GB2286674A

GB2286674A - Tagging substances or items

Info

Publication number: GB2286674A
Application number: GB9425924A
Authority: GB
Inventors: Ramadan Arbi Abuknesha
Original assignee: GEC Marconi Ltd; Marconi Co Ltd
Current assignee: BAE Systems Electronics Ltd
Priority date: 1993-12-23
Filing date: 1994-12-22
Publication date: 1995-08-23
Also published as: GB9326277D0; GB2286044A; GB9425925D0; JPH09508199A; GB9425922D0; JPH09509372A; DE4446042A1; FR2714474A1; WO1995017669A1; GB2286672A; JPH07203987A; GB2286673A; GB9425923D0; JPH09506972A; WO1995017670A1; WO1995017667A1; GB9425924D0

Description

1 - 1 2286674 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 convertible species.

According to another 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 convertible species and performing a step by means of which a convertible species may be used to provide an 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 convertible species, performing a step by means of which a convertible species may be used to provide an identifiable species, and performing a detection step to establish the presence or absence of an 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 a convertible species. By way of example, one type of convertible species may be used in accordance with the present invention or, optionally, more than one type of convertible species may be used in accordance with the present invention.

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 step by means of which a convertIble species may be used to provide an 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 step by means of which a convertible species may be used to produce an identifiable species, and performing a detection step to establish the presence or absence of an identifiable species.

An identifiable species may be, for example, any species the presence of which may be detected.

It will be appreciated, by way of example, that a step by means of which a convertible species may be used to produce an identifiable species may be performed whether or not a convertible species is present. Where, for example, such a step is performed in the absence of a convertible species a detection step may indicate the absence of identifiable species which may indicate the absence of a convertible species. Where, for example, such a step is performed in the presence of a convertible species a detection species may indicate the presence of identifiable species which may indicate the presence of a convertible species.

Thus, for example, it is to be understood that by providing a chosen substance or a chosen item with a selected convertible species it may be possible,for example, to distinguish that substance or item from other 1 % substances and items by testing to ascertain whether, or not, the selected convertible species is present.

Thus, for example, if performing a step by means of which a convertible species may produce an identifiable species results in the production of an identifiable species (as may be detected, for example, in a detection step) this may indicate that the substance or item tested is a chosen substance or chosen item. However, if performing a step by means of which a convertible species may produce an identifiable species does not result in the production of identifiable species, this may indicate that the substance or item tested is not a chosen substance or item.

Any suitable species may be utilised as a convertible species in accordance with the present invention, for example, if the said suitable species may be used to produce an identifiable species.

By way of example, a convertible species may be a species which may be converted thereby to become an identifiable species or a convertible species may be a species which is capable of giving rise to identifiable species. Thus, for example, a convertible species may be a species which may itself be converted into an identifiable species, or a convertible species may be a species which is capable of producing a product (e.g. by being treated in an appropriate manner) which product is an identifiable species which may be detected either directly or indirectly in any suitable manner.

By way of example, where a convertible species may be converted to become an identifiable species, any suitable first species which may be converted into a second species (which is identifiable) may be utilised.

An example of a convertible species which may be converted to an identifiable species is a ligand which has a selected specific binding capability thereof substantially inhibited such that the ligand cannot bind with a selected specific binding species. An example of 1 such a ligand which has a selected specific binding capability substantially inhibited is a ligand to which a "blocking" entity has been added. Such a ligand with a blocking entity may be considered to be a "masked" ligand. Removal of the blocking entity (which may be considered to be "unmasking" of the ligand) renders operative the capability of the ligand to bind with the selected specific binding species.

Thus, for example, where the convertible species is a ligand which has a selected specific binding capability inhibited, conversion of the convertible species into a ligand which has selected specific binding capability converts the convertible species into a ligand which may be an identifiable species. For example, a ligand which has a specific binding capacity may be detected by use of, inter alla, a specific binder for the ligand.

By way of further example, a convertible species may be a substrate for an enzyme and such a substrate may be acted upon by an enzyme to give products which may be identifiable species (e.g. species which may be detected directly or indirectly by any suitable method).

By way of further example, a convertible species may be a species which is a combination of a substrate moiety and a signal moiety. Such a combination may be considered, for example, to be a synthetic substrate for an enzyme. For example upon action of an enzyme upon such a substrate, removal of the substrate moiety may lead to a signal of the signal moiety becoming detectable.

By way of further example, a convertible species may be a "blocked" cofactor, the conversion of which to an unblocked co-factor gives co-factor which may be an identifiable species. Such an unblocked co-factor may be detected by any suitable means (e.g. enzymologically).

