ANALYTE ASSAY DEVICE

Abstract: The present invention relates to an analyte assay device for a liquid sample, the device comprising a carrier along which liquid sample may travel by capillary action, said carrier having, in the direction of liquid flow, a first region to which a sample may be applied, and an electrochemical detection arrangement, said device further comprising signal processing means and indicator means operatively associated with the electrochemical detection arrangement to indicate the result of the assay and methods of use thereof. The present invention also relates to a pregnanediol assay device and use thereof.

Description: ANALYTE ASSAY DEVICE The present invention relates to assay devices for determining the presence of one or more analytes in a liquid sample by specific binding. Particularly, though not exclusively, the invention relates to analytical devices for use in the home, doctor's surgery or clinic by unskilled users to allow for self diagnosis of certain conditions or disease states, for example pregnancy. Analytes present in a liquid sample may be detected and quantified by immunoassay. Generally immunoassay methods utilise an antibody which will bind with the analyte to be determined. The amount of analyte bound antibody present is determined by labelling the antibody or providing a labelled analyte which competes with the unlabelled analyte for binding to the antibody and determining the amount of label present. Hand held devices for the detection of analytes in a sample of, for example, urine, are known. The use of such devices as home tests for pregnancy and fertility is now commonplace and a wide variety of test devices and kits are available commercially. The use of strip material along which a liquid sample will travel by capillary action in devices for the detection of analytes by specific binding assays, such as immunoassays, has previously been proposed. Generally, a liquid sample is applied to the strip material and permeates throughout the strip material to a region impregnated with a specific binding partner for the analyte under test. The analyte bound to the specific binding partner migrates further along the strip where it is immobilised at an indicator region impregnated with an immobilising agent specific for the analyte bound specific binding partner. The extent to which the analyte present in the sample becomes immobilised at the indicator region is determined by labelled reagents either incorporated in the strip or applied subsequently thereto. Generally, the presence of immobilised analyte at the indicator region provides a colour change at the indicator region and it is the detection of that colour change by the user that indicates the presence of the particular analyte in the sample. Such devices are widely used as home pregnancy and fertility tests. A problem associated with known analyte assay devices is that there is scope for error associated with the reading of the colour change. Generally the specific binding partner for the analyte is labelled with a visible coloured label, for example latex particle impregnated with a dye solution. The indicator region of the test strip in a known device generally would contain an amount of an immobilising agent which stops the analyte within the indicator zone so that the amount of label at the indicator zone builds up to give a visible colour change. The presence of this immobilising agent may provide a slight amount of colour to the indicator region and it is this colour that is intensified when a positive result is observed. For example, the strip at the indicator region may be pale blue. When the test has been used and the analyte has bound a labelled antibody which is immobilised at the indictor region the strip at the indicator region may be a darker blue. The colour change is not easily reproducible or accurately readable, especially under varying light conditions Furthermore, a user of an analyte assay device which works by colour indication may have a preferred outcome to the assay in mind when reading the assay results and this may cloud their interpretation of the colour change. For example, a user of a pregnancy test kit which indicates pregnancy by an intensification of the colour at the indicator region may see an intensification in colour more readily if they wanted to be pregnant than if they did not want to be pregnant. This leads to error in determining the presence of the analyte and the condition which is associated with the presence of that analyte, in this case pregnancy. A further problem associated with known analyte assay devices is that although they show the presence of an analyte in a sample they do not quantify the levels of that analyte as compared to expected levels of the analyte and interpret the results. Therefore the analyte to be detected may need to be present in hugely elevated levels in a positive test if in a normal sample the analyte to be determined is generally present, usually at low levels so that a colour change may be seen. Having information about the amount of analyte present in a sample may provide indications, for example, of the progression of a condition. It is an object of the present invention to obviate or mitigate the aforementioned disadvantages. According to a first aspect of the present invention there is provided an analyte assay device for a liquid sample, the device comprising a first carrier along which the liquid sample may travel by capillary action, said carrier having, in direction of liquid flow, a first region to which the sample may be applied and an electrochemical detection arrangement, said device further comprising signal processing means and indicator means operatively associated with the electrochemical detection arrangement to indicate the result of the assay. The invention provides an analyte assay device which overcomes the problems referred to in association with known analyte assay devices and provides an analyte assay device which may be readily usable by an unskilled person in a non-laboratory setting such as at home, at a doctor's surgery or at a clinic. The present invention provides a simple, non-invasive, rapid and accurate assay device whose results may be clearly readable by eye and which may provide