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WO1993002363A1 - Procede de detection d'anticorps diriges contre le virus de l'hepatite c, et trousses pour sa mise en ×uvre - Google Patents

Procede de detection d'anticorps diriges contre le virus de l'hepatite c, et trousses pour sa mise en ×uvre Download PDF

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Publication number
WO1993002363A1
WO1993002363A1 PCT/IT1992/000082 IT9200082W WO9302363A1 WO 1993002363 A1 WO1993002363 A1 WO 1993002363A1 IT 9200082 W IT9200082 W IT 9200082W WO 9302363 A1 WO9302363 A1 WO 9302363A1
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WO
WIPO (PCT)
Prior art keywords
amino acid
detect
hcv virus
sequence
antibodies
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Ceased
Application number
PCT/IT1992/000082
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English (en)
Inventor
Carlo Rosa
Silvia Griva
Fabrizio Bonelli
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sorin Biomedica SpA
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Sorin Biomedica SpA
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Filing date
Publication date
Application filed by Sorin Biomedica SpA filed Critical Sorin Biomedica SpA
Publication of WO1993002363A1 publication Critical patent/WO1993002363A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis

Definitions

  • This invention relates to a method to detect antibodies against the env protein of the hepatitis C virus and to a kit for the use thereof.
  • this invention relates to a method to detect antibodies against any variant of the envelope (env) protein of the hepatitis C virus (HCV) for employment in the preparation of in vitro diagnostic assays either as marker of HCV infection, or of viral activity in chemical or pharmaceutical preparations, or for identifying new serotypes.
  • env envelope protein of the hepatitis C virus
  • the virus HCV is believed to be responsible for the hepatites classified as non-A/non-B (PT-NANB) (1).
  • PT-NANB non-A/non-B
  • the existence of an etiological agent for NANB hepatitis has been also proved by Alter et al. (2).
  • the virus has been identified as an RNA virus, of positive polarity, and the genome, in the form of cDNA, has been wholly cloned and sequenced. From an analysis of the sequence it turned out that the sequence in question consists of about 10,000 ribonucleotides and forms a single reading frame that potentially codes for a single amino acid chain. This same organization is also present in other viral families such as those of flavivirus and of Pestivirus; however, other structural characteristics make it uncertain to set forth a precise taxonomic position of HCV.
  • HCV hepatitis A
  • the preparation of immunological tests requires the availability of synthetic peptides capable of mimicking the immunological activity of viral antigens.
  • synthetic peptides capable of mimicking the immunological activity of viral antigens.
  • the identification of specific protein portions, denominated epitopes, capable of reacting with antibodies is necessary due to the short length of synthetic peptides.
  • tests which employ just the epitope of the protein are more sensitive and more accurate.
  • RNA viruses are characterized by a high frequency of spontaneous mutation.
  • variable and hypervariable domains have been identified in the sequences corresponding to the surface proteins (5 and EP 004191.82A1), possibly related to viral mechanisms of escaping of the immune response.
  • NANB hepatitis becomes a chronic disease in about 50 % of patients. It is therefore very useful to identify epitopes of surface proteins both for diagnosis and for prognosis purposes.
  • the Authors of this invention have identified variable regions with a high antigenic activity of the amino acid sequence of the env protein, and they have found that such regions correspond to epitopes of said protein.
  • the Authors also have identified some variants of such regions by means of amplification of nucleic acids from serum samples; among such regions, one is coded by a HCV variant not disclosed before.
  • the endemic distribution of the different viral variants of HCV virus makes it necessary to prepare assays able to detect epitopes of the different variants.
  • the Authors have synthesized such epitopes in vitro for immunological assays on serum samples.
  • a so-called "first-generation" assay makes use of a fusion protein of 363 amino acids (protein c100), which is expressed in S.cerevisiae, coded by the genome portion which has been called NS4.
  • protein c100 protein c100
  • the employment of such protein in an indirect ELISA assay allows anti-HCV antibodies to be identified in 80-85 % of chronic PT-NANB hepatites and in 15 X of acute PT-NANB (6).
  • said reaction step of the HCV env protein epitopes, with said antibodies comprises the adhesion of env synthetic peptides to a solid phase, the incubation of said peptides in the sample; and said detection step of said reaction comprises the incubation with a detecting system which is selected from the following group: an enzymatic tracer with incubation with chromogen and measurement of the optical density, a radioisotopic system.
  • said sample is a serum sample, alternatively said sample is a chemical product, and alternatively said sample is a pharmaceutical product of natural origin or recombinant.
