[go: up one dir, main page]

WO1996039630A1 - Detection d'anticorps du htlv a l'aide de proteines de recombinaison - Google Patents

Detection d'anticorps du htlv a l'aide de proteines de recombinaison Download PDF

Info

Publication number
WO1996039630A1
WO1996039630A1 PCT/US1996/008537 US9608537W WO9639630A1 WO 1996039630 A1 WO1996039630 A1 WO 1996039630A1 US 9608537 W US9608537 W US 9608537W WO 9639630 A1 WO9639630 A1 WO 9639630A1
Authority
WO
WIPO (PCT)
Prior art keywords
htln
htlv
sequence
recombinant
antigen
Prior art date
Application number
PCT/US1996/008537
Other languages
English (en)
Inventor
Sean F. Nowlan
Keith R. Hickman
Jessie W. Shih
Michael B. Cerney
Sushil G. Devare
Original Assignee
Abbott Laboratories
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Priority to EP96917970A priority Critical patent/EP0848820A1/fr
Priority to JP50110697A priority patent/JP2001510553A/ja
Publication of WO1996039630A1 publication Critical patent/WO1996039630A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/14011—Deltaretrovirus, e.g. bovine leukeamia virus
    • C12N2740/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates generally to human T-lymphotrophic virus (HTLV) types I and ⁇ , and more particularly, relates to an immunoassay and reagents for the detection of HTLV-I antibodies and/or HTLV-II antibodies in a test sample.
  • HTLV human T-lymphotrophic virus
  • HTLV types I and Ii are retroviruses of type C oncoviruses.
  • HTLV-I the first discovered human retrovirus, was isolated in 1978 from a patient with cutaneous T-cell lymphoma.
  • B. Poiesz et al. Proc. Natl. Acad. Sci. USA 77:7415-7419 (1980).
  • HTLV- ⁇ was discovered in 1982 in a patient with hairy cell leukemia.
  • V. Kalyanarana et al. Science 218:571-573 (1982).
  • HTLV-I HUT 102.B2 also termed HTLV-I CR
  • HTLV-H Wil-NRA also termed HTLV-IIb
  • the entire HTLV- II Wil-NRA has been sequenced and published (see H. Lee et al., Virology 196:57-69 [1993]), and also is disclosed in U.S. patent application Serial No.
  • a phylogenetic tree for HTLV has been determined with three separate HTLV-I families designated as A, B and C, and two HTLV-II groups designated as HTLV-L a and HTLV-IIb.
  • HTLV-I HTLV associated myelopathy or tropical spastic paraparesis
  • ATL adult T cell lymphoma/leukemia
  • HAM/TSP HTLV associated myelopathy or tropical spastic paraparesis
  • HTLV-II The diseases associated with HTLV-II are not as well characterized. Although HTLV-LI initially was isolated from a patient with T-lymphocytic hairy cell leukemia, a leukemia pathogenesis is not well established. Large granular lymphocyte leukemia has been reportedly associated with HTLV-II infection. Several recent reports associate neurodegenerative diseases with HTLV-II infection.
  • Diagnosis of HTLV infection can be made either by detecting the virus (by virus isolation or by a DNA amplification method such as polymerase chain reaction [PCR]) or by serologically detecting antibody to the virus.
  • PCR polymerase chain reaction
  • Currently, screening of blood donor samples for antibody to HTLV-I is mandated in the United States.
  • a majority of government-approved assays for use in clinical laboratories utilize nativ&HTLV-I viral lysate antigens which detect HTLV-I antigens and cross-react with HTLV-II antigens.
  • HTLV-II viral lysate antigens now are available, they are not utilized in any known commerical test kit.
  • HTLV-II testing has not been mandated in the United States, even though more than 50% of the blood donors who are confirmed repeat reactive for HTLV are HTLV-II infected and not HTLV-I infected.
  • Specific recombinant antigens and synthetic peptide antigens for HTLV-I and synthetic peptide antigens for HTLV-II have been described and used in both diagnostic and screening assays for detection of HTLV-I and/or HTLV-II antibodies. See, for example, Lai et al., WO 92/04046; Shih et al., WO 93/01316; and J. J. Lipka et al., J. Inf. Pis.
  • HTLV-I gp46 Some portions of HTLV-I gp46 sequence have been described and HTLV-I gp46 is known to ' have a major and a minor epitope (T. Palker et al., J. of Immunology 142:971-978 [1989]; P. Horal et al., Proc. Natl. Acad. Sci. USA 88:5754-5758 -[1991]).
  • the present invention provides an assay for detecting the presence or amount of antibodies to HTLV-I and/or HTLV-II in a test sample.
  • This assay comprises the steps of contacting the test sample with a capture reagent attached to a solid phase wherein said capture reagent comprises at least one recombinant HTLV-I env and/or HTLV-II env antigen, and at least one recombinant HTLV-I gag and/or HTLV-II gag antigen, and incubating said capture reagent and test sample for a time and under sufficient conditions to form antigen/antibody complexes; contacting said antigen/antibody complexes with a first and a second indicator reagent, wherein said first indicator reagent comprises a recombinant HTLV-I env and/or HTLV-II env antigen labeled with a signal generating compound, and said second indicator reagent comprises a recombinant HTLV-
  • the first capture reagent is selected from the group consisting of recombinant HTLV-I gp46, HTLV-I gp21 , HTLV-JJ gp21 antigen and any combination thereof.
  • the second capture reagent is selected from the group consisting of HTLN-I p24 and HTLN-II p24.
  • the recombinant antigen HTLN-I g ⁇ 46 is SEQUENCE ID. NO. 9; the recombinant antigen HTLN-I gp21 is SEQUENCE ID. NO. 6; the recombinant HTLV-II gp21 is SEQUENCE I.D.NO.
  • the recombinant antigen HTLN-I p24 is SEQUENCE I.D. NO. 3; and the recombinant antigen HTLN-II p24 is SEQUENCE ID. NO. 12.
  • the antigen(s) may be produced in different organisms (i.&., in heterologous sources) or in the same organisms (i.e., in homologous sources).
  • the present invention also provides an assay to detect the presence or amount of antibodies to HTLV-I and/or HTLV-II in a test sample comprising the steps of contacting a first aliquot of the test sample with a first capture reagent attached to a solid phase, wherein the capture reagent is a recombinant HTLV-I env and/or HTLV-II env antigen and contacting a second aliquot of the test sample with a second capture reagent attached to a solid phase, wherein the capture reagent is a recombinant comprising HTLV-I gag_and/or HTLV-II gag antigen for a time and under conditions sufficient to form first capture reagent/test sample complexes and or second capture reagent/test sample complexes; contacting the first capture reagent/test sample complexes and the second capture reagent/test sample complexes with an indicator reagent comprising an anti-human IgG antibody labeled with a signal
  • the first capture reagent is selected from the group consisting of recombinant antigen HTLV-I gp46, recombinant antigen HTLN-I gp21, recombinant antigen HTLV-II gp21 antigen and any combination thereof.
  • the second capture reagent is selected from the group consisting of HTLV-I p24 and HTLV-II p24.
  • the recombinant antigen HTLV-I g ⁇ 46 is SEQUENCE I.D. NO.
  • the recombinant antigen HTLV-I gp21 is SEQUENCE I.D. NO. 6; the recombinant HTLV-II gp21 is SEQUENCE LD.NO. 15; the recombinant antigen HTLV-I p24 is SEQUENCE ID. NO. 3; and the recombinant antigen HTLV-II ⁇ 24 is SEQUENCE I.D. NO. 12.
  • the present invention also provides a test kit comprising a container containing a first capture reagent attached to a solid phase, wherein the first capture reagent is a recombinant HTLV-I env and/or HTLV-JJ eny antigen; and a container containing a second capture reagent attached to a solid phase, wherein the second capture reagent is a recombinant HTLV-I gag and/or HTLV-II gag antigen.
  • the first capture reagent is selected from the group consisting of recombinant antigen HTLV-I g ⁇ 46, HTLV-I gp21, HTLV-II gp21 and any combination thereof.
  • the second capture reagent is selected from the group consisting of recombinant antigen HTLV-I p24 and HTLV-II p24.
  • the recombinant antigen of the first capture reagent is selected from the group consisting of SEQUENCE I.D. NO. 9, SEQUENCE ID. NO. 6 and SEQUENCE ID. NO. 15.
  • the recombinant antigen of the second capture reagent is selected from the group consisting of SEQUENCE I.D. NO. 3 and SEQUENCE ID. NO. 12.
  • the first capture reagent and the second capture reagent may be combined in one container.
  • Plasmid p24ICKS A.T.C.C. deposit No. ; plasmid ⁇ 46ICKS, A.T.C.C. deposit No. ; plasmid p24_3CKS,
  • the recombinant antigen HTLV-I gp46 is SEQUENCE I.D. NO. 9; the recombinant antigen HTLV-I gp21 is SEQUENCE ID. NO. 6; the recombinant HTLV- ⁇ gp21 is SEQUENCE I.D.NO. 15; the recombinant antigen HTLV-I p24 is SEQUENCE I.D. NO. 3; and the recombinant antigen HTLV- ⁇ ⁇ 24 is SEQUENCE I.D. NO. 12
