EP0929811A1 - EPREUVE POUR LA DETECTION D'ANTICORPS CONTRE p53 - Google Patents

EPREUVE POUR LA DETECTION D'ANTICORPS CONTRE p53

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Publication number
EP0929811A1
EP0929811A1 EP97944306A EP97944306A EP0929811A1 EP 0929811 A1 EP0929811 A1 EP 0929811A1 EP 97944306 A EP97944306 A EP 97944306A EP 97944306 A EP97944306 A EP 97944306A EP 0929811 A1 EP0929811 A1 EP 0929811A1
Authority
EP
European Patent Office
Prior art keywords
peptide
immobilized
seq
peptides
binding
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Withdrawn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
EP97944306A
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German (de)
English (en)
Inventor
Daniel T. Mytych
Steven J. Swanson
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.)
Merck Sharp and Dohme Corp
Original Assignee
Schering Corp
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 Schering Corp filed Critical Schering Corp
Publication of EP0929811A1 publication Critical patent/EP0929811A1/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Withdrawn legal-status Critical Current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
    • G01N33/57484—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/82—Translation products from oncogenes

Definitions

  • the present invention relates to a method for the detection of antibodies capable of binding to p53 protein, particularly human p53 protein.
  • Wild-type p53 gene is a tumor suppressor gene encoding cellular wild- type p53 protein. Endogenous wild-type p53 protein has been found to be lacking (either absent or mutated) in a variety of cancer cells, such as breast carcinoma cells and lung carcinoma cells, and it has been found that administration of wild-type p53 gene into cancer cells lacking endogenous wild- type p53 protein can be used to treat cancer by suppression of the neoplastic phenotype. See U.S. Patent 5,532,220.
  • Soussi et al. disclosed an assay for using biotinylated peptides in an enzyme linked immunosorbent assay ("ELISA") format to detect p53 antibodies in patient serum samples.
  • ELISA enzyme linked immunosorbent assay
  • the major areas where improvement is needed are as follows. First, there is a critical need for an assay that is better suited for detecting lower affinity antibodies. Methods employing ELISAs have difficulty in this regard because
  • ELISAs require multiple incubation steps followed by wash cycles during which lower affinity antibodies can wash away.
  • ELISAs require multiple incubation steps followed by wash cycles, and therefore the materials used in the assay cannot be regenerated for analysis of subsequent samples.
  • the present invention meets the foregoing needs by providing a method for detecting antibodies capable of binding to p53 protein comprising:
  • a plurality of selected immunogenic peptides are used, each immobilized directly on its own separate flowcell.
  • a first peptide is selected from the amino terminal region of the p53 protein and a second peptide is selected from the carboxy terminal region of the p53 protein.
  • the present invention employs one or more of the following four peptides (or peptides having substantial sequence identity thereto): a) a peptide comprising SEQ ID NO: 1 (C11-35, corresponding to residues 11-
  • a peptide comprising SEQ ID NO: 2 (C40-65, corresponding to residues 40- 65 of the human p53 protein: H-CMDDLMLSPDDIEQWFTEDPGPDEAPR- Amide), or a peptide having substantial sequence identity thereto;
  • a peptide comprising SEQ ID NO: 4 (C371-390, corresponding to residues 371-390 of the human p53: protein H-CSKKGQSTSRHKKLMFKTEGP- Amide), or a peptide having substantial sequence identity thereto.
  • Figure 1 is a graphical depiction of response units vs. dilution factor to show the relative sensitivity of the four noted peptides to antibodies in normal human serum.
  • Figure 2 is a graphical depiction of response units vs. dilution factor to show the relative sensitivity of the four noted peptides to antibodies in normal pig serum.
  • Figures 3A and 3B are graphical depictions showing antibody binding (to the four noted peptides) for normal human serum samples.
  • the abbreviation NHS pooled normal human serum
  • Pos sheep polyclonal Ab 1 :200 into NHS.
  • peptide C11-35 the mean is 41.1 and the standard deviation (SD) is 29.8; for peptide C40-65 the mean is 30.6 and SD is 27.0; for peptide C346-370 the mean is 38.9 and SD is 30.9; and for peptide C371-390 mean is 38.0 and SD is 18.7.
  • Figures 4A and 4B are graphical depictions showing antibody binding (to the four noted peptides) for serum samples from normal pigs.
  • NPS pooled normal pig serum 1 :40 dilution
  • Pos sheep polyclonal Ab 1 :200 into NPS.
  • peptide C11-35 the mean is 50.9 and SD is 17.7; for peptide C40-
  • SD is 16.6; and for peptide C371-390 mean is 38.8 and SD is 14.8.
  • the direct immobilization technique comprises the use of amine and/or thiol chemistry to directly immobilize N-labelled cysteine peptides.
