WO1990006512A1

WO1990006512A1 - Small analyte assays

Info

Publication number: WO1990006512A1
Application number: PCT/GB1989/001417
Authority: WO
Inventors: Colin Henry Self
Original assignee: Cambridge Patent Developments Ltd.
Priority date: 1988-11-25
Filing date: 1989-11-27
Publication date: 1990-06-14
Also published as: AU4749690A; GB8827522D0; CA1340488C

Abstract

A homogeneous non-competitive assay for a hapten is described which comprises: (i) contacting the hapten with a primary binding partner of the hapten which binding partner is associated with a member of a signal generating pair which generate a signal when in proximity of each other, (ii) contacting the unbound binding partner with a secondary binding partner therefore, (iii) contacting the complex of the hapten and primary binding partner with an antibody which binds the primary binding partner in said complex but does not bind the primary binding partner which has bound thereto the secondary binding partner which antibody is associated with the other member of said signal generating pair, (iv) determining signal produced by said signal generating pair. Kits for performing the method are also disclosed.

Description

SMALL ANALYTE ASSAYS

This invention relates to a method of determining haptens and Kits useful in that method.

At present there are a number of commercially available methods of determining haptens. However such methods frequently suffer from the disadvantage of producing an increasing signal as the concentration of the hapten decreases. This is often inconvenient and can be a source of variability, noise or error. I believe that it would be desirable to provide a method that enables haptens to be determined in a manner that allows the determination to produce a response which increases as the concentration of the haptens increase (as opposed to the inverse ratio which often applies). It would be an added advantage if such a method could employ excess reagents such as certain antibodies, especially labelled antibodies, so that precision would not be adversely effected by small variations in the amount of such reagents employed.. It would be -a considerable advantage if a homogeneous system could be employed which would avoid the need for separation steps and which would thereby offer ease of automation. It would be an even greater advantage if the assay could be non-competitive, that is not require competition between hapten and a hapten anologue such as labelled hapten.

The systems described in European Patent Application Nos 85901495.3, 85903019.9 and 87308829.8 in which antibodies are described which bind a complex of a small molecule and an antibody thereto but do__ not bind the small molecule or the antibody thereto alone offer many advantages. Unfortunately preparing the antibodies disclosed in those Patent Applications is believed to be frequently tedious with considerable repetitive procedures to be followed if antobodies having the desired level of specificity are to be achieved. It is believed that high backgrounds can result if specificity is sacrificed to ease of preparation. UK Patent Application No 8700461 discloses a further class of antibodies which can bind a complex of small molecule and an antibody thereto and can bind the small molecule alone. These could be used in determination methods and can be readily produced. Unfortunately they were not said to allow the determination of haptens other than by limited methods such as, for example, by using labelled haptens in competitive assays.

US Patent No 4233401 (Maggio et al) entitled "Reagents and Method Employing Channeling" discloses an assay system whereby an analyte, typically a protein such as hlgG, is determined as a result of its facilitating the bringing into the same vacinity (i) as chemical entity (hereinafter termed "CE") which provides a means for chemically changing the concentration of a compound which acts as a signal mediator (hereinater termed "SM"), (ii) a second reagent which is the signal mediator precursor and (iii) a third component (hereinafter termed "TC") which is a member of a signal producing system of which system SM is a member. This system was illustrated by employing two enzymes the product of the first being the substrate for the second. The enzymes are .chosen so that the product of the first enzyme has a long half life in the bulk solution so that if the two enzymes are not close to each other no detectable change is produced. Maggio et al illustrate their invention using large molecules but also envisage that small molecules can be determined if involved in competition with a hub system which requires tedious conversion of small molecule into large molecules. The method also suffers from the other drawbacks of competitive assays such as producing an increasing signal as the concentration of hapten decreases. US Patent No 4233402 is incorporated herein by cross reference.

I have now found a method whereby channeling can be used on small molecules without the aforementioned disadvantages. Amongst the advantages of the present invention is that it enables the assay to be "homogeneous", that is there is no necessary (mandatory) separation step after the addition of the labelled components and before the termination of signal. A further advantage is that the assay is not a competitive assay in that it does not require competition between analyte labelled analyte so that disadvantages of such systems such as an inverse ratio of signal to analyte concentration are avoided.

