EP1301798A2 - Mild enrichment of foetal cells from peripheral blood and use thereof - Google Patents
Mild enrichment of foetal cells from peripheral blood and use thereofInfo
- Publication number
- EP1301798A2 EP1301798A2 EP01964843A EP01964843A EP1301798A2 EP 1301798 A2 EP1301798 A2 EP 1301798A2 EP 01964843 A EP01964843 A EP 01964843A EP 01964843 A EP01964843 A EP 01964843A EP 1301798 A2 EP1301798 A2 EP 1301798A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- fetal
- antibody
- antibodies
- blood
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
Definitions
- the invention relates to a method for the enrichment of fetal cells as a product from, preferably peripheral, maternal, blood, wherein in step a) after the removal of maternal blood in the presence of a solution containing one or more anti oagulates to provide a blood fraction with the blood an isotonic solution, and in step b) the blood fraction is centrifuged for the enrichment of a cell fraction with fetal cells containing cell nuclei, the use of the products for the treatment of diseases and for the determination of gene and / or genome analyzes.
- the isolated fetal cells and their genome can be used to analyze hereditary damage, such as point mutations, chromosome abnormalities, chromosomal aberrations, without the need, in contrast to the prior art, to first remove cells from the amniotic fluid or the uterine apparatus, so that the Risks of abortion can be excluded.
- hereditary damage such as point mutations, chromosome abnormalities, chromosomal aberrations
- the ceramide compounds are surface molecules of progenitor cells of red blood cells.
- the polylactosaminoglycans are believed to branch during postnatal development. This maturation process of the carbohydrate chains, which also occurs in precursors of the erythrocytes in the bone marrow of adults, leads to a reduction in the lacto-N-nor-hexaosyl-ceramide compound as i-antigen (or also called antigen-i) and to an increase in lacto -N-iso-octaosyl-ceramide compound called antigen-I or I-antigen. Antigen-I is therefore weak, if any, at birth. Only at around 18 months does it reach the amount and concentration that it has in adults.
- the i-antigen is fully developed in the newborn. It is therefore lost in the first 18 months after birth and is replaced by Antigen-I.
- Antigen-I There is now a need to provide methods which enable a high concentration of fetal cells while eliminating maternal blood cells and a gentle concentration thereof.
- the DNA of the highly enriched fetal cells can provide information about the primary structure through to quaternary structures of the gene sequences, without the analysis results being influenced or even changed by the genome of the maternal blood cells.
- US Pat. No. 5,437,987 describes a method in which fetal red blood cells are enriched from maternal blood and complexes of polyclonal antibodies and fetal cells are separated off after the anti-i antibody has been added; the polyclonal antibodies bind to the surface antigens in the fetal cells.
- Antibodies are used whose antigen binding region with the Gal ß-4-GlcNAc ß-l-3-Gal ß-l-4-GlcNAc ß-l-3-Gal ß-l-4-Glc chain of the antigen binds specifically as antigenic determinates.
- This antibody directed against the linear carbohydrate chain requires at least two repeating N-acetyl-lactosarr ⁇ i units for binding.
- the simplest i-active structure is therefore lacto-N-nor-hexaosyl-ceramide.
- the antibody which is able to bind to lacto-N-nor-hexaosyl-ceramide or derivatives thereof as an antigen is called anti-i in the sense of the invention.
- magnetically activated cell sorting methods are used, which, however, are only able to enrich the fetal cells to an insufficient extent, since the fetal cells in the antibody antigen are in a negligible ratio and extent Complex are present.
- the cells sought are extremely rare in an excess of maternal cells, so that the fetal cells are enriched only to a small degree, but not to the point of purity. Due to the lack of fetus-specific properties of the previously available antibodies, noteworthy, so-called cellular contamination with maternal, essentially blood cells cannot be prevented. In addition, it is not possible to separate cells with magnetically activated line sorting methods.
- fetal cells can be used as products in a diagnostic procedure.
- the invention relates to a method for high enrichment of fetal cells as a product from, preferably peripheral, maternal blood, wherein in Step a) after taking a mother's blood in the presence of a solution containing anticoagulants to provide a blood fraction, the blood is diluted with an isotonic solution and
- Step c to the cell fraction comprises an antibody cocktail of antibodies of the polyclonal and / or monoclonal type, preferably at RT for 15 to 30 min, more preferably 10 to 20 min
- Antibodies anti-w that specifically bind to antigens of the intracellular structures of white blood cells and / or the intracellular molecules of white blood cells and / or surfaces of white blood cells,
- Antibody Anti-r which specifically binds to antigens of the intracellular structures of nucleated progenitor cells of red blood cells and / or of the intracellular molecules of nucleated progenitor cells of red blood cells and / or the surfaces of nucleated progenitor cells of red blood cells,
- Antibodies anti-i of the polyclonal and / or monoclonal type which specifically bind to antigen-i as antigens of the surfaces of nucleated progenitor cells of red blood cells,
- Antibody anti i plus of the polyclonal and / or monoclonal type ⁇ which specifically binds to intracellular structures of fetal progenitor cells and / or the intracellular molecules of fetal progenitor cells and / or membrane fragments of fetal progenitor cells,
- Step d) from the Zeil approach fetal cells with antibodies anti-r and / or antibodies anti-i, preferably and / or antibodies anti-i plus, coupled to them, on the basis of their scattered light and fluorescence properties, the properties of surfaces and of the cell interior and the size distribution of the fetal cells with a 10 to 10-fold enrichment of the fetal cells, preferably until their cellular presentation is clear.
