CA2179381A1

CA2179381A1 - Differentiation of htlv-i and htlv-ii using synthetic peptides

Info

Publication number: CA2179381A1
Application number: CA 2179381
Authority: CA
Inventors: Jessie W. Shih; John D. Burczak; Helen H. Lee; Debra L. O'donnell
Original assignee: Individual
Current assignee: Abbott Laboratories
Priority date: 1993-12-20
Filing date: 1994-12-20
Publication date: 1995-06-29
Also published as: JPH09510009A; AU1443695A; WO1995017678A2; EP0736179A1; WO1995017678A3

Abstract

Methods of differentiation of sera containing antibodies to HTLV-I from sera containing antibodies to HTLV-II are provided, along with peptides for their differentiation. Articles of manufacture containing these peptides are provided which allow for the differentiation of HTLV-I from HTLV-II infected sera.

Description

~ WO 95/17678 2 1 7 q 3 8 1 PCT/US94/]4815 DIFFERENTIATION OF HTLV-I AND
EITLV-II USING SYNTHETIC pEpTrnE~s This is a continuation in part application based on parent application, Serial No. 07/727,765, filed July l0, 199l.
.

BA('KC~ROUND OF Tl~ INVE~TION
This invention relates generally to a method for the detection of antibodies to Human T-Cell Lymphotropic Virus types I and II (HTLV-I
lo 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. Fri~ mi~ gical 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approximately 50% of U.S. blood donors confirmed seropositive for HTLV-I are in fact infected with HTLV-II (Lee et al., unpublished observation).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 zo 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) z5 and R~lioimmunoprecipitation 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 Iymphocytes 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 SUBSTITUTE SHEET ~RULE 26) usefulness in that they are time consuming, expensive, require specialized facilities and are not easily ~lltnm~t~
United States Patent Nos. 4,525,300 and 4,804,476 to Yoshida teach methods for preparing antibodies to human leukemia virus related 5 peptides. The antibodies disclosed are capable of binding to human leukemia virus.
Palker et al., T. Immunol, 135 (1):247-254 (1985) report the dldliOll of mnnnrlnn~l antibodies reactive with HTLV-I which were raised to synthetic peptides ~ g sections of the pl9 ~g internal o 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 HTLV-I
and HTLV-II in samples by means of in~llh~tin~ samples with synthetic 5 or cloned polypeptides 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 rl~t~rmin~tinn by methods such as SDS PAGE electrophoresis. However, 20 SDS PAGE electrophoresis is not easily ~lltnm~ted 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 ~nnho~ c specific to HTLV in test samples.
2~ European Patent Application No. 0 267 622 to Masanori, published May 18, 1988, teaches a device comprising a fused HTLV .g~ and env 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.

SUESTITUTE SHEET ~RULE 26) ~ wo 95/17678 2 1 7 9 3 8 1 pCTlus94~14XlS
Palker et al. T. Immunol, 142:g71-978 (1989) report the mapping of the immunogenic regions of the HTLV-I gp46 and gp21 env proteins and the synthesis of peptides which are useful in the generation of specific monoclonal antibodies. These peptides may be used in immllnn~cc~ys to s detect ~n~ihorliPc to HTLV. ~rlrli~inn~lly, 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. W089/08664 (PCT/SE89/00126) to Vahlne et ~o al., published September 21, 1989, teaches of further synthetic peptides, derived from the env region of the HTLV-I genome, which may be used in the detection of Antihnr~iPc 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 Binmr-r~ir~l Inc., published July 26,1990, describes synthetic peptides for the detection of H~LV-I reactive antibodies and diagnosis of ATL (adult T cell leukemia/lymphoma). These peptides are from the tr~ncmpmhrane (p21e) and external (gp46) segments of the envelope protein of HTLV-I.
Also described are immllnn~cc~ys using these peptides. The peptide(s) 20 described are used in the SynthEIA~9 (Olympus Corp., Lake Success, NY) for HTLV-I.
PCT Publication No. WO90/10231 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 2~ derived from the g~g and env 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. Peptides disclosed in Blomberg also show si~nifir~n~ cross-reactivity. Blomberg improved the 30 [1icrrimin7~ion of infected sera by compiling all results and multiplying SU~STITUTE SHEET (RULE 26) the absorbances with weights according to the relative abi~ity of each peptide to ~1icrriminAtP between HTLV-I and HTLV-II. The weighted absorbances were then input into a computer program to calculate "points" for either HTLV-I or HTLV-II, ~ iv~ly. According to Blomberg, using this serotyping technique, no false typing results were obtained, but a small number were found to be "not typable."
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
0 specific antibodies. Several of the peptides are capable of distinguishing between HTLV-I and HTLV-II infection.
Recently, R. B. Lal et al. desçribed the serologic discrimination of HTLV-I from HTLV-~ using syntheFlc peptides which would be used to differentiate between HTLV-I and HTLV-II. R. B. Lal et al., T. Infeçtious Diseases 163:41~6 (Tanuary, 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 ~istinr~inn between HTLV-I and HTLV-II. With the exception of this recent article and the two patent applications which describe differentiation (Vahlne, WO90/15820 and Blomberg, WO90/10231), 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 detPrminAn~c on the two viruses must be i(iPn~ifierl Antigenic de~PrminAn~q on proteins have been predicted by the i~iPn~ifirAtit7n of hydrophilic regions using the method of Hopp and Woods, Proc. Natl. Acad. Sci. U.S.A. 78:3824.~1981) as well as i~lPntifirA~irn of flexible regions using the method of Karplus SIJBS~ITUTE SHEET ~RULE 26) and Schultz, Naturwissenschaften 72:212-213 (~985). Antigenic detf~rmin~nt~c appear to be located at hydrophilic as well as flexible regions of protein SeqllPnrPC Antigenic ~lPt~rmin~n~c have also been empirically identified by immunological e~min~tinn of peptides 5 produced by protein degradation or in vitro synthesis.
Prior art methods for differentiating between HTLV-I infection and HTLV-II infection using peptide sequences from HTLV-I or HTLV-II
have had the problem that HTLV-I derived peptides have been cross-reactive with sera infected with HTLV-II, and HTLV-II derived peptides o have been cross-reactive with sera infected with HTLV-I. Prior art methods have also demonstrated cignifi~-Ant sensitivity problems with respect to detecting antibody in sera. The present inventors believe that the addition or deletion of amino acids to a peptide cignifif~ntly inflll~nrf~c its ability to bind to antibodies. As a result, it is possible to 15 ci~nifir~ntly improve the ,u~lru~ -llce of assays by altering the length of peptides from unique imml]nr)~l~minant regions of E~TLV-I and HTLV-II. What were believed to be previously unknown antigenic regions as well as known antigenic regions were surveyed and systematically examined to identify peptide sequences which represent ci~nifi~-~nt 20 antigenic epitopes, which are strain specific between HTLV-I and HTLV-II and which show little cross-reactivity between strains.
SUMM ~R~ OF Tl T~ J~VENTION
It is an object of the present invention to provide a superior 25 peptide assay for dirr~ il.g HTLV-I and HTLV-II.
It is another object of the present invention to provide an assay for differentiating HTLV-I and HTLV-II using peptides having increased selectivity and thus decreased cross-reactivity between HTLV-I and HTLV-II.

