WO2007103538A2 - Procedes de marquage d'anticorps multiples et utilisations desdits procedes - Google Patents

Procedes de marquage d'anticorps multiples et utilisations desdits procedes Download PDF

Info

Publication number
WO2007103538A2
WO2007103538A2 PCT/US2007/005996 US2007005996W WO2007103538A2 WO 2007103538 A2 WO2007103538 A2 WO 2007103538A2 US 2007005996 W US2007005996 W US 2007005996W WO 2007103538 A2 WO2007103538 A2 WO 2007103538A2
Authority
WO
WIPO (PCT)
Prior art keywords
cell
target
antibodies
sample
methods
Prior art date
Application number
PCT/US2007/005996
Other languages
English (en)
Other versions
WO2007103538A3 (fr
Inventor
David N. Krag
Edward A. Manna
Original Assignee
University Of Vermont And State Agricultural College
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 University Of Vermont And State Agricultural College filed Critical University Of Vermont And State Agricultural College
Publication of WO2007103538A2 publication Critical patent/WO2007103538A2/fr
Publication of WO2007103538A3 publication Critical patent/WO2007103538A3/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
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • G01N33/57492—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 compounds localized on the membrane of tumor or cancer cells
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus

Definitions

  • the invention relates, in part, to methods of using two or more antibodies to label and identify cells, organisms, and/or additional targets of interest.
  • the invention additionally relates, in part, to diagnostic assays for disorders such as cancer, identification of organisms or compounds in samples or on surfaces, and to assays to assess cell status, such as cell differentiation and cell lineage.
  • Rare cell events are difficult to assess and confirm using currently available assay methodologies. For example, the detection of one or a few metastatic cancer cells in lymph node or other tissue is extremely difficult, which make it difficult to accurately diagnose metastases in patients. Identification of the existence of metastases in a patient can be of critical importance to providing an accurate diagnose and selection of appropriate treatment strategies for cancer patients. Inaccurate results in diagnostic assays for cancer and in other rare cell events, may be the result of false positives. A false positive result may indicate that a subject has cancerous cells and results in treatment, when in fact the cells were not cancerous and the treatment unnecessary. Presently available diagnostic assays using brightfield immunocytochemistry have been demonstrated to result in the detection of false positives (Krag, D.
  • results from such assays may be erroneously "read" as positive when the samples tested are healthy samples or are samples that are being tested using irrelevant (e.g., negative control) antibodies.
  • the invention relates in part to the discovery that two or more antibodies or antigen- binding fragments thereof that are raised in different species can be used in assays for specific and sensitive detection of cells, organisms, compounds, or other targets of interest.
  • the methods of the invention may be used to detect a cell, organism, compound, or other target in a sample, including targets that are rare and only make up a small proportion of the total sample.
  • the methods of the invention allow detection of a target when only one, two, three, or more of the target entity is present in the sample.
  • the target is a cell that is associated with a disease or condition and the methods of the invention may be used to diagnose or assess the presence of a disease or condition in a sample or a subject.
  • the methods of the invention may also be useful to quantify how much of a target entity is present in a sample.
  • the invention may also be used to test two or more samples, which may be obtained at different times, to compare the amount of a target in the samples as a measure of the change in amount of the target over time. The comparison can be useful to assess the onset, progression, or regression of a disease or changes in a condition associated with the target.
  • the methods of the invention can also be used to monitor response to a treatment of a disease associated with a target and thus may also be useful for screening compounds for therapeutic use or for assessing the efficacy of a treatment of a target-associated disease or condition in a subject. According to one aspect of the invention, methods of identifying a target in a biological sample are provided.
  • the methods include (1) contacting the biological sample with two test antibodies and one negative control antibody, wherein: (a) the two test antibodies are antibodies that specifically bind the target; (b) the two test antibodies are antibodies that are derived from different species, and (c) the negative control antibody is an antibody that does not specifically bind the target, (2) determining the presence of specific binding of the test and negative control antibodies to a common entity in the biological sample, wherein specific binding to the common entity of the two test antibodies derived from different species and no specific binding of the negative control antibody to the common entity, identifies the common entity as the target in the biological sample.
  • the methods also include contacting the biological sample with one or more additional test antibodies and/or one or more additional negative control antibodies.
  • the target is a cell.
  • the target is an organism. In some embodiments, the target is a compound. In some embodiments, the compound is a contaminant or a toxin. In certain embodiments, the biological sample is from a subject. In some embodiments, the target is a cancer cell. In some embodiments, the cancer cell is an estrogen/progesterone- (ER/PR) and/or HER2-positive cell. In certain embodiments, the target is a virally infected cell. In some embodiments, the virally infected cell is infected with human papillomavirus. In some embodiments, the target in the sample from the subject is a cancer cell and its presence is diagnostic for cancer in the subject, hi some embodiments, the target is a stem cell.
  • the target is a cancer stem cell.
  • the cancer stem cell is a CD44 + CD24 '/low Lineage " cancer cell.
  • the biological sample includes cells from culture.
  • the biological sample is an in vivo sample, hi some embodiments, the biological sample is a blood, tissue, lymph, or body fluid sample.
  • the methods also include contacting the cell with a positive-control antibody, hi certain embodiments, the specific binding is determined using immunodetection means.
  • the immunodetection means includes detecting a detectable label that is either directly or indirectly attached to the test and/or control antibodies.
  • the test and/or control antibodies includes a detectable label, hi some embodiments, the test and/or control antibody is contacted with a secondary antibody that includes a detectable label.
  • the detectable label is a fluorescent, enzyme, radioactive, metallic, biotin, chemiluminescent, bioluminescent, or chromophore label.
  • the methods also include determining the amount of the target of interest in the sample.
  • one or more of the species is a non-mammalian species.
  • the non-mammalian species is an avian species.
  • the avian species is chicken.
  • methods of identifying a phenotype of a cell include contacting the cell with (a) a negative-control antibody that does not specifically bind to a cell with the phenotype, and (b) two test antibodies that specifically bind a cell with the phenotype, wherein the two test antibodies are derived from different species; and determining the specific binding of the test and negative control antibodies to the cell, wherein specific binding to a common cell of the two test antibodies derived from different species and no significant binding of the negative control antibody to the common cell identifies the common cell as having the phenotype.
  • the methods also include contacting the cell with one or more additional test antibodies and/or one or more additional negative control antibodies.
  • the cell is obtained from a biological sample.
  • the biological sample is from a subject.
  • the cell is an in vivo cell.
  • the cell phenotype is cancer.
  • the cancer cell is an estrogen/progesterone- (ER/PR) and/or HER2-positive cell.
  • the presence of a cell with a cancer phenotype in the sample is diagnostic for cancer.
  • the target is a virally infected cell.
  • the virally infected cell is infected with human papillomavirus.
  • the biological sample includes cells from culture.
  • the biological sample is a blood, tissue, lymph, or body fluid sample.
  • the methods also include contacting the cell with a positive-control antibody.
  • the phenotype is a stem cell phenotype.
  • the phenotype is a cancer stem cell phenotype.
  • the cancer stem cell phenotype is: CD44 + CD24 "/low Lineage " .
  • the binding is determined using immunodetection.
  • the immunodetection includes detecting a detectable label that is either directly or indirectly attached to the test and/or control antibodies.
  • the test and/or control antibody includes a detectable label.
  • the test and/or control antibody is contacted with a secondary antibody that includes a detectable label.
