JP2018050559A - Method of isolating fetal cells from peripheral blood collected from pregnant mother, antibody set, and prenatal genetic testing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel method of isolating fetal cells using antibodies from peripheral blood collected from a pregnant mother.SOLUTION: The method of isolating fetal cells comprises: a step of contacting at least two kinds of antibodies selected from the group consisting of anti-HBG antibody, anti-ALAS 2 antibody, anti-HEMGN antibody, anti-C9orf5 antibody, anti-GYPB antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody, anti-C4orf3 antibody, anti-IGF2 antibody, anti-GYPE antibody, anti-CLIC6 antibody and anti-DDX3Y antibody with a blood cell population and marking the cells with at least two kinds of antibodies; and a step of isolating the cells marked with the at least two kinds of antibodies. An antibody set comprising the at least two kinds of antibodies and an application thereof are also provided.SELECTED DRAWING: None

Description

  The present invention relates to a method for isolating fetal cells from peripheral blood collected from a pregnant mother, an antibody set, and a prenatal genetic testing method.

  Conventionally, as a prenatal genetic test, an amniotic fluid chromosome test has been performed in which fetal cells are isolated from amniotic fluid collected by amniocentesis and the chromosomes in the fetal cells are examined. However, amniocentesis can cause miscarriage.

  On the other hand, a test that detects fetal chromosomal abnormality by analyzing fetal cell-free DNA present in the blood of the pregnant mother or chromosomal DNA of fetal cells present in the blood of the pregnant mother is non-invasive. Known as prenatal genetic testing.

  Among non-invasive prenatal genetic tests, tests that analyze fetal cell-free DNA have already been put to practical use. On the other hand, tests for analyzing chromosomal DNA of fetal cells have not yet been put to practical use. The reason is that fetal cells (for example, fetal nucleated red blood cells) are rare cells that are present only in about 1 maternal blood count. However, if the chromosomal DNA of fetal cells can be analyzed, a noninvasive prenatal genetic test with better test accuracy can be expected.

  By the way, a method for immunologically identifying and isolating cells using an antibody is an isolation method with high cell specificity and is widely used in clinical practice. Against this background, as a method for isolating fetal cells contained in the peripheral blood of a pregnant mother, a method for identifying and isolating fetal cells using an antibody that binds to a protein unique to fetal cells is, for example, patented. It is disclosed in Documents 1-4. Most of the hemoglobin expressed in fetal erythrocytes is hemoglobin F (fetal hemoglobin) consisting of two subunits α and two subunits γ. Expression is dominant and most of hemoglobin expressed in adult erythrocytes is hemoglobin A (adult hemoglobin) composed of two subunits α and two subunits β. Using this phenomenon, Patent Documents 1 to 4 disclose that fetal erythrocytes are identified and isolated using an antibody against subunit γ.

JP 2000-039433 A JP-T-2001-502805 Japanese Patent Laying-Open No. 2014-223082 Special table 2014-533509 gazette

  In order to improve the accuracy of prenatal genetic testing, there is a need for an immunological method that reliably isolates fetal cells from the peripheral blood of a pregnant mother.

The embodiment of the present disclosure has been made under the above circumstances.
An object of the present disclosure is to provide a novel method for isolating fetal cells from peripheral blood collected from a pregnant mother using an antibody.
Moreover, the subject of this indication is providing the novel antibody set used in order to isolate a fetal cell.
Another object of the present disclosure is to provide a prenatal genetic testing method for fetuses that is performed by obtaining chromosomal DNA from isolated fetal cells.

Specific means for solving the above problems include the following aspects.
[1] A method for isolating fetal cells from peripheral blood collected from a pregnant mother, comprising anti-HBG antibody, anti-ALAS2 antibody, anti-HEMGN antibody, anti-C9orf5 antibody, anti-GYPB antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody At least two antibodies selected from the group consisting of an anti-C4orf3 antibody, an anti-IGF2 antibody, an anti-GYPE antibody, an anti-CLIC6 antibody, and an anti-DDX3Y antibody are contacted with the blood cell population, and the cells are marked with at least two antibodies. A method of isolating fetal cells, comprising: isolating cells marked with at least two antibodies from a blood cell population.
[2] The method for isolating fetal cells according to [1], wherein the at least two kinds of antibodies comprise an anti-HBG antibody.
[3] The method further comprises the step of subjecting peripheral blood to density gradient centrifugation to obtain a fraction containing nucleated red blood cells, wherein the fraction containing nucleated red blood cells is a blood cell population [1] or [2] A method for isolating fetal cells as described in 1. above.
[4] The method further comprises a step of contacting an anti-CD45 antibody with a blood cell population to mark the cells with the anti-CD45 antibody, and a step of isolating cells not marked with the anti-CD45 antibody from the blood cell population. And [1] to [3]. The method for isolating fetal cells according to any one of [1] to [3].
[5] Further, the method includes a step of contacting a nuclear staining dye with a blood cell population to stain the cell nucleus, and a step of isolating a nucleated cell having a nucleus stained with the nuclear staining dye from the blood cell population. [1] The method for isolating fetal cells according to any one of [4].
[6] The method for isolating fetal cells according to any one of [1] to [5], wherein the cells are isolated from the blood cell population by flow cytometry.
[7] Anti-HBG antibody, anti-ALAS2 antibody, anti-HEMGN antibody, anti-C9orf5 antibody, anti-GYPB antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody, anti-C4orf3 antibody, anti-IGF2 antibody, anti-GYPE antibody, anti-CLIC6 antibody, and anti-DDX3Y An antibody set for use in isolating fetal cells, comprising at least two types of antibodies selected from the group consisting of antibodies.
[8] The antibody set according to [7], wherein the at least two kinds of antibodies include an anti-HBG antibody.
[9] A part or all of at least two kinds of antibodies are antibodies labeled with at least one kind selected from a fluorescent dye, a fluorescent protein, an enzyme, biotin, and a magnetic bead, [7] or [8] The antibody set described in 1.
[10] Using the fetal cell isolation method according to any one of [1] to [6], fetal cells are isolated from peripheral blood collected from a pregnant mother and chromosomal DNA is isolated from the isolated fetal cells. A prenatal genetic testing method for fetuses.

According to the present disclosure, a novel method for isolating fetal cells using antibodies from peripheral blood collected from a pregnant mother is provided.
Moreover, according to this indication, the novel antibody set used in order to isolate a fetal cell is provided.
In addition, according to the present disclosure, there is provided a prenatal genetic test method for a fetus, which is performed by obtaining chromosomal DNA from an isolated fetal cell.

