JP2000506608A - Methods for analyzing fetal cells - Google Patents

Abstract

  (57) [Summary] Methods for analyzing fetal cells are provided, as well as kits used in such methods and the use of suitable binding agents to isolate fetal cells from maternal blood.

Description

DETAILED DESCRIPTION OF THE INVENTION                             Methods for analyzing fetal cells   The present invention relates to a method for analyzing fetal cells and a kit used for such a method. Is what you do.   Prenatal identification of the fetus to detect fetal chromosomal aneuploidy, disease or condition. It is often desirable to analyze cells. And where desired and appropriate Unwanted aneuploidy, disease or condition can cause the pregnancy to stop, It is also desirable to be detected as early as possible in child development. Selective vignette E.g. elective chorionic villus sampling (CVS) Known techniques involve taking tissue, such as trophoblast tissue, directly from the fetus Things. The main drawback of such a technique is that it can result in fetal loss. And   Therefore, it is non-invasive in nature and without the obvious risk of fetal loss A method is needed.   We have determined that reliable analysis of fetal cells from maternal blood samples It has been found that this can be done by isolating at least two types. Womb By appropriately labeling the offspring cells, they can be easily and easily isolated and Can be used for the analysis of   Thus, according to a first concept, the present invention provides a method for preparing nucleated fetal cells from a maternal sample. A method of analyzing fetal cells, comprising isolating at least two types of Things. Preferably, the cell type is nucleated red blood cells and trophoblast Yes, the maternal sample is a peripheral blood sample.   Preferably, the cells are isolated from maternal peripheral blood and the different cell types are the same Desirably, it is isolated from a maternal sample.   Preferably, the maternal blood has a gestation period of 8 to 16 weeks, preferably 10 weeks of gestation. Sampled from a human being.   Preferably, the fetal trophoblast cells are isolated prior to isolation of nucleated red blood cells . Trophoblasts can be used to trophy maternal blood before subsequent labeling, identification and / or removal. It may be isolated by contact with a hoblast binder. For example, the binder Antibodies, such as monoclonal, polyclonal or genetically engineered antibodies, May consist of active derivatives of any of these. Or other suitable A ligand may be used.   Preferably, the binding agent is a 170 kD epidermal growth factor receptor (EGFr) anti- Binds to the original or its 150 kD cleavage product. According to one particularly preferred embodiment Then, the binding agent is a monoclonal antibody Mab340 (monoclonal antibody Mab3). 40) (Durrant et al., Prenatal Diagnosis, 14: 131-140 (1994)) or its EGFr binding derivative.   The Mab340 antigen is provisional accession number 9702428 and is available from European   European Collection of CellCult ures), CAMR (Center for Applied Microbiology and   Research (Center for Applied Microbiology & Research) (ECACC) Deposited.   Therefore, the present invention also provides for peripheral blood or other tissue To binding cells and / or cell fragments or clusters from a body sample It provides an antibody to EGFr to be used. Antibodies described herein Mab340 (antibody Mab 340) and those functionally equivalent to Mab are preferred . An antibody having binding specificity to EGFr, or a functional equivalent thereof, It will be obvious to one skilled in the art that it will be useful in this application. Antibodies are a perfect immunological Roblin molecule For example, monovalent or divalent Ig entities such as Fab fragments, single-chain It may be an Ig fragment such as Fv (single chain Fv's). Such as Mab340 Antibodies that recognize epitopes against the extracellular domain of EGFr are preferred antibodies is there. EGFr generally present in the cell membrane or on the cytoplasmic surface of the membrane Antibodies having binding specificity for other domains are specific examples in the present invention. It will be obvious to those skilled in the art.   Also, EGFr that binds to EGFr and is used for diagnostic screening before childbirth Enrichment and enrichment of cells, cell fragments or clusters with Peptides that can function to facilitate purification and / or purification Tides or other synthetic mimetic molecules are also encompassed by the present invention.   