quantitative, rather than qualitative results and allows for self diagnosis of certain conditions or disease states. The use of signal processing means which are operatively linked to indicator means has the advantage that it provides a system for determining the level of analyte in the test sample and comparing this with expected levels known for certain disease states or conditions, thereby providing for rapid diagnosis in a non-invasive manner. The device according to the present development does not require that a specific binding partner providing a label for the analyte is provided on the carrier. This provides the advantage that the device can be used in an assay where the binding partner providing a label for the analyte and the analyte are pre-mixed and subsequently applied to the assay device. According to a preferred embodiment of the present invention the analyte assay device further comprises a second region incorporating a specific binding partner for an analyte, said specific binding partner having a label which is directly or indirectly electrochemically detectable. A typical embodiment of the invention is an analytical test device comprising a hollow liquid-impermeable casing containing the carrier which communicates directly or indirectly with the exterior of the casing such that a liquid test sample can be applied to the first region of the carrier. The liquid sample may be applied at a proximal end of the carrier and migrates towards a distal end by capillary action. During the migration the liquid sample moistens the previously dry carrier allowing migration of dissolved or dispersed agents present on the carrier (for example, as provided at the second region) along with the liquid. The aforementioned second region of the carrier incorporates a binding partner specific for an analyte which may be present in the sample. This specific binding partner has a label which is either directly or indirectly detectable by the electrochemical detection arrangement. By directly detectable we mean that it is the label itself which is detected at the electrochemical detection arrangement. By indirectly detectable we mean that the label participates in a reaction generating an electrochemically detectable species which is then detected at the electrochemical detection arrangement. The analyte assay device is arranged such that only electrochemically detectable label that has resulted directly or indirectly from the analyte binding to a reagent specific for that analyte is measured by the electrochemical detection arrangement. Unbound analyte, label free in solution or labelled or unlabelled specific binding partner that has not bound analyte are not detected. Accordingly, it is preferred that the analyte assay device further comprises a capture region having a immobilising agent which acts, when the analyte assay device is used in an assay in which the sample is treated with a specific binding partner incorporating a label which is directly or indirectly electrochemically detectable at the electrochemical detection arrangement, to immobilise any unbound label such that only label due to the presence of analyte can reach the electrochemical detection arrangement. Such an arrangement provides a true indication of the amount of analyte under test and does not provide false results due to the presence of unbound label. At least some of the analyte present in the sample binds to its labelled specific binding partner on the carrier. Preferably, it is the label that is electrochemically detectable and therefore both labelled specific binding partner and analyte bound labelled specific binding partner may be detectable by the electrochemical detector. To prevent signal generation by label that has not bound the analyte, unbound labelled specific binding partner itself may be immobilised at the second region and is therefore prevented from migrating through the carrier to the electrochemical detection apparatus unless it binds analyte in a competition reaction. Alternatively, both analyte bound and unbound labelled specific binding partner may migrate along the carrier but there is a capture region on the carrier prior to the electrochemical detection arrangement such that any labelled specific binding partner that has not bound to the analyte, passing through this region is "captured" by a specific binding partner for the unbound labelled or unlabelled specific binding partner and prevented from reaching the electrochemical detection arrangement. Another alternative to prevent signal generation due to non analyte bound labelled specific binding partner or label is to make it so that binding of the analyte to its labelled specific binding partner produces a conformational change such that only analyte bound labelled specific binding partner is detectable by the electrochemical detection arrangement. For example, the label may be internalised in the labelled specific binding partner but binding of analyte may externalise the label so that it may be electrochemically detectable. Examples of specific binding partners for an analyte include specific immunoglobulins which are antibodies to that analyte or fragments of that antibody that have analyte binding capacity, antigens that bind the analyte and are then bound by antigen specific antibodies, receptors for the analyte, for example if the analyte is a hormone the specific binding reagent may be its receptor or a binding fragment or variant thereof, an enzyme which may bind the analyte, for example if the analyte is a metabolite of an enzymatic reaction the specific binding reagent may be the enzyme or an analyte binding fragment or variant thereof, any other protein capable of recognising and binding to the analyte or a non-proteinaceous compound having analyte binding capacity. In a preferred embodiment the specific binding partner for an analyte is an anti- analyte antibody. The anti-analyte antibody has specificity for a particular epitope present on the analyte. Preferably the anti-analyte antibody can freely migrate within the carrier in the presence of a liquid sample, both when analyte bound or as free anti- analyte antibody. While free anti-analyte antibody is migrating through the carrier it can bind to any analyte present in the