It is a graph which shows the ratio of the antibody positive cell which occupies for all the cells of a nucleated erythrocyte fraction.

  Embodiments will be described below. These descriptions and examples illustrate the embodiments and do not limit the scope of the embodiments.

  In the present disclosure, the term “process” is not only included in an independent process, but is included in the term if the intended purpose of the process is achieved even when it cannot be clearly distinguished from other processes.

  In the present disclosure, when referring to the amount of each component in the composition, when there are a plurality of substances corresponding to each component in the composition, the plurality of the substances present in the composition unless otherwise specified. It means the total amount of substance.

  In the present disclosure, numerical ranges indicated using “to” indicate ranges including numerical values described before and after “to” as the minimum value and the maximum value, respectively.

<Method of isolating fetal cells>
The isolation method of the present disclosure is a method of isolating fetal cells from peripheral blood collected from a pregnant mother. In the present disclosure, blood includes blood samples such as blood itself and blood diluted with physiological saline; stored blood obtained by adding an additive such as glucose or an anticoagulant to the blood; fractions thereof; It is. In the present disclosure, the blood cell population refers to a population of cells constituting blood.

  The isolation method of the present disclosure isolates fetal cells, preferably fetal nucleated cells, from peripheral blood collected from a pregnant mother. Since nucleated cells have chromosomes, fetal chromosomes and fetal genes can be obtained by isolating fetal nucleated cells. Examples of fetal nucleated cells contained in the peripheral blood of the pregnant mother include nucleated red blood cells.

  Fetal nucleated red blood cells migrate through the placenta into the maternal blood. Fetal nucleated red blood cells are said to be present in maternal blood from about 6 weeks after pregnancy. Therefore, the blood used for the isolation method of the present disclosure is a blood sample prepared from peripheral blood collected from a pregnant mother about 6 weeks after pregnancy or from peripheral blood collected from a pregnant mother after about 6 weeks after pregnancy. Preferably there is.

When the mother is pregnant, fetal red blood cells can be nucleated. Fetal nucleated red blood cells are said to be present in a ratio of about 1 in 10 ⁶ cells in the maternal blood of the pregnancy, and the probability of existence is very low in the peripheral blood of the pregnant mother. In addition to fetal nucleated red blood cells, maternal white blood cells (eosinophils, neutrophils, basophils, monocytes, lymphocytes); maternal mature red blood cells without a nucleus; Blood cells such as maternal nucleated red blood cells;

  In order to efficiently isolate fetal cells, preferably fetal nucleated red blood cells, from the blood cell population containing the various cells described above, the isolation method of the present disclosure includes anti-HBG antibody, anti-ALAS2 antibody, anti-HEMGN antibody. Using at least two antibodies selected from the group consisting of anti-C9orf5 antibody, anti-GYPB antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody, anti-C4orf3 antibody, anti-IGF2 antibody, anti-GYPE antibody, anti-CLIC6 antibody, and anti-DDX3Y antibody Sort the cells. Details of the human proteins to which the 12 types of antibodies bind are shown in Table 1.

  There are two human hemoglobin subunit gamma (HBG) genes, which express HBG1 (hemoglobin subunit gamma 1) and HBG2 (hemoglobin subunit gamma 2), respectively. In the present disclosure, HBG is a term including HBG1 and HBG2, and the anti-HBG antibody means an antibody that binds to at least one of HBG1 and HBG2.

In the present disclosure, the antibody includes not only an immunoglobulin molecule itself (intact immunoglobulin) but also a fragment thereof (for example, Fab, F (ab ′) ₂ , Fab ′) having a binding ability to an antigen. It is a term to do.

  According to the analysis of the inventor, HBG, ALAS2, HEMGN, C9orf5, GYPB, SLC1A5, EIF1AY, C4orf3, IGF2, GYPE, CLIC6, and DDX3Y are highly expressed in human umbilical cord blood compared to human adult bone marrow blood. confirmed. By using at least two types of antibodies that bind to these human proteins, fetal cells can be efficiently isolated from the blood cell population.

  12 antibodies (anti-HBG antibody, anti-ALAS2 antibody, anti-HEMGN antibody, anti-C9orf5 antibody, anti-GYPB antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody, anti-C4orf3 antibody, anti-IGF2 antibody, anti-GYPE antibody, anti-CLIC6 antibody, and Combinations of at least two types selected from anti-DDX3Y antibody (hereinafter the same)) include a combination of an anti-HBG antibody and at least one antibody selected from the other 11 types of antibodies. By using an anti-HBG antibody, the probability that cells isolated from a blood cell population are fetal erythrocytes is increased. A preferred combination is a combination of an anti-HBG antibody and an anti-ALAS2 antibody, anti-C9orf5 antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody, anti-IGF2 antibody, anti-CLIC6 antibody, and anti-DDX3Y antibody. More preferable combinations include a combination of an anti-HBG antibody and at least one antibody selected from an anti-ALAS2 antibody, an anti-C9orf5 antibody, an anti-SLC1A5 antibody, an anti-EIF1AY antibody, and an anti-CLIC6 antibody, and A preferable combination includes a combination of an anti-HBG antibody and at least one antibody selected from an anti-ALAS2 antibody, an anti-C9orf5 antibody, an anti-EIF1AY antibody, and an anti-SLC1A5 antibody.

  Each of the 12 types of antibodies is a method of purifying an antigen from the blood of an immunized non-human animal; a hybridoma that is a fusion cell of B cells and myeloma cells of an immunized non-human animal is prepared, and a monoclonal antibody is prepared from the hybridoma. It can be produced by a method of obtaining an antibody; The 12 types of antibodies may be commercially available products.

  Each of the 12 types of antibodies may be a labeled antibody labeled with at least one selected from fluorescent dyes, fluorescent proteins, enzymes, biotin, and magnetic beads.

  Examples of fluorescent dyes for labeling antibodies include FITC (fluorescein isothiocyanate), Texas Red, Cy3, Cy5, Alexa Fluor 488, Alexa Fluor 647, and the like. Examples of fluorescent proteins for labeling antibodies include phycocyanin, allophycocyanin, phycoerythrin, phycoerythrocyanin, and the like. Examples of the enzyme labeling antibody include peroxidase and alkaline phosphatase.

