EP0621802A1 - Supports magnetiques a phase solide - Google Patents

Supports magnetiques a phase solide

Info

Publication number
EP0621802A1
EP0621802A1 EP93924712A EP93924712A EP0621802A1 EP 0621802 A1 EP0621802 A1 EP 0621802A1 EP 93924712 A EP93924712 A EP 93924712A EP 93924712 A EP93924712 A EP 93924712A EP 0621802 A1 EP0621802 A1 EP 0621802A1
Authority
EP
European Patent Office
Prior art keywords
msps
magnetisable
particles according
magnetisable particles
particles
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Withdrawn
Application number
EP93924712A
Other languages
German (de)
English (en)
Inventor
Ian James Bruce
Martin John Davies
Geoffrey Eric Scopes
Diane Elizabeth Smethurst
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
WILLIAMS John Francis
Original Assignee
WILLIAMS John Francis
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by WILLIAMS John Francis filed Critical WILLIAMS John Francis
Publication of EP0621802A1 publication Critical patent/EP0621802A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/291—Gel sorbents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28009—Magnetic properties
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/3007—Moulding, shaping or extruding
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
    • B01J20/3293—Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
    • G01N2400/32—Galactans, e.g. agar, agarose, agaropectin, carrageenan
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/20—Magnetic particle immunoreagent carriers the magnetic material being present in the particle core
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/30—Magnetic particle immunoreagent carriers the magnetic material being dispersed in the polymer composition before their conversion into particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
    • G01N2446/84—Polymer coating, e.g. gelatin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
    • G01N2446/90—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids characterised by small molecule linker used to couple immunoreagents to magnetic particles