As mentioned above, Mab 340 is a particularly preferred binder used in the method of the present invention. It is a mixture. We now describe the sequence of the variable region of both the heavy and light chains of the antibody. Thus, the present invention provides that the EGFR binding derivative of Mab340 is One having at least one CDR region of a light chain or heavy chain variable region It also includes the law. In particular, the EGFR binding derivative of Mab340 is Mab3 One or more framework regions of one or more of the 40 light or heavy chain variable regions Will have more zones.   In addition, those skilled in the art will recognize that this sequence information can be used to mimic Mab340, eg, M Although not having the entire sequence of ab340, the CDRs and, if necessary, the Mab340 For constructing peptides or proteins containing the framework region of the variable part You will understand that it can be used.   According to another embodiment, the affinity medium, eg, an EGFr binding agent, binds Solid media may be used. For example, the medium is a paramagnetic bead. Or a magnetic colloid such as 51 (Cr) -sodium chromate-labeled MG63 cells May be. Thus, for example, the paramagnetic beads can then be It may be coated with Mab340 that binds to blast cells. Next, this cell is It can be isolated using a net. Or a substance that binds the beads themselves to an EGFr binder Bead, for example, if the EGFr binding agent is an antibody, the beads It may be labeled with rabbit anti-mouse antiserum. Therefore, in this example, the anti-EG After the Fr antibody was bound to the trophoblast, the beads bound to the antibody and The cells can be isolated by using gnet.   Magnetic colloid concentrations are on the order of 1/80 and 1 μg / ml antibody. Preferred Alternatively, the magnetic colloid is pre-labeled with a substance before being introduced into the blood sample. Preferably, the colloid / sample mixture is incubated at approximately room temperature for about 30 minutes. To   In addition, for example, two or more antibodies such as an antibody capable of binding to The use of the above EGFr binding agents in combination is also included in the present invention.   This mixed use allows the simultaneous use of two or more binding agents for different epitope clusters. Or binding agents to different epitope clusters They may be used sequentially. Monoclonal antibodies, preferably used as binders Or an EGFr surface for mAb 340 or mAb 340 or a derivative thereof. Other co-existing or future monoclonal antibodies with different binding specificities on And the combination used together. As a combination of binding agents, antibodies Alternatively, a combination of an antibody derivative and another antibody or antibody derivative may be used. No. The combination of the binding agent may be an antibody or a derivative thereof and a functionalized domain. A non-antibody molecule, which may be a synthetic ligand with in, or a non-covalent agent It may be a combination with a functional group. The combination of binders can bind to EGFr And may be entirely composed of non-antibody molecules.   Erythrocytes are labeled using erythrocyte labeling agents, such as antibodies that selectively bind to fetal erythrocytes. May be isolated. One example of such an antibody is the CD71 antibody. Next In addition, labeled erythrocytes are directly or indirectly attracted by the label, e.g., reversible. May be separated by passing the blood through a medium that binds to the blood. Preferably, a label Include anti-transferrin antibodies, preferably monoclonal antibodies. The medium is Consists of paramagnetic beads coated with an antibody attractant, such as rabbit anti-mouse antiserum Is also good.   Alternatively or additionally, the label is an anti-transferrin antibody, preferably a monoclonal antibody. It may consist of ferromagnetic particles coated with a lonal antibody, the medium being a MACS column It may consist of   Fetal erythrocytes are sampled on a density gradient, such as a triplet gradient. Initial separation by passing through a filter may be performed.   The method of the present invention is primarily aimed at detecting chromosomal aneuploids, diseases or conditions. Further comprising a biochemical and / or genetic analysis of the isolated fetal cells. There may be.   