CN111781360A

CN111781360A - Free cell capture probes and related products and uses

Info

Publication number: CN111781360A
Application number: CN202010714367.5A
Authority: CN
Inventors: 徐天宏; 高学娟
Original assignee: Baylor Zhilin Hangzhou Biotechnology Co ltd
Current assignee: Baylor Zhilin Hangzhou Biotechnology Co ltd
Priority date: 2020-07-23
Filing date: 2020-07-23
Publication date: 2020-10-16

Abstract

The invention discloses an isolated cell capture probe for specifically identifying and separating cells expressing placenta-like Chondroitin Sulfate (CSA) on the surface, and related products and application thereof, in particular to fetal trophoblast cells and malignant tumor cells. The method has the advantages of ingenious conception, safety, reliability, and high sensitivity and specificity.

Description

Free cell capture probes and related products and uses

Technical Field

The invention relates to the field of biomedicine, in particular to an isolated cell capture probe of CSA (surface-expressed protein A) cells, and a related product and application thereof. .

Background

Prenatal diagnosis in the early gestation stage is important to detect genetic defects such as genetic or chromosomal abnormalities in the fetus. Currently, the three prenatal screening and detecting methods widely used mainly include: amniotic fluid puncture, chorionic villus sampling, and fetal free dna (cfdna) detection. However, the three current technologies have certain limitations. Amniocentesis: a professional inserts a needle into a fetal amniotic sac, takes 20-30ml of amniotic fluid, and performs cytogenetic inspection on fetal cells in the amniotic fluid, usually at 16 weeks of gestation; chorion sampling: the skilled artisan takes a small biopsy sample of the placenta and performs a genetic test analysis, typically at 8-12 weeks. The two detection means can provide accurate information for clinical decision, but the detection means are invasive detection means, and have adverse factors of increasing miscarriage risk (0.6-2%), causing trauma to pregnant women and the like.

In a non-invasive prenatal diagnosis method, the detection of plasma free fetal DNA has been shown to have good sensitivity in the diagnosis of fetal aneuploidy diseases (21-trisomy, 18-trisomy, 13-trisomy). The second generation Non-invasive Prenatal Testing (Non-invasive Prenatal Testing) NIPT which is started in the last 10 years is based on the principle that a small amount of fetal fragment DNA exists in the peripheral blood of a pregnant woman, adopts a second generation sequencing technology to eliminate the interference of maternal DNA by depending on a statistical method, and detects whether a fetus has trisomy 21 or trisomy 13 and trisomy 18. The sensitivity and the specificity for trisomy 21, namely Down syndrome, are 90 percent and 96 percent respectively, and the sensitivity and the specificity for trisomy 13 and trisomy 18 are both about 90 percent respectively.

However, according to research analysis, cfDNA accounts for only about 3.4% and 6.2% of total plasma DNA in the early and late gestations, respectively, and its content is affected by the age, body weight, and time of pregnancy of the pregnant woman, and it is usually necessary to reach a ratio of 5% of peripheral plasma before 10-12 weeks after gestation to detect cfDNA. These factors limit its use in the diagnosis of other genetic diseases such as microdeletions, microduplications, point mutations, etc., which presents a great challenge for accurate analysis, making the detection of plasma-free fetal DNA a screening means rather than a diagnostic means.

The highly fragmented fetal free DNA and the vast majority of maternal DNA occupying the sequenced sample limit further improvement in NIPT accuracy and also limit the method to detect tens of thousands of other genetic diseases. Currently, NIPT tests are directed almost exclusively to down syndrome. At present, about 80-120 ten thousand birth defects of infants are born in China every year, wherein Down syndrome only accounts for about 2%, and 98% of newborn defects are not covered at all.

The isolation of foetal cells from maternal blood is a very advantageous method. The fetal cells in maternal blood are mainly two types, fetal nucleated red blood cells and fetal trophoblast cells. However, the fetal nucleated red blood cells are very rare in blood, only 1 fetal nucleated red blood cell may be contained in 106-107 maternal cells, the cellular characteristics of the fetal nucleated red blood cells are basically consistent with those of blood cells, the separation is very difficult, and the methods can only detect the fetal nucleated red blood cells at a later time (12-26 weeks) in pregnancy, so that the method has limitations. Trophoblast cells are smaller individual cells that begin to differentiate and spread and line up the inner wall of the zona pellucida after the embryo develops into morula, which further develops into morula, and placenta. Early studies showed that fetal trophoblast cells were present in the blood of pregnant women starting at 5 weeks gestation, and although the number of these cells increased with the week of pregnancy, the increase was not significant. Therefore, the method has great significance for the earliest prenatal screening and diagnosis by effectively separating the fetal trophoblast cells and obtaining fetal DNA.

