WO2008059553A1 - Method for detecting hiv-1 without being affected by anti-mouse protein antibody existing in human and kit to be used for the same - Google Patents

Method for detecting hiv-1 without being affected by anti-mouse protein antibody existing in human and kit to be used for the same Download PDF

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Publication number
WO2008059553A1
WO2008059553A1 PCT/JP2006/322535 JP2006322535W WO2008059553A1 WO 2008059553 A1 WO2008059553 A1 WO 2008059553A1 JP 2006322535 W JP2006322535 W JP 2006322535W WO 2008059553 A1 WO2008059553 A1 WO 2008059553A1
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hiv
antibody
detecting
antigen
human
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PCT/JP2006/322535
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French (fr)
Japanese (ja)
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Masami Moriyama
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Masami Moriyama
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present invention relates to a method for detecting human immunodeficiency virus (HIV-1) from human serum, human serum-derived blood products or blood transfusion serum, and a kit used therefor, in particular, HIV-
  • HIV-1 human immunodeficiency virus
  • the present invention relates to a method for detecting 1 without being affected by an anti-mouse protein antibody present in human blood, a method for detecting an anti-HIV-1 antibody, and a kit used therefor.
  • AIDS is an abbreviation for Acquired Immunodeficiency Syndrome, a cell that controls immunity by infection with human immunodeficiency virus (HIV-1).
  • HIV-1 human immunodeficiency virus
  • HIV is a disease that affects immunity, which is the most basic function for humans to live, and overcoming this disease is considered to be an important issue that modern medicine must solve.
  • AIDS is a kind of disease caused by a viral infection called retrovirus. That is, the AIDS pathogen is an RNA virus belonging to the Retroviridae family, and since HIV-1 prefers to infect lymphocytes and directly or indirectly destroys lymphocytes, the cells resulting from the reduction Sexual immune deficiency (acquired) occurs and AIDS develops.
  • the power of a wide range of genetic dispersibility exists to date, various HIV-1 strains isolated from patients in the United States, Yotsuno, Haiti, Asia, and Africa Has an antigenic site. This antigenic site is retained in the major proteins, namely core protein p24, envelope glycoprotein gpl20 and transmembrane protein gp41.
  • the first retroviral LAV strain that has been confirmed to be the cause of AIDS development is an antibody against all HIV-1 class viruses in all carriers that are not related to their hometown. It can be used as a virus strain of an antigen for detection of. Therefore, this strain is currently widely used to detect HIV-1 antibodies in blood donors and patients, particularly using immunofluorescence, Western plot, RIPA (immunoprecipitation assay) methods known as ELISA methods. in use.
  • the AIDS test performed today is an antibody test against HIV-1, and how to evaluate this antibody test is a problem.
  • antibody evaluation methods vary widely depending on the type of virus, and two indicators, sensitivity and specificity, are used to evaluate antibody tests.
  • Sensitivity is the percentage of true infected persons who test positive
  • specificity is the percentage of true non-infected persons who test negative. Therefore, a highly sensitive test can more reliably identify an infected person as positive, There is also a high possibility of false positives, which are determined as positive even in people who have not been infected with the virus.
  • a highly specific test method can more reliably identify a non-infected person as negative, but it also increases the possibility of a false negative in which an infected person is mistakenly determined to be negative.
  • Non-patent document 1 "How to save AIDS-the forefront of research and treatment” by Shinji Harada, Chuko Shinsho, July 1997
  • the present invention is influenced by an anti-mouse protein antibody present in human blood, which can be used for diagnosis of HIV-1 infection simply and reliably. It is an object of the present invention to provide a reliable detection method without using a kit and a kit used for the method.
  • the present inventor has intensively studied to solve the above-mentioned problems! ⁇ , focusing on viremia in which a large amount of virus is detected in the blood in the early stage of HIV-1 infection.
  • P 24 antigen in the blood at the growth phase was examined whether or not detectable (HIV-1 p24) directly.
  • the present inventor succeeded in producing a hybridoma that produces an antibody that specifically recognizes and binds to HIV-1 p24 in the virus growth phase.
  • mouse-derived anti-mouse protein antibody exists in human blood. Because it binds to the force SHIV-1 p24, HIV-1 p24 cannot be detected with high sensitivity. That is, there was a false positive due to a reaction with nonspecific HIV-1 p24 in the test serum sample.
  • HIV-1 p24 can be directly detected in the virus growth phase by using a specific antibody produced by the above-mentioned hyperidoma in a state not affected by the anti-mouse protein antibody.
  • a sandwich ELISA kit capable of directly detecting and quantifying HIV-1 p24 can be prepared.
  • this kit can be used to diagnose the presence or absence of antibodies to HIV-1 p24 (that is, infectious conditions in which antibodies are present even if the p24 value is low).
  • HIV-1 body ie, the virus body.
  • HIV-1 antibody was detected instead of detecting HIV-1, and its sensitivity and specificity were problematic.
  • Sensitivity can diagnose HIV-1 infection, which can be said to be particularly excellent.
  • it is necessary to determine whether the serum of the sample suppresses the detection of p24 by this kit. The present invention has been completed as a result of intensive novel studies.
  • the present invention for solving a problem to be solved is, as one aspect thereof,
  • HIV-1 p24 the p24 antigen expressed in the human immunodeficiency virus (HIV-1) during the growth phase by suppressing nonspecific reactions of anti-mouse protein antibodies present in the blood
  • the present invention provides:
  • a kit for detecting HIV-1 from human serum, human serum-derived blood products or blood transfusion serum by ELISA, and for detecting human-derived p24 antibody in the serum comprising using an antibody that recognizes and binds to a p24 antigen (HIV-1 p24) expressed in HIV-1 (anti-HIV-1 p24 antibody);
  • Anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is inactivated in mice.
  • HIV-1 p24 as described in 8 above which is produced by cell fusion of myeloma cells, screening of the obtained fused cell force-infected cells, and further cloning of the hybridoma obtained by cloning.
  • kit (12) The ELISA kit for detecting an HIV-1 p24 antigen / antibody according to the above 10, wherein the capture antibody for capturing HIV-1 p24 is an RT-N-48 antibody;
  • the ELISA kit for detecting HIV-1 p24 antigen / antibody according to the above 13, wherein the RT-C-50 antibody, which is a detection antibody for detecting HIV-1 p24, is a peroxidase (PO D) labeled antibody; .
  • PO D peroxidase
  • the method and kit for detecting HIV-1 provided by the present invention, the influence of non-specific reaction of anti-mouse protein antibody present in blood on human serum, human serum-derived blood products or blood transfusion serum It is possible to diagnose the presence or absence of HIV-1 infection easily and with high sensitivity.
  • methods and kits provided by the present invention which can detect the P 24 antigen in the blood in virus growth stages of HIV-1 infection early (HIV-1 p24) directly, the measurement sensitivity is 10 This is a 30-fold enhancement, and can be detected without the problems of false positives and false negatives, which have been regarded as problems in the past.
  • the p24 value in the serum decreases and the anti-P24 antibody increases in inverse proportion to the course after infection, the presence or absence of the antibody can be diagnosed with high specificity by an antibody binding competition test.
  • the hybridoma used in the method and kit of the present invention is a hybridoma that produces an antibody that can be used to more accurately detect the presence or absence of HIV-1 infection.
  • an antibody By using an antibody, it is possible to easily determine the presence or absence of HIV-1 infection, and the amount of virus can be quantified with force.
  • Ability that could be quantified but could not be quantified The kit provided by the present invention has the advantage that it can be easily performed.
  • the present invention is particularly excellent in that HIV-1 p24 can be directly detected.
  • p24 antigen (HIV-1 p24) expressed in proliferating human immunodeficiency virus (HIV-1) is recognized.
  • a binding antibody (anti-HIV-1 p24 antibody) is used.
  • this anti-HIV-1 p24 antibody is produced by the antibody that specifically produces an antibody that recognizes the p24 antigen (HIV-1 p24) expressed in proliferating HIV-1 (HIV-1 p24).
  • the p24 antigen (HIV-1 p24) expressed during the HIV-1 proliferative phase in the early stages of HIV-1 infection is an infectious antigen, and in general, antibodies against it were thought to interfere with the detection system.
  • the hybridoma used in the present invention is a hyperidoma that produces an antibody capable of effectively recognizing HIV-1 p24 itself without interfering with the detection system.
  • HIV-1 p24 p24 antigen
  • the present invention uses a hyperidoma that produces an antibody that directly recognizes the p24 antigen at the time when viremia after the initial infection of HIV-1 is observed, and the proliferation produced by this hybridoma.
  • HIV-1 p24 expressed in the HIV-1 stage the diagnosis of HIV-1 infection can be carried out directly, with higher sensitivity and with ease.
  • conventional diagnosis of HIV-1 infection includes, for example, an antibody that recognizes core protein p24 as an antigenic site common to HIV-1 of various infected persons, or envelope glycoprotein gpl20 and transmembrane protein gp41.
  • an antibody capable of recognizing proliferative HIV-1 p24 after HIV-1 infection is used, thereby diagnosing HIV-1 infection. Therefore, it can be said that it is an excellent one that directly detects the virus itself.
  • antibody binding competition tests are quite specific Anti-HIV-1 antibody can be detected with sex.
  • Such a hybridoma that specifically produces HIV-1 p24 is basically inoculated with HIV-1, for example, to a mouse so that the mouse has an immune function against HIV-1, and as a result, Mouse tissue cells, preferably spleen cells, that produce anti-HIV-1 antibodies are collected and fused with commonly used mouse myeloma cells (mouse melanoma cells). It can be obtained by screening by culturing with a HAT selective medium that can be used for cloning of normal hyperidoma.
  • Operations such as powerful cell fusion, high-pridoma cloning, and screening can be performed by appropriately applying generally used techniques (see, for example, Examples described later).
  • Neubridoma obtained by force is a hybridoma that produces an antibody that recognizes HIV-1 p24 extremely effectively and with high sensitivity, that is, an anti-HIV-1 p24 antibody.
  • the antibody produced by hyperidoma is, for example, a capture antibody that captures HIV-1 p2 4, and another antibody binds to other sites in p24. -1 It functions as a detection antibody that detects p24.
  • the present inventors have detected a capture antibody that captures HIV-1 p24, named RT-N-48 antibody, and HIV-1 p24, named RT-C-50 antibody. Functions as a detection antibody.
  • the present invention particularly specifically relates to an RT-N-48 antibody that captures HIV-1 p24, and a method for detecting HIV-1 using RT-C-50 antibody that detects HIV-1 p24. As well as kits.
  • the antibody that directly recognizes HIV-1 p24 produced from the above-mentioned ibridoma functions as an antibody that captures and detects HIV-1 p24. Therefore, these antibodies can be used to directly diagnose HIV-1 infection with high sensitivity by using the ELISA kit for HIV-1 antigen / antibody detection, that is, the FLISA sandwich method. It can.
  • the conventional p24 antigen detection method uses a mouse monoclonal antibody as a capture antibody for p24 antigen capture, and a polyclonal antibody derived from an HIV-1 infected person for detection. However, this method does not completely suppress the appearance of nonspecific positive reactions. like this From the background, the specificity of this kit is worthy of evaluation.
  • RT-N-48 antibody as a capture antibody against HIV-1 p24 is applied to, for example, a well microphone mouth plate for HIV-1 detection.
  • an RT-C-50 antibody as a detection antibody to which peroxidase (POD) as a detection dye is bound (labeled) is prepared, and the detection antibody is added together with the detection sample on the above plate. It is possible to diagnose the presence or absence of HIV-1 infection by developing a color with a coloring agent.
