WO2000043785A1

WO2000043785A1 - Improvements in or relating to displacement assays

Info

Publication number: WO2000043785A1
Application number: PCT/EP1999/010456
Authority: WO
Inventors: Steven Howell; Gerhard Nebe Von Caron
Original assignee: Unilever Plc; Unilever Nv; Hindustan Lever Limited
Priority date: 1999-01-25
Filing date: 1999-12-23
Publication date: 2000-07-27
Also published as: AU2103600A

Abstract

A method and assay device for performing displacement assays having increased sensitivity. In particular, the assay device includes a first solid support having reversibly immobilised thereon a binding partner having specific binding activity for an analyte of interest, the binding partner capable of being displaced from the first solid support in the presence of the analyte of interest and forming a binding partner/analyte complex; a second solid support bearing a capture moiety, the capture moiety having a specific binding affinity for the binding partner which is greater than that of the analyte of interest for the binding partner, such that the binding partner may be captured by the capture moiety and the analyte of interest displaced from the binding partner/analyte complex; means for conducting the binding partner/analyte complex from the first solid support to the second solid support; and a means for conducting the displaced analyte from the second solid support to the first solid support.

Description

Improvements in or Relating to Displacement Assays

Field of the Invention This invention relates to a method of performing an assay and to apparatus for performing the assay method of the invention.

Background of the Invention

Numerous assays have been described which make use of the specific binding properties of certain molecules to detect the presence of an analyte of interest in a sample. Typically such assays involve the specific binding between immunoglobulins (such as antibodies or functional binding fragments thereof) and haptens or antigens to which the immunoglobulins bind. Examples of such assays include enzyme-linked immunosorbent assays (ELISAs) and radio-immunoassay (RIA).

Generally, in order to detect binding between the analyte of interest and a binding partner having specific binding affinity therefor, it is usual for the binding partner to be labelled. Known labels include enzymes, radio- labels, fluorescent or chemiluminescent labels, electroactive labels (such as redox labels) and coloured particles (e.g. latex beads).

A refinement of assays of the general nature outlined above relates to "displacement" assays. In such assays, the presence of an analyte of interest in a sample typically causes the displacement either of a labelled binding partner or a labelled ligand from a pre-existing binding partner/ ligand complex. Generally speaking the amount of displaced labelled substance will be proportional to the concentration of the analyte of interest in the sample.

"True" displacement occurs where two different molecules have different binding affinities for a single binding site on a particular ligand molecule, such that introduction of one molecule with a high binding affinity will displace from the binding site a molecule with a lower binding affinity for the ligand. Assays based on this principle are termed "displacement assays" . However displacement of a sort can also occur where two molecules have the same binding affinity for a binding site. For example, if a labelled molecule is bound to a ligand molecule, introduction of a large amount of the same, but unlabelled, molecule will, due to equilibrium or "mass action" considerations, result in displacement of at least some of the labelled molecule from the ligand. Assays based on this principle may be termed "competition assays".

Several assay methods relying on competition and/ or displacement are described in the prior art. For example, EP 0,324,540 discloses assays designed to measure the amount of free ligand (rather than complexed ligand, which complexed ligand is typically protein-bound) in biological samples such as plasma or serum. The assay method requires the use of a

"signal reagent", which is labelled monoclonal antibody. The monoclonal binds to free ligand, which is in competition with a ligand analogue (which analogue does not bind to the natural ligand complexing proteins present in the sample). Typically the analogue is immobilised (e.g. on particles or beads). The analogue is selected to have a lower affinity than the ligand for the anti-ligand monoclonal antibody. The assay thus works on the principle of immuno-competition, the presence of free ligand in the sample serving to decrease the amount of labelled antibody which becomes associated with the ligand analogue.

WO 91 /05262 discloses a device and method for detecting the presence of molecular analytes in a fluid (especially e.g. steroids, and other low molecular weight analytes). Typically, aqueous biological samples are drawn along a test strip by capillary action. As the sample advances, it carries a labelled analyte from an area of storage at one end of the strip to a first binding means, which is an anti-analyte antibody. In the absence of free analyte in the sample, the labelled analyte (e.g. analyte /enzyme conjugate) will remain bound to the first binding means. However, if free analyte is present in the sample it will tend to displace the labelled analyte

(or at least, compete therewith for binding sites on the first binding means) such that some labelled analyte will be bound to the second binding means, which is an anti-enzyme antibody. Colour is developed by placing the strip in an appropriate substrate solution. The present invention relates to an improvement of displacement type assays. In particular, the invention provides a method and assay device for performing displacement assays having increased sensitivity (i.e. the ability to detect lower concentrations of analyte of interest and/ or to detect the same concentration of analyte within a shorter time period) compared to prior art methods and devices.