It is reported that 10 to 180 fetal trophoblast cells per 10ml of blood are present in the blood of pregnant women between 5 and 26 weeks of gestation. Although there are various methods for detecting fetal trophoblast cells from maternal blood reported so far, there are various methods, such as microfluidic methods, which are based on the size difference between fetal cells and maternal blood cells, but these detection methods have not been clinically applied due to the defects of insufficient sensitivity and specificity, complicated operation, poor stability, etc. In addition, the proportion of fetal trophoblasts in maternal blood cells is particularly low, and no method is available at present for accurately marking the fetal trophoblasts in a large number of maternal blood cells, so that the fetal trophoblasts can be accurately separated from the maternal blood cells more difficultly. All methods reported so far are basically: only 1-5 fetal trophoblast cells can be detected per 10ml of maternal blood.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, there is a need for a method with high selectivity and specificity and relatively simple operation. Plasmodium falciparum expresses on the surface of infected erythrocytes an adhesion protein VAR2CSA which specifically binds to placental cells (i.e. fetal trophoblasts). The research finds that the VAR2CSA is specifically combined with a unique Chondroitin Sulfate (CS) glycosaminoglycan Chain (CSA) expressed by trophoblast cells, and the combination sequence is placenta chondroitin sulfate combined peptide (VAR2CSA-FCR 3). The invention discovers that CSA is only expressed on the surfaces of fetal cells and malignant tumor cells, and normal cells do not express CSA. The invention skillfully utilizes the characteristic that after the peripheral blood of a normal pregnant woman is treated by breaking red blood cells, trophoblast cells from a fetus are the only cell type for expressing CSA, and blood cells in the peripheral blood such as leucocytes or a small amount of endothelial cells do not express CSA. Therefore, when the peripheral blood of the pregnant woman is mixed with the VAR2CSA-FCR3 modified magnetic beads, the fetal trophoblast cells in the peripheral blood sample of the pregnant woman can be specifically bound and separated.

In addition to fetal trophoblast cells, since CSA is also expressed on the surface of malignant tumor cells, this method is also applicable to the specific identification and isolation of circulating tumor cells in the peripheral blood of malignant tumor patients.

The placental cells express a unique chondroitin sulfate CSA on their surface, enabling them to invade the mother and immobilize the placenta, the specific presence of 4-o-sulfur (C4S) on most of the GalNAc residues of a given CS chain. C4S can specifically bind to VAR2 CSA. The binding site of the VAR2CSA and the CSA is a polypeptide consisting of 28 amino acid residues, the polypeptide is called as the VAR2CSA-FCR3, the affinity binding coefficient is up to KD 15nM, and the CS expressed by normal tissues cannot be combined with the VAR2CSA-FCR3 because of no C4S modification. Earlier studies found that by combining synthetic VAR2CSA-FCR3 with drug-encapsulated liposomes, the drug could be successfully delivered to the placenta for treatment of gestational disorders.

In one aspect, the present invention provides a probe for identifying and/or isolating cells expressing placental-like Chondroitin Sulfate (CSA) on their surface, the probe being a magnetic bead modified with a binding peptide fragment of a plasmodium-infected erythrocyte surface antigen (VAR2 CSA).

Further, the CSA-surface expressing cells are selected from fetal trophoblast cells or malignant tumor cells.

Further, the binding peptide segment is a minimum binding peptide (VAR2CSA-FCR3) with the sequence of EDVKDINFDTKEKFLAGCLIVSFHEGKC SEQ ID NO.1

Further, the binding peptide is a longer polypeptide sequence comprising VAR2CSA-FCR 3.

