent of the measurement kit (2), and the concentration of the insoluble carrier in the first reagent of the measurement kit (3) are , 0.001 mg / mL or more, 0.01 mg / mL or more, 0.02 mg / mL or more, 0.03 mg / mL or more, 0.04 mg / mL or more, 0.05 mg / mL or more, 0.06 mg / mL or more, 0.07 mg / mL or more, 0.08 mg / mL or more, 0.09 mg / mL or more, 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0.4 mg / mL or more, or It may be 0.5 mg / mL or more. Further, the concentration of the insoluble carrier in the second reagent of the measurement kit (1), the concentration of the insoluble carrier in the first reagent of the measurement kit (2), and the insoluble carrier in the first reagent of the measurement kit (3). The concentration is 0.6 mg / mL or less, 0.7 mg / mL or less, 0.8 mg / mL or less, 0.9 mg / mL or less, 1 mg / mL or less, 2 mg / mL or less, 3 mg / mL or less, 4 mg / mL or less. It may be 5, 5 mg / mL or less, 6 mg / mL or less, 7 mg / mL or less, 8 mg / mL or less, 9 mg / mL or less, or 10 mg / mL or less.
　前記の数値は、それぞれの測定キットにおいて自由に組み合わせることができる。例えば、前記測定キット（１）の第２試薬における不溶性担体の濃度、前記測定キット（２）の第１試薬における不溶性担体の濃度、及び、前記測定キット（３）の第１試薬における不溶性担体の濃度は、０．０１ｍｇ／ｍＬ～１０ｍｇ／ｍＬであってもよく、０．０３ｍｇ／ｍＬ～５ｍｇ／ｍＬであってもよく、０．１ｍｇ／ｍＬ～２ｍｇ／ｍＬであってもよい。 The above values can be freely combined in each measurement kit. For example, the concentration of the insoluble carrier in the second reagent of the measurement kit (1), the concentration of the insoluble carrier in the first reagent of the measurement kit (2), and the insoluble carrier in the first reagent of the measurement kit (3). The concentration may be 0.01 mg / mL to 10 mg / mL, 0.03 mg / mL to 5 mg / mL, or 0.1 mg / mL to 2 mg / mL.
　前記測定キット（１）の第２試薬におけるＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片の濃度、前記測定キット（２）の第１試薬におけるＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の濃度、及び、前記測定キット（３）の第２試薬におけるＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の濃度は、０．０１μｇ／ｍＬ以上、０．０２μｇ／ｍＬ以上、０．０３μｇ／ｍＬ以上、０．０４μｇ／ｍＬ以上、０．０５μｇ／ｍＬ以上、０．０６μｇ／ｍＬ以上、０．０７μｇ／ｍＬ以上、０．０８μｇ／ｍＬ以上、０．０９μｇ／ｍＬ以上、０．１μｇ／ｍＬ以上、０．２μｇ／ｍＬ以上、０．３μｇ／ｍＬ以上、０．４μｇ／ｍＬ以上、０．５μｇ／ｍＬ以上、０．６μｇ／ｍＬ以上、０．７μｇ／ｍＬ以上、０．８μｇ／ｍＬ以上、０．９μｇ／ｍＬ以上、１μｇ／ｍＬ以上、２μｇ／ｍＬ以上、３μｇ／ｍＬ以上、又は４μｇ／ｍＬ以上であってよい。また、前記測定キット（１）の第２試薬におけるＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片の濃度、前記測定キット（２）の第１試薬におけるＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の濃度、及び、前記測定キット（３）の第２試薬におけるＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の濃度は、５μｇ／ｍＬ以下、６μｇ／ｍＬ以下、７μｇ／ｍＬ以下、７．５μｇ／ｍＬ以下、８μｇ／ｍＬ以下、９μｇ／ｍＬ以下、１０μｇ／ｍＬ以下、１１μｇ／ｍＬ以下、１２μｇ／ｍＬ以下、１３μｇ／ｍＬ以下、１４μｇ／ｍＬ以下、１５μｇ／ｍＬ以下、１６μｇ／ｍＬ以下、１７μｇ／ｍＬ以下、１８μｇ／ｍＬ以下、１９μｇ／ｍＬ以下、２０μｇ／ｍＬ以下、３０μｇ／ｍＬ以下、４０μｇ／ｍＬ以下、５０μｇ／ｍＬ以下、又は１００μｇ／ｍＬ以下であってよい。 The concentration of the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (1), and the first antibody or its antibody that binds to the DUPAN-2 antigen in the first reagent of the measurement kit (2). The concentration of the fragment and the concentration of the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (3) are 0.01 μg / mL or more, 0.02 μg / mL or more, and 0. .03 μg / mL or more, 0.04 μg / mL or more, 0.05 μg / mL or more, 0.06 μg / mL or more, 0.07 μg / mL or more, 0.08 μg / mL or more, 0.09 μg / mL or more, 0. 1 μg / mL or more, 0.2 μg / mL or more, 0.3 μg / mL or more, 0.4 μg / mL or more, 0.5 μg / mL or more, 0.6 μg / mL or more, 0.7 μg / mL or more, 0.8 μg It may be / mL or more, 0.9 μg / mL or more, 1 μg / mL or more, 2 μg / mL or more, 3 μg / mL or more, or 4 μg / mL or more. Further, the concentration of the antibody or antibody fragment thereof that binds to the DUPAN-2 antibody in the second reagent of the measurement kit (1), the first antibody that binds to the DUPAN-2 antigen in the first reagent of the measurement kit (2), or The concentration of the antibody fragment and the concentration of the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (3) are 5 μg / mL or less, 6 μg / mL or less, and 7 μg / mL. Below, 7.5 μg / mL or less, 8 μg / mL or less, 9 μg / mL or less, 10 μg / mL or less, 11 μg / mL or less, 12 μg / mL or less, 13 μg / mL or less, 14 μg / mL or less, 15 μg / mL or less, 16 μg It may be / mL or less, 17 μg / mL or less, 18 μg / mL or less, 19 μg / mL or less, 20 μg / mL or less, 30 μg / mL or less, 40 μg / mL or less, 50 μg / mL or less, or 100 μg / mL or less.
　前記の数値は、それぞれの測定キットにおいて自由に組み合わせることができる。例えば、前記測定キット（１）の第２試薬におけるＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片の濃度、前記測定キット（２）の第１試薬におけるＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の濃度、及び、前記測定キット（３）の第２試薬におけるＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の濃度は、０．０１μｇ／ｍＬ～１００μｇ／ｍＬであってもよく、０．１μｇ／ｍＬ～３０μｇ／ｍＬであってもよく、６μｇ／ｍＬ～３０μｇ／ｍＬであってもよく、１０μｇ／ｍＬ～２０μｇ／ｍＬであってもよい。 The above values can be freely combined in each measurement kit. For example, the concentration of the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (1), the first antibody that binds to the DUPAN-2 antigen in the first reagent of the measurement kit (2), or Even if the concentration of the antibody fragment and the concentration of the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (3) are 0.01 μg / mL to 100 μg / mL. It may be 0.1 μg / mL to 30 μg / mL, 6 μg / mL to 30 μg / mL, or 10 μg / mL to 20 μg / mL.
　ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片がマイクロタイタープレートに結合しており、且つ、水性媒体を含んでおらず乾燥状態である場合、マイクロタイタープレートの１ウェルにおける、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の量は、０．００１μｇ以上、０．０１μｇ以上、０．０２μｇ以上、０．０３μｇ以上、０．０４μｇ以上、０．０５μｇ以上、０．０６μｇ以上、０．０７μｇ以上、０．０８μｇ以上、０．０９μｇ以上、０．１μｇ以上、０．２μｇ以上、０．３μｇ以上、０．４μｇ以上、又は０．５μｇ以上であってよい。また、前記マイクロタイタープレートの１ウェルにおける、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の量は、０．６μｇ以下、０．７μｇ以下、０．８μｇ以下、０．９μｇ以下、１μｇ以下、２μｇ以下、３μｇ以下、４μｇ以下、５μｇ以下、６μｇ以下、７μｇ以下、７．５μｇ以下、８ｇ／ｍＬ以下、９μｇ以下、１０μｇ以下、１１μｇ以下、１２μｇ以下、１３μｇ以下、１４μｇ以下、１５μｇ以下、１６μｇ以下、１７μｇ以下、１８μｇ以下、１９μｇ以下、２０μｇ以下、３０μｇ以下、４０μｇ以下、５０μｇ以下、又は１００μｇ以下であってよい。 When the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen is bound to the microtiter plate and does not contain an aqueous medium and is in a dry state, DUPAN-2 in one well of the microtiter plate. The amount of the first antibody or antibody fragment thereof that binds to the antigen is 0.001 μg or more, 0.01 μg or more, 0.02 μg or more, 0.03 μg or more, 0.04 μg or more, 0.05 μg or more, 0.06 μg or more, It may be 0.07 μg or more, 0.08 μg or more, 0.09 μg or more, 0.1 μg or more, 0.2 μg or more, 0.3 μg or more, 0.4 μg or more, or 0.5 μg or more. The amount of the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in one well of the microtiter plate is 0.6 μg or less, 0.7 μg or less, 0.8 μg or less, 0.9 μg or less, 1 μg. 2 μg or less, 3 μg or less, 4 μg or less, 5 μg or less, 6 μg or less, 7 μg or less, 7.5 μg or less, 8 g / mL or less, 9 μg or less, 10 μg or less, 11 μg or less, 12 μg or less, 13 μg or less, 14 μg or less, 15 μg or less. , 16 μg or less, 17 μg or less, 18 μg or less, 19 μg or less, 20 μg or less, 30 μg or less, 40 μg or less, 50 μg or less, or 100 μg or less.
　前記の数値は自由に組み合わせることができる。例えば、前記マイクロプレートの１ウェルにおける、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片の量は、０．０１μｇ～３０μｇであってもよく、０．１μｇ～１０μｇであってもよく、０．３μｇ～３μｇであってもよく、０．３μｇ～１μｇであってもよく、０．５μｇ～１μｇであってもよい。 The above values can be freely combined. For example, the amount of the first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in one well of the microplate may be 0.01 μg to 30 μg, or 0.1 μg to 10 μg. It may be 0.3 μg to 3 μg, 0.3 μg to 1 μg, or 0.5 μg to 1 μg.
