The present invention concerns an improved lateral flow test strip for use in personal home- based testing, as well as methods, kits and applicators for personal use in detecting change, or the rate of change of an analyte associated with a health condition, disease or illness, particularly in the field of fertility. In particular, there is disclosed a novel monitoring system consisting of a lateral flow assay for use with an analyser to improve accuracy in home- based health testing and which is suitable for measuring change in a health status or condition or the rate of change over a period of time, for example, as it concerns fertility and pregnancy determination, or disease/illness detection, as well as ongoing monitoring thereof.

BACKGROUND

Early detection of a modified biological condition or state, such as when a woman becomes pregnant or in the case of a negative change in health condition of a person, such as a newly developed illness or disease can be very useful. Early intervention in all such cases can enable a better outcome for the person as compared to if the changed state remains undetected for a further period of weeks or months.

Personalised home testing may therefore play a significant role in the ability to enable early detection of those health changes, since it is often the first significant warning to a person that they should seek medical intervention, whether diagnostic and/or treatment.

Around 5% of women of fertile age decide to try for a baby each year. 70% of these women fall pregnant within the first 6 months, 84% get pregnant within 1 year and about 91% within 3 years. Medical intervention usually commences after 12 months of trying to fall pregnant unsuccessfully. Women who have failed to conceive after 12 months of trying and with no clinical cause found for failure to get pregnant, are classified under "unexplained infertility". Many of these women go on to conceive naturally although this can take up to several months or even years. It is estimated that 1 in 7 heterosexual couples have difficulty conceiving, these are approximately 3.5 million people in the UK (https://www.nhs.uk/conditions/infertility). According to UK NICE guidelines one of the main causes of infertility (25%) is unexplained infertility with no identified male or female cause (NICE Guidelines February 2013). At least 1 in 4 (potentially up to half) women with unexplained infertility have issues with their fertility hormones (i.e. estrogen, Luteinising Hormone and Progesterone). There are multiple conditions and lifestyle choices that can affect key fertility hormone levels and thus women ^' s fertility, for example excessive exercise can suppress FSH and as such reduce fertility, or Polycystic Ovary Syndrome (PCOS) may lead to increased LH levels and irregular cycles. Understanding if there are hormonal issues at an early stage allows time to take corrective action and thereby improve hormonal health and fertility.

During pregnancy, medical issues often occur in the first trimester (0-13 weeks) but many of the complications can be treated and/or resolved if identified early. It is estimated that 1 in 4 pregnancies in the UK alone resulted in miscarriage or ectopic pregnancy in 2017, with 3 out of 4 of these incidences occurring in the first trimester so it remains a significant concern. However, before occurrence of complications it is difficult to identify a woman who may be prone to ectopic pregnancy, because many of the diseases are caused by bacterial infections which often exhibit no noticeable symptoms. However, if clearly detected at an early stage, medication can be used to stop the egg developing and limited little long term physical effect is experienced. Similarly, miscarriage is also common during the first trimester and early identification of an abnormal pregnancy is desirable as it allows for further tests and the provision of a treatment regime for hormone imbalances can be rectified and a healthy viable pregnancy established. Other issues during the first trimester include multiple pregnancies. If multiple pregnancies are detected in a woman very early in the first trimester, the support given can be adapted to reduce the risks of miscarriage, anaemia, high blood pressure, pre-eclampsia, gestational diabetes, haemorrhage, and early labour. Again, accurately determining abnormality in a pregnancy, once that pregnancy is established is therefore useful.

Detections of analytes, including hormones, in lateral flow test strips previously typically relied upon a qualitative result, with the presence or absence of said analyte being detected. Calibration of such assays is simple; assays are produced using a pre-defined technical standard to make each batch produced fit a required specification. This is the case, for example, for standard ovulation tests that detect the presence of luteinising hormone (LH). The limitation of these immunoassays is that they can only give a yes/no response and cannot reliably/ accurately quantify the amount of LH present in the sample as they provide results when the LH concentration in a urine sample equals or exceeds the "average" level. Research shows that many women can have a high LH baseline level (e.g. PCOS) and many have a low level (many possible causes), therefore, classic ovulation tests may not work for them.

There are studies (Menstrual cycle hormonal profiles Allente et a I, fertility and sterility (2002), 78: 90 - 95) to show that the hormonal profiles differ significantly from the classic mean curves, in terms of length, height and shape (one peak and two peak surges) frequently found in healthy fertile women. This work further supports the need for longitudinal evaluation in order to accurately and noninvasively, test and measure hormonal levels and obtain an accurate representation of an individual's personal profile.

In their appreciation of this issue, the present applicant previously disclosed a device which may quantify all the key fertility hormones that regulate a menstrual cycle and present a personal profile to the end user. The device analyses a biological liquid sample from a woman by measuring parameters indicative of a quantity of Follicle Stimulating hormone (FSH), luteinizing hormone (LH), progesterone and human chorionic gonadotropin hormone (hCG). Test strips for use with such devices are the means by which they may be analysed. Such a device accumulates a personal set of data within the device relating to FSH, LH, progesterone and/or hCG hormone levels over a number of days and even multiple times a day to provide a personal baseline measurement. In this manner the device can provide a personal profile with a personal ovulation date (and/or time depending on the number of samples) because the calculation is performed and measured against a woman's own normalised hormone base line. The user also will build a longitudinal profile of their hormones and obtain a visual reference for predicting ovulation.

