WO2001018212A2 - Produit et procede - Google Patents

Produit et procede Download PDF

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Publication number
WO2001018212A2
WO2001018212A2 PCT/GB2000/003430 GB0003430W WO0118212A2 WO 2001018212 A2 WO2001018212 A2 WO 2001018212A2 GB 0003430 W GB0003430 W GB 0003430W WO 0118212 A2 WO0118212 A2 WO 0118212A2
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WO
WIPO (PCT)
Prior art keywords
sequence
polypeptide
nucleic acid
binding
paratuberculosis
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PCT/GB2000/003430
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English (en)
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WO2001018212A3 (fr
Inventor
Ingrid Olsen
Harald Wiker
Original Assignee
Veso As
Jones, Elizabeth, Jones
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Publication date
Application filed by Veso As, Jones, Elizabeth, Jones filed Critical Veso As
Priority to AU70233/00A priority Critical patent/AU7023300A/en
Publication of WO2001018212A2 publication Critical patent/WO2001018212A2/fr
Publication of WO2001018212A3 publication Critical patent/WO2001018212A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Mycobacteriaceae (F)
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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

Definitions

  • the present invention relates to polypeptides derivable from Mycobac terium avium ssp . paratuberculosis and related molecules, e.g. from other bacteria, fragments of these molecules and the nucleic acid molecules encoding such molecules, and antibodies specific to such polypeptides and the identification or use of these molecules for detection, diagnosis and/or prevention of the disease termed Paratuberculosis in ruminants, and also for the diagnosis and/or prevention of Crohn ' s disease in humans, and kits for performing such methods.
  • the invention also provides processes for preparing the polypeptides and nucleic acid molecules of the invention, which are brought to a state of biological purity such that they may constitute part of a diagnostic or prophylactic reagent system.
  • Paratuberculosis is an infectious, enteric disease of ruminants also known under the name Johne ' s disease.
  • the condition is caused by infection with the microorganism Mycobac terium avium ssp . paratuberculosis .
  • the disease is chronic and incurable when attacking cattle, characterised by a slow development involving a long subclinical incubation time before the first symptoms develop, typically diarrhoea, emaciation, cachexy and finally death. Histological examination shows oedema, infiltration and thickening of the ileal mucosa, and hypertrophy and necrosis of intestinal lymph nodes.
  • a miliary syndrome with diffused parenchima granuloma in liver, spleen and lungs also appears rather frequently.
  • Paratuberculosis is considered to be one of the most serious diseases affecting the world cattle industry.
  • the economical losses following infection are related to decreased production of milk, shortening of life and decreased slaughter weight.
  • the disease was first diagnosed in cattle in Germany in 1895, and has since then been spread to most of the world. Although the condition has been periodically brought under control in some countries, there has been a tendency of reinfection from imported cattle. Over all, the disease is an increasing problem in many countries.
  • Diagnosis of paratuberculosis is essential, especially in the absence of clinical symptoms. Elimination of hidden bacteria shedders is essential to limit the spread of the disease, and the only reliable method by which the disease could be brought under proper control, is the periodic use of a sensitive and specific diagnostic screening analysis to animal populations considered to be at risk of infection.
  • diagnosis of the disease in the subclinical phase is difficult due to the lack of reliable, early indicators of the disease. Identification of the etiologic agent, which is a slow grower, is a lengthy process often lasting up to three months . Histological examination of biopsy material is difficult and expensive and not suited for routine screening purposes.
  • Mycobacterium avium ssp . avium, Mycobacterium bovis , and Mycobacterium tuberculosis is essential. Taking into account the close relation between the Mycobacteria species, it is a priori a difficult task to find antigens containing epitopes specific for the desired organism, and to derive reagents from the antigens constituting reliable diagnostic tools for the analysis of Mycobacterium avium ssp . paratuberculosis .
  • Mycobacterium avium ssp . paratuberculosis is also suspected to play an etiological role in at least some fraction of the cases with Crohn's disease in humans.
  • Crohn's disease was originally described as a chronic ileitis producing hyperplastic granulomata of the intestine and lymph nodes.
  • the syndrome as it is described today entails inflammatory alterations of different organs of the intestinal tract.
  • the motive apparatus joints, muscles and bones
  • isolates of Mycobacterium avium ssp . paratuberculosis from patients with Crohn's disease have been reported.
  • the antigens 101 and 102 have been demonstrated to react with the majority of sera from cattle suffering from Paratuberculosis. Thus, their applicability in the diagnosis of Paratuberculosis is evident.
  • the full amino acid and nucleotide sequence of 101 has been determined ( Figure 1) and the N-terminal sequence of 102 has been determined (Example 1) .
  • 102 has a molecular weight of approximately 19,000 Da and appears not to be attached to other subunits by disulfide linkages.
  • 101 appears to be composed on two disulfide-linked subunits of approximately 23-24,000 Da which have some heterogeneity.
  • the antigens 101 and 102 can be observed in a sonicate of Mycobacterium avium ssp . paratuberculosis that is run through a column with absorbed polyclonal and polyvalent antiserum directed against Mycobacterium avium ssp . avium . In this procedure, all antibodies cross-reacting with antigens in Mycobacterium avium ssp . avium are removed. The dominant proteins that pass such a column are 101 and 102.
  • Homologous proteins are found in other mycobacteria, but no cross-reaction occurs as shown in Figure 2.
  • 101 and 102 show homology to proteins in other Mycobacteria such as Mycobacterium tuberculosis which are identified as alkyl hydroxyperoxide-reductase C and alkyl hydrogen peroxide reductase D, respectively.
  • the genes coding for the proteins that are homologous to 101 and 102, are located next to each other in the same operon in Mycobacterium tuberculosis .
  • the present invention provides a polypeptide comprising
  • sequence (i) (according to the test described hereinafter) which provides a functionally equivalent protein, or (iii) the amino acid sequence SVENLKEALPEYAKDLKLN (102) , or
  • sequence (iii) (according to the test described hereinafter) which provides a functionally equivalent protein fragment, or (v) a functionally equivalent fragment of any of sequences (i) to (iv) .
  • polypeptides of the invention are obtainable or obtained from the sub-species Mycobacterium avium ssp . paratuberculosis .
  • the antigens have a molecular weight in the order of 19,000 or 23-24,000 Da under reducing conditions.
  • the polypeptides are isolated or purified from the source material, e.g. using methods described herein.
  • purified refers to a preparation in which the protein is enriched relative to the majority of proteins derived from the same source, e.g. with a specific activity (e.g. measured as ⁇ g of the protein of interest ⁇ / ⁇ g total protein ⁇ or ⁇ units of enzymatic activity ⁇ / ⁇ g total protein ⁇ ) increased by more than 10, e.g. more than 100 relative to the starting material, especially preferably wherein the protein of interest is substantially free of contaminating source (e.g. M. a. paratuberculosis) proteins .