SlJBSTITU~E SHEET (RU~E

wo 95/17678 2 1 7 9 3 8 1 PCT/US94/1481~ ~
It is another object of the present invention to provide an assay for differentiating HTLV-I and HTLV-II which shows superior sensitivity for detecting antibody in sera.
It is yet another object of the present invention to provide a metkod which would be a useful tool to tke physician, allowing a more detailed diagnosis of the disease and therefore more appropriate patient counseling and treatment.
It is yet another object of the present invention to provide a simple but reproducible HTLV-I and HTLV-II differentiation assay which 0 facilitates routine laboratory usage and generates sensitive and specific results.
The amino acid sequences according to the present invention 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
ls 80:3618-3622 (1983). The peptides specific for HTLV-I include the following: HTLV-I env-1 (SEQ. ID. NO. 1), HTLV-I env-2 (SEQ. ID. NO.
2), HTLV-I env-3 (SEQ. ID. NO. 3), HTLV-I env-4 (SEQ. ID. NO. 4), HTLV-I env-5 (SEQ. ID. NC~. 5), and HTLV-I env-6 (SEQ. ID. NO. 6). The peptides specific for HTLV-I further includes HTLV-I ~g-1 (SEQ. ID. NO.
7), HTLV-I ~g-2 (SEQ. ID. NO. 8), HTLV-I g~-3 (SEQ. ID. NO. 9), HTLV-I
g~-5 (SEQ. ID. NO. 10l, HTLV-I g~g-6 (SEQ. ID. NO. 11), and HTLV-I ~a~7 (SEQ. ID. NO. 12).
The amino acid sequences according to the present invention for tke HTLV-II synthetic peptides were obtained from the predicted amino acid sequences af tke two HTLV-II IJlUlULy~s. ~The amino acid sequence of tke Mo HTLV-II was published by ~hinnn~nhnn et al., Proc. Natl. Acad.
Sci. USA 82:3101-3105 (1985). Tke sequence of the NRA HTLV-II
prototype is unpublished data. The peptides specific for HTLV-II include the following: HTLV-II env-1 ~SEQ. ID. NO.: 13), HT~V-II env-lA (SEQ.
ID. NO. 14), HTLV-II env-2 (SEQ. ID. NO. 15), HTLV-II env-2A (SEQ. ID.

SUBSTITUTE SHEET (RULE 26) WO 95/17678 2 1 7 9 3 8 1 PCT/llS94/14815 NO. 16), HTLV-II env-3 (SEQ. ID. NO. 17), HTLV-II env-4 (SEQ. ID. NO.
18), HTLV-II env-5 (SEQ. ID. NO. 19), HTLV-II env-5A (SEQ. ID. NO. 20), HTLV-II env-6 (SEQ. ID. NO. 21) and HTLV-II env-8 (SEQ. ID. NO. 22).
The peptides specific for HTLV-II further include HTLV-II gag-2 (SEQ. ID.
NO. 23), HTLV-II ~-3 (SEQ. ID. NO. 24), HTLV-II g~g-4 (SEQ. ID. NO.
25). Peptides HTLV-II env-lA, HTLV-II env-2A, and HTLV-II env 5A are NRA seql7Pnl P~. All the other remaining HTLV-II sequences are Mo sequences.
According to the present invention, a method for differentiating o antibodies against HTLV-I from antibodies against HTLV-II in a test sample is provided comprising: cnntA~tin~ the test sample with at least one peptide specific for HTLV-I to form a mixture, the peptide(s) specific for HTLV-I being 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 and SEQ. ID. NO. 12; inrllhAt;n~ the mixture for a time and under ~ nllitinn~ sufficient for antigen/antibody complexes to form;
fnntA. tin~ the r~mrlPYPC with an indicator reagent comprising a signal ~PnPrAtin~ compound attached to an anti-human IgG antibody to form a second mixture; incubating the second mixture for a time and under rnnfiition~ sufficient for antigen/antibody/antibody complexes to form;
and determining the presence of antibodies against HTLV-I by detecting the measurable signal.
In another embodiment of the invention, a method for differentiating antibodies against HTLV-I from antibodies against HTLV-II in a test sample is provided comprising dP~ the presence of antibodies against HTLV-II in the test sample by fontArtin~ the test sample with at least one peptide specific for HTLV-II selected from the group consisting of SEQ. ID. NO. 13, 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.

SUE;STITUTE SHEET (RULE 26) 2l 7q381 ID. NO. 20, SEQ. ID. NO. 21, SEQ. ID. NO. 22, SEQ. ID. NO. 23, SEQ. ID.
NO. 24 and SEQ. ID. NO. 25 to forrn a mixture; inrllhAhn~ the mixhure for a time and under rr~nr1itir~nc sufficient for antigen/antibody complexes to form; contacting the rr~mrleYpc with an indicator reagent comprising a s signal generating compound attached to an anti-human IgG antibody, to form a second mixture; inrllhAt;n~ said second mixture for a time and under rr~nflitir~nc sufficient for antigen/antibody/antibody rrlmrl~Yrc to form; and determining the presence of Anhh~rlirc against HTLV-II by detecting the measurable signal.
0 In a preferred Pmhor~imr-nt of the invention, a first and a second assay are performed as above, the first assay romrrisin~ rr,ntArfin~ a test sample with a peptide from HTLV-I according to the invention, and a second assay rr,mrricin~ rr~ntAr~ing a test sample with a peptide from HTLV-II according to the invention.
A still more preferred embodiment of the invention comprises rnntArtin~ said test sample with SEQ. ID. NO. 1 in the first assay, and rrlntArtin~ said test sample with one of SEQ. ID. NOS. 15 or 16 in the second assay.
A most preferred method according to the present invention provides for differentiation of HTLV-I and HTLV-II infected sera by performing two assays, as above, the first assay comprising rcmtArhn~ a test sample cim~lltAnrously with two peptides derived from HTLV-I, wherein the first peptide is chosen from the group consisting of SEQ. ID.
NOS. 1, 2 and 6, and the second peptide is chosen from the group 2s consisting of SEQ. ID. NOS. 3, 4 and 5. The second assay comprises cimlll~AnrQusly rrlntArtin~ the test sample with two peptides derived from HTLV-II, wherein the first peptide is chosen from the group consisting of SEQ. ID. NOS. 13, 14,15 and 16 and the second peptide is SEQ. ID. NO. 22. Results from both assays are analyzed to determine the ~UBST~T~)TE SHFET r~U! ' 2 WO 95/17678 PcT/US94/1481~i 2 ~ 793~ 1 pattern of reaction of the test sample for antibodies against HTLV-I and HTLV-II to disting ush between HTLV-I and HTLV-II infections.
The invention also provides for an article of manufacture comprising pArkA~in~ material ron~Ainin~ a first and second container, the first container including a solid phase having attached thereto a peptide of the present invention specific for HTLV-I, and the second container including a solid phase having attached thereto a peptide of the present invention specific for HTLV-II, wherein the packaging material comprises a label on each of said ~ illl'lh which indicates that lo the contents thereof may be used to differentiate sera infected with HTLV-I from sera infected with HTLV-II.
The invention, together with further objects and attendant advantages, will best be understood by reference to the following description, examples and tables. However, the invention is not limited thereto.
DE~T.ATT.T;n DF~('RTT~IION OF T~TF. INVFNTION
The present 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 novel and unique synthetic peptides disclosed herein.
The present invention identifies novel and unique peptide sequences in regions of HTLV-I and HTLV-II which are useful in assays to detect and differentiate serum which contains antibodies to either of these viruses. For these purposes, unique peptide sequences are provided in several antigenic regions of HTLV-I env and g~ that are specific for HTLV-I and do not ci~nifil-~nily cross-react with sera having antibodies to 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 80:3618-22 (1983).

