FR2775690A1 - Monoclonal antibodies useful for detecting and/or quantifying hepatitis C virus core protein - Google Patents
Monoclonal antibodies useful for detecting and/or quantifying hepatitis C virus core protein Download PDFInfo
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- FR2775690A1 FR2775690A1 FR9803087A FR9803087A FR2775690A1 FR 2775690 A1 FR2775690 A1 FR 2775690A1 FR 9803087 A FR9803087 A FR 9803087A FR 9803087 A FR9803087 A FR 9803087A FR 2775690 A1 FR2775690 A1 FR 2775690A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
- C07K16/109—Hepatitis C virus; Hepatitis G virus
-
- 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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Communicable Diseases (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
La présente invention concerne l'obtention d'anticorps dirigés contre des déterminants antigéniques de l'extrémité N-terminale de la protéine Core du virus de l'hépatite C (VHC), et l'utilisation de ceux-ci notamment pour le diagnostic de la maladie à un stade précoce, et pour le suivi de la charge virale, chez les malades chroniques. The present invention relates to obtaining antibodies directed against antigenic determinants of the N-terminal end of the hepatitis C virus (HCV) Core protein, and the use thereof especially for the diagnosis of early stage disease, and viral load monitoring in chronic patients.
Conformément au document EP-O 569 309, au nom de la Demanderesse, on connaît un peptide s'étendant depuis l'acide aminé en position 2 jusqu'à l'acide aminé en position 45 de l'extrémité N-terminale de la protéine Core du VHC, protéine dont la séquence est identifiée par
SEQ ID NO: 1. Ce peptide détermine une région immunodominante qui suffit, à elle-seule, pour obtenir les mêmes sensibilité et spécificité en terme de détection des anticorps dirigés contre le VHC, qu'avec la protéine Core dans son entier.According to EP-0 569 309, in the name of the Applicant, there is known a peptide extending from the amino acid in position 2 to the amino acid at position 45 of the N-terminus of the protein Core of HCV, protein whose sequence is identified by
SEQ ID NO: 1. This peptide determines an immunodominant region which alone is sufficient to achieve the same sensitivity and specificity in terms of detection of antibodies against HCV as with the entire Core protein.
A la suite de ces travaux, la Demanderesse a maintenant déterminé des anticorps capables de reconnaître l'antigène naturel de la protéine Core du VHC. As a result of this work, the Applicant has now determined antibodies capable of recognizing the natural antigen of the HCV core protein.
Selon la présente invention, on a obtenu des anticorps monoclonaux ou fragments fonctionnels d'anticorps monoclonaux, susceptibles de se lier spécifiquement, pour former un complexe immun, à un composé antigénique de la protéine Core du virus de l'hépatite C, dont l'antigénicité ne résulte que de la présence, dans sa séquence peptidique, d'un ou plusieurs déterminants antigéniques identiques, que la Demanderesse a caractérisés. According to the present invention, monoclonal antibodies or functional fragments of monoclonal antibodies, capable of binding specifically to form an immune complex, to an antigenic compound of the core protein of hepatitis C virus, have been obtained. Antigenicity results only from the presence, in its peptide sequence, of one or more identical antigenic determinants, which the Applicant has characterized.
Lesdits déterminants antigéniques sont définis par l'une quelconque des séquences suivantes : celle commençant à l'acide aminé 18 et se terminant à l'acide aminé 25 de ladite protéine Core, celle commençant à l'acide aminé 29 et se terminant à l'acide aminé 36 de ladite protéine, et celle commençant à l'acide aminé 57 et se terminant à l'acide aminé 64 de ladite protéine. Said antigenic determinants are defined by any of the following sequences: that starting at amino acid 18 and terminating at the amino acid of said Core protein, that starting at amino acid 29 and terminating at amino acid 36 of said protein, and that starting at amino acid 57 and terminating at amino acid 64 of said protein.
Des composés antigéniques répondant à la définition énoncée ci-dessus sont notamment ceux dont la séquence peptidique comprend une séquence choisie parmi
SEQ ID NO: 2, SEQ ID NO: 3 et SEQ ID NO: 4, et de préférence ceux dont la séquence peptidique est choisie parmi SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 et
SEQ ID NO: 8.Antigenic compounds as defined above are in particular those whose peptide sequence comprises a sequence chosen from
SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and preferably those whose peptide sequence is selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and
SEQ ID NO: 8.
Par fragment fonctionnel d'un anticorps, on entend toute partie d'un anticorps comprenant ou consistant en au moins un site de combinaison antigénique, permettant audit fragment de se lier à au moins un déterminant antigénique d'un composé antigénique. A titre d'exemple, on peut citer le fragment F(ab)2 d'un anticorps. By functional fragment of an antibody is meant any part of an antibody comprising or consisting of at least one antigenic combination site, allowing said fragment to bind to at least one antigenic determinant of an antigenic compound. By way of example, mention may be made of the F (ab) 2 fragment of an antibody.
Un anticorps monoclonal de l'invention peut en particulier être obtenu par fusion entre la lignée de myélomes SP2/O-Agl-14 et des splénocytes murins de l'espèce Balb/c et de la souche JYco, immunisés par une protéine recombinante de la protéine Core du VHC. A monoclonal antibody of the invention may in particular be obtained by melting between the SP2 / O-Agl-14 myeloma line and murine splenocytes of the Balb / c species and the JYco strain, immunized with a recombinant protein of the Core protein of HCV.
