JP2018105645A - Rare cell detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve a detection rate for detecting rare cells from a biological sample in a rare cell detection method using an antibody.SOLUTION: The rare cell detection method for detecting a rare cell from a biological sample containing a rare cell and contaminating cells other than the rare cell, includes the steps of: marking cells with a first labeled antibody in contact with the biological sample in which an antibody against a protein present in the rare cell is labeled with a first labeling substance; marking cells with a second labeled antibody in contact with the biological sample in which an antibody against a protein absent in the rare cell but present in the contaminating cells is labeled with a second labeling substance; and detecting from the biological sample a cell marked with the first labeled antibody and not marked with the second labeled antibody, so that the antibody against the protein present in the rare cell is at least one selected from Fab antibodies and Fab' antibodies.SELECTED DRAWING: None

Description

  The present disclosure relates to a method for detecting rare cells.

  Detecting and isolating rare cells present in a biological sample is useful for disease diagnosis, genetic testing, clinical research of rare cells, and the like. As a method for detecting rare cells, methods for immunologically identifying and detecting rare cells using antibodies against proteins expressed in the rare cells are disclosed in, for example, Patent Documents 1 to 4.

Special table 2008-507286 Special table 2008-500558 Japanese translation of PCT publication No. 2002-504992 Japanese Patent No. 2644029

  The immunological detection method using an antibody is a detection method with relatively high cell specificity, but when the rare cells to be detected are very few cells in the biological sample, the antibody against the rare cells is contaminated cells. The probability of non-specific binding to can not be ignored and miscellaneous cells may be misdetected as rare cells. In order to avoid this false detection, a technique is known that combines the identification of contaminating cells with an antibody against the contaminating cells and the exclusion of the contaminating cells. However, when this technique is used, rare cells may not be detected for the reasons described below.

Where an immunoglobulin molecule has one Fc region, the Fc region of an antibody against rare cells may bind to contaminating cells. In particular, when the rare cells to be detected are cells in blood, neutrophils present in large numbers in the blood express Fc receptors, so the Fc region of the antibody against rare cells becomes neutrophils. Easy to combine.
Further, when an immunoglobulin molecule has two Fab regions containing an antigen binding site, when the rare cells to be detected are very few cells in the sample, one Fab region of an antibody against the rare cells is a rare cell. And the other Fab region may bind nonspecifically to contaminating cells.

  As described above, when a rare cell and a contaminated cell are connected via one antibody against the rare cell, the rare cell is excluded together with the contaminated cell by excluding the contaminated cell, and the rare cell is not detected. End up.

The embodiment of the present disclosure has been made under the above circumstances.
The subject of this indication is improving the detection rate which detects a rare cell from a biological sample in the detection method of the rare cell using an antibody.

  Specific means for solving the above problems include the following aspects.

[1] A detection method for detecting rare cells from a biological sample containing rare cells and contaminating cells other than rare cells, wherein the antibody against a protein present in the rare cells is labeled with a first labeling substance. A step in which a labeled antibody is brought into contact with a biological sample, and a cell is marked with the first labeled antibody; and a second labeled substance is labeled with an antibody against a protein that is not present in a rare cell but present in a contaminated cell. Contacting the labeled antibody with a biological sample, marking the cells with the second labeled antibody, and, from the biological sample, marking cells with the first labeled antibody and not marked with the second labeled antibody. A method for detecting a rare cell, wherein the antibody to the protein present in the rare cell is at least one selected from a Fab antibody and a Fab ′ antibody.
[2] The method for detecting a rare cell according to [1], wherein the antibody against the protein present in the rare cell is a Fab ′ antibody.
[3] Detection of a rare cell according to [1] or [2], wherein the first labeled antibody is a labeled antibody in which a first labeling substance is covalently bound to a thiol group of an antibody against a protein present in the rare cell. Method.
[4] A step in which the rare cell is a nucleated cell, and further, a step of staining the cell nucleus by bringing a nuclear staining dye into contact with the biological sample; and a step of detecting a cell in which the nucleus is stained with the nuclear staining dye from the biological sample; The method for detecting a rare cell according to any one of [1] to [3].
[5] The method for detecting a rare cell according to any one of [1] to [4], wherein the biological sample is a fraction containing rare cells obtained by subjecting blood to density gradient centrifugation.
[6] The method for detecting a rare cell according to any one of [1] to [5], wherein the biological sample is a biological sample derived from pregnant maternal peripheral blood, and the rare cell is fetal nucleated red blood cell.
[7] The method for detecting a rare cell according to any one of [1] to [5], wherein the biological sample is a biological sample derived from peripheral blood of a cancer patient, and the rare cell is a circulating tumor cell in the blood. .
[8] In any one of [1] to [7], the first labeling substance and the second labeling substance are chemical substances selected from fluorescent dyes, fluorescent proteins, enzymes, biotin, and magnetic beads, respectively. The detection method of the rare cell of description.
[9] The method for detecting a rare cell according to any one of [1] to [8], wherein the cell is detected from the biological sample by flow cytometry.

  According to the present disclosure, in the method for detecting rare cells using an antibody, the detection rate for detecting rare cells from a biological sample can be improved.

  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.

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

  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.

<Rare cell detection method>
The detection method of the present disclosure is a method for detecting rare cells from a biological sample including rare cells and contaminating cells other than rare cells.

