EP1183327A1

EP1183327A1 - Metal chelating filters and metal chelate filters

Info

Publication number: EP1183327A1
Application number: EP00926556A
Authority: EP
Inventors: Edward Hanna Kachab; Graeme Ross Barnett; Martin Smith
Original assignee: Panbio Pty Ltd; Whatman International Ltd
Current assignee: Abbott Rapid Diagnostics Pty Ltd; Whatman International Ltd
Priority date: 1999-05-14
Filing date: 2000-05-15
Publication date: 2002-03-06
Also published as: AUPQ034399A0; WO2000070012A1

Abstract

A method of making a metal chelate filter or metal chelating species filter including the following step of: treating a filter having a pore size of 0.01 to 1000 microns and accessible functional groups with a metal chelate or metal chelating species to provide the metal chelate filter or metal chelating filter species.

Description

TITLE

"METAL CHELATING FILTERS AND METAL CHELATE FILTERS"

BACKGROUND OF THE INVENTION

Field of the invention

The present invention relates generally to the separation and analysis of molecules. In particular, 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

whereby the porous membrane is in a microplate format, tube, vial,

syringe filter housing, column or similar arrangement. The captured

molecules may then be analysed using a wide variety of methodologies including the employment of specific bioaffinity molecules, specific

chemical reactions, specific hybridisation probes, or interrogation using

lasers, UV light, IR, etc. Alternatively, the molecules may be released

from the porous membrane and collected for analysis.

Description of the background art

Molecular separation based on affinity interaction has

proven to be a valuable tool for the purification of biological or related

molecules (Affinity Chromatography: a practical approach, P. D. G. Dean, W. S. Johnson, and F. A. Middle (eds.). IRL Press, Oxford, 1986. Affinity

Separations: a practical approach, P. Matejtschuk (ed.) IRL Press,

Oxford, 1997). The principle of affinity separation relies on the selective

recognition and interaction of the desired biological or related substance,

present in a fluid phase, with its complementary ligand immobilised on a solid support as it passes over the support in a column. The desired material is retained on the column whilst most impurities are flushed through. The binding is reversible allowing for the recovery of the desired

molecules by applying an appropriate eluting solution to the column

which disrupts the ligand-binding interaction, by either changing the ionic

strength, the pH, or adding a competing reagent, thereby releasing the desired molecules from the column. The types of ligands which may be

used in affinity separation is very broad and include antibodies, antigens, enzymes, peptides, oligonucleotides, isolated receptors, carbohydrates,

and recombinant proteins. There is now a wide range of support matrices that have been used in affinity chromatography, e.g. Agarose (Sepharose

(Pharmacia)), cross-linked dextran (sephadex (Pharmacia)), cross-linked

cellulose (Matrex Cellufine (Amicon)), controlled pore glass (CPG

(Pierce)), and silica (Hypersil WP 300 (Shandon)) beads.

Microporous membranes as support matrices have also

been used in the art. Ligand immobilised membranes provide a compact,

easy to manipulate system allowing for the capture of the desired

molecule and the removal of unwanted components in a fluid phase at

higher throughput and faster processing times than possible with column chromatography. This is due to the fast diffusion rates possible on

membranes. Ligands have been immobilised on support materials in the

form of microporous membranes used in affinity chromatography. This

has occurred either through simple adsorption or through a chemical

reaction between complementary reactive groups present on the membrane and on the ligand resulting in the formation of a covalent bond

between the ligand and membrane In some instances, membranes have been initially coated with a water-insoluble protein such as Zein,

collagen, fibnnogen, etc , followed by the immobilisation of the ligand of interest (e g U S Pat No 4407943)

Porous membrane materials used for non-covalent ligand immobilisation in affinity chromatography have included materials such

as nylon, nitrocellulose, and hydrophobic polyvinylidene fluoride (PVDF)

Non-covalent binding results in random orientation of the ligand and can

also be a problem with small molecules such as peptides,

oligonucleotides or oligosacchaπdes or where competing ions may wash

away the adsorbed ligand To circumvent this problem, peptides and oligonucleotides have been synthesised directly onto membranes as a

means of generating the desired membrane affinity supports in which the

peptides and the oligonucleotides are not only covalently bound to the

support but are also placed in the correct orientation (e g U S Pat No 4923901 ) This method is limited, as individual membranes would need

to be generated, every time, for different peptide and ohgonucleotide

sequences More often, peptides may be coupled to a solid support via

the C- or N-terminal ends of the peptide which may also result in random

coupling via reactive groups on the side chains of the peptide A number of methods and reagents have been developed to also allow the direct

coupling of oligonucleotides onto solid supports (e g J M Coull et al ,

Tetrahedron Lett 27 3991 , B A Conolly, 1987, Nucleic Acids Res , 15 3131 ; B. A. Conolly and P. Rider, 1985, Nucleic Acids Res., 12 4485)_.

UV cross-linking of DNA (Church et al., 1984, PNAS, 81 1991 ) and RNA

(Khandjian, et al., 1986, Anal. Biochem., 159 227) to nylon membranes

have also been reported.

Many chemical methods have been developed for the immobilisation of proteins as ligands on microporous membranes. For example, activated paper (TransBind.TM., Schleicher & Schuell Ltd., Keene, N.H.) carbodimidazole-activated hydrogel-coated PVDF

membrane (Immobilin-IAV ™, Millipore Corp., Bedford, Mass.), activated

nylon (BioDyne. TM., Pall Corp., (Glen Cove, N.Y.), DVS- and cyanogen bromide-activated nitrocellulose. Membranes bound with specific ligands

are also known such as the SAM2TM Biotin Capture Membrane (Promega) which binds biotinylated molecules based on their affinity to

streptavidin or MAC affinity membrane system (protein A/G) (Amicon).

