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EP1654539A1 - Utilisation de la proteine sahh comme marqueur du cancer colorectal - Google Patents

Utilisation de la proteine sahh comme marqueur du cancer colorectal

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Publication number
EP1654539A1
EP1654539A1 EP04763805A EP04763805A EP1654539A1 EP 1654539 A1 EP1654539 A1 EP 1654539A1 EP 04763805 A EP04763805 A EP 04763805A EP 04763805 A EP04763805 A EP 04763805A EP 1654539 A1 EP1654539 A1 EP 1654539A1
Authority
EP
European Patent Office
Prior art keywords
sahh
colorectal cancer
diagnosis
protein
sample
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Withdrawn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
EP04763805A
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German (de)
English (en)
Inventor
Michael Tacke
Peter Berndt
Marie-Luise Hagmann
Johann Karl
Hanno Langen
Stefan Palme
Markus Roessler
Wolfgang Rollinger
Werner Zolg
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.)
F Hoffmann La Roche AG
Roche Diagnostics GmbH
Original Assignee
F Hoffmann La Roche AG
Roche Diagnostics GmbH
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 F Hoffmann La Roche AG, Roche Diagnostics GmbH filed Critical F Hoffmann La Roche AG
Priority to EP04763805A priority Critical patent/EP1654539A1/fr
Publication of EP1654539A1 publication Critical patent/EP1654539A1/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Withdrawn legal-status Critical Current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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon

Definitions

  • CRC colorectal cancer
  • the earlier cancer can be detected/diagnosed, the better is the overall survival rate.
  • More than one third of the patients will die from progressive disease within five years after diagnosis, corresponding to a survival rate of about 40% for five years.
  • CRC colorectal cancer
  • a protein encoded by a rare mRNA may be found in very high amounts and a protein encoded by an abundant mRNA may nonetheless be hard to detect and find at all. This lack of correlation between mRNA-level and protein level is due to reasons like mRNA stability, efficiency of translation, stability ofthe protein, etc.
  • WO 02/078636 reports about nine colorectal cancer-associated spots as found by surface-enhanced laser desorption and ionization (SELDI). These spots are seen more frequently in sera obtained from patients with CRC as compared to sera obtained from healthy controls. However, the identity ofthe molecule(s) comprised in such spot, e.g., its (their sequence), is not known.
  • a new diagnostic marker as a single marker should be at least as good as the best single marker known in the art. Or, a new marker should lead to a progress in diagnostic sensitivity and/or specificity either if used alone or in combination with one or more other markers, respectively.
  • the diagnostic sensitivity and/or specificity of a test is best assessed by its receiver-operating characteristics, which will be described in detail below.
  • CEA carcinoembryonic antigen
  • a tumor-associated glycoprotein a tumor-associated glycoprotein
  • Samples taken from stool have the advantage that such sampling is easily possible by non-invasive means.
  • the guaiac test is currently most widely used as a screening assay for CRC from stool.
  • the guaiac test however, has both poor sensitivity as well as poor specificity.
  • the sensitivity of the guaiac-based fecal occult blood tests is ⁇ 26%, which means 74% of patients with malignant lesions will remain undetected (Ahlquist, D.A., Gastroenterol. Clin. North Am. 26 (1997) 41-55).
  • the hemoglobin assay has an unsatisfactory sensitivity for the detection of colorectal neoplasms. Whereas cancer in its progressed carcinoma stage is detected with a sensitivity of about 87% the earlier tumor stages are not detected with a sufficient sensitivity.
  • the hemoglobin-haptoglobin complex assay was more sensitive in the detection of earlier stages of CRC. This more sensitive detection was accompanied by a poor specificity. Since poor specificity, however, translates to a high number of unnecessary secondary investigations, like colonoscopy, an assay with a poor accuracy also does not meet the requirements of a generally accepted screening assay.
  • the present invention therefore relates to a method for the diagnosis of colorectal cancer comprising the steps of a) providing a stool sample obtained from an individual, b) contacting said sample with a specific binding agent for SAHH under conditions appropriate for formation of a complex between said binding agent and SAHH, and c) correlating the amount of complex formed in (b) to the diagnosis of colorectal cancer.