By selecting and combining at least two antibodies from the twelve antibodies, an antibody set for use in isolating fetal cells from a blood cell population is provided. One embodiment of this antibody set includes at least an anti-HBG antibody, and includes an anti-HBG antibody and at least one antibody selected from the other 11 antibodies. A preferred embodiment of this antibody set is an anti-HBG antibody and at least one selected from an anti-ALAS2 antibody, an anti-C9orf5 antibody, an anti-SLC1A5 antibody, an anti-EIF1AY antibody, an anti-IGF2 antibody, an anti-CLIC6 antibody, and an anti-DDX3Y antibody. Antibody. A more preferred embodiment of this antibody set comprises an anti-HBG antibody and at least one antibody selected from anti-ALAS2 antibody, anti-C9orf5 antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody, and anti-CLIC6 antibody. Further preferred embodiment examples of the antibody set include an anti-HBG antibody and at least one antibody selected from an anti-ALAS2 antibody, an anti-C9orf5 antibody, an anti-EIF1AY antibody, and an anti-SLC1A5 antibody.
A part or all of at least two kinds of antibodies included in the present antibody set may be labeled antibodies labeled with at least one selected from fluorescent dyes, fluorescent proteins, enzymes, biotin, and magnetic beads.

  Hereinafter, the isolation method of the present disclosure will be described in detail by explaining the steps.

[Fetal cell marking process, fetal cell isolation process]
The isolation method of the present disclosure includes:
It consists of anti-HBG antibody, anti-ALAS2 antibody, anti-HEMGN antibody, anti-C9orf5 antibody, anti-GYPB antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody, anti-C4orf3 antibody, anti-IGF2 antibody, anti-GYPE antibody, anti-CLIC6 antibody, and anti-DDX3Y antibody Contacting a blood cell population with at least two antibodies selected from the group and marking the cells with at least two antibodies (referred to as “fetal cell marking step”);
Isolating cells marked with at least two types of antibodies from the blood cell population (referred to as “fetal cell isolation step”).

  Since HBG, ALAS2, HEMGN, C9orf5, GYPB, SLC1A5, EIF1AY, C4orf3, IGF2, GYPE, CLIC6, and DDX3Y are highly expressed in human umbilical cord blood compared to human adult bone marrow blood, at least 12 kinds of antibodies The two types of positive cells have a high probability of being fetal cells. Therefore, fetal cells are isolated with high probability from the blood cell population derived from peripheral blood collected from the pregnant mother by the fetal cell marking step and the fetal cell isolation step.

  The fetal cell marking step is a step in which at least two types of antibodies selected from 12 types of antibodies are brought into contact with the blood cell population, and the cells are marked with at least two types of antibodies. Not limited. The contact between the antibody and the blood cell population may be performed by, for example, adding the antibody to a cell suspension of the blood cell population or adding the blood cell population to a phosphate buffer or physiological saline containing the antibody at 4 ° C. Performed by incubating at room temperature for 30 minutes to 120 minutes. At least two antibodies selected from 12 types of antibodies may be contacted simultaneously or sequentially with the blood cell population. When some or all of at least two kinds of antibodies selected from 12 kinds of antibodies are not labeled with a fluorescent dye, fluorescent protein, enzyme, biotin, magnetic beads, etc., labeled secondary antibodies for these antibodies are used. The blood cell population may be further contacted. Examples of secondary antibody labels include fluorescent dyes, fluorescent proteins, enzymes, biotin, and magnetic beads.

  The blood cell population to which the antibody is contacted in the fetal cell marking step is preferably a blood cell population that has been subjected to cell membrane permeabilization for the purpose of allowing the antibody to pass through the cell membrane and reach the cytoplasm. The cell membrane permeabilization treatment is performed, for example, by mixing a phosphate buffer solution or physiological saline containing a surfactant such as Triton X100 with a blood cell population and incubating at 4 ° C. to room temperature for 1 to 30 minutes. . When a blood cell population is subjected to a cell membrane permeabilization treatment, the blood cell population may be a blood cell population that has been subjected to an immobilization treatment prior to the cell membrane permeation treatment for the purpose of suppressing protein loss from within the cells. preferable. The cell immobilization treatment is performed, for example, by mixing a phosphate buffer solution or physiological saline containing glutaraldehyde and a blood cell population and incubating at 4 ° C. to room temperature for 1 to 30 minutes.

The fetal cell isolation step is a step of isolating cells marked with at least two kinds of antibodies, and the isolation method is not limited.
A label in which an antibody (at least two kinds of antibodies selected from 12 kinds of antibodies or a secondary antibody to these antibodies) used in the fetal cell marking step is labeled with a fluorescent dye, fluorescent protein, enzyme, biotin, magnetic beads, etc. In the case of an antibody, the fetal cell isolation step is performed by an optical method, a chemical method, an electrical method, or a magnetic method according to the label. The fetal cell isolation step is preferably performed by flow cytometry from the viewpoint of efficiency. Therefore, antibodies used in the fetal cell marking step (at least two antibodies selected from twelve antibodies, or two antibodies against these antibodies). The secondary antibody) is preferably an antibody labeled with a fluorescent dye, fluorescent protein, or biotin.

[Nucleated red blood cell concentration step]
The blood cell population to which the antibody is contacted in the fetal cell marking step is preferably a cell population concentrated from peripheral blood collected from a pregnant mother to a population rich in nucleated red blood cells. Therefore, the isolation method of the present disclosure further includes a step of subjecting peripheral blood to density gradient centrifugation to obtain a fraction containing nucleated red blood cells (referred to as a “nucleated red blood cell concentration step”). It is preferable that the fraction containing erythrocytes is a blood cell population in the fetal cell marking step.

  Nucleated red blood cells can be separated from plasma components and other blood cells present in the blood by density gradient centrifugation. A known method may be applied to density gradient centrifugation for separating nucleated red blood cells. For example, nucleated red blood cells can be fractionated and concentrated by layering and centrifuging blood diluted with physiological saline on a discontinuous density gradient in which two types of media having different densities are layered on a centrifuge tube.

  WO 2012/023298 describes the density of maternal blood cells including fetal nucleated red blood cells. According to the description, the expected density of fetal nucleated red blood cells is about 1.065 g / mL to 1.095 g / mL, and the density of mother's blood cells is about 1.070 g / mL to 1.120 g / mL of red blood cells. About 1.090 g / mL to 1.110 g / mL for spheres, about 1.075 g / mL to 1.100 g / mL for neutrophils, about 1.070 g / mL to 1.080 g / mL for basophils, 1.060 g / mL for lymphocytes About 1.080 g / mL and monocytes are about 1.060 g / mL to 1.070 g / mL.