According to one embodiment, one or more gene sequences of the fetal cells are used to facilitate analysis. For example, using the polymerase chain reaction (PCR) or other suitable amplification technique And amplify. Amplify specific sequences on the Y chromosome when analyzing fetal sex Is also good. Preferably, fetal hemoglobin and / or fetal red blood cells MRN for human chorionic gonadotropin (HCG) in trophoblast A fluorescence in situ PCR (fluorescence in situ PCR) is used.   Preferably, the hybridization technique is used to detect fetal sequences For example, fluorescence in situ hybridization (fluorescence in situ hybridization) (FISH) There is art.   The present invention further provides a binding and / or labeling substance used in the method of the present invention. To provide.   The above substances may be monoclonal, polyclonal or genetically engineered antibodies Or an active derivative of any of these. Or The substance can associate and preferably bind to the surface of the fetal cell. Ligands or chemicals. According to a preferred embodiment, The quality is determined by the 170 kD epidermal growth factor receptor (EGFr) antigen or its 15 Will bind to the 0 kD cleavage product. As already mentioned herein, Ma b340 is a particularly preferred substance and therefore Mab340 or its activity Derivatives (eg, CDR regions of the light or heavy chain variable region of Mab340) Are particularly preferred.   In addition, the binding / labeling reagent may include a fetal red blood cell binding agent.   According to a further concept, the present invention relates to the amplification of target nucleotide sequences in fetal cells. It provides the primers used.   The above primers have a partial or complete sequence encoding fetal hemoglobin May be hybridizable for the amplification of Above Indicates that the additional primer encodes a human chorionic gonadotropin May be hybridizable for amplification of all sequences No.   According to a further concept, the present invention provides: i) Mab340, in the method of isolating at least two types of fetal cells, Or the use of an active derivative thereof; ii) EGFr binding agent in a method for isolating fetal trophoblasts Use of; iii) from at least one trophoblast binder and, if necessary, a labeling agent A kit for use in the method of the invention. Preferably, the trophoblast binder is M ab340 or an active derivative thereof, wherein the kit is an erythrocyte binding agent such as an antibody. May further be included; iv) A method for analyzing fetal cells comprising:   a) obtaining a maternal sample;   b) contacting the sample of a) with a trophoblast binder;   c) contacting the sample with an erythrocyte binding agent; and   d) one or more of the cells isolated in b) and c) Performs a genetic analysis step.   The present invention relates to the enrichment of small cell populations in the peripheral circulation. ) Methods and their subsequent analysis at the molecular genetic level. According to a preferred embodiment, the present invention relates to a method for treating a disease, preferably using mAb340. Used to capture and analyze circulating fetal cells for prenatal diagnosis of You. mAb340 recognizes and binds to an epitope in the extracellular domain of EGFr Then, it is disclosed that it is most conceivable. Thus, one further embodiment As a small group of malignant cells such as squamous cell carcinoma cells or EGFr Enrichment of tumor cells expressing There is use of technology.   EGFr is a 170 kD transmembrane glycoprotein with tyrosine kinase activity It is. The latter is activated after ligand binding, which results in the automatic reactivation of the receptor. It causes autophosphorylation and eventually stimulation of cell growth. E Overexpression of GFr includes cancers of the breast, brain, bladder, head and neck, pancreas and lung. Have been reported in many human malignancies. ing. For histological and biological studies of biological tissues and cell lines of various human tumors Thus, over-expression of the receptor may result in the receptor, so-called TGFα and / or Or often associated with the production of one or more known ligands for EGF Indicated. With such considerations, the autocrine loop It was suggested that it may respond to the type of tumor growth.   