  • the detectable label is a fluorescent, enzyme, radioactive, metallic, biotin, chemiluminescent, bioluminescent, or chromophore label.
  • one or more of the species is a non-mammalian species.
  • the non- mammalian species is an avian species.
  • the avian species is chicken.
  • the methods of the invention permit specific and sensitive detection of a target of interest in a sample.
  • the assay methods of the invention can be used to detect target-associated diseases or conditions and may be used in diagnostic methods, drug screening methods, and monitoring methods, etc. and are useful both in research and for clinical use.
  • the methods of the invention include the detection of a target using two or more test antibodies or antigen-binding fragments thereof that specifically bind the target.
  • An important aspect of the methods of the invention is the use of two or more test antibodies that are raised in different animal species and specifically bind the same target.
  • the assay methods described herein include antibody-based assays for determining the presence or absence of a target that is specifically bound by the two or more antibodies or antigen- binding fragments thereof raised in different species against the same target.
  • the invention includes contacting a sample with two or more test antibodies or antigen-binding fragments thereof and also includes contacting the sample with one or more negative-control antibodies or antigen-binding fragments thereof.
  • the assay also includes one or more positive-control antibodies or antigen- binding fragments thereof.
  • negative control antibody means an antibody that does not specifically bind a target of interest.
  • positive control antibody means an antibody that specifically binds a constituent known to be present in the sample being tested. The detection of binding of a positive-control antibody serves to verify that the assay conditions are operational.
  • the term "common entity” means the same entity. Thus, if two antibodies bind to a common entity, they bind to the same entity.
  • an entity may be a cell, a molecule, a tissue, a contaminant, etc.
  • test antibody means an antibody that is known to specifically bind a target of interest.
  • at least two of the two or more test antibodies are antibodies that are derived from different species or different isotypes of the same species, and the one or more negative control antibodies are antibodies that do not specifically bind the target of interest.
  • the methods of the invention also include determining whether there is specific binding of the test antibodies and negative-control antibodies to a common target in the biological sample. Detection of specific binding of two or more test antibodies derived from different species to a common target with no significant specific binding of a negative control antibody to the common target, identifies the common target as the target of interest.
  • three or more test antibodies that are raised in different species can be used in the methods of the invention.
  • the invention involves, in part, antibodies and antigen-binding fragments thereof of numerous size and type that bind specifically to targets of interest.
  • an antibody that is raised against a target in mouse can be contacted with a sample or surface along with an antibody raised against the same target in rabbit and if a common entity is detected in the sample or on the surface, to which both antibodies bind, and there is no significant specific binding of a negative-control antibody, then the assay identifies the target of interest as being present in the sample or on the surface.
  • test antibodies raised in different species are antibodies that are raised against the same target, but may or may not be raised against the same antigen.
  • two or more test antibodies used in the methods of the invention need not recognize the same epitope.
  • the two or more test antibodies raised in different species may recognize the same epitope, may recognize overlapping epitopes, or may not recognize the same epitope. Those of skill in the art will understand that although the two or more test antibodies may not recognize the same epitopes, they do specifically bind to the same target for use in the methods of the invention.
  • the two or more test antibodies that are raised in different species may be the same isotype or may be different isotypes.
  • three or more test antibodies that are raised in different species can be used in the methods of the invention, hi some embodiments, each test antibody is an antibody raised in a different species than the other test antibodies.
  • An example, though not intended to be limiting is the use of three test antibodies with each test antibody raised in a different species than the other test antibodies.
  • the methods of the invention may include combinations of test antibodies raised in the same species with test antibodies raised in different species, as long as at least two antibodies raised in different species are utilized in the methods.
  • the two or more antibodies that specifically bind the target of interest are able to simultaneously bind the same target.
  • the two or more antibodies that are raised in two or more different species do not compete with each other or interfere with each other's specific binding to the target.
  • a target is a biological or chemical entity that is recognized and specifically bound by an antibody or antigen-binding fragment thereof.
  • a target can be recognized and bound by an antibody or antigen-binding fragment thereof because the target includes a specific epitope on a molecule, an overlapping epitope on a molecule, an antigen, a marker for a specific phenotype, etc.
  • targets include, but are not limited to: a cell with a specific phenotype (e.g., a disease-associated cell, a cell of a specific developmental stage; a cell of a specific lineage or type); an organism; a molecule; a compound; a toxin; etc.
  • the binding of an antibody to a target that is a cell may be predictive of a phenotype in that cell.
  • a target may be an entity that is of interest to detect or identify using a method of the invention.
  • targets in the context of the invention include, but are not limited to, a cell with a phenotype or phenotypes of interest, an organism, a compound, or other entity that can be specifically bound by an antibody and is detectable using the methods of the invention.
  • the methods of the invention can be used to identify whether or not a cell with a phenotype of interest, for example a stem cell, is present in a sample.
  • the sample can be contacted with a cocktail of multiple antibodies, including antibodies that specifically bind hematopoetic cells and additional sets of antibodies that specifically bind stem cells, plasma cells, and/or cells of erythrocyte origin. Detection of binding of two or more antibodies, raised in different species, and that specifically bind stem cells and no significant specific binding of a negative-control antibody (e.g., an antibody that specifically binds a hematopoetic cell) indicates the presence of one or more stem cells in the sample.
  • a negative-control antibody e.g., an antibody that specifically binds a hematopoetic cell
  • target-associated disease or condition means an illness or state that is associated with a target of interest.
  • a particular development stage or state of a tissue or cell may be associated with the presence or absence of a target in a cell or tissue.
  • the developmental state or stage would be considered to be a target- associated condition.
  • a form of cancer may be associated with the presence of target that is a cancer cell in a sample.
  • the cancer would be considered to be a target- associated disease.
  • a target-associated disease is considered a illness but a target-associated condition may be a developmental stage, or other normal stage or state is not an indication of disease or of an abnormal physiological process.
  • a cell target may be a cell that has a phenotype of interest such as a stem cell, a cancer cell, or other specific type of cell or disease-associated cell.
  • a cell that can be identified using the methods of the invention may be associated with a disease.
  • Cells with a cancer-associated phenotype are non-limiting examples of cells that have a phenotype of interest that can be identified using the methods of the invention.
  • Other cells with a disease- associated phenotype include, but are not limited to: "decoy" cells in the urothelium indicating human polyoma virus, atypical squamous cells in cervicovaginal epithelium indicating Human Papillomavirus (HPV) infection or pre-cancerous changes or lupus erythematosis (LE) cells indicating Lupus Erythematosis, Reed-Stemberg cells indicating Hodgkin's Disease.
  • Additional types of cells that may be targets according to the methods of the invention are cells with specific lineages, cells that are at a particular developmental stage, or cells that have some other characteristic that allows specific binding of an antibody to the cell.
  • a cell with a lineage-specific phenotype may be detected using two or more antibodies raised in different species that specifically bind to a cell that has the lineage-specific phenotype of interest.
  • a cell at a particular stage of development may express a polypeptide that is specifically bound by two or more antibodies using methods of the invention and thus can be identified.
