Lateral flow test device

Abstract

Provided herein is a lateral flow test device for determining the amount of a plurality of hormones in a test sample such as a blood sample. The lateral flow test device is suitable for self-testing and provides rapid and accurate results.

Description

[0001] Lateral Flow Test Device

[0002] Field of Invention

[0003] The present invention relates to a lateral flow test device for determining the amount of a plurality of hormones in a sample, a method for determining the amount of a plurality of hormones in a sample, and a kit comprising the lateral flow test device.

[0004] Background

[0005] Lateral flow test

[0006] A lateral flow test enables rapid detection of an analyte in a liquid sample. Lateral flow tests are widely used in medical diagnostics for point of care testing (for example, home testing) or laboratory use. A typical lateral flow test device includes the following components: 1) sample pad; 2) conjugate pad; 3) nitrocellulose membrane; and 4) absorbent pad. The components may be housed in a cassette or provided on a backing card for ease of handling.

[0007] A common type of lateral flow test is based on a ‘sandwich’ immunoassay. In this format, the conjugate pad typically comprises a primary antibody conjugated to a label which is capable of binding to a target analyte in a sample. The labelled antibody becomes mobilised (released) when the sample flows through the conjugate pad. The nitrocellulose membrane typically includes a test region in the form of a line on which a capture antibody capable of binding to another epitope on the target analyte is immobilised. The nitrocellulose membrane may also include a control region (for example, a control line) comprising a secondary immobilised antibody which is capable of binding to the labelled antibody.

[0008] In use, a test sample comprising a target analyte is added to the sample pad of the lateral flow test device and the sample flows through to the conjugate pad, thereby mobilising the labelled primary antibody. The target analyte and the labelled antibody come into contact. The labelled antibody, bound to analyte, migrates towards nitrocellulose membrane by capillary action. The capture antibody which is immobilised on the

[0009] 14245329-1 nitrocellulose membrane captures labelled antibody bound to analyte by binding to another epitope on the analyte. This “sandwiches” the analyte between the capture antibody and the labelled antibody, thereby producing a visible line indicative of a positive response. If the test sample does not comprise any target analyte (or the target analyte is present in an amount which is below the limit of detection of the assay), a visible line will not appear on the test region. Thus, the signal intensity of the test line is proportional to the amount of analyte in the test sample. Any labelled antibody which has not bound to the target analyte may bind to the secondary antibodies immobilised in the control region. A detectable signal in the control region indicates that the labelled antibody has flowed correctly, regardless of the result of the test. In other words, the signal intensity of the control line is independent of the signal intensity of the test line, and serves only to indicate that the lateral flow test is viable.

[0010] Another type of lateral flow test is based on a “competitive” immunoassay. In competitive assays, the conjugate pad may comprise a labelled antibody which specifically binds to a target analyte in a test sample. The test line may comprise an immobilised capture molecule which mimics the target analyte such that it binds only to “free” labelled antibodies which are not bound to analyte. Therefore, in use, when target analyte is present in the test sample, the labelled antibody binds to the target analyte, and this, in turn, inhibits the binding of the labelled antibody to the immobilised capture molecule of the test line. When there is no target analyte in the test sample, the labelled antibody binds to the immobilised capture molecule, producing a detectable signal at the test region. Thus, the signal intensity of the test line is inversely proportional to the amount of analyte in the sample.

[0011] Fertility

[0012] Currently, patients undergo hormone testing during in-vitro fertilisation (IVF) and fertility treatments for many reasons. These include: assessing ovarian reserve before treatment, monitoring follicle growth during ovarian stimulation, monitoring ovulation status, determining the optimal time for triggering ovulation during treatment and monitoring the receptivity of the endometrium for embryo transfer.

[0013] Most fertility clinics require patients to attend the clinic to undergo ultrasound scans and blood hormone tests, and typically, blood samples are sent away to centralized

[0014] 14245329-1 laboratories for hormone analysis. Hormone results are returned by pathology services typically from 4 to 8 hours later, and sometimes as much as 24 hours later. Furthermore, pathology services operate within normal working hours and are often unavailable at weekends.

[0015] The slow turnaround of hormone results means that patients are not in the clinic when results are returned but may be required to the clinic in the evening if particular intervention is required. Additionally, clinicians are sometimes forced to make decisions on treatment without the hormone results being available which could subsequently require reversal when the hormone results do arrive.

[0016] The uncertainty surrounding the availability of hormone test results also results in the need to periodically check electronic medical records throughout the day or to call the pathology service to see when results become available. Both of these tasks are timeconsuming and result in significant inefficiencies for the clinic.

[0017] Currently, point-of-care hormone tests exist which can be carried out in the clinic. These tests may examine a single hormone with each test, and require approximately 100 pL of either serum or plasma, each necessitating venous blood collection of at least 0.5mL and a centrifugation step to extract the serum / plasma from whole blood. These procedures are not suitable for conducting outside of a clinic or laboratory setting. However, attending a clinic may be undesirable. For example, travelling to the clinic may be expensive, take time, and cause undue stress during fertility treatment.

[0018] Accordingly, there remains a need to provide an improved hormone test device which can be used to determine the amount of multiple hormones in a sample, particularly a blood sample, during fertility treatment. In particular, it would be desirable to provide a device that is suitable for use with a small amount of whole blood, removing the need for venipuncture, sample centrifugation and clinic visits. In particular, it would be desirable to detect different hormones simultaneously. The device should be easy to use, practical, and suitable for self-testing in a non-clinical setting.

[0019] The present invention seeks to achieve one or more of the above objectives.

[0020] 14245329-1 Summary of Invention

[0021] In a first aspect of the invention, there is provided a lateral flow test device for determining the amount of a plurality of hormones in a test sample comprising at least a first solid support structure, the first solid support structure comprising a sample-receiving region, a conjugate pad, and at least first, second and third test regions, wherein the first solid support structure is configured to permit liquid to flow sequentially from the samplereceiving region to each of the test regions via the conjugate pad, and wherein: i) the conjugate pad comprises a first labelled conjugate comprising an estradiol- binding moiety conjugated to a detectable label, a second labelled conjugate comprising a progesterone-binding moiety conjugated to a detectable label, and a third labelled conjugate comprising a luteinizing hormone (LH)-binding moiety conjugated to a detectable label, wherein each of the first, second and third labelled conjugates are capable of mobilisation on contact with the sample; ii) the first test region comprises immobilised estradiol or a derivative thereof which is capable of binding to the estradiol-binding moiety of the first labelled conjugate; iii) the second test region comprises immobilised progesterone or a derivative thereof which is capable of binding to the progesterone-binding moiety of the second labelled conjugate; iv) the third test region comprises an immobilised LH-binding element which is capable of binding to LH; and v) the detectable label of the first, second and third labelled conjugates is capable of producing a signal having a signal intensity in the first, second and third test regions, respectively, wherein the amount of estradiol, progesterone and LH in the test sample is determined based on the signal intensity in the first, second and third test regions, respectively.

[0022] Preferably, the estradiol-binding moiety of the first labelled conjugate comprises an antiestradiol antibody, the progesterone-binding moiety of the second labelled conjugate comprises an anti-progesterone antibody, and / or the LH-binding moiety of the third labelled conjugate comprises an anti- LH antibody.

[0023] 14245329-1 Advantageously, the detectable label of the first, second and third labelled conjugates is selected from: a fluorescent particle, a dye particle, a latex particle, a nanoparticle, a gold particle, and a platinum particle. Preferably, the detectable label comprises europium.

[0024] Conveniently, the first solid support structure comprises, in addition to the first test region, a first supplementary test region comprising immobilised estradiol or a derivative thereof which is capable of binding to the estradiol-binding moiety of the first labelled conjugate, wherein the first test region and the first supplementary test region are configured to detect different amounts of estradiol.

[0025] Optionally, the first test region is configured to detect a higher minimum level of estradiol than the first supplementary test region, and the solid support structure is configured to permit liquid to flow sequentially from the first test region to the first supplementary test region.

[0026] Preferably, the first test region is configured to detect estradiol in an amount of from 200- 8000 pg / mL, and the first supplementary test region is configured to detect estradiol in an amount of from 20-2000 pg / mL.

[0027] Conveniently, the first solid support structure comprises, in addition to the second test region, a second supplementary test region comprising immobilised progesterone or a derivative thereof which is capable of binding to the progesterone-binding moiety of the second labelled conjugate, wherein the second test region and the second supplementary test region are configured to detect different levels of progesterone.

[0028] Optionally, the second test region is configured to detect a higher minimum level of progesterone than the second supplementary test region, and the solid support structure is configured to permit liquid to flow sequentially from the second test region to the second supplementary test region.

[0029] Preferably, the second test region is configured to detect progesterone in an amount of from 3,000-60,000 pg / mL, and the second supplementary test region is configured to detect progesterone in an amount of from 50-3,000 pg / mL.

[0030] 14245329-1 In a second aspect of the invention, there is provided a lateral flow test device for determining the amount of each of a plurality of hormones in a test sample comprising at least a first solid support structure, the first solid support structure comprising a sample-receiving region, a conjugate pad, and at least first, second and third test regions, wherein the solid support structure is configured to permit liquid to flow sequentially from the sample-receiving region to the test regions via the conjugate pad, and wherein: i) the conjugate pad comprises a first labelled hormone conjugate comprising estradiol or a derivative thereof conjugated to a detectable label, a second labelled hormone conjugate comprising progesterone or a derivative thereof conjugated to a detectable label, and a third labelled conjugate comprising a luteinizing hormone (LH)- binding moiety conjugated to a detectable label, wherein each of the first labelled hormone conjugate, second labelled hormone conjugate and third labelled conjugate is capable of mobilisation on contact with the sample; ii) the first test region comprises an immobilised estradiol-binding element which is capable of binding to estradiol of the first labelled hormone conjugate and estradiol in the test sample; iii) the second test region comprises an immobilised progesterone-binding element which is capable of binding to progesterone of the second labelled hormone conjugate and progesterone in the test sample; iv) the third test region comprises an immobilised LH-binding element which is capable of binding to LH; and v) the detectable label of the first labelled hormone conjugate, second labelled hormone conjugate and the third labelled conjugate is capable of producing a signal having a signal intensity in the first, second and third test regions, respectively, wherein the amount of estradiol, progesterone and LH in the test sample is determined based on the signal intensity in the first, second and third test regions, respectively.

[0031] Advantageously, the estradiol-binding element comprises an anti-estradiol antibody, the progesterone-binding element comprises an anti-progesterone antibody, the LH-binding moiety comprises an anti- LH antibody, and / or the LH-binding element comprises an anti-LH antibody.

[0032] Preferably, the detectable label of the first and second labelled hormone conjugate, and the third labelled conjugate, is selected from: a fluorescent particle, a dye particle, a latex

[0033] 14245329-1 particle, a nanoparticle, a gold particle, and a platinum particle, preferably, wherein the detectable label comprises europium.