Further, the VAR2CSA-FCR3 sequence or a longer polypeptide sequence comprising VAR2CSA-FCR3 may be chemically or biologically synthesized. The identification of glycosaminoglycan binding regions of plasmodium falciparum-encoding placental isolating ligand VAR2CSA, published at 104, stage 7 of malaria journal, 2008, by Mafalda resude, et al, at the university of copenhagen, succeeded in finding the 28-residue sequence constituting VAR2CSA-FCR3, by constructing a phage display library based on the entire VAR2CSA coding region. The nanoparticle targeted trophoblast specific drug delivery mouse placenta which is published by Zhangbazhen et al in biomedical and biotechnological research institute biomedical health laboratory of Shenzhen advanced technology institute of Chinese academy of sciences in J.theranostics 2018, No. 8, No. 10, 2765 and page 2781, modifies the nanometer by VAR2CSA-FCR3, and successfully carries the drug to mouse trophoblast cells.

The preparation of polypeptide-modified magnetic beads is a well-established prior art method, and a commonly used method for polypeptide-modified magnetic beads is given in example 1 of the present patent.

In a second aspect, the invention provides a method for isolating fetal trophoblast cells from a maternal blood sample during pregnancy using the probe described above and its use in fetal genetic diagnosis.

Further, the blood sample is a peripheral blood sample.

Further, the blood sample may be of mammalian origin, which may be a human, such as a pregnant woman.

Further, the blood sample is subjected to a pretreatment, i.e., to remove red blood cells from the sample.

Further, the method comprises:

1) adding the probe to the pretreated blood sample;

2) incubating for a period of time greater than 2 hours;

3) the target cells labeled with the probe are separated with a magnet.

In a third aspect, the invention provides a method for detecting or isolating circulating tumor cells from a body fluid using the probe and its use in tumor screening, diagnosis, detection of tumor progression or prognosis, and the like.

Further, the body fluid is blood or other biological fluid samples, such as urine, ascites, cerebrospinal fluid, pleural fluid, etc.

Further, the body fluid sample may be of mammalian origin, which may be a human.

Further, the method comprises:

1) adding the probe to the pretreated blood sample;

2) incubating for a period of time greater than 2 hours;

3) the target cells labeled with the probe are separated with a magnet.

Drawings

FIG. 1 shows a photograph taken by confocal microscope, FIG. 1A is blood of 5 males with prostate cancer cell line PC3, FIG. 1B is blood of 5 pregnant women carrying male infants, FIG. 1C is blood of 5 pregnant women carrying female infants, and FIG. 1D is blood of 5 non-pregnant females.

FIG. 2 shows the state of magnetic bead-cell binding.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring harbor LABORATORY Press, 1989 and Third edition, 2001; ausubel et al, Current PROTOCOLS Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; methodsin Enzymology, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1 preparation of free cell Capture probes that specifically recognize and isolate CSA surface-expressing cells

1. A biotin-labeled VAR2CSA-FCR3 minimal binding peptide (Zhongtai Biochemical Co., Ltd.) was chemically synthesized.

2. Commercial streptavidin magnetic beads (Shanghai medical Venai technology) were purchased.

3. Mixing the VaR2CSA-FCR3 with biotin label with streptavidin magnetic beads to obtain a free cell capture probe modified by the VAR2CSA-FCR3, and washing the free cell capture probe with PBS for 3 times under a high magnetic field for later use.

Example 2 free cell capture probes capture and isolate fetal trophoblast cells in the peripheral blood of pregnant women

1. Respectively extracting 10ml of blood from 5 pregnant women of 12 weeks old and 5 pregnant women of 12 weeks old by EDTA anticoagulation tube, respectively extracting 10ml of blood from 5 non-pregnant women, respectively extracting 10ml of blood from 5 male, respectively extracting 1 × 10ml of blood from 5 male³Epithelial tissue-derived cancer cell (male prostate cancer cell line PC 3); mixing erythrocyte lysate and blood volume of 1:1 respectively, and standing for 10 minutes at 4 ℃ (operating in sterile environment) to break red blood; gently rinsed 3 times with 1ml of DPBS;

2. after being respectively placed in a 37 ℃ cell culture box by 2ml of 2 mul for 30min and resuspended in a trophoblast cell culture medium containing 10 percent fetal calf serum, each sample is respectively transferred to 1 hole of a 6-hole plate for marking, and is cultured in the 37 ℃ cell culture box for one week;

