WO1997026324A1 - Enrichissement en phase solide de cellules intactes a l'aide de constituants intracellulaires - Google Patents

Enrichissement en phase solide de cellules intactes a l'aide de constituants intracellulaires Download PDF

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Publication number
WO1997026324A1
WO1997026324A1 PCT/AU1997/000020 AU9700020W WO9726324A1 WO 1997026324 A1 WO1997026324 A1 WO 1997026324A1 AU 9700020 W AU9700020 W AU 9700020W WO 9726324 A1 WO9726324 A1 WO 9726324A1
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Prior art keywords
cells
cell
agent
detecting agent
solid phase
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PCT/AU1997/000020
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English (en)
Inventor
Bill Kalionis
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Flinders Technologies Pty. Ltd.
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Priority to JP9525529A priority Critical patent/JP2000504213A/ja
Priority to EP97900153A priority patent/EP0928327A1/fr
Priority to AU13608/97A priority patent/AU1360897A/en
Publication of WO1997026324A1 publication Critical patent/WO1997026324A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation

Definitions

  • the present invention relates to the field of enrichment of a specific cell type present in a mixed cell population.
  • the invention provides for concentration and identification of a target cell type, based on intracellular constituents, utilizing a solid phase support.
  • the invention also provides a method for increasing the sensitivity of enrichment of target cells in a mixed cell population by amplifying a selected intracellular constituent present in the target cell.
  • the technique of in situ hybridization is a powerful method for the detection and quantitation of nucleic acids and proteins at the level of a single cell.
  • the ability to detect the presence or absence of a specific gene product is important not only for genetic, biochemical, and molecular biological characterization of normal cell metabolism and differentiation, but also for the identification and detection of genetic markers for disease and infection.
  • genetic diseases are characterized by the presence or absence of a specific gene product in the cell which is not present in normal cells.
  • cells infected by infectious agents also express nucleic acids and proteins that are not expressed in normal cells.
  • a problem with current methods for in situ hybridization is the inability to readily detect hybridized cells when they are in very low abundance in the cell population.
  • Manual detection of cells requires scanning of microscope slides by skilled technicians which is laborious, time consuming and inefficient.
  • Automated means for scanning large populations of cells for rare cells identified by in situ hybridization requires sophisticated and expensive computer-controlled optical or fluorescent scanning devices.
  • specific cell types identified by in situ hybridization typically cannot be studied in isolation. Further, genetic, biochemical and molecular biological studies on the specific cells detected takes place in the presence of the entire population of cells.
  • Enrichment of rare cells is possible.
  • Current methods for cell enrichment following in situ hybridization fall into two categories: immunomagnetic or immunoaffinity separation based on solid phase supports and flow cytometry employing fluorescence activated cell sorting.
  • In situ hybridization followed by concentration using solid phase supports is a common method for enrichment of cells based on detection of extracellular constituents such as the outer cell membrane or cell wall proteins or antigens.
  • an extracellular protein or antigen is bound by an antibody that may be directly or indirectly coupled to a solid phase support.
  • Cells bound by antibody, coupled directly or indirectly to a solid phase support are then enriched from the mixture of cells in suspension using immunomagnetic or immunoaffinity methods.
  • Intracellular gene products can be used to characterize specific cells. During development and differentiation, normal cells typically express distinguishing nucleic acids and proteins in the cell cytoplasm. Many genetic diseases have been shown to be caused by, or can be diagnosed by, the appearance or disappearance of specific gene products located only in the cytoplasm or nucleus of the cell. Pathogenic and non-pathogenic viruses express specific viral nucleic acids and proteins found only in the cytoplasm. In addition, bacterial, insect and animal cells can harbor plasmids or viral vectors which contain recombinant DNA or expression products found only in the cytoplasm. Detection of these nucleic acids and proteins can be achieved by in situ hybridization. However, current methods do not provide a means by which to enrich these cells following in situ hybridization.
  • intracellular nucleic acids or protein ⁇ are detected by in situ hybridization using probes that are directly labelled, or indirectly labelled, with a fluorochrome.
  • the suspended cells are brought, one by one, to a detector by means of a flow channel where fluorescence is detected by a fluorescence cytometric sensor and the cells that fluoresce are sorted and subsequently analyzed.
  • flow cytometry followed by sorting requires skilled technicians and expensive equipment that is not portable.
  • the present invention provides a method for enrichment of a specific cell type in a mixed cell population.
  • the invention also provides an enriched cell complex which is formed during enrichment of a specific cell type.
  • the components used in the invention may be combined into a kit for enrichment of a specific cell according to the method of the inventio .
  • enrichment of a "specific" or “target” cell, which is present in a mixed cell population is performed utilizing a solid phase support system.
  • the solid phase support system provides separation of an enriched cell complex from non-target cells present in the mixed cell population.
  • an "enriched cell complex” refers to the combination of a target cell, a detecting agent hybridized to an intracellular constituent of the target cell, and a solid phase support.
  • the method of the invention may include fixing a mixed cell population with a fixing agent, permeabilizing the mixed cell population with a permeabilizing agent, detecting a "selected” or “target” intracellular constituent of a target cell using a detecting agent and concentrating the target cells using a solid phase support.
  • Fixing agents suitable for the invention are known in the art.
  • Preferred fixatives include those which act as cross ⁇ linking fixatives or precipitating fixatives.
  • Cross-linking fixatives include, for example, formaldehyde, formalin, glutaraldehyde, formaldehyde-glutaraldehyde mixtures, ⁇ - hydroxyadipaldehyde, acrolein, dimethylsuberimidate and ethyldimethylamino-propylcarbodiimide.
  • Precipitating fixatives include, for example, ethanol or methanol mixed with acetic acid or acetone and alcohol-ether mixtures.
  • a suitable permeabilizing agent is any compound which facilitates access of the below-described detecting agent to the cytoplasm of the cell and which does not inhibit enrichment of an intact and viable target cell.
  • Preferred permeabilizing agents include those which unmask nucleic acids from associated proteins, form pores that allow access of the detecting agent to the cytoplasm or extract lipid from the outer cell membrane and allow access of the detecting agent to the underlying cytoplasm.
  • Preferred permeabilizing agents for unmasking nucleic acids from proteins include, for example, Proteinase K, pronase E, dispase, diastase, papain, trypsin and pepsin/HCl for animal cells; cellulase or pectinase for plant cells; and lysozyme for bacterial cells.
  • Permeabilizing agents that extract lipid from the outer cell membrane include, for example, alcohol such as ethanol or methanol in combination with other compounds including acids such as acetic acid, or acetone.
  • permeabilizing agents suitable for the invention include, for example, detergents such as sodium dodecyl sulphate, CHAPSTM, Triton-XlOOTM, Brij35TM and Brij58TM.
  • some fixatives such as formaldehyde and alcohol-based fixatives, also act as permeabilization agents and may make further permeabilization unnecessary. Permeabilization may also be accomplished using mechanical means such as freeze-thaw methods.
  • Another embodiment of the invention provides for increasing the sensitivity or detection of a target cell by amplification of an intracellular constituent when the cell, or selected intracellular constituent, is in low abundance.
  • amplification of a desired intracellular constituent preferably is performed subsequent to permeabilization of the mixed cell population and prior to contacting permeabilized cells with a detecting agent.
  • the mixed cell population is contacted with at least one detecting agent which has specificity for a target intracellular constituent which is present in a target cell of the mixed cell population.
  • detecting agents may include genetic probes, antibodies, proteins, peptides, amino acids, sugars, polynucleotides, enzymes, co-enzymes, co-factors, antibiotics, steroids, hormones or vitamins.
  • a detecting agent of the invention attaches to an intracellular constituent in a manner which is sufficiently stable for concentration of the target cell using the below described solid phase support system.
  • the solid phase support system is used to concentrate the detected target cell.
  • Target cells are concentrated by coupling the detecting agent to the solid phase support system.
  • the detecting agent may be labeled such that the solid phase support system can detect and couple to the label of the detecting agent.
  • a bound detecting agent may be detected and coupled to the solid phase support system without the need for labeling.
  • a solid phase support system includes a solid phase support and other components which may be necessary to separate the target cells from non-target cells in the cell population.
  • Solid phase support suitable for a solid phase support system of the invention are known in the art and include, for example, magnetizable particles, silica, agarose, glass, dextran, fiber supports, cellulose and synthetic polymers, and similar supports.
  • Preferred solid phase supports include superparamagnetic particles.
  • a solid phase support system may also include a mechanism for separating the enriched cell complex from other cells in a mixed cell population, for example, in the case of a magnetizable particle solid phase support, a solid phase support system can include a magnetic field.
  • target cells are eluted from the ⁇ olid phase support. Because target cell enrichment may lack complete specificity, eluted target cells may be further identified by using an identification system and visualized using a signal generating system.
  • an identification system identifies the target cell using an identifying agent coupled directly or indirectly to a signal generating system.
  • An identifying agent identifies a target cell by binding to a bound detecting agent or by directly binding to an intracellular or extracellular constituent of the target cell.
  • an identification system may be the detecting agent which hybridized to an intracellular constituent prior to solid phase concentration.
  • a signal generating system provides vi ⁇ ualization of an enriched target cell.
  • the ⁇ ignal generating system may be incorporated into the identifying agent, or incorporated into a compound which binds to the identifying agent. If the identifying agent is the detecting agent, the signal generating system may or may not incorporated into the detecting agent prior to solid phase enrichment.
  • kits for enrichment of at least one specific cell from a mixed cell population using a detecting agent specific for an intracellular constituent of a target cell may include a fixing agent, a permeabilizing agent, a detecting agent and a solid phase support system, as previously described.
  • the kit may further include an identifying agent and signal generating system.
  • the kit may include a single detecting agent for identification of a single specific cell, or alternatively, multiple detecting agents for detecting multiple specific cell types or detecting a single specific cell type based on the presence of multiple intracellular constituents.
  • a solid phase support for concentration of a specific cell in a mixed population of cells provides a simple and co ⁇ t effective means for enrichment of the specific cell from the mixed cell population.
  • the cells enriched are intact and viable for use in subsequent procedures.
  • the present invention provides an "enriched cell complex” and a method for enrichment of at least one specific cell, which may be present in a mixed population of cells, using a solid phase support system.
  • a "specific” or “target” cell is enriched utilizing a solid phase support to concentrate cells having an intracellular constituent which is detected by a herein described detecting agent. Once a target cell is bound to the detecting agent, the cells may be separated from the mixed cell population using a solid phase support system.
  • the combination of a target cell, a detecting agent and a solid phase support is referred to as an "enriched cell complex".
  • the invention not only yields an enriched population of target cells but also produces a mixed population of cells depleted of a specific cell type. The benefit of target cell depletion in a mixed cell population is further discussed below.
  • the invention also discloses a method for enhancing sensitivity of detection of a target cell by amplifying a "selected" or "target” intracellular constituent of the specific cell.
