Method for detecting CEACAM1

Abstract

Provided is a technique for detecting, with high sensitivity, a specific protein that can be used as a cancer marker. More specifically, the present invention provides a method and the like for detecting CEACAM1, the method comprising: (1) a step for modifying CEACAM1 in a sample containing CEACAM1 to produce modified CEACAM1; and (2) a step for measuring and detecting CEACAM1 by a sandwich method using both a capture antibody having the ability to recognize modified CEACAM1 and a labeled antibody.

Description

Technical Field

[0001] The present invention relates to a method for detecting CEACAM1, etc. Background Art

[0002] CEACAM1 is a protein belonging to the carcinoembryonic antigen (CEA) family. It is known that there are 12 isoforms of CEACAM1, of which 3 are secretory and exist in body fluids such as serum. In addition, it is known that secretory CEACAM1 can be used as a marker for cancers such as melanoma, pancreatic cancer, and urothelial bladder cancer. In pancreatic cancer, CEACAM1 has been confirmed to show higher diagnostic sensitivity and specificity than CA19-9, a universal cancer marker. Therefore, CEACAM1 is expected to be a cancer marker.

[0003] Regarding CEACAM1, some prior art technologies have been disclosed. For example, Patent Document 1 discloses an antibody capable of recognizing a CEACAM1-specific epitope (if necessary, an antibody capable of binding to other subtypes of the CEACAM protein family). Patent Document 2 discloses a method for diagnosing the presence or absence of extrahepatic bile duct cancer, intrahepatic bile duct cancer, or gallbladder cancer using an antibody capable of specifically binding to CEACAM1. Patent Document 3 discloses a method for detecting carcinoembryonic antigen (CEA) by immunoassay after heating human serum or plasma under acidic conditions to separate unnecessary substances.

[0004] Prior art literature Patent Literature Patent Document 1: Japanese Patent Application No. 2015-502138 Patent Document 2: Japanese Patent Application Publication No. 2018-17723 Patent Document 3: Japanese Patent Publication No. 2-503716 Summary of the invention

[0005] Technical problem to be solved by the invention An object of the present invention is to detect with high sensitivity a specific protein that can be used as a cancer marker.

[0006] Another object of the present invention is to detect a specific protein that can be used as a cancer marker with high sensitivity and specificity.

[0007] The present inventors have conducted intensive studies and, as a result, have found that CEACAM1, which can be used as a cancer marker, can be detected with high sensitivity (and specificity) by using a sandwich assay that selects CEACAM1 as a specific protein that can be used as a cancer marker and uses both a capture antibody capable of recognizing the modified CEACAM1 and a labeling antibody after CEACAM1 is modified, thereby completing the present invention. The prior art neither describes nor suggests the use of such a sandwich assay after CEACAM1 is modified.

[0008] That is, the present invention is as follows.

[0009] [1] A method for detecting CEACAM1, comprising: (1) a step of modifying CEACAM1 in a sample containing CEACAM1 to produce modified CEACAM1; and (2) A step of detecting CEACAM1 by a sandwich assay using both a capture antibody capable of recognizing modified CEACAM1 and a labeled antibody.

[0010] [2] The method according to [1], wherein the modification is selected from the group consisting of a modifier, heating, and a combination thereof.

[0011] [3] The method according to [2], wherein the modifier is selected from the group consisting of alkaline substances, surfactants, reducing agents, and combinations of two or more thereof.

[0012] [4] The method according to any one of [1] to [3], wherein as one of the capture antibody and the labeling antibody, (a) an antibody capable of recognizing an epitope represented by the amino acid sequence of PANSGRETIY (SEQ ID NO: 1) in CEACAM1 is used.

[0013] [5] The method according to [4], wherein as one of the capture antibody and the labeling antibody, (b) an antibody capable of recognizing an epitope represented by the amino acid sequence of TESMP (SEQ ID NO: 2) in CEACAM1 is further used.

[0014] [6] The method according to [4] or [5], wherein as the other of the capture antibody and the labeling antibody, (c) an antibody capable of recognizing an epitope represented by the amino acid sequence of DTTYLWWINN (SEQ ID NO: 3) in CEACAM1 is used.

[0015] [7] The method according to [4] or [5], wherein as the other of the capture antibody and the labeling antibody, (d) an antibody capable of recognizing an epitope represented by the amino acid sequence of EATGQFHVYP (SEQ ID NO: 4) in CEACAM1 is used.

[0016] [8] A method for detecting cancer, comprising: (1) a step of measuring the amount of CEACAM1 in a sample collected from a subject by the method of any one of [1] to [7]; and (2) A step of comparing the measured CEACAM1 amount with a reference value.

[0017] [9] The method according to [8], wherein the cancer is selected from the group consisting of colorectal cancer, liver cancer, pancreatic cancer, and a combination of two or more thereof.

[0018]

[10] A method for treating cancer, comprising: (1) a step of measuring the amount of CEACAM1 in a sample collected from a subject by the method of any one of [1] to [7]; (2) a step of comparing the measured CEACAM1 amount with a reference value; (3) a step of selecting a subject in which the amount of CEACAM1 measured is higher than a reference value; and (4) A step of administering an anticancer drug to the selected subject.

[0019]

[11] A kit for detecting CEACAM1, comprising: (1) Modifiers; (2) a capture antibody capable of recognizing modified CEACAM1; and (3) A labeled antibody capable of recognizing modified CEACAM1.

[0020] Effects of the Invention The method for detecting CEACAM1 of the present invention is useful, for example, for highly sensitive and specific detection of CEACAM1.

[0021] The method for detecting cancer of the present invention can measure CEACAM1, which is a cancer marker, with high sensitivity and specificity, and is therefore useful for detecting cancer with high diagnostic sensitivity and high diagnostic specificity.

[0022] The therapeutic approaches of the present invention are useful in the context of effective treatments based on anticancer agents.

[0023] In addition, according to the methods of the present invention, the influence of autoantibodies can be reduced or avoided.

[0024] The kit of the present invention is useful, for example, in conveniently carrying out the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The diagram shows the amino acid sequences of the expressed proteins (pAT-TrpE-His-CEACAM1-N, pAT-TrpE-His-CEACAM1-N-m3C3d, pAT-TrpE-His-CEACAM1-A1, and pAT-TrpE-His-CEACAM1-A1-m3C3d).

[0026] Figure 2 It is a graph showing the cross-reactivity of anti-CEACAM1 monoclonal antibodies.

[0027] Figure 3 Figure 1 is a diagram showing the amino acid sequence comparison of the CEACAM family. The amino acid sequences of the N domain and the A1 domain of the signal sequence of the CEACAM family retrieved from the UniProt database are compared. Amino acids with high homology are indicated by black blanks. The epitopes of each antibody are shown on the sequence of CEACAM1, and the amino acids considered to be hot spots of each antibody epitope are indicated by black boxes.

[0028] Figure 4 This is a graph showing the blood concentration distribution of various cancer samples: colon cancer samples (n=42), liver cancer samples (n=50), pancreatic cancer samples (n=40), and healthy human samples (Normal, n=36).

[0029] Figure 5A This is a graph showing the ROC (Receiver Operating Characteristic) analysis of colorectal cancer samples.

[0030] Figure 5B Graph showing ROC analysis in liver cancer samples.

[0031] Figure 5C Graph showing ROC analysis in pancreatic cancer samples.

[0032] Figure 6 Graph showing the difference in effect of SDS reduction heat treatment and non-SDS reduction heat treatment. m g / mL) was reacted with A3054 and A3073 on a co-solid phase plate and detected using ALP-labeled A7004 antibody.

[0033] Figure 7 This is a graph showing the effect of sample modification treatment by alkali treatment (NaOH) (part 1).

[0034] Figure 8This is a graph showing the effect of sample modification treatment by alkali treatment (NaOH) (part 2).

[0035] Figure 9 This is a graph showing the blood concentration distribution of various cancer samples: colon cancer samples (n=42), liver cancer samples (n=50), pancreatic cancer samples (n=40), and healthy human samples (Normal, n=36).

[0036] Figure 10A The figure shows the ROC analysis of colorectal cancer samples relative to healthy human samples.

[0037] Figure 10B Graph showing ROC analysis of liver cancer samples relative to healthy human samples.

[0038] Figure 10C Graph showing ROC analysis of pancreatic cancer samples relative to healthy human samples.

[0039] Figure 11 This is a graph showing the correlation between SDS reduction heat treatment and alkali treatment.

[0040] Figure 12 Graphs showing the effects of modification treatment on autoantibody model samples: A) measurement system without modification treatment, B) measurement system with modification treatment. DETAILED DESCRIPTION

[0041] The present invention provides a method for detecting CEACAM1, comprising: (1) a step of modifying CEACAM1 in a sample containing CEACAM1 to produce modified CEACAM1; and (2) A step of detecting CEACAM1 by a sandwich assay using both a capture antibody capable of recognizing modified CEACAM1 and a labeled antibody.

[0042] In the above step (1), CEACAM1 is modified in a sample containing CEACAM1, thereby generating modified CEACAM1.

[0043] In the present invention, in order to improve recognition by antibodies, the linear epitope of CEACAM1 that is not exposed in the natural state (i.e., non-modified state) is modified to be exposed. This is because, for example, when CEACAM1 is in an aggregated state or forms a multimer or a complex, the linear epitope is covered and thus cannot be fully exposed.

[0044] The modification can be performed by a modifier, heating, or a combination thereof. Examples of the modifier include alkaline substances, surfactants, chaotropic modifiers (eg, urea, guanidine), reducing agents, and combinations of two or more thereof.

[0045] As the alkaline substance, for example, inorganic substances (for example, metal ions) and organic substances can be cited. As the alkaline substance, there are various substances, among which hydroxides of alkali metals (for example, sodium, potassium) or alkaline earth metals (for example, magnesium, calcium) are generally used. Therefore, such hydroxides can be preferably used.

