WO1993011432A1

WO1993011432A1 - Reagent system and method of analysis

Info

Publication number: WO1993011432A1
Application number: PCT/GB1992/002201
Authority: WO
Inventors: Ramadan Arbi Abuknesha
Original assignee: GEC Marconi Ltd; Marconi Co Ltd
Current assignee: BAE Systems Electronics Ltd
Priority date: 1991-11-27
Filing date: 1992-11-27
Publication date: 1993-06-10
Anticipated expiration: 1994-05-27
Also published as: GB9125204D0; GB2261948A; GB9224897D0

Abstract

The invention relates to analysis and more particularly to a reagent system, a reagent material, an apparatus, a method and a test-kit which find application in analysis. The invention provides, $i(inter alia), a reagent system, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which reagent system includes an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent system being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction. The invention also provides, $i(inter alia), a reagent material, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which reagent material includes an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent material being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction.

Description

REAGENT SYSTEM AND METHOD OF ANALYSIS

The present invention relates to analysis and more particularly to a reagent system, a reagent material, an apparatus, a method and a test-kit which find application in analysis.

According to one aspect of the present invention there is provided a reagent system, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which reagent system includes an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent system being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction.

It will be appreciated that a reagent system includes one or more constituents suitable for use in a method for detecting an analyte species in which method a specific binding reaction is utilised.

According to another aspect of the present invention there is provided a reagent material, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which reagent material includes an auxiliary entity, said auxiliary entity being in a substantially inactive form

SUBSTITUTE SHEET and said reagent material being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction.

A reagent material in accordance with the immediately foregoing aspect of the present invention may be suitable for use in a reagent system in accordance with the present invention.

The auxiliary entity may be, for example, an auxiliary species; an auxiliary species is a species which is not itself capable of taking part in a primary binding reaction with an analyte species or with an authentic analyte species.

The present invention may thus provide, for example, a reagent material, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which reagent material includes an auxiliary species, said auxiliary species being in a substantially inactive form and said reagent material being such that it may be activated so as to permit the auxiliary species to undergo a specific binding reaction.

More than one auxiliary species may be utilised in accordance with the present invention.

Thus, the present invention may, for example, provide a reagent material, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which reagent material includes a first auxiliary species, said first auxiliary species being in a substantially inactive form and said reagent material being such that it may be activated so as to permit binding between the first auxiliary species and a second auxiliary species.

The first auxiliary species and the second auxiliary species may, for example, be included in the reagent material. Alternatively, by way of example, a first auxiliary species may be included in a reagent material and a second auxiliary species may be provided separately (e.g. on a support material as is further disclosed hereinafter) ; it will be appreciated that the first auxiliary species may be such that, upon activation of the reagent material, the first auxiliary species may undergo binding with the second auxiliary species provided separately.

Where the first auxiliary species is an auxiliary ligand, the second auxiliary species may be an auxiliary binder being a binding species capable of binding with the auxiliary ligand; where the first auxiliary species is an auxiliary binder, the second auxiliary species may be an auxiliary ligand being a ligand species capable of binding with the auxiliary binder.

Examples of auxiliary ligands are antigenic ligands such as 2,4 dinitrophenol, fluorescein, digitoxin, coumarin and cibacron blue and examples of auxiliary binders are antibodies such as anti-2,4 dinitrophenol antibody, anti-fluorescein antibody, anti-digitoxin antibody, anti-coumarin antibody and anti-cibacron blue antibody.

An example of a non-antigenic ligand which may be used as an auxiliary ligand is a ligand of a specific ligand-binder pair (e.g. biotin) . Where an auxiliary ligand is a non-antigenic ligand of a specific ligand- binder pair the auxiliary binder may be the binder of a specific ligand binder pair (e.g. avidin) .

As was hereinbefore disclosed an auxiliary species is a species which is not itself capable of taking part in a primary binding reaction with an analyte species or with an authentic analyte species. An example of a primary binding reaction is a primary immune specific binding reaction; a further example of a primary binding reaction is a non-immune protein binding reaction.

By way of example, a primary immune specific binding reaction (which may also be called a primary immune binding reaction) is one in which an analyte species undergoes, or an authentic analyte species undergoes, a specific binding reaction, or an analyte species and an authentic analyte species 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.)

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.

A species capable of taking part in a primary binding reaction may be considered to be a primary species. Thus, for example, a species capable of taking part in a primary immune reaction may be considered to be a primary species. A primary species may be, for example, a primary antibody or a ligand (e.g. an antigen) . It is also to be understood that, for example, a primary species may be an antibody to an analyte species or an antibody to an authentic analyte species; 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. It is also to be understood that a primary species may be, for example, an analyte species or an authentic analyte species. An analyte species may be, for example, a ligand or an antibody.

A further example of a primary species is a non- immune binding protein (e.g. cortisol binding protein) .

By way of further example, the auxiliary entity may be a "mixed" primary/auxiliary species (i.e. a species which has a part which provides a species capable of interaction with an auxiliary species and a part which provides a primary species being a binder for a primary species; an example of such a mixed primary/auxiliary species is an antibody having more than one function (e.g. a bifunctional antibody) . It is to be understood that the part of a mixed primary/auxiliary species which provides a species for interaction with an auxiliary species may be regarded as an "auxiliary function" in that it may interact with an auxiliary species.

The present invention may thus provide a reagent material suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which reagent material includes a mixed primary/auxiliary species, said mixed primary/auxiliary species being in a substantially inactive form and said reagent material being such that it may be activated so as to permit the mixed primary/auxiliary species to undergo a specific b.nding reaction. reagent material in accordance with the present invention may include, for example, any species suitable for taking p r in a given analysis. Thus, for example, where an auxl " ary species is used, the reagent material may include ar« auxiliary species (e.g. a first auxiliary species) and, optionally, any other species (e.g. a primary species and a detectable species or a tracer species) as may be desired for carrying out a given analysis. By way of further example, where a mixed primary/auxiliary species is used, the reagent material may include a mixed primary/auxiliary species and, optionally, any other species (e.g. a primary species and a detectable specie? ^r a tracer species) as may be desired for "rying out a given analysis. Any species in the reage* material may be, for example, retained in an in, ;tive state until a desired time. Where more than one kind of species are present in a reagent material the reagent material may be a physical combination of species; in such a combination the species may be separate or mixed together to any degree such that, upon activation of the reagent material any necessary interaction or interactions between the species of the reagent material, and/or between the species of the regent material and any other species, may take place to permit a desired analysis to be effected.

According to a further aspect of the present invention there is provided a reagent system, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which reagent system includes a second antibody, a support material for the second antibody, and a reagent material which includes a primary antibody, said second antibody and said primary antibody being in substantially inactive form and said second antibody and said primary antibody being such that the reagent system may be activated so that the second antibody may be provided on the support material and so as to permit binding between the second antibody and the primary antibody.

The second antibody may be regarded as an "auxiliary entity" in that it does not take part in a primary binding reaction with an analyte species or an authentic analyte species. A second antibody may be provided on a support material by activation of inactive second antibody already on the support material.

Where the reagent material is suitable for use in a reagent system, such as a reagent system immediately hereinbefore disclosed, the reagent material may include, for example, any species suitable for taking part in a given analysis. Thus, for example, a reagent material suitable for use in a reagent system, such as immediately hereinbefore disclosed, may include a primary antibody and, optionally, any other species (e.g. other primary species and a detectable species or a tracer species) as may be desired for carrying out a given analysis. Any species in a reagent material suitable for use in a reagent system as immediately hereinbefore disclosed may be, for example, retained in an inactive state until a desired time. When more than one kind of species is present in a reagent material the reagent material may be a physical combination of species; in such a combination the species may be separate or mixed together to any degree such that upon activation of the reagent material any necessary interaction or interactions between the species of the reagent material, and/or between the species of the reagent material and any other species, may take place to permit a desired analysis to be effected.