[0034] Advantageously, in the first and / or second aspects, the conjugate pad further comprises a primary antibody conjugated to a detectable label (primary antibody-label conjugate), wherein the primary antibody-label conjugate is capable of mobilisation on contact with the sample, and wherein the primary antibody does not bind to any of estradiol, progesterone, and LH; and wherein the lateral flow test device further comprises a control region, wherein the control region comprises an immobilised secondary antibody which is capable of binding to the primary antibody of the primary antibody-label conjugate, and wherein the detectable label of the primary antibody-label conjugate is capable of producing a detectable signal in the control region.

[0035] Alternatively, in the first and / or second aspects, the lateral flow test device further comprises a control region, wherein the control region comprises an immobilised secondary antibody which is capable of binding to one or more of the following: the first, second and third labelled conjugates, first labelled hormone conjugate, and second labelled hormone conjugate.

[0036] Preferably, the detectable label of the primary antibody-label conjugate is selected from: a fluorescent particle, a dye particle, a latex particle, a nanoparticle, a gold particle, and a platinum particle, preferably wherein the detectable label of the primary antibody-label conjugate comprises europium.

[0037] Conveniently, the lateral flow test device according to the first aspect comprises a second solid support structure, wherein the second solid support structure optionally comprises a supplementary sample-receiving region, and further comprises a supplementary conjugate pad, and at least first and second supplementary test regions, wherein the second solid support structure is configured to permit liquid to flow sequentially from the sample-receiving region or the supplementary sample-receiving region to the supplementary test regions via the supplementary conjugate pad, and wherein:

[0038] 14245329-1 i) the supplementary conjugate pad comprises a first labelled conjugate comprising an estradiol-binding moiety conjugated to a detectable label, and a second labelled conjugate comprising a progesterone-binding moiety conjugated to a detectable label, wherein each of the first and second labelled conjugates are capable of mobilisation on contact with the sample; ii) the first supplementary test region comprises immobilised estradiol or a derivative thereof which is capable of binding to the estradiol-binding moiety of the first labelled conjugate; iii) the second supplementary test region comprises immobilised progesterone or a derivative thereof which is capable of binding to the progesterone-binding moiety of the second labelled conjugate; iv) the detectable label of the first and second labelled conjugates is capable of producing a detectable signal having a signal intensity in the supplementary first and second supplementary test regions, respectively; v) the first test region and the first supplementary test region comprising immobilised estradiol are configured to detect different amounts of estradiol, vi) the second test region and the second supplementary test region comprising immobilised progesterone are configured to detect different amounts of progesterone; and wherein the amount of estradiol in the test sample is determined based on the signal intensity of the first test region and / or first supplementary test region; and wherein the amount of progesterone in the test sample is determined based on the signal intensity of the second test region and / or second supplementary test region.

[0039] Advantageously, the first and second labelled conjugates are as described above.

[0040] Conveniently, the lateral flow test device according to the second aspect comprises a second solid support structure, wherein the second solid support structure optionally comprises a supplementary sample-receiving region, and further comprises a supplementary conjugate pad, and at least first and second supplementary test regions, wherein the second solid support structure is configured to permit liquid to flow sequentially from the sample-receiving region or the supplementary sample-receiving region to the supplementary test regions via the supplementary conjugate pad, and wherein:

[0041] 14245329-1 i) the supplementary conjugate pad comprises a first labelled hormone conjugate comprising an estradiol or a derivative thereof conjugated to a detectable label, and a second labelled hormone conjugate comprising progesterone or a derivative thereof conjugated to a detectable label, wherein each of the first and second labelled hormone conjugates are capable of mobilisation on contact with the sample; ii) the first supplementary test region comprises an immobilised estradiol-binding element which is capable of binding to estradiol of the first labelled hormone conjugate or estradiol in the test sample; iii) the second supplementary test region comprises an immobilised progesterone-binding element which is capable of binding to progesterone of the second labelled conjugate or progesterone in the test sample; iv) the detectable label of the first and second labelled hormone conjugates is capable of producing a detectable signal having a signal intensity in the first and second supplementary regions, respectively; v) the first test region and the first supplementary test region are configured to detect different amounts of estradiol; vi) the second test region and the second supplementary test region are configured to detect different amounts of progesterone; wherein the amount of estradiol in the test sample is determined based on the signal intensity of the first test region and / or first supplementary test region; and wherein the amount of progesterone in the test sample is determined based on the signal intensity of the second test region and / or second supplementary test region.

[0042] Preferably, the first and second labelled hormone conjugates, the estradiol-binding element of the first supplementary test region, the progesterone-binding element of the second supplementary test region, and the detectable label of the first and second labelled hormone conjugates is as defined above in respect of the second aspect.

[0043] Advantageously, the conjugate pad of the second solid support structure comprises a primary antibody conjugated to a detectable label as defined in above, and the second solid support structure further comprises a control region as defined above.

[0044] Preferably, the capillary flow rate through the first solid support structure is different from the capillary flow rate through the second solid support structure.

[0045] 14245329-1 Advantageously, the lateral flow test device as described herein is configured to detect estradiol in an amount of from 20- 8,000 pg / mL, progesterone in an amount of from 50 - 60,000 pg / ml and LH in an amount of from 0.2-150 I ll / L.

[0046] Preferably, the lateral flow test device as described herein is suitable for determining the amount of a plurality of hormones in a blood sample, and more preferably, a whole blood sample.

[0047] In a third aspect of the invention, there is provided a method for determining the amount of a plurality of hormones in a test sample, the method comprising the following steps i) to iii): i) providing the lateral flow test device according to the first aspect, ii) applying the test sample to the sample-receiving region of the first solid support structure, and if present, the supplementary sample-receiving region of second solid support structure, and allowing the test sample to migrate to the conjugate pad, and if present, the supplementary conjugate pad, and mix with the first, second and third labelled conjugates, thereby enabling the labelled conjugates to become mobilised, iii) allowing the mobilised first, second and third labelled conjugates to migrate to the first, second and third test regions, and if present, the first and second supplementary test regions, to contact the respective immobilised molecules in each of the test regions, or wherein the method comprises the following steps iv) to vi): iv) providing the lateral flow test device according to the second aspect, v) applying the test sample to the sample-receiving region of the first solid support structure, and if present, to the supplementary sample-receiving region of the second solid support structure, and allowing the test sample to migrate to the conjugate pad, and if present, to the supplementary conjugate pad, and mix with the first and second labelled hormone conjugates, and third labelled conjugate, thereby enabling the labelled conjugates to become mobilised, vi) allowing the mobilised first and second labelled hormone conjugates and third labelled conjugate to migrate to the first, second and third test regions, and if present, the first and second supplementary test regions, to contact the respective immobilised molecules in each of the test regions; and vii) measuring the signal intensity at one or more of the first, second and third test regions, and if present, first and second supplementary test regions, and determining the amount of estradiol in the test sample based on the signal intensity at the first test region

[0048] 14245329-1 and / or first supplementary test region, determining the amount of progesterone in the test sample based on the signal intensity at the second test region and / or second supplementary test region, and determining the amount of LH in the test sample based on the signal intensity at the third test region.

[0049] Conveniently, step vii) is carried out using an optical signal reader.

[0050] Advantageously, step vii) is computer-implemented and comprises outputting an indication of the amount of estradiol, progesterone and LH in the test sample.

[0051] In the above method, preferably, the test sample comprises blood, and more preferably, whole blood.

[0052] In a fourth aspect of the present invention, there is provided a kit of parts comprising a lateral flow test device as described above, and an optical signal reader configured to measure the intensity of a signal in one or more of the first, second and third test regions, and if present, the first and second supplementary test regions.

[0053] 14245329-1 Brief Description of the Figures

[0054] Figure 1 is a schematic representation of a lateral flow test device according to one embodiment of the invention.

[0055] Figure 2 is a schematic diagram illustrating ranges of concentration of a hormone which may be detected by a test region and a supplementary test region.

[0056] Figure 3 is a flow chart illustrating the steps that may be taken to quantify the amount of a hormone in a test sample using a lateral flow test device in which the test region and supplementary test region are provided on different solid support structures.

[0057] Figure 4 is a schematic representation of a computer system for quantifying an amount of a hormone in a test sample based on signal intensity according to one embodiment of the invention.

[0058] Figure 5 is a schematic representation of computer system comprising multiple apparatuses for quantifying and recording an amount of a hormone in a test sample according to one embodiment of the invention.

[0059] Definitions

[0060] The term “amount”, as used herein, means a quantity of a particular entity, and may refer to the concentration of a particular entity (for example, within a test sample).

[0061] The term “immobilised”, as used herein, means that a molecule is retained or fixed on the relevant part of the device using any suitable means known to the person skilled in the art. The immobilised molecule is not released or mobilised upon contact with the test sample or by the flow of the test sample. In particular, an “immobilised” capture molecule is one which will normally be retained in the test region in use, and will hence bind to relevant complementary molecules contained within a liquid contacting the test region.

[0062] The term “mobilisable” or “capable of mobilisation”, as used herein, means that the relevant molecule is releasably retained or fixed on the relevant part of the device prior

[0063] 14245329-1 to use of the device, and is released from the part of the device upon contact with the test sample. A molecule which is capable of mobilisation can be carried away from the original point of retention / fixation by the lateral flow of the test sample.

[0064] Detailed Description

[0065] The invention generally relates to a lateral flow test device for determining the amount of a plurality of hormones in a test sample. The device comprises at least a first solid support structure, the first solid support structure comprising a sample-receiving region, a conjugate pad, and at least first, second and third test regions, wherein the first solid support structure is configured to permit liquid to flow sequentially from the samplereceiving region to each of the test regions via the conjugate pad. The conjugate pad comprises a first labelled conjugate comprising an estradiol-binding moiety conjugated to a detectable label, a second labelled conjugate comprising a progesterone-binding moiety conjugated to a detectable label, and a third labelled conjugate comprising a luteinizing hormone (LH)-binding moiety conjugated to a detectable label, wherein each of the first, second and third labelled conjugates are capable of mobilisation on contact with the sample. The first test region comprises immobilised estradiol or a derivative thereof which is capable of binding to the estradiol-binding moiety of the first labelled conjugate. The second test region comprises immobilised progesterone or a derivative thereof which is capable of binding to the progesterone-binding moiety of the second labelled conjugate. The third test region comprises an immobilised LH-binding element which is capable of binding to LH. The detectable label of the first, second and third labelled conjugates is capable of producing a signal having a signal intensity in the first, second and third test regions, respectively, wherein the level of estradiol, progesterone and LH in the test sample is determined based on the signal intensity in the first, second and third test regions, respectively.