3. sucking the upper culture solution containing cells into a 15ml centrifuge tube by using a pipette, digesting adherent cells for 3min by using trypsin, transferring the cells into the same centrifuge tube, centrifuging for 10min by 500g, removing a supernatant, lightly blowing and uniformly beating the cells by using 3ml of DPBS buffer solution, centrifuging for 10min by 500g, removing the supernatant, adding 500 mu l of trypsin for digesting for 5min, centrifuging for 10min by 500g, removing the supernatant, lightly blowing and uniformly beating the cells by using 3ml of DPBS buffer solution, centrifuging for 10min by 500g, removing the supernatant, and repeating the steps twice;

4. fully scattering and resuspending the cells by using 1ml of DPBS buffer solution, adding 200 mu l of prepared separated magnetic bead suspension with the surface connected with a VAR2CSA-FCR3 polypeptide sequence, and placing the suspension at 37 ℃ for incubation for 2h by inclined shaking;

5. washing the magnetic bead-cell combination incubated in the step 4 with DPBS buffer solution for 2 times, wherein 1ml of DPBS solution is used each time;

6. installing the separation column into a magnetic field, adding 0.5ml of DPBS buffer solution, and naturally draining under the action of gravity to pretreat the separation column;

7. connecting a speed limiting needle at the tail of the separation column, continuously passing the cells through the separation strain twice, and collecting the flowing-down cells.

8. Making collected cells into cell smear, and fixing with 50 μ l of 4% paraformaldehyde for 30 min; taking out paraformaldehyde, and gently washing with 100 μ l DPBS buffer solution for 3 times;

9. after treating the cells according to the instructions for use of the antibody specific to the sex-determining gene SRY and treating with the secondary antibody in red, gently washing 3 times with 100. mu.l of PBS buffer;

10. as shown in table 1, which is basic information corresponding to a pregnant woman who had been pregnant for a 12-week period, fig. 1 is a photograph taken by a confocal microscope, fig. a is blood of a 5-bit male containing a prostate cancer cell line PC3, fig. B is blood of a 5-bit pregnant woman who had been pregnant with a male, fig. C is blood of a 5-bit pregnant woman who had been pregnant with a female, and fig. D is blood of a 5-bit non-pregnant woman, and the photograph processed by the above steps, the free cell capturing probe can separate cells from all of the blood of the male containing tumor cells and blood of the pregnant woman, but can not separate cells from blood of the non-pregnant woman; cells isolated from both the blood of a male containing the prostate cancer cell line PC3 and the blood of a pregnant woman carrying a male infant can be stained with the sex-determining gene specific antibody SRY specific to the male, while cells isolated from the blood of a pregnant woman carrying a female infant cannot be stained with the sex-determining gene SRY specific antibody and cells cannot be isolated from the blood of an infertile female. Since only trophoblast cells in the blood of pregnant women were fetal cells after the erythrolysis treatment, it was determined that all cells isolated by the free cell capture probe were fetal trophoblast and prostate cancer cell line PC3 cells.

TABLE 1

Example results illustrate that:

in the embodiment, the pregnant woman blood cells of the girl baby of 12 weeks of pregnancy are selected as the negative control of the SRY antibody, because the cells contain fetal trophoblast cells and do not contain male cells, and the specific immunofluorescent staining for determining the SRY antibody does not stain female cells, so that the interference of maternal cells in the following results is eliminated; selecting a male specific tumor cell prostate cancer cell line PC3 containing a sex determination gene SRY, mixing the tumor cell prostate cancer cell line PC3 in male blood to serve as positive quality control of an SRY antibody, and determining the biological function of the SRY antibody; and (3) using the blood cells of the non-pregnant women to perform negative quality control on the free cell capture probe, and judging that the free cell capture probe cannot separate normal adult blood cells.

The results show that: in the blood cell group of the pregnant women who carry the baby girls for 12 weeks, the smear is provided with cells after the treatment, but the cells are not stained by the SRY antibody; in the blood cell group of the pregnant women who carry male infants for 12 weeks, the cells are smeared after the treatment, and the cells are all stained by an SRY antibody; in a male blood cell group added with a male specific tumor cell prostate cancer cell line PC3, after the treatment, cells are smeared, and the cells are all stained by an SRY antibody; the blood group of the non-pregnant women is coated without cells after the treatment. Thus, it was confirmed that all the cells separated by the cell-free capture probe in this example were fetal cells or tumor cells, and that all the cells separated by the cell-free capture probe in the blood of the pregnant woman were fetal trophoblast cells because only the fetal trophoblast cells in the blood of the pregnant woman were fetal cells after the erythrolysis treatment.

