JP2016114360A - Method for detecting human t-cell leukemia virus hbz protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means capable of sensitively detecting or quantifying HBZ (HTLV-1 basic leucine zipper factor) protein which plays an important role in the onset of an HTLV-1 related disease.SOLUTION: A detection reagent of HBZ protein contains at least one type of antibody selected from the group consisting of an antibody specifically binding to peptide composed of an amino acid sequence represented by SEQ ID NO:4, an antibody specifically binding to peptide composed of an amino acid sequence represented by SEQ ID NO:5, and an antibody specifically binding to peptide composed of an amino acid sequence represented by SEQ ID NO:6. A diagnostic drug of an HTLV-1 related disease contains at least two types of the antibodies. A method for detecting an HTLV-1 related disease includes the steps of: (1) bringing a blood sample derived from a subject and the antibody into contact to form a composite body of HBZ protein contained in the blood sample and the antibody; and (2) detecting HBZ protein in the composite body formed in the step (1).SELECTED DRAWING: None

Description

  The present invention relates to a method for detecting human T cell leukemia virus HBZ protein, and relates to the field of diagnosis of adult T cell leukemia and human T cell leukemia virus-related myelopathy.

  Human T-lymphotropic virus type 1 (HTLV-1) is the world's first retrovirus that has been found to be associated with human diseases. 1Develops HTLV-1-associated myelopathy (HAM) and adult T-cell leukemia (ATL). Although most HTLV-1 infected patients have passed as lifetime asymptomatic carriers, there are currently more than 1 million HTLV-1 infected persons in Japan, so HTLV-1 as soon as possible It is awaited to elucidate the pathophysiology of related diseases and develop preventive and novel treatments.

  HTLV-1 basic leucine zipper factor (HBZ), a gene encoded by the minus strand of HTLV-1 provirus, is significantly positive in terms of the amount of provirus in HAM patients, the degree of movement disorder, and the concentration of inflammatory markers in cerebrospinal fluid. (Non-patent document 1), which is expressed in leukemia cells of all ATL patients and can suppress the proliferation of ATL cells by suppressing the expression of HBZ (non-patent document 2). It is considered as a causative gene for the onset of HTLV-1 infection because it exhibits T cell infiltration into the body and develops an ATL-like T cell lymphoma (Non-patent Document 3).

  As a technique for diagnosing an HTLV-1-related disease, a diagnostic instrument for diagnosing an ATL patient or an asymptomatic carrier of HTLV-1 infection using an antibody against TSLC1, which is a lung cancer suppressor gene, is known (Patent Document 1). In addition, as a technique for detecting an HTLV-1 antigen using an antibody, a technique that targets a core antigen (p24, p53) of HTLV-1 (Patent Document 2) and an envelope protein (gp46) of HTLV-1 (Patent Document 3) and HTLV-1 envelope protein (Patent Document 4) are known.

  The present inventors have used monoclonal antibodies against HBZ protein to detect anti-HBZ antibodies present in the plasma of HAM patients and asymptomatic carriers of HTLV-1 infection, and HBZ protein in peripheral blood mononuclear cells. Attempts have been made to detect anti-HBZ antibody in plasma and HBZ protein contained in peripheral blood mononuclear cells because they are very small amounts (Non-patent Document 4). In addition, the present inventors prepared a monoclonal antibody (mouse B6-15) against HBZ protein, constructed a sandwich ELISA method, and developed HBZ forced expression cell line (293T / HBZ-SI) and some HTLV-1 HBZ protein has been successfully detected using HBZ highly expressing cells such as an infected cell line (SLB-1) (Non-patent Document 5).

JP 2006-317220 A US5164293A US5614366A US6270959B1

Retrovirology 6:19 (2009). Proc Natl Acad Sci USA 103: 720-725, (2006). PLoS Pathog 7: e1001274 (2011). Retrovirology, 8 (Suppl 1), A244 (2011). The 61st Annual Meeting of the Japanese Society for Virology Vol.61st, p.358 (2013.10.29)

However, the expression level of HBZ protein in vivo is extremely low, and since there was no excellent antibody against HBZ protein, there was no sensitive detection and quantification system for HBZ, especially regarding the onset of HBZ and HTLV-1-related diseases. Research using clinical specimens has not progressed.
An object of the present invention is to produce a monoclonal antibody that reacts with HBZ protein, which plays an important role in the development of HTLV-1-related diseases, with high sensitivity, and to provide a means capable of sensitively detecting or quantifying HBZ protein. .

  The present inventors performed Blast search on the amino acid sequence of the HBZ protein to select a candidate peptide by avoiding a sequence region showing homology with a human protein, and selected three candidates as regions for which antigenicity is predicted. And the antibody was obtained. From the antibodies obtained, a combination of antibodies particularly excellent for detection of HBZ protein was found, and the present invention was completed.

The present invention provides the following.
[1] An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4;
An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5; and at least one antibody selected from the group consisting of an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6. Detection reagent for HBZ protein.
[2] An antibody produced by administering a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 as an antigen to a non-human animal, and a polypeptide containing the amino acid sequence shown in SEQ ID NO: 5 as an antigen in a non-human animal The detection according to [1], which is an antibody selected from the group consisting of an antibody produced by administration and an antibody produced by administering a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 as an antigen to a non-human animal reagent.
[3] An antibody produced by administering a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 to a non-human animal as an antigen, and administering a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 to a non-human animal as an antigen The antibody according to [1] or [2], wherein the antibody is selected from the group consisting of an antibody produced by administration and an antibody produced by administering a peptide comprising the amino acid sequence shown in SEQ ID NO: 6 as an antigen to a non-human animal. Detection reagent.
[4] The detection reagent according to any one of [1] to [3], wherein the antibody is a monoclonal antibody.
[5] The detection reagent according to any one of [1] to [4], which contains at least two of the antibodies.
[6] A combination of an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 and an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 5,
A combination of an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 and an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 6, or a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 The detection reagent according to any one of [1] to [5], which is a combination of an antibody that specifically binds to and an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6.
[7] A combination of an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 and an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 6. [1] -Detection reagent in any one of [6].
[8] The monoclonal antibody produced by a hybridoma selected from the group consisting of the hybridoma represented by the reception number NITE ABP-01967 and the hybridoma represented by the reception number NITE ABP-01968, [1] to [7 ] The detection reagent in any one of.
[9] An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5, and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 A diagnostic agent for HTLV-1-related diseases, comprising at least two antibodies selected from the group consisting of antibodies that specifically bind.
[10] The diagnostic agent according to [9], wherein the HTLV-1-related disease is adult T-cell leukemia or HTLV-1-related myelopathy.
[11] An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 A kit for detecting an HBZ protein or diagnosing an HTLV-1-related disease, comprising at least two types of antibodies selected from the group consisting of antibodies that specifically bind to each other in separate containers.
[12] The kit according to [11], wherein one kind of the antibody is immobilized on a solid phase.
[13] The kit according to [11] or [12], wherein the HTLV-1-related disease is adult T-cell leukemia or HTLV-1-related myelopathy.
[14] The monoclonal antibody produced by the hybridoma selected from the group consisting of the hybridoma represented by the reception number NITE ABP-01967 and the hybridoma represented by the reception number NITE ABP-01968, [9] or [10 Or the kit according to any one of [11] to [13].
[15] Monoclonal antibody produced by a hybridoma selected from the group consisting of a hybridoma represented by receipt number NITE ABP-01967, a hybridoma represented by receipt number NITE ABP-01968, and a hybridoma represented by receipt number NITE ABP-01969 .
[16] The following steps:
(1) a blood sample derived from a subject;
An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 Contacting with an antibody selected from the group consisting of antibodies to bind to form a complex of HBZ protein and antibody contained in the blood sample; and (2) HBZ in the complex formed in step (1) Detecting the protein;
A method for detecting an HTLV-1-related disease, comprising:
[17] The detection method according to [16], wherein the step (2) detects the HBZ protein in the complex using an antibody that specifically binds to a peptide different from the antibody used in the step (1). .
[18] The antibody used in step (1) is a capture antibody immobilized on a solid phase, and the antibody used in step (2) is an antibody that specifically binds to a peptide different from the capture antibody, (2) The detection method according to [16], wherein the HBZ protein in the complex is detected by a sandwich method.
[19] The detection method according to [18], wherein the antibody used in the step (2) is enzyme-labeled, and the step (2) detects by a sandwich ELISA method.
[20] The monoclonal antibody produced by a hybridoma selected from the group consisting of the hybridoma represented by the reception number NITE ABP-01967 and the hybridoma represented by the reception number NITE ABP-01968, [16] to [19 ] The detection method in any one of.

