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WO1991007510A1 - Methode de detection d'anticorps htlv-i dans les fluides corporels humains - Google Patents

Methode de detection d'anticorps htlv-i dans les fluides corporels humains Download PDF

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Publication number
WO1991007510A1
WO1991007510A1 PCT/US1990/006647 US9006647W WO9107510A1 WO 1991007510 A1 WO1991007510 A1 WO 1991007510A1 US 9006647 W US9006647 W US 9006647W WO 9107510 A1 WO9107510 A1 WO 9107510A1
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WIPO (PCT)
Prior art keywords
env
moles
htlv
gag
antibody
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PCT/US1990/006647
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English (en)
Inventor
David L. Hare
Gary L. Kieft
Edward P. Lau
Marie A. Renick
Susan A. Yancik
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Amgen Inc.
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Publication of WO1991007510A1 publication Critical patent/WO1991007510A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

  • the present invention relates generally to immunoassays for the detection of HTLV-I antibodies in body fluids and, more particularly, to highly accurate and reliable HTLV-I screening immunoassays based on the use of multiple HTLV-I recombinant antigenic
  • HTLV-I Human T-Cell Leukemia Virus
  • HTLV-I infection is endemic in some parts of the world, including southern Japan, Okinawa, and the Caribbean basin. The prevalence of infection in these endemic areas is about 5 to 10% and may be higher in specific locations.
  • HTLV-I proteins which are or may be immunogenic are encoded by the gag, env and tax genes of the HTLV-I genome.
  • the immunogenic proteins encoded by these genes are
  • infected mammalian cells may be impractical and contaminating mammalian proteins may lead to unreliable assay results.
  • Assays which use the live virus as part of the manufacturing process also create a safety hazard for workers because they may be exposed to infected cells or infected cell products when assembling the assay. The development of a practical, specific, and sensitive assay for HTLV-I is thus burdened because of the difficulties of working with infected cells and infected cell products.
  • HTLV-I proteins derived from infected mammalian cells may be overcome by applying recombinant DNA methods and techniques to develop antigenic polypeptides in non- mammalian host cells.
  • HTLV-I antigenic polypeptides as fusion products or transforming heterologous cells with
  • antigenic polypeptides expressed in E. coli transformed with portions of the gag gene may be used in an
  • antigenic polypeptides require antigens which are readily available and which are
  • antigenic polypeptides must be readily purified in order to avoid or eliminate non-specific binding to contaminating host cells proteins by cross-reactive antibodies which may be present in body fluid samples.
  • polypeptides must also retain their immunological activity when they are used to prepare immunoassay apparatus which typically involve adsorption of the antigenic polypeptides onto a solid support and
  • a sensitive HTLV-I assay requires more than one antigen in a single assay.
  • the use of more than one antigen in a single assay allows the detection of individuals exposed to HTLV-I that have differing antibody profiles. For any given sera positive population, individuals will exhibit different immunogenic responses to viral antigens. Thus, an assay employing only one antigen may not detect all the exposed individuals.
  • a single screening assay employing more than one antigen is needed to ensure all exposed individuals are detected.
  • the present invention provides a HTLV-I assay which meets the criteria referred to above.
  • This invention encompasses a method for detecting exposure to HTLV-I comprising combining a body fluid containing an antibody with an amount of one or more recombinant polypeptide antigens selected from the group consisting of polypeptides encoded by all or part of the env, tax, and gag genes of HTLV-I effective to maximize the sensitivity and selectivity of an immunological assay, wherein the polypeptides are bound to a synthetic polymeric solid support; forming a detectable antibodyantigen binding pair between the antibody and
  • a preferred solid support is a microtiter well.
  • Preferred polypeptide antigens include env B, p40 x , and p24 gag.
  • the preferred polypeptide, env B is a polypeptide that has a molecular weight of about
  • the preferred polypeptide, p40 x is a protein that has a molecular weight of about 40,000 and has an amino acid sequence which includes the amino acid sequence encoded by the native HTLV-I tax gene.
  • the preferred polypeptide, p24 gag is a protein that has a molecular weight of about 24,000 and has an amino acid sequence which includes a portion of the amino acid sequence encoded by the native HTLV-I gag gene.
  • a preferred embodiment of this invention uses at least three recombinant polypeptides in a single assay, where the three polypeptides are selected from the group consisting of polypeptides encoded by all or part of the env, tax, and gag genes of HTLV-I.
  • This invention also encompasses an assay kit for detecting HTLV-I antibodies comprising a solid support coated with an amount of at least three
  • a preferred assay kit includes the three polypeptides, env B, p40 x , and p24 gag, bound to a microtiter well.
  • the kit may also include additional reagents which may provide various diluents or buffers needed to perform the assay.
  • a preferred detection reagent is an enzyme-conjugated goat antihuman antibody.
