GB2286044A - Plurality of labels - Google Patents

Description

1 1 Labellino 2286044 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 an entity suitable for use in labelling of a substance or an item which entity provides a plurality of identifiable species.

In an embodiment of the present invention there is provided an entity suitable- for use in labelling of a substance or an item which entity provides a plurality of separately 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 an entity which entity provides a plurality of identifiable species.

In an embodiment of the present invention there is provided a method suitable for use in labelling of a substance or an item which method includes providing the substance or item with an entity which entity provides a plurality of separately identifiable species.

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

The entity may provide a plurality of identifiable species (e.g. a plurality of separately identifiable species) In any suitable manner. Thus. for example, the entity may comprise a plurality of identifiable species (e.g. separately identifiable species) linked together in any suitable manner. By way of example, each of the identifiable species (e.g. separately identifiable species) inay be attached to a common carrier material. Thus, for example, each of the identifiable species (e.g. separately identifiable species) may be chemically attached to a carrier material. Examples of suitable carrier materials are polymeric structures such as a protein, a polysaccharide or a polyalcohol. By way of example-, identifiable species nay be treated as necessary to facilitate attachment to a carrier material. Thus. for example, an identifiable species may be derivatised to introduce chemical arms and suitable reactive functional groups, the resulting derivatives may be activated into intermediates able to react with functional groups on a carrier material to effect covalent links. it is to be understood, by way of example, that, if appropriate, a carrier material itself may provide an identifiable species.

By way of example. an identifiable species may be a species which is detectable as such, or may be a species which may be formed into a species which may be detected. Thus. for example, in this Specification, where the context allows, 'identifiable species" embraces species which may be detected as such. and species which may be formed Into species which may be detected.

Accordingly, for example, in this specification, where the context allows, "an entity which provides a plurality of identifiable species" in accordance with the present invention may be, for example, an entity which provides a plurality of identifiable species, which species are detectable as such, or muy be, for example, an entity which may provide, or is capable of providing, a plurality of species which may be formed into detectable species (e.g. by forming species into species 1 0! 1 which may be detected), or may be, for example, an entity which provides identifiable species. which are detectable as such, and species which may be formed into detectable species. Further details regarding identifiable species which may be formed into a detectable species are disclosed hereinafter.

An entity may, for example, provide a plurality of the same identifiable species or an entity may, for example, provide different identifiable species.

In accordance with the present invention there is provided a method for the preparation of an entity, which entity provides a plurality of identifiable species, which method includes attaching a plurality of identifiable species to a common carrier material.

For example, in accordance with an embodiment of the present invention a method is provided for the preparation of an entity, which entity provides a plurality of identifiable species, which method includes attaching a plurality of the same identifiable species to a common carrier material.

By way of further example, in accordance with another embodiment of the present invention there is provided a method for the preparation of an entity, which entity provides a plurality of identifiable species, which method includes attaching a plurality of different identifiable species to a common carrier material.

An identifiable species may be any suitable species. For example, an identifiable species may be any suitable chemical species or any suitable biochemical species.

For example, an identifiable species may be any suitable species examples of which are ligands (e.g. antigenic ligands or non-antigenic ligands, binders (e.g. binders for antigenic ligands and binders for nonantigenic ligands), and any other suitable identifiable species (e.g. nucleic acids such as DNA or RNA). It will be appreciated that a binder for an antigenic ligand nay be an antibody.

1 1 BY way of example an identifiable species may be a suitable species which may be capable of binding with a reaction partner therefor (hereinafter referred to as "identifiable species reaction partner') such that the presence of a given identifiable species 'may be detected.

Thus, for example, an identifiable species and an identifiable species reaction partner may be capable of undergoing a specific interaction (e.g. specific binding).

Examples of identifiable species are species which are capable of undergoing immunological binding; further examples of identifiable species are species which are capable of undergoing non-immunological binding.

Examples of species capable of undergoing immunological binding (which is specific binding) are antigenic ligands (e.g. haptens and protein antigens (such as macromolecular protein antigens)) and binders therefor. Thus. where, for example, an identifiable species is an antigenic ligand, the antigenic species reaction partner may be an antibody to the ligand; conversely, where, for example, an identifiable species is an antibody. the identifiable species reaction partner may be an antigenic ligand. An example of an antigenic ligand is fluorescein.

It will be appreciated that, by way of further example. an antibody to an antibody may be used as an identifiable species or as an identifiable species reaction partner.

Examples of identifiable species capable of undergoing non-immunological binding are non-antigenic ligands and binders for non-antigenic ligands. Examples of such identifiable species are ligands and binders capable of undergoing non-immunological specific protein binding. Thus, for example, where an identifiable species is a non-antigenic ligand (e.g. biotin or vitamin D) the identifiable species reaction partner may be a binder for a non-antigenic ligand (e.g. avidin or vitamin 4 1 D binding protein); conversely, for example, where the identifiable species is a binder (e.g. avidin or vitamin D binding protein) for a non- antigenic ligand the identifiable species reaction partner may be a nonantigenic ligand (e.g. biotin or vitamin D).

By way of further example, an identifiable species 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. 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 an enzyme molecule.

Further examples of identifiable species and identifiable species reaction partners capable of undergoing non-immunological binding are species capable of undergoing specific interactions. Examples of such species are nucleic acids (e.g. DNA or RNA) and species capable of undergoing specific interaction with nucleic acids. Thus, for example. a DNA sequence and a species capable of specific interaction with the sequence may be utilised in accordance with the present invention.

An entity which provides a plurality of identifiable species may provide any suitable combination of identifiable species. Thus. for example, an entity may provide only species capable of undergoing immunological interactions, or the entity may provide only species capable of undergoing non-immunological interactions, or the entity may provide any desired combination of species (e.g. one or more species capable of undergoing immunological Interaction and one or more species capable of undergoing non-immunological interaction).

It is to be understood that an entity in accordance with the present invention may, for example, provide a plurality of identifiable species of the same type; thus, for example, the identifiable species may be substantially the same as each other (e--9- the same 1 ligand). Also, it is to be understood that an entity in accordance with the present invention may. for example, provide identifiable species of different types and such different types nay, for example, be separately identifiable.

By way of example, one type of entity may be used in accordance with the present invention or. if desired. more than one type of entity may be used in accordance with the present Invention.

By way of further example, as hereinbefore disclosed, an ideritif iable species may be a species which is detectable as such, or may be a species which may be formed (e.g. converted) into a species which may be detected. Thus, for example, a ligand (e.g. an antigenic ligand or a non-antigenic ligand) may be "masked" (e.g. by chemical means or biochemical means) and used as an identifiable species. The masked species nay be "inactive" in the sense of being substantially unreactive towards a corresponding binder. Upon "unTnasking" the ligand nay become reactive towards its corresponding binder and may therefore be detectable by reaction with the corresponding binder.

A ligand may be, for example, "masked" by any suitable means. Thus, for example. a ligand may be "masked" by chemical introduction of suitable species (e.g. sugar species or ester species).

