[go: up one dir, main page]

WO1994010575A1 - Procede et trousse de diagnostic pour le cancer - Google Patents

Procede et trousse de diagnostic pour le cancer Download PDF

Info

Publication number
WO1994010575A1
WO1994010575A1 PCT/US1993/010411 US9310411W WO9410575A1 WO 1994010575 A1 WO1994010575 A1 WO 1994010575A1 US 9310411 W US9310411 W US 9310411W WO 9410575 A1 WO9410575 A1 WO 9410575A1
Authority
WO
WIPO (PCT)
Prior art keywords
mutant
proteins
assay
protein
cancer
Prior art date
Application number
PCT/US1993/010411
Other languages
English (en)
Inventor
Varda Rotter
Original Assignee
Yeda Research And Development Co. Ltd.
Rycus, Avigail
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 Yeda Research And Development Co. Ltd., Rycus, Avigail filed Critical Yeda Research And Development Co. Ltd.
Priority to AU55434/94A priority Critical patent/AU5543494A/en
Publication of WO1994010575A1 publication Critical patent/WO1994010575A1/fr

Links

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
    • G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds

Definitions

  • the present invention is generally in the field of cancer diagnosis and provides a method and a kit for the diagnosis of cancer based on the detection of anti-p53 antibodies in suspected individuals.
  • p53 is a nuclear protein having a molecular weight of about 53 kilodalton and is a cell cycle suppressor. When p53 was first discovered, more than a decade ago, it was described as a cellular phosphoprotein that is overproduced in tumor cells (1-4) and forms a complex with the viral large T antigen. (1-2) Analysis of a variety of cell lines and primary tumors, have indicated that p53 is ove ⁇ roduced in a large number of primary tumors and established cell lines and thus it was suggested that p53 could be defined as a tumor-specific marker. (5)
  • mutant p53 proteins hereinafter "m-p53" which are accumulated in the tumor cells are immunogenic and induce an immune reaction resulting in the production of specific anti-p53 antibodies that can be detected in the serum by means of interaction with mutant p53 proteins.
  • an assay method for the diagnosis of cancer in an individual based on the detection of an ⁇ i-p53 antibodies comprising:
  • the immune reaction which is being determined is an antibody-antigen reaction ("antibody embodiment”) while by another embodiment the immune reaction which is being determined is a cellular immune reaction (“cellular embodi ⁇ ment").
  • antibody embodiment is currently preferred in accordance with the present invention as it is generally easier to carry out than the cellular embodiment and involves techniques which are more familiar and more readily available to technicians in clinical laboratories. Both the antibody embodiment and the cellular embodiment can be performed by a large number of standard procedures readily familiar to the artisan.
  • said test sample is contacted with said assay composition and the binding of the antibodies in the test sample to the proteins in the assay composition is measured.
  • the m-p53 in the assay composition may be suspended in an aqueous medium or bound to a solid support such as beads, microbeads, gel particles, the wall of microwells, etc.
  • Measurement of binding of antibodies in the test sample to the m-p53 in the assay composition may be carried out by any number of means known per se such as immunoprecipitation, radio-immunoassay (RIA), fluorescence-immuno- assay (FIA) enzyme-linked immuno-sorbent assay (ELISA), etc.
  • the chosen means for the measurement will determine the exact nature of the assay composition (whether the m-p53 are in suspension or bound to a solid support, and in the latter case or the nature of the solid support), and vice versa.
  • the assy composition may comprise a single m-p53 type or a plurality of different types of m-p53.
  • the assay composition comprises a single type of m-p53, typically a plurality of test samples from the same individual will be used, each contacted with a different assay composition, each of which comprises a different type of m-p53.
  • the assay composition comprises a plurality of different types of m-p53, a single assay composition may at times be sufficient for carrying out the test.