[0046] The concentration of the alkaline substance is not particularly limited as long as it can modify CEACAM1, and may be, for example, 0.1 M or more, preferably 0.2 M or more, more preferably 0.3 M or more, and even more preferably 0.35 M or more. The concentration of the alkaline substance may also be 5 M or less, 2 M or less, or 1 M or less.

[0047] The alkaline substance can be used together with a surfactant. As the surfactant, nonionic surfactants, anionic surfactants, cationic surfactants, and zwitterionic surfactants are preferred. Nonionic surfactants are particularly preferred. As nonionic surfactants, ether surfactants such as polyoxyethylene (23) lauryl ether (Brij35) and polyethylene glycol cetyl ether (Brij58), polyoxyethylene sorbitan monolaurate (Tween20), polyoxyethylene sorbitan monopalmitate (Tween40), polyoxyethylene sorbitan monooleate (Tween80) and other polyoxyethylene sorbitan fatty acid esters (for example, Tween (trade name, registered trademark) series), polyoxyethylene Polyoxyethylene alkylphenyl ethers such as octylphenyl ether (for example, Triton (trade name, registered trademark) series), secondary alcohol polyoxyethylene ethers such as Tergitol (registered trademark) 15-S-7 (for example, TERGITOL (trade name, registered trademark) series), copolymers of ethylene oxide and propylene oxide such as poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic F108) (for example, Pluronic (trade name, registered trademark) series), etc. The concentration of the surfactant is not particularly limited and can be appropriately set.

[0048] In the case of modification using a modification treatment liquid containing an alkaline substance, after the modification treatment, in order to neutralize or adjust the pH, a process of adding a neutralizing solution containing an acidulant or a buffer having a buffering capacity may be further included. Examples of the acidulant include hydrochloric acid, sulfuric acid, acetic acid, and the like. In addition, in the case where the process of adding a neutralizing solution is not included, for example, in the following reaction process, the alkaline substance in the modification treatment liquid can be neutralized by adjusting the buffering capacity and pH of the mixed buffer, thereby alleviating the influence in the reaction process. The pH in the reaction process can be appropriately set according to the components included in the reaction process. For example, the amount of the acidulant or buffer contained in the liquid of the reaction process can be appropriately set so as to have a pH of 5.5 to 9.5.

[0049] As the surfactant, for example, anionic surfactants, cationic surfactants, zwitterionic surfactants and nonionic surfactants can be cited. As the surfactant, one or more surfactants can be used. As the surfactant, anionic surfactants can be used alone, and in addition, a combination of anionic surfactants and other surfactants can also be used. As the anionic surfactant, for example, sulfate surfactants (for example, sodium dodecyl sulfate (SDS)), carboxylic acid surfactants (for example, sodium N-decanoyl sarcosinate (NDS), sodium N-lauroyl sarcosinate hydrate (NLS)), sulfonic acid surfactants (for example, sodium 1-nonane sulfonate (NSS), sodium dodecylbenzene sulfonate (SDBS)), and carboxylic acid-sulfonic acid surfactants (for example, sodium chondroitin sulfate (CSSS)) can be cited. Examples of zwitterionic surfactants include C10APS (N-decyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), C12APS (N-dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), C14APS (N-tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), and C16APS (N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate). Examples of cationic surfactants include decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium chloride, and hexadecyltrimethylammonium chloride (C16TAC), decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, and hexadecyltrimethylammonium bromide (CTAB), laurylpyridinium chloride, tetradecylpyridinium chloride, and hexadecylpyridinium chloride.

[0050] The concentration of the surfactant is not particularly limited as long as it can modify CEACAM1, and can be, for example, 0.5 wt % or more, preferably 1 wt % or more, more preferably 2 wt % or more, and even more preferably 5 wt % or more. The concentration of the surfactant can also be 20 wt % or less, or 15 wt % or less.

[0051] As the reducing agent, a reagent that can cleave the disulfide bond present in CEACAM1 can be used. Examples of such reducing agents include tricarboxyethylphosphine (TCEP), cysteine, dithiothreitol, reduced glutathione, β -Mercaptoethanol. Examples of the protease include trypsin, chymotrypsin, Lys-C, Asp-N, Glu-C, Arg-C, asparaginyl endopeptidase, arginyl endopeptidase, and V8 protease.

[0052] The concentration of the reducing agent is not particularly limited as long as it can cleave the disulfide bonds present in CEACAM1, and can be, for example, 0.5 mM or more, preferably 1 mM or more, more preferably 2 mM or more, and even more preferably 5 mM or more. The concentration of the reducing agent can also be 100 mM or less or 50 mM or less.

[0053] It should be noted that the concentration of the above-mentioned modifier (alkaline substance, surfactant and reducing agent) is a value based on the weight or volume of the modified treatment solution containing the modifier. In addition to the modifier, the modified treatment solution also contains a solvent or a dispersion medium (for example, water) and additives as needed. As additives, chelating agents such as EDTA can be mentioned.

[0054] The heating may be, for example, 35°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, 65°C or higher, 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, 90°C or higher, 95°C or higher, or about 100°C (i.e., boiling). The heating may also be 100°C or lower.

[0055] In certain embodiments, the modification can be performed by combining a surfactant and a reducing agent. The modification can also be performed by combining a surfactant, a reducing agent and heating (e.g., SDS reduction heat treatment).

[0056] The treatment time for modifying CEACAM1 (e.g., incubation time after adding a modifying agent to a sample, heating treatment time) varies, for example, depending on factors such as the type and degree of the modification treatment (e.g., concentration, heating temperature), and may be, for example, 1 second or more, 5 seconds or more, 10 seconds or more, preferably 30 seconds or more, more preferably 1 minute or more, and even more preferably 2 minutes or more, 3 minutes or more, or 5 minutes or more. Such a time may also be less than 1 hour, less than 30 minutes, or less than 20 minutes.

[0057] The CEACAM1-containing sample is any sample containing CEACAM1. The CEACAM1-containing sample is preferably a biological fluid sample. The CEACAM1-containing sample may also be subjected to other treatments before being used in the method of the present invention. Such treatments include, for example, centrifugation, extraction, filtration, precipitation, heating, freezing, refrigeration, and stirring.

[0058] In one embodiment, the CEACAM1-containing sample may be a liquid sample derived from an animal. Examples of animals from which the liquid sample may be derived include mammals (e.g., primates such as humans and monkeys; rodents such as mice, rats, and rabbits; ungulates such as cattle, pigs, goats, horses, and sheep; carnivores such as dogs and cats; and birds (e.g., chickens)). Preferably, the animal is a mammal, preferably a human. The liquid sample derived from an animal may be a body fluid sample derived from the above-mentioned animal. Examples of body fluid samples include blood samples (e.g., whole blood, serum, and plasma), urine, saliva, lymph, tissue fluid, cerebrospinal fluid, ascites, sweat, semen, tears, mucus, milk, pleural fluid, bronchoalveolar lavage fluid, and amniotic fluid. The body fluid is preferably a blood sample, urine, or saliva.

[0059] In other embodiments, the CEACAM1-containing sample may also be a culture supernatant sample. The culture supernatant sample may be a cell culture supernatant sample or a tissue culture supernatant sample. Examples and preferred examples of animals from which the cultured cells or tissues are derived are as described above.

[0060] CEACAM1 can be determined based on the type of animal from which it originates. Examples and preferred examples of animals from which CEACAM1 originates are the same as those described above. Therefore, CEACAM1 is mammalian CEACAM1, preferably human CEACAM1. CEACAM1 also has 12 isoforms, of which 3 are secretory (CEACAM1-4C1, CEACAM1-3, CEACAM1-3C2). Therefore, when a body fluid sample is used, CEACAM1 may also include such secretory CEACAM1.

[0061] After the above step (1), the sample containing modified CEACAM1 may be diluted with a solution (e.g., a buffer solution). Thus, when the sample obtained in the above step (1) contains a high concentration of the modifying agent, the concentration of the modifying agent can be diluted, and the sandwich assay in the above step (2) can be performed well.

[0062] In the above step (2), CEACAM1 is detected by sandwich assay. In the sandwich assay, both a capture antibody and a labeled antibody having the ability to recognize modified CEACAM1 can be used. The sandwich assay can be performed in any manner using such an antibody. Examples of such a manner include chemiluminescent immunoassay (CLIA) [e.g., chemiluminescent enzyme immunoassay (CLEIA)], enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), and immunochromatography.

[0063] The capture antibody can be solid-phased on a solid phase. In addition, the antibody against CEACAM1 can also be an antibody that can be solid-phased on a solid phase in the reaction process. In this specification, antibodies that are solid-phased on a solid phase and antibodies that can be solid-phased on a solid phase in the reaction process are sometimes referred to as solid-phase antibodies. The capture antibody is an antibody used to capture the target antigen (modified CEACAM1 in the case of the present invention) on a solid phase. As a solid phase, for example, particles (for example, agarose beads (sepharose beads), agarose beads (agarose beads), magnetic particles), supports (for example, membranes), containers (for example, plates such as plastic plates, tubes, microfluidics) can be cited. The fixation of antibodies to the solid phase can utilize existing known methods. For example, it can be performed by physical adsorption, covalent bonding, methods using affinity substances (for example, biotin, streptavidin), and ionic bonding methods, with covalent bonds or non-covalent bonds. The capture antibody may be an antibody obtained by fixing one capture antibody on one solid phase (for example, particles such as magnetic particles), or an antibody obtained by fixing two or more capture antibodies on one solid phase (for example, particles such as magnetic particles). In addition, it may be an antibody obtained by fixing two or more capture antibodies on two or more solid phases (for example, particles such as magnetic particles), respectively.