  • the stool sample is processed to obtain a processed sample liquid which is more convenient to handle than a stool specimen. Such processed sample is then incubated with the specific binding agent for SAHH.
  • the present invention therefore also relates to a method for the diagnosis of colorectal cancer comprising the steps of a) providing a stool sample obtained from an individual, b) processing said sample to obtain a processed liquid sample, c) contacting said processed liquid sample with a specific binding agent for SAHH under conditions appropriate for formation of a complex between said binding agent and SAHH, and d) correlating the amount of complex formed in (c) to the diagnosis of colorectal cancer.
  • a preferred method uses a stool sample obtained from an individual.
  • Another preferred embodiment of the invention is a method for the diagnosis of colorectal cancer comprising the steps of a) processing a stool sample obtained from an individual to obtain a processed liquid sample b) contacting said processed liquid sample with a specific binding agent for SAHH under conditions appropriate for formation of a complex between said binding agent and SAHH, and c) correlating the amount of complex formed in (b) to the diagnosis of colorectal cancer.
  • the stool sample is processed to retrieve colonycytes which are then smeared on a microscopic slide. Such processed sample is then incubated with the specific binding agent for SAHH.
  • the present invention therefore also relates to a method for the diagnosis of colorectal cancer comprising the steps of a) providing a stool sample obtained from an individual, b) processing said sample to retrieve colonycytes, c) contacting said processed sample with a specific binding agent for SAHH under conditions appropriate for formation of a complex between said binding agent and SAHH, and d) correlating the amount of complex formed in (c) to the diagnosis of colorectal cancer.
  • the protein SAHH (S-adenosylhomocysteine hydrolase; SWISS-PROT: P23526) is characterized by the sequence given SEQ ID NO: 1.
  • the corresponding cloned human cDNA encodes for a 48-kDa protein which catalyzes the following rerversible reaction: S-adenosyl-L-homocysteine + H 2 O ⁇ adenosine + L- homocysteine (Cantoni, G.L., Annu. Rev. Biochem. 44 (1975) 435-451).
  • Hershfield Hershfield,
  • S-adenosyl-L-homocysteine is formed by the donation of a methyl group of S- adenosylmethionine, a universal methyl donor, to a methyl acceptor (e.g. DNA). Subsequently, it is hydrolyzed to adenosine and L-homocysteine by SAHH. Inhibition of SAHH results in accumulation of cellular S-adenosyl-L-homocysteine, which acts as feedback inhibitor on most methylation reactions (Chiang, P.K.,
  • a deficiency in SAHH is one of the different causes of hypermethioninemia, a disease associated with failure to thrive, mental and motor retardation, facial dysmorphism, and myocardiopathy (Labrune, P., et al, J. Pediatr. 117 (1990) 220-
  • the present invention shall not be construed to be limited to the full-length protein SAHH of SEQ ID NO:l.
  • SAHH are also encompassed by the present invention.
  • Artificial fragments preferably encompass a peptide produced synthetically or by recombinant techniques, which at least comprises one epitope of diagnostic interest consisting of at least 6 contiguous amino acids as derived from the sequence disclosed in SEQ ID NO:l. Such fragment may advantageously be used for generation of antibodies or as a standard in an immunoassay. More preferred the artificial fragment comprises at least two epitopes of interest appropriate for setting up a sandwich immunoassay.
  • the novel marker SAHH may be used for monitoring as well as for screening purposes. Its use for screening purposes is most preferred.
  • the diagnostic method according to the present invention may help to assess tumor load, efficacy of treatment and tumor recurrence in the follow-up of patients.
  • Increased levels of SAHH are directly correlated to tumor burden. After chemotherapy a short term (few hours to 14 days) increase in SAHH may serve as an indicator of tumor cell death. In the follow-up of patients (from 3 months to 10 years) an increase of SAHH can be used as an indicator for tumor recurrence in the colorectum.
  • the diagnostic method according to the present invention is used for screening purposes. I.e., it is used to assess subjects without a prior diagnosis of CRC by measuring the level of SAHH in a stool sample and correlating the level measured to the presence or absence of CRC.
  • the staging of cancer is the classification of the disease in terms of extent, progression, and severity. It groups cancer patients so that generalizations can be made about prognosis and the choice of therapy.