Some of the disadvantages of covalent attachment of

biomolecules as ligands onto activated membranes are:-

(a) Most of the chemistries used for covalent ligand attachment couple the ligand randomly through amino residues on the protein which often results in

partial or complete loss of binding capacity due to the reduced efficiency of ligand-product binding.

This is mostly due to multi-site attachment of the

ligand when the activated support contains an

excess of activating groups, making the binding site on the ligand inaccessible due to steric hindrance

(Spitznagel, T. M. and Clark, D. S., 1993,

Biotechniques, 11 825).

(b) Ligand immobilisation is often slow requiring 20-180 minutes for reaction completion.

(c) High ligand concentration is needed for fast ligand immobilisation.

(d) Constant agitation is needed during the

immobilisation process that may result in further ligand denaturation and deactivation.

(e) Unsuitable for the immobilisation of unstable recombinant proteins due to immobilisation

chemistry, mechanical agitation, and the long

incubation times needed at room temperature.

(f) Once the immobilisation process is complete, often a blocking (capping) step is required to remove

residual covalent binding capacity.

(g) Covalently bound ligands can not be retrieved from

the membrane.

The technique of immobilised metal affinity

chromatography (IMAC) has found the widest application in affinity purification of recombinant proteins (e.g. J. Porath, Prot. Express. And

Purif., 1992, 3 263; Vosters et al., 1992, Protein Expr. Purif., 3 18;

Alnemri et al., 1993, Proc. Natl Acad. Sci., 90 6839; C. Tang, and H. L. Henry, 1993, J Biol Chem , 268 5069) In this technique, a metal chelate

possessing suitable co-ordination sites is immobilised on a solid support

(e g Iminodiacetic acid immobilised on sepharose beads (IDA-

Sepharose , Pharmacia) or nitπlotπacetic acid immobilised on sepharose (NTA-Sepharose)) Only those proteins with two or three suitable donor ligands in a conformationally favourable arrangement will bind and form

a stable complex (E Sulkowski, 1989, BioEssays, 10, 170, K J Petty,

1996, Metal-chelate affinity chromatography In Current protocols in

molecular biology, F M Ausubel (ed ), vol 2, John Wiley and Sons, New

York) In theory, a wide variety of metal ions with high affinity for electron

donor groups can be utilised Of these, Nι²⁺ and Zn²⁺ are the most widely

used 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

purification purposes via IMAC (e g Sharma et al , 1992, In Methods A

companion to Methods in enzymology, F Arnold (ed ) 457-67, Academic

Press, New York) The most popular tag is the hexahistidine tag although

many other tags utilising a combination of histidine and other ammo

acids such as aspartic acid or tryptophan residues have been used (e g

Kasher ef a/ , 1993, Bio/Techniques, 14630)

Metal chelating ligands have been immobilised on solid

supports exclusive of 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

epoxy resin and incorporated into a microporous plastic sheet (MPS)

matrix (Acti-Disk, Whatman) and used for the selective purification of proteins (U.S. Patent Nos. 3862030, 4102746, 4169014 and 4689302).

The MPS matrix is an inert polymeric microporous sheet that contains

silica that can either be fuπctionalised with ion exchange groups or affinity ligands solely for the purpose of protein purification.

SUMMARY OF THE INVENTION

Unexpectedly, it has now been discovered that filter media

when processed in accordance with the invention to provide a metal chelate filter or metal chelating species filter may provide a number of

surprising advantages and applications as described hereinafter which

would not have been contemplated from the prior art discussed above.

Thus use of the filter media of the invention may now provide advantages

of faster processing of fluids as well as multiple processing of fluids.

Thus the invention provides a ligand immobilisation

procedure for peptides, oligonucleotides and recombinant proteins, which involves site specific attachment of the ligand onto the membrane via use

of a metal chelating species or metal chelate thus avoiding the use of harsh chemical reactions and long incubation times as was the case in

the prior art. In particular, the invention provides for the capture and

separation of these biomolecules from a mixture in a fluid or liquid phase

onto a microporous filter matrix. Such a filter matrix processed in accordance with the invention would have valuable and widespread

applications in the diagnostics, high-throughput screening, and biotechnology industries. In particular the invention has particular

application to fluid phase assay systems and methods described in

International Publications WO 9932884 and/or WO 9932885which are totally incorporated herein by reference.

The process of the invention includes the following variants:-

(i) reaction of a filter with a linker and metal chelating species to produce a metal chelating filter;

(ii) reaction of a filter with a linker and metal chelating species and a metal to produce a metal chelate

filter;

(iii) reaction of a filter with a metal chelating species to produce a metal chelating filter; and

(iv) reaction of a filter with a metal chelate to produce a

metal chelate filter. The invention includes within its scope products produced

by the above mentioned alternative variants (i) to (iv).