La protéine recombinante utilisée pour obtenir l'anticorps monoclonal est préférentiellement choisie parmi la C22-GST120 constituée par la séquence commençant à l'acide aminé 1 et se terminant à l'acide aminé 120 de la protéine Core couplée, à l'extrémité N-terminale, à la glutathione S-transférase < GST), et la C22-120-Histidine constituée par la séquence commençant à l'acide aminé 1 et se terminant à l'acide aminé 120 de la protéine Core couplée, à l'extrémité N-terminale, à une séquence de 6 résidus histidine. The recombinant protein used to obtain the monoclonal antibody is preferably chosen from C22-GST120 consisting of the sequence starting at amino acid 1 and terminating at amino acid 120 of the coupled core protein, at the N-terminus. terminally, with glutathione S-transferase (GST), and C22-120-Histidine consisting of the sequence starting at amino acid 1 and terminating at amino acid 120 of the coupled core protein, at the N-terminus -terminal, to a sequence of 6 histidine residues.
L'invention a aussi pour objet les applications des anticorps ou fragments définis ci-dessus. The invention also relates to the applications of the antibodies or fragments defined above.
Ainsi un anticorps ou un fragment d'anticorps présente une utilité pour détecter et/ou quantifier des antigènes de la protéine Core du VHC, dans un échantillon biologique. Conformément à une telle application, on utilise au moins undit anticorps et/ou fragment, et avantageusement au moins deux anticorps et/ou fragments, susceptibles de se lier spécifiquement à des déterminants antigéniques respectivement différents. Thus, an antibody or an antibody fragment has utility for detecting and / or quantifying antigens of the HCV core protein in a biological sample. According to such an application, at least one antibody and / or fragment is used, and advantageously at least two antibodies and / or fragments capable of binding specifically to different antigenic determinants respectively.
L'invention concerne aussi un procédé pour détecter et/ou quantifier des antigènes de la protéine
Core du VHC, dans un échantillon biologique, selon lequel
- on dispose d'un premier et d'un second anticorps et/ou fragments de l'invention, le premier anticorps ou fragment étant susceptible de se lier à un premier composé antigénique comprenant un premier déterminant antigénique ou plus, le second anticorps ou fragment étant susceptible de se lier à un second composé antigénique comprenant un second déterminant antigénique ou plus, et ledit premier déterminant antigénique étant différent du second déterminant antigénique,
- on met l'échantillon biologique en contact avec les premier et second anticorps et/ou fragments précités, et
- on détecte, directement ou indirectement, la formation des complexes immuns formés respectivement entre le premier anticorps ou fragment et le premier déterminant antigénique d'une part, et entre le second anticorps ou fragment et le second déterminant antigénique d'autre part.The invention also relates to a method for detecting and / or quantifying antigens of the protein
Core of HCV, in a biological sample, according to which
a first and a second antibody and / or fragments of the invention are available, the first antibody or fragment being capable of binding to a first antigenic compound comprising a first or more antigenic determinant, the second antibody or fragment being capable of binding to a second antigenic compound comprising a second or more antigenic determinant, and said first antigenic determinant being different from the second antigenic determinant,
the biological sample is brought into contact with the first and second antibodies and / or fragments mentioned above, and
the formation of the immune complexes formed respectively between the first antibody or fragment and the first antigenic determinant on the one hand, and between the second antibody or fragment and the second antigenic determinant, on the other hand, is detected, directly or indirectly.
Avantageusement, on traite l'échantillon biologique par un agent dénaturant, avant la détection, et de préférence, avant sa mise en contact avec les premier et second anticorps et/ou fragments. Cet agent dénaturant pourra notamment être un détergent, une solution acide. Advantageously, the biological sample is treated with a denaturing agent, prior to detection, and preferably before it is brought into contact with the first and second antibodies and / or fragments. This denaturing agent may especially be a detergent, an acid solution.
Selon la technique de détection employée, notamment immunoenzymatique, le premier anticorps ou fragment peut être fixé sur un support solide et/ou le second anticorps ou fragment peut être marqué. Depending on the detection technique used, in particular immunoenzymatic, the first antibody or fragment can be fixed on a solid support and / or the second antibody or fragment can be labeled.
Comme cela sera illustré dans l'Exemple ci-après, on utilisera avantageusement la technique "sandwich" dans laquelle le premier anticorps ou fragment est fixé sur un support solide et le second anticorps ou fragment est marqué. As will be illustrated in the Example below, the "sandwich" technique in which the first antibody or fragment is fixed on a solid support and the second antibody or fragment is advantageously used.
Divers supports et marqueurs peuvent être envisagés selon l'invention. Ils sont bien connus de l'homme du métier. Various supports and markers may be envisaged according to the invention. They are well known to those skilled in the art.
