WO2007085459A2 - Procédé de diagnostic in vitro d'infections à chlamydophila pneumoniae - Google Patents

Procédé de diagnostic in vitro d'infections à chlamydophila pneumoniae Download PDF

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Publication number
WO2007085459A2
WO2007085459A2 PCT/EP2007/000650 EP2007000650W WO2007085459A2 WO 2007085459 A2 WO2007085459 A2 WO 2007085459A2 EP 2007000650 W EP2007000650 W EP 2007000650W WO 2007085459 A2 WO2007085459 A2 WO 2007085459A2
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WIPO (PCT)
Prior art keywords
proteins
derivatives
hydrolase
derived
phosphatase
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PCT/EP2007/000650
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German (de)
English (en)
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WO2007085459A3 (fr
Inventor
Stefan Russwurm
Dirk Osterloh
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Sirs-Lab Gmbh
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Priority to EP07703040A priority Critical patent/EP1974217A2/fr
Publication of WO2007085459A2 publication Critical patent/WO2007085459A2/fr
Publication of WO2007085459A3 publication Critical patent/WO2007085459A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56927—Chlamydia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/295—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Chlamydiales (o)

Definitions

  • the present invention relates to a method for in vitro diagnosis of a
  • Chlamydophila pneumoniae Chlamydia pneumoniae specific infection by means of the DnaJ and / or hydrolase / phosphatase homologous and / or derivative derived derivates thereof according to claim 1, the use of said biomarkers for diagnostic and therapeutic purposes according to claims 11, 12 and 13, a kit for in vitro diagnosis of a
  • the invention relates to a method for the in vitro diagnosis of a Chlamydophila pneumoniae specific infection using the proteins having the amino acid sequences according to SEQ ID 1 and SEQ ID 2 as biomarkers.
  • Chlamydophila pneumoniae and Chlamydia pneumoniae is used below only the term Chlamydia pneumoniae.
  • Chlamydia are very small Gram-negative, obligate intracellular bacteria. There are nine different species today. To date, three human pathogenic Chlamydia species have been described: Chlamydia trachomatis, Chlamydophila pneumoniae and Chlamydophila psittaci. Chlamydia are bacteria that multiply only in the interior of eukaryotic cells. They go through a development cycle that begins with the incorporation of so-called infectious elementary bodies (extracellular form of chlamydia) into a phagosome of a host cell. The Chlamydia are not killed, but the organelle in such a way that it serves as a niche for propagation.
  • the dividable stages are called reticulate bodies, they revert to elementary bodies when the cell threatens to burst under the pressure of bacterial load.
  • the elementary bodies are released into the environment and infect new cells.
  • persistence the chlamydia remain as reticulate bodies within the cell without continuing growth and lysing the cell.
  • Chlamydia pneumoniae and Chlamydia trachomatis are the medically most important species. Almost every person is infected by Chlamydia pneumoniae during his lifetime. The pathogen causes diseases of the upper respiratory tract and about 10% of community-acquired pneumonia. Chlamydia trachomatis is the causative agent of Chlamydia pneumoniae and Chlamydia trachomatis as well as Chlamydophila psittaci. Almost every person is infected by Chlamydia pneumoniae during his lifetime. The pathogen causes diseases of the upper respiratory tract and about 10% of community-acquired pneumonia. Chlamydia trachomatis is the causative agent of
  • Chlamydia have been known as pathogens for almost 100 years now. Chlamydia infections are also prevalent worldwide. According to WHO estimates, the incidence of Chlamydia trachomatis infections was 92 million in 1999 (see eg www.medac.de/fachnik/diagnostik/chlamydienserologie.php). Chlamydia colonize the mucous membranes of the eye and urogenital tract (C. trachomatis) as well as the respiratory tract (Chlamydophila pneumoniae, Chlamydophila psittaci). Chlamydia can also be taken up by cells of the mononuclear system and thus spread throughout the organism. Viable pathogens of C. trachomatis and C. pneumoniae have also been demonstrated in joints synovia.
  • Chlamydia trachomatis may cause trachoma (serotype A-C), lymphogranuloma venereum (serotype L1-L3) and non-specific infections of the urogenital tract (serotype D-K).
