EP0573630A1 - Immunoassay unter verwendung von synthetischen htlv-ii-peptiden - Google Patents

Immunoassay unter verwendung von synthetischen htlv-ii-peptiden

Info

Publication number
EP0573630A1
EP0573630A1 EP92925329A EP92925329A EP0573630A1 EP 0573630 A1 EP0573630 A1 EP 0573630A1 EP 92925329 A EP92925329 A EP 92925329A EP 92925329 A EP92925329 A EP 92925329A EP 0573630 A1 EP0573630 A1 EP 0573630A1
Authority
EP
European Patent Office
Prior art keywords
htlv
solid phase
sequence
amino acids
peptide
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP92925329A
Other languages
English (en)
French (fr)
Other versions
EP0573630A4 (de
Inventor
Dinesh O. Shah
Nrapendra Nath
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.)
Dade International Inc
Original Assignee
Baxter Diagnostics Inc
Dade International 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, Dade International Inc filed Critical Baxter Diagnostics Inc
Publication of EP0573630A1 publication Critical patent/EP0573630A1/de
Publication of EP0573630A4 publication Critical patent/EP0573630A4/de
Withdrawn legal-status Critical Current

Links

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
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14022—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 and method to use these synthetic peptides in an improved immunoassay for antibodies to HTLV-II.
  • HTLV Human T-Cell Lymphotropic Viruses
  • Env-5 peptide may allow for a categorical distinction between the closely related HTLV-I and
  • the present invention relates to certain peptides corresponding to a portion of the glyco- protein gp-46 encoded by HTLV-II env gene, having the basic sequence disclosed by Lai et al.
  • the improvement discovered by the inventors involves adding three amino acids homologous to HTLV-I prior to amino acid 187 of the Lai et al., HTLV-II peptide. Serine was also added at amino acid 183 of HTLV-II i The amino acid residue numbering is from the amino terminus of each protein.
  • certain amino acids may be added to the modified peptide to facilitate coupling to a solid phase. Surprisingly, this peptide modified to increase sensitivity to HTLV-I resulted in a highly sensitive solid phase assay for HTLV-II.
  • this invention relates to an antigen peptide of the formula: (Sequence Id. No. 1) .
  • tyrosine, cysteine or lysine may be added at either the N or C terminal end of the peptide.
  • These modified peptides have the amino acid sequences shown in Sequence Id. Nos. 2-7. -
  • These peptides can be adsorbed or attached to a solid phase for use in an assay for the detection of HTLV-II specific antibodies.
  • the method involves contacting the sample with the peptide under conditions such that an im unological complex will form between the peptide and antibodies to HTLV-II 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 HTLV-II in the sample. This method is described in Example 3.
  • Renu Lai et al. have published the following amino acid sequences for part of the envelope gene for the HTLV-I and HTLV-II respectively, Sequence Id. No. 8 (corresponding to amino acid 191 to 214 of the natural peptide) ; and Sequence Id. No. 9 (corresponding to amino acid 187 to 210 of the natural peptide) .
  • the amino acid residue numbering is from the amino terminus of each protein. Renu Lai et al. claimed that these sequences were important in distinguishing infection and immune response to HTLV-I and HTLV-II.
  • Sequence Id. No. 10 (numbered from amino acid 187 to 214) published by Seiki et al. , Human Adult T-Cell Leukemia Virus: Complete Nucleotide Sequence of the Provirus Genome Integrated in Leukemia, Cell DNA,- Proc. Nat'l Acad. Sci. (USA) 80, 3618-3622 (June 1983) . Sequence Id. No. 10 was used to construct the sequence shown in Sequence Id.
  • amino acids at the N-terminus of the Lai et al. HTLV-II peptide was extended to include a short sequence of amino acids common with the amino acid sequence of the HTLV-I peptide. Additionally, amino acid Serine was added to the Lai et al. peptide.
  • lysine was added to the N-terminus of the peptide to facilitate coupling or adsorption to a solid phase such as a microparticle.
  • Tyrosine (Y) or cysteine (C) may also be added instead of lysine (K) at either N or C terminal end of the peptide.
  • this peptide modified to increase sensitivity to HTLV-I resulted in a highly sensitive solid phase assay for HTLV-II.
  • the new peptides shown by Sequence Id. Nos. 1-7 can be coupled or adsorbed to a solid phase such as a microparticle. It is understood that the microparticle may be magnetic.
  • This immunoreactant can react with antibodies to HTLV-II in a blood sample. The extent of the reaction between the synthetic antigen and the antibody in solution can be detected with a marked antibody that immunologically reacts with human HTLV-II.
  • the term marked means either directly or indirectly. To be indirectly marked a substrate for the bound enzyme is added.
