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AU660752B2 - Immunoassay using synthetic HTLV-11 peptides - Google Patents

Immunoassay using synthetic HTLV-11 peptides Download PDF

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AU660752B2
AU660752B2 AU31425/93A AU3142593A AU660752B2 AU 660752 B2 AU660752 B2 AU 660752B2 AU 31425/93 A AU31425/93 A AU 31425/93A AU 3142593 A AU3142593 A AU 3142593A AU 660752 B2 AU660752 B2 AU 660752B2
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htlv
sequence
solid phase
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amino acids
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Nrapendra Nath
Dinesh O. Shah
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Dade International Inc
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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
    • 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
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    • 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

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Description

t tf WO 93/11431 PCT/US92/09973 1 .IMMUNOASSAY USING SYNTHETIC HTLV-II PEPTIDES BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION The present invention relates to synthetic peptides and method to use these synthetic peptides in an improved immunoassay for antibodies to HTLV-II.
2. pESCRIPTION OF THE PRIOR ART Human T-Cell Lymphotropic Viruses (HTLV) types I and II are closely related human retroviruses.
HTLV-I is associated with adult T-Cell Leukemaia, while HTLV-II although not associated with a specific disease, is found in many intravenous drug users. R. Lal et al., Serologic Discrimination of Human T Cell Lymphotropic Virus Infection by Using a Synthetic Peptide Based Enzyme Immunoassay, J. of Inf. Dis. 163:41-46 (January, 1991). Generally, serological assays cannot reliably distinguish between HTLV-I and HTLV-II infections. To serologically distinguish between these two viruses Lal et al., have disclosed synthetic peptides corresponding with unique regions of the envelope glycoprotein (gp46) of human T-Cell lymphotropic viruses. In the Lal et al. study two synthetic HTLV-I sequence derived peptides, Env-1 (amino acids 191-215) and Env-5 (amino acids 242-257), reacted with 92% and 100% of serum specimens from HTLV-I infected person; although a small percentage of serum specimens from persons infected with HTLV- II cross-reacted with Env-1, none of these specimens 1 0 0 WO 93/11431 PCT/US92/09973 2 reacted with Env-5. Peptide Env-2 encoded by the envelope region of HTLV-II (amino acids 187-210) reacted with serum specimens from both HTLV-I (94%) and HTLV-II infected patients, whereas as Env-6, another HTLV-II peptide (amino acids 238-254) reacted with less than 6% of the specimens. Based on this work Lal et al., concluded that the peptide represents an immunodominant domain of HTLV- I that is recognized by serum antibodies from all HTLV-I infected persons. Thus, an assay using the Lal et al. Env-5 peptide may allow for a categorical distinction between the closely related HTLV-I and II infections. In the Lal et al. paper the peptides were coated onto polyvinyl plates to perform the serological assessment of antibodies to HTLV-I or II. The HTLV-II peptide (Env-2) was attached to a microparticle by the present inventors. This solid phase, however, resulted in a serological assay for HTLV-YI with low sensitivity. As such, a need exists to devise a peptide that could be used with microparticles to obtain a high sensitivity serological assay for HTLV-II. In the context of blood banking a serological test for HTLV-II would be useful.
SUMMARY OF THE INVENTION The present invention relates to certain peptides corresponding to a portion of the glycoprotein gp-46 encoded by HTLV-II env gene, having the basic sequence disclosed by Lal et al. The improvement discovered by the inventors involves adding three amino acids homologous to HTLV-I prior to amino acid 187 of the Lal et al., HTLV-II WO 93/11431 PCT/US92/09973 3 peptide. Serine was also added at amino acid 183 of HTLV-II. The amino acid residue numbering is from the amino terminus of each protein. Additionally, 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.
In particular, this invention relates to an antigen peptide of the formula: (Sequence Id. No.
In addition 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 (Sequence Id. Nos. 1-7) 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 immunological 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.
DETAILED DESCRIPTION OF THE INVENTION Renu Lal 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 WO 93/11431 PCT/US92/09973 4 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 Lal et al. claimed that these sequences were important in distinguishing infection and immune response to HTLV-I and HTLV-II.