A ligand may be "unmasked", for example, in any suitable manner (e.g. enzymatically, or by use of extreme pH conditions, or by physical means (e.g. UV irradiation))- Iz is to be understood that the "unmasking" of a "masked" ligand may be considered, f or example, to be the "switching on" of its reactivity towards its corresponding binder. It is also to be understood that a #$masked#% ligand may be considered to be a precursor for an "unmasked" ligand.

it is to be understood that where an identifiable species is, for example, a binder, a corresponding # 1 3 Imaokedct ligand may be usea in the detection of such a binder, said detection may then include, for example, "unmasking" of the "maskedg' ligand to permit it to react with the binder.

By way of example, any suitable means may be utilised for converting an "inactive" ligand into an active ligand which may be detected. Thus, for example, "Switching onn of interacting capability of a species may be effected by adding to a species, or by removing something from a species, or by any other suitable means (e.g. chemical means).

By way of further example, an antigen sub-unit is a further example of a species which may be formed (e.g. converted) into a species which may be detected. Thus, for exanple, an antigen sub-unit may have added thereto another antigen sub-unit to form an antigen which is a species which may be detected. Thus, for example, addition of a sub-unit to form an antigen may form part of a inethod for detection.

It will be appreciated that an entity which provides a plurality of identifiable species (e.g. separately identifiable species) may be considered to be.a label, and a label in accordance with the present invention may, for example, be an entity which provides a plurality of Identifiable species (e.g. separately identified species).

in accordance with the present invention a substance or an item may be provided with a label, comprising an entity in accordance with the present invention, in any suitable manner.

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

^8^ Thus for example, a label which comprises an entity which provides a plurality of identifiable species (e.g. separately identifiable species) may be incorporated into a substance.

where an item is to be provided with a label comprising an entity the label may be provided in any suitable manner (e.g. by application to the item). Thus, for example, an tntity which provides a plurality of identifiable species (e.g. separately identifiable species) may be applied to an item in any suitable manner.

By way of example, a label comprising an entity may be Incorporated into a composition for application to an item. A composition may be, for example, an ink or ink medium or an ink composition or a paint composition for application to an item by suitable means. Thus, for example, a label which comprises an entity which provides a plurality of identifiable entities (e.g. separately identifiable species) may be incorporated into an ink or ink medium or a paint composition suitable for application to an item. The ink or ink medium or ink composition 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 entity which entity provides a plurality of identifiable species. The composition may be, for example, an ink composition which includes an entity which entity provides a plurality of identifiable species.

it will be appreciated that constituents of a composition may be chosen such that there is no unacceptable interference between a label compricing an entity and other constitutions of a composition such as to cause unacceptable deterioration in properties of the composition; also constituents of the composit-ion nay be such as to cause no unacceptable deterioration in 1 0 1 -g- properties of the label comprising an entity. Also the label comprising an entity may be such that it nay still be detected after application to an item, and after exposure of the item to such conditions as the item nay encounter (e.g. in shipping andlor storage).

It is to be understood that the present invention may be utilised to provide a substance or an item with a label comprising-an entity in s uch 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).

By way of example, use of more than one type of identifiable species may increase the difficulty which an unauthorised person may encounter in relation to ascertaining the presence andlor identity of a label or labels andlor in relation to detection of a label or labels.

The use of a "masked" ligand (as hereinbefore disclosed), may, for example, lead to an increase in the difficulty which an unauthorised person may encounter in relation to ascertaining the presence andfor identity of a label andlor in relation to detection of a label.

in accordance with the present invention, a substance or an item may be provided with a label, 1 comprising an entity which provides a plurality of identifiable species (e.g. separately detectable species) 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 by "conventional' methods (e.g. chemico-physical methods such at 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 an entity (in accordance with the present invention) at a concentration of 0.1-80,000 Agfl.

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 purpose 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 1 is to be understood that 'labelling" may also be considered to be "tagging" and that a "label" nay 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 suit-able, or nay be rendered suitable, for labelling or anything whatsoever that is suitable, or may be rendered suitable, for labelling.

Thus, for example, "substance or itenll 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 nay 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 or items by testing to detect the selected label or labels. Thus, for example, the presence of a selected label or labels may indicate that the 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 a 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 andlor "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 (e.g. an entity which entity provides a plurality of identifiable species) 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 waterjorganic solvent combination) directly to items, or 1 (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 (a) 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 as a 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. reaction sites to which a label may covalently bond, applying the ink vehicle composition to an item and treating the ink vehicle 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 Inay 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- 1 -14 It will be appreciated that a label as immediately hereinbefore disclosed in (a) to (j) may comprise an entity which provides a plurality of identifiable species (e.g. separately identifiable species). As was hereinbefore disclosed, In one embodiment of the present invention an entity suitable for use in labelling of a substance or an item may be, for example. an entity which-provides a plurality of separately identifiable species. Thus, for example, an entity may provide two or more different types of identifiable species, said different types of identifiable species being capable of being detected separately or such that each zype of identifiable species may he separately identified. Thus, for example, it may be arranged that different types of identifiable species are capable of being detected independently of one another. Prom the foregoing it will be appreciated that, for example, an entity which provides a plurality of identifiable species in accordance with the present invention may be arranged to' provide any suitable combination of identifiable species. Thus, for example, an entity may provide: (a) a plurality of the same non-immunological ligands or non-immunological binders; or (b) a plurality of different non-immunological ligands or nonimmunological binders; or (c) a plurality of the same immunological ligands or immunological binders; or (d) a plurality of different immunological ligands or immunological binders; or (e) a non-immunological ligand or a non-Lmmunological binder (or a plurality of such ligands or binders) and an immunological ligand or an immunological binder (or a plurality of such ligands or binders); or 1 1 (f) a ligand andlor a binder, and a DNA sequence andlor a DNA sequence marker; or (g) 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 (h) a plurality of "masked" ligands of the same type; or (i) a plurality of "masked" ligands of different types; or a "masked" ligand or "masked" ligands and a ligand which is not "masked" or ligands which are not "masked'; or (k) a plurality of different enzyme molecules, or of different fragments of enzyme molecules, or of different binders for enzyme molecules or of different binders for fragments of enzyme molecules; or (1) a ligand and 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 (m) a binder and 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 an embodiment of the present invention an entity which entity provides a plurality of identifiable species may be formed by attaching at least one type of antigenic ligand and at least one type of non-antigenic ligand to a common carrier material. in another embodiment of the present invention an entity which entity provides a plurality of identifiable species nay be formed by attaching two different types of non-antigenic ligands to a common carrier material. An entity, such as hereinbefore disclosed in the two immediately preceding paragraphs, may be used, for example, in the labelling of a substance or an item and subsequently t-he identifiable species provided by the entity may be used to identify the substance or item in any suitable manner (e.g. by an immunological procedure andlor by a nonimmunological procedure).

By way of example a non-antigenic ligand may take part in a specific binding reaction with a non-antibody protein.

It is to be understood that some type of ligands, for example, may serve as an antigenic llgand (if used in conjunction with a reaction partner which is an antibody) or as a non-antigenic ligand (if used with a reaction Partner which is a non-immunological binding protein).

