EP2277051A1 - Method for detecting the existence of renal calculi and/or inflammation of the excretory urinary tracts - Google Patents

Method for detecting the existence of renal calculi and/or inflammation of the excretory urinary tracts

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Publication number
EP2277051A1
EP2277051A1 EP09741736A EP09741736A EP2277051A1 EP 2277051 A1 EP2277051 A1 EP 2277051A1 EP 09741736 A EP09741736 A EP 09741736A EP 09741736 A EP09741736 A EP 09741736A EP 2277051 A1 EP2277051 A1 EP 2277051A1
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detection
urine
tff2
urinary tract
detecting
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French (fr)
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Werner Hoffmann
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Otto Von Guericke Universitaet Magdeburg
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Otto Von Guericke Universitaet Magdeburg
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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
    • G01N33/68—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
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/345—Urinary calculi

Definitions

  • a method for detecting the presence of kidney stones and / or inflammations of the urinary tract according to the preamble of the claims. It is suitable for the detection of specific diseases.
  • DE 44 44 533 A1 discloses an apparatus and a method for carrying out a test for the detection of particles in the urine (test for determining the susceptibility of a subject for kidney stones).
  • DE 38 87 314 T2 discloses a method for detecting basic fetal proteins in urine and a suitable analytical kit.
  • DE 18 12 584 discloses a rapid and sensitive detection of bacteria in blood products, urine and other fluids.
  • Fucose such as gastric ulcers, liver cirrhosis and carcinomas in humans, are related. This procedure consists in a Comparison of the concentrations of free L-fucose with their normal value in a sample from the subject.
  • TFF detection is performed on biopsy-derived cells or in the blood by conventional bioanalytical detection methods, i. it is always necessary to proceed invasively in order to remove cells or blood of the affected patient.
  • TFF Plant Factor Fa mily
  • TFF1 5 TFF2 and TFF3 have been described so far.
  • the physiological occurrence of TFF1 (former names: pS3, BCEI, pNR-2, pNR-105) and TFF2 is mainly confined to the stomach, whereas TFF3 (formerly called Intestinal Trefoil Factor / TTF, hPl.B) is restricted to many mucous Epithelia is synthesized.
  • TFF2 (formerly known as pancreatic spasmolytic polypeptides / PSP, spasmolytic polypeptides / SP, human spasmolytic polypeptides / hSP) is a component of gastric juice and occurs in both a glycosylated and a non-glycosylated form.
  • gastric TFF2 is bound to muzzine (Kouznetsova et al., (2007) Cell Physiol Biochem 20: 899-908).
  • TFF3 Overexpression of TFF3 is known to be associated with prostate cancer.
  • TFF peptides are present only in very low concentrations.
  • TFF1 nor TFF2 nor TFF3 can be detected by Western blot analysis.
  • the invention has for its object to provide a method for the detection of diseases of the urinary tract, which is non-invasively feasible and thereby relatively quickly and inexpensively first hints, for. with regard to the presence of kidney stones and / or inflammation (e.g., after UTI) of the urinary tract, in order to prevent u.a. To clarify caused by inflammation microhematuria quickly and inexpensively.
  • TFF2 (and to a lesser extent TFF1 and sometimes also TFF3) is present in urine of kidney stone carriers and patients with UTIs in a significantly increased concentration compared to healthy individuals and is detectable.
  • the TFF2 concentration in HWI patients is increased compared to kidney stone carriers, making differential diagnosis possible.
  • TFF2 quantitative fluorescent FF2
  • ECL system chemiluminescence
  • TFF2 A corresponding immunoassay for TFF2 is known in the art (Vestergaard et al., (2004) Scand. J Clin. Lab., Invest., 64: 146-156).
  • the advantage of the biochemical method according to the invention is that it represents a cost-effective rapid test, the results of which provide a basis for decision with regard to possibly further required applications / examinations, which are often expensive and stressful for the patient. This is an economic use of z.Z. limited financial resources in the health sector, while at the same time avoiding unnecessary and stressful examinations.