- One object of the invention also relates to the use of the cell fraction as a product, which can be produced by the method according to the invention, for the treatment of hereditary diseases, preferably in fetal age, for use in diagnostic methods, for example by genome examination.
- Another object of the invention includes fetal cells from venous blood, which can be produced by im
- Step c Incubation of the cell fraction with an antibody cocktail of antibodies of the polyclonal and / or monoclonal type at RT for 15 to 30 min, preferably 10 to 20 min, for which
- Antibodies anti-w that specifically bind to antigens of the intracellular structures of white blood cells and / or the intracellular molecules of white blood cells and / or surfaces of white blood cells,
- Antibody Anti-r which specifically binds to antigens of the intracellular surictures of nucleated progenitor cells of red blood cells and / or of the intracellular molecules of nucleated progenitor cells of red blood cells and / or the surfaces of nucleated progenitor cells of red blood cells,
- Antibodies anti-i of the polyclonal and / or monoclonal type which specifically bind to antigen-i as antigens of the surfaces of nucleated progenitor cells of red blood cells,
- Antibodies Anti i plus of the polyclonal and / or monoclonal type _ which bind specifically to intracellular structures of fetal progenitor cells and / or the intracellular molecules of fetal progenitor cells and / or membrane fragments of fetal progenitor cells,
- Anti i plus from the Zeil approach due to its scattered light and fluorescence Shafts, the properties of surfaces and the interior of the cell, as well as the size distribution with 10 5 to 10 7 -fold enrichment of the fetal cells, preferably up to their cellular clean presentation.
- the intracellular structures of white blood cells or fetal progenitor cells e.g. Understand cell organelles such as mitochondria, endoplasmic reticulum, and cell nuclei.
- the intracellular molecules of white blood cells or of fetal progenitor cells e.g. cell-specific products or compounds, for example proteins, such as enzymes, protein derivatives, such as fetal hemoglobin.
- antigens of the surfaces of nucleated progenitor cells of red blood cells e.g. Understand transferrin receptor molecules.
- adding or adding is understood to mean the addition of antibody cocktail and antibody anti i and antibody anti i plus simultaneously or successively.
- the antibody cocktail of the cell fraction can first be added in succession, then the anti-i antibodies added and then the anti-i plus antibodies added.
- adding or adding the antibody cocktail simultaneously or successively means that, for example in step c), the antibody cocktail is first added to the cell fraction as a mixture of antibody anti-w and antibody anti-r, or in step c) the antibody Anti-w is first added to the cell fraction and then antibody Anti-r,
- step c) the anti-r antibody is added to the cell fraction and then the anti-w antibody.
- the incubation is the simultaneous or sequential addition of antibody cocktail and anti-i antibody and anti-i plus antibody, in step c) first the antibody cocktail as a mixture of antibody
- Anti-w and antibody Anti-r are added to the cell fraction or in step c) the antibody Anti-w is added to the cell fraction and then antibody Anti-r or in step c) the antibody Anti-r to the cell fraction and then antibody can be added by anti-w.
- the antibody cocktail, the antibody Anti-i or the antibody Anti-i-plus or mixtures of the antibodies Anti-i or the antibody Anti-i-plus can be added simultaneously or in succession.
- Mixtures can also contain antibodies anti-i of the polyclonal and / or monoclonal type and / or antibodies anti i plus of the polyclonal and / or monoclonal type.
- a blood fraction is taken from maternal, preferably peripheral, such as venous and / or arterial, blood, for example from the arm vein, and transferred to a vessel which has one or more conventional complexing agents as anticoagulants.
- complexing agents ethylenediaminetetraacetic acid compounds, heparin, citric acid compounds and / or salts thereof and / or derivatives thereof can be used.
- Eu ⁇ ylenediamine tetraacetate compounds or their derivatives in concentrations known to those skilled in the art serve as suitable anticoagulants without any interactions in the subsequent workup steps.