  • FIGURE 1 is a flow chart which shows the strategy for construction of the HTLV-I p24 CKS expression vector (p24ICKS) (SEQUENCE ID. NO. 3).
  • FIGURE 2 is a flow chart which shows the strategy for construction of the HTLV-I gp21 CKS expression vector (p21ICKS) (SEQUENCE ID. NO. 6).
  • FIGURE 3 is a flow chart which shows the strategy for construction of the HTLV-I gp46 CKS expression vector (p46ICKS) (SEQUENCE ID. NO. 9).
  • FIGURE 4 is a flow chart which shows the strategy for construction of the HTLV- ⁇ p24 CKS expression vector (p24HCKS) (SEQUENCE ID. NO. 12).
  • FIGURE 5 is a flow chart which shows the strategy for construction of the HTLV- ⁇ gp21e CKS expression vector (p21HCKS) (SEQUENCE ID. NO. 15).
  • FIGURE 6 is a photograph of a slot blot showing immunoreactivity of recombinant HTLV-I and HTLV- ⁇ antigens with seroreactive samples.
  • FIGURE 7 is a bar graph showing the immunoreactivity of recombinant HTLV-I p24 (SEQUENCE ID. NO. 3) antigen coated on polystyrene microparticles with seroreactive samples.
  • FIGURE 8 is a ?ar graph showing the immunoreactivity of recombinant HTLV-I gp21 (SEQUENCE ID. NO. 6) antigen coated on polystyrene microparticles with HTLV seroreactive samples.
  • FIGURE 9 is a bar graph showing the immunoreactivity of recombinant HTLV-I gp46 (SEQUENCE ID. NO. 9) antigen coated on polystyrene microparticles with seroreactive samples.
  • FIGURE 10 is a bar graph showing the immunoreactivity of recombinant
  • HTLV- ⁇ p24 SEQUENCE ID. NO. 12
  • antigen coated on polystyrene microparticles with HTLV seroreactive samples SEQUENCE ID. NO. 12
  • FIGURE 11 is a bar graph showing the immunoreactivity of recombinant HTLV- ⁇ gp21 (SEQUENCE ID. NO. 15) antigen coated on polystyrene microparticles with HTLV seroreactive samples.
  • FIGURE 12 is a bar graph showing the immunoreactivity of HTLV seroreactive samples against combinations of polystyrene microparticles coated individually with either recombinant p24 (SEQUENCE ID. NO. 3), gp46 (SEQUENCE ID. NO. 9) or gp21 (SEQUENCE ID. NO. 6) HTLV-I antigens.
  • FIGURE 13 is a bar graph showing the immunoreactivity of HTLV seroreactive samples against combinations of polystyrene microparticles coated individually with either recombinant p24 (SEQUENCE ID. NO. 12) or g ⁇ 21 (SEQUENCE ID. NO. 15) '3 HTLV- ⁇ antigens.
  • a “recombinant polypeptide” or “recombinant antigen” (which terms may be used interchangeably) as used herein means a polypeptide of genomic, semisynthetic or synthetic origin which by virtue of its origin or manipulation is not associated with all or a portion of the polypeptide with which it is associated in nature or in the form of a library and/or is linked to a polynucleotide other than that to which it is linked in nature.
  • Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the original progeny of the original cell which has been transfected.
  • replicon means any genetic element, such as a plasmid, a chromosome or a virus, that behaves as an autonomous unit of polynucleotide replication within a cell. That is, it is capable of replication under its own control.
  • a “vector” is a replicon in which another polynucleotide segment is attached, such as to bring about the replication and/or expression of the attached segment.
  • control sequence refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they -are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, such control sequences generally include promoter, ribosomal binding site and terminators; in eukaryotes, such control sequences generally include promoters, terminators and, in some instances, enhancers.
  • control sequence thus is intended to include at a minimum all components whose presence is necessary for expression, and also may include additional components whose presence is advantageous, for example, leader sequences.
  • operably linked refers to a situation wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a manner that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • a "coding sequence” is a polynucleotide sequence which is transcribed into mRNA and or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5' -terminus and a translation stop codon at the 3' -terminus.
  • a coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.
  • a polypeptide or antigen is "immunologically reactive" with an antibody when it binds to an antibody due to antibody recognition of a specific epitope contained within the polypeptide. Immunological reactivity may be determined by antibody binding, more particularly by the kinetics of antibody binding, and/or by competition in binding using as competitor(s) a known polypeptide(s) containing an epitope against which the antibody is directed. The methods for determining whether a polypeptide is immunologically reactive with an antibody are known in the art.
  • transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction, or f-mating are included.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • the term "individual” as used herein refers to vertebrates, particularly members of the mammalian species and includes but is not limited to domestic animals, sports animals, primates and humans; more particularly the term refers to primates/simians and humans.
  • the term "antibody containing body component"(or "test sample”) refers to a component of an individual's body which is the source of the antibodies of interest. These components are well known in the art.
  • test samples include biological samples which can be tested by the methods of the present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitorurinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supematants; fixed tissue specimens; and fixed cell specimens.
  • human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitorurinary tracts, tears, saliva, milk, white blood cells, myelomas and the like
  • biological fluids such as cell culture supematants
  • fixed tissue specimens fixed cell specimens.
  • the purified recombinant antigens of the present invention can be used to develop unique assays as described herein to detect or confirm the presence of antigen or antibody to HTLN-I and/or HTLV- ⁇ . These recombinant antigens also can be used to develop monoclonal and/or polyclonal antibodies with a specific recombinant protein as the immunogen.
  • the purified antigens also may be used in combination with native viral antigens such as viral lysates made from either the HTLN-I or HTLN-H cell lines.
  • the purified antigens may be used in combination with HTLN-I and/or HTLN-H synthetic peptides such as the synthetic peptide sequences disclosed in U. S. patent application Serial No.
  • the purified recombinant antigens also may be used in combination with both HTLV viral lysates, other specific peptide sequences and yet other HTLV recombinant antigens. It is contemplated that the reagent(s) employed for the assay may be provided in the form of a test kit with one or more containers 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 recombinant antigen of the present invention, and the like.
  • a test kit would also include instructions which indicate that the contents thereof may be used to detect/confirm the presence of HTLV-I and/or HTLV-H antibodies and/or antigens and/or differentiate between HTLN-I and HTLV-H.
  • solid phase is not critical and may be any variety of materials which may be selected by one skilled in the art without undue experimentation.
  • 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.
  • porous or nonporous materials, latex or polystyrene particles, magnetic or non-magnetic microparticles, beads, membranes, plastic tubes, walls of microtiter wells and tanned sheep red blood cells are all suitable examples.
  • the size, dimensions, and shape of the solid phase generally are not critical in practicing the methods of the invention.
  • the present invention preferably uses microparticles or beads with one or more recombinant antigen(s) specific for HTLV-I as disclosed herein or one or more recombinant antigen(s) specific for HTLV-H as disclosed herein or a combination of at least one HTLV-I and one HTLV-H antigen recombinant antigen disclosed herein, which are immobilized on a solid phase.
  • Suitable methods for immobilizing antigens on solid phases include ionic, hydrophobic, covalent interactions and the like. Those skilled in the art will recognize the variety of methodologies which may be applied relative to the application of useful solid phases.
  • Linking agents known in the art also may be utilized to secure attachment of the recombinant antigen to the solid phase.
  • the linking agent can be incorporated as part of, or derivatized onto, the solid phase before the antigens are added.
  • test sample is 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 supernatants which may contain the antibodies or antigens of interest.
