GB2286673A

GB2286673A - Tagging substances or items

Info

Publication number: GB2286673A
Application number: GB9425923A
Authority: GB
Inventors: Ramadan Arbi Abuknesha
Original assignee: GEC Marconi Ltd; Marconi Co Ltd
Current assignee: BAE Systems Electronics Ltd
Priority date: 1993-12-23
Filing date: 1994-12-22
Publication date: 1995-08-23
Also published as: GB2286672A; JPH09509372A; FR2714474A1; WO1995017667A1; WO1995017669A1; JPH07203987A; GB9326277D0; GB2286044A; GB9425924D0; DE4446042A1; JPH09506972A; WO1995017670A1; GB9425923D0; GB9425925D0; GB9425922D0; JPH09508199A; GB2286674A

Description

i 2286673 Labelling The present invention relates to labelling of a

substance or an item and finds an application in labelling of a substance or an item for the purpose of identifying said substance or item.

According to one aspect of the present invention there is provided a method suitable for use in labelling of a substance or an item which method includes providing a substance or an item with an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

According to a further aspect of the present invention there is provided a method suitable for use in identifying a substance or an item, which method includes providing a substance or an item with an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule and performing a detection step to establish the presence or absence of an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

By way of example, the present invention may find application in covert labelling of a substance or an item.

It is to be understood that, in accordance with the present invention, a label may be an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

According to a further aspect of the present invention there is provided a method suitable for identifying a substance or an item which method includes performing a detection step to establish the presence or absence of an enzyme molecule, or of a fragment of an enzyme molecule, or of a binder for an enzyme molecule, or of a binder for a fragment of an enzyme molecule.

By way of example, it is to be understood that an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, in accordance with the immediately preceding aspect of the invention, may be, for example, "selected" in the sense that one, or more, of them may be a label.

It will be appreciated that an antibody to an enzyme molecule is a binder for an enzyme molecule and that an antibody to a fragment of an enzyme molecule is a binder for a fragment of an enzyme molecule. Also, it will be appreciated that an enzyme molecule and a binder therefor may be considered to be specific binding partners and that a fragment of an enzyme molecule and a binder therefor may be considered to be specific binding partners.

Many enzyme molecules and fragments of enzyme molecules are available. By way of example, any suitable enzyme molecule, fragment of an enzyme molecule, binder for an enzyme molecule, or binder for a fragment of an enzyme molecule, may be utilised in accordance with the present invention.

Alkaline phosphatase and horseradish peroxidase are examples of enzyme molecules which may find application in accordance with the present invention. Antibodies to alkaline phosphatase and to horseradish peroxidase are 41 0 1 examples of binders which may find application in accordance with the present invention.

It is to be noted that, for example, enzyme molecules and fragments of enzyme molecules may be more stable (e.g. less susceptible to unacceptable change) when bound with their respective binders (antibodies).

Thus, for example, it may be advantageous in some circumstances to provide a substance or an item with an enzyme molecule bound with a binder of an enzyme molecule, or with a fragment of an enzyme molecule bound with a binder for the fragment of an enzyme molecule, since bound enzyme molecules or bound fragments of enzyme molecules may be more stable than unbound forms, (for example, to processing and storage).

Detection of an enzyme molecule bound with its binder, or a fragment of an enzyme molecule bound with its binder, may be effected, for example, enzymologically.

Accordingly, for example, in this Specification, where the context allows, "enzyme molecule" may indicate an enzyme molecule as such, or may indicate an enzyme molecule bound with a specific binder (antibody) for the enzyme molecule, and "fragment of an enzyme molecule" may indicate a fragment of an enzyme molecule as such, or may indicate a fragment of an enzyme molecule bound with a specific binder (antibody) for the fragment of an enzyme molecule.

In accordance with the present invention a substance or an item may be provided with a label, said label being an enzyme molecule, a fragment of an enzyme molecule, a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, in any suitable manner.

Thus, for example, where a substance is a fluid, such as a liquid, a label may be introduced to the fluid by mixing therein. By way of further example, where the substance is a paste or a solid (e.g. a chemical product) i i / 1 a label may be introduced during manufacture of the paste or solid.

Thus for example, a label which comprises an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be incorporated into a substance.

Where an item is to be provided with a label the label may be provided in any suitable manner (e.g. by application to the item). Thus, for example, an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be applied to an item in any suitable manner.

By way of example, a label may be incorporated into a composition for application to an item. A composition may be, for example, an ink, or ink medium, or i. nk composition, or a paint composition for application to an item by suitable means. Thus, for example, a label which comprises an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be incorporated into an ink, or ink medium, or ink composition, or a paint composition suitable for application to an item. The ink or ink medium may be, for example, an invisible ink or invisible ink medium, or invisible ink composition.

In accordance with a further aspect of the present invention there is provided a composition which includes an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule. The composition may be, for example, an ink composition which includes an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

R, It will be appreciated that constituents of a composition may be chosen such that there is no unacceptable interference between a label and other constitutions of a composition such as to cause unacceptable deterioration in properties of the composition; also constituents of the composition may be such as to cause no unacceptable deterioration in properties of the label. Also the label may be such that it may still be detected after application to an item, and after exposure of the item to such conditions as the item may encounter (e.g. in shipping and/or storage).

It is to be understood that the present invention may be utilised to provide a substance or an item with a label in such a manner that the nature of the label is not known to unauthorised persons; also it may be arranged that the amount of a label provided is unknown to unauthorised persons. If desired it may be arranged that the existence of a label may be unknown to unauthorised persons. Where an item is labelled it may be arranged that a location of a label is unknown to an unauthorised person.

It is also to be understood that the nature of a label may only need to be known by essential staff since knowledge of the nature of a label is not necessarily required by staff involved in providing a substance or an item with a label, nor by staff involved in quality control testing, nor by an individual in conducting a test to see if a particular label is present (since test reagents need not be identified other than by, for example, a reference code).