[0064] The labeled antibody is an antibody labeled with a labeling substance. Examples of the labeling substance include enzymes (e.g., peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (e.g., streptavidin, biotin), fluorescent substances or fluorescent proteins (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent or light-absorbing substances (e.g., luciferin, acridinium, ruthenium), radioactive substances (e.g., 3 H. 14 C. 32 P. 35 S. 125 I). The labeling of antibodies with labeling substances can be performed by covalent bonding or non-covalent bonding.

[0065] In the present invention, an antibody is a protein containing a portion (e.g., a variable region or a portion equivalent thereto) that has the ability to bind to a target antigen. Therefore, examples of antibodies in capture antibodies and labeled antibodies include full-length antibodies (e.g., IgG, IgM, IgA, IgD, IgE, and IgY) and antigen-binding fragments of antibodies (e.g., F(ab′) 2 The antibody may be a polyclonal antibody or a monoclonal antibody.

[0066] In the present invention, as the capture antibody and the labeling antibody, an antibody capable of recognizing modified CEACAM1 can be used. The antibody capable of recognizing modified CEACAM1 can be an antibody capable of specifically recognizing modified CEACAM1 or an antibody capable of non-specifically recognizing modified CEACAM1. As an antibody capable of non-specifically recognizing modified CEACAM1, for example, (i) an antibody capable of recognizing not only modified CEACAM1 but also natural (i.e., non-modified) CEACAM1, (ii) an antibody capable of recognizing not only modified CEACAM1 but also other CEACAM1 (e.g., CEACAM3), and (iii) an antibody capable of recognizing not only modified CEACAM1 but also natural (i.e., non-modified) CEACAM1 and other CEACAM1 (e.g., CEACAM3). At least one of the capture antibody and the labeling antibody is preferably an antibody capable of specifically recognizing modified CEACAM1.

[0067] In the present invention, as the capture antibody and the labeled antibody as described above, (a) an antibody capable of recognizing an epitope represented by the amino acid sequence of PANSGRETIY (SEQ ID NO. 1) in CEACAM1, (b) an antibody capable of recognizing an epitope represented by the amino acid sequence of TESMP (SEQ ID NO. 2) in CEACAM1, (c) an antibody capable of recognizing an epitope represented by the amino acid sequence of DTTYLWWINN (SEQ ID NO. 3) in CEACAM1, or (d) EATGQFHVYP (SEQ ID NO. 4) in CEACAM1 can be used. Each of these epitopes is specifically exposed by the modification as described above, and therefore is an epitope that reacts better with modified CEACAM1 than with non-modified CEACAM1. According to an experiment using a peptide represented by the amino acid sequence of TESMP (SEQ ID NO. 2), the antibody of (b) above cross-reacts not only with CEACAM1, but also with CEACAM3, CEACAM5 and / or CEACAM6, and therefore is not a CEACAM1-specific antibody (see Examples and Figure 3 ). However, the present inventors have found that the detection sensitivity of CEACAM1 is improved by using the antibody (b) as an auxiliary in sandwich assay. In addition, it has been confirmed that the detection of CEACAM3, CEACAM5 and / or CEACAM6 can be avoided by using other antibodies capable of recognizing CEACAM1 in addition to the antibody (b) above (see Examples).

[0068] In a specific embodiment, as one of the capture antibody and the labeling antibody, the antibody of (a) or a combination of the antibody of (a) and the antibody of (b) can be used, and as the other, the antibody of (c), the antibody of (d), or a combination of the antibody of (c) and the antibody of (d) can be used. Thus, CEACAM1 can be detected specifically and with high sensitivity. Preferably, as the capture antibody, the antibody of (a) or a combination of the antibody of (a) and the antibody of (b) can be used, and as the labeling antibody, the antibody of (c), the antibody of (d), or a combination of the antibody of (c) and the antibody of (d) can be used.

[0069] When the antibody of (a) and the antibody of (b) are combined, the weight ratio of (a):(b) can be, for example, 1:9 to 9:1, preferably 2:8 to 8:2, more preferably 3:7 to 7:3, further preferably 4:6 to 6:4, and most preferably 4.5:5.5 to 5.5:4.5.

[0070] Antibodies capable of recognizing the above epitopes can be prepared by using CEACAM1 or a partial protein thereof as an antigen. In fact, hybridomas producing the above antibodies (a), (b), (c) and (d) were successfully produced at probabilities of 2 / 500 (0.4%), 4 / 500 (0.8%), 1 / 800 (0.1%) and 1 / 120 (0.8%), respectively. In addition, by using a peptide containing an epitope recognized by the above antibodies (a), (b), (c) and (d), or a polypeptide rich therein (a polypeptide containing a plurality of the above epitopes) as an antigen (for example, fusion with a carrier protein such as albumin), hybridomas producing antibodies capable of recognizing the above epitopes can be efficiently prepared. Alternatively, antibodies capable of recognizing the above epitopes can be efficiently obtained by selecting from an antibody library using the above epitopes.

[0071] The present invention also provides a method for detecting cancer, comprising: (1) a step of measuring the amount of CEACAM1 in a sample collected from a subject using the above-mentioned method for detecting CEACAM1; and (2) A step of comparing the measured CEACAM1 amount with a reference value.

[0072] The step (1) in the method for detecting cancer can be performed in the same manner as the method for detecting CEACAM1. The subject from which the sample is collected is the same as the above-mentioned animal, preferably a mammal, and more preferably a human. The sample is the same as the liquid sample (e.g., body fluid sample) derived from the above-mentioned animal.

[0073] The above-mentioned step (2) in the method for detecting cancer compares the measured CEACAM1 amount with a reference value. When the measured CEACAM1 amount is higher than the reference value, it can be used as an indicator that the subject may suffer from cancer. It is known that CEACAM1 such as secretory CEACAM1 can be used as a marker for various cancers such as melanoma, pancreatic cancer, urothelial bladder cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, bladder cancer, melanoma, etc. Therefore, the cancer detection method of the present invention is useful in the detection of such various cancers (for example, colorectal cancer, liver cancer and pancreatic cancer shown in the reference examples, and combinations of two or more thereof).

[0074] The present invention further provides a method for treating cancer, comprising: (1) a step of measuring the amount of CEACAM1 in a sample collected from a subject using the above-mentioned method for detecting CEACAM1; (2) a step of comparing the measured CEACAM1 amount with a reference value; (3) a step of selecting a subject in which the amount of CEACAM1 measured is higher than a reference value; and (4) A step of administering an anticancer drug to the selected subject.

[0075] The steps (1) and (2) in the method for treating cancer can be performed in the same manner as the steps (1) and (2) in the method for detecting cancer.

[0076] The step (3) in the method of treating cancer can be performed by selecting a subject in which the amount of CEACAM1 measured is higher than the reference value based on the comparison result of the above (2).

[0077] The above step (4) in the method for treating cancer is to administer an anticancer agent to the selected subject. The anticancer agent to be administered can be appropriately determined according to the type of cancer. In addition, other treatment methods such as radiotherapy can also be used in combination.

[0078] The present invention further provides a CEACAM1 detection kit, which comprises: (1) Modifiers; (2) a capture antibody capable of recognizing modified CEACAM1; and (3) A labeled antibody capable of recognizing modified CEACAM1.

[0079] The modifying agent, the capture antibody capable of recognizing the modified CEACAM1, and the labeled antibody capable of recognizing the modified CEACAM1 are the same as those described above. The capture antibody capable of recognizing the modified CEACAM1 and the labeled antibody capable of recognizing the modified CEACAM1 can be provided in the form of a sandwich assay reagent using these antibodies. The kit of the present invention is useful, for example, in the simple and rapid implementation of the method of the present invention.

[0080] Example The present invention will be described in more detail by the following examples, but the present invention is not limited to the following examples. It should be noted that the % indicated in the components in the examples described below means % by weight.

[0081] Example 1: Expression and purification of immunizing antigens and screening antigens (A) Construction of antigen expression plasmid In order to prepare TrpE-His-CEACAM1-N-m3C3d and TrpE-His-CEACAM1-A1-m3C3d as antigens for immunization and TrpE-His-CEACAM1-N and TrpE-His-CEACAM1-A1 as antigens for screening, antigen expression plasmids were constructed. Using cDNA corresponding to human CEACAM1 as a template, two primers (5′-GACTCGTACGCAGCTCACTACTGAATCCATG-3′ (SEQ ID NO: 16), 5′-GACTGTCGACTTACGGGTATACATGGAACTGTCC-3′ (SEQ ID NO: 17)) were used to construct the pAT-TrpE-His-CEACAM1-N expression plasmid, and two primers (5′-GACTCGTACGCAGCTCACTACTGAATCCATG-3′ (SEQ ID NO: 18), 5′-GACTCTCGAGCCCGGGTATACATGGAACTGTCC-3′ (SEQ ID NO: 19)) were used to construct the pAT-TrpE-His-CEACAM1-N-m3C3d expression plasmid. 19)), two primers (5′-GACTCGTACGCCCAAGCCCTCCATCTCCAGC-3′ (SEQ ID NO: 20), 5′-GACTGTCGACTTAATTCAAGGTGACTGGGTCACT-3′ (SEQ ID NO: 21)) were used to construct the pAT-TrpE-His-CEACAM1-A1 expression plasmid, and two primers (5′-GACTCGTACGCCCAAGCCCTCCATCTCCAGC-3′ (SEQ ID NO: 22), 5′-GACTCTCGAGCCATTCAAGGTGACTGGGTCACT-3′ (SEQ ID NO: 23)) were used to construct the pAT-TrpE-His-CEACAM1-A1-m3C3d expression plasmid for PCR. PCR was performed using the KAPA TaqEXtra kit (Genetics, Japan) under 25 cycles of the following conditions: DNA modification at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and DNA synthesis at 72°C for 45 seconds. The resulting DNA fragments were separated by 1.2% agarose gel electrophoresis and purified by Freeze′N Squeeze. TM DNA Gel Extraction Spin Columns (BIO-RAD) were used for purification. In the construction of TrpE-His-CEACAM1-N or TrpE-His-CEACAM1-A1, the restriction enzyme reaction solution was 20 m l〔100mM NaCl, 50mM Tris-HCl, 10mM MgCl 2In the construction of pAT-TrpE-His-CEACAM1-N-m3C3d or pAT-TrpE-His-CEACAM1-A1-m3C3d, 20 units of BsiWI and 10 units of SalI were added to a solution prepared by adding 10 units of BsiWI and 10 units of SalI ... m l〔100mM NaCl, 50mM Tris-HCl, 10mM MgCl 2 10 units of BsiWI and 10 units of XhoI were added to a solution prepared by adding 1 mM DTT, pH 7.9, and 0.5% of the amplified CEACAM1 gene fragment was added. m g was digested at 37°C for 1 hour and then subjected to 1.2% agarose gel electrophoresis, and the BsiWI-SalI fragment or BsiWI-XhoI fragment of the CEACAM1 gene was purified by the same method as the above PCR product.