SLIBSTITUTE SHEET (RULE 26) ~ l / t~ 8 1 The present invention also identifies novel and unique peptide sequences in HTLV-II gp46 env and~g that are specific for HTLV-II and do not cross-react with serum having antibodies to H~LV-I. The amino acid sequences for the HTLV-II synthetic peptides were obtained from the 5 predicted amino acid sequences of two HTLV-II ~-uLuly~e~, Mo and NRA. 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, and is included in pending patent application Serial No. 08/086,415 filed o July 1, 1993, assigned to Abbott Laboratories and the Regents of the Ur~vers~lty of California.
In general, the method of the invention comprises rr~ntArtin~ a test sample with a solid phase to which at least one HTLV-I or at least one HTLV-II peptide is bound, to form a mixture. The mixture is 15 incubated for a time and under conditions sufficient for antigen/antibody . ..I.,r,l,~ 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 fnnrlitir,ns sufficient to form antigen/antibody/antibody complexes. The presence of immobilized antibody is fiPtPrminpcl 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 may be any variety of materials which may be selected by one skilled in the art without undue eXperimPntAti-n The term "solid phase" is used in a broad sense and refers to any material which is insoluble, or may be made insoluble by a subsequent reaction. Thus, porous or non-porous materials, latex or polystyrene particles, microparticles, beads, membranes, plastic tubes, SUBST~TUTE SHEET (RULE 2~) W0 95/17678 2 1 7 ~ 3 8 I PCT/US94114815 walls of microtiter wells and tanned sheep red blood cells are all suitable examples. The size, rlimPncirmq, and shape of the solid phase are not generally aitical in the methods of the invention. However, the present invention preferably envisions the use of miaoparticles when more than one peptide specific for HTLV-I or more than one peptide specific for HTLV-II is imm~bili7~d on a solid phase.
Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like. Those ski~led in the art will recognize the scope of mPth~ lngies which may be o applied relative to the application of useful solid phases. Linking agents known in the art may also be utilized to secure 7~ -hnn~nt of a peptide to the solid phase. The linking agent may be incorporated as part of, or derivatized onto, the solid phase before the peptides are added.
The "test sample" may be a sample of human or animal biological 15 fluid, such as serum, plasma, ascites, urine, cerebral spinal fluid or any other body c~nc~ n~c, or any tissue culture supernatants which may contain antibodies of interest.
A suitable "indicator reagent" may be a signal generating compound (label) which is capable of generating a measurable signal 20 detectable by external means ronj~ ~d (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 may be a member of any specific binding pair, including either hapten-anti-hapten such as biotin 25 or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a ~-r)mrl~nn~n~ry 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) ~r)n~pmr~ d 30 include chromogens, catalysts such as enzymes, Illmin~c~;~n~ compounds Il S~lscTlTuTE SHECT (RULE 20!

