GB2270976A

GB2270976A - Immunoassay/separation process using an auxiliary species on a support

Info

Publication number: GB2270976A
Application number: GB9219743A
Authority: GB
Inventors: Ramadan Arbi Abuknesha
Original assignee: GEC Marconi Ltd; Marconi Co Ltd
Current assignee: BAE Systems Electronics Ltd
Priority date: 1992-09-18
Filing date: 1992-09-18
Publication date: 1994-03-30
Also published as: GB9219743D0

Abstract

A separation method, for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species as defined, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner for the auxiliary species being capable of binding with the auxiliary species and said specific reaction partner for the auxiliary species being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type (as defined), to the specific reaction partner for the auxiliary species, is described.

Description

Separation Method The present invention relates to a separation method which finds application in the detection of species (e.g.

in immunoassay and in immunosensors), to a method suitable for use in detection, to a sensor and to a testkit.

According to one aspect of the present invention there is provided a separation method, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner for the auxiliary species being capable of binding with the auxiliary species and said specific reaction partner for the auxiliary species being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner for the auxiliary species.

In this Specification "a selected specific binding type" as used with reference to a link to a specific reaction partner for an auxiliary species indicates: (A) a link which is formed by a specific binding reaction between the specific reaction partner for the auxiliary species and an antibody for the specific reaction partner, which antibody has been purposely prepared, or (B) where the specific reaction partner for the auxiliary species is a biologically produced antibody having more than one function (e.g. a bifunctional antibody), a link formed by specific binding between the antibody or part of the antibody and a primary species (said antibody or said part thereof not being a primary antibody), or (C) where the auxiliary species is a binder and the specific reaction partner is a biologically produced antibody having more than one function (e.g. a bifunctional antibody), part of which antibody comprises a primary species being a binder for a primary species, a link formed by specific binding between the antibody, or part of the antibody, and a primary species, or (D) where the auxiliary species is a binder and a specific reaction partner for the auxiliary species has a ligand function and a binder function, a link formed by specific binding between the binder function and a further species comprising a ligand, said ligand not being a primary species.

(E) where the auxiliary species is a ligand and the specific reaction partner is a biologically produced antibody having more than one function (e.g. a bifunctional antibody), part of which antibody comprises a primary species being a binder for a primary species, a link formed by specific binding between the antibody, or part of the antibody, and a primary species, or (F) where the specific reaction partner is a species which has more than one ligand function, one of which functions may bind with an auxiliary species which is a binder, a link formed by a binding reaction between another of said functions and a binder which may be associated with a primary species.

The antibody to which reference is made hereinbefore in (A) may be any suitable antibody. Such an antibody may be purposely prepared in any suitable manner; thus, for example, the antibody may be purposely induced (e.g.

purposely raised in animals). One example of such an antibody is a second antibody.

Furthermore, the antibody to which reference is made hereinbefore in (A) may be, for example, an antibody having one type of antibody function; alternatively, if desired, the antibody to which reference is hereinbefore made in (A) may be, for example, an antibody having more than one antibody function (e.g. a bifunctional antibody), or may be, for example, part of an antibody having more than one antibody function (e.g. a bifunctional antibody).

An antibody having more than one antibody function may be produced, for example, biologically (e.g. a chimeric antibody) or may be, for example, formed by linking two antibodies together.

By way of example, where the auxiliary species is a binder and the specific reaction partner is a biologically produced antibody having more than one function (as hereinbefore disclosed in (C)) the binder may be a second antibody which is capable of binding with part of the biologically produced antibody.

By way of further example, where the specific reaction partner for the auxiliary species is a species which has more than one ligand function, the specific reaction species may have two ligand functions by means of one of which functions the specific reaction partner may be linked by specific binding with a binder (e.g. an antibody) (which binder, in turn, may be linked to a primary species (e.g. a ligand or an antibody)) and by the other of which functions the specific reaction partner may undergo specific binding with an auxiliary species, comprising a binder, provided on a support material.

A specific reaction partner having two ligand functions may be, for example, a species comprising two ligands linked together by a suitable means (e.g. by covalent bonding with linear linker arms or by covalent coupling to a carrier). The two ligands may be, for example, of essentially the same type, in which case the auxiliary species provided on the support material and the binder to be linked with a primary species may be essentially the same type of binder.

Alternatively, by way of example, the two ligands may be different in which case the auxiliary species provided on the support material and the binder to be linked with a primary species may be different binders.

By way of example the present invention provides a separation method, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being capable of undergoing a specific binding reaction with the auxiliary species and said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a specific binding type, to the specific reaction partner, said link of a specific binding type being a link of the type formed by specific binding between the specific reaction partner and a purposely prepared antibody for the specific reaction partner.

By way of further example, the present invention provides a separation method, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being an antibody which antibody is not a primary species and which antibody is a biologically produced antibody which has more than one function such that said specific reaction partner is capable of undergoing specific binding with the auxiliary species and such that said specific reaction partner is capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

By way of yet further example, the present invention provides a separation method, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species, said auxiliary species being a binder and said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being a biologically produced antibody having more than one function, part of which antibody comprises a primary species being a binder for a primary species, said specific reaction partner being capable of undergoing specific binding with the auxiliary species and said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

By way of yet further example, the present invention provides a separation method, suitable for use in an immunoassay method for the detection of an analyte species which separation method includes the use of an auxiliary species comprising a binder, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner having a ligand function and binder function such that the specific binder partner is capable of undergoing specific binding with the auxiliary species and such that said specific reaction partner is capable of being linked with a primary species via a third species by means of a linkage which involves a link, of a selected specific binding type to the specific reaction partner.

By way of yet further example the present invention provides a separation method, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species, said auxiliary species being a ligand and said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being a biologically produced antibody having more than one function, part of which antibody comprises a primary species being a binder for a primary species, said specific reaction partner being capable of undergoing specific binding with the auxiliary species and said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

By way of yet further example, the present invention provides a separation method suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species, said auxiliary species being a binder and said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being a species which has more than one ligand function, one of which functions may bind with the auxiliary species and another of which functions may bind with a third species, which third species may be associated with a primary species, such that the said specific reaction partner is capable of being linked with a primary species, by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

A specific reaction partner for an auxiliary species is a species capable of undergoing specific binding with the auxiliary species. Thus, for example, where the auxiliary species is a ligand, the specific reaction partner for the auxiliary species may be a binder and, conversely, by way of example, where the auxiliary species is a binder, the specific reaction partner for the auxiliary species may be a ligand.

The auxiliary species may be considered to be "auxiliary" in the sense that it does not take part in a primary immune reaction as is further disclosed hereinafter. It is to be understood that a primary species is a species which may take part in a primary immune reaction.

It is to be understood that where the specific reaction partner for the auxiliary species is also one which does not take part in a primary immune reaction, then such a specific reaction partner may be considered to be an auxiliary specific reaction partner in that it does not take part in a primary immune reaction.

In accordance with the present invention a specific reaction partner for the auxiliary species may be linked with a primary species (by means of a linkage, which involves a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner) in any suitable manner for example directly or indirectly (e.g. via other species).

Thus, for example, where the specific reaction partner is arranged to be linked directly to a primary species the linkage may be a link of a selected specific binding type between the specific reaction partner and the primary species, said link being of an appropriate selected specific binding type being as hereinbefore defined.

By way of further example, where the specific reaction partner is arranged to be linked indirectly to a primary species the linkage may include one or more other species, and one or more links, as required, at least one of which links being a link, of a selected specific binding type, to the specific reaction partner, said link being of an appropriate selected specific binding type being as hereinbefore disclosed.

It is to be understood that where one or more links are involved in the linkage, one of the links will be a link of an appropriate selected specific binding type, as hereinbefore disclosed, and any further link or links in the linkage may be of any suitable type (e.g. nonspecific binding type or specific linking type); thus, for example, the linkage may include a further link or links of a non-specific binding type, or may include a further link or links of a specific binding type (e.g.

ligand-binder type), or a mixture of types of links (e.g.

non-specific binding type and specific binding type).

Where the specific reaction partner for the auxiliary species is linked to a primary species via other species, said other species may be, for example, a second antibody or a ligand or a binder and, for example, the specific reaction species may be linked to said other species by a link of an appropriate selected specific binding type as hereinbefore disclosed.

A binder for use as an auxiliary species or as a specific reaction partner in accordance with the present invention may be, for example, an antibody.

Antibodies for use as auxiliary species or specific reaction partners in accordance with the present invention may be purposely prepared by any suitable technique; it is to be understood that "purposely prepared" indicates that an antibody has been purposely prepared as opposed to being naturally occurring.

Thus, for example, antibodies which are capable of specifically binding to a selected chemical or biochemical species (i.e. a selected ligand) may be purposely prepared by inducing such antibodies by repeatedly immunising animals with given amounts (e.g. 20 pg-1000 pg) of selected chemical or biochemical species (i.e. selected ligand) as immunogens in accordance with procedures known in the art; affinity chromatography purification may be used as desired in the purification of induced antibodies.

Second antibodies may be used, for example, to form a link of a selected specific binding type to a specific reaction partner comprising a binder (e.g. an antibody) in accordance with the present invention. In these circumstances the binder will be acting as a ligand for the second antibody.

Second antibodies may be, for example, generated with a broad specifity to different animal species immunoglobulin classes, or to a single class of immunoglobulins (e.g. IgG, IgM or others), or with specificity for a particular immunoglobulin molecule.

Reference was hereinbefore made, by way of example, to a type of specific reaction partner which has more than one ligand function.

Thus, a specific reaction partner may have two, or more, ligand functions. If desired a polyvalent ligand structure (e.g. either synthetic or naturally occurring (such as a protein antigen)) may be used.

Synthetic polyvalent ligands may be prepared by covalent coupling of multiple ligands (either all of the same sort or of two or more different sorts) to, for example, a protein, a polysaccharide or a synthetic polymer.

Naturally occurring protein antigens have several different antigenic sites on a given molecule and it is possible to introduce (e.g. by covalent attachment) other ligands thereby to increase the number of types of ligands provided by the antigen.

Reference was hereinbefore made to antibodies having more than one function.

Genetic manipulation of the biosynthetic processes of immunoglobulins has been successful in generating antibody producing cells capable of secreting immunoglobulin molecules made up of two half molecules each of which halves has a different specificity; thus, for example, one half has specificity towards a first antigen and the other half has specificity towards a second antigen. Such immunoglobulin molecules are known as "bifunctional" antibodies, or "dual specificity" antibodies, or "chimeric" antibodies. Antibodies produced in this way may be considered to be "biologically produced" antibodies having more than one function; such antibodies may be used to form bridges between two species.

Bifunctional antibodies or dual specificity antibodies may, alternately, be formed artificially, for example by linking two antibodies, or two Fab fragments of two antibodies, together (e.g. by covalent linkage).

It is to be understood that separation in accordance with the present invention may be effected by arranging for the auxiliary species and the specific reaction partner for the auxiliary species to bind together.

According to another aspect of the present invention there is provided a method, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner for the auxiliary species being capable of binding with the auxiliary species and said specific reaction partner for the auxiliary species being capable of being linked with a primary species by means of a linkage which involves a link of a selected specific binding type (as hereinbefore defined), to the specific reaction partner for the auxiliary species.

By way of example, the present invention provides a method, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being capable of undergoing a specific binding reaction with the auxiliary species and said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a specific binding type, to the specific reaction partner, said link of a specific binding type being a link of the type formed by specific binding between the specific reaction partner and a purposely prepared antibody for the specific reaction partner.

By way of further example, the present invention provides a method, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being an antibody which antibody is not a primary species and which antibody is a biologically produced antibody which has more than one function such that said specific reaction partner is capable of undergoing specific binding with the auxiliary species and such that said specific reaction partner is capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

By way of yet further example, the present invention provides a method, suitable for use in the detection of an analyte species, which method includes the use of an auxiliary species, said auxiliary species being a binder and said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being a biologically produced antibody having more than one function, part of which antibody comprises a primary species being a binder for a primary species, said specific reaction partner being capable of undergoing specific binding with the auxiliary species and said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

By way of yet further example, the present invention provides a method, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species comprising a binder, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner having a ligand function and binder function such that the specific binder partner is capable of undergoing specific binding with the auxiliary species and such that said specific reaction partner is capable of being linked with a primary species via a third species by means of a linkage which involves a link, of a selected specific binding type to the specific reaction partner.

By way of yet further example, the present invention provides a method, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species, said auxiliary species being a ligand and said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being a biologically produced antibody having more than one function, part of which antibody comprises a primary species being a binder for a primary species, said specific reaction partner being capable of undergoing specific binding with the auxiliary species and said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, or a selected specific binding type, to the specific reaction partner.

By way of yet further example, the present invention provides a method, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species, said auxiliary species being a binder and said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being a species which has more than one ligand function, one of which functions may bind with the auxiliary species and another of which functions may bind with a third species, which third species may be associated with a primary species, such that the said specific reaction partner is capable of being linked with a primary species, by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

According to a further aspect of the present invention there is provided a sensor, suitable for use in the detection of an analyte species, which sensor includes an auxiliary species provided on a support material and which sensor is constructed such as to be capable of effecting a method in accordance with the present invention.

In accordance with one embodiment of the present invention there is provided a sensor, suitable for use in the detection of an analyte species, which sensor includes an auxiliary species provided on a support material and the use of a specific reaction partner for the auxiliary species capable of undergoing a specific binding reaction with the auxiliary species, said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner.

By way of example, in accordance with the present invention, an analyte species may be detected as such, or an analyte species may be part of an entity to be detected (e.g. the analyte species may be part of an entity which is an analyte species-containing entity).

Also, by way of example, the present invention may utilise an antibody to an analyte species or an antibody to an entity. to be detected.