sample. To prevent any free anti-analyte antibody from being detected at the detector preferably carrier comprises an capture region which contains an anti-anti-analyte antibody. Preferably, the anti-anti-analyte antibody binds a specific epitope on the anti-analyte antibody which is available for binding on the free anti-analyte antibody but is masked on the analyte-bound anti- analyte antibody (possibly because the analyte binds at a region overlapping with the epitope or binding of the analyte induces a conformational change in the anti-analyte antibody structure which internalises a previously available epitope). Therefore only analyte-bound anti-analyte antibody reaches the electrochemical detection arrangement so that its label can be directly or indirectly electrochemically detected. Preferred directly electrochemically detectable labels are electroactive species which promote a change in potential at the electrochemical detection arrangement. Examples of electroactive species which may directly change the potential at the electrochemical detection arrangement are metals and metal ions, such as sodium and potassium, as well as calcium ions, hydrogen ions, fluoride ions and ammonium ions. A preferred directly electrochemically detectable label is gold. The conductive properties of gold will directly provide a change in potential when present at the electrochemical detection arrangement. Examples of electroactive species which may indirectly change the potential at the electrochemical detection arrangement are those that undergo redox reactions, for example hydrogen peroxide may generate hydrogen ions which may change the potential at the electrochemical detection arrangement. Preferred indirectly electrochemically detectable labels are reagents that induce at least one reaction at a reaction region that produce a species that is directly or indirectly electrochemically detectable. A preferred indirectly electrochemically detectable label is an enzyme which catalyses a reaction at a reaction region on the carrier which produces a species which is directly or indirectly electrochemically detectable. To reduce artificial results for being generated, only indirect label due to analyte binding to specific binding partner is able to reach the reaction region, unbound labelled specific binding partner remaining either immobilised in the second region or at a capture region as described above. The enzyme label preferably catalyses one reaction that produces a species that is electrochemically detectable label or may initiate a cascade of reactions which at a point in the cascade produces a species that is electrochemically detectable label. An example of an enzyme which is a suitable indirectly electrochemically detectable label is the enzyme PI nuclease. PI nuclease initiates a cascade reaction which activates the enzyme glucose oxidase to catalyse the reaction of glucose to gluco 1,5, lactone with the release of hydrogen peroxide. The levels of hydrogen peroxide generated may be indirectly measured by the electrochemical detection arrangement. Alternatively, horseradish peroxidase may also be present and this may catalyse the reduction of hydrogen peroxide to water by reductant, the water also being indirectly detectable at the electrochemical detection apparatus. Steps in the PI nuclease catalysed cascade reaction are shown schematically below. An advantage of using PI nuclease as an indirectly electrochemically detectable label is that the cascade reaction initiated by PI nuclease amplifies the effect of the presence of analyte at the electrochemical detection apparatus, in that for each molecule of analyte present in the sample up to 10 6 molecules of hydrogen peroxide may be produced to be detected as hydrogen ions at the electrochemical detection arrangement. This allows low levels of analyte in a sample to be detected. FADP PI Nuclease 4Λ-PP FAD Apo Glucose Oxidase -» Glucose Oxidase I Glucose - Gluco 1,5, lactone H 2 O 2 ^PRODUCT (MEASURE) Horseradish (MEASURE) Peroxidase The electrochemical detection arrangement preferably comprises an amperometric, a potentiometric or a coulometric detector. An amperometric detector determines the amount of a substance by means of an oxidation-reduction reaction involving that substance. Electrons are transferred as a part of the reaction, so that the electrical current through the detector is related to the amount of the substance seen by the detector. A potentiometric detector is a chemical detector that measures the concentration of a substance by determining the electrical potential between a specially prepared surface and a solution containing the substance being measured. A coulometric detector detects differences in charge. The electrochemical detection arrangement may be an ion specific electrode. In general an electrochemical detector comprises two electrodes, a reference electrode which is typically silver-coated with silver chloride and immersed in a concentration of potassium chloride that communicates with the sample solution via a porous plug or gel, and an ion specific electrode, typically immersed in a concentrated solution of the ion of interest and separated from the sample solution by a membrane that is selectively permeable to the ion under test. Preferably the electrochemical detector comprises an ion specific electrode arranged to be responsive to an increase in hydrogen ions. Preferably, the reference electrode is at a fixed potential. The electrochemical detector may be a pH electrode. Preferably, the electrochemical detection arrangement comprises an electrode which is printed onto the carrier as a printed circuit board (PCB). Preferably, adjacent the electrode of the electrochemical detection arrangement is a funnel region adapted to funnel electrochemically detectable label onto the electrode of the electrochemical detection arrangement. The electrochemical detection arrangement may further comprise an integral power source. Power sources suitable for use in the analyte assay device of the present invention include batteries, small solar cells, induction coils (stand and shake) and crushable acid vials, containing an acid (preferably citric acid) and dissimilar metal plates arranged so as to produce a potential difference