WO1995017667A1

WO1995017667A1 - Labelling

Info

Publication number: WO1995017667A1
Application number: PCT/GB1994/002805
Authority: WO
Inventors: Ramadan Arbi Abuknesha
Original assignee: Gec-Marconi Limited
Priority date: 1993-12-23
Filing date: 1994-12-22
Publication date: 1995-06-29
Also published as: FR2714474A1; JPH09506972A; WO1995017670A1; JPH09508199A; JPH09509372A; GB9425922D0; JPH07203987A; DE4446042A1; GB2286672A; GB9425924D0; GB9425923D0; GB2286674A; WO1995017669A1; GB9326277D0; GB9425925D0; GB2286673A; GB2286044A

Abstract

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. In 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 a non-immunological identifiable species. In another 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 a non-immunological identifiable species and with an immunological identifiable species.

Description

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 a non-immunological identifiable species.

According to another 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 a non-immunological identifiable species and with an immunological identifiable species.

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 a non-immunological identifiable species and performing a detection step to establish the presence or absence of a non-immunological identifiable species.

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 a non-immunological identifiable species and with an immunological identifiable species and performing a detection step to establish the presence or absence of a non-immunological identifiable species and an immunological identifiable species.

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 a non-immunological identifiable species.

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 a non-immunological identifiable species and the presence or absence of an immunological identifiable species.

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, for example, one kind of non-immunological identifiable species, or a plurality of kinds of non-immunological identifiable species, or one kind of non-immunological identifiable species and one kind of immunological identifiable species, or one kind of non-immunological identifiable species and a plurality of different kinds of immunological identifiable species, or a plurality of different kinds of non-immunological identifiable species and one kind of immunological identifiable species, or a plurality of different kinds of non-immunological identifiable species and a plurality of different kinds of immunological identifiable species.

By way of example, any suitable identifiable species that may be detected may find application as an identifiable species in accordance with the present invention. In accordance with the present invention a substance or an item may be provided with a label (as hereinbefore mentioned) in any suitable manner.

A non-immunological identifiable species may be any species capable of taking part in non-immunological specific interaction. Thus, for example, a non- immunological identifiable species may be a species capable of taking part in a non-immunological specific binding reaction. Accordingly, an identifiable species may be, for example, a non-antigenic ligand or a binder species for a non-antigenic ligand. Examples of such species are ligand and binder species capable of undergoing non-immunological specific protein binding. Examples of non-antigenic ligands are steroids, carbohydrates, immunoglobulins, vitamin D and biotin. Examples of binder species for non-antigenic ligands are serum proteins, receptors, lectins, protein A, protein G, vitamin D binding protein and avidin.

An immunological identifiable species may be any species capable of taking part in an immunological specific interaction. Thus, for example, an immunological identifiable species may be a species capable of taking part in an immunological specific binding reaction. Accordingly, an identifiable species may be, for example, an antigenic ligand or a binder species for an antigenic ligand. Examples of such species are ligand and binder species capable of undergoing immunological binding. Examples of antigenic ligands are peptides, proteins (e.g. protein antigens), haptens, enzyme molecules and fragments of enzyme molecules. Examples of binder species for antigenic ligands are polyclonal antibodies, monoclonal antibodies and fragments of antibodies. An example of an antigenic ligand is fluorescein and an example of a binder species for fluorescein is anti-fluorescein-antibody.

It will be appreciated that, by way of example, a non-antigenic ligand may be detected by a method which includes tat use* of its corresponding binder species and a binder species for a non-antigenic ligand may be detected by a method which includes the use of its corresponding non-antigenic ligand.

It will also be appreciated that, by way of example, an antigenic ligand may be detected by a method which includes the use of its corresponding binder species and a binder species for an antigenic ligand may be detected by a method which includes the use of its corresponding antigenic ligand.

It will be appreciated that a binder species may be considered to be a specific reaction partner for a ligand and that a ligand may be considered to be a specific reaction partner for a binder species.

Any suitable method of detection may be employed such as those known in the non-immunological field and those known in the immunological field. Thus, for example, any form of suitable assay may be employed. It will be appreciated that a detection method may include the use of a tracer.