  • a suitable "indicator reagent” comprises 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 to HTLV derived from the test sample.
  • label a signal generating compound
  • Specific binding member means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule.
  • the indicator reagent also may be a member of any specific binding pair, including either hapten-anti-hapten such as biotin or antibiotin, 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.
  • An immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to HTLV as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
  • labels include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as luminol, dioxetances, acridinium compounds and phenanthridinium compounds, radioactive elements and direct visual labels.
  • luminescent compounds such as fluorescein and rhodamine
  • chemiluminescent compounds such as luminol, dioxetances, acridinium compounds and phenanthridinium compounds
  • radioactive elements include direct visual labels.
  • 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.
  • the reaction mixture is incubated for a time and under conditions sufficient for HTLV antigen/antibody complexes to form.
  • the selection of testing conditions such as appropriate incubation times, temperature, and other conditions and reagents of the assay are well within the skill of the routineer.
  • the present invention provides a method for the detection of antibodies against either HTLV-I and/or HTLV-H by detecting the binding of the antibodies to novel and unique HTLV-I and/or HTLV-H recombinant antigens disclosed herein.
  • the present invention identifies novel and unique antigen sequences in regions of HTLV-I and HTLV-H which are useful in assays to detect and/or differentiate samples which contain antibodies to either of these viruses.
  • unique antigen sequences are provided in the env and gag regions of HTLV-I and HTLV-H.
  • the HTLV-I p24 SEQUENCE ID. NO. 3
  • HTLV-I gp21 SEQUENCE I . NO.
  • recombinant antigens are specifically reactive with samples having antibodies to HTLV-I and are cross-reactive with samples having antibodies to HTLV-H.
  • the HTLV-H p24 (SEQUENCE ID. NO. 12) and HTLV- ⁇ gp21 (SEQUENCE ID. NO. 15) recombinant antigens are specifically reactive with samples having antibodies to HTLV- ⁇ and are cross- reactive with samples having antibodies to HTLV-I.
  • a unique antigenic sequence is provided in the env region (gp46) of HTLV-I that is virus- specific, cross-reacting only with high-titered HTLV-H samples.
  • the method of the invention comprises contacting a human test sample with a solid phase to which at least one HTLV-I or at least onerHTLV-H recombinant antigen disclosed herein, or a combination of at least one HTLN-I and one HTLN-H recombinant antigen disclosed herein is bound, to form a mixture.
  • This mixture is incubated for a time and under conditions sufficient for antigen/antibody complexes to form.
  • these so-formed complexes are contacted with an indicator reagent comprising an anti-human antibody (or other appropriate specific binding pair member) 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/indicator reagent complexes.
  • the presence of immobilized antibody to HTLV in the test sample is determined by detecting the measurable signal generated.
  • the assay for antibodies against HTLN-I and/or antibodies against HTLV-H provides an effective method for detecting the presence an HTLV infection caused by either or both of these viruses.
  • recombinant antigen(s) of the invention which are specific for antibodies against HTLV-I or HTLV-H or a combination of HTLV-I and HTLV-H are immobilized on a solid phase such as polystyrene beads.
  • the beads then are incubated with a human test sample (diluted or undiluted) of human serum, plasma or other body fluid, and incubated under suitable conditions and for an appropriate period of time such that HTLV antibodies present in the test sample will bind specifically to the immobilized HTLV antigens on the bead.
  • the bead then is washed to remove any unbound proteins which may be present.
  • a second incubation is performed in which the bead is incubated with an indicator reagent comprising anti-human antibodies labelled with a signal generating compound.
  • an indicator reagent comprising anti-human antibodies labelled with a signal generating compound.
  • the amount of labelled anti-human antibody complex immobilized on the bead is determined by measuring the detectable generated signal.
  • the presence of specific antibodies against HTLN-I or HTLN-H, or the presence of a combination of HTLN-I and HTLN-H antibodies in the test sample is determined.
  • a one-step sandwich assay is performed wherein a recombinant antigen of the invention specific for HTLN-I and/or HTLN-H of the invention is immobilized on a solid phase such as polystyrene beads.
  • the beads then are incubated with a diluted or undiluted test sample and an appropriate indicator reagent comprising a signal generating compound attached to anti-human IgG, under conditions and for ah appropriate period of time sufficient to allow complexes of recombinant antig£n/antibody/indicator reagent to form.
  • the amount of anti-HTLN antibody present in the test sample is determined by detecting the measurable signal generated by the indicator reagent.
  • the presence of specific antibodies against HTLN-I or HTLN-H, or antibodies against a combination of HTLN-I and HTLN-H, in the test sample is determined.
  • At least one of the recombinant antigens of the invention specific for antibodies against HTLN-I, HTLN-H or a combination of HTLN-I and HTLN-H recombinant antigens are immobilized on a nitrocellulose membrane.
  • the recombinant antigen(s) also may be conjugated or crosslinked to itself, other antigens (recombinant or synthetic) or to various carrier proteins such as BSA, keyhole limpet hemocyanin, ovalbumin, and the like, before immobilization onto the nitrocellulose membrane.
  • the test sample is diluted and then incubated on the membrane for a time and under conditions sufficient for antigen/antibody complexes to form on the membrane.
  • the membrane surface then is washed to remove unbound proteins.
  • an indicator reagent comprising anti-human antibodies labelled with (attached to) a signal generating compound, and incubated.
  • the antibodies against either HTLV- I and/or HTLN-H, as the case may be depending upon what antigens were immobilized on the nitrocellulose membrane, are determined by detecting the measurable signal generated with a suitable detection system. Quantification of the level of signal recognized by the detection system allows the amount of specific antibody to HTLN-I and/or HTLN-H present in a test sample to be determined.
  • a sandwich assay comprises contacting a test sample with a solid phase to which at least one HTLN- I recombinant antigen provided herein, or at least one HTLV-H recombinant antigen provided herein or a combination of at least one HTLN-I and at least one HTLN-H recombinant antigen provided herein are bound, to form a mixture.
  • This mixture is incubated for a time and under conditions sufficient to allow antigen/antibody complexes to form.
  • These complexes then are contacted with an indicator reagent comprising HTLN-I and/or HTLN-H antigen(s) previously conjugated to a signal generating compound, to form a second rnixture.
  • This second mixture is incubated for a time and under conditions sufficient for antigen/antibody indicator reagent complexes to form.
  • the presence oMie antigen/antibody/indicator reagent complexes is deterrnined by detecting the measurable signal generated.
  • a first antigen which can be a recombinant antigen provided herein specific to the antibody to be detected is immobilized on a solid phase, a test sample suspected of containing the antibody is added to the solid phase, and a second antigen which can be a recombinant antigen of the invention having a label affixed thereto then is contacted with the solid phase.
  • two recombinant antigens which are specific to a single binding pair member are used in one assay as a capture phase and a part of the indicator reagent.
  • These antigens are the same and may be made in different, e.g., heterologous, sources. These sources could be bacterial and yeast, for example. It also is within the scope of the present invention that one recombinant antigen provided herein could be used as the capture reagent or as part of the indicator reagent, and the other antigen in this assay could be a synthetic peptide, or viral lysate, or obtained from other antigenic sources known to the routineer.