  • said epitopes are synthetic peptides which are preferably cyclized between 2 residues od cysteine.
  • Figures 1A, 1B, and 1C represent the hydrophilic profiles respectively of the env 1, env 2 and env 3 variants.
  • the variant env 1 and env 2 are comprised in viral variants respectively known by those skilled in the art as HCV A1 (american isolate) and HCV J1 (japan isolate).
  • the variant env 3 is coded by a viral variant which is not included in any HCV isolate disclosed up to the present invention, denominated HCV 3.
  • Such variant differentiates mainly by the insertion of a histidine residue into a region delimited by 2 cysteines, which modifies the hydrophilic profile of the genie product ( Figures 1A, 1B and 1C).
  • Such modification is of particular relevance for the analogy with the transmembrane region of the HIV1 surface protein (8, 9).
  • Oligopeptides comprising respectively the sequence of the env protein from the amino acid 13 to the amino acid 32 of the SEQ ID N1 (the env 1 variant); the sequence of the env protein from the amino acid 13 to the amino acid 32 of SEQ ID N2 (the env 2 variant); the sequence of the env protein from the amino acid 13 to the amino acid 33 of SEQ ID N3 (the env 3 variant) are synthesized according to Merrifield's method (10), employing as the solid phase a polyamide resin "Pepsin K polyamide Kieselguliz" (Milligen, Novato, California), which had been previously functionalized with ethilendiamine and with 4-(alpha-Fmoc-amino- 2',4'-dimethoxybenzyl)phenoxyacetic acid.
  • Merrifield's method 10
  • the amino acids employed for the synthesis are protected on the side chains by tert-butyl groups and on the alpha-amino position with the F-moc group (9-fluoro-methyloxycarbonyl group).
  • the guanidinium group of arginine and the imidazole group of histidine is respectively protected with the substituents consisting of the 2,2,5,7,8-pentamethylchroman- 6-sulfonyl and trityl groups.
  • the carboxy group of the amino acids employed is activated by the formation of an ester-type bond with the pentafluorophenyl group.
  • the synthesis is performed with the Milligen 9050 synthesizer (Novato, California) employing the continuous flow method.
  • the removal of protection and the separation of the peptides from the resin are carried out by treatment with trifluoroacetic acid.
  • the peptide sequence is checked with an automatic microsequencer (Portan Instruments).
  • Oligopeptides comprising respectively the sequence of the env protein from the amino acid 21 to the amino acid 30 of the SEQ ID N1 (the env l variant); the sequence of the env protein from the amino acid 21 to the amino acid 30 of SEQ ID N2 (the env 2 variant); the sequence of the env protein from the amino acid 21 to the amino acid 31 of SEQ ID N3 (the env 3 variant) are synthesized according to Example 1.
  • the cyclization of a fraction of the peptides is carried out in the following way: the peptide is dissolved in water to a concentration of 0.1 mg/ml. The pH value is adjusted to 7 with 1M NH 4 OH. Potassium ferricyanide is then added slowly to the solution (400 mg K 3 Fe(CN) 6 in 200 ml of water) till persistence of the yellow colour. The disappearance of the free SH groups is obtained employing the method of Edman (11).
  • the peptide is dissolved at 0.2 mg/ml in distilled/deionized water (Milliq) and the pH is adjusted to pH 8 using a solution of 3M NH 4 Cl. The solution is allowed to stir for four days and the loss of the free sulphide groups is monitored using the
  • the cyclic and linear peptides are dissolved in 50 mM carbonate buffer, pH 9.6 at a concentration of 5 ⁇ g/ml.
  • 200 ⁇ l/well of a microtitration plate is dispensed and incubated for 1 hr at 37°C.
  • the overcoating of the wells is performed by coating to the empty wells 300 ⁇ l of a solution containing 50 mM Tris-HCl pH 7.4 and 0.2% bovine serum albumin (BSA, Sigma, Fraction V). The plates are incubated for 2 hrs at room temperature. Finally 300 ⁇ l/well of a solution containing 10% sucrose, 4% polyvinylpirrolidone and 9% NaCl is added and left for 1 hr at room temperature.
  • the ELISA assay is performed by dispensing 200 ⁇ l/well of sera, previously diluted, using a HCV negative serum, as sample diluent. The samples are incubated for 1 hr at 37°C. The plates are then washed five times with a solution containing 0.05% Tween-20, 0.1% BSA in 50mM phosphate buffer pH 7.4 (washing buffer) and incubated for 1 hr at 37°C with 200 ⁇ l of a solution containing goat IgG anti-human IgGs, conjugated with horse radish peroxidase (HRP).
  • HRP horse radish peroxidase
  • the serum utilized (21) belongs to the panel BBI mixed HCV (Boston Biomedica Inc.).
  • the control HCV negative serum gives constantly values lower than 0.002.
  • MOLECULAR TYPE cDNA from genomic RNA
  • EXPERIMENTAL SOURCE genic library from viral isolate
  • CHARACTERISTICS coding for a portion of env protein variant env 1
  • PROPERTY coding sequence
  • Trp Val Ala lie Thr Pro Thr Val Ala Thr
  • SEQUENCE TYPE Nucleotide with corresponding protein LENGTH OF THE SEQUENCE: 153 base pairs
  • MOLECULAR TYPE cDNA from genomic RNA
  • CHARACTERISTICS coding for a portion of env protein env 2 variant
  • PROPERTY coding sequence
  • MOLECULAR TYPE cDNA from genomic RNA
  • CHARACTERISTICS coding for a portion of the env protein env 3 variant
  • PROPERTY coding sequence