- fetal cells which can be enriched and can be enrichable by the method according to the invention are understood to mean fetal progenitor cells of the erythrocytes containing nucleus.
- the fetal cells to be enriched have a cell nucleus and a transferrin receptor.
- transferrin receptors are understood to mean those compounds which serve the function of the iron transmitter in the cell and can no longer be found on the cell surfaces of the mature erythrocytes. Transferrin is an iron carrier in the cell.
- the mature cells from the precursor cells of the red blood cells namely the seedless erythrocytes, have essentially no transferrin receptors on their surface.
- the blood fraction is subjected to a density gradient centrifugation step.
- the nucleus-containing fetal precursor cells can be enriched by the other cells from the fraction due to their different density, however, erythocytes are also found in the cell fraction because of the so-called streaking that occurs in all fractions.
- the nucleated cells concentrate under the influence of gravity according to their density in a boundary layer, also called a buffy-coat layer, between the upper fraction, the diluted blood plasma of the blood fraction, and the lower fraction with a portion of sucrose as a pillow.
- a boundary layer also called a buffy-coat layer
- the sucrose-polymer portion can be, for example, a synthetic polymer made from sucrose with a molecular weight of 70,000 to 400,000 daltons, preferably 400,000 daltons, for example Ficoll, preferably colloidal, polyvinylpyrrolidone-coated silica particles, such as Percoll, or the like.
- the sucrose-polymer portion is in an isotonic solution of buffered physiological saline with pH values between 7.2 and 7.4, preferably 7.2.
- Conventional sodium chloride-containing ones can be used as buffers, such as potassium phosphate and / or sodium phosphate buffers, NaH 2 P0 4 , Na 3 HP0 4 with or without NaN 3 .
- the pillow of isotonic sucrose polymer solution with the blood fraction if necessary after its previous dilution with a volume ratio of 1: 1 (VokVol), can be overlaid with phosphate-buffered saline buffer solution; 1 volume of blood fraction is added to 1 volume of sucrose-polymer solution.
- the red, but physically denser erythrocytes sediment into the sucrose cushion.
- the less dense nucleated fetal cells accumulate as a so-called buffy-coat layer at the boundary layer between the diluted blood fraction located above the pillow and the denser lower pillow.
- the buffy-coat layer regularly visible as a white ring, is transferred to a vessel and the cell fraction of the buffy-coat layer is washed with phosphate-buffered saline buffer solution.
- the washed cell fraction is then fed to an antibody incubation.
- the cell fraction can contain: fetal progenitor cells of the red blood cells containing nucleus, which have i-antigen and antigen-r and / or antigen-i-plus, some fetal red cell nucleus-free blood cells, i.e. mature, which contain i-antigen and no transferrin receptors,
- Cells in the cell fraction generally have a high number of maternal cells (1 to 4 ⁇ 10 7 cells) compared to 20 to 50 fetal cells in the cell fraction.
- an antibody cocktail of different antibodies of the monoclonal type and / or polyclonal type is added in order to provide a Zeil approach from antibody complexes.
- the antibody cocktail as a mixture of antibody anti-w and antibody anti-r of the cell fraction can also be used first, or in step c) first the antibody anti-w of the cell fraction and then antibody anti-r or in step c) first the antibody anti-r of the cell fraction and then antibody anti-w are added.
- a method for high enrichment of fetal cells can be provided as a product from, preferably peripheral, maternal blood, wherein in the
- Step b) the blood fraction is enriched for the enrichment of a cell fraction with fetal cells containing cell nuclei, preferably by means of density gradients, which is characterized in that in the
- Antibodies anti-w that can specifically bind to antigens on the surface of white blood cells
- Antibodies anti-r which can specifically bind to transferrin receptor molecules as antigens of the surfaces of nucleated progenitor cells of red blood cells, and / or
- Antibodies anti-i that specifically bind to antigen-i as antigens of the surfaces of nucleated progenitor cells of red blood cells, and preferably
- Anti i plus which can specifically bind to membrane fragments of fetal progenitor cells, is added to form a Zeil approach which contains antibody complexes, which may include antibodies anti-r and anti-i, and optionally anti-i plus, coupled to fetal cells, and
- Step d from the Zeil approach fetal cells with antibodies anti-r and / or antibodies anti-i, preferably and / or antibodies anti-i plus, coupled to the same, due to their scattered light and fluorescence properties, according to the properties of surfaces and / or is separated from the interior of the cell and / or the size distribution of the fetal cells with a 10 to 10-fold enrichment of the fetal cells, preferably until their cellular presentation is pure.
- the cell fraction as a product that can be produced by the method according to the invention for the treatment of hereditary diseases, preferably in fetal age, for use in diagnostic methods, for example by genome examination.
