WO2005076002A1 - Antibody assay method - Google Patents

Abstract

There has been required a reagent for judging the occurrence or non-occurrence of the onset of an HTLV-1-associated disease and the onset of superinfection with an infections disease other than HTLV-1 in an avirulent HTLV-1 carrier. It is intended to provide an assay method and an assay reagent for anti-gp46-197 antibody; and the above reagent to be used in judging the occurrence or non-occurrence of the onset of an HTLV-1-associated disease and the onset of superinfection with an infections disease other than HTLV-1 in an avirulent HTLV-1 carrier. It is also intended to provide a kit for judging the occurrence or non-occurrence of the onset of an HTLV-1-associated disease and the onset of superinfection with an infections disease other than HTLV-1 in an avirulent HTLV-1 carrier which comprises a reagent for assaying anti-gp46-197 antibody combined with a known assay reagent for an antibody binding to HTLV-1. It is further intended to provide a diagnostic for an HTLV-1-associated disease and a method of judging the onset thereof by which the presence or absence of anti-gp46-197 antibody is examined, and a diagnostic for the superinfection with an infections disease other than HTLV-1 in an avirulent HTLV-1 carrier and a method of judging the occurrence or non-occurrence of the onset thereof. Moreover, it is intended to provide a solid phase to be used in assaying anti-gp46-197 antibody.

Description

 Description Antibody measurement method

 The present invention uses a peptide (hereinafter, referred to as gp46-197) corresponding to the region of Aspl97-Leu216 of glycoprotein gp46 of human T lymphocyte tropic virus 1 (hereinafter, referred to as HTLV-1) coat A method and a reagent for measuring an antibody binding to gp46-197 in a sample (hereinafter referred to as an anti-gp46-197 antibody) for determining the presence or absence of an HTLV-1-related disease The present invention also relates to a method and a reagent for measuring an anti-gp46-197 antibody in a sample for determining the presence or absence of an infectious disease other than HTLV-1 in which a non-onset HTLV-1 carrier is co-infected. Further, the present invention provides a kit for determining the presence or absence of an HTLV-1-related disease, which comprises a reagent for measuring an anti-gp46-197 antibody and a reagent for measuring an antibody that binds to HTLV-1, and a kit for determining the absence of HTLV-1 related disease. — 1) A kit for determining the presence or absence of infectious diseases other than HTLV-1 co-infected carriers. Further, the present invention is characterized by detecting an anti-gp46-197 antibody in a sample, a diagnostic agent for an HTLV-1-related disease and a method for determining the presence or absence of the disease, and an anti-gp46-197 antibody in the sample. The present invention relates to a diagnostic agent for an infectious disease other than HTLV-1 co-infected with an asymptomatic HTLV-1 carrier and a method for determining the presence or absence of a disease, characterized in that the disease is detected. The present invention also relates to a solid phase on which gp46-197 is immobilized and which has been blocked with a substance not containing HSC70. Background art

 Human T, lymphocytic virus (hereinafter referred to as HTLV) is a type of retrovirus and has two types, type 1 and type 2. Among them, HTLV-1 was identified as the cause of adult T-cell leukemia / lymphoma (hereinafter referred to as ATLL) with the use of Hinozuma et al. In 1981 [Proceeding of the National Academy of Sciences of the United States of America, 78, 6476 (1981), Proceeding of the National Academy of Sciences of the United States of America, 79, 2031 (1982), Sciences, 219, 856 (1983)]. HTLV-1 infection in humans is mainly vertical transmission from mother to child and horizontal transmission from husband to wife, but iatrogenic transmission by blood transfusion has also been known. Transgenic iatrogenic infection was prevented by testing for anti-HTLV-1 (or 2) antibodies, which began in 1986. Epidemiological studies of iatrogenic infection by blood transfusion indicate that HTLV-1 infection is mediated by blood cell components.

Recognition of the blood cell receptor targeted by the virus in virus infection TJP2005 / 002103 A function involved in the binding of virus to the receptor is provided in the glycoprotein of the viral coat. The protein of the HTLV-1 envelope is synthesized as a precursor protein of 6 lkDa, and is treated by cellular proteases with gp46, an N-terminal membrane surface glycoprotein consisting of Metl-Arg312, Ala313—cleaved into the C-terminal transmembrane glycoprotein gP21 consisting of Leu488. gp46 and gp21 form a complex by non-covalent bonding, and gp21 is present on the virus surface as an anchor protein of gp46. After that, three regions, HT111, lys111 on gp46, a region corresponding to Asp197-Leu216 on gp46, and a region corresponding to Cys400-Leu429 on gp21 were HT. It was found to be an area involved in LV-1 infection [Journal of Virology, 70, 1564 (1996)].

 As HTLV-1 related diseases (hereinafter referred to as HTLV-1 related diseases), HTLV-1 related myelopathy (HAM / TSP), uveitis (HU) and the like are known in addition to ATLL. When infected with HTLV-1, an anti-HTLV-1 antibody is produced in the living body. Thus, by measuring the anti-HTLV-1 antibody, the infection of HTLV-1 can be known. Known methods for measuring anti-HTLV-1 antibody include immunological methods, such as gelatin particle agglutination (PA) and fluorescent antibody.

(FA method), indirect fluorescent antibody method (IF method), enzyme immunoassay. (ELISA method) ', estanbroth method (WB method) and the like are known. The PA method is used for primary screening because the PA method is simple and can measure many samples. However, the PA method has problems such as false positives or nonspecific reactions due to low antibody titers in serum, and the conventional ELISA method has problems such as nonspecific reactions due to autoantibodies. This non-specific reaction is thought to be due to the fact that the antigen used for antibody detection uses a non-single antigen derived from cultures of HTLV-1-infected cell lines. As an ELISA method using a single antigen, an enzyme immunoassay using a peptide corresponding to the region from Phe175 to I1e199 on gp46 (hereinafter referred to as gp46-175) is used. [1990 Ministry of Health and Welfare adult T-cell leukemia

Study on the prevention of mother-to-child transmission of (ATL), '174]. In this method, antibodies reactive with gp46-175 can be detected in 90% or more of blood donors, but all HTLV-1 infections can be detected. It is not a method that can detect the antibody from a blood donor.

 Judgment of HTLV-1 infection by a single method is not reliable. Even if the primary screening is positive, it is necessary to perform secondary screening such as the FA method and the WB method. Furthermore, in order to finally make a diagnosis that the subject is infected with HTLV-1, the Southern Protocol method should be used to confirm that HTLV-1 proviral DNA is integrated into the lymphocyte genome of peripheral blood or lymph node cells. Alternatively, it must be genetically verified using the PCR method.

At present, methods for determining the presence or absence of HTLV-1-related disease are mainly based on clinical findings. HTLV-1 provirus in lymphocyte genome In the PCR method that detects the incorporation of DNA, the PCR method is based on the difference in the staining pattern of the non-onset HTLV-1 carrier and the patient who developed the HTLV-1 related disease on electroperfusion. — 1 Although it may be an index for determining the presence or absence of related diseases, the PCI method involves complicated operations.

 So far, factors that can be used to determine the onset of HTLV-1 related disease have been extensively searched for using simple methods, but HTLV has only been substantiated based on the amount of virus in vivo and history of onset in families. — 1 Factors that can be used to determine the onset of related diseases have not been identified.

 In addition, patients infected with HTLV-1 are more likely to develop special opportunistic infections that are rarely seen in healthy people, such as viral infections, fungal infections, pneumonia caused by potassium biprotozoa, and nematode disease. The risk of superinfection with infections other than HTLV-1 is increased [Journal of Infectious Disease, 184, 1114 (2001)]. Evidence of invention

An object of the present invention is to provide a method for measuring anti-gp46-197.antibody in a sample, which comprises using 'gp46-197, for determining the presence or absence of an HTLV-1-related disease. To provide a measuring reagent and a method for measuring an anti-gp46-197 antibody in a sample and a test assay for determining the presence / absence of an infection other than HTLV-1 in which a non-onset HTLV-1 carrier is superinfected. It is in. Another object of the present invention is to provide a kit for determining the presence or absence of an HTLV-1-related disease, comprising a reagent for measuring an anti-gp46-197 antibody and a reagent for measuring an antibody that binds to HTLV-1. Onset HTLV-1 'To provide a kit for determining the presence or absence of an infection other than HTLV-1 with which the carrier was co-infected. Further, an object of the present invention is to detect an anti-gp46-197 antibody in a sample, comprising a diagnostic agent for HTLV-1 related disease and a method for determining the presence or absence of the onset, and an anti-gp46 in the sample. An object of the present invention is to provide a diagnostic agent for an infectious disease other than HTLV-1 co-infected with a non-onset HTLV-1 carrier and a method for determining the presence or absence of the onset, characterized by detecting the 197 antibody. Another object of the present invention, g _P 46- 197 is immobilized, it is to provide a an included no material solid that blocking is made by the phase HSC70. The present invention relates to the following (1) to (27).

