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WO2005015223A1 - Utilisation de la proteine ribosomale acide p0 (rla-0) comme marqueur dans le cancer colorectal - Google Patents

Utilisation de la proteine ribosomale acide p0 (rla-0) comme marqueur dans le cancer colorectal Download PDF

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Publication number
WO2005015223A1
WO2005015223A1 PCT/EP2004/008763 EP2004008763W WO2005015223A1 WO 2005015223 A1 WO2005015223 A1 WO 2005015223A1 EP 2004008763 W EP2004008763 W EP 2004008763W WO 2005015223 A1 WO2005015223 A1 WO 2005015223A1
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Prior art keywords
rla
colorectal cancer
protein
diagnosis
marker
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Inventor
Michael Tacke
Peter Berndt
Marie-Luise Hagmann
Johann Karl
Hanno Langen
Stefan Palme
Markus Roessler
Wolfgang Rollinger
Werner Zolg
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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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
    • G01N33/53—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 prognosis in advanced stages of tumor is poor. 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. 15 Current treatment is only curing a fraction of the patients and clearly has the best effect on those patients diagnosed in an early stage of disease.
  • 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 of the 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 of the 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
  • serum CEA determination possesses neither the sensitivity nor the specificity to enable its use as a screening test for colorectal cancer in the asymptomatic population (Reynoso, G., et al., JAMA 220 (1972) 361- 365; Sturgeon, C, Clinical Chemistry 48 (2002) 1151-1159).
  • 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 RLA-0 under conditions appropriate for formation of a complex between said binding agent and RLA-0, 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 RLA-0.
  • 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 RLA-0 under conditions appropriate for formation of a complex between said binding agent and RLA-0, 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 RLA-0 under conditions appropriate for formation of a complex between said binding agent and RLA-0, 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 RLA-0.
  • 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 RLA-0 under conditions appropriate for formation of a complex between said binding agent and RLA-0, and d) correlating the amount of complex formed in (c) to the diagnosis of colorectal cancer.
  • Ribosomes the organelles that catalyze protein synthesis, consist of a small 40S subunit and a large 60S subunit. Together these subunits are composed of 4 RNA species and approximately 80 structurally distinct proteins (Uechi, T., et al, Genomics (2001) 223-230).
  • RLA0 60S acidic ribosomal protein P0; SWISS-PROT: P05388
  • P-proteins the family of acidic ribosomal phosphoproteins
  • ribosomal proteins are synthesized stoichiometrically to produce equimolar supply of ribosomal components (Mager, W.H., Biochim. Biophys. Acta 949 (1988) 1-15).
  • regulation of transcriptional activity of the ribosomal protein genes is reported to be less regulated than expected (Bortoluzzi, S., et al., Bioinformatics 17 (2001) 1152- 1157).
  • recent studies provide growing evidence, that ribosomal proteins can also function in various cellular processes such as replication, transcription, DNA repair, and inflammation, independent of their involvement in protein biosynthesis (Wool, I.G., Trends Biochem. Sci. 21 (1996) 164-165; Yamamoto, T., Pathol. Int. 50 (2000) 863-871).
  • ribosomal proteins not including RLA0 (P0) were identified to be overexpressed in neoplastic tissue as compared to healthy control tissue, e. g. in esophageal (Wang, Q., et al., Gene 263 (2001) 205-209) or prostate cancer (Vaarala, M.H., et al, Int. J. Cancer 78 (1998) 27-32).
  • RLA0 P0
  • it is reported that it was found to be overexpressed in colorectal cancer and colorectal polyps Pogue-Geile, K., et al, Mol. Cell. Biol. 11 (1991) 3842-3849).
  • RLA0 RLA0
  • hepatocellular carcinoma Kondoh, N., et al., Cancer Res. 59 (1999) 4990-4996
  • colon carcinoma Barnard, G.F., et al., Cancer Res. 52 (1992) 3067-3072
  • in colorectal carcinoma expression of RLA0 (P0) was increasing with increasing Dukes' stage (Barnard, G.F., et al., Cancer Res. 52 (1992) 3067-3072).
  • all of these studies were based on analyses carried out on the transcriptional level. Most recently, Kasai, H., et al., J. Histochem. Cytochem.
  • the present invention shall not be construed to be limited to the full-length protein RLA-0 of SEQ ID NO:l.
  • Physiological or artificial fragments of RLA-0, secondary modifications of RLA-0, as well as allelic variants of RLA-0 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 RLA-0 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 RLA-0 are directly correlated to tumor burden. After chemotherapy a short term (few hours to 14 days) increase in RLA-0 may serve as an indicator of tumor cell death. In the follow-up of patients (from 3 months to 10 years) an increase of RLA-0 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 RLA-0 in a stool sample and correlating the level measured to the presence or absence of CRC.
  • Colorectal cancer most frequently progresses from adenomas (polyps) to malignant carcinomas.
  • 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 of the 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, T ⁇ s , NO, M0 or Tl-4; NO; M0 are present.
  • T; s denotes carcinoma in situ.
  • the detection of RLA-0 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 RLA-0 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 RLA-0, a lectin binding to RLA-0 or an antibody to RLA-0.
  • 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 RLA-0.
  • 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 RLA-0.
  • 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 of the art procedures, e.g., as described in Tijssen (Tijssen, P., Practice and theory of enzyme immunoassays 11 (1990) the whole book, especially pages 43-78; Elsevier, Amsterdam).
  • Tijssen Tejssen, P., Practice and theory of enzyme immunoassays 11 (1990) the whole book, especially pages 43-78; Elsevier, Amsterdam.
  • the skilled artisan is well aware of methods based on immunosorbents that can be used for the specific isolation of antibodies. By these means the quality of polyclonal antibodies and hence their performance in immunoassays can be enhanced. (Tijssen, P., supra, pages 108-115).
  • polyclonal antibodies raised in rabbits have been used. However, clearly also polyclonal antibodies from different species , e.g. rats or guinea pigs, as well as 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 RLA-0 in a method according to the present invention is yet another preferred embodiment.
  • RLA-0 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 RLA-0 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.
  • 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 RLA-0.
  • An aliquot of the processed stool sample is incubated with the specific binding agent for RLA-0 under conditions appropriate for formation of a binding agent RLA-0-complex. Such conditions need not be specified, since the skilled artisan without any inventive effort can easily identify such appropriate incubation conditions.