In accordance with the present invention, it is to be noted that a step by means of which a convertible species may be used to produce an identifiable species may include one stage or any number of stages as may be appropriate. Also, it is to be noted that, for example, a detection step may involve one stage or any number of stages as may be appropriate.

Some convertible species may be utilised in more than one way.

Thus, for example, where a "masked" ligand is also a synthetic substrate for an enzyme (and said substrate includes a signal moiety) the "unmasked" ligand may be detected by its own signal (e.g. fluorescence) or may be detected immunologically (e.g. by a method which includes a corresponding binding species).

Further details regarding a convertible species are now given by way of example.

For example, a convertible species may be a species which may have something added to it to convert it into an identifiable species, or it may be a species which may have something removed from it to convert it to an identifiable species, or it may be a species which can be changed in any suitable manner to convert it to an identifiable species.

Thus, for example, a convertible species may be, for example, regarded as a precursor for an identifiable species. For example, the convertible species may be formed into an identifiable species by means of chemical synthesis. Alternatively, by way of example, a convertible species may be essentially similar to an identifiable species with the exception that the convertible species carries a "blocking" entity which inhibits its capability of interacting (e.g. reacting) with a species which may take part in a detection step On removal of the blocking entity (i.e. the "unblocking" of the convertible species) the first convertible species may be formed into an identifiable species which may be then capable of interacting (e.g. reacting) with species which may take part in a detection step.

1 It will be appreciated that "unblocking" of a convertible species may be considered to be, for example, "unmasking" of a "masked" species. Thus, for example, where the convertible species is a "masked" ligand the ligand may be "unmasked to give an identifiable species.

It is to be understood that the removal of a blocking entity may be considered to be "unmasking" of a "masked" species or the "switching on" of an interacting (e.g. reacting) capability of a species.

By way of example, any suitable structural change may be utilised in accordance with the present invention to obtain an identifiable species by use of a convertible species.

For example, any suitable structural change may be utilised in accordance with the present invention to obtain an identifiable species from a convertible species.

It will be appreciated that a structural change may result in species having new functions.

By way of example, any substance (e.g. an organic substance) which can act as a ligand, and which can exist in more than one stable form or structure may be suitable to provide a convertible species and an identifiable species in accordance with the present invention.

Thus, by way of example, to obtain identifiable species being a ligand any suitable convertible species (which may be considered to be a pro- ligand) capable of forming the ligand may be utilised.

For example, an antigenic ligand (e.g. a hapten) may be selected as the identifiable species and antibodies (either monoclonal or polyclonal) to the ligand may be raised (e.g. in any suitable manner such as those known in the art) said antibodies being species which may take part in a detection step. It will be appreciated that the antibodies to the ligand are binder species which may react with the ligand.

1 The identifiable species may be formed from the convertible species by any suitable means, for example by chemical means or biochemical (e.g. enzymatic) means.

It is to be understood that formation of the identifiable species from the convertible species may be effected in any suitable manner (i.e. "switching on" of an interacting (e.g. reacting) capability of a convertible species (e.g. interacting capability with respect to a species which may take part in a detection step) may be effected in any suitable manner), for example, by adding something to a convertible species, or by removing something from a convertible species, or by exposing a ligand (e.g. an antigenic determinant) of a convertible species (e.g. by a conformational change).

By way of example, the convertible species may be chosen or arranged such as to have stability which is satisfactory under conditions involved in its storage and use.

By way of example, any suitable ligand may be utilised as an identifiable species, examples of which ligands are antigenic ligands and nonantigenic ligands.

Examples of antigenic ligands are peptides, proteins and haptens. 7hydroxy4-methyl coumarin-3-propionic acid is an example of an antigenic ligand. Examples of non-antigenic ligands are ligands of specific ligandbinder species paris (e.g. the ligand biotin in the case of the specific ligand-binder species pair biotinavidin).

By way of example, any suitable binder species for a ligand may be utilised in a detection step; the binder species may be, for example, a binding protein (e.g. an antibody or a binding partner for a nonantigenic ligand).

For example, where the identifiable species is an antigenic ligand a binder species may be an antibody to the ligand.

-8 Accordingly where, for example, the identifiable species is fluorescein, a binder species may be anti-7hydroxy-4-methyl coumarin-3-propionic acid antibody.

Wherel for example, the identifiable species is a non-antigenic ligand, the ligand may be, for example, such that the binder species is a binding partner that is a non-immunoglobulin (e.g. a naturally-occurring protein); the binding partner may be considered to be the binder of the ligand. An example of such a binding partner is avidin in the specific ligand-binder species pair comprising a biotin-avidin complex.

