DIFFERENTIATION OF HTLV-I AND HTLV-II USING SYNTHETIC PEPTIDES

Abstract: Method for differentiating HTLV-I antibodies from HTLV-II antibodies, and peptides which enable the differentiation. Kits containing these peptides which allow for the differentiation between anti-HTLV-I antibodies and anti-HTLV-II antibodies.

Description: DIFFERENTIATION OF HTLV-I AND HTLV-II USING SYNTHETIC PEPTIDES Background of the Invention. This invention relates generally to a method useful for the detection of antibodies to Human T-Cell Lymphotropic Virus types I and II (HTLV-I and HTLV-II) in a test sample, and more particularly, relates to synthetic peptides specific for HTLV-I and HTLV-II, respectively, and methods useful for the differential detection of antibodies to HTLV-I and HTLV-II, thereby allowing the differential diagnosis of HTLV-I and HTLV-II infections. HTLV-I is known to cause disease in humans, whereas HTLV-II is not clearly associated with disease. Epidemiological data indicate that approximately 50% of U.S blood donors confirmed seropositive for HTLV-I are in fact infected with HTLV-II (Lee et al., unpublished observation). Therefore, there is a critical need to be able to distinguish between the two viruses for appropriate donor notification and counseling. Current screening immunoassays for HTLV-I infection detect antibodies against HTLV-I and to a lesser extent HTLV-II. Serological methods which are more specific than EIA, such as Western blot (WB) and Radioimmunoprecipitation assays (RIPA) cannot distinguish between the two viruses. To date, differentiation between HTLV-I and HTLV-II is achievable only by use of molecular genetic techniques such as restriction mapping or DNA sequencing of the provirus, and by Polymerase Chain Reaction (PCR) using specific primers for HTLV-I or HTLV-II. These procedures require the use of lymphocytes from patients to be tested which are clearly less convenient to collect and store than serum or plasma test samples. These techniques thus are limited in their usefulness in that they are time consuming, expensive, require specialized facilities and are not easily automated. United States Patent Nos. 4,525,300 and 4,804,746 to Yoshida teach methods for preparing antibodies to human leukemia virus related peptides. The antibodies disclosed are capable of binding to human leukemia virus. Palker et al.. J. Immunol. 135 (1 ): 247-254 (1985) report the preparation of monoclonal antibodies reactive with HTLV-I which were raised to synthetic peptides representing sections of the p19 gag internal core protein. Some of the clones generated were reported to bind to HTLV-I virus isolates but not to bind to HTLV-II. PCT Application No. PCT/US85/01803 to Slamon, published March 27, 1986, teaches a method for the detection of antibodies to HTLV-I and HTLV-II in samples by means of incubating samples with synthetic or cloned poiypeptides and proteins derived from the HTLV genome and immobilized on a solid support. The teachings include methods for differentially diagnosing HTLV-I and HTLV-II, which require immunoprecipitation of proteins followed by molecular mass determination by methods such as SDS PAGE electrophoresis. However, SDS PAGE electrophoresis is not easily automated or convertible to a form suitable for routine laboratory use. U.S. Patent No. 4,689,398 to Wu teaches a further group of synthetic peptides, derived from the HTLV genome sequence, which may be used to detect antibodies specific to HTLV in test samples. European Patent Application No. 0 267 622 to Masanori, published May 18, 1988, teaches a device comprising a fused HTLV gas and suy gene protein immobilized on a solid phase which may be used to detect antibodies to these proteins in a sample. However, this device is unable to distinguish between antibodies to HTLV-I and HTLV-II. Palker et al. J. Immunol. 142:971-978 (1989) report the mapping of the immunogenic regions of the HTLV-I gp46 and gp21 8ΩX proteins and the synthesis of peptides which are useful in the generation of specific monoclonal antibodies. These peptides may be used in immunoassays to detect antibodies to HTLV. Additionally, the report suggests the presence of, but does not identify, a region of the gp46 protein which is not shared by HTLV-I and HTLV-II and may therefore be used to differentially detect antibodies to the two viruses. PCT Publication No. WO89/08664 (PCT/SE89/00126) to Vahlne et al., published September 21 , 1989, teaches of further synthetic peptides, derived from the QBX region of the HTLV-I genome, which may be used in the detection of antibodies to the HTLV-I virus. No mention is made of differentiation between antibodies against HTLV-I and HTLV-II. PCT Publication No. WO90/08162 to United Biomedical Inc., published July 26, 1990, describes synthetic peptides for the detection of HTLV-I reactive antibodies and diagnosis of ATL (adult T cell leukemia/ lymphoma). These peptides are from the transmembrane (p21e) and external (gp46) segments of the envelope protein of HTLV-I. Also described are immunoassays using these peptides. The peptide(s) described are used in the SynthEIA® (Olympus Corp., Lake Success, NY) for HTLV-I. PCT Publication No. WO 90/10231 to Blomberg, published March 5, 1990, teaches a method for differentially detecting antibodies to HTLV-I and HTLV-II by detecting binding of such antibodies to synthetic peptides derived from the flag_and ≤QY regions of HTLV-I and HTLV-II. The method described requires the performance of at least four immunoassays on each sample and would therefore be inconvenient for the routine screening of a large number of samples. PCT publication No. WO90/15820 to Vahlne et al., published December 27, 1990, describes peptides and antibodies derived from the disclosed peptides which are immunologically reactive with HTLV-I specific antibodies. Several of the peptides are capable of distinguishing between HTLV-I and HTLV-II