  • the present invention further provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide as defined above or a complementary sequence thereof, preferably comprising
  • nucleotide sequence encoding the amino acid sequence SVENLKEALPEYAKDLKLNL, or (iv) a nucleotide sequence which has more than
  • sequence (iii) (according to the test described hereinafter) or a sequence which hybridizes to sequence (iii) under conditions of 2 x SSC, room temperature, preferably 65°C (wherein
  • SCC 0.15M NaCl, 0.015M sodium citrate, pH 7.2) which encodes a functionally equivalent protein fragment to the amino acid sequence SVENLKEALPEYAKDLKLNL, or (v) a fragment of any of sequences (i) or (iv) encoding a functionally equivalent protein fragment, or a complementary sequence thereof .
  • polypeptides or nucleic acid molecules of the invention are identical to those described in Figure 1, or the amino acid sequence SVENLKEALPEYAKDLKLNL or a nucleic acid molecule encoding the same, optionally with only minor modifications, e.g. variations in less than 10 bases or 3 amino acid residues, e.g. less than 5 (such as 1 or 2) base changes and/or 1 or 2 amino acid residue alterations.
  • polypeptide of the invention comprises
  • a nucleic acid molecule of the invention comprises a nucleotide sequence encoding a polypeptide as defined hereinbefore or a complementary sequence thereof, preferably comprising
  • Polypeptides as referred to herein includes both full-length proteins and shorter length peptide sequences, e.g. protein fragments as described herein. Such polypeptides may be prepared by any convenient means, e.g. by isolation from a source prokaryote or by recombinant means by expression of the appropriate nucleic acid molecule in a host cell operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule. Preferably polypeptides of the invention are in recombinant form.
  • polypeptide refers to any molecule having its desired polypeptide chain produced by inserting a nucleotide sequence in the genome of a suitable cellular host or a plasmid which is capable of expression in that host, which through transcription and translation and optional post-translational modifying processes is able to produce said polypeptide in a controlled environment.
  • polypeptides or fragments thereof may be produced by a different process, for instance classical chemical synthesis according to methods used for peptide synthesis, purification processes using the natural biological source (s) for the said polypeptides as starting material, or chemical or proteolytic fragmentation methods to produce fragments from any of the said polypeptides.
  • polypeptides are obtained which have an appropriate biological purity.
  • biological purity means the grade of purity such that the said polypeptides or nucleotide sequences or said fragments from any of these, may be used in an assay and have the appropriate properties for application to routine diagnoses of diseased individuals, or in individuals suspected of carrying the disease.
  • Nucleic acid molecules may be single or double stranded DNA, cDNA or RNA, preferably DNA, and include homologous and hybridising sequences as defined above and degenerate and complementary sequences thereof . Derivatives of nucleotide sequences capable of encoding functionally- equivalent polypeptides may be obtained by using conventional methods well known in the art. As referred to herein the term “complementary” refers to DNA sequences with bases which match the DNA sequence of interest insofar as they can hybridize with - li the stated sequence.
  • sequence identity refers to the value obtained when assessed using the BESTFIT program of Computer Genetics Group Inc., University Research Park, Madison, Wisconsin, USA (Deuereaux et al . , 1984, Nucl . Acid. Res., 12, p387-395) .
  • sequence homology refers to sequences which have the stated value when assessed using the SWISS-PROT protein sequence databank using FASTA pep.cmp with a variable pamfactor, and gap creation penalty set at 12.0 and gap extension penalty set at 4.0. Sequence identity at a particular residue is intended to include identical residues which have simply been derivatized.
  • Proteins or protein fragments refers to proteins or fragments related to, or derived from the amino acid sequence of Figure 1 or the N-terminal sequence SVENLKEALPEYAKDLKLNL, where the amino acid sequence has been modified by single or multiple amino acid (e.g. at 1 to 50, e.g. 10 to 30, preferably 1 to 5 residues) substitution and/or addition and/or deletion but which nonetheless retains functional activity, i.e. are recognised by the same antibodies that recognize the corresponding intact protein antigens, but not recognised by antibodies raised against other Mycobacteria.
  • functionally equivalent proteins or fragments are recognized specifically and selectively using serum raised against the protein comprising the full-length protein sequence of 101 (ie. as given in Figure 1) or 102.
  • Selective and specific reaction may be assessed by determining whether a positive signal is identified in Western blotting using antiserum described above and in the examples. Testing using 5-10 ⁇ g, e.g. 6 ⁇ g of a sample preparation e.g. a bacterial homogenate or 22,000xg supernatant is considered appropriate. Homologues in for example Mycobacterium avium ssp . avium do not give a positive reaction according to this test.
  • the absence of a positive signal may reflect either the absence of the antigen (or its homologue) from a preparation or the presence of the antigen (or its homologue) but at undetectable levels in the detection method. Even if the latter is the case, marked differences in expression still allow 101 and/or 102 of the invention to be used as specific and selective markers of the presence of Mycobacterium a. paratuberculosis .
  • functional equivalents may be determined on the basis of alkyl hydroperoxidase reductase C or D activity. It will however be appreciated that some molecules having enzymatic activity might not be antigenic in methods of the invention and vice versa .
  • addition variants are included amino and/or carboxyl terminal fusion proteins or polypeptides, comprising an additional protein or polypeptide fused to the polypeptide sequence.
  • Naturally occurring equivalents such as biological variations, e.g. allelic, geographical or allotypic variants and derivatives prepared using known techniques.
  • functionally-equivalent proteins or fragments may be prepared either by chemical peptide synthesis or in recombinant form using the known techniques of site- directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.
  • the invention is particularly directed to homologues and related molecules from Mycobacterium avium ssp . paratuberculosis, e.g. homologues from M. a . paratuberculosis ATCC 19698, 173p, 186p and 2E.
  • Such sequences may themselves be modified, particularly derivatized providing they still retain functionality.
  • Derivatives of the proteins may be prepared by post-synthesis/isolation modification or by modification during synthesis, e.g. using modified residues or expression of modified nucleic acid molecules, where appropriate .
  • Functionally-equivalent fragments according to the invention may be made by truncation, e.g. by removal of a peptide from the N and/or C-terminal ends or by selection of an appropriate active domain region, e.g. an epitopic region which retains its functionality.
  • Such fragments may be derived from the amino acid sequence of Figure 1 or from the sequence SVENLKEALPEYAKDLKLNL or may be derived from a functionally equivalent protein or peptide to those sequences.
  • fragments are between 6 and 400 residues in length, e.g. 6 to 100 residues, preferably 6 to 30 or 10 to 25 residues.
  • Particularly preferred fragments are those derived from surface regions of the naturally occurring protein.