The term "filter" as used herein means a porous material or

filter media having an average pore size in the region of 0.01 to 1000 microns and more preferably 0.1-5 microns. Viral antigen immune

complexes would generally be retained by a pore size of 0.05-3 microns while a larger size may be more appropriate for bacterial antigen immune complexes. The filter may be formed from either fully or partly from

glass, silica or quartz including their beads, fibres or derivatives thereof,

having bound thereto 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, -NH₂ and other amine

containing groups, including alkylamino, thiols, cyanobromides, aldehydes, carboxylates, sulfonylchlorides, ketones, halogens,

maleimido, hydrazine, acetyl or other groups combining acetyl including

haloacetyl inclusive of chloroacetyl, iodoacetyl or bromoacetyl and

epoxy groups. It will also be appreciated that the functional groups may be attached directly to the filter or by an appropriate spacer arm or linker.

It will further be appreciated that porous filter materials comprising

polyamides inclusive of nylon, cellulose acetate, nitrocellulose,

polyvinylidene fluoride, or other fluoropolymers, polysulfone, paper, or

combinations thereof, may also be suitable media for the attachment of linkers and/or a metal chelating species or a metal chelate.

The term "linker" as used herein means any spacer group

which can be utilised to link the various entities described above. As

such, the linker, prior to use has an appropriate functional group as

described above at each end. One group is appropriate for the

attachment to the filter and the other group is appropriate for attachment

to the metal chelate or metal chelating species. Any suitable linking

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 Thus for example the linking group may be variable comprising a single methylene or a plurality of methylene groups The

linking group may also comprise peptides or branched peptides inclusive

of lysine Other examples of linkers include, for example, both linear,

cyclic, and branched polymers of polysacchandes, phospho pids and peptides having either alpha- beta-, or omega-ammo acids, heteropolymers, polyurethanes, polyesters, polycarbonates, polyureas,

polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes,

polyimides, polyacetates, dendπmers and dendrimeπc-like molecules or

other polymers as will be readily apparent to those skilled in the art

It will also be appreciated that the term linker as used

herein may also include cross-linking species or reagents used to couple the metal chelate or metal chelating species to accessible functional

groups either on the filter or which have been produced by deπvatisation Such cross-linking species are referred to by way of example to the

Pierce Chemical Company Product Catalog 1999-2000 which is totally

incorporated herein by reference

In a preferred embodiment of the invention, microfibre

glass filters made from silica are especially adapted for use in the

invention Such filters have silanol (Si-OH) functional groups as part of

the filter matrix These may be derivatised using a suitable reagent to

provide appropriate functional groups such as those described above for the attachment of the metal chelating species or metal chelate to the filter or by use of a linker.

In this regard it will be appreciate that such reagents may have an in-built linker or the linker may be pre-attached to the metal

chelating species prior to attachment to the functional groups on the

filter. It may further be appreciated that these reagents may also

comprise the various functional groups inclusive of amine, halogen, or

epoxy as described above. Alternatively the metal chelating species may

be reacted with the Si-OH groups on the filter if the metal chelating species includes a functional group capable of reacting with Si-OH groups such as triethoxysilane or trimethoxysilane. Such functional

groups may also be attached to the metal chelating species by a linker.

Examples of silanisation reagents which may be used for the

derivatisation of the glass fibre filters are: 3-aminopropyl triethoxysilane, 3-aminopropyltrimethoxysilane, 3-(2-aminoethylamino) propyltrimethoxysilane and 3-glycidoxypropyitrimethoxysilane.

In relation to nitrocelluose or cellulose paper such porous

filters comprise accessible amine groups on the surface of the filter. In

this case the linker or metal chelating species with linker attached may

be attached to the filter directly without the need for prior derivatisation using for example standard peptide and conjugation chemistries such as

those described in Frank, et al., 1991 , "Peptides", Giralt and Andrew Eds,

ESCOM Science Pub, pp 151-152, which is directed to facile and rapid

"spot synthesis" of large numbers of peptides as linkers on porous membrane filters. This reference is incorporated herein by reference.

Reference also may be made to Furka et al., 1991 , Int. J.

Peptide Protein Res. 37: 487-493, which is totally incorporated herein by

reference with regard to use of peptides as linkers. Usually such filters may be treated with a reagent such as ethylenediamine which may be

considered as a short linker in a suitable solvent such as acetonitrile and usually in the presence of a catalyst such as triethylamine.

In the case of fluoropolymers, there are no existing

functional groups suitable or otherwise for the attachment of a linker of a metal chelating species. Suitable reactive groups may need to be

introduced onto the filter by a process which is described in the following

publications each of which are incorporated herein by reference ie Vargo

et al., 1992, Langmuir 8:130; Vargo et al., 1992, J. Polym. Sci. Part A:

Poly. Chem. 29:555; and Hook et al., 1991 , Langmuir 7:142.

Briefly these processes are called radiofrequency glow

discharge (RFGD) plasma using hydrogen gas and methanol vapour

which facilitates the introduction of hydroxyl groups to the filter surface.

Such hydroxyl groups can be directly used for the attachment of a filter,

metal chelating species or a metal chelate using standard coupling

chemistries and protocols such as those described above. Alternatively

the hydroxyl groups may be further derivatised with silanisation reagents

as described above to introduce the more versatile amino group. This

can then be used for the attachment of the linker, metal chelating species

or metal chelate. It will be appreciated from standard texts such as a publication entitled "Affinity Membranes in Bioseparations" by E. Klein

which is part of "Membrane Processes in Separation and Purification"

1994, by Crespo and Boddeker Eds, published by Kluwer Academic Publishers which is totally incorporated herein by reference that there is

a full description of various microporous membranes which may be

utilised in the current invention and also reference to various reagents

and processes which may attach the above mentioned functional groups to the filter. Such reagents for example may include cyanogen bromide, tosyl chloride, N-hydroxy succinamide, diamines, hydrazine and its

derivatives, dialdehydes such as glutaraldehyde, carbodiimides, and

diepoxides. This reference also described the preparation of polyamide

filters having accessible functional groups.