  • the present invention encompasses a competition immunoassay to detect the presence of an antibody to a HTLV-I antigen comprising preparing identical first and second dilutions of a body fluid containing an antibody; generating a detectable signal using the first dilution and a recombinant polypeptide antigen, wherein the antibody forms a detectable antigen-antibody binding pair and the antigen is selected from the group consisting of purified env B, p40 x , and p24 gag polypeptides; adding a known amount of a recombinant polypeptide antigen selected from the group consisting of purified env B, p40 x , and p24 gag polypeptides to the second dilution, wherein the antigen is the antigen used with the first dilution; generating a detectable signal using the second dilution, wherein the HTLV-I antibody forms a detectable antigen-antibody binding pair with the antigen used with the first d
  • both the screening and competition assays incorporate recombinant polypeptides produced by transformed E. coli.
  • the preferred polypeptides are p24 gag, env B, and p40 x . These polypeptides are preferably purified in order to provide the optimized sensitivity and selectivity for the assay.
  • purified refers to polypeptides that are free from contaminating materials which would interfere in an immunoassay. For example, contaminating E. coli
  • each antigenic polypeptide for use in an immunoassay or assay kit either individually or as a combination of two or more antigenic polypeptides bound to the same support or to the same microtiter well are about 1.89 x 10 -13 to 3.78 x 10 -11 moles env B, 1.25 x 10 -13 to 2.50 x 10 -11 moles p40 x , or 2.09 x 10 -13 to 8.34 x 10 -11 moles p24 gag.
  • antigenic polypeptides for the above assays are about 3.78 x 10 -13 to 9.45 x 10 -12 moles env B, 2.5 x 10 -13 to 6.25 x 10 -12 moles p40 x , or 4.17 x 10 -13 to 2.08 x 10 -11 moles p24 gag.
  • antigenic polypeptides are 1.89 x 10 -12 moles env B, 1.25 x 10 -12 moles p40 x , or 1.04 x 10 -11 moles p24 gag.
  • the preferred amounts of antigenic peptide may be added to a microtiter well as a solution.
  • a solution having a concentration of about 0.95 to 189 nmolar env B, 0.625 to 125 nmolar p40 x , or 1.05 to 417 nmolar p24 gag may be added to a microtiter well.
  • Particular preferred concentrations for a 200 microliter solution are 1.89 to 47.3 nmolar env B, 1.25 to 31.3 nmolar p40 x , or 2.08 to 104.3 nmolar p24 gag.
  • the most preferred concentrations for a 200 microliter solution are 9.5 nmolar env B, 6.25 nmolar p40 x or 52.0 nmolar p24 gag.
  • Figures 1A-1C illustrates a composite synthetic/ native DNA sequence encoding the amino acid sequence of the antigenic polypeptide env B
  • Figures 2A- 2B illustrates a synthetic DNA sequence encoding the amino acid sequence of the antigenic polypeptide p24 gag
  • Figures 3A-3C illustrates a composite
  • the HTLV-I genome encodes at least three proteins that may be antigenic in humans, gag, env, and tax proteins.
  • a test for exposure to HTLV-I includes antigenic proteins encoded by these genes in order to provide a very sensitive and specific test.
  • Table 1 illustrates various immunogenic responses identified from 46 positive serum samples.
  • the 46 samples were initially identified as positive using a multiple antigen assay containing three antigenic polypeptides, p24 gag, env B, and p40 x . Each of the samples were then assayed using a single antigen assay containing three antigenic polypeptides, p24 gag, env B, and p40 x . Each of the samples were then assayed using a single
  • Recombinant HTLV-I polypeptides suitable for use in this invention may be prepared by employing known recombinant DNA technologies. Briefly, synthetic DNA sequences to encode all or part of the antigenic
  • polypeptides were designed and prepared to optimize expression of the encoded polypeptides in E. coli.
  • the actual DNA sequences employed were derived from the native HTLV-I amino acid sequences rather than from the native nucleic acid sequences. This derivation
  • the DNA sequences used to express the desired polypeptides may include
  • fragments of native DNA that were isolated from natural sources as well as fragments of synthetic DNA that were laboratory-made.
  • the complete sequences were assembled from the different fragments using well established techniques.
  • the complete DNA sequences were incorporated into expression vectors and the vectors were transformed into E. coli cells. The transformed cells were then used to express the desired polypeptides encoded by the complete DNA sequences. Upon isolation using known methods, the identities of the expressed recombinant polypeptides were confirmed both biochemically and immunologically.
  • HTLV-I recombinant antigens suitable for use in this invention may be obtained from Triton Biosciences, Alameda, California. The
  • env polypeptide p21e/gp46
  • p21e/gp46 is a recombinant fusion protein with a molecular weight of 38,000 which includes a majority of the p21e amino acid sequence and a substantial part of the gp46 amino acid sequence.
  • the commercially available polypeptide, fusion p24 gag is a fusion protein with a molecular weight of 40,000 which includes the entire p24 gag amino acid sequence.
  • the commercially available polypeptide, fusion p40 x is a fusion protein with a molecular weight of 42,000.
  • An assay according to this invention may use about 5 to 2000 ng of purified antigen bound to a suitable solid support.