  • TNM the most widely used classification of the anatomical extent of cancer. It represents an internationally accepted, uniform staging system. There are three basic variables: T (the extent ofthe primary tumor), N (the status of regional lymph nodes) and M (the presence or absence of distant metastases).
  • TNM criteria are published by the UICC (International Union against Cancer), Sobin, L.H., Wittekind, Ch. (eds): TNM Classification of Malignant Tumours, fifth edition, 1997.
  • early diagnosis of CRC refers to a diagnosis at a pre-malignant state (adenoma) or at a tumor stage where no metastases at all (neither proximal nor distal), i.e., adenoma, Tj s , NO, M0 or Tl-4; NO; M0 are present.
  • T ⁇ s denotes carcinoma in situ.
  • the detection of SAHH is used to diagnose CRC as early as in the adenoma stage.
  • the diagnostic method according to the present invention is based on a stool sample which is derived from an individual.
  • the stool sample is extracted and SAHH is specifically measured from this processed stool sample by use of a specific binding agent.
  • a specific binding agent is, e.g., a receptor for SAHH, a lectin binding to SAHH or an antibody to SAHH.
  • a specific binding agent has at least an affinity of 10 7 1/mol for its corresponding target molecule.
  • the specific binding agent preferably has an affinity of 10 8 1/mol or even more preferred of 10 9 1/mol for its target molecule.
  • specific is used to indicate that other biomolecules present in the sample do not significantly bind to with the binding agent specific for SAHH.
  • the level of binding to a biomolecule other than the target molecule results in a binding affinity which is only 10%, more preferably only 5% of the affinity of the target molecule or less.
  • a most preferred specific binding agent will fulfill both the above minimum criteria for affinity as well as for specificity.
  • a specific binding agent preferably is an antibody reactive with SAHH.
  • the term antibody refers to a polyclonal antibody, a monoclonal antibody, fragments of such antibodies, as well as to genetic constructs comprising the binding domain of an antibody.
  • Antibodies are generated by state ofthe art procedures, e.g., as described in
  • polyclonal antibodies raised in rabbits have been used.
  • polyclonal antibodies from different species e.g. rats or guinea pigs
  • monoclonal antibodies can also be used. Since monoclonal antibodies can be produced in any amount required with constant properties, they represent ideal tools in development of an assay for clinical routine.
  • the generation and use of monoclonal antibodies to SAHH in a method according to the present invention is yet another preferred embodiment.
  • SAHH has been identified as a marker which is useful in the diagnosis of CRC
  • alternative strategies to generate antibodies may be used. Such strategies comprise amongst others the use of synthetic peptides, representing an epitope of SAHH for immunization.
  • DNA Immunization also known as DNA vaccination may be used.
  • the stool sample is obtained from an individual. An aliquot of the stool sample may be used directly. Preferably an aliquot of the stool sample is processed to yield a liquid sample.
  • the stool sample is preferably used or processed directly after sampling or stored cooled or more conveniently stored frozen.
  • Frozen stool samples can be processed by thawing, followed by dilution in an appropriate buffer, mixing and centrifugation. Supernatants are used as liquid sample for subsequent measurement of marker SAHH.
  • the amount of complex is measured and correlated to the diagnosis of CRC.
  • CRC CRC-binding agent
  • SAHH is detected in a sandwich type assay format.
  • a first specific binding agent is used to capture SAHH on the one side and a second specific binding agent, which is labeled to be directly or indirectly detectable is used on the other side.
  • SAHH can be measured from a stool sample obtained from an individual sample. No tissue and no biopsy sample is required to apply the marker SAHH in the diagnosis of CRC.
  • Antibodies to SAHH with great advantage can also be used in established procedures, e.g., to detect colorectal cancer cells in situ, in biopsies, or in immunohistological procedures.
  • an antibody to SAHH is used in a qualitative (SAHH present or absent) or quantitative (SAHH amount is determined) immunoassay.
  • the present invention relates to use of protein SAHH as a marker molecule in the diagnosis of colorectal cancer from a stool sample obtained from an individual.
  • marker molecule is used to indicate that an increased level of the analyte SAHH as measured from a bodily fluid or especially a processed stool sample obtained from an individual marks the presence of CRC.