According to a further aspect of the present invention there is provided a reagent material, suitable for use in a method of analysis for detecting an analyte species by homogeneous assay in which method a specific binding reaction is utilised, which reagent material includes a primary species comprising a primary binder and a primary species comprising a primary ligand, said primary binder and said primary ligand being in a substantially inactive form and said reagent material being such that it may be activated so as to permit specific binding between the primary binder and the primary ligand.

It is to be understood that the primary species comprising the primary binder (hereinafter referred to as "primary binder") and the primary species comprising the ligand (hereinafter referred to as "primary ligand") in the reagent material are "inactive" in the sense that they are inhibited from undergoing specific binding until the reagent material is activated, whereupon specific binding may occur between the primary binder and the primary ligand; thus the inactive primary binder and inactive primary ligand are such that they are potentially capable of specific binding but they are maintained in a state in which specific binding is inhibited until the reagent material is activated.

Where a reagent material, in accordance with the present invention, is suitable for use in a homogeneous immunoassay, the reagent material may be considered to be a "reagent material" in that it may be a physical combination of, for example, inactive primary binder and inactive primary ligand. In such a reagent material the inactive primary binder and inactive primary ligand may be, for example, separate or mixed together to any degree, provided that, upon activation of the reagent material, specific binding may occur between the primary binder and the primary ligand.

From the foregoing disclosure it will be appreciated that analysis for detecting an analyte species in accordance with the present invention may include one, or more, specific binding reactions. Thus, for example, such a method may include a primary binding reaction (e.g. a primary immune binding reaction) . Alternatively, for example, such a method may include a primary binding reaction (e.g. a primary immune binding reaction) and a further specific binding reaction or reactions (e.g. a reaction between a first auxiliary species and a second auxiliary species, or a reaction between a mixed primary/ uxiliary species and an auxiliary species, or a reaction between a primary antibody and a second antibody) .

According to yet a further aspect of the present invention there is provided an apparatus, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which apparatus includes a reagent system and a means for maintaining, in operation, a sample to be subjected to analysis and the reagent system in sufficient proximity to permit interaction thereof, said reagent system including an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent system being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction.

According to yet a further aspect of the present invention there is provided an apparatus, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which apparatus includes a reagent material and ?. means for maintaining, in operation, a s? le to be objected to analysis and the reagent mater, 1 in sufficient proximity to permit interaction thereof, said reagent material including an auxiliary entity, said auxiliary entity being in a substantially i- stive form and said reagent material being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction.

The auxiliary entity of the immediately foregoing aspect of the present invention may be, for example, an auxiliary species (as hereinbefore defined) or, for example, a mixed primary/auxiliary species (as hereinbefore defined) .

According to yet a further aspect of the present invention there is provided an apparatus, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which apparatus includes a reagent system, said reagent system including a second antibody, a support material for the second antibody and a reagent material, which includes a primary antibody, and a means for maintaining, in operation, a sample to be subjected to analysis and the reagent system in sufficient proximity to permit interaction thereof, said second antibody and said primary antibody being in a substantially inactive form and said second antibody and said primary antibody being such that the reagent system may be activated so that the second antibody may be provided on the support material and so as to permit binding between the second antibody and the primary antibody.

According to yet a further aspect of the present invention there is provided an apparatus, suitable for use in a method of analysis for detecting an analyte species by homogeneous assay in which method a specific binding reaction is utilised, which apparatus includes a reagent material and a means for maintaining, in operation, a sample to be subjected to analysis and the reagent material in sufficient proximity to permit interaction thereof, said reagent material including a primary species comprising a primary binder and a primary species comprising a primary ligand, said primary binder and said primary ligand being in a substantially inactive form and said reagent material being such that it may be activated so as to permit specific binding between the primary binder and the primary ligand.

The means for maintaining a sample and a reagent system, or a sample and a reagent material, in sufficient proximity to permit inter-setion thereof may be, for example, a vessel. The vessel may be, for example, a reaction vessel such as a tube having a closed end and an open end (e.g. a test tube) .

In accordance with one embodiment of the present invention there is provided an apparatus which includes a tube and, within the tube, a reagent system said reagent system including an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent system being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction.

In accordance with one embodiment of the present invention there is provided an-apparatus which includes a tube and, within the tube, a reagent material said reagent material including an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent material being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction.

In accordance with another embodiment of the present invention there is provided an apparatus which includes a tube and, within the tube, a reagent system which includes a second antibody, a support material for the second antibody, and a reagent material which includes a primary antibody, said second antibody and said primary antibody being in a substantially inactive form and said second* antibody and said primary antibody being such that the reagent system may be activated so that the second antibody may be provided on the support material and so as to permit binding between the second antibody and the primary antibody.

In accordance with a further embodiment of the present invention there is provided an apparatus which includes a tube and, within the tube, a reagent material which includes a primary antibody, an internal surface of said tube being capable of providing a second antibody, said second antibody and said primary antibody being such that the reagent system may be activated so that the second antibody may be provided on an internal surface of the tube and so as to permit binding between the second antibody and the primary antibody.

In accordance with a further embodiment of the present invention there is provided an apparatus which includes a tube, and within the tube a reagent material, s id reagent material including a primary binder and a ^: _ϋmary ligand, said primary binder and said primary ligand being in a substantially inactive form and said reagent material being such that it may be activated so as to permit specific binding between the primary binder and the primary ligand.

According to a further aspect of the present invention there is provided a test-kit, which test-kit includes an apparatus in accordance with the present invention.

The present invention may be utilised in the detection of any suitable analyte species.

An analyte species may be, for example, a ligand or an antibody.

Examples of ligands are antigens which may be considered to be haptens, and non-antigenic ligands. Thus, for example, a ligand may be an analyte species which analyte species may be, for example, a drug of abuse (e.g. cocaine, tetrahydrocannabinol and morphine) , a therapeutic drug (e.g. digoxin, theophylline and phenytoin) , a steroid (e.g. estradiol, progesterone and cortisol) , or a water pollutant (e.g. a herbicide or a pesticide) .

Examples of binders are antibodies (e.g. antibodies to the ligands, immediately hereinbefore disclosed (i.e. anti-cocaine antibody, anti-tetrahydrocannibol antibody, anti-morphine antibody, anti-digoxin antibody, anti- theophylline antibody, anti-phenytoin antibody, anti- estradiol antibody, anti-progesterone antibody, an anti- herbicide antibody and an anti-pesticide antibody)) and non-immune binding proteins such as cortisol binding proteins. It will be appreciated that antibodies (for example 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 the analyte species and immunogenic carrier proteins such as bovine serum albumin or keyhole limpet haemocyanin.

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

It will be appreciated that the examples of primary binders and primary ligands herein disclosed are also examples of the types of analyte species which may be detected in accordance with the present invention.

The present invention may find application in, for example, qualitative or quantitative detection of drugs of abuse, therapeutic drug monitoring, screening or monitoring of organic pollutants or toxins in water or food stuffs, steroid hormone measurement in human or animal samples, thyroid hormone measurement, peptide or protein hormone measurement in human or animal samples. Samples to be analysed for analyte species may therefore include, inter alia , whole blood samples, blood plasma samples, blood serum samples, urine samples and samples of water.