　前記測定キット（２）の第２試薬におけるＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の濃度、及び、前記測定キット（３）の第３試薬におけるＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の濃度は、０．００１μｇ／ｍＬ以上、０．０１μｇ／ｍＬ以上、０．０２μｇ／ｍＬ以上、０．０３μｇ／ｍＬ以上、０．０４μｇ／ｍＬ以上、０．０５μｇ／ｍＬ以上、０．０６μｇ／ｍＬ以上、０．０７μｇ／ｍＬ以上、０．０８μｇ／ｍＬ以上、０．０９μｇ／ｍＬ以上、又は０．１μｇ／ｍＬ以上であってよい。また、前記測定キット（２）の第２試薬におけるＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の濃度、及び、前記測定キット（３）の第３試薬におけるＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の濃度は、０．２μｇ／ｍＬ以下、０．３μｇ／ｍＬ以下、０．４μｇ／ｍＬ以下、０．５μｇ／ｍＬ以下、０．６μｇ／ｍＬ以下、０．７μｇ／ｍＬ以下、０．８μｇ／ｍＬ以下、０．９μｇ／ｍＬ以下、１μｇ／ｍＬ以下、２μｇ／ｍＬ以下、３μｇ／ｍＬ以下、４μｇ／ｍＬ以下、５μｇ／ｍＬ以下、６μｇ／ｍＬ以下、７μｇ／ｍＬ以下、７．５μｇ／ｍＬ以下、８ｇ／ｍＬ以下、９μｇ／ｍＬ以下、１０μｇ／ｍＬ以下、１１μｇ／ｍＬ以下、１２μｇ／ｍＬ以下、１３μｇ／ｍＬ以下、１４μｇ／ｍＬ以下、１５μｇ／ｍＬ以下、１６μｇ／ｍＬ以下、１７μｇ／ｍＬ以下、１８μｇ／ｍＬ以下、１９μｇ／ｍＬ以下、２０μｇ／ｍＬ以下、３０μｇ／ｍＬ以下、４０μｇ／ｍＬ以下、５０μｇ／ｍＬ以下、又は１００μｇ／ｍＬ以下であってよい。 The concentration of the second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (2), and the second that binds to the DUPAN-2 antigen in the third reagent of the measurement kit (3). The concentration of the antibody or its antibody fragment is 0.001 μg / mL or more, 0.01 μg / mL or more, 0.02 μg / mL or more, 0.03 μg / mL or more, 0.04 μg / mL or more, 0.05 μg / mL or more. , 0.06 μg / mL or more, 0.07 μg / mL or more, 0.08 μg / mL or more, 0.09 μg / mL or more, or 0.1 μg / mL or more. Further, the concentration of the second antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (2) and the DUPAN-2 antigen in the third reagent of the measurement kit (3). The concentration of the second antibody or its antibody fragment is 0.2 μg / mL or less, 0.3 μg / mL or less, 0.4 μg / mL or less, 0.5 μg / mL or less, 0.6 μg / mL or less, 0.7 μg / mL or less. mL or less, 0.8 μg / mL or less, 0.9 μg / mL or less, 1 μg / mL or less, 2 μg / mL or less, 3 μg / mL or less, 4 μg / mL or less, 5 μg / mL or less, 6 μg / mL or less, 7 μg / mL Below, 7.5 μg / mL or less, 8 g / mL or less, 9 μg / mL or less, 10 μg / mL or less, 11 μg / mL or less, 12 μg / mL or less, 13 μg / mL or less, 14 μg / mL or less, 15 μg / mL or less, 16 μg It may be / mL or less, 17 μg / mL or less, 18 μg / mL or less, 19 μg / mL or less, 20 μg / mL or less, 30 μg / mL or less, 40 μg / mL or less, 50 μg / mL or less, or 100 μg / mL or less.
　前記の数値は、それぞれの測定キットにおいて自由に組み合わせることができる。例えば、前記測定キット（２）の第２試薬におけるＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の濃度、及び、前記測定キット（３）の第３試薬におけるＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片の濃度は、０．０１μｇ／ｍＬ～１００μｇ／ｍＬであってもよく、０．０３μｇ／ｍＬ～３０μｇ／ｍＬであってもよく、０．１μｇ／ｍＬ～２０μｇ／ｍＬであってもよい。 The above values can be freely combined in each measurement kit. For example, the concentration of the second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (2), and the DUPAN-2 antigen that binds to the DUPAN-2 antigen in the third reagent of the measurement kit (3). The concentration of the second antibody or antibody fragment thereof may be 0.01 μg / mL to 100 μg / mL, 0.03 μg / mL to 30 μg / mL, or 0.1 μg / mL to 20 μg / mL. May be.
　本開示の試料中のＤＵＰＡＮ－２抗原の測定キットを用いて、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定する場合、例えば、前述の方法により測定することができる。この際、不溶性担体、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片、第３抗体、腹水由来のＤＵＰＡＮ－２抗原、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原、及び標識としては、例えば前述の不溶性担体、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片、第３抗体、腹水由来のＤＵＰＡＮ－２抗原、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原、及び標識を用いることができる。 When the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the DUPAN-2 antigen measurement kit in the sample of the present disclosure, for example, the measurement is carried out by the above-mentioned method. can do. At this time, an insoluble carrier, an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, the first 3 Antigen, DUPAN-2 antigen derived from ascites, DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and as a label, for example, the above-mentioned insoluble carrier, an antibody that binds to DUPAN-2 antigen or an antibody fragment thereof, DUPAN-2. First antibody or antibody fragment thereof that binds to the antigen, second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, third antibody, DUPAN-2 antigen derived from ascites, DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF , And signs can be used.
　本開示の試料中のＤＵＰＡＮ－２抗原の測定キットを用いて、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定する場合、前記反応液中の不溶性担体、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片、及び第３抗体の濃度としては、例えば前述の濃度が挙げられる。
　本開示の試料中のＤＵＰＡＮ－２抗原の測定キットを用いて、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定する場合、前記反応温度としては、例えば前述の反応温度が挙げられ、前記反応時間としては、例えば前述の反応時間が挙げられる。 When the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF are measured using the DUPAN-2 antigen measurement kit in the sample of the present disclosure, the insoluble carrier in the reaction solution is used. , Concentration of antibody or antibody fragment thereof that binds to DUPAN-2 antigen, first antibody or antibody fragment thereof that binds to DUPAN-2 antigen, second antibody or antibody fragment thereof that binds to DUPAN-2 antigen, and third antibody. For example, the above-mentioned concentration can be mentioned.
When the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the DUPAN-2 antigen measurement kit in the sample of the present disclosure, the reaction temperature may be, for example. The above-mentioned reaction temperature is mentioned, and examples of the said reaction time include the above-mentioned reaction time.
　本開示の、試料中のＤＵＰＡＮ－２抗原の測定キットは、前記方法に従い、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定した場合に、下記式（Ｘ）を満たす。このようなＤＵＰＡＮ－２抗原の測定キットは、高感度なＤＵＰＡＮ－２抗原の測定キットとなる。
　０．８≦（Ｂ）／（Ａ）≦１．２　（Ｘ）
（式中、（Ａ）は、腹水由来のＤＵＰＡＮ－２抗原の測定値、（Ｂ）は、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値を意味し、ここで、腹水由来のＤＵＰＡＮ－２抗原とヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原との抗原濃度比は、１：１である。） The DUPAN-2 antigen measurement kit in the sample of the present disclosure has the following formula when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF are measured according to the above method. X) is satisfied. Such a DUPAN-2 antigen measurement kit is a highly sensitive DUPAN-2 antigen measurement kit.
0.8 ≤ (B) / (A) ≤ 1.2 (X)
(In the formula, (A) means the measured value of DUPAN-2 antigen derived from ascites, and (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites. The antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
　腹水由来のＤＵＰＡＮ－２抗原の測定値（Ａ）及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値（Ｂ）は、前述の吸光度、発光強度又は蛍光強度のようなシグナルであってもよく、前述のＤＵＰＡＮ－２抗原の濃度であってもよい。 The measured value of DUPAN-2 antigen derived from ascites (A) and the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF (B) may be a signal such as the above-mentioned absorbance, luminescence intensity or fluorescence intensity. It may be the concentration of the above-mentioned DUPAN-2 antigen.
　（Ｂ）／（Ａ）値は０．７以上、０．８以上、０．９以上であってよい。また、（Ｂ）／（Ａ）値は１．１以下、１．２以下であってよい。前記の数値は自由に組み合わせることができる。例えば、（Ｂ）／（Ａ）値は０．７～１．２であってもよく、０．８～１．２であってもよく、０．９～１．２であってもよい。 The (B) / (A) value may be 0.7 or more, 0.8 or more, and 0.9 or more. Further, the (B) / (A) value may be 1.1 or less and 1.2 or less. The above numerical values can be freely combined. For example, the (B) / (A) value may be 0.7 to 1.2, 0.8 to 1.2, or 0.9 to 1.2.
　試料中のＤＵＰＡＮ－２抗原の測定試薬及び測定キットは、試料中のＤＵＰＡＮ－２抗原を測定する方法に使用できる。
　試料中のＤＵＰＡＮ－２抗原を測定する方法としては、例えば、以下の（１）～（３）を含む方法が挙げられる。
（１）ＤＵＰＡＮ－２抗原を含む試料と、水性媒体とを混合する工程；
（２）試料中のＤＵＰＡＮ－２抗原に、不溶性担体と、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、を反応させ、前記不溶性担体と、前記ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原と、を含む免疫複合体を生成させる工程；及び
（３）工程（２）で生成された免疫複合体を測定する工程。 The reagent and measurement kit for measuring the DUPAN-2 antigen in the sample can be used in the method for measuring the DUPAN-2 antigen in the sample.
Examples of the method for measuring the DUPAN-2 antigen in the sample include methods including the following (1) to (3).
(1) A step of mixing a sample containing the DUPAN-2 antigen with an aqueous medium;
(2) The DUPAN-2 antigen in the sample is reacted with the insoluble carrier and the antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and the insoluble carrier and the antibody that binds to the DUPAN-2 antigen or the antibody thereof. A step of producing an immune complex containing the antibody fragment and the DUPAN-2 antigen; and (3) a step of measuring the immune complex produced in step (2).
