Classifications

• • 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
• • 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
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
  • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• HIV polypeptide its preparation and use in detection of HIV antibodies.
• the present invention relates to immunochemical detection 5. of human immunodeficiency virus type 1 (HIV-1). Especially the invention relates to a new polypeptide to be used in the detection.
• HAV-1 human immunodeficiency virus type 1
• HIV-1 human immunodeficiency virus type 1
• Myers G. et al.. Human Retroviruses and AIDS A Compilation and analysis of nucleic acid and amino acid sequences. Theoretical and Bio-- physics Group, Los Alamos, 1987, p. 11-53). In most infected individuals antibodies, which are reactive with the envelope proteins gpl20 and gp41, are produced.
• So far detection of antibodies against HIV-1 has been mainly based on: a) whole virus immunoassays , b) gene technologically produced virus protein immuno ⁇ assays, or c) i munoblotting by separating the viral proteins first in SDS-PAGE (either gene technologically produced viral proteins or by cocultivati ⁇ n the HIV-virus) and transferring the separated proteins to nitrocellulose sheet and detecting the separated proteins with HIV-1 antibody-positive sera using conventional immunoblotting techniques.
• test reagents prepared with the inactivated virus can be contaminated with live virus.
• persons who handle the reagents may be subjected to the risk of HIV-1 infection.
• the peptide is highly sensitive and specifically immunoreactive with HIV-1 infected sera.
• the sensitivity and specificity of tests employing this peptide are on the same level as those of tests employing whole virus.
• the new peptide can be prepared conviniently by conventional synthetic methods with no infectious risks. Because of the shortness of the peptide, sequence recombinant technics need not be used.
• the invented peptide is an envelope peptide developed from an "immuno- dominant" epitope region of the transmembrane protein gp41 in HIV-1.
• the env-peptide does not react with antibodies to HIV-2.
• a small synthetic env-peptide SGKLICTTAVPWNAS has been synthesized.
• This peptide is derived from env-residues 599-613 of the transmembrane protein gp41 of HIV-1.
• This epitope is located in a hydrophobic domain of gp41 but, nevertheless, it has turned out to be highly immunogenic epitope in eliciting an immune response to HIV-1. Especially it seems to elicit the primary responses at the early stages of the infection and the antibodies formed persist through the different stages of the infection.
• the peptide is therefore most suitable for the detection 'of HIV-1 infections.
• the detection can be carried out by using any suitable immunochemical technics e.g. enzyme immunoassay (EIA) , radioimmunoassay (RIA) or fluoroimmunoassay (FIA) .
• EIA enzyme immunoassay
• RIA radioimmunoassay
• FFA fluor
• the env-peptide can be coupled to a protein carrier e.g. albumin or transferrin.
• This peptide-conjugate can be immunochemically identified with rabbit antibody to the peptide or with HIV-1 antibody positive human sera.
• the sensitivity and specificity of this test are the same as those of whole virus enzyme-linked immunosorbent assays or whole virus immunoblottings.
• the specificity of the reaction can be confirmed by addition of unconjugated env-peptide to the sample incubation mixture.
• the test results can be made readable with a naked eye.
• env-peptide its immuno ⁇ chemically equivalent homologues can be used.
• These related peptides can be easily detected by polyclonal or monoclonal antibodies or HIV-1 antibody positive sera directed against the env-peptide.
• a related peptide is e.g. SGKLICTAVP- W AS.
• the env-peptide can be synthetized by conventional methods starting from the individual amino acids. Such methods are described e.g. by Barany G., Merrifield B., in The Peptides, ed. by Gross E. and Meienkoffer J. ? Academic Press, New York, 1979, p. 1-284.
• FIG. 1 shows the reactivity of the env-peptide EIA in a reference polulation and in patients with HIV-1 infection.
• Fig. 2 shows reactivity of the env-peptide EIA in different stages of HIV-1 infection.
• Figs. 3A and 3B show comparison of the sensitivity of the env-peptide EIA with whole virus EIA
• Fig. 4 shows immunoblot analysis of HIV-1 proteins and the env-peptide-BSA-conjugates with different antibody samples.
• the env-peptide SGKLICTTAVPWNAS (later EP-peptide) and related peptide SGKLICTAVPWNAS (later RP-peptide) were chemically synthesized on a t-butyloxycarbonyl (BOO- methylbenzyl-cysteine-phenyl-acetamidomethyl (PAM) polystyrene/divinylbenzene resin (Applied Biosystems, Inc.). Benzyl based side chain protections and the alfa-amino group t-butyloxycarbonyl (t-Boc) protections were used in amino acids (Applied Biosystems) .
• the peptides were cleaved from resin and protecting groups were removed by conventional procedures.
• the synthetic peptides were purified with reversed phase high performance liquid chromatography.
