HK1226814A1 - Improved pregnancy test device and method - Google Patents

Description

Improved pregnancy testing device and method

Technical Field

The present invention relates to a pregnancy test device, and to a method of performing a pregnancy test, and to a method of manufacturing the device.

Background

Simple lateral flow immunoassay devices have been developed and commercialized for detecting analytes in fluid samples, see e.g. EP 291194. Such devices typically comprise a porous carrier comprising a dried mobilisable labelled binding reagent capable of binding to the analyte in question; and an immobilized binding reagent that is also capable of binding to the analyte provided at a detection zone downstream of the labeled binding reagent. Detection of the immobilised labelled binding reagent at the detection zone provides an indication of the presence of analyte in the sample.

Alternatively, where the analyte of interest is a hapten, the immunoassay device may employ a competition reaction in which a labelled analyte or analyte analogue competes with analyte present in the sample for binding to an immobilised binding reagent at a detection zone. Alternatively, an assay device may employ an inhibition reaction in which an immobilised analyte or analyte analogue is provided at a detection zone, the assay device comprising a mobilisable labelled binding reagent for the analyte.

One assay device may be capable of detecting the presence and/or amount of more than one analyte. For example, in the case of an assay that detects the presence of drug abuse, the device may be capable of assaying an entire set of drugs. Such lateral flow immunoassay devices are typically provided with a plurality of detection zones, such zones being provided on a single or multiple lateral flow carriers within the assay device.

Determination of the results of the assay has traditionally been performed by eye. However, such devices require interpretation of the results by the user, which introduces an undesirable degree of subjectivity, particularly at lower analyte levels when the intensity of the detection zone is weak.

Accordingly, digital devices have been developed which comprise an optical detection means arranged to determine the result of the assay; and a display device for displaying the result of the measurement. Digital assay readers for use in combination with assay test strips for determining the concentration and/or amount of an analyte in a fluid sample are known, as are assay devices comprising an integrated digital assay reader. An example of such a device is disclosed in EP 1484601.

Light from a light source, such as a Light Emitting Diode (LED), is directed onto a portion of the porous support and reflected or transmitted light is detected by a light detector. Typically, the reader will have more than one LED to illuminate different regions of the carrier, and a respective light detector is provided for each of the plurality of LEDs. EP1484601 discloses an optical arrangement for a lateral flow test strip digital reading device comprising a baffle arrangement allowing the possibility of reducing the number of light detectors in the device.

Assay techniques of the above type have been implemented into "self-testing" pregnancy test devices. These are typically devices used by women who suspect that they may be pregnant. Therefore, they must be designed in such a way that they are easy to use (without any medical or technical training) and can typically be discarded after a single use. The device is typically a lateral flow immunoassay device, and testing is typically initiated by contacting a sampling portion of a lateral flow assay stick with a urine sample. The sampling portion of the wand may be immersed in a urine sample in a container, and more typically the user may urinate directly onto the sampling portion. The assay is then run without the woman having to perform any further steps and the results are indicated and read by eye or, in a digital device, the results are determined by an assay result reading means and displayed to the user by a display, such as for example a Liquid Crystal Display (LCD).

This routine pregnancy test is performed by measuring human chorionic gonadotropin (hCG) in a sample. hCG is produced by developing embryos and hCG concentrations in the sample above a certain threshold will trigger a positive (i.e., "pregnancy") result.

There is a need for an improved pregnancy test, in particular an improved self-testing pregnancy test: many women wish to know as soon as possible whether they are pregnant and therefore there is a need for a very sensitive test which is able to detect hCG in samples such as urine at very low concentrations. However, this creates a problem because hCG may sometimes be present in urine at relatively low concentrations for reasons other than pregnancy, such that a very sensitive pregnancy test may give a false positive result (i.e. the specificity of the assay is diminished).

As an illustration of this, non-pregnancy related hCG may be present in a urine sample. More particularly, hCG can be present in urine from perimenopausal and postmenopausal women and is derived from the pituitary rather than the developing embryo. In a pregnancy test, detection of such pituitary-derived hCG or other non-pregnancy related hCG will result in a false positive result for pregnancy. In many countries, women are delayed from home, for example due to work or other liability, until late in life, and so a smaller but significant market for "home" or self-test pregnancy test devices is constituted by older women who may fall into the peri-or post-menopausal age group and are therefore prone to false positive results if a sensitive hCG assay is used. According to one study, up to 10% of the OTC pregnancy tests have been marketed for women > 40 years old (Leavitt SA2006, "personal revolution: bulletin of the household pregnancy test in American culture" medical history (Bull. Hist. Med.) 2006; 80: 317-45).

According to the world health organization (world health organization), the accepted definitions of "menopause" and "peri-menopause" are as follows:

menopause (natural menopause) -is defined as the permanent cessation of the menstrual period caused by the loss of ovarian follicular activity. Natural menopause is thought to occur after 12 consecutive months of amenorrhea for which there are no other obvious pathological or physiological causes. Menopause occurs with the last menstrual period (FMP), which is known for certain only when reviewed for one year or more after the event.

Perimenopause-the term perimenopause includes the period immediately before menopause (when the endocrine, biological, and clinical features are near the beginning of menopause) and the first year after menopause.

Thus, for the purposes of the present invention, perimenopausal women are defined as those women WHO are also peri-menopausal according to the WHO definition above, and postmenopausal women are defined as those WHO have undergone menopause according to the WHO definition above.

Self-test or "home" pregnancy tests need to be reliable in order to ensure that women take appropriate action on receiving their test results. One desirable reliability target is 99% accuracy (i.e., a combined false positive and false negative rate of 1% or less). Currently available self-test or home test equipment is capable of detecting urinary hCG at concentrations of 25mIU/ml or higher with a sensitivity of 99% or more. Such devices are capable of achieving the desired accuracy target of > 99%, but are only used on or after the day the subject expects to start his menstrual period (i.e. the first day of expected menstrual bleeding), since from this point in time almost all pregnant women will have a urinary hCG concentration of 25mIU/ml or more, and the level of non-pregnancy related hCG will not normally reach this level. However, it follows that if a woman uses a self-test device before the day of their expected menstruation period, a false negative result is possible because the urinary hCG concentration has not reached a level detectable by the test. Thus, currently available conventional self-test pregnancy test devices are not 99% accurate when used prior to the day of the expected menstrual period. Indeed, the median level of hCG in urine at 10 days post-ovulation is about 8.4mIU/ml, and only about 10% of samples from that day will have hCG concentrations > 25 mIU/ml. Thus, very low pregnancy detection rates will be observed early in pregnancy using a conventional self-test device with a sensitivity of 25 mIU/ml. At 11 days post-ovulation, the median level rose to 19.8mIU/ml, so less than 50% of women at this stage would likely receive a positive result. Based on the 25mIU/ml test sensitivity, the detection rate for subsequent tests would be about 70% (day 12), 80% (day 13), and almost 100% (day 14). The use of only a more sensitive test is not a solution as this increases the risk of a false positive result, since the test is still not ≧ 99% accurate due to the increased likelihood of detection of non-pregnancy related hCG.

Therefore, there is a need for a pregnancy testing device, in particular a self-test or home test device, which is capable of detecting pregnancy with an accuracy of ≧ 99% even when used at a point in pregnancy earlier than the day of the expected menstruation period.

In a different context, in many countries, female patients of almost all childbearing ages are routinely tested for serum hCG before any medical intervention that may harm a developing fetus is performed. In perimenopausal and postmenopausal women, the problem of elevated serum hCG levels due to "pituitary" hCG is identified. Snyder et al, (clinical chemistry)2005511830-1835) the change in serum hCG concentration with age of non-pregnant women was examined and the use of serum Follicle Stimulating Hormone (FSH) measurements as a help to explain the higher than expected hCG results was investigated. They suggest that a combination of serum hCG measurements, knowledge of the age of the subject, and serum FSH measurements may be used to reduce or avoid "false positive" pregnancy outcomes. However, these workers are not concerned with self-test pregnancy tests, and in particular, with detecting pregnancy at a very early stage.

Summary of The Invention

In a first aspect, the present invention provides a test device for detecting pregnancy in a human female subject, the test device comprising:

an assay device for measuring the absolute or relative amount of hCG in a sample from the subject;

an assay device for measuring the absolute or relative amount of FSH in a sample from the subject;

and an assay device for measuring the absolute or relative amount of one or more progesterone metabolites in a sample from the subject.

The sample may be any suitable bodily fluid, such as whole blood, plasma, serum or urine. However, urine samples are strongly preferred, as such samples are readily available and do not require an invasive procedure. In addition, the use of urine samples facilitates self-testing by the user.

In this context, the amount of an analyte present in a sample may be determined in an absolute sense (e.g., in terms of a number per unit volume) or in a relative sense (e.g., by reference to a predetermined threshold). In particular, "relative amounts of one or more progesterone metabolites" andis notIt is intended to mean that the concentrations of different progesterone metabolites in a sample are compared to each other, but that the concentration of one or more such analytes can be compared to a predetermined threshold. These assay devices will be adapted and configured to measure their corresponding analytes, but as explained below, one assay device may be adapted and configured to measure the amount of two or more different analytes.