Accordingly the present invention provides a homogeneous non-competitive assay for a hapten which method comprises:

(i) contacting the hapten with a primary binding partner of the hapten which binding partner is associated with a member of a signal generating pair which generate a signal when in proximity cf each other

(ii) contacting the unbound binding partner with a secondary .._. binding partner therefore, (iii) contacting the complex of the hapten and primary binding partner with an antibody which binds the primary binding partner in said complex but does not bind the primary binding partner which has bound thereto the secondary binding partner Triiich antibody is associated with the other member of said signal generating pair

(iv) determining signal produced by said signal generating pair.

The antibody referred to in (iii) and (iv) above will .often be referred to as the "selective antibody" hereinafter. The selective antibody may be polyclonal although I prefer to employ a monoclonal selective antibody.

The binding partner for the hapten may be any suitable binding material such as a specific binding protein or antibody but I believe best results are obtained when a monoclonal antibody is employed as the binding partner for the hapten.

When the term "antibody" is used it should be realised that the antibody can be the whole binding molecule normally a whole immunoglobulin or fragments thereof which containing the binding site (such as Fab, Ffab¹ )₂, Fv or dAb). The antibody may also be an aggregate or hybrid but this is usually less preferable for the primary binding partner or selective antibody. Using a fragment as the binding partner of the hapten (such as a Fab fragment) can be particularly useful. When used herein the term "hapten" has the normal meaning of a small molecule analyte which is not itself normally immunogenic. The skilled art worker will appreciate that low molecular weight materials such as small molecules for determination by this invention are noπήally non-immunogenic but that antibodies against these haptens can be obtained by immunising an animal with a conjugate of the non-immunogenic molecule (or sometimes a very close analogue) and an immunogenic material (such as bovine serum ablumin or an equivalent agent) or by their special methods.

It will be understood that haptens are small molecules. Such haptens aptly have a molecular weight of for example 100 to 1500, more suitably from 120 to 1200 and favourably from 200 to 1000. Haptens with molecular weights of less than 1000 are often of particular interest for this invention. European Patent Application No 85901495.3 and 85903019.9 and UK Patent Application No 8700461 may be consulted for the types of haptens which are most suitable for determination by the method of this invention. European Patent Application No 87308829.8 and US Patent No 4233402 may similarly be consulted.

Haptens of particular interest for determination may be selected from amongst groups such as medicaments, drugs of abuse, metabolites, industrial chemicals (such as pollutants and agrochemicals), toxins and the like.

In order to carry out the determination of the amount of selective antibody bound to the binding partner the signal generating pair are —-_ employed. The signal generating pair may be any pair of means which co-operate to produce a signal when in proximity. Such a pair may be considered to be in proximity when they are close together on the level of antibody molecules. This means that the signal generating pair will be acting within the same micro-environment (as opposed to in a bulk solution).

The signal generating pair may be carried by the primary binding partner and selective antibody directly or indirectly. The association may be produced by covalent bonding or by non-covalent bonding such as antibody-antigen binding. When a member of the signal generating is an enzyme covalent bonding is preferred. Methods of binding materials to one another are well documented and methods described in the patents referred to hereinbefore may be employed. The primary binding partner may be associated with its member of the primary binding pair by immobilising both in proximity, for example on a surface. It follows that the term "associated" means that the components are maintained within the same microenvironment.

One of the signal generating pair (referred to hereinbefore as the chemical entity) provides means for chemically changing the concentration of a compound which acts as a signal mediator. This chemical entity mav be termed as Reactant Label and is aptly a catalyst and is preferably an enzyme. This may act either to destroy a compound (signal mediator) or more suitably may produce a compound (signal mediator) in solution.

Normally, the signal mediator will be produced from a precursor so that the initial concentration of the signal mediator may be zero or very small. It is desirable that the signal mediator and signal mediator precursor are not present to any significant extent in the sample to be analysed so that in a preferred apsect of this invention the reactant label is an enzyme which removes or more preferably generates a compound not found in biological tissue to any significant extent.

The other of the signal producing pair (referred to briefly hereinbefore as third component) may be termed the Signal Producing Label). The signal producing label acts together with the signal mediator and as appropriate ancilliary reagents) to produce a signal. The observed signal is related to the concentration of the signal mediator which in turn is effected by the Reactant Label. The signal mediation may provide the signal directly or indirectly. The signal may be any convenient determinable change but is aptly the absorption or more aptly the emission of electromagnetic energy in the ultraviolet or preferably the visable range but can also be ^"an electrochemical or other change.

US Patent No 4233402 may be inspected for appropriate Reactant Labels, Signal Producing Labels, Signal Mediators and Signal Mediator Precursors, the disclosure of said patent being incorporated herein.

