WO2003014736A1

WO2003014736A1 - Biological molecules comprising glycosaminoglycans

Info

Publication number: WO2003014736A1
Application number: PCT/AU2002/001069
Authority: WO
Inventors: Warren Charles Kett; Deirdre Roma Coombe; Colin John Sanderson
Original assignee: Meditech Reserach Limited
Priority date: 2001-08-10
Filing date: 2002-08-09
Publication date: 2003-02-20

Abstract

The present invention relates generally to a capture device for peptides, polypeptides or proteins or other biological or synthetic molecules, alone in a sample or associated with a cell or virus, cell or virus wall or cell membrane or viral core or an extract or fraction thereof. More particularly, the present invention provides a device useful for facilitating the identification of peptides, polypeptides or proteins or other biological or synthetic molecules alone in a sample or associated with a cell or virus, cell or viral wall, cell membrane or viral core or a portion or fraction thereof immobilized to the device. In a particular embodiment, the present invention comprises a carbohydrate molecule of one or more saccharide monomers associated with a polysaccharide molecule which in turn is immobilized to a solid support useful in the capture of peptides, polypeptides or proteins or other biological or synthetic molecules alone or associated with a cell or virus, cell or viral wall or cell membrane or viral coat or an extract or fraction thereof. The capture device is useful in combination with detection assays such as immune-based detection assays including enzyme-linked immunoassays.

Description

Solid binding support for biological molecules comprising glycosaminoglycans

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Bibliographic details of references provided in the subject specification are listed at the end of the specification.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

Immunoassay procedures are widely used to quantify specific component molecules in biological samples such as blood samples from patients for prognostic, diagnostic or other clinical purposes. Immunoassays can be designed to either detect antigens or host antibodies reactive against antigens. Several immunoassays are commercially available for the detection of antibodies or antigens in sera or other biological fluids.

One of the more widely used and traditional immunoassays is the enzyme-linked immunosorbent assay (ELISA). ELISAs normally comprise a solid-phase support such as a polystyrene microplate coated with a specific antibody. However, whilst these types of ELISAs are very useful, they have some associated problems. For example, there is a requirement for specific antibodies or antigens to be produced for each specific application and can be complex and labour intensive. Moreover, these types of pre-coated ELISA microplates are not readily storable for long periods of time.

There is a need, therefore, to develop alternative technologies to immobilize biological and synthetic molecules which can then be identified by binding partners having specificity or other discriminating properties.

One alternative type of microplate assay employs the glycosaminoglycan, heparin. The use of heparin as a capture molecule in ELISA plates has been previously proposed using a variety of means to immobilize the heparin. For example, heparin can be directly coupled to hydrazide activated polystyrene (Saphire et al, EMBO J. 18(23): 6771-6785, 1999) or amino-styrene surface (Nadkarni & Linhardt, Biotechniques 23(3): 382-385, 1997). A reducing-end modified heparin has been coupled to a lipid-like moiety which is then passively absorbed (Sugiura et al, J. Biol Chem. 268(21): 15779-15787, 1993) and heparin fragments have been coupled to styrene monomers with subsequent polymerization to yield heparinized polystyrene which is then passively absorbed to ELISA plates (Ishihara et al, J. Biomed. Mater Res. 50(2): 144-152, 1998).

Beads are also another common immunoassay components, particularly for automated instrumentation. For example, side-ways attachment of heparin to beads with subsequent use in an immunoassay to detect bacteria (Aleljung et al, FEMS Immunol. Med. Microbiol. 13(4): 303-309, 1996) and random polymerization of monomers being heparin side chains (Jaulin et al, J. Drug Target 8(3): 165-172, 2000) have been proposed. However, despite these proposals, there has not been wide usage of heparin coated solid- phase supports because of a number of technical problems. One particular problem is that the heparin may "leach" from the solid-phase support. Another problem is that of homogeneity, in that many of the methods of coating heparin to solid-phase supports are unable to differentiate between end-point attachment and side-ways attachment, with end- point attachment being preferable.

In accordance with the present invention, the interaction between certain carbohydrate moieties and biological or synthetic molecules to capture the molecules to a solid support has been exploited. These carbohydrate moieties enable presentation of captured molecules for analysis or purification.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The present invention is predicated in part on the specificity or generalized non-specificity of certain carbohydrate moieties including carbohydrate moieties which interact with particular biological or synthetic molecules. Particular biological or synthetic molecules include peptides, polypeptides or proteins, carbohydrate or lipid molecules or chemical molecules identified from natural product screening or produced by chemically synthetic techniques. In a preferred embodiment, the carbohydrate molecules are saccharides such as but not limited to glycosaminoglycans (GAGs) which includes heparin-like GAG (HL- GAG). Such GAG molecules or their derivatives or homologs are coated to a solid support such as in the form of a planar surface, bead or polymeric structure (such as nitrocellulose or glass) or cellulose (e.g. photographic cellulose) having pores or a surface co-continuous within an external environment. The GAG molecules preferably have generalized non- specificity for a range of molecules which are capturable via the GAG moieties. Once captured, the molecules may be eluted off and subjected to purification protocols or may be identified by the ability of the captured molecules to interact with a binding partner such an immunoglobulin. The latter may be coupled to a reporter molecule or another immunoglobulin labeled with a reporter molecule and specific to the first mentioned immunoglobulin may be brought into contact with the first immunoglobulin.

Generally, the carbohydrate moeities such as GAG are immobilized to the solid support via an intermediate polymer such as but not limited to a polysaccharide such as dextran or its derivatives or homologs. The preferred carbohydrate moieties comprise repeating disaccharide units, are negatively charged and may optionally be sulfated. Examples include heparin, heparan sulfate, keratin sulfate, chondroitin sulfate, dermatan sulfate, dextran sulfate and hyaluronic acid. The present invention provides, therefore, an assay device comprising a carbohydrate moiety capable of interacting with and binding to one or more types of biological or synthetic molecules immobilized to a solid support via an intermediate polymer.

The assay device may also be used to capture biological or synthetic molecules or cells or viruses having biological molecules associated thereto. The captured molecules or cells may then be subjected to identification protocols, or the molecules or cells or viruses may be eluted and subjected to subsequent purification and/or further analysis.

The present invention further provides a method for detecting a biological or synthetic molecule or a cell or virus associated with a particular biological molecule by immobilizing the biological or synthetic molecule or cell or virus and subjecting same to detection means.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a diagrammatic representation of carbohydrate moieties (heparin) associated with an intermediate polymer (dextran). The short, wavy lines represent the heparin, while the thick, snaking lines represent the dextran.

Figure 2 is a representation of the partial oxidation of dextran.

Figure 3 is a diagrammatic representation of the coupling of oxidized dextran to amine plates.

Figure 4 is a diagrammatic representation of the coupling of adipic dihydrazide to oxidized dextran.

Figure 5 is a diagrammatic representation of the coupling of heparin to hydrazide linker.

Figure 6 is a graphical representation of a calibration curve from heparin-based ELISA for lactoferrin.

Figure 7 is a graphical representation of a calibration curve from heparin-based ELISA for CD31.

Figure 8 is a graphical representation showing binding of europium labeled lactoferrin (Eu-DTPA-lactoferin) to heparinized microtitre plate wells and control wells.

Figure 9 is a graphical representation showing binding of avidin-FITC to heparinized magnetic beads and control beads. The nett binding (heparinized beads - control beads) is also shown.