Definitions

  • This invention relates to the isolation and purification of molecules of biological interest eg proteins, nucleic acids, and other biopolymers.
  • An affinity chromatography step is often the final or penultimate step. Ideally this step should be employed as early as possible in the process to maximise the yield of desired product and minimise the time required to generate it. Usually this is not feasible as the substance of interest (ligate) is present in a medium not readily amenable to conventional column affinity chromatography. Often the ligate may be present in a mixture of substances containing particulate and/or semi-solid or colloidal material such as cell debris and denatured cell components and/or in a mixture which is highly viscous.
  • the present invention is concerned with the development of relatively simple techniques for use with complex mixtures of high viscosity and/or those containing particulate matter and which are based on the use of magnetisable solid phase support (MSPS) materials.
  • MSPS magnetisable solid phase support
  • the present invention comprises magnetisable polymer based particles derivatised with ligands possessing direct-binding affinities for nucleic acids and other types of biomolecule, and the use of these particles for nucleic acid (and other biomolecular) purification and speciation, including double or single stranded nucleic acids, genomic DNA, plasmid DNA, and cellular RNA.
  • the direct binding of the ligand to the target molecule is important to achieve the benefits of this invention as contrasted with prior proposals based on indirect linking of the target molecules eg. through intercalating agents as described in EP 301,899 A.
  • the present invention comprises particulate support material having magnetic properties and bearing a ligand which binds to molecules of a specific type. From another aspect the present invention comprises a method of separating biomolecules of various types by binding such molecules to ligands which are selective therefore and which are attached to magnetisable particulate support materials. The invention is further apparent from the appended claims.
  • the magnetic susceptibility of MSPS materials in accordance with the present invention is a function of the weight and size of the particulate material and the quantity of paramagnetic material incorporated therein. This property may therefore be adjusted as desired in order to provide an appropriate selection parameter.
  • a product range of MSPS materials may be manufactured in order to cater for the wide range of separations encountered in both analytical and preparative procedures.
  • the MSPS materials in accordance with this invention are preferably gels and are conveniently in the form of beads. Such gels may be stabilised by crosslinking eg with epichlorohydrin, leading to greater particle stability especially at high temperatures.
  • Many polymers may be used for the purposes of this invention including cellulose, dextran, polyacrylamide and the synthetic material trisacryl.
  • One of the most preferred materials is agarose.
  • the positive benefits of this polymer are gel strength and biological inertness.
  • the abundance of hydroxyl groups possessed by the agarose matrix allows for easy covalent attachment of various ligands desirable for molecular biology applications and its non-covalent secondary structure also engenders structural stability, porosity and ease of shaping into spheres. Since many molecular biology methodologies are performed at temperatures above that at which agarose melts it would also be desirable to introduce thermal stability into the agarose solid phase support.
  • MSPS materials consist of at least two separate components a non-magnetisable component which may be chemically derivatised with suitable ligands and a magnetisable component or core.
  • Paramagnetic properties can be introduced into the solid phase support by addition of powdered paramagnetic iron oxide (Fe 3 0 4 ) to the agarose during the preparation of the MSPS, which leads to entrapment of the paramagnetic component within the solid phase matrix and gives the particles their magnetisability.
  • MSPS particles should be within a size range which allows the greatest particle surface area exposure to ligate, but at the same time allows the best particle magnetic sedimentation rate.
  • the magnetisable component should also have a high degree of magnetisable susceptibility to optimise magnetic sedimentation rates.
