WO2007054515A1

WO2007054515A1 - Method for detecting microorganisms

Info

Publication number: WO2007054515A1
Application number: PCT/EP2006/068233
Authority: WO
Inventors: Malik Merza
Original assignee: Boehringer Ingelheim Vetmedica Gmbh
Priority date: 2005-11-14
Filing date: 2006-11-08
Publication date: 2007-05-18
Also published as: EP1957664A1

Abstract

The present invention relates to a detection method for microorganism that is based on the Proximity ligation Assay (PLA). According to a preferred embodiment, the present invention relates to a solid-phase PLA. Moreover, the present invention relates to a PLA, preferably solid-phase PLA for the detection of Lawsonia intracellularis.

Description

Method for detecting microorganisms

FIELD OF THE INVENTION

The present invention relates to the field of microbiology. In particular, the invention relates to a method for the detection of microorganism infections in samples taken from an infected animal. More particularly, the present invention relates to a method for the detection of Lawsonia intracellularis infections in animals.

BACKGROUND OF THE INVENTION There is an urgent need for efficient and reliable methods for detection of pathogens and monitoring of infectious diseases. Infectious diseases are the second-leading cause of deaths with more than ten million people, 90% of whom live in the developing world, killed by infections worldwide each year according to the United Nations (emerging infectious diseases. Center for Disease Control and Prevention; World health organization. The world health report 2004-changing history. Geneva: The organization; 2004) Infectious diseases in livestock animals cause major economical loss and there is a renewed interest in defence against biological warfare. Sensitive, accurate and easy-to-use methods are thus needed for detection of pathogens of microbiological origin in food, water, environmental samples, and livestock as well as the human body.

Along with the increasing demand from governments world -wide for a control system of different animal diseases, reliable techniques with high sensitivity as well as specificity for the detection of pathogenic organisms are in great need. Recently major efforts have been put into developing techniques that detects the nucleic acid of the organisms, mainly different PCR assays, while in order to detect viral or bacterial proteins you are mainly referred to either different agglutination tests or, more recently developed, capture Elisa's.

Proximity ligation is an assay that has proven to be very successful for sensitive protein detection, mainly cytokine detection, in complex biological samples (Gullberg et al, Rollman et al). Proximity ligation (see Figure 1 ) in solution is performed in three steps. Target proteins are incubated with pairs of proximity probes consisting of oligonucleotides with either free 5' or 3' ends conjugated to a target specific antibody. In the second step, a mix containing components necessary for ligation and PCR amplifiation is added, and the free DNA ends on pairs of probes bound to the same target are hybridized by a common connector-oligonucleotide The probe ends are instantly ligated by DNA ligase. The final step in the homogenous-phase PLA protocol, is the amplification and detection of the newly formed DNA strand in real time.

Lawsonia intracellularis is the causative agent of proliferative enteropathy (PE), a Gram negative obligate intracellular bacterium in the Desulfovibho family. Infection of pigs with this bacterium is consistently linked with the presence of proliferative lesions of the mucosa of the ileum and large intestine, hyperplasia of crypt enterocytes along with a decrease in goblet cells in association with the presence of intracellular, curved or S-shaped Lawsonia bacteria. The chronic forms of PE lead to clinical or sub clinical effects on weight gain, feed conversion and faecal consistency. Clinical observations generally include diarrhoea, with "variation" in the weights of growing pigs. It also may present as an acute form with sudden death or bloody diarrhoea due to massive haemorrhage within infected proliferative mucosa, particularly in late finishing pigs and replacement gilts. Lawsonia infection may persist in some pigs for at least 10 weeks. The bacteria may be viable outside the host for at least two weeks under certain conditions. Lawsonia is today treated with antibiotics and new vaccines are under development. A more detailed description of Lawsonia intracellularis is given for example in WO97/39629, WO97/20050, WO03/00665, WO04/033631.

SUMMARY OF THE INVENTION

The present invention relates to a method for detecting the presence of a microorganism in a sample , comprising the steps: a. Incubating a sample that comprises a microorganism with two antibody- oligonucleotide probes, wherein the first and the second antibody of the two antibody-oligonucleotide probes specifically recognize and bind to said microorganism, and wherein one of oligonucleotides of the first and tie second antibody-oligonucleotide probes comprises a free 5' end and wherein the other oligonucleotide of the two antibody-oligonucleotide probes comprises a free 3' end; b. Incubating the mixture of step a) with a mixture that comprises: i. a connector oligonucleotide, wherein the 5' end of said connector oligonucleotide overlaps and hybridize with the free 3' end of one oligonucleotide of the two antibody-oligonucleotide probes and wherein the 3' end of said connector oligonucleotide overlaps and hybridize with the free 5' end of the second oligonucleotide of the antibody-oligonucleotide probes; ii. a DNA Ligase and a DNA Polymerase; iii. two oligonucleotides that bind to the hybrid consisting of the oligonucleotides of the of the two antibody-oligonucleotide probes and the connector olignucleotide; c. amplifying a nucleotide sequences that is generate by the hybrid consisting of the oligonucleotides of the two antibody-oligonucleotide probes and the connector olignucleotide; d. detecting the amplification product of step c).

Steps a) and b) of the method as described supra can be performed simultaneously or .one after the other, wherein in the latter event, step b) is performed after step a). The method described supra is called "Proximity ligation Assay" (PLA).

According to another embodiment of the present invention, the PLA as described supra is performed as an solid phase PLA. This means that the sample comprising the microorganism is first incubated with an immobilized antibody that specifically recognizes and binds to said microorganism. It has been found that this pre-selection step surprisingly increase the accuracy of the PLA and to allow detection of a single- copy of microorganism in a sample. Electively, this first incubation step is followed by an wash step in order to remove non-binding or non-specific binding contaminants within the reaction vessel. The method, that comprises such a wash step is preferably used.