  • the methods of the invention allow the detection of the presence or absence of a target cell, and also may be used to quantify the number of the target cell type in a sample or per unit volume of a sample.
  • a target may be a cell that has a disease- associated phenotype.
  • the term "disease-associated phenotype" of a cell means that the cell has a phenotype that is associated with a disease.
  • a cancer cell has a cancer-associated phenotype.
  • a sample can be contacted with a combination of antibodies that will specifically bind to a cell with the disease-associated phenotype and the presence or absence of specific binding of the antibodies to a common cell in the sample can be used to detect whether or not a cell with the disease-associated phenotype is present in the sample.
  • two or more cytokeratin antibodies that are raised in at least two different species can be contacted with a sample to detect the presence or absence of a cancer cell in the sample.
  • diseases in which cells have detectable phenotypes that are indicative of the disease include cancers, infections, Alzheimer's disease, auto-immune diseases, allergic reactions, arthritis, anemias, and benign neoplasms.
  • a target may be a cell that has a developmental stage-associated phenotype.
  • developmental-stage- associated phenotype in a cell means that the cell has a phenotype that is associated with a particular stage of development.
  • a sample can be contacted with a combination of antibodies that will specifically bind to a cell with the a developmental stage-associated phenotype and the presence or absence of specific binding of the antibodies to a common cell in the sample can be used to detect whether or not a cell with the developmental stage-associated phenotype is present in the sample.
  • one or more cells that have a phenotype that is associated with a developmental stage can be detected.
  • Non-limiting examples of cells with developmentally associated phenotypes include, but are not limited to, progenitor cells, precursor cells, immature cells, mature cells,, etc.
  • one or more cells that have a phenotype that is associated with a particular disease or cell type can be detected.
  • diseases or cell types that can be detected using methods of the invention, include, but are not limited to: leukemias, lymphomas, sarcomas, well-differentiated versus poorly differentiated carcinomas.
  • Methods of the invention can be used to diagnose types of lymphomas and leukemias in which the identification of certain cell types or mixes of cell types are important for treatment and prognosis.
  • a non-limiting example of such diseases are lymphomas.
  • ACL Anaplastic large cell lymphoma
  • lymphoma diagnostic assays using the methods of the invention is an assay that include the use of antibodies such as ALKl, which specifically binds the formalin-resistant epitope of native anaplastic lymphoma kinase (ALK) protein as well as the fusion of the t(2;5)(p23;q35), nucleophosmin.
  • ALKl antibody is known to specifically bind 50% to 60% of CD30+ ALCL.
  • the ALKl antibody is not known to be effective as a marker for Hodgkin's disease (Reed-Sternberg cells).
  • ALKl antibodies raised in two or more species can be use in the methods of the invention to help identify anaplastic large cell lymphomas.
  • the ALKl antibodies can be used in conjunction with one or more of additional antibodies that specifically bind CD15, CD30, TIA-I, and/or endomysial tissue (EMA antibodies).
  • the methods of the invention may include the use of antibodies that specifically bind to bcl-x as a marker for Reed-Sternberg cells that include the nodular lymphocyte predominance subset of Hodgkin's.
  • the Ki-I (CD30) antigen is expressed in mononuclear Hodgkin's and multinucleated
  • Reed-Sternberg cells in Hodgkin's disease It is expressed by the tumor cells of a majority of anaplastic large cell lymphomas, and by a varying proportion of activated T and B cells.
  • CD30 is also expressed on embryonal carcinomas.
  • the CD30 monoclonal antibody from the Ber-H2 cell line can be used to distinguish large cell lymphomas derived from activated lymphoid cells, from histiocytic malignancies and lymphomas derived from resting and precursor lymphoid cells, or from anaplastic carcinomas.
  • CD30 and CDl 5 primary antibodies may be used in tandem to differentiate between anaplastic large cell lymphoma and Hodgkin's disease (Reed-Sternberg cells).
  • the methods of the invention may include the use of antibodies raised in two or more species to detect cancer stem cells.
  • cancer stem cells For example, in breast cancer, Al- Hajj et al, 2003 have found that tumorigenic cells can be identified and isolated using flow cytometry as CD44 + CD24 " ⁇ ow Lineage " cells.
  • the methods of the invention can be used to accurately detect the presence of such cells using antibodies raised in different species that specifically bind to cancer stem cell markers such as the CD44, CD24 markers, etc.
  • specific cancer stem cells can be identified even when they make up only a very small portion of a sample for analysis. Additional cancer stem cells that are recognized in the art can also be detected using the methods of the invention.
  • a target may be a cell that has a cell type- associated phenotype.
  • the term "cell type-associated phenotype" of a cell means that the cell has a phenotype that is associated with a particular cell type.
  • a sample can be contacted with a combination of antibodies that will specifically bind to a cell with a cell type-associated phenotype and the presence or absence of specific binding of the antibodies to a common cell in the sample can be used to detect whether or not a cell with the cell type-associated phenotype is present in the sample.
  • one or more cells that have a phenotype that is associated with a target cell type can be detected.
  • the methods of the invention can be used to identify cells that have a phenotype that indicates the identity of the type of cell. For example, in early development, differentiation decisions are made and a cells fate may be determined to be either a neuronal cell or a epithelial cell.
  • a cell can be contacted with antibodies that permit the identification of the cell as having a phenotype associated with a neuronal cell type versus an epithelial cell type, a stem cell phenotype, a blood cell phenotype, etc.
  • stem cell development and cell fate determination are examples of cells that have cell-type-associated phenotypes that can be determined using the assay methods of the invention.
  • methods of the invention may include assays to detect the presence or absence of specific features on cancer cells.
  • the methods of the invention can be used to detect cells that have a phenotype of HER2 expression, are ER/PR cells, etc.
  • the methods of the invention that utilize antibodies that are raised in two or more species can be used to detect additional specific phenotypic features of cells, including, but not limited to cancer cells.
  • phenotypic markers that can be identified using the antibody-binding methods of the invention
  • it is a phenotypic characteristic of a cell that permits the cell's identification and its detection as a target using the assay methods of the invention.
  • Some phenotypes are associated with disease and the methods of the invention are also useful to identify normal stages and developmental characteristics of cells. It will be clear to those of skill in the art that not all cell type- or developmental stage-associated phenotypes are indicative of an abnormality of the cell or are indicative of illness.
  • Some developmental stage- or cell type-associated phenotypes represent a normal state of a cell or tissue in development, growth, healing, and day-to-day cellular operations.
  • a disease-associated phenotype in a cell may indicate an illness, injury, or other abnormal status in a cell.
  • the disease or condition is associated with a phenotype that can be detected using the methods of the invention.
  • Diseases, cell types, and developmental stages that have phenotypes that may be detectable using methods of the invention include, but are not limited to cancer, neurodegenerative diseases (e.g., Parkinson's disease (PD), Alzheimer's disease, etc.), normal cell and tissue development, normal cell and tissue aging, stroke, cardiovascular disease, bacterial infection, toxin exposure, CNS diseases, metabolic disorders, infections, and prion disease, etc.
  • the methods of the invention may also be used to identify the presence or absence of an organism.
  • types of organisms that can be detected using the methods of the invention may be microorganisms, infectious organisms, parasitic organisms, or other organisms whose presence or absence is desirable to detect.
  • Bacteria are a non-limiting example of organisms that can be targets in some embodiments.
  • a blood or tissue sample may be assayed using the methods of the invention to determine the presence or absence of a specific strain of bacterium.
  • the methods may also be used to quantify the number of bacteria in a sample or per unit volume of a sample.
  • a non-limiting example of a particular strain of bacterium that may be detected using methods of the invention is the strain of E.