A titre d'exemple, le support peut être sous la forme d'une plaque, telle qu'une plaque de microtitration, d'une feuille, d'un cône, d'un puits, d'une barrette, par exemple de polystyrène, ou d'un copolymère à base de styrène, d'un tube de verre ou analogue. By way of example, the support may be in the form of a plate, such as a microtiter plate, a sheet, a cone, a well, a bar, for example polystyrene , or a styrene-based copolymer, a glass tube or the like.
Le marqueur peut être toute molécule biologique ou chimique, et peut par exemple être choisi parmi les isotopes radioactifs, des enzymes, en particulier des enzymes susceptibles d'agir sur un substrat chromogène, fluorigène ou luminescent (notamment une peroxydase ou une phosphatase alcaline), des composés chimiques chromophores, des composés chromogènes, fluorigènes ou luminescents, des analogues de bases nucléotidiques, et des ligands tels que la biotine. The marker may be any biological or chemical molecule, and may for example be chosen from radioactive isotopes, enzymes, in particular enzymes capable of acting on a chromogenic, fluorogenic or luminescent substrate (in particular a peroxidase or an alkaline phosphatase), chromophoric chemical compounds, chromogenic, fluorogenic or luminescent compounds, nucleotide base analogs, and ligands such as biotin.
De préférence, le support solide est une plaque de microtitration, et le marqueur est la biotine. Preferably, the solid support is a microtiter plate, and the label is biotin.
La Demanderesse a déterminé des couples préférés d'anticorps pour mettre en pratique le procédé de détection de l'invention. The Applicant has determined preferred pairs of antibodies to practice the detection method of the invention.
Ainsi, les couples d'anticorps préférés sont ceux qui comprennent à titre de premier anticorps ou fragment, un anticorps ou fragment susceptible de se lier spécifiquement au déterminant antigénique défini par
SEQ ID NO: 2 ou SEQ ID NO: 4, et à titre de second anticorps ou fragment, un anticorps ou fragment susceptible de se lier spécifiquement au déterminant antigénique défini par SEQ ID NO: 3 ; le premier ou second anticorps ou fragment servant à la capture ou à la détection, de préférence, le premier anticorps ou fragment servant à la capture, le second anticorps ou fragment servant à la détection. Thus, the preferred antibody pairs are those which comprise as the first antibody or fragment, an antibody or fragment capable of binding specifically to the antigenic determinant defined by
SEQ ID NO: 2 or SEQ ID NO: 4, and as the second antibody or fragment, an antibody or fragment capable of binding specifically to the antigenic determinant defined by SEQ ID NO: 3; the first or second antibody or fragment for capture or detection, preferably the first antibody or fragment for capture, the second antibody or fragment for detection.
D'autres objets de l'invention sont définis ci après
- un réactif pour la détection et/ou quantification des antigènes de la protéine Core du VHC, comprenant au moins deux anticorps et/ou fragments de l'invention, et susceptibles de se lier spécifiquement à des déterminants antigéniques différents ; et
- un kit de détection et/ou quantification des antigènes de la protéine Core du VHC, comprenant un réactif tel que défini ci-dessus, un support et un marqueur ; dans ce kit l'un des anticorps et/ou fragments peut avoir été préalablement fixé sur le support et/ou l'autre avoir été marqué.Other objects of the invention are defined below
a reagent for the detection and / or quantification of antigens of the HCV core protein, comprising at least two antibodies and / or fragments of the invention, and capable of binding specifically to different antigenic determinants; and
a kit for detecting and / or quantifying antigens of the HCV core protein, comprising a reagent as defined above, a support and a marker; in this kit one of the antibodies and / or fragments may have been previously fixed on the support and / or the other have been labeled.
Les différents objets de l'invention sont illustrés par les exemples 1 à 3 ci-après, à l'appui de l'unique figure qui représente l'influence du traitement d'un échantillon de sérum sur la formation des complexes immuns pour deux couples d'anticorps de l'invention, à savoir le couple d'anticorps constitué d'un premier anticorps reconnaissant SEQ ID NO: 2 et d'un second anticorps reconnaissant SEQ ID NO: 3 (+), et le couple d'anticorps constitué d'un premier anticorps reconnaissant
SEQ ID NO: 4 et d'un second anticorps reconnaissant
SEQ ID NO: 3 (N). The various objects of the invention are illustrated by Examples 1 to 3 below, in support of the single figure which represents the influence of the treatment of a serum sample on the formation of immune complexes for two couples. of the invention, namely the antibody pair consisting of a first antibody recognizing SEQ ID NO: 2 and a second antibody recognizing SEQ ID NO: 3 (+), and the pair of antibodies constituted by of a first recognizing antibody
SEQ ID NO: 4 and a second recognizing antibody
SEQ ID NO: 3 (N).