  In order to separate fetal nucleated red blood cells having a density of about 1.065 g / mL to 1.095 g / mL from other blood cells, the density of the medium to be stacked is set. Since the density of the center of fetal nucleated red blood cells is about 1.080 g / mL, a fraction having fetal nucleated red blood cells at its interface can be obtained by layering two different density media adjacent to each other. It becomes possible to collect. Preferably, the density of the lower layer medium is 1.08 g / mL to 1.10 g / mL (more preferably 1.080 g / mL to 1.095 g / mL), and the density of the upper layer medium is 1.06 g / mL to 1.08 g / mL (more Preferably, it is 1.065 g / mL to 1.080 g / mL. In the isolation method of the present disclosure, the lower layer medium and the upper layer medium may be of the same type or different types, and the same type of medium is preferably used.

  As the medium, Percoll (registered trademark), which is a dispersion of 15 to 30 nm in diameter of silicate colloid coated with polyvinylpyrrolidone, Ficoll-, which is a neutral hydrophilic polymer rich in side chains made from sucrose, is used. Examples include Paque (registered trademark), Histopaque (registered trademark) containing polysucrose and sodium ditrizoate. In the isolation method of the present disclosure, it is preferable to use Percoll and / or Histopaque. Percoll has a commercial product with a density of 1.130 g / mL, and it is possible to prepare a density gradient by diluting with water. Histopaque can prepare density gradients using commercially available media with a density of 1.077 g / mL and media with a density of 1.119 g / mL and water.

  The two-layer discontinuous density gradient is formed in the centrifuge tube as follows, for example. First, the lower layer medium in a temperature state not lower than the freezing point and not higher than 14 ° C. (preferably not higher than 8 ° C.) is accommodated in the bottom part of the centrifuge tube, or after the lower layer medium is accommodated in the bottom part of the centrifuge tube, It is preferably stored and cooled at a temperature of 8 ° C. or lower. Next, the upper layer medium is overlaid on the lower layer medium.

[Leukocyte marking process, leukocyte exclusion process]
From the viewpoint of improving the isolation efficiency of red blood cells, the isolation method of the present disclosure further includes
A step of contacting an anti-CD45 antibody with a blood cell population and marking the cell with the anti-CD45 antibody (referred to as a “leukocyte marking step”);
Preferably, a step of isolating cells that are not marked with an anti-CD45 antibody from the blood cell population (referred to as a “leukocyte exclusion step”).

  Since CD45, which is a leukocyte common antigen, is expressed on leukocytes in general, ie, eosinophils, neutrophils, basophils, monocytes, and lymphocytes, CD45 negative cells contained in the blood cell population are: There is a high probability that it is a red blood cell. Therefore, red blood cells are isolated with high probability from the blood cell population derived from peripheral blood collected from the pregnant mother by the leukocyte marking step and the leukocyte exclusion step.

  The leukocyte marking step is a step in which an anti-CD45 antibody is brought into contact with the blood cell population and the cells are marked with the anti-CD45 antibody, and the method of bringing the anti-CD45 antibody into contact with the blood cell population is not limited. The contact between the anti-CD45 antibody and the blood cell population is, for example, adding the anti-CD45 antibody to a cell suspension of the blood cell population, or the blood cell population in a phosphate buffer or physiological saline containing the anti-CD45 antibody. And is incubated at 4 ° C. to room temperature for 30 minutes to 120 minutes. The leukocyte marking step may be performed simultaneously with the fetal cell marking step, or may be performed before or after the fetal cell marking step. For example, the fetal cell marking step and the leukocyte marking step may be performed simultaneously by adding at least two antibodies selected from 12 antibodies and an anti-CD45 antibody to a cell suspension of a blood cell population, or 12 This is carried out by adding a blood cell population to a phosphate buffer or physiological saline containing at least two antibodies selected from these antibodies and an anti-CD45 antibody.

  The anti-CD45 antibody is labeled with a fluorescent dye, fluorescent protein, enzyme, biotin, magnetic beads or the like from the viewpoint of performing the leukocyte exclusion step by an optical method, a chemical method, an electrical method, or a magnetic method. From the viewpoint of performing the leukocyte exclusion step by flow cytometry, an antibody labeled with a fluorescent dye, a fluorescent protein, or biotin is preferable.

  The leukocyte exclusion step is performed by an optical method, a chemical method, an electrical method, or a magnetic method according to the labeling of the anti-CD45 antibody. The leukocyte exclusion step may be performed before the fetal cell isolation step or after the fetal cell isolation step.

[Cell nucleus staining process, nucleated cell isolation process]
From the viewpoint of improving the isolation efficiency of nucleated cells, the isolation method of the present disclosure further includes:
A step of staining a cell nucleus by contacting a nuclear staining dye with a blood cell population (referred to as a “cell nucleus staining step”);
It is preferable to have a step of isolating nucleated cells whose nuclei are stained with a nuclear staining dye (referred to as “nucleated cell isolation step”) from a blood cell population.

  Nucleated cells are isolated with high probability from a blood cell population derived from peripheral blood collected from a pregnant mother by the cell nucleus staining step and the nucleated cell isolation step.

  The cell nucleus staining step is a step of staining a cell nucleus by bringing a nucleus staining dye into contact with a blood cell population, and a method for contacting the nucleus staining dye with a blood cell population is not limited. The contact between the nuclear staining dye and the blood cell population can be achieved, for example, by adding the nuclear staining dye to the cell suspension of the blood cell population, or in the phosphate buffer or physiological saline containing the nuclear staining dye. And is incubated at 4 ° C. to room temperature for 30 minutes to 120 minutes. The cell nucleus staining step may be performed before the fetal cell marking step, may be performed after the fetal cell marking step and before the fetal cell isolation step, or may be performed after the fetal cell isolation step. When the isolation method of the present disclosure includes a leukocyte marking step and a leukocyte exclusion step, the cell nucleus staining step may be performed before the leukocyte marking step, or after the leukocyte marking step and before the leukocyte exclusion step. It may be performed after the leukocyte exclusion step.

  The nuclear staining dye may be any dye that stains chromatin, nucleoprotein, DNA, and the like. Specifically, DAPI (4 ′, 6-diamidino-2-phenylindole), propidium iodide, 7-AAD (7 -Amino-Actinomycin D), Hoechst 33342, DRAQ5, methylene blue, hematoxylin, thionine and the like.