Summary of the Invention

In a first aspect the invention provides a method of detecting the presence of an analyte of interest in a sample, the method comprising the steps of:

(a) contacting the sample with a first solid support having reversibly immobilised thereon a binding partner having specific binding activity for the analyte of interest, the binding partner being reversibly immobilised on the first solid support by means of an interaction having a lower binding affinity than the binding affinity of the binding partner for the analyte, such that the presence of the analyte in the sample causes specific displacement of the binding partner from the first solid support and formation of a binding partner/analyte complex;

(b) contacting the binding partner/analyte complex with a second solid support bearing a capture moiety, the capture moiety having a specific binding affinity for the binding partner greater than that of the analyte of interest for the binding partner, such that the analyte of interest is displaced from the binding partner/analyte complex, and the binding partner is captured at the second solid support by the capture moiety;

(c) contacting the displaced analyte with the first solid support, so as to displace a further binding partner and form a new binding partner/analyte complex, and contacting the new binding partner/analyte complex with the second solid support so as to capture the further binding partner at the second solid support; and (d) detecting the presence of binding partner molecules captured by the capture moiety and/ or detecting displacement of binding partner molecules from the first support.

In short, the method is such that the presence of one molecule of the analyte of interest can cause the specific displacement from the first support of a plurality of molecules of binding partner. Indeed, by repeatedly "recycling" the analyte molecule between the first and second supports, a large amount of binding partner can, in effect, be transferred from the first support to the second support, such that there is a great amplification of the "signal" provided by a single analyte molecule, such that the present invention confers greatly improved sensitivity compared with prior art assays. Those skilled in the art will recognise that steps (b) and (c) will therefore preferably be effected a plurality of times during performance of the invention.

In a second aspect the invention provides an assay device for detecting the presence of an analyte of interest in a sample, the device comprising: a first solid support having reversibly immobilised thereon a binding partner having specific binding activity for the analyte of interest, the binding partner being displaced from the first solid support in the presence of the analyte of interest and forming a binding partner/analyte complex; a second solid support bearing a capture moiety, the capture moiety having a specific binding affinity for the binding partner which is greater than that of the analyte of interest for the binding partner, such that the binding partner may be captured by the capture moiety and the analyte of interest displaced from the binding partner/analyte complex; means for conducting the binding partner/analyte complex from the first solid support to the second solid support; and means for conducting the displaced analyte from the second solid support to the first solid support.

The method and device of the invention may be used qualitatively or quantitatively. The sample will generally be in fluid form, conveniently (but not necessarily) as a liquid (e.g. aqueous solution, or comprise a sample of body fluid, such as blood, serum, plasma, urine, saliva, sweat, semen or tears) . The analyte of interest may be any molecule, such as a drug, hormone, protein, carbohydrate, nucleic acid (DNA, RNA or chimera), enzyme, antibody, antigen and the like. Alternatively, the analyte may be of a macromolecular or particulate nature, such as bacteria or virus particles, or other micro-organism (fungus, yeast, chlamydia, fungal or bacterial spore, etc), or allergen (e.g. house dust mite faeces). Of particular interest are those analytes which may be present in the human body or body fluids, such as hormones, especially sex and/ or fertility hormones and analogues thereof, such as estrone-3-glucuronide (E3G), pregnanediol-3-glucuronide (P3G), human chorionic gonadotrophin (hCG), luteinising hormone (LH), and follicle stimulating hormone (FSH). Also of interest are antigens that may be present on solid surfaces that may, or may not, be taken up into solution (e.g. biofilms in plant and machinery, or residues left on surfaces - e.g. after cleaning, especially in the food processing industry).

The reversibly immobilised binding partner and the analyte are conveniently members of a specific binding pair. Numerous examples of such specific binding pairs are known (e.g. DNA and DNA-binding proteins; complementary strands of nucleic acids; ligands and their receptors; antigens and antibodies thereto). Typically the binding partner is a protein, preferably an immunoglobulin (e.g. antibody) or a functional binding fragment or variant thereof, which term relates to, inter alia, Fv, scFv, Fab, Fab₂ HCV, bispecific antibody, and chimeric molecules comprising one or more of the aforementioned binding fragments, and the like.