WO 95tl7678 PCTI~JS94/14815 21793~1 --such as fluorescein and rhodamine, chemi~-lminPcrf~nt compounds, radioactive elements, and direct visual labels. Examples of enzymes include alkaline rh~SphAtACi', horseradish peroxidase, beta-~AlAr~Ci~AC~, and the like. The selection of a particular label is not critical, but it will 5 be capable of producing a signal either by itself or in conjunction with one or more AA~i~ionAl substances.
The reaction mixture is incubated for a time and under conditions sufficient for HTLV antigen/antibody complexes to form. Selecting a~ u~lidl~ times, temperature, and other f~n-liti~-nq of the in~ hAIion lo are well within the skill in the art.
The methods employed in the description and examples desaibed below were performed according to standard molecular genetics techniques known in the art, such as those desaibed in Maniatis et al., Molecular (~lonin~ A l aboratsry ~anual Cold Spring Harbor (1982). In 5 one embodiment of the present invention, a synthetic peptide of the invention which is specific for Antihor1i~c against HTLV-I 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 con~ nc and for an appropriate period of time during which 20 time Antih~-liPs 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 in~ llhAtinn then is performed in which the bead is incubated with an indicator reagent comprising anti-human antibodies labelled with an appropriate signal ~ ,Ling compound. By 2s 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 in the test sample is determined. The innmllnoAccAy is then repeated as stated above, except that a synthetic 30 peptide according to the present invention which is specific for ~ WO 95/17678 2 1 7 9 3 ~ I PCT/US94114815 antibodies against HTLV-II is immobilized on the solid phase. A
r~ of the pattern of reactivity of the test sample for antibodies agamst HTLV-I and antibodies against HTLV-II allows differentiation between infections with the two viruses.
In another embodiment, a synthetic peptide of the invention specific for HTLV-I is immobilized on polystyrene beads. The beads are then in~l~hAtP11 with a test sample or a diluted test sample and an appropriate indicator reagent romrriSin~ a signal generating compound attached to anti-human IgG, under t~-nllitinnc and for an appropriate period of time to allow antibodies to bind specifically to the immobilized peptides on the bead and simultaneously to the indicator reagent. The amount of labelled anti-human antibody complex immobilized on the bead may be ~l~ptprminpd by detecting the measurable signal gPnPrAtP~l Thus, the presence of antibodies against HTLV-I in the test sample may be determined by a single incubation. The immunoassay is then repeated, as stated above, with a synthetic peptide according to the invention which is specific for HTLV-II. A ~ . ;c.~" of the pattern of reactivity of the test sample to AntihQ-liPc against HTLV-I and antibodies against HTLV-II allows differentiation between infections with the two viruses.
In yet another embodiment, at least one of the synthetic peptides of the invention specific for antibodies against HTLV-I or at least one of the synthetic peptides of the invention specific for antibodies against HTLV-II, are immnbili7P(l on a nitrocellulose membrane. The peptide also may 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 rnn~1iti~nc sufficient for antigen/antibody ~`~mrl~PyPC
to form. The membrane surface is then washed to remove unbound Sl,P,STITUTE SHEET (RULE 26) WO 95/17678 2 1 ~ ~ 3 3 1 PCTNS94/14815 proteins, and in a second in~llhAfi~n, 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, and thus the presence of 5 antibodies against either HTLV-I or:HTLV-II, is 11Pt~rmin~l by detecting the measurable signal generated with a suitable detection system.
QIlAntififAti~n of the level of signal recognized by the detection system allows the qll~ntifi~Atilm of the amount of specific antibody present in a test sample. A ( .""~ ic(", of the pattern of reactivity of the test sample o to antibodies against HTLV-I and antibodies against HTLV-II allows differentiation between infections with the two viruses.
In still another embodiment, a sandwich assay is utilized. This method comprises contacting a test sample with a solid phase to which at least one HTLV-I peptide or at least one HTLV-II peptide is bound to 5 form a mixture. The mixture is inrllhAt.od for a time and under ,-nn~litif)n~ sufficient to allow antigen/antibody complexes to form. Then the ~lmrl~c are contacted with antigen to which as been ~njll~At~ri a signal generating compound, to form a second mixture. The second mixture is incubated and the presence of the antigen/antibody/antigen 20 complex is ~ tPrminl~l by detecting the measurable signal generated. A
comparison of the pattern of reactivity of the test sample for antibodies against HTLV-I and antibodies against HTLV-II allows differentiation between infections with the two viruses.
In still another embodiment, a combination of two peptides of the 25 invention specific for HTLV-I are co-coated on a single solid support, for example, by immobilization on polystyrene beads. The procedure for co-coating of peptides is essentially the same as coating a single peptide, as described above, and in the examples, infra. Briefly, the peptides are individually dissolved into a stock solution at a suitable concentration.
30 Aliquots of the two peptides are then added together into the coating l4 SU~STITUTE SHEET (RULE 26) ~ W095/17678 2~ 7~381 pCT/US9U14815 solution for the beads. Preferably, the two peptides are added at equal concentrations, however, differing proportions of each peptide may also be used. The coated beads are then used in an immllnnAsc~y according to any of the above-methods. The procedure is then repeated with beads 5 having co-coated thereon two peptides according to the invention which are specific for HTLV-II. A "~ I of the pattern of reactivity of the test sample to antibodies against HTLV-I and antibodies against HTLV-II
allows differentiation between infections with the two viruses.
A preferred embodiment comprises the use of two peptides 0 specific for HTLV-I or two peptides specific for HTLV-II being bound to a microparticle solid phase. Microparticle EIA (MEIA) are preferably conducted with the use of polystyrene microparticles. The size of these particles is ~l~r~ldbly between 0.19-5 microns. The protein may be bound either passively or actively on the particle. Passive coating is intended to 15 mean non-covalent bonding or Aff~hm~nf between the peptide and the microparticle~ An example of passive coating involves dissolving peptides in a stock solution with sterile water. The peptides are then diluted in a suitable coating buffer at twice the desired final coating concentration. The microparticles are washed and resuspended in a 20 buffered salt coating solution. The microparticle and peptide solutions are mixed in equal proportions and incubated at a suitable temperature and length of time. At the end of the coating procedure, the coated microparticles are isolated by centrifugation, washed, and resuspended in a microparticle diluent. Due to the nature of the microparticle, the 25 peptides become bound to the microparticle through electrostatic interactions or the like.
Active coating is intended to mean the effecting of a covalent bond between the peptide and the solid support. Generally, such a covalent bond is formed by either the carboxy or the amino terminal end of the 30 peptide binding to an appropriate functional group on the surface of the SU~STI~UTE SHEET (RU~E ~5?

217~381 microparticle. Microparticles having sucl~ functional groups are termed derivatized microparticles. An example of a derivatized microparticle has a carboxy functional group on its surface. The carboxy derivatized microparticle is then treated with 1-ethyl-3-(dimethyl-5 aminopropyl)carbodumide hydrochloride (EDAC). Subsequently, themicroparticle is processed in a similar fashion to the passively coated microparticle procedure except that the pH of the solution should be 4.5.
The EDAC may be added to the final coating solution simultaneously with the peptides and microparticles, or to either the peptides or 10 microparticles prior to mixing. The resulting coated microparticle has the peptides bound thereto bçcause Qf reaction between the amino terminal ends of the peptides and the carboxy group on the microparticles. The use of EDAC with a non-derivatized microparticle will also result in an active coating.
Two approaches are envisioned for the use of microparticles coated with two peptide sequences in the same assay. First, the microparticles may be co-coated. In co-coating, the peptides are individually dissolved into a stock solution at a suitable concentration and subsequently added together. To the peptide mixture, the microparticles are added. Preferably, however, a first quantity of microparticles is coated with a first peptide sequence, and a second quantity of microparticles is coated with a second peptide sequence independently of each other. Sl~h~q~ nt to individual coating, the first and second quantity of coated microparticles are combined for use in an assay. This preferred technique facilitates qll~ntifirAtilm of the amount of each peptide actually present on the microparticle beads.
The methods of the present invention may be adapted for use in systems which utilize automated and semi-~llt~m~ d 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 SUBSl ITUTE SHEE~ (RULE 2f.

~ W095117678 2 1 7 9 ` 8;1 PCT/US94J14815 correspond to published EPO applications Nos. 0 425 633 and 0 424 634, ,e~liv~ly, which are incorporated herein by reference.
Other embodiments which utilize various other solid phases also are contPmrl~tPd and are within the scope of this invention. For 5 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 EPO publication 0 326 100, and U.S. Patent Application Serial No. 375,029 (EPO publication 0 406 473) both of which enjoy common ownership and 0 are incorporated herein by reference, may 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 negatively charged poly-anion/immune complex and the previously treated, 15 positively charged porous matrix and detected by using various signal generating systems previously described, including those described in rhPmill~minPcrPnt signal me.. ,~ m~ 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 20 incorporated herein by reference.
The use of scanning t-~nnPIIin~ microscopy for immllnrl~ccays 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 25 peptides of the invention, is adhered to a solid phase and a scanning probe microscope is utilized to detect antigen/antibody r~mrlPYPc which may be present on the surface of the solid phase. The use of scanning tl-nn~lling microscopy Plimin~tPc the need for labels which normally must be utilized in many immunoassay systems to detect 30 antigen/antibody complexes. Such a system is described in pending U.S.
.