When the nuclear staining dye is a non-fluorescent dye, the nucleated cell isolation step is performed, for example, by observing a cell population with an optical microscope and isolating cells with stained nuclei.
When the nuclear staining dye is a fluorescent dye, the nucleated cell isolation step is performed, for example, by flow cytometry.

  The nucleated cell isolation step may be performed before the fetal cell isolation step or after the fetal cell isolation step. When the isolation method of the present disclosure includes a leukocyte marking step and a leukocyte exclusion step, the nucleated cell isolation step may be performed before the leukocyte exclusion step or after the leukocyte exclusion step.

  Examples of the isolation method of the present disclosure include a fetal cell marking step, a leukocyte marking step, a cell nucleus staining step, a fetal cell isolation step, a leukocyte exclusion step, and a nucleated cell isolation step, Examples include performing a leukocyte marking step (the order is not limited), followed by a cell nucleus staining step, followed by three isolation steps sequentially by flow cytometry (the order is not limited). According to this embodiment, fetal nucleated red blood cells can be isolated efficiently.

<Prenatal genetic testing methods for fetuses>
The test method of the present disclosure is a method for prenatal genetic testing of a fetus that is performed by isolating fetal cells from peripheral blood collected from a pregnant mother and obtaining chromosomal DNA from the isolated fetal cells. The test method of the present disclosure is a test for clarifying genetic information inherently possessed by a human individual. For example, detection of the presence or absence of chromosome aneuploidy, detection of gene polymorphism, and detection of gene mutation It is an inspection that performs at least one of them. The test method of the present disclosure is applied to detection of the presence or absence of chromosome aneuploidy such as, for example, trisomy of chromosomes 13, 18, and 21; excess of sex chromosomes;

  The testing method of the present disclosure may be performed by applying a known genetic testing method and a known genetic analysis technique. The test method of the present disclosure preferably includes a step of performing whole genome amplification (WGA) from fetal cells for the purpose of facilitating analysis of chromosomal DNA. Furthermore, the inspection method of the present disclosure includes, for example, an amplification step of amplifying a target region on a chromosome by a polymerase chain reaction (PCR) using an amplification product of whole genome amplification as a template, and the amplified target region. And a sequence analysis step for determining the base sequence and amount. Hereinafter, an exemplary embodiment of the inspection method of the present disclosure will be described in detail by describing the above-described steps.

[Whole genome amplification]
The method of whole genome amplification is not particularly limited, and may be performed by a known method. Whole genome amplification has, for example, a step of lysing cells using a surfactant and a proteolytic enzyme, and a step of amplifying DNA by DNA polymerase using genomic DNA eluted from the cells as a template.

  Whole genome amplification may be performed by applying commercially available reagents. Examples of the PCR-based reagent include PicoPLEX WGA Kit (New England Biolabs), GenomePlex Single Cell Whole Genome Amplification Kit (Sigma-Aldrich), and MALBAC method (Multiple Annealing and Looping disclosed in International Publication No. 2012/166425). -Based Amplification Cycles). Examples of the reagent based on the strand displacement type DNA synthesis reaction include GenomiPhi DNA Amplification Kit (GE Healthcare, GenomiPhi is a registered trademark) and REPLI-g Single Cell Kit (QIAGEN, REPLI-g is a registered trademark). .

  It is preferable to confirm the presence or absence of amplification by performing agarose gel electrophoresis after the completion of whole genome amplification. The amplification product of whole genome amplification is preferably purified. Purification is performed using, for example, a QIAquick PCR Purification Kit (QIAGEN, QIAquick is a registered trademark). The amount of the amplification product can be confirmed by measuring the concentration using, for example, NanoDrop (registered trademark, Thermo Fisher Scientific), BioAnalyzer (Agilent), or Quantus Fluorometer (Promega).

[Amplification process of target region by PCR]
This amplification step is a step of amplifying the target region by PCR using the amplification product obtained by whole genome amplification as a template. The target region (region to be amplified) is a region on the chromosome that is selected according to the purpose of genetic testing. The base sequence of the primer pair used for PCR is designed according to the base sequence of the target region and the analysis principle of the sequencer. The amplification step may be multiplex PCR (multiplex PCR) for performing multiplex amplification of a plurality of target regions using a plurality of primer pairs for the purpose of analyzing a plurality of regions on the chromosome. In the amplification step, PCR may be performed once or two or more times according to the analysis principle of the sequencer.

  PCR reagents include, for example, Multiplex PCR Assay Kit (Takara Bio), Multiplex PCR Assay Kit ver2 (Takara Bio), KAPA Library Amplification Kit (Nippon Genetics), Platinum Multiplex PCR Master Mix Kit (Life Technologies, Platinum is a registered trademark). When performing multiplex PCR, it is preferable to examine the reaction conditions because the optimum temperature for annealing may differ for each primer pair. The number of cycles of PCR may be set based on the result of a pre-examination by real-time PCR. PCR may change reaction temperature and / or reaction time in the middle.

  The amplification product obtained in the amplification step is preferably purified, and purification is performed using, for example, QIAquick PCR Purification Kit (QIAGEN) or AMPure XP Kit (BECKMAN COULTER). The amount of amplification product is confirmed by measuring the concentration using, for example, NanoDrop (Thermo Fisher Scientific), BioAnalyzer (Agilent), Quantus Fluorometer (Promega), KAPA Library Quantification Kits (Nippon Genetics). obtain.

[Sequence analysis process]
The sequence analysis step is a step of determining the base sequence and amount of the amplified target region. The sequence analysis step is performed by various sequencers. All or part of the amplification product obtained in the amplification step by PCR is a substance that can be applied to the sequencer in the sequence analysis step, and the amplification product obtained in the amplification step by PCR is a sequence library.

  The sequence analysis step is preferably performed by a next-generation sequencer in terms of the accuracy and speed of analysis, the number of samples that can be processed at one time, and the like. The next generation sequencer (NGS) means a sequencer classified in comparison with a capillary sequencer (called a first generation sequencer) using the Sanger method. Next generation sequencers include second generation, third generation, fourth generation, and sequencers that will be developed in the future. The most popular next-generation sequencer at present is a sequencer based on the principle of determining a base sequence by capturing fluorescence or light emission linked to complementary strand synthesis by DNA polymerase or complementary strand binding by DNA ligase. Specific examples include MiSeq (Illumina), HiSeq2000 (Illumina, HiSeq is a registered trademark), Roche454 (Roche).