By way of explanation, after conception, further cycles of ovulation are not necessary, as the woman is already pregnant. Thus, after conception, folliculogenesis (folliculturargensis) is inhibited by suppression of FSH production. Thus, in a pregnant female, elevated hCG levels and low FSH levels would be expected to be observed. Therefore, measurement of FSH can be used to help explain the significance of detection of slightly elevated hCG levels, especially where it is suspected that this may be due to a non-pregnancy related source.

By way of further explanation, during the first 10-12 weeks of pregnancy, progesterone produced by the corpus luteum supports the endometrium, allowing pregnancy to continue. Progesterone levels are elevated during the luteal portion of the menstrual cycle, but if pregnancy does not occur (i.e., if the subject is menstruating), the levels will fall back to baseline levels. However, if pregnancy occurs, the level of progesterone (and its urine metabolites) will remain elevated and will continue to rise during pregnancy, such that progesterone (and its urine metabolites) can be used as an adjunct to hCG as an additional confirmation of pregnancy.

The test device will preferably further comprise means for interpreting the assay results in order to determine the outcome of the pregnancy test, and preferably a display means for displaying the outcome of the test.

In a second aspect, the present invention provides a method of detecting pregnancy in a human female subject, the method comprising the steps of: contacting a sample from the subject with a test device according to the first aspect defined above. As mentioned above, the sample is preferably a urine sample.

Preferred features of the invention will be described in further detail below. It should be clear that when these preferred features are described in relation to the test device of the first aspect, they will apply equally to the method of the second aspect (and vice versa), unless the context indicates otherwise.

The test device may comprise a microfluidic-based assay having one or more fabricated capillary channels (typically with defined pores) along which a liquid sample may flow; but more preferably the test device comprises a lateral flow assay. The test device may comprise both a microfluidic assay and a lateral flow assay. In one embodiment, the test device will comprise three lateral flow assays, wherein each assay is directed to a corresponding one of each of the three analytes (i.e., hCG; FSH; and a progesterone metabolite).

In some embodiments, it may be desirable to have more than one assay for a particular analyte. For example, one assay may be a microfluidic assay and another assay may be a lateral flow assay. Alternatively, both assays for the analyte may be microfluidic assays or may be lateral flow assays. Where both assays for an analyte are lateral flow assays, they may be on a single lateral flow test strip or on separate lateral flow test strips.

For example, hCG levels increase exponentially during early pregnancy, and thus the concentration of hCG in a urine sample from a pregnant subject may vary significantly, depending on how long the subject has been pregnant. It may therefore be desirable to provide an assay which is a relatively high sensitivity assay for hCG and an assay which is a relatively low sensitivity assay for hCG, such that the concentration of hCG in a sample can be determined over an extended concentration range. (by way of explanation, high levels of analyte may cause complications known as the hook effect. this may lead to inaccuracies in high sensitivity assays due to limited assay range, and thus it may be beneficial to provide an assay with reduced sensitivity, allowing accurate measurements to be made at very high analyte levels).

Typically, progesterone is not present in urine in detectable amounts. Instead, it is metabolized and various progesterone metabolites are excreted in the urine. The assay of the invention is thus suitable for measuring one or more progesterone metabolites in urine. Progesterone metabolites can be divided into 4 groups: pregnanolone, pregnanedione, and a final group containing compounds more polar than pregnanediol. In theory, one or more metabolites from any of these four groups may be suitable for use in the measurements of the present invention. Preferred examples include pregnenolone (3 α -hydroxy-5 β -pregn-20-one) and pregnanediol (5 β -pregn-3 α -20 α -diol). The latter compound is particularly preferred for testing since it is the progesterone metabolite which is usually present in urine at the highest concentration (Cooke, I.D.1976, progesterone and its metabolites (Progesteron and peptides) in Loran (Lorraine), J.A. and Bell (Bell), E.T. (ed) "hormone assay and their use (Hormone assay) page 447-508). It will be clear to the person skilled in the art that, unless the context indicates otherwise, reference to, for example, pregnanediol and pregnenolone encompasses their commonly occurring derivatives. In particular, progesterone metabolites are usually present in urine as glucuronides or occasionally as sulfates. Thus, reference herein to, for example, a pregnanediol specifically encompasses pregnanediol-3-glucuronide ("P-3-G"), sometimes also referred to as PdG. Furthermore, unless the context indicates otherwise, references herein to "progesterone" or "progesterone assay" are intended to specifically encompass progesterone metabolites and assays for progesterone metabolites, respectively.

It will be appreciated that the degree of structural homology, and thus antigenic similarity, between the various metabolites of progesterone can be very high. Thus, in an immunology-based assay, an antibody that binds a particular progesterone metabolite with a first binding affinity may bind a different progesterone metabolite with a second binding affinity, which may not be significantly lower than the first binding affinity, such that the antibody may cross-react to some extent. Alternatively, a highly specific antibody may be employed that has a much higher (e.g., at least 10-fold or higher) binding affinity for a particular progesterone metabolite than for other progesterone metabolites present in human urine. Thus, an assay for "a progesterone metabolite" can frequently detect a variety of different progesterone metabolites. The degree of cross-reactivity of any agent for different progesterone metabolites is not critical to the present invention, but it will be understood by those skilled in the art that this, along with other characteristics, may need to be reflected in the choice of agent and the choice of an appropriate "threshold", as explained below.

Similarly, several variants (e.g., fragments, such as the isolated β chain) and different isoforms of FSH (see walton W.J. (walton w.j.), et al, journal of clinical endocrine and metabolism (jclinendocrinolometab.), 86, 3675-85, 2001; dall k.d. (Dahl, k.d.) and stethon M.P. (stonem.p.) journal of andrology (janogray) exist in humans,1311, 1992; and betzger J.U (Baenziger, J.U.) and green E.D (greene.d) biochem. acta,947287-306, 1988) (e.g., with varying degrees of glycosylation), and an immunological-based assay can utilize relative specificityAn antibody that binds a single variant, fragment or isotype of FSH, or a less specific antibody that binds several different human FSH variants with acceptably high affinity may be utilized. Again, this may have an impact on the choice of reagents and/or the choice of a threshold value selected in the assay, but is generally not critical to the operation of the invention. Similar comments apply to hCG and variants thereof.

In summary, the underlying concept of the present invention is to achieve that a very high sensitivity hCG assay can be used to detect very early pregnancy and that by additional measurements of FSH and progesterone metabolites the specificity of the test can be maintained at an acceptably high level (i.e. avoiding a large number of false positive results due to the detection of hCG from non-pregnancy related sources) without knowing the age of the subject. The assay device is particularly suitable as a simple PoC or more specifically self-testing device without any medical or technical training for its use. In particular, the assay device is preferably disposable after a single use. Furthermore, the device is desirably a simple lateral flow or microfluidic based device in which various assays can be performed automatically without any further user intervention once the device has been contacted with a sufficient volume of urine sample. It is further preferred that the device is a digital device, i.e. the outcome of the determination is read and displayed to the user.

In particular, the assay device of the invention is preferably a self-test or home test device. In a preferred embodiment, the assay device has an accuracy of > 99%, even if used before the day of the expected menstruation period. More particularly, the assay device of the invention may achieve an accuracy of ≧ 99% even if used 2 days before the day of the expected menstruation, preferably even if used 3 days before the day of the expected menstruation, preferably even if used 4 days before the day of the expected menstruation, and preferably even if used 5 days before the day of the expected menstruation and more preferably even if used 6 days before the day of the expected menstruation (e.g., as calculated with reference to the LH spike or LH surge).

Tests for hCG may indicate positive (pregnant) results (i.e. those samples in which urinary hCG is above a predetermined threshold), which can be confirmed (or negated) by the results of FSH and progesterone metabolite assays. Thus, for example, a level of hCG that is just above a predetermined threshold (with an elevated FSH level and a lower progesterone metabolite level) is indicative of a subjectIs prepared fromPregnancy (and suggesting that elevated hCG levels have a source not associated with pregnancy).

In this manner, the present invention provides a pregnancy test that is capable of detecting pregnancy with high sensitivity (i.e., detecting 99% or more pregnant subjects), with high specificity (i.e., a false positive rate of 1% or less), and moreover is capable of achieving these results even when these subjects include perimenopausal and postmenopausal women; and even more surprisingly, these results could be achieved at a single point in time (day) in the very early stages of pregnancy (i.e. even before the day of the expected menstruation) and without any other external information like for example the age of the subject.

Another advantage of the present invention is that it can avoid false negative results that can occur when a subject has very high levels of hCG β core fragment in the case of point-of-care hCG-based pregnancy test devices this problem has been recognized in the art and is described, for example, by Glonoviski (Gronowski) et al (2009 clinical chemistry)551389-1394) because the artificially low signal of hCG caused by the inhibition of the excess β core fragment would be compensated by the measurement of FSH and progesterone metabolites that would not be affected by the excess β core fragment.

Advantageously, one single urine sample will be used to provide a test sample for each of the (at least) three analyte assays performed by the assay device/method of the invention. Suitably, all three analyte assays will be performed substantially simultaneously, preferably using one single urine sample applied to the assay device. This may conveniently be achieved using (inter alia) the embodiments described below.