The present invention will be described with reference to the following examples, which limit the present invention. It is understood that it does not do.   The examples refer to the following figures:   FIG. 1 shows the results of immunoaffinity chromatography. FIG. 4 shows silver-stained SDS-PAGE analysis of purified 40 antigen;   FIG.¹²⁵Radioligand binding showing that EGF binds to 719T cells Figure 2 shows the saturation curve and Scatchard plot of the assay;   FIG. 3 shows that I versus 719 T cells.¹²⁵EGF and Mab340 using EGF 3 shows the results of a competitive binding assay.Example 1   The trophoblast cells are transformed with a specific monoclonal antibody, 340 (Duran). rant) et al., supra), isolated from whole blood of usually one-third of women at the beginning of pregnancy. Incubate with liquid samples and paramagnetic coated with rabbit anti-mouse antiserum It was passed through a separating agent containing beads. Nucleated erythrocytes were subjected to a three-phase density gradient (triplet density g radient), and the whole blood is separated and coated with anti-transferrin monoclonal antibody. Dye with ferromagnetic particles separated on a mini MACS column or Coated with anti-transferrin monoclonal antibody and rabbit anti-mouse antiserum Paramagnetic bee And isolated by staining.   These two isolated cell types are identified on the Y chromosome (only detected in males). Sex is identified using nested PCR for specific sequences of Sex of chorionic villus samples by displaying metaphase chromosomes (karyotyping) confirmed.   Selective chorionic villus sample for fetal cells to detect fetal aneuploids 10-14 weeks gestation undergoing elective chorionic villus sampling Isolated from 19 patients in between. Selective chorionic villus sampling (elective chorionic villus sampling) is a trophoblast for analysis of human fetal DNA. This is a known method for obtaining a strike tissue. The chorion can be located in the femoral neck or transabdominal route. It was collected at a distance. The main disadvantages of the above-mentioned known techniques that are not performed in the present invention are as follows. However, direct biopsy can damage the fetus.   When both trophoblasts and nucleated red blood cells were isolated, the fetal sex was 92% of patients Is exactly what is expected, which includes an accurate diagnosis of 5 out of 6 male pregnancies Met. One pregnancy was diagnosed with both trophoblasts and nucleated red blood cells and two were Only detected with trophoblast, two were detected with nucleated red blood cells alone. false positive( The boy's signal from a girl's pregnancy is highly sensitive and nested Whether using trophoblasts or nucleated red blood cells, even using PCR (nested PCR) Was also not determined. From these results, trophobra from the same maternal blood sample It is possible to isolate both test and nucleated red blood cells, and both isolation and analysis techniques Can significantly increase the sensitivity of prenatal decisions on fetal aneuploidy, condition and disease Is shown.   In the second study, 46 peripheral blood samples were collected from a selective chorionic villus sample. Collected after aging. Table 1 shows the results obtained, Although this shows a slight decrease in sensitivity and specificity compared to the initial study, By isolating two fetal cell types from maternal blood in accordance with the present invention. Results in increased sensitivity in detecting fetal aneuploidy, condition, or disease. The argument is still confirmed.Table 1 : Sensitivity and features to select different fetal cell types from maternal blood         Comparison of opposite sex  Sensitivity in determining pregnancy in boys in a second study using nucleated red blood cells alone Was 38% and 39% when trophoblast was used. Separation and analysis revealed that male pregnancies were accurately detected in boys with a sensitivity of 10 or 56% of 18 cases. Anticipated pregnancy. False positive, i.e. determined by incorrect diagnosis as a boy's pregnancy Specificity (83-86%) is very similar regardless of the method of fetal cell selection Was. Of 10 correctly diagnosed boy pregnancies, 3 are diagnosed with nucleated red blood cells alone Three were diagnosed only with trophoblasts and four were positive for both cell types there were.   The concentration of fetal cells in maternal blood is particularly low. 20 per 20 ml of maternal blood A small number of fetal cells, this is the number of fetuses in Down's pregnancy It is thought that it will increase due to board malformation, but it exists You.   