In accordance with the present invention, a substance or an item may be provided with a label, comprising an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, in any suitable concentration. A suitable concentration may be such that, for example, the amount of label present would -6 render it difficult, or substantially impossible, for an unauthorised person to identify the label by "conventional" methods (e.g. chemico- physical methods such as chromatography) but such that the amount of label used would be sufficient to facilitate detection by an authorised person using authorised detection reagents.

By way of example, a composition, suitable for use in labelling of a substance or an item, may contain a label at a concentration of 0.1-80, 000 gg/1.

The present invention may find application in, for example, labelling for any suitable purpose; thus, for example, labelling may be carried out in order that a substance or an item may be identified so as to authenticate the substance or item.

Thus, for example, the present invention may find application in identifying a substance or an item for the purpose of distinguishing a genuine substance or a genuine item from a counterfeit substance or a counterfeit item. By way of further example, the present invention may find application in identifying a substance or an item for the purpose of monitoring movement of a substance or an item (e.g. in a chain or network); thus, for example, the movement of a substance or an item may be monitored for the purpose of monitoring the performance of a distribution chain or network, or, for example, the movement of a substance or an item may be monitored for the purposes of detecting diversion of goods (e.g. by an intermediate agent in a marketing chain or network).

The present invention may find application in, for example, labelling of any suitable substance or item. It is to be understood that "labelling" may also be considered to be "tagging" and that a "label" may also be considered a "tag".

A substance which it is desired to label may be, for example, any suitable substance (e.g. a solid, a liquid or any other suitable substance).

An item which it is desired to label may be, for example, any suitable item. It is to be understood that in this Specification the term "item" embraces "article"; thus any suitable item or article may be labelled.

It will be appreciated that the present invention may find application, for example, in labelling of any matter whatsoever that is suitable, or may be rendered suitable for labelling, or anything whatsoever that is suitable, or may be rendered suitable, for labelling.

Thus, for example, "substance or item" as used in this Specification may be construed as embracing any matter whatsoever that is suitable, or may be rendered suitable, for labelling and anything whatsoever that is suitable, or may be rendered suitable, for labelling.

It will be appreciated that by providing a chosen substance or a chosen item with a selected label or labels it may be possible, for example, to distinguish that substance or item from other substances and items by testing to detect the selected label or labels. Thus, for example, the presence of a selected label or labels may indicate that a substance or item tested is the chosen substance or chosen item whereas the absence of such selected label or labels may indicate that a substance or item is not the chosen substance or chosen item.

Examples of substances and items which may be labelled are: perfume, bank notes, art work, documents of realisable value, fashion clothes, watches, electrical goods, books, passports, medicines, any high value goods (e.g. luxury goods), any high volume sales items, prestige high value articles, chemical formulations, meats and meat products (e.g. Kosher meats and foods), and packaging for various goods.

It is to be understood that the present invention may find application in, for example, labelling of a substance or an item in a manner which is aimed at inhibiting fraudulent sales of forgeries, unauthorised copies and bogus goods. Such labelling may be used, for example, to permit a genuine manufacturer to identify, unequivocally, a substance or an item produced by the manufacturer and distinguish such a substance or item from a non-genuine substance or item. This offers the possibility of a genuine manufacturer to seek to inhibit loss of revenue which may be caused by the presence of non-genuine substances or items in a given market. For example, manufacturers may be able to discover substances of items which, although not provided by them, bear their brand names andlor packaging. If desired the present invention may, for example, be utilised to label a substance or an item to indicate batch number and/or "best before" date. By way of example, a label may be applied to an article in any suitable arrangement; thus, for example, a composition (e.g. an ink) containing a label comprising an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be applied to an item in the form of a desired set of markings (e.g. numbers or letters or shapes or designs). Labelling of substances and items in accordance with the present invention may be effected, by way of example, by: (a) mixing a label with an ink or an ink vehicle composition (e.g. at 0.1-80,000 gg/1) and printing directly on to an item, or (b) applying a solution of a label (e.g. in water or in a water/organic solvent combination) directly to items, or dipping part of an area of an item into a solution of a label to attach (e.g. adsorb) the label to the item, or (d) mixing a label with colouring matter or paint-stuff for use with items or packaging, or (e) adding a label directly to solutions or formulations of chemical goods (e.g. medicines, paints, foods, etc.), or (f) mixing a label with an adhesive substance which may then be used to attach tags or paper to items, or (g) marking (e.g. during printing) paper certification which is to accompany goods, or (h) applying an ink vehicle composition to an item and treating (e.g. by drying) the ink vehicle composition to form a firmly attached hydrophobic layer and applying a label (e.g. in solution or suspension) to the hydrophobic layer so that label may become associated with the layer (e.g. by adsorption) and treating (e.g. by drying) to provide a label on the item, or (i) activating (e.g. by periodate oxidation) a component of an ink vehicle composition to provide reactive sites to which a label may covalently bond, applying the ink vehicle composition to an item aAd treating the ink composition (e.g. by drying) to form a firmly attached layer, and introducing label to the layer so as to attach label to the layer by covalent bonding, or (j) applying an ink vehicle composition to an item and treating (e.g. by drying) the ink vehicle composition to form a firmly attached layer, activating (e.g. by periodate oxidation) the layer to generate reactive sites thereon to which a label may covalently bond in order to effect attachment of label to the layer and introducing label to the layer to attach label to the layer by covalent bonding. It will be appreciated that a label as immediately hereinbefore disclosed in (a) to (j) may comprise an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

In accordance with the present invention an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be detected in any suitable manner.

By way of example, an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be detected, in situ (e.g. whilst still attached to an item).

Alternatively, by way of example, an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule may be retrieved from a substance or item and subjected to detection.

Thus, for example, an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be subjected to detection in situ (e.g. whilst still attached to an item), or alternatively, may be retrieved (e.g. by extraction by a suitable method) from a substance or item and subjected to detection.