  • Enrichment of cells provides an enriched cell product which is useful in many areas of research and clinical use.
  • the term “enrich”, and derivatives thereof, refer to a process for the treatment, detection and concentration of a target cell which may be present in a population of cells.
  • the population of cells on which the method of the invention is used will be a mixed population of cells.
  • a “mixed” cell population means a population of cells wherein one or more cells of the population has an identifiable characteristic which distinguishes the cell from one or more other cells in the population.
  • mixed populations of cells include mixtures such as fetal cells and maternal blood cells; virally, bacterially, or fungally infected cells and non-infected cells; oncogenic cells and non-oncogenic cells; leukemic and non-leukemic cells; hematopoietic cells in bone marrow and blood; recombinantly transformed cells and non-transformed cells; differentiated and non-differentiated cells; cells expressing mutated genes and wild-type cells; and other cell populations which contain one or more cells with distinguishable intracellular characteristics.
  • Mixed cell populations may be naturally occurring or artificially created for example, fetal cells isolated from placental material and mixed into xenogenous blood or other fluids; human or animal cells from cell culture medium mixed into xenogenous blood or other fluids; or other artificial combinations of cells in liquid medium.
  • a mixed cell population of the invention is in a liquid suspension.
  • examples include cells present in blood, lymph, bone marrow, synovial fluid, amniotic fluid, cerebrospinal fluid, seminal fluid, ventricular fluid, nasopharyngeal mucous, sputum, semen, urine, water, effluent, sewage, feces, animal and plant cell culture media, bacterial and fungal enrichment media, tissue samples and tumors or organs disaggregated by physical, chemical or enzymatic means or other combination of cells in a liquid medium.
  • a “target” or “specific” cell include any cell which can be enriched according to the invention.
  • a target cell will have one or more detectable intracellular characteristics which distinguishes the target cell from other cells present in a mixed population of cells.
  • RNA for example, is generally produced in the cell nucleus and transported to the cytoplasm in eukaryotes. DNA is found primarily in the nucleus in eukaryotes.
  • the present invention does not rely on extracellular or outer membrane characteristics to enrich a target cell. Rather, the present invention provides for enrichment of a cell based on detection of one or more intracellular constituents.
  • intracellular it is meant that the target constituent is on the intracellular aspect of the outer cell membrane or cell wall. However, it is not required that the intracellular component be found exclusively intracellular.
  • an intracellular constituent includes, for example, nucleic acids, amino acids, proteins, peptides, polypeptides, lipids, metabolites, cofactors, polysaccharides, hormones or other intracellular component which may be detectable by a herein described detecting agent.
  • the intracellular constituent need not be endogenous to the cell but rather, may come from an exogenous intracellular source, such as a plasmid, phage, virus, bacteria, protozoa, parasite, mycoplasma, fungus, or other similar source.
  • Deoxyribonucleic acid (DNA) , ribonucleic acid (RNA) , peptides, polypeptides and proteins are the preferred intracellular constituents for target cell enrichment according to the invention.
  • problems that need to be overcome for enrichment of intact and viable cells based on intracellular constituents include, for example, maintenance of cell morphology, maintenance of nucleic acid integrity, maintenance of peptide or protein antigenicity, and accessibility of the solid phase support to the intracellular constituent.
  • the present invention teaches that cell morphology, nucleic acid integrity and protein antigenicity can be maintained during fixation, permeabilization, in situ detection, and subsequent concentration using a solid phase support system.
  • a further teaching is that following permeabilization of intact cells and in situ detection, there is sufficient access to the cytoplasm and sufficient detection agent in the expo ⁇ ed cytopla ⁇ m underlying the outer cell membrane or cell wall, to permit concentration of ⁇ pecific cell ⁇ from a mixed population using a solid phase support.
  • cells enriched according to the method of the invention are morphologically intact.
  • the enriched cells are viable.
  • a "viable" cell i ⁇ a cell which i ⁇ suitable for further study of anatomic, genetic, biological, morphological, physiological, pharmacological or other purpose for which the cell was enriched.
  • the specificity and sen ⁇ itivity of the invention provides for enrichment of a low number of specific cells from large populations of mixed cells.
  • one embodiment of the invention provides for enhancement of detection of target cells by amplification of a selected intracellular constituent.
  • enrichment of a specific cell present in a mixed population of cells includes the steps of pretreating a mixed population of cells, fixing a mixed population of cells, permeabilizing the fixed cells, contacting the permeabilized cell ⁇ with a detecting agent which binds to a selected intracellular constituent of a target cell and concentrating the target cell bound by the detecting agent using a ⁇ olid pha ⁇ e ⁇ upport system.
  • Cells of the invention may be pretreated in many ways.
  • the cells to be treated are in su ⁇ pension.
  • a cell suspension can be prepared using methods known in the art. For example, culturing cells in an appropriate medium including bacterial or animal cell culture media.
  • Cell su ⁇ pen ⁇ ion ⁇ can al ⁇ o be prepared from body fluids such as blood, bone marrow, lymph fluid, and synovial fluid or can be prepared by disaggregating tissues, organs or tumors using known physical (eg. cutting, mincing, ⁇ hearing, ⁇ ieving or scratching adherent cells) , chemical (eg.
  • the cell ⁇ prepared by the ⁇ e techniques may or may not be alive.
  • Peroxidases When an enzyme is to be used as a label, it may be necessary to inactivate the endogenous enzyme during pretreatment of cells.
  • Peroxidases for example, are inactivated with 1% hydrogen peroxide (v/v) in methanol for 30 min. Treatment of cell ⁇ with 0.2M HCl for 30 min is sometimes used to improve the signal to noise ratio.
  • the cell suspension is pelleted by centrifugation at 100 x g to 4000 x g, preferably about 400 x g, at 0°C to 25°C, preferably about 4°C, for 1-60 min, preferably about 15 minutes.
  • the supernatant is removed and the cells are then fixed as described below.
  • a mixed population of cell ⁇ i ⁇ fixed with a fixing agent A ⁇ u ⁇ ed herein a fixing agent i ⁇ any compound which serve ⁇ to provide a viable cell after enrichment (eg., prevention of o ⁇ motic damage, autolysis, etc.).
  • the fixative will result in the cell maintaining an accurate representation of the structure of the cell in vivo, and the cell will retain its original size with minimal los ⁇ of cellular materials during fixation. It is also preferred that the reactivity of intracellular constituent ⁇ remain ⁇ sufficiently high to enable them to be detected.
  • the fixative chosen will depend on the material and probe being used and the level of sensitivity required. Any fixing agent which does not prevent enrichment and which fixes a mixed cell population such that the enriched cell ⁇ are intact and viable for the purpose for which the cells were enriched is suitable for the invention.
  • Preferred fixatives include those which act as cross ⁇ linking fixatives or precipitating fixative ⁇ .
  • Preferred cro ⁇ -linking fixing agent ⁇ include, but are not limited to, formaldehyde, formalin, glutaraldehyde, formaldehyde- glutaraldehyde mixtures, ⁇ -hydroxyadipaldehyde, acrolein, dimethylsuberimidate and ethyldimethylamino- propylcarbodiimide.
  • Preferred precipitating fixatives include, but are not limited to, ethanol or methanol mixed with acetic acid at a preferred ratio of 3:1, methanol mixed with acetone at a preferred ratio of 1:1, ab ⁇ olute methanol, 95% alcohol, and alcohol-ether mixture ⁇ .
  • PBS pho ⁇ phate buffered ⁇ aline
  • the cells can be resuspended in an isotonic buffer such as PBS to a volume of 5-500/il, preferably lOO ⁇ l at a concentration of about 10 s to 10 12 cell ⁇ per ml, preferably about 10* to 10 9 cells per ml.
  • an isotonic buffer such as PBS to a volume of 5-500/il, preferably lOO ⁇ l at a concentration of about 10 s to 10 12 cell ⁇ per ml, preferably about 10* to 10 9 cells per ml.
  • cells may be stored at 2°C to 12°C, preferably 4°C, for treatment at a later date.
  • a permeabilizing agent is any compound which facilitates access of a below-described detecting agent to the cytoplasm of the cell.
  • a permeabilizing agent does not inhibit enrichment of a target cell by the solid phase support.
  • Preferred permeabilizing agents include those which unmask nucleic acids from associated proteins, form pores that allow access of the below described detecting agent to the cytoplasm, or that extract lipid from the outer cell membrane and allow acces ⁇ of the detecting agent to the underlying cytoplasm.
  • Particularly preferred permeabilizing agents that unmask nucleic acid from protein include Proteinase K, prona ⁇ e E, dispase, diastase, papain, trypsin and pepsin/HCl for animal cells; cellulase or pectinase for plant cells,- and lysozyme for bacterial cells.
  • Non-chemical means such as cycles of freezing followed by thawing of cells or microwave irradiation can also be used for permeabilization.
  • Permeabilizing agents that form pores that allow access of the detecting agent to the cytoplasm include detergent ⁇ such as sodium dodecyl ⁇ ulphate, CHAPSTM, Triton-X100TM, Brij35TM and Brij58TM.
  • Permeabilizing agents that extract lipid from the outer cell membrane include, for example, alcohols such as ethanol or methanol which may be used in combination with other compounds including acids such as acetic acid, or acetone.
  • Some fixative ⁇ such as formaldehyde and alcohol-ba ⁇ ed fixatives also act as permeabilization agents and make further permeabilization unnecessary, but in general permeabilization is recommended.
  • fixed cell ⁇ may be permeabilized with Proteina ⁇ e K in PBS buffer at a concentration of l-500 ⁇ g/ml, preferably about 10-100 ⁇ g/ml for 1-180 min, preferably about 10 min, at 15°C-42°C, preferably about 37°C.
  • a ⁇ with other permeabilizing agent ⁇ , the concentration of Proteinase K, the time of incubation and temperature used are optimized for each cell type.
  • Permeabilization i ⁇ ⁇ topped by replacing the Proteina ⁇ e K solution with 0.02%-2% (w/v), preferably 0.2% (w/v) glycine in PBS for 1-20 min, preferably 2 min at 18°C-42°C, preferably room temperature. Stopping the reaction with glycine is an optional treatment.
  • Preferred permeabilizing agents for use with antibody detecting agents include alcohols such as ethanol and methanol which may be used in combination with compounds known in the art including acids such as acetic acid, or acetone (eg. methanol-acetone solution (1:1) .
  • fixation of the cells may be repeated.
  • Post-fixation is optional.
  • the fixatives and methods previously described may be used for post-fixation of cell ⁇ .
  • a preferred fixing agent i ⁇ 0.4%-10% (w/v), preferably 4% (w/v) paraformaldehyde in PBS for about 1-180 minutes, preferably about 10 minutes at 0°C-50°C, preferably room temperature. Removal of the fixative can be accomplished by a series of 4-5 washes for 2-20 min each, preferably 5 min each wash, in PBS. Cells are pelleted by centrifugation between wa ⁇ he ⁇ .
  • Another embodiment of the invention provide ⁇ for increasing the sensitivity of detection of a target cell by amplification of an intracellular constituent when the cell, or a selected intracellular constituent, is in low abundance. If increased sensitivity of detection is desired, preferably, amplification is performed at this stage of treatment.
  • the invention provides for amplification of a ⁇ elected nucleic acid which is present in low copy number in a specific cell.
  • the selected nucleic acid is amplified using the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • In situ PCR amplification on intact cells increases the amount of a selected nucleic acid and increases the ⁇ ensitivity of detection of an intracellular nucleic acid.
  • PCR Amplification take ⁇ place between two oligonucleotide sequences that are complementary to a defined segment of the selected nucleic acid sequence. Sss. eg. , G.R. Taylor, PCR: A Practical Approach, 1-13, (M.S. McPherson et al. eds. 1991) IRL Press, Oxford, England.
  • cell ⁇ that have been fixed and permeabilized may be re ⁇ uspended in 50 ⁇ l-200 ⁇ l, preferably about lOO ⁇ l, of a buffer suitable for PCR amplification.
  • a buffer suitable for PCR amplification.
  • a buffer will contain lOmM Tris-HCl (pH 8.4), lmM-lOmM MgCl 2 , preferably l.5mM MgCl 2 , 5mM to 250mM KCl, preferably 50mM KCl, 50 ⁇ M-200 ⁇ M each dNTP, 1-2 units Taq polymerase, lOO ⁇ g/ml gelatin and 0.l ⁇ M-0.5 ⁇ M, preferably 0.25 ⁇ M, each oligonucleotide primer (Tm >55°C, see below) .
  • the sample is overlayed with 75 ⁇ l of mineral oil and the temperature raised to 90°C-95°C for 5-10 min preferably 5 min, to denature nucleic acids in the cells.
  • the cells are then subjected to 25-35 cycles of 90°C-95°C for 15 sees to 1 min, preferably about 1 min,- followed by 40°C to 60°C, preferably 55°C, for 30 sees to 5 min, preferably about 1 min; followed by 70°C to 75°C for 30 sec ⁇ to 5 min, preferably about 1.5 min.