Isolated fetal cells are swept away in numbers by over 1000 times maternal cells Therefore, fluorescence in situ hybridization ridization) analysis makes cell identification very difficult to determine. In red blood cells Human chorionic gonadotropin in fetal hemoglobin and trophoblast ( HCG) Fluorescence of mRNA for in situ polymerase chain reaction (fluores Identification of fetal cells by cence in situ polymerase chain reaction (PCR) Therefore, more accurate fluorescence in situ hybridization (fluorescenc) e in situ hybridization) analysis (FISH) becomes possible.   Another technique is to further increase the sensitivity and achieve fluorescence in situ hybridization. Chromosomal aneuploids, diseases and conditions are more easily detected by solubilization staining In an attempt to isolate cells, magnetic colloids were used to isolate cells. First Studies have shown that 51 (Cr) -cloth expressing high levels of 340 trophoblast antigens. This was performed using sodium oxalate-labeled MG63 cells. The optimal isolation and analysis is 1 / The 80 and 1 μg / ml 340 monoclonal antibody ferrofluid uid) concentration. Optimum incubation is 30 minutes at room temperature, with antibody Pre-labeling ferrofluid is the most effective labeling protocol I found it. Using these parameters, 74-80% of MG63 Cells were isolated and analyzed from blood.   Furthermore, fluorescent in situ hybridization (fluorescent in situ hybridization)  hybridization) was successfully performed on cells isolated using magnetic colloids . After isolation using magnetic colloids, PCR and F ISH may be performed.   Isolation and analysis of at least two fetal cell types is performed using chromosomal aneuploids, Could be used to analyze cell types for disease, condition or other characteristics U. For example, cells can be analyzed for diseases such as Down's syndrome.Example 2 -340 antibody sequencing   5 × 10⁶Of hybridoma 340 cells and mRNA Instructions (Life Technologies, Inc.) Inc.), Paizley Scotland) Isolation using 1 ™ reagent. DNA / RNA hybrids are ready -To-Go ™ T-Primed Fast-Stand Kit (Ready-T o-Go^TM T-primed First-St and kit) (Pharmacia, Sweden) ). PCR was performed using primers specific for the mouse heavy and light chain variable regions. Performed using Marset (Jones, S.T.) and Bende Bing, M., Bio / Technology, 9: 8 8 (1991)). The PCR product was cloned into pCRII (Invitrogen). , The Netherlands) and the sequences of six other clones were Ride Biosystems Automated Sequencer Model 37 Determined using 3A (Applied Biosystems automated sequencer model 373A) . The VH and VL sequences are shown below; using the one-letter amino acid code, Underline. Identify the variable region genes used in the sequence and establish its uniqueness For this purpose, Kabat datab ase).   Antibody chains are separated by reducing SDS-PAGE to support sequence data Applied Biosystems Protein Sequencer Model 4 73A (Applied Biosystems protein sequencer model 473A) Western brochure on PVDF paper according to protocol And sequence analysis was performed on each strand 10 times. Protein sequence analysis Data was confirmed by DNA sequence analysis as described below. Example 3Identification of antigen recognized by -340 antibodyMethod for analyzing protein sequence of 340 antigen:   Wash 30-40 g placenta in phenylmethylsulfonyl-containing PBS Chop, homogenize, dissolve in 1% NP-40 lysis buffer, Centrifuged at 000 × g for 10 minutes. The supernatant was collected and the CNBr-activated mAb 340 Loaded on a Faroose column (2 mg mAb / ml gel). About the eluted fraction 340 antigen content was determined by SDS-PAGE and protein analysis. FIG. 1 shows a concentrated, immunoaffinity separated by SDS-PAGE and silver stained. FIG. 3 shows a sample of an immuno-affinity purified 340 antigen preparation. . The molecular weights of the proteins are 170 and 150 KDa.   