[0066] Solid Support Structure

[0067] With reference to Figure 1 , the lateral flow test device (100) comprises at least one solid support structure (101), upon or within which the other components and reagents of the lateral flow test are placed. For example, the solid support structure (101) may comprise a backing card upon which are arranged the other components and reagents for the

[0068] 14245329-1 lateral flow test (referred to as a “test strip”). The backing card may then be contained within a housing. In some instances, the solid support structure may itself comprise a housing (for example, a test cartridge or cassette), within which are arranged the other components and reagents for the lateral flow test. The solid support structure (101) may be made of any suitable material known to the person skilled in the art, such as plastic. Preferably, the solid support structure (101) is arranged to allow at least a portion of the sample-receiving region and each test region to be visible (for example, by means of one or more apertures) to enable sample deposition and signal detection, respectively. In some embodiments, the solid support structure is configured to be incorporated into an optical signal reader, allowing optimal alignment with the reader for optical measurements.

[0069] The solid support structure (101) is configured to allow liquid (for example, a test sample, and any other liquid reagents or components added to or within the device) to flow sequentially from the sample-receiving region (102) to the conjugate pad (103), and subsequently to each of the test regions (105a, 105b, 105c), in turn. The first, second and third test regions (105a, 105b, 105c) may be provided in any order on the solid support structure (101). For example, the liquid may flow through the test regions (105a, 105b, 105c) (from the conjugate pad) in the following orders: first, second and third test regions; or second, third and first test regions; or third, first and second test regions; or third, second and first test regions, etc. It is advantageous to provide each of the test regions (105a, 105b, 105c) on a single support structure (for example, as a single test strip) to facilitate manufacturing of the device and to minimise any variation that could otherwise occur in sample application if each of the test regions were provided on separate structures. Additionally, such an arrangement is convenient for the user as it requires only a single finger-prick blood sample. Moreover, if one or two hormones out of estradiol, progesterone and LH need to be quantified at any given time, no selection of test strips or cartridges would be required, facilitating testing by the user.

[0070] The lateral flow test device is configured to allow the test sample to contact and mix with the first labelled conjugate, the second labelled conjugate and the third labelled conjugates of the conjugate pad, and subsequently, to contact the immobilised capture molecules of the test regions.

[0071] 14245329-1 In some embodiments of the present invention, the lateral flow test device comprises a second support structure which may generally be arranged as defined above. This is discussed in further detail below.

[0072] Sample-receiving region

[0073] With further reference to Figure 1 , the sample-receiving region (102) may be configured to receive a test sample which is preferably in liquid form. Preferably, the liquid sample is a blood sample, and more preferably, a whole blood sample. The sample-receiving region may also be configured to receive any other liquids, such as liquid reagents or buffers. In some embodiments, the sample is mixed with a liquid reagent or buffer prior to applying to the device. The liquid reagent or buffer may be selected to optimise binding between the hormone analyte and the mobilisable components of the conjugate pad.

[0074] In some embodiments, the sample-receiving region (102) comprises a sample pad. The sample pad may be made of any suitable material known to the person skilled in the art. For example, the sample pad may be composed of glass fibre, cellulose, or cotton. In preferred embodiments, the sample-receiving region is composed of a blend of cotton and rayon. The composition of the sample pad may be varied according to the test sample medium.

[0075] In some embodiments, the sample pad does not undergo any pre-treatment. In alternative embodiments, the sample pad is pre-treated with buffer components to enhance assay performance. The pre-treatment may serve to mitigate sample variability (pH, viscosity, protein concentration, salt concentration etc.), and to improve flow and reproducibility. Sample pad treatment can be performed by any suitable means known to the person skilled in the art, such as by spraying or immersion. The buffer components may be dried onto the sample pad, such that addition of a liquid, such as a liquid test sample, causes the buffer components to be released from the sample-receiving region and to mix with the test sample. Any suitable buffer components known to the person skilled in the art, such as salts, sucrose and glycerol, may be used. In some embodiments, the buffer comprises an anti-coagulant to prevent a blood sample clotting on application to the device.

[0076] 14245329-1 The sample pad may be a part of a single structure comprising the conjugate pad (103), and optionally, also the first, second and third test regions (105a, 105b, 105c), or more preferably, the sample pad may be a separate structure from each of the conjugate pad (103) and the first, second and third test regions (105a, 105b, 105c). In some embodiments, the sample pad may be a region of the conjugate pad, such that there is no distinction between the sample pad and the conjugate pad (i.e. the test sample is added to the conjugate pad).

[0077] Conjugate pad

[0078] In some embodiments, the conjugate pad (103) comprises a first labelled conjugate comprising an estradiol-binding moiety conjugated to a detectable label, a second labelled conjugate comprising a progesterone-binding moiety conjugated to a detectable label, and a third labelled conjugate comprising a luteinizing hormone (LH)-binding moiety conjugated to a detectable label.

[0079] The first, second and third labelled conjugates are capable of mobilisation on contact with the sample. The labelled conjugates are disposed on or within the conjugate pad (103) so that they are released by, and mix with, a test sample which contacts the conjugate pad. For example, the labelled conjugates may be deposited and dried onto the conjugate pad, so that they are released or mobilised by, and become mixed with, the test sample upon contact. The labelled conjugates may be deposited on the conjugate pad using an air jet dispenser. Alternatively, the conjugates may be deposited on the conjugate pad by immersing the pad into a solution comprising the conjugates followed by drying.

[0080] It may be necessary to pre-treat the conjugate pad before dispensing or depositing the labelled conjugates. The pre-treatment components may include a buffer for pH adjustment (for example, T ris buffer). Proteins, polymers and surfactants may also added to aid in the release of the labelled conjugate and flow of the assay. When running the assay, these components may move through the device faster than the conjugate and thus may help block protein-binding sites in the test regions prior to conjugate interaction. This reduces non-specific interactions.

[0081] 14245329-1 The conjugate pad may be composed of any suitable material known to the person skilled in the art. There are many materials commercially available for use as a conjugate pad from suppliers such as Millipore™ and Ahlstrom™. The material selected will determine the volume of conjugate that can be adsorbed, as well as the speed of release. Therefore, the selection can be made according to the type of sample to be tested. A preferred conjugate pad is composed of polyester.

[0082] In some embodiments, the conjugate pad (103) is a region of the same structure comprising the test regions (105a, 105b, 105c). Alternatively, it may be a separate structure from the test regions (105a, 105b, 105c). When the conjugate pad (103) is a separate structure from the test regions (105a, 105b, 105c), it may be attached to the structure comprising the test regions by any suitable means known to the person skilled in the art, for example, by means of a backing card.

[0083] Labelled conjugates

[0084] In some embodiments, the estradiol-binding moiety of the first labelled conjugate comprises an anti-estradiol aptamer. Aptamers are short, single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can selectively bind to a specific target, including proteins, peptides, carbohydrates, small molecules, toxins. In preferred embodiments, the estradiol-binding moiety comprises an anti-estradiol antibody. Similarly, the progesterone-binding moiety of the second labelled conjugate may comprise an antiprogesterone aptamer or preferably, an anti-progesterone antibody. The LH-binding moiety may comprise an anti-LH aptamer or preferably, an anti-LH antibody.

[0085] Each of the hormone-binding moieties (estradiol-binding moiety, progesterone-binding moiety and LH-binding moiety) are conjugated to a detectable label.

[0086] The detectable label of the first, second and third labelled conjugates is capable of producing a signal having a signal intensity in the first, second and third test regions, respectively, wherein the level of estradiol, progesterone and LH in the test sample is determined based on the signal intensity in the first, second and third test regions, respectively.

[0087] 14245329-1 The detectable labels may be any suitable molecule known to the person skilled in the art which produces a detectable signal, the intensity of which can be detected or measured. For example, the detectable label may be a dye particle, a carbon particle, a fluorescent label, a latex particle, a gold particle, a magnetic particle, a nanoparticle or the like. In some embodiments, the detectable label produces an optical signal, such as a fluorescent signal or a colour change. Preferably, the detectable label comprises a fluorescent label such as europium. In alternative embodiments, the detectable label comprises quantum dots (for example, cadmum mixed with selenium and coated with zinc sulphide), a DyLight dye or an Atto dye, typically comprising a rhodamine derivative. In some embodiments, the detectable label comprises a nanoparticle, preferably a gold or platinum nanoparticle.

[0088] The hormone-binding moiety, preferably anti-hormone antibody, may be conjugated to a detectable label using any suitable means known to the person skilled in the art. The detectable label continues to be detectable (that is to say, capable of emitting a signal that can be measured) whilst the hormone-binding moiety is conjugated thereto. In some embodiments, passive adsorption is used to conjugate a label such as a gold nanoparticle to the moiety. The mechanism of passive adsorption is based on Van der Waals forces, hydrophobic interactions, and ionic interactions between the hormone- binding moiety and the detectable label (for example, the surface of nanoparticles) to enable spontaneous coupling of the moiety to the label. The resulting interactions between the moiety and the label molecule may be influenced by the coupling environment. Thus, for example, the pH may be adjusted to optimise the efficiency of the adsorption. An excess of the hormone-binding moiety with respect to the label is generally typically used to ensure dense surface binding and high stability postconjugation.

[0089] In other embodiments, the hormone-binding moiety is conjugated to the detectable label through covalent attachment. Covalent attachment may be accomplished using routine methods. In some embodiments, the moiety is covalently conjugated to the detectable label via a linker. In other embodiments, the detectable label may be conjugated via a thiol group or amine group present on the antibody. The linker may be any suitable linker known to the person skilled in the art. For example, the linker may comprise biotin and / or BSA. In some embodiments, the linker is biotin-BSA.

[0090] 14245329-1 In preferred embodiments, the hormone-binding moiety may be conjugated to metal fluorophores such as europium via a metal chelator. Conjugation kits are commercially available.

[0091] Once the conjugation is complete, labelled conjugates may be re-suspended in an appropriate solution for deposition onto the conjugate pad, as described above.

[0092] Test Regions

[0093] As described above, and with further reference to Figure 1 , the lateral flow device (100) comprises at least first, second and third test regions (105a, 105b, 105c). A signal may be detected in each of these regions to determine the amount of a hormone in the test sample. Each of the test regions (105a, 105b, 105c) comprises an immobilised capture molecule which is capable of binding to the relevant labelled conjugate of the conjugate pad. In some embodiments, the first test region (105a) comprises immobilised estradiol or a derivative thereof which is capable of binding to the estradiol-binding moiety of the first labelled conjugate. The second test region (105b) may comprise immobilised progesterone or a derivative thereof which is capable of binding to the progesterone- binding moiety of the second labelled conjugate. The third test region (105c) may comprise an immobilised LH-binding element which is capable of binding to LH.

[0094] Examples of derivatives include but are not limited to Hapten-carrier conjugates such as Estradiol-BSA, Estradiol-Ovalbumin (OVA) and Estradiol-globulin, and corresponding progesterone derivatives.

[0095] In preferred embodiments, the first test region comprises immobilised estradiol, the second test line comprises immobilised progesterone, and the LH-binding element of the third test region comprises an anti-LH antibody.