  The present invention enables sensitive and quantitative detection of HBZ protein, which was impossible in the past. By applying the results of the present invention to research using clinical specimens, testing, diagnosis and pathology of HTLV-1-related diseases It is expected to be applicable to grasping.

The HBZ gene and its splicing isoform are shown. The result of the Western blot analysis using an anti-HBZ monoclonal antibody is shown. An example of flow cytometry analysis using an anti-HBZ monoclonal antibody is shown. The result of the fluorescence microscope analysis using an anti-HBZ monoclonal antibody is shown. The sandwich ELISA system (1) using a combination of anti-HBZ monoclonal antibodies is shown. The results of examining the amount of HBZ protein expressed in various cell lines using the sandwich ELISA system (1) are shown. CEM, Jurkat: HTLV-1 non-infected human T cell line; C5MJ, HUT102, MT-1, MT-2, MT-4, SLB-1, TLOm1: HTLV-1-infected human T cell line; 293T-HA-Mock : Negative control; 293T-HA-HBZ: Positive control. The sandwich ELISA system (2) using a combination of anti-HBZ monoclonal antibodies is shown. The results of examining the amount of HBZ protein expressed in various cell lines using the sandwich ELISA system (2) are shown. CEM, Jurkat: HTLV-1 non-infected human T cell line; C5MJ, HUT102, MT-1, MT-2, MT-4, SLB-1, TLOm1: HTLV-1-infected human T cell line; 293T-HA-Mock : Negative control; 293T-HA-HBZ: Positive control. The results of examining the amount of HBZ protein expressed in various cell lines and patient peripheral blood mononuclear cells (PBMC) using a sandwich ELISA system (2) are shown. Non-infected PBMC-1, Non-infected PBMC-2, ATL-1, ATL-2, ATL-3: Patient peripheral blood mononuclear cells; CEM, Jurkat: HTLV-1 non-infected human T cell line; C5MJ, HUT102, MT -1, MT-2, MT-4, SLB-1, TLOm1, ATL-43Tb, ED: HTLV-1-infected human T cell line; 293T-HA-Mock: negative control; 293T-HA-HBZ (3-fold dilution) ): Positive control.

Definitions In the present invention, HTLV-1 is human T-lymphotropic virus type 1 (Human T-lymphotropic virus 1 type 1), and is a pathogenic virus for human T-cell leukemia virus (HTLV-1) -related diseases.

In the present invention, HTLV-1-related diseases refer to adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy (HAM). ATL is a type of leukemia / lymphoma that occurs in about 5% of HTLV-1-infected individuals, and is classified into four types, acute, lymphoma, chronic, and smoldering. Symptoms and abnormalities such as swollen lymph nodes, hepatosplenomegaly, skin lesions, opportunistic infections, elevated serum LDH and calcium levels are observed. If such a patient is positive for an HTLV-1 antibody test and falls under any of the following, ATL is diagnosed:
1) When abnormal cells that increase in the blood are T cells
2) When enlarged lymph nodes or skin lesions are caused by T cell cancer cells
HAM is a neurological intractable disease characterized by chronic progressive spastic spinal cord palsy that occurs in some HTLV-1 infected individuals (approximately 0.3%). At the core of clinical symptoms are slowly progressive gait disturbance and urinary dysfunction such as dysuria, frequent urination, and constipation. Unlike ATL, life prognosis is good, but progression makes about 40% of patients unable to walk and severely restricts daily life. Neurological examinations showed findings suggestive of pyramidal tract disorders such as spastic paraparesis of both lower limbs, increased deep tendon reflexes, pathological reflexes (Babinsky signs), and anti-HTLV in serum (or plasma) and cerebrospinal fluid If -1 antibody is positive, it is diagnosed as HAM after distinguishing other diseases such as spinal cord compression lesions, spinal cord tumors, and multiple sclerosis that cause myelopathy.

The HTLV-1 basic leucine zipper factor (HBZ) gene is a gene encoded by the minus strand of the HTLV-1 provirus (see FIG. 1), and several splicing isoforms are known. HBZ mRNA or protein is involved in cell proliferation (Proc Natl Acad Sci USA 103: 720-725, 2006), and the protein has been reported to be a target for killer T cells (J Infect Dis 203: 529-536, 2011). The amino acid sequence of HBZ and the nucleotide sequence encoding it are registered with NCBI as follows.
Nucleotide sequence of HBZ (Accession number DQ273132 (version DQ273132.1)) (SEQ ID NO: 1)
Amino acid sequence (Accession number ABB89742.1) (SEQ ID NO: 2)
The unspliced (unspliced) isoform of HBZ has its structure and N-terminal amino acid sequence disclosed in Retrovirology 2009, 6:71, and its entire amino acid sequence is shown in SEQ ID NO: 3.

  In the present invention, the level of HBZ protein represents the content of HBZ protein in a certain amount of sample. The level of HBZ protein can be expressed in a manner known to those skilled in the art according to the measurement method described below, for example, it may be expressed as the concentration of HBZ protein, or it is a relative value based on the measured value of the standard sample. It may be expressed as

In the present invention, an antibody includes all immunoglobulin classes and subclasses and forms of functional fragments of an antibody. In addition to a natural antibody, the antibody is produced using a gene recombination technique. The resulting antibodies, antibody fragments, and binding fragments thereof are also included, but are not limited to these. In addition, polyclonal antibodies and monoclonal antibodies are included, but antibody preparations containing different antibodies against different epitopes from polyclonal antibodies. Monoclonal antibodies are antibodies obtained from a substantially homogeneous antibody population (including antibody fragments). ). The binding fragment means a partial region of the aforementioned antibody, specifically, for example, F (ab) ₂ , Fab ', Fab, Fv (variable fragment of antibody), scFv (single chain Fv), dsFv (disulphide stabilized Fv), sdAb (single domain antibody) and the like (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996).

  In the present invention, determining the presence or absence of an HTLV-1-related disease includes the evaluation and examination against a certain standard, and the present invention alone or in combination with another examination comprehensively. It also includes determining.

