Compositions and methods for isolating, detecting and analyzing fetal cells

Abstract

Compositions, kits, and methods for isolating, detecting, and analyzing fetal cells are provided. Methods for preparing a fetal cell sample and for performing a fetal genetic test are also provided herein. The compositions, kits, and methods can comprise or use an anti-TREML2 antibody. Alternatively or additionally, the compositions, kits, and methods comprise or use an antibody conjugated to a colloidal magnetic particle and / or an exogenous aggregation enhancing factor.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 874,306, filed on July 15, 2019, the disclosure of which is incorporated by reference in its entirety. Background Art

[0003] For the past four decades, researchers have been trying to isolate fetal cells from pregnant women to develop prenatal diagnostic tools. Amniocentesis was first developed in the early 1970s, followed by chorionic villus sampling (CVS) in the 1980s. Amniocentesis and chorionic villus sampling (CVS) are two invasive procedures used in routine clinical practice to diagnose chromosomal abnormalities such as common fetal aneuploidies (extra copies of chromosomes), such as trisomy of chromosomes 13, 18, and 21 (which causes Down syndrome).

[0004] The ability to isolate fetal cells and fetal DNA from maternal blood during pregnancy has opened up exciting opportunities for improving non-invasive prenatal testing. Recently, cell-free DNA-based screening (cfDNA), known as non-invasive prenatal testing (NIPT), has been introduced in prenatal screening and has been recognized to be highly predictive for trisomy 21. However, screening performance is lower than that of invasive diagnostic tools, and confirmatory testing is still required. In addition, according to professional associations (Practice bulletin n163 Obstet Gynecol. 2016; 127(5)979-981), NIPT cannot predict copy number variations (CNVs) or microdeletions / duplications. Therefore, current cell-free NIPT is not sufficient to detect subchromosomal deletions and duplications with high specificity, sensitivity, and positive predictive value.

[0005] In view of the scarcity of fetal cells in maternal blood, direct analysis of fetal cells from maternal circulation has been challenging so far. Many different enrichment methods have been tested, including filters, density gradients, fluorescence activated cell sorting (FACS), microfluidics and immunomagnetic beads. Although circulating fetal cells can be reclaimed, these methods lack consistency and reproducibility. This is because extremely low amounts of circulating fetal cells (1ml contains 0.1-10 cells in the maternal blood of approximately 1-5 million cells) have hindered the establishment of reproducible schemes so far. The challenge is to eliminate all contaminating nucleated blood cells without losing the very few circulating fetal cells in early pregnancy.

[0006] Given these limitations, and the fact that amniocentesis and chorionic villus sampling (CVS) are procedures with associated risks of miscarriage, there is a need to develop new cell-based NIPD (non-invasive prenatal diagnostic) procedures to screen for birth defects and genetic diseases from the maternal blood of pregnant women, selecting fetal cells.

[0007] Fetal nucleated red blood cells (nRBCs) and trophoblast cells are known to be present in the maternal circulation, but it has been difficult to develop a reliable cell-based NIPT form. Recently, the possibility of developing a cell-based NIPT form capable of detecting abnormalities with similar accuracy to that currently available through amniocentesis and CVS has been proposed (Amy M. Breman et al., Prenatal Diagnosis, 2016, 36(11): 1009-1019).

[0008] Disclosed herein are fetal cell markers and reagents that bind thereto. Further disclosed herein are compositions, kits, and methods for isolating, detecting, and analyzing fetal cells based on fetal cell markers. SUMMARY

[0009] Disclosed herein is a method for detecting a fetal cell in a sample from a pregnant subject, the method comprising: (a) contacting the sample with a first antibody, wherein the sample comprises a plurality of cells; (b) isolating cells bound to the first antibody to produce an enriched sample; (c) contacting the enriched sample with a second antibody; and (d) identifying cells bound to the second antibody as fetal cells, wherein the first antibody or the second antibody: (i) is an antibody that binds to a Triggering Receptor Expressed on Myeloid Cells Like 2 (TREML2) protein; or (ii) comprises an antigen-binding fragment that binds to a TREML2 protein.

[0010] In some embodiments, the fetal cell is a fetal nucleated red blood cell (fnRBC). In some embodiments, the fetal cell is a trophoblast cell.

[0011] In some embodiments, the first antibody is conjugated to one or more magnetic particles. In some embodiments, the magnetic particles are colloidal magnetic particles. In some embodiments, the colloidal magnetic particles are ferrofluid magnetic particles. In some embodiments, the magnetic particles are coupled to a first exogenous aggregation enhancing factor (EAEF), wherein the first EAEF comprises a member selected from the following specific binding pairs: biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analogue-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin and iminobiotin-avidin.

[0012] In some embodiments, step (a) comprises adding a second EAEF comprising the other member of the specific binding pair to induce aggregation of the magnetic particles.

[0013] In some embodiments, step (b) comprises subjecting the sample to a magnetic field.

[0014] In some embodiments, step (b) comprises adding a member of the specific binding pair to the enriched sample to reverse aggregation of the magnetic particles in the enriched sample.

[0015] In some embodiments, the method further comprises, before step (a), adding at least one aggregation inhibitor selected from a reducing agent, an immune complex, a chelating agent, and diaminobutane to the sample. In some embodiments, the aggregation inhibitor is a chelating agent. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA).

[0016] In some embodiments, the second antibody is an antibody that binds to a TREML2 protein or comprises an antigen-binding fragment that binds to a TREML2 protein. In some embodiments, the TREML2 protein comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the TREML2 protein comprises, consists of, or consists essentially of the amino acid sequence set forth in any one of SEQ ID Nos: 2-5.

[0017] In some embodiments, the method further comprises isolating single fetal cells prior to step (d).

[0018] In some embodiments, single fetal cells are isolated by isolating single fetal cells that bind to the second antibody.

[0019] In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP) and biotin.

[0020] In some embodiments, the isolation of single fetal cells is based on immunofluorescence techniques. In some embodiments, the isolation of single fetal cells is performed by fluorescence activated cell sorting (FACS). In some embodiments, the isolation of single cells is performed by DEPArray.

[0021] In some embodiments, step (d) comprises performing a sequencing analysis. In some embodiments, the sequencing analysis comprises a short tandem repeat (STR) analysis.

[0022] In some embodiments, the method further comprises analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises performing genomic or genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

[0023] In some embodiments, the first antibody is an antibody that binds to a TREML2 protein or comprises an antigen-binding fragment that binds to a TREML2 protein. In some embodiments, the TREML2 protein comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the TREML2 protein comprises, consists of, or consists essentially of the amino acid sequence set forth in any one of SEQ ID Nos: 2-5.

[0024] In some embodiments, the antibody or antigen-binding fragment that binds to the TREML2 protein comprises one or more CDRs selected from the following: (i) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (iii) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (v) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the antibody or antigen-binding fragment that binds to the TREML2 protein comprises 2, 3, 4, 5, or 6 CDRs selected from (i) to (vi).

[0025] In some embodiments, the antibody that binds to the TREML2 protein is an anti-TREML2 antibody. In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbpi-70737-20ul and BD563661.

[0026] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting the sample with a magnetic reagent, wherein the sample comprises a plurality of cells, wherein the magnetic reagent comprises magnetic particles conjugated to a first antibody, and wherein the first antibody binds to a protein selected from the group consisting of EpCAM, CD105, and CD71; (b) contacting the sample with an anti-TREML2 antibody or an antigen-binding fragment thereof; and (c) identifying cells that bind to the anti-TREML2 antibody as fetal cells.

[0027] In some embodiments, the method further comprises, prior to step (c), isolating cells bound to the first antibody. In some embodiments, isolating cells comprises subjecting the sample to a magnetic field to enrich the sample for cells bound to the first antibody.

[0028] In some embodiments, the magnetic particles are colloidal magnetic particles. In some embodiments, the colloidal magnetic particles are ferrofluid magnetic particles. In some embodiments, the colloidal magnetic particles are less than 200 nm. In some embodiments, the colloidal magnetic particles are between about 80 and 200 nm. In some embodiments, the colloidal magnetic particles are between about 90 and 150 nm. In some embodiments, the colloidal magnetic particles have a magnetic mass of at least 50%. In some embodiments, the colloidal magnetic particles have a magnetic mass of at least 60%. In some embodiments, the colloidal magnetic particles have a magnetic mass of between 70% and 90%. In some embodiments, the colloidal magnetic particles comprise a crystalline core of superparamagnetic material surrounded by coating molecules.

[0029] In some embodiments, the magnetic particles are further coupled to a first extrinsic aggregation enhancing factor (EAEF), which comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analogue-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0030] In some embodiments, the method comprises adding a second EAEF to increase aggregation of the particles during step (a), wherein the second EAEF comprises another member of the specific binding pair.

[0031] In some embodiments, the method further comprises adding a member of the specific binding pair (a third EAEF) to the enriched sample to reverse aggregation of the magnetic reagent in the sample, thereby facilitating identification of the cells.

[0032] In some embodiments, the method further comprises, before step (a), adding at least one aggregation inhibitor selected from a reducing agent, an immune complex, a chelating agent, and diaminobutane to the sample. In some embodiments, the aggregation inhibitor is a chelating agent. In some embodiments, the chelating agent is EDTA.

[0033] In some embodiments, the method further comprises, before step (c), isolating the cells using the anti-TREML2 antibody or a second antibody. In some embodiments, the second antibody is selected from an anti-cytokeratin antibody and an anti-HLAG antibody.

[0034] In some embodiments, the anti-TREML2 antibody or the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP) and biotin.

[0035] In some embodiments, the cells are separated by immunofluorescence techniques. In some embodiments, the cells are separated by fluorescence activated cell sorting (FACS). In some embodiments, the cells are separated by DEPArray.

[0036] In some embodiments, identifying the cell comprises performing a sequencing analysis.

[0037] In some embodiments, the sequencing analysis comprises short tandem repeat (STR) analysis.

[0038] In some embodiments, the method further comprises analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises performing genomic or genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

[0039] In some embodiments, the fetal cell is a fetal erythroblast or a fetal trophoblast.

[0040] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises one or more complementarity determining regions (CDRs) selected from the group consisting of: (i) a heavy chain variable region (HCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (iii) a HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (v) a LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) a LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0041] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0042] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting the sample with a first antibody, wherein the sample comprises a plurality of cells, and wherein the first antibody binds to a triggering receptor-like transcription factor on myeloid cells 2 (TREML2) protein (anti-TREML2 antibody) or an antigen-binding fragment thereof; and (b) identifying cells that bind to the first antibody as fetal cells.

[0043] In some embodiments, the fetal cell is a fetal nucleated red blood cell (fnRBC).

[0044] In some embodiments, the first antibody is conjugated to one or more magnetic particles. In some embodiments, the magnetic particles are colloidal magnetic particles. In some embodiments, the colloidal magnetic particles are ferrofluid magnetic particles. In some embodiments, the colloidal magnetic particles are less than 200 nm. In some embodiments, the colloidal magnetic particles are between about 80 and 200 nm. In some embodiments, the colloidal magnetic particles are between about 90 and 150 nm. In some embodiments, the colloidal magnetic particles have a magnetic mass of at least 50%. In some embodiments, the colloidal magnetic particles have a magnetic mass of at least 60%. In some embodiments, the colloidal magnetic particles have a magnetic mass of between 70% and 90%. In some embodiments, the colloidal magnetic particles comprise a crystalline core of superparamagnetic material surrounded by coating molecules.

[0045] In some embodiments, the method further comprises subjecting the sample to a magnetic field.

[0046] In some embodiments, the magnetic particles are coupled to a first extrinsic aggregation enhancing factor (EAEF), wherein the first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analogue-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0047] In some embodiments, the method further comprises, prior to step (b), adding a second EAEF to increase aggregation of the magnetic particles, wherein the second EAEF comprises another member of the specific binding pair.

[0048] In some embodiments, the method further comprises isolating cells that bind to the first antibody to produce an enriched sample.

[0049] In some embodiments, the method further comprises adding a third EAEF to the enriched sample to reverse the aggregation of the magnetic particles in the enriched sample, wherein the third EAEF is capable of binding to the first EAEF or the second EAEF. In some embodiments, the third EAEF is a member of the specific binding pair.

[0050] In some embodiments, the method further comprises, before step (a), adding to the sample at least one aggregation inhibitor selected from a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibitor is a chelating agent. In some embodiments, the chelating agent is EDTA.

[0051] In some embodiments, the first antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP) and biotin.

[0052] In some embodiments, the method further includes separating the cells bound to the first antibody before step (b), wherein separating the cells is based on immunofluorescence technology. In some embodiments, the cells bound to the first antibody are separated by fluorescence activated cell sorting (FACS). In some embodiments, the cells bound to the first antibody are separated by DEPArray.

[0053] In some embodiments, step (b) comprises performing a sequencing analysis. In some embodiments, the sequencing analysis comprises a short tandem repeat (STR) analysis. In some embodiments, the method further comprises analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises performing a genomic or genetic analysis. In some embodiments, performing a genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

[0054] In some embodiments, the first antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more CDRs selected from: (a) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the first antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 2, 3, 4, 5, or 6 CDRs selected from (a)-(f).

[0055] In some embodiments, the first antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0056] Further disclosed herein is a method for cell-based fetal genetic testing, comprising: (a) contacting a sample obtained from a pregnant subject with an anti-TREML2 antibody or an antigen-binding fragment thereof, wherein the sample comprises a plurality of cells; (b) isolating cells that bind to the anti-TREML2 antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells that bind to the anti-TREML2 antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of the one or more nucleic acid molecules, wherein the report provides a likelihood that the fetus has one or more genetic abnormalities.

[0057] In some embodiments, the cell that binds to the anti-TREML2 antibody or antigen-binding fragment thereof is a fetal cell.

[0058] In some embodiments, the fetal cell is a fetal erythroblast. In some embodiments, the fetal cell is a fetal trophoblast.

[0059] In some embodiments, analyzing the one or more nucleic acid molecules comprises performing karyotyping.

[0060] In some embodiments, analyzing the one or more nucleic acid molecules comprises performing a sequencing analysis. In some embodiments, the sequencing analysis comprises a short tandem repeat (STR) analysis.

[0061] In some embodiments, the one or more genetic abnormalities are selected from trisomy, sex chromosome abnormality and structural abnormality. In some embodiments, the trisomy is selected from trisomy 3, trisomy 4, trisomy 6, trisomy 7, trisomy 8, trisomy 9, trisomy 10, trisomy 11, trisomy 12, trisomy 13, trisomy 16, trisomy 17, trisomy 18, trisomy 20, trisomy 21 and trisomy 22. In some embodiments, the sex chromosome abnormality is selected from X monosomy, X trisomy and Klinefelter syndrome. In some embodiments, the structural abnormality is copy number variation (CNV). In some embodiments, the structural abnormality is a deletion of CNV or a duplication of CNV.

[0062] In some embodiments, the anti-TREML2 antibody is conjugated to a magnetic particle. In some embodiments, the magnetic particle is a colloidal magnetic particle. In some embodiments, the colloidal magnetic particle is a ferrofluid magnetic particle.

[0063] In some embodiments, step (b) comprises subjecting the sample to a magnetic field.

[0064] In some embodiments, the method further comprises, prior to step (a), contacting the sample with a first antibody, wherein the first antibody binds to a protein selected from the group consisting of EpCAM, CD105, and CD71.

[0065] In some embodiments, the method further comprises isolating cells that bind to the first antibody before step (a).

[0066] In some embodiments, the first antibody is conjugated to a magnetic particle. In some embodiments, the magnetic particle is a colloidal magnetic particle. In some embodiments, the colloidal magnetic particle is a ferrofluid magnetic particle.

[0067] In some embodiments, the colloidal magnetic particles are less than 200 nm. In some embodiments, the colloidal magnetic particles are between about 80 and 200 nm. In some embodiments, the colloidal magnetic particles are between about 90 and 150 nm.

[0068] In some embodiments, the colloidal magnetic particles have a magnetic quality of at least 50%. In some embodiments, the colloidal magnetic particles have a magnetic quality of at least 60%. In some embodiments, the colloidal magnetic particles have a magnetic quality of between 70% and 90%. In some embodiments, the colloidal magnetic particles comprise a crystalline core of superparamagnetic material surrounded by coating molecules.

[0069] In some embodiments, isolating cells bound to the first antibody comprises subjecting the sample to a magnetic field. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the label is a fluorescent label.