The device also monitors for deviations from predetermined statistical data and the woman's own personal data. Some conclusions can be drawn from statistically relevant deviations in the user's data over time, as compared with pre-determined values. For example, during a normal pregnancy the measured quantity of hCG is expected to double in the two to three days immediately after conception. Identified deviations in the measured quantity of hCG may be a guide for recognising abnormality. The device and system is described with reference to the disclosure in published patent application WO2015/121661, which is incorporated by way of reference.

However, the value of this testing has some limitation; whilst some quantifiable determination has been made, home-based systems including fertility and pregnancy testing, would benefit from further improvement. Currently quantification of hCG and other hormones or biological analytes is only possible under laboratory conditions using a blood sample. The ability to quantitatively measure for change, or the rate of change, after initial detection of any condition marker is useful to review progress of that condition. Accurate and reliable testing based on one's own quantitative personalised data (baseline calculation) is vital in such cases. However, this is largely unavailable outside a lab setting.

Being able to better understand the menstrual cycle from an early age empowers women to achieve a natural conception and healthy condition. It would be useful for women to personally test and measure their hormone levels in real-time and with improved accuracy.

It would be further useful to present such information in a way that is easy to understand and utilise.

The present disclosure therefore arises from a continued need to provide a home testing solution to enable accurate determination, early or otherwise, when a significant change in a person's biological condition or health state occurs.

In particular, personalised testing which more accurately determines and/or monitors: either a change in a woman's biological condition, as required in home fertility or pregnancy testing; or early stage indication of health deterioration caused by a particular condition, illness or disease would be very useful. SUMMARY OF INVENTION

According to a first aspect, the present disclosure relates to an improved lateral flow test or assay strip for use in quantitatively detecting and/or monitoring an analyte in a biological liquid sample, the test strip comprising a backing layer supporting: a dry porous membrane comprising two zones: a first zone comprising mobile labelled reagent capable of specifically binding with said analyte to form a first complex and a detection zone, spatially distinct from the first zone, comprising a non-labelled, immobile means for binding said first complex; a sample pad for receiving the biological fluid a portion of which overlaps at the first end of the dry porous member; an absorbent pad, partially overlapping a second end of the porous membrane, which is distal to the first end of the porous membrane, to encourage lateral flow and draw liquid through the membrane from the first to the second end; and characterised by a laminate layer, which covers the overlapping portion of the sample pad and extends partially over the porous membrane, such that wherein migration of the liquid sample through said membrane mobilises the first complex comprising labelled reagent and said analyte to said detection zone and the non-labelled, immobile binding means binds said first complex to form a second complex which can be analysed to quantitatively determine the analyte in the sample.

The applicant has been able through extensive testing able to determine that this novel arrangement strongly improves direction and force of sample flow through porous membrane and ultimately this efficiency surprisingly improves the accuracy of detection such that the data can be quantitatively determined with a greater accuracy than has been possible with methods and processing of test or assay strips used in the art.

The applicant has further noted that applying such a laminate layer is not a universal solution since its precise positioning in relation to the overlap of other layers of the assay assembly is required to achieve the improvement in accuracy and/or results in a non working product.

Following successful execution and application of the novel test strip when used with an analysis device it is considered by the applicants that such an improvement in accuracy of the test data has wider implications than the field of fertility. The lateral flow test strip of the invention may enable personal determination of a particular negative health state, or a change in that particular health state, or a prediction for a change in that particular health state, with greater accuracy than has previously been possible. The ability to analyse and monitor such data with inherently improved accuracy derived from the quantitative measurement of other biological analytes, such as other hormones, permits home testing to extend to the early warning of different types of disease or condition including, for example, markers of hormone imbalance and conditions associated therewith or known markers of specific types of disease such as cancer.

In an age where early diagnosis is crucial and primary health care providers are often saturated in their resources, improved accuracy and reliability in home testing, such as enabled by the solutions according to the present invention can provide vital early indications of whether the person should seek intervention. This solution may enable diagnosis and/or treatment to allow a person return to a healthy state more quickly, or at least improve that state to a manageable condition, reducing the chance of more serious health complications. As well as the personal benefit, the cost implications and further burden on the health service associated with late diagnosis are reduced. Further, where stigma is attached to consulting the GP, particularly in men, and or where the person is unsure whether to consult their GP and using their time up unnecessarily, such testing solutions can indicate whether that course of action should be followed.

The laminate is typically a clear polypropylene film, but in other instances it may not be clear. In some embodiments the laminate is a (preferably co-extruded bi-axially oriented) transparent corona-treated polypropylene film.

In embodiments the analyte to be tested may be another hormone analyte, such as thyroid stimulating hormone (TSH), as a marker for Hypo or Hyperthyroidism. In such cases the sample is blood, serum or plasma.

Other examples concern other hormone analytes, particularly for example, where the field concerns fertility, the hormone analyte maybe selected from luteinizing hormone (LH) and human chorionic gonadotropin hormone (hCG), Anti-Mullerian hormone (AMH), prolactin, progesterone (PDG), Estrogen (ESG), Inhibin B, Testosterone and follicle-stimulating hormone (FSH).

Conditions or diseases which require a multitude of indicators to diagnose would benefit greatly from the invention. Usefully, where a combination of factors is required for diagnosis; accurate change within each of those indicators facilitates need to obtain quick diagnosis. For example, where different test strips can be identified and used with the same analyser to quickly, specifically and accurately measure relevant hormone analyte, the plethora of personal data that results can be used to make an early warning of conditions such as polycystic ovary syndrome (PCOS), which can then be followed up with a medical practitioner facilitating earlier stage diagnosis. Earlier intervention can be crucial in conditions, like PCOS, in which a delay in diagnosis or misdiagnosis, leads to infertility.