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Food Science & Technology (AREA)
  • Communicable Diseases (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Procédé immunologique de détection d'anticorps aptes à réagir avec les épitopes de la protéine env du virus de l'hépatite C (HCV). Lesdits épitopes sont de préférence cyclisés.
PCT/IT1992/000082 1991-07-19 1992-07-16 Procede de detection d'anticorps diriges contre le virus de l'hepatite c, et trousses pour sa mise en ×uvre Ceased WO1993002363A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ITRM91A000547 1991-07-19
ITRM910547A IT1249685B (it) 1991-07-19 1991-07-19 Medoto per la rivelazione di anticorpi contro il virus dell'epatite c e corredi per la sua realizzazione

Publications (1)

Publication Number Publication Date
WO1993002363A1 true WO1993002363A1 (fr) 1993-02-04

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PCT/IT1992/000082 Ceased WO1993002363A1 (fr) 1991-07-19 1992-07-16 Procede de detection d'anticorps diriges contre le virus de l'hepatite c, et trousses pour sa mise en ×uvre

Country Status (3)

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AU (1) AU2370692A (fr)
IT (1) IT1249685B (fr)
WO (1) WO1993002363A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995018382A1 (fr) * 1993-12-27 1995-07-06 Euro-Diagnostica Ab Antigene diagnostique et procede pour diagnostiquer in vitro une infection active causee par le virus de l'hepatite c

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0388232A1 (fr) * 1989-03-17 1990-09-19 Chiron Corporation Diagnostics et vaccins de NANBV

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0388232A1 (fr) * 1989-03-17 1990-09-19 Chiron Corporation Diagnostics et vaccins de NANBV

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CLINICAL CHEMISTRY vol. 37, no. 6, 1 August 1991, WINSTON-SALEM NC USA pages 1024 - 1025 D.E. POLLET ET AL. 'Development of a screening ELISA and a confirmatory assay for hepatitis C antibodies based on synthetic peptides.' *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995018382A1 (fr) * 1993-12-27 1995-07-06 Euro-Diagnostica Ab Antigene diagnostique et procede pour diagnostiquer in vitro une infection active causee par le virus de l'hepatite c

Also Published As

Publication number Publication date
IT1249685B (it) 1995-03-09
AU2370692A (en) 1993-02-23
ITRM910547A1 (it) 1993-01-19
ITRM910547A0 (it) 1991-07-19

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