  • serotype A-C trachoma
  • L1-L3 lymphogranuloma venereum
  • D-K non-specific infections of the urogenital tract
  • Chlamydophila pneumoniae causes usually easily flowing, but often protracted infections of the respiratory organs. After an untreated respiratory disease, tedious and severe extrapulmonary diseases often follow.
  • the clinical picture of infection with Chlamydophila pneumoniae includes: atypical pneumonia, sinusitis, pharyngitis, bronchitis, chronic obstructive
  • the direct detection of the pathogen requires a fresh infection.
  • Antigen detection is performed using direct or indirect immunofluorescence (IFT) tests and microscopic evaluation.
  • Species-specific monoclonal antibodies are used, which are fluorescently labeled and predominantly directed against the outer membrane proteins of the pathogen.
  • the method allows the examination of smear material, but also of non-invasively obtained sample material.
  • the technique is labor intensive and the evaluation of the test is subjective. The method therefore requires trained laboratory personnel.
  • EIAs enzyme immunoassays
  • LPS lipopolysaccharide
  • IFT enzyme immunoassays
  • the nucleic acid-based amplification techniques Polymerase Chain Reaction (PCR) and the Ligase Chain Reaction (LCR) allow a highly specific and highly sensitive detection of the DNA of bacterial pathogens.
  • PCR Polymerase Chain Reaction
  • LCR Ligase Chain Reaction
  • the method allows pathogen detection in a variety of different clinical samples. Theoretically, a single elementary body can be detected (sensitivity ⁇ 1 inclusion forming unit (IFU)).
  • IFU inclusion forming unit
  • Serological tests For the detection of chronic infections, as they are characteristic of these pathogens, serological tests are given preference. Chlamydia are often no longer detectable at the portal of entry very soon after the primary infection and have retreated to deeper areas of the respiratory or genitourinary tract where they can persist. The bacteria thus removed from direct detection can only be detected indirectly by serological methods. Serological tests are also used to validate the results of direct detection procedures. Serology also plays an important role in the therapy control of antibiotic treatments.
  • MIF microimmunofluorescence test
  • the MIF is the only serological test in addition to a species-specific Chlamydia detection also allows for simultaneous differentiation of the detected immunoglobulins into individual isotypes (IgG, IgA and IgM).
  • the specificity of the test is ensured by the use of purified elementary bodies of the human pathogenic Chlamydia species.
  • the test format involves a fixation of the purified elementary bodies on a slide. The slide is incubated with the stepwise diluted patient serum and the bound immunoglobulins are detected with a fluorescently labeled secondary antibody.
  • the test is not automatable, the evaluation of the test is subjective and requires appropriately trained laboratory personnel. In addition, cross-reactivities can not be ruled out. Standardization of the method has not yet been achieved.
  • species-specific chlamydial antibody ELISAs are the method of choice. These methods use synthethical or highly purified peptides or proteins as antigens. For this purpose, epitopes from immunodominant proteins of the respective species, typically proteins of the outer cell wall in question. So far, epitopes of the proteins MOMP (Major Outer Membrane Protein), Omp2 (a cysteine-rich protein of the outer cell membrane) and HSP60 (Heat Shock
  • Antibody ELISA tests also allow differentiation of the detected immunoglobulins into the individual isotypes. Antibody ELISAs are automatable, reproducible and can be objectively evaluated.
  • Chlamydia infections are highly clinically relevant. Therefore, there is an urgent need for the development of sensitive and species-specific methods for the reliable detection of Chlamydia-related infections in order to be timely
  • a kit according to claim 14 solves the problem as well.
  • Another solution to the above problem is a pharmaceutical composition according to claim 18.
  • the invention relates to a method for in vitro diagnosis of a Chlamydia pneumoniae specific infection based on an in vitro determination of the presence of at least one of the biomarkers DnaJ and hydrolase / phosphatase homologous and / or at least one directed against these proteins and / or derived derivatives antibodies in a patient sample.
  • the invention further enables new detection possibilities based on the use of at least one of the biomarkers DnaJ and Hydrolase / phosphatase homologous and / or at least one directed against these proteins and / or derived derivatives antibody.