[0096] In some embodiments, the first, second and third test regions (105a, 105b, 105c) may be different areas of a single structure or may each be separate structures. When the test regions are separate structures, the structures may be fixed to one another using any suitable means known to the person skilled in the art (for example, by means of a

[0097] 14245329-1 backing card) to allow liquid to flow from the conjugate to each of the test regions, in turn. The order of the test regions is not limited as described above.

[0098] In preferred embodiments, the test regions (105a, 105b, 105c) are separate areas of a single structure (104), which may be a permeable or semi-permeable membrane allowing liquid to flow on or through it. Preferably, first, second and third test regions (105a, 105b, 105c) are provided on separate areas of a single nitrocellulose membrane.

[0099] The test regions (105a, 105b, 105c) may be any suitable shape, such as a square, rectangle, circle, triangle, oval or line. Preferably, each test region is a line formed by immobilising the relevant capture molecule in a line. Preferably, each test region stretches across the entire width of the flow path of the liquid along / through the membrane (i.e. perpendicular to the direction of liquid flow). This provides the advantage that the labelled conjugate which is bound to the hormone cannot circumvent the relevant test region before migrating further along the device, thereby ensuring an accurate detection of the hormones.

[0100] Competitive and Sandwich Style Assays

[0101] The present invention is based on a combination of sandwich and competitive assays. A competitive assay is used for estradiol and progesterone detection whilst a sandwich assay is used for LH detection. Estradiol and progesterone are small molecules and thus may not allow simultaneous binding of multiple molecules (for example, a labelled antibody and immobilised antibody) which would be required in a sandwich assay.

[0102] In use, when the test sample comes into contact with the conjugate pad, the first labelled conjugate comprising an estradiol-binding moiety conjugated to a detectable label, becomes mobilised and migrates towards the test regions. Any estradiol in the sample will bind to the mobilised first labelled conjugate. Once bound to estradiol originating from the sample, the first labelled conjugate is unable to bind to estradiol (or a derivative thereof) which is immobilised in the first test region. This prevents the detectable label of the first labelled conjugate from producing a detectable signal at the first test region. Thus, the intensity of the signal that is observed in the first test region is inversely proportional to the amount of estradiol in the test sample.

[0103] 14245329-1 Similarly, when the test sample comes into contact with the conjugate pad, the second labelled conjugate comprising a progesterone-binding moiety conjugated to a detectable label, becomes mobilised and migrates towards the test regions. Any progesterone in the sample will bind to the second labelled conjugate. Once bound to progesterone originating from the sample, the first labelled conjugate is unable to bind to progesterone (or a derivative thereof) which is immobilised in the second test region. This prevents the detectable label of the first labelled conjugate from producing a detectable signal in the second test region. Thus, the intensity of the signal that is observed in the second test region is inversely proportional to the amount of progesterone in the test sample.

[0104] The third labelled conjugate comprising a LH-binding element conjugated to a detectable label becomes mobilised on contact with the test sample and binds to any LH present in the sample. The complex of the LH-binding moiety and LH migrates to the third test region where the immobilised LH-binding element binds to the LH in the complex, and immobilises the complex in a sandwich-style format. Consequently, a detectable signal is observed in the third test region, confirming the presence of the LH in the test sample. In this sandwich assay, the signal intensity on the test region is proportional to the amount of LH present in the sample.

[0105] Control

[0106] In some embodiments of the present invention, the conjugate pad (103) further comprise a primary antibody conjugated to a detectable label (primary antibody-label conjugate), wherein the primary antibody-label conjugate is capable of mobilisation on contact with the sample, and wherein the primary antibody does not bind to any of estradiol, progesterone, and LH. In these embodiments, the first support structure further comprises a control region (105d), wherein the control region comprises an immobilised secondary antibody which is capable of binding to the primary antibody of the primary antibody-label conjugate, and wherein the detectable label of the primary antibody-label conjugate is capable of producing a detectable signal in the control region. The secondary antibody is preferably specific to the species from which the primary antibody originates. Thus, if the primary antibody is a mouse antibody, the secondary antibody is preferably an anti-mouse antibody.

[0107] 14245329-1 A signal will be detectable in the control region regardless of whether or not there is hormone in the sample. The primary antibody-label conjugate in the conjugate pad and secondary antibody in the control region may collectively serve as a control for the assay which demonstrates that the assay is functional and that the results are valid.

[0108] The detectable label of the primary antibody-label conjugate may be as defined above with respect to the labelled conjugates of the conjugate pad.

[0109] In other embodiments, the control region comprises an immobilised secondary antibody which is capable of binding to one or more of the first, second and third labelled conjugates. Excess, unbound labelled conjugates which do not bind to the test regions are captured by the immobilised secondary antibody. Thus, for example, if the estradiol- binding moiety, progesterone-binding moiety, and / or LH-binding moiety comprises a mouse anti-estradiol antibody, then the immobilised secondary antibody may be an antimouse antibody.

[0110] The lateral flow test device may be configured to detect estradiol in an amount of from 20 to 8,000 pg / ml, 40-7,000 pg / mL, or 40-6,000 pg / mL. The lateral flow test device may be configured to detect progesterone in an amount of from 50-60,000 pg / mL, 100-50,000 pg / mL or 100-40,000 pg / mL. The lateral flow test device may be configured to detect LH in an amount of from 0.2-150 lll / L, or 1-150 lll / L.

[0111] Dual-range assays - “single-strip solution"

[0112] In some embodiments, the solid support structure comprises, in addition to the first test region, a first supplementary test region comprising immobilised estradiol or a derivative thereof which is capable of binding to the estradiol-binding moiety of the first labelled conjugate. The first test region and the first supplementary test region may be configured to detect different concentrations of estradiol. In preferred embodiments, the first test region is configured to detect a higher minimum level of estradiol than the first supplementary test region, and the solid support structure is configured to permit liquid to flow sequentially from the first test region to the first supplementary test region. In other words, the first supplementary test region, which is positioned further away from the conjugate pad than the first test region, has a greater sensitivity to estradiol detection.

[0113] 14245329-1 Without wishing to be bound by theory, in the above embodiment, when the concentration of estradiol is low in a test sample, there is a high concentration of free labelled antibody molecules (i.e. those which have not bound to estradiol) reaching the first test region. Only a fraction of these molecules may bind to first test region (for example, due to a limited amount of immobilised antigen). Thus, a portion of the free labelled antibody molecules become depleted at the first test region. As such, there remains an excess amount of free labelled antibody molecules that are available to bind to the first supplementary test region whilst the amount of labelled antibody bound to the sample antigen ([total labelled antibody] - [antigen in sample]) remains the same. Accordingly, the ratio of the amount of bound antibody molecules to the amount of labelled antibody molecules reaching, and available to bind to, the first supplementary test region increases as the sample flows through the device.

[0114] In the above embodiment, when the concentration of estradiol is high in a test sample, there is a low concentration of free labelled antibody molecules (i.e. those which have not bound to estradiol) reaching the first test region. As such, an excess amount of immobilised antigen at the first test region may deplete a large portion of the free labelled antibody molecules, and there remains only a small excess of, or a negligible amount of labelled antibody molecules that are available to bind to the first supplementary test region.

[0115] Therefore, it is evident that the sensitivity of detection of the first supplementary test region is greater than the first test region due to the depletion of the labelled antibody after passing through the first test region.

[0116] Similarly, in some embodiments, the support structure comprises, in addition to the second test region, a second supplementary test region comprising immobilised progesterone or a derivative thereof which is capable of binding to the progesterone- binding moiety of the second labelled conjugate. The second test region and the second supplementary test region are configured to detect different concentrations of progesterone. In preferred embodiments, the second test region is configured to detect a higher minimum level of progesterone than the second supplementary test region, and the solid support structure is configured to permit liquid to flow sequentially from the second test region to the second supplementary test region. In other words, the second supplementary test region, which is positioned further away from the conjugate pad than

[0117] 14245329-1 the second test region, has a greater sensitivity to progesterone detection. The above considerations for estradiol apply by analogy.

[0118] The amount of estradiol in the test sample may be determined based on the signal intensity of the first test region and / or first supplementary test region. The amount of progesterone in the test sample may be determined based on the signal intensity of the second test region and / or second supplementary test region.

[0119] The first test region may be configured to detect estradiol in an amount of 200-8,000 pg / mL, 400-8,000 pg / mL, 300-8000 pg / mL, pg / mL, or 300-4000 pg / mL. The first supplementary test region may be configured to detect estradiol in an amount of 20-300 pg / mL, 20-400 pg / mL, or 40-400 pg / mL.

[0120] In some embodiments, the first test region and the first supplementary test region may be configured to detect estradiol in the amounts shown in Table 1 below:

[0121] Table 1 - exemplary concentration ranges of estradiol

[0122] The second test region may be configured to detect progesterone in an amount of 1 ,000- 60,000 pg / mL, 800-60,000 pg / mL, or 1 ,000-40,000, 3,000-60,000. The second supplementary test region may be configured to detect progesterone in an amount of 50- 2,000 pg / mL, 50-1 ,000 pg / mL, 100-2000 pg / mL, or 50-3000 pg / mL.

[0123] In some embodiments, the second test region and the second supplementary test region may be configured to detect progesterone in the amounts shown in Table 2 below:

[0124] 14245329-1 Table 2 - exemplary concentration ranges of progesterone

[0125] It can be difficult to optimise the same level of precision in lateral flow test assays at the low and high end of a large range of concentrations of hormone, particularly given the increased complexity of measurement in whole blood. Taking estradiol as an example, the clinical range of interest in blood is, approximately, from 20-8,000 pg / mL (quantitation range of 400x), where clinical decisions at made at both the low end (above or below 55pg / mL) and the high end (above or below 2,000 pg / mL). It may also be necessary to determine whether estradiol levels are dangerously elevated (>2,700 pg / mL). Similar clinical decisions are made on progesterone across a large range of values. It is therefore advantageous to provide two or more test regions which are configured to detect different concentration ranges of estradiol and progesterone as described above to ensure precise and accurate measurements.

[0126] The above configurations of the test regions and supplementary test regions may be achieved by routine modification of design parameters including the amount of antigen immobilised in the test regions and supplementary test regions, amount of labelled conjugate deposited onto the conjugate pad and supplementary conjugate pad, and the properties of the hormone-binding moieties (preferably, antibodies) that are selected.

[0127] Dual range assays (“two-strip solution")

[0128] In an alternative embodiment, the first and second supplementary test regions described above may be provided on a separate (second) support structure. Thus, further provided is a lateral flow test device further comprising a second solid support structure, wherein the second solid support structure comprises an optional supplementary sample-

[0129] 14245329-1 receiving region, a supplementary conjugate pad, and at least first and second supplementary test regions, wherein the second solid support structure is configured to permit liquid to flow sequentially from the sample-receiving region or the supplementary sample-receiving region to the supplementary test regions via the supplementary conjugate pad.

