KR101837768B1 - Protein for dianosing porcine proliferative enteritis and application thereof - Google Patents

Abstract

The present invention relates to a protein and its application for diagnosing porcine proliferative ileitis, and more particularly, to a method for diagnosing porcine proliferative pseudomonas overexpression in a LI0147 ORF which is expected to be an outer membrane protein of Lawsonia intracellularis And a method for diagnosing porcine proliferative renalositis by using the protein fragment as an antigen and expressing the 95 amino acid sequence protein fragment having high immunogenicity.

Description

Protein for dianosing porcine proliferative enteritis and application thereof.

The present invention relates to a protein and its application for diagnosing porcine proliferative ileitis, and more particularly, to a method for diagnosing porcine proliferative pseudomonas overexpression in a LI0147 ORF which is expected to be an outer membrane protein of Lawsonia intracellularis And a method for diagnosing porcine proliferative renalositis by using the protein fragment as an antigen and expressing the 95 amino acid sequence protein fragment having high immunogenicity.

Porcine proliferative pneumonitis is caused by gram negative intracellular parasitic bacteria called Lawsonia intracellularis. Farm surveys in Europe, Asia and North America have shown that farms that are one-quarter to one-half of pig farms are seriously infected, and in Korea there are more than 50% of farms diagnosed with ileitis. Serological surveys have shown that over 90% of swine farms have L. intracellularis in most countries. In particular, the disease is a disease that has a tendency to increase in the incidence rate even in the advanced countries where the hygiene control is thorough.

The spread of the disease is caused by vertical infestation through the feces of the infected sow and horizontal infestation through the feces of infected money.

This proliferative pyelonephritis is a major problem in the swine industry including Korea and the world. Numerous swine farms suffer from economical damage due to the proliferative pancreatitis of swine, but it is difficult to separate and cultivate the causative organism. . In the United Kingdom and the United States, Lawsonia intracellularis, a causative organism of proliferative pneumonitis, has been successfully isolated and purified, and genome information of Lawsonia intracellularis has been registered with the NCBI gene bank ACCESSION: NC008011).

In the past, pig annulitis (causative bacteria: Lawsonia intracellularis method) was diagnosed by clinical symptoms and histopathological findings, identification of pathogenic bacteria, and individual diagnosis by PCR. Has come.

However, in order to identify microorganisms, it is necessary to confirm whether the microorganisms are infiltrated by the existing clinicopathologic methods or identification of pathogenic microorganisms by microscopic examination after tissue sectioning and staining. The condition is required and the diagnosis takes a lot of time.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a kit capable of diagnosing swine- will be.

Another object of the present invention is to provide a method for diagnosing porcine proliferative ileitis, which is a major disease occurring in pigs in a short time in a single reaction.

In order to achieve the above object, the present invention provides a protein for diagnosing porcine proliferative ileum having the amino acid sequence of SEQ ID NO: 1.

The present invention also provides an antibody specifically recognizing the protein of the present invention.

The present invention also provides a composition for diagnosing porcine proliferative ileum comprising the protein of the present invention.

Also, the present invention provides a kit for diagnosing porcine proliferative ileum comprising the protein of the present invention.

(A) providing a biological sample; (b) contacting the biological sample with a protein of claim 1 of Lawsonia intracellularis when Lawsonia intracellularis is present, Reacting a biological sample with the protein of claim 1 under conditions that allow for the formation of an antibody / antigen complex; (c) detecting the presence of said complex to detect the presence of an antibody of Lawsonia intracellularis A method for detecting an antibody or an antigen against Lawsonia intracellularis in a biological sample, including detecting the presence of the antibody.

The present invention also relates to a method for the treatment and prophylaxis of porcine proliferative ileitis, comprising reacting an antibody according to the present invention with a sample comprising Rhodia intracellularis and determining a sample exhibiting a positive response to the antibody as a proliferative porphyria Provides a diagnostic method.

Hereinafter, the present invention will be described.

An exemplary embodiment of the present invention provides a method for efficiently diagnosing the infection of Lawsonia intracellularis with LI0147 in pigs.

Another exemplary embodiment provides a recombinant protein having the amino acid sequence of SEQ ID NO: 1.