  • POD peroxidase
  • the concentration applied to the well microphone mouth plate is appropriately diluted stepwise.
  • the sandwich method color development with a color former, measurement of absorbance at a wavelength of 450 nm, for example, and qualitative and quantitative determination of HIV-1 p24 or antibodies against it by comparison with a standard curve with a standard (See the examples below).
  • the present invention will be described below with reference to preparation of a specific hyperidoma, production of an HIV-1 p24 capture antibody and detection antibody from the hybridoma, actual detection of HIV-1 using these antibodies, and a specific kit.
  • the present invention will be described in more detail by describing one embodiment, but the present invention is not limited to these.
  • the present invention can be variously modified without departing from the object thereof, and it goes without saying that powerful modifications are also included in the technical scope of the present invention.
  • Test Example 1 Creation of hybridoma
  • the mouse is first immunized with HIV-1 as a first step.
  • V ⁇ ⁇ ⁇ ⁇ immune tissue cells (spleen Cells).
  • Concentrated HIV-1 was obtained by the following method.
  • HIV-1 clinical isolate virus
  • PBMC peripheral blood mononuclear cells
  • IL-2 interleukin-2
  • Concentrated HIV-1 obtained in (a) above is solubilized with a surfactant, mixed with an equal volume of Complete Freund Adjuvant (CFA), and inoculated subcutaneously in a mouse in the state of emulsion. , Sensitized.
  • CFA Complete Freund Adjuvant
  • CFA Freund's complete adjuvant
  • the mouse spleen excised above was lined with a stainless mesh to obtain a cell suspension, and this cell suspension and the proliferated mouse myeloma cells SP2 / 0 cells at a ratio of 4: 1 polyethylene glycol 4000 ( Cell fusion was performed using PEG4000).
  • fused cells (fused cells) were seeded on a 96-well microphone mouthplate, and selection of high-pridoma was performed.
  • the selection of the cells was performed using a commercially available HAT selection medium. That is, the medium was changed every 3 days, and the culture supernatant of the fused cells that appeared during the culture 14 to 21 days after the fusion was screened by the following fluorescent antibody method.
  • HIV-1 infected cell line Molt4 / IIIB and non-infected cell Molt4 cells are smeared on separate 15-well slide glass (Dainippon Pharmaceutical Co., Ltd.), air-dried, solidified with 100% ethanol at 20 ° CZ for 10 minutes, then E7 .
  • HIV-1 infected activated human PBMC and non-infected control cells are smeared on separate 15-well slide glasses (Dainippon Pharmaceutical Co., Ltd.), air-dried, and then solidified with 100% ethanol for 10 minutes at 20 ° CZ for 7 minutes.
  • FITC label (note: fluorescein isothiocyanate: antibody fluorescent labeling reagent) goat anti-mouse IgG ( 1/100 dilution) was further reacted at room temperature for 30 minutes.
  • the infected cells were stained using an fluorescence microscope, and antibodies that did not stain uninfected cells were screened.
  • Hypridoma in antibody-producing wells was cloned twice by the limiting dilution method using mouse thymocytes as feeder cells according to a conventional method.
  • the determination of the Ig class and subclass of the antibody contained in the culture supernatant after the final cloning was determined by ELISA using a commercially available goat anti-mouse Ig kit.
  • a commercially available SCID mouse (CB-17) was administered 0.5 mL of pristane intraperitoneally, and one week later, 5 million hyperpridoma obtained above were intraperitoneally transplanted. Ascites was obtained after about 10 days. The ascites is pooled, the cells are precipitated by centrifugation at 15,000 rpm for 10 minutes, the resulting supernatant is further filtered through a 10-m filter, and the filtrate is precipitated with 50% aqueous ammonium sulfate solution. It was. The obtained sediment was dissolved in phosphate buffered saline (PBS), and an IgG fraction was prepared by gel filtration using Sephadex G200. Antibody concentration is OD280 Estimated.
  • PBS phosphate buffered saline
  • the following combination made it possible to measure HIV-1 p24 with high sensitivity by sandwich ELISA.
  • Test Example 2 Preparation of standard solution of HIV-1 D 24 Hanghara used for sandwich ELISA
  • the kit of the present invention comprises the following.
  • HIV-1 p24 standard 1% Triton-X100, solubilized virus l, 000pg / mL [2.0mL Z kit]
  • Example 2 HIV-1 D24 Hanghara and pile body extraction (fixed, quantitative) Actual use of ELISA kit
  • the kit described in Example 1 is specifically used for detection of HIV-1 p24 antigen and antibody detection by the following method.
  • sample serum plasma
  • sample diluent preferably in the most measurable range at 1/10, 1/100, or 1 / 1,000 dilution
  • HIV-1 p24 antibody For detection of HIV-1 p24 antibody, add 0.05 mL of 250 pg / mL HIV-1 p24 solution (standard diluted 1: 4 with sample diluent) to the well containing the sample. The standard-filled uel remains.
  • HIV-1 p24 Atsei is positive for an antigen if the color of the sample such as serum (plasma) is colored blue.
  • HIV-1 p24 antibody Atsei is positive for antibodies if color development is blocked.
  • Example 3 Usage example of an actual kit
  • the HIV-1 p24 standard was diluted from 1 000 pg / mL to 16 pg / mL, and 50 L was added to each well (column 1 to 4 from the left column). In the fourth row of left force and other wells, 50 L of plasma from HIV-1-infected individuals was added to a final concentration of 20%.
  • the concentration of HIV-1 p24 can be determined with the naked eye.
  • the plasma of HIV-1 infected individuals was found to be completely inhibited from detection of HIV-1 p24 at 16 to 1 000 pg / mL and is understood to be antibody positive.
  • a calibration curve for HIV-1 p24 can be drawn by measuring the absorbance of this plate.
  • the present invention provides a method and kit for detecting HIV-1 using an antibody that directly recognizes HIV-1 p24 as a method for easily and reliably diagnosing HIV-1 infection.
  • the hybridoma provided by the present invention produces an anti-HIV-1 antibody for detecting HIV-1 p24 for diagnosing HIV-1 infection, and produces an antibody capable of detecting the virus body. It is.
  • the hybridoma provided by the present invention produces HIV-1 p24, that is, an antibody that can detect the virus main body.
  • HIV-1 p24 that is, an antibody that can detect the virus main body.
  • a strong antibody By using a strong antibody, the presence or absence of HIV-1 infection is detected. Diagnosis is extremely accurate, and in addition to the presence or absence of infection, Can be used for quantification.
  • the conventional diagnostic methods have detected HIV-1 antibody, the sensitivity and specificity of the diagnosis are not necessarily sufficient.
  • the detection method and kit using the antibody produced by the provided hyperidoma makes it possible to detect the virus itself and to diagnose HIV-1 infection more reliably.
  • the antibody produced by the hyperidoma provided by the present invention is used in a sandwich ELISA kit for diagnosing the presence or absence of HIV-1 infection, so that the kit for diagnosing HIV-1 infection can be made inexpensively and in a large amount. It can be manufactured. Therefore, even in developing countries or countries where the incidence of AIDS is constant, there is an advantage that it is possible to more easily diagnose AIDS infection, which is an intractable disease, and to help with treatment.
  • FIG. 1 is a photograph showing the results of performing HIV-1 p24 assembly using the kit of the present invention in Example 3 in practice.
  • HTLV-I Human T-Lymphotoropic Virus-I
  • ATL adult T-cell leukemia

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Abstract

A method for detecting HIV-1 reliably that can be used for the diagnosis of HIV-1 infection simply and reliably and a kit to be used for the same are provided. The method for detecting HIV-1 in human serum, a human serum-derived blood preparation or serum for transfusion by an ELISA method characterized by using an antibody (anti-HIV-1 p24 antibody) that recognizes and binds to p24 antigen (HIV-1 p24) that is expressed in human immunodeficiency virus (HIV-1) in the growth phase without being affected by an anti-mouse protein antibody existing in human blood, a method for detecting a human-derived p24 antibody in the serum and an ELISA kit for detecting HIV-1 p24 antigen/antibody to be used for the method are provided.

Description

明 細 書  Specification
ヒト血中に存在する抗マウスタンパク抗体の影響を受けない HIV—1を検 出する方法及びそれに使用するキット  Method for detecting HIV-1 unaffected by anti-mouse protein antibody present in human blood and kit used therefor
技術分野  Technical field
[0001] 本発明は、ヒト血清、ヒト血清由来血液製剤または輸血用血清からヒト免疫不全ウイ ルス (HIV-1)を検出する方法、及びそれに使用するキットに関し、詳細には ELISA法 による HIV-1を、ヒト血中に存在する抗マウスタンパク抗体の影響を受けないで検出 する方法、及び抗 HIV-1抗体を検出する方法、並びにそれに使用するキットに関す る。  [0001] The present invention relates to a method for detecting human immunodeficiency virus (HIV-1) from human serum, human serum-derived blood products or blood transfusion serum, and a kit used therefor, in particular, HIV- The present invention relates to a method for detecting 1 without being affected by an anti-mouse protein antibody present in human blood, a method for detecting an anti-HIV-1 antibody, and a kit used therefor.
背景技術  Background art
[0002] エイズ（AIDS)は、後天'性免疫不全症候群 (Acquired Immunodeficiency Syndrome) の略称であって、ヒト免疫不全ウィルス（HIV- 1： Human Immunodeficiency Virus Type -1)の感染によって免疫を司る細胞の一種、ヘルパー T細胞が破壊された結果、免 疫能が極端に低下することにより生ずる日和見感染症候群である。エイズは、ヒトが 生きていくための最も基本的な機能である免疫を冒す疾患であり、この疾患の克服 は、現代医学が解決すべき重要な一つの課題であるとされて 、る。  [0002] AIDS (AIDS) is an abbreviation for Acquired Immunodeficiency Syndrome, a cell that controls immunity by infection with human immunodeficiency virus (HIV-1). A kind of opportunistic infection syndrome caused by extremely reduced immune immunity as a result of destruction of helper T cells. AIDS is a disease that affects immunity, which is the most basic function for humans to live, and overcoming this disease is considered to be an important issue that modern medicine must solve.
[0003] エイズは、 1980年代力も短期間の内に全世界に蔓延し、今日においても、未だ広く 流行している。現代では、 WHO (世界保険機関）では、毎日およそ 5,000〜6,000人の 新たな感染者が発生しているであろうと推定しており、また、英国保険省の推定では 、 16,000人 Z日の新たな感染者が発生しているとされている。 UNICEFの推計によれ ば、 2010年までにおよそ 4千万人の子供力 エイズで親を失うとさえいわれている。取 り分け、エイズ治療と HIV-1感染予防が不十分な東アフリカでは、およそ 4割の子供 力 15歳になるまでに、片親をエイズで失うという悲惨な状況にある。  [0003] AIDS has spread throughout the world within a short period of time in the 1980s, and is still widely prevalent today. In modern times, the World Health Organization (WHO) estimates that approximately 5,000 to 6,000 new cases of infection will occur each day, and that the UK Department of Insurance estimates that 16,000 new Z days It is said that there are some infected people. According to UNICEF estimates, by 2010, about 40 million child-powered AIDS are even said to lose their parents. In particular, in East Africa, where AIDS treatment and HIV-1 infection prevention are inadequate, about 40% of children have a disastrous situation where one parent is lost by AIDS by the age of 15.