  • Cycling i ⁇ concluded with a final exten ⁇ ion at 65°C to 80°C, preferably 72°C for 5-20 min, preferably about 5 min.
  • the reaction i ⁇ terminated by chilling to 4°C and/or by addition of EDTA to lOmM final concentration.
  • RNA targets are converted to DNA prior to PCR amplification by employing a reverse transcripta ⁇ e enzyme ⁇ uch as Avian Myelobla ⁇ t Viru ⁇ (AMV) reverse transcripta ⁇ e or Moloney Murine Leukemia virus (MoMuLV)reverse transcriptase.
  • AMV Avian Myelobla ⁇ t Viru ⁇
  • MoMuLV Moloney Murine Leukemia virus
  • Rever ⁇ e tran ⁇ criptase synthesizes a complementary DNA (cDNA) at the 3• -end of the poly(A) -mRNA strand with an oligo-p(dT)15 primer, or at non-specific points along the mRNA template with a random primer p(dN)6, or at a site determined by a specific primer.
  • Reverse transcriptase synthesis is carried out using methods known in the art.
  • such a buffer may contain 50mM KCl, 20mM Tris-HCl (pH 8.4), 2.5mM MgCl 2 , O.lmg/ml bovine serum albumin, ImM each dNTP, RNasin inhibitor (Promega Corporation, USA) at 1 unit/ml, lOOpmol random hexamer oligonucleotides and 200 units of MoMuLV (or AMV) reverse transcripta ⁇ e.
  • the reaction i ⁇ incubated at room temperature for 10 min and then at 37°C to 42°C for 30-60 min. The reaction is terminated by heating at 95C for 5-10 min.
  • the cells are pelleted at 400 x g and a PCR reaction is carried out using specific primer ⁇ to amplify the DNA sequence of interest as described above.
  • a PCR reaction is carried out using specific primer ⁇ to amplify the DNA sequence of interest as described above.
  • the cell ⁇ are fixed as described above.
  • labels can be incorporated into nucleic acids during the amplification process by substituting one of the nucleotides (dNTP) for a labelled nucleotide.
  • dNTP nucleotides
  • Labelled nucleotide ⁇ include, but are not limited to, for example, digoxigenin-nucleotides (eg.
  • the below described detecting agent is the amplified nucleic acid sequence including a label.
  • Target cells containing labelled nucleic acids can then be concentrated using a solid phase support sy ⁇ tem a ⁇ de ⁇ cribed below.
  • a below described detecting agent such as a ⁇ a genetic probe, will typically be necessary.
  • An increase in detection sen ⁇ itivity may be beneficial, for example, to detect and enrich cells infected with viruses such as HIV (human immunodeficiency virus) from mixed populations of cells.
  • viruses such as HIV (human immunodeficiency virus)
  • the presence of actively infected cells can be detected by amplifying viral RNA in the cytoplasm using reverse transcripta ⁇ e and in ⁇ itu PCR.
  • the cells are sufficiently permeable to permit in situ detection of a selected intracellular constituent by a detecting agent.
  • in situ detection refers to detection of an intracellular constituent in an intact cell.
  • an intracellular constituent is detected by using a detecting agent.
  • a "detecting agent” is any compound presently known or later discovered which preferentially attaches to a "selected” or “target” intracellular constituent over other cellular constituents and which itself can be detected for coupling to a solid pha ⁇ e support.
  • a detecting agent of the invention attaches to an intracellular constituent in a manner which is sufficiently stable for the below described solid phase support system to effectively concentrate the specific cells.
  • the attachment may be reversible or irreversible, preferably reversible. Methods of attachment include, but are not limited to, ionic bonding, hydrogen bonding, covalent bonding, van der Waals interactions, and electrostatic interaction.
  • Suitable detecting agents include genetic probes, antibodies, proteins, peptide ⁇ , amino acids, sugars, polynucleotides, enzymes, coenzyme ⁇ , cofactors, antibiotic ⁇ , ⁇ teroids, hormones or vitamins.
  • a detecting agent is preferably detectable by the below described solid phase support ⁇ y ⁇ tem.
  • a selected cellular constituent is detected by binding (hybridization) with a genetic probe as the detecting agent.
  • a genetic probe is a substance that is used to identify a gene or a gene product and can be a genetic material such as DNA (deoxyribonucleic acid), RNA (ribonucleic acid) or synthetic oligonucleotides. Genetic probes may contain natural or chemical derivatives of the normal components of nucleic acids that include guanine, adeno ⁇ ine, uridine, thymidine, or cytosine. Utilizing a genetic probe, methods of in situ hybridization known in the art may be used to detect a selected intracellular nucleic acid. S ⁇ eg. r U.S. Patent No. 5,225,326 issued to Bresser et al.
  • the previously fixed cells are prehybridized (described below) to block any non-specific hybridization. Hybridization is then carried out by replacing the prehybridization solution with the same solution but now containing the hybridization probe.
  • the probe may be rendered detectable by the use of a label prior to attaching to a ⁇ elected nucleic acid or after attaching to the ⁇ elected nucleic acid.
  • Suitable label ⁇ to render a genetic probe detectable include, but are not limited to, digoxigenin, photodigoxigenin, biotin, photobiotin, 2-acetylaminoflourene, sulphone groups, mercury, fluorochromes, dinitrophenol, and psoralen.
  • Other labels include enzyme substrates, enzyme inhibitors and coenzymes.
  • Genetic probes suitable for the invention can come from many ⁇ ource ⁇ .
  • Genetic probe ⁇ may be cloned nucleic acid ⁇ .
  • a DNA fragment of interest may be inserted into a vector and amplified inside an appropriate host cell.
  • the amplified DNA is then extracted and purified for use as a probe.
  • Common vectors include bacterial plasmids, bacterial viru ⁇ e ⁇ , yea ⁇ t artificial chromosomes and cosmids.
  • Genetic probes may also be synthetic oligonucleotides.
  • a synthetic oligonucleotide of 15 to 50 base pairs, in length may be prepared using a DNA synthesizer.
  • Genetic probes can also be prepared by amplification using the polymerase chain reaction, a process that relies on the use of suitable oligonucleotide primers that flank the DNA to be used as a probe.
  • Probes can be double- ⁇ tranded probes such as double- stranded DNA or complementary DNA (cDNA) or single-stranded probes ⁇ uch as single-stranded DNA or RNA or oligonucleotides.
  • a detecting agent is detectable by the below-de ⁇ cribed ⁇ olid phase support system.
  • Genetic probes may be made detectable by using label ⁇ .
  • Label ⁇ can be incorporated into probes by enzymatic or chemical means.
  • Double-stranded probes can be labelled by using DNA polymerases using known methods ⁇ uch a ⁇ random primed DNA labelling, nick translation, labelling with Taq DNA polymerase in the polymerase chain reaction.
  • Single stranded probes M13 can be prepared by in vitro transcription for RNA probes using RNA polymerase ⁇ ⁇ uch a ⁇ T3, T7, SP6.
  • Oligonucleotides can also be labelled by end-labelling or tailing.
  • Nucleic acid probes can be labelled enzymatically with a variety of labels. These include, but are not limited to, nucleotide derivatives of digoxigenin, biotin, and fluorochromes. In addition, nucleic acid probes can be labelled chemically with a variety of labels including but not limited to, photodigoxigenin, photobiotin, 2- acetylaminoflourene, sulphone group ⁇ , mercury, fluorochrome ⁇ , dinitrophenol, and psoralen. Other types of labels include but are not limited to, enzyme sub ⁇ trate ⁇ , enzyme inhibitor ⁇ , and coenzymes chemiluminescer ⁇ and bioluminescers.
  • the probe i ⁇ labelled with a fluorochrome-nucleotide.
  • a fluorochrome-nucleotide incorporated into the each genetic probe allow ⁇ ⁇ imultaneous detection of more than one genetic probe in the same cell by use of an appropriate emis ⁇ ion wavelength filter and epifluorescence microscopy.
  • the above methods involve the labelling of the genetic probe prior to hybridization with an intracellular constituent of the target cell.
  • the genetic probe can be labelled following hybridization of the probe to the target using the technique of primed in situ labelling (PRINS) ⁇ ££ ⁇ .. Koch J. et al. , Genet. Anal. Techniques Applications £.:171 (1991) .
  • PRINS primed in situ labelling
  • DNA probes in the form of oligonucleotides, PCR products or DNA fragments are hybridized to a target cell nucleic acid.
  • the hybridized DNA then acts as a primer for the incorporation of labelled nucleotides in situ.
  • DNA polymerase is used.
  • reverse transcriptase is used to synthesize nucleic acid along the RNA template. Labels that can be incorporated into nucleic acid probes by enzymatic means have been described above.
  • Prehybridization is an optional treatment used to minimize hybridization of the probe to non- ⁇ pecific target molecules.
  • the prehybridization solution i ⁇ generally identical to the hybridization ⁇ olution de ⁇ cribed below but lacks the probe.
  • Blocking DNA is heterologous DNA (or RNA) that reduces non- ⁇ pecific hybridization by binding to molecules in the cytoplasm or nucleus that would otherwise bind probe or detection reagents.
  • Other hybridization solutions suitable for the process of in situ hybridization are described below.
  • Prehybridization is preferably carried out at the same temperature as the hybridization reaction described below and is in the range of 30°C to 50°C, preferably about 37°C, for 30 min to 16 hours, preferably about 2 hours, to block non- specific binding of the probe.
  • the temperature and duration of the prehybridization treatment are varied depending on the cell type and probe used. Following prehybridization, the prehybridization solution is removed after pelleting the cells.
  • the preferred method for denaturation according to the invention i ⁇ combined denaturation of the double- ⁇ tranded nucleic acid probe ⁇ and chromosomal DNA in the pre ⁇ ence of a hybridization buffer that contain ⁇ a chaotropic agent ⁇ uch as formamide (see below) .
  • Denaturation occurs at approximately 30°C above the melting temperature (T , usually at 70°C to 90°C, preferably 80°C, for 2-20 min, preferably 10 min, to denature the probes and chromosomal DNA in the nucleus.
  • T melting temperature
  • the ⁇ m is defined as the temperature at which half the nucleic acids are present in single-stranded form.
  • the T B and reannealing of the nucleic acids is affected by temperature, pH, concentration of monovalent cations and the presence of organic solvents.
  • hybridization solution ⁇ may contain the following component ⁇ .
  • An example of a chaotropic agent which decreases the T m of nucleic acid hybrids and allows hybridizations to be performed at lower temperatures is formamide.
  • Hybridizations are generally performed at 30°C to 45°C with 30%-60% formamide present in the hybridization mixture.
  • formamide also allows the denaturation of probes and cellular nucleic acids by heating to approximately 30°C above the T m .
  • Other chaotropic agents that can used include sodium iodide, urea, thiocyanate, guanidine, and perchlorate.
  • a monovalent cation which stabilizes the hybrids once formed.
  • Monovalent cations such as sodium ion, interact through electrostatic forces with the phosphate groups in nucleic acids. Electrostatic repulsion decreases with increasing salt concentration. High salt concentrations will stabilize mismatched hybrids and allow the detection of cross-hybridizing species.
  • a hybridization buffer typically contains a 20-50mM phosphate, pH 6.5-7.5 buffer. Provided the hybridization reaction is carried out in the pH range of 5 to 9, the rate of hybridization is independent of pH.
  • Blocking DNA which is non ⁇ specific DNA (or RNA) , to reduce non-specific hybridization by binding to molecules in the cytoplasm or nucleus that would otherwise bind probe or detection reagents.
  • DNA used for this purpose is fragmented by physical or chemical means to an average of 100-200 base pair fragments. Commonly used blocking DNA includes calf thymus DNA and fi ⁇ h ⁇ perm DNA. Oligonucleotides such as Poly(C) and Poly(A) can also be used.
  • the hybridization solution may also contain the following optional components:
  • Example ⁇ include dextran ⁇ ulphate, polyethylene glycol and ⁇ imilar polymers.
  • Non- polymers such as phenol can also be used to increase the hybridization rate.
  • Detergents such as ⁇ odiu dodecyl ⁇ ulphate (SDS), CHAPSTM, Triton-XlOOTM, Brij35TM and Brij58 w which act as wetting agents and as permeabilizing agents to as ⁇ ist in probe penetration to the cytoplasm.
  • SDS ⁇ odiu dodecyl ⁇ ulphate
  • CHAPSTM Triton-XlOOTM
  • Brij35TM Brij58 w
  • Bovine ⁇ erum albumin BSA
  • Denhardt's reagent 0.02% Ficoll, 0.02% polyvinyl pyrrolidone and 0.02% BSA
  • a hybridization solution suitable for this invention includes 30%-60%, preferably 50% formamide, 0.1x-6xSSC, preferably 2xSSC, 5%-10% (w/v) , preferably 10% (w/v) dextran sulphate, O.l ⁇ g-lO ⁇ g/ ⁇ l, preferably l ⁇ g/ ⁇ l fish sperm blocking DNA, l-10ng/ ⁇ l, preferably 5ng/ ⁇ l of each labelled probe.
  • Other hybridization solutions suitable for in situ hybridization can be used and are known to skilled in the art See eg. f A.R. Leitch et al., In Situ Hybridization, Bios Scientific, (1994) Oxford, England; B.D. Hames et al., Nucleic Acid Hybridization: A Practical Approach, IRL Press, (1988) Oxford, England.
  • hybridization conditions will depend on the type of hybrids to be formed (ie DNA:DNA or DNA:RNA or RNA:RNA) and whether the sequences are closely related or distantly related.
  • Hybridization conditions suitable for this invention are those that promote the formation of well matched hybrids using conditions of high stringency.
  • Nucleic acid probes suitable for this invention are lOObp-lOOObp, preferably 200bp-400bp in length.