  • Nucleic acid molecules of the invention may consist only of sequences derived from Figure 1 or encoding SVENLKEALPEYAKDLKLNL (or related functionally equivalent sequences or sequences encoding polypeptides of the invention), or may comprise additional sequences, e.g. sequences which control transcription and/or expression or sequences to prepare fusion proteins .
  • the nucleic acid molecule may be in the form of a vector containing a nucleic acid molecule described herein.
  • nucleic acid molecules of the invention include cloning and expression vectors containing or consisting of the nucleic acid molecules of the invention and methods for preparing recombinant nucleic acid molecules according to the invention, comprising inserting a nucleic acid molecule as described herein into vector nucleic acid, e.g. vector DNA.
  • Appropriate expression vectors include appropriate control sequences such as for example translational (e.g. start and stop codons, ribosomal binding sites) and transcriptional control elements (e.g. promoter-operator regions, termination stop sequences) linked in matching reading frame with the nucleic acid molecules of the invention.
  • Vectors according to the invention may include plasmids and viruses (including both bacteriophage and eukaryotic viruses) according to techniques well known and documented in the art, and may be expressed in a variety of different expression systems, also well known and documented in the art .
  • Suitable viral vectors include baculovirus and also adenovirus and vaccinia viruses . Many other viral vectors are described in the art .
  • a variety of techniques are known and may be used to introduce such vectors into prokaryotic or eukaryotic cells for expression, or into germ line or somatic cells to form transgenic animals. Suitable transformation or transfection techniques are well described in the literature .
  • the present invention thus also includes transformed or transfected prokaryotic or eukaryotic host cells or transgenic organisms containing a nucleic acid molecule according to the invention as defined above .
  • the present invention also extends to antibodies (monoclonal or polyclonal) and their antigen-binding fragments (eg. F(ab) 2 , Fab and Fv fragments ie . fragments of the "variable" region of the antibody, which comprises the antigen binding site) directed to polypeptides as defined hereinbefore, ie. which bind to epitopes present on the polypeptides and thus bind selectively and specifically to such polypeptides.
  • antigen-binding fragments eg. F(ab) 2 , Fab and Fv fragments ie . fragments of the "variable" region of the antibody, which comprises the antigen binding site
  • the present invention further extends to methods for isolating polypeptides or nucleic acid molecules of the invention.
  • the present invention provides a method of isolating an antigen specific to a particular bacterial subspecies comprising at least the steps of: i) obtaining a sample of said bacterial subspecies, ii) contacting said sample with antiserum directed to a different bacterial subspecies, in which the antibodies of said antiserum are preferably attached to a solid support , iii) removing from said sample the moieties (ie. antigens) which bind to said antiserum, and optionally iv) isolating said antigens remaining in said sample.
  • the above method results in antigens which are not cross-reactive (under the conditions used) to the second subspecies, thus allowing the identification of antigens unique to the first subspecies or significantly differentially expressed.
  • the invention also extends to products identified by the above method and their uses, particularly antigens specific to Mycobacterium avium ssp . avium.
  • polypeptides and nucleic acid molecules according to the invention have particular uses in preventing and/or diagnosing Paratuberculosis and Crohn's disease.
  • Diagnosis of Paratuberculosis may be achieved in a number of different ways. For example evidence of the presence in a sample of one or more of the polypeptides described herein, nucleic acid molecules encoding such polypeptides or antibodies thereto may be sought.
  • the present invention provides a method of diagnosing infection by Mycobacterium avium ssp . paratuberculosis in an individual characterised by analysing the presence or quantity of at least one of the antigens 101 or 102, or the presence or quantity of any antibodies to at least one of the antigens 101 or 102, or the presence or quantity of one or more nucleic acid sequences corresponding to at least one of the antigens 101 or 102.
  • the present invention provides a method of diagnosing and/or monitoring infection of a human or non-human animal by Mycobacterium avium ssp . paratuberculosis , wherein said method comprises at least the step of assessing the presence or concentration of one or more polypeptides, antibodies or antigen-binding fragments thereof or nucleic acid molecules as defined hereinbefore, or the presence or concentration of infection markers (e.g. IFN- ⁇ ) which result from in vi tro presentation of one or more polypeptides described hereinbefore, in a sample from said human or non-human animal.
  • said method and methods described hereinafter are directed to the diagnosis and/or monitoring and/or prevention of Paratuberculosis or Crohn's disease.
  • diagnosis refers to determination of the presence of the bacteria in a test animal relative to a normal animal. This includes diagnosis of an infection that will ultimately lead to the development of symptoms resulting from infection, ie. a disease state, but also includes diagnosis of the type of infection e.g. the serotype of the infecting bacteria.
  • Monitoring refers to establishing the extent of infection, particularly when an individual is known to be infected, for example to monitor the effects of treatment or the development of disease.
  • Assessing refers to both quantitation in the sense of obtaining an absolute value for the amount of bacteria in a sample, and also obtaining a semi- quantitative assessment or other indication, e.g.
  • an index or ratio, of the amount of bacteria in the sample is prepared using control samples spiked with different amounts of said bacterial protein, nucleic acid molecule or antibody. The test sample result may then be compared to the standard curve to determine the amount of bacteria which are present.
  • sample refers to any material obtained from the human or non-human animal under investigation which is suitable for measurement of infectious bacteria per se or the biological responses to their presence, including, but not limited to, blood, urine, saliva, spinal fluid, sperm, lymph, expectorated matter (pulmonary patients) , placenta, faeces or tissue samples obtained by biopsy or surgical interventions .
  • Non-human animals which may be diagnosed include, but are not limited to mammals, particularly primates, domestic animals and livestock, particularly preferably cattle.
  • the expression "cattle”, as used herein, shall mean ruminants, such as bovines, sheep, goat, and cervidae, but also some non-ruminant animals may be infected by Paratuberculosis (horses, monkeys) and may be the subjects of application of the diagnostic tools described herein.
  • preferred animals for diagnosis include mice, rats, guinea pigs, cats, dogs, pigs, cows, goats, sheep, horses.
  • the disease state of cows and/or humans is diagnosed and/or monitored and/or prevented.
  • Imaging markers refers to markers of lymphocyte activation through antigenic stimulation and includes for example the increased or decreased secretion of cytokines or lymphokines, increased metabolism and other immune responses.
  • diagnostic methods refers to any suitable method known in the art, and includes the use of immunoassays to identify the presence of polypeptide antigens or antibodies which bind specifically to said polypeptide antigens, in materials from the animal (s) investigated (ie. samples), or methods relying on detection of the presence of genetic sequences coding for at least part of said polypeptide antigens.
  • the presence or concentration of polypeptides as described herein may be examined. In simplified form, this may involve the use of a binding partner to said polypeptide (e.g. an antibody) , which may be immobilized, to separate said polypeptide from the sample and the amount of polypeptide may then be determined.
