WO1995000545A1 - Peptides synthetiques pour la detection d'anticorps contre le virus de l'hepatite c dans le serum de personnes infectees - Google Patents

Peptides synthetiques pour la detection d'anticorps contre le virus de l'hepatite c dans le serum de personnes infectees Download PDF

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Publication number
WO1995000545A1
WO1995000545A1 PCT/ES1993/000051 ES9300051W WO9500545A1 WO 1995000545 A1 WO1995000545 A1 WO 1995000545A1 ES 9300051 W ES9300051 W ES 9300051W WO 9500545 A1 WO9500545 A1 WO 9500545A1
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Prior art keywords
peptides
peptide
resin
arg
amino
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PCT/ES1993/000051
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English (en)
Spanish (es)
Inventor
Carmen Berasain Lasarte
José Ignacio RIEZU BOJ
Jesús PRIETO VALTUEÑA
Francisco Borras Cuesta
Original Assignee
Instituto Cientifico Y Tecnologico De Navarra, S.A.
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Priority to ES09101525A priority Critical patent/ES2051152B1/es
Application filed by Instituto Cientifico Y Tecnologico De Navarra, S.A. filed Critical Instituto Cientifico Y Tecnologico De Navarra, S.A.
Priority to PCT/ES1993/000051 priority patent/WO1995000545A1/fr
Priority to AU43277/93A priority patent/AU4327793A/en
Publication of WO1995000545A1 publication Critical patent/WO1995000545A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to synthetic peptides that are useful as detection agents, in the serum of infected persons, of antibodies against the hepatitis C virus, as well as to methods of synthesis of such peptides.
  • the invention also encompasses an improved test for the diagnosis of high-sensitivity hepatitis C and a test kit or set of test components, an expression that corresponds to what in English is called a "test kit", intended for carrying out the cited improved trial.
  • Hepatitis C virus is a single-stranded and positive polar RNA virus, with a diameter of 50-60 nm, provided with envelope, related to the Flaviviridae family that encompasses the genera Pestivirus and Flavivirus (Miller, R and Purcell, R. Proc Nati Acad Sci USA 87: 2057-2061 (1990)).
  • the genome has 5 'and 3' non-coding ends and a region of around 9,400 nucleotides that encodes a polyprotein that is subsequently processed to give rise to the different proteins of the virus that by homology with those of Flavivirus and Pestivirus have been classified as structural (core, El, E2 / NS1) and non-structural (NS2, NS3, NS4 and NS5).
  • diagnostic test 01 allowed clarifying the etiology of a large part of NANB hepatitis.
  • a low sensitivity of the assay in acute hepatitis has been described, because the anti-c100 antibodies detected by this assay appear late in the infection (Alter, H., Purcell, R., Shih, J. , Melpolder, J., ASCP, M., Houghton, M., Choo, Q. and Kuo, G. N Engl J Med. 321: 1494-500 (1989)) (Courouce, A., Jullien, A., Vedrenne, J. and Habibi, B. Vox Sang. 58: 226-7 (1990)).
  • trial 01 is responsible for false positive results in patients with anti-SOD antibodies.
  • patients with anti-SOD antibodies Ikeda, Y., Toda, G., Hashimoto, N. and Kurokawa, K. Lancet. 335: 1345-6 (1990)
  • patients with chronic autoimmune active hepatitis McFarlane, I., Smith, H., Johnson, P., Bray, G., Vergani, D. and Williams, R. Lancet. 335: 754-7 (1990)
  • paraproteinemia Bodart, D., Lucas, J., Muller, J. , Le Carrer, D., Planchón, B.
  • Recombinant proteins because of their size, have the advantage of being able to contain a large number of epitopes potentially recognizable by the antibodies present in people infected with HCV. But for the same reason, they are also more likely to contain epitopes with sequence homology with those present in other agents, so that cross-reactions with non-induced antibodies to HCV can occur.
  • the applicant's laboratory has a equipment that allows the simultaneous synthesis of 96 different peptides in amounts of the order of 2 to 3 mg of each peptide (F. Borras Cuesta, Spanish patent application N2 P9000764, filed on March 14, 1990).
  • This equipment in the course of the works that have led to the present invention, more than 300 peptides 15 amino acids long have been synthesized, representing different regions of all proteins encoded by the HCV genome.
  • These peptides have been synthesized based on different sequences: HCV-1 sequence (Houghton, M., Choo, Q. and Kuo, G. European patent application. 88: 310,922: 5; Publication 318,216.