- Fetal cells from venous blood which can be produced by im
- Step c) Incubation of the cell fraction with an antibody cocktail of antibodies of the polyclonal and / or monoclonal type at RT for 5 to 30 min, preferably 10 to 20 min, or 5 or 10 min, whichever
- Antibodies anti-w that can specifically bind to antigens on the surface of white blood cells
- Antibodies anti-r which can specifically bind to transferrin receptor molecules as antigens of the surfaces of nucleated progenitor cells of red blood cells, and
- Antibodies anti-i which can specifically bind to antigen-i as antigens of the surfaces of nucleated progenitor cells of red blood cells, and / or
- Antibody Anti i plus which can bind specifically to membrane fragments of fetal precursor cells, contains, with the formation of a Zeil approach from antibody complexes, which antibodies coupled to fetal cells anti-r and anti-i and / or anti-i- plus can include, contains,
- Step d Separation of fetal cells with those coupled to them
- the antibody cocktail can comprise antibodies of the monoclonal type and / or polyclonal type:
- Antibodies anti-w against surface antigens specific for white blood cells such as leukocytes, lymphocytes and
- Antibodies anti-r against surface antigens here against the transferrin receptors for red still nucleated precursor cells of the red blood cells are specific
- Antibody anti-i plus against membrane fragments of fetal progenitor cells that are specific for fetal cells containing nuclei are specific for fetal cells containing nuclei.
- nucleus-containing leukocytes are hereby labeled with the antibodies Anti-w, by binding the antibodies Anti-w to white blood cells, such as leukocytes, lymphocytes, which are discarded.
- an antibody cocktail of various monoclonal antibodies Type and / or polyclonal type added to provide a Zeil approach from antibody complexes.
- the antibody cocktail of the method according to the invention can comprise antibodies of the monoclonal type and / or polyclonal type:
- Antibodies anti-w that specifically bind to antigens of the intracellular structures of white blood cells and / or the intracellular molecules of white blood cells and / or surfaces of white blood cells, such as leukocytes, lymphocytes,
- Antibodies anti-r that specifically bind to antigens of the intracellular structures of nucleated progenitor cells of red blood cells and / or the intracellular molecules of nucleated progenitor cells of red blood cells and / or the surfaces of nucleated progenitor cells of red blood cells, e.g. against surface antigens, such as against the transferrin receptors, for red precursor cells of the red blood cells that still contain nuclei.
- polyclonal and / or monoclonal antibodies anti-i against the antigen-i
- the antibody cocktail can be added to the antibody cocktail before it is added to the cell fraction if, for example, it is to be incubated simultaneously with the cell fraction.
- only the anti-i antibodies can be added to the antibody cocktail if, for example, the incubation with the cell fraction is to take place simultaneously.
- only the anti-i plus antibodies can be added to the antibody cocktail if, for example, the incubation with the cell fraction is to take place simultaneously.
- the antibodies Anti-i plus or the antibodies Anti-i are added to the cell fraction-antibody cocktail
- fetal, coreless red blood cells are marked by the binding of the antibodies anti-i to the antigen-i, which can be discarded.
- the fetal precursor cells of the red blood cells that still contain nuclei become enriched
- the antibodies Anti-r because of their binding, for example, to antigens of the intracellular structures of nucleated progenitor cells of red blood cells and / or antigens of the intracellular molecules of nucleated progenitor cells of red blood cells and / or antigens of the surfaces of nucleated progenitor cells of red blood cells, such as to the transferrin receptors or their binding to antigens of the surfaces which are specific for nucleated precursor cells of red blood cells, and by the binding of the antibodies anti-i to the antigen-i and / or, if appropriate, preferably by the specific am-enrichment of increasing the fetal cells visibly, marked by the binding of the anti-i plus antibodies to intracellular structures which bind specifically to fetal progenitor cells and / or the intracellular molecules of fetal progenitor cells and / or membrane fragments of fetal progenitor cells.
- the fetal precursor cell of a red blood cell to be enriched may have both anti-i and anti-r antibodies on and / or on its surface.
- the fetal precursor cell of a red blood cell to be enriched, which still contains nuclei can have, for example on and / or on its surface, both anti-i antibodies, anti-r antibodies and anti-i plus antibodies, in order, for example, to separate the specific cells in the subsequent step d) increase.
- the maternal mature erythrocytes cannot be marked by the anti-w, anti-r, anti-i and / or anti i plus antibodies due to the lack of transferrin receptors and antigen i and / or antigen i plus. to these the antibodies Anti-w, Anti-r, Anti-i and / or Anti i plus cannot specifically bind which should be discarded.
- this method of magnetic enrichment or depletion of cells uses commercially available antibodies, some of which are among the anti-bodies described, for example -r and anti-w fall; however, the anti-i or anti-i-plus species cannot be contained in this method.
