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 [Aoki A et al. (1996) MoI Hum Reprod 2: 489-497]. 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].

The biologically active and circulating form of uteroglobin in human blood has been isolated from human blood filtrate using a technique established for large volumes of blood filtrate [Forssmann K et al. (1986) Klin Wochenschr 64: 1276- 1280; Forssmann WG et al. (1993) In: Yanaihara IM (ed), Peptide Chemistry 1992. Leiden, Escom, pp 553-557; Kuhn M et al. (1993) FEES Lett 318: 205-209; Schulz-Knappe P et al. (1996) J Exp Med 183: 295-299].

Various techniques to identify this peptide by chemical or immunochemical assays were developed. Surprisingly, 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.

Surprisingly, 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.

Preferably 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 is also within the scope of the present invention, said method 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.

Preferably 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. Even more preferably 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 according to the present invention 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 according to the invention 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.

Typically, 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. However, other body fluids are also suitable for use'^in the present invention. Furthermore tissue samples 'San be isolated using conventional methods known in the art and their uteroglobin content can be analyzed.

Preferably, 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].

Subsequently the uteroglobin content of the samples is determined using techniques as outlined above. The term "content" according to the invention comprises the uteroglobin concentration of a sample as well as its total amount in a sample.

Preferably 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. For example in 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). For example in seminal fluid, 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 1 Immunocytochemistry of uteroglobin expression in endometrial biopsies at different dilutions and in relation to progesterone levels in chlamydia infected patients with fertility problems. Significant increase of uteroglobin expression in chlamydia infected patients.

Figure 2 Uteroglobin immunohistochemistry showing the positive reaction after 24 hours inoculation.

Figure 3 Uteroglobin immunohistochemistry showing the positive reaction after 30 days inoculation.

Figure 4 Significant increase of uteroglobin content in seminal fluid of male patients with Chlamydia infections.

Figure 5 Strict correlation of PX(+) (Klebsiella pneumoniae positive) cells and uteroglobin in seminal fluid. Figure 6 Increase of prostate binding protein (PBP) identical member of the uteroglobin family in prostate gland infected with E. coli.

Figure 7 Uteroglobin as a marker of the functional activity of the respiratory system in induced sputum of asthma patients and smokers.

Figure 8 Same as Figure 5, asthmatic patients suffering from chronic asthma and chronic obstructive respiratory disease after treatment.

Figure 9 Endometrial uteroglobin in patients related to chlamydia IgG antibodies.

Figure 10 Mass spectrometric analysis of purified uteroglobin by MALDI-TOF MS.

Example 1

Preparation of antibodies against uteroglobin

Whereas several routine methods of preparing antibodies have been adapted, surprisingly the production of monoclonal antibodies using native uteroglobin or synthetic fractions thereof is very effective. 10 NZW X NZB mice were immunized during 70 days using 6 injections of uteroglobin fraction 48/989420 Pool 2 from hemofiltrate for each mouse, resulting in immunization. The popliteal lymphnodes were recovered and then fusioned using the PEG method with X63-Ag8.653. The seed of the fusion was effected using 24 well plates to which mouse peritoneal macrophages were transferred 2 days before. The screening of the hybridomas was carried out with a direct ELISA using 200 ng antigen per well. 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. Also 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. (1998) Cell Biology: A Laboratory Handbook, Second Edition, VoI 2, pp 398-403]. For the production of antibodies with the synthetic fragments, the method of multiple antigen peptides (MAP) published earlier by Tarn JP et al. [Tarn JP et al. (1988) Proc Natl Acad Sci USA 85: 5409-5413] was mainly used for various regions of the molecules, namely the link region of the elongated part of the dimeric molecule, which provides a further possibility of adapting the assay by using the monoclonal antibodies with polyclonal antibodies for a sandwich assay.

Example 2

Immunochemical test for uteroglobin

An ELISA technique for uteroglobin was developed for serum, biological fluids and tissues. For semen the samples are diluted with saline and spun down to remove sperm as well as other cells and debris. Induced sputum in patients with respiratory diseases is obtained with nebulization of increasing concentrations of saline solutions and the samples are centrifuged to separate cells and debris.

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+H2O2). 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).

0 Example 3 Mass spectrometric determination of purified uteroglobin by MALDI-TOF MS Sample solutions were spotted on a stainless steel sample plate according to the dried-droplet technique using equal volumes (1 μl) of sample and matrix solution. 5 The matrix compound α-cyano-4-hydroxycinnamic acid was dissolved in 50% (v/v) ACN/0.05% (v/v) TFA in a concentration of 5 mg/ml. MALDI-TOF MS measurements were performed in the linear positive ion mode using a Voyager DE-Pro instrument (1.2 m flight tube, 337 nm laser, Applied Biosystems, Darmstadt, Germany) supported by the accompanying Biospectrometry 0 Workstation 5.1 software for controlling and the Data Explorer 4.0 program for -5f data analysis. Internal calibration was carried out with lysozyme (from chicken egg white; Sigma, St. Louis, MO, USA). The molecular weight of uteroglobin was determined to be 15,858 ± 2 Da corresponding to the bi-oxidized form.

5 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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