Example 3 testing of separation efficiency of free cell Capture probes to separate cells

1. 10ml of blood was collected from 3 women who were not pregnant and had no cancer history by using EDTA anticoagulant tubes, and after mixing the blood volume 1:1 with erythrocyte lysate, the mixture was left to stand at 4 ℃ for 10 minutes (operation was performed in a sterile environment); gently rinsed 3 times with 1ml of DPBS;

2. after being resuspended by 2ml of DPBS buffer solution, the mixture is respectively placed into a centrifuge tube of 5ml for marking, and after cell counting, 1000 fetal trophoblast cell lines HTR8-Svneo cells which are transfected and stably express GFP are added into each tube;

3. adding 200 mu l of prepared separated magnetic bead suspension with the surface connected with a VAR2CSA-FCR3 polypeptide sequence, placing at 37 ℃, and incubating for 2h by inclined shaking;

4. washing the magnetic bead-cell combination incubated in the step 3 with DPBS buffer solution for 2 times, wherein 1ml of DPBS solution is used each time;

5. installing the separation column into a magnetic field, adding 0.5ml of DPBS buffer solution, and naturally draining under the action of gravity to pretreat the separation column;

6. connecting an upper speed limiting needle at the tail of the separation column, continuously passing the cells through the separation strain twice, and collecting the flowing-down cells; the cells in the beads were observed and resolved under a fluorescent microscope and photographed, and the number of cells was counted using a blood cell counting plate, and the number of cells was averaged 3 times for each count, and the results are shown in Table 2, and the state of the bead-cell combination is shown in FIG. 2.

TABLE 2

Example results illustrate that:

since the blood cells themselves do not fluoresce, the efficiency and accuracy of separation of fetal trophoblasts from our free cell capture probes can be determined by the fact that only our mixed fetal trophoblast cell line HTR8-SVneo fluoresces green, observed under a fluorescence microscope, counted, and statistically analyzed as if all cells were green-fluorescent, and the percentage of recovered cells to the original cells in the whole mixture.

The results show that: all magnetic bead-bound cells fluoresced green, and 98.79% of fetal trophoblast cells HTR8-Svneo were recovered, indicating that our free cell capture probe had a sensitivity of 98.79% and a specificity of 100%.

Example 4 Whole genome sequencing of fetal trophoblast cells isolated from maternal peripheral blood

We amplified the whole genome of a single fetal trophoblast cell using a single cell multiple annealing circular cycle amplification (MALBAC) technique. The method comprises the following specific steps:

1. to 55. mu.l of Cell lysine Buffer was added 1.1. mu.l of CellLysis Enzyme according to the instructions for use of the MALBAC kit, and the mixture was gently swirled to mix well.

2. Each tube was filled with 4.5. mu.l of the cell lysis mixture in 200. mu.l of PCR tubes, and from the 10 fetal trophoblasts isolated in example 1, individual cells were picked up and labeled in the cell lysis mixture, and centrifuged by flicking the tube wall 10 times or so with the index finger.

3. And (3) incubating the mixed solution in the step 2 in a preheated PCR instrument according to the steps of 50 ℃ for 50min and 80 ℃ for 10 min.

4. Mu.l of Pre-Amp Enzyme Mix was added to 330. mu.l of Pre-Amp Buffer, and gently mixed by pipetting.

5. Mu.l of each of the mixtures obtained in step 4 was added to the PCR tube incubated in step 3, and pre-amplification was carried out according to the procedure shown in Table 3.

TABLE 3

6. To 330. mu.l of Amplification Buffer was added 8.8. mu.l of Amp Enzyme Mix, and the mixture was gently pipetted to Mix well.

7. Mu.l of each tube was added to the reaction system after amplification in step 5, and amplification was performed according to the procedure in Table 4.

TABLE 4

8. The above amplification products were subjected to whole genome sequencing by two-generation sequencing.

Example 5 isolation of circulating tumor cells in peripheral blood of patients with Lung cancer

1. 10ml of blood was collected from 4 patients with confirmed lung cancer by EDTA anticoagulation tube, mixed with erythrocyte lysate in a volume of 1:1, and left to stand at 4 ℃ for 10 minutes (operation was performed in sterile environment); gently rinsed 3 times with 1ml of DPBS;

2. the remaining steps were the same as in example 1 to isolate cells.