The term "metal chelating species" (MCS) as used herein

can be any species with electron donating groups such as -NH₂, -COOH,

-SH, -OH and heterocyclic moieties comprising nitrogen atoms, capable

of coordinating with metal ions. Examples of such metal chelating species include iminodiacetic acid, nitrilotriacetic acid, diethylenetriamine

- N,N,N',N" - pentoacetic acid and branched superstructures such as dendrimers or dendrimeric - like molecules or polymeric structures with

the appropriate donating groups, triethylenetetramine, ethylenediamine,

glycine, o-phenanthroline, 4,4-bipyridyl, 2,2-bipyridyl, pyridine and 6-

hydroxynicotinic acid

The term "metal chelate" (MC) as used herein 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

metals Fe, Co, Ru, Rh, Rh, Pd, Os, Ir, Pt, Pb, Sn, Ge, Sc, Y, lanthanides and actinides, B, Al, Ga, In, Tl, Li, Na, K, Rb, Cs, Fr and Be, Mg, Ca, Sr, Ba, Ra, Cu, Ni, Zn and transition metals.

It will be appreciated that the above mentioned products of the invention which incorporate the metal chelating species can be utilised for immobilisation of metal containing ligands or metal ions. On

the other hand metal chelate filters of the invention which include the

metal can be used for immobilisation of ligands that coordinate with the

metal.

In the above it will be appreciated that the metal chelating

species may be attached to the filter by a covalent bond, charge

interaction or metal chelation. It will also be appreciated that the metal chelating species may be attached to the filter via a linker by either of the

following:-

(i) attaching the linker first to the filter followed by attachment of the metal chelating species followed

by addition of the metal; (ii) alternatively, the linker may be pre-attached to the metal chelating species followed by attachment of

the linker - metal chelate species moiety on to the

filter (Ji er a/., 1996, Analy. Biochem., 240 197);

(iii) pre-attachment of a linker moiety on the filter in which one of its reactive functional groups is protected by a suitable protecting group will allow

for unreacted functional groups on the filter to be

blocked. Examples of suitable protecting groups include Fmoc, Boc, trityl groups and any other

protecting groups as is known in the art. For example, attachment of an Fmoc group protected linker will allow for uncoupled amine groups on the

filter to be blocked by an acetic anhydride/pyridine/DMF blocking procedure. It will

also be appreciated that reaction of the linker with

branched species such as lysine or branched polymeric species such as dendrimers may increase

the number of functional groups on the filter. This

may then be reacted with a MCS thereby increasing

the metal chelating capacity of the filter. Examples of suitable protecting groups are disclosed in The

Peptides, Vol 3. (eds. Gross, E., and J. Meienhofter,

Academic Press, 1981 ).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a derivatised glass fibre filter with

imidodiacetic acid metal chelate attached to the filter with a linker.

FIG. 2 shows a polyacrylamide gel with a low range

molecular weight marker in lane 1 and various molar ratios of E5 dendπmer-horseradish peroxidase (HRP) conjugates in lanes 2 to 5. In

lanes 2 and 3 free HRP is detected at around 45 kDa. In lane 4 the

dendrimer-HRP conjugate at about 73 kDa is readily apparent in the

absence of free HRP. In lane 5 the concentration of the dendrimer-HRP conjugate at a 1 :1 ratio has reduced noticeably.

FIG. 3 shows results of an experiment using Zn²⁺ immobilised on the filter in which HRP, HRP-antibody conjugate and HRP-dendrimer conjugate were passed through metal chelate filter discs

at varying concentrations. At 1 :1600, only the dendrimer-HRP conjugate

was detectable. Traces of HRP-antibody conjugates were detectable at 1 :400 to 1 :1200 and free HRP was detectable at 1 :400 only. HRP was

detected by applying an HRP substrate (3,3',5,5'-Tetramethylbenzidine

(TMB), insoluble) for 2 minutes after which the discs were then removed,

washed with doubly deionised water and dried on filter paper.

FIG. 4 shows free HRP, HRP-antibody and E5 dendrimer-

HRP conjugates diluted 1 :100 and passed through different derivatised

metal chelate glass fibre filters (Whatman GF/B, GF/C and GF/F). The

metal ion used was Zn²⁺.

FIG. 5 shows results of an experiment in which E5-

dendrimer conjugate, diluent only, E5 dendπmer only and E5 dendrimer- antibody conjugate were passed through Zn²⁺ metal chelate filter discs

in duplicate. For one set of discs, phosphate buffered saline with Tween 20 only was used to wash the discs to remove unbound reagent. For the

other set of discs, 500 mM imidazole was added to the washing buffer. Then a 1 1000 dilution of anti-mouse immunoglobulin conjugated to HRP

was added to the discs and incubated for 30 minutes at 37JC After this the discs were washed as before and TMB substrate was applied and washed as described above

FIG 6 of a polyacrylamide gel shows that metal chelate filter discs efficiently capture a 6 x histidine tagged protein from a

solution 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

E X P E R I M E N T A L

EXAMPLE 1 Functionalization of glass fibre membranes with

3-amin opropyltrieth oxysilane

Twenty sheets (10 5 cm x 14 7 cm) of GF/F glass microfibre filters, as obtained from Whatman, Maidstone, England, were

placed in a flat dish and covered with 300 mL of a 10% solution of 3-

aminopropyltπethoxysilane in ethanol (99 7-100% v/v) The reaction was

allowed to proceed for 48 hours with rocking The sheets were then

washed with ethanol and air dried before they were activated at 110DC

for 3 hours The presence of amine groups was confirmed with a 2, 4, 6-

trmitrobenzynesulfonic acid (TNBS) test A few drops of a 1 % TNBS solution in N,N-dιmethylformamιde (DMF) were added to a small piece

of filter membrane and the colour change recorded after I minute EXAMPLE 2 Coupling of linker (Fmoc α-aminocaproic acid).