In accordance with the present invention there is also provided a separation method, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species provided on a support material, the use of a specific reaction partner for the auxiliary species capable of undergoing a specific binding reaction with the auxiliary species, said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner, and the use of an antibody to an analyte species or the use of an antibody to an entity to be detected.

An antibody to an analyte species, or an antibody to an entity to be detected, may be prepared by any suitable method, for example those known for the raising of polyclonal or monoclonal antibodies; thus, antibodies may be raised, for example, by immunising animals.

In accordance with an embodiment of the present invention there is provided a separation method, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species provided on a support material, the use of a specific reaction partner capable of undergoing a specific binding reaction with the auxiliary species, said specific reaction partner being linked to an entity to be detected, and the use of an antibody to the entity to be detected, said specific reaction partner being linked to an entity to be detected by a linkage which includes a link, of a selected specific binding type (as hereinbefore defined) to the specific reaction partner.

The present invention also provides a method, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species provided on a support material, the use of a specific reaction partner capable of undergoing a specific binding reaction with the auxiliary species, said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner, and the use of an antibody to an analyte species or the use of an antibody to an entity to be detected.

In accordance with an embodiment of the present invention there is provided a method, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species provided on a support material, the use of a specific reaction partner capable of undergoing a specific binding reaction with the auxiliary species, said specific reaction partner being linked to an entity to be detected, and the use of an antibody to the entity to be detected, said specific reaction partner being linked to the entity to be detected by means of a linkage which involves a link, of a selected specific binding type (as hereinbefore defined) to the specific reaction partner.

The present invention also provides a sensor for the detection of an analyte species which sensor includes an auxiliary species provided on a support material, said auxiliary species being for undergoing a specific binding reaction with a specific binding partner which specific binding partner may be associated with an entity to be detected, said specific reaction partner being linked to the entity to be detected by means of a linkage which involves a link of a selected specific binding type (as hereinbefore defined) to the specific reaction partner.

By way of example, a sensor in accordance with the present invention may include a surface suitable for acting as a support material for an auxiliary species.

In accordance with an embodiment of the present invention a sensor may include a support material having a surface upon which there is immobilised the auxiliary species.

The surface may be, for example, glass, quartz or an electrode material.

According to a further aspect of the present invention there is provided a process for the preparation of a sensor for use in the detection of an analyte species or an entity to be detected by a method in accordance with the present invention which process includes the step of providing, upon a surface of a support material, an immobilised auxiliary species.

The specific reaction partner may be arranged to be associated with the entity to be detected in any suitable manner and at any suitable time. Thus, the specific reaction partner may, for example, be arranged to be associated with the entity either before or after a specific binding reaction has taken place between the auxiliary species and the specific reaction partner.

A primary species in accordance with the present invention is a species capable of taking part in a primary immune binding reaction. By way of example, a primary immune binding reaction in accordance with the present invention is one in which an analyte species undergoes, or an entity to be detected (containing an analyte species) undergoes, a specific binding reaction or an authentic analyte species (as hereinafter defined) undergoes, or an authentic entity to be detected (as hereinafter defined) undergoes, a specific binding reaction or an analyte species and an authentic analyte species undergo specific binding reactions or an entity to be detected and an authentic entity to be detected undergo specific binding reactions. (It will be appreciated that the analyte species and the authentic analyte species undergo specific binding reactions with other species and not with each other and that the entity to be detected and the authentic entity to be detected undergo specific binding reactions with other species and not with each other.) By way of example, a primary species may be a primary antibody or a ligand (e.g. an antigen).It is to be understood that, for example, a primary species may be an antibody to an analyte species, an antibody to an authentic analyte species, an antibody to an entity to be detected or an antibody to an authentic entity to be detected; it will be appreciated that, for a given immunoassay, the antibody to the analyte species and the antibody to the authentic analyte species will be the same antibody; similarly, it will be appreciated that,for a given immunoassay, the antibody to the entity to be detected and the antibody to the authentic entity to be detected will be the same antibody. It is also to be understood that a primary species may be, for example, an analyte species, an authentic analyte species, an entity to be detected or an authentic entity to be detected.

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

Also it is to be understood that an "authentic analyte species" is a species which is capable of reacting in a substantially similar manner as an analyte species to be detected under substantially similar conditions; similarly, it is to be understood that an "authentic entity to be detected" is an entity which is capable of reacting in a substantially similar manner as an entity to be detected under substantially similar conditions.

It will be appreciated that an authentic analyte species may be used, inter alia, as a calibrator or a standard; similarly, it will be appreciated that an authentic entity to be detected may be used, inter alia, as a calibrator or a standard.

The auxiliary species is a species which does not itself take part in a primary specific binding immune reaction with an analyte species (or an authentic analyte species) or an entity to be detected (or an authentic entity to be detected).

The auxiliary species may be, for example, provided on a support material in any suitable manner, and it is to be understood that in this Specification "providing on a support material" and "provided on a support material" embrace, for example, situations where the support material itself, or a part of the support material itself, provides auxiliary species and situations where the support material carries an auxiliary species.

Thus, for example, an auxiliary species comprising a ligand may be provided by chemical groups or units of the support material or an auxiliary species comprising a ligand or a binder may be, for example, attached to the support material (e.g. by covalent linkage or adsorption). Where the support material is, for example, a polymer, units of the polymer may act as auxiliary species comprising a ligand. Also, by way of example, surface groups present on a support material, such as polystyrene or modified silica, may act as auxiliary species comprising a ligand.

By way of example, the support material may, if desired, provide oligomers or polymers of auxiliary species comprising an auxiliary ligand.

Where an auxiliary species comprising a ligand or a binder is attached to a support material the auxiliary species may be directly attached to the support material or indirectly attached to the support material via other species (e.g. a carrier protein).

In one embodiment the present invention provides a method which also includes the step of attaching, either directly, or indirectly, an auxiliary species to a support material.

By way of further example, the surface of a support material may be activated thereby to permit attachment of auxiliary species; for example, the surface of a suitable support material may be activated by chemical treatment to provide free amino groups to which auxiliary species carrying ligands or binders may be linked.

Further, by way of example, instead of linking single auxiliary species to free amino groups, (produced as immediately hereinbefore disclosed) oligomers of auxiliary species or polymers of auxiliary species may be attached directly or indirectly to a support material.

Thus, for example, oligomers or polymers of an auxiliary species may be attached to free amino groups on a support material.

Also, by way of example, an auxiliary species may be linked (e.g. covalently or otherwise) to a further species (e.g. a carrier protein or a polymer) and the further species may be associated with the support material such that the auxiliary species may become indirectly provided on the support material.

By way of further example,instead linking single auxiliary species indirectly to a support material it is possible to link (e.g. covalently or otherwise) oligomers or polymers of auxiliary species to a further species (e.g. a carrier protein or a polymer) and the further species may be associated with the support material such that oligomers of auxiliary species or polymers of auxiliary species may become indirectly provided on the support material.

Where an auxiliary species is attached to a support material, attachment of the auxiliary species may be effected at any desired time. Thus, for example, the auxiliary species may be allowed to undergo a specific binding reaction with the specific reaction partner before, or after, the auxiliary species is attached to the support material; however, in general, it may be more convenient to attach the auxiliary species to the support material before bringing together the auxiliary species and the specific reaction partner.

It is to be understood that the use of oligomers or polymers of auxiliary species may be advantageous in certain circumstances. Thus, for example, by use of an oligomer or a polymer of an auxiliary species more auxiliary species may be provided than it is possible to provide when using only single auxiliary species.

Accordingly, for example, the provision of more auxiliary species offers the possibility of faster reactions since more auxiliary species are available to undergo reaction with available specific reaction partner therefor.

The specific reaction partner may be arranged to bind with the auxiliary species in any suitable manner and at any suitable time. Thus, for example, the specific reaction partner may be arranged to be linked (either directly or indirectly) with a primary species either before, or after, undergoing a specific binding reaction with the auxiliary species or may be arranged to be linked simultaneously with a primary species and with an auxiliary species.

The auxiliary species may be a suitable ligand or a suitable binder. Examples of suitable ligands are antigenic ligands (such as haptens) and non-antigenic ligands.

Examples of antigenic ligands are 2,4 dinitrophenol, fluorescein, digitoxin, coumarin and cibacron blue.

Examples of non-antigenic ligands are ligands of specific ligand-binder pairs (e.g. the ligand biotin in the case of the ligand-binder pair biotin-avidin).

Further examples of auxiliary species which may be attached to a support material (e.g. by covalent linkage or non-covalent attachment (adsorption)) are soluble materials for example soluble molecules (e.g. polymers) such as polypeptides, proteins, polysaccharides and conducting polymers; haptenic functions may be covalently coupled to such soluble materials (e.g. polymers). By way of example, an auxiliary species may be provided as a coating of a polymer on a support material.

Where the auxiliary species is a ligand, the specific reaction partner therefor may be any suitable binder capable of binding with the auxiliary species.

By way of example, the specific reaction partner may be a binding protein (e.g. an antibody or a binding partner for a ligand).

Thus, for example, where the auxiliary species is an antigenic ligand, the specific reaction partner may be an antibody to the ligand. An antibody to the auxiliary species may be considered to be an anti-auxiliary species antibody.

Where, for example, the auxiliary species is fluorescein, digitoxin, coumarin, 2,4 dinitrophenol or cibacron blue, the specific reaction partner may be, respectively, anti-fluorescein antibody, anti-digitoxin, anti-coumarin antibody, anti-2,4 dinitrophenol antibody or anti-cibacron blue antibody.

It will be appreciated that antibodies such as those immediately hereinbefore disclosed may be prepared by any suitable method, for example those known for the raising of polyclonal or monoclonal antibodies; thus, antibodies may be raised, for example, by immunising animals with conjugates made of suitable derivatives of a ligand and an immunogenic carrier protein such as bovine serum albumin or key-hole limpet haemocyanin; the product obtained by immunising animals may be purified as desired (e.g. by the use of affinity chromatography) to obtain the required antibodies.

Where, for example, the auxiliary species is a nonantigenic ligand, the ligand may be, for example, such that the specific reaction partner 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 pair comprising a biotin-avidin complex.

Where the auxiliary species is a binder, the auxiliary species may be for example any suitable binder, examples of which are antibodies to antigenic ligands and binders for non-antigenic ligands.

Examples of antibodies to antigenic ligands, which antibodies may be used as auxiliary species, are antifluorescein antibody, anti-coumarin antibody, anti-2,4 dinitrophenol antibody and anti-cibacron blue antibody.

Examples of auxiliary species which are binders for non-antigenic ligands are binding partners of specific ligand binder pairs (e.g. the binder avidin in the specific ligand-binder pair comprising a biotin-avidin complex).

Where the auxiliary species is a binder, the specific reaction partner may be, for example, any suitable ligand which may undergo a specific reaction with the auxiliary species.

Thus, for example, where the auxiliary species is an antibody, the specific reaction partner may be an antigenic ligand to the antibody and when the auxiliary species is a binder of a specific ligand-binder pair the specific reaction partner may be the corresponding nonantigenic ligand of the pair.

By way of example, where the auxiliary species is a binder comprising anti-fluorescein antibody, antidigitoxin antibody, anti-coumarin antibody, anti-2,4 dinitrophenol antibody or anti-cibacron blue antibody, the corresponding specific reaction partners may be respectively fluorescein, digitoxin, coumarin, 2,4 dinitrophenol or cibacron blue.

Also, by way of example, where the auxiliary species is avidin, the specific reaction partner may be the nonantigenic ligand biotin.

The separation method of the present invention may, therefore, for example, utilise separation systems comprising fluorescein/anti-fluorescein antibody, digitoxin/anti-digitoxin antibody, coumarin/anti-coumarin antibody, 2,4 dinitrophenol/anti-2,4 dinitrophenol antibody, cibacron blue/anti-cibacron blue antibody or a biotin-avidin complex.

Reference has hereinbefore been made to nonantigenic ligands and binders therefor. It will be appreciated that although such a non-antigenic ligand and binder therefor may be non-antigenic with respect to each other, they may be antigenic with respect to antibodies raised against them. Thus, for example, it is possible to prepare an antibody for biotin and an antibody for avidin if required.

The present invention may find application, for example, in the detection of an analyte species, or in the detection of an entity to be detected, in any suitable sample. Thus, for example, samples of water, soil, living species (such as plants (e.g. vegetables) or animals) or air may provide an analyte species, or an entity to be detected, for detection in accordance with the present invention. Examples of biological samples in which an analyte species, or an entity to be detected, may be detected in accordance with the present invention are blood, plasma, serum, urine, saliva and milk. An analyte species, or an entity to be detected, may be, for example, present in water, an aqueous preparation or a fluid extract. An analyte species may be, for example, a ligand (e.g. a hapten) or a binder (e.g. an antibody).

Examples of analyte species which may be detected in accordance with the present invention are: (a) steroid hormones such as progesterone, 17a-hydroxy progesterone or estradiol (e.g. in a sample of blood, serum, saliva, urine or milk), (b) hormones such as a thyroid hormone (e.g. thyroxine or triiodothyronine), (c) steroids in extracts (e.g. extracts of solids or liquids), (d) drugs such as drugs of abuse (e.g. phenobarbital) and therapeutic drugs (e.g. digoxin) (in for example, a sample of blood, serum, saliva or urine), (e) polypeptide hormones (e.g. hCG) in, for example, a sample of blood or urine, (f) tumour markers such as marker proteins (e.g. in a sample of blood or serum), (g) protein antigens, (h) blood proteins (e.g. human serum albumin, immunoglobulins (e.g.IgG), enzyme markers or receptors), (i) marker proteins (j) pesticides such as insecticides, or herbicides (e.g.

in water or soil), (k) toxins (such as those extracted from feeds and food stuffs), (1) micro-organisms (e.g. viruses and bacteria), and (m) antibodies to micro-organisms.