SUBSTITUTE SHEET (RULE 26) Patent Application Serial No. 662,14~, which enjoys common ownership and is incorporated herein by reference.
While the present invention discloses a preference for the use of solid phases, it is rr~nt~nnrl~t~ll that the peptides of the present invention 5 may 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.
Another embodiment is envisioned in which only peptides specific for HTLV-I or pepfiaes specific for HTLV-II are utilized in an 0 immunoassay according to any of the methods described above. The specificity of the peptides disclosed herein, and the lack of cross-reactivity shown to be exhibited by these peptides, enables the use of these peptides in a single assay which will provide superior results. Thus, a single immunoassay using either one or more peptides according to the 5 invention specific for HTLV-I or one of more peptides according to the invention specific for HTLV-II, results in increased specificity and selectivity with respect to infection by either HTLV-I or HTLV-II.
However, because of the superior differentiation results obtained, it is preferred that two assays be performed, a first assay utilizing one or more 20 peptides specific for HTLV-I and a second assay utilizing one or more peptides specific for HTLV-II, when differentiating between sera infected with HTLV-I or HTLV-II.
Accordingly, tests which detect specific antibodies against HTLV-I
separately from antibodies against HTLV-rl may be designed by selecting 25 appropriate synthetic peptides of each of these viruses, and coating them onto solid phases, thereby f~rjljt~tin~ the differential diagnosis of the two viral infections. Peptides suitable for the specific detection of antibodies against HTLV-I are specified herein and comprise the peptides of the HTLV-I env region and the peptides of the HTLV-I g~ region which are 30 (1~ci~n~t~1 as SEQ. ID. NO. I through SEQ. ID. NO. 12. Peptides suitable St,'~SrlTL~E S~EET '~U~E "6 WO 95/17678 PCTiUS94/14815 for the specific detection of antibodies against HTLV-II also are specified herein and comprise the peptides of the HTLV-II env region, and the peptides of the HTLV-II g~g region, and are (lt~ci~n~h~cl as SEQ. ID. NO. 13 through SEQ. ID. NO. 25.
It is rnnt~mrl~tf~d that the reagent employed for the assay may be provided in the form of a kit with one or more ron~inPr.c such as vials or bottles. Each container or vial contains 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.
0 The kit would also include instructions which indicate that the contents thereof may be used to differentiate between HTLV-I and HTLV-II
infection.
The following examples and related tables are intended to further illustrate the invention. It will be understood, however, that the invention is not limited to these specific examples or the embodiments expressed therein.
FXAMPT .F~
EXAMPJ.F 1 DP~P~ tinn of AnfihodiPc ;~ nct HI~LV-I
Peptides were ~y~ LI by stepwise addition of amino acids to a solid phase using procedures known in the art and described in Merrifield T. Am. Chem Soc 85:2149-2154 (1963) and Barany and M~rrifi~ 1 in E. Gross and J. Meienhofer, eds., 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 performed starting with the C terminus and progressing to the N
terminus. N-protected amino acids were used, with N being t-butylo;~yL~IlbL~llyl (t-Boc). The C t~rminl1m t-Boc amino acid was ~9 SUBSrlTUTE SHEET (RULE 26) WO 95/17678 PCT/US9411481~ ~
217~38 l 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'-dicyclohexy~carbodiimide) and then d~ ,L~.L~d 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 a polystyrene bead solid support for capture of antibodies against HTLV-I
0 using procedures known in the art. Briefly, the polystyrene beads were washed with distilled water and incubated at 40C for two (2) hours with between 0.01 llg/ml and 50 llg/ml of peptide(s) in a phosphate buffered saline (PBS) solution. The beads were washed once with PBS ~nf~ining 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 thèn dried.
Beads used im this example as the solid phase for the detection of antibodies to HTLV-I were coated with peptide HTLV-I env-1 (SEQ. ID.
NO. 1) corresponding to amino acids 174-204.
Anti-human IgG antiserum was prepared by imml~ni7ing goats with purified human IgG, according to known methods. The resulting antiserum was affinity purified and labelled with Horseradish Peroxidase ~RPO).
~h~
Samples were diluted in sample diluent buffer between dilution factors of 1:2 and 1:1000. 200 Ill of the diluted sample was incubated with a coated bead in a reaction tray for 60 minutes at 40C. After thorough washing, the beads were incubated with goat anti-human HRPO diluted in a suitable diluent, for 30 minutes at 40C. The beads were again SUBSTITUTE SHEET (RULE 26 ~ wo 95/17678 2 1 7 q 3 8 1 PCT/US91/1481~i thoroughly washed. The amount of HRPO immobilized on the beads was quantified by inrllh~tin~ with an O-phenyl~nPrli~minl~ ~CI (OPD) reagent for 30 minutes at ambient room l~ . At the end of this incubation 1.0 ml of 1 N sulfuric acid was added to stop the color generating reaction. The degree of color generation was ~1~tPrmin.od by measuring the absorbance of the resulting solution at 492/600nm.
The method of the invention was applied to a panel of 100 test 0 samples that previously had been ~r~nfirm~d to be positive for antibodies against HTLV by the method of D.W. Anderson et al., Blood 74:2585-91 (1989~, with 50 samples confirmed positive for HTLV-I and 50 samples ~-rmfirme~l positive for HTLV-II by Polymerase Chain Reaction (PCR).
The method of the invention detected 48 of 50 (96%) samples positive for }5 antibodies against HTLV-I. ~fir1ition~11y, no .ci~nifir~nt cross-reactivity was detected with the 50 samples of HTLV-II infected sera.
F~MPLF 2 IC-' - ' of ,~nh'h~ c ~ ct HTLV-IT
~
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 inrllh~tf~l at 40C for two (2) hours with between 0.01 llg/ml and 50 llg/ml of peptide(s) in a PBS solution. The beads were washed once with PBS f~n~ining 0.15% Triton X-100(1~ for one (1) hour and blocked for one (1) hour with 2% BSA in PBS, then overcoated with 5% sucrose in PBS for 20 minutes at room temperature and then dried.

SUBSrlTUTE SHEEr (RULE 26) WO 95117678 PCTIUS94114~15 ,1 2 ~ 7938 ~
For the detection of antibodies against HTLV-II in this example, beads were coated with peptide HTLV-II env-2 (SEQ. ID. NO. 15) corrPcponrlin~ to amino acids 171-198 of the HTLV-II gp46 env protein.
Anti-human IgG antiserum was prepared by imml]ni7in~ goats 5 with purified human IgG. The resulting antiserum was affinity purified and labelled with HRPO according to standard methods known in the art.
~h~
Samples were diluted in sample diluent buffer between a dilution factor of 1:2 and 1:1000. 200 ~1 of the diluted sample was incubated with a coated bead in a reaction tray for 60 minutes at 40C. After thorough washing, the beads were incubated for 30 minutes at 40C with goat anti-human HRPO diluted in a suitable diluent. The beads were again thoroughly washed. The amount of HRPO immobilized on the beads was quantified by inrllh~ting with an OPD reagent for 30 minutes at ambient room temperature. At the end of this inrllh~tinn 1.0 ml of 1 N sulfuric acid was added to stop the color generating reaction. The degree of color generation was ~letprminpcl by mP lcllrin~ the absorbance of the substrate at 492/600nm.
~g~
When this method was applied to a panel of test samples which had been r~nfirmPd to be positive for antibodies against HTLV by the 25 method of Anderson (cited supra) and positive for HTLV-II by PCR, the test was able to detect 72 of 74 samples (97.3%). ~ lifir,n~lly, when tested against 50 samples confirmed positive for HTLV-I, cross-reactivity was detected for only 1 sample. This sample, however, was also borderline HTLV-II positive.
z SUBSTlTUrE SYEE~ (RULE 26) WO 9!i117678 2 ~ 7 9 3 8 1 PCTtUS94tl4815 F~AMPI.F. 3 RP~tivity ~f pPpti~1pc With HTLV-I An~ HTI.~V-II TnfP~`tP~ SPr:~
Reagents were prepared as for example 2 except that peptides for SEQ. ID. NO. I through SEQ. ID. NO. 25 were individually coated onto beads. The data presented below in Table 1 is a ~nmril~inn of the data generated for a panel of 28 r~nfirmPd HTLV positive samples. 14 of the samples were ~onfirmPd HTLV-I positive and the other 14 samples were l~nfirm~Pcl HTLV-II positive. The ~ùll~ulldillg ~Pci~n~inn of the SEQ.
10 ID. NO. is indicated in parenthesis.
As shown in Table 1, no cross-reactivity was observed with HTLV-Irsera for any of the peptides specific for HTLV-I, and no ci~nifir7n~
cross-reactivity was observed for HTLV-I sera with peptides specific for HTLV-II.
-SIJBSTITIJTE SHEEr ~ULE 26~

