JP7807250B2 - DUPAN-2 antigen measurement method, measurement reagent and measurement kit - Google Patents

Description

The present invention relates to a method, reagent, and kit for measuring DUPAN-2 antigen.

The monoclonal antibody DUPAN-2 was produced using the human pancreatic cancer cultured cell line HPAF as an immunogen, and has been reported to react with a mucin-like protein (antigen) produced at high rates by pancreatic cancer cells (Non-Patent Documents 1 and 2). This antigen is called the DUPAN-2 antigen and is used as a cancer marker for pancreatic cancer, biliary tract cancer, and other cancers.

Methods reported for measuring DUPAN-2 antigen include competition radioimmunoassay (Non-Patent Document 1) and enzyme-linked immunosorbent assay (Patent Document 1).

International Publication No. 2019/189874

Metzgar et al., “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 Sakurabayashi et al., "Study on pancreatic cancer-associated glycoprotein antigen, DU-PAN-2 I. Basic studies in enzyme immunoassay and distribution of measured values in healthy subjects," Clinical Pathology, 1986, Vol. 34, pp. 705-710

When measuring DUPAN-2 antigen in a sample, the concentration of DUPAN-2 antigen in the sample is determined based on the measured value of a standard substance. Standard substances used for measuring DUPAN-2 antigen include DUPAN-2 antigen derived from ascites of human pancreatic cancer patients and DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF. However, when different types of standard substances are measured, the measured values sometimes differ even though the standard substances have the same concentration. Therefore, the concentration of DUPAN-2 antigen in a sample sometimes varies depending on the type of standard substance used. Therefore, there has been a need for a measurement method and a measurement reagent that can eliminate or reduce the difference in measured values when different types of standard substances are measured, and accurately measure DUPAN-2 antigen in a sample.
An object of the present invention is to provide a measurement method, a measurement reagent, and a measurement kit that can accurately measure DUPAN-2 antigen in a sample.

As a result of extensive research to solve the above-mentioned problems, the inventors discovered that by mixing the first antibody that recognizes the DUPAN-2 antigen and the second antibody that recognizes the DUPAN-2 antigen in a predetermined ratio when reacting the DUPAN-2 antigen in a sample with an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen, it is possible to eliminate or reduce the difference in measurement values when measuring different types of standard substances, thereby enabling accurate measurement of the DUPAN-2 antigen in a sample, and thus completed the invention disclosed herein.

That is, in one aspect, the present disclosure provides the following.
[1] A method for detecting DUPAN-2 antigen comprising preparing a mixture containing an aqueous medium, a DUPAN-2 antigen in a sample, an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen;
In the mixture, the insoluble carrier and a first antibody that recognizes the DUPAN-2 antigen are bound to each other,
a method for measuring DUPAN-2 antigen in a sample, wherein the amount of the first antibody that recognizes the DUPAN-2 antigen in the mixed solution is 3 to 100 times by weight greater than the amount of the second antibody that recognizes the DUPAN-2 antigen.

[2] (1) reacting, in an aqueous medium, an insoluble carrier with a DUPAN-2 antigen in a sample, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen, to form an immune complex on the insoluble carrier, the immune complex consisting of the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen; and (2) measuring the immune complex formed in the step (1).
Including,
In step (1), the insoluble carrier is bound to a first antibody that recognizes the DUPAN-2 antigen;
A method for measuring DUPAN-2 antigen in a sample, wherein in step (1), the amount of the first antibody that recognizes the DUPAN-2 antigen is 3 to 100 times by weight the amount of the second antibody that recognizes the DUPAN-2 antigen.

[3] (1) reacting an insoluble carrier and a first antibody that recognizes the DUPAN-2 antigen with the DUPAN-2 antigen in a sample in an aqueous medium to form an immune complex 1 on the insoluble carrier, the immune complex 1 comprising the first antibody that recognizes the DUPAN-2 antigen and the DUPAN-2 antigen;
(2) reacting a second antibody that recognizes the DUPAN-2 antigen with the immune complex 1 produced in step (1) in an aqueous medium to produce an immune complex 2 on the insoluble carrier, the immune complex 2 consisting of the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen; and (3) measuring the immune complex 2 produced in step (2).
Including,
In step (1), the insoluble carrier is bound to a first antibody that recognizes the DUPAN-2 antigen;
A method for measuring DUPAN-2 antigen in a sample, wherein in step (2), the amount of the first antibody that recognizes the DUPAN-2 antigen is 3 to 100 times by weight the amount of the second antibody that recognizes the DUPAN-2 antigen.

[4] (1) adding an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen to a sample containing the DUPAN-2 antigen;
(2) reacting the DUPAN-2 antigen with the insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen in an aqueous medium to form an immune complex on the insoluble carrier, the immune complex consisting of the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen; and (3) measuring the immune complex formed in step (2).
Including,
In step (2), a first antibody that recognizes the DUPAN-2 antigen is bound to the insoluble carrier;
A method for measuring DUPAN-2 antigen in a sample, wherein in step (1), the amount of the first antibody that recognizes the DUPAN-2 antigen added is 3 to 100 times by weight the amount of the second antibody that recognizes the DUPAN-2 antigen added.

[5] (1) adding an insoluble carrier and a first antibody that recognizes the DUPAN-2 antigen to a sample containing the DUPAN-2 antigen;
(2) reacting the insoluble carrier and a first antibody that recognizes the DUPAN-2 antigen with the DUPAN-2 antigen in the sample in an aqueous medium to form an immune complex 1 on the insoluble carrier, the immune complex 1 consisting of the first antibody that recognizes the DUPAN-2 antigen and the DUPAN-2 antigen;
(3) adding a second antibody that recognizes the DUPAN-2 antigen to the immune complex 1 produced in step (2);
(4) reacting a second antibody that recognizes the DUPAN-2 antigen with the immune complex 1 produced in step (2) in an aqueous medium to produce an immune complex 2 on the insoluble carrier, the immune complex 2 comprising the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen; and (5) measuring the immune complex 2 produced in step (4).
Including,
In step (2), a first antibody that recognizes the DUPAN-2 antigen is bound to the insoluble carrier;
A method for measuring DUPAN-2 antigen in a sample, wherein the amount of a first antibody that recognizes the DUPAN-2 antigen added in step (1) is 3 to 100 times (by weight) the amount of a second antibody that recognizes the DUPAN-2 antigen added in step (3).
[6] The method according to any one of [1] to [5], wherein the insoluble carrier and the first antibody that recognizes the DUPAN-2 antigen can be bound via two affinity substances, one of the two affinity substances being bound to the insoluble carrier, and the other of the two affinity substances being bound to the first antibody that recognizes the DUPAN-2 antigen.
[7] The method according to any one of [1] to [6], wherein a label is bound to the second antibody that recognizes the DUPAN-2 antigen.
[8] The method according to [7], wherein the label is alkaline phosphatase.

[9] A method for detecting a DUPAN-2 antigen comprising: an insoluble carrier; a first antibody that recognizes the DUPAN-2 antigen; and a second antibody that recognizes the DUPAN-2 antigen;
the insoluble carrier and the first antibody that recognizes the DUPAN-2 antigen can be bound to each other via two affinity substances;
one of two affinity substances is bound to the insoluble carrier, and the other of the two affinity substances is bound to the first antibody that recognizes the DUPAN-2 antigen;
A reagent for measuring DUPAN-2 antigen in a sample, wherein the amount of a first antibody that recognizes the DUPAN-2 antigen is 3 to 100 times by weight the amount of a second antibody that recognizes the DUPAN-2 antigen.

[10] A method for detecting a DUPAN-2 antigen comprising: a first reagent containing an insoluble carrier; a second reagent containing a first antibody that recognizes the DUPAN-2 antigen; and a third reagent containing a second antibody that recognizes the DUPAN-2 antigen;
the insoluble carrier and the first antibody that recognizes the DUPAN-2 antigen can be bound to each other via two affinity substances;
one of two affinity substances is bound to the insoluble carrier, and the other of the two affinity substances is bound to the first antibody that recognizes the DUPAN-2 antigen;
A kit for measuring DUPAN-2 antigen in a sample, wherein the amount of a first antibody that recognizes the DUPAN-2 antigen in the second reagent is 3 to 100 times by weight greater than the amount of a second antibody that recognizes the DUPAN-2 antigen in the third reagent.

According to the present disclosure, even when different types of standard substances are measured, the differences in the measurement values of each standard substance are eliminated or reduced. Therefore, it is possible to provide a method, reagent, and kit that can accurately measure DUPAN-2 antigen in a sample.

Hereinafter, embodiments for carrying out the present disclosure will be described in detail. Note that the embodiments described below are examples of typical embodiments of the present disclosure, and should not be construed as narrowing the scope of the disclosure.
Note that numerical ranges indicated using "to" indicate ranges that include the numerical values before and after "to" as the minimum and maximum values, respectively. Furthermore, when multiple substances corresponding to each component are present in the solution, the amount of each component in the solution means the total amount of the multiple substances present in the reagent, unless otherwise specified.
In the present disclosure, "% (w/v)" means the percentage of mass (g) based on volume (100 mL).

In this disclosure, when an antibody is expressed as "binding" to or "reacting with" the DUPAN-2 antigen, or as an antibody "recognizing" the DUPAN-2 antigen, these terms include the meanings commonly used in the field of this disclosure and are all used synonymously. Methods for confirming the "binding" of an antibody to the DUPAN-2 antigen include competitive ELISA, sandwich ELISA, and methods utilizing the principle of surface plasmon resonance, all of which are well known to those skilled in the art.

"Aqueous medium"
Examples of aqueous media in the present disclosure include deionized water, distilled water, and buffer solutions, with buffer solutions being preferred. Examples of buffering agents used to prepare buffer solutions include acetate buffer solutions and Good's buffer solutions. One type of buffering agent may be used alone, or two or more types may be used in combination.
The aqueous medium may contain salts, metal ions, sugars, proteins, surfactants, and the like.
Examples of salts include lithium chloride, sodium chloride, potassium chloride, calcium chloride, and magnesium chloride. Examples of metal ions include sodium ions, magnesium ions, manganese ions, and zinc ions. Examples of sugars include mannitol and sorbitol. Examples of proteins include bovine serum albumin (hereinafter referred to as BSA). Examples of surfactants include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants.

"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 examples thereof include any one or more biological samples selected from the group consisting of whole blood, plasma, serum, urine, ascites, cerebrospinal fluid, saliva, amniotic fluid, urine, sweat, and pancreatic juice, and preferably includes whole blood, plasma, serum, ascites, etc. Furthermore, the sample may be a cell lysate of cultured human pancreatic cancer cells HPAF, or a culture supernatant obtained when cultured human pancreatic cancer cells HPAF.
The sample according to the present disclosure may be a DUPAN-2 antigen contained in an aqueous medium, such as a DUPAN-2 antigen diluted with phosphate-buffered saline (10 mmol/L phosphate buffer containing 0.15 mol/L sodium chloride, pH 7.2, hereinafter referred to as PBS). The DUPAN-2 antigen contained in the aqueous medium may be a DUPAN-2 antigen purified from the biological sample, the cell lysate, or the culture supernatant.

