WO2008059553A1 - Procédé de détection du vih-1 sans être affecté par l'anticorps de la protéine anti-souris existant chez l'homme et trousse destinée à une utilisation dans ce procédé - Google Patents

Procédé de détection du vih-1 sans être affecté par l'anticorps de la protéine anti-souris existant chez l'homme et trousse destinée à une utilisation dans ce procédé Download PDF

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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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English (en)
Japanese (ja)
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Masami Moriyama
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Masami Moriyama
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Publication of WO2008059553A1 publication Critical patent/WO2008059553A1/fr

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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

L'invention concerne un procédé fiable de détection du VIH-1 pouvant être utilisé pour le diagnostic d'une infection au VIH-1 de manière simple et fiable, et une trousse destinée à une utilisation dans ce procédé. Le procédé de détection du VIH-1 dans du sérum humain, une préparation de sang dérivée de sérum humain ou du sérum pour transfusion par un procédé ELISA sont caractérisés par l'utilisation d'un anticorps (anticorps anti-VIH-1 p24) qui reconnaît et se lie à l'antigène p24 (VIH-1 p24) exprimé dans le virus de l'immunodéficience humaine (VIH-1) dans la phase de croissance sans être affecté par un anticorps de protéine anti-souris existant dans le sang humain. Cette invention concerne aussi un procédé de détection d'un anticorps p24 d'origine humaine dans le sérum et un kit ELISA destiné à la détection d'un antigène/anticorps VIH-1 p24 à utiliser dans le procédé.
PCT/JP2006/322535 2006-11-13 2006-11-13 Procédé de détection du vih-1 sans être affecté par l'anticorps de la protéine anti-souris existant chez l'homme et trousse destinée à une utilisation dans ce procédé WO2008059553A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
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CN102608312A (zh) * 2012-02-28 2012-07-25 李洪 一种应用于微量明胶颗粒凝集hiv-1近期感染检测法的血清或血浆样品稀释液
JP2015532589A (ja) * 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 投与に関する形質導入ｔ細胞の適合性の評価方法

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JP2004536568A (ja) * 2000-12-06 2004-12-09 アボット・ラボラトリーズ ヒト免疫不全ウイルスに対するモノクローナル抗体およびその用途

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EP0345461B1 (fr) * 1988-06-10 1994-09-28 Abbott Laboratories Anticorps monoclonaux de souris contre VIH-IP24 et leur utilisation dans des tests de diagnostic
JP2004536568A (ja) * 2000-12-06 2004-12-09 アボット・ラボラトリーズ ヒト免疫不全ウイルスに対するモノクローナル抗体およびその用途

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DI MARZO V.F. ET AL.: "Monoclonal Antibodies Specific for p24, the Major Core Protein for Human T-Cell Leukemia Virus Type III", PROC. NATL. ACAD. SCI. USA, vol. 82, 1985, pages 5199 - 5202, XP003024472 *
LEE K.-B. ET AL.: "The use of nanoarrays for highly sensitive and selective detection of human immunodeficiency virs type 1 in plasma", NANO LETTERS, vol. 4, no. 10, 2004, pages 1869 - 1872, XP003024471 *
LEE S.K. ET AL.: "Relative Epitope Mapping of Monoclonal Antibodies against HIV Gag p24 for Sandwich ELISA of HIV-1 Detection", MOL. CELLS, vol. 3, 1993, pages 43 - 46, XP003024469 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102608312A (zh) * 2012-02-28 2012-07-25 李洪 一种应用于微量明胶颗粒凝集hiv-1近期感染检测法的血清或血浆样品稀释液
JP2015532589A (ja) * 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 投与に関する形質導入ｔ細胞の適合性の評価方法

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