[0082] Next, in the construction of pAT-TrpE-His-CEACAM1-N or pAT-TrpE-His-CEACAM1-A1, in the restriction enzyme reaction solution 20 m l [100mM NaCl, 50mM Tris-HCl, 10mM MgCl 2 In the construction of pAT-TrpE-His-CEACAM1-N-m3C3d or pAT-TrpE-His-CEACAM1-A1-m3C3d, 20 units of BsiWI and 10 units of SalI were added to a solution prepared by adding 10 units of BsiWI and 10 units of SalI ... m l [100mM NaCl, 50mM Tris-HCl, 10mMgCl 2 10 units of BsiWI and 10 units of XhoI were added to the solution prepared by adding 1 mM DTT, pH 7.9, and the DNA of pAT-trpE-His or pAT-trpE-His-m3C3d as the expression vector was added. m g were digested at 37°C for 1 hour and then subjected to 1.2% agarose gel electrophoresis using Freeze′N Squeeze TM The vector was purified by treatment with BsiWI-SalI or BsiWI-XhoI using DNA Gel Extraction Spin Columns (BIO-RAD).

[0083] An equal amount of DNA Ligation Kit was added to the DNA solution prepared by mixing the obtained BsiWI-SalI or BsiWI-XhoI treated vector DNA and the CEACAM1 fragment treated with the restriction enzyme to the same weight.<Mighty Mix> (TAKARA BIO Co., Ltd.), and incubated at 16°C for 30 minutes to perform a ligation reaction. In order to obtain an expression plasmid, the ligation reaction solution was used to transform Escherichia coli XL1-blue MRF (Stratagen Co., Ltd.).

[0084] The sensitive E. coli strain used for transformation was prepared by Mix&Go E. coli Transformation Kit&BufferSet. The transformed E. coli was plated in a 100 μl m g / ml ampicillin on LB plates (1% tryptone, 0.5% NaCl, 1.5% agar) and incubate at 37°C overnight. Use a sterile toothpick to pick up the bacterial colonies produced on the plate and transfer them to a plate containing 100 μg / ml ampicillin. m The cells were cultured in 2YT medium containing 100 g / ml ampicillin at 37°C overnight.

[0085] 1.5 ml of bacterial culture was centrifuged and collected, and plasmid DNA was prepared in minipreparation using Wizard (R) Plus SV Minipreps DNA Purification Systems (Promega). In the construction of TrpE-His-CEACAM1-N or A1, the restriction enzyme reaction solution was 20 m l〔100mM NaCl, 50mM Tris-HCl, 10mM MgCl 2 In the construction of TrpE-His-CEACAM1-N-m3C3d or A1-m3C3d, 20 units of restriction enzyme reaction solution were added to a solution prepared by adding 10 units of BsiWI and 10 units of SalI to 1 mM DTT, pH 7.9. m l〔100mM NaCl, 50mM Tris-HCl, 10mM MgCl 2 , 1 mM DTT, pH 7.9〕, 10 units of BsiWI and 10 units of XhoI were added to the solution, and the obtained plasmid DNA was 1 mg was digested at 37°C for 1 hour, followed by 1.2% agarose gel electrophoresis, and expression plasmids producing a fragment of about 450 bp were selected from pAT-TrpE-His-CEACAM1-N or pAT-TrpE-His-CEACAM1-N-m3C3d, and expression plasmids producing a fragment of about 400 bp were selected from pAT-TrpE-His-CEACAM1-A1 or pAT-TrpE-His-CEACAM1-A1-m3C3d.

[0086] (B) Antigen expression and purification Escherichia coli transformed with pAT-TrpE-His-CEACAM1-N or pAT-TrpE-His-CEACAM1-N-m3C3d and pAT-TrpE-His-CEACAM1-A1 or pAT-TrpE-His-CEACAM1-A1-m3C3d were cultured in a 100 μl m The cells were cultured overnight at 37°C in LB medium containing 100 μg / ml ampicillin. m g / ml ampicillin M9-CA, cultured overnight at 37°C. After the culture, the cells were collected by centrifugation, resuspended in 50ml of Lysis solution [50mMTris-HCl (pH8.5), 30mM NaCl, 5mM EDTA], 1ml of lysozyme solution (10mg / ml Lysozyme) was added, and treated at 37°C for 1 hour. The suspension was subjected to ultrasonic treatment (150W, 90 seconds twice) to destroy the cells. Centrifuged at 15000rpm at 4°C for 30 minutes, and the insoluble fraction was recovered. The insoluble fraction was resuspended in 50ml of A solution [50mM Tris-HCl (pH8.5)] containing 1% NP40. The suspension was centrifuged at 15000rpm at 4°C for 30 minutes, and the insoluble fraction was recovered. The insoluble fraction was resuspended in 50ml of A solution containing 2M urea. The suspension was centrifuged at 15,000 rpm at 4°C for 30 minutes to recover the insoluble fraction. The insoluble fraction was resuspended in 50 ml of solution A containing 6 M urea. The suspension was centrifuged at 15,000 rpm at 4°C for 30 minutes to recover the soluble fraction.

[0087] The antigen proteins TrpE-His-CEACAM1-N, TrpE-His-CEACAM1-N-m3C3d, TrpE-His-CEACAM1-A1, and TrpE-His-CEACAM1-A1-m3C3d were purified from the antigen solution solubilized with a solution containing 6 M urea by an ion exchange method using an S Sepharose HP column (GE Healthcare) and an IMAC method using a HisTrap HP column (GE Healthcare).

[0088] Example 2: Obtaining anti-CEACAM1 monoclonal antibodies (A) Immunity The antigen protein TrpE-His-CEACAM1-N-m3C3d or TrpE-His-CEACAM1-A1-m3C3d prepared by the above method was dissolved in 6 M urea, diluted in 10 mM phosphate buffer (pH 7.3) (PBS) containing 0.15 M NaCl to a final concentration of 1.0 mg / ml, mixed with an equal amount of Freund's adjuvant, and subcutaneously administered to 4- to 6-week-old BALB / c mice at 50 to 100 μg / ml. m The same booster immunization was performed every 2 weeks, and 100 g of antigen protein dissolved in PBS was further added. m g was administered intraperitoneally as the final immunization.

[0089] (B) Cell fusion On the third day after the final immunization, the spleen was aseptically removed from the mouse, and the spleen was disassembled into single cells using forceps and a cell strainer, and suspended in RPMI1640 medium (no serum added). The cells precipitated by centrifugation (1000rpm, 5 minutes, room temperature) were pipetted using 1ml of Red Blood Cell Lysing Buffer (red blood cell lysis buffer, MERCK) and left to stand at room temperature for 1 minute. 20mL of RPMI1640 medium (no serum added) was added to the cells and fully suspended. The cells precipitated by centrifugation (1000rpm, 5 minutes, room temperature) were suspended in RPMI1640 medium (no serum added). Myeloma cells (mouse myeloma cell line Sp2 / 0 Ag14) in the logarithmic growth phase were recovered from the culture flask, precipitated by centrifugation (1000rpm, 5 minutes, room temperature), and suspended in RPMI1640 medium (no serum added). Myeloma cells and spleen cells were mixed at a cell number ratio of 1:1 so that the total cell number was 3.4×10 7The cell mixture was centrifuged (1000 rpm, 5 minutes, room temperature), the supernatant was removed, and the mixture was washed twice with ECF buffer (0.3 M mannitol, 0.1 mM calcium chloride, 0.1 mM magnesium chloride solution). The cell fusion device ECFG21 (NEPA GENE) and the MS holder type chamber platinum electrode CUY497P2 (0.8 mL) were used, with a liquid volume of 0.35 ml, an AC voltage of 40 Vrms, an AC time of 10 sec, a DC voltage of 350 V, and a pulse width of 30 m sec, pulse interval 0.5sec, pulse number 3 times, decay rate 10%, polarity switching + (no switching), post-fusion 7sec, decay sine wave ON, the mixed cell solution finally suspended in 0.35ml ECF buffer was subjected to cell fusion. Immediately after fusion, it was suspended in RPMI1640 medium containing serum and allowed to stand for 10 minutes to restore the cell membrane. Then, it was suspended in RPMI-1640 medium containing 10% fetal bovine serum and hypoxanthine, aminopterin and thymidine (HAT), and inoculated in a 96-well cell culture plate. After culturing for about 10 days, only the hybridoma was proliferated, and the culture supernatant was recovered for screening described below.