1. HBZ protein detection reagent and diagnostic agent for HTLV-1-related diseases "Anti-HBZ antibody" contained as an active ingredient in the detection reagent and diagnostic agent of the present invention is particularly limited as long as it is an antibody that can detect HBZ protein Instead, a part of the antibody having antigen-binding properties such as a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, or a fragment generated by a Fab fragment or a Fab expression library is included.

The detection reagent and diagnostic agent of the present invention are used as anti-HBZ antibodies.
An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4; an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5; and a peptide specific to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 At least one antibody selected from the group consisting of antibodies that bind to. For detection with higher sensitivity, it is preferable to contain at least two kinds of the antibodies. When two or three types are contained, two or three types may be contained in one detection reagent or diagnostic agent, or a combination of two or three types of detection reagent or diagnostic agent containing one antibody. It may be.

  A polyclonal antibody or a monoclonal antibody can be produced by a known general production method. For example, an HBZ protein or a part thereof is used as an antigen, optionally together with Freund's Adjuvant, and in the case of a mammal such as a polyclonal antibody, a mouse, rat, hamster, guinea pig, rabbit, cat, dog, pig, Goats, horses, cows, etc., preferably mice, rats, hamsters, guinea pigs, goats, horses or rabbits, and in the case of monoclonal antibodies, mice, rats, hamsters are immunized.

  In one embodiment, for example, a nucleotide encoding a peptide comprising the amino acid sequence of SEQ ID NO: 2 or a part thereof, optionally a nucleotide encoding another protein (eg, glutathione-S-transferase (GST), His tag). Using an expression vector linked to, a purified protein expressed in a host as a fusion protein with a recombinant protein or other protein by a conventional method, and purified can be used as an antigen. When producing HBZ protein in large quantities, it is also desirable to use a wheat cell-free protein expression system.

The present inventors performed a Blast search on the amino acid sequence of the HBZ protein, selected candidate peptides while avoiding sequence regions showing homology with human proteins, and selected three candidates as regions for which antigenicity was predicted. In the present invention, the following three peptides are preferably used as antigens.
Peptide 1: LRRGPPGEKAPPRGETHRD (SEQ ID NO: 4)
Peptide 2: KAKQHSARKEKMQELGIDG (SEQ ID NO: 5)
Peptide 3: KEDLMGEVNYWQGRLEAMW (SEQ ID NO: 6)

  In a preferred embodiment, the anti-HBZ antibody is an antibody produced by administering a polypeptide containing the amino acid sequence shown in SEQ ID NO: 4 as an antigen to a non-human animal, and a polypeptide containing the amino acid sequence shown in SEQ ID NO: 5 as a non-antigen. An antibody selected from the group consisting of an antibody produced by administration to a human animal and an antibody produced by administering a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 as an antigen to a non-human animal.

  In another preferred embodiment, the anti-HBZ antibody is an antibody produced by administering a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 to a non-human animal as an antigen, and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 as a non-antigen. An antibody selected from the group consisting of an antibody produced by administration to a human animal and an antibody produced by administering a peptide comprising the amino acid sequence shown in SEQ ID NO: 6 as an antigen to a non-human animal.

  In a further preferred embodiment, the anti-HBZ antibody is a monoclonal antibody produced by a specific hybridoma. The applicant, on November 19, 2014, received the receipt number NITE ABP-01967 from the National Institute for Product Evaluation Technology Patent Microorganisms Deposit Center (2-5-8, Kazusa Kamashiri, Kisarazu City, Chiba Prefecture, Japan) We have deposited three types of hybridomas, receipt number NITE ABP-01968 and receipt number NITE ABP-01969. The three hybridomas NITE ABP-01967, NITE ABP-01968 and NITE ABP-01969 were obtained by using peptide 2, peptide 3, and HBZ protein produced in a wheat cell-free protein expression system as antigens, respectively. The monoclonal antibodies produced by these hybridomas are referred to as pep # 2 # 20-H12, pep # 3 # 91-1 and α wheat HBZ # 7-1, respectively. In the present invention, it is preferable to use monoclonal antibodies produced by these deposited hybridomas from the viewpoint of low background noise.

  Specifically, the polyclonal antibody can be produced as follows. That is, 1 to several antigens in mice, rats, hamsters, guinea pigs, goats, horses or rabbits, preferably goats, horses or rabbits, more preferably rabbits subcutaneously, intramuscularly, intravenously, in a food pad or intraperitoneally. Immunization is given by multiple injections. Usually, immunization is performed 1 to 5 times about every 1 to 14 days from the first immunization, and serum is obtained from the immunized mammal about 1 to 5 days after the final immunization.

  Serum can be used as a polyclonal antibody, but preferably, ultrafiltration, ammonium sulfate fractionation, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), anti-immunoimmune It is isolated and / or purified by affinity column chromatography using a globulin column, a protein A / G column, a column with an antigen cross-linked, or the like.

  Monoclonal antibodies were prepared from the antibody-producing cells obtained from the above immunized animals and myeloma cells (myeloma cells) having no autoantibody-producing ability, and the hybridomas were cloned and used for immunization of mammals. Produced by selecting clones that produce monoclonal antibodies that exhibit specific affinity for the antigen.

  Specifically, the monoclonal antibody can be produced as follows. That is, the antigen can be administered subcutaneously, intramuscularly, intravenously in mice, rats or hamsters (including transgenic animals created to produce antibodies from other animals such as human antibody-producing transgenic mice) Immunization is performed by injection or implantation in the Hood pad or intraperitoneally 1 to several times. Usually, immunization is carried out 1 to 4 times about every 1 to 14 days from the first immunization, and antibody producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization.

  Hybridomas (fused cells) that secrete monoclonal antibodies can be prepared according to the method of Köhler and Milstein et al. (Nature, Vol. 256, p.495-497, 1975) and modification methods according thereto. That is, spleen, lymph nodes, bone marrow or tonsils obtained from a mammal immunized as described above, preferably antibody-producing cells contained in the spleen, and preferably mouse, rat, guinea pig, hamster, rabbit or human It is prepared by cell fusion with a myeloma cell having no autoantibody-producing ability derived from a mammal such as mouse, rat or human.

  Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (653; ATCC No. CRL1580), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8 .U1 (P3U1), SP2 / 0-Ag14 (Sp2 / 0, Sp2), PAI, F0 or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., Human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15 can be used.

  For screening of hybridoma clones producing monoclonal antibodies, the hybridoma is cultured in, for example, a microtiter plate, and the reactivity of the culture supernatant of wells in which proliferation has been observed to the antigen used in the above-described immunization is determined, for example, by ELISA. It can carry out by measuring by enzyme immunoassay methods.

  The hybridoma can be cultured using a medium (for example, DMEM containing 10% fetal bovine serum), and the centrifuged supernatant of the culture can be used as a monoclonal antibody solution. In addition, ascites can be generated by injecting the hybridoma into the abdominal cavity of an animal from which the hybridoma is derived, and the obtained ascites can be used as a monoclonal antibody solution. The monoclonal antibody is preferably isolated and / or purified in the same manner as the polyclonal antibody described above.

  Chimeric antibodies can be produced with reference to, for example, “Experimental Medicine (Special Issue), Vol. 6, No. 10, 1988”, Japanese Patent Publication No. 3-73280, and the like. A single-chain antibody is produced by ligating the DNA encoding the monoclonal antibody obtained as described above so as to be expressed as a single-chain antibody by gene recombination technology, incorporating it into an expression vector, introducing it into a host, and producing it. Is obtained.