The binding partner may be reversibly immobilised to the first solid support in any one of a number of ways, which will be apparent to the person skilled in the art (e.g. as described in "Protein Immobilisation" 1991, [ed. R. F. Taylor] Marcel Dehler, Inc. New York). The binding partner may conveniently be removed from the solid support by application of particular chemicals (e.g. solutions, such as 50mM glycine, buffered to a very low pH [pH2] or 50mM diethylamide buffered to a very high pH [pH 12] and the like) but, under conditions in which the assay is performed (such as those generally found in biological systems (e.g. about 0-50°C, more preferably 10-40°C, typically pH 5-9)), will be released from the solid support only by the presence of the analyte of interest. It is a requirement of the invention that the interaction by which the binding partner is reversibly immobilised on the first solid support is of lower affinity than that of the binding partner for the analyte of interest. It will be apparent to those skilled in the art that this condition can readily be met by judicious selection of an appropriate binding partner molecule and/ or selection of the manner in which the binding partner is reversibly immobilised to the first solid support. Conveniently this is achieved by immobilising on the first support (e.g. via covalent interactions) an analogue of the analyte of interest. Methods suitable for accomplishing this are well known to those skilled in the art. The binding partner is then allowed to bind (comparatively loosely) to the analogue of the analyte (e.g. via non-covalent interactions), so as to immobilise reversibly the binding partner to the first solid support.

Conversely, it is a requirement that the binding affinity between the binding partner and the capture moiety is greater than that between the binding partner and the analyte of interest. Again, judicious selection of the capture moiety and binding partner, and/ or optimisation of the assay conditions, will readily allow this condition to be met.

Binding of the binding partner to the capture moiety preferably excludes the possibility of simultaneous binding of the binding partner to the analyte. Conveniently this is achieved by requiring the capture moiety to bind to the same binding site on the binding partner (but with greater affinity) as that occupied by the analyte or to an overlapping site. Alternatively, binding of the binding partner to the capture moiety may induce a conformational change in the binding partner, which conformational change alters or abolishes the binding site occupied by the analyte.

In preferred embodiments, the capture moiety is an analogue of the analyte of interest. The capture moiety is advantageously immobilised on the second solid support, typically by means of covalent interactions and/or by adsorption. Again, methods of achieving this are well known to those skilled in the art. Accordingly, in preferred embodiments, the invention will involve the use of two analogues of the analyte of interest: a "low affinity analogue" immobilised on the first solid support and a "high affinity analogue" (the capture moiety) borne on the second solid support. Desirably, the affinity of the binding partner for the analyte of interest is at least about 5, more desirably at least about 10, and no more than about 100, more desirably no more than about 20 times greater than the affinity of the binding partner for the "low affinity analogue" on the first solid support. Desirably, the affinity of the binding partner for the "high affinity analogue" (i.e. the capture moiety) is at least about 5, more desirably at least about 10, and no more than about 100, more desirably no more than about 20 times greater than the affinity of the binding partner for the analyte of interest. Typical useful affinity values (dissociation constant, KD) for the various interactions are 10^"^M (low affinity analogue /binding partner), 10^"^M (binding partner/analyte) and 10"^M (binding partner/ high affinity analogue) respectively.

Conveniently, the binding partner may be reversibly immobilised on the first solid support by interaction (with relatively low affinity) with a mimotope, the mimotope itself immobilised (by conventional means) on the first solid support. Similarly, the capture moiety at the second solid support may conveniently take the form of a mimotope (although one having a relatively high affinity interaction with the binding partner). Mimotopes are molecules (generally peptides) which "mimic" the structure of epitopes recognised by antibodies. A useful discussion of mimotopes, their synthesis, and their uses, is provided by Cortese et al, (1995 Current Opinion in Biotechnology 6, 73-80).

The first and second solid supports may be substantially similar or identical in nature, or may be different. Solid supports which may be of use in the invention are generally well known and include, for example synthetic plastics materials, microtitre assay plates, latex beads, filters comprising cellulose or synthetic polymeric materials, glass or plastics slides, dipsticks, capillary fill devices and the like.