Examples of non-antigenic ligands are steroids, carbohydrates, immunoglobulinr>, Fc fragments of immunoglobulins, glycoproteins, vitamin D and blotin.

Examples of non-antigenic binders are serum proteins, receptors, lectins, protein A, protein G, vitamin D binding protein and avidin. It is to be understood that receptors inay be prepared from animal tissues according to known procedures. Serum proteins inay be isolated from sera by affinity chromatography.

By way of example, any suitable substance which can be used to prepare an antibody may be used as an antigenic ligand. Examples of antigenic ligands are peptides, proteins, haptens, enzyme molecules and fragments of enzyme molecules.

Examples of binders for antigenic ligands are polyclonal antibodies,monoclonal antibodies and fragments of antibodies.

In accordance with the present invention an identifiable species may be detected in any suitable manner- By way of example, an identifiable species may be detected, in situ (e.g. whilst still attached to an item).

Alternatively, by way of example, an identifiable species may be retrieved from a substance or iteM (e-gby means of extraction by use of a suitable method (e.g.

by use of a suitable solvent)) and subjected to detection.

Thus, for example, an entity which entity provides a plurality of identifiable species, 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 an entity nay be effected in any suitable manner.

ror example, where an entity is to be retrieved from a liquid, the liquid nay be evaporated to leave a residue and the residue may be treated with a suitable liquid inedium (e.g. a buffer solution) to give a liguid sample which may be subjected to detection.

By way of further example, where an entity is to be retrieved from a substance which is not a liquid, the substance nay be dissolved or otherwise treated to give a liquid containing an entity 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 entity nay be extracted from an item (e.g. to give a liquid containing the entity) and then subjected to detection in any suitable manner.

By way of further example, where extraction involving a solvent is used, solvent may be removed after extraction and a suitable but fer 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 entity andlor identifiable species to be detected. Thus, for example, a solvent or solvent system may be chosen such that the use thereof does not cause -is- unacceptable changes in the entity andlor identifiable species to be detected.

Where an entity (which provides a plurality of identifiable species) or identifiable species has been retrieved (e.g. extracted) it may be ixmobilised 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 assay procedure).

Thus, f or example, a non-specif ic chemical method may be used to immobilise an entity on a suitable support material (e.g. on nitrocellulose paper); for example, an entity which entity provides a plurality of identifiable species nay be retrieved (e.g. extracted) by use of a suitable solvent. solvent may be removed, buffer added, and the entity may be immobilised on a suitable support by means of a nonspecific chemical method.

Alternatively, by way of example, an entity may be retrieved (e.g. by extraction), solvent removed, buffer added (as hereinbefore disclosed) and then the entity may be immobilised on a suitable support material (e. g. polystyrene) by non-specifi c adsorption.

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

Conversely where an identifiable species is a binder (e.g. an antibody or a non-immunological binder), a ligand may be utilised to bring about attachment to a support material- It will be appreciated that an identiúiable species may be retrieved, for example, by retrieving an entity which provides the identifiable species.

Where. by way of example. an entity, which entity provides a plurality of identifiable species is to be Immobilised by specific binding, identifiable species may be used In conjunction with an identifiable species reaction partner to bring about attachment to a support material.

Where, by way of example, an entity, which entity provides a plurality of separately identifiable species is to be Immobilised by specific binding, one of the plurality of separately identified species nay be used in conjunction with an identifiable species reaction partner to bring about attachment to a support material.

Thus, for example, an entity may provide more than one type of identifiable species and one of the identifiable species nay be used to undergo a specific interaction with a corresponding reaction partner. In such circumstances it will be appreciated that one of the identifiable species is lid entifiablell at least in the sense that It can be recognised by a corresponding reaction partner and used to bring about immobilisation.

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

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

An identifiable species reaction partner 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, where an identifiable species reaction partner is a binder, it may be linked to a support material by a second binder (being an antibody) which second binder is binder for the identifiable species 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 identifiable species reaction partner before or after the identifiable species reaction partner has undergone specific interaction with the identifiable species.

Alternatively, by way of example, an identifiable species reaction partner may be arranged to be associated with a support material (before or after reaction with a corresponding 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, suitable identifiable species reaction partners may be used which reaction partners may be arranged to be associated with a tracer species capable of giving rise to a detectable signal.

It will be appreciated that, by way of examplee where an entity which entity provides a plurality of separately identifiable species is utilised. one type (or more than one type) of identifiable species may be used to effect attachment of the entity to a support material whilst leaving a different type (or more than one different types) of identifiable species provided by the entity available for detection (e,g. by any suitable immunological means or any suitable non-ii=unological means) which may include the use of a tracer species capable of giving rise to a detectable signal.

BY way of example any suitable tracer species nay be utilised as desired in accordance with the present invention; examples of such tracer species are enzynes, úluorescent compounds, chemiluninescent componentsl bioluminescent compounds, radioisotopes and dyes.

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

By way of further example, a liquid or liquid sample containing an entity 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 the thin film to provide an 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 glass rod) a thin film of liquid or liquid sample may be provided thereon by dipping the solid surface into a 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 an entity in a liquid or liquid sample may be detected by adding suitable identification reagents to the liquid or liquid sample.

Detection in sltu of an identifiable species (e.g. an identifiable species provided by an entity which entity provides a plurality of identifiable species) may be carried out using any suitable procedure, for example procedures known in the i-mmunological 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 identifiable species may be effected using an identifiable species reaction partner which may be associated with a suitable tracer species. For example. any tracer species signal may be detected in situ.

An entity which provides a plurality of identifiable species may be used, 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 entity).

Thus, for example. where the identifiable species, provided by an entity, are of the same type, an antibody (e.g. which is or nay be attached to a support material) nay be used to attach an entity to a support material, via the antibody and some of the identifiable species, and remaining identifiable species of the entity may be arranged to bind with an essentially similar antibody (which in turn may be directly or indirectly associated with a tracer species) thereby to facilitate detection of Identifiable species.

Subsequently, if desiredi the antibodies may be denatured and the entity recovered for use in a further (e.g. competitive) assay which may be used to confirm results of the non-competitive assay.

Where, for example, an entity provides a plurality of separately identifiable species (e.g. where the entity provides Taore than one type of species) more than one non-competitive assay may be effected with the same entity. Thus. for example, one type of identifiable species nay be used, in combination with a corresponding identifiable species reaction partner, to attach the entity to a support material and one, or more, different Identifiable species provided by the entity may be used in combination with a corresponding identifiable species reaction partner, or partners, and suitable tracer species to effect a non-competitive sandwich assaySubsequently antibodies involved in the assay may be denatured to permit the entity to be recovered and subjected to a further non-competitive assay in which a different type of identifiable species provided by the entity is used to effect attachment of the entity to a support material. The number of different noncompetitive assays it is possible to conduct may depend upon the number of different types of identifiable species provided by the entity. It will be appreciated that the possibility of conducting nore- than one assay on a given sample of entity may offer an advantage in terms of confirming assay results and improving the reliability of results. The use of an entity which provides a plurality of separately identifiable species may offer the possibility of: (a) providing a plurality of independent detections which may lead to improved reliability of detection results; (b) utilizing more than one method of detection (e.g. sequentially) in a given detection procedure; (c) repeating detection in different configurations (e.g. one detection may be carried out using a first identifiable species and a first identifiable species reaction partner to effect immobilisation on a support material and a second identifiable reaction species/second identifiable species reaction partner combination to effect detection and then desorbing the entity from the support material and subsequently using the second identifiable species and second identifiable species reaction partner to effect immobilisation and the first identifiable species and first identifiable species reaction partner to effect detection); (d) overcoming interference with one identifiable species (e. g. by substances from a given environment) since other identifiable species may not be affected by a given interference (e.g. if an immunological reaction is inhibited by environmental influences, non-immunological reactions may still permit detection); (e) high sensitivity of detection by use of identifiable specieslidentifiable species reaction partner combinations which have high affinity (e.g.

picogramme quantities may be detected).