[0004] エイズ感染の診断方法は種々開発され、実用化されており、発展途上国において もエイズの診断が可能になってきている。また、エイズ治療薬の開発も積極的に行わ れており、感染後におけるエイズ発症の進行を遅らせることが可能となったが、診断 にかかる費用が高いこと、また、治療薬の薬価が高いことから、発展途上国では一部 の人たちにしかその恩恵が及んでいないのが現状である。また一方、先進国におい てもこの副作用の多い疾患の治療を完全に解明したものではなぐそのうえ、現在行 われているエイズ感染の診断方法は、かなり煩雑なものであり、安価にその感染を診 断できる診断方法の開発が望まれている。 [0004] Various methods for diagnosing AIDS infection have been developed and put into practical use, and it is now possible to diagnose AIDS in developing countries. In addition, the development of AIDS treatments has been actively carried out, and it has become possible to delay the progression of AIDS after infection, but the cost of diagnosis is high and the price of the treatment is high. In developing countries It is the present situation that only the people of that have the benefit. On the other hand, even in developed countries, the treatment of this disease with many side effects is not completely elucidated, and the current methods for diagnosing AIDS infection are quite complicated, and the infection is diagnosed at low cost. It is desired to develop a diagnostic method that can be used.
[0005] ところでエイズは、レトロウイルス（Retrovirus)というウィルス感染によって引き起こさ れる一種の疾患である。すなわち、エイズの病原体は、レトロウイルス科（Retrovividae )に属する RNAウィルスであり、この HIV-1はリンパ球に好んで感染して直接または間 接的にリンパ球を破壊するため、その減少による細胞性免疫不全 (後天性)が生じ、 エイズが発症する。  [0005] By the way, AIDS is a kind of disease caused by a viral infection called retrovirus. That is, the AIDS pathogen is an RNA virus belonging to the Retroviridae family, and since HIV-1 prefers to infect lymphocytes and directly or indirectly destroys lymphocytes, the cells resulting from the reduction Sexual immune deficiency (acquired) occurs and AIDS develops.
[0006] ウィルス中には、力なり広い遺伝分散性が存在する力 今日までアメリカ、ョ一口ッ ノ^ハイチ、アジア及びアフリカ人の患者から単離された種々の HIV-1株は、共通の 抗原部位を有している。この抗原部位は、主要タンパク質、すなわちコアタンパク質 p 24、エンベロープ糖タンパク質 gpl20及びトランスメンブランタンパク質 gp41に保持さ れている。このため、例えば、エイズ発症の原因であることが確認された最初のレトロ ウィルス LAV (始原型 LAV)株を、キヤリャの出身地に関わりなぐすべてのキヤリャに おける全ての HIV-1クラスウィルスに対する抗体の検出のための抗原のウィルス株と して使用することが可能となる。したがって、この株は現在では、特に ELISA法として 公知の免疫蛍光法、ウェスタンプロット法、 RIPA (免疫沈降アツセィ)法等を用いて、 血液提供者、患者における HIV-1抗体を検出するために広く使用されている。  [0006] Among viruses, the power of a wide range of genetic dispersibility exists To date, various HIV-1 strains isolated from patients in the United States, Yotsuno, Haiti, Asia, and Africa Has an antigenic site. This antigenic site is retained in the major proteins, namely core protein p24, envelope glycoprotein gpl20 and transmembrane protein gp41. Thus, for example, the first retroviral LAV strain that has been confirmed to be the cause of AIDS development is an antibody against all HIV-1 class viruses in all carriers that are not related to their hometown. It can be used as a virus strain of an antigen for detection of. Therefore, this strain is currently widely used to detect HIV-1 antibodies in blood donors and patients, particularly using immunofluorescence, Western plot, RIPA (immunoprecipitation assay) methods known as ELISA methods. in use.
し力しながら、西アフリカ出身の患者の HIV-1溶解液について行った血清学的研究 によれば、これらの溶解液は臨床的並びに免疫学的にはエイズの徴候を示したにも 拘わらず、血清反応は陰性または極めて弱 、陽性でしかな力つた。  However, serological studies conducted on HIV-1 lysates from patients from West Africa showed that these lysates showed signs of AIDS both clinically and immunologically. Serum reaction was negative or very weak and positive.
[0007] 今日行われて 、るエイズの検査は、 HIV-1に対する抗体検査であり、この抗体検査 を如何に評価するかの問題である。一般的に抗体の評価法は、ウィルスの種類によ つて多種多様であり、抗体検査の評価には感度と特異性という二つの指標が使われ ている。感度とは、真の感染者のうち検査により陽性となった人の比率であり、特異性 とは、真の非感染者の中でその検査によって陰性となった人の比率である。したがつ て、感度の高い検査法では、より確実に感染者を陽性と認定することができるが、逆 に感染していない人でも陽性と判定される偽陽性の可能性も高くなる。一方、特異性 の高い検査法では、より確実に非感染者を陰性と認定し得るが、感染者が誤って陰 性と判定される偽陰性の可能性も高くなる。 [0007] The AIDS test performed today is an antibody test against HIV-1, and how to evaluate this antibody test is a problem. In general, antibody evaluation methods vary widely depending on the type of virus, and two indicators, sensitivity and specificity, are used to evaluate antibody tests. Sensitivity is the percentage of true infected persons who test positive, and specificity is the percentage of true non-infected persons who test negative. Therefore, a highly sensitive test can more reliably identify an infected person as positive, There is also a high possibility of false positives, which are determined as positive even in people who have not been infected with the virus. On the other hand, a highly specific test method can more reliably identify a non-infected person as negative, but it also increases the possibility of a false negative in which an infected person is mistakenly determined to be negative.
[0008] 生物化学的化学反応を利用する抗体検査法では、感度 100%であり、かつ特異性  [0008] Antibody detection methods using biochemical reactions have 100% sensitivity and specificity
100%というものは存在しない。感度の高い検査法では特異性が低下し、特異性を 高めると感度が低下する。したがって、今日行われているエイズの検査では、確実に 感染者を陽性として捕捉するために、感度の高 、検査法をスクリーニングとして使用 し、特性の高いものを確認検査として利用しているのが現状である。具体的には、ゼ ラチン粒子を用いた粒子凝集反応法（Particle Agglutination： PA法）をスクリーニング として使用し、この方法により陽性と判定された場合に、ウェスタンプロット (WB)法あ るいは IF法 (蛍光抗体法)等により確認検査を行って 、る (総説としての非特許文献 1 There is no such thing as 100%. Specificity decreases with high-sensitivity testing methods, and sensitivity increases with increasing specificity. Therefore, today's AIDS tests use highly sensitive and test methods for screening, and those with high characteristics as confirmation tests, in order to ensure that infected persons are positively detected. Currently. Specifically, the particle agglutination method (Particle Agglutination: PA method) using gelatin particles is used as a screening, and if this method is determined to be positive, the Western plot (WB) method or IF method is used. Confirmation by (fluorescent antibody method), etc. (non-patent document 1 as a review)
) o ) o
したがって、より正確に HIV-1感染を検出し得る、簡便な診断方法の確立が望まれ て!、るのもまた別の現状でもある。  Therefore, the establishment of a simple diagnostic method that can detect HIV-1 infection more accurately is desired!
非特許文献 1 :「エイズをどう救うか-研究と治療の最前線」原田信志著、中公新書、 1997年 7月  Non-patent document 1: "How to save AIDS-the forefront of research and treatment" by Shinji Harada, Chuko Shinsho, July 1997
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0009] 本発明は、力かる現状に鑑み、簡便に、かつ確実に HIV-1感染の診断に使用する ことができる、 HIV-1をヒト血中に存在する抗マウスタンパク抗体の影響を受けないで 確実に検出する方法、及びそれに使用するキットを提供することを課題とする。  [0009] In light of the current situation, the present invention is influenced by an anti-mouse protein antibody present in human blood, which can be used for diagnosis of HIV-1 infection simply and reliably. It is an object of the present invention to provide a reliable detection method without using a kit and a kit used for the method.
[0010] すなわち本発明者は、上記課題を解決するために、鋭意検討を行!ヽ、 HIV-1感染 初期における血液中にウィルスが多量に検出されるウィルス血症に着目し、このウイ ルス増殖段階での血中の P24抗原（HIV-1 p24)を直接検出できないか否かを検討し た。 [0010] That is, the present inventor has intensively studied to solve the above-mentioned problems! ヽ, focusing on viremia in which a large amount of virus is detected in the blood in the early stage of HIV-1 infection. P 24 antigen in the blood at the growth phase was examined whether or not detectable (HIV-1 p24) directly.
その結果、本発明者はウィルス増殖期における HIV-1 p24を特異的に認識し、結合 する抗体を産生するノ、イブリドーマを作成することに成功した。  As a result, the present inventor succeeded in producing a hybridoma that produces an antibody that specifically recognizes and binds to HIV-1 p24 in the virus growth phase.
しかしながら、ヒト血中にはマウス由来の抗マウスタンパク抗体（IgM)が存在し、これ 力 SHIV-1 p24と結合してしまうため、 HIV-1 p24の検出が高感度でできないものとなつ ている。すなわち、被験血清サンプル中の非特異的な HIV-1 p24との反応による疑 陽性が生じていた。 However, mouse-derived anti-mouse protein antibody (IgM) exists in human blood. Because it binds to the force SHIV-1 p24, HIV-1 p24 cannot be detected with high sensitivity. That is, there was a false positive due to a reaction with nonspecific HIV-1 p24 in the test serum sample.
そこで本発明者は、抗マウスタンパク抗体の影響を受けない状態とし、上記のハイ プリドーマが産生する特異的な抗体を用いて、ウィルス増殖期における HIV-1 p24を 直接検出し得るを確認した。  Therefore, the present inventor confirmed that HIV-1 p24 can be directly detected in the virus growth phase by using a specific antibody produced by the above-mentioned hyperidoma in a state not affected by the anti-mouse protein antibody.
[0011] そして、これらのノ、イブリドーマが産生する特異的な抗体を用いて、 HIV-1 p24を直 接検出し、定量し得るサンドイッチ ELISAキットを作成することができるものであり、こ のキットにより、 HIV-1感染を高感度に、かつ簡便に診断し得ることを確認し、本発明 を完成させるに至った。さらにこのキットを用いれば HIV-1 p24に対する抗体の有無（ つまり p24値が低くても抗体が存在する感染状態)も診断できる。  [0011] Then, using these specific antibodies produced by these cells, hybridomas, a sandwich ELISA kit capable of directly detecting and quantifying HIV-1 p24 can be prepared. As a result, it was confirmed that HIV-1 infection could be diagnosed with high sensitivity and ease, and the present invention was completed. Furthermore, this kit can be used to diagnose the presence or absence of antibodies to HIV-1 p24 (that is, infectious conditions in which antibodies are present even if the p24 value is low).
[0012] HIV-1感染の有無を診断するにあたって、 HIV-1 p24を直接検出することは、 HIV- 1本体、すなわちウィルス本体を直接検出するものである。従来の診断法では、 HIV- 1の検出に替え、 HIV-1抗体を検出していたものであり、その感度と特異性が問題とさ れていたが、本発明により、特異的、かつ高感度に HIV-1感染を診断し得ることとなり 、特に優れたものであるといえる。このキットで HIV-1 p24に対する抗体の有無を診断 するには、検体の血清が本キットによる p24の検出を抑制するかどうかを見ればよい。 本発明は、力かる新規な検討の結果、完成されたものである。  [0012] In diagnosing the presence or absence of HIV-1 infection, directly detecting HIV-1 p24 directly detects the HIV-1 body, ie, the virus body. In conventional diagnostic methods, HIV-1 antibody was detected instead of detecting HIV-1, and its sensitivity and specificity were problematic. However, according to the present invention, it is specific and highly sensitive. Sensitivity can diagnose HIV-1 infection, which can be said to be particularly excellent. In order to diagnose the presence of antibodies against HIV-1 p24 with this kit, it is necessary to determine whether the serum of the sample suppresses the detection of p24 by this kit. The present invention has been completed as a result of intensive novel studies.