  • the invention forms the basis for the development of new therapeutic agents against infections with Chlamydia pneumoniae.
  • the invention describes a method for the diagnosis of a Chlamydia pneumoniae specific infection, wherein in a human sample the presence of at least one of the biomarkers DnaJ and hydrolase / phosphatase is homologous and / or at least one directed against these proteins and / or derivatives derived therefrom Antibodies and derivatives derived therefrom and concluded on the basis of the result on the presence and / or the therapeutic success of a Chlamydia pneumoniae specific infection.
  • a Chlamydia pneumoniae specific infection may be inferred if the presence of at least one of the biomarkers or derivatives derived therefrom and / or at least one antibody directed against these proteins and / or derivatives thereof is present in infected patients.
  • the presence of at least one of the biomarkers DnaJ and hydrolase / phosphatase homologous and / or derived derivatives indicate the presence of the pathogen Chlamydia pneumoniae.
  • the biomarkers DnaJ and hydrolase / phosphatase have homologous amino acid sequences according to Seq-ID. 1 or Seq-ID. 2 on.
  • derivatives will be understood to include any modifications of the biomarkers, as well as peptides or nucleic acids that may be derived from the biomarkers
  • Such modications may be, for example, pre- or post-translational, such as glycosylation, proteolytic Cleavage of proteins, methylation, phosphorylation, sulfation, and prenylation.
  • biomarkers DnaJ and hydrolase / phosphatase homologous or derivatives derived therefrom and / or an antibody directed against these proteins and / or derivatives thereof is obtained from a sample which may be whole blood, blood components, urine, smears or tissue, certainly.
  • a preferred method is to determine the presence of at least one of the DnaJ and hydrolase / phosphatase biomarkers homologous to the plasma or serum of the patients.
  • Biomarkers DnaJ and hydrolase / phosphatase homologous or derivatives derived therefrom and / or an antibody directed against these proteins and / or derivatives derived therefrom various methods known to those skilled in the art are used. Immunodiagnostic methods are preferably used. In this case usually antibody assays are used, which against at least one of Biomarkers DnaJ and hydrolase / phosphatase homologous or derivates derived therefrom and / or an antibody directed against these proteins and / or derivatives derived antibodies directed antibodies include. Examples of such measuring methods are ELISA, Western blot, immunoprecipitation or FACS analyzes.
  • biomarkers DnaJ and hydrolase / phosphatase homologous or derivatives derived therefrom and / or an antibody directed against these proteins and / or derivatives derived therefrom also parallel determination of other biomarkers Diagnose a Chlamydia pneumoniae specific infection.
  • additional biomarkers may represent additional protein markers which are determined by protein chips or immunochromatographic measuring devices.
  • the methods according to the invention can be evaluated with computer-assisted evaluation units known to the person skilled in the art.
  • the measured values can be the presence or absence of at least one of the biomarkers DnaJ and hydrolase / phosphatase homologous derivatives derived therefrom and / or an antibody directed against these proteins and / or derived derivatives directly or via a converted signal as input to the evaluation unit Serve, processed therein data can be displayed via a graphical or other representation for evaluation.
  • the invention also encompasses the use of the biomarkers or derivatives derived therefrom for the recovery of antibodies which are homologous to these biomarkers DnaJ and hydrolase / phosphatase and / or derivatives derived therefrom.
  • derived derivatives are for example (mono, bi, tri) Fab fragments, Fv fragments, SFv (single chain antibodies), di-, tri- or tetrabodies.
  • the antibodies may be monoclonal or polyclonal antibodies.
  • Such antibodies and / or derivatives derived therefrom can be obtained by various methods known to those skilled in the art, such as hybridoma technology or recombinant methods such as phage display technology.
  • the antibodies obtained on this basis can be used in diagnostic test systems or for therapeutic use. Such therapeutic use may be accomplished, for example, by use of such antibodies and / or derivatives derived therefrom in prophylactic and / or therapeutic vaccinations, injection or gene therapy.
