EP1238087A2 - Schweinendoviren gag und env und deren diagnostische verwendungen - Google Patents

Schweinendoviren gag und env und deren diagnostische verwendungen

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Publication number
EP1238087A2
EP1238087A2 EP00953333A EP00953333A EP1238087A2 EP 1238087 A2 EP1238087 A2 EP 1238087A2 EP 00953333 A EP00953333 A EP 00953333A EP 00953333 A EP00953333 A EP 00953333A EP 1238087 A2 EP1238087 A2 EP 1238087A2
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EP
European Patent Office
Prior art keywords
poerv
polypeptide
gag
fragment
polypeptide fragment
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EP00953333A
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English (en)
French (fr)
Inventor
Daniel Galbraith
Helena Kelly
Kenneth Smith
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Quip Technology Ltd
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Quip Technology Ltd
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C12N2740/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates inter alia to porcine endogenous retrovirus (PoERV) fragments, in particular to gag and env fragments of PoERV.
  • the invention relates further to use of such fragments in detection of PoERV or detection of exposure to PoERV.
  • Porcine endogenous retrovirus is an endogenous Gammaretrovirus present typically as a provirus found in several loci in the porcine genome.
  • the proviral genome can be silent or is expressed.
  • Expression of the virus was found to be associated with leukaemic pigs (Strandstro et al, 1974) and some continuous porcine cell lines produce PoERV (Todaro et al, 1974) .
  • Virus from these cells has been shown to infect non-porcine cell-types including human cells (Galbraith et al, 1997; Patience et al, 1997).
  • PoERV A, B and C dependent on the tropism of the virus and the related envelope gene structure (Onions et al, 1998) . Only subtypes A and B have been shown to be capable of consistently infecting human cells in vitro. Subgroup C PoERV from mini-pigs has only been shown to infect one human cell line and this may reflect a low capacity for infection of human cells. Since PoERV is expressed in pigs there is the potential for virus to be present in material prepared from pigs. Furthermore, as a consequence of xenotransplantation using porcine donor organs, there is the possibility that the endogenous virus will be expressed in vivo and be a potential risk of PoERV infection of the patient and the general population thereafter.
  • PoERV A number of different types of PoERV are known, based on their genetic makeup. Types designated PERV A, PERV B, and PoEVl are described in International Patent Application O97/40167, while types designated PERV MSL and Tsukuba are described in International Patent Application 097/21836.
  • PoERV viruses comprise three genes: gag, pol, and env, generating GAG, POL and ENV polypeptides. It has been observed that the gag region of the genome appears to be substantially conserved among different viruses, as well as between PoERV virus types, while the env region contains both conserved and non-conserved regions, which non- conserved regions are observed to vary between viral types. It is among the objects of the present invention to provide means whereby patients and/or samples may be monitored for viral infection. It is further among the objects of the present invention to provide means whereby the viral type may be determined. According to one aspect of the present invention there is provided a PoERV polypeptide fragment, wherein said polypeptide fragment has PoERV specific antigenic or immunogenic activity.
  • Antigenic or immunogenic activity is to be understood as capable of eliciting a PoERV specific immune response when introduced into a normal mammalian host.
  • PoERV specific antibodies are produced as a consequence.
  • a fragment of a PoERV GAG polypeptide wherein said fragment has PoERV specific antigenic or immunogenic activity.
  • a fragment will be referred to hereinafter as a GAG peptide, it being understood that this is distinct from native GAG protein, and may comprise only a fragment thereof, provided the GAG peptide has antigenic activity.
  • the consensus PoERV GAG polypeptide sequence is shown in Figure 1; in preferred embodiments of the invention, the GAG peptide may be selected from within this sequence.
  • a fragment of a PoERV ENV polypeptide wherein said fragment has PoERV specific antigenic or immunogenic activity.
  • a fragment will be referred to hereinafter as an ENV peptide, it being understood that this is distinct from native ENV protein, and may comprise only a fragment thereof, provided the ENV peptide has antigenic activity.