  • This method may be performed in a competitive or non-competitive assay, ie . with or without molecules which may interfere with binding between the binding partners .
  • a sandwich type assay e.g. immunoassays such as an ELISA
  • an antibody specific to the polypeptide and carrying a label may be bound to the binding pair (e.g. the first antibody :polypeptide pair) and the amount of label detected.
  • the competing molecules may be labelled or not labelled to provide an indication of the extent of binding in the presence of competing molecules. Similar considerations apply with respect to assays in which the presence or concentration of antibodies are determined.
  • the present invention provides a method of diagnosing and/or monitoring infection of a human or non-human animal by Mycobacterium avium ssp . paratuberculosis , wherein said method comprises at least the steps of : a) contacting a sample of said human or non-human animal with a binding partner capable of binding to a polypeptide or antibody or antigen-binding fragment thereof as defined hereinbefore present in said sample to form a first binding pair, wherein said binding partner is optionally immobilized, b) optionally conducting step a) in the presence of a competing molecule which competes with the binding between the binding pair, optionally forming a second binding pair, c) assessing the presence or concentration of the first and/or second binding pair to determine the presence or concentration of said polypeptide or antibody.
  • binding refers to any suitable technique which allows the binding partner to have access, and thus the possibility of binding, to polypeptides or antibodies in the sample, e.g. by mixing aqueous solutions.
  • a "binding partner" to said polypeptide or antibody or antigen-binding fragment thereof refers to a moiety which recognizes and binds specifically (ie. in preference to binding to other molecules) to the polypeptide or antibody or fragment thereof . Such binding pairs when bound together form a complex.
  • a binding partner is an antibody to the polypeptide or an antigen of the antibody.
  • binding partners may however also be used, such as antibodies which recognise and bind to the antibody to be detected or other non-antibody proteins which recognize the polypeptides .
  • the competing molecule may compete with binding between the polypeptide or antibody to be detected and its binding partner by binding to one or other of that binding pair or simply by interfering with the formation of an appropriate interaction. Conveniently the competing molecule corresponds to a polypeptide or antibody as described herein.
  • a detection step is performed.
  • a technique which allows identification/visualization (use of a signalling means) of the bacterial protein or antibody is required.
  • said assessment is made by detecting the presence of a signalling means.
  • said signalling means is a label .
  • the label is introduced into or onto the first or second binding pair and its presence is assessed.
  • the amount of unbound competing molecule may be assessed.
  • the assay requires the use of a moiety (or signalling means) being detectable by chemical or physical means.
  • moiety being detectable by chemical or physical means shall mean any chemical compound which directly or indirectly may produce a signal that can be read either visually or by a suitable instrument .
  • moieties may be, but are not limited to, radioactive isotopes, enzymes able to convert an added substrate to a detectable product, dyes, fluorescent compounds, particles containing a colour or fluorescent compound such as coloured latex, metal colloids, colloids of metal salts, liposomes containing coloured substances or fluorescent compounds. It is to be understood that any moiety detectable by its ability to absorb or emit electromagnetic radiation is included, including compounds absorbing or emitting radiation in the UV, visible or IR-range.
  • chemical or physical means shall mean any detection method able to determine, on a qualitative, semi-quantitative or quantitative basis, the presence or amount of said moiety, such method including, but not limited to, inspection by the naked eye, photometers, fluorometers , reflectometers , particle counters, turbidometers, and counters able to detect radioactive materials.
  • the signalling means is carried on the competing molecule or on a secondary binding agent which may be bound to the first or second binding pairs.
  • a labelled antibody may be used to detect the presence of the first binding pair.
  • said secondary binding agent is added to the binding partner (which may be in solution or immobilized) simultaneously with or after addition of said specimen sample, said secondary binding agent being a conjugate between a compound capable of binding to e.g. said antibodies from the species from which said specimen is derived, and a chemical moiety being detectable by physical or chemical means as described hereinbefore .
  • the binding partner of the antibody or polypeptide to be detected is immobilized on a solid support although alternatively the binding step may be performed in solution.
  • solid support shall mean any solid material able to bind (poly) peptide (s) , antibodies or nucleic acids by hydrophobic, ionic or covalent bridges.
  • solid supports suitable as immobilizing moieties according to the invention are well known in the art and widely described in the literature and generally speaking, the solid support may be any of the well-known supports or matrices which are currently widely used or proposed for immobilization, separation etc. in chemical or biochemical procedures.
  • Such materials include, but are not limited to, any synthetic organic polymer such as polystyrene, polyvinylchloride, polyethylene; or nitrocellulose and cellulose acetate; or tosyl activated surfaces; or any surface carrying an array of amino- or carboxyl groups suited for covalent coupling of peptides, or nucleic acids.
  • the immobilizing moieties may take the form of particles, sheets, gels, filters, membranes, microfibre strips, tubes or plates, fibres or capillaries, made for example of a polymeric material e.g. agarose, cellulose, alginate, teflon, latex or polystyrene. Particulate materials, e.g. beads, are generally preferred.
  • the immobilizing support may carry further moieties for attachment of the binding partner which will attach to the molecule to be detected in the sample .
  • This may be achieved through affinity binding, e.g. in which the binding partner is a fusion protein and can thus be attached to the solid support through interactions such as avidin: streptavidin or biotin, DNA:DNA binding protein, antibody : antigen etc.
  • Solid supports presenting appropriate moieties for attachment of the binding partner may be produced by routine methods known in the art .
  • polypeptides as described herein, e.g. derivable from Mycobacterium avium ssp . paratuberculosis of desired biological purity to a solid support, contacting said solid phase containing said polypeptide (s) with a mixture of i) sample of animal material, and ii) a known amount of (a) competing molecule (s) which is able to block the binding between said immobilised polypeptide (s) and antibodies in said sample specific to said polypeptides, said competing molecule (s) carries a moiety being detectable by chemical or physical means; under conditions where any antibodies in said animal material will compete with said competing molecule for the binding to said polypeptides.
  • polypeptides as described herein e.g. obtainable from Mycobacterium avium ssp . paratuberculosis of desired biological purity with ii) a sample of animal material under conditions that lead any antibodies in said animal material to bind to said polypeptides, and iii) a known amount of one or more competing molecules carrying a moiety being detectable by chemical or physical means, said competing molecules being able to bind to said polypeptide (s) in competition with said antibodies; said competing molecules, may be added to the said polypeptide (s) together with, after, or before said animal material; and finally measuring the amount of detectable chemical moiety in isolated or precipitated complexes between said polypeptide (s) and said competing molecules.
  • the presence of antigens or antibodies indicating the presence of Mycobacterium avium ssp . paratuberculosis can be performed by the methods known in the art such as blotting methods.