[0070] In some embodiments, the cells bound to the anti-TREML2 antibody or its antigen-binding fragment are separated by immunofluorescence. In some embodiments, the cells bound to the anti-TREML2 antibody or its antigen-binding fragment are separated by fluorescence activated cell sorting (FACS). In some embodiments, the cells bound to the anti-TREML2 antibody or its antigen-binding fragment are separated by DEPArray.

[0071] In some embodiments, the method further comprises contacting the cell bound to the anti-TREML2 antibody or antigen-binding fragment thereof with a second antibody or antigen-binding fragment thereof.

[0072] In some embodiments, the second antibody is an anti-TREML2 antibody or an antigen-binding fragment thereof. In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label.

[0073] In some embodiments, the method further comprises separating the cells bound to the second antibody or its Fab. In some embodiments, separating the cells bound to the second antibody or its Fab is based on immunofluorescence technology. In some embodiments, the cells bound to the second antibody or its Fab are separated by fluorescence activated cell sorting (FACS). In some embodiments, the cells bound to the second antibody or its Fab are separated by DEPArray.

[0074] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0075] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more CDRs selected from the group consisting of: (a) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 2, 3, 4, 5, or 6 CDRs selected from (i)-(vi).

[0076] Further disclosed herein is a method for preparing a fetal cell sample from a maternal sample obtained from a pregnant subject, the method comprising: (a) contacting the maternal sample comprising fetal cells and maternal cells with a first antibody conjugate, wherein the first antibody conjugate comprises (i) a first antibody; and (ii) colloidal magnetic particles, wherein the first antibody is conjugated to the colloidal magnetic particles; and (b) separating cells bound to the first antibody conjugate by subjecting the maternal sample to a magnetic field, thereby preparing a fetal cell sample.

[0077] In some embodiments, the magnetic particles are colloidal magnetic particles. In some embodiments, the colloidal magnetic particles are ferrofluid magnetic particles. In some embodiments, the colloidal magnetic particles are less than 200 nm. In some embodiments, the colloidal magnetic particles are between about 80 and 200 nm. In some embodiments, the colloidal magnetic particles are between about 90 and 150 nm. In some embodiments, the colloidal magnetic particles have a magnetic mass of at least 50%. In some embodiments, the colloidal magnetic particles have a magnetic mass of at least 60%. In some embodiments, the colloidal magnetic particles have a magnetic mass of between 70% and 90%. In some embodiments, the colloidal magnetic particles comprise a crystalline core of superparamagnetic material surrounded by coating molecules.

[0078] In some embodiments, the magnetic particles are coupled to a first extrinsic aggregation enhancing factor (EAEF), wherein the first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analogue-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0079] In some embodiments, the method further comprises adding a second EAEF to the maternal sample, wherein the second EAEF comprises the other member of the specific binding pair.

[0080] In some embodiments, the first antibody is an anti-TREML2 antibody.

[0081] In some embodiments, the first antibody is an anti-CD71 antibody.

[0082] In some embodiments, the first antibody binds to a protein selected from EpCAM and CD105.

[0083] In some embodiments, preparing the fetal cell sample further comprises contacting the cells isolated from the maternal sample with a second antibody.

[0084] In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label.

[0085] In some embodiments, preparing the fetal cell sample further comprises isolating cells that bind to the second antibody.

[0086] In some embodiments, separation is based on immunofluorescence technology.In some embodiments, separation is based on immunofluorescence technology.In some embodiments, separation is based on fluorescence activated cell sorting (FACS) separation is based on fluorescence activated cell sorting (FACS).In some embodiments, separation is based on DEPArray separation is based on DEPArray separation.

[0087] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting the sample with a first antibody conjugate, wherein the sample comprises a plurality of cells, and wherein the first antibody comprises a first antibody conjugated to colloidal magnetic particles; (b) separating cells that bind to the first antibody by subjecting the sample to a magnetic field, thereby producing an enriched sample; (c) contacting the enriched sample with a second antibody, wherein the second antibody binds to a marker on the surface of fetal cells; and (d) identifying cells that bind to the second antibody as fetal cells.

[0088] In some embodiments, the fetal cell is a fetal nucleated red blood cell (fnRBC).In some embodiments, the fetal cell is a fetal trophoblast cell.

[0089] In some embodiments, the colloidal magnetic particles are ferrofluid magnetic particles. In some embodiments, the colloidal magnetic particles are less than 200 nm. In some embodiments, the colloidal magnetic particles are between about 80 and 200 nm. In some embodiments, the colloidal magnetic particles are between about 90 and 150 nm. In some embodiments, the colloidal magnetic particles have a magnetic mass of at least 50%. In some embodiments, the colloidal magnetic particles have a magnetic mass of at least 60%. In some embodiments, the colloidal magnetic particles have a magnetic mass of between 70% and 90%. In some embodiments, the colloidal magnetic particles comprise a crystalline core of superparamagnetic material surrounded by coating molecules.

[0090] In some embodiments, the magnetic particles are coupled to a first extrinsic aggregation enhancing factor (EAEF), wherein the first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analogue-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0091] In some embodiments, step (a) comprises adding a second EAEF comprising the other member of the specific binding pair to increase aggregation of the magnetic particles.

[0092] In some embodiments, step (b) comprises adding a member of the specific binding pair to the enriched sample to reverse aggregation of the magnetic particles in the enriched sample.

[0093] In some embodiments, the method further comprises, before step (a), adding to the sample at least one aggregation inhibitor selected from a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibitor is a chelating agent. In some embodiments, the chelating agent is EDTA.

[0094] In some embodiments, the second antibody is an antibody that binds to a TREML2 protein or comprises an antigen-binding fragment that binds to a TREML2 protein.

[0095] In some embodiments, the method further comprises, prior to step (d), isolating a single fetal cell. In some embodiments, the single fetal cell is isolated by isolating a single fetal cell that binds to the second antibody.

[0096] In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP) and biotin.

[0097] In some embodiments, the isolation of single fetal cells is based on immunofluorescence techniques. In some embodiments, the isolation of single fetal cells is performed by fluorescence activated cell sorting (FACS). In some embodiments, the isolation of single cells is performed by DEPArray.

[0098] In some embodiments, step (d) comprises performing a sequencing analysis. In some embodiments, the sequencing analysis comprises a short tandem repeat (STR) analysis.

[0099] In some embodiments, the method further comprises analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises performing genomic or genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

[0100] In some embodiments, the first antibody is an antibody that binds to a TREML2 protein or comprises an antigen-binding fragment that binds to a TREML2 protein.

[0101] In some embodiments, the antibody or antigen-binding fragment that binds to a TREML2 protein comprises, consists of, or consists essentially of one or more CDRs selected from: (i) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (iii) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iv) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (v) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody or antigen-binding fragment that binds to a TREML2 protein comprises 2, 3, 4, 5, or 6 CDRs selected from (i) to (vi). In some embodiments, any one of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0102] In some embodiments, the antibody that binds to TREML2 protein is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0103] Further disclosed herein are anti-TREML2 antibodies. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more CDRs selected from the group consisting of: (a) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises two or more CDRs selected from (a)-(f). In some embodiments, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises three or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises four or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises five or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises all CDRs in (a)-(f).

[0104] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0105] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to magnetic particles. In some embodiments, the magnetic particles are colloidal magnetic particles. In some embodiments, the colloidal magnetic particles are ferrofluid magnetic particles. In some embodiments, the colloidal magnetic particles are less than 200 nm. In some embodiments, the colloidal magnetic particles are between about 80 and 200 nm. In some embodiments, the colloidal magnetic particles are between about 90 and 150 nm. In some embodiments, the colloidal magnetic particles have a magnetic mass of at least 50%. In some embodiments, the colloidal magnetic particles have a magnetic mass of at least 60%. In some embodiments, the colloidal magnetic particles have a magnetic mass of between 70% and 90%. In some embodiments, the colloidal magnetic particles comprise a crystalline core of superparamagnetic material surrounded by coating molecules.

[0106] In some embodiments, the magnetic particles are coupled to a first extrinsic aggregation enhancing factor (EAEF), wherein the first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analogue-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0107] Disclosed herein is an anti-TREML2 antibody conjugate comprising (a) an anti-TREML2 antibody or an antigen-binding fragment thereof; and (b) a magnetic particle, wherein the magnetic particle is conjugated to the anti-TREML2 antibody.

[0108] In some embodiments, the magnetic particles are colloidal magnetic particles. In some embodiments, the colloidal magnetic particles are ferrofluid magnetic particles. In some embodiments, the colloidal magnetic particles are less than 200 nm. In some embodiments, the colloidal magnetic particles are between about 80 and 200 nm. In some embodiments, the colloidal magnetic particles are between about 90 and 150 nm. In some embodiments, the colloidal magnetic particles have a magnetic mass of at least 50%. In some embodiments, the colloidal magnetic particles have a magnetic mass of at least 60%. In some embodiments, the colloidal magnetic particles have a magnetic mass of between 70% and 90%. In some embodiments, the colloidal magnetic particles comprise a crystalline core of superparamagnetic material surrounded by coating molecules.

[0109] In some embodiments, the magnetic particles are coupled to a first extrinsic aggregation enhancing factor (EAEF), wherein the first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analogue-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0110] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more CDRs selected from the group consisting of: (a) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of two or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or its antigen-binding fragment comprises, consists of, or consists essentially of three or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or its antigen-binding fragment comprises, consists of, or consists essentially of four or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or its antigen-binding fragment comprises, consists of, or consists essentially of five or more CDRs selected from (a)-(f). In some embodiments, the anti-TREML2 antibody or its antigen-binding fragment comprises, consists of, or consists essentially of all CDRs in (a)-(f).

[0111] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0112] Further disclosed herein are kits for isolating, detecting, and / or analyzing fetal cells. In some embodiments, the kit comprises, consists of, or consists essentially of: (a) an antibody that binds to triggering receptor-like transcription factor 2 on myeloid cells (TREML2) protein (anti-TREML2 antibody) or an antigen-binding fragment thereof; and (b) a magnetic reagent comprising colloidal magnetic particles.

[0113] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0114] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more complementarity determining regions (CDRs) selected from: (a) a heavy chain variable region (HCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0115] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a label to produce a conjugated antibody. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP) and biotin.

[0116] In some embodiments, the colloidal magnetic particles are less than 200 nm in size. In some embodiments, the colloidal magnetic particles are ferrofluid particles.

[0117] In some embodiments, the colloidal magnetic particles are conjugated to antibodies or antigen-binding fragments thereof.

[0118] In some embodiments, the antibody is an anti-TREML2 antibody. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more CDRs selected from the group consisting of: (a) an HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) an HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) an LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 2, 3, 4, 5, or 6 CDRs selected from (i)-(vi).

[0119] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0120] In some embodiments, the kit further comprises, consists of, or consists essentially of an inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the kit further comprises, consists of, or consists essentially of a chelating agent. In some embodiments, the chelating agent is EDTA.

[0121] In some embodiments, the kit further comprises, consists of, or consists essentially of an exogenous aggregation enhancing factor (EAEF). In some embodiments, the EAEF comprises, consists of, or consists essentially of a member of a specific binding pair selected from: biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0122] Further disclosed herein is a kit comprising (a) a first antibody capable of binding to a protein expressed on the surface of fetal cells, wherein the first antibody is bound to colloidal magnetic particles; and (b) an anti-TREML2 antibody or an antigen-binding fragment thereof.

[0123] In some embodiments, the first antibody binds to a protein selected from the group consisting of EpCAM, CD105, and CD71.

[0124] In some embodiments, the colloidal magnetic particles are ferrofluid particles.

[0125] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of one or more CDRs selected from: (a) a HCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (d) a LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, any of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0126] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661.

[0127] In some embodiments, the kit further comprises, consists of, or consists essentially of an inhibitor selected from the group consisting of a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the chelating agent is EDTA.

[0128] In some embodiments, the kit further comprises, consists of, or consists essentially of an extrinsic aggregation-enhancing factor (EAEF), wherein the EAEF comprises, consists of, or consists essentially of one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] FIG1 depicts an exemplary method for isolating, detecting, and analyzing rare cells.

[0130] FIG2 depicts an exemplary ferrofluid structure.

[0131] FIG3 depicts an exemplary method for detecting rare cells.

[0132] FIG4 depicts an exemplary method for isolating and detecting rare cells.

[0133] Figures 5A-5E depict gating of cells using a FACS instrument.

[0134] FIG6 depicts an exemplary method for isolating, detecting, and analyzing rare cells.

[0135] FIG. 7 depicts a schematic diagram of ferrofluid aggregation via controlled aggregation.

[0136] Figure 8: Schematic workflow for fetal cell enrichment: Enrichment and staining of fetal cells from maternal whole blood. TM Isolate pure single cells for whole genome amplification and genomic analysis.

[0137] Figure 9 Gating strategy for erythroblasts isolated from fetal blood samples: (1) FSC-A / SSC-A gate for major cell populations (2) Gate for Sytox Green negative live cells (3) FSC-H / W exclude doublet cells (4) Gate for double positive GPA / Hoechst (5) Gate for CD71 positive / CD45 negative (6) Gate for TLS1 / TREML2 and overlay with isotype control to determine the % of TREML2 positive cells.

[0138] Figure 10 Gating strategy for erythroblasts isolated from bone marrow samples: (1) FSC-A / SSC-A gate for major cell populations (2) Gate for Sytox Green negative live cells (3) FSC-H / W exclude doublet cells (4) Gate for double positive GPA / Hoechst (5) Gate for CD71 positive / CD45 negative (6) Gate for TLS1 / TREML2 and overlay with isotype control to determine the % of TREML2 positive cells.

[0139] Figures 11A-11J show TLS1 / TREML2 expression on fetal erythroblasts isolated from various fetal blood (FB) samples from various clones.

[0140] Figures 12A-12L show TLS1 / TREML2 expression on adult human erythroblasts isolated from various bone marrow (BM) samples from various clones.

[0141] Figure 13 shows the enrichment of CD105-FF and EpCAM-FF by DEPArray. TM Scatter plot analysis of identified TLS1 / TREML-2 positive trophoblast cells.

[0142] Figure 14 shows Image Gallery: Trophoblast cells showing positive staining for TREML-2-PE antibody, CK-APC, and nuclei.

[0143] Figure 15A shows a scatter plot analysis of Draq5 / Hoechst positive erythroblasts spiked with healthy donor blood and enriched with CD71-FF. Figure 15B shows Image Gallery: Erythroblasts show positive staining with CD71-PE antibody, Draq5 and Hoechst nuclear staining, and negative staining with CD45-FITC antibody.

[0144] Figure 16A shows a scatter plot analysis of Draq5 / Hoechst positive erythroblasts spiked with healthy donor blood and enriched with TLS1 / TREML-2-FF. Figure 16B shows Image Gallery: Erythroblasts show positive staining with CD71-PE antibody, Draq5 and Hoechst nuclear staining, and negative staining with CD45-FITC antibody.

[0145] Figure 17 shows STR analysis of single fetal cells isolated from maternal blood.

[0146] FIG18 shows the results of CNV analysis of single fetal cells.

[0147] FIG19 shows the results of CNV analysis of single cells from healthy donors.

[0148] FIG. 20 depicts an exemplary method for isolating and detecting rare cells. DETAILED DESCRIPTION

[0149] Disclosed herein are compositions, kits, and methods for separating, detecting, and / or analyzing rare cells in a sample. Typically, the compositions, kits, and methods disclosed herein comprise reagents that bind to a protein called triggering receptor-like transcription factor 2 (TREML2) on myeloid cells (this protein is also referred to as TLS1 throughout the application). Alternatively or additionally, the compositions, kits, and methods disclosed herein comprise antibody conjugates. The antibody conjugates comprise antibodies conjugated to colloidal magnetic particles. The rare cells may be fetal cells. The sample may be a sample from a pregnant subject.

[0150] Methods for isolating, detecting and / or characterizing rare cells

[0151] Disclosed herein are methods for isolating, detecting, and / or characterizing rare cells. In some embodiments, the rare cells are fetal cells. In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs). In some embodiments, the fetal cells are trophoblasts. Typically, the method includes using an anti-TREML2 antibody or an antigen-binding fragment thereof to identify the cells as fetal cells. Alternatively or additionally, the method includes using an antibody conjugated to colloidal magnetic particles to isolate fetal cells.

[0152] Disclosed herein are methods for detecting fetal cells in a sample from a pregnant subject, the methods comprising: (a) contacting the sample with an anti-TREML2 antibody or an antigen-binding fragment thereof, wherein the sample comprises a plurality of cells; and (b) identifying cells that bind to the anti-TREML2 antibody as fetal cells.

