JP2016008195A - Anti-human cardiac muscle troponin t monoclonal antibody, its production method, hybridoma, hybridoma production method, human cardiac muscle troponin t measuring method and human cardiac muscle troponin t measuring kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anti-human cardiac muscle troponin T monoclonal antibody, a hybridoma producing the antibody, and a human cardiac muscle troponin T measuring method and a human cardiac muscle troponin T measuring kit using the antibody which make it possible to accurately measure human cardiac muscle troponin T.SOLUTION: An anti-human cardiac muscle troponin T monoclonal antibody has an amino acid sequence comprising Glu-Leu-Gln-Ala-Leu-Ile-Glu-Ala-His-Phe-Glu-Asn-Arg-Lys-Lys-Glu-Glu-Glu-Glu-Leu-Val-Ser-Leu-Lys-Asp as an epitope.

Description

  The present application relates to an anti-human cardiac troponin T monoclonal antibody, a method for producing the same, a hybridoma producing the antibody, a method for producing the hybridoma, a method for measuring human cardiac troponin T using the antibody, and a kit for measuring human cardiac troponin T.

Human cardiac troponin T (hereinafter abbreviated as “TnT”) is one of the proteins constituting the myofibrils of the myocardium, and the TnT concentration in the blood increases with the onset of myocardial infarction. To rise. Therefore, the TnT concentration in blood is used as a diagnostic index for myocardial infarction.
Conventionally, the concentration of TnT is measured by an immunoassay. For example, Patent Document 1 discloses an anti-human cardiac troponin T monoclonal antibody used for an immunoassay.

Japanese Patent Laid-Open No. 03-7597

  In the conventional technique described above, a technique capable of measuring TnT with higher accuracy has been demanded. Non-limiting exemplary embodiments of the present application provide an anti-human cardiac troponin T monoclonal antibody capable of accurately measuring TnT, a method for producing the same, a hybridoma producing the antibody, a method for producing the hybridoma, A method for measuring human cardiac troponin T and a kit for measuring human cardiac troponin T are provided.

  The anti-human cardiac troponin T monoclonal antibody according to one aspect of the present application is an anti-human cardiac troponin T monoclonal antibody produced from a hybridoma whose accession number is NITE P-01863 at the National Institute of Technology and Evaluation for Microorganisms. .

  According to an anti-human cardiac troponin T monoclonal antibody according to one embodiment of the present application, a method for producing the same, a hybridoma that produces the antibody, a TnT measurement method using the antibody, and a TnT measurement kit, TnT can be measured with high accuracy. .

It is a figure which shows an example of the measurement result of the binding ability with respect to TnT of a Tn-P15 antibody. It is a figure which shows the amino acid sequence (sequence number: 1-29) of 29 types of peptides used for identification of the epitope of Tn-P15 antibody. It is a figure which shows an example of the measurement result of the binding ability with respect to each of 29 types of peptides used for identification of the epitope of Tn-P15 antibody. It is a figure which shows an example of the immunoassay result using TnT-P15 antibody. It is a figure which shows the amino acid sequence (sequence number: 30) of TnT (wild type). It is a figure which shows the amino acid sequence (sequence number: 32) of TnT (isoform 3).

TnT is known to have at least 6 types of isoforms (Isforms) and contributes to various changes in TnT (eg, J. Biol. Chem. 2002; 277: 35341). 35349).
TnT (wild type) has a molecular weight of about 39,000, and its amino acid sequence is as shown in FIG. 5 (SEQ ID NO: 30). On the other hand, TnT (isoform) shows a specific change in the 1-40th region from the N-terminal side of TnT (wild-type), and isoform 3 is a major measure in adults. Isoform 3 has a molecular weight of about 37,000, and its amino acid sequence (SEQ ID NO: 31) is as shown in FIG. Isoform 3 differs from TnT (wild type) in that the 24-33th amino acid is deleted from the N-terminal side of TnT (wild type).

  On the other hand, it has been reported that the structure of TnT changes in the blood of patients with acute myocardial infarction depending on the time from the onset of myocardial infarction (for example, Clinical Biochemistry. 2007; 40: 851- 855.). Specifically, the TnT concentration in the blood rapidly increases with the damage of the myocardium due to myocardial infarction or the like, but the TnT in the blood decomposes depending on the elapsed time since the onset of the myocardial infarction. Subdivided. For example, there is almost no TnT (isoform 3) having a molecular weight of about 37,000 in blood in a patient 12 hours after the onset of myocardial infarction, and the molecular weight is about 25,000 after being fragmented. And other fragments. The fragmented fragment having a molecular weight of about 25,000 has been confirmed to correspond to the 1-200th region from the N-terminal side of the amino acid sequence of TnT (isoform 3).

  Based on these facts, the present inventor considered that it is necessary to use an antibody that specifically binds to the amino acid sequence of a stable structural site of TnT in order to accurately measure TnT in blood. Specifically, in the case of isoform 3, it is an antibody that specifically binds to the amino acid sequence in the 41-200th region from the N-terminal side.

  Therefore, the present inventor has conducted intensive studies on an antibody that specifically binds to an amino acid sequence of a stable structural site, and has developed an anti-human cardiac troponin T monoclonal antibody, a hybridoma that produces the antibody, a method for producing the hybridoma, the antibody The inventors have completed an invention relating to a method for measuring human cardiac troponin T and a kit for measuring human cardiac troponin T. An anti-human cardiac troponin T monoclonal antibody according to an embodiment of the present application, a hybridoma that produces the antibody, a method for producing the hybridoma, a method for measuring human cardiac troponin T using the antibody, and a measurement kit for human cardiac troponin T include the following: Street.

  The anti-human cardiac troponin T monoclonal antibody according to one aspect of the present application is a Glu-Leu-Gln-Ala-Leu-Ile-Glu-Ala-His-Phe-Glu-Asn-Arg-Lys-Lys-Glu-Glu-Glu -An amino acid sequence containing Glu-Leu-Val-Ser-Leu-Lys-Asp (SEQ ID NO: 32) is used as an epitope.

  The anti-human cardiac troponin T monoclonal antibody according to one embodiment of the present application can measure TnT with high accuracy. This is because the antibody has an antigen recognition site for the amino acid sequence 101-125 from the N-terminal side in the case of isoform 3 and the 111-135th amino acid sequence from the N-terminal side in the wild type. Therefore, even a fragment having a molecular weight of about 25,000 can be bound and does not depend on the isoform, so that it can cope with various changes in TnT.

  In addition, the antigen recognition site of the anti-human cardiac troponin T monoclonal antibody according to one aspect of the present application does not recognize either slow muscle type or fast muscle type skeletal muscle troponin T (Skeletal Troponin T). This is because both slow muscle type and fast muscle type skeletal muscle troponin T is Glu-Leu-Gln-Ala-Leu-Ile-Glu-Ala-His-Phe-Glu-Asn-Arg-Lys-Lys-Glu- This is because it does not have an amino acid sequence corresponding to Glu-Glu-Glu-Leu-Val-Ser-Leu-Lys-Asp (SEQ ID NO: 32). Therefore, the anti-human cardiac troponin T monoclonal antibody according to one embodiment of the present application can measure TnT with high accuracy.