  Burrows-Wheeler Aligner (BWA) is mentioned as a means for aligning the sequence data obtained by the sequencer, and it is preferable to map the sequence data to a known human genome sequence by BWA. Examples of means for analyzing genes include SAMtools and BEDtools, and it is preferable to analyze gene polymorphisms, gene mutations, and / or chromosome numbers by these analysis means.

  Presence / absence of chromosome aneuploidy, gene polymorphism, and / or gene mutation are detected from the base sequence and amount of the amplified target region analyzed in the sequence analysis step. An example of a method for detecting chromosome aneuploidy will be described below.

  For chromosomes obtained from fetal nucleated red blood cells, the amount of amplification product is analyzed with a sequencer. As a standard (or reference), the amount of amplification product is analyzed with a sequencer for chromosomes obtained from maternal cells. If the fetus has no chromosomal aneuploidy, it is expected that the amount of amplification product from the mother and the amount of amplification product from the fetus will be approximately 1: 1. When the chromosome in which the amplification region is located is trisomy in the fetus, the amount of the amplification product derived from the mother and the amount of the amplification product derived from the fetus are approximately 1.0: 1.5 (or 2: 3). Expected to be

  Hereinafter, embodiments of the present invention will be described in detail by way of examples, but the embodiments of the present invention are not limited to these examples.

  In the following, regarding the substance concentration, “M” represents a molar concentration, and 1M = 1 mol / L. Regarding the substance concentration, “%” is “v / v%” unless otherwise specified.

  In the following, “EDTA” means ethylenediaminetetraacetic acid, “BSA” means bovine serum albumin, and “PBS” means phosphate buffer (phosphate). buffered saline), and “PE” means phycoerythrin. PBS has a pH of 7.4 unless otherwise noted.

<Example 1>
Transcriptome analysis was performed for the purpose of searching for genes with high expression frequency in fetal cells.

[Blood sample]
Human umbilical cord blood (product number C-CB101) was purchased from VERITAS.

  As an anticoagulant, 7.0 mL of peripheral blood was collected from two pregnant volunteers in a 7 mL blood collection tube containing 10.5 mg of sodium salt of EDTA. Blood collection from pregnant volunteers was performed after obtaining informed consent.

  Each blood was diluted 2-fold with physiological saline and subjected to the following steps.

[Total RNA extraction]
Total RNA was collected from each blood using NucleoSpin RNA Blood kit (MACHEREY-NAGEL). The recovered amounts of total RNA were 2340 ng from cord blood (1.2 mL), 5985 ng from maternal peripheral blood A (7.0 mL), and 5355 ng from maternal peripheral blood B (7.0 mL). The RIN values (RNA Integrity Numbers) indicating the degree of degradation of RNA are 9.5 (umbilical cord blood), 7.9 (maternal peripheral blood A), and 7.7 (maternal peripheral blood B), respectively. Confirmed that there is no.

[RNA sequence analysis]
The sequencing library was prepared using TruSeq RNA Sample Preparation Kit v2 (Illumina) using 1 μg of total RNA extracted from each blood as a starting material. The length of the polynucleotide in the prepared library was 382 bp to 385 bp (including the adapter sequence). Sequence analysis was performed using the next-generation sequencer HiSeq2500 (Illumina), and sequence information was obtained with a read length of 101 bp, Paired-end, and Multiplex.

[Bioinformatics analysis]
The sequence information obtained by the next generation sequencer was analyzed according to the following flow.

Flow: (1) Read data quality check, (2) Read data cleaning, (3) Removal of poly-A / T tail, (4) Mapping to reference sequence, (5) Junction structure prediction, (6) Assembly And expression level comparison

Details of each flow are as follows.
(1) Read data quality check, (2) Read data cleaning
The cutadapt program performed the following procedure.
-Remove the adapter sequence that has been contaminated at the 3 'end.
-Removal of bases with a quality value at the 3 'end of less than 5.
・ Removal of low quality leads.
-Removal of reads with more than 10% of all bases with a quality value of less than 5.
-Removal of reads where N is greater than 10% of total bases.
・ Removal of leads in which one type of base occupies more than “total number of bases −3”.
-Removal of a 6 bp adapter sequence that frequently appears at the 3 'end.
-Removal of reads whose base length is less than 20 bp.
-Extract only Paired-end leads.

(3) Removal of poly-A / T tail
The PRINSEQ program was followed by the following procedure.
-Remove A or T that is 10 bases or more continuous at the 3 'end or 5' end.
-Removal of reads whose base length is less than 20 bp.
-Extract only Paired-end leads.

(4) Mapping to reference sequence, (5) Junction structure prediction
The TopHat program was executed with the command "tophat -o $ SAMPLE -G ref_genes.gtf -p 2 --solexa1.3-quals bowtie-indexed-genome fastq1 fastq2".

(6) Assembly and expression level comparison
The Cufflinks program was executed in the following procedure.
Cufflinks: Assemble transcripts for each sample. Mitochondrial transcripts and ribosomal RNA are removed. Command `` cufflinks -o $ SAMPLE -M mask.gtf -g ref_genes.gtf -b genome.fasta -u -p 2 $ SAMPLE.bam ''
Cuffmerge: Merge transcripts from all samples to create a consensus transcript. Command `` cuffmerge -o merge_all -g ref_genes.gtf -s genome.fasta -p 8 gtflist ''
Cuffcompare: Compares the transcript for each sample with the consensus transcript. Command `` cuffcompare -o $ SAMPLE -r merged.gtf $ SAMPLE.transcripts.gtf ''
Cuffquant: Calculate the expression level of the consensus transcript for each sample. Command `` cuffquant -o $ SAMPLE -b genome.fasta -u merged.gtf $ SAMPLE.bam ''
Cuffdiff: Compare expression levels and structure between samples. Command `` cuffdiff merged.gtf $ SAMPLE1.bam $ SAMPLE2.bam -M mask.gtf -b genome.fasta -u -p 2 -o $ GROUP1_ $ GROUP2 -L $ GROUP1, $ GROUP2 ''
-CummeRbund: graphs the statistical results of cuffdiff.

