Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
  • B01D67/0081—After-treatment of organic or inorganic membranes
  • B01D67/0093—Chemical modification
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
  • B01D69/14—Dynamic membranes
  • B01D69/141—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes
• • 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
  • B01J45/00—Ion-exchange in which a complex or a chelate is formed; Use of material as complex or chelate forming ion-exchangers; Treatment of material for improving the complex or chelate forming ion-exchange properties

Definitions

• the present invention relates generally to the separation and analysis of molecules.
• the invention relates to the isolation of these molecules from a mixture in a fluid phase on a porous membrane comprising a metal chelating filter or metal chelate filter
• porous membrane is in a microplate format, tube, vial
• bioaffinity molecules may then be analysed using a wide variety of methodologies including the employment of specific bioaffinity molecules, specific
• the molecules may be released
• Microporous membranes as support matrices have also
• Ligand immobilised membranes provide a compact
• Ligands have been immobilised on support materials in the
• membranes have been initially coated with a water-insoluble protein such as Zein,
• Porous membrane materials used for non-covalent ligand immobilisation in affinity chromatography have included materials such
• PVDF polyvinylidene fluoride
• Non-covalent binding results in random orientation of the ligand and can
• peptides and the oligonucleotides are not only covalently bound to the
• peptides may be coupled to a solid support via
• biomolecules as ligands onto activated membranes are:-
• N ⁇ 2+ and Zn 2+ are the most widely.
• Suitable donor ligands include ammo acids histidine, cysteine and tryptophan Since natural proteins rarely contain suitably arranged donor
• ligands recombinant proteins are engineered with a metal chelate
• binding tag at either the C- or N- terminal ends of the protein for
• microporous filter materials such as Sepharose (IDA-sepharose, Pharmacia), magnetic beads (Ni-NTA Magnetic Agarose Beads, Qiagen) and microtitre plates (Ni-NTA HisSorb, Qiagen).
• Iminodiacetic acid (IDA) has also been covalently attached to a stable IDA
• MPS microporous plastic sheet
• the MPS matrix is an inert polymeric microporous sheet that contains
• silica that can either be fu ⁇ ctionalised with ion exchange groups or affinity ligands solely for the purpose of protein purification.
• metal chelate filter or metal chelating species filter when processed in accordance with the invention to provide a metal chelate filter or metal chelating species filter may provide a number of
• the invention provides a ligand immobilisation
• the invention provides for the capture and
• the invention has particularity in the diagnostics, high-throughput screening, and biotechnology industries.
• the invention has particularity in the diagnostics, high-throughput screening, and biotechnology industries.
• the invention has particularity in the diagnostics, high-throughput screening, and biotechnology industries.
• the invention has particularity in the diagnostics, high-throughput screening, and biotechnology industries.
• the invention has particularity in the diagnostics, high-throughput screening, and biotechnology industries.
• the process of the invention includes the following variants:-
• the invention includes within its scope products produced
• filter means a porous material
• filter media having an average pore size in the region of 0.01 to 1000 microns and more preferably 0.1-5 microns.
• the filter may be formed from either fully or partly from
• accessible functional groups such as -Si-OH groups or which may be treated with a reagent to provide accessible functional groups including -OH, -Si-OH, -NH 2 and other amine
• groups including alkylamino, thiols, cyanobromides, aldehydes, carboxylates, sulfonylchlorides, ketones, halogens,
• haloacetyl inclusive of chloroacetyl, iodoacetyl or bromoacetyl and
• epoxy groups may be attached directly to the filter or by an appropriate spacer arm or linker.
• porous filter materials comprising
• polyamides inclusive of nylon, cellulose acetate, nitrocellulose,
• polyvinylidene fluoride or other fluoropolymers, polysulfone, paper, or
• linkers and/or a metal chelating species or a metal chelate may also be suitable media for the attachment of linkers and/or a metal chelating species or a metal chelate.
• linker as used herein means any spacer group
• the linker prior to use has an appropriate functional group as
• linker can be utilised which may include substituted or
• unsubstituted alkyl groups having from one to twenty, or more preferably one to six carbons, and wherein the alkyl groups may be linear or