  • the ENV peptide may be selected from within a conserved region of the various PoERV sequences, as illustrated in Figures 2 and 3.
  • conserved regions are those which comprise identical and/or highly conserved amino acid sequences in different PoERV virus types; wholly conserved amino acids are indicated in Figures 2 and 3 by an asterisk beneath the amino acid, with highly conserved amino acids being indicated by a colon.
  • the ENV peptide may be selected from within the non-conserved regions of the various PoERV sequences of Figures 2 and 3 , in which case the ENV peptide will be specific for a particular type of PoERV.
  • the ENV peptide may comprise both a conserved and a non-conserved region of the PoERV ENV protein, from either adjacent or non-adjacent regions of the ENV protein. Such peptides may be considered useful in simultaneous detection of any PoERV virus and a specific viral type.
  • a fusion GAG/ENV peptide which peptide comprises both GAG peptide sequences and ENV peptide sequences.
  • Such peptide may be considered useful in simultaneous detection of any PoERV virus, by means of the GAG peptide, and a specific viral type, by means of the ENV peptide.
  • antibodies specific to either GAG or ENV peptides may be polyclonal or monoclonal.
  • Such antibodies can include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs) , humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti- Id) antibodies, and epitope-binding fragments of any of the above.
  • the Ig tails of such antibodies can be modified to reduce complement activation and Fc binding, (See, for example, European Patent No. 239400 Bl, Aug. 3, 1994).
  • various host animals can be immunized by injection with a peptide, or a portion thereof.
  • host animals can include but are not limited to rabbits, mice, and rats, to name but a few.
  • Various adjuvants can be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Gurein) and Corynebacterium parvum .
  • BCG Bacille Calmette-Gurein
  • Corynebacterium parvum bacille Calmette-Gurein
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof.
  • an antigen such as target gene product, or an antigenic functional derivative thereof.
  • host animals such as those described above, can be immunized by injection with a gene product supplemented with adjuvants as also described above.
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, can be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milsrein, (1975, Nature 256:495- 497; and US Pat. No.
  • Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAb of this invention can be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
  • chimeric antibodies In addition, techniques for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454; U.S. Pat. No. 4,816,567) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • Antibody fragments which recognize specific epitopes can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries can be constructed (Huse et al. , 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • Further aspects of the present invention provide methods of screening serum or tissue from humans or animal recipients of porcine tissue for exposure to PoERV. These methods include: use of antibodies to GAG or ENV peptides in the capture and/or detection of PoERV antigens; use of antibodies to GAG or ENV peptides in the detection of PoERV gene expression in virus infected cells by indirect immunofluorescence staining; the use of antibodies to GAG or ENV peptides in the visualisation of PoERV virions in a sample by immuno-electron microscopy; use of GAG or ENV peptides in Western blotting for the detection of PoERV antibodies in samples from recipients of porcine-derived materials; and the use of GAG or ENV peptides in an enzyme- linked immunosorbent assay (ELISA) for the detection of PoERV antibodies.
  • ELISA enzyme- linked immunosorbent assay
  • Each of these methods may non-specifically detect any PoERV type (if GAG peptides, conserved ENV peptides or antibodies are used) or specific PoERV types, if type- specific non-conserved ENV peptides or antibodies are used.
  • the present invention also encompasses assay kits including GAG or ENV peptides or antibodies to such peptides, for use in the abovementioned assays.
  • the kits may further comprise any or all necessary preparative reagents, washing reagents, detection reagents and signal producing reagents commonly known in the art.
  • a number of distinct peptides or antibodies may be used, either sequentially or simultaneously, and differently labelled, in order to detect a number of different PoERV types in a single assay.
  • Diagnostic assays based upon the present invention may be used to determine the presence or absence of PoERV infection, and the PoERV type involved.
  • an assay for the diagnosis of viral infection there are basically three distinct approaches that can be adopted, involving the detection of viral nucleic acid, viral antigen or viral antibody respectively.