infection. Recently, R. B. Lai et al. described the serologic discrimination of HTLV-I from HTLV-II using synthetic peptides which could be used to differentiate between HTLV-I and HTLV-II. R. B. Lai et al.. J. Infectious Diseases 163:41-46 (January, 1991 ). In particular, they reported that HTLV-I "Env-5" (amino acids 242-257) represented an immunodominant domain of HTLV-I, and that ENV-5- based ELISA allowed distinction between HTLV-I and HTLV-II. With the exception of this recent article and the two most recent patent applications which describe differentiation, all of the above disclosed techniques are aimed at the detection of specific antibodies to HTLV-I and HTLV-II. To date, however, no detailed methods have been described which would provide a simple method of effectively detecting, and distinguishing between, antibodies to HTLV-I and HTLV-II. In order to detect and distinguish between antibodies against HTLV-I and HTLV-II, unique antigenic determinants on the two viruses must be identified. Antigenic determinants on proteins have been predicted by the identification of hydrophilic regions using the method of Hopp and Woods, Proc. Natl Acad Sci U.S.A 78:3824 (1981 ) as well as identification of flexible regions using the method of Karplus and Schultz, Naturwissenschaften 72:212- 213 (1985). Antigenic determinants appear to be located at hydrophilic as well as flexible regions of protein sequences. Antigenic determinants have also been empirically identified by immunological examination of peptides produced by protein degradation or in vitro synthesis. It therefore would be advantageous to provide a method to both detect the presence of antibodies to HTLV-I or HTLV-II, and to effectively distinguish between antibodies to each virus. Such a method would be a useful tool to the physician, allowing a more detailed diagnosis of the disease and therefore more appropriate patient counseling and treatment. The method preferably would be in a simple but reproducible form to facilitate routine laboratory usage and generate sensitive and specific results. Summary of the Invention This invention provides a method for differentiating antibodies against HTLV-I from antibodies against HTLV-II in a test sample. A combination of the above described techniques were used to identify antigenic regions in the gp46 env and p19 flag proteins of HTLV-I and HTLV-II. Since the addition or deletion of amino acids to a peptide significantly influences its ability to bind to antibodies, antigenic regions were systematically examined to identify antigenic sequences which are unique and superior to previously described peptides. The invention provides a method comprising (a) determining the presence of antibody against HTLV-I in a test sample by (i) contacting a test sample with at eas one pep e spec c or - , o orm a m x ure, ncu a ng e mixture for a time and under conditions sufficient for antigen/antibody complexes to form, (iii) contacting said complexes with an indicator reagent comprising a signal generating compound attached to an anti-human IgG antibody to form a second mixture, (iv) incubating said second mixture for a time and under conditions sufficient for antigen/antibody/antibody complexes to form, (v) determining the presence of antibody against HTLV-I by detecting the measurable signal; (b) determining the presence of antibodies against HTLV-II in the test sample, by (i) contacting a test sample with at least one peptide specific for HTLV-II , to form a mixture, (ii) incubating the mixture for a time and under conditions sufficient for antigen/antibody complexes to form, (iii) contacting said complexes with an indicator reagent comprising a signal generating compound attached to an anti-human IgG antibody, to form a second mixture, (iv) incubating said second mixture for a time and under conditions sufficient for antigen/antibody/antibody complexes to form, (v) determining the presence of antibodies against HTLV-II by detecting the measurable signal; (c) determining the pattern of reaction of the test sample for HTLV-I and HTLV-II to distinguish between HTLV-I and HTLV-II. The amino acid sequence for the HTLV-I synthetic peptides were obtained from the predicted amino acid sequence as published by Seiki et al.. Proc. Natl. Acad. Sci.. USA 80:3618-3622 (1983). The peptides specific for HTLV-I include the following: HTLV-I ≤nγ-1 (SEQ. ID. NO. 1), HTLV-I env-2 (SEQ. ID. NO. 2), HTLV-I an -3 (SEQ. ID. NO.3), HTLV-I env-4 (SEQ. ID. NO. 4), HTLV-I a tδ (SEQ. ID. NO. 5) and HTLV-I ≤n*-6 (SEQ. ID. NO. 6). The peptides specific for HTLV-I further include HTLV-I oaq-1 (SEQ. ID. NO. 7), HTLV-I gag-2 (SEQ. ID. NO. 8), HTLV-I gag-3 {SEQ. ID. NO. 9), HTLV-I gag-4 (SEQ. ID. NO. 10), HTLV-I gag-5 (SEQ. ID. NO. 11), HTLV-I gag-6 (SEQ. ID. NO. 12) and HTLV-I gag-7 (SEQ. ID. NO. 13). The amino acid sequences for the HTLV-II synthetic peptides were obtained from the predicted amino acid sequences of the two HTLV-II prototypes. The amino acid sequence of the Mo HTLV-II prototype was published by Shimotohno et al., Proc. Natl. Acad. Sci. USA 82:3101-3105 (1985). The sequence of the NRA HTLV-II prototype is unpublished data. The peptides specific for HTLV-II include the following: HTLV-II sny-1 (SEQ. ID. NO. 14), HTLV-II env-1 A (SEQ. ID. NO. 15), HTLV-II ≤n -2 (SEQ. ID. NO. 16), HTLV- II ≤nγ-2A (SEQ. ID. NO. 17), HTLV-II env-3 (SEQ. ID. NO. 18), HTLV-II ≤nγ-4 (SEQ. ID. NO. 19), HTLV-II env-5 (SEQ. ID. NO. 20), HTLV-II an - 5A (SEQ. ID. NO. 21), HTLV-II env-6 (SEQ. ID. NO. 22), HTLV-II aα -7 (SEQ. ID. NO. 23) and HTLV-II env-8 (SEQ. ID. NO. 24). The peptides specific for HTLV-II further include HTLV-II gag-1 (SEQ. ID. NO. 25), HTLV-II gag-2 (SEQ. ID. NO. 26), HTLV-II gag-3 (SEQ. ID. NO. 27), HTLV-II gag-4 (SEQ. ID. NO. 28). Peptides HTLV-II sntt-1 A, HTLV-II ≤ny.