(1) The onset of HTLV-1 related disease, characterized by using a peptide corresponding to the region of Aspl97-Leu216 of gp46 glycoprotein gp46 (hereinafter referred to as gp46-197). A method for measuring an antibody that binds to gp46-197 (hereinafter referred to as anti-gp46-197 antibody) in a sample to determine the presence or absence of gp46-197. JP2005 / 002103

(2) Anti-gp46-197 in f 贪 body for determining the presence or absence of an infectious disease other than HTLV-1 co-infected with asymptomatic HTLV-1 carrier, characterized by using gp46-197. How to measure antibodies.

 (3) The method according to (2), wherein the infectious disease other than HTLV-1 is hepatitis C virus (hereinafter referred to as HCV) infectious disease.

 (4) gp46-l97 is a peptide having the amino acid sequence shown in SEQ ID NO: 1 or one or more amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 1; The method according to any one of (1) to (3), which is a peptide capable of binding to the 197 antibody.

 (5) The method according to any one of (1) to (4), wherein the method for measuring the antibody is an immunological assay.

 (6) The method according to (5), wherein the immunological assay is a method using a solid phase on which p46-197 is immobilized.

 (7) An immunoassay using a solid phase on which gp46-197 has been immobilized and which has been blocked with a substance that does not contain a heat-shock-like protein of 71 kDa (hereinafter referred to as HSC70). The method according to (5) or (6).

 (8) The method according to (7), wherein the substance not containing HSC 70 is a synthetic polymer.

 (9) The method according to (8), wherein the synthetic polymer is a surfactant.

 (10) The method according to any one of (5) to (9), wherein the immunological assay is an enzyme immunoassay (ELISA).

 (11) The measurement method according to (10), comprising the following steps.

 (a) a step of immobilizing gp46-197 on a carrier to produce a solid phase in which 46-197 is immobilized;

 (b) Step of reacting the sample with gp46-197 immobilized on the solid phase (primary reaction step)

 (c) a step of reacting a labeled secondary antibody with a complex consisting of gp46-197 immobilized on a solid phase and an anti-gp46-197 antibody bound to the peptide (secondary reaction step)

 (d) measuring the amount of labeled secondary antibody bound to a complex consisting of gp46-197 immobilized on a solid phase and an anti-gp46-197 antibody bound to the peptide

 (e) From the calibration curve showing the relationship between the anti-gp46-197 antibody concentration and the labeling amount prepared using the standard substance containing the anti-gp46-197 antibody, and the measurement value measured in (d), Determining anti-gp46-197 antibody concentration

(12) A reagent for measuring an anti-gp46-197 antibody in a sample, which is characterized by using gp46-197, for determining the presence or absence of an HTLV-1-related disease. (13) Anti-gp46-in a sample, for determining the presence or absence of an infectious disease other than HTLV-1 and co-infected HTLV-1 carriers, characterized by using gp46-197. Assay reagent for 197 antibody.

 (14) The reagent according to (13), wherein the infection other than HTLV-1 is HCV infection.

(15) gp46-197 is a peptide having the amino acid sequence shown in SEQ ID NO: 1 or one or more amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 1, and the anti-gp46-197 antibody Peptides that can bind (12)-

The reagent according to any one of (14).

 (16) The reagent according to any one of (12) to (15), wherein the method for measuring an antibody is an immunoassay. '

 (17) The reagent according to (16), wherein the immunological assay is a method using a solid phase on which gp46-197 is immobilized.

 (18) The reagent according to (16) or (17), wherein the immunological assay is a method using a solid phase on which gp46-197 is immobilized and which has been blocked with a substance not containing HSC70. .

 (19) The reagent according to (18), wherein the substance not containing HSC70 is a synthetic polymer. (20) The reagent according to (18), wherein the synthetic polymer is a surfactant.

 (21) The reagent according to any one of (16) to (20), wherein the immunoassay is an enzyme immunoassay (ELISA).

 (22) The reagent for measuring an antibody according to any one of (12) to (21), which comprises an anti-gp46-197 human antibody as a standard substance.

 (23) To determine the presence or absence of an HTLV-1-related disease, comprising a combination of a reagent for measuring an antibody that binds to HTLV-1 and the reagent according to any one of (12) to (22). Kit.

 (24) An HTLV-1 non-symptomatic HTLV-1 carrier, which is obtained by combining a reagent for measuring an antibody that binds to HTLV-1 and the reagent according to any one of (12) to (22). — A kit to determine the presence or absence of an infection other than 1.

 (25) The key according to (24), wherein the infection other than HTLV-1 is HCV infection.

(26) An antibody that binds to HTLV-1 antibody (hereinafter referred to as gp46-175) that binds to a peptide corresponding to the region of Phe 175-11 e 199 of glycoprotein gp46 of HTLV-1 coat (hereinafter referred to as gp46-175) The kit according to any one of (23) to (25), which is referred to as an anti-gp46-175 antibody.

(27) a peptide in which gp46-175 has the amino acid sequence shown in SEQ ID NO: 2, or one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 are deleted, added, or substituted, and an anti-gp46-175 antibody The reagent according to (26), which is a peptide capable of binding. (28) A solid phase on which gp46-197 has been immobilized and which has been blocked with a substance not containing HSC70.

 (29) The solid phase according to (28), wherein the substance not containing HSC70 is a synthetic polymer. (30) The solid phase according to (29), wherein the synthetic polymer is a surfactant.

 (31) A diagnostic agent for an HTLV-1 related disease, characterized by detecting an anti-gp46-197 antibody in a sample.

 (32) A diagnostic agent for an infectious disease other than HTLV-1 in which a non-onset HTLV-1 carrier is co-infected, characterized by detecting an anti-gp46-197 antibody in a sample.

 (33) The diagnostic agent according to (32), wherein the infection other than HTLV-1 is HCV infection.

 (34) A method for determining the presence or absence of an HTLV-11-related disease, comprising detecting an anti-gp46-197 antibody in a sample.

 (35) A method for determining the presence or absence of an infectious disease other than HTLV-1 in which a non-onset HTLV-1 carrier is co-infected, comprising detecting an anti-gp46-197 antibody in a sample.

(36) The determination and method according to (35), wherein the infectious disease other than HTLV-1 is HCV infectious disease. Hereinafter, the present invention will be described in detail. This application claims the priority of Japanese Patent Application No. 2004-31431 filed on Feb. 6, 2004 and includes the contents described in the description and drawings of the patent application. This ^ Akira is characterized by the use of g _P 46- 197, HTLV- 1 for determining the presence or absence of the onset of associated diseases, anti-gp 46 in the sample - 197 method for measuring the antibody, no onset parallel beauty The present invention relates to a method for measuring an anti-gp46-197 antibody in a sample for determining the presence or absence of an infection other than HTLV-1 in which HTLV-1 carrier is co-infected. gp46-197 is a peptide corresponding to Asp97-Leu216 of glycoprotein gp46 of HTLV-1 coat, and has an amino acid sequence represented by SEQ ID NO: 1.

 In the present invention, gp46-197 of the present invention also includes a peptide in which one or more amino acids of the amino acid sequence represented by SEQ ID NO: 1 has been deleted, added or substituted, and has an activity capable of binding to an anti-gp46-197 antibody. You. Anti-gp46-197 antibody refers to an antibody that binds to p46-197.

The number of amino acids to be deleted, added or substituted is one or more, and the number is not particularly limited. However, Molecular 'Cloning Second Edition, / Rent' Protocols 'in Molecular' biology , Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids 2005/002103

Natl. Acad. Sci USA, 82, 488 (1985), etc.), and can be deleted, added or substituted by well-known techniques such as site-directed mutagenesis. The number is, for example, one to several tens, preferably one to twenty, more preferably one to ten, and even more preferably one to five.

 Deletion, addition or substitution of one or more amino acid residues in an amino acid sequence is defined as deletion, addition or substitution of one or more amino acid residues in any one or more amino acid sequences in the same sequence. Deletion, addition, or substitution may occur simultaneously, and the amino acid residue to be deleted, added, or substituted may be either natural or non-natural. Natural amino acid residues include L-alanine, L-asparagine, L-asparaginic acid, L-glutamic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L -Methionine, L-phenylalanine, L-.proline, L-serine, 'L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.

 Preferred examples of mutually substitutable amino acid residues are shown below. Amino acid residues included in the same group below can be substituted for each other.

 Group A: leucine, isoleucine, norleucine, valine, norparin, alanine, 2-aminobutanoic acid, methionine, 0-methylserine, t-butylglycine, t-butylylalanine, cyclohexylalanine

 , Group ：: Aspartic acid Glutamic acid, Isoaspartic acid, Isoglutamic acid, 2-Aminodipic acid, 2-Aminosveric acid

 Group C: Asparagine, Glutamine.