Also, the use of an entity which provides a plurality of separately identifiable species may, for example, otter the possibility of increasing the difficulty which an unauthorized person nay encounter in relation to ascertaining the presence andfor identity of identifiable species utilised.

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 entity, which entity provides a plurality of identifiable species, and performing a detection step to establish the presence or absence of 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 an Identifiable species provided by an entity.

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 an identifiable species reaction partner for undergoing specific interaction with an identifiable species, said identifiable species being provided by an entity which entity provides a plurality of 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 an entity which entity provides a plurality of 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 a 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 also provided a combination comprising a label associated with a solubilising agent, said label being an entity which entity provides a plurality of identifiable species.

The solubilising agent may be a surfactant.

Thus in an embodiment of the present invention there is provided a combination comprising a label associated with a surfactant, said label being an entity which provides a plurality of identifiable species.

An entity may be associated with a surfactant in any suitable manner. For example, an identifiable species of an entity may be conjugated to a reactive site of a surfactant. Alternatively, for example, an identifiable species of different entities may be conjugated to reactive sites of a surfactant. By way of further example, a common carrier material of an entity may be conjugated to a surfactant so that an entity may be associated with a surfactant. A common carrier material may be, for example, associated with a solubilising agent before or after the common carrier material is provided with a plurality of identifiable species (i.e. before or after formation of an entity in accordance with the present invention). It will be appreciated that conjugation may be by covalent bending. It will be appreciated that examples of common carrier materials are hereinbefore disclosed.

r_ PO1YethYlene glycol is an example of a surfactant with which an entity may be associated (e.g. conjugated).

A combination of an entity associated with a surfactant may be soluble in both aqueous and organic liquids. This may offer advantages, for examplef when seeking to introduce an entity to an ink which has organic components (e.g. an organic solvent).

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

In accordance with the present invention there is also provided a combination comprising an entity associated with an insolubilising medium, said entity providing a plurality of identifiable species.

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 any entity associated with the insolubilising inedium is also rendered substantially insoluble in chosen organic or aqueous solvents.

The insolubillsing medium may be, for example, microparticles of micron and sub-micron size (e.g. latex particles, polystyrene nicroparticles, 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.

By way of example, where an entity includes a common carrier material (e. g. a carrier of a polymeric structure) the common carrier material may be attached to an insolubilising medium whereby identifiable species associated with common carrier material become attached to the insolubilising medium.

By way of example, any suitable method of attachment may be utilised, for example those known in the art for attaching species (0.9. biochemical species) to a microparticle.

Examples of attachment are adsorption and covalent bonding.

Thus, for example, a combination comprising an entity associated with an insolubilising medium may comprise an entity, having a plurality of identifiable species attached to a common carrier material, attached by means of the common carrier material to an insolubilising medium comprising a microparticle.

By way of example. a combination of an entity and an insolubilising 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% hydroxypropylmethyl cellulose solution) and such a suspension may offer advantages in, for example, assisting in providing substantially uniform concentration of entity when applied as a label.

It will be appreciated that examples of common carrier materials are hereinbefore disclosed.

As hereinbefore disclosed, an entity may provide, 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, as an identifiable species. Thus, for example, an entity for use as a label nay provide an enzyme molecule or molecules. or a fragment of an enzyme molecule or fragments of enzyme molecules, or a binder for an enzyme molecule or binders for enzyme molecules, or a binder for a fragment of an enzyme molecule, or binders for fragmenr_s of an enzyme molecule. For example, a carrier material may have attached thereto an enzyme molecule or molecules, or a fragment of an enzyme molecule or fragments of enzyme molecules, or a binder for an enzyme molecule or binders for enzyme molecules, or a binder for a fragment of an enzyme molecule. or binders for fragments of an enzyme molecule.

Prom the foregoing disclosure it will be appreciated that even it 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 frhgments of enzyme molecules may still be detectable even though they have lost some or all of choir enzymacic activity.

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

This is possible since structural specificity may be independent of enzymatic activity.

Thus, for example, a competitive inmunoassay 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-conpetitive Immunoassay.

By way of further example, an identifiable species which is an enzyme molecule (e.g. a denatured enzyme molecule) or a fragmenz of an enZY1Tte molecule, may be detected by use of an antibody or an antibody fragment (i.e. (Fab),), which has two binding sites- Thus, for exanple, it nay be arranged that one binding site binds wlzn zne enzyme moiecuie te.g. cenazurea enzyme molecule) or fragment of an enzyme molecule and that the other 1 binding site binds with an enZymatically active enzyme molecule, said enzymatically active enzyme nolecule 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 enzyme molecule or fragment of enzyme molecule (e.g. by any suitable enzymological method). The enzyme molecule or fragment of enzyme molecule nay 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 enzyme molecule or fragment of enzyme molecule nay he 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.

Altnougn reference has been made hereinbefore to the detection of an enzyme molecule, or a fragment of an enzyme molecule. by enzymological nethode it will bo appreciated that a binder for an enzyme molecule or a binder for a fragment of an enzyme molecule may be detected by any suitable inunological method.

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

Prom 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 by any suitable method such as an immunological method. Also, from the foregoing, it will be appreciated that where an identifiable species has suitable enzymatic activity any suitable enzymatic method may be utilised in detection in solution or on a solid surface as appropriateThus, for example, an enzyme may be detected by use of direct detection by application of a suitable substrate (e.g. in a suitable buffer)r or by detection by application of a substrate and a chromogenic substance, or by detection by coupled enzyme reactions, or by detection by enzyme cycling methods (i.e. enzyme amplification methods).

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 sltu 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.

Prom 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 andlor any appropriate enzymological nethod.

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 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, it may be advantageous in some circumstances to use an enzyme bound with its binder, or a fragment of an enzyme molecule bound with its binder, since bound enzyme molecules and bound fragments of enzyme molecules may be more stabIQ -than unbound forms, for Gsxample in proceGeing 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.

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 identifiable species provided by an entity, said identifiable species 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.

By way of yet further example, where an enzyme fragment is, for example, a sub-unit of an enzyme molecule it may be immunologically detectable, but not enzymologically detectable. However, f or example, such a sub- unit may have added thereto a further enzyme molecule sub-unit or sub- units thereby to render it detectable by immunological means.