• branched Linkers may also comprise substituted or unsubstituted aryl or aryl alkyl groups
• the linking group may be variable comprising a single methylene or a plurality of methylene groups
• linking group may also comprise peptides or branched peptides inclusive
• linkers include, for example, both linear,
• polyamides polyethyleneimines, polyarylene sulfides, polysiloxanes,
• polyimides polyacetates, dend ⁇ mers and dendrime ⁇ c-like molecules or
• linker as used
• cross-linking species or reagents used to couple the metal chelate or metal chelating species to accessible functional
• cross-linking species either on the filter or which have been produced by de ⁇ vatisation
• Such cross-linking species are referred to by way of example to the
• microfibre In a preferred embodiment of the invention, microfibre
• glass filters made from silica are especially adapted for use in the
• Such filters have silanol (Si-OH) functional groups as part of
• the filter matrix may be derivatised using a suitable reagent to
• such reagents may have an in-built linker or the linker may be pre-attached to the metal
• the metal chelating species may be any metal chelating species.
• the metal chelating species includes a functional group capable of reacting with Si-OH groups such as triethoxysilane or trimethoxysilane.
• silanisation reagents which may be used for the
• filters comprise accessible amine groups on the surface of the filter.
• linker or metal chelating species with linker attached may
• peptides as linkers.
• filters may be treated with a reagent such as ethylenediamine which may be
• a short linker in a suitable solvent such as acetonitrile and usually in the presence of a catalyst such as triethylamine.
• Suitable reactive groups may need to be
• Such hydroxyl groups can be directly used for the attachment of a filter
• hydroxyl groups may be further derivatised with silanisation reagents
• Such reagents for example may include cyanogen bromide, tosyl chloride, N-hydroxy succinamide, diamines, hydrazine and its
• dialdehydes such as glutaraldehyde, carbodiimides, and
• MCS metal chelating species
• metal chelating species include iminodiacetic acid, nitrilotriacetic acid, diethylenetriamine
• metal chelate means the metal chelating species having a metal coordinated thereto. Chelation type associations make use of metal ions such as but not limited to, the
• metal chelate filters of the invention which include the
• metal can be used for immobilisation of ligands that coordinate with the
• species may be attached to the filter by a covalent bond, charge
• metal chelating species may be attached to the filter via a linker by either of the
• the linker may be pre-attached to the metal chelating species followed by attachment of
• suitable protecting groups include Fmoc, Boc, trityl groups and any other protecting groups.
• branched species such as lysine or branched polymeric species such as dendrimers may increase
• FIG. 1 shows a derivatised glass fibre filter
• imidodiacetic acid metal chelate attached to the filter with a linker.
• FIG. 2 shows a polyacrylamide gel with a low range
• dendrimer-HRP conjugate at about 73 kDa is readily apparent in the
• FIG. 3 shows results of an experiment using Zn 2+ immobilised on the filter in which HRP, HRP-antibody conjugate and HRP-dendrimer conjugate were passed through metal chelate filter discs
• FIG. 4 shows free HRP, HRP-antibody and E5 dendrimer-
• metal chelate glass fibre filters (Whatman GF/B, GF/C and GF/F).
• metal ion used was Zn 2+ .
• FIG. 5 shows results of an experiment in which E5-
• dendrimer conjugate, diluent only, E5 dend ⁇ mer only and E5 dendrimer- antibody conjugate were passed through Zn 2+ metal chelate filter discs
• FIG 6 of a polyacrylamide gel shows that metal chelate filter discs efficiently capture a 6 x histidine tagged protein from a
• the concentration of the tagged protein was found to be approximately five times higher in material eluted from the discs than material that flowed through the discs
• TNBS trmitrobenzynesulfonic acid
• HBTU Benzotriazole-N,N,N',N',-Tetramethyl-Uronium-Hexafluorophosphate
• HOBt N-Hydroxybenzothazole
• DIPEA diisopropylethylamine
• the Fmoc group was then tested with a TNBS test.
• the deprotected filters were treated with nitrilotriacetic acid
• Nitrilotriacetic acid derivatised filter discs were each placed on a sintered glass funnel connected to a vacuum pump. The discs were
• FIG. 1 shows an example of a molecular structure showing the derivatised filter, linker(s) and the metal chelate.
• FIG. 2 shows examples of different molar ratios of E5 dendrimer-HRP conjugations.
• FIG. 3 shows results of an experiment using Zn 2+
• HRP-dendrimer conjugate were passed through metal chelate filter discs
• FIG. 4 shows free HRP, HRP-antibody and E5 dendrimer-
