This invention relates generally to the field of cell seperation. In particular, the invention provides processes for isolating a target cell, cellular organelle or virus from a sample, using inter alia, nonspecific binding between a target cell, cellular organelle or virus with a magnetic microbead modified to comprise hydroxyl, carboxyl or epoxy groups.

Description of the Invention

DISCLOSURE OF THE INVENTION

The present invention relates to the separation of target cells, cellular organelles or viruses (such as leukocyte, virus, epithelial cell and cultured cell) containing target biological molecules (such as nucleic acid and protein) from various sources, e.g., whole blood, saliva, serum, marrow, saliva, urine and culture solution of cells and tissues, using nonspecific or low-specificity adsorption and the paramagnetism of the magnetic micro-beads. Under the appropriate buffer, the magnetic micro-beads can be separated from the bio-conjugates. The separated cells can be used in cell culture, drug screening, bio-chemical reactions and biological analysis. This simple and rapid separation method can be used in sample preparation of different scales, especially for small-quantity and microscale samples and it is easy to build up automatic and micromatic device.

In one aspect, the present invention is directed to a process for isolating a target cell, cellular organelle or virus from a sample, which process comprises: a) contacting a sample containing or suspected of containing a target cell, cellular organelle or virus with a magnetic microbead, said magnetic microbead not comprising a moiety that binds to said target cell, cellular organelle or virus with high specificity; b) allowing said target cell, cellular organelle or virus, if present in said sample, to bind to said magnetic microbead nonspecifically or with low specificity to form a conjugate between said target cell, cellular organelle or virus and said magnetic microbead; and c) separating said conjugate from other undesirable constituents via a magnetic force to isolate said target cell, cellular organelle or virus from said sample.

In another aspect, the present invention is directed to a kit for isolating a target cell, cellular organelle or virus from a sample, which kit comprises in a same or different container(s): a) a magnetic microbead for contacting a sample containing or suspected of containing a target cell, cellular organelle or virus, said magnetic microbead not comprising a moiety that binds to said target cell, cellular organelle or virus with high specificity; b) means for allowing said target cell, cellular organelle or virus, if present in said sample, to bind to said magnetic microbead nonspecifically or with low specificity to form a conjugate between said target cell, cellular organelle or virus and said magnetic microbead; and c) means for separating said conjugate from other undesirable constituents via a magnetic force to isolate said target cell, cellular organelle or virus from said sample.

In still another aspect, the present invention is directed to a process for isolating a virus or bacteriophage from a sample, which process comprises: a) removing cells from a sample containing or suspected of containing a target virus or bacteriophage; b) contacting said cell-free sample with a magnetic microbead, said magnetic microbead not comprising a moiety that binds to said target virus or bacteriophage with high specificity; c) allowing said target virus or bacteriophage, if present in said sample, to bind to said magnetic microbead nonspecifically or with low specificity to form a conjugate between said target virus or bacteriophage and said magnetic microbead; and c) separating said conjugate from other undesirable constituents via a magnetic force to isolate said target virus or bacteriophage from said sample.

In yet another aspect, the present invention is directed to a kit for isolating a virus or bacteriophage from a sample, which kit comprises in a same or different container(s): a) means for removing cells from a sample containing or suspected of containing a target virus or bacteriophage; b) a magnetic microbead for contacting said cell-free sample, said magnetic microbead not comprising a moiety that binds to said target cell, cellular organelle or virus with high specificity; c) means for allowing said target virus or bacteriophage, if present in said cell-free sample, to bind to said magnetic microbead nonspecifically or with low specificity to form a conjugate between said target virus or bacteriophage and said magnetic microbead; and d) means for separating said conjugate from other undesirable constituents via a magnetic force to isolate said target virus or bacteriophage from said sample.

 METHODS AND KITS FOR ISOLATING A TARGET CELL, CELLULAR ORGANELLE OR VIRUS

In one aspect, the present invention is directed to a process for isolating a target cell, cellular organelle or virus from a sample, which process comprises: a) contacting a sample containing or suspected of containing a target cell, cellular organelle or virus with a magnetic microbead, said magnetic microbead not comprising a moiety that binds to said target cell, cellular organelle or virus with high specificity; b) allowing said target cell, cellular organelle or virus, if present in said sample, to bind to said magnetic microbead nonspecifically or with low specificity to form a conjugate between said target cell, cellular organelle or virus and said magnetic microbead; and c) separating said conjugate from other undesirable constituents via a magnetic force to isolate said target cell, cellular organelle or virus from said sample.