The amino acid sequence of SEQ ID NO: 1 is obtained by analyzing the antigenic and hydrophobic regions of the LI0147 ORF protein of Lawsonia intracellularis and selecting the region having a different sequence. The amino acid sequence of SEQ ID NO: 1 is the 59th to 154th amino acids of LI0147 ORF It is an immunogenic antigen specific to Lawsonia intracellularis. The above-mentioned SEQ ID NO: 1 is a specific immunogenic antigen against porcine proliferative ileum and can be useful for diagnosing porcine proliferative ileitis.

The amino acid sequence of SEQ ID NO: 1 was selected by analyzing the antigenic and hydrophobic regions of Lawsonia intracellularis LI0147 ORF protein. The antigenicity and hydrophobicity analysis can be performed using known methods, preferably the antigenicity can be analyzed according to Kolaskar & Tongaonkar Antigenicity method, and the hydrophobicity can be analyzed according to the Parker method. The amino acid sequence of SEQ ID NO: 1 may be synthesized using a known method, preferably an oligonucleotide synthesizer, or may be made of a recombinant protein using the oligonucleotide synthesizer.

Another embodiment of the present invention provides a recombinant protein having the amino acid sequence of SEQ ID NO: 1. The recombinant protein has immunogenicity to Lawsonia intracellularis and can be used to specifically diagnose porcine proliferative ileitis.

In order to select a Lawsonia antigen protein, the genome sequence of Lawsonia intracellularis was determined from NCBI GenBank data (ACCESSION: NC008011), and LI0147, which is an ORF expected to encode an outer membrane protein, was selected. From the amino acid sequence of this gene, a specific sequence capable of detecting Lawsonia intracellularis was selected by analyzing the antigenicity and hydrophilicity, and this gene was cloned into an E. coli expression vector and expressed. In addition, insoluble proteins were successfully purified using a buffer containing 8 M urea. This purified protein provides a method for screening pigs infected with Lawsonia intracellularis and pigs not infected using direct enzyme immunoassay.

In the present invention, an immunogenic antigen or fragment means a fragment of a protein having at least one epitope that can be recognized by an antibody to the protein of the present invention.

In order to accomplish another object of the present invention, the present invention provides an agent for diagnosing porcine proliferative ileum comprising an antibody which specifically binds to the protein of the present invention or an immunogenic fragment thereof as an active ingredient.

The antibody of the present invention may be a polyclonal antibody, but is preferably a monoclonal antibody.

Polyclonal antibodies can be prepared by injecting an external host with a fragment of the immunogenic protein of the invention according to conventional methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, and rabbits. The immunogen is injected intraperitoneally, intraperitoneally or subcutaneously, and is generally administered with an adjuvant to increase the antigenicity. Blood is routinely collected from an external host to collect the sera showing improved titer and specificity for the antigen or isolating and purifying the antibody therefrom.

Monoclonal antibodies can be prepared by immortalized cell line generation techniques by fusion as disclosed in the art [Koeher and Milstein 1975, Nature, 256: 495). The manufacturing method will be briefly described as follows. First, 20 ug of pure protein is obtained to immunize Balb / C mice, or peptide is synthesized and bound to bovine serum albumin to immunize mice. Then hybridizing the antigen-producing lymphocytes isolated from the mice with the myeloma of a human or mouse to produce immortalized hybridomas, and using hybridoma (ELISA) method to generate the desired monoclonal antibody After selective proliferation, the monoclonal antibody is separated and purified from the culture.

In the method of the present invention, the detecting step may be performed by directly detecting the presence of the protein or its immunogenic fragment by two-dimensional (2-D) electrophoresis from the liver blood solution of the individual, or by contacting the blood with the antibody of the present invention, Indirectly confirming the presence of the protein or the protein fragment of the present invention through the reaction.

The most widely known immunoassay method for antigen-antibody reaction is enzyme-linked immunosorbent assay (ELISA, Coated tube), antibody-conjugated magnetic particles are bound to a tube, and antigen-tracer and degradation-resistant contaminants are then competitively reacted with each other, Magnetic particle method, and latex particle method using antibody-bonded latex particles.

The diagnostic kits of the present invention are prepared by conventional methods known to those skilled in the art and typically include lyophilized forms of antibodies and buffers, stabilizers, inert proteins, and the like. The antibody may be labeled with a radionuclide, a fluorescent source, an enzyme, or the like.