  • Passive coating means non-covalent bonding or non-covalent attachment between the recombinant antigen and the microparticles. Passive coating involves, for example, diluting the recombinant antigen(s) into a suitable coating buffer at twice the desired final coating concentration, washing and resuspending the microparticles in a buffered salt coating solution, mixing the microparticle and antigen solutions in equal proportions and incubating the solution with mixing under suitable temperature and time conditions. The coated microparticles then are isolated by centrifugation, washed, and resuspended in a microparticle diluent. Due to the nature of the microparticle, the antigens become bound to the microparticle through electrostatic interactions or the like.
  • Active coating means effecting a covalent bond between the antigen and the solid support.
  • a covalent bond is formed either by the carboxy or the amino terminal end of the antigen or by a free carboxy or amino group within the protein binding to an appropriate functional group on the surface of the microparticle.
  • Such microparticles having such functional groups are termed derivatized microparticles.
  • An example of a derivatized microparticle is one which has a carboxy functional group on its surface. The carboxy derivatized microparticle then is treated with l-emyl-3(dimemyl-a ⁇ nopropyl)carbodiimide hydrochloride (EDC).
  • EDC l-emyl-3(dimemyl-a ⁇ nopropyl)carbodiimide hydrochloride
  • the microparticle then is processed in a manner similar to the passively coated microparticle procedure, except that the pH of the solution preferably is about 4.5.
  • the EDC may be added to the final coating solution simultaneously with the recombinant antigens and the microparticles, or to either the recombinant antigens or the microparticles prior to mixing.
  • the resulting coated microparticles have the recombinant antigens bound thereto due to the reaction between the amino terminal ends of the recombinant antigens and the carboxy group on the microparticles.
  • the use of EDC with a non-derivatized microparticle also will result in an active coating.
  • Active coating also can encompass the use of linker compounds to effect the formation of a covalent bond between the antigen and the solid support.
  • linker compounds to effect the formation of a covalent bond between the antigen and the solid support.
  • heterobifunctional such as sulfo-SMCC (sutfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-l-carboxylate) or sulfo-SIAB (sulfosuccinmidyl(4- iodoacetyl)aminobenzoate) or homobifunctional compounds such as sulfo-DST (disufosuccinimidyl tartarate) or sulfo-EGS (ethylene glycolbis[sulfosucch ⁇ idyl- succinate]) allows more specific/site directed covalent bonding to occur.
  • linker compound also can include a spacer arm which would allow the antigen to more freely interact with antibodies while attached to the solid phase.
  • Linker group chemistry is'well known to the skilled artisan.
  • the use of microparticles coated with two recombinant antigen preparations provided herein in the same assay can be effected in two different ways. In the first method, the microparticles are co-coated by dissolving each separate antigen preparation into a stock solution at a suitable concentration and then, mixing them together. Next, the microparticles are added to the antigen solution.
  • a first quantity of microparticles is coated with a first antigen preparation
  • a second quantity of microparticles is coated with a second antigen preparation, and so on; independently of each other.
  • the first and second quantity of coated microparticles are combined, in whole or in part, for use in an assay.
  • T s preferred technique facilitates quantification of the amount of each antigen actually present on the microparticle beads by having the ability to vary quantity of antigen by the amount of coated microparticles of each antigen added to the mixture.
  • the methods of the present invention may be adapted for use in systems which utilize 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.0425 633 and 0424634, respectively, which are incorporated herein by reference.
  • an irnmobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged poly-arrion/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. 0273 115, which enjoys common ownership and which is incorporated herein by reference.
  • scanning tunnelling microscopy for immunoassays also is a technology to which the methods of the present invention are easily adaptable.
  • the capture phase for example, a selected antigen or antigens of the invention
  • 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 incorporated herein by reference.
  • FIA flow immunoassay
  • This approach uses, for example, several different size microparticles, e.g. 5, 7, 10, 15 ⁇ m, each of which is coated individually with a specific recombinant antigen of the invention. A mixture of these particles is added to the samples, incubated, washed and a fluorochrome-tagged anti-human IgG added to detect binding of specific human antibodies in the sample.
  • fluorochromes also could be used to additionally differentiate between the different antibodies.
  • Analysis is performed on a flow cytometer, which allows for simultaneous detection of the binding of the fluorochrome-labeled detection reagent to the different sized particles in the sample.
  • Flow cytometry methods that sense electronic and optical signals from cells which are illuminated allows deterrnination of cell surface characteristics, volume and cell size.
  • Antibodies present in a test sample- are bound to the capture reagent (for example, the recombinant antigens of the invention) and detected with a fluorescent dye which is either directly conjugated to the capture reagent or added via a second reaction.
  • Different dyes which may be excitable at different wavelengths, can be used with more than one capture reagent specific to different antibodies such that more than one antibody can be detected from one sample.
  • a suspension of particles is transported through a flowcell where the individual particles in the sample are illuminated with one or more focused light beams.
  • One or more detectors detect the interaction between the light beam(s) and the labeled particles flowing through the flowcell. Commonly, some of the detectors are designed to measure fluorescence emissions, while other detectors measure scatter intensity or pulse duration.
  • each particle that passes through the flowcell can be mapped into a feature space whose axes are the emission colors, light intensities, or other properties, i.e., scatter, measured by the detectors.
  • the different particles in the sample map into distinct and non-overlapping regions of the feature space, allowing each particle to be analyzed based on its mapping in the feature space.
  • To prepare a test sample for flow cytometry analysis the operator manually pipettes a volume of test sample from the sample tube into an analysis tube. A volume of the desired fluorochrome labeled capture reagent is added. The sample/capture reagent mixure then is incubated for a time and under conditions sufficient to allow antibody/recombinant antigen bindings to take place. After incubation, and if necessary, the operator adds a volume of RNS lyse to destroy any RBCs in the sample.
  • the sample After lysis, the sample is centrifuged and washed to remove any left-over debris from the lysing step. The centrifuge/wash step may be repeated several times. The sample is resuspended in a volume of a fixative and the sample then passes through the fluorescence flow cytometry instrument.
  • a method and apparatus for performing flow automated analysis is described in co-owned U.S. Patent application Serial No. 08/283,379, which is incorporated herein by reference. It is within the scope of the present invention that microspheres can be utilized in the methods described herein, tagged or labeled, and employed for in vitro diagnostic applications. It also is within the scope of the present invention that other cells or particles, including bacteria, viruses, durocytes, etc., can be tagged or labeled with the recombinant antigens described by the present invention and used in flow cytometric methods.
  • analytes which are members of specific binding pairs are quantified by mixing an aliquot of test sample with microparticles coated with a capture reagent capable of binding to the antibody of interest as the other member of the specific binding pair. If the nucleic acid is present in the test sample, it will bind to some of the microparticles coated with the capture reagent and agglutinates will form.
  • the analyte concentration is inversely proportional to the unagglutinated particle count. See, for example, Rose et al., eds., Manual of Clinical Laboratory Immunology. 3rd edition, Chapter 8, pages 43-48, American Society for Microbiology, Washington, D. C. (1986).