  • 1 volume (eg 500 ⁇ L) of a urine sample (eg morning midstream urine) of a patient with suspected UTI is mixed with 4 volumes (eg 2000 ⁇ L) of cold acetone (-2O 0 C) and the proteins precipitated at -20 0 C for at least 2 hours , Then the precipitated proteins are centrifuged for 10 minutes at 4 ° C and 12,000xg. The supernatant is discarded; the pellet is air-dried and then taken up in 0.5% SDS (in half of the original urine volume, eg in 250 ⁇ L).
  • the proteins are transferred to a nitrocellulose membrane, fixed with 0.1% glutaraldehyde and counterstained in PBS with an affinity purified polyclonal antiserum
  • TFF2 e.g., anti-hTFF2-I, Jagla W, Wiede A, Dietzmann K, Rutkowski
  • TFF2 forms are evaluated separately.
  • the calibration can be done via recombinant TFF2.
  • Significantly increased TFF2 values compared to the control values of healthy persons are an indication of an inflammation, eg by UTI. 2nd embodiment
  • TFF2 concentration may also be a differential diagnosis to distinguish HWI or kidney stones.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention relates to methods for detecting diseases of the excretory urinary tracts. The aim of the invention is to provide a method for detecting the existence of diseases of the excretory urinary tracts, which can be carried out non-invasively, thus allowing first indications of disease, e.g. in relation to the existence of renal calculi and/or inflammation (e.g. after urinary tract infections) of the excretory urinary tracts, to be rapidly and cost-effectively detected, in order to resolve e.g. microhaematuria caused by inflammation rapidly and cost-effectively. To achieve this, the presence or concentration of TFF-peptides is determined in the urine.

Description

Verfahren zum Nachweis des Vorkommens von Nierensteinen und/oder Entzündungen der ableitenden Harnwege Method for detecting the presence of kidney stones and / or inflammation of the urinary tract
Verfahren zum Nachweis des Vorkommens von Nierensteinen und/oder Entzündungen der ableitenden Harnwege gemäß der Gattung der Patentansprüche. Es ist zur Detektion spezifischer Erkrankungen geeignet.A method for detecting the presence of kidney stones and / or inflammations of the urinary tract according to the preamble of the claims. It is suitable for the detection of specific diseases.
Verschiedene Erkrankungen der ableitenden Harnwege [bspw. verschiedene Nierensteine oder Harnwegsinfekte (HWI)] lassen sich zur Zeit nur mit relativ teuren Methoden und auch oft nicht sicher diagnostizieren (z.B. Ultraschall, Anlegen von Kulturen). Besonders bei Steinen ist eine sichere Diagnose oft problematisch und erfordert teure Geräte und sehr erfahrene Ärzte.Various diseases of the draining urinary tract [eg. various kidney stones or urinary tract infections (UTI)] can currently be diagnosed only with relatively expensive methods and also often not sure (for example, ultrasound, creating cultures). Especially with stones, a reliable diagnosis is often problematic and requires expensive equipment and very experienced doctors.
DE 696 19 416 T2 offenbart ein Verfahren und Reagenzien zum immunologischen Nachweis von Cortisol im UrinDE 696 19 416 T2 discloses a method and reagents for the immunological detection of urinary cortisol
Aus DE 695 23 707 T2 ist ein Verfahren zum Nachweis von Kreatinin und von in Urin enthaltenen Proteinen bekannt.From DE 695 23 707 T2 a method for detecting creatinine and proteins contained in urine is known.
DE 44 44 533 Al offenbart eine Vorrichtung und ein Verfahren zur Durchfuhrung eines Tests zum Nachweis von Partikeln im Urin (Test zur Bestimmung der Anfälligkeit einer Testperson für Nierensteine).DE 44 44 533 A1 discloses an apparatus and a method for carrying out a test for the detection of particles in the urine (test for determining the susceptibility of a subject for kidney stones).
Aus DE 38 87 314 T2 sind eine Methode zum Nachweis basischer fötaler Proteine im Urin und ein dafür geeigneter Analysesatz bekannt.DE 38 87 314 T2 discloses a method for detecting basic fetal proteins in urine and a suitable analytical kit.