 Group D: lysine, arginine, orditin, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid

 Group ：: Proline, 3-hydroxyproline, 4-hydroxyproline

 Group F: serine, threonine, homoserine

 Group G: Phenylalanine, Tyrosine

 The gp46-197 used in the present invention is obtained by introducing and expressing an expression vector containing cDNA encoding the amino acid sequence of the peptide into E. coli, yeast, insect cells, animal cells, etc. It can be obtained as a type peptide. Alternatively, a synthetic peptide having the sequence can be used.

 A cysteine can be added to the end of the synthetic peptide to crosslink with the carrier protein. In addition, the synthetic peptide may be acetylated at the N-terminus and amidated at the C-terminus as necessary.

 Synthetic peptides can be synthesized by general liquid-phase and solid-phase peptide synthesis methods, by appropriately combining them, or by methods analogous thereto.

Peptides, Analysis, Synthesis, Biology, Vol. 1, 1979; Vol. 2, 1980; Vol. 3, 1981, Academic Press; Basics and experiments on peptide synthesis, Maruzen, 1985; Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991; International Journal of Peptide & Protein Research, 35, 161-214, 1990).

 Synthetic peptides can be synthesized using an automatic peptide synthesizer. Peptide synthesis by peptide synthesizer is carried out by peptide synthesizer manufactured by Shimadzu Corporation, peptide synthesizer manufactured by AppliedBiosystems, Inc. (hereinafter referred to as ABI), Advanced ChemTech Inc. (hereinafter ACT) On a commercially available peptide synthesizer such as a peptide synthesizer, use No: -Fmoc-amino acid or Να-Boc-amino acid with appropriately protected side chains, etc., according to the synthesis program for each. be able to.

 Protected amino acids and carrier resins used as raw materials can be obtained from ABI, Shimadzu, Kokusan Chemical, NovaBiochem, Watanabe Chemical, ACT, or Peptide Research Laboratories. Protected amino acids, protected organic acids, and protected organic amines as raw materials can be synthesized according to or according to the reported synthetic methods (The Peptides, Analysis, Synthesis, Biology, Vol. 1, 1979; Vol.2, 1980; Vol.3, 1981, Academic Press; Basics and experiments of peptide synthesis, Maruzen, 1985; Development of Continuing Pharmaceuticals, Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991; International Journal of Peptide & Protein Research, 35, 161-214, 1990).

 Anti-gp46-197 antibody is specifically expressed in patients with HTLV-1 related disease and in patients with infections other than HTLV-1 with co-infection of asymptomatic HTLV-1 carrier Therefore, measurement of anti-gp46-197 antibody in a sample can be used to determine the presence or absence of HTLV-1 related disease and to develop non-HTLV-1 carriers other than HTLV-1 that have been co-infected with a carrier. Can be used to determine the presence or absence of

 As the specimen of the present invention, any biological sample derived from a subject that may contain an anti-gp'46-197 antibody can be used. Examples include blood, plasma, serum, saliva, amniotic fluid, urine, sweat, semen, semen, breast milk, etc., and plasma and serum are preferably used.

 As a method for measuring the anti-gp46-197 antibody in a sample of the present invention, any method may be used as long as it uses gp64-197, but an immunological measurement method is preferably used.

Immunoassays include any known immunoassays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA or ELISA), and fluorescence immunoassay (FIA) indirect. Fluorescent antibody method (Ind irect Fluo resc en ceassay), luminescence immunoassay method (Lumi η esce η t immunoassay), physicochemical assay method (TIA, LAP IA, PCIA) _N Western blotting The ELISA method is preferably used [Monoclonal Antibody Experiment Manual (Kodansha Scientific, 1987), Seikagaku Laboratory Course 5 Immunobiochemical Research Method (Tokyo Kagaku Dojin, 1986) )]. Labels used in radioimmunoassay (RIA) include radioisotopes commonly used in biochemical experiments. For ^{example, 1 4 C, 3 2 P} , 1 2 5 E and Ru mentioned.

 As a label used in the enzyme immunoassay, any known enzyme label (enzyme immunoassay, edited by Eiji Ishikawa et al., Medical Shoin) can be used. For example, alkaline phosphatase label, peroxidase label, luciferase label and the like can be used.

 As a label used in the fluorescent immunoassay or the indirect fluorescent antibody method, any known fluorescent label (by Akira Kawai, fluorescent antibody method, Soft Science) can be used. For example, ^ such as FITC label and RITC label can be used.

 The label used in the luminescence immunoassay may be any known luminescence [Biol and Chemiluminescence, edited by Kazuhiro Imai, Hirokawa Shoten; Clinical Examination (1998)]. For example, an acridinium ester label, a mouth fin label, and the like can be used. The physicochemical measurement method (TIA, LAPIAPCIA) is a method of detecting an immune complex formed by an antigen-antibody reaction by increasing turbidity. Specifically, the detection is performed by using an antibody that binds to the antigen and detecting an aggregate formed by binding the antigen and the antibody. Examples of other physical measurement methods include a capillary method, a one-dimensional immunodiffusion method, an immunoturbidimetric method, and a latex immunoturbidimetric method. 1998)].

 In the latex immunoturbidimetry, for example, a carrier such as polystyrene latex having a particle size of about 0.1 to 1 / m sensitized with an antibody or an antigen is used to cause an antigen-antibody reaction with the corresponding antigen or antibody. Then, the scattered light in the reaction solution increases and the transmitted light decreases. By detecting this change as absorbance or turbidity of the integrating sphere, the amount of the antigen or antibody can be measured.

 As the immunological measurement method of the anti-gp46-197 antibody of the present invention, an immunological measurement method using an antigen-antibody reaction on a solid phase is used. Then, gp46-197 is immobilized on a carrier by a known method, and the peptide immobilized on a solid phase is allowed to react with a sample, whereby anti-gp46-197 bound on the solid phase is reacted. 7 A method of detecting an antibody by any method can be used, but preferably, an ELISA method is used.

 The ELISA method involves reacting an antibody immobilized on a solid phase with an antibody, and then reacting the antibody bound to the antigen with a secondary antibody that has been labeled with an enzyme such as peroxidase or alfa phosphatase. Then, the enzyme label is measured by an appropriate method, for example, a competitive method, a sandwich method [Immunology Illustrated, 5th Edition (Nankodo)].

 Examples of the sandwich method include a method in which a first antigen is immobilized on a solid phase, an antibody is trapped, and a labeled second antibody is reacted.

In the competitive method, for example, the same antigen as the first antigen is used simultaneously with the antibody of the above-mentioned sandwich method. PT / JP2005 / 002103 This is a method of adding and reacting, and is a method of measuring an antibody by comparing the measured value with and without the addition of an antigen.

 Hereinafter, the measurement method of the anti-46-197 antibody of the present invention and the use of the measurement method will be described in detail, taking the measurement by the ELISA method as an example.

 1. Measurement method of anti-gp46-197 antibody in sample

 The measurement of the anti-gp46-197 antibody in the sample can be carried out by using gp46-197 according to the San Germanti method or the competition method shown below.

Measurement method 1 (sandwich method)

 (1) immobilizing gp46-l97 on a carrier to produce a solid phase on which gp46-197 is immobilized;

 (2) reacting the sample with gp46-197 immobilized on a solid phase (primary reaction step);

 (3) a step of reacting a labeled secondary antibody with a complex comprising gp46-197 immobilized on a solid phase and an anti-gp46-197 antibody bound to the peptide (secondary reaction step) ;

 (4) the step of measuring the amount of labeled secondary antibody bound to a complex consisting of gp46-197 immobilized on a solid phase and an anti-46-197 antibody bound to the peptide;

 (5) From the calibration curve representing the relationship between the concentration of the anti-gp46- · 197 antibody and the amount of the label prepared using the standard substance containing the anti-gp46-197 antibody, and from the measurement value measured in (4), Determining the concentration of anti-gp46-197 antibody in the sample;

Measurement method consisting of:

Measurement method 2 (Competition method-1)

 (1) immobilizing p46-197 on a carrier to produce a solid phase having gp46_197 immobilized thereon;

 (2) a step of reacting the sample and the labeled anti-gp46-197 antibody with gp46-197 immobilized on a solid phase (primary reaction step);

 (3) a step of measuring the amount of labeling in the labeled anti-gp46-197 antibody bound to gp46-197 immobilized on the solid phase and the peptide;

 (4) From the calibration curve showing the relationship between the concentration of anti-gp46-197 antibody and the amount of label prepared using a standard substance containing anti-gp46-197 antibody, and the measurement value measured in (3), Determining the anti-gp46-197 antibody concentration of the antibody;

Measurement method consisting of:

Measurement method 3 (Competition method 1)

(1) immobilizing the anti-gp46-1.97 antibody on the carrier to prepare a solid phase on which the anti-gp46-197 antibody is immobilized; 02103

(2) reacting the sample, labeled 46-197 and anti-gp46-197 antibody immobilized on a solid phase (primary reaction step);

 (3) a step of measuring the amount of labeling in a complex comprising an anti-gp46-197 antibody immobilized on a solid phase and a labeled gp46-197 bound to the antibody;

 (4) From the calibration curve showing the relationship between the concentration of the anti-gp46-197 antibody and the amount of label prepared using the standard substance containing the anti-gp46-197 antibody, and the measurement value measured in (3), Determining the anti-gp46-197 antibody concentration of the antibody;

Measurement method consisting of:

 The solid phase having gp46-197 immobilized thereon, which is used in the above-described measurement methods 1 to 3, can be prepared by a method including the following steps.