  • Viral nucleic acid is generally regarded as the best indicator of the presence of the virus itself and would identify materials likely to be infectious.
  • the detection of nucleic acid is not usually as straightforward as the detection of antigens or antibodies since the level of target can be very low.
  • Viral antigen is used as a marker for the presence of virus and as an indicator of infectivity. Depending upon the virus, the amount of antigen present in a sample can be very low and difficult to detect.
  • Antibody detection is relatively straightforward because, in effect, the host immune system is amplifying the response to an infection by producing large amounts of circulating antibody.
  • the nature of the antibody response can often be clinically useful, for example IgM rather than IgG class antibodies are indicative of a recent infection, or the response to a particular viral antigen may be associated with clearance of the virus.
  • a diagnostic assay may embody any one or a combination of these three approaches.
  • the method may comprise contacting a test sample with a peptide of the present invention or a polyclonal or monoclonal antibody against the peptide and determining whether there is any antigen-antibody binding contained within the test sample.
  • a test kit may be provided comprising a peptide, as defined herein, or a polyclonal or monoclonal antibody thereto and means for determining whether there is any binding with antibody or antigen respectively contained in the test sample to produce an immune complex.
  • the test sample may be taken from any appropriate tissue or physiological fluid, such as blood (e.g. serum or plasma), saliva, urine, cerebrospinal fluid, sweat, tears or tissue exudate. If a physiological fluid is obtained, it may optionally be concentrated for any viral antigen or antibody present.
  • the peptide can be used to capture selectively antibody against PoERV from solution, to label selectively the antibody already captured, or both to capture and label the antibody.
  • the peptide may be used in a variety of homogeneous assay formats in which the antibody reactive with the peptide is detected in solution with no separation of phases.
  • the types of assay in which the peptide is used to capture antibody from solution involve immobilization of the peptide on to a solid surface.
  • This surface should be capable of being washed in some way.
  • suitable surfaces include polymers of various types (moulded into microtitre wells; beads; dipsticks of various types; aspiration tips; electrodes; and optical devices) , particles (for example latex; stabilized red blood cells; bacterial or fungal cells; spores; gold or other metallic or metal-containing sols; and proteinaceous colloids) with the usual size of the particle being from 0.02 to 5 microns, membranes (for example of nitrocellulose; paper; cellulose acetate; and high porosity/high surface area membranes of an organic or inorganic material) .
  • the attachment of the peptide to the surface can be by passive adsorption from a solution of optimum composition which may include surfactants, solvents, salts and/or chaotropes; or by active chemical bonding.
  • Active bonding may be through a variety of reactive or activatable functional groups which may be exposed on the surface (for example condensing agents; active acid esters, halides and anhydrides; amino, hydroxyl, or carboxyl groups; sulphydryl groups; carbonyl groups; diazo groups; or unsaturated groups) .
  • the active bonding may be through a protein (itself attached to the surface passively or through active bonding) , such as albumin or casein, to which the viral peptide may be chemically bonded by any of a variety of methods.
  • a protein in this way may confer advantages because of isoelectric point, charge, hydrophilicity or other physico-chemical property.
  • the viral peptide may also be attached to the surface (usually but not necessarily a membrane) following electrophoretic separation of a reaction mixture, such as immunoprecipitation.
  • the captured antibody After contacting the surface bearing the peptide with a test sample (in the presence of a blocking mixture if required) , allowing time for reaction, and, where necessary, removing the excess of the sample by any of a variety of means, (such as washing, centrifugation, filtration, magnetism or capillary action) the captured antibody is detected by any means which will give a detectable signal. For example, this may be achieved by use of a labelled molecule or particle as described above which will react with the captured antibody or any molecule containing an epitope contained in the peptide. In one embodiment, it is preferred to add an anti-human IgG conjugated to horseradish peroxidase and then to detect the bound enzyme by reaction with a substrate to generate a colour.