  • Peptides were synthesized in the amide form on a Milligen-Biosearch 9600 model peptide synthesizer using fluorenyl ethoxy 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 (Sequence Id. No. 2) were passively coated onto paramagnetic microparticles according to the following procedure: 1 ml of 2.5% of weight/volume approx. 5 ⁇ paramagnetic particles 0 consisted of a polystyrene surface 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. 5 The particles were then separated as before and supernatant was removed. The particles were resuspended in 1 ml of 50 mM carbonate buffer, pH 8.3. The particles were separated as before and supernatant was removed.
  • washing procedure 0 with carbonate buffer was repeated twice as before and supernatant removed.
  • To the slurry of particles was added 100 ⁇ l of peptide solution (1 mg/ml in 50 mM carbonate buffer, pH 8.3) and 900 ⁇ l of 50 mM carbonate buffer. The particles were resuspended and then tumbled for approximately 5 hours, at room temperature.
  • the passively adsorbed peptide particles were then separated on a magnetic separator, supernatant removed and particles 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 are then resuspended in isotonic buffered saline at final particle concentration of 0.25% weight to volume.
  • Peptides (Sequence Id. No. 2) were covalently coupled to carboxyl functionalized paramagnetic microparticles according to the following procedure: 1 ml of 2.5% weight/volume approx. 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 were 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 50 mM carbonate buffer, pH 8.3. The particles were separated as before and supernatant was removed.
  • washing procedure with carbonate buffer was repeated twice as before and supernatant removed.
  • To the slurry of particles was added 5 mg of 1- Ethyl-3- (3-Dimethylaminopropyl) carbodiimide hydrochloride and 1 ml of carbonate buffer. The particles were mixed thoroughly and then tumbled for 40 minutes. The activated particles then separated on a magnetic separator Supernatant was removed and the particles were resuspended in 1 ml of peptide solution (0.1% mg/ml in carbonate buffer, pH 8.3). The particles were mixed thoroughly and then added 4 mg of sulfo-N-hydroxy succini ide. Again, the particles were mixed thoroughly and then tumbled for approximately 5 hours.
  • 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 suspended in isotonic buffered saline at final concentration of 0.25% weight to volume.
  • Example 3 A paramagnetic particle assay using particles coated with peptide as shown in Examples 1-2 was performed as follows: Human serum or plasma was diluted 1:100 in well buffer (20% neonate calf serum, 1.06 M sodium chloride 0.015 M Tris-HCL, pH 7.4, 0.018 M Phosphate buffer, pH 7.4 + 0.1, 0.09% sodium azide, and 0.45% NP-40 ). 50 ul of the diluted samples were added to each well of a Pandex black microtiter plate. Samples were tested in replicates of at least 2. Paramagnetic particles, coated with peptides as described in example 1 or 2, were added to each well (20 ⁇ l) . The plate was then placed at 42° C for 30 minutes.
  • the particles in the wells were washed with 100 ⁇ l phosphate buffered 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 l.Og sodium azide per liter; pH 7.4).
  • the paramagnetic particles were held in the microtiter plate well via a magnetic field applied to the bottom of the plate. Particles were washed in this manner five times.
  • Particles in each well were resuspended in 30 ⁇ l of particle resuspension buffer (4.346 g sodium phosphate dibasic, 0.524 g sodium phosphate monobasic, 8.76 g sodium chloride, and 1 g sodium azide per liter; pH 7.4).
  • particle resuspension buffer 4.346 g sodium phosphate dibasic, 0.524 g sodium phosphate monobasic, 8.76 g sodium chloride, and 1 g sodium azide per liter; pH 7.4
  • 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 + .3) was then added to the wells. After incubation with conjugate for 15 minutes at 42° C the particles in the wells were washed five times with phosphate buffered saline and Tween-20 as described above to remove essentially all of the unbound conjugate. The Tween-20 in the wash solution enhanced the ' washing process and removed nonspecifically bound conjugate.
  • 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 + .3 was then added to the wells. After incubation with conjugate for 15 minutes at 42° C the particles in the well
  • Fluorescence (excitation wavelength 400 nm/emmision wavelength 450 nm) was measured at two timed 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.
  • MOLECULE TYPE peptide
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO:5: Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His Val 1 5 10
  • MOLECULE TYPE peptide

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • AIDS & HIV (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
EP92925329A 1991-11-25 1992-11-20 Immunoassay unter verwendung von synthetischen htlv-ii-peptiden. Withdrawn EP0573630A4 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US79755791A 1991-11-25 1991-11-25
US797557 1991-11-25

Publications (2)

Publication Number Publication Date
EP0573630A1 true EP0573630A1 (de) 1993-12-15
EP0573630A4 EP0573630A4 (de) 1995-07-12

Family

ID=25171175

Family Applications (1)

Application Number Title Priority Date Filing Date
EP92925329A Withdrawn EP0573630A4 (de) 1991-11-25 1992-11-20 Immunoassay unter verwendung von synthetischen htlv-ii-peptiden.