[0153] Further disclosed herein are methods for detecting fetal cells in a sample from a pregnant subject, the methods comprising: (a) contacting the sample with a first antibody or an antigen-binding fragment thereof, wherein the sample comprises a plurality of cells; (b) isolating cells that bind to the first antibody or an antigen-binding fragment thereof to produce an enriched sample; (c) contacting the enriched sample with a second antibody or an antigen-binding fragment thereof; and (d) identifying cells that bind to the second antibody as fetal cells, wherein the first antibody or the second antibody is an antibody that binds to a triggering receptor-like transcription factor 2 (TREML2) protein.

[0154] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting the sample with a magnetic reagent, wherein the sample comprises a plurality of cells, wherein the magnetic reagent comprises magnetic particles conjugated to a first antibody or an antigen-binding fragment thereof, and wherein the first antibody or antigen-binding fragment thereof binds to a protein selected from the group consisting of EpCAM, CD105, and CD71; (b) contacting the sample with an anti-TREML2 antibody or an antigen-binding fragment thereof; and (c) identifying cells that bind to the anti-TREML2 antibody as fetal cells.

[0155] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting the sample with a magnetic reagent, wherein the sample comprises a plurality of cells, wherein the magnetic reagent comprises colloidal magnetic particles conjugated to a first antibody or an antigen-binding fragment thereof, and wherein the first antibody or antigen-binding fragment thereof binds to a protein selected from the group consisting of EpCAM, CD105, and CD71; (b) contacting the sample with a second antibody or antigen-binding fragment thereof; and (c) identifying cells that bind to the second antibody as fetal cells.

[0156] Further disclosed herein is a method for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting the sample with a magnetic reagent and a second exogenous aggregation-enhancing factor (EAEF), wherein the sample comprises a plurality of cells, wherein the magnetic reagent comprises colloidal magnetic particles conjugated to a first antibody or an antigen-binding fragment thereof, wherein the colloidal magnetic particles are conjugated to the first EAEF, and wherein the first antibody or antigen-binding fragment thereof binds to a protein selected from the group consisting of EpCAM, CD105, and CD71; (b) contacting the sample with a second antibody or antigen-binding fragment thereof; and (c) identifying cells bound to the second antibody as fetal cells. In some embodiments, the first EAEF comprises a first member of a specific binding pair and the second EAEF comprises a second member of the specific binding pair, wherein the specific binding pair is selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0157] Further disclosed herein are methods for detecting fetal cells in a sample from a pregnant subject, the method comprising: (a) contacting the sample with a first antibody conjugate, wherein the sample comprises a plurality of cells, and wherein the first antibody conjugate comprises a first antibody, or an antigen-binding fragment thereof, conjugated to colloidal magnetic particles; (b) isolating cells that bind to the first antibody by subjecting the sample to a magnetic field, thereby producing an enriched sample; (c) contacting the enriched sample with a second antibody, or an antigen-binding fragment thereof, wherein the second antibody binds to a marker on the surface of fetal cells; and (d) identifying cells that bind to the second antibody as fetal cells.

[0158] Further disclosed herein is a method for preparing a fetal cell sample from a maternal sample obtained from a pregnant subject, the method comprising: (a) contacting the maternal sample comprising fetal cells and maternal cells with a first antibody conjugate, wherein the first antibody conjugate comprises (i) a first antibody or an antigen-binding fragment thereof; and (ii) colloidal magnetic particles, wherein the first antibody is conjugated to the colloidal magnetic particles; and (b) separating cells bound to the first antibody conjugate by subjecting the maternal sample to a magnetic field, thereby preparing a fetal cell sample.

[0159] Further disclosed herein is a method for preparing a fetal cell sample from a maternal sample obtained from a pregnant subject, the method comprising: (a) contacting the maternal sample comprising fetal cells and maternal cells with a first antibody conjugate and a second extrinsic aggregation-enhancing factor (EAEF), wherein the first antibody conjugate comprises (i) a first antibody or an antigen-binding fragment thereof; (ii) colloidal magnetic particles; and (iii) a first EAEF, wherein the first antibody is conjugated to the colloidal magnetic particles, and wherein the first EAEF is conjugated to the colloidal magnetic particles; and (b) separating cells bound to the first antibody conjugate by subjecting the maternal sample to a magnetic field, thereby preparing a fetal cell sample. In some embodiments, the first EAEF comprises a first member of a specific binding pair and the second EAEF comprises a second member of the specific binding pair, wherein the specific binding pair is selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0160] Further disclosed herein is a method for preparing a fetal cell sample from a maternal sample obtained from a pregnant subject, the method comprising: (a) contacting the maternal sample comprising fetal cells and maternal cells with a first antibody conjugate, wherein the first antibody conjugate comprises (i) a first antibody or an antigen-binding fragment thereof; and (ii) colloidal magnetic particles, wherein the first antibody is conjugated to the colloidal magnetic particles and wherein the first antibody is an anti-TREML2 antibody; and (b) separating cells bound to the first antibody conjugate by subjecting the maternal sample to a magnetic field, thereby preparing a fetal cell sample.

[0161] Further disclosed herein is a method for preparing a fetal cell sample from a maternal sample obtained from a pregnant subject, the method comprising: (a) contacting the maternal sample comprising fetal cells and maternal cells with a first antibody conjugate and a second extrinsic aggregation-enhancing factor (EAEF), wherein the first antibody conjugate comprises (i) a first antibody or an antigen-binding fragment thereof; and (ii) colloidal magnetic particles, wherein the first antibody is conjugated to the colloidal magnetic particles, and wherein the colloidal magnetic particles are conjugated to a first EAEF; and (b) separating cells bound to the first antibody conjugate by subjecting the maternal sample to a magnetic field, thereby preparing a fetal cell sample. In some embodiments, the first EAEF comprises a first member of a specific binding pair and the second EAEF comprises a second member of the specific binding pair, wherein the specific binding pair is selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0162] In some embodiments, the fetal cell is a fetal nucleated red blood cell (fnRBC). In some embodiments, the fetal cell is an erythroblast. In some embodiments, the fetal cell is a trophoblast.

[0163] In some embodiments, any of the methods disclosed herein further comprises isolating cells that bind to the anti-TREML2 antibody or the first antibody, wherein isolating the cells occurs prior to identifying the cells.

[0164] In some embodiments, any of the methods disclosed herein include the use of a first antibody. In some embodiments, the first antibody is conjugated to one or more magnetic particles. In some embodiments, the magnetic particles are colloidal magnetic particles. In some embodiments, the magnetic particles are ferrofluid magnetic particles.

[0165] In some embodiments, any of the methods disclosed herein comprises isolating cells bound to the first antibody or antigen-binding fragment thereof. In some embodiments, isolating the cells comprises subjecting the sample to a magnetic field.

[0166] In some embodiments, the magnetic particles are coupled to a first extrinsic aggregation enhancing factor (EAEF) comprising one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analogue-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0167] In some embodiments, any of the methods disclosed herein comprises adding a second EAEF comprising the other member of the specific binding pair to induce aggregation of the magnetic particles.

[0168] In some embodiments, isolating cells bound to the first antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of adding a member of the specific binding pair to the enriched sample to reverse aggregation of the magnetic particles in the enriched sample.

[0169] In some embodiments, any of the methods disclosed herein include adding at least one aggregation inhibitor selected from a reducing agent, an immune complex, a chelating agent, and diaminobutane to the sample. In some embodiments, the aggregation inhibitor is a chelating agent. In some embodiments, the chelating agent is EDTA. The reducing agent can be mercaptoethanesulfonic acid. The aggregation inhibitor can be bovine serum albumin (BSA).

[0170] In some embodiments, any of the methods disclosed herein utilize a second antibody. In some embodiments, the second antibody is an antibody that binds to a TREML2 protein, or comprises, consists of, or consists essentially of an antigen-binding fragment that binds to a TREML2 protein.

[0171] In some embodiments, any of the methods disclosed herein comprises isolating a single fetal cell. In some embodiments, the single fetal cell is isolated by isolating a single fetal cell that binds to the second antibody.

[0172] In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the isolation of single fetal cells is based on immunofluorescence techniques. In some embodiments, single fetal cells are isolated by fluorescence activated cell sorting (FACS). In some embodiments, single cells are isolated by DEPArray.

[0173] In some embodiments, any of the methods disclosed herein comprises performing sequencing analysis on one or more nucleic acid molecules isolated from fetal cells. In some embodiments, the sequencing analysis comprises short tandem repeat (STR) analysis.

[0174] In some embodiments, any of the methods disclosed herein include analyzing fetal cells. In some embodiments, analyzing the fetal cells includes performing genomic or genetic analysis. In some embodiments, performing genetic analysis includes detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

[0175] In some embodiments, the first antibody is an antibody that binds to a TREML2 protein or comprises an antigen-binding fragment that binds to a TREML2 protein.

[0176] In some embodiments, the antibody or antigen-binding fragment that binds to a TREML2 protein comprises 1, 2, 3, 4, 5, or 6 CDRs selected from: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID Nos: 6-11 independently comprises one or more substitutions, additions, or deletions.

[0177] In some embodiments, the anti-TREML2 antibody is conjugated to one or more magnetic particles. In some embodiments, the magnetic particles are colloidal magnetic particles. In some embodiments, the colloidal magnetic particles are ferrofluid magnetic particles. In some embodiments, separating the cells comprises placing the sample in a magnetic separator. In some embodiments, separating the cells comprises subjecting the sample to a magnetic field.

[0178] In some embodiments, the anti-TREML2 antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, separating cells comprises flow cytometry. In some embodiments, flow cytometry is fluorescence activated cell sorting (FACS).

[0179] In some embodiments, isolating the cells comprises performing a DEPArray.

[0180] In some embodiments, identifying the cell comprises performing a sequencing reaction.

[0181] In some embodiments, the sample is a sample enriched for fetal cells prior to contacting the sample with the anti-TREML2 antibody. In some embodiments, fetal cells in the sample are enriched by contacting the sample with a ferrofluid reagent, wherein the ferrofluid comprises an antibody coupled to the ferrofluid.

[0182] In some embodiments, the antibody binds to a protein selected from EpCAM, CD105, and CD71.

[0183] In some embodiments, the methods disclosed herein further comprise isolating cells bound by the ferrofluid-coupled antibody, thereby generating a sample enriched for fetal cells.

[0184] In some embodiments, any of the methods disclosed herein further comprises performing sequencing analysis. In some embodiments, the sequencing analysis comprises short tandem repeat (STR) analysis.

[0185] In some embodiments, any of the methods disclosed herein further comprise analyzing the fetal cells. In some embodiments, analyzing the fetal cells comprises detecting the presence or absence of one or more fetal abnormalities. In some embodiments, analyzing the fetal cells comprises performing a genomic analysis. In some embodiments, analyzing the fetal cells comprises performing a genetic analysis. In some embodiments, performing a genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells. In some embodiments, performing a genetic analysis comprises detecting the presence or absence of a chromosomal abnormality in the fetal cells. In some embodiments, the chromosomal abnormality is trisomy 21, trisomy 18, or trisomy 13.

[0186] In some embodiments, any of the methods disclosed herein further comprise performing genetic testing on the fetal cells. In some embodiments, performing genetic testing on the fetal cells comprises detecting the presence or absence of one or more fetal abnormalities. In some embodiments, performing genetic testing on the fetal cells comprises performing genomic analysis. In some embodiments, performing genetic testing on the fetal cells comprises performing genetic analysis. In some embodiments, performing genetic analysis comprises detecting the presence or absence of a chromosomal abnormality in the fetal cells. In some embodiments, the chromosomal abnormality is trisomy 21, trisomy 18, or trisomy 13.

[0187] In some embodiments, any of the methods disclosed herein further comprise providing a treatment recommendation based on the results of the analysis of the fetal cells. In some embodiments, any of the methods disclosed herein further comprise providing a treatment recommendation based on the results of the genetic testing of the fetal cells.

[0188] In some embodiments, any of the methods disclosed herein further comprise administering a therapy to the subject based on the results of the analysis of the fetal cells. In some embodiments, any of the methods disclosed herein further comprise administering a therapy to the subject based on the results of the genetic testing of the fetal cells.

[0189] In some embodiments, any of the methods disclosed herein further comprises recommending additional monitoring of the subject or fetus based on the results of the analysis of the fetal cells. In some embodiments, any of the methods disclosed herein further comprises recommending additional monitoring of the subject or fetus based on the results of the genetic testing of the fetal cells.

[0190] Figure 1 depicts an exemplary method for isolating, detecting, and / or analyzing rare cells. The methods disclosed herein may include, consist of, or consist essentially of one or more of the steps shown in Figure 1. In some embodiments, the method includes, consists of, or consists essentially of: (a) obtaining a sample comprising a plurality of cells from a subject (101); and (b) isolating rare cells (110). In some embodiments, the method includes, consists of, or consists essentially of: (a) obtaining a sample comprising a plurality of cells from a subject (101); (b) isolating rare cells (110); and (c) analyzing the rare cells (120). In some embodiments, the method includes, consists of, or consists essentially of: (a) obtaining a sample comprising a plurality of cells from a subject (101); (b) isolating rare cells (110); (c) analyzing the rare cells (120); and (d) generating one or more reports based on the analysis of the rare cells (106).

[0191] As shown in FIG1 , in some embodiments, the method comprises, consists of, or consists essentially of: (a) obtaining a sample comprising a plurality of cells from a subject (101); (b) isolating rare cells (110) by: (i) depleting non-rare cells from the sample to produce an enriched rare cell sample (102); and (ii) isolating rare cells from the enriched rare cell sample (103); (c) analyzing the rare cells (120) by: (i) purifying nucleic acid molecules from the rare cells (104); and (ii) sequencing one or more nucleic acid molecules (105); and (f) generating one or more reports (106). In some embodiments, the rare cells are fetal cells. In some embodiments, enriching rare cells (102) comprises, consists of, or consists essentially of: contacting the sample with a ferrofluid, wherein the ferrofluid comprises antibodies coupled to magnetic particles, and wherein the antibodies bind to a marker on the rare cells. In some embodiments, the marker on the rare cells is any marker disclosed herein. In some embodiments, the marker on the rare cells is TREML2 protein. In some embodiments, the antibody is any antibody disclosed herein. In some embodiments, the antibody is an anti-TREML2 antibody. In some embodiments, the antibody is any anti-TREML2 antibody disclosed herein. In some embodiments, the ferrofluid comprises, consists of, or consists essentially of the ferrofluid structure depicted in Figure 2. In some embodiments, enriching rare cells (102) further comprises, consists of, or consists essentially of applying an external gradient magnetic separator to the sample to remove cells that are not bound to the ferrofluid. In some embodiments, the method further comprises, consists of, or consists essentially of contacting the rare cells with one or more additional antibodies, wherein the one or more additional antibodies are conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the rare cells are contacted with one or more additional antibodies before separating the rare cells (103). In some embodiments, isolating rare cells (103) comprises, consists of, or consists essentially of selecting a single cell bound by an antibody that binds to a marker on the rare cell. In some embodiments, the antibody is an anti-TREML2 antibody. In some embodiments, the anti-TREML2 antibody is any anti-TREML2 antibody disclosed herein.In some embodiments, the anti-TREML2 antibody comprises (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, isolating rare cells (103) comprises, consists of, or consists essentially of sorting rare cells from the enriched cell sample. In some embodiments, isolating rare cells (103) comprises, consists of, or consists essentially of performing fluorescence activated cell sorting (FACS). In some embodiments, isolating rare cells (103) comprises, consists of, or consists essentially of performing a DEPArray. In some embodiments, purifying nucleic acid molecules (104) from the rare cells comprises, consists of, or consists essentially of performing nucleic acid amplification. In some embodiments, purifying nucleic acid molecules (104) from the rare cells comprises, consists of, or consists essentially of generating a nucleic acid library.

