Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56966—Animal cells
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/962—Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/968—High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/825—Pretreatment for removal of interfering factors from sample
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
  • Y10T436/2525—Stabilizing or preserving

Definitions

• This invention relates to methods for labeling intracytoplasmic molecules.
• Detection of the labeled intracytoplasmic molecule in a biological sample can be used (1) to determine whether a cell containing the intracytoplasmic molecule is present in the sample, and (2) to facilitate isolation of a cell containing the intracytoplasmic target molecule from the sample for further analysis.
• the invention features a method of labeling a cell containing an intracytoplasmic target molecule.
• the plasma membrane of the cell is permeabilized so that: (1) a reagent capable of detectably labeling the intracytoplasmic target molecule can traverse the plasma membrane into the cytoplasm of the cell, and (2) substantially all of the intracytoplasmic target molecule and the DNA of the cell remain in the cell.
• the cell is then contacted with the reagent to label the intracytoplasmic target molecule.
• substantially all of the intracytoplasmic target molecule and the DNA of the cell is meant that preferably 50% or greater, more preferably 75% or greater, more preferably 85% or greater, more preferably 90% or greater, more preferably 95% or greater, and most preferably 99% or greater, of the intracytoplasmic target molecule and the DNA of the cell remain in the cell.
• the method further involves detecting the labeled intracytoplasmic molecule in the cell using standard methods, e.g., flow cytometry.
• the method further involves isolating the cell on the basis of detection of the labeled intracytoplasmic molecule in the cell. This can be achieved using standard methods in the art, e .g. , fluorescence-activated cell sorting.
• a blood sample from e .g. , a pregnant female
• a tissue homogenate obtained from, e.g. , any mammal including, but not limited to, humans, cows, horses, dogs, cats, sheep, goats, rabbits, rats, guinea pigs, hamsters, and mice.
• Cells that can be labeled and, optionally, detected and/or isolated, using the methods of the invention include, but are not limited to, fetal nucleated erythrocytes (NRBC) , fetal erythrocyte precursor cells, fetal hematopoietic stem cells, trophoblasts, fetal granulocytes, fetal leukocytes, tumor cells, cancer cells, and adult blood cells (e.g., leukocytes and red blood cells) .
• NRBC fetal nucleated erythrocytes
• fetal erythrocyte precursor cells include, but are not limited to, fetal erythrocyte precursor cells, fetal hematopoietic stem cells, trophoblasts, fetal granulocytes, fetal leukocytes, tumor cells, cancer cells, and adult blood cells (e.g., leukocytes and red blood cells) .
• trophoblasts fetal granulocytes
• Reagents that can be used to label the intracytoplasmic molecules of the invention include, but are not limited to, antibodies, non-antibody proteins, and nucleic acids (such as DNA and/or RNA probes) .
• the reagents either contain a label (e.g., a fluorescent molecule, such as fluorescein or rhodamine, a chemiluminescent tag, or biotin) , or can be labeled by a secondary reagent (e.g. , a secondary antibody) that contains a label.
• the target molecules of the invention include any intracytoplasmic molecule (e.g., a protein or a nucleic acid) that is diagnostic for a target cell type.
• a fetal cell-specific molecule or a molecule present in both maternal and fetal cells, but characteristic of a fetal cell (e.g., fetal hemoglobin)
• a target molecule for detecting the presence of a fetal cell (e.g., a fetal nucleated erythrocyte (NRBC)) in a biological sample (e.g., a maternal blood sample) .
• the intracytoplasmic target molecule can be hemoglobin or a hemoglobin variant. Hemoglobin variants that can be the target molecules of the invention include, but are not limited to, fetal hemoglobin, hemoglobin S, hemoglobin Lepore, hemoglobin H, and hemoglobin M (see, e.g.,
• target molecules of the invention are fetal hemoglobin and ⁇ -globin.
• Other molecules that can be used as intracytoplasmic target molecules in the invention include cancer cell-specific molecules, or molecules that are present in both cancer cells and normal cells, but characteristic of cancer cells (e.g., terminal deoxynucleotidyl transferase (TDT) and the c-erbB2 protein) .
• intracellular molecules that are diagnostic of infection by, e.g., bacteria or viruses (e.g., cy omegalovirus (CMV) pp65) may also be used as target molecules in the invention.
• the plasma membrane of the cell is permeabilized by (1) incubating the cell at 25°C to 40 ⁇ C, preferably at 36 ⁇ C to 38 ⁇ C, and most preferably at 37°C; in about 2% to 8% by weight/volume, or preferably 4% by weight/volume, of paraformaldehyde; for 10 minutes to 4 hours, or preferably for 30 minutes to one hour.
• the cell is then permeabilized using standard methods by incubation in a solution containing alcohol.