In some embodiments, the mobile, labelled reagent is an antibody, antibody fragment (such as scFv and Fab fragments), antibody derivative, chimeric antibody or an aptamer, or an antigen, or protein labelled antigen. The labelled reagent is specific for the analyte, such as where the hormone analyte is LH or hCG. In some embodiments, the mobile, labelled reagent is selected from an anti-LH antibody and an anti-hCG antibody.

In some embodiments, the immobile, non-labelled binding means is an antibody, antibody fragment (such as scFv and Fab fragments), antibody derivative, chimeric antibody or an aptamer, or an antigen, or protein labelled antigen. The non-labelled binding means is specific for the analyte, such as the hormone analytes LH or hCG. In some embodiments, the non- labelled binding means is selected from an anti-LH antibody and an anti-hCG antibody. Alternatively, the non-labelled binding means can be specific for the complex of the analyte and the mobile labelled reagent, but not for the mobile labelled reagent in the absence of the analyte. In some embodiments the label provided on the labelled reagent is selected from particles, including for example, nano-gold particles or coloured latex particles. In some embodiments the sample pad comprises a wicking material. In some embodiments the porous membrane is nitrocellulose. The biological liquid sample may be selected from a urine sample, a blood based sample such as blood plasma or serum, an interstitial fluid sample, a saliva sample, or a gingival fluid sample.

In some embodiments the invention extends to an applicator comprising of a shell or casing for use with the test strip of the invention. The casing may comprise of a base portion and a cover portion which are releasably fixed as a single unit to form the applicator and together house the lateral flow test strip described herein within. The applicator may further comprise and elongate handle or holding tad. The cover of the casing typically may comprise an aperture through which a portion of the sample pad of the test strip is accessible. This aperture enables the sample to be deposited accurately on the test strip. The aperture may be circular is shape and bevelled to create a funnelled access to the strip. The cover of the casing typically further comprises a detection window. The window may be elongate and rectangular in form. In some embodiments the cover of the casing comprises at least one or two bar codes, including QR codes, to identify the type of lateral flow test strip that it houses. The bar codes may be positioned on the cover one or both sides of the detection window and each extend substantially along the length of the window. This enables detection, e.g. at the detection window and data reading, e.g. from the bar codes to be captured in a single defined section/area of the applicator during use of the device.

In a further aspect the disclosure concerns the above applicator for use with a device comprising an analyser, such as an electrochemical analyser which may be coupled with an ion selective electrode. In some embodiments the device is a fertility and/or pregnancy testing/monitoring device. In other embodiments the device may be a personal health device configured to determine a new diseased state or condition based on the type of strip that is utilised and the analyte tested. Such a device may be programmed or programmable (e.g. wirelessly) to identify the lateral flow assay that has been under taken from the applicator barcoding, perform the necessary detection and quantitative personal calculations to provide a patient output reading in order to determine a biological condition, or warn or indicate illness or disease. The patient output maybe displayed on the device and/ or it can be transmitted via a wireless connection, for example Bluetooth or Wi-Fi, to a Device, such as a Smart Phone, Laptop, Tablet or Virtual Assistant, where the outputs may be displayed or presented and stored, using an App.

In a further aspect the disclosure concerns the calibration of an assay performed on the lateral flow test strip, in order to enable quantitative measurement of the analyte, optionally a hormone analyte. The information to allow calibration of the lateral flow test strip is provided on the casing in the bar codes. The device may therefore read the bar code and calibrate the assay using the information provided therein.

The invention further concerns a kit for use in determining a health condition, illness or disease characterised by one or multiple biological marker analytes, the kit including either two or more applicators according to those disclosed above wherein the lateral flow test strip is specific to the one biological marker analyte; or two or more applicators wherein at least two of said applicators comprise lateral flow test strips which are specific to a different one of said multiple biological marker analytes. In embodiments the personal health analyser is configured to receive the applicator(s), quantitatively measure the biological marker analyte present on the lateral flow test strip to determine a test result and convert the result for visual display. The personal health analyser may be configured to process a device-readable data code on the applicator to automatically identify the test type needed to quantitatively measure the biological marker analyte.

The personal health analyser may be further configured to compare one or more test results against a standard reference, particular to a health status, condition, illness or disease and to yield a status outcome and communicate the status outcome on the visual display.

Usefully the personal health analyser may comprise wireless connectivity capability to transmit said assay result for remote display on a further electronic device.

In a further aspect there is disclosed a personal testing method for a condition-specific health status comprising: conducting a lateral flow assay by applying a biological liquid sample analyte to the test strip of applicator, to obtain an assay result; and applying the applicator to a lateral flow assay analyser configured to identify the assay type from the applicator, quantitatively measure the assay result; and determine the condition-specific health status. The device may provide visual output communicating the condition-specific health status.

The method of the invention may further include repeating steps described above, to provide a series of quantitative assay results such that the device determines a personally calibrated result and the personally calibrated result is used to determine and/or monitor a change and/ or rate of change in the condition-specific health status.

In that case the device communicates the change or the rate of change in the condition- specific health status to the user on a visual display. Where the device is pre-programmed with data corresponding to the condition-specific health status, on the basis of that change or rate of change, the device may determine the onset of a health condition or pre-empt it and communicates that determination on a visual display to the user.