  • the ideal magnetisable component can be selected on the basis of the above equation, but in practice relatively few paramagnetic molecules have been used in MSPS. Iron oxide, barium ferrite and nickel oxide may be used.
  • MSPS construction since it possesses a high magnetic susceptibility, is readily available, inexpensive and non toxic.
  • a highly preferred system for our purposes entails iron oxide as the magnetisable component for
  • MSPS and agarose as the polymer matrix in which to embed it.
  • other natural and synthetic polymers may be used as MSPS matrix material including cellulose, alginate, dextran and perfluorocarbon based supports.
  • MSPS may be prepared either by encapsulation of the magnetisable component during the preparation of the particle, or by addition of the magnetisable component subsequent to particle formation.
  • the magnetisable component can be mixed in an aqueous suspension of cellulose or agarose and allowed to cure, the water is removed and the dried material ground to the size range required.
  • MSPS may be prepared by spraying or droplet formation and this leads to beaded particles.
  • MSPS may be prepared by addition of a suspension of sodium alginate and iron oxide dropped into a solution of calcium chloride. Droplet formation can be brought about through emulsification techniques and thee have been employed for the formation of spherical magnetisable dextran, albumin, acrylates, acrolein and polyglutaraldehyde. In this system an aqueous emulsion of the matrix polymer and magnetisable component is prepared. This is added to mineral oil and the two phase system stirred. A precipitate is formed of beaded particles.
  • the particle is coated with an aqueous solution of polyacrylamide containing ferric oxide.
  • the resulting particles tend to be large >250 ⁇ and very porous. However, they are amenable to activation via either the polysaccharide or polyacrylamide component.
  • agarose MSPS In one method we have used to prepare agarose MSPS an aqueous suspension is extruded into an immiscible organic phase.
  • a suspension of paramagnetic iron oxide, Fe 3 0 4 , (4% w/v) in molten agarose (2% w/v) was extruded into vegetable oil stirred with an overhead paddle stirrer using a specially adapted plastic syringe. The end of the syringe was sealed, then drilled with a small hole to allow the molten agarose/iron oxide mixture to be extruded into the oil phase.
  • the MSPS was collected, washed with water and sized by sieving, then checked under a light microscope set up for K ⁇ hler illumination for morphological homogeneity. The particles formed were highly bead like and uniformly spherical, a consequence of both the extrusion technique and the immiscibility of the oil and water.
  • the highly spherical nature of the particles produced is particularly desirable because it prevents local concentration change effects that might occur with particles whose surface is more heterogeneous. It is particularly important to maintain uniform ionic concentrations around the surface of a particle in order that local absorption and/or desorption does not occur and alter the bulk properties of the support.
  • MSPS When the MSPS was viewed under the light microscope, small yellow inclusions were observed incorporated within the agarose matrix which were believed to be vegetable oil retained in the matrix of the agarose during the preparation of the MSPS. To remove these inclusions the MSPS was washed following preparation in an acetone/water series.
  • MSPS size, quantity and quality of the MSPS produced.
  • a range of differently sized MSPS (>500 ⁇ m - ⁇ 150 ⁇ m) were obtained when stir rate, hole bore diameter and extrusion rate were altered. Results indicated that the faster the stir rate of the oil phase, the narrower the bore size of the pierced hole and the higher the extrusion rate, the greater the proportion of smaller diameter MSPS (less than 150 ⁇ m) was produced.
  • Small particles are sought for affinity chromatography as they possess a greater surface area/volume ratio, significantly enhancing their ligand loading capacity and subsequent ability to take up and release macromolecules during chromatography.
  • cross-linked MSPS In order to confer thermal and chemical stability on the MSPS following manufacture, it may be cross-linked with epichlorohydrin under basic conditions using known methods.
  • the cross-linking reaction covalently binds together the polymeric agarose strands by a three-carbon link, instilling a physical rigidity in the particles that reduces the risk of shear damage.