Further examples of analyte species which may be detected in accordance with the present invention are complexes of metals.

Thus, the present invention may find application in, for example, the detection of complexes of metals such as strong metal complexes which may be regarded as toxic (e.g. in biological terms when present in the environment).

An example of a metal complex which may be detected in accordance with the present invention is methyl mercury.

By way of further example, metal ions may be formed in to a complex of a metal by use of a complexing agent (e.g. a chelating agent) and the complex thus formed may act as an analyte species in accordance with the present invention.

Also, by way of further example, metals may be detected in accordance with the present invention as is further disclosed hereinafter.

Examples of support materials which may find application in accordance with the present invention are solid phase materials such as a reaction vessel wall, insoluble polysaccharides, microparticles (e.g.

particulate microcellulose), polystyrene (e.g. in the form of wells, beads, micro-titre plates, discs, sticks or tubes), cross-linked dextran (e.g. Sephadex), insoluble polymer structures, glass surfaces, derivatised silica surfaces (e.g. having silyl groups with chemical functions attached), soluble polymers attached to a suitable surface (e.g. a glass surface), microparticulate materials with entrapped ferrous oxide (magnetisable particles), nylon and polyamides.

It will be appreciated that some types of support materials may be inappropriate for use with some analyte species and some entities to be detected. Thus, for example, where the analyte species is a metal ion, or the entity to be detected contains a metal, the use of some types of support materials may be inappropriate (e.g.

support materials containing entrapped iron oxide may give rise to unacceptable interference).

The present invention may find application in, for example, label-free or detectable species-dependent (e.g.

tracer species-dependent) assay methods such as enzymeimmunoassay, fluoro-immunoassay and radio-immunoassay.

By way of example, the present invention may find application in antibody-labelled or antigen-labelled as says.

By way of example, any suitable detectable species may be used in accordance with the present invention in the detection of an analyte species or an entity to be detected. It is to be understood that a detectable species may be, for example, a detectable structure.

Examples of detectable species are enzymes, fluorophores (or polymeric fluorophores), radioisotopes, ligands (or polymers of a ligand), and binders.

By way of example, an enzyme may be detected by a corresponding substrate, fluorophores and radioisotopes may be detected directly with suitable detectors, ligands may be detected by use of binders therefor, said binders being associated with tracer species, and binders may be detected by use of ligands therefor, said ligands being associated with tracer species.

The tracer species may be, for example, any suitable tracer species such as those known in the art relating to protein binding assays (e.g. immunoassays). (It is to be understood that a tracer species may also be considered to be a signal species or a labelling species.) Examples of tracer species are enzymes (e.g.

alkaline phosphatase, P-galactosidase and horse-radish peroxidase), fluorophores (e.g. fluoresceins, coumarins or rhodamin), chemiluminescent compounds, bioluminescent compounds, radioisotopes and dyes.

Detection or measurement of a signal from a detectable species may be carried out in any suitable manner such as those known in the immunochemical field.

Where, for example, a ligand or a binder is used (as hereinbefore disclosed) as a detectable species any suitable ligand or binder may be utilised.

Examples of ligands which may be utilised as detectable species are antigens (e.g. 2,4 dinitrophenol, fluorescein, digitoxin, coumarin and cibacron blue), which may be considered to be haptens, and non-antigenic ligands such as the ligand of a specific ligand-binder pair (e.g. biotin).

Examples of binders which may be utilised as detectable species are antibodies (e.g. anti-2,4 dinitrophenol antibody, anti-fluorescein antibody, antidigitoxin antibody, anti-coumarin antibody and anticibacron blue antibody) and binders of a specific ligandbinder pair (e.g. avidin). It will be appreciated that antibodies such as those immediately hereinbefore disclosed may be prepared by any suitable method such as those known for the raising of polyclonal or monoclonal antibodies; thus antibodies may be raised, for example, by immunising animals.

The present invention may find application in any suitable form of immunological detection or immunoassay examples of which are competitive-immunoassay methods, non-competitive immunoassay methods and sandwich immunoassay methods.

Where a detectable species is used in accordance with the present invention, the detectable species may be, for example, arranged to be associated, in any suitable manner and at any suitable time, with any chosen species or entity; thus, for example, a detectable species may be associated (as appropriate to an immunoassay being utilised) with a specific reaction partner for an auxiliary species, or with an authentic analyte species, or with an authentic entity species (being an authentic entity to be detected), or with a primary antibody, or with a second antibody.

The present invention may find application, for example, in any suitable immunoassay method or configuration; the present invention will now be further described, by way of example only, with reference to a number of immunoassay configurations in accordance with the invention: (i) In one example the following may be utilised; a support material, an auxiliary species (being, in this example, a ligand) provided on the support material, a specific reaction partner for the auxiliary species, said specific reaction partner being, in this example, an antibody to the auxiliary species, a primary species being an antibody for an analyte species or for an entity to be detected, a second antibody being an antibody both for the specific reaction partner and for the primary species.

The support material, auxiliary species, specific reaction partner, primary species (which here may be considered to be a primary antibody) and second antibody as disclosed in the immediately preceding paragraph may be used in a competitive immunoassay.

In carrying out such a competitive immunoassay an excess of auxiliary species may be provided on a support material, and an excess of specific reaction partner is contacted therewith and specific binding is allowed to take place such that the specific reaction partner becomes attached to the support material via the auxiliary species.

Washing may then be carried out to remove unbound, excess specific reaction partner.

Subsequently, second antibody is added in excess and allowed to react with the specific reaction partner attached to the support material such that second antibody becomes indirectly attached to the support material (via the specific reaction partner and the auxiliary species).

Washing may be carried out to remove unbound, excess second antibody. The specific reaction partner (which is an antibody in this example) and the second antibody need not necessarily be affinity purified to a high degree in this example.

Separately from the above a primary immune reaction may be arranged to take place by contacting the primary species being, in this example, a primary antibody with authentic analyte species linked to a tracer species or with authentic entity to be detected linked to a tracer species and with any "unknown" analyte species, or "unknown" entity to be detected, or with any known amounts of analyte species or entity to be detected (i.e. standards) in as much as they are capable of taking part in a primary immune reaction.

It will be appreciated that the authentic analyte species, or authentic entity, or analyte species or entity may also be regarded as primary species.

It will also be appreciated that "unknown" is used in this Specification with reference to analyte species or entity to be detected to indicate analyte species or entity of unknown concentration (e.g. in a sample).

In the absence of analyte species or entity to be detected any authentic analyte species carrying a tracer species or authentic entity to be detected carrying a tracer species will bind with the primary antibody. However, in the presence of analyte species or entity (e.g.

either "unknown" or known standard), the analyte species or entity will compete with the authentic analyte species or authentic entity for binding with the primary antibody with the result that less tracer species will be associated with the primary antibody.

The minimum amount of tracer species will be associated with the primary antibody when the analyte species or entity to be detected is at a maximum.

After the primary immune reaction has proceeded to a desired degree (e.g. equilibrium) the resulting primary immune reaction mixture may be contacted with second antibody, which is attached to the support material via the specific reaction partner and the auxiliary species as disclosed earlier in this example.

Accordingly, primary antibody may be removed from the primary immune reaction mixture by being bound by the second antibody.

Thus, after washing if required, the amount of tracer species associated with the support material (via, inter alia, the primary antibody) may be measured, by any suitable method such as those known in the art, to enable the amount of "unknown" analyte species if any, or the amount of "unknown" entity if any, in a sample to be determined by comparison with calibration results obtained using known amounts of "standard" analyte species or "standard" entity.

(ii) In another example the following may be utilized; a support material, an auxiliary species (being in this example a ligand) provided on the support material, a specific reaction partner (being in this example an antibody to the auxiliary species), a second antibody capable of undergoing specific binding with the specific reaction partner, a primary species (being a ligand in the form of an authentic analyte species or an authentic entity to be detected) covalently linked to the second antibody,a primary antibody, capable of undergoing a primary immune binding reaction with the authentic analyte species or with the authentic entity to be detected and capable of undergoing a primary immune binding reaction with any "unknown" analyte species or any "unknown" entity to be detected in a sample or with any known amounts of analyte species or entity to be detected (i.e. standards), and a tracer species associated with the primary antibody.

The support material, auxiliary species, specific reaction partner, second antibody, primary species and the primary antibody as disclosed in the immediately preceding paragraph may be used in a competitive immunoassay.

In carrying out such a competitive immunoassay the second antibody to which is covalently linked the primary species (ligand) is contacted with the primary antibody, which is associated with a tracer species, and with any analyte species or entity to be detected so as to permit a primary immune binding reaction to occur.

In the absence of any analyte species or entity to be detected the primary antibody carrying the tracer species will undergo a primary immune binding reaction only with primary species linked to the second antibody and hence tracer species will become associated with the second antibody.

However, in the presence of analyte species or entity to be detected (e.g. either the "unknown" or a known standard) the analyte species or entity to be detected will compete with the primary species for binding with the primary antibody; as the concentration of an analyte species or entity to be detected increases the amount of primary antibody which may be associated with the second antibody decreases.

Accordingly, as the concentration of analyte species or entity to be detected increases, the amount of tracer species associated with the second antibody decreases.

When the primary immune reaction has proceeded to a desired degree (e.g. equilibrium) second antibody may be removed from the resulting primary immune reaction mixture by attachment to the support material.

This may be done by exposing the primary reaction mixture to the support material, which has provided thereon the auxiliary species, and introducing the specific reaction partner for the auxiliary species.

Thus, the second antibody (and any primary antibody carrying tracer species associated with the second antibody) may bind with the specific reaction partner and the specific reaction partner may bind with the auxiliary species.

Accordingly, the specific reaction partner may act as a "bridge" by means of which the second antibody may be linked with the support material.

Subsequently, after any necessary washing, any tracer species associated with the support material (via the primary antibody, the primary species, the second antibody, the specific reaction partner and the auxiliary species) may be measured, by any suitable method such as those known in the art, to enable the amount of "unknown" analyte species if any, or entity if any, in a sample to be determined by comparison with calibration results obtained using known amounts of "standard" analyte species or "standard" entity.

In one embodiment of an immunoassay in accordance with this example, the primary immune reaction may be allowed to proceed to the desired degree in the presence of the support material upon which is provided the auxiliary species and subsequently the specific reaction partner may be introduced thereby to effect separation of secondary antibody (and any indirectly associated tracer species) from the primary immune reaction mixture.

This immediately foregoing embodiment offers the advantage of conducting a two-step immunoassay in one vessel.

(iii) In another example the following may be utilised; a support material, an auxiliary species (being in this example a non-antigenic ligand (e.g.

biotin)) provided on the support material, a specific reaction partner (being in this example a binder for the non-antigenic ligand (e.g. a binder such as avidin) for the auxiliary species), a specific reaction partner antibody capable of undergoing specific binding with the specific reaction partner, a primary species (being a ligand in the form of an authentic analyte species or an authentic entity to be detected) covalently linked to the specific reaction partner antibody, a primary antibody, capable of undergoing a primary immune binding reaction with the authentic analyte species or with the authentic entity to be detected and capable of undergoing a primary immune binding reaction with any "unknown" analyte species or any "unknown" entity to be detected in a sample or with any known amounts of analyte species or entity to be detected (i.e. standards), and a tracer species associated with the primary antibody.

The support material, auxiliary species, specific reaction partner, specific reaction partner antibody, primary species and the primary antibody as disclosed in the immediately preceding paragraph may be used in a competitive immunoassay.

In carrying out such a competitive immunoassay the specific reaction partner antibody to which is covalently linked the primary species (ligand) is contacted with the primary antibody, which is associated with a tracer species, and with any analyte species or entity to be detected so as to permit a primary immune binding reaction to occur.

In the absence of any analyte species or entity to be detected the primary antibody carrying the tracer species will undergo a primary immune binding reaction only with primary species linked to the specific reaction partner antibody and hence tracer species will become associated with the specific reaction partner antibody.

However, in the presence of analyte species or entity to be detected (e.g. either the "unknown" or a known standard) the analyte species or entity to be detected will compete with the authentic analyte species or authentic entity for binding with primary antibody; as the concentration of an analyte species or entity to be detected increases the amount of primary antibody which may be associated with the specific reaction partner antibody decreases.

Accordingly, as the concentration of analyte species or entity to be detected increases, the amount of tracer species associated with the specific reaction partner antibody decreases.

When the primary immune reaction has proceeded to a desired degree (e.g. equilibrium) specific reaction partner antibody may be removed from the resulting primary immune reaction mixture by attachment to the support material.

Thus, the specific reaction partner antibody (and any primary antibody carrying tracer species associated with the specific reaction partner antibody) may bind with the specific reaction partner and the specific reaction partner may bind with the auxiliary species. Accordingly, the specific reaction partner may act as a "bridge" by means of which the specific reaction partner antibody may be linked with the support material.

Subsequently, after any necessary washing, any tracer species associated with the support material (via the primary antibody, the primary species, the specific reaction partner antibody, the specific reaction partner and the auxiliary species) may be measured, by any suitable method such as those known in the art, to enable the amount of "unknown" analyte species if any, or entity if any, in a sample to be determined by comparison with calibration results obtained using known amounts of "standard" analyte species or "standard" entity.

In one embodiment of an immunoassay in accordance with this example, the primary immune reaction may be allowed to proceed to the desired degree in the presence of the support material upon which is provided the auxiliary species and subsequently the specific reaction partner may be introduced thereby to effect separation of specific reaction partner (and any indirectly associated tracer species) from the primary immune reaction mixture.