"Insoluble carrier"
In the present disclosure, the insoluble carrier is not particularly limited as long as it enables the measurement method for DUPAN-2 antigen of the present disclosure or is usable in the measurement reagent or measurement kit of the present disclosure, and examples thereof include a slide glass, an ELISA plate (microtiter plate), beads, latex particles, magnetic particles, a filter, a film, a membrane, etc. Examples of materials for the insoluble carrier include those made of an inorganic substrate, a polysaccharide substrate, a polymer substrate, etc., such as glass, silicon, ceramic, cellulose, nitrocellulose, nylon, polycarbonate, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, and polyurethane.

"DUPAN-2 antigen"
The DUPAN-2 antigen in the present disclosure refers to a mucin-like protein (glycoprotein) to which the monoclonal antibody "DUPAN-2" binds (Metzgar et al., "Antigens of Human Pancreatic Adenocarcinoma Cells Defined by Murine Monoclonal Antibodies", CANCER RESEARCH (1982), 42:601-608), a sugar chain to which the monoclonal antibody "DUPAN-2" binds, or a peptide containing the sugar chain. Specifically, the DUPAN-2 antigen refers to a sialyl Lewis C sugar chain to which the monoclonal antibody "DUPAN-2" has been reported to bind (Kawa et al., "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), a protein having such a sugar chain, or a peptide having such a sugar chain. The DUPAN-2 antigen may be a DUPAN-2 antigen contained in the ascites of a human pancreatic cancer patient or in the culture supernatant of the human pancreatic cancer cultured cell line HPAF. Human pancreatic cancer cultured cells HPAF can be obtained from the American Type Culture Collection (ATCC) under the name "HPAF-II."

"Antibody that recognizes DUPAN-2 antigen"
In the present disclosure, an antibody that recognizes the DUPAN-2 antigen (a first antibody that recognizes the DUPAN-2 antigen, a second antibody that recognizes the DUPAN-2 antigen) refers to an antibody that recognizes the sialyl Lewis C of the DUPAN-2 antigen and is capable of binding to the DUPAN-2 antigen.

An antibody that recognizes the DUPAN-2 antigen may be a mouse antibody, rat antibody, rabbit antibody, human antibody, humanized antibody, or chimeric antibody, or may be an antibody derived from another species. Furthermore, an antibody that recognizes the DUPAN-2 antigen may be of any class of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

Antibodies that recognize the DUPAN-2 antigen include monoclonal antibodies, polyclonal antibodies, dimers, multimers, etc., with monoclonal antibodies being preferred from the standpoints of homogeneity and stability.

In the present disclosure, the antibody that recognizes the DUPAN-2 antigen may be an antibody fragment. An antibody fragment refers to a portion of an antibody that recognizes the DUPAN-2 antigen, which comprises the variable domain of the antibody or at least the antigen-binding region. Examples of antibody fragments in the present disclosure include Fab, Fab', F(ab') ₂ , Fv fragments, linear antibodies, single-chain antibodies (scFv), sc(Fv) ₂ , Fab ₃ , domain antibodies (dAb), diabodies, triabodies, tetrabodies, and minibodies. An "Fv fragment" is the smallest antibody fragment and comprises the complete antigen-recognition region and antigen-binding region.

The first antibody that recognizes the DUPAN-2 antigen of the present disclosure and the second antibody that recognizes the DUPAN-2 antigen may bind to the same or different sites on the DUPAN-2 antigen. Furthermore, the first antibody that recognizes the DUPAN-2 antigen and the second antibody that recognizes the DUPAN-2 antigen may be the same antibody or different antibodies.

Antibodies that recognize the DUPAN-2 antigen in the present disclosure can be obtained by conventional antibody production methods using the DUPAN-2 antigen or cancer cells that produce the DUPAN-2 antigen as an immunogen. For example, they can be obtained by immunizing an animal with the immunogen to obtain antibody-producing cells, fusing these cells with myeloma cells to produce cells (hybridomas), and then obtaining monoclonal antibodies produced by the hybridomas. Examples of monoclonal antibodies that recognize the DUPAN-2 antigen include the aforementioned monoclonal antibody "DUPAN-2" (Metzgar et al., "Antigens of Human Pancreatic Adenocarcinoma Cells Defined by Murine Monoclonal Antibodies," CANCER RESEARCH (1982), 42:601-608). In this disclosure, the monoclonal antibody DUPAN-2 is also referred to as the "DUPAN-2 antibody."

"Method for measuring DUPAN-2 antigen in a sample"
One embodiment of the method for measuring DUPAN-2 antigen in a sample according to the present disclosure is a method for measuring DUPAN-2 antigen, which comprises preparing a mixed solution containing an aqueous medium, the DUPAN-2 antigen in the sample, an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen.

The DUPAN-2 antigen in the sample can be measured by preparing a mixed solution containing an aqueous medium, the DUPAN-2 antigen in the sample, an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen, and then performing the following steps.
(1) a step of reacting a DUPAN-2 antigen with an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen to form an immune complex on the insoluble carrier, the immune complex consisting of the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen; and (2) a step of measuring the immune complex formed in step (1).

In the step (1), the insoluble carrier and the first antibody that recognizes the DUPAN-2 antigen are bound to each other, and the first antibody that recognizes the DUPAN-2 antigen is generated and bound to the insoluble carrier.
The method for binding the first antibody that recognizes the DUPAN-2 antigen to the insoluble carrier is not particularly limited as long as it enables the method of measuring the DUPAN-2 antigen of the present disclosure, and examples thereof include physical adsorption, chemical bonding, etc. Examples of physical adsorption include electrostatic bonding, hydrogen bonding, hydrophobic bonding, etc. Examples of chemical bonding include covalent bonding, coordinate bonding, etc.

The first antibody that recognizes the DUPAN-2 antigen may be bound directly or indirectly to an insoluble carrier using the aforementioned physical adsorption and/or chemical binding. Indirect binding methods include a method in which the first antibody that recognizes the DUPAN-2 antigen is bound to an insoluble carrier using the specific binding between a pair of affinity substances such as biotin and avidin (avidin, streptavidin, neutravidin, etc.), and a method in which the first antibody is bound to an insoluble carrier by a covalent bond via a linker.

When a pair of affinity substances is used, a first antibody that recognizes the DUPAN-2 antigen bound to one of the affinity substances (A) of the pair can be bound to an insoluble carrier by binding the first antibody to the DUPAN-2 antigen bound to one of the affinity substances (a) of the pair, thereby producing a first antibody that recognizes the DUPAN-2 antigen bound to an insoluble carrier.

Examples of the combination of Aa include the following combinations.
- Combinations of biotin and avidins (avidin, neutravidin, streptavidin, etc.);
- Combinations of avidins (avidin, neutravidin, streptavidin, etc.) with biotin;
A combination of the Fc region of a first antibody that recognizes the DUPAN-2 antigen and an antibody that binds to the Fc region.

Examples of linkers include molecules capable of covalently bonding a functional group on the surface of the insoluble carrier to a functional group possessed by the first antibody that recognizes the DUPAN-2 antigen. For example, a molecule that simultaneously possesses a first reactive group capable of reacting with a functional group possessed by the first antibody that recognizes the DUPAN-2 antigen and a second reactive group capable of reacting with a functional group on the surface of the insoluble carrier, wherein the first reactive group and the second reactive group are different groups, is preferably used. Examples of functional groups possessed by the first antibody that recognizes the DUPAN-2 antigen and functional groups possessed on the surface of the insoluble carrier include carboxyl groups, amino groups, glycidyl groups, sulfhydryl groups, hydroxyl groups, amide groups, imino groups, N-hydroxysuccinyl groups, and maleimide groups. Examples of reactive groups in linkers include allyl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, maleimide, N-hydroxysuccinimide ester, pentafluorophenyl (PFP) ester, psoralen, pyridyl disulfide, and vinyl sulfone.

After the step (2), the concentration of the DUPAN-2 antigen in the sample can also be determined by carrying out the following steps (3) and (4).
(3) performing the steps (1) and (2) using a DUPAN-2 antigen of known concentration as a sample to prepare a calibration curve showing the relationship between the DUPAN-2 antigen concentration and the measured value;
(4) A step of determining the concentration of DUPAN-2 antigen in the sample from the calibration curve prepared in step (3) and the measured value obtained by the measurement in step (2).

Examples of DUPAN-2 antigen of known concentration include DUPAN-2 antigen derived from ascites of a human pancreatic cancer patient (hereinafter simply referred to as "DUPAN-2 antigen derived from ascites") or a DUPAN-2 antigen solution obtained by diluting DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF with PBS. Multiple concentrations of DUPAN-2 antigen of known concentration may be prepared.

The concentration of DUPAN-2 antigen in a DUPAN-2 antigen of known concentration is not particularly limited as long as it is a concentration that enables measurement of the DUPAN-2 antigen, and may be 10 U/mL or more, 20 U/mL or more, 30 U/mL or more, 40 U/mL or more, 50 U/mL or more, 60 U/mL or more, 70 U/mL or more, 80 U/mL or more, 90 U/mL or more, or 100 U/mL or more. Furthermore, the concentration of DUPAN-2 antigen in the DUPAN-2 antigen of known concentration may be 200 U/mL or less, 300 U/mL or less, 350 U/mL or less, 400 U/mL or less, 500 U/mL or less, 600 U/mL or less, 700 U/mL or less, 800 U/mL or less, 900 U/mL or less, 1000 U/mL or less, 1100 U/mL or less, 1200 U/mL or less, 1400 U/mL or less, 1600 U/mL or less, 1800 U/mL or less, 2000 U/mL or less, 3000 U/mL or less, 4000 U/mL or less, 5000 U/mL or less, 6000 U/mL or less, 7000 U/mL or less, 8000 U/mL or less, 9000 U/mL or less, 10000 U/mL or less, or 15000 U/mL or less.
The concentration unit of DUPAN-2 antigen, "U/mL," is defined as "the concentration of DUPAN-2 antigen contained in serum of a specific pancreatic cancer patient measured by competitive radioimmunoassay, is 1000 U/mL" (Metzgar et al., "Detection of a pancreatic cancer-associated antigen (DU-PAN-2 antigen) in serum and associates of patients with adenocarcinoma," Proceedings of the National Academy of Sciences of the United States of America, pp. 111-115, 2013). America, 1984, Vol. 81, pp. 5242-5246).

The above values can be freely combined, for example, 10 U/mL to 10,000 U/mL, 20 U/mL to 5,000 U/mL, 30 U/mL to 1,600 U/mL, 40 U/mL to 400 U/mL, or 40 U/mL to 350 U/mL.
In the present disclosure, any numerical range obtained by combining any numerical value selected from the examples of upper and lower limits for a certain concentration is also considered to be exemplified in the present disclosure. The same applies to other numerical values such as weight multiple, molar equivalent multiple, temperature, and time.