[Comparison of expression levels]
As a result of the above analysis, 515,217 types of umbilical cord blood, 583,632 types of maternal peripheral blood A, 579,043 types of transcripts of maternal peripheral blood B were assembled, and 631,932 types of consensus transcripts. As an index for comparing the expression level between samples, FPKM (fragments per kilobase of exon per million fragments mapped) was used. Of the consensus transcripts, 15,157 expression levels were compared between umbilical cord blood and maternal peripheral blood A, 14,436 expression levels were compared between umbilical cord blood and maternal peripheral blood B, and maternal peripheral blood A and maternal blood. The expression levels of 20,508 species were compared among peripheral blood B. Extraction of a gene specifically expressed in cord blood was performed by the following procedure.

First, the following requirement (a) and requirement (b) are set, and a transcript satisfying the requirement (a) and a transcript satisfying the requirement (b) are separated between the cord blood and the maternal peripheral blood A, and the cord blood. Extracted from between maternal peripheral blood B.
Requirement (a): FPKM value is 5 or more for cord blood and 0.1 or less for maternal peripheral blood (expression is observed only in cord blood).
Requirement (b): The FPKM value is 200 or more for umbilical cord blood and 50 or less for maternal peripheral blood (expressed in maternal peripheral blood but highly expressed in umbilical cord blood).

  Next, among the extracted genes, transcripts common to two groups were extracted. As a result, 68 types of genes satisfying the requirement (a) and 123 types of genes satisfying the requirement (b) were extracted, a total of 191 types. Among 191 genes, 16 were selected which have a particularly high FPKM value in cord blood and are commercially available for antibodies against the protein encoded by the gene. Table 2 shows the names of proteins encoded by the genes and the FPKM values for these 16 species.

<Example 2>
Using antibodies against the 16 proteins selected in Example 1, fetal cells were isolated from human umbilical cord blood and human adult bone marrow blood. The antibodies shown in Table 3 were prepared as antibodies against 16 proteins.

[Blood sample]
Human umbilical cord blood (product number C-CB101) and human adult bone marrow blood (product number ABM001) were purchased from VERITAS. Each blood was diluted 2-fold with physiological saline and subjected to the following steps.

[Concentration of nucleated red blood cells by density gradient centrifugation]
Using a Percoll solution (registered trademark, Sigma-Aldrich) and water, a liquid having a density of 1.070 g / mL and a liquid having a density of 1.095 g / mL were prepared. A centrifuge tube was charged with 2 mL of a liquid having a density of 1.095 g / mL, and cooled at 4 ° C. for 30 minutes. Next, 2 mL of a liquid having a density of 1.070 g / mL was slowly layered on the liquid having a density of 1.095 g / mL so as not to disturb the interface. Next, 11 mL of diluted blood was slowly layered on the liquid having a density of 1.070 g / mL, and centrifugation was performed at 2000 rpm for 20 minutes. Next, a fraction deposited between a liquid having a density of 1.070 g / mL and a liquid having a density of 1.095 g / mL was collected using a pipette. The collected fraction was washed twice with a washing solution (2 mM EDTA, 0.1% BSA-PBS) to obtain a fraction containing nucleated red blood cells.

[Immunostaining]
The number of cells in the fraction obtained above was measured with a cell counter (Bio-Rad Laboratories, TC20 fully automatic cell counter), and 0.1% BSA-PBS so that the cell concentration was 1 × 10 ⁷ cells / mL. Diluted with To this 1 mL of cell suspension, 10 μL of BV421-labeled anti-CD45 antibody (host animal: mouse, BD biosciences) was added for the purpose of staining leukocytes, and incubated at 4 ° C. for 30 minutes. Subsequently, the cells were washed once with 0.1% BSA-PBS, 1 mL of 0.025% glutaraldehyde-PBS was added, and the cells were fixed by inversion mixing at 25 ° C. for 10 minutes. Next, the cells after the fixation treatment were washed once with 0.1% BSA-PBS, suspended in 1 mL of 0.1% BSA-PBS, 100 μL of 0.1% Triton X100-PBS was added, and 25 ° C. The cell membrane permeabilization treatment was performed by allowing to stand for 5 minutes. Next, the cells after the cell membrane permeabilization treatment were washed once with 0.1% BSA-PBS, suspended in 1 mL of 0.1% BSA-PBS, and dispensed at 100 μL each into a 1.5 mL tube. To each tube, add each antibody shown in Table 3 (primary antibody. As anti-HBG antibody, an antibody labeled with FITC was used) so that the recommended amount or dilution rate described in the package insert was obtained, Incubated at 25 ° C. for 60 minutes. The cells after the primary antibody treatment were washed once with 0.1% BSA-PBS and suspended in 100 μL of 0.1% BSA-PBS. When the primary antibody was directly fluorescently labeled (HBG, C4orf3, GYPE, CD71), it proceeded to the nuclear staining step described later. When the primary antibody was not labeled, a labeling reaction was performed using a fluorescently labeled secondary antibody. PE labeled anti-rabbit IgG (Thermo Fisher Scientific) was used as a secondary antibody corresponding to the host animal species of the primary antibody. A labeling reaction was performed by adding 200 μL of a secondary antibody solution diluted to an antibody concentration of 10 μg / mL to each tube and incubating at 25 ° C. for 30 minutes.

[Nuclear staining]
The antibody-treated cells were washed once with 0.1% BSA-PBS, replaced with 1 mL of 0.1% BSA-PBS containing 5 μM DRAQ5 (Cell Signaling Technology) as a nuclear stain, and allowed to stand for 5 minutes. Cell nuclei were stained. The cell suspension after nuclear staining was subjected to the next step.

[Isolation of nucleated red blood cells]
Using a flow cytometer (SH800ZP manufactured by Sony Corporation), cells were gated from the cell suspension with forward scattered light and side scattered light. Subsequently, white blood cells were removed from the gated cell fraction by gating CD45 negative cells to obtain a CD45 negative fraction mainly composed of red blood cells. Subsequently, DRAQ5 positive cells were gated from the CD45 negative fraction to obtain a nucleated red blood cell fraction.

[Isolation of antibody positive cells]
Using a flow cytometer (SH800ZP manufactured by Sony Corporation), antibody-positive cells are gated from the nucleated erythrocyte fraction, and the ratio of antibody-positive cells to all cells in the nucleated erythrocyte fraction (%, number basis) Asked. The results are shown in Table 4 and FIG. The horizontal axis in FIG. 1 is the ratio of antibody-positive cells to the total cells of the nucleated red blood cell fraction when umbilical cord blood is used as the sample, and the vertical axis in FIG. 1 is the case when adult bone marrow blood is used as the sample. The ratio of antibody-positive cells to the total cells of the nucleated red blood cell fraction.