-2A and HTLV-II sm 5A are NRA sequences. All the other remaining HTLV-II sequences are Mo sequences. The invention also provides kits containing the peptides of the invention for differentiating between anti-HTLV-I antibody and anti-HTLV-II antibody. Detailed Description of The Invention. The invention provides a method for the detection of antibodies against either HTLV-I or HTLV-II by means of detecting the binding of the antibodies to synthetic peptides. With the exception of recently published PCT WO90/15820 (cited sjiβia), previously described peptides for HTLV-I gp46 are in a common region. We identify unique and superior peptide sequences in this region as well as unique peptide sequences in a second antigenic region of HTLV-I nx that are specific for HTLV-I and do not cross-react with HTLV-II. The amino acid sequence for the HTLV-I synthetic peptides were obtained from the predicted amino acid sequence as published by Seiki et al.. Proc. Nat'l. Acad. Sci.. USA 80:3618- 3622 (1983). We have identified a unique peptide sequence in a previously non-identified region of HTLV-II gp46 ej that is specific for HTLV-II, as well as unique and superior peptide sequences in a previously identified antigenic region. The amino acid sequences for the HTLV-II synthetic peptides were obtained from the predicted amino acid sequences of the two HTLV-II prototypes. The amino acid sequence of the Mo HTLV-II prototype was published by Shimotohno et al., Proc. Nat'l. Acad. Sci. USA 82:3101-3105 (1985). The sequence of the NRA HTLV-II prototype is unpublished data . We also have identified unique peptide sequences in antigenic regions of HTLV-I p19 gag and HTLV-II p19 gag that are specific for HTLV-I and HTLV-II, respectively. In general, the method of the invention comprises contacting a test sample with a solid phase to which at least one of the HTLV-I or HTLV-II peptides are bound, to form a mixture. The mixture is incubated for a time and under conditions sufficient for antigen / antibody complexes to form. Then the complexes are contacted with an indicator reagent comprising an anti-human antibody attached to a signal generating compound, to form a second mixture. The second mixture is incubated for a time and under conditions sufficient to form antigen / antibody / antibody complexes. The presence of immobilized antibody is determined by detecting the measurable signal generated. The investigation of a test sample separately for antibodies against HTLV-I and antibodies against HTLV-II allows an effective method for distinguishing between infections with these two viruses. The "solid phase" is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, beads, membranes, plastic tubes, walls of microtiter wells and tanned sheep red blood cells are all suitable examples. Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like. Those skilled in the art will recognize the scope of methodologies which may be applied relative to the application of useful solid phases. The "test sample" can be a sample of human or animal biological fluid, such as serum, plasma, ascites, urine, cerebral spinal fluid or any other body constituents, or any tissue culture supematants which might contain the antibodies of interest. A suitable "indicator reagent" can be a signal generating compound (label) which is capable of generating a measurable signal detectable by external means conjugated (attached) to a specific binding member for antibodies derived from the test sample. In addition to being an antibody member of a specific binding pair for test sample-derived antibodies, the indicator reagent also can be a member of any specific binding pair, including either hapten-anti-hapten such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme and the like. The various "signal generating compounds"(labels) contemplated include chromogens, catalysts such as enzymes, luminescent compounds such as fluoroscein and rhodamine, chemiluminescent compounds, radioactive elements, and direct visual labels. Examples of enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances. In one embodiment of the present invention, at least one of the synthetic peptides of the invention, specific for antibodies against HTLV-I or HTLV-II is immobilized on polystyrene beads. The beads are then incubated with a diluted test sample of human serum, plasma or other body fluid, and incubated under conditions and for an appropriate period of time during which time antibodies will bind specifically to the immobilized peptides on the bead. The bead then is washed to remove any unbound proteins which may be present . A second incubation then is performed in which the bead is incubated with an indicator reagent comprising anti-human antibodies labelled with an appropriate signal generating compound. By means of a suitable detection system, the amount of labelled anti-human antibody complex immobilized on the bead may be determined by measuring the detectable signal. Hence, the presence of specific antibodies against HTLV-I or HTLV-II in the test sample is determined. A comparison of the pattern of reactivity of the test sample to antibodies against HTLV-I and antibodies against HTLV-II allows distinction between infections with the two viruses. In a second embodiment of the present invention, at least one of the synthetic peptides of the invention specific for HTLV-I or HTLV-II is immobilized on polystyrene beads. The beads are then incubated with a test sample or a diluted test sample and an appropriate indicator reagent comprising signal generating compound attached to anti-human IgG, under conditions and for an appropriate period of time, during which time antibodies will bind specifically to the immobilized peptides on the bead and simultaneously, thus bind to the indicator reagent. The amount of labelled anti-human antibody complex immobilized on the bead may e eterm ned by detecting t e measura e signal generated. Thus, the presence of antibodies against HTLV-I or HTLV-II in the test sample can be determined by a