By way of example, where a substance is a fluid, such aε 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) a label may be introduced during manufacture of the paste or solid.

Thus, for example, a label 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 a label 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 ink composition, or a paint composition for application to an item by suitable means. Thus, for example, a label 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 one or more non-immunological identifiable species, or one or more non-immunological identifiable species and one or more immunological identifiable species. The composition may be, for example, an ink composition which includes one or more non-immunological identifiable species, or one or more non-immunological identifiable species and one or more immunological identifiable species.

It is to be understood that "one or more non- immunological identifiable species, or one or more non- immunological identifiable species and one or more immunological identifiable species" may indicate "one kind of non-immunological identifiable species, or a plurality of kinds of non-immunological identifiable species, or one kind of non-immunological identifiable species and one kind of immunological identifiable species, or one kind of non- immunological identifiable species and a plurality of different kinds of immunological identifiable species, or a plurality of different kinds of non-immunological identifiable species and one kind of immunological identifiable species, or a plurality of different kinds of non-immunological identifiable species and a plurality of different kinds of immunological identifiable species".

It will be appreciated that constituents of a composition may be chosen such that there is no unacceptable interference between an identifiable species or a plurality of identifiable species included in 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^" an identifiable species or a plurality of identifiable species included in the label. Also the label may be such that an identifiable species or plurality of identifiable species included therein 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 (e.g. the nature of an identifiable species or of a plurality of identifiable species included therein) 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 (e.g. the nature of an identifiable species or of a plurality of identifiable species included therein) 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 (e.g. a particular species therein) 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, in any suitable concentration. A suitable concentration may be such that, for example, the amount of label present would render it difficult, or substantially impossible, for an unauthorised person to identify the label (e.g. to identify identifiable species or plurality of -1 - identifiable species included therein) 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 μg/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".

By way of example, any suitable substance (e.g. a solid, a liquid or any other suitable substance) may be labelled. By way of example, any suitable item may be labelled. 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 it may be possible, for example, to distinguish that substance or item from other substances and items by testing to detect the selected label. Thus, for example, the presence of a selected label 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 and/or 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 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. with identifiable species each at 0.1-80,000 μg/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

(c) 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 and 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 one or more non-immunological identifiable species or one or more non-immunological identifiable species and one or more immunological identifiable species.

It will be appreciated that, for example, when a label comprises more than one identifiable species a substance or an item may be provided with such species either in any appropriate order, or simultaneously (e.g. where a label comprising more than one identifiable species is in a composition) .

In accordance with the present invention one or more non-immunological identifiable species, or one or more non-immunological identifiable species and one or more immunological identifiable species may be detected in any suitable manner.

By way of example, one or more non-immunological identifiable species, or one or more non-immunological identifiable species and one or more immunological identifiable species may be detected, in situ (e.g. ₍whilst still attached to an item) .

Alternatively, by way of example, one or more non- immunological identifiable species, or one or more non- immunological identifiable species and one or more immunological identifiable species may be retrieved from a substance or item and subjected to detection.

Thus, for example, one or more non-immunological identifiable species, or one or more non-immunological identifiable species and one or more immunological identifiable species may be subjected to detection in situ (e.g. whilst still attached to an item) , or alternatively, one or more non-immunological identifiable species, or one or more non-immunological identifiable species and one or more immunological identifiable species may be retrieved (e.g. by extraction by a suitable method) from a substance or item and subjected to detection..

Retrieval of a label may be effected in any suitable manner.

For example, where an identifiable species or a plurality of identifiable species included in a label is or are 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

SBSmUlESHEET (RULE26) εolution) to give a liquid sample which may be subjected to detection.

By way of further example, where an identifiable species or a plurality of identifiable species included in a label is or are to be retrieved from a substance which is not a liquid, the substance may be dissolved or otherwise treated to give a liquid containing an identifiable species or a plurality of identifiable species, which liquid may then be subjected to detection or treated as immediately hereinbefore disclosed by evaporation and treatment with a liquid medium to give a liquid sample which may be subjected to detection.