  • the ELISA test basically consists of the following: (1) adsorb the peptides in the bottom of wells of special plastic plates; (2) sequester those immunoglobulins present in the serum that specifically recognize the peptide; (3) reacting human immunoglobulins, sequestered by the peptides, with an anti-human anti-human body that is biotinylated and with streptavidin-peroxidase; (4) develop a color reaction by adding a substrate that reacts with peroxidase.
  • P an assay, which will be called P, based on the detection of antibodies against the mixture of the 3 peptides.
  • the results obtained were compared with tests P, 01, 02 and an unmarketed prototype developed by The Wellcome (W) laboratories, which present recombinant proteins from the nucleocapsid and NS5 region, in 3 sera panels: sera from healthy controls, sera from patients with chronic NANB hepatitis and sera from patients with hepatitis B.
  • HCV positive HCV positive
  • the developed test P has a high sensitivity, much higher than that of test 01 and similar to that of test 02.
  • the developed test P has a significantly higher specificity than that of tests 01 and 02 which they present a high number of false positives in patients with chronic hepatitis B, as already described in other studies (Nakatsuji, Y., Matsumoto, A., Tanaka, E., Ogata, H. and Kiyosawa, K. Hepatol. 16: 300-305 (1992)).
  • each trunk (1 ⁇ g per well of each of the 3 individual trunks and 3 ⁇ g per well of the trunk containing all 3 peptides) was treated with PBS and centrifuged, the supernatant being used in adsorption.
  • the two types of peptide derivatives were adsorbed to ELISA plates as follows: a first group of plates was incubated with a solution in sodium carbonate containing 1 ⁇ g / well of each of the 3 peptides with sequences 1 to 3 previously transcribed, bound to random BSA. A The second group was incubated with a solution in sodium carbonate containing 1 ⁇ g / well of each of the 3 peptides of said BSA-linked sequences through its carboxyl terminus.
  • the P and HP tests have a very good specificity, significantly higher (p ⁇ 0.001) than that of tests 01 and 02. As mentioned above, the low specificity of both tests is explained by the existence of false positive results in the sera panel of patients with chronic hepatitis B. These false positives are the result of the use of large recombinant proteins that contain homologous epitopes inside those existing in other antigens.
  • the P and HP assays are based on 3 short peptides that have been selected for not presenting cross reactions.
  • test P significantly higher than that of test 01, has been optimized with the use of heteropolymers (HP);
  • HP test a sensitivity similar to that of test 02 is obtained, with a significantly higher specificity.
  • the increase in sensitivity obtained with the HP assay may be due to the fact that the 3 peptides would be present in the heteropolymer forming different combinations, of different sizes, so that the competition problems of the 3 peptides in adsorption to the plaque and would ensure the presence of repeated epitopes several times.
  • the existence of combinations of different lengths, and the random binding of the peptides through their amino or carboxyl terminus, would ensure the proper presentation of each of the peptides.
  • the HP test also has a higher sensitivity than the P test, in the early or early detection of infection.
  • the fundamental aspect of this invention is in using, in an assay for the diagnosis of hepatitis C, synthetic peptides whose general formula is as follows: R 1 -aa 1 -aa 2 -aa 3 -aa 4 -aa 5 -aa 6 -aa 7 -aa 8 -aa 9 -aa 10 -aa 11 -aa 12 -aa 13 -aa 14 ⁇
  • R 1 and R 2 are polypeptide chains of 15 amino acids, represented by the above formula, may be equal to each other, or different, and repeated n times, the formula represents homopolymers or heteropolymers.
  • aa 4 represents Ser or Arg;
  • aa 5 represents Arg, Pro or Lys
  • aa 6 represents Gly, Gln or Thr
  • aa 7 represents Asn, Asp or Lys
  • aa 10 represents Ser, Phe or Thr
  • aa 11 represents Pro or Asn
  • aa 12 represents Thr, Gly or Arg
  • aa 1 , aa 2 , aa 3 , aa 8 , aa 13 , aa 14 and aa 15 each represent some of the 20 possible natural amino acids.
  • the detection of antibodies against hepatitis C virus in the serum of infected individuals can be carried out by ELISA assays or other type of immunological assay, using each of the 3 peptides separately, either in its simple form, or as a homopolymer; using mixtures containing the different combinations of the 3 peptides either in their simple form or as homopolymers; or using heteropolymers containing all 3 peptides.