WO 9S/17678 , PCIIUS94/1481S
2~7938i ~k~
tivjty of Inl1;vi~ p~ ~ti-lPc HTLV-I HTLV-II
5Pe~tide .~mir~ A~ tivjty l~ tivjty SEQ. ID. 1 (HTLV-I env-l) 174-204 11/14 0/14 SEQ. ID. 2 (HTLV-I env-2) 180-213 10/14 0/14 SEQ. ID. 3 (HTLV-I env-3) 227=257 7/14 0/14 10SEQ. ID. 4 (HTLV-I env-4) 230-260 12/14 0/14 SEQ. ID. 5 (HTLV-I env-5) 237-260 9/14 0/14 SEQ. ID. 6 (HTLV-~env-6)' 190-213 4~14 0114 SEQ. ID. 7 (HTLV-I g~-l) 100-129 14/14 0~14 SEQ. ID. 8 (HTLV-I ~-2) I04-129 14/14 0/14 lsSEQ. ID. 9 (HTLV-I g~g-3) 109-129 5/14 0/14 SEQ.ID.lO(HTLV-Ig~-5) 100-119 10/14 0/14 SEQ. ID. 11 (HTLV-Ig~-6~ 100-127 13/14 0/14 SEQ. ID. 12 (HTLV-I g~g-7) 100-126 13/14 0/14 20SEQ. ID. 13 ~HTLV-I env-l) 167-198 0/14 9/14 SEQ. ID. 14 (HTLV-I env-lA)~ 167-198 0/14 9/14 SEQ. ID. 15 (HTLV-I env-2) 171-198 0/14 11/14 SEQ. ID. 16 (HTLV-I env-2A)' 171~-198 0/14 12/14 SEQ. ID. 17 (HTLV-I env-3) 173-200 0/14 7/14 2sSEQ. ID. 18 (HTLV-I env-4) 173-204 0/14 8/14 SEQ. ID. 19 (HTLV-I env-5) 176-209 0/14 8/14 SEQ. ID. 20 (HTLV-I env-5A)' 176-209 0/14 8/14 SEQ. ID. 21 (HTLV-I env-6) 228-257 0/14 3/14 SEQ. ID. 22 (HTLV-I env-8) 83-108~ 0/14 6/14 30SEQ. ID. 23 (HTLV-I g~g-2) 111-129 0/14 4/14 SEQ. ID. 24 (HTLV-I ~-3) 109-127 0/14 5/14 SEQ. ID. 25 (HTLV-I g~-4) 107-125 0/14 3/14 'These three sequences were derived from the NRA HTLV-II prototype.

SUE,STITUTE SHEET (RULE 26) WO 9~117678 2 1 7 9 3 8 1 PCT/US94J1481 FX~MPI.~ 4 Detection Of Antibodies Against HrLV-I And HTLV-II ~Jcir~ Co-('~ c 5 I:cial~
. Usmg the procedure as described in Example 1, beads were co-coated with a solution rnnt~inin~ a mixture of peptides according to the invention specific for HTLV-I. By co-coating beads, the present inventors discovered that it was possible to increase the sensitivity of the assay to 0 detect either HTLV-I or HTLV-II, while m~int~ining the level of selectivity against cross-reactivity. For example, antibodies in a given test sample may be detected by one peptide, but not by another peptide. The same two peptides, however, may give the opposite results for another sample. Thus, a co-coated bead with both peptides would detect antibodies in both samples. For this assay, the peptides were coated as in Example 1, except that the peptide coating solution included equal r~nrrntr~tir)nc of SEQ. ID. NOS. 2 and 5. An assay was c-~n~llrtrrl using the same panel of 28 sera samples as in Example 3. As illustrated in Table 2, when applied to the 28 member panel, the method of the invention was able to correctly identify 14 out of 14 (100%) of the samples positive for HTLV-I, with no ci~nifir~nt cross reactivity for 14 samples positive for HTLV-II. Similar results were obtained for assays run with combinations of SEQ. ID. NOS. 1 and 4, and SEQ. ID. NOS. 2 and 5.
Using the ~JlU~dlllt~ described in Example 1, peptides prepared as described above which were specific for HTLV-II were co-coated on polystyrene beads. Peptide rr,mhin~ti~nc used were SEQ. ID. NOS. 15 and 13, SEQ. ID. NOS. 15 and 14, SEQ. ID. NOS. 15 and 18, SEQ. ID. NOS. 15 and 19, SEQ. ID. NOS. 15 and 21, and SEQ. ID. NOS. 15 and 22. Results are tabulated in Table 2. As shown in Table 2, assays run with a ~-r,mhin~tjlm of HTLV-II specific peptides showed no ci~nifir~nt cross-reactivity with SUBSTIME SHEET ~RULE 26) WO 95/17678 PCT/US94/~4815 ~
2 1 7938 ~
samples c~-nfirm~d positive for HTLV-I, and excellent results with respect to detection of HTLV-II.
~k~2 1` " ' ' Of HTLV-I A;^-~1 HTLV-TT Wifh CO_('~
~ HlLV-I R~f;vify ~ilLV-IT i~ fivif~y SEQ. ID. NOS. 1 and 4 14/14 0/14 SEQ. ID. NOS. 1 and 5 14/14 0/14 SEQ. ID. NOS. 2 and 5 14/14 0/14 SEQ. ID. NOS. 15 and 13 0/14 11/14 SEQ. ID. NOS. 15 and 14 0/14 9/14 SEQ. ID. NOS. 15 and 18 0/14 11/14 SEQ. ID. NOS. 15 and 19 0/14 7/14 SEQ. ID. NOS. 15 and 21 0/14 11/14 SEQ. ID. NOS. 15 ar~d 22 0/14 8/14 An assay using co-coated beads was prepared for ~ iitir)n~l sample 20 testing. Peptides prepared according to the present invention were coated on polystyrene beads as the solid support for the capture of antibodies against HTLV-II using procedures known in the art.
Polystyrene beads were prepared by washing with 15% N-propyl alcohol and incubated at 40C for two hours with between 0.01 llg/ml and 50 25 llg/ml of peptide in a phosphate buffered solution. The beads were washed once with PBS t~lm~ining 0.15% Triton X-100 for one hour at 40C and blocked for one hour at 40C with 2% BSA in PBS, and then overcoated with 5% sucrose in PBS for 20 minutes at room l~
The beads are then drained and dried with nitrogen gas heated to 37C