(iv) In a further example the following may be utilised; a support material, an auxiliary species (being, in this example, a ligand) provided on the support material, a primary species (being a primary antibody capable of undergoing a primary immune binding reaction with an authentic analyte species or with an authentic entity to be detected and capable of undergoing a primary immune binding reaction with any "unknown" analyte species or any "unknown" entity to be detected in a sample or with any known amounts of analyte species or entity to be detected (i.e. standards), a specific reaction partner (being a biologically produced bifunctional antibody which antibody has one function capable of specific binding with the auxiliary ligand and another function which is capable of specific binding with the primary species), and an authentic analyte species associated with a tracer species or an authentic entity associated with a tracer species.

The support material, auxiliary species, specific reaction partner, primary species and authentic analyte species associated with a tracer species or authentic entity associated with a tracer species as disclosed in the immediately foregoing paragraphs may be used in a competitive immunoassay.

In carrying out such a competitive immunoassay a primary immune binding reaction may be arranged to occur by contacting primary antibody with authentic analyte species associated with a tracer species or with authentic entity associated with a tracer species and with any "unknown" analyte species or "unknown" entity or with any known amounts of analyte species or entity to be detected (i.e. "standards").

In the absence of any analyte species or entity to be detected only the authentic analyte species (associated with tracer species) or authentic entity (associated with tracer species) will bind with the primary antibody.

However, in the presence of analyte species or entity to be detected (e.g. either "unknown" or a known standard) the analyte species or entity to be detected will compete with authentic analyte species or authentic entity, for binding with the primary antibody; as the concentration of an analyte species or entity to be detected increases, the amount of authentic analyte species or authentic entity bound to the primary antibody decreases and hence the amount of tracer species associated with primary antibody decreases.

When the primary immune reaction has proceeded to a desired degree (e.g. equilibrium) primary antibody (and any associated tracer species) may be removed from the resulting primary immune reaction mixture by attachment to the support material.

This may be done by exposing the primary immune reaction mixture to the support material, which has provided thereon the auxiliary species, and introducing the specific reaction partner whereupon one function of the specific reaction partner binds specifically with the primary species and the other function of the specific reaction partner binds with the auxiliary species.

Thus, the primary antibody (and any authentic analyte species (and associated tracer species) or any authentic entity (and associated tracer species) associated with the primary antibody) becomes associated with the support material; it will be appreciated that the specific reaction partner (in this case a bifunctional antibody) may act as a "bridge" by means of which the primary antibody may be linked with the support material.

Subsequently, after any necessary washing, any tracer species associated with the support material (via authentic analyte species (or authentic entity), or the specific reaction partner, and the auxiliary species) may be measured, by any suitable method such as those known in the art, to enable the amount of "unknown" analyte species if any, or "unknown" entity if any, in a sample to be determined by comparison with calibration results obtained using known amounts of "standard" analyte species or "standard" entity.

In one embodiment of an immunoassay in accordance with this example, the primary immune reaction may be allowed to proceed to the desired degree in the presence of the support material upon which is provided the auxiliary species and subsequently the specific reaction partner may be introduced thereby to effect separation of primary antibody (and any indirectly associated tracer species) from the primary immune reaction mixture.

Thus the immediately foregoing embodiment offers the advantage of conducting a two-step immunoassay in one vessel.

(v) In a further example, the following may be utilised; a support material, an auxiliary species (being, in this example, a ligand) provided on the support material. a specific ~ (being a blunder) reaction partnerLfor the auxiliary species, a bifunctional antibody, which antibody has one function capable of specific binding with the specific reaction partner and another function which is capable of acting as a primary antibody in that it is capable of specific binding with an authentic analyte species or with an analyte species (e.g. either "unknown" or a known standard) or an authentic entity or an entity to be detected (e.g. either "unknown" or a known standard), and a tracer species associated with an authentic species or with an authentic entity.

The support material, auxiliary species, specific reaction partner, bifunctional antibody, authentic analyte species having attached thereto a tracer species or authentic entity having attached thereto a tracer species as disclosed in the immediately foregoing paragraph may be used in a competitive assay.

In carrying out such a competitive immunoassay a primary immune binding reaction may be arranged to occur by contacting the bifunctional antibody with authentic analyte species carrying a tracer species or with authentic entity carrying a tracer species and with any "unknown" analyte species, or "unknown" entity or with any known amounts of analyte species or entity to be detected (i.e. "standards").

In the absence of any analyte species or entity to be detected only the authentic analyte species carrying tracer species or authentic entity carrying tracer species will bind with the function of the bifunctional antibody which is capable of acting as a primary antibody.

However, in the presence of analyte species or entity to be detected (e.g. either "unknown" or a known standard) the analyte species or entity to be detected will compete with authentic analyte species or authentic entity for binding with the function of the bifunctional antibody which is capable of acting as a primary antibody; as the concentration of an analyte species or entity to be detected increases, the amount of authentic analyte or authentic entity bound to the antibody decreases and hence the amount of tracer species associated with the antibody decreases.

When the primary immune reaction has proceeded to a desired degree (e.g. equilibrium) the antibody may be removed from the resulting primary immune reaction mixture by attachment to the support material.

This may be done by exposing the primary immune reaction mixture to the support material, which has provided thereon the auxiliary species, and introducing the specific reaction partner.

Thus, the function of the bifunctional antibody which is capable of binding with the specific reaction partner may bind with the specific reaction partner and the specific reaction partner may bind with the auxiliary species.

Accordingly any authentic analyte species (and associated tracer species) or any authentic entity (and associated tracer species) associated with the bifunctional antibody will become associated with the support material; it will be appreciated that the specific reaction partner may act as a "bridge" by means of which any tracer species may be linked with the support material.

Subsequently, after any necessary washing, any tracer species associated with the support material may be measured, by any suitable method such as those known in the art, to enable the amount of "unknown" analyte species if any, or entity if any, in a sample to be determined by comparison with calibration results obtained using known amounts of "standard" analyte species or "standard" entity.

In one embodiment of an immunoassay in accordance with this example, the primary immune reaction may be allowed to proceed to the desired degree in the presence of the support material upon which is provided the auxiliary species and subsequently the specific reaction partner may be introduced thereby to effect separation of bifunctional antibody (and any indirectly associated tracer species) from the primary immune reaction mixture.

(vi) In a further example the following may be utilised; a support material, an auxiliary species (being, in this example, a ligand) provided Qn the support material, a specific - iDelnq a nder) reaction partner, a primary species (comprising a primary antibody capable of undergoing a primary immune binding reaction with an authentic analyte species or with an authentic entity to be detected and capable of undergoing a primary immune binding reaction with any "unknown" analyte species or any "unknown" entity to be detected in a sample or with any known amounts of analyte species or entity to be detected (i.e.

standards)), a bifunctional antibody, which antibody has one function capable of specific binding with the specific reaction partner and another function which is capable of specific binding with the primary species, and an authentic analyte species associated with a tracer species or an authentic entity associated with a tracer species.

The support material, auxiliary species, specific reaction partner, primary species, bifunctional antibody and authentic analyte species associated with a tracer species or authentic entity associated with a tracer species as disclosed in the immediately foregoing paragraph may be used in a competitive immunoassay.

In carrying out such a competitive immunoassay, a primary immune reaction may be arranged to occur by contacting primary antibody with authentic analyte species associated with a tracer species or authentic entity associated with a tracer species and with any "unknown" analyte species or "unknown" entity, or with any known amounts of analyte species or entity to be detected (i.e. "standards").

In the absence of any analyte species or entity to be detected only the authentic analyte species (associated with tracer species) or entity (associated with tracer species) will bind with the primary antibody.

However in the presence of analyte species or entity to be detected (e.g. either "unknown" or a known standard) the analyte species or entity to be detected will compete with the authentic analyte species, or authentic entity, for binding with the primary antibody; as the concentration of analyte species or entity to be detected increases, the amount of authentic analyte species or authentic entity bound to primary antibody decreases and hence the amount of tracer associated with the primary antibody decreases.

When the primary immune reaction has proceeded to a desired degree (e.g. equilibrium) primary antibody (and any associated tracer species) may be removed from the resulting primary immune reaction mixture by attachment to the support material. This may be done by introducing the bifunctional antibody such that it binds with primary antibody (which may be indirectly associated with some of the tracer species), subsequently exposing the bifunctional antibody to the support material, which has provided thereon auxiliary species, and then introducing the specific reaction partner.

The bifunctional antibody is able to bind with the specific reaction partner and the specific reaction partner is able to bind with the auxiliary species such that any tracer species associated with the primary antibody may become indirectly attached to the support material.

Subsequently, after any necessary washing, any tracer species associated with the support material may be measured, by any suitable method such as those known in the art, to enable (by reference to calibration results obtained using known amounts of "standard" analyte species or "standard" entity) the amount of "unknown" analyte species if any, or "unknown" entity if any, in a sample to be determined.

In one embodiment of an immunoassay in accordance with this example, the primary immune reaction may be allowed to proceed to the desired degree in the presence of the support material upon which is provided the auxiliary species, and subsequently the bifunctional antibody and the specific reaction partner may be used to effect separation and attachment of any tracer species to the support material.

As an alternative, by way of example, a further embodiment of the primary immune reaction may be allowed to proceed to the desired degree in the presence of the support material upon which is provided the auxiliary species and in the presence of the bifunctional antibody and the specific reaction partner may be subsequently used to effect attachment.

The immediately foregoing embodiments offer the advantage of conducting a two-step immunoassay in one vessel.

(vii) In a further example the following may be utilised; a support material, an auxiliary species (being, in this example, a binder) provided on the support material, a specific reaction partner having two ligand functions (said specific reaction partner being two ligands of the same type covalent linked together), a specific reaction partner binding antibody, an authentic analyte species, or an authentic entity associated with the specific reaction partner binding antibody, a primary species (being a primary antibody capable of undergoing a primary immune binding reaction with the authentic analyte species or with the authentic entity to be detected and capable of undergoing a primary immune binding reaction with any "unknown" analyte species or any "unknown" entity to be detected in a sample or with any known amounts of analyte species or entity to be detected (i.e.

standards)), and a tracer species associated with the primary species.

The support material, auxiliary species, specific reaction partner, specific reaction partner binding antibody, authentic analyte species, or authentic entity, primary species and tracer species as disclosed in the immediately preceding paragraph may be used in a competitive immunoassay.

In carrying out such a competitive immunoassay, a primary immune reaction may be arranged to occur by contacting the primary species (having tracer species associated therewith) with specific reaction partner binding antibody (having associated therewith authentic analyte species or authentic entity) and with any "unknown" analyte species or "unknown" entity, or with any known amounts of analyte species or entity to be detected (i.e. "standards").

In the absence of any analyte species or entity to be detected the primary species will bind only with the authentic analyte species or authentic entity linked to the specific reaction partner binding antibody.

However, in the presence of analyte species or entity to be detected (e.g. either "unknown" or a known standard) the analyte species or entity to be detected will compete with the authentic analyte species, or authentic entity, for binding with the primary antibody; as the concentration of analyte species or entity to be detected increases, the amount of primary antibody which becomes associated with the specific reaction partner binding antibody decreases and hence the amount of tracer species associated with the specific reaction partner binding antibody decreases.

When the primary immune reaction has proceeded to a desired degree (e.g. equilibrium) specific reaction partner binding antibody (and indirectly associated tracer species) may be removed from the resulting primary immune reaction mixture by attachment to the support material.

This may be done by exposing the primary immune reaction mixture to the support material, upon which is provided the auxiliary species, and introducing the specific reaction partner.

Thus, the specific reaction partner is able to be bound by the auxiliary species on the support material and by the specific reaction partner binding antibody.

Accordingly, any tracer species indirectly associated with the specific reaction partner binding antibody may be attached, indirectly, to the support material.

Subsequently, after any necessary washing, any tracer species associated with the support material may be measured, by any suitable method such as those known in the art, to enable the amount of "unknown" analytic species if any, or "unknown" entity if any, in a sample to be determined by comparison with calibration results obtained using known amounts of "standard" analyte species or "standard" entity.

In one embodiment of an immunoassay in accordance with this example, the primary immune reaction may be allowed to proceed to the desired degree in the presence of the support material upon which is provided the auxiliary species and subsequently the specific reaction partner is introduced to effect separation and attachment of any tracer species to the support material.

The immediately foregoing embodiment offers the advantage of conducting a two-step immunoassay in one vessel.

It will be appreciated that since the specific reaction partner in this example has two ligand functions of the same type, then the auxiliary species and the specific reaction partner binding antibody may be binders of the same type.

(viii) In a further example, the following may be utilised; a support material, an auxiliary species (being in this example, a binder) provided on the support material, a specific reaction partner having two ligand functions (said specific reaction partner being two ligands of different types covalent linked together), a specific reaction partner binding antibody, an authentic analyte species, or an authentic entity associated with the specific reaction partner binding antibody, a primary species (being a primary antibody capable of undergoing a primary immune binding reaction with the authentic analyte species or with the authentic entity to be detected and capable of undergoing a primary immune binding reaction with any "unknown" analyte species or any "unknown" entity to be detected in a sample or with any known amounts of analyte species or entity to be detected (i.e.

standards)), and a tracer species associated with the primary species.

In the absence of. any analyte species or entity to be detected the primary species will bind only with the authentic analyte species or authentic entity linked to the specific reaction partner binding antibody.

It will be appreciated that since the specific reaction partner in the example has ligand functions of different types, the auxiliary species will be a binder for one type of ligand function and the specific reaction partner binding antibody will be a binder for the other type of ligand function.

(ix) In a further example, the following may be utilised; a support material, an auxiliary species (being in this example a binder), provided on the support material, a specific reaction partner (in this example said specific reaction partner having a ligand function and a binder function), a ligand (capable of binding with the binding function of the specific reaction partner), an authentic analyte species linked to the ligand, or an authentic entity linked to the ligand, a primary antibody (being an antibody for the analyte species or for the authentic entity and capable of binding with analyte species or entities (e.g. "unknown" or standards)) said primary antibody being associated with a tracer species.

A competitive immunoassay may be carried out in accordance with this example.