Preferably the method and device of the invention are such that the "capture" step may be performed using generic components. Thus, simply by making suitable modification to the binding partner and the first solid support, the method and device of the invention can be used to test for the presence and/ or concentration of any analyte of interest. Preferably, this may be achieved by use of a chimeric binding partner, in which one portion is retained (regardless of the identity of the analyte of interest) which interacts with the generic capture moiety. Such a generic, modular arrangement facilitates production of the assay device and reduces cost. Performance of the method of the invention and/ or use of the apparatus of the invention, in the presence of the analyte of the interest, leads to a transfer of binding partner molecules from the first solid support to the second solid support. Accumulation of the binding partner at the second support, and/ or its displacement from the first support, can be detected in any of a number of conventional methods. For example, the binding partner may comprise a detectable label (e.g. enzyme, radioactive label, fluorescent or chemiluminescent label, or electroactive (redox) label or a particulate, coloured or uncoloured, e.g. latex bead or gold sol). Alternatively, presence or absence of the binding partner at the first and/ or second support can be detected indirectly - if the binding partner is unlabelled it can compete with labelled binding partner added at the end of the assay, the presence of analyte /interest being indicated in such an embodiment by reduction in binding of labelled binding partner to the second support, or by an increase of labelled binding partner binding to the first support.

In another embodiment, the first and/ or second support may comprise part of a mass-dependent biosensor (e.g. acoustic wave or evanescent wave type sensors, or surface plasmon resonance ["SPR"] detectors, all of which are known to those skilled in the art - see, for example, EP 0,341,927; EP 0,416,730; EP 0,453,224; and Jonsson et al, 1991 Bio/Techniques II, 620- 627).

The apparatus of the invention may take any one of numerous forms, depending on the precise nature of the assay being performed (e.g. the identity of the analyte of interest, the binding partner and capture moiety; the type of solid support; whether the binding partner is labelled; etc). For example, in some embodiments the first and second surfaces may be physically very close to each other (of the order of a millimetre or so apart), possibly inter-digitated, during performance of at least some steps of the assay. In one embodiment the apparatus may comprise a capillary-fill test device in which a liquid sample may be drawn into the device by capillary action along a suitably-proportioned capillary inlet. Capillary-fill devices which may be adapted for use in the present invention are disclosed, for example, in US Patent 5, 141 ,868.

In another embodiment, the first solid support comprises a synthetic plastics peg which is proportioned as to fit within a well of a microtitre plate, there being only a small separation between the peg and the sides of the well. The second support is formed by the microtitre plate, the well being coated with the capture moiety. Alternatively, as described in the Examples below, a microtitre plate may constitute the first support and a peg provides the second support. Advantageously, an array of a plurality of pegs may be used, the spacing of the pegs being such that the array can be inserted into a corresponding plurality of wells in the microtitre plate. The presence of the analyte of interest in a liquid sample in the well causes displacement of binding partner from the first support. The displaced binding partner may then diffuse across the small gap between the pegs and the side of the well, so as to be captured by the capture moiety. Similarly, analyte displaced from the binding partner/analyte complex can readily diffuse across the narrow gap, so as to displace a further binding partner molecule. After a suitable period (which can readily be determined by routine trial-and-error), the pegs are removed from the wells. The wells and/ or pegs are then typically washed with appropriate washing buffer (e.g. PBS) and the presence of binding partner detected. In an embodiment such as this, the binding partner may conveniently be labelled (e.g. with an enzyme label), and detection can thus be accomplished very simply (e.g. by means of an ELISA-type protocol).

In other embodiments, the physical separation between the first and second supports is too great for diffusion to provide a reasonably practicable means for conducting the binding partner from the first to the second support and/ or the analyte from the second support to the first support. In such embodiments it is preferred to provide some motive force or active transport means. This could be anything which facilitates transport of the relevant molecules. Conveniently, for fluid (especially, liquid) samples, there may be provided a duct, channel, tube, pipe or the like which provides fluid communication between the first and second supports. The direction of fluid movement may be bi-directional (e.g. pulsatile, with fluid movement within the fluid communication means being from the first support to the second support for a period of time, then with flow in the opposite direction for a time, with as many changes of direction as deemed appropriate). More preferably, the fluid movement within the fluid communication means is uni-directional.

Conveniently, the fluid communication means forms a loop, in one part of which displaced binding partner is transported from the first support to the second support, and in the other part of which displaced analyte is "recycled" by transport from the second support to the first support. In a particular embodiment of this type of apparatus, the first and /or second support forms part of a mass-dependent biosensor (e.g. SPR or evanescent wave-type biosensor).