  • the methods of the invention can be used to identify whether or not the strain of bacterium is present in a sample such as a tissue sample, body fluid sample, water sample, soil sample, air sample, etc.
  • a sample such as a tissue sample, body fluid sample, water sample, soil sample, air sample, etc.
  • a target may be a compound that can be specifically bound by an antibody.
  • a compound may be, for example, a peptide toxin whose presence in a biological sample desirable to detect.
  • An example of such a peptide though not intended to be limiting is a spider or other animal venom.
  • the methods of the invention can be used to detect the presence or absence of such a venom in a sample.
  • Those of ordinary skill in the art will recognize compounds that can be identified using the methods of the invention include compounds to which an antibody will specifically bind, thus, there are numerous compounds to which the assay methods of the invention can be applied.
  • the invention includes methods to detect a cell, organism, compound, or other target in a sample, including, but not limited to, targets that are rare and make up a only small proportion of the total sample.
  • the methods of the invention allow detection of a target when only one, two, three, or more of the target entity is present in the sample.
  • the target is a cell that is associated with a disease or disorder and the methods of the invention may be used to diagnose or assess the presence of a disease or disorder in a sample or a subject. The methods of the invention may also be useful to determine how much of the target is present in a sample.
  • the methods of the invention may be used to quantify the presence of a cell with a phenotype of interest that is in a sample, or the number of a target organism in a sample, or the amount of a target compound in a sample.
  • the determination of the amount of a target allows the use of the methods of the invention to comparative samples obtained a separate times to assess change in the presence and/or amount of a target over time. Change in the presence of amount of a target can be assessed by detecting the presence and/or amount of the target in multiple samples that have been obtained at separate times. This is useful to assess change in the target over time and allows determination of the onset, progression, or regression of a disease or condition associated with the target cell, organism, compound, contaminant, etc.
  • the methods of the invention can also be used to monitor response to a treatment of a condition associated with a target cell, organism, or contaminant and thus may also be useful for screening compounds for treatment or for assessing the efficacy of a treatment in a subject.
  • An additional aspect of the invention includes the detection of targets such as microorganisms or toxins on surfaces.
  • the methods of the invention include detecting the presence or absence and/or amount of a target on a surface.
  • the methods may be used to assay a surface for targets, e.g., for microorganisms or compounds, or cells, and may be applied to detect a target on a surface or material.
  • an example, although not intended to be limiting is the use of the methods of the invention to monitor for prion-associated contamination of tissue (e.g., prior to transplantation), biological samples (e.g., blood), and tissue for use in the food or cosmetic industries.
  • Assays of the invention that utilize antibodies that specifically bind to a prion target can also be used to monitor objects such as, but not limited to: surgical instruments and animal-processing equipment, food, and cosmetics for prion contamination.
  • the methods of the invention may be used to monitor objects for targets in addition to the use of the methods for assaying biological samples and other liquid samples.
  • a cocktail of different antibodies made against a target as well as negative and/or positive control antibodies may be contacted with a sample to give colored (e.g., fluorescent) readouts that indicate the presence or absence of the target in the sample.
  • colored (e.g., fluorescent) readouts that indicate the presence or absence of the target in the sample.
  • the presence of specific binding of two or more antibodies that are raised in different species and recognize the same target, and the lack of significant non-specific binding by a control antibody to the target indicates that the target of interest is present in the sample.
  • These methods can be used in diagnostic methods for clinical application and are also useful as research tools to study the cell development, differentiation, or disease and the effect on the target by contact with a pharmaceutical agent.
  • the invention includes, in part, reliable and sensitive assays and methods to determine the presence or absence of a target of interest in a sample and the use of the assays of the invention for diagnostic and screening methods.
  • the methods of the invention include, in part, the determination of the amount of a target of interest and are carried out on a sample. In addition to identifying the presence or absence of the target of interest in a sample, the methods can be used to determine the amount of a target in a sample and/or the change in the amount over time. The methods of the invention can also be used to determine the amount of a target in a cell, tissue, subject, and may be used to diagnose the state of cell differentiation, the identity of cell lineage, or the presence of disease- associated cells.
  • the methods also may be used to determine the amount of a target in a sample such as a water sample, a soil sample, an air sample, etc., hence the sample need not be a "biological" sample but may also include other environmental, materials, chemical, etc. samples.
  • the methods of the invention involve determining the amount of a target in a sample and that amount is then compared to a control amount of the target.
  • a sample is a biological sample obtained from a subject.
  • a sample can be synthetic or (e.g., laboratory prepared) and not obtained from a subject.
  • the term "subject" includes vertebrate and invertebrate organisms. Examples of invertebrate organisms include, but are not limited to, drosophila, nematodes, etc. Vertebrate subjects may include fish, birds, and mammals. Subjects include but are not limited to: humans, non-human primates, cats, dogs, sheep, pigs, horses, cows, rodents such as mice, rats, hamsters, gerbils, etc.
  • a subject is known to have, or is considered to be at risk of having, a disease or condition associated with a particular target — e.g., a cell development-associated phenotype, a disease-associated phenotype, a cell-type associated phenotype, a microorganism infection, or the presence of another target compound.
  • a subject is a mammal that is an animal model for a particular cell phenotype or other condition that is detectable using the methods of the invention.
  • animal models of a cell development-associated phenotype, a disease-associated phenotype, or a cell-type associated phenotype may be generated by genetic engineering or by chemical or physical treatment or infection to alter the cell phenotype and/or the presence of other targets in the animal.
  • a "biological sample” encompasses a variety of sample types obtained from an individual through invasive or non-invasive approaches (e.g., urine collection, blood drawing, needle aspiration, and other procedures).
  • the definition also includes samples that have been manipulated in any way after their procurement (through invasive or non-invasive approaches), such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides.
  • biological sample includes, but is not limited to, any body tissue or body fluid sample obtained from a subject. Body fluids include: urine, blood, saliva, lacrimal fluid, synovial fluid, cerebrospinal fluid, sweat, pulmonary secretions (sputum), seminal fluid, and feces.
  • Body tissues may be from skin, nerve, CNS tissue, tumor tissue, etc.
  • a biological sample may be cells or tissue in and obtained from culture as well as cells or tissues in or obtained from a subject. Examples of cells or tissues in culture that may be used in the methods of the invention as test cells tissues or as control cells or tissues include cells or tissues known to be afflicted with a condition associated with a target that can be detected using the methods of the invention.
  • cells may not be afflicted with a condition that is associated with the target of interest and may serve as control cells or tissues.
  • Cells and tissues that are free of a specific target of interest may be examined in parallel with a test cell or tissue and may serve as a control cell or tissue.
  • Such control cells and tissues may be useful to determine a "normal" level of a target in a sample.
  • a cancer-free sample may be a control for a cancer test sample.
  • the presence or absence and/or amount of a target of interest may be determined in a cell and/or tissue that is in vivo, e.g., in a subject.
  • a biological sample may be a cell or tissue located in vivo.
  • the invention provides a method for detecting the presence and/or amount of a target of interest in cells and/or tissue in vivo. The methods include, in part, administering to a subject two or more antibodies that selectively bind a target of interest, (with two or more of the antibodies raised in different species), and one or more negative-control antibodies.