  In the present disclosure, the biological sample includes an extract from the living body (raw sample) and a diluted sample obtained by diluting the raw sample with physiological saline or the like; a preservation sample obtained by adding an additive to the raw sample; Examples include a fraction, a dried product, or a frozen product; a sample in which cells contained in these samples are fixed with a fixing agent (for example, glutaraldehyde, paraformaldehyde); and the like.

The rare cell which is the target of the detection method of the present disclosure refers to a cell having a low existing ratio in a biological sample. A contaminated cell means a cell other than a rare cell that constitutes a biological sample containing a rare cell that is a target of the detection method of the present disclosure. In a raw sample (for example, peripheral blood), the abundance ratio of rare cells to contaminating cells (number basis) is, for example, rare cells: contaminated cells = 1: 1 × 10 ⁵ to 1: 1 × 10 ⁹ .

  Examples of rare cells that are targets of the detection method of the present disclosure include fetal nucleated red blood cells contained in peripheral maternal blood of pregnancy, circulating tumor cells in blood contained in peripheral blood of cancer patients, and the like.

  Fetal nucleated red blood cells (fNRBC) are precursors of erythrocytes that pass through the placenta and are present in the peripheral blood of the pregnant mother. It is said that fNRBC is present at a rate of about 1 per mL of peripheral maternal blood in pregnancy. Since fNRBC has chromosomes, fetal chromosomes and fetal genes can be obtained by isolating fNRBC. Therefore, by isolating fNRBC, a prenatal genetic test that is less invasive to the pregnant mother can be performed.

  fNRBC is said to be present in the peripheral blood of pregnant mothers from about 6 weeks after pregnancy. Also, fetal erythrocytes can be nucleated when the mother is pregnant. Therefore, when the target of the detection method of the present disclosure is fNRBC, the biological sample to be used for the detection method of the present disclosure is peripheral blood collected from a pregnant mother about 6 weeks after pregnancy or blood prepared from this peripheral blood A sample is preferred.

  In addition to fNRBC, maternal white blood cells (eosinophils, neutrophils, basophils, monocytes, lymphocytes); maternal mature red blood cells without nuclei; maternal nucleated red blood cells And so on. These cells other than fNRBC correspond to the contaminating cells in the present disclosure.

  Circulating tumor cells (CTC) are circulating tumor cells in the blood of cancer patients. It is said that CTC is present at a rate of about 1 in peripheral blood counts of cancer patients. By measuring the number of CTCs in peripheral blood, it is expected that determination of therapeutic effect, early diagnosis of metastatic cancer, determination of necessity of treatment, prediction of patient prognosis, and the like can be performed. In addition, by isolating CTC, it is possible to obtain tumor cell chromosomes and examine gene mutations.

  The peripheral blood of cancer patients includes cells such as leukocytes (eosinophils, neutrophils, basophils, monocytes, lymphocytes); erythrocytes; in addition to CTC. These cells other than CTC are the contaminating cells in the present disclosure.

The detection method of the present disclosure includes:
A step of marking a cell with a first labeled antibody by contacting a first labeled antibody in which an antibody against a protein present in a rare cell is labeled with a first labeling substance with a first sample (“first labeling step”) And)
A step of contacting a biological sample with a second labeled antibody in which an antibody against a protein not present in a rare cell but present in a contaminated cell is labeled with a second labeling substance, and marking the cell with the second labeled antibody ( "Second labeling process"),
A step of detecting a cell marked with the first labeled antibody and not marked with the second labeled antibody from the biological sample (referred to as “rare cell detection step”);
Have In the detection method of the present disclosure, the first labeled antibody is a labeled antibody in which at least one selected from a Fab antibody and a Fab ′ antibody is labeled with a first labeling substance.

When the target of the detection method of the present disclosure is a nucleated cell, the detection method of the present disclosure further includes:
A step of contacting a biological sample with a nuclear staining dye to stain a cell nucleus (referred to as a “cell nucleus staining step”);
A step of detecting cells whose nuclei are stained with a nuclear staining dye (referred to as “nucleated cell detection step”) from a biological sample;
It is preferable to have.

  Hereinafter, the antibody, the labeling substance, and the process of the detection method of the present disclosure will be described in detail.

[First labeled antibody, second labeled antibody]
The antibody constituting the first labeled antibody and the antibody constituting the second labeled antibody are different antibodies. The antibody constituting the first labeled antibody is an antibody against a protein present in a rare cell, and the antibody constituting the second labeled antibody is an antibody against a protein present in a contaminated cell but not present in a rare cell.

  The first labeled antibody may be either an antibody obtained by labeling an antibody against one kind of protein with one kind of chemical substance; an antibody obtained by labeling a set of antibodies against a plurality of kinds of proteins with one kind of chemical substance, The former is preferred. The antibody may be a monoclonal antibody or a polyclonal antibody. The protein that is the target of the first labeled antibody is preferably a protein that is expressed on the cell membrane surface of a rare cell.

  The second labeled antibody may be either an antibody obtained by labeling an antibody against one kind of protein with one kind of chemical substance; an antibody obtained by labeling a set of antibodies against a plurality of kinds of proteins with one kind of chemical substance, The former is preferred. When a sample contains a plurality of types of contaminating cells, the second labeled antibody may be in any of the following forms (a) and (b), with form (a) being preferred. The antibody may be a monoclonal antibody or a polyclonal antibody. The target protein of the second labeled antibody is preferably a protein expressed on the cell membrane surface of a contaminated cell.