A washing step can also be performed before step (2). The washing solution used to wash the insoluble carrier is not particularly limited as long as it allows for measurement of the DUPAN-2 antigen, and examples include PBS and PBS containing a surfactant. Examples of surfactants include nonionic surfactants such as Tween (registered trademark) 20.

When preparing the mixture, the DUPAN-2 antigen in the sample, the insoluble carrier, the first antibody that recognizes the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen may be mixed in any order. For example, a first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen in a sample, and a second antibody that recognizes the DUPAN-2 antigen may be mixed with an insoluble carrier; a first antibody that recognizes the DUPAN-2 antigen may be mixed with the DUPAN-2 antigen in a sample, an insoluble carrier, and a second antibody that recognizes the DUPAN-2 antigen; a second antibody that recognizes the DUPAN-2 antigen may be mixed with the DUPAN-2 antigen in a sample, an insoluble carrier, and the first antibody that recognizes the DUPAN-2 antigen; or a DUPAN-2 antigen in a sample may be mixed with an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen.

In the mixture, the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 times or more, 2.7 times or more, 2.8 times or more, 2.9 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, or 9 times or more by weight of the amount of the second antibody that recognizes the DUPAN-2 antigen. Furthermore, in the mixed solution, the amount of the first antibody that recognizes the DUPAN-2 antigen may be 10 times or less, 12 times or less, 13 times or less, 14 times or less, 15 times or less, 20 times or less, 25 times or less, 30 times or less, 35 times or less, 40 times or less, 45 times or less, 50 times or less, 60 times or less, 70 times or less, 80 times or less, 90 times or less, 100 times or less, 150 times or less, or 200 times or less by weight of the amount of the second antibody that recognizes the DUPAN-2 antigen.

The above values can be freely combined. For example, in the mixed solution, the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 to 200 times by weight, 2.8 to 150 times by weight, 3 to 100 times by weight, 3 to 30 times by weight, 5 to 30 times by weight, or 5 to 10 times by weight.

In the mixture, the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 molar equivalents or more, 2.7 molar equivalents or more, 2.8 molar equivalents or more, 2.9 molar equivalents or more, 3 molar equivalents or more, 4 molar equivalents or more, 5 molar equivalents or more, 6 molar equivalents or more, 7 molar equivalents or more, 8 molar equivalents or more, or 9 molar equivalents or more of the amount of the second antibody that recognizes the DUPAN-2 antigen. Furthermore, in the mixed solution, the amount of the first antibody that recognizes the DUPAN-2 antigen may be 10 molar equivalents or less, 12 molar equivalents or less, 13 molar equivalents or less, 14 molar equivalents or less, 15 molar equivalents or less, 20 molar equivalents or less, 25 molar equivalents or less, 30 molar equivalents or less, 35 molar equivalents or less, 40 molar equivalents or less, 45 molar equivalents or less, 50 molar equivalents or less, 60 molar equivalents or less, 70 molar equivalents or less, 80 molar equivalents or less, 90 molar equivalents or less, 100 molar equivalents or less, 150 molar equivalents or less, or 200 molar equivalents or less of the amount of the second antibody that recognizes the DUPAN-2 antigen.

The above values can be freely combined. For example, in the mixed solution, the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 to 200 molar equivalents, 2.8 to 150 molar equivalents, 3 to 100 molar equivalents, 3 to 30 molar equivalents, 5 to 30 molar equivalents, or 5 to 10 molar equivalents, relative to the amount of the second antibody that recognizes the DUPAN-2 antigen.

The concentration of the insoluble carrier in the mixture is not particularly limited as long as it is a concentration that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 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, 0.4 mg/mL or more, or 0.5 mg/mL or more. Furthermore, the concentration of the insoluble carrier in the mixture may be 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, 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 above values can be freely combined. For example, the concentration of the insoluble carrier in the mixed 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/mL.

The concentration of the first antibody that binds to the DUPAN-2 antigen in the mixture is not particularly limited as long as it is a concentration that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 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, 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 μg/mL or more, 9 μg/mL or more, or 10 μg/mL or more. Furthermore, the concentration of the first antibody that binds to the DUPAN-2 antigen in the mixture may be 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 or less.

The above values can be freely combined. For example, the concentration of the first antibody that binds to the DUPAN-2 antigen in the mixture may be 0.01 μg/mL to 100 μg/mL, 0.1 μg/mL to 30 μg/mL, 6 μg/mL to 30 μg/mL, or 10 μg/mL to 20 μg/mL.

The concentration of the second antibody that binds to the DUPAN-2 antigen in the mixture is not particularly limited as long as it is a concentration that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 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, 0.1 μg/mL or more, 0.2 μg/mL or more, 0.3 μg/mL or more, 0.33 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, or 0.9 μg/mL or more. Furthermore, the concentration of the second antibody that binds to the DUPAN-2 antigen in the mixture may be 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 or less, 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/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.

The above values can be freely combined. For example, the concentration of the second antibody that binds to the DUPAN-2 antigen in the mixed solution may be 0.01 μg/mL to 100 μg/mL, 0.03 μg/mL to 30 μg/mL, 0.1 μg/mL to 20 μg/mL, or 0.1 μg/mL to 1 μg/mL.
In this specification, the amount or concentration of a second antibody that binds to a DUPAN-2 antigen means not only the amount or concentration of the second antibody itself, but also the amount or concentration of a labeled antibody formed by binding a label described below to the second antibody.

The concentration of DUPAN-2 antigen in the mixture is not particularly limited as long as it allows measurement of the DUPAN-2 antigen, and may be 1 U/mL to 1000 U/mL, 2 U/mL to 500 U/mL, 3 U/mL to 160 U/mL, 4 U/mL to 160 U/mL, 4 U/mL to 40 U/mL, or 6 U/mL to 35 U/mL.

Another aspect of the method for measuring DUPAN-2 antigen in a sample according to the present disclosure includes:
(1) reacting an insoluble carrier with a DUPAN-2 antigen in a sample in an aqueous medium, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen, to form an immune complex on the insoluble carrier, the immune complex consisting of the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen; and (2) measuring the immune complex formed in step (1).
The method for measuring DUPAN-2 antigen in a sample comprises:

Furthermore, after step (2), the concentration of DUPAN-2 antigen in the sample can also be determined by carrying out the following steps (3) and (4).
(3) performing the steps (1) and (2) using a DUPAN-2 antigen of known concentration as a sample to prepare a calibration curve showing the relationship between the DUPAN-2 antigen concentration and the measured value;
(4) A step of determining the concentration of DUPAN-2 antigen in the sample from the calibration curve prepared in step (3) and the measured value obtained by the measurement in step (2).

Examples of DUPAN-2 antigens of known concentrations include DUPAN-2 antigen solutions obtained by diluting DUPAN-2 antigen derived from ascites fluid or DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF with PBS. A plurality of concentrations of DUPAN-2 antigens of known concentrations may be prepared.
The concentration of DUPAN-2 antigen in the known concentration of DUPAN-2 antigen is not particularly limited as long as it is a concentration that allows measurement of the DUPAN-2 antigen, and examples thereof include the concentrations mentioned above.

A washing step can also be performed before step (2). The washing solution used to wash the insoluble carrier is not particularly limited as long as it allows for measurement of the DUPAN-2 antigen, and examples include PBS and PBS containing a surfactant. Examples of surfactants include nonionic surfactants such as Tween 20.

<Step (1)>
Step (1) is a step of reacting a DUPAN-2 antigen in a sample with an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen in an aqueous medium, to form an immune complex on the insoluble carrier, the immune complex consisting of the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen.

In step (1), the reaction between the DUPAN-2 antigen, the insoluble carrier, the first antibody that recognizes the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen is not particularly limited, as long as an immune complex consisting of the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen is formed on the insoluble carrier. For example, the DUPAN-2 antigen may be reacted with the insoluble carrier and the first antibody that recognizes the DUPAN-2 antigen to form an immune complex of the first antibody that recognizes the DUPAN-2 antigen and the DUPAN-2 antigen on the insoluble carrier, and then the second antibody that recognizes the DUPAN-2 antigen may be reacted therewith. Alternatively, the DUPAN-2 antigen may be reacted with the insoluble carrier, the first antibody that recognizes the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen, all mixed together.

When an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen are mixed and reacted with the DUPAN-2 antigen, the order in which the insoluble carrier, the first antibody that recognizes the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen are added may be any order. For example, the insoluble carrier may be added first, followed by the first antibody that recognizes the DUPAN-2 antigen and the second antibody that recognizes the DUPAN-2 antigen; the first antibody that recognizes the DUPAN-2 antigen may be added first, followed by the insoluble carrier and the second antibody that recognizes the DUPAN-2 antigen; or the second antibody that recognizes the DUPAN-2 antigen may be added first, followed by the insoluble carrier and the first antibody that recognizes the DUPAN-2 antigen.

Step (1) may be carried out separately into the following steps (1-1) and (1-2), and then step (2) may be carried out.
(1-1) a step of reacting an insoluble carrier and a first antibody that recognizes the DUPAN-2 antigen with the DUPAN-2 antigen in a sample in an aqueous medium to form an immune complex 1 on the insoluble carrier, the immune complex 1 comprising the first antibody that recognizes the DUPAN-2 antigen and the DUPAN-2 antigen;
(1-2) reacting a second antibody that recognizes the DUPAN-2 antigen with the immune complex 1 produced in step (1) in an aqueous medium to produce an immune complex 2 on the insoluble carrier, the immune complex 2 comprising the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen; and (2) measuring the immune complex 2 produced in step (1-2).

A washing step can be performed between step (1-1) and step (1-2). A washing step can also be performed between step (1-2) and step (2). The washing solution used to wash the insoluble carrier is not particularly limited as long as it allows measurement of the DUPAN-2 antigen, and examples include PBS and PBS containing a surfactant. Examples of surfactants include nonionic surfactants such as Tween (registered trademark) 20.

In the steps (1) and (1-1), the insoluble carrier and the first antibody that recognizes the DUPAN-2 antigen are bound to each other, thereby producing the first antibody that recognizes the DUPAN-2 antigen bound to the insoluble carrier.
The method for binding the first antibody that recognizes the DUPAN-2 antigen to the insoluble carrier is not particularly limited as long as it enables the method for measuring the DUPAN-2 antigen of the present disclosure, and examples thereof include the binding methods described above.

The first antibody that recognizes the DUPAN-2 antigen may be bound directly or indirectly to an insoluble carrier using the aforementioned physical adsorption and/or chemical binding. Indirect binding methods include a method in which the first antibody that recognizes the DUPAN-2 antigen is bound to an insoluble carrier using the specific binding of the aforementioned pair of affinity substances, and a method in which the first antibody is bound to an insoluble carrier by a covalent bond via a linker.