  The anti-human cardiac troponin T monoclonal antibody according to one embodiment of the present application is IgG, but includes not only the IgG itself but also a functional fragment that retains its binding characteristics (for example, F (ab) and F (ab ') 2). Typical examples of such binding properties include Glu-Leu-Gln-Ala-Leu-Ile-Glu-Ala-His-Phe-Glu-Asn-Arg-Lys-Lys-Glu-Glu-Glu-Glu-Leu-Val. -An amino acid sequence containing Ser-Leu-Lys-Asp (SEQ ID NO: 32) is used as an epitope.

  Furthermore, the anti-human cardiac troponin T monoclonal antibody according to one embodiment of the present application includes a variant of the above IgG antibody. Such a variant may be one in which one to several, or one to several tens of amino acids are substituted, deleted, or added in the amino acid sequence of the IgG antibody. Such variants may be mammalian antibodies, humanized antibodies or human antibodies. Such variants also retain the binding properties of the IgG antibody.

A hybridoma according to one embodiment of the present application (Pep. 3 No. 15), which produces the above-mentioned anti-human cardiac troponin T monoclonal antibody, is defined in the Budapest Treaty concerning the international recognition of the deposit of microorganisms in the patent procedure. Accordingly, it was deposited with the Patent Microorganism Depositary Center for Product Evaluation Technology, 2-5-8, Kazusa Kamashika, Kisarazu City, Chiba Prefecture, and was given the deposit number NITE P-01863 as of June 17, 2014.

  Therefore, the anti-human cardiac troponin T monoclonal antibody according to one embodiment of the present application may be an antibody produced by the hybridoma. In a related aspect, the present invention provides a method for producing an anti-human cardiac troponin T monoclonal antibody, which comprises culturing the above hybridoma.

  The method for producing a hybridoma according to one aspect of the present application is configured with a predetermined number of consecutive amino acid sequences among the 41-200th amino acid sequences from the N-terminal side of isoform 3 of human cardiac troponin T to a mammal. Step A for immunization with a peptide, Step B for removing spleen cells from a mammal immunized with the peptide, Step C for cell fusion of the spleen cells and a cell line derived from the myeloma of the mammal to obtain a hybridoma Including. Preferably, in the method, the monoclonal antibody produced from the hybridoma is evaluated by the step D of evaluating the binding ability to human cardiac troponin T, and the hybridoma producing the monoclonal antibody having the binding ability to human cardiac troponin T. And a step E of sorting.

  In the method for producing a hybridoma according to one aspect of the present application, the peptide that immunizes a mammal is preferably a peptide composed of an amino acid sequence composed of 10 to 30 amino acids. It is not limited to. However, a peptide composed of an amino acid sequence composed of more than 30 amino acids may be used, or the 41st to 200th amino acid sequence itself from the N-terminal side of isoform 3 of human cardiac muscle troponin T may be used. . However, when a peptide composed of an amino acid sequence composed of less than 10 amino acids is used, the peptide may not be recognized as an antigen in the living body of a mammal, so a peptide composed of 10 or more amino acids Preferably there is. More preferably, the peptide has the amino acid sequence Glu-Leu-Gln-Ala-Leu-Ile-Glu-Ala-His-Phe-Glu-Asn-Arg 101-125 from the N-terminal side in isoform 3. -It contains Lys-Lys-Glu-Glu-Glu-Glu-Leu-Val-Ser-Leu-Lys-Asp (SEQ ID NO: 32). In addition, in the amino acid sequence of SEQ ID NO: 32, the peptide has 1 to several (for example, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions. It may be a mutated peptide comprising an added or deleted amino acid sequence. The hybridoma obtained using the peptide comprising the amino acid sequence of SEQ ID NO: 32 or a mutant peptide thereof can produce a monoclonal antibody capable of measuring TnT with high accuracy. In a related aspect, the present invention provides a method for producing an anti-human cardiac troponin T monoclonal antibody, which comprises culturing the above hybridoma.

  The measurement method of TnT which concerns on 1 aspect of this application is an immunoassay method, Comprising: Glu-Leu-Gln-Ala-Leu-Ile-Glu-Ala-His-Phe-Glu-Asn-Arg-Lys-Lys-Glu -An anti-human cardiac troponin T monoclonal antibody having an epitope of an amino acid sequence containing Glu-Glu-Glu-Leu-Val-Ser-Leu-Lys-Asp (SEQ ID NO: 32) is used.

  A method for measuring TnT according to one embodiment of the present application is, for example, an enzyme immunoassay represented by an enzyme-linked immunosorbent assay (hereinafter referred to as “ELISA method”) (Enzyme Immunoassay: Any one of “EIA method”, radioimmunoassay (hereinafter referred to as “RIA method”), or fluorescent antibody method (Fluorescent Antibody Technique) may be used. Alternatively, a chemiluminescence assay or an electrochemiluminescence assay may be used.

  As a measurement format, a typical method includes a direct adsorption method, a sandwich method, and a competitive method, and any of them may be used.

  The measurement kit for TnT according to one embodiment of the present application includes Glu-Leu-Gln-Ala-Leu-Ile-Glu-Ala-His-Phe-Glu-Asn-Arg-Lys-Lys-Glu-Glu-Glu-Glu- An anti-human cardiac troponin T monoclonal antibody having an amino acid sequence containing Leu-Val-Ser-Leu-Lys-Asp (SEQ ID NO: 32) as an epitope is included. Usually, an instruction manual is attached to the kit.

In the present application, amino acids are represented by abbreviations as follows. In addition, when there are optical isomers with respect to amino acids, the L-form is shown unless otherwise specified.
(1) A, Ala: Alanine
(2) R, Arg: Arginine
(3) N, Asn: Asparagine
(4) D, Asp: Aspartic acid (Asparagine Acid)
(5) C, Cys: Cysteine
(6) Q, Gln: Glutamine
(7) E, Glu: Glutamic acid (Glutamic Acid)
(8) G, Gly: Glycine
(9) H, His: Histidine
(10) I, Ile: Isoleucine
(11) L, Leu: Leucine
(12) K, Lys: Lysine
(13) M, Met: Methionine
(14) F, Phe: Phenylalanine
(15) P, Pro: Proline
(16) S, Ser: Serine
(17) T, Thr: Threonine
(18) W, Trp: Tryptophan
(19) Y, Tyr: Tyrosine
(20) V, Val: Valine

  The following shows an example of an anti-human cardiac troponin T monoclonal antibody, a hybridoma producing the antibody, a TnT measurement method using the antibody, and a TnT measurement kit according to one embodiment of the present application. A detailed description will be given. However, the examples are for illustrative purposes and should not be construed as limiting the scope of the present invention.

Example 1 Preparation of Anti-Human Cardiac Troponin T Monoclonal Antibody and Hybridoma The inventor of the present application obtained an anti-human cardiac troponin T monoclonal antibody and a hybridoma according to one embodiment of the present application as follows.