 (1) a step of immobilizing gp46-197 on a carrier;

 (2) a step of blocking the surface of the carrier on which gp46-197 is immobilized;

(3) A step of washing the carrier after blocking.

 In the above method for preparing a solid phase, steps (2) and (3.) may be performed simultaneously using a washing solution containing a substance used for blocking in the washing solution used for washing. As the carrier, any carrier can be used as long as it can bind and retain a .gp46-197 or anti-gp46-197 antibody, but a material obtained by molding various polymer materials to suit the application is used. .

 Specifically, the shape of the carrier includes tubes, beads, plates, fine particles such as latex, and sticks, and the materials include polystyrene, polycarbonate, polyvinyl toluene, polypropylene, polyethylene, polyvinyl chloride, nylon, and the like. Examples include polymer materials such as polymethacrylate, gelatin, agarose, cellulose, and polyethylene terephthalate, glass, ceramics, magnetic particles and metals.

 As a method for immobilizing the gp46-l97 or anti-gp46-197 antibody, a known method such as a physical method and a chemical method, or a combination thereof can be used. Examples of the physical binding include physical adsorption. Examples of the chemical bond include a covalent bond and a non-covalent bond. Examples of the non-covalent bond include an electrostatic bond, a hydrogen bond, a hydrophobic bond, and a coordinate bond. For example, a polystyrene 96-well immunoassay microplate having a hydrophobic solid phase of peptide or the like can be used.

As the gp46-197 or anti-gp46-197 antibody to be immobilized, the gp46-197 or anti-gp46-197 antibody may be directly immobilized on a solid phase, or the gp46-197 or anti-gp46-197 antibody may be immobilized. Once it is bound to a carrier substance such as a protein, it may be immobilized on a solid phase. Carrier substances to which gp46-197 or anti-gp46-197 antibody is bound include, for example, 血清 serum derived albumin (hereinafter referred to as BSA) ゃ keyhole phosphate hemocyan (hereinafter referred to as KLH), etc. But Used.

The anti-gp46-197 antibody immobilized in the above-mentioned measurement method 1 or 3 or the anti-gp46-197 antibody used in the production of the labeled anti-gp46-197 antibody in the measurement method 2 includes antibodies capable of binding to gp46-197. Any antibody can be used. The antibody may be a polyclonal antibody or a monoclonal antibody, or Fab, Fab 'ヽ F (ab,) ₂ , a single-chain antibody and a disulfide antibody. Stabilized antibodies and the like can be used.

 The solid phase on which the gp46-197 or anti-gp46-197 antibody is immobilized protects the functional groups remaining on the carrier by blocking. Proteins, surfactants, commercially available blocking reagents, and the like are usually used as the substances used for blocking, but substances that do not contain HSC70 [Nucleic Acids Res. 15 (13), 5181-5197 (1987).] It is preferably used. The substance not containing HSC70 includes, for example, a synthetic polymer, and the synthetic polymer includes, for example, a surfactant. Examples of the surfactant include a nonionic surfactant, a cationic surfactant, an anionic surfactant, an amphoteric surfactant and the like, and a nonionic surfactant is preferably used. Examples of the nonionic surfactant include an acid amide type, a polyoxyethylene derivative ^, and a fatty acid ester type. Examples of the polyoxyethylene derivative-type surfactant include a polyoxyethylene alkylamine type, a polyoxyethylene alkyl ether type, a polyoxyethylene aryl ether type, and a polyoxyethylene / polystyrene. Oxypropylene mixed type and the like can be mentioned. Examples of the fatty acid ester type surfactant include a polyoxyethylene ester type of fatty acid and a polyoxyethylene sorbin ester type of fatty acid. As the substance used in the blocking of the present invention, a fatty acid ester surfactant of a polyoxyethylene sorbitan ester type of a fatty acid is preferably used, and specifically, Tween 20 ™ can be mentioned. '

 Procking can be performed, for example, by reacting at 4 to 37 ° C for 30 minutes or more.

 In the method for measuring an anti-gp46-197 antibody in a sample of the present invention, the reaction conditions in the primary reaction step or the secondary reaction step may be any conditions under which a normal antigen-antibody reaction is performed. The reaction temperature may be any range as long as the reactivity of the antibody does not change. For example, the reaction temperature can be freely set in the range of 4 ° C to 40 ° C. The reaction time can be set according to the reaction temperature.For example, when the reaction temperature is 4 ° C, the reaction time is 1 hour or more, and when the reaction temperature is room temperature, the reaction time is 10 minutes to 10 minutes. Preferably 8 hours. .

In the above measurement method, the sample is used as it is, but may be used after being diluted with a sample diluent or the like. TJP2005 / 002103 As the secondary antibody used for detection, any antibody can be used as long as it can bind to a human antibody. The antibody may be a polyclonal antibody, a monoclonal antibody, or a Fab antibody. And Fab 'ヽ F (ab') ₂ , single-chain antibodies and disulfide-stabilized antibodies, and the like. Preferably, a polyclonal antibody is used preferably.

The secondary antibody, anti-gp46-197 antibody or gp46-197 used for detection is labeled and used for detection. As a substance for labeling the peptide, the antibody or the antibody fragment, any known enzyme label (enzyme immunoassay, edited by Eiji Ishikawa et al., Medical School), luminescent label, fluorescent label or radioisotope can be used. Avidin, streptavidin, or biotin can also be used. For example, an enzyme label may be an alkaline phosphatase label, a peroxidase label, a luciferase label, etc., a luminescent label may be an acridinium ester label, a mouth fin label, or a fluorescent label. For example, green fluorescein protein label, red fluorescein protein label, fluorescein 4-isocyanate (FITC) label, and radioactive isotopes such as ¹⁴ C, ³² P, and ¹²⁵ I radioactive isotopes Used. .

 As a method for labeling the secondary antibody, anti-gp46-197 antibody or gp46-197 used for detection with the above-mentioned label, a method of binding by genetic engineering or a method of binding chemically is used. Can be The method of genetic engineering is described in the literature

Natl. Acad. Sci. USA, 93, 974 (1996); Proc. Natl. Acad. Sci. USA, 93, 7826 (1996). The method of chemically bonding can be performed according to the method described in the literature [Science, 261, 212 (1993)]. The method of chemically bonding radioisotopes can be performed according to the method described in the literature [Antibody Immunoconjugates and Radiopharmaceuticals, 3, 60 (1990); Anticancer Research, 11, 2003 (1991)]. .

 In the above measurement method, the solid phase during the reaction is washed as necessary during the operation, and unreacted gp46-197, anti-gp46-197, food, labeled gp46-197, and labeled anti-gp46 are used. -The 197 antibody and the like may be removed. Washing is performed by a washing method in a known immunological reaction using a washing solution described later. For example, when a 96-well plate is used for the solid phase, a commercially available plate washer or the like can be used.

Methods for measuring the amount of label include an absorbance method (colorimetric method), a fluorescence method, a luminescence method, and a radioactivity method. When the label is an enzyme, the amount of the label can be measured by reacting the enzyme substrate with the enzyme and measuring the produced substance. The reaction conditions between the enzyme substrate and the enzyme can be set for each enzyme used for labeling. The reaction temperature may be within a range where the reactivity of the enzyme does not change, and may be freely set within a range of 4 ° C or more. The reaction time can be set according to the reaction temperature, For example, when the reaction temperature is 4 ° C., the reaction time is 1 hour or more, and when the reaction temperature is room temperature, the reaction time is preferably 10 minutes to 8 hours.

 When the enzyme is peroxidase, the amount of peroxidase can be measured by, for example, an absorbance method or a fluorescence method. As a method for measuring the amount of peroxidase by the absorbance method, for example, peroxidase is reacted with a combination of its substrate, hydrogen peroxide and an oxidative chromogen, and the absorbance of the reaction solution is measured with a spectrophotometer or the like. There is a measuring method. Examples of the oxidative color-forming chromogen include a leuco-type chromogen and a chemical coupling-forming chromogen.