[0192] Figure 3 depicts an exemplary method for detecting rare cells (e.g., fetal cells). In some embodiments, the method comprises, consists of, or consists essentially of: (a) contacting a sample (301) comprising a plurality of cells (302, 303) with a first antibody or antigen binding fragment thereof (304); and (b) identifying cells (303) bound by the first antibody or antigen binding fragment thereof (304) as fetal cells. In some embodiments, the first antibody (304) is an antibody that binds to a TREML2 protein. In some embodiments, the antigen binding fragment (304) binds to a TREML2 protein. In some embodiments, the first antibody or antigen binding fragment thereof comprises, consists of, or consists essentially of any of the anti-TREML2 antibodies disclosed herein. In some embodiments, the first antibody or antigen binding fragment thereof comprises, consists of, or consists essentially of: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. The first antibody or antigen binding fragment thereof can be coupled to a magnetic particle. For example, the first antibody or antigen binding fragment can be in the form of a ferrofluid. Alternatively, the first antibody or antigen binding fragment can be conjugated to a label. The label can be any of the labels disclosed herein. For example, the first antibody or antigen binding fragment can be conjugated to a fluorescent label. The cells can be identified by any of the identification techniques disclosed herein. In some embodiments, identifying cells bound to the first antibody or antigen binding fragment thereof comprises isolating cells bound to the first antibody or antigen binding fragment thereof. Isolating the cells can comprise any of the cell isolation techniques disclosed herein. In some embodiments, isolating the cells comprises magnetic isolation. In some embodiments, identifying the cells can comprise using a microscope. Identifying the cells can comprise fluorescence microscopy. In some embodiments, identifying the cells comprises or is based on FACS. Alternatively or additionally, identifying the cells comprises or is based on DEPArray.

[0193] Figure 4 depicts an exemplary method for separating and detecting rare cells. In some embodiments, the method for detecting rare cells comprises, consists of, or consists essentially of: (a) contacting a sample (401) comprising a plurality of cells (402, 403) with an antibody conjugate (406), wherein the antibody conjugate (406) comprises a first antibody or antigen-binding fragment (404) coupled to a magnetic particle (405); (b) enriching rare cells (403) by subjecting the sample to a magnetic field (407) and removing cells (402) that are not bound to the antibody conjugate, thereby producing an enriched rare cell sample (411); (c) contacting the enriched rare cell sample (411) with an antibody conjugate (410), wherein the antibody conjugate (410) comprises a second antibody or antigen-binding fragment (408) conjugated to a label (409); and (d) identifying cells that bind to the antibody conjugate as rare cells (403).

[0194] In some embodiments, the rare cells are fetal cells. In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs). In some embodiments, the enriched rare cell sample (411) comprises rare cells (403) bound to the antibody conjugate (406), wherein the antibody conjugate comprises a first antibody (404) or an antigen-binding fragment thereof (404) conjugated to the magnetic particles (405). Alternatively or additionally, the enriched rare cell sample (411) is further processed to separate the rare cells (403) from the antibody conjugate (406). In some embodiments, the first antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment both bind to the TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment or (b) the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment binds to a protein selected from EpCAM, CD105, and CD71; and (b) the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment binds to EpCAM; and (b) the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment binds to CD105; and (b) the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment binds to CD71; and (b) the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, the antibody or antigen-binding fragment that binds to TREML2 is any anti-TREML2 antibody or antigen-binding fragment disclosed herein.In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 1, 2, 3, 4, 5, or 6 CDRs selected from: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the second antibody is an antibody that binds to the first antibody. For example, if the first antibody is a goat IgG antibody, the second antibody can be a mouse anti-goat IgG antibody. In some embodiments, the label is any label disclosed herein. In some embodiments, the label is a fluorescent label. In some embodiments, the magnetic particles are colloidal magnetic particles. In some embodiments, the colloidal magnetic particles are ferrofluid magnetic particles. In some embodiments, the magnetic particles are further conjugated to a first exogenous aggregation enhancing factor (EAEF). In some embodiments, the method further includes contacting the sample (401) with a second EAEF capable of binding to the first EAEF during step (A). In some embodiments, adding the second EAEF induces aggregation of the antibody conjugate (406). In some embodiments, the method further includes adding a third EAEF capable of binding to the first or second exogenous aggregation enhancing factor. In some embodiments, adding the third EAEF reverses the aggregation of the first EAEF. In some embodiments, the method further includes adding an aggregation inhibitor to the sample before step (a). The cells can be identified by any identification technique disclosed herein. In some embodiments, identifying the cells can include using a microscope. Identifying the cells can include fluorescence microscopy. In some embodiments, identifying the cells comprises or is based on FACS.Alternatively or additionally, identifying the cells comprises or is based on DEPArray.

[0195] Figure 20 depicts another exemplary method for isolating and detecting rare cells. As shown in Figure 20, in some embodiments, the method for detecting rare cells comprises, consists of, or consists essentially of: step (A1): contacting a sample (2001) comprising a plurality of cells (2002, 2003) with a first antibody conjugate (2006) and a second exogenous aggregation enhancing factor (EAEF) (2011), wherein the first antibody conjugate (2006) comprises a first antibody or antigen binding fragment (2004) coupled to a magnetic particle (2005), wherein the magnetic particle (2005) is further conjugated to the first EAEF (2012); and step (B1): enriching rare cells (2003) by subjecting the sample to a magnetic field (2007) and removing cells (2002) that are not bound to the antibody conjugate (2006)-second EAEF (2011) complex, thereby producing an enriched rare cell sample (2011). As shown in step (A2), adding the second EAEF (2011) induces aggregation of the first antibody conjugate (2006). In some embodiments, the method further includes step (B2): adding a third EAEF (2013) to the enriched rare cell sample (2011). As shown in step (B3), adding the third EAEF (2013) reverses the aggregation of the first antibody conjugate (2006). In some embodiments, the method further includes step (C): contacting the enriched rare cell sample (2011) with a second antibody conjugate (2010), wherein the second antibody conjugate (2010) comprises a second antibody or antigen-binding fragment (2008) conjugated to a label (2009). In some embodiments, the method further includes step (D): identifying cells bound to the first antibody conjugate (2006) as rare cells (2003). In some embodiments, the method further comprises step (D): identifying cells bound to the second antibody conjugate (2010) as rare cells (2003). In some embodiments, the rare cells are fetal cells. In some embodiments, the fetal cells are fetal nucleated red blood cells (fnRBCs). In some embodiments, the enriched rare cell sample (2011) comprises rare cells (2003) bound to the first antibody conjugate (2006), wherein the first antibody conjugate comprises a first antibody (2004) or an antigen-binding fragment thereof (2004) conjugated to the magnetic particle (2005), wherein the magnetic particle (2005) is further conjugated to the first EAEF (2012). Alternatively or additionally, the enriched rare cell sample (2011) is further processed to separate the rare cells (2003) from the antibody conjugate (2006).In some embodiments, the first antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment both bind to the TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment or (b) the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment binds to a protein selected from EpCAM, CD105, and CD71; and (b) the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment binds to EpCAM; and (b) the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment binds to CD105; and (b) the second antibody or antigen-binding fragment binds to the TREML2 protein. In some embodiments, (a) the first antibody or antigen-binding fragment binds to CD71; and (b) the second antibody or antigen-binding fragment binds to TREML2 protein. In some embodiments, the antibody or antigen-binding fragment that binds to TREML2 is any anti-TREML2 antibody or antigen-binding fragment disclosed herein. In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 1, 2, 3, 4, 5, or 6 CDRs selected from: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the second antibody is an antibody that binds to the first antibody. For example, if the first antibody is a goat IgG antibody, the second antibody can be a mouse anti-goat IgG antibody. In some embodiments, the label is any label disclosed herein. In some embodiments, the label is a fluorescent label. In some embodiments, the magnetic particles are colloidal magnetic particles.In some embodiments, the colloidal magnetic particles are ferrofluid magnetic particles. In some embodiments, the method further comprises adding an aggregation inhibitor to the sample before step (A1). In some embodiments, the first EAEF (2012) is desthiobiotin. In some embodiments, the second EAEF (2011) is streptavidin. In some embodiments, the third EAEF (2013) is biotin. The cells can be identified by any identification technique disclosed herein. In some embodiments, identifying the cells may include using a microscope. Identifying the cells may include fluorescence microscopy. In some embodiments, identifying the cells includes or is based on FACS. Alternatively or additionally, identifying the cells includes or is based on DEPArray. In some embodiments, identifying the cells includes or is based on an immunoassay.

[0196] Although the methods disclosed herein may cite the use of anti-TREML2 antibodies or antigen-binding fragments thereof, or antibody conjugates comprising such anti-TREML2 antibodies, any of these methods may be performed using any agent that can bind to a TREML2 protein, or a conjugate comprising an agent that can bind to a TREML2 protein. Therefore, the methods disclosed herein are not limited to the use of anti-TREML2 antibodies or antigen-binding fragments thereof, or antibody conjugates comprising such anti-TREML2 antibodies.

[0197] Methods for cell-based fetal genetic testing

[0198] The identification of novel fetal cell markers (such as TREML-2) allows the separation and / or detection of fetal cells and the subsequent analysis of such cells. Therefore, disclosed herein is a method for cell-based fetal genetic testing. In some embodiments, the method includes (a) using an anti-TREML2 antibody to separate fetal cells from a sample from a pregnant subject; and (b) analyzing one or more nucleic acid molecules from the fetal cells to determine the possibility that the fetus has one or more genetic abnormalities. Alternatively, the method includes separating fetal cells using any method disclosed herein for separating or detecting fetal cells, and analyzing one or more nucleic acid molecules from the separated or detected fetal cells to determine the possibility that the fetus has one or more genetic abnormalities. In some embodiments, the method includes analyzing fetal cells separated and / or detected by any method disclosed herein. In some embodiments, the method includes analyzing fetal cells prepared by any method disclosed herein.

[0199] Disclosed herein are methods for cell-based fetal genetic testing, comprising: (a) contacting a sample obtained from a pregnant subject with an anti-TREML2 antibody or an antigen-binding fragment thereof, wherein the sample comprises a plurality of cells; (b) isolating cells that bind to the anti-TREML2 antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells that bind to the anti-TREML2 antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of the one or more nucleic acid molecules, wherein the report provides a likelihood that the fetus has one or more genetic abnormalities.

[0200] Disclosed herein are methods for cell-based fetal genetic testing, the methods comprising: (a) contacting a sample obtained from a pregnant subject with a first antibody or antigen-binding fragment thereof, wherein the sample comprises a plurality of cells, wherein the first antibody or antigen-binding fragment thereof is conjugated to colloidal magnetic particles, and wherein the first antibody or antigen-binding fragment thereof binds to a marker on fetal cells; (b) isolating cells that bind to the first antibody or antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells that bind to the first antibody or antigen-binding fragment thereof; and (d) generating a report based on the analysis of the one or more nucleic acid molecules, wherein the report provides a likelihood that the fetus has one or more genetic abnormalities.

[0201] Disclosed herein are methods for cell-based fetal genetic testing, the methods comprising: (a) contacting a sample obtained from a pregnant subject with a first antibody, or an antigen-binding fragment thereof, and a second exogenous aggregation-enhancing factor (EAEF), wherein the sample comprises a plurality of cells, wherein the first antibody, or the antigen-binding fragment thereof, is conjugated to colloidal magnetic particles, wherein the colloidal magnetic particles are conjugated to a first EAEF, and wherein the first antibody, or the antigen-binding fragment thereof, binds to a marker on fetal cells; (b) isolating cells that bind to the first antibody, or the antigen-binding fragment thereof; (c) analyzing one or more nucleic acid molecules from the cells that bind to the first antibody, or the antigen-binding fragment thereof; and (d) generating a report based on the analysis of the one or more nucleic acid molecules, wherein the report provides a likelihood that the fetus has one or more genetic abnormalities. In some embodiments, the first EAEF comprises a first member of a specific binding pair and the second EAEF comprises a second member of the specific binding pair, wherein the specific binding pair is selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0202] In some embodiments, the first antibody is an anti-TREML2 antibody. In some embodiments, the first antibody is an anti-CD71 antibody. In some embodiments, the first antibody is an anti-EpCAM antibody. In some embodiments, the first antibody is an anti-CD105 antibody. In some embodiments, when the first antibody is an anti-ECAM antibody or an anti-CD105 antibody, the method further includes contacting the separated cells with a second antibody or its antigen-binding fragment, wherein the second antibody binds to a marker on the fetal cells. In some embodiments, the second antibody is an anti-TREML2 antibody. In some embodiments, the second antibody is an anti-CD71 antibody. In some embodiments, the second antibody or its antigen-binding fragment is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the method further includes isolating cells bound to the second antibody. In some embodiments, the method further includes analyzing nucleic acid molecules from cells bound to the second antibody or its antigen-binding fragment.

[0203] In some embodiments, the cell bound to the anti-TREML2 antibody or antigen-binding fragment thereof is a fetal cell. In some embodiments, the fetal cell is a fetal erythroblast. In some embodiments, the fetal cell is a fetal nucleated red blood cell (fnRBC). In some embodiments, the fetal cell is a fetal trophoblast.

[0204] In some embodiments, analyzing the one or more nucleic acid molecules comprises performing karyotyping.Karyotyping can be performed using any technique known in the art.

[0205] In some embodiments, analyzing the one or more nucleic acid molecules comprises performing a sequencing analysis. Sequencing analysis can be performed using any technique known in the art. In some embodiments, the sequencing analysis comprises short tandem repeat (STR) analysis.

[0206] In some embodiments, analyzing the one or more nucleic acid molecules includes performing one or more amplification reactions. Nucleic acid amplification can be performed by any technology known in the art. In some embodiments, nucleic acid amplification is performed by polymerase chain reaction (PCR).

[0207] In some embodiments, the one or more genetic abnormalities are selected from trisomy, sex chromosome abnormality and structural abnormality. In some embodiments, the genetic abnormality is trisomy. In some embodiments, the trisomy is selected from trisomy 3, trisomy 4, trisomy 6, trisomy 7, trisomy 8, trisomy 9, trisomy 10, trisomy 11, trisomy 12, trisomy 13, trisomy 16, trisomy 17, trisomy 18, trisomy 20, trisomy 21 and trisomy 22. In some embodiments, the genetic abnormality is a sex chromosome abnormality. In some embodiments, the sex chromosome abnormality is selected from X monosomy, X trisomy and Klinefelter syndrome. In some embodiments, the genetic abnormality is a structural abnormality. In some embodiments, the structural abnormality is a copy number variation (CNV). In some embodiments, the structural abnormality is a deletion of a CNV or a duplication of a CNV.

[0208] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof is conjugated to a magnetic particle. In some embodiments, the magnetic particle is a colloidal magnetic particle.

[0209] In some embodiments, isolating cells bound to the anti-TREML2 antibody or antigen-binding fragment thereof comprises subjecting the sample to a magnetic field.

[0210] In some embodiments, the methods disclosed herein further include contacting the sample with a first antibody before contacting the sample with the anti-TREML2 antibody, wherein the first antibody binds to a protein selected from EpCAM, CD105, and CD71. In some embodiments, the methods disclosed herein further include separating cells bound to the first antibody before contacting the sample with the anti-TREML2 antibody. In some embodiments, the first antibody is conjugated to magnetic particles. In some embodiments, the magnetic particles are colloidal magnetic particles. In some embodiments, separating cells bound to the first antibody includes subjecting the sample to a magnetic field.

[0211] In some embodiments, the anti-TREML2 antibody or its antigen-binding fragment is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the cells bound to the anti-TREML2 antibody or its antigen-binding fragment are separated based on immunofluorescence technology. In some embodiments, the cells bound to the anti-TREML2 antibody or its antigen-binding fragment are separated by fluorescence activated cell sorting (FACS). In some embodiments, the cells bound to the anti-TREML2 antibody or its antigen-binding fragment are separated by DEPArray.

[0212] In some embodiments, the methods disclosed herein further include contacting cells bound to the anti-TREML2 antibody or its antigen-binding fragment with a second antibody or its antigen-binding fragment. In some embodiments, the second antibody is an anti-TREML2 antibody or its antigen-binding fragment. In some embodiments, the second antibody is conjugated to a label. In some embodiments, the label is a fluorescent label. In some embodiments, the methods disclosed herein further include separating cells bound to the second antibody or its antigen-binding fragment. In some embodiments, separating cells bound to the second antibody or its antigen-binding fragment is based on immunofluorescence technology. In some embodiments, cells bound to the second antibody or its antigen-binding fragment are separated by fluorescence activated cell sorting (FACS). In some embodiments, cells bound to the second antibody or its antigen-binding fragment are separated by DEPArray.

[0213] In some embodiments, the anti-TREML2 antibody is selected from sc-109096, ARP49877_P050, OACA04996, AF3259, MA5-30973, PA5-47471, ABIN634968, ABIN928294, 30-552, ABIN2463297, ABIN19999041, 11655-r001, ABIN749888, bs-2737r, ABIN1999045, 11655-rp02, ABIN293207, ABIN2387613, t8282-40, ABIN4249314, nbp1-70737-20ul and BD563661. Alternatively or additionally, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 1, 2, 3, 4, 5 or 6 CDRs selected from: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions. In some embodiments, any one of SEQ ID Nos: 6-11 independently comprises two or more amino acid substitutions, additions, or deletions.