An identifiable species may be formed from a convertible species in any suitable manner as hereinbefore disclosed. For example a convertible species may be a "masked" ligand comprising an enzyme substrate (e.g. a substrate for a hydrolase) which may be converted, enzymatically, into constituents or product fragments which may act as an identifiable species. A "masked" ligand comprising an enzyme substrate and constituents produced by enzymatic action may be arranged, for example, to differ structurally so as to have large differences in affinity for a given binder.

Examples of enzymes suitable f or use in an enzyme/ enzyme substrate system are glactosidases, glucosidases and phosphatases; thus, for example, galactosyl-7-hydroxy coumarin may be formed into galactose + 7hydroxy coumarin, or glycosyl-7-hydroxy coumarin may be formed into glucose + 7-hydroxy counarin, or phosphate-7-hydroxy coumarin may be formed into phosphate + 7-hydroxy coumarin. By way of further example, an enzymelenzyme substrate system which yields nitrophenol (e.g. by conversion of nitrophenol derivatives) may be used. In the immediately foregoing examples it will be appreciated that the enzymatic substrates constitute convertible species (being ligand precursors) and the ligands 7hydroxy coumarin and nitrophenol constitute identifiable species.

k 9_ It will be appreciated that, for example, a ligand and a corresponding binder may be considered to be specific binding partners.

By way of example, it is preferred that any affinity of a convertible species for a given binder species is very much lower (e.g. three-fold lower and preferably 1% or lower) than affinity between identifiable species formed from a convertible species and the given binder species.

Reference has hereinbefore been made, by way of example, to convertible species which may be acted upon to give products which may be identifiable species. Further details of such convertible species are now given.

Thus, for example, a convertible species may be a substrate for an enzyme such that upon interaction with the enzyme the substrate produces products which are identifiable species in that, for example, the products may react with other reagents (e.g. chromogenic or fluorogenic substances, to produce a colour signal or to produce a fluorescent signal), or may take part in a coupled enzyme reaction (in which the products react with another enzyme to produce a final detectable product) or may take part in enzyme cycling methods (enzyme amplification methods) where the product act as cofactors for a second enzyme-substrate system and the cofactors are regenerated by a third enzyme to re-enter the second enzyme system and so on.

Reference has hereinbefore been made, by way of example, to convertible species which is a combination of a substrate moiety and a signal moiety. Further details of such a convertible species are now given.

Thus, a convertible substance may be, for example, a species which is a combination of a substrate moiety and a signal moiety. As hereinbefore disclosed such a combination may, for example, be considered to be a synthetic substrate.

The substrate moiety may be, for example, any suitable substrate which a selected enzyme may act upon and the signal moiety may be, for example, any suitable signal moiety, examples of which are chromogenic and fluorogenic moieties.

It will be appreciated that action of an enzyme on such a combination may lead to removal of substrate moiety such that signal of the signal moiety becomes detectable. Thus a colour signal or a fluorescence signal may become detectable.

It will be appreciated that, for example, the substrate comprising a combination of substrate moiety and signal moiety is a convertible species which may be formed into a detectable species by means of removal of the substrate moiety and thus, the combination may be considered, for example, to be a species in which the signal moiety (identifiable species) is "masked".

Reference has hereinbefore been made, by way of example, to a convertible species which is a "blocked" co-factor. Further details of such a convertible species are now given.

Thus, a convertible species may be a co-factor which is "blocked" in the sense that it is not available for reaction with a selected enzyme. However, such a cofactor may be unblocked, for example, by action of another enzyme so as to be available to detection by any suitable method which may include, for example, interaction with the selected enzyme.

By way of further example, a co-factor system such as Cofactor I-Cofactor II may be utilised in accordance with the present invention to provide a convertible species and an identifiable species. Thus, for example, NADNADH or NADP-NAD may be utilised.

Also, by way of example, a system involving reduced chromophore dye oxidised chromophore dye may be utilised in accordance with the present invention to 11- provide a convertible species and an identifiable species.

Examples of further systems which may be used to produce a convertible species and an identifiable species are ultraviolet (UV) light-sensitive precursor substances that may be influenced by photolysis and chelating agent/ chelate-metal complex systems.

Also, by way of example, it may be possible to use a sub-unit of an antigen as a convertible species and identifiable species comprising an antigen may be formed by adding a further sub-unit or sub-units to the said sub-unit.