  • the detectable signal may be produced by any means known in the art such as optical or radioactive or physico- chemical and may be provided directly by labelling the molecule or particle with, for example, a dye, radiolabel, fluorescent, luminescent, chemiluminescent, electroactive species, magnetically resonant species or fluorophore, or indirectly by labelling the molecule or particle with an enzyme itself capable of giving rise to a measurable change of any sort.
  • the detectable signal may be obtained using, for example, agglutination, or through a diffraction or birefrigent effect if the surface is in the form of particles.
  • Assays in which a peptide itself is used to label an already captured antibody require some form of labelling of the peptide which will allow it to be detected.
  • the labelling may be direct by chemically or passively attaching for example a radiolabel, magnetic resonant species, particle or enzyme label to the peptide; or indirect by attaching any form of label to a molecule which will itself react with the peptide.
  • the chemistry of bonding a label to the peptide can be directly through a moiety already present in the peptide, such as an amino group, or through an intermediate moiety, such as a maleimide group.
  • Capture of the antibody may be on any of the surfaces already mentioned in any reagent including passive or activated adsorption which will result in specific antibody or immune complexes being bound.
  • capture of the antibody could be by anti- species or anti-immunoglobulin-sub-type, by rheumatoid factor, proteins A, G and the like, or by any molecule containing an epitope contained in the peptide.
  • the labelled peptide may be used in a competitive binding fashion in which its binding to any specific molecule on any of the surfaces exemplified above is blocked by antigen in the sample. Alternatively, it may be used in a non-competitive fashion in which antigen in the sample is bound specifically or non-specifically to any of the surfaces above and is also bound to a specific bi- or poly-valent molecule (e.g. an antibody) with the remaining valencies being used to capture the labelled peptide.
  • a specific bi- or poly-valent molecule e.g. an antibody
  • Figure 1 is the consensus amino acid sequence of the PoERV GAG protein
  • Figure 2 is a comparison of amino acid sequences of five different PoERV ENV proteins
  • Figure 3 is a comparison of amino acid sequences of the variable region of five different PoERV ENV proteins, showing the six different ENV peptides (peptides D-H and J) referred to in the following examples.
  • Human 293 cells (American Type Culture Collection [ATCC] # CRL1573) and Raji cells (ATCC # CCL 86 ) were infected with PoERV by exposure to polybrene (Sigma-Aldrich Co. Ltd.) and continued incubation with cell-free filtered supernatant from PK-15 (ATCC # CCL 33) cells previously shown to be infected with all three subgroups of PoERV.
  • the 293 cells allow replication of type B PoERV (POEV-1) .
  • POEV-1 type B PoERV
  • the 293 cells were shown to be infected after passage by measurement of the reverse transcriptase activity of the cell supernatant and by a PoERV GAG-specific Polymerase Chain Reaction (PCR) .
  • PCR PoERV GAG-specific Polymerase Chain Reaction
  • the resulting virus particles were isolated from the cell line supernatant as follows. Supernatant from exponentially growing cells was layered onto a 20/40% (w/v) discontinuous sucrose density gradient and centrifuged at 100,000 g for 150 min. The viral material at the sucrose interface was harvested, and viral particles pelleted by further ultracentrifugation at 100,000g for 60 min, followed by resuspension in DMEM (Life Technologies Ltd. , UK) .
  • Control retroviruses To provide retroviral controls for cross reactivity with PoERV GAG and ENV, Squirrel monkey retrovirus, Murine leukaemia virus, Maedi-Visna virus and Equine infectious anemia virus virions were prepared from the appropriate infected cell line as described by Shepherd and Smith (1999) .
  • Peptides from the GAG protein can provide a capture antigen and a means to generate positive control antisera.
  • the antisera can be directed against conserved polypeptides present in the PoERV virion core likely to induce an immune response in recipients of the virus.
  • These reagents would be useful diagnostic tools for immunosurveillance of recipients of porcine material or tissues for exposure to PoERV. Therefore, peptides encompassing potential antigenic regions of PoERV GAG were selected from the translated amino-acids derived from the sequence of the gag region of PoERV based on three criteria; hydrophilicity, potential ⁇ -turns and K, D, R and E charged residues.