  • the present inventors have found that their method of direct immobilization has several critical advantages, including far better detection of lower affinity antibodies (resulting in significantly more precise analyses).
  • the present invention significantly reduces analysis time, allowing for high throughput analyses.
  • the assay of the present invention with its direct immobilization feature permits high throughput by virtue of the fact that, after a given sample is analyzed, the materials used in the assay can quickly be regenerated for analysis of subsequent samples.
  • the direct immobilization techniques of the present invention permit fast and easy washing with reagents such as a HCI-SDS solution (preferably 25 mM HCI-0.25% SDS).
  • the present invention also provides an assay that requires only a small amount of serum, e.g., less than 5 ⁇ l in order to carry out an analysis.
  • Previous assays using a mircotiter plate ELISA format can require much more serum from the patient, e.g., when there is a need to characterize the antibodies produced by the patient.
  • the present inventors have also found that their new method allows for the isotypic identification of the antibodies that are detected without requiring additional samples. (The assay of Soussi et al requires multiple applications of samples to obtain isotype determination).
  • Preferred peptides for use in the method of the present invention are selected from the group consisting of:
  • substantially sequence identity means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more.
  • residue positions which are not identical differ by conservative amino acid substitutions. For example, the substitution of amino acids having similar chemical properties such as charge or polarity are not likely to effect the properties of a protein. Examples include glutamine for asparagine or glutamic acid for aspartic acid.
  • a plurality of selected immunogenic peptides can be directly immobilized, each on its own separate flowcell.
  • a first peptide can be selected from the amino terminal region of the p53 protein and a second peptide can be selected from the carboxy terminal region of the p53 protein.
  • the first peptide comprises SEQ ID NO: 1 (H-CEPPLSQETFSDLWKLLPENNVLSPL-Amide)
  • the second peptide comprises SEQ ID NO: 4 (H-CSKKGQSTSRHKKLMFKTEGP- Amide).
  • a third peptide comprising SEQ ID NO: 2 H- CMDDLMLSPDDIEQWFTEDPGPDEAPR-Amide
  • a fourth peptide comprising SEQ ID NO: 3 H-CEALELKDAQAGKEPGGSRAHSSHLK-Amide
  • Phosphorylated peptides can also be used in the method of the present invention. Specifically, a serine residue can be replaced by a phosphoserine residue, and a threonine residue can be replaced by a phosphothreonine residue.
  • peptides that have been used successfully in the method of the present invention have been selected from the group consisting of:
  • the biosensor used in the present invention is preferably a BIAcore
  • the BIAcore 2000TM operates on a principle of surface plasmon resonance and allows for high throughput analyses. (See, e.g., Hodgson, Bio/Technology vol. 12, Jan. 1994 for a review of biosensors). Other biosensors, e.g., a BIAcore X from BIAcore or a lAsys biosensor from Fisons, could be used in connection with the present invention. In analyzing the results of the assay, it is preferred that the amount of antibody that binds to each peptide is directly proportional to the biosensor signal (e.g., the response units that are reported by the BIAcore 2000TM biosensor).
  • the serum samples to be analyzed are diluted within a range of 1 :2 through 1 :500 (more preferably within a range of 1 :2 through 1 :500, more preferably still at a dilution of 1 :40) in a HEPES buffered saline solution containing the detergent P-20 and carboxymethylated dextran, prior to the step of simultaneously contacting the serum samples with the immobilized peptides.
  • a HEPES buffered saline solution containing the detergent P-20 and carboxymethylated dextran prior to the step of simultaneously contacting the serum samples with the immobilized peptides.
  • each peptide is directly immobilized onto the surface of separate flowcells of a sensorchip in the BIAcore 2000TM (BIAcore, Uppsalla, Sweden).
  • Serum samples are diluted (1 :40 in a HEPES buffered saline solution containing the detergent P-20 and carboxymethylated dextran in this example) and allowed to bind to each immobilized peptide simultaneously.
  • the amount of antibody that binds to each peptide is directly proportional to the response units that are reported by the instrument.
  • the flowcells are regenerated simultaneously using 25 mM HCI- 0.25% SDS to remove the bound antibody and the next sample is then analyzed.
  • p53 antibodies can be precisely detected in human and pig serum samples, and further that the assay can be performed in an automated format that allows a high throughput of sample analysis.