Fluid movement within the fluid communication means is typically assisted by means of one or more pumps of a conventional nature (e.g. peristaltic pump or the like) . The fluid communication means may also comprise one or more valves and one or more inlet ports for intake of sample, reagents, buffers etc., and one or more outlet ports for removal of fluids. The apparatus of the invention may also comprise computerised control means and/ or computerised data processing means.

The invention will now be further described by way of illustrative examples, and with reference to the accompanying drawings, in which:

Figures 1 and 4 are schematic illustrations of two embodiments of performing the method of the invention;

Figures 2 and 3 are sensorgrams (arbitrary Response Units against time) of results obtained using apparatus in accordance with the invention; and

Figure 5 is a bar chart showing results obtained in an Assay performed in accordance with the method of the invention. EXAMPLES

Example 1 Detecting Estrone 3-Sulphate (E3S) using a mass- dependent biosensor In summary, this example shows how E3S (analyte) can be detected using two displacement events at respective first and second supports. The example is illustrated schematically in Figure 1. Two surfaces are prepared as follows. A first support (2) has attached to its surface a low affinity analogue (4) of the analyte (6). A binding partner (8), (in this case a monoclonal antibody raised against the analyte) is then reversibly immobilised on the first surface (2) by interaction with the low affinity analogue (4). A second support (10) has attached to its surface a high affinity analogue (12) of the analyte which acts as a capture moiety. The assay is performed by passing sample containing the analyte (6) across the first support (2), where it displaces the binding partner (8), as it has a higher affinity for it compared with the low affinity analogue (4). The binding partner/analyte complex (14) so formed is then contacted with the second surface (10) where a second displacement event occurs, as the high affinity analogue (12) has a higher affinity for the binding partner (8) than does the analyte (10). In this way, binding partner molecules are transferred from the surface of the first support (2) to the surface of the second support (10) where they are detected; free, unbound analyte molecules (6) are regenerated. The solution is then re-contacted with the first support (2) to displace further binding partner molecules. In this way a single analyte molecule (6) can cause the displacement of a plurality of binding partner molecules (8) from the first support (2) .

Coupling estradiol 3-glucuronide and estrone 3-glucuronide to a biosensor CM5 chip

A CM5 biosensor chip (Biacore AB) was docked in the Bialite biosensor (an SPR-type device) fitted with an upgrade kit (Biacore AB) and the flow rate of hepes buffered saline (HBS, Biacore AB) was set to 10 μl/min. The sensor chip surface was then activated by two sequential injections (40 μl and 20 μl) of a 1 , ethyl-3-[dimethylaminopropyl] carbodiimide (EDC) N- hydroxysuccinimide (NHS) activating solution made up as described by the manufacturer (Biacore AB). A solution of ethylenediamine (20 % v/v) was injected (60 μl) across flow cells 1 and 2. The flow path was then changed to flow across flow cell 2 and estrone 3-glucuronide (E3G, preincubated in EDC/NHS activating solution for 7 min) was injected (60 μl). The flow path was again changed to flow across flow cell 1 and estradiol 3-glucuronide (ED3G, preincubated in EDC/NHS activating solution for 7 min) was injected (60 μl). 60 μl of estrone 3-glucuronide (preincubated in EDC/NHS activating solution for 7 min) was then injected across flow cell 2 and a further 60 μl of estradiol 3-glucuronide (preincubated in EDC/NHS activating solution for 7 min) was injected across flow cell 1. The flow path was once again changed to flow across flow cells 1 and 2 and excess activated binding sites were blocked by a 60 μl injection of 1 M ethanolamine.

Assay Procedure

The monoclonal antibody (MAb) used as a binding partner in the following experiment (MAb 4101) was prepared according to procedures known in the prior art. A method of generating monoclonal antibodies is described by Gani et al (1994 J. Steroid Biochem. Molec. Biol. 48, 277-282) and this method can be adapted to produce a relevant antibody. A suitable monoclonal antibody can be selected on the basisof its relative affinity for the analyte and analyte analogues. This can be undertaken, for example, by performing standard kinetic determinations using a BiacoreTM 2000 biosensor (Biacore AB, Sweden), as described in the manufacturer's protocol (BIApplications handbook, Biacore AB), using a panel of closely related analogues (e.g. steroid analogues commercially available from Sigma Chemical Co.). Additionally, a commercially available anti-estrone glucuronide monoclonal antibody (from Wallaceville Animal Research Centre, New Zealand) is described in Linscott's Directory of Immunological and Biological Reagents (9th edition, 1996-7). MAb 4101 used was raised against E3G but showed cross reactivity to a number of E3G analogues. The order of cross reactivity was such that MAb 4101 bound to E3G > E3S > ED3G. Accordingly, with E3S as the analyte of interest, E3G could be used as the capture moiety and ED3G as the low affinity analogue.