  • the present invention discloses a preference for the use of solid phases, it is contemplated that the antigens of the present invention 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.
  • only recombinant antigens specific for HTLN- I or recombinant antigens specific for HTLN-H are utilized in an immunoassay according to any of the methods described above.
  • the specificity of the antigens disclosed herein enables the use of these recombinant antigens in a single assay.
  • a single immunoassay using either one or more recombinant antigens according to the present invention specific for HTLN-I or one or more antigens specific for HTLN-H results in increased specificity and selectivity with respect to detection of antibody to HTLN-I or HTLN- ⁇ in a patient sample, thus indicating infection by HTLN-I and/or HTLN-H.
  • the fusion protein is produced by an E. coli enzyme, CKS (CTP:CMP-3- deoxy-manno-octulosonate cytidylyl transferase or CMP-KDO synthetase), and a heterologous protein is expressed in cells transformed with a cloning vehicle which has a D ⁇ A insert coding for CKS and the heterologous protein.
  • CKS CTP:CMP-3- deoxy-manno-octulosonate cytidylyl transferase or CMP-KDO synthetase
  • the recombinant antigens of the present invention can be used in immunodot blot assay systems.
  • the immunodot blot assay system uses a panel of purified recombinant antigens provided herein placed in an array on a nitrocellulose solid support.
  • the prepared solid support is contacted with a test sample and captures specific antibodies (specific binding member) to the recombinant antigen (the other specific binding member) to form specific binding member pairs.
  • the captured antibodies are detected by reaction with an indicator reagent.
  • the conjugate specific reaction is quantified using a reflectance optics assembly within an instrument which has been described in U.S. Patent Application Serial No.07/227,408 filed August 2, 1988.
  • Patent Application Serial No.07/227,586 and 07/227,590 (both of which were filed on August 2, 1988) further described specific methods and apparatus useful to perform an immunodot assay, as well as U. S. Patent No. 5,075,077 (U.S. Serial No. 07/227,272 filed August 2, 1988), which enjoys common ownership and is incorporated herein by reference. Briefly, a nitrocellulose-base test cartridge is treated with multiple antigenic polypeptides. Each polypeptide is contained within a specific reaction zone on the test cartridge. After all the antigenic polypeptides have been placed on the nitrocellulose, excess binding sites on the nitrocellulose are blocked.
  • test cartridge then is contacted with a test sample such that each recombinant antigen in each reaction zone will react if the test sample contains the appropriate antibody.
  • test cartridge is washed and any antigen-antibody reactions are identified using suitable well-known reagents. As described herein, the entire process is amenable to automation.
  • nucleic acid sequences permits the construction of expression vectors encoding antigenically active regions of the polypeptide encoded in either strand.
  • antigenically active regions may be derived from structural regions of HTLV-I and or HTLV-H, including, for example, envelope (coat) or core antigens, in addition to nonstructural regions of HTLV-I and/or HTLV-H, including, for example, polynucleotide binding proteins, polynucleotide polymerase(s), and other viral proteins necessary for replication and/or assembly of the viral particle.
  • Fragments encoding the desired polypeptides are derived from the genomic or cDNA clones using conventional restriction digestion or by synthetic methods, and are ligated into vectors which may, for example, contain portions of fusion sequences such as beta-galactosidase ( ⁇ -gal) or superoxide dismutase (SOD) or CMP-KDO synthetase (CKS).
  • fusion sequences such as beta-galactosidase ( ⁇ -gal) or superoxide dismutase (SOD) or CMP-KDO synthetase (CKS).
  • CKS CMP-KDO synthetase
  • the Lambda PL system could be used. Methods and "vectors which are useful for the production of polypeptides which contain fusion sequences of SOD are described in EPO 0196056, published October 1, 1986, and those of CKS are described in EPO Publication No. 0331961, published September 13, 1989.
  • the nucleic acid sequence encoding the desired polypeptide may be ligated into expression vectors suitable for any convenient host. Both eucaryotic and prokaryotic host systems are used in the art to form recombinant proteins, and some of these are listed herein.
  • the polypeptide then is isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Purification can be performed by techniques known in the art, and include salt fractionation, chromatography on ion exchange and sizing resins, affinity chromatography, centrifugation, among others. Such polypeptides may be used as diagnostic reagents, or for passive immunotherapy.
  • the immunogenic recombinant antigens prepared as described herein are used to produce antibodies, either polyclonal or monoclonal.
  • a selected mammal for example, a mouse, rabbit, goat, horse and the like
  • a recombinant antigen of the invention having an immunogenic polypeptide bearing at least one HTLV epitope.
  • Serum from the immunized animal is collected after an appropriate incubation period and treated according to known procedures. If serum containing polyclonal antibodies to an HTLV epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by, for example, immunoaffinity chromatography.
  • polyclonal antibodies are known in the art and are described in, among others, Mayer and Walker, eds., Immunochemical Methods In Cell and Molecular Biology. Academic Press, London (1987). Polyclonal antibodies also may be obtained from a mammal previously infected with HTLV. An example of a method for purifying antibodies to HTLV epitopes from serum of an individual infected with HTLV using affinity chromatography is provided herein. Monoclonal antibodies directed against HTLV epitopes also can be produced by one skilled in the art. The general methodology for producing such antibodies is well-known and has been described in, for example, Kohler and Milstein, Nature 256:494 (1975) and reviewed in J.G.R.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See also, M. Schreier et al., Hybridoma Techniques. Scopes (1980) Protein Purification. Principles and Practice.2nd Edition, Springer-Verlag, New York (1984); Hammerling et al., Monoclonal Antibodies and T-Cell Hvbridomas (1981); Kennet et al., Monoclonal Antibodies (1980). Examples of uses and techniques of monoclonal antibodies are disclosed in U.S. patent applications Serial Nos. 748,292; 748,563;610,175, 648,473; 648,477; and 648,475, which are incorporated herein by reference.
  • Monoclonal and polyclonal antibodies thus developed, directed against HTLV epitopes, are useful in diagnostic and prognostic applications, and also, those which are neutralizing are useful in passive immunotherapy.
  • Monoclonal antibodies especially can be used to produce anti-idiotype antibodies. These anti- idiotype antibodies are immunoglobulins which carry an "internal image" of the antigen of the infectious agent against which protection is desired. See, for example, A. Nisonoff et al., Clin. Immunol. Immunopath. 21:397-406 (1981), and Dreesman et al., J. Infect. Pis. 151:761 (1985).
  • Transformation may be by any known method for introducing polynucleotides into a host cell, including packaging the polynucleotide in a virus and transducing a host cell with the virus, and by direct uptake of the polynucleotide.
  • the transformation procedures selected depends upon the host to be transformed.
  • Bacterial transformation by direct uptake generally employs treatment with calcium or rubidium chloride. Cohen, Proc. Natl. Acad. Sci. USA 69:2110 (1972).
  • Yeast transformation by direct uptake may be conducted using the calcium phosphate precipitation r ⁇ ethod of Graham et al., Virology 52:526 (1978), or modification thereof.
  • Vector construction employs methods known in the art. Generally, site- specific DNA cleavage is performed by treating with suitable restriction enzymes under conditions which generally are specified by the manufacturer of these commercially available enzymes. Usually, about 1 microgram ( ⁇ g) of plasmid or DNA sequence is cleaved by 1-10 units of enzyme in about 20 ⁇ l of buffer solution by incubation at 37°C for 1 to 2 hours. After incubation with the restriction enzyme, protein is removed by phenol/chloroform extraction and the DNA recovered by precipitation with ethanol. The cleaved fragments may be separated using polyacrylamide or agarose gel electrophoresis methods, according to methods known by the routineer.