課題を解決するための手段  Means for solving the problem
[0013] したがって、力かる課題を解決するための本発明は、その一つの態様として、 [0013] Therefore, the present invention for solving a problem to be solved is, as one aspect thereof,
(1)血中に存在する抗マウスタンパク抗体の非特異的反応を抑制し、増殖期のヒト免 疫不全ウィルス (HIV-1)に発現する p24抗原 (HIV-1 p24)を認識して結合する抗体（ 抗 HIV-1 p24抗体)を用いることにより、反応感度を 10〜30倍に増強させる ELISA法 によるヒト血清、ヒト血清由来血液製剤または輸血用血清から、 HIV-1の検出する方 法、及び血清中のヒト由来の p24抗体を検出する方法；  (1) Recognizes and binds to the p24 antigen (HIV-1 p24) expressed in the human immunodeficiency virus (HIV-1) during the growth phase by suppressing nonspecific reactions of anti-mouse protein antibodies present in the blood To detect HIV-1 from human sera, human serum-derived blood products or blood transfusion sera by ELISA, which enhances reaction sensitivity by 10 to 30 times by using anti-HIV-1 antibody (anti-HIV-1 p24 antibody) And a method for detecting human-derived p24 antibody in serum;
(2)血中に存在する抗マウスタンパク抗体の非特異的反応の抑制を、非特異的マウ ス IgGを産生するノ、イブリドーマによりヒト血中に存在する抗マウスタンパク抗体の測 定の阻害を抑制することにより行う上記 1に記載の HIV-1を検出する方法； (3) HIV-1 p24を認識して結合する抗 HIV-1 p24抗体が、マウスに不活性ィ匕 HIV-1を 接種し得られた HIV-1に対する免疫能を有するマウス脾臓細胞と、マウスミエローマ 細胞を細胞融合し、得られた融合細胞力 感染細胞をスクリーニングし、さらにクロー ニングして得られたノヽイブリドーマ力 産生されたものであることを特徴とする上記 1ま たは 2に記載の HIV-1を検出する方法； (2) Suppression of non-specific reactions of anti-mouse protein antibodies present in blood, and inhibition of measurement of anti-mouse protein antibodies present in human blood by non-specific mouse IgG-producing and hybridomas. The method for detecting HIV-1 according to the above 1, which is carried out by suppressing; (3) An anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is inactivated in mice. 3. The fusion cell force obtained by cell fusion of myeloma cells, screened for the infected cells, and further cloned to produce a neurobrima force obtained by the method according to 1 or 2 above A method for detecting HIV-1;
(4) HIV-1 p24を認識して結合する抗 HIV-1 p24抗体が、 HIV-1 p24を捕獲する捕獲 抗体である上記 1、 2または 3に記載の HIV-1を検出する方法；  (4) The method for detecting HIV-1 according to 1, 2 or 3, wherein the anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is a capture antibody that captures HIV-1 p24;
(5) HIV-1 p24を認識して結合する抗 HIV-1 p24抗体が、 HIV-1 p24を検出する検出 抗体である上記 1、 2または 3に記載の HIV-1を検出する方法；  (5) The method for detecting HIV-1 according to 1, 2 or 3, wherein the anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is a detection antibody that detects HIV-1 p24;
(6) HIV-1 p24を捕獲する捕獲抗体が RT-N-48抗体である上記 4に記載の HIV-1を 検出する方法;及び  (6) The method for detecting HIV-1 as described in 4 above, wherein the capture antibody that captures HIV-1 p24 is an RT-N-48 antibody; and
(7) HIV-1 p24を検出する検出抗体が RT-C-50抗体である上記 5に記載の HIV-1を 検出する方法；  (7) The method for detecting HIV-1 according to 5 above, wherein the detection antibody for detecting HIV-1 p24 is an RT-C-50 antibody;
である。 It is.
本発明は、また別の態様として、  As another aspect, the present invention provides:
(8) ELISA法によるヒト血清、ヒト血清由来血液製剤または輸血用血清から HIV-1を検 出するため、及び血清中のヒト由来の p24抗体を検出するためのキットであって、増殖 期の HIV-1に発現する p24抗原（HIV-1 p24)を認識して結合する抗体 (抗 HIV-1 p24 抗体)を用いることを特徴とする HIV-1 p24抗原'抗体検出用 ELISAキット；  (8) A kit for detecting HIV-1 from human serum, human serum-derived blood products or blood transfusion serum by ELISA, and for detecting human-derived p24 antibody in the serum. An ELISA kit for detecting an HIV-1 p24 antigen 'antibody, comprising using an antibody that recognizes and binds to a p24 antigen (HIV-1 p24) expressed in HIV-1 (anti-HIV-1 p24 antibody);
(9) HIV-1 p24を認識して結合する抗 HIV-1 p24抗体が、マウスに不活性ィ匕 HIV-1を 接種し得られた HIV-1に対する免疫能を有するマウス脾臓細胞と、マウスミエローマ 細胞を細胞融合し、得られた融合細胞力 感染細胞をスクリーニングし、さらにクロー ニングして得られたノヽイブリドーマ力 産生されたものであることを特徴とする上記 8 に記載の HIV- 1 p24抗原'抗体検出用 ELISAキット；  (9) Anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is inactivated in mice. HIV-1 p24 as described in 8 above, which is produced by cell fusion of myeloma cells, screening of the obtained fused cell force-infected cells, and further cloning of the hybridoma obtained by cloning. ELISA kit for antigen 'antibody detection;
(10) HIV-l p24を認識して結合する抗 HIV-1 p24抗体が、 HIV-1 p24を捕獲する捕 獲抗体である上記 8または 9に記載の HIV-1 p24抗原'抗体検出用 ELISAキット； (10) The ELISA for detecting an HIV-1 p24 antigen 'antibody according to 8 or 9 above, wherein the anti-HIV-1 p24 antibody that recognizes and binds to HIV-l p24 is a capture antibody that captures HIV-1 p24. kit;
(11) HIV-l p24を認識して結合する抗 HIV-1 p24抗体が、 HIV-1 p24を検出する検 出抗体である上記 8または 9に記載の HIV-1 p24抗原'抗体検出用 ELISAキット； (12) HIV-1 p24を捕獲する捕獲抗体が RT-N-48抗体である上記 10に記載の HIV-1 p24抗原 ·抗体検出用 ELISAキット； (11) The ELISA for detecting an HIV-1 p24 antigen ′ antibody according to 8 or 9 above, wherein the anti-HIV-1 p24 antibody that recognizes and binds to HIV-l p24 is a detection antibody that detects HIV-1 p24. kit; (12) The ELISA kit for detecting an HIV-1 p24 antigen / antibody according to the above 10, wherein the capture antibody for capturing HIV-1 p24 is an RT-N-48 antibody;
(13) HIV-1 p24を検出する検出抗体が RT-C- 50抗体である上記 11に記載の HIV-1 p24抗原 ·抗体検出用 ELISAキット;及び  (13) The ELISA kit for detecting an HIV-1 p24 antigen / antibody according to the above 11, wherein the detection antibody for detecting HIV-1 p24 is an RT-C-50 antibody; and
(14) HIV-1 p24を検出する検出抗体である RT-C-50抗体が、ペルォキシダーゼ（PO D)標識抗体である上記 13に記載の HIV- 1 p24抗原 ·抗体検出用 ELISAキット； である。  (14) The ELISA kit for detecting HIV-1 p24 antigen / antibody according to the above 13, wherein the RT-C-50 antibody, which is a detection antibody for detecting HIV-1 p24, is a peroxidase (PO D) labeled antibody; .
発明の効果  The invention's effect
[0015] 本発明が提供する HIV-1を検出する方法並びにキットにより、ヒト血清、ヒト血清由 来血液製剤または輸血用血清について、血中に存在する抗マウスタンパク抗体の非 特異的反応の影響を受けないで、 HIV-1感染の有無を、簡便に、かつ高感度に診断 することができる。特に、本発明が提供する方法及びキットは、 HIV-1感染初期のウイ ルス増殖段階での血中の P24抗原 (HIV-1 p24)を直接検出できるものであり、測定感 度が 10〜30倍増強するものであり、従来問題とされている偽陽性、偽陰性の問題の ない検出を行うことができる。また、感染後の経過により血清中の p24値が下がり、反 比例して抗 P24抗体が上昇した後でも、抗体結合競合試験により高!ヽ特異性で抗体 の有無を診断できる。 [0015] By the method and kit for detecting HIV-1 provided by the present invention, the influence of non-specific reaction of anti-mouse protein antibody present in blood on human serum, human serum-derived blood products or blood transfusion serum It is possible to diagnose the presence or absence of HIV-1 infection easily and with high sensitivity. In particular, methods and kits provided by the present invention, which can detect the P 24 antigen in the blood in virus growth stages of HIV-1 infection early (HIV-1 p24) directly, the measurement sensitivity is 10 This is a 30-fold enhancement, and can be detected without the problems of false positives and false negatives, which have been regarded as problems in the past. In addition, even after the p24 value in the serum decreases and the anti-P24 antibody increases in inverse proportion to the course after infection, the presence or absence of the antibody can be diagnosed with high specificity by an antibody binding competition test.
[0016] また、本発明の方法並びにキットに使用するハイブリドーマは、より正確に HIV-1感 染の有無を検出するのに使用し得る抗体を産生するハイブリドーマであり、このハイ プリドーマ力 産生される抗体を用いることにより、 HIV-1感染の有無を簡便に、より 高感度に診断することができるば力りでなぐウィルス量の定量が行い得るものであり 、これまでのキットでは HIV-1の定性はできても定量ができないものであった力 本発 明が提供するキットによりそれを簡便に行い得る利点を有している。  [0016] The hybridoma used in the method and kit of the present invention is a hybridoma that produces an antibody that can be used to more accurately detect the presence or absence of HIV-1 infection. By using an antibody, it is possible to easily determine the presence or absence of HIV-1 infection, and the amount of virus can be quantified with force. Ability that could be quantified but could not be quantified The kit provided by the present invention has the advantage that it can be easily performed.
したがって、ヒト血清、ヒト血清由来血液製剤または輸血用血清の HIV-1感染を直 接的に検出することが可能となり、 HIV-1感染の蔓延を効果的に防止し得る。  Therefore, it becomes possible to directly detect HIV-1 infection in human serum, human serum-derived blood products or blood transfusion serum, and can effectively prevent the spread of HIV-1 infection.
[0017] すなわち、本発明が提供する HIV-1を検出方法における非特異的マウス IgGを産生 するハイプリドーマから産生されるマウス IgGは、 HIV-1 p24、及び HIV-1 p24に対す る抗体には結合しないものであり、したがって、 HIV-1 p24に関与した抗原 抗体系 には影響を与えないものである。 That is, mouse IgG produced from a hyperidoma that produces non-specific mouse IgG in the method for detecting HIV-1 provided by the present invention is an antibody against HIV-1 p24 and HIV-1 p24. Does not bind and is therefore an antigen-antibody system involved in HIV-1 p24 It has no effect.