  • These peptides can also be used attached to a resin or to a macromolecule (BSA, lysine logs, etc.) that allows "presentation" for an ELISA or for flow cytofluorometry.
  • peptides can also be used to induce antibodies for the detection of hepatitis C virus, or its fragments, in human or animal biological tissues or liquids.
  • the peptides described above were experimentally chosen from more than 300 synthetic peptides representing different regions of the viral genome.
  • the sarcosine functionalized polyamide gel resin is treated with ethylenediamine for about 10 hours. It is then washed with abundant dimethylformamide (DMF) and the resin is reacted with an activated arm (linkage agent in the Anglo-Saxon literature).
  • the activated arm may be the 4-hydroxy-methyl-phenoxyacetic acid pentafluorophenyl ester (for common peptides) or the 4-hydroxy-methyl-methoxy-phenoxyacetic acid pentafluorophenyl ester (to prepare peptides intended to obtain polymers or to bind them to trunks of plants or to BSA through their carboxyl end).
  • the first amino acid in the chain is attached to the arm in the resin through an ester bond.
  • the symmetric anhydride of the first amino acid is prepared, which has the ⁇ -amino group protected with the fluorenyl-methyl-oxycarbonyl group (Fmoc) and any other side chain group capable of reacting, protected with a group compatible with Fmoc technology, reacting, for each equivalent of resin used, 2 equivalents of the Fmoc-amino acid with an equivalent of carbodiimide.
  • the binding of the first amino acid of the synthesis to the binding arm is one of the delicate stages and it needs to be controlled by amino acid analysis in order to ensure that the binding took place in good yield. If the multiple synthesis apparatus is used, and in order to avoid the large amount of amino acid analysis that would be necessary, it is preferable to add an amino acid (such as valine for example) to an important amount of resin, then aliquot this resin and use it as a starting material for each of the syntheses. This has the disadvantage that an additional foreign amino acid is introduced. However, in the applicant's experience, this does not usually affect the biological activity of the peptides. If you want to avoid this procedure, it is possible to buy different batches of resin with the first amino acid already incorporated.
  • an amino acid such as valine for example
  • dimethyl-amino-pyridine which acts as a catalyst and is allowed to react with the resin between 80 and 100 minutes. At the end of the reaction, wash with plenty of DMF. To ensure the entry of the first amino acid, the reaction of adding the symmetric anhydride is repeated. After the DMF washes, the Fmoc group is removed with 20% piperidine in DMF. This operation exposes the ⁇ -amino group of the first amino acid to react with the carboxyl group of the second amino acid.
  • the other amino acids in the sequence are added, one by one, using the method of either symmetric anhydrides or active esters.
  • Symmetric anhydrides are prepared from conveniently protected amino acids, activating them with dicyclohexyl carbodiimide in dichloromethane (DCM).
  • DCM dichloromethane
  • the dicyclohexyl urea that is formed is removed by filtration, the dichloromethane is removed in a rotary evaporator and the residue (symmetric anhydride) is dissolved in DMF and added to the resin. It is reacted for 60 to 90 minutes until the ninhydrin test shows absence of free amino groups; otherwise this stage is repeated.
  • active esters In the case of using active esters, they are dissolved directly in DMF together with the same molar amount of 1-hydroxy-benzotriazole (HOBT) and added to the reaction mixture.
  • HOBT 1-hydroxy-benzotriazole
  • the ninhydrin test is performed at the end of each amino acid binding reaction (either by means of symmetric anhydrides or active esters) at the end of each amino acid binding reaction. If this is negative, the Fmoc protective group is removed with 20% piperidine in DMF in order to add the following amino acid. This process is repeated for the successive amino acids of the sequence, whereby the polypeptide chain grows from the C-terminal to the N-terminal.
  • the first amino acid of the synthesis (the C-terminal amino acid) is attached to the arm forming an ester bond that is labile acid. For this reason it is possible to selectively cut the resin peptide, under conditions of complete stability for the peptide bond.
  • TFA trifluoroacetic acid
  • the use as a 1% TFA acid cutting solvent in DCM allows the cutting of the resin peptide without deprotecting the groups of the side chains, so that the peptides can be reacted to form polymers or to bind to lysine logs, to BSA, etc.
  • the reaction time of the cutting mixtures with the peptides depends on the amino acid composition of the peptides, being 6 hours per arginine. present in the sequence.