Five sheets (10 5 cm x 14 7 cm) of functionalised GF/F filters were treated with Fmoc α-aminocaproic acid (4.0 mmol), O-

Benzotriazole-N,N,N',N',-Tetramethyl-Uronium-Hexafluorophosphate (HBTU) (3.9 mmol), N-Hydroxybenzothazole (HOBt) (4.5 mmol), and

diisopropylethylamine (DIPEA) (5.0 mmol) in 40 mL of dry DMF overnight with rocking. The sheets were washed with methanol, dichloromethane

and then air-dried. The extent of coupling was measured with an Fmoc test and gave an Fmoc loading of 0.016 mmol/g of filter. Briefly, accurately weighed filter discs (16 mg) were treated with a 0.4 mL of a

50% solution of piperidine in dichloromethane for 10 minutes with

sonication. This volume was then made up to 2.4 mL with dichloromethane and the absorbance of the liberated Fmoc group

measured at 301 nm (extinction coefficient at 7,800).

EXAMPLE 3 Coupling of metal chelating species.

Eighty filter discs (5 mm diameter) were treated with a 20%

solution of piperidine in DMF for 5 min with sonication to effect the deprotection of the Fmoc group on the linker moiety. The filters were then

washed with methanol, dichloromethane and then air-dried. Removal of

the Fmoc group was then tested with a TNBS test.

The deprotected filters were treated with nitrilotriacetic acid

(1.0 mmol), HBTU (0.9 mmol), HOBt, (1.2 mol), and DIPEA (0.26 mmol) in 6 mL of dry DMF overnight with rocking. The filters were washed with

DMF, methanol, and dichloromethane and air-dried. TNBS test indicated

efficient coupling of the nitrilotriacetic acid. EXAMPLE 4 Immobilisation of Zn²⁺ on filter discs. Nitrilotriacetic acid derivatised filter discs were each placed on a sintered glass funnel connected to a vacuum pump. The discs were

each initially washed with 100 μL of doubly deionised water, 100 μL of

50 mM phosphate buffered saline, pH 7.5, containing 50 mM sodium

chloride, and followed by 100 μL of doubly deionised water. Fifty μL of

a θ.1 M solution of Zinc sulphate were then allowed to pass through each filter discs followed by washing with a 100 μL of doubly deionised water.

FIG. 1 shows an example of a molecular structure showing the derivatised filter, linker(s) and the metal chelate.

EXAMPLE 5 Preparation of enzyme dendrimer conjugates.

To a solution of HRP (4.3 mg) made up in 30 mM sodium

acetate buffer containing 150 mM sodium chloride, pH 4.5, was added 80

μL of a freshly prepared solution of sodium periodate (Nal0₄) and the reaction mixture allowed to incubate for 5 hours in the dark. The mixture

was then loaded onto a Sephadex G-25 PD-10 column equilibrated with

20 mM phosphate buffer, pH 6.0, and the activated enzyme eluted with

the same buffer. Thirteen μL of a 50 mg/ml solution of generation five dendrimer (E5) (Michigan Molecular Institute) was then incubated with

1.1 mg of the activated HRP enzyme in the presence of 0.70 mg of sodium cyanoborohydride (NaBH₃CN) at 4°C for 16 hours. The reaction

mixture was then dialysed against 0.1 M NaCI and doubly deionised

water. Polyacrylamide gel electrophoresis (PAGE) on a linear gradient

gel (4-15%) under SDS reducing conditions revealed the main product

to be a 1 :1 conjugate of HRP to E5. FIG. 2 shows examples of different molar ratios of E5 dendrimer-HRP conjugations.

EXAMPLE 6 HRP-E5 conjugate capture on a glass microfibre metal chelate filter

Metal chelate glass microfibre disc filters prepared as in EXAMPLE 4 were used for the capture of HRP-E5 conjugates. The discs

were each washed with 100 μL of 50 mM phosphate buffered saline, pH 7.5, containing 500 mM NaCI and 500 mM imidazole. Fifty μL of either

diluted HRP, HRP-antibody conjugate, or HRP-E5 conjugate were then

allowed to pass through the filter followed by a washing step of 100 μL of the imidazole, buffer. The filter discs were then incubated in the

presence of an HRP substrate (3,3',5,5'-Tetramethylbenzidine (TMB)

insoluble) for 2 minutes after which the discs were then removed, washed

with doubly deionised water and dried on filter paper.

FIG. 3 shows results of an experiment using Zn²⁺

immobilised on the filter in which HRP, HRP-antibody conjugate and

HRP-dendrimer conjugate were passed through metal chelate filter discs

at varying concentrations. At 1 :1600, only the dendrimer-HRP conjugate

was detectable. Traces of HRP-antibody conjugates were detectable at

1 :400 to 1 :1200 and free HRP was detectable at 1 :400 only.