Such a testing method provides accurate data and allows the user to easily visualise that health data and/or monitor their own condition, i.e. ovulation "Signature" or profile, with real time and comprehensive results and also predict the status of a condition, such as ovulation events, in the forthcoming months.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

BRIEF DESCRIPTION

Figure la displays an exploded perspective view of the components of a non-assembled lateral flow test strip according to a first aspect of the invention. Figure lb displays an exploded side view of the components of the same non-assembled lateral flow test strip according to a first aspect of the invention.

Figure 2a displays a side view of an assembled test strip according to the invention.

Figure 2b displays a perspective view of the same assembled test strip shown in Figure 2a.

Figure 3 shows a plan view of the test strip of the invention shown in the previous figures.

Figure 4a shows a non-assembled applicator cassette for use with the test strip of the invention.

Figures 4b-d show a detailed view of the user identifiable cover variations based on the type of lateral flow assay strip housed within.

Figure 5a shows a plan view of the applicator of Figure 4a housing the test strip according to an embodiment of the invention.

Figure 5b shows an exploded view of a non-assembled applicator cassette including a test strip according to an embodiment of the invention.

Figure 6 displays graphically the results of the laminated test strip arranged as per the invention compared to a non-laminated test strip.

DETAILED DESCRIPTION

An example lateral flow assay or test strip in accordance with the invention and embodiments thereof is shown in detail in Figures 1 through to 3.

Specifically, Figures la and lb provides a lateral flow test strip 10 according to the invention. In this exploded view the various components or layers of the assembly can be seen clearly. In this example the assay or test strip assembly is shown in deconstructed form comprising first a backing layer 1, which supports the remaining layers or features of the test strip. The backing may be made of card or having a similar structure with sufficient strength to support the layers of the assembly.

The features of the test strip assay further include a dry porous membrane 2, comprising first and second ends 2a, 2b and two distinct and spatially separated zones a first zone (Zl) and a detection zone (Z2). The first zone Zl typically comprises a mobile labelled reagent which is capable of specifically binding to the relevant analyte to be tested so it may to form a first complex. In some embodiments, the mobile, labelled reagent is an antibody, antibody fragment (such as scFv and Fab fragments), antibody derivative, chimeric antibody or an aptamer or an antigen, or protein labelled antigen. The labelled reagent is specific for the analyte, such as where the hormone analyte is LH or hCG. In some embodiments, the mobile, labelled reagent is selected from an anti-LH antibody and an anti-hCG antibody.

The label provided on the labelled reagent is typically selected from particles, such as nanoparticles which are gold or coloured. In examples where gold particles are used these may be approximately 40nm in size.

The second zone Z2 is a detection zone and comprises a non-labelled, immobile means for binding to said first complex once it is formed. In some embodiments, the immobile, non- labelled binding means is an antibody, antibody fragment (such as scFv and Fab fragments), antibody derivative, chimeric antibody or an aptamer, or an antigen, or protein labelled antigen. The non-labelled binding means is specific for the analyte, such as the hormone analytes LH or hCG. In some embodiments, the non-labelled binding means is selected from an anti-LH antibody and an anti-hCG antibody. Alternatively, the non-labelled binding means can be specific for the complex of the analyte and the mobile labelled reagent, but not for the mobile labelled reagent in the absence of the analyte.

The dry porous membrane is positioned substantially centrally along the length of the backing layer. The porous membrane is typically made of nitrocellulose of other suitable porous material. A sample pad 5 for receiving the biological fluid containing the analyte sits directly on top of the backing layer 1. The pad is generally thicker than the membrane and comprises a partially recessed portion 5a, the area of the recess equating to the thickness of the membrane at a first end 2a. When assembled, as shown in Figure 2a and 2b, the sample pad interlocks with the backing layer and the first end of the membrane is sandwiched there between such that the non-recessed part of the pad (full thickness) sits directly on the backing layer whilst its recessed portion 5a overlaps the first end of the porous membrane, securely surrounding it. In some embodiments the sample pad is made of a wicking material.

The test strip also includes an absorbent pad 7 which partially overlaps a second end 2b of the porous membrane positioned distally to the first end 2a of the porous membrane. The absorbent pad has a recessed portion 7a and, in a similar manner to the sample pad, is generally thicker than the porous membrane. The partially recessed area equates to the thickness of the membrane at a second end 2b of the membrane. When assembled, as shown in Figure 2a and 2b, the end of the porous membrane is interlocked between the backing layer and the absorbent pad such that the full thickness of the pad is able to sit directly on the backing layer whilst the recessed portion overlaps the second end of the porous membrane, securely surrounding it. The absorbent member encourages lateral flow and in use draws liquid through the porous membrane in the direction of the second end. The strip assembly 10 further includes a laminate layer 9. The laminate layer is positioned over part of the sample pad 5 particularly extending entirely over the recessed portion 5a. The laminate further includes a stepped portion 9a which steps down, extends along and directly covers a short, limited section of the porous membrane. The laminate layer therefore contacts both the recessed portion 5a of the sample pad and the porous membrane to create a novel layered arrangement. The laminate layer may be adhered to the test strip with adhesive such as a permanent acrylic adhesive, which maybe pre-existing on one side of the laminate. The adhesive is typically a clear permanent adhesive featuring excellent UV resistance and weather ability together with good adhesion performance, even with polar substrates. The adhesive complies with the European food directives and legislations, for example FDA 175.105. In production, batches of improved lateral flow test strips in accordance with assembly provided are manufactured together, and each batch of assay strips for the present invention is calibrated to enable the quantitative measurement of the relevant analyte. General application of a laminate layer per se is not considered particularly useful within this type of structure. The applicant has found that applying laminate in the position and arrangement disclosed herein, as it relates to other layers of the assay assembly, is required to achieve the improvement in accuracy of the product.