EXEMPLE 1: Clonage. expression et Durification des protéines recombinantes Core
L'ARN du VHC a été extrait à partir du sérum d'un patient ayant une hépatite non A non B et 1'ADN complémentaire a été obtenu par RT-PCR d'après Li et al., 1992. L'ADNc d'une partie du gène de la protéine Core située dans la région codante du VHC de génotype la (acides aminés 1-120) a été amplifié par PCR en utilisant des amorces nucléotidiques correspondant respectivement aux extrémités 5' et 3' du gène de la protéine Core 120 plus respectivement les sites BamH1 and EcoR1. Les produits d'amplification ont été ensuite clonés dans les plasmides d'expression pGEX-3 (Pharmacia, France) et pET 21b (Novagen, UK) digérés par les enzymes de restriction BamH1 and EcoR1. Les plasmides résultants, pGEx-Core and pET-Core, codent chacun pour la protéine Core de 120 acides aminés fusionnée soit à la glutathione-Stransférase (GST) (Smith and Johnson, 1988) soit à un peptide composé de 6 résidus histidine. Ces deux protéines recombinantes ont été respectivement appelées C22-GST 120 et M120-His.EXAMPLE 1 Cloning Core expression and hardening of recombinant proteins
HCV RNA was extracted from the serum of a patient with non-A non-B hepatitis and the complementary DNA was obtained by RT-PCR according to Li et al., 1992. The cDNA of a portion of the Core protein gene located in the genotype 1a (HCV 1-120) coding region was amplified by PCR using nucleotide primers corresponding to the 5 'and 3' ends of the Core protein gene, respectively 120 plus BamH1 and EcoR1 sites respectively. The amplification products were then cloned into the expression plasmids pGEX-3 (Pharmacia, France) and pET 21b (Novagen, UK) digested with the BamH1 and EcoR1 restriction enzymes. The resulting plasmids, pGEx-Core and pET-Core, each encode the 120 amino acid core protein fused to either glutathione-transferase (GST) (Smith and Johnson, 1988) or a peptide composed of 6 histidine residues. These two recombinant proteins were respectively called C22-GST 120 and M120-His.
Les protéines recombinantes ont été ensuite exprimées dans la souche B1 21(DE3) d'Escherichia coli après induction par ss-D-thiogalactopyranoside (IPTG,
Gibco-BRL).The recombinant proteins were then expressed in Escherichia coli strain B1 21 (DE3) after induction by ss-D-thiogalactopyranoside (IPTG,
Gibco-BRL).
La protéine C22-GST 120 a été ensuite purifiée sur une colonne Gluthatione Sepharose 4B (Pharmacia, France) tandis que la protéine M120-His a été purifiée par chromatographie d'affinité par métal-chelate (Ni-NTA
Resin, Quiagen, France).The C22-GST 120 protein was then purified on a Gluthatione Sepharose 4B column (Pharmacia, France) while the M120-His protein was purified by metal-chelate affinity chromatography (Ni-NTA).
Resin, Quiagen, France).
L'homogénéité des protéines purifiées a été caractérisée par SDS-PAGE et leur immunoréactivité analysée par Western blot à l'aide d'un lot de sérums positifs vis-à-vis de VHC, d'un sérum polyclonal anti-GST de lapin ou anticorps anti hexa-histidine (Dianova,
France).The homogeneity of the purified proteins was characterized by SDS-PAGE and their immunoreactivity analyzed by Western blot using a batch of HCV-positive sera, a rabbit anti-GST polyclonal serum or anti-hexa-histidine antibody (Dianova,
La France).
EXEMPLE 2 : Préparation d'anticorPs de l'invention
Des souris femelles Balb/c de la souche JYco âgées de 4 à 6 semaines (IFFA Credo, Les Oncins, l'Arbresle,
France) ont été immunisées par voie intrapéritonéale avec 10 Ag de la protéine recombinante C22-GST 120 émulsifiée avec un volume égal d'adjuvant complet de Freund. Six injections d'immunogène ont été ensuite effectuées toutes les deux semaines en utilisant de l'adjuvant incomplet de
Freund. Quatre jours après la dernière injection, les cellules spléniques des souris récupérées et fusionnées avec des cellules du myélome de souris Sp2/O-Agl-14 suivant la technique de Kohler et Milstein (1975, 1976).EXAMPLE 2 Preparation of AnticorPs of the Invention
Balb / c female mice of the strain JYco aged 4-6 weeks (IFFA Credo, Oncins, Arbresle,
France) were immunized intraperitoneally with 10 Ag of the recombinant protein C22-GST 120 emulsified with an equal volume of complete Freund's adjuvant. Six immunogen injections were then performed every two weeks using incomplete adjuvant
Freund. Four days after the last injection, the spleen cells of the mice recovered and fused with Sp2 / O-Agl-14 mouse myeloma cells according to the technique of Kohler and Milstein (1975, 1976).
Douze à quatorze jours plus tard, les surnageants de culture ont été analysés par un test ELISA où l'antigène ayant servi pour l'immunisation des souris est déposé au fond des puits. Les colonies positives sont sous clonées deux fois par dilution limitante.Twelve to fourteen days later, the culture supernatants were analyzed by an ELISA test where the antigen used for the immunization of the mice is deposited at the bottom of the wells. Positive colonies are subcloned twice by limiting dilution.
Les ascites ont été obtenus à partir de souris dans lesquelles ont été successivement injectés par voie intrapéritonéale 0,5 ml de Pristane puis 106 hybridomes. Ascites were obtained from mice in which 0.5 ml Pristane and then 106 hybridomas were successively injected intraperitoneally.
Les immunoglobulines G ont été ensuite purifiées sur une colonne de ProteinA-Sepharose 4FF suivant le protocole fourni par le fabricant (Pharmacia, France). Immunoglobulins G were then purified on a ProteinA-Sepharose 4FF column following the protocol provided by the manufacturer (Pharmacia, France).