Figure 10 is a graphical representation of the nett binding of avidin-FITC to a second batch of heparinized beads, and shows the nett binding data of Figure 10 for comparison. Both batches were prepared by the same operator.

Figure 11 is a graphical representation showing inhibition of binding of avidin-FITC to heparinized magnetic beads by exogenous soluble heparin.

Figure 12 is a graphical representation showing the nett binding of avidin-FITC to a third batch of heparin-dextran beads. This batch was prepared by a different operator.

Figure 13 is a graphical representation showing binding of europium labeled lactoferrin (Eu-DTPA-lactoferin) to heparinized magnetic beads and control beads.

The following are abbreviations used in the subject specification:

ABBREVIATIONS

The term "GAG" includes "HLGAG".

An example of glycosaminoglycan is heparin DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the use of carbohydrate moieties associated with a larger polymer which is in turn immobilized to a solid support in a device which is capable of capturing biological or synthetic molecules. The latter molecules may be the same or different and are capable of interacting or otherwise associating with the carbohydrate moieties. Consequently, the biological or synthetic molecules bind to the carbohydrate moieties which in effect captures the molecules on a polymeric molecule which in turn is immobilized to a solid support. Once captured, the anchored biological or synthetic molecules may be subjected to detection methods to identify same and/or are eluted off the carbohydrate moieties and subjected to purification protocols or, in the case of cells having biological molecules on their surface or co-continuous with the external environment, subjected to culturing conditions. The term "polymeric molecule" may also be referred to as an "intermediate polymer" and effectively is a bridging molecule between a solid support and the carbohydrate moieties.

Accordingly, one aspect of the present invention contemplates a solid support comprising a polymeric molecule immobilized to all or discrete regions of the solid support and wherein the polymeric molecule comprises carbohydrate moieties which are capable of interacting with a binding partner.

Suitable binding partners may be biological or chemical molecules. A chemical molecule may also be a chemical analog of a biological molecule. All such biological and chemical molecules are encompassed by the term "agent".

Accordingly, the present invention provides a solid support comprising a polymeric molecule immobilized to all or discrete regions of the solid support and wherein the polymeric molecule comprises carbohydrate moieties which are capable of interacting with an agent.

More particularly, the present invention provides a capture device for biological or synthetic molecules, said device comprising a solid support having a polymeric molecule attached to all or discrete regions thereon and wherein the polymeric molecule comprises carbohydrate moieties which are capable of interacting with an agent.

Peptides, polypeptides or proteins, lipids or carbohydrate molecules, or glycoproteins or proteoglycans, other complex or simple molecules which are naturally occurring or exist within or on a biological entity such as a cell or virus are encompassed by the term "biological molecule". The cell or virus may be naturally occurring or in a purified state or may be generated following recombinant, mutagenic or selection procedures. The biological molecules may also be receptors or other surface or sub-surface moieties on a cell or virus. A sub-surface molecule is one, for example, which is in a pore or channel and which is co-continuous with an external environment. A molecule "co-continuous" with an external environment includes any molecule which comes into contact with culture fluid or other medium external to a cell. The co-continuity may be naturally occurring or genetically treated with chemical (e.g. an enzyme) or physical stress means (e.g. pressure, sonic disruption, electricity). Furthermore, the biological molecule may be within a cell or on an internal membrane including an organelle and is captured following subjecting a cell or virus or virus or culture of cells or viruses to disruption means. The latter includes sonic or pressure disruption, heat, enzyme-mediated lysis or other form of cell or virus lysis protocol.

Accordingly, another aspect of the present invention contemplates a cell or virus capture device, said cell or virus capture device comprising a solid support comprising a polymeric molecule attached to all or discrete regions thereon and wherein the polymeric molecule comprises carbohydrate moieties which are capable of acting as binding partners for an agent.

The captured cells or viruses may then be eluted off and cultured or subjected to other processes such as detection processes. Alternatively, the captured cells or viruses are identified while immobilized to the solid support. As stated above, agents of the present invention extends to synthetic molecules. Such molecules include molecules synthesized following human intervention and may exist alone or as part of a chemical library or mixture. The synthetic molecules may also be derivatized naturally occurring molecules.

Whether the agents to be captured are natural or synthetic, they need to be able to bind, associate or otherwise reversibly or irreversibly interact with the carbohydrate moieties on the polymeric molecule. The carbohydrate moieties have, therefore, a biological or synthetic molecule as a binding partner.

The preferred carbohydrate moieties comprise monomeric or multimeric carbohydrate structures. A multimeric carbohydate includes a polysaccharide of, preferably, repeating monomeric or multimeric carbohydrate units. In this regard, a "multimeric" carbohydrate comprises two or more of the same or different carbohydrate units.

The preferred carbohydrate moieties are glycosaminoglycans (GAGs) selected from heparin, heparan sulfate, chondroitin sulfate (type A, B or C), keratan sulfate or hyaluronic acid (HA) or synthetic or derivative forms thereof. Reference to a "GAG" includes all synthetic forms and parts, fragments and derivatives thereof. However, the present invention extends to any carbohydrate moieties which specifically or preferentially interact and bind to protein molecules.

Accordingly, another aspect of the present invention is directed to a solid support comprising a polymeric molecule immobilized to all or discrete parts thereon and wherein the polymeric molecule comprises GAG molecules which are capable of interacting with an agent.

More particularly, the present invention provides a capture device for biological or synthetic molecules, said device comprising a solid support having a polymeric molecule attached to all or discrete regions thereon and wherein the polymeric molecule comprises GAG molecules which are capable of interacting with an agent. Yet another embodiment contemplates a cell or virus capture device, said cell or virus capture device comprising a solid support comprising a polymeric molecule attached to all or discrete regions thereon and wherein the polymeric molecule comprises GAG molecules which are capable of interacting with an agent.

The polymeric molecule to which the GAG molecules are attached include complex carbohydrate molecules (e.g. polysaccharides) such as dextran, or polyethylene glycol. Generally, any polymer which is linear or branched and exhibits at least one property selected from being hydrophilic, uncharged and/or non-anti genie may be used. The polymer may be attached to the solid support using a variety of standard chemical processes such as coupling oxidized dextran to amine coated plates.

The GAG molecules may be bound to the polymeric molecule directly or may be associated via a linker. The function of the linker is to incorporate groups into the polymeric molecule that react specifically with the reducing terminus of GAG. A linker is not necessary if the polymers used already possess an appropriate reactive group for coupling to GAG. For example, oxidized dextran may be converted to amino dextran and this complex coupled to a suitable surface. The remaining amino groups can then be used to couple with GAG using reductive amination. Alternatively, amongst other choices, chitosan, polyethyleneimine and animated polyethylene glycols and polyvinyl alcohols could also be substituted for the amino dextran. It is also possible to modify the reducing terminus of heparin prior to introducing it to the microplate. For example, oxidation of the reducing terminus of a GAG to an acid, which subsequently forms a lactone. The lactone can then readily couple with, for example, an amine, hydrazine or hydrazide on the intermediate polymer. The reducing terminus may also be modified by reductive amination or hydrazone formation with the resulting nucleophile being used to couple to suitable polymers.