According to a further embodiment, the present invention relates to an PLA or solid- phase PLA for the detection of Lawsonia intracelluaris. It has been surprisingly found, - A - that the PLA, and prefereably the solid-phase PLA are superior over those assays known in the prior art. It has been surprisingly found that the antibodies 301 :39, antibody 287:6, antibody 268:29, antibody 1 10:9, antibody 1 13:2 and/or antibody 268:18, as described more in detail under section "DETAILED DESCRIPTION" are preferably used for the detection of Lawsonia intracellularis via PLA or solid-phase PLA.

DESCRIPTION OF THE FIGURES

Figure"! . The homogenous phase PLA is performed in three steps. In the first step one microliter of a sample containing a microbe target is incubated with a pair of proximity probes in a volume of 5 uL for at least one hour at 37C. The proximity probes are antibody-DNA conjugates with either a free 5'end (grey ribbon) or a free 3' end (black ribbon). In the second step, a common ligation and PCR mix (45 microliters) is added to the incubation. The proximity probes that are bound to the same microbe target are lined up by hybridization of a splint-oligonucleotide (comb- like) to the free 5' and 3' ends. The free ends are then instantly ligated by T4 DNA ligase. In the third step, the ligated proximity probes are amplified, and detected by real-time PCR.

Figure 2. The solid-phase PLA. The top figure displays the binding of a microbial target to an immobilized antibody in a microtiter well. The non-bound particles are washed away, and the proximity probes are subsequently added to the well. After at least one hour of incubation at 37C, proximity probes that are not bound to the target are washed away. The common ligation and PCR mix is added to the well, and the target-bound proximity probes are ligated and amplified with real-time detection and quantification of the products (bottom figure).

Figure 3A. Measurements of dilutions of Porcine Parvovirus (PPV) by three different methods, solid-phasePLA (purple squares), qPCR (blue diamonds) and ELISA (green triangles). The analyses were performed in the presence of a negative tissue sample. The x-axis displays the total number of viral infectious units (TCI D ₅₀) present in 50 uL sample (solid-phase PLA), 1 uL sample (qPCR), and 100 uL (ELISA). The left yaxis displays the signal to noise ratio for PLA and qPCR, and the right yaxis displays the absorbance at 450 nM for ELISA. Figure 3B. PLA (purple bars), qPCR (blue bars) and HA (grey bars) of lysed pig embryos, positive (1 -10) and negative (1 -10) for the PPV virus. The left y-axis shows the results of the PLA and qPCR analysis, the number of PPV infectious units per uL of sample.

Figure 4A. Measurements of dilutions of the bacterium Lawsonia intracellularis by three different methods, homogenous-phase PLA (pink squares), qPCR (blue diamonds) and ELISA (green triangles). The analyses were performed in the presence of a negative faeces sample. The x-axis displays the total number of bacteria present in 1 uL sample (homogenous-phase PLA), 1 uL sample (qPCR) and 100 uL (ELISA). The left y-axis displays signal to noise ratio for the PLA and qPCR, and the right y-axis displays the absorbance at 450 nM for ELISA.

Figure 4B. Twenty faeces samples, positive (1 -10) and negative (1 -10) for Lawsonia intracellularis , were analyzed with PLA (purple bars), qPCR (blue bars) and ELISA (grey bars). On the left y-axis, the number of bacteria per 1 uL faeces sample as analyzed by PLA and qPCR Shown are triplicate measurements with standard deviations. The y-axis displays the absorbance at 450 nM for ELISA

Figure 5 A displays the advantages of combining different antibodies for sensitive microbe detection by PLA. A series of dilutions of Lawsonia intracellularis bacteria were analyzed by one monoclonal antibody (green triangles), two different monoclonal antibodies (blue diamonds and grey circles) and three different antibodies (purple squares). The assays with one and two different antibodies were performed in a homogenous format, the one with three different antibodies is a solid phase assay with one capturing antibody and two different antibodies for detection. The y-axis displays C_τ values from the real-time PCR. A C_τ value of 35 corresponds roughly to 8 copies of PCR templates formed by ligation of the proximity probes, and CT 21 corresponds to around 130,000 templates. Shown are triplicate measurements with standard deviations.

Figure 5B shows a comparison of the signal (from a single datapoint in a standardcurve) to noise ratio of different antibodies and combinations of those in homogenous-phase PLA and ELISA. Standardcurves with two of the antibody combinations are shown in A (triangles).

DETAILED DESCRIPTION Reliable as well as simple methods are of great importance to diagnose different organisms that cause disease, preferably in the veterinary field. As clinical signs could be similar between different diseases, it is of crucial importance to know which organism is responsible for the signs in order to give the correct treatment of the animal or to take the necessary precautions.

Until today the field of detecting microorganisms has been more or less limited to cultivation on cell-culture, different agglutination tests or ELISA for the detection of the viral or bacterial proteins, while quite large efforts has been put into developing new techniques for the detection of nucleic acid (PCR). To have a correct diagnose it is important that the laboratory test results is reflecting the current status of the disease and not a previous one. Certain pathogens, e.g. herpes viruses, establish latent infections which mean in reality carrying viral DNA throughout life. In such a case the PCR that detects parts of the genome of the pathogen (DNA, RNA) will not be the method of choice, since one will not be able to distinguish between the acute or latent phase. Hence preferred method would be a test system that detects the viral proteins that exist only in the acute phase of the disease.