In making the synthetic peptides (Sequence Id.
Nos. 1-7) of this invention the inventors combined the sequence published by Lal et al. for HTLV-II (Sequence Id. No. with a portion of a sequence for HTLV-I: 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) 3618-3622 (June 1983). Sequence Id. No. 10 was used to construct the sequence shown in Sequence Id. No.
1. In particular one can see homology between amino acids 2-4 of Sequence Id. No. 10 and amino acids 2-4 Sequence Id. No. 1. The incorporation of a peptide sequence corresponding to HTLV-I (env) in an assay for anti-HTLV-I would be expected to broaden the antigenic spectrum to detect both HTLV-I and II.
Thus the amino acids at the N-terminus of the Lal 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 Lal et al. peptide.
This was thought to help discriminating HTLV-I from HTLV-II samples. Finally, lysine was added to the N-terminus of the peptide to facilitate coupling or WO 93/11431 PCT/US92/09973 adsorption to a solid phase such as a microparticle.
Tyrosine or cysteine may also be added instead of lysine at either N or C terminal end of the peptide. Surprisingly, however, this peptide modified to increse 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. In the context of this invention 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 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 tree acid form. Activated amino acids were coupled to a 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, WO 93/11431 PCT/US92/09973 6 which comprises trifluoroacetic acid, thioanisole, ethanedithiol and anisol in a volume;ric ratio of 90:5:3:2.
Peptides cleaved from resins were purified by high performance liquid chromatography (HPLC), and characterized by Porton P1 20 90 E Integrated Micro Sequencing system to confirm the correct sequence.
Purity was ascertained by HPLC on a reverse phase column using a linear gradient 0.1 trifluoroacetic acid in H 2 0 trifluoroacetic acid in CH 3 CN) of 5% to 60% in 45 minutes.
Absorbance was followed at 230 nm.
Example 1 Peptides (Sequence Id. No. 2) were passively coated onto paramagnetic microparticles according to the following prrcedure: 1 ml of 2.5% of weight/volume approx. 5 Am paramagnetic particles 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.
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. Further, washing procedure with carbonate buffer was repeated twice as before and supernatant removed.
WO 93/11431 PCT/US92/09973 7 To the slurry of particles was added 100 Al of peptide solution (1 mg/ml in 50 mM carbonate buffer, pH 8.3) and 900 pl 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.
Example 2 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 pm 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 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.
Further, washing procedure with carbonate buffer was repeated twice as before and supernatant removed.
WO 93/11431 PCT/US92/09973 8 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 mg/ml in carbonate buffer, pH 8.3).
The particles were mixed thoroughly and then added 4 mg of sulfo-N-hydroxy succinimide. 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 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 WO 93/11431 PCr/US92/09973 9 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 Al). The plate was then placed at 420 C for minutes.
Upon completion of the incubation, the particles in the wells were washed with 100 gl 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 1.0g sodium azide per liter; pH During the wash steps, 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 gl 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 20 pl of goat anti-human IgG (H L) conjugate with B-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 was then added to the wells. After incubation with conjugate for 15 minutes at 420 C the particles in the wells were washed five times with phosphate buffered saline and Tween-20 as described above to WO 93/11431 PCT/US92/9973 remove essentially all of the unbound conjugate.
The Tween-20 in the wash solution enhanced the washing process and removed nonspecifically bound conjugate.
Finally the sixth wash with substrate solution followed by addition of 50 gl of a substrate solution of 4-methyl-umbelliferyl-B-D galactoside (MUG' was added to each well (0.178 MUG, 3.58 g tricine, 5.1 ml dimethyl sulfoxide, 30 ml methyl alcohol. 0.20 g sodium azide, 0.5 ml Tween-20, per liter, pH The presence of B-galactosidase conjugate) in the wells triggered the cleavage of MUG to generate a fluorescent coumarin 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 timed intervals 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.
I WO 93/11431 PCT/US92/09973 11 TABLE 1 PEPTIDES PASSIVELY ADSORBED ON PARTICLES: SAMPLE ID ASSAY VALUE Peptide of Peptide of Sequence Id. Sequence Id.