After SDS-PAGE of the immunoprecipitated protein, the gel was applied with a blotting membrane. PVDF (Problot Applied Biosystems) according to the supplied protocol. Western blot on Thames (ProBlot Applied Biosystems). This bro Stained with Coomassie blue, destained with 10% (v / v) methanol / acetic acid Was. The band is excised from the blot and the protein sequence is Stems Protein Sequencer Model 473A (Applied Biosystems Protein sequencer model 473A).Results of protein sequence analysis of 340 antigen:   The N-terminal amino acid sequence was LEEKKKVCQG. This peptide is Consistent with the mature N-terminal sequence of skin growth factor receptor (EGFr). Iodized E Establish a competitive binding assay using GFr ligand (EGF) and mAb340 This confirmed the findings from peptide sequence analysis.I- ¹²⁵ EGF binding assay method:   Cells are harvested with trypsin / EDTA and 5 × 10^FiveRP before resuspension in Washed twice with MI 1640 medium. 100 μl of cell suspension at various concentrations 100 μl of I¹²⁵EGF (Amersham; 100 μCi / μg, 50 μCi / ml) and incubated at 37 ° C. for 1 hour. Cells and binding E GF was separated by centrifugation and 0.1% (v / w) bovine serum albumin (BSA) Was washed twice with ice-cold phosphate buffered saline (PBS) containing 1 for non-specific binding It was measured by adding more than 00 times cold EGF.I- ¹²⁵ EGF binding and mAb340 inhibition assay methods:   5 × 10 per test tube^FourCells were incubated at 4 ° C. for 30 minutes with EGF and m, respectively. The mixture was mixed while increasing the concentration of Ab340. Next, 20 pg of I-¹²⁵EGF (2 . 2 × 10^Fivecpm / ng) was added to each tube and the mixture was allowed Cubbed. The other steps were the same as described above.Binding assay results:   From the radioligand binding assay, I¹²⁵EGF can bind to 791 T cells Is shown. A saturation curve was obtained, and the non-linear Scatchard plot The presence of a population of receptors with altered affinity for GF is shown (FIG. 2).   Data are from 791 T cells with 20,000 binding sites per cell. This is consistent with the measured EGFr density.   Data from the inhibition experiments indicate that 340 mAb antagonizes EGF binding to EGFr. Consistent with the description that it is a harmful agent.   The EGF receptor derived from A431 cells was analyzed by affinity chromatography. Was first purified in 1980 by using This purified receptor Had an apparent molecular weight of 150 KDa; However, from the following purification method that removes calcium from the buffer, the actual molecular weight is It was shown to be 170 KDa. Most cell homogenates are 170K Calcium-activating protein that cleaves the original molecule of Da to a lower molecular weight of 150 KDa Contains thease. I-¹²⁵EG with different affinity from EGF binding assay It was shown that there were two sub-populations of Fr.   The experimental results presented herein show that the 340 antigen is EGFr and mAb34 Consistent with the conclusion that 0 is a competitive inhibitor of EGF for this receptor.Example 4 -Selection of fetal cells by internal antigenMethod for selecting fetal cells by internal antiaen:   The ferrofluid was diluted before use (specifically, 1/4 0/1/80). After fixing the cells in 1% paraformaldehyde for 10 minutes, Infiltrated in 0% ethanol for 20 minutes. Ferrofluid, Incubate with fixed and infiltrated cells for 5 minutes at 37 ° C and 55 minutes at room temperature Before labeling, the cells were labeled with antibodies (anti-HbF, anti-G6PDH) at room temperature for 30 minutes. Sign Cells are selected with an Immunicon magnet for 5 minutes before washing. Different.Combined immunocytochemistry / FISH method:   Immunicon ferrofluid reduced to 1/40 to 1/80 Diluted and labeled with biotinylated antibody for 30 minutes at room temperature. Sort cells for another 30 minutes After the addition over a period of time, the mixture was selected with an Immunicon magnet. Selection Separated cells in acetone (neat) or paraformaldehyde (1%) For 10 minutes, dropped on a microscope slide, and air-dried. Slide 3 0% for 20 minutes in Tris Buffered Saline (TBS) With normal rabbit serum After incubation together, they were washed twice with TBS for 5 minutes. Avid antibody binding Detection using a biotin / biotin peroxidase complex and a DAB substrate Was. A specific chromosome is subjected to fluorescence in situ hybridization (fluoresce nce in situ hybridization (FISH) technology and provided by the manufacturer Detected by commercial fluorescent hybridization probes and protocols .How to check Trophoblast:   The HCG is encoded for the fetal trophoblast selected by the above method. PCR analysis was performed for the presence of transforming DNA. The primers used are as follows Was as follows: (1) 5'hcg5'CTG GCT GTG GAG AAG 3 '; (2) 3'hcg3'AGT CGG GAT GGG CTT 5 '; (3) 3'hcg3'GAG TGC ACA TTG ACA G5 '.   