It has been surprisingly found that the Proximity ligation assay (PLA) can be used to detect microbial infections in an animal via sensitive and specific detection of microbial surface proteins. The detection method as described herein combines the techniques of the classical antibody-capture assay and the high potential of the nucleic acid amplification assays. This combination results in a very sensitive and highly accuracy detection assay for the detection of microbial infection in an animal, preferably in an non-human animal. However, the PLA overcomes the disadvantages of nucleic acid amplification assays, because the PLA allows discrimination between acute and latent infection phase.

Therefore, the present invention relates to a method for detecting the presence of a microorganism in a sample , comprising the steps: a. Incubating a sample that comprises a microorganism with two antibody- oligonucleotide probes, wherein the first and the second antibody of the two antibody-oligonucleotide probes specifically recognize and bind to said microorganism, and wherein one of oligonucleotides of the first and the second antibody-oligonucleotide probes comprises a free 5' end and wherein the other oligonucleotide of the two antibody-oligonucleotide probes comprises a free 3' end; b. Incubating the mixture of step a) with a mixture that comprises: i. a connector oligonucleotide, wherein the 5' end of said connector oligonucleotide overlaps and hybridize with the free 3' end of one oligonucleotide of the two antibody-oligonucleotide probes and wherein the 3' end of said connector oligonucleotide overlaps and hybridize with the free 5' end of the second oligonucleotide of the antibody-oligonucleotide probes; ii. a DNA Ligase and a DNA Polymerase; iii. two oligonucleotides that bind to the hybrid consisting of the oligonucleotides of the of the two antibody-oligonucleotide probes and the connector olignucleotide; c. amplifying a nucleotide sequences that is generate by the hybrid consisting of the oligonucleotides of the two antibody-oligonucleotide probes and the connector olignucleotide; d. detecting the amplification product of step c)

The PLA technique has been developed with the aim to detect two different infectious organisms i.e. a virus and a bacterium. The PLA assay is therefore applicable to different viral and bacterial pathogens. According to a preferred embodiment, the present detection method for microorganisms is suitable for the detection of infections, for example with, Bovine Respiratory Syncytial Vaccine (BRSV), Bovine Rhinotracheitis (IBR) Bovine Virus Diarrhea (BVD), Brachyspira hyodysentehae, , Parovirus, Porcine parovirus (PPV), Porcine reproductive and respiratory syndrome virus (PRRS) Herpesvirus, Rotavirus , Enterovirus, Coronovirus, Rabiesvirus, Adenovirus , Astrovirus, Actinobacillus spp., Actinobacillus lignieresii, Actinobacillus pleuropneumoniae, Actinomyces spp., Actinomyces pyogenes, Ascaris suum, Ascarops strongylina, Lawsonia intracellulars, Mannheimia spp., Mannheimia haemolytica (formerly Pasteurella haemolytica), Pasteurella spp., Pasteurella multocida, Haemophilus spp., Haemophilus somnus (Histophilus ovis and Haemophilus agni), Clamydia spp., Chlamydia psittaci, Camphylobacter spp., Campylobacter fetus venerealis and Campylobacter fetus fetus (formerly C fetus intestinalis), Leptospira spp., Leptospira interrogans, Leptospira pomona, and Leptospira ghppotyphosa, Leptospira canicola, Leptospira ghppotyphosa, Leptospira hardjo (Leptospira hardjoprajitno and Leptospira hardjo-bovis), Brucella spp., Brucella abortus, Brucella suis and Brucella melitensis, Listeria spp., Listeria monocytogenes, Clostridium spp., Clostridium chauvoei, Clostridium septicum, Clostridium haemolyticum, Clostridium novyi, Clostridium sordellii, Clostridium perfringens, Clostridium tetani, Moraxella spp., Moraxella bovis, Klebsiella spp, Klebsiella pneumoniae, Salmonella spp., Salmonella typhimuhum; Salmonella newport, Mycobacterium spp., Mycobacterium avium paratuberculosis, Staphylococcus spp., Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus uberus, Mycoplasma dispar, Mycoplasma bovis, and Ureaplasma spp., Tritrichomonas foetus, Trichophyton spp., Trichophyton verrucosum, Trichophyton mentagrophytes, Trichophyton sarkisovii, Neospora caninum (formerly Toxoplasma gondii), Cryptspohdium spp., Cryptsporidium parvum, Cryptspohdium hominis, Babesia spp., Babesia bigemina, Babesia bovis, Dictyocaulus viviparous (Lungworm disease). According to a preferred embodiment, the method described herein is used for the detection of Lawsonia intracelularis. As described herein, it has been surprisingly shown that the PLA allows the detection of only one copy of Lawsonia intracellulahs in a sample. The present method is therefore more sensitive than any other protein based detection methods for Lawsonia intracellulahs.

The PLA analysis of the microorganism were performed both in solution and on a solid phase with the aim to investigate the optimal sensitivity and specificity of the assays. In the solid phase assay (Figure 2), the microbial target is bound to immobilized antibodies in microtiter wells. The sample and the antibody- oligonucleotide probes (Proximity probes) are added to the well and allowed to react in two steps. Washing steps are included between the different incubations to remove any unbound material. Finally the combined mix containing the ligation components as well as the PCR components is added. The present invention also relates to a method for detecting the presence of a microorganism in a sample , comprising the steps:

(a) incubating a sample that comprises a microorganism with an immobilized antibody, that specifically recognize and binds to said microorganism; (b) washing the mixture of step a);

(c) Incubating the mixture of step b) with two antibody-oligonucleotide probes, wherein the first and the second antibody of the two antibody- oligonucleotide probes specifically recognize and bind to said microorganism, and wherein one of oligonucleotides of the first and the second antibody-oligonucleotide probes comprises a free 5' end and wherein the other oligonucleotide of the two antibody-oligonucleotide probes comprises a free 3' end;