1. Immunization to mammals A peptide (ELQALIEAHFENRKKEEEELVSLKD (SEQ ID NO: 32)) corresponding to the amino acid sequence 101-125 from the N-terminal side of TnT (isoform 3) is prepared, and the mammal is immunized with this peptide. Antibody-producing cells were produced in the mammalian body.
As mammals to be immunized, mice, rats, cows, rabbits, goats and the like can be used, but the present inventor used mice. Examples of immunized mouse systems include A / J strain, BALB / C strain, DBA / 2 strain, C57BL / 6 strain, C3H / He strain, SJL strain, NZB strain, and CBA / JNCrj strain. It is done. BALB / C strain mice are known to show high antibody titers (Titer Of Antibody) in the serum after immunization, and it is expected that monoclonal antibodies with high affinity with TnT will be obtained. The present inventor decided to use a mouse of this strain because it is generally used in the production of a large amount of antibody by ascites after the establishment of a cell line (Cell Line).

  The age of the mouse to be immunized is not particularly limited, but generally it is 4 to 12 weeks old, preferably 6 to 10 weeks old, more preferably 7 weeks old Age. The inventor of the present application used a female BALB / C mouse that was 7 weeks old.

  When the peptide used for immunization is a synthetic peptide, it is difficult to be recognized as an antigen in the living body of a mammal. Therefore, it is desirable to use a synthetic peptide bound to a carrier protein for immunization. Examples of the carrier protein include keyhole limpet hemocyanin (hereinafter abbreviated as “KLH”), bovine serum albumin (hereinafter abbreviated as “BSA”) and egg white albumin (Ovaalbuminn; “OVA”). Such as albumin, Thyroglobin (sometimes abbreviated as “THY”), and the like. The inventor of the present application used KLH as a carrier protein. Synthetic peptides used for immunization usually have a purity of 70% or more with respect to the total protein, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more. is there.

  The synthetic peptide used for immunization is preferably used in combination with an adjuvant. This is because the use of a mixture of a synthetic peptide and an adjuvant can be expected to enhance the immune response to mammalian antigens.

  Examples of adjuvants include water-in-oil emulsions (eg, incomplete Freund's adjuvant), water-in-oil-in-water emulsions, oil-in-water emulsions, liposomes, aluminum hydroxide gels, silica adjuvants, powdered bentonite and tapioca adjuvants. . In addition, cells such as BCG and Propionbacterium acnes, cell components such as cell walls and trehalose dicholate (TDM), lipopolysaccharide (LPS) which is an endotoxin of Gram-negative bacteria, lipid A fraction, β-glucan (polysaccharide) Body), synthetic compounds such as muramyl dipeptide (MDP), bestatin, and levamisole, thymic hormones, thymic hormone humoral factors, and proteins or peptide substances derived from biological components such as tuftsin can also be used as an adjuvant. Further, a mixture of these (for example, complete Freund's adjuvant) may be used as an adjuvant.

  These adjuvants can enhance or suppress the immune response depending on the route of administration, dose, timing of administration, etc. to the mammal. Furthermore, depending on the type of adjuvant, there are differences in antibody production against antigens, induction of cellular immunity, and immunoglobulin classes. Therefore, it is necessary to select an appropriate adjuvant depending on the intended immune response. The inventor used complete Freund's adjuvant.

  Mammal immunization is performed, for example, by injecting a synthetic peptide subcutaneously, intradermally, intravenously or intraperitoneally into the mammal. Since the immune response varies depending on the type and strain of the target mammal, the immunization schedule needs to be appropriately set according to the mammal to be used. Further, after the first immunization of the mammal, additional immunization is performed multiple times.

≪Reagent≫
The following reagents (a) and (b) were prepared.
(A) Phosphate Buffer Saline (hereinafter abbreviated as “PBS”)
PBS was prepared by dissolving NaCl, KCl, Na ₂ HPO ₄ · 12H ₂ O, and KH ₂ PO ₄ in water to final concentrations of 137 mM, 2.7 mM, 8.1 mM, and 1.47 mM, respectively.

(B) First antigen solution A synthetic peptide (ELQALIEAHF ENRKKEEEELVSLKD (SEQ ID NO: 32)) conjugated with KLH as a carrier protein is prepared, and the synthetic peptide is added with PBS so that the final concentration is 1 mg / ml. It melt | dissolved and the 1st antigen solution was prepared. In addition, the synthetic peptide which combined KLH used what requested the synthesis | combination to SIGMA.

≪Procedure≫
The BALB / C strain mice were immunized by the following procedures (1) to (4).
(1) To the first antigen solution, the same volume of human tuberculosis-killed complete Freund's adjuvant (H37Rv, manufactured by Wako Pure Chemical Industries, Ltd.) is added as an adjuvant, and sufficiently emulsified with a homogenizer (homogenizer rotation speed is 1000 rpm). Adjuvant emulsions (Adjuvant Emulsion) were prepared.
(2) 50 μl of the prepared adjuvant emulsion was intraperitoneally injected into each of five female mice of the 7-week-old BALB / C strain.
(3) Two weeks after the initial injection of the adjuvant emulsion, each of these five mice was injected intraperitoneally with 50 μl of the adjuvant emulsion.
(4) Thereafter, similarly, 4 weeks, 6 weeks and 8 weeks after the first injection, 50 μl of the adjuvant emulsion was injected intraperitoneally into each of these 5 mice.

2. Confirmation of antibody production in the mouse body After immunization to mammals, it was confirmed that anti-human cardiac troponin T antibody was produced in vivo. Specifically, blood is collected from an immunized mammal, and the obtained blood is used to measure TnT binding activity (Binding Activity), thereby producing an anti-human cardiac troponin T antibody in the body of the mammal. Confirmed that. It was also confirmed that IgM to IgG class switching (Immunoglobulin Class Switching) occurred at the end of immunization. The present inventor made these evaluations one week after the second injection and one week after the fourth injection to the BALB / C strain mice.

  The measurement of the binding activity can be confirmed by, for example, ELISA method, RIA method or fluorescent antibody method. The inventor of the present application employs the ELISA method for measuring the binding activity of TnT.

≪Reagent≫
The following reagents (c) to (h) were newly prepared.
(C) PBS-Az solution Sodium azide was added to PBS to a final concentration of 0.04% by weight to prepare a PBS-Az solution.
(D) TnT solution TnT (manufactured by Fitzgerald, 30C-CP3037) was dissolved in a PBS-Az solution to a final concentration of 0.1 μg / ml to prepare a TnT solution.
(E) Blocking solution A skim milk was added to the PBS-Az solution so that it might become 5 weight%, and the blocking solution was prepared.
(F) PBST solution Tween 20 (manufactured by Wako Pure Chemicals, 167-11515) was added to PBS to a final concentration of 0.05% by volume to prepare a PBST solution.
(G) BSA solution Bovine Serum Albumin (hereinafter abbreviated as “BSA”) (manufactured by Sigma, A7888) was added to the PBS-Az solution so that the final concentration was 1% by weight, and the BSA solution was added. Prepared.
(H) Labeled antibody solution A peroxidase-labeled goat-derived anti-mouse IgG antibody (manufactured by Southern Biotech, 1034-05) was dissolved in a BSA solution to a final concentration of 0.2 μg / ml to prepare a labeled antibody solution.