さらに、また、マウスモノクローナル抗体に対しても結合しないで、血清中に存在す る抗マウスタンノ ク抗体をブロックするものである。したがって、本発明は、 HIV-1 p24 を直接検出し得る点で特に優れたものである。  Furthermore, it also blocks anti-mouse tank antibody present in serum without binding to mouse monoclonal antibody. Therefore, the present invention is particularly excellent in that HIV-1 p24 can be directly detected.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0018] 本発明が提供する HIV-1の検出方法においては、前記したように、増殖期のヒト免 疫不全ウィルス (HIV-1)に発現する p24抗原 (HIV-1 p24)を認識して結合する抗体（ 抗 HIV-1 p24抗体）を用いることを特徴とする。この抗 HIV-1 p24抗体は、本発明にあ つては、増殖期の HIV-1に発現する p24抗原 (HIV-1 p24)を認識する抗体を特異的 に産生するノ、イブリドーマ力ら産生される。  [0018] In the method for detecting HIV-1 provided by the present invention, as described above, p24 antigen (HIV-1 p24) expressed in proliferating human immunodeficiency virus (HIV-1) is recognized. A binding antibody (anti-HIV-1 p24 antibody) is used. In the present invention, this anti-HIV-1 p24 antibody is produced by the antibody that specifically produces an antibody that recognizes the p24 antigen (HIV-1 p24) expressed in proliferating HIV-1 (HIV-1 p24). The
HIV-1感染の初期における HIV-1の増殖期に発現する p24抗原（HIV-1 p24)は、感 染性のある抗原であり、一般にこれに対する抗体はその検出系を妨害するとされてい た。し力しながら、本発明で使用するハイブリドーマは、検出系を妨害することなぐこ の HIV-1 p24自体を効果的に認識し得る抗体を産生するハイプリドーマである。  The p24 antigen (HIV-1 p24) expressed during the HIV-1 proliferative phase in the early stages of HIV-1 infection is an infectious antigen, and in general, antibodies against it were thought to interfere with the detection system. However, the hybridoma used in the present invention is a hyperidoma that produces an antibody capable of effectively recognizing HIV-1 p24 itself without interfering with the detection system.
[0019] 一般に HIV-1に感染すると、その感染初期段階においてウィルス血症が明らかにな る、すなわち、血液中にウィルスが多量に検出される。そのため血中に p24抗原（HIV -1 p24)が大量に存在することとなる。この HIV-1 p24を確実に検出すれば、より効果 的かつ高感度で HIV-1感染の診断を行うことが可能となる。  [0019] In general, infection with HIV-1 reveals viremia at an early stage of infection, that is, a large amount of virus is detected in the blood. Therefore, a large amount of p24 antigen (HIV-1 p24) is present in the blood. If this HIV-1 p24 is reliably detected, HIV-1 infection can be diagnosed more effectively and with high sensitivity.
[0020] 本発明は、まさに HIV-1の初感染後のウィルス血症が認められる時期における p24 抗原を、直接認識する抗体を産生するハイプリドーマを用いるものであり、このハイブ リドーマが産生する、増殖期の HIV-1に発現する HIV-1 p24を使用することにより、 HI V-1感染の診断を直接的に、より高感度でありながら、簡便に行いうるものとなる。  [0020] The present invention uses a hyperidoma that produces an antibody that directly recognizes the p24 antigen at the time when viremia after the initial infection of HIV-1 is observed, and the proliferation produced by this hybridoma. By using HIV-1 p24 expressed in the HIV-1 stage, the diagnosis of HIV-1 infection can be carried out directly, with higher sensitivity and with ease.
[0021] したがって、従来の HIV-1感染の診断は、例えば、種々の感染者の HIV-1に共通 する抗原部位としてのコアタンパク質 p24、あるいはエンベロープ糖タンパク質 gpl20 及びトランスメンブランタンパク質 gp41を認識する抗体を検出していたものであるが、 本発明においては、 HIV-1の感染後における増殖期の HIV-1 p24を認識することが できる抗体を使用し、それにより HIV-1感染を診断する点で、ウィルス本体を直接検 出するという優れたものであるといえる。同時に抗体結合競合試験ではかなりの特異 性をもって抗 HIV-1抗体を検出できる。 [0021] Therefore, conventional diagnosis of HIV-1 infection includes, for example, an antibody that recognizes core protein p24 as an antigenic site common to HIV-1 of various infected persons, or envelope glycoprotein gpl20 and transmembrane protein gp41. In the present invention, an antibody capable of recognizing proliferative HIV-1 p24 after HIV-1 infection is used, thereby diagnosing HIV-1 infection. Therefore, it can be said that it is an excellent one that directly detects the virus itself. At the same time, antibody binding competition tests are quite specific Anti-HIV-1 antibody can be detected with sex.
[0022] かかる HIV-1 p24を特異的に産生するハイブリドーマは、基本的には HIV-1を、例 えばマウスに接種することによりそのマウスに HIV-1に対する免疫能を機能させ、その 結果、抗 HIV-1抗体を産生するマウスの組織細胞、好ましくは脾臓細胞を採取し、一 般的に汎用されているマウスミエローマ細胞 (マウス黒色腫細胞）と細胞融合させ、得 られた融合細胞を、通常のハイプリドーマのクローユングに使用することができる HAT 選択培地による培養でスクリーニングを行うことにより得ることができる。 [0022] Such a hybridoma that specifically produces HIV-1 p24 is basically inoculated with HIV-1, for example, to a mouse so that the mouse has an immune function against HIV-1, and as a result, Mouse tissue cells, preferably spleen cells, that produce anti-HIV-1 antibodies are collected and fused with commonly used mouse myeloma cells (mouse melanoma cells). It can be obtained by screening by culturing with a HAT selective medium that can be used for cloning of normal hyperidoma.
力かる細胞融合、ハイプリドーマのクローユング、並びにスクリーニング等の操作は 、一般的に汎用されている技術を適宜応用し、行うことができる（例えば、後記する実 施例を参照)。  Operations such as powerful cell fusion, high-pridoma cloning, and screening can be performed by appropriately applying generally used techniques (see, for example, Examples described later).
[0023] 力べして得られたノヽイブリドーマは、その特性として、 HIV-1 p24を極めて効果的に、 かつ高感度に認識する抗体、すなわち抗 HIV-1 p24抗体を産生するハイブリドーマ である。そのなかでも、ハイプリドーマが産生する抗体は、その一つとして、 HIV-1 p2 4を捕獲する捕獲抗体であり、また、別の抗体は、 p24の他の部位に結合することによ り HIV-1 p24を検出する検出抗体として機能するものである。  [0023] Neubridoma obtained by force is a hybridoma that produces an antibody that recognizes HIV-1 p24 extremely effectively and with high sensitivity, that is, an anti-HIV-1 p24 antibody. Among them, the antibody produced by hyperidoma is, for example, a capture antibody that captures HIV-1 p2 4, and another antibody binds to other sites in p24. -1 It functions as a detection antibody that detects p24.
[0024] 具体的には、本発明者によって RT-N-48抗体と命名された、 HIV-1 p24を捕獲する 捕獲抗体、並びに RT-C-50抗体と命名された HIV-1 p24を検出する検出抗体として 機能する。  [0024] Specifically, the present inventors have detected a capture antibody that captures HIV-1 p24, named RT-N-48 antibody, and HIV-1 p24, named RT-C-50 antibody. Functions as a detection antibody.
したがって、本発明は、特に具体的には、この HIV-1 p24を捕獲する RT-N-48抗体 、並びに HIV-1 p24を検出する RT-C-50抗体を使用する HIV-1の検出方法、並びに キットを提供するものである。  Therefore, the present invention particularly specifically relates to an RT-N-48 antibody that captures HIV-1 p24, and a method for detecting HIV-1 using RT-C-50 antibody that detects HIV-1 p24. As well as kits.
[0025] すなわち、本発明においては、当該ノ、イブリドーマより産生された HIV-1 p24を直接 認識する抗体は、 HIV-1 p24を捕獲、並びに検出する抗体として機能する。したがつ て、これらの抗体を HIV-1抗原 ·抗体検出用の ELISAキット使用することにより、すな わち FLISAサンドイッチ法により HIV-1感染の診断を直接的に、高感度で行うことがで きる。これまでの p24抗原検出方法は、 p24抗原捕獲用には、捕獲抗体をマウスの単 クロン抗体を、検出用には HIV-1感染者由来のポリクローナル抗体を用いている。し かし、この方法では非特異的な陽性反応の出現を完全には抑えられない。このような 背景からも本キットの特異性は評価に値する。 [0025] That is, in the present invention, the antibody that directly recognizes HIV-1 p24 produced from the above-mentioned ibridoma functions as an antibody that captures and detects HIV-1 p24. Therefore, these antibodies can be used to directly diagnose HIV-1 infection with high sensitivity by using the ELISA kit for HIV-1 antigen / antibody detection, that is, the FLISA sandwich method. it can. The conventional p24 antigen detection method uses a mouse monoclonal antibody as a capture antibody for p24 antigen capture, and a polyclonal antibody derived from an HIV-1 infected person for detection. However, this method does not completely suppress the appearance of nonspecific positive reactions. like this From the background, the specificity of this kit is worthy of evaluation.
[0026] 具体的には、 HIV-1 p24に対する捕獲抗体としての RT-N- 48抗体を、例えば HIV-1 検出用のウェルマイク口プレートに塗布する。一方、例えば検出色素としてのペルォ キシダーゼ (POD)を結合 (標識)させた検出抗体としての RT-C- 50抗体を作成し、上 記のプレート上で、検出サンプルと共に検出抗体を加え、サンドイッチ法による反応 を行い、発色剤による発色をおこない、 HIV-1感染の有無を診断することができる。  Specifically, RT-N-48 antibody as a capture antibody against HIV-1 p24 is applied to, for example, a well microphone mouth plate for HIV-1 detection. On the other hand, for example, an RT-C-50 antibody as a detection antibody to which peroxidase (POD) as a detection dye is bound (labeled) is prepared, and the detection antibody is added together with the detection sample on the above plate. It is possible to diagnose the presence or absence of HIV-1 infection by developing a color with a coloring agent.
[0027] また、本発明にお 、ては、当該ハイプリドーマが産生する HIV-1 p24捕獲抗体なら びに検出抗体について、例えばウェルマイク口プレートに塗布するその濃度を適宜 段階的な希釈を行うことにより、サンドイッチ法による反応を行い、発色剤による発色 をおこない、例えば 450nm波長で吸光度を測定し、スタンダードとの検量線の対比に より、 HIV-1 p24またはそれに対する抗体を定性及び定量することができるのである（ 後記する実施例を参照)。  [0027] Further, in the present invention, for the HIV-1 p24 capture antibody and detection antibody produced by the hyperidoma, for example, the concentration applied to the well microphone mouth plate is appropriately diluted stepwise. By using the sandwich method, color development with a color former, measurement of absorbance at a wavelength of 450 nm, for example, and qualitative and quantitative determination of HIV-1 p24 or antibodies against it by comparison with a standard curve with a standard (See the examples below).
実施例  Example
[0028] 以下に本発明を、具体的ハイプリドーマの作成、該ハイブリドーマからの HIV-1 p24 捕獲抗体ならびに検出抗体の産生、それら抗体を使用した HIV-1の検出の実際、具 体的キットの一実施例を説明することにより、さらに詳細に説明するが、本発明はこれ らのものに限定されるものではない。本発明は、その目的を逸脱しない限りの種々の 変形も可能であり、力かる変形も本発明の技術的範囲に包含されるものであることは いうまでもない。  [0028] The present invention will be described below with reference to preparation of a specific hyperidoma, production of an HIV-1 p24 capture antibody and detection antibody from the hybridoma, actual detection of HIV-1 using these antibodies, and a specific kit. The present invention will be described in more detail by describing one embodiment, but the present invention is not limited to these. The present invention can be variously modified without departing from the object thereof, and it goes without saying that powerful modifications are also included in the technical scope of the present invention.