  • the assay is preferably performed with several dilutions of the assay composition which is necessary since the antibody titer in the sample is as a rule, not known beforehand.
  • the present invention also provides assay compositions comprising a single m-p53 protein or a mixture of different m-p53 proteins, for use in the above method.
  • the assay composition may be provided as an aqueous suspension, as a dry composition, e.g. a lyophilizate, which is supplemented with an aqueous medium prior to performing the assay or may be provided as bound to a solid support, such as walls of microwells, beads, microbeads, gel particles etc.
  • the m-p53 to be used in accordance with the invention may be any mutant p53 protein, such as those isolated from bacterial, e.g. E.Coli, cells, as described in Shohat-Foord et al. (30).
  • mutant p53 proteins can be expressed by Vaccinia viral vectors as described hereinbelow and in
  • Ronen et al. (33). More common and less common m-p53's can be used.
  • cDNA encoding m-p53 may be isolated from different tumors and then cloned and expressed by various known cloning methods.
  • wild type or m-p53 gene may be isolated and site directed mutated in random by various means and then cloned and expressed.
  • the m-p53's are isolated and then used in accor ⁇ dance with the invention. It is appreciated that by the last approach, the m- p53 mixture may include also a large number of such proteins which may not occur naturally, but this by itself should have no adverse affect on the assay.
  • the present invention further provides a serological kit for the diagnosis of cancer in accordance with the above method comprising:
  • kits for use in said method.
  • An example of the kit is one comprising microtiter plates having a plurality of wells, the wells of which are coated with m-p53.
  • the wells may be coated by a mixture of m-p53 or each one as a group of wells may be coated by a different m-p53.
  • the serological anti-p53 defecting kit comprises a substantially purified and preferably defined mixture of m-p53 proteins expressed in E.Coli bacterial vector or vaccinia viral vectors.
  • the complexes generated as a result of reaction of the assay composition with a positive serum can be visualized by specific anti-human immunoglobulin reagents.
  • Fig. 1 shows the expression of p53 protein by recombinant vaccinia viruses.
  • HeLa cells were infected for 60 min., at M.O.I. 5 PFU per cell, with either wild type vaccinia virus (WR), vaccinia p53WT (p53CD) which is a recombinant vaccinia vector coding for wild type p53, or vaccinia p53mut (p53M8) which is a recombinant vaccinia vector coding for a mutant p53 protein.
  • WR wild type vaccinia virus
  • p53CD vaccinia p53WT
  • p53M8 vaccinia p53mut
  • Fig. 2 shows the results of a Western blot analysis.
  • HeLa cells were infected with either vaccinia p53WT or vaccinia p53mut and at indicated time points, cells were harvested and reacted with the anti-p53 monoclonal antibody PAb-242.
  • UI cell lysates obtained from uninfected HeLa cells
  • BV cell lysates obtained from HeLa cells 24 hrs post-infection with WT vaccinia virus
  • M markers indicating the positions of molecular weight standards.
  • RNA is extracted from different fresh tumor biopsies. An alliquot of 5 ⁇ g of RNA is reverse transcribed in a 20 ⁇ l reaction vessel using reverse transcriptase (Molecular Genetics Resources, Tampa Florida). 5 ⁇ l of cDNA are added to lOO ⁇ l primer extension reaction mixture which contains 0.25mM of each primer (the primers are oligonucleotides 17-mer in size synthesized according to known p53 sequence), 200 mM of each deoxynucleotide triphosphate with 2 units of Vent DNA polymerase with appropriate buffers (N.E. Biolabs, Bevery, MA). 3-35 PCR cycles are carried out.
  • lO ⁇ l of the reaction mixture are then run on a 1% agarose gel to visualize amplified DNA and to estimate yield.
  • lO ⁇ l of the amplified reaction mixture are incubated at 37°C for 30 min with 1 unit of T4 DNA polymerase (Boehringer Mannheim, GmbH, Germany) in order to create blunt ends in the amplified DNA.
  • the primer may also be designed to code for specific cloning sites such as BamHI direct cloning sites.
  • Amplified DNA is electrophoresed on a 1% agarose gel, the appropriate band is cut out and DNA is extracted and purified using GenecleanTM Kit (Bio 101, La Jolla CA).