Leuco-type chromogens are substances that are converted to dyes alone in the presence of hydrogen peroxide and peroxidatively active substances such as peroxidase. Specifically, o-phenylenediamine, 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -1H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7bis (dimethylamino) -1 10H- phenothiazine (MCDP), N-(carboxymethyl § iminocarbonyl) one 4, 4 ⁵ one bis (Jimechiruamino) Jifuenirua Min sodium salt (DA- 64)ヽ4, 4, one-bis (Jimechiruamino) Jifue two Ruamin, Bis [3-bis (4-chlorophenyl) methyl-1-dimethylaminophenyl] amine (BCMA).

 An oxidative coupling chromogen is a substance in which two compounds are oxidatively coupled to form a dye in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Examples of the combination of the two compounds include a combination of a coupler and an aniline (Trinda reagent), a combination of a coupler and a phenol. Examples of couplers include 4-aminoantipyrine (4-AA), 3-methyl-2-benzothiazolinone hydrazine and the like. Examples of anilines include N- (3-sulfopropyl) aniline, N-ethyl-1N- (2-hydroxy-3-sulfopropyl) -13-methylaniline ('TOOS), N-ethyl-N- (2-hydrogen) Mouth xyl 3-sulfopropyl) 1,3,5-dimethylaniline (MAOS), N-ethyl-1-N- (2-hydroxy-13-sulfopropyl) 1,3-dimethoxyaniline (DA0S), N-ethyl — N— (3-Sulfopropyl) -1-3-methylaniline

(TOPS) s N— (2-hydroxy-13-sulfopropyl) -1,3-dimethoxyaniline (HDAOS), N, N-dimethyl-3-methylaniline, N, N-di

(3-Sulfoprovir) 1,3,5-Dimethoxyaniline, 1 ^ _Ethyl-1-! ^-(3-Sulfopropyl) -13-Methoxyaniline, N-Ethyl-1-N- (3-Sulfopropyl) aniline,

— N— (3-sulfopropyl) _3,5-dimethoxydiamine, N— (3-sulfopropyl) -1,3,5-dimethoxyaniline, N-ethyl —N— (3-sulfopropyl) -3,5-dimethyl Aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -13-methoxyaniline, N-ethyl-N

(2-Hydroxy-1-3-sulfopropyl) aniline, N-ethyl-1N— (3-methyl Rufueniru) one N, one succinyl ethylene § Min (EMSE), N- Echiru N i (3-methylphenyl) - N ⁵ - § Cetyl ethylene § Min, N- Echiru one N-(2-hydroxy-one 3- sulfo Propyl) -14-fluoro-3,5-dimethoxyaniline (F-DAOS). Examples of phenols include phenol, 4-methylphenol, 3-methylphenol, and 3-hydroxy-1,2,4,6-triiodobenzoic acid (HTIB).

 Examples of a method for measuring the amount of peroxidase by a fluorescence method include a method of reacting peroxidase with a combination of its substrate, hydrogen peroxide and a fluorescent substance, and measuring the intensity of generated fluorescence. Examples of the fluorescent substance include 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, and coumarin.

When the enzyme is alkaline phosphatase, the amount of alkaline phosphatase can be measured by, for example, a luminescence method. Methods for measuring the amount of alkaline phosphatase by a luminescence method include, for example, a method of inverting an alkaline phosphatase and its substrate and measuring the luminescence intensity of the generated luminescence with a luminescence intensity meter or the like. Can be exacerbated. Substrates for alkaline phosphatase include, for example, 3- (2,1-spiradamantane) -1,4-methoxy-14- (3,1-phosphoryloxy) phenyl-1,1,2-dioxenyl disodium salt (AMPPD) , 2-black mouth 5-{4-methoxyspiro [1,2-dioxenone 1-3, 2, 1 (5, 1 mouth) tricyclo [3.3.1.13,7] decane]-4 —Yl} phenyl phosphate 'disodium salt (CDP-Star ™), 3- {4-methoxyspiro [1,2-dioxetane-1,3,2']-(5, one-mouth) tricyclo [3. 3. 1. 13,7] decane] _4-—yl} phenyl phosphate 'disodium salt (CSPD ^™ ), [10-methyl 9 (10H) -acridinyliden] phenoxymethyl phosphate' Disodium salt (Lumigen ^™ APS-5).

 When the enzyme is 5-D-galactosidase, the amount of D-galactosidase can be measured by, for example, an absorbance method (colorimetric method). As a method of measuring the amount of /?-D-galactosidase by the absorbance method (colorimetric method), for example, reacting /?-D-galactosidase with its substrate, and measuring the absorbance of the reaction solution with a spectrophotometer And the like. Examples of the substrate for D-galactosidase include 2-chloro-1,412-drofurdinyl-D-galactoside.

 In the case of performing the above-mentioned measurement using R A using a radioisotope as a label, the amount of the radioisotope can be determined by measuring the radioactivity using a scintillation counter or the like.

 The calibration curve can be obtained by preparing an anti-gp46-197 antibody solution of known concentration as a standard substance, diluted in several steps, and performing the above-described measurement method.

2. Anti-46-197 antibody assay reagent The measurement reagent for the anti-gp46-197 antibody of the present invention is used in the above-described measurement method, and is essentially the same as each component of the measurement reagent for the antibody including the component capable of performing the method, or a part thereof. As long as it contains a substance that is essentially the same as the above, it is included in the reagent of the present invention, even if the composition or form is different.

 The components include a solid phase on which gp46-197 or anti-gp46-197 antibody is immobilized, a labeled secondary antibody or an antibody fragment thereof used for detection, an anti-gp46-l97 antibody or gp46-197. 197, etc., and if necessary, also include the sample diluent, reaction buffer, washing solution, label detection reagent, anti-gp46-197 human antibody standard used in the above measurement method, etc. It is.

 Examples of the sample diluent include an aqueous solution containing a stabilizer in a surfactant, a buffer, or the like. When whole blood is used as the sample, the aqueous solution contains salts, sugars, etc. for the purpose of preventing a change in the concentration of components in serum due to swelling or contraction of blood cells such as red blood cells.

 6

Preferably, the isotonic solution is prepared with a buffer or the like. The salts are not particularly limited, and examples thereof include alkali metal halides such as sodium chloride and potassium chloride. The saccharide is not particularly limited, and examples thereof include sugar alcohols such as mannitol and sorbitol.

 (2) Any reaction buffer may be used as long as it provides the solvent conditions for the antigen-antibody reaction. If necessary, a surfactant, a buffer, a preservative, a stabilizer, an enzyme activity regulator or an enzyme stabilizer may be added.

Any washing solution can be used as long as it can remove and wash unreacted substances and does not affect the antigen-antibody reaction. If necessary, add buffering agents, surfactants, preservatives or stabilizers! You may. For example, phosphate buffered saline containing 0.05% Tween20 ^™ (hereinafter referred to as PBS) is usually used.

 The buffer is not particularly limited as long as it has a buffering ability. For example, lactate buffer, citrate buffer, acetate buffer, succinate buffer, fluoric acid buffer, phosphoric acid having ρΗ1 to 11 Buffer (except when the label is alcoholic phosphatase), triethanolamine buffer, genolamine buffer, lysine buffer, barbituric buffer, tris (hydroxymethyl) aminome Even buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer, borate buffer, carbonate buffer, glycine buffer, good buffer and the like. Good buffers include, for example, MES (2-morpholinoenesulfonic acid) buffer, bis-tris [bis (2-hydroxyethyl) imino tris (hydroxymethyl) methane] buffer, ADA

[N- (2-acetamido) iminoniacetic acid] buffer, PIPES [piperazine-N, N'-bis (2-enesulfonic acid)] buffer, ACES {2- [N- (2-acetamido) Amino] ethanesulfonic acid} buffer, MOPSO (3-morpholino-2-hydroxypropanesulfonic acid) buffer, BES {2- [N, 'N-bis (2-H Droxityl) Amino] ethanesulfonic acid} buffer, MOPS (3-morpholinopropanesulfonic acid) buffer, TES <2- {N- [tris (hydroxymethyl) methyl] amino} sulfonic acid> buffer, HE PES [N-(2-hydroxy Echiru) Single N'(2 Suruhoechiru) piperidines Rajin] buffer, DIPSO {3- [N ₃ N- bis (2-hydroxy E chill) Amino] - 2-hydroxy propane sulfonic Acid) buffer, TAP SO '<2-Hydroxy-3-{[N-tris (hydroxymethyl) tyl] amino} propanesulfonic acid> buffer, POP SO [piperazine-N, N ⁵ -bis (2-hydroxypropane) — 3-Sulfonic acid)] buffer, HEPPSO [N- (2-hydroxyethyl) 1 N, 1 (2-hydroxy-13-sulfopropyl) piperazine] buffer, EPPS [N— (2-hydroxyethyl) Chill) one N, one (3-sul hoplo) Pill) piperazine] buffer, tricine [N-tris (hydroxymethyl) methylglycine] buffer, bicine [N, N-bis (2-hydroxyethyl) glycine] buffer, TAPS {3- [N-tris Hydroxymethyl) methyl] aminopropanesulfonic acid} buffer, CHES [2- (N-cyclohexylamino) ethanesulfonate] buffer, CAPS0 [3- (N-cyclohexylamino) 1-2-hydroxypropanesulfonic acid] Buffers, CAPS [3- (N-cyclohexylamino) pulpsulfonic acid] buffer, and the like.