[0029] 試験例 1：ハイブリドーマの作成  [0029] Test Example 1: Creation of hybridoma
本発明で使用する特異的な HIV-1 p24捕獲抗体ならびに検出抗体を産生するハイ ブリドーマを得るために、最初のステップとしてマウスを用いて HIV-1により免疫を行 Vヽ、免疫組織細胞 (脾臓細胞）を摘出した。  In order to obtain a hybridoma producing a specific HIV-1 p24 capture antibody and detection antibody for use in the present invention, the mouse is first immunized with HIV-1 as a first step. V ス テ ッ プ, immune tissue cells (spleen Cells).
[方法]  [Method]
[0030] (a)濃縮 HIV- 1の調製:  [0030] (a) Preparation of concentrated HIV-1:
以下の方法により濃縮 HIV-1を得た。  Concentrated HIV-1 was obtained by the following method.
すなわち、臨床分離ウィルス (HIV-1)株を、抗 CD3抗体とインターロイキン— 2 (IL- 2)で刺激したヒト末梢血液単核球 (PBMC)で増殖させ、 AT-2薬剤で不活化させた後 、超遠心分離することにより濃縮した分画として、濃縮 HIV-1を得た。 That is, clinical isolate virus (HIV-1) strain was grown on human peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 antibody and interleukin-2 (IL-2) and inactivated with AT-2 drug. After As a fraction concentrated by ultracentrifugation, concentrated HIV-1 was obtained.
[0031] (b)マウスへの免疫： [0031] (b) Immunization to mice:
上記 (a)で得た濃縮 HIV-1を、界面活性剤により可溶ィ匕し、フロイント完全アジュバ ント（Complete Freund Adjuvant: CFA)と等量混合し、ェマルジヨンの状態で、マウス 皮下に接種し、感作を行った。  Concentrated HIV-1 obtained in (a) above is solubilized with a surfactant, mixed with an equal volume of Complete Freund Adjuvant (CFA), and inoculated subcutaneously in a mouse in the state of emulsion. , Sensitized.
さらに 2週間後に、同量の濃縮 HIV-1可溶ィ匕物をフロイント完全アジュバント（CFA) と等量混合して、マウス皮下に免疫をした。この免疫操作を 2週間おきに 3回繰り返し た。  Two weeks later, the same amount of concentrated HIV-1 soluble yeast was mixed with Freund's complete adjuvant (CFA) in an equal amount and immunized subcutaneously in mice. This immunization was repeated 3 times every 2 weeks.
この最終免疫の 2週間後に、同量の濃縮 HIV-1可溶ィ匕物を免疫マウスの腹腔内に 接種して、その 3日後にマウスより免疫脾臓を摘出した。  Two weeks after this final immunization, the same amount of concentrated HIV-1 soluble yeast was inoculated into the peritoneal cavity of the immunized mouse, and three days later, the immune spleen was removed from the mouse.
[0032] (c)雄 : [0032] (c) Male:
マウスミエローマ（マウス骨髄腫)細胞 SP2/0を、 10%FCS (胎児ゥシ血清）添加 RPMI- 1培地で、対数的に増殖させ、洗浄した。  Mouse myeloma (mouse myeloma) cells SP2 / 0 were grown logarithmically and washed in RPMI-1 medium supplemented with 10% FCS (fetal urine serum).
一方、上記で摘出したマウス脾臓をステンレスメッシュで裏ごしし、細胞浮遊液を得 、この細胞浮遊液と、増殖したマウスミエローマ細胞 SP2/0細胞とを 4 : 1の比率で、ポ リエチレングリコール 4000 (PEG4000)を用いて細胞融合した。  On the other hand, the mouse spleen excised above was lined with a stainless mesh to obtain a cell suspension, and this cell suspension and the proliferated mouse myeloma cells SP2 / 0 cells at a ratio of 4: 1 polyethylene glycol 4000 ( Cell fusion was performed using PEG4000).
[0033] (d)ハイブリドーマの選択： [0033] (d) Selection of hybridoma:
融合細胞（融合した細胞）を 96ウェルマイク口プレートに播き込み、ハイプリドーマ の選択を行った。  The fused cells (fused cells) were seeded on a 96-well microphone mouthplate, and selection of high-pridoma was performed.
ノ、イブリドーマの選択には、市販の HAT選択培地を用いて行った。すなわち、 3日 ごとに培地交換を行い、融合後 14〜21日に培養中に出現した融合細胞の培養上 清を、以下の蛍光抗体法でスクリーニングした。  The selection of the cells was performed using a commercially available HAT selection medium. That is, the medium was changed every 3 days, and the culture supernatant of the fused cells that appeared during the culture 14 to 21 days after the fusion was screened by the following fluorescent antibody method.
[0034] (e)スクリーニング（正）： [0034] (e) Screening (positive):
HIV-1感染細胞株 Molt4/IIIB、及び非感染細胞 Molt4細胞を、別々の 15ウェルスラ イドグラス（大日本製薬社製）に塗抹し、風乾後、 100%エタノールで 20°CZ10分間 固 ¾Eし 7こ。  HIV-1 infected cell line Molt4 / IIIB and non-infected cell Molt4 cells are smeared on separate 15-well slide glass (Dainippon Pharmaceutical Co., Ltd.), air-dried, solidified with 100% ethanol at 20 ° CZ for 10 minutes, then E7 .
スライドグラスの各ゥエルにアツセィサンプルを乗せ、室温にて 30分反応させ、洗浄 後、市販の FITC標識 (注：フルォレイセンイソチオシァネート：抗体の蛍光標識試薬） ャギ抗マウス IgG (1/100希釈）を室温で 30分さらに反応させた。 Place a sample on each slide glass well, react at room temperature for 30 minutes, wash, and then commercially available FITC label (note: fluorescein isothiocyanate: fluorescent labeling reagent for antibodies) Goat anti-mouse IgG (1/100 dilution) was further reacted at room temperature for 30 minutes.
洗浄後、蛍光顕微鏡を用いて、 MoltVlIIB細胞（注:感染細胞）を染色し、 Molt4細 胞（注：非感染細胞）は染色しな 、抗体をスクリーニングした。  After washing, using a fluorescence microscope, MoltVlIIB cells (Note: infected cells) were stained, and Molt4 cells (Note: non-infected cells) were not stained.
HIV-1 p24に対する反応性は、 Molt4/IIIB、及び Molt4細胞の可溶物を抗原として 作成したウェスタンプロット (WB)法で確認した。  The reactivity to HIV-1 p24 was confirmed by Western plot (WB) method using Molt4 / IIIB and a soluble product of Molt4 cells as antigens.
[0035] (f)スクリーニング（副）： [0035] (f) Screening (secondary):
HIV-1感染活性化ヒト PBMC、及び非感染コントロール細胞を、別々の 15ウェルスラ イドグラス（大日本製薬社製）に塗抹し、風乾後、 100%エタノールで 20°CZ10分間 固 ¾Eし 7こ。  HIV-1 infected activated human PBMC and non-infected control cells are smeared on separate 15-well slide glasses (Dainippon Pharmaceutical Co., Ltd.), air-dried, and then solidified with 100% ethanol for 10 minutes at 20 ° CZ for 7 minutes.
スライドグラスの各ゥエルにアツセィサンプルを乗せ、室温にて 30分反応させ、洗浄 後、市販の FITC標識 (注：フルォレイセンイソチオシァネート：抗体の蛍光標識試薬） ャギ抗マウス IgG (1/100希釈）を室温で 30分さらに反応させた。  Place the Atsy sample on each slide glass well, react at room temperature for 30 minutes, wash, and then commercially available FITC label (note: fluorescein isothiocyanate: antibody fluorescent labeling reagent) goat anti-mouse IgG ( 1/100 dilution) was further reacted at room temperature for 30 minutes.
洗浄後、蛍光顕微鏡を用いて、感染細胞を染色し、非感染細胞は染色しない抗体 をスクリーニングした。  After washing, the infected cells were stained using an fluorescence microscope, and antibodies that did not stain uninfected cells were screened.
HIV-1 p24に対する反応性は、同様に、 Molt4/IIIB、及び Molt4細胞の可溶物を抗 原として作成したウェスタンプロット (WB)法で確認した。  Similarly, the reactivity to HIV-1 p24 was confirmed by the Western plot (WB) method using Molt4 / IIIB and a soluble product of Molt4 cells as an antigen.
[0036] (g)クローニング Igクラスの決定： [0036] (g) Cloning Ig class determination:
抗体産生ゥエル中のハイプリドーマは、常法にしたがって、マウス胸腺細胞をフィー ダー細胞とする限界希釈法で 2回クローユングした。最終クローユング後の培養上清 に含まれる抗体の Igクラスとサブクラスの決定は、市販のャギ抗マウス Igキットを用い て ELISA法で判定した。  Hypridoma in antibody-producing wells was cloned twice by the limiting dilution method using mouse thymocytes as feeder cells according to a conventional method. The determination of the Ig class and subclass of the antibody contained in the culture supernatant after the final cloning was determined by ELISA using a commercially available goat anti-mouse Ig kit.
[0037] (h)杭体の大量調製 精製: [0037] (h) Mass preparation of pile bodies Purification:
市販の SCIDマウス (C.B-17)に 0.5mLのプリスタンを腹腔内に投与し、一週間後に上 記で得たハイプリドーマの 500万個を腹腔移植した。約 10日間後に腹水を得た。こ の腹水をプールし、 15, 000回転 Z10分間の遠心により細胞を沈殿させ、得られた 上清をさらに 10 mのフィルターで濾過し、濾液を 50%硫酸アンモ -ゥム水溶液に より沈殿させた。得られた沈查をリン酸緩衝生理食塩水（PBS)に溶解させ、セフアデ ックス G200を用いるゲル濾過法により、 IgG分画を調製した。抗体の濃度は、 OD280 にて推定した。 A commercially available SCID mouse (CB-17) was administered 0.5 mL of pristane intraperitoneally, and one week later, 5 million hyperpridoma obtained above were intraperitoneally transplanted. Ascites was obtained after about 10 days. The ascites is pooled, the cells are precipitated by centrifugation at 15,000 rpm for 10 minutes, the resulting supernatant is further filtered through a 10-m filter, and the filtrate is precipitated with 50% aqueous ammonium sulfate solution. It was. The obtained sediment was dissolved in phosphate buffered saline (PBS), and an IgG fraction was prepared by gel filtration using Sephadex G200. Antibody concentration is OD280 Estimated.
[0038] 以上の方法により 20数種類の抗 HIV-lp24抗体を産生するノ、イブリドーマを得た。  [0038] By the above method, nobridoma producing 20 or more kinds of anti-HIV-lp24 antibodies was obtained.
その中で、サンドイッチ ELISA法により高感度に HIV-1 p24の測定を可能としたのは 、次の組み合わせであった。  Among them, the following combination made it possible to measure HIV-1 p24 with high sensitivity by sandwich ELISA.
捕獲抗体: RT-N-48  Capture antibody: RT-N-48
検出抗体： RT- C-50  Detection antibody: RT-C-50
その両者とも、その IgGクラスは、マウス IgGであった。  In both cases, the IgG class was mouse IgG.