• the amino acid sequences were confirmed by automated Ed an degradation with a gas-phase sequencer (Model 470A, Applied Biosystems).
• the purity of the peptides was approximately 99 % based on amino acid sequence data.
• the chemically synthezised EP-peptide and RP-peptide were coupled to a carrier protein e.g. albumin by using hetero- bifunctional cross linkers e.g. -maleimidobenzoic acid N- hydroxysuccinimide ester (MBS) (Liu F.-T., et al.. Bio ⁇ chemistry, 1979, 18:690-697) .
• MBS -maleimidobenzoic acid N- hydroxysuccinimide ester
• the coated plates were incubated for two hours at 37 °C with 100 yl of serum specimens (diluted 1:40 in PBS containing 1 % BSA and 0.02 % Tween 20 (TM)) and the unbound antibodies were removed by three washes (200 ⁇ l) with 0.02 % Tween 20.
• the plates were then reacted for one hour at 37 °C with swine anti-human IgG alkaline phosphatase conjugate (Labsystems Oy) followed by three washes as above and exposure to paranitrophenyl phosphate (Sigma) as substrate.
• the absorbance values were measured at 405 nm using a microtitration plate reader (Multiskan, Labsystems).
• the positivity and negativity of the selected serum samples were studied in two antibody EIA tests, Vironostika HTLV-III (Organon Teknika) and Labsystems whole virus lysate kits, and confirmed by immunoblottning using whole virus antigen strips prepared by Labsystems.
• the reactivity of the sera with each peptide was defined as the reciprocal of antibody dilution at half-saturating serum dilution.
• the EP-peptide and RP-peptide reacted with all antibody-positive sera. The positive reactions could be totally inhibited by addition of the EP-peptide to the incubation mixtures (100 ⁇ g/ml).
• EIU enzyme immuno- assay units
• EP-peptide-BSA conjugate was tested for its immunoreactivity with rabbit anti-BSA antiserum'(Cappel Laboratories). Bound antibodies were detected by rabbit anti-human IgG-peroxidase conjugate (DAKO) and by swine anti-rabbit Ig-peroxidase conjugate (Liu F.-T. et al.. Biochemistry 1979, 18:690-697).
• DAKO rabbit anti-human IgG-peroxidase conjugate
• swine anti-rabbit Ig-peroxidase conjugate Liu F.-T. et al.. Biochemistry 1979, 18:690-697.
• HIV-1-infected H9 cells were attached to glass slides coated with poly-L-lysine (Sigma). The cells were fixed with 3.5 % parafo ⁇ aldehyde (Riedel-de-Haen) in PBS. The fixed cells were permeabilized with 0.05 % saponin (Merck) in PBS and treated with rabbit serum in PBS-saponin. Cells were stained with the unfractionated HIV-1 serum pool or with a HIV-1-negative human serum diluted 1:50 or with anti-peptide Ig fraction (23 g/ml) for 30 min at 37 °C.
• the cells were washed three times PBS-saponin and the bound antibodies were detected by incubating for 30 min at 37 °C with biotinylated sheep anti-human IgG with fluorescein-streptavidin conjugate (Amersham) .
• the coupled EP-peptide (about lUg EP-peptide/5 u g BSA in 100 yi) and also the UP-peptide are absorbed for instance as small spots to a " solid support, for instance to nitro ⁇ cellulose.
• the unabsorbed peptide-co jugate is washed off using for instance 50 mM sodium phosphate pH 7.2 (PBS). Thereafter the solid support is incubated, about 1 h, in a protein solution, for instance albumin (about 5 mg/100 ml) in 50 mM sodium phosphate, pH 7.2, containing 0.2 % Triton- X-100 (TM) .
• the solid support containing the EP- conjugate and the UP-peptide is incubated with the studied serum sample about 1 h.
• the unbound sample is washed off with PBS.
• the absorbed antibodies to the EP-peptide conjugate can then be detected by incubating the ' solid support with anti-human IgG conjugated with an enzyme marker, the presence of which can be determined by addition of the enzyme-substrate.
• an enzyme marker can be used for instance horse-radish-peroxidase. (HRP) , which can be determined for instance by adding o-phenylene diamine.
• the result is determined as positive by visual examination, when the spot containing the EP-conjugate develops a colored reaction while the spot containing the UP-conjugate remains uncolored.
• the result is determined as negative when the spot containing EP-conjugate do not develope a colored reaction or develops equal color as the spot containing UP-conjugate.

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• Proteomics, Peptides & Aminoacids (AREA)
• Virology (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

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• 1987
  • 1987-05-29 FI FI872409A patent/FI872409A0/fi not_active Application Discontinuation
• 1988
  • 1988-05-27 ES ES8801687A patent/ES2009280A6/es not_active Expired
  • 1988-05-30 WO PCT/FI1988/000083 patent/WO1988009339A1/en not_active Application Discontinuation
  • 1988-05-30 EP EP19880904541 patent/EP0317595A1/en not_active Withdrawn
  • 1988-05-30 JP JP50447288A patent/JPH02500590A/ja active Pending
  • 1988-05-30 AU AU17958/88A patent/AU614971B2/en not_active Ceased
• 1989
  • 1989-01-27 DK DK036989A patent/DK36989D0/da not_active Application Discontinuation

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