For the purposes of the present invention, when the three analyte tests are performed using urine samples derived from the same urination event, the tests will be performed "substantially simultaneously" and the results of the assays are read within a period of 10 minutes of each other, preferably within 5 minutes of each other, more preferably within 3 minutes of each other, and most preferably within 60 seconds of each other. Desirably, all three analyte tests are initiated by the user applying a urine sample to a sample contacting portion of the testing device at substantially the same time (i.e., within 60 seconds of each other).

In a preferred embodiment, the various aspects of the invention have one or more (desirably all) of the following features:

(i) sensitivity of 99% or higher;

(ii) a specificity of 99% or more;

(iii) (iii) is capable of achieving (i) and (ii) even on the test day early in pregnancy (i.e. before the day of the expected menstruation period, as defined herein);

(iv) (iii) enable (i) and (ii) even when testing/using the assay device once at a single point in time (i.e. on a single day);

(v) the assay device/method does not require any external information (e.g. the age of the female or any history of the subject, such as previous measurements of hCG, FSH or progesterone); and

(vi) (iii) and (ii) are achieved even when the subject comprises a perimenopausal and/or postmenopausal woman (as defined herein).

For the purposes of this specification, "early" in pregnancy means prior to the day of the expected menstrual period, as calculated by reference to the peak level of luteinizing hormone [ "LH peak" ] detected in women at or near ovulation time. By way of explanation, the expected day of the menstrual period is generally considered to be 15 days after the LH peak.

The inventors have calculated that using the apparatus and method of the invention it should be possible to establish true pregnancy status from an assay at a single time point, even in perimenopausal and postmenopausal women, with a sensitivity of 99% or more and a specificity of 99% or more.

The expected day of the menstruation period can be calculated by referring to certain time points. In particular, the expected day of menstruation can be calculated by reference to the day of LH surge (i.e. the day on which a significant increase in LH level is first detected in a cycle), which typically occurs about 12-24 hours before the LH peak. The day of the expected menstrual period can also be calculated by adding the usual cycle length (days) to the date of the last menstrual period. Other ways of calculating the expected menstrual period include adding 28 days to the date of the last menstrual period.

Generally, the result of the analyte determination will be determined after a particular time has elapsed (typically, but not necessarily, by reference to the time at which the sample is in contact with a sampling area of the assay device). The time at which the analyte determination is determined may be referred to as t_E. The assay result reading device may include some sort of integrated timing means to determine the attainment of t_ETime of (d). The timing means may be actuated automatically by contacting the sample with the assay device (e.g. by a liquid sample allowing a current to flow), or may be triggered by the user (e.g. pressing a switch or the like) or by any other convenient means. The assay reaction may suitably be at t_EEquilibrium is achieved, but this is not required. For all analyte determinations carried out simultaneously, t_ECan be reached at the same time, or t_EMay be different for different analytes.

In some embodiments, if an analyte determination signal is at t_EStill below an upper threshold, the result of the assay is negative (where it is the presence of the analyte of interest that results in a signal)Among those forms formed). The end point of the assay may not necessarily be at the completion of the reaction. Indeed, end point t_EWill generally be considered to have been reached before the reaction is complete.

t_EThe endpoint may conveniently refer to a particular point in time (i.e., t)_ECan be considered to occur a specified amount of time after the assay begins, e.g., a specified time interval after activation of the reader and/or insertion of an assay stick into the reader and/or application of a sample to the test stick) is determined by the reader. For purposes of illustration, t_EWill typically occur between 1 and 10 minutes, preferably between 1 and 5 minutes after the start of the assay.

Desirably, the assay result reader will be programmed to repeat the test measurement if a moderate signal is obtained. In a simple embodiment, at t_EThe measurement was repeated. However, it is preferred to repeat the measurement one or more times before the endpoint. Most preferably, the reader device is programmed to repeat the measurement at regular time intervals (e.g. 1 or 5 second time intervals) until the signal exceeds an upper threshold or until t is reached_EWhichever occurs first.

It is desirable to include a clock or other timing device in the assay result reader so that the reader can automatically make measurements at predetermined points in time without further user input.

Thus, for example, the reader may be programmed to be at an initial point in time t. Making measurements and, if necessary, repeating the measurements at any desired time interval thereafter until the signal exceeds the upper threshold or t is reached_EAs described above.

In addition, a clock or other timing device assists the reading device in determining the rate of signal accumulation. The rate of signal accumulation can be easily calculated if the measurement of the amount of signal is made at two or more points in time (with a known time separation).

It should be noted that the rate or amount of signal accumulation can be measured in an absolute sense or as a relative value (e.g., as compared to a control or other comparative value, optionally obtained from a substantially contemporaneous reaction).

Specifically, in some embodiments, if one or more analyte assay readings are all well above or below (as appropriate) a particular relevant threshold level, the assay result reader may be at t_EThe assay results (i.e., pregnant or not pregnant) were previously determined. An "early" determination of the results of the measurements carried out in this way is described in EP 1484613. In this example, for example, if the amount of FSH in a sample is above a predetermined upper threshold (at a level at which pregnancy has never been observed), the FSH assay signal will develop very rapidly, and this may allow the assay result reader (or a human observer) to read at t_EThe outcome of the assay was previously determined as an "not pregnant" result, and it may not be necessary to wait for the results of the assay to analyse hCG and/or progesterone metabolites. Similarly, if the level of progesterone metabolite in the sample is very low (e.g., less than 1 μ g/ml), a strong progesterone metabolite assay signal will be at the time of reaching the progesterone metabolite assay t_EThe former is formed very rapidly, allowing the assay result to be determined early as "not pregnant", again possibly without reading the hCG assay and/or FSH assay.

The "early" determination that the assay result can be at t for any of the three analytes (i.e., hCG, progesterone metabolite, and FSH) is achieved_EThe method is carried out before; or may be at t to any combination of two of these three analytes (i.e., hCG and progesterone; hCG and FSH; or progesterone metabolite and FSH)_EBefore; or on reaching t for only one of the three analytes_EBefore (i.e., t at the other two of the three analytes)_EOr at the t_EThereafter) is performed.

Similarly, there may be a lower threshold, an intermediate threshold, and an upper threshold for all three analyte determinations or any one of the three analyte determinations or for any combination of two of the three analyte determinations.

The test device will comprise at least one flow path, preferably at least two flow paths, and typically three or even four flow paths.

For the purposes of the present invention, the term "flow path" refers to a substrate capable of transporting a liquid from a first location to a second location and may be, for example, a capillary channel, a microfluidic path, or a porous support such as a lateral flow porous support. The porous support may comprise one or more porous support materials that may overlap or be fluidly connected in a straight line or stacked arrangement. These porous support materials may be the same or different. The various assays of the test device may be provided on separate substrates, or they may be provided on a common substrate, such that liquid conveyed along one flow path of one assay cannot pass over to a flow path of a different assay. For example, the first assay and the second assay may be disposed on the same porous carrier such that the first flow path and the second flow path are separated from each other. This can be achieved, for example, by: laser cutting portions of the porous support to render the porous support non-porous, thereby separating the first assay and the second assay. Alternatively, a non-porous barrier material may be applied along a strip to provide two or more (typically substantially parallel) flow paths on the same porous support. In other embodiments, one single flow path may accommodate testing for two or even three different analytes. For example, a single flow path may include reagents for testing hCG, and may also include reagents for testing a progesterone metabolite. Alternatively, one single flow path may comprise reagents for testing hCG and FSH; or a single flow path may comprise reagents for testing FSH and a progesterone metabolite. In particular, a flow path may include two detection zones, one for each analyte, where a labeled reagent may tend to accumulate in a manner that is generally proportional (directly or inversely) to the concentration of the corresponding analyte in the sample.

One or more flow paths in the test device may include a lateral flow porous carrier. Suitable materials that may be used as a porous support include nitrocellulose, cellulose acetate, cellulose or cellulose derivatives, polyesters, polyamides, polyolefins or glass fibres. The porous carrier may comprise nitrocellulose. This has the following advantages: a binding agent can be firmly immobilized without prior chemical treatment. For example, if the porous solid phase material comprises paper, immobilization of one antibody may be performed by chemical coupling using, for example, CNBr, carbonyldiimidazole or trifluoroethanesulfonyl chloride.

The assay devices/methods of the present invention typically utilize one or more binding reagents. Typically, hCG assays utilize one binding agent that binds hCG, FSH assays utilize one binding agent that binds FSH, and progesterone metabolite assays utilize yet another binding agent that binds one progesterone metabolite. In particular, the assay may comprise the use of a labelled binding reagent. As explained elsewhere, one labelled binding reagent that binds both hCG and FSH may be used, as the two molecules have some common structure.

For the purposes of the present invention, the term "binding agent" refers to one member of a binding pair, i.e. two different molecules, wherein one of these molecules is chemically and/or physically bound to the second molecule. These two molecules are related in the following sense: their binding to each other is such that they are able to distinguish their binding partners from other assay components having similar characteristics. Members of a binding pair are referred to as ligands and receptors (anti-ligands), binding pair members and binding pair partners, and the like. A molecule can also be a binding pair member of an aggregate of molecules; for example, an antibody directed against an immune complex of a second antibody and its corresponding antigen may be considered a binding pair member of the immune complex.