  • Preferred amounts of each antigen are 10 to 250 ng of env B, 10 to 250 ng of p40 x and 10 to 500 ng of p24 gag, the most preferred amounts are 50 ng of env B, 50 ng of p40 x and 250 ng of p24 gag.
  • Amounts of bound antigen less than 10 ng appear to be about the lower limit of sensitivity for detection of HTLV-I antibodies in routine samples.
  • Amounts of bound antigen exceeding 250 ng appear to be about the upper limit of selectivity because the binding specificity of HTLV-I antibodies, compared to other antibodies in the sample, begins to significantly decrease.
  • concentrations that are used to coat the support as a 200 microliter solution are preferably 0.95 to 189 nmolar env B, 0.625 to 125 nmolar p40 x , and 1.05 nmolar to 417 nmolar p24 gag,
  • concentrations are 1.89 to 47.3 nmolar env B, 1.25 to 31.3 nmolar p40 x , and 2.08 to 104.3 nmolar p24 gag, the most preferred concentrations are 9.5 nmolar env B, 6.25 nmolar p40 x , and 52.0 nmolar p24 gag.
  • Examples 1 to 4 describe the preparation of recombinant polypeptide antigen coated microtiter wells and procedures to test sera samples for HTLV-I antibodies using these coated wells.
  • Example 5 describes competition assays which preferably provide confirmatory results for the presence of a particular HTLV-I antibody in a given sample.
  • Example 6 describes immunoblot assay protocols in which the antigenic polypeptides are bound to a nitrocellulose solid support.
  • Examples 7 to 9 describe procedures to prepare p24 gag, env B, and p40 x recombinant antigenic polypeptides, respectively.
  • Examples 10 to 12 describe procedures to purify the HTLV-I antigenic polypeptides used in this invention.
  • Example 1
  • Example 8 The env B polypeptide described in Example 8 was expressed in E. coli and purified as described infra. Either env polypeptide was placed in 8 M Guanidine-HCl and 50 mM Glycine, pH 3.0, at a concentration of 2 mg per ml. This concentrate was then diluted to 0.25 ng/ ⁇ l in 4 M Guanidine-HCl and 50 mM Tris-HCl, pH 7.4, to make an immunoassay microtiter well adsorption solution and 200 ⁇ l of the adsorption solution was added to each microtiter well of an Immulon IV Removawell strip (Dynatech Laboratories, Inc.,
  • microtiter wells containing the adsorption solution were covered and incubated for 12 hours at 37°C in a
  • a blocking solution 250 ⁇ l, 2% alkaline casein, 2% glycerol, 10% sucrose in TEN buffer: 150 mM NaCl, 10 mM tetrasodium EDTA, 1:10000 (w/v) thimerosal, and 50 mM Tris-HCl, pH 7.2 to 7.6, as described in Clinica Chemica ACT 193:123 (1982)) was added to each well.
  • the microtiter wells were covered and incubated with the blocking solution for 12 hours at 37°C in a humidified incubator. After blocking, the coated and blocked microtiter wells were decanted and patted as above.
  • microtiter wells were then inverted and dried for 12 hours at 4°C. Once the coated and blocked microtiter wells had been dried, they were stored at -20°C until used. Antibodies to the HTLV-I env polypeptide in human serum or plasma were detected with an enzyme linked detection antibody by adding 4 ⁇ l of the serum or plasma to 2 ml of sample diluent (25% normal goat serum, 0.1% polyoxyethylenesorbitan monolaurate (Tween-20), in TEN buffer) and 150 ⁇ l of the diluted serum or plasma was added to each microtiter well at room temperature. The microtiter wells were covered and incubated for 2 hours at 37°C in a humidified incubator. The diluted serum or plasma was decanted from the microtiter well and the microtiter wells were patted on an absorbent pad to remove any remaining solution.
  • sample diluent 25% normal goat serum, 0.1% polyoxyethylenesorbitan monolaurate (Tween-20), in TEN buffer
  • wash solution 0.01% Tween-20 in TEN buffer.
  • Each wash step involved squirting the wash solution from a 500 ml squirt bottle into the microtiter well until full, decanting the wash solution and patting the well on an absorbent pad to remove the excess wash solution.
  • HTLV-I positive serum or plasma samples were examined. Of these samples, twenty were positive for antibodies to the HTLV-I env protein by radioimmunoprecipitation analysis (RIPA).
  • the RIPA analysis was performed following procedures described by Slamon, et al., Science,
  • Table 2 illustrates the preferred range of the amount of antigenic polypeptide env B to incorporate in the assay to provide the greatest sensitivity and selectivity.
  • the p24 gag polypeptide described in Example 7 was expressed in E. coli and purified as described
  • the polypeptide was placed in 1 mM dithiotheitol (DTT) and 50 mM Tris-HCl, pH 7.5, at a concentration of 2 mg per ml. This concentrate was then diluted to 1.25 ng/ ⁇ l in 100 mM NaCl and 50 mM Tris-HCl, pH 7.4, to make a immunoassay microtiter well adsorption solution and 200 ⁇ l of the adsorption solution was added to each microtiter well of an Immulon IV Removawell strip
  • microtiter wells A blocking solution (200 ⁇ l, 2% alkaline-treated casein, 2% glycerol, 10% sucrose in TEN buffer: 150 mM NaCl, 10 mM tetrasodium EDTA, 1:10000 (w/v) thimerosal, and 50 mM Tris-HCl, pH 7.2 to 7.6) was added to each well.