  • Once cross- linked MSPS can be autoclaved (120°C, 15psi, 15 min) without damage or alteration of its physical properties, and is resistant to a whole range of chemical reagents used in further derivatisations.
  • Cross-linked MSPS can be stored quite satisfactorily at room temperature for several months as a suspension in 20% aqueous methanol (to act as an anti-bacterial agent) without any deterioration. No leaching of iron oxide from the agarose matrix was observed.
  • DEAE-, ECTEOLA-, spermine-, TAEA-, EHEP, and HDA-MSPS can all be used for the general isolation of nucleic aids and are listed in decreasing order of affinity for nucleic acids.
  • the degree of ligand loading may be expressed as the ability of a known weight of MSPS to adsorb a known amount of DNA, and the adsorption measured by ultra ⁇ violet spectroscopy. This serves as a method of quality control for the MSPS to check that surface derivatisation had proceeded satifactorily.
  • a suspension of DEAE-MSPS 100 mg ml was incubated with a solution of salmon sperm DNA (50 ⁇ g ml -1 ) at room temperature in an Eppendorf tube. After 30 minutes the MSPS was immobilised with a magnetic concentrator, the supernatant removed and its adsorbance at 260nm and 280nm recorded, and compared with that of the original stock solution of DNA to calculate the level of nucleic acid uptake. Typically, for DEAE-MSPS a figure of at least 95% uptake of salmon sperm DNA was obtained. Similar results were obtained for ECTEOLA-, TAEA- and spermine-MSPS (Table 1).
  • tertiary amines are more basic than secondary or primary amines, solid phase supports derivatised with tertiary amine-containing ligand have a higher positive charge density and therefore a greater affinity for nucleic acids.
  • the adsorbed DNA could be eluted from all samples with at least 80% efficiency (based on A 250 readings) by incubation for up to 30 minutes with 1M NaCl/50mM arginine free base at 65°C.
  • the derivatised MSPS can be stored at 4°C for several months in suspensions of 20% aqueous methanol without any decrease in performance or capacity for nucleic acids.
  • MSPS magnetisable solid phase support
  • the majority of the cleared oil phase is decanted and the aqueous phase containing the beaded MSPS re-washed with deionised water (100ml).
  • the suspension of MSPS is initially sized by sieving through a series of Endecott sieves of mesh size 500 ⁇ m, 250 ⁇ m, 200 ⁇ m, 180 ⁇ m, and 150 ⁇ m using a Fritsch sieve shaker.
  • the fraction containing particles of less than 150 ⁇ m diameter is further sieved with sieves of mesh size lOO ⁇ m, 50 ⁇ m, 32 ⁇ m and 20 ⁇ m.
  • Cross-linked MSPS (0.7 g moist weight) is suspended in 40% sodium hydroxide (3ml) and cooled to 0°C in an ice- water bath. A solution of triethanolamine (0.4ml) in epichlorohydrin (0.7ml) is added drop-wise with occasional shaking, maintaining the temperature at 0°C. After 30 minutes the suspension is allowed to warm 23°C, shaken vigorously using a flask shaker and left to stand for 16 hours. The mixture is then poured into a large volume of 1M hydrochloric acid with stirring, filtered, then washed successively with 1M sodium hydroxide, sterile distilled water, 20% aqueous methanol then finally water. The ECTEOLA-MSPS is stored in 20% aqueous methanol to give a particle density of 100 mg/ml.
  • Cross-linked MSPS (0.5 g moist weight) is suspended in 1M sodium hydroxide (0.4ml). Sodium borohydride (l g) is added followed by diglycidylbutane-1,4-diol (0.5ml). The suspension is shaken at 23°C for 6 hours, then filtered, washed thoroughly with deionised water and immediately resuspended in 5M tris(2-aminoethyl)amine (l l) and shaken at 30"c for 2 hours. The suspension is 0 washed successively with sterile distilled water, 1M sodium chloride, sterile distilled water and finally 0.05M phosphate buffer (pH7). The TAEA-MSPS is stored in 20% aqueous methanol to a final particle density of 100 mg/ml. 5
  • MSPS (2g moist weight) is activated with oxirane groups as described for TAEA-MSPS, then suspended in 5M
  • Cross-linked MSPS (0.9 g moist weight) is suspended in 3040% sodium hydroxide (4ml) and cooled to 0°C in an ice- water bath.
  • the mixture is then poured into a large volume of 1M hydrochloric acid with stirring, filtered, then washed successively with 1M sodium hydroxide, sterile distilled water, 20% aqueous methanol and finally sterile distilled water.
  • the EHEP-MSPS is stored in 20% aqueous methanol to give a final particle density of 100 mg/ml.