　試料中のＤＵＰＡＮ－２抗原を測定する別の方法としては、例えば、以下の（１）及び（２）を含む方法が挙げられる。
（１）試料中のＤＵＰＡＮ－２抗原に、不溶性担体と、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片と、を反応させ、前記不溶性担体上に、前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原と、前記ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片と、を含む免疫複合体を生成させる工程；及び
（２）工程（１）で生成された免疫複合体を測定する工程。 As another method for measuring the DUPAN-2 antigen in the sample, for example, a method including the following (1) and (2) can be mentioned.
(1) The DUPAN-2 antigen in the sample is reacted with an insoluble carrier, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof. Then, on the insoluble carrier, a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, a DUPAN-2 antigen, and a second antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen are placed. A step of producing an immune complex containing; and (2) a step of measuring the immune complex produced in step (1).
　不溶性担体、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、及びＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片としては、例えば前述の不溶性担体、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、及びＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片が挙げられる。 Examples of the insoluble carrier, an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, a first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and a second antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof include, for example. Examples thereof include the above-mentioned insoluble carrier, an antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen, and a second antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen. Be done.
　本開示の試料中のＤＵＰＡＮ－２抗原の測定試薬、及び本開示の試料中のＤＵＰＡＮ－２抗原の測定キットには、前述の水性媒体、塩類、金属イオン、糖類、蛋白質、界面活性剤、ポリエチレングリコール誘導体等が含有されてもよい。 The DUPAN-2 antigen measuring reagent in the sample of the present disclosure and the DUPAN-2 antigen measuring kit in the sample of the present disclosure include the above-mentioned aqueous medium, salts, metal ions, saccharides, proteins, surfactants and polyethylene. A glycol derivative or the like may be contained.
　水性媒体としては、脱イオン水、蒸留水、緩衝液等が挙げられ、緩衝液が好ましい。緩衝液の調製に使用される緩衝剤としては、酢酸緩衝液、グッド緩衝剤等が挙げられる。緩衝剤は、１種を単独で使用してもよいし、２種以上を併用してもよい。水性媒体には、前述の塩類、金属イオン、糖類、蛋白質、界面活性剤等が含有されてもよい。 Examples of the aqueous medium include deionized water, distilled water, a buffer solution and the like, and a buffer solution is preferable. Examples of the buffer used for preparing the buffer include acetate buffer and Good's buffer. One type of buffer may be used alone, or two or more types may be used in combination. The aqueous medium may contain the above-mentioned salts, metal ions, saccharides, proteins, surfactants and the like.
　ポリエチレングリコール誘導体の存在下でＤＵＰＡＮ－２抗原の測定を行うと、ＤＵＰＡＮ－２抗原の検出感度が向上する。本開示おいて、ポリエチレングリコール誘導体とは、本開示のＤＵＰＡＮ－２抗原の測定方法を可能とし、又は本開示の測定試薬若しくは測定キットに使用可能なポリエチレングリコール誘導体であれば特に制限はなく、例えばポリエチレングリコール、ポリオキシエチレンアルキルフェニルエーテル、ポリオキシエチレンアルキルエーテル、ポリオキシエチレンソルビタン脂肪酸エステル、ポリオキシエチレン多環フェニルエーテル等が挙げられる。ポリオキシエチレンアルキルフェニルエーテル及びポリオキシエチレン多環フェニルエーテルは、フェニル基を有するポリオキシエチレン系非イオン性界面活性剤である。 When the DUPAN-2 antigen is measured in the presence of the polyethylene glycol derivative, the detection sensitivity of the DUPAN-2 antigen is improved. In the present disclosure, the polyethylene glycol derivative is not particularly limited as long as it is a polyethylene glycol derivative that enables the method for measuring the DUPAN-2 antigen of the present disclosure or can be used in the measuring reagent or measuring kit of the present disclosure. Examples thereof include polyethylene glycol, polyoxyethylene alkyl phenyl ether, polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, and polyoxyethylene polycyclic phenyl ether. Polyoxyethylene alkyl phenyl ether and polyoxyethylene polycyclic phenyl ether are polyoxyethylene nonionic surfactants having a phenyl group.
　ポリエチレングリコールとしては、例えば数平均分子量１００～２５，０００のポリエチレングリコール等が挙げられ、数平均分子量２００～２０，０００のポリエチレングリコールが好ましく、数平均分子量６，０００～２０，０００のポリエチレングリコールが特に好ましい。 Examples of the polyethylene glycol include polyethylene glycol having a number average molecular weight of 100 to 25,000, preferably polyethylene glycol having a number average molecular weight of 200 to 20,000, and polyethylene glycol having a number average molecular weight of 6,000 to 20,000. Especially preferable.
　ポリオキシエチレンアルキルフェニルエーテルにおけるアルキルとしては、例えば炭素数８～９のアルキル等が挙げられる。炭素数８～９のアルキルとしては、例えばオクチル、ノニル等が挙げられる。ポリオキシエチレンアルキルフェニルエーテルの市販品としては、例えばトリトン（登録商標）Ｘ－１００（ポリオキシエチレンオクチルフェニルエーテル）（シグマアルドリッチジャパン合同会社製）、エマルゲン（登録商標）８１０（ポリオキシエチレンオクチルフェニルエーテル）、エマルゲン（登録商標）９１０、エマルゲン（登録商標）９３０（以上、ポリオキシエチレンノニルフェニルエーテル）（以上、花王株式会社製）、ニッコール（登録商標）ＯＰ－１０（ポリオキシエチレンオクチルフェニルエーテル）、ニッコール（登録商標）ＮＰ－１０（ポリオキシエチレンノニルフェニルエーテル）（以上、日光ケミカルズ株式会社製）、ノニオンＨＳ－２１０、ノニオンＨＳ－２１５、ノニオンＨＳ－２２０（以上、ポリオキシエチレンオクチルフェニルエーテル）、ノニオンＮＳ－２１０、ノニオンＮＳ－２１５、ノニオンＮＳ－２２０（以上、ポリオキシエチレンノニルフェニルエーテル）（以上、日油株式会社製）、ＢＬＡＵＮＯＮ　ＮＫ－８１０（青木油脂工業株式会社製）等が挙げられる。 Examples of the alkyl in the polyoxyethylene alkyl phenyl ether include an alkyl having 8 to 9 carbon atoms. Examples of the alkyl having 8 to 9 carbon atoms include octyl and nonyl. Commercially available products of polyoxyethylene alkyl phenyl ether include, for example, Triton (registered trademark) X-100 (polyoxyethylene octylphenyl ether) (manufactured by Sigma Aldrich Japan GK) and Emargen (registered trademark) 810 (polyoxyethylene octylphenyl). Ether), Emargen (registered trademark) 910, Emargen (registered trademark) 930 (above, polyoxyethylene nonylphenyl ether) (above, manufactured by Kao Co., Ltd.), Nikkor (registered trademark) OP-10 (polyoxyethylene octylphenyl ether) ), Nikkor® NP-10 (polyoxyethylene nonylphenyl ether) (above, manufactured by Nikko Chemicals Co., Ltd.), nonion HS-210, nonion HS-215, nonion HS-220 (above, polyoxyethylene octylphenyl). Ether), Nonion NS-210, Nonion NS-215, Nonion NS-220 (above, polyoxyethylene nonylphenyl ether) (above, manufactured by Nichiyu Co., Ltd.), BLAUNON NK-810 (manufactured by Aoki Oil & Fat Industry Co., Ltd.), etc. Can be mentioned.