 Examples of the surfactant include the surfactants described in 1. above.

 Examples of the stabilizer include proteins such as BSA, gelatin, and casein.

 Examples of the enzyme activity regulator and the enzyme stabilizer include metal ions such as magnesium ion, manganese ion and zinc ion. The content of these metal ions in the reagent is not particularly limited as long as the enzyme is stabilized in the measurement. Examples of preservatives include sodium azide, antibiotics and the like. The content of these preservatives in the reagent is not particularly limited as long as the content of the analyte in the sample can be measured appropriately in the measurement.

 The reagent for detecting the labeled substance may be any substance as long as the enzyme can react, including a coloring substrate corresponding to the labeling enzyme described in 1 above, and if necessary, a surfactant, a buffer, a preservative, and the like. An agent, a stabilizer, a reaction accelerator, an enzyme activity regulator or an enzyme stabilizer may be added.

As a standard substance of the anti-gp46-197 antibody, it is sufficient that anti-gp46-197 human antibody obtained from a biological sample such as a patient's serum having HTLV-1 related disease or a patient's serum is contained at a known concentration. . The antibody used as the standard substance can be obtained from the patient serum by a combination of ordinary protein purification methods, a method of obtaining the antibody by purification by affinity chromatography using p46-197, or a method of producing an ordinary antibody. It can be obtained by a method of obtaining from antibody-producing cells that have been monocloned by the production method. 3. Use of anti-gp46-197 antibody assay

 The method of measuring the anti-gp46-197 antibody of the present invention can measure the anti-gp46-197 antibody that is specifically observed in patients who have developed an HTLV-1 related disease. It can be used to determine the presence or absence of a related disease.

 In addition, the method for measuring the anti-gp46-197 antibody of the present invention is a method for measuring the occurrence of infection other than HTLV-1 which is a non-onset HTLV-1 carrier that has not developed 11 Because it is possible to measure the anti-gp46-197 antibody that specifically appears in patients who have been diagnosed, it is possible to determine the presence or absence of an infectious disease other than HTLV-1 which is a non-infected HTLV-1 carrier co-infected with a carrier Can be.

 The HTLV-1 related diseases include any diseases caused by HT.LV-1 infected cells in infected persons. Examples of HTLV-1 related diseases include adult T-cell leukemia Z lymphoma (ATLL) that developed in the bloodstream and lymphatic organs, and HTLV-1 related myelopathy (HAM / TSP) that developed in the spinal cord. ) Or Pudo's meningitis (HU) that developed in the eyeball.

 In the present invention, “onset” means that HTLV-1 infected cells cause a disease in an infected person, and clinical findings may or may not be recognized. HTLV-1 infected cells cause pathology in various parts of the patient.

 Examples of infectious diseases other than HTLV-1 include Staphylococcus aureus such as MRSA, Pseudomonas aeruginosa, pathogenic Escherichia coli, Candida infection, cytomegalovirus, herpes virus, and human acquired immunodeficiency virus. And viral infections such as EB virus or hepatitis C virus (hereinafter referred to as HCV), and protozoal infections such as Carinii pneumonia or Toxoplasma gondii.

 The anti-gp46-197 antibody measurement reagent of the present invention can be used as a kit in combination with the HTLV-1 binding antibody measurement reagent used for screening HTLV-1 infected persons. Such kits can be used to determine the presence or absence of HTLV-1 infection, as well as the presence or absence of HTLV-1 related diseases or the occurrence of infections other than TLV-1 that have been superinfected with HTLV-1. Can also be used. In other words, if the antibody that binds to HTLV-1 is detected with the reagent for measuring HTLV-1 antibody and the anti-gp46-197 antibody is not detected with the reagent for measuring anti-gp46-197 antibody, no symptoms occur. HTLV—determined as one carrier. When an antibody that binds to HTLV-1 is detected in the reagent for binding to HTLV-1 and an anti-gp46-197 antibody is detected in the reagent for binding to anti-gp46-197 antibody, It is determined that an HTLV-1 related disease has developed, or that an asymptomatic HTLV-1 carrier has developed an infection other than HTLV-1.

As a reagent for measuring an antibody that binds to HTLV-1, any assay that can measure an antibody that binds to HTLV-1 may be used, but a reagent using an immunological technique is preferably used. You can. Specifically, the cellodia HTLV-1 (manufactured by Fujirebio), Atest (manufactured by Enzy), and a peptide (hereinafter referred to as gp46-175 and gp46-175) corresponding to the region of Phe75_Ilel99 of gp46 (Hereinafter referred to as the gp46-175 antibody) [Research on the prevention of mother-to-child transmission of adult T-cell leukemia (ATL), Ministry of Health and Welfare, 1990, p. 174].

 When an anti-gp46-175 antibody measurement reagent is used as the measurement reagent for the antibody that binds to HTLV-1 used in the kit of the present invention, the measurement using the anti-gp46-175 antibody is performed using the anti-gp46-antibody described in 1. above. — Can be performed in the same manner as the method for measuring the 197 antibody.

 gp46-175 is a peptide having an amino acid sequence corresponding to 175th Phe to 99th I1e counted from the N-terminus of gp46, and specifically, an amino acid represented by SEQ ID NO: 2 It is a peptide represented by the sequence.

 In place of gp46-175, a peptide in which one or more amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 2 and which can bind to an antibody that binds to gp46-175 can also be used.

 The number of amino acids to be deleted, added or substituted is one or more, and the number is not particularly limited. Molecular 'Cloning 2nd edition, current' Protocols 'in molecular' biology, Nucleic Acids Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), etc.), which is a number that can be deleted, added or substituted by a well-known technique such as a site-directed mutagenesis method. , Preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.

 As the kit of the present invention, the measurement reagent for the antibody that binds to HTLV-1 and the measurement reagent for the anti-gp46-197 antibody of the present invention may be combined with independent kits, or may be included in the same kit. . In addition, among the reagents included in the same kit, a common reagent among the respective measurement reagents can be shared. Common reagents include, for example, a diluent of a biological sample, a reaction buffer, a washing solution, a labeled secondary antibody or an antibody fragment thereof, and a labeled product of a biological sample contained in the anti-gp46-197 antibody measurement reagent of the present invention. And a detection reagent. Alternatively, a kit may be prepared by combining a reagent for measuring the anti-gp46-197 antibody of the present invention with a device suitable for measurement.

 4. Diagnostic agents for HTLV-1 related diseases and methods for determining the presence / absence of the disease, as well as diagnostic agents for non-HTLV-1 carrier-infectious diseases other than HTLV-1 and a method for determining the presence / absence of the disease

In the present invention, since the anti-gp46-197 antibody is an antibody that is specifically detected in a patient who has developed an HTLV-1 related disease, the measurement and detection of the antibody enables the diagnosis of an HTLV-1 related disease. Can be used as medicine. Since the anti-gp46-l97 antibody is an asymptomatic HTLV-1 carrier and is an antibody that is specifically detected in patients who developed infections other than HTLV-1 that had been co-infected, the antibody was measured. Alternatively, by detecting it, the asymptomatic HTLV-1 carrier can be used as a diagnostic agent for infections other than HTLV-1 that have been co-infected.

 When used as a diagnostic for HTLV-1 related diseases, for example, (1) measure the concentration of anti-gp46197 antibody contained in a sample derived from a subject, and (2) measure the concentration above a certain standard value. By determining whether or not the subject has an HTLV-1 related disease, even if the subject is an HTLV-1 infected person for whom no clinical findings have been obtained, it can be determined.

 Furthermore, when judging the presence or absence of an infectious disease other than HTLV-1 in which a non-onset HTLV-1 carrier was co-infected, for example, (1) measuring the concentration of anti-46-197 antibody contained in a sample derived from a subject, (2) By determining whether or not the concentration exceeds a certain reference value, the subject is free of HTLV that has no clinical findings, even if he / she is a single carrier, but has a co-infected HTLV— The presence / absence of an infection other than 1 can be determined.

 These reference values may be different for each disease to be determined, even when the same substance is measured. The reference value can also be calculated statistically. For example, a value obtained by doubling the measured value of the anti-gp46-197 antibody concentration in a negative control of a healthy person or the like is used. Hereinafter, the present invention will be described in more detail with reference to Examples, but these do not limit the scope of the present invention in any way. In this example, the following reagents were used. For peptides having an amino acid sequence equivalent to gp46-197 (peptides equivalent to gp46-197), an automatic peptide synthesizer 43 1A peptide synthesiser (ABI Applied Biosystems) Was used.