Retrieval of a label with is an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, nay be effected in any suitable manner.

For example, where such a label is to be retrieved from a liquid, the liquid may be evaporated to leave a residue and the residue may be treated with a suitable liquid medium (e.g. a buffer solution) to give a liquid sample which may be subjected to detection.

By way of further example, where a label is to be retrieved from a substance which is not a liquid, the substance may be dissolved or otherwise treated to give a liquid containing a label, which liquid may then be subjected to detection or trea4--e,--5 as immediately hereinbefore disclosed by evaporation and treatment with A a liquid medium to give a liquid sample which may be subjected to detection.

By way of further example, a label may be extracted from an item (e.g. to give a liquid containing the label) and subjected to detection in any suitable manner.

By way of example, there may be circumstances where solvent extraction may not be applicable in the recovery of enzymatically active materials. However, where solvent extraction is applicable, solvent may be removed after extraction and a suitable buffer may be added (e.g. an immunoassay buffer such as PBS pH 7.4).

It will be appreciated that any solvent or solvent system used may be chosen such as to be appropriate to the label to be detected. Thus, for example, a solvent or a solvent system may be chosen such that the use thereof does not cause unacceptable changes in the label.

Where an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, has been retrieved (e.g. extracted) it may be immobilised in any suitable manner so as to permit detection (e.g. by a specific protein binding assay (e.g. an immunoassay procedure) or by any other suitable detection procedure (e.g. by an enzymological method)).

Thus, for example, a non-specific chemical method may be used to immobilise an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule on a suitable support material (e.g. on nitrocellulose paper); for example, an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be retrieved, provided in a suitable liquid medium (e.g. a buffer solution), and the enzyme molecule, or fragment of enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be immobilised on a suitable support by means of a non- specific chemical method.

Alternatively, by way of example, an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be retrieved, provided in a suitable liquid medium (e.g. a buffer solution), and then an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be immobilised on a suitable support material (e.g. polystyrene) by nonspecific adsorption.

By way of further example, specific interaction (e.g. specific binding) nay be used to immobilise a retrieved enzyme molecule, or fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule. Thus, for example, a specific reaction partner (being an antibody) for an enzyme molecule, or a specific reaction partner (being an antibody) for a fragment of an enzyme molecule, may be used to effect attachment to a support material by use of specific interaction with a corresponding enzyme molecule, or fragment of an enzyme molecule. For example, a binder (being an antibody) for an enzyme molecule, or a binder (being an antibody) for a fragment of an enzyme molecule may be utilised to bring about immobilisation of an enzyme molecule, or a fragment of an enzyme molecule on a support material.

Alternatively, by way of example, an enzyme molecule, or a fragment of an enzyme molecule, may be used to effect attachment to a support material of a binder (being an antibody to an enzyme molecule, or a fragment of an enzyme molecule may be used to effect attachment to a support material of a binder (being an antibody to a fragment of an enzyme molecule). For example, an enzyme molecule, or a fragment of an enzyme molecule may be utilised to bring about immobilisation, on a support material, of a binder (being an antibody) for an enzyme molecule or a binder (being an antibody) for a fragment of an enzyme molecule.

It will be appreciated that a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, or an enzyme molecule, or a fragment f or an enzyme molecule, may be used to bring about immobilisation of respectively an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, in any convenient manner.

Thus. for example, a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, or an enzyme molecule, or a fragment of an enzyme molecule, may be associated with a support material before or after undergoing specific interaction respectively with an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

A binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, or an enzyme molecule, or a fragment of an enzyme molecule, may be directly associated with a support material (e.g. by covalent linkage) or may be indirectly associated with a support material via another species and links (e.g. specific or non-specific in nature).

Thus, for example, a reaction partner being a binder, may be linked to a support material by a second binder (being an antibody) which second binder is binder for the reaction partner; in this configuration the second binder may be attached to a support material in any convenient manner and at any convenient time and may be allowed to undergo specific binding with the reaction partner before or after the reaction partner has undergone specific interaction with an enzyme molecule or with a fragment of an enzyme molecule.

Alternatively, by way of example, a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, or an enzyme molecule, or a fragment of an enzyme molecule, may be arranged to be associated with a support material (before or after reaction with an enzyme molecule or with a fragment of an enzyme molecule, or with a binder for an enzyme molecule, or with a binder for a fragment of an enzyme molecule) by means of an auxiliary binder or an auxiliary ligand system (e.g. by means of a further ligand-binder system).

Once an enzyme molecule or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, has been immobilised on a support material it may be detected in any suitable way such as those known in the immunological field and in the non-immunological field. Thus, for example, a suitable binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, or an enzyme molecule, or a fragment of an enzyme molecule, may be used which binder for an enzyme molecule, binder for a fragment of an enzyme molecule, enzyme molecule, or fragment of enzyme molecule, may be arranged to be associated with a tracer species capable of giving rise to a detectable signal.

By way of example any suitable tracer species may be utilised as desired in accordance with the present invention; examples of such tracer species are enzymes, fluorescent compounds, chemiluminescent components, bioluminescent compounds, radioisotopes and dyes.

A signal from a tracer species may be determined, for example, by any suitable chemical or biochemical method or in any suitable manner.

By way of further example, a liquid or liquid sample containing a label may be subjected to detection by means of providing a thin film of the liquid or liquid sample on a suitable solid surface and drying this film to provide on the solid surface a thin layer of material to be subjected to detection.

The solid surface may be, for example, a glass microscope slide, a glass rod or a petri dish. Where the solid surface is in a suitable form (e.g. a microscope slide or a glass rod) a thin film of liquid or liquid sample may be provided thereon by dipping the solid surface into the liquid or liquid sample.