The monoclonal antibody of the present invention can be used not only in an immunoassay kit (ELISA, antibody coated tube test, lateral-flow test, potable biosensor), but also in various pig breeding Can also be used for the development of protein chips having a sex sphincter detection spectrum.

As described above, the protein of the present invention can be used as a diagnostic agent to detect the presence of a reactive antibody of Lawsonia intracellularis in a biological sample to determine the presence of a Lawsonia intracellularis infection. For example, the presence of antibodies reactive with the proteins of the invention can be detected using standard electrophoresis and immunodiagnostic techniques, including immunoassays such as competition, direct response or sandwich type assays. Such assays include, but are not limited to, Western blots; Coagulation test; Enzyme labeled and mediated immunoassays such as ELISA; Biotin / avidin type assay; Radioimmunoassay; Immunoelectrophoresis; And immunoprecipitation. Such reactions generally involve the uncovering of labels such as fluorescence, chemiluminescence, radioactive activity, enzymatic labeling or dye molecules, or other methods of detecting the formation of complexes between an antigen and an antibody or antibodies that react therewith .

The assays described above generally involve isolating unbound antibody in a liquid phase from a solid phase support to which the antigen-antibody complex is bound. Solid supports that may be used in the practice of the invention include substrates such as nitrocellulose (e.g., in the form of membranes or microtiter wells); Polyvinyl chloride (e.g., sheet or microtiter wells); Polystyrene latex (e.g., beads or microtiter plate); Polyvinylidene fluoride; Diazo paper; nylon membrane; And activated beads, magnetically reactive beads, and the like.

Typically, the solid support first reacts with a solid phase component (e. G., One or more proteins of Lawsonia intracellularis) under suitable binding conditions, so that the component is sufficiently immobilized on the support. Occasionally, immobilization of an antigen to a support may be enhanced by binding the antigen to a peptide with better binding properties. Suitable binding proteins include, but are not limited to, macromolecules such as serum albumin, including bovine serum albumin (BSA), keyhole limpethemomycin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins known to those skilled in the art . Other molecules that can be used to bind an antigen to a support include polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers and the like. Such molecules and methods for conjugating such molecules to antigens are known to those skilled in the art [Brinkley, M.A. Bioconjugate Chem. (1992) 3: 2-13; Hashida et al., J. Appl. Biochem. (1984) 6: 56-63; and Anjaneyulu and Staros, International J. of Peptide and Protein Res. (1987) 30: 117-124.

After the solid support is reacted with the solid phase component, any unfixed solid phase component is removed from the support by washing and the support bound component is then added to the reaction mixture containing the ligand residue (e.g., antibody to the immobilized antigen) under suitable binding conditions Contact with the expected biological sample. After washing to remove any unbonded ligand, a secondary binder residue is added under suitable bonding conditions, wherein the secondary binder can be selectively bound to the bound ligand. The presence of the secondary binder can then be detected by using techniques known in the art.

More particularly, an ELISA method is used, wherein the wells of the microtiter plate can be coated with the desired protein. Biological samples expected to contain or contain anti-protein immunoglobulin molecules are then added to the coated wells. After incubation sufficient to allow the antibody to bind to the immobilized antigen, the plate is washed to remove the unbound residue and the added detectably labeled secondary binding molecule. The secondary binding molecules are allowed to react with any captured sample antibodies, the plates are washed, and the presence of secondary binding molecules is detected using methods known in the art.

Thus, in one particular embodiment, the presence of bound anti-antigen ligands from a biological sample can be readily detected by using a secondary binding agent comprising an antibody directed against the antibody ligand. A number of anti-porcine immunoglobulin (Ig) molecules that can readily be conjugated to detectable enzyme labels, such as horseradish peroxidase, alkaline phosphatase, or urease, are known to those skilled in the art using methods known in the art . A suitable enzyme substrate is then used to generate a detectable signal. In other related embodiments, competitive ELISA techniques may be practiced by those skilled in the art using known methods.