It is particularly apt that the member of the signal producing pair associated with the primary.binding partner is an enzyme. Favourably the enzyme is carried by the primary binding partner and is preferably .._.„ covalently linked thereto. It is particularly apt that the member of the member of the signal producing pair carried by the selective antibody is covalently linked thereto.

The use of enzymes in such manner avoids the use of more difficult to prepare reagents and can call upon well.understood techniques already available to the skilled worker. More iπportantly it can give rise to more readilly detectable systems especially by employing highly active enzymes.

A preferred pair of enzymes to use is a peroxidase such as horse radish peroxidase and an oxide such as glucose oxidase or other hydrogen peroxide producing enzyme.

In order to determine the amount of selective antibody bound to the binding partner (and hence determine the hapten) the labelled selective antibody or the labelled binding may be immobilized if desired but in a particularly apt form of the invention the determination takes place in solution. If a component is to be immobilized then it may be by any suitable method for example on the surface of a plate, tube, dip-stick, capillary, paper, gel, particle or the like and the associated member of the signal producing pair may be similarly immobilized.

The secondary binding partner for the haptens binding partner may be any which will bind to that binding partner and which once bound wi . prevent significant binding of the antibody thereto. Generally the secondary binding partner will be an analogue or derivative of the hapten. Most suitable the secondary binding partner will be a moiety which is sufficiently large to prevent binding of the antibody by έteric effects. I believe that a particularly favoured class of secondary binding partners are large binding partners and that these are preferably large derivatives of the hapten, for example conjugates of the hapten with a large molecule or of a close analogue of the hapten with a large molecule. Such large molecules will most suitably have a molecule weight of greater than 5000 and will preferably be macromolecules, for example proteins. lypically these macromolecules will have molecular weights greater than 10000, more aptly greater than 20000 and preferably greater than 40000. I believe conventional macromolecules such as albumin (for example boven serum albumin) are suitable for conjucation to the hapten.

If a conjugate of a hapten (or a close analogue) and large molecule is used to raise the binding partner, then that conjugate is often a particularly suitable secondary binding partner. It is often advantageous that the conjugate or secondary binding partner so employed are highly water soluble (although those ocnjugates of lower solubility can be erπployed). The skilled art worker will be aware that such conjugates are readily available and may be obtained by many conventional methods of conjugation.

The secondary binding partner can advantageously be an antibody against the binding partner of the hapten, for example against the hapten binding site, for example an anti-idiotypic antibody.. It can be ..._ particularly advantageous to use a fragment of the aiiti-idiotypic antibody as previously indicated. The skilled art worker will appreciate that the binding partners will usually be selected to bind with as high an affinity as conveniently obtainable but that the affinity of the selective antibody for the secondary binding partner binding partner complex should be as low as conveniently obtainable.

The binding partner for the hapten may be any suitable binding material such as a specific binding protein or antibody but I believe best results are obtained when a monoclonal antibody is employed as the binding partner for the hapten. The binding partner should have as high a binding affinity for the hapten as is readily achievable as should the labelled binding partner if one is employed.

The amount of binding partner for the hapten (generally a monoclonal antibody) employed will normally be such that it is not saturated on exposure to the hapten; an excess is advantageously present with the resulting benefit that the assay is not unduly effected by the size of that excess within reasonable limits. This is easily achieveable by using an excess of binding partner over the likely amount of sample to be tested. In practice this will provide the skilled worker with little difficulty as the likely concentration of hapten in a sample is generally known (or estimateable) within broad limits and ranging experiments can be carried out if necessary. Similarly an excess of secondary binding partner will also normally be employed and this can be estimated by the skilled worker without difficulty although ranging experiments can be carried out if necessary. The source of the hapten for diagnostic tests may be blood, serum, saliva, urine or other source suspected of containing the haptens. Other* sources such as food samples, industrial samples, laboratory samples are also envisaged. The source may be purified or concentrated before use if desired. Thus the hapten being introduced into the test method may be in its original medium or in a subsequent medium. Such sample handling is conventional and does not form part of the present invention.

The selective antibody can have varying degrees of affinity for the primary binding partner. In the case where the binding of the selective antibody is high compared to the secondary binding then the secondary binding partner and the selective antibody may be added simultaneously. When the ratio is lower, as is generally the case, it will be advantageous to add the secondary binding partner first and then subsequently add the selective antibody.

Since it is easier to produce selective antibodies with some residual binding I prefer to use a method of determination in which the selective antibody is introduced, after the secondary binding partner has been introduced.