  • Oligonucleotide probe ⁇ suitable for thi ⁇ invention are preferably about 15bp-50bp in ⁇ ize.
  • the probe concentration affects the rate of the nucleation reaction which is the rate limiting step in hybridization and refers to the formation of the first few base pairs. Once nucleation occurs, adjacent base pairs will form to give a zippering effect.
  • a probe concentration which increa ⁇ e ⁇ the annealing rate without excessive background signal is preferred.
  • Preferred probe concentrations are at about 1 to lOng/ ⁇ l, preferably about 5ng/ ⁇ l.
  • Hybridization temperature Hybridization depends on the ability of denatured nucleic acid ⁇ to reanneal with complementary strands in a hybridization solution maintained at a temperature that is just below their melting point (TJ .
  • TJ melting point
  • the broad maximum rate for nucleic acid reannealing occurs from 20 D C to 30°C below the ⁇ « .
  • Hybridization buffers suitable for this invention contain the chaotropic agent formamide (at 50%-60% v/v)and employ hybridization temperatures for DNA:DNA and DNA:RNA hybrids of 30°C to 45°C, preferably 37°C to 42°C, and for RNA:RNA hybrids, temperatures of 50°C to 60°C, preferably 55°C.
  • cells are concentrated by centrifugation at 100 x g to 4000 x g, preferably 400 x g, at 0°C to 25°C, preferably 4°C, for 1-60 min, preferably 15 minutes and then resuspended in an appropriate volume of PBS (see below) .
  • an antibody can be a detecting agent.
  • An antibody is a protein that is produced by blood plasma cells in response to an antigen or a hapten as ⁇ ociated with a ⁇ uitable carrier.
  • Antigen ⁇ include proteins, peptides, polypeptides, carbohydrates, nucleic acids, amino acids, lipids, metabolite ⁇ , and protein label ⁇ .
  • Antibodies are of two types; polyclonal antibodies that react with different parts of the ⁇ ame antigen molecule or monoclonal antibodies that are specific for a single antigenic determinant. Both types of antibodies are suitable for this invention. Methods for the preparation of both type ⁇ of antibodie ⁇ are known to those of skill in the art. See eg. , Antibodie ⁇ : A Laboratory Manual (Harlow E. et al. , eds. 1988), Cold Spring Harbor Pres ⁇ , New York, USA; Monoclonal Antibodies: Principles and Practice (Goding, JW. ed. 1986) Academic Pre ⁇ , London, England.
  • Antibodies act as detecting agents by binding to a selected intracellular constituent in a target cell. Accordingly, as used in the present invention, the term
  • hybridization and derivatives thereof, may be used to refer to the attachment of a detecting agent to an intracellular constituent, whether the detecting agent i ⁇ an antibody, genetic probe or other agent which performs the function of a detecting agent as described herein.
  • Antibody detecting agent ⁇ are preferably detectable by the below-described ⁇ olid pha ⁇ e ⁇ upport ⁇ y ⁇ tem.
  • Blocking ⁇ olutions are known in the art and include, for example, lOOmM Tris/HCl at pH 7.5, 150mM NaCl or PBS or SSC containing a blocking reagent such as 0.5%-2% bovine ⁇ erum albumin, 0.5%-5% normal ⁇ erum or 0.5%-5% non-fat dry milk and may also contain a detergent such as 0.05%-0.2% Tween20TM or SDS or TritonX-lOOTM.
  • Antibody detecting agents can be used individually or as mixture of several antibodies.
  • the antibodie ⁇ may be used at dilutions ranging from undiluted to 1:10,000.
  • the dilution used is determined empirically for each antibody but typically polyclonal antibodies are used as dilutions of 1:10 to 1:100 whereas monoclonal antibodies are used at dilutions of 1:500 or greater.
  • the antibodies are incubated with permeabilized cells for a period of 30 min to 24 hours, preferably 30min-3 hours at 0°C to 37°C, preferably 4°C.
  • the antibody hybridization solution contains lOOmM Tris/HCl; pH 7.5, 150mM NaCl or PBS or SSC and may contain a blocking reagent such as 0.5%-2% bovine serum albumin, 0.5%-5% normal serum or 0.5%-5% non-fat dry milk and may al ⁇ o contain a detergent ⁇ uch a ⁇ 0.05%-0.2% Tween20TM or SDS or TritonX-100TM.
  • the cell ⁇ are wa ⁇ hed 4-5 times for 2-20 min each, preferably 5 min each wash, using lOOmM Tris/HCl; pH 7.5 or PBS or SSC containing 0.5%-5% normal ⁇ erum or 0.5%-2% bovine serum albumin.
  • the washing buffer may also contain a detergent such as 0.05%-0.2% Tween20TM or SDS or TritonX-100TM. Cells are pelleted by centrifugation between washe ⁇ .
  • Cell ⁇ of the mixed cell population which are detected by hybridization of a detecting agent are concentrated u ⁇ ing a ⁇ olid pha ⁇ e ⁇ upport sy ⁇ tem (SPSS) .
  • the solid phase support system of the invention concentrates a target cell by separating an enriched cell complex from non-complexed cells in a mixed population.
  • An "enriched cell complex" refers to the combination of a target cell, a detecting agent and a solid phase support.
  • an enriched cell complex includes an intracellular constituent hybridized to a detecting agent which is coupled to a solid phase support.
  • a solid phase support system includes a solid phase ⁇ upport which can couple to a detecting agent hybridized to an intracellular con ⁇ tituent.
  • a ⁇ olid pha ⁇ e support system may include a ligand or other component for coupling the detected target cell to the solid phase.
  • a detecting agent may couple to the solid phase support directly.
  • a solid phase support ⁇ y ⁇ tem may also include a mechanism for separating the enriched cell complex from other cells in a mixed cell population, for example, in the case of a magnetizable particle solid phase ⁇ upport, a SPSS can include a magnetic field.
  • Solid Phase Support Systems 1. Solid Phase Supports
  • Solid pha ⁇ e ⁇ upport ⁇ suitable for the invention include, for example, magnetizable particles (see eg. , Pourfarzaneth et al. , The Use of Magnetizable Particles in Solid Phase Immunoassay, in Methods of Biochemical Analysis 28, 267-295 (D. Glick ed. 1981) John Wiley, New York) , silica, agarose, glass, dextran, fibre supports, cellulose and synthetic polymers, and similar supports (see eg. Affinity Chromatography 12-145, (Lowe CR. et al. , eds. 1974) Wiley and Sons, London, England).
  • magnetizable particles see eg. , Pourfarzaneth et al. , The Use of Magnetizable Particles in Solid Phase Immunoassay, in Methods of Biochemical Analysis 28, 267-295 (D. Glick ed. 1981) John Wiley, New York
  • silica silica
  • a preferred solid phase support is a superparamagnetic particle.
  • Methods for preparing ⁇ uperparamagnetic particle ⁇ ⁇ uitable for this invention are known to those skilled in the art. £L_n eg. , S. Miltenyi et al. , Cytometry 11:231-238 (1990); E.V. Groman et al., Biotechniques. 156-160 (1985); J.T. Kemshead et al. , Mol. Cell. Biochem 67:11-18 (1985); Pourfarzaneth et al. , Methods of Biochemical Analysi ⁇ 28, 267-295 (D. Glick ed. 1981) , John Wiley, New York)
  • a ⁇ olid phase support system may include a ligand for coupling a detecting agent to the solid phase support.
  • the ligand preferably exhibits specific binding affinity for the detecting agent.
  • the ligand is typically immobilized by al ⁇ o binding to the ⁇ olid pha ⁇ e ⁇ upport.
  • the ligand can have chemically modifiable groups that allow it to be attached to the solid phase without de ⁇ troying it ⁇ binding activity.
  • Chemically modifiable group ⁇ include amino, aldehyde, carboxyl, thiol, hydroxyl and mercurated ba ⁇ es. Where there is no information on the location of chemically modifiable groups in the ligand, a sy ⁇ tematic trial and error approach i ⁇ u ⁇ ed to identify a modifiable group that doe ⁇ not de ⁇ troy the binding activity of the ligand.
  • the ligand can be a protein, peptide, amino acid, ⁇ ugar, polynucleotide enzyme, coenzyme, cofactor, antibiotic, ⁇ teroid, antibody, nucleic acid, hormone or vitamin.
  • ligands include; antigen:antibody interactions where the ligand is an antibody which can couple to a solid phase support such as a synthetic polymer; glycoprotein:lectin interactions where the ligand is lectin which can couple to a synthetic polymer; receptor:ligand interactions where the ligand can couple to a synthetic polymer, for example, SepharoseTM.
  • the ligand can couple to the solid phase support through an amino group.
  • a component of the solid phase support system may include a secondary antibody (the ligand) directly coupled to the solid phase support which binds to the detecting agent (the primary antibody) .
  • the ligand may be coupled to a superparamagnetic particle (the solid phase support) .
  • a superparamagnetic particle the solid phase support
  • Methods for coupling ligands to superparamagnetic particles are known in the art. (£L££ eg.. S. Miltenyi et al. Cytometry 11:231-238 (1990); E.V. Groman et al. ,
  • An enriched cell complex includes a target cell having a detecting agent attached to an intracellular con ⁇ tituent of the target cell which i ⁇ coupled to the solid pha ⁇ e support.
  • the ligand can be eliminated if the detecting agent is directly coupled to the solid phase support.
  • the target intracellular constituent in the target cell is a protein and the detecting agent is an antibody raised to that protein, the antibody may be directly coupled to the solid support through chemical means.
  • the detecting agent may be a genetic probe that is directly coupled to the solid support through, for example, chemical means. Direct coupling of the detecting agent to the solid phase support may reduce the efficiency of hybridization to the intracellular constituent when using a genetic probe or antibody as a detecting agent because of the possibility of stearic hindrance of the solid phase.
  • Non-magnetic solid phase supports may be used. These support ⁇ are mo ⁇ t often packed into a chromatography column and equilibrated by pa ⁇ ing through 10 volume ⁇ of a column buffer. The volume of ⁇ olid support will depend on the number of total cells added to the column and the binding capacity of the solid support. Cell populations of 10 2 -10 6 are separated with 2-5ml of solid pha ⁇ e ⁇ upport containing about lmg/ml of a protein ligand ⁇ uch as an antibody or lectin.
  • Tris Tri ⁇ [hydroxymethyl]aminomethane
  • HEPES HEPES
  • N-2-hydroxyethylpiperazine-N' -2-ethane ⁇ ulfonic acid sodium azide(0.02% w/v) and a protective colloid ⁇ uch as Ficoll70 (0.3% w/v), or human ⁇ erum albumin (0.3%) or gelatin (0.25%). Separations are usually carried out at temperatures below 37°C.
  • the mixed cell population is added to the column in a volume of 30% or les ⁇ of the volume of the ⁇ olid phase support.
  • This percentage is not ab ⁇ olute but rather, is a recommendation to prevent overloading of the column which may reduce the efficiency of the enrichment.
  • the cells are passed into the solid phase support and the buffer flow stopped. Cells are allowed to be in contact with the solid phase support preferably, for about 2-20min to form the enriched cell complex, the precise time of contact may vary, longer incubation times (ie. 15-20min) enhances efficient removal of a ⁇ pecific cell type wherea ⁇ ⁇ horter times (ie. 2- lOmin) les ⁇ ens the risk of contamination with other cells.
  • the column After incubation of the cells with the ⁇ olid pha ⁇ e ⁇ upport, the column is washed. Washing is carried out using approximately 20 volumes of the column at a rate of 2-10ml/min buffer wash to remove non-specifically bound material prior to removing cells from the solid phase support (elution) .
  • solid phase ⁇ upports suitable for the invention include magnetizable particle ⁇ .
  • Superparamagnetic ⁇ olid pha ⁇ e ⁇ upports generally require no equilibration, however, superparamagnetic particles can be washed in buffer prior to use.
  • the amount of ⁇ uperparamagnetic particle ⁇ used for cell enrichment depends on the cell type to be i ⁇ olated and the total number of cell ⁇ in the population.
  • superparamagnetic particles fall into two size ranges: >l ⁇ m diameter particles; and ⁇ 150nm diameter particles.
  • Particles >l ⁇ m in diameter require low gradient magnetic systems, for example, cobalt samarium permanent magnets.
  • Particles ⁇ 150nm in diameter require high gradient magnetic sy ⁇ tem ⁇ where magnetic ⁇ teel wire me ⁇ h i ⁇ packed into a column. When ⁇ uch columns are placed between the poles of a strong permanent magnet (0.6 Telsa) , a very large field gradient is generated next to the wire and cells coated with ⁇ 150nm particles are attracted to the wire.
  • the ratio of particles to total cells ranges from 2:1 to 20:1 depending on the cell type, with a total cell number of 10 s - 10 9 cells, in volumes ranging from 5 ⁇ l (for 10 s total cells) to 5.0 ml (for 10 9 total cells) .
  • Buffers such a ⁇ PBS are u ⁇ ed and may contain sodium azide (0.01%) and bovine serum albumin (0.1%) or fetal calf serum(1.0%) . Magnetic separations are typically carried out at temperatures below 37°C.
  • Superparamagnetic particles are combined with the cell population using the ratios described above and then incubated for 5-30min at 2°C-12°C with constant agitation.
  • the ligand which i ⁇ coupled to the ⁇ olid phase support system binds to the detecting agent which is hybridized to the selected intracellular constituent of the target cell to form an enriched cell complex.
  • the mixture is then placed into a magnetic field. The magnetic field will attract the enriched cell complex.
  • Non-target cells are separated by washing the column.
  • Washing in a low gradient magnetic system is carried out by aspirating the excess solution thilecells bound by the magnetic particle ⁇ are kept on the wall of the tube. The tube is then removed from the magnet and the cell ⁇ are resu ⁇ pended in washing buffer (PBS containing 0.1% w/v bovine serum albumin) . The washing process is repeated 4-5 times to remove non-target cells. The total volume of buffer used for the washes should be 5-10 times the volume in which the particle:cell complex was originally formed.