Accordingly, the present invention may find application in, for example, clinical diagnosis, agricultural diagnosis, veterinary diagnosis, environmental monitoring (e.g. by water industries, food agencies and research laboratories) ; the invention may thus find application in, for example, field testing (e.g. of pollutants ir water or soil), the physician's surgery, hospital emergency units, health centres and home-use test products.

By way of example, an auxiliary entity or a second antibody may be utilised in bringing about a separation of bound and unbound fractions in an immunological detection procedure. Thus, for example, an auxiliary entity or a second antibody may be utilised in heterogeneous immunological detection procedures.

Thus, for example, by arranging for a second auxiliary species to be provided on a support material separation may be effected by allowing binding between a first auxiliary species and the second auxiliary species whereby the first auxiliary species (and any species associated therewith) may become associated with the support material. Thus, for example, a first auxiliary species may comprise an auxiliary ligand and a second auxiliary species may comprise an auxiliary binder, said second auxiliary species being provided on a support material. By allowing binding to occur between the auxiliary ligand and the auxiliary binder, the auxiliary ligand (and any species associated with the auxiliary ligand) becomes associated with the support material. Alternatively, by way of example, a first auxiliary species may comprise an auxiliary binder and a second auxiliary species may comprise an auxiliary ligand, said second auxiliary species being provided on a support material. By allowing binding to occur between the auxiliary binder and the auxiliary ligand, the auxiliary binder (and any species associated with the auxiliary binder) becomes associated with the support material.

By way of further example, a separation may be effected using an auxiliary species provided on a support material and a mixed primary/auxiliary species. Thus, separation may be effected by binding between the auxiliary species (in this case an auxiliary ligand) and a part of the mixed primary/auxiliary species which can act as an auxiliary function (as hereinbefore disclosed) whereby the mixed primary/auxiliary species (and any species associated therewith) may become associated with the :§μpport material.

Also, by way of example, a separation may be effected by using a second antibody provided on a support material; thus, upon binding occurring between the second antibody and a primary antibody, the primary antibody (and any species associated therewith) may become associated with the support material.

By way of example, where the nature of the support material permits, a support material (e.g. microspheres) may form part of a reagent material in accordance with the present invention and an auxiliary species or a second auxiliary species, or a second antibody, as appropriate, may be provided on the support material. Alternatively, by way of example, a support material may be provided by a reaction vessel surface and an auxiliary species or second auxiliary species, or a second antibody, as appropriate, may be provided on the reaction vessel surface.

Where, for example, an auxiliary species is provided on a support material an auxiliary species or a second auxiliary species may be provided on a support material in any convenient manner and at a convenient time. Thus, for example, a support material itself, or a part thereof, may provide an auxiliary ligand or a support material may have attached thereto (either directly or indirectly) in any suitable manner, an auxiliary ligand or an auxiliary binder. If desired, by way of example, oligomers and polymers of an auxiliary ligand or of an auxiliary binder may be utilised.

By way of example, a first auxiliary species may be associated with any suitable other species. Thus, for example, a first auxiliary species may be linked either directly (e.g. conjugated) or indirectly with any other suitable species such as a primary species (e.g. a primary antibody or an authentic analyte species) .

By way of further example, a mixed primary/auxiliary species may be associated with any suitable species such as a primary species which is capable of binding with part of the mixed primary/auxiliary species which has a part which provides a primary species being a binder for a primary species.

In view of the foregoing disclosure it will be appreciated that, by way of example, any suitable species may be provided on a support material such as to facilitate separation of immunochemical assay fractions.

Examples of support materials which may find application in accordance with the present invention are solid phase material such as a reaction vessel wall, insoluble polysaccharides, microparticles (e.g. particulate icrocellulose) , polystyrene (e.g. in the form of wells, beads, discs and microtitre plates, 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) , inso¹ able polysaccharides incorpor. iing entrapped i n oxide (e.g. magnetisable particles) , soluble polymers attached to a suitable surface (e.g. a glass surface), nylon and polyamides.

Known assay methods include, for example, displacement assay methods and competitive assay methods. Displacement assay methods, in principle, have an advantage in being convenient to use inasmuch as all that a user is required to do is to add a sample to be analysed to a complex of a ligand and a binder (one of which may carry a tracer species) and allow re- equilibration to take place. However, compared with competitive assay methods, displacement assay methods suffer the disadvantage of lacking sensitivity.

Known competitive assay methods suffer from the disadvantage that, in order to seek to achieve the full potential of such methods, certain conditions have to be satisfied and it is normally necessary to adhere to rather rigid protocols; although such conditions and protocols may be relatively easy to meet in laboratory operations, they place serious constraints on non- laboratory use (e.g. use in field tests) .

The present invention may be used substantially to overcome or avoid the above disadvantages of competitive assay methods and facilitate the use of competitive protein binding analysis, one example of which is competitive immunoassay.

Thus, for example, the present invention may find application in any suitable form of immunological detection or immunoassay (such as heterogeneous immunoassay and homogeneous immunoassay) . The present invention finds application in, for example, competitive- immunoassay methods, non-competitive immunoassay methods, sandwich immunoassay methods and homogeneous immunoassay methods. For example, the present invention may find application in label-free, or in detectable species- dependent, or tracer species-dependent assay methods such as enzyme-immunoassay, fluoro-immunoassay and radio- im unoassay. By way of example, the present invention may find application in antibody-labelled or antigen- labelled assays. Thus, for example, a primary binder or a primary ligand may be associated with a detectable species or a tracer species. By way of example, any suitable detectable species may be used in accordance with the present invention in the detection of an analyte species. 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 uch as those known in the art relating to protein binding assays (e.g. im unoassays) . (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, jS-galactosidase and horse-radish peroxidase) , fluorophores (e.g. fluoresceins, coumarins or rhodamine) , 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 antigenic ligands (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, anti- digitoxin antibody, anti-coumarin antibody and anti- cibacron blue antibody) and binders of a specific ligand- binder 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.

It is to be understood that in a competitive assay, actual analyte species (e.g. analyte species from a sample) is arranged to compete with authentic analyte species (e.g. a binder or a ligand, as is appropriate) which will bind to a ligand or a binder in substantially the same manner as an analyte species to be detected.

As was hereinbefore disclosed a reagent material in accordance with the present invention may include, for example, any species suitable for taking part in a given analysis. Thus, for example, it is to be understood that the reagent material may include, as desired, another component or other components in addition to, for example, a primary binder and/or a primary ligand.

Also, optionally, the reagent material may, if desired, also include, for example, a buffer or buffers and/or a stabilising agent or agents (e.g. phosphate, sodium chloride, sodium azide and mannitol) , and/or a serum binding protein blocking reagent, and/or a colour marker and/or a standard analyte species.

It will be appreciated that, for example, a reagent material may have a component which is chosen on the basis of the type of analysis it is intended to perform.

Thus, for example, a reagent material may include, as may be appropriate, a component or components selected from the following list, which list is not intended to be considered as exhaustive: a primary species (e.g. an antibody or a ligand) , a tracer species (which may be, for example, associated with a primary species) and/or an auxiliary entity.

According to a yet further aspect of the present invention, there is provided a method of analysis for detecting an analyte species which method includes the use of a reagent system in accordance with the present invention.

According to a yet further aspect of the present invention, there is provided a method of analysis for detecting an analyte species which method includes the use of a reagent material in accordance with the present invention.