  Antibody production by phage display can easily obtain antibodies such as Fab by collecting and concentrating phages having affinity for antigen by biopanning from a phage library prepared for antibody screening. it can.

F (ab ′) ₂ and Fab ′ can be produced by treating immunoglobulin with pepsin or papain, which are proteolytic enzymes, respectively. Fab can be produced by screening a Fab-expressing phage library in the same manner as the antibody production method using phage display.

The detection reagent and diagnostic agent of the present invention may be composed only of the above-mentioned antibody or may contain a pharmaceutically acceptable carrier. As a pharmaceutically acceptable carrier, when preparing the diagnostic agent of the present invention as a detection reagent and a liquid, various carriers conventionally used as pharmaceutical materials, such as diluents, solvents, solubilizers, suspending agents, It may contain an isotonic agent, a buffering agent and the like. Furthermore, in order to prevent antibody aggregation, it is preferable to add a surfactant such as Tween 20 (registered trademark) or Tween 80 (registered trademark).
Diluents include water, physiological saline and the like.
Examples of the solvent include water, physiological saline, ethanol and the like.
Examples of the solubilizer include cyclodextrins.
Examples of the suspending agent include gum arabic and carmellose.
Examples of the isotonic agent include inorganic salts such as sodium chloride and potassium chloride, and carbohydrates such as glycerin, mannitol and sorbitol.
Examples of the buffer include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, and the like.
Those skilled in the art can appropriately determine the mixing ratio of these carriers.

  Using the detection reagent or diagnostic agent of the present invention, the presence or absence of an HTLV-1-related disease (ATL or HAM) is more easily determined by examining the blood sample of the subject by the detection method of the present invention described later. It becomes possible.

2. Kit for detecting HBZ protein and kit for diagnosing HTLV-1-related disease The anti-HBZ antibody contained in the kit of the present invention is the antibody described in “1. Detection reagent for HBZ protein and diagnostic agent for HTLV-1-related disease”. It is the same.

  The kit of the present invention may contain other antibodies or reagents in addition to the above-described antibodies, and these antibodies or reagents may be pre-mixed with the above-described antibodies or in separate containers. May be stored. Examples of antibodies or reagents include secondary antibodies, substrate agents, labeling substances (eg, fluorescent dyes, enzymes), solid phases, reaction vessels, treatment solutions, buffers for diluting antibodies, and positive controls (eg, , Recombinant HBZ), negative controls, instructions describing the protocol, and the like. These elements can be mixed in advance if necessary.

  In the kit of the present invention, the antibody may be immobilized in advance, and the antibody may be labeled in advance. The solid phase that can be used in the kit of the present invention is not particularly limited, and examples thereof include polymers such as polystyrene, glass beads, magnetic particles, microplates, immunochromatographic filter paper, glass filters, and insoluble carriers. . Preferably, it is a microplate used for sandwich ELISA.

  The form of the kit of the present invention is not particularly limited, but for the purpose of simple diagnosis, it can be an integrated kit in which the components of the kit of the present invention are integrated. Examples of the form of the integrated kit include a cassette type using an immunochromatography method.

  By using the kit of the present invention, detection of HBZ protein and determination of the presence or absence of HTLV-1-related disease (ATL or HAM) can be performed more easily.

3. Method for detecting HTLV-1-related disease The method for detecting an HTLV-1-related disease of the present invention comprises the following steps:
(1) contacting a blood sample derived from a subject with an anti-HBZ antibody to form a complex of HBZ and the antibody; and (2) detecting the HBZ protein in the complex formed in step (1). (Preferably quantitatively detecting) step;
(Hereinafter also referred to as the detection method of the present invention).

In the detection method of the present invention, after the step (2),
(3) a step of comparing the level of HBZ protein detected in step (2) with a reference value; and (4) whether the subject suffers from an HTLV-1-related disease based on the comparison result of step (3) Determining whether or not
It is preferable to contain.

  The subject in the detection method of the present invention is a human suspected of being infected with HTLV-1.

  The biological sample to be measured by the detection method of the present invention is not particularly limited as long as it can be collected from humans, and examples thereof include body fluid samples such as blood, lymph and urine, and biopsies of tissues such as lymph nodes. It is done. A blood sample is preferable because the burden on the subject is small, and examples of the blood sample include whole blood, plasma, and serum.

  In order to detect HBZ protein, the protein can be isolated from a biological sample (peripheral blood mononuclear cells, mononuclear cells separated from organs such as lymph nodes, etc.) according to a conventional method. General methods for extracting proteins are well known in the art and are described in standard molecular biology textbooks such as Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). It is disclosed. In addition, protein isolation can be performed according to the manufacturer's instructions using a purification kit, a buffer solution set, and a protease inhibitor obtained from the manufacturer.

  A biological sample derived from a subject is brought into contact with an anti-HBZ antibody to form a complex of HBZ protein and antibody.

The antibody may be directly or indirectly labeled with a labeling substance. Examples of labeling substances include fluorescent substances (eg, FITC, rhodamine), radioactive substances (eg, ³² P, ³⁵ S, ¹⁴ C, ³ H), enzymes (eg, alkaline phosphatase, peroxidase), colored particles (eg, metal colloids) Particles, colored latex), biotin and the like.

  The antibody can be used in a soluble state in which nothing else is bound, but may be bound to a solid phase. “Solid phase” includes plates (eg, microwell plates), tubes, beads (eg, plastic beads, magnetic beads), chromatographic carriers (eg, filter paper, water-absorbing substrates such as nitrocellulose membrane, Sepharose) , Membranes (eg, nitrocellulose membrane, PVDF membrane), gels (eg, polyacrylamide gel), metal membranes (eg, gold membrane) and the like. Of these, plates, beads, chromatographic carriers and membranes are preferably used, and plates are most preferably used from the viewpoint of easy handling. Examples of the bond include covalent bond, ionic bond, physical adsorption, and the like, and are not particularly limited. However, covalent bond and / or physical adsorption are preferable because sufficient bond strength can be obtained. The solid phase may be directly bonded to the solid phase or indirectly bonded to the solid phase using a substance known per se.

  In order to suppress non-specific adsorption and non-specific reaction, a phosphate buffer solution such as bovine serum albumin (BSA) or bovine milk protein is brought into contact with the solid phase, and the solid surface portion not coated with the antibody is removed. Blocking with BSA, bovine milk protein or the like is generally performed.

  The contact between the anti-HBZ antibody and the biological sample derived from the subject is not particularly limited as long as it is a method in which these antibodies can interact with the HBZ protein or the like in the biological sample. The contact can be made, for example, by adding a biological sample to a plate on which an anti-HBZ antibody is immobilized. Alternatively, for example, the biological sample may be separated by means such as SDS-PAGE, transferred to a membrane and fixed, and then contacted with an antibody.

  The time for maintaining such contact is not particularly limited as long as it is sufficient for the antibody to bind to the HBZ protein or the like contained in the biological sample derived from the subject to form a complex. It is a few seconds to a dozen hours. Moreover, as temperature conditions which perform a contact, it is 4 to 50 degreeC normally, 4 to 37 degreeC is preferable and the room temperature of about 15 to 30 degreeC is the most preferable. Furthermore, the pH condition for carrying out the reaction is preferably 5.0 to 9.0, particularly preferably the neutral range of 6.0 to 8.0.

  Step (2) is a step of detecting the HBZ protein in the complex formed in the step (1).