[0214] In some embodiments, any of the methods disclosed herein further comprises providing a treatment recommendation based on the genetic testing results of the fetal cells.

[0215] In some embodiments, any of the methods disclosed herein further comprise administering a therapy to the subject based on the results of the genetic testing of the fetal cells.

[0216] In some embodiments, any of the methods disclosed herein further comprises recommending additional monitoring of the subject or fetus based on the genetic testing results of the fetal cells.

[0217] While the methods disclosed herein can recite the use of an anti-TREML2 antibody or antigen-binding fragment thereof or an antibody conjugate comprising the same, any of these methods can be carried out by using any agent that can bind to a TREML2 protein or a conjugate comprising an agent that can bind to a TREML2 protein. Thus, the methods disclosed herein are not limited to the use of an anti-TREML2 antibody or antigen-binding fragment thereof or an antibody conjugate comprising the same.

[0218] Agents that bind to a rare cell marker

[0219] Disclosed herein are agents that bind to a rare cell marker. As used herein, a “rare cell marker” is a marker (e.g., a cell surface protein) on a rare cell (e.g., a fetal cell). The rare cell marker can be a cell surface protein that is expressed at a higher level on a rare cell than another type of cell in a sample. The rare cell marker can be a Triggering Receptor Expressed On Myeloid Cells Like Transmembrane Protein 2 (TREML2) protein. The rare cell marker can be a human TREML2 protein. The human TREML2 protein can have the amino acid sequence of SEQ ID NO: 1. Alternatively, the rare cell marker can be CD71. In some embodiments, the rare cell marker is not CD71.

[0220] As used herein, the terms “TREML2” and “TLS1” refer to the same protein and are used interchangeably. TLS1 and TREML2 refer to the same marker having an amino acid sequence corresponding to SEQ ID NO: 1 and comprising domains and fragments having the amino acid sequences of SEQ ID Nos: 2-5.

[0221] As used herein, a “rare cell” refers to a cell that is present in a sample from a subject at a concentration of less than 10% of the total cell population, wherein the sample is an unpurified or unenriched sample. In some embodiments, the rare cell is present in the sample at a concentration of less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the total cell population. In some embodiments, the rare cell is present in the sample at a concentration of less than 1% of the total cell population. In some embodiments, the rare cell is a fetal cell and the sample is from a pregnant subject.

[0222] As used herein, the terms "unpurified sample" or "unenriched sample" may be used interchangeably and refer to a sample obtained from a subject that has not been processed in a manner to remove or isolate cells from the sample. Alternatively or additionally, an unpurified sample or unenriched sample refers to a sample obtained from a subject that has not been depleted of one or more cells. Alternatively or additionally, an unpurified or unenriched sample refers to a sample obtained from a subject that contains a plurality of different cell types.

[0223] In some embodiments, the reagent that binds to a rare cell marker is selected from an antibody, an antibody fragment, a receptor, and a ligand. In some embodiments, the antibody fragment comprises an antigen-binding domain of an antibody. In some embodiments, the antibody fragment is selected from a monovalent antigen-binding fragment (Fab or Fab'), a divalent antigen-binding fragment ((Fab)2 or (Fab')2), a variable fragment (Fv), a single-chain variable fragment (scFv), a divalent diabody, a triabody, a tetrabody, a minibody, and a bispecific scFv (bis-scFv).

[0224] Typically, a monovalent Fab fragment has one antigen-binding site, while a bivalent (Fab)2 fragment has two antigen-binding regions linked by disulfide bonds. H ) and light chain variable region (V L ) and heavy chain 1 constant region (C H 1 ) and light chain 1 constant region (C L 1 ) composition. V The fragment has a heavy chain variable region (V H ) and light chain variable region (V L ) composed of an antigen-binding site but lacking the constant region (C H 1 and C L ). V H and V L In F V The fragments are held together by non-covalent interactions. Fab can be a dimer (Fab2) or a trimer (Fab3), which allows binding to two or three different antigens, respectively.

[0225] The orientation of the V domains and the length of the linker can be varied to produce different forms of Fv molecules. In general, when the linker is at least 12 residues long, the scFv fragment is primarily monomeric. Linkers 3-11 residues long produce fragments that cannot fold into a functional Fv. V These molecules associate with a second scFv molecule, which creates a bivalent diabody. If the linker is less than three residues long, triabodies or tetrabodies can be formed. Minibodies are scFv-C molecules assembled into bivalent dimers.H 3 Fusion proteins. The bis-scFv fragment consists of an scFv fragment with two different variable domains and is able to bind to two different epitopes simultaneously.

[0226] The antibody may be a polyclonal antibody. Alternatively or additionally, the antibody may be a monoclonal antibody. The antibody may be an immunoglobulin gamma (IgG) antibody. The IgG antibody may be an IgG1 antibody. The IgG antibody may be an IgG2 antibody. The IgG antibody may be an IgG3 antibody. The IgG antibody may be an IgG4 antibody. The antibody may be an immunoglobulin mu (IgM) antibody. The antibody may be an immunoglobulin epsilon (IgE) antibody. The antibody may be an immunoglobulin delta (IgD) antibody. The antibody may be an immunoglobulin alpha (IgA) antibody. The IgGA antibody may be an IgGA1 antibody. Alternatively, the IgG antibody is an IgGA2 antibody.

[0227] In some embodiments, the agent is an antibody or antibody fragment that binds to a TREML2 protein. In some embodiments, the antibody or antibody fragment binds to the extracellular domain of the TREML2 protein. In some embodiments, the extracellular domain has the amino acid sequence of SEQ ID NO: 2. Alternatively, the antibody or antibody fragment can bind to a fragment of the extracellular domain of TREML2. The fragment of the extracellular domain has the amino acid sequence of SEQ ID NO: 3-4. The antibody or antibody fragment can bind to the N-terminal domain of the TREML2 protein.

[0228] In some embodiments, the anti-TREML2 antibody is a polyclonal antibody. The polyclonal antibody can be selected from an anti-TREML2 antibody selected from sc-109096 (Santa Cruz Biotechnology, Inc.), ARP49877_P050 (Aviva Systems Biology), OACA04996 (Aviva Systems Biology), AF3259 (R&D Systems), PA5-47471 (Thermo Fisher), ABIN634968 (Antibodies-online.com), ABIN928294 (Antibodies-online.com), 30-552 (ProSci), ABIN2463297 (antibodies-online.com), ABIN749888 (antibodies-online.com), bs-2737r (Bioss), ABIN1999045 (antibodies-online.com), 11655-rp02 (Sino Biological), ABIN293207 (antibodies-online.com), ABIN2387613 (antibodies-online.com), t8282-40 (USBio), ABIN4249314 (antibodies-online.com) and nbp1-70737-20ul (Novus Biologicals).

[0229] The anti-TREML2 antibody may be a monoclonal antibody. The monoclonal antibody may be selected from MA5-30973 (Thermo Fisher), ABIN19999041 (antibodies-online.com), 11655-r001 (SinoBiological) and BD563661 (Fisher Scientific).

[0230] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 1, 2, 3, 4, 5, or 6 CDRs selected from: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11.

[0231] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 1, 2, or 3 CDRs selected from: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; and (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8.

[0232] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of 1, 2, or 3 CDRs selected from: (a) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (b) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (c) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11.

[0233] In some embodiments, the anti-TREML2 antibody or antigen-binding fragment thereof comprises, consists of, or consists essentially of: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (b) a HCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; (c) a HCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8; (d) a light chain variable region (LCVR) CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; and (e) a LCVR CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (f) a LCVR CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11.

[0234] In some embodiments, any one of SEQ ID NO: 6-11 independently comprises one, two, or three or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 6 comprises one, two, or three or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 7 comprises one, two, or three or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 8 comprises one, two, or three or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 9 comprises one, two, or three or more amino acid substitutions, additions, or deletions. In some embodiments, SEQ ID NO: 10 comprises one amino acid substitution, addition, or deletion. In some embodiments, SEQ ID NO: 11 comprises one, two, or three or more amino acid substitutions, additions, or deletions.

[0235] In some embodiments, the anti-TREML2 antibody is conjugated to a label to produce a conjugated antibody. In some embodiments, the label is selected from the group consisting of a fluorescent label, a radionuclide, an enzymatic label, a chemiluminescent label, and a hapten. In some embodiments, the detectable label is a hapten. In some embodiments, the hapten is selected from the group consisting of DCC, biotin, nitrophenylpyrazole, thiazolesulfonamide, benzofurazan, and 2-hydroxyquinoxaline. In some embodiments, the detectable label is biotin. In some embodiments, the label is a fluorescent molecule. In some embodiments, the fluorescent molecule is selected from the group consisting of a fluorophore, a cyanine dye, and a near-infrared (NIR) dye. In some embodiments, the fluorescent molecule is fluorescein. In some embodiments, the fluorescent molecule is fluorescein isothiocyanate (FITC). In some embodiments, the label is selected from the group consisting of phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin. In some embodiments, the conjugated antibody is selected from the group consisting of ABIN6070559 (antibodies-online.com), abx307664 (Abbbexa, polyclonal), ABIN6070561 (antibodies-online.com), abx307665 (Abbbexa, polyclonal), ABIN2662892 (antibodies-online.com), bld-351203 (BioLegend), ABIN2662891 (antibodies-online.com), bld-351204 (BioLegend), ABIN2662890 (antibodies-online.com, monoclonal), and bld-351104 (BioLegend).

[0236] magnetic particles

[0237] The methods, compositions, and kits disclosed herein can comprise or use magnetic particles. For example, any of the antibodies (or more generally, any agent that binds to a rare cell marker) disclosed herein can be conjugated to a magnetic particle. In some embodiments, the agent that binds to a rare cell marker (e.g., TREML2) is conjugated to a magnetic particle. The magnetic particle can be a colloidal magnetic particle. The colloidal magnetic particle can be a ferrofluid.

[0238] As used herein, the term “magnetic particle” refers to a particle that can be manipulated using a magnetic field. Magnetic particles comprise a metal. Examples of metals include, but are not limited to, iron, nickel, cobalt, and copper.

[0239] As used herein, the term “colloidal magnetic particle” refers to a magnetic particle that is coated with a non-magnetic material. An example of a non-magnetic particle is bovine serum albumin (BSA).

[0240] As used herein, the term "ferrofluid magnetic particles" refers to colloidal magnetic particles containing iron.

[0241] In some embodiments, the magnetic particles are characterized by their submicron particle size. In some embodiments, the diameter of the particles is typically less than about 300 nanometers (nm), 275 nm, 250 nm, 225 nm, 200 nm, 190 nm, 180 nm, 170 nm, 160 nm, 150 nm, 140 nm, 130 nm, 120 nm, 110 nm, or 100 nm. In some embodiments, the diameter of the particles is typically at least 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, or 120 nm or larger. In some embodiments, the particle has a diameter between about 40 nm and 250 nm, 40 nm and 200 nm, 50 nm and 200 nm, 50 nm and 190 nm, 50 nm and 180 nm, 50 nm and 170 nm, 60 nm and 200 nm, 70 nm and 200 nm, 80 nm and 200 nm, 90 nm and 200 nm, 90 nm and 175 nm, or 90 nm and 150 nm.

[0242] In some embodiments, the particles have a magnetic quality of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% or more. In some embodiments, the particles have a magnetic quality of between about 40% and 95%, 45% and 95%, 50% and 90%, 55% and 90%, 60% and 90%, or 70% and 90%.

[0243] In some embodiments, particles in the range of 90-150 nm and having a magnetic quality between 70%-90% may be used.

[0244] In some embodiments, the particles are characterized in that they are resistant to gravity separation from a solution. The particles can resist gravity separation for an extended period of time. The particles can resist gravity separation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, or 120 minutes or longer. The particles can resist gravity separation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, or 120 hours or longer. The particles may resist gravity separation for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, or 120 days or more.

[0245] In some embodiments, magnetic particles are composed of a crystalline core of a superparamagnetic material surrounded by coating molecules, which are bound (e.g., physically adsorbed or covalently linked) to the magnetic core and impart stable colloidal properties. The coating material can be applied in an amount that effectively prevents the nonspecific interactions between the biomacromolecules found in the sample and the magnetic core. Such biomacromolecules can include sialic acid residues, lectins, glycoproteins, and other membrane components on the surface of non-target cells. In addition, the coating material can contain a magnetic mass / nanoparticle ratio as high as possible. The size of the magnetic crystals constituting the core is sufficiently small so that they do not contain complete magnetic domains. The size of the nanoparticles is such that their Brownian energy exceeds their magnetic moment. Therefore, even in a magnetic field of moderate intensity, the north pole-south pole arrangement of these colloidal magnetic particles and subsequent mutual attraction / repulsion will not occur, which contributes to their solution stability.

[0246] The magnetic particles can be separated in a high magnetic gradient external field separator. This feature facilitates sample handling and offers an economic advantage over more complex internal gradient columns loaded with ferromagnetic beads or steel wool.

[0247] Magnetic particles may be prepared by modification of base materials as described in EP0842042, which is incorporated by reference in its entirety.

[0248] The magnetic particles can be coated with an Ab (or more generally any reagent) that recognizes a differentially expressed protein corresponding to the top candidate identified in Example 1. In some embodiments, the magnetic particles can be coated with a reagent that binds to a rare cell marker (e.g., TREML2). The magnetic particles can be coated with any antibody or reagent disclosed herein.

[0249] Coating of the magnetic particles can be performed by any method known in the art. For example, the magnetic particles can be coated with antibodies as described in US6365362B1, which is incorporated by reference in its entirety.

[0250] Figure 2 depicts an exemplary ferrofluid magnetic particle structure. The ferrofluid magnetic particles disclosed herein may include, consist of, or consist essentially of the ferrofluid magnetic particle structure shown in Figure 2. In some embodiments, the ferrofluid magnetic particles disclosed herein have the ferrofluid magnetic particle structure shown in Figure 2. As shown in Figure 2, the exemplary ferrofluid magnetic particle structure includes, consists of, or consists essentially of iron atoms surrounded by bovine serum albumin (BSA). BSA is connected to streptavidin (SA), which is connected to biotin (BT). BT can be connected to another BSA, which is connected to an exogenous aggregation enhancing factor (e.g., desthiobiotin (Dt-BT)). BT can also be connected to an antibody (Y) that binds to a marker on a rare cell. In some embodiments, the rare cell is a fetal cell. In some embodiments, the marker is TREML2. Alternatively, the marker is EpCAM, CD105, or CD71.

[0251] Figure 7 depicts a schematic diagram of magnetic particle aggregation via controlled aggregation. As shown in Figure 7, magnetic particles (such as the ferrofluid magnetic particles of Figure 2) are coupled with an exogenous aggregation enhancing factor (EAEF, such as desthiobiotin (Dt-BT)). Adding a second EAEF (e.g., streptavidin (SA)) capable of binding to the first EAEF promotes the aggregation of the antibody-magnetic particle conjugate. In some embodiments, the aggregation of the antibody-magnetic particle conjugate is reversed by adding a third EAEF, wherein the third EAEF is capable of binding to the first EAEF or the second EAEF. In some embodiments, the third EAEF is identical to the first EAEF. Alternatively, the third EAEF is identical to the second EAEF. In another embodiment, the third EAEF is a binding partner (e.g., biotin) of the first or second EAEF.

[0252] Compositions and kits

[0253] Disclosed herein are compositions and kits comprising any of the anti-TREML2 antibodies or antigen-binding fragments thereof disclosed herein. The composition or kit may further comprise one or more components selected from the group consisting of a magnetic agent, one or more additional antibodies or antibody conjugates, an aggregation inhibitor, and an aggregation factor.

[0254] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or an antigen-binding fragment thereof; and (b) a magnetic reagent.

[0255] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or an antigen-binding fragment thereof; and (b) colloidal magnetic particles.

[0256] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen-binding fragment thereof; and (b) one or more additional antibodies or antigen-binding fragments thereof.

[0257] Further disclosed herein are kits comprising (a) an anti-TREML2 antibody or an antigen-binding fragment thereof; and (b) a second antibody or an antigen-binding fragment thereof, wherein the second antibody binds to a protein expressed on the surface of fetal nucleated red blood cells (fnRBCs).