[0130] In this alternative embodiment, the supplementary conjugate pad comprises a first labelled conjugate comprising an estradiol-binding moiety conjugated to a detectable label, and a second labelled conjugate comprising a progesterone-binding moiety conjugated to a detectable label, wherein each of the first and second labelled conjugates are capable of mobilisation on contact with the sample. The estradiol-binding moiety, progesterone-binding moiety and detectable label may be as described above.

[0131] The first supplementary test region and second supplementary test region may be as described above. Analogously to the “single-strip solution”, the first test region (of the first solid support structure) and the first supplementary test region (of the second solid support structure) are preferably configured to detect different amounts of estradiol. The second test region (of the first solid support structure) and the second supplementary test region (of the second solid support structure) comprising immobilised progesterone are preferably configured to detect different amounts of progesterone. The amount of estradiol in the test sample may be determined based on the signal intensity of the first test region and / or first supplementary test region. The amount of progesterone in the test sample may be determined based on the signal intensity of the second test region and / or second supplementary test region. In the case of estradiol detection, either of the first test region and first supplementary test region may be configured to detect the lower concentration range (i.e. having a lower minimum concentration of detection) of estradiol. In the case of progesterone detection, either of the second test region and second supplementary test region may be configured to detect the lower concentration range of progesterone. The “two-strip solution” may be preferred over the “single-strip solution” if the test regions and supplementary test regions cannot be positioned with appropriate or sufficient spacing on a single support structure.

[0132] In some embodiments, the capillary flow rate through the first solid support structure is different from the capillary flow rate through the second solid support structure. Reducing the capillary flow rate is known to increase the sensitivity of the device as the analyte

[0133] 14245329-1 hormone molecules are present within the test region for a longer period of time during the flow. Therefore, preferably, the capillary flow rate is reduced through the solid support structure comprising the test regions which are configured to detect lower concentration ranges of estradiol or progesterone. This would lead to an earlier signal detection in the solid support structure comprising the test regions which are configured to detect higher concentration ranges of estradiol or progesterone.

[0134] In preferred embodiments, the first test region on the first support structure (which also comprises a third test region configured to detect LH) is configured to detect a higher minimum concentration of estradiol than the first supplementary test region on the second support structure. Similarly, in preferred embodiments, the second test region on the first support structure is configured to detect a higher minimum concentration of progesterone than the second supplementary test region on the second support structure. In these embodiments, the capillary flow rate of the second support structure is preferably lower than the capillary flow rate of the first support structure to provide increased sensitivity for the lower range of detection. This also ensures that a detectable signal appears in the third test region (configured to detect LH) at a faster rate than would be achieved if the capillary flow rate were lower in the first support structure.

[0135] The capillary flow rate through the test regions may be altered by routine methods known to the skilled person in the art. These include, but are not limited to, the selection of membrane properties such as pore size, pore size distribution, porosity and viscosity.

[0136] Table 3 below illustrates exemplary capillary flow rates for various High-Flow™ Plus nitrocellulose membranes which may be used in accordance with the invention.

[0137] 14245329-1 Table 3 (adapted from Merck Millipore)

[0138] The membrane comprising the test regions which are configured to detect lower concentration ranges of estradiol or progesterone may have a capillary flow rate of more than 120 seconds / 4cm (assessed using water). The membrane comprising the test regions which are configured to detect higher concentration ranges of estradiol or progesterone may have a capillary flow rate of less than 120 seconds / 4cm (assessed using water).

[0139] With reference to Figure 2, in some embodiments, the lateral flow test device enables measurement of a signal at the first and second test regions (205) which are configured to detect a higher concentration range of estradiol or progesterone (range C+D) before the supplementary test regions (225) which are configured to detect a lower concentration range of estradiol or progesterone (range B+C). If the first reading of the first or second test region (205) (Rhi) is greater than the upper limit of quantitation (IILOQ) of the corresponding supplementary test region (ULOQio) (range D), the result can be reported immediately reducing the time to result. If the reading of the first or second test region (Rhi) is below the IILOQ of the corresponding supplementary test region (ULOQio), then it is necessary to wait for the supplementary test regions to produce a signal. If the reading of the supplementary test region (R|O) and test region (Rhi) falls between the lower limit of quantitation (LLOQhi) of the test region and the ULOQ of the supplementary test region (ULOQio) (for example, between 200 and 2000 pg / mL for estradiol according to the examples above) (range C) then a mean average of Rhi and R|Ocan be taken and the

[0140] 14245329-1 result reported. If the reading of the supplementary test region(Rio) is below the LLOQ of the test region (LLOQhi) (range B), then the measurement from the supplementary test region (R|O) is reported. If the reading of the first or second test region (205) (Rhi) is greater than the upper limit of quantitation of the test region (ULOQhi) (range E), then the reading is out of range and the concentration of hormone cannot be accurately quantified. Similarly, if the reading of the first or second supplementary test region (225) (Rio) is lower than the lower limit of quantitation of the supplementary test region (ULOQio) (range A), then the reading is out of range and the concentration of hormone cannot be accurately quantified.

[0141] The above method of quantification may be applied to both the “single strip” solution in which the test regions and supplementary test regions are provided on the same solid support structure, and the alternative “two-strip solution” in which the test regions are provided on a first solid support structure, and the supplementary test regions are provided on a second solid support structure. Figure 3 illustrates the sequence of steps which may be taken to quantify estradiol or progesterone in a test sample when the lateral flow test device comprises test regions on a first solid support structure, and supplementary test regions on a second solid support structure, wherein the test regions and supplementary test regions are configured to detect different concentration ranges of hormone in accordance with the above.

[0142] It is to be noted that, in some embodiments, the test regions and corresponding supplementary test regions may be configured to detect ranges of concentration of estradiol or progesterone which are non-overlapping. In this case, with reference to Figure 2, region C would be absent.

[0143] As mentioned above, in some embodiments of the “two-strip solution”, the supplementary test regions, as provided on the second support structure, may be configured to detect a higher concentration range of the hormone whilst the test regions, as provided on the first support structure, may be configured to detect the lower concentration range of hormone. (As described above, the first solid support structure may also comprise a third test region comprising an immobilised LH-binding element.) Nevertheless, the above description of quantifying the hormone concentration applies by analogy.

[0144] 14245329-1 Typically, it is not necessary or useful to provide a supplementary test region which is configured to detect an amount of LH which is different from the third test region. This is because the concentration of LH in blood does not normally vary as much as estradiol and progesterone. Therefore, a single test region may be sufficient to quantify LH with precision and accuracy across the entire range of concentrations that would be observed in users. As such, in some embodiments, the lateral flow test device comprises only a single test region configured to detect LH.

[0145] Further arrangements

[0146] In a further embodiment of the present invention, the components of the lateral flow test device may be arranged differently. In this embodiment, the conjugate pad comprises a first labelled hormone conjugate comprising an estradiol or a derivative thereof conjugated to a detectable label, a second labelled hormone conjugate comprising progesterone or a derivative thereof conjugated to a detectable label, and a third labelled conjugate comprising a LH-binding moiety conjugated to a detectable label. Each of the first labelled hormone conjugate, second labelled hormone conjugate and third labelled conjugate is capable of mobilisation on contact with the sample. In this embodiment, the first test region comprises an immobilised estradiol-binding element which is capable of binding to estradiol of the first labelled hormone conjugate and estradiol in the test sample. The second test region comprises an immobilised progesterone-binding element which is capable of binding to progesterone of the second labelled hormone conjugate and progesterone in the test sample. The third test region comprises an immobilised LH-binding element which is capable of binding to LH. The detectable label of the first labelled hormone conjugate, second labelled hormone conjugate and the third labelled conjugate is capable of producing a signal having a signal intensity in the first, second and third test regions, respectively, wherein the amount of estradiol, progesterone and LH in the test sample is determined based on the signal intensity in the first, second and third test regions, respectively.

[0147] The estradiol derivative, progesterone derivative, and LH- binding moiety of the conjugate pad may, and detectable label, may be as described above. The estradiol- binding element, progesterone binding element, and LH-binding element of the test regions, may also be as described above.

[0148] 14245329-1 In use, when the test sample comes into contact with the conjugate pad, the labelled estradiol conjugate will become mobilised from the conjugate. Subsequently, the estradiol conjugate will migrate to the first test region, together with any estradiol in the sample. The labelled estradiol conjugate will compete with estradiol analyte in the sample for binding to the immobilised estradiol-binding element (preferably an antiestradiol antibody) in the first test region. The higher the concentration of estradiol in the test sample, the greater the competition with the labelled estradiol conjugate, and the less labelled estradiol conjugate available to binding to the immobilised estadiol-binding element. Thus, the intensity of the signal that is observed at the first test region is inversely proportional to the amount of estradiol in the test sample.

[0149] Similarly, when the test sample comes into contact with the conjugate pad, the labelled progesterone conjugate will become mobilised from the conjugate. Subsequently, the progesterone conjugate will migrate to the second test region, together with any progesterone in the sample. The labelled progesterone conjugate will compete with progesterone analyte in the sample for binding to the immobilised progesterone-binding element (preferably an anti-progesterone antibody) in the second test region. The higher the concentration of progesterone in the test sample, the greater the competition with the labelled progesterone conjugate, and the less labelled progesterone conjugate available to binding to the immobilised progesterone -binding element. Thus, the intensity of the signal that is observed at the second test region is inversely proportional to the amount of progesterone in the test sample.

[0150] Other aspects of the use of the lateral flow test device relating to the third labelled conjugate may be as described above.

[0151] In this further embodiment, the lateral flow test device may comprise a second solid support structure comprising first and second supplementary test regions. Specifically, the second solid support structure may optionally comprise a supplementary samplereceiving region, and may further comprise a supplementary conjugate pad, and at least first and second supplementary test regions. Preferably, the second solid support structure is configured to permit liquid to flow sequentially from the sample-receiving region or the supplementary sample-receiving region to the supplementary test regions via the supplementary conjugate pad. In this embodiment, the supplementary conjugate pad comprises a first labelled hormone conjugate comprising an estradiol or a derivative

[0152] 14245329-1 thereof conjugated to a detectable label, and a second labelled hormone conjugate comprising progesterone or a derivative thereof conjugated to a detectable label, wherein each of the first and second labelled hormone conjugates are capable of mobilisation on contact with the sample. The first supplementary test region may comprise an immobilised estradiol-binding element and the second supplementary test region may comprise an immobilised progesterone-binding element as provided in the first solid structure. Preferably, the first test region and the first supplementary test region are configured to detect different concentrations of estradiol, and the second test region and the second supplementary test region are configured to detect different concentrations of progesterone. In accordance with the methods described above which apply by analogy, the amount of estradiol in the test sample may be determined based on the signal intensity of the first test region and / or first supplementary test region, and the amount of progesterone in the test sample may be determined based on the signal intensity of the second test region and / or second supplementary test region.

