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WO1993009252A1 - Peptides synthetiques correspondant a des portions du virus vih-2 et procedes d'utilisation de ces peptides dans une analyse amelioree - Google Patents

Peptides synthetiques correspondant a des portions du virus vih-2 et procedes d'utilisation de ces peptides dans une analyse amelioree Download PDF

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Publication number
WO1993009252A1
WO1993009252A1 PCT/US1992/009376 US9209376W WO9309252A1 WO 1993009252 A1 WO1993009252 A1 WO 1993009252A1 US 9209376 W US9209376 W US 9209376W WO 9309252 A1 WO9309252 A1 WO 9309252A1
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WO
WIPO (PCT)
Prior art keywords
peptide
hiv
antibodies
sample
sequence
Prior art date
Application number
PCT/US1992/009376
Other languages
English (en)
Inventor
Dinesh O. Shah
Andrew Schneider
Original Assignee
Baxter Diagnostics Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Baxter Diagnostics Inc. filed Critical Baxter Diagnostics Inc.
Priority to AU30598/92A priority Critical patent/AU657590B2/en
Priority to JP5508647A priority patent/JPH06503843A/ja
Publication of WO1993009252A1 publication Critical patent/WO1993009252A1/fr

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • 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

  • the present invention relates to synthetic peptides
  • HIV-2 is a virus related to HIV-1. Guyader et al.. Nature 326: 662-669 (1987). The complete nucleotide
  • antigenic peptides can have at its N-terminus either a H of the amino terminal NH 2 group of the peptide or an additional amino acid bonded to the amino terminal NH 2 group of the peptide, the
  • a peptide may by its self be antigenic, in coupling the peptide to various solid phases the antigenicity may be effected. This change in antigenicity may result in a solid phase that it not useful for conducting an immunoassay. In conducting an immunoassay the ability of the solid phase immunoreactant to detect HIV-2 antibodies in low concentrations is required, as these antibodies may exist in low concentration in the bodily fluid.
  • the present invention relates to certain synthetic peptides corresponding to a portion of the glyco-protein gp-41 encoded by HIV-2 env gene, having the basic sequence disclosed by Vahlne et al.
  • the improvement discovered by the inventors involves adding a Lysine substantially adjacent to either terminus of the peptide for coupling on to the solid phase and by adding a Glycine between the added Lysine and -Aspartic acid of the Vahlne et al. peptide, if Lysine is added to the N-terminus of the peptide.
  • this peptide modified for handling convenience showed improved sensitivity in an immunoassay.
  • this invention relates to an antigenic'peptide of the formula: (Sequence Id. No:l) wherein the improvement involves adding a Lysine substantially adjacent to either terminus of said peptide and adding a Glycine between the added Lysine and Aspartic acid, if Lysine is added to the N-terminus.
  • this invention relates to an antigenic peptide of the formula: (Sequence Id. No:2) wherein the improvement involves adding a Lysine substantially adjacent to either terminus of said peptide and adding a Glycine between the added Lysine and Aspartic acid if Lysine is added to the N-terminus.
  • the invention can be further defined as peptides having: a sequence of 26 amino acids (Sequence Id. No:3) 27 amino acids having (Sequence Id. No:4) as described in the sequence listing; 25 amino acids (Sequence Id. No:5) ; 26 amino acids (Sequence Id. No:6).
  • peptides can be used for detecting antibodies to HIV-2 in a sample.
  • the method involves contacting the sample with the peptide under conditions such that an immunological complex will form between the peptide and antibodies to HIV-2 present in the sample, if such antibodies are present in the sample, and measuring the formation, if any of the•immunological complex to determine the presence of antibodies to HIV-2 in the sample..
  • the present invention provides improved peptides corresponding to a region of the transmembrane glyco-protein gp-41 of HIV-2 which has been synthesized and tested for immunoreactivity to HIV-2 positive serum samples as shown by Vahlne et al., (U.S. Patent No. 4,812,556 incorporated by reference).
  • the Vahlne et al. peptide has the following amino acid sequence: (Seq. Id. No:l) (Seq. Id.
  • No:2 can have at its N-terminus either a H of the amino * terminal NH 2 group of the peptide or an additional amino acid bonded to the amino terminal NH 2 group of the peptide, the additional amino acid being selected to facilitate coupling of the peptide to a carrier protein and at C-terminus either a OH or NH 2 .
  • Lysine can be incorporated at either end of the peptide for covalent coupling to the solid phase.
  • the Lysine is positioned to be substantially adjacent to either terminus.
  • substantially adjacent means within one or two amino acids of the terminus.
  • the modified peptides may have the amino acid sequences as shown in (Sequence Id. Nos:3-6)
  • Peptides were synthesized in the amide form on a Milligen-Biosearch 9600 model peptide synthesizer using fluorenylmethoxy carbonyl (FMOC) amino protection scheme and 1-3 diisopropyl carbodiimide coupling chemistry.
  • the amide form of the sequence was adopted because it could be expected to more closely mimic the biologically active analogue than the free acid form.
  • Activated amino acids were coupled to a 2,4- dimethoxy benzhydrylamine resin.
  • Peptide synthesis was monitored by ninhydrin analysis for all amino acids except proline for which an Isatin test was performed.
  • the synthesized peptide was cleaved from the resin by Reagent R, which comprises trifluoroacetic acid, thioanisole, ethanedithiol and anisol in a volumetric ratio of 90:5:3:2.
  • Peptides cleaved from resins were purified by high performance liquid chromatography (HPLC) , and characterized by Porton PI 20 90 E Integrated Micro Sequencing system to confirm the correct sequence. Purity was ascertained by HPLC on a reverse phase colu n using a linear gradient ((A) 0.1 trifluoroacetic acid in H J, (B)0.1% trifluoroacetic acid in CH-»CN) of 5% to 60% (B) in 45 minutes. Absorbance was followed at 230 nm. The peptides made by this process have the amino acid sequence as shown in (Sequence Id. No:3).