Assays are performed in solution to allow antibodies specific for the protein of the invention to form complexes under precipitation conditions. In one particular embodiment, the proteins of the invention can be conjugated to solid phase particles (e. G., Agarose beads, etc.) by using coupling techniques known in the art, such as by direct chemical bonding or indirect bonding. have. The antigen-coated particle is then contacted with a biological sample that is expected to contain an antibody to the protein under suitable binding conditions. Cross-linking between bound antibodies leads to the formation of a particle-antigen-antibody complex aggregate that can be precipitated and separated from the sample by using washing and / or centrifugation methods. The reaction mixture can be analyzed to determine the presence or absence of the antibody-antigen complex by using any of a number of standard methods, such as the immunoassay described above.

In another embodiment, an immunoaffinity matrix is provided wherein a group of polyclonal antibodies from a biological sample suspected of containing an antibody to the desired peptide of the invention is immobilized to the substrate. In this regard, the initial affinity purification of the sample can be performed by using a fixed antigen.

Many methods of immobilizing immunoglobulins (either as free or specific fragments) at high yields of antibody binding activity and at good retention levels are known in the art. Although not limited in any particular way, a fixed protein A or protein G can be used to immune the immunoglobulin.

Thus, when an immunoglobulin molecule is immobilized to provide an immunoaffinity matrix, the protein fragment is contacted with the bound antibody under suitable binding conditions. After any non-specifically bound antigen has been washed from the immunoaffinity support, the presence of the bound antigen can be determined by assaying the label using methods known in the art.

In addition, an antibody to be induced against a protein, not the protein itself, of the present invention can be used in the assay described above to detect the presence of an antibody to the protein in a given sample. Such assays are performed essentially as described above and are well known to those skilled in the art.

As can be seen from the present invention, the protein fragments of the present invention can be used for efficiently diagnosing the infection of Lawsonia intracellularis in pigs and can be used for efficiently detecting the presence of Lawsonia intracellularis antibodies, It can be used for diagnosis.

1 is a diagram showing the antigenicity (Kolaskar & Tongaonkar Antigenicity method) (FIG. 1A) and the hydrophilicity analysis (Parker method) (FIG. 1B) of LI0147.
FIG. 2 is a view showing the result of expressing lawsonia antigen in E. coli and then purifying it.
Figure 3 is a diagram showing direct ELISA results of a recombinant Lawsonia-His fusion protein.

The following examples are intended to illustrate the present invention and are not to be construed as limiting the scope of the present invention.

Example  One. Lawsonia antigen Antigenicity and Hydrophilicity Analysis

In order to select Lawsonia antigen protein, genome sequence of Lawsonia intracellularis was confirmed from NCBI GenBank data (NCBI GenBank data, ACCESSION: NC008011), and LI0147, which is an ORF expected to encode outer membrane protein, was selected. Based on the nucleotide sequence of LI0147, the amino acid sequence was known. From this information, the antigenicity was analyzed by Kolaskar & Tongaonkar Antigenicity method and the hydrophilicity was analyzed by Parker method. The results are shown in Fig. According to Fig. 1, the 59th to 154th amino acid portions showed high hydrophilicity and antigenicity, and this portion was selected as an antigen of the present invention.

Example  2. lawsonia antigen  Preparation of expression vector

(A) Preparation of synthetic lawsonia antigen gene

The nucleotide sequence of LI0147, the antigenic protein of lawsonia, was obtained from NCBI GenBank data (ACCESSION: NC008011) and the oligonucleotide of the synthetic gene was synthesized in Bionics, Korea (Table 1).

name The sequence (5 '- > 3') Other LI0147F GAATTCATTCTAGGTACTATTACTGGT (SEQ ID NO: 3) EcoR I LI0147R GTCGACATTGAAGATACTCGAGCCG (SEQ ID NO: 4) Sal I

Table 1 is information on oligonucleotides used in the manufacture of lawsonia antigens.

Nucleotide sequence Amino acid sequence 5-attctaggt actattactg gtggagctgt tggtggtgtt cttggtagtt taatcggtgg aggatcagga agaatattat caactgttgt tggtgctgga gcaggggctg ttgctggaaa cattgctgaa agaaaaatta caacgcaaca aggtctcgaa atagaagtga aacttgataa tggtcaaatt atttctatag ttcaaggtgc tgatcaatct tttagtcctg gcgaacgtgt tcgagtacta cgtggaagtg atggttcggc tcgagtatct tcaata-3 (SEQ ID NO: 2) 1-ILGTITGGAVGGVLGSLIGGGSGRILSTVVGAGAGAVAGNIAERKITTQQGLEIEVKLDNGQIISIVQGADQSFSPGERVRVLRGSDGSARVSSI-95 (SEQ ID NO: 1)

Table 2 shows the nucleotide sequences and amino acid sequences encoding Lawsonia antigens amplified by PCR.