Form (a): An antibody obtained by labeling an antibody against one kind of protein commonly expressed in plural kinds of contaminating cells with one kind of chemical substance.
Form (b): An antibody in which a set of antibodies against each of a plurality of types of proteins expressed in at least one type of a plurality of types of contaminating cells is labeled with one type of chemical substance.

  The first labeling substance constituting the first labeled antibody is distinguished from the second labeling substance constituting the second labeled antibody by an optical method, a chemical method, an electrical method, or a magnetic method. It is another chemical substance.

  Examples of the first labeling substance include known fluorescent dyes, fluorescent proteins, enzymes, biotin, magnetic beads and the like for labeling antibodies. Examples of the second labeling substance include known fluorescent dyes, fluorescent proteins, enzymes, biotin, magnetic beads and the like for labeling antibodies.

  Examples of fluorescent dyes for labeling antibodies include Alexa Fluor series (Alexa Fluor 488, Alexa Fluor 647, etc.), Brilliant Violet series (BV421, BV570, etc.), BODIPY series (BODIPY FL, BODIPY TR, etc.), Cy3, Cy5, FITC (fluorescein isothiocyanate), a coumarin fluorescent dye, etc. are mentioned. Among these, Alexa Fluor series or Brilliant Violet series is preferable from the viewpoint of low background, strong fluorescence intensity, and difficulty in fading. 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.

  The antibody constituting the first labeled antibody is at least one selected from Fab antibody and Fab ′ antibody, which are fragments of immunoglobulin molecules. The antibody constituting the first labeled antibody is a Fab antibody or Fab ′ antibody that is an antibody having one antigen-binding site and no Fc region from the viewpoint of not forming a bridge between rare cells and contaminating cells. Since the antibody constituting the first labeled antibody is unlikely to form a cross-link between cells, the target of the antibody constituting the first labeled antibody is present not only in rare cells but also in contaminated cells. It may be a protein. However, from the viewpoint of improving the detection rate of rare cells, the target of the antibody constituting the first labeled antibody is preferably a protein that does not exist in contaminating cells but exists in rare cells.

  The first labeled antibody can be produced, for example, by covalently binding a labeling substance to the thiol group or amino group of the Fab antibody or Fab ′ antibody. A method for covalently binding a labeling substance to an antibody via a thiol group or an amino group is known. Since amino groups generally exist randomly in immunoglobulin molecules, labeling an antibody via an amino group may cause the labeling substance to bind to or close the antibody's antigen binding site or its vicinity. There is. On the other hand, the thiol group is a group formed by reducing the disulfide bond of the hinge part of an immunoglobulin molecule, so when the antibody is labeled through the thiol group, the labeling substance binds to the hinge part. Activity is difficult to deactivate. Accordingly, the first labeled antibody is preferably a labeled antibody in which a labeling substance is covalently bonded to a thiol group of the antibody.

  For the above reasons, as the antibody constituting the first labeled antibody, Fab ′ antibody, which is an antibody having a hinge part, is preferable to Fab antibody which is an antibody having no hinge part.

  A Fab antibody is a fragment containing an antigen binding site formed by cleaving an immunoglobulin molecule with papain. Methods for preparing Fab antibodies from immunoglobulin molecules are known. The Fab antibody may be a commercially available product.

  The Fab 'antibody is a fragment containing an antigen binding site in which an immunoglobulin molecule is cleaved by pepsin and a disulfide bond in the hinge region is reduced. Methods for preparing Fab 'antibodies from immunoglobulin molecules are known. The Fab 'antibody may be a commercially available product.

  The immunoglobulin molecule used for the preparation of Fab antibody or Fab ′ antibody is a method for purifying an antigen from the blood of an immunized non-human animal; a fusion cell of B cells and myeloma cells of an immunized non-human animal It can be produced by a method of producing a hybridoma and obtaining a monoclonal antibody from the hybridoma; The immunoglobulin molecule may be a commercially available product.

Antibodies constituting the second labeled antibody (antibodies to proteins present in contaminated cells but not in rare cells) are immunoglobulin molecules themselves (intact immunoglobulin), Fab antibodies, Fab ′ antibodies, F (ab ′) ₂ antibodies Either of these may be used. Based on the ratio of rare cells and contaminated cells, the antibody that constitutes the second labeled antibody binds nonspecifically only to rare cells, or nonspecifically binds to rare cells and also binds to contaminated cells. However, the probability of forming a bridge between the two is low. Therefore, the antibody constituting the second labeled antibody may be an antibody having an Fc region or an antibody having two Fab regions.

  The antibody constituting the second labeled antibody and the second labeled antibody may be produced by the production method described above for the first labeled antibody, or may be a commercially available product.

  When the target of the detection method of the present disclosure is fNRBC contained in the maternal peripheral blood, examples of the target protein of the first labeled antibody include CD71, CD235a, fetal hemoglobin (HbF), Examples of the target protein of the labeled antibody include CD2, CD3, CD14, CD16, CD19, CD38, CD45, CD56, and CD61.

  When the target of the detection method of the present disclosure is CTC contained in the peripheral blood of a cancer patient, examples of the target protein of the first labeled antibody include EpCAM, cytokeratin, and vimentin. Examples of the protein that is a target of the antibody include CD2, CD3, CD14, CD16, CD19, CD36, CD38, CD45, CD56, CD61, CD66, CD71, and CD235a.

[First labeling step, second labeling step]
The first labeling step is a step of marking cells with the first labeled antibody by bringing the first labeled antibody into contact with the biological sample. In this step, for example, the first labeled antibody is added to the biological sample, or the biological sample is added to a phosphate buffer solution or physiological saline containing the first labeled antibody, and then at 4 ° C. to room temperature for 30 minutes. By incubating for ~ 120 minutes.