  • the microtiter wells were covered and incubated with the blocking solution for 12 hours at 37°C in a humidified incubator. After blocking , the p24 gag coated and blocked microtiter wells were then inverted and dried for 12 hours at 4°C. Once the p24 gag coated and blocked microtiter wells had been dried, they were stored at -20°C until used.
  • Antibodies to p24 gag in human serum or plasma were detected using the same assay protocol as that for detection of antibodies to env polypeptides described in Example 1.
  • Table 3 illustrates the preferred range of the amount of antigenic polypeptide 24 gag to incorporate in the assay to provide the greatest sensitivity and selectivity.
  • Table 4 also illustrates the preferred range of the amount of antigenic polypeptide used to provide the optimal sensitivity and selectivity in an assay. The data clearly illustrates lesser amounts, 10 ng or less, suffer from lack of sensitivity where as higher amount show decreasing selectivity.
  • the different optical density values were then normalized to a background level by subtracting the optical density measurement for a microtiter well containing only developed and stopped substrate reagent (the substrate reagent was stopped with 2.5 M H 2 SO 4 ) no added serum or secondary conjugate.
  • Example 9 The p40 x polypeptide described in Example 9 was expressed in E. coli and purified as described
  • the polypeptide was placed in 8 M Guanidine-HCl, 100 mM dithiotheitol (DTT) and 50 mM Tris-HCl, pH 7.5, at a concentration of 2 mg per ml. This concentrate was then diluted to 0.34 ng/ ⁇ l in 4 M Guanidine-HCl and 50 mM Tris-HCl, pH 7.4, to make an immunoassay microtiter well adsorption solution and 200 ⁇ l of the solution was added to each microtiter well of an Immulon IV
  • microtiter wells containing the p40 x solution were covered and incubated for 12 hours at 4°C in a
  • a blocking solution (200 ⁇ l, 2% alkaline-treated casein, 2% glycerol, 10% sucrose in TEN buffer: 150 mM NaCl, 10 mM tetrasodium EDTA, 1:10000 (w/v) thimerosal, and 50 mM Tris-HCl, pH 7.2 to 7.6) was added to each well.
  • the microtiter wells were covered and incubated with the blocking solution for 12 hours at 4°C in a humidified incubator. After blocking, the coated and blocked microtiter wells were decanted and patted as above. The microtiter wells were then
  • Table 5 illustrates the preferred range of the concentration of antigenic polypeptide p40 x to
  • the p24 gag polypeptide, env B polypeptide, p21e/gp46, and the p40x polypeptide prepared as
  • microtiter wells containing the adsorption solution were incubated for 12 hours at 37°C. After the adsorption, the adsorption solution in the microtiter wells was decanted and the microtiter wells were inverted and patted on an
  • a blocking solution 250 ⁇ l, 2% alkaline-treated casein, 2% glycerol, 10% sucrose in TEN buffer: 150 mM NaCl, 10 mM tetrasodium EDTA, 1:10000 (w/v) thimerosal, and 50 mM Tris HC1, pH 7.2 to 7.6) was added to each well.
  • the microtiter wells were covered and incubated with the blocking solution for 12 hours at 37°C in a humidified
  • the antigen coated wells were decanted and patted as above.
  • the microtiter wells were then inverted and dried for 12 hours at 4°C. Once the antigen coated and blocked wells had been dried, they were stored at -20°C until used.
  • Antibodies to the HTLV-I antigens in human serum or plasma were detected using the same protocol as that for detection of env antibodies in human serum or plasma. Fifty-two HTLV-I positive sera containing antibodies to p24 gag, env B, or p40 x were examined for reactivity in the multiple antigen assay. All fifty-two samples were identified by the multiple antigen assay.
  • Table 6 illustrates the optical density measurements for a multiple antigen assay using the three antigenic polypeptides, p24 gag, env B, and p40 x .
  • Optical density measurements for assays using each antigenic polypeptide individually are also given in Table 6.
  • recombinant p24 gag reduced the signal to 0.294 O.D.
  • the O.D. signals for normal human serum were 0.123 without the addition of p24 gag and 0.114 O.D. with the addition of recombinant p24 gag.
  • recombinant HTLV-I antigenic polypeptides resulted in a significant visible decrease in the intensity of the associated antigen band on the gel. This indicated that the recombinant polypeptides added during the serum incubation competed with the radiolabelled HTLV-I antigen in the lysate for serum HTLV-I antibodies. This also indicated that the recombinant polypeptides are immunologically equivalent to the viral-derived antigens using HTLV-I positive serum.
  • the antigenic polypeptides are placed in a suitable buffer such as 8 M Guanidine-HCl in distilled water, 4 M Guanidine-HCl in distilled water, or
  • concentrations are selected to optimize the assay selectivity and sensitivity, typical concentrations are about 20 ng/ ⁇ l to 500 ng/ ⁇ l.