  • Sticky end cleavage fragments may be blunt ended using E. coli DNA polymerase 1 (Klenow)-in the presence of the appropriate deoxynucleotide triphosphates (dNTPs) present in the mixture. Treatment with S 1 nuclease also may be used, resulting in the hydrolysis of any single stranded DNA portions.
  • E. coli DNA polymerase 1 Klenow
  • dNTPs deoxynucleotide triphosphates
  • Ligations are performed using standard buffer and temperature conditions using T4 DNA ligase and ATP. Sticky end ligations require less ATP and less ligase than blunt end ligations.
  • the vector fragment often is treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase to remove the 5'- phosphate and thus prevent religation of the vector.
  • BAP bacterial alkaline phosphatase
  • restriction enzyme digestion of unwanted fragments can be used to prevent ligation.
  • Ligation mixtures are transformed into suitable cloning hosts such as E. coli and successful transformants selected by methods including antibiotic resistance, and then screened for the correct construction.
  • Synthetic oligonucleotides may be prepared using an automated oligonucleotide synthesizer such as that described by Warner, DNA 3:401 (1984). If desired, the synthetic strands may be labelled with 32 P by treatment with polynucleotide kinase in the presence of 32 P-ATP, using standard conditions for the reaction. DNA sequences including those isolated from genomic or cDNA libraries, may be modified by known methods which include site directed mutagenesis as described by Zoller, Nucleic Acids Res. 10:6487 (1982).
  • the DNA to be modified is packaged into phage as a single stranded sequence, and converted to a double stranded DNA with DNA polymerase using, as a primer, a synthetic oligonucleotide complementary to the portion of the DNA to be modified, and having the desired modification included in its own sequence.
  • Culture of the transformed bacteria, which contain replications of each strand of the phage are plated in agar to obtain plaques. Theoretically, 50% of the new plaques contain phage having the mutated sequence, and the remaining 50% have the original sequence.
  • Replicates of the plaques are hybridized to labelled synthetic probe at temperatures and conditions suitable for hybridization with the correct strand, but not with the unmodified sequence. The sequences which have been identified by hybridization are recovered and cloned.
  • E. coli strain XL-1 Blue or other suitable host and successful transformants selected by antibiotic resistance or other markers. Plasmids from the transformants then are prepared according to the method of Clewell et al., Proc. Natl. Acad. Sci. USA 62: 1159 (1969) usually following chloramphenicol amplification as reported by Clewell et al., J. Bacteriol. 110:667 (1972). The DNA is isolated and analyzed usually by restriction enzyme analysis and or sequencing. Sequencing may be by the well-known dideoxy method of Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977) as further described by Messing et al., Nucleic Acid Res.
  • HUT 102.B2 cells Total nucleic acid was isolated from HUT 102.B2 cells by boiling 10 6 cells for 15 min in a total volume of 1 ml phosphate buffered saline (PBS). Typically, 10 ⁇ l was used for each PCR amplification.
  • the HUT 102.B2 cell line is a subclone of HUT 102 (A.T.C.C. deposit No. TIB 162, American Type Culture Collection, 10231 Parklawn Drive, Rockville, MD 20852).
  • primers were designed (SEQUENCE ID. NOS. 1 AND 2) which flanked the entire coding region for this core protein.
  • an EcoR I site was added to the 5' primer (SEQUENCE ID. NO. 1) while a BamH I site was incorporated in the 3' (SEQUENCE ID. NO..2) primer.
  • An artificial stop codon (TAG) also was added to the 3' primer (SEQUENCE ID. NO. 2) allowing for translational termination.
  • TAG artificial stop codon
  • Using these primers allowed for the amplification of a 657 bp fragment which then was ligated into the CKS expression vector, pJO201 , using the restriction enzymes EcoR I and BamH I.
  • the EcoR I site translated into Glu Phe at amino acid numbers 245 and 246 in the-CKS protein followed by the intact p24 gene (SEQUENCE ID. NO. 3).
  • This plasmid was designated p24ICKS.
  • gp21 antigen Preparation of gp21 antigen.
  • primers were designed (SEQUENCE ID. NOS 4 AND 5) which flanked the entire coding region for this envelope protein. Since a BamH I site already existed at the junction between gp46 and 21e, an artificial restriction site was not included in the 5' primer (SEQUENCE ID. NO.4); however, a HinD HI site plus stop codons were incorporated into the 3' primer (SEQUENCE ID. NO. 5). Using these primers allowed for the amplification of a 546 base pair fragment. Ligation was performed into the CKS expression vector pJO201 using the BamH I and HinD HI restriction sites. This procedure fused the entire gp21 protein (SEQUENCE ID. NO. 6) to amino acid numbers 252 and 253 of the CKS protein (see FIGURE 2), creating the plasmid designated as pSN784.
  • primers were designed (SEQUENCE ID. NOS.7 and 8) which flanked the C terminal half of this envelope protein.
  • the 5" primer utilized a Sal I restriction site which was unique to gp46, while the 3' primer (SEQUENCE ID. NO. 8) contained both a stop codon and a HinD HI site. Since there were two Sal I restriction sites in the pJO201 expression vector, an additional site, Xba I, was incorporated into the 5' primer (SEQUENCE ID. NO. 7) which allowed for proper alignment with the pJO201 polylinker.
  • p46ICKS SEQUENCE ID. NO. 9
  • Total nucleic acid was isolated from HTLN-H Wil- ⁇ RA cells (ATCC Deposit No. CLR 11580, disclosed in U.S. patent application Serial No. 08/086,415, filed July 1, 1993, previously incorporated herein by reference) by boiling 10 6 cells for 15 min in a total volume of 1 ml of phosphate buffered saline (PBS). Typically, 10 ⁇ l of cells were used for each PCR amplification. i. Preparation of p24 antigen. As shown in FIGURE 4, primers were designed (SEQUENCE ID. NOS. 10 AND 11) which flanked the entire coding region for this core protein. For ease in cloning, an EcoR I site was added to the 5' primer (SEQUENCE ID. NO.
  • E. coli XL1 Blue, available from Stratagene, La Jolla Ca.
  • a recombinant antigen construct were prepared in 500 ml sterile Excell Terrific Broth (available from Sigma Chemical Corp., St. Louis Mo.) supplemented with 5 ⁇ g/ml Tetracycline-HCl and 100 ⁇ g/ml sodium ampicillin, and placed in a shaking orbital incubator at 37°C.
  • One hundred ml (100 ml) inoculums from seed cultures were transferred to flasks containing 1 liter sterile Excell Terrific Broth supplemented with 100 ⁇ g/ml sodium ampicillin and incubated at 37° C until the cultures reached rrid-logarithmic growth.
  • HTLN-I/TI antigens i.e. HTLN-I ⁇ 24 and HTLN- ⁇ p24t produced in E. coli
  • Frozen E. coli cells prepared as described in Example 1 (C) hereinabove were resuspended in cold lysis buffer (50 mM Tris (tris[hydroxymemyl]ammomethane) pH 8, 10 mM ⁇ a EDTA (sodium ethylenediaminetetraacetic acid), 150 mM ⁇ aCl (sodium chloride), 1 mM PMSF (phenylmethylsulfonylflouride) and 1 ⁇ M pepstatin A by homogenization (Virtus Company Inc., Gardiner, ⁇ .Y.). Lysozyme (Sigma Chemical Co., St.
  • Soluble p24 antigens in the clarified homogenates were precipitated by ammonium sulfate (Scientific Products Division, Baxter Diagnostics Inc., McGaw P , IL.) fractionation at 20%-35% saturation.
  • the precipitated proteins were pelleted by centrifugation.
  • the pelleted proteins were resuspended in 50 mM Tris pH 8, 50 mM NaCl and stored at 4° C until purified by chromatography.
  • Frozen E. coli cells prepared as described in Example 1 (C) hereinabove were resuspended by homogenization in cold lysis buffer comprising 50 mM Tris pH 8, 10 mM ⁇ a EDTA , 150 mM ⁇ aCl , 8% (w/v) sucrose, 5% Triton X-100 ® (v/v), 1 mM PMSF and 1 ⁇ M pepstatin A. Lysozyme was added to the homogenates at a concentration of 1.3 mg per gram of cells processed, and incubated for 30 minutes on ice to lyse the cells. Inclusion bodies were separated from soluble proteins by centrifugation.
  • pelleted inclusion bodies were washed and pelleted sequentially in 1) Lysis Buffer; 2) 10 mM Na EDTA pH 8, 30% (w/v) sucrose; and 3) water.
  • the washed inclusion bodies were resuspended in 50 mM Tris pH 8, 10 mM Na EDTA, 150 mM NaCl and 3 M urea, and incubated on ice for 1 hour.
  • the inclusion bodies then were separated from the solubilized proteins by centrifugation.
  • pelleted inclusion bodies were fully solubilized in 7 M guanidine-HCl 50, mM Tris pH 8, 0.1% (v/v) beta- mercaptoethanol (BME) overnight at 4°C
  • solubilized recombinant antigens were clarified by centrifugation, passed through a 0.2 ⁇ m filter and stored at ⁇ -20° C until purified by chromatography.