In addition to antigen and antibody binding pair members, other binding pairs include (by way of example and not limitation), biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, a peptide sequence and an antibody specific for the sequence or the entire protein, polymeric acids and bases, dyes and protein binding agents, peptides and specific protein binding agents (e.g., ribonuclease, S-peptide, and ribonuclease S-protein), and the like. In addition, specific binding pairs may include members that are analogs of the original specific binding member.

When used in the context of a label binding agent, "label" refers to any substance capable of producing a signal that is detectable by visual or instrumental means. Various labels suitable for use in the present invention include labels that generate a signal by either chemical or physical means, such as optically detectable labels. Such labels include enzymes and substrates, chromogens, catalysts, fluorescent compounds, chemiluminescent compounds, electroactive species, dye molecules, radioactive labels, and particle labels. The particle labels may comprise magnetic or charged labels, which may be detected magnetically or electrochemically. The label may be covalently attached to the binding agent. In particular, the label may be derived from an optically detectable label. Preferred optically detectable labels include colloidal metal particle labels and particles with dyes, as described below.

The label may comprise a colloidal metal particle, such as gold, silver, platinum, silver-enhanced gold sol, carbon sol or carbon nanoparticles; colloidal metalloids or non-metallic particles, such as tellurium and selenium; or dyed or coloured particles, such as a polymer particle incorporating a dye or a dye sol. The dye may be of any suitable colour, for example blue. The dye may be fluorescent or comprise a quantum dot. Suitable fluorescent materials are well known to those skilled in the art. Dye sols can be prepared from commercially available hydrophobic dyes such as ForonBlueSRP (Sandoz) and ResolinBlueBBLS (Bayer). Suitable polymer labels may be selected from a range of synthetic polymers such as polystyrene, polyvinyltoluene, polystyrene-acrylic acid, and polyacrolein. The monomers used are generally not water-soluble and are emulsified in an aqueous surfactant so that monomer micelles are formed, and then polymerization of these monomer micelles is induced by adding an initiator to the emulsion. Resulting in substantially spherical polymer particles. One desirable size range for such polymer particles is from about 0.05 μm to about 0.5 μm. According to an exemplary embodiment, the label is a gold colloid having a preferred average particle diameter in the range of 0.02 μm to 0.25 μm.

The dried binding reagent may be disposed in the flow path of a microfluidic device or on a porous support material provided upstream of a porous support material comprising a detection zone in a lateral flow device. The upstream porous support material may be macroporous. The macroporous support material should be low or non-protein binding, or should be easily blockable by reagents such as BSA or PVA in order to minimize non-specific binding and to facilitate free movement of the labeling reagent after the macroporous support has become wetted by the liquid sample. The macroporous support material may be pre-treated with a surfactant or solvent (if desired) in order to make the macroporous support material more hydrophilic and to promote rapid absorption of the liquid sample. In addition, one or more sugars (e.g., sucrose, trehalose) may be used to stabilize and help mobilize the labeling reagents. These may conveniently be applied to the flow path and/or porous support material as part of a solution to which the labelling reagent is applied. Suitable materials for a macroporous support include plastic materials such as polyethylene and polypropylene; or other materials such as paper or fiberglass. Where the labeled binding reagent is labeled with a detectable particle, the macroporous support may have a pore size at least ten times larger than the maximum particle size of the particle label. Larger pore sizes result in better release of the labeling reagent.

The test apparatus will suitably comprise three or more flow paths: one corresponding flow path for each analyte assay. However, it is possible that two or more analyte assays may share a common flow path (possibly in addition to an optional common sample application zone, as described below). The flow path will typically comprise a capillary or other microfluidic flow channel, or one or more porous members, along which an aqueous liquid (such as a urine sample) can be transported.

In one embodiment, the test device comprises three separate lateral flow assay strips, one for each analyte assay. In another embodiment, the test device may comprise two lateral flow assay strips; one of the strips is used to make an assay for one of the analytes (e.g. hCG) and the other strip is used to make a corresponding assay for each of the other two analytes. An embodiment is also envisaged in which one flow path or lateral flow test strip is used to test all three analytes.

In particular, the test device may comprise a sample application zone. The sample application zone is a zone of porous (typically absorbent) material to which an aqueous sample such as urine can be applied. The sample application zones may be one common zone. That is, one liquid sample applied to a common sample application zone may be delivered to two or more different assay flow paths.

Typically, the sample is applied to the sample application zone by the user urinating directly onto the sample application zone.

In a preferred embodiment, the assay device comprises a housing containing most or all of the functional components of the assay and the assay reagents. The housing is conveniently formed from a waterproof synthetic plastics material and is preferably substantially opaque. An opacifier may be added to the plastic material to achieve the desired level of opacity. Specifically, suitable synthetic plastic materials for forming the housing include polycarbonate, polystyrene, copolymers of polystyrene, polyolefins, polypropylene, polyethylene, and acrylonitrile. The housing may desirably be formed in two or more parts which are joined together with most (or all) of the remainder of the assay device components housed within or between the assembled parts of the housing. These parts of the housing may be joined and fastened by conventional fastening means, such as snap-fit action or by plastic welding or the like.

In a preferred embodiment, the assay device comprises a housing, (typically having the features described above), and wherein a sample application portion or zone extends beyond the housing, so as to facilitate the application of a urine sample to the sample application portion or zone. The sample application portion or zone extending beyond the housing may be covered by a removable cap prior to use. The cap may also preferably be formed from a synthetic plastics material which may be opaque or transparent or translucent. Typically, the cap is replaced once the sample has been applied to the sample application zone.

An absorbent "sink" may be provided at a distal downstream end of the assay flow path. A common slot may be provided or a slot may be provided at the distal end of each assay. The absorbent sink may preferably comprise a highly absorbent material, such as, for example, CF7 Whatman (Whatman) paper, and should provide sufficient absorbent capacity to remove any unbound label from the vicinity of the detection zone. As an alternative to such a slot, it may be sufficient to have a length of porous solid phase material extending beyond the detection zone. One advantage of providing a highly absorbent sink is that it removes or substantially removes excess labeled binding reagent from the flow paths of these respective assays. This has the effect of minimising the extent of unbound labelled binding reagent in the vicinity of the corresponding zone and thus enables the assay flow path to be used in devices which may have different amounts of labelled binding reagent.

The test device will conveniently include other features known to those skilled in the art and common in conventional self-test or home test pregnancy test devices, including (but not limited to) sample sufficiency indicators (e.g. as described in PCT/EP 2013/061178), a "flood protection" pad (e.g. as disclosed in WO 2012/069610), the use of sample flow as an assay control (e.g. as disclosed in EP1,484,611; US6,194,222); an "early" determination of a positive or negative result (e.g., as disclosed in EP1,484,613; US5,679,584); automatic "wake-up" of the electronic device (e.g., automatic actuation upon wetting of the device by the sample, which forms a circuit); and using a digital assay schedule (e.g. as in european community design (european community design) accession number 1367304) or a "colour change core" or the like which gives the user a visual indication when the sample has been applied to the test device and/or a visual indication that the assay has commenced (e.g. WO 2003/058245). The test device will advantageously be presented to the consumer in a waterproof package, optionally further comprising a desiccant, such as a sachet of silica gel.

These assays will conveniently be supported on some sort of support or backing layer to provide mechanical strength and a suitable degree of rigidity. The one or more assay plus support may conveniently be referred to and provided as a test stick. All three assays can be on a single stick, or on two, three, or even four (i.e., more than one stick for one analyte) test sticks. The one or more test sticks may be adapted and configured to be inserted into the testing device by a user, or the one or more test sticks may more preferably form an integral part of the testing device purchased by a consumer, with the one or more test sticks pre-inserted or loaded in the testing device.

The results of these assays may be read by an external assay result reading device or directly by a user, but more preferably the test device will comprise an integral assay result reader for reading the results of an assay. The assay result reader preferably reads the results of all three analyte assays. The measurement result reader will advantageously comprise an electronic component, in particular a digital electronic component, such as a microprocessor. Typically (but not necessarily), these assays are read by optical means, i.e., measuring the amount of light reflected and/or transmitted by a detection zone, where an optically labeled reagent tends to accumulate in a manner that is proportional (directly or inversely) to the concentration of the analyte in the sample. Alternatively, these assays may be read by, for example, magnetic or electrochemical measurements. Clearly, the manner in which the assay is read may depend on the nature of the label or labels used to label the assay reagent or reagents.

An external assay result reading device may comprise a dedicated result reading apparatus (e.g. similar to that described in EP 1066530). Alternatively, the external assay result reader may be "non-dedicated", such as a mobile phone or other portable electronic device (e.g. a tablet computer), preferably provided with a camera, wherein the assay result is read by measuring the signal strength generated by a visible marker.

The assay result reader (whether external or forming part of an integrated assay device/assay result reader) may read and interpret the assay results, or may transmit the assay result data to a remotely located device for interpreting the assay data. The measured data may be transmitted to the remotely located device in real time. Data may be transmitted via an internet connection, or may be stored on a memory device (such as a "flash" drive, etc.), physically transferred to a remote device, or the data may be transmitted via wireless communication means (e.g., bluetooth, near field communication [ NFC ], etc.).

A microprocessor may control the operation of the optical reading or other assay reading means and will conveniently be programmed with or have access to the relevant assay signal threshold for each analyte, compare the actual assay signal value with a predetermined threshold and interpret the assay result to determine the outcome of the pregnancy test.