  • the antibodies may include a detection means, such as a detectable label, and the subject is exposed to a means for detecting the detectable label in the cells and/or tissues of the subject — e.g., via NMR, confocal microscopy, tomography, etc, and the presence or absence and/or amount of the target in the subject is determined.
  • a detection means such as a detectable label
  • two or more antibodies that bind specifically to a target can be prepared and used to identify and quantify the amount of the target in a sample.
  • binding specifically to or “specifically binds” mean capable of distinguishing the identified material from other materials sufficient for the purpose to which the invention relates.
  • “specifically binds" the target means that the antibody(or antigen- binding fragment thereof) has the ability to bind to and distinguish target from other polypeptides and proteins.
  • An antibody that specifically binds to a target may preferentially bind to target with an affinity that is at least two-fold, 50-fold, 100-fold, or greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the target.
  • a non-specific antigen e.g., BSA, casein
  • detection of specific binding of two or more antibodies that bind to the target (with two or more of the antibodies raised in different species) and no significant specific binding of a negative-control antibody to the target indicates the presence of the target in the sample.
  • no significant specific binding means that the negative control antibody does not specifically bind to the target.
  • Methods to determine the presence or absence and/or amount of a target may include the use of antigen-binding fragments of antibodies that bind to the target to detect presence or amounts of the target as described herein.
  • An antigen-binding fragment of an antibody is a fragment of the antibody that retains the specific binding function of the whole antibody and has the ability to specifically bind to the same antigen target as the antibody.
  • antibody means an antibody or an antigen-binding fragment thereof.
  • An antigen-binding fragment of an antibody that is useful in the methods of the invention can also be used in the methods of the invention.
  • Antibodies and antigen-binding fragments thereof can be used in the methods of the invention to determine the presence or absence and/or amount of a target using known methods including, but not limited to, immunocytochemistry, flow cytometry, enzyme linked immunosorbent (ELISA) assays, immunoprecipitations, electrophoretic methods, chromotographic methods, and Western blots, etc.
  • Antibodies or antigen-binding fragments thereof may be used to determine the presence or absence and/or amounts of a target using additional standard methods known to those of ordinary skill in the art.
  • Antibodies useful in the methods of the invention may be conjugated to a solid support.
  • the antibodies of the present invention maybe prepared using a variety of methods, including administering protein, fragments of protein, cells expressing the protein or fragments thereof and the like to an animal to induce polyclonal antibodies.
  • the production of monoclonal antibodies is according to techniques well known in the art. As detailed herein, such antibodies or antigen-binding fragments thereof may be used for example to identify the presence or absence and/or amount of a target.
  • the antibodies of the invention include monoclonal and polyclonal antibodies.
  • species suitable for raising antibodies for use in the methods of the invention include, but are not limited to, rabbit, mouse, rat, guinea pig, sheep, bovine, canine, avian (e.g., chicken), donkey, feline, goat, hamster, horse, human, porcine, Camelidae (e.g., llama, alpaca, and camel, etc.), ungulates, non-human primate, etc.
  • avian e.g., chicken
  • donkey feline
  • goat goat
  • hamster horse
  • human porcine
  • Camelidae e.g., llama, alpaca, and camel, etc.
  • ungulates non-human primate, etc.
  • the invention includes the use of antibodies raised in birds (e.g., chickens) or other organisms, wherein the antibodies have different morphologic characteristics than antibodies that raised in a mammalian species. Morphological differences between antibodies such as avian antibodies and mammalian antibodies, may affect nonspecific binding affinity and may increase sensitivity of the assays and be advantageous in methods of the invention.
  • chicken antibodies which are IgY and not IgG, may be used in some embodiments of the invention.
  • IgY antibodies have a different structure than IgG antibodies and the IgY structure is not recognized by human monocytes, which can bind mouse (or any other mammal) IgG.
  • the Fc region of chicken IgY is sufficiently different from mammalian IgG so that the use of chicken antibodies in methods of the invention may result in increased sensitivity and reduced background, in part because the chicken antibodies do not bind to rheumatoid factors, human anti-mouse IgG antibodies, or bacterial and human Fc receptors.
  • avian antibodies see: AgriSera, Vannas, Sweden; Immune Therapy Research Laboratory, Los Angeles, CA; and Gallus Immunotech Inc., Gary, NC).
  • the invention includes the use of antibodies raised in birds (e.g., chickens) or other organism, wherein the antibodies have different morphologic characteristics than antibodies that are used in methods of the invention and have been raised in a mammalian species. Morphological differences between the antibodies such as avian antibodies and mammalian antibodies, may affect nonspecific binding affinity of the non- mammalian and mammalian antibodies.
  • chicken antibodies which are IgY and not IgG, may be used in some embodiments of the invention.
  • IgY antibodies have a different structure that is not recognized by human monocytes, which can bind mouse (or any other mammal) IgG.
  • the Fc region of chicken IgY is sufficiently different from mammalian IgG so that the use of chicken antibodies in methods of the invention may result in increased sensitivity and reduced background, in part because the chicken antibodies do not bind to rheumatoid factors, human anti-mouse IgG antibodies, or bacterial and human Fc receptors.
  • antibodies such as avian antibodies (e.g., chicken antibodies), which have a greater phylogenetic distance between the immunized animal and the antigen-producing species, may be used in the methods of the invention and may provide increased sensitivity and reduced background.
  • avian antibodies see: AgriSera, Vannas, Sweden; Immune Therapy Research Laboratory, Los Angeles, CA, and Gallus Immunotech Inc., Gary, NC).
  • the antibodies may be coupled to specific detectable labels for detecting and/or imaging of binding to a target.
  • Antibodies may be coupled to specific labeling agents, for example, for imaging of cells and tissues with according to standard coupling procedures.
  • Methods for detecting bound antibodies in the assays of the invention may include the use of labeling methods known in the art.
  • labeled secondary antibodies e.g., fluorescent, or colorimetric
  • fluorescent secondary antibodies may be used such as: Neutravidin Alexa Fluor 350, which binds to the biotin labeled rabbit cytokeratin; goat anti-mouse Alexa Fluor 488 IgG2a, which labels the CAM5.2 mouse cytokeratin; and goat anti-mouse Alexa Fluor 568 IgGl, which identifies all the white blood cell (WBC) antibodies.
  • WBC white blood cell
  • the rabbit cytokeratin may be in the far red spectrum — e.g., with Alexa 647 rather than the Neutravidin Alexa Fluor 350 as described above and in the Examples section.
  • cell nuclei in samples assayed may be stained with a Harris hematoxylin (Zymed, San Francisco, CA) solution; a DAPI solution; or with another suitable, art-recognized nuclei-staining solution.
  • fluorescent labels may be used as detectable labels in the methods of the invention.
  • Additional detectable labels useful in the invention include, but are not limited to: an enzyme label, a radioactive label, visual label (e.g., a metallic label such as ferritin or gold), a nuclear magnetic resonance active label, an electron spin resonance label, a positron emission tomography label, a luminescent label, and a chromophore label.
  • Other labeling agents useful in the invention will be apparent to one of ordinary skill in the art.
  • the detectable labels of the invention can be attached to the antibodies or antigen-binding fragments thereof using standard protocols known in the art.
  • the detectable labels may be covalently attached to an antibody or antigen-binding fragment thereof of the invention.
  • the covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules.
  • a detectable label may be attached to an antibody or antigen-binding fragment thereof of the invention using genetic methods.
  • more than one type of detectable label may be attached to an antibody or antigen-binding fragment thereof for use in the methods of the invention.
  • an antibody from which the pFc 1 region has been enzymatically cleaved, or which has been produced without the pFc' region designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd Fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDRl through CDR3 complementarity determining regions
  • non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody.