≪Procedure≫
<Measurement of TnT binding activity using mouse blood>
TnT binding activity was measured using the blood of the immunized mouse according to the following procedures (1) to (11).
(1) Blood was collected from each of the mice (5 mice) one week after the second injection and one week after the fourth injection, and serum was obtained from the blood.
(2) TnT solution was injected into a microplate (manufactured by Nunc, immunoplate maxisorp round bottom U96, 449824) at a rate of 100 μl per well and stored overnight in saturated water vapor at room temperature.
(3) The TnT solution in the microplate stored overnight was removed with an aspirator, 200 μl of blocking solution was injected into each well for each well, and left at room temperature for 60 minutes.
(4) The blocking solution in the microplate was discarded, each well was washed 3 times with PBST solution, and then the PBST solution remaining in each well was removed with an aspirator.
(5) For each well, 50 μl of the serum obtained per well was injected and left at room temperature for 90 minutes.
(6) The serum in the microplate was discarded, each well was washed 3 times with PBST solution, and then the PBST solution remaining in each well was removed with an aspirator.
(7) For each well, 50 μl of the labeled antibody solution was injected into one well and left at room temperature for 30 minutes.
(8) The labeled antibody solution in the microplate was discarded, washed with PBST solution three times, and the PBST solution remaining in each well was removed with an aspirator.
(9) For each well, 50 μl of a coloring reaction reagent (Thermo TMB ELISA, 1 STEP Ultra) was injected into each well and allowed to react at room temperature for 5 minutes.
(10) After the reaction for 5 minutes, 50 μl of 4N sulfuric acid was injected into each well for each well to stop the color reaction.
(11) After stopping the color development reaction, the absorbance (wavelength of 450 nm) was measured for the solution in each well using a microplate reader (manufactured by TECAN, Infinite 200).

<Confirmation of class switch to IgG>
The class switch to IgG is based on the binding reactivity to the anti-mouse IgG antibody (manufactured by Southern Biotech, 1034-05) against the serum obtained from the mouse one week after the fourth injection to the mouse obtained as described above. confirmed.

≪Result≫
<Measurement of TnT binding activity using mouse blood>
The TnT binding activity of the obtained blood was evaluated based on whether or not a clear color was observed in the absorbance (wavelength of 450 nm) of the solution in the well. That is, if clear color development is observed, it means that antibody production is carried out in the living body of a BALB / C strain female mouse corresponding to the obtained blood.

  As a result, production of anti-human cardiac troponin T antibody was observed in all 5 mice immunized. In particular, since the titer of two mice was high, these two mice were used in the experiments described later.

<Confirmation of class switch to IgG>
As a result of confirming the binding reactivity to the anti-mouse IgG antibody (manufactured by SouthernBiotech, 1034-05) against the serum obtained from the mouse one week after the fourth injection to the mouse obtained as described above, any mouse It was confirmed that a class switch to IgG was also occurring in the blood of the blood.

3. Additional Injection of Immunogen After confirming that the anti-human cardiac troponin T antibody was produced in the living body by measuring the binding activity of TnT as described above, the mouse was further injected with the immunogen (hereinafter referred to as “boost”). ), The mouse spleen was enlarged.

  The amount of the synthetic peptide administered in the boost is not particularly limited, but is preferably 4 to 5 times the amount of the synthetic peptide immunized first. In addition, an emulsion in which a synthetic peptide and an adjuvant are emulsified is usually used for boosting, but in the final immunization (additional injection performed several days before cell fusion by preparing a hybridoma described later), an adjuvant is not used and only the synthetic peptide is used. It is preferable to use it. Moreover, as a route of administration of TnT to a mouse, there is a possibility that an antibody that recognizes a different site with respect to TnT can be obtained depending on each of subcutaneous, intradermal, intravenous, or intraperitoneal.

≪Reagent≫
The following reagent (i) was newly prepared.
(I) Second antigen solution Prepare a synthetic peptide (ELQALIEAHF ENRKKEEEEL VSLKD) to which KLH is bound as a carrier protein, dissolve the synthetic peptide by adding PBS so that the final concentration is 0.5 mg / ml, A second antigen solution was prepared.

≪Procedure≫
For each of the three mice, a booster injection of the immunogen was performed by injecting 100 μl of the second antigen solution intraperitoneally 3 months after the first immunization.

4). Preparation of Hybridoma After final immunization of mice, spleen cells were extracted from the mice, and the extracted spleen cells were fused with myeloma-derived cell lines to obtain hybridomas.
Since the growth ability of the hybridoma depends on the type of myeloma-derived cell line used at the time of cell fusion, it is necessary to select a cell line derived from myeloma with excellent growth ability. In addition, it is necessary to consider the compatibility of the cell line derived from myeloma with the origin of the fused spleen cells. Examples of mouse myeloma-derived cell lines include P3X63-Ag8.653, Sp2 / O-Ag14, FO · 1, S194 / 5. XX0 BU. l, P3 / NS1 / 1-Ag4-1. The inventor of the present application selected P3X63-Ag8.653, which is known to have an excellent ability to grow a hybridoma, as a cell line derived from myeloma.

  Examples of techniques for cell fusion of spleen cells and myeloma-derived cell lines include the polyethylene glycol method, a method using Sendai virus, and a method using electric current. The inventor of the present application employs a polyethylene glycol method with low cytotoxicity, easy fusion operation, and high reproducibility.

≪Procedure≫
A hybridoma was prepared by the following procedures (1) to (4).
(1) Three days after the final immunization, spleen cells of the target mice were removed.
(2) Spleen cells were fused with P3X63-Ag8.653 using polyethylene glycol (MERCK, 807589, average molecular weight 1,500) to prepare a hybridoma.
(3) Hypoxanthine / Aminopterin / Thymidine medium prepared in Ishikov medium containing 15% by weight of fetal calf serum (hereinafter abbreviated as “FCS”) Hereinafter, the hybridoma was suspended in “HAT medium”.
(4) After dispensing the hybridoma on the microplate, the cells were cultured for 1 week in a CO ₂ incubator (manufactured by Sanyo Electric Co., Ltd., MCO-40AIC). At this time, the same mouse solid spleen cells were used as feeder cells, which are growth factors at the start of hybridoma culture. The hybridoma culture conditions are a CO ₂ concentration of 5% by volume, a temperature of 37 ° C., and a humidity of 95%. In the following experiments, hybridomas are cultured under these conditions unless otherwise specified.

5. Cell Selection and Cloning By evaluating the ability of antibodies produced from the prepared hybridomas to bind to TnT, hybridomas producing antibodies with the desired specificity were selected, and the selected hybridomas were cloned.

The evaluation of the binding ability can be confirmed by ELISA method, RIA method, and fluorescent antibody method, but the present inventor adopted the ELISA method.
The anti-human cardiac troponin T monoclonal antibody used for the measurement of TnT needs to have a high binding ability to TnT. On the other hand, the anti-human cardiac troponin T monoclonal antibody used for the measurement of TnT needs to have no binding ability or low binding ability to skeletal muscle troponin T. Therefore, the inventor of the present application evaluates the binding ability to TnT and the binding ability to skeletal muscle troponin for the antibodies produced from each of the obtained hybridomas, and selects the hybridoma that produces the antibody having the target specificity. did.