When a pair of affinity substances is used, a first antibody that recognizes the DUPAN-2 antigen bound to one of the affinity substances (A) of the pair can be bound to an insoluble carrier to produce a first antibody that recognizes the DUPAN-2 antigen bound to an insoluble carrier.
Examples of the combination of Aa include the combinations described above.
Examples of the linker include the molecules described above.

In steps (1), (1-1) and (1-2), the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 times or more, 2.7 times or more, 2.8 times or more, 2.9 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, or 9 times or more by weight of the amount of the second antibody that recognizes the DUPAN-2 antigen. Furthermore, in steps (1), (1-1), and (1-2), the amount of the first antibody that recognizes the DUPAN-2 antigen may be 10 times or less by weight, 12 times or less by weight, 13 times or less by weight, 14 times or less by weight, 15 times or less by weight, 20 times or less by weight, 25 times or less by weight, 30 times or less by weight, 35 times or less by weight, 40 times or less by weight, 45 times or less by weight, 50 times or less by weight, 60 times or less by weight, 70 times or less by weight, 80 times or less by weight, 90 times or less by weight, 100 times or less by weight, 150 times or less by weight, or 200 times or less by weight of the amount of the second antibody that recognizes the DUPAN-2 antigen.

The above numerical values can be freely combined. For example, in steps (1), (1-1), and (1-2), the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 to 200 times by weight, 2.8 to 150 times by weight, 3 to 100 times by weight, 3 to 30 times by weight, 5 to 30 times by weight, or 5 to 10 times by weight.

In steps (1), (1-1), and (1-2), the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 molar equivalents or more, 2.7 molar equivalents or more, 2.8 molar equivalents or more, 2.9 molar equivalents or more, 3 molar equivalents or more, 4 molar equivalents or more, 5 molar equivalents or more, 6 molar equivalents or more, 7 molar equivalents or more, 8 molar equivalents or more, or 9 molar equivalents or more of the amount of the second antibody that recognizes the DUPAN-2 antigen. Furthermore, in steps (1), (1-1), and (1-2), the amount of the first antibody that recognizes the DUPAN-2 antigen may be 10 molar equivalents or less, 12 molar equivalents or less, 13 molar equivalents or less, 14 molar equivalents or less, 15 molar equivalents or less, 20 molar equivalents or less, 25 molar equivalents or less, 30 molar equivalents or less, 35 molar equivalents or less, 40 molar equivalents or less, 45 molar equivalents or less, 50 molar equivalents or less, 60 molar equivalents or less, 70 molar equivalents or less, 80 molar equivalents or less, 90 molar equivalents or less, 100 molar equivalents or less, 150 molar equivalents or less, or 200 molar equivalents or less of the amount of the second antibody that recognizes the DUPAN-2 antigen.

The above numerical values can be freely combined. For example, in steps (1), (1-1), and (1-2), the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 to 200 molar equivalents, 2.8 to 150 molar equivalents, 3 to 100 molar equivalents, 3 to 30 molar equivalents, 5 to 30 molar equivalents, or 5 to 10 molar equivalents, of the amount of the second antibody that recognizes the DUPAN-2 antigen.

The concentration of the insoluble carrier in step (1) and step (1-1) is not particularly limited as long as it is a concentration that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 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, 0.4 mg/mL or more, or 0.5 mg/mL or more. Furthermore, the concentration of the insoluble carrier in step (1) and step (1-1) may be 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, 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 above values can be freely combined. For example, the concentration of the insoluble carrier in steps (1) and (1-1) 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/mL.

The amount of the first antibody that recognizes the DUPAN-2 antigen in steps (1), (1-1), and (1-2) is not particularly limited as long as it is an amount that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 0.5 ng or more, 1 ng or more, 1.5 ng or more, 2 ng or more, 2.5 ng or more, 3 ng or more, 3.5 ng or more, 4 ng or more, 4.5 ng or more, 5 ng or more, 10 ng or more, 15 ng or more, 20 ng or more, 25 ng or more, 30 ng or more, 35 ng or more, 40 ng or more, 45 ng or more, 50 ng or more, 100 ng or more, 150 ng or more, 200 ng or more, 250 ng or more, 300 ng or more, 350 ng or more, 400 ng or more, 450 ng or more, or 500 ng or more. Furthermore, the amount of the first antibody that recognizes the DUPAN-2 antigen in steps (1), (1-1), and (1-2) may be 550 ng or less, 600 ng or less, 650 ng or less, 700 ng or less, 750 ng or less, 800 ng or less, 850 ng or less, 900 ng or less, 950 ng or less, 1 μg or less, 1.5 μg or less, 2 μg or less, 2.5 μg or less, or 5 μg or less.

The above values can be freely combined. For example, the amount of the first antibody that recognizes the DUPAN-2 antigen in steps (1), (1-1), and (1-2) may be 0.5 ng to 5 μg, 5 ng to 1.5 μg, 150 ng to 1 μg, 300 ng to 1 μg, or 500 ng to 1 μg.

The concentration of the first antibody that recognizes the DUPAN-2 antigen in steps (1), (1-1), and (1-2) is not particularly limited as long as it is a concentration that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 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, 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 μg/mL or more, 9 μg/mL or more, or 10 μg/mL or more. Furthermore, the concentration of the first antibody that binds to the DUPAN-2 antigen in steps (1), (1-1), and (1-2) may be 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 or less.

The above values can be freely combined. For example, the concentration of the first antibody that binds to the DUPAN-2 antigen in steps (1), (1-1), and (1-2) may be 0.01 μg/mL to 100 μg/mL, 0.1 μg/mL to 30 μg/mL, 6 μg/mL to 30 μg/mL, or 10 μg/mL to 20 μg/mL.

The amount of the second antibody that recognizes the DUPAN-2 antigen in steps (1) and (1-2) is not particularly limited as long as it is an amount that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 0.1 ng or more, 1 ng or more, 2 ng or more, 3 ng or more, 4 ng or more, 5 ng or more, 6 ng or more, 7 ng or more, 8 ng or more, 9 ng or more, 10 ng or more, 20 ng or more, 30 ng or more, 40 ng or more, 50 ng or more, 60 ng or more, 70 ng or more, 80 ng or more, or 90 ng or more. Furthermore, the amount of the second antibody that recognizes the DUPAN-2 antigen in steps (1) and (1-2) may be 100 ng or less, 200 ng or less, 300 ng or less, 400 ng or less, 500 ng or less, 600 ng or less, 700 ng or less, 800 ng or less, 900 ng or less, 1 μg or less, 1.1 μg or less, 1.2 μg or less, 1.3 μg or less, 1.4 μg or less, 1.5 μg or less, 1.6 μg or less, 1.7 μg or less, 1.8 μg or less, 1.9 μg or less, 2 μg or less, 3 μg or less, 4 μg or less, 5 μg or less, or 10 μg or less.

The above values can be freely combined. For example, the amount of the second antibody that recognizes the DUPAN-2 antigen in steps (1) and (1-2) may be 1 ng to 10 μg, 3 ng to 3 μg, 10 ng to 2 μg, or 10 ng to 100 ng.

The concentration of the second antibody that binds to the DUPAN-2 antigen in step (1) and step (1-2) is not particularly limited as long as it is a concentration that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 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, 0.1 μg/mL or more, 0.2 μg/mL or more, 0.3 μg/mL or more, 0.33 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, or 0.9 μg/mL or more. Furthermore, the concentration of the second antibody that binds to the DUPAN-2 antigen in steps (1) and (1-2) may be 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 or less, 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/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.

The above values can be freely combined. For example, the concentration of the second antibody that binds to the DUPAN-2 antigen in steps (1) and (1-2) may be 0.01 μg/mL to 100 μg/mL, 0.03 μg/mL to 30 μg/mL, 0.1 μg/mL to 20 μg/mL, or 0.1 μg/mL to 1 μg/mL.

The steps (1), (1-1) and (1-2) are preferably carried out in an aqueous medium, such as the aqueous medium described above.
Furthermore, the steps (1), (1-1) and (1-2) may contain the above-mentioned salts, metal ions, sugars, proteins, surfactants and the like.

The reaction temperature for steps (1), (1-1), and (1-2) is not particularly limited as long as it allows for measurement of the DUPAN-2 antigen, and is, for example, 0 to 50°C, preferably 4 to 45°C, and particularly preferably 20 to 40°C. Furthermore, the reaction time for steps (1), (1-1), and (1-2) is not particularly limited as long as it allows for measurement of the DUPAN-2 antigen, and is, for example, 5 minutes to 3 hours, and preferably 10 minutes to 2 hours.

<Step (2)>
In step (2), the immune complex produced in step (1) or immune complex 2 produced in step (1-2) is measured using the following method to determine the measured value of DUPAN-2 antigen in the sample.

[When a label is not bound to the second antibody that recognizes the DUPAN-2 antigen]
The immune complex or the immune complex 2 can be measured using a labeled third antibody (hereinafter referred to as the third antibody), in which a label is bound to an antibody that binds to the second antibody that recognizes the DUPAN-2 antigen. That is, the immune complex is reacted with the labeled third antibody to form a complex 3 between the immune complex or the immune complex 2 and the labeled third antibody, and the label of the complex 3 is measured by the method described below, thereby measuring the amount of the immune complex produced in step (1) or the amount of the immune complex 2 produced in step (1-2). Examples of the third antibody include an antibody that binds to the Fc region of the second antibody that recognizes the DUPAN-2 antigen.

The reaction temperature for the reaction between the labeled third antibody and the immune complex or immune complex 2 is not particularly limited as long as it allows for measurement of the DUPAN-2 antigen, and may be, for example, 0 to 50°C, 4 to 45°C, or 20 to 40°C. The reaction time for the reaction is not particularly limited as long as it allows for measurement of the DUPAN-2 antigen, and may be, for example, 1 minute to 4 hours, 5 minutes to 3 hours, or 10 minutes to 2 hours. The concentration of the labeled third antibody in the reaction solution is not particularly limited as long as it allows for measurement of the DUPAN-2 antigen, and may be, for example, 0.01 to 100 μg/mL, or 0.1 to 20 μg/mL.

[When a label is bound to the second antibody that recognizes the DUPAN-2 antigen]
Step (2) can be carried out by measuring the immune complex or the immune complex 2 using a label.
Methods for binding a label to a second antibody that recognizes the DUPAN-2 antigen are known in the technical field of immunoassays. For example, a label can be bound to an antibody via one or several amino acids, or via one or several amino acids and a linker. Various kits for binding a label to a protein are also commercially available.

Examples of labels include those used in conventional immunoassays, such as enzymes, radioisotopes, fluorescent substances, luminescent substances, DNA, RNA, coenzymes or substances that specifically bind to coenzymes (biotin, avidin), tags, substances that absorb in the ultraviolet to infrared range, chromogenic microparticles, fluorescent microparticles, metallic microparticles, magnetic substances, and substances that have properties as spin labeling agents.