  • Soluble recombinant HTLN-I and HTLN-H p24 antigens were purified by a two-step method, as follows. Ammonium sulfate fractionated proteins were loaded onto a cation exchange column (S-Sepharose available from Pharmacia Biotech Inc., Piscataway, N.J.) and equilibrated with 50 mM Tris pH 8. Proteins not binding to the cation exchange matrix were collected and loaded onto a anion exchange column (Q-Sepharose, available from Pharmacia Biotech Inc., Piscataway, N.J.) and equilibrated with 50 mM Tris pH 8.
  • Proteins were eluted from the anion exchange column using-a 0-1 M NaCl gradient. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the fractions. Those fractions containing recombinant p24 antigen (SEQUENCE ID. NOS. 3 and 12, respectively) were pooled and then concentrated by ultrafiltration using a 10,000 MW cutoff spin filter (Amicon Inc., Beverley, MA). The pooled fractions were treated with 4% SDS and 5% BME at 100°C for 5 min. Heat-treated recombinant HTLN-I and HTLN-H p24 antigens (SEQUENCE ID. NOS.
  • HTLN-I gp46 HTLN-I gp21 and HTLN-H gp21 antigens Insoluble HTLV recombinant antigens HTLN-I gp46 (SEQUENCE ID.
  • HTLN-I gp21 SEQUENCE LD. NO. 6
  • HTLV-H gp21 SEQUENCE ID. NO. 15
  • Guanidine-HCl extracts of insoluble antigens were purified by size exclusion chromatography on a Sephacryl S-300 column equilibrated with 50 mM Tris pH 8, 8 M Urea and 0.1 % BME (v/v).
  • SDS-polyacrylamide electrophoresis was used to analyze fractions. Fractions containing HTLV recombinant antigen were pooled and then concentrated by ultrafiltration.
  • the pooled fractions were treated with 4% SDS (w/v) and 5% BME (w/v) at room temperature for three hours.
  • SDS treated recombinant antigens were further purified by size exclusion chromatography on a Sephacryl S-300 column equilibrated with 25 mM Tris pH 8, 0.15 M NaCl, 0.1% v/v BME, 0.1% SDS (w/v).
  • SDS-polyacrylamide electrophoresis was used to analyze the fractions. Fractions containing purified HTLV recombinant antigen were pooled, passed through a 0.2 ⁇ m filter and stored at -70° C.
  • HTLV-recombinant antigens prepared as described hereinabove in Example 1 were coated onto a nitrocellulose solid support using procedures known in the art, as follows. Briefly, HTLV recombinant antigens (see TABLE 1) HTLV-I p24 (SEQUENCE LD. NO. 3), HTLV-H p24 (SEQUENCE ID. NO. 12), HTLV-I gp46 (SEQUENCE LD. NO. 9), HTLV-I gp21 (SEQUENCE ID. NO. 6) and HTLV-H gp21 (SEQUENCE ID. NO. 15) or human IgG (available from Sigma Chemical Co., St.
  • Goat anti-human IgG antibody labeled with alkaline phosphatase available from Sigma Chemical Co., St. Louis, MO
  • substrate NBT/BCIP available from Sigma Chemical Co., St. Louis, MO
  • Strips were added to 3 ml of sample diluent buffer (PBS with 5% wt/v NFDM and 10% v/v heat inactivated goat serum). Thirty microliters (30 ⁇ l) of test sample were added to each strip and incubated at room temperature for 16-20 hours. The strips were washed with PBS containing 0.025% Tween-20® (available from Sigma Chemical Co., St. Louis, MO). Three ml of conjugate (goat anti-human IgG antibody labeled with alkaline phosphatase) diluted in sample diluent buffer were added to the strips and incubated at room temperature for 60 minutes. The strips again were washed with PBS containing 0.025% Tween-20®.
  • sample diluent buffer PBS with 5% wt/v NFDM and 10% v/v heat inactivated goat serum.
  • the amount of goat anti-human alkaline phosphatase conjugate immobilized on the nitrocellulose strip was determined by the deposition of an insoluble blue precipitate by developing the strips with 2 ml of NBT/BCIP phosphatase substrate for 15 minutes at room temperature. The reactions were stopped with 2 ml of 1 M sodium acetate buffer pH 4. The strips then were rinsed with distilled water and dried at room temperature.
  • the method of invention described hereinabove was applied to a 25- member test panel, which included neat and diluted plasma samples.
  • the test panel contained seven confirmed HTLV-I, eight confirmed HTLV-H, and five negative samples. Seventeen of twenty HTLV positive test samples were obtained from Serologicals, Specialty Products, 780 Park N. Blvd., Clarkston, GA 30021). All samples obtained were confirmed positive by a combination of Western blot, RIPA and PCR. The remaining three positive samples were either donor samples (2) or sex partner study samples (1) tested at Abbott Laboratories and confirmed positive by a combination of Westem blot, RJPA and PCR. The five seronegative samples were from internal research studies.
  • the immuno-slot blot assay described herein detected 20 of 20 (1 * 00%) members of the antibody positive sample panel.
  • Each seropositive sample reacted to at least one gag gene product (HTLV-I p24 [SEQUENCE ID. NO. 3] and/or HTLV-H ⁇ 24 [SEQUENCE ID. NO. 12]) and one env gene product (HTLV-1 gp21 [SEQUENCE ID. NO. 6] and/or HTLN-I gp46 [SEQUENCE ID. NO. 9] and/or HTLN-H g ⁇ 21 [SEQUENCE ID. NO. 15]).
  • the five negative samples were nonreactive against the recombinant HTLV antigens coated on the strips (see FIGURE 6). These data also are presented in TABLE 2.
  • Example 3 Detection of Antibodies to HTLN-I and/or HTLN-H bv ELIS A
  • Reagents Purified HTLV recombinant antigens prepared as described in the preceeding examples were coated on polystyrene microparticle solid supports (available from Seradyn, Indianapolis, IN) for the capture of antibodies to HTLV- I and/or HTLV-H using procedures known in the art, as follows. Microparticles used in this example were coated with either recombinant HTLV-I p24 [SEQUENCE ID. NO. 3], HTLV-H p24 [SEQUENCE ID. NO. 12], HTLV-I gp46 [SEQUENCE ID. NO.
  • HTLV-I gp21 [SEQUENCE ID. NO. 6] or HTLV-H gp21 [SEQUENCE ID. NO. 15].
  • 0.5 ⁇ m polystyrene microparticles were washed with distilled water. The washed microparticles at 1% solids were incubated overnight at room temperature with 200 ⁇ g/ml of recombinant antigen in PBS pH 7.4. The microparticles were washed with PBS and then resuspended in microparticle diluent ( 65 mM Tris pH 8, 100 mM NaCl, 0.4 M sucrose and 0.2% wt/v sodium azide) at 1% solids. Microparticles were diluted in microparticle diluent with 30% (v/v) heat inactivated goat serum and incubated overnight at 4° C before use.
  • Plasma samples were diluted 1:10 or 1:20 in sample MEIA dilue buffer and run in an IM ⁇ ® assay format following manufacturer's directions and utilizing manufacturer's buffers and indicator reagents (available from Abbott Laboratories, Abbott Park, IL), except as noted herein. Briefly, 15 ⁇ l of diluted test sample was aspirated with 115 ⁇ l of MEIA buffer, mixed with 100 ⁇ l of coated microparticles in diluent (prepared as described in the preceeding examples) and incubated at 37" C. After washing, 60 ⁇ l of goat anti-human alkaline phosphatsase cojugate was added to the microparticles. The amount of goat anti- human alkaline phosphatase conjugate immobilized was quantified by turnover of the fluorogenic substrate reagent 4-methylumbelliferyl phosphate (MUP).
  • MUP fluorogenic substrate reagent 4-methylumbelliferyl phosphate
  • a HTLN-I combination assay with HTLN-I ⁇ 24, HTLN-I gp46 and HTLN-I gp21 microparticles detected ten of ten HTLN-I and eight often HTLN-H positive samples, for a total of 18 / 20 (90%) of the positive test panel samples, as shown in FIGURE 12.
  • the HTLN-I combination assay detected eight of eight HTLN-I and five of six HTLN-H neat (undiluted) seropositive samples, for a total of 13 / 14 (93%).
  • HTLN-H combination assay with HTLN-H ⁇ 24 and HTLN-H gp21 recombinant antigens detected five of ten HTLN-I and nine of ten HTLN-H positive samples, for a total of 14 / 20 (70%) of the positive test panel samples, as shown in FIGURE 13.
  • the HTLN- ⁇ combination assay detected five of eight HTLN-I and six of six HTLN-H neat (undiluted) seropositive samples, for a total of 11 / 14 (79%).
  • plasmid ⁇ 46ICKS received A.T.C.C. deposit No.
  • plasmid p24HCKS received A.T.C.C. deposit No.
  • plasmid p21HCKS received A.T.C.C. deposit No. .
  • NAME Pore ⁇ ibski, Priscilla E.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CAAACTGGAA TCACCCTGGT CGCGCTACTC CTTCTTGTTA TCCTTGCAGG ACCATGCATC 420
  • CTCCGTCAGC TACGACACCT CCCCTCGCGC GTCAGATACC CCCATTACTC TCTTATAAAA 480
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CTATCCACTT GGCACGTCCT ATACTCTCCC AACGTCTCTG TTCCATCCCC TTCTTCTACC 300
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • ATCACCGAGG CTGAGACTCG CGGGGTGACA GGCTACAACC CCATGGCAGG GCCCCTAAGA 300 ATGCAGGCTA ATAACCCCGC CCAACAAGGT CTTAGACGGG AGTACCAGAA CCTTTGGCTG 360
  • AAAGAATGCC AAAAAATCTT ACAAGCCCGT GGACACACTA ACAGCCCCCT CGGGGAGATG 600
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • AAAAATCATC AAAACATCCT CCGGGTTGCA CAATATGCAG CCCAGAATAG ACGAGGATTA 180