- a magnetically activated line sorting method with antibodies of the above-mentioned type, eg anti-w and anti-r after a centrifugation the method according to the invention can be accelerated during the sorting.
- the use of magnetically activated cell sorting in combination with the above-mentioned method steps of the method according to the invention or with the representation of fetal cells according to the invention also enables the cleaning of fetal cells to be accelerated.
- the Zeil batch flow cytometer process is subjected.
- different cells labeled by means of antibodies can be distinguished and separated from one another on the basis of the labeled antibodies and the cells with their own different light scattering and / or different emitted fluorescence properties.
- the cells marked accordingly with antibodies are excited in the jacket current method as a single cell suspension in a measuring chamber with laser light and the light is subsequently characterized by means of transmitted light, scattered light and fluorescence detectors.
- the liquid flow also passes through baffles, and the cells characterized as fetal using the properties given above receive an electrical impulse that deflects them laterally out of the liquid jet. This allows the fetal cells to be sorted to purity as individual cells in appropriate containers such as microtitre plates. Due to the flow cytometer method, fetal erythrocyte progenitor cells as fetal cells are sufficiently and gently enriched with the surface antigen i and / or surface antigen i plus.
- the antibodies of the polyclonal and / or monoclonal type can be induced by isolated or purely represented from cells, tissues or organs, such as native, antigens, and / or by chemically synthesized antigens.
- the antibodies to be provided for the antibody cocktail step are e.g. those who are provided for the antibody cocktail step.
- lacto-N-nor-hexaosyl-ceramide compounds of the native type (isolatable from cells) and / or of the synthetic type (chemically producible) or with derivatives of the same type (isolatable from cells) and / or of the synthetic type (chemically producible) and / or
- the antibodies with specificity for anti-i can belong to the IgM class.
- an antibody is also understood to mean that which can specifically react with the antigen i.
- Specific antibodies can represent immunoglobulins produced as a result of antigenic stimulation.
- the polyclonal antibodies can be produced from vertebrates of any species, such as horses, sheep, mice and / or rabbits or the like.
- the monoclonal antibodies can be produced from e.g. B-lymphocytes from vertebrates of any species such as horses, sheep, mice and / or rabbits or the like.
- monoclonal antibodies also include those where the purposes of the invention.
- Immunoglobulins understood, which are specifically directed only against a single antigenic determinant, such as the lacto-N-nor-hexaosyl-ceramide compounds and / or derivatives thereof, which can be chemically synthesized or can be produced purely from cells.
- the monoclonal antibodies Anti-w, Anti-r, and Anti-i, Anti-i plus can be synthesized using conventional hybridoma techniques.
- a single antibody-producing B-lymphocyte is cloned in culture, so that monoclonal antibodies Anti i, Anti-r or Anti-w or Anti-i plus can be obtained in large quantities.
- the B lymphocytes of a mouse immunized against the lacto-N-norhexaosyl-ceramide compound as antigen are fused with cells of an unlimitedly divisible B lymphocyte tumor.
- those hybrid cells are selected from the resulting fusion products which, on the one hand, produce the anti-i antibody and on the other hand have acquired the ability to divide indefinitely in culture.
- Each of these hybridoma cells is the starting point for one individual cell clones that grow continuously and produce a specific monoclonal antibody.
- the monoclonal antibodies Anti-w, Anti-r, Anti-i and / or Anti-i plus can simultaneously be coupled with fluorescein isothiocyanate compounds (FITC), PE or the like to provide fluorochrome antibodies. Even with a large dilution, the antibodies, which can be coupled to the antigen, can be determined on the basis of the fluorescent labeling and increase the yield of the cell sorting method.
- FITC fluorescein isothiocyanate compounds
- anti-w antibodies anti-r antibodies, anti-i antibodies, anti-i plus antibodies, polyclonal antibodies can also be used, which are found in vertebrates such as mammals such as mice, rats, rabbits, sheep, horses, etc., according to the person skilled in the art Methods can be induced and isolated.
- the anti-i antibody by adding lacto-N-nor-hexaosyl-ceramide compound of the native and / or synthetic type and / or their derivatives of the native and / or synthetic type in vertebrates, e.g. Mammals, such as mice, rats, rabbits, sheep, horses, etc., are induced and isolated by the methods known to those skilled in the art.
- polyclonal antibodies Anti-i plus can be a mixture of different species of antibodies, these being administered by administering intracellular structures of fetal progenitor cells and / or the intracellular molecules which are specific for fetal progenitor cells and / or membrane fragments which are suitable for fetal progenitor cells are specific, for example membrane fragments of fetal, preferably nucleated, cells, such as fetal precursor rotor cells of the red blood cells, in vertebrates, for example mammals such as mice, rats, rabbits, sheep, horses etc., can be induced and enriched or can be represented purely in a conventional manner.