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

It will be appreciated that, for example, where a plurality of identifiable species is to be retrieved from a substance or an item, identifiable species may be retrieved separately, or together or, if appropriate, in any suitable combination.

By way of example, there may be circumstances where solvent extraction may not be applicable in the recovery of an identifiable species or a plurality of identifiable species. 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 an identifiable species or to a plurality of identifiable species included in a label. Thus, for example, a solvent or a solvent system may be chosen such that the use thereof does not cause unacceptable changes in an identifiable species or plurality of identifiable species included in a label.

Where an identifiable species has, or a plurality of identifiable species have been retrieved (e.g. extracted) the identifiable species or a plurality of identifiable species may be immobilised in any suitable manner so as to permit detection (e.g. by a specific protein binding assay (e.g. a non-immunoassay procedure or an immunoassay procedure) by any other suitable detection procedure.

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

Alternatively, by way of example, an identifiable species or plurality of identifiable species may be retrieved, provided in a suitable liquid medium (e.g. a buffer solution) , and then may be immobilised on a suitable support material (e.g. polystyrene) by non¬ specific adsorption.

By way of further example, specific interaction (e.g. specific binding) may be used to immobilise a retrieved identifiable species or a plurality of identifiable species. Thus, for example, a specific reaction partner for an identifiable species may be used to effect attachment to a support material by use of specific interaction with an identifiable species. For example, where an identifiable species is a ligand, a binder species (e.g. an antibody or a non-immunological binder species) may be utilised to bring about immobilisation of an identifiable species on a support material. Alternatively, by way of example, where an identifiable species is a binder species, a ligand may be used to effect attachment of an identifiable species to a support material.

It will be appreciated that a plurality of specific reaction partners may be utilised to bring about immobilisation of a plurality of identifiable species.

It will be appreciated that a specific reaction partner may be used to bring about immobilisation of an identifiable species in any convenient manner.

Thus, for example, a specific reaction partner may be associated with a support material before or after undergoing specific interaction with a specific reaction partner.

A specific reaction partner for an identifiable species 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 linfts (e.g. specific or non-specific in nature).

Thus, for example, where a specific a reaction partner is a binder species, may be linked to a support material by a second binder species (being an antibody) which second binder species is binder species for the reaction partner; in this configuration the second binder species 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 identifiable species.

Alternatively, by way of example, a specific reaction partner for an identifiable species may be arranged to be associated with a support material (before or after reaction with an identifiable species ) by means of an auxiliary binder or an auxiliary ligand system (e.g. by means of a further ligand-binder system) . Once an identifiable species 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, where an identifiable species is a ligand a suitable binder species for an identifiable species may be arranged to be associated with a tracer species capable of giving rise to a detectable signal.

Alternatively, for example, where an identifiable species is a binder species for a ligand a suitable ligand 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.

It will also be appreciated that where detection involves detection of an enzyme (e.g. as a tracer in a binding assay or in any other method where it is desired to detect an enzyme) , an enzyme may be detected by any suitable technique such as direct detection, detection by substrate plus chromogenic substance, detection by coupled enzyme reactions or detection by enzyme cycling methods (e.g. enzyme amplification) .

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 an identifiable species or a plurality of identifiable species 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) .

By way of further example, an identifiable species or a plurality of identifiable species 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 identifiable species or a plurality of identifiable species may be carried out using any suitable procedure, for example procedures known in the immunological field or non-immunological 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 identifiable species or a plurality of identifiable species may be effected using a specific reaction partner or specific reaction partners, which may be associated with suitable tracer species. For example, tracer species may be detected in situ .

An identifiable species or a plurality of identifiable species 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 identifiable species or a plurality of identifiable species) .

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

Subsequently, if desired, and the configuration of assay is appropriate, antibody may be denatured and the identifiable species recovered for use in a further (e.g. competitive) assay which may be used to confirm results of the non-competitive assay.

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

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 specific reaction partner for a non- immunological identifiable species, or a specific reaction partner for a non-immunological identifiable species and a specific reaction partner for an immunological identifiable species.

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 a non-immunological identifiable species, or a non-immunological identifiable species and an immunological identifiable species.