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

It desired, by way of example, an entity in accordance with the present invention may provide nore 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, and may be used to label a substance or an item.

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- In view of the foregoing disclosure 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.

By way of example. it may be noted that, optionally, for certain applications it may be possible to arrange for a label to become attachedto 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 an entity, which entity provides a plurality of identifiable species.

It will 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) inay 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) in the field of specific interaction assay and, if appropriate, in the field of enzymology (e.g. substrate based systems)-

Thust for example, an identifiable species may be detected by a method which includes immunological binding or by a method which includes nonimmunological binding.

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.

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).

From the foregoing disclosure it will be appreciated that, for example, an entity, or entities, in accordance with the present invention may be used to effect labelling alone. However, if desired, such an entity or entities may be used with other identifiable species (e.g. separate identifiable species) which do not form part of an entity or entities.

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

Reference may be made, for example, to co-pending application (Agent's Reference P160539/HRF) in relation to enzyme molecules and fragments of enzyme molecules and binders therefor in connection with labelling. Reference may be made, for example, to co-pending Application (Agent's Reference P160540/HRF) in relation to llrnaslcecl" ligands in connection with labelling. Reference may be made, for example, to copending Application (Agent's Reference P160541/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:

ú2cainple 1 An entity which provides a plurality of identifiable species was prepared.

The entity was prepared by attaching three different types of identifiable species to a common carrier material.

in this Example the common carrier material was BSA (bovine scrum albumin) and the three different identifiable species were 5(6)-carboxyfluorescein. 2- C(amido-heniglutaryl) phenyl]-6-methyl benzothiazole, and (+)camphorcarboxylic acid.

Before attaching the identifiable species to the common carrier material each of the identifiable species immediately hereinbefore disclosed was activated with Nhydroxysuccinimide and 1,3-dicyclohexylcarbodiinide.

BSA (250 ng; 4.1 g moles) was dissolved in coupling buffer (50 m.M aqueous sodium bicarbonate (pH 8.6)) (50 ml) and dimethyl-fornamide (DMF) (10 ml) and activated (+)-camphorcerboxylic acid (3.2 mg; 16 g moles) was added while stirring.

After stirring for 30 min activated 2-[(amido- hemiglutaryl) phenyl] -6 -methyl benzothiazole A12 Tag; 32 g moles) was added and stirring was continued for a further 30 min, after which activated 5(6)-carboxyfluorescein (24 mg; 64 p moles) was added and the resulting reaction mixture was left with mixing for 16 hours before dialysis for three days to remove unreacted reagents.

An entity was thus obtained which comprised a common carrier material having attached thereto three different identifiable species (in this Example ligands).

Example -2

TO demonstrate detection of individual detectable species in the entity prepared in Example 1 specific binding partners for the identifiable species were used; thus antibodies to the detectable species were used.

1 The antibodies were rabbit anti-5(6)carboxyfluorescein antibody, rabbit anti-2-[(amidohemiglutaryl) phenyl] -6-methyl benzothyiazole antibody and =abb!C anti-(+)-camphorcarboxylic acid antibody.

These antibodies were prepared following known general procedures for producing antibodies adapted as necessary to produce the particular antibodies required.

Wells of nicrotitre plates were coated with 150 gl volumes of serially diluted samples of the entity prepared in Example 1. The serial dilution was effected with coating buffer which was 0. 05 M sodium tetraborate buffer (pH 9.5).

After washing, separate plates were reacted either with anti-5(6)carboxyfluroescein antibody, or with anti2[(amido-hemiglutaryl) phenyl) 6-methyl benzothiazole antibody. or with anti-(+)-camphoricarboxylic acid antibody, suitably diluted in binding buffer. The binding buffer was PBS containing 0.2% gelatin, 10 mg/ml BSA, 0.05 vIv Tween 20, o.ooi% thinerosal and 20 ng/1 rhodamine B base.

The plates were developed using goat anti-rabbit horse radish peroxidase conjugate (ex Sigma) and an HRP substrate mixture (0.5 inglnl of ABTS in 150 mm sodium acetatelcitric acid buffer (pH 4.1) containing 60 g11100 nl of 34% VIV H202) in accordance with known general procedures for developing plates.

Table 1 shows the Optical Density (at 405 nm) results wherein Column A gives the results for the identifiable species 5(6)-carboxyfluorescein, column B gives the results for the identifiable species 2[amidoheniglutaryl) phenyl]-6-methyl benzathiazole and Column C gives the results for the identifiable species(+) camphorcarboxylic acid.

The results show that different detectable species provided by the entity prepared in Example 1 are separately detectable.

r-. - - --i - -, 6 J&ble 1 Amount of entity added per optical Density (OD) at well (ng) 405 nm A B c 6250 3.00 2.97 3.00 1562 3.00 3.00 2.95 390 2.85 2.26 2.15 98 2.15 1.76 1.47 24' 1.71 1.12 0.91 6 0.81 0.63 0.42 j Example 3

An entity which provides a plurality of identifiable species was prepared.

The entity was prepared by attaching three difterent types of identifiable species to a common carrier material.

BSA (250 mg; 4.1 p moles) were dissolved in coupling buffer (50 ml) (see Example 1) and DMF (10 nl), and an activated detectable species was added while stirring; the activated detectable species was biotinamidocaproate N-hydroxysuccinimide ester C15 ng; 33 g Moles) (ex Sigma).

After 30 rnin of stirring activated 5(6)carboxyfluroescein (12 ing) (see Example 1) was added and stirring was continued for a further 30 min after which activated 2-[(amido-hemiglutaryl) phenyl]-6-inethyl benzothiazole (24 mg) (see Example 1) was added and the resulting Tnixture was left with mixing for 16 hours at rooTa temperature before purification by dialysis.

An entity was thus obtained which comprised a common carrier material having attached thereto three different identifiable species (in the Example ligands).

Exa,mPle 4 Detectable species of an entity as prepared in Example 3 were individually detected in a manner similar to that in Example 2; in tests for the detection of biotinamidocaproate, development involved the use of 1 1 3 avidin-peroxidase conjugate (ex Signa). it will be appreciated that avidin is a specific binding partner for biotin.

optical Density results indicated that the three identifiable species of the entity prepared in Example 3 are separately detectable.

Example 5

A combination comprising an entity (providing a plurality of identifiable species) associated with a surfactant was prepared.

In this Example a common carrier material was associated with a surfactant before identifiable species were associated with the common carrier material.

Polyethylene glycol (PEG) (5 9) (ex Sigma) of average molecular weight 20, 000 was dissolved in 1,4dioxan (80 ial) and 1,11-carboxyldiinadazole (CD1) (1.5 g) (ex Signa) was added. After 24 hr at room temperature activated PEG was precipitated from dioxan by the addition of cold diethyl ether (300 M1).

After standing the res'ulting suspension on ice for 15 min solid product was collected by filtration.