  • nucleic acids to be adsorbed by the MSPS can be in a variety of forms, including aqueous solutions of one, or more than one, species of nucleic acid, or semi-crude cell lysate mixtures. Nucleic acids are adsorbed non- specifically by the MSPS at ambient temperature (23°C) from solution at pH7 or below. Nucleic acids can be eluted specifically in pure form from the MSPS by treatment with elution solutions of differing ionic strengths.
  • RNA and DNA can be adsorbed simultaneously by the DEAE-derivatised beads, the RNA eluted using 0.lMNaCl/50mM arginine (free base) at 65°C for 30 minutes.
  • Plasmid DNA can be adsorbed by the MSPS from a cell lysis mixture (boiling lysis) which has been treated with RNAse. Any remaining small fragments of oligoribonucleotide which co-adsorb with the plasmid DNA can be eluted using 0.1M/50mM arginine (free base), then the pure plasmid DNA can be eluted using 1.0M/50mM arginine (free base) at 65°C for 15 minutes.
  • RNA could be adsorbed to and eluted from the DEAE- MSPS, but elution occurred at much lower ionic concentrations and temperatures and at a significantly faster rate.
  • DEAE-MSPS was incubated with a solution of total RNA (50 ⁇ g ml "1 ) at 23°C for 30 minutes, magnetically immobilised and the supernatant removed. Comparison of the A 2 go values of the supernatant and original stock solution of RNA showed that, on average, 90 % of the RNA had been adsorbed by the MSPS.
  • the RNA could be eluted with an efficiency of 80-85% at salt concentrations of 0.1M NaCl up to 1.0M NaCl (50mM arginine free base) at 23°C.
  • DEAE-MSPS can also be used for differentiating plasmid DNA and total cellular RNA.
  • a solution of plasmid pUC 18 was prepared by standard boiling lysis, which contained amounts of RNA carried over from the lysis step.
  • DEAE-MSPS was incubated with this solution under the same conditions used for other protocols, magnetically immobilised, then treated with 0.1M NaCl, to remove adsorbed oligoribonucleotides, then the plasmid DNA eluted using 1.0M/50mM arginine free base. Eluted samples were analysed by agarose gel electrophoresi ⁇ , which showed firstly that the MSPS will adsorb and release plasmid DNA satisfactorily using the standard uptake and elution protocol, and secondly, that RNA was eluted at 0.05M-0.1 NaCl, whilst the plasmid DNA was eluted at a salt concentration of 1.0M NaCl. Therefore, DEAE-MSPS can be used to separate unwanted RNA resulting from cell lysis from plasmid DNA, and provide plasmid DNA of a purity and quality suitable for further applications.
  • DEAE-MSPS appears to selectively release them according to size upon application of elution solutions.
  • the DEAE-MSPS was non-specific in its uptake of a mixtures of nucleic acids from solution, but by application of different concentrations of an elution solution, DNA and RNA could be eluted specifically from the DEAE-MSPS, a procedure which can be scaled down to a microtitre plate format without loss of efficiency.
  • plasmid DNA and RNA could be speciated on the DEAE-MSPS.
  • Total RNA can be isolated from a mixture of RNA and DNA using a method similar to that described above. 250 ⁇ l of a suspension of DEAE-MSPS (100 mg ml -1 ) is aliquotted into an Eppendorf tube and washed twice with sterile distilled water. The nucleic acid solution containing RNA and DNA (up to 50 ⁇ g total) is added to the MSPS and the suspension mixed by end-over-end rotation for 30 minutes at 23°C. The MSPS is magnetically immobilised and the supernatant removed, then 1ml of an elution solution (0.1M NaCl, 50mM arginine free base) is added.
  • an elution solution 0.1M NaCl, 50mM arginine free base
  • 1.5 ml of cell culture is spun in a microcentrifuge for 20 seconds in an Eppendorf tube.
  • the supernatant is aspirated and the cell pellet resuspended in 350 ⁇ l STET buffer (0.1M NaCl, lOmM Tris-HCl pH 8, l EDTA, 5% triton X-100) containing 2 ⁇ l of RNAse A solution (lOmg ml "1 ).
  • 25 ⁇ l of a freshly prepared solution of lysozyme (lOmg ml "1 ) is added in lOmM tris-HCl, pH8.
  • the solution is placed in a boiling water bath for 40 seconds, then spun in a centrifuge at 13,000 rpm for 10 minutes.
  • the pelleted cell debris is removed using a disposable pipette tip, and the lysate solution added to 25mg of DEAE-MSPS (250 ⁇ l of 100 mg ml "1 suspension) in an Eppendorf tube.
  • the suspension is mixed by end-over- end rotation for 30 minutes at 23°C, the MSPS magnetically immobilised and the supernatant removed.
  • the DEAE-MSPS is washed twice with sterile distilled water, and then washed two times with O.lmM NaCl/50mM arginine free base to remove any oligoribonucleotides present in the lysis mixture which may become adsorbed to the MSPS.