  • the present invention relates to the use of SAHH as a marker molecule for colorectal cancer in combination with one or more other marker molecules for colorectal cancer in the diagnosis of colorectal cancer from a stool sample obtained from an individual.
  • Preferred selected other CRC markers with which the measurement of SAHH may be combined are hemoglobin and/or the hemoglobin-haptoglobin complex.
  • the marker SAHH is used in combination with hemoglobin.
  • the marker SAHH is used in combination with the hemoglobin-haptoglobin complex.
  • Diagnostic reagents in the field of specific binding assays like immunoassays, usually are best provided in the form of a kit, which comprises the specific binding agent and the auxiliary reagents required to perform the assay.
  • the present invention therefore also relates to an immunological kit comprising at least one specific binding agent for SAHH and auxiliary reagents for measurement of SAHH.
  • ROC receiver-operating characteristics
  • the clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease or benign versus malignant disease.
  • the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds.
  • sensitivity or the true-positive fraction [defined as (number of true-positive test results) (number of true-positive + number of false- negative test results)] .
  • positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup.
  • false-positive fraction or 1 - specificity [defined as (number of false- positive results) / (number of true-negative + number of false-positive results)]. It is an index of specificity and is calculated entirely from the unaffected subgroup.
  • the ROC plot is independent of the prevalence of disease in the sample.
  • Each point on the ROC plot represents a sensitivity/-specificity pair corresponding to a particular decision threshold.
  • a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity).
  • the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes.
  • One convenient goal to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number.
  • Figure 1 shows a typical example of a 2D-gel, loaded with a tumor sample (left side), and a gel, loaded with a matched control sample (right side) obtained from adjacent healthy mucosa.
  • the molecular weight inferred from the position of the protein spot in the gel is about 48 kDa, the isoelectric point is at about pH 6 (less).
  • the circle in the enlarged section of these gels indicates the position for the protein SAHH. This protein was not detectable by the same method in healthy mucosa.
  • Figure 2 Typical example of a Western-Blot.
  • a polyacrylamide gel was loaded with tissue lysates from colorectal tumor tissue and adjacent healthy control tissue from 3 patients (subject 4: colon ca (carcinoma), Dukes C; subject 7: colon ca, Dukes C; and subject 13: colon ca, Dukes B) and after electrophoresis the proteins were blotted onto a nitrocellulose membrane. Presence of SAHH in the samples was tested using a polyclonal rabbit anti-SAHH serum. Lanes containing tumor lysates are indicated with "T", lanes containing normal control tissue with "N". The arrow indicates the position in the gel ofthe SAHH band. All tumor samples give a strong signal at the position of SAHH, whereas only a weak signal can be detected in the lysates from adjacent normal control tissue.
  • tissue specimen from 10 patients suffering from colorectal cancer are analyzed. From each patient three different tissue types are collected from therapeutic resections: tumor tissue (> 80% tumor) (T), adjacent healthy tissue (N) and stripped mucosa from adjacent healthy mucosa (M). The latter two tissue types serve as matched healthy control samples. Tissues are immediately snap frozen after resection and stored at - 80°C before processing. Tumors are diagnosed by histopathological criteria.
  • 0.8-1.2 g of frozen tissue are put into a mortar and completely frozen by liquid nitrogen.
  • the tissue is pulverized in the mortar, dissolved in the 10-fold volume (w/v) of lysis buffer (40 mM Na- itrate, 5 mM MgCl 2 , 1% Genapol X-080, 0.02% Na-azide, Complete ® EDTA-free [Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 1 873 580]) and subsequently homogenized in a Wheaton ® glass homogenizer (20 x loose fitting, 20 x tight fitting). 3 ml of the homogenate are subjected to a sucrose-density centrifugation (10-60% sucrose) for 1 h at 4500 g. After this centrifugation step three fractions are obtained. The fraction on top of the gradient contains the soluble proteins and is used for further analysis.
  • the samples are concentrated in an Amicon" Ultra-15 device (Millipore GmbH, Schwalbach, Germany) and the protein concentration is determined using the Bio- Rad R protein assay (Cat.No. 500-0006; Bio-Rad Laboratories GmbH, Munchen, Germany) following the instructions of the supplier's manual.
  • Bio- Rad R protein assay Cat.No. 500-0006; Bio-Rad Laboratories GmbH, Munchen, Germany
  • IPG strips pH 4-7 (Amersham Biosciences, Freiburg, Germany) overnight.