Generally the incubation period used for this part of the invention are from about 1 minute to 2 hours, more usually from 2 minutes to .100 minutes, for example 10 minutes to 60 minutes. Obviously it is desirable that the times are as short as possible. - 12 -

The method of determining the amount of antibody which becomes bound to the hapten's binding partner will employ the aforementioned signal generating pair. Both the antibody and the binding partner will be in the same microenvironment as one of the members of the pair and generally both the antibody and binding partner will be labelled with a member of the signal generating pair. These members of the pair may be present in situ thoughout the method or may be added subsequently, for example by a "Sandwich¹¹ technique such as using another antibody which is itself labelled to bind to antibody or binding partner. However, I prefer to use a label which has been present on the antibody or binding partner thoughout the method so that the pair may be brought as close together as practicable.

in the determination system a signal mediator precursor acts together with one of the signal generating pair to produce a signal mediator which then causes the other member of the signal generating pair to give rise to a signal. Although in some cases the signal mediator precursor can be present before the binding of the selective antibody has taken place it is generally preferred to add the signal mediator precursor to the system after the selective antibody, for example 2 minutes to 120 minutes later, more aptly 5 minutes to 100 minutes later, for example 10 minutes to 60 minutes later. In some cases it is convenient to add it in the same solution as the secondary binding partner. Any other necessary components of signal generating systems should have been introduced by this time. (It will be appreciated that the signal mediator precursor need not be a substance which is converted_^ to the signal mediator but is a substance which causes a member of the pair to effect a change in the concentration of the signal mediator, for example it could be a cofactor to activate a member of the signal generating pair, a substrate for an enzyme which causes the enzyme to produce a different entity (for example glucose causes glucose oxidase to produce hydrogen peroxide) or the like.

The signal generation may employ reagents and conditions such as those described in US Patent No 4233402. Generally I prefer to use luminescence (including chemiluminescence and fluorescence) in the determination. An apt method of generating luminescence is that of H. Arakawa et al, Clin Chem 31/3, 430-434 (1985) or H. Arakawa et al, Immunoenzymatic Techniques, S. Arrameas et al Eds, Elsevier, 1983, pp 233-237.

From the foregoing it will be appreciated that preferably one of the signal generating pair will generate hydrogen peroxide, for example an oxidase such as glucose oxidase. Aptly this member of the pair will be carried by the selective antibody and preferably will be covalently bound thereto. The other member of the signal generating pair will produce a signal when the hydrogen peroxide is introduced to its microenvironment. The other member of the pair may be an enzyme, for example a proxidase such as horse radish peroxidase which causes a substance such as luminol to emit light, or it may be a non-enzymatic compound or system, for example bis (2, 4, 6-tricholorophyenyl) oxalate and a fluorescent dye such as 8-anilino-l-naphthalene sulphonic acid. This other member, will aptly be associated with the primary binding — partner. The determination step and indeed the whole assay is normally carried out under non-extreme conditions, for example at 4-40°C aptly 15-27°C and more aptly at ambient temperature, under conditions of non-extreme pH. The skilled worker if fully familiar with the conditions best suited to immunoassays.

The present invention also provides a kit which may be used in carrying out the method of this invention for the determination of a hapten. The kit will comprise a selective antibody for use in deter ing the hapten which selective antibody is associated with a member of a signal generating pair and a primary binding partner for the hapten which primary binding partner is associated with the other member of the signal generating pair. The kit will also aptly contain a secondary binding partner. The kit will also aptly contain the signal mediator precursor and aptly any other components used by either member of the signal generating pair (other than the signal mediator).

The reagents may be provided in dry form and the kit may contain suitable solution for their reconstitution. If a solid matrix is required this may also be included in the kit, for example microtitre well, tubes, dip-sticks and the like.

The primary binding partner, the selective antibody and the^" secondary binding partner may be obtained and labelled as described in a copending patent application originally entitled Determination Method, •— Use and Components and eventually published as UK Patent Application No 2214295A which is incorporated herein by cross reference. Selective antibodies for use in this invention may also be raised by immunising with a primary binding partner labelled with a member of the signal generating means.

This invention further provides an assay for a small analyte which comprises (a) contacting said analyte with one of a primary binding partner associated with one of CE and TC where CE is a chemical entity which provides a means for chemically changing the concentration of a compound which acts as a signal mediator and where TC is a third component which is a member of a signal producing system of which system the signal mediator is a member; (b) a secondary binding partner is contacted with primary unbound sites of the primary binding partner; (c) selective antibody bound to the other of the CE TC pair is contacted with the primary binding partner binding only that bound by analyte and (d) signal mediator precursor is acted upon by CE and the detectable change produced by TC is measured.