Les anticorps monoclonaux purifiés ont été biotinylés à l'aide de Sulfo-NHS-LC-Biotin (Merck,
Rockford, Illinois) suivant la méthode de Gretch et al.The purified monoclonal antibodies were biotinylated using Sulfo-NHS-LC-Biotin (Merck,
Rockford, Ill.) Following the method of Gretch et al.
EXEMPLE 3 : Détection d'antigènes de la protéine
Core du vHC dans un échantillon biologique
Le premier anticorps monoclonal est déposé sur la phase solide à la concentration finale de 5 pg/ml en PBS et incubé une nuit à 40C. Les plaques sont encore lavées 3 fois avec du PBS-T puis un conjugué avidine-peroxydase (Jackson ImmunoResearch Laboratories) est ensuite ajouté à la dilution 1:5000 en PBS-T- sérum de chèvre et laissé pendant une heure à 370C. Après avoir lavé une dernière fois les plaques avec du PBS-T, la réaction colorimétrique est obtenue en utilisant le kit commercial de bioMérieux (Color kit) qui contient de l'ortho-phénylène-diamine de l'eau oxygénée. Après 10 min d'incubation, la réaction est stoppée avec de l'acide sulfurique est les plaques sont lues à 492 nm avec un lecteur de plaque d'ELISA. Les valeurs sont exprimées par la moyenne de points expérimentaux effectués en triple.EXAMPLE 3 Detection of antigens of the protein
Core of vHC in a biological sample
The first monoclonal antibody is deposited on the solid phase at the final concentration of 5 μg / ml in PBS and incubated overnight at 40C. The plates are washed 3 more times with PBS-T and then an avidin-peroxidase conjugate (Jackson ImmunoResearch Laboratories) is then added to the 1: 5000 dilution in PBS-T-goat serum and left for one hour at 370C. After washing the plates for one more time with PBS-T, the colorimetric reaction is obtained using the bioMérieux commercial kit (Color kit) which contains ortho-phenylenediamine from hydrogen peroxide. After 10 min of incubation, the reaction is stopped with sulfuric acid and the plates are read at 492 nm with an ELISA plate reader. The values are expressed as the average of experimental points made in triplicate.
La protéine M120-His purifiée (50 pg/ml) a été utilisée dans une gamme étalon pour quantifier l'antigène
Core dans les sérums de malades VHC. Différentes dilutions (de 500ng/ml à 0,5pg/ml) de la protéine ont été effectuées dans du sérum humain négatif et traitées de la même façon que les sérums de patient VHC à tester. La protéine C22 120GST a été aussi testée et a donné des résultats comparables.Purified M120-His protein (50 μg / ml) was used in a standard range to quantify the antigen
Core in the sera of HCV patients. Different dilutions (from 500 ng / ml to 0.5 μg / ml) of the protein were performed in negative human serum and treated in the same way as the HCV patient sera to be tested. C22 120GST protein was also tested and gave comparable results.
Ainsi que le montre la figure, les traitements chimiques effectués sur les sérums affectent peu la sensibilité de détection du couple utilisant l'anticorps dirigé contre la séquence SEQ ID NO: 2 pour la capture de l'antigène tandis qu'ils affectent fortement la sensibilité de détection du couple utilisant l'anticorps dirigé contre la séquence SEQ ID NO: 4 pour la capture de l'antigène. As shown in the figure, the chemical treatments carried out on the sera do not affect the detection sensitivity of the pair using the antibody directed against the sequence SEQ ID NO: 2 for the capture of the antigen while they strongly affect the sensitivity. detection of the pair using the antibody directed against the sequence SEQ ID NO: 4 for the capture of the antigen.
Ainsi le traitement de 5 sérums virémiques par du
Triton x100 à la concentration finale de 1%, et acidification à pH 4, suivis d'une neutralisation après 45 min. a permis une détection d'antigène Core dont les qualités sont montrées dans le tableau suivant.Thus the treatment of 5 viremic sera with
Triton x100 at the final concentration of 1%, and acidification at pH 4, followed by neutralization after 45 min. has allowed detection of Core antigen whose qualities are shown in the following table.