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. In accordance with the present invention there is provided a combination comprising a non-immunological identifiable species associated with a solubilising agent.

In accordance with the present invention there is also provided a combination comprising a non- immunological identifiable species, associated with a first solubilising agent and an immunological identifiable species associated with a second solubilising agent.

The first solubilising agent and the second solubilising agent may be, for example, the same kind of solubilising agent or different kinds of solubilising agent. Where, for example, the first solubilising species and the second solubilising species are the same kind of species, a non-immunological identifiable species may be associated with one sample of the solubilising agent and an immunological identifiable species may be associated with another sample of solubilising agent.

An solubilising agent may be a surfactant.

Thus, in one embodiment of the present invention there is provided a combination comprising a non- immunological identifiable species associated with a surfactant.

In another embodiment of the present invention there is provided a combination of a non-immunological identifiable species associated with a first surfactant and an immunological identifiable species associated with a second surfactant.

The first surfactant and the second surfactant may be the same kind of surfactant or different kinds of surfactant.

A non-immunological identifiable species or a non- immunological identifiable species and immunological identifiable species may be associated with a surfactant in any suitable manner, for example by conjugation with a reactive site or sites of a surfactant. It will be appreciated that conjugation may be by covalent bonding.

Polyethylene glycol is an example of a surfactant which may be utilised in accordance with the present invention. Thus, for example, a non-immunological identifiable species may be associated (e.g. conjugated) with polyethylene glycol.

Also, for example, a non-immunological identifiable species may be associated (e.g. conjugated) with one sample of polyethylene glycol, and an immunological identifiable species may be associated with another sample of polyethylene glycol.

A combination as immediately hereinbefore disclosed (which combination includes a solubilising agent) may be soluble in both aqueous and organic liquids. This may offer advantages, for example, when seeking to introduce a non-immunological identifiable species or a non- immunological identifiable species and an immunological identifiable species to an ink which has organic components (e.g. an organic solvent).

It will be appreciated that a combination as immediately hereinbefore disclosed may also find application in labelling using water-based solvents.

In accordance with the present invention there is also provided a combination comprising a non- immunological identifiable species associated with an insolubilising medium.

In accordance with the present invention there is also provided a combination comprising a non- immunological identifiable species associated with a first insolubilising medium and an immunological identifiable species associated with a second insolubilising medium.

The first insolubilising medium and the second insolubilising medium, for example, may be the same kind of insolubilising medium or different kinds of insolubilising medium. Where, for example, the first insolubilising medium and the second insolubilising medium are the same kind of insolubilising medium, a non- immunological identifiable species may be associated with one sample of insolubilising medium and an immunological identifiable species may be associated with another sample of insolubilising medium.

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 a non-immunological identifiable species or a non-immunological identifiable species and an immunological identifiable species associated with insolubilising medium is also rendered substantially insoluble in chosen organic or aqueous solvents.

The insolubilising medium may be, for example, microparticles 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 non-immunological identifiable species, or a non-immunological identifiable species and an immunological identifiable species with 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.

By way of example, a combination of a label associated with an insolubilising medium may comprise a non-immunological identifiable species attached to an insolubilising medium comprising a microparticle or microparticles, or may comprise a non-immunological identifiable species attached to an insolubilising medium comprising a microparticle or microparticles and an immunological identifiable species attached to an insolubilising medium comprising a microparticle or microparticles.

By way of example, a combination as immediately hereinbefore disclosed (which combination includes an insolubilising medium) may be substantially insoluble in chosen organic or aqueous solvents_., and 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 non-immunological identifiable species or non- immunological identifiable species and immunological identifiable species when included in a label.

By way of example, if more than one identifiable species is used in accordance with the present invention this may increase the difficulty which an unauthorised person may encounter in relation to ascertaining the presence and/or identity thereof and/or in relation to the detection thereof.

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

By way of example, it may be noted that, optionally, for certain applications it may be possible to arrange for a non-immunological identifiable species or a non- immunological identifiable species and an immunological identifiable species 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 non-immunological identifiable species or a non- immunological identifiable species and an immunological identifiable species.

