EP1395827A2 - Utilisation d'isocitrate-deshydrogenase cytosolique nadp-dependante dans des fluides corporels et des selles, pour detecter une maladie maligne, et kit d'essai utilise a cet effet - Google Patents

Utilisation d'isocitrate-deshydrogenase cytosolique nadp-dependante dans des fluides corporels et des selles, pour detecter une maladie maligne, et kit d'essai utilise a cet effet

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Publication number
EP1395827A2
EP1395827A2 EP02745117A EP02745117A EP1395827A2 EP 1395827 A2 EP1395827 A2 EP 1395827A2 EP 02745117 A EP02745117 A EP 02745117A EP 02745117 A EP02745117 A EP 02745117A EP 1395827 A2 EP1395827 A2 EP 1395827A2
Authority
EP
European Patent Office
Prior art keywords
receptor
isocitrate dehydrogenase
test kit
cytosolic nadp
dependent isocitrate
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.)
Withdrawn
Application number
EP02745117A
Other languages
German (de)
English (en)
Inventor
Erich Eigenbrodt
Sybille Mazurek
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.)
ScheBo Biotech AG
Original Assignee
ScheBo Biotech AG
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by ScheBo Biotech AG filed Critical ScheBo Biotech AG
Publication of EP1395827A2 publication Critical patent/EP1395827A2/fr
Withdrawn legal-status Critical Current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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)