Abstract

Des biomolécules sont séparées à l'aide de particules magnétisables à base de polymères, formant un dérivé avec un ligand présentant une affinité de liaison directe aux acides nucléiques et à d'autres types de molécules biopolymères. Ces particules sont utilisées pour la spécification et la purification d'acides nucléiques (et d'autres éléments biomoléculaires), y compris des acides nucléiques double-brin ou simple-brin, l'ADN génomique, l'ADN plasmidique et l'ADN cellulaire.
EP93924712A 1992-11-06 1993-11-05 Supports magnetiques a phase solide Withdrawn EP0621802A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9223334 1992-11-06
GB929223334A GB9223334D0 (en) 1992-11-06 1992-11-06 Magnetic solid phase supports
PCT/GB1993/002289 WO1994011103A1 (fr) 1992-11-06 1993-11-05 Supports magnetiques a phase solide

Publications (1)

Publication Number Publication Date
EP0621802A1 true EP0621802A1 (fr) 1994-11-02

Family

ID=10724683

Family Applications (1)

Application Number Title Priority Date Filing Date
EP93924712A Withdrawn EP0621802A1 (fr) 1992-11-06 1993-11-05 Supports magnetiques a phase solide

Country Status (4)

Country Link
EP (1) EP0621802A1 (fr)
AU (1) AU5427394A (fr)
GB (1) GB9223334D0 (fr)
WO (1) WO1994011103A1 (fr)

Families Citing this family (46)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2127605A1 (fr) * 1993-12-23 1995-06-24 Peter J. Degen Methode de separation par affinite
WO1996003653A1 (fr) * 1994-07-27 1996-02-08 Silica Gel Ges.Mbh Absorptionstechnik, Apparatebau Particules superparamagnetiques, leur procede de production et leur utilisation
GB9425138D0 (en) 1994-12-12 1995-02-08 Dynal As Isolation of nucleic acid
JP2965131B2 (ja) * 1995-07-07 1999-10-18 東洋紡績株式会社 核酸結合用磁性担体およびそれを用いる核酸単離方法
EP1260595B1 (fr) * 1995-07-07 2006-09-13 Toyo Boseki Kabushiki Kaisha Support magnétique pour acides nucléiques et procédé pour leur isolation en utilisant le dit support
WO1999015023A2 (fr) * 1997-09-22 1999-04-01 Kiwi Co-Operative Dairies Limited Procede de recuperation
US6607829B1 (en) 1997-11-13 2003-08-19 Massachusetts Institute Of Technology Tellurium-containing nanocrystalline materials
US6207392B1 (en) 1997-11-25 2001-03-27 The Regents Of The University Of California Semiconductor nanocrystal probes for biological applications and process for making and using such probes
US6914137B2 (en) * 1997-12-06 2005-07-05 Dna Research Innovations Limited Isolation of nucleic acids
GB9821783D0 (en) * 1998-10-06 1998-12-02 Bioprocessing Ltd Adsorbent medium
AU2001250882A1 (en) 2000-03-20 2001-10-03 Massachusetts Institute Of Technology Inorganic particle conjugates
EP1266385B1 (fr) 2000-03-24 2008-12-31 QIAGEN GmbH Particules de verre ferro- ou ferrimagnetiques poreuses servant a isoler des molecules
US6692700B2 (en) 2001-02-14 2004-02-17 Handylab, Inc. Heat-reduction methods and systems related to microfluidic devices
US8895311B1 (en) 2001-03-28 2014-11-25 Handylab, Inc. Methods and systems for control of general purpose microfluidic devices
US6852287B2 (en) 2001-09-12 2005-02-08 Handylab, Inc. Microfluidic devices having a reduced number of input and output connections
US7829025B2 (en) 2001-03-28 2010-11-09 Venture Lending & Leasing Iv, Inc. Systems and methods for thermal actuation of microfluidic devices
US7010391B2 (en) 2001-03-28 2006-03-07 Handylab, Inc. Methods and systems for control of microfluidic devices