  • the amount of complex is measured and correlated to the diagnosis of CRC.
  • CRC CRC-binding agent
  • RLA-0 is detected in a sandwich type assay format.
  • a first specific binding agent is used to capture RLA-0 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.
  • RLA-0 can be measured from a stool sample obtained from an individual sample. No tissue and no biopsy sample is required to apply the marker RLA-0 in the diagnosis of CRC.
  • Antibodies to RLA-0 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 RLA-0 is used in a qualitative (RLA-0 present or absent) or quantitative (RLA-0 amount is determined) immunoassay.
  • the present invention relates to use of protein RLA-0 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 RLA-0 as measured from a bodily fluid or especially a processed stool sample obtained from an individual marks the presence of CRC.
  • novel marker RLA-0 in the early diagnosis of colorectal cancer.
  • the use of protein RLA-0 itself represents a significant progress to the challenging field of CRC diagnosis from stool. Combining measurements of RLA-0 with other known markers, like hemoglobin or the hemoglobin-haptoglobin complex, or with other markers of CRC yet to be discovered, leads to further improvements. Therefore in a further preferred embodiment the present invention relates to the use of RLA-0 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 RLA-0 may be combined are hemoglobin and/or the hemoglobin-haptoglobin complex. Very preferred the marker RLA-0 is used in combination with hemoglobin. Also very preferred the marker RLA-0 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 RLA-0 and auxiliary reagents for measurement of RLA-0.
  • 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)].
  • This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup.
  • the 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.
  • Two distinctive spots are shown which both represent RLA-0. Both fit the theoretical size of 34.3 kDa; one spot fits the theoretical isoelectric point at pH 5.72, the other spot probably corresponds to phosphorylated RLA-0, most likely due to a post-translational modification.
  • Each circle in the enlarged section of these gels indicates the position for the a RLA-0 protein form. RLA-0 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 4 patients (subject 36: rectum ca (carcinoma), Dukes B; subject 37: rectum ca, Dukes A; subject 39: colon ca, Dukes A; and subject 40: colon ca, Dukes B) and after electrophoresis the proteins were blotted onto a nitrocellulose membrane. Presence of RLA-0 in the samples was tested using a polyclonal rabbit anti-RLA-0 serum. Lanes containing tumor lysates are indicated with "T", lanes containing normal control tissue with "N”. The marker lane containing a molecular weight protein standard is indicated by "M”.
  • the lane containing recombinant RLA-0 is indicated by "300".
  • the arrow indicates the position in the gel of the RLA-0 (RLA0) band. All tumor samples give a strong signal at the position of RLA-0, whereas only a weak signal can be detected in the lysates from adjacent normal control tissue.
  • BSA bovine serum albumin cDNA complementary DNA CHAPS 3 - [ ( 3 -Cholamidopropyl) -dimethylammonio] - 1 -propane- sulfonate
  • 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-citrate, 5 mM MgCl 2 , 1% Genapol X-080, 0.02% Na-azide, Complete R 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).
  • lysis buffer 40 mM Na-citrate, 5 mM MgCl 2 , 1% Genapol X-080, 0.02% Na-azide, Complete R EDTA-free
  • 3 ml of the homogenate are subjected to a sucrose-density centrifugation (10-60% sucrose) for 1 h at 4500 x 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.
  • 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 all viation 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, M ⁇ nchen).
  • 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 RLA-0 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 RLA-0 is generated for further use of the antibody in the measurement of serum and plasma and blood and stool levels of RLA-0 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.
  • His-RLA-0 fusion protein Purification of His-RLA-0 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-RLA-0 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 RLA-0. This is followed after 6 weeks by two further intraperitoneal immunizations at monthly intervals. In this process each mouse is administered 100 ⁇ g RLA-0 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 RLA-0 in PBS buffer for each. I - 19 -
  • 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 of the 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/l 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.
  • RLA-0- 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 lxlO 5 cells per ml in RPMI 1640 medium containing 10% FCS and proliferated for 7 days in a fermenter (Thermodux Co., Wertheim/Main, Model MCS-104XL, Order No. 144-050). On average concentrations of 100 ⁇ g monoclonal antibody per ml are obtained in the culture supernatant. Purification of this antibody from the culture supernatant is carried out by conventional methods in protein chemistry (e.g. according to Brack, G, et al, Methods in Enzymology 121 (1986) 587-695). Generation of polyclonal antibodies
  • a fresh emulsion of the protein solution (lOO ⁇ g/ml protein RLA-0) and complete Freund's adjuvant at the ratio of 1:1 is prepared.
  • Each rabbit is immunized with 1 ml of the emulsion at days 1, 7, 14 and 30, 60 and 90. Blood is drawn and resulting anti- RLA-0 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 x 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 NaCl). The fraction containing biotinylated IgG are collected. Monoclonal antibodies are biotinylated according to the same procedure.
  • 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-RLA-0 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.
  • the membranes are washed 6 times in PBS/0.05% Tween- 20.
  • the membrane is incubated with the Lumi-Light PLUS Western Blotting Substrate (Order-No. 2015196, Roche Diagnostics GmbH, Mannheim, Germany) and exposed to an autoradiographic film.
  • a sandwich ELISA For detection of RLA-0 in human a processed stool sample, a sandwich ELISA is developed. For capture and detection of the antigen, aliquots of the anti-RLA-0 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-RLA-0 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 RLA-0, plates are washed three times with 0.9% NaCl, 0.1% Tween-20.
  • wells are incubated with 100 ⁇ l of digoxygenylated anti- RLA-0 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.
  • wells are incubated with 100 ⁇ l ABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 11685767) and OD is measured after 30-60 min at 405 nm with an ELISA reader.
  • 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. L'invention se rapporte à l'utilisation de la protéine RLA-0 (Protéine ribosomale acide 60S P0) dans le diagnostic du cancer colorectal. L'invention porte sur un procédé qui permet de diagnostiquer le cancer colorectal à partir d'un prélèvement de selles issu d'un individu en mesurant la RLA-0 dans ledit prélèvement. La mesure de RLA-0 peut, par exemple, être utilisée pour la détection ou le diagnostic précoce du cancer colorectal.
PCT/EP2004/008763 2003-08-07 2004-08-05 Utilisation de la proteine ribosomale acide p0 (rla-0) comme marqueur dans le cancer colorectal Ceased WO2005015223A1 (fr)

