AU3016700A - Methods for labeling intracytoplasmic molecules - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): NEW ENGLAND MEDICAL CENTER HOSPITALS, INC.

Invention Title: METHODS FOR LABELING INTRACYTOPLASMIC MOLECULES The following statement is a full description of this invention, including the best method of performing it known to me/us: 1A METHODS FOR LABELING INTRACYTOPLASMIC MOLECULES Background of the Invention This invention relates to methods for labeling intracytoplasmic molecules.

Current methods for prenatal diagnosis at cellular and genetic levels involve invasive procedures such as amniocentesis and chorionic villus sampling (CVS).

Because these methods involve small but significant chances of miscarriage, the current standard of care is to offer invasive prenatal diagnosis only to those women whose risk of a chromosomal or genetic abnormality is greater than or equal to the risk of a diagnostic procedure-related loss.

Thus, prenatal diagnosis of chromosomal and genetic Sabnormalities is limited to a small portion of pregnant 15 women. This is unfortunate, as the majority of abnormal "newborns are born to women who were considered to have low .o risks (Geifman-Holtzman et al., Seminars in Perinatology 18(4):366-375, 1994; D'alton et al., Curr. Probl. Obstet.

Gynecol. Fertil. 17(2):44-78, 1994).

Summary of the Invention We have developed a method for permeabilizing the plasma membrane of a cell so that a molecule within the cell can be labeled. Detection of the labeled 25 intracytoplasmic molecule in a biological sample can be used to determine whether a cell containing the intracytoplamic 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.

Accordingly, in one aspect, the invention provides a method for isolating a fetal erythroid cell in suspension from a blood sample of a pregnant female, said fetal erythroid cell containing an intracytoplasmic target molecule said method comprising the steps of: permeabilizing plasma membrane of said fetal erythroid cell such that E:\Simeona\Keep\Speci\48668-96.doc 27/04/00 1B a directly or indirectly labeled reagent capable of specifically binding to said intracytoplasmic target molecule can traverse said plasma membrane into the cytoplasm of said fetal erythroid cell, and (ii) substantially all of said intracytoplasmic target molecule and DNA of said fetal erythroid cell remain in said fetal erythroid cell; contacting said permeabilized fetal erythroid cell with said directly or indirectly labeled reagent; and isolating said fetal erythroid cell from said blood sample on the basis of detecting said label. In this method, the o O *o E:\Simeona\Keep\Speci\48668-96.doc 27/04/00 2 plasma membrane of the cell is permeabilized so that: 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. By "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 15 cell remain in the cell.

In one embodiment, the method further involves detecting the labeled intracytoplasmic molecule in the cell using standard methods, flow cytometry.

In another embodiment, 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.

The cells labeled using the methods of the 25 invention can be obtained from any biological sample a blood sample (from a pregnant female) or a tissue homogenate), which can obtained from, 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, 3 trophoblasts, fetal granulocytes, fetal leukocytes, tumor cells, cancer cells, and adult blood cells leukocytes and red blood cells). However, any cell containing an intracellular target molecule that can be labeled may be isolated using the methods of the invention.

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 a fluorescent molecule, such as fluorescein or rhodamine, a chemiluminescent tag, or biotin), or can be labeled by a 5secondary reagent a secondary antibody) that 15 contains a label.

The target molecules of the invention include any Sintracytoplasmic molecule a protein or a nucleic acid) that is diagnostic for a target cell type. For example, a fetal cell-specific molecule, or a molecule present in both maternal and fetal cells, but characteristic of a fetal cell fetal hemoglobin), can be used as a target molecule for detecting the presence of a fetal cell a fetal nucleated erythrocyte (NRBC)) in a biological sample a 25 maternal blood sample). In addition, 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., Rubenstein et al., (eds.) Scientific American Medicine, Scientific American, Inc. (New York, 1978), and references therein). Preferred target molecules of the invention are fetal hemoglobin and y-globin. Other molecules that can be used as intracytoplasmic target 4 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 terminal deoxynucleotidyl transferase (TDT) and the c-erbB2 protein). In addition, intracellular molecules that are diagnostic of infection by, e.g., bacteria or viruses cytomegalovirus (CMV) may also be used as target molecules in the invention.

In a preferred embodiment, the plasma membrane of the cell is permeabilized by incubating the cell at 250C to 400C, preferably at 36 0 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 15 to one hour. The cell is then permeabilized using standard methods by incubation in a solution containing alcohol. For example, the cell can be incubated at 1°C to 8OC 4 0 C) in methanol:acetone at a ratio of 0.1:1 to 1:0.1 volume/volume or in 20% to S" 20 90% by volume/volume methanol for at least minutes for 1 to 2 hours).