when the vial is crushed. The electrochemical detection arrangement is associated with signal processing means which outputs data to an indicator for indicating the assay result. Preferably, the signal processing means comprises software which compares data output from the electrochemical detection arrangement with known levels of analyte present to determine the signal to be shown on the indicator. The signal processing means provides a specific signal at the indicator means depending upon the assay results. Preferably, the signal at the indicator is clearly visible or may alternatively be audible. Suitable indicator means comprise liquid crystals or light emitting diode displays. The signal generated at the indicator means may show that the test is positive for analyte, negative for analyte or that the test results are inconclusive and that another test should be undertaken. A preferred indicator means is a traffic light signal, whereby a positive test is indicated by a green signal, a negative test is indicated by a red signal and an inconclusive test is indicated by an amber or orange signal. Furthermore it is desirable that the indicator be adapted to provide more information about the assay results than just that analyte is present or not in the sample under test. Software in the signal processing means may contain standard data relating to certain levels of the analyte under test and their implications in the progression or a disease state or condition. The indicator means may be adapted to display a diagnosis based on the software's interpretation of the test results as compared to standard data. For example, if a certain condition is associated with a particular concentration of analyte the indicator may display a diagnosis of that condition. Preferably, such a diagnosis would be provided on a print-out, either on a liquid crystal display or as a paper print out. An important preferred embodiment of the present invention is the choice of nitro-cellulose as the carrier. Nitro-cellulose has a considerable advantage over conventional strip materials such as paper because it has a natural ability to bind proteins without prior sensitisation. Specific binding reagents such as immunoglobulins can be applied directly to nitro-cellulose and immobilised thereon without chemical treatment of the nitro-cellulose. Furthermore, nitro-cellulose is readily available in a range of pore sizes and this facilitates the selection of a carrier to suit requirements such as sample flow rate. Preferably, the carrier has a pore size of at least one micron. Preferably, the porous carrier has a pore size of not greater than about 20 microns. Generally a porous carrier having a pore size of between about 8-12 microns is preferred. It is preferable that the flow rate of the sample through the carrier should be at a rate of 1cm in not more than 2 minutes but slower flow rates may be used if desired. The reagents (for example, specific binding partners and immobilising agent) may be applied to the carrier material by "printing" with micro-syringes, by direct printing or by ink jet printing. The spatial separation between the specific regions on the carrier, first region, second region, any capture region, any reaction region and the electrochemical detection arrangement and the flow rate characteristics of the carrier can be selected to allow adequate reaction times during which specific binding to analyte may occur. If desired, the analyte assay device according to the present invention can incorporate two or more discrete bodies of carrier material, as sheets or strips separated by a moisture impermeable barrier. These discrete bodies can be arranged in parallel, for example, such that a single application of liquid sample to the device initiates migration of the sample in each discrete body. At least one of the bodies comprises a carrier strip in accordance with the present invention. If one of other bodies does not comprise reagents but does comprise an electrochemical detection arrangement, the analyte assay device provides a control against which to compare the assay strip results to take account of any artefacts in the liquid sample which may affect the level of label detected by the electrochemical detection arrangement. Alternatively, if different reagents are used on the different bodies, the simultaneous determination of a plurality of analytes in a single sample can be made. A prefened embodiment of the present invention is a pregnancy testing device for a liquid urine sample, the pregnancy testing device comprising a carrier along which the liquid sample will travel by capillary action, said carrier having, in the direction of liquid flow, a first region to which the urine sample may be applied, a second region incorporating a specific anti-pregnanediol antibody having a label which is directly or indirectly electrochemically detectable, and an electrochemical detection arrangement, said device further comprising signal processing means and indicator means operatively associated with the electrochemical detection arrangement to indicate the results of the assay. Pregnanediol is an inactive form of progesterone which is excreted in the urine conjugated to glucuronic acid. It has been shown that levels of serum progesterone correspond to the levels of pregnanediol in the urine. In women, progesterone is produced mainly by the corpus luteum following ovulation. Some progesterone is also produced by the adrenal cortex. During pregnancy, progesterone is synthesised mainly by the placenta. The main function of progesterone appears to be to increase the secretory phase of endometrial development, which prepares the uterus for possible implantation by a fertilised egg. After fertilisation, progesterone is necessary for the development and maintenance of the placenta. Different levels of progesterone may indicate pregnancy and furthermore various stages of pregnancy are associated with different levels of progesterone. As urine levels of pregnanediol are said to correspond to serum levels of progesterone, an assay for pregnanediol levels in urine may allow diagnosis of pregnancy and furthermore the prognosis for that pregnancy. The signal processing means of the pregnancy test kit may comprise data on the