　ポリオキシエチレンアルキルエーテルにおけるアルキルとしては、例えば炭素数８～２４のアルキルが挙げられ、これらの中では炭素数１０～２０のアルキルが好ましい。炭素数８～２４のアルキルとしては、例えばオクチル、イソオクチル、ノニル、デシル、イソデシル、ウンデシル、ドデシル（ラウリル）、トリデシル、テトラデシル（ミリスチル）、ペンタデシル、ヘキサデシル（セチル）、ヘプタデシル、オクタデシル（ステアリル）、オレイル、ノナデシル、イコシル、ヘネイコシル、ドコシル（ベヘニル）、トリコシル、テトラコシル等が挙げられる。炭素数１０～２０のアルキルとしては、例えばデシル、イソデシル、ウンデシル、ドデシル（ラウリル）、トリデシル、テトラデシル（ミリスチル）、ペンタデシル、ヘキサデシル（セチル）、ヘプタデシル、オクタデシル（ステアリル）、オレイル、ノナデシル、イコシル等が挙げられる。ポリオキシエチレンアルキルエーテルの市販品としては、例えばＢｒｉｊ（登録商標）３５（ポリオキシエチレンラウリルエーテル）、Ｂｒｉｊ（登録商標）９９（ポリオキシエチレンオレイルエーテル）、Ｂｒｉｊ（登録商標）５６（ポリオキシエチレンセチルエーテル）、Ｂｒｉｊ（登録商標）７８（ポリオキシエチレンステアリルエーテル）（以上、シグマアルドリッチジャパン合同会社製）、ニッコール（登録商標）ＢＣ－２３（ポリオキシエチレンセチルエーテル）（日光ケミカルズ株式会社製）、ノニオンＰ－２１０、ノニオンＰ－２１３（以上、ポリオキシエチレンセチルエーテル）、ノニオンＫ－２２０（ポリオキシエチレンラウリルエーテル）、ノニオンＥ－２０５、ノニオンＥ－２１５、ノニオンＥ－２３０（以上、ポリオキシエチレンオレイルエーテル）、ノニオンＳ－２１５、ノニオンＳ－２２０（以上、ポリオキシエチレンステアリルエーテル）（以上、日油株式会社製）、エマルゲン（登録商標）１２０（ポリオキシエチレンラウリルエーテル）、エマルゲン（登録商標）２２０（ポリオキシエチレンセチルエーテル）、エマルゲン（登録商標）３２０Ｐ（ポリオキシエチレンステアリルエーテル）、エマルゲン（登録商標）４２０（ポリオキシエチレンオレイルエーテル）（以上、花王株式会社製）、アデカトールＬＡ－８７５、アデカトールＬＡ－９７５（以上、ポリオキシエチレンラウリルエーテル）（以上、株式会社ＡＤＥＫＡ製）、エマルミンＮＬ－１００、エマルミンＮＬ－１１０、エマルミンＬＳ－８０、エマルミンＬＳ－９０、（以上、ポリオキシエチレンラウリルエーテル）（以上、三洋化成工業株式会社製）、ＢＬＡＵＮＯＮ　ＥＬ－１５０９、ＢＬＡＵＮＯＮ　ＥＬ－１５１２Ｐ、ＢＬＡＵＮＯＮ　ＥＬ－１５１５、ＢＬＡＵＮＯＮ　ＥＬ－１５２１（以上、ポリオキシエチレンラウリルエーテル）、ＢＬＡＵＮＯＮ　ＣＨ－３１０、ＢＬＡＵＮＯＮ　ＣＨ－３１０Ｌ、ＢＬＡＵＮＯＮ　ＣＨ－３１３（以上、ポリオキシエチレンセチルエーテル）、ＢＬＡＵＮＯＮ　ＳＲ－７１１（ポリオキシエチレンステアリルエーテル）（以上、青木油脂工業株式会社製）、ノイゲンＳＤ－７０、ノイゲンＳＤ－８０、ノイゲンＳＤ－１１０、ノイゲンＳＤ－１５０（以上、ポリオキシエチレンイソデシルエーテル）、ノイゲンＴＤＳ－８０、ノイゲンＴＤＳ－１００、ノイゲンＴＤＳ－２００Ｄ（以上、ポリオキシエチレントリデシルエーテル）（第一工業製薬株式会社製）等が挙げられる。 Examples of the alkyl in the polyoxyethylene alkyl ether include alkyl having 8 to 24 carbon atoms, and among these, alkyl having 10 to 20 carbon atoms is preferable. Examples of alkyl having 8 to 24 carbon atoms include octyl, isooctyl, nonadecylic, decyl, isodecyl, undecylic, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), and oleyl. , Nonadecylic, Icosyl, Heneikosyl, Docosyl (Behenil), Tridecylic, Tetracosyl and the like. Examples of alkyl having 10 to 20 carbon atoms include decyl, isodecyl, undecylic, dodecyl (lauryl), tridecylic, tetradecyl (myristyl), pentadecyl, hexadecyl (cetyl), heptadecyl, octadecyl (stearyl), oleyl, nonadecylic, and icosyl. Can be mentioned. Commercially available products of polyoxyethylene alkyl ether include, for example, Brij (registered trademark) 35 (polyoxyethylene lauryl ether), Brij (registered trademark) 99 (polyoxyethylene oleyl ether), and Brij (registered trademark) 56 (polyoxyethylene). Cetyl ether), Brij (registered trademark) 78 (polyoxyethylene stearyl ether) (above, manufactured by Sigma Aldrich Japan GK), Nikkor (registered trademark) BC-23 (polyoxyethylene cetyl ether) (manufactured by Nikko Chemicals Co., Ltd.) , Nonion P-210, Nonion P-213 (above, polyoxyethylene cetyl ether), Nonion K-220 (polyoxyethylene lauryl ether), Nonion E-205, Nonion E-215, Nonion E-230 (above, poly) Oxyethylene oleyl ether), Nonion S-215, Nonion S-220 (above, polyoxyethylene stearyl ether) (above, manufactured by Nichiyu Co., Ltd.), Emargen (registered trademark) 120 (polyoxyethylene lauryl ether), Emargen (above) Registered trademark) 220 (polyoxyethylene cetyl ether), Emargen (registered trademark) 320P (polyoxyethylene stearyl ether), Emargen (registered trademark) 420 (polyoxyethylene oleyl ether) (all manufactured by Kao Co., Ltd.), Adecator LA -875, Adecator LA-975 (above, polyoxyethylene lauryl ether) (above, manufactured by ADEKA Co., Ltd.), Emalmin NL-100, Emalmin NL-110, Emalmin LS-80, Emalmin LS-90, (above, polyoxy) Ethylene lauryl ether) (above, manufactured by Sanyo Kasei Kogyo Co., Ltd.), BLAUNON EL-1509, BLAUNON EL-1512P, BLAUNON EL-1515, BLAUNON EL-1521 (above, polyoxyethylene lauryl ether), BLAUNON CH-310, BLAUNON CH-310L, BLAUNON CH-313 (above, polyoxyethylene cetyl ether), BLAUNON SR-711 (polyoxyethylene stearyl ether) (above, manufactured by Aoki Oil & Fat Industry Co., Ltd.), Neugen SD-70, Neugen SD-80, Neugen SD-110, Neugen SD-150 (above, polyoxyethylene isodecyl ether), Neugen TDS-80, Neugen TDS-100, Neugen Examples thereof include TDS-200D (above, polyoxyethylene tridecyl ether) (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.).
　ポリオキシエチレンソルビタン脂肪酸エステルにおける脂肪酸としては、例えば炭素数８～２４の飽和又は不飽和脂肪酸が挙げられ、炭素数１２～１８の飽和又は不飽和脂肪酸が好ましい。炭素数８～２４の飽和又は不飽和脂肪酸としては、例えばオクタン酸、デカン酸、ラウリン酸、ミリスチン酸、パルミチン酸、ステアリン酸、オレイン酸、リノール酸、リノレン酸、エイコサトリエン酸、アラキドン酸、イコサン酸、エイコサテトラエン酸、エイコサペンタエン酸、ドコサン酸、ドコサヘキサエン酸、テトラドコサン酸、テトラコサペンタエン酸等が挙げられる。炭素数１２～１８の飽和又は不飽和脂肪酸としては、例えばラウリン酸、ミリスチン酸、パルミチン酸、ステアリン酸、オレイン酸、リノール酸、リノレン酸等が挙げられる。ポリオキシエチレンソルビタン脂肪酸エステルとしては、例えばポリオキシエチレンソルビタンモノラウレート、ポリオキシエチレンソルビタンモノオレエート、ポリオキシエチレンソルビタンモノステアレート、ポリオキシエチレンソルビタンモノパルミテート等が挙げられる。ポリオキシエチレンソルビタン脂肪酸エステルの市販品としては、例えばＴｗｅｅｎ（登録商標）２０（ポリオキシエチレンソルビタンモノラウレート；シグマアルドリッチジャパン合同会社製）、Ｔｗｅｅｎ（登録商標）４０（ポリオキシエチレンソルビタンモノパルミテート；富士フイルム和光純薬株式会社製）、Ｔｗｅｅｎ（登録商標）６０（ポリオキシエチレンソルビタンモノステアレート；シグマアルドリッチジャパン合同会社製）、Ｔｗｅｅｎ（登録商標）８０（ポリオキシエチレンソルビタンモノオレエート；富士フイルム和光純薬株式会社製）、レオドール（登録商標）ＴＷ－Ｌ１０６（ポリオキシエチレンソルビタンモノラウレート；花王株式会社製）、レオドール（登録商標）ＴＷ－Ｌ１２０（ポリオキシエチレンソルビタンモノラウレート；花王株式会社製）、レオドール（登録商標）ＴＷ－Ｏ１０６Ｖ（ポリオキシエチレンソルビタンモノオレエート；花王株式会社製）、レオドール（登録商標）ＴＷ－Ｏ１２０Ｖ（ポリオキシエチレンソルビタンモノオレエート；花王株式会社製）、レオドール（登録商標）ＴＷ－Ｐ１２０（ポリオキシエチレンソルビタンモノパルミテート；花王株式会社製）、レオドール（登録商標）ＴＷ－Ｓ１０６Ｖ（ポリオキシエチレンソルビタンモノステアレート；花王株式会社製）、レオドール（登録商標）ＴＷ－Ｓ１２０Ｖ（ポリオキシエチレンソルビタンモノステアレート；花王株式会社製）、ＢＬＡＵＮＯＮ　ＯＴ－１０６（ポリオキシエチレンソルビタンモノオレエート；青木油脂工業株式会社製）、ＢＬＡＵＮＯＮ　ＯＴ－２１（ポリオキシエチレンソルビタンモノオレエート；青木油脂工業株式会社製）、ニューコール（登録商標）２５（ポリオキシエチレンソルビタンラウレート；日本乳化剤株式会社製）、ニューコール（登録商標）６５（ポリオキシエチレンソルビタンステアレート；日本乳化剤株式会社製）、ニューコール（登録商標）８５（ポリオキシエチレンソルビタンオレエート；日本乳化剤株式会社製）等が挙げられる。 Examples of the fatty acid in the polyoxyethylene sorbitan fatty acid ester include saturated or unsaturated fatty acids having 8 to 24 carbon atoms, and saturated or unsaturated fatty acids having 12 to 18 carbon atoms are preferable. Saturated or unsaturated fatty acids having 8 to 24 carbon atoms include, for example, octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linolenic acid, linolenic acid, eicosatrienoic acid, arachidonic acid, and the like. Examples thereof include icosanoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosanoic acid, docosahexaenoic acid, tetradocosanic acid, tetracosapentaenoic acid and the like. Examples of saturated or unsaturated fatty acids having 12 to 18 carbon atoms include lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. Examples of the polyoxyethylene sorbitan fatty acid ester include polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, and polyoxyethylene sorbitan monopalmitate. Commercially available products of polyoxyethylene sorbitan fatty acid ester include, for example, Tween (registered trademark) 20 (polyoxyethylene sorbitan monolaurate; manufactured by Sigma Aldrich Japan LLC) and Tween (registered trademark) 40 (polyoxyethylene sorbitan monopalmitate). Fujifilm Wako Junyaku Co., Ltd., Tween (registered trademark) 60 (polyoxyethylene sorbitan monostearate; Sigma Aldrich Japan LLC), Tween (registered trademark) 80 (polyoxyethylene sorbitan monooleate; Fuji) Film Wako Junyaku Co., Ltd., Leodor (registered trademark) TW-L106 (polyoxyethylene sorbitan monolaurate; Kao Co., Ltd.), Leodor (registered trademark) TW-L120 (polyoxyethylene sorbitan monolaurate; Kao) Leodor (registered trademark) TW-O106V (polyoxyethylene sorbitan monooleate; manufactured by Kao Co., Ltd.), Leodor (registered trademark) TW-O120V (polyoxyethylene sorbitan monooleate; manufactured by Kao Co., Ltd.) , Leodor (registered trademark) TW-P120 (polyoxyethylene sorbitan monopalmitate; manufactured by Kao Co., Ltd.), Leodor (registered trademark) TW-S106V (polyoxyethylene sorbitan monostearate; manufactured by Kao Co., Ltd.), Leodor (registered) Trademarks) TW-S120V (polyoxyethylene sorbitan monostearate; manufactured by Kao Co., Ltd.), BLAUNON OT-106 (polyoxyethylene sorbitan monooleate; manufactured by Aoki Yushi Kogyo Co., Ltd.), BLAUNON OT-21 (polyoxyethylene sorbitan) Monooleate; manufactured by Aoki Oil & Fat Industry Co., Ltd.), Newcol (registered trademark) 25 (polyoxyethylene sorbitan laurate; manufactured by Japan Emulsifier Co., Ltd.), Newcol (registered trademark) 65 (polyoxyethylene sorbitan stearate; Japan) Examples thereof include Newcol (registered trademark) 85 (polyoxyethylene sorbitan oleate; manufactured by Nippon Embroidery Co., Ltd.).