  • the TFA acid is evaporated in a rotary evaporator and the peptide is precipitated with ether.
  • the peptides are purified by high pressure liquid chromatography (HPLC), using a C18 column of solvent-balanced reverse phase (0.1% w / v TFA acid in water) and making a linear gradient, up to 70%, of a solution of acetonitrile containing 0.1% w / v of TFA acid.
  • HPLC high pressure liquid chromatography
  • the peptides were synthesized according to the synthesis protocol described above using 4-hydroxymethylmethoxyphenoxyacetic acid as the binding arm to the resin.
  • This arm allows the resin peptide to be cut without deprotecting the groups of the side chains, by using mild cutting mixtures (1% trifluoroacetic acid (TFA) in dichloromethane (DCM).
  • TFA trifluoroacetic acid
  • DCM dichloromethane
  • 5 ⁇ moles of each peptide was dissolved in 200 ⁇ l of dimethylformamide (DMF).
  • the pH was adjusted to 8-9 with N-ethyl morpholine and 5 ⁇ moles of dicyclohexylcar were added.
  • heteropolymer 2.5 ⁇ moles of each peptide was dissolved in 200 ⁇ l of DMF. The pH was adjusted to 8-9 with N-ethyl morpholine and 7.5 ⁇ moles of DCC and 7.5 ⁇ moles of HOBT in 100 ⁇ l of DMF were added. The reaction mixtures were allowed to stir for 115 hours.
  • Fmoc-L-Ala-PepSyn KA Resin, Millipore Lysines were incorporated in the form of symmetric anhydride.
  • As an amino acid Fmoc-L-Lys (Fmoc) -OH (Neosystem) was used. Incorporation of the plant: for each equivalent of resin, 12 equivalents of Fmoc-L-Lys (Fmoc) -OH were reacted with 6 equivalents of DCC, in a concentration not exceeding 0.1 M in DCM. It was allowed to react for 15 minutes.
  • the DCM was evaporated in a rotary evaporator, the corresponding volume of DMF was added and filtered in vacuo to remove the DCU.
  • the anhydride was added to the reactor and allowed to react for an hour and a half. The washes and deprotections were done as described in the manual synthesis section.
  • the incorporation of a peptide into the lysine trunk was done by adding 24 equivalents of peptide, 24 equivalents of DCC, 24 equivalents of HOBT and 24 equivalents of N-ethyl-morpholine per equivalent of resin, in 300 ⁇ l of DMF.
  • 24 equivalents of DCC, 24 equivalents of HOBT and 24 equivalents of N-ethyl-morpholine per equivalent of resin in 300 ⁇ l of DMF. It was allowed to react with stirring for 115 hours. After the reaction time, 10 DMF washes were performed and the ninhydrin test was carried out to verify that the peptides had bound.
  • the cutting of the resin logs and the deprotection of the side groups of the peptides was done by incubation under stirring for 65 hours with the corresponding cutting mixture (5% phenol, 95% TFA w / v). After cutting, the TFA was evaporated on the rotary evaporator, precipitated with ether and lyophilized.
  • the peptides were reacted with BSA in a 30: 1 molar ratio, in the presence of 10 mg / ml of l-ethyl-3- (3 dimethylaminopropyl) carbodiimide (Sigma), in a 1: 1 DMF / water solution .
  • the peptides were dissolved in 0.5 ml of DMF and the pH was adjusted to 8-9 by the addition of N-ethyl-morpholine.
  • Carbodiimide was added, dissolved and the solution was mixed with an equal volume of BSA in water. It was allowed to react under stirring for 20 hours.
  • the peptide and BSA reaction mixtures were diluted to 10 ml with distilled water, dialyzed against water for 24 hours to remove unbound peptide and lyophilized. Peptides bound to BSA "ad libitum" were stored frozen for direct use in the ELISA. Peptides bound to BSA by the carboxyl terminus were treated to deprotect the amino group.
  • the lyophilisate of the peptides bound to BSA by the carboxyl terminus was treated with 1 ml of 20% piperidine in DMF for 15 minutes, then diluted with 10 ml of distilled water, dialyzed for 24 hours and lyophilized. Subsequently, the groups of the side chains were checked out.
  • Lyophilisates obtained after removal of the Fmoc group (deprotection of the amino group) were treated with 10 ml of the 5% phenol, 95% TFA (w / v) mixture for 6 hours for each arginine present in its sequence.