In another aspect, the invention features a cell that contains an intracytoplasmic target molecule and is permeabilized so that: a reagent capable of 25 detectably labeling the intracytoplasmic target molecule can traverse the plasma membrane into the cytoplasm of the cell, and substantially all of the intracytoplasmic target molecule and the DNA of the cell remain in the cell. The cell may be, a fetal cell, such as a fetal nucleated erythrocyte, a fetal erythrocyte precursor, or a fetal hematopoietic stem cell. Other examples of cells included in the invention, intracytoplasmic target molecules within these cells, and reagents and labels used to label the intracytoplasmic target molecules within the cells, are listed above.

5 The methods of the invention allow single-cell genetic and chromosomal analysis which can be used for, 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. Thus, 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. This is particularly important considering that pregnant women who are characterized as .being at low risk, and thus are not routinely evaluated for these abnormalities, give birth to the majority of abnormal newborns. For example, 80% of infants having Downs Syndrome are born to women who are under the age of 35, and thus are not routinely tested for this abnormality.

Other features and advantages of the invention will be apparent from the following description of thepreferred embodiments thereof, and from the claims.

Detailed DescriDtion 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 6 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 (preferably for 30 minutes to 1 hour) at about 25 0 C-400C (preferably at about 36°C to 38 0 C, more preferably at 37 0 and then is incubated permeabilized in a solution containing alcohol. For example, the cell may be 15 permeabilized by incubation in methanol:acetone at a volume/volume ratio of 0.1:1 to 1:0.1 or in 20% to 90% volume/volume methanol for around 1 to 24 hours at about 3 0 C to 5 0 C (preferably about 4°C).

The cells are then washed and contacted with a reagent if.' 20 an antibody) that labels the intracellular target molecule. 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.

25 Any intracytoplasmic molecule a protein or a nucleic acid) that is diagnostic for a target cell type may be used as the intracytoplasmic target molecule in the methods of the invention. For example, a fetal cellspecific molecule, or a molecule present in both maternal and fetal cells, but characteristic of a fetal cell fetal hemoglobin), can be used as a target molecule for detecting the presence of a fetal cell a fetal nucleated erythrocyte (NRBC)) in a biological sample a maternal blood sample).

Detection of the labeled target molecule allows isolation 7 of the fetal cell and subsequent genetic analysis of the cell, which can be used for prenatal diagnosis.

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. Detection of these molecules in a biological sample blobd, lymph, or a cell suspension derived from a tissue sample) can be used to diagnose cancer or to monitor the progress of anti-cancer treatment. For example, terminal deoxynucleotidyl transferase (TDT) can be used as a target molecule for diagnosis of acute myeloid leukemia, while the c-erbB2 protein can be used as a target molecule for diagnosis of breast cancer.

Infectious diseases caused by, bacteria or viruses may also be diagnosed and/or monitored using the method of the invention. For example, 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 bone marrow) being used for 25 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 fluorescein isothiocyanate (FITC)) or rhodamine, biotin, or magnetic particles. The reagent a primary antibody) that binds to the intracytoplasmic molecule may not itself contain a detectable label, but may be detected by using 8 a labeled secondary reagent a secondary antibody) that binds to the primary reagent (see, Coligan et al. Current Protocols in Immunology, John Wiley Sons, Inc., 1994).

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 Parks et al., Flow Cytometry and Fluorescence-Activated Cell Sorting, In Fundamental Immunology, second edition Paul, Ed.) Raven Press, New York, 1989, 781-802; Coligan et al.

Current Protocols in Immunology, John Wiley .Sons, Inc., 1994). Isolation of cells containing the 15 labeled intracytoplasmic molecules can be carried out 0 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, magnetic-activated cell sorting (MACS). In 20 addition, immunomagnetic beads and antibody-conjugated columns can be used (see, Parks et al., supra; Zheng et al., J. Med. Genet. 30:1051-1056, 1993).

In the case of, a cancer-specific intracytoplasmic target molecule, detection of a cell 25 containing the target molecule in a biological sample may be by itself diagnostic. In the case of, detecting a fetal cell in a maternal blood sample, further analysis genetic, chromosomal, or morphologic analysis) of the isolated cell may be required in order to reach a diagnosis.

Genetic analysis of isolated cells can be carried out using standard methods in the art, including, but not limited to, the polymerase chain reaction (PCR) (see, Geifman-Holtzman et al., supra). This method can be used to detect any type of genetic or chromosomal 9 change that results in a disease, 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 aneuploidy, chromosomal rearrangements, and chromosomal deletions) in an isolated cell, a fetal NRBC, can be detected using fluorescence in situ hybridization (FISH; Geifman- Holtzman et al., supra). 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 trisomy 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 25 al., supra). Similarly, nucleic acid probes may be used in this method to detect chromosomal rearrangements or deletions. Thus, any condition characterized by aneuploidy trisomy 21, trisomy 18, trisomy 13, and Kleinfelter syndrome), chromosomal rearrangement, or chromosomal deletion, may be diagnosed using this method.

The following example is meant to illustrate, but not to limit, the methods of the invention.