According to yet a further aspect of the present invention there is provided a method of analysis for detecting an analyte species which includes contacting a sample to be subjected to analysis with a reagent system, said reagent system including an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent system being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction, said contacting being under conditions such that the reagent system is activated.

According o yet a further aspect of the present invention ther is provided a. method of analysis for detecting an analyte species which includes contacting a sample to be subjected to analysis with a reagent material, said reagent material including an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent material being such that it may be activated so as to srmit t ϋ auxiliary entity to une?argo a binding reactio said contacting being under conditions such that the reagent material is activated.

According to yet a further aspect of the present invention there is provided a method of analysis for detecting an analyte species which includes contacting a sample to be subjected to analysis with a reagent system, said reagent system including a second antibody, a support material, and a reagent material which includes a primary antibody, said second antibody and said primary antibody being in a substantially inactive form and said second antibody and said primary antibody being such that the reagent system may be activated so that the second antibody may be provided on the support material and so as to permit binding between the second antibody and the primary antibody, said contacting being under conditions such that the reagent system is activated.

According to yet a further aspect of the present invention there is provided a method of analysis for detecting an analyte species by homogeneous immunoassay which method includes contacting a sample to be subjected to analysis with a reagent material, said reagent material including a primary species comprising a binder and a primary species comprising a ligand, said primary binder and said primary ligand being in a substantially inactive form and said reagent material being such that it may be activated so as to permit specific binding between the primary binder and the primary ligand, said contacting being under conditions such that the reagent material is activated.

It is to be understood that, if desired, the reagent material may be contacted with a sample in a reaction vessel (e.g. in a tube) and the reagent material activated therein such as to permit any analyte species present to react simultaneously in a competitive reactio . Thus, the present invention offers, inter alia , the possibility of initiating a competitive assay in a one step operation.

Where a reaction vessel is, for example, a tube, the present invention offers the possibility of conducting a single tube assay.

It will be appreciated that the present invention may be utilised in qualitative analysis, which may also be considered to be qualitative detection of an alyte species or in quantitative analysis, whicn may also be considered to be quantitative detection (i.e. measurement or determination) of an analyte species.

Also, it will be appreciated that the present invention may be utilised with samples which contain analyte species and with samples which contain substantially no analyte species (e.g. a "standard" sample containing substantially no analyte species or an "unknown" sample which, upon subjecting to detection in accordance with the present invention, is found to contain substantially no analyte species) .

The present invention may, for example, find application in the detection of one kind of analyte species, or in the detection of more than one kind of analyte species. Thus, for example, by appropriate choice of reagent material, a single kind of analyte species may be probed for in a given sample.

Alternatively, by way of further example, if desired, by appropriate choice of reagent material more than one kind of analyte species may be probed for in a given sample (i.e. the present invention may find application in multi-analyte species detection (which may also be considered to be multi-analyte detection) ) .

By way of example, in applying the present invention to the detection of two or more analyte species, two or more auxiliary species pairs may be used. For example, two or more auxiliary ligand-auxiliary binder pairs may be used. Thus, for example, an auxiliary ligand of a first auxiliary ligand-auxiliary binder pair may be provided at a first position on a support material and an auxiliary ligand of a second auxiliary ligand-auxiliary binder pair may be provided at a second position on a support material such that, upon binding between the auxiliary ligand and auxiliary binder of the first pair, any species associated with the auxiliary binder of the first pair will be retained at the first position on the support material, and such that, upon binding between the auxiliary ligand and auxiliary binder of the second pair, any species associated with the auxiliary binder of the second pair will be retained at the second position on the support material.

Any detectable species retained at the first and/or second position may be detected in any suitable manner thereby to enable detection of different analyte species at different positions on the support material.

It will be appreciated that as an alternative, given by way of example, auxiliary binders of different auxiliary ligand-auxiliary binder pairs may be provided at different positions on a support material such that, upon binding between respective auxiliary ligands and auxiliary binders of the pairs, any detectable species associated with the auxiliary ligands may be retained at different positions on the support material.

It will be appreciated that, for example, any number of different auxiliary ligand-auxiliary binder pairs may be used as desired such that any chosen number of separations may be effected at any given number of positions or zones on a support material.

Also it will be appreciated that as an alternative, given by way of example, two or more different second antibodies may be provided at different positions on a support material to enable separation to be effected such that different analyte species may be detected at the different positions. By way of example, multi-analyte analysis may be used, if desired, to probe for two or more analyte species at the same time.

The present invention may be used, for example, in quantitative analysis by utilising a standard or standards containing known amounts of standard analyte species. Thus, for example, a series of standards may be prepared containing from 0% standard analyte species to the maximum concentration of analyte species it is expected to encounter.

The separate addition of such standards, and a sample or samples to be analysed for analyte species, each to separate quantities of reagent material (e.g. contained in a reaction vessel such as a tube) permits the sample or samples to be compared with known standards under substantially the same conditions; thus, a quantitative analysis of a sample or samples may be effected.

The reagent material may be prepared, for example, by freezing a chosen component or chosen components (e.g. a primary binder and/or a primary ligand and/or an auxiliary entity and/or any other chosen component or components (if any)) separately (but such that they are in proximity) and then freeze-drying (lyophilising) the component or components; it will be appreciated that the component is, or the components are, thus rendered substantially inactive in the sense of being dehydrated and prevented from undergoing any substantial interaction. It will be understood that activation of the reagent material may be such as to permit a component or components to undergo any desired interaction. By way of example, where a reagent material is to contain more than one component, components may be frozen separately, but arranged so that they are in proximity. By "in proximity" it is meant the components are such that in operation the components may be activated, under suitable conditions, so as to be capable of taking part in any desired interaction (e.g. a specific binding reaction where this is a desired reaction) .

According to yet a further aspect of the present invention there is provided a method for the preparation of a reagent system which includes freezing a chosen constituent or constituents and/or a chosen component or components.

According to yet a further aspect of the present invention there is provided a method for the preparation of a reagent material which includes freezing a chosen component or components for the reagent material and freeze-drying the resulting frozen component or components.

Where a component of a reagent material has, or components of a reagent material have, been rendered inactive by being dried, it is to be understood that such a component or components may be reactivated (and thus the reagent material may be activated) by rehydration or solubilisation which may be achieved in any suitable manner (e.g. by the introduction of an aqueous sample or a suitable reconstituting medium) .

By way of example, a chosen component in a suitable medium may be introduced into a vessel (e.g. a tube) and frozen. Subsequently, a further chosen component (which may be associated with a further component comprising a tracer species) in a suitable medium may be introduced into the vessel and frozen. It will be appreciated that any suitable temperature which achieves freezing of a medium containing a chosen component may be used; where such a medium is an aqueous medium a temperature of, for example, -10°C may be convenient. The amount of medium containing a chosen component introduced to a vessel may be_r for example, 10-100 μl and amounts of medium containing chosen components introduced to the vessel may be, for example, respectively 10-100 μl each. A layer of a component or layers of components thus formed in the vessel may be kept frozen and subjected to freeze-drying (lyophilisation) to produce reagent material in the form of a powder.

Additional components such as buffers, stabilisers, serum binding protein blocking reagents, colour markers or standard analyte species (i.e. "calibrator") may be similarly included in a reagent material if desired.

It is to be understood that by way of example, during preparation of a reagent material containing more than one component, in order to inhibit interaction between components to be< incorporated in the reagent material, freezing may be effected after the addition of each component to a vessel, and freezing condition_^ may be maintained until freeze-drying has been carried out.