BSA (1 9) was dissolved in aqueous sodium bicarbonate solution (85 ml; 100 mM, pH 9.6) and CD1 activated PEG (2.5 g) was added. Af tor 16 hr at room temperature the resulting reaction mixture was dialysed against water for 3 days.

A BSA-PEG material was precipitated f rom water by the addition of 4 volumes of cold acetone and leaving the resulting suspension at -2occ for 4 hr. The BSA-PEG material was collected by centrifugation.

BSA-PEG material (300 ng) was dissolved in coupling buffer (50 ml) (see Example 1) and DMF (10 nl) and activated biotinamidocaproate (15 mg) (see Example 3) was added followed after 30 minutes bv activate 5(6) carboxvf luroescein (24 mg) After dialysis a product was obtained, said product being a common carrier material linked to two identifiable species (two ligands in this Example) and to a surfactant (PEG).

Zx"ple 6 Detectable species of a combination (as prepared in Example 5) comprising an entity (which provides detectable species) associated with a surfactant were individually detected in a manner similar to that in Example 2; in tests for bictinamidocaproate, development involved the use of avidin-peroxidase conjugate (ex Sigma).

The OD results are shown in Table 2,wherein Column A gives results for the identifiable species bictinamidocaproate and Column B gives results for the identifiable species 5(6)-carboxyfluoresceiri.

Table 2

Amount of combination Optical Density (OD) at added per well (ng) 405 nm A B 1000 2.85 2.26 333 2.56 2.08 ill 1.97 1.81 37 1.47 1.46 12 1.20 0.79 4 0.67 0.37 Exam,Ple 7 Detectable species of an entity of a combination (as prepared in Example 5) comprising an entity (which provides detectable species) associated with a surfactant were detected sequentially.

Decreasing amounts of the combination were added to microtitre plate wells in a manner similar to that of Example 2. Biotinamidocaproate was first detected by a method involving the use of avidin-peroxidase conjugate (ex Signa) as hereinbefore disclosed. The Optical Density was measured and the wells were washed. Suitably J diluted (in binding buffer (see Example 2)) rabbit anti- 5(6)-carboxyfluorescein antibody was added to the wells. left to react and the wells were washed. Goat antirabbit-alkaline phosphatase (ex Sigma), suitable diluted in 50 iuM Tris-HCl buffer (pH 7.8) containing loo mM NaCI, 0.5 zam MgC12, 10 Tag/M1 BSA and 0,i% sodium azide, was added. After 1 hr. the wells were washed and 6 mM pnitrophenyl phosphate (dissolved in 10% diethanolamine butter (pH 9.8) containing M9C12) was added. The Optical Density was read at 405 nm after 30 min incubation.

The OD results are shown in Table 3 wherein Column A gives results for the Identifiable species biotinamidiocaproate and Column B gives results for the identifiable species 5(6)carboxyfluorescein.

Table 3

Amount of combination optical Density (OD) at added per well (ng) 405 nm A B 1000 3.00 3.00 333 2.71 2.58 ill 2.10 2.00 37 1.61 1.71 12 1.43 0.93 4 0.71 0.45 Example 8

An entity which provides a plurality of identifiable species was prepared.

Hydroxypropylmethyl cellulose (hpui-cellulose) was first activated. Thus hpn-cellulose (5 g) (ex Signa) was suspended in dry acetone (120 ml) and 1,11carbonyldiinidazole (CD1) (2 g) (ex Signa) was added. After mixing for 24 hours the resulting suspension was cooled to -200C and filtered to allow recovery of activated hpmcellulose.

Diaminooctane (6 g) was dissolved in water (80 ml) and the pH was adjusted to about 9.6 with conc. HCl.

After cooling sodium bicarbonate was added and the pH was adjusted to 9.6. 1,4-dioxan (20 ml) was added after which activated hPm-cellulose (4.5 9) was added and the resulting reaction mixture was stirred for 24 hr. After dialysis against water for 3 days, hpm-cellulose-8diaminooctane product was brought into solution by addition of 3 volumes of 96 ethanol.

in order to prepare an entity having a plurality of identifiable species, hpm-cellulose-S-diaininooctane (1.6 g) was dissolved in water (20 Tal) and ethanol (60 nl)t sodium bicarbonate (0.7 9) was added and the pH adjusted to 8.6. Activated bictinanidocaproate (15 mg), activated 5(6)carboxyfluorescein (24 ng) and activated 2-[(amidoherniglutaryl) phenyl]6-methyl benzothiazole (25 ng) were added in that order leaving reactions to proceed for 30 minutes after each addition. The resulting reaction mixtures were dialysed against 50 mM sodium bicarbonate, then against water. The volume was reduced to about 30 nI and then 2 volumes of 96% ethanal were added to bring the resulting product into solution. It will be appreclated that the product was an entity comprising hpm cellulose-Sdianinocctane having three identifiable species (ligands in this Example) attached thereto.

Example 9

An entity which provides a plurality of identifiable species was prepared.

Ethylcellulose (e-cellulose) was activated with CD1 in a manner similar to that disclosed in Example 8 and ecellulose-B-diaminooetane was prepared in a manner similar to that disclosed for the preparation of hpmcellulose-S-diaminooctane in Example B. Three types of ligands (the types disclosed in Example 8) were attached to e-cellulose-S-dianinooctane in a manner similar to that described in Example 8 to give an entity having three identifiable species.

EXaMD1-e io A combination comprising an entity associated with a surfactant was prepared.

CD1-activated PEG (0.6 g) was added to hpmcellulose-e-diaininooczane (1.8 g) (prepared as disclosed in Example 8) dissolved in so mM sodium bicarbonate (6o ml; pH 9.6) and 1,4-dioxan (30 ml) and the resulting reaction mixture stirred for 16 hr at room temperature.

The resulting product (hpm-cellulose-8diaminooctane-PEG) was recovered in a manner similar to that disclosed in Example 8 and treated in a manner similar to that disclosed in Example 8 to attach to hpmcellulose-adianinooctane-PEC three types of ligand (the types disclosed in example S).

Example 11

Solubility of entities and combinations in organic solvent systems was tested.

The entities prepared as disclosed in Examples 1, 3r 8 and 9 and combinations prepared as disclosed in Examples 5 and 10 were dissblved in 50% ethanol/water and added to 96% ethanol to various concentrations ranging from 0.6 mglml to 6 mgllnl.

All gave homogeneous solutions. After standing for 72 hr, solutions prepared using the entities of Examples 1 and 3 appeared to show fine precipitates. NO precipitates were observed in solutions prepared using the other entitles and the combinations.

Example 12

Entities prepared as disclosed in Examples 1, 3, 8 and 9 and combinations prepared as disclosed in Examples 5 and 10 were added to a commercially available ink vehicle composition (to give ink vehicle compositions each containing a different entity or combination) at concentrations ranging from about 0.8 mg/1 to 80 mgjI to give "labelled" ink vehicle coMpositions. The ink vehicle composition was a liquid containing a mixture of substances normally found in quick drying ink formulations except for a colourant dye. (a) 50 g 1 quantities of ink vehicle compositions each containing an entity (prepared as described In Examples 1, 3, 8 or 9) or a combination (prepared as described in Examples 5 or 10) were added to wells of microtitre plates and left to dry at 400C. Control test wells having ink vehicle compositions without added entities or combinations were included in all plates.