No. 9 No. 2 HTLV-II 1 9 #3 9 13 #8 63 77 11 22 52 12 43 189 13 36 74 16 10 292 17 37 2755 18 9 221 HIV-I POS 6 9 NEG. CONTROL 6 SAMPLE DILUENT 1 4 Particles at 0.02% in a suspension buffer; Goat anti Human IgG B-Galactosidase labeled conjugate at 1:2000 dilution. An assay value of one hundred was set as the assay cutoff, and values less then one hundred were thus considered negative in this evalution.
WO 93/11431 PCT/US92/09973 12 TABLE 2
PEPTIDES
SAMPLE ID COVALENTLY COUPLED PARTICLES: ASSAY VALUE HTLV-II 1 3 8 11 12 13 16 17 18 HIV-I POS NEG. CONTROL SAMPLE DILUENT Peptide of Sequence Id.
No. 9 27 26 31 150 306 388 164 124 70 21 18 3 Peptide of Sequence Id.
No. 2 138 277 108 158 836 771 1203 5000 769 11 11 2 Particles at 0.02% in a suspension buffer; Goat anti Human IgG B-Galactosidase labeled conjugate at 1:2000 dilution. An assay valua of one hundred was set as the assay cutoff, and values less than one hundred were thus considered negative in this evalution.
I I WO 93/11431 PCT/US92/09973 13 These examples show that synthetic peptide of Sequence Id. No. 2 attached or associated with a solid phase, such as a microparticle, results in a more sensitive assay for HTLV-II than the HTLV-II peptide reported by Lal et al.
Although the invention has been described primarily in connection with special and preferred embodiments, it will be understood that it is capable of modification without departing from the scope of the invention. The following claims are intended to cover all variations, uses, or adaptations of the invention, following, in general, the principles thereof and including such departures from the present disclosure as come within known or customary practice in the field to which the invention pertains,, or as are obvious to persons skilled in the field.
WO 93/11431 PCT/US92/09973 14 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Shah, Dinesh 0.
Nath, Nrapendra (ii) TITLE OF INVENTION: Synthetic Peptides Corresponding to a Portion of HTLV-II Virus and Method To Use the Same in an Improved Immunoassay (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Baxter Diagnostics Inc.
STREET: One Baxter Parkway, DF2-2E CITY: Deerfield STATE: Illinois COUNTRY: USA ZIP: 60015 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US FILING DATE:
CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION: NAME: Barta, Kent REGISTRATION NUMBER: 29,042 REFERENCE/DOCKET NUMBER: PA-4196 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 708/948-3308 TELEFAX: 708/948-2642 WO 93/11431 PCT/US92/09973 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 28 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His Val 1 Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys Ile Leu Lys 20 Phe Ile 28 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 29 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Lys Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His 1 Val Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys Ile Leu 20 Lys Phe Ile 29 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 29 amino acids TYPE: amino acid WO 93/11431 PCT/US92/09973 16 STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His Val 1 5 Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys Ile Leu Lys 20 Phe Ile Lys 29 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 29 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Tyr Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His 1 5 Val Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys Ile Leu 20 Lys Phe Ile 29 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 29 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His Val WO 93/11431 PCI US92/09973 17 1 5 Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys Ile Leu Lys 20 Phe Ile Tyr 29 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 29 amino acids TYPE: amino acid .0 STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Cys Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His 1 Val Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys Ile Leu 20 Lys Phe Ile 29 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 29 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ IO NO:7: Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His Val 1 5 Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys Ile Leu Lys 20 Phe Ile Cys 29 WO 93/11431 PCT/US92/09973.
18 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Leu Pro His Ser Asn Leu Asp His Ile Leu Glu Pro Ser 1 5 Ile Pro Trp Lys Ser Lys Leu Leu Thr Leu Val INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Val His Asp Ser Asp Leu Glu His Val Leu Thr Pro Ser 1 5 Thr Ser Trp Thr Thr Lys Ile Leu Lys Phe Ile INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 28 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID WO 93/1 1431 PO'/ US92/09973 19 Ala Pro Pro Leu Leu Pro His Ser Asn Leu Asp His Ile 1 5 Leu Glu Pro Ser Ile Pro Trp Lys Ser Lys Leu Leu Thr 20 Leu Val 28