Country Status (5)

Country Link
EP (1) EP0573630A4 (de)
JP (1) JPH06506479A (de)
AU (1) AU660752B2 (de)
CA (1) CA2100883A1 (de)
WO (1) WO1993011431A1 (de)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2117378C (en) * 1992-02-24 2003-04-15 Steven K. H. Foung Htlv-i/htlv-ii assay and method
US7166439B2 (en) * 2002-03-01 2007-01-23 Gmp Endotherapeutics, Inc. Assay for anti-INGAP antibodies

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4070246A (en) * 1976-04-09 1978-01-24 Abbott Laboratories Reactive matrices
US4070247A (en) * 1977-03-30 1978-01-24 Indiana University Foundation Diagnostic media
US5039604A (en) * 1987-08-21 1991-08-13 Cellular Products, Inc. Test device and method of preparing same, assay kit and method for the simultaneous detection of two HTLV or HIV antibodies
US4847325A (en) * 1988-01-20 1989-07-11 Cetus Corporation Conjugation of polymer to colony stimulating factor-1
SE8900721D0 (sv) * 1989-03-02 1989-03-02 Blomberg Jonas Methods for detection of antibodies to
FI910245L (fi) * 1990-01-24 1991-07-25 United Biomedical Inc Syntetiska peptidkompositioner med immunoreaktivitet mot htlv-antikroppar.
NZ238855A (en) * 1990-07-18 1994-03-25 Iaf Biochem Int Peptides, mixtures thereof and compositions useful for detecting htlv-i and htlv-ii infections

Also Published As

Publication number Publication date
AU660752B2 (en) 1995-07-06
EP0573630A4 (de) 1995-07-12
AU3142593A (en) 1993-06-28
CA2100883A1 (en) 1993-05-26
JPH06506479A (ja) 1994-07-21
WO1993011431A1 (en) 1993-06-10

Similar Documents

Publication Publication Date Title
CA2102301C (en) Process for the determination of peptides corresponding to immunologically important epitopes and their use in a process for determination of antibodies or biotinylated peptides corresponding to immunologically important epitopes, a process for preparing them and compositions containing them
EP0199438B1 (de) HTLV III Polypeptide
CA2166291C (en) Htlv-iinra compositions and assays for detecting htlv infection
US5017687A (en) Peptides for the detection of HTLV-1 infection
CA1340097C (en) Synthetic antigen for detection of aids-related disease
JP2994031B2 (ja) Ｈｉｖ−１又はｈｉｖ−２抗体の存在又は量の確定方法、免疫特異的試薬、合成ペプチド、診断用キット、ｈｉｖ−１又はｈｉｖ−２抗体の調製方法、免疫原及び抗体
US5260189A (en) Synthetic HIV-like peptides their compositions and uses
WO1992022571A1 (en) Immunoassay for non-a non-b hepatitis
US6649735B1 (en) Process for the determination of peptides corresponding to immunologically important epitopes and their use in a process for determination of antibodies or biotinylated peptides corresponding to immunologically important epitopes, a process for preparing them and compositions containing them
SE467542B (sv) Syntetiska peptidantigener, immuniserande komposition och immunanalys foer htlv-1 antikroppar
AU660752B2 (en) Immunoassay using synthetic HTLV-11 peptides
US5283320A (en) Peptides for HTLV-2 infection diagnosis of, therapy for, vaccination against, for distinguishing between HTLV-1 and HTLV-2 infections and antibodies derived therefrom
AU657590B2 (en) Synthetic peptides corresponding to portions of HIV-2 virus and methods of using in an improved assay
JP3560987B2 (ja) グループｏ ｈｉｖ−１ウイルスによる感染検出用の生物学的アッセイに有用な合成ペプチド
EP0532746B1 (de) Nicht-a, nicht-b hepatitis test
JP2002501930A (ja) ペプチド構造物およびそれらの単純ヘルペスウイルス２型の診断における使用
CA2004287A1 (en) Peptides
DE DK et al. HCV MOSAIK ANTIGEN ZUSAMMENSETZUNG COMPOSITION D’ANTIGENE MOSAIQUE DU VIRUS DE L’HEPATITE C (VHC)

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19930702

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): BE CH DE ES FR GB IT LI

RHK1 Main classification (correction)

Ipc: G01N 33/569

A4 Supplementary search report drawn up and despatched
AK Designated contracting states

Kind code of ref document: A4

Designated state(s): BE CH DE ES FR GB IT LI

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: DADE INTERNATIONAL INC.

17Q First examination report despatched

Effective date: 19960918

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19970402