Detection of various identifiable species was carried out as appropriate, using procedures similar to those disclosed in Examples 2, 4 and 6.

It was observed that wells to which labelled ink vehicle compositions had been added showed clearly visible blue colour, the highest colour intensity corresponding to the highest concentration of entity or combination.

Control test wells with unlabelled ink compositions showed no observable colour during a 15 observation period after development. _ (b) Glass rods (4 mm diameter, 80 nin length) were dipped to 15 ran depth for about 5 seconds into an ink vehicle composition labelled with the entity prepared in Example 8.

Idenzical glass rods were dipped into an unlabelled ink vehicle composition and used as controls.

After drying, individual rods were dipped (for 2 min) into solutions containing, diluted in binding buffer, specific binding partners for the three identifiable species. (Reference may be nade to Examples 2 and 4 regarding specific binding partners for the identifiable species used in this Example.) After washinS, the rods were subjected t-G test development, for the detection of identifiable 7 -43^ species by transferring the rods to test tubes containing 1.5 ml quantities of appropriate reagents. it will be appreciated that reference may be inade to Examples 2 and 4 regarding appropriate reagents.

Colour development was observed within about 4 minutes from contact with final reagent (peroxidase substrate) for rods dipped into labelled ink vehicle compositions. Unlabelled controls showed considerably less colour. After 10 min contact. with the final reagent. the rods were removed and the developed colour measured in a spectrophotometer at 405 nn.

The results are shown in Table 4 wherein column A gives results for the rods treated with labelled ink vehicle composition and column B gives results for the controls.

T!b-1-e-4.

Detectable species Optical Density (OD) at.405 nm A B Biotinamidocaproate 0.86 0.24 5(6)-carboxyfluorescein 1.04 0.21 2f(amido-hemiglutaryl) phenyl)-6- 0.43 0.2 methyl benzothiazole (c) A thin film of ink vehicle composition labelled with the entity prepared in Example 8 was formed on the outer surface of the bottom of tin cans by applying the ink vehicle composition by a dropping pipette and withdrawing excess liquid. The applied composition covered an area of about 25 mm diameterAfter drying with hot air, appropriate test reagents for detecting the identifiable species of the entity were applied. It will be appreciated that reference may be made to Examples 2 and 4 regarding appropriate reagents.

Colour was observed with the cans within the first 5 min of contact with final reagent (peroxidase substrate). Substantially less colour was observed after 15 min in the case of control tests with cans which had been treated with unlabelled ink vehicle composiZion.

ExaYnple 13 A combination comprising an entity associated with an insolubilising medium was prepared.

In this Example, the entity used to prepare the combination was, as it happens, associated with a surfactant (PEG). However, it is to be understood that the presence of PEG associated with the common carrier does not affect the insolubilising aspects demonstrated In this Example.

* Microparticles (microcellulose powder) (1.5 g) were suspended in water (30 ml) and methanol (30 inl) and sodium periodate (1 9) in w:ater (15 ml) was added. After mixing in-the dark for 6 hr, the periodate activated microparticles thus prepared were collected by centrifugation and washed 3 times.

The combination prepared in Example 5 (40 ing) in 50 mm phosphate burrer (12 mi; pH 6.7) was mixec for 16 hr with approximately 0.5 g periodate activated microparticles.

The particles were washed and stored in 50% ethanol.

Example 14

The activity of identifiable species in the combination (cf entity associat-ed with an insolubilising medium) prepared in Example 13 was investigated- Thus, in test experiments 50 pl volumes of inicrocelluiese particles having attached thereto an entity which Drovides a plurality of identifiable species were suspended in 250 pl, of binding buffer containing 0.8k hydroxypropylmethyleellulose and reacted either with avidin-peroxidaze conjugate or with rabbit antifluorescein antibody.

Detection of biotinamidocaproate was effected by avid in-peroxidase conjugate (using ABTS and H20. to detect HRP activity in accordance with known procedures) and detection of fluorescein was effected by use of goat anti-rabbit-alkaline phosphatase and a suitable substraze (in accordance with known procedures).

Control experimenCe were conaucted in which the procedures were the same except for the fact that avidin- peroxidase and rabbit anti-fluorescein antibody were OTAitted.

The results (in terms of optical Density) are given in Table 5 wherein Colunn A gives results for the test experiments and Column B gives results for the control experiments.

Table 5

Identifiable species optical Density (OD) at 405 nm A B Bi,Otin 1.65 0.17 5(6)-carboxytluorescein 1.53 0.16 Exapple-15 The activity of identifiable species in the combination prepared in Example 13 when in a dried ink vehicle composition was investigated.

A commercially available ink vehicle composition was added to various amounts of the combination prepared in Example 13 (i.e. a combination of an entity associated with insolubilising medium) to give labelled ink vehicle compositions- 1 volumes of 100 P1 of the labelled ink vehicle compositions were spread on petri dishes and allowed to dry at 400C. Control experiments were conducted with equal volumes of unlabelled ink vehicle compositions.

The resulting dried samples were subject to detection by procedures similar to those disclosed in Example 14.

Dishes whi.ch were originally treated with labelled ink vehicle compositions showed colour after 4 to 5 min and were distinguishable from control experiments by eye.

Example 16

The enzyme alkaline phosphatase (AP) (EC3.1.3.l.) Was coupled to activated hpm-cellulose. The activated hpm-cellulose was prepared as in Example 8.

CD1-activated hpm-cellulose (100 mg) was added to AP 1.5 mg dissolved In 50 TaM sodium tetraborate buffer (2 ml; pH 9.6) and the resulting mixture was mixed for 16 hr at room temperature after which 4 volumes of 96% ethanol were added to bring constituents into solution. Volumes of the hpm-cellulose-AP weri- added to a commercially available ink vehicle composition to give labelled ink vehicle compositions.

Dried layers of the ink vehicle compositions were found to have enzyinatic activity when tested using indloyl phosphate substrate in an appropriate buffer medium.

Claims (53)

Claims

1. An entity suitable for use in labelling of a substance or an item which entity provides a plurality of identifiable species.

2. An entity as claimed in Claim 1 suitable for use in labelling of a substance or an item which entity provides a plurality of-separately identifiable species.

3. A method suitable f or use in labelling of a substance or an item which method includes providing a substance or an item with an entity which entity provides a plurality of identifiable species.

4. A method as claimed in Claim 3 suitable f or use in labelling of a substance or an item which method includes providing the substance or item with.an entity which entity provides a plurality of separately identifiable species.

5. An entity or a method as claimed in any one of the preceding Claims wherein an entity comprises a plurality of identifiable species linked together.

6. An entity or a method as claimed in any one of the preceding claims wherein the entity comprises a plurality of identifiable species attached to a common carrier material.

7. A.method as claimed in any one of Clains 3 to 6 wherein an entity which provides a plurality of identifiable species is incorporated into a substance.