  • p53 proteins which are mutated in the central part of the p53 molecule may be used.
  • human p53 cDNA that t ⁇ ive been isolated e.g. from a ⁇ gtlO library of SV40 transformed cells, may be cloned as BamHI fragments into the pBluescript KSII+ vector (Stratagene). Single-stranded DNA isolated from the library serve as a template for mutagenesis.
  • Oligonucleotide- directed site-specific mutagenesis may be performed essentially according to the Bio-RAD MUTAGENETM kit protocol, using single-stranded DNA, which may be produced in the E.Coli strain, CJ236, as a template.
  • the specific oligonucleotides for mutagenesis may be synthesized and purified by fractionation on a polyacrylamide gel.
  • the extension reaction may be carried out using T4 DNA polymerase and T4 gene 32 product (Boehringer Mannheim, FRG), transfected into E.Coli TGI competent bacteria. Mutated plasmids are analyzed by DNA sequencing using the Sequenase V.2 system (USB Cleaveland, OH).
  • Construction of a p53 expression piasmid can be performed, for example, by insertion of p53 cDNAs into the pET-8c piasmid (26-27) in which case the expression of the inserted p53 cDNA is controlled by the
  • T7 polymerase promoter As another example, p53 cDNA may be inserted into a modified pET12 vector.
  • Bacterial cells e.g. XL-1 blue (Stratagene) are transformed with the plasmids.
  • Such bacterial cells containing the different plasmids may be grown in 37°C LB medium containing ampicillin (50 mg/ml).
  • IPTG at a final concentration of 0.4mM is added when the cells reach about 1
  • DNA in the piasmid A certain time after the induction, e.g three hours, cells are harvested, e.g. by centrifugation, and the pellet is washed and resuspended, e.g. in TE containing 50mM NaCl and lOmg/ml lysozyme.
  • the lysate is sonicated, e.g. for 3 x 15 sec, and subjected to low speed centrifugation.
  • the pellet contained the total membrane fraction, and the supernatant contained the soluble fraction.
  • Insoluble material containing the p53 protein is washed, e.g. three times with 4M urea 0.1M Tris-HCl pH 8.5, and is solubilized, e.g. by dissolving in 7M guanidine-HCl, 50mM Tris-HCl pH 9.0, 2mM EDTA. Finally, the cell lysates are clarified and dialyzed against 50mM NaCl, lOmM Tris-HCl pH 7.8 and ImM EDTA.
  • E.Coli strains expressing mutants of the p53 proteins are obtained as reported in Shohat- Foord et al. (30), the context of which is inco ⁇ orated herein by reference.
  • Vaccinia expression vector pSVL-CD coding for the wild type p53 protein and pSVL-M8 coding for the mutant p53 protein, were used as sources for construction of p53 recombinant vaccinia viruses.
  • the two cDNA inserts were isolated, e.g. by digestion with BamHI, and subcloned, e.g into a BamHI site of vaccinia virus vector, such as pgpt-ATA-18 carrying a xanthine guanine phosphoribosyl transferase gene (gpt) for selection (the vector is available from D. H. Stunnenberg of EMBL).
  • TK " 143 cells are infected with wild type vaccinia virus WR strain at a multiplicity of infection (M.O.I.) of 0.1 PFU per cell.
  • the - cells were transfected by the calcium phosphate precipitation procedure (29), with l ⁇ g of pgpt-ATA-18 piasmid DNA either carrying p53CD or p53M8 coding sequences and 2 days later, recombinant viruses were collected from the infected cells and plaque-purified on RK 13 cells overlaid with 1% agarose in a growth selective medium (DMEM supplemented with 10% FCS and 20 ⁇ g/ml mycophenolic acid, 150 ⁇ g/ml xanthine and 15 ⁇ g ml hypoxanthine); Individual recombinant plaques were purified by three rounds of plaque purification and amplified on RK 13 cells.
  • DMEM calcium phosphate precipitation procedure
  • mutant p53 cDNA designated vaccinia p53mut (containing the p53M8 coding for mutant p53 protein) were prepared from the amplified cultures and titrated for the amount of virus PFU before use. The viral stocks were successfully utilized in the transfection of wells to produce wild type and mutant p53 proteins.
  • Monolayer cultures were infected with vaccinia p53 WT, vaccinia p53 mut or with parental vaccinia virus WR vaccinia vector at a M.O.I, of 5 PFU per cell of each virus. 24 h post-infection, the cells were metabolically labelled with 0.125 mCi of 35 S-methionine (Amersham) for 1 h at 37°C in methionine-deficient (met " ) Eagle's modified medium supplemented with 10% heat-inactivated dialyzed fetal calf serum.