Examples of enzymes include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase, etc. Examples of radioisotopes include ³ H, ¹⁴ C, ³⁵ S, ³² P, ¹²⁵ P, ¹³¹ I, etc. Examples of fluorescent substances include FITC (fluorescein isothiocyanate) and RITC (rhodamine B-isothiocyanate). Examples of luminescent substances include acridinium and its derivatives, ruthenium complex compounds, lophine, etc.

By detecting the signals generated from these labels, the immune complex or immune complex 2 can be measured.
The method for measuring the signal generated from the label may be appropriately selected depending on the label used.
When the label is a chromogenic substance, that is, a substance that absorbs light of a certain wavelength, the label can be measured by measuring absorbance using a spectrophotometer, a multiwell 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 multiwell 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 multiwell plate reader, or the like.
When the label is a radioisotope, the label can be measured by measuring the radioactivity using 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, by reacting the enzyme's substrate with the enzyme and measuring the product.

The measurement method according to the present disclosure may include the following addition step (A) prior to the step (1).
(A) A step of adding an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen to the DUPAN-2 antigen in a sample in an aqueous medium.

The measurement method according to the present disclosure may include the following addition step (A1) prior to the step (1-1).
(A1) A step of adding an insoluble carrier and a first antibody that recognizes the DUPAN-2 antigen to a sample containing the DUPAN-2 antigen.
The step (A1) may be a step of adding a sample containing the DUPAN-2 antigen to an insoluble carrier and a first antibody that recognizes the DUPAN-2 antigen.

The measurement method according to the present disclosure may include the following addition step (A2) prior to the step (1-2).
(A2) A step of adding a second antibody that recognizes the DUPAN-2 antigen to the immune complex 1 produced in the step (1-1).
The step (A2) may be a step of adding the immune complex 1 produced in the step (1-2) to a second antibody that recognizes the DUPAN-2 antigen.

In steps (A) and (A1), the amount of the first antibody that recognizes the DUPAN-2 antigen added may be 2.6 times or more by weight, 2.7 times or more by weight, 2.8 times or more by weight, 2.9 times or more by weight, 3 times or more by weight, 4 times or more by weight, 5 times or more by weight, 6 times or more by weight, 7 times or more by weight, 8 times or more by weight, or 9 times or more by weight of the amount of the second antibody that recognizes the DUPAN-2 antigen added. Furthermore, in steps (A) and (A1), the amount of the first antibody that recognizes the DUPAN-2 antigen added may be 10 times or less by weight, 12 times or less by weight, 13 times or less by weight, 14 times or less by weight, 15 times or less by weight, 20 times or less by weight, 25 times or less by weight, 30 times or less by weight, 35 times or less by weight, 40 times or less by weight, 45 times or less by weight, 50 times or less by weight, 60 times or less by weight, 70 times or less by weight, 80 times or less by weight, 90 times or less by weight, 100 times or less by weight, 150 times or less by weight, or 200 times or less by weight of the amount of the second antibody that recognizes the DUPAN-2 antigen added.

The above numerical values can be freely combined. For example, in steps (A) and (A1), the amount of the first antibody that recognizes the DUPAN-2 antigen added may be 2.6 to 200 times by weight, 2.8 to 150 times by weight, 3 to 100 times by weight, 3 to 30 times by weight, 5 to 30 times by weight, or 5 to 10 times by weight.

In steps (A) and (A1), the amount of the first antibody that recognizes the DUPAN-2 antigen added may be 2.6 molar equivalents or more, 2.7 molar equivalents or more, 2.8 molar equivalents or more, 2.9 molar equivalents or more, 3 molar equivalents or more, 4 molar equivalents or more, 5 molar equivalents or more, 6 molar equivalents or more, 7 molar equivalents or more, 8 molar equivalents or more, or 9 molar equivalents or more of the amount of the second antibody that recognizes the DUPAN-2 antigen added. Furthermore, in steps (A) and (A1), the amount of the first antibody that recognizes the DUPAN-2 antigen added may be 10 molar equivalents or less, 12 molar equivalents or less, 13 molar equivalents or less, 14 molar equivalents or less, 15 molar equivalents or less, 20 molar equivalents or less, 25 molar equivalents or less, 30 molar equivalents or less, 35 molar equivalents or less, 40 molar equivalents or less, 45 molar equivalents or less, 50 molar equivalents or less, 60 molar equivalents or less, 70 molar equivalents or less, 80 molar equivalents or less, 90 molar equivalents or less, 100 molar equivalents or less, 150 molar equivalents or less, or 200 molar equivalents or less of the amount of the second antibody that recognizes the DUPAN-2 antigen added.

The above numerical values can be freely combined. For example, in steps (A) and (A1), the amount of the first antibody that recognizes the DUPAN-2 antigen added may be 2.6 to 200 molar equivalents, 2.8 to 150 molar equivalents, 3 to 100 molar equivalents, 3 to 30 molar equivalents, 5 to 30 molar equivalents, or 5 to 10 molar equivalents, of the amount of the second antibody that recognizes the DUPAN-2 antigen added.

The amount of the first antibody that recognizes the DUPAN-2 antigen added in step (A) and step (A1) is not particularly limited as long as it enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 0.5 ng or more, 1 ng or more, 1.5 ng or more, 2 ng or more, 2.5 ng or more, 3 ng or more, 3.5 ng or more, 4 ng or more, 4.5 ng or more, 5 ng or more, 10 ng or more, 15 ng or more, 20 ng or more, 25 ng or more, 30 ng or more, 35 ng or more, 40 ng or more, 45 ng or more, 50 ng or more, 100 ng or more, 150 ng or more, 200 ng or more, 250 ng or more, 300 ng or more, 350 ng or more, 400 ng or more, 450 ng or more, or 500 ng or more. Furthermore, the amount of the first antibody that recognizes the DUPAN-2 antigen added in step (A) and step (A1) may be 550 ng or less, 600 ng or less, 650 ng or less, 700 ng or less, 750 ng or less, 800 ng or less, 850 ng or less, 900 ng or less, 950 ng or less, 1 μg or less, 1.5 μg or less, 2 μg or less, 2.5 μg or less, or 5 μg or less.

The above values can be freely combined. For example, the amount of the first antibody that recognizes the DUPAN-2 antigen added in step (A) and step (A1) may be 0.5 ng to 5 μg, 5 ng to 1.5 μg, 150 ng to 1 μg, 300 ng to 1 μg, or 500 ng to 1 μg.

The concentration of the first antibody that recognizes the DUPAN-2 antigen added in steps (A) and (A1) is not particularly limited as long as it is an addition concentration that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 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 or above, 0.2 μg/mL or above, 0.3 μg/mL or above, 0.4 μg/mL or above, 0.5 μg/mL or above, 0.6 μg/mL or above, 0.7 μg/mL or above, 0.8 μg/mL or above, 0.9 μg/mL or above, 1 μg/mL or above, 2 μg/mL or above, 3 μg/mL or above, 4 μg/mL or above, 5 μg/mL or above, 6 μg/mL or above, 7 μg/mL or above, 7.5 μg/mL or above, 8 μg/mL or above, 9 μg/mL or above, or 10 μg/mL or above. Furthermore, the concentration of the first antibody that binds to the DUPAN-2 antigen added in step (A) and step (A1) may be 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 or less.

The above values can be freely combined. For example, the concentration of the first antibody that binds to the DUPAN-2 antigen added in step (A) and step (A1) may be 0.01 μg/mL to 100 μg/mL, 0.1 μg/mL to 30 μg/mL, 6 μg/mL to 30 μg/mL, or 10 μg/mL to 20 μg/mL.

The amount of the second antibody that recognizes the DUPAN-2 antigen added in steps (A) and (A2) is not particularly limited as long as it enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 0.1 ng or more, 1 ng or more, 2 ng or more, 3 ng or more, 4 ng or more, 5 ng or more, 6 ng or more, 7 ng or more, 8 ng or more, 9 ng or more, 10 ng or more, 20 ng or more, 30 ng or more, 40 ng or more, 50 ng or more, 60 ng or more, 70 ng or more, 80 ng or more, or 90 ng or more. Furthermore, the amount of the second antibody that recognizes the DUPAN-2 antigen added in steps (A) and (A2) may be 100 ng or less, 200 ng or less, 300 ng or less, 400 ng or less, 500 ng or less, 600 ng or less, 700 ng or less, 800 ng or less, 900 ng or less, 1 μg or less, 1.1 μg or less, 1.2 μg or less, 1.3 μg or less, 1.4 μg or less, 1.5 μg or less, 1.6 μg or less, 1.7 μg or less, 1.8 μg or less, 1.9 μg or less, 2 μg or less, 3 μg or less, 4 μg or less, 5 μg or less, or 10 μg or less.

The above values can be freely combined. For example, the amount of the second antibody that recognizes the DUPAN-2 antigen added in steps (A) and (A2) may be 1 ng to 10 μg, 3 ng to 3 μg, 10 ng to 2 μg, or 10 ng to 100 ng.

The concentration of the second antibody that binds to the DUPAN-2 antigen added in step (A) and step (A2) is not particularly limited, as long as it is a concentration that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 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, 0.1 μg/mL or more, 0.2 μg/mL or more, 0.3 μg/mL or more, 0.33 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, or 0.9 μg/mL or more. Furthermore, the concentration of the second antibody that binds to the DUPAN-2 antigen added in steps (A) and (A2) may be 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 or less, 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/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.

The above values can be freely combined. For example, the concentration of the second antibody that binds to the DUPAN-2 antigen added in steps (A) and (A2) may be 0.01 μg/mL to 100 μg/mL, 0.03 μg/mL to 30 μg/mL, 0.1 μg/mL to 20 μg/mL, or 0.1 μg/mL to 1 μg/mL.

"Reagent for measuring DUPAN-2 antigen in a sample"
The measurement reagent for DUPAN-2 antigen in a sample of the present disclosure is a reagent used in the measurement method for DUPAN-2 antigen in a sample of the present disclosure, and one embodiment of the measurement reagent is a measurement reagent for DUPAN-2 antigen in a sample, comprising an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen.

In the measurement reagent disclosed herein, the insoluble carrier and the first antibody that recognizes the DUPAN-2 antigen can be bound via two affinity substances. Methods for binding the first antibody that recognizes the DUPAN-2 antigen to the insoluble carrier include, for example, the binding methods described above.

The first antibody that recognizes the DUPAN-2 antigen may be bound directly or indirectly to an insoluble carrier using the aforementioned physical adsorption and/or chemical binding. Indirect binding methods include a method in which the first antibody that recognizes the DUPAN-2 antigen is bound to an insoluble carrier using the specific binding of the aforementioned pair of affinity substances, and a method in which the first antibody is bound to an insoluble carrier by a covalent bond via a linker.

When a pair of affinity substances is used, a first antibody that recognizes the DUPAN-2 antigen bound to one of the affinity substances (A) of the pair can be bound to an insoluble carrier to produce a first antibody that recognizes the DUPAN-2 antigen bound to an insoluble carrier.
Examples of the combination of Aa include the combinations described above.
Examples of the linker include the molecules described above.