- the mixture can be, for example, a species as an antibody which is specific against antigen-i or binds with antigen-i and contains one or more species which are specific to a surface molecule or to surface molecules of the membrane fragments of fetal cells, such as fetal precursor cells the red blood cells, can bind or react with them.
- the antibody cocktail can be fluorescein, phycoerythrin and / or peridinin chlorophyll protein (PerCP) labeled antibodies (20 ⁇ l per antibody) of the monoclonal type, the polyclonal type or mixtures thereof with 0.1 to 10.00 ⁇ g, preferably 0 , 2 to 5.0, even more preferably 0.2 ⁇ g, protein / ⁇ l antibody cocktail, preferably in PBS.
- PerCP peridinin chlorophyll protein
- the antibody cocktail can contain for simultaneous incubation with the cell fraction:
- the mixing ratio of antibody cocktail to Zeil approach can be 0.1 to 100.0 ⁇ l, preferably 10.0 to 60.0 ⁇ l, preferably 20.0 to 40.0 ⁇ l, even more preferably 20.0 ⁇ l, antibody Cocktail with essentially 10 5 - 10 9 , preferably 10 7 cell nucleus-containing cells in the Zeil approach.
- the antibody cocktail can contain for simultaneous incubation with the cell fraction:
- Membrane pieces of fetal red progenitor cells that are specific for fetal nuclei-containing cells are specific for fetal nuclei-containing cells.
- the antibodies can also be added in succession to the cell fraction in the concentrations and proportions given above; for example, as additions in step c) first the Antikö ⁇ er cocktail as a mixture of Antikö ⁇ er Anti-w and Antikö ⁇ er Anti-r and then Anti-i and / or Anti-i-plus, or in step c) first the Antikö ⁇ er cocktail as a mixture from anti-w antibodies and anti-r antibodies and then anti-i-plus and / or anti-i, or in step c) first the anti-w antibodies and then anti-r antibodies, then anti-i and / or anti -i-plus, or in step c) first the anti-w antibody and then anti-r antibody, then anti-i-plus and / or anti-i, or in step c) first the anti-r antibody and then antibody anti-w, then anti-i and / or anti-i, or in step c) first the anti-r antibody and then antibody anti-w, then anti-i and / or anti-i-plus, or in step c)
- Antibodies Anti-i plus can react specifically with Antigen-i; Likewise, Antibody Anti-i plus can be a mixture of different species of antibodies, where, for example, the one antibody specifically against Antigen-i and other antibodies specifically bind to surface molecules of the membrane fragments of fetal cells, such as fetal precursor cells of the red blood cells, or to bind with them.
- the mixing ratio of antibody cocktail to Zeil batch 0.1 to 100.0 ul, preferably 10.0 to 60.0 ul, preferably 20.0 to 40.0 ul can be even more preferred 20.0 .mu.l, antibody cocktail to 10 5 - 10 9 , preferably 10 7 cell nucleus-containing cells in the Zeil approach.
- lacto-N-nor-hexaosyl-ceramide compound is a naturally occurring substance and a surface antigen which can either be isolated from progenitor cells as a compound of the native type or can be prepared purely or can be chemically synthesized as a compound of the synthetic type and is an indicator of fetal Is cells that cannot be detected in the blood of healthy adult people.
- Lacto-N-nor-hexaosyl-ceramide compound is usually designated with the designation i. It is a chain of sugar molecules, which is a linear, unbranched carbohydrate chain made up of repeating N-acetyllactosamine units.
- the simplest i-active glucosphingolipid is lacto-N-nor-hexoosyl-ceramide. It is also possible to use antibodies in the antibody cocktail, which reacts specifically as against lacto-N-iso-octaosyl-ceramide compounds and / or against their derivatives.
- antigen i on cells of erythropoiesis ie lacto-N-nor-hexaosyl-ceramide compound
- this antigen i is a suitable fetal marker for cells of the peripheral blood. All experiments show that adults do not have antigen i on cells of erythropoiesis, but fetuses, newborns and small children, but also in the blood of pregnant women, antigen i can be found as a surface connection.
- the antibodies contained in the antibody cocktail are monoclonal
- Antibodies with uniform monospecificity are synthesized according to conventional steps known to those skilled in the art. Since that
- Antigen i as a sugar molecule can have limited immunogenic activity, antibodies against antigen i are produced in a test animal as follows:
- pure cells are isolated from the blood of a newborn using a conventional high-performance cell sorter in a time-consuming manner (for example Becton Dickinson, Vantage SE), which have i as the antigen lacto-N-nor-hexaosyl-ceramide compound on the surface of the cells.
- These cells are used both for the production of polyclonal antisera, e.g. B. in rabbits, as well as for the production of monoclonal antibodies, e.g. B. used in the mouse.