In another aspect, the present invention is directed to a kit for isolating a target cell, cellular organelle or virus from a sample, which kit comprises in a same or different container(s): a) a magnetic microbead for contacting a sample containing or suspected of containing a target cell, cellular organelle or virus, said magnetic microbead not comprising a moiety that binds to said target cell, cellular organelle or virus with high specificity; b) means for allowing said target cell, cellular organelle or virus, if present in said sample, to bind to said magnetic microbead nonspecifically or with low specificity to form a conjugate between said target cell, cellular organelle or virus and said magnetic microbead; and c) means for separating said conjugate from other undesirable constituents via a magnetic force to isolate said target cell, cellular organelle or virus from said sample. The kit can further comprise an instruction for using the kit for isolating a target cell, cellular organelle or virus from a sample.

The present processes and kits can be used to isolate any suitable target cell, cellular organelle or virus from a sample. Exemplary target cells include animal cells, plant cells, fungus cells, bacterium cells, recombinant cells and cultured cells. Exemplary target cellular organelles include nuclei, mitochondria, chloroplasts, ribosomes, ERs, Golgi apparatuses, lysosomes, proteasomes, secretory vesicles, vacuoles and microsomes. Exemplary target viruses include eucaryotic cell viruses and bacteriophages.

The magnetic microbeads can be prepared by any suitable methods. For example, the methods disclosed in CN 01/109870.8 or WO02/075309 can be used. Any suitable magnetizable substance can be used to prepare the magnetic microbeads useful in the present processes and kits. No-limiting examples of the magnetizable substances include ferrimagnetic substance, ferromagnetic substance, paramagnetic substance or superparamagnetic substances. In a specific embodiment, the magnetic microbeads comprise a paramagnetic substance, e.g., a paramagnetic metal oxide composition. Preferably, the paramagnetic metal oxide composition is a transition metal oxide or an alloy thereof. Any suitable transition metals can be used, such as iron, nickel, copper, cobalt, manganese, tantalum (Ta), zinc and zirconium (Zr). In a preferred embodiment, the metal oxide composition is Fe.sub.3O.sub.4 or Fe.sub.2O.sub.3. In another example, the magnetizable substance used in the magnetic microbeads comprises a metal composition. Preferably, the metal composition is a transition metal composition or an alloy thereof such as iron, nickel, copper, cobalt, manganese, tantalum, zirconium and cobalt-tantalum-zirconium (CoTaZr) alloy.

The magnetic microbeads may be prepared from the available primary beads, from raw materials or from metal oxides that are encapsulated by monomers which when crosslinked form rigid, polymeric coatings as disclosed in U.S. Pat. No. 5,834,121. As used herein, "rigid" refers to a polymeric coating that is cross linked to the extent that the polymeric coating stabilizes the metal oxide particle within the coating (i.e. the coating essentially does not swell or dissolve) so that the particle remains enclosed therein. As used herein, "microporous" refers to a resinous polymeric matrix that swells or expands in polar organic solvent. As used herein, "load" is used to mean the capacity of the bead for attachment sites useful for functionalization or derivatization.

Suitable substances which may be incorporated as magnetizable materials, for example, include iron oxides such as magnetite, ferrites of manganese, cobalt, and nickel, hematite and various alloys. Magnetite is the preferred metal oxide. Frequently, metal salts are taught to be converted to metal oxides then either coated with a polymer or adsorbed into a bead comprising a thermoplastic polymer resin having reducing groups thereon. When starting with metal oxide particles to obtain a hydrophobic primary bead, it is necessary to provide a rigid coating of a thermoplastic polymer derived from vinyl monomers, preferably a cross-linked polystyrene that is capable of binding or being bound by a microporous matrix. Magnetic particles may be formed by methods known in the art, e.g., procedures shown in Vandenberge et al., J. of Magnetism and Magnetic Materials, 15-18:1117-18 (1980); Matijevic, Acc. Chem. Res., 14:22-29 (1981); and U.S. Pat. Nos. 5,091,206; 4,774,265; 4,554,088; and 4,421,660. Examples of primary beads that may be used in this invention are shown in U.S. Pat. Nos. 5,395,688; 5,318,797; 5,283,079; 5,232,7892; 5,091,206; 4,965,007; 4,774,265; 4,654,267; 4,490,436; 4,336,173; and 4,421,660. Or, primary beads may be obtained commercially from available hydrophobic or hydrophilic beads that meet the starting requirements of size, sufficient stability of the polymeric coating to swell in solvents to retain the paramagnetic particle, and ability to adsorb or absorb the vinyl monomer used to form the enmeshing matrix network. Preferably, the primary bead is a hydrophobic, polystyrene encapsulated, paramagnetic bead. Such polystyrene paramagnetic beads are available from Dynal, Inc. (Lake Success, N.Y.), Rhone Poulonc (France), and SINTEF (Trondheim, Norway). The use of toner particles or of magnetic particles having a first coating of an unstable polymer which are further encapsulated to produce an exterior rigid polymeric coating is also contemplated.