(d) Incubating the mixture of step a) with a mixture that comprises: i. a connector oligonucleotide, wherein the 5' end of said connector oligonucleotide overlaps and hybridize with the free 3' end of one oligonucleotide of the two antibody-oligonucleotide probes and wherein the 3' end of said connector oligonucleotide overlaps and hybridize with the free 5' end of the second oligonucleotide of the antibody-oligonucleotide probes; ii. a DNA Ligase and a DNA Polymerase; iii. two oligonucleotides that bind to the hybrid consisting of the oligonucleotides of the of the two antibody-oligonucleotide probes and the connector olignucleotide;

(e) amplifying a nucleotide sequences that is generate by the hybrid consisting of the oligonucleotides of the two antibody-oligonucleotide probes and the connector olignucleotide;

(f) detecting the amplification product of step e).

According to a preferred embodiment of the PLA described herein, the immobilized antibody is bound to a 96 well microtiter plate. This allows the analyses of a high number of samples in parallel and makes the PLA as described herein applicable for the commercial use. The coating of the microtiter plates with monoclonal antibodies is well known in the art, and could be exemplary done according to the method as described below under section "Examples". Several oligonucleotides can be used to prepare the antibody-oligonucleotide probes and the connector oligonucleotide. The criteria to be fulfilled are given in the description of the assay. However, sensitivity of the PLA may depend on the specific oligonucleotides sued as connector oligonucleotide or for the preparation of the antibody-oligonucleotide probe. In order to realize the method as described herein, the oligonucleotides 5' STV : 5'P-TCG TGT CTA AAG TCC GTT ACC TTG ATT CCC CTA ACC CTC TTG AAA AAT TCG GCA TCG GTG A (SEQ ID NO:1 ), 3' STV : CGC ATC GCC CTT GGA CTA CGA CTG ACG AAC CGC TTT GCC TGA CTG ATC GCT AAA TCG TG-3'OH (SEQ ID NO:2) and the connector oligonucleotide 5'- TAC TTA GAC ACG ACA CGA TTT AGT TT -3' (SE ID NO:3) were used. It has been found, that the use of these oligonucleotides allows a very specific and sensitive detection of the bound microorganism. According to a further embodiment, the present invention relates to a PLA or solid-phase PLA, wherein, the oligonucleotides of the antibody- oligonucleotide probes having the sequences of SEQ ID NO:1 and SEQ ID NO:2;, and the connector oligonucleotide having the sequence of SEQ ID NO:3.

According to a further embodiment of the present invention, it has been shown that the hybrid comprising the two oligonucleotides of antibody-olignucleotide probes and the connector oligonucleotide can be amplified by using the oligonucleotides 5'- CAT CGC CCT TGG ACT ACG A -3 (SEQ ID NO:4), and 5'- GGG AAT CAA GGT AAC GGA CTT TAG -3' (SEQ ID NO:5).

Specific Lawsonia intracellularis antibodies described herein were generated to be used in the PLA, preferably in the solid-phase PLA. The antibodies have the following reference numbers: 301 :39, 287:6, 268:29, 1 10:9, 1 13:2 and 268:18. All antibodies are specific for antigens of L. intracellularis bacteria.

The antibodies as used herein are produced by hybridoma cells. Said hybhdoma cells are deposited at the Centre for Applied Microbiology and Research (CAMR) and European Collection of Cell Cultures (ECACC)", Salisbury, Wiltshire SP4 OJG, UK, as patent deposit according to the Budapest Treaty. The date of deposit was May 1 1 , 2004. HYBRIDOMA CELL LINE 110:9 is successfully deposited under ECACC Ace. No. 04092204. HYBRIDOMA CELL LINE 113:2 is successfully deposited under ECACC Ace. No. 04092201. HYBRIDOMA CELL LINE 268:18 is successfully deposited under ECACC Ace. No. 04092202. HYBRIDOMA CELL LINE 268:29 is successfully deposited under ECACC Ace. No. 04092206. HYBRIDOMA CELL LINE 287:6 is successfully deposited under ECACC Ace. No. 04092203. HYBRIDOMA CELL LINE 301 :39 is successfully deposited under ECACC Ace. No. 04092205.

According to further embodiment of the present invention, use of the monoclonal antibodies 287:6, antibody 110:9 and antibody 113:2 for the detection of Lawsonia intracllularis infections is most preferred. It has been surprisingly shown that these antibodies are highly suitable for the use in the PLA. Specific combinations of these antibodies result in high assay accuracy. As described under section "Example", the use of the monoclonal antibody 1 10:9 as immobilized capture antibody and use of the antibodies 113:2 and 287:6 for the preparation of the antibody-oligonucleotide probes results in a highly sensitive and specific detection assay for the detection of Lawsonia intracellularis . Therefore, a further embodiment of the present invention relates to PLA, preferably a solid-phase PLA, wherein the antibody 1 10:9 is used as immobilized capture antibody and the antibodies 113:2 and 287:6 are sued to prepare the antibody-oligonucleotide probes.

The results as provided herein clearly show that the PLA is a very sensitive test system detecting single particles of infectious bacteria and viruses, meaning an assay as sensitive as the Quantitative PCR and far better than the traditional capture ELISA's used in the prior art. Very high specificity can be achieved using two or three imAb^'s that specifically bind to specific protein epitopes and could thereby easily detect minor variants of the protein in question (e.g. strain differentiation).