[0039] 試験例 2：サンドイッチ ELISA法に使用する HIV-1 D24杭原の標準液の作成 [0039] Test Example 2: Preparation of standard solution of HIV-1 D 24 Hanghara used for sandwich ELISA
(a) (正） Molt4/IIIB細胞の培養上清に、最終濃度 1%の Triton Χ-100を添加し、氷中 で 30分反応させた。エツペンドルフ型のチューブに小分けし、— 20°Cで保存した。 (a) (Correct) Triton Χ-100 with a final concentration of 1% was added to the culture supernatant of Molt4 / IIIB cells and allowed to react in ice for 30 minutes. Aliquot into Eppendorf type tubes and store at -20 ° C.
(b) (副）臨床分離ウィルス株を抗 CD3抗体と IL-2で刺激した人の末梢血液単核球 P BMCで増殖させた培養上清に最終濃度 10%の Triton Χ-100を添加し、氷中で 30分 反応させた。エツペンドルフ型のチューブに小分けし、— 20°Cで保存した。 (b) Add 10% final concentration of Triton Χ-100 to the culture supernatant grown in peripheral blood mononuclear cells P BMC of humans who were stimulated with (CD) antibody and IL-2. The mixture was reacted for 30 minutes in ice. Aliquot into Eppendorf type tubes and store at -20 ° C.
[0040] ¾施例 i : HTV- 1 η24杭原及び杭体檢出用（定件、定畺) RUSAキット  [0040] ¾ Example i: HTV-1 η24 Hanghara and pile body extraction (fixed, fixed) RUSA kit
本発明のキットとしては、以下のものからなる。  The kit of the present invention comprises the following.
(1) HIV-l p24獲得抗体（RT-N- 48抗体）を塗布した 96ウェルマイク口プレート [1プレ ート Zキット]  (1) 96-well microphone mouthplate coated with HIV-l p24-acquired antibody (RT-N-48 antibody) [1 plate Z kit]
(2)サンプル希釈液（1% Triton-X100、 0.2%ゥシ血清アルブミン含有リン酸緩衝生 理食塩水） [30mLZキット]  (2) Sample diluent (phosphate buffered saline containing 1% Triton-X100, 0.2% ushi serum albumin) [30mLZ kit]
(3) HIV- 1 p24スタンダード（1% Triton- X100、可溶化ウィルス） l,000pg/mL[2.0mL Zキット]  (3) HIV-1 p24 standard (1% Triton-X100, solubilized virus) l, 000pg / mL [2.0mL Z kit]
(4)ペルォキシダーゼ (POD)標識 HIV-1 p24検出抗体 (RT-C-50抗体）（100 X濃度 ) [0.07mLZキット]  (4) Peroxidase (POD) -labeled HIV-1 p24 detection antibody (RT-C-50 antibody) (100 X concentration) [0.07mLZ kit]
(5)プレート洗浄液 (20 X濃度、 Tween-20含有リン酸緩衝生理食塩水） [20mLZキッ 卜]  (5) Plate washing solution (20 X concentration, phosphate buffered saline containing Tween-20) [20 mLZ kit]
(6)ペルォキシダーゼ（POD)基質液 [15mLZキット]  (6) Peroxidase (POD) substrate solution [15mLZ kit]
(7)発色剤（100 X濃度、 TMB :トリパンブルー） [0.15mLZキット]  (7) Color former (100 X concentration, TMB: Trypan blue) [0.15mLZ kit]
(8)発色停止液 (2N-硫酸） [15mLZキット] (9)プレートシール [3枚 Zキット] (8) Color stop solution (2N-sulfuric acid) [15mLZ kit] (9) Plate seal [3 pieces Z kit]
[0041] 実施例 2 : HIV-1 D24杭原及び杭体枪出用（定件、定量) ELISAキットの使用の実際 [0041] Example 2: HIV-1 D24 Hanghara and pile body extraction (fixed, quantitative) Actual use of ELISA kit
実施例 1に記載したキットは、具体的には以下の方法により HIV-1 p24抗原及び抗 体検出の検出に使用される。  The kit described in Example 1 is specifically used for detection of HIV-1 p24 antigen and antibody detection by the following method.
[0042] [方法]  [0042] [Method]
(1) HIV-l p24スタンダードの希釈：  (1) Dilution of HIV-l p24 standard:
別の 96ウェルマイク口プレート（U底型）またはエツペンドルフチューブで、サンプル 希釈液を O.lmLずつ分注し、 1,000 pg/mLの HIV- 1 p24スタンダード O.lmLを 2倍段階 希釈する。通常は、最終濃度 7段階までとする (最終濃度 : 8pg/mL)  Using a separate 96-well microphone mouthplate (U-bottom) or Eppendorf tube, aliquot sample dilutions and dilute 1,000 pg / mL HIV-1 p24 standard O.lmL in 2-fold increments. . Usually, the final concentration is up to 7 levels (final concentration: 8 pg / mL).
(2)必要に応じて、検体となる血清 (血漿)をサンプル希釈液で希釈する (好ましくは 、ほとんどの場合、 1/10、 1/100または 1/1, 000希釈で測定可能な範囲）。  (2) If necessary, dilute the sample serum (plasma) with the sample diluent (preferably in the most measurable range at 1/10, 1/100, or 1 / 1,000 dilution) .
(3)段階希釈スタンダードを 0.05mLずつ各ゥエルに入れる。ブランクとなるゥエルに はサンプル希釈液だけを 0.05mL入れる。  (3) Add 0.05mL serial dilution standard to each well. Add 0.05 mL of the sample diluent only to the blank well.
(4)検体を 0.05mLずつゥエルに入れる。  (4) Put 0.05mL sample into the well.
(5) HIV-1 p24抗体検出の場合には、検体の入ったゥエルに 250pg/mLの HIV-1 p24 液 (スタンダードをサンプル希釈液で 1:4に希釈した溶液）を 0.05mL入れる。スタンダ ードの入ったゥエルはそのまま。  (5) For detection of HIV-1 p24 antibody, add 0.05 mL of 250 pg / mL HIV-1 p24 solution (standard diluted 1: 4 with sample diluent) to the well containing the sample. The standard-filled uel remains.
[0043] (6)ペルォキシダーゼ（POD)標識 HIV-1 p24検出抗体（RT- C- 50抗体）をサンプル 希釈液で 1/100に希釈する。  [0043] (6) Dilute the peroxidase (POD) labeled HIV-1 p24 detection antibody (RT-C-50 antibody) to 1/100 with the sample diluent.
(7)各ゥエルに希釈 POD標識 HIV-1 p24検出抗体（RT- C- 50抗体）を 0.05mL入れる  (7) Add 0.05 mL of diluted POD-labeled HIV-1 p24 detection antibody (RT-C-50 antibody) to each well
(8)プレートをシールする。 (8) Seal the plate.
(9)室温（20°C以上）、または 37°C孵卵器で 1時間反応させる。  (9) Incubate for 1 hour at room temperature (over 20 ° C) or 37 ° C incubator.
(10)希釈したプレート洗浄液を精製水で作成し、プレートをよく洗浄する。  (10) Prepare diluted plate washing solution with purified water and wash the plate thoroughly.
(11)必要なペルォキシダーゼ (POD)基質液を清浄な試験管に移し、 1/100量の発 色剤を加え、静かに混和した後、各ゥエルに O.lmLずつ分注する。  (11) Transfer the necessary peroxidase (POD) substrate solution to a clean test tube, add 1/100 amount of colorant, mix gently, and dispense 0.1 mL to each well.
(12)室温で 20〜30分遮光静置し、スタンダードの列が段階的に青色に発色するの を確認する。 [0044] (13)この時点で定性は可能である。すなわち、 HIV-1 p24アツセィでは、血清（血漿） 等の検体のゥエルが青色に発色していれば、抗原陽性である。また、 HIV-1 p24抗体 アツセィでは、発色が阻止されていれば、抗体陽性である。 (12) Let stand for 20-30 minutes at room temperature in the dark and check that the color of the standard row gradually turns blue. [0044] (13) Qualitative is possible at this point. In other words, HIV-1 p24 Atsei is positive for an antigen if the color of the sample such as serum (plasma) is colored blue. In addition, the HIV-1 p24 antibody Atsei is positive for antibodies if color development is blocked.
(14)発色停止液を各ゥエルに O.lmL加え、溶液を黄色にする。プレート吸光度計で 450nm波長（可能であれば 2波長の reference 540nm)で吸光度を測定する。スタンダ ードより検量線を引き、 HIV-1 p24抗原または抗体を定性及び定量する。  (14) Add O.lmL of color stop solution to each well to make the solution yellow. Measure absorbance at 450nm wavelength (2 wavelengths reference 540nm if possible) with a plate absorptiometer. A standard curve is drawn from the standard to qualify and quantify HIV-1 p24 antigen or antibody.
[0045] 実施例 3 :実際のキットの使用例 [0045] Example 3: Usage example of an actual kit
HIV-1 p24スタンダードを、 l,000pg/mLから 16pg/mLまで希釈し、 50 Lずつ各ゥェ ル（縦列左から 1〜4列）まで入れた。左力ら 4列目のゥエルには、 HIV-1感染者の血 漿を 50 Lずつ最終濃度 20%に入れた。  The HIV-1 p24 standard was diluted from 1 000 pg / mL to 16 pg / mL, and 50 L was added to each well (column 1 to 4 from the left column). In the fourth row of left force and other wells, 50 L of plasma from HIV-1-infected individuals was added to a final concentration of 20%.
次に、各ゥエルに POD標識 HIV-1 p24検出抗体（RT-C-50抗体）を 50 /z Lずつ入れ 、 1時間反応させた。洗浄後、発色液を 100 Lずつ入れ、 20分暗所で反応させた。 その結果を図 1に示す。  Next, 50 / zL of POD-labeled HIV-1 p24 detection antibody (RT-C-50 antibody) was added to each well for 1 hour. After washing, 100 L of coloring solution was added and allowed to react for 20 minutes in the dark. The results are shown in Fig. 1.
[0046] 図中、縦列左から 1〜4列まで発色 (青色）の濃淡は、硫酸を入れる前、その他の発 色 (黄色)の濃淡硫酸を入れた後の様子である。 [0046] In the figure, the shades of blue (blue) from left to right in the column are the states after adding sulfuric acid of other colors (yellow) before adding sulfuric acid.
HIV-1 p24の濃度により肉眼でも判定可能であることが判明する。特に HIV-1感染 者の血漿は、 16〜l,000pg/mLの HIV-1 p24の検出を完全に阻害していることが判明 し、抗体陽性であることが理解される。  The concentration of HIV-1 p24 can be determined with the naked eye. In particular, the plasma of HIV-1 infected individuals was found to be completely inhibited from detection of HIV-1 p24 at 16 to 1 000 pg / mL and is understood to be antibody positive.
このプレートの吸光度を測定することにより HIV-1 p24の検量線を引くことができる。 産業上の利用可能性  A calibration curve for HIV-1 p24 can be drawn by measuring the absorbance of this plate. Industrial applicability
[0047] 以上記載のように、本発明により HIV-1感染を簡便かつ確実に診断する方法として 、 HIV-1 p24を直接認識する抗体を使用した HIV-1の検出方法及びキットが提供され る。本発明が提供するハイブリドーマは、 HIV- 1感染を診断するための、 HIV- 1 p24を 検出するための抗 HIV-1抗体を産生するものであり、ウィルス本体を検出し得る抗体 を産生するハイブリドーマである。  [0047] As described above, the present invention provides a method and kit for detecting HIV-1 using an antibody that directly recognizes HIV-1 p24 as a method for easily and reliably diagnosing HIV-1 infection. . The hybridoma provided by the present invention produces an anti-HIV-1 antibody for detecting HIV-1 p24 for diagnosing HIV-1 infection, and produces an antibody capable of detecting the virus body. It is.