GB0116358D0 (en) * 2001-07-04 2001-08-29 Genovision As Preparation of polymer particles
US20140011201A1 (en) * 2003-05-13 2014-01-09 Ibis Biosciences, Inc. Method for the purification of targeted nucleic acids from background nucleic acids
US7964343B2 (en) * 2003-05-13 2011-06-21 Ibis Biosciences, Inc. Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US8158354B2 (en) 2003-05-13 2012-04-17 Ibis Biosciences, Inc. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
WO2005011867A2 (fr) 2003-07-31 2005-02-10 Handylab, Inc. Traitement d'echantillons contenant des particules
US8546082B2 (en) 2003-09-11 2013-10-01 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US8097416B2 (en) 2003-09-11 2012-01-17 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US20120122099A1 (en) 2003-09-11 2012-05-17 Rangarajan Sampath Compositions for use in identification of bacteria
US8852862B2 (en) 2004-05-03 2014-10-07 Handylab, Inc. Method for processing polynucleotide-containing samples
CA2575446C (fr) * 2004-08-03 2014-03-25 Becton, Dickinson And Company Utilisation d'un materiau magnetique permettant de proceder a l'isolement de composes et le fractionnement d'echantillons constitues de plusieurs parties
US8883490B2 (en) 2006-03-24 2014-11-11 Handylab, Inc. Fluorescence detector for microfluidic diagnostic system
US11806718B2 (en) 2006-03-24 2023-11-07 Handylab, Inc. Fluorescence detector for microfluidic diagnostic system
DK3088083T3 (en) 2006-03-24 2018-11-26 Handylab Inc Method of carrying out PCR down a multi-track cartridge
US7998708B2 (en) 2006-03-24 2011-08-16 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
US10900066B2 (en) 2006-03-24 2021-01-26 Handylab, Inc. Microfluidic system for amplifying and detecting polynucleotides in parallel
EP2091647A2 (fr) 2006-11-14 2009-08-26 Handylab, Inc. Système microfluidique utilisé pour amplifier et détecter des polynucléotides en parallèle
US8324372B2 (en) 2007-07-13 2012-12-04 Handylab, Inc. Polynucleotide capture materials, and methods of using same
US9618139B2 (en) 2007-07-13 2017-04-11 Handylab, Inc. Integrated heater and magnetic separator
US8105783B2 (en) 2007-07-13 2012-01-31 Handylab, Inc. Microfluidic cartridge
US8182763B2 (en) 2007-07-13 2012-05-22 Handylab, Inc. Rack for sample tubes and reagent holders
US9186677B2 (en) 2007-07-13 2015-11-17 Handylab, Inc. Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
US8133671B2 (en) 2007-07-13 2012-03-13 Handylab, Inc. Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
US8287820B2 (en) 2007-07-13 2012-10-16 Handylab, Inc. Automated pipetting apparatus having a combined liquid pump and pipette head system
USD787087S1 (en) 2008-07-14 2017-05-16 Handylab, Inc. Housing
BR112013026451B1 (pt) 2011-04-15 2021-02-09 Becton, Dickinson And Company sistema e método para realizar ensaios de diagnóstico molecular em várias amostras em paralelo e simultaneamente amplificação em tempo real em pluralidade de câmaras de reação de amplificação
USD692162S1 (en) 2011-09-30 2013-10-22 Becton, Dickinson And Company Single piece reagent holder
JP6117217B2 (ja) 2011-09-30 2017-04-19 ベクトン・ディキンソン・アンド・カンパニーＢｅｃｔｏｎ， Ｄｉｃｋｉｎｓｏｎ Ａｎｄ Ｃｏｍｐａｎｙ ユニット化された試薬ストリップ
EP2773892B1 (fr) 2011-11-04 2020-10-07 Handylab, Inc. Dispositif de préparation d'échantillons de polynucléotides
US10822644B2 (en) 2012-02-03 2020-11-03 Becton, Dickinson And Company External files for distribution of molecular diagnostic tests and determination of compatibility between tests