The assay result reader therefore comprises the necessary components for reading the results of these assays. The test device will also advantageously include a measurement result display for displaying the outcome of the pregnancy test to the user. Typically, the display will comprise an LCD, but other types of displays are possible (e.g., using "electronic ink"). In those embodiments where the assay device (or more precisely a component thereof) requires a power source to operate, then the assay device will preferably be provided with an integrated power source, such as a battery. Very small and inexpensive batteries are readily commercially available. The device may also be provided with a switch to connect the integrated power supply and activate the device.

In alternative embodiments, the results of these assays may be read directly by the user in a manner known from conventional "self-test" pregnancy tests, e.g., by the user examining one or more windows overlying the assay detection zones to determine the presence or absence of a detectable signal at one or more (or all) detection zones. Each detection zone may be provided with a separate window in an opaque housing to allow a user to inspect the detection zone. Alternatively, one large window may accommodate two or more (or all) detection zones. Typically, in such user-read devices, the user will directly examine the detection zone of a lateral flow or microfluidic assay. In other forms, the user may determine the result by reference to a color chart or indicator. Conveniently, the test device will be provided with instructions or guidance for reading the assay results (if the device is not intended for the user to interpret the assay results). For example, a printed color chart may be provided to the user to facilitate interpretation of such direct-read visual tests.

A combined pregnancy test stick/assay result reader device with display may be referred to as a "digital pregnancy test" and such digital test devices are commercially available and may be adapted with the benefit of the present disclosure to provide a test device according to the present invention.

The microprocessor will desirably be programmed to cause the assay result reading means to read the results of one or more assays; interpreting the results of these measurements; and displays the conclusion to the user.

The means for reading the results of these assays will preferably comprise at least one light source and at least one light detector. The at least one light source is preferably a Light Emitting Diode (LED). The at least one photodetector is preferably a photodiode or a phototransistor. The light source illuminates a detection zone on the assay that tends to accumulate a labeling substance during the course of conducting the assay in a manner that depends on the concentration of the analyte of interest in the sample applied to the assay. The label material may accumulate in a positive correlation with the concentration of the analyte (i.e., the higher the concentration of the analyte, the greater the amount of label accumulated in the detection zone). Typically, a sandwich assay format is employed, which is well known to those skilled in the art. Alternatively, the accumulation of the label substance may be inversely related to the concentration of the analyte (i.e., the higher the concentration of the analyte, the smaller the amount of label accumulated in the detection zone). This negative correlation is a characteristic feature of a competitive or inhibitory type of assay, which is typically employed when the analyte of interest is a hapten and/or too small to accommodate simultaneous binding of two different antibodies (e.g., as in the case of progesterone metabolites).

Both FSH and hCG are heterodimeric molecules comprising α and β subunits. The alpha subunits of FSH and hCG are substantially identical, such that antibodies directed against the alpha subunits can bind both FSH and hCG. An assay for both molecules may thus potentially utilize a common reagent, which may be, for example, a mobilisable directly labelled (i.e. optically detectable) antibody. The corresponding detection zone may comprise an immobilized capture antibody specific for the beta subunit (which differs between FSH and hCG). Obviously, this involves a sandwich immunological assay, but other assay formats are well known and may be used instead.

It is possible that if a sample contains high levels of hCG (e.g. because the sample is provided by a subject with a relatively advanced pregnancy), an antibody specific for the common alpha subunit antibody may be "engulfed" by the high levels of hCG, effectively reducing the amount of antibody available to bind FSH and may thereby cause an underestimation of the amount of FSH present in the sample. In practice this is probably unlikely to cause any problems, as such high levels of hCG will almost always (> 99.9%) be due to pregnancy, which will be correctly detected and interpreted by the test device regardless of any FSH assay results. It is also possible to use a dual beta subunit specific antibody pair for the hCG assay and a dual beta specific antibody pair for the FSH assay. This format would negate the above-described effects observed at high analyte levels.

In view of the need to minimize costs, (especially in those embodiments where the device is discarded after a single use), it is preferred to use one single light detector to detect light emanating from the detection zones of at least two, preferably three, different assays. It will be appreciated that light does not actually originate from the detection zones from which it originates from the light source, but light is reflected by and/or transmitted through the detection zones (as the case may be) such that it appears to emanate from the detection zones. Typically, but not necessarily, each detection zone is illuminated by a corresponding light source (e.g., an LED).

A single light source (e.g. an LED) may be used to illuminate the detection zone for at least two different assays and, if possible, three different assays. However, it is also possible to provide a plurality of LEDs. For example, one LED may be provided for illuminating each corresponding detection zone. Where multiple LEDs are provided, these may produce illumination of the same color, or may produce illumination of different wavelengths. Embodiments may exist in which the number of LEDs (or other light sources) equals or even exceeds the number of detection zones. Alternatively, different geometries may be used, wherein one LED illuminates two detection areas, such that the number of LEDs is less than the number of detection areas.

The device may typically utilize a reference region that is part of a microfluidic or lateral flow assay flow path for referencing a reading obtained from a detection region. The use of a reference region is well known to those skilled in the art. In particular, the device may utilise a "shared" reference zone (as disclosed in EP2,031,376), wherein a reference zone serves as a reference for two or more detection zones, at least one of which is located on a different flow path.

The microprocessor or computerized control may cause the one or more light sources to sequentially illuminate the one or more detection zones so as to distinguish between light reflected by and/or transmitted through the corresponding detection zones. In one embodiment, the light sources emit light at different wavelengths at different times, and the one or more light detectors distinguish between the different wavelengths. Additionally or alternatively, optical baffles (fixed or adjustable) may be used to control the area illuminated by a particular light source. More details of the kind of optical arrangements that can be used are disclosed in e.g. EP1,484,601, US6,055,060 and US5,889,585. For the avoidance of doubt, the term "light" as used herein is not intended to refer only to radiation in that part of the electromagnetic spectrum which is visible to a human observer, and also covers, for example, ultraviolet and infrared radiation. However, operating components in the visible portion of the spectrum and sensitivity to the visible portion of the spectrum may be preferred.

The microprocessor or computerized control means will preferably include a plurality of stored analyte thresholds against which the assay results are compared in order to allow the assay result reading device to interpret the results and display an appropriate conclusion (e.g. pregnant or not pregnant) to the user. The microprocessor or control device will advantageously be programmed with an algorithm to measure the test results, compare them to predetermined thresholds and display a conclusion.

In one embodiment, the microprocessor or control device will first determine the hCG measurement and compare that measurement to a predetermined lower hCG threshold. If the measured hCG measurement is below the predetermined lower hCG threshold, it can be immediately determined that the subject is not pregnant, and this result can be indicated to the user via a display (e.g., by forming the word not pregnant (NOTPREGNANT), or its equivalent in any language; or by a visual symbol, such as a minus sign or a zero).

However, if the measured hCG assay result is above the predetermined lower threshold, the assay result reader may continue to measure the results of the FSH assay. If the FSH assay result indicates that the FSH concentration in the sample is greater than its predetermined threshold, it can be determined that the subject is not pregnant and the result displayed to the user as described above.

However, if the measured FSH level in the sample is below its corresponding predetermined threshold, the assay result reader may then measure the result of the progesterone metabolite assay (in this example, one assay for P3G). If the assay result indicates that the concentration of P3G in the sample is below its corresponding predetermined threshold, it can be determined that the subject is not pregnant. If the concentration of P3G in the sample is above a predetermined threshold, the subject is pregnant and the assay result reader will display an appropriate conclusion to the user via the display.

The threshold value may be stored in the device of the present invention as an absolute analyte concentration (i.e. in terms of mass per unit volume or IU) and/or as an absorbance value or in any other convenient way.

In one embodiment, the device may be provided with an upper threshold and a lower threshold for one, two or all three of the hCG/FSH/progesterone analytes. For example, in one embodiment, the device may have an upper hCG threshold and a lower hCG threshold. If the measured hCG concentration exceeds the upper hCG threshold, the device may declare the subject pregnant without analysing the results of the FSH/progesterone assay. If it is apparent that the hCG analyte assay result will exceed the upper threshold, this declaration can be made "early" by the apparatus (e.g., before the assay has reached equilibrium). In other embodiments, the "pregnancy" result is still confirmed by examining the results of the FSH and progesterone assays.

If the hCG assay is below the lower hCG threshold, the device declares the subject not pregnant (with or without confirmation by the FSH and progesterone assays). If the hCG measurement is above the lower hCG threshold but not above the upper hCG threshold, the device will require FSH and progesterone measurements in order to obtain a pregnancy or non-pregnancy determination. The device may examine those FSH and progesterone assays sequentially (e.g., first one analyte, then the other, in any order) or in parallel.

In particular, the device may have an upper or lower threshold value for FSH and/or progesterone assay. It is possible that the choice of which of the multiple thresholds to apply will depend on the absolute or relative concentration of analyte detected in the other two assays, in one particular example, such that a "weighting" or "compensation" scheme can be employed. For example, an hCG concentration determined to be at the upper end of the mid-range between the upper hCG threshold and the lower hCG threshold may be "compensated" for a relatively low progesterone concentration by causing the device to apply a lower progesterone threshold in obtaining a pregnancy/non-pregnancy determination. Conversely, low FSH and/or high progesterone, for example, may compensate for relatively low hCG concentrations in the sample.