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • AIDS & HIV (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention porte sur des techniques recourant à des antigènes de recombinaison de l'HTLV-I et de l'HTLV-II pour détecter les anticorps du HTVL dans un échantillon test. Les antigènes utilisés comprennent les HTLV-Igp46, HTLV-Igp21, HTLV-Ip24, HTLV-IIgp21 et HTLV-IIgp24 ou l'une de leurs combinaisons. L'invention porte également sur les trousses qui comprennent les antigènes de l'HTLV-I et de l'HTLV-II, et les plasmides à partir desquels ces antigènes de recombinaison ont été produits.
PCT/US1996/008537 1995-06-05 1996-06-04 Detection d'anticorps du htlv a l'aide de proteines de recombinaison WO1996039630A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP96917970A EP0848820A1 (fr) 1995-06-05 1996-06-04 Detection d'anticorps du htlv a l'aide de proteines de recombinaison
JP50110697A JP2001510553A (ja) 1995-06-05 1996-06-04 組換え蛋白質を用いたｈｔｌｖ抗体の検出法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US46209795A 1995-06-05 1995-06-05
US08/462,097 1995-06-05

Publications (1)

Publication Number Publication Date
WO1996039630A1 true WO1996039630A1 (fr) 1996-12-12

Family

ID=23835158

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/008537 WO1996039630A1 (fr) 1995-06-05 1996-06-04 Detection d'anticorps du htlv a l'aide de proteines de recombinaison

Country Status (4)

Country Link
EP (1) EP0848820A1 (fr)
JP (1) JP2001510553A (fr)
CA (1) CA2223235A1 (fr)
WO (1) WO1996039630A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1477495A1 (fr) * 2003-04-25 2004-11-17 Development Center For Biotechnology Fragments antigéniqes de la protéine gp21 du virus lymphotropique humain HTLV
US7033751B2 (en) 2002-12-12 2006-04-25 Development Center For Biotechnology Antigenic fragment of human T-lymphotropic virus
EP2913338A1 (fr) * 2014-02-28 2015-09-02 Roche Diagniostics GmbH Variants et immunoréactif soluble de HTLV antigène capsidique p24

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0181107A1 (fr) * 1984-10-26 1986-05-14 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Production de fragments de protéine enveloppante de rétrovirus (HTLV-1) (lymphotropique) de leucémie de T-cellules humaines en bactéries et utilisation pour l'examen séroépidémiologique de la malignité lymphoide humaine
WO1991007510A1 (fr) * 1989-11-17 1991-05-30 Amgen Inc. Methode de detection d'anticorps htlv-i dans les fluides corporels humains
WO1993001316A1 (fr) * 1991-07-10 1993-01-21 Abbott Laboratories Differentiation de htlv-i et htlv-ii a l'aide de peptides synthetiques
WO1995001457A1 (fr) * 1993-07-01 1995-01-12 Abbott Laboratories Compositions de virus htlv-iinra et essais utiles pour detecter l'infection par des virus htlv

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0181107A1 (fr) * 1984-10-26 1986-05-14 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Production de fragments de protéine enveloppante de rétrovirus (HTLV-1) (lymphotropique) de leucémie de T-cellules humaines en bactéries et utilisation pour l'examen séroépidémiologique de la malignité lymphoide humaine
WO1991007510A1 (fr) * 1989-11-17 1991-05-30 Amgen Inc. Methode de detection d'anticorps htlv-i dans les fluides corporels humains
WO1993001316A1 (fr) * 1991-07-10 1993-01-21 Abbott Laboratories Differentiation de htlv-i et htlv-ii a l'aide de peptides synthetiques
WO1995001457A1 (fr) * 1993-07-01 1995-01-12 Abbott Laboratories Compositions de virus htlv-iinra et essais utiles pour detecter l'infection par des virus htlv

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
P. HORAL ET AL.: "Identification of type-specific linear epitopes in the glycoproteins gp46 and gp21 of human T-cell leukemia viruses type I and type II using synthetic peptides", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, vol. 88, no. 13, 1 July 1991 (1991-07-01), WASHINGTON DC USA, pages 5754 - 5758, XP000215690 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7033751B2 (en) 2002-12-12 2006-04-25 Development Center For Biotechnology Antigenic fragment of human T-lymphotropic virus
EP1477495A1 (fr) * 2003-04-25 2004-11-17 Development Center For Biotechnology Fragments antigéniqes de la protéine gp21 du virus lymphotropique humain HTLV
EP2913338A1 (fr) * 2014-02-28 2015-09-02 Roche Diagniostics GmbH Variants et immunoréactif soluble de HTLV antigène capsidique p24
JP2017512200A (ja) * 2014-02-28 2017-05-18 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ ＨＴＬＶキャプシド抗原ｐ２４の可溶性かつ免疫反応性バリアント
US10466242B2 (en) 2014-02-28 2019-11-05 Roche Diagnostics Operations, Inc. Soluble and immunoreactive variants of HTLV capsid antigen p24
US11567079B2 (en) 2014-02-28 2023-01-31 Roche Diagnostics Operations, Inc. Soluble and immunoreactive variants of HTLV capsid antigen P24

Also Published As

Publication number Publication date
JP2001510553A (ja) 2001-07-31
EP0848820A1 (fr) 1998-06-24
CA2223235A1 (fr) 1996-12-12

Similar Documents

Publication Publication Date Title
AU601175B2 (en) Peptides for the diagnosis of htlv-iii antibodies, their preparation and use
KR100847586B1 (ko) Ｃ형 간염 바이러스의 측정방법
US6406841B1 (en) Methods for the detection of HTLV-II antibodies employing novel HTLV-II NRA envelope peptides
EP0860504A1 (fr) Polypeptides de l'antivirus lymphptropiques de cellules T du chat
US6383765B1 (en) Polypeptides of feline T-cell lymphotropic lentivirus
WO1995001457A9 (fr) Compositions de virus htlv-iinra et essais utiles pour detecter l'infection par des virus htlv
JP2679973B2 (ja) 免疫学的に反応性のウイルス蛋白質の発現
EP0424748A1 (fr) Peptides d'enveloppe synthétiques d'HTLV-I
US6924095B2 (en) Retrovirus isolated from mantle histiocytes in mantle cell lymphoma
JP3506252B2 (ja) Ｈｔｌｖ−ｉ／ｈｔｌｖ−ｉｉの分析及び方法
WO1996039630A1 (fr) Detection d'anticorps du htlv a l'aide de proteines de recombinaison
JP3421336B2 (ja) 合成ペプチドを用いてのｈｔｌｖ−▲ｉ▼とｈｔｌｖ−▲ｉｉ▼との区別
EP0453563A4 (en) Diagnostic proteins to test for more than one antibody
AU627738B2 (en) Htlv-i / hiv-1 fusion proteins
EP0951646B1 (fr) DOSAGE IMMUNO-ENZYMATIQUE POUR DIAGNOSTIQUER L'ANEMIE INFECTIEUSE EQUINE AU MOYEN DE LA PROTEINE RECOMBINANTE (gp90) DERIVEE DU VIRUS DE L'ANEMIE INFECTIEUSE EQUINE
DE19919121B4 (de) Multimeres CMV-Antigen
WO1998029747A1 (fr) Reactifs et procedes permettant de detecter un antigene du virus de l'hepatite gb
AU2002313771A1 (en) Retrovirus isolated from mantle histiocytes in mantle cell lymphoma
WO1992007961A1 (fr) Detection specifique d'anticorps contre les virus du syndrome immunodeficitaire acquis

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2223235

Country of ref document: CA

Ref country code: CA

Ref document number: 2223235

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref country code: JP

Ref document number: 1997 501106

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1996917970

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1996917970

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97501106

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97501106

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97501106

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1997501106

Format of ref document f/p: F

WWW Wipo information: withdrawn in national office

Ref document number: 1996917970

Country of ref document: EP