A particularly yet small analyte is theophylline. The following Examples illustrate the invention. The abbreviation Selab is an abbreviation for selective antibody.

EXAMPLES

Example 1

Method for the Determination of Theophylline

Primary anti-theophylline monclonal antibody is obtained by standard means and a secondary (Selab) monoclonal antibody is obtained as described in Description 1. The Secondary Binding Partner anti-idiotypic antibody is also obtained according to the application.

The Selab is conjugated to horse radish peroxidase (HRP) giving HRP-Selab and the anti-theophylline monoclonal antibody is conjugated to glucose oxidase (GO) giving GO-anti-T employing the methods described in U.S. patent number 4233402 (Maggio et al. Reagents and Method Employing Channeling) November 11, 1980. These are then employed in a determination of theophylline as follows:

The amounts of GO-anti-T and HRP-Selab which give rise to an appropriate enzymatic signal are determined following the methods of Maggio et al and a concentration of the secondary binding partner is established which gives clear inhibition of the production of the final enzyme signal which inhibition is increasingly reduced by exposure of the GO-anti-T to-_, increasing concentrations of theophylline within the range to be tested. The effect of a range of theophylline standard preparations in preventing the inhibition otherwise caused by the secondary binding partner is determined. Unknown standards are then put in place of the standard preparations and their theophylline concentrations determined with respect to their effect in comparison to the standards.

Example 2

Method for the Determination of Oestradiol

Using a selective antibody of Description 2 the above Example is repeated for the determination of oestradiol

Example 3

Method for the Determination of Tetrahydrocannibal

Using the selective antibody of Description 3 Example 2 is repeated for the determination of tetrahydrocannibal.

Example 4

Method for Determination of Theophylline

A Nunc microtitre plate is taken and into each well is placed 200ul of a solution of 50mM bicarbonate buffer pH 9.5 containing 5 ug/ml of an anti-theophylline antibody against theophylline and 5 ug tnl of a solution of Horse Raddish Peroxidase (Boehringer Mannheim Cat No 814407) and "- incubated overnight at 4C. The solution is then discarded and the plate - 18 -

washed four times with 50mM Bicarbonate buffer pH 9.5 containing 2% bovine serum albumin. The plate is then washed with 50mM Tris pH 7.4 containing 0.05% Tween 20 (TT).

A Selab antibody is prepared for the anti-theophylline monoclonal antibody according to Description 1. It is conjugated to Glucose Oxidase by the Method described in US Patent number 4233402 (Maggio et al) Example 2.

An anti-idiotypic antibody secondary binding partner is also prepared for the theophylline antibody following Description 1.

Twelve standard solutions of theophylline are prepared from a solution containing 10ug/ml down in five-fold serial dilutions in phosphate buffered saline and also five unknown samples containing theophylline are obtained. 150ul of each are added to separate wells in the prepared microtitre plate and incubated for 30 minutes -at room temperature. 25ul of a solution of phosphate buffered saline containing 500ugtol of the anti-idiotypic antibody is added with mixing to each of the wells and the plate incubated for two further minutes followed by the addition of 25ul of a solution of phosphate buffered saline containing lOOug/ml Selab-glucose oxidase conjugate. The plate is incubated for thirty minutes and then washed four times with TT. The Selab-glucose oxidase in each well is then measured for each standard as in US Patent 4233402 by means of the chemiluminescent signal it produces from^*glucose and ' luminol. A standard curve of luminescence against theophylline concentration is drawn and the concentrations of theophylline in the unknown samples then calculated from this standard curve and the chemiluminescent signals obtained from the wells to which they were added.

Example 5

The procedure of Example 1 is repeated using the enhanced chemiluminescent detection system described in M. Pazzagli et al J. Immum methods, 114, 1988,, 61-68.

Description 1

A murine monoclonal antibody is obtained by conventional means against theophylline.