　ポリオキシエチレン多環フェニルエーテルにおける多環フェニルとは、基内に１つの芳香環を有する基（置換基）が２つ以上置換したフェニル基、基内に２つ以上の芳香環を有する基（置換基）が１つ又は複数置換したフェニル基等が挙げられる。基内に１つの芳香環を有する基としては、例えばベンジル、１－（フェニル）エチル等が挙げられる。基内に２つ以上の芳香環を有する基としては、例えばナフチル等が挙げられる。ポリオキシエチレン多環フェニルエーテルの市販品としては、例えばニューコール（登録商標）７０４、ニューコール（登録商標）７０６、ニューコール（登録商標）７０７、ニューコール（登録商標）７０８、ニューコール（登録商標）７０９、ニューコール（登録商標）７１０、ニューコール（登録商標）７１１、ニューコール（登録商標）７１２、ニューコール（登録商標）７１４、ニューコール（登録商標）７４０、ニューコール（登録商標）６１０、ニューコール（登録商標）２６０７、ニューコール（登録商標）２６０９、ニューコール（登録商標）２６１４（以上、日本乳化剤株式会社製）等が挙げられる。 The polycyclic phenyl in the polyoxyethylene polycyclic phenyl ether is a phenyl group in which two or more groups having one aromatic ring (substituent) are substituted in the group, and a group having two or more aromatic rings in the group (substituent). Examples thereof include a phenyl group in which one or more substituents) are substituted. Examples of the group having one aromatic ring in the group include benzyl, 1- (phenyl) ethyl and the like. Examples of the group having two or more aromatic rings in the group include naphthyl and the like. Commercially available products of polyoxyethylene polycyclic phenyl ether include, for example, Newcol (registered trademark) 704, Newcol (registered trademark) 706, Newcol (registered trademark) 707, Newcol (registered trademark) 708, and Newcol (registered). Trademark) 709, New Call (Registered Trademark) 710, New Call (Registered Trademark) 711, New Call (Registered Trademark) 712, New Call (Registered Trademark) 714, New Call (Registered Trademark) 740, New Call (Registered Trademark) 610, New Call (registered trademark) 2607, New Call (registered trademark) 2609, New Call (registered trademark) 2614 (all manufactured by Nippon Embroidery Co., Ltd.) and the like can be mentioned.
（試料）
　本開示における試料としては、ＤＵＰＡＮ－２抗原を含有する可能性がある試料であれば特に制限はなく、全血、血漿、血清、尿、腹水、髄液、唾液、羊水、尿、汗及び膵液からなる群から選択されるいずれか１以上の生体試料等が挙げられ、好ましくは全血、血漿、血清、腹水等が挙げられる。また、ヒト膵癌培養細胞ＨＰＡＦの細胞可溶化物（ｃｅｌｌ　ｌｙｓａｔｅ）、ヒト膵癌培養細胞ＨＰＡＦを培養した際に得られる培養上清であってもよい。また、前記生体試料、前記細胞可溶化物又は前記培養上清から精製されたＤＵＰＡＮ－２抗原であってもよい。 (sample)
The sample in the present disclosure is not particularly limited as long as it is a sample that may contain the DUPAN-2 antigen, and is not particularly limited. Examples thereof include any one or more biological samples selected from the group consisting of, preferably whole blood, plasma, serum, ascites and the like. Further, it may be a cell lysate of cultured human pancreatic cancer cells HPAF, or a culture supernatant obtained when cultured human pancreatic cancer cells HPAF are cultured. Further, it may be the DUPAN-2 antigen purified from the biological sample, the cell solubilized product or the culture supernatant.
　以下、本開示を実施例により、更に詳細に説明するが、これらは本開示の範囲を何ら制限するものではない。なお、以下の実施例においては、次のメーカーの試薬類を使用した。 Hereinafter, the present disclosure will be described in more detail by way of examples, but these do not limit the scope of the present disclosure at all. In the following examples, reagents from the following manufacturers were used.
　リン酸水素二ナトリウム（無水）（関東化学株式会社製）、リン酸二水素ナトリウム（無水）（関東化学株式会社製）、トリス（トリスヒドロキシメチルアミノメタン；関東化学株式会社製）、塩化ナトリウム（富士フイルム和光純薬株式会社製）、ＭＥＳ（２－モルホリノエタンスルホン酸；株式会社同仁化学研究所製）、ＭＯＰＳ（３－モルホリノプロパンスルホン酸；株式会社同仁化学研究所製）、ＮＰ－４０（シグマアルドリッチ社製）、Ｔｗｅｅｎ（登録商標）　２０（関東化学株式会社製）、Ｍｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ　Ｅａｇｌｅ（シグマアルドリッチ社製）、ウシ胎仔血清（サーモフィッシャーサイエンティフィック社製）、ウシ血清アルブミン（ＢＳＡ）（Ｂｏｖｏｇｅｎ社製）、「デタミナー（登録商標）ＤＵＰＡＮ－２　Ｎ」（日立化成ダイアグノスティックス・システムズ株式会社製）の構成試薬「検体希釈液」を使用した。 Sodium hydrogen phosphate (anhydrous) (manufactured by Kanto Chemical Co., Ltd.), Sodium dihydrogen phosphate (anhydrous) (manufactured by Kanto Chemical Co., Ltd.), Tris (Trishydroxymethylaminomethane; manufactured by Kanto Chemical Co., Ltd.), Sodium chloride ( Fujifilm Wako Junyaku Co., Ltd.), MES (2-morpholinoetan sulfonic acid; manufactured by Dojin Chemical Research Institute Co., Ltd.), MOPS (3-morpholinopropane sulfonic acid; manufactured by Dojin Chemical Research Institute Co., Ltd.), NP-40 ( Sigma Aldrich (manufactured by Sigma Aldrich), Tween (registered trademark) 20 (manufactured by Kanto Chemical Co., Ltd.), Minimum Essential Medium Eagle (manufactured by Sigma Aldrich), fetal bovine serum (manufactured by Thermofisher Scientific), bovine serum albumin (BSA) The constituent reagent "specimen diluent" of "Detaminer (registered trademark) DUPAN-2 N" (manufactured by Hitachi Chemical Diagnostics Systems Co., Ltd.) was used.
［実施例１］
　ＤＵＰＡＮ－２抗体結合マイクロプレート、及びペルオキシダーゼ（ＰＯＤ）標識ＤＵＰＡＮ－２抗体溶液を含む、ＤＵＰＡＮ－２抗原の測定キット１及びキット２を作製した。なお、下記のＤＵＰＡＮ－２抗体結合マイクロプレートの作製時において、ＤＵＰＡＮ－２抗体溶液におけるＤＵＰＡＮ－２抗体濃度を１０μｇ／ｍＬとしたものを測定キット１に、２０μｇ／ｍＬとしたものを測定キット２に使用した。 [Example 1]
DUPAN-2 antigen measurement kits 1 and 2 containing a DUPAN-2 antibody-binding microplate and a peroxidase (POD) -labeled DUPAN-2 antibody solution were prepared. At the time of preparing the following DUPAN-2 antibody-binding microplate, the measurement kit 1 had a DUPAN-2 antibody concentration of 10 μg / mL in the DUPAN-2 antibody solution, and the measurement kit 2 had a DUPAN-2 antibody concentration of 20 μg / mL. Used for.
＜ＤＵＰＡＮ－２抗体結合マイクロプレート＞
　９６ウェルポリスチレンプレート（サーモフィッシャーサイエンティフィック社製）の各ウェルに、結合用緩衝液［０．１４ｍｏｌ／Ｌの塩化ナトリウムを含有する０．０１ｍｏｌ／Ｌリン酸緩衝液（ｐＨ７．３）］で調製したＤＵＰＡＮ－２抗体溶液を５０μＬ添加し、２５℃で１６時間インキュベートした。次いで、当該ＤＵＰＡＮ－２抗体溶液を除去し、ブロッキング液［１％（ｗ／ｖ）ＢＳＡ、０．０５％（ｗ／ｖ）Ｔｗｅｅｎ（登録商標）　２０を含むリン酸緩衝液（ｐＨ７．２）］を各ウェルに２５０μＬ加えて洗浄した後、当該ブロッキング液を各ウェルに２５０μＬ加えて、４℃で１６時間インキュベートした。その後、当該ブロッキング液を除去し、ＤＵＰＡＮ－２抗体結合マイクロプレートを調製した。 <DUPAN-2 antibody binding microplate>
In each well of a 96-well polystyrene plate (manufactured by Thermo Fisher Scientific), use a binding buffer [0.01 mol / L phosphate buffer (pH 7.3) containing 0.14 mol / L sodium chloride]. 50 μL of the prepared DUPAN-2 antibody solution was added, and the mixture was incubated at 25 ° C. for 16 hours. The DUPAN-2 antibody solution is then removed and a phosphate buffer (pH 7.2) containing a blocking solution [1% (w / v) BSA, 0.05% (w / v) Tween® 20). ] To each well and wash, then 250 μL of the blocking solution was added to each well and incubated at 4 ° C. for 16 hours. Then, the blocking solution was removed to prepare a DUPAN-2 antibody-binding microplate.