Modifications of the conditions and parameters set forth 10 below that are apparent to one skilled in the art are included in the invention.

Example Isolation of Fetal Nucleated Erythrocvtes (NRBCs) for Prenatal Diagnosis 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.

Isolation and preparation of mononuclear cells Mononuclear cells can be isolated from maternal blood using, a Ficoll-Paque (Pharmacia, Piscataway, New Jersey) density gradient, or similar standard methods (see, Coligan et al. (eds.), Current Protocols in Immunology, John Wiley Sons, Inc., 1994; Kumar et al., Pathology 16:53-62, 1984).

2. 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 j 0 6 and 20 x 106 cells/ml, the cells are ready intracellular staining (step 3, below). If the cell count is less than x 106,- the cells may be spun down and resuspended in a smaller volume in order to bring the concentration to the .i 25 correct range. If the count is greater than 20 x 106 cells/ml, the cells may be diluted in order to bring the concentration to the correct range.

Intracellular Staining Protocol 3. Aliquots of 2 x 106 cells (about 100li each) are put into separate tubes (10 tubes, according to the above volumes and cell counts).

4. 0.5 mls of 4% by weight/volume paraformaldehyde (or 3.5% to 8% weight/volume) are added 11 to each tube, and the tubes are then vortexed vigorously.

After incubation for about 30 minutes to 1 hour at 37 0

C,

the cells are washed.

0.5 mls 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 0 C. The cells are then washed.

6. Approximately lg/2 x 106 cells of antigamma Hb FITC is added to each tube, which is then vortexed, and incubated for about 30 minutes at 4 0 C. The cells are then pelleted and washed.

7. The cells are then resuspended in 0.5 mls 2ug/ml Hoechst 33342 in PBS, and stored at 4 0 C until being sorted.

S* Throughout the description and claims of this m specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", means "including but not limited to", and is not intended to exclude other additives, components, integers or steps".

o *fe The entire disclosure in the complete specification of our Australian Patent Application No. 48668/96 is by this cross-reference incorporated into the present specification.

E:\Simeona\Keep\Speci\48668-96.doc 27104/00

Claims (11)

1. A method for isolating a fetal erythroid cell in suspension from a blood sample of a pregnant female, said fetal erythroid cell containing an intracytoplasmic target molecule said method comprising the steps of: permeabilizing plasma membrane of said fetal erythroid cell such that a directly or indirectly labeled reagent capable of specifically binding to said intracytoplasmic target molecule can traverse said plasma membrane into the cytoplasm of said fetal erythroid cell, and (ii) substantially all of said 15 intracytoplasmic target molecule and DNA of said fetal erythroid cell remain in said fetal erythroid cell; contacting said permeabilized fetal erythroid cell with said directly or indirectly labeled reagent; and o" 20 isolating said fetal erythroid cell from said blood sample on the basis of detecting said label.

2. A method according to claim 1, wherein said intracytoplasmic target molecule is fetal haemoglobin.

3. A method according to claim 1, wherein said intracytoplasmic molecule is y-globulin.

4. A method according to any one of claims 1 to 3, wherein said label in said fetal erythroid cell is detected by flow cytometry. A method according to any one of claims 1 to 4, wherein said fetal erythroid cell is isolated by fluorescence-activated cell sorting.

E:\Simeona\Keep\Speci\48668- 9 6 .doc 27/04/00 13

6. A method according to any one of claims 1 to wherein said directly or indirectly labeled reagent comprises an antibody.

7. A method according to any one of claims 1 to 6, wherein said directly or indirectly labeled reagent comprises a fluorescent label or biotin.

8. A method according to any one of claims 1 to 7, wherein permeabilizing step comprises: a. incubating said fetal erythroid cell at 25 0 C to 40 0 C in 2-8% weight/volume parafomaldehyde for minutes to 4 hours; and then 15 b. incubating said fetal erythroid cell in a Sa solution comprising alcohol 15 minutes to 3 hours. 0*

9. A method according to claim 8, wherein said incubating in step is carried out at 37 0 C in 4% weight/volume paraformaldehyde for 30 minutes to 1 hour. A fetal erythroid cell being permeabilized so that: a directly or indirectly labeled reagent can 25 traverse said plasma membrane into the cytoplasm of said cell, said reagent being capable of binding an intracellular target molecule present in said fetal erythroid cell but not present in an adult erythroid cell; and substantially of said intracytoplasmic target molecule and the DNA of said fetal erythroid cell remain in said cell, said fetal erythroid cell containing said reagent capable of binding said intracellular target molecule.

E:\Simeona\Keep\Speci\48668- 9 6 .doc 27/04/00 14

11. A method according to claim 1 substantially as hereinbefore described with reference to any one of the examples. Dated this 27th day of April 2000 NEW ENGLAND MEDICAL CENTER HOSPITALS, INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and E:\Simeona\Keep\Speci\48668-96.doc 27/04/00