  • nitrocellulose solid supports which may be used include: 1) nitrocellulose 0.45 ⁇ m, 0.2 ⁇ m, 0.1 ⁇ m, (Schleicher and Schuell);
  • each antigenic polypeptide concentrate is contacted with the solid support and allowed to dry.
  • the coated supports are further treated with a blocking solution (3% Carnation nonfat dry milk, 0.3% Tween-20, in Dulbecco's phosphate-buffered saline, PBS, Gibco Laboratories, Grand Island, NY) by placing the supports and blocking solution in a multiple well tray.
  • the tray is typically shaken at 60 RPM on a rotary shaker (Junior Orbit Shaker, Labline) at room temperature for one hour.
  • the blocking solution is then aspirated from the tray wells.
  • the strips may be removed and placed antigen side up on blotting paper, such as 3MM, Whatman, for approximately 30 minutes until dry.
  • the strips may then be stored sandwiched between blotting paper at 4°C in the dark.
  • a sample is diluted in the same solution as the blocking solution and then added to each tray well containing a coated and blocked support.
  • the sample and support are shaken on a rotary shaker generally at room temperature for about one to five hours.
  • the excess sample containing the serum or plasma is then aspirated.
  • a wash solution such as 0.3% Tween-20 in PBS is added to each well, the wells are rocked back and forth several times and the wash solution is then aspirated. This wash and
  • the support is shaken on a rotary shaker generally for about one hour at room temperature.
  • the supports are then aspirated and washed, if desired.
  • the supports are washed an additional time with water and aspirated.
  • a substrate developing solution such as 78.6 ml of deionized water added to 1.334 ml of 3 M ammonium acetate, pH 5.5, 80 ⁇ l of 30% hydrogen peroxide, 2 ml of 10 mg/ml 3,3'-diaminobenzidine tetrahydrochloride
  • the support is then added to the support.
  • the support is incubated for about 5 to 30 minutes at room temperature until adequate color develops.
  • the substrate developing reaction is stopped by rinsing the strip in the well with deionized water.
  • HTLV-I positive sera Six HTLV-I positive sera were tested using antigenic polypeptides bound to nitrocellulose. All six HTLV-I positive sera reacted visibly with purified p24 gag at all concentrations while HTLV-I negative sera did not react significantly at any concentration. Two HTLV- I positive sera reacted best with respect to negative serum at the 10 ng concentration for env B. Other concentrations of the purified env B gave a visible signal for the negative serum. Three HTLV-I positive sera reacted significantly for p40 x at concentrations of 10 and 50 ng while the negative sera reacted only weakly. In all cases, when the antigen is initially taken up in 4 M or 8 M Guanidine-HCl the signal was improved over presentation of the antigen to the solid support in distilled water.
  • the DNA sequence encoding the antigenic polypeptide was constructed from three laboratory produced oligonucleotides.
  • the nucleotide fragments were synthesized on an Applied BioSystems 380B DNA synthesizer following operating parameters established by the manufacturer. In general, the oligonucleotide fragments were
  • acetonitrile The washed polymer was rinsed with dry acetonitrile, placed under argon and then treated in the condensation step as follows: a solution of 10 mg tetrazole in 0.5 ml of acetonitile was added to the reaction vessel containing polymer; 30 mg of a protected nucleoside phosphoramidite in 0.5 ml of acetonitrile was added; the mixture was agitated and allowed to react for 2 minutes; the reactants were removed by suction; and the polymer was rinsed with acetonitrile. This was followed by an oxidation step wherein a solution
  • the oligonucleotide was cleaved from the polymer using concentrated ammonia and the oligonucleotide solution was extracted four times with 1-butanol. The solution was loaded into a 20% polyacrylamide 7 M urea
  • oligomers were treated with polynucleotide kinase to introduce 5' phosphate moieties as described in Maniatis, et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory
  • Oligomer duplexes were formed by mixing equal molar quantities of the appropriate single-strand oligomers in water, placing the mixture in a boiling water bath for 5 minutes, and then slowly cooling to 4°C. The hybridized oligomer duplexes were then covalently connected with bacteriophage T4 ligase to form the correct duplex structures for molecular cloning. The duplex synthetic DNA constructs were isolated by 8-10% PAGE and passive elution.
  • the synthetic DNA sequence was then inserted as a Hindlll/BamHI fragment into plasmid pCFM1156, described below, to give plasmid pADK1001.
  • the plasmid pCFM1156 was digested with restriction enzymes Hindlll and BamHI, and isolated by agarose gel electrophoresis using a NA-45 membrane (as described in Schleicher & Schuell Applications Update No. 364).
  • the purified vector was mixed with an equal molar quantity of the above synthetic DNA segment, ligated using T4 ligase, and transformed into E. coli host cell FM6, described below. Transformants were plated onto Luria Broth/kanamycin agar plates and grown for approximately 24 hours at 30°C.