  • Cells were lysed in lysis bufer: 50 M Tris pH 7.5; 150 mM NaCl; 0.5% NP40; 0.5% deoxycholate; 0.01% SDS; 2mM PMSF, and pre-cleared with 10% fixed Staphylococcus aureus. Equal amounts of TCA-insoluble radioactive material were reacted with specific antibodies for 2 h at 4°C. The immune complexes were precipitated with 10% fixed Staphylococcus aureus and washed 3X in PLB buffer; 10 mM NaH,HPO pH 7.5; 100 mM NaCl; 1% Triton X100; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate.
  • the immune complexes were separated on SDS-PAGE (Lammli, 1971).
  • cells were lysed in sample buffer and subjected to PAGE, as above.
  • the fractionated proteins were electrotransferred to nitrocellulose membranes and the proteins were detected using the Protoblot western blot Ap system (Promega).
  • HeLa cells that lack almost any detectable endogenous p53 proteins, were chosen as a convenient cell system for measuring the expression of p53 protein introduced by the viral infection.
  • HeLa cells were infected with either vaccinia p53WT, vaccinia p53M8 or the native vaccinia virus vector devoid of any foreign DNA.
  • Fig. 1 represents cellular and viral specific proteins synthesized in the cells infected with the various recombinant viruses. Lanes A in Fig. 1 show the pattern of total protein expressed in the cells. At that level of resolution it was clear that the cells infected by vaccinia p53WT or vaccinia p53mut, expressed an additional band of the expected p53 product.
  • the rate of p53 synethesize at various time intervals after infection was measured.
  • HeLa cells were infected with recombinant wild type p53 or mutant p53 vaccinia viruses. At times indicated in Fig. 2, the cells were harvested, cell lyses were analyzed by the western blot technique using PAb-242 specific anti-p53 antibodies. As can be seen in Fig. 2, the synthesis of recombinant wild type p53, as well as that of recombinant mutant p53 proteins, is detectable as early as 8 hrs after infection. 30 hours following infection, at the level of p53 proteins platode and remained as after 24 hrs (data not shown).
  • Secreted m-p53 produced in vectors such as the pET 12 vector commercially available (Novagene) is purified as a correctly folded protein, using either immunoaffinity or an alternative route, or a combination of the two.
  • Non-secreted m-p53 produced in vectors such as the pET3 vector commercially available (Novagene) is purified as inclusion bodies. These are dissolved using 6.5M urea and the denatured m-p53 protein is correctly refolded by dialysing the solution against decreasing concentrations of urea and NaCl and increasing DTT concentration. Incorrectly folded molecules are removed by ultracentrifugation. The correctly folded protein may be further purified using either immunoaffinity or a combination of immunoaffinity with other chromatographic procedures:
  • Immunoaffinity purification may be carried out by utilizing three anti-p53 monoclonal antibodies. Ascites fluids from female C 57 Bl 6 mice injected inte ⁇ eritonally with hybridoma lines may be used as a source for the antibodies. Antibody levels in these ascites fluids can reach 10-20 mg/ml.
  • the following monoclonal antibodies (MAb) may be used: monoclonal anti-p53 PAb240, PAb-242, PAb-246 (36,37); PAb-421 (38); 200.47 (39), and RA3— 2C2 (40). MAb can be initially purified by affinity chromatography on Protein A-Sepharose columns.
  • Purified MAbs can be bound covalently to an activated matrix (Affi-prep, BioRad), or to Protein A-Sepharose.
  • p53 proteins from difference sources can be affinity purified by the use of one or more antibodies, as needed. Conditions for elution of the p53 protein from each antibody are in accordance with standard procedures.
  • Kits and assay method The kit may consist of a microtiter plate whose wells are coated with a "broad p53 antigen", i.e. a plurality of different m-p53 proteins. Alternatively, each well or a group of wells may be coated with a different type of m-p53.
  • the kit may contain serum from p53-antibody positive and negative patients and purified anti-p53 Ab for quantitative pu ⁇ oses.