＜ＰＯＤ標識ＤＵＰＡＮ－２抗体溶液＞
　Ｐｅｒｏｘｉｄａｓｅ　Ｌａｂｅｌｉｎｇ　Ｋｉｔ－ＮＨ２（株式会社同仁化学研究所製）を用いて、当該キットの取扱説明書に従い、ＤＵＰＡＮ－２抗体をＰＯＤで標識し、ＰＯＤ標識ＤＵＰＡＮ－２抗体を作製した。得られたＰＯＤ標識ＤＵＰＡＮ－２抗体を用いて、以下の組成のＰＯＤ標識ＤＵＰＡＮ－２抗体溶液を調製した。 <POD-labeled DUPAN-2 antibody solution>
Using Peroxidase Labeling Kit-NH 2 (manufactured by Dojin Kagaku Kenkyusho Co., Ltd.), the DUPAN-2 antibody was labeled with POD according to the instruction manual of the kit to prepare a POD-labeled DUPAN-2 antibody. Using the obtained POD-labeled DUPAN-2 antibody, a POD-labeled DUPAN-2 antibody solution having the following composition was prepared.
　ＭＥＳ（ｐＨ６．５）　　　　　　　　　　　　　　　　０．１ｍｏｌ／Ｌ
　ＰＯＤ標識ＤＵＰＡＮ－２抗体　　　　　　　　　　　　０．９μｇ／ｍＬ
　ＢＳＡ　　　　　　　　　　　　　　　　　　　　　　　０．１％（ｗ／ｖ）
　ＮＰ－４０　　　　　　　　　　　　　　　　　　　　　０．２％（ｗ／ｖ）
　塩化ナトリウム　　　　　　　　　　　　　　　　　　　０．１５ｍｏｌ／Ｌ MES (pH 6.5) 0.1 mol / L
POD-labeled DUPAN-2 antibody 0.9 μg / mL
BSA 0.1% (w / v)
NP-40 0.2% (w / v)
Sodium chloride 0.15 mol / L
［比較例］
　前記のＤＵＰＡＮ－２抗体結合マイクロプレートの作製方法において、ＤＵＰＡＮ－２抗体溶液におけるＤＵＰＡＮ－２抗体濃度を５μｇ／ｍＬとした、ＤＵＰＡＮ－２抗体結合マイクロプレートを作製した。当該ＤＵＰＡＮ－２抗体結合マイクロプレートと、実施例１記載の方法で調製したＰＯＤ標識ＤＵＰＡＮ－２抗体溶液とを組み合わせた、測定キットＡを比較例として作製した。 [Comparison example]
In the above-mentioned method for preparing a DUPAN-2 antibody-binding microplate, a DUPAN-2 antibody-binding microplate was prepared with a DUPAN-2 antibody concentration of 5 μg / mL in the DUPAN-2 antibody solution. A measurement kit A in which the DUPAN-2 antibody-binding microplate and the POD-labeled DUPAN-2 antibody solution prepared by the method described in Example 1 were combined was prepared as a comparative example.
［実施例２］
＜試料中のＤＵＰＡＮ－２の測定方法１＞
　ヒト腹水由来のＤＵＰＡＮ－２抗原を、抗原希釈液［１％（ｗ／ｖ）ＢＳＡ、０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０５ｍｏｌ／Ｌトリス緩衝液（ｐＨ８．０）］で４０Ｕ／ｍＬ、１００Ｕ／ｍＬ、３５０Ｕ／ｍＬに希釈して、ヒト腹水由来ＤＵＰＡＮ－２抗原溶液を調製した。同様に、ヒト膵癌培養細胞ＨＰＡＦ－ＩＩの培養上清由来のＤＵＰＡＮ－２抗原を、前記抗原希釈液で４０Ｕ／ｍＬ、１００Ｕ／ｍＬ、３５０Ｕ／ｍＬに希釈して、ヒト膵癌培養細胞ＨＰＡＦ－ＩＩ由来のＤＵＰＡＮ－２抗原溶液を調製した。
　なお、公知の方法（Ｍｅｔｚｇａｒら、“Ａｎｔｉｇｅｎｓ　ｏｆ　Ｈｕｍａｎ　Ｐａｎｃｒｅａｔｉｃ　Ａｄｅｎｏｃａｒｃｉｎｏｍａ　Ｃｅｌｌｓ　Ｄｅｆｉｎｅｄ　ｂｙ　Ｍｕｒｉｎｅ　Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ”、ＣＡＮＣＥＲ　ＲＥＳＥＡＲＣＨ（１９８２），４２（２）：６０１－６０８）に従い、ヒト膵癌培養細胞ＨＰＡＦ－ＩＩを培養し、当該培養で得られた培養上清を、ヒト膵癌培養細胞ＨＰＡＦ－ＩＩ由来のＤＵＰＡＮ－２抗原として用いた。 [Example 2]
<Measurement method 1 of DUPAN-2 in the sample>
DUPAN-2 antigen derived from human ascites is 40 U / mL in an antigen diluted solution [1% (w / v) BSA, 0.05 mol / L Tris buffer (pH 8.0) containing 0.15 mol / L sodium chloride]. , 100 U / mL, 350 U / mL to prepare human ascites-derived DUPAN-2 antigen solution. Similarly, the DUPAN-2 antigen derived from the culture supernatant of human pancreatic cancer cultured cell HPAF-II is diluted with the antigen diluted solution to 40 U / mL, 100 U / mL, 350 U / mL, and human pancreatic cancer cultured cell HPAF-II. A DUPAN-2 antigen solution of origin was prepared.
In addition, a known method (Metsgar et al., "Antigens of Human Pancreatic Adenocarcinoma Cells Defined by Murine Monoclonal Antibodies", CANCE R RESEARCH (1982), 60 (1982) , The culture supernatant obtained in the culture was used as a DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF-II.
　ＤＵＰＡＮ－２抗体結合マイクロプレートの各ウェルに、検体希釈液を１００μＬ添加した。その後、当該各ウェルに、ヒト腹水由来ＤＵＰＡＮ－２抗原溶液、又はヒト膵癌培養細胞ＨＰＡＦ－ＩＩ由来のＤＵＰＡＮ－２抗原溶液２０μＬをそれぞれ添加し、２５℃１時間インキュベートした（１次反応）。１次反応後のプレートの各ウェルを、洗浄液［０．０５％（ｗ／ｖ）Ｔｗｅｅｎ（登録商標）　２０、０．１５ｍｏｌ／Ｌ塩化ナトリウムを含む０．０１ｍｏｌ／Ｌリン酸緩衝液（ｐＨ７．４）］で洗浄した後、各ウェルに、ＰＯＤ標識ＤＵＰＡＮ－２抗体溶液１００μＬを添加し、２５℃で１時間反応を行った（２次反応）。２次反応後のプレートの各ウェルを前記洗浄液で洗浄した後、各ウェルに、ＴＭＢ（３，３’，５，５’－テトラメチルベンジジン）水溶液を１００μＬ添加し、２５℃で３０分間インキュベートし、０．５ｍｏｌ／Ｌ　硫酸５０μＬを添加し反応を停止した（発色反応）。発色反応で得られた反応液の吸光度（主波長４５０ｎｍ、副波長６５０ｎｍ）を測定した。
　腹水由来のＤＵＰＡＮ－２抗原溶液を測定したときの吸光度を（Ａ）、ヒト膵癌培養細胞ＨＰＡＦ－ＩＩ由来のＤＵＰＡＮ－２抗原溶液を測定したときの吸光度を（Ｂ）とした場合の（Ｂ）／（Ａ）値を算出した。当該値、及び、プレートに結合したＤＵＰＡＮ－２抗体の結合量を表１に示す。 100 μL of sample diluent was added to each well of the DUPAN-2 antibody-bound microplate. Then, 20 μL of a human ascites-derived DUPAN-2 antigen solution or a human pancreatic cancer cultured cell HPAF-II-derived DUPAN-2 antigen solution was added to each well and incubated at 25 ° C. for 1 hour (primary reaction). After the primary reaction, each well of the plate was subjected to a washing solution [0.05% (w / v) Tween® 20, 0.15 mol / L sodium chloride-containing 0.01 mol / L phosphate buffer (pH 7. After washing with 4)], 100 μL of POD-labeled DUPAN-2 antibody solution was added to each well, and the reaction was carried out at 25 ° C. for 1 hour (secondary reaction). After washing each well of the plate after the secondary reaction with the washing solution, 100 μL of TMB (3,3', 5,5'-tetramethylbenzidine) aqueous solution was added to each well, and the mixture was incubated at 25 ° C. for 30 minutes. , 0.5 mol / L Sulfuric acid 50 μL was added to terminate the reaction (color development reaction). The absorbance (main wavelength 450 nm, sub-wavelength 650 nm) of the reaction solution obtained by the color development reaction was measured.
(B) when the absorbance when measuring the DUPAN-2 antigen solution derived from ascites is (A) and the absorbance when measuring the DUPAN-2 antigen solution derived from human pancreatic cancer cultured cells HPAF-II is (B). / (A) The value was calculated. The value and the amount of DUPAN-2 antibody bound to the plate are shown in Table 1.
Figure JPOXMLDOC01-appb-T000003
 　表１の結果より、キット１及びキット２の（Ｂ）／（Ａ）値は１．３より小さかった。
このような（Ｂ）／（Ａ）値を示すキット１及びキット２は、比較例キットＡと比べて、ＤＵＰＡＮ－２抗体の結合量が多い傾向に有り、検出感度の高い測定キットであることが判明した。
Figure JPOXMLDOC01-appb-T000003
 From the results in Table 1, the (B) / (A) values of Kit 1 and Kit 2 were smaller than 1.3.
Kit 1 and Kit 2 showing such (B) / (A) values tend to have a larger amount of DUPAN-2 antibody bound than Comparative Example Kit A, and are measurement kits with high detection sensitivity. There was found.