[0153] Other arrangements of the lateral flow test device are also envisaged. For example, each of the first, second and third test regions may be provided on separate solid support structures. In this arrangement, the first test region and first supplementary test region (each configured to detect estradiol as described above) may be provided on a first solid support structure, the second test region and second supplementary test region (each configured to detect progesterone as described above) may be provided on a second solid support structure, and the third test region (configured to detect LH) may be provided on a third solid support structure. Each of the solid support structures (for example, test strips) may be incorporated into a single housing for ease of handling.

[0154] In some embodiments, the lateral flow test device of the invention may include additional supplementary test regions which are configured to detect additional concentration ranges of estradiol or progesterone. The lateral flow test device may also include additional test regions which are configured to detect one or more other hormones selected from follicle-stimulating hormone, anti-mullerian hormone, prolactin and beta- hCG. The additional test regions or supplementary test regions may be provided on the first and second solid support structures as described above, or on one or more additional support structures. In these embodiments, the conjugate pad(s) may be adapted accordingly.

[0155] 14245329-1 Immobilisation of molecules to the test regions

[0156] The capture molecules of the test regions (namely, estradiol or a derivative thereof, progesterone or a derivative thereof, estradiol-binding element, progesterone-binding element and LH-binding element in the embodiments described above) and secondary antibody of the control region, may be immobilised onto an appropriate membrane (preferably, a nitrocellulose membrane) by routine methods known to a person skilled in the art. For example, striping of test and control lines onto a nitrocellulose membrane may be accomplished with the use of a dispensing instrument. Such instruments are commercially available (for example, Kinematic™, Biodot™, and Imagene™) which may use contact or non-contact dispensing. Handling of the membrane prior to and after striping is important for the performance. For example, nitrocellulose membranes that are too dry may result in spotty, non-uniform lines, whilst nitrocellulose membranes that are too damp may result in a widened test line that results in a decreased signal intensity. Therefore, the nitrocellulose membrane is preferably equilibrated to a controlled humidity environment of suitably humidity level. After striping, the capture molecule or secondary antibody is fixed to the membrane by standard drying and curing protocols. Once the nitrocellulose has been striped and dried, it is important that the membranes are stored in a moisture-free environment. Dried nitrocellulose will readily absorb any moisture in the environment, and this can result in proteins detaching from the striped lines. Accordingly, the lateral flow test device is preferably stored in a sealed pouch with a desiccant prior to use.

[0157] Absorbent Region

[0158] With reference to Figure 1 , in some embodiments, the lateral flow test device comprises an absorbent region (6), which may be an absorbent pad. The absorbent region (6) absorbs excess sample containing reagents that have not been bound or immobilised in the assay, whilst maintaining capillary flow through the test regions to clear the background and optimise signal intensity. In a preferred embodiment, an absorbent pad is composed of cotton or cellulose.

[0159] Further Components

[0160] 14245329-1 The lateral flow test device of the present invention may include further components for the optimisation of the lateral flow test device, in line with conventional practice in the art. These additional components may be any suitable components known in the art for use with lateral flow tests or ELISA assays. For example, the lateral flow test device may comprise a blocking agent for reducing non-specific binding of hormone-labelled conjugate and reducing background signals. The blocking agent may be any suitable blocking agent known in the art, such as a blocking agent comprising BSA or PBS.

[0161] In preferred embodiments, the lateral flow test device is suitable for determining the amount of estradiol, progesterone and / or LH in a blood sample, optionally, a whole blood sample. The lateral flow test device may be configured specifically to receive a whole blood sample. For example, whole blood contains a variety of cells which may obstruct the flow of sample through the device. Thus, in some embodiments, the lateral flow test device may comprise a filter such that blood cells are trapped by the filter before the sample enters the conjugate pad. In other embodiments, the lateral flow test device may comprise an anticoagulant to prevent the blood from clotting.

[0162] Signal detection

[0163] The signal produced by the detectable label of each of the labelled conjugates at each of the test regions, and in the control region, may be detected by any suitable means for the type of signal to be detected. In some embodiments, the detectable label produces an optical signal (for example, a fluorescent signal) and the signal is detected, and the intensity thereof measured, using an optical reader. For example, an optical signal may be detected, and the intensity thereof measured, using a smartphone (e.g. using the camera of a smartphone). Alternatively, the optical signal may be detected, and the intensity thereof measured, using a reader which comprises a lateral low test device holder configured to receive the lateral flow test device for measurement. The holder may include one or more apertures configured to enclose the lateral flow test device (e.g. to block external light) whilst allowing the test regions to be exposed so that the signal in the test regions can be detected by the reader. The reader may include a light source to illuminate the lateral flow test device during measurement. Thus, in the case of a fluorescent label, the lateral flow test device may be illuminated with a first wavelength(s) with a first light source, and the light emitted at a second wavelength(s) may be measured

[0164] 14245329-1 using one or more photodetectors or a camera. Further discussion of the means of signal detection is provided below with reference to Figure 4.

[0165] The detection of the signal and measurement of the intensity thereof in each test region may be performed at a minimum time after the test sample has been applied to the sample-receiving region. It will be understood that the detection of the signal intensities can be performed using the test regions themselves (i.e. direct detection from the detection region) or using a reproduction, such as a photograph, of each detection region. In particular, sufficient time must elapse before the detection of the signal is performed in order to permit the test sample to migrate to the conjugate pad, and then to the test regions. The detection of the signal in each test region may be performed after any suitable minimum time from the point of applying the sample to the sample-receiving region (and if present, supplementary sample-receiving region), for example, after at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes or at least 30 minutes. Preferably, the detection of the signal and optional measurement of the intensity thereof is performed at least 15 minutes, for example 15 to 20 minutes, after the test sample has been applied to the sample-receiving region.

[0166] The detection of the signal and measurement of the intensity thereof in each test region may be based on the signal present in the test region before a maximum time has elapsed from the application of the test sample to the sample-receiving region. For example, the detection of the signal and intensity thereof may be based on the signal present at the detection region(s) no more than 1 hour, no more than 50 minutes, no more than 45 minutes, no more than 40 minutes, no more than 35 minutes or no more than 30 minutes after the test sample has been applied to the sample-receiving region. In some embodiments, the detection of the signal and intensity thereof may be based on the signal present in each detection region no more than one hour after the test sample has been applied to the sample-receiving region.

[0167] The quantitation of hormone molecules in the test sample is carried out by measuring the signal intensity in the test regions. This may be achieved by obtaining one or more photographs of the test regions. The location of each test region within each of the one or more photographs may be obtained through user input or using an image recognition method, such as edge detection and / or machine learning. The signal intensity of each

[0168] 14245329-1 detection region may be determined based on the intensity of one or more pixels representing the test regions and supplementary test regions. This may be based on one pixel, or may be an accumulation of intensity values of multiple pixels (e.g. an average intensity). Typically, signal intensities are compared to a calibration standard and converted to a hormone concentration value for estradiol, progesterone and LH. Relevant calibration data may be encoded into the appropriate software. In some embodiments, the concentration of hormone is determined based on the ratio of signal intensity at the test regions to the signal intensity at the control region. This is to ensure that any differences in the performance between devices used for detection of the hormone (for example, smart phone, camera etc.) do not result in variations in the quantification of the hormone.

[0169] Computer system

[0170] Figure 4 shows a schematic representation of a computing system (490) for quantifying the amount of estradiol, progesterone and / or LH in a test sample based on the signal intensities at the relevant tests regions, and optionally, the control region, according to an embodiment of the invention.

[0171] The computing system (490), which may be an integrated apparatus or distributed system, comprises a processor (410), volatile memory (420) (e.g RAM such as DRAM), non-volatile memory (430) (e.g. ROM such as Flash memory) and at least one input and / or output (I / O) interface (440), for example at least one user interface (e.g. a display, touch screen or keyboard) and / or a communications interface such as to communicate via Bluetooth (RTM or via the internet). The computing system (490) is controlled by the processor (410). A bus (410a) may connect the processor to the memory and other components of the computing system such as I / O interface(s). The processor (410) is configured to quantify the level of estradiol, progesterone and LH in a test sample using executable code for processing the data from an optical reader (450). The code may be stored in the non-volatile memory (430) and executed using the non-volatile memory (430).

[0172] The computing system may be a dedicated single apparatus, e.g. a single user mobile device or computer. Alternatively, referring to Figure 5, the computing system (500) may comprise multiple apparatuses, such as computer(s) (590), an optical reader device

[0173] 14245329-1 (550), server(s) and / or mobile device(s) (570) such as a smartphone, tablet, PDA, etc.. Such server(s) may comprise cloud server(s) and / or may be within backend infrastructure (595), which is optionally connected via a cloud network. Such a cloud network may comprise cloud storage (580).

[0174] The computing system (490) may comprise or connect to an optical reader (450) having e.g. a digital camera. The reader, which when provided separately for connection to the system may be referred to as a reader device, is for obtaining an image of the test regions, supplementary test regions (if present) and optionally, the control region, and generating or providing image data - and / or measurement data based on the image - to the computing system.

[0175] The computing system (490) may connect to a clinician computer system or device by a said I / O interface (440).

[0176] The amount of the hormones in a test sample is determined based on one or more measurements of signal intensity at the test regions (and supplementary test regions, if present) and optionally, the control region. These measurements may be obtained, by the computing system using the reader, from one or more images (e.g. photograph(s)) of the lateral flow test device showing each of the test regions. As mentioned above, the optical reader may be integrated within or external to the computing system. That is, the computing system may be configured to obtain one or images of the lateral flow test device (or data derived from such image(s)) through an integrated optical reader, or may be configured to obtain such image(s) or data from an external reader device. The transfer of data from such an external reader device may be over a network, such as the internet and / or a Bluetooth (RTM) link, e.g. using BT or BTLE. It may be preferred that the reader communicates via Bluetooth (RTM) with a user mobile device of the computing system.

[0177] Where the user of the computing system makes use of the reader preferably in the form of a mobile device (e.g. smartphone or tablet) to obtain an image of the lateral flow test device, the mobile device and / or other part(s) of the computing system may be operable, through firmware and / or software, to perform the steps described herein to quantify the concentration of a hormone in the test sample. In some embodiments, each image generated by the reader - or representative data and / or measurements based on the

[0178] 14245329-1 image - may be transferred to another part of the computing system to perform the quantification, optionally wherein that part is remote from the reader, e.g. is within or connected via a cloud network.

[0179] The quantified amount of hormone determined by the processor may be output via an I / O interface (e.g. a display, or a network interface to an external system; optionally via the internet). The amount may be utilised by the processor for further calculations, and / or may be stored in non-volatile memory for later use. The quantified concentration is preferably transferred to a clinician computer optionally via a cloud network.