  • the molecular constituents of a sample applied to a suitable solid medium like a gel, a sheet or similar are separated from each other in an electrical field, followed by transfer of the molecules to a solid surface in which they are immobilised, followed by detection of the presence of certain molecular species by application of a binder (e.g. a secondary binding agent) specific to such molecular species, said binder being conjugated with a chemical moiety capable of being detectable by physical or chemical means as described hereinbefore .
  • a binder e.g. a secondary binding agent
  • the present invention provides diagnostic/monitoring methods in which said binding partner is a polypeptide (preferably 101 or 102 or fragments thereof as defined herein) or an antibody as defined hereinbefore, and preferably said molecules are immobilized.
  • a competing molecule is used which prevents binding of the first binding pair and which itself binds to one of said binding pair.
  • said signalling means is present on the secondary binding agent or on the competing molecule.
  • the method of diagnosis/monitoring may alternatively be performed by detecting the presence of nucleic acid molecules as defined herein which express the antigens 101 and 102. In this second type of assay, the presence of such nucleic acid molecules is identified for example by the use of appropriate probes to such molecules, optionally employing amplification to enhance signal production.
  • the present invention provides a method of diagnosing and/or monitoring infection of a human or non-human animal by Mycobacterium avium ssp. paratuberculosis, wherein said method comprises at least the steps of : a) preparing a nucleic acid molecule preparation from a sample of said human or non-human animal, b) contacting said nucleic acid molecule preparation with a nucleic acid probe comprising a sequence which is complementary to a nucleic acid molecule as described hereinbefore under conditions appropriate to allow hybridization to a complementary sequence (target molecules) in said sample nucleic acid preparation, and c) detecting the presence or amount of hybridisation between said nucleic acid probe and target molecules in said sample .
  • a nucleic acid molecule preparation refers to any preparation in which the nucleic acid molecules are accessible to exogenously added nucleic acid probes.
  • the preparation may comprise e.g. a lysed homogenate or the nucleic acid material may be enriched in, or isolated from, said sample.
  • a “probe” as used herein refers to an oligonucleotide capable of binding to a target nucleic acid molecule.
  • said probe comprises nucleotides in a sequence selected from the group consisting of any of the sequences coding for any of the antigens 101 and 102 as described herein and said nucleic acid probe contains at least 15 nucleotides.
  • Such probes form further aspects of the invention and indeed are considered nucleic acid molecules of the invention due to their ability to discriminate 101 or 102 from other proteins .
  • sufficient time is allowed for hybridisation to occur between said nucleic acid probe and any complementary sequences in said specimen. Appropriate hybridizing conditions are as described hereinbefore .
  • the probes may be labelled with a signalling means which may be detected.
  • methods of detection may be based on amplification and in this case the probe or oligonucleotide may itself act as a primer for amplification or the probe may be used to identify amplified products.
  • amplification techniques may be used, e.g. PCR.
  • two primers may be used which hybridize to opposing strands on the target DNA.
  • PCR may also be carried out using a single specific primer by ligating a standard sequence or tail to the target DNA (e.g. after restriction cleavage) to provide a hybridisation site for a standard PCR primer.
  • Other techniques such as Self-sustained Sequence Replication (3SR) using probe/primers carrying polymerase binding sites to permit the action of reverse transcriptase may also be used.
  • 3SR Self-sustained Sequence Replication
  • an immobilized probe may be used to capture one strand of target DNA and thereafter this sequence may hybridize with an RNA probe carrying a tertiary structure for an RNA-directed RNA polymerase.
  • the Ligase Amplification Reaction uses two oligonucleotide probes that hybridize at adjacent positions on the target nucleic acid molecule which may then be ligated and form a template for further hybridisation and ligation.
  • an assay may be performed by contacting a sample material suspected to contain material from Mycobacterium avium ssp . paratuberculosis with a primer nucleotide sequence known to be present in said Mycobacterium avium ssp.
  • paratuberculosis increasing the temperature in the reaction mixture to more than 90°C in the presence of a thermostable DNA-polymerase and the four essential nucleotide bases in order to allow the DNA-polymerase to copy the primer-specific nucleotide sequence (s), reducing the temperature to below 50°C to allow corresponding strands of DNA to hybridise, and repeating the sequence of heating and addition of primers in a number of cycles sufficient to generate a detectable amount of primer-related DNA. Detection may be performed by subjecting the resulting material to electrophoretic separation, and identification of any visible DNA product (s) .
  • Detection may also be performed by contacting the amplified sample material with an immobilised nucleotide sequence corresponding to a part of one or more
  • DNA-strands suspected to be present in the sample heating to at least 90°C and decreasing the temperature to below 50°C to allow DNA to separate the strands and to hybridise with said nucleotide sequence.
  • Any DNA molecule specifically attached to a further DNA molecule may be detected by applying a second nucleotide sequence corresponding to said DNA (e.g. another part of the sequence coding for 101 or 102) , in which said second nucleotide sequence is conjugated to a moiety able to create a signal detectable by any chemical or physical means as described hereinbefore.
  • the invention further extends to methods as described above, with the additional step that said target molecule is amplified before or after hybridization of said probe (which may itself function as a primer) using an amplification system, such as PCR.
  • said amplification step is conducted prior to hybridisation of said probe by a method comprising; selecting oligonucleotide primers designed on the basis of the sequence of nucleotides coding for any of the antigens 101 and 102 as described herein, and amplifying said nucleic acid sequence which codes for any of the antigens 101 and 102 using the polymerase chain reaction with said oligonucleotide primers.
  • tests measuring cellular immune responses to in vi tro presented antigen can be applied.
  • the oldest and most extensively used test measuring cellular immune responses is the skin test which is equivalent to the tuberculin test in tuberculosis. Johnin made from a filtrate of Mycobacterium paratuberculosis is injected intracutaneously . The skin thickness is measured before, and 72 hrs after the injection. An increase in thickness of the skin is due to a delayed hypersensitivity reaction and indicates infection. This skin test is not very specific, and some animals with advanced Paratuberculosis will be test negative.
  • Lymphocyte-proliferation test Another method founded on cell-mediated immunity, is the Lymphocyte-proliferation test (LPT) .
  • Plasma is drawn from the animal or human being to be tested using heparin for anticoagulation.
  • Peripheral blood monocytes (PBMC) are isolated by density centrifugation on Lymphoprep gradient (Nycomed; Oslo, Norway) .
  • the test can be performed on whole blood.
  • Freshly isolated cells are cultivated with and without antigen in 96 well flat bottomed microculture plates. 2 x 10 5 cells in 200 ⁇ l RPMI 1640 with 10% FCS (fetal calf serum) supplemented with L-Glut, Penicillin, and streptomycin are applied to each well.
  • Antigen is added in three parallels to the desired final concentration in each well.