levels of pregnanediol associated with unpregnant females, pregnant females and post menopausal females to provide an indication of pregnancy. Furthermore as levels of progesterone associated with safe pregnancy, ovarian cysts, eptopic pregnancy, pre- eclampsia, toxaemia of pregnancy, ovarian cancer, amenonhea, placental failure, foetal death and threatened spontaneous abortion are known the test, via the signal processing means may diagnose these conditions based on determining the levels of pregnanediol in a urine sample and the indicator means as well as indicating a problem, may provide a print out of that diagnosis. The analyte assay device may furthermore be adapted to determine fertility by determining levels of lutenising hormone in urine. The analyte assay device according to the present invention may be used to determine a wide variety of analytes by choice of specific binding reagents. The analytes can be, for example, proteins, antigens, immunoglobulins, hormones, polynucleotides, drugs, steroids and infectious agents, for example infectious agents of bacterial origin such as Chlamydia, Streptococcus. The analyte assay device may be used to diagnose diseases, genetic mutations causing a pre-disposition to a disease and alcohol or drug abuse. To adapt the analyte assay device to determine the presence of an analyte known to be associated with a particular condition it is necessary to adapt the reagent to be specific for that analyte. According to the present invention in a second aspect there is provided a method of analysing the presence of analyte in a liquid sample, the method comprising: (a) adding a specific binding partner incorporating a label which is directly or indirectly electrochemically detectable to a liquid sample to provide a labelled liquid sample; (b) applying the labelled liquid sample to an analyte assay device according to the first aspect of the invention; and (c) determining the result of the assay. The invention will be further described by way of example only with reference to the accompanying drawings, in which: Figure 1 is a schematic plan view of a first embodiment of an analyte assay device in accordance with the invention; Figure 2 is a schematic plan view of a second embodiment of an analyte assay device in accordance with the invention; Figure 3 is a schematic plan view of a third embodiment of an analyte assay device in accordance with the invention; and Figure 4 illustrates a perspective side view of a fourth embodiment of an analyte assay device in accordance with the invention. Figure 5 is a schematic diagram of circuitry that may be used to convert the input of the electrochemical detection arrangement into output. Referring to Figure 1, the analyte assay device 10 is seen as a rectangular carrier strip 12 of a material along which a liquid sample will travel by capillary action. Strip 12 extends longitudinally from a proximal end 14 to a distal end 16. Adjacent the proximal end 14 of the strip 12 is a first region 18 extending across the entire width of the strip 12. Adjacent the first region 18 is a second region 20 which also extends across the entire width of the strip 12. At approximately two thirds of the way along the strip 12 is a third region 22, similarly extending across the entire width of the strip 12. The spatial separation along the strip 12 between regions 20 and 22 can be of any length as long as the two regions 20 and 22 are separated. Between the third region 22 and the distal end 16 of the strip 12 is a funnel region 24, followed by a electrochemical detection arrangement 26 which is operatively linked via signal processing means (not shown) to indicator means 28 adjacent the distal end 16 of the strip 12. The electrochemical detection arrangement 26, signal processing means and indicator means 28 are also linked to a power source (not shown). The first region 18 of the strip 12 is the region of the strip to which the liquid sample (not shown) is applied. The strip 12 at the second region 20 is either surface printed or impregnated with a specific binding partner for the analyte, in this particular embodiment an anti-analyte antibody (not shown) having an electrochemically detectable label. This labelled antibody can migrate along the strip 12 by virtue of the capillary action of the liquid sample. In an alternative arrangement specific binding partner for the analyte may be added to the sample before application to the first region 18 of the strip 12. In this arrangement the second region 20 is not necessary. Third region 22 is impregnated with a specific binding partner for the anti-analyte antibody, in this case an anti-anti-analyte antibody (not shown) which binds only labelled anti- analyte antibody that has not bound to analyte, thus "capturing" label not due to the presence of analyte in the sample. When liquid sample is applied to the strip 12 at region 18, adjacent the proximal end 14 of the strip 12, it migrates longitudinally along the strip 12 by capillary action. The liquid sample first migrates to the second region 20 and dissolves the labelled anti-analyte antibody present at the second region 20, drawing the labelled anti-analyte antibody along the strip 12 along with the liquid sample by capillary action. Whilst migrating along the strip 12 analyte in the liquid sample may bind to the labelled anti-analyte antibody to produce a complex (not shown) comprising the analyte bound to the anti-analyte antibody having an electrochemically detectable label. The complex migrates along the strip 12 to the third region 22, as does any remaining labelled anti-analyte antibody, allowing more opportunity for complex to be formed. On reaching the third region 22, any remaining free labelled anti-analyte antibody is immobilised by binding to the anti-anti-analyte antibody, thus preventing further migration of free labelled antibody. However, the complex comprised of analyte bound labelled anti-analyte antibody is able to continue its migration along the strip 12 by capillary action. As the complex approaches the distal end 16 of the strip it encounters the funnel region 24 which concentrates the complex ("by funnelling") into a mid-section 30 of the strip 12. The complex then migrates to an electrode 32 of the electrochemical