As used herein, “an antibody having a high binding ability to TnT” refers to an antigen-antibody reaction with TnT in an ELISA method (detailed “inhibition ELISA method”) shown in the experimental procedure described later. This means that the inhibition is applied and the calculated half value of the inhibition is 1.0 × 10 ⁻⁹ M or less.
In addition, “inhibition takes place” as used in the present application means that the amount of an antibody that binds to TnT immobilized on a solid phase is greater in the presence of a competitor (Inhibitor) than in the absence of a competitor. It means to decrease. On the other hand, “inhibition does not occur” means that the amount of antibody that binds to TnT immobilized on the solid phase is the same in the presence and absence of the competitor (including the case where it is substantially equivalent). It means that.
As used herein, “half-value of inhibition” refers to the concentration of an inhibitor at which the absorbance at half of the absorbance (reflecting the amount of antibody bound) in the absence of a competitor is measured.

  Hybridomas can be selected by, for example, a limiting dilution method or a soft agar method. The inventor of the present application has cloned a hybridoma by a limiting dilution method that is easy to operate and highly reproducible. In order to efficiently select useful cells from many hybridomas obtained by cell fusion, it is preferable to select cells from the initial stage of cloning. The finally selected hybridoma can be stored semi-permanently in liquid nitrogen.

≪Reagent≫
The following reagent (j) was newly prepared.
(J) Cell preservation solution 10 parts by volume of FCS and 90 parts by volume of dimethyl sulfoxide were mixed to prepare a cell preservation solution.

≪Procedure≫
Cell sorting and cloning were performed according to the following procedure.
<First stage selection>
(1) One week after culturing the hybridoma, 100 μl of the culture supernatant in each well of the microphone plate was collected. On the other hand, the culture solution remaining in each well was subcultured to a 24-well plate, and 1 ml of hypoxanthine / thymidine (HT) medium containing 15% by weight of FCS was added to each well.
(2) Four days after the passage of the hybridoma in a 24-well plate, 150 μl of the culture supernatant was collected per well. The culture supernatant that was subcultured 1 week after the culture of the hybridoma and 4 days later was used for measuring the binding ability to TnT by the ELISA method shown in the following (3) to (12).
(3) 100 μl of TnT solution was injected into a microplate (manufactured by Nunc, immunoplate maxisorp round bottom U96, 449824) per well, and stored overnight in saturated water vapor at room temperature.
(4) The TnT solution in the microplate stored overnight was removed with an aspirator, 200 μl of blocking solution was injected into each well for each well, and left at room temperature for 60 minutes.
(5) The blocking solution in the microplate was discarded, each well was washed three times with PBST solution, and then the PBST solution remaining in each well was removed with an aspirator.
(6) For each well, 50 μl of the serum obtained per well was injected and left at room temperature for 90 minutes.
(7) The serum in the microplate was discarded, each well was washed three times with PBST solution, and then the PBST solution remaining in each well was removed with an aspirator.
(8) For each well, 50 μl of the labeled antibody solution was injected into one well and left at room temperature for 30 minutes.
(9) The labeled antibody solution in the microplate was discarded, washed with PBST solution three times, and the PBST solution remaining in each well was removed with an aspirator.
(10) For each well, 50 μl of a color reaction reagent (Thermo TMB ELISA, 1 STEP Ultra) was injected into each well and allowed to react at room temperature for 5 minutes.
(11) After the reaction for 5 minutes, 50 μl of 4N sulfuric acid was injected into each well for each well to stop the color reaction.
(12) After stopping the color reaction, the absorbance (wavelength of 450 nm) was measured for the solution in each well using a microplate reader (manufactured by TECAN, Infinite 200).
(13) Based on the measurement results of absorbance, wells having high binding ability to TnT and having a good growth state were selected.
(14) The hybridomas in the selected wells were subcultured to 6-well plates, and 4 ml each of hypoxanthine / thymidine (HT) medium containing 15% by weight of FCS was added to each well and cultured for 2 days.

<Second stage selection>
(1) Two days after passage of the hybridoma to a 6-well plate, 150 μl of the culture supernatant was collected per well. This culture supernatant was used for measuring the binding ability to TnT by the inhibition ELISA method shown in the following (2) to (12).
(2) TnT solution was injected into a microplate (manufactured by Nunc, immunoplate maxisorp round bottom U96, 449824) at a rate of 100 μl per well and stored overnight in saturated water vapor at room temperature.
(3) The TnT solution in the microplate stored overnight was removed with an aspirator, 200 μl of blocking solution was injected into each well for each well, and left at room temperature for 60 minutes.
(4) The blocking solution in the microplate was discarded, each well was washed 3 times with PBST solution, and then the PBST solution remaining in each well was removed with an aspirator.
(5) For each well, 50 μl of the serum obtained per well was injected and left at room temperature for 90 minutes.
(6) The serum in the microplate was discarded, each well was washed 3 times with PBST solution, and then the PBST solution remaining in each well was removed with an aspirator.
(7) For each well, 25 μl of TnT solution as an inhibitor was injected into each well, 25 μl of the labeled antibody solution was further injected, and allowed to stand at room temperature for 1 hour.
(8) The labeled antibody solution in the microplate was discarded, washed with PBST solution three times, and the PBST solution remaining in each well was removed with an aspirator.
(9) For each well, 50 μl of a coloring reaction reagent (Thermo TMB ELISA, 1 STEP Ultra) was injected into each well and allowed to react at room temperature for 5 minutes.
(10) After the reaction for 5 minutes, 50 μl of 4N sulfuric acid was injected into each well for each well to stop the color reaction.
(11) After stopping the color development reaction, the absorbance (wavelength of 450 nm) was measured for the solution in each well using a microplate reader (manufactured by TECAN, Infinite 200).
(12) Inhibited wells were selected based on the absorbance measurement results.
(13) The cells in each selected well were subcultured into medium flasks (50 ml). As the medium, 45 ml of HT medium containing 15% by weight of FCS was added and cultured for 3 days.

<Measurement of binding ability to TnT and skeletal muscle troponin T>
The measurement of the binding ability to TnT is the same as the inhibition ELISA method performed in the above-described second stage of selection, and thus the description thereof is omitted. The measurement of the binding ability to skeletal muscle troponin T is omitted because the antigen and inhibitor immobilized in the microplate are merely changed from TnT to skeletal muscle troponin T.

<Isolation and selection of hybridomas by limiting dilution method>
(1) The hybridoma contained in each selected well is limit-diluted to a concentration containing 2 cells per well using HT medium containing 15% by weight of FCS, and each diluted solution is added to a 96-well microplate. Dispense into each well.
(2) The early growth of hybridomas was promoted using feeder cells from breast cells of 4-week-old BALB / C female mice.
(3) The hybridoma in each well is appropriately subcultured to increase the culture scale. At each subculture, measurement of the binding ability to TnT performed in the above-mentioned “first stage selection” (ELISA) Screening) was repeated.
(4) Screening was performed by repeating subculture, and finally, the hybridomas in 200 ml of the medium were grown until they reached about 5.0 × 10 ⁵ cells / ml.
(5) By repeating the screening, a hybridoma having the target function was selected.