FIG. 4 shows free HRP, HRP-antibody and E5 dendrimer-

HRP conjugates diluted 1 :100 and passed through different derivatised

metal chelate glass fibre filters (Whatman GF/B, GF/C and GF/F). The

metal ion used was Zn²⁺. EXAMPLE 7 An immunoassay to detect the presence of

mouse antibody on a metal chelate filter

E5-dendrimer conjugate, diluent only, E5 dendrimer only and E5 dendrimer-antibody conjugate were passed through Zn²⁺ metal

chelate filter discs in duplicate. For one set of discs, phosphate buffered

saline with Tween 20 only was used to wash the discs to remove unbound reagent. For the other set of discs, 500 mM imidazole was added to the washing buffer. Then a 1 :1000 dilution of anti-mouse

immunoglobulin conjugated to HRP was added to the discs and incubated for 30 minutes at 37°C. After this the discs were washed as

before and TMB substrate was applied and washed as described above.

FIG. 5 shows the results. It can be seen that the presence

of 500 mM imidazole inhibited non-specific binding of HRP-conjugated

anti-mouse immunoglobulin from the metal chelate filters.

EXAMPLE 8 Recombinant 6 X histidine tagged protein capture on Zn²⁺ immobilised metal chelate filter

Two metal chelate filter discs (5 mm diameter) immobilised

with Zn²⁺ prepared as in EXAMPLE 4 were incubated with 30 μg of 6 X histidine tagged protein in 400 μL of phosphate buffer for 20 minutes.

The protein solution was then removed and concentrated (flow through)

and the filters washed with 400 μL of phosphate buffer. Bound protein

was then eluted from the filter by incubation with 400 μL of 500 mM imidazole in phosphate buffer for 20 minutes (eluate). This fraction was

concentrated and along with the flow through fraction was subjected to PAGE under SDS denaturating conditions

FIG 6 of a polyacrylamide gel shows that metal chelate filter discs efficiently captured a hexahistidme tagged protein from a

solution and that the concentration of the tagged protein was

approximately five times higher in filter eluates than in the flow through

APPLICATIONS

MC and MCS filters may be incorporated into an immunoassay filter plate, column, syringe filter housing or tube for a wide

range of diagnostics, high-throughput screening, recombinant protein assays, and research applications The MC and MCS filters may also be embodied with a second type filter such as an FTA™ Gene Guard

System (Fitzco Inc, a member of the Whatman Group) filter which will

perform a complementary function to the MC or MCS filters The MC and MCS filters may further embodied with a second filter such as to restrict

the flow of fluids through the MC and MCS filters

Molecular and immunoassay diagnostics

(1 ) FTA coated filters are known to capture nucleic

acids after spontaneously lysmg cells introduced to it Once captured the genetic material is maintained

in a bound form for any length of time without damage 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

dendnmer-bound gene specific capture probes, to which genes of interest will bind and ultimately be detected

(2) Immobilisation of oligonucleotide probes for genomic

assays Such 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

dendrimers (Dendntech Inc, Midland, Michigan,

USA) or dendrimeπc-like structure for the

hybridization capture of genomic DNA E g traditional methodology (other than FTA) of purification yields genomic DNA as a soluble fraction After restriction digest, or in vitro

transcription, template nucleic acid can be produced that is in a position to be captured by such

oligonucleotide probes Alternatively, these probes may be mixed with the genomic DNA whereby the

DNA/probe hybrids formed can be captured on MC

filter through their metal chelate tail on an MC filter

A second probe conjugated to a detector label, e g

an enzyme can then be added

(3) Oligonucleotides, mRNA and cDNA coupled to dendrimers or other species capable of acting as a ligand, for example a hexahistidine tag, can be

immobilized onto a MC filter. Incorporation of metal

chelate filters into multi-well filterplates provides a

platform for microarray analysis. It will also be appreciated that nucleic acids tagged with

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.

(4) Recombinant proteins may be directly immobilized

on a metal chelate without the need for a purification step. Proteins with a hexahistidine tag

will be captured on the filter while the culture

supernatant is washed away. Such proteins may then be detected or utilised in an immunoassay or

similar type of analysis.

(5) Site-directed immobilisation of recombinant proteins

for use in immunoassays. Recombinant fusion

proteins including a hexahistidine tail may be immobilised on a metal chelate filter. This will

ensure that the majority of the proteins have their

active site(s) available for ligand binding resulting in

more efficient antigen usage and more sensitive

immunoassays. (6) Unstable recombinant protein may be immobilized on MC filters quickly and at room temperature

without the need for covalent bond formation involving harsh chemical reactions which may damage the protein

High-throughput screening (HTS)

(1 ) Microplates and filterplates in a 96-well or greater

format are widely used in HTS A filterplate

incorporating an MC filter would enable the user to rapidly immobilize potential drugs or target

molecules labelled with a specific tag, e g hexahistidine

(2) "DELphi" (Cytos Biotechnology Ag, Zurich, Switzerland) is a virus-based expression and

screening system that allows reproduction of all

proteins from a chosen tissue or cell type using

cDNA libraries converted into alphaviral expression

libraries that are used to infect cell cultures expressing the original proteins in a "one-gene-per- cell" or "one-gene-per-plaque" format (WO

99/50432, WO 99/25876, C Blaser, et al , Genetic

Engineering News 2000,20(6) 32-35) If the cDNA were co-expressed with 6-hιs this would permit rapid

capture of the expressed protein The method could also be combined with a FTA filter for simultaneous

capture of the amplified viral DNA for sequencing

either after or without the need for RT-PCR. The

MCF-captured proteins may then be tested for function in bioassays.