In the DUPAN-2 antigen measurement reagent of the present disclosure, the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 times or more by weight, 2.7 times or more by weight, 2.8 times or more by weight, 2.9 times or more by weight, 3 times or more by weight, 4 times or more by weight, 5 times or more by weight, 6 times or more by weight, 7 times or more by weight, 8 times or more by weight, or 9 times or more by weight of the amount of the second antibody that recognizes the DUPAN-2 antigen. Furthermore, in the DUPAN-2 antigen measurement reagent of the present disclosure, the amount of the first antibody that recognizes the DUPAN-2 antigen may be 10 times or less by weight, 12 times or less by weight, 13 times or less by weight, 14 times or less by weight, 15 times or less by weight, 20 times or less by weight, 25 times or less by weight, 30 times or less by weight, 35 times or less by weight, 40 times or less by weight, 45 times or less by weight, 50 times or less by weight, 60 times or less by weight, 70 times or less by weight, 80 times or less by weight, 90 times or less by weight, 100 times or less by weight, 150 times or less by weight, or 200 times or less by weight of the amount of the second antibody that recognizes the DUPAN-2 antigen.

The above values can be freely combined. For example, in the DUPAN-2 antigen measurement reagent of the present disclosure, the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 to 200 times by weight, 2.8 to 150 times by weight, 3 to 100 times by weight, 3 to 30 times by weight, 5 to 30 times by weight, or 5 to 10 times by weight.

In the DUPAN-2 antigen measurement reagent of the present disclosure, the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 molar equivalents or more, 2.7 molar equivalents or more, 2.8 molar equivalents or more, 2.9 molar equivalents or more, 3 molar equivalents or more, 4 molar equivalents or more, 5 molar equivalents or more, 6 molar equivalents or more, 7 molar equivalents or more, 8 molar equivalents or more, or 9 molar equivalents or more of the amount of the second antibody that recognizes the DUPAN-2 antigen. Furthermore, in the DUPAN-2 antigen measurement reagent of the present disclosure, the amount of the first antibody that recognizes the DUPAN-2 antigen may be 10 molar equivalents or less, 12 molar equivalents or less, 13 molar equivalents or less, 14 molar equivalents or less, 15 molar equivalents or less, 20 molar equivalents or less, 25 molar equivalents or less, 30 molar equivalents or less, 35 molar equivalents or less, 40 molar equivalents or less, 45 molar equivalents or less, 50 molar equivalents or less, 60 molar equivalents or less, 70 molar equivalents or less, 80 molar equivalents or less, 90 molar equivalents or less, 100 molar equivalents or less, 150 molar equivalents or less, or 200 molar equivalents or less of the amount of the second antibody that recognizes the DUPAN-2 antigen.

The above values can be freely combined. For example, in the DUPAN-2 antigen measurement reagent of the present disclosure, the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 to 200 molar equivalents, 2.8 to 150 molar equivalents, 3 to 100 molar equivalents, 3 to 30 molar equivalents, 5 to 30 molar equivalents, or 5 to 10 molar equivalents, of the amount of the second antibody that recognizes the DUPAN-2 antigen.

The concentration of the insoluble carrier in the measurement reagent for the DUPAN-2 antigen of the present disclosure is not particularly limited as long as it is a concentration that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 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, 0.4 mg/mL or more, or 0.5 mg/mL or more. Furthermore, the concentration of the insoluble carrier in the measurement reagent may be 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, 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 above values can be freely combined. For example, the concentration of the insoluble carrier in the measurement reagent 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/mL.

The concentration of the first antibody that recognizes the DUPAN-2 antigen in the DUPAN-2 antigen measurement reagent of the present disclosure is not particularly limited as long as it is a concentration that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 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 or above, 0.2 μg/mL or above, 0.3 μg/mL or above, 0.4 μg/mL or above, 0.5 μg/mL or above, 0.6 μg/mL or above, 0.7 μg/mL or above, 0.8 μg/mL or above, 0.9 μg/mL or above, 1 μg/mL or above, 2 μg/mL or above, 3 μg/mL or above, 4 μg/mL or above, 5 μg/mL or above, 6 μg/mL or above, 7 μg/mL or above, 7.5 μg/mL or above, 8 μg/mL or above, 9 μg/mL or above, or 10 μg/mL or above. Furthermore, in the DUPAN-2 antigen measurement reagent of the present disclosure, the concentration of the first antibody that binds to the DUPAN-2 antigen may be 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 or less.

The above values can be freely combined. For example, in the DUPAN-2 antigen measurement reagent of the present disclosure, the concentration of the first antibody that binds to the DUPAN-2 antigen may be 0.01 μg/mL to 100 μg/mL, 0.1 μg/mL to 30 μg/mL, 6 μg/mL to 30 μg/mL, or 10 μg/mL to 20 μg/mL.

The concentration of the second antibody that binds to the DUPAN-2 antigen in the measurement reagent for the DUPAN-2 antigen of the present disclosure is not particularly limited, as long as it is a concentration that enables measurement of the DUPAN-2 antigen of the present disclosure, and may be 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, 0.1 μg/mL or more, 0.2 μg/mL or more, 0.3 μg/mL or more, 0.33 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, or 0.9 μg/mL or more. Furthermore, in the DUPAN-2 antigen measurement reagent of the present disclosure, the concentration of the second antibody that binds to the DUPAN-2 antigen may be 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 or less, 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/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.

The above values can be freely combined. For example, in the DUPAN-2 antigen measurement reagent of the present disclosure, the concentration of the second antibody that binds to the DUPAN-2 antigen may be 0.01 μg/mL to 100 μg/mL, 0.03 μg/mL to 30 μg/mL, 0.1 μg/mL to 20 μg/mL, or 0.1 μg/mL to 1 μg/mL.

The measurement reagent of the present disclosure may be in a freeze-dried state or in a liquid state. When a freeze-dried measurement reagent is used, it is dissolved in an aqueous medium before measurement to form a liquid, which is then used for measurement.
In the liquid test reagent, at least one antibody selected from the group consisting of an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen is dissolved in an aqueous medium.

Examples of aqueous media include the aqueous media described above. The aqueous media may contain the salts, metal ions, sugars, proteins, surfactants, etc. described above.

"DUPAN-2 antigen measurement kit in samples"
The reagent for measuring DUPAN-2 antigen in a sample according to the present disclosure can be in the form of a kit from the viewpoint of storage, transportation, distribution, etc.
Preferred embodiments of the kit for measuring DUPAN-2 antigen in a sample according to the present disclosure are exemplified below. Note that in the following embodiments, the insoluble carrier, the first antibody that recognizes DUPAN-2 antigen, and the second antibody that recognizes DUPAN-2 antigen may be, for example, the insoluble carrier, the first antibody that recognizes DUPAN-2 antigen, and the second antibody that recognizes DUPAN-2 antigen described above.

[Measurement kit (1)]
a first reagent comprising an insoluble carrier;
a second reagent comprising a first antibody that recognizes the DUPAN-2 antigen; and a third reagent comprising a second antibody that recognizes the DUPAN-2 antigen.
Including, a measurement kit.

[Measurement kit (2)]
a first reagent comprising an insoluble carrier; and a second reagent comprising a first antibody recognizing the DUPAN-2 antigen and a second antibody recognizing the DUPAN-2 antigen.
Including, a measurement kit.

In the measurement kit of the present disclosure, the insoluble carrier and the first antibody that recognizes the DUPAN-2 antigen can be bound via two affinity substances. Methods for binding the first antibody that recognizes the DUPAN-2 antigen to the insoluble carrier include, for example, the binding methods described above.

The first antibody that recognizes the DUPAN-2 antigen may be bound directly or indirectly to an insoluble carrier using the aforementioned physical adsorption and/or chemical binding. Indirect binding methods include a method in which the first antibody that recognizes the DUPAN-2 antigen is bound to an insoluble carrier using the specific binding of the aforementioned pair of affinity substances, and a method in which the first antibody is bound to an insoluble carrier by a covalent bond via a linker.

When a pair of affinity substances is used, a first antibody that recognizes the DUPAN-2 antigen bound to one of the affinity substances (A) of the pair can be bound to an insoluble carrier to produce a first antibody that recognizes the DUPAN-2 antigen bound to an insoluble carrier.
Examples of the combination of Aa include the combinations described above.
Examples of the linker include the molecules described above.

The measurement kits (1) and (2) may include a reagent containing an aqueous medium as a reagent for diluting the sample. Examples of aqueous media include the aqueous media described above. The aqueous medium may contain the salts, metal ions, sugars, proteins, surfactants, etc. described above. The measurement kits (1) and (2) may also include the washing solution described above, DUPAN-2 antigen of known concentrations as a standard substance, and an instruction manual describing the measurement method of the present disclosure.

In the measurement kit (1), the amount of the first antibody that recognizes the DUPAN-2 antigen in the second reagent may be 2.6 times by weight or more, 2.7 times by weight or more, 2.8 times by weight or more, 2.9 times by weight or more, 3 times by weight or more, 4 times by weight or more, 5 times by weight or more, 6 times by weight or more, 7 times by weight or more, 8 times by weight or more, or 9 times by weight or more of the amount of the second antibody that recognizes the DUPAN-2 antigen in the third reagent. Furthermore, in the measurement kit (1), the amount of the first antibody that recognizes the DUPAN-2 antigen in the second reagent may be 10 times or less by weight, 12 times or less by weight, 13 times or less by weight, 14 times or less by weight, 15 times or less by weight, 20 times or less by weight, 25 times or less by weight, 30 times or less by weight, 35 times or less by weight, 40 times or less by weight, 45 times or less by weight, 50 times or less by weight, 60 times or less by weight, 70 times or less by weight, 80 times or less by weight, 90 times or less by weight, 100 times or less by weight, 150 times or less by weight, or 200 times or less by weight of the amount of the second antibody that recognizes the DUPAN-2 antigen in the third reagent.

The above values can be freely combined. For example, in the measurement kit (1), the amount of the first antibody that recognizes the DUPAN-2 antigen in the second reagent may be 2.6 to 200 times by weight, 2.8 to 150 times by weight, 3 to 100 times by weight, 3 to 30 times by weight, 5 to 30 times by weight, or 5 to 10 times by weight.