  • a preferred therapeutic use of antibodies thus obtained and / or derivatives derived therefrom are infectious diseases, inflammatory diseases or use in controlled gene therapy, e.g. TET-ON / TET-OFF system.
  • kits for diagnosis, a Chlamydia pneumoniae specific infection may contain at least one of the biomarkers DnaJ and hydrolase / phosphatase homologous and / or components which homologous recognize at least one of the biomarkers DnaJ and hydrolase / phosphatase and / or its derivatives specifically.
  • Such components may be, for example, antibodies, antibody fragments, nucleic acids, peptide nucleotides or aptamers.
  • kits may contain reagents needed for sample preparation or detection.
  • Such reagents may be, for example, dyes, enzymes, primers, radioactive substances, buffers or carrier materials.
  • the kit is part of a device which performs homologous semi-automatic or fully automatic homologous different steps of the determination of at least one of the biomarkers DnaJ and hydrolase / phosphatase.
  • Such devices may be designed to perform the processing of the patient samples in vitro semi-automatically or fully automatically, for example, the separation of the serum of residual blood components, the dilution of the blood sample, etc., and the metrologically determined content of the biomarkers in these patient samples via an appropriate Display is displayed numerically or graphically.
  • the invention further encompasses the use of the biomarkers specific for Chlamydia pneumoniae infections and / or their derivatives or antibodies raised against these biomarkers in a pharmaceutical composition.
  • Such pharmaceutical compositions can be used, for example, as vaccines.
  • the embodiment describes the identification of novel Chlamydia pneumoniae specific immunogenic proteins in isolated elementary bodies by serological proteome analysis (SERPA), which are applicable to the methods and uses of the invention.
  • SERPA serological proteome analysis
  • the proteome of the purified elementary bodies was solubilized and the proteins separated by means of two-dimensional gel electrophoresis (2D electrophoresis). The separate proteins were transferred to a membrane and immobilized. The immobilized proteins were then contacted with the sera of two selected patients afflicted with a proven Chlamydia pneumoniae infection and the specific immune responses visualized. The immunogenic proteins were identified by mass spectrometry and a database search.
  • Chlamydia pneumoniae (strain TWAR 183).
  • Host cells stock BGM cells were grown in tissue culture flasks 250 ml and 550 ml with OptiMEM and 10% FCS, respectively; Seed density 1, 5x10 6 .
  • 48 tissue culture bottles 50 ml were applied.
  • As a parallel culture 4-10 tubes were inserted with inserted coverslip; 0.6 ml of cell suspension were seeded per tube.
  • tissue culture flasks 550 ml
  • the cell lawn was trypsinized and the cell pellet was taken up in 500 ml OptiMEM + 10% FCS. Of these, 8 ml each were added to the 50 ml tissue culture flask. The incubation was carried out in the incubator at 37 ° C and 5% CO 2 for about 3 days. After the cell lawn was closed, the Chlamydia infection was performed.
  • the culture fluid was aspirated and into each tissue culture flask was added 6 ml of PBS with Chlamydophila pneumoniae (TWAR 183 strain, commercially available from LGC Protochem under No. ATCC VR-2282, [see http://www.lgcpromochem-atcc.com/common / catalog / numSearch / numResults.cfm, see also: GRAYSTON (JT), KUO (CC), CAMPBELL (LA) and WANG (SP): Chlamydia pneumoniae sp.nov for Chlamydia sp. strain TWAR, Int. J.
  • the parallel tubes were given with 400 ⁇ l of the Chlamydia batch to control the infection rate.
  • 2 tubes each with 10 .mu.l of Chlamydienansatzes for titre control were inoculated and also centrifuged. Chlamydophila pneumoniae was fixed and stained after 2 days. After centrifugation, the supernatant in the bottles was aspirated and replaced with panserin (6 ml / bottle).
  • the culture supernatant was sucked off (a part of the supernatant was stored as a rinse solution).
  • the cell lawn was gently scraped off and the chlamydia-containing liquid collected in a culture flask.
  • the scraped culture bottles were washed with repealed rinse solution and the wash then added to the chlamydia-containing suspension.
  • the culture flask was tightly closed, sealed with parafilm and the harvest was then disrupted in an ultrasonic bath for about 30 sec.