  • the TFA was evaporated in the rotary evaporator and lyophilized. Lyophilisates were used in ELISA assays.
  • ABTS 2,2'-azinobis-3-ethylbenzothiazolin-6-sulfonic acid
  • a serum was considered to have antibodies against a peptide, only when the ELISA result was positive for the two serum dilutions studied (1/20 and 1/100). That is, when, at each dilution, the absorbance corresponding to this peptide was a minimum of 2 times higher than the average optical density of the 30 peptides with lower reading.
  • the antigens are adsorbed to ELISA plates by adding 200 ⁇ l of a 0.1 M sodium carbonate solution containing 1 ⁇ g of each peptide (for the P test), or 0.1 ⁇ g of the heteropolymer (for the HP test) and incubating for 2 hours at room temperature or overnight at 4 ° C. The wells are washed 3 times, after incubation, with PBST. To each well of the ELISA plate 200 ⁇ l of PLT and 10 ⁇ l of serum are added. 2 wells for a positive control serum and 3 wells for a negative control serum are included. It is allowed to incubate for 45 minutes at 37 ° C.
  • the wells are emptied and 3 washes are performed with enough PBST in order to fill the wells. It is then incubated for 30 minutes at 37 [ deg.] C. with 200 ⁇ l of a 1/2000 solution of biotinylated goat anti-human IgG complete antibody and 1/500 streptavidin-peroxidase in PBST.
  • the wells are emptied, washed 3 times with PBST and then 100 ⁇ l of a substrate solution (0.8 mg / ml o-phenylenediamine (OPD) in 0.05 M phosphate citrate buffer with 0.014% peroxide is added of hydrogen, pH 5). It is allowed to incubate at room temperature for 15 minutes.
  • a substrate solution 0.8 mg / ml o-phenylenediamine (OPD) in 0.05 M phosphate citrate buffer with 0.014% peroxide is added of hydrogen, pH 5
  • reaction is stopped by adding 75 ⁇ l of 4 N sulfuric acid. It is read at 492 nm in a Titertek Multiskan PLUS MK II reader or equivalent. Sera whose absorbance is higher than the average of the 3 negative controls plus 0.4 are considered positive.
  • the invention is illustrated below with the
  • Example 1 The results of tests 01 were compared,
  • the results of trials 01, 02, W, P and HP were compared in a panel of 98 sera from patients with Chronic hepatitis B.
  • the 2nd generation RIBA of Chiron and PCR were performed in sera with discordant results between trials.
  • the criteria followed to designate a serum as HCV positive or negative were the following:
  • HCV positive HCV positive
  • the results obtained with the P test are shown, with the heteropolymer, the peptides bound to BSA by the carboxyl terminal end, the sum of homopolymers, the peptides bound to BSA "at random", the sum of individual trunks and the trunk with the mix of the three peptides
  • This case basically consists of the following components:

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Abstract

Sont décrits des peptides capables de détecter des anticorps antivirus de l'hépatite C (VHC) dans le sérum d'individus infectés, ces peptides ayant la formule générale: R1-aa1-aa2-aa3-aa4-aa5-aa6-aa7-aa8-aa9-aa10-aa11-aa12-aa13-aa14-aa15-R2, dans laquelle, si R1 est H et R2 est OH, alors la formule représente un pentadécapeptide dans lequel les aminoacides à terminaison N et à terminaison C sont respectivement aa1 et aa15, et si R1 et R2 sont des chaînes polypeptidiques de 15 aminoacides, représentées par la formule antérieure, pouvant être identiques ou différentes, et répétées n fois, la formule représente alors des homopolymères ou des hétéropolymères. Dans la formule, aa4 représente Ser ou Arg; aa5 représente Arg, Pro ou Lys; aa6 représente Gly, Gln ou Phr; aa7 représente Asn, Asp ou Lys; aa10 représente Ser, Phe ou Phr; aa11 représente Pro ou Asn; aa12 représente Phr, Gly ou Arg; et aa1, aa2, aa3, aa8, aa13, aa14 et aa15 représentent chacun l'un des 20 aminoacides naturels possibles. Sont également décrits des procédés de synthèse de ces peptides. Les peptides et les polymères peuvent s'utiliser individuellement ou dans des mélanges afin de détecter au moyen de dosages ELISA ou d'autres types de dosages immunologiques la présence d'anticorps anti-VHC dans le sérum de personnes infectées. Ces peptides peuvent également s'utiliser unis à une résine ou à une macromolécule (BSA, troncs de lysines, etc.) qui permette la 'présentation' pour un dosage ELISA ou pour une cytofluorimétrie de flux. Ces peptides peuvent également s'utiliser pour induire des anticorps afin de détecter le virus de l'hépatite C, ou ses fragments, dans des tissus ou des liquides biologiques humains ou animaux.
