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WO2006003209A1 - Utilisation de l'utéroglobine comme marqueur dans les maladies oncologiques et infectieuses humaines - Google Patents

Utilisation de l'utéroglobine comme marqueur dans les maladies oncologiques et infectieuses humaines Download PDF

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Publication number
WO2006003209A1
WO2006003209A1 PCT/EP2005/053223 EP2005053223W WO2006003209A1 WO 2006003209 A1 WO2006003209 A1 WO 2006003209A1 EP 2005053223 W EP2005053223 W EP 2005053223W WO 2006003209 A1 WO2006003209 A1 WO 2006003209A1
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WIPO (PCT)
Prior art keywords
uteroglobin
diseases
content
samples
mammal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2005/053223
Other languages
English (en)
Inventor
Augustin Aoki
Wolf-Georg Forssmann
Ludger STÄNDKER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pharis Biotec GmbH
Original Assignee
Pharis Biotec GmbH
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Filing date
Publication date
Application filed by Pharis Biotec GmbH filed Critical Pharis Biotec GmbH
Publication of WO2006003209A1 publication Critical patent/WO2006003209A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4718—Lipocortins

Definitions

  • Uteroglobin is a 15,826 Da peptide in natural circulating form containing two chains of identical amino acid sequence of 70 residues which are arranged in an antiparallel dimer to the disulfide bonds of two cysteins in position 3 and 69
  • Uteroglobin is shown to be regulated by several hormonal mechanisms such as progesterone expression as described for the equine endometrium [Beier-Hellwig K et al. (1995) Reprod Dom Anim 30:295-298].
  • uteroglobin does not only exist in human plasma but also in all body fluids investigated so far.
  • Chlamydia are gram-negative bacteria which are intracellular ⁇ immobile and dependent on the reproduction in intact living host cells. As the cycle of reproduction occurs in membrane-bound intracellular vacuoles, chlamydia are distinct from most bacteria, particularly forming persistent infections rarely resulting in antibody formation. Furthermore developing forms which are metabolically active as well as resistant to antibiotics are difficult to detect. Thus, the detection of chlamydial infection or a marker assay for chlamydial disease is an important tool in infection biology of human diseases.
  • Chlamydia are known to occur in several forms. The most important pathogenic forms are Chlamydia trachomatis and Chlamydia pneumoniae, which are the most frequent germs of sexually transmitted urogenital infections and which are the prime causality of acquired female infertility. Furthermore, chlamydia are important in acquired pneumonia, coronary heart diseases, and vascular inflammations. Thus the development of a marker assay for chlamydial infection is an urgent necessity of medical prevention and medical care.
  • the problem to be solved by the present invention is to improve the quality of diagnosis of mammalian diseases, especially oncological and inflammatory diseases.
  • this problem is solved by using the content of native uteroglobin as a diagnostic marker for the detection of diseases selected from the group consisting of inflammatory diseases and oncological diseases in mammals according to claim 1.
  • the increase of uteroglobin content in samples such as e.g. plasma, urine, vaginal fluid, seminal fluid and others is directly correlated with diseases such as chlamydial infection or inflammation of the respiratory tract.
  • the content of uteroglobin in samples from a mammal is used as diagnostic marker for diseases of the male and female urogenital tract, the cardiovascular system, the respiratory system and the gastrointestinal tract as well as a marker for bacterial infections, especially infections with chlamydia.
  • the present inventiopt ⁇ «utilizes different chemical and immunochemical assays #5 well as different mass spectrometric techniques to detect uteroglobin.
  • a method for diagnosing diseases selected from the group consisting of inflammatory diseases and oncological diseases in a mammal comprising a) isolating samples from said mammal, b) determining the uteroglobin content in said sample and c) comparing the uteroglobin content with a healthy control.
  • the diseases to be diagnosed by the method according to the invention comprise diseases of the male and female urogenital tract, the cardiovascular system, the respiratory system and the gastrointestinal tract as well as bacterial infections.
  • the bacterial infection to be diagnosed by the method according to the invention is an infection with chlamydia bacteria.
  • the method according to the invention can be applied using chemical and immunochemical assays as well as mass spectrometric techniques.
  • Preferred chemical and immunochemical detection techniques use antibodies developed against uteroglobin and include radioimmunoassay, ELISA, immunocytochemistry and immunohistochemistry.
  • a semi-analytical procedure applying Dot Blot densitometric analysis both of samples in serum and biological samples as tissue homogenates can also be used.
  • Preferred mass spectrometric techniques comprise MALDI, coupled liquid chromatography/mass spectrometry and a combination of a separation technique like micro-HPLC or capillary zone electrophoresis coupled on-line to an ESI mass spectrometer.
  • samples of body fluids such as venous blood, bronchioalveolar lavage, vaginal and seminal fluid, plasma or urine are obtained using conventional methods known to a person skilled in the art.