<Preservation of hybridoma>
(1) The selected hybridoma was centrifuged (1600 rpm, 5 minutes), and the culture supernatant was removed.
(2) The hybridoma from which the culture supernatant was removed by centrifugation was suspended in a cell preservation solution so that the hybridoma was about 1.0 × 10 ⁷ cells / ml. The number of cells per ml of hybridoma was adjusted by counting the number of cells per unit area with a hemocytometer.
(3) The hybridoma suspended in the cell preservation solution was pre-frozen at −80 ° C. and then stored in liquid nitrogen.

≪Result≫
<First stage selection>
Of the culture supernatants collected twice, hybridomas in 45 wells that had a high binding ability to TnT and had a good growth state by qualitative judgment were used for the second stage selection.

<Second stage selection>
The well in which an antibody that binds to TnT is present in the culture supernatant binds to TnT added as an inhibitor, and the binding to TnT immobilized on the microplate is inhibited. Color development is suppressed. Therefore, 18 wells that had been subjected to inhibition were selected by the inhibition ELISA method and used to measure the binding ability to TnT and skeletal muscle troponin T.

<Measurement of binding ability to TnT and skeletal muscle troponin T>
About the hybridoma in 18 wells selected by the second stage selection, the binding ability to TnT and skeletal muscle troponin T is measured, and the inhibition is applied only to TnT, and the inhibition is caused by skeletal muscle troponin T. The 8 wells that did not exist were selected and used to isolate hybridomas by limiting dilution.

<Isolation and selection of hybridomas by limiting dilution method>
Finally, one hybridoma producing a monoclonal antibody having high binding ability to TnT and not binding ability to skeletal muscle troponin T was selected.
This selected hybridoma is referred to as Pep. 3 No. No. 15 was deposited at the Patent Microorganisms Depositary Center for Product Evaluation Technology, 2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, and the accession number NITE P on June 17, 2014 -01863. In the following description, Pep. 3 No. The monoclonal antibody produced by 15 will be referred to as TnT-P15 antibody.

6). Purification of antibody The hybridoma obtained by the above-described cell selection and cloning was cultured in large quantities, and the anti-cardiac troponin T monoclonal antibody produced from the hybridoma cultured in large quantities was purified.

  As a method for culturing a hybridoma in large quantities, there are a method performed in vivo and in vitro. An example of a large-scale culture method in vivo includes a method in which a hybridoma is injected into the abdominal cavity of a mouse and the hybridoma is proliferated in the mouse ascites. Thereby, a large amount of anti-cardiac troponin T monoclonal antibody can be produced in mouse ascites. On the other hand, in vitro culture includes a method of culturing hybridomas in a serum-free medium (ASF medium, ASF medium). As a result, a large amount of anti-cardiac troponin T monoclonal antibody can be produced in the culture solution. The inventor of the present application performed large-scale doubling in vivo.

  Anti-cardiac troponin T monoclonal antibody was purified using the obtained ascites. As a method for purifying the antibody, DEAE (abbreviation of “Diethylamino Ethyl”) anion exchange chromatography, affinity chromatography (Affinity Chromatography), ammonium sulfate fractionation method, polyethylene glycol (hereinafter referred to as “Polyethylene glycol”). And abbreviated as “PEG”.) Fractionation method (PEG Fractionation), ethanol fractionation method (Ethanol Fractionation), etc. These purification methods can also be used in appropriate combination. The inventor of the present application purified the anti-cardiac troponin T monoclonal antibody by affinity chromatography.

  In the purification of the antibody, the purity of the anti-cardiac troponin T monoclonal antibody with respect to the total protein is preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more.

≪Reagent≫
The following reagents (k) and (l) were newly prepared.
(K) Binding Buffer A binding buffer was prepared by dissolving in water such that the final concentrations of glycine and NaCl were 1.5 M and 3 M, respectively, and adjusting to pH 8.9.
(L) Elution buffer The elution buffer was prepared by dissolving citric acid in water to a final concentration of 100 mM and adjusting the pH to 4.0.

≪Procedure≫
Each of the TnT-P15 antibody and the TnT-P32 antibody was purified by the following procedures (1) to (8).
(1) Each of the hybridomas (Pep. 3 No. 15) was injected into the abdominal cavity of a 7-week-old female mouse of BALB / C strain.
(2) Accumulated ascites was collected sequentially from the surviving mice while confirming the ascites accumulation from intraperitoneal injection.
(3) A column packed with protein A binding gel (GE Healthcare, Protein A Sepharose 4FF) was equilibrated with binding buffer.
(4) The collected ascites was diluted 3-fold with a binding buffer and applied to an equilibrated column.
(5) The eluate from the column was monitored by absorbance at a wavelength of 280 nm, and the column was washed with a binding buffer until the elution of impurities was completed.
(6) After washing the column, an elution buffer was applied to the column (linear flow rate was 20 cm / hour), and an IgG-containing eluate was collected.
(7) Absorbance at a wavelength of 280 nm was measured for each purified fraction of the collected IgG-containing eluate, and the antibody concentration was calculated by converting the measured absorbance into an extinction coefficient.
(8) Each of the purified fractions of the IgG-containing eluate was subjected to SDS polyacrylamide gel electrophoresis and compared with a standard protein (protein marker).

≪Result≫
As a result of SDS polyacrylamide gel electrophoresis, it was confirmed that all of the purified fractions of the TnT-P15 antibody were IgG composed of an H chain with a molecular weight of about 50,000 and an L chain with about 25,000.

Example 2 Antibody TnT-P15 antibody obtained by purification was measured for its ability to bind to TnT.
≪Procedure≫
Since this is the same as the inhibition ELISA method performed in the “second stage selection” in the “cell selection and cloning” described above, the description is omitted.

≪Result≫
FIG. 1 is a graph showing the measurement results of the binding ability of TnT-P15 antibody to TnT.
In the graph shown in FIG. 1, the vertical axis represents absorbance at a wavelength of 450 nm (in FIG. 1, “OD450”), and the horizontal axis represents the logarithmic value of TnT concentration (M).

As a result of calculating the half value of the inhibition of the TnT-P15 antibody based on the result of FIG. 1, it was 1.0 × 10 ⁻⁹ . As described above, an antibody having an inhibition half value of 1.0 × 10 ⁻⁹ or less is an antibody having a high binding ability to TnT, and thus has a high binding ability to TnT.

8). Epitope identification The antigen recognition site of the TnT-P15 antibody was identified. First, a plurality of peptides consisting of about 10 to 20 amino acids contained in the amino acid sequence of TnT were prepared, and epitopes were identified by ELISA using these peptides. To identify the epitope, 29 types of peptides shown in FIG. 2 and the peptide sequence used for the immunogen (the peptide corresponding to the amino acid sequence 101-125 from the N-terminal side of TnT (isoform 3) (ELQALIEAHFENRKKEEEELVSLKD (sequence The peptide fragment of number: 32)) was used.