  • Candidate colonies were then picked and grown in 10 ml of Luria Broth/ kanamycin (50- ⁇ g/ml) overnight at 30°C. Candidates were diluted 1:50 into fresh Luria Broth/kanamycin and grown until the optical density at 600 nm was 0.2. At that time, the cultures were heated to 42°C and grown for an additional 5 hours. Samples of the induced whole cells were run on SDS-PAGE to look for protein expression. Cells expressing the desired protein had a band at the correct molecular weight which was not present in cells not containing the desired construct.
  • the plasmid pCFM1156 is prepared from the known plasmid pCFM836.
  • the preparation of plasmid pCFM836 is described in U.S. Patent 4,710,473, the relevant portions of the specification, particularly examples 1 to 7, are hereby incorporated by reference.
  • Ndel sites are cut, the exposed ends are filled with T4 polymerase and the filled ends are blunt-end ligated.
  • the resulting plasmid is then digested with Clal and Kpnl and the excised DNA fragment is replaced with a DNA oligionucleotide of the following sequence:
  • AGC 5' The construction of the DNA sequence coding for the polypeptide p24 gag required preparing, as described above, a second oligonucleotide having the following sequence.
  • This second oligonucleotide was inserted into plasmid pADK1001 by digesting the oligonucleotide with Xhol and BamHI and ligating the XhoI/BamHI fragment into plasmid pADK1001 which had also been digested with Xhol and BamHI to give the plasmid pADK1007.
  • the conditions to transform the host strain, FM6, with pADK1007 were similar to the conditions used with pADK1001, described above.
  • the construction of the DNA sequence coding for the polypeptide p24 gag was completed by inserting a third oligonucleotide into plasmid pADK1007.
  • the third oligonucleotide of the following sequence was prepared as described above.
  • the third oligonucleotide was initially replicated in plasmid M13mp19.
  • Plasmid M13mp19 was purchased from New England BioLabs, Inc. Ligation was as described above.
  • the construct was transfected into host cell JM103 (commercially available from many sources, including the ATCC). Transformants were plated onto Luria Broth plates using soft agar containing untransfected JM103 cells, 5-bromo-4-chloro-3-indolyl- ⁇ - galactoside in dimethylformamide (1:120) and 333 ⁇ M isopropylthiogalactoside.
  • the plasmid M13mp19 was digested with Xbal and
  • Hindlll and the Xbal/Hindlll fragment was isolated and then inserted into plasmid pADK1007 by digesting the plasmid with Xbal and Hindlll and removing the existing Xbal/Hindlll fragment and inserting the desired
  • the new plasmid was designated pADK1026.
  • the conditions to transform the host strain, FM6, with pADK1026 were similar to the conditions used with pADK1001, described above.
  • the directly expressed p24 gag polypeptide was purified as described in Example 10 or by high pressure liquid chromatography and the purified polypeptide was analyzed by protein microsequencing and amino acid composition.
  • the antigenic polypeptide p24 gag was found to react with commercially available monoclonal antibodies (available from DuPont and Sigma) which are immunoreactive with virally derived antigenic proteins.
  • the DNA sequence encoding the antigenic polypeptide, env B was prepared by digesting the native env DNA sequence with Sail and BamHI to give the following Sall/BamHI fragment. 10 20 30 40 50
  • the native HTLV-I DNA sequence used for the construction of env B was obtained from Irvin Chen at the University of California, Los Angeles, California.
  • the DNA obtained from Chen was sequenced using the dideoxynucleotide technique of Sanger, (Proc. Natl.
  • 5'-CATTAACTGGACCCACTGC-3' was synthesized on an Applied BioSystems 380B DNA synthesizer and isolated by
  • the mutated DNA sequence was modified by fusing it to a DNA sequence encoding the first 82 amino acids of alpha consensus interferon (described in U.S. Patent 4,695,623). This required the synthesis of a DNA duplex to insert as an in-frame coding sequence between the coding sequence for alpha consensus interferon (Hindlll) and the coding sequence for this portion of the HTLV-I env protein (Sail).
  • the sequence of the oligomer duplex was as follows: 5'AGCTTCGGTATGGGTTTCCCGTTCTCTCTGCTGG3'
  • linker oligomers were synthesized on an Applied BioSystems 380B DNA Synthesizer and the
  • oligomers were annealed by mixing in a 1:1 ratio in sterile water, placing the mixture in boiling water for 5 minutes and allowing the solution to slowly cool to 4°C.
  • the 446 nucleotide insert Sail to BamHI DNA sequence encoding amino acids 158 through 308 of the HTLV-I env protein was isolated from the HTLV-I native sequence using NA-45 membrane
  • the three pieces of DNA were ligated together in a molar ratio of 1:3:3 (vector: insert: linker) following procedures described in Maniatis, et al., p. 126, transformed into E. coli strain FM6 as described above and selected for growth on Luria Broth agarose plates containing 50 ug/ml kanamycin at 28-30°C.