A thin layer of material on a suitable solid surface obtainable as immediately hereinbefore disclosed may be subjected to detection in any suitable manner, for example, by adding identification reagents or by any suitable method (e.g. an assay method or an enzymological method).

By way of further example, a label in a liquid or liquid sample may be detected by adding suitable identification reagents to the liquid or liquid sample.

Detection in situ of an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be carried out using any suitable procedure, for example procedures known in the immunological field or nonimmunological protein binding field; it will be appreciated that in an in situ procedure, an item itself may act as a support material in a detection procedure.

By way of example, in situ detection of an enzyme molecule, or of a fragment of an enzyme molecule, or of a binder for an enzyme molecule, or of a binder for a fragment of an enzyme molecule, may be effected using a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, or an enzyme molecule, or a fragment of an enzyme molecule, which may be associated with a suitable tracer species. For example, a tracer species may be detected in Situ.

An enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be detected, for example, in any suitable form of assay.

In one form of assay a non-competitive sandwich assay configuration may be used (e.g. after retrieval of an enzyme molecule or a fragment of an enzyme molecule).

Thus, for example, an antibody for an enzyme molecule, or for a fragment of an enzyme molecule (e.g. which antibody is or may be attached to a support material) may be used to attach an enzyme molecule or a fragment of an enzyme molecule to a support material and another antibody (which in turn may be directly or indirectly associated with a tracer species) to the enzyme molecule, or to the fragment of an enzyme molecule, may be utilised to facilitate detection of the enzyme molecule or the f ragment of an enzyme molecule.

Subsequently, if desired, the antibody may be denatured and the enzyme molecule or fragment of an enzyme molecule recovered for use in a further (e.g. competitive) assay which may be used to confirm results of the noncompetitive assay.

It will be appreciated that the possibility of conducting more than oneassay on a given sample may offer an advantage in terms of confirming assay results and improving the reliability of results.

Prom the foregoing disclosure it will be appreciated that even if enzyme molecules, or fragments of enzyme molecules, may, for example, become denatured by being introduced to a substance or an item, such enzyme molecules, or fragments of enzyme molecules, may still be detectable even though they have lost some or all of their enzymatic activity.

Thus, for example, denatured enzyme molecules or fragments of enzymenolecules-may be recognised by an antibody which also recognises whole, active enzyme molecules.

This is possible since structural specificity may be independent of envna-L-_ic activity.

Thus, for example, a competitive immunoassay may be conducted in which a label, comprising an enzyme molecule or fragment of.an enzyme molecule. competes with active enzyme molecules (which may be used as a tracer species) for binding with an antibody (which may be provided on a support material).

Although reference was hereinbefore made to competitive immunoassay in relation to detection of an enzyme molecule or a fragment of an enzyme molecule, it will be appreciated that an enzyme molecule or a fragment of an enzyme molecule may be detected in any suitable manner, thus for example, detection may be by means of a non-competitive immunoassay.

By way of further example, a label which is an enzyme molecule (e.g. a denatured enzyme molecule) or a fragment of an enzyme molecule may be detected by use of an antibody or an antibody fragment (i.e. (Fab)2), which has two binding sites. Thus, for example, it may be arranged that one binding site binds with the label (said label being an enzyme molecule (e.g. a denatured enzyme molecule) or a fragment of an enzyme molecule) and that the other binding site binds with an enzymatically active enzyme molecule, said enzymatically active enzyme molecule being of the same type of enzyme molecule as that used to provide the label. The enzymatically active enzyme molecule may then be used, for example, to detect the label. The label may thus be detected in situ by use of the antibody or antibody fragment having two binding sites and the enzymatically active enzyme molecule, or the label may be detected by being retrieved and associated with a support material before the antibody or antibody fragment having two binding sites and the enzymatically active enzyme molecule are used. The enzymatically active enzyme molecule may be detected in any suitable manner (e.g. by enzymological methods as hereinafter disclosed).

It will be appreciated that an enzyme moleculeantibodyenzyme chain or a (fragment of an enzyme molecule)-antibody-enzyme chain which may be formed by use of an antibody having two binding sites may be utilised, for example, to extend sensitivity of detection. Also, it may be possible, for example, to utilise an enzyme nolecule-antibody-ligand- antibodyligand chain or a (fragment of an enzyme molecule)antibody-ligandantibody-ligand chain.

It will also be appreciated that if an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, is used as a label, any suitable immunological detection method may be used to detect such a label, either in sitv or after retrieval.

According to a further aspect of the present invention there is provided a test-kit suitable for use in identifying a substance or an item, which test-kit includes a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, or an enzyme molecule, or a fragment of an enzyme molecule.

According to a further aspect of the present invention there is provided a composition suitable for use in labelling of a substance or an item which composition includes an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

By way of example, a composition in accordance with the immediately preceding aspect of the present invention may be an ink composition, or an ink vehicle composition, or paint composition.

An ink vehicle composition may be a composition which contains a mixture of substances normally found in ink compositions except for a colourant dye. An ink vehicle composition may have a dye added to it in order to make a coloured ink.

-19 In accordance with the present invention there is provided a combination comprising a label associated with a solubilising agent, said label being an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

The solubilising agent may be a surfactant.

Thus, in one embodiment of the present invention there is provided a combination comprising a label associated with a surfactant, said label being an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

An enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule or a binder for a fragment of an enzyme molecule, may be associated with a surfactant in any suitable manner. For example, a reactive site or sites of a surfactant may be conjugated with an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, or any appropriate combination thereof.

It will be appreciated that conjugation may be by covalent bonding.

Polyethylene glycol is an example of a surfactant with which an enzyme molecule, a fragment of an enzyme molecule, a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be associated (e.g. conjugated).

A combination comprising a label in accordance with the present invention associated with a surfactant may be soluble in both aqueous and organic liquids. These may offer advantages, for example, when seeking to introduce a label to an ink which has organic components (e.g. an organic solvent).