Definitions

  • the present invention relates to a detection of malignant processes or diseases in humans or animals and a test kit for performing the detection according to the invention. It also relates to the use of the test kit to determine the extent and degree of the disease and to determine a promising treatment regimen, in particular with chemotherapeutic agents.
  • Tumor markers are macromolecules or antigens that occur in blood, serum or other body fluids, and their change in concentration is related to the development and growth of an individual's malignant tumors.
  • An ideal tumor marker should have the following properties: high specificity, i.e. not detectable in benign diseases and healthy people; high sensitivity, i.e. he can in a high
  • Percentage of tumor patients are detected; - good correlation with the tumor stages or the
  • Tumor mass it should provide information about the metabolism of the tumor; its concentration should correlate well with tumor malignancy; Relationship to forecast; reliable predictive values.
  • test principles are not very specific, since they can be disturbed by a large number of influencing factors (false positive / false negative, e.g. due to non-compliance with urgently necessary dietary requirements on the part of the patient and from a number of medicinal products, as well as through excessive vitamin C administration ( e.g. in vegetables, fruit juices etc.) (Thomas, L., Labor und Diagnose, 5th edition, 1998).
  • a positive test for occult blood in the stool must be clarified until the source of the bleeding is located or the cause of the bleeding has been found. In any case, the clinical finding justifies a prompt further diagnostics to be carried out, for example by endoscopy, sonography, X-ray.
  • endoscopy, sonography, X-ray the presence of blood in the stool is not always due to a tumorous event and the other way around, the lack of blood in the stool does not mean that there is no tumor in the intestine.
  • Isocitrate dehydrogenase is an enzyme of the glutaminolysis metabolism and occurs in humans and animals in the form of two different isoenzymes, namely as cytosolic NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) and as mitochondrial NAD-dependent isocitrate dehydrogenase (EC 1.1.1.41).
  • cytosolic NADP-dependent isocitrate dehydrogenase EC 1.1.1.42
  • mitochondrial NAD-dependent isocitrate dehydrogenase EC 1.1.1.41
  • An overexpression of the NADP-dependent has already been found in histochemical studies on tumor tissues such as breast cancer
  • the tumor tissue had to be opened histologically, i.e. the cell structure of the tissue and the individual cells had to be destroyed so that the substances enclosed in the cell were released and made accessible for analysis.
  • Tumor tissues themselves are detectable, but are not suitable as tumor markers.
  • the present invention aims to continue
  • Another object of the invention is to provide an easy-to-carry out specific method or a test with which a neoplastic process in the gastrointestinal tract can be carried out particularly early and
  • the aim of the invention is in particular to detect a malignant process or tumor with respect to the problem of the so-called adenoma-carcinoma sequence in polyps (polyposis coli) before the submucosa.
  • .5 has infiltrated and has infiltrated neighboring tissue.
  • cytosolic NADP-dependent isocitrate dehydrogenase or parts thereof is determined qualitatively and / or quantitatively in a stool and / or body fluid sample $ 0.
  • the content of cytosolic NADP-dependent isocitrate dehydrogenase in body fluids and / or in $ 5 excrement, e.g. B. can be determined in the stool of tumor patients, the content of the degree of malignancy in the gastrointestinal tract. It is usually directly proportional to this.
  • the cytosolic NADP-dependent isocitrate dehydrogenase remains quantitatively detectable even in the case of intensely homogenized, longer-stored samples, for example when sending samples. This also applies if the sample has been extracted beforehand. A clear reaction can be obtained even if the body fluid samples or stool are very diluted. It was surprisingly found here that the detection takes place selectively even without prior extraction in the sample. However, the sample is preferably extracted. Such an extraction is carried out in particular with detergents such as CHAPS.
  • Suitable body fluids with which the method according to the invention can be carried out include, for example, plasma, serum, cerebrospinal fluid, ejaculate, saliva, sputum and / or urine.
  • the method according to the invention is particularly preferred using plasma and / or serum and
  • the marker enzyme according to the invention can be detected in the urine, but not in the serum, this shows that there is a malignant process in the bladder or kidney. So if it is detectable both in the urine and in the serum, then the tumor has already infiltrated neighboring tissue that is in contact with the bloodstream and the lymphatic system or has infiltrated into it. The same is true in the case of bronchial and lung cancer Sputum to and in the ejaculate on malignant processes of the prostate and testicle. This also applies to the cerebrospinal fluid. If only the marker in fluid is found here, this means that tissue in contact with the cerebrospinal fluid is affected, but not adjacent tissue.
  • the detection according to the invention and the associated test system provide a further general tumor marker which can be used to detect generally malignant neoplastic tumorous events in a living body without having to take tissue samples from the body to be examined in a surgical manner.
  • neoplastic processes or tumors can be recorded, such as, for example, esophageal, stomach and / or colon cancer, in particular colon tumors, breast cancer, ovarian cancer, uterine cancer and