It will also be appreciated that an identifiable species 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 an identifiable species 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) .

The term "antibody" as used in this Specification embraces whole antibody and antibody fragments such as Fab and (Fab), 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 antigenic ligand may be prepared by raising antibodies respectively to an antigenic ligand.

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 a ligand and a binder which combination has a high affinity (e.g. picogra me quantities may be detected) . Fro the foregoing disclosure it will be appreciated that, for example, one or more non-immunological identifiable species or one or more non-immunological identifiable species and one or more immunological identifiable species may be used to effect labelling. However, if desired, such a species or species 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, one or more non- immunological identifiable species or one or more non- immunological identifiable species and one or more immunological identifiable species, in accordance with the present invention, may be used together with one or a plurality or any combination of other identifiable species such as, for example, a "masked" ligand, an enzyme molecule, a fragment of an enzyme molecule, a binder for an enzyme molecule, a binder for a fragment of an enzyme molecule, 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 P/60539/HRF) in relation to enzyme molecules, fragments of enzyme molecules, and binders therefore in connection with labelling.

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

crffit -Tj IT Example 1

Biotin (Vitamin H) was added (100 ng/ml) to a commercially available ink vehicle composition to provide a labelled ink vehicle composition.

A sample (0.1 ml) of the labelled ink vehicle composition was applied to a petri dish and dried at 40°C.

A volume (0.5 ml) of an extraction liquid (100 mM sodium bicarbonate containing 100 mM NaCl, 0.05% Tween 20 and 10% methanol) were added to the petri dish and after 5 min, aliquots of 0.1 ml were taken and incorporated in a competitive protein binding assay to detect extracted biotin. Control experiments were also conducted in which unlabelled ink vehicle composition was treated in the manner described above.

Competitive assay was carried out on microtitre plates.

Thus, wells of microtitre plates were coated with a biotinamidocaproate-BSA conjugate and an avidin- peroxidaεe conjugate was used as a labelled binder species.

Thus, in the assay biotin of the biotinamido¬ caproate-BSA conjugate competed with biotin (if any) in extraction liquid samples for binding with the avidin of the avidin-peroxidase conjugate.

The fraction bound to the plates was measured. Signal was developed by use of known procedures using peroxidase reaction with ABTS as a chromogen.

Signals were measured by means of Optical Density readings at 405 n .

The results were as follows:

(a) Test on extraction liquid contacted with labelled ink vehicle composition: O.D. = 0.12;

(b) Control (extraction liquid and appropriate assay reagents) : O.D. = 1.51;

(c) Control (extraction liquid contacted with unlabelled ink vehicle composition) : O.D. = 1.43; (d) Peroxidaεe reaction blank: O.D. = 0.12. This Example demonstrates that the non-immunological identifiable species biotin may be applied in an ink vehicle composition, retrieved from such a composition and detected.

Example 2

The procedures disclosed in Example 1 were generally followed with the exception that an ink vehicle composition was provided with the antigenic ligand carboxyfluorescein (80 ng/ml) in addition to biotin as in Example 1.

Samples of extraction liquid were subjected to detection for biotin as in Example 1.

Samples of extraction liquid (0.1 ml) were also incorporated into competitive immunoassay for carboxyfluorescein.

Thus, for the carboxyfluorescein assay microtitre plates were coated with an egg-albumin-carboxyfluorescein conjugate. Rabbit anti-fluorescein antibody was used and carboxyfluorescein of the egg-albumin-carboxyfluorescein conjugate was allowed to compete with carboxyfluorescein (if any) in the extraction liquid for binding to the antibody.

Signal was detected on the plates by use of goat- anti rabbit-alkaline phoεphatase conjugate and p- nitrophenylphosphate and signal was measured by means of Optical Density readings at 405 nm.

The results of biotin were as follows:

(a) Test on extraction liquid contacted with labelled ink vehicle composition: O.D. = 0.14;

(b) Control (extraction liquid and appropriate assay reagents) : O.D. = 1.48;

(c) Control (extraction liquid contacted with unlabelled ink vehicle composition) : O.D. = 1.43;

(d) Peroxidase reaction blank: O.D. = 0.12.