  • Viable clones were screened by restriction digestion. Clones which were determined to contain the correct insert were induced by growing the clones at 28°C in Luria Broth containing 50 ug/ml kanamycin until they reached an optical density at 600 nm of 0.3. The culture was then heated to 42°C and grown for an
  • the cells were harvested by spinning at 8000 rpm for 20 minutes in a Beckman J2-21 centrifuge .
  • the isolated env B antigenic polypeptide is expressed as a fusion polypeptide where the leader sequence is encoded by the following DNA sequence which expresses the amino acid sequence shown below.
  • the leader amino acid sequence may be cleaved from the antigenic polypeptide sequence using CNBr because the desired antigenic polypeptide amino acid sequence contains no internal methionines.
  • the native HTLV-I sequence used for the construction of expression of p40 x was obtained from Irvin Chen at the University of California, Los Angeles, California.
  • the vector containing the p40 x coding sequence was digested with AceI and EcoRI and the 2.9 kb fragment containing the desired portion of the tax gene was isolated using agarose gel purification with NA-45 membrane pcper (Schleicher and Schuell).
  • the expression vector pCFM1156 was cleaved with Xbal and EcoRI and also purified using agarose gel electrophoresis and NA-45 membrane paper (Schleicher and Schuell).
  • a synthetic DNA linker with the following sequence was synthesized:
  • the resulting composite synthetic/native tax gene was isolated using agarose gel electrophoresis and NA-45 membrane paper (Schleicher and Schuell). This Xbal/EcoRI DNA fragment was ligated into pCFM1156 cleaved as described above. No expression was observed of any candidate clones. Upon dideoxynucleotide DNA sequencing of one of the clones, nucleotides near the Xbal site were found to be deleted.
  • the synthetic sequence contains a Clal restriction site for ligation to the Clal site of the plasmid, pCFM1156, and encodes the native amino acid sequence for the p40 x polypeptide up to Clal site of the native p40 x gene.
  • the synthetic sequence was designed to use the optimal codon usage for E. coli and minimized undesired
  • containing the modified Xbal site was cleaved with Clal and agarose gel purified.
  • the synthetic Clal/Clal DNA sequence was ligated into the Clal-cleaved vector.
  • Transformants were screened for correct orientation of the inserted synthetic DNA fragment and those with the fragment in the correct orientation were induced as above.
  • a polypeptide band of the predicted molecular weight was seen in the inclusion body fraction of induced cells containing the proper construct when analyzed on SDS-PAGE.
  • the desired recombinant polypeptide, p40 x was immunologically identified by a Western blot using antiserum generated to a peptide containing amino acids 269 to 353 of the carboxy terminus of the native HTLV-I tax protein developed jointly by Amgen and Dennis Slamon at the University of California, Los Angeles, California.
  • the recombinant polypeptide was negative by Western blot using normal rabbit serum.
  • the recombinant p40 x polypeptide was identified immunologically by
  • the crude polypeptide prepared as described in Example 7 is diluted to 50 ml with 4 M Guanidine HCl: 100 mM DTT:50 mM Tris-HCl, pH 8.0, stirred briefly, and transferred into a pre-washed Spectra/Por 1, 6-8K molecular weight cutoff dialysis tubing (51mm).
  • the solution is dialyzed against 4 liters of 50 mM Tris-HCl pH 8.0:1 mM DTT twice (6 h and 12 h), then 4 liters of 10 mM Tris-HCl, pH 8.0:1 mM DTT twice (6 h and 18 h). Dialyzed material is centrifuged at 8K for 10 minutes.
  • CM-52 75 ml bed volume
  • CM-52 75 ml bed volume
  • This elution pool is lyophilized, resolubilized in 100 ml of 2 M Guanidine HCl:50 mM Tris HC1 pH 8.0:100 mM DTT, dialyzed in
  • the crude polypeptide prepared as described in Example 9 is added to 10 ml of 8 M Guanidine HCl:100 mM DTT: 50 mM Tris-HCl, pH 8.0 and stirred for 2 hours.
  • the mixture is diluted to 2.5 M Guanidine HCl with 22 ml of 50 mM Tris-HCl, pH 8.0, and centrifuged at 5K for 5 minutes.
  • the supernatant is decanted away and the pellet is solubilized in 30 ml of 45% aqueous CH 3 CN:55% 0.1% TFA with sonication (high power, 2 to 3 minutes).
  • the mixture is centrifuged at 15K for 10 minutes, filtered through a 0.45 micron filter, and chromato- graphed.
  • a first purification is performed on a Vydac WPC 4 semi-prep column (1.0 x 25 cm) using a linear gradient of 40-60% (0.05% TFA in CH 3 CN, 0.1% aqueous TFA) over 20 minutes. The proper fractions are combined and lyophilized.
  • the dry material is resolubilized in 15 ml of 45% CH 3 CN:55% 0.1% aqueous TFA and reprepped on a Vydac WPC 18 semi-prep column (1.0 x 25cm) using a linear gradient of 45-55% over 20 minutes (0.05% TFA in CH 3 CN, 0.1% aqueous TFA).
  • the proper fractions are collected and lyophilized.