[0048] すなわち、本発明が提供するハイブリドーマは、 HIV-1 p24、すなわちウィルス本体 を検出し得る抗体を産生するものであり、力かる抗体を使用することにより、 HIV-1感 染の有無の診断は極めて正確なものであり、さらに感染の有無以外にウィルス量の 定量にまで使用し得る。 [0048] That is, the hybridoma provided by the present invention produces HIV-1 p24, that is, an antibody that can detect the virus main body. By using a strong antibody, the presence or absence of HIV-1 infection is detected. Diagnosis is extremely accurate, and in addition to the presence or absence of infection, Can be used for quantification.
したがって、ヒト血清、ヒト血清由来血液製剤または輸血用血清の HIV-1感染を直 接的に検出することが可能となり、 HIV-1感染の蔓延を効果的に防止し得るものであ り、その産業上の貢献度は多大なものである。  Therefore, it is possible to directly detect HIV-1 infection in human serum, human serum-derived blood products or blood transfusion serum, and effectively prevent the spread of HIV-1 infection. Industrial contribution is tremendous.
[0049] すなわち、これまでの診断方法は、 HIV-1抗体を検出していたことから、その診断の 感度及び特異性は必ずしも十分なものとは 、えな 、ものであつたが、本発明が提供 するハイプリドーマが産生する抗体を使用する検出方法並びにキットにより、ウィルス 本体を検出することが可能となり、 HIV-1感染をより確実に診断することができる。  [0049] That is, since the conventional diagnostic methods have detected HIV-1 antibody, the sensitivity and specificity of the diagnosis are not necessarily sufficient. The detection method and kit using the antibody produced by the provided hyperidoma makes it possible to detect the virus itself and to diagnose HIV-1 infection more reliably.
[0050] また、本発明が提供するハイプリドーマが産生する抗体を、 HIV-1感染の有無を診 断するサンドイッチ ELISAキットに使用し、 HIV-1感染診断用キットを安価に、かつ大 量に製造することが可能となる。したがって、発展途上国、あるいはエイズの発生が 恒常化している国においても、この難病であるエイズ感染をより簡便に診察し、治療 の一助としうる利点を有して 、る。  [0050] In addition, the antibody produced by the hyperidoma provided by the present invention is used in a sandwich ELISA kit for diagnosing the presence or absence of HIV-1 infection, so that the kit for diagnosing HIV-1 infection can be made inexpensively and in a large amount. It can be manufactured. Therefore, even in developing countries or countries where the incidence of AIDS is constant, there is an advantage that it is possible to more easily diagnose AIDS infection, which is an intractable disease, and to help with treatment.
図面の簡単な説明  Brief Description of Drawings
[0051] [図 1]図 1は、実施例 3における本発明のキットを実際に使用して HIV-1 p24アツセィ を行った結果を示した写真である  [0051] FIG. 1 is a photograph showing the results of performing HIV-1 p24 assembly using the kit of the present invention in Example 3 in practice.
[0052] HTLV-I (Human T- Lymphotoropic Virus - I)は成人 T細胞白血病（ATL)の原因ゥ ィルスです。 HTLV-Iは 1980年に発見されたウィルスで、ヒトの癌の原因ウィルスとし て初めて明らかになったものです。比較的日本人に多ぐ国内でも地域差があり九州 地方に多くみられます。潜伏期が長ぐ発症の年齢は平均 50才代です。 [0052] HTLV-I (Human T-Lymphotoropic Virus-I) is the causative virus of adult T-cell leukemia (ATL). HTLV-I was discovered in 1980 and was the first to reveal human cancer causing virus. There are regional differences in Japan, which is relatively large among Japanese people, and it is common in the Kyushu region. The age of onset with a long incubation period is on average 50s.
日本では約 120万人のウィルス保有者がおり、このうち発症するのは年間に約 0.1% と非常に少ない割合です  There are about 1.2 million virus holders in Japan, of which only about 0.1% per year is very small.
Η-ρ24  Η-ρ24

Claims

請求の範囲 The scope of the claims
[1] 血中に存在する抗マウスタンパク抗体の非特異的反応を抑制し、増殖期のヒト免疫 不全ウィルス (HIV-1)に発現する p24抗原 (HIV-1 p24)を認識して結合する抗体 (抗 HIV-1 p24抗体)を用いることにより、反応感度を 10〜30倍に増強させる ELISA法に よるヒト血清、ヒト血清由来血液製剤または輸血用血清から、 HIV-1の検出する方法、 及び血清中のヒト由来の p24抗体を検出する方法。  [1] Suppresses nonspecific reaction of anti-mouse protein antibody present in blood, recognizes and binds to p24 antigen (HIV-1 p24) expressed in proliferating human immunodeficiency virus (HIV-1) A method for detecting HIV-1 from human serum, human serum-derived blood products or blood transfusion serum by ELISA, which uses an antibody (anti-HIV-1 p24 antibody) to enhance reaction sensitivity by 10 to 30 times; And a method for detecting human-derived p24 antibody in serum.
[2] 血中に存在する抗マウスタンパク抗体の非特異的反応の抑制を、非特異的マウス I gGを産生するハイブリドーマによりヒト血中に存在する抗マウスタンパク抗体の測定の 阻害を抑制することにより行う請求項 1に記載の HIV-1を検出する方法。  [2] Suppression of non-specific reaction of anti-mouse protein antibody present in blood, and inhibition of measurement of anti-mouse protein antibody present in human blood by hybridoma producing non-specific mouse IgG The method for detecting HIV-1 according to claim 1, which is carried out by:
[3] HIV-1 p24を認識して結合する抗 HIV- 1 p24抗体が、マウスに不活性ィ匕 HIV- 1を接 種し得られた HIV-1に対する免疫能を有するマウス脾臓細胞と、マウスミエローマ細 胞を細胞融合し、得られた融合細胞力 感染細胞をスクリーニングし、さらにクロー二 ングして得られたノ、イブリドーマ力も産生されたものであることを特徴とする請求項 1ま たは 2に記載の HIV-1を検出する方法。  [3] An anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is a mouse spleen cell that has immunogenicity against HIV-1 obtained by inoculating mice with inactive HIV-1; The fusion cell force obtained by cell fusion of mouse myeloma cells, the infected cells are screened, and further cloned and cloned, and the strength of the hybridoma is also produced. The method for detecting HIV-1 according to 2.
[4] HIV-1 p24を認識して結合する抗 HIV-1 p24抗体が、 HIV-1 p24を捕獲する捕獲抗 体である請求項 1、 2または 3に記載の HIV-1を検出する方法。  [4] The method for detecting HIV-1 according to claim 1, 2 or 3, wherein the anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is a capture antibody that captures HIV-1 p24. .
[5] HIV-1 p24を認識して結合する抗 HIV-1 p24抗体が、 HIV-1 p24を検出する検出抗 体である請求項 1、 2または 3に記載の HIV-1を検出する方法。  [5] The method for detecting HIV-1 according to claim 1, 2 or 3, wherein the anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is a detection antibody that detects HIV-1 p24. .
[6] HIV-1 p24を捕獲する捕獲抗体が RT-N-48抗体である請求項 4に記載の HIV-1を 検出する方法。  6. The method for detecting HIV-1 according to claim 4, wherein the capture antibody that captures HIV-1 p24 is an RT-N-48 antibody.
[7] HIV-1 p24を検出する検出抗体が RT-C- 50抗体である請求項 5に記載の HIV-1を 検出する方法。  [7] The method for detecting HIV-1 according to claim 5, wherein the detection antibody for detecting HIV-1 p24 is an RT-C-50 antibody.
[8] ELISA法によるヒト血清、ヒト血清由来血液製剤または輸血用血清から HIV-1を検出 するため、及び血清中のヒト由来の p24抗体を検出するためのキットであって、増殖期 の HIV-1に発現する p24抗原（HIV-1 p24)を認識して結合する抗体 (抗 HIV-1 p24抗 体)を用いることを特徴とする HIV-1 p24抗原'抗体検出用 ELISAキット。  [8] A kit for detecting HIV-1 from human serum, human serum-derived blood products or blood transfusion serum by ELISA, and for detecting human-derived p24 antibody in the serum, which is in the proliferative phase An ELISA kit for detecting an HIV-1 p24 antigen 'antibody, comprising using an antibody (anti-HIV-1 p24 antibody) that recognizes and binds to a p24 antigen (HIV-1 p24) expressed in -1.
[9] HIV-1 p24を認識して結合する抗 HIV- 1 p24抗体が、マウスに不活性ィ匕 HIV- 1を接 種し得られた HIV-1に対する免疫能を有するマウス脾臓細胞と、マウスミエローマ細 胞を細胞融合し、得られた融合細胞力 感染細胞をスクリーニングし、さらにクロー二 ングして得られたノ、イブリドーマ力も産生されたものであることを特徴とする請求項 8 に記載の HIV-1 p24抗原'抗体検出用 ELISAキット。 [9] An anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is a mouse spleen cell that has immunogenicity against HIV-1 obtained by inoculating mice with inactive HIV-1; Mouse myeloma thin The cell fusion of the vesicles, the obtained fused cell force, the infected cells were screened, and further cloned and cloned, and the hybridoma force was also produced. 1 ELISA kit for detecting p24 antigen 'antibody.
[10] HIV-1 p24を認識して結合する抗 HIV-1 p24抗体が、 HIV-1 p24を捕獲する捕獲抗 体である請求項 8または 9に記載の HIV-1 p24抗原'抗体検出用 ELISAキット。 [10] The anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is a capture antibody that captures HIV-1 p24. ELISA kit.
[11] HIV-1 p24を認識して結合する抗 HIV-1 p24抗体が、 HIV-1 p24を検出する検出抗 体である請求項 8または 9に記載の HIV-1 p24抗原'抗体検出用 ELISAキット。 [11] The anti-HIV-1 p24 antibody that recognizes and binds to HIV-1 p24 is a detection antibody that detects HIV-1 p24. ELISA kit.
[12] HIV-1 p24を捕獲する捕獲抗体が RT-N-48抗体である請求項 10に記載の HIV-1 p[12] The HIV-1 p of claim 10, wherein the capture antibody that captures HIV-1 p24 is an RT-N-48 antibody.
24抗原 ·抗体検出用 ELISAキット。 24 antigen · ELISA kit for antibody detection.
[13] HIV-1 p24を検出する検出抗体が RT-C- 50抗体である請求項 11に記載の HIV-1 p[13] The HIV-1 p according to claim 11, wherein the detection antibody for detecting HIV-1 p24 is an RT-C-50 antibody.
24抗原 ·抗体検出用 ELISAキット。 24 antigen · ELISA kit for antibody detection.
[14] HIV-1 p24を検出する検出抗体である RT-C-50抗体が、ペルォキシダーゼ（POD) 標識抗体である請求項 13に記載の HIV-1 p24抗原 ·抗体検出用 ELISAキット。 [14] The ELISA kit for detecting HIV-1 p24 antigen / antibody according to claim 13, wherein the RT-C-50 antibody which is a detection antibody for detecting HIV-1 p24 is a peroxidase (POD) -labeled antibody.
PCT/JP2006/322535 2006-11-13 2006-11-13 Method for detecting hiv-1 without being affected by anti-mouse protein antibody existing in human and kit to be used for the same WO2008059553A1 (en)

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CN102608312A (en) * 2012-02-28 2012-07-25 李洪 Blood serum or blood plasma diluting solution applied to detection method of trace gelatin particle agglutinated HIV-1 (Human Immunodeficiency Virus-1) recent infection
JP2015532589A (en) * 2012-07-13 2015-11-12 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア Method for evaluating the suitability of transduced T cells for administration

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