≪Reagent≫
The following reagents (m) and (n) were newly prepared.
(M) Antigen solution 29 types of peptides and peptide fragments corresponding to human-derived troponin T and immunogenic peptide sequences (peptides corresponding to the 101st to 125th amino acid sequences from the N-terminal side of TnT (isoform 3) (ELQALIEAHFENRKKEEEELVSLKD) Was prepared with a PBS-Az solution to a final concentration of 0.1 μM.
(N) TnT-P15 antibody solution TnT-P15 antibody was added to a PBS solution so that the final concentration was 0.1 μM to prepare a TnT-P15 antibody solution.

≪Procedure≫
The epitope of TnT-P15 antibody was identified by the following procedures (1) to (10).
(1) 100 μl of each antigen solution was injected into a microplate (manufactured by Nunc, immunoplate maxisorp round bottom U96, 449824) per well, and stored overnight in saturated water vapor at room temperature.
(2) The antigen solution in the microplate stored overnight was removed with an aspirator, 200 μl of blocking solution was injected into each well for each well, and left at room temperature for 60 minutes.
(3) The blocking solution in the microplate was discarded, each well was washed 3 times with PBST solution, and then the PBST solution remaining in each well was removed with an aspirator.
(4) For each well, 50 μl of the TnT-P15 antibody solution was injected into each well and left at room temperature for 90 minutes.
(5) The antibody solution in the microplate was discarded, each well was washed three times with PBST solution, and then the PBST solution remaining in each well was removed with an aspirator.
(6) For each well, 50 μl of the labeled antibody solution was injected into one well and left at room temperature for 30 minutes.
(7) The labeled antibody solution in the microplate was discarded, washed with PBST solution three times, and the PBST solution remaining in each well was removed with an aspirator.
(8) For each well, 50 μl of a color reaction reagent (Thermo TMB ELISA, 1 STEP Ultra) was injected into each well and allowed to react at room temperature for 5 minutes.
(9) After reaction for 5 minutes, 50 μl of 4N sulfuric acid was injected into each well for each well to stop the color reaction.
(10) After stopping the color reaction, the absorbance (wavelength of 450 nm) was measured for the solution in each well using a microplate reader (manufactured by TECAN, Infinite 200).

≪Result≫
FIG. 3 is a graph showing the measurement results of the binding ability of the TnT-P15 antibody to each peptide and troponin. In the graph shown in FIG. 3, the vertical axis represents absorbance at a wavelength of 450 nm, and the horizontal axis represents the type of antigen (such as peptide numbers of 29 types of peptides). The peptide numbers on the horizontal axis in FIG. 3 correspond to the peptide numbers in FIG. Further, TnT on the horizontal axis is a control.

As is clear from the results in FIG. 3, the TnT-P15 antibody has a specific binding ability to troponin T and the immunized peptide sequence (ELQALIEAHFENRKKEEEELVSLKD), while binding ability to any other peptide fragment. I didn't have it.
Peptides corresponding to the 101st to 125th amino acid sequences from the N-terminal side of TnT (isoform 3) correspond to a part of peptide numbers 12 and 13 and 11 and 14 of the peptides used in the evaluation.

  As is apparent from the results of FIG. 3, the TnT-P15 antibody has binding ability to the peptide used for the immunogen (amino acid sequence 101 to 125 from the N-terminal side of TnT (isoform 3)). On the other hand, it does not show binding ability to any of peptide numbers 11 to 14, and at least the epitope of TnT-P15 antibody is the amino acid sequence of 101-106 from the N-terminal side of TnT (isoform 3) and 122 It can be seen that the amino acid sequence is not limited to the 125th amino acid sequence and is not limited to the region of peptide number 12 or 13. From this, the epitope of TnT-P15 antibody is estimated to correspond to the amino acid sequence containing the 101st to 125th amino acids from the N-terminal side of TnT (isoform 3).

  From these facts, the TnT-P15 antibody can bind even if it is a fragment with a molecular weight of about 25,000 which has been subdivided with TnT over time, and is not dependent on isoforms. It can be said that TnT can be measured with high accuracy by using a TnT-P15 antibody.

  Further, the TnT-P15 antibody does not specifically bind to skeletal muscle troponin T of either slow muscle type or fast muscle type. This is because neither slow muscle type nor fast muscle type skeletal muscle troponin T has an amino acid sequence corresponding to the 101st to 125th amino acid sequence from the N-terminal side of cardiac muscle TnT (isoform 3). This is because it was also shown that it did not have the ability to bind to skeletal muscle troponin T in “cell sorting and cloning”.

  Note that the anti-human cardiac troponin T monoclonal antibody according to one embodiment of the present application is IgG, but includes not only IgG itself but also functional fragments retaining its binding characteristics (for example, F (ab), F (ab ′ 2). Moreover, the anti-human cardiac troponin T monoclonal antibody according to one embodiment of the present application includes a mutant antibody that retains its binding characteristics.

Example 3 Measurement of TnT by TnT-P15 Antibody The present inventor measured TnT by the EIA method using the TnT-P15 antibody. Specifically, the EIA method performed in this example is an ELISA method (colorimetric measurement, sandwich method).

≪Reagent≫
The following reagents were newly prepared.
(O) Solid phase antibody solution The TnT-P15 antibody of the present invention was prepared as a solid phase antibody for sandwich reaction using a PBS solution so that the final concentration was 0.1 μM.
(P) Liquid Phase Antibody As a liquid phase antibody for a sandwich reaction, troponin T antibody (manufactured by Fitzgerald, 10-T85A) was biotinylated by the following procedure.
2 μL of NHS-Biotin (Pierce, 21425) prepared in a PBS solution to a final concentration of 20 mM was added to 1 ml of troponin T antibody solution prepared to 2 μM in PBS, and mixed by inverting at room temperature for 30 minutes. Then, 1 ml of blocking buffer (0.5 M glycine / 0.5 M NaCl pH 8.3) was added, and the mixture mixed by inverting at room temperature for 30 minutes was used as a biotinylated antibody. (Glycine: Wako Pure Chemical, 077-00735, NaCl: Wako Pure Chemical, 191-01665)
This biotinylated antibody prepared using a PBS solution to a final concentration of 0.1 μM was used as a liquid phase antibody solution.
(Q) Antigen solution for reaction As an antigen solution, TnT (manufactured by Fitzgerald, in a 1% BSA / PBS solution so that the final concentration is 0.0001, 0.001, 0.01, 0.1, 1, 10 nM, 30C-CP3037) was dissolved to prepare TnT solutions having six concentrations.