DE 18 12 584 offenbart einen schnellen und empfindlichen Nachweis von Bakterien in Blutprodukten, Urin und anderen Flüssigkeiten.DE 18 12 584 discloses a rapid and sensitive detection of bacteria in blood products, urine and other fluids.
DE 38 38 718 Al offenbart ein Verfahren zum Aufspüren vonDE 38 38 718 A1 discloses a method for detecting
Krankheiten, die in Verbindung mit Stoffwechselabnormalitäten von L-Diseases associated with metabolic abnormalities of
Fucose, wie Magengeschwüren, Leberzirrhosen und Karzinomen in Menschen, in Verbindung stehen. Dieses Verfahren besteht in einem Vergleich der Konzentrationen an freier L-Fucose mit ihrem normalen Wert in einer Probe aus dem Subjekt.Fucose, such as gastric ulcers, liver cirrhosis and carcinomas in humans, are related. This procedure consists in a Comparison of the concentrations of free L-fucose with their normal value in a sample from the subject.
Die Publikation "Williams, G.R.; Wright, N.A.: Trefoil factor family domain peptides. Virchows Archiv (1997), Vol. 431, Nr. 5, Seiten 299- 304" offenbart die prognostische Bedeutung der pathologischen Expression von TFF-Peptiden bei verschiedenen Karzinomen, u.a. auch bei Tumoren der ableitenden Harnwege (speziell der Blase).The publication "Williams, GR; Wright, NA: Trefoil factor family domain peptides, Virchow's Archives (1997), Vol. 431, No. 5, pp. 299-304" discloses the prognostic significance of the pathological expression of TFF peptides in various carcinomas , among others also in tumors of the urinary tract (especially the bladder).
Die Publikation „Vestergaard, E. M.: Plasma Levels of Trefoil Factors are Increased in Patients with Advanced Prostate Cancer. Clinical Cancer Research (2006), Vol. 12, Nr. 3, Seiten 807 - 812" offenbart ein ELISA-Immunoassay zum TFF-Nachweis im Zusammenhang mit der Diagnose von Prostatakrebs.The publication "Vestergaard, E.M .: Plasma Levels of Trefoil Factors Are Increased in Patients with Advanced Prostate Cancer. Clinical Cancer Research (2006), Vol. 12, No. 3, pp. 807-812 discloses an ELISA immunoassay for TFF detection in connection with the diagnosis of prostate cancer.
Gemäß dieser beiden voran stehend aufgeführten Publikationen erfolgt der TFF-Nachweis an per Biopsy entnommenen Zellen oder im Blut vermittels herkömmlicher bioanalytischer Nachweismethoden, d.h. es muss immer invasiv vorgegangen werden, um Zellen oder Blut des betroffenen Patienten zu entnehmen.According to these two publications listed above, TFF detection is performed on biopsy-derived cells or in the blood by conventional bioanalytical detection methods, i. it is always necessary to proceed invasively in order to remove cells or blood of the affected patient.
TFF (Trefoil Factor Fa mily) -Peptide stellen eine Familie von luminalen Schutzpeptiden dar, die u.a. eine Schlüsselrolle für den Schutz, die Regeneration und die Reparatur muköser Epithelien spielen.TFF (Trefoil Factor Fa mily) peptides represent a family of luminal protective peptides, which i.a. play a key role in the protection, regeneration and repair of mucosal epithelia.