The present invention provides a means for screening a sample for the presence of a cell or virus or biological or synthetic chemical. The term "sample" includes a biological and/or environmental sample. The term "biological sample" is used in its broadest sense and includes a sample of tissue or cells from a tissue or organ isolated by, for example, surgical intervention, lumbar punctures (e.g. cerebral spinal fluid), bone marrow (e.g. stem cells or progenitor cells) biopsy, blood collection procedures or invasive or passive collection procedures. Furthermore, a biological sample may comprise cells maintained in in vitro culture or suspension. A biological sample is, therefore, a collection or population of cells which may comprise a single cell type or comprise a mixed population of two or more cell types. In a particularly preferred embodiment, the biological sample is a biopsy of tissue or other form of tissue sample or an in vitro culture comprising erythrocytes, endothelial cells, epithelial cells, muscle cells, leukocytes (e.g. lymphocytes, macrophages, dendritic cells), platelets, stem cells or their progenitors, NK cells or non-lineage cells as well as inflammatory cells and cancer cells.

An environmental sample is generally a sample from a hospital, industrial, domestic site, food processing or preparation site or an environmental location such as any terrestrial location (e.g. rivers, waterways, land areas) which may contain microorganisms or viruses.

Cells contemplated herein from a biological or environmental sample include prokaryotic and eukaryotic cells. Prokaryotic cells include any bacterial or microbial cell such as present in an environmental or biological sample. Such prokaryotic organisms include Pseudomonas sp., E. coli, Enterobacter sp., Salmonella sp., Klebsiella sp., Acetobacter sp., Porphroymonas sp., Staphylocous sp., Streptococcus sp., Bacillus sp., Proteus sp., Helicobacter sp., Camphylobacter sp. or Legionella sp. amongst many others. Viruses include hepatitis virus, a retrovirus, an ADDS virus (e.g. HIV), foot and mouth disease virus or polio virus amongst many others. Εukaroytic cells include eukaryotic organisms such as yeast, fungi, amoeba, and other single cell organisms as well as cells from higher plants or animals. Particularly useful cells are those in animals and mammals such as humans cells which form part of tissues or organisms or cells associated with a disease condition. The assay device including the cell or virus or other biological entity capture device of the present invention preferably comprises a solid support having a flat, planar, round or curved surface. Examples of suitable solid supports include membranes, plastic cover slips, glass slides or the wells of microtitre trays and magnetic beads. Nitrocellulose and magnetic beads are particularly preferred. Cellulose and in particular photographic cellulose may also be used. The solid support may also comprise polymer coated regions such as in the form of spots or other geometric patterns. The spots are generally discrete and surrounded by regions not containing any polymeric molecules.

The immobilized polymer associated with a GAG or other carbohydrate moiety is then contacted by an environmental, biological or chemical sample. As stated above, an environmental sample includes a sample from an aquatic or air or terrestrial environment as well as samples from plants, microorganisms and coral. An environmental sample includes a sample for screening natural products for use, for example, as pharmaceutical or diagnostic agents. It also includes samples such as soil, water and air which may contain a contaminant. A biological sample also includes a tissue biopsy or other biological specimen.

A chemical sample may comprise chemical molecules produced, for example, by combinatorial synthesis or maintained in a library. In one embodiment, the sample contains a mixed population of agents. The contact is for a time and under conditions sufficient for an agent to be captured by the immobilized carbohydrate moiety. The captured agents may then be detected by any convenient means such as biochemically, immunologically or microscopically. Cells, viruses, biological or chemical molecules identified may also be removed (e.g. eluted) and transferred to cultures or maintenance media.

Examples of biochemical detection includes the use of binding partners of the captured molecules, cells or viruses as well as enzymes which catalyse to a product which can be readily detected, such as by visual color or spectroscopically. A binding partner may also be labeled with a reporter molecule capable of providing an identifiable signal. Such signals include fluorophores and radioactive isotopes amongst many others. Immunological detection is particularly preferred. For example, an immunoglobulin specific for a captured molecule, labeled with a reporter molecule, may be added. The identification of the reporter molecule indicates that the molecule is captured. Alternatively, after the immunoglobulin is added and it forms a complex with the captured molecule, an anti-immunoglobulin immunoglobulin labeled with a reporter molecule is added and the presence of a signal from the reporter molecule determined.

Accordingly, another aspect of the present invention contemplates a method for detecting a biological or synthetic molecule or a cell or virus comprising same, said method comprising capturing said biological or synthetic molecule or cell or virus on a capture device comprising a solid support having a polymeric molecule attached to all or discrete regions thereon and wherein the polymeric molecule comprises carbohydrate moieties which are capable of acting as binding partners of said biological or chemical molecules or cell or virus comprising same and then subjecting same to detection means.

Preferably, the polymeric molecule is a polysaccharide such as dextran, or polyethylene glycol.

Preferably, the carbohydrate moiety is a GAG such as heparin, heparan sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, dextran sulfate or hyaluronic acid.

The detection means may be biochemical, macro- or micro-scopic or immunological. Immunologically-based detection means are particularly convenient, amongst others.

Accordingly, in a particularly preferred embodiment, the present invention is directed to a method for detecting a biological or synthetic molecule or a cell or virus comprising same, said method comprising capturing said biological or synthetic molecule or cell or virus or a capture device comprising a solid support having a polymeric molecule attached to all or discrete regions thereon and wherein the polymeric molecule comprises GAG molecules which are capable of acting as binding partners of said biological or chemical molecules or cell or virus comprising same and then identifying the captured entities by immunological means.

Monoclonal or polyclonal antibodies may be used in the immunological detection of captured entities. The use of monoclonal antibodies in an immunoassay is particularly convenient because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981; Kohler and Milstein, Nature 256: 495-499, 1975; Kohler and Milsten, European Journal of Immunology 6: 511-519, 1976).

Another aspect of the present invention contemplates a method for detecting a captured molecule in a biological or chemical sample, said method comprising contacting said sample with an antibody specific for said entity or its derivatives or homologues for a time and under conditions sufficient for an antibody-entity complex to form, and then detecting said complex. The term "antibody" includes an immunoglobulin and antigen-binding portions, parts and fragments thereof.

The presence of the captured entity may be accomplished in a number of ways including immunoassays. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. Furthermore, once the captured entities are eluted off the solid support, ELISAs may be performed to identify the molecules.

In one method, an antibody specific to the captured entity, labeled with a reporter molecule capable of producing a detectable signal, is added and incubated, allowing time sufficient for the formation of a complex of antibody bound to the entity, the entity being immobilized to the solid support via the carbohydrate moiety (e.g. GAG) attached to the intermediate polymer. Any unreacted material is washed away, and the presence of the entity is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of captured entity. Variations of this assay include any assay in which both sample and labeled antibody are added simultaneously to the capture device which comprises the captured entity attached to the carbohydrate moiety. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. As stated above, similar procedures may be used to identify captured entities once they have been eluted off the solid support.

As stated above, the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking, covalently binding or physically adsorbing the polymer to the solid surface. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. from room temperature to about 37°C including 25°C) to allow for binding. Following the incubation period, the antibody specific for the captured entity is added. The antibody is generally linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.

An alternative method involves using an antibody not labeled with a reporter molecule. A second antibody, directed to an immunoglobulin and labeled with a reporter molecule is then added. The complex is detected by the signal emitted by the reporter molecule.

In the case of an enzyme immunoassay, an enzyme is conjugated to the antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β- galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the captured entity allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex comprising entity-antibody. The substrate will react with the enzyme linked to the antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

By "reporter molecule", as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of immunoglobulin bound antigen. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes). In the case of an enzyme immunoassay, an enzyme is conjugated to the second or third immunoglobulin, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist which are readily available to one skilled in the art. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. It is also possible to employ fluorogenic substrates which yield a fluorescent product.