  • One sensorchip can be used to analyze pre-dose and post-dose samples from multiple individuals along with a positive control (e.g., sheep polyclonal anti-p53 antibody from Oncogene Sciences or the equivalent) and a negative control (pooled serum) analyzed at the beginning and at the end of each assay.
  • a positive control e.g., sheep polyclonal anti-p53 antibody from Oncogene Sciences or the equivalent
  • a negative control pooled serum
  • Initial criteria for samples to be considered "POSITIVE" for the presence of anti-human p53 antibodies are: 1) binding above threshold to any of the immobilized peptides and 2) that the binding is verified to be due to immunoglobulins.
  • isotyping reagents anti-human lgG1 , anti-human lgG2, anti-human lgG3, anti-human lgG4 anti-human IgA, and anti-human IgM (from ICN or equivalent) could be added in sequence and the binding monitored.
  • An individual is considered to have developed antibodies against p53 if there is binding above threshold and at least a 2-fold increase in response units when comparing a sample obtained after administration of a delivery vehicle (e.g., vector) containing the p53 gene with a sample obtained from the same individual prior to administration.
  • a delivery vehicle e.g., vector
  • construction of an adenovirus p53 gene therapy vector has been described by Wills et al. Human Gene Therapy 5 : 1079 (1994)).
  • Peptide C371-390 was immobilized using amine coupling. For this immobilization, the peptide was diluted to a concentration of 100 ⁇ g/ml into 20 mM borate buffer at pH 8. The remaining three peptides were immobilized using thiol coupling. Peptide C11-35 was diluted to a concentration of 700 ⁇ g/ml into 10 mM sodium acetate buffer pH 4; peptide C40-65 was diluted to a concentration of 1 mg/ml into 10 mM sodium acetate buffer at pH 4.0; and peptide C346-370 was diluted to a concentration of 500 ⁇ g/ml into 10 mM MES buffer at pH 5.
  • Table 1 A summary of the peptide immobilizations is shown in Table 1. (Tables 1 through 7 are grouped together after section X below). The data in Table 1 indicate that there is variability in the peptide immobilizations. This variability does not effect the precision of the assay (discussed in section V below). These data suggest that it is important to verify that the amount of peptide immobilized is sufficient to allow binding of the positive control antibody at a 1 :200 dilution. The data on stability during regeneration with 25 mM HCI and 0.25% SDS (Table 1) indicate that the immobilized peptide surfaces are stable through at least 147 regeneration cycles since the binding among replicates of the positive control assayed after multiple regeneration cycles was within 20.4 % coefficient of variability ("CV”) for all peptides.
  • CV % coefficient of variability
  • the linearity of this assay was determined by assaying the sheep positive control antibody at various dilutions and plotting the response units ("RU") versus the dilution factor of the sample.
  • RU response units
  • 1000 response units (or resonance units) is equivalent to 1 ng/mm 2 ; see, e.g., BIAcore Methods Manual).
  • Figure 1 the response of the positive control diluted in pooled normal human serum is dependent on the concentration of the antibody from a 1 :10 initial dilution through a 1 :2560 dilution.
  • the C346-370 peptide is not recognized as strongly by the positive control as are the remaining three peptides.
  • the precision of this assay for human and pig serum samples was determined by adding the sheep anti-p53 positive control antibody (Oncogene Sciences) at three dilutions (1 :50, 1 :100, and 1 :200) into 2.5% pooled normal human serum and pooled normal pig serum (diluted into HEPES buffered saline containing the detergent P-20 and soluble carboxymethylated dextran) and then assaying multiple aliquots during the same assay (intra-assay precision) and for multiple assays (inter-assay precision). At least 4 aliquots of each dilution were analyzed per day for 5 days.
  • the results for intra-assay precision in human serum samples can be found in Table 2, and the results for inter-assay precision are in Table 3.
  • the intra-assay and inter-assay precision results from pig serum samples can be found in Tables 4 and 5 respectively.
  • the results from the samples analyzed for intra-assay precision in human serum all ranged within 2% CV for each dilution of positive control binding to each peptide.
  • the samples analyzed for inter-assay precision in human serum had CVs that ranged within 16.3% for all dilutions for the three peptides that are fully recognized by the positive control.
  • the CVs from peptide C346-370 ranged from 22.6% to 37.7% and therefore the assay is less precise for this peptide due to the low binding to that peptide by this polyclonal sheep antibody.
  • the samples analyzed for intra-assay precision in pig serum were all analyzed with CVs in the range of 2.6% to 16.1%.
  • the CVs from the samples analyzed for inter-assay precision were all within the range of 3.6% to 17.0%.