[0258] Further disclosed herein is a kit comprising (a) an anti-TREML2 antibody or an antigen-binding fragment thereof; and (b) a second antibody or an antigen-binding fragment thereof, wherein the second antibody is conjugated to a label.

[0259] Further disclosed herein is a kit comprising (a) a first anti-TREML2 antibody or an antigen-binding fragment thereof, wherein the first anti-TREML2 antibody or an antigen-binding fragment thereof is conjugated to magnetic particles; and (b) a second anti-TREML2 antibody or an antigen-binding fragment thereof, wherein the second anti-TREML2 antibody is conjugated to a label.

[0260] In some embodiments, the composition or kit comprises an anti-TREML2 antibody or antigen-binding fragment thereof, wherein the anti-TREML2 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region (HCVR) comprising, consisting of, or consisting essentially of: (i) a complementarity determining region (CDR) 1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6; (ii) a CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7; and (iii) a CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 8; and (b) a light chain variable region (LCVR) comprising, consisting of, or consisting essentially of: (i) a CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 9; (ii) a CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10; and (iii) a CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11. In some embodiments, any one of SEQ ID Nos: 6-11 independently comprises one or more amino acid substitutions, additions, or deletions.

[0261] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen binding fragment thereof; and (b) a buffer comprising an aggregation inhibitor.

[0262] In some embodiments, the kit comprises (a) an anti-TREML2 antibody or antigen binding fragment thereof; and (b) an exogenous aggregation enhancing factor.

[0263] Any of the compositions, kits, or methods disclosed herein can comprise one or more magnetic reagents. The magnetic reagents can comprise one or more magnetic particles. The magnetic reagents can comprise ferromagnetic particles, superparamagnetic particles. The magnetic reagents can comprise ferrofluid reagents.

[0264] As used herein, the term "ferromagnetic particle" refers to a particle that can be permanently magnetized.

[0265] The magnetic reagents can comprise superparamagnetic particles. As used herein, the term "superparamagnetic particle" can refer to a particle that is a magnetically responsive particle. Superparamagnetic particles are particles that exhibit magnetic behavior only when subjected to a magnetic field. In some embodiments, the colloidal magnetic particles are superparamagnetic particles.

[0266] In some embodiments, the magnetic reagents comprise magnetic particles. In some embodiments, the magnetic particles have a size of about 1.5 to about 50 microns, 0.7-1.5 microns, or less than 200 nm. In some embodiments, the magnetic particles have a size of less than 200 nm. In some embodiments, the magnetic reagents comprise magnetic particles conjugated to an antibody. In some embodiments, such an antibody conjugated to a magnetic particle is an antibody that binds to a protein selected from the group consisting of epithelial cell adhesion molecule (EpCAM) and endoglin (CD105). Alternatively, such an antibody conjugated to a magnetic particle binds to CD147. In further embodiments, such an antibody conjugated to a magnetic particle binds to CD45. In another embodiment, such an antibody conjugated to a magnetic particle binds to a protein expressed on the surface of a fetal cell.

[0267] In some embodiments, the magnetic reagent comprises a ferrofluid reagent. As used herein, the term "ferrofluid reagent" refers to a liquid suspension comprising magnetic particles. In some embodiments, the ferrofluid reagent comprises a liquid suspension containing magnetic particles conjugated to the anti-TREML2 antibody. Alternatively, the ferrofluid reagent comprises a liquid suspension containing magnetic particles conjugated to one or more antibodies disclosed herein. In some embodiments, the ferrofluid reagent comprises a liquid suspension containing magnetic particles conjugated to anti-EpCAM antibodies. In some embodiments, the ferrofluid reagent comprises a liquid suspension containing magnetic particles conjugated to anti-CD105 antibodies. In some embodiments, the ferrofluid reagent comprises a liquid suspension containing magnetic particles conjugated to antibodies that bind to proteins expressed on the surface of fetal cells. In some embodiments, the ferrofluid reagent comprises a liquid suspension containing magnetic particles conjugated to anti-CD147 antibodies.

[0268] In some embodiments, the kit further comprises one or more staining reagents. In some embodiments, the one or more staining reagents comprise one or more antibody conjugates. In some embodiments, the antibody conjugate of the one or more antibody conjugates is an antibody conjugated to a label. In some embodiments, the antibody binds to a protein selected from CD71, glycophorin A (GPA), and CD45.

[0269] In some embodiments, any of the antibodies disclosed herein (e.g., anti-TREML2 antibodies or one or more additional antibodies) further comprises a label. In some embodiments, the label is conjugated to the antibody. In some embodiments, the label is selected from phycoerythrin (PE), allophycocyanin (APC), horseradish peroxidase (HRP), and biotin.

[0270] Any kit disclosed herein may comprise one or more antibodies or fragments thereof. The one or more antibodies may bind to a protein expressed on the surface of fetal cells. Alternatively or additionally, the one or more antibodies may bind to a protein expressed on the surface of maternal cells. The one or more antibodies may bind to a protein selected from the group consisting of EpCAM, CD105, CD147, CD15, CD71, GPA, and CD45. The one or more antibodies may bind to a protein selected from the group consisting of CD15, CD71, GPA, and CD45.

[0271] Any kit disclosed herein may comprise one or more antibodies or fragments thereof, wherein the one or more antibodies bind to a protein expressed on the surface of fetal nucleated red blood cells (fnRBC) or trophoblast cells. The antibody may bind to a protein selected from the group consisting of EpCAM, CD105, CD71, and CD147.

[0272] Any kit disclosed herein may include one or more aggregation inhibitors. The kit disclosed herein may include 1, 2, 3, 4 or 5 or more aggregation inhibitors. The aggregation inhibitor can inhibit endogenous ferrofluid aggregation factors. In some embodiments, the aggregation inhibitor is selected from a reducing agent, an immune complex, a chelating agent and diaminobutane. The reducing agent can be mercaptoethanesulfonic acid. The aggregation inhibitor can be bovine serum albumin (BSA). The chelating agent can be EDTA.

[0273] The aggregation inhibitor may comprise an antibody or a fragment thereof, wherein the antibody is of the same isotype as the anti-TREML2 antibody. The antibody may be a non-specific antibody. In some embodiments, the antibody is a mouse antibody.

[0274] Any kit disclosed herein may include an anti-TREML2 antibody, wherein the anti-TREML2 antibody may be coupled to a ferrofluid. Any kit disclosed herein may include an anti-TREML2 antibody, wherein the anti-TREML2 antibody is conjugated to magnetic particles. The magnetic particles may be colloidal magnetic particles. The magnetic particles may be ferrofluid magnetic particles.

[0275] Any kit disclosed herein can include exogenous aggregation enhancing factor (EAEF). In some embodiments, the kit disclosed herein includes 1, 2, 3, 4 or 5 kinds or more EAEF. In some embodiments, the magnetic particles disclosed herein are coupled with one or more EAEF. In some embodiments, the EAEF includes a member selected from the following specific binding pairs: biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analogue-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin and iminobiotin-avidin.

[0276] In some embodiments, the kits disclosed herein comprise two or more EAEFs. In some embodiments, the first EAEF comprises one member of a specific binding pair selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin, and the second EAEF comprises the other member of the specific binding pair.

[0277] In some embodiments, the kit disclosed herein further comprises a third EAEF. In some embodiments, the third EAEF is identical to the first EAEF. Alternatively, the third EAEF is identical to the second EAEF. In another embodiment, the third EAEF is capable of interacting with the first EAEF. In another embodiment, the third EAEF is capable of interacting with the second EAEF. By having a third EAEF that is identical to the first or second EAEF or that is capable of interacting with the first or second EAEF, the addition of the third EAEF results in a reversal of the aggregation of the magnetic particles.

[0278] In some embodiments, the kit disclosed herein further comprises one or more aggregation inhibitors. In some embodiments, the aggregation inhibitor is selected from a reducing agent, an immune complex, a chelating agent, and diaminobutane. In some embodiments, the aggregation inhibitor is a chelating agent. In some embodiments, the chelating agent is EDTA. The reducing agent can be mercaptoethanesulfonic acid. The aggregation inhibitor can be bovine serum albumin (BSA).

[0279] Example

[0280] Example 1: Identification of novel markers for fetal cells

[0281] This example describes the identification of novel markers for fetal cells.

[0282] Preparation of nucleated red blood cells (nRBCs)

[0283] Pregnant women who underwent ultrasound-guided procedures for planned surgical termination of pregnancy (10 +0 15 +6 Fetal whole blood was obtained at gestational age (n=5).

[0284] 20 mL of peripheral blood was collected from pregnant women at delivery (n=2) or before surgical termination of pregnancy (n=1).

[0285] After collection, fetus and maternal blood were diluted with an equal volume of phosphate buffered saline (PBS) and slowly layered onto a Percoll gradient. Samples were centrifuged at room temperature for 10 minutes at 1800 rpm. The interphase containing fetus or adult erythroblasts was collected and washed twice with PBS.

[0286] For maternal blood, a depletion step of CD45 / CD15 positive cells was performed by labeling cells with anti-CD45 and anti-CD15 microbeads (Miltenyi Biotec) using LD columns (Miltenyi Biotec).

[0287] For maternal blood, microfluidic devices were also used to remove RBC contaminating cells and enrich for adult erythroblasts.

[0288] Enrichment and cell sorting by flow cytometry

[0289] To prepare samples for FACS sorting, enriched cells from fetal and maternal blood were stained with anti-CD71 antibody (Miltenyi Biotec), anti-GPA antibody (BD Bioscience), anti-CD45 antibody (Miltenyi Biotec), Hoechst (nuclear stain), and Sytox Green dye (live / dead cells) for 30 minutes at room temperature.

[0290] FACS sorting

[0291] Erythroblasts were gated and sorted as shown in Figures 5A-5E. Figure 5B: Gate FSC-H / W and exclude doublets. Figure 5C: Gate Sytox Green-negative live cells. Figure 5D: Gate dp GPA / Hoechst. Figure 5E: Gate CD71-positive / CD45-negative cells.

[0292] For fetal blood samples, fewer than 200,000 target erythroblasts were sorted.

[0293] For maternal blood samples, the maternal erythroblast count never exceeded 1,000.

[0294] RNA extraction from sorted populations

[0295] The treated and sorted cells were used for total RNA extraction.

[0296] Total RNA was extracted from sorted cells using the Picopure RNA isolation kit (Applied Biosystems) and quantified by the Quant-iT RiboGreen RNA assay kit (Thermo Fisher), and quality control was analyzed on an Agilent 2100 bioanalyzer using the RNA 6000 Pico kit.

[0297] RNAseq preparation and library preparation

[0298] cDNA libraries were prepared according to Illumina sequencing (Appendix A; RNA-Seq protocol) and sequenced on a HiSeq 2000 at 20 million reads per sample.

[0299] Sequencing

[0300] The quality of reads obtained by next-generation (Illumina) sequencing was first checked using the FastQC protocol using standard procedures, then mapped to the reference genome and subsequently quantified using STAR software version 2.5. The size of the resulting read-count matrix (i.e., a table of read counts for all detected features (coding or non-coding RNA) in each sample) was reduced using a data reduction step, retaining only genes with at least a single count in a single sample.

[0301] Data analysis using bioinformatics tools

[0302] The resulting data were further processed for differential expression using the DESeq2 R / Biocondutor package to find genes that were significantly more or less expressed between the two sample types being compared.

[0303] A default DESeq2 differential expression analysis was performed consisting of the following steps: for each sample, the size factor was estimated using the “median ratio method” (Anders and Huber, 2010), for each gene, an estimate of the dispersion was found using a fitting procedure that optimizes the dispersion of negative binomial distributed data, and finally the obtained size factor and dispersion estimates were used to test the significance of the coefficients of the fitted distribution.

[0304] Finally, the result table from the DESeq2 analysis was extracted to obtain the base mean, log2 fold change, standard error, test statistic, p-value, and adjusted p-value across samples for the 20,205 features (genes) with non-zero total read counts. Differentially expressed genes were filtered based on an adjusted p-value (Benjamini-Hockberg / FDR method) cutoff of 0.01 and an expression cutoff of 2-fold change. Using these parameters, 3,233 differentially expressed genes were selected, the majority of which (2,961) were upregulated in fetal blood (independent of the fold change cutoff).

[0305] The resulting gene list was modified using annotations and functional descriptions extracted from the Ensembl database. Genes associated with the "plasma membrane" annotation were annotated according to the Gene Ontology term GO:0005886 and were additionally labeled as "transmembrane." Data were obtained from the Uniprot database. Of these, 366 plasma membrane genes were differentially expressed, with the majority (336) being upregulated in fetal cells (independent of the fold-change cutoff).

[0306] In parallel with the differential expression analysis, DESeq2 was used to normalize and transform the read counts so that selection could be made according to more stringent expression criteria: a first selection was made starting from a list of genes expressed only in fetal samples, i.e., those with zero reads in all three maternal samples (12,187 genes, list "ALL DATA"), according to the following criteria: 1) genes detected (i.e., expressed) in all fetal samples were selected; 2) genes with an expression mean / standard deviation ratio higher than 1 were selected; 3) genes that were both associated with the plasma membrane GO tag and labeled as "transmembrane prediction" by the Uniprot database were selected. See the selection scheme below. The resulting 77 genes were then ranked according to descending fetal average expression (list "RANKED"). Final selection (16 genes, list "Selected") was made manually, taking into account known biological functions, the stability of expression levels between samples, and a rough estimate of absolute expression levels (read segment compared to gene length), antibody availability, and other biological considerations.

[0307] Select a plan

[0308] The following is a selection of potential novel markers for identifying fetal cells:

[0309] 1) Genes not expressed in any maternal sample: 12187

[0310] 2) Genes expressed only in all fetal samples: 2079

[0311] 2.1) Genes annotated as related to GO plasma membrane terms: 213

[0312] 2.2) Genes predicted as transmembrane by UniProt: 305

[0313] 3) Genes that are both plasma membrane-related and predicted to be transmembrane: 89

[0314] 4) Genes with mean / standard deviation ratio higher than 1: 77

[0315] Ranking of differentially expressed genes from gene lists

[0316] A further final manual selection and ranking procedure was performed taking into account transcript length, read number and other biologically relevant criteria, and the resulting top candidates were as follows:

[0317]

[0318] Identification of antibodies specific for selected target molecules

[0319] Testing of erythroblastic Ab candidates by FACS analysis

[0320] To determine whether differential expression at the RNA level was reflected at the level of the corresponding protein, immunostaining was performed with commercially available antibodies (n=13) for flow cytometry and DEPArray analysis.

[0321] As a negative control, an isotype-matched Ab conjugated to the same fluorochrome as the commercial antibody was used at the same concentration.

[0322] All 13 antibodies shown in Table 2 were first tested on frozen fetal blood.

[0323] Antibodies that were positively expressed only on fetal erythroblasts were tested on frozen maternal blood samples.

[0324] In addition to the specific antibodies or isotype controls used for testing, the antibodies used for staining included CD71 Ab (Miltenyi Biotec), GPA Ab (BD Bioscience), CD45 Ab (Miltenyi Biotec), and Hoechst for erythroblast identification. Briefly, cells (2.5-5x10 5 ) were incubated with Ab in the presence of FcR blocking reagent (Miltenyi Biotec) at room temperature for 30 minutes. After washing away unbound Ab, the cell pellet was resuspended in AutoMACS running buffer (Miltenyi) containing Sytox Green dye.

[0325] Table 2: FACS analysis results

[0326]

[0327] Ab 1TLT2 for TREML2 (the latter is also referred to as TLS-1 in this article)

[0328] Example 2: Ferrofluid Technology for Cell Capture and Selection

[0329] In this example, an antibody selected to be expressed only by fetal cells was used for ferrofluid conjugation. TREML2-FF (also known as TLS1-FF) refers to an antibody conjugated to ferrofluid that binds to a protein expressed by the TLS1 gene.

[0330] The size of FF-Ab was checked by using a NanoBrook Zeta Plus particle size analyzer, and the concentration was checked using a spectrophotometer.