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EP03017560 2003-08-07
EP03017560.8 2003-08-07

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WO2005015223A1 true WO2005015223A1 (fr) 2005-02-17

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110819715A (zh) * 2019-11-26 2020-02-21 华夏帮服科技有限公司 用于结直肠癌检测的免疫基因标志物及试剂盒

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BARNARD G F ET AL: "INCREASED EXPRESSION OF HUMAN RIBOSOMAL PHOSPHOPROTEIN P0 MESSENGER RNA IN HEPATOCELLULAR CARCINOMA AND COLON CARCINOMA", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, BALTIMORE, MD, US, vol. 52, no. 11, 1 June 1992 (1992-06-01), pages 3067 - 3072, XP001145714, ISSN: 0008-5472 *
POGUE-GEILE K ET AL: "RIBOSOMAL PROTEIN GENES ARE OVEREXPRESSED IN COLORECTAL CANCER ISOLATION OF A COMPLEMENTARY DNA CLONE ENCODING THE HUMAN S3 RIBOSOMAL PROTEIN", MOLECULAR AND CELLULAR BIOLOGY, vol. 11, no. 8, 1991, pages 3842 - 3849, XP002299999, ISSN: 0270-7306 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110819715A (zh) * 2019-11-26 2020-02-21 华夏帮服科技有限公司 用于结直肠癌检测的免疫基因标志物及试剂盒

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