It will be appreciated that a combination of a label associated with a surfactant also may find application in labelling using water-based solvents.

In accordance with the present invention there is also provided a combination comprising a label associated with an insolubilising medium, said label being an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

The insolubilising medium may be, for example, a medium which is of a type, and of sufficient particle size, such that it does not dissolve in chosen organic or aqueous solvents whereby label associated with the insolubilising medium is also rendered substantially insoluble in chosen organic or aqueous solvents.

The insolubilising medium may be, for example, inicroparticles of micron and sub-micron size (e.g. latex particles, polystyrene microparticles, microcellulose particles and glass powder particles). Microparticles of micron and sub-micron size are commercially available.

By way of example, any suitable means may be utilised to associate a label with an insolubilising medium.

Thus, for example, any suitable method of attachment may be utilised, for example, those known in the art for attaching species (e.g. biochemical species) to a microparticle.

Examples of attachment are adsorption and covalent bonding.

Thus, for example, a combination comprising a label associated with an insolubilising medium may comprise a label attached to an insolubilising medium comprising a microparticle or microparticles.

By way of example, a combination of a label in accordance with the present invention, and an insclubilising medium, which combination may be substantially insoluble in chosen organic or aqueous solvents, may prove advantageous in certain circumstances; for example, such a combination may be formed as a homogeneous suspension in organic or aqueous media of high viscosity (e.g. 0.8% hydroxylpropylmethyl cellulose solution) and such a suspension may offer advantages in, for example, assisting providing substantially uniform concentrations of an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, when applied as a label.

In view of the foregoing it will be appreciated that, for example, the present invention may provide, inter alla, for the labelling or tagging of a substance or an item by means of a label which is biodetectable.

Where enzyme molecules, or fragments of enzyme molecules, are not denatured to an unacceptable extent by being introduced to a substance or an item, enzyme molecules, or fragments of enzyme molecules, may be detected (e.g. in situ or after retrieval) by use of a suitable procedure and a suitable substrate.

Reference has hereinbefore been made to immunological detection of a label in accordance with the present invention.

Also, from the foregoing disclosure, it will be appreciated that where an enzyme molecule, or a fragment of an enzyme molecule, has suitable enzymatic activity, any suitable enzymological method may be utilised in detection in solution or on a solid surface as appropriate.

From the foregoing disclosure it will be appreciated that an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be detected, for example, by any appropriate immunological method and/or by any appropriate enzymological method.

Thus, for example, an enzyme molecule which has suitable enzymatic activity, or a fragment of an enzyme molecule which has suitable enzymatic activity, may be detected (e.g. whether gn a colid surface or in solution) by methods such as, for example:

(i) direct detection by application of a suitable substrate in a suitable buffer system. (The substrate may be synthetic or natural. Synthetic substrates may be chromogenic (capable of giving soluble or insoluble coloured products) or fluorogenic (capable of giving soluble or insoluble fluorescent products)); detection by the application of a substrate plus chromogenic substance mixture. (Action on the substrate produces product (intermediate) which acts on the chromogenic substance converting it to a coloured soluble or insoluble product); detection by coupled enzyme reactions in which a second enzyme (or enzymes) act on a product from a first enzyme to produce a final detectable product (e.g. a soluble or insoluble coloured or fluorescent product); detection by enzyme cycling methods in which the product of a first enzyme acts as a cofactor for a second enzyme-substrate system. (The co-factor may be regenerated by a third enzyme system which co- factor enters the second enzyme system and so on. Enzyme cycling is a biochemical amplification method which may permit detection of very small amounts of a first enzyme.) It will be appreciated that where, for example, a bivalent antibody "bridges" between an immobilised enzyme and an active homologous enzyme molecule, the active enzyme molecule may be detected by any suitable method such as those immediately hereinbefore disclosed in (i) to (iv).

(ii) (iii) (iv) It will be appreciated that, by way of example, a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be detected (e.g. in situ or after retrieval) by means of an enzymological method using an appropriate and suitably enzymatic active enzyme molecule, or suitably enzymatically active fragment of an enzyme molecule.

According to a further aspect of the present invention there is provided a test-kit suitable for use in identifying a substance or an item, which test-kit includes selected reagents for enzymological detection of an enzyme molecule, or a fragment of an enzyme molecule, or of a binder for an enzyme molecule, or of a binder of a fragment for an enzyme molecule.

By way of example, enzyme molecules as such, or fragments of enzyme molecules as such, or a binder for an enzyme molecule as such, or a binder for a fragment of an enzyme molecule as such, may be used to label a substance or an item; alternatively, by way of further example, enzyme molecules, or fragments of enzyme molecules, or a binder for enzyme molecules, or a binder for fragments of enzyme molecules, may be attached to a carrier material.

If desired, by way of example, more than one type of enzyme molecule, or more than one type of fragment of an enzyme molecule, or more than one type of binder for an enzyme molecule, or more than one type of binder for a fragment of an enzyme molecule, or any suitable combination thereof, may be used to label a substance or an item.

By way of example, use of more than one type of enzyme molecule, or more than one type of fragment of an enzyme molecule, or more than one type of binder for an enzyme molecule, or more than one type of binder for a fragment of an enzyme molecule, or any suitable combination thereof may increase the difficulty which an unauthorised person may encounter in relation to ascertaining the presence and/or identity thereof andlor in relation to detection thereof.

By way of example, if desired, an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be used in combination with any other suitable identifiable species. Thus, an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be used to provide a first label and another label or labels may be provided by any other suitable identifiable species (e.g. a chemical species or a biochemical species or an entity which provides identifiable species).

It will be appreciated that an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, which may be detected, may be considered, for example, to be an identifiable species.

It is to be understood that where, for example, a fragment of an enzyme molecule is a sub-unit of an enzyme molecule, the fragment may be detectable by immunological methods. However, such a sub-unit may not, for example, be detectable by enzymological methods. However, for example, by addition of a further enzyme molecule subunit or sub-units, as appropriate, to the sub-unit the fragment may be rendered detectable by enzymological means.