  The second labeling step is a step of marking cells with the second labeled antibody by bringing the second labeled antibody into contact with the biological sample. In this step, for example, the second labeled antibody is added to the biological sample, or the biological sample is added to a phosphate buffer or physiological saline containing the second labeled antibody, and the reaction is performed at 4 ° C. to room temperature for 30 minutes. By incubating for ~ 120 minutes.

  The first labeling step and the second labeling step may be performed simultaneously or sequentially. Simultaneous execution of the first labeling step and the second labeling step includes, for example, adding the first labeled antibody and the second labeled antibody to the biological sample, or containing the first labeled antibody and the second labeled antibody. This is done by adding a biological sample to a phosphate buffer or physiological saline. When the first labeling step and the second labeling step are sequentially performed, either may be performed first.

The biological sample to be contacted with the antibody in the first labeling step and the second labeling step has a total cell concentration of, for example, 1 × 10 ² to 1 × 10 ¹⁰ cells / mL, preferably 1 × 10 ³ to 1 × 10. ^{It is 9} / mL, and more preferably 1 × 10 ⁴ to 1 × 10 ⁸ / mL.

  The amount of the biological sample brought into contact with the antibody in the first labeling step and the second labeling step is usually 1000 μL or less, preferably 500 μL or less, more preferably 300 μL from the viewpoint of reducing the time required for the rare cell detection step. It is as follows.

[Rare cell detection process]
The rare cell detection step is performed by an optical method, a chemical method, an electrical method, or a magnetic method according to the first labeling substance and the second labeling substance.

  The rare cell detection step is preferably performed by flow cytometry from the viewpoint of not only detecting but also isolating cells. Flow cytometry is a technique for measuring cells by spectroscopic techniques by flowing a cell suspension so that the cells are aligned. According to flow cytometry, it is also possible to sort specific cells by controlling the flow direction by charging droplets containing cells. From the viewpoint of performing the rare cell detection step by flow cytometry, the first labeling substance and the second labeling substance are preferably an antibody labeled with a fluorescent dye, a fluorescent protein, or biotin. When the rare cell detection step is performed by flow cytometry, scattered light corresponding to a form specific to the rare cell can be detected, and the detection rate of the rare cell can be further improved.

  The rare cell detection step may be a step of circulating a biological sample through a microcapillary and separating the cells by an optical method, a chemical method, an electrical method, or a magnetic method. If the rare cell detection step is a step of capturing cells circulating in the microcapillary by an electric method or a magnetic method, not only the detection of the rare cells but also the isolation can be performed.

[Cell nucleus staining process, nucleated cell detection process]
The cell nucleus staining step is a step of staining cell nuclei by bringing a nuclear staining dye into contact with a biological sample. The cell nucleus staining step is preferably provided when the target of the detection method of the present disclosure is a nucleated cell.

  The nuclear staining dye may be any dye that stains chromatin, nucleoprotein, DNA, and the like. Specifically, DRAQ5 (Deep Red Anthraquinone 5), DAPI (4 ′, 6-diamidino-2-phenylindole), iodine Propidium iodide, 7-AAD (7-amino-actinomycin D), Hoechst 33342, methylene blue, hematoxylin, thionine and the like.

  In the cell nucleus staining step, for example, a nuclear staining dye is added to a biological sample, or a biological sample is added to a phosphate buffer or a physiological saline containing the nuclear staining dye, and then at 4 ° C. to room temperature for 30 minutes to 120 minutes. This is done by incubating.

  The cell nucleus staining step, the first labeling step, and the second labeling step may be performed simultaneously or sequentially. The simultaneous execution of the three steps includes, for example, adding the first labeled antibody and the second labeled antibody and the nuclear staining dye to the biological sample, or contains the first labeled antibody, the second labeled antibody and the nuclear staining dye. This is done by adding a biological sample to a phosphate buffer or physiological saline. When the three steps are sequentially performed, the order is not limited.

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

  The rare cell detection step and the nucleated cell detection step may be performed simultaneously or sequentially. When both steps are performed by flow cytometry, both steps can be performed simultaneously.

Examples of embodiments of the detection method of the present disclosure include a first labeling step, a second labeling step, a cell nucleus staining step, a rare cell detection step, and a nucleated cell detection step,
The first labeling step, the second labeling step, and the cell nucleus staining step are performed (the order of the three steps is not limited, and the three steps may be performed simultaneously), and then the rare cell detection step and the nucleated cell detection step are flowed Examples are given by cytometry (the order of the two steps is not limited and the two steps may be performed simultaneously).

  When the labeled antibody used in the detection method of the present disclosure targets a protein present in the cell, or when the nuclear staining dye used in the detection method of the present disclosure is poor in cell membrane permeability, a living body to be subjected to a series of labeling steps The sample is preferably a sample that has been subjected to cell membrane permeabilization. The cell membrane permeabilization treatment is performed, for example, by mixing a biological sample with a phosphate buffer or physiological saline containing a surfactant such as Triton X100 and incubating at 4 ° C. to room temperature for 1 to 30 minutes. In the case of subjecting a biological sample to a cell membrane permeation treatment, the biological sample is preferably a biological sample that has been subjected to an immobilization treatment prior to the cell membrane permeation treatment for the purpose of suppressing loss of protein from within the cell. The biological sample is immobilized by, for example, mixing a biological sample with a phosphate buffer or physiological saline containing glutaraldehyde or paraformaldehyde, and incubating at 4 ° C. to room temperature for 1 to 30 minutes. Is called.

[Preparation of biological sample by density gradient centrifugation]
When the target of the detection method of the present disclosure is a rare cell contained in blood (for example, fNRBC, CTC), a biological sample subjected to a series of labeling steps is a rare cell obtained by subjecting blood to density gradient centrifugation. It is preferable that it is a fraction containing. A biological sample enriched with rare cells can be obtained by density gradient centrifugation, whereby the detection rate of rare cells can be further improved.

  fNRBC or CTC 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 fNRBC or CTC. For example, fNRBC or CTC can be fractionated and concentrated by layering and centrifuging blood diluted with physiological saline on a discontinuous density gradient in which at least two types of media with different densities are layered on a centrifuge tube. .

  Hereinafter, an example embodiment of a method for concentrating fNRBC by density gradient centrifugation will be described.

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

  In order to separate fNRBC 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 fNRBC is about 1.080 g / mL, it is possible to collect fractions having fNRBC at the interface by adjacently stacking two different density media sandwiching this density. 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. The lower layer medium and the upper layer medium may be 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. Of these, Percoll and / or Histopaque are preferable. 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.

  Then, blood that has been diluted, for example, twice with physiological saline is layered on the upper layer medium, centrifuged, and the fraction deposited between the lower layer medium and the upper layer medium is collected and collected. The fraction is washed with phosphate buffer. Since the fraction containing nucleated red blood cells thus obtained contains concentrated fNRBC, it is preferable to subject this fraction containing nucleated red blood cells to a series of labeling steps.

  The detection method of the present disclosure can be applied to, for example, prenatal genetic testing of a fetus. That is, by the detection method of the present disclosure, fNRBC can be detected from a biological sample derived from pregnant maternal peripheral blood, and chromosomal DNA can be obtained from the detected fNRBC to perform prenatal genetic testing. In the following, exemplary embodiments of prenatal genetic testing of the fetus are described.

<Prenatal genetic testing of the fetus>
The test method of the present disclosure is a method for prenatal genetic testing of a fetus that is performed by detecting fNRBC from a biological sample derived from peripheral maternal blood of a pregnancy and obtaining chromosomal DNA from the detected fNRBC. 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. In order to analyze chromosomal DNA simply and efficiently, the inspection method of the present disclosure includes an amplification step of amplifying a target region on a chromosome by a polymerase chain reaction (PCR), a base sequence of the amplified target region, and And a sequence analysis step for determining the amount. In order to facilitate analysis of chromosomal DNA, first, whole genome amplification (WGA) is performed from fNRBC, and the target region on the chromosome is amplified by PCR using the amplification product of whole genome amplification as a template. An amplification step for performing the sequence, and a sequence analysis step for determining the base sequence and amount of the amplified target region may be performed. Hereinafter, an exemplary embodiment of the inspection method of the present disclosure will be described in detail by describing the above-described steps.

[Amplification process of target region by PCR]
This amplification step is a step of amplifying the target region by PCR. 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.

[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).

[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 PCR amplification step is a substance that can be applied to the sequencer in the sequence analysis step, and the amplification product obtained in the PCR amplification step is a library for sequencing.

  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 the chromosome obtained from fNRBC, 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 disclosure will be described in detail by way of examples, but the embodiments of the present disclosure 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 based on mass unless otherwise specified.

  The water used in the following Reference Examples or Examples is water purified by Merck Milli-Q equipment unless otherwise specified. (Milli-Q is a registered trademark)

Abbreviations such as chemical substances used in the following Reference Examples or Examples are as follows.
BSA: bovine serum albumin
BV421: Brilliant Violet 421
DRAQ5: Deep Red Anthraquinone 5
EDTA: ethylenediaminetetraacetic acid
FITC: fluorescein isothiocyanate
fNRBC: fetal nucleated red blood cell, fetal nucleated red blood cell PBS: phosphate buffered saline
PBMC: peripheral blood mononuclear cell, peripheral blood mononuclear cell PCR: polymerase chain reaction
SDS: sodium dodecyl sulfate
TCEP-HCL: tris- (2-Carboxyethyl) phosphine hydrochloride

[Reference Example 1]
(1) Preparation of fragmented antibody An anti-CD71 antibody (BD biosciences, product number 555534) was prepared as an antibody that recognizes and binds to fNRBC.

Anti-CD71 antibody was fragmented using F (ab ′) 2 Micro Preparation Kit (Pierce). That is, the anti-CD71 antibody was digested with immobilized pepsin at 37 ° C. for 3 hours, the digest was passed through a protein A sepharose column, and the unbound product was purified to obtain F (ab ′) ₂ .

Next, ₂ μL of 10 mM TCEP-HCL (Thermo Fisher Scientific) solution was added to 20 μL of F (ab ′) ₂ solution having a concentration of 0.1 to 5 mg / mL, and the mixture was allowed to stand at 25 ° C. for 30 minutes. Subsequently, TCEP-HCL was removed with an Antibody conjugate purification Kit for 50-100 μg (Thermo Fisher Scientific), and Fab ′ was purified.

(2) Fluorescent labeling of fragmented antibody 4 μL of 25 mM Alexa Fluor 488 C5 maleimide (Thermo Fisher Scientific) (dimethyl sulfoxide solution) was added to 50 μL of Fab ′ solution at a concentration of 0.1 to 5 mg / mL, and at 25 ° C. For 6 hours. Subsequently, the unreacted Alexa Fluor 488 C5 maleimide was removed with an antibody conjugate purification kit for 50-100 μg (Thermo Fisher Scientific), and Fab ′ labeled with a fluorescent dye was purified. Hereinafter, this antibody is referred to as fluorescently labeled fragmented antibody (1).

(3) Preparation of model sample containing rare cells Human umbilical cord blood (VERITAS, product number C-CB101) was prepared as a biological sample containing rare cells, and fNRBC was collected from the umbilical cord blood by the following steps.

  Using Percoll liquid (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 blood diluted twice with physiological saline 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.

The number of cells in the nucleated red blood cell fraction obtained above was measured with a cell counter (Bio-Rad Laboratories, TC20 fully automatic cell counter), diluted with 0.1% BSA-PBS, and a cell concentration of 1.0 × 10. ^{A 7} / mL nucleated red blood cell suspension was obtained. 3 μL of Fc Block Reagent for Human (BD biosciences) was added to 100 μL of nucleated red blood cell suspension for the purpose of blocking Fc receptors expressed on the cell surface such as leukocytes and left at 25 ° C. for 10 minutes. did. Next, 1 μL of BV421-labeled anti-CD45 antibody (BD biosciences, product number 563879, antibody part is an intact immunoglobulin molecule) as a leukocyte marker, 4 μL of fluorescently labeled fragmented antibody (1) as a fNRBC marker, nuclear staining 100 μL of DRAQ5 (Cell Signaling) diluted to 0.1% with 0.1% BSA-PBS was added as a dye, and left at 4 ° C. for 30 minutes or more.

Separately, PBMC (Precision Bioservices, product number 93000-10M) is prepared as a model of contaminated cells, diluted with 0.1% BSA-PBS, and suspended at a cell concentration of 1.0 × 10 ⁷ cells / mL. A liquid was prepared.

The nucleated red blood cell suspension after reacting with the antibody and the nuclear staining dye is applied to a flow cytometer (Sony Corporation, SH800ZP), and in a single cell mode, CD45 negative, CD71 positive and DRAQ5 positive cells are separated. The collected cells were collected in 100 cells and 100 μL of PBMC suspension (PBMC concentration 1.0 × 10 ⁷ cells / mL, 100 μL containing 1.0 × 10 ⁶ PBMCs).

  CD45-negative, CD71-positive, and DRAQ5-positive cells collected by a series of operations can be regarded as fNRBC. Thus, a model sample having a cell abundance ratio of fNRBC: PBMC = 1: 10000 was obtained.

(4) Labeling of cell population 3 μL of Fc Block Reagent for Human (BD biosciences) was added to 100 μL of the model sample and left at 25 ° C. for 10 minutes. Next, DRAQ5 (Cell Signaling) diluted to 0.1% with 0.1% BSA-PBS, 1 μL of BV421-labeled anti-CD45 antibody (BD biosciences, product number 563879), 4 μL of fluorescently labeled fragmented antibody (1) 100 μL was added and left at 4 ° C. for 30 minutes or longer.

(5) Detection of rare cells A model sample after reacting with an antibody and a nuclear staining dye is subjected to a flow cytometer (Sony Corporation, SH800ZP), and in a semi-yield mode, CD45 negative, CD71 positive and DRAQ5 positive Cells were collected and collected one by one in 4 μL of 0.1% BSA-PBS.

  After the model sample was supplied to the flow cytometer in total, the sample tube was washed with 200 μL of DRAQ5 diluted to 0.1% with 0.1% BSA-PBS, and the washing solution was supplied again to the flow cytometer. Then, CD45-negative, CD71-positive and DRAQ5-positive cells were collected and collected one by one in 4 μL of 0.1% BSA-PBS. After using the whole amount in a flow cytometer, the same operation was performed again, and cells were collected one by one in 4 μL of 0.1% BSA-PBS.

  From the number of fNRBC contained in the model sample (number of CD45 negative, CD71 positive and DRAQ5 positive cells) and the number of recovered fNRBC (number of CD45 negative, CD71 positive and DRAQ5 positive cells), the detection rate (%) Was calculated. The results are shown in Table 1.

[Reference Example 2]
In Reference Example 1, “(3) Preparation of model sample containing rare cells” and “(4) Labeling of cell population”, except that fluorescently labeled fragmented antibody (1) was changed to fluorescently labeled fragmented antibody (2) In the same manner as in Reference Example 1, rare cells were detected. The results are shown in Table 1.
The fluorescently labeled fragmented antibody (2) was prepared by changing “(2) fluorescent labeling of the fragmented antibody” in Reference Example 1 as follows.

(2) Fluorescent labeling of fragmented antibody 10 μL of 1 M aqueous sodium hydrogen carbonate solution was added to 100 μL of Fab ′ solution having a concentration of 0.1 to 5 mg / mL. 100 μL of this solution was added to the reactive dye of Alexa fluor 488 labeling kit (Thermo Fisher Scientific) and left at 25 ° C. for 1 hour. Subsequently, unreacted reactive dye was removed with an Antibody conjugate purification Kit for 50-100 μg (Thermo Fisher Scientific), and Fab ′ labeled with a fluorescent dye was purified to obtain a fluorescently labeled fragmented antibody (2).

[Reference Example 3]
Except for changing the fluorescently labeled fragmented antibody (1) to the fluorescently labeled fragmented antibody (3) in “(3) Preparation of model sample containing rare cells” and “(4) Labeling of cell population” in Reference Example 1 In the same manner as in Reference Example 1, rare cells were detected. The results are shown in Table 1.
The fluorescently labeled fragmented antibody (3) was prepared by changing “(1) Preparation of fragmented antibody” and “(2) Fluorescent labeling of the fragmented antibody” in Reference Example 1 as follows.

(1) Preparation of fragmented antibody Anti-CD71 antibody (BD biosciences, product number 555534) was fragmented using Fab Micro Preparation Kit (Pierce). That is, the anti-CD71 antibody was digested with immobilized papain at 37 ° C. for 6 hours, the digest was passed through a protein A sepharose column, and the unbound product was purified to obtain Fab.

(2) Fluorescent labeling of fragmented antibody 10 μL of 1M aqueous sodium hydrogen carbonate solution was added to 100 μL of Fab solution having a concentration of 0.1 to 5 mg / mL. 100 μL of this solution was added to the reactive dye of Alexa fluor 488 labeling kit (Thermo Fisher Scientific) and left at 25 ° C. for 1 hour. Subsequently, the unreacted reactive dye was removed with an Antibody conjugate purification Kit for 50-100 μg (Thermo Fisher Scientific), and the Fab labeled with the fluorescent dye was purified to obtain a fluorescently labeled fragmented antibody (3).

[Reference Example 4]
In “(3) Preparation of model sample containing rare cells” and “(4) Labeling of cell population” in Reference Example 1, FITC-labeled anti-CD71 antibody (BD biosciences, product number 555536, the antibody part is an intact immunoglobulin molecule), and the detection of rare cells was performed in the same manner as in Reference Example 1 except that the amount of this antibody added was changed to 15 μL with respect to 100 μL of the cell suspension. went. The results are shown in Table 1.

[Reference Example 5]
In “(3) Preparation of model sample containing rare cells” in Reference Example 1, the concentration of PBMC suspension for recovering CD45-negative, CD71-positive, and DRAQ5-positive cells was set to a PBMC concentration of 1.0 × 10 ⁵ cells / mL. Rare cells were detected in the same manner as in Reference Example 1 except that the abundance ratio of cells in the model sample was changed to fNRBC: PBMC = 1: 100. The results are shown in Table 1.

[Reference Example 6]
In Reference Example 2, “(3) Preparation of model sample containing rare cells”, the concentration of PBMC suspension for recovering CD45-negative, CD71-positive, and DRAQ5-positive cells was set to a PBMC concentration of 1.0 × 10 ⁵ cells / mL. Rare cells were detected in the same manner as in Reference Example 2 except that the cell abundance ratio in the model sample was changed to fNRBC: PBMC = 1: 100. The results are shown in Table 1.

[Reference Example 7]
In Reference Example 3, “(3) Preparation of model sample containing rare cells”, the concentration of PBMC suspension for recovering CD45-negative, CD71-positive, and DRAQ5-positive cells was set to a PBMC concentration of 1.0 × 10 ⁵ cells / mL. Rare cells were detected in the same manner as in Reference Example 3 except that the cell abundance ratio in the model sample was changed to fNRBC: PBMC = 1: 100. The results are shown in Table 1.

[Reference Example 8]
In “(3) Preparation of model sample containing rare cells” in Reference Example 4, the concentration of PBMC suspension for recovering CD45-negative, CD71-positive, and DRAQ5-positive cells was set to a PBMC concentration of 1.0 × 10 ⁵ cells / mL. Rare cells were detected in the same manner as in Reference Example 4 except that the cell abundance ratio in the model sample was changed to fNRBC: PBMC = 1: 100. The results are shown in Table 1.

[Example 1]
Peripheral blood was obtained from a pregnant volunteer at 12 weeks of gestation, and fNRBC detection from the pregnant maternal peripheral blood was attempted. Blood collection from pregnant volunteers was performed after obtaining informed consent. The fetus is known to be a boy.

  In a 7 mL blood collection tube containing 10.5 mg of EDTA disodium salt as an anticoagulant, 7.0 mL of peripheral blood was collected from one pregnant volunteer, diluted twice with physiological saline, and subjected to the following steps.

(1) Density gradient centrifugation of nucleated red blood cells
Using Percoll liquid (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.

(2) Labeling of cell population The number of cells of the nucleated red blood cell fraction obtained above was measured with a cell counter (Bio-Rad Laboratories, TC20 fully automatic cell counter), diluted with 0.1% BSA-PBS, A nucleated red blood cell suspension with a cell concentration of 1.0 × 10 ⁷ cells / mL was obtained. 3 μL of Fc Block Reagent for Human (BD biosciences) was added to 100 μL of nucleated red blood cell suspension for the purpose of blocking Fc receptors expressed on the cell surface such as leukocytes and left at 25 ° C. for 10 minutes. did. Next, 1 μL of BV421-labeled anti-CD45 antibody (BD biosciences, product number 563880) as a leukocyte marker, 4 μL of fluorescently labeled fragmented antibody (1) as an fNRBC marker, and 0.1% BSA-PBS as a nuclear staining dye. 100 μL of DRAQ5 (Cell Signaling) diluted to 1% was added and left at 4 ° C. for 30 minutes or more.

(3) Detection of rare cells The nucleated red blood cell suspension after reacting with the antibody and the nuclear staining dye is subjected to a flow cytometer (Sony Corporation, SH800ZP), and is CD45 negative and CD71 positive in a semi-yield mode. In addition, DRAQ5-positive cells were collected and collected on a PCR plate into which 4 μL of 0.1% BSA-PBS was dispensed, one cell per well. Ten DNAs (10 wells) arbitrarily selected from the collected cells were subjected to the following DNA analysis.