  • This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. See, e.g., U.S. patents 4,816,567, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.
  • PCT International Publication Number WO 92/04381 teaches the production and use of murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies.
  • Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.
  • HAMA human anti-mouse antibody
  • the present invention also provides for F(ab')2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or nonhuman sequences.
  • the present invention also includes so-called single chain antibodies.
  • the invention also involves a variety of assays based upon determining the presence or absence and/or the amount of a target of interest in subjects.
  • the assays may include, but are not limited to, (1) identifying the presence or absence of a cell of a specific phenotype such as cancer, or a developmental phenotype, etc.
  • samples and subjects can be characterized, treatment regimens can be monitored, treatments can be selected and diseases and conditions (e.g., developmental stages) can be better understood using the assays of the present invention.
  • diseases and conditions e.g., developmental stages
  • the invention provides in one aspect a method for detecting the presence or absence and/or amount of a target in a sample from a subject that can be used as a diagnostic or other measure of the target in the subject.
  • the methods of the invention can also be used in two or more samples obtained from a subject at separate times to determine whether a change in the presence or amount of a target over time has occurred. Alterations in the presence or amount of a target in a subject are in some instances indicative of normal cell, tissue, or systemic changes and in other instances are indicative of abnormal changes in cells, tissues or in the subject.
  • the comparison of amounts of a target in a test sample compared to a control amount of the target can be used to correlate the amount of the target in the test sample with a disease or conditions including either normal or abnormal conditions.
  • a disease or condition for example cancer
  • the amount of a target that is associated with the disease or condition can be determined to be statistically higher than a normal amount of the target, e.g., that of a normal control.
  • detection of an abnormal amount may be diagnostic for a disease or condition that is characterized by abnormal amounts of the target.
  • a disease or condition is indicated by an increased amount of the target.
  • an increase of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more (including all percentages in between) of target than in a normal control indicates the presence of the disease or condition in the subject.
  • a control level may be zero and any increase from zero detected in a test sample is diagnostic for a disorder.
  • a normal control for a cancer test may have zero cancer cell targets and a finding of one or more cancer cell targets in a test sample indicates the presence of cancer in the sample and a subject from whom the sample was obtained.
  • a decrease in the amount of a target associated with a disease or condition compared to a control amount may indicate the presence of the disease or condition.
  • a reduction in the amount of target of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, (including all percentages in between) in a sample compared to a normal control may indicate the presence of the disease or condition in the sample and may be diagnostic of the disease or condition in a subject from whom the sample was obtained.
  • Assays described herein involve measuring levels of a target of interest. Levels of a target of interest can be determined in a number of ways when carrying out the various methods of the invention.
  • the amount of a target of interest is measured and compared to a control amount of a target.
  • the measurement is a relative measure between the test and control amounts of target.
  • the measurement of target may be presented as an absolute level of target. This could be expressed, for example, in terms of weight per volume of sample, or number of molecules per cell, etc.
  • Another measurement of the amount of target is a measurement of the change in the amount of target over time. This may be expressed in an absolute amount or may be expressed in terms of a percentage increase or decrease over time.
  • the control may be a predetermined value, which can take a variety of forms. It can be a single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as in groups (e.g., of cells, tissues, or subjects) having normal amounts of a target of interest and groups having abnormal amounts of the target of interest. Another example of comparative groups would be groups (e.g., of cells, tissues of subjects) having a particular disease, condition or symptoms and groups without the disease, condition or symptoms. Another comparative group would be a group (e.g., of cells or tissues, or subjects) having a family history of a condition and a group without such a family history.
  • Another comparative group would be a group (e.g., of cells or tissues, or subjects) having a family history of a condition and a group without such a family history.
  • the predetermined value can be arranged, for example, where a tested population is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group or into quadrants or quintiles, the lowest quadrant or quintile being individuals with the lowest risk or lowest amount of a target of interest and the highest quadrant or quintile being individuals with the highest risk or highest amount of the target of interest.
  • groups such as a low-risk group, a medium-risk group and a high-risk group or into quadrants or quintiles
  • the lowest quadrant or quintile being individuals with the lowest risk or lowest amount of a target of interest and the highest quadrant or quintile being individuals with the highest risk or highest amounts of the target.
  • the predetermined value will depend upon the particular population selected. For example, an apparently healthy population will have a different 'normal' range than will a population that is known to have a condition related to the target, for example cancer or a developmental stage associated condition. Accordingly, the predetermined value selected may take into account the category in which a cell, tissue, and/or subject falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art. By abnormally high it is meant high relative to a selected control. Typically the control will be based on apparently healthy normal cell, tissue, and/or subject.
  • controls according to the invention may be, in addition to predetermined values, samples of materials tested in parallel with the experimental materials. Examples include samples from control populations or control samples generated through manufacture to be tested in parallel with the experimental samples.
  • a subject “at risk” is a subject who is considered more likely to develop a disease state or a physiological state than a subject who is not at risk.
  • a subject “at risk” may or may not have detectable symptoms indicative of the target-associated disease or physiological condition, and may or may not have displayed detectable disease prior to the treatment methods (e.g., therapeutic intervention) described herein.
  • At risk denotes that a subject has one or more so-called risk factors.
  • a subject having one or more of these risk factors has a higher probability of developing one or more disease(s) or physiological condition(s) than a subject without these risk factor(s).
  • These risk factors can include, but are not limited to, history of family members developing one or more diseases (e.g., cancer), related conditions, or pathologies, history of previous disease, age, sex, race, diet, presence of precursor disease, genetic (i.e., hereditary) considerations, and environmental exposure.
  • the level of risk can be assessed using standard methods known to those in the art. For example, based on factors such as medical history, family medical history, and current medical condition, a health care professional may assess a percentage chance that a subject will have or will develop a target-associated disease or condition.
  • a health care professional may determine that a subject who has a family history of cancer or who has a cancer-associated genetic mutation, may have a 10%, 20%, 30%, 40%, 50%, 60%, 70% or more chance of developing cancer than an individual with no family history of the disorder or a cancer-associated genetic mutation, respectively.
  • a subject's level of risk for target-associated diseases or conditions can also be evaluated using standard methods.
  • a target can monitor the presence and/or amount of a target over time to determine if the amount of the target in a tissue or subject is changing.
  • Changes in the amounts of a target of greater than 0.1% may indicate a change in a target-associated disease or condition.
  • the change in the presence and/or amount of a target that indicates a target-associated disease or condition is greater than 0.2%, greater than 0.5%, greater than 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 7.0%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more.
  • a first sample may be obtained from a subject and serve as a baseline of the amount of a target of interest.