  • the IEF is performed using the following gradient protocol: 1.) 1 minute to 500 V; 2.) 2 h to 3,500 V; 3.) 22 h at constant 3,500 V giving rise to 82 kVh. After IEF, strips are stored at -80°C or directly used for SDS-PAGE.
  • the strips Prior to SDS-PAGE the strips are incubated in equilibration buffer (6 M urea, 50 mM Tris/HCl, pH 8.8, 30% glycerol, 2% SDS), for reduction DDT (15 min, + 50 mg DTT/10 ml), and for alkylation IAA (15 min, + 235 mg iodacetamide/10 ml) is added.
  • the strips are put on 12.5% polyacrylamide gels and subjected to electrophoresis at 1 W/gel for 1 h and thereafter at 17 W/gel. Subsequently, the gels are fixed (50% methanol, 10% acetate) and stained overnight with No vex Colloidal Blue Staining Kit (Invitrogen, Düsseldorf, Germany, Cat No. LC6025, 45- 7101).
  • Each patient is analyzed separately by image analysis with the ProteomeWeaver® software (Definiens AG, Germany, Munchen).
  • all spots of the gel are excised by a picking robot and the proteins present in the spots are identified by MALDI-TOF mass spectrometry (Ultraflex TM Tof/Tof, Bruker Daltonik GmbH, Bremen, Germany).
  • MALDI-TOF mass spectrometry Ultraflex TM Tof/Tof, Bruker Daltonik GmbH, Bremen, Germany.
  • 4 gels from the tumor sample are compared with 4 gels each from adjacent normal and stripped mucosa tissue and analyzed for distinctive spots corresponding to differentially expressed proteins.
  • protein SAHH is found to be specifically expressed or strongly overexpressed in tumor tissue and not detectable or less strongly expressed in healthy control tissue. It therefore - amongst many other proteins - qualifies as a candidate marker for use in the diagnosis of colorectal cancer.
  • Polyclonal antibody to the colorectal cancer marker protein SAHH is generated for further use of the antibody in the measurement of serum and plasma and blood and stool levels of SAHH by immunodetection assays, e.g. Western Blotting and ELISA.
  • recombinant expression of the protein is performed for obtaining immunogens.
  • the expression is done applying a combination of the RTS 100 expression system and E.coli.
  • the DNA sequence is analyzed and recommendations for high yield cDNA silent mutational variants and respective PCR-primer sequences are obtained using the "ProteoExpert RTS E.coli HY” system. This is a commercial web based service (www.proteoexpert.com).
  • the "RTS 100 E. coli Linear Template Generation Set, His-tag” (Roche Diagnostics GmbH, Mannheim, Germany, Cat.No.
  • coli BL 21 (DE 3) (Studier, F.W., et al., Methods Enzymol. 185 (1990) 60-89) and the transformed bacteria are cultivated in a 1 1 batch for protein expression.
  • Purification of His-SAHH fusion protein is done following standard procedures on a Ni-chelate column. Briefly, 1 1 of bacteria culture containing the expression vector for the His-SAHH fusion protein is pelleted by centrifugation. The cell pellet is resuspended in lysis buffer, containing phosphate, pH 8.0, 7 M guanidium chloride, imidazole and thioglycerole, followed by homogenization using a Ultra-Turrax ® .
  • Insoluble material is pelleted by high speed centrifugation and the supernatant is applied to a Ni-chelate chromatographic column.
  • the column is washed with several bed volumes of lysis buffer followed by washes with buffer, containing phosphate, pH 8.0 and Urea. Finally, bound antigen is eluted using a phosphate buffer containing SDS under acid conditions.
  • mice 12 week old A/J mice are initially immunized intraperitoneally with 100 ⁇ g SAHH. This is followed after 6 weeks by two further intraperitoneal immunizations at monthly intervals. In this process each mouse is administered 100 ⁇ g SAHH adsorbed to aluminum hydroxide and 10 9 germs of Bordetella pertussis. Subsequently the last two immunizations are carried out intravenously on the 3rd and 2nd day before fusion using 100 ⁇ g SAHH in PBS buffer for each.