During production, each lateral flow test strip 10 may be housed in a cassette 20 or application for ease of use in the sampling and analysing processes. In examples, the invention therefore extends to an applicator as shown in Figures 4a-d and 5a-b in which the applicator comprises a shell or casing and is used in combination with before herein described test strip to form a useful consumable for easy personal health testing and use with analysing means.

With reference to Figure 4a, the applicator 20 is typically made from a two-part casing comprising a base portion 22 and a cover portion 24 which are releasably or permanently fixed as a single unit applicator. The lateral flow test strip 10, as described herein, is therefore positioned within the applicator prior to assembly as shown in Figures 5a and 5b and then secured therein when the cover and base are joined together. The applicator 10 may further comprise an elongate handle or holding tab 26.

The cover 24 of the casing typically may comprise an aperture 28 through which a portion of the sample pad 5 of the test strip 10 is accessible. This aperture 28 enables the sample to be applied to directly onto the test strip 10 while being held by the user. The aperture may be generally annular in shape and have internal bevelled edge(s) defined in its thickness to create a funnelled access slope to permit a small reservoir of sample liquid to be temporarily held and accurately deposited centrally on the sample pad 5 as it absorbs thereon.

The cover further typically comprises a detection window 30 which may be elongate and rectangular in form. In some embodiments the cover of the casing comprises at least one or two identification and/or calibrations area in the form of bar codes 32, 34, including QR codes, to identify the type of lateral flow test strip that it houses and provide calibration data that is needed. For example, bar codes 32, 34 may be positioned on the cover adjacent one or both sides of the detection window 30. In these examples each extends substantially along the length of the window. This enables detection, e.g. at the detection window and data reading, e.g. from the bar codes to be captured in a single defined section/area of the applicator during use of the device.

Furthermore, to aid visual identification and prevent user error, a specific symbol S relevant to the type of strip that is housed and thus the type of assay that is being conducted, may also be present/displayed on the cover, for example the cover may display "LH" or "hCG" as appropriate, as shown in Figures 4b and 4c, respectively.

In a further aspect the disclosure concerns the above applicator for use with a device comprising an analyser, such as an electrochemical analyser coupled which may be coupled with an ion selective electrode. In some embodiments the device is a fertility and/or pregnancy testing/monitoring device. In other embodiments the device may be a personal health device configured to determine a new diseased state or condition based on the type of strip that is utilised and the analyte tested. Such a device may be programmed or programmable (e.g. wirelessly) to identify the lateral flow assay that has been under taken from the applicator barcoding, perform the necessary detection and quantitative personal calculations to provide a patient output reading in order to determine a biological condition, or the rate of change of a biological condition, or indicate illness or disease.

When utilised, the user holds the applicator and applies a biological liquid sample to the accessible section of the sample pad 5 via aperture 28. The sample may be selected from a different biological fluid dependent on the condition that is being tested for. For example in pregnancy testing this may be a urine sample but in other cases this could be a blood-based sample such as blood plasma or serum. It is also possible the fluid is an interstitial sample, saliva, or a gingival fluid.

Once placed on the sample pad 5 the fluid migrates efficiently into and through the porous membrane 2 mobilising the first complex comprising labelled reagent and the analyte towards the detection zone Z2 in direction D, as shown in Figure 3. The non-labelled, immobile binding means in the detection zone then binds said first complex to form a second complex, which can be analysed by a detection device to quantitatively determine the analyte in the sample. Each batch of assay strips is specific for a particular analyte, such as a specific hormone analyte and may be identified easily by the means described above both for user purposes and relevant analysis detection. The components included on the lateral flow test strip includes mobile labelled reagents, such as anti-analyte antibodies or fragments thereof and immobilised non-labelled binding means, such as anti analyte/complex antibodies or fragments thereof, both the binding means and the reagent together forming the detection means.

The lateral flow test or assay strip is therefore useful in reliably enabling quantitative detection, monitoring and determining the rate of change (if several assays are conducted and analysed) of any particular analyte in a biological liquid sample. The method of calibration of the detection of an analyte in a biological liquid enables that the result provided is reliably quantitative. The improvements discussed herein enable the particular measurement of analyte concentration, and therefore calibration systems utilised must also ensure that the detection device can determine the results from the lateral flow test strip accurately.

Since, using the improved lateral flow test strip of the present invention, it is possible to always detect the specific level of the particular analyte; a more refined calibration of the assay is required, such that the quantitative result can be produced. Although some quantitative determination has been possible in the past it is the improved arrangement of the test strip that always permits the more accurate and reliable quantitative measurement which is so useful in personalised testing described herein.

Each batch may therefore require testing of the binding capacity and/or detection capability of the detection means. Such testing may require the use of standards of the relevant analyte being tested. Quantitative immunoassays using antibody detection generally refer to the World Health Organization (WHO) International Reference Preparations for calibration purposes. Various standard solutions of the analytes may be applied to the lateral flow test strip and the resulting complex formation measured. The readings are plotted as a standard curve parameter, optionally using the 4 or 5 parameter logistic models. Preferred is the five- parameter logistic equation, which is an alternative name for the standard dose-response curve, which has a classic S shape. 5 parameters can be taken from the curve; these are (a) estimated response at zero concentration; (b) slope factor; (c) mid-range concentration (C50); (d) estimated response at infinite concentration; and (g) asymmetry factor. Data from at least three of these parameters for each batch is taken and encoded onto the barcode; the at least three parameters being preferably a, c and d. The parameters b and g can be fixed as appropriate for the region in which the lateral test strips will be used; these are encoded into the barcodes referred to above. Alternatively, the parameters b and/or g may also be taken from the curve and encoded onto the barcode.