Beim Menschen wurden bis jetzt die Peptide TFFl 5 TFF2 und TFF3 beschrieben. Das physiologische Vorkommen von TFFl (frühere Namen: pS3, BCEI, pNR-2, pNR-105) und TFF2 ist vor allem auf den Magen beschränkt, während TFF3 (frühere Namen: Intestinal Trefoil Factor /TTF, hPl.B) von vielen mukösen Epithelien synthetisiert wird. So ist TFF2 (frühere Namen: Pancreatic Spasmolytic Polypeptide/PSP, Spasmolytic Polypeptide/SP, human Spasmolytic Polypeptide/hSP) ein Bestandteil des Magensafts und kommt da sowohl in einer glykosylierten als auch in einer nichtglykosylierten Form vor. Außerdem ist gastrales TFF2, im Unterschied zu TFFl, an Müzine gebunden (Kouznetsova et al. (2007) Cell. Physiol. Biochem. 20:899-908).In humans, the peptides TFF1 5 TFF2 and TFF3 have been described so far. The physiological occurrence of TFF1 (former names: pS3, BCEI, pNR-2, pNR-105) and TFF2 is mainly confined to the stomach, whereas TFF3 (formerly called Intestinal Trefoil Factor / TTF, hPl.B) is restricted to many mucous Epithelia is synthesized. For example, TFF2 (formerly known as pancreatic spasmolytic polypeptides / PSP, spasmolytic polypeptides / SP, human spasmolytic polypeptides / hSP) is a component of gastric juice and occurs in both a glycosylated and a non-glycosylated form. In addition, unlike TFFI, gastric TFF2 is bound to muzzine (Kouznetsova et al., (2007) Cell Physiol Biochem 20: 899-908).
Eine Überexpression von TFF3 ist im Zusammenhang mit Prostata- Krebs bekannt.Overexpression of TFF3 is known to be associated with prostate cancer.
Im normalen menschlichen Urin sind TFF-Peptide nur in sehr geringer Konzentration vorhanden. So sind in der Regel weder TFFl noch TFF2 noch TFF3 über Westernblot- Analysen nachweisbar.In normal human urine, TFF peptides are present only in very low concentrations. As a rule, neither TFF1 nor TFF2 nor TFF3 can be detected by Western blot analysis.
Der Erfindung liegt die Aufgabe zugrunde, ein Verfahren zum Nachweis von Erkrankungen der ableitenden Harnwege anzugeben, das nichtinvasiv durchführbar ist und dabei relativ schnell und kostengünstig erste Hinweise z.B. bezüglich des Vorkommens von Nierensteinen und/oder Entzündungen (z.B. nach HWI) der ableitenden Harnwege ermöglicht, um u.a. durch Entzündungen hervorgerufene Mikrohämaturien schnell und kostengünstig abzuklären.The invention has for its object to provide a method for the detection of diseases of the urinary tract, which is non-invasively feasible and thereby relatively quickly and inexpensively first hints, for. with regard to the presence of kidney stones and / or inflammation (e.g., after UTI) of the urinary tract, in order to prevent u.a. To clarify caused by inflammation microhematuria quickly and inexpensively.
Gemäß der Erfindung wird die Aufgabe durch die kennzeichnenden Merkmale des ersten Patentanspruchs gelöst. In den Unteransprüchen sind vorteilhafte Ausgestaltungen der Erfindungen angegeben.According to the invention the object is achieved by the characterizing features of the first claim. In the subclaims advantageous embodiments of the inventions are given.
Es wurde festgestellt, dass TFF2 (und in geringerem Maße auch TFFl sowie mitunter auch TFF3) im Urin von Nierensteinträgern und Patienten mit HWI gegenüber gesunden Personen in stark erhöhter Konzentration vorliegt und nachweisbar ist. Dabei ist die TFF2-Konzentration in HWI-Patienten gegenüber Nierensteinträgern erhöht, so dass dadurch eine Differentialdiagnostik möglicht ist.It has been found that TFF2 (and to a lesser extent TFF1 and sometimes also TFF3) is present in urine of kidney stone carriers and patients with UTIs in a significantly increased concentration compared to healthy individuals and is detectable. The TFF2 concentration in HWI patients is increased compared to kidney stone carriers, making differential diagnosis possible.