The measurement of the assay and/or interpretation of the assay results may include one or more data processing steps in which the assay data is subjected to one or more calculations or other types of processing. Such processing will conveniently be performed by a digital electronic device (such as a microprocessor or the like) which will typically form part of an external or integral assay result reader. For example, the data processing may include calculation of a ratio. In particular, the processing step may comprise calculating FSH: ratio of progesterone metabolites, or vice versa. The ratio may be based on relative signal intensities or on a calculation of the concentration of the corresponding analyte derived from measured signal intensities or other suitable correlation parameters. More specifically, a "border" hCG signal intensity or derived hCG concentration can be determined by calculating the FSH: progesterone metabolite (or vice versa) ratios for validation or examination.

Furthermore, the device may measure other analytes in addition to hCG, FSH and progesterone. These other analytes may include, in particular, hormones, such as, for example, LH, hPL (human placental lactogen) and/or relaxin and/or estrogen or a metabolite thereof. Another example of such a hormone is Thyroid Stimulating Hormone (TSH). TSH is related to hCG, FSH (and LH) in that all these hormones comprise an alpha subunit that is very closely similar to the alpha subunits of other hormones. TSH also contains a beta subunit unique to TSH. TSH levels in urine have been previously measured (see, e.g., Yoshida et al, 1988, journal of the japanese endocrine (jpn.) 35, 733-739), but at rather low concentrations. The concentration of such one or more additional analytes may also be taken into account by the device in obtaining a pregnancy/non-pregnancy determination, possibly by influencing the choice of applicable thresholds for one or more of the hCG/FSH/progesterone analytes. The ratio of the one or more additional analytes to one or more of hCG, FSH and progesterone may be measured. In particular, the ratio of TSH to hCG (or vice versa) can be measured. Measurement of other analytes may be particularly useful in the improvements of the present invention described below.

In a refinement of the basic principles described above, it may be desirable to indicate to the user not only whether the subject is pregnant, but also (if pregnant) the degree of pregnancy (i.e. gestational age) in terms of the amount of time elapsed since conception. This may be indicated by displaying for several days or more preferably for several weeks. Conveniently, one of three time intervals may be displayed: 1-2 weeks; 2-3 weeks; and > 3 weeks. To facilitate this, the test device may advantageously be provided with a plurality of different hCG concentration thresholds (or, more precisely, hCG assay test result thresholds) corresponding to corresponding numbers of weeks since conception. A method of achieving this is disclosed in WO 2009/147437. Again, to facilitate this embodiment, it may be desirable for the test device to be able to test hCG concentration over an extended range (e.g. by including both a relatively high sensitivity test and a relatively low sensitivity test for hCG), and suitable methods of achieving this are described in WO 2008/122796. Estimation of gestational age can be facilitated by measuring the concentration of other analytes than hCG (e.g. hPL) (see WO2012/055355) and/or one or more progesterone metabolites.

In some embodiments, it will be desirable for the test equipment to also include some sort of control functionality. This is common in self-test devices to provide some indication that the test has run correctly.

Typically, a control function will involve the use of a control zone in which a labelled reagent will tend to accumulate in the event that sufficient sample has been applied to the sample application zone of the test device. Conventionally, the labeling reagent will be a labeled antibody or other reagent that is releasably deposited in dry form at an upstream or proximal portion of a test strip and mobilized after rehydration by the sample, and captured by a specific capture agent immobilized in the control region. The control indicates whether sufficient sample has been applied to the test device, and whether the test reagent retains its binding properties within a reasonable range, and whether the labeling reagent has been mobilized to a sufficient extent.

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

FIG. 1 is an exploded view of one embodiment of an apparatus according to the present invention;

FIGS. 2-5 show different forms of assay test strips for use in different embodiments of an apparatus according to the invention; and is

Fig. 6 schematically shows an embodiment of an algorithm/logic tree for use in an apparatus according to the invention.

Examples of the invention

Example 1

In an initial experiment, urine hCG, urine FSH and urine P3G were measured in 119 non-pregnant volunteers in a peri-and post-menopausal (PP, age 41-90) group (of which a total of 50 samples had [ hCG ]]≧ 2.5mIU/ml), and urinary hCG, urinary FSH, and urinary P3G were measured from 72 pregnant volunteers (age 21-40) from EMP at day-7 (expected missed menstrual period) to EMP at day +3 of successful pregnancy. Available samples were tested to generate a total of 589 early pregnancy samples ("EPS"), of which 434 had [ hCG]More than or equal to 2.5mIU/ml, such as oneThe measured values are determined.

Analysis of these results showed that true pregnancy can be referred to as 100% positive prediction (no false positives in the non-pregnant group) using a combination of a threshold level of urinary hCG of at least 2.5mIU/ml with both a FSH threshold of 10mIU/ml or lower and a P3G threshold of at least 4 μ g/ml to define pregnancy. The threshold levels of these three analytes used to define pregnancy (as established in this study) may vary over a larger data set, but these results show that by using hCG in combination with FSH and P3G, pregnancy-derived hCG can be distinguished from pituitary-derived peri-and post-menopausal hCG.

The results using these thresholds are presented in table 1 below.

The results in table 1 are very significant. These results show that in group a, a very sensitive hCG test (detecting hCG as low as 2.5mIU/ml) gave zero false positive rates in statistically significant female samples when combined with tests for FSH and a progesterone metabolite (P3G), while all pregnant subjects were successfully identified in group B.

Example 2

In one embodiment of the invention, a pregnancy test device will comprise a lateral flow immunoassay constructed in the form of a two-strip Nitrocellulose (NC) in which an hCG sandwich assay is provided to a first strip. An FSH sandwich assay and a competitive P3G assay will be provided on a second separate strip having two distinct capture zones. Both strips will run simultaneously via a common sample application zone contacting a porous medium that will contact both nitrocellulose strips. The device will digitally measure the signal response (bound label) on these NC capture zones in response to the amount of analyte of interest. A built-in algorithm will give a digitally displayed response (pregnant/not pregnant and/or gestational age) to the end user on the screen depending on the level of analyte.

An example of one possible algorithm is schematically shown in fig. 6. It should be noted that the algorithm presented in fig. 6 is only one embodiment, and many other embodiments may be used. It should also be noted that the absolute analyte concentrations presented in fig. 6 are merely illustrative, and other analyte thresholds may be employed. Example 3

Referring to fig. 1, which shows an exploded view of one embodiment, an apparatus according to the present application includes a two-part housing formed of a synthetic plastic material. The housing has a top portion 2 and a bottom portion 4. The housing is formed from an opaque plastics material such as polycarbonate or polypropylene. If desired, a sunscreen may be included.

Within the housing is a power source, such as a small button cell 6 that delivers power to the components mounted on a Printed Circuit Board Assembly (PCBA) 8. These components include, in particular, one or more LEDs and photodiodes, and a liquid crystal display 10. The components mounted on PCBA8 include those necessary to read the results of assays performed on two lateral flow assay strips mounted within the housing. One 12 of the bands is for performing an hCG assay and the other 14 is for performing both an FSH assay and a pregnanediol-3-glucuronide (P3G) assay.

The top and bottom portions 2 and 4 of the housing cooperate to form a substantially moisture tight seal around the components. A urine sample passes through a sampling wick 16 to the lateral flow assay strips 12, 14. An end region of the sampling core 16 is in liquid flow communication with an adjacent end region of each of the assay strips 12, 14 (such that the sampling core acts as a common sample application zone).

An opposite end region of the core 16 projects through and beyond an aperture in one end of the housing, allowing sample to be applied to the core. The protruding portion of the sampling core is protected by a removable cap 18 shaped and dimensioned to mate with an end of the housing to thereby receive and form a snug, tight-fitting engagement therewith.

In the embodiment shown, to apply a urine sample to the sampling core 16, the user removes the cap 18 and urinates directly onto the core. The core is made of absorbent material and the sample is thus absorbed into and flows passively along the core and into the assay strips 12, 14. The user then optionally replaces the cap 18. To facilitate sample flow into and along the assay strips, the assay strips are in liquid flow communication at their distal ends (i.e., the ends further from the sampling core 16) with a "trough" pad 20 of superabsorbent material.

The lateral flow assays are performed in a conventional manner, resulting in accumulation of a labeled binding reagent at one or more detection zones on test strips 12, 14 in an analyte concentration dependent manner (either proportionally or inversely, as the case may be), which is detected and read by an assay reading component mounted on PCBA 8. A microprocessor, ASIC or the like analyzes and interprets the readings and displays the assay result on the LCD10, which is visible to the user via a window or aperture 21 formed at a suitable location in the top portion 2 of the housing.

It is important that the lateral flow test strips 12, 14 remain dry prior to use, and for this reason the PCBA is provided with a container for a desiccant 22 which absorbs moisture from the interior of the housing.

Example 4

Referring to fig. 2, this example relates to an embodiment in which two different lateral flow assay strips are used in combination with or form an integral part of a device according to the present invention.