As an alternative, by way of example, when preparing a reagent material containing more than one component, instead of forming frozen layers of components in a vessel and subjecting these to freeze drying, a component in a suitable medium and another component in a suitable medium may be separately subjected to freeze-drying and subsequently brought together to form the reagent material.

The freeze-dried reagent material may be kept for considerable periods in sealed tubes at, for example, -20°C to +25 °C without serious deterioration of properties.

By way of example, it will be appreciated that, if appropriate, in accordance with an assay procedure it is desired to effect, a detectable species (e.g. a tracer species) may be carried by a primary binder or by a primary ligand. It will be appreciated that, for example ^rhen a reager^4* is to contain more than one compo components jy be introduced t a vessel and frozen _.., any suitable rder. Also it will be appreciated that any number of components may be introduced and frozen and subseqp ly freeze-dried to give a reagent material having any desired number of components. A variety of separation methods may be used in heterogeneous assays (e.g. support material immobilised reagent methods) and these methods may be utilised in accordance with the present invention.

Thus, for example, any suitable support material immobilised reagent separation method (i.e. any suitable solid phase separation method) involving an auxiliary species or a second antibody may be utilised in accordance with the present invention. Examples of support material immobilised reagent separation methods are:

(i) those involving the use of an immobilised second binder (second antibody) , (ii) those involving the use of an immobilised auxiliary binder (e.g. an antibody such as anti-fluorescein antibody or anti-7- hydroxycou arin antibody) and an auxiliary ligand-antianalyte species binder conjugate, (iii) those involving the use of an immobilised auxiliary ligand and an auxiliary binder- antianalyte species binder dimer (e.g. an auxiliary antibody-antibody dimer) , (iv) those involving the use of an immobilised auxiliary binder (e.g. antibody) and an auxiliary ligand-analyte species conjugate, (v) those involving the use of an immobilised auxiliary ligand and an auxiliary binder (e.g. antibody)-analyte species conjugate, (vi) those involving the use of an auxiliary protein binder and a ligand (e.g. an avidin-biotin system) . It will be appreciated that in any of the foregoing examples (i-v) , a polyclonal or a monoclonal whole antibody or an antibody f agment may be used as appropriate. It will be appreciated that where the chosen support material immobilised reagent separation method allows, a support material carrying an immobilised reagent may be included in the reagent material.

Also, it will be appreciated that, alternatively, where appropriate, a support material carrying an immobilised reagent (for effecting separation) may be added after binding has occurred between a primary species (e.g. a primary binder and primary ligand) in a reagent material following activation of the reagent material. Thus,-for example, an analysis for an analyte species may be carried out as hereinbefore disclosed by adding a sample to a reagent material in accordance with the present invention and any resulting binding reaction may be allowed to proceed to a chosen point; subsequently a support material carrying a reagent may be added to effect a separation.

As was hereinbefore disclosed, the present invention may be utilised in an homogeneous competitive assay. Thus, for example, where detectable species activity (e.g. tracer species activity) is influenced (e.g. increased or decreased) by binding of a binder and a ligand-detectable species conjugate (e.g. a ligand-tracer species conjugate) an homogeneous competitive assay may be used since it is unnecessary to separate bound and unbound fractions before detectable species activity or tracer species activity is measured. Changes in detectable species activity, or tracer species activity, may be monitored in homogeneous assays as a result of, for example, fluorescence polarisation, fluorescence quenching, or modulation of catalytic activity of an enzyme.

Accordingly, for example, by use of a reagent material in accordance with the present invention in an homogeneous competitive assay, it is possible to provide a "single addition" assay (i.e. it is possible to provide an assay where only a sample may need to be added to a reagent material in accordance with the present invention) .

Examples of combinations of substances which may be used to prepare reagent materials, suitable for use in a labelled-analyte species competitive immunoassay, in accordance with the present invention include:

(a) For use in an homogeneous assay, 100 μl of antiserum reagent in 10 mg/ml IgG carrier protein isolated from non-immune sera, 100 μl of buffer + ι% mannitol and 50 μl tracer solution;

(b) For use in an homogeneous assay, 100 μl of antiserum reagent in 10 mg/ml IgG, 100 μl of buffer + 1% mannitol, 50 μl of tracer solution and 50 μl of standard analyte species ("calibrator") solution;

(c) 100 μl of auxiliary antibody-solid phase separation reagent suspended in 10 mg/ml IgG, 100 μl of antianalyte antibody-auxiliary ligand conjugate reagent in 10 mg/ml IgG, 100 μl of buffer + 1% mannitol, 50 μl tracer solution;

(d) 100 μl of auxiliary antibody-solid phase separation reagent suspended in 10 mg/ml IgG, 100 μ antianalyte antibody-auxiliary ligand conjugate reagent in 10 mg/ml IgG, 100 μl of buffer + 1% mannitol, 50 μl tracer solution and 50 μl of standard analyte species ("calibrator") solution;

(e) 100 μl of auxiliary ligand-solid phase separation reagent suspended in 10 mg/ml IgG, 100 μl antianalyte antibody-auxiliary antibody binder conjugate reagent in 10 mg/ml IgG, 100 μl of buffer + 1% mannitol and 50 μ tracer solution;

(f) 100 μl of auxiliary ligand-solid phase separation reagent suspended in 10 mg/ml IgG, 100 μl antianalyte antibody-auxiliary antibody binder conjugate reagent in 10 mg/ml IgG, 50 μl tracer solution and 50 μl of standard analyte species ("calibrator") solution. In relation to the foregoing examples (a) to (f) above, the following should be noted:-

(i) the buffer solution is 0.05M phosphate, pH 7.0 + 0.1M sodium chloride + 0.1% sodium azide in water; (it will be appreciated that, in general, any suitable buffer solution such as those known in the field of immunoassay may be used) ;

(ii) an antiserum reagent is a solution of an antibody to the analyte species in buffer solution; the antibody may be a whole immunoglobulin (e.g. IgG or IgM) or polyclonal or monoclonal antiserum or may be an antibody fragment such as (Fab)₂ or Fab;

(iii) the term "tracer solution" as used in examples (a) to (f) above is used to indicate a solution containing an authentic analyte species associated with a detectable species; an authentic analyte species is a species which will bind with an antibody to be used in an assay in a substantially similar manner as an analyte species to be detected. The authentic analyte species may be modified, as appropriate, as known in the immunochemical field to permit a detectable species to be linked thereto; thus, it may be considered that the tracer solution contains an authentic analyte species derivative linked to a detectable species. It will be appreciated that the authentic analyte species or a derivative thereof may be recognised and bound by antianalyte antibody to substantially the same degree as an analyte species (e.g. as may be found in a calibrator solution or in a sample under test) ;

(iv) an auxiliary antiserum-solid phase separation reagent is a reagent for use in the separation of bound and unbound fractions in an immunoassay said reagent being an auxiliary antibody attached to a support material; and (v) an auxiliary ligand-solid phase separation reagent is a reagent for use in the separation of bound and unbound fractions in an immunoassay said reagent being an auxiliary ligand attached to a support material, (vi) As hereinbefore disclosed, the examples (a) to (f) above relate to reagent materials suitable for use in labelled-analyte species competitive immunoassay; it will be appreciated, however, that by suitable choice of substances, such as is appropriate in accordance with immunoassay procedures, reagent materials may be prepared which are suitable for use in labelled-antibody competitive im unoassays. It will be appreciated that relative amounts of species which are to undergo binding reactions may be adjusted according to titration experiments conducted in accordance with known procedures and techniques.