Landscapes

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

Abstract

On décrit un procédé de titrage, concernant le diagnostic du cancer chez une personne, avec lequel des échantillons de sérums humains sont mis en contact avec des compositions de titrage comprenant chacune une seule protéine p53 mutante ou plusieurs protéines p53 mutantes différentes. Une réponse immunitaire, qui se produit alors dans les sérums contenant la protéine mutante, fait l'objet d'un titrage qui établit une base permettant de diagnostiquer un éventuel cancer chez cette personne.
PCT/US1993/010411 1992-10-30 1993-10-29 Procede et trousse de diagnostic pour le cancer WO1994010575A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU55434/94A AU5543494A (en) 1992-10-30 1993-10-29 Method and kit for cancer diagnosis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IL103600 1992-10-30
IL103600A IL103600A0 (en) 1992-10-30 1992-10-30 Method and kit for cancer diagnosis

Publications (1)

Publication Number Publication Date
WO1994010575A1 true WO1994010575A1 (fr) 1994-05-11

Family

ID=11064167

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/010411 WO1994010575A1 (fr) 1992-10-30 1993-10-29 Procede et trousse de diagnostic pour le cancer

Country Status (3)

Country Link
AU (1) AU5543494A (fr)
IL (1) IL103600A0 (fr)
WO (1) WO1994010575A1 (fr)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996018906A1 (fr) * 1994-12-16 1996-06-20 Orga-Med Detection d'anticorps anti-p53 dans des liquides organiques
US5670325A (en) * 1996-08-14 1997-09-23 Exact Laboratories, Inc. Method for the detection of clonal populations of transformed cells in a genomically heterogeneous cellular sample
US5741650A (en) * 1996-01-30 1998-04-21 Exact Laboratories, Inc. Methods for detecting colon cancer from stool samples
US5928870A (en) * 1997-06-16 1999-07-27 Exact Laboratories, Inc. Methods for the detection of loss of heterozygosity
US5952178A (en) * 1996-08-14 1999-09-14 Exact Laboratories Methods for disease diagnosis from stool samples
US6020137A (en) * 1996-08-14 2000-02-01 Exact Laboratories, Inc. Methods for the detection of loss of heterozygosity
US6100029A (en) * 1996-08-14 2000-08-08 Exact Laboratories, Inc. Methods for the detection of chromosomal aberrations
US6146828A (en) * 1996-08-14 2000-11-14 Exact Laboratories, Inc. Methods for detecting differences in RNA expression levels and uses therefor
US6203993B1 (en) 1996-08-14 2001-03-20 Exact Science Corp. Methods for the detection of nucleic acids
US6268136B1 (en) 1997-06-16 2001-07-31 Exact Science Corporation Methods for stool sample preparation
US6280947B1 (en) 1999-08-11 2001-08-28 Exact Sciences Corporation Methods for detecting nucleotide insertion or deletion using primer extension
US6300077B1 (en) 1996-08-14 2001-10-09 Exact Sciences Corporation Methods for the detection of nucleic acids
US6849403B1 (en) 1999-09-08 2005-02-01 Exact Sciences Corporation Apparatus and method for drug screening
US6919174B1 (en) 1999-12-07 2005-07-19 Exact Sciences Corporation Methods for disease detection
US6964846B1 (en) 1999-04-09 2005-11-15 Exact Sciences Corporation Methods for detecting nucleic acids indicative of cancer
CN1300580C (zh) * 2004-12-31 2007-02-14 中国人民解放军第306医院 检测肝癌血清特征蛋白的质谱模型及其制备方法和应用
US7208313B2 (en) * 1999-05-28 2007-04-24 United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Combined growth factor-deleted and thymidine kinase-deleted vaccinia virus vector
US7368233B2 (en) 1999-12-07 2008-05-06 Exact Sciences Corporation Methods of screening for lung neoplasm based on stool samples containing a nucleic acid marker indicative of a neoplasm
US9109256B2 (en) 2004-10-27 2015-08-18 Esoterix Genetic Laboratories, Llc Method for monitoring disease progression or recurrence
US9777314B2 (en) 2005-04-21 2017-10-03 Esoterix Genetic Laboratories, Llc Analysis of heterogeneous nucleic acid samples