In figure 2, one of the strips 30 was used to perform a high sensitivity assay for hCG. The other strip 32 was used to perform an assay for FSH and for P3G. Urine sample enters both assay strips 30, 32 from a common sampling member (omitted for clarity) and flows along the strips in the direction indicated by arrow 34. At its proximal end (i.e., the end first encountered by a urine sample), both strips have a glass fiber conjugate pad 36, and towards the distal end, both strips are in liquid flow communication with a highly absorbent "trough" 38 that facilitates sample flow along the assay strips. Both strips also include a pair of alignment holes 40 outside the "slots" 38. These facilitate correct positioning of the strips within the device, in particular so that the assay strips can be correctly read by assay reading means of the device. Note that the pairs of alignment holes 40 are not symmetrical and therefore the strips cannot be inadvertently replaced with each other.

Referring to strip 30, conjugate pad 36 is loaded with a mobilizable anti- α hCG subunit monoclonal antibody conjugated to a gold sol (42). The conjugate dries on the conjugate pad and is released after wetting by the sample.

The detection zone 44 includes an immobilized monoclonal antibody specific for the β subunit of hCG. Thus, any hCG present in the sample binds to the labelled conjugate and forms a sandwich with the "capture" anti-hCG β antibody at the detection zone in a manner that will be familiar to those skilled in the art.

The area 46 defined by the dashed line indicates a reference area that is used by the assay result reading device to calibrate the assay reading. The same reference zone 46 may be used to calibrate both hCG assay readings and also readings obtained for FSH and P3G assays on the strip 32 (i.e. a common reference zone may be used).

Referring to strip 32, the glass fiber conjugate pad has two different conjugates 48: one was gold sol conjugated to an anti-FSH alpha subunit monoclonal antibody and the other was gold sol conjugated to an anti-P3G monoclonal antibody. (in this example, anti-hCG. alpha. and anti-FSH. alpha. are the same antibody clone).

An immobilized anti-FSH β -subunit monoclonal antibody was used as the capture antibody at the FSH detection zone 50, while an immobilized P3G-ovalbumin conjugate was used as the capture molecule at the P3G detection zone 52.

The body of each assay strip 30, 32 is formed of nitrocellulose, as indicated by reference numeral 54.

Although in this embodiment, the P3G detection zone 52 is shown downstream of the FSH detection zone 50, it is contemplated that the relative positions of the two detection zones may be reversed.

Another variant is a format in which the hCG and P3G assays are performed on one strip and a separate strip is used for the FSH assay.

Example 4A

Another assay format can be envisaged. The assay format is essentially the same as that depicted in fig. 2 and described in example 4, except that in this variation, one of the mobile conjugates deposited on the conjugate pad 36 is a gold sol labeled with P3G-ovalbumin conjugate (instead of anti-P3 GmAb), and the immobilized capture molecules at the P3G detection zone 52 are immobilized anti-P3 GmAb. In this arrangement, P3G in the sample competes with the labeled conjugate for binding to the capture antibody at the detection zone 52.

Example 5

Referring to fig. 3 (where like components share the reference numbers used in fig. 2), in this example, all 3 assays were performed using a single assay strip. The glass fiber conjugate pad 36 is loaded with a common anti-hCG α and anti-FSH α gold sol conjugate and an anti-P3 GmAb gold sol conjugate (42, 48, respectively).

The hCG detection zone 44 includes an immobilized anti-hCG β mAb. The FSH detection zone 50 includes an immobilized anti-FSH β mAb and the P3G detection zone 52 includes an immobilized P3G-ovalbumin conjugate.

As before, the relative positions of these detection zones may be changed. Further, as in example 4A, the mobilizable P3G reagent may be a gold sol labeled with a P3G-ovalbumin conjugate, and the P3G detection zone 52 may include an immobilized anti-P3G antibody.

Yet another variant can be easily envisaged in which the mobilizable conjugate can comprise a first gold sol conjugated to a monoclonal antibody specific for one beta subunit of hCG and a second gold sol conjugated to a monoclonal antibody specific for one beta subunit of FSH. These corresponding detection zones include immobilized anti-hCG α antibodies and anti-FSH α antibodies. Another potential variation is that an anti-hCG β subunit antibody is used both in the mobile conjugate and as an immobilized capture antibody, provided that the two antibodies bind different epitopes and therefore do not interfere with or compete with each other for binding to hCG. FSH can also be assayed in a similar manner, with anti-FSH β subunit-specific antibodies being used both on the mobile conjugate and as immobilized capture antibodies.

Example 6

In the example shown schematically in fig. 4, a separate lateral flow assay strip is provided for each analyte. hCG was measured on strip 100, FSH was measured on strip 102, and P3G was measured on strip 104. Like components share the same reference numerals as in fig. 2.

The sample is applied to all three assay strips via one common sample application means (omitted for clarity). A common "slot" pad 38 is in fluid flow contact towards the distal end of each assay strip. In contrast to fig. 2, in this embodiment, each assay strip 100, 102, 104 has its own reference region 46. In addition, each assay strip has a program control zone 60 which indicates whether the assay has been performed correctly in the sense that the binding reagents retain their binding characteristics.

Example 7

Yet another embodiment is shown in fig. 5. This example also utilizes three assay strips. However, two of the three assay strips were used to measure hCG levels, so one of the strips 110 was a high sensitivity hCG assay (for detecting low levels of hCG), one of the strips 112 was a low sensitivity hCG assay (for detecting high levels of hCG), and the third strip 114 was used to determine both FSH and P3G. Like parts are indicated by common reference numerals with figure 2.

To reduce the sensitivity of the hCG assay on the strip 112, the conjugate pad 36 is loaded (42') with not only a mobilizable gold sol conjugated to an anti-hCG α mAb, but also a free (unlabeled) anti-hCG β mAb that binds hCG present in the sample and thus competes with the immobilized anti-hCG β capture antibody located at the detection zone 44. Thus, at high levels of hCG analyte in the sample, the assay system is not engulfed. In this example, the unlabeled anti-hCG β monoclonal antibody acts as a "scavenger". The scavenger need not be an antibody but may be any unlabelled agent that binds hCG and prevents hCG from (indirectly) depositing the labelled reagent at the detection zone. The scavenger may for example be immobilized on/in the flow path, or may be mixed (in a mobile form) with the labeled conjugate.

The use of both a high sensitivity hCG assay and a low sensitivity hCG assay allows the device to accurately measure hCG over an extended concentration range, and this arrangement is suitable for those embodiments in which the device is capable of displaying a result that not only indicates that the subject is pregnant, but also is capable of indicating to the user a quantitative estimate of how long the subject has been pregnant (e.g. in terms of weeks since conception).

Although the above examples illustrate the use of different lateral flow assay formats, it will be clear that a similar assay arrangement may utilize a microfluidic-based assay, or an assay based on both lateral flow and microfluidics.

Example 8

This example describes the steps in creating a lateral flow assay strip (as shown in fig. 4) suitable for use in a device according to the present invention.

Generation of assay reagents:

A. preparing a gold sol labeled antibody:

1.P3G determination:

the method comprises the following steps:

borate buffer (20ml, 20mM, pH8.5) was added to 80nm gold sol solution (20ml, A)₅₅₀OD6.85, BBI international (bbiitematic)) to obtain a final solution containing OD3.425 in 10mM borate bufferAnd (4) gold sol.

A solution of anti-P3G antibody (clone # 5806: 2, Elite san Diego, 40ml, 160. mu.g/ml in 10mM borate buffer) was rapidly mixed with the gold sol solution on a magnetic stirrer at room temperature for 30 minutes.

After 30 minutes mixing, 610 μ l of a 65.6mg/ml beta casein solution was added to the reaction mixture and mixing was continued for another 30 minutes at room temperature.

The final concentration of beta-casein in the reaction mixture was 0.5 mg/ml.

The sol solution was poured into a centrifuge tube (50ml) and the solution was centrifuged (4,000g, 10 min, 15 ℃).

The clear supernatant was decanted and the precipitated sol was vortexed and sonicated.

The sol solution was transferred to an eppendorf tube and centrifuged (4,000g, 7 min, 15 ℃).

The supernatant was carefully removed and the precipitated sol was vortexed and sonicated, and wash buffer was added (1ml, 0.5mg/ml beta casein in 10mM borate buffer) to the resuspended sol. After resuspension, the solution was centrifuged (4,000g, 7 min, 15 ℃).

The supernatant was carefully removed and the precipitated sol was vortexed and sonicated, and wash buffer was added (1ml, 0.5mg/ml beta casein in 10mM borate buffer) to the resuspended sol. After resuspension, the solution was centrifuged (4,000g, 7 min, 15 ℃).

The supernatant was removed and the precipitated sol was vortexed and sonicated, the sol was resuspended in a small volume of storage buffer (0.5mg/ml bsa in PBS + azide [ PBSA ]) and the final volume was adjusted to 2 ml. Although the initial experiment used 0.5mg/ml BSA in storage buffer, the presence of BSA was found to interfere with the P3G assay. Therefore, 0.5mg/ml casein was subsequently used in place of BSA in the storage buffer. In embodiments of the invention where the P3G assay components may be in contact with components of other assays (e.g. where one P3G assay and one hCG and/or FSH assay are carried out on a single assay flow path or lateral flow strip), then it would be desirable for these other assays to also avoid the use of BSA in order to prevent the P3G assay from being affected.