Alternatively, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to immunoglobulins without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled immunoglobulin adsorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable microscopically or using other imaging devices such as a confocal microscope or 2 dimensional laser scanner (e.g. Fluorolmager or Typhoon, Molecular Dynamics, Inc., Sunnyvale, USA).

The present invention further contemplates the use of the assay device as herein described in the manufacture of a biological- or chemical-molecule or cell- or virus-capture device for the capture of particular agents in a sample.

The device of the present invention may also be adapted for use as a biochip or microchip which includes a matrix support comprising an array of carbohydrate moieties immobilized to a solid support via intermediate polymers. The carbohydrate moieties may be ligands or binding partners of the moieties in a sample to be assayed.

The present invention further comprises a kit useful for capturing and optionally identifying or detecting particular agents in a sample, said kit comprising a capture device for biological or synthetic molecules, said device comprising a solid support having a polymeric molecule attached to all or discrete regions thereon and wherein the polymeric molecule comprises carbohydrate moieties which are capable of acting as binding partners of said biological or chemical molecules.

In a preferred embodiment, the kit comprises a capture device for biological or synthetic molecules, said device comprising a solid support having a polymeric molecule attached to all or discrete regions thereon and wherein the polymeric molecule comprises GAG molecules which are capable of acting as binding partners of said biological or chemical molecules.

The kit is conveniently in compartmental form and may further comprise compartments adapted to confer reagents for screening for interaction between a binding partner and an agent. The kit or assay device therein is also conveniently adapted for automation or for computer-assisted reading of potential interaction between an agent and an immobilized carbohydrate moiety and is readily adapted for high through-put screening.

Preferably, the surface is substantially planar. In another preferred embodiment, the surface is substantially columnar.

With respect to the latter embodiment, the method of this aspect of the present invention can at least be partly implemented using a suitably programmed computer. In particular, the preferred data processing means comprises a suitably programmed computer and the steps of the method are preferably performed using the suitably programmed computer. In various forms of the invention, the input information may take the form of values, identifiers or other data in respect of the identity of interaction with the immobilized binding partners. The input data may be digitized. Alternatively, for implementation of the instant invention, a dedicated Fast Fourier transform chip can be employed as at least part of the processing means.

In a preferred form of the subject invention, representative measurements are made identifying or valuing the presence of an interaction event between an immobilized binding partner and an agent. The presence of the interaction may optionally be recorded on the cell or virus capture or other biological entity device.

Accordingly, another aspect of the subject invention is directed to data processing means for assessing the interaction between an immobilized carbohydrate moiety and its binding partner wherein said data processing means executes the steps of (in any order):-

1. optionally identifying the position of discrete spots of binding partners immobilized to a solid surface via the carbohydrate moiety;

2. identifying interaction between carbohydrate moieties and the binding partners in

(i); 3. analyzing the data obtained in (2) to identify the immobilized binding partners;

4. optionally analyzing the data to identify agents in a sample; and

5. optionally controlled removal of the solid phase to a culture or maintenance medium.

The input program and general methodology of this aspect of the present invention are useful in a range of applications including the rapid determination of the presence of particular agents or the resistance or sensitivity of microbial or eukaryotic cells to particular agents.

The present invention is particularly useful in the development of appropriate and efficacious medical or therapeutic protocols. In one embodiment, it is used to identify cells or viruses resistant or otherwise exhibiting reduced sensitivities to particular agents. The present invention also permits the rapid determination of whether a substantially homogenous population of cells is resistant or sensitive to particular agents.

The present invention provides, therefore, a method of treatment of a subject comprising capturing cells or viruses or other biological entity from a sample from said subject and determining whether said cells or viruses or other biological entity are sensitive or resistant to particular therapeutic agents, and then selecting an agent to which said cell or virus or other biological entity is sensitive and using this agent to treat said subject.

The capture device also enables rapid purification of cell types such as stem cells from bone marrow or spinal fluid.

The present invention is further described by the following non-limiting Examples. EXAMPLE 1 Techniques used for GAG/intermediate polymer immobilization

The GAG/intermediate polymer may be immobilized on a solid carrier by a variety of methods known in the -art, including covalent coupling. For references describing these methodologies, see Silman and Katchalski (Annual Review of Biochemistry 35: 873, 1966); Melrose (Review of Pure and Applied Chemistry 21: 83, 1971) and Cuatrecasas and Anfinsen (Methods in Enzymology, Vol. 22: 1971).

The surface of microtitre plates are non-porous. The attachment of the intermediate polymer effectively mimics a porous surface, hence, increasing the effective surface area of the support. There are many possible polymers that may be used including oxidized dextran or polyethylene glycol. Preferably the polymer is hydrophilic, uncharged and non- antigenic, although this is not an essential feature of the present invention. The polymer may be linear or branched.

In one embodiment, a linker is used to contact the immobilized intermediate polymer to the GAG. The function of the linker is to incorporate groups into the intermediate polymer that react specifically with the reducing terminus of GAG. If a polymer already has an appropriate reactive group for coupling to GAG, the linker is not required. For example, oxidized dextran can be converted to amino dextran and this complex coupled to a suitable surface. The remaining amino groups can then be used to couple with GAG using reductive amination. Alternatively, amongst other choices, chitosan, polyethyleneimine and animated polyethylene glycols and polyvinyl alcohols could also be substituted for the amino dextran. Finally, it is also possible to modifying the reducing terminus of heparin prior to introducing it to the microplate. For example, oxidation of the reducing terminus of a GAG to an acid, which then subsequently rearranges to a lactone. The lactone can then readily couple with, for example, an amine, hydrazine or hydrazide on the intermediate polymer. The reducing terminus may also be modified by reductive amination or hydrazone formation with the resulting nucleophile being used to couple to suitable polymers. EXAMPLE 2 Antibody capture technique

The immobilized GAG/intermediate polymer complex that has bound the protein of interest is put into contact with a biological sample suspected of containing antibody that recognizes the GAG binding protein of interest (antigen). In the case of aqueous samples such as blood or urine, the solution may be buffered and ionic salt may be present at optimum concentrations for the antigen-immobilized GAG/intermediate polymer complex. The ionic salts may be Tris, borate or other like salts. The inert surface with antigen immobilized GAG/intermediate polymer complex thereon is next put into contact with an antibody conjugated to a chromophormic molecule and this second antibody recognizes the antibody in the sample. After careful rinsing under water or with suitable surfactants such as Tween 20 to remove excess coloured antibody, the inert surface is inspected for colour, fluorescence or luminescence directly or after addition of colour-developing agents.

In an alternate procedure, the assay for a GAG degrading compound (e.g. an enzyme) is based on the ELISA. In this situation, the biological sample would contain an unknown amount of enzyme activity. The biological sample would be added to the microplate containing the immobilized GAG. Modification of the GAG occurs because enzyme is present in the biological sample and the extent to which the immobilized GAG is modified is monitored by the ability of the immobilized GAG to bind a protein or peptide. The protein or peptide may or may not be labeled. If the protein or peptide is not labeled, the level of protein or peptide binding is then detected by the binding of a specific antibody that may or may not be labeled. If the antibody is not labeled a second antibody that is conjugated to a label must be added as an additional step.

In yet another alternative procedure, GAG-ELISA is used to measure the amount of ligand/receptor that binds a GAG binding protein. In this situation, the GAG immobilized intermediate polymer complex is used to capture the GAG binding protein. The sample would contain an unknown amount of a ligand/receptor that binds the protein. The ligand/receptor may or may not be labeled. If the ligand/receptor is not labeled the quantity of ligand or receptor that binds the captured protein can then be monitored by a specific antibody that recognizes the ligand or the receptor. The antibody may be labeled or unlabeled.