Thus, for example, the authentic analyte species linked to the ligand, or the authentic entity linked to the ligand, may be allowed to compete with analyte species if present or entity to be detected if present, for binding with the primary antibody in a manner similar to that hereinbefore disclosed.

Subsequently tracer species may be removed from any resulting primary immune reaction mixture, for subsequent measurement, by indirect attachment to the support material.

This may be done by exposing the primary immune reaction mixture to the support material, upon which there is provided an auxiliary species (being a binder in this example) and introducing the specific reaction partner. Thus, the ligand function of the specific reaction partner may be bound by the auxiliary species and the ligand (to which any tracer species may be linked) may be bound by the binder function of the specific reaction partner.

By carrying out a process in accordance with this example in a manner disclosed in the immediately preceding paragraphs, a two-step immunoassay may be conducted in one vessel.

The use of calibration results obtained by use of "standards" may be utilised to determine "unknown" analyte species or entities as hereinbefore disclosed.

(x) In a further example, the following may be utilised; a support material, an auxiliary species (being in this example, a binder) provided on the support material, a specific reaction partner (in this example said specific reaction partner having a ligand function and a binder function), a ligand (capable of binding with the binding function of the specific reaction partner), a "capture" antibody linked to the ligand, a "developing" antibody and a tracer species associated with the "developing" antibody.

A non-competitive immunoassay of the "sandwich" type may be carried out in accordance with this example.

Thus, for example, a primary immune reaction may be allowed to occur whereby an analyte species if present, or an entity to be detected, if present, (e.g. "unknown" or standards), binds with the "capture" antibody and subsequently the developing antibody is applied and allowed to bind with analyte species if any, or entity, if any, bound to the capture antibody.

It will be appreciated that in the absence of analyte species, or entity to be detected, substantially no tracer species will become associated with the capture antibody; also it will be appreciated that as an analyte species or entity increases in concentration then an increasing amount of tracer species becomes associated with the capture antibody.

Reference may be made to results obtained using "standard" analyte species or "standard" entities in order to determine an amount of analyte species if any or entity if any in a sample.

Indirect tracer species attachment to the support material may be achieved substantially as hereinbefore described in example (ix).

(xi) In this example, the following may be utilised; a support material, an auxiliary species (being, in this example, a ligand) provided on the support material, a specific reaction partner being, in the example, a biologically produced bifunctional antibody, a "developing" antibody and a tracer species associated with the "developing" antibody.

A non-competitive immunoassay of the "sandwich" type may be carried out in accordance with the example.

Thus, for example, a primary immune reaction may be allowed to occur whereby an analyte species if present, or an entity to be detected if present, (e.g. "unknown" or standards), binds with the one function of the bifunctional antibody (which function may be regarded as acting as a "capture" antibody) and subsequently, developing antibody is applied and allowed to bind with analyte species if any, or entity if any, bound to the function of the bifunctional antibody which is acting as a "capture" antibody.

It will be appreciated that in the absence of analyte species, or entity to be detected, substantially no tracer will become associated with the bifunctional antibody; also it will be appreciated that as an analyte species or entity increases in concentration then an increasing amount of tracer species becomes associated with the capture antibody.

Indirect tracer species attachment to the support material may be achieved by introducing support material upon which there is provided auxiliary species (being, in this example, a ligand) such that the other function of the bifunctional antibody may bind with the auxiliary species.

It will be appreciated that in heterogenous immunochemical analysis (immunoassay) where there is a requirement for the separation of bound and unbound fractions of detectable species (e.g. a tracer species) in order to permit assessment of the distribution of detectable species (e.g. tracer species) between bound and unbound states. It may be considered that this separation step in an immunoassay method is one of the most important features of such a method.

The practical importance of a separation step is based upon the fact that the efficiency of the step, the simplicity (or otherwise) thereof, and the speed thereof may influence the general properties of an assay method and may influence the performance of an assay method.

Separation of bound and unbound fractions of detectable species (e.g. tracer species) may be effected using a solid phase immobilised primary antibody technique in which primary antibody is adsorbed onto a surface of a reaction vessel (e.g. polystyrene tubes) or covalently linked to a micro-particulate material such as microcellulose, Sephadex or micro-particulate material with entrapped ferrous oxide (magnetisable particles).

The use of immobilised primary antibodies in separation in an immunoassay may suffer from several disadvantages; for example interaction between primary antibody and analyte species (or authentic analyte species) may be considerably slower (e.g. due to diffusion considerations) than that which takes place with the same primary antibody and analyte species (or authentic analyte species) in solution (i.e. a longer time may be required to reach equilibrium).

Also, the immobilisation of primary antibody on a solid phase is not a problem-free procedure; thus, for example, it can be difficult to control or reproduce and also serious protein leakage may occur.

The present invention may offer the advantages of reactions in solution with the convenience of a rapid solid-phase separation technique.

Thus, for example, a given primary immune reaction may be expected to approach equilibrium in solution in a short period of time (e.g. at a temperature of 370C to 420C).

Also, by way of example, when using a separation process in accordance with the present invention, in some circumstances, but not all, as much as 99% of a desired material may become bound to the support material.

Reference has hereinbefore been made to an entity to be detected; such an entity to be detected may be, for example, an analyte species-containing species (i.e. a species containing or carrying an analyte species) formed by interaction of an analyte species with a suitable agent.

Thus, in an embodiment of the present invention there is provided a method which includes the use of an agent, said agent being capable of interacting with an analyte species, or with an authentic analyte species, to form an entity to be detected, the use of an auxiliary species, the use of a specific reaction partner for the auxiliary species, the use of a support material, and the use of an antibody to the entity to be detected, said specific reaction partner being capable of being associated with the entity to be detected by a linkage which involves a link, of a selected specific type (as hereinbefore defined), to the specific reaction partner.

In a further embodiment of the present invention there is provided a method which includes the use of an agent, said agent being capable of interacting with an analyte species, or with an authentic analyte species, to form an entity to be detected, the use of a specific reaction partner capable of being associated with the agent, by a linkage which includes a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner, the use of an auxiliary species, said specific reaction partner and said auxiliary species being capable of binding together, the use of a support material, and the use of an antibody to the entity to be detected.

It is to be understood that the analyte species, or authentic analyte species, the agent, the specific reaction partner, the auxiliary species, the support material and the antibody may be brought together in any suitable manner and at any suitable time.

Thus, by way of example, the present invention provides a method which includes the steps of bringing together an analyte species and an agent capable of interaction with the analyte species, thereby to effect interaction of the analyte species with the agent so as to form an entity to be detected, bringing together a specific reaction partner and an auxiliary species provided on a support material, so as to cause binding between the specific reaction partner and the auxiliary species, arranging for an antibody, said antibody being an antibody to the entity to be detected, to bind with the entity to be detected and also arranging for the antibody to be associated with the specific reaction partner by a linkage which involves a link of a selected specific binding type (as hereinbefore defined) to the specific reaction partner.

By way of further example, the present invention provides a method which includes the steps of bringing together an analyte species and specific reaction partner (for an auxiliary species), said specific reaction partner being associated (by a linkage which includes a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner) with an agent capable of interaction with the analyte species, thereby to effect interaction of the analyte species with the agent so as to form an entity to be detected, bringing together the specific reaction partner associated with the entity to be detected thus formed, and an auxiliary species provided on a support material, so as to cause binding between the specific reaction partner and the auxiliary species, and applying an antibody, said antibody being an antibody to the entity to be detected, such that the antibody binds with the entity to be detected.

By way of further example, as an alternative to the example immediately hereinbefore disclosed, the auxiliary species and the specific reaction partner, associated with an agent capable of interaction with an analyte species, may be arranged to bind together before an analyte species interacts with the agent to form the entity to be detected.

It will be appreciated that the antibody to the entity to be detected may be an antibody that is a specific antibody to the entity to be detected.

In accordance with the present invention an antibody to an entity to be detected may, optionally, be linked to a detectable species (as hereinbefore disclosed); the detectable species may be, for example, a tracer species (e.g. an enzyme tracer, a fluorescence tracer or a radioactive tracer).

Accordingly, in accordance with an embodiment of the present invention there is provided a method which comprises the steps of bringing together a specific reaction partner, and an auxiliary species provided on a support material, so as to cause binding between the specific reaction partner and the auxiliary species, arranging for the specific reaction partner to be associated with an entity to be detected (by means of a linkage which involves a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner), applying an antibody, said antibody being an antibody to the entity to be detected and said antibody being linked with a tracer species, such that the antibody binds with the entity to be detected and detecting by means of the tracer species the binding of the antibody and the entity.

The specific reaction partner may be arranged to be associated with an entity to be detected in any suitable manner and at any suitable time (e.g. before or after binding between the specific reaction partner and the auxiliary species).

Any suitable agent capable of forming an entity to be detected may be used in accordance with the present invention. An agent capable of forming a complex with the analyte species is an example of an agent which may find application in the formation of an entity to be detected in accordance with the present invention.

Where the analyte species is a metal (e.g. in the form of a metal ion) the agent, which may be associated with the specific reaction partner, may be, for example, an agent capable of forming a metal-containing species (e.g. a complex of the metal).

Examples of agents for forming a complex with a metal are chelating agents.

By way of example, the antibody may be linked with a detectable species comprising an enzyme tracer and detecting the binding of the antibody and the metalchelate complex may be by means of an enzyme induced change.

The enzyme induced change may be, for example, a colour change, or a fluorescence change, or a luminescence change or an electro-chemical change.

It will be appreciated that the antibody may be such that it recognizes the metal-chelate complex but not chelating agent molecules which are not complexed with a metal ion.

Thus, the antibody may be specific with respect to the metal-chelate complex. Also, the antibody may be such as to be specific to a complex of a particular metal ion with a chelating agent such that it does not bind significantly with complexes comprising other metal ions complexed with the same chelating agent.

Examples of chelating agents for forming a metal chelate are: ethylene diamine tetra acetate (EDTA), diethylene triamine penta acetate (DTPA), cyclohexylenedinitrilo tetra acetic acid (CDTA), 1-benzyl-EDTA, derivatives of 1-benzyl-EDTA, 8-hydroxyquinoline, derivatives of 8 hydroxyquinoline, and deferoxamine.

The agent may be associated with the specific reaction partner in any suitable manner (which involves a link of a selected specific binding type to the specific reaction species); by way of example one unit of an agent may be associated with the specific reaction partner or more than one unit of an agent may be associated with the specific reaction partner. Thus, for example, one molecule of an agent may be associated with a specific reaction partner or, for example, more than one molecule of an agent may be associated with a specific reaction partner.

From the foregoing disclosure it will be appreciated that in an embodiment of the present invention a method may include the step of forming a complex of an analyte species said complex thus formed providing an entity to be detected.

Also, from the foregoing disclosure, it will be appreciated that in an embodiment of the present invention a method may include the step of associating an agent and a specific reaction partner.

The present invention may find application in, for example, detection of metals (e.g. in the form of free, hydrated metal ions) in environmental, or clinical, or industrial samples.

It will be understood that the present invention may find application in, for example, the detection and determination of metals which can be considered to be toxic in the environment. Accordingly, the present invention may find application in environmental monitoring.

Thus, for example, a level of concentration of a metal ion (e.g. the concentration of a heavy metal ion), which may typically be in the ppb range, may be detected and determined in any suitable sample. Thus, for example, samples of water, soil, living products (such as plants (e.g. vegetables) or animals) or air may provide an analyte species, or an entity to be detected, for detection in accordance with the present invention.

Preferably sample collection procedures and any concentration procedures should be such as to minimise contamination from external sources.

By way of example, samples to be subjected to detection in accordance with the invention may be, conveniently, aqueous samples.

The following are examples of metals which may be detected: calcium (CaII), iron (FeII, FeII), cobalt (CoII, CoIII), aluminium (AlII), zinc (ZnII), lead (PbII), copper (cuII, CuI), cadmium (CdII), vanadium (VII, VIII, silver (AgI, AIIo), mercury (HgI, HgII), indium (InIII), manganese (MnII) and nickel (NiII).

It will be appreciated that an entity to be detected and an antibody to the entity may be brought together in any suitable manner; thus, for example, the bringing together of an entity to be detected and an antibody in accordance with the present invention may be effected in an aqueous medium.

As hereinbefore disclosed an antibody to an entity to be detected may be prepared by any suitable method, for example those known for the raising of polyclonal or monoclonal antibodies; thus, antibodies may be raised, for example, by immunising animals.

For example, an analyte species may be reacted with an agent so as to form an entity (e.g. a complex of the analyte species) and the entity may be conjugated to a carrier protein prior to being used to prepare an antibody to the entity.

It is to be understood that when being used to prepare an antibody, the entity may be regarded as a hapten.

By way of further example, an agent may be conjugated to a carrier protein and then an analyte species introduced so as to react with the agent to form an entity (e.g. a complex of the analyte species) such that the carrier protein carrying the entity may be used to prepare an antibody to the entity.

Thus, for example, a chelating agent such as p-amino benzyl-EDTA may be conjugated to bovine serum albumin (BSA) or keyhole limpet haemocymin (KLH) via a thiocyanate-derivative or diazo-derivative of the p-amino function and the product resulting from this conjugation allowed to react with excess of a salt of a metal (e.g.

lead nitrate or cobalt nitrate) so that a metal ion-EDTA BSA species forms or a metal ion-EDTA-KLH species forms.

Following removal of excess metal salt (e.g. by gel filtration or dialysis) such a species may be used to raise an antibody to the metal ion-EDTA complex, for example, by immunising animals over a period of 4-6 months.- It will be appreciated that after a specific binding reaction between a specific reaction partner for an auxiliary species and an auxiliary species provided on the support material, the specific reaction partner is also, in effect, attached to the support material.Thus, where, for example, a specific reaction partner is associated with an analyte species, or an entity to be detected (by means of a linkage which includes a link, of a selected specific binding type (as hereinbefore defined) to the specific reaction partner), and an antibody to the analyte species, or to the entity to detected, is applied, bound antibody may be, in effect, attached to the support material (via, in turn, the analyte species or the entity to be detected, the specific reaction partner and the auxiliary species).