detection arrangement 26 where electrochemically detectable label is detected and compared with data from a reference electrode (not shown) to generate an output signal (not shown). Details concerning the production of output signal from the electrochemical detection system are provided later, in relation to Figure 5. The output signal from the electrochemical detection arrangement 26 is fed to signal processing means (not shown) which compares data output from the electrochemical detection arrangement 26 with previously input data concerning expected analyte levels and the prognosis associated with such analyte levels. The assay result is indicated, in this case, by one of three coloured lights 34, 36 and 38 or the indicator means 28 being illuminated. Light 34 is coloured red indicating that the test was negative for a particular condition, light 36 is coloured amber indicating that the test results were inconclusive and the test should be repeated and light 38 is coloured red indicating that the test was positive for a particular condition Figure 2 shows a similar anangement to that described with reference to Figure 1 with an additional strip 40 of carrier material added, running parallel to the strip 12 and separated from strip 12 by a plastic barrier 42. The strip 40 has a funnel region 44 similar to that of the strip 12 and a reference electrode 46, linked to the electrochemical detector arrangement 26 of strip 12. A difference in potential between the reference electrode 46 of the strip 40 and the electrode 32 of the strip 12 provides data to the signal processing means (not shown) and ultimately to the indicator means 28. Again details concerning the production of an output signal from the electrochemical detection system are provided later in relation to Figure 5. The arrangement shown in Figure 2 allows more accurate detection of analyte levels in a liquid sample compared to the arrangement shown in Figure 1. More particularly, any agent in the liquid sample which is an interferant of the electrochemical detection arrangement 26 is allowed for in the analysis of the test results since its effect is the same at the electrode 32 of the strip 12 and at the reference electrode 46 of the strip 40. Figure 3 shows an arrangement in which the binding of an analyte to a specific binding partner having an enzyme label which is not itself electrochemically detectable but which is capable of generating an electrochemically detectable species may be assayed. The arrangement shown in Figure 3 is described with reference to the detection of a particular analyte, pregnanediol, whose presence in the urine is associated with pregnancy. Furthermore quantification of the amount of pregnanediol present in a sample allows a prognosis for the likelihood of any pregnancy detected from proceeding to birth of a healthy child. The first region 18 adjacent the proximal end 14 of the strip 12 is, as in Figures 1 and 2, the region to which the liquid sample is to be applied. The second region 20, adjacent the first region 18, is printed or impregnated with a specific anti-pregnanediol antibody which is labelled with PI nuclease (an enzyme). PI nuclease itself is not electrochemically detectable. Approximately midway along the length of the strip 12 is the third region 22 which is impregnated with an anti-anti-pregnanediol antibody which only binds free labelled anti-pregnanediol antibody. Approximately two thirds of the way along the length of the strip 12 is a reaction region 48 which comprises an enzyme pad comprising all the substrates and enzymes (except PI nuclease) necessary for a cascade reaction whose initial reaction is catalysed by PI nuclease. The cascade reaction produces hydrogen peroxide. The cascade reaction amplifies the effect of the presence of pregnanediol in a sample by up to 10 6 times, in that up to 10 6 molecules of hydrogen peroxide are produced for every molecule of analyte that binds the PI nuclease labelled anti-analyte antibody, thus allowing detection of even low levels of pregnanediol. The funnel region 24 concentrates hydrogen peroxide into the mid section 30 and the hydrogen peroxide migrates by capillary action to the electrochemical detection anangement 26 where it causes a potential difference between the electrode 32 and a reference detector (not shown) due to the production of protons. The electrochemical detection arrangement 26 detects the change in potential and feeds output data to the signal processing means (not shown) which is linked to indicator means 28 to display the test results. A preferred use of the analyte assay device 10 will now be described with reference to Figure 4 in relation to determining pregnancy and furthermore the prognosis for that pregnancy. Referring to Figure 4 there is shown a hand-held pregnancy testing kit having a plastic casing 50 enclosing strip 12 as described with reference to Figure 3. The casing 50 terminates at its proximal end 51 in a small integral receptacle 52 which can hold a predetermined volume of a liquid sample, for example urine. The proximal end 14 of the strip 12 protrudes into the receptacle 52. The receptacle 521 is especially adapted to be able to receive urine samples directly from the urine stream. Referring to Figure 5 there is shown an example of appropriate circuitry for converting the input to the electrochemical detection arrangement 26 into output. In use, due to the presence of charged label, charge in the sample will accumulate at electrode 32 and at a reference electrode, indicated by 54 (this is also electrode 46 as shown in Figure 2). This forms the input to the electrochemical detection arrangement. The input leads to an op amp or comparator system where the input signals from electrodes 32 and 54 are compared, to produce an output signal. If there is no analyte in the sample, there will be no difference in the charge between electrodes 32 and 54 and thus the comparator system will provide an output signal showing this result. If analyte is present in the sample more label will reach electrode 32 than electrode 46. Thus the comparator will note a difference in its two inputs and indicate that analyte was present in the sample in its output signal. The electrochemical detection arrangement may be