 Sodium carbonate (manufactured by Wako Pure Chemical Industries), sodium bicarbonate (manufactured by Wako Pure Chemical Industries), 96-well plate Maxisorp I marauding unomodule (manufactured by Nunc), Tween 20 (manufactured by Katayama Chemical), horseradish Peroxidase-labeled anti-human I0 antibody (manufactured by ^ 81 ^), o-phenylenediamine (manufactured by Sigma), cunic acid (manufactured by Wako Pure Chemical Industries, Ltd.) ', peroxidase hydrogen water (Wako Pure Chemical Industries, Ltd.) Best Mode for Carrying Out the Invention), Sulfuric Acid (Wako Pure Chemical Industries, Ltd.)

Example 1. Preparation of gp46-197 fixed plate

A plate on which gp46-197 was fixed was prepared as follows. The gp 46- 197 lyophilization _{10 mm o 1 / L Na 2} C0 3 - NaHCO

3 Dissolve in buffer (pH 9.55) and prepare a 5 μg / mL solution of gp46-197. Made. The solution was dispensed into a 96-well plate, Maxisorp Immunoniodule, at 100 L / well, and allowed to stand at 4 ° C. After removing the solution in each well, Tween was added to phosphate buffered saline (pH 7.2 10 mmol / L phosphate buffer containing 0.15 mol 1 / L sodium chloride; hereinafter, referred to as PBS). The wells were washed using a washing solution containing 0.05% of en20 ^™ (hereinafter referred to as Tween-PBS). This washing operation was repeated four times in total. Example 2 · Measurement of anti-gp46-197 antibody in sample

 The measurement of the anti-gp46-197 antibody in the sample was performed as follows.

 4. For each well of the plate prepared in Example 1, 100 μL of serum sample diluted 100-fold with PBS containing 6-197 of 〃 ^ / ^^ was added and allowed to react at 37 ° C for 1 hour (hereinafter, gp46-197). Absorption test in presence). At this time, a basic reaction test was performed in which 100 L of a serum sample diluted 100-fold with PBS containing no gp46-197 was simultaneously added to each well of the plate, and allowed to stand at 37 ° C for 1 hour to react. (Hereinafter referred to as a reaction test in the absence of gp46-197). After the reaction, the washing operation with Tween-PBS was repeated four times to completely remove the water remaining in each well. Next, 100 μL of PBS solution containing l〃gZmL of horseradish peroxidase-labeled anti-human IgG antibody and 5% Block Ace (Dainippon Pharmaceutical) was added to each well. Stood at 45 ° C for 45 minutes. After the reaction, the washing operation with Tween-PBS was repeated four times to completely remove the water remaining in each well. Next, a color-forming reagent obtained by adding 1/2 volume of 0.5% hydrogen peroxide solution to 0.7 lmmol / L citrate buffer solution of 0.7 mg / mL o-phenylenediamine was added per 1 liter. After adding 150 L and leaving the mixture to stand at 37 ° C for 10 minutes, 50 L of a color stop solution (2.5 mmo 1 / L sulfuric acid) was added per 1 L, and the absorbance at 492 nm was measured. Example 3. Determining the onset of HTLV-1 related disease in a subject

 Samples included (1) serum from asymptomatic HTLV-1 carrier, (2) serum from adult T-cell leukemia (ATLL) patient, (3) serum from HTLV-1-associated myelopathy (HAM) patient, (4 ) Anti-gp46-197 antibody in each sample was measured in the same manner as in Example 2, using serum from non-symptomatic H TLV-1 / HC V carrier and (5) serum from non-symptomatic HCV carrier. . Each sample is as follows.

(1) Symptom-free HTLV-1 serum from carrier

Blood serum obtained by donating blood from subjects who did not develop HTLV-1 related diseases was analyzed by indirect fluorescence assay using cellodia HTLV-1 (Fujirebio) and HTLV-1 infected cell lines. 85 specimens that were positive due to HTLV-1 carrier were asymptomatic. (2) Serum from ATLL patients

 Forty-seven sera collected from ATLL patients were used.

 (3) serum from HAM patients,

 32 sera collected from HAM patients were used.

 (4) Asymptomatic HTLV—1 carrier ZH CV Carrier-derived serum

 Twenty samples positive for Serodia HTLV-1 (manufactured by Fujirebio Co., Ltd.) were used for serum of blood obtained by donating blood from a person with hepatitis C.

 (5) Serum from a non-onset HCV carrier

 Seventy samples positive for HCV.PHA (manufactured by Dynabot) for blood serum obtained by donating blood from subjects who did not develop an HCV-related disease were used.

 A sample whose absorbance in the basic reaction test was greater than 0.300 and whose absorbance in the absorption reaction test decreased by 30% or more with respect to the absorbance in the basic reaction test was determined to be positive. For the anti-gp46-197 antibody titer in each sample, a dilution factor indicating an absorbance of 0.5 was calculated from the absorbance of each diluted sample prepared by diluting the sample at each magnification, and the reciprocal was used to calculate the antibody titer. And The antibody retention rate (%) is the number of positive donations Z total donations.

The results are shown in Tables 1 and 2.

 Prevalence of anti-gp46-197 antibody in patients with HTLV-1 related disease

 Asymptomatic

 HTLV-1 ATLL patient HAM patient

 Career

Antibody retention rate (%) 5.9 95.7 100

 (5/85) (45/47) (32/32)

 Price 5-68 692-10520

(median SD) (41 ± 25) (1784 ± 1077) (3855 ± 2705)

Table 2

 HTLV—1 carrier and asymptomatic HCV carriers

 Retention rate of anti-gp46_l 97 antibody

From the results shown in Table 1, anti-gp46-197 antibody was found in almost all patients with HTLV-1-related disease, and by measuring the presence or absence of anti-gp46-197 antibody, HTLV It is possible to determine the presence or absence of —1 related diseases.

 Furthermore, in the asymptomatic HTLV-1 carrier group shown in Table 1 and in the HCV single infection group indicated by the asymptomatic HCV carrier shown in Table 2, the prevalence of anti-gp46-197 antibody was very low. In contrast, the prevalence of this antibody is extremely high at 95% in the HCV-developed group in which non-developed HTLV-1 carriers are supercontaminated. Therefore, by measuring the anti-gp46-197 antibody in the sample, it becomes possible to determine the presence or absence of the onset other than HTLV-1 which is a co-infected HTLV-1 carrier without the onset.

 Ninety-six patients who developed HCV without HTLV-1 infection were considered negative. This suggests that the method of measuring anti-gp46_197 antibody has high specificity for HTLV-1 infection and does not determine that patients infected only with viruses other than HTLV-1 are considered HTLV-1 infected. Indicated. Example 4. Comparison between anti-gp46-197 antibody and anti-gp46_175 antibody

 (1) serum-free HTLV-1 serum from a carrier; (2) serum from an adult T-cell leukemia (ATLL) patient; (3) anti-gp46-197 in serum from a HTLV-1-associated myelopathy (HAMZTSP) patient. The antibody holding ratio and the antibody titer of the antibody and the anti-gp46-175 antibody were calculated in the same manner as in Examples 2 and 3.

Table 3 shows the results. Table 3

 HTLV-1 related disease

 As shown in Table 3, serum from ATLL patients and serum from patients with HAM / TSP possess anti-gp46-197 antibodies. Antibodies of anti-gp46-197 antibody in serum from 1 carrier are low. Therefore, the presence or absence of HTLV-1-related disease can be determined by measuring the anti-gp46_197 antibody. — Furthermore, a comparison of the antibody prevalence of anti-gp46-19.7 antibody and anti-gp46-175 antibody in the sera of asymptomatic HTLV-1 carriers and ATLL patients showed that in the asymptomatic HTLV-1 carrier, anti-gp46 — The 175 antibody was positive and the anti-gp46-197 antibody was negative. On the other hand, anti-gp46-175 antibody and anti-gp46-197 antibody were positive in ATLL patients. Therefore, the presence or absence of the above two types of antibodies in the serum indicates that the subject is: 1. non-infected with HTLV-1; 2. asymptomatic HTLV-1 carrier; and 3. developing HTLV-1 related disease. It can be classified into three categories.

 When sera from 96 healthy persons infected with HTLV-1 were used in the same manner, no anti-gp46-197 antibody was detected in any of the sera. Example 5. Validation of ¾¾gp46-l 97 antibody assay

 After washing the 96-well plate prepared in Example 1, blocking with a protein used in the usual ELISA was attempted.