  • peptides were covalently coupled to carboxyl functionalized magnetic particles according to the following procedure: 1 ml of 2.5% weight/volume approximately 5 ⁇ m paramagnetic particles were separated on a magnetic separator in a 5 ml size disposable sterile cryogenic vial (corning, cat #25708). The supernatant was removed and.the particles resuspended with 1 ml of 70% ethanol for 10 minutes. The particles were then separated as before and supernatant was removed. The particles were resuspended in 1 ml of 0.1 M phosphate buffer, pH 5.5. The particles were separated as before and supernatant was removed. Further washing procedure with phosphate buffer was repeated twice as before and supernatant removed.
  • the covalently coupled peptide particles were then separated on a magnetic separator. Supernatant was removed and the particles were resuspended in isotonic buffered saline with 0.05% Tween 20 detergent. The particles were further separated and resuspended three times in isotonic buffered saline. The coated particles were then resuspended in isotonic buffered saline at final particles concentration of 0.25% weight to volume. It should be noted that the C-terminus can be comprised of either amide or acid groups depending on the desired end use.
  • Peptide were also passively coated onto paramagnetic microparticles according to the following procedure: 1 ml of 2.5% of weight/volume approximately 5 ⁇ m paramagnetic particles were separated on a magnetic separator in a 5 ml size disposable sterile cryogenic vial (corning, cat #25708) . The supernatant was removed and the particles resuspended with 1 ml of 70% ethanol for 10 minutes. The particles were then separated as before and supernatant was removed. The particles were resuspended in 1 ml of 0.1 M CHES buffer (2-(N-Cyclohexylamino) ethansulfonic acid) at pH 9.3. The particles were separated as before and supernatant was removed. Further, washing procedure with CHES buffer was repeated twice as before and supernatant removed.
  • EXAMPLE 1 A paramagnetic particle assay using particles coated with peptide prepared as previously described was performed as follows: Human serum or plasma was diluted 1:100 in well buffer (20% Neonate Calf Serum, 1.06 M Sodium Chloride, 0.03 M Tris-HCL, pH 7.4, 0.018 ° M Phosphate buffer, pH 7.4 + or - 0.3, 0.09% Sodium Azide, and 1.01% NP-40) .
  • the particles in the wells were washed with 100 ⁇ l phosphate buffered 0 saline and Tween-20 (2.06 g sodium phosphate dibasic, 0.318 g sodium phosphate monobasic 0.5 ml Tween-20, 8.76 g sodium chloride, and 1.0 g sodium azide per liter; pH 7.4).
  • the paramagnetic particles were held in the microtiter 5 plate well via a magnetic field applied to the bottom of the plate. Particles were washed in this manner six times.
  • Particles in each well were resuspended in 30 ⁇ l of particles resuspension buffer (4.346 g sodium 0 phosphate dibasic, 0.524 g sodium phosphate monobasic, 8.76 g sodium chloride, and 1 g sodium azide per liter; pH 7.4) 20 ⁇ l of goat antihuman IgG (H+L) conjugated ,9-Galactosidase (conjugate) and diluted 1:2000 in conjugate dilution buffer (0.1 M Tris-HCL pH 7.5, 0.5 M sodium chloride, 5% glycerol, 5.25 mM magnesium chloride, 0.1% sodium azide and 20% neonate calf sera pH 7.5 + or - 0.3) was then added to the wells.
  • particles resuspension buffer 4.346 g sodium 0 phosphate dibasic, 0.524 g sodium phosphate monobasic, 8.76 g sodium chloride, and 1 g sodium azide per liter; pH 7.4
  • 4-methyl-umbelliferyl-3-D-galactoside was added to each well (0.178 MUG, 3.58 g tricine, 5.1 ml dimethyl sulfoxide, 30 ml methyl alcohol, 0.2 g sodium azide, 0.5 ml Tween-20, per liter, pH 8.5).
  • the presence of ,9-galactosidase (ie: conjugate) in the wells triggered the cleavage of MUG to generate a fluorescent cou arin product.
  • This reagent and conjugate were used as a sensitive detection system. Fluorescence (excitation wavelength 400 nm/emmision wavelength 450 nm) was measured at two time intervals (i.e. 2 and 14 minutes) post MUG addition.
  • the difference between the two values was a kinetic measurement of fluorescent product generation and is a direct measurement of conjugate and human IgG/IgM bound to the particles. Fluorescent values were converted to nM coumarin values using various concentrations of coumarin itself and its resultant fluorescence to establish a standard curve.
  • HIV-2 POS HIV-2 POS
  • HIV-2 POS HIV-2 POS
  • HIV-2 POS HIV-2 POS
  • HIV-2 POS HIV-2 POS
  • the modified peptide passively as well as covalently coupled on magnetic particles, show significantly better sensitivity compared to unmodified peptide coated onto particles under identical coating and test conditions. This better sensitivity would allow the detection of HIV-2 antibody in lower concentrations in the patent sample.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • AIDS & HIV (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des peptides synthétiques du virus VIH-2 ainsi qu'un procédé d'utilisation de ces peptides synthétiques dans une immunoanalyse améliorée de détection d'un anticorps contre VIH-2. L'invention concerne également le peptide antigénique ayant la formule (séquence n° 1 ou 2), l'amélioration consistant à ajouter de la lysine à un endroit sensiblement adjacent à la terminaison N ou C du peptide et à ajouter un glycine entre la lysine et l'acide aspartique si la lysine et ajoutée sur la terminaison N du peptide antigénique.
PCT/US1992/009376 1991-11-04 1992-11-04 Peptides synthetiques correspondant a des portions du virus vih-2 et procedes d'utilisation de ces peptides dans une analyse amelioree WO1993009252A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU30598/92A AU657590B2 (en) 1991-11-04 1992-11-04 Synthetic peptides corresponding to portions of HIV-2 virus and methods of using in an improved assay
JP5508647A JPH06503843A (ja) 1991-11-04 1992-11-04 Ｈｉｖ−２ウイルスの一部分に対応する合成ペプチドおよび改良されたイムノアッセイにおけるその使用方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US78991491A 1991-11-04 1991-11-04
US789,914 1991-11-04