[0331]

[0332] Controlled enrichment

[0333] Before the ferrofluid is added to the blood, the blood sample is pre-incubated with a buffer containing one or more inhibitors that inhibit endogenous ferrofluid aggregation factors (as described in EP1311820, which is incorporated by reference in its entirety). One of the inhibitors can be a reducing agent, such as 100mM mercaptoethanesulfonic acid, which can invalidate the aggregation induced by IgM without affecting the ligand used to label the cells. The reducing agent can be added to the blood as a single reagent. The second inhibitor can be bovine serum albumin, which can be included in the buffer at 10mg / ml and will neutralize any HABAA. The third inhibitor can be a non-specific mouse antibody, particularly an appropriate isotype that matches the antibody on the ferrofluid. This can be included in the buffer at a concentration of 0.5-5mg / ml to neutralize even the most severe HAMA. If necessary, the fourth inhibitor can be streptavidin contained in the buffer to neutralize any anti-streptavidin antibodies present in the plasma. The blood can be pre-treated with the above-mentioned buffer and reducing agent for 15-30 minutes to neutralize all endogenous aggregation factors. After neutralizing all endogenous aggregation factors, an exogenous ferrofluid aggregation factor is added to the sample, followed by the ferrofluid. The ferrofluid is coupled to an antibody specific for the target and another ligand specific for the exogenous aggregation factor. After optimal labeling of target cells with the ferrofluid and induction of ferrofluid aggregation with the exogenous aggregation factor, the sample is magnetically separated to enrich the target.

[0334] The sample is placed in a magnetic separator (Immunicon catalog number QS-012) for 10 minutes. The sample is taken out from the magnet, and the sample is mixed by vortexing and put back into the magnetic separator for 10 minutes to collect magnetically labeled cells. The sample not collected is sucked out, and the magnetically collected cells are resuspended in 0.75ml washing dilution buffer and separated again for 10 minutes in the magnetic separator. The sample not collected is discarded, and after removing the tube from the magnetic separator, the collected cells are resuspended. After removing all non-targets, the target of the magnetic label and free ferrofluid are resuspended in buffer. In some cases, the ferrofluid aggregation of exogenous mediation should be reversed. This can be achieved by resuspending the final sample in a buffer containing a disaggregation factor combined with an exogenous aggregation factor. The disaggregation factor disaggregates all ferrofluid aggregates, making cells easy to further analyze.

[0335] Example 3: Detection and Analysis of Fetal Cells

[0336] This example describes the isolation and analysis of single fetal cells.DEPArray can be performed as described in EP2152859, which is incorporated by reference in its entirety.

[0337] Pregnant women and healthy volunteers

[0338] Peripheral blood samples were drawn by venipuncture from 14 pregnant women within 12 to 17+2 weeks of gestation into 10 mL CellSave preservative tubes (Menarini Silicon Biosystems, Huntington Valley, PA, USA). For spiking experiments, peripheral blood was drawn from healthy donors. All donors provided written informed consent and the study protocol was approved by the Medical Ethics Committee of San Gerardo Hospital, Monza (Italy). All samples were processed 1-4 days later.

[0339] Fetal trophoblast cells were enriched from contaminating cells using antibodies against epithelial cell adhesion antigen (EpCAM), vascular endothelial marker (CD105), and / or TREML2 coupled to ferrofluid. Enriched cells were labeled with anti-TREML2 monoclonal antibody (mAb) labeled with phycoerythrin (PE). Enriched cells were also fluorescently labeled with anti-cytokeratin mAb C11 labeled with allophycocyanin (APC), anti-HLA-G mAb labeled with APC, and anti-CD45 mAb labeled with fluorescein isothiocyanate (FITC) to identify leukocytes.

[0340] These enrichment procedures of target cells (e.g., trophoblast cells) are necessary because the frequency of said cells in maternal blood is known to be extremely low, only 1-10 cells in 1 ml of total blood, which contains more than a billion cells.

[0341] CVS and cord blood for spiking.

[0342] Whole blood from healthy volunteers was spiked with fetal trophoblast cells derived from chorionic villus sampling (CVS) or fetal erythroblasts derived from cord blood.

[0343] CVS cultures were selected for their CD105 / EpCAM expression. Cells were grown in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin (Gibco), and L-glutamine (Gibco) at 37°C and 5% CO2. Prior to spiking, cells were detached from flasks, resuspended in 10 ml PBS (Gibco), and placed in CellSave preservative tubes for at least 1 day.

[0344] Cord blood samples obtained by San Gerardo Hospital were collected into CellSave preservative tubes.

[0345] Spiking experiments were performed to demonstrate the specificity of the selection procedure when fetal cells are captured using ferrofluid-conjugated antibodies.

[0346] Fetal blood and bone marrow samples

[0347] Pregnant women who underwent ultrasound-guided procedures for planned surgical termination of pregnancy (10 +0 15 +6 Fetal whole blood was obtained at gestational age (n=3).

[0348] All donors provided written informed consent, and the study protocol was approved by the Medical Ethics Committee of KK Women's and Children's Hospital, Singapore.

[0349] After collection, fetal blood was diluted with an equal volume of PBS and slowly layered onto a Percoll gradient. Samples were centrifuged at room temperature for 10 min at 1800 rpm. The interphase containing fetal erythroblasts was collected and washed twice with PBS.

[0350] Cryopreserved bone marrow mononuclear cells containing adult erythroblasts were purchased (Lonza, catalog number 2M-125C). Following the manufacturer's instructions, the cells were thawed and treated with DNase I and washed. The cells were then placed in RPMI medium provided with 10% FBS, penicillin / streptomycin, L-glutamine at 37 degrees for 1 hour and used as a negative control (three different donors were tested).

[0351] Four clones of commercially available TREML2 antibodies were tested in the samples by using flow cytometry.

[0352] Isotype-matched Abs conjugated to the same fluorescent dye as the commercial TREML2 Ab were used at the same concentration. In addition to TREML2 Ab or isotype control, Abs used for staining included CD71 Ab (Miltenyi Biotec), GPA Ab (BD Bioscience), CD45 Ab (Miltenyi Biotec), and Hoechst. Briefly, cells (2.5-5x10 5 ) were incubated with Ab in the presence of FcR blocking reagent (Miltenyi Biotec) at room temperature for 30 min. After washing away unbound Ab, the cell pellet was resuspended in running buffer containing Sytox Green dye to gate live cells for FACS analysis.

[0353] Preparation of Desthiobiotin Ferrofluid Antibodies for Controlled Aggregation

[0354] In some embodiments, the ferrofluid for implementing the present invention is a particle that behaves as a colloid. The characteristic of such particles is that they are generally less than 200 nanometers (nm) of submicron particle size and their resistance to gravity separation from solution over a long period of time. Use particles within the range of 90-150nm and with a magnetic quality between 70%-90%. Suitable magnetic particles are composed of a crystalline core of a superparamagnetic material surrounded by coating molecules, which are bound (for example, physically adsorbed or covalently linked) to the magnetic core and impart stable colloidal properties. The coating material should preferably be applied in an amount that effectively prevents the nonspecific interaction between the biomacromolecules found in the sample and the magnetic core. Such biomacromolecules can include sialic acid residues, lectins, glycoproteins and other membrane components on the surface of non-target cells. In addition, the coating material should contain a magnetic quality / nanoparticle ratio as high as possible. The size of the magnetic crystals constituting the core is small enough so that they do not contain a complete magnetic domain. The size of the nanoparticles is such that their Brownian energy exceeds their magnetic moment. In a preferred embodiment of the present invention, magnetic particles coated with anti-CD105 antibodies are prepared by modifying the base material described in EP0842042. In a preferred embodiment of the present invention, magnetic particles coated with anti-CD105 antibodies are prepared by modifying the base material described in EP0842042. In a preferred embodiment of the present invention, magnetic particles coated with anti-CD105 antibodies are prepared by modifying the base material described in EP084204204204205. In a preferred embodiment of the present invention, magnetic particles coated with anti-CD105 antibodies are prepared by modifying the base material described in EP08 ...

[0355] Recombinant human antibodies for CD105 antigen are obtained from hybridoma numbering 166707 (R&D Systems) and coupled to base material by standard coupling chemistry, as described in U.S. Patent Application No. 09 / 248,388. The CD105 Ab ferrofluid is then resuspended in 20mM HEPES (pH 7.5) to be conjugated with desthiobiotin using N-hydroxysuccinimide-DL-dethiobiotin (NHS-dethiobiotin) (Sigma, catalog number H-2134). The stock solution of NHS dethiobiotin is prepared in DMSO at 1mg / ml. NHS-dethiobiotin (5mg) is added to 1mg CD105Ab ferrofluid and incubated at room temperature for 2 hours. Unreacted NHS-dethiobiotin is removed by washing three times with 20mM HEPES (pH 7.5) containing 1mg / ml BSA, 0.05% Proclin 300 using a high gradient magnet. After the final wash, the desthiobiotin / CD105 Ab ferrofluid was resuspended in water / BSA / Proclin 300 and filtered through a 0.2 μm syringe filter. The iron concentration of the CD105 Ab ferrofluid was determined spectrophotometrically and adjusted to 0.22 mg / ml. Particle size was determined using a NanoBrook 90 Plus particle size analyzer (Brookhaven Instruments Corporation).

[0356] Anti-CD71, anti-TREML2, and anti-EpCAM antibodies were conjugated to ferrofluids by using the same method.

[0357] Processing blood

[0358] A 7.5 ml aliquot of blood was diluted with 6.5 ml dilution buffer (Menarini Silicon Biosystems).

[0359] Before the ferrofluid is added to the blood, the blood sample (7.5 ml aliquot) is pre-incubated with 6.5 ml of dilution buffer (Menarini Silicon Biosystems) containing one or more inhibitors that inhibit endogenous ferrofluid aggregation factors (as described in EP1311820, which is incorporated by reference in its entirety). One of the inhibitors can be a reducing agent, such as 100 mM mercaptoethanesulfonic acid, which can invalidate the aggregation induced by IgM without affecting the ligand used to label the cells. The reducing agent can be added to the blood as a single reagent. The second inhibitor can be bovine serum albumin, which can be included in the buffer at 10 mg / ml and will neutralize any HABAA. The third inhibitor can be a non-specific mouse antibody, particularly an appropriate isotype that matches the antibody on the ferrofluid. This can be included in the buffer at a concentration of 0.5-5 mg / ml to neutralize even the most severe HAMA. If desired, the fourth inhibitor can be streptavidin included in the buffer to neutralize any anti-streptavidin antibodies present in the plasma. The blood can be pretreated with the above buffer and reducing agent for 15-30 minutes to neutralize all endogenous aggregation factors. During this incubation time, the diluted blood is centrifuged at 800 g for 10 minutes at room temperature without braking to remove the plasma.

[0360] After neutralizing all endogenous aggregation factors, an exogenous ferrofluid aggregation factor (streptavidin) is added to the sample, followed by the ferrofluid. The ferrofluid is coupled to an antibody specific for the target and another ligand specific for the exogenous aggregation factor, such as desthiobiotin (binding pair desthiobiotin-streptavidin).

[0361] Anti-CD105 ferrofluids, anti-EpCAM ferrofluids, and / or anti-TREML2 ferrofluids are used to enrich fetal trophoblasts. Anti-CD71 ferrofluids and / or anti-TREML2 ferrofluids are used to enrich fetal erythroblasts. After optimal labeling of target cells with the ferrofluids and induction of ferrofluid aggregation with exogenous aggregation factors, the sample is magnetically separated to enrich the target.

[0362] The sample is placed in a magnetic separator (Immunicon catalog number QS-012) for 10 minutes. From the magnet, take out the sample, and by vortex mixing sample and put back in the magnetic separator for 10 minutes. The sample is taken out from the magnet, and mixed again and put back in the magnetic separator for 20 minutes to collect magnetically labeled cells. The sample that is not collected is sucked out, and the cells collected by magnetism are resuspended in 3ml washing dilution buffer and separated again in the magnetic separator for 10 minutes. Abandon the sample that is not collected, and after taking out the tube from the magnetic separator, the cells collected are resuspended. After removing all non-targets, the target of the magnetic label and free ferrofluid are resuspended in buffer. In some cases, the ferrofluid of reversal exogenous mediation is aggregated. The reversal of aggregation (in the case of combining desthiobiotin-streptavidin, the exogenous reagent that reverses aggregation can be biotin) can be realized by the final sample being resuspended in the buffer containing the depolymerization factor combined with exogenous aggregation factor. Without wishing to be bound by theory, the disaggregating factor disaggregates all ferrofluid aggregates, leaving the cells for further analysis.

[0363] For trophoblasts, enriched cells were fluorescently labeled with anti-TREML2 monoclonal antibody (mAb) labeled with phycoerythrin (PE). For trophoblasts, enriched cells were also fluorescently labeled with a nucleic acid dye (Hoechst 33342) for DNA staining, anti-cytokeratin mAb C11 labeled with allophycocyanin (APC), anti-HLA-G mAb labeled with APC, and / or anti-CD45 mAb labeled with fluorescein isothiocyanate (FITC) to identify leukocytes.

[0364] For erythroblasts, the enriched cells were fluorescently labeled with anti-TREML2 monoclonal antibody (mAb) labeled with phycoerythrin (PE). For erythroblasts, the enriched cells were also fluorescently labeled with a nucleic acid dye (Hoechst 33342) for DNA staining, anti-CD71 monoclonal antibody (mAb) labeled with phycoerythrin (PE), and / or anti-CD45 mAb labeled with fluorescein isothiocyanate (FITC).

[0365] The stained cells were fixed with 2% paraformaldehyde (PFA) for 20 min at room temperature, then washed and resuspended in the appropriate buffer and volume for DEPArray. TM NxT system (Menarini Silicon Biosystems) or FACS analysis.

[0366] DEPArray analysis

[0367] DEPArray TMNxT is a semiconductor-based technology for precise isolation of pure single cells. TM The system consists of a control unit and disposable cartridge, which combines state-of-the-art microfluidics and silicon biochip technologies to gently manipulate each single target cell in the enriched sample.

[0368] The phenomenon that allows cells to be manipulated within the chip is called "dielectrophoresis" and is based on the ability to polarize particles within a liquid suspension medium through the action of an electric field. This polarization generates a force field that can be used to trap each individual particle in an array of potential wells, allowing the particle's position to be controlled. Each potential well can be controlled by modifying the chip's programming to move one or more particles from their initial location to their final destination for retrieval.

[0369] DEPArray TM Allows selection and isolation of rare cells with extremely high resolution (down to single cells) and very high purity; cells are selected by multiparametric analysis of fluorescence signals and morphological features obtained by processing brightfield or fluorescence images.

[0370] This technology has been used to isolate and select single circulating tumor cells in the blood of tumor patients (as described in EP1311820, which is incorporated by reference in its entirety).

[0371] Whole blood samples from healthy volunteers were spiked with chorionic villus cultures containing fetal trophoblasts with trisomy 21. Samples were enriched and stained as previously described.

[0372] In DEPArray TM Trophoblast cells were analyzed on the NxT system. Trophoblast cells showed positive staining for TREML2. In addition, cells that showed positive pan-cytokeratin (CK) staining and undetectable CD45 marker and positive nuclear staining were classified as fetal trophoblast cells and isolated into single cells.

[0373] Whole blood samples from healthy volunteers were spiked with umbilical cord blood containing fetal erythroblasts pre-labeled with Draq5 nuclear dye. Samples were enriched with CD71-Ab ferrofluid and TREML2-Ab ferrofluid and stained as described previously. TM Cells enriched for erythroid cells were analyzed on the NxT system. Cells showing positive staining for CD71, undetectable CD45 marker, and positive nuclear staining for Hoechst / Draq5 were classified as fetal erythroblasts.

[0374] Proof of fetal cell origin by short tandem repeat (STR) analysis

[0375] DEPArray was used according to the manufacturer's instructions. TM The isolated cells were lysed using LysePrep kit (MSB, Italy).

[0376] DNA from single cells was PCR amplified using the PowerPlex Fusion 6c Human DNA Amplification Kit (Promega TMD045), which consists of multiplex primer sets targeting 27 loci in the human genome.

[0377] As a control, genomic DNA was also isolated from 200 μl of maternal whole blood using the QIAgen DSP Blood Mini Kit (QIAgen).When available, fetal genomic DNA obtained from direct or cultured CVS tissue or amniotic fluid was also analyzed.

[0378] STR was performed according to the manufacturer's recommendations, and fragment analysis was performed using a ThermoFisher Scientific 3500 Genetic Analyzer (POP-4 and 36 cm capillary array); Analysis was performed using ID-X v1.4 software. The allelic patterns of isolated single cells were then compared with fetal and parental genomic DNA patterns to assess allelic dropout and expected inheritance patterns.