Mice are then immunised with the intact monoclonal antibody by multiple intra-peritoneal injections, their spleens are taken and IgG class monoclonal antibodies against the monoclonal antibody are raised. These are screened for anti-idiotypic activity as follows:

Nunc microtitre plates are coated per well with lOO l of 50mM bicarbonate buffer pH 9.6 containing l_//g anti-mouse IgG antibody by overnight incubation at room temperature. The solutions are removed and the wells glazed with 200/1 per well of 0.2% casein in the same buffer for one hour at room temperature. The solutions are removed and the wells washed four times with 50mM Tris pH 7.4. Quadruplicate wells then each received Iμq of an antibody to be tested in 100/1 Tris pH 7.4. They are incubated for two hours at room temperature. The wells are then washed four times with 50mM Tris 7.4 containing 0.02% Tween 20 (TT). A conjugate of the anti-theophylline monoclonal antibody is then made with alkaline phosphatase according to the method of Voller A, E. Bidwell and Ann Barlett, Bull. World Health Organ., 53, 55 (1976). A dilution of 1:500 is made of this. Two 200,yl alliquots are taken and 50 1 of a lmg/ml solution of theophylline in 50mM Tris pH 7.4 added to one while buffer alone is added to the other. Two of the quadruplicate wells then receive lOOμl of the theophylline containing solution while the other duplicate pair receive the solution without

SUBSTITUTESHEET theophylline. The solutions are incubated for a further hour at room temperature. The wells are then washed four times with TT and each well given 100/1 of lOmM para-nitrophenol phosphate in 50mM bicarbonate buffer pH 10.3. Their phosphatase content is assessed by the rate of change of optical density at 405nm. Screened antibodies in which the addition of theophylline very significantly reduces the amount of phophatase conjugate bound (such as reading of 2.0 optical density units without theophylline with 0.7 optical density units with theophylline) are considered anti-idiotypic antibodies of use in the following system.

Another group of mice are immunised intro-splenically with one immunisation each consisting of 100_/c/g of the anti-theophylline monoclonal antibody mixed with an equal quantity of theophylline in lOO l 50mM Tris pH 7.4. Over the next four days the mice receive three further injections of lOOg of theophylline introperitoneally.

One the fourth day their spleens are removed and hybridomas made by conventional procedure with myeloma cell line NSO. The hybridomas are screened for the required (Selab) antibody as follows:

SUBSTITUTE SHEET Microtitre plates are taken and the wells coated each with 100//1 of 50mM bicarbonate buffer pH 9.6 containing l_/g of anti-mouse IgM antibody by leaving them overnight at room temperature. The solutions are removed and the wells filled with 0.2% casein in the same buffer and left for one hour at room temperature. They are then washed four times with TT. Quadruplicate wells then receive lOOμl each of the same hybridoma culture fluid to be tested. They are incubated for 2 hours at room temperature 100/ l of a solution of 2 μq mouse IgM is then mixed into each well and a further 30 minute incubation carried out. The wells are then washed four times with TT.

Two 200/1 aliquots of a 1:500 dilution of the anti-theophylline monoclonal antibody - alkaline phosphatase conjugate in 50mM Tris pH 7.4 buffer containing 0.1% bovine serum albumin (TBSA) are taken. To one is added 50μl of the buffer containing 100_//g theophylline and to the other buffer alone. The solutions are mixed and incubated for ten minutes at room temperature. To each is then added 50ul of TBSA containing lOyu-g of a selected anti-idiotypic antibody. The solutions are then incubated for a further ten minutes at room temperature. 100/1 of each is then added in duplicate to the microtitre wells which have been exposed to the antibody to be tested. A further

SUBSTITUTE SHEET incubation of half an hour is made at room temperature. The solutions are then discarded and the wells washed four times with TT. Each well then receives 100_//1 of lOmM para-nitrophenyl phosphate in 50mM bicarbonate buffer pH 10.3 and containing 3.3mM MgCl₂. The alkaline phosphatase activity is then determined by the optical density change at 405nm. Where the optical density change with those duplicates which received the addition of theophylline is significantly larger than those which did not (i.e. such as 2.0 OD to 1.0 OD units) the antibody tested is considered to be a selective antibody and the hybridoma from which it derived purified free of contaminating clones and used to raise ascites by conventional techniques. The monoclonal antibody thus obtained is purified by means of Protein A fractionation.

SUBSTITUTE SHEET 24

Description 2

A urine monoclonal antibody is raised against oestradiol by conventional procedure employing as immunogen beta-oestradiol 6-(0-carboxy-methyl) omimerBSA (Sigma Chemical Company Ltd cat number E5630).