  • Spleen cells of the mice immunized according to a) are fused with myeloma cells according to Galfre, G., and Milstein, C, Methods in Enzymology 73 (1981) 3-46.
  • ca. 1*10 8 spleen cells ofthe immunized mouse are mixed with 2xl0 7 myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged (10 min at 300 x g and 4°C).
  • the cells are then washed once with RPMI 1640 medium without fetal calf serum (FCS) and centrifuged again at 400 x g in a 50 ml conical tube.
  • FCS fetal calf serum
  • the sedimented cells are taken up in RPMI 1640 medium containing 10% FCS and sown in hypoxanthine-azaserine selection medium (lOO mmol/1 hypoxanthine, 1 ⁇ g/ml azaserine in RPMI 1640+10% FCS).
  • Interleukin 6 at 100 U/ml is added to the medium as a growth factor.
  • SAHH- positive primary cultures are cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter. In this process again interleukin 6 at 100 U/ml is added to the medium as a growth additive.
  • the hybridoma cells obtained are sown at a density of lxl 0 5 cells per ml in RPMI
  • a fresh emulsion of the protein solution (100 ⁇ g/ml protein SAHH) and complete Freund's adjuvant at the ratio of 1:1 is prepared.
  • Each rabbit is immunized with 1 ml ofthe emulsion at days 1, 7, 14 and 30, 60 and 90. Blood is drawn and resulting anti-SAHH serum used for further experiments as described in examples 3 and 4.
  • IgG immunoglobulin G
  • rabbit serum is diluted with 4 volumes of acetate buffer (60 mM, pH 4.0). The pH is adjusted to 4.5 with 2 M Tris-base. Caprylic acid (25 ⁇ l/ml of diluted sample) is added drop-wise under vigorous stirring. After 30 min the sample is centrifuged (13,000 g, 30 min, 4°C), the pellet discarded and the supernatant collected. The pH of the supernatant is adjusted to 7.5 by the addition of 2 M Tris-base and filtered (0.2 ⁇ m).
  • the immunoglobulin in the supernatant is precipitated under vigorous stirring by the drop-wise addition of a 4 M ammonium sulfate solution to a final concentration of 2 M.
  • the precipitated immunoglobulins are collected by centrifugation (8,000 x g, 15 min, 4°C).
  • the supernatant is discarded.
  • the pellet is dissolved in 10 mM NaH 2 PO /NaOH, pH 7.5, 30 mM NaCl and exhaustively dialyzed.
  • the dialysate is centrifuged (13,000 x g, 15 min, 4°C) and filtered (0.2 ⁇ m).
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl. Per ml IgG solution 50 ⁇ l Biotin -N-hydroxysuccinimide (3.6 mg/ml in DMSO) are added. After 30 min at room temperature, the sample is chromatographed on Superdex 200 (10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, 30 mM NaCl, pH 7.5.
  • Per ml IgG solution 50 ⁇ l digoxigenin-3-O-methylcarbonyl- ⁇ - aminocaproic acid-N-hydroxysuccinimide ester (Roche Diagnostics, Mannheim, Germany, Cat. No. 1 333 054) (3.8 mg/ml in DMSO) are added. After 30 min at room temperature, the sample is chromatographed on Superdex® 200 (10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl). The fractions containing digoxigenylated IgG are collected. Monoclonal antibodies are labeled with digoxigenin according to the same procedure.
  • Tissue lysates from tumor samples and healthy control samples are prepared as described in Example 1, "Tissue preparation”.
  • SDS-PAGE and Western-Blotting are carried out using reagents and equipment of Invitrogen, Düsseldorf, Germany.
  • 10 ⁇ g of tissue lysate are diluted in reducing NuPAGE ® (Invitrogen) SDS sample buffer and heated for 10 min at 95°C.
  • Samples are run on 4-12% NuPAGE ® gels (Tris-Glycine) in the MES running buffer system.
  • the gel-separated protein mixture is blotted onto nitrocellulose membranes using the Invitrogen XCell II Blot Module (Invitrogen) and the NuPAGE" transfer buffer system.
  • the membranes are washed 3 times in PBS/0.05% Tween-20 and blocked with Roti®-Block blocking buffer (A151.1; Carl Roth GmbH, Düsseldorf, Germany) for 2 h.