adapted to use a potentiometric system (to detect a potential difference between the electrodes) or to use an amperometric system (to detect the difference in accumulation of charge between electrodes). In operation, referring to Figures 3 and 4, the plastic casing 50 of the analyte assay device is held directly in the urine stream so that liquid fills the receptacle 52. From the receptacle 52 the liquid sample is absorbed into the strip 12 at region 18 and migrates along the strip 12 by capillary action, through the second region 20 and the third region 22 as described above. During migration along the strip 12 pregnanediol present in the urine sample should bind to the anti-pregnanediol antibody labelled with PI nuclease provided at the second region 20 to form a complex. The capillary action of the liquid sample on the strip 12 draws with it the specific anti-pregnanediol antibody labelled with PI nuclease from the second region 20 where it is impregnated or printed to the third region 22 which is impregnated with an anti-anti-pregnanediol antibody which only binds free anti-pregnanediol antibody labelled with PI nuclease but not the complex of pregnanediol bound anti-pregnanediol antibody labelled with PI nuclease. Capillary action draws the complex along the strip 12 to the reaction region 48 which comprises an enzyme pad including all the substrates and enzymes (except PI nuclease) necessary for a cascade reaction whose initial reaction is catalysed by PI nuclease. The cascade reaction produces hydrogen peroxide. The cascade reaction amplifies the effect of the presence of pregnanediol in a sample by up to 10 6 times, in that up to 10 6 molecules of hydrogen peroxide are produced for every molecule of analyte that binds the PI nuclease labelled antibody, thus allowing detection of even low levels of pregnanediol. Hydrogen peroxide generated by the cascade reaction at the reaction region 48 is drawn along the strip 12 to the funnel region 24 which concentrates the hydrogen peroxide into the mid section 30 and the hydrogen peroxide migrates by capillary action to the electrochemical detection arrangement 26 where it causes a potential difference between the electrode 32 and a reference detector (not shown) due to the production of protons. The electrochemical detection arrangement 26 detects the change in potential and feeds output data to the signal processing means (not shown). Ideally, the signal processing means contains data concerning expected levels of pregnanediol and their relation to the prognosis of a pregnancy. It is known that the levels of serum progesterone correspond to the levels of pregnanediol in the urine. In women, progesterone is produced mainly by the corpus luteum following ovulation. Some progesterone is also produced by the adrenal cortex. During pregnancy, progesterone is synthesised mainly by the placenta. The main function of progesterone appears to be to increase the secretory phase of endometrial development, which prepares the uterus for possible implantation by a fertilised egg. After fertilisation, progesterone is necessary for the development and maintenance of the placenta. In a non-pregnant woman, after ovulation serum progesterone levels decrease as indicated generally as urine pregnanediol levels of between 0.2 to 7.0 milligrams per 24 hours (for both fertile and post-menopausal women). In pregnant women the pregnanediol levels are elevated. It is estimated that up to 50 percent of all fertilised eggs die and are lost (aborted) spontaneously, usually before the woman knows that she is pregnant. Among known pregnancies, the rate of spontaneous abortion is approximately 10 percent and usually occurs between the 7th and 12th weeks of the pregnancy. On occasion, the uterus does not expel all of the foetal tissue, in which case it is considered an incomplete spontaneous abortion. Such an abortion may require surgical removal of the retained tissue. If foetal death occurs that is not accompanied by uterine expulsion of the dead tissue, it is considered as a missed abortion. Signs of pregnancy decrease and the uterus begins to shrink back to its original size. If spontaneous abortion does not occur to remove dead tissue a D & C (dilation and curettage) may have to be performed or labour induced to expel the dead foetal tissue otherwise it may cause infection. This can be very distressing for the woman and it is desirable to provide for an early prognosis of the likelihood of a pregnancy proceeding to term. Pregnanediol levels have been linked with the prognosis for a pregnancy going to term and may indicate the likelihood of foetal death and other disorders which may affect the pregnancy. In pregnancy, higher than expected levels of pregnanediol in the urine are characteristic of the presence of ovarian cysts, adrenocortical hyperplasia, arrhenoblastoma of ovary, choriocarcinoma of ovary, hyperadrenocorticism. Lower than expected levels of pregnanediol in the urine are characteristic of amenonhea, ovarian cancer, ovarian hypofunction (decreased function) foetal death, placental failure (including eptopic implantation), preeclampsia, toxaemia of pregnancy or threatened abortion. The analyte assay device according to the present invention, may be adapted such that the signal processing means contains data concerning predetermined preganediol levels associated with certain disorders associated with pregnancy. The signal processing means then compares the results of the assay with the known levels and can provide a prognosis on the likelihood of the pregnancy going to term, at the indicator means 28. The indicator means 28 may simply indicate that the pregnancy is proceeding well or that a doctor should be consulted or that the test results are inconclusive and the test should be repeated, for example. It may be possible however to provide printing means as part of the indicator means so that the device may provide a print out of the test results and the prognosis for the pregnancy. Therefore, the analyte assay device according to a preferred embodiment of the present invention provides a non invasive diagnostic test which may have application in the clinical laboratory but more particularly in the doctor's surgery and as an OTC (over the counter) product.