It has been further demonstrated that PLA can clearly differentiate between positive and negative cases, using field samples representative for detection of the relevant disease. The background noise of such a negative sample was in most cases surprisingly less than 1 infectious particle, which is very specific. Quantification of the bacterial as well as the viral copies used to exemplahly demonstrate the benefit of the PLA, particularly the solid-phase PLA, shows a very good correlation with reference protein detection methods (HA and Capture ELISA). AII together it has been surprisingly shown that the PLA is very useful for the detection of particles from infectious agents such as viral or bacterial proteins. The assays were proven to be as sensitive and specific as the quantitative PCR that was tested in parallel and has proven to be a simple and reliable tool for detection of viral or bacterial infection without the need for preparation of samples.

The PLA is also well suited for large-scale applications running hundred of samples at one time. A future area for this technique would be a multiplexed screening system for several different infectious agents simultaneously.

The PLA techniques as described herein also can apply on detection of fairly new organisms as PLA has the advantage over PCR that it only needs antibodies directed to the organism, while in the case of PCR the nucleic acid sequence has to be known for the development of the specific assay.

REFERENCES

1. Belak S et.al (1998) Detection of challenge virus in fetal tissues by nested PCR as a test of the potency of a porcine parvovirus vaccine. Vet Res Com; 22, 139-146. 2. Dinter Z. (1989) Diagnostic Virology, A review of methods at the National Veterinary Institute. Edited by J. Moreno-Lopez. Swedish University of Agricultural Sciences, National Veterinary Institute, Uppsala, Sweden, Swedish International Development Authority. ISBN 91 -576-3814-4

3. Fredriksson, S., et al., Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol, 2002. 20(5): p. 473-7.

4. Gullberg, M., et al., Cytokine detection by antibody-based Proximity ligation. Proc Natl Acad Sci U S A, 2004. 101 (22): p. 8420-4.

5. Juntti N. et al. (1986) Use of monoclonal antibody against hemagglutinin in ELISA for the diagnostics of porcine parvovirus. Proc. of the International Pig Veterinary Society, Barcelona. Proc 9th IPVS Congress, Barcelona

6. Kim, J. and C. Chae, Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome. Can J Vet Res, 2003. 67(2): p. 133-7. 7. Lindecrona, R.H., et al., Application of a 5' nuclease assay for detection of Lawsonia intracellularis in fecal samples from pigs. J Clin Microbiol, 2002. 40(3): p. 984-7.

8. Mengeling W. L. (1986) Porcine Parvovirus infection. In Diseases of Swine 6th Edition. Edited by A.D. Leman, B. Straw, R. D. Glock, W.L. Mengeling, R.H. Penny and E. Scholl, Iowa State Uni. Press, pp. 41 1 -424.

9. Rivera E et. al (2005) The Rb 1 fraction of ginseng elicits a balanced Th1 and Th2 immune response. In press

10. Rivera E et al. (1986) Porcine parvovirus: propagation in microcarrier cell culture and immunogenic evaluation in pregnant gilts. Research in Veterinary Science; 41 , 391 -396. 1 1. Rollman et al. (2003) Platelet derived growth factor (PDGF) responsive epidermis formed from human keratinocytes transduced with the PDGF beta receptor gene. J Invest. Dermatol 120(5) :p. 742-749.

12. Rockborn G. et al. (1990) Diagnostic Virology, Second Part: Guidebook to Procedures. Edited by J. Moreno-Lopez. Swedish University of Agricultural Sciences, National Veterinary Institute, Uppsala, Sweden, Swedish International Development Authority

13. Rivera E et. al (1986). A solid Phase Flourescent Immunoassay for the Rapid Detection of Virus Antigen or Antibodies in Fetuses Infected with Porcine Parvovirus. Arch Virol; 88, 19-26.

EXAMPLES

The present invention is further described in the following examples which are provided for illustrative purposes only and are not to be construed as limiting. Indeed, other variants of the invention will be readily apparent to one of ordinary skill in the art.

MATERIAL AND METHODS PPV and a-PPV mAb

The Porcine parvovirus (PPV) strains NADL-2 and the anti-PPV monoclonal antibody 5B was used in this study. The virus has a titre of 10⁸ TCID₅₀/ml and was propagated on roller bottles according to Rivera et.al. (Research in Veterinary Science 1986).

Purification of virus

Supernatants of virus-infected cell cultures were clarified by low-speed centhfugation. Virus particles in the supernatants were concentrated by pelleting in a centrifuge (x) at 16000 g for 4 h. Thepelleted virus was re-suspended in PBS layered on a linear 20 to 60% sucrose gradient and centrifuged at 100 00Og for 16 h. The virus band was collected. This partially purified virus was used as an immunogen for the preparation of monoclonal antibodies.

Lawsonia intracellularis and a-Lawsonia mAb s

The Lawsonia intracellularis strain EU-01 used was kindly provided by Dr. Keller, Bioscreen, Germany. The bacterial cultivation of Lawsonia intracellularis was prepared by inoculation of the bacteria on McCoy cells as described in WO96/39629. The bacterial suspension used as a standard throughout the experiments has a titre of 10⁷'⁶⁸⁸ TCIDso/ml. Three anti-Lawsonia monoclonal antibodies, namely 1 10:9, 287:6 and 1 13:2 were used in this study. The antibodies were described more in detail under section "Detailed Description".

Purification of Bacteria The bacteria in the supernatant were concentrated by centhfugation at 3700Og for 15 minutes. The pellet was re-suspended in Percoll and the solution was centrifuged at 3700Og for 1 hour. The white band containing the Lawsonia intracellularis was collected. This material was used as an immunogen for the preparation of the monoclonal antibodies.