  • the primary antibody, polyclonal rabbit anti-SAHH serum (generation described in Example 2), is diluted 1:10,000 in
  • Roti®-Block blocking buffer and incubated with the membrane for 1 h.
  • the membranes are washed 6 times in PBS/0.05% Tween-20.
  • the specifically bound primary rabbit antibody is labeled with a POD-conjugated polyclonal sheep anti- rabbit IgG antibody, diluted to 10 mU/ml in 0.5 x Roti®-Block blocking buffer. After incubation for 1 h, the membranes are washed 6 times in PBS/0.05% Tween-
  • the membrane is incubated with the Lumi-Light s Western Blotting Substrate (Order-No. 2015196, Roche Diagnostics GmbH, Mannheim, Germany) and exposed to an autoradiographic film.
  • Lumi-Light s Western Blotting Substrate Order-No. 2015196, Roche Diagnostics GmbH, Mannheim, Germany
  • Example 4 ELISA for the measurement of SAHH in human stool specimen.
  • a sandwich ELISA For detection of SAHH in human a processed stool sample, a sandwich ELISA is developed. For capture and detection of the antigen, aliquots of the anti-SAHH polyclonal antibody (see example 2) are conjugated with biotin and digoxygenin, respectively.
  • Streptavidin- coated 96-well microtiter plates are incubated with 100 ⁇ l biotinylated anti-SAHH polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween-20. After incubation, plates are washed three times with 0.9% NaCl , 0.1% Tween-20. Wells are then incubated for 2 h with either a serial dilution of the recombinant protein (see Example 2) as standard antigen or with diluted stool samples from patients. After binding of SAHH, plates are washed three times with 0.9% NaCl, 0.1% Tween-20.
  • wells are incubated with 100 ⁇ l of digoxygenylated anti- SAHH polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween-20. Thereafter, plates are washed three times to remove unbound antibody.
  • wells are incubated with 20 mU/ml anti-digoxigenin-POD conjugates (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1633716) for 60 min in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween-20. Plates are subsequently washed three times with the same buffer.
  • ABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 11685767) and OD is measured after 30-60 min at 405 nm with an
  • Accuracy is assessed by analyzing individual stool samples obtained from well- characterized patient cohorts, i.e., 30 patients having undergone colonoscopy and found to be free of adenoma or CRC, 30 patients diagnosed and staged as Tl-3, NO,

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Abstract

La présente invention concerne le diagnostic du cancer colorectal et plus précisément l'utilisation de la protéine SAHH (S-adénosylhomocystéine hydrolase) pour le diagnostic du cancer colorectal. L'invention concerne une méthode de diagnostic du cancer colorectal qui consiste à prélever un échantillon de selles chez un individu et à mesurer la concentration de SAHH dans ledit échantillon. Les mesures de la concentration de SAHH peuvent être utilisées, par exemple, pour un dépistage ou un diagnostic précoce du cancer colorectal.
EP04763805A 2003-08-07 2004-08-05 Utilisation de la proteine sahh comme marqueur du cancer colorectal Withdrawn EP1654539A1 (fr)

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EP04763805A EP1654539A1 (fr) 2003-08-07 2004-08-05 Utilisation de la proteine sahh comme marqueur du cancer colorectal

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EP03017554 2003-08-07
EP04763805A EP1654539A1 (fr) 2003-08-07 2004-08-05 Utilisation de la proteine sahh comme marqueur du cancer colorectal
PCT/EP2004/008758 WO2005015221A1 (fr) 2003-08-07 2004-08-05 Utilisation de la proteine sahh comme marqueur du cancer colorectal

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WO2007071366A1 (fr) * 2005-12-21 2007-06-28 Roche Diagnostics Gmbh Méthode d'évaluation du cancer colorectal par mesure de l'hémoglobine et de la pyruvate-kinase m2 dans un echantillon de selles
CN112881692B (zh) * 2021-01-08 2022-11-22 深圳华大基因股份有限公司 一种用于结直肠癌及腺瘤早期筛查的蛋白定量检测方法

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JPH07500411A (ja) * 1991-02-05 1995-01-12 ファロク　セィディ 癌胎児性抗原を検出するための簡易検査法

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WO2005015221A1 (fr) 2005-02-17

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