Employing standard procedures monoclonal antibodies are then raised against lots of 100/ of the anti-oestradiol monoclonal antibody mixed with 200// oestradiol. The hybridoma clones are screened for production of the required (SelAb) monoclonal antibody as follows:-

SUBSTITUTE SHEET

25

Microtitre plates (Nunc Immunoplate 1 code 4-39454) are taken and the wells coated each with 100/1 of 50 mM bicarbonate buffer pH 9.6 containing Iμq of anti-mouse IgG antibody (Sigma cat no. M 8642) by leaving them overnight at room temperature. The solutions are removed and the wells filled with 0.2% casein in the same buffer and left for one hour at room temperature. They are then washed four times with 50mM Tris pH 7.4 containing 0.02% Tween 20 (TT). Quadruplicate, wells then receive 100μl each of the same hybridoma culture fluid to be tested. They are incubated for 2 hours at room temperature 10 μl of a solution of 2 ug mouse IgG is then mixed into each well and a further 30 minute incubation carried out. The wells are then washed four times with TT.

A conjugate of the anti-oestradiol monoclonal antibody is then made with alkaline phosphatase employing 1.5mg of the monoclonal antibody and 5mg of alkaline phosphatase according to the method of Voller A, .E. Bidwell and Ann Barlett, Bull. World Health Organ., 53, 55 (1976). A dilution of 1:200 is made of. this. To 40ml of this is added lOOug of beta-oestradiol 6-(0-carboxy-methyl)oxime:BSA (solution I) and to another 40ml the same amount of BSA (Solution II). Each quadruplicate group of wells are taken and

SUBSTITUTE SHEE 26

duplicates receive 100/1 of solution I. The remaining duplicate pair receive 100/1 of solution II. The wells are then incubated for a further hour at room temperature. The solutions are then discarded and the wells washed four times with TT. Each well then receives 100 μl of lOmM para-nitrophenyl phosphate in 50 mM bicarbonate buffer pH 10.3 and containing 3.3mM MgCl2. The alkaline phosphatase activity is hen recorded at 405nm.

The hybridoma clones are selected for those which produce culture fluid providing the following result in the above screen: duplicated wells containing solution I - low alkaline phosphatase activity? duplicate wells containing solution II - high alkaline phosphatase activity.

The clones are purified free of contaminating clones and used to raise ascites by conventional techniques. The monoclonal antibody thus obtained is purified by means of Protein A fractionation. It is then conjugated to alkaline phosphatase as above (giving._^ 'SelAb-Conj ' ) . This conjugate is used in an assay for oestradiol as follows:

Microtitre plate wells are coated by the addition into each of 100/1 of 50mM bicarbonate buffer pH 9.6.

.SUBSTITUTE SHEET containing l g of the anti-oestradiol monoclonal antibody. They are left overnight at room temperature. The wells are then glazed with 100 μl of 0.2% casein in the same buffer and left for a further hour at room temperature. They are then washed four times with TT. A serial 1:2 dilution of the oestradiol:BSA conjugate is then made in phosphate buffered saline starting with a concentration of l_/g/ml. lOO l of each of these are added to individual wells in duplicate and incubated for one hour at room temperature and a duplicate pair of wells receive buffer alone. The solutions are discarded and the wells are washed four times with TT. 100/1 of a 1:400 dilution of the SelAb-Conj preparation is mixed into each well and the wells incubated at room temperature for a further one hour. The solutions are then discarded and the wells washed four times with TT. 100_/1 of 10mM para-nitrophenyl phosphate in 50mM bicarbonate pH 10.3 and containing 3.3mM MgCl₂ is then added to each well and the alkaline phosphatase activity followed at 402nm. A graph of oestradiol concentraton against phosphatase activity is then drawn which shows the power of added oestradiol-conjugate to inhibit binding of the primary and secondary antibodies. The lowest concentration of the conjugate which gives more than 90% inhibition is identified and used in the following determination.

SUBSTITUTE SHEET The microtitre plates are coated with anti-oestradiol monoclonal antibody, glazed and washed as in the preceding paragraph. 1:2 serial dilutions of oestradiol are made from l_/g/rol in 50mM Tris pH 7.4. lOOμl of each of these are added to duplicate wells and the wells incubated for one hour at room temperature. 10_//1 of a solution of the oestrogen-conjugate at ten fold higher concentration than identified above is then mixed into each well. The wells are incubated for a further hour at room temperature. The solutions are discarded and the wells washed four times with TT. 100_/1 of the SelAb-Conj is then added to each well and incubated for a further hour at room temperature. The solution is then discarded and the wells washed four times with TT. Each well then receives lOO l of lOmM para-nitrophenol phosphate in 50mM bicarbonate pH 10.3 and containing 3.3mM MgCl₂ and the alkaline phosphate activity followed at 402nm. Thus a standard curve of oestradiol concentration against phosphatase activity is obtained.