The thermal cycle condition using the primers (1) and (2) is 5 minutes at 80 ° C. After starting with heating, Taq polymerase was added and 1 minute at 94 ° C and 1 minute at 55 ° C. And 30 cycles of 1 minute at 72 ° C. As a result, a 230 bp production The primers (1) and (3) and the same conditions as described above were obtained. HCG DNA from fetal trophoblasts A product of 230 bp was obtained, suggesting the presence ofExample 5 -Optimal selection of trophoblasts and nucleated red blood cells   Mononuclear cells were obtained on percol from whole female blood during the first third of pregnancy. Isolated. Trophoblast is coated on ferrofluid Isolate using biotinylated monoclonal antibody 340 and incubate with mononuclear cells. I was vate. Labeled cells are sorted with a magnet, washed, and dropped on a slide. Down.   Fix and permeate unsorted mononuclear cells in 1% paraformaldehyde / 70% ethanol Let it. Nucleated red blood cells, biotin coated on ferrofluid Using anti-HbF, anti-G6PDH . The labeled cells are sorted with a magnet, washed, and further dropped on a slide. Next Next, the isolated fetal cells are stained by immunohistochemistry / FISH.Method for sorting cells by internal antigen using fetal cells instead:   These experiments were performed as surrogates of fetal cells, in this case tumor cell lines. Cytokeratin 18 (C1) as COLO and internal antigen 8).   Collect and measure COLO 205 cells and measure viability by Tryphan Blue X It was evaluated and measured by inclusion (tryphan blue exclusion). 5 × 10⁶ Cells were converted to 2 MBqs of tritiated thymidine (^ThreeH) (Amersham) , UK) and incubated at 37 ° C for at least 6 hours (generally overnight). I did it. Cells are collected and placed in an RPMI medium (Life Technologies logies), Paisley Scotland). Fine Re-measure vesicles and measure viability by tryphanblue  exclusion). Cells (10^FiveWere collected by centrifugation and 1 ml Resuspend in 1% (w / v) paraformaldehyde and incubate at 4 ° C for 20 minutes. Fixed by heating. After fixation, cells were collected by centrifugation and 1 ml Resuspend in 70% ethanol and incubate at 4 ° C for another 20 minutes By infiltration. Cells are harvested by centrifugation and resuspended in 500 μl RPMI. Suspended.   Ferrofluid (Immunocon Corporation (Immunocon C orp.), Huntingdon Valley,   PA), USA) if necessary (generally 1/80) and 500 μl Anti-cytokeratin 18 antibody (C18, Sigma) at room temperature for 30 minutes at room temperature , Pool, Dorset). Is the antibody concentration 1/50? To 1/200 as a part of the confirmation experiment. Ferrofluid / antibody Add the mixture to the immobilized cells, mix by gentle shaking, For 5 minutes, followed by another 55 minutes at room temperature. Fine Cells are sorted using an immunocon sorting system Using a scintillation counter^ThreeLumaplate for H measurement (Pipetted in Canbarra Packard) .Results of cell sorting using internal antigens instead of fetal cells (Table 2):   All experiments were performed in triplicate. Data are for C18 antibody and 1/100 dilution. And experiments using 1/80 ferrofluid. Control Data on cells (C18 negative) showed a sorting efficiency of less than 10% (data Data not shown).   Sorting efficiency (%) = number of sorted cells / total number of counts = 73%

──────────────────────────────────────────────────続 き Continued on the front page (51) Int. Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/68 C12N 5/00 E G01N 33/577 15/00 ZNAA (81) Designated countries EP (AT, BE) , CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN) , ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM) , AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL , PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN (72) Inventor Liu, David, Techjung, Nottingham Energy 5 1PBY, Hacknall Road, City Hospital, Department of Tetrix and Gina Ecology

Claims (1)