  • body fluids such as venous blood, bronchioalveolar lavage, vaginal and seminal fluid, plasma or urine are obtained using conventional methods known to a person skilled in the art.
  • other body fluids are also suitable for use' ⁇ in the present invention.
  • tissue samples 'San be isolated using conventional methods known in the art and their uteroglobin content can be analyzed.
  • the samples to be analyzed are prepurified by techniques known in the art, e.g. ultrafiltration and reverse phase chromatography where the fractions containing uteroglobin appear as described in the publication of Aoki A et al. [Aoki A et al. (1996) MoI Hum Reprod 2: 489-497].
  • the uteroglobin content of the samples is determined using techniques as outlined above.
  • content according to the invention comprises the uteroglobin concentration of a sample as well as its total amount in a sample.
  • an uteroglobin content between 0.1 and 1000 ng per ml or per mg of total protein as measured by ELISA, dot blot, radioimmunoassay or quantitative mass spectrometry is the range of concentration which can be assumed in human body fluids or tissue extracts, e.g. plasma or seminal fluid.
  • a concentration of 5 - 15 ng uteroglobin per mg total protein is the concentration for healthy volunteers and for Chlamydia trachomatis negative careers, whereas a concentration of > 25 ng uteroglobin per mg total protein is indicative for a bacterial, especially chlamydial infection (Figure 4).
  • a concentration of 5 - 15 ng uteroglobin per mg total protein is the concentration for healthy volunteers and for Klebsiella pneumoniae negative careers, whereas a concentration of > 50 ng uteroglobin per mg total protein is indicative for a bacterial, especially Klebsiella pneumoniae infection (Figure 5).
  • Figure 5 Strict correlation of PX(+) (Klebsiella pneumoniae positive) cells and uteroglobin in seminal fluid.
  • Figure 6 Strict correlation of PX(+) (Klebsiella pneumoniae positive) cells and uteroglobin in seminal fluid.
  • PBP prostate binding protein
  • Uteroglobin as a marker of the functional activity of the respiratory system in induced sputum of asthma patients and smokers.
  • Endometrial uteroglobin in patients related to chlamydia IgG antibodies Endometrial uteroglobin in patients related to chlamydia IgG antibodies.
  • the subsequent cloning of the uteroglobin antibody producing hybridoma cells was carried out using 96 well plates. After repetitive tests, the direct ELISA revealed selected clones on 24 and further on 6 well plates which were being expanded and subsequently cryoconserved.
  • the monoclonal antibodies are useful for various immunochemical detection techniques including radioimmunoassay, ELISA, and immunohistochemistry.
  • the best results were obtained with the clones L14-7a-EI (42), L14-7a-F7 (46), L5- 18b-F4 (37) and LII-12b-A3, with LII-12b-A3 displaying superior quality.
  • Further experiments showed that the purification of polyclonal antibodies or of the supematants from LII-12b-A3 are of superior quality for the assay.
  • the assay was carried out with an increased lower limit of detection as described in Example 2.
  • the method of immunization was adapted to the description previously published [Niebuhr K et al.
  • the ELISA technique is performed using antibodies raised against human uteroglobin isolated and purified from hemofiltrate of renal patients submitted to dialysis.
  • a solid phase 96 well microplate is prepared with trapping antibodies.
  • Control and test sera are diluted to an appropriate dilution range and added to each well. After this, the antigen is added to each well. After one hour of incubation at 37°C with shaking, the plates are washed and detecting antibody substrate /chromogen is added. The plates are washed and conjugate at a dilution previously tested is added. The plates are incubated with shaking for 30 minutes at 37°C. Then the plates are washed three times with wash buffer and subsequently horseradish peroxidase conjugate is added for 30 min, followed by washing and 5 addition of the substrate/chromogen (OPD+H 2 O 2 ). The optical density values are determined in a plate reader at 492 nm wavelength. Immunocytochemistry and immunohistochemistry were carried out using published standard procedures (Elia J et al., Histochem. Cell. Biol. 2000, 113, 125-133).
  • Sample solutions were spotted on a stainless steel sample plate according to the dried-droplet technique using equal volumes (1 ⁇ l) of sample and matrix solution. 5
  • the matrix compound ⁇ -cyano-4-hydroxycinnamic acid was dissolved in 50%
  • Figure 10 shows (a) the purified uteroglobin (m/z 15,859: [M+H] + ; m/z 7931: [M+2H] 2+ ; m/z 5289: [M+3H] 3+ ) demonstrating its homogeneity and (b) the purified uteroglobin (m/z 15,859; 7931 and 5289) mixed with lysozyme as internal standard (m/z 14,306 as [M+ H] + ) for improved mass accuracy.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne l'utilisation d'utéroglobine comme marqueur diagnostique pour la détection de maladies choisies dans le groupe composé des maladies inflammatoires et oncologiques chez les mammifères. L'invention porte en outre sur un procédé qui permet de diagnostiquer une maladie choisie dans le groupe composé des maladies inflammatoires et oncologiques en mesurant la teneur en utéroglobine chez un mammifère, selon lequel: a) on isole des échantillons chez ledit mammifère; b) on mesure la teneur en utéroglobine desdits échantillons; et c) on compare la teneur en utéroglobine avec un témoin en bonne santé.
PCT/EP2005/053223 2004-07-06 2005-07-06 Utilisation de l'utéroglobine comme marqueur dans les maladies oncologiques et infectieuses humaines Ceased WO2006003209A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP04015867.7 2004-07-06
EP04015867 2004-07-06