Unknown samples are determed by adding them to the assay in place of the oestradiol serial dilutions in. the above protocol. The degree of phosphatase activity being finally found in the wells which had received the unknowns is then related to the standard curve to concentration of oestradiol in these unknown samples.

SUBSTITUTE SHEET Description 3

Preparation and use of a selective antibody against tetrahydrocannibal.

By methods analogous to thos described in European Patent No 0264219: (1) a monoclonal antibody is obtained against tetrahydrocannibal (THC); (2) it is affinity labelled with THC derivative as described to form a delta-8-THC conjugate; (3) a monoclonal antibody is prepared against this conjugate. Although this antibody will bind a complex of the anti-THC monoclonal antibody and THC it also binds the anti-THC antibody on its own to a not insignificant extent so that high backgrounds result if it is used in the assays described in European Patent No 0264219; however it may be employed effectively for the determination of THC by the method of this present invention as a selective antibody. It is employed as in Description 1 of the present specification with the following changes: the hapten to be determined is THC; the primary binding partner is the anti-THC monoclonal antibody; the selective antibody is as made as described above; the secondary binding partner is a conjugate of 11 carboxymethyloxime derivative of delta-9-THC and bovine serum albumin prepared in conventional manner.

SUBSTJTUTE SHEET

Claims

Claims 3 0

1, A homogeneous non-competative assay for a hapten which method comprises:

(i) contacting the hapten with a primary binding partner of the hapten which binding partner is associated with a member of a signal generating pair which generate a signal when in proximity of each other

(ii) contacting the unbound binding partner with a secondary binding partner therefore,

(iii) contacting the complex of the hapten and primary binding partner with an antibody which binds the primary binding partner in said complex but does not bind the primary binding partner which has bound thereto the secondary binding partner which antibody is associated with the other member of said signal generating pair

(iv) determining signal produced by said sijgnal generating pair.

2. An assay as claimed in Claim 1 wherein the primary binding partner has bound thereto a member of the signal operating pair which is an enzyme.

3. An assay as claimed in Claim 1 wherein the antibody has bound thereto a member of the signal generating pair which is an enzyme.

4. An assay as claimed in Claim 1 wherein the primary binding partner has bound thereto a member of the signal generating pair which is an enzyme and the antibody has bound thereto a member of the signal generating pair which is an enzyme.

5. An assay as claimed in Claim 4 wherein one enzyme produces a signal mediator precursor not found in biological tissue which signal mediator precursor causes the other enzyme to produce a signal.

6. An assay as claimed in any of Claims 1 to 5 for the determination of a molecule of molecular weight 200 to 1000.

7. An assay as claimed in any of Claims 1 to 6 • wherein the primary binding partner and the antibody are both monoclonal antibodies.

8. An assay as claimed in any of Claims 1 to 7 wherein the secondary binding partner is an antibody.

9. An assay as claimed in any of Claims 1 to 8 wherein the steps (i), (ii) and (iii) are effected before all components of the signal generating system are present.

10. An assay as claimed in Claim 9 wherein all components of the signal generating system are present during effecting steps (i), (ii) and (iii) except the signal mediator precursor.

11. An assay as claimed in any of Claims 1 to 10 for theophylline.

12. An assay for a small analyte which comprises (a) contacting said analyte with one of a primary binding partner associated with one of CE and TC where CE is a chemical entity which provides a means for chemically changing the concentration of a compound which acts as a signal mediator and where TC is a third component which is a member of a signal producing system of which system the signal mediator is a member; (b) a secondary binding partner is contacted with primary unbound sites 3.3

of the primary binding partner; (c) selective antibody bound to the other of the CE/TC pair is contacted with the primary binding partner binding only that bound by analyte and (d) signal mediator precursor is acted upon by CE and the detectable change produced by TC is measured.

13. An assay as claimed in Claim 12 for theophylline in which the primary binding partner has bound thereto glucose oxidase and the selective antibody has bound thereto horse radish peroxidase.

14. A kit which comprises a selective antibody for a hapten which selective antibody is associated with a member of a signal generating pair and a primary binding partner for the hapten which primary binding partner is associated with the other member of the signal generating pair.

15. A kit as claimed in Claim 14 which also comprises a secondary binding partner.

16. A kit as claimed in Claims 14 or 15 which also comprises a signal mediator precursor.