  • Peptide 1 was from the C-terminus of p30-GAG at residue 437-451 of the polypeptide, nucleotides 1896-1940 of the gag open reading frame (ORF) .
  • Peptide 2 was from the start of the plO segment of the GAG polypeptide at residue 502-515 of the polypeptide, nucleotides 2091- 2132 of the gag ORF.
  • the peptides shown below were chemically synthesised by Genosys Biotechnologies Inc.
  • bracketed amino-acid is not in sequence - 5 • position is from next residue (R) .
  • a BLAST search (Altschui et al., 1997) of the non- redundant GenBank coding sequences with GAG peptide 1 showed homology with seven sequences all from the gag ORF. Of the seven, three were with PoERV sequences with accessions gi 3116446 (100% match) , emb CAA7651 (100% match) , gi 3116442 (86% match) . The remainder were against the closely related Gibbon ape leukaemia virus (gi 3033415, 92% match) and Simian sarcoma virus (sp PO3330, 86% match).
  • the remaining two sequences were from murine viruses, including a virus from Rattus norvegicus (emb CAA24514; 92% match) , and one against Mus dunni endogenous retrovirus (gi 3309124, 93% match).
  • a similar BLAST search with GAG peptide 2 showed 100 - 99% homology with only the PoERV sequences listed above.
  • Peptide F RKTGKYSKVDKWYELGNS
  • amino acid residues of all peptides are identified by the standard one letter abbreviations .
  • PoERV p30- GAG and an abbreviated ENV polypeptides were designed and produced for use as capture antigens and to produce anti- polypeptide sera.
  • the required polypeptide portions of the gag and env genes were produced by PCR amplification, molecularly cloned into a prokaryotic expression vector and expressed as described below using standard techniques (Maniatis et al, 1982) .
  • a fragment encompassing the p30 region of the gag ORF from nucleotide 1173-1949 of the PoERV genome was amplified by PCR from cDNA generated from PK15 mRNA using ligation independent cloning oligonucleotide primers (pET-32 Ek/LIC cloning and expression vector; Novagen Inc. Catalogue # 69076-3) .
  • the oligonucleotides were: p30 forward 5' GAC GAC GAC AAG CTG CGC ACC TAT GGC C 3' p30 reverse 5" GAG GAG AAG CCC GGG TCT AGG CCA AGA TCT
  • the nucleotides in bold are viral specific.
  • the PCR conditions were 30 cycles of 95°C for 1 min, 58°C for 1 min and 72°C for 1 min.
  • the resulting 776 base pair fragment was molecularly cloned into the appropriate LIC site of the pET-32 LIC vector following the manufacturer's instructions (Novagen Inc. 69076-3 instruction manual) , transfected into competent NovoblueTM Escherichia coli cells and plated on solid LB medium containing ampicillin. The transformed colonies were selected by resistance to ampicillin.
  • a fragment encompassing the region of the env ORF from nucleotide 5616- 6304 of the PoERV genome was amplified by PCR from cDNA generated from PK15 and PoERV-infected 293 cells (PoERV B) RNA using ligation independent cloning oligonucleotide primers (pET-32 Ek/LIC cloning and expression vector; Novagen Inc. Catalogue # 69076-3) .
  • the oligonucleotides were:
  • the nucleotides in bold are viral specific.
  • the PCR conditions were 30 cycles of 95°C for 1 min, 60°C for 1 min and 72°C for 1 min.
  • the resulting 688 base pair fragment was molecularly cloned into the appropriate LIC site of the pET-32 LIC vector following the manufacturer's instructions as described above.
  • plasmid DNA was isolated from the ampicillin resistant NovoblueTM clones carrying the gag or env fragment in the correct orientation for expression as determined by restriction endonuclease mapping.
  • the plasmid DNAs were each transfected into competent E. coli AD494 (DE3) trx B- .