Most of the red blood cells in cord blood are fetal nucleated red blood cells, and most of the red blood cells in adult bone marrow blood are mature, anucleated red blood cells,
Anti-HBG antibody, anti-ALAS2 antibody, anti-HEMGN antibody, anti-C9orf5 antibody, anti-GYPB antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody, anti-C4orf3 antibody, anti-IGF2 antibody, anti-GYPE antibody, anti-CLIC6 antibody, and anti-DDX3Y antibody are The percentage of antibody-positive cells was higher in umbilical cord blood than in adult bone marrow blood.
On the other hand, anti-CD71 antibody, anti-SNCA antibody, anti-AHSP antibody, and anti-HMBS antibody did not differ in the proportion of antibody-positive cells between umbilical cord blood and adult bone marrow blood.

<Example 3>
Two antibodies shown in Table 3 were combined to try to isolate fetal cells from human umbilical cord blood and human adult bone marrow blood.

  In the same manner as in Example 2, a fraction containing nucleated red blood cells was obtained from human umbilical cord blood and human adult bone marrow blood by density gradient centrifugation. Subsequently, the fraction containing nucleated red blood cells was treated with a BV421-labeled anti-CD45 antibody in the same manner as in Example 2. Next, the treatment with the primary antibody was carried out in the same manner as in Example 2, except that two kinds of antibodies in the combinations shown in Table 5 were used. At this time, as an anti-HBG antibody, when combined with an anti-C4orf3 antibody or anti-GYPE antibody, an antibody labeled with RPE is used. When combined with an antibody other than the above, an antibody labeled with FITC is used. Using. Subsequently, PE labeled anti-rabbit IgG (Thermo Fisher Scientific) was reacted as a secondary antibody to the unlabeled primary antibody in the same manner as in Example 2. Next, in the same manner as in Example 2, staining of cell nuclei was performed with DRAQ5, and the cell suspension after nuclear staining was subjected to the next step.

  In the same manner as in Example 2, cells were gated from the cell suspension using a flow cytometer (SH800ZP manufactured by Sony Corporation). Subsequently, white blood cells were removed from the gated cell fraction by gating CD45 negative cells to obtain a CD45 negative fraction mainly composed of red blood cells. Subsequently, DRAQ5 positive cells were gated from the CD45 negative fraction to obtain a nucleated red blood cell fraction. Subsequently, antibody positive cells were gated from the nucleated red blood cell fraction. The ratio (%, number basis) of antibody (1) positive and antibody (2) positive cells in all cells of the nucleated red blood cell fraction was measured. The results are shown in Table 5.

Anti-HBG antibody, anti-ALAS2 antibody, anti-HEMGN antibody, anti-C9orf5 antibody, anti-GYPB antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody, anti-C4orf3 antibody, anti-IGF2 antibody, anti-GYPE antibody, anti-CLIC6 antibody, and anti-DDX3Y antibody In the case of immunostaining with any one, the ratio of cells positive for antibody (1) and antibody (2) is higher in umbilical cord blood than in adult bone marrow blood, and umbilical cord blood / adult bone marrow blood is 2.8-39. .2.
On the other hand, when immunostaining with anti-CD71 antibody and any one of anti-SNCA antibody, anti-AHSP antibody, and anti-HMBS antibody, antibody (1) positive and antibody (2) positive with umbilical cord blood and adult bone marrow blood There was no difference in the proportion of cells.

  The results of Examples 1 to 3 are as follows: anti-HBG antibody, anti-ALAS2 antibody, anti-HEMGN antibody, anti-C9orf5 antibody, anti-GYPB antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody, anti-C4orf3 antibody, anti-IGF2 antibody, anti-GYPE antibody, anti Demonstrate that fetal cells can be isolated from peripheral blood of a maternal mother by immunostaining cells with at least two antibodies selected from the group consisting of CLIC6 antibody and anti-DDX3Y antibody Yes.

Claims (10)

A method for isolating fetal cells from peripheral blood collected from a pregnant mother,
It consists of anti-HBG antibody, anti-ALAS2 antibody, anti-HEMGN antibody, anti-C9orf5 antibody, anti-GYPB antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody, anti-C4orf3 antibody, anti-IGF2 antibody, anti-GYPE antibody, anti-CLIC6 antibody, and anti-DDX3Y antibody Contacting at least two antibodies selected from the group with a blood cell population and marking the cells with said at least two antibodies;
Isolating cells marked with the at least two antibodies from the blood cell population;
A method for isolating fetal cells.   The method for isolating fetal cells according to claim 1, wherein the at least two kinds of antibodies comprise an anti-HBG antibody. Further, the step of subjecting the peripheral blood to density gradient centrifugation to obtain a fraction containing nucleated red blood cells,
The fraction containing the nucleated red blood cells is the blood cell population;
The method for isolating fetal cells according to claim 1 or 2. further,
Contacting an anti-CD45 antibody with the blood cell population and marking the cells with the anti-CD45 antibody;
Isolating cells not marked with the anti-CD45 antibody from the blood cell population;
The method for isolating fetal cells according to any one of claims 1 to 3, comprising: further,
Contacting the blood cell population with a nuclear staining dye to stain the cell nucleus;
Isolating nucleated cells having nuclei stained with the nuclear staining dye from the blood cell population;
The method for isolating fetal cells according to any one of claims 1 to 4, comprising:   The method for isolating fetal cells according to any one of claims 1 to 5, wherein the cells are isolated from the blood cell population by flow cytometry.   It consists of anti-HBG antibody, anti-ALAS2 antibody, anti-HEMGN antibody, anti-C9orf5 antibody, anti-GYPB antibody, anti-SLC1A5 antibody, anti-EIF1AY antibody, anti-C4orf3 antibody, anti-IGF2 antibody, anti-GYPE antibody, anti-CLIC6 antibody, and anti-DDX3Y antibody An antibody set for use in isolating fetal cells, comprising at least two antibodies selected from the group.   The antibody set according to claim 7, wherein the at least two kinds of antibodies comprise an anti-HBG antibody.   The part or the whole of the at least two kinds of antibodies is an antibody labeled with at least one kind selected from a fluorescent dye, a fluorescent protein, an enzyme, biotin, and a magnetic bead, respectively. Antibody set.   A fetal cell is isolated from peripheral blood collected from a pregnant mother by the method for isolating fetal cells according to any one of claims 1 to 6, and chromosomal DNA is obtained from the isolated fetal cells. A prenatal genetic testing method for the fetus.