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

Example 1

Preparation of anti-atrazine-antiseru

Atrazine was selected as an analyte species to illustrate, by way of example, the use of a reagent material in accordance with the present invention, said reagent material containing, inter alia, a first auxiliary species.

Accordingly, it was necessary to prepare an antibody (for use as a primary binder) to atrazine.

An atrazine immunogen was prepared by conjugating atrazine-6-caproic acid to keyhole limpet haemocyanin (KLH) using an N-hydroxysuccinimide ester derivative of atrazine-6-caproic acid. Atrazine-6-caproic acid N-hydroxysuccinimide ester (10 mg) in dimethylformamide (2 ml) was added to KLH (30 mg) in 0.1M NaHCOj (5 ml, pH 8.6) and dimethyl formamide (1 ml) .

After mixing for 4 hours at room temperature the resulting mixture (containing conjugate immunogen) was dialysed, for 3 days, against 3 changes of 4 litres of 0.5% NaHC0₃ solution.

Rabbit antiserum to atrazine was generated by the injection of rabbits with 0.5 mg quantities of conjugate immunogen at monthly intervals for 9 months.

The antibody binding activity of the resulting antibody was assayed by ELISA (enzyme labelled immunosorbent assay) using antiserum dilution response curve procedures; a plate coating antigen for this was prepared from atrazine-6-caproic acid conjugated to ovalbumin.

Example 2

Preparation of antiserum to an auxiliary species

In this Example a first auxiliary species being an antibody (a binding species_)_Ξ to a second auxiliary species (being a ligand) was prepared. Thus, 7-amino 4- methyl coumarin-3-propionic acid (7AMCPA) was synthesised and coupled to bovine serum using active ester derivative to form an immunogen conjugate. Sheep antibody to 7- a ino 4-methyl coumarin-3-propionic acid was generated by the injection of 1.5 mg quantities of the immunogen conjugate at monthly intervals for 12 months. 7-amino 4-methyl coumarin-3-propionic acid ester derivative, as hereinbefore disclosed, was coupled to egg albumin (ovalbumin) to provide a ligand-protein conjugate for use in the coating of polystyrene microtitre plates. Antibody binding activity in the generated antiserum was assessed by using a dilution response curve procedure involving ELISA. It will be appreciated that the antibody to the second auxiliary species is a first auxiliary species and is a binding species for the second auxiliary species.

Example 3

Coupling of analyte species derivative to the irst auxiliary species

The IgG fraction of sheep anti-7AMCPA antibody was prepared by ion exchange chromatography on DEAE-Sephadex A50. Atrazine-6-caproic acid N-hydroxysuccinimide ester was coupled to the IgG fraction by standard conjugation methods. Thus, 2.0 mg of atrazine-6-caproic acid active ester (in 200 μl of dimethylformamide) were added to 120 mg of the IgG fraction in 15 ml of 0.1M NaHC0₃ (pH 8.6) to form a first auxiliary species-authentic analyte species conjugate. The conjugate was removed and purified by dialysis and treatment with 1% charcoal.

Example 4

Purification of first auxiliary species-authentic analyte species conjugate

First auxiliary species-authentic analyte species conjugate, prepared by coupling atrazine-6-caproic acid active ester to the IgG fraction of sheep anti-7AMCPA antibody, as disclosed in Example 3, contained non- antibody IgG conjugates as contaminants. These contaminants were removed by affinity chromatography purification using the auxiliary species disclosed in Example 2 coupled to Sepharose-6B as the affinity matrix. Thus an affinity matrix was prepared by coupling a 7AMCPA-egg albumin (ovalbumin) conjugate (20 mg) to cyanogen bromide-activated Sepharose-6B (5 ml) .

All steps in the treatment of the affinity matrix, its washing, and the affinity chromatography procedure were carried out in the cold (5°C) and a cold chromatography cabinet was used throughout. The affinity matrix was first washed extensively according to standard procedure in affinity chromatography. In order to remove loosely bound ligand from the matrix, the matrix was mixed with 5 ml of anti- 7AMCPA antiserum (diluted with assay buffer to 20 ml) , and subsequently the matrix was washed with 100 ml of eluting reagent (20% acetonitrile and 1% propionic acid in distilled water) . The affinity matrix was washed thoroughly with assay buffer to remove traces of the eluting reagent. The assay buffer had the following composition: 50mM Tris-HCl, pH 7.4, containing 0.1M NaCl, 0.1% gelatin, 0.05% Tween 20 and 0.01% thimerosal.

It will be appreciated that the affinity matrix thus prepared may be referred to as 7AMCPA-ovalbumin- Sepharose-6B gel.

Anti-7AMCPA antibody-atrazine-6-caproic acid conjugate (105 mg in 20 ml of buffer, 0.1M phosphate buffer, pH 7.1, containing 0.1M NaCl, 0.1% sodium azide and 0.05% Tween 20) was passed through 2 ml of the affinity matrix in a column at a flow rate of about 0.1 ml/min. After all of the conjugate had been exposed to the matrix in the column, by being introduced in solution to the column, the column was washed with 50 ml of wash buffer (0.1M phosphate buffer, pH 7.5, containing 0.5M NaCl, 0.05% Tween 20 and 0.1% sodium azide) to remove unbound materials. When the optical density (at 280 nm) of eluate from the column fell to 0.05, the column was washed with 20 ml of ice-cold distilled water, the flow rate of which was increased to 0.5 ml/min during washing steps. After the water wash, a gradient of the eluting reagent was applied to the column. ( eluting reagent was a mixture of 20% acetonitrile and ; propionic acid in distilled water.) The eluting reagent was kept in ice to maintain low temperature. Eluted fractions (2 ml per fraction) were received in 0.5 ml of 0.2M phosphate buffer (pH 8.0) to neutralise the acid. The elution was followed by measuring O.D. at 280 nm (protein absorption) . The first auxiliary species-authentic analyte species conjugate was recovered after dialysis in the cold for 3 days against PBS.

Example 5

Attachment of second auxiliary species to a support material

Polystyrene assay tubes (9 mm x 70 mm) were coated with second auxiliary species - ovalbumin conjugate (in this Example an auxiliary ligand-ovalbumin conjugate) by incubating the tubes with^=£- ml of a solution of 7-amino- 4-methyl coumarin-3-propioπic acid-ovalbumin conjugate (10 μg/ l) in 0.05M sodiu itetraborate buffer (pH 9.0) for 16 hours at 4°C. -s-

The tubes were emptied and phosphate buffer solution (PBS) (laml) containing ovalbumin (200 μg) and D-mannitol (100 μg) was added and left in the tubes at room temperature for 4 hours.

The tubes were emptied and washed three times with wash buffer (1% NaHC0₃, 0.1M NaCl and 0.05% (v/v) Tween 20) . r

The washed, empty tubes were stored at -20°C awaiting further use.

Example 6

Introduction of a reagent material component to assay tubes

Affinity chromatography purified sheep anti-7AMCPA IgG conjugated with atrazine-6-caproic acid (i.e. first auxiliary species-authentic analyte species conjugate) prepared as in Example 4 was diluted to a suitable concentration with assay buffer (50mM sodium phosphate buffer, pH 7.5, containing lOOmM NaCl, 5 mg/ml ovalbumin, 10% D-mannitol, 80 μg/ml rhodamine B base and 0.01% thimerosal) .

The resulting solution was left on ice for 30 minutes before dispensing 50 μl aliquots into each of the assay tubes (prepared as described in Example 5) placed on solid C0₂.