  • a subsequent sample from the subject can be compared to the initial baseline "control" amount for the subject as an indication of change in the amount of target in the subject.
  • the invention in another aspect provides a diagnostic method to determine the effectiveness of treatments for a target-associated disease or condition.
  • the "evaluation of treatment” as used herein means the comparison of a subject's amount of target measured in samples collected from the subject at different sample times, preferably at least one day apart.
  • the preferred time to obtain a second sample from the subject is at least one day after obtaining the first sample, which means the second sample is obtained at any time following the day of the first sample collection.
  • a second sample is obtained preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days or weeks after the time of a first sample collection.
  • the comparison of levels of a target in two or more samples, taken at different times, or on different days, is a measure of level of the subject's amount of target over time and allows evaluation of a treatment the cell, tissue, or subject is undergoing to regulate the target-associated disease or condition.
  • the evaluation of the treatment also may be based upon an evaluation of the symptoms or clinical end points of the associated disease, such as the complications of cancer.
  • the methods of the invention also provide for determining the onset, progression, and/or regression of a disease or condition that is characterized by amounts of a target that differ from that of a control amount of the target.
  • the subjects, tissues, and or cells to which the methods of the invention are applied are already diagnosed as having a particular target-associated condition or disease.
  • the measurement will represent the diagnosis of the target- associated condition or disease.
  • the subjects will already be undergoing drug therapy for regulating a target-associated disease or condition, while in other instances the subjects will be without present drug therapy for regulating the target-associated disease or condition.
  • kits that include materials to carry out the methods of the invention and instructions for use.
  • the kits may include antibodies or antigen-binding fragments thereof as described herein and can further contain at least one additional reagent, such as a negative-control antibody or antigen-binding fragment thereof, a positive-control antibody or antigen-binding fragment thereof, etc.
  • Kits of the invention can be prepared for in vitro or in vivo diagnosis, prognosis and/or monitoring the amount of a target and determination of the presence of a target-associated disease or condition.
  • Kits of the invention may include antibodies or antigen-binding fragments thereof or other binding agents that specifically bind a cell with a phenotype of interest — e.g., a cancer cell, or a cell at a particular developmental stage. Kits may include antibodies or antigen-binding fragments thereof that specifically bind to an organism or compound of interest.
  • the components of the kits can be packaged either in aqueous medium or in lyophilized form.
  • a kit of the invention in some embodiments, may further comprise a container containing a target of interest. Some or all of the kit components may be frozen.
  • a kit of the invention may also include control compounds and solutions for testing the binding activity of the antibodies.
  • Such materials may include, buffer, a non-limiting example of which is sodium phosphate buffer, etc.
  • a kit may also include materials and instructions for detectably labeling an antibody or antigen-binding fragment thereof.
  • a kit of the invention may comprise a carrier being compartmentalized to receive in close confinement therein one or more container means or series of container means such as test tubes, vials, flasks, bottles, syringes, or the like.
  • a kit of the invention may also include vials, cuvettes, pipet tips, transfer pipets, solutes, sterile and/or distilled water, one or more control samples, (e.g., blank control, test control), printed graphs, tables, figures, or diagrams, which may be used for interpretation and/or analysis of results or for instructional purposes.
  • a kit of the invention may also include equipment and/or supplies for determining the presence and/or amount of a target.
  • kits may include ELISA assay materials, gel preparation materials (e.g., solutions, agarose, acrylamide, control markers, dyes and/or labels, etc,).
  • gel preparation materials e.g., solutions, agarose, acrylamide, control markers, dyes and/or labels, etc.
  • a kit may also include materials for chromatographic analysis, e.g., beads, solvents, solutes, control samples, columns, etc.
  • kits for analysis of the presence and/or amount of a target are provided in a ready-to-use format.
  • the kits provide materials that can be utilized for determining the presence and/or amount of a target in a sample and will be assembled for use by the operator.
  • Some kits of the invention will include all materials necessary for determining the presence and/or amount of a target in a sample, and other kits of the invention will include some, but not all of the materials for the determination of the presence and/or amount of the target in a sample.
  • additional materials will be provided by the operator and may include: pipets, tubes, gel apparatus, flasks, solutions, enzymes, target polypeptides, etc.
  • Antibodies raised in different species were utilized in combination with control antibodies to determine the presence of targets for identification.
  • mouse CK-detecting antibody was used without rabbit CK-detecting antibody.
  • a mix of white blood cell (WBC) IgGl mouse antibodies (referred to in results as CD) were also applied with the cytokeratins to identify WBCs.
  • WBC white blood cell
  • CD- in results included CD45 (Lab Vision/Neomarkers, Fremont, CA), CD34, CD38 (Dako, Carpintaria, CA) and glycophorin A (CALTAG, Burlingame, CA).
  • the WBC antibodies were used at a 1:100 dilution. All antibodies were applied for 30 minutes.
  • the slides were incubated with a goat anti- rabbit biotin conjugate antibody (Santa Cruz Biotechnology, Santa Cruz,CA) at 1 :400 dilution for 30 min to recognize the primary rabbit cytokeratin.
  • a cocktail of fluorescent antibodies (Invitrogen/Molecular Probes) were applied and the mixture incubated for 30 min.
  • the cocktail included Neutravidin Alexa Fluor 350, which binds to the biotin labeled rabbit cytokeratin; goat anti-mouse Alexa Fluor 488 IgG2a, which labels the CAM5.2 mouse cytokeratin; and goat anti-mouse Alexa Fluor 568 IgGl, which identifies all the WBC antibodies.
  • the staining method was evaluated by spiking BT474, SKBR3, or MCF7 breast cancer cells into healthy blood and bone marrow samples. Unspiked healthy blood and bone marrows were used to assess specificity of the method.
  • Alexa Fluor excites and emits in a. specific wavelength of light.
  • the Alexa Fluors used in these methods were chosen to match with the available microscope filter sets and to minimize overlapping of the different fluorescent colors.
  • a cell was interpreted as a tumor cell if it was positive for both cytokeratins, negative for the WBC markers, contained a hematoxylin-stained nucleus and/or had morphology consistent with a tumor cell.
  • Tests were performed and images, including results and images of a tumor cell from a spiked blood sample that was positive for rabbit cytokeratin; a tumor cell from a spiked blood sample that was positive for mouse cytokeratin; a tumor cell from a spiked blood sample that was negative for WBC; and a tumor cell in spiked blood sample that was positive for hematoxylin.
  • results from bone marrow from breast cancer patients showed that 42 cells were stained with the mouse antibody (mouse CK+); 24 cells were positive for both mouse and rabbit CK (dual CK+); and one cell had dual CK+ staining and was negative for staining with the CD markers.
  • Table 1 Results of bone marrow analysis from 25 healthy controls and 14 breast cancer atients usin dual c tokeratin.
  • results are shown in Table 2 and indicate that of the healthy blood samples: 4 cells were stained with the mouse CK antibody (mouse CK+); no cells were stained with both mouse and rabbit CK antibodies (dual CK+); and no cells had dual CK+ staining and were negative for the CD markers. Results of testing blood from breast cancer patients showed that there were 33 cells that were stained with the mouse antibody (mouse CK+); 24 cells were positive for both mouse and rabbit CK (dual CK+); and 17 cells had dual CK+ and were negative for staining with the CD markers.
  • Table 2 Results of blood analysis from 6 healthy controls and 20 breast cancer patients using multicolor dual cytokeratin.
  • CK mouse antibodies
  • the methods described herein provide verification that dual labeling with anti-CK antibodies raised in different species is due to true CK binding and not due to species sensitivity.
  • a WBC cocktail of antibodies provides even an additional level of confidence that the positive cells are not of WBC origin but are truly epithelial.
  • the methods of the invention provide increased sensitivity and accuracy of the detection of true tumor cells in the samples.