single incubation. A comparison of the pattern of reactivity of the test sample to antibodies against HTLV-I and antibodies against HTLV-II allows distinction between infections with the two viruses. In a third embodiment of the present invention, at least one of the peptides of the invention specific for antibodies against HTLV-I or antibodies against HTLV-II, are immobilized on a nitrocellulose membrane. The peptide also can be conjugated or crosslinked to itself, other peptides or to various carrier proteins such as BSA, keyhole limpet hemocyanin, ovalbumin, and the like, before immobilization on the nitrocellulose membrane. The test sample is diluted and incubated on the membrane for a time and for conditions sufficient for antigen / antibody complexes to form. The membrane surface is then washed to remove unbound proteins, and in a second incubation, the membrane is incubated with an indicator reagent comprising anti-human antibodies labelled with a signal generating compound. The amount of labelled anti-human antibody immobilized on the membrane, thus the presence of antibodies against either HTLV-I or HTLV-II, is determined by a detecting the measurable signal generated with suitable detection system. Quantification of the level of signal recognized by the detection system allows the quantification of the amount of specific antibody present in a test sample. A comparison of the pattern of reactivity of the test sample to antibodies against HTLV-I and antibodies against HTLV-II allows distinction between infections with the two viruses. Other embodiments which utililize various other solid phases also are contemplated and are within the scope of this invention. For example, ion capture procedures for immobilizing an immobilizable reaction complex with a negatively charged polymer, described in co-pending U. S. Patent Application Serial No. 150,278 corresponding to EP publication 0326100, and U. S. Patent Application Serial No. 375,029 (EP publication no. 0406473) both of which enjoy common ownership and are incorporated herein by reference, can be employed according to the present invention to effect a fast solution-phase immunochemical reaction. An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negat ve y charged poly-anion immune complex and the previously treated, positively charged porous matrix and detected by using various signal generating systems previously described, including those described in chemiluminescent signal measurements as described in co-pending U.S. Patent Application Serial No.921 ,979 corresponding to EPO Publication No. 0 273,115, which enjoys common ownership and which is incorporated herein by reference. Also, the methods of the present invention can be adapted for use in systems which utilize microparticle technology including in automated and semi-automated systems wherein the solid phase comprises a microparticle. Such systems include those described in pending U. S. Patent Applications 425,651 and 425,643, which correspond to published EPO applications Nos. EP 0 425 633 and EP 0 424 634, respectively, which are incorporated herein by reference. The use of scanning tunnelling microscopy for immunoassays also is a technology to which the methods of the present invention are easily adaptable. In scanning probe microscopy, in particular in atomic force microscopy, the capture phase, for example, a selected peptide or peptides of the invention, is adhered to a solid phase and a scanning probe microscope is utilized to detect antigen/antibody complexes which may be present on the surface of the solid phase. The use of scanning tunnelling microscopy eliminates the need for labels which normally must be utilized in many immunoassay systems to detect antigen/antibody complexes. Such a system is described in pending U. S. patent application Serial No. 662,147, which enjoys common ownership and is incoφorated herein by reference. While the present invention discloses the preference for the use of solid phases, it is contemplated that the peptides of the present invention can be utilized in non-solid phase assay systems. These assay systems are known to those skilled in the art, and are considered to be within the scope of the present invention. We have been able to design tests which detect specific antibodies against HTLV-I separately from antibodies against HTLV-II by selecting appropriate synthetic peptides of each of these viruses, and coating them onto , infections. Peptides suitable for the specific detection antibodies against HTLV- I are specified herein and comprise the peptides of the HTLV-I sni region and the peptides of the HTLV-I gagjegion which are designated as SEQ. ID. No. 1 through SEQ. ID. NO. 13. Peptides suitable for the specific detection of antibodies against HTLV-II also are specified herein and comprise the peptides of the HTLV-II a region, and the peptides of the HTLV-II gag region, and are designated as SEQ. ID. NO. 14 through SEQ. ID. NO. 28. It is contemplated that the reagent employed for the assay can be provided in the form of a kit with one or more containers such as vials or bottles, with each container or vial containing a separate reagent such as a diluent, indicator reagent, signal generating compound, assay reagents comprising at least one peptide of the present invention, and the like. The present invention will now be described by way of examples, which are meant to illustrate, but not to limit, the spirit and scope of the invention. EXAMPLES Example 1 Detection of Antibodies against HTLV-I Peptides were synthesized by stepwise addition of amino acids to a solid phase using procedures known in the art and described in Merrifield * Am.Chem Soc 85: 2149-2154 (1963) and Barany and Merrifield in E. Gross and J. Meienhofer, βds., The Peptides. Vol. 2:1-284 (1979), Academic Press, New York, which are incorporated herein by reference. Briefly, the procedure was as follows. The