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BRITISH JOURNAL OF CANCER, Volume 65 (Supplement 16), issued December 1992, J.A. GREEN, "Serum Autoantibody to p53 in Breast Cancer Patients: Relationship to Tumor Differentiation", page 15. *
CANCER RESEARCH, Volume 52, issued 01 August 1992, S.T. WINTER et al., "Development of Antibodies Against p53 in Lung Cancer Patients Appears to be Dependent on the Type of p53 Mutation", pages 4168-4174. *
CLINICAL BIOCHEMISTRY, (Canada), Volume 25, No. 6, issued December 1992, S. HASSAPOGLIDOU et al., "Antibodies to the p53 Tumor Suppressor Gene Product Quantified in Cancer Patient Serum with a Time-Resolved Immunofluorometric Technique", pages 445-449. *
PROC ANNU MEET AM ASSOC CANCER RES, Volume 33, issued 1992, G.E. TRIVERS et al., "Detection of Anti-p53 Antibodies in Lung Cancer Cases and Controls", Abstract No. A1745. *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4444969C1 (de) * 1994-12-16 1996-10-24 Orga Med Dr Med Erwin Klopfer Nachweis von Antikörpern gegen p53 in Körperflüssigkeiten
WO1996018906A1 (fr) * 1994-12-16 1996-06-20 Orga-Med Detection d'anticorps anti-p53 dans des liquides organiques
US5741650A (en) * 1996-01-30 1998-04-21 Exact Laboratories, Inc. Methods for detecting colon cancer from stool samples
US6300077B1 (en) 1996-08-14 2001-10-09 Exact Sciences Corporation Methods for the detection of nucleic acids
US5670325A (en) * 1996-08-14 1997-09-23 Exact Laboratories, Inc. Method for the detection of clonal populations of transformed cells in a genomically heterogeneous cellular sample
US6303304B1 (en) 1996-08-14 2001-10-16 Exact Laboratories, Inc. Methods for disease diagnosis from stool samples
US5952178A (en) * 1996-08-14 1999-09-14 Exact Laboratories Methods for disease diagnosis from stool samples
US6020137A (en) * 1996-08-14 2000-02-01 Exact Laboratories, Inc. Methods for the detection of loss of heterozygosity
US6100029A (en) * 1996-08-14 2000-08-08 Exact Laboratories, Inc. Methods for the detection of chromosomal aberrations
US6146828A (en) * 1996-08-14 2000-11-14 Exact Laboratories, Inc. Methods for detecting differences in RNA expression levels and uses therefor
US6203993B1 (en) 1996-08-14 2001-03-20 Exact Science Corp. Methods for the detection of nucleic acids
US6268136B1 (en) 1997-06-16 2001-07-31 Exact Science Corporation Methods for stool sample preparation
US5928870A (en) * 1997-06-16 1999-07-27 Exact Laboratories, Inc. Methods for the detection of loss of heterozygosity
US6964846B1 (en) 1999-04-09 2005-11-15 Exact Sciences Corporation Methods for detecting nucleic acids indicative of cancer
US7208313B2 (en) * 1999-05-28 2007-04-24 United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Combined growth factor-deleted and thymidine kinase-deleted vaccinia virus vector
US8506947B2 (en) 1999-05-28 2013-08-13 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Vaccinia virus expression vector for selective replication in a tumor cell and introduction of exogenous nucleotide sequence into a tumor cell
US6280947B1 (en) 1999-08-11 2001-08-28 Exact Sciences Corporation Methods for detecting nucleotide insertion or deletion using primer extension
US6849403B1 (en) 1999-09-08 2005-02-01 Exact Sciences Corporation Apparatus and method for drug screening
US6919174B1 (en) 1999-12-07 2005-07-19 Exact Sciences Corporation Methods for disease detection
US7368233B2 (en) 1999-12-07 2008-05-06 Exact Sciences Corporation Methods of screening for lung neoplasm based on stool samples containing a nucleic acid marker indicative of a neoplasm
US9109256B2 (en) 2004-10-27 2015-08-18 Esoterix Genetic Laboratories, Llc Method for monitoring disease progression or recurrence
CN1300580C (zh) * 2004-12-31 2007-02-14 中国人民解放军第306医院 检测肝癌血清特征蛋白的质谱模型及其制备方法和应用
US9777314B2 (en) 2005-04-21 2017-10-03 Esoterix Genetic Laboratories, Llc Analysis of heterogeneous nucleic acid samples