  • the chlamydial harvest was then filled into 2 centrifuge tubes (50 ml tubes) and centrifuged for 3 min at 4000 x g.
  • the supernatant was then filled with a peristaltic pump into tubes for the ultracentrifuge.
  • the cell pellet was washed once more in the used medium and then centrifuged again.
  • Reticulum (RK) by density gradient centrifugation was Visipaque 320 [Amersham Health (GE Healthcare)] used. The desired dilution for the gradients was done with PBS.
  • centrifuge tube was filled with 24 ml chlamydia suspension. Then it was underlaid with 2 ml 8%, 3 ml 15% and 15 ml 30% Visipaque. After welding the centrifuge tubes was centrifuged in the ultracentrifuge at 40 000 xg for 50 min at 8 0 C. After centrifugation, the tubes were cut open, the supernatants aspirated and discarded. The sediment was taken up in PBS, homogenized and placed in a new tube for ultracentrifugation.
  • a further sub-stratification of the preparation with Visipaque 6 ml of Chlamydiensuspension were underlaid with 8%, 15%, 30%, 40% and 47% Visipaque, the tubes are welded and centrifuged at 50,000 xg for 50 min at 10 0 C. After opening the tubes, the contents were pumped from the bottom. At the border between 47% and 40% Visipaque were the EC, while the RK rallied in the range of 36% Visipaque. The EC were collected in a new ultracentrifuge tube. This was filled with PBS, sealed and the preparation was xg for 50 min at 4 0 C again centrifuged at 30 000th The pelleted Ek were added to PBS, homogenized, aliquoted and frozen at -80 0 C.
  • the finished IPG strips were removed from the focusing chamber, briefly immersed in H 2 O and frozen at -2O 0 C.
  • SDS-PAGE Prior to SDS-PAGE, the thawed IPG strips were incubated 2 x 15 min in SDS-PAGE equilibration buffer I (6 M urea (Sigma U 6504), 0.375 M Tris-HCl pH 8.8, 2% (w / v) SDS, 20% (v / v) glycerol, 2% (w / v) DTT (Sigma D-9779)) and then immediately 2 x 15 min in SDS-PAGE equilibration buffer II (6 M urea (Sigma U 6504 ), 0.375M Tris-HCl pH 8.8, 2% (w / v) SDS, 20% (v / v) glycerol, 2.5% (w / v) iodoacetamide (1-1149)).
  • SDS-PAGE equilibration buffer II 6 M urea (Sigma U 6504 ), 0.375M Tris-HCl pH 8.8, 2% (w / v)
  • the IPG strips were then rinsed briefly in 1 ⁇ SDS-PAGE running buffer and immediately applied to the SDS-PAGE gels mounted in the electrophoresis chamber.
  • the IPG strips applied to the stacking gel were embedded with hot agarose in 1 ⁇ SDS running buffer.
  • Electrophoresis was carried out at a constant temperature of 16 0 C (Lauda WK 1400).
  • a programmable voltage source was used (Power Pac 1000, Bio-Rad): The samples entered the gel at 50V. Thereafter, the voltage was set to 100V. After reaching the separating gel limit 300V were set and the proteins were separated at this voltage. The electrophoresis was stopped after the added dye bromophenol blue had migrated out of the gel.
  • the finished gels were dyed or processed as follows: GeI 1 (700 ⁇ g protein): staining with Coomassie Brilliant Blue G250 (60 min on shaker). Decolorization with 20% methanol / 10% glacial acetic acid.
  • the finished SDS-PAGE gels were incubated for 60 minutes in a Coomassie staining solution (6.25 g Coomassie Brilliant Blue R 250, 900 ml methanol, 180 ml glacial acetic acid, H 2 O ad 21) (Schüttler, RT) and then with Decolorizing solution destained.
  • a Coomassie staining solution (6.25 g Coomassie Brilliant Blue R 250, 900 ml methanol, 180 ml glacial acetic acid, H 2 O ad 21) (Schüttler, RT) and then with Decolorizing solution destained.