Publications (1)

Publication Number Publication Date
WO1993009252A1 true WO1993009252A1 (fr) 1993-05-13

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PCT/US1992/009376 WO1993009252A1 (fr) 1991-11-04 1992-11-04 Peptides synthetiques correspondant a des portions du virus vih-2 et procedes d'utilisation de ces peptides dans une analyse amelioree

Country Status (5)

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EP (1) EP0565704A4 (fr)
JP (1) JPH06503843A (fr)
AU (1) AU657590B2 (fr)
CA (1) CA2099367A1 (fr)
WO (1) WO1993009252A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023973A3 (fr) * 1994-03-02 1995-09-28 Abbott Lab Dosage immunologique vih a l'aide de proteines recombinantes et de reactifs peptidiques de synthese
EP0920443B1 (fr) * 1997-04-07 2003-07-09 Syva Company Peptides actives et leurs conjugues

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4812556A (en) * 1987-05-18 1989-03-14 Virovahl Synthetic peptide antigen for the detection of HIV-2 infection

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5075211A (en) * 1986-03-26 1991-12-24 Genetic Systems Corporation Synthetic antigen for the detection of AIDS-related disease
GB9005829D0 (en) * 1990-03-15 1990-05-09 Proteus Biotech Ltd Synthetic polypeptides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4812556A (en) * 1987-05-18 1989-03-14 Virovahl Synthetic peptide antigen for the detection of HIV-2 infection

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0565704A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023973A3 (fr) * 1994-03-02 1995-09-28 Abbott Lab Dosage immunologique vih a l'aide de proteines recombinantes et de reactifs peptidiques de synthese
EP0920443B1 (fr) * 1997-04-07 2003-07-09 Syva Company Peptides actives et leurs conjugues

Also Published As

Publication number Publication date
EP0565704A4 (en) 1995-10-25
EP0565704A1 (fr) 1993-10-20
AU3059892A (en) 1993-06-07
CA2099367A1 (fr) 1993-05-05
AU657590B2 (en) 1995-03-16
JPH06503843A (ja) 1994-04-28

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