PCT/ES1993/000051 1991-03-20 1993-06-18 Peptides synthetiques pour la detection d'anticorps contre le virus de l'hepatite c dans le serum de personnes infectees WO1995000545A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
ES09101525A ES2051152B1 (es) 1991-03-20 1991-06-28 Procedimiento de sistesis de peptidos con capacidad de detectar anticuerpos anti-virus de la hepatitis c (vhc) en suero de individuos afectados.
PCT/ES1993/000051 WO1995000545A1 (fr) 1991-03-20 1993-06-18 Peptides synthetiques pour la detection d'anticorps contre le virus de l'hepatite c dans le serum de personnes infectees
AU43277/93A AU4327793A (en) 1991-03-20 1993-06-18 Synthetic peptides for the detection of antibodies against the virus of hepatitis c in the serum of infected persons

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB919105871A GB9105871D0 (en) 1991-03-20 1991-03-20 Synthetic peptides
PCT/ES1993/000051 WO1995000545A1 (fr) 1991-03-20 1993-06-18 Peptides synthetiques pour la detection d'anticorps contre le virus de l'hepatite c dans le serum de personnes infectees

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5670310A (en) * 1994-07-29 1997-09-23 The United States Of America As Represented By The Department Of Health And Human Services Methods and compositions for differential diagnosis of acute and chronic hepatitis c virus infection

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0484787A2 (fr) * 1990-11-03 1992-05-13 BEHRINGWERKE Aktiengesellschaft Peptides spécifiques pour HCV, procédé pour les synthétiser et leur emploi
WO1992012171A1 (fr) * 1990-12-27 1992-07-23 Instituto Cientifico Y Tecnologico De Navarra, S.A. Nouveaux peptides synthetiques avec activite imunomodulatrice et leur preparation
WO1992012992A2 (fr) * 1991-01-14 1992-08-06 James N. Gamble Institute Of Medical Research Polypeptides immunogeniques structuraux de base ayant des epitopes pour vhc, anticorps, sequences de polynucleotides, vaccins et methodes
WO1992017493A2 (fr) * 1991-04-05 1992-10-15 Biochem Pharma Inc. Peptides et melanges de peptides servant a detecter les anticorps diriges contre le virus de l'hepatite c
WO1993006488A1 (fr) * 1991-09-16 1993-04-01 Genelabs Technologies, Inc. Dosages immunologiques virus de l'hepatite c, a base de peptides

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0484787A2 (fr) * 1990-11-03 1992-05-13 BEHRINGWERKE Aktiengesellschaft Peptides spécifiques pour HCV, procédé pour les synthétiser et leur emploi
WO1992012171A1 (fr) * 1990-12-27 1992-07-23 Instituto Cientifico Y Tecnologico De Navarra, S.A. Nouveaux peptides synthetiques avec activite imunomodulatrice et leur preparation
WO1992012992A2 (fr) * 1991-01-14 1992-08-06 James N. Gamble Institute Of Medical Research Polypeptides immunogeniques structuraux de base ayant des epitopes pour vhc, anticorps, sequences de polynucleotides, vaccins et methodes
WO1992017493A2 (fr) * 1991-04-05 1992-10-15 Biochem Pharma Inc. Peptides et melanges de peptides servant a detecter les anticorps diriges contre le virus de l'hepatite c
WO1993006488A1 (fr) * 1991-09-16 1993-04-01 Genelabs Technologies, Inc. Dosages immunologiques virus de l'hepatite c, a base de peptides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BERASAIN, C. ET AL: "Detection of anti-hepatitis C virus antibodies by Elisa using synthetic peptides", JOURNAL OF HEPATOLOGY, vol. 18, no. 1, April 1993 (1993-04-01), AMSTERDAM, pages 80 - 84 *
TAM, J.P. ET AL: "Multiple antigen peptide", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 124, 1989, AMSTERDAM, pages 53 - 61 *

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ES2051152B1 (es) 1994-12-16
GB9105871D0 (en) 1991-05-08
AU4327793A (en) 1995-01-17

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