The magnetic microbeads used in the present processes and kits can have any suitable size, e.g., having a diameter ranging from about 5 to about 50,000 nanometers.

The magnetic microbeads used in the present processes and kits can be untreated or can be modified, e.g., modified with an organic molecule. In a specific embodiment, the magnetic microbead is modified to comprise a hydroxyl, a carboxyl or an epoxy group.

The present processes can further comprise washing the separated conjugate to remove the undesirable constituents and/or can further comprise recovering the target cell, cellular organelle or virus from the separated conjugate. For example, the target cell, cellular organelle or virus can be released from the separated conjugate with a suitable buffer solution into the buffer and the magnetic microbead is removed from the solution via a magnetic force.

The present processes and kits can be used to isolate any suitable target cell, cellular organelle or virus from any suitable sample. For example, the present processes and kits can be used to isolate any suitable target cell, cellular organelle or virus from a clinical sample. In another example, the present processes and kits can be used to isolate any suitable target cell, cellular organelle or virus from serum, plasma, whole blood, sputum, cerebral spinal fluid, amniotic fluid, urine, gastrointestinal contents, hair, saliva, sweat, gum scrapings, marrow, tissue or cell culture.

The present processes can further comprise recovering a biological material from the isolated target cell, cellular organelle or virus. Exemplary biological materials include amino acids, peptides, proteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, vitamins, monosaccharides, oligosaccharides, carbohydrates, lipids and a complex thereof. Preferably, the biological material to be recovered from the isolated target cell, cellular organelle or virus is an oligonucleotide or a nucleic acid. The present processes can further comprise amplifying the recovered oligonucleotide or nucleic acid. The biological materials can be recovered and/or amplified from the isolated target cell, cellular organelle or virus using any suitable methods (See e.g., Ausabel et al., eds., Current Protocol of Molecular Biology, John Wiley and Sons, Inc (2000)).

The present processes can be performed manually. Preferably, the present processes are automated. Any, some or all steps of the present processes can be automated. For example, the sample contacting, binding, separating, as well as any other additional steps such as washing, target cell, cellular organelle or virus releasing, and biological material recovering or amplifying step(s) can be automated.

The present processes can be performed within any suitable time frame. For example, the present processes can be performed within a time ranging from about 1 minute to about 20 minutes.

The present processes can be performed at any suitable temperature. For example, the present processes can be performed at an ambient temperature ranging from about 0.degree. C. to about 35.degree. C.

The present processes can be performed in an EPPENDORF.TM. tube. The present processes can be performed in the absence of a precipitation procedure. The present processes can be performed in the absence of a poisonous agent.

In one specific embodiment, the present process is used to isolate a leukocyte from whole blood, marrow or lympha, e.g., fresh or low-temperature conserved whole blood, marrow or lympha. The leukocyte can be contacted with the magnetic microbead in a suitable chemical environment having the following characteristic(s): a) a pH ranging from about 3 to about 7; and/or b) a suitable concentration or amount of an anticoagulant, e.g., acid citrate dextrose (ACD), sodium citrate and sodium heparin, e.g., 23 mM citric acid, 80 mM dextrose and 45 mM sodium citrate.

The process can further comprise washing the separated leukocyte-magnetic-microbead conjugate with a washing buffer to remove the undesirable constituents. Any suitable washing buffer can be used. For example, the washing buffer can be a physiological salt water having a pH at about 6.5 or a phosphate buffer (PBS) having a pH at about 6.5. The leukocyte can be released from the separated leukocyte-magnetic-microbead conjugate with a suitable separation buffer solution into the buffer and the magnetic microbead is removed from the solution via a magnetic force.

In another specific embodiment, the present process is used to isolate a target cell, e.g., an epithelia cast-off cell or a bacteria cell, cellular organelle or virus from saliva, urine and tissue culture. The saliva, urine and tissue culture can be fresh or low-temperature conserved saliva, urine and tissue culture. The target cell, cellular organelle or virus can be contacted with the magnetic microbead in a suitable chemical environment having a pH ranging from about 3 to about 7. The process can further comprise washing the separated conjugate between the target cell, cellular organelle or virus and the magnetic microbead with a washing buffer to remove the undesirable constituents. Any suitable washing buffer can be used. For example, the washing buffer can be a physiological salt water having a pH at about 6.5 or a phosphate buffer (PBS) having a pH at about 6.5. The target cell, cellular organelle or virus can be released from the separated conjugate between the target cell, cellular organelle or virus and the magnetic microbead with a suitable separation buffer solution into the buffer and the magnetic microbead is removed from the solution via a magnetic force. Any suitable separation buffer can be used. For example, the separation buffer can be a Tris-EDTA buffer having a pH ranging from about 6.5 to about 8 and a detergent at a concentration about less than 1% (w/w).