Thus, any required washing steps (e.g. with appropriate buffers) may be used to remove unbound materials.

Tracer species left on the support material, the amount of which is directly proportional to the concentration of the analyte species, or the entity to be detected, may be measured by any suitable method such as those known in the art.

It is to be understood that by use of an antibody to an entity to be detected (e.g. a metal complex), the binding of antibody and entity may be specific such that the detection thus achieved is specific and sensitive to the entity and thus to any analyte species which is part of the entity.

By way of example, analyte species concentrations in a nanomolar to picomolar (i.e. 109 to 10.12 M) range may be detected in accordance with the present invention (e.g.

using an immunoassay procedure such as an enzyme-labelled immunoassay procedure).

Sensitivity of detection in accordance with the present invention may be expressed as a "detection limit" (also known in the art as a "quantification limit").

This limit relates to the relation between a change in a response and a concentration of an analyte species causing the change in response.

The "detection limit" is the lowest concentration or amount of an analyte species (usually expressed in concentration units) which can be detected by an analytical method.

The "detection limit" should, in general, have at least 95% confidence to be meaningful.

In one example of a method for detection in accordance with the present invention, which example includes the use of a labelled immunoassay procedure, factors which affect sensitivity of detection include: (a) affinity of an antibody with respect to an analyte species or an entity to be detected, (b) specific activity of any assay tracer used, (c) signal species nature (i.e. the type of signal species used (e.g. radioactive tracer, enzymatic tracer or fluorescence tracer)), (d) assay background (i.e. background "noise") and (e) instrumentation used to measure assay signal.

Where, for example, an entity to be detected is to be detected in accordance with the present invention, it will be appreciated that the entity to be detected should be sufficiently stable to permit satisfactory detection to be effected. It will also be appreciated that the entity may be considered to be acting as a "hapten" with respect to the antibody to the entity.

Where in accordance with the present invention an entity to be detected and an antibody to the entity are utilised, specificity in relation to detection of analyte species, which forms part of the entity to be detected, in accordance with the present invention may be defined as the degree to which the detection distinguishes between the analyte species (e.g. metal ion) and other species (e.g. other metal ions) when a sample to be subjected to detection contains a mixture of species.

The specificity of detection may, therefore, depend upon the specificity of the antibody to the entity to be detected in the presence of other species (e.g. a complex or complexes which may give rise to interfering reactions).

For example, an agent capable of forming a complex with an analyte species may also be capable of forming a complex with species other than the analyte species.

In such circumstances there may be some "crossreaction" of antibody (i.e. antibody to the complex of the analyte species) with complexes of an agent with species other than the analyte species.

By way of example, one procedure for determining cross-reaction of an antibody makes use of an immobilised primary antibody and labelled complex of the analyte species in a competitive immunoassay method. Thus, a calibration curve (i.e. a standard curve) may be constructed using a range of analyte species concentrations and a "limited" amount of immobilised antibody. Binding of a limited amount of labelled complex of the analyte species is monitored in the absence of a competing standard complex of the analyte species and in the presence of increasing levels of such a standard.

A calibration curve (a "dose response curve") is constructed by plotting the level of bound tracer (as % bound) against the concentration of standard. It is to be understood that the binding of the tracer may be taken as 100% (when 0% of the standard is bound) or the total amount of tracer may be taken as 100%.

The above steps are repeated replacing the standard with a range of known concentrations of complexes comprising the same agent (as used in complexing the analyte species) complexed with species other than the analyte species; all other parameters are kept substantially unchanged.

Thus, a series of dose response curves of standard and possible "cross-reacting" complexes may be co-plotted on a graph.

Cross-reaction values (CR) are calculated as 100 x the ratio of molar concentrations of standard and possible cross-reacting complexes which cause a decrease of tracer binding by 50% (taking the binding value in the absence of competing entities as 100%).

Accordingly, From the foregoing disclosure it will be understood that, by way of example, detection of an analyte species CR = [moles of standard complex] 50% X 100.

moles of cross-reacting compl ex] 508 in accordance with the present invention may be conducted using an immunochemical detection procedure, for example, an immunoassay procedure.

Where, for example, an agent capable of interacting with an analyte species is used to form an entity to be detected, an analyte species (e.g. a metal ion) may be, for example, extracted from a sample by interaction with the agent which may be associated with a specific binding partner thereby to form an entity to be detected.

Subsequently, the specific binding partner may be bound to an auxiliary species provided on a support material and then a labelled antibody is applied; the amount of entity present (and hence the amount of analyte species present in the sample) may be determined by methods known for immunoassay.

The present invention may also offer for sensors, for example immunosensors such as optical immunosensors and electrochemical immunosensors, the possibility that a sensor surface carrying an auxiliary species may be the same for a variety of, or all, analyte species thereby providing a generic sensor construction.

A combination comprising a sensor surface carrying an auxiliary species may also, for example, be such as to permit regeneration for multiple reuse; for example, such a combination of a sensor surface carrying an auxiliary species may be regenerated by removal of bound substances by washing with a suitable buffer preparation (e.g. 0.1M glycine-HCl at pH 2.5).

Also, by way of further example, the auxiliary species may be selected so as to be stable to multiple treatment with low or high pH organic solvents and/or chaiotropic reagents thereby to permit regeneration for multiple reuse.

The present invention thus offers the possibility of constructing a non-dedicated sensor such as an immunosensor or immunoelectrode.

Thus, by way of example, an electrochemical immunosensor may be provided in which an electrode surface is coated with a conducting polymer (e.g. a polypyrrole polymer) which may be arranged to act as an auxiliary species. For use in such a sensor antibodies to the conducting polymer (e.g. polypyrrole polymer), or segments thereof, may be generated by suitable techniques such as those known in the art; a combination comprising an electrode surface and a conducting polymer coating may be, for example, such as to permit regeneration for multiple reuse.

Any suitable methods of competitive and noncompetitive immunoassay may be used in an electrochemical immunosensor as immediately hereinbefore described. A tracer species for use in such an electrochemical immunosensor may be arranged to provide an electroactive species.

In conventional electrochemical immunosensors known in the art specific primary antibodies may be immobilised on an electrode surface thereby to provide a dedicated immunosensor; such immunosensors may have inherent constraints when multiple electrodes with the same properties are required. Thus, disadvantages may become apparent since regeneration of the sensor may be difficult or regeneration may lead to deterioration of primary antibody activity.

The present invention may offer the possibility of avoiding such disadvantages in that a sensor in accordance with the present invention may not need to be a dedicated sensor.

It will be appreciated from the foregoing that, by way of further example, detection of an analyte species in accordance with the present invention may use an immunosensor technique.

For example, the auxiliary species may be immobilised on a surface of a sensor device (e.g. on a glass surface, on a quartz surface, or on the surface of an electrode).

For example, in the use of direct optical immunosensors no label is used and the binding of an analyte species or entity to be detected and antibody may give rise to signal generation to permit detection (e.g.

by surface plasmon resonance).

By way of further example, in the use of a labelled antibody immunosensor, signal generation may be detected by a suitable method appropriate to the nature of the signal generated by the label (e.g. a means for measuring fluorescent emission may be used when the label is a fluorescence producing entity).

According to a further aspect of the present invention there is provided a test-kit which test-kit includes the use of an auxiliary species provided on a support material, said test-kit being arranged to be capable of effecting a method of detection in accordance with the present invention; such a test-kit may include, for example, an auxiliary species provided on a support material and a specific reaction partner.

It is to be understood that the present invention may find application in, for example, qualitative detection, which may also be considered to be qualitative analysis, or in quantitative detection which may also be considered to be quantitative analysis (i.e. measurement or determination); it will also be appreciated that quantitative detection may be used to determine a level of concentration.

It will be appreciated that, for example, the present invention may be utilised with samples which contain analyte species or entity to be detected and with samples which contain substantially no analyte species or no entity to be detected (e.g. such as in the case where a "blank" standard sample or an "unknown" sample which, upon subjecting to detection in accordance with the present invention, is found to contain substantially no analyte species or entity to be detected). Thus, it will be appreciated that the present invention may be used to "probe" for an analyte species or an entity whether, or not, such an analyte species or an entity is present in a particular sample.

Further it will be appreciated, by way of example, that by use of standard quantities of authentic analyte species, or authentic entity to be detected, calibration may be effected such that, subsequently, unknown quantities of analyte species, or unknown quantities of entity to be detected may be determined.

It will also be appreciated that, in a competitive immunoassay, as the amount of analyte species or entity to be detected increases, the amount of tracer species retained on the support material decreases and, conversely, as the amount of analyte species or entity to be detected decreases the amount of tracer species retained on the support material increases, reaching a maximum when no analyte species or entity to be detected is present.

It will be appreciated also that washing of a support material with appropriate buffers may be used to remove unbound materials as required; also where a tracer species is used any tracer species activity associated with the support material may be measured by any suitable method such as these known in the art.

Optionally, where a tracer species is used any tracer species activity may be eluted into solution using appropriate buffers to facilitate measurement of tracer species.

In some embodiments of the present invention (e.g.

embodiments involving the use of an optical immunosensor based on the surface plasmon resonance principle or on evanescent wave fluorescence) a washing step may not be required; thus, where a tracer species is used any tracer activity associated with the support material (which may be, for example, a sensor surface) may be measured in the presence of unbound material.

By way of further example, an immunoassay (e.g. an enzyme immunoassay (EIA)) may comprise the following steps: Step 1 An auxiliary species, being for example an auxiliary antibody, may be adsorbed onto a support material. Thus, for example, an affinity chromatography purified auxiliary antibody solution (in PBS) may be added to wells of a micro-titre plate (e.g. at 10 Ag/ml) and left to adsorb for 24 hours at 4 C.

Blocking of excess sites may be effected, for example, by use of horse haemoglobin.

The resulting plate may be stored dry in the cold ( < 00C).

SteD 2 Analyte species standards (or entity standards) (e.g. at 0, and 2 up to 200 ng/ml) and/or an "unknown" sample, or samples, may be added to the plate, followed by serum blocking agents if desired.

Subsequently, to the wells may be added (a) a specific reaction partner antibody (being an antibody for a specific reaction partner) conjugated to an authentic analyte species (or a specific reaction partner antibody (being an antibody for a specific reaction partner) conjugated to an authentic entity) and (b) an analyte species antibody (being an antibody for an analyte species) associated with an enzyme tracer species (or an entity antibody (being an antibody for an entity to be detected) associated with an enzyme tracer).

The plates may be incubated for 1 hour with shaking.

Subsequently, a specific reaction partner being a ligand with more than one ligand function may be added to initiate a separation step whereby tracer species, if associated (indirectly) with the anti-specific reaction partner antibody, may become associated (indirectly) with the support material.

Incubation and shaking of the plates may be continued for i hr.

It will be appreciated that one of the ligand functions may bind with the specific reaction partner antibody and another of the ligand functions may bind with the auxiliary species.

Ste 3 The plates may be washed, an enzyme substrate may be added and, after colour development, absorbance may be read.

Using "standards" a plot of OD against analyte species concentration (or entity concentration) may be made such that subsequently the concentration of analyte species (or entity to be detected) in "unknown" samples may be determined by subjecting such samples to Steps 1 to 3 as immediately hereinbefore disclosed and comparing the OD obtained with such samples with the plot obtained using standards.

An immunoassay as immediately hereinbefore disclosed, with reference to Steps 1 to 3, may be applied to the immunoassay of any suitable analyte species (e.g.

L-thyroxine) or any suitable entity to be detected.

The present invention will now be further described, by way of example only, as follows: Example 1 Preparation of L-thyroxine antibody L-thyroxine was coupled to a mixture of immunogenic proteins as follows: L-thyroxine (free acid) (0.776 mg) was dissolved in dimethyl formamide (5 ml) and the resulting solution was heated to approximately 500C.

Glutaric anhydride (0.342 mg) was added to the thyroxine solution and the resulting mixture was kept at approximately 50"C for 30 minutes; subsequently the mixture was added to cold water (100 ml) where a white precipitate appeared. The resulting suspension was stirred for 1 hour and then was extracted twice with 200 ml of ethyl acetate.

The resulting ethyl acetate extracts were combined and washed with water (three times with 400 ml), then with 0.1M HCl (three times with 400 ml) and finally with water (three times with 400 ml).

After drying on anhydrous sodium sulphate, ethyl acetate was removed by rotary evaporation under reduced pressure. The resulting residue was washed with diethyl ether (about 100 ml) and left under vacuum to dry and solidify.

The resulting white material, designated Lthyroxine-amido-glutaric acid (i.e. L-thyroxine-NH-(CO (CH2)3-COOH)), was examined by TLC and compared with Lthyroxine. The comparison indicated that the material was pure, did not contain free amino groups and was different from L-thyroxine.

An N-hydroxysuccinimide ester of L-thyroxine-amidoglutaric acid was prepared as follows: L-thyroxine-amido-glutaric acid (216 mg) was added to dimethyl formamide (4 ml) together with N-hydroxy succinimide (50 mg) and dicyclohexylcarbodiimide (60 mg).

The resulting mixture was left at 40C for 3 days before use (by which time a fine precipitate had appeared) to give an ester-containing solution.

A volume of 1 ml of the ester-containing solution was added, over a period of 2 hours, to a mixture of bovine serum albumin (BSA) (30 mg), keyhole-limpet haemocyanin (KLH) (30 mg), and fetuin (30 mg) dissolved in 0.1M sodium bicarbonate buffer (12 ml; pH 8.6) and dioxane (3 ml).