• the cell can be incubated at 1°C to 8°C (e.g., 4°C) in methanol:acetone at a ratio of 0.1:1 to 1:0.1 volume/volume (e.g., 1:1), or in 20% to 90% by volume/volume methanol (e.g., 70%), for at least 15 minutes (e.g., for 1 to 2 hours).
• 1°C to 8°C e.g., 4°C
• methanol:acetone at a ratio of 0.1:1 to 1:0.1 volume/volume (e.g., 1:1)
• 20% to 90% by volume/volume methanol e.g., 70%
• the invention features a cell that contains an intracytoplasmic target molecule and is permeabilized so that: (1) a reagent capable of detectably labeling the intracytoplasmic target molecule can traverse the plasma membrane into the cytoplasm of the cell, and (2) substantially all of the intracytoplasmic target molecule and the DNA of the cell remain in the cell.
• the cell may be, e.g., a fetal cell, such as a fetal nucleated erythrocyte, a fetal erythrocyte precursor, or a fetal he atopoietic stem cell.
• the methods of the invention allow single-cell genetic and chromosomal analysis which can be used for, e.g., prenatal diagnosis.
• Prenatal diagnosis carried out using the methods of the invention allows screening for chromosomal and genetic abnormalities without incurring the risks, costs, and relative discomfort of invasive procedures, such as amniocentesis and CVS.
• the methods of the invention allow prenatal diagnosis of chromosomal and genetic abnormalities to be offered to all pregnant women, rather than being limited to high risk pregnancies.
• the invention provides methods for detecting a rare cell (a target cell) in a biological sample based on labeling a target molecule within the cell. Once a cell is detected by these methods, it can be isolated using standard methods, and subject to further analysis.
• a central feature of the invention is the method used to permeabilize the plasma membrane of the target cell so that the intracytoplasmic target molecule can be labeled.
• a cell treated by this method has two characteristics that are essential for the efficacy of the invention. First, the plasma membrane is sufficiently permeable so that a reagent capable of detectably labeling the target molecule is able to traverse the plasma membrane into the cytoplasm. Second, the plasma membrane is sufficiently intact so that substantially all of the intracytoplasmic target molecule and the DNA of the target cell remain in the cell.
• the permeabilization method is carried out briefly as follows.
• a cell preparation containing (or suspected of containing) a target cell is incubated in about 2% to 8% by weight/volume (preferably 4% by weight/volume) paraformaldehyde for around 10 minutes to 4 hours
• the cell may be permeabilized by incubation in methanol:acetone at a volume/volume ratio of 0.1:1 to 1:0.1 (e.g., 1:1), or in 20% to 90% volume/volume methanol (e.g., 70%), for around 1 to 24 hours at about 3°C to 5°C (preferably about 4 ⁇ C) .
• a reagent e.g., an antibody
• Detection of the label bound to the intracytoplasmic target molecule can then be used for detecting and/or isolating the target cell from the cell preparation.
• Any intracytoplasmic molecule e.g., a protein or a nucleic acid
• Any intracytoplasmic molecule that is diagnostic for a target cell type may be used as the intracytoplasmic target molecule in the methods of the invention.
• a fetal cell-specific molecule or a molecule present in both maternal and fetal cells, but characteristic of a fetal cell (e.g., fetal hemoglobin), can be used as a target molecule for detecting the presence of a fetal cell (e.g., a fetal nucleated erythrocyte (NRBC) ) in a biological sample (e.g., a maternal blood sample). Detection of the labeled target molecule allows isolation of the fetal cell and subsequent genetic analysis of the cell, which can be used for prenatal diagnosis.
• a fetal cell e.g., a fetal nucleated erythrocyte (NRBC)
• a biological sample e.g., a maternal blood sample.
• TDT terminal deoxynucleotidyl transferase
• c-erbB2 protein can be used as a target molecule for diagnosis of breast cancer.
• Infectious diseases caused by, e.g., bacteria or viruses may also be diagnosed and/or monitored using the method of the invention.
• CMV cytomegalovirus
• detection of cytomegalovirus (CMV) pp65 in white blood cells can be used to diagnose CMV infection.
• Other uses for the methods of the invention include identification of carriers of recessive genetic diseases characterized by abnormal intracytoplasmic molecules, and characterization of tissues (e . g. , bone marrow) being used for transplantation.
• Reagents used to label the intracytoplasmic target molecules include, but are not limited to, antibodies, non-antibody proteins, and nucleic acids.
• the reagents can contain any of a number of labels that are known in the art including, but not limited to, fluorescent labels, such as fluorescein (e.g., fluorescein isothiocyanate (FITC)) or rhodamine, biotin, or magnetic particles.
• the reagent e.g., a primary antibody
• that binds to the intracytoplasmic molecule may not itself contain a detectable label, but may be detected by using a labeled secondary reagent (e .g.
• Detection of a labeled intracytoplasmic target molecule, and isolation of a cell containing it, can be carried out using standard methods in the art. For example, detection of the labeled cell can be carried out using flow cytometry methods (see e.g., Parks et al . , Flow Cytometry and Fluorescence-Activated Cell Sorting, In Fundamental Immunology, second edition (W.E.