  • the threshold is defined as the mean + 3x standard deviation from the binding of the normal human serum samples (at a specific serum sample dilution). Any sample with binding greater than the threshold for any of the immobilized peptides will be considered reactive for the presence of antibodies against human p53.
  • the binding of serum samples from normal pigs is shown in Figures 4A and 4B.
  • the threshold is again defined as mean + 3x the standard deviation from the binding observed with the normal pig samples.
  • Samples will be considered positive for the presence of antibodies capable of binding to human p53 if the following conditions are met: 1) there is binding greater than threshold to any of the immobilized peptides and 2) the binding is found to be a result of immunoglobulin (verified by an increase in binding when an anti-species immunoglobulin is added to the captured anti-p53 antibody).
  • individuals will be considered to have generated antibodies in response to treatment with a p53-containing vector if the following conditions occur: 1) there is binding greater than threshold to any of the immobilized peptides, 2) the binding is found to be a result of immunoglobulin (verified by an increase in binding when an anti-species immunoglobulin is added to the captured serum component), and 3) there is at least a 2-fold increase in binding observed from a sample obtained after dosing with the vector compared with a sample obtained prior to dosing.
  • the human or pig serum samples will be considered to be positive if there is binding to any of the 4 synthetic peptides greater than threshold and that binding is shown to be a result of immunoglobulin by increased binding with an anti-species specific immunoglobulin).
  • Serum samples from cancer patients were tested. As shown in Table 7, two of the 7 samples were verified to contain antibodies capable of binding to human p53. The verification was performed by re-assaying initially positive samples and testing whether the binding to a synthetic peptide could be repeated and also whether there was additional binding upon addition of anti-human IgG/lgA/lgM (Kirkegaard and Perry, Gaithersburg, MD) (or equivalent).
  • Stability was determined by assaying aliquots of the sheep anti-p53 antibody at 1 :50 into 2.5% normal pooled human serum after multiple regeneration cycles using 25 mM HCI and O. 25% SDS
  • NEGATIVE no initial reactivity above threshold to any peptide
  • 2 POSITIVE reactivity against 3/4 peptides, confirmed as Ab
  • 3 NEGATIVE initial reactivity against peptide 4 could not be confirmed as Ab
  • 5 NEGATIVE initial reactivity against peptide 2 could note confirmed as Ab)
  • 6 NEGATIVE no initial reactivity above threshold to any peptide
  • 7 NEGATIVE no initial reactivity above threshold to any peptide
  • Amine Coupling Kit- BIAcore (Code BR-1000-50 ) consisting of:
  • EDC N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide @ 75 mg/ml 1 M Ethanolamine-HCL pH 8.
  • NHS and EDC See Amine coupling Kit ( Pharmacia Biosensor ) above 100 mM Sodium Borate pH 8.5 : Fisher or equivalent
  • PDEA Pharmacia Biosensor (Code BR-1000-58 ) Prepare 80 mM PDEA by dissolving 0.036 gms. into 2.0 ml 0.1 M Borate pH 8.5 (Note: This must be prepared immediately before use and Discarded after 2 hours after preparation) (Also note: one can use other equivalent reagents (besides PDEA for introducing reactive disulfide groups onto amino groups).
  • Formic Acid Aldrich or equivalent Sodium Formate: Aldrich or equivalent; Prepare a 100 mM formate by dissolving 0.061 gms. sodium formate into 25 ml deionized water and adjust pH with Formic acid to pH 4.3 Sodium chloride (NaCI) : Fisher or equivalent;
  • Cysteine Sigma Chemical or equivalent; Prepare a 50 mM cysteine / 1 M sodium chloride by dissolving 0.061 gms. cysteine and 0.5844 gms. NaCI into 10.0 ml 0.1 M formate pH 4.3 ( Note: This must be prepared immediately before use- Discard 2 hours after preparation)
  • HEPES buffered saline containing P-20 surfactant( per 1 L) 2.38 g HEPES (Fisher or equivalant)
  • Re-pH and adjust to pH 7.4 if necessary 7.
  • Regeneration solution 25 mM HCL / 0.25 % SDS
  • Sample Diluent HBS w/ P-20 and CM-D;
  • Negative Control Serum to match matrix of samples analyzed and 0.2 ⁇ filtered. 5.
  • Positive Control Sheep polyclonal anti-p53 antibody (Oncogene
  • Sample response units (RU) measured for each peptide are then determined to be "Positive" by the following criteria: 1- If the sample RU is less than each peptide threshold of all four peptides for that specific sample dilution, then the sample is considered
  • Threshold RU for that peptide for that specific-sample dilution then the sample is repeated.
  • the repeat sample is confirmed by the addition of a anti- species specific antibody to verify that the RU increase measured is due to antibody.
  • the sample is considered "Positive" for p53 antibodies.
  • the confirming anti-species specific antibody or isotyping reagents can be added after the initial serum sample has bound (prior to addition of the regeneration solution)).