EXAMPLE 3 Enzyme-linked immunoassay-ELISA

A biological sample suspected of containing a molecule such as a protein to be detected is put into contact with GAG molecules attached to intermediate polymers immobilized to a solid support such as PVC, paper strip or glass bead. Antibodies to the captured molecules are conjugated to an enzyme capable of catalyzing a reaction giving a detectable product, such as a colored product. The substrate of the enzyme is added and the assay involves detecting the product of catalysis of the substrate. Such product is indicative of an antibody bound to a captured entity. In an alternative embodiment, a specific antibody to the molecules to be detected is first added followed by an anti-immunoglobulin antibody which is labeled with a reporter molecule such as an enzyme.

EXAMPLE 4 Means of immobilizing heparin

Unmodified heparin does not passively absorb to polystyrene microplates and, therefore, other routes to immobilize heparin are necessary. The inventors initially focused upon end- point attachment of heparin via an intermediate scaffold of dextran to the microplate surface. This scenario is depicted in Figure 1.

The preparation of heparin coated microplates involves four steps:

1. preparation of oxidized dextran;

2. coupling of oxidized dextran to amine plates; 3. coupling of intermediate di-functional linker to the remaining aldehyde groups of oxidized dextran; and

4. coupling heparin to the remaining reactive end of the intermediate linker.

Step 1: Preparation of oxidized dextran

Dextran is ostensibly a linear polysaccharide comprised of 1,6-linked glucose units. During oxidation, some of the hydroxyl groups are converted to aldehyde groups as shown in Figure 2. For the immobilization, 500 kDa dextran was used (approximately 3,000 glucose units per chain) and the oxidation was limited to approximately ¹/₁₀ of the glucose units.

The inventors found that the oxidized dextran produced by this procedure was not suitable for long term storage, although storage for approximately 1-2 months was achievable after lyophilization and protection from light.

Initially, the inventors dissolved 2 g of dextran (500 kDa) in 40 mL of water, then added 0.28 g of sodium periodate (NaIO₄). This was covered and incubated overnight at 4°C. After ultrafiltation with a 30 kDa cutoff Amicon filter which reduced the volume down to ~20 mL, the sample was diluted to 160 mL and ultrafiltration was repeated.

The inventors found that this material could either be used directly for the next step by adding buffer, or freeze-dried for storage. The oxidized dextran could also be precipitated with ethanol

Step 2: Coupling of oxidized dextran to amine plates

The oxidized dextran as prepared in step 1 above was coupled to the amine surface of a polystyrene microplate (Costar amine) using reductive amination with sodium cyanoborohydride. Figure 3 shows this procedure pictorially. In this reaction, the aldehyde reacts with a primary amine to generate an imine. Imines are easily hydrolysed, but reduction to an secondary amine results in a stable linkage.

During this step, a high concentration of oxidized dextran was used so as to increase the likelihood that many chains of dextran would bind to the surface at a few sites along the chain, rather than a few chains binding at many sites along the chain. While no attempt to vary or optimize the concentration of dextran used during this step was undertaken, it is possible to alter these conditions.

The inventors first prepared a solution of partially oxidized dextran at 40 mg/mL in 0.2 M sodium phosphate pH 6.3 and aliquots of 100 μL were dispensed into each well. 20 μL of 25 mg/mL NaCNBH₃ in water was then added to each well. After incubation for 4 hr at room temperature, the microplate was washed 3 times with 300 μL of water.

Step 3: Coupling of intermediate di-functional linker

To attach heparin to the dextran surface,a di-fiinctional linker was used. One end of the linker reacts with the remaining aldehyde groups of the oxidized dextran on the surface, whilst the other was used in subsequent steps to react with heparin. Adipic dihydrazide was used as the linker, exploiting the classic reaction of an aldehyde with a hydrazide (Figure 4).

Initially a solution of adipic dihydrazide at 10 g/mL was prepared. 100 μL aliquots were dispensed into well and incubated for 3 hr at room temperature. The microplate was then washed 3 times with 300 μL of water.

Step 4: Coupling heparin to the linker

As depicted in Figure 5, in the final step, the reducing terminus of heparin was reacted with the free hydrazide end of the linker. Sodium cyanoborohydride was added to the resulting hydrazone conjugate in an attempt to reduce the hydrazone double-bond and make this bond resistant to hydrolysis.

A solution of 5 mg/mL heparin in 0.5 M sodium citrate pH 6.3 was prepared and 100 μL was added to each well except control wells. Buffer only was added to control (non- heparinized) wells. After incubation overnight at room temperature, 100 μL of 0.5 M sodium citrate (Na₂C₂O₄) pH 6.3 was added to each well.

A solution of NaCNBH at 25 mg/mL in water was prepared and 20 μL aliquots were added to each well. After incubation at room temperature for 4 hr the microplate was washed 3 times with 300 μL of water. The microplate was then washed 3 times with 300 μL of 2.5 M NaCl in 10 mM tris pH 7.5 and then washed 3 times with 300 μL of water. The microplate was then used directly or stored until required.

EXAMPLE 5

Evaluation of the prepared microplates

The present system has been tested with two heparin binding proteins, lactoferrin and CD31.

Lactoferrin ELISA

Lactoferrin is an 80 kDa glycoprotein implicated in infection and inflammation. Lactoferrin and lactoferrin antibodies are commercially available.

Lactoferrin was obtained from Sigma and a 0.8 mg/mL stock solution was prepared. The lactoferrin was then diluted in a mixture of 5% v/v fetal calf serum and 2% w/v BSA in PBS/0.05% v/v Tween to give concentrations in the range 0-100 pmol/100 μL. 100 μL aliquots were then added to each well of a microplate prepared in Example 3 as required. The microplate was then covered and incubated at room temperature for 1 hr. After washing the microplate 3 times with 300 μL of wash buffer [PBS/0.05% v/v Tween] 100 μL of rabbit anti-human lactoferrin polyclonal (V₅₀₀0 dilution in blocking buffer) was added into each well. The microplate was then incubated, covered at room temperature for 1 hr. The microplate was then washed 3 times with wash buffer and 100 μL of goat anti- rabbit IG-HRP conjugate (V₂ooo dilution in blocking buffer) was added and the microplate was covered and incubated at room temperature for 1 hr. The microplate was then washed 3 times with 300 μL of wash buffer.

TMB substrate was then prepared in accordance with the manufacturer's instructions (Kirkgaard & Perry TMB Micro well Peroxidase Substrate System). 100 μL aliquots were then added to each well and the microplate was incubated at room temperature for 30 min, or until colour development was detected. Figure 6 shows a typical calibration curve from the lactoferrin ELISA. The color developed is linearly related to the amount of lactoferrin added to the well. Significantly, the control wells (non-heparinized) have developed less color. Fetal calf serum contains many proteins that bind heparin other than the exogenous lactoferrin (e.g. fibronectin, thrombin and antithrombin). Moreover, these proteins are at far higher concentration than the exogenous lactoferrin. This experiment demonstrated that there was sufficient binding capacity of the microplates to overcome the potential negative interference caused by competitive binding of other heparin-binding proteins present in the sample.