  • the crude polypeptide prepared as described in Example 8 is suspended in 20 ml of water and centrifuged at 15K for 10 minutes. The supernatant is decanted away and the pellet solubilized in 10 ml 8 M Guanidine HCl: 100 mM DTT:50 mM Tris-HCl, pH 8.0, with stirring for 2 hours. The solution is diluted to 2 M Guanidine HCl with 30 ml of 50 mM Tris-HCl, pH 8.0, with formation of a precipitate which is pelletized by centrifugation at 5K for 5 minutes.
  • the supernatant is decanted away and the pellet is solubilized immediately in 40 ml of 45% CH 3 CN:55% 0.1% aqueous TFA with sonication (high power, 2-3 minutes).
  • the solution is centrifuged at 15K for 10 minutes, filtered through 0.45 micron filter and HPLC purif ied on Vydac WPC 18 semi-prep column (1.0 X 25cm) using a linear gradient of 35-60% over 25 minutes (0.05% TFA in CH 3 CN, and 0.1% aqueous TFA). The proper fractions are combined and lyophilized.

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Abstract

Une immunoanalyse utilisant des peptides antigéniques recombinants codés par les gènes env, tax et gag du VLHT-I (virus de la leucémie humain à cellules en T). L'utilisation d'une combinaison de ces polypeptides antigènes permet de réaliser une analyse de dépistage de la présence d'anticorps du VLHT-I dans les fluides corporels par: combinaison de fluides corporels renfermant des anticorps avec un ou plusieurs antigènes de polypeptides recombinants sélectionnés parmi le groupe qui consiste en des polypeptides codés par l'ensemble ou une partie des gènes env, tax et gag du VLHT-I; la formation d'une paire de liaison d'anticorps-antigène détectable et détection de la présence de la paire de liaison.
PCT/US1990/006647 1989-11-17 1990-11-14 Methode de detection d'anticorps htlv-i dans les fluides corporels humains WO1991007510A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994018322A1 (fr) * 1993-02-05 1994-08-18 Genelabs Technologies, Inc. Peptides derives de gp21 de htlv-i et htlv-ii utilises dans des diagnostics
WO1996039630A1 (fr) * 1995-06-05 1996-12-12 Abbott Laboratories Detection d'anticorps du htlv a l'aide de proteines de recombinaison
WO2002008761A1 (fr) * 2000-07-21 2002-01-31 Cuno Incorporated Produits chimiques de blocage ameliores pour membrane de nylon
US6689879B2 (en) 1998-12-31 2004-02-10 Chiron Corporation Modified HIV Env polypeptides
US7211659B2 (en) 2001-07-05 2007-05-01 Chiron Corporation Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US7282364B2 (en) 2001-08-31 2007-10-16 Novartis Vaccines And Diagnostics, Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof
US7348177B2 (en) 1998-12-31 2008-03-25 Novartis Vaccines And Diagnostics, Inc. Expression of HIV polypeptides and production of virus-like particles
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0136798A2 (fr) * 1983-08-25 1985-04-10 Biotech Research Laboratories Inc. Plaque de titration et trousse d'analyse pour détecter des anticorps dans le sérum humain, leur préparation et utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0136798A2 (fr) * 1983-08-25 1985-04-10 Biotech Research Laboratories Inc. Plaque de titration et trousse d'analyse pour détecter des anticorps dans le sérum humain, leur préparation et utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MOLECULAR AND CELLULAR RPOBES, Volume 2, issued 1988, KITAJIMA et al., "Synthesis of proteins in Esherichia coli immunoreactive with sera grom individuals infected with human T-cell leukemia virus type 1", pages 39-46. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5643714A (en) * 1986-12-31 1997-07-01 Genelabs Technologies, Inc. Method and assay for HTLV
WO1994018322A1 (fr) * 1993-02-05 1994-08-18 Genelabs Technologies, Inc. Peptides derives de gp21 de htlv-i et htlv-ii utilises dans des diagnostics
AU690540B2 (en) * 1993-02-05 1998-04-30 Genelabs Technologies, Inc. HTLV-I and HTLV-II gp21 derived peptides for use in diagnostics
WO1996039630A1 (fr) * 1995-06-05 1996-12-12 Abbott Laboratories Detection d'anticorps du htlv a l'aide de proteines de recombinaison
US6689879B2 (en) 1998-12-31 2004-02-10 Chiron Corporation Modified HIV Env polypeptides
US7348177B2 (en) 1998-12-31 2008-03-25 Novartis Vaccines And Diagnostics, Inc. Expression of HIV polypeptides and production of virus-like particles
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
WO2002008761A1 (fr) * 2000-07-21 2002-01-31 Cuno Incorporated Produits chimiques de blocage ameliores pour membrane de nylon
US7211659B2 (en) 2001-07-05 2007-05-01 Chiron Corporation Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
US9598469B2 (en) 2001-07-05 2017-03-21 Novartis Vaccines And Diagnostics, Inc. HIV-1 south african subtype C env proteins
US7282364B2 (en) 2001-08-31 2007-10-16 Novartis Vaccines And Diagnostics, Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof

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