- antigen i lacto-N-nor-hexaosyl-ceramide compound by methods known to the person skilled in the art, antigen i being able to be prepared in pure form and used as described under 1.
- Withdrawal of venous maternal blood is carried out in an isotonic phosphate buffer solution containing anticoagulants, pH 7.2, 0.9% (w / v) NaCl, 150 mM NaH 2 P0 4 / Na 3 HP0 4 , preferably with NaN 3 for preservation (PBS ) diluted at 25 ° Celsius to provide a blood fraction.
- the blood fraction will be centrifuged at 800 xg for 45 seconds to enrich the cell fraction with nucleated fetal cells as a buffy coat layer at room temperature.
- the whitish buffy-coat layer is incubated as a cell fraction (eg 1 - 4 x 10 cells) with an antibody cocktail of monoclonal type antibodies at room temperature for 20 minutes to show the Zeil approach.
- the antibody cocktail contains fluorescein, phycoerythrin and / or peridinin chlorophyll protein (PerCP) labeled antibodies (20 ⁇ l per antibody) of the monoclonal type with 0.2 ⁇ g protein / ⁇ l antibody cocktail in PBS.
- the antibody cocktail contains the antibody as an antibody for simultaneous incubation with the cell fraction:
- the mixing ratio is 20.0 .mu.l, antibody cocktail to 10 5 - 10 9 , preferably 10 7 cell-containing cells in the Zeil approach.
- the antibody cocktail for simultaneous incubation with the cell fraction contains: 20 ⁇ l of anti-w antibodies with 0.2 ⁇ g protein / ⁇ l of antibody solution, preferably in PBS, which specifically bind to antigens on the surface of white blood cells,
- the antibody cocktail contains only anti-w antibodies and anti-r antibodies, which is first incubated with the cell fraction, with the additions of the antibodies subsequently
- Antispring ⁇ er Cocktail 20 ⁇ l of anti-w antibodies with 0.2 ⁇ g protein / ⁇ l of antibody solution, preferably in PBS, which specifically bind to antigens on the surface of white blood cells,
- antibody-cocktail cell fraction either 20 ⁇ l of anti-i antibody with 0.2 ⁇ g of protein / ⁇ l of antibody solution, preferably in PBS, which specifically bind to the fetal antigen-i on red precursor cells,
- 20 ⁇ l anti-i antibody with 0.2 ⁇ g protein / ⁇ l antibody solution preferably in PBS, which specifically bind to the fetal antigen-i red precursor cells
- 20 ⁇ l anti-i plus antibody with 0.2 ⁇ g protein / ⁇ l antibody solution preferably in PBS against, preferably also containing antigen-i on the membrane surface, membrane pieces of fetal progenitor cells which are specific for fetal cells containing cells.
- the Antikö ⁇ er Anti-i is an Antikö ⁇ er with a specific binding to
- Lacto-N-nor-hexaosylceramide and an anti-i antibody which is provided from B-lymphocytes of a vertebrate immunized against lacto-N-nor-hexaosylceramide of the native type and against membrane pieces of fetal cells, such as mouse and / or rabbit, and another which is provided from B lymphocytes of a mouse and / or rabbit immunized against lacto-N-nor-hexaosylceramide of the chemically synthesized type.
- the anti-i-plus antibody is an antibody with a specific binding to membrane fragments, intracellular structures and / or intracellular molecules of fetal red precursor cells and an anti-i-plus antibody which consists of B-lymphocytes against membrane membrane pieces, intracellular Structures and / or intracellular molecules of fetal red progenitor cells immunized mouse and / or other species from eg Vertebrate comes.
- Layer as a cell fraction (eg 1 - 4 x 10 7 cells) with an antibody cocktail incubated from antibodies of the polyclonal type at room temperature for 20 min to show the Zeil approach.
- the antibody cocktail with polyclonal antibodies enriched or isolated from rabbits, sheep or horses contains fluorescein, phycoerythrin and / or peridinin chlorophyll protein (PerCP) labeled antibodies (20 ⁇ l per antibody) of the polyclonal type with 0.2 ⁇ g protein / ⁇ l
- Antibody cocktail in PBS with the above-mentioned compositions and the incubation conditions are as above for those with monoclonal antibodies.
- the cell line After incubation with polyclonal and / or monoclonal antibodies, the cell line is transformed with the aid of a flow cytometer (eg type Becton Dickinson, Vantage SE) using the transmitted light, scattered light and fluorescence properties of the fetal cells bound by means of fluorescence-labeled antibodies and also depending on their cell size and their existing cell compartments, separated by up to 10 to 10 times pure representation of the same.