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

L'invention a trait à une méthode de détection d'anticorps capables de se fixer à la protéine p53 faisant intervenir un biocapteur à résonance de plasmon de surface porteur de peptides de p53 immobilisés.
EP97944306A 1996-10-07 1997-10-01 EPREUVE POUR LA DETECTION D'ANTICORPS CONTRE p53 Withdrawn EP0929811A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US2853396P 1996-10-07 1996-10-07
US28533P 1996-10-07
PCT/US1997/016132 WO1998015834A1 (fr) 1996-10-07 1997-10-01 EPREUVE POUR LA DETECTION D'ANTICORPS CONTRE p53

Publications (1)

Publication Number Publication Date
EP0929811A1 true EP0929811A1 (fr) 1999-07-21

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EP97944306A Withdrawn EP0929811A1 (fr) 1996-10-07 1997-10-01 EPREUVE POUR LA DETECTION D'ANTICORPS CONTRE p53

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EP (1) EP0929811A1 (fr)
JP (1) JP2001502419A (fr)
KR (1) KR20000048833A (fr)
CN (1) CN1240028A (fr)
AU (1) AU4583397A (fr)
BR (1) BR9712504A (fr)
CA (1) CA2267200A1 (fr)
HU (1) HUP9904222A3 (fr)
IL (1) IL129323A0 (fr)
NZ (1) NZ334809A (fr)
WO (1) WO1998015834A1 (fr)

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EP1090111A1 (fr) * 1998-06-25 2001-04-11 Institut Pasteur Analyse exhaustive d'interactions de proteines virales au moyen d'un criblage a deux hybrides et selection de polypeptides a interaction virale correctement plies
AUPP932199A0 (en) * 1999-03-19 1999-04-15 St Vincent's Hospital Sydney Limited Anti-p53 antibodies
CN1217194C (zh) * 2001-01-04 2005-08-31 上海数康生物科技有限公司 蛋白芯片及其制备方法和使用方法
EP1914550A1 (fr) * 2001-05-07 2008-04-23 HiPep Laboratories Substrat immobilisé à peptide et procédé de mesure d'une protéine cible l'utilisant
KR20030092462A (ko) * 2002-05-30 2003-12-06 박영미 암의 진행 정도를 진단하는 방법 및 키트
ES2610806T3 (es) * 2005-12-22 2017-05-03 Abbott Molecular Inc. Métodos y combinaciones de marcadores para la detección de la predisposición al cáncer de pulmón
CN102066937A (zh) 2008-01-25 2011-05-18 P53股份有限公司 P53生物标记物
WO2013042603A1 (fr) * 2011-09-20 2013-03-28 コニカミノルタホールディングス株式会社 Liquide pour diluer un spécimen, kit et procédé fluorométrique l'utilisant
WO2013116455A1 (fr) * 2012-01-31 2013-08-08 Bio-Rad Laboratories, Inc. Sensibilité et spécificité améliorées pour le cancer des ovaires
LT3201234T (lt) * 2014-09-30 2019-03-12 Diadem S.R.L. Antikūnas, surišantis linijinį žmogaus p53 epitopą, ir jo panaudojimas diagnostikai
CN112964870B (zh) * 2021-02-03 2024-01-26 军事科学院军事医学研究院军事兽医研究所 一种基于spr生物传感器的流感病毒快速检测方法

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FR2698367B1 (fr) * 1992-11-02 1995-02-17 Eurobio Lab Fragments de la protéine p53 et leurs utilisations dans la détection et le suivi d'états pathologiques.
IL108726A (en) * 1994-02-22 1999-12-31 Yissum Res Dev Co Electrobiochemical method and system for the determination of an analyte which is a member of a recognition pair in a liquid medium and electrodes therefor

Non-Patent Citations (1)

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Title
See references of WO9815834A1 *

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HUP9904222A3 (en) 2000-08-28
CN1240028A (zh) 1999-12-29
CA2267200A1 (fr) 1998-04-16
JP2001502419A (ja) 2001-02-20
WO1998015834A1 (fr) 1998-04-16
KR20000048833A (ko) 2000-07-25
IL129323A0 (en) 2000-02-17
BR9712504A (pt) 1999-10-19
HUP9904222A2 (hu) 2000-04-28
NZ334809A (en) 2000-12-22
AU4583397A (en) 1998-05-05

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