  For the detection, any method capable of detecting the HBZ protein can be used. For example, a method of detecting the HBZ protein based on its molecular weight or a method of detecting using an antibody is used.

  Methods for detecting HBZ protein based on its molecular weight include gel electrophoresis (eg, SDS-PAGE), various separation and purification methods (eg, size exclusion chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography). , Reverse phase chromatography, etc.) and mass spectrometers (eg, double-focusing mass spectrometer, quadrupole analyzer, time-of-flight mass spectrometer (TOF MS), Fourier transform mass spectrometer, ion cyclotron mass) It can be used for measurement by an analyzer, etc., as well as methods for combining them, and by comparing with the molecular weight of a predetermined marker peptide and detecting a band, spot, or peak of the target molecular weight. It is not limited.

  Prior to detection of the HBZ protein, the complex may be isolated from the reaction solution in step (1). Such isolation can be performed by, for example, an isolation method using affinity with heparin, or an immunoprecipitation method, and an appropriate label (for example, biotin) added to the antibody used in step (1). May be used.

  You may perform the said detection in the state in which the complex was formed. Alternatively, the formed complex may be isolated from the sample, and the complex may be dissociated to release the HBZ protein.

  When the HBZ protein is detected in step (2), it can be determined that the subject has a high probability of suffering from an HTLV-1-related disease. If HBZ protein is not detected or below the detection limit, it can be determined that the subject has a low probability of not suffering from an HTLV-1-related disease.

  When adopting a method for detecting an HBZ protein using an antibody, the step (2) of the present invention is such that the complex formed in the step (1) remains as a complex or the complex is dissociated to form an HBZ protein. Can be contacted with the antibody in a free state. For the description of the labeling of the antibody, the contact method with the HBZ protein and the contact conditions, the corresponding description of the labeling of the antibody, the contact method with the biological sample and the contact conditions in the above step (1) can be applied.

  Detection is performed by detecting the HBZ protein contained in the complex or the anti-HBZ antibody used in step (1). Such detection can be performed in the state of the complex or in a state where the complex is dissociated and the HBZ protein is released.

  The detection can be performed by enzyme immunoassay (EIA method), fluorescence immunoassay (FIA), immunochromatography, Western blotting, radioimmunoassay and the like, and these analysis methods are well known to those skilled in the art.

  When the EIA method is selected as the detection method in step (2), the detection method of the present invention is performed by sandwich ELISA using the anti-HBZ antibody used in step (1) and another antibody that recognizes the HBZ protein. It is preferable to do. Such a sandwich ELISA method is suitable for practicing the present invention because of its high specificity for an antigen.

The combination of antibodies used in the sandwich ELISA method is as follows, and one of the antibodies is used as the immobilized antibody in step (1), and the other antibody is used as the detection antibody in step (2). .
1) a combination of an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 and an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5;
2) A combination of an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 and an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 6, or
3) Combination of an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 and an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 6

  A preferred antibody combination is pep # 3 # 21-1 (capture antibody) and pep # 2 # 20-H12 (detection antibody).

  For example, the sandwich ELISA method is performed as follows. First, the antibody in step (1) is immobilized on the well surface of an ELISA plate. Next, after blocking to prevent nonspecific adsorption to the well surface, a biological sample is added, and the HBZ protein in the sample is brought into contact with the antibody to form a complex. After washing away the protein that did not bind to the antibody, another labeled anti-HBZ antibody was added to the well and contacted with the HBZ protein to form a complex sandwiched between the two antibodies and detected by the label.・ Quantify.

  An enzyme-labeled antibody can be used in the sandwich ELISA method. Examples of the enzyme used for labeling include peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase and the like. As the substrate agent used for the detection of the enzyme, an appropriate one is selected according to the selected labeling enzyme. For example, when peroxidase is selected as an enzyme, o-phenylenediamine (OPD), tetramethylbenzidine (TMB) or the like is used. When alkaline phosphatase is selected, p-nitrophenyl phosphate (PNPP) or the like is used. used. As the reaction stop solution and substrate solution, conventionally known ones can be appropriately used without particular limitation depending on the selected enzyme.

  A method utilizing an avidin-biotin reaction is applicable as a kind of sandwich ELISA method. In this method, for example, an HBZ protein in a sample is captured with the anti-HBZ antibody used in the solid phase step (1), and an antigen is detected between the captured HBZ protein and another anti-HBZ antibody labeled with biotin. An antibody reaction is performed. Next, enzyme-labeled streptavidin is added to cause the avidin-biotin reaction. Subsequently, the HBZ protein is detected by detecting this enzyme.

  The anti-HBZ antibody labeled with biotin can be produced by binding biotin and anti-HBZ antibody by a method known per se. Such labeling can be performed, for example, using a commercially available biotin labeling kit. A commercially available enzyme-labeled streptavidin can also be used.

  In addition to the above method, a method using a secondary antibody is also applicable. In this method, a complex is formed between the antibody used in step (1) and a secondary antibody that recognizes the Ig domain of the antibody. The secondary antibody is labeled with an enzyme and the presence of the HBZ protein in the complex is determined by detecting this enzyme. A commercially available enzyme-labeled secondary antibody can be used. Such a method using a secondary antibody can enhance the sensitivity of the detection method of the present invention.

When the FIA method is selected as the detection method in step (2), the enzyme used as a label in the EIA method is replaced with a fluorescent substance, thereby detecting the HBZ protein by the same method as the sandwich ELISA method described above. Can be done.
As the fluorescent substance, chemical substances such as APC, PE, Cy2, Cy3, Cy5, ECD, FITC, PerCP, Alexa (registered trademark) Fluor, fluorescein, and rhodamine can be preferably used. Labeling with these chemical substances can be carried out by a method known per se.
The fluorescence can be detected using a commercially available measuring instrument, a fluorescence microscope, or the like.

  When the immunochromatography method is selected as the detection method of the step (2), for example, the antibody of the step (2) solidified in a line form on a water-absorbing substrate such as a nitrocellulose membrane is developed on the membrane. A complex is formed between the biological sample and the labeled antibody in the step (1). The labeled antibody in step (1) may be premixed with the biological sample, or on the water-absorbing substrate so that the biological sample contacts the biological sample before contacting the antibody used in step (2). It may be supplied. The formation of the complex can be detected by a technique corresponding to the label. For example, when colloidal gold particles are used as a label, a region where the labeled antibody is accumulated exhibits a red color, and is thus detected.

  Western blotting can also be selected as the detection method in step (2). Western blotting (also called immunoblotting) is a method for detecting a specific protein in a sample. Gel electrophoresis is performed to separate proteins according to the length of the polypeptide under denaturing conditions or according to the three-dimensional structure of the protein under non-denaturing conditions. The protein is then transferred to a membrane (typically nitrocellulose or PVDF) and immobilized, after which the target protein is detected using a specific antibody.

  The detection step in the present invention is preferably a step of quantitatively detecting the HBZ protein. The quantification can be performed by a method known per se, such as quantifying the signal of a biological sample detected based on the label used using a calibration curve prepared using a standard sample of HBZ protein. The HBZ protein standard sample may be an HBZ protein prepared from a biological sample, or can be prepared by a genetic engineering technique known per se based on gene information encoding HBZ.