[0090] (C) Screening of anti-CEACAM1 antibody-producing hybridomas Hybridomas producing the target antibodies were searched by ELISA using the above antigen proteins TrpE-His-CEACAM1-N and TrpE-His-CEACAM1-A1. In order to select antibodies that recognize linear epitopes, various antigens were mixed with TBS containing 1 mM TCEP and 6 M urea at 1 m g / mL. m L was added to a Nunc multi-module plate and allowed to stand overnight at 4-8°C. After removing the antigen diluent, 100 μL of m L blocking solution (0.1% casein, 1 mM EDTA, PBS) was added and allowed to stand at room temperature for 1 hour to fix the antigen to the plate.

[0091] 50% of the culture supernatant diluted with the reaction solution (0.1% casein, 1 mM EDTA, PBS) was added to each well. m L, and allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween 20, 50 m L of horseradish peroxidase (HRP)-labeled anti-mouse IgG Fc specific antibody was allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween20, 50% TMB solution was added to each well. mL, induced color development. Then, 2M H 2 SO 4 Add to each well to stop the reaction, and measure the absorbance at a wavelength of 450 nm.

[0092] Since the culture supernatant of the hybridoma that reacts with both TrpE-His-CEACAM1-N and TrpE-His-CEACAM1-A1 is considered to recognize the TrpE-His sequence or the E. coli-derived confounding protein, it was eliminated to obtain an antibody-producing hybridoma having the desired reaction specificity.

[0093] The hybridomas obtained from the spleen of mice immunized with the antigen protein TrpE-His-CEACAM1-N-m3C3d were named A3029, A3048, A3054, A3057, A3067, A3073, and A6056, and the hybridoma obtained from the spleen of mice immunized with the antigen protein TrpE-His-CEACAM1-A1-m3C3d was named A7004.

[0094] The epitopes of the antibodies produced by the obtained hybridomas are shown in Example 3. Hybridomas A3054 and A3067 that produce antibodies with the same epitope are two clones obtained from 500 hybridoma-positive wells, hybridomas A3029, A3048, A3057, and A3073 that produce antibodies with the same epitope are four clones obtained from 500 hybridoma-positive wells, A7004 is a clone obtained from 800 hybridoma-positive wells, and A6056 is a clone obtained from 120 hybridoma-positive wells.

[0095] (D) Cloning of hybridomas The obtained hybridomas were single cloned by the limiting dilution method to establish antibody-producing hybridomas.

[0096] (E) Preparation of anti-CEACAM1 monoclonal antibody The established mouse hybridoma was acclimated and cultured in a serum-free medium (Hybridoma-SFM, GIBCO). The culture was expanded to 50 mL in a T75 flask, and when the cell density was about 5E5 cells / ml, it was transplanted into a culture bag filled with 500 mL of serum-free medium (Nipro). Cultured for 2-4 weeks, the culture supernatant was recovered. The culture fluid was applied to a column filled with Protein G Sepharose (GE Healthcare), and the bound antibody was eluted with a pH 3 buffer. The eluate was quickly neutralized with 2M Tris (pH 8), and the buffer was exchanged with PBS using a desalting column. 5-20 mg of anti-CEACAM1 monoclonal antibody can be obtained from 500 mL of culture fluid.

[0097] Example 3: Epitope analysis of anti-CEACAM1 monoclonal antibodies In order to analyze the epitope of the obtained anti-CEACAM1 monoclonal antibody, a deletion protein obtained by truncating 10 or 20 amino acids from the C-terminus of the N domain protein or the A1 domain protein of CEACAM1 and a deletion protein obtained by truncating 3 amino acids from the N-terminus of the N domain protein were expressed in Escherichia coli, and analysis was performed by dot blot using the E. coli disrupted solution.

[0098] (A) Construction of deletion protein expression plasmid The construction of the protein deletion expression plasmid was carried out using the KOD-Plus-Mutagenesis Kit (TOYOBO). As a template, pAT-GST-His-CEACAM1-N (Nfull) was used to construct the N domain deletion protein expression plasmid, and pAT-TrpE-His-CEACAM1-N-A1 (A1full) was used to construct the A1 domain deletion protein expression plasmid. The primers used were set outside the regions to be deleted. The amino acid sequences corresponding to the various deleted proteins are shown in Table 1. According to the manufacturer's instructions, reverse PCR, decomposition of the template plasmid by DpnI, phosphorylation and self-circularization of the PCR product were performed in sequence. The self-circularized PCR product was used to transform Escherichia coli XL1-blue MRF (Stratagen). The transformed Escherichia coli was spread on a plate containing 100 m g / ml ampicillin on LB plates (1% tryptone, 0.5% NaCl, 1.5% agar) and incubate at 37°C overnight. Use a sterile toothpick to pick up the bacterial colonies produced on the plate and transfer them to a plate containing 100 μg / ml ampicillin. m The cells were cultured in 2YT medium containing 100 μg / ml ampicillin at 37°C overnight. m g / ml ampicillin in M9-CA and cultured overnight at 37°C. After the culture was completed, the cells were collected by centrifugation and solubilized with 1× SDS-PAGE sample buffer (62.5mM Tris-HCl, 10% Glycerol, 2% SDS, 20mM DTT) to prepare E. coli fragments of each protein deletion. SDS-PAGE of the E. coli fragments was performed, and the expression of the target protein was confirmed by Coomassie Brilliant Blue staining and immunoblotting using anti-His-Tag antibodies.

[0099] [Table 1]

[0100] (B) Dot blot The above-mentioned various E. coli lysates were spotted on the nitrocellulose membrane for 1 m After air drying, the cells were immersed in transfer buffer (25 mM Tris, 192 mM glycine, 20% MetOH) for 10 minutes at room temperature and then blocked with a reaction solution (0.1% sodium caseinate, 1 mM EDTA, PBS). m Each antibody diluted in the manner of 100 g / mL was reacted with the spotted nitrocellulose membrane (spot membrane) and allowed to stand at room temperature for 1 hour. The spot membrane was washed with a cleaning solution (0.05% Tween20, PBS), reacted with a horseradish peroxidase (HRP)-labeled anti-mouse IgG Fc specific antibody diluted with the reaction solution, and allowed to stand at room temperature for 1 hour. The spot membrane was washed with a cleaning solution, reacted with a luminescent substrate SuperSignal West Pico (Thermo scientific company), and allowed to stand at room temperature for 5 minutes before detecting chemiluminescence using a CCD imager.

[0101] (C) Results The results of epitope analysis based on dot blot showed that the amino acid sequence of TESMP (SEQ ID NO. 2) in the N domain of A3054 and A3067 antibodies was an epitope. The amino acid sequence of PANSGRETIY (SEQ ID NO. 1) in the N domain of A3029, A3048, A3057, and A3073 antibodies was an epitope. The amino acid sequence of EATGQFHVYP (SEQ ID NO. 4) in the N domain of A6056 antibody was an epitope. The amino acid sequence of DTTYLWWINN (SEQ ID NO. 3) in the A1 domain of A7004 antibody was an epitope.

[0102] Example 4: Cross-reactivity evaluation using recombinant antigens The cross-reactivity of the obtained anti-CEACAM1 monoclonal antibodies (one antibody was selected for antibodies with the same epitope) was evaluated using commercially available recombinant antigens CEACAM1 (R&D Systems), CEACAM3 (R&D Systems), CEACAM5 (R&D Systems), and CEACAM6 (R&D Systems).

[0103] (A) Antigen solid phase ELISA Each antigen was diluted to 1 in TBS containing 1 mM TCEP and 6 M urea. m g / mL. Dilute the antigen at 50 m L was added to the Nunc multi-module plate and allowed to stand overnight at room temperature. The antigen diluent was removed and washed with PBS. 100 μL of mL blocking solution (0.1% sodium casein, 150 mM NaCl, 1 mM EDTA, PBS) was added and allowed to stand at room temperature for 1 hour.

[0104] The blocking solution was removed, and 50% of each antibody diluted in a reaction solution (0.1% sodium caseinate, 150 mM NaCl, 1 mM EDTA, PBS) was added to each well. m L, and allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween 20, 50 m L of horseradish peroxidase (HRP)-labeled anti-mouse IgG Fc specific antibody was allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween20, 50% TMB solution was added to each well. m L, induced color development. Then, 2MH 2 SO 4 Add to each well to stop the reaction, and measure the absorbance at a wavelength of 450 nm.

[0105] (B) Results The results of the cross-reactivity evaluation of CEACAM1 antibodies are shown in Figure 2 . The reactivity to CEACAM3, CEACAM5, and CEACAM6 is expressed as a relative value to CEACAM1 (relative binding rate (%)). The A3054 antibody showed a cross-reactivity of about 18% to CEACAM5, but less than 5% to CEACAM3 and CEACAM6. The A3073 antibody showed no cross-reactivity to CEACAM3, CEACAM5, and CEACAM6, with the highest specificity to CEACAM1. The A6056 antibody showed no cross-reactivity to CEACAM5, but showed a cross-reactivity of about 50% to CEACAM6 and a cross-reactivity of 250% to CEACAM3. The A7004 antibody showed a cross-reactivity of about 5% to CEACAM3 and CEACAM6, and a cross-reactivity of 150% to CEACAM5.

[0106] Example 5: Sequence comparison with the CEACAM family The epitopes of each antibody were compared with the amino acid sequences of the CEACAM family ( Figure 3 ) showed that the A3054 antibody and the A3073 antibody recognized sequences containing amino acids inherent to CEACAM1 (threonine residue (T) in TESMP (SEQ ID NO. 2) and asparagine residue (N) in PANSGRETIY (SEQ ID NO. 1), respectively). Figure 3Indicated by black boxes in the figure). According to the evaluation results of cross-reactivity, these two antibodies have low cross-reactivity with CEACAM5 and CEACAM6, so these CEACAM1-specific amino acids are considered to be epitope hot spots. In the evaluation results of cross-reactivity of the A6056 antibody, CEACAM6 showed moderate cross-reactivity, while CEACAM5 did not cross-react, so the histidine residue (H) in EATGQFHVYP (SEQ ID NO. 4) is considered to be the epitope hot spot ( Figure 3 This sequence is also present in CEACAM3 and CEACAM4, so the A6056 antibody may cross-react with them. In the evaluation results of cross-reactivity, the A7004 antibody strongly cross-reacted with CEACAM5 but not with CEACAM6, so the aspartic acid residue (D) in DTTYLWWINN (SEQ ID NO. 3) is considered to be the hot spot of the epitope ( Figure 3 This sequence is only shared by CEACAM1 and CEACAM5, and therefore is not considered to cross-react with other CEACAM family members.