Thus, addition of an enzyme molecule sub-unit or sub-units may form part of a method of detection.

It will also be appreciated that an enzyme molecule, or a fragment of an enzyme molecule, which has enzymatic activity, may be considered to be a catalytic species in that such an enzyme molecule, or fragment of an enzyme molecule, may catalyse a reaction.

By way of example, it may be noted that, optionally, for certain applications it nay be possible to arrange 1 for a label to become attached to a component of a composition (e.g. a component of an ink vehicle composition).

According to a further aspect of the present invention there is provided a labelled substance or a labelled item wherein the substance or item is labelled with a label which is an enzyme molecule, a fragment for an enzyme molecule, a binder f or an enzyme molecule, or a binder for a fragment of an enzyme molecule.

It will also be appreciated that a label may be detected qualitatively or quantitatively; it will be understood that a quantitative detection (which may be considered, for example, to be measurement) may be carried out in conjunction with a calibration curve.

Detection of a label may be effected by any suitable technique such as those known in the field, for example in the field of protein binding assay (e.g. immunological assay and non-immunological assay) and in the field of enzymology (e.g. substrate based systems).

By way of example, it will be appreciated that identifiable species used separately may also be detected in situ or after retrieval as is appropriate.

The term "antibody" as used in this Specification embraces whole antibody and antibody fragments such as Fab and (Fab)2 and, accordingly, the term "antibodies" as used herein embraces whole antibodies and antibody fragments as may be appropriate.

It will be appreciated that antibodies may be prepared as desired by any suitable method, for example those known for the raising of polyclonal or monoclonal antibodies.

Thus, for example, a binder comprising an antibody for an enzyme molecule or a binder comprising an antibody for a fragment of an enzyme molecule, may be prepared by raising antibodies respectively to an enzyme molecule, or a fragment of an enzyme molecule.

From the foregoing disclosure it will be understood that any suitable enzyme molecule, or fragment of an enzyme molecule, may find application in accordance with the present invention. If appropriate, by way of example, a thermophilic enzyme, or an enzyme which is active in organic media may find application in accordance with the present invention.

Where a support material is used in accordance with the present invention any suitable support material such as those known in the art may be utilised; examples of support materials are polystyrene (which may be used in any convenient form) and nitro-cellulose (which may be used in any convenient form).

The present invention may offer, for example, an advantage of utilising more than one method of detection (e.g. sequentially) in a given detection procedure; also the present invention may offer an advantage of high sensitivity of detection by use of a combination of an enzyme molecule, or a fragment of an enzyme molecule and a reaction partner therefor which combination has a high affinity (e.g. picogramme quantities may be detected).

From the foregoing disclosure it will be appreciated that, for example, an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may, or any combination thereof may be used alone to effect labelling. However, if desired, an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, may be used with other identifiable species (e.g. separate identifiable species or those which form part of an entity or entities).

Thus, if desired, for example, an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, in accordance with the present invention, may be used together with one or a p-lural-Jty or any combination of separate identifiable species such as, for example, a ligand (e.g. antigenic or non-antigenic), a binder (e.g. for an antigen ligand or for a non-antigenic ligand), a "masked" ligand, a nucleic acid, or a species capable of undergoing specific interaction with a nucleic acid, or with an entity which provides a plurality of identifiable species.

Reference may be made, for example, to co-pending application (Agent's Reference P/60090/HRF) in relation to entities which provide a plurality of identifiable species in connection with labelling. Reference may be made,for example, to co-pending Application (Agent's Reference P/60540/HRF) in relation to "masked" ligands in connection with labelling. Reference may be made, for example, to co-pending Application (Agent's Reference HRF/60541/HRF) in relation to ligands and binders in connection with labelling.

The present invention will now be further described, by way of example, as follows:

Example 1

The enzyme alkaline phosphatase (EC3.1.3.1) was introduced to wells of a microtitre plate in various quantities and subjected to enzymological detection.

Thus, various quantities of alkaline phosphatase (S.A. 2290 units/mg) in 100 Al samples of aqueous coating buffer solution (50 mM sodium tetraborate (pH 9.5) containing 0.5 mM MgC12) were added to wells of a microtitre plate and left at room temperature for 4 hr. The wells were then emptied and washed (3 times with 100 mM sodium bicarbonate containing 100 mM NaCl at 0.05%-' Tween 20).

Also to some wells samples containing no enzyme were added, thereby to provide "controls".

To carry out detection a synthetic alkaline phosphatase substrate solution was used. In this Example the synthetic alkaline phosphatase substrate solution was a solution of the disodium salt of p-nitrophenyl phosphate (pNPP) (6 mM) in 10% diethanalomine buffer (pH 9.8) containing 0.5 MM M9C12- Thus, to each of the wells of the washed microtitre plate 100 gl of the synthetic alkaline phosphatase substrate solution was added.

Yellow colour was seen to be developing within 3 to 4 minutes; Optical Density (OD) was read at 405 nm after 15 'minutes. The results are given in Table 1.

Table 1

Amount of alkaline phosphatase added Optical Density per well (ng) (OD) at 405 nm 3.00 3.00 12.5 2.56 6.25 1.91 3.12 1.65 1.56 1.12 0.78 0.61 NIL 0.12 Example 2

Alkaline phosphatase (EC3.1.3.1) in coating buffer solution (see Example 1) (125 nglml) was applied (250 g 1) to the bottom outer surfaces of polystyrene screw cap containers and allowed to air-dry at room temperature for 16 hours.