Claims (8)

1. A serological method for detection of antibodies to HTLV-II in a blood sample comprising: a) covalently attaching a synthetic peptide selected from the group consisting of amino acids shown in Sequnce Id. Nos. 1, 2, 3, 4, 5, 6, and 7, to a solid phase; b) combining said solid phase with a blood sample; c) detecting antibodies to HTLV-II bound to said solid phase.
2. The method of Claim 1 wherein said solid phase is a microparticle.
3. A serological method for detection of antibodies to HTLV-II in a blood sample comprising: a) adsorbing a synthetic peptide selected from the group consisting of amino acids shown in Sequence Id. Nos. 1, 2, 3, 4, 5, 6, and 7 to a solid phase; b) combining said solid phase with a blood sample; c) detecting antibodies to HTLV-II bound to said solid phase.
4. The method of Claim 3 wherein said solid phase is a microparticle.
5. A synthetic peptide consisting of the amino acids shown in Sequence Id. No. 1.
6. An immunological reagent comprising a synthetic peptide compound capable of binding to antibodies to HTLV-II coupled or adsorbed to a solid phase said peptide selected from the group of peptides represented by the following N-terminal to C- terminal amino acid sequences coupled or adsorbed to a, solid phase said: -21- a. Sequence Id No. 2 b. Sequence Id No. 0 c. Sequence Id No. 4 d. Sequence Id No. e. Sequence Id No. 6 f. Sequence Id No. 7
7. The method of claim 8 wherein said solid phase is a microparticle.
8. A diagnostic test kit when used for the detection of HTLV-II specific antibodies according to the method of any *e one of claims 1 to 4, said kit comprising: a) microparticles coated with synthetic peptides selected from the group consisting of amino acids shown in Sequence Id. Nos. 1, 2, 3, 4, 5, 6 and 7; b) a vial of marked antibody that immunologically V. C reacts with human HTLV-II antibody; c) a positive control; d) a negative control; and Coo. e) microtiter plates. e. DATED this 24th day of March, 1995. DADE INTERNATIONAL INC. Patent Attorneys for the Applicant: PETER MAXWELL ASSOCIATES Id .ci 1 .4 WNUATIONAL SEARCH REPORT International application No. IPCTIUS92/09973 A. CA DCI04OFf SUBJECT MAT1ER IP() aM=W, 331W66 CO7K 17/00 US CL :43K, 524; 530139,402, 403, 326; 435/71 Accodi8g 10 10111 Pwd Claification (IP or iz both nuarmal claasication and IPC B. F7ELD6 UKARCRID Min==u doemum.~l Searched (Chlifcatioe system followed by classification symbols) U.S. :436/501, 524; 530/395, 402, 403, 826; 435/7.1 Doumntatin 2*=he other than minimuin documentatonl to extent that such documents are included in the fields seched F-1ectronic data base consulted during the interniational search (name of data base and, where practiable, search terms used) APS, MEDLINE search tem: HTLV, immunoassay, magnectic, lysine C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Re-atto claimNo Y EP, A, 0,439,077 (CHANG YI WANG) 31 JULY 1991, entire document. 