  • washing buffer PBS containing 0.1% w/v bovine serum albumin
  • High gradient magnetic systems Superparamagnetic particles of size ⁇ 150nm are used at a higher ratio of to cells because of their very small size. Typically, ratio ⁇ of 1000:1 or greater are preferred.
  • Total cell number ⁇ range from 10 6 -10 xo , with wire mesh areas of 10cm 2 (for 10 ⁇ -10 7 cells) to 10cm 3 (for 10 9 -10 10 cells) in volumes ranging from 0.5ml (for 10 6 -10 7 cell ⁇ ) to 50ml (for 10 9 -10 10 ) .
  • Buffers described above for low gradient magnetic systems can be used. Magnetic separations are usually carried out at temperatures below 37°C. Superparamagnetic particles are combined with the cell population, in the ratio ⁇ described above, and then incubated for 5-30min at 2°C to 12°C with constant agitation to form the enriched cell complex.
  • the mixture is loaded onto a column containing wire mesh in the presence of a magnetic field and the sample is passed slowly through the column.
  • the column is washed with 3-5 times the column volume with PBS containing 0.1% w/v bovine serum albumin.
  • the flow of buffer is stopped, the column removed from the magnetic field and cells are back-flushed with buffer.
  • the column is again placed in the magnetic field and the flow of buffer continued.
  • the flow is stopped again, the column removed from the magnetic field and the "magnetic" cell fraction is collected from the column and concentrated by pelleting the cells at 400 x g.
  • the cells are resu ⁇ pended in about lOO ⁇ l PBS.
  • Specific cells of the enriched cell complex may be removed from the solid phase support and used for further diagnostic, therapeutic or research purposes.
  • Methods for eluting the concentrated specific cells to provide an enriched cell population are determined by the type of solid phase support used, the type of ligand coupled to the solid phase support and the type of coupling of the ligand to the solid phase support.
  • elution agents are used to remove the concentrated target cells from the solid pha ⁇ e support sy ⁇ tem. Removal of the ⁇ pecific cell ⁇ from the solid phase support is a compromise between the harshness of the eluent needed for elution and the risk of denaturing or destroying the bioactivity of the eluted material.
  • Elution agents include compounds that weaken ionic bonding, hydrogen bonding, covalent bonding, van der Waals interactions or electrostatic forces that maintain the ligand:detecting agent complex. Elution may invoke changes in pH changes, change ⁇ in ionic strength, changes in polarity of the eluant, deforming agents or electrophoretic desorption. Another common means of elution is affinity elution where the eluting agent competes for binding to the detecting agent, or for binding to the ligand. Elution can occur either by a concentration gradient of a single eluant or by pulse elution using several elution agents. (See eg. , Affinity Chromatography, 52-70, C . et al. , eds.
  • the ligand can be de ⁇ troyed by physical, chemical or enzymatic means to release the target cell.
  • the solid phase support is a superparamagnetic particle.
  • Particles in the ⁇ 150nm range are biodegradable, are not visible by light microscopy or flow cytometry and do not interfere with either the post enrichment identification sy ⁇ tem or ⁇ ignal-generating ⁇ y ⁇ tem ( ⁇ ee below) , therefore it i ⁇ unnecessary to carry out removal of the magnetic particle. If removal is required, incubation overnight at 37°C can be used to remove the particles from the cells.
  • Particles in the >l ⁇ m range can be removed from the cell ⁇ by heating in 95% formamide at temperatures of at least 65°C for at least 2 min, or by boiling in the presence of 0.1% SDS for 5 min, or by incubation overnight at 37°C.
  • biotin:avidin (or streptavidin) identification ⁇ y ⁇ tem ⁇ ee below
  • the u ⁇ e of biotin-nucleotide analogue ⁇ that incorporate a cleavable linker arm into the biotin molecule can be used to dissociate the detecting agent from the superparamagnetic particle.
  • Target cell enrichment may not yield a completely pure target cell population. Accordingly, once the enriched cells are eluted from the solid pha ⁇ e ⁇ upport, preferably, target cells are identified using the below de ⁇ cribed identification ⁇ y ⁇ tem and signal generating sy ⁇ tem.
  • an "identification ⁇ y ⁇ tem” identifies the target cell using an “identifying agent” coupled directly or indirectly to a signal generating system.
  • an identifying agent identifies a target cell by binding to a detecting agent (hybridized to an intracellular constituent prior to enrichment) or by binding directly to an intracellular or extracellular constituent of the target cell.
  • an identifying agent includes all compounds which were previously described as a detecting agent.
  • an identifying agent such as a nucleic acid or antibody
  • binds directly to an intracellular constituent it may bind to the same, or a different intracellular constituent than that bound by the detecting agent.
  • the identification system may also include a label which is incorporated into the detecting agent, intracellular constituent or extracellular constituent, that is bound by the identifying agent.
  • an identification system may be the detecting agent which hybridized to an intracellular constituent prior to solid pha ⁇ e concentration.
  • a signal generating sy ⁇ tem provide ⁇ vi ⁇ ualization of a target cell.
  • the ⁇ ignal generating ⁇ ystem may be incorporated into an identifying agent, or incorporated into a compound which binds to an identifying agent. If the identifying agent is the detecting agent, the signal generating system may or may not be incorporated into the detecting agent prior to solid phase enrichment.
  • the methods for identification and vi ⁇ ualization of enriched target cells will depend on the type of identification system used. If, for example, the identifying agent is to identify a target cell by binding to a detecting agent which is a nucleic acid with a label such as digoxigenin, 2-acetylaminofluorence, a sulphone group, mercury/trinitrophenol, or biotin then an immunocytochemical identifying agent may be u ⁇ ed with a fluorochrome, enzyme- generated precipitate or metal ⁇ ignal generating system.
  • a detecting agent which is a nucleic acid with a label such as digoxigenin, 2-acetylaminofluorence, a sulphone group, mercury/trinitrophenol, or biotin
  • an immunocytochemical identifying agent may be u ⁇ ed with a fluorochrome, enzyme- generated precipitate or metal ⁇ ignal generating system.
  • an avidin (or ⁇ treptavidin) identifying agent may be u ⁇ ed with a fluorochrome, enzyme-generated precipitate or metal signal-generating system. If the detecting agent used was a nucleic acid probe with a fluorochrome-nucleotide incorporated, no further identification or signal generating system may be needed. Visualization can be direct, by fluorescence. Alternatively, identification can be indirect by using an immunocytochemical identifying agent and a fluorochrome, enzyme-generated precipitate or metal signal- generating.
  • immunocytochemical identification and visualization can be carried out in a one stage or two stage process.
  • the signal generating system is coupled to an antibody which binds to a detecting agent, a label incorporated into a detecting agent, an intracellular constituent or an extracellular constituent.
  • Identification occurs by the antibody (identifying agent) , with the coupled signal generating system, binding to the antigen.
  • identification and visualization includes a primary and secondary antibody.
  • the primary antibody binds to an antigen, such as a detecting agent, a label incorporated into a detecting agent, an intracellular constituent or an extracellular constituent.
  • the secondary antibody which carries the signal generating system binds to the primary antibody.
  • a two stage detection system is preferred because several secondary antibodies carrying the signal-generating sy ⁇ tem can bind to the primary antibody which provides for significant amplification of the signal thereby enhancing the sensitivity of visualization.
  • Biotin.avidin or streptavidin ⁇ identification system
  • a biotin:avidin (or streptavidin) identification system uses a biotin label.
  • the biotin label may be incorporated into the detecting agent prior to solid phase enrichment or the biotin may be incorporated into an identifying agent.
  • the avidin (or streptavidin) is conjugated to a signal-generating sy ⁇ tem.
  • the avidin (or streptavidin) then binds to biotin.
  • Amplification of this biotin-avidin complex can be obtained by adding a biotinylated anti-avidin (or streptavidin) antibody followed by another layer of avidin (or streptavidin) conjugated to a signal-generating system.
  • immunocytochemical or biotin:avidin (or streptavidin) sy ⁇ tem ⁇ are suitable for identifying a target cell.
  • Methods for employing immunocytochemical identification sy ⁇ tem ⁇ and the biotin:avidin (or streptavidin) identification sy ⁇ tems are known in the art See eg. r Leitch, et al. , In Situ Hybridization, Bios Scientific (1994) , Oxford, England; B.D. Hames, et al. , In Nucleic Acid Hybridization: A Practical Approach, (1988) IRL Pre ⁇ , Oxford, England. B. Signal Generating Systems
  • visualization of a target cell identified by an identifying agent may be accomplished through use of a signal-generating sy ⁇ tem.
  • Signal generating systems suitable for the invention include any compound which can couple to an identifying agent for visualization of a target cell.
  • Preferred signal generating sy ⁇ tem ⁇ include, for example, fluorochrome ⁇ , enzyme-generated precipitate or metals.
  • a fluorochrome is a chemical compound that emits fluorescence at a specific emis ⁇ ion wavelength when excited by light of the appropriate excitation wavelength.
  • an identifying agent such as a genetic probe
  • Fluorochromes can al ⁇ o be attached to avidin (or ⁇ treptavidin) , or to antibodies, which allows them to be used in the immunocytochemical identification systems and biotin- avidin (or streptavidin) identification systems described above.
  • Two commonly used enzyme ⁇ for immunocytochemi ⁇ try are alkaline pho ⁇ phata ⁇ e and horse radish peroxidase.
  • the enzymes allow visualization by catalyzing the localized precipitation of a colored product at the site where the probe has bound, following addition of the appropriate substrate.
  • endogenous alkaline phosphata ⁇ es and peroxidases are preferably inactivated prior to immunocytochemistry.
  • Endogenous alkaline phosphatases found in placental and intestinal tissue may be inactivated by, for example, the addition of levamisole or 20% acetic acid.
  • Endogenou ⁇ peroxidases, which may be found in various cell types in the blood, can be inactivated by borohydride, periodate or phenyl hydrazine.
  • Colloidal gold conjugated to an antibody or to avidin (or streptavidin) can be used with both of the above types of identification systems.
  • the metal allows the target cell, bound to an identifying agent, to be visualized either by light microscopy or electron microscopy.
  • Target cells that have been enriched and identified according to the invention are suitable for further analysi ⁇ .
  • the type ⁇ of analy ⁇ is fall into two broad categories: analysis of intact cells and analy ⁇ i ⁇ of intracellular component ⁇ .
  • cells may be depo ⁇ ited on ⁇ lide ⁇ and visualized either with a light microscope when employing enzyme-generated precipitate or metal signal generating sy ⁇ tem ⁇ .
  • identifying agents that are either directly or indirectly labelled with a fluorescent label
  • the results can be visualized on a fluorescence microscope, employing a wavelength emis ⁇ ion filter appropriate for the particular fluorochrome to be visualized.
  • Cells may also be automatically analyzed using a computer-controlled fluorescence-based image analysis system.
  • the target cell of interest may express several intracellular specific nucleic acids or protein products that can be used to provide additional means for enriching a particular cell type of interest.
  • the method of the invention can al ⁇ o be combined with known method ⁇ of extracellular hybridization. Accordingly, the enriched cell population can be ⁇ ubjected to further rounds of in situ hybridization and solid phase enrichment u ⁇ ing detecting agents for other intracellular or extracellular constituents.
  • the enriched cells can also be subjected to further in situ hybridization analysi ⁇ or to fluore ⁇ cence activated cell sorting using other detecting agents ⁇ pecific for a ⁇ elected intracellular constituent.
  • in situ hybridization analysi ⁇ or to fluore ⁇ cence activated cell sorting using other detecting agents ⁇ pecific for a ⁇ elected intracellular constituent.
  • detecting agents ⁇ pecific for a ⁇ elected intracellular constituent For example, when fetal cells have been enriched from maternal blood, the use of labelled chromo ⁇ ome-specific probes and in situ hybridization can provide information on the chromosome complement of the fetus and in doing so identify chromosomal aneuploidy.
  • Thi ⁇ can be achieved by u ⁇ ing another round of in ⁇ itu hybridization and ⁇ olid pha ⁇ e enrichment u ⁇ ing detecting agent ⁇ specific for a selected intracellular constituent of other cell types in the cell population.
  • Cell ⁇ that have been concentrated by in situ hybridization and solid phase enrichment are suitable for biochemical and genetic analy ⁇ is.
  • nucleic acids extracted from the cells can be analyzed by molecular biological techniques, ⁇ uch a ⁇ Northern and Southern blotting analy ⁇ i ⁇ , to identify specific nucleic acid ⁇ equence ⁇ that are pre ⁇ ent in the enriched cell population.