［実施例３］
　以下の磁性粒子懸濁液、ビオチン結合ＤＵＰＡＮ－２抗体溶液、アルカリホスファターゼ標識ＤＵＰＡＮ－２抗体溶液を含む、ＤＵＰＡＮ－２抗原の測定キット３を作製した。 [Example 3]
A DUPAN-2 antigen measurement kit 3 containing the following magnetic particle suspension, biotin-conjugated DUPAN-2 antibody solution, and alkaline phosphatase-labeled DUPAN-2 antibody solution was prepared.
＜磁性粒子懸濁液＞
　磁性粒子として、ストレプトアビジンが結合した市販の磁性粒子［Ｄｙｎａｂｅａｄｓ　ＭｙＯｎｅ　Ｓｔｒｅｐｔａｖｉｄｉｎ　Ｔ１（サーモフィッシャーサイエンティフィック社製）］を用いて、以下の組成の磁性粒子懸濁液を調製した。 <Magnetic particle suspension>
As the magnetic particles, a commercially available magnetic particle [Dynabeads MyOne Streptavidin T1 (manufactured by Thermo Fisher Scientific Co., Ltd.)] to which streptavidin was bound was used to prepare a magnetic particle suspension having the following composition.
　ＭＥＳ（ｐＨ６．５）　　　　　　　　　　　　　５０ｍｍｏｌ／Ｌ
　ストレプトアビジン結合磁性粒子　　　　　　　　０．４ｍｇ／ｍＬ
　ＢＳＡ　　　　　　　　　　　　　　　　　　　　０．１％（ｗ／ｖ）
　塩化ナトリウム　　　　　　　　　　　　　　　　０．１ｍｏｌ／Ｌ MES (pH 6.5) 50 mmol / L
Streptavidin-bound magnetic particles 0.4 mg / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
＜ビオチン結合ＤＵＰＡＮ－２抗体溶液＞
　Ｂｉｏｔｉｎ　Ｌａｂｅｌｉｎｇ　ｋｉｔ－ＮＨ２（株式会社同仁化学研究所製）を用いて、当該キットの取扱説明書に従い、ＤＵＰＡＮ－２抗体にビオチンが結合した、ビオチン結合ＤＵＰＡＮ－２抗体を作製した。得られたビオチン結合ＤＵＰＡＮ－２抗体を用いて、以下の組成のビオチン結合ＤＵＰＡＮ－２抗体溶液を調製した。 <Biotin-bound DUPAN-2 antibody solution>
Using Biotin Labeling kit-NH 2 (manufactured by Dojin Kagaku Kenkyusho Co., Ltd.), a biotin-bound DUPAN-2 antibody in which biotin was bound to the DUPAN-2 antibody was prepared according to the instruction manual of the kit. Using the obtained biotin-bound DUPAN-2 antibody, a biotin-bound DUPAN-2 antibody solution having the following composition was prepared.
　ＭＥＳ（ｐＨ６．５）　　　　　　　　　　　　　５０ｍｍｏｌ／Ｌ
　ビオチン結合ＤＵＰＡＮ－２抗体　　　　　　　　７．５μｇ／ｍＬ
　ＢＳＡ　　　　　　　　　　　　　　　　　　　　０．１％（ｗ／ｖ）
　塩化ナトリウム　　　　　　　　　　　　　　　　０．１ｍｏｌ／Ｌ MES (pH 6.5) 50 mmol / L
Biotin-conjugated DUPAN-2 antibody 7.5 μg / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
＜アルカリホスファターゼ標識ＤＵＰＡＮ－２抗体溶液＞
　Ａｌｋａｌｉｎｅ　Ｐｈｏｓｐｈａｔａｓｅ　Ｌａｂｅｌｉｎｇ　ｋｉｔ－ＳＨ２（株式会社同仁化学研究所製）を用いて、当該キットの取扱説明書に従い、ＤＵＰＡＮ－２抗体にアルカリホスファターゼが結合した、アルカリホスファターゼ標識ＤＵＰＡＮ－２抗体を作製した。得られたアルカリホスファターゼ標識ＤＵＰＡＮ－２抗体を用いて、以下の組成のアルカリホスファターゼ標識ＤＵＰＡＮ－２抗体溶液を調製した。 <Alkaline phosphatase-labeled DUPAN-2 antibody solution>
Alkaline Phosphatase Labeling kit-SH 2 (manufactured by Dojin Kagaku Kenkyusho Co., Ltd.) was used to prepare an alkaline phosphatase-labeled DUPAN-2 antibody in which alkaline phosphatase was bound to DUPAN-2 antibody according to the instruction manual of the kit. Using the obtained alkaline phosphatase-labeled DUPAN-2 antibody, an alkaline phosphatase-labeled DUPAN-2 antibody solution having the following composition was prepared.
　ＭＥＳ（ｐＨ６．５）　　　　　　　　　　　　　　５０ｍｍｏｌ／Ｌ
　アルカリホスファターゼ標識ＤＵＰＡＮ－２抗体　　０．３μｇ／ｍＬ
　ＢＳＡ　　　　　　　　　　　　　　　　　　　　　０．１％（ｗ／ｖ）
　塩化ナトリウム　　　　　　　　　　　　　　　　　０．１ｍｏｌ／Ｌ MES (pH 6.5) 50 mmol / L
Alkaline phosphatase-labeled DUPAN-2 antibody 0.3 μg / mL
BSA 0.1% (w / v)
Sodium chloride 0.1 mol / L
［実施例４］
＜試料中のＤＵＰＡＮ－２の測定方法２＞
　実施例２で調製した、３５０Ｕ／ｍＬの腹水由来ＤＵＰＡＮ－２抗原溶液、及び、３５０Ｕ／ｍＬのヒト膵癌培養細胞ＨＰＡＦ－ＩＩ由来のＤＵＰＡＮ－２抗原溶液を測定用試料とした。 [Example 4]
<Measurement method 2 of DUPAN-2 in the sample>
The 350 U / mL ascites-derived DUPAN-2 antigen solution prepared in Example 2 and the 350 U / mL human pancreatic cancer cultured cell HPAF-II-derived DUPAN-2 antigen solution were used as measurement samples.
　測定用試料１０μＬに、実施例３で調製した磁性粒子懸濁液、ビオチン結合ＤＵＰＡＮ－２抗体溶液、及び、アルカリホスファターゼ標識ＤＵＰＡＮ－２抗体溶液を各３０μＬ加えて攪拌し、３７℃で２０分間反応させた。磁性粒子を磁力で集めて、磁性粒子以外の反応溶液を除去すると共に、洗浄液［０．１％（ｗ／ｖ）Ｔｗｅｅｎ（登録商標）　２０を含有する５０ｍｍｏｌ／Ｌ　ＭＯＰＳ緩衝液（ｐＨ７．３５）］で磁性粒子を５回洗浄した。その後、９－（４－クロロフェニルチオホスホリルオキシメチリデン）－１０－メチルアクリダン・二ナトリウム塩を主成分とする発光基質液を１００μＬ加えて攪拌し、生じた発光量を測定した。
　腹水由来のＤＵＰＡＮ－２抗原溶液を測定したときの発光量を（Ａ）、ヒト膵癌培養細胞ＨＰＡＦ－ＩＩ由来のＤＵＰＡＮ－２抗原溶液を測定したときの発光量を（Ｂ）とした場合の（Ｂ）／（Ａ）値を算出した。当該値、及び、磁性粒子に結合したＤＵＰＡＮ－２抗体の結合量を表２に示す。 To 10 μL of the measurement sample, add 30 μL each of the magnetic particle suspension prepared in Example 3, the biotin-conjugated DUPAN-2 antibody solution, and the alkaline phosphatase-labeled DUPAN-2 antibody solution, stir, and react at 37 ° C. for 20 minutes. I let you. A 50 mmol / L MOPS buffer (pH 7.35) containing magnetic particles magnetically collected to remove reaction solutions other than the magnetic particles and containing a cleaning solution [0.1% (w / v) Tween® 20). ], The magnetic particles were washed 5 times. Then, 100 μL of a luminescent substrate solution containing 9- (4-chlorophenylthiophosphoryl oxymethylidene) -10-methylacridan / disodium salt as a main component was added and stirred, and the amount of luminescence generated was measured.
When the luminescence amount when the DUPAN-2 antigen solution derived from ascites was measured was (A), and the luminescence amount when the DUPAN-2 antigen solution derived from human pancreatic cancer cultured cells HPAF-II was measured was (B) ( B) / (A) values were calculated. Table 2 shows the value and the amount of DUPAN-2 antibody bound to the magnetic particles.
　また、比較例キットＡを用いて、実施例２記載の試料中のＤＵＰＡＮ－２の測定方法１に従い、腹水由来ＤＵＰＡＮ－２抗原溶液、及びＨＰＡＦ－ＩＩ由来のＤＵＰＡＮ－２抗原溶液を測定した。その後、実施例２記載の方法で算出した（Ｂ）／（Ａ）値、及びプレートに結合したＤＵＰＡＮ－２抗体の結合量を表２に示す。 Further, using Comparative Example Kit A, the DUPAN-2 antigen solution derived from ascites and the DUPAN-2 antigen solution derived from HPAF-II were measured according to the measurement method 1 of DUPAN-2 in the sample described in Example 2. Then, the (B) / (A) values calculated by the method described in Example 2 and the binding amount of the DUPAN-2 antibody bound to the plate are shown in Table 2.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　表２の結果より、キット３の（Ｂ）／（Ａ）値は１．３より小さかった。このような（Ｂ）／（Ａ）値を示すキット３は、比較例キットＡと比べて、ＤＵＰＡＮ－２抗体の結合量が多い傾向に有り、検出感度の高い測定キットであることが判明した。 From the results in Table 2, the (B) / (A) values of Kit 3 were smaller than 1.3. Kit 3 showing such (B) / (A) values tended to have a larger amount of DUPAN-2 antibody bound than Comparative Example Kit A, and was found to be a measurement kit with high detection sensitivity. ..
　本開示により、膵癌、胆道系癌などの癌診断に有効な、高感度且つ正確な試料中のＤＵＰＡＮ－２抗原の測定試薬及び測定キットが提供される。 The present disclosure provides a highly sensitive and accurate measurement reagent and measurement kit for DUPAN-2 antigen in a sample, which is effective for cancer diagnosis such as pancreatic cancer and biliary tract cancer.