(3) A step of determining the presence or absence of HTLV-1-related disease from the level of HBZ protein detected in step (2). Using the result obtained in detection step (2), in step (2) The presence or absence of HTLV-1-related disease can be determined from the level of the detected HBZ protein. If the detection of step (2) is obtained as a qualitative result, it is determined that HBZ-positive is likely to have HTLV-1-related disease, and HBZ-negative (below detection limit) is afflicted with HTLV-1-related disease It is possible to determine that the probability of the image is low. If detection of step (2) is obtained as a quantitative result by sandwich ELISA etc., it is only possible to determine the presence or absence of HTLV-1-related disease in the subject from the concentration of HBZ protein in the biological sample (blood sample) First, a subject having an HBZ protein concentration of a certain level or higher in a biological sample can also estimate the severity of an HTLV-1-related disease. The same subject can also be compared to previously measured levels.

  The standard HBZ protein level (cutoff value) is an appropriate standard set according to the purpose. The cut-off value is a value that can satisfy both high diagnostic sensitivity (prevalence of correct diagnosis) and high diagnostic specificity (prevalence of non-diagnostic diagnosis) when a disease is determined based on that value. Usually, if it is below the detection limit, it can be determined that the patient is not suffering from an HTLV-1-related disease.

  When a simple test such as immunochromatography is performed, the presence or absence of the HBZ protein in the biological sample can be determined based on the presence or absence of a visually observed signal (coloring or the like).

  If the HBZ protein level in the subject's biological sample is above the cutoff value, the subject is determined to be likely to be “afflicted with an HTLV-1-related disease”. If the cut-off value or less, the subject is determined to be less likely to be “afflicted with an HTLV-1-related disease”.

  The method of the present invention can also be used to evaluate the effects of treatment on patients undergoing treatment for HTLV-1-related diseases. A biological sample is collected from such a patient before treatment, during treatment and / or after treatment, and the change in the concentration of HBZ protein is examined to know the effect of treatment. For example, if the concentration of HBZ protein in the later biological sample is lower than that in the previous biological sample, the treatment can be evaluated as being effective.

  When a simple test such as immunochromatography is performed, if the signal (color development, etc.) is confirmed by visual observation, the subject is determined to have a high probability of being “afflicted with an HTLV-1-related disease,” and other diagnoses. Methods can be combined to determine the presence or absence of HTLV-1-related disease.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these in any meaning.

Example 1 Acquisition of a novel anti-HBZ monoclonal antibody
Peptide antigen candidates that immunize animals were selected from the amino acid sequence of the HBZ protein (SEQ ID NO: 2). That is, Blast search was performed on the reference sequence protein of Homo sapiens, and the search was performed avoiding the sequence region showing homology. Three candidate peptides shown below were selected from the region where the turn structure and antigenicity were predicted.
Peptide # 1: LRRGPPGEKAPPRGETHRD (SEQ ID NO: 4)
Peptide # 2: KAKQHSARKEKMQELGIDG (SEQ ID NO: 5)
Peptide # 3: KEDLMGEVNYWQGRLEAMW (SEQ ID NO: 6)

Peptides 1 to 3 were recovered by performing deprotection and cleaving under acidic conditions after chemical synthesis using a fully automated synthesizer by the Fmoc solid phase synthesis method, and using reverse-phase HPLC. Separated and purified (consignee: Sigma Aldrich Japan GK). On the other hand, a wheat cell-free protein expression system (Proc Natl Acad Sci USA. 97 (2): 559-64, 2000.) using an N-terminal His-HBZ vector (provided by Professor Akihide Liang, Department of Microbiology, Yokohama City University School of Medicine) The recombinant HBZ protein was synthesized in large quantities by That is, amino acids and energy molecules (ATP and GTP) for cell-free extracts containing wheat-derived translation components (ribosome, tRNA, aminoacylated tRNA synthetase, translation initiation factor, translation elongation factor, translation termination factor, etc.) , Energy regeneration system, salts (Mg ^2+, etc.) and template mRNA were added to synthesize proteins in vitro (contractor: Cell Free Science Co., Ltd.).

  After immunizing animals (C / 57BL / 6 mice and WKA rats) three times with the above antigens (three peptides and recombinant HBZ protein), spleen cells and myeloma cells (SP2 / 0) were fused with polyethylene glycol. After selection using a hypoxanthine-aminopterin-thymidine medium (HAT), screening was performed using the culture supernatant of the hybridoma. Positive colonies were cloned by limiting dilution. The positive hybridoma cells were inoculated into the abdominal cavity of the mouse to make it ascites, and the immunoglobulin (immunoglobulin: Ig) was purified by gel filtration. As a result, the following monoclonal antibodies were newly obtained.

A. Peptide # 1-recognizing antibody / pep # 1 P6-A7 (Mouse, IgG2b, κ chain)
・ Pep # 1 P4-E12 (Mouse, IgG2b, κ chain)

B. Peptide # 2 recognition antibody ・ pep # 2 P3-E3 (Mouse, IgM, κ chain)
・ Pep # 2 P1-A12 (Mouse, IgM, κ chain)
・ Pep # 2 # 20-H12 (Mouse, IgG1)

C. Peptide # 3 recognition antibody ・ pep # 3 # 21-1 (Rat, IgG2a)
・ Pep # 3 # 91-1 (Rat, IgG1)

D. Recombinant HBZ protein recognition antibody / α wheat HBZ # 1-1 (Mouse, IgG2a)
・ Α wheat HBZ # 2-1 (Mouse, IgM)
・ Α wheat HBZ # 5-1 (Mouse, IgM)
・ Α wheat HBZ # 6-1 (Mouse, IgG1)
・ Α wheat HBZ # 7-1 (Mouse, IgG2b)
・ Α wheat HBZ # 8-1 (Mouse, IgM)
・ Α wheat HBZ # 10-1 (Mouse, IgM)
・ Α wheat HBZ # 12-1 (Mouse, IgM)
・ Α wheat HBZ # 14-1 (Mouse, IgM)

  Among the obtained hybridomas, pep # 2 # 20-H12 strain was founded on November 19, 2014 at the Japan Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2-5 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan). -8) is deposited with the receipt number NITE ABP-01967. Similarly, the pep # 3 # 91-1 strain is deposited under the receipt number NITE ABP-01968, and the α wheat HBZ # 7-1 strain is deposited under the receipt number NITE ABP-01969.

Example 2 Western blot analysis using a novel anti-HBZ monoclonal antibody The specificity of the monoclonal antibody obtained in Example 1 was confirmed by Western blotting. 293T cells (ATCC: obtained from American Type Culture Collection) were transformed with HBZ expression vector and control vector to obtain 293T-HA-HBZ (positive control) and 293T-HA-Mock (negative control), respectively. It was.
1 × 10 ⁸ 293T-HA-HBZ and 293T-HA-Mock were treated with RIPA buffer supplemented with protein inhibitors to obtain cell lysates. Using the anti-HBZ monoclonal antibody obtained in the examples, it was subjected to Western blotting by a conventional method. As secondary antibodies, anti-mouse IgG-HRP (Affinity Purified Secondary Antibodies against Mouse IgG (H & L) adsorbed against Rat, Human, Bovine, and Horse and conjugated to Horseradish Peroxidase: A106PU, American Qualex International, Inc.) or anti-rat IgG -HRP (Affinity Purified antibodies goat anti-rat IgG (H & L) Horseradish Peroxidase Labeled: A103PN, American Qualex International, Inc.) was used. The results are shown in FIG.
FIG. 2 shows that the obtained anti-HBZ monoclonal antibody specifically recognizes the HBZ protein.