It will be appreciated that, by way of example, an identifiable species comprising an antigenic ligand may be detected (either directly or indirectly) by a detection step which includes the use of any suitable immunological binding method which includes the use of a corresponding binder species (linked directly or indirectly) to a tracer species. Also it is to be noted that, by way of example, a non-immunological ligand may be detected (directly or indirectly) by a detection step which includes any suitable non-immunological binding method which includes the use of a corresponding nonimmunological binder species (linked directly or indirectly) to a trace species. Detection by an immunological or by a nonimmunological binding method may, for example, be carried out in solution or using a support material.

It will also be appreciated that where, for example, a convertible species is a substrate which is a combination of a substrate moiety and a signal -moiety, formation of identifiable species by removal of the substrate moiety, is converting the convertible species to an identifiable species. It will be appreciated that, for example, where such conversion reveals a signal species, such that a signal may be detected, then it may not be necessary to carry out any other step to render an identifiable species detectable.

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).

In accordance with the present invention a substance or an item may be provided with a label, said label being a convertible species, in any suitable manner.

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

Thus for example, a label which comprises a convertible species may be incorporated into a substance.

Where an item is to be provided with a label the label may be provided in any suitable manner (e.g. by application to the item). Thus, for example, a convertible species 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 which comprises a convertible species 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 a convertible species. A composition in accordance with the present invention may be, for example, an ink composition which includes a convertible species or an ink vehicle composition which includes a convertible species or a paint composition which includes a convertible species.

where, for example, a convertible species is an enzyme substrate which upon interaction with an enzyme may produce products which may interact, directly or indirectly, with a reagent or reagents to produce a signal, such a reagent or reagents may be incorporated, for example, into a composition together with the convertible species, such as to be available for signal production.

Chromogenic species and fluoragenic species are examples of such reagents.

By way of example, the reagent or reagents may interact with products produced by action of enzyme upon the enzyme substrate, or the reagent or reagents may interact with products produced in a further enzyme system or further enzyme systems as a result of action of products produced by the enzyme substrate.

In accordance with the present invention there is provided a composition which includes a convertible species and a reagent which may be capable of producing a signal.

Such a composition which includes a convertible species and a reagent which may be capable of producing a signal may be, for example, an ink composition or an ink vehicle composition.

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

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

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

In accordance with the present invention, a substance or an item may be provided with a label, comprising a convertible 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 as chromatography) but such that the amount of label used would be sufficient to facilitate detection by an authorised person using authorised detection reagents.

-is- 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 Mg/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 aii item may be identified so as to authenticate the substance or item.

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

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

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

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

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

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

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 andbogus goods. Such labelling may be used, for example, to permit a genuine manufacturer to identify, unequivocally, a substance or an item produced by the manufacturer and distinguish such a substance or item from a non-genuine substance or item. This offers the possibility of a genuine manufacturer to seek to inhibit loss of revenue which may be caused by the presence of. non-genuine substances or items in a given market. For example, manufacturers may be able to discover substances of items which, although not provided by them, bear their brand names andlor packaging.

If desired the present invention may, for example, be utilised to label a substance or an item to indicate batch number and/or "best before" date.

By way of example, a label may be applied to an article in any suitable arrangement; thus, for example, a composition (e.g. an ink) containing a label comprising a convertible 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 0.180,000 Mg/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 solut ion 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 a convertible species. In accordance with the present invention an identifiable species produced by use of a convertible species may be detected in any suitable manner. By way of example, identifiable species may be detected, in situ (e.g. whilst still attached to an item). Thus, for example, a convertible species may be treated to produce an identifiable species in situ (e.g. whilst still attached to an item) and the identifiable species may be subjected to detection in situ (e.g. whilst still attached to an item). Alternatively, by way of example, a convertible species may be retrieved from a substance or item, treated to produce an identifiable species, and the identifiable species subjected to detection. By way of further example, a convertible species may be treated in situ (e.g. whilst still attached to an item) to produce an identifiable species and the identifiable species may be retrieved and subjected to detection.

Retrieval of a convertible species or an identifiable species may be effected in any suitable manner.

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

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

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

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

Retrieval may, for example, include solvent extraction, however, there may be circumstances where solvent extraction may not be applicable in the recovery of a convertible species or an 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 the convertible species or identifiable species to be detected. Thus, for example, a solvent or a solvent system may be chosen such that the use thereof does not cause unacceptable changes in the convertible species or the identifiable species.

Where a convertible species or an identifiable species has been retrieved (e.g. extracted) the convertible species or identifiable species may be immobilised in any suitable manner so as to permit detection (e.g. by a specific protein binding assay (e.g. an immunoassay procedure) or by any other suitable detection procedure). It will be appreciated that the detection procedure used may be dependent upon the identifiable species to be detected.