C. PROCESSES AND KITS FOR ISOLATING A VIRUS OR BACTERIOPHAGE

In still another aspect, the present invention is directed to a process for isolating a virus or bacteriophage from a sample, which process comprises: a) removing cells from a sample containing or suspected of containing a target virus or bacteriophage; b) contacting said cell-free sample with a magnetic microbead, said magnetic microbead not comprising a moiety that binds to said target virus or bacteriophage with high specificity; c) allowing said target virus or bacteriophage, if present in said sample, to bind to said magnetic microbead nonspecifically or with low specificity to form a conjugate between said target virus or bacteriophage and said magnetic microbead; and c) separating said conjugate from other undesirable constituents via a magnetic force to isolate said target virus or bacteriophage from said sample.

In yet another aspect, the present invention is directed to a kit for isolating a virus or bacteriophage from a sample, which kit comprises in a same or different container(s): a) means for removing cells from a sample containing or suspected of containing a target virus or bacteriophage; b) a magnetic microbead for contacting said cell-free sample, said magnetic microbead not comprising a moiety that binds to said target cell, cellular organelle or virus with high specificity; c) means for allowing said target virus or bacteriophage, if present in said cell-free sample, to bind to said magnetic microbead nonspecifically or with low specificity to form a conjugate between said target virus or bacteriophage and said magnetic microbead; and d) means for separating said conjugate from other undesirable constituents via a magnetic force to isolate said target virus or bacteriophage from said sample.

The present processes and kits can be used to isolate a virus or bacteriophage from any suitable sample. Exemplary samples include saliva, urine or serum. Preferably, the saliva, urine or serum is fresh or low-temperature conserved saliva, urine or serum.

The present processes and kits can be used to isolating a virus or bacteriophage from any suitable cells. Exemplary cells include epithelia cast-off cells and bacteria cells. The cells can be from the sample by any suitable methods, e.g., centrifugation.

In one specific embodiment, the virus or bacteriophage is contacted with the magnetic microbead in the presence of: a) a highly hydratable compound at a concentration ranging from about 10% (v/v) to about 100% (v/v); and/or b) a salt at a concentration ranging from about 2.5 M to about 5.0 M.

Any suitable highly hydratable compound, e.g., organic compound, can be used in the present processes and kits. Exemplary high-hydrability organic compounds include ethanol, acetone and polyethylene glycol. Any suitable salt can be used in the present processes and kits. Exemplary salt includes sodium chloride.

The process can further comprise washing the separated conjugate between the target virus or bacteriophage and the magnetic microbead with a washing buffer to remove the undesirable constituents. Any suitable washing buffer can be used. For example, the washing buffer can be a physiological salt water having a pH at about 6.5 or a phosphate buffer (PBS) having a pH at about 6.5.

The process can further comprise releasing the target virus or bacteriophage from the separated conjugate between the target virus or bacteriophage and the magnetic microbead with a suitable separation buffer solution into the buffer and the magnetic microbead is removed from the solution via a magnetic force.

The general teachings of the above Section B, e.g., target cells, samples, properties of the magnetic microbeads, washing and separation buffers and procedures, biological materials to be released, various aspects of automation, time, temperature and locations wherein the isolation are conducted, the absence of certain procedure or substance, etc., are also applicable to this Section C.
 

Claim 1 of 38 Claims

1. A process for isolating a target cell, cellular organelle or virus from a sample, which process comprises: a) contacting a sample containing or suspected of containing a target cell, cellular organelle or virus with a magnetic bead, said magnetic bead not comprising a moiety that can form a specific binding pair with said target cell, cellular organelle or virus; b) allowing said target cell, cellular organelle or virus, if present in said sample, to bind to said magnetic bead nonspecifically to form a conjugate between said target cell, cellular organelle or virus and said magnetic bead; and c) separating said conjugate from unbound constituents via a magnetic force to isolate said target cell, cellular organelle or virus from said sample, wherein the magnetic bead has a diameter of 200 nm and is modified to comprise a hydroxyl, a carboxyl or an epoxy group.

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