Example 3 Flow cytometry analysis using a novel anti-HBZ monoclonal antibody Flow cytometry analysis was performed using the monoclonal antibody obtained in Example 1 and the 293T-HBZ and 293T-Mock prepared in Example 2. That is, after washing the cells, the anti-HBZ monoclonal antibody was reacted at 4 ° C. for 20 minutes at a final concentration of 1 μg / ml. After washing, anti-mouse IgG (Fab) ₂ -Alexa488 antibody (4408S: Cell Signaling Technology) diluted 100-fold was further reacted at 4 ° C for 20 minutes, and after washing, analyzed with a BD FACSCanto ^™ II flow cytometer (BD Biosciences) . The results are shown in FIG.
FIG. 3 shows that the novel anti-HBZ monoclonal antibody specifically binds to the intracellular HBZ protein expressing the HBZ protein and can be detected by flow cytometry.

Example 4 Fluorescence microscopy analysis using a novel anti-HBZ monoclonal antibody Staining of HBZ using the monoclonal antibody obtained in Example 1 and the HBZ-overexpressing cells 293T-HA-HBZ prepared by the method described in Example 2 Went. That is, after washing the cells, the anti-HBZ monoclonal antibody was reacted at 4 ° C. for 20 minutes at a final concentration of 1 μg / ml. After washing, anti-mouse IgG (Fab) ₂ -Alexa488 antibody (4408S: Cell Signaling Technology) diluted 100 times was reacted at 4 ° C for 20 minutes, washed, and then fluorescence microscope (confocal laser scanning microscope LSM 700: ZEISS) Analyzed with The results are shown in FIG.
FIG. 4 shows that the novel anti-HBZ monoclonal antibody specifically binds to the intracellular HBZ protein in which the HBZ protein was expressed, and can be detected with a fluorescent microscope by the fluorescent antibody method.

Example 5 Construction of sandwich ELISA using novel anti-HBZ monoclonal antibody (1)
A sandwich ELISA was constructed using anti-HBZ monoclonal antibodies pep # 3 # 21-1 (capture antibody) and pep # 2 # 20-H12 (detection antibody). The solid phase used was a 96 well microtiter plate (Nunc immunomodule maxi soap: Nunc 467466).

[Experimental procedure]
5 μg / mL of pep # 3 # 21-1 (anti-HBZ monoclonal antibody for antigen capture) is immobilized (room temperature, 1 hour).
↓ Washing (PBS)
Block with 5% skim milk / PBS-.
↓ Washing (PBS)
The diluted antigen is reacted (room temperature, 1 hour, 50 μg / well).
↓ Washing (PBS)
PRP # 2 # 20-H12 (detection antibody) labeled with HRP (horseradish peroxidase) is reacted (room temperature, 1 hour).
↓ Washing (PBS)
Color by adding substrate TMB (tetramethylbenzidine) solution (3 minutes, room temperature, 100 μL / well)
↓ Measured with reaction-stopped spectrophotometer (OD450nm) with 1N H ₂ SO ₄ (OD620nm as reference wavelength).

The cells used for the measurement are as follows.
293T-HA-HBZ (positive control)
293T-HAMock (negative control)
CEM, Jurkat: HTLV-1 non-infected human T cell line
C5MJ, HUT102, MT-1, MT-2, MT-4, SLB-1, TLOm-1: HTLV-1-infected human T cell lines The results are shown in FIGS.
5 and 6, the sandwich ELISA system constructed using pep # 3 # 21-1 as an antigen-capturing antibody and pep # 2 # 20-H12 as a detection antibody can quantitatively detect intracellular HBZ protein. I understood it. OD450 = 0.048 was set as a cut-off value from the measured values of CEM cells and Jurkat cells, which are HTLV-1 non-infected cells. The measurement range is 40 ng / ml to 3000 ng / ml.

Example 6 Construction of sandwich ELISA using novel anti-HBZ monoclonal antibody (2)
A sandwich ELISA was constructed in the same manner as in Example 5 except that pep # 3 # 91-1 was used instead of pep # 3 # 21-1 as the capture antibody. The results are shown in FIGS.
7-9, the sandwich ELISA system constructed using pep # 3 # 91-1-1 as the antigen capture antibody and pep # 2 # 20-H12 as the detection antibody can quantitatively detect intracellular HBZ protein. I found out that OD450 = 0.060 was set as the cut-off value from the measured values of CEM cells and Jurkat cells, which are HTLV-1 non-infected cells. The measuring range is 50 ng / ml to 2500 ng / ml. In addition, since the HBZ protein in the patient's peripheral blood mononuclear cells can also be detected quantitatively, this sandwich ELISA can be used to quickly evaluate the dynamics of the pathological condition-related protein HBZ in HTLV-1-related disease patient samples. it can.

The present invention enables sensitive and quantitative detection of HBZ protein, which was impossible in the past. By applying the results of the present invention to research using clinical specimens, testing, diagnosis and pathology of HTLV-1-related diseases It is expected to be applicable to grasping.
According to the diagnostic agent and diagnostic kit of the present invention, the presence or absence of HTLV-1-related disease can be determined specifically and simply. According to the detection method of the present invention, it is possible to appropriately determine the presence or absence of a HTLV-1-related disease and the presence or absence of a therapeutic effect alone or in combination with other diagnostic methods.

  Although several specific embodiments of the present invention have been described in detail, those skilled in the art will recognize that various modifications may be made to the specific embodiments shown without departing from the teachings and advantages of the invention. It is possible to make changes. Accordingly, all such modifications and changes are intended to be included within the spirit and scope of the invention as claimed in the following claims.