The results for carboxyfluorescein were as follows: (a) Test on extraction liquid contacted with labelled ink vehicle composition: O.D. = 0.25;

(b) Control (extraction liquid and appropriate assay reagents) : O.D. = 1.12;

(c) Control (extraction liquid contacted with unlabelled ink vehicle composition) : O.D. = 0.96;

(d) Peroxidase reaction blank: O.D. = 0.12.

This Example demonstrates that the non-immunological identifiable species biotin and the immunological identifiable species carboxyfluorescein may be applied in an ink vehicle composition, retrieved from such a composition and detected independently of each other.

Example 3

Polyethylene glycol (PEG) was activated, a diaminooctane derivative was formed and a ligand was coupled to the derivatives. Thus, polyethylene glycol of average MW 20,000 (ex Sigma) (5 g) was dissolved in 1,4 dioxan (80 ml) and of 1,1' carbonyldiimidazole (CDI) (ex Sigma) (1.5 g) was added.

After standing at room temperature for 24 hr, activated PEG was precipitated from dioxan by addition of cold diethyl ether (300 ml) . After standing on ice for 15 min solid product was recovered by filtration.

CDI-activated PEG (2.5 g) was added to a solution of diaminooctane (0.5 g in 30 ml sodium bicarbonate/ carbonate buffer (pH 9.6) and 20 ml 1,4-dioxan) . After mixing for 24 hr the resulting reaction mixture was dialysed to remove unreacted diamine. Thus, PEG- diaminooctane derivative was prepared.

5 (6) carboxyfluorescein (12 mg) (previously activated with N-hydroxysuccinimide, and 1,3 dicyclohexyl- carbodiimide) , was added to PEG-dia inooctane derivative (150 mg) in 15 ml of sodium bicarbonate buffer (pH 8.6) and 5 ml pf DMF.

After a suitable reaction time the resulting reaction mixture was dialysed.

SyBSTITUTE SHEET (RULE 26) The resulting product was a combination comprising PEG-diaminooctane-5 (6) -carboxyfluorescein.

Example 4

The product of Example 3 was followed with the exception that biotinamidocaproate N-hydroxysuccinimide ester (ex Sigma) (10 mg) waε used in place of 5(6)- carboxyfluoreεcein.

Thus, a product was obtained which was a combination comprising PEG-diaminooctane-biotina idocaproate.

Example 5

The combination prepared in Example 3 was added (at a concentration of 50 mg/1) to a commercially available ink vehicle composition to give a labelled ink vehicle composition.

The labelled ink vehicle composition (200 μl) was applied to a petri dish, spread to form a thin layer, and allowed to dry at 37°C for 2 hr to form a "test" dish. A "control" dish was prepared in the same way using unlabelled ink vehicle composition.

The dry ink layers were treated for about 5 minutes with a conjugate comprising anti-5 (6) -carboxyfluorescein antibody-HRP (400 μl) in a binding buffer.

(The binding buffer was PBS containing 0.2% gelation, 10 mg/ml BSA, 0.05% Tween 20, 0.001% thimerosal and 20 mg/ml rhodamine B base.)

After washing peroxidase substrate solution (400 μl) was added to the dishes.

(The peroxidase substrate solution was 0.5 mg/ml of 2,2 azino-bis (3-ethylbenz-thiazaline-6-sulfonic acid [ABTS], in 150 mM sodium acetate/citric acid buffer (pH 4.1) containing 60 μl of 34% v/v hydrogen peroxide/100 ml of buffer. )

After a fev; minutes colour was observed to be developing in the test dishes; considerably less colour was noted in the control dish. This Example demonstrates the use of a combination in accordance with the present invention for labelling an ink vehicle combination.

Example 6

The procedure of Example 5 was followed with the exception that the combination prepared as in Example 4 was also added to an ink vehicle combination and thus an avidin-HRP conjugate was also used in addition to anti- 5(6) -carboxyfluorescein antibody to detect identifiable species.

The results were similar to those reported in Example 5.

This Example demonstrates the use of two combinations in accordance with the present invention for labelling an ink vehicle composition.