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

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

-21 By way of further example, if appropriate, specific interaction (e.g. specific binding) may be used to immobilise a retrieved convertible species or identifiable species. Thus, for example, a specific reaction partner (being a binding species such as an antibody or a binder for a non-antigenic ligand) for a convertible species or an identifiable species may be used to effect attachment to a support material by use of specific interaction.

By way of example, a binder species for a convertible species or an identifiable species may be associated with a support material before or after undergoing specific interaction respectively with the convertible species or identifiable species.

A binder 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 links (e.g. specific or non-specific in nature).

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

Alternatively, by way of example, a binder species may be arranged to be associated with a support material (before or after reaction with a convertible species or an identifiable species) by means of an auxiliary binder or an auxiliary ligand system (e.g. by means of a further ligandbinder system).

Once a convertible species or an identifiable species has been immobilised on a support material it ma be treated to produce an identifiable species and the identifiable species may be detected in any suitable way such as those known in the immunological field and in the non-immunological field. Alternatively, once an identifiable species produced by treating a convertible 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, a suitable binder for an identifiable species may be arranged to be associated with a tracer species capable of giving rise to a detectable signal.

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

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

By way of further example, a liquid or liquid sample containing a convertible species or an identifiable species may be provided as a thin film of the liquid or liquid sample on a suitable solid surface and this film may be dried to provide on the solid surface a thin layer of material which then may be subjected to detection where an identifiable species is present or treated to_ produce an identifiable species (by use of the convertible species) and then 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.

By way of example, a thin layer of material (which includes a convertible material) on a suitable solid 11 surface obtainable as immediately hereinbefore disclosed may be treated to produce an identifiable species and the identifiable species subjected to detection in any suitable manner, for example, by adding a reagent or reagents capable of producing an identifiable species by use of the convertible species and adding an identification reagent or reagents or by any suitable method (e.g. an assay method).

By way of further example, a thin layer of material (which includes an identifiable species provided by use of a conventional species) on a solid surface obtainable as immediately hereinbefore disclosed may be subjected to detection in any suitable manner, for example, by adding an identification reagent or reagents or by any other suitable method (e.g. an assay method).

By way of further example, a convertible species in a liquid or liquid sample may be treated to produce an identifiable species by use of the convertible species and the identifiable species subjected to detection by adding to the liquid or liquid sample a reagent or reagents capable of producing an identifiable species by use of the convertible species and the identifiable species may be detected by adding suitable identification reagents to a liquid or liquid sample.

By way of further example, an identifiable species (produced by use of a convertible species) in a liquid or liquid sample may be detected by adding suitable identification reagents to the liquid or liquid sample.

It will be appreciated that a convertible species in a liquid or liquid sample may be treated so as to produce identifiable species by adding suitable reagents to the liquid or liquid sample.

Detection in situ of an identifiable species produced by use of a convertible 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 -24 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 produced by use of a convertible species may be effected using a binder species for an identifiable species, which binding species may be associated with a suitable tracer species. For example, a tracer species may be detected in situ.

An identifiable species produced by use of a convertible 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 provided by use of a convertible species).

Thus, for example, an antibody for an identifiable species (e.g. which antibody 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, the 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.

It is to be understood that, for example, a competitive immunoassay or a non-competitive immunoassay may be utilised to detect an identifiable species produced using a convertible species.

According to a further aspect of the present invention there is provided a tes---.-kit suitable for use in identifying a substance or an item, which test-kit includes means for performing a step by means of which a convertible species may be used to produce an identifiable species.

A test-kit in accordance with the immediately preceding aspect of the present invention may, for example, also include means for performing a detection step to establish the presence or absence of an 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 convertible 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 label associated with a solubilising agent, said label being a convertible species.

The solubilising agent may be a surfactant.

Thus, in one embodiment of the present invention there is provided a combination comprising a label associated with a surfactant, said label being a convertible species.

A convertible species may be associated with a surfactant in any suitable manner. For example, a reactive site or sites of a surfactant may be conjugated with a convertible species.

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

1 Polyethylene glycol is an example of a surfactant with which a convertible species may be associated (e.g. conjugated).

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

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

In accordance with the present invention there is also provided a combination comprising a label associated with an insolubilising medium, said label being a convertible 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 label associated with the 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 label with an insolubilising medium.

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

Examples of attachment are adsorption and covalent bonding.