The present invention provides the following.
[1] An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4;
An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5; and at least one antibody selected from the group consisting of an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6. Detection reagent for HBZ protein.
[2] An antibody produced by administering a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 as an antigen to a non-human animal, and a polypeptide containing the amino acid sequence shown in SEQ ID NO: 5 as an antigen in a non-human animal The detection according to [1], which is an antibody selected from the group consisting of an antibody produced by administration and an antibody produced by administering a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 as an antigen to a non-human animal reagent.
[3] An antibody produced by administering a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 to a non-human animal as an antigen, and administering a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 to a non-human animal as an antigen The antibody according to [1] or [2], wherein the antibody is selected from the group consisting of an antibody produced by administration and an antibody produced by administering a peptide comprising the amino acid sequence shown in SEQ ID NO: 6 as an antigen to a non-human animal. Detection reagent.
[4] The detection reagent according to any one of [1] to [3], wherein the antibody is a monoclonal antibody.
[5] The detection reagent according to any one of [1] to [4], which contains at least two of the antibodies.
[6] A combination of an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 and an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 5,
A combination of an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 and an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 6, or a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 The detection reagent according to any one of [1] to [5], which is a combination of an antibody that specifically binds to and an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6.
[7] A combination of an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 and an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 6. [1] -Detection reagent in any one of [6].
[8] the hybridoma antibody is selected from the group consisting of hybridoma represented by Accession No. NITE BP-01993 hybridoma and Accession No. NITE BP -01 968 represented is a monoclonal antibody produced, [1] to [7 ] The detection reagent in any one of.
[9] An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5, and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 A diagnostic agent for HTLV-1-related diseases, comprising at least two antibodies selected from the group consisting of antibodies that specifically bind.
[10] The diagnostic agent according to [9], wherein the HTLV-1-related disease is adult T-cell leukemia or HTLV-1-related myelopathy.
[11] An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 A kit for detecting an HBZ protein or diagnosing an HTLV-1-related disease, comprising at least two types of antibodies selected from the group consisting of antibodies that specifically bind to each other in separate containers.
[12] The kit according to [11], wherein one kind of the antibody is immobilized on a solid phase.
[13] The kit according to [11] or [12], wherein the HTLV-1-related disease is adult T-cell leukemia or HTLV-1-related myelopathy.
[14] a monoclonal antibody hybridoma antibody is selected from the group consisting of hybridoma represented by Accession No. NITE BP-01993 hybridoma and Accession No. NITE BP -01968 represented is produced, [9] or [10 Or the kit according to any one of [11] to [13].
[15] Accession No. NITE BP-01993 hybridoma represented by Accession No. NITE BP hybridoma represented by -01968 and Accession No. NITE monoclonal antibody hybridoma produced selected from the group consisting of hybridoma represented by BP -01969 .
[16] The following steps:
(1) a blood sample derived from a subject;
An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 Contacting with an antibody selected from the group consisting of antibodies to bind to form a complex of HBZ protein and antibody contained in the blood sample; and (2) HBZ in the complex formed in step (1) Detecting the protein;
A method for detecting an HTLV-1-related disease, comprising:
[17] The detection method according to [16], wherein the step (2) detects the HBZ protein in the complex using an antibody that specifically binds to a peptide different from the antibody used in the step (1). .
[18] The antibody used in step (1) is a capture antibody immobilized on a solid phase, and the antibody used in step (2) is an antibody that specifically binds to a peptide different from the capture antibody, (2) The detection method according to [16], wherein the HBZ protein in the complex is detected by a sandwich method.
[19] The detection method according to [18], wherein the antibody used in the step (2) is enzyme-labeled, and the step (2) detects by a sandwich ELISA method.
[20] a hybridoma antibody is selected from the group consisting of hybridoma represented by Accession No. NITE BP-01993 hybridoma and Accession No. NITE BP -01968 represented is a monoclonal antibody produced, [16] - [19 ] The detection method in any one of.

In a further preferred embodiment, the anti-HBZ antibody is a monoclonal antibody produced by a specific hybridoma. On November 19, 2014, the present applicant was assigned to the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Kazusa Kamashizu 2-5-8, Kisarazu, Chiba, Japan) with accession numbers NITE BP- 01968 and was deposited with the two types of hybridoma of accession number NITE BP -01969, on January 22, 2015, it has been deposited with the hybridoma of accession number NITE BP-01993. The three hybridomas NITE BP-01993 , NITE BP- 01968 and NITE BP- 01969 were obtained by using the HBZ protein produced by peptide 2, peptide 3 and wheat cell-free protein expression system as antigens, respectively. The monoclonal antibodies produced by these hybridomas are referred to as pep # 2 # 20-H12, pep # 3 # 91-1 and α wheat HBZ # 7-1, respectively. In the present invention, it is preferable to use monoclonal antibodies produced by these deposited hybridomas from the viewpoint of low background noise.

Among the resulting hybridomas, pep # 2 is # 20-H12 strain, 201 to 5 January 22, National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (Japan Kisarazu, Chiba Kazusa legs bowed in 2- 5-8), deposit number NITE BP-01993 . Similarly, the pep # 3 # 91-1 strain was deposited under the accession number NITE BP- 01968 and the α wheat HBZ # 7-1 strain was deposited under the accession number NITE BP- 01969 on November 19, 2014 .

Claims (20)

An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4;
An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5; and at least one antibody selected from the group consisting of an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6. Detection reagent for HBZ protein.   An antibody produced by administering a polypeptide containing the amino acid sequence shown in SEQ ID NO: 4 as an antigen to a non-human animal, and administering a polypeptide containing the amino acid sequence shown in SEQ ID NO: 5 as an antigen to a non-human animal The detection reagent according to claim 1, which is an antibody selected from the group consisting of an antibody produced by administering the produced antibody and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 as an antigen to a non-human animal.   The antibody is produced by administering a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 as an antigen to a non-human animal, and produced by administering a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 as an antigen to a non-human animal. The detection reagent according to claim 1 or 2, which is an antibody selected from the group consisting of an antibody and an antibody produced by administering a peptide comprising the amino acid sequence shown in SEQ ID NO: 6 as an antigen to a non-human animal.   The detection reagent according to any one of claims 1 to 3, wherein the antibody is a monoclonal antibody.   The detection reagent according to any one of claims 1 to 4, comprising at least two kinds of the antibodies. A combination of an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 and an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5;
A combination of an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4 and an antibody that specifically binds to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 6, or a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 The detection reagent according to any one of claims 1 to 5, which is a combination of an antibody that specifically binds to a peptide and an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6.   The antibody is a combination of an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 and an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6. The detection reagent of any one.   The antibody according to any one of claims 1 to 7, wherein the antibody is a monoclonal antibody produced by a hybridoma selected from the group consisting of a hybridoma represented by the receipt number NITE ABP-01967 and a hybridoma represented by the receipt number NITE ABP-01968. The detection reagent according to item.   An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 A diagnostic agent for HTLV-1-related diseases, comprising at least two antibodies selected from the group consisting of antibodies that bind.   The diagnostic agent according to claim 9, wherein the HTLV-1-related disease is adult T-cell leukemia or HTLV-1-related myelopathy.   An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 A kit for detecting an HBZ protein or diagnosing an HTLV-1-related disease, comprising at least two types of antibodies selected from the group consisting of antibodies that bind to each other in separate containers.   The kit according to claim 11, wherein one of the antibodies is immobilized on a solid phase.   The kit according to claim 11 or 12, wherein the HTLV-1-related disease is adult T-cell leukemia or HTLV-1-related myelopathy.   The diagnosis according to claim 9 or 10, wherein the antibody is a monoclonal antibody produced by a hybridoma selected from the group consisting of a hybridoma represented by receipt number NITE ABP-01967 and a hybridoma represented by receipt number NITE ABP-01968. A drug or a kit according to any one of claims 11 to 13.   A monoclonal antibody produced by a hybridoma selected from the group consisting of a hybridoma represented by receipt number NITE ABP-01967, a hybridoma represented by receipt number NITE ABP-01968, and a hybridoma represented by receipt number NITE ABP-01969. The following steps:
(1) a blood sample derived from a subject;
An antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, an antibody that specifically binds to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 and a peptide consisting of the amino acid sequence shown in SEQ ID NO: 6 Contacting with an antibody selected from the group consisting of antibodies to bind to form a complex of HBZ protein and antibody contained in the blood sample; and (2) HBZ in the complex formed in step (1) Detecting the protein;
A method for detecting an HTLV-1-related disease, comprising:   The detection method according to claim 16, wherein the step (2) detects the HBZ protein in the complex using an antibody that specifically binds to a peptide different from the antibody used in the step (1).   The antibody used in the step (1) is a capture antibody fixed on a solid phase, the antibody used in the step (2) is an antibody that specifically binds to a peptide different from the capture antibody, and the step (2) The detection method according to claim 16, wherein the HBZ protein in the complex is detected by a sandwich method.   The detection method according to claim 18, wherein the antibody used in the step (2) is enzyme-labeled, and the step (2) detects by a sandwich ELISA method.   20. The monoclonal antibody produced by a hybridoma selected from the group consisting of a hybridoma represented by the receipt number NITE ABP-01967 and a hybridoma represented by the receipt number NITE ABP-01968. The detection method according to item.