[0107] Example 6: Cross-reactivity evaluation using peptides Cross-reactivity evaluation using synthetic peptides was performed for the A3054 and A3067 antibodies and the A3029, A3048, A3067, and A3073 antibodies that are thought to recognize CEACAM1-specific sequences.

[0108] (A) Peptide solid phase ELISA Each synthetic peptide was diluted to 20% in TBS containing 1 mM TCEP and 6 M urea. m g / mL. Dilute the antigen at 50 m L was added to the Nunc multi-module plate and allowed to stand overnight at room temperature. The antigen diluent was removed and washed with PBS. 100 μL of m L blocking solution (0.1% sodium casein, 150 mM NaCl, 1 mM EDTA, PBS) was added and allowed to stand at room temperature for 1 hour.

[0109] The blocking solution was removed, and 50% of each antibody diluted in a reaction solution (0.1% sodium caseinate, 150 mM NaCl, 1 mM EDTA, PBS) was added to each well. m L, and allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween 20, 50 m L of horseradish peroxidase (HRP)-labeled anti-mouse IgG Fc specific antibody was allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween20, 50% TMB solution was added to each well. m L, induced color development. Then, 2MH 2 SO 4 Add to each well to stop the reaction, and measure the absorbance at a wavelength of 450 nm.

[0110] (B) Results The cross-reactivity of each antibody is shown as a ratio (%) to the sequence of CEACAM1 in Tables 2 and 3. A3054 and A3067 antibodies showed cross-reactivity to CEACAM3, CEACAM5, and CEACAM6. A3073, A3029, A3048, and A3057 did not show cross-reactivity to any sequence of the CEACAM family and showed high specificity for CEACAM1.

[0111] [Table 2]

[0112] [Table 3]

[0113] Example 7: Construction of CEACAM1 Sandwich Assay (A) Preparation of biotin-labeled antibodies The buffer of the antibody solution was replaced with PBS using a desalting column, and the antibody concentration was adjusted to 0.5 mg / mL. Sulfo-NHS-LC-Biotin (Thermo Scientific) was added to the antibody solution to make it about 70 m M, reacted at room temperature for 90 minutes, and then 1 / 20 volume of 1M Tris (pH 7) was added to stop the reaction as a biotin marker.

[0114] (B) Sandwich ELISA antibody combination Each antibody was diluted with PBS to 5 or 10 m g / mL. Dilute the antibody at 50 μg / mL per well. m L was added to the Nunc multi-module plate and left to stand overnight at 4-8°C. The antibody diluent was removed and washed with PBS. 100 μL of m L of blocking solution (0.1% sodium caseinate, 150 mM NaCl, 1 mM EDTA, PBS) was added and allowed to stand at room temperature for 1 hour to prepare an antibody solid phase plate. The sample was diluted with LUMIPULSE (registered trademark) (Fuji Rebio Co., Ltd.) to a concentration of 1 or 0. m The commercially available CEACAM1 antigen (R&D Systems) was diluted at 50 g / mL. m L and 50 mThe modified sample was prepared by mixing 20 μL of modified solution (10% SDS, 3.75% EDTA2Na, 10 mM TCEP), shaking at 1000 rpm, and heating at 80°C for 10 minutes. m L and 100 m 100 mM Tris-HCl, 200 mM NaCl, 20 mM EDTA3Na, 0.1% ProClin300, 2% BSA, pH 7.5) were mixed and 50 m L was added to the wells of the antibody solid phase plate from which the blocking solution was removed, and the plate was left to stand at room temperature for 1 hour. The wells were washed with PBS containing 0.05% Tween 20, and a marker diluent (50 mM MES, 100 mM NaCl, 0.3 mM ZnCl) was added to the wells. 2 , 1 mM MgCl 2 , 2% BSA, 0.10% NaN 3 , pH 6.8) to become 1 m The biotin-labeled antibody diluted in the manner of 100 g / mL was allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween20, alkaline phosphatase (ALP)-labeled streptavidin diluted with the labeling agent diluent was added to the wells and allowed to stand at room temperature for 30 minutes. After washing the wells with PBS containing 0.05% Tween20, LUMIPULSE substrate solution (Fuji Rebio Co., Ltd.) was added to measure the chemiluminescence amount based on the enzyme reaction.

[0115] Table 4 shows the results of the study of the combination of solid phase antibody and labeled antibody in sandwich ELISA. D 0 count value is from CEACAM1 antigen 1 m Subtract 0 from the measured value of g / mL m When the A3073 antibody, which has the highest specificity for CEACAM1, was used for the solid phase and the A7004 antibody or the A6056 antibody was used as a labeling substance, high reactivity was shown.

[0116] [Table 4]

[0117] (C) Solid-phase antibody combination The effect of combining the A3054 antibody (or A3067 antibody) and the A3073 antibody (or A3029, A3048, A3057) as solid phase antibodies was investigated. The assay method was the same as in the above-mentioned "(B) Sandwich ELISA antibody combination". PBS was used as the solid phase condition, and the solid phase was 5 or 10 m g / mL, and the co-solid phase was diluted to 5 m The samples were mixed at a concentration of 1 or 0 using LUMIPULSE sample diluent (Fuji Rebio Co., Ltd.). m The samples were obtained by diluting commercially available CEACAM1 antigen (R&D Systems) at 1 g / mL.

[0118] Table 5 shows the results of the solid phase antibody combinations. D 0 count value is from CEACAM1 antigen 1 m g / mL Subtract 0 from each measured value m The difference in the measured value of g / mL was obtained. The co-solid phase showed high reactivity compared with the individual solid phases of the A3054 antibody (or A3067 antibody) and the A3073 antibody (or A3029, A3048, A3057). This value was greater than the sum of the individual solid phases, so it was considered that the CEACAM1 binding ability showed a synergistic effect.

[0119] [Table 5]

[0120] (D) Evaluation of cross-reactivity in sandwich assays The cross-reactivity when the A3054 antibody (or A3067 antibody) and the A3073 antibody (or A3029, A3048, A3057) are combined as solid phase antibodies was confirmed. The measurement method was the same as the above-mentioned "(B) Sandwich ELISA antibody combination". PBS was used as the solid phase condition, and the A3054 antibody (or A3067 antibody) and the A3073 antibody (or A3029, A3048, A3057) were 5 m The samples were mixed at a concentration of 1 or 0 using LUMIPULSE sample diluent (Fuji Rebio Co., Ltd.). m The samples were prepared by diluting commercially available CEACAM1 antigen (R&D Systems), CEACAM3 antigen (R&D Systems), CEACAM5 antigen (R&D Systems), and CEACAM6 antigen (R&D Systems) at a concentration of 1 g / mL.

[0121] Table 6 shows the results of the cross-reactivity evaluation in the sandwich assay system. D 0 count value is from CEACAM1 antigen 1 m Subtract 0 from the measured value of g / mL mg / mL. No significant cross-reactivity was shown for CEACAM3, CEACAM5, and CEACAM6 in any solid phase antibody combination. This indicates that the cross-reactive A3054 antibody (or A3067 antibody) does not affect the CEACAM1 specificity of the A3073 antibody (or A3029, A3048, A3057).

[0122] [Table 6]

[0123] Example 8: Evaluation of CEACAM1 assay system using a fully automated chemiluminescent enzyme immunoassay system (A) Preparation of anti-CEACAM1 antibody-conjugated ferrite particles Anti-CEACAM1 antibodies (A3054 and A3073) were added to magnetic particles in 10 mM MES buffer (pH 5.0) to obtain a suspension containing 0.04 mg / mL anti-CEACAM1 antibodies (A3054 and A3073) and 5 mg / mL magnetic particles. The suspension was incubated at 25°C for 1 hour while slowly stirring to solidify the anti-CEACAM1 antibodies (A3054 and A3073) and magnetic particles. Then, the magnetic particles were collected using a magnet and washed with a washing solution (50 mM Tris buffer, 150 mM NaCl, 2.0% BSA, pH 7.2) to obtain anti-CEACAM1 antibodies (A3054 and A3073) solid-phase particles.

[0124] (B) Preparation of alkaline phosphatase (ALP)-labeled anti-CEACAM1 antibody The desalted alkaline phosphatase and N-(4-maleimidobutyryloxy)-succinimide (GMBS) (final concentration 0.3 mg / mL) were mixed in 0.1 M phosphate buffer (pH 7.0) and allowed to stand at 30°C for 1 hour for maleimidation. After desalting with coupling buffer, Fab′ anti-CEACAM1 monoclonal antibody A7004 was mixed with maleimidized alkaline phosphatase at a molar ratio of 1:1 and allowed to stand at 25°C for 30 minutes for coupling. The antibody and ALP mixture after the reaction was concentrated and eluted with ALP buffer (1 mM MgCl 2 , 0.1 mM ZnCl 2 、0.1%NaN 3 , 0.15 M NaCl, 0.1 M MES, pH 6.8) was used for gel filtration purification to obtain ALP-labeled anti-CEACAM1 antibody.

