WO1993011431A1

WO1993011431A1 - Immunoassay using synthetic htlv-ii peptides

Info

Publication number: WO1993011431A1
Application number: PCT/US1992/009973
Authority: WO
Inventors: Dinesh O. Shah; Nrapendra Nath
Original assignee: Baxter Diagnostics Inc.
Priority date: 1991-11-25
Filing date: 1992-11-20
Publication date: 1993-06-10
Also published as: AU660752B2; JPH06506479A; CA2100883A1; EP0573630A1; AU3142593A; EP0573630A4

Abstract

The present invention relates to a serological method for detection of antibodies to HTLV-II in a blood sample using a synthetic peptide coated on a solid phase consisting of the amino acids of Sequence Id. Nos. 1-7. Additionally, this invention relates to a diagnostic test kit for the detection of HTLV-II.

Description

it

JHMUJiOASSAY 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.

10

2. DESCRIPTION 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 Leukemia,

15 while HTLV-II although not associated with a specific disease, is found in many intravenous drug users. R. Lai et al., Serologic Discrimination of Human T Cell Lymphotropic Virus Infection by Using a Synthetic Peptide - Based Enzyme Immunoassay. J. of

20 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 Lai et al., have disclosed synthetic peptides

25 corresponding with unique regions of the envelope glycoprotein (gp46) of human T-Cell lymphotropic viruses. In the Lai et al. study two synthetic HTLV-I sequence derived peptides, Env-1 (amino acids 191-215) and Env-5 (amino acids 242-257) , reacted

30 with 92% and 100% of serum specimens from HTLV-I infected person; although a small percentage (8.6%) of serum specimens from persons infected with HTLV- II cross-reacted with Env-1, none of these specimens 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 (74%) - 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 Lai et al. , concluded that the Env-5 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

Lai et al. Env-5 peptide may allow for a categorical distinction between the closely related HTLV-I and

II infections. In the Lai 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--II 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 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. 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. 1) . 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 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.

DETAILED DESCRIPTION OF THE INVENTION 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.

In making the synthetic peptides (Sequence Id. Nos. 1-7) of this invention the inventors combined the sequence published by Lai et al. for HTLV-II (Sequence Id. No. 8) , 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) 80, 3618-3622 (June 1983) . Sequence Id. No. 10 was used to construct the sequence shown in Sequence Id. No. l. In particular one can see homology between amino acids 2-4 of Sequence Id. No. iθ 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 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.

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 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. Surprisingly, however, 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. 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 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.

^"5 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 0 column using a linear gradient (A) 0.1 trifluoroacetic acid in H₂0 (B)0.1% trifluoroacetic acid in CH₃CN) of 5% to 60% (B) in 45 minutes. Absorbance was followed at 230 nm.

5 ^'Example 1

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 μ 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. Further, washing procedure 0 with carbonate buffer was repeated twice as before and supernatant removed. To the slurry of particles was added 100 μl of peptide solution (1 mg/ml in 50 mM carbonate buffer, pH 8.3) and 900 μl 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 μm 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.

Further, 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 μl) . The plate was then placed at 42° C for 30 minutes.

Upon completion of the incubation, the particles in the wells were washed with 100 μl 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). 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 μl 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). 20 μl of goat anti-human IgG (H + L) conjugate with β-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 + .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.

Finally the sixth wash with substrate solution followed by addition of 50 μl of a substrate solution of 4-methyl-umbelliferyl-β-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 8.5). The presence of β-galactosidase (i.e. : 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 (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.

TABLE 1

PEPTIDES PASSIVELY ADSORBED ON PARTICLES:

Particles at 0.02% in a suspension buffer; Goat anti Human IgG (H+L) β-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.

TABLE 2

PEPTIDES COVALENTLY COUPLED PARTICLES:

Particles at 0.02% in a suspension buffer; Goat anti Human IgG (H+L) β-Galactosidase labeled conjugate at 1:2000 dilution. An assay value of one hundred was set as the assay cutoff, and values less than one hundred were thus considered negative in this evalution.

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 Lai 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.

SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: Shah, Dinesh O. 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: 10 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Baxter Diagnostics Inc.

(B) STREET: One Baxter Parkway, DF2-2E

(C) CITY: Deerfield

(D) STATE: Illinois (E) COUNTRY: USA

(F) ZIP: 60015 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US (B) FILING DATE:

(C) CLASSIFICATION: (viii) ATTORNE /AGENT INFORMATION:

(A) NAME: Barta, Kent

(B) REGISTRATION NUMBER: 29,042 (C) REFERENCE/DOCKET NUMBER: PA-4196

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 708/948-3308

(B) TELEFAX: 708/948-2642 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His Val 1 5 10

Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys lie Leu Lys

15 20 25

Phe He 28 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown (D) 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 5 10 Val Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys He Leu 15 20 25

Lys Phe He 29 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: unknown

(D) 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 10

Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys He Leu Lys

15 20 25

Phe He Lys 29

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE .TYPE: peptide ( i) SEQUENCE DESCRIPTION: SEQ ID NO:4: Tyr Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His 1 5 10

Val Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys He Leu

15 20 25

Lys Phe He 29 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide ( i) SEQUENCE DESCRIPTION: SEQ ID NO:5: Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His Val 1 5 10

Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys He Leu Lys

15 20 25

Phe He Tyr 29

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: unknown

(D) 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 5 10

Val Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys He Leu

15 20 25

Lys Phe lie 29 (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ser Pro Pro Leu Val His Asp Ser Asp Leu Glu His Val 1 5 10 Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys He Leu Lys 15 20 25

Phe He Cys 29 (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Leu Pro His Ser Asn Leu Asp His He Leu Glu Pro Ser 1 5 10

He Pro Trp Lys Ser Lys Leu Leu Thr Leu Val

20 (2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS: ' (A) LENGTH: 24 amino^"acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown

(D) 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 10

Thr Ser Trp Thr Thr Lys He Leu Lys Phe He 15 20 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

( i) SEQUENCE DESCRIPTION: SEQ ID NO:10: Ala Pro Pro Leu Leu Pro His Ser Asn Leu Asp His He

1 5 10

Leu Glu Pro Ser He Pro Trp Lys Ser Lys Leu Leu Thr

15 20 25

Leu Val

28

Claims

WE CLAIM:

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:

7. The method of Claim 8 wherein said solid phase is a microparticle.

8. A diagnostic test kit for the detection of HTLV- II specific antibodies comprising: a) microparticles coated with synthetic peptides selected from the group consisting of amino acids shown in Sequence Id. Nos. l, 2, 3, 4, 5, 6, and 7; b) a vial of marked antibody that immunologically reacts with human HTLV-II antibody; c) a positive control; d) a negative control; and e) microtiter plates.