  • Cervical carcinoma liver carcinoma, prostate and / or testicular cancer, kidney and / or bladder cancer, lung and / or bronchial carcinoma, in particular small-cell bronchial carcinoma and tumors of the nervous tissue, in particular brain tumors.
  • the detection according to the invention is combined with further tumor tests. According to the invention, it is preferred to use a method to detect the NADP-dependent isocitrate dehydrogenase
  • a further detection of malignancy that is to be carried out together with the method according to the invention is the detection of malate decarboxylase (EC1.1.1.40), specifically as described above in body fluids and in Chair.
  • malate decarboxylase EC1.1.1.40
  • the method according to the invention is particularly selective, it has been found that when the method is combined with one or both of the previously described proofs, the probability of false positive and false negative results is significantly reduced. In this way, the reliability of the detection according to the invention can be further increased.
  • Chemotherapy is accessible. This is particularly true if this is carried out together with the malate decarboxylase and / or the tumor M2 pyruvate kinase. It has been found that when the tumor markers mentioned above, namely tumor M2 pyruvate kinase, NADP-dependent isocitrate dehydrogenase and malate decarboxylase are present in the tumor cell, a detoxification of certain chemotherapeutic agents or cytostatics, such as cis-platinum, with other chemotherapeutic agents such as mitomycin being enhanced in their effect. It is thus possible with the detection 5 according to the invention to also select a corresponding chemotherapeutic agent with which the tumor can be combated without valuable time being lost due to lengthy trial and error in vivo until it is recognized whether a tumor is responding to the treatment.
  • the detection according to the invention is preferably carried out immunologically.
  • the marker enzyme is bound by means of a receptor, which is immobilized on a solid phase, and preferably after
  • .0 can be demonstrated quantitatively or qualitatively. Detection using other biosensors is also possible. Further detection is possible using mass spectroscopy, in particular using MALDI-TOF (matrix-assisted laser desorption / ionization flight time
  • the invention thus also relates to a device or a test kit for carrying out the detection according to the invention.
  • Such a test kit usually comprises at least one first receptor immobilized on a solid phase, which selectively binds the marker enzyme to be detected or the tumor marker. It also preferably includes
  • the & 5 at least one biosensor, which indicates the binding of the marker enzyme to be detected to the receptor.
  • the first receptor bound to the solid phase any agent binding the marker enzyme or tumor marker to be used is used.
  • the receptor is particularly preferably an antibody.
  • receptor or “antibody” is also intended to include those parts or fragments of receptors or antibodies which still mediate the necessary binding to cytosolic isocitrate dehydrogenase. It is also possible to use a single antibody instead
  • the antibodies used according to the invention preferably react selectively with the tumor marker to be detected and discriminate against other, also similar isoenzymes. In particular, they show no cross-reactions with other substances found in the samples.
  • the biosensor is a further receptor which has a detectable detection marker and which reacts with the tumor marker or marker enzyme bound to the solid phase. It is preferably an antibody. Detection markers of this type are known per se and are generally used in so-called immunassays, in particular in ELISA or other sandwich assays. These are in particular second antibodies which are bindable with the tumor marker enzyme such as isocitrate dehydrogenase, the detection being carried out via a further labeled third, in particular soluble antibody which is directed against the Fc part of the second soluble receptor and which bears a detectable label or again is coupled to a detectable molecule.
  • This Conjugate formation from two antibodies is intended to be included in the context of the present invention. The same applies to the binding of the receptor to the solid phase. This binding can also be carried out via solid-phase-coupled antibodies to which the Fc part of the receptor binds.
  • One of the two receptors preferably mediates the binding to a solid phase, so that a separation of the liquid phase containing the body fluid sample or stool and the solid phase is made possible, on which the cytosolic NADP-dependent isocitrate dehydrogenase by means of the receptor (first receptor ) is bound.
  • this solid phase is also part of the test kit.
  • the sample to be examined is then brought into contact with the preferably dissolved protein and bound to the solid phase by means of the receptor in a suitable buffer system.
  • another detectable labeled receptor (second receptor), e.g.
  • the test kit expediently contains reference material of known content of cytosolic NADP-dependent isocitrate dehydrogenase for quantitative determination.
  • the analyte concentration can in principle also be determined using biosensors, e.g. amperometric sensors, potentiometric, ion-selective potentiometric or photometric sensors, or also by means of semiconductor electrodes, such as field effect transistors (FET), chemosensitive field effect transistors (CHEMFET), suspended gate field effect transistors (SGFET) or ion sensitive field effect transistors.
  • biosensors e.g. amperometric sensors, potentiometric, ion-selective potentiometric or photometric sensors, or also by means of semiconductor electrodes, such as field effect transistors (FET), chemosensitive field effect transistors (CHEMFET), suspended gate field effect transistors (SGFET) or ion sensitive field effect transistors.