The solution appeared to freeze as soon as it was placed in the assay tubes.

The tubes were left on solid C0₂ awaiting further use.

Example 7

Introduction of a further reagent material component to assay tubes

A primary species comprising rabbit-anti atrazine antiserum (antibody) , prepared as disclosed in Example 1, was diluted to a suitable concentration with assay buffer of the composition given in Example 6 and the resulting solution left to stand on ice for 30 minutes. Subsequently the 50 μl of the solution was introduced into each of th* assay tubes (containing frozen anti- 7AMCPA-atrazine-6-caproic acid conjugate) prepared as in Example 6.

The solution appeared to freeze as soon as it was placed into the assay tubes.

The contents of the tubes were lyophilised by freeze-drying.

At this point the tubes contained a reagent material comprising, in an inactive form, a first auxiliary species-authentic analyte species conjugate and a primary species (being in this Example a primary antibody) .

The assay tubes were stoppered and stored at 4^βC awaiting use.

Example 8

Detection of an analyte species

In accordance with the present invention atrazine was detected as an analyte species in accordance with the present invention. Tap water was treated twice with 1% activated Norrit A charcoal to remove atrazine and the treated water thus obtained was used to prepare standards.

Thus, a plain sample of the treated water was used as a zero concentration standard and other samples of the treated water were used to make up, according to conventional procedures, atrazine standards of 0.05, 0.10, 0.25, 0.50, 0.75 and 1.00 ng/ml.

An enzyme-immunoassay calibration graph or curve for atrazine was prepared. Thus, 1 ml sample volumes of the zero standard and the other atrazine standards hereinbefore disclosed in this Example were added to the assay tubes (in duplicate) prepared as disclosed in Example 7.

The contents of the tubes were mixed and the tubes were left at 37°C for 30 mins after which the tubes were emptied and washed three times with wash buffer having the composition given in Example 5. A 1 ml volume of solution, prepared by diluting with assay buffer of the composition given in Example 4 commercially available anti-rabbit-HRP conjugate (donkey anti-rabbit-HRP conjugate, ex Sigma) to the recommended concentration, was added to each of the assay tubes and, after an incubation period of 30 min, the tubes were emptied and washed three times with a wash buffer having the composition given in Example 5.

Activity of the bound HRP (horse radish peroxidase) was measured by adding to each tube 0.9 ml of a solution of 1.3mM H₂0₂ in 0.1M sodium acetate/sodium citrate buffer, pH 4.1, containing 0.5 mg/ml of 2,2'-azino-bis(3- ethyl benzothiazoline-6-sulphonic acid) diammonium salt (ABTS) .

Enzyme reaction was stopped after 25 min by the addition of 100 μl of 0.1% sodium azide in distilled water. >

Optical Density (O.D.) at 415 nm of the contents of the tubes was measured in a standard spectrophotometer. The O.D. values obtained are shown in Table I.

Table I

The O.D. values are inversely proportional to the concentration of atrazine in the sample volumes added to the assay tubes. A calibration graph or curve may be constructed by plotting O.D. values against atrazine concentrations. Such a calibration graph or curve may be used to determine atrazine concentrations in "unknown" samples (e.g. samples of river water or tap water) by carrying out suitable immunoassay procedures.

It will be appreciated that, in this Example, an auxiliary ligand-auxiliary binder system was used to effect separation of bound and unbound assay tracer species fraction in an ELISA immunoassay. Also it will be appreciated that this Exa le demonstrates which may be considered to be a "single reagent material assay tube" enzyme immunoassay for an analyte species.

Claims

1. A reagent system, ^suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which reagent system includes an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent system being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction.

2. A reagent material, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which reagent material includes an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent material being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction. -

3. A reagent material as claimed in Claim 2 wherein the auxiliary entity is an auxiliary species.

4. A reagent material as claimed in Claim 3, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which reagent material includes a first auxiliary species, said first auxiliary species being in a substantially inactive form and said reagent material being such that it may be activated so as to permit binding between the first auxiliary species and a second auxiliary species.

5. A reagent material as claimed in Claim 4 wherein the first auxiliary species and the second auxiliary species are included in the reagent material.

6. A reagent material as claimed in Claim 4 wherein the first auxiliary species is included in the reagent material and is such that, upon activation of the reagent material, the first auxiliary species may undergo binding with a second auxiliary species provided separately.

7. A reagent material as claimed in any one of Claims 4, 5 or 6 wherein the first auxiliary species is an auxiliary ligand and the second auxiliary species is an auxiliary binder.

8. A reagent material as claimed in any one of Claims 4, 5 or 6 wherein the first auxiliary species is an auxiliary binder and the second auxiliary species is an auxiliary ligand.

9. A reage' material as claimed in Claim 7 or Claim 8 wherein the auxiliary ligand is 2,4 dinitrophenol, fluorescein, digitoxin, coumarin or cibacron blue.

10. A reagent material as claimed in Claim 7 or Claim 8 wherein the auxiliary binder is anti-2,4 dinitrophenol antibody, anti-fluorescein antibody, anti-digitoxin antibody, anti-coumar^n antibody or anti-cibacron blue antibody.

11. A reagent mater i 1 as claimed in Claim 7 or Claim 8 wherein the auxiliary ligand is biotin.

12. A reagent material as claimed in Claim 7 or Claim 8 wherein the auxiliary binder is avidin.

13. A reagent material as claimed in Claim 2, suitable for use in a method of analysis for dete,. -ing an analyte species in which method a specific binding reaction is utilised, which-reagent material includes a mixed primar /auxiliary species, said mixed primary/auxiliary speciϊ , being in a substantially inactive form and said reagent material being such that it may be activated so as to permit the mixed primary/auxiliary species to undergo a specific binding reaction.

14. A reagent system, suitable for use in a method of analysis for detecting an analyte species in which method a specific bidding reaction is utilised, which reagent system includes a second ant dy, a support material for the second antibody, and a regent material which includes a primary antibody, said second antibody and said primary antibody being in substantially inactive form and said second antibody and said primary antibody being such that the reagent system may be activated so that the second antibody may be provided on the support material and so as to permit binding between the second antibody and the primary antibody.

15. A reagent material, suitable for use in a method of analysis for detecting an analyte species by homogeneous assay in which method a specific binding reaction is utilised, which reagent material includes a primary species comprising a primary binder and a primary species comprising a primary ligand, said primary binder and said primary ligand being in a substantially inactive form and said reagent material being such that it may be activated so as to permit specific binding between the primary binder and the primary ligand.

16. An apparatus, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which apparatus includes a reagent system and a means for maintaining, in operation, a sample to be subjected to analysis and the reagent system in sufficient proximity to permit interaction thereof, said reagent system including an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent system being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction.

17. An apparatus, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which apparatus includes a reagent material and a means for maintaining, in operation, a sample to be subjected to analysis and the reagent material in sufficient proximity to permit interaction thereof, said reagent material including an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent material being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction.

18. An apparatus, suitable for use in a method of analysis for detecting an analyte species in which method a specific binding reaction is utilised, which apparatus includes a reagent system, said reagent system including a second antibody, a support material for the second antibody and a reagent material, which includes a primary antibody, and a means for maintaining, in operation, a sample to be subjected to analysis and the reagent system in sufficient proximity to permit interaction thereof, said second antibody and said primary antibody being in a substantially inactive form and said second antibody and said primary antibody being such that the reagent system may be activated so that the second antibody may be provided on the support material and so as to permit binding between the second antibody and the primary antibody.