Claims (14)

  1. 　試料中のＤＵＰＡＮ－２抗原の測定試薬であって、
    　不溶性担体と、ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、を含み、
    　前記測定試薬を用いて、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定した場合に、下記式（Ｘ）を満たす、試薬。
    　０．８≦（Ｂ）／（Ａ）≦１．２　（Ｘ）
    （式中、（Ａ）は、腹水由来のＤＵＰＡＮ－２抗原の測定値、（Ｂ）は、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値を意味し、ここで、腹水由来のＤＵＰＡＮ－２抗原とヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原との抗原濃度比は、１：１である。）     A reagent for measuring the DUPAN-2 antigen in a sample.
    It comprises an insoluble carrier and an antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
    A reagent that satisfies the following formula (X) when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the measurement reagent.
    0.8 ≤ (B) / (A) ≤ 1.2 (X)
    (In the formula, (A) means the measured value of DUPAN-2 antigen derived from ascites, and (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites. The antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
  2. 　前記腹水由来のＤＵＰＡＮ－２抗原及び前記ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定する方法が、
    （１）前記腹水由来のＤＵＰＡＮ－２抗原又は前記ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原に、前記不溶性担体と、前記ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、を反応させ、
    　前記不溶性担体上に、前記ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原と、を含む免疫複合体を生成させる工程；及び
    （２）工程（１）で生成された免疫複合体を測定する工程、
    を含む方法である、請求項１に記載の試薬。     A method for measuring the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from the cultured human pancreatic cancer cell HPAF is
    (1) The insoluble carrier and an antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof are reacted with the DUPAN-2 antigen derived from ascites or the DUPAN-2 antigen derived from the human pancreatic cancer cultured cell HPAF.
    A step of producing an immune complex containing the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen and the DUPAN-2 antigen on the insoluble carrier; and (2) produced in step (1). The process of measuring immune complexes,
    The reagent according to claim 1, which is a method comprising the above.
  3. 　前記ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片が、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片、及び、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片である、請求項１又は２に記載の試薬。 Claimed that the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen is a first antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen and a second antibody or an antibody fragment thereof that binds to the DUPAN-2 antigen. Item 2. The reagent according to Item 1 or 2.
  4. 　前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片が前記不溶性担体に結合している、請求項３に記載の試薬。 The reagent according to claim 3, wherein the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof is bound to the insoluble carrier.
  5. 　前記不溶性担体には２つの親和性物質のうちの片方が結合しており、前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片には２つの親和性物質のうちのもう一方が結合している、請求項３又は４に記載の試薬。 One of the two affinity substances is bound to the insoluble carrier, and the other of the two affinity substances is bound to the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen. The reagent according to claim 3 or 4.
  6. 　前記腹水由来のＤＵＰＡＮ－２抗原の濃度、及び前記ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の濃度が４０Ｕ／ｍＬ～４００Ｕ／ｍＬである、請求項１～５のいずれか１項に記載の試薬。 The one according to any one of claims 1 to 5, wherein the concentration of the DUPAN-2 antigen derived from the ascites and the concentration of the DUPAN-2 antigen derived from the human pancreatic cancer cultured cell HPAF are 40 U / mL to 400 U / mL. reagent.
  7. 　試料中のＤＵＰＡＮ－２抗原の測定キットであって、
    　水性媒体を含む第１試薬、並びに、不溶性担体、及びＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片を含む第２試薬を含み、
    　前記測定キットを用いて、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定した場合に、下記式（Ｘ）を満たす、キット。
    　０．８≦（Ｂ）／（Ａ）≦１．２　（Ｘ）
    （式中、（Ａ）は、腹水由来のＤＵＰＡＮ－２抗原の測定値、（Ｂ）は、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値を意味し、ここで、腹水由来のＤＵＰＡＮ－２抗原とヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原との抗原濃度比は、１：１である。）     A measurement kit for DUPAN-2 antigen in a sample.
    It comprises a first reagent comprising an aqueous medium and a second reagent comprising an insoluble carrier and an antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
    A kit that satisfies the following formula (X) when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the measurement kit.
    0.8 ≤ (B) / (A) ≤ 1.2 (X)
    (In the formula, (A) means the measured value of DUPAN-2 antigen derived from ascites, and (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites. The antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
  8. 　腹水由来のＤＵＰＡＮ－２抗原及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定する方法が、
    （１）腹水由来のＤＵＰＡＮ－２抗原又はヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原に、前記不溶性担体と、前記ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、を反応させ、前記不溶性担体上に、前記ＤＵＰＡＮ－２抗原に結合する抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原と、を含む免疫複合体を生成させる工程；及び
    （２）工程（１）で生成された免疫複合体を測定する工程、
    を含む方法である、請求項７に記載のキット。     A method for measuring DUPAN-2 antigen derived from ascites and DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF is
    (1) The insoluble carrier is reacted with the DUPAN-2 antigen derived from ascites or the DUPAN-2 antigen derived from the cultured human pancreatic cancer cell HPAF, and the antibody or antibody fragment thereof that binds to the DUPAN-2 antigen is reacted with the insoluble. A step of producing an immune complex containing the DUPAN-2 antigen or an antibody fragment thereof and the DUPAN-2 antigen on a carrier; and (2) the immune complex produced in step (1). The process of measuring the body,
    7. The kit according to claim 7, which is a method comprising the above.
  9. 　試料中のＤＵＰＡＮ－２抗原の測定キットであって、
    　不溶性担体、及びＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片を含む第１試薬、並びに、ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片を含む第２試薬を含み、
    　前記測定キットを用いて、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定した場合に、下記式（Ｘ）を満たす、キット。
    　０．８≦（Ｂ）／（Ａ）≦１．２　（Ｘ）
    （式中、（Ａ）は、腹水由来のＤＵＰＡＮ－２抗原の測定値、（Ｂ）は、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値を意味し、ここで、腹水由来のＤＵＰＡＮ－２抗原とヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原との抗原濃度比は、１：１である。）     A measurement kit for DUPAN-2 antigen in a sample.
    It comprises an insoluble carrier, a first reagent containing a first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, and a second reagent that contains a second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen.
    A kit that satisfies the following formula (X) when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the measurement kit.
    0.8 ≤ (B) / (A) ≤ 1.2 (X)
    (In the formula, (A) means the measured value of DUPAN-2 antigen derived from ascites, and (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites. The antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
  10. 　試料中のＤＵＰＡＮ－２抗原の測定キットであって、
    　不溶性担体を含む第１試薬、ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片を含む第２試薬、及びＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片を含む第３試薬を含み、
    　前記測定キットを用いて、腹水由来のＤＵＰＡＮ－２抗原、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定した場合に、下記式（Ｘ）を満たす、キット。
    　０．８≦（Ｂ）／（Ａ）≦１．２　（Ｘ）
    （式中、（Ａ）は、腹水由来のＤＵＰＡＮ－２抗原の測定値、（Ｂ）は、ヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の測定値を意味し、ここで、腹水由来のＤＵＰＡＮ－２抗原とヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原との抗原濃度比は、１：１である。）     A measurement kit for DUPAN-2 antigen in a sample.
    Includes a first reagent containing an insoluble carrier, a second reagent containing a first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, and a third reagent that contains a second antibody or antibody fragment thereof that binds to the DUPAN-2 antigen. ,
    A kit that satisfies the following formula (X) when the DUPAN-2 antigen derived from ascites and the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF are measured using the measurement kit.
    0.8 ≤ (B) / (A) ≤ 1.2 (X)
    (In the formula, (A) means the measured value of DUPAN-2 antigen derived from ascites, and (B) means the measured value of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF, and here, DUPAN derived from ascites. The antigen concentration ratio between the -2 antigen and the DUPAN-2 antigen derived from cultured human pancreatic cancer HPAF is 1: 1).
  11. 　腹水由来のＤＵＰＡＮ－２抗原及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原をそれぞれ測定する方法が、
    （１）腹水由来のＤＵＰＡＮ－２抗原又はヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原に、前記不溶性担体と、前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片と、を反応させ、前記不溶性担体上に、前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片と、前記ＤＵＰＡＮ－２抗原と、前記ＤＵＰＡＮ－２抗原に結合する第２抗体又はその抗体断片と、を含む免疫複合体を生成させる工程；及び
    （２）工程（１）で生成された免疫複合体を測定する工程、
    を含む方法である、請求項９又は１０に記載のキット。     A method for measuring DUPAN-2 antigen derived from ascites and DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF is
    (1) The insoluble carrier, the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof, and the DUPAN-2 to the DUPAN-2 antigen derived from ascites or the DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF. The first antibody or antibody fragment thereof that binds to the DUPAN-2 antigen, the DUPAN-2 antigen, and the DUPAN- 2 A step of generating an immune complex containing a second antibody or an antibody fragment thereof that binds to an antigen; and (2) a step of measuring the immune complex produced in step (1).
    The kit according to claim 9 or 10, wherein the method comprises.
  12. 　前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片が前記不溶性担体に結合している、請求項９又は１１に記載のキット。 The kit according to claim 9 or 11, wherein the first antibody that binds to the DUPAN-2 antigen or an antibody fragment thereof is bound to the insoluble carrier.
  13. 　前記不溶性担体には２つの親和性物質のうちの片方が結合しており、前記ＤＵＰＡＮ－２抗原に結合する第１抗体又はその抗体断片には２つの親和性物質のうちのもう一方が結合している、請求項９～１１のいずれか１項に記載のキット。 One of the two affinity substances is bound to the insoluble carrier, and the other of the two affinity substances is bound to the first antibody or the antibody fragment thereof that binds to the DUPAN-2 antigen. The kit according to any one of claims 9 to 11.
  14. 　腹水由来のＤＵＰＡＮ－２抗原の濃度、及びヒト膵癌培養細胞ＨＰＡＦ由来のＤＵＰＡＮ－２抗原の濃度が４０Ｕ／ｍＬ～４００Ｕ／ｍＬである、請求項７～１３のいずれか１項に記載のキット。 The kit according to any one of claims 7 to 13, wherein the concentration of DUPAN-2 antigen derived from ascites and the concentration of DUPAN-2 antigen derived from human pancreatic cancer cultured cell HPAF are 40 U / mL to 400 U / mL.
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