The surfaces were then washed to remove loosely attached material and allowed to dry. A synthetic alkaline phosphatase substrate solution was then applied and within a few minutes blue colour began to appear and an intense blue precipitate was formed. The synthetic alkaline phosphatase substrate solution used in this Example was a 0.5 mg/ml solution of the disodium salt of 5-bromo-4 chloro-3 indolyl phosphate (BCIP) in 10% diethanclamine buffer (pH 9.8) containing 0.5 mM MaCl., Example 3

In this Example enzymatic activity of denatured alkaline phosphatase was investigated.

Thus, various quantities of alkaline phosphatase (EC3.1.3.1) were introduced to the wells of a microtitre plate in a manner similar to that disclosed in Example 1. Methanol (100 M1) was then added to each of the wells in one half of the microtitre plate and coating buffer solution (see Example 1) was added to the wells in the other half of the microtitre plate.

After 1 hr at room temperature, the wells were emptied and washed 3 times. Subsequently, pNPP solution (see Example 1) (100 Al) was added to each of the wells.

Yellow colour was observed to develop in the wells to which coating buffer was originally added whereas no colour was observed in the methanol treated wells. Optical Density was measured at 405 nm after 15 minutes and the results are given in Table 2. Column A gives results for the wells treated with methanol and Column B gives results for the other wells. Table 2 Amount of alkaline phosphatase added optical Density per well (ng) (OD) at 405 nm A B 0.05 3.00 0.05 2.93 12.5 0.06 2.46 6.26 0.05 1.95 3.12 0.06 1.34 1.65 0.06 0.97 0.78 0.06 0.54 Example 4

Alkaline phosphatase (EC3.1.3.1) was added to the wells of a microtitre plate as in Example 3; half of the wells were treated with methanol and the other half were treated with buffer as in Example 3.

To investigate the antibody binding activity of the Alhgline phosphatase in the wells suitably diluted rabbit anti-alkaline phosphatase antibody in binding buffer was used. (The binding buffer was 50 m14 Tris-HCl buffer (pH 7.4) containing 0.1 M NaCl, 0.2% gelatin, 0.001% thimerosal, 10 mg/ml BSA and 20 mg11 rhodamine base B.) The antibody was prepared by following known general procedures for producing antibodies adapted as necessary to produce the particular antibody required.

Signal was developed with goat-anti-rabbithorseradish peroxidase (HRR) conjugate (ex. Sigma) and peroxidase substrate solution (0.5 mg/ml of the disodium salt of 2,21-azino-bis(3-ethylben---thiazoline-6-sulfonic acid (ABTS) in 150 mM sodium acetate/citric acid buffer (pH 4.1) containing 60 g11100 ml of 34% v/v hydrogen peroxide) in accordance with known procedures.

Colour was produced in all wells. Optical Density was read at 405 nm after 30 minutes and results are given in Table 3 wherein Column A gives results for the wells treated with methanol and Column B gives results for the other wells.

Table 3

Amount of alkaline phosphatase added Optical Density per well (ng) (OD) at 405 nm A B so 2.96 3.00 2.13 2.56 12.5 1.87 2.12 6.26 1.12 1.32 3.12 0.86 0.97 1.65 0.45 0.65 0.78 0.24 0.36 comparing the results obtained in Examples 3 and 4 it will be noted that although methanol denatured alkaline phosphatase shows little or no enz,,, ,,atic activity (Column A, Table 2) the denatured alkaline phosphatase still retains antLI.-jcdv bindina Example 5

Alkaline phosphatase (EC3.1.3.1) was introduced to wells of a microtitre plate in Example 1, then treated with methanol (see Example 3) and then reacted with rabbit anti-alkaline phosphatase antibody (see Example 4). Serum from uninmunised rabbit was added to a set of "control" wells containing equal amounts of alkaline phosphatase as the other wells and treated with methanol as were the other wells.

After washing, excess native alkaline phosphatase (i.e., active, undenatured enzyme) was added to the wells (in 50 mM Tris (pH 7.8) containing 10 mg/inl BSA and 0.5 MM M9C12) and lef t f or 3 0 min. Af ter washing to remove unbound enzyme BC1P solution (see Example 2) was added to the wells and it was noted that in the wells which had been treated with rabbit anti-alkaline phosphatase antibody, blue colour developed (the intensity of which was related to the amount of alkaline phosphatase introduced to the wells) whereas the "control" wells showed no colour development.

In the wells treated with rabbit anti-alkaline phosphatase antibody, antibody had bound to the adsorbed alkaline phosphatase and to active enzyme thereby attaching active enzyme to the microtitre plate such that it could be detected by use of substrate solution.

Example 6

Alkaline phosphatase (EC3.1.3.1) was added to a commercially available ink vehicle composition and mixed to produce a homogeneous mixture comprising a labelled ink vehicle composition containing approximately 50 AgIM1 alkaline phosphatase.

The ink vehicle composition to which the alkaline phosphatase was added, was a liquid containing a mixture of substances normally found in quick-drying ink formulations except for a colourant dye.

gls of labelled ink vehicle composition were added to wells of a microtitre plate and allowed to dry at 370C in an incubator for 2 hr. To some wells of the microtitre plate only unlabelled ink vehicle composition was added (in the same amount as the labelled ink vehicle composition in the other wells) and treated in the same manner as the wells containing labelled ink vehicle composition, thereby to give "control" wells.

Rabbit anti-alkaline phosphatase antibody (100 M1), suitably diluted in binding buffer (see Example 4), was added to all of the wells. After standing for 30 rain at room temperature, the wells were emptied and washed three times. Goat-anti-rabbit-HFP conjugate (100 gl) was added to the wells and after 1 hour incubation the wells were washed (three times) and peroxidase substrate solution (see Example 4) was added.

After 10 min reaction, colour was observed developing in the wells to which labelled ink vehicle composition had been added originally.

After 30 min reaction, the resulting reaction solutions were transferred to an unused microtitre plate and the Optical Density (OD) was measured at 405 nm.

For the wells to which labelled ink composition had been added originally the OD was 0.65 and for the "control" wells the OD was 0.16.