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

By way of example, a combination of a convertible species in accordance with the present invention, 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% hydroxylpropylinethyl cellulose solution) and such a suspension may offer advantages in, for example, assisting providing substantially uniform concentrations of a convertible substance when applied as a label.

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

It was hereinbefore disclosed that, optionally, more than one type of convertible species may be used in accordance with the present invention.

By way of example, use of more than one type of convertible species may increase the difficulty which an unauthorised person may encounter in relation to ascertaining the presence andlor identity thereof andlor in relation to detection of identifiable species which nay be produced using said convertible species.

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

By way of example, it may be noted that, optionally, for certain applications it may be possible to arrange for a label comprising convertible 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 label which is a convertible species.

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

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

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

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

Thus, for example, a binder comprising an antibody for an identifiable species which is a ligand, may be prepared by raising antibodies respectively to the ligand.

Where a support material is used in accordance with the present invention any suitt-able support material such as those known in the art may be utilised; examples of support materials are polystyrene (which may be used in 1 any convenient form) and nitro-cellulose (which may be used in any convenient form).

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

It will be appreciated that, in accordance with the present invention, when performing a step by means of which a convertible species may be used to provide an identifiable species, and a reagent is used, or reagents are used, in such a step, such a reagent or reagents may be, for example, a specific reagent or specific reagents selected so as to be appropriate for bringing about provision of identifiable species in a manner specific to the convertible species.

By way of example, where an identifiable species is to be detected by use of immunological binding or nonimmunological binding, a specific binding partner for an identifiable species may be, for example, selected so as to be appropriate for specific binding with the identifiable species.

From the foregoing disclosure it will be appreciated that, for example, a convertible species or an y combination of convertible species may be used alone to effect labelling. However, if desired, a convertible species or a combination of convertible 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, a convertible species, 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, a ligand (e.g. antigenic or non-antigenic), a binder (e.g. for an antigen ligand or for a non-antigenic ligand), an enzyme molecule, a fragment of an enzyme molecule, or binder for an enzyme molecule, a binder for a fragment of an enzyme moleculef 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/60539/HRF) in relation to enzyme molecules and fragments of enzyme molecules and binders therefore in connection with labelling. Reference may be made, for example, to co-pending 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:

Example 1

In accordance with this Example 7-hydroxy-4-methyl coumarin-3-propionic acid was prepared as an example of a ligand for use as an identifiable species in accordance with the present invention.

Thus, 7-hydroxy-4-methyl coumarin-3-propionic acid ethyl ester was prepared by condensing resorcinol (11 g) and diethyl 2-acetyl glutarate (23 g) in concentrated sulphuric acid (50 ml). After 24 hr standing at room temperature the resulting reaction mixture was poured into 2 1 of cold distilled water thereby to form a creamy-white precipitate. The creamy-white precipitate was washed with distilled water (4 1) and collected as an intermediate product. Thin layer chromatography on 4 silica plates showed the intermediate product to be highly pure.

7-hydroxy-4-methyl coumarin-3-propionic acid was prepared from the intermediate product by removing ethyl ester with potassium hydroxide in methanol (5%). The final product was isolated by standard acid-based precipitation-dissolving procedures.

Example 2

In accordance with this Example, a precursor was prepared for a convertible species from the identifiable species prepared as in Example 1.

The convertible species was 0-galactopyranoside-o(4-methyl coumarin-3propionic acid).

Thus, tetraacetyla-D-galactopyranosyl bromide (5 g) was added to a solution of 7-hydroxy-4-methyl coumarin-3propionic acid ethyl ester (0.75 9) in acetone (50 ml) with 0.5 9 of anhydrous potassium carbonate. The resulting mixture was refluxed for 10 hr to give a postreaction mixture. The product, the tetraacetyl galactopyranoside derivative of 7-hydroxy-4methyl coumarin-3-propionic acid ethyl ester, was isolated by standard extraction steps. Thus, chloroform (300 ml) solution of the postreaction mixture was washed repeatedly with 0.5M sodium hydroxide until all trace of starting materials were removed from the chloroform layer. The product was recovered from the chloroform layer. The acetyl groups and the ethyl ester were removed by 5% potassium hydroxide in methanol.

Thus the tetraacetyl-O-D-galactopyranoside-o-(4methyl coumarin-3propionic acid ethyl ester (0.7 g) was left for 16 hr.at 501C in 40 ml of 5% potassium hydroxide in methanol.

The free glycoside product was recovered and purified by preparative liquid chromatography on silica gel plates.