In the measurement kit (1), the amount of the first antibody that recognizes the DUPAN-2 antigen in the second reagent may be 2.6 molar equivalents or more, 2.7 molar equivalents or more, 2.8 molar equivalents or more, 2.9 molar equivalents or more, 3 molar equivalents or more, 4 molar equivalents or more, 5 molar equivalents or more, 6 molar equivalents or more, 7 molar equivalents or more, 8 molar equivalents or more, or 9 molar equivalents or more of the amount of the second antibody that recognizes the DUPAN-2 antigen in the third reagent. Furthermore, in the measurement kit (1), the amount of the first antibody that recognizes the DUPAN-2 antigen in the second reagent may be 10 molar equivalents or less, 12 molar equivalents or less, 13 molar equivalents or less, 14 molar equivalents or less, 15 molar equivalents or less, 20 molar equivalents or less, 25 molar equivalents or less, 30 molar equivalents or less, 35 molar equivalents or less, 40 molar equivalents or less, 45 molar equivalents or less, 50 molar equivalents or less, 60 molar equivalents or less, 70 molar equivalents or less, 80 molar equivalents or less, 90 molar equivalents or less, 100 molar equivalents or less, 150 molar equivalents or less, or 200 molar equivalents or less, of the amount of the second antibody that recognizes the DUPAN-2 antigen in the third reagent.

The above values can be freely combined. For example, in the measurement kit (1), the amount of the first antibody that recognizes the DUPAN-2 antigen in the second reagent may be 2.6 to 200 molar equivalents, 2.8 to 150 molar equivalents, 3 to 100 molar equivalents, 3 to 30 molar equivalents, 5 to 30 molar equivalents, or 5 to 10 molar equivalents, of the amount of the second antibody that recognizes the DUPAN-2 antigen in the third reagent.

In the second reagent of the measurement kit (2), the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 times or more by weight, 2.7 times or more by weight, 2.8 times or more by weight, 2.9 times or more by weight, 3 times or more by weight, 4 times or more by weight, 5 times or more by weight, 6 times or more by weight, 7 times or more by weight, 8 times or more by weight, or 9 times or more by weight of the amount of the second antibody that recognizes the DUPAN-2 antigen. Furthermore, in the second reagent of the measurement kit (2), the amount of the first antibody that recognizes the DUPAN-2 antigen may be 10 times or less by weight, 12 times or less by weight, 13 times or less by weight, 14 times or less by weight, 15 times or less by weight, 20 times or less by weight, 25 times or less by weight, 30 times or less by weight, 35 times or less by weight, 40 times or less by weight, 45 times or less by weight, 50 times or less by weight, 60 times or less by weight, 70 times or less by weight, 80 times or less by weight, 90 times or less by weight, 100 times or less by weight, 150 times or less by weight, or 200 times or less by weight of the amount of the second antibody that recognizes the DUPAN-2 antigen.

The above values can be freely combined. For example, in the second reagent of the measurement kit (2), the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 to 200 times by weight, 2.8 to 150 times by weight, 3 to 100 times by weight, 3 to 30 times by weight, 5 to 30 times by weight, or 5 to 10 times by weight.

In the second reagent of the measurement kit (2), the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 molar equivalents or more, 2.7 molar equivalents or more, 2.8 molar equivalents or more, 2.9 molar equivalents or more, 3 molar equivalents or more, 4 molar equivalents or more, 5 molar equivalents or more, 6 molar equivalents or more, 7 molar equivalents or more, 8 molar equivalents or more, or 9 molar equivalents or more of the amount of the second antibody that recognizes the DUPAN-2 antigen. Furthermore, in the second reagent of the measurement kit (2), the amount of the first antibody that recognizes the DUPAN-2 antigen may be 10 molar equivalents or less, 12 molar equivalents or less, 13 molar equivalents or less, 14 molar equivalents or less, 15 molar equivalents or less, 20 molar equivalents or less, 25 molar equivalents or less, 30 molar equivalents or less, 35 molar equivalents or less, 40 molar equivalents or less, 45 molar equivalents or less, 50 molar equivalents or less, 60 molar equivalents or less, 70 molar equivalents or less, 80 molar equivalents or less, 90 molar equivalents or less, 100 molar equivalents or less, 150 molar equivalents or less, or 200 molar equivalents or less of the amount of the second antibody that recognizes the DUPAN-2 antigen.

The above values can be freely combined. For example, in the second reagent of the measurement kit (2), the amount of the first antibody that recognizes the DUPAN-2 antigen may be 2.6 to 200 molar equivalents, 2.8 to 150 molar equivalents, 3 to 100 molar equivalents, 3 to 30 molar equivalents, 5 to 30 molar equivalents, or 5 to 10 molar equivalents, of the amount of the second antibody that recognizes the DUPAN-2 antigen.

The concentration of the insoluble carrier in the first reagent of the measurement kit (1) and the measurement kit (2) may be 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 0.5 mg/mL or more. Furthermore, the concentration of the insoluble carrier in the first reagent of the measurement kit (1) and the measurement kit (2) may be 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, 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 above values can be freely combined. For example, the concentration of the insoluble carrier in the first reagent of measurement kit (1) and measurement kit (2) 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 first antibody that recognizes the DUPAN-2 antigen in the second reagent of the measurement kit (1) and the measurement kit (2) is 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. It may be 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 μg/mL or more, 9 μg/mL or more, or 10 μg/mL or more. Furthermore, the concentration of the first antibody that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (1) and the measurement kit (2) may be 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 or less.

The above values can be freely combined. For example, the concentration of the first antibody that binds to the DUPAN-2 antigen in the second reagent of the measurement kit (1) and the measurement kit (2) may be 0.01 μg/mL to 100 μg/mL, 0.1 μg/mL to 30 μg/mL, 6 μg/mL to 30 μg/mL, or 10 μg/mL to 20 μg/mL.

The concentration of the second antibody that recognizes the DUPAN-2 antigen in the third reagent of the measurement kit (1) and the second reagent of the measurement kit (2) may be 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, 0.1 μg/mL or more, 0.2 μg/mL or more, 0.3 μg/mL or more, 0.33 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, or 0.9 μg/mL or more. Furthermore, the concentration of the second antibody that binds to the DUPAN-2 antigen in the third reagent of Measurement Kit (1) and the second reagent of Measurement Kit (2) may be 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 or less, 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/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.

The above values can be freely combined. For example, the concentration of the second antibody that binds to the DUPAN-2 antigen in the third reagent of measurement kit (1) and the second reagent of measurement kit (2) may be 0.01 μg/mL to 100 μg/mL, 0.03 μg/mL to 30 μg/mL, 0.1 μg/mL to 20 μg/mL, or 0.1 μg/mL to 1 μg/mL.

The constituent reagents of the measurement kit of the present disclosure may be in a lyophilized state or in a liquid form.
When the reagents constituting the assay kit are in a freeze-dried state, they are dissolved in an aqueous medium before assay to form a liquid, which is then used for assay. Examples of the aqueous medium include the aqueous media described above. The aqueous medium may contain the aforementioned salts, metal ions, sugars, proteins, surfactants, etc.

When the constituent reagents of the measurement kit are in liquid form, at least one selected from the group consisting of an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen is dissolved in an aqueous medium. Examples of aqueous media include those mentioned above. The aqueous medium may also contain the aforementioned salts, metal ions, sugars, proteins, surfactants, etc.

Examples of DUPAN-2 antigen of known concentrations as a standard substance include DUPAN-2 antigen solutions prepared by diluting DUPAN-2 antigen derived from ascites fluid or DUPAN-2 antigen derived from cultured human pancreatic cancer cells HPAF with PBS. The kit of the present disclosure may also contain multiple DUPAN-2 antigens of known concentrations.

The concentration of DUPAN-2 antigen in a known concentration of DUPAN-2 antigen used as a standard substance can be, for example, the concentrations mentioned above.

The present disclosure will be explained in more detail below using examples, but these are not intended to limit the scope of the present disclosure in any way. In the following examples, reagents from the following manufacturers were used.

The following reagents were used: disodium hydrogen phosphate (anhydrous) (Kanto Chemical), sodium dihydrogen phosphate (anhydrous) (Kanto Chemical), trishydroxymethylaminomethane (Kanto Chemical), sodium chloride (Fujifilm Wako Pure Chemical Industries, Ltd.), MES (Dojindo Laboratories, Ltd.), MOPS (Dojindo Laboratories, Ltd.), NP-40 (Sigma-Aldrich), Tween 20 (Kanto Chemical), Minimum Essential Medium Eagle (Sigma-Aldrich), fetal bovine serum (Thermo Fisher Scientific), bovine serum albumin (BSA) (Bovogen), and the "specimen diluent" component of "Determiner DUPAN-2 N" (Hitachi Chemical Diagnostics Systems Co., Ltd.).

[Example 1]
A DUPAN-2 antigen assay kit 1 was prepared, which contained the following magnetic particle suspension, biotin-conjugated DUPAN-2 antibody solution, and alkaline phosphatase-labeled DUPAN-2 antibody solution.

<Magnetic particle suspension>
Commercially available streptavidin-bound magnetic particles [Dynabeads MyOne Streptavidin T1 (manufactured by Thermo Fisher Scientific)] were used as the magnetic particles to prepare a magnetic particle suspension having the following composition.

MES (pH6.5) 50 mmol/L
Streptavidin-coupled magnetic particles 0.4 mg/mL
BSA 0.1% (w/v)
Sodium chloride 0.1 mol/L

<Biotin-conjugated DUPAN-2 antibody solution: first antibody>
Biotin-conjugated DUPAN-2 antibody was prepared by conjugating biotin to the DUPAN-2 antibody using Biotin Labeling kit-NH ₂ (Dojindo Laboratories, Inc.) according to the kit's instructions. Using the obtained biotin-conjugated DUPAN-2 antibody, a biotin-conjugated DUPAN-2 antibody solution having the following composition was prepared.

MES (pH6.5) 50 mmol/L
Biotin-conjugated DUPAN-2 antibody 7.5 μg/mL
BSA 0.1% (w/v)
Sodium chloride 0.1 mol/L

<Alkaline phosphatase-labeled DUPAN-2 antibody solution: secondary antibody>
Alkaline phosphatase-labeled DUPAN-2 antibody was prepared by conjugating alkaline phosphatase to DUPAN-2 antibody using Alkaline Phosphatase Labeling kit-SH ₂ (Dojindo Laboratories, Inc.) according to the kit's instructions. 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) 50 mmol/L
Alkaline phosphatase-labeled DUPAN-2 antibody 0.3 μg/mL
BSA 0.1% (w/v)
Sodium chloride 0.1 mol/L

[Comparative Example]
As a comparative example, a DUPAN-2 antigen measurement kit A was prepared, which included a DUPAN-2 antibody-bound microplate and a peroxidase (POD)-labeled DUPAN-2 antibody solution. When preparing the DUPAN-2 antibody-bound microplate described below, the DUPAN-2 antibody concentration in the DUPAN-2 antibody solution was set to 5 μg/mL.

<DUPAN-2 antibody-bound microplate: first antibody>
50 μL of a DUPAN-2 antibody solution prepared in binding buffer [0.01 mol/L phosphate buffer (pH 7.3) containing 0.14 mol/L sodium chloride] was added to each well of a 96-well polystyrene plate (manufactured by Thermo Fisher Scientific) and incubated at 25°C for 16 hours. Next, the DUPAN-2 antibody solution was removed, and 250 μL of a blocking solution [phosphate buffer (pH 7.2) containing 1% (w/v) BSA and 0.05% (w/v) Tween 20] was added to each well for washing, followed by addition of 250 μL of the blocking solution to each well and incubation at 4°C for 16 hours. The blocking solution was then removed to prepare a DUPAN-2 antibody-bound microplate.