SUBSTITUTE SHE~T (RULE 26) ~ W0 95/17678 2 1 7 9 3 8 I PCT/US91/1481~
until dry, approximately one to two hours. The beads are stored cicr~tPrl at 2-8C until use.
For detection of antibodies against HTLV-II in this example, beads were co-coated with peptides according to SEQ. ID. NOS. 15 and 22 at 5 equal concentrations. Anti-human IgG antiserum was prepared by imml~ni7ing, goats with purified human IgG. The resulting antiserum was affinity purified and labeled with HRPO according to standard methods. A similar assay was performed using polystyrene beads co-coated with SEQ. ID. NOS. 1 and 5.
Results Beads prepared according to the method of this example were used in an immllnn~c~y against a panel of 100 test samples which had been rr)nfirmi-d positive for HTLV by the method of Anderson, supra, with 50 5 samples ~-mfirme~l positive for HTLV-I and 50 samples confirmed positive for HTLV-II by PCR In that assay, the beads coated with SEQ. ID.
NOS. 15 and 22 correctly identified 47 out of 50 samples as HTLV-II
positive. Additionally, in this immunoassay, the beads showed no ~i~nifir~nt cross-reactivity with sera samples confirmed positive for 20 HTLV-I.
For the assay performed utilizing beads co-coated with SEQ. ID.
NOS 2 and 5, 55 of 57 samples confirmed positive for ~TLV-I were detected, with no ci~,rnific;~n~ cross-reactivity for HTLV-II infected sera.
F~CAMPT F. 5 Eff~f of P.opt~ S~p nn Acc~y r Reagents were prepared as for example 1 except that beads were coated individually with HTLV-I env-l (SEQ. ID. NO. 1)(174-204), HTLV-I
env-6 (SEQ. ID. NO. 6)(190-213), HTLV-I ~-1 (SEQ. ID. NO. 7)(100-129), SUBSTITUTE SHEET (RULE 26) wo 95/17678 PCT/USg4/14815 217938~ --HTLV~ 3 (SEQ. ID. NO. 9)(109-129), HTLV-II env-2 (SEQ. ID. NO.
15)(171-198) and HTLV-II~-2 (SEO. ID. NO. 23)~111-129). ~d~ if~n~l1y, peptides were prepared which were taken from the same antigenic region as the present peptides, but contain several fewer or more amino acid residues. These peptides correspond to HTLV-I g~-4 (103-116~ (SEQ.
ID. NO. 26), HTLV-II g~g-1 (115-135) (SEQ. ID. NO. 28) and HTLV-II env-7 (186-195) (SEQ. ID. NO. 27). The data from these assays was tabulated in Table 3.
~k~
0 C.. l- ~ - Of Peptide Arnino Acid Sf~ ~ nd Sqrfllflvif,ql Rq.~l;vjl;Pc HTLV-I HTLV-II
~Qh~ A ~f;d~ R qq~f;v;ty ~ ;vity HTLV-I gp46 190-213 4/14 0/14 HTLV-I gp46 174-204 11/14 0/14 HTLV-II gp46 186-195 0/14 0/14 20HTLV-II gp46 171-198 0/14 11/14 HTLV-I pl9 103-116 0/14 0/14 HTLV-I pl9 100-129 14~14 0/14 HTLV-I pl9 109-129 5/14 0/14 HTLV-II p19 115-135 6/14 3/14 HTLV-II pl9 111-129 ~ 0/14 4/14 This data clearly demonstrates that the addition, or removal, of small numbers of amino acids to or from a peptide may ~ignififqnfly influence its ability to bind to an antibody, and the specificity with which it binds.

SUBSTITUTE SHEET (RULE 26) ~ WO 95/17678 2 1 7 9 3 & 1 PCT/US9~/14815 FXAMPT.F. 6 ~'''~~~ ~"~' Of ElTLV-I ~n~1 HTLV-TT With Prinr ~rt p.-p~
TmmllnrlaccAys were conducted as in Examples 1 and 2, and 5 applied to the same 28 member panel used in Example 3. Beads were coated with peptides disclosed in Blomberg, ~, and Vahlne, supra.
The Vahlne peptides tested were HTLV-I "H" and "O" and HTLV-II "H"
and "O". The Blomberg peptides tested were the four disclosed preferred peptides, lGB, 2GB, lEA and 2EA, in approximately the same resion as o the peptides of this disclosure. Each of the comparative methods were rr~n~ rtP~I according to the method of the present invention with the substitution of the prior art peptides. In the case of the commercially available SynthEIA, the assay was performed according to the mAn1lfArhlrer~s rerrmm~nr~rd protocol. Results are tabulated in Table 4.
~k~
RP:~tivity of Prinr Art Pe~ti-1~c HTLV-I l~LV-II
20 Pevtide Aminrl Arid ReactiVity Reactivity Vah ne H~-.V- "O" :~TLV-I 9-110 /~
Vahlle H.-_V-. "H" :~LV--~~ -:.99 / ,1 (/-~.
Vahlre H . V-: "H" : : I V-: 7 - ~5 / .
Va~lne H _V-. "O" -:~,V-.I -~, / ~
25omer~ . " -. .'.V-. 1 ,-.~ 2 / .
om r~ -.~ V-:: 1 . '- /.- / -om ~r~" ~" -::.V~
. oml r~ "' ,A" --. LV-~
S~nth~~A S~s-em 1 /1- /14 I sample was ~
21 sample was in.~
31 additional HTLV-II sample was identified as HTLV-I

r~ T~ JE~T 'Rr.l', ~ ~6~

Wo 95/17678 PCT/US94/14815 2 ~ 7938 1 F.~ l~le 7 Dif~ Between HTLV-I ~n/l HTLV-II (('I
The method of Examples 1 and Z was used to investigate a panel of 5 test samples which had been previously classified by PCR. The present invention was compared with the method of Blomberg. The Blomberg peptides tested were the four disclosed preferred peptides, lGB, 2GB, lEA
and ZEA, which fall in approximately the same region as several of the peptides of the present invention. Each of these ~ ,pa~aliv~ methods 0 were rnnrlllo~l according to the method of the present invention with the substitution of the prior art peptides. The results are shown in Table 5.
Table 5 D;rr~ ., ..1. ~i.,.. Of HTLV-I ~ntl HTLV-II (C~

SEO. Il). NO. Bll 1 HTLV-II 1/50~ 7/50 0/50 34/50 0/50 2/50 34/50 40/50 2 This sample was strongly HTLV-I positive and borderline HTLV-II positive.
This sample was strongly gp HTLV-II positive and borderline HTLV-I positive.
Of course, it should be understood that a wide range of changes 25 and m-)riifif~ti~-nc may be made to the preferred Pmho~lim~n~ described above. It is therefore intended that the foregoing detailed description be understood that it is the following claims, including all equivalents, which are intended to define the scope of this invention.