  • GAG peptide 1 and peptide 2 the peptides were conjugated with keyhole limpet hemacyanin carrier protein and each of two rabbits was inoculated six times at fourteen day intervals. The animals were bled out at day seventy seven after the first inoculation. The p30-GAG polypeptide was inoculated three times at fourteen day intervals into a rabbit. The animal was bled out at day seventy seven.
  • ENV peptides D-H and J the peptides were conjugated with keyhole limpet hemacyanin carrier protein and one sheep was inoculated three times at twenty eight day intervals. The ENV polypeptide was inoculated three times at fourteen day intervals into a rabbit.
  • Virions purified from PK15 cells were inoculated three times at fourteen day intervals into each of two guinea pigs. Indirect immunofluorescence staining
  • Recombinant p30-GAG polypeptide and ENV polypeptide were prepared, harvested and purified from an E . coli vector. The recombinant proteins were tested to determine an appropriate dilution of protein which yielded a positive result in the immunoassay. In addition, extracts from PoERV-infected 293 cells, PoERV-infected Raji cells or purified PoERV virions were used as antigens.
  • Samples were prepared in a Class 2 safety cabinet or other clean environments .
  • a typical negative control was prepared by making up to a 1:200 dilution of normal sera in blocking reagent.
  • a typical positive control was prepared by making a 1: 500, 1: 1000 or greater dilution of anti-PoERV p30-GAG polypeptide, peptide serum or anti - recombinant ENV serum.
  • a typical test serum was prepared by making up to a 1:200 dilution of sera.
  • membrane strips each were placed in a 15 ml centrifuge tube and 2 ml blocking reagent (2.5 g skimmed dried milk in 50 ml PBS/ 0.5% v/v Tween-20TM) added. The strips were placed on a rotary shaker such that the strip moved slightly on each revolution and were incubated for 30 min at ambient temperature. The blocking reagent was removed and replaced with 5-10 ⁇ l of the diluted serum. The membrane was incubated with shaking for 1 h at ambient temperature. To stop incubation the strip was removed from diluted serum and placed into PBS/ Tween-20TM and washed with three changes of PBS/ Tween-20TM at ambient temperature with shaking.
  • 2 ml blocking reagent 2.5 g skimmed dried milk in 50 ml PBS/ 0.5% v/v Tween-20TM
  • the appropriate species specific secondary antiserum conjugated to alkaline phosphatase was used as detector e.g. if human serum was being tested, an anti-human IgG alkaline phosphatase (AP) conjugate was used.
  • AP anti-human IgG alkaline phosphatase
  • the p30-GAG positive control required anti-rabbit IgG AP conjugate for detection and the anti ENV required anti-sheep IgG AP conjugate.
  • the detection was done as follows; each strip was placed in an unused 15 ml centrifuge tube, 2 ml of 1:1000 dilution of secondary sera in blocking reagent was added and incubated with shaking at ambient temperature for 1 h.
  • the strip was removed from the centrifuge tube, placed in PBS/Tween-20TM and washed with 3 changes of PBS/Tween- 20TM, at ambient temperature with shaking. The strips were then put into a 15 ml centrifuge tube and 2 ml of bromochlorindoyl phosphate/ nitroblue tetrazolium (BCIP/NBT, Sigma-Aldrich Co. Ltd.) solution was added to each tube. The strips were shaken gently and allowed to develop for 5 min. The reaction was stopped by rinsing the membrane strip in purified water and the strips were removed from the water and allowed to air dry.
  • the unbound antigen and coating solution were then removed from the wells with a pipette and washed three times with PBS / 0.05% Tween-20TM. Any remaining PBS / 0.05% Tween-20TM was removed by blotting on a tissue.
  • the substrate was prepared as follows: one 0- phenylenediamine tablet (Sigma-Aldrich Co. Ltd.) and one urea/H 2 0 2 tablet were dissolved in 20 ml of purified water. An aliquot of 50 ⁇ l of substrate was added to each well and the plate incubated at ambient temperature in the dark for 30 min. The reaction was then stopped by adding 50 ⁇ l of 3N HCl or 3 M H 2 S0 4 to each well. The colour development in the wells was measured at 490 nm using a Dynex MRX microplate reader.