The sensor chip prepared as described above was docked in the Bialite biosensor fitted with an upgrade kit and the flow rate of HBS was set to 10 μl/min. The flow path was set to cross flow cell 1 and MAb 4101 was injected across the sensor surface (40 μl of a 10 μg/ml solution). The Bialite biosensor was primed in 1.5 ml HBS in a closed system. The closed system comprised the waste outlet pipe being fed back into the buffer reservoir used to supply the syringe pump for the instrument. This ensured that after liquid had passed through the biosensor it was fed back into the filling reservoir of the syringe pump, thus, when the syringe was next filled the same solution passed through the instrument once more (i.e. was recycled back across the flow cells). E3S was then added to the buffer reservoir (50 μl of 1 mg/ml) and the Bialite system was primed again. The flow rate was set to 10 μl/min and sensorgram recording started. Data was collected for over 3 hours during which time the syringe was refilled 4 times. This ensured that E3S was allowed to recycle across both displacement surfaces. The sensorgram of this is shown in figure 2. Figure 2 shows the SPR sensorgram traces obtained at the first surface ("Fcl") and at the second surface ("Fc2"). The arrows indicate the points at which the syringe pump was refilled. Displacement of the MAb 4101 by E3S can be observed from flow cell 1 and displaced MAb 4101 (occurring as a MAb4101-E3S complex) can be seen to undergo a second displacement such that the MAb 4101 is displaced from E3S and binds to E3G at flow cell 2. The MAb 4101 accumulates at surface 2 where it is detected.

The initial displacement of the MAb 4101 from flow cell 1 (ED3G) is dependent on the presence of E3S. This can be seen in figure 3 as, when the method was repeated but no E3S was introduced into the system, MAb 4101 was substantially retained at the surface of flow cell 1.

Example 2 Detecting Estrone 3-Sulphate (E3S) in an Assay Involving Diffusion

In summary, this example shows how E3S (analyte) can be detected using two displacement events occurring in close proximity to each other. The example is illustrated schematically in Figure 4. The principle is essentially similar to that illustrated in Figure 1 , and corresponding components are denoted by use of the same reference numerals. Two surfaces are prepared as follows. A first support (2) has attached to its surface a low affinity analogue (4) of the analyte (6) . A binding partner (8) , (in this case a monoclonal antibody raised against the analyte) is then reversibly immobilised on the first support (2). A second support (10) has attached to its surface a high affinity analogue (12) of the analyte (6). The high affinity analogue (12) acts as a capture moiety. The two supports (2) and (10) are set up in such a way that there is a small gap between them. The assay is performed by placing a sample containing the analyte (6), (E3S) in between the two supports. At the first support (2) analyte (6) displaces the binding partner (8) as it has a higher affinity for it compared with the low affinity analogue (4). The binding partner/analyte complex (14) so formed then diffuses across to the second support (10) where a second displacement event occurs as the high affinity analogue (12) has a higher affinity for the binding partner (8) than the analyte (6). The analyte molecule (6) is regenerated in a free unbound state and can diffuse back to the first support (2) where it can once again displace a binding partner molecule (8). In this way, binding partner molecules (8) are transferred from the first to the second support, where they can be detected by conventional means. In this particular embodiment, the second support is a nylon peg, which fits in the wells of a microtitre plate (the first support) .

Preparation of ED3G- and E3G-ovalbumin conjugates

l-Ethyl-3-(3-dimethylaminopropyl) carboiimide (EDC) at 10 mg/ml in dimethylformamide was slowly added dropwise to ED3G (10 mg/ml also in dimethylformamide) whilst stirring. N-hydroxysuccinimide (NHS)(12 mg/ml in dimethylformamide) was then slowly added dropwise to the ED3G/EDC mixture and left to stir for 40 minutes at room temperature. The NHS/EDC/ED3G mixture was slowly added dropwise to an ovalbumin solution at 4 °C with constant stirring and then incubated overnight under the same conditions. The conjugate was desalted into phosphate buffered saline (PBS) using a PD 10 column. An E3G-ovalbumin conjugate was prepared using the same protocol except that E3G (10 mg/ml in dimethylformamide) was used in place of ED3G.