Therefore, each lateral flow test strip from a particular batch, once placed into the casing and marked with a barcode, contains all the relevant information on the binding capacity and/or detection capabilities of the particular lateral flow test strip and enables quantification of the results obtained. It is preferred that the assay device is programmed to enable the comparison of the reading to the relevant standard dose response curve, the instructions for which are included on the barcode for that batch. Calibration of the lateral flow test strip is therefore enabled to allow for quantification of the bioassay.

Examples

A first set of test strips 10, as described with the general configuration shown in Figures 1-3, were placed in a cassette or applicator 20, as shown in Figures 4 and 5, to create applicators having test strips with the laminate feature and novel configuration of the invention.

The specific lateral flow assay strip construction was therefore as follows:

Backing layer/support structure: matt vinyl material thickness 0.07mm-0.254mm

Dry porous membrane: nitrocellulose material with a capillary flow rate 60-100 s/4cm Mobile labelled reagent: mouse anti-LH monoclonal antibody lmg/ml labelled with 40 nm gold nanoparticles Non-labelled, immobile means for binding: mouse anti-LH monoclonal antibody, no label lmg/mIControl line binding: rabbit anti-mouse IgG lmg/ml

Sample pad: glass fibre base having a thickness 355pm at 53kPA

Absorbent/sink pad: cotton linter thickness 954pm at 53 kPA absorption 50.9 mg/cm2

Laminate: layer of clear polypropylene with/without self-adhesion properties

A second set of applicators were constructed in an identical manner, however in this case the set was constructed without the laminate layer.

A series of biological liquid samples, in this case urine, was prepared comprising the analyte of interest LH at varying concentrations. In each case the sample to be tested was added to the sample pad of the relevant test strip applicator.

The urine flows up the test strip carrying the mobile gold nanoparticle labelled antibodies with it. The hormones are detected using known flow technology, for example as published in USlO/990,946 of Inverness Medical Switzerland GmbH. As previously described the test strip has a dry porous nitrocellulose membrane with two separate zones. The first zone contains highly specific anti-LH antibodies bearing the gold label, the labelled antibodies being freely mobile within the porous carrier when in moist state (therefore when in contact with a liquid sample). The second zone, which is the detection zone, contains unlabelled highly specific anti-LH antibodies permanently immobilised on the carrier material and therefore not mobile in the moist state. The labelled and unlabelled antibodies have specificities for different LH epitopes. The urines permeates via the first zone through to the second zone, and by doing so, migrate the freely mobile labelled antibodies to the second zone where they become bound, via the LH, to the immobilised unlabelled antibodies. Any excess of mobile labelled reagents from the first zone not bound to the LH (and therefore not participating in any binding reaction in the second detection zone) is flushed away from the detection zone by the continuing flow of the sample. When LH hormone is present in the urine, in sufficient concentration, the gold labelled antibodies and the immobilized phase antibodies bind to the LH molecules, forming a sandwich and a red positive test line forms. In addition, a quality control is included via deposition of second line of antibodies in the detection zone, which will always form a red control line to check the test has run correctly. A "myLotus" device similar to that described in the applicant's previous application uses this reads the result of this colorimetric lateral flow assay on the first and second set of test strips. The test strip is inserted into the device and the completed assay is viewed by a camera to detect the test line, check for the presence of a control line and then communicate a positive or negative result based on a predefined threshold. The device may also give a quantitative result providing the actual concentration of the LH present in the sample if the assay is sensitive enough.

Several known hCG urine sample concentrations across the range 1000-6.25 IU/L were used for the first set of applicators (having test strips according to the invention) and a second set of test strips (without the test strips of the invention). The result was measured for each set of applicator test strips 5 times with a repeat at each the different concentrations to determine the Co-efficient Variation (CV).

The results are shown in Tables 1 (laminated strips) and Table 2 (non-laminated strips).

Table 1

Table 2

As regards the CV, the applicants assert that the lower the integer observed, the more accurate the determination for any given test concentration, especially where the concentration in the sample is lower. As shown above in Table 1 and as further displayed graphically in Figure 6, the CV is a much lower figure at all the concentrations shown, meaning that whatever baseline concentration is measured (as this varies from woman to woman) the accuracy of the data for both the personal baseline tests and then further the accuracy between a first test and then further tests another remains constantly reliable. As expected the lower the concentration the more error occurs in the deviation. However, when this is compared to the result of the non-laminated test the CV is constantly higher and demonstrates that the laminate is able to retain a far improved consistent accuracy even at very low concentrations as compared to the data seen in the comparative example. The strength of the signal received by the device at the point of detection was also measured (Table 3). Table 3

Furthermore, there was a consistently improved strength in signal received across all concentration results in the laminated test strips, as compared to the non-laminated strips. The applicants believe this confirms that for any concentration in the realistically measurable range of analyte (such as this test of LH or similar) the measurement read from the laminated test strip of the invention permits a more likely accurate quantitative result to be processed from the signal received.