3. The basic information and isolated cell status of the patients are shown in Table 5.

TABLE 5

Example results illustrate that:

in the process of diagnosing lung cancer, imaging data are required, and for some patients with early stage and distant metastasis, the benign and malignant tumors and the primary focus can be determined even by sampling through an operation and then carrying out pathological detection. Not only is time-consuming and labor-consuming, but also brings great physical and mental damage to patients. Especially for patients who are resected by operation, the judgment of whether the recurrence happens or not needs to be carried out by a plurality of examinations, the parameter to be determined changes to be diagnosed, and the recurrence focus forms a certain scale at this time, thereby greatly increasing the medical difficulty and shortening the survival time of the patients. The diagnosis of tumors is ideal in that the benign or malignant tumor, the primary focus, or whether the tumor has recurred or not can be judged by blood test. However, this examination requires extremely high sensitivity and specificity to accurately isolate a very small number of tumor cells from a large number of blood cells. There are many methods for detecting circulating tumor cells, but they cannot be successfully applied to clinical diagnosis because of insufficient sensitivity and specificity of diagnostic probes.

As shown in Table 5, our free cell capture probe can accurately separate cancer cells from the blood of a patient with lung cancer without being affected by parameters such as the stage of the tumor and whether the patient is treated by surgery, and the separated cells are viable cells, which can be used not only for diagnosis of the tumor, but also for obtaining a large amount of tumor cells by cell culture in a short time. The cells can detect the gene mutation condition of a patient by methods such as second-generation sequencing and the like so as to determine the condition of the patient receiving targeted therapy, and can further study the drug dosage by a drug test of the cells so as to achieve the aim of accurate therapy.

Other embodiments

The foregoing is a detailed description and embodiments of the invention, and certain changes may be made in conjunction with the prior art within the scope of the claims without departing from the scope of the patent. The various features disclosed in this specification and drawings, both as to their own use and in various combinations with those of prior art, may be varied considerably without thereby affecting the technical advantages achieved. The embodiments disclosed in the specification are merely characteristic embodiments, and not all embodiments are intended to be limited.

Sequence listing

<110> Beile Zhi Lin (Hangzhou) Biotechnology Ltd

<120> free cell capture probes and related products and uses

<141>2020-07-22

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>28

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Glu Asp Val Lys Asp Ile Asn Phe Asp Thr Lys Glu Lys Phe Leu Ala

1 5 10 15

Gly Cys Leu Ile Val Ser Phe His Glu Gly Lys Cys

20 25

Claims

1. A probe for identifying and/or isolating cells that express placental-like Chondroitin Sulfate (CSA) on their surface, the probe comprising: magnetic beads modified with conjugated peptide fragments of plasmodium infected erythrocyte surface antigen (VAR2 CSA).

2. The probe of claim 1, wherein: the CSA-surface expressing cells are selected from fetal trophoblast cells or malignant tumor cells.

3. The probe of claim 1, wherein: the sequence of the binding peptide fragment is the minimum binding peptide (VAR2CSA-FCR 3): EDVKDINFDTKEKFLAGCLIVSFHEGKC SEQ ID the number of the carbon atoms in the carbon atoms is EDVKDINFDTKEKFLAGCLIVSFHEGKC SEQ ID NO.1,

or a polypeptide sequence comprising a minimal binding peptide of VAR2CSA-FCR 3.

4. Use of a probe according to any of claims 1 to 3 for the preparation of a product for identifying and/or isolating CSA-surface expressing cells.

5. Use according to claim 4, characterized in that: the CSA-surface-expressing cells are fetal trophoblast cells or malignant tumor cells.

6. A method of detecting and/or isolating CSA-surface expressing cells, the method comprising: adding a probe according to any one of claims 1 to 3 to a sample of cells to be detected; incubating for a period of time; isolating the cells of interest.

7. The method of claim 6, wherein: the CSA-surface expressing cells are selected from fetal trophoblast cells or malignant tumor cells.

8. The method of claim 6, wherein: the sample to be detected in the step 1) is a blood sample subjected to pretreatment, wherein the pretreatment is to remove red blood cells in the sample.

9. The method of claim 6, wherein: the sample to be detected in the step 1) is other biological body fluid samples except blood, such as urine, ascites, cerebrospinal fluid, pleural effusion and the like.

10. Use of a probe according to any one of claims 1 to 9 as a detection reagent or tool for prenatal screening or diagnosis, tumour progression or prognosis.