Tableau
<tb> S. <SEP> virémi <SEP> ues <SEP> <SEP> Core <SEP> / <SEP> ml <SEP> sérum
<tb> <SEP> sérum <SEP> 1 <SEP> | <SEP> 2,2 <SEP> ng/ml <SEP>
<tb> <SEP> sérum <SEP> 2 <SEP> 4 <SEP> ng/ml
<tb> <SEP> sérum <SEP> 3 <SEP> 2 <SEP> ng/ml
<tb> <SEP> sérum <SEP> 4 <SEP> 20 <SEP> pg/ml
<tb> <SEP> sérum <SEP> 5 <SEP> 92 <SEP> pg/ml <SEP>
<tb>
LI8TAGE DE SEQUENCES (1) INFORMATIONS GENERALES:
(i) DEPOSANT:
(A) NOM: BIO MERIEUX
(B) RUE: Chemin de l'Orme
(C) VILLE: MARCY L'ETOILE
(E) PAYS: FRANCE
(F) CODE POSTAL: 69280
(ii) TITRE DE L'INVENTION: Anticorps monoclonal et utilisations pour détecter des antigènes de la protéine Core du VHc
(iii) NOMBRE DE SEQUENCES: 8 (2) INFORMATIONS POUR LA SEQ ID NO:1
(i) CARACTERISTIQUES DE LA SEQUENCE:
(A) LONGUEUR: 191 résidus d'acide aminé
(B) TYPE: acides aminés
(ii) TYPE DE MOLECULE: protéine Core du VHC
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO:1 protéine Core (dont la séquence est notamment décrite dans EP 0 569 309) (2) INFORMATIONS POUR LA SEQ ID NO: 2:
(i) CARACTERISTIQUES DE LA SEQUENCE:
(A) LONGUEUR: 8 résidus d'acide aminé
(B) TYPE: acides aminés
(ii) TYPE DE MOLECULE: peptide de l'extrémité N-terminale de la protéine Core du VHC (18-25)
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 2:
Arg Pro Gln Asp Val Lys Phe Pro
1 5 (2) INFORMATIONS POUR LA SEQ ID NO: 3:
(i) CARACTERISTIQUES DE LA SEQUENCE:
(A) LONGUEUR: 8 résidus d'acide aminé
(B) TYPE: acides aminés
(ii) TYPE DE MOLECULE: peptide de l'extrémité N-terminale de la protéine Core du vHC (29-36)
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 3: Gln Ile Val Gly Gly Val Tyr Leu
1 5 (2) INFORMATIONS POUR LA SEQ ID NO: 4:
(i) CARACTERISTIQUES DE LA SEQUENCE:
(A) LONGUEUR: 8 résidus d'acide aminé
(B) TYPE: acides aminés
(ii) TYPE DE MOLECULE: peptide de l'extrémité N-terminale de la protéine Core du vHC (57-64)
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 4:
Pro Arg Gly Arg Arg Gln Pro Ile
1 5 (2) INFORMATIONS POUR LA SEQ ID NO: 5:
(i) CARACTERISTIQUES DE LA SEQUENCE:
(A) LONGUEUR: 44 résidus d'acide aminé
(B) TYPE: acides aminés
(ii) TYPE DE MOLECULE: peptide de l'extrémité N-terminale de la protéine Core du vHC (2-45)
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 5:
Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg
1 5 10 15
Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly
20 25 30
Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly
35 40 (2) INFORMATIONS POUR LA SEQ ID NO: 6:
(i) CARACTERISTIQUES DE LA SEQUENCE:
(A) LONGUEUR: 20 résidus d'acide aminé
(B) TYPE: acides aminés
(ii) TYPE DE MOLECULE: peptide de l'extrémité N-terminale de la protéine Core du VHC (2-21)
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 6:
Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg
1 5 10 15
Arg Pro Gln Asp
20 (2) INFORMATIONS POUR LA SEQ ID NO: 7:
(i) CARACTERISTIQUES DE LA SEQUENCE:
(A) LONGUEUR: 24 résidus d'acide aminé
(B) TYPE: acides aminés
(ii) TYPE DE MOLECULE: peptide de l'extrémité N-terminale de la protéine Core du VHC (22-45)
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 7:
Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu
1 5 10
Pro Arg Arg Gly Pro Arg Leu Gly
20 (2) INFORMATIONS POUR LA SEQ ID NO: 8:
(i) CARACTERISTIQUES DE LA SEQUENCE:
(A) LONGUEUR: 44 résidus d'acide aminé
(B) TYPE: acides aminés
(ii) TYPE DE MOLECULE: peptide de l'extrémité N-terminale de la protéine Core du VHC (38-81)
(xi) DESCRIPTION DE LA SEQUENCE: SEQ ID NO: 8:
Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser
1 5 10 15
Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg
20 25 30
Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr
35 40
BIBLIOGRAPHIE
- Kohler, G, and C. Milstein. 1975. Continuous culture of fused cells secreting antibody of predefined specificity. Nature, 256: 495-497.<tb> S. <SEP> viremia <SEP> ues <SEP><SEP> Core <SEP> / <SEP> ml <SEP> serum
<tb><SEP> Serum <SEP> 1 <SEP> | <SEP> 2.2 <SEP> ng / ml <SEP>
<tb><SEP> serum <SEP> 2 <SEP> 4 <SEP> ng / ml
<tb><SEP> serum <SEP> 3 <SEP> 2 <SEP> ng / ml
<tb><SEP> serum <SEP> 4 <SEP> 20 <SEP> pg / ml
<tb><SEP> serum <SEP> 5 <SEP> 92 <SEP> pg / ml <SEP>
<Tb>