Gemäß der vorliegenden Erfindung erfolgt der semiquantitative Nachweis von TFF2 durch Westernblotanalysen von Urinproben nach Acetonfällung und nachfolgender Detektion mit Hilfe der Chemilumineszenz (ECL-System) sowie digitaler Auswertung gemäß dem Stand der Technik. - A -According to the present invention, semiquantitative detection of TFF2 is performed by Western blot analysis of urine samples after acetone precipitation and subsequent detection by chemiluminescence (ECL system) and prior art digital evaluation. - A -
Dies stellt eine Kombination von biochemischen Standardmethoden dar (siehe z.B. Kouznetsova et al. (2007) Cell. Physiol. Biochem. 20:899-908). Dadurch wird sichergestellt, dass nur glykosyliertes TFF2 in die Analyse einbezogen wird, das im Urin dieser Patienten vorliegt.This represents a combination of standard biochemical methods (see, e.g., Kouznetsova et al., (2007) Cell Physiol. Biochem. 20: 899-908). This ensures that only glycosylated TFF2 is included in the analysis that is in the urine of these patients.
Neben diesem hochspezifischen quantitativen Nachweisverfahren für TFF2 im Urin, können auch andere Detektionsmethoden im erfindungsgemäßen Verfahren angewendet werden, wie bspw. ELISA, Streifentest, RIA, IRMA oder verschiedene chromatographische Verfahren.In addition to this highly specific quantitative detection method for TFF2 in urine, other detection methods can also be used in the method according to the invention, such as ELISA, strip test, RIA, IRMA or various chromatographic methods.
Ein entsprechender Immunoassay für TFF2 gehört zum Stand der Technik (Vestergaard et al. (2004) Scand. J Clin. Lab. luvest. 64: 146-156).A corresponding immunoassay for TFF2 is known in the art (Vestergaard et al., (2004) Scand. J Clin. Lab., Invest., 64: 146-156).
Der Vorteil des erfindungsgemäßen, biochemischen Verfahrens besteht darin, dass es einen kostengünstigen Schnelltest darstellt, dessen Ergebnisse eine Entscheidungsgrundlage hinsichtlich ggf. weiterer erforderlicher Anwendungen / Untersuchungen, die oft teuer und für den Patienten belastend sind, liefern. Dadurch ist eine ökonomische Verwendung der z.Z. besonders begrenzten finanziellen Ressourcen auf dem Gesundheitssektor möglich, wobei gleichzeitig unnötige und für den Patienten belastende Untersuchungen vermieden werden können.The advantage of the biochemical method according to the invention is that it represents a cost-effective rapid test, the results of which provide a basis for decision with regard to possibly further required applications / examinations, which are often expensive and stressful for the patient. This is an economic use of z.Z. limited financial resources in the health sector, while at the same time avoiding unnecessary and stressful examinations.
Die Erfindung soll nachfolgend an Hand der Ausfuhrungsbeispiele näher erläutert werden, ohne dadurch auf diese beschränkt zu sein.The invention will be explained in more detail below with reference to the exemplary embodiments, without thereby being limited to these.
1. Ausfuhrungsbeispiel1st exemplary embodiment
(Bestimmung der TFF2-Konzentration im Urin von Patienten mit vermutetem HWI)(Determination of TFF2 concentration in urine of patients with suspected UTI)
1 Volumen (z.B. 500 μL) einer Urinprobe (z.B. morgendlicher Mittelstrahlurin) eines Patienten mit vermutetem HWI wird mit 4 Volumina (z.B. 2000 μL) kaltem Aceton (-2O0C) versetzt und die Proteine bei -200C für mindestens 2 Stunden gefällt. Dann werden die gefällten Proteine für 10 Minuten bei 4°C und 12.000xg abzentrifugiert. Der Überstand wird verworfen; das Pellet wird an der Luft getrocknet und dann in 0.5% SDS aufgenommen (in der Hälfte des ursprünglichen Urinvolumens, z.B. in 250 μL).1 volume (eg 500 μL) of a urine sample (eg morning midstream urine) of a patient with suspected UTI is mixed with 4 volumes (eg 2000 μL) of cold acetone (-2O 0 C) and the proteins precipitated at -20 0 C for at least 2 hours , Then the precipitated proteins are centrifuged for 10 minutes at 4 ° C and 12,000xg. The supernatant is discarded; the pellet is air-dried and then taken up in 0.5% SDS (in half of the original urine volume, eg in 250 μL).