• metal chelate glass fibre filters (Whatman GF/B, GF/C and GF/F).
• E5-dendrimer conjugate, diluent only, E5 dendrimer only and E5 dendrimer-antibody conjugate were passed through Zn 2+ metal
• TMB substrate was applied and washed as described above.
• FIG. 5 shows the results. It can be seen that the presence
• FIG 6 of a polyacrylamide gel shows that metal chelate filter discs efficiently captured a hexahistidme tagged protein from a
• MC and MCS filters may be incorporated into an immunoassay filter plate, column, syringe filter housing or tube for a wide
• the MC and MCS filters may also be embodied with a second type filter such as an FTATM Gene Guard
• the MC and MCS filters may further embodied with a second filter such as to restrict
• the bound genetic material such as human genomic DNA can be ultimately removed by use of reagents such as restriction endonucleases
• the fragments of the restriction digest can be incubated in the presence of a MC filter harbouring
• probes could be derivatised at their 5' or 3' ends with a metal chelate tail such as a hexahistidme tag or dendrimer e g PAMAM
• genomic DNA E g traditional methodology (other than FTA) of purification yields genomic DNA as a soluble fraction After restriction digest, or in vitro
• template nucleic acid can be produced that is in a position to be captured by such
• oligonucleotide probes may be mixed with the genomic DNA whereby the
• DNA/probe hybrids formed can be captured on MC
• a second probe conjugated to a detector label e g
• Oligonucleotides, mRNA and cDNA coupled to dendrimers or other species capable of acting as a ligand, for example a hexahistidine tag, can be
• dendrimers or a hexahistidine may be added directly to a MC filter sheet to form a microarray suitable for analysis without the need for a multi-well plate format.
• proteins including a hexahistidine tail may be immobilised on a metal chelate filter. This will
• Unstable recombinant protein may be immobilized on MC filters quickly and at room temperature
• incorporating an MC filter would enable the user to rapidly immobilize potential drugs or target
• the method could also be combined with a FTA filter for simultaneous
• MCF-captured proteins may then be tested for function in bioassays.
• An indicator of cellular events is the change in kinase activity. Interaction at the cell surface is
• a synthetic kinase substrate is incubated with the kinase sample of
• the substrate was engineered to contain a 6xHis
• a MC filter will have greater specificity than Promega's streptavidin filter that is commonly used together with biotin tagged peptides.
• the isolated library compound may then be further interrogated to
• target molecule may be conjugated to a dendrimer or hexahistidine tag either of which
• dendrimer or hexahistidine may then be further
• Overlapping peptide sequences may be tagged with a hexahistidme tail or dendrimers and immobilised onto metal chelate filters for subsequent screening
• metal chelate filter bottoms could be used to capture hexahistidine or dendrimer tagged or target molecules
• product may be engineered to bear a hexahistidme sequence This product may then readily be captured onto a metal chelate filter
• a deep well multiwell plate encapsulating a MC filter at the bottom of each well may be constructed
• each well may be carried out mammalian, yeast or bacterial culture without fluid dripping through the filter bottoms Protein
• FTA and MC filter materials within the same device may be constructed.
• the cells will lyse, the cells
• episomal and/or genomic DNA will bind to the FTA, and the recombinant protein containing an
• the recombinant protein can be harvested and assayed. If the protein collected is of interest
• Metalloproteases are implicated with the onset of
• metastasis being part of the mechanism utilised by
• Recombinant proteins and enzymes expressing a tag such as a hexahistidine tail may be assayed directly from culture to determine a) levels of
• each tube, column or well will amplify the capacity
• microorganisms or parts of microorganisms such as antigens or nucleic acids

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• Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Inorganic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Manufacturing & Machinery (AREA)
• Organic Chemistry (AREA)
• Dispersion Chemistry (AREA)
• Solid-Sorbent Or Filter-Aiding Compositions (AREA)
• Treatment Of Liquids With Adsorbents In General (AREA)

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• 1999
  • 1999-05-14 AU AUPQ0343A patent/AUPQ034399A0/en not_active Abandoned
• 2000
  • 2000-05-15 WO PCT/AU2000/000477 patent/WO2000070012A1/en not_active Application Discontinuation
  • 2000-05-15 EP EP00926556A patent/EP1183327A1/de not_active Withdrawn

Non-Patent Citations (1)

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