synthesis was performing starting with the C terminus and progressing to the N terminus. N-protected amino acids were used, with N being t-butyloxycarbonyl (t-Boc). The C terminus t-Boc amino acid was attached to the solid phase. The t-Boc protecting group was removed with trifluoroacetic acid, leaving a free amino group to couple to the next amino acid. Successive t-Boc amino acids were added, coupled using a reagent such as DCC (N-N'-dicyclohexylcarbodiimide) and then deprotected. Once the peptide was completed, the final t-Boc protecting group was removed and the peptide was cleaved from the polymer by using anhydrous hydrogen fluoride. Peptides prepared as described hereinabove were coated on polystyrene beads as the solid support for capture of antibodies against HTLV-I using procedures known in the art . Briefly, the polystyrene beads were washed with distilled water and incubated at 40°C for two (2) hours with between 0.01μg/ml and 50 μg/ml of peptide(s) in a phosphate buffered saline (PBS) solution. The beads were washed once with PBS containing 0.1% Triton X- 100® for one (1) hour, blocked for one (1) hour with 2% bovine serum albumin (BSA) in PBS, overcoated with 5% sucrose in PBS for 15 minutes, and then dried. Beads used in this example as the solid phase for the detection of antibodies to HTLV-I were co-coated with peptide HTLV-I env-1 (SEQ. ID NO. 1 ) corresponding to amino acids 174-204 and peptide HTLV-I env-4 (SEQ. ID NO. 5) corresponding to amino acids 237-260 of the HTLV-I gp46 aΩX protein. Anti-human IgG antiserum was prepared by immunizing goats with purified human IgG, according to known methods. The resulting antiserum was affinity purified and labelled with Horseradish Peroxidase (HRPO). Method Samples were diluted in sample diluent buffer between dilution factors of 1 :2 and 1 :1000. 200 μl of the diluted sample was incubated with a coated bead in a reaction tray for 60 minutes at 40°C. After thorough washing, the beads were incubated with goat anti-human HRPO diluted in a suitable diluent, for 30 minutes at 40 °C. The beads were again thoroughly washed. The amount of HRPO immobilized on the beads was quantified by incubating with an O- phenylenediamine:2HCI (OPD) reagent for 30 minutes at ambient room temperature. At the end of this incubation 1.0 ml of 1 N suifuric acid was added to stop the color generating reaction. The degree of color generation was determined by measuring the absorbance of the resulting solution at 492/600nm. esuts When the method of the invention was applied to a panel of test samples that previously has been confirmed to be positive for antibodies against HTLV by the method of D.W. Anderson et al., Blood 74:2585-2591 (1989) and confirmed HTLV-I positive by PCR, the method of the invention detected 55 of 57 (96.5%) samples positive for antibodies against HTLV-I. Example 2 Detection of Antibodies against HTLV-II Baag__D_s Peptides prepared as described hereinabove in Example 1 were coated on polystyrene beads as the solid phase for capture of antibodies against HTLV-II, as follows. The polystyrene beads were washed with 15% v/v isopropanol and incubated at 40°C for two (2) hours with between 0.01μg/ ml and 50 μg/ml of peptide(s) in a phosphate buffered saline (PBS) solution. The beads were washed once with PBS containing 0.15% Triton X-100® for one hour and blocked for one hour with 2% bovine serum albumin (BSA) in PBS, and then overcoated with 5% sucrose in PBS for 20 minutes at room temperature and then dried. For the detection of antibodies against HTLV-II in this example, beads were coated with peptide HTLV-II env-2 (SEQ. ID NO. 16) corresponding to amino acids 171-198 of the HTLV-II gp46 any protein. Anti-human IgG antiserum was prepared by immunizing goats with purified human IgG. The resulting antiserum was affinity purified and labelled with Horseradish Peroxidase (HRPO) according to standard methods known in the art. Method Samples were diluted in sample diluent buffer between dilution factors of 1 :2 and 1 :1000. 200 μl of the diluted sample was incubated with a coated bead in a reaction tray for 60 minutes at 40°C. After thorough washing, the beads were incubated for 30 minutes at 40°C with goat anti-human HRPO diluted in a suitable diluent. The beads were again thoroughly washed. The amount of HRPO immobilized on the bead was quantified by incubating with an O- phenylenediamine:2HCI (OPD) reagent for 30 minutes at ambient room temperature. At the end of this incubation, 1.0 ml of 1 N suifuric acid was added to stop the color generating reaction. The degree of color generation was determined by measuring the absorbance of the substrate at 492/ 600nm. Results. When this method was applied to a panel of test samples which had been confirmed to be positive for antibodies against HTLV by the method of Anderson (cited sjjgta) and positive for HTLV-II by PCR, the test was able to detect 72 of 74 samples (97.3%). Example 3 Detection of Antibodies Aoainst HTLV-I or HTLV-II Reagents were prepared as for example 2 except that peptides HTLV-I gag-1 (SEQ. ID. NO. 7), HTLV-II gag3 (SEQ. ID. NO. 27), HTLV-II ≤nγ-8 (SEQ. ID. NO. 7) and HTLV-I soy-1 (SEQ. ID. NO. 1) were individually coated onto beads. The beads then were used in the assay method of the invention following the procedure of Examples 1 and 2. The data presented below in Table 1 is a compilation of the data generated in examples 1 ,2 and 3. It also was shown that by using the peptides of examples 1 ,2 and 3, with the method of the invention, we were able to detect 57/57 (100%) test samples confirmed to be positive for antibodies against HTLV-I. In addition, the method of the invention was able to detect 73 / 74 (98.6%) of samples confirmed to be positive for antibodies against HTLV-II. Example 4 Comparison of Present Invention With Other Tests. The methods of examples 1 ,2 and 3 were used to investigate a panel of test samples