[0125] (C) CEACAM1 assay The CEACAM1 assay was performed using an automatic chemiluminescent enzyme immunoassay system according to the following method. m L and modified treatment solution (10% SDS, 3.75% EDTA2Na, 10mM TCEP) 50 m L were mixed, shaken at 1000 rpm, and heated at 80°C for 10 minutes to prepare a modified sample. m 50 μL of magnetic particle solution (100 mM Tris-HCl, 200 mM NaCl, 20 mM EDTA3Na, 0.1% ProClin300, 2% BSA, pH 7.5) containing magnetic particles obtained by co-binding anti-CEACAM1 antibodies A3054 and A3073 m The magnetic particles were collected and washed to remove the components not bound to the magnetic particles, and a marker diluent (50 mM MES, 100 mM NaCl, 0.3 mM ZnCl) was added. 2 , 1mM MgCl 2 , 2% BSA, 0.10% NaN3, pH 6.8) to 0.2 m ALP labeled A7004 antibody diluted in the manner of 50 g / mL m The magnetic particles were collected and washed to remove the components not bound to the magnetic particles, and 200 μL of substrate solution containing AMPDD (LUMIPULSE substrate solution, manufactured by Fuji Rebio Co., Ltd.) was added. m L. The amount of luminescence generated by the enzyme reaction was counted, and the CEACAM1 value in the sample was calculated from the above counts using a standard curve. The calibration curve was prepared by measuring CEACAM1 (R&D Systems) standard solutions corresponding to 0, 1.6, 8, 40, 200, and 1000 ng / mL of CEACAM1 in the same manner as the sample, and based on the luminescence obtained for each standard solution. All steps after the sample modification treatment in this example were performed using an automatic analyzer LUMIPULSE L2400 (registered trademark, manufactured by Fuji Rebio Co., Ltd.).

[0126] (D)Disease sample measurement The CEACAM1 assay using the fully automated chemiluminescent enzyme immunoassay system was performed on colorectal cancer samples (purchased from TRINA BIOREACTIVES AG) (n=42), liver cancer samples (purchased from TRINA BIOREACTIVES AG) (n=50), pancreatic cancer samples (purchased from TRINA BIOREACTIVES AG) (n=40), and healthy human samples (n=36). Figure 4Figure 5 shows the ROC (Receiver Operating Characteristic) analysis of each cancer sample. The average CEACAM1 concentration of healthy samples was 125.7±49.4ng / mL, showing a relatively stable value. The average CEACAM1 concentrations of colorectal cancer samples, liver cancer samples, and pancreatic cancer samples were 216.1±108.4ng / mL, 399.9±119.3ng / mL, and 430.9±259.4ng / mL, respectively, which were significantly higher than those of healthy samples. As a result of the ROC analysis, in colorectal cancer samples, the area under the curve was 0.836, the specificity was 0.778 when the critical value was set to 154.4ng / mL, and the sensitivity was 0.762. In liver cancer samples, the area under the curve was 0.996, the specificity was 0.994 when the critical value was set to 201.6ng / mL, and the sensitivity was 1.0. In pancreatic cancer samples, the area under the curve was 0.969, the specificity was 0.917 when the cutoff value was set at 181.9 ng / mL, and the sensitivity was 0.925.

[0127] (E) Evaluation of CEACAM1 specificity based on CEA uptake In order to evaluate the specificity of the CEACAM1 measurement system, the anti-CEA monoclonal antibody-bound particle solution of LUMIPULSE Presto CEA Assay Reagent (manufactured by Fuji Xerbio Co., Ltd.) was used to compare the CEACAM1 measurement values ​​of samples that absorbed CEA [absorption (+)] and untreated samples [absorption (-)] to confirm that there was no influence of the coexisting CEA.

[0128] The anti-CEA monoclonal antibody concentrated to 2 times was combined with the particle solution 50 m L and sample 50 m L was mixed, shaken at 1000 rpm, and reacted at 37°C for 30 minutes. The magnetic particles were collected and the supernatant was recovered to prepare a sample that absorbed CEA [absorption (+)]. m L and sample 50 m L was mixed, shaken at 1000 rpm, and reacted at 37°C for 30 minutes to prepare an untreated sample [absorption (-)]. These samples were used for CEACAM1 and CEA assays. CEACAM1 assays were performed according to the above-mentioned CEACAM1 assay method using a fully automatic chemiluminescent enzyme immunoassay system, and CEA assays were performed using an automatic analyzer LUMIPULSE L2400 (manufactured by Fuji Rebio Co., Ltd.) according to the attachment of LUMIPULSE Presto CEA assay reagent (manufactured by Fuji Rebio Co., Ltd.).

[0129] The evaluation results of CEACAM1 specificity based on CEA absorption are shown in Table 7. In samples 1 and 2 where the CEA measurement values ​​exceeded 100 ng / mL, the CEACAM1 measurement values ​​hardly changed despite the absorption (+) condition that allows absorption of approximately 90% of CEA. This indicates that the constructed CEACAM1 measurement system can specifically detect CEACAM1 without being affected by CEA.

[0130] [Table 7]

[0131] Example 9: Study on the method of revealing the epitope of anti-CEACAM1 antibody The anti-CEACAM1 antibody obtained in the present invention recognizes a linear epitope. Therefore, in order to improve the antigen-antibody reaction, an antigen modification process is required. In the above-mentioned embodiment, SDS reduction heat treatment is used as the antigen modification process. Figure 6 As shown, the epitope was exposed by heat treatment of the antigen with SDS reduction, and the CEACAM1-specific signal increased by about 1000 times. As a different modification method, modification with alkali was studied.

[0132] (A) Alkali treatment studies PBS was used to make 5 m Dilute A3054 and A3073 at 50 μg / mL. m L was added to the Nunc multi-module plate and left to stand overnight at 4-8°C. The antibody solution in the wells was removed and washed with PBS. 100 μL of m The plate was incubated with 50 μL of blocking solution (0.1% sodium caseinate, 150 mM NaCl, 1 mM EDTA, PBS) and allowed to stand at room temperature for 1 hour to prepare an antibody solid phase plate. A sample prepared by diluting a commercially available CEACAM1 antigen (R&D Systems) to 1000 or 0 ng / mL using LUMIPULSE (registered trademark) sample diluent (Fuji Rebio) was added. m L and 50 m L of modified solution (0-0.5M NaOH) was mixed and kept at 37°C for 7 minutes to prepare a modified sample. m L and 50 m Mix 50 L of the reaction solution (1.5 M Tris-HCl, 100 mM NaCl, 10 mM EDTA3Na, 0.1% ProClin300, 5% BSA, pH 7.5) and mL was added to the wells of the antibody solid phase plate from which the blocking solution was removed, and the plate was left to stand at room temperature for 1 hour. The wells were washed with PBS containing 0.05% Tween 20, and a marker diluent (50 mM MES, 100 mM NaCl, 0.3 mM ZnCl) was added to the wells. 2 , 1 mM MgCl 2 , 2% BSA, 0.10% NaN 3 , pH 6.8) to become 1 m The ALP-labeled A7004 antibody diluted to 100 g / mL was left to stand at room temperature for 1 hour. The wells were washed with PBS containing 0.05% Tween 20, and then LUMIPULSE substrate solution (Fuji Rebio) was added to measure the chemiluminescence amount based on the enzyme reaction.

[0133] Figure 7 The results of the study on the modification treatment by alkali are shown. In the alkali treatment by NaOH, the CEACAM1-specific signal increased in a concentration-dependent manner. This result shows that not only the SDS reduction heat treatment but also the alkali treatment is effective for the exposure of the epitope.

[0134] (B) Optimization of NaOH concentration for alkali treatment According to the above determination method, the NaOH concentration of the modified treatment solution was optimized to 0-1 M. The results are shown in Figure 8 The results showed that the CEACAM1-specific signal increased in a concentration-dependent manner after NaOH treatment, and the effect of the alkali treatment almost reached a plateau when the concentration was above 0.25 M during the modification treatment.

[0135] Example 10: Determination of CEACAM1 based on alkali modification (A) Detection of CEACAM1 using a fully automated chemiluminescent enzyme immunoassay system Sample 25 m L and modified treatment solution (0.8M NaOH, 0.5% Brij35 (polyoxyethylene 23 lauryl ether)) 25 m L were mixed and subjected to a modification treatment at 37°C for 6.5 minutes. 50 m After neutralization with 1 L of neutralizing solution (1.5 M Tris, 10 mM EDTA3Na, 2% BSA, 0.1% ProClin300, pH 7.5), the cells were mixed with a magnetic particle solution (1.5 M Tris-HCl, 20 mM EDTA3Na, 0.1% ProClin300, 2% BSA, pH 7.5) containing magnetic particles to which anti-CEACAM1 antibodies A3054 and A3073 were co-bound for 50 min. mThe magnetic particles were collected and washed to remove the components not bound to the magnetic particles, and a marker diluent (50 mM MES, 100 mM NaCl, 0.3 mM ZnCl) was added. 2 , 1mM MgCl 2 , 2% BSA, 0.10% NaN3, pH 6.8) to 0.2 m ALP labeled A7004 antibody diluted in the manner of 50 g / mL m The magnetic particles were collected and washed to remove the components not bound to the magnetic particles, and 200 μL of substrate solution containing AMPDD (LUMIPULSE substrate solution, manufactured by Fuji Rebio Co., Ltd.) was added. m L. The amount of luminescence generated by the enzyme reaction was counted, and the CEACAM1 value in the sample was calculated from the above counts using a standard curve. The calibration curve was prepared by measuring CEACAM1 (R&D Systems) standard solutions corresponding to 0, 8, 40, 200, and 1000 ng / mL of CEACAM1 in the same manner as the sample, and based on the luminescence obtained for each standard solution. All the steps of this example were performed using an automatic analyzer LUMIPULSE L2400 (manufactured by Fuji Rebio Co., Ltd.).