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4335094A (en) 1979-01-26 1982-06-15 Mosbach Klaus H Magnetic polymer particles
WO1983003426A1 (fr) 1982-03-29 1983-10-13 SCHRÖDER, Ulf Particules magnetiques d'hydrate de carbone utilisees comme support a des fins de separation par affinite, par exemple la separation de cellules
US4452773A (en) 1982-04-05 1984-06-05 Canadian Patents And Development Limited Magnetic iron-dextran microspheres
WO1986003136A1 (fr) 1984-11-28 1986-06-05 University Patents, Inc. Chromatographie par affinite par utilisation d'agents de separation entre la magnetite et l'alginate de calcium seche dans un lit fluidise et stabilise magnetiquement
EP0184710A2 (fr) 1984-12-08 1986-06-18 Bayer Ag Microsphères magnétiques
EP0234083A2 (fr) 1986-02-25 1987-09-02 Tosoh Corporation Support pour une composante biologiquement active pour les essais immunologiques ou réactions enzymatiques

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4335094A (en) 1979-01-26 1982-06-15 Mosbach Klaus H Magnetic polymer particles
WO1983003426A1 (fr) 1982-03-29 1983-10-13 SCHRÖDER, Ulf Particules magnetiques d'hydrate de carbone utilisees comme support a des fins de separation par affinite, par exemple la separation de cellules
US4452773A (en) 1982-04-05 1984-06-05 Canadian Patents And Development Limited Magnetic iron-dextran microspheres
WO1986003136A1 (fr) 1984-11-28 1986-06-05 University Patents, Inc. Chromatographie par affinite par utilisation d'agents de separation entre la magnetite et l'alginate de calcium seche dans un lit fluidise et stabilise magnetiquement
EP0184710A2 (fr) 1984-12-08 1986-06-18 Bayer Ag Microsphères magnétiques
EP0234083A2 (fr) 1986-02-25 1987-09-02 Tosoh Corporation Support pour une composante biologiquement active pour les essais immunologiques ou réactions enzymatiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9411103A1

Also Published As

Publication number Publication date
GB9223334D0 (en) 1992-12-23
AU5427394A (en) 1994-06-08
WO1994011103A1 (fr) 1994-05-26

Similar Documents

Publication Publication Date Title
EP0621802A1 (fr) Supports magnetiques a phase solide
US6783962B1 (en) Particulate material for purification of bio-macromolecules
DE60012318T2 (de) pH ABHÄNGIGE IONTAUSCHERMATRIX UND ANWENDUNGSVERFAHREN IN DER ISOLIERUNG VON NUKLEINSÄUREN
DE69830999T2 (de) Verfahren zur adsorption/trennung
Augusto et al. New materials and trends in sorbents for solid-phase extraction
Levison et al. Recent developments of magnetic beads for use in nucleic acid purification
Pang et al. Bovine serum albumin-imprinted polyacrylamide gel beads prepared via inverse-phase seed suspension polymerization
EP0538350B1 (fr) Conglomerat de transport de substances
US4675113A (en) Affinity chromatography using dried calcium alginate-magnetite separation media in a magnetically stabilized fluidized bed
US8288169B2 (en) Surface mediated self-assembly of nanoparticles
US7833796B2 (en) Method for the concentration and purification of biological compounds
Li et al. Efficient on‐chip proteolysis system based on functionalized magnetic silica microspheres
US5053135A (en) Composite polymers, their preparation and their use in liquid chromatography
JP2005532799A (ja) 標的物質の結合
JP2002510787A (ja) 小型高密度微多孔性固体支持物質、その調製、ならびに大型高分子および生体粒子の精製のための使用
JP2022515769A (ja) 大孔径アガロース
Vergara-Barberan et al. Recent advances in aptamer-based miniaturized extraction approaches in food analysis
EP2236529A1 (fr) Procédé de fabrication d'un matériau sorbant composite pour la séparation chromatographique de biopolymères
DE60033505T2 (de) Partikelmaterial für wirbelbettreinigung von biomakromolekülen wie plasmid-dns, chromosomen-dns, rns,virale dns, bakterien und viren
Tanyolaç et al. A new low cost magnetic material: magnetic polyvinylbutyral microbeads
JP4551568B2 (ja) 常磁性粒子を用いた集細胞及びライセート清澄化
Pang et al. Soft-wet polyacrylamide gel beads with the imprinting of bovine serum albumin
JP2003524680A (ja) 刺激応答性高分子を用いた親和力制御型材料および該材料を用いた分離精製方法
JP4198461B2 (ja) シラン処理シリカ基質を用いた溶解物クリアランスおよび核酸単離
Ebrahimpour et al. Adsorption strategy of plasmid DNA nanoparticulate: Preparative purification by a simple custom expanded bed column

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19940705

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): DE DK ES FR GB IT NL SE

17Q First examination report despatched

Effective date: 19961122

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19970603