CD31 ELISA

The plate was blocked by incubation with 300 μL/well 0.5% Amersham membrane blocking agent NIF833 (Amersham) in 10 mM sodium phosphate buffer, 20 mM NaCl, pH 6.3. After 1 hr at room temperature, the wells were washed with 3 x 300 μL/well washing buffer (10 mM sodium phosphate buffer, 20 mM NaCl, pH 6.3/0.05% v/v Tween-20). CD31 (Recombinant human CD31-Fc fusion protein expressed in COS cells) was diluted to 0-5 pmol mL in wash buffer and 100 μL/well were incubated for 1 hr at room temperature. The wells were washed with 3 x 300 μL/well washing buffer. Mouse anti- human CD31 (Clone JC70A, Dako) was diluted 1:1000 in wash buffer and 100 μL/well was incubated for 1 hr at room temperature. The wells were washed with 3 x 300 μL/well washing buffer. Sheep anti-mouse Ig-HRP conjugate (Amersham) was diluted 1 :2400 in wash buffer and 100 μL/well was incubated for 1 hr at room temperature. The wells were washed with 3 x 300 μL/well washing buffer.

TMB substrate was then prepared in accordance with the manufacturer's instructions (Kirkgaard & Perry TMB Microwell Peroxidase Substrate System). 100 μL aliquots were then added to each well and the microplate was incubated at room temperature for 30 min, or until color development was detected. Figure 7 shows a typical calibration curve from the CD31 ELISA demonstrating a linear response between the amount of CD31 added and the color developed. Moreover, the development of color is greater in the heparinized wells than in the control (non-heparinized) wells.

Europium labelled Lactoferrin

There are significant advantages in using labeled proteins as probes for the assays as a substitute for the combination of antibody and secondary antibody-enzyme conjugates for detection, such as those described in the preceding assays. The most significant of which is that amplification afforded by peroxidase is not required to enable detection of the bound protein, thus extending the possible applications beyond merely diagnostic assays and creating the opportunity for high-throughput screening applications using the heparinized plates. Other advantages in using labeled proteins include the following:-

1. faster assays (incubate, wash and detect as opposed to the multiple incubations with detection and secondary antibodies and then subsequent color development);

2. eliminates losses due to desorption of bound protein during the washing and incubation steps involved with anitbody based detection;

3. quantitative estimation of the amount of protein absorbed onto the heparinized surface. Calibration with the labeled protein can be performed, whereas enzyme based techniques require assumptions about the activity of surface-absorbed enzyme. 4. elimination of concerns with non-specific absorption of the detection and secondary antibodies; and

5. Long term stability. The long term storage stability of the detection and secondary antibodies is eliminated and, by virtue of the calibration with the labeled protein binding can be expressed as a percentage of the input.

Lactoferrin was labelled with the chelating agent diethylenetriamine pentacetic acid and europium and purified according to the general instructions of Degan et al. (Mol. Biotechnol. 13(3): 215-22, 1999). An incorporation rate of -10 moles Eu: 1 mole lactoferrin was achieved. The europium labelled lactoferrin was used in a binding assay analagous to the lactoferrin ELISA described above, with modifications allowing for the different detction methods.

The results are presented in Figure 8 which shows binding of europium labeled lactoferrin (Eu-DTPA-Lactoferin) to heparinized microtitre plate wells and control wells. Binding was conducted in 10 mM Tris pH 7.4 containing 150 mM NaCl and 0.05% v/v Tween 20 (binding buffer). Eu-DTPA-Lactoferin was prepared at the specified concentrations in binding buffer containing 0.2% w/v BSA and 100 μL contacted with the wells for 60 mins. After washing (3 x 200 Degan et al. (Mol. Biotechnol. 13(3): 215-22, 1999L binding buffer), the fluorescence signal was developed using DELFIA enhancement solution (Wallac) in accordance with the manufacturer's directions.

EXAMPLE 6

Measurement of binding capacity of immobilized GAG

A GAG binding protein or peptide was labeled with a fluorogenic moiety. Ideally, the

GAG binding protein or peptide binds GAG by electrostatic interactions which may or may not rely upon a specific sequence within the GAG. Suitable GAG binding protein or peptides includes the protein like protamine, lysozyme, tryptase, thrombin and lactoferrin and the synthetic peptides (Arg-gly)_nArg (where n=integer). After incubation of the GAG binding protein or peptide with the immobilized GAG surface and requisite washing, a suitable agent is added to enable measurement of the amount of immobilized GAG binding protein or peptide by homogenous fluorescent assay. A suitable agent may be a salt solution to release the GAG binding protein or peptide from the heparin and also contain additives to maintain the solubility of the GAG binding protein or peptide. Another suitable agent may be a development solution such as the Wallac enhancement solution for time-resolved fluorescence of lanthanides. As the final measurement is a homogenous assay (i.e. in solution only) and is not reliant upon a catalytic activity of the GAG binding protein or peptide, the response can be calibrated using solutions containing known amounts of the labelled GAG binding protein or peptide.

Avidin-FITC was dissolved in PBST to generate a stock solution at 1 mg/ml and stored at 4°C. This solution was centrifuged prior to each use. The incorporation rate of FITC varied between batches within the range 2-3.4 moles FITC/mole avidin. Dilute solutions of avidin-FITC in the range 5-600 nM were prepared in binding buffer of the appropriate pH containing 0.2% w/v BSA and 0.05% v/v Tween-20. The binding buffer was either PBS, 20 mM Bistris containing 150 mM NaCl (pH 5.5 -7.5) or 20 mM ethanolamine containing 150 mM NaCl (pH 7.5-10). Aliquots (100 μl) of the avidin-FITC solutions were placed into heparinized and control wells and incubated at room temperature for 1 hr. The plates were washed with 3 x 300 μl binding buffer of the appropriate pH, containing 0.05% v/v Tween-20. Bound avidin-FITC was resolubilized by addition of 200 μl 10 mM Tris, pH 7.4, containing 2.5 M NaCl. After shaking for 5 mins at room temperature, fluorescence was measured in a Wallac Victor 1420 Plate Reader (Wallac, Turku, Finland). The amount of avidin-FITC was quantified by external calibration using standard solutions of avidin- FITC dissolved in 10 mM Tris, pH 7.4, containing 2.5 M NaCl. Avidin- AlexaFluor488 (moles AlexaFluor488: moles avidin, 3.5:1) was substituted for avidin-FITC in some experiments.

For GAG inhibition studies, aliquots of either heparin or BSA-heparin conjugate were added to solutions of 300 nM avidin-FITC prepared in binding buffer containing 0.2% w/v BSA and 0.05% v/v Tween-20. The solutions were incubated at room temperature for 15 minutes, prior to aliquoting 100 μl into heparinized and control wells. After incubation at room temperature for 1 hr, the remainder of the assay was completed as described above.

For salt inhibition studies, 30 nM and 300 nM avidin-FITC were prepared in 20 mM Bistris, pH 5.5, containing 0.2 % w/v BSA, 0.05% v/v Tween-20 and between 150-1.5 M NaCl. Aliquots of 100 μl of the avidin-FITC solutions were placed into heparinized and control wells and incubated at room temperature for 1 hr. The plates were washed with 3 x 300 μl 20 mM Bistris, pH 5.5, containing 0.05% v/v Tween-20 and the bound avidin-FITC quantified as described above.

The binding capacity assay is a functional assay, designed to account for factors which may limit the binding capacity of the immobilized GAG, e.g. possible steric exclusions to binding.

EXAMPLE 7 Scaffold polymers

Polymers other than oxidized dextran can serve as suitable scaffold for attaching GAG. The following two examples illustrate this concept.