Titration TCID50 was performed as described by Rivera et al (10). The virus were serially diluted and added to flasks containing confluent PK-15 cells. The titer was determined as the highest dilution in which full cytopahtic effect could be seen.

Haemaggluttination test was performed as described by Rivera et al (13). Supematants from organ suspensions were tested for HA activity using guinea pig red blood cells (RBC). The HA test was performed in microplates at 4C uning 0.5% RBC suspension in PBS pH 7.2 and 0.05% bovine serum albumine.

Field samples, PPV. A total of 10 PPV positive and 10 PPV negative samples were used throughout this study. The samples were randomly chosen and originated from a larger study where pregnant gilts were exposed to PPV. Tissue samples were collected from foetuses, and classified as PPV positive or negative by the use of different techniques such as Haemaggluttination (HA) test, Immunofluorescence (IF) assay and nested PCR. The animal experiments and the previous results are presented in Belak et al (1998 Vet Res).

Field samples, Lawsonia intracellularis . A total of 10 Lawsonia positive and 10 Lawsonia negative faecal samples collected at pig farms in Sweden were used. Most of the positive field samples were kindly supplied by Dr. Magdalena Forsberg at the University of Agricultural Sciences in Sweden. The faecal samples were classified as Lawsonia positive or negative by nested PCR as well as by an in-house Capture ELISA. The samples were prepared by making a 1/10 suspension in PBS. The supematants were then used for the PLA assay as well as the qPCR and Capture ELISA.

Preparation of monoclonal antibodies

Six Balb/C mice were immunized subcutaneously twice, six weeks apart with 70 μg of purified PPV virus or Lawsonia intracellularis bacteria that was mixed with an equal volume of Freunds complete or incomplete adjuvant. The animals were sacrificed 4 days after the second immunization. Spleen cells were collected and fused with the myeloma cell line SP 2/0 (x). Supernatants from the hybridoma cell clones were tested by an indirect ELISA using purified virus or bacteria as coating material. Bound antibodies were detected by a HRP anti-mouse conjugate(x) diluted 1 :1000. Positive clones were sub-cloned several times, re-tested by ELISA and stored at -135⁰C. Selected clones were cultured and the produced imAb's were purified as described previously by Johnstone ATR, Affinity chromatography and immunoprecipitation. 2^nd ed. 1990, London, UK, Blackwell Scientific. 30-48).

Biotinylation of the monoclonal antibodies The biotinylation was performed according to the instructions of the manufacturer

(Roche Diagnostics Corp, Germany). Briefly, D-Biotin-N-hydroxysuccinimide ester was mixed with the antibody in a 10fold molar excess and with a volume ratio of 1 :10.

The mixtures were incubated for 4 hours at room temperature under constant mixing.

Thereafter the biotinylated imAb^'s were extensively dialysed against Phosphate buffered Saline (PBS) to remove any unbound biotin.

Functionality test of the biotinylated monoclonal antibody

The biotinylated monoclonal antibodies were tested in commercially available PPV

ELISA kit (Svanovir® PPV-Ab test) and in an in-house Lawsonia capture ELISA according to the manual of the kit/in-house method respectively, but with the following exceptions. The biotinylated PPV/Lawsonia imAb^'s were used instead of the HRP conjugated imAb included in the standard test system. To visualise the binding of the biotinylated imAb an HRP labelled streptavidin (Dakopatts, Roskilde, Denmark) was used in a dilution of 1/5000. The plate was read in a Spectrophometer (Flow Laboratories, Irvine, UK) at 450nm.

Preparation of proximity probes

Proximity probes were prepared according to Gullberg et al. 2004. Thiol modified oligonucleotides were coupled to malemide-dehvatized streptavidin, creating streptavidin-oligonucleotide conjugates with free 3' and 5'ends respectively (5' STV : 5'P-TCG TGT CTA AAG TCC GTT ACC TTG ATT CCC CTA ACC CTC TTG AAA AAT TCG GCA TCG GTG A (SEQ ID NO:1 ) and 5' STV : CGC ATC GCC CTT GGA CTA CGA CTG ACG AAC CGC TTT GCC TGA CTG ATC GCT AAA TCG TG-3'OH (SEQ ID NO:2). The biotinylated monoclonal antibodies were combined with the STV -oligonucleotide conjugations as follows. The biotinylated antibodies were diluted in PBS with 1 % Bovine Serum Albumine (BSA) (Sigma, St Louis, MO, USA) to a final concentration of 3OnM. The antibodies were then reacted with the streptavidin- oligonculeotide conjugations in a 1 :1 ratio in a volume of 5 uL at room temperature for 1 hour. Thereafter the antibody-oligonucleotide probes were further diluted to a concentration of 1.2nM in a probe-dilution buffer (PBS, 1 % BSA, 16μg/ml sheared polyA bulk nucleic acid (Sigma), 1 mM D-biotin (Molecular Probes, Eugene, USA)), and stored at 4°C until usage.