Publications (1)

Publication Number Publication Date
WO2006003209A1 true WO2006003209A1 (fr) 2006-01-12

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PCT/EP2005/053223 Ceased WO2006003209A1 (fr) 2004-07-06 2005-07-06 Utilisation de l'utéroglobine comme marqueur dans les maladies oncologiques et infectieuses humaines

Country Status (1)

Country Link
WO (1) WO2006003209A1 (fr)

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
AOKI A ET AL: "Isolation of human uteroglobin from blood filtrate.", MOLECULAR HUMAN REPRODUCTION. JUL 1996, vol. 2, no. 7, July 1996 (1996-07-01), pages 489 - 497, XP008053437, ISSN: 1360-9947 *
BERNARD A ET AL: "Clara cell protein in serum and bronchoalveolar lavage", EUROPEAN RESPIRATORY JOURNAL, vol. 5, no. 10, 1992, pages 1231 - 1238, XP008053473, ISSN: 0903-1936 *
BROECKAERT F ET AL: "Clara cell secretory protein (CC16): Characteristics and perspectives as lung peripheral biomarker", CLINICAL AND EXPERIMENTAL ALLERGY, vol. 30, no. 4, April 2000 (2000-04-01), pages 469 - 475, XP002347708, ISSN: 0954-7894 *
GIOLDASSI X M ET AL: "Clara cell secretory protein: Determination of serum levels by an enzyme immunoassay and its importance as an indicator of bronchial asthma in children.", JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, vol. 34, no. 4, 1 March 2004 (2004-03-01), pages 823 - 826, XP002347711, ISSN: 0731-7085 *
HAYASHIDA SHINYA ET AL: "Regulation and function of CCSP during pulmonary Pseudomonas aeruginosa infection in vivo", AMERICAN JOURNAL OF PHYSIOLOGY, vol. 279, no. 3 Part 1 of 2, September 2000 (2000-09-01), pages L452 - L459, XP008053474, ISSN: 0002-9513 *
LENSMAR C ET AL: "Decreased pulmonary levels of the anti-inflammatory Clara cell 16 kDa protein after induction of airway inflammation in asthmatics", CMLS CELLULAR AND MOLECULAR LIFE SCIENCES, vol. 57, no. 6, June 2000 (2000-06-01), pages 976 - 981, XP002347710, ISSN: 1420-682X *
LINNOILA R I ET AL: "PERIPHERAL AIRWAY CELL MARKER EXPRESSION IN NON-SMALL CELL LUNG CARCINOMA. ASSOCIATION WITH DISTINCT CLINICOPATHOLOGIC FEATURES", AMERICAN JOURNAL OF CLINICAL PATHOLOGY, PHILADELPHIA, PA, US, vol. 97, no. 2, February 1992 (1992-02-01), pages 233 - 243, XP000946537, ISSN: 0002-9173 *
NORD MAGNUS ET AL: "Decreased serum and bronchoalveolar lavage levels of Clara cell secretory protein (CC16) is associated with bronchiolitis obliterans syndrome and airway neutrophilia in lung transplant recipients", TRANSPLANTATION (BALTIMORE), vol. 73, no. 8, 27 April 2002 (2002-04-27), pages 1264 - 1269, XP002347709, ISSN: 0041-1337 *
PEREZ ALZAA J A ET AL: "High level of expression of endometrial uteroglobin in patients with Chlamydia trachomatis IgG antibodies", HUMAN REPRODUCTION (OXFORD), vol. 15, no. Abstract Book 1, June 2000 (2000-06-01), & 16TH ANNUAL MEETING OF THE EUROPEAN SOCIETY OF HUMAN REPRODUCTION AND EMBRYOLOGY; BOLOGNA, ITALY; JUNE 25-28, 2000, pages 179, XP008053475, ISSN: 0268-1161 *
SIGNOR LUCA ET AL: "Two-dimensional electrophoresis protein profiling and identification in rat bronchoalveolar lavage fluid following allergen and endotoxin challenge", PROTEOMICS, vol. 4, no. 7, 1 July 2004 (2004-07-01), pages 2101 - 2110, XP008053483, ISSN: 1615-9853 *

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