-32 Cross-reaction of O-D-galactopyranoside-o-(4-methy1 couinarin-3-propionic acid) with anti-7-hydroxy-4-methyl coumarin-3-propionic acid antiserum was assessed using competitive ELISA.

Taking the binding of 7-hydroxy-4-methyl coumarin-3propionic acid with anti-7-hydroxy-4-methyl coumarin-3propionic acid antiserum as 100% the cross-reaction of D-galactopyranoside-o-(4-methyl coumarin3-propionic acid) with the antiserum was found to be 0.016%.

Example 3

Convertible species prepared in Example 2 galactopyranoside-o-(4-methyl coumarin-3-propionic acid)) was added (400 ng/ml) to a commerciallyavailable ink vehicle composition to give a labelled ink vehicle composition.

Samples (0.1 ml) of the labelled ink vehicle composition were applied to petri dishes and dried to 400C. Volumes (0.5 nl) of an extraction liquid (100 mM sodium bicarbonate containing 100 mM NaCl, 0.05% Tween 20 and 10% methanol) were added to each petri dish and after 5 min 0.2 ml aliquots were transferred to test-tubes containing 2 units of galactosidase (in 0. 05 ml of PBS (pH 7.8) containing 10 mM MgC1,) and reaction was allowed to proceed to give a reaction mixture.

At this point the convertible species had been converted to an identifiable species.

The convertible species in this Example was, in fact, a synthetic substrate for an enzyme and also a "masked" ligand. Thus, it was possible to detect the identifiable species in two ways as is disclosed in Example 4 and Example 5.

Example 4

0.1 ml aliquots of the reaction mixture found in Example 3 were viewed under a U7 lamp and showed a clearly visible blue fluorescence.

1.

1 These test results demonstrate that removal of a substrate moiety from a synthetic substrate comprising a substrate moiety and a signal moiety may expose the signal moiety which may be detected as such. Thus, it has been demonstrated that a convertible species has been converted to an identifiable species. Control experiments with unlabelled ink vehicle composition showed no visible fluorescence.

Example 5

In this Example a binder species comprising rabbit anti-7-hydroxy-(4methyl coumarin-3-propionic acid) antibody was used. This antibody was prepared in accordance with known general procedures for producing antibodies adapted as necessary to produce the particular antibody required.

The treatment of the convertible species of Example 2 by galactosidase as in Example 3 resulted in the "unmasking" of a ligand. That this is so was demonstrated by an immunological detection method.

Thus, 0.1 ml aliquots of the reaction mixture prepared as described in Example 3 were taken to conduct test experiments.

Competitive immunoassay was carried out using microtitre plates, the wells of which had adsorbed thereon a conjugate of albumin and 7-hydroxy4-methyl coumarin-3-propionic acid. Rabbit-anti-7-hydroxy-4methyl coumarin-3-propionic acid antibody was used in the competitive assay in which 7-hydroxy-4-methyl coumarin-3propionic acid in aliquots of the reaction mixture was allowed to compete, with adsorbed 7-hydroxy-4-methyl counarin-3-propionic acid adsorbed to the microtitre plates, for binding with the antibody.

Goat anti-rabbit antibody conjugated to alkaline phosphatase together with p-nitrophenylphosphate substrate was used to produce detection results which were read at 405 nm.

For the reaction mixture produced by the action of galactosidase on convertible species extracted-from the dried ink vehicle compositions the optical Density (O.D.) at 405 nm was 0.25. Results (at 405 nm) for other samples were as follows: (a) Extraction liquid not contacted with labelled ink vehicle composition, I..D. = 2.08; (b) Sample of extracted convertible species not treated with galactosidase, O.D. = 1.85; Sample of extracting liquid contacted with unlabelled ink vehicle composition, O. D. = 1.93; (d) Alkaline phosphatase substrate background O.D. 0.09. This Example demonstrates the immunological detection of an identifiable species produced by conversion of a convertible species.

T claims

Claims

1. A method suitable for use in labelling of a substance or an item which

method includes providing a substance or an item with convertible species.

2. A method suitable for use in identifying a substance or an item, which method includes providing a substance or an item with a convertible species and performing a step by means of which a convertible species may be used to produce an 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 convertible species, performing a step by means of which a convertible species may be used to produce an identifiable species, and performing a detection step to establish the presence or absence of an identifiable species.

4. A method suitable for identifying a substance or an item which method includes performing a step by means of which a convertible species may be used to produce an identifiable species.

5. A method suitable for identifying a substance or an item which method includes performing a step by means of which a convertible species may be used to produce an identifiable species, and performing a detection step to establish the presence or absence of an identifiable species.