  • IFN ⁇ interferon gamma
  • the present invention provides a method of diagnosing and/or monitoring infection of a human or non-human animal by Mycobacterium avium ssp . paratuberculosis, wherein said method comprises at least the step of assessing the presence or concentration of infection markers after in vi tro presentation of one or more polypeptides described hereinbefore to a sample from said human or non-human animal.
  • the biological response to contact with said polypeptides is recorded.
  • the response which is recorded may be generated after contacting (presenting) antigens subcutaneously to an individual and said marker which is measured is the thickness of the skin at the application site after a period of time.
  • the infection marker which is recorded is the cell ' s incorporation of increased amounts of nucleotides in response to antigen presentation, or the ability of said cells to secrete material, e.g. interferon-gamma.
  • the presence or concentration of infection markers is determined by recording the biological response to contact with said polypeptide and said biological response is selected from the group consisting of: i) hypersensitivity after subcutaneous presentation of said polypeptide to an individual wherein the thickness of the skin is measured at the application site, or ii) increased metabolism wherein a cell's incorporation of nucleotides is measured, or iii) secretion of particular molecules, preferably interferon-gamma is measured.
  • the method described above may be used for diagnosis of infection of an animal, the method may also be used to assess the levels/presence of bacteria in samples not derived from an animal, e.g. quality control testing of water or food samples or testing for contamination of biological samples .
  • the present invention provides a method of assessing the presence or concentration of Mycobacterium avium ssp. paratuberculosis in a sample, wherein said method comprises at least the step of assessing the presence or concentration of one or more polypeptides, antibodies or antigen- fragments thereof, nucleic acid molecules or infection markers as described hereinbefore, in said sample according to a method as described hereinbefore.
  • polypeptides or antibodies described herein also have prophylactic uses.
  • the present invention provides a pharmaceutical composition comprising a polypeptide or antibody or antigen-binding fragment thereof as described hereinbefore, and a pharmaceutically acceptable excipient, diluent or carrier.
  • “Pharmaceutically acceptable” refers to ingredients that are compatible with other ingredients of the compositions as well as physiologically acceptable to the recipient.
  • compositions according to the invention may be formulated in conventional manner using readily available ingredients.
  • the active ingredient ie. the protein/peptide or antibody or fragment thereof
  • the active ingredient may be incorporated, optionally together with other active substances, with one or more conventional carriers, diluents and/or excipients, to produce conventional galenic preparations such as tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile packaged powders, and the like.
  • compositions may additionally comprise molecules which assist or augment the action of the antibodies or polypeptides described hereinbefore.
  • Preventative preparations may be formulated to include one or more suitable adjuvants, e.g. aluminium hydroxide, saponin, quil A, or more purified forms thereof, muramyl dipeptide, mineral or vegetable oils, Novasome or non-ionic block co-polymers or DEAE dextran, in the presence of one or more pharmaceutically acceptable carriers or diluents.
  • suitable adjuvants e.g. aluminium hydroxide, saponin, quil A, or more purified forms thereof, muramyl dipeptide, mineral or vegetable oils, Novasome or non-ionic block co-polymers or DEAE dextran, in the presence of one or more pharmaceutically acceptable carriers or diluents.
  • suitable carriers include liquid media such as saline solution.
  • Suitable carriers, excipients, and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, aglinates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol , water/glycol , water/polyethylene glycol , propylene glycol , methyl cellulose, methylhydroxybenzoates , propyl hydroxybenzoates, talc, magnesium stearate, mineral oil or fatty substances such as hard fat or suitable mixtures thereof.
  • compositions may additionally include lubricating agents, wetting agents, emulsifying agents, suspending agents, preserving agents, sweetening agents, flavouring agents, and the like.
  • the compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures well known in the art .
  • compositions may be in an appropriate dosage form, for example as an emulsion or in liposomes, niosomes, microspheres, nanoparticles or the like.
  • compositions may also contain targeting moieties attached to the active ingredient, e.g. a ligand which binds specifically and selectively to an endogenous receptor to allow targeting to a particular cell type or location.
  • targeting moieties attached to the active ingredient, e.g. a ligand which binds specifically and selectively to an endogenous receptor to allow targeting to a particular cell type or location.
  • compositions have utility in the prophylaxis of infection by M. avium ssp . paratuberculosis .
  • the present invention provides pharmaceutical compositions, such as vaccine compositions as described herein for use as a medicament, preferably for use in preventing infection by M. avium ssp . paratuberculosis in a human or non- human animal .
  • the present invention provides a method of preventing infection by M. avium ssp . paratuberculosis in a human or non-human animal, wherein said animal is administered a pharmaceutical composition as described hereinbefore.
  • the invention further provides a method of stimulating an immune response against M. avium ssp . paratuberculosis in a human or non-human animal, comprising administering to said animal a vaccine composition as defined hereinbefore.
  • the present invention provides the use of a polypeptide or antibody as defined hereinbefore in the preparation of a medicament for preventing infection by M. avium ssp . paratuberculosis in a human or non-human animal.
  • Prevention refers to delaying or preventing the onset of the condition (e.g. Paratuberculosis or Crohn's disease) or reducing the severity resulting from infection by M. avium ssp. paratuberculosis , as assessed by the appearance or extent of one or more symptoms of said condition.
  • compositions of the invention may take place by any of the conventional routes, e.g. orally, rectally or parenterally, such as by intramuscular, subcutaneous, intraperitoneal or intravenous injection, optionally at intervals, e.g. 3 or more applications at 3-5 day intervals.
  • Prophylaxis by topical application of a composition, e.g. an ointment, to the skin is also possible.
  • composition of the invention may comprised from about 0.01% to about 99% by weight of the formulation, preferably from about 0.1 to about 50%, for example 10%.
  • the compositions are preferably formulated in a unit dosage form, e.g. with each dosage containing from about O.Olmg to about lg of the active ingredient, e.g. 0.05mg to 0.5g, for a human, e.g. 1-lOOmg.
  • an effective dose may lie in the range of from about l ⁇ g/kg to about lOmg/kg, e.g. from about lmg to 0.2g of polypeptide per day, depending on the animal to be treated and the dosage form, taken as a single dose.
  • an appropriate daily dose for an adult may be from 0.5mg to 0.5g per day, e.g. 1 to lOOmg of the polypeptide per day.
  • kits for performing the assay methods of the invention furthermore extends to kits for performing the assay methods of the invention.
  • the present invention provides a kit for diagnosing and/or monitoring infection of a human or non-human animal by Mycobacterium avium ssp . paratuberculosis or assessing the presence or concentration of Mycobacterium avium ssp . paratuberculosis in a sample, comprising at least the following: i) a polypeptide, antibody or antigen-binding fragment thereof, nucleic acid molecule, or primer or probe as defined hereinbefore, optionally attached to a solid support and optionally carrying a signalling means .