Also Published As

Publication number Publication date
IL103600A0 (en) 1993-03-15
AU5543494A (en) 1994-05-24

Similar Documents

Publication Publication Date Title
WO1994010575A1 (fr) Procede et trousse de diagnostic pour le cancer
EP0241961B1 (fr) Essai pour la détection de carcinogénèses causées par des oncogènes
JP3989528B2 (ja) Ｄｎａ配列及びエンコードされた乳房に特異的な乳癌たんぱく質
US5053489A (en) Genetically engineered polypeptides with determinants of the human DF3 breast carcinoma-associated antigen
EP1876241A2 (fr) Compositions et procédés pour le traitement et le diagnostic du cancer du sein
EP0152477B1 (fr) Anticorps monoclonaux d'oncoproteines induits par des polypeptides
US5030565A (en) Polypeptide-induced monoclonal receptors to protein ligands
US5888751A (en) Method for diagnosis and treating cancers, and methods for identifying pathogenic markers in a sample of normal cells
US5756668A (en) Hypermethylated in cancer polypeptide, HIC-1
JP4270523B2 (ja) 乳癌の処置および診断のための組成物および方法
US5717067A (en) Substrate for the epidermal growth factor receptor kinase
Yen et al. Bacteriophage f1 gene II and X proteins. Isolation and characterization of the products of two overlapping genes.
WO1997025431A1 (fr) Compositions et procedes de traitement et de diagnostic du cancer
JP2005245469A (ja) 腫瘍サプレッサ遺伝子、並びに癌の検出、腫瘍進行のモニタおよび癌処置のための方法
US5002870A (en) Plastin isoforms and their use
EP0318179B1 (fr) Utilisation de recepteurs monoclonaux diriges contre des oncoproteines pour le suivi d'un traitement contre le cancer
US6329198B1 (en) Production and use of human nm23 protein and antibodies therefor
EP0354808B1 (fr) Récepteurs monoclonaux de ligands protéiques induits par les polypeptides
Van den Ouweland et al. Characteristics of a multicopy gene family predominantly consisting of processed pseudogenes
JP2003507050A (ja) 乳癌のヒト内在性レトロウイルス
Berthon et al. A microdissection approach to detect molecular markers during progression of prostate cancer
Yeh et al. Competition radioimmunoassay for mason‐pfizer monkey virus: Comparison with recent isolates
US6177080B1 (en) Polypeptides encoded by Kaposi sarcoma-associated herpes virus the use thereof in diagnosis and therapy
US7067635B2 (en) Nucleotide and deduced amino acid sequences of tumor gene Int6
CA2387576C (fr) Fragments immuno-interactifs de la sous-unite .alpha.c de l'inhibine

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR BY CA CZ FI GB HU JP KP KR KZ LK LV MG MN MW NO NZ PL PT RO RU SD SK UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA