WO2023284795A1 - Method and kit for isolating fetal trophoblast cells - Google Patents

Abstract

A method and kit for isolating fetal trophoblast cells, in particular, the present invention relates to an antibody combination for isolating and purifying fetal trophoblast cells, said combination comprising antibodies for negative screening and/or antibodies for positive screening, and also relates to a method and kit for isolating fetal trophoblast cells. The method comprises the following steps: (1) obtaining a maternal tissue sample containing trophoblast cells; (2) preparing a single cell suspension of the tissue sample; (3) enriching fetal trophoblast cells by means of an immunomagnetic bead method; and (4) sorting fetal trophoblast cells by using a cell sorting instrument. The kit comprises the following reagent(s): (a) a reagent for enriching fetal trophoblast cells by using an immunomagnetic bead method; and/or (b) a reagent for sample washing.

Description

Method and kit for isolating fetal trophoblast cells technical field

The invention relates to a cell separation technology combining immunomagnetic bead enrichment with cell sorter sorting. Specifically, the present invention relates to a set of antibody combinations for isolating and purifying fetal trophoblast cells, including antibodies for negative screening and antibodies for positive screening. The invention also relates to a method for isolating fetal trophoblast cells in a maternal tissue sample and a kit suitable for the method.

Background technique

During the development of the embryo into a morula and further development, a kind of individual smaller cells that differentiate and expand and arrange along the inner wall of the zona pellucida begin to appear, which are trophoblast cells. During the development of the inner cell mass, the chorion is composed of trophoblast cells and the parietal layer of the extraembryonic mesoderm. With the embryonic development, the dense chorion and decidua basal together constitute the placenta, while the smooth chorion and decidua capsule gradually fuse with the parietal decidua. During this process, the exfoliated trophoblast cells (Extravillous trophoblast, EVT) enter the cervix.

Fetal trophoblast cells can be used as a source of fetal genetic material for noninvasive prenatal diagnosis ¹ . Therefore, purifying high-purity trophoblast cells has very important clinical application significance.

It has also been reported that trophoblast cells can be enriched from maternal blood ² ; however, the number of fetal trophoblast cells in maternal blood is very rare, about 10 ⁸ maternal cells contain 1 fetal trophoblast cell, isolated from maternal blood Fetal trophoblast cells are very difficult.

The experiment using the specific surface antibody anti-HLAG staining of trophoblast cells showed that the proportion of trophoblast cells in cervical scrape samples was about 1/2000 ³ . Currently, there are mainly two techniques for purifying trophoblast cells from cervical samples: microdissection and ^{microfluidics4,5,6} . However, the operation period of the two methods is very long, the cost is high, and the operation skills of equipment and experimenters are very high, and the feasibility of technology and clinical application has not been fully verified.

Sorting with a cell sorter is a routine and mature cell sorting technology, but due to the interference of a large number of background cells on specific cell staining, it is still very difficult to separate cells with a purity of less than 0.1% or even 1%. Even in the case of very specific antibodies, the separation purity by flow cytometry can only reach about 20% or even ^lower7 . Due to factors such as many cell debris in the cervical scrape sample and bacterial infection in the cervix itself, the purity of trophoblast cells in actual flow sorting may be much less than 1/2000. The interference of cervical mucus in cervical smear samples brings more difficulties to the purification of trophoblast cells. Therefore, before sorting with a cell sorter, it is very important to effectively enrich trophoblast cells in maternal tissue samples such as cervical samples.

The immunomagnetic bead method uses cell surface-specific antigen and antibody binding, and uses magnetic force to directly enrich target cells (positive selection) or remove non-target cells (negative selection). Positive screening can generally achieve an enrichment effect of several to several tens of ^times8 , while negative screening can generally achieve an enrichment effect of several to hundreds of ^times9 . HLAG is specifically expressed on trophoblast cells, and anti-HLAG can be used for positive screening of trophoblast cells. Positive selection using multiple antibodies will increase the yield of trophoblast cells. So far, there is no report on the enrichment of trophoblast cells by immunomagnetic bead negative selection method.

Contents of the invention

The purpose of the present invention is to solve the problems in the prior art that the separation of fetal trophoblast cells is complicated, the cycle is long, the cost is high, and the technical requirements for experimenters are high.

First, the present invention relates to an antibody combination comprising:

A) primary antibody; or

B) a second antibody; or

C) a first antibody and a second antibody; wherein,

The first antibody comprises one or more of anti-CD44, anti-CD45, anti-CD66c, anti-CD82, anti-CD114, anti-CD227 and anti-E-cadherin; preferably, the first The antibody contains two or more of anti-CD44, anti-CD45, anti-CD66c, anti-CD82, anti-CD114, anti-CD227 and anti-E-cadherin.

The second antibody comprises one or more of anti-HLAG, anti-EpCAM and anti-Trop2.

In some embodiments, the first antibody comprises one or more of anti-CD44, anti-CD45, anti-CD227, anti-CD66c. In a preferred embodiment, the first antibody comprises a combination of anti-CD44, anti-CD45 and anti-CD227; or a combination of anti-CD45 and anti-CD66c.

In some embodiments, the second antibody comprises anti-HLAG, anti-EpCAM, or a combination thereof. In preferred embodiments, said second antibody comprises anti-HLAG.

Another aspect of the present invention relates to a kit for isolating fetal trophoblast cells, which includes: (a) reagents for enriching fetal trophoblast cells by immunomagnetic bead method; wherein said enrichment of fetal trophoblast cells The reagent for laminar cells comprises any antibody combination according to the present invention. Preferably, the reagent (a) further includes immunomagnetic beads.

In some embodiments, the kit further includes (b) reagents for sample washing. Preferably, the reagent (b) is selected from PBS, HBS or a combination thereof; optionally, EDTA, BSA or a combination thereof is further included.

Another aspect of the present invention relates to a method for isolating fetal trophoblast cells, characterized in that it comprises the following steps:

(1) Provide maternal tissue samples containing fetal trophoblast cells;

(2) preparing a single cell suspension of the sample;

(3) Enrichment of fetal trophoblast cells by immunomagnetic bead method; and

(4) cell sorting fetal trophoblast cells; wherein,

In the described step (3), the immunomagnetic bead method comprises:

3.1) Negative screening step; or

3.2) a positive screening step; or

3.3) Negative screening step and positive screening step; Wherein,

The negative screening step includes: incubating negative screening antibodies and magnetic beads to remove non-trophoblast cells; the positive screening step includes: incubating positive screening antibodies and magnetic beads to enrich fetal trophoblast cells.

In some embodiments, the negative screening antibody is selected from any one of the above-mentioned first antibodies according to the present invention; the positive screening antibody is selected from the above-mentioned second antibodies according to the present invention.

In a specific embodiment, in the immunomagnetic bead method described above, the incubation includes the following steps: adding antibodies for incubation, and then incubating the magnetic beads; or directly adding antibody-magnetic bead complexes for incubation.

According to the method for isolating fetal trophoblast cells described herein, wherein said maternal tissue sample is selected from a cervical sample.

According to the method described herein, the cell sorting uses a cell sorter or a cell sorting column, preferably a flow cytometric sorter or a cell sorter based on a microfluidic method.

In another aspect of the present invention, the product obtained according to the method described above of the present invention.

Another aspect of the present invention relates to the use of any antibody combination as described herein for isolating fetal trophoblast cells.

The invention provides an antibody combination for isolating and purifying fetal trophoblast cells in maternal tissue samples, including antibodies for negative screening and antibodies for positive screening. As shown in Figure 1, first obtain (5-20 gestational weeks) maternal tissue samples (preferably such as cervical samples), and prepare a single cell suspension; then use the immunomagnetic bead method to perform a negative screening step to remove the non-trophoblast layer in the sample Cells, such as cervical epithelial cells or inflammatory cells, etc.; and/or a positive screening step to further enrich the fetal trophoblast cells; finally, the purpose of obtaining fetal trophoblast cells by sorting is achieved by a cell sorter.

Although the combinations and methods described herein specifically refer to human mothers and human fetuses, they are not limited to humans, and fetal cells of other species can be similarly isolated and analyzed.

A method or product according to aspects of the present invention, comprising obtaining a maternal cervical sample containing fetal extravillous trophoblast cells from a subject, isolating fetal trophoblast cells therefrom, and lysing the isolated fetal trophoblast cells.

In the prior art, fetal trophoblast cells are separated by immunomagnetic bead purification. Usually, maternal cervical samples are taken and incubated with PE-HLAG antibodies. PE-beads specifically bind to antibodies, and then the samples incubated with antibodies and magnetic beads are subjected to cell sorting. Under the action of an external magnetic field, the target cells will be adsorbed to the sorting column to achieve the purpose of enriching fetal trophoblast cells, but this method takes a long time, and the secondary antibodies on the surface of the magnetic beads and the cells in the sample are non-specific Combined, the accuracy of detection is reduced, and the enrichment efficiency is low; and the method of sample fixation also has a great impact on the enrichment of fetal trophoblast cells.

The inventors of the present invention have unexpectedly discovered that a "negative selection" antibody that can specifically bind to surface antigens of a large number of negative cells (such as cervical epithelial cells or inflammatory cells) in a cervical sample can be utilized by a "negative selection" method, and Incubation of the preliminarily processed cervical sample to sort out the large number of negative cells (ie, non-trophoblast cells) and remove the sorted cells from the sample can significantly improve the "fetal trophoblast" in the remaining sample. "Proportion of cells: the concentration in the sample can be enriched to more than 40 times only by negative screening alone; and the positive screening enrichment efficiency can be improved by combining positive screening (positive screening can enrich the concentration to 8 times) Above), the two-step screening combination can enrich the concentration to more than 370 times, far exceeding the immunomagnetic bead method of the prior art (which can only be enriched to about 5.8 times).

Thus, according to the first aspect, the present invention relates to an antibody combination that can be used to isolate and purify fetal trophoblast cells (such as from maternal cervical samples); it is characterized in that the antibody combination comprises:

A) primary antibody; or

B) a second antibody; or

C) Primary and secondary antibodies.

Wherein, the first antibody can be used in the negative screening step; the second antibody can be used in the positive screening step.

As used herein, the term "antibody" refers to a functional component of serum and is generally considered to be a collection of molecules (antibody or immunoglobulin, or a single molecule (antibody molecule or immunoglobulin molecule). Antibody molecules can Combine or react with a specific antigenic determinant (antigen or antigenic epitope) to trigger an immune effect. Each antibody molecule is usually specific, and the composition of antibody molecules can be monoclonal antibodies (that is, composed of the same antibody molecules) or polyclonal antibodies Clonal antibody (that is, composed of two or more different antibody molecules that bind or react with the same or different epitopes on the same antigen or even different antigens). Each antibody molecule has a specificity that enables it to be specific to the corresponding antigen Antibodies are also collectively referred to as immunoglobulins. "Antibody" as used herein not only covers complete antibody molecules, but also antibody fragments and variants thereof (such as derivative structures). As used herein, "antibody" also includes : chimeric and single chain antibodies; binding fragments of antibodies, such as Fab, Fv fragments or scFv fragments; and multimeric antibodies, such as dimers (such as dimeric IgA) or pentamers (such as pentameric IgM). Antibodies can be human, murine, chimeric, humanized or reshaped antibodies.

As used herein, antibodies of the present invention, such as anti-CD44, anti-CD45, anti-CD66c, anti-CD82, anti-CD114, anti-CD227, anti-E-cadherin, anti-HLAG, anti-EpCAM and anti- Trop2 is any antibody molecule or active fragment thereof that can recognize and bind to the above-mentioned corresponding antigens (ie, CD44, CD45, CD66c, CD82, CD114, CD227, E-cadherin, HLAG, EpCAM and Trop2); herein, the above-mentioned An antibody is not limited or intended to be limited to one or a specific antibody or fragment thereof to the corresponding antigen.

As used herein, the terms "comprising", "comprising" or "containing", "having" or similar expressions thereof, are non-exclusive inclusions. For example, a combination, step, method, article, or device comprising listed elements is not necessarily limited to the corresponding elements, and may include other elements not explicitly listed or elements inherent to the combination, step, method, article, or apparatus. The term "consisting of" excludes any unlisted elements, steps or components. When used in a claim, it will make the claim closed, ie containing no material other than the stated material except for the customary impurities associated therewith. When the phrase "consisting of" appears in a clause of the subject of a claim rather than immediately following the subject matter, it only defines the elements described in that clause; other elements are not excluded from the claims as a whole. beyond the claims. Unless otherwise stated or defined, the schemes, combinations, methods, products, etc. referred to in the present invention as "comprising", "comprising" or "comprising", "having" all include schemes, combinations consisting of the listed elements , method, product.

As used herein, the term "combination" refers to a fixed combination in the form of a dosage unit, e.g., wherein an antibody of the invention and its combined "partner" (e.g., another antibody of the invention) are administered separately or within a certain time interval. use. The individual components (antibodies) can be packaged together in the kit or packaged separately, or mixed. One or more components (antibodies) may be formulated or diluted to the desired dosage prior to use. Terms such as "combined use" or "combined use" as described herein include simultaneous, separate or sequential use of selected antibodies and combined antibodies.

As used herein, the term "antibody combination" refers to a product resulting from the association of more than one antibody; it includes fixed or non-fixed combinations of antibodies.

"Fixed combination" refers to an antibody (such as a component of a primary antibody) and antibodies in combination (such as another component of a primary antibody), both used as separate entities or doses simultaneously or in admixture (such as for negative screening step).

"Non-fixed combination" refers to antibodies (such as primary antibodies (single or multiple components)) and antibodies in combination (such as secondary antibodies (single or multiple components)), both as separate Entities or doses, used sequentially or sequentially (eg for negative and positive selection steps, respectively).

Further, the above-mentioned "use" is also applicable to the combination of multiple antibodies, for example, the use of three or more antibodies, including the simultaneous use of multiple antibodies in one step (such as the simultaneous use of two or more negative screening antibodies for the negative selection step), and the sequential use of multiple antibodies in different steps (eg, using three negative selection antibodies for the negative selection step and two positive selection antibodies for the positive selection step).

As used herein, unless otherwise stated, "a", "an" and "the" do not specifically refer to one or a certain kind, and "plural forms" are included. For example, "an antibody" or "an antibody" may encompass a plurality of antibodies including mixtures thereof.

The term "one or more" refers to one, one or more in the modified target population; specifically, "one or more" herein refers to 1 of the antibodies , 2, 3, 4, 5, 6, 7, 8, 9 or 10 types.

In the context of the present invention, "negative screening" means that by incubating negative cell surface markers in the sample and binding to their corresponding magnetic beads, the sample is passed through, for example, a cell sorting column, and only the group that is not bound by the sorting column is collected. To achieve the purpose of removing a large number of negative cells (ie, non-trophoblast cells) and increase the proportion of trophoblast cells in the sample.

In the context of the present invention, "positive screening" means that by incubating positive cell surface markers in the sample and binding to their corresponding magnetic beads, the sample is passed through, for example, a cell sorting column, and the positive cells bound by the specific sorting column are collected. Cells, namely fetal trophoblast cells, achieve the purpose of enrichment.

In a specific embodiment, the negative screening step includes: incubating negative screening antibodies and magnetic beads to remove non-trophoblast cells; the positive screening step includes: incubating positive screening antibodies and magnetic beads to enrich fetal trophoblast cells. Wherein, the negative screening antibody is the first antibody described above in the present invention, and the positive screening antibody is the second antibody described above in the present invention.

In some embodiments, the first antibody is selected from one or more of anti-CD44, anti-CD45, anti-CD66c, anti-CD82, anti-CD114, anti-CD227 and anti-E-cadherin; Preferably, the first antibody is selected from two, three or more of anti-CD44, anti-CD45, anti-CD66c, anti-CD82, anti-CD114, anti-CD227 and anti-E-cadherin.

The second antibody is selected from one or more of anti-HLAG, anti-EpCAM and anti-Trop2.

In a preferred embodiment, the first antibody used for negative screening is selected from one, two, three or more of anti-CD44, anti-CD45, anti-CD227, and anti-CD66c; further , the first antibody is selected from the combination of anti-CD44, anti-CD45 and anti-CD227; or the first antibody is selected from the combination of anti-CD45 and anti-CD66c.

In a preferred embodiment, wherein the second antibody used for positive screening comprises anti-HLAG, anti-EpCAM or a combination thereof; further, the second antibody comprises anti-HLAG.

According to another aspect of the present invention, the present invention also relates to a method for isolating fetal trophoblast cells, the method comprising the following steps:

(1) obtaining a maternal tissue sample containing fetal trophoblast cells;

(2) preparing a single cell suspension of the tissue sample;

(3) Enrichment of fetal trophoblast cells by immunomagnetic bead method;

(4) Cell sorting of fetal trophoblast cells.

In some embodiments, in the method, the maternal tissue sample is from a mother at 5-20 weeks of gestation; preferably, from a mother at 5-12 weeks of gestation.

In some embodiments, the maternal tissue sample is selected from a cervical sample.

In some embodiments, in the step (2), the single cell suspension of the sample is prepared by first resuspending the obtained sample in the TCT sample cell preservation solution of Xinpai; filtering the sample with a 100 μm cell sieve, and centrifuging , discard the supernatant, wash several times with sample wash reagent, and resuspend in the wash reagent. The centrifugation process is preferably performed at 400 g for 5-10 min; the sample cleaning reagent is preferably PBS or HBS, more preferably PBS; optionally, EDTA and/or BSA can also be added to the reagent.

In some embodiments, the immunomagnetic bead method comprises:

a) a negative screening step; or

b) a positive screening step; or

c) a negative screening step and a positive screening step; wherein, in a preferred embodiment, the immunomagnetic bead method includes a negative screening step and a positive screening step.

As described herein, in the step of enriching fetal trophoblast cells, the "negative selection" step or the "positive selection" step can be performed independently, that is, only the "negative selection" step or only the "positive selection" step is performed ; or sequentially, said sequentially refers to the first "negative screening" and then "positive screening", or first "positive screening" and then "negative screening".

In a specific embodiment, in the above immunomagnetic bead method, the negative screening step includes: incubating the negative screening antibody and magnetic beads to remove non-trophoblast cells; the positive screening step includes: incubating the positive screening antibody and magnetic beads Beads, enriched for trophoblast cells.

In a preferred embodiment, in the immunomagnetic bead method described therein, the incubation step includes adding an antibody for incubation, and then incubating the magnetic beads; or directly adding the antibody-magnetic bead complex for incubation.

In a preferred embodiment, the incubation process of the present invention is incubation in the dark; the temperature range of antibody incubation is 4°C-25°C; the incubation time range is 30min-120min.

In this article, the term "immunomagnetic bead (IMB)" is also called biomagnetic beads or magnetic beads. "Immunomagnetic separation (IMS)" refers to a method for cell sorting, such as binding antibodies to magnetic microspheres or the surface of magnetic spheres through a coupling reaction to form immunomagnetic microspheres (such as That is, the antibody-magnetic bead coupling complex described herein); the antibody coated on the surface of the magnetic bead undergoes an antigen-antibody reaction, and an "antigen-antibody-magnetic bead" immune complex is formed on the cell surface. When the cells bound to the magnetic beads are placed under a strong magnetic field, they will move in a direction, so that the immune complexes can be separated from other unbound cells. When the magnetic beads leave the magnetic field, the magnetism disappears immediately, so as to achieve the purpose of positive or negative selection of specific cells. The use of immunomagnetic bead sorting to separate target cells from the complex environment of biological samples requires specific biomarkers, good stability and dispersion, and no aggregation. At the same time, the particle size of the immunomagnetic beads should not be too large, otherwise it will compress the target cells. In this paper, the immunomagnetic beads, for example, use nano-magnetic materials as a solid phase carrier, and add a polymer coating layer on the surface to introduce active functional groups (such as carboxyl, amino, mercapto, aldehyde, hydroxyl, etc.). The immunomagnetic beads have a small particle size, nanometer level, and a large specific surface area, which can capture more analytes. In the present invention, it is more conducive to the sorting of subsequent cells.

The "antibody-magnetic bead coupling complex" described above can be obtained by adding a coupling agent and an antibody that can specifically capture the corresponding cells to the immunomagnetic beads and incubating; For magnetic beads, the antibody-coupled immunomagnetic beads (ie, antibody-magnetic bead complex) are stored in a preservation solution for later use. Specifically, in the present invention, "antibodies capable of specifically capturing corresponding cells" include negative screening antibodies that can specifically capture "non-trophoblast cells" in the negative screening step, and negative screening antibodies that can specifically capture "non-trophoblast cells" in the positive screening step. Positive screening antibody that captures "fetal trophoblast cells".

In this paper, it is also possible to first add antibodies to the sample for incubation, and then add immunomagnetic beads and corresponding coupling reagents for incubation to realize the coupling of antibodies and magnetic beads. The specific size and type of the immunomagnetic beads can be selected according to the actual situation. Preferably, the immunomagnetic beads described herein can be selected from amino magnetic beads, carboxyl magnetic beads, epoxy-based magnetic beads, silicon-based magnetic beads, tosyl-based magnetic beads, superparamagnetic microbeads composed of polysaccharides and iron oxide. Beads; wherein superparamagnetic microbeads composed of polysaccharides and iron oxide are preferred, such as German Miltenyi cell sorting magnetic beads (for example, Anti-APC MicroBeads or Anti-PE Microbeads). The particle size range of the magnetic beads is preferably: 50nm-2000nm, more preferably 50-800nm, further preferably 50-200nm, most preferably 50nm. The coupling agent is a coupling agent corresponding to the type of immunomagnetic beads, and the coupling method is consistent with the type of immunomagnetic beads purchased.

In a preferred embodiment, the negative screening antibody is selected from the primary antibody of the present invention. Specifically, the first antibody is selected from one or more of anti-CD44, anti-CD45, anti-CD66c, anti-CD82, anti-CD114, anti-CD227 and anti-E-cadherin; preferably, The first antibody is selected from two, three or more of anti-CD44, anti-CD45, anti-CD66c, anti-CD82, anti-CD114, anti-CD227 and anti-E-cadherin; more preferably, One, two, three or more selected from anti-CD44, anti-CD45, anti-CD227, anti-CD66c; further preferably, the first antibody is selected from anti-CD44, anti-CD45 and A combination of anti-CD227; or a combination selected from anti-CD45 and anti-CD66c.

As used herein, the above-mentioned antibodies used for negative screening are just examples, which can achieve sorting and separation by specifically binding to the surface antigen of negative cells (ie, non-trophoblast cells) in maternal cervical samples. Therefore, it should be understood that for any other surface antigens of negative cells contained in the maternal sample, or other surface Antigens can also be used as cell markers, and antibodies that can specifically bind to the above markers can also be used as primary antibodies for negative screening.

In a preferred embodiment, the positive screening antibody is selected from the second antibody described in the present invention; specifically, the second antibody is selected from one or more of anti-HLAG, anti-EpCAM and anti-Trop2 kind. Preferably, selected from anti-HLAG, anti-EpCAM or a combination thereof. Further preferably, selected from anti-HLAG.

As used herein, the term "cell sorting" refers to a method for separating cells according to their type and/or characteristics. Typically, cells are screened and isolated based on differences in cell size, morphology, and/or expression of surface proteins or markers. Cell sorting can rely on different strategies known to those skilled in the art, such as single cell sorting, fluorescent cell sorting, magnetic cell sorting or buoyancy activated cell sorting. Specifically, in the cell sorting methods described herein, cells bound to the antibody are sorted from unbound cells.

In a specific embodiment, the cell sorting step is performed using a cell sorting column; further, during the sorting process of the cell sorting column, the sorting column is placed in a magnetic frame, and the cells are washed with a buffer Separation column, the sample is added to the washed cell separation column, the sample flows through the separation column under the action of gravity, the target cells bound to the magnetic beads are adsorbed to the separation column by the magnetic force, and the unbound sample or Sample bound to separation column.

Alternatively, the cell sorting step in the present invention can be performed using a cell sorter, wherein preferably, the cell sorter is selected from a flow cytometer and a cell sorter based on a microfluidic method. More preferably, a flow cytometer is used.

According to another aspect of the present invention, the present invention also relates to a kit for isolating fetal trophoblast cells, characterized in that the kit includes:

(a) Reagents used for enrichment of fetal trophoblast cells by immunomagnetic bead method.

Wherein, the reagent (a) used for immunomagnetic bead method to enrich fetal trophoblast cells comprises: an antibody used for negative screening; or an antibody used for positive screening; or an antibody used for negative screening and an antibody used for positive screening Combinations of antibodies screened.

In a specific embodiment, the negative screening antibody is selected from the first antibody described in the present invention. Specifically, the first antibody is selected from one or more of anti-CD44, anti-CD45, anti-CD66c, anti-CD82, anti-CD114, anti-CD227 and anti-E-cadherin; preferably, The first antibody is selected from two, three or more of anti-CD44, anti-CD45, anti-CD66c, anti-CD82, anti-CD114, anti-CD227 and anti-E-cadherin; more preferably, One, two, three or more selected from anti-CD44, anti-CD45, anti-CD227, anti-CD66c; further preferably, the first antibody is selected from anti-CD44, anti-CD45 and A combination of anti-CD227; or a combination selected from anti-CD45 and anti-CD66c.

In a preferred embodiment, the positive screening antibody is selected from the second antibody of the present invention. Specifically, the second antibody is selected from one or more of anti-HLAG, anti-EpCAM and anti-Trop2; preferably, selected from anti-HLAG, anti-EpCAM or a combination thereof; further preferably, selected from Self-anti-HLAG.

In some embodiments, the reagent (a) further includes immunomagnetic beads. In some preferred embodiments, the antibody and the immunomagnetic beads in the reagent (a) are stored separately and not coupled together; in other preferred experimental schemes, the antibody and the magnetic beads are coupled together to form an antibody - Magnetic bead complex.

Further, the kit described herein also includes (b) reagents for sample cleaning. In some embodiments, the reagent (b) used for sample washing is selected from PBS, HBS or a combination thereof, preferably selected from PBS. Optionally, the reagent (b) further includes EDTA, BSA or a combination thereof.

According to another aspect of the present invention, it also relates to the product obtained by the method described above in the present invention.

According to another aspect of the present invention, the present invention also relates to the use of the antibody combination described above for isolating fetal trophoblast cells.

In some embodiments, the isolating is, isolating from a maternal tissue sample containing fetal trophoblast cells; further, isolating from a maternal cervix sample; more preferably, isolating from a maternal cervix sample.

In some embodiments, the isolating is from a maternal cervical sample at 5-20 weeks' gestation; preferably, from a maternal cervical sample at 5-12 weeks' gestation.

The excellent technical effects of the method and kit of the present invention mainly lie in the following aspects:

(1) The purification process is rigorous. A large number of negative cells in cervical samples, including cervical epithelial cells and inflammatory cells, are removed by negative screening; subsequent positive screening can be used to further enrich the proportion of target trophoblast cells, which is beneficial to the purification of cell sorters to obtain trophoblast cells;

(2) It can be applied to real cervical smear samples. In real cervical scraping samples, the purity of trophoblast cells obtained by the method of the present invention can reach 48%;

(3) The operation process is simple, and multi-sample automatic operation can be realized;

(4) Wide range of uses. The cell sorter can realize multi-cell sorting and single-cell sorting, and operators can choose flexibly according to subsequent needs.

Description of drawings

Fig. 1 is a flowchart of purifying trophoblast cells from cervical samples according to the method of the present invention.

Fig. 2 is the result of staining and flow cytometric analysis of the trophoblast cell line JEG3 and cervical samples by using the negative screening antibody according to the method in Example 1.

Fig. 3 is the result of staining and flow cytometric analysis of the trophoblast cell line JEG3 and cervical samples by using the positive screening antibody according to the method in Example 1.

Fig. 4 is a verification of the efficiency of JEG3 incorporation of trophoblast cells in cervical samples by magnetic bead positive screening (4A) and negative screening combined with positive screening (4B and 4C) according to the method in Example 2.

Fig. 5 is a graph showing the results of purifying a single trophoblast cell from a real cervical sample and performing high-throughput sequencing verification according to the method of the present invention.

detailed description

The content of the present invention can be more easily understood in conjunction with the following preferred embodiments of the present invention. Unless otherwise specified, all techniques and terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Unless otherwise indicated, the techniques employed and covered herein are standard methods well known to those of skill in the art to which the invention pertains. The materials, methods, and examples are presented for purposes of illustration only and do not limit the scope of the invention in any way.

Example 1: Using the antibody of the present invention to stain human cervical epithelial cell line HcerEpic, human chorionic trophoblast cell line JEG3 and cervical scraping samples

Step 1: Prepare Single Cell Suspension

The cultured HcerEpic and JEG3 cell lines were digested with 0.25% trypsin, a single cell suspension was prepared according to 5× ^{10 5} cells/ml, and placed in PBS solution, and 100 μl cells were taken for each staining reaction.

Use a cell brush to take a maternal cervical scrape sample at 5-20 weeks of pregnancy, and resuspend it in 20ml of TCT sample cell preservation solution. Filter the sample with a 100 μm cell sieve, centrifuge at 400 g for 5 min, discard the supernatant and wash twice with PBS, then resuspend in 5 ml of PBS, and take 100 μl of cells for each staining reaction.

Step 2: Flow Cytometry Antibody Staining Analysis

Add 5 μl of antibody to 100 μl of cells, and incubate in the refrigerator for 30 minutes in the dark. Centrifuge at 400g for 5min, discard the supernatant and wash twice with PBS. The cells were resuspended in 300 μl PBS and the antibody efficiency was detected by flow cytometry.

Step 3: Analysis of flow cytometry antibody staining results

The results of flow cytometry negative screening antibody and positive screening antibody are shown in Figure 2 and Figure 3, respectively. The staining efficiencies of different antibodies in different cells compared to the blank control are summarized in Table 1. Antibody criteria that can be used for negative screening are positive staining in cervical smear samples, negative staining in JEG3 cells, or much higher staining efficiency in cervical smear samples than JEG3 cells. The standard that can be used for positive screening antibody is more than 80% positive staining in JEG3 cells, and basically negative staining in cervical smear samples.

Table 1: List of negative screening and positive screening antibodies of the method of the present invention and their staining efficiency statistics in trophoblast cells JEG3 and cervical samples.

Example 2: Using the immunomagnetic bead method of the present invention to enrich JEG3 trophoblast cells incorporated in cervical samples

Step 1: Preparation of cervical sample and JEG3 cell suspension

According to the method of Example 1, cervical samples and JEG3 single cell suspensions were respectively prepared and counted under a microscope. Take the JEG3 single-cell suspension, mix it into the cervical sample single-cell suspension at a ratio of 1:1000 and mix well.

Step 2: Positive screening of enriched JEG3 cells by immunomagnetic bead method

(1) Take 1ml of mixed cells, add 20μl anti-HLAG antibody, and incubate at 4°C for 1h in the dark.

(2) The sample was washed twice with 1ml of PBS solution, centrifuged at 400g for 5min at 4°C, and resuspended in 250μl of PBS solution.

(3) Add 20 μl of magnetic beads (Miltenyi Cell Sorting Magnetic Beads Anti-PE MicroBeads) to the sample, and incubate at 4°C in the dark for 30 min.

(4) Add the sample to the Miltenyi MS cell separation column after washing with PBS, and collect the bound cells on the separation column.

(5) The purity of the enriched JEG3 cells was analyzed by flow cytometry.

Step 3: Enrich JEG3 cells by immunomagnetic bead negative selection combined with positive selection

(1) Take 1ml of mixed cells, add negative selection antibody combination 1 anti-CD44+anti-CD45+anti-CD227, or negative selection antibody combination 2 anti-CD45+anti-CD66c, and incubate at 4°C in the dark. 1h.

(2) The sample was washed twice with 1ml of PBS solution, centrifuged at 400g for 5min at 4°C, and the sample was resuspended with 1ml of PBS solution.

(3) Add 100 μl of magnetic beads (Miltenyi Cell Sorting Magnetic Beads Anti-APC MicroBeads) to the sample, and incubate at 4°C in the dark for 30 min.

(4) Add the sample to the Miltenyi LD cell separation column after washing with PBS, and collect the flow-through after the column is over-separated.

(5) Centrifuge at 400g for 5min at 4°C, and resuspend the sample with 250μl PBS solution. Add 5 μl anti-HLAG antibody, and incubate for 1 h at 4°C in the dark.

(6) The sample was washed twice with 1ml of PBS solution, centrifuged at 400g for 5min at 4°C, and resuspended in 250μl of PBS solution.

(7) Add 20 μl of magnetic beads (Miltenyi Cell Sorting Magnetic Beads Anti-PE MicroBeads) to the sample, and incubate at 4°C in the dark for 30 min.

(8) Add the sample to the Miltenyi MS cell separation column after washing with PBS, and collect the bound cells on the separation column.

(9) The purity of the enriched JEG3 cells was analyzed by flow cytometry.

Step 4: Analyze the enrichment effect of JEG3 trophoblast cells by flow cytometry

The efficiency of enriching JEG3 trophoblast cells by the immunomagnetic bead method was analyzed by flow cytometry, and the results are shown in FIG. 4 . The initial ratio of JEG3 was 0.11%, and after anti-HLAG positive screening, the concentration was increased to 0.64%, enriched by 5.8 times (A in Figure 4). After anti-CD44+anti-CD45+anti-CD227 negative screening, the concentration was 4.4%, enriched 40 times, combined with anti-HLAG positive screening, the concentration was increased to 41.3%, a total enrichment of 375.5 times (B in Figure 4 ). After anti-CD45+anti-CD66c negative screening, the concentration was 6.3%, enriched 57.3 times, combined with anti-HLAG positive screening, the concentration increased to 51.6%, a total of 469.1 times enriched (C in Figure 4). These results indicate that negative selection using the antibody combination anti-CD44+anti-CD45+anti-CD227 or anti-CD45+anti-CD66c can obtain a good effect of enriching trophoblast cells; further positive selection combined with anti-HLAG can Significantly improve the enrichment efficiency.

Example 3: Utilizing the method of the present invention to purify trophoblast cells in cervical samples

Step 1: Obtain a cervical sample containing trophoblast cells

Use a cell brush to take a maternal cervical scrape sample at 5-20 weeks of pregnancy, and resuspend it in 20ml of TCT sample cell preservation solution.

Step 2: Prepare cervical sample single cell suspension

Filter the sample with a 100 μm cell sieve, centrifuge at 400 g for 5 min, discard the supernatant, wash twice with PBS, and resuspend in 1 ml of PBS.

Step 3: Enrichment of fetal trophoblast cells by immunomagnetic bead method

(1) Add the negative selection antibody combination anti-CD44+anti-CD45+anti-CD227 to the cervical cells resuspended in 1ml PBS, and incubate for 1 hour at 4°C in the dark.

(2) The sample was washed twice with 1ml of PBS solution, centrifuged at 400g for 5min at 4°C, and the sample was resuspended with 1ml of PBS solution.

(3) Add 100 μl of magnetic beads (Miltenyi Cell Sorting Magnetic Beads Anti-APC MicroBeads) to the sample, and incubate at 4°C in the dark for 30 min.

(4) Add the sample to the Miltenyi LD cell separation column after washing with PBS, and collect the flow-through after the column is over-separated.

(5) Centrifuge at 400g for 5min at 4°C, and resuspend the sample with 250μl PBS solution. Add 5 μl anti-HLAG antibody, and incubate for 1 h at 4°C in the dark.

(6) The sample was washed twice with 1ml of PBS solution, centrifuged at 400g for 5min at 4°C, and resuspended in 250μl of PBS solution.

(7) Add 20 μl of magnetic beads (Miltenyi Cell Sorting Magnetic Beads Anti-PE MicroBeads) to the sample, and incubate at 4°C in the dark for 30 min.

(8) Add the sample to the Miltenyi MS cell separation column after washing with PBS, and collect the bound cells on the separation column.

Step 4: Cell sorter sorts fetal trophoblast cells

The HLAG-positive cells were enriched by the flow cytometry gate magnetic bead method, and a single cell was collected into a PCR tube for subsequent Y chromosome identification.

Example 4: Detection and purity identification of trophoblast cells

Three 12-week-gestational cervical smear samples of male fetuses verified by NIPT were collected from Peking Union Medical College Hospital. Referring to the steps of magnetic bead enrichment combined with flow cytometry sorting in Example 3, a single trophoblast cell was obtained. Using Keyunan PGT-A Preimplantation Chromosomal Aneuploidy Detection Kit (Beijing Berry Hekang Biotechnology Co., Ltd.) The ratio of reads of the X chromosome to the ratio of the reads of the Y chromosome was analyzed to determine whether the purified single cells still had the Y chromosome. If it contains a Y chromosome, it indicates that the cell is a fetal trophoblast and not derived from a maternal background. Representative sequencing results are shown in the Manhattan diagram of Figure 5. Male control cells contain 1 copy of X chromosome and 1 copy of Y chromosome at the same time, and female control cells contain 2 copies of X chromosome.

The purity identification of trophoblast cells is shown in Table 2. Sample #1 was purified to obtain 81 single cells, 39 of which were trophoblast cells, with a purity of 48.2%; sample #2 was purified to obtain 124 single cells, of which 60 were trophoblast cells cells, with a purity of 48.4%; sample #3 was purified to obtain 60 single cells, 30 of which were trophoblast cells, with a purity of 50%; the average purity of trophoblast cells obtained from the three samples was 48.9%. These results indicate that trophoblast cells can be enriched from cervical smear samples using the method described in the invention.

Table 2: Purification of three cases of cervical smear samples using the method of the present invention, the statistics of the number of cells obtained and the purity of trophoblast cells.

It should be noted that although some features of the present invention have been clarified through the above examples, they cannot be used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. The antibody combinations, reaction reagents, and reaction conditions involved in the enrichment of fetal trophoblast cells by the immunomagnetic bead method can be adjusted and changed according to specific needs. Therefore, for those skilled in the art, without departing from the idea and principle of the present invention, some simple replacements can also be made, and these should be included in the protection scope of the present invention.

references

[1] Farideh Z Bischoff, Joe Leigh Simpson. Endocervical fetal trophoblast for prenatal genetic diagnosis. Curr Opin Obstet Gynecol. 2006 Apr; 18(2):216-20.