  • the kit also contains a competing molecule or secondary binding agent which may optionally carry a signalling means.
  • the kit also comprises compounds or solutions necessary for the development of an identifiable signal from the signalling means.
  • the kit may also include means for standardization of the assay or for comparative purposes .
  • Figure 1 shows the nucleotide and amino acid sequence of 101
  • Fi ⁇ ure 2 shows SDS-PAGE with Western Blotting of various Mycobacterium species.
  • A) Incubated with antiserum against 101 B) Incubated with antiserum against 102 1) Purified 101, 2) Purified 102, 3) M. a. paratuberculosis 2E, 4) M. a . paratuberculosis ATCC 19698, 5) M. a. paratuberculosis 173p, 6) M. a . paratuberculosis 186p, 7) M. a. avium D4 , 8) M. intracellulare, 9) M. scrofelaceum, 10) M. kansasii , 11) M. gordonae, 12) M. fortuitum, 13) M. phlei , 14) M. bovis AN5, 15) M. tuberculosis H37rv, 16) BCG;
  • Fi ⁇ ure 3 shows crossed immunoelectrophoresis of M. a. paratuberculosis homogenates that have been absorbed with polyclonal and polyvalent antiserum against Mycobacterium avium (see description in Example 3) .
  • the figure shows the precipitation lines for 101 and 102 A. M. a. paratuberculosis separated with polyclonal anti M. a. paratuberculosis antiserum incorporated in the top gel and NaCl (0.9%) in the middle gel, B. M. a . paratuberculosis separated with polyclonal anti M. a. paratuberculosis antiserum incorporated in the top gel and polyclonal anti M. a. avium antiserum incorporated in the middle gel ; and
  • Figure 4 shows SDS-PAGE with Coomassie Blue staining of purified 101 and 102 under reducing and non-reducing conditions .
  • the antigens were obtained as follows:
  • M. a. paratuberculosis (strain 2E) culture fluid was separated by ion exchange chromatography.
  • the culture fluid had a concentration of 4 mg/mL as determined by
  • Antigen 102 was eluted in fractions with 0.4 mol/L NaCl.
  • the fractions containing 102 were separated on a 6 mL Resource Q column (Pharmacia, Uppsala, Sweden) . Buffers were the same as above .
  • the samples were diluted 1 : 5 with Buffer A.
  • the gradient was from 0 - 0.4 mol/L NaCl for Ag 10 and 0.2 - 0.7 mol/L NaCl for 102 in 30 min. Flow was 3 mL/min. The resolution was high with several sharp peaks which were confirmed by staining patterns. Some bands were only detected in 1-2 fractions.
  • the fractions containing 102 were diluted 1:1 with 30 mmol/L Tris/HCl pH 7.5 with 2mol/L Ammonium sulphate giving a salt concentration of 1 mol/L Ammonium sulphate. pH was adjusted to 7.5. The sample was run through the column, and the flow-through fraction containing 102 was dialysed against Tris/HCl buffer pH 7.5 overnight. The bound protein was eluted with 30 mmol/L Tris/HCl pH 7.5 and mixed with the fractions from QSFF containing 102.
  • Buffer A 30 mmol/L Tris/HCl pH 7.5.
  • Buffer B 30 mmol/L Tris/HCl (pH 7.5) with lmol/L Ammonium sulphate.
  • the gradient was 0-50 % buffer B over 20 minutes.
  • the flow was 3mL/min and 3 mL fractions were collected. 102 was eluted in two fractions at a salt concentration of approximately 0.25 mol/L Ammonium sulphate.
  • the fractions containing the 19kD protein were separated on a Superdex 75 26/60 column for gel filtration. Two mL samples were applied, and the buffer was PBS (0.1 mol/L Phosphate buffer pH 7.5 with 0.15 mol/L NaCl) . The flow was 2mL/min and 10 mL fractions were collected.
  • PBS 0.1 mol/L Phosphate buffer pH 7.5 with 0.15 mol/L NaCl
  • M. a. paratuberculosis (strain 2E) culture fluid was separated by ion-exchange chromatography .
  • the culture fluid had a concentration of 4 mg/mL determined by
  • Antigen 101 was eluted in several fractions in the range 0.3 - 0.6 mol/L NaCl
  • Source phenyl The fractions containing antigen 101 were diluted 1:1 with 30 mmol/L Tris/HCl pH 7.5 with 2 mol/L Ammonium sulphate giving a salt concentration of 1 mol/L in the sample. PH was adjusted to 7.5 and the fractions were separated on a HIC column. Six mL Source phenyl medium (Pharmacia, Uppsala, Sweden) were packed in a XK16 column. Buffer A was 30 mmol/L Tris/HCl pH 7.5 with 1 mol/L Ammonium sulphate. Buffer B was 30 mmol/L Tris/HCl pH 7.5. The flow was 4 mL/min and 8 mL fractions were collected. The proteins were eluted in a gradient from 0-100% buffer B over 30 min + 15 min buffer B. 101 was eluted at the end of the gradient from 0.1- 0 mol/L Ammonium sulphate.
  • Buffer A 30 mmol/L Tris/HCl pH 7.5.
  • Buffer B 30 mmol/L Tris/HCl pH 7.5 with 1 mol/L Ammonium sulphate.
  • the gradient was 0-50 % buffer B over 20 minutes.
  • the flow was 3mL/min and 3 mL fractions were collected. 101 was eluted at a salt concentration of approximately 0.3 mol/L Ammonium sulphate.
  • Buffer A was 30 mmol/L Tris/HCl pH 7.5 with 1 mol/L Ammonium sulphate.
  • Buffer B was 30 mmol/L Tris/HCl pH 7.5. The flow was 2mL/min and fractions of 4mL were collected.
  • the N-terminal sequences of the above antigens were determined by automatic Edman degradation, and the full sequence of 101 was determined.
  • High molecular weight DNA from M. a . paratuberculosis ATCC19698 was purified according to standard procedures using sonication, SDS and proteinase K treatment followed by CTAB/NaCl treatment. DNA was extracted using phenol/chloroform isoamylalcohol and dissolved in TE buffer.
  • PCR was performed in a Perkin Elmer thermocycler using 35 cycles with an annealing temperature of 58°C.
  • the PCR product was sequenced using "BigDye Terminator Cycle Sequencing Ready Reaction kit” (Perkin Elmer) and analysed on an automated sequencer (ABI 377; Perkin Elmer) with standard sequencing gels. The sequence was determined using the SequencherTM software.
  • the characteristics of the antigens 101 and 102 which were obtained are as follows:
  • 102 has an apparent molecular weight of 19,000 as determined by SDS-PAGE and compared to a molecular mass standard. The molecular weight estimation is identical in samples treated with a reducing agent such as dithiothreitol . Thus, 102 contains no disulfide linked subunits, but is rather a single unit, or is part of a system of non-covalently linked protein subunits.