  • the gels were incubated in 50% methanol, 5% glacial acetic acid for 20 min at RT on a shaker. This was followed by incubation of the gels in 50% methanol for 10 minutes.
  • the staining reaction was stopped by decanting the staining solution and immediately adding 5% glacial acetic acid.
  • the gels were stored in 1% glacial acetic acid at 4 ° C.
  • the separated proteins from gels 2 and 4 were transferred to PVDF membranes by electroblotting.
  • the gel was first equilibrated for 15 minutes in the transfer buffer (48 mM Tris, 39 mM glycine, 1.3 mM SDS, 20% (v / v) methanol).
  • the equilibrated SDS-GeI was then contacted with the previously methanol-activated PVDF membrane free of air bubbles.
  • the transfer was carried out with the aid of the Trans-Blot apparatus (Bio-Rad) for 1 h at 20 V. After the transfer, the membrane was removed from the blotting apparatus and dried in air. Immediately prior to immunochemical detection, the dried membrane was reactivated with methanol.
  • the unspecific binding sites were first saturated in TBST [Tris-Buffered Saline Tween-20] with 4% skimmed milk powder (1 h, shaker, RT). Subsequently, the saturated membranes were sequentially contacted with the patient sera.
  • serum 2 (SE 118715) was mixed with TBST with 2% skimmed milk powder at a dilution of 1: 5000 and the membranes were incubated for 1 h on the shaker at RT.
  • a TBST wash (5 min, 15 min and 40 min, shaker, RT)
  • the blot was incubated with the secondary antibody (Rabbit anti-human IgG (DAKO, Denmark) (dilution 1: 10,000 with TBST with 2% skimmed milk powder, 1 h, shaker, RT)
  • the serum IgGs bound to the immobilized chlamydial proteins were detected by chemoluminescence (ECL kit, Amersham) .
  • the emitted chemiluminescence was detected by X-ray film (Exposure time: 10 min, 60 min).
  • the membranes Prior to immunoassay with serum 1 (SE 170026), the membranes were first stripped of bound antibodies derived from serum 2 (SE 118715) (stripping buffer (0.2 M glycine-HCl, pH 2.5; , 05% TWEEN 20), 1 h, 65 ° C) and the membranes are then saturated again with TBST with 4% skimmed milk powder.
  • Immunoassay with serum 1 was carried out on the basis of 4 times the titer with a dilution of 1: 10,000 (TBST with 2% skimmed milk powder). The membranes were first incubated with the diluted serum for 1 h on the shaker at RT.
  • the membrane was incubated with the secondary antibody (Rabbit anti-human IgG (DAKO, Denmark) (dilution 1: 10,000 with TBST with 2% skimmed milk powder, 1 h, shaker, RT) After the final washing step (5 min, 15 min and 40 min, shaker, RT), the serum IgGs binding to the immunogenic proteins were analyzed with the aid of
  • the silver-stained SDS-GeI shows the positions of the immunogenic proteins. These were cut out of the gel and analyzed by mass spectrometry (FIG. 1).
  • MALDI-PMF MALDI Peptides Mass Fingerprint
  • the signals on the X-ray films were compared to the spot pattern of the stained gels. Spots whose coordinates coincided with the signals on the X-ray film were excised from the Coomassie or silver-stained gel, respectively, for identification by mass spectrometric analysis.
  • the protein spots cut out of the gel were first decolorized (1.51 methanol, 1, 1 1 glacial acetic acid, H 2 O ad 101) and the contained therein
  • STR Applied Biosystems
  • peptide mass fingerprints were compared with the database entries of the NCBI database.
  • the database search revealed the following
  • the peptide masses found were compared with the NCBI database. For identification, the highest levels were found for proteins from Chlamydophila pneumoniae (strain CWL029).
  • the calculated molecular weights and isoelectric points (17.5 kDa / 5.13 for hydrolase / phosphatase homologous and 42.6 kDa / 7.04 for DnaJ) correspond to the values estimated for the respective protein spots from the 2D gels.
  • the analyzes shown thus clearly show that the identified proteins DnaJ and hydrolase / phosphatase are homologous as specific markers for Chlamydia pneumoniae infections and thus applicable for the inventive method.