Examnle 7 A commercially available ink vehicle composition was applied to plastic petri dishes and allowed to air-dry to form layers attached to the surface of the petri dishes.

Alkaline phosphatase (EC3.1.3.1) (200 gl of 125 ng/ml) in a coating buffer(see Example 1) was applied to the dry ink layers and left to air- dry at room temperature. Subsequently the petri dishes were placed in an incubator at 370C for 10 min. A plIPP solution (see Example 1) was applied to the areas of the petri dishes A to which ink vehicle composition and enzyme had been applied.

Yellow colour was observed in the dishes immediately. This Example demonstrates that ink vehicle composition layers are capable of adsorbing active enzyme species and that the enzyme species remain active after drying.

Claims

1. A method suitable for use in labelling of a substance or an item which method includes providing a substance or an item with an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

2. A method suitable for use in identifying a substance or an item, which method includes providing a substance or an item with an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule and performing a detection step to establish the presence or absence of an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

3. A method as claimed in Claim 1 or Claim 2 wherein a substance or an item is provided with an enzyme molecule bound with a binder for an enzyme molecule, or a substance or an item is provided with a fragment of an enzyme molecule bound with a binder for a fragment of an enzyme molecule.

4. A method suitable for identifying a substance or an item which method includes performing a detection step to establish the presence or absence of an enzyme molecule, or of a fragment of an enzyme molecule, or of a binder for an enzyme molecule, or of a binder for a fragment of an enzyme molecule.

5. A method as claimed in any one of Claims 2 to 4 wherein an enzyme molecule or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, is detected by immunological detection.

6. A method as claimed in any one of Claims 2 to 4 wherein an enzyme molecule, or a fragment of an enzyme molecule, is detected by an enzymological method.

7. A method as claimed in Claim 5 wherein an immunological method includes the use of an enzyme molecule, or a fragment of an enzyme molecule, in combination with a binder for the enzyme molecule, or a binder for the fragment of an enzyme molecule, or the use of a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule in combination with an enzyme molecule, or a fragment of an enzyme molecule.

8. A method as claimed in Claim 6 wherein an enzyme molecule, or a fragment of an enzyme molecule, is detected by application of a substance, application of a substrate and a chromogenic substance, detection by coupled enzyme reactions, or detection by an enzyme cycling method.

9. A method as claimed in any one of Claims 2, 3, 4, 6, 7 or 8 wherein detection of an enzyme molecule, or a fragment of an enzymenolecule, includes the use of an antibody having two binding sites and the use of an enzymatically active enzyme molecule.

10. A method as claimed in any one of Claims 2 to 9 wherein an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, is detected in situ whilst still attached to an item.

11. A method as claimed in any one of Claims 2 to 9 wherein an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, is retrieved from a substance or an item before being subjected to detection.

12. A method as claimed in any one of Claims 2 to 8 or Claim 11 wherein a liquid or a liquid sample containing an entity is subjected to detection by means of providing a thin film of the liquid or liquid sample on a suitable solid surface and drying the thin film to provide on the solid surface a thin layer of material to be subjected to detection.

13. A method as claimed in any one of Claims 1 to 3 or 5 to 9 or Claim 11 or Claim 12 wherein an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, is incorporated into a substance.

14. A method as claimed in any one of Claims 1 to 3 or 5 to 10 or Claim 12 wherein an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, is applied to an item.

15. A method as claimed in any one of Claims 1 to 3 or 5 to 14 wherein a substance or an item which is labelled is perfume, a bank note, an art work, a document of realisable value, an item of fashion clothes, a watch, an item of electrical goods, a book, a passport, a medicine, high value goods, a high volume sales item, a prestige high value article, a chemical formulation, meat, a meat product or packaging for goods.

16. A method as claimed in any one of Claims 1 to 3 or 5 to 15 wherein labelling a substance or an item is effected by mixing a label with an ink or ink vehicle composition and printed onto an item, applying a solution of a label directly to an item, dipping part of an area of an item into a solution of a label to attach label to the item, mixing a label with colouring matter or paintstuff for use with an item or packaging, adding a label directly to solutions or formulations of chemical goods, mixing a label with an adhesive substance which may then be attached-to tags or papers or items, marking paper certification which is to accompany goods, or applying an ink vehicle composition to an item to form a layer and applying a label to the layer.

17. A composition suitable for use in labelling of a substance or an item which composition includes an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

71

18. A composition as claimed in Claim 17 wherein the composition is an ink composition, or an ink vehicle composition, or a paint composition.

19. A combination comprising a label associated with a solubilising agent, said label being an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

20. A combination as claimed in Claim 19 wherein the solubilising agent is a surfactant.

21. A combination as claimed in Claim 20 wherein the surfactant is polyethylene glycol.

22. A combination comprising a label associated with an insolubilising medium, said label being an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

23. A combination as claimed in Claim 22 wherein the insolubilising medium is microparticles.

24. A test-kit suitable for use in identifying a substance or an item, which test-kit includes selected reagents for enzymological detection of an enzyme, or a fragment of an enzyme.

25. A test-kit suitable for use in identifying a substance or an item, which test-kit includes a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule, or an enzyme molecule, or a fragment of an enzyme molecule.

26. A labelled substance or a labelled item wherein the substance or item is labelled with a label which is an enzyme molecule, or a fragment of an enzyme molecule, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

27. A method suitable for use in labelling of a substance or an item substantially as hereinbefore described with reference to Example 2, Example 6 or Example 7.

28. A method suitable tor use in identifying a substance or an item substantially as hereinbefore described with reference to Example 2, Example 6 or Example 7.

29. A composition, suitable for use in labelling of a substance or an item, substantially as hereinbefore described with reference to Example 2. Example 6 or Example 7.

30. A labelled substance or labelled item substantially as hereinbefore described with reference to Example 2, Example 6 or Example 7.

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