SEQUENCE LINKAGE (1) GENERAL INFORMATION:
(i) DEPOSITOR:
(A) NAME: BIO MERIEUX
(B) STREET: Chemin de l'Orme
(C) CITY: MARCY THE STAR
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 69280
(ii) TITLE OF THE INVENTION: Monoclonal Antibody and Uses for Detecting VHc Core Protein Antigens
(iii) NUMBER OF SEQUENCES: 8 (2) INFORMATION FOR SEQ ID NO: 1
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 191 amino acid residues
(B) TYPE: amino acids
(ii) MOLECULE TYPE: HCV Core Protein
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 Core protein (the sequence of which is especially described in EP 0 569 309) (2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 8 amino acid residues
(B) TYPE: amino acids
(ii) MOLECULE TYPE: N-terminal peptide of the HCV core protein (18-25)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
Arg Pro Gln Val Val Lys Phe Pro
1 (2) INFORMATION FOR SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 8 amino acid residues
(B) TYPE: amino acids
(ii) MOLECULE TYPE: N-terminal peptide of the vHC Core protein (29-36)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: Gln Val Gly Island Gly Val Tyr Leu
1 (2) INFORMATION FOR SEQ ID NO: 4:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 8 amino acid residues
(B) TYPE: amino acids
(ii) MOLECULE TYPE: N-terminal peptide of the vHC Core protein (57-64)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
Pro Arg Gly Arg Arg Gln Pro Ile
(5) INFORMATION FOR SEQ ID NO: 5
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 44 amino acid residues
(B) TYPE: amino acids
(ii) MOLECULE TYPE: N-terminal peptide of the vHC Core protein (2-45)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg
1 5 10 15
Arg Pro Gln Asp Val Lys Phe Gly Glly Gly Gln Val Gly Gly
20 25 30
Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly
40 (2) INFORMATION FOR SEQ ID NO: 6:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 20 amino acid residues
(B) TYPE: amino acids
(ii) MOLECULE TYPE: N-terminal peptide of the HCV core protein (2-21)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn Arg
1 5 10 15
Arg Pro Gln Asp
(2) INFORMATION FOR SEQ ID NO: 7:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 24 amino acid residues
(B) TYPE: amino acids
(ii) MOLECULE TYPE: N-terminal peptide of the HCV core protein (22-45)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:
Val Lys Phe Pro Gly Gly Gly Gln Val Gly Island Gly Val Tyr Leu Leu
1 5 10
Pro Arg Arg Gly Pro Arg Leu Gly
(2) INFORMATION FOR SEQ ID NO: 8:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 44 amino acid residues
(B) TYPE: amino acids
(ii) MOLECULE TYPE: N-terminal peptide of the HCV core protein (38-81)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:
Pro Arg Arg Gly Pro Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5 10 15
Glu Arg Ser Gln Arg Pro Arg Gly Arg Arg Gln Pro Pro Lys Ala Arg
20 25 30
Arg Pro Glu Gly Arg Tr Thr Ala Gln Pro Gly Tyr
35 40
BIBLIOGRAPHY
- Kohler, G, and C. Milstein. 1975. Continuous culture of fused cells secreting antibody of predefined specificity. Nature, 256: 495-497.
- Li, J.S. Vitvitski, S.P. Tong, and C. Trepo. Li, J. S. Vitvitski, S. P. Tong, and C. Trepo.
1992. PCR detection of HCV RNA among French non-A, non-B hepatitis patients. Archives of Virology 4: 234-237.1992. PCR detection of HCV RNA among French non-A, non-B hepatitis patients. Archives of Virology 4: 234-237.
- Kohler, G., and C. Milstein. 1976. Derivation of specific antibody producing tissue, culture and tumor lines by cells fusion. Eur. J. Immunol. 6: 511-519. - Kohler, G., and C. Milstein. 1976. Derivation of a specific antibody producing tissue, culture and tumor lines by fusion cells. Eur. J. Immunol. 6: 511-519.
- Gretch, D.R., M. Suter, and M.F. Stinski. 1987. - Gretch, D.R., M. Suter, and M.F. Stinski. 1987.
The use of biotinylated monoclonal antibodies and steptavidin affinity chromatography to isolate herpes virus hydrophobic proteins or glycoproteins. Anal.The use of biotinylated monoclonal antibodies and steptavidin affinity chromatography to isolate herpes virus hydrophobic proteins or glycoproteins. Anal.
Biochem. 163, 270-277. Biochem. 163, 270-277.