Abstract

La présente invention concerne un procédé de diagnostic in vitro d'une infection spécifique à Chlamydia pneumoniae, caractérisé en ce qu'il consiste à mettre en évidence la présence d'au moins une des protéines dnaJ et hydrolase/phosphatase homologue et/ou de dérivés de celles-ci et/ou d'au moins un anticorps dirigé contre ces protéines et/ou ces dérivés dans un échantillon d'un patient. L'invention concerne également une trousse pour mettre en oeuvre ce procédé de diagnostic in vitro, ainsi que l'utilisation prophylactique et thérapeutique des biomarqueurs susmentionnés.
PCT/EP2007/000650 2006-01-26 2007-01-25 Procédé de diagnostic in vitro d'infections à chlamydophila pneumoniae WO2007085459A2 (fr)

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DE200610003814 DE102006003814A1 (de) 2006-01-26 2006-01-26 Verfahren zur in-vitro-Diagnose von Chlamydophila pneumoniae Infektionen
DE102006003814.2 2006-01-26

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999027105A2 (fr) * 1997-11-21 1999-06-03 Genset Sequence genomique et polypeptides de chlamydia pneumoniae, leurs fragments et leurs utilisations, en particulier pour le diagnostic, la prevention ou le traitement d'une infection
WO2000046359A2 (fr) * 1999-02-05 2000-08-10 Neutec Pharma Plc Medicament
US6489122B1 (en) * 1994-09-20 2002-12-03 Hitachi Chemical Company, Ltd. Method of detecting anti-Chlamydia pneumoniae antibody using Chlamydia pneumoniae-specific antigens

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6489122B1 (en) * 1994-09-20 2002-12-03 Hitachi Chemical Company, Ltd. Method of detecting anti-Chlamydia pneumoniae antibody using Chlamydia pneumoniae-specific antigens
WO1999027105A2 (fr) * 1997-11-21 1999-06-03 Genset Sequence genomique et polypeptides de chlamydia pneumoniae, leurs fragments et leurs utilisations, en particulier pour le diagnostic, la prevention ou le traitement d'une infection
WO2000046359A2 (fr) * 1999-02-05 2000-08-10 Neutec Pharma Plc Medicament

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIENDO M ET AL: "LIMITS OF THE MICROIMMUNOFLUORESCENCE TEST AND ADVANTAGES OF IMMUNOBLOTTING IN THE DIAGNOSIS OF CHLAMYDIOSIS" CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, Bd. 3, Nr. 6, November 1996 (1996-11), Seiten 706-709, XP000853580 ISSN: 1071-412X *
DATABASE UniProt [Online] EMBL-EBI; 5. Juli 2005 (2005-07-05), UNIPROT: "Hydrolase/phosphatase homolog" XP002444727 gefunden im EMBL-EBI accession no. Q9Z6Y9 Database accession no. Q9Z6Y9 *
KALMAN S ET AL: "COMPARATIVE GENOMES OF CHLAMYDIA PNEUMONIAE AND C. TRACHOMATIS" NATURE GENETICS, NEW YORK, NY, US, Bd. 21, Nr. 4, April 1999 (1999-04), Seiten 385-389, XP000856693 ISSN: 1061-4036 *
PEREZ MELGOSA M ET AL: "Isolation and characterization of a gene encoding a Chlamydia pneumoniae 76-kilodalton protein containing a species-specific epitope" EMBO JOURNAL, OXFORD UNIVERSITY PRESS, SURREY, GB, März 1994 (1994-03), XP002076845 ISSN: 0261-4189 *
See also references of EP1974217A2 *
VANDAHL B B ET AL: "PROTEOME ANALYSIS OF THE CHLAMYDIA PNEUMONIAE ELEMENTARY BODY" ELECTROPHORESIS, WILEY-VCH VERLAG, WEINHEIM, DE, Bd. 22, Nr. 6, April 2001 (2001-04), Seiten 1204-1223, XP008001234 ISSN: 0173-0835 *

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EP1974217A2 (fr) 2008-10-01
WO2007085459A3 (fr) 2007-10-11

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