(3) An indicator of cellular events is the change in kinase activity. Interaction at the cell surface is

translated to gene expression via a multitude of pathways consisting of discrete quinces. This process is known as signal transduction, and is the

mechanism by which a cell interacts with its

environment. Kinases are therefore regarded as useful drug targets as their (de)activation can alter

the behaviour of a cell. To this end many assay techniques to detect kinase activity exist. In the

microplate format, typically, a synthetic kinase substrate is incubated with the kinase sample of

interest and the level of phosphorylation of the substrate measured post incubation. Difficulty has

always arisen when attempting to isolate the substrate in a purified form to facilitate detection. If

the substrate was engineered to contain a 6xHis

tag, it could therefore be readily captured on a MC

filterplate that is incorporated within a multiwell device. A MC filter will have greater specificity than Promega's streptavidin filter that is commonly used together with biotin tagged peptides.

(4) Antibodies, enzymes and other molecules (targets)

required to be tested against solid phase or liquid phase combinatorial chemistry libraries may be tagged with hexahistadine or dendrimers. Following

an incubation, the reacted mixtures are passed

through a MC filter and washed. The hexahistidine or dendrimer tagged targets are retained by the MC

filter together with any combinatorial library

compound of "hit" compound. The isolated library compound may then be further interrogated to

determine its identity as is known in the art. As an

alternative the target molecule may be conjugated to a dendrimer or hexahistidine tag either of which

may be itself tagged with a dye, fluorescent

compound, enzyme, etc. Following an incubation, the "hit" library compounds either on a resin particle

or in free solution, are labeled by the tagged

dendrimer or hexahistidine and may then be further

interrogated.

(5) Peptide immobilisation for epitope mapping.

Overlapping peptide sequences may be tagged with a hexahistidme tail or dendrimers and immobilised onto metal chelate filters for subsequent screening

(6) Filterplates with 96, 384, 1536 or more wells with

metal chelate filter bottoms could be used to capture hexahistidine or dendrimer tagged or target molecules

(7) When searching for reporter genes, le those that

switch on or off in response to a drug, the gene

product may be engineered to bear a hexahistidme sequence This product may then readily be captured onto a metal chelate filter

Biotechnology applications

(1 ) A deep well multiwell plate encapsulating a MC filter at the bottom of each well may be constructed

with a hydrophobic filter layer placed directly

beneath the chelate filter to act as a liquid retention

barrier Within each well may be carried out mammalian, yeast or bacterial culture without fluid dripping through the filter bottoms Protein,

recombinant or otherwise, either secreted from the

cells in culture, obtained via peπplasmic harvesting

or lysis will bind to the metal chelate filter at the

bottom of the well if it has a hexahistidine tag

Application of vacuum to the well will remove cellular debris; leaving recombinant protein bound to the filter. After washing steps the captured protein may be released with an elution procedure as is known in the art.

(2) Simultaneous capture of library DNA and

recombinant protein. A device encapsulating both

FTA and MC filter materials within the same device (tube, column or multiwell) may be constructed.

Upon application of a cellular population containing

an expression library, the cells will lyse, the

episomal and/or genomic DNA will bind to the FTA, and the recombinant protein containing an

engineered hexahistidine tag will bind to the chelate

filter. With the addition of imidazole elution buffer, the recombinant protein can be harvested and assayed. If the protein collected is of interest

(desired level, activity, interaction, etc.), the exact

DNA that produced it can be collected using the

FTA techniques.

(3) Immobilization of metalloproteases.

Metalloproteases are implicated with the onset of

metastasis, being part of the mechanism utilised by

transformed cells during the break through of epithelial layers. The action of metalloprotease is currently of interest to the drug discovery community as they provide a good anti-cancer target. The ability to rapidly harvest and assay for

metalloproteases is advantageous. Such proteases

will specifically bind to MC or MCS filters.

(4) Recombinant proteins and enzymes expressing a tag such as a hexahistidine tail, may be assayed directly from culture to determine a) levels of

expression, b) enzyme activity, c) the best construct,

d) best culture medium to use thereby eliminating tedious column chromatography work for simple assay determinations.

(5) Immobilisation of metalloproteins for purification and

quantitation.

(6) Western blot and dot blot applications.

(7) Immunoassays where it is desirable to capture, for example, dendrimer or hexahistidine tagged

peptides, proteins, nucleic acids or other

substances onto a MC or MCS filter.

Other applications

(1 ) Multi-layer MC or MCS filterplates, tubes or

columns. Several layers of MC or MCS filter within

each tube, column or well will amplify the capacity

to isolate and harvest compounds tagged with dendrimers or hexahistidine or other suitable substances

(2) Environmental samples incubated with specific dendrimer or hexahistidme conjugated reagents

may be passed through columns containing large rolled up sheets of MC or MCS filters This

approach allows for large volumes processing of

fluids such as water supplies Captured pollutants

and microorganisms or parts of microorganisms such as antigens or nucleic acids may then be

assayed using methods well known in the art

(3) The inclusion of a flow restriction filter beneath the MC or MCS filter may be used to control flow

through of fluids

(4) Large and small scale filter devices for heavy metal

removal systems using the MC or MCS filter

Claims

1. A method of making a metal chelate filter or metal chelating species filter including the following step of:

treating a filter having a pore size of 0.01 to 1000 microns and

accessible functional groups with a metal chelate or metal chelating

species to provide the metal chelate filter or metal chelating filter species.