which had previously been classified by Polymerase Chain Reaction (PCR). The present invention was compared with three other methods (PCR, SynthEIA™[available from Olympus Corp., Lake Success, NY] and a method by Svennerholm [cited supra as Vahlne, EP Publication No. , w , against HTLV-I and HTLV-II. The Svennerholm peptides tested were HTLV-I H, O, T, V, and HTLV-II H, O, and T. The Blomberg peptides tested were the four disclosed preferred peptides (1GB, 2GB, 1EA and 2EA), in approximately the same region as the peptides of this disclosure. Each of the comparative methods were conducted according to the disclosed preferred method, or in the case of the commercially available SynthEIA, according to the manufacturers' recommended protocol. The results of the comparison are shown in Tables 1 and 2. TABLE 1 Comparison of Disclosed Method With Other Tests. Method Pr s nt Method SvnthEIA Svennerholmt 57/ 57 (100%) 10/14 (71.4%) 8/ 11 (72.7%) * I 73/74(98.6%) 9/31 (29.0%) 5/12(41.7%) Indeterminate HTLV-I 6/ 8 (75%) 1/8(12.5%) 1/3(33.3%) Indeterminate HTLV-II 3/9(33.3%) 1/9(11.1%) 0/2(0%) Sero negative 0/144(0%) 5/44(11.4%) * One sample discrepant with PCR results; fpreviously cited. * In the present method, two HTLV-I samples cross-reacted with this peptide. In the Blomberg method, 24 HTLV-I samples cross-reacted; tpreviously cited. Using the method described in this example, we have demonstrated the serological differentiation of antibodies against HTLV-I from antibodies against HTLV-II using a panel of highly pedigreed samples. When compared with existing methods, which use peptides with other sequences, the peptides described herein show superior sensitivity and specificity. In comparison to the Polymerase Chain Reaction (PCR) reference method, the described method of the invention provides high efficacy and much improved ease of use. Example 5 Effect of Peotide Sequence on Assay Performance Reagents were prepared as for examples 1 ,2 and 3 except that beads were coated individually with HTLV-I snγ-6 (SEQ. ID. NO. 6) (190-213), HTLV-I gag-4 (SEQ. ID. NO. 10) (103-116), HTLV-I gag-3 (SEQ. ID. NO. 9) (109-129) and HTLV-II env-7 (SEQ. ID. NO. 23) (186-195). The data from these experiments, performed as the methods of examples 1 ,2 and 3, was tabulated (Table 3) and compared to the data previously generated for experiments 1 ,2 and 3. Comparison Of Peptide Amino Acid Sequences and Serological Reactivities HTLV-II Reactivity 0/ 9 9/ 9 This data clearly demonstrates that the addition, or removal, of small numbers of amino acids to or from a peptide may significantly influence its ability to bind to an antibody. Example 6 Reactivity of Peptides Reagents were prepared as for example 2 except that peptides SEQ. ID 1 THROUGH SEQ. ID 28 were individually coated onto beads. The data presented below in Table 4 is a compilation of the data generated . The corresponding designation of the SEQ. ID. No. is indicated in parenthesis next to the SEQ. ID. NO. It was shown that by using the peptides SEQ. ID 1 through SEQ. ID 13 with the method of the invention, we were able to detect 14/14 (100%) test samples confirmed to be positive for antibodies against HTLV-I, with no cross- reactivity observed for HTLV-II. In addition the method of the invention was able to detect 9/14 (64.3%) of samples confirmed to be positive for antibodies against HTLV-II by using a single peptide selected from SEQ. ID 14 through SEQ. ID 28. Cross-reactivity was observed with only one peptide of the group SEQ. ID 14 to SEQ. ID. 28: SEQ. ID. 25. TABLE 4 Reactivity of Individual Peptides Peotide Amino Acid HTLV-I HTLV-II Reactivit y Reactivity SEQ. ID.1 (HTLV-I flny-1 ) 174-204 11/14 0/14 SEQ. ID.2 (HTLV-I any-2) 180-213 13/14 0/14 SEQ. ID.3 (HTLV-I eny-3) 227-257 7/14 0/14 SEQ. ID.4 (HTLV-I ≤ny-4) 230-260 12/14 0/14 SEQ. ID.5 (HTLV-I n -5) 237-260 9/14 0/14 SEQ. ID.6 (HTLV-I snγ-6) 190-213 4/14 0/14 SEQ. ID.7 (HTLV-I gag-1) 100-129 14/14 0/14 SEQ. ID.8 (HTLV-I gag-2) 104-129 14/14 0/14 SEQ. ID.9 (HTLV-I gag-3) 109-129 5/14 0/14 SEQ. ID.10 (HTLV-I gag-4. 103-116 0/14 0/14 SEQ. ID 11 (HTLV-I gag-5) 100-119 10/14 0/14 SEQ. ID 12 (HTLV-I gag-6. 100-127 13/14 0/14 SEQ. ID 13 (HTLV-I gag-7. 100-126 13/14 0/14 SEQ. ID.14 (HTLV-II any-1) 167-198 0/14 8/14 SEQ. ID.15 (HTLV-II ≤ΠU-1 A) * 167-198 0/14 9/14 SEQ. ID.16 (HTLV-II env-2. 171 -198 0/14 6/14 SEQ. ID.17 (HTLV-II £flγ-2A) * 171 -198 0/14 9/14 SEQ. ID.18 (HTLV-II en -3) 173-200 0/14 5/14 SEQ. ID.19 (HTLV-II env-4. 173-204 0/14 8/14 SEQ. ID.20 (HTLV-II an^-5) 176-209 0/14 4/14 SEQ. ID.21 (HTLV-II ≤n -5A) * 176-209 0/14 9/14 SEQ. ID.22 (HTLV-II fin -6) 228-257 0/14 3/14 SEQ. ID.23 (HTLV-II sm-7) 186-195 0/14 0/14 SEQ. ID.24 (HTLV-II a____-8) 83-108 0/14 5/14 SEQ. ID.25 (HTLV-II gag-1) 115-135 6/14 3/14 SEQ. ID.26 (HTLV-II gag-2) 111-129 0/14 4/14 SEQ. ID.27 (HTLV-II gag-3) 109-127 0/14 5/14 SEQ. ID.28 (HTLV-II gag-4) 107-125 0/14 2/14 * These three sequences were derived from the NRA HTLV-II prototype. er va atons an mo catons o t e spec ic em o ments of t e invention as set forth herein will be apparent to those skilled in the art. Accordingly, the invention is intended to be limited only in accordance with the appended claims. (1) GENERAL INFORMATION: (i) APPLICANT: Shi , Jessie W. Burczak, John D. Lee, Helen H. O'Donnell, Debra L. (ii) TITLE OF INVENTION: DIFFERENTIATION OF HTLV-I AND HTLV-II USING SYNTHETIC PEPTIDES ( ii) NUMBER OF SEQUENCES: 28 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Abbott Laboratories (B) STREET: One Abbott Park Road (C) CITY: Abbott Park (D) STATE: Illinois (E) COUNTRY: USA (F) ZIP: 60064-3500 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vi ) ATTORNEY/AGENT INFORMATION: (A) NAME: Porembski , Priscilla E. (B) REGISTRATION NUMBER: 33,207 (C) REFERENCE/DOCKET NUMBER: 5013.US.01 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 708-937-4884 (B) TELEFAX: 708-937-9556 (2) INFORMATION FOR SEQ ID