[Claims]   1. From isolating at least two types of nucleated fetal cells from a maternal sample A method for analyzing fetal cells.   2. The claim 1 wherein the fetal cell type is red blood cells and trophoblast. The method described in.   3. 3. The method according to claim 1, wherein the cells are isolated from maternal peripheral blood. The described method.   4. 2. The method of claim 1, wherein said two cell types are isolated from the same sample. A method according to any one of the preceding claims.   5. The sample is taken from a human female of 8-16 weeks gestation, preferably 10 weeks. A method according to any one of claims 1 to 4 taken.   6. Wherein the fetal trophoblast is isolated prior to isolation of fetal nucleated red blood cells. 6. The method according to any of paragraphs 1 to 5 above.   7. The trophoblast contacts the maternal sample with a trophoblast binder. 7. The method according to any of claims 2 to 6, wherein the method is isolated by:   8. The method of claim 7, wherein the trophoblast binding agent comprises an antibody. .   9. The antibody is a monoclonal, polyclonal or genetically engineered antibody Or an active derivative of any of these. Method. 10. The binder also binds to a 170 kD epidermal growth factor receptor (EGFR) antigen Binds to the 150 kD cleavage product of any of claims 7-9. The method described in Crab. 11. The binding agent is the monoclonal antibody Mab340 or its EGFr binding induction 11. The method of claim 10, wherein the method is a body. 12. The EGFr-binding derivative of Mab340 can be a light chain or heavy chain of Mab340. 12. The method according to claim 11, which comprises at least one or the other CDR region of the variable region. The described method. 13. 13. The method of claim 7, wherein said binding agent is itself associated with the labeling agent. The method according to any of the above. 14． 14. The labeling agent according to claim 13, wherein the labeling agent is a solid phase and / or has magnetism. The method described in. 15. 15. The labeling agent according to claim 14, wherein the labeling agent is a paramagnetic bead or a magnetic colloid. The method described in. 16. The binder itself is labeled or can be labeled or associated with a signaling agent A method according to any of claims 7 to 15, wherein the method comprises: 17． After the binder is biotinylated, the avidin / biotin peroxidase complex 17. The method according to claim 16, which can be labeled with coalescence and DAB. 18. 3. The method of claim 2, wherein the red blood cells are isolated using one or more red blood cell binding agents. 18. The method according to any of claims 17 to 17. 19. At least one of the one or more red blood cell binding agents is an anti-binding agent that binds to fetal red blood cells. 19. The method of claim 18, wherein the method is a body. 20. Passing the blood sample through a density gradient, eg, a three phase gradient, of the fetal red blood cells 20. A method according to claim 18 or claim 19, wherein an initial separation according to 21. Performing one or more biochemical and / or genetic analysis steps on the isolated fetal cells 21. A method according to any of the preceding claims, further comprising: 22. 22. The method of claim 21, wherein the method comprises analyzing fetal sex. Law. 23. A coupling for use in a method according to any of claims 1 to 22. And / or labels and / or signaling agents. 24. Modifications according to one or more aspects of claims 7 to 17 or 19. 24. The agent according to claim 23, which is decorated. 25. Primer pairs used to amplify fetal cell target nucleotide sequences. 26. Amplification of some or all sequences encoding fetal hemoglobin The primer pair according to claim 25, which is capable of hybridizing. 27. Amplification of some or all sequences encoding human chorionic gonadotropin The primer according to claim 25, which is capable of hybridizing with respect to versus. 28. Mab34 in a method of isolating at least two types of nucleated fetal cells Use of 0 or an active derivative thereof. 29. According to one or more aspects of one or more of claims 12 to 17, Use according to claim 28, which is modified. 30. Use of an EGFr binding agent in a method for fetal trophoblast isolation. 31. At least one trophoblast binding agent and, if necessary, a labeling agent and / or Or a signal agent according to any one of claims 1 to 22. Kit used for the method. 32. The trophoblast binder is Mab340 or an active derivative thereof. 32. The kit of claim 31, wherein 33. 33. The method according to claim 31, further comprising an erythrocyte binding agent such as an antibody. The kit according to the item. 34. A method for analyzing fetal cells comprising:   a) obtaining a maternal sample;   b) contacting the sample of a) with a trophoblast binder;   c) contacting the sample with a red blood cell binding agent; and   d) one or more of the cells isolated in b) and c) Performs a genetic analysis step.