A blocking reagent containing 0.5% milk casein, 0.05% Tween 20 and 10% inactivated goat serum was dispensed at 100 L per 1 L into the 96-well plate after washing, and the blocking operation was performed by allowing to stand at room temperature. After performing the washing operation described in Example 1, the serum of a healthy person as a negative control and anti- Serum from ATLL patients confirmed to contain the gp46-197 antibody in Example 3 was measured. As a result, no color development was observed in both samples. In addition, blocking was performed using a commonly used substance derived from glycerol such as porcine serum albumin and gelatin as a blocking reagent, but no color formation was observed. As described above, the anti-gp46-197 antibody cannot be measured by the method including the usual blocking operation, and the 96-well plate was washed only with the Tween-PBS described in Examples 1 to 4. Only in the plate, the anti-gp46-197 antibody could be measured.

 Since gp46-197 and HSC70 protein can bind [Journal of Virology, 72, 535 (1998)], HSC70 contained in the blocking reagent was considered to be the cause of color loss in serum from ATLL patients. Therefore, the following study was conducted to examine the effect of HSC 7.0 on the measurement of anti-gp46-197 antibody using purified HSC70.

 The purified series of HSC70 dilution series was reacted with the 96-well plate prepared in Example 1, added, and left at 37 ° C for 1 hour. The washing operation with Tween-PBS was repeated four times to completely remove the water remaining in each gel. Next, 100 µL of PBS solution containing 0.5 µg / mL rat anti-HSC70 antibody (manufactured by Stress Gen) was added to each well, and the mixture was allowed to stand at 37 ° C for 1 hour. Was placed. After the reaction, the washing operation with Tween-PBS was repeated four times to completely remove water remaining in each well. Next, 100 μL of a PBS solution containing 0.5 μg / mL of horseradish peroxidase-labeled anti-rat IgG antibody and 10% Proc Ace (Dainippon Pharmaceutical Co.) was added to each well. The mixture was allowed to stand at ° C for 1 hour. After the reaction, the washing operation with Tween-PBS was repeated four times to completely remove the water remaining in each gel. Next, add 150 mg / well of a color-forming reagent obtained by adding 1/2 volume of 0.5% hydrogen peroxide solution to 0.1 mmo 1 / L citrate buffer solution of 0.7 mg / mL of 0-phenylenediamine. After adding L, the mixture was allowed to stand at 37 ° C for 10 minutes, and then a color stop solution (2.5 mmol / L sulfuric acid) was added at 50 L per 1 L, and the absorbance at 492 nm was measured.

 The above operation confirmed the concentration-dependent color development of HSC70, and thus revealed that HSC70 has an effect on the measurement of anti-gp46-197 antibody by the present ELISA method. Therefore, in the measurement of the anti-gp46-197 antibody, the solid phase blocking solution or washing solution, and the reaction buffer used when reacting gp46-197 with the anti-gp46-197 antibody, are considered to contain HSC70. It was clarified that commonly used pest-derived serum serum albumin, gelatin or milk casein could not be used.

Claims

The scope of the claims

1. Use of a peptide corresponding to the Asp 197-Leu216 region of the HTLV-1 envelope glycoprotein gp46 (hereinafter referred to as gp46-197) to determine the presence or absence of an HTLV-1 related disease A method for measuring an antibody that binds to gp46-197 (hereinafter referred to as an anti-gp46-197 antibody) in a sample.

 2. An anti-gp46-197 antibody in a sample for determining the presence or absence of an infectious disease other than HTLV-1 which is a co-infected HTLV-1 carrier, characterized by using gp46-l97 Measurement method.

 3. The method according to claim 2, wherein the infectious disease other than HTLV-1 is a hepatitis C virus (hereinafter referred to as HCV) infectious disease.

 4. gp46-l97 is a peptide having the amino acid sequence shown in SEQ ID NO: 1 or one or more amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 1, and the anti-gp46-197 antibody The method according to any one of claims 1 to 3, which is a peptide capable of binding.

 5. The method according to any one of claims 1 to 4, wherein the measuring method of the antibody is an immunological measuring method.

 6. The method according to claim 5, wherein the immunological assay is a method using a solid phase to which 46_197 has been immobilized.

 7. The immunoassay is a method using a solid phase on which gp46-197 is immobilized and blocked by a substance that does not contain a heat-shock-like protein (HSC70) of 71 kDa. The method according to claim 5 or 6.

 8. The method according to claim 7, wherein the substance not containing HSC70 is a synthetic polymer.

9. The method according to claim 8, wherein the synthetic polymer is a surfactant.

 10. The method according to any one of claims 5 to 9, wherein the immunoassay is an enzyme immunoassay (ELISA).

 11. The measuring method according to claim 10, comprising the following steps.

(a) immobilizing gp46-197 on a carrier to produce a solid phase on which gp46-197 is immobilized;

 (b) reacting the sample with gp46-197 immobilized on a solid phase (primary reaction step),

 (c) a step of reacting a labeled secondary antibody with a complex comprising gp46-197 immobilized on a solid phase and an anti-gp46-197 antibody bound to the peptide (secondary reaction step)

(d) Measuring the amount of labeled secondary antibody bound to a complex consisting of gp46-197 immobilized on a solid phase and an anti-gp46197 antibody bound to the peptide ■ ■ (e) From the calibration curve showing the relationship between the concentration of anti-gp46-197 antibody and the amount of label prepared using a standard substance containing the irLgp46-197 antibody, and the measured value measured in (d), The step of determining the anti-gp46-197 antibody concentration

 12. A reagent for measuring anti-gp46-197 antibody in a sample, which is characterized by using gp46-197, for determining the presence or absence of an HTLV-1-related disease.

 13. An anti-gp46-197 antibody in a sample for determining the presence or absence of an infectious disease other than HTLV-1 co-infected with a non-onset HTLV-1 carrier, characterized by using gp46-l97 Measuring reagent.

 14. The method according to claim 13, wherein the infection other than HTLV-1 is HCV infection.

B expression.

 15. p46-197 is a peptide having the amino acid sequence shown in SEQ ID NO: 1 or one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 are deleted, substituted or substituted, and anti-gp46- Claim 1 which is a peptide capable of binding to the 197 antibody

15. The reagent according to any one of 2 to 14.

 16. The reagent according to any one of claims 12 to 15, wherein the measuring method of the antibody is an immunological measuring method.

 17. The reagent according to claim 16, wherein the immunological assay is a method using a solid phase on which gp46-197 is immobilized.

 18. The reagent according to claim 16 or 17, wherein the immunological assay is a method using a solid phase on which gp46-197 is immobilized and blocked with a substance not containing HSC70.

 19. The reagent according to claim 18, wherein the substance not containing HSC70 is a synthetic polymer.

20. The reagent according to claim 18, wherein the synthetic polymer is a surfactant.

 21. The reagent according to any one of claims 16 to 20, wherein the immunoassay is an enzyme immunoassay (ELISA).

 22. The reagent for measuring an antibody according to any one of claims 12 to 21, wherein the reagent comprises an anti-gp46_197 human antibody as a standard substance.

 23. A kit for determining the presence or absence of an HTLV-1 related disease, comprising a combination of a reagent for measuring an antibody that binds to HTLV-1 and the reagent according to any one of claims 12 to 22. .

 24. Except for HTLV-1 which is a non-symptomatic HTLV-1 carrier that is obtained by combining a reagent for measuring an antibody that binds to HTLV-1 with the reagent according to any one of claims 12 to 22. A kit for determining the presence or absence of an infectious disease.

 25. The kit according to claim 24, wherein the infection other than HTLV-1 is HCV infection.

26. An antibody that binds to HTLV-1 is a peptide corresponding to the Phel 75-Ile 99 region of the HTLV-1 envelope glycoprotein gp46 (hereinafter gp46_175 and gp46_175). 26. The kit according to any one of claims 23 to 25, which is an antibody that binds to (hereinafter, referred to as an anti-gp46-175 antibody). .

27. gp46-175 has a peptide having the amino acid sequence shown in SEQ ID NO: 2 or has one or more amino acids deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 1, and has an anti-gp46-175 antibody 27. The reagent according to claim 26, which is a peptide capable of binding.

 28. Solid phase with gp46-197 immobilized thereon and blocked with HSC70-free material.

 29. The solid phase according to claim 28, wherein the substance not containing HSC70 is a synthetic polymer.

30. The solid phase according to claim 29, wherein the synthetic polymer is a surfactant.

 31. HTLV— characterized by detecting anti-gp46—197 antibody in the sample

1 Diagnostic agent for related diseases. '

 32. A diagnostic agent for an infectious disease other than HTLV-1 in which a non-onset HTLV-1 carrier is co-infected, characterized by detecting an anti-gp46-197 antibody in a sample.

 33. The diagnostic agent according to claim 32, wherein the infection other than HTLV-1 is HCV infection.

 34. A method for determining the presence or absence of an HTLV-1-related disease, comprising detecting an anti-gp46-197 antibody in a sample.

 35. A method for determining the presence or absence of an infectious disease other than HTLV-1 co-infected with a non-onset HTLV-1 carrier, characterized by detecting anti-gp46-197 antibody in the sample o

 36. The method according to claim 35, wherein the infection other than HTLV-1 is HCV infection.