N0:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 174-204 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: Phe Leu Asn Thr Glu Pro Ser Gin Leu Pro Pro Thr Ala Pro Pro Leu 174 175 180 185 Leu Pro His Ser Asn Leu Asp His He Leu Glu Pro Ser He Pro 190 195 200 (2) INFORMATION FOR SEQ ID N0:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 180-213 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2: Ser Gin Leu Pro Pro Thr Ala Pro Pro Leu Leu Pro His Ser Asn Leu 180 185 190 195 Asp His lie Leu Glu Pro Ser lie Pro Trp Lys Ser Lys Leu Leu Thr 200 205 210 Leu Val (2) INFORMATION FOR SEQ ID N0:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: Cys He Asp Arg Ala Ser Leu Ser Thr Trp His Val Leu Tyr Ser Pro 227 230 235 240 Asn Val Ser Val Pro Ser Ser Ser Ser Thr Pro Leu Leu Tyr Pro 245 250 255 (2) INFORMATION FOR SEQ ID N0:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 230-260 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4: Arg Ala Ser Leu Ser Thr Trp His Val Leu Tyr Ser Pro Asn Val Ser 230 235 240 245 Val Pro Ser Ser Ser Ser Thr Pro Leu Leu Tyr Pro Ser Leu Ala 250 255 260 (2) INFORMATION FOR SEQ ID N0:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 237-260 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5: His Val Leu Tyr Ser Pro Asn Val Ser Val Pro Ser Ser Ser Ser Thr Pro Leu Leu Tyr Pro Ser Leu Ala 255 260 (2) INFORMATION FOR SEQ ID N0:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 190-213 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6: Leu Pro His Ser Asn Leu Asp His He Leu Glu Pro Ser He Pro Trp 190 195 200 205 Lys Ser Lys Leu Leu Thr Leu Val 210 (2) INFORMATION FOR SEQ ID N0:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 100-129 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Pro Pro Pro Ser Ser Pro Thr His Asp Pro Pro Asp Ser Asp Pro Gin 100 105 110 115 He Pro Pro Pro Tyr Val Glu Pro Thr Ala Pro Gin Val Leu 120 125 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 104-129 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8: Ser Pro Thr His Asp Pro Pro Asp Ser Asp Pro Gin He Pro Pro Pro 104 105 110 115 Tyr Val Glu Pro Thr Ala Pro Gin Val Leu 120 125 (2) INFORMATION FOR SEQ ID N0:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 109-129 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9: Pro Pro Asp Ser Asp Pro Gin He Pro Pro Pro Tyr Val Glu Pro Thr 109 110 115 120 Ala Pro Gin Val Leu 125 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 103-116 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: Ser Ser Pro Thr His Asp Pro Pro Asp Ser Asp Pro Gin He 103 105 110 115 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 100-119 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:11: Pro Pro Pro Ser Ser Pro Thr His Asp Pro Pro Asp Ser Asp Pro Gin 100 105 110 115 He Pro Pro Pro (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 100-127 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12: 100 105 110 115 He Pro Pro Pro Tyr Val Glu Pro Thr Ala Pro Gin 120 125 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 100-126 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13: Pro Pro Pro Ser Ser Pro Thr His Asp Pro Pro Asp Ser Asp Pro Gin 100 105 110 115 He Pro Pro Pro Tyr Val Glu Pro Thr Ala Pro 120 125 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 167-198 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Pro Leu Trp Phe He Thr Ser Glu Pro Thr Gin Pro Pro Pro Thr Ser 167 170 175 180 Pro Pro Leu Val His Asp Ser Asp Leu Glu His Val Leu Thr Pro Ser 185 190 195 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 167-198 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15: Pro Leu Trp Phe He Thr Ser Glu Pro Thr Arg Pro Pro Pro Thr Pro 167 170 175 180 Pro Pro Leu Val His Asp Ser Asp Leu Glu His Val Leu Thr Pro Ser 185 190 195 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 171-198 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: He Thr Ser Glu Pro Thr Gin Pro Pro Pro Thr Ser Pro Pro Leu Val 171 175 180 185 His Asp Ser Asp Leu Glu His Val Leu Thr Pro Ser 190 195 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 171-198 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17: He Thr Ser Glu Pro Thr Arg Pro Pro Pro Thr Pro Pro Pro Leu Val 171 175 180 185 His Asp Ser Asp Leu Glu His Val Leu Thr Pro Ser 190 195 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 173-200 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:18: Ser Glu Pro Thr Gin Pro Pro Pro Thr Ser Pro Pro Leu Val His Asp Ser 173 175 180 185 Asp Leu Glu His Val Leu Thr Pro Ser Thr Ser 190 195 200 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 173-204 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:19: Ser Glu Pro Thr Gin Pro Pro Pro Thr Ser Pro Pro Leu Val His Asp 173 175 180 185 Ser Asp Leu Glu His Val Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys 190 195 200 (2) INFORMATION FOR SEQ ID N0:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 176-209 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20: Thr Gin Pro Pro Pro Thr Ser Pro Pro Leu Val His Asp Ser Asp Leu 176 180 185 190 Glu His Val Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys He Leu Lys 195 200 205 Phe He (2) INFORMATION FOR SEQ ID N0:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 176-209 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21: Thr Arg Pro Pro Pro Thr Pro Pro Pro Leu Val His Asp Ser Asp Leu 176 180 185 190 Glu His Val Leu Thr Pro Ser Thr Ser Trp Thr Thr Lys Met Leu Lys 195 200 205 Phe He (2) INFORMATION FOR SEQ ID N0:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( i) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 228-257 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22: Ser Leu Ser Ser Trp His Val Leu Tyr Thr Pro Asn He Ser He Pro 228 230 235 240 Gin Gin Thr Ser Ser Arg Thr He Leu Phe Pro Ser Leu Ala 245 250 255 (2) INFORMATION FOR SEQ ID N0:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 186-195 