  • the technique of PCR can also be used to identify specific nucleic acid ⁇ equences in enriched cell populations.
  • Episomal elements such as eukaryotic or bacterial plasmid ⁇ or viral nucleic acids can also be isolated from the enriched cells and reintroduced into an appropriate host for further analysis. Proteins extracted from the enriched cells can be analyzed by Western blotting.
  • the ability to reliably detect ⁇ pecific cells present in low concentration may, for example, provide a safe and cost effective means for diagnosi ⁇ of fetal genetic abnormalitie ⁇ by enrichment of fetal cell ⁇ from maternal blood (See eg. M. Adinolfi, Prenatal Diagnosis 15:889-896 (1995)).
  • the method may also provide for the early diagnosis of oncogenic disea ⁇ e ba ⁇ ed on the pre ⁇ ence of target cells in blood, lymph, bone marrow or other body fluid or tissue, as well as provide screening for HIV, or other infectious viral, bacterial or parasitic di ⁇ ea ⁇ e.
  • the invention may al ⁇ o provide a non- inva ⁇ ive method for detecting an individual' ⁇ genetic predi ⁇ position to various condition ⁇ , for example, heart disease, prostate cancer, breast cancer, leukemia and other conditions where genetic predisposition may be a factor.
  • the method may also provide for detecting cells shed from tumor ⁇ , carcinoma ⁇ , ⁇ arcomas and melonamas into lymph fluid and into the circulatory system and in so doing, may provide a prediction for the likelihood of metastasis.
  • the ease and safety of the method further provides for minimally invasive evaluation of therapeutic efficacy of, for example, anti-bacterial, anti-viral, and anti-cancer treatments.
  • target cell enrichment also cau ⁇ es target cell depletion of a mixed cell population
  • the present invention may lead to new methods for treatment of disea ⁇ e ⁇ such as leukemia, AIDS, blood-borne parasitic disease ⁇ and ⁇ imilar di ⁇ ea ⁇ es which may be ameliorated through depletion of selected cells.
  • the inventor also foresee ⁇ the u ⁇ e of the present invention in combination with other methods, such as extracellular ba ⁇ ed solid phase enrichment, fluorescence activated cell sorting, and molecular biological methods.
  • the invention further provides a portable method for enrichment of at least one specific cell from a mixed cell population using a solid phase support system.
  • a portable enrichment system may be included into a kit which may be as ⁇ embled and packaged for use in enriching one or more specific cells.
  • a kit may include, at least, a fixing agent, a permeabilizing agent, a detecting agent and a solid phase support system as described previously.
  • the solid phase support system may include a portable solid phase support, for example, magnetizable particle ⁇ , silica, agarose, dextran, fibre supports, cellulose, synthetic polymer ⁇ and similar ⁇ upports .
  • the kit may further include an identifying agent and a ⁇ ignal generating ⁇ ystem.
  • the fixing agent and permeabilizing agent included with a kit may be any of those agents previously described.
  • a single detecting agent may be included for identification of a single specific cell.
  • multiple detecting agents may be included for detecting multiple specific cell types or detecting a single specific cell type based on the presence of multiple intracellular constituents .
  • EXAMPLES Example 1 Enrichment of fetal cells using intracellular messenger RNA (mRNA) gene products from maternal blood in a model system.
  • mRNA messenger RNA
  • Enrichment of fetal cells from maternal blood was performed in a model sy ⁇ tem where known numbers of fetal trophoblast cell ⁇ (called ⁇ yncytiotrophoblast sprout ⁇ ) were prepared and then added to maternal blood and then recovered using an enrichment procedure.
  • ⁇ yncytiotrophoblast sprout ⁇ known numbers of fetal trophoblast cell ⁇
  • a 1.2kb partial cDNA fragment encoding the human placental lactogen hormone (hPL) gene was excised from a plasmid cDNA clone pN202 (18) (S. Latham et al. , Prenatal Diagnosis. 16_:813-821 (1995)) by restriction digestion with EcoRI.
  • a 0.9kb partial cDNA fragment encoding the PSG1 gene was excised from a plasmid cDNA clone pSPIO.9 (S. Latham and B. Kalionis unpubli ⁇ hed data) by re ⁇ triction digestion with EcoRI. Each DNA fragment was labelled separately with digoxigenin-11-dUTP, using the random-primed labelling kit from Boehringer Mannheim (DIG-High Prime kit, 1995 Catalogue No. 1-585-606) and following the manufacturers instruction ⁇ included in the kit.
  • the total number of syncytiotrophoblast sprout ⁇ in each aliquot was counted microscopically to determine an average number per aliquot. For example, the count ⁇ obtained in a typical ⁇ eries of five aliquots were 68, 70, 71, 71 & 72. From this, the volume of su ⁇ pension containing approximately 50 syncytiotrophoblast sprouts wa ⁇ calculated (eg. 35 ⁇ l) .
  • 50 ⁇ yncytiotrophoblast ⁇ prout ⁇ were added to 20 ml of human peripheral blood cells in 10ml tube ⁇ and then incubated with ly ⁇ i ⁇ buffer (0.1M NH 4 C1, 15mM NaHC0 3 , O.lmM Na 2 EDTA) to ly ⁇ e the red blood cell ⁇ , centrifuged at 400 x g for 5 min, incubated a second time in lysis buffer and then washed with 50 ml cold saline. The white blood cells were centrifuged at 400 x g for 10 minutes in a clinical centrifuge (yielding a total of about 10 ⁇ cells) .
  • ly ⁇ i ⁇ buffer 0.1M NH 4 C1, 15mM NaHC0 3 , O.lmM Na 2 EDTA
  • the cells were fixed by resuspending in 4% paraformaldehyde in PBS for 10 minutes at room temperature. Centrifugation was repeated at 400 x g to pellet the cells. The excess fixative was withdrawn by aspiration. The cell pellet was washed in PBS for 5 min and again centrifuged to pellet the cells. The PBS was removed by aspiration and the cells washed once more.
  • the cells were then permeabilized with Proteina ⁇ e K at 100 ⁇ g/ml for 10 min at 37°C Permeabilization wa ⁇ stopped with 0.2% (w/v) glycine in PBS for 2 min at room temperature.
  • the cells were pelleted at 400 x g and excess glycine removed.
  • the cells were then post-fixed in 4% paraformaldehyde as described above.
  • the cells were washed twice with PBS as described above with saline and the cell pellet was resuspended in hybridization buffer containing one or more of the genetic probes.
  • the hybridization buffer contained 60% formamide, 2xSSC, 25mM NaH 2 P0 ⁇ pH 7.4, 5% dextran sulphate, 250 ⁇ g/ml sonicated, denatured salmon sperm DNA and a detection agent comprising one or more digoxigenin-labelled hybridization genetic probes at a concentration of 5ng/ ⁇ l.
  • the labelled genetic probe Prior to re ⁇ u ⁇ pending the cell ⁇ , the labelled genetic probe, in hybridization buffer, was denatured by incubating the mixture at 80°C for 10 min followed by snap chilling on ice for 5 min. Hybridization was carried out for 16 hour ⁇ at 37°C Cells were pelleted at 400 x g in a clinical centrifuge and the excess hybridization solution was removed. The cells were washed twice in 0.5XSSC at room temperature for 5 min and then washed a third time in 0.5xSSC at 37°C for lOmin. The cells were again pelleted and washed in PBS for 5min.
  • mice anti-digoxigenin antibody (approx. 0.4 ⁇ g) (1995 Cat No. 1-333-062, Boehringer Mannheim, Germany) was added to the cells. The cells were incubated in the presence of the antibody for 2 to 3 hours at 37°C The cells were then pelleted and exces ⁇ antibody removed by a ⁇ piration and the cells washed twice in PBS as de ⁇ cribed above. The cell pellet wa ⁇ re ⁇ u ⁇ pended in 20 ⁇ l of Rat-anti-mouse IgGl-Microbeads (1992 Cat No. 271-01, Miltenyi Biotec Gmbh, Germany) and incubated at 4 ⁇ C for 15 min.
  • Rat-anti-mouse IgGl-Microbeads (1992 Cat No. 271-01, Miltenyi Biotec Gmbh, Germany
  • the column was removed from the magnetic field and backwashed with 2 ml of the PBS/0.01% sodium azide/l%BSA buffer to dislodge any cells that were non ⁇ specifically bound to the column.
  • the column was placed back into the magnetic field and the cells allowed to migrate back onto the wire-mesh.
  • the concentrated cells retained by the magnet after washing and backwa ⁇ hing were eluted by removing the column from the magnet and passing 10 ml of PBS buffer through the column. This elution was repeated once more. The eluted cells were then centrifuged at 400 x g for 5 min to pellet the cells.
  • enriched cells were then identified and visualized.
  • Pelleted cells were resuspended in approximately 50 ⁇ l of PBS buffer and then deposited onto a microscope ⁇ lide and air dried.
  • Cell ⁇ on the ⁇ lide ⁇ were incubated in 20% normal sheep serum for 30 min at 20°C to block non-specific binding of the ⁇ econdary antibody.
  • Slide ⁇ were briefly rinsed in PBS.
  • a labelled secondary antibody, anti-digoxigenin-rhodamine Fab fragments from sheep (1995 Cat No. 1-207-750, Boehringer Mannheim, Germany) wa ⁇ applied to the cell ⁇ at a dilution of 1:10 and incubated for 60 min at 20°C for target cell identification and vi ⁇ ualization.
  • the ⁇ lides were washed twice in PBS containing 0.1% v/v Nonidet P40 (Boehringer
  • Anti-fade mountant (90% v/v glycerol 0.1% v/v p-phenylenediamine) wa ⁇ added to the cell samples and then a coverslip was applied. Cells were then detected with fluorescence microscopy using a 615nm emis ⁇ ion wavelength filter and the total number of fluore ⁇ cent cell ⁇ determined.
  • the entire sample was scanned under the microscope and the fluorescent syncytiotrophoblast sprout ⁇ counted.
  • the percentage recovery of contaminating white blood cells (WBC) wa ⁇ calculated from the number recovered in the magnetic activated cell sorting (MACS) eluate compared to the total WBC count of the sample (average 10 7 cells/ml) and was ⁇ 0.001% in all cases (data not shown).
  • Table 1 ⁇ hows that syncytiotrophoblast sprout ⁇ were recovered from a mixed population of cell ⁇ in maternal blood u ⁇ ing genetic probe ⁇ as detecting agents and a superparamagnetic particle as solid pha ⁇ e ⁇ upport for enrichment. The efficiency of recovery of syncytiotrophoblast sprout ⁇ was improved by the use of multiple genetic probes.
  • Example 2 Enrichment of 5T33 myeloma cells using an intracellular messenger RNA (mRNA) for the IgH gene in a model system.
  • mRNA messenger RNA
  • Myelomas are a tumor of cells that are derived from the hematopoietic tissue of bone marrow. Myeloma tumors are clonal in origin and secrete large amounts of a single species of antibody.
  • In vitro cultured cell lines have been establi ⁇ hed from 5T33 myeloma tumors. See L.S. Manning et al., Br. J. Cancer ££:1088-1093 (1992) .
  • myeloma cell lines the immunoglobulin heavy chain (IgH) gene is expres ⁇ ed at high level ⁇ . PCR primer ⁇ were used to specifically amplify a segment of the IgH gene to be used as the genetic probe.
  • IgH immunoglobulin heavy chain
  • the PCR primeers used for amplification were FR1 5'-(GC) AGGT(CG) (AC)A(AG)CTGCAG(CG)AGTCT-3' (SEQ ID N0:1); and FR4 5'-GGAGACTCTGAGAGTGGTG-3' (SEQ ID NO:2) .
  • the IgH PCR fragment was labelled with digoxigenin-11- dUTP, using the random-primed labelling kit from Boehringer Mannheim (DIG-High Prime kit, 1995 Catalogue No. 1-585-606) and following the manufacturers in ⁇ truction ⁇ included in the kit.
  • DIG-High Prime kit 1995 Catalogue No. 1-585-606
  • In situ hybridization experiments with the IgH genetic probe have shown this probe to be specific for 5T33 myeloma cells and the IgH genetic probe does not significantly cross- hybridize with other cell types in human maternal peripheral blood (H. Su ⁇ kin and B. Kalioni ⁇ , unpubli ⁇ hed data) .
  • a genetic probe to the 3 ⁇ HSD gene was used as a control.
  • 5T33 myeloma cell culture lines were grown and harvested using methods known in the art. ( &s. eg. , Culture of Animal Cells: A Manual of Basic Technique (R.I., Freshney ed. 1987), Wiley-Lis, New York, USA) .
  • Approximately 1000 5T33 myeloma cells were added to 20 ml of human maternal peripheral blood cells and then incubated with lysi ⁇ buffer (0.1M NH 4 C1, 15mM NaHC0 3 , O.lmM Na 2 EDTA) to ly ⁇ e the red blood cell ⁇ , centrifuged at 400 x g for 5 min, incubated a ⁇ econd time in lysis buffer and then washed with 50 ml cold saline. The white blood cells were centrifuged at 400 x g for 10 minutes in a clinical centrifuge (yielding a total of about 10 8 cells) .
  • lysi ⁇ buffer 0.1M NH 4 C1, 15mM NaHC0 3 , O.lmM Na 2 EDTA
  • the cells were fixed by resuspending in 4% paraformaldehyde in PBS for 10 minutes at room temperature. Centrifugation was repeated at 400 x g to pellet the cells. The excess fixative was withdrawn by aspiration. The cell pellet was washed in PBS and again centrifuged to pellet the cells. The PBS was removed by aspiration and the cells washed once more. The cells were then permeabilized with Proteinase K at 100 ⁇ g/ml for 10 min at 37°C Permeabilization was stopped with 0.2% (w/v) glycine in PBS for 2 min at room temperature. The cells were then post-fixed in 4% paraformaldehyde as de ⁇ cribed above. The cell ⁇ were washed two more times with PBS and pelleted as described above.
  • hybridization buffer containing 60% formamide, 2xSSC, 25mM NaH 2 P0 « pH 7.4, 5% dextran sulphate, 250 ⁇ g/ml ⁇ onicated, denatured salmon sperm DNA and the detection agent comprising a digoxigenin-labelled hybridization genetic probe at a concentration of 5ng/ ⁇ l.
  • the labelled genetic probe Prior to resuspending the cells, the labelled genetic probe, in hybridization buffer, wa ⁇ denatured by incubating the mixture at 80°C for 10 min followed by snap chilling on ice for 5 min. Hybridization was carried out for 16 hours at 37°C Cells were pelleted at 400 x g in a clinical centrifuge and excess hybridization solution was removed. The cells were washed twice in 0.5XSSC twice at room temperature for 5 min and then washed a third time in 0.5xSSC at 37°C for lOmin. The cells were again pelleted and washed in PBS for 5min.