1-8 Y Journal of Infectious Diseases, Volume 163, Issued January 1991, R.B. Lal et 1,3,5,6 'Serologic Discrimination of Human T Cell Lymphotropic Virus Infection by using a syrthetic peptide-based ynzyme immunoassay',pages 41-46. entire document, especially abstract, page 43, paragraph bridging columns, and paragraph bridging pages 42-43. Y Proceedings of the Nationa Academy of Sciences, Vol. 82, Issued May 19B5, K. 1,3,5,6 Shimotobno et aI., 'Complete nucleotide sequence of an infectious clone of human T-cll leukemia virus type II: An open reading .fn for the prtease gene%, pages 3101-3105, we page 3103. Y WO, A, 90110231 (Dlomberg et aQ 09 SEPTEMBER 199-0, see Peptide 2EA, page 10, 1,3,5,6 page 4, second paragraph from bottom of page, Table on p. 13, page 19. §3. Further documents arm listed in the continuation of Box C. see patent family antnex. Special u of cisad doassaaftict dmcm pub 6C aner rbc kwAse~oW fitimg data or pronot: des ad am oict wide dw eppliain but cb so undsami bW .domaeinieg do Swers mass ofdes art wWis s m csieM P. or asory undyia des iooe to be pan of pelcar rehrsc 'F aster r j 1 b~ fih W docuiaw of parbaukr ,,skvmoe4 dw ckwood awmaaic mo; be aditr o p as a shw A im ag de waded leovel ort casb odriddto inyotre as muveve .ue, L documnt w" stly dtrow double a priority clw(s) or whichb wholk do O sM a aso apecld to mw des ulidedewaodw o Y. dow of paricular reladvm do ckmsd inventon tma*4b specil areiod) owkiend w kyWi' as hivdY. W1 whta d doc"md is .0 damawd slaciiin to as orl disclosure, vtm ezhifas or odwr *6 will ow or owni oher wick docuxnw. nic ocin-teio amm beiag obvrious lo a as sid in *4 ad Daeof the actual completion of the international search Date of mailing of the international hr Jainuary 1993 27 JAN 19a Name and mailing address of the ISA/ Commissioner of Ftwot and Trademarks IBox PcI Washington. D.C. 2023I Aijitorized officer LORRAINE M. SPECTOR, Telephone No. 74~ J-1793 Form PCTIISAr21O (seccond shect)(July 1992)* PIMNTONAL SEARCH REPORT lnecinatianal appUiation No. PCT/U592/09M7 C (Congiaunon). DOCUMENT CO2NSIDERED TO BE REL.EVANT Ca Y ckio of &meunmu, with indiczin, whers approiate, of the rclcvant paSag Relevant to claim No. USA 4,647,=2 (SHADLE EiT AL) 11 JULY 199 ENTIRE DOCUMENT, capeiay ooL 4, How. 45-55. USSA, 4,070,246 (KENNEDY EiT AL) 24 JANUARY 1978, ENTIRE DOCUMENT. 2,4,7,8 2,4,7,8 Form PCTIISAI21O (conti~zoion of second shcct)(July 1992)*
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US7166439B2 (en) * 2002-03-01 2007-01-23 Gmp Endotherapeutics, Inc. Assay for anti-INGAP antibodies

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US4070247A (en) * 1977-03-30 1978-01-24 Indiana University Foundation Diagnostic media
US4847325A (en) * 1988-01-20 1989-07-11 Cetus Corporation Conjugation of polymer to colony stimulating factor-1
EP0439077A2 (en) * 1990-01-24 1991-07-31 United Biomedical Inc. Synthetic peptide compositions with immunoreactivities to antibodies to HTLV

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US4070246A (en) * 1976-04-09 1978-01-24 Abbott Laboratories Reactive matrices
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
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US4070247A (en) * 1977-03-30 1978-01-24 Indiana University Foundation Diagnostic media
US4847325A (en) * 1988-01-20 1989-07-11 Cetus Corporation Conjugation of polymer to colony stimulating factor-1
EP0439077A2 (en) * 1990-01-24 1991-07-31 United Biomedical Inc. Synthetic peptide compositions with immunoreactivities to antibodies to HTLV

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