  • the substrate used was p-Nitrophenyl phosphate (pNPP; Sigma-Aldrich Co. Ltd.) and the plates were read at 405 nm.
  • pNPP p-Nitrophenyl phosphate
  • Negative stain electron microscopy (NSEM) (Doane, 1980) was used to identify the presence of PoERV virions.
  • Supernatant from PoERV-infected pK15 cells was layered onto a 20/40% (w/v) discontinuous sucrose density gradient and centrifuged at 100,000 g for 150 min.
  • the viral material at the sucrose interface was harvested, and viral particles pelleted by further ultracentrifugation at 100,000g for 60 min, followed by resuspension in DMEM (Life Technologies Ltd.). The sample was then applied to pioloform-coated copper 300 mesh EM grids and allowed to air dry.
  • Grids were fixed with 2.5% glutaraldehyde (Agar Scientific), stained with 5% uranyl acetate (Agar Scientific) and allowed to air dry. Grids were examined on a Philips EM-400 transmission electron microscope. Immuno- Electron Microscopy
  • NSEM samples were applied to pioloform-coated nickel 300 mesh EM grids and allowed to air dry. Grids were fixed with modified immunofix, post fixed with 0.5 M NH 4 C1, then incubated with 2% bovine serum albumin (Sigma-Aldrich Co. Ltd.). Samples were then incubated with rabbit anti-PoERV (rabbits immunised with whole PoERV) or rabbit anti-PoERV p30-GAG antibody, washed in modified immunobuffer followed by incubation with anti- rabbit IgG gold conjugate (Sigma-Aldrich Co. Ltd.). Grids were stained with 5% uranyl acetate, and allowed to air dry. Samples were visualised on a Philips EM-400 transmission electron microscope.
  • Anti-GAG peptide 1 antisera and sera from rabbits inoculated with the recombinant p30-GAG polypeptide detected the expected protein of approximately 30kd in extracts of PoERV-infected 293 or PoERV-infected Raji cells, purified PoERV virions and recombinant p30-GAG.
  • the PoERV antibody could be detected at a dilution of 1:1000.
  • PoERV virion preparations by negative stain revealed particles showing the characteristic size and structure of a Gammaretrovirus of approximately 90-120 nm with a dark inner core and double membraneous outer region.
  • Patience C Takeuchi Y and Weiss R. (1997) . Infection of human cells by an endogenous virus of pigs. Nature Medicine , 3 , 282-286 .
EP00953333A 1999-08-18 2000-08-16 Schweinendoviren gag und env und deren diagnostische verwendungen Withdrawn EP1238087A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9919604 1999-08-18
GBGB9919604.0A GB9919604D0 (en) 1999-08-18 1999-08-18 Retrovirus assay
PCT/GB2000/003159 WO2001012816A2 (en) 1999-08-18 2000-08-16 Porcine endovirus gag and env and diagnostic uses thereof

Publications (1)

Publication Number Publication Date
EP1238087A2 true EP1238087A2 (de) 2002-09-11

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EP00953333A Withdrawn EP1238087A2 (de) 1999-08-18 2000-08-16 Schweinendoviren gag und env und deren diagnostische verwendungen

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AU (1) AU6584600A (de)
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WO (1) WO2001012816A2 (de)

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GB0313132D0 (en) * 2003-06-06 2003-07-09 Ich Productions Ltd Peptide ligands

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EP0907739A1 (de) * 1996-04-19 1999-04-14 Q-One Biotech Limited Schweineretrovirus

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See references of WO0112816A3 *

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GB9919604D0 (en) 1999-10-20
AU6584600A (en) 2001-03-13
WO2001012816A3 (en) 2002-07-11
WO2001012816A2 (en) 2001-02-22
WO2001012816A9 (en) 2002-12-12

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