Detection of PPV and Lawsonia intracellularis by Proximity ligation in solution phase

To perform the homogenous-phase assay, 1 μl of sample was incubated with 4μl mix of the pair of proximity probes, each diluted to a concentration of 24 pM in the probe- dilution buffer in optical PCR tubes (Applied Biosystems, Foster City, CA) at +37⁰C. After one hour of incubation, 45μl of a combined mix for ligation and amplification was added (5OmM KCI, 1 OmM Ths-HCI, pH 8.3, 3.15mM MgCI₂, 0.4 Weiss-units T4 DNA ligase (Fermentas, Hanover, MD, USA), 40OnM connector oligonucleotide (5'- TAC TTA GAC ACG ACA CGA TTT AGT TT -3' (SEQ ID NO:3)), 80μM ATP, 200 uM dNTPs, 100 nM primers (biomers.net GmbH) (forward: 5'- CAT CGC CCT TGG ACT ACG A -3' (SEQ ID NO:4); reverse: 5'- GGG AAT CAA GGT AAC GGA CTT TAG -3' (SEQ ID NO:5)), 100 nM TaqMan® MGB probe (5'fam- TGACGAACCGCTTTGCCTGACTGA-MGBNFQ-3'(SEQ ID NO:10)) (Applied Biosystems), and 1.5 units Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA)). After the addition of the combined mix the tubes were sealed with optical PCR lids (Applied Biosystems) and transferred to a real-time PCR instrument (Stratagene's MX 3000P, ABI 7700 or ABI 7000 from Applied Biosystems). The reactions were cycled at 95°C for 2 minutes, followed by 45 cycles at 95°C for 15 seconds and 60°C for 1 minute. The results are presented as Cr values, signal to noise where the number of ligations of the pairs of proximity probes occurred in the sample is divided by the number of ligations performed in the negative control, or as infectious doses where the data is correlated to a series of standards. Detection of viral and bacterial proteins by Proximity ligation in solid phase

Streptavidin coated PCR tubes (Roche Diagnostics GmbH, Mannheim, Germany) were coated with 30ng of the biotinylated capture antibody in a total volume of 50 uL for 1 hour at 37 ⁰C. After washes with PBS/0.05%Tween20 (Labkemi, Stockholm, Sweden), the tubes were blocked with an appropriate blocking solution for 1 hour at 37⁰C. The tubes used for PPV detection were blocked with PBS, 10% sheep sera and 10% sucrose After washes with PBS, 0.05% Tween, the sample, if necessary diluted in PBS/0.1 % BSA, was added in a volume of 50μl_, and incubated for one hour at +37⁰C. Unbound material was removed by washing. Thereafter a mix of the proximity probes (24 pM) was added and incubated for another one hour at +37⁰C. The washing step was repeated and 45μl of the combined ligation and PCR mix was added, and the real-time PCR was performed according the protocol described for the solution phase assay.

qPCR Porcine Parvovirus: qPCR analysis was performed on PPV infected samples, with primers used for traditional PCR as described by Kim et al. Samples were denatured for 10 minutes at 95C and subsequently analysed by qPCR.1 uL of each sample was analysed in a total volume of 5OuL, containing I xPCR buffer (5OmM KCI, 1 OmM Tris-HCI, pH 8.3 (Invitrogen)), 3.15 imM MgCI₂ (Invitrogen), 200 uM dNTPs, 10OnM primers (Biomers.net) (Fwd: AGC AAC AGC AAT TAG GCC AG (SEQ ID NO:6); Rev: GGT CCA CCA TTG GAG TAT TCA (SEQ ID NO:7)) 0.15x SYBR (FMC Byproducts, Rockland, ME USA) and 1.5 units of Platinum Taq Polymerase. The reaction was cycled 45 times (95C 15 sec, 55C 1 minute) with an initial denaturing step of 95C for 2 minutes. Organ samples were diluted 3Ox in PBS prior to analysis.

qPCR Lawsonia intracellularis :

A TaqMan assay for Lawsonia intracellularis detection and quantification has been developed and described by Lindecrona et al Samples were denatured for 10 minutes at 95C and subsequently analysed by qPCR. 1 uL of each sample, diluted 1/100 in PBS, was analysed in a total volume of 5OuL containing I xPCR buffer (5OmM KCI, 1 OmM Tris-HCI, pH 8.3 (Invitrogen)), 3.15 mM MgCI₂ (Invitrogen), 200 uM dNTPs, 10OnM primers (Biomers.net) (Fwd: 5'- GCG CGC GTA GGT GGT TAT AT -3' (SEQ ID NO:8); Rev: 5'- GCC ACC CTC TCC GAT ACT CA -3' (SEQ ID NO:9)) 0.15x SYBR(FMC Bioproducts) and 1.5 units of Platinum Taq Polymerase. The reaction was cycled 45 times (95 ⁰C 15 sec, 55 ⁰C 1 minute) with an initial denaturing step of 95C for 2 minutes.

RESULTS

Functionality test of the biotinylated monoclonal antibody

The imAb's were biotinylated and thereafter tested in their respective Elisa's to examine the level of successful biotinylation. It was found that the biotinylated imAb's could be diluted up to 10-12000 times and still obtaining a positive signal (data not shown).

Detection of PPV

The PPV cell culture of a known concentration was serially diluted in order analyzed in the presence of a negative field sample to expolore the sensitivity of the assay. As it can be seen in figure 3A, single copies of viral particles could be detected by the assay which is way beyond the detection limit of the capture ELISA used for comparison of protein detection, and equivalent to the detection limit of the PCR. 20 field samples (foetuses) were analyzed by solid-phase PLA, real-time PCR (figure 3B) and homogenous-phase PLA (data not shown). The samples have previously been classified as 10 PPV positive and 10 negative by different techniques (xx). The solid-phase PLA was chosen for the presentation of the results, as the limit of detecion in the solid-phase assay is lower than in the homogenous -phase PLA. Other benefits of the solid-phase assay include the washing steps, that allow the analysis of very complex samples containing substances that inhibit the PLA assay. The samples contained substances that inhibited the PLA reaction, All samples scored correctly positive or negative in PLA. The assay obtained a high sensitivity with low background noise of the negative samples (less than the signal obtained by 1 viral copy). The number of virus particles in the positive samples varied from 10,000 copies to 10,000,000 copies per 50 uL sample volume. The quantification was not found to be in correlation with the HA-titers, still all the samples showed the same level of positivity when compared within each test system. Detection of Lawsonia

As in the case of PPV, Lawsonia bacterial culture of a known concentration was serially diluted and analysed in the presence of a negative field sample in order to obtain the sensitivity of the test. The results are presented in figure 4A, single bacteria could be detected by the homogenous-phase PLA assay. The sensitivity of the Capture ELISA was much lower, clearly showing that Proximity ligation is superior to the ELISA for detection of Lawsonia. The qPCR used for DNA detection showed similar sensitivity as the PLA test.