[0379] Point-of-care: A clinical study of 20 pregnant women in early pregnancy

[0380] Peripheral blood samples (20 ml) were collected from 14 pregnant women between 12 and 17+2 weeks of gestation by venipuncture into 10 mL CellSave preservative tubes (Menarini Silicon Biosystems, Huntington Valley, Pennsylvania, USA). All samples were processed after 1-4 days. Fetal trophoblasts were successfully isolated from the 14 pregnant women (Table X). An average of 1.4 fetal trophoblasts were isolated from the 14 positive pregnant women.

[0381] Copy Number Variation Analysis (CNV)

[0382] Whole blood samples from healthy volunteers were spiked with chorionic villus cultures containing fetal trophoblasts. Samples were enriched and stained as described previously.

[0383] From DEPArray TM Single fetal trophoblast cells recovered from NxT were subjected to whole genome amplification (Ampli1WGA, Menarini Silicon Biosystems).

[0384] Ampli1 products were purified according to manufacturer's instructions with 1.8X SPRIselect beads (Beckman Coulter) and eluted in 12.5 μΐ TE buffer for library preparation. TM WGA products and Ampli1 TM Lowpass kits (Menarini Silicon Biosystems) were eluted in 12.5 μΐ TE buffer for library preparation.

[0385] FASTQ files from 13 Ampli1 TM Lowpass libraries were aligned on the hg19 reference genome using BWA. Copy number profiles were calculated using Control-FREEC (without control samples and with GC normalization). Copy number plots were obtained using a custom python script.

[0386] Results

[0387] Figure 8 shows a schematic of the workflow for fetal cell enrichment. As shown in Figure 8, the workflow consists of 3 separate steps: 1. Collection of the sample and capture of target cells using ferrofluid conjugated antibodies that specifically select for the target cells. 2. Labeling of the target cells with the selected antibodies and loading into the DEPArray cartridge for screening and selection. Selected single cells are then sorted using the DEPArray instrument. 3. Analysis of the sorted single cells by STR (short tandem repeat) technology to prove their fetal cell origin.

[0388] In this example, fetal cells were enriched and stained from whole blood of a pregnant woman. By using the DEPArray TM Isolated pure single cells for whole genome amplification and genomic analysis.

[0389] Figures 9-10 demonstrate the specificity of the novel TREML2 antibodies as evidenced by flow cytometric analysis of TREML2 (i.e., TLS) expression on erythroblasts isolated from fetal blood (FB) (Figure 9) and bone marrow samples (BM) (Figure 10). As shown in Figures 9-10, erythroblasts isolated from fetal blood or bone marrow samples were gated by: (1) FSC-A / SSC-A gating the main cell population, (2) gating Sytox Green negative live cells, (3) FSC-H / W excluding doublet cells, (4) gating double positive GPA / Hoechst, (5) gating CD71 positive / CD45 negative, and (6) gating TLS and overlaying with isotype control to determine the % of TREML2 positive cells.

[0390] Figure 11A-Figure 11J shows TLS expression on the erythroblasts of various fetal blood (FB) samples isolated from various clones. Figure 12A-Figure 12L shows TLS expression on the erythroblasts of various bone marrow (BM) samples isolated from various clones. As shown in Figure 11A-Figure 11J, fetal erythroblasts from fetal blood show TREML2 antibody staining positive, while adult erythroblasts isolated from bone marrow can not detect expression (Figure 12A-Figure 12L).

[0391] Table 3. Size Measurement Ab Ferrofluid. The average diameter (nm) of CD105-FF is 128.70 nm.

[0392]

[0393] Table 4: Dimensional measurements of Ab ferrofluids. The average diameter (nm) of TREML-2-FF was 189.57, which is within the range of colloidal particles.

[0394]

[0395] Feeder blast cells derived from CVS cultures were used to demonstrate the specificity of CD105-FF and EpCAM-FF capture and enrichment.

[0396] Figure 13 shows the enrichment of CD105-FF and EpCAM-FF by DEPArray. TM Figure 14 shows a scatter plot analysis of the identified TREML-2 positive trophoblast cells. Image Gallery: Trophoblast cells showing positive staining for TREML-2-PE antibody, CK-APC, and nuclei.

[0397] Erythroblasts derived from umbilical cord blood were used to demonstrate the specificity of CD71 or TREML-2 capture and enrichment.

[0398] Figure 15A shows a scatter plot analysis of Draq5 / Hoechst positive erythroblasts spiked with healthy donor blood and enriched with CD71-FF. Figure 15B shows Image Gallery: Erythroblasts show positive staining with CD71-PE antibody, Draq5 and Hoechst nuclear staining, and negative staining with CD45-FITC antibody.

[0399] Figure 16A shows a scatter plot analysis of Draq5 / Hoechst positive erythroblasts spiked with healthy donor blood and enriched with TREML-2-FF. Figure 16B shows Image Gallery: Erythroblasts show positive staining with CD71-PE antibody, Draq5 and Hoechst nuclear staining, and negative staining with CD45-FITC antibody.

[0400] Sorted single cells were analyzed by STR (short tandem repeat) technology to prove their fetal cell origin (compared with maternal DNA and fetal DNA analysis derived from amniocentesis procedures). The same locus distribution was detected. Figure 17 shows STR analysis from single fetal cells.

[0401] In a preliminary clinical study, 14 pregnant women at various gestational weeks were recruited, and fetal cells obtained from blood samples from the pregnant women tested positive as shown by STR analysis.

[0402] Table 5 shows the summary of STR analysis.

[0403]

[0404] To demonstrate that we can detect chr21 trisomy (VK) from single-cell recovery of fetal cells from chorionic villus sampling, we performed copy number variation (CNV) analysis.

[0405] The results of CNV analysis of fetal cells are shown in Figure 18. As shown in Figure 18, single cell recoveries from DEPArray (e.g., from recovery 1 (R1), recovery 3 (R3), and recovery 6 (R6)) confirmed the presence of chr21 trisomy on the library from chorionic villus sampling (VK).

[0406] The results of CNV analysis of healthy donors are shown in Figure 19. As shown in Figure 19, healthy donors (HD) exhibited a flat copy number distribution, similar to those obtained by PBMC single cells isolated from DEPArray.

[0407] Example 4: Workflow Procedure for Selecting nRBCs from Maternal Blood

[0408] This example describes a method for selecting nucleated red blood cells (nRBCs) from a blood sample of a pregnant subject. As shown in FIG6 , a blood sample is collected from a pregnant subject ( 601 ). The blood sample is collected in a CellSave tube ( 601 ). nRBCs can be enriched by magnetic separation ( 602 , such as ferrofluid enrichment). Alternatively or additionally, a The system processes the sample (603). A DEPArray based on the control unit image can be used TMNxT technology is used to visualize and isolate individual cells (604). Once the cells are isolated, nucleic acids are purified from the isolated cells (605). Genomic and / or genetic analysis is performed (606). For example, nucleic acid molecules are sequenced to detect chromosomal abnormalities.

[0409] Example 5: RNA sequencing protocol

[0410] Nucleic acid molecules (such as RNA) can be isolated from rare cells (e.g., fetal cells).This example provides an exemplary method for sequencing RNA from fetal cells.

[0411] SMART-Seq V2

[0412] For RT-PCR, some modifications were made to Smartseq version 2.

[0413] For fetal erythroblasts (EBs), 2 ng of total RNA input was used for the reverse transcription reaction. For maternal EBs, due to the limited number of maternal EBs that could be sorted, all RNA was concentrated and used for the reverse transcription reaction.

[0414] (1) Reverse transcription

[0415] Add 1 ul of oligo dT 30VN primer (10 uM) and 1 ul of dNTP mixture (10 mM each) to the sample tube.

[0416] The samples were incubated at 72°C for 3 min and immediately placed on ice.

[0417] Reverse transcription mix was prepared on ice as follows and 5.7 ul was added to each sample.

[0418]

[0419]

[0420] Incubate the reaction in a thermal cycler as follows:

[0421]

[0422] (2) PCR pre-amplification

[0423] Prepare PCR mix on ice as follows and add 15 ul to each sample.

[0424] Components Volume (ul) Nuclease-free water 2.25 KAPA HiFi HotStart ReadyMix(2X) 12.5 IS PCR primers (10 μM) 0.25 sample 10

[0425] Place samples in a thermal cycler and run the following program.

[0426]

[0427] The number of PCR cycles depends on the cell type and can be increased (for cells with low RNA content) or decreased (for cells with more RNA).

[0428] (3) PCR purification

[0429] The amplified cDNA product was purified twice using AMPure XP beads (Beckman Coulter) in a 0.5X reaction volume. The purified cDNA was quantified on an Agilent 2100 Bioanalyzer using a High Sensitivity DNA Kit.

[0430] (4) Illumina Nextera XT DNA sample preparation

[0431] Libraries were prepared using the Illumina NEXTERA XT DNA kit with modifications (cDNA sample volume, reagents, and reaction volumes were optimized to 1 / 4 of the manufacturer's instructions)

[0432] The cDNA was diluted accordingly to obtain 300 pg.

[0433] 1.25ul cDNA (300pg) was aliquoted into 0.2 PCR tubes.

[0434] Add 2.5ul Tagment DNA buffer and 1.25ul Amplicon Tagment Miz.

[0435] The labeling reaction was incubated in a thermal cycler at 55°C for 5 min.

[0436] Immediately add 1.25 μl of NT and incubate at room temperature for 5 min.

[0437] 1.25ul Index 1, 1.25ul Index 2 and 3.75ul Nextera PCR Master Mix (NPM) were added to the labeled DNA.

[0438] Amplification was performed using the following procedure:

[0439]

[0440] (5) Library DNA cleanup (purification):

[0441] AMPure XP beads (0.6X reaction volume) were added to the library DNA.

[0442] Discard the beads and keep the supernatant for the first cleanup.

[0443] In a second cleanup, AMPure XP beads (0.7X reaction volume) were added.

[0444] The beads are retained and the DNA fragments are eluted.

[0445] Successful libraries (average 400 bp) were quantified on an Agilent 2100 Bioanalyzer using a High Sensitivity DNA Kit.

[0446] To pool libraries, each library sample was adjusted to 10 nM and pooled by volume.

[0447] (6) Library DNA sequencing:

[0448] The library was sent to the sequencing facility and sequenced using Illumina HiSeq TM Paired-end sequencing (2x101 bp) was performed using the High Output v3 System.

[0449] Example 6: Trophoblast cell detection

[0450] This example describes a method for detecting trophoblast cells. In this example, ferrofluid technology is used for cell capture and selection, and DEPArray technology is used for cell sorting.

[0451] Trophoblast cells are captured by ferrofluid technology and controlled aggregation. A blood sample from a pregnant subject is contacted with a ferrofluid containing colloidal magnetic particles (EpCAM-FF) conjugated to an anti-EpCAM antibody or colloidal magnetic particles (CD105-FF) conjugated to an anti-CD105 antibody. The blood sample contains multiple cells (fetal cells and maternal cells). A first exogenous aggregation enhancing factor (such as desthiobiotin) is conjugated to the colloidal magnetic particles. A second exogenous aggregation enhancing factor (such as streptavidin) is added to the sample. Without wishing to be bound by theory, adding a second exogenous aggregation enhancing factor induces the aggregation of colloidal magnetic particles, thereby making it easier to separate fetal cells and reduce contamination of non-fetal cells. The sample is applied to a magnetic separator, and cells bound to EpCAM-FF or CD105-FF are separated.

[0452] To help facilitate further analysis of cells that bind EpCAM-FF or CD105-FF, a third exogenous aggregation enhancing factor (such as biotin) is added to the isolated cells. Without wishing to be bound by theory, the addition of the third exogenous aggregation enhancing factor reverses the aggregation of the colloidal magnetic particles, which makes it easier to analyze single cells.

[0453] DEPArray technology for cell sorting: TLS1 (i.e., TREML2) is used as a candidate for staining of trophoblast cells. Isolated cells are stained with a fluorescently labeled anti-TLS antibody (i.e., an anti-TREML2 antibody), an anti-HLA-G antibody, and cytokeratin. The isolated and stained cell sample is applied to a DEPArray cartridge and analyzed using a DEPArray instrument. If the cells stain positively for TLS, HLA-G, and cytokeratin staining, they are identified as having trophoblast cells.

[0454] Example 7: Diagnosing fetal abnormalities

[0455] Fetal cells isolated or identified by any of the methods disclosed herein are further analyzed to diagnose fetal abnormalities. The fetal cells are subjected to karyotyping to detect chromosomal abnormalities. If a chromosomal abnormality is detected, the fetus is diagnosed with the corresponding disorder. For example, if three copies of chromosome 21 are detected, the fetus is diagnosed with Down syndrome. In another example, if three copies of chromosome 18 are detected, the fetus is diagnosed with Edwards syndrome.

[0456]

Claims

1. Use of a first antibody and a second antibody in the preparation of a kit for use in a method for detecting fetal erythroblasts or fetal trophoblasts in a sample from a pregnant subject, the method comprising: (a) contacting the sample with the first antibody, wherein the sample comprises a plurality of cells; (b) isolating cells that bind to the first antibody to produce an enriched sample; (c) contacting the enriched sample with the second antibody; and (d) identifying cells that bind to the second antibody as fetal cells, The first antibody: (i) an antibody that binds to triggering receptor-like transcription factor 2 protein on myeloid cells; or (ii) comprising an antigen-binding fragment that binds to a TREML2 protein; and wherein the second antibody: (i) an antibody that binds to triggering receptor-like transcription factor 2 protein on myeloid cells; or (ii) comprising an antigen-binding fragment that binds to the TREML2 protein.

2. The use according to claim 1, wherein the first antibody is conjugated to one or more magnetic particles.

3. The use according to claim 2, wherein the magnetic particles are colloidal magnetic particles.

4. The use according to claim 3, wherein the colloidal magnetic particles are ferrofluid magnetic particles.

5. Use according to any one of claims 2 to 4, wherein step (b) comprises subjecting the sample to a magnetic field.

6. The use according to claim 5, wherein the magnetic particles are coupled to a first exogenous aggregation enhancing factor EAEF, the first exogenous aggregation enhancing factor comprising a member of a specific binding pair comprising: Biotin-streptavidin, antigen-antibody, receptor-ligand, agonist-antagonist, protein A-antibody Fc, avidin-biotin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin or iminobiotin-avidin.

7. Use according to claim 6, wherein step (a) comprises adding a second EAEF to induce aggregation of the magnetic particles, the second EAEF comprising the other member of the specific binding pair.

8. The use according to claim 7, wherein step (b) comprises adding a member of the specific binding pair to the enriched sample to reverse aggregation of the magnetic particles in the enriched sample.

9. The use according to any one of claims 1 to 4, wherein the method further comprises, before step (a), adding at least one aggregation inhibitor to the sample, wherein the aggregation inhibitor is selected from a reducing agent, an immune complex, a chelating agent and diaminobutane.

10. The use according to claim 9, wherein the aggregation inhibitor is a chelating agent, and wherein the chelating agent is EDTA.

11. The use according to claim 1, further comprising, before step (d), isolating single fetal cells.

12. The use according to claim 11, wherein the single fetal cells are isolated by isolating single fetal cells that bind to the second antibody.

13. The use according to claim 12, wherein the second antibody is conjugated to a label.

14. The use according to claim 13, wherein the label is a fluorescent label.

15. Use according to claim 14, wherein the isolation of single fetal cells is based on immunofluorescence technology.

16. Use according to claim 15, wherein single fetal cells are isolated by fluorescence activated cell sorting.

17. The use according to claim 15, wherein single cells are isolated by DEPArray.

18. The use according to any one of claims 1 to 4, wherein step (d) comprises performing sequencing analysis.

19. The use according to claim 18, wherein the sequencing analysis comprises short tandem repeat analysis.

20. The use according to any one of claims 1 to 4, further comprising analyzing the fetal cells.

21. The use of claim 20, wherein analyzing the fetal cells comprises performing genomic or genetic analysis.

22. The use of claim 21, wherein performing genetic analysis comprises detecting the presence or absence of one or more genetic abnormalities in the fetal cells.

23. The use according to any one of claims 1 to 4, wherein the first antibody binds to a protein selected from the group consisting of EpCAM, CD105 and CD71.

24. The use according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment that binds to TREML2 protein comprises one or more CDRs selected from the group consisting of: (i) a heavy chain variable region HCVR complementarity determining region CDR1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) HCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 7; (iii) an HCVR CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iv) a light chain variable region LCVR CDR1 comprising the amino acid sequence of SEQ ID NO: 9; (v) an LCVR CDR2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi) an LCVR CDR3 comprising the amino acid sequence of SEQ ID NO:

11.

25. The use according to claim 6, wherein the receptor-ligand is a receptor-hormone.

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