Anschließend werden 15 μL dieser so vorbereiteten Probe mit 5 μL 4- fach Probenpuffer für die Gelelektrophorese versetzt, 4 Minuten im kochenden Wasserbad erhitzt und sofort einer SDS-Polyacrylamid- Gelektrophorese (SDS-PAGE; 15%) unterzogen. Alle nachfolgend aufgeführten Schritte erfolgen dann analog Kouznetsova et al. (Kouznetsova I, Laubinger W, Kaibacher H, Kalinski T5 Meyer F, Roessner A, Hoffmann W (2004) Biosynthesis of gastrokine-2 in the human gastric mucosa: restricted spatial expression along the antral gland axis and differential interaction with TFFl, TFF2 and mucins. Cell. Physiol Biochem. 20:899-908):Subsequently, 15 μL of this sample thus prepared are mixed with 5 μL 4-fold gel buffer gel samples, heated for 4 minutes in a boiling water bath and immediately subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 15%). All of the steps listed below are then carried out analogously to Kouznetsova et al. (Kouznetsova I, Laubinger W, Kaibacher H, Kalinski T 5 Meyer F, Roessner A, Hoffmann W (2004) Biosynthesis of gastrokin-2 in the human gastric mucosa: restricted spatial expression along the anterior gland axis and differential interaction with TFF1, TFF2 and mucins, Cell Physiol Biochem 20: 899-908):
Nach der SDS-PAGE werden die Proteine auf eine Nitrozellulosemembran übertragen, mit 0.1% Glutaraldehyd fixiert und in PBS mit einem affinitätsgereinigten polyklonalen Antiserum gegenAfter SDS-PAGE, the proteins are transferred to a nitrocellulose membrane, fixed with 0.1% glutaraldehyde and counterstained in PBS with an affinity purified polyclonal antiserum
TFF2 (z.B. anti-hTFF2-l; Jagla W, Wiede A, Dietzmann K, RutkowskiTFF2 (e.g., anti-hTFF2-I, Jagla W, Wiede A, Dietzmann K, Rutkowski
K, Hoffmann W (2000) Co-localization of TFF3 peptide and Oxytocin in the human hypothalamus. FASEB J. 14: 1126-1131) inkubiert. Die TFF2- enthaltenden Banden werden mit einem kommerziellen ECL-K, Hoffmann W (2000) Co-localization of TFF3 peptide and oxytocin in the human hypothalamus. FASEB J. 14: 1126-1131). The TFF2-containing bands are probed with a commercial ECL
Detektionssystem (z.B. GE Healthcare) sichtbar gemacht. Das Bild dieser Westernblotanalyse wird dann elektronisch gespeichertDetection system (e.g., GE Healthcare). The image of this western blot analysis is then stored electronically
(GeneGnome System, Syngene). Die semi-quantitative Auswertung der(GeneGnome System, Syngene). The semi-quantitative evaluation of
Banden erfolgt mit der GeneTools Gel-Analyse-Software (Syngene). Dabei können entweder die glykosylierten oder die nicht-glykosyliertenBands are made with the GeneTools gel analysis software (Syngene). In this case, either the glycosylated or the non-glycosylated
TFF2-Formen getrennt ausgewertet werden.TFF2 forms are evaluated separately.