The oligonucleotides amplified by the above primers by the PCR method were prepared to contain restriction sites for cloning into the expression vector pET28a (+). When the expression vector pET28a (+) is used, the forward oligonucleotide contains the EcoRI restriction site and the reverse oligonucleotide contains the Sail restriction site.

When PCR was performed using primers LI0147F and LI0147R, a total of 288 bp (96 amino acids, 15 kDa) of LI0147-His gene capable of being introduced into pET28a (+) was synthesized (Table 2).

The PCR reaction (50 ul volume) was set as follows.

Each oligonucleotide sense primer and anti-sense primer (Table 1) were used with 20 pmol each of dNTPs (dATP, dCTP, dGTP and dTTP) at 25 uM with DNA polymerase (1 U) and 1X buffer. The reaction was carried out at 95 ° C. for 5 minutes, amplified by 30 cycles of 95 ° C. for 15 seconds, 60 ° C. for 20 seconds and 72 ° C. for 60 seconds, and then left at 72 ° C. for 10 minutes. The PCR product was purified using a Qiagen PCR spin column.

 (B) Cloning of PCR products

The PCR products amplified for introduction into pET28a (+) (Novagen, USA) as described above were digested with restriction endonucleases EcoRI + SalI (New England Biolab, USA) and gel- pET28a (+).

The conjugated product is used to transform DH5-alpa (Invitrogen, USA) recipient cells. The transformed cells were cultured in LB medium (DIFCO, USA) supplemented with 50 μg / ml of kanamycin. DNA for miniprep was obtained by culturing overnight from the colony medium and digested with ECoRI + SalI to select the desired clones.

Example  3. Lawsonia  Growth and expression of Escherichia coli strains with antigen plasmids

Each Escherichia coli strain was inoculated overnight in a shaking orbital incubator at 37 占 폚 in 50 ml of sterile LB supplemented with 50 占 퐂 / ml kanamycin. 50 ml of the inoculum were transferred from the seeds to a flask containing 0.5 liter of aseptic LB supplemented with 50 μg / ml of kanamycin and cultured at 37 ° C. until they were medium-logarithmic and cultured in 0.2 mM IPTG (isopropylthiogalactoside) Followed by incubation at 37 ° C for 8 hours. After the induction period, cells were pelleted in a centrifuge and collected according to standard procedures. Pelleted cells were analyzed by 12% SDS-PAGE after sonication with Lysis- Equilibration buffer / Wash (LEW, 50 mM NaH2PO4, 300 mM NaCl, pH to 8.0) and were found to be insoluble And stored at -70 DEG C until the next step (Fig. 2).

Example  4. Lawsonia  Production of antigens

Frozen cells obtained from Example 3 were prepared according to the standard procedure for purification of each polyhistidine-tagged protein. Each standard procedure is as follows.

Lawsonia-His fusion protein was resuspended by addition of Lysis-Equilibration buffer / Wash (LEW, 50 mM NaH2PO4, 300 mM NaCl, pH to 8.0) and the cells were pulverized by ultrasonication. An insoluble lysate was prepared by centrifugation of the cell-lysate (10,000 rpm, 30 min). The protein was dissolved again in Denaturing Solublilization buffer (50 mM NaH2PO4, 300 mM NaCl, 8M Urea, pH to 8.0) Lt; / RTI > syringe filter (Sartorius). The antigen protein was purified by nickel affinity chromatography and the insoluble cell lysate was loaded onto a Ni-NTA His Bind Resin (Novagen) column equilibrated with Denaturing Solublilization buffer. After washing the column with 3 column volume denaturing solublilization buffer, Lawsonia-His protein was eluted with buffer solution of Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 8M Urea, 250 mM Imidazole, pH 8.0) . The eluted fractions were analyzed on 12% SDS-PAGE. The purified fusion protein was buffered overnight in PBS buffer and stored in a refrigerator through a 0.2-μm filter.

Example  5. Recombination Lawsonia - His  Direct enzyme immunoassay of fusion proteins ( direct ELISA )

ELISA analysis was carried out using the fusion protein antigen of the present invention prepared in Example 3.