The applicant has therefore determined that the novel arrangement of the invention strongly improves detection and measurement in such assays. The applicant considers and hypothesises but is not limited to the understanding that the laminate enhances direction and force of the sample flow through porous membrane with greater efficiency. Ultimately this efficiency is converted into a surprising technical improvement, since that the data resulted possesses greater accuracy compared to test strips without the configuration and construction of the invention. Quantitative determinations can be made routinely with this reliable, accurate measuring ability.

It was further clarified during testing that introduction of lamination on a larger, non specific area of the strip resulted in poor performance. This was deemed to be due to reflectance within the analyser device when the test strip is inserted for analysis. Thus, the specific solution arrived at by the applicant is both novel and not an obvious modification which could only be achieved with experimental burden (as determined by the results section herein). Analyte variation

Other embodiments of the invention may extend to the use of other hormone analytes in such assays, particularly for example, where the field concerns fertility. The hormone analyte maybe specifically selected from luteinizing hormone (LH) and human chorionic gonadotropin hormone (hCG), Anti-Mullerian hormone (AMH), prolactin, progesterone (PSG), Estrogen (ESG), Inhibin B, Testosterone and follicle-stimulating hormone (FSH).

In particular, conditions or diseases concerning fertility which require a multitude of indicators to diagnose would benefit greatly from the invention. Usefully, where a combination of factors is required for diagnosis; accurate change within each of those indicators facilitates need to obtain quick diagnosis. For example, where different test strips can be identified and used with the same analyser to quickly, specifically and accurately measure any particular hormone analyte relevant to that diagnosis would be very useful and the combination of accurate quantitative personally derived data that results can be used to make an early warning of conditions such as polycystic ovary syndrome (PCOS). Such results can be used to follow up with a medical practitioner facilitating earlier stage diagnosis. Earlier intervention can be crucial in multi-faceted conditions, like PCOS, in which a delay in diagnosis or misdiagnosis, leads to infertility.

As described above, as well as LH and HCG testing, other analytes may be tested for in the lateral flow assay method or strip of the invention using the following marker examples:

Follicle-stimulating hormone (FSH)

FSH is excreted by the pituitary gland. During the follicular phase of the menstrual cycle, FSH fosters the growth and maturation of a woman's eggs. If a woman experiences infertility or approaches menopause, it may be a sign of a low ovarian egg supply - and this is indicated by a high level of FSH. With infertility issues, the FSH hormone is released in higher-than- normal amounts to stimulate the ovaries into producing a mature egg and more estrogen. Reference range:

FSH can be used for detection of menopause or infertility issues as it maybe used as a marker for confirmation of the LH surge. Testing should begin on cycle day 3. Follow up tests to confirm should be used on consecutive days following (cycle day 4 and 5). Cycle day 3 is defined as the third day of menstrual bleeding. The ratio of LH (Luteinizing hormone) to FSH (Follicle-stimulating hormone), when measured in international units, is elevated in women with PCOS. Common cut-offs to designate abnormally high LH/FSH ratios are 2:1 or 3:1 as tested on Day 3 of the menstrual cycle. The applicant developed the following FSH assay and exemplifies an embodiment of the invention in which LH is the analyte for use in determination of confirmation of LH surge. Sample used was urine, such as first morning urine and is tested in a sandwich assay in conjunction with the previously described myLotus device. FSH Assay Range is determined as OmIU/ml - 80mlU/ml:

my Lot: us FSH Assay

Progesterone (PPG)

This hormone is released by the ruptured follicle (one that has released an egg). After the egg is released from the follicle, the follicle closes and becomes a corpus luteum. The corpus luteum secretes increasing amounts of progesterone. This rise in the level of progesterone typically causes a rise in body temperature. If no pregnancy occurs, the levels of progesterone falls and this along with the decreasing amount of oestrogen, helps the built- up lining of the uterus to separate and for menstruation to begin.

Reference range of progesterone:

• Women at the beginning of their menstrual cycle: 1 ng/mL or under

• Before ovulation, progesterone levels are usually below 10 ng/ml

• In the middle of the second half of the cycle, midcycle, about 7-10 days after ovulation, progesterone levels are usually above 8-10 ng/ml.

• Women in the middle of their menstrual cycle: 5 to 20 ng/mL

• A low progesterone level especially a level below 10 ng/ml may indicate no ovulation

The breakdown product of progesterone in urine is pregnanediol glucuronide (PdG). Urine PDG demonstrates excellent agreement with P4 in both serum and urine. A rise in urine PDG above a certain threshold (typically 5 pg/mL) is required to confirm ovulation. Sample is urine; preferably a first morning urine sample.

The applicant has developed a working competitive PcG assay. In an embodiment of the invention PcG is therefore utilised the analyte and a marker for confirmation of ovulation as well as detection of luteal phase defect.

An assay Range: 0 pg/ml - 20pg/ml was determined as shown below:

Estrogen (E3G) Rising levels of estrogen are responsible for the build-up of the uterine lining (endometrium). This build-up of the lining gets the uterus ready to accept a fertilized egg. As the cycle continues, and no pregnancy occurs, the levels of estrogen decrease. Decreasing estrogen loosens the support for the built-up lining and helps to make it separate and prepare for menses. This demonstrates a characteristic peak approximately 1 day before the LH surge and 37 h before ovulation.