  • mice anti-digoxigenin antibody (approx. 0.4 ⁇ g) (1995 Cat No. 1-333-062, Boehringer Mannheim, Germany) was added to the cells. The cells were incubated in the presence of the antibody for 2 to 3 hours at 37°C The cells were then pelleted and excess antibody removed by aspiration and the cells washed twice in PBS as described above. The cell pellet was resuspended in 20 ⁇ l of Rat-anti-mouse IgGl-Microbeads (1992 Cat No. 271-01, Miltenyi Biotec Gmbh, Germany) and incubated at 4°C for 15 min. Magnetic columns (1992 Cat No.
  • the column was removed from the magnetic field and backwashed with 2 ml of the PBS/0.01% sodium azide/l%BSA buffer to di ⁇ lodge any cell ⁇ that were non ⁇ specifically bound to the column.
  • the column was placed back into the magnetic field and the cells allowed to migrate back onto the wire-mesh.
  • the washing and backwashing step was repeated four times. Cells retained by the magnet after washing and backwashing were eluted by removing the column from the magnet and passing 10 ml of PBS buffer through the column. This elution was repeated once more.
  • the eluted cells were then centrifuged at 400 x g for 5 min to pellet the cells. r 1
  • the enriched cells were then identified and visualized. Pelleted cells were resu ⁇ pended in approximately 50 ⁇ l of PBS buffer and then depo ⁇ ited onto a micro ⁇ cope ⁇ lide and air dried. Cell ⁇ on the ⁇ lide ⁇ were incubated in 20% normal ⁇ heep ⁇ erum for 30 min at 20°C to block non-specific binding of the secondary antibody. Slides were briefly rinsed in PBS. A labelled secondary antibody, anti-digoxigenin-rhodamine Fab fragments from sheep (1995 Cat No.
  • the percentage recovery of fluorescent 5T33 myeloma cells following in situ hybridization with the genetic probe and solid phase enrichment Each number represents percentage recovery per individual sample.
  • the percentage recovery of contaminating white blood cells (WBC) was calculated from the number recovered in the MACS eluate compared to the total WBC count of the sample (average 10' cells/ml) and was ⁇ 0.001* in all cases (data not shown) .
  • Table 2 ⁇ hows that 5T33 myeloma cells were recovered from a mixed population of cell ⁇ in maternal blood u ⁇ ing a genetic probe as a detecting agent and a superparamagnetic particle as the solid phase support for enrichment. Recovery of cells was dependent on the addition of 5T33 myeloma cells to maternal blood.
  • a 3 ⁇ HSD genetic probe which is not expres ⁇ ed either in maternal cells or 5T33 myeloma cells, also gives very low recovery of cells indicating that recovery of cells was dependent on the addition of a specific genetic probe.
  • EXAMPLE 3 In situ PCR amplification in liquid to amplify intracellular nucleic acid target sequences prior to enrichment using a model system.
  • HTR8 The human cytotrophoblast cell line, HTR8 (CH. Graham et al., Experimental Cell Research. 2 :204-211 (1993)) expre ⁇ e ⁇ the tran ⁇ cription factor gene Dlx-4 (L. Quinn and B. Kalioni ⁇ , Gene, in pre ⁇ s (1997)) .
  • HTR8 cells are grown in culture and harvested (CH. Graham et al. , Experimental Cell Research. 2QJ2.:204-211 (1993)). Cells (approx. total of 10 ⁇ -10 ⁇ cells) are then fixed by resuspending in 4% paraformaldehyde in PBS for 10 minutes at room temperature. The cells are centrifuged at 400 x g to pellet the cells. Excess fixative is withdrawn by aspiration and the cell pellet is washed in PBS for 5 min and again centrifuged to pellet the cells.
  • the cells are then permeabilized by treatment with Proteinase K at 100 ⁇ g/ml for 10 min at 37°C
  • Permeabilization is stopped with 0.2% (w/v) glycine in PBS for 2 min at room temperature.
  • the cells are pelleted at 400 x g and excess glycine is removed.
  • the cells are then post-fixed in 4% paraformaldehyde in PBS for 10 minutes at room temperature.
  • Cells are pelleted at 400 x g and exces ⁇ fixative is withdrawn by aspiration.
  • the cell pellet is washed in PBS for 5 min and again centrifuged to pellet the cells.
  • Cells are resuspended in lOO ⁇ l of buffer containing 50mM KCl, 20mM Tris-HCl (pH 8.4), 2.5mM MgCl 2 , O.lmg/ l bovine serum albumin, ImM each dNTP, RNasin inhibitor (Promega Corporation, USA) at 1 unit/ ⁇ l, lOOpmol random hexamer oligonucleotides and 200 units of MoMuLV (or AMV) reverse transcriptase.
  • the reaction is incubated at room temperature for 10 min and then at 37°C for 60 min.
  • the reaction i ⁇ terminated by heating at 95°C for 5 min.
  • the cell ⁇ are then pelleted at 400 x g, and excess solution is removed by aspiration.
  • Cells are resuspended in lOO ⁇ l of buffer containing lOmM Tris-HCl (pH 8.4), 1.5mM MgCl 2 , 50mM KCl, 200 ⁇ M each of dGTP, dCTP, dATP, 190 ⁇ M dTTP, lO ⁇ M digoxigenin- 11-dUTP, 1-2 units Taq polymerase, lOO ⁇ g/ml gelatin and 0.25 ⁇ M each of Dlx-4 specific 31ks primer (5' -AGTCTTCCGGGTGGAGC-3 • ) (SEQ ID NO:3) and Dlx-4 specific 31sk primer (5'-
  • GTCACTATCAGCGCTGC-3 1 GTCACTATCAGCGCTGC-3 1 ) (SEQ ID NO:4) (L. Quinn and B. Kalionis, Gene, in pres ⁇ (1997) .
  • the sample is overlayed with 75 ⁇ l of mineral oil and the temperature raised to 95°C for 5 min, to denature nucleic acids in the cells.
  • the cells are then subjected to 30 cycles of 95°C for 1 min, 52°C for 1 min and 72°C for 1.5 min. Cycling is concluded with a final extension at 72°C for 10 min.
  • the reaction is terminated by chilling to 4°C and addition of EDTA to 10 mM.
  • the cells are then pelleted at 400 x g and resu ⁇ pended in 4% paraformaldehyde in PBS and incubated for 10 minute ⁇ at room temperature to fix the cell ⁇ .
  • HTR8 cell ⁇ are added to 20 ml of human peripheral blood cell ⁇ , incubated with ly ⁇ i ⁇ buffer (0.1M NH 4 C1, 15mM NaHC0 3 , 0.ImM Na 2 EDTA) to ly ⁇ e the red blood cells, centrifuged at 400 x g for 5 min, incubated a second time in lysis buffer and then washed with 50 ml cold saline.
  • the cells are centrifuged at 400 x g for 10 minute ⁇ in a clinical centrifuge (yielding a total of about 10* cell ⁇ ) .
  • the cells are fixed by resu ⁇ pending in 4% paraformaldehyde in PBS for 10 minute ⁇ at room temperature.
  • the cells are then permeabilized by treatment with Proteinase K at 10-100 ⁇ g/ml for 10 min at 37°C Permeabilization is stopped with 0.2% (w/v) glycine in PBS for 2 min at room temperature.
  • the cells are then post-fixed in 4% paraformaldehyde as described above.
  • the cell ⁇ are washed two more times with PBS and pelleted as described above.
  • Target cells are then concentrated.
  • a one in 100 dilution of a mouse anti-digoxigenin antibody (approx. 0.4 ⁇ g) (1995 Cat No. 1-333-062, Boehringer Mannheim, Germany) is added to the cells.
  • the cells are incubated in the presence of the antibody for 2 to 3 hours at 37C
  • the cells are then pelleted and excess antibody is removed by aspiration and the cells are washed twice in PBS as described above.
  • the cell pellet is resu ⁇ pended in 20 ⁇ l of Rat-anti-mouse IgGl- Microbead ⁇ (1992 Cat No. 271-01, Miltenyi Biotec Gmbh, Germany) and incubated at 4°C for 15 min. Magnetic column ⁇ (1992 Cat No.
  • the column is removed from the magnetic field and backwashed with 2 ml of the PBS/0.01% ⁇ odium azide/l%BSA buffer to dislodge any cell ⁇ that non- ⁇ pecifically bind to the column.
  • the column is placed back into the magnetic field and the cells allowed to migrate back onto the wire-mesh.
  • the washing and backwashing step is repeated four times.
  • Cell ⁇ retained by the magnet after washing and backwashing are eluted by removing the column from the magnet and then passing 10 ml of buffer through the column. This elution is repeated once more.
  • the eluted cells are then centrifuged at 400 x g for 5 min to pellet the cells.
  • Pelleted cells are resu ⁇ pended in approximately 50 ⁇ l of PBS buffer and then depo ⁇ ited onto a micro ⁇ cope slide and air dried. Cells on the slide ⁇ are incubated in 20% normal ⁇ heep serum for 30 min at 20°C to block non-specific binding of the secondary antibody. Slides are briefly rinsed in PBS. A labelled secondary antibody, anti-digoxigenin-rhodamine Fab fragments from sheep, (1995 Cat No.
  • Example 4 Enrichment of cells using an intracellular messenger RNA (mRNA) expressed in prostate cells, in a model system and from patient samples.
  • mRNA messenger RNA
  • LNCaP cells (American Type Culture Collection CC CRL-1740 LNCaP.FGC Metastatic prostate adenocarcinoma, human) are an in vitro cultured cell line, originally derived from cells isolated from a needle aspiration biopsy of the supraventricular lymph node, from a patient with metastatic prostate carcinoma (Gibaz, Z. et al. , Cancer Genet. Cytogenet. 11:399-404, 1984) . The cells express the gene for Androgen Receptor (AR) at high levels.
  • AR Androgen Receptor
  • PCR primers were used to amplify a segment of the AR gene to be used as the genetic probe.
  • the 750bp AR PCR fragment was prepared by PCR amplification using primers: ARCS1 5'- TGAAGCAGGGATGACTCTGGG-3' (SEQ ID NO:5) and ARCAS4 5'- CTCGCAATAGGCTGCACGGAG-3' (SEQ ID NO:6) [position 2016 to 2766; Tilley et al, Proc. Natl. Acad. Sci. USA ££(D :327-331
  • the 750bp fragment generated with the ⁇ e primers was subcloned into the Smal site of Bluescript pla ⁇ mid vector (Stratagene, USA) and pla ⁇ mid DNA prepared.
  • the in ⁇ ert wa ⁇ i ⁇ olated following restriction digestion with EcoRI and BamHI and the isolated insert fragment was labelled with digoxigenin-11-dUTP, using the random-primed labelling kit from Boehringer Mannheim (DIG-High Prime kit, 1995 Catalogue No. 1-585-606) and following the manufacturers instructions included in the kit.
  • LNCaP cell culture lines were grown and harvested using known methods (In ⁇ Culture of Animal Cells : a manual of basic technique" (1987), Freshney, R.I. (ed., Wiley-Li ⁇ , New York, USA) .
  • LNCaP cells were added to a 20 ml sample of normal human male blood cells.
  • Three other human male blood samples from patients with benign prostatic hyperpla ⁇ ia were used (approx. 10 mis each) , but no LNCaP cell ⁇ were added to the ⁇ e ⁇ amples.
  • the blood sample ⁇ were then incubated with ly ⁇ is buffer (0.1M NH 4 C1, 15mM NaHC0 3 , O.lmM Na 2 EDTA) to lyse the red blood cells, centrifuged at 400g for 5 min, incubated a second time in lysi ⁇ buffer and then washed with 50 ml cold saline.
  • the white blood cells were centrifuged at 400g for 10 minutes in a clinical centrifuge.
  • the cells were fixed by resu ⁇ pending in 4% paraformaldehyde in PBS for 10 minute ⁇ at room temperature. Centrifugation was repeated at 400g to pellet the cells. The excess fixative was withdrawn by aspiration. The cell pellet was washed in PBS and again centrifuged to pellet the cells. The PBS was removed by aspiration and the cells washed once more.
  • the cells were then permeabiulized by treatment with Proteinase K at 100 ⁇ g/ml for 5 min at 37 ⁇ C Permeabilization wa ⁇ stopped with 0.2% (w/v) glycine in PBS for 2 min at room temperature and then the cells were washed twice in PBS as described above. The cells were then po ⁇ t-fixed in 4% paraformaldehyde as de ⁇ cribed above. The cell ⁇ were wa ⁇ hed 5 two more times with PBS and pelleted as described above.
  • the detection agent comprising a digoxigenin-labelled hybridization genetic probe at a concentration of 5 ng/ ⁇ l.
  • the labelled genetic probe Prior to resuspending the cells, the labelled genetic probe, in hybridization buffer, wa ⁇ denatured by incubating the mixture at 80°C for 10 min followed by ⁇ nap chilling on ice
  • Hybridization was carried out for 16 hours at 37 ⁇ C Cells were pelleted at 400g in a clinical centrifuge and excess hybridization ⁇ olution wa ⁇ removed. The cells were washed twice in 0.5xSSC twice at room temperature for 5 min and then washed a third time in 0.5xSSC at 37 D C for 15 min.
  • mice anti-digoxigenen antibody (approx. 0.4 g) (1995 Cat No. 1-333-062, Boehringer Mannheim, Germany)
  • Cells retained by the magnet after washing and backwashing were eluted by removing the column from the magnet and passing 10 ml of buffer through the column. This elution was repeated once more. The eluted cells were then centrifuged at 400g for 5 min to pellet the cells.
  • Pelleted cells were resuspended in approximately 50 ⁇ l of PBS buffer and then deposited onto a microscope slide and air dried. Cells on the ⁇ lides were incubated in 20% normal sheep serum for 30 min at 20 ⁇ C to block non-specific binding of the secondary antibody. Slides were briefly rinsed in PBS. A labelled secondary antibody, anti-digoxigenin-rhodamine Fab fragments from sheep (1995 Cat No. 1-207-750, Boehringer Mannheim, Germany) , was applied to the cells at a dilution of 1:10 and incubated for 60 min at 20°C The slides were washed twice in PBS containing 0.1% v/v Nonidet P40 (Boehringer Mannheim, Germany) .
  • Anti-fade mountant (90% v/v glycerol 0.1% v/v p-phenylenediamine) was added to the cell samples and then a coverslip was applied. Cells were then detected with fluorescence microscopy using a 615nm emission wavelength filter and the total number of strongly fluorescent cells determined.
  • Table 3 shows that LNCaP cells were recovered from a mixed population of cells in normal human male blood using the 750bp AR digoxigenin-labelled genetic probe as a detecting agent and a superparamagnetic particle as the solid phase ⁇ upport for enrichment.
  • Patient ⁇ ample ⁇ 981, 982 and 983 ⁇ how that AR positive cells could be detected in peripheral blood from human males with prostatic disease.
  • All patents and publication ⁇ in the specification are indicative of the level of ordinary ⁇ kill in the art to which the invention pertains. All patents and publications herein are incorporated by reference to the same extent a ⁇ if each individual patent and publication wa ⁇ specifically and individually indicated by reference.
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO:3 :