[0136] (B) Disease sample measurement The CEACAM1 assay using the fully automated chemiluminescent enzyme immunoassay system was used to measure colorectal cancer samples (purchased from TRINA BIOREACTIVES AG) (n=42), liver cancer samples (purchased from TRINA BIOREACTIVES AG) (n=50), pancreatic cancer samples (purchased from TRINA BIOREACTIVES AG) (n=41), and healthy human samples (n=36). Figure 9Figure 10 shows the ROC (Receiver Operating Characteristic) analysis of each cancer sample. The average CEACAM1 concentration of healthy samples was 166.3±64.9ng / mL, showing a relatively stable value. The average CEACAM1 concentrations of colorectal cancer samples, liver cancer samples, and pancreatic cancer samples were 276.7±124.7ng / mL, 458.716±111.2ng / mL, and 424.3±237.7ng / mL, respectively, which were significantly higher than those of healthy samples. As a result of the ROC analysis, in colorectal cancer samples, the area under the curve was 0.838, the specificity was 0.583 when the critical value was set to 166.2, and the sensitivity was 0.952. In liver cancer samples, the area under the curve was 0.994, the specificity was 0.944 when the critical value was set to 262.9, and the sensitivity was 1.0. In pancreatic cancer samples, the area under the curve was 0.944, the specificity was 0.944 when the critical value was set to 262.2, and the sensitivity was 0.756.

[0137] (C) Comparison with SDS reduction heat treatment Using 168 human blood samples, we compared CEACAM1 concentrations in off-board SDS-reduced heat treatment (X-axis) and on-board alkaline treatment (Y-axis). Figure 11 ). The Pearson's correlation coefficient was r = 0.967, confirming a high positive correlation. The slope of the regression based on the Passing-Bablok method was 1.04.

[0138] SDS reduction heat treatment requires sample modification at high temperature (80°C), so the modification process cannot be automated and the operation is complicated. By introducing an alkali treatment that does not require high temperature treatment, all steps including the modification process can be automated, providing a simpler CEACAM1 measurement method.

[0139] Example 11: Effects of Modification Treatment Using Autoantibody Model Samples In order to demonstrate the usefulness of the CEACAM1 assay system involving modification of the present invention, an assay system not involving modification was constructed using antibodies used in an ELISA kit of R&D Systems, and the reactivity with autoantibody model samples was compared.

[0140] (A) CEACAM1 assay using RD antibody Sample 10 m50 μL and a magnetic particle solution (100 mM Tris-HCl, 20 mM EDTA3Na, 0.1% ProClin300, 2% BSA, pH 7.5) containing magnetic particles bound with anti-CEACAM1 antibody 283324 (R&D Systems) m The magnetic particles were collected and washed to remove the components not bound to the magnetic particles, and a marker diluent (50 mM MES, 100 mM NaCl, 0.3 mM ZnCl) was added. 2 , 1 mM MgCl 2 , 2% BSA, 0.10% NaN3, pH 6.8) diluted 0.1 m g / mL biotinylated anti-CEACAM1 antibody BAF2244 (R&D Systems) and 0.2 m g / mL alkaline phosphatase (ALP)-labeled streptavidin mixture 50 m The magnetic particles were collected and washed to remove the components not bound to the magnetic particles, and 200 μL of substrate solution containing AMPDD (LUMIPULSE substrate solution, manufactured by Fuji Rebio Co., Ltd.) was added. m L. The luminescence amount based on the enzyme reaction was counted, and the CEACAM1 value in the sample was calculated from the above counts using a standard curve. The calibration curve was prepared by measuring CEACAM1 (R&D Systems) standard solutions corresponding to 0, 8, 40, 200, and 1000 ng / mL of CEACAM1 in the same manner as the sample, and based on the luminescence amount obtained for each standard solution. All the steps of this example were performed using an automatic analyzer LUMIPULSE L2400 (manufactured by Fuji Rebio Co., Ltd.).

[0141] (B) Competitive evaluation of CEACAM1 polyclonal antibodies The commercially available recombinant antigen CEACAM1 (R&D Systems) was added to PBS at a concentration of 1. m g / mL. m L was added to the Nunc multi-module plate and allowed to stand overnight at room temperature. The antigen diluent was removed and washed with PBS. 100 μL of m L blocking solution (0.1% sodium casein, 150 mM NaCl, 1 mM EDTA, PBS) was incubated at room temperature for 1 hour.

[0142] The blocking solution was removed and the reaction solution (0.1% sodium caseinate, 150 mM NaCl, 1 mM EDTA, PBS) was added to each well to make 0.1 mThe various antibody solutions were diluted to 0.1 g / mL or m 10 g / mL of each antibody was added m A solution of 50 μg / mL CEACAM1 polyclonal antibody (R&D Systems, AF2244) was prepared. m L, and allowed to stand at room temperature for 1 hour. After washing the wells with PBS containing 0.05% Tween 20, 50 m The wells were washed with PBS containing 0.05% Tween20 and 50% AMPDD solution was added to each well. m L, measure the chemiluminescence.

[0143] Table 8 shows the binding rates of various antibodies after adding the anti-CEACAM1 polyclonal antibody. The binding rates of BAF2244 and 283324 of the R&D Systems antibodies decreased, and the binding rate of the antibody A3054 of the present invention increased slightly, but the binding rates of A3073 and A7004 decreased. These results indicate that both the assay system using the R&D Systems antibodies and the assay system using the antibodies of the present invention were inhibited by the anti-CEACAM1 polyclonal antibody.

[0144] [Table 8]

[0145] (C) Comparison of assay systems using autoantibody model samples Among cancer samples with high CEACAM1 expression, 30 m g / mL, anti-CEACAM1 polyclonal antibody (R&D Systems, AF2244) was added to prepare autoantibody model samples. CEACAM1 measurement was performed "without modification" or "with modification" for samples without anti-CEACAM1 polyclonal antibody (-) and samples with anti-CEACAM1 polyclonal antibody (+). "With modification" was measured according to the above-mentioned CEACAM1 measurement based on alkali modification, and "without modification" was measured according to the above-mentioned CEACAM1 measurement method using R&D Systems antibodies. The results are shown in Figure 12 In the R&D Systems antibody combination without modification, the quantitative value of adding (+) decreased to about 3% compared to the non-addition of polyclonal antibody (-). On the other hand, the modified assay system of the present invention was not greatly affected, and the quantitative value of the added (+) sample was also about 90%.

[0146] There are some reports of autoantibodies against CEA, and the frequency of occurrence in cancer patients is high (references 1, 2). CEACAM1 belongs to the CEA family, so it is believed that the possibility of the occurrence of autoantibodies is high. In addition, the CEA family including CEACAM1 also has high homology in sequence and structure, so autoantibodies against CEA also have the possibility of affecting the CEACAM1 measurement system. From this point of view, the modified CEACAM1 measurement system of the present invention can avoid the influence of blocking substances such as autoantibodies, so it is possible to perform high-precision diagnostic measurements.

[0147] (References) 1.K Albanopoulos et al., Am J Gastroenterol.2000, Apr;95(4):1056-61, PMID:10763959, DOI:10.1111 / j.1572-0241.2000.01982.x 2.D Haidopoulos et al., Eur J Surg Oncol.2000, Dec;26(8):742-6, PMID:11087638, DOI:10.1053 / ejso.2000.0996

Claims

1. A method for detecting CEACAM1, characterized in that: include: (1) a step of modifying CEACAM1 in a sample containing CEACAM1 to generate modified CEACAM1; as well as (2) A step of detecting CEACAM1 by a sandwich assay using both a capture antibody capable of recognizing modified CEACAM1 and a labeled antibody.

2. The method according to claim 1, wherein: The modification is selected from the group consisting of a modifying agent, heating, and combinations thereof.

3. The method according to claim 2, wherein: The modifier is selected from alkaline substances, surfactants, reducing agents, and combinations of two or more thereof.

4. The method according to claim 1, wherein: As one of the capture antibody and the labeling antibody, (a) an antibody capable of recognizing an epitope represented by the amino acid sequence of PANSGRETIY (SEQ ID NO: 1) in CEACAM1 is used.

5. The method according to claim 4, wherein: As one of the capture antibody and the labeling antibody, (b) an antibody capable of recognizing an epitope represented by the amino acid sequence of TESMP (SEQ ID NO: 2) in CEACAM1 is further used.

6. The method according to claim 4, wherein: As the other of the capture antibody and the labeling antibody, (c) an antibody capable of recognizing an epitope represented by the amino acid sequence of DTTYLWWINN (SEQ ID NO: 3) in CEACAM1 was used.

7. The method according to claim 4, wherein: As the other of the capture antibody and the labeling antibody, (d) an antibody capable of recognizing an epitope represented by the amino acid sequence of EATGQFHVYP (SEQ ID NO: 4) in CEACAM1 was used.

8. A method for detecting cancer, characterized in that: include: (1) a step of measuring the amount of CEACAM1 in a sample collected from a subject by the method according to any one of claims 1 to 7; and (2) A step of comparing the measured CEACAM1 amount with a reference value.

9. The method according to claim 8, wherein: The cancer is selected from the group consisting of colorectal cancer, liver cancer, pancreatic cancer, and a combination of two or more thereof.

10. A method for treating cancer, characterized in that: include: (1) a step of measuring the amount of CEACAM1 in a sample collected from a subject by the method according to any one of claims 1 to 7; (2) a step of comparing the measured CEACAM1 amount with a reference value; (3) a step of selecting a subject in which the amount of CEACAM1 measured is higher than the reference value; and (4) A step of administering an anticancer drug to the selected subject.

11. A kit for detecting CEACAM1, characterized in that: include: (1) Modifiers; (2) a capture antibody capable of recognizing modified CEACAM1; as well as (3) A labeled antibody capable of recognizing modified CEACAM1.

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