Reference range: the reference range estradiol in women varies by menstrual cycle and menopausal status, as follows: before menopause, estradiol levels are widely variable throughout the menstrual cycle:

Mid-follicular phase: 27-123 pg/mL

Periovulatory: 96-436 pg/mL

Mid-luteal phase: 49-294 pg/mL

Postmenopausal: 0-40 pg/mL

Following menopause: Under 10 pg/mL

Detection of elevated E3G concentrations, typically at concentrations between 20 and 30 ng/mL, demonstrates a characteristic peak approximately 1 day before the LH surge and 37h before ovulation. The same would typically be first morning urine. Estrogen may be used as a marker for prediction of LH surge and ovulation and thus in one embodiment of the invention is the selected analyte for testing and measuring via competitive assay.

Prolactin

This is one of several hormones produced by the pituitary gland. Prolactin has many different roles throughout the body. Perhaps the most important role of prolactin is to stimulate milk production in women after the delivery of a baby. High prolactin levels may cause infertility. Another term for high prolactin levels is hyperprolactinemia. Women who are not pregnant and are not breastfeeding should have low levels of prolactin. If a non pregnant woman has abnormally high levels of prolactin, it may cause her difficulty in becoming pregnant. Prolactin may cause infertility in several different ways. First, prolactin may stop a woman from ovulating. If this occurs, a woman's menstrual cycles will stop. In less severe cases, high prolactin levels may only disrupt ovulation once in a while. This would result in intermittent ovulation or ovulation that takes a long time to occur. Women in this category may experience infrequent or irregular periods. Women with the mildest cases involving high prolactin levels may ovulate regularly but not produce enough of the hormone progesterone after ovulation. This is known as a luteal phase defect. Deficiency in the amount of progesterone produced after ovulation may result in a uterine lining that is less able to have an embryo implant. Some women with this problem may see their period come a short time after ovulation. For unknown reasons, some women with PCOS may have slightly high prolactin levels.

In an embodiment of the invention Prolactin could therefore be used as an analyte marker for and detection of preeclampsia and/or infertility.

Reference range:

Normal levels for females are less than 25ng/ml

Normal levels for men are less than 15 ng/mL

Sample: Serum (or urine)

Format: Sandwich assay Anti-Mullerian Hormone (AMH)

This hormone has emerged as a marker of ovarian reserve and a possible surrogate measure of reproductive aging. AMH steadily decreases with age and generally becomes undetectable in the menopause transition. Anti-Mullerian Hormone levels are naturally lower in older women (particularly over the age of 40) and higher in women with Polycystic Ovaries (PCO) or Polycystic Ovary Syndrome (PCOS). In an embodiment of the invention AMH may be used for as the marker for ovarian function as well as PCOS indicator.

Reference range: A typical AMH level for a fertile woman is 1.0-4.0 ng/ml; under 1.0 ng/ml is considered low and indicative of a diminished ovarian reserve.

Samples would be whole blood, serum or plasma used in a sandwich assay Inhibin B

Inhibin B is a protein hormone produced by your ovaries. It works to inhibit FSH, which is responsible for helping your follicles to develop. Levels of Inhibin B decrease with age. Inhibin B is actually secreted directly by small, developing follicles in your ovaries. During the follicular phase of ovulation, small follicles eventually develop into mature eggs, ready for fertilization.

In embodiments, inhibin B maybe therefore be used an analyte for determining the quality and quantity of ovarian reserve. Reference range: a normal result is anything above 45 - 200 pg/mL; an abnormal result is anything below 45 pg/mL Sample: Whole blood, serum or plasma in a sandwich assay.

Testosterone is just one of several hormones known as androgens. High androgen levels in women are known as hyperandrogenism. Possible causes of high testosterone levels in women include: PCOS: Polycystic ovarian syndrome is a common cause of infertility in women. In embodiments of the invention, testosterone maybe used as an analyte for detection of PCOS and/or infertility. Reference range with whole blood or saliva for women are: 15 to 70 ng/dL total testosterone and 3 to 1.9 ng/dL free testosterone and for men are within 300 - 1,110 ng/dL.

Following successful execution and application of the novel test strip when used with an analysis device it is considered by the applicants that such an improvement in accuracy of the test data has even wider implications than the field of fertility. The lateral flow test strip of the invention enables personal determination of a negative health state with greater accuracy than has previously been possible. The ability to analyse and monitor such data with inherently improved accuracy derived from the quantitative measurement of other biological analytes, such as other hormones, permits home testing to extend to the early warning of different types of disease or condition including, for example, markers of hormone imbalance and conditions associated therewith or known markers of specific types of disease such as cancer.

Non-fertility marker

The analyte selected and utilised in the embodiment of the invention may be a different type of hormone analyte, such as thyroid stimulating hormone (TSH), as a marker for Hypo or Hyperthyroidism. In such cases, the sample is blood, serum or plasma. For example, TSH is often checked to assess the function of the thyroid, a gland responsible for metabolism regulation. Normal function of the thyroid is needed to optimize successful conception and maintenance of pregnancy. For example, women with hypothyroidism (underactive thyroid) have elevated levels of TSH. This hormone is frequently checked in women with infertility or recurrent pregnancy loss. A TSH level of 5mll/l or greater is indicative of hypothyroidism. A TSH level less than 5 mU/l but greater than 2.5mll/l is associated with implantation failure and early pregnancy loss. In such a case the reference range for thyroid stimulating hormone (TSH) is 0.3 to 3.0 mlU/l. However it is clearly established that reliable quantitative testing of this marker and determination of an under active or over active thyroid provides a greater analytical tool than application in fertility.

TSH can therefore also be used as a potential marker for the detection of Hypothyroid or Hyperthyroid using whole blood, serum or plasma in sandwich assay test strip in accordance with the examples of the invention.