<POD-labeled DUPAN-2 antibody solution: second antibody>
Using Peroxidase Labeling Kit-NH ₂ (manufactured by Dojindo Laboratories, Inc.), DUPAN-2 antibody was labeled with POD according to the kit's instructions 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) 0.1 mol/L
POD-labeled DUPAN-2 antibody 1 μg/mL
BSA 0.1% (w/v)
NP-40 0.2% (w/v)
Sodium chloride 0.15 mol/L

[Example 2]
<Preparation of DUPAN-2 antigen solution>
An ascites-derived DUPAN-2 antigen solution was prepared by adjusting DUPAN-2 antigen derived from the ascites of a human pancreatic cancer patient (hereinafter simply referred to as "ascites-derived DUPAN-2 antigen") to 350 U/mL with an antigen diluent [0.05 mol/L Tris buffer (pH 8.0) containing 1% (w/v) BSA and 0.15 mol/L sodium chloride]. Similarly, a DUPAN-2 antigen solution derived from the culture supernatant of human pancreatic cancer cultured cells HPAF-II was prepared by adjusting DUPAN-2 antigen derived from the cultured human pancreatic cancer cells HPAF-II to 350 U/mL with the antigen diluent.
According to a known method (R. S. Metzgar et al. CANCER RESEARCH (1982), 42 (2): pp. 601-608), human pancreatic cancer cultured cells HPAF-II were cultured, and the culture supernatant obtained by the culture was used as the DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF-II.

<Measurement of DUPAN-2 in a sample using measurement kit 1>
A 350 U/mL solution of DUPAN-2 antigen derived from ascites and a 350 U/mL solution of DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF-II were used as measurement samples.
To 10 μL of the measurement sample, 30 μL each of the magnetic particle suspension, biotin-conjugated DUPAN-2 antibody solution, and alkaline phosphatase-labeled DUPAN-2 antibody solution prepared in Example 1 was added, stirred, and allowed to react at 37°C for 20 minutes. The magnetic particles were collected by magnetic force, and the reaction solution other than the magnetic particles was removed. The magnetic particles were then washed five times with a washing solution [50 mmol/L MOPS buffer (pH 7.35) containing 0.1% (w/v) Tween 20]. Subsequently, 100 μL of a luminescent substrate solution containing 9-(4-chlorophenylthiophosphoryloxymethylidene)-10-methylacridan disodium salt as the main component was added and stirred. The amount of luminescence generated was measured using a fully automated chemiluminescence immunoassay system "CL-JACK NX" (manufactured by Hitachi Chemical Diagnostics Systems Co., Ltd.).
The (B)/(A) value was calculated by taking the luminescence intensity when measuring the DUPAN-2 antigen solution derived from ascites as (A) and the luminescence intensity when measuring the DUPAN-2 antigen solution derived from human pancreatic cancer cultured cells HPAF-II as (B). The (B)/(A) value is shown in Table 1.

<Measurement of DUPAN-2 in a sample using measurement kit A>
A 350 U/mL solution of DUPAN-2 antigen derived from ascites and a 350 U/mL solution of DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF-II were used as measurement samples.
100 μL of sample dilution solution was added to each well of the DUPAN-2 antibody-bound microplate. Then, 20 μL of a measurement sample 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 washed with a washing solution [0.01 mol/L phosphate buffer (pH 7.4) containing 0.05% (w/v) Tween 20 and 0.15 mol/L sodium chloride], and then 100 μL of a POD-labeled DUPAN-2 antibody solution was added to each well and allowed to react at 25°C for 1 hour (secondary reaction). After the secondary reaction, each well of the plate was washed with the washing solution, and then 100 μL of a TMB (3,3',5,5'-tetramethylbenzidine) aqueous solution was added to each well and incubated at 25°C for 30 minutes. The reaction was stopped by adding 50 μL of 0.5 mol/L sulfuric acid (color development reaction). The absorbance (main wavelength 450 nm, secondary wavelength 650 nm) of the reaction solution obtained by the color reaction was measured using a microplate reader "Emax" (Molecular Devices Japan Co., Ltd.).
The (B)/(A) value was calculated by taking the absorbance (A) when measuring the DUPAN-2 antigen solution derived from ascites and the absorbance (B) when measuring the DUPAN-2 antigen solution derived from human pancreatic cancer cultured cells HPAF-II. The (B)/(A) value is shown in Table 1.

The results in Table 1 show that the (B)/(A) value for Kit 1 was close to 1.0. Compared to the comparative Kit A, Kit 1, which exhibits such a (B)/(A) value, tends to have a smaller difference between the measured values of DUPAN-2 antigen derived from ascites and the measured values of DUPAN-2 antigen derived from human pancreatic cancer cultured cells HPAF-II, demonstrating that accurate measurements are possible.

This disclosure provides a method, reagent, and kit for measuring DUPAN-2 antigen in a sample that is effective and accurate for diagnosing cancers such as pancreatic cancer and biliary tract cancer.

Claims (9)

(1) reacting an insoluble carrier with a DUPAN-2 antigen in a sample in an aqueous medium, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen, to form an immune complex on the insoluble carrier, the immune complex consisting of the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen ;
(2) washing the insoluble carrier with a washing solution; and
(3) after step (2), measuring the immune complex produced in step (1);
Including,
In step (1), the insoluble carrier is bound to a first antibody that recognizes the DUPAN-2 antigen;
A method for measuring DUPAN-2 antigen in a sample, wherein in step (1), the amount of the first antibody that recognizes the DUPAN-2 antigen is 3 to 100 times by weight the amount of the second antibody that recognizes the DUPAN-2 antigen. (1) reacting an insoluble carrier with a first antibody that recognizes the DUPAN-2 antigen in a sample in an aqueous medium to form an immune complex 1 on the insoluble carrier, the immune complex 1 comprising the first antibody that recognizes the DUPAN-2 antigen and the DUPAN-2 antigen;
(2) reacting a second antibody that recognizes the DUPAN-2 antigen with the immune complex 1 produced in step (1) in an aqueous medium to produce an immune complex 2 on the insoluble carrier, the immune complex 2 comprising the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen ;
(3) washing the insoluble carrier with a washing solution; and
(4) after step (3), measuring the immune complex 2 produced in step (2);
Including,
In step (1), the insoluble carrier is bound to a first antibody that recognizes the DUPAN-2 antigen;
A method for measuring DUPAN-2 antigen in a sample, wherein in step (2), the amount of the first antibody that recognizes the DUPAN-2 antigen is 3 to 100 times by weight the amount of the second antibody that recognizes the DUPAN-2 antigen. (1) adding an insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen to a sample containing the DUPAN-2 antigen;
(2) reacting the DUPAN-2 antigen with the insoluble carrier, a first antibody that recognizes the DUPAN-2 antigen, and a second antibody that recognizes the DUPAN-2 antigen in an aqueous medium to form an immune complex on the insoluble carrier, the immune complex consisting of the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen ;
(3) washing the insoluble carrier with a washing solution; and
(4) after step (3), measuring the immune complex produced in step (2);
Including,
In step (2), a first antibody that recognizes the DUPAN-2 antigen is bound to the insoluble carrier;
A method for measuring DUPAN-2 antigen in a sample, wherein in step (1), the amount of the first antibody that recognizes the DUPAN-2 antigen added is 3 to 100 times by weight the amount of the second antibody that recognizes the DUPAN-2 antigen added. (1) adding an insoluble carrier and a first antibody that recognizes the DUPAN-2 antigen to a sample containing the DUPAN-2 antigen;
(2) reacting the insoluble carrier and a first antibody that recognizes the DUPAN-2 antigen with the DUPAN-2 antigen in the sample in an aqueous medium to form an immune complex 1 on the insoluble carrier, the immune complex 1 consisting of the first antibody that recognizes the DUPAN-2 antigen and the DUPAN-2 antigen;
(3) adding a second antibody that recognizes the DUPAN-2 antigen to the immune complex 1 produced in step (2);
(4) reacting a second antibody that recognizes the DUPAN-2 antigen with the immune complex 1 produced in step (2) in an aqueous medium to produce an immune complex 2 on the insoluble carrier, the immune complex 2 comprising the first antibody that recognizes the DUPAN-2 antigen, the DUPAN-2 antigen, and the second antibody that recognizes the DUPAN-2 antigen ;
(5) washing the insoluble carrier with a washing solution; and
(6) after step (5), measuring the immune complex 2 produced in step (4);
Including,
In step (2), a first antibody that recognizes the DUPAN-2 antigen is bound to the insoluble carrier;
A method for measuring DUPAN-2 antigen in a sample, wherein the amount of a first antibody that recognizes the DUPAN-2 antigen added in step (1) is 3 to 100 times (by weight) the amount of a second antibody that recognizes the DUPAN-2 antigen added in step (3). The method according to any one of claims 1 to 4, wherein the insoluble carrier and the first antibody that recognizes the DUPAN-2 antigen can be bound via two affinity substances, one of the two affinity substances being bound to the insoluble carrier and the other of the two affinity substances being bound to the first antibody that recognizes the DUPAN- 2 antigen. The method according to any one of claims 1 to 5 , wherein a label is bound to the second antibody that recognizes the DUPAN-2 antigen. The method of claim 6 , wherein the label is alkaline phosphatase. The method comprises: an insoluble carrier; a first antibody that recognizes the DUPAN-2 antigen; a second antibody that recognizes the DUPAN-2 antigen; and a washing solution used to wash the insoluble carrier ;
the insoluble carrier and the first antibody that recognizes the DUPAN-2 antigen can be bound to each other via two affinity substances;
one of two affinity substances is bound to the insoluble carrier, and the other of the two affinity substances is bound to the first antibody that recognizes the DUPAN-2 antigen;
A reagent for measuring DUPAN-2 antigen in a sample, wherein the amount of a first antibody that recognizes the DUPAN-2 antigen is 3 to 100 times by weight the amount of a second antibody that recognizes the DUPAN-2 antigen. a first reagent containing an insoluble carrier; a second reagent containing a first antibody that recognizes the DUPAN-2 antigen; a third reagent containing a second antibody that recognizes the DUPAN-2 antigen; and a washing solution used to wash the insoluble carrier ;
the insoluble carrier and the first antibody that recognizes the DUPAN-2 antigen can be bound to each other via two affinity substances;
one of two affinity substances is bound to the insoluble carrier, and the other of the two affinity substances is bound to the first antibody that recognizes the DUPAN-2 antigen;
A kit for measuring DUPAN-2 antigen in a sample, wherein the amount of a first antibody that recognizes the DUPAN-2 antigen in the second reagent is 3 to 100 times by weight greater than the amount of a second antibody that recognizes the DUPAN-2 antigen in the third reagent.