[0180] Appropriate computing software, for example on a user’s mobile device, may guide the user (for example, a patient) through the steps of taking the sample (for example, a blood sample) and using the lateral flow test device. The clinician may have access to data through a software portal, which also may be accessed through an interface to the computing system, e.g. via a cloud network. The portal may allow the clinician to view and manage quantification results, image data and / or patient information, and / or results of diagnostic analysis steps based thereon. The clinician may also use the portal as a means of communication between the clinician and the patient.

[0181] The computing system may also be configured to handle identification and / or security of the hormone data results, such that only those with permission may access the data. It is further noted that the computing system may have an application programming interface (API) for integration with another application, for example an application that provides electronic medical records of a hospital or surgery.

[0182] Method of use

[0183] In another aspect of the invention, there is provided is a method for determining the amount of a plurality of hormones in a test sample. The method may comprise the following steps i) to iii): i) providing a lateral flow test device as described above; ii) applying the test sample to the sample-receiving region of the first solid support structure, and if present, to the supplementary sample-receiving region of the second solid support structure, and allowing the test sample to migrate to the conjugate pad and

[0184] 14245329-1 to mix with the first, second and third labelled conjugates thereby enabling the labelled conjugates to become mobilised; and iii) allowing the mobilised first, second and third labelled conjugates to migrate to the first, second and third test regions, and if present, the first and second supplementary test regions, to contact the respective immobilised molecules in each of the test regions.

[0185] In an alternative embodiment, the method comprises the following steps iv) to vi): iv) providing a lateral flow test device as described above (see “further arrangements” above) , v) applying the test sample to the sample-receiving region of the first solid support structure, and if present, to the supplementary sample-receiving region of the second solid support structure, and allowing the test sample to migrate to the conjugate pad and to mix with the first and second labelled hormone conjugates, and third labelled conjugate, thereby enabling the labelled conjugates to become mobilised; and vi) allowing the mobilised first and second labelled hormone conjugates and third labelled conjugate to migrate to the first, second and third test regions, and if present, to the first and second supplementary test regions, to contact the respective immobilised molecules in each of the test regions.

[0186] Subsequently, the signal intensity at one or more of the first, second and third test regions, and if present, first and second supplementary test regions is measured. The amount of estradiol in the test sample is determined based on the signal intensity at the first test region and / or first supplementary test region, the amount of progesterone in the test sample is determined based on the signal intensity at the second test region and / or second supplementary test region, and / or the amount of LH in the test sample is determined based on the signal intensity at the third test region, in accordance with the methods described above.

[0187] In some embodiments, the test sample comprises a blood sample, and optionally, a whole blood sample. Advantageously, the lateral flow test device according to the invention may require only a small volume of blood (for example, obtained by a fingerprick method). The blood may be applied directly to the sample-receiving region (for example, as whole blood, and without centrifugation and / or filtration). In other

[0188] 14245329-1 embodiments, the blood sample may be subjected to a centrifugation and / or filtration step to remove blood cells.

[0189] Kit of parts

[0190] The present invention further provides a kit of parts comprising the lateral flow test device described in any of the embodiments above, and an optical signal reader configured to measure the intensity of a signal in one or more of the first, second and third test regions, and if present, the first and second supplementary test regions. The kit may also comprise instructions for using the lateral flow test device, and / or further components for carrying out the method of the present invention. The further components may be, for example, a capillary tube for collecting blood and a buffer for diluting the blood.

[0191] Clinical applications

[0192] It is advantageous to measure estradiol, progesterone and LH, as these hormones are tested frequently during an in vitro-fertilisation (IVF) programme. Other hormones such as AMH and FSH are typically tested at the start of IVF treatment to determine ovarian status.

[0193] Estradiol can be tested as much as every day during the follicular phase. It is used to as a secondary measure to monitor maturation of follicles and to check for the risk of ovarian hyper-stimulation syndrome. LH is also monitored regularly to check that the patient undergoing IVF is not at risk of ovulating naturally. If this occurs then an opportunity to collect the eggs may be missed. Daily testing, particularly towards the end of ovarian stimulation is advised. Progesterone can be monitored throughout treatment, but is monitored more frequently towards the end of ovarian stimulation. If the concentration of progesterone is higher than a threshold (e.g. 4.5 nmol / L) then a clinical decision may be made to freeze the eggs rather than proceed to fresh embryo transfer. During the embryo transfer phase of an IVF programme, a patient is typically given progesterone supplements to support the luteal phase and ensure a receptive endometrium. Progesterone levels above a certain threshold (e.g. 28 nmol / L) on the day of embryo transfer are considered to give a greater chance of clinical pregnancy, and

[0194] 14245329-1 therefore, regular progesterone testing is required in order to monitor levels to ensure this threshold is surpassed.

[0195] On some days, only one hormone may need to be tested. On other days, two hormones may need to be tested, and on some days, all three hormones may need to be tested. It is feasible that additional hormones may need to be tested. The present invention simplifies hormone monitoring by the patient and reduces the risk of error by combining multiple tests into a single lateral flow device. Additionally, the sensitivity of the device can be improved by incorporating at least two support structures with different capillary flow rates and / or different ranges of detection to improve the sensitivity and accuracy of the device.

[0196] The fast turn-around time of the present invention allows the clinician to have the hormone results as soon as the test is completed. If the patient is instructed to test their hormones each morning, the clinician may be able to access the results at the start of the day, streamlining the whole clinical decision-making process.

[0197] It has been shown that serum hormones can be released in a pulsatile manner and that there can be significant variation in hormone levels throughout a day. A diurnal profile of hormone levels can be observed, where, for example, progesterone levels are higher in the morning compared with the evening. Advantageously, by allowing the patient to take measurements in the comfort of their home or wherever they might be during a day, the time of blood collection and measurement can be more easily controlled, thus controlling the diurnal dependence. The present invention allows multiple measurements to be taken more practically which helps to alleviate the issue related to pulsatile release of hormones into the bloodstream.

[0198] Embodiments of the invention will be described by the following Example.

[0199] 14245329-1 Example

[0200] Provided below are exemplary steps for using the lateral flow test device according to the present invention, one or more of which may be taken in combination.

[0201] 1. User guide: The user may have a mobile app which guides them through the process.

[0202] 2. Blood sampling: The user may prepare the entry site on their finger by wiping with an alcohol wipe and allowing the alcohol to dry. They may puncture their skin with a supplied lancet to ensure a good flow of blood. The first drop of blood is wiped away and a second drop is allowed to form. A capillary tube of known volume may be used to collect the capillary blood sample.

[0203] 3. Sample processing: In some implementations the capillary blood sample may be mixed with a known volume of buffer solution. A buffer solution may be provided in a separate container / tube and the capillary blood sample may deposited into the tube and mixed with the buffer solution. The buffer solution may comprise components to facilitate the assay and stabilise the pH. For example, the buffer solution may include an anticoagulant, blocking agent, and / or a salt, in addition to standard buffer components (for example, a mixture of a weak acid and its conjugate base or a mixture of a weak base and it conjugate acid).

[0204] 4. Addition to test cartridge: In a preferred embodiment, the capillary blood sample is applied directly to the test cartridge via the sample pad. Direct contact is made between the capillary tube and the sample pad of the test cartridge and the blood sample is wicked by capillary action into the sample pad. This may be followed by the dispensing of a known volume of buffer solution on to the sample pad. Alternatively, if the capillary blood sample has been mixed with a buffer solution, a known volume of the blood-buffer solution will be dispensed onto the sample pad of the test cartridge.

[0205] 5. Measurement: After addition of the blood sample (and buffer) to the test cartridge, the test cartridge is inserted into an optical reader. A timer is triggered in the optical reader and a timed incubation is started. After the incubation time has elapsed, an optical measurement of the test cartridge will be taken and the intensity of the test and control lines will be extracted. The line intensities will be converted using a calibration function to generate hormone concentrations.

[0206] 6. Communication: In a preferred embodiment, the optical reader is connected by BTLE to a mobile phone with a companion software app. The test result is then

[0207] 14245329-1 communicated to the mobile phone, uploaded to cloud storage and downloaded to the electronic medical record for the clinician to view the result.

[0208] 14245329-1

Claims

44CLAIMS1 . A lateral flow test device for determining the amount of a plurality of hormones in a test sample comprising at least a first solid support structure, the first solid support structure comprising a sample-receiving region, a conjugate pad, and at least first, second and third test regions, wherein the first solid support structure is configured to permit liquid to flow sequentially from the sample-receiving region to each of the test regions via the conjugate pad, and wherein: i) the conjugate pad comprises a first labelled conjugate comprising an estradiol- binding moiety conjugated to a detectable label, a second labelled conjugate comprising a progesterone-binding moiety conjugated to a detectable label, and a third labelled conjugate comprising a luteinizing hormone (LH)-binding moiety conjugated to a detectable label, wherein each of the first, second and third labelled conjugates are capable of mobilisation on contact with the sample; ii) the first test region comprises immobilised estradiol or a derivative thereof which is capable of binding to the estradiol-binding moiety of the first labelled conjugate; iii) the second test region comprises immobilised progesterone or a derivative thereof which is capable of binding to the progesterone-binding moiety of the second labelled conjugate; iv) the third test region comprises an immobilised LH-binding element which is capable of binding to LH; and v) the detectable label of the first, second and third labelled conjugates is capable of producing a signal having a signal intensity in the first, second and third test regions, respectively, wherein the amount of estradiol, progesterone and LH in the test sample is determined based on the signal intensity in the first, second and third test regions, respectively.

2. The lateral flow test device according to claim 1 , comprising one or more of the following: i) the estradiol-binding moiety of the first labelled conjugate comprises an antiestradiol antibody; ii) the progesterone-binding moiety of the second labelled conjugate comprises an anti-progesterone antibody;14245329-145 iii) the LH-binding moiety of the third labelled conjugate comprises an anti- LH antibody; and iv) the LH-binding element comprises an anti-LH antibody.

3. The lateral flow test device according to claim 1 or claim 2, wherein the detectable label of the first, second and third labelled conjugates is selected from: a fluorescent particle, a dye particle, a latex particle, a nanoparticle, a gold particle, and a platinum particle, preferably, wherein the detectable label comprises europium.

4. The lateral flow test device according to any preceding claim, wherein the first solid support structure comprises, in addition to the first test region, a first supplementary test region comprising immobilised estradiol or a derivative thereof which is capable of binding to the estradiol-binding moiety of the first labelled conjugate, wherein the first test region and the first supplementary test region are configured to detect different amounts of estradiol; and optionally, wherein the first test region is configured to detect a higher minimum level of estradiol than the first supplementary test region, and the solid support structure is configured to permit liquid to flow sequentially from the first test region to the first supplementary test region.

5. The lateral flow test device according to claim 4, wherein the first test region is configured to detect estradiol in an amount of from 200-8000 pg / mL, and the first supplementary test region is configured to detect estradiol in an amount of from 20-2000 pg / mL.