SUBSTITUTE SHFET JRULF 29

Claims

Cl a i s

1. A method suitable for use in labelling of a substance or an item which method includes providing a substance or an item with a non-immunological identifiable species.

2. A method suitable for use in .labelling of a substance or an item which method includes providing a substance or an item with a non-immunological identifiable species and with an immunological identifiable species.

3. A method suitable for use in identifying a substance or an item, which method includes providing a substance or an item with a non-immunological identifiable species and performing a detection step to establish the presence or absence of a non-immunological identifiable species.

4. A method suitable for use in identifying a substance or an item which method includes providing a substance or an item with a non-immunological identifiable species and with an immunological identifiable species and performing a detection step to establish the presence or absence of a non-immunological identifiable species and an immunological identifiable species.

5. A method suitable for identifying a substance or an item which method includes performing a detection step to establish the presence or absence of a non-immunological identifiable species.

6. A method suitable for identifying a substance or an item, which method includes performing a detection step to establish the presence or absence of a non- immunological identifiable species and the presence or absence of an immunological identifiable species.

7. A method as claimed in any one of Claims 1 to 4 wherein a substance or an item is provided with one kind of non-immunological identifiable species, or with a plurality of kinds of non-immunological identifiable species, or with one kind of non-immunological identifiable species and with one kind of immunological identifiable species, or with one kind of non- immunological identifiable species and with a plurality of different kinds of immunological identifiable species, or with a plurality of different kinds of non- immunological identifiable species and with one kind of immunological identifiable species, or with a plurality of different kinds of non-immunological identifiable species and with a plurality of different kinds of immunological identifiable species.

8. A method as claimed in any one of Claims 1 to 4 or Claim 7 wherein a non-immunological identifiable species is a non-antigenic ligand or a binder species for a non- antigenic ligand.

9. A method as claimed in any one of Claims 1 to 4 or Claim 7 wherein an immunological identifiable species is an antigenic ligand or a binder species for an antigenic ligand.

10. A method as claimed in any one of Claims 3 to 9 wherein a non-immunological identifiable species is, or a non-immunological identifiable species and an immunological identifiable species are, detected in situ whilst still attached to an item.

11. A method as claimed in any one of Claims 3 to 9 wherein a non-immunological identifiable species is, or a non-immunological identifiable species and an immobilised identifiable species are, retrieved from a substance or an item before being subjected to detection.

12. A method as claimed in any one of Claims 3 to 9 or Claim 11 wherein a liquid or a liquid sample containing an identifiable species or a plurality of identifiable species 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 4, or 7 to 9 or Claim 11 or Claim 12 wherein a label is incorporated into a substance.

14. A method as claimed in any one of Claims 1 to 4 or 7 to 12 wherein a label is applied to an item.

15. A composition suitable for use in labelling of a substance or an item which composition includes one or more non-immunological identifiable species, or one or more non-immunological identifiable species and one or more immunological identifiable species.

16. A composition as claimed in Claim 15 wherein the composition is an ink composition, or an ink vehicle composition, or a paint composition.

17. A combination comprising a non-immunological identifiable species associated with a solubilising agent.

18. A combination comprising a non-immunological identifiable species associated with a first solubilising agent and an immunological identifiable species associated with a second solubilising agent.

19. A combination as claimed in Claim 18 wherein the surfactant is polyethylene glycol.

20. A combination comprising a non-immunological identifiable species associated with an insolubilising medium.

21. A combination comprising a non-immunological identifiable species associated with a first insolubilising medium and an immunological identifiable species associated with a second insolubilising medium.

22. A combination as claimed in Claim 20 or Claim 21 wherein the insolubilising medium is microparticles.

23. A test-kit suitable for use in identifying a substance or an item, which test-kit includes a specific reaction partner for a non-immunological identifiable species or a specific reaction partner for a non- immunological identifiable species and a specific reaction partner for an immunological identifiable species.

SUBSTITUTE SHFFT R _LE 26}

24. A labelled substance or a labelled item wherein the substance or item is labelled with a non-immunological identifiable species or with a non-immunological identifiable species and with an immunological identifiable species.