Die Eichung kann über rekombinantes TFF2 erfolgen. Deutlich erhöhte TFF2-Werte gegenüber den Kontrollwerten gesunder Personen sind ein Hinweis auf eine Entzündung, z.B. durch HWI. 2. AusführungsbeispielThe calibration can be done via recombinant TFF2. Significantly increased TFF2 values compared to the control values of healthy persons are an indication of an inflammation, eg by UTI. 2nd embodiment
(Bestimmung der TFF2-Konzentration im Urin von Patienten mit Mikrohämaturie)(Determination of TFF2 concentration in the urine of patients with microhematuria)
10 mL einer Urinprobe (z.B. morgendlicher Mittelstrahlurin) eines Patienten mit ungeklärter Mikrohämaturie werden für 15 Minuten bei 5.000xg zentrifugiert und ein Aliquot des Überstands wird dann einem spezifischen ELISA zur quantitativen Bestimmung der TFF2- Konzentration unterzogen. Dabei werden entsprechende Verdünnungen entsprechend dem Protokoll von Vestergaard et al. (Vestergaard EM, Brynskov J5 Ejskjaer, Clausen JT, Thim L, Nexo E, Poulsen SS (2004) Immunoassays of human trefoil factors 1 and 2: measured on serum from patients with inflammatory bowel disease. Scand. J. Clin. Lab. luvest. 64:146-156) behandelt.10 mL of a urine sample (eg morning midstream urine) from a patient with unexplained microhematuria is centrifuged for 15 minutes at 5,000xg and an aliquot of the supernatant is then subjected to a specific ELISA to quantify the TFF2 concentration. Corresponding dilutions are made in accordance with the protocol of Vestergaard et al. (Vestergaard EM, Brynskov J 5 Ejskjaer, Clausen JT, Thim L, Nexo E, Poulsen SS (2004) Immunoassays of human trefoil factors 1 and 2: Measured on serum from patients with inflammatory bowel disease Scand. J. Clin. Luv. 64: 146-156).
Deutlich erhöhte TFF2- Werte gegenüber den Kontrollwerten gesunder Personen sind ein Hinweis auf eine Entzündung im Bereich der ableitenden Harnwege (z.B. durch HWI oder Nierensteine) und könnten die Mikrohämaturie erklären. Bei Vorliegen von Daten aus einem größeren Patientenkollektiv könnte in Zukunft über die Bestimmung derSignificantly increased levels of TFF2 compared to healthy controls are indicative of inflammation in the urinary tract (such as UTIs or kidney stones) and may explain microhematuria. In the future, if data from a larger group of patients could be found on the determination of
TFF2-Konzentration auch eine Differentialdiagnose zur Unterscheidung von HWI bzw. Nierensteinen möglich sein.TFF2 concentration may also be a differential diagnosis to distinguish HWI or kidney stones.
3. Ausführungsbeispiel3rd embodiment
[Bestimmung der TFF2-Konzentration im Urin von Patienten mit Nephrolithiasis nach extrakorporaler Stoßwellenlithotripsie (ESWL)] Die Behandlung von hochgradiger Nephrolithiasis erfolgt heute zunehmend durch extrakorporale Stoßwellenlithotripsie (ESWL). Dadurch werden die Steine zerkleinert und dann ausgespült. Allerdings kommt es dadurch auch zu Läsionen des Nierenepithels bzw. der ableitenden Harnwege sowie zu temporären Entzündungen. Deshalb wird in der Regel nach einer ESWL-Behandlung ein Anstieg der TFF2- Konzentration im Urin beobachtet. Dieser Anstieg spiegelt den Grad der Entzündung wider und erreicht normalerweise nach einigen Tagen wieder einen Basalwert. Mitunter kommt es aber auch zu Komplikationen durch nachfolgende Entzündungen. Durch wiederholte Messung des TFF2-Spiegels (z.B. im Abstand von 2 Tagen mit Hilfe eines ELISA; siehe 2. Ausführungsbeispiel) kann nun die Abheilung nach ESWL-Behandlung verfolgt werden und so bei Bedarf rechtzeitig einer Infektion vorgebeugt werden. Alle in der Beschreibung und den Ausfiihrungsbeispielen dargestellten Merkmale können einzeln oder in beliebiger Kombination miteinander erfindungswesentlich sein. [Determination of TFF2 Concentration in the Urine of Patients with Nephrolithiasis After Extracorporeal Shockwave Lithotripsy (ESWL)] Today, the treatment of high grade nephrolithiasis is increasingly due to extracorporeal shockwave lithotripsy (ESWL). As a result, the stones are crushed and then rinsed out. However, this also leads to lesions of the renal epithelium or the draining urinary tract and to temporary inflammation. Therefore, an increase in TFF2 concentration in the urine is usually observed after ESWL treatment. This increase reflects the degree of inflammation and usually returns to basal levels after a few days. Sometimes it comes to complications from subsequent inflammation. Repeated measurement of the TFF2 level (eg at intervals of 2 days with the help of an ELISA, see example 2) allows the follow-up of the healing after ESWL treatment to be prevented in good time if necessary. All features shown in the description and the exemplary embodiments can be essential to the invention individually or in any combination with one another.