- a flow cytometer eg type Becton Dickinson, Vantage SE
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DE10035433 | 2000-07-20 | ||
DE10035433A DE10035433C2 (en) | 2000-07-20 | 2000-07-20 | Gentle high enrichment of fetal cells from peripheral blood and use of the same |
PCT/DE2001/002723 WO2002008751A2 (en) | 2000-07-20 | 2001-07-17 | Mild enrichment of foetal cells from peripheral blood and use thereof |
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ES2375724T3 (en) | 2002-09-27 | 2012-03-05 | The General Hospital Corporation | MICROFLUDE DEVICE FOR SEPERATION OF CELLS AND ITS USES. |
ES2337702T3 (en) * | 2004-03-31 | 2010-04-28 | Adnagen Ag | MONOCLONAL ANTIBODIES WITH SPECIFICITY FOR FETAL Erythrocytes. |
US20070196820A1 (en) | 2005-04-05 | 2007-08-23 | Ravi Kapur | Devices and methods for enrichment and alteration of cells and other particles |
US8921102B2 (en) | 2005-07-29 | 2014-12-30 | Gpb Scientific, Llc | Devices and methods for enrichment and alteration of circulating tumor cells and other particles |
US8774488B2 (en) | 2010-03-11 | 2014-07-08 | Cellscape Corporation | Method and device for identification of nucleated red blood cells from a maternal blood sample |
SG194959A1 (en) * | 2011-06-30 | 2013-12-30 | Univ Singapore | Foetal nucleated red blood cell detection |
US20130130266A1 (en) * | 2011-11-17 | 2013-05-23 | James Stone | Methods and devices for obtaining and analyzing cells |
PT2925776T (en) | 2012-11-27 | 2018-07-30 | Biomarin Pharm Inc | Targeted therapeutic lysosomal enzyme fusion proteins and uses thereof |
US11493428B2 (en) | 2013-03-15 | 2022-11-08 | Gpb Scientific, Inc. | On-chip microfluidic processing of particles |
US20150064153A1 (en) | 2013-03-15 | 2015-03-05 | The Trustees Of Princeton University | High efficiency microfluidic purification of stem cells to improve transplants |
US10324011B2 (en) | 2013-03-15 | 2019-06-18 | The Trustees Of Princeton University | Methods and devices for high throughput purification |
US10976232B2 (en) | 2015-08-24 | 2021-04-13 | Gpb Scientific, Inc. | Methods and devices for multi-step cell purification and concentration |
EP3675876A4 (en) | 2017-09-01 | 2021-06-02 | GPB Scientific, Inc. | Methods for preparing therapeutically active cells using microfluidics |
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US5629147A (en) * | 1992-07-17 | 1997-05-13 | Aprogenex, Inc. | Enriching and identifying fetal cells in maternal blood for in situ hybridization |
US5275933A (en) * | 1992-09-25 | 1994-01-04 | The Board Of Trustees Of The Leland Stanford Junior University | Triple gradient process for recovering nucleated fetal cells from maternal blood |
US5731156A (en) * | 1996-10-21 | 1998-03-24 | Applied Imaging, Inc. | Use of anti-embryonic hemoglobin antibodies to identify fetal cells |
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2000
- 2000-07-20 DE DE10035433A patent/DE10035433C2/en not_active Expired - Fee Related
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2001
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- 2001-07-17 WO PCT/DE2001/002723 patent/WO2002008751A2/en not_active Application Discontinuation
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- 2001-07-17 EP EP01964843A patent/EP1301798A2/en not_active Withdrawn
Non-Patent Citations (3)
Title |
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EDDLEMAN KEITH A. ET AL: "Circulating hematopoietic stem cell populations in human fetuses: Implications for fetal gene therapy and alterations with in utero red cell transfusion", FETAL DIAGNOSIS AND THERAPY, vol. 11, no. 4, 1996, pages 231 - 240, XP009045823 * |
LAN N. ET AL: "Ribozyme-mediated repair of sickle beta-globin mRNAs in erythrocyte precursors", SCIENCE, vol. 280, no. 5369, 5 June 1998 (1998-06-05), UNITED STATES, pages 1593 - 1596, XP002101539 * |
OPIE TIM M. ET AL: "Cell-surface antigen expression in early and term gestation fetal hematopoietic progenitor cells", STEM CELLS, vol. 16, no. 5, 1998, MIAMISBURG, pages 343 - 348, XP009045821 * |
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DE10035433C2 (en) | 2002-07-18 |
JP2004504818A (en) | 2004-02-19 |
WO2002008751A8 (en) | 2002-06-20 |
WO2002008751A3 (en) | 2002-08-15 |
AU2001285683A1 (en) | 2002-02-05 |
WO2002008751A2 (en) | 2002-01-31 |
DE10035433A1 (en) | 2002-02-07 |
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