19. An apparatus, suitable for use in a method of analysis for detecting an analyte species by homogeneous assay in which method a specific binding reaction is utilised, which apparatus includes a reagent material and a means for maintaining, in operation, a sample to be subjected to analysis and the reagent material in sufficient proximity to permit interaction thereof, said reagent material including a primary species comprising a primary binder and a primary species comprising a primary ligand, said primary binder and said primary ligand being in a substantially inactive form and said reagent material being such that it may be activated so as to permit specific binding between the primary binder and the primary ligand.

20. An apparatus as claimed in any one of Claims 16 to 19, wherein the means for maintaining a sample and a reagent system, or a sample and a reagent material in sufficient proximity is a vessel.

21. An apparatus as claimed in Claim 20 wherein the vessel is a tube.

22. An apparatus as claimed in Claim 21 which includes a tube and, within the tube, a reagent system said reagent system including an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent system being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction.

23. An apparatus as claimed in Claim 21 which includes a tube and, within the tube, a reagent material said reagent material including an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent material being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction.

24. An apparatus as claimed in Claim 21 which includes a tube and, within the tube, a reagent system which includes a second antibody, a support material for the second antibody, and a reagent material which includes a primary antibody, said second antibody and said primary antibody being in a substantially inactive form and said second antibody and said primary antibody being such that the reagent system may be activated so that the second antibody may be provided on the support material and so as to permit binding between the second antibody and the primary antibody.

25. An apparatus as claimed in Claim 21 which includes a tube and, within the tube, a reagent material which includes a primary antibody, an internal surface of said tube being capable of providing a second antibody, said second antibody and said primary antibody being such that the reagent system may be activated so that the second antibody may be provided on an internal surface of the tube and so as to permit binding between the second antibody and the primary antibody.

26. An apparatus as claimed in Claim 21 which includes a tube and, within the tube, a reagent material, said reagent material including a primary binder and a primary ligand, said primary binder and said primary ligand being in a substantially inactive form and said reagent material being such that it may be activated so as to permit specific binding between the primary binder and the primary ligand.

27. A reagent system, a reagent material, or an apparatus as claimed in any one of the preceding Claims which includes a primary species.

28. A reagent system, a reagent material or an apparatus claimed in Claim 27 wherein a primary species is an antibody or a ligand.

29. A reagent system, a reagent material, or an apparatus as claimed in Claim 28 wherein the antibody is anti-cocaine antibody, anti-tetrahydrocannabinol antibody, anti-morphine antibody, anti-digoxin antibody, anti-theophylline antibody, anti-phenytoin antibody, anti-estradiol antibody, anti-progesterone antibody, an anti-herbicideantibody or an anti-pesticide antibody.

30. A :reagenl£ system, a reagent material, or an apparatus as claimed in Claim 27 wherein a primary species is a non-immune binding protein.

31. A reagent system, a reagent material or an apparatus as claimed in Claim 30 wherein the non-immune binding protein is cortisol binding protein.

32. A reagent system, a reagent material, or an apparatus as claimed in any one of the preceding Claims wherein an analyte species is a drug of abuse, a therapeutic drug, a steroid, a water pollutant, a hormone or a peptide.

33. A reagent system, a reagent material, or an apparatus as claimed in Claim 32 wher- the analyte species is cocaine, tetrahydrorannabi. morphine, digoxin, theophylline, phenytoin, estϊ ol, progesterone, cortisol, a herbicide or a pesticide.

34. A reagent system, a reagent material, or an apparatus as claimed in any one of Claims l to 14, 16 to 18, 20 to 25, or 27 to 33 wherein an auxiliary entity or a second antibody is utilised to bring about a separation.

35. A reagent system, a reagent material, or an apparatus as claimed in Claim 34 wherein a first auxiliary species and a second auxiliary species, or a mixed primary/auxiliary and an auxiliary species, or a second antibody and a primary antibody, are used to effect a separation.

36. A reagent system, a reagent material, or an apparatus as claimed in Claim 35 wherein the second auxiliary species is provided on a support material, or the auxiliary species is provided on a support material, or the second antibody is provided on a support material.

37. A reagent system, a reagent material, or an apparatus as claimed in Claim 36 wherein the support material is a reaction vessel wall, an insoluble polysaccharide, a microparticle, polystyrene, a cross- linked dextran, an insoluble polymer structure, a glass surface, a derivatised silica surface, an insoluble polysaccharide incorporating entrapped iron oxide, a magnetisable particle, a polymer attached to a surface, nylon or a polyamide.

38. A reagent system, a reagent material, or an apparatus as claimed in any one of the preceding Claims which includes a detectable species or a tracer species.

39. A reagent system, a reagent material, or an apparatus as claimed in Claim 38 wherein a detectable species is an enzyme, a fluorophore, a radioisotope, a ligand or a binder.

40. A reagent system, a reagent material, or an apparatus as claimed in Claim 38 wherein a tracer species is an enzyme, a fluorophore, a chemiluminescent compound, a bioluminescent compound, a radioisotope or a dye.

41. A reagent system, a reagent material, or an apparatus as claimed in Claim 40 wherein the enzyme is alkaline phosphatase, jβ-galactosidase or horse-radish peroxidase.

42. A reagent system, a reagent material, or an apparatus as claimed in Claim 40 wherein the fluorophore is a fluorescein, a coumarin or a rhodamine.

43. A method of analysis for detecting an analyte species which includes contacting a sample to be subjected to analysis with a reagent system, said reagent system including an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent system being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction, said contacting being under conditions such that the reagent system is activated.

44. A method of analysis for detecting an analyte species which includes contacting a sample to be subjected to analysis with a reagent material, said reagent material including an auxiliary entity, said auxiliary entity being in a substantially inactive form and said reagent material being such that it may be activated so as to permit the auxiliary entity to undergo a binding reaction, said contacting being under conditions such that the reagent material is activated.

45. A method of analysis for detecting an analyte species which includes contacting a sample to be subjected to analysis with a reagent system, said reagent system including a second antibody, a support material, and a reagent material which includes a primary antibody, said second antibody and said primary antibody being in a substantially inactive form and said second antibody and said primary antibody being such that the reagent system may be activated so that the second antibody may be provided on the support material and so as to permit binding between the second antibody and the primary antibody, said contacting being under conditions *-nch that the reagent system is activated.

46. A method of analysis for detecting an analyte species by homogeneous assay which method includes contacting a sample to be subjected to analysis with a reagent material, said reagent material including a primary species comprising a binder and a primary species comprising a ligand, said primary binder and said primary ligand being in a substantially inactive form and said reagent material being such that it may be activated so as to permit specific binding between the primary binder and the primary ligand, said contacting being under conditions such that the reagent material is activated.

47. A test-kit which includes apparatus as claimed in any one of Claims 16 to 26.

48. A method of analysis for detecting an analyte species which method includes the use of a reagent system as claimed in Claim 1, or Claim 14, or any one of Claims 27 to 42.

49. A method of analysis for detecting an analyte species which method includes the use of a reagent material as claimed in any one of Claims 2 to 13, or Claim 15, or any one of Claims 27 to 42.

50. A method for the preparation of a reagent system which includes freezing a chosen constituent or constituents and/or a chosen component or components.

51. A method for the preparation of a reagent material which includes freezing a chosen component or components and freeze-drying the resulting frozen component or components.

52. A method as claimed in any one of Claims 43 to 46, or Claim 49 wherein a competitive binding assay is used.

53. A method as claimed in Claim 52 wherein the competitive assay is a competitive immunoassay.