SU~ST1TUTE SHEET (RULE 26)

Claims

IN THE CLAIMS:

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 said test sample, said determination comprising:
i. contacting said test sample with at least one peptide specific for HTLV-I to form a mixture, the peptide(s) specific for HTLV-I being 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;
ii. incubating said 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 antihuman 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 in said test sample by detecting the measurable signal;
b. determining the presence of antibodies against HTLV-II in said test sample, said determination comprising:
i. contacting said test sample with at least one peptide specific for HTLV-II selected from the group consisting of SEQ.
ID. NO. 13, 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 to form a mixture;

ii. incubating said 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 antihuman 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 in said test sample by detecting the measurable signal;
c. determining the pattern of reaction of said 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 HTLV-I is selected from the group consisting of SEQ. ID. NOS. 1, 2, 4, 5, 7 and 8.

3. The method of claim 1, wherein said peptide specific for HTLV-II is selected from the group consisting of SEQ. ID. NOS. 15 and 16.

4. The method of claim 1, wherein said test sample is contacted in said step (a)(i) with two of said peptides specific for HTLV-I.

5. The method of claim 4, wherein the first of said two peptides is chosen from the group consisting of SEQ. ID. NOS. 1, 2 and 6 and the second of said two peptides is chosen from the group consisting of SEQ.ID.NOS.3,4 and 5.

6. The method of claim 4, wherein said first peptide is SEQ. ID.
NO. 1 and said second peptide is SEQ. ID. NO. 5.

7. The method of claim 4, wherein said test sample is contacted with said first and second peptides concurrently.

8. The method of claim 7, wherein said first and second peptides are coated on polystyrene beads.

9. The method of claim 7, wherein said first and second peptides are coated on microparticle beads.

10. The method of claim 1, wherein said test sample is contacted in said step (b)(i) with two of said peptides specific for HTLV-II.

11. The method of claim 10, wherein the first of said two peptides is chosen from the group consisting of SEQ. ID. NOS. 13,14,15 and 16 and the second of said two peptides is SEQ. ID. NO. 22.

12. The method of claim 11, wherein said test sample is contacted with said two peptides concurrently.

13. The method of claim 12, wherein said first and said second peptides are coated on polystyrene beads.

14. The method of claim 12, wherein said first and said second peptides are coated on microparticle beads.

15. The method of claim 1, wherein said test sample is contacted in said step (a)(i) concurrently with a first and second peptide specific for HTLV-I, wherein said first peptide is chosen from the group consisting of SEQ. ID. NOS. 1, 2 and 6 and said second peptide is chosen from the group consisting of SEQ. ID. NOS. 3, 4 and 5, and said test sample is contacted in step (b)(i) concurrently with a third and fourth peptide specific for HTLV-II, wherein said third peptide is chosen from the group consisting of SEQ. ID. NOS. 13, 14, 15 and 16 and said fourth peptide is SEQ. ID. NO. 22.

16. An article of manufacture comprising packaging material comprising:
a first container comprising a peptide specific for HTLV-I
attached to a solid phase, said peptide specific for HTLV-I being selected from the group consisting of SEQ. ID. NOS. 1 through 12;
a second container comprising a peptide specific for HTLV-II
attached to a solid phase, said peptide specific for HTLV-II being selected from the group consisting of SEQ. ID. NOS. 13 through 25;
wherein the packaging material comprises a label on said containers which indicates that the contents thereof may be used to differentiate sera infected with HTLV-I from sera infected with HTLV-II.

17. The article of claim 16, wherein said peptide specific for HTLV-I is selected from the group consisting of SEQ. ID. NOS. 1, 2, 4, 5, 7 and 8 and said peptide specific for HTLV-II is selected from the group consisting of SEQ. ID. NOS. 15 and 16.

18. The article of claim 16, wherein said first container comprises a first peptide specific for HTLV-I selected from the group consisting of SEQ. ID. NOS. 1, 2 and 6 and a second peptide specific for HTLV-II selected from the group consisting of SEQ. ID. NOS. 3, 4 and 5, and said second container comprises a first peptide specific for HTLV-II
selected from the group consisting of SEQ. ID. NOS. 13,14, 15 and 16 and a second peptide specific for HTLV-II which is SEQ. ID. NO 22.

19. 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 said test sample, said determination comprising:
i. contacting said test sample with at least one peptide specific for HTLV-I to form a mixture, the peptide(s) specific for HTLV-I being 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;
ii. incubating said 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 a peptide antigen which can bind to said antigen/antibody complex, to form a second mixture;
iv. incubating said second mixture for a time and under conditions sufficient for antigen/antibody/antigen complexes to form;
V. determining the presence of antibodies against HTLV-I in said test sample by detecting the measurable signal;

b. determining the presence of antibodies against HTLV-II
in said test sample, said determination comprising:
i. contacting said test sample with at least one peptide specific for HTLV-II selected from the group consisting of SEQ.
ID. NO. 13, 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 to form a mixture;
ii. incubating said 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 a peptide antigen which can bind to said antigen/antibody complex, to form a second mixture;
iv. incubating said second mixture for a time and under conditions sufficient for antigen/antibody/antigen complexes to form;
v. determining the presence of antibodies against HTLV-II in said test sample by detecting the measurable signal;
c. determining the pattern of reaction of said test sample for antibodies against HTLV-I and HTLV-II to distinguish between HTLV-I and HTLV-II infections.

20. The method of claim 19, wherein said peptide specific for HTLV-I is selected from the group consisting of SEQ. ID. NOS. 1, 2, 4, 5, 7 and 8.

21. The method of claim 19, wherein said peptide specific for HTLV-II is selected from the group consisting of SEQ. ID. NOS. 15 and 16.

22. The method of claim 19, wherein said test sample is contacted in said step (a)(i) with two of said peptides specific for HTLV-I.

23. The method of claim 22, wherein the first of said two peptides is chosen from the group consisting of SEQ. ID. NOS. 1, 2 and 6 and the second of said two peptides is chosen from the group consisting of SEQ, ID. NOS. 3, 4 and 5.

24. The method of claim 19, wherein said test sample is contacted in said step (b)(i) with two of said peptides specific for HTLV-II.

25. The method of claim 24, wherein the first of said two peptides is chosen from the group consisting of SEQ. ID. NOS. 13,14,15 and 16 and the second of said two peptides is SEQ. ID. NO. 22.