  • FET field effect transistors
  • CHEMFET chemosensitive field effect transistors
  • SGFET suspended gate field effect transistors
  • biosensors are summarized in E.A. H. Hall and G. Hummel in “Biosensors”, Springer Verlag Heidelberg, Germany, 1995. Further
  • ISFET ion-sensitive field effect transistors
  • optical detectors include by F. Aberl and H. Wolf in “Current Trends in Immune Sensing", Labor 2000, pp. 70-96 (1993).
  • the method according to the invention is also suitable for
  • Electrochemiluminescence is the process in which light generation occurs when a low voltage is applied to an electrode that triggers a cyclic redox reaction of a ruthenium metal ion (Bruno, G. (1997) Rec. Rp pp. 175-179; Williams R. (1996) Amer. Biotech., P. 26).
  • Antibodies which can be used according to the invention can, according to
  • Methods known in the art can be produced. Methods for producing specific monoclonal antibodies are well known to those skilled in the art.
  • the antigen in the present case the cytosolic isocitrate dehydrogenase or the malate decarbxylase or parts thereof, is used in a customarily purified form to produce antibodies.
  • the method described for the first time by Köhler and Milstein can be used for this purpose, modifications and further developments of these methods being known to the person skilled in the art. Production is not critical as long as specific antibodies are obtained, which can be determined by selection. The selection takes place for antibodies which specifically bind the cytosolic isocitrate dehydrogenase or parts thereof, but not one of the other isoenzymes of the isocitrate dehydrogenase.
  • the antibodies used in the detection or test according to the invention can be obtained using methods known per se.
  • the cytosolic NADP-dependent isocitrate dehydrogenase for example from colon tumors, human liver and breast tumors, isolated.
  • the purification of the isoenzyme consists of the following
  • Steps An ammonium sulfate precipitation, QAE Sephadex A-50, 2 '5' -ADP-Sepharose 4B and a Sephacryl-S300 chromatography as described by Oluyinka O. Irukera (dissertation at the Justus Liebig University in G funnelen , July 1998), in analogy to other NADH-dependent enzymes (such as malate decarboxylase).
  • the purified fraction gave a band after SDS gel electrophoresis.
  • a test animal is then immunized with the cytosolic NADP-dependent isocitrate dehydrogenase obtained in this way or with fragments thereof which have the corresponding epitopes, and the antibodies formed are isolated.
  • fragments used for immunization can e.g. originate from a protease digestion of the purified cytosolic NADP-dependent isocitrate dehydrogenase or consist of synthetic partial peptides thereof. The preparation of such partial peptides is known per se to the person skilled in the art. From the total sequence, e.g. with the aid of computer programs, partial sequences are selected which contain corresponding epitopes. These sequences are then tested for their usability for the production of specific antibodies.
  • Monoclonal antibodies are preferably produced by the method of Koehler, Milstein (Nature 256, 495-497 (1975).
  • BALB / c mice are immunized with isolated isocitrate dehydrogenase, for example, and the spleen cells of these animals are immunized with a myeloma cell line, for example PA I
  • the antibodies secreted in the ascites are tested for their specificity, for example in the ELISA or RIA (radio immunoassay) and isolated.
  • the antibodies thus obtained usually belong to the IgGl class.
  • the second receptor is preferably a soluble, labeled antibody or a soluble antibody which is bound to an enzyme, which in turn can be determined via a detection reaction.
  • the test kit is designed in such a way that it is suitable for a sandwich, ELISA, quartz crystal, microbalance or
  • Electrochemiluminescence test contains necessary reagents and / or devices.
  • the test kit preferably comprises an extractant for the sample to be examined, such as CHAPS.
  • an extractant for the sample to be examined such as CHAPS.
  • it preferably contains a reference standard for comparison or calibration of the test results.
  • the test according to the invention also includes an evaluation scale for determining or calibrating the size, the extent and / or the degree of the tumor or the malignant process.
  • the test contains a calibration or scale and / or table for determining and defining a therapy scheme with suitable, promising chemotherapeutic agents such as, for example, B. Cytostatics.
  • the table, scale or curve is designed so that the respective therapeutic agent to be used results directly from it.
  • test kit is designed as an array in which the isocitrate dehydrogenase, the malate decarboxylase and the tumor M2 pyruvate kinase can be detected side by side or also one after the other.
  • the test kit is in the form of a test strip in which the sample liquid passes through different areas in which the first and second receptors are in soluble form. It is preferred that the test kit is in the form of a test strip which has an application zone for the sample and has further sections which are designed such that a liquid sample applied in the sample application area travels to a detection zone in which the test result is read can be.
  • the different areas must support the migration of the sample, for example by capillary action.
  • the application zone is followed by an area in which a soluble labeled antibody is present which corresponds to the second receptor.
  • the first receptor is preferably already in immobilized form.
  • the first receptor can also be present both in a zone before and in a zone after the region and can be present there in soluble form.
