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EP0621802A1 - Supports magnetiques a phase solide - Google Patents

Supports magnetiques a phase solide

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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Withdrawn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.)
Individual
Original Assignee
Individual
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Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0621802A1 publication Critical patent/EP0621802A1/fr
Withdrawn legal-status Critical Current

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    • 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 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • 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.

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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)

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

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GB9223334D0 (en) 1992-12-23
WO1994011103A1 (fr) 1994-05-26

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