Landscapes

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

Abstract

La présente invention concerne, en partie, des procédés d'utilisation d'anticorps multiples produits dans deux espèces différentes ou plus, pour marquer et identifier des cellules, des organismes et des cibles supplémentaires. En outre, l'invention a trait, en partie, à des dosages de diagnostic pour des maladies telles que le cancer, à l'identification d'organismes ou de contaminants dans des échantillons, et à des dosages permettant d'évaluer le statut des cellules, notamment la différenciation des cellules et le lignage des cellules.
PCT/US2007/005996 2006-03-08 2007-03-08 Procedes de marquage d'anticorps multiples et utilisations desdits procedes WO2007103538A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US78033106P 2006-03-08 2006-03-08
US60/780,331 2006-03-08

Publications (2)

Publication Number Publication Date
WO2007103538A2 true WO2007103538A2 (fr) 2007-09-13
WO2007103538A3 WO2007103538A3 (fr) 2007-12-06

Family

ID=38372382

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/005996 WO2007103538A2 (fr) 2006-03-08 2007-03-08 Procedes de marquage d'anticorps multiples et utilisations desdits procedes

Country Status (1)

Country Link
WO (1) WO2007103538A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012047481A (ja) * 2010-08-24 2012-03-08 Tanaka Kikinzoku Kogyo Kk 免疫測定法による尿中抗原の検出方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683197A (en) * 1981-07-16 1987-07-28 Hoffmann-La Roche Inc. Detection method for occult blood

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683197A (en) * 1981-07-16 1987-07-28 Hoffmann-La Roche Inc. Detection method for occult blood

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DAVID N KRAG ET AL: "The Detection of Isolated Tumor Cells in Bone Marrow Comparing Bright-Field Immunocytochemistry and Multicolor Immunofluorescence" ANNALS OF SURGICAL ONCOLOGY, SPRINGER-VERLAG, NE, vol. 12, no. 9, 1 September 2005 (2005-09-01), pages 753-760, XP019369729 ISSN: 1534-4681 cited in the application *
M. LAGRANGE ET AL: "Non-specifically labelled cells that simulate bone marrow metastases in patients with non-metastatic breast cancer." JOURNAL OF CLINICAL PATHOLOGY MAR 1997, vol. 50, no. 3, March 1997 (1997-03), pages 206-211, XP002448051 England ISSN: 0021-9746 cited in the application *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012047481A (ja) * 2010-08-24 2012-03-08 Tanaka Kikinzoku Kogyo Kk 免疫測定法による尿中抗原の検出方法

Also Published As

Publication number Publication date
WO2007103538A3 (fr) 2007-12-06

Similar Documents

Publication Publication Date Title
JP7080899B2 (ja) 同じヒト対象に由来する少なくとも２つの試料に対して早期バイオマーカーを使用する、外傷性脳損傷の超急性の診断及び決定の一助となるための方法
RU2744836C2 (ru) Прямой иммуногистохимический анализ
JP2021509475A (ja) 外傷性脳損傷を診断及び査定するための、新規のバイオマーカー及び方法
KR20150008842A (ko) 순환 종양 세포를 확인하기 위한 장치, 시스템 및 방법
KR102198869B1 (ko) 잠재적 염증, 특히 이식 거부반응과 연관된 잠재적 염증, 신경변성 장애 또는 우울증의 조기 검출을 위한 시험관 내 방법
JP2013507641A (ja) 黒色腫腫瘍細胞の識別のためのバイオマーカー
CN109975549B (zh) 肿瘤来源IgG在胰腺癌诊断或预后中的用途
US20070292887A1 (en) Immunohistochemical Methods
US10928398B2 (en) Method for double staining colocalized nuclear-markers in histological lymphoid or bone marrow tissue sample
KR101495225B1 (ko) Ast 양의 측정을 통한 간 질환 진단, 예후 또는 모니터링 키트 및 방법
US11486878B2 (en) Compositions and methods for identifying cancer cells
CN116559462A (zh) 用于肿瘤患者预后的生物标志物组及其用途
JP2021534381A (ja) パーキンソン病の判定
US20090226933A1 (en) Quantification of fusion proteins and their activity from chromosomal translocation
WO2009014342A2 (fr) Marqueurs et kit de diagnostic de la maladie d'alzheimer
WO2007103538A2 (fr) Procedes de marquage d'anticorps multiples et utilisations desdits procedes
US20100047830A1 (en) Compositions and methods for detecting cancers in a subject
US10139398B2 (en) Methods and combinations of signaling markers for assessment of disease states
KR20110115292A (ko) 폐암 진단용 Ｘａｇｅ－１ｄ 마커 및 이를 이용한 진단 키트
US10753935B2 (en) Prognostic method and kits useful in said method
Santangelo et al. Special Staining Techniques: Application and Quality Assurance
US20140113385A1 (en) Compositions and Methods for Identifying Single Antigens or Other Molecules in Cell Preparations
EP1507147A1 (fr) Procédé de détection de faibles niveaux de protéine de fusion
KR20230150221A (ko) c-Met 발현 CTC를 이용한 유방암 환자의 예후 예측 방법
JP2022076410A (ja) 悪性末梢神経鞘腫瘍の診断マーカー

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07752679

Country of ref document: EP

Kind code of ref document: A2