  • a solid phase must then be present on the test kit, in which the first receptor binds to the solid phase and thereby a complex of the first receptor, isocitrate dehydrogenase and second receptor with the label or the detectable molecules is bound to the solid phase ,
  • the presence of the cytosolic NADP-dependent isocitrate dehydrogenase can then again be determined via the label, this label being able to be designed such that it can be recognized without further addition of substances, such as a gold label or a fluorescent label, or else can be designed such that the addition of further reagents enables the determination.
  • an enzyme substrate can be applied to the test strip in a second step, or in one separate area of the test strip, through which the sample liquid including the second receptor passes.
  • the test strip is arranged such that a filter is arranged between the application zone and the further areas of the test strip, which retains insoluble constituents of the sample.
  • a liquid-absorbing medium can be applied behind the detection area, which can absorb essentially the entire amount of liquid of the applied sample.
  • the soluble second receptor is preferably a labeled antibody.
  • An approximately 12 ml disposable tube and a microbiological inoculation loop (e.g. Sarstedt) are balanced to 0 using a sensitive digital laboratory balance.
  • the stool is then weighed in with the inoculation loop by pricking the stool sample with the eyelet and the amount of stool remaining at the tip (approx. 100 mg) into the Disposable tube.
  • a toothpick can also be used instead of the inoculation loop.
  • the volumes of the extraction buffer to be added are varied according to the weighed stool sample mass (e.g. 100 mg stool + 10 ml buffer or 75 mg stool + 7.5 ml buffer). Final concentration: 10 mg stool / ml extraction buffer.
  • the stool sample suspension is mixed vigorously at room temperature with a test tube shaker (e.g. VORTEX).
  • the chairs must be well homogenized.
  • CHAPS 3- [(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate, 10 mM (Sigma) to the phosphate-buffered extraction buffer.
  • An ELISA plate is coated with a monoclonal antibody that only recognizes the cytosolic isocitrate dehydrogenase.
  • the monoclonal antibody that only recognizes the cytosolic isocitrate dehydrogenase.
  • Antibody was produced by means of purified cytosolic NADP-dependent isocitrate dehydrogenase according to the method known from Köhler and Milstein and screened for selectivity.
  • the cytosolic NADP-dependent isocitriate dehydrogenase from dilute stool samples and from standards binds to the antibody and is thus immobilized on the plate.
  • a second monoclonal antibody, which is biotinylated, binds to the in the next incubation step cytosolic NADP-dependent isocitrate dehydrogenase.
  • a conjugate of POD (peroxidase) and streptavidin then binds to the biotin unit.
  • the peroxidase oxidizes ABTS (2,2-azino-bis- (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt) to form a dark green color.
  • concentration of oxidized ABTS is then determined photometrically in a manner known per se.
  • a patient's serum which was obtained in the usual way, is tested with the same ELISA plate.
  • the sample was diluted 1:10 in a series of dilutions and the diluted samples were examined for their detection limit, showing a high sensitivity of the test according to the invention.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
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  • Urology & Nephrology (AREA)
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  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
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  • Microbiology (AREA)
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  • General Physics & Mathematics (AREA)
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  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé destiné à détecter une maladie maligne chez des êtres humains ou chez des animaux, ainsi qu'un kit d'essai permettant la mise en oeuvre du procédé de l'invention, et l'utilisation du kit d'essai pour la détection sélective d'isocitrate-déshydrogénase cytosolique NADP-dépendante dans des fluides corporels ou selles humains ou animaux, et pour la détermination de l'ampleur de la maladie maligne et la mise en place du schéma de traitement.
EP02745117A 2001-06-01 2002-06-01 Utilisation d'isocitrate-deshydrogenase cytosolique nadp-dependante dans des fluides corporels et des selles, pour detecter une maladie maligne, et kit d'essai utilise a cet effet Withdrawn EP1395827A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE10126763 2001-06-01
DE10126762 2001-06-01
DE10126763 2001-06-01
DE10126762 2001-06-01
PCT/DE2002/002020 WO2002099431A2 (fr) 2001-06-01 2002-06-01 Utilisation d'isocitrate-deshydrogenase cytosolique nadp-dependante dans des fluides corporels et des selles, pour detecter une maladie maligne, et kit d'essai utilise a cet effet

Publications (1)

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EP1395827A2 true EP1395827A2 (fr) 2004-03-10

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EP02745117A Withdrawn EP1395827A2 (fr) 2001-06-01 2002-06-01 Utilisation d'isocitrate-deshydrogenase cytosolique nadp-dependante dans des fluides corporels et des selles, pour detecter une maladie maligne, et kit d'essai utilise a cet effet

Country Status (3)

Country Link
EP (1) EP1395827A2 (fr)
DE (1) DE10292477D2 (fr)
WO (1) WO2002099431A2 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5952177A (en) * 1997-12-03 1999-09-14 Incyte Pharmaceuticals, Inc. Human cytosolic isocitrate dehydrogenase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO02099431A3 *

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WO2002099431A3 (fr) 2003-02-20
DE10292477D2 (de) 2004-04-29

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