The resulting mixture was dialysed at 40C for 3 days against 3 changes each of 4 1 of 1% aqueous sodium bicarbonate.

The resulting immunogen solution was recovered, made up to 40 ml with distilled water and stored below OCC until required for use.

The resulting immunogen solution was used to immunise sheep for 9 months according to standard procedures known in the art.

A plate coating antigen, suitable for use in enzyme immunoassay of L-thyroxine using polystyrene ELISA microtitre plates, was prepared by coupling L-thyroxine-amidoglutaric acid to ovalbumin. Ovalbumin (30 mg) was dissolved in 0.1M sodium bicarbonate buffer (3 ml; pH 8.6) and dioxane (2 ml); to the resulting solution was added 0.12 ml of L-thyroxine-amido-glutaric acid-Nhydroxysuccinimide ester (50 mg/ml) and the resulting mixture was immediately vigorously stirred to ensure good distribution of the ester.

The mixture was left at room temperature for 24 hours and then dialysed at 40C for 3 days against 3 changes each of 4 1 of 1% aqueous sodium bicarbonate.

The resulting conjugate solution was recovered, made up to 5 ml with distilled water and treated with 1% Nor it A activated charcoal (by mixing and leaving to stand for 1 hour, and centrifuging).

The resulting conjugate, which may be used as a plate coating conjugate, was stored below OOC.

The conjugate was used to titrate L-thyroxine antiserum in accordance with processes known in the art such as to permit the construction of a competitive quantitative EIA (enzyme immunoassay) method for the detection of L-thyroxine.

Antibody titres of 1/120,000 were obtained by standard ELISA procedures.

Example 2 Purification of anti-L-thyroxine antibody by affinity chromatography An immunoadsorbent for anti-L-thyroxine antibodies was prepared by coupling L-thyroxine-amido-glutaric acid ovalbumin conjugate (as prepared in Example 1) to CNBr activated Sepharose-4B; (Sepharose-4B is a dextran product of Pharmacia).

The conjugate (5 mg in 10 ml of 0.1N sodium bicarbonate buffer, pH 8.0) was added to 7.5 ml of swollen CNBr activated Sepharose-4B to effect coupling of the conjugate to the activated Sepharose-4B.

After the coupling step was completed, the resulting immunoadsorbent was washed with buffer (0.1M sodium bicarbonate at pH 8.0, > 4 1) then with an eluting mixture (of 20% acetonitrile and 1% propionic acid in distilled water) and washed again with buffer (approximately 1 1) to remove the eluting mixture.

The IgG fraction (prepared by ion exchange chromatography) of sheep anti-L-thyroxine antibody (100 mg in 20 ml PBS) was passed through a 2 ml column of the immunoadsorbent (prepared as immediately hereinbefore disclosed) at a rate of 0.5 ml/min.

After washing the column with 0.1M sodium phosphate buffer (pH 8.0), containing 0.5M NaCl, to remove nonspecifically adsorbed protein, the anti-L-thyroxine antibody was eluted by gradient application of the eluting mixture (as hereinbefore mentioned in this Example) into 20 ml of distilled water in a mixing chamber.

The pH of the eluted antibody peak was adjusted to neutral (with phosphate buffer) and the resulting solution dialysed in the cold against PBS in a manner substantially similar to that disclosed hereinbefore in Example 1.

Antibody (approximately 8 mg) was recovered and was shown to contain a high level of binding activity with conventional ELISA.

An aliquot (2 mg) of the antibody was labelled with the enzyme horse radish peroxidase (4 mg), using known standard periodate oxidation.

Unconjugated free enzyme was separated from the antibody enzyme-complex by gel filtration on Sephacryl S300 column; (Sephacryl is a product of Pharmacia).

Example 3 Conjugation of L-thyroxine to sheep anti-rabbit Pc fragment Sheep antiserum (to act as a second antibody (i.e.

an antibody to an antibody for the auxiliary species) in accordance with the present invention), raised against rabbit IgG Fc fragment, was obtained from a commercial source (Dako).

The IgG fraction of this antiserum was conjugated with L-thyroxine-amido-glutaric acid in a manner substantially similar to that described in Example 1 for the conjugation of L-thyroxine-amido-glutaric acid to ovalbumin.

The resulting conjugate was purified by affinity chromatography using Sepharose 4B-rabbit Fc as an immunoadsorbent.

The binding activity of the conjugate was tested by conventional ELISA and the L-thyroxine content of the conjugate was examined using the conjugate as a plate coating antigen.

Example 4 Preparation of specific reaction partner for the auxiliary species In this Example an antibody to the auxiliary species was prepared as follows: 7-hydroxycoumarin-3-propionic acid (CPA) was conjugated to BSA to prepare an immunogen as follows: CPA (244 mg) was added to dioxane (12 ml) together with N-hydroxysuccinimide (120 mg) and dicyclo hexylcarbodiimide (220 mg).

The resulting mixture was left at room temperature for 24 hours (after which a fine precipitate had appeared) to form an ester-containing mixture.

An aliquot (1 ml) of the ester-containing mixture was added (over a period of 1 hour) with mixing to BSA (30 mg) in 2 ml of 0.1M sodium bicarbonate buffer (pH 8.6) and 1 ml of dimethylsulphoxide.

The resulting mixture was allowed to react to give CPA-BSA immunogenic conjugate and the conjugate thus formed was treated by dialysis in a manner substantially similar to that disclosed in relation to the purification of the immunogen prepared as disclosed in Example 1.

Standard antibody producing rabbits were immunised with the CPA-BSA immunogen over a period of 9 months.

A plate coating antigen for 7-hydroxycoumarin-3propionic acid was prepared by coupling a derivative thereof to ovalbumin. Thus, 7-hydroxycoumarin-3propioamido-caproic acid was prepared and coupled to ovalbumin using steps similar to the preparation of the immunogenic conjugate hereinbefore disclosed in this Example.

The plate coating antigen was used to titrate rabbit serum, prepared as hereinbefore disclosed in the Example, by a conventional ELISA procedure. Titres of 1/20,000 were obtained.

Purification of anti-7-hydroxycoumarin-3-propionic acid antibody by affinity chromatography was carried out in a manner analogous to that described for the purification of anti-L-thyroxine antibody as hereinbefore described.

The anti-7-hydroxycoumarin-3-propionic acid antibody is a specific reaction partner for the auxiliary species 7-hydroxycoumarin-3-propionic acid.

Example 5 Enzyme immunoassay for measurement of serum thyroxine (total T4) (i) Auxiliary species (being in this Example an auxiliary ligand) was adsorbed to polystyrene micro-titre ELISA plates.

Thus, a conjugate of 7-hydroxycoumarin-3 prop ionic acid and ovalbumin (prepared as disclosed in Example 4) in 1% aqueous sodium bicarbonate (at 10 Ag/ml) was added to wells of a micro-titre plate (at 200 1 per well) and left to adsorb at room temperature for 24 hours.

The wells were emptied and 200 p1 of a 0.1% aqueous horse haemoglobin solution was added to each well to block any excess sites on the surface of the wells.

After 2 hours, the wells were washed six times (by filling and emptying) using aqueous washing buffer (being an aqueous solution of 1% sodium bicarbonate, 0.1M NaCl and 0.05% Tween 20).

(ii) (a) To micro-titre plate wells prepared as immediately disclosed in (i) a range of calibration standards of analyte species L-thyroxine (in stripped serum) were added to different wells. A volume of 25 41 of each standard was added to each well; the concentration of analyte species in the standards were 0 ng/ml (i.e. a "blank") and 2 up to 200 ng/ml.

(b) Subsequently a blocking reagent solution (50 1 per well) was added to each well; the blocking reagent solution was 150 pg of ANS and 100 4g of rhodamine B base in 50 p1 of assay buffer. The assay buffer was an aqueous solution of 0.05M Tris, pH 8.0, 0.1M NaCl, 0.1% gelatin and 0.01% thimerosal.

(c) Subsequently second antibody-L-thyroxine conjugate (prepared as in Example 3) was added to the wells; 5041 of conjugate solution, suitably diluted in assay buffer in accordance with immunoassay procedures, were used in each well.

(d) Enzyme-labelled L-thyroxine antibody (prepared as in Example 2) was also added to the wells; 50 41 of labelled antibody, suitably diluted in assay buffer in accordance with immunoassay procedures, was used in each well.

(e) Following (d) the resulting assay mixture in each well was mixed by shaking the plate at room temperature of approximately 220C for 1 hour.

(f) Subsequently, to each well, 25 4 of anti auxiliary species antibody as prepared in Example 4 (and suitably diluted in assay buffer in accordance with immunoassay procedures) was added to initiate separation by means of binding with the auxiliary species on the support material (the micro-titre plates) and by means of binding with the second antibody. Shaking was resumed for 30 minutes.

(iii) The wells were next washed with buffer solution (1% aqueous sodium bicarbonate) by filling and emptying six times.

Subsequently, a substrate for HRP enzyme was added to the wells (200 41).

The enzyme substrate was H2O2 at 1.3mM and ABTS prepared in acetate/citrate buffer (pH 4.1) at 0.5 mg/ml.

The plate was shaken for 25 minutes, after addition of the substrate, at room temperature and the absorbance was read in an ELISA micro titre plate reader.

A plot of OD (at 405 nm) was made against standard analyte species concentration.

The OD was 1.7 at a T4 concentration of 0 ng/ml; the OD fell as T4 concentration increased and had a value of 0.15 to 0.2 at a T4 concentration of 200 ng/ml.

It will be appreciated that an "unknown" sample or samples may replace the standards applied to the plate as in (ii) (a) of Example 5 and by measuring the OD obtained with such a sample or samples the amount of analyte species in a sample may be determined by reference to a plot (which may be considered to be a calibration plot) obtained using "standards".

Claims

1. A separation method, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner for the auxiliary species being capable of binding with the auxiliary species and said specific reaction partner for the auxiliary species being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner for the auxiliary species.

2. A separation method as claimed in Claim 1 wherein the link of a selected specific binding type is a link which is formed by a specific binding reaction between the specific reaction partner for the auxiliary species and an antibody for the specific reaction partner, which partner has been purposely prepared.

3. A separation method as claimed in Claim 1 wherein the-specific reaction partner for the auxiliary species is a biologically produced antibody having more than one function and the link of a selected specific binding type is a link which is formed by specific binding between the antibody or part of the antibody and a primary species, said antibody or said part thereof not being a primary antibody.

4. A separation method as claimed in Claim 1 wherein the auxiliary species is a binder and the specific reaction partner is a biologically produced antibody having more than one function, part of which antibody comprises a primary species being a binder for a primary species, and the link of a selected specific binding type is a link formed by specific binding between the antibody, or a part of the antibody, and a primary species.

5. A separation method as claimed in Claim 1 wherein the auxiliary species is a binder and a specific reaction partner for the auxiliary species has a ligand function and a binder function, and the link of a selected specific binding type is a link which is formed by specific binding between the binder function and a further species comprising a ligand, said ligand not being a primary species.

6. A separation method as claimed in Claim 1 wherein the auxiliary species is a ligand and the specific reaction partner is a biologically produced antibody having more than one function, part of which antibody comprises a primary species being a binder for a primary species, and the link of a selected specific binding type is a link which is formed by specific binding between the antibody, or part of the antibody, and a primary species.

7. A separation method as claimed in Claim 1 wherein the specific reaction partner is a species which has more than one ligand function, one of which functions may bind with an auxiliary species which is a binder and the link of a selected specific binding type is a link which is formed by a binding reaction between another of said functions and a binder which may be associated with primary species.

8. A separation method as claimed in Claim 1, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being capable of undergoing a specific binding reaction with the auxiliary species and said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a specific binding type, to the specific reaction partner, said link of a specific binding type being a link of the type formed by specific binding between the specific reaction partner and a purposely prepared antibody for the specific reaction partner.

9. A separation method as claimed in Claim 1, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being an antibody which antibody is not a primary species and which antibody is a biologically produced antibody which has more than one function such that said specific reaction partner is capable of undergoing specific binding with the auxiliary species and such that said specific reaction partner is capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

10. A separation method as claimed in Claim 1, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species, said auxiliary species being a binder and said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being a biologically produced antibody having more than one function, part of which antibody comprises a primary species being a binder for a primary species, said specific reaction partner being capable of undergoing specific binding with the auxiliary species and said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

11. A separation method as claimed in Claim 1, suitable for use in an immunoassay method for the detection of an analyte species which separation method includes the use of an auxiliary species comprising a binder, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner having a ligand function and binder function such that the specific binder partner is capable of undergoing specific binding with the auxiliary species and such that said specific reaction partner is capable of being linked with a primary species via a third species by means of a linkage which involves a link, of a selected specific binding type to the specific reaction partner.

12. A separation method as claimed in Claim 1 suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species, said auxiliary species being a ligand and said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being a biologically produced antibody having more than one function, part of which antibody comprises a primary species being a binder for a primary species, said specific reaction partner being capable of undergoing specific binding with the auxiliary species and said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

13. A separation method as claimed in Claim 1 suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species, said auxiliary species being a binder and said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being a species which has more than one ligand function, one of which functions may bind with the auxiliary species and another of which functions may bind with a third species, which third species may be associated with a primary species, such that the said specific reaction partner is capable of being linked with a primary species, by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

14. A method, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner for the auxiliary species being capable of binding with the auxiliary species and said specific reaction partner for the auxiliary species being capable of being linked with a primary species by means of a linkage which involves a link of a selected specific binding type (as hereinbefore defined), to the specific reaction partner for the auxiliary species.

15. A method as claimed in Claim 14, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being capable of undergoing a specific binding reaction with the auxiliary species and said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a specific binding type, to the specific reaction partner, said link of a specific binding type being a link of the type formed by specific binding between the specific reaction partner and a purposely prepared antibody for the specific reaction partner.