Claims: CLAIMS 1. An analyte assay device for a liquid sample, the device comprising a carrier along which liquid sample may travel by capillary action, said carrier having, in the direction of liquid flow, a first region to which a sample may be applied, and an electrochemical detection anangement, said device further comprising signal processing means and indicator means operatively associated with the electrochemical detection arrangement to indicate the result of the assay. 2. An analyte assay device according to claim 1, the device further comprising a second region incorporating a specific binding partner for an analyte, said specific binding partner having a label which is directly or indirectly electrochemically detectable. 3. An analyte assay device according to claim 2, in which a specific binding partner is an antibody to the analyte or an antibody fragment having analyte binding capacity, an antigen that binds the analyte, a receptor for the analyte or a receptor fragment; or an enzyme which binds the analyte. 4. An analyte assay device according to claim 3, in which the specific binding partner is an analyte specific antibody. 5. An analyte assay device according to claim 4, in which the analyte specific antibody is an anti-pregnanediol antibody. 6. An analyte assay device according to any one of claims 2 to 5, in which the electrochemically detectable label is directly electrochemically detectable and comprises an ionic species. 7. An analyte assay device according to any one of claims 2 to 5, further comprising a third region between the second region and the electrochemical detection arrangement, said third region providing a reaction region. 8. An analyte assay device according to claim 7, in which electrochemically detectable label is indirectly electrochemically detectable and comprises one or more reagents that induce at least one reaction at the reaction region that produces a species that is directly or indirectly electrochemically detectable. 9. An analyte assay device according to claim 8, in which the indirectly electrochemically detectable label is an enzyme. 10. An analyte assay device according to claim 9, in which the enzyme is PI nuclease. 11. An analyte assay device as claimed in claim 1, the device further comprising a capture region having an immobilising agent which acts, when the analyte assay device is used in an assay in which the sample is treated with a specific binding partner incorporating a label which is directly or indirectly electrochemically detectable at the electrochemical detection arrangement, to immobilise any unbound label such that only label due to the presence of analyte can reach the electrochemical detection arrangement. 12. An analyte assay device according to any preceding claim, in which the electrochemical detection arrangement comprises an ion specific electrode. 13. An analyte assay device according to claim 12, in which the ion specific electrode is a pH electrode. 14. An analyte assay device according to any preceding claim, in which the electrochemical detection arrangement comprises an electrode which is printed onto the carrier as a printed circuit board. 15. An analyte assay device according to claim 14, in which adjacent the electrode is a funnel region adapted to funnel electrochemically detectable moieties onto the electrode. 16. An analyte assay device according to any preceding claim further comprising an integral power supply. 17. An analyte assay device according to any preceding claim, in which the signal processing means comprises software to compare data output from the electrochemical detection arrangement with known analyte levels to provide an output to the indicator means. 18. An analyte assay device according to any preceding claim, in which the indicator means provides a visible signal. 19. An analyte assay device according to claim 18, in which the indicator means comprises light emitting diodes or liquid crystals. 20. An analyte assay device according to claim 18, in which the indicator means comprises a plurality of differently coloured light emitting diodes or liquid crystals. 21. An analyte assay device according to claim 18, in which the indicator means comprises a text or numeric display. 22. An analyte assay device according to any preceding claim, in which the carrier comprises nitro-cellulose. 23. An analyte assay device according to any preceding claim, adapted such that the flow rate of sample along the carrier is at least 1cm in 2 minutes. 24. An analyte assay device according to claim 1 comprising, in addition to the first carrier at least one additional carrier separated from the first carrier by a moisture impermeable barrier. 25. An analyte assay device according to claim 24, in which the additional carrier(s) provide reference assays. 26. An analyte assay device according to claim 2 comprising, in addition to the first carrier at least one additional carrier separated from the first carrier by a moisture impermeable barrier. 27. An analyte assay device according to claim 26, in which the additional carrier(s) comprise binding partners specific for an analyte other than that for which the specific binding partner on the first carrier is specific. 28. An pregnanediol assay device for a urine sample, the device comprising a carrier along which the liquid sample may travel by capillary action, said carrier having, in the direction of liquid flow, a first region to which the sample may be applied, a second region incorporating a anti-pregnanediol specific antibody having a PI nuclease label, a third region comprising a reaction region comprising all of the substrates and enzymes, other than PI nuclease, necessary to generate hydrogen peroxide in a reaction catalysed by PI nuclease, and an electrochemical detection arrangement, said device further comprising signal processing means and indicator means operatively associated with the electrochemical detection arrangement to indicate the result of the assay. 29. A method of analysing the presence of analyte in a liquid sample, the method comprising: (a) adding a specific binding partner incorporating a label which is directly or indirectly electrochemically detectable to a liquid sample to provide a labelled liquid sample; (b) applying the labelled liquid sample to the first region of the analyte assay device as claimed in claim 1 ; and (c) determining the result of the assay. 30. A method of analysing the presence of analyte in a liquid sample, the method comprising: (a) applying a liquid sample to the first region of the analyte assay device as claimed in claim 2, and (b) determining the result of the assay. 31. Use of the analyte assay device according to any preceding claim to measure the levels of pregnanediol in the urine of a woman to detect pregnancy. 32. Use of the analyte assay device according to any preceding claim to measure the levels of pregnanediol in the urine of a woman to provide a prognosis for a pregnancy proceeding to term.

Inventors: MINTER STEPHEN JOHN, MINTER TIMOTHY JOHN, MINTER, STEPHEN, JOHN, MINTER, TIMOTHY, JOHN
Applicants: MINTER STEPHEN JOHN, MINTER TIMOTHY JOHN, MOORLODGE BIOTECH VENTURES LIM, MINTER, STEPHEN, JOHN, MINTER, TIMOTHY, JOHN, MOORLODGE BIOTECH VENTURES LIMITED
Application year: 1999
Main classification: G01N33
IPC codes: G01N33