2. A process is claimed in claim 1 wherein the accessible functional groups are bound directly to the filter.

3. A process as claimed in claim 1 wherein the accessible functional

groups are attached to a linker which is bound to the filter.

4. A process as claimed in claim 3 wherein the metal chelate is

attached to the linker.

5. A process as claimed in claim 1 wherein the filter is derivatised or

treated with a reagent to provide the accessible functional groups

on its surface.

6. A process as claimed in claim 3 wherein the linker is treated with a reagent to provide the linker with accessible functional groups.

7. A process as claimed in claim 3 wherein the linker is pre-attached

to the metal chelating species to form a linker - metal chelating species moiety followed by attachment of the moiety to the filter.

8. A process as claimed in claim 1 wherein a linker is pre-attached to

the filter in the form of a linker moiety in which at least one of the

reactive functional groups of the linker is protected by a suitable protecting group to allow for unreacted functional groups to be blocked.

9. A process as claimed in any preceding claim wherein the metal

chelating species is selected from the group consisting of:

iminodiacetic acid, nitrilotriacetic acid, diethylenet amine -

N,N,N',N" - pentoacetic acid, branched superstructures or polymers, triethylenetetramine, ethylenediamine, glycine, o- phenanthroline, 4,4-bipyridyl, 2,2-bipyridyl, pyridine and 6-

hydroxynicotinic acid.

10. A process as claimed in claim 8 wherein the branched superstructures or polymers are selected from the group consisting of: dendrimers, dendrimeric - like molecules or polymeric structures

with appropriate election donating groups capable of coordinating

with metal ions.

11. A process as claimed in claim 9 wherein the electron donating

groups are selected from the group consisting of: -NH₂, -COOH, -SH, -OH and heterocyclic moieties comprising nitrogen atoms.

12. A process as claimed in claim 1 wherein the metal chelate is a

metal chelating species having a metal coordinated thereto which

is selected from the group consisting of: Fe, Co, Ru, Rh, Rh, Pd, Os, Ir, Pt, Pb, Sn, Ge, Sc, Y, lanthanides and actinides, B, Al, Ga,

In, Tl, Li, Na, K, Rb, Cs, Fr and Be, Mg, Ca, Sr, Ba, Ra, Cu, Ni, Zn

and transition metals.

13. A process as claimed in claim 1 wherein the functional groups are selected from the group consisting of -OH, -Si-OH, -NH₂,

canobromides, hydrazides and other amine containing groups, thiols, aldehydes, carboxylates, sulfonyl chlorides, ketones,

halogens, acetyl, epoxy groups, maleimido, hydrazzme, groups containing acetyl and epoxy

A process as claimed in claim 1 wherein the linker is selected from the group consisting of substituted or unsubstituted alkyl groups,

substituted or unsubstituted aryl groups, substituted or

unsubstituted arylalkyl groups, peptides and branched peptides, linear, cyclic, and branched polymers of polysacchandes,

phospholipids and peptides having either alpha-, beta-, or omega-

ammo acids, heteropolymers, polyurethanes, polyesters,

polycarbonates, polyureas, polyamides, polyethyleneimines,

polyarylene sulfides, polysiloxanes, polyimides, polyacetates,

dendrimers and dendπmeπc-like molecules A process as claimed in claim 14 wherein the substituted or

unsubstituted alkyl groups have 1 to 6 carbons A process as claimed in claim 15 wherein the alkyl group comprises

a single methylene or plurality of methylene groups A process as claimed in claim 1 wherein the filter is filter media

formed either fully or partly from glass, silica or quartz, including

their fibre or derivatives thereof A process as claimed in claim 1 wherein the filter comprises a porous material selected from polyvinyhdene fluoride and other fluoropolymer, polyamide, cellulose acetate, nitrocellulose, polyv yl chloride, polysulfone, polyamide, paper or combinations thereof

A process as claimed in claim 1 wherein the metal chelate filter incorporates a metal which is selected from Fe, Co, Ru, Rh, Rh,

Pd, Os, Ir, Pt, Pb, Sn, Ge, Sc, Y, lanthanides and actinides, B, Al, Ga, In, Tl, Li, Na, K, Rb, Cs, Fr and Be, Mg, Ca, Sr, Ba, Ra, Cu, Ni, Zn and transition metals

A process as claimed in claim 1 wherein the metal chelating species is attached to the filter by a covalent bond, charge interaction or metal chelation

A process is claimed in claim 1 wherein the linker is reacted with a branched species inclusive of lysme or a branched polymeric

species inclusive of dendrimers and dendπmer-like species to

increase the number of functional groups on the filter

A process as claimed in claim 20 wherein the reacted linker is

treated with a metal chelating species to increase the metal

chelating capacity of the filter A process as claimed in claim 1 wherein the filter is glass or glass

fibre A metal chelate filter or metal chelating species filter when prepared

by a process as claimed in any preceding claim A metal chelate filter or metal chelating species filter comprising a filter incorporating a metal chelating species bound thereto by accessible functional groups located on the filter.

26. A filter incorporating a metal chelate bound thereto by accessible functional groups located on the filter.

27. A filter having a linker bound thereto by accessible functional groups located on the filter and a metal chelating species attached

to the linker.

28. A filter having a linker bound thereto by accessible functional groups located on the filter and a metal chelate attached to the

linker.

29. A filter as claimed in claim 24, 25, 27, 27 or 28 which is a porous

membrane in the form of a filterplate, microplate, tube, vial, syringe

filter housing, column or similar arrangement.