8. A method as claimed in any one of Claims 3 to 6 wherein an entity which provides a plurality of identifiable species is applied to an item.

9. A method for the preparation of an entity, which entity provides a plurality of identifiable species, which method includes attaching a plurality of identifiable species to a common carrier material.

10. A method as claimed in Claim 9 which method includes attaching a plurality of the same identifiable species to a common carrier material.

11. A method as claimed in Claim 9 which method includes attaching a plurality of different identifiable species to a comon carriei material.

12. A method as claimed in claim 9 or Claim 11 wherein an entity Is formed by attaching at least one type of antigenic ligand and at least one type or non-antigenic ligand to a common carrier material.

13. A method as claimed in Claim 9 or Claim 11 wherein an entity is formed by attaching at least two different types of non-antigenic ligands to a common carrier material.

14. An entity or a method as claimed in any one of Claims 1 to 11 wherein the identifiable species is a ligand or a binder.

15. An entity or a method as claimed in claim 14 wherein the ligand is an antigenic ligand.

16. An entity or a method as claimed in claim 14 wherein the entity is a non-antigenic ligand.

17. An entity or a method as claimed in Claim 14 wherein the binder is a binder for-an antigenic ligand.

18. An entity or a method as claimed in Claim 14 wherein the binder is a binder for a non-antigenic ligand.

19. An entity or a method as claimed in any one of Claims 1 to 11 wherein the identifiable species is 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.

20. An entity or a method as claimed in any one of Claims 1 to 12 or Claim 14 or Claim 15 wherein the identifiable species is an antigenic ligand which is a peptide, a protein, a hapten, an enzyme molecule, or a fragment of an enzyme molecule.

21. An entity or a method as claimed in any one of Claims 1 to 12 or Claim 14 or Claim 17 wherein a binder is an antibody.

22. An entity or a method as claimed in any one of Claims 1 to 13 or Claim 14 or claim 16 wherein the 1 2 identifiable species is a non-antigenic ligand which is a steroid, a carbohydrate, an immunoglobulin, Fc fragments of immunoglobulins, glycoproteins, Vitamin D or biotin.

23. An entity or a method as claimed in any one of Claims 1 to 13 or Claim 14 or claim 18 wherein the binder is a serum protein, a receptor, a lectin, protein A, protein G, vitamin D binding protein or avidin.

24. An entity or a method as claimed in any one of Claims 1 to 19 wherein the identifiable species is detectable as such.

25. An entity or a method as claimed in any one of claims 1 to 19 wherein the identifiable species is a species which may be formed into a species which may be detected.

26. An entity which entity provides a plurality of the sama non-immunological ligands or non-immunological binders; or a plurality of different non-immunological ligands or non- immunological binders; or a plurality of the same immunological ligands or immunological binders; or a plurality of different-immunological ligands or immunological binders; or a non-immunological ligand or a non-immunological binder (or a plurality of such ligands or binders) and an immunological ligand or an immunological binder (or a plurality of such ligands or binders); or a ligand andlor a binder, and a DNA sequence andlor a DNA sequence marker; or 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 a plurality of "masked" ligands of the same type; or a plurality of "masked" ligands of different types; or a "masked" ligand or "masked" ligands and a ligand which is not "masked" or ligands which are not "masked"; or a plurality of different enzyme molecules, or of different fragments of enzyme molecules, or of different binders for enzyme molecules, or of different binders for fragments of enzyme molecules; or a ligand and an enzyme molecule or a fragment of an enzyme -so- molecule, or a binder for an enzyme molecule, or a binder 14 for a fragment of an enzyme i.,nolecule,.(r a binder and an enzyme molecule or a fragment of an enzyme molecules, or a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

27. A method suitable for use in identifying a substance or an item, which method Includes providing a substance or an item with an entity, which entity provides a plurality of identifiable species, and performing a detection step to establish the presence or absence of identifiable species.

28. 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 identifiable species provided by an entity.

29. A method as claimed in Claim 27 or Claim 28 wherein an identifiable species is detected by a method which includes immunological binding.

30. A method as claimed in claim 27 or Claim 28 wherein an identitlable species is detected by a methad which includes non-immunologicallinding.

31. A method as claimed in Claim 27 or claim 28 wherein an identifiable species is detected by an enzymological method.

32. A method as claimed in any one of Claims 27 to 31 wherein an entity is subjected to detection in s!Lu whilst still attached to an item.

33. A method as claimed in any one of claims 27 to 31 wherein an entity is retrieved from a substance or an item before being subjected to detection.

34. A method as claimed in any one of Claims 27 to 31 or Claim 33 wherein a liquid or 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.

35. A composition suitable for use in labelling of a substance or an item which composition includes an entity which entity provides a plurality of identifiable species.

36. A composition as claimed in Claim 35 wherein the composition is an ink composition, or an ink vehicle composition. or a paint composition.

37. A method as claimed in any one of Claims 3 to 27 wherein a substance or item 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, high volume sales item, a prestige high value article, chemical formulation, meat, a meat product or packing for goods -

38. A method as claimed in any one of Claims 3 to 27 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 paint-stuff for use with an item or packaging, adding a label directly solutions or formulations o ú 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.

39. A combination comprising a label associated with a solubilising agent, said label being an entity which entity provides a plurality of identifiable species.

40. A combination as claimed in Claim 39 wherein the solubilising agent is a surfactant.

41. A combination as claimed in Claim 40 wherein the surfactant is polyethylene glycol.

42. A combination comprising a label associated with an Insolubilising medium, said label being an entity which entity provides a plurality of identifiable species.

43. A combination as claimed in Claim 42 Wherein the insolubilising medium is microparticles.

44. A test-kit suitable for use in identifying a substance or an item, which test-kit includes an identifiable species reaction partner for undergoing specific Interaction with an identifiable species. said identifiable species being provided by an entity which entity provides a plurality of identifiable species.

45. Attest-kit suitable for use in identifying a substance or an item, which test-kit includes selected reagents for enzymological d.etection of an identifiable species provided by an entity said identifiable species being an enzyme molecule, or a fragment of an enzyme molecule. a binder for an enzyme molecule, or a binder for a fragment of an enzyme molecule.

46. A labelled substance or a labelled item wherein the substance or item is labell.ed with an entity which provides a plurality of Identifiable species.

47. An entity substantially as hereinbef ore- described with reference to Example 1, Example 3, Example 8, Example 9 or Example 16.

48. A method suitable for use in the labelling of a substance or an item substantially as hereinbefore described with reference to Example 12 or Example 15.

49. A method for the preparation of an entity substantially as hereinbefore described with reference to Example 1, Example 3, Example 8. Example 9 or Example 16.

50. A method suitable for use in identifying a substance or item substantially as hereinbefore described with reference to Example 12, Example 15, or Example 16.

51. A composition substantially as hereinbefore described with Reference to Example 12, Example 15, or Example 16.

1 1

52. A combination substantially as hereinbefore described with reference to Example 5, Example 10 or Example 13.

53. A labelled substance or a labelled item subs,tantially as hereinbefore described with reference to Example 12. or Example 15.