• Isolation of cells containing the labeled intracytoplasmic molecules can be carried out using any of a number of standard methods in the art, including, but not limited to, fluorescence-activated cell sorting (FACS) and magnetic systems for isolating cells, e.g., magnetic-activated cell sorting (MACS).
• FACS fluorescence-activated cell sorting
• MCS magnetic-activated cell sorting
• immunomagnetic beads and antibody-conjugated columns can be used (see, e.g., Parks et al . , supra; Zheng et al . , J. tied. Genet . 30:1051-1056, 1993).
• detection of a cell containing the target molecule in a biological sample may be by itself diagnostic.
• further analysis e.g., genetic, chromosomal, or morphologic analysis
• further analysis e.g., genetic, chromosomal, or morphologic analysis
• PCR polymerase chain reaction
• This method can be used to detect any type of genetic or chromosomal change that results in a disease, e .g. , Cystic Fibrosis, Tay-Sachs disease, Gaucher disease, hemoglobinopathies, Duchenne muscular dystrophy, Lesch-Nyhan syndrome, and Sickle cell anemia.
• This method can also be used for determining the sex or rhesus factor status of a fetus (Geifman-Holtzman et al . , supra) .
• Chromosomal abnormalities e.g., aneuploidy, chromosomal rearrangements, and chromosomal deletions
• an isolated cell e.g., a fetal NRBC
• FISH fluorescence in situ hybridization
• This technique relies on the hybridization of chromosome-specific nucleic acid probes to a particular chromosome of interest. For example, when used in conjunction with a fluorescent dye, chromosome-specific probes can be used to determine the numbers of copies of a given chromosome in an interphase nucleus.
• Each chromosome is detected as a colored dot, and the number of dots indicates the number of copies present of the specific chromosome. For example, a cell from an individual with triso y 21 will show three dots after hybridization with a chromosome 21 probe set (Bianchi, Fetal Cells in Maternal Blood: Prospects for Noninvasive Prenatal Diagnosis , In Annals of the New York Academy of Sciences 731:92-102, 1994; Geifman-Holtzman et al . , supra) .
• nucleic acid probes may be used in this method to detect chromosomal rearrangements or deletions.
• any condition characterized by aneuploidy e.g., trisomy 21, trisomy 18, trisomy 13, and Kleinfelter syndrome
• chromosomal rearrangement chromosomal deletion
• the method of the invention can be used to isolate a fetal NRBC from a maternal blood sample for genetic analysis.
• the maternal blood sample is obtained using standard methods in the art, preferably, in the range of 4 weeks to 20 weeks gestation.
• Mononuclear cells can be isolated from maternal blood using, e.g., a Ficoll-Paque (Pharmacia, Piscataway, New Jersey) density gradient, or similar standard methods (see, e.g., Coligan et al . (eds.),
• the mononuclear cells are resuspended in 1 ml 2% fetal calf serum (FCS) in PBS, and counted with a Coulter Counter. If the cell count is between 10 x 10 6 and 20 x 10 6 cells/ml, the cells are ready intracellular staining (step 3, below). If the cell count is less than 10 x 10 6 , the cells may be spun down and resuspended in a smaller volume in order to bring the concentration to the correct range. If the count is greater than 20 x 10 6 cells/ml, the cells may be diluted in order to bring the concentration to the correct range.
• FCS fetal calf serum
• 0.5 is 1:1 by volume/volume methanol:acetone (or 70% methanol) are added to each tube while vortexing vigorously, and the tubes are then incubated for 1 hour to overnight at 4°C. The cells are then washed.
• the cells are then resuspended in 0.5 mis 2 ⁇ g/ml Hoechst 33342 in PBS, and stored at 4°C until being sorted.

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• 1995
  • 1995-02-14 US US08/388,533 patent/US5648220A/en not_active Expired - Fee Related
• 1996
  • 1996-02-09 EP EP99125132A patent/EP0992578A1/en not_active Withdrawn
  • 1996-02-09 AU AU48668/96A patent/AU4866896A/en not_active Abandoned
  • 1996-02-09 WO PCT/US1996/001806 patent/WO1996025486A1/en not_active Ceased
  • 1996-02-09 EP EP96904607A patent/EP0813594B1/en not_active Expired - Lifetime
  • 1996-02-09 CA CA002212796A patent/CA2212796A1/en not_active Abandoned
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  • 1996-02-09 DK DK96904607T patent/DK0813594T3/da active
  • 1996-02-09 ES ES96904607T patent/ES2149453T3/es not_active Expired - Lifetime
  • 1996-02-09 JP JP8525034A patent/JPH11500010A/ja active Pending
  • 1996-02-09 PT PT96904607T patent/PT813594E/pt unknown
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• 2000
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