Polyvinylalcohol is aminated according to the guidelines described by Prigent-Richard et al. (J. Biomed. Mater. Res. 40(2): 275-281, 1998). Amine surface plates (Costar amine and Nunc covalink) are activated with cyanuric chlorides and reacted with aminated polyvinyl alcohol according to the manufacturer's guidelines (Nunc). After washing, a solution of 5 mg/mL heparin in 0.5 M sodium citrate pH 6.3 is prepared and 100 μL is added to each well except control wells. After incubation overnight at room temperature, a solution of NaCNBH₃ at 25 mg/mL in water is prepared and 20 μL aliquots are added to each well. After incubation for 4 hr at room temperature, the microplate is washed 3 times with 300 μL of water. The microplates aree then washed 3 times with 300 μL of 2.5 M NaCl in 10 mM Tris pH 7.5 and then washed 3 times in 300 μL of water. The microplate is then ready to be used.

A solution containing 20 mg/mL MMAC dissolved in 0.5% w/v pyridine in DMSO is prepared and 100 μL is added to each well. The plate is sealed and the plate incubated at room temperature for 30 mins. A solution of adipic dihydrazide at 10 mg/mL in 10 mM sodium phosphate pH 6.5 is prepared and 100 μL is added to each well. After 2 hr at room temperature, the wells are washed 3 times with 300 μL of water. After washing a solution of 5 mg/mL heparin in 0.5 M sodium citrate pH 6.3 is prepared and 100 μL is added to each well except control wells. After incubation overnight at room temperature, a solution of NaCNBH₃ at 25 mg/mL in water is prepared and 20 μL aliquots are added to each well. After incubation for 4 hr at room temperature, the microplate is washed 3 times with 300 μL of water. The microplates are then washed 3 times with 300 μL of 2.5 M NaCl in 10 mM Tris pH 7.5 and then washed 3 times in 300 μL of water. The microplate is then used in the lactoferrin ELISA described in Example 5. This demonstrate that effective protein binding surfaces can be generated with these intermediate polymers.

EXAMPLE 8 Multi-analyte assays

As heparin and other GAGs capture a wide variety of analytes, the ELISA system disclosed herein may be adapted readily to the "one-pot", simultaneous detection of two or more analytes. In traditional ELISA systems, multiple analyte detection usually requires multiple sample application to different plates.

A plate is blocked with 2% w/v BSA in PBS/0.05% v/v Tween for 1 hr at room temperature and then washed with 300 μL of wash buffer [PBS/0.05% v/v Tween]. Mixed standards containing both lactoferrin (0-100 pmol/100 μL) and IL-2 (0-100 pmol/100 μL) are prepared in blocking buffer [2% v/v BSA in PBS/0.05% v/v Tween]. 100 μL aliquots were then added to each well of a microplate prepared in Example 4 as required. The microplate was then covered and incubated at room temperature for 1 hr. After washing the microplate 3 times with 300 μL of wash buffer [PBS/0.05% v/v Tween] 100 μL of a solution containing both Rabbit anti-human lactoferrin polyclonal ( /₅ooo dilution in blocking buffer) and mouse anti-human IL-2 (7₂₅oo dilution in blocking buffer) polyclonal antibody was added into each well. The microplate was then incubated, covered at room temperature for 1 hr. The microplate was then washed 3 times with wash buffer and 100 μL of a solution containing a mixture of Eu labeled anti-rabbit Ig (7₂ooo dilution in blocking buffer) and Sm-labelled anti-mouse Ig (7₂ooo) dilution in blocking buffer was added and the microplate was covered and incubated at room temperature for 1 hr. The microplate was then washed 3 times with 300 μL of wash buffer. Wallac enhancement solution (200 μL/well) was then added, the plate shaken for 30 min and the europium and samarium time-resolved fluorescence signals measured using a Victor 1420 plate reader (Wallac) according to the manufacturer's instructions.

The example illustrates that due to the broad specificity of the GAG, two analytes may be assayed simultaneously in the same well of a microtitre plate. Moreover, the presence of one analyte does not inhibit the second analyte.

EXAMPLE 9 Heparinized magnetic beads

Modern automated, clinical immunoassay analysers use magnetic particle technology in preference to microplates. Magnetic beads afford faster assays than plate based systems due to enhanced mass transfer characteristics that enable incubations to be reduced from 1 hr to less than 15 min. The auto analyzers are routinely used for cell or protein capture.

Amine-surface polystyrene magnetic microspheres (Spherotech) of 4 μm diameter (2 mL at 2.5% w/v, 0.05 g) were washed according to the manufacturer's instructions using magnetic separation. The beads were re-suspended in 5 mL of a solution of partially oxidized dextran at 40 mg/mL in 0.2 M sodium phosphate pH 6.3 was added and 25 mg of NaCNBH was added. The suspension was shaken at room temperature for 4 hr before washing 3 times with 5 mL of 10 mM sodium phosphate pH 6.5 according to the manufacturer's instructions. The beads were re-suspended in 5 mL of a solution of adipic dihydrazide at 10 mg/mL in 10 mM sodium phosphate pH 6.5 and shaken for 3 hr at room temperature. The beads were washed 3 times with 5 mL of 10 mM sodium phosphate pH 6.5. The beads were re-suspended in a solution of 5 mg/mL heparin in 0.5 M sodium citrate pH 6.3 and shaken overnight at room temperature. 25 mg of NaCNBH₃ was added and shaking continued for a further 4 hr. The beads were washed 3 times with 5 mL of PBS. The beads were then washed 3 times with 5 mL of 2.5 M NaCl in 10 mM Tris pH 7.5 and then washed 3 times with 5 mL of PBS. The beads were re-suspended in 2 mL of PBS/Tween and stored at 4°C until used. Another aliquot of beads were treated similarly, although heparin was not added to the 0.5 M sodium citrate pH 6.3 buffer.

A third aliquot of beads were washed and then re-suspended in a solution of 5 mg/mL heparin in 0.5 M sodium citrate pH 6.3 and shaken overnight at room temperature. 25 mg of NaCNBH₃ was added and shaking continued for a further 4 hr. The beads were washed with 3 times with 5 mL of PBS. The beads were then washed 3 times with 5 mL of 2.5 M NaCl in 10 mM Tris pH 7.5 and then washed 3 times with 5 mL of PBS. The beads were re-suspended in 5mL ice-cold saturated NaHCO₃ and 200 μL of acetic anhydride added. The beads were shaken for 15 min at room temperature and the beads magnetically separated. The supernatant was withdrawn and a further 5 mL of ice-cold saturated NaHCO and 200 μL of acetic anhydride added. The beads were re-suspended in 5 mL ice- cold saturated NaHCO₃ and 200 μL of acetic anhydride added. The beads were shaken for 15 min at room temperature and the beads magnetically separated. This procedure was repeated a third time. The beads were re-suspended in 2 mL of PBS/Tween and stored at 4°C until use. This third aliquot of heparinized beads were referred to as directly coupled heparin beads (i.e. no intermediate polymer layer). In addition, a fourth aliquot of beads were treated to the three rounds of NaHCO₃/acetic anhydride only and served as control beads for the directly coupled beads.