Claims

Patentansprüche claims
1. Verfahren zum Nachweis von entzündlichen Erkrankungen der ableitenden Harnwege und/oder zum Nachweis von Nierensteinen bei dem die TFF-Peptid-Anwesenheit eines oder mehrerer TFF-1. A method for the detection of inflammatory diseases of the urinary tract and / or for the detection of kidney stones in which the TFF peptide presence of one or more TFF
Peptide (TFFl, TFF2, TFF3) im Urin durch Westernblot- Analyse, ELISA, Streifentest, RIA oder IRMA bestimmt wird.Peptides (TFF1, TFF2, TFF3) in urine are determined by Western blot analysis, ELISA, streak test, RIA or IRMA.
2. Verfahren zum Nachweis von entzündlichen Erkrankungen der ableitenden Harnwege und/oder zum Nachweis von Nierensteinen gemäß Anspruch 1, dadurch gekennzeichnet, dass die TFF2- Anwesenheit im Urin durch Westernblot-Analyse, ELISA, Streifentest, RIA oder IRMA bestimmt wird.2. A method for the detection of inflammatory diseases of the urinary tract and / or for the detection of kidney stones according to claim 1, characterized in that the TFF2 presence in the urine by Western blot analysis, ELISA, strip test, RIA or IRMA is determined.
3. Verfahren zum Nachweis von entzündlichen Erkrankungen der ableitenden Harnwege und/oder zum Nachweis von Nierensteinen gemäß Anspruch 1, dadurch gekennzeichnet, dass TFFl- und/ oder TFF3 -Anwesenheit im Urin durch Westernblot- Analyse, ELISA, Streifentest, RIA oder IRMA bestimmt wird.3. A method for the detection of inflammatory diseases of the urinary tract and / or for the detection of kidney stones according to claim 1, characterized in that TFFl and / or TFF3 presence in the urine by Western blot analysis, ELISA, strip test, RIA or IRMA is determined ,
4. Verfahren zum Nachweis von entzündlichen Erkrankungen der ableitenden Harnwege und/oder zum Nachweis von Nierensteinen bei dem die Konzentration von einem oder mehrer TFF-Peptide (TFFl, TFF2, TFF3) im Urin durch Westernblot-Analyse, ELISA, Streifentest, RIA oder IRMA bestimmt wird.4. A method for detecting urinary tract inflammatory diseases and / or for detecting kidney stones, wherein the concentration of one or more TFF peptides (TFFI, TFF2, TFF3) in urine by Western blot analysis, ELISA, streaking test, RIA or IRMA is determined.
5. Verfahren zum Nachweis von entzündlichen Erkrankungen der ableitenden Harnwege und/oder zum Nachweis von Nierensteinen gemäß Anspruch 4, dadurch gekennzeichnet, dass die TFF2- Konzentration im Urin durch Westernblot-Analyse, ELISA,5. A method for the detection of inflammatory diseases of the urinary tract and / or for the detection of kidney stones according to claim 4, characterized in that the TFF2 concentration in the urine by Western blot analysis, ELISA,
Streifentest, RIA oder IRMA bestimmt wird.Strip test, RIA or IRMA is determined.
6. Verfahren zum Nachweis von entzündlichen Erkrankungen der ableitenden Harnwege und/oder zum Nachweis von Nierensteinen gemäß Anspruch 4, dadurch gekennzeichnet, dass TFFl- und/oder TFF3- Konzentration im Urin durch Westernblot- Analyse, ELISA, Streifentest, RIA oder IRMA bestimmt wird. 6. A method for the detection of inflammatory diseases of the urinary tract and / or for the detection of kidney stones according to claim 4, characterized in that TFFl- and / or TFF3 concentration in urine is determined by Western blot analysis, ELISA, streak test, RIA or IRMA.
EP09741736A 2008-05-05 2009-04-28 Method for detecting the existence of renal calculi and/or inflammation of the excretory urinary tracts Ceased EP2277051A1 (en)

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