(4) DNA analysis In each well of the PCR plate, cell lysis buffer (mixture of 10 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 20 mM KCl, 0.007% SDS, 13.3 μg / mL Proteinase K) 5 μL was added, incubated at 50 ° C. for 60 minutes, then incubated at 95 ° C. for 5 minutes to lyse the cells in the PCR plate.

  For the purpose of performing DNA amplification from the cell lysate, a multiplex PCR reaction was performed using a multiplex PCR assay kit (Takara Bio Inc.). The reaction solution was prepared by mixing 9 μL of cell lysate, 4 μL of 647 mixed primers, 0.125 μL of Multiplex PCR Mix1, 12.5 μL of Multiplex PCR Mix2, and 9.375 μL of water. The PCR reaction was denatured at 94 ° C./60 seconds, followed by 32 cycles of 94 ° C./30 seconds, 56.7 ° C./600 seconds, and 72 ° C./30 seconds.

  The obtained PCR product was purified using magnetic beads (AMPure XP Kit, BECMAN COULTER). 45 μL of magnetic bead dispersion (AMPure XP) was added to 25 μL of the PCR reaction solution, and the PCR reaction product was bound to the magnetic beads. Magnetic beads were separated by magnetic force of MagnaStand (Nippon Genetics) to remove impurities. After washing with 70 v / v% ethanol, the nucleic acid bound to the magnetic beads was eluted with Tris-EDTA buffer.

  Using the obtained eluate as a template, PCR reaction was performed again using Multiplex PCR Assay kit (Takara Bio Inc.). 2 μL of the eluate, 2 μL of the forward primer (1.25 μM) of the Y chromosome partial region, 2 μL of the reverse primer (1.25 μM) of the Y chromosome partial region, 0.125 μL of Multiplex PCR Mix1, and 12.5 μL of Multiplex PCR Mix2 And 6.375 μL of water were mixed to prepare a reaction solution. The PCR reaction was denatured at 94 ° C./3 minutes, followed by 5 cycles of 94 ° C./45 seconds, 50 ° C./60 seconds, 72 ° C./30 seconds, and then 94 ° C./45 seconds, 55 ° C./60 seconds, Eleven cycles of 72 ° C./30 seconds were performed.

  After the PCR reaction, agarose gel electrophoresis was performed to confirm the presence or absence of DNA amplification. Since amplification bands were observed in 3 out of 10 cells, it was confirmed that fNRBC could be detected from maternal peripheral blood using fluorescently labeled fragmented antibody (1) and BV421-labeled anti-CD45 antibody as labeled antibodies. It was done.

[Comparative Example 1]
In “(2) Labeling of cell population” in Example 1, the fluorescence-labeled fragmented antibody (1) was changed to FITC-labeled anti-CD71 antibody (BD biosciences, product number 555536), and the amount of this antibody added was changed to cell suspension. Detection of fNRBC was attempted in the same manner as in Example 1 except that the amount was 15 μL with respect to 100 μL of the turbid liquid. Since no amplification band was observed in all 10 cells, it was judged that fNRBC could not be detected from peripheral maternal blood.

Claims (9)

A detection method for detecting the rare cells from a biological sample containing rare cells and contaminating cells other than the rare cells,
Contacting the biological sample with a first labeled antibody in which an antibody against a protein present in the rare cell is labeled with a first labeling substance, and marking the cell with the first labeled antibody;
A second labeled antibody in which an antibody against a protein that is not present in the rare cell but present in the contaminated cell is labeled with a second labeling substance is brought into contact with the biological sample, and the cell is bound with the second labeled antibody. Marking process;
Detecting from the biological sample cells marked with the first labeled antibody and not marked with the second labeled antibody;
Have
The method for detecting a rare cell, wherein the antibody against the protein present in the rare cell is at least one selected from Fab antibody and Fab ′ antibody.   The method for detecting a rare cell according to claim 1, wherein the antibody against the protein present in the rare cell is a Fab 'antibody.   The detection of a rare cell according to claim 1 or 2, wherein the first labeled antibody is a labeled antibody in which the first labeling substance is covalently bound to a thiol group of an antibody against a protein present in the rare cell. Method. The rare cell is a nucleated cell, and
Contacting a nuclear staining dye with the biological sample to stain cell nuclei;
Detecting a cell having a nucleus stained with the nuclear staining dye from the biological sample;
The method for detecting a rare cell according to any one of claims 1 to 3, comprising:   The method for detecting a rare cell according to any one of claims 1 to 4, wherein the biological sample is a fraction containing the rare cell obtained by subjecting blood to density gradient centrifugation.   The method for detecting a rare cell according to any one of claims 1 to 5, wherein the biological sample is a biological sample derived from pregnant maternal peripheral blood, and the rare cells are fetal nucleated red blood cells.   The method for detecting a rare cell according to any one of claims 1 to 5, wherein the biological sample is a biological sample derived from peripheral blood of a cancer patient, and the rare cell is a circulating tumor cell in the blood.   The first labeling substance and the second labeling substance are chemical substances selected from fluorescent dyes, fluorescent proteins, enzymes, biotin, and magnetic beads, respectively. For detecting rare cells.   The method for detecting a rare cell according to any one of claims 1 to 8, wherein cells are detected from the biological sample by flow cytometry.