[2] Xinming Zhuo, Qun Wang, Liesbeth Vossaert, Roseen Salman, Adriel Kim, Ignatia Van den Veyver, Amy Breman, Arthur Beaudet. Use of amplicon-based sequencing for testing fetal identity and monogenic traits with Single Circulating as CT one form of cell-based NIPT.PLOS ONE.2021 April; 16(4):e0249695.

[3]Anthony N Imudia, Yoko Suzuki, Brian A Kilburn, Frank D Yelian, Michael P Diamond, Roberto Romero, D Randall Armant. Retrieval of trophoblast cells from the cervical canal for prediction proof of abnormal pregnancy. 2009Sep;24(9):2086-92.

[4] Chung-Er Huang, Gwo-Chin Ma, Hei-Jen Jou, Wen-Hsiang Lin, Dong-Jay Lee, Yi-Shing Lin, Norman A Ginsberg, Hsin-Fu Chen, Frank Mau-Chung Chang, Ming Chen .Noninvasive prenatal diagnosis of fetal aneuploidy by circulating fetal nucleated red blood cells and extravillous trophoblasts using silicon-based nanostructured microfluidics. Mol Cytogenet. 2017 Dec 2; 10:44.

[5]Christina M Bailey-Hytholt,Sumaiya Sayeed,Morey Kraus,Richard Joseph,Anita Shukla,Anubhav Tripathi.A Rapid Method for Label-Free Enrichment of Rare Trophoblast Cells from Cervical Samples Sci Rep.2019 (1 Aug ) :12115.

[6]Yifang Huang,Bo Situ,Liping Huang,Yingsi Cao,Hong Sui,Xinyi Ye,Xiujuan Jiang,Aifen Liang,Maliang Tao,Shihua Luo,Ye Zhang,Mei Zhong,Lei Zheng.Nondestructive Identification of Rare Trophoblastic Cells by Endoplasmic Reticulum Staining for Noninvasive Prenatal Testing of Monogenic Diseases. Adv Sci(Weinh). 2020 Feb 13;7(7):1903354.

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Claims (16)

1. Antibody combination, characterized in that it comprises:

   A) primary antibody; or

   B) a second antibody; or

   C) a first antibody and a second antibody; wherein,

   The first antibody comprises one or more of anti-CD44, anti-CD45, anti-CD66c, anti-CD82, anti-CD114, anti-CD227 and anti-E-cadherin;

   The second antibody comprises one or more of anti-HLAG, anti-EpCAM and anti-Trop2.
2. The antibody combination according to claim 1, wherein the first antibody comprises one or more of anti-CD44, anti-CD45, anti-CD227, and anti-CD66c.
3. The antibody combination according to claim 2, wherein the first antibody comprises a combination of anti-CD44, anti-CD45 and anti-CD227; or a combination of anti-CD45 and anti-CD66c.
4. The antibody combination according to any one of claims 1-3, wherein the second antibody comprises anti-HLAG, anti-EpCAM or a combination thereof.
5. The antibody combination according to any one of claims 1-3, wherein the second antibody comprises anti-HLAG.
6. A kit for isolating fetal trophoblast cells, characterized in that it comprises:

   (a) a reagent for enriching fetal trophoblast cells by immunomagnetic bead method; wherein,

   The reagent for enriching fetal trophoblast cells comprises the antibody combination according to any one of claims 1-5.
7. The kit according to claim 6, wherein said reagent (a) further comprises immunomagnetic beads.
8. The kit according to claim 6 or 7, wherein the kit further comprises (b) reagents for sample cleaning.
9. The kit according to claim 8, wherein the reagent (b) is selected from PBS, HBS or a combination thereof; optionally, further comprising EDTA, BSA or a combination thereof.
10. The method for isolating fetal trophoblast cells is characterized in that it comprises the following steps:

    (1) Provide maternal tissue samples containing fetal trophoblast cells;

    (2) preparing a single cell suspension of the sample;

    (3) Enrichment of fetal trophoblast cells by immunomagnetic bead method; and

    (4) cell sorting fetal trophoblast cells; wherein,

    Described step (3) immunomagnetic bead method comprises:

    3.1) Negative screening step; or

    3.2) a positive screening step; or

    3.3) Negative screening step and positive screening step; Wherein,

    The negative screening step includes: incubating negative screening antibodies and magnetic beads to remove non-trophoblast cells;

    The positive screening step includes: incubating positive screening antibodies and magnetic beads to enrich fetal trophoblast cells.
11. The method according to claim 10, characterized in that,

    The negative screening antibody is selected from the first antibody described in any one of claims 1-5; the positive screening antibody is selected from the second antibody described in any one of claims 1-5.
12. The method according to claim 10 or 11, wherein, in the immunomagnetic bead method, the incubation comprises the steps of:

    Add antibody to incubate, then incubate beads; or,

    Add antibody-magnetic bead complexes directly for incubation.
13. The method according to claim 10 or 11, wherein the maternal tissue sample in step (1) is selected from a cervical sample.
14. The method according to claim 10 or 11, wherein said step (4) cell sorting adopts a cell sorter or a cell sorter column, preferably a flow cytometer or a cell sorter based on a microfluidic method .
15. The product obtained by the method according to any one of claims 10-14.
16. Use of the antibody combination according to any one of claims 1-5 for isolating fetal trophoblast cells.

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