Claims (18)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9803087A FR2775690B1 (en) | 1998-03-09 | 1998-03-09 | MONOCLONAL ANTIBODIES AND USES TO DETECT CORE PROTEIN ANTIGENS OF HCV |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9803087A FR2775690B1 (en) | 1998-03-09 | 1998-03-09 | MONOCLONAL ANTIBODIES AND USES TO DETECT CORE PROTEIN ANTIGENS OF HCV |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| FR2775690A1 true FR2775690A1 (en) | 1999-09-10 |
| FR2775690B1 FR2775690B1 (en) | 2001-12-14 |
Family
ID=9524000
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| FR9803087A Expired - Fee Related FR2775690B1 (en) | 1998-03-09 | 1998-03-09 | MONOCLONAL ANTIBODIES AND USES TO DETECT CORE PROTEIN ANTIGENS OF HCV |
Country Status (1)
| Country | Link |
|---|---|
| FR (1) | FR2775690B1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1083428A2 (en) * | 1999-08-19 | 2001-03-14 | Kyowa Medex Co., Ltd. | Method and reagent for the detection or determination of HCV core antigens |
| EP1308507A2 (en) | 2001-11-05 | 2003-05-07 | Ortho Clinical Diagnostics Inc. | HCV anti-core monoclonal antibodies |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2239245A (en) * | 1989-12-18 | 1991-06-26 | Wellcome Found | Post-transfusional non-A non-B hepatitis viral polypeptides |
| WO1993011158A2 (en) * | 1991-12-06 | 1993-06-10 | Akzo Nobel N.V. | Non-a, non-b peptides |
| WO1993017111A1 (en) * | 1992-02-28 | 1993-09-02 | The Wellcome Foundation Limited | Hepatitis c virus peptides |
| EP0582243A2 (en) * | 1992-08-07 | 1994-02-09 | Roche Diagnostics GmbH | HCV peptide antigens and a method of testing for the hepatitis C virus (HCV) |
| US5443965A (en) * | 1990-04-06 | 1995-08-22 | Genelabs Incorporated | Hepatitis C virus epitopes |
| EP0717104A2 (en) * | 1994-07-12 | 1996-06-19 | The Tokyo Metropolitan Institute Of Medical Science | Immunoassay of non-A, non-B hepatitis virus-related antigens, monoclonal antibodies for use therein, and hybridomas producing the antibodies |
| EP0754704A2 (en) * | 1990-12-14 | 1997-01-22 | Innogenetics N.V. | Synthetic antigens for the detection of antibodies to hepatitis C virus |
| FR2760458A1 (en) * | 1997-03-05 | 1998-09-11 | Bio Merieux | ANTIGENIC STRUCTURAL PEPTIDE, ANTIGENIC AND IMMUNOGENIC COMPOUNDS AND USES FOR THE DETECTION, PREVENTION AND TREATMENT OF HCV INFECTION |
-
1998
- 1998-03-09 FR FR9803087A patent/FR2775690B1/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2239245A (en) * | 1989-12-18 | 1991-06-26 | Wellcome Found | Post-transfusional non-A non-B hepatitis viral polypeptides |
| US5443965A (en) * | 1990-04-06 | 1995-08-22 | Genelabs Incorporated | Hepatitis C virus epitopes |
| EP0754704A2 (en) * | 1990-12-14 | 1997-01-22 | Innogenetics N.V. | Synthetic antigens for the detection of antibodies to hepatitis C virus |
| WO1993011158A2 (en) * | 1991-12-06 | 1993-06-10 | Akzo Nobel N.V. | Non-a, non-b peptides |
| WO1993017111A1 (en) * | 1992-02-28 | 1993-09-02 | The Wellcome Foundation Limited | Hepatitis c virus peptides |
| EP0582243A2 (en) * | 1992-08-07 | 1994-02-09 | Roche Diagnostics GmbH | HCV peptide antigens and a method of testing for the hepatitis C virus (HCV) |
| EP0717104A2 (en) * | 1994-07-12 | 1996-06-19 | The Tokyo Metropolitan Institute Of Medical Science | Immunoassay of non-A, non-B hepatitis virus-related antigens, monoclonal antibodies for use therein, and hybridomas producing the antibodies |
| FR2760458A1 (en) * | 1997-03-05 | 1998-09-11 | Bio Merieux | ANTIGENIC STRUCTURAL PEPTIDE, ANTIGENIC AND IMMUNOGENIC COMPOUNDS AND USES FOR THE DETECTION, PREVENTION AND TREATMENT OF HCV INFECTION |
Non-Patent Citations (1)
| Title |
|---|
| CERINO A ET AL: "A HUMAN MONOCLONAL ANTIBODY SPECIFIC FOR THE N TERMINUS OF THE HEPATITIS C VIRUS NUCLEOCAPSID PROTEIN", JOURNAL OF IMMUNOLOGY, vol. 151, no. 12, 15 December 1993 (1993-12-15), pages 7005 - 7015, XP002048542 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1083428A2 (en) * | 1999-08-19 | 2001-03-14 | Kyowa Medex Co., Ltd. | Method and reagent for the detection or determination of HCV core antigens |
| EP1083428A3 (en) * | 1999-08-19 | 2001-05-16 | Kyowa Medex Co., Ltd. | Method and reagent for the detection or determination of HCV core antigens |
| EP1308507A2 (en) | 2001-11-05 | 2003-05-07 | Ortho Clinical Diagnostics Inc. | HCV anti-core monoclonal antibodies |
| EP1308507A3 (en) * | 2001-11-05 | 2003-10-22 | Ortho Clinical Diagnostics Inc. | HCV anti-core monoclonal antibodies |
| US7049060B2 (en) | 2001-11-05 | 2006-05-23 | Ortho-Clinical Diagnostics, Inc. | HCV anti-core monoclonal antibodies |
| EP1881064A2 (en) * | 2001-11-05 | 2008-01-23 | Ortho-Clinical Diagnostics, Inc. | HCV-Anti-core monoclonal antibodies |
| EP1881064A3 (en) * | 2001-11-05 | 2008-04-09 | Ortho-Clinical Diagnostics, Inc. | HCV-Anti-core monoclonal antibodies |
| EP2186884A3 (en) * | 2001-11-05 | 2010-09-08 | Ortho-Clinical Diagnostics, Inc. | HCV-anti-core monoclonal antibody |
Also Published As
| Publication number | Publication date |
|---|---|
| FR2775690B1 (en) | 2001-12-14 |
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