  • the antigenic protein 101 In the absence of a reducing agent under SDS-PAGE, the antigenic protein 101 demonstrates two bands with apparent molecular weights ranging from 43-45,000, in addition to a weak band at 24,000, when compared to a molecular weight mass standard. In the presence of a reducing agent, two separate bands representing antigens with apparent molecular weights of 23,000 and 24,000 are observed ( Figure 4) . Thus, 101 appears to be composed of two disulfide-linked subunits of a certain heterogeneity. These two types of subunits appears to be randomly associated in the total pool of 101. All the different bands seen in SDS-PAGE reacted with a specific antiserum raised against 101.
  • N-terminal amino acid sequence of 102 was determined as in as follows:
  • N-terminal amino acid sequence of 101 has been determined and is as follows:
  • EXAMPLE 2 SPECIFICITY OF 101 AND IQ2 FOR M. A .
  • EXAMPLE 3 ABSORPTION OF M. A . PARATUBERCULOSIS ANTIGENS WITH M. AVIUM ANTIBODIES
  • Cross-reacting antigens in Mycobacterium avium ssp paratuberculosis were removed by absorption using a polyclonal and polyvalent anti -M. a. avium antiserum with a column chromatographic technique.
  • Anti-M. a. avium antibodies were adsorbed to a HiTrap protein G column.
  • M. a . paratuberculosis sonicate was run through the column in order to remove cross-reactive antigens.
  • M. a. avium antiserum 800 ⁇ l, 45mg Ig/ml
  • Bound IgG/antigen complexes were eluted with glycine/HCl buffer (pH 2.7) . The primary effluent fraction and eluate were collected. Between each step the column was washed with 0.20 mM Na-phosphate buffer pH 7.0. The protein content in the output was monitored by measuring OD at 280 nm.
  • the lines representing these peptides are the only bands visible in SDS-PAGE with Coomassie Brilliant Blue staining and the two most distinct precipitation lines visible in crossed immunoelectrophoresis with a polyclonal M. a. paratuberculosis antiserum in the second dimension ( Figure 4) .
  • Interferon- ⁇ (IFN- ⁇ ) production after stimulation with the antigens were measured in experimentally infected goats.
  • heparinised blood was added to 24 -well tissue culture trays.
  • the samples were stimulated with purified antigen at a concentration of 2.5 ⁇ g/ml, purified antigen 101 (2.5 ⁇ g/ml), Johnin Purified Protein Derivative (PPD) (lO ⁇ g/ml) and incubated at 37°C in 5% C0 2 in air for 24 hours.
  • the plasma was removed and assayed for IFN- ⁇ using the bovine IFN- ⁇ EIA (CSL, Australia) . According to the manufacturer's instructions, a test is considered negative when corrected OD values are below 0.05.
  • heparinised blood from experimentally infected goats were stimulated with antigen 101 and antigen 102. After 24 hours incubation, the plasma was tested for IFN- ⁇ production.
  • the 101 -102 -ELISA method was performed by coating wells in microtiter plates with lOO ⁇ l solutions of 101 and 102 (0.5 ⁇ g protein /mL in Na-carbonate buffer (pH 9,6)), purified as described in Example 1 and stored overnight at 4°C.
  • the microtiter plates were blocked with 200 ⁇ l 1% BSA for one hour at room temperature. Serum samples from the infected animals were applied in duplicate to respective wells and incubated for one hour at 20°C. Bound antibodies were recognised by adding solutions of protein-G conjugated to biotin. After 1 hour of incubation, streptavidin-peroxidase was added, followed by orthophenylene-diamine as substrate for the peroxidase. Between each step the plates were washed with Phosphate-buffered saline (pH 7.4) with 0.1% Tween.
  • Results The results are shown in tables 2 and 3. Table 2. Fraction of animals with the diagnosis of Paratuberculosis that are correctly diagnosed by two commercially available ELISA-kits for Paratuberculosis, IDEXX and CSL, and the internally made ELISA detecting antibodies to 101 and 102 as described herein. The animals were selected from different countries.
  • Table 3 shows that serum from one of the four animals tested false positively using the IDEXX-kit, whereas all the sera tested correctly negative using the I01-I02-ELISA. In addition, 15 healthy animals where tested, all of which tested negative in both assays.

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Abstract

L'invention concerne des procédés de diagnostic et/ou de surveillance d'une infection chez un individu par Mycobacterium avium ssp. paratuberculosis dans lesquels la présence ou la concentration de (i) polypeptides comportant la séquence d'acide aminé décrite Figure 1 ou Svenlkealpeyakdlklnl ou des molécules associées, ou (ii) des anticorps dirigés vers ces mêmes polypeptides, ou (iii) des molécules d'acide nucléique codant ces polypeptides, ou (iv) des réactions à l'infection provenant de la présentation de ces polypeptides, sont détectées. L'invention concerne également des compositions pharmaceutiques comprenant de telles entités à des fins prophylactiques, des utilisations des mêmes, de nouveaux polypeptides, des molécules d'anticorps et des molécules d'acide nucléique et des lots servant à réaliser les procédés de cette invention.
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EP1400532A1 (fr) * 2002-09-18 2004-03-24 Gerald-F. Prof. Dr. Gerlach ABC-transporter operon, specifique pour le microoranisme M. paratuberculosis
WO2008020185A1 (fr) * 2006-08-15 2008-02-21 Institute For Animal Health Diagnostic des infections mycobactériennes par la détermination de l'ifn-gamma
CN108864294A (zh) * 2017-08-09 2018-11-23 芜湖英特菲尔生物制品产业研究院有限公司 一种由牛白蛋白与牛干扰素γ组成的融合蛋白及其制备方法和一种重组牛长效干扰素γ

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1400532A1 (fr) * 2002-09-18 2004-03-24 Gerald-F. Prof. Dr. Gerlach ABC-transporter operon, specifique pour le microoranisme M. paratuberculosis
WO2004026901A1 (fr) * 2002-09-18 2004-04-01 Gerlach Gerald-F Operon de transporteur abc specifique de m. paratuberculosis
WO2008020185A1 (fr) * 2006-08-15 2008-02-21 Institute For Animal Health Diagnostic des infections mycobactériennes par la détermination de l'ifn-gamma
US8455201B2 (en) 2006-08-15 2013-06-04 The Pirbright Institute Diagnose of mycobacterial infections by determination of IFN-gamma
CN108864294A (zh) * 2017-08-09 2018-11-23 芜湖英特菲尔生物制品产业研究院有限公司 一种由牛白蛋白与牛干扰素γ组成的融合蛋白及其制备方法和一种重组牛长效干扰素γ

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