16. A method as claimed in Claim 14, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being an antibody which antibody is not a primary species and which antibody is a biologically produced antibody which has more than one function such that said specific reaction partner is capable of undergoing specific binding with the auxiliary species and such that said specific reaction partner is capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

17. A method as claimed in Claim 14 suitable for use in the detection of an analyte species, which method includes the use of an auxiliary species, said auxiliary species being a binder and said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being a biologically produced antibody having more than one function, part of which antibody comprises a primary species being a binder for a primary species, said specific reaction partner being capable of undergoing specific binding with the auxiliary species and said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

18. A method as claimed in Claim 14, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species comprising a binder, said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner having a ligand function and binder function such that the specific binder partner is capable of undergoing specific binding with the auxiliary species and such that said specific reaction partner is capable of being linked with a primary species via a third species by means of a linkage which involves a link, of a selected specific binding type to the specific reaction partner.

19. A method as claimed in Claim 14, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species, said auxiliary species being a ligand and said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being a biologically produced antibody having more than one function, part of which antibody comprises a primary species being a binder for a primary species, said specific reaction partner being capable of undergoing specific binding with the auxiliary species and said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, or a selected specific binding type, to the specific reaction partner.

20. A method as claimed in Claim 14, suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species, said auxiliary species being a binder and said auxiliary species being provided on a support material, and the use of a specific reaction partner for the auxiliary species, said specific reaction partner being a species which has more than one ligand function, one of which functions may bind with the auxiliary species and another of which functions may bind with a third species, which third species may be associated with a primary species, such that the said specific reaction partner is capable of being linked with a primary species, by means of a linkage which involves a link, of a selected specific binding type, to the specific reaction partner.

21. A method as claimed in any one of the preceding Claims wherein the linkage also includes a further link.

22. A method as claimed in Claim 21 wherein the further link is of a non-specific binding type or the further link is of a specific binding type.

23. A method as claimed in any one of Claims 1, 2, 8 or 15 wherein the purposely prepared antibody is a second antibody.

24. A method as claimed in any one of Claims 1, 2, 8, 15 or 23 wherein the purposely prepared antibody is an antibody having more than one function.

25. A sensor suitable for use in the detection of an analyte species, which sensor includes an auxiliary species provided on a support material and which sensor is constructed such as to be capable of effecting a method in accordance with the present invention as claimed in any one of the preceding Claims.

26. A sensor as claimed in Claim 25 suitable for use in the detection of an analyte species, which sensor includes an auxiliary species provided on a support material and the use of a specific reaction partner for the auxiliary species capable of undergoing a specific binding reaction with the auxiliary species, said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner.

27. A separation method as claimed in Claim 1, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species provided on a support material, the use of a specific reaction partner for the auxiliary species capable of undergoing a specific binding reaction with the auxiliary species, said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner, and the use of an antibody to an analyte species or the use of an antibody to an entity to be detected.

28. A separation method as claimed in Claim 1, suitable for use in an immunoassay method for the detection of an analyte species, which separation method includes the use of an auxiliary species provided on a support material, the use of a specific reaction partner capable of undergoing a specific binding reaction with the auxiliary species, said specific reaction partner being linked to an entity to be detected, and the use of an antibody to the entity to be detected, said specific reaction partner being linked to an entity to be detected by a linkage which includes a link, of a selected specific binding type (as hereinbefore defined) to the specific reaction partner.

29. A method as claimed in Claim 14 suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species provided on a support material, the use of a specific reaction partner capable of undergoing a specific binding reaction with the auxiliary species, said specific reaction partner being capable of being linked with a primary species by means of a linkage which involves a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner, and the use of an antibody to an analyte species or the use of an antibody to an entity to be detected.

30. A method as claimed in Claim 14 suitable for use in the detection of an analyte species by immunoassay, which method includes the use of an auxiliary species provided on a support material, the use of a specific reaction partner capable of undergoing a specific binding reaction with the auxiliary species, said specific reaction partner being linked to an entity to be detected, and the use of an antibody to the entity to be detected, said specific reaction partner being linked to the entity to be detected by means of a linkage which involves a link, of a selected specific binding type (as hereinbefore defined) to the specific reaction partner.

31. A sensor as claimed in Claim 25 which sensor includes an auxiliary species provided on a support material, said auxiliary species being for undergoing a specific binding reaction with a specific binding partner which specific binding partner may be associated with an entity to be detected, said specific reaction partner being linked to the entity to be detected by means of a linkage which involves a link of a selected specific binding type (as hereinbefore defined) to the specific reaction partner.

32. A method or a sensor as claimed in any one of the preceding Claims wherein the support material, or a part thereof, provides the auxiliary species.

33. A method or a sensor as claimed in Claim 32 wherein the auxiliary species is attached directly, or indirectly, to the support material.

34. A method or a sensor as claimed in Claim 32 or Claim 33 wherein an oligomer of an auxiliary species or a polymer of an auxiliary species is provided on the support material.

35. A method or a sensor as claimed in any one of the preceding Claims wherein the auxiliary species is a ligand.

36. A method or a sensor as claimed in Claim 35 wherein the auxiliary species is an antigenic ligand or a nonantigenic ligand.

37. A method or a sensor as claimed in Claim 36 wherein the ligand is 2,4 dinitrophenol, fluorescein, digitoxin, coumarin or cibacron blue.

38. A method or a sensor as claimed in Claim 36 wherein the ligand is biotin

39. A method or a sensor as claimed in Claim 32 or Claim 33 wherein the auxiliary species is a binder.

40. A method or a sensor as claimed in Claim 39 when the binder is an antibody or a binder for a non-antigenic ligand.

41. A method or a sensor as claimed in Claim 40 wherein the antibody is anti-2,4 dinitrophenol antibody, antifluorescein antibody, anti-digitoxin antibody, anticoumarin antibody or anti-cibacron blue antibody.

42. A method or a sensor as claimed in Claim 40 wherein the binder for a non-antigenic ligand is avidin.

43. A method or a sensor as claimed in any one of the preceding Claims wherein the auxiliary species is provided as a coating of a polymer on a support material.

44. A method or a sensor as claimed in any one of Claims 1 to 31 wherein the specific reaction partner is a ligand or a binder.

45. A method or a sensor as claimed in Claim 44 wherein the ligand is an antigenic ligand or a non-antigenic ligand.

46. A method or a sensor as claimed in Claim 45, wherein the ligand is 2,4 dinitrophenol, fluorescein, digitoxin, coumarin or cibacron blue.

47. A method or a sensor as claimed in Claim 45 wherein the ligand is biotin.

48. A method or a sensor as claimed in Claim 44 wherein the binder is an antibody or a binder for a non-antigenic ligand.

49. A method or a sensor as claimed in Claim 48 wherein the antibody is anti-2,4 dinitrophenol antibody, antifluorescein antibody, anti-digitoxin antibody, anticoumarin antibody or anti-cibacron blue antibody.

50. A method or a sensor as claimed in Claim 48 wherein the binder for a non-antigenic ligand is avidin.

51. A method or a sensor as claimed in any one of the preceding Claims wherein the primary species is a primary antibody or a ligand.

52. A method or a sensor as claimed in any one of the preceding Claims wherein the analyte species is detectable as such or wherein the analyte species is part of an entity to be detected.

53. A method or a sensor as claimed in Claim 52 wherein the analyte species is a hormone, a drug, a steroid, a tumour marker, a protein antigen, a blood protein, a marker protein, a pesticide, a toxin, a micro-organism, an antibody to a micro-organism, a metal complex or a metal ion.

54. A method or a sensor as claimed in Claim 53 wherein the metal complex is methyl mercury.

55. A method or a sensor as claimed in Claim 53 wherein the metal ion is a calcium ion, an iron ion, a cobalt ion, an aluminium ion, a zinc ion, a lead ion, a copper ion, a cadmium ion, a vanadium ion, a silver ion, a mercury ion, an indium ion, a manganese ion or a nickel ion.

56. A method or a sensor as claimed in any one of the preceding Claims wherein an entity to be detected is formed by interaction of an analyte species with an agent.

57. A method or a sensor as claimed in Claim 56 wherein the agent is an agent capable of forming a complex.

58. A method or a sensor as claimed in Claim 56 or Claim 57 which includes the step of forming a complex of an analyte species said complex providing an entity to be detected.

59. A method or a sensor as claimed in any one of Claims 56 to 57 which includes the use of an agent, said agent being capable of interacting with an analyte species, or with an authentic analyte species, to form an entity to be detected, the use of an auxiliary species, the use of a specific reaction partner for the auxiliary species, the use of a support material, and the use of an antibody to the entity to be detected, said specific reaction partner being capable of being associated with the entity to be detected by a linkage which involves a link, of a selected specific type (as hereinbefore defined), to the specific reaction partner.

60. A method or a sensor as claimed in any one of Claims 56 to 58 which includes the use of an agent, said agent being capable of interacting with an analyte species, or with an authentic analyte species, to form an entity to be detected, the use of a specific reaction partner capable of being associated with the agent, by a linkage which includes a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner, the use of an auxiliary species, said specific reaction partner and said auxiliary species being capable of binding together, the use of a support material, and the use of an antibody to the entity to be detected.

61. A method or a sensor as claimed in any one of Claims 56 to 58 which method includes the steps of bringing together and analyte species and an agent capable of interaction with the analyte species, thereby to effect interaction of the analyte species with the agent so as to form an entity to be detected, bringing together a specific reaction partner and an auxiliary species provided on a support material, so as to cause binding between the specific reaction partner and the auxiliary species, arranging for an antibody, said antibody being an antibody to the entity to be detected, to bind with the entity to be detected and also arranging for the antibody to be associated with the specific reaction partner by a linkage which involves a link of a selected specific binding type (as hereinbefore defined) to the specific reaction partner.

62. A method or a sensor as claimed in Claim 56 or Claim 57 which comprises the steps of bringing together a specific reaction partner, and an auxiliary species provided on a support material, so as to cause binding between the specific reaction partner and the auxiliary species, arranging for the specific reaction partner to be associated with an entity to be detected (by means of a linkage which involves a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner), applying an antibody, said antibody being an antibody to the entity to be detected and said antibody being linked with a tracer species, such that the antibody binds with the entity to be detected and detecting by means of the tracer species the binding of the antibody and the entity.

63. A method or a sensor as claimed in any one of Claims 56 to 58 which includes the steps of bringing together an analyte species and specific reaction partner (for an auxiliary species), said specific reaction partner being associated (by a linkage which includes a link, of a selected specific binding type (as hereinbefore defined), to the specific reaction partner) with an agent capable of interaction with the analyte species, thereby to effect interaction of the analyte species with the agent so as to form an entity to be detected, bringing together the specific reaction partner associated with the entity to be detected thus formed, and an auxiliary species provided on a support material, so as to cause binding between the specific reaction partner and the auxiliary species, and applying an antibody, said antibody being an antibody to the entity to be detected, such that the antibody binds with the entity to be detected.

64. A method or a sensor as claimed in any one of Claims 56 to 63 wherein a chelating agent is used to form a complex as an entity to the detected.

65. A method or a sensor as claimed in Claim 64 wherein the chelating agent is ethylene diamine tetra acetate, diethylene triamine penta acetate, cyclohexylenedinitrilo tetra acetic acid, 1-benzyl-ETDA, a derivative of 1benzyl-ETDA, ss-hydroxyquinoline, a derivative of Phydroxyquinoline, or deferoxamine.

66. A method or sensor as claimed in any one of the preceding Claims wherein the support material is a solid phase material.

67. A method or a sensor as claimed in Claim 66 wherein the solid phase material is a reaction vessel wall, an insoluble polysaccharide, a microparticle, polystyrene, cross-linked dextran, an insoluble polymer structure, a glass surface, a derivatised silica surface, a polymer attached to a surface, a microparticulate material with entrapped ferrous oxide, nylon or a polyamide.

68. A method or a sensor as claimed in any one of the preceding Claims wherein a detectable species is used.

69. A method or a sensor as claimed in Claim 68 wherein the detectable species comprises an enzyme, a fluorophore, a polymeric fluorophore, a radioisotope, a ligand, a polymer of a ligand, or a binder.

70. A method or a sensor as claimed in Claim 69 wherein a detectable species is a ligand and the ligand is detected by use of a binder therefor, or the detectable species is a binder and the binder is detected by use of a ligand therefor.

71. A method or a sensor as claimed in any one of the preceding Claims wherein a tracer species is used.

72. A method or a sensor as claimed in Claim 71 wherein the tracer species is an enzyme, a fluorophore, a chemiluminescent compound, a bioluminescent compound, a radioisotope or a dye.

73. A method or a sensor as claimed in any one of the preceding Claims wherein a sample of water, soil, a living species, or air provides an analyte species, or an entity to be detected, for detection.

74. A method or a sensor as claimed in any one of the preceding Claims wherein a biological sample is subjected to detection.

75. A method of a sensor as claimed in any one of the preceding Claims wherein an analyte species, or an entity to be detected, is present in water, an aqueous preparation, or a fluid extract.

76. A process for the preparation of a sensor for use in the detection of an analyte species or an entity by a method as claimed in any one of Claims 14 to 24 or 29 to 75 which process includes the step of providing, upon a surface of a support material, an immobilised auxiliary species.

77. A test-kit which includes the use of an auxiliary species provided on a support material, said test-kit being arranged to be capable of effecting a method of detection as claimed in any one of Claims 14 to 24 or 29 to 75.

78. A method as claimed in any one of Claims 14 to 24 or 29 to 75 which includes attaching an auxiliary species to a support material.

79. A competitive immunoassay method which includes the use of a separation method as claimed in Claim 1.

80. A non-competitive immunoassay method which includes the use of a separation method as claimed in Claim 1.

81. A separation method substantially as hereinbefore described with reference to Example 5.