6. A method as claimed in any one of Claims 1 to 3 wherein a convertible species is a ligand which has a specific binding capability thereof substantially inhibited, or is a substrate for an enzyme, or is a synthetic substrate for an enzyme or is a blocked cofactor.

7. A method as claimed in any one of the preceding Claims wherein an identifiable species is detected by an immunological binding method or by a non-immunological binding method.

8. A method as claimed in any one of the preceding claims wherein a convertible species is treated to produce an identifiable species in situ whilst still attached to an item, and the identifiable species is subjected to detection in situ whilst still attached to an item.

9. A method as claimed in any one of-Claims 1 to 7 wherein a convertible species is retrieved from a substance or item, treated to produce an identifiable species and the identifiable species is subjected to detection.

10. A method as claimed in any one of Claims 1 to 7 wherein a convertible species is treated in situ whilst still attached to an item to produce an identifiable species and the identifiable species is retrieved and subjected to detection.

11. A method as claimed in any one of Claims 1 to 8 wherein an identifiable species is detected in situ whilst still attached to an item.

12. A method as claimed in any one of Claims 1 to 7 wherein a thIn LE-11m of liquid or 'Liquid sample containing a convertible species or an entity is provided on a suitable solid surface and the thin film is dried to provide on the solid surface a thin layer of material which may be subjected to detection where identifiable species is present or treated to produce identifiable species which is then subjected to detection.

13. A method as claimed in any one of Claims 1 to 3 or Claims 6, 7, 9 or 12 wherein a convertible species is incorporated into a substance.

14. A method as claimed in any one of Claims 1 to 3 or Claims 6, 7, 8, 10, 11 or 12 wherein a convertible species is applied to an item.

15. A method as claimed in any one of Claims 1 to 3 or 6 to 14 wherein a substance or an item which is labelled is perfume, a bank note, an art work, a document of realisable value, an item of fashion clothes, a watch, an 0 7 k_ c.

1 item of electrical goods, a book, a passport, a medicine, high value goods, a high volume sales item, a prestige high value article, a chemical formulation, meat, a meat product or packaging for goods.

16. A method as claimed in any one of Claims 1 to 3 or 6 to 15 wherein labelling a substance or an item is effected by mixing a label with an ink or ink vehicle composition and printed onto an item, applying a solution of a label directly to an item, dipping part of an area of an item into a solution of a label to attach label to the item, mixing a label with colouring matter or paintstuff for use with an item or packaging, adding a label directly to solutions or formulations of chemical goods, mixing a label with an adhesive substance which may then be attached to tags or papers or items, marking paper certification which is to accompany goods, or applying an ink vehicle composition to an item to form a layer and applying a label to the layer.

17. A composition suitable for use in labelling of a substance or an item which composition includes a convertible species.

18. A composition as claimed in Claim 17 which composition includes a convertible species and a reagent which may be capable of producing a signal.

19. A composition as claimed in Claim 18 wherein the reagent capable of producing a signal is a chromogenic species or a fluorogenic species.

20. A composition as claimed in Claims 17, 18 or 19 wherein the composition is an ink composition, or an ink vehicle composition, or a paint composition.

21. A combination comprising a label associated with a solubilising agent, said label being a convertible species.

22. A combination as claimed in claim 21 wherein the solubilising agent is a surfactant.

23. A combination as claimed in Claim 22 wherein the surfactant is polyethylene glycol.

24. A combination comprising a label associated with an insolubilising medium, said label being a convertible species.

25. A combination as claimed in claim 24 wherein the insolubilising medium is microparticles.

26. A test-kit suitable for use in identifying a substance or an item, which test-kit includes means for performing a step by means of which a convertible species may be used to produce an identifiable species.

27. A test-kit suitable for use in identifying a substance or an item, as claimed in Claim 26 which testkit includes means for performing a detection step to establish the presence or absence of an identifiable species.

28. A labelled substance or a labelled item wherein the substance or item is labelled with a label which is a convertible species.

29. A method suitable for use in labelling of a substance or an item substantially as hereinbefore described with reference to Claim 3.

30. A method suitable for identifying a substance or an item which method includes performing a step by means of which a convertible species may be used to produce an identifiable species substantially as hereinbefore described with reference to Example 3.

31. A method suitable for use in identifying a substance or an item substantially as hereinbefore described with reference to Example 4 or Example 5.

32. A composition suitable for use in labelling of a substance or an item substantially as hereinbefore described with reference to Example 3.

33. A labelled substance or labelled item substantially as hereinbefore described with reference to Example 3.

1