20 field samples (faeces) were analysed with homogenous-phase PLA and with realtime PCR. The samples had previously been classified as positive (10) and negative (10) by an in-house capture ELISA and nested PCR (personal communication). Figure 4B shows that all samples scored correctly positive or negative in PLA with bacterial particles varying from 1 ,000 up to almost 100,000 in the positive samples and less than 1 copy in the negative samples, apart from 2 samples, that showed to be weakly positive too when analysed by qPCR. As in the case of PPV the particle number did not fully correlate with the results obtained from qPCR (differs by a factor of around 10) but as in the former case the level of positivity agreed in both tests.

Choice of reagents, PLA vs ELISA

It is of great importance to select imAb's (single, or in combination) with high affinity and specificity when setting up PLA reactions. (Gullberg et al Cytokine detection, Gullberg et al, sense of closeness). The source of antibodies seems to be important to the outcome. Ascites fluid from immunized mice as a source of antibodies for PLA has proven to be a poorer choice than monoclonal antibodies produced by hybridoma cell lines. In order to select the best candidates for the Lawsonia Proximity ligation we used existing information from the selection of imAbs for the in-house capture ELISA. We found that the most promising imAb's for the capture ELISA were also the best choice for PLA. These imAb's obtained the highest specific signal in correlation to the background noise, that can be translated into high sensitivity, in both test systems. This is illustrated in figure 5B. Combining antibodies for higher sensitivity and specificity

Combining different antibodies has two major advantages for the Proximity ligation assay, increased specificity and sensitivity. By requiring two or more binding events, the specificity of the assay increases as the number of specific epitopes that have to be recognized on the target is increased. Combining different antibodies for protein detection by PLA has an other benefit, namely an increased sensitivity. More required binding events lowers the likelihood of non-specific probe ligation which results in a lower background signal, and thereby an elevated sensitivity. As shown in figure 5A, series of Lawsonia intracellularis standards were analysed by PLA performed with one, two or three imAb's. The PLA's with one and two antibodies we carried out in a homogenous-phase assay, and the PLA with three antibodies was performed in a solid-phase setup with one capture antibody, and two different antibodies for detection. It is clearly shown that the sensitivity of the assay is enhanced when using two or three imAb's. Despite the good results with three imAbs we decided to perform the experiments on Lawsonia with only two imAb's as these two imAb's were available in larger amounts which was necessary for the study.

Claims

Claims:

1 . A method for detecting the presence of a microorganism in a sample, comprising the steps: (a) Incubating a sample that comprises a microorganism with two antibody- oligonucleotide probes, wherein the first and the second antibody of the two antibody-oligonucleotide probes specifically recognize and bind to said microorganism, and wherein one of oligonucleotides of the first and the second antibody-oligonucleotide probes comprises a free 5' end and wherein the other oligonucleotide of the two antibody-oligonucleotide probes comprises a free 3' end;

(b) Incubating the mixture of step a) with a mixture that comprises: i. a connector oligonucleotide, wherein the 5' end of said connector oligonucleotide overlaps and hybridize with the free 3' end of one oligonucleotide of the two antibody-oligonucleotide probes and wherein the 3' end of said connector oligonucleotide overlaps and hybridize with the free 5' end of the second oligonucleotide of the antibody-oligonucleotide probes; ii. a DNA Ligase and a DNA Polymerase; iii. two oligonucleotides that bind to the hybrid consisting of the oligonucleotides of the of the two antibody-oligonucleotide probes and the connector olignucleotide;

(c) amplifying a nucleotide sequences that is generate by the hybrid consisting of the oligonucleotides of the two antibody-oligonucleotide probes and the connector olignucleotide;

(d) detecting the amplification product of step c)

2. The method according to claim 1 , characterized in thatihe steps a) and b) are performed simultaneously.

3. The method according to claim 1 or 2, characterized in that the sample comprising the microorganism is incubated with an immobilized antibody before step a), and wherein said immobilized antibody specifically recognizes and binds to said microorganism.

4. The method according to any one of claims 1 to 3, characterized in that

(a) the oligonucleotides of the antibody-oligonucleotide probes having the sequences of SEQ ID NO:1 and SEQ ID NO:2;

(b) the connector oligonucleotide having the sequence of SEQ ID NO:3; and (c) the olignucleotides for the amplification of the hybrid olignucleotide consisting of the oligonucleotides of the of the two antibody-oligonucleotide probes and the connector olignucleotide having the sequence of SEQ ID NO:4 and SEQ ID NO:5.

5. The method according to any one of claims 1 to 4, characterized in thatthe microorganismus is Lawsonia intracellularis or Porcine Parvovirus.

6. The method according to claim 5, characterized in thatthe microorganism is Lawsonia intracellularis .

7. The method according to claim 6, characterized in that antibodies used are antibody 301 :39, antibody 287:6, antibody 268:29, antibody 1 10:9, antibody 1 13:2 and/or antibody 268:18.

8. The method according to claim 6, characterized in thatthe antibodies that are used are antibody 287:6, antibody 110:9 and antibody 1 13:2.

9. The method according to claim 8, characterized in thatthe antibody 1 10:9 was used as immobilized antibody.