The stock solutions of heparinized beads were diluted 10 fold in PBS/Tween and 50 μL of the resulting suspension aliquoted into wells of a microtitre plate. The lactoferrin ELISA described in Example 5 was conducted with the modification that incubations were stopped after 30 min and a magnetic separation plate (Spherotech) was used for washing. The beads were re-suspended between each washing step. The results demonstrate a linear response between the amount of lactoferrin added and the signal developed. Moreover, the development of signal is greater in the heparinized wells than in the control (non- heparinized) wells indicative of a selective binding of the lactoferrin by the polymer GAG complex. In contrast, the directly coupled beads showed only marginally more selective binding of the lactoferrin than the relevant control beads.

Figure 9 shows binding of avidin-FITC to heparinized magnetic beads and control beads. Binding was conducted in 20 mM Bistris pH 5.5 containing 150 mM NaCl and 0.05% v/v Tween 20 (binding buffer). Avidin-FITC was prepared at the specified concentrations in binding buffer containing 0.2% w/v BSA and 100 μL contacted with the beads for 15 minutes. After washing (3 x 200 μL binding buffer), bound avidin-FITC was resolubihzed in 200 μL 10 mM Tris pH 8.5 containing 2 M NaCl and the fluorescence measured in a plate reader. Eπor bars represent a single standard deviation from three replicates. The amount of beads in each well was 50 μL of a 0.025% (w/v) suspension of 4 μm beads with a calculated surface area of 0.3 cm². The beads were blocked with 2% w/v BSA in binding buffer prior to the binding experiment.

A second set of beads generated according to the same method, yielded identical results (Figure 10).

Figure 11 shows inhibition of binding of avidin-FITC to heparinized magnetic beads by exogenous soluble heparin. Binding to control beads is also shown. Binding was conducted in 20 mM Bistris pH 5.5 containing 150 mM NaCl and 0.05% v/v Tween 20 (binding buffer). Avidin-FITC (300 nM) was incubated with known amounts of heparin for 15 minutes prior to aliquoting 100 μL into microtitre plate wells containing the magnetic beads. Binding was conducted in 20 mM Bistris pH 5.5 containing 150 mM NaCl. After washing, bound avidin-FITC was resolubihzed in 200 μL 10 mM Tris pH 8.5 containing 2 M NaCl and the fluorescence measured in a plate reader. Error bars represent a single standard deviation from three replicates. The amount of beads in each well was 50 μL of a 0.025% (w/v) suspension of 4 μm beads with a calculated surface area of 0.3 cm . The beads were blocked with 2% w/v BSA in binding buffer prior to the binding experiment.

Yet another set of beads was prepared, as described. This set was generated by a third operator. These beads also gave good assay performance, as may be seen in Figure 12.

Figure 13 shows binding of europium labelled lactoferrin (Eu-DTPA-Lactoferin) to heparinized magnetic beads and control beads. Binding was conducted in 10 mM Tris pH 7.4 containing 150 mM NaCl and 0.05% v/v Tween 20 (binding buffer). Eu-DTPA- Lactoferin was prepared at the specified concentrations in binding buffer containing 0.2% w/v BSA and 100 μL contacted with the beads for 15 minutes. After washing (3 x 200 μL binding buffer), the fluorescence signal was developed using DELFIA enhancement solution (Wallac) in accordance with the manufacturer's directions. The amount of beads in each well was 50 μL of a 0.025% (w/v) suspension of 4 μm beads with a calculated surface area of 0.3 cm². The beads were blocked with 2% w/v BSA in binding buffer prior to the binding experiment.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds refeπed to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. BIBLIOGRAPHY

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Claims

1. A solid support comprising glycosaminoglycan (GAG) molecules immobilized to all or discrete regions of the solid support wherein said GAG molecules are capable of acting as binding partners for an agent.

2. The solid support of Claim 1, wherein the GAG molecules are selected from the list consisting of heparin, heparan sulfate, condroitin sulfate, keratan sulfate and hyaluronic acid or a fraction thereof.

3. The solid support of Claim 2, wherein the GAG is heparin or a fraction thereof.

4. The solid support of Claim 1, wherein the agent is selected from the list consisting of polypeptides, lipids and carbohydrates.

5. The solid support of Claim 4, wherein the agent is naturally occurring in a cell or virus.

6. The solid support of Claim 5, wherein the agent is a receptor on or in said cell or virus.

7. The solid support of Claim 1, wherein the GAG molecules are immobilized to the solid support via an intermediate polymer.

8. The solid support of Claim 7, wherein the intermediate polymer is selected from the list consisting of dextran and polyethylene glycol.

9. A capture device for an agent, said device comprising a solid support having GAG molecules immobilized to all or discrete regions thereof wherein said GAG molecules are capable of interacting with said agent.

10. The capture device of Claim 9, wherein the GAG molecules are selected from the list consisting of heparin, heparan sulfate, condroitin sulfate, keratan sulfate and hyaluronic acid or a fraction thereof.

11. The capture device of Claim 10, wherein the GAG is heparin or a fraction thereof.

12. The capture device of Claim 9, wherein the agent is selected from the list consisting of polypeptides, lipids and carbohydrates.

13. The capture device of Claim 12, wherein the agent is naturally occurring in a cell or virus.

14. The capture device of Claim 13, wherein the agent is a receptor on or in said cell or virus.

15. The capture device of Claim 9, wherein the GAG molecules are immobilized to the solid support via an intermediate polymer.

16. The capture device of Claim 15, wherein the intermediate polymer is selected from the list consisting of dextran and polyethylene glycol.

17. The capture device of Claim 9, wherein the said device is adapted to capture a cell or virus.

18. The capture device of Claim 17, wherein the captured cell or virus is subsequently identified.

19. The capture device of Claim 18, wherein the said cell or virus is eluted off prior to identification.

20. Use of a GAG in the manufacture of a capture device for capturing a biological or chemical molecule or a cell or a virus.

21. A method of detecting an agent, said method comprising capturing said agent on the solid support of Claim 1 and then subjecting said agent to detection means.

22. The method of Claim 21, wherein the solid support comprises GAG molecules immobilized to all or discrete regions thereof.

23. The method of Claim 22, wherein the GAG molecules are selected from the list consisting of heparin, heparan sulfate, condroitin sulfate, keratan sulfate and hyaluronic acid or a fraction thereof.

24. The method of Claim 23, wherein the GAG is heparin or a fraction thereof.

25. The method of Claim 21, wherein the agent is selected from the list consisting of polypeptides, lipids and carbohydrates.

26. The method of Claim 25, wherein the agent is naturally occurring in a cell or virus.

27. The method of Claim 26, wherein the agent is a receptors on or in said cell or virus.

28. The method of Claim 21, wherein the GAG molecules are immobilized to the solid support via an intermediate polymer.

29. The method of Claim 28, wherein the intermediate polymer is selected from the list consisting of dextran and polyethylene glycol.

30. The method of any one of Claims 21 to 29, wherein the detection means are biochemical or immunological.

31. The method of Claim 30, wherein the detection means is immunological.

32. A kit useful for capturing and optionally identifying or detecting a particular agent in a sample, said kit comprising a capture device for biological or chemical molecules, wherein the capture device comprises the solid support of any one of Claims 1 to 8.

33. A data processing means for assessing the interaction between an immobilized carbohydrate moiety and its binding partner wherein said data processing means executes the steps of (in any order) :-

(i) optionally identifying the position of discrete spots of binding partners immobilized to a solid surface via the carbohydrate moiety;

(ii) identifying interaction between carbohydrate moieties and the binding partners in (i);

(iii) analyzing the data obtained in (ii) to identify the immobilized binding partners;

(iv) optionally analyzing the data to identify agents in a sample; and

(v) optionally controlled removal of the solid phase to a culture or maintenance medium.