6. The lateral flow test device according to any preceding claim, wherein the first solid support structure comprises, in addition to the second test region, a second supplementary test region comprising immobilised progesterone or a derivative thereof which is capable of binding to the progesterone-binding moiety of the second labelled conjugate, wherein the second test region and the second supplementary test region are configured to detect different levels of progesterone; and optionally,14245329-146 wherein the second test region is configured to detect a higher minimum level of progesterone than the second supplementary test region, and the solid support structure is configured to permit liquid to flow sequentially from the second test region to the second supplementary test region.

7. The lateral flow test device according to claim 6, wherein the second test region is configured to detect progesterone in an amount of from 3,000-60,000 pg / mL, and the second supplementary test region is configured to detect progesterone in an amount of from 50-3,000 pg / mL.

8. A lateral flow test device for determining the amount of each of a plurality of hormones in a test sample comprising at least a first solid support structure, the first solid support structure comprising a sample-receiving region, a conjugate pad, and at least first, second and third test regions, wherein the solid support structure is configured to permit liquid to flow sequentially from the sample-receiving region to the test regions via the conjugate pad, and wherein: i) the conjugate pad comprises a first labelled hormone conjugate comprising estradiol or a derivative thereof conjugated to a detectable label, a second labelled hormone conjugate comprising progesterone or a derivative thereof conjugated to a detectable label, and a third labelled conjugate comprising a luteinizing hormone (LH)- binding moiety conjugated to a detectable label, wherein each of the first labelled hormone conjugate, second labelled hormone conjugate and third labelled conjugate is capable of mobilisation on contact with the sample; ii) the first test region comprises an immobilised estradiol-binding element which is capable of binding to estradiol of the first labelled hormone conjugate and estradiol in the test sample; iii) the second test region comprises an immobilised progesterone-binding element which is capable of binding to progesterone of the second labelled hormone conjugate and progesterone in the test sample; iv) the third test region comprises an immobilised LH-binding element which is capable of binding to LH; and v) the detectable label of the first labelled hormone conjugate, second labelled hormone conjugate and the third labelled conjugate is capable of producing a signal having a signal intensity in the first, second and third test regions, respectively, wherein14245329-1the amount of estradiol, progesterone and LH in the test sample is determined based on the signal intensity in the first, second and third test regions, respectively.

9. The lateral flow test device according to claim 8, comprising one or more of the following: i) the estradiol-binding element comprises an anti-estradiol antibody; ii) the progesterone-binding element comprises an anti-progesterone antibody; iii) the LH-binding moiety comprises an anti- LH antibody; and iv) the LH-binding element comprises an anti-LH antibody.

10. The lateral flow test device according to claim 8 or claim 9, wherein the detectable label of the first and second labelled hormone conjugate, and the third labelled conjugate, is selected from: a fluorescent particle, a dye particle, a latex particle, a nanoparticle, a gold particle, and a platinum particle, preferably, wherein the detectable label comprises europium.

11. The lateral flow test device according to any preceding claim, wherein the conjugate pad further comprises a primary antibody conjugated to a detectable label (primary antibody-label conjugate), wherein the primary antibody-label conjugate is capable of mobilisation on contact with the sample, and wherein the primary antibody does not bind to any of estradiol, progesterone, and LH; and wherein the lateral flow test device further comprises a control region, wherein the control region comprises an immobilised secondary antibody which is capable of binding to the primary antibody of the primary antibody-label conjugate, and wherein the detectable label of the primary antibody-label conjugate is capable of producing a detectable signal in the control region, or wherein the lateral flow test device further comprises a control region, wherein the control region comprises an immobilised secondary antibody which is capable of binding to one or more of the following: the first, second and third labelled conjugates, first labelled hormone conjugate, and second labelled hormone conjugate.14245329-112. The lateral flow test device according to claim 11 , wherein the detectable label of the primary antibody-label conjugate is selected from: a fluorescent particle, a dye particle, a latex particle, a nanoparticle, a gold particle, and a platinum particle, preferably wherein the detectable label of the primary antibody-label conjugate comprises europium.

13. The lateral flow test device according to any one of claims 1 to 3, wherein the lateral flow test device comprises a second solid support structure, wherein the second solid support structure optionally comprises a supplementary sample-receiving region, and further comprises a supplementary conjugate pad, and at least first and second supplementary test regions, wherein the second solid support structure is configured to permit liquid to flow sequentially from the sample-receiving region or the supplementary sample-receiving region to the supplementary test regions via the supplementary conjugate pad, and wherein: i) the supplementary conjugate pad comprises a first labelled conjugate comprising an estradiol-binding moiety conjugated to a detectable label, and a second labelled conjugate comprising a progesterone-binding moiety conjugated to a detectable label, wherein each of the first and second labelled conjugates are capable of mobilisation on contact with the sample; ii) the first supplementary test region comprises immobilised estradiol or a derivative thereof which is capable of binding to the estradiol-binding moiety of the first labelled conjugate; iii) the second supplementary test region comprises immobilised progesterone or a derivative thereof which is capable of binding to the progesterone-binding moiety of the second labelled conjugate; iv) the detectable label of the first and second labelled conjugates is capable of producing a detectable signal having a signal intensity in the supplementary first and second supplementary test regions, respectively; v) the first test region and the first supplementary test region comprising immobilised estradiol are configured to detect different amounts of estradiol, vi) the second test region and the second supplementary test region comprising immobilised progesterone are configured to detect different amounts of progesterone; and14245329-149 wherein the amount of estradiol in the test sample is determined based on the signal intensity of the first test region and / or first supplementary test region; and wherein the amount of progesterone in the test sample is determined based on the signal intensity of the second test region and / or second supplementary test region.

14. The lateral flow test device according to claim 13, wherein the first and second labelled conjugates are as defined in claim 2 or claim 3.

15. The lateral flow test device according to any of claims 8 to 12 , wherein the lateral flow test device comprises a second solid support structure, wherein the second solid support structure optionally comprises a supplementary sample-receiving region, and further comprises a supplementary conjugate pad, and at least first and second supplementary test regions, wherein the second solid support structure is configured to permit liquid to flow sequentially from the sample-receiving region or the supplementary sample-receiving region to the supplementary test regions via the supplementary conjugate pad, and wherein: i) the supplementary conjugate pad comprises a first labelled hormone conjugate comprising an estradiol or a derivative thereof conjugated to a detectable label, and a second labelled hormone conjugate comprising progesterone or a derivative thereof conjugated to a detectable label, wherein each of the first and second labelled hormone conjugates are capable of mobilisation on contact with the sample; ii) the first supplementary test region comprises an immobilised estradiol-binding element which is capable of binding to estradiol of the first labelled hormone conjugate or estradiol in the test sample; iii) the second supplementary test region comprises an immobilised progesterone-binding element which is capable of binding to progesterone of the second labelled conjugate or progesterone in the test sample; iv) the detectable label of the first and second labelled hormone conjugates is capable of producing a detectable signal having a signal intensity in the first and second supplementary regions, respectively; v) the first test region and the first supplementary test region are configured to detect different amounts of estradiol; vi) the second test region and the second supplementary test region are configured to detect different amounts of progesterone;14245329-150 wherein the amount of estradiol in the test sample is determined based on the signal intensity of the first test region and / or first supplementary test region; and wherein the amount of progesterone in the test sample is determined based on the signal intensity of the second test region and / or second supplementary test region.

16. The lateral flow test device according to claim 15, comprising one or more of the following: i) the first and second labelled hormone conjugates are as defined in claim 9 or claim 10; ii) the estradiol-binding element of the first supplementary test region comprises an anti-estradiol antibody; iii) the progesterone-binding element of the second supplementary test region comprises anti-progesterone antibody; and iv) the detectable label of the first and second labelled hormone conjugates is as defined in claim 10.

17. The lateral flow test device according to any one of claims 12 to 16, wherein the conjugate pad of the second solid support structure comprises a primary antibody conjugated to a detectable label as defined in claim 11 or claim 12, and the second solid support structure further comprises a control region as defined in claim 11.

18. The lateral flow test device according to any one of claims 13 to 17, wherein the capillary flow rate through the first solid support structure is different from the capillary flow rate through the second solid support structure.

19. The lateral flow test device according to any preceding claim, wherein the device is configured to detect estradiol in an amount of from 20 - 8,000 pg / mL, progesterone in an amount of from 50 - 60,000 pg / ml and LH in an amount of from 0.2 - 150 lll / L.

20. The lateral flow test device according to any preceding claim, wherein the test sample comprises blood, preferably, whole blood.21 . A method for determining the amount of a plurality of hormones in a test sample, the method comprising the following steps i) to iii):14245329-151 i) providing the lateral flow test device of any of claims 1 to 7, 11 to 14, 17 to 20, ii) applying the test sample to the sample-receiving region of the first solid support structure, and if present, the supplementary sample-receiving region of second solid support structure, and allowing the test sample to migrate to the conjugate pad, and if present, the supplementary conjugate pad, and mix with the first, second and third labelled conjugates, thereby enabling the labelled conjugates to become mobilised, iii) allowing the mobilised first, second and third labelled conjugates to migrate to the first, second and third test regions, and if present, the first and second supplementary test regions, to contact the respective immobilised molecules in each of the test regions, or wherein the method comprises the following steps iv) to vi): iv) providing the lateral flow test device of any of claims 8 to 20, v) applying the test sample to the sample-receiving region of the first solid support structure, and if present, to the supplementary sample-receiving region of the second solid support structure, and allowing the test sample to migrate to the conjugate pad, and if present, to the supplementary conjugate pad, and mix with the first and second labelled hormone conjugates, and third labelled conjugate, thereby enabling the labelled conjugates to become mobilised, vi) allowing the mobilised first and second labelled hormone conjugates and third labelled conjugate to migrate to the first, second and third test regions, and if present, the first and second supplementary test regions, to contact the respective immobilised molecules in each of the test regions; and vii) measuring the signal intensity at one or more of the first, second and third test regions, and if present, first and second supplementary test regions, and determining the amount of estradiol in the test sample based on the signal intensity at the first test region and / or first supplementary test region, determining the amount of progesterone in the test sample based on the signal intensity at the second test region and / or second supplementary test region, and determining the amount of LH in the test sample based on the signal intensity at the third test region.

22. The method according to claim 21 , wherein step vii) is carried out using an optical signal reader.

23. The method according to claim 21 or claim 22, wherein step vii) is computer- implemented and comprises outputting an indication of the amount of estradiol, progesterone and LH in the test sample.14245329-1M&C PN853397W05224. The method according to any one of claims 21 to 23, wherein the test sample comprises blood, preferably, whole blood.

25. A kit of parts comprising a lateral flow test device according to any one of claims1 to 20, and an optical signal reader configured to measure the intensity of a signal in one or more of the first, second and third test regions, and if present, the first and second supplementary test regions.14245329-1

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