The recombinant Lawsonia-His fusion protein antigen was diluted to 10 μg / ml using PBS, and two kinds of peptides were coated at a concentration of 100 μl / well in each well of a 96-well plate (Maxisorp, Nunc, Denmark).

The plate was stored at 4 ° C for one day, washed three times with PBST (1X PBS + 0.1% Tween-20), blocked with PBS supplemented with 5% skim milk for 30 minutes at room temperature, 3 times.

Ten pigs infected with Lawsonia intracellularis and 10 infected sera were used as pig samples for diagnosis. The serum was diluted 1: 100 with PBS, reacted at 100 μl / well at room temperature for 2 hours, and washed 5 times with PBST. HRP-conjugated goat anti-pig IgG (Serotec, UK) diluted 1: 500 was used as the secondary antibody. The secondary antibody was also reacted at room temperature for 1 hour and then washed seven times with PBST. The bound secondary antibody was reacted with TMB substrate (BioFX, SurModics, USA) for 10 minutes at room temperature, and the reaction was monitored by measuring the reaction at 450 nm after 5N HCl was added to the well / 20.

Based on the above results, it can be confirmed that Lawsonia intracellularis infection can be diagnosed effectively in pig serum through the diagnosis method using Lawsonia-His fusion protein proposed in the present invention (FIG. 3).

<110> Konkuk University Industrial Cooperation Corp. <120> Protein for dianosing porcine proliferative enteritis and          application thereof <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 95 <212> PRT <213> Lawsonia intracellularis <400> 1 Ile Leu Gly Thr Ile Thr Gly Gly Ala Val Gly Gly Val Leu Gly Ser   1 5 10 15 Leu Ile Gly Gly Gly Ser Gly Arg Ile Leu Ser Thr Val Val Gly Ala              20 25 30 Gly Ala Gly Ala Val Ala Gly Asn Ile Ala Glu Arg Lys Ile Thr Thr          35 40 45 Gln Gln Gly Leu Glu Ile Glu Val Lys Leu Asp Asn Gly Gln Ile Ile      50 55 60 Ser Ile Val Gln Gly Ala Asp Gln Ser Phe Ser Pro Gly Glu Arg Val  65 70 75 80 Arg Val Leu Arg Gly Ser Asp Gly Ser Ala Arg Val Ser Ser Ile                  85 90 95 <210> 2 <211> 285 <212> DNA <213> Lawsonia intracellularis <400> 2 attctaggta ctattactgg tggagctgtt ggtggtgttc ttggtagttt aatcggtgga 60 ggatcaggaa gaatattatc aactgttgtt ggtgctggag caggggctgt tgctggaaac 120 attgctgaaa gaaaaattac aacgcaacaa ggtctcgaaa tagaagtgaa acttgataat 180 ggtcaaatta tttctatagt tcaaggtgct gatcaatctt ttagtcctgg cgaacgtgtt 240 cgagtactac gtggaagtga tggttcggct cgagtatctt caata 285 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gaattcattc taggtactat tactggt 27 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtcgacattg aagatactcg agccg 25

Claims (7)

An antigen protein fragment for diagnosing a porcine proliferative ileum comprising the amino acid sequence of SEQ ID NO: 1. An antibody that specifically recognizes the protein fragment of claim 1. A composition for diagnosing porcine proliferative ileum comprising the protein fragment of claim 1. A kit for diagnosing porcine proliferative ileum comprising the protein fragment of claim 1. A kit for the diagnosis of porcine proliferative ileum comprising the antibody of claim 2. (a) providing a biological sample;
(b) contacting the biological sample with a sample of the strain of Lawsonia intracellularis in the presence of Lawsonia intracellularis under conditions that allow binding to the protein fragment of Clause 1 of the Lawsonia intracellularis to form an antibody / antigen complex , Reacting the biological sample with the protein of claim 1;
(c) detecting the presence or absence of said complex to detect the presence of an antibody of Lawsonia intracellularis in said sample, wherein the antibody to Lawsonia intracellularis in a biological sample / RTI &gt; A method for diagnosing porcine proliferative ileitis, comprising reacting an antibody according to claim 2 with a sample comprising rosonic acid intraceluris, and determining a sample exhibiting a positive reaction against said antibody as a porcine proliferative ileum.

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