≪Procedure≫
TnT was measured by the following procedures (1) to (12).
(1) 50 μl of solid phase antibody solution was injected into a microplate (manufactured by Nunc, immunoplate maxisorp round bottom U96, product number 449824) per well, and stored overnight in saturated water vapor at room temperature.
(2) The antibody solution in the microplate stored overnight was removed with an aspirator, 200 μl of blocking solution was injected into each well for each well, and allowed to stand at room temperature for 60 minutes.
(3) The blocking solution in the microplate was discarded, each well was washed 3 times with PBST solution, and then the PBST solution remaining in each well was removed with an aspirator.
(4) For each well, 50 μl of each of 12 types of antigen solutions for reaction was injected into one well and left at room temperature for 30 minutes.
(5) The antigen solution in the microplate was discarded, each well was washed 3 times with PBST solution, and then the PBST solution remaining in each well was removed with an aspirator.
(6) For each well, 50 μl of the liquid phase antibody solution was injected into each well and allowed to stand at room temperature for 30 minutes.
(7) The antibody solution in the microplate was discarded, each well was washed three times with PBST solution, and then the PBST solution remaining in each well was removed with an aspirator.
(8) For each well, 50 μl of a streptavidin-labeled antibody solution was injected into one well and left at room temperature for 30 minutes.
(9) The streptavidin-labeled antibody solution in the microplate was discarded, washed with PBST solution three times, and the PBST solution remaining in each well was removed with an aspirator.
(10) For each well, 50 μl of a color reaction reagent (Thermo TMB ELISA, 1 STEP Ultra) was injected into each well and allowed to react at room temperature for 5 minutes.
(11) After the reaction for 5 minutes, 50 μl of 4N sulfuric acid was injected into each well for each well to stop the color reaction.
(12) After stopping the color reaction, the absorbance (wavelength of 450 nm) was measured for the solution in each well using a microplate reader (manufactured by TECAN, Infinite 200).

≪Result≫
FIG. 4 is a graph showing the measurement results of the sandwich reaction using the TnT-P15 antibody. In the graph shown in FIG. 4, the vertical axis represents absorbance at a wavelength of 450 nm (“OD450” in FIG. 4), and the horizontal axis represents the logarithmic value of TnT concentration (nM).

From the results of FIG. 4, it is possible to detect troponin T from 0.01 nM by sandwich reaction between the TnT-P15 antibody of the present invention and the commercially available troponin T antibody (Fitzgerald, 10-T85A) used this time. Using the calibration curve obtained from FIG. 4, it can be seen that the troponin T concentration in a solution containing an unknown concentration of troponin T can be quantitatively calculated.

As described above, it can be seen that highly sensitive detection and quantification of troponin T is possible by using the antibody of the present invention.

In this example, TnT measurement using TnT-P15 antibody was performed by ELISA (colorimetric measurement, sandwich method), but the present application is not limited to this.
Examples of the signal detection method include not only the system using the colorimetric reaction by the enzyme shown in the present example, but also, for example, RIA method, fluorescent antibody method, chemiluminescence method, and electrochemiluminescence method.
Further, as the measurement format, the sandwich method is shown in the present embodiment, but the present application is not limited to this. For example, a direct adsorption method or a competitive method in which a measurement specimen is adsorbed on a solid phase and measured. May be used.

Furthermore, as shown in this Example, the TnT-P15 antibody was used as an antibody immobilized on a solid phase, and a commercially available antibody was used for an antigen-antibody reaction against TnT bound to the solid phase antibody. For example, the TnT-P15 antibody may be used in a liquid phase.
Furthermore, the liquid phase antibody used in this example was biotinylated, but other labeling may be performed. Examples of the substance labeled with the antibody include enzymes such as peroxidase, and signal substances such as chemiluminescent substances and electrochemiluminescent substances.

  According to the anti-human myocardial troponin T monoclonal antibody according to one embodiment of the present application, its production method, hybridoma, TnT measurement method and TnT measurement kit, TnT can be measured with high accuracy.

NITE P-01863

SEQ ID NOs: 1 to 29 are amino acid sequences of peptides used for specifying an epitope of TnT-P15 antibody.
SEQ ID NO: 30 is the amino acid sequence of TnT (wild type).
SEQ ID NO: 31 is the amino acid sequence of TnT (isoform 3).
SEQ ID NO: 32 is an amino acid sequence contained in the epitope of TnT-P15 antibody.

Claims (11)

  Glu-Leu-Gln-Ala-Leu-Ile-Glu-Ala-His-Phe-Glu-Asn-Arg-Lys-Lys-Glu-Glu-Glu-Glu-Leu-Val-Ser-Leu-Lys-Asp ( An anti-human cardiac troponin T monoclonal antibody having an amino acid sequence comprising SEQ ID NO: 32) as an epitope.   The anti-human myocardial troponin T monoclonal antibody according to claim 1, which is produced from a hybridoma having an accession number of NITE P-01863 at the Patent Microorganism Depositary, National Institute of Technology and Evaluation.   A hybridoma whose accession number at the Patent Microorganism Depositary Center is NITE P-01863. Immunizing a mammal with a peptide composed of a predetermined number of consecutive amino acid sequences among the 41-200th amino acid sequences from the N-terminal side of isoform 3 of human cardiac troponin T;
Removing spleen cells from the mammal immunized with the peptide;
Step C for obtaining a hybridoma by cell fusion of the spleen cells and a cell line derived from the mammalian myeloma;
A method for producing a hybridoma comprising:   The method for producing a hybridoma according to claim 4, wherein the peptide is a peptide composed of an amino acid sequence composed of 10 to 30 amino acids.   The peptide is Glu-Leu-Gln-Ala-Leu-Ile-Glu-Ala-His-Phe-Glu-Asn-Arg-Lys-Lys-Glu-Glu-Glu-Glu-Leu-Val-Ser-Leu- The method for producing a hybridoma according to claim 4, which is a peptide containing Lys-Asp (SEQ ID NO: 32).   A method for producing an anti-human cardiac troponin T monoclonal antibody, comprising culturing the hybridoma according to claim 2 or the hybridoma obtained by the production method according to any one of claims 4 to 6.   An immunoassay method comprising: Glu-Leu-Gln-Ala-Leu-Ile-Glu-Ala-His-Phe-Glu-Asn-Arg-Lys-Lys-Glu-Glu-Glu-Glu-Leu-Val-Ser A method for measuring anti-human cardiac troponin T, comprising using an anti-human cardiac troponin T monoclonal antibody having an amino acid sequence containing Leu-Lys-Asp (SEQ ID NO: 32) as an epitope.   9. The anti-human myocardial troponin T monoclonal antibody is a monoclonal antibody produced from a hybridoma having a deposit number of NITE P-01863 in the Patent Microorganism Depositary Center of National Institute of Technology and Evaluation, the anti-human cardiac troponin T monoclonal antibody. Method for measuring T.   Glu-Leu-Gln-Ala-Leu-Ile-Glu-Ala-His-Phe-Glu-Asn-Arg-Lys-Lys-Glu-Glu-Glu-Glu-Leu-Val-Ser-Leu-Lys-Asp ( A human cardiac troponin T assay kit comprising an anti-human cardiac troponin T monoclonal antibody having an amino acid sequence comprising SEQ ID NO: 32) as an epitope.   The anti-human myocardial troponin T monoclonal antibody is a monoclonal antibody produced from a hybridoma having an accession number of NITE P-01863 in the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation, the anti-human cardiac troponin T monoclonal antibody. T measurement kit.