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Abstract

La présente invention a pour objet un procédé et un kit simples et présentant un bon rapport coût-efficacité pour enrichir une ou plusieurs cellules cibles provenant d'une population de cellules mélangées. Selon l'invention, les cellules cibles sont détectées par un agent de détection qui se fixe à un constituant intracellulaire de la cellule cible, par exemple, un acide nucléique, un peptide, une protéine, etc., dans le cytoplasme placé en dessous de la membrane cellulaire externe ou de la paroi cellulaire externe. Les cellules détectées sont ensuite concentrées à partir de la population de cellules mélangées à l'aide d'un système de support en phase solide qui peut comprendre un système d'affinité ou de magnétisme immunitaires. Les cellules enrichies peuvent ensuite être identifées et visualisées par un agent d'identification et un système de génération de signaux. La présente invention concerne également un procédé pour augmenter la sensibilité de l'enrichissement des cellules à partir d'une population mélangée de cellules en amplifiant un constituant intracellulaire sélectionné de la cellule cible avant son enrichissement.
PCT/AU1997/000020 1996-01-17 1997-01-17 Enrichissement en phase solide de cellules intactes a l'aide de constituants intracellulaires WO1997026324A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP9525529A JP2000504213A (ja) 1996-01-17 1997-01-17 細胞内成分を用いる無傷細胞の固相強化
EP97900153A EP0928327A1 (fr) 1996-01-17 1997-01-17 Enrichissement en phase solide de cellules intactes a l'aide de constituants intracellulaires
AU13608/97A AU1360897A (en) 1996-01-17 1997-01-17 Solid phase enrichment of intact cells using intracellular constituents

Applications Claiming Priority (2)

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US1011396P 1996-01-17 1996-01-17
US60/010,113 1996-01-17

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WO1997026324A1 true WO1997026324A1 (fr) 1997-07-24

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000065092A2 (fr) * 1999-04-22 2000-11-02 Science And Technology Corporation Blocage de la fixation non specifique des granulocytes dans la detection de micro-organismes
WO2002097129A2 (fr) * 2001-05-31 2002-12-05 Peter und Traudl Engelhorn-Stiftung zur Förderung der Biotechnologie und Gentechnik Procedes de tri de cellules
US7659063B2 (en) 1998-07-02 2010-02-09 High Throughput Genomics, Inc. High throughput assay system
US8187805B2 (en) 2004-02-09 2012-05-29 Fuso Pharmaceutical Industries, Ltd. Method of detecting nucleic acid and utilization thereof
WO2024026364A3 (fr) * 2022-07-26 2024-03-07 Salk Institute For Biological Studies Procédés et compositions pour une analyse multiplexée unicellulaire et spatiale 3d d'expression de gène dans un tissu végétal

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1688503A4 (fr) * 2003-10-20 2007-10-31 Sysmex Corp Methode de traitement de cellules
DE102007043281A1 (de) * 2007-09-11 2009-05-28 Sebastian Dr. med. Chakrit Bhakdi Vorrichtung, Materialien und Verfahren zur Hochgradientenmagnetseparation biologischen Materials

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993022053A1 (fr) * 1992-05-01 1993-11-11 Trustees Of The University Of Pennsylvania Structures de detection micro-usinees

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993022053A1 (fr) * 1992-05-01 1993-11-11 Trustees Of The University Of Pennsylvania Structures de detection micro-usinees

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ANALYTICAL BIOCHEMISTRY, Volume 228, 1995, GIBELLINI D. et al., pages 252-258. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7659063B2 (en) 1998-07-02 2010-02-09 High Throughput Genomics, Inc. High throughput assay system
WO2000065092A2 (fr) * 1999-04-22 2000-11-02 Science And Technology Corporation Blocage de la fixation non specifique des granulocytes dans la detection de micro-organismes
WO2000065092A3 (fr) * 1999-04-22 2001-07-19 Science & Technology Corp Blocage de la fixation non specifique des granulocytes dans la detection de micro-organismes
WO2002097129A2 (fr) * 2001-05-31 2002-12-05 Peter und Traudl Engelhorn-Stiftung zur Förderung der Biotechnologie und Gentechnik Procedes de tri de cellules
WO2002097129A3 (fr) * 2001-05-31 2003-10-09 Peter Und Traudl Engelhorn Sti Procedes de tri de cellules
US8187805B2 (en) 2004-02-09 2012-05-29 Fuso Pharmaceutical Industries, Ltd. Method of detecting nucleic acid and utilization thereof
WO2024026364A3 (fr) * 2022-07-26 2024-03-07 Salk Institute For Biological Studies Procédés et compositions pour une analyse multiplexée unicellulaire et spatiale 3d d'expression de gène dans un tissu végétal

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CA2243197A1 (fr) 1997-07-24
AU1360897A (en) 1997-08-11
EP0928327A1 (fr) 1999-07-14
JP2000504213A (ja) 2000-04-11

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