Preparation of E3G-ovalbumin pegs

Approximately 200 nylon coated cavity pegs were mixed with 200 ml of PBS containing 0.01 % sodium azide (PBSA) for 30 min. The pegs were drained and 200 ml glutaraldehyde (2 % (v/v)) was added and mixed for 60 min. The pegs were then drained and 200 ml dH₂O was added and the pegs were mixed for 5 min. This dH₂O washing step was repeated twice more and then the pegs were washed three times in PBSA. The pegs were drained and 200 ml E3G-ovalbumin at 20 μg/ml in PBSA was added and the pegs mixed. After 30 min the pegs were drained and 200 ml BSA (2 % (w/v) in PBSA) was added and mixed for 30 min. The pegs were drained and 200 ml PBSA was added, mixed for 5 min, drained and a further 200 ml of PBSA added. Pegs were stored at 4 °C until required.

Testing E3G-ovalbumin pegs bind MAb 4101

The monoclonal antibody used (MAb) in the following experiment (MAb 4101) was prepared according to procedures known in the prior art. The MAb 4101 was raised to E3G but showed cross reactivity to a number of E3G analogues. The order of cross reactivity was such that MAb 4101 bound to E3G > E3S > ED3G.

PBSA containing 0.1 % Tween 20 (PBSTA) was added to the wells (200 μl per well) of a microtitre plate (Greiner HB) and incubated for 1 hour at room temperature. The solution in the wells was replaced with 200 μl MAb 4101 (1 :500 in PBSTA) and E3G-ovalbumin pegs were then inserted into the wells and incubated for 1 hour at room temperature. As a control 200 μl PBSTA was incubated with pegs. Pegs were then washed in PBSTA and inserted into wells, pretreated with PBSTA, containing 200 μl anti-mouse IgG-alkaline phosphatase conjugate (1 : 1000 in PBSTA). Following a 1 hour incubation at room temperature the pegs were washed in PBSTA and inserted into wells containing 200 μl Sigma 104 phosphatase substrate solution. The absorbance of the substrate solution in the wells was measured at 405 nm after 1 hour at room temperature. It was found that the MAb 4101 bound satisfactorily to the E3G-ovalbumin coated pegs.

A solution of ED3G-ovalbumin conjugate (200 μl) was incubated in the wells of a microtitre plate (Greiner HB) for 2 h at room temperature. The wells were then washed with PBSTA and the MAb 4101 (100 μl of a 1 : 1000 dilution in PBSTA) was incubated in the ED3G-ovalbumin adsorbed wells for 16 h at 4 °C. The wells of the plate were then washed with PBSTA. Detection of estrone 3-sulphate (E3S) by double displacement of a monoclonal antibody

E3S (100 μl of 1 mg/ml in PBSTA) was incubated in ED3G-ovalbumin/MAb 4101 loaded wells each containing an E3G-ovalbumin peg for 2 hours at room temperature. The pegs were removed from the wells and both the wells and the pegs were washed with PBSTA. A solution (100 μl) of rabbit anti-mouse IgG-alkaline phosphatase conjugate (1 : 1000 in PBSTA) was incubated in the wells for 1 hour at room temperature. Meanwhile, the pegs were placed in wells of a separate microtitre plate (which had been blocked by pretreatment for 1 hour with 200 μl PBSTA) containing 100 μl of rabbit anti-mouse IgG-alkaline phosphatase conjugate (1 : 1000 in PBSTA). After a 1 hour incubation at room temperature the rabbit anti-mouse IgG- alkaline phosphatase conjugate solution was removed from the original wells and the pegs and original wells were washed with PBSTA. 100 μl Sigma 104 phosphatase substrate solution was then added to the original wells and to the wells of a new microtitre plate into which the pegs were placed. The absorbance at 405 nm was determined after incubating the wells for 4 hours at room temperature. In this way the MAb 4101 could be located, either still bound to the original well or displaced at the E3G- ovalbumin peg.