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:23: Val His Asp Ser Asp Leu Glu His Val Leu 186 190 195 (2) INFORMATION FOR SEQ ID N0:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 83-108 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24: Trp He Lys Lys Pro Asn Arg Gin Gly Leu Gly Tyr Tyr Ser Pro Ser 83 85 90 95 Tyr Asn Asp Pro Cys Ser Leu Gin Cys Pro 100 105 (2) INFORMATION FOR SEQ ID N0:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 115-135 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: Pro Pro Ser Pro Glu Ala His Val Pro Pro Pro Tyr Val Glu Pro Thr 115 120 125 130 Thr Thr Gin Cys Phe 135 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 111-129 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:26: Pro Pro Pro Pro Pro Pro Ser Pro Glu Ala His Val Pro Pro Pro Tyr 111 115 120 125 Val Glu Pro (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 109-127 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:27: Thr Pro Pro Pro Pro Pro Pro Pro Ser Pro Glu Ala His Val Pro Pro 109 110 115 120 Pro Tyr Val 125 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ( ) MOLECULE TYPE: peptide (viii) POSITION IN GENOME: (B) MAP POSITION: 107-125 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:28: Thr Thr Thr Pro Pro Pro Pro Pro Pro Pro Ser Pro Glu Ala His Val 107 110 115 120 Pro Pro Pro 125

Claims: What is claimed is: 1 . A method for differentiating antibodies against HTLV-I from antibodies against HTLV-II in a test sample, comprising: a determining the presence of antibodies against HTLV-I in a test sample, said determination comprising: i . contacting a test sample with at least one peptide specific for HTLV-I, to form a mixture; ii . incubating the mixture for a time and under conditions sufficient for antigen/antibody complexes to form; iii. contacting said complexes with an indicator reagent comprising a signal generating compound attached to an anti-human IgG antibody, to form a second mixture; iv. incubating said second mixture for a time and under conditions sufficient for antigen/antibody/antibody complexes to form; v. determining the presence of antibodies against HTLV-I by detecting the measurable signal; b. determining the presence of antibodies against HTLV-II in the test sample, said determination comprising: i. contacting the test sample with at least one peptide specific for HTLV- II, to form a mixture; i i . incubating the mixture for a time and under conditions sufficient for antigen/antibody complexes to form; iii. contacting said complexes with an indicator reagent comprising a signal generating compound attached to an anti-human IgG antibody, to form a second mixture; iv. incubating said second mixture for a time and under conditions sufficient for antibody/antigen/antibody complexes to form; v. determining the presence of antibodies against HTLV-II by detecting the measurable signal c. determining the pattern of reaction of the test sample for antibodies against HTLV-I and HTLV-II to distinguish between HTLV-I and HTLV-II infections. 2. The method of claim 1 wherein said peptide specific for HJLV-I comprises at least one peptide selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. . , . . . , . . . , . . . , . . . , . . . 7, SEQ. ID. NO.8, SEQ. ID. NO. 9, SEQ. ID. NO. 10, SEQ. ID. NO. 11 , SEQ. ID. NO. 12 and SEQ. ID. NO. 13. 3. The method of claim 1 wherein said peptide specific for HTLV-II comprises at least one peptide selected from the group consisting of SEQ. ID. NO. 14, SEQ. ID. NO. 15, SEQ. ID. NO. 16, SEQ. ID. NO. 17. SEQ. ID. NO. 18, SEQ. ID. NO. 19, SEQ. ID. NO. 20, SEQ. ID. NO. 21, SEQ. ID. NO. 22, SEQ. ID. NO.23, SEQ. ID. NO.24, SEQ. ID. NO.25, SEQ. ID. NO.26, SEQ. ID. NO.27 and SEQ. ID. NO. 28. 4. The method of claim 1 wherein said peptide specific for HTLV-I comprises at least one peptide selected from the group consisting of SEQ. ID. NO. 1 , SEQ. ID. NO. 5 or SEQ. ID. NO. 7 and wherein said peptide specific for HTLV-II comprises at least one peptide selected from the group consisting of SEQ. ID. NO. 16, SEQ. ID. NO. 17, SEQ. ID. NO. 24 and SEQ. ID. NO. 27. 5. A peptide specific for HTLV-I having SEQ. ID. NO. 1 , SEQ. ID. NO. 5, or SEQ. ID. NO.7. 6. A peptide specific for HTLV-II having SEQ. ID. NO. 16, SEQ. ID. NO. 17, SEQ. ID. NO.24 or SEQ. ID. NO.27. 7. An assay kit for differentiating HTLV-I from HTLV-II, comprising: a container containing at least one peptide specific for HTLV-I attached to a solid phase; and a container containing at least one peptide specific for HTLV-II attached to a solid phase. 8. The kit of claim 7 wherein said peptide specific for HTLV-I comprises at least one peptide selected from the group consisting of SEQ. ID. NO. 1 , SEQ. ID. NO. 2, SEQ. ID. NO.3, SEQ. ID. NO. 4, SEQ. ID. NO.5, SEQ. ID. NO. 6, SEQ. ID. NO.7, SEQ. ID. NO. 8, SEQ. ID. NO. 9, SEQ. ID. NO. 10, SEQ. ID. NO. 11 , SEQ. ID. NO. 12 and SEQ. ID. NO. 13. 9. The kit of claim 7 wherein said peptide specific for HTLV-II comprises at least one peptide selected from the group consisting of SEQ. ID. NO. 14, SEQ. ID. NO. 15, SEQ. ID. NO. 16. SEQ. ID. NO. 17. SEQ. ID. NO. 18. SEQ. ID. NO. 19, SEQ. ID. NO.20, SEQ. ID. NO. 21 , SEQ. ID. NO. 22, SEQ. ID. NO. 23, SEQ. ID. NO.24. SEQ. ID. NO.25, SEQ. ID. NO.26. SEQ. ID. NO.27 and SEQ. ID. NO.28. 1 0. The kit of claim 7 wherein said peptide specific for HTLV-I comprises at least one peptide selected from the group consisting of SEQ. ID. NO. 1 , SEQ. ID. NO. 5 or SEQ. ID. NO. 7 and wherein said peptide specific for HTLV-II comprises at least one peptide selected from the group consisting of SEQ. ID. NO. 16, SEQ. ID. NO. 17. SEQ. ID. NO. 24 and SEQ. ID. NO. 27.

Inventors: BURCZAK JOHN D, LEE HELEN H, O'DONNELL DEBRA L, SHIH JESSIE W, BURCZAK, JOHN, D., LEE, HELEN, H., O'DONNELL, DEBRA, L., SHIH, JESSIE, W.
Applicants: ABBOTT LAB, ABBOTT LABORATORIES
Application year: 1992
Main classification: C12Q1
IPC codes: C07K7, C07K14, C07K16, C07K17, G01N33