The results are shown in Figure 5, which is a bar chart, showing % of the MAb 4101 bound in the presence (left hand side) or absence (right hand side) of E3S substrate. The MAb 4101 still bound to the wells of the original microtitre plate (i.e. undisplaced from the first solid support) is indicated by the solid bars. The MAb 4101 bound to the pegs (i.e. displaced from the first support and captured on the second support) is denoted by the partially shaded bars.

It can be seen that, in the presence of the analyte of interest, about 90% of the MAb (binding partner) is displaced, whereas this is greatly reduced in the absence of E3S analyte.

Claims

1. A method of detecting the presence of an analyte of interest in a sample, the method comprising the steps of:

(c) contacting the displaced analyte with the first solid support, so as to displace a further binding partner and form a new binding partner/analyte complex, and contacting the new binding partner/analyte complex with the second solid support so as to capture the further binding partner at the second solid support; and

(d) detecting the presence of binding partner molecules captured by the capture moiety and/ or detecting displacement of binding partner molecules from the first support.

2. A method according to claim 1 , wherein steps (b) and (c) are performed a plurality of times.

3. A method according to claim 1 or 2, wherein the binding partner comprises an immunoglobulin or a functional binding fragment or variant thereof.

4. A method according to any one of claims 1 , 2 or 3, wherein the binding partner comprises a label.

5. A method according to claim 4, wherein the label is an enzyme label, radiolabel, fluorescent label, a chemiluminescent label, an electroactive label, or a particulate.

6. A method according to any one of the preceding claims, wherein the binding partner is reversibly immobilised to the first solid support by interaction with a low affinity analogue of the analyte of interest present on the first solid support, said interaction having a lower affinity than an interaction between the binding partner and the analyte of interest.

7. A method according to claim 6, wherein the binding partner has a binding affinity for the analyte of interest at least about 5, more desirably at least about 10, and no more than about 100, more desirably no more than about 20 times greater than the binding affinity for the low affinity analogue.

8. A method according to any one of the preceding claims, wherein the binding partner has a binding affinity for the capture moiety at least about

5, more desirably at least about 10, and no more than about 100, more desirably no more than about 20 times greater than the binding affinity for the analyte of interest.

9. A method according to any one of the preceding claims, wherein the first and /or second solid support forms part of a mass-dependent biosensor.

10. A method according to any one of claims 1-8, wherein the first or second solid support comprises a microtitre plate.

11. A method according to any one of the preceding claims, wherein the analyte of interest is selected from the group consisting of the following: drugs; hormones; proteins; nucleic acids (DNA, RNA or chimera); carbohydrates; enzymes; antibodies; antigens; bacteria; viruses.

12. A method according to any one of the preceding claims, wherein the analyte of interest is selected from the group consisting of: estrone-3- glucuronide (E3G); pregnanediol-3-glucuronide (P3G); human chorionic gonadotrophin (hCG); luteinising hormone (LH); follicle stimulating hormone (FSH).

13. An assay device for detecting the presence of an analyte of interest in a sample, the device comprising: a first solid support having reversibly immobilised thereon a binding partner having specific binding activity for the analyte of interest, the binding partner capable of being displaced from the first solid support in the presence of the analyte of interest and forming a binding partner/analyte complex; a second solid support bearing a capture moiety, the capture moiety having a specific binding affinity for the binding partner which is greater than that of the analyte of interest for the binding partner, such that the binding partner may be captured by the capture moiety and the analyte of interest displaced from the binding partner/analyte complex; means for conducting the binding partner/analyte complex from the first solid support to the second solid support; and means for conducting the displaced analyte from the second solid support to the first solid support.

14. Apparatus according to claim 13, wherein the first and /or second support is provided on one of the following: capillary-fill device; microtitre assay plate; latex bead; filter comprising cellulose or synthetic polymeric materials; glass or plastics slide; dipstick; mass-dependent biosensor.

15. Apparatus according to any one of claims 13 or 14, wherein the means for conducting the binding partner/analyte complex from the first solid support to the second solid support and/ or the means for conducting the displaced analyte from the second solid support to the first solid support, comprises diffusion in a fluid medium.

16. Apparatus according to any one of claims 13 or 14, wherein the means for conducting the binding partner/analyte complex from the first solid support to the second solid support and/ or the means for conducting the displaced analyte from the second solid support to the first solid support, comprises fluid communication means between the first and second solid supports and fluid pumping means to pump fluid through the fluid communication means.

17. Apparatus according to any one of claims 13- 16, comprising computerised control means and/or computerised data processing means.