The final OD of the sol formulation was determined by measuring the absorbance at 550 nm.

2.hCG assay and FSH assay:

the method comprises the following steps:

borate buffer (20ml, 20mM, pH8.5) was added to 80nm gold sol solution (20ml, A)₅₅₀OD6.85, BBI international corporation) to obtain a final solution containing the gold sol at OD3.425 in 10mM borate buffer.

A solution of anti- α -hCG antibody (clone # 3299: 4, Elite san Diego, 40ml, 20 μ g/ml in 10mM borate buffer) was rapidly mixed with the gold sol solution on a magnetic stirrer at room temperature for 30 minutes.

After 30 minutes mixing, 610 μ l of a 65.6mg/ml beta casein solution was added to the reaction mixture and mixing was continued for another 30 minutes at room temperature. The final concentration of beta-casein in the reaction mixture was 0.5 mg/ml.

The sol solution was poured into a centrifuge tube (50ml) and the solution was centrifuged (4,000g, 10 min, 15 ℃).

The supernatant was decanted and the precipitated sol was vortexed and sonicated.

The sol solution was transferred to an eppendorf tube and centrifuged (4,000g, 7 min, 15 ℃).

The supernatant was carefully removed and the precipitated sol was vortexed and sonicated, and wash buffer was added (1ml, 0.5mg/ml beta casein in 10mM borate buffer) to the resuspended sol. After resuspension, the solution was centrifuged (4,000g, 7 min, 15 ℃).

The supernatant was carefully removed and the precipitated sol was vortexed and sonicated, and wash buffer was added (1ml, 0.5mg/ml beta casein in 10mM borate buffer) to the resuspended sol. After resuspension, the solution was centrifuged (4,000g, 7 min, 15 ℃).

The supernatant was removed and the precipitated sol was vortexed and sonicated, and the sol was resuspended in a small volume of storage buffer: (0.5mg/ml BSA in PBSA)Medium and adjust the final volume to 2 ml.

The final OD of the sol formulation was determined by measuring the absorbance at 550 nm.

B. Preparation of the reagent to be immobilized on nitrocellulose:

although the following examples describe the preparation and use of P3G conjugates of ovalbumin, it is contemplated that other protein and synthetic polymer conjugates of P3G may be employed. Protein conjugates of P3G include, but are not limited to, bovine serum albumin, immunoglobulin G, gelatin, and beta casein. Representative examples of polymeric carriers include polyallylamine, polyvinyl alcohol, polylysine, and polyethyleneimine.

P3G determination:

preparation of P3G conjugate of ovalbumin (P3G to ovalbumin 10: 1 molar ratio)

Method of producing a composite material

Preparation of NHS activated P3G ester (10mgP3G, 10% DMSO ratio)

Water soluble carbodiimide, EDC was used to prepare NHS activated P3G esters, compare to P3G (2.0136 × 10)^-5Mole P3G) molar excess EDC (1.1x) and NHS (1.5 x).

The total volume used to carry out the reaction was 600. mu.l.

A 14.153mg/ml solution of EDC was prepared by dissolving 48mg of EDC (1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride, zemer scientific, catalog No. 77149, molecular weight 191.7) in 3.39ml of DMSO (99.7% extra dry, Acros, catalog No. 3484400).

A 11.6mg/ml solution of NHS was prepared by dissolving 29mg of NHS (N-hydroxysuccinimide, sigma aldrich, cat # 130672, molecular weight 115.09) in 2.5ml of DMSO.

10mg of P3G (5 β -pregnan-3 α, 20 α -diol glucuronide, P3G, Sigma (Sigma), catalog No. P3635, molecular weight 496.63) was weighed in a glass vial, and 300 μ l of EDC solution and 300 μ l of NHS solution prepared above were added to obtain a solution containing more EDC and 1.5x more NHS than p3g1.1x. The reaction mixture was stirred at room temperature in the dark for 4 hours and the reaction was allowed to proceed overnight (approximately 16 hours) at 20 ℃.

Dry DMSO (400. mu.l) was added to the above solution so that the total volume of the solution was made up to 1000. mu.l.

Assuming 100% conversion of P3G to P3G-NHS ester, there will be 2.0136 × 10 in 1000. mu.l^-5Thus, 100. mu.l of this solution will contain 2.0136 × 10^-6And (3) mol.

Preparation of P3G-ovalbumin conjugate

100 μ l of active ester (2.0136 × 10) was used^-6Moles).

A 4.43mg/ml solution of ovalbumin in PBS buffer (PBS tablets, sigma, cat # P4417) was prepared by dissolving 92.4mg of ovalbumin (sigma, cat # a5503 > 98%, molecular weight 44, 000) in 20.858ml of PBS and 2ml of this solution was used for 10-fold preparation.

DMSO (100 μ Ι) was added to the ovalbumin solution just prior to the addition of NHS activated P3G solution (100 μ Ι). Thus, the final concentration of DMSO in the reaction mixture was maintained at 10% (v/v).

The reaction solution was stirred at room temperature for 3.5 hours. After 3.5 hours of reaction, the reaction was quenched by adding 1M Tris buffer (pH7.4) (100. mu.l) to the solution.

P3G-ovalbumin was then centrifuged and the clear supernatant removed and purified on a PD-10 column (GEHealthcare, Cat. No. 17-0851-01) pre-equilibrated with PBSA.

Apply conjugate solution (2.5ml) to the top of the equilibration column and let all solution flow into the gel bed.

-subsequently applying elution buffer (PBSA, 3ml) to the column and collecting the effluent into a clean glass vessel. The effluent contained purified P3G-ovalbumin conjugate.

Extinction coefficient (A) of 0.7₂₈₀ ^0.1％) For determining protein concentration. The P3G-ovalbumin conjugate was then concentrated to 3mg/ml for immobilization on nitrocellulose.

1.hCG assay:

anti- β -hCG antibody (clone # 3468: 2, Elite san Diego) was diluted to 3mg/ml in PBSA before being fixed to nitrocellulose.

2.FSH assay:

anti-beta-FSH antibody (clone # 5948: 2, Elite san Diego) was diluted to 3mg/ml in PBSA before being immobilized onto nitrocellulose.

C. Formulations and devices for locating and immobilizing specific binding substances:

method of producing a composite material

1.P3G determination:

1.1A PVA blocking buffer (pH9) was prepared (Tris base 20mM (Sigma), PVA 1% w/v (PVA 80% hydrolysed, 9-10KMW (Sigma), Tween 200.05% w/v (Sigma), and NaCl150mM (Sigma)).

1.2A PVA blocking solution was prepared by adding 2% w/v sucrose (Sigma) to 47.5ml PVA blocking buffer, adding 2.5ml ethanol (Sigma).

1.3 a white backed sheet of cellulose nitrate (waterman) was cut into 35cm x 40mm strips using a pressure cutter and punched at one end of the strip to a 6mm pitch (many different pitch sizes could be used, however for the examples presented herein a 6mm pitch was used).

1.4A biodot plotter was set to plot P3G-ovalbumin conjugate at the desired position on the nitrocellulose strip at a concentration of 3mg/ml and a plotting rate of 1. mu.l/cm.

1.5 after plotting, the tapes were dried at 55 ℃ and blocked with a PVA blocking solution, and subsequently dried at 65 ℃ and stored overnight at room temperature together with the desiccant in sealed aluminum foil pouches.

2.hCG assay:

according to section c.1, steps 1.1 to 1.3 are included.

A biodot plotter was set to plot the 3468 antibody at a desired position on the nitrocellulose strip at a concentration of 3mg/ml and a plotting rate of 1. mu.l/cm.

After plotting, the strips were dried at 55 ℃ and blocked with PVA blocking solution, and subsequently dried at 65 ℃ and stored overnight at room temperature with desiccant in sealed aluminum foil bags.

3.FSH assay:

according to section c.1, steps 1.1 to 1.3 are included.

A biodot plotter was set to plot 5948 antibody at the desired position on the nitrocellulose strip at a concentration of 3mg/ml and a plotting rate of 1. mu.l/cm.

After plotting, the strips were dried at 55 ℃ and blocked with PVA blocking solution, and subsequently dried at 65 ℃ and stored overnight at room temperature with desiccant in sealed aluminum foil bags.

D. Formulation and apparatus for immobilization of gold sol labelled binding reagents (for use in the example of example 1):

method of producing a composite material

1.P3G determination:

the 5806 coated sol conjugate prepared in section a.1 was spun down in a centrifuge and the supernatant removed. The resulting precipitate was vortexed and sonicated, and then the precipitate was reconstituted in a gold sol conjugate spray buffer to gold of the desired OD (OD 80 in the high example). The gold sol conjugate spray buffer (ph7.6) used in the following examples contained 10mm tris (sigma), 5% w/v sucrose (sigma), and 0.5% (w/v) BSA (proliant biologicals, SKU # 68700). However, other examples of spray buffers that may also be used may have additional substances in the diluent solution and may also have higher or lower levels of the ingredients listed in the examples above. (subsequent change to 0.5% w/v casein).

Mixing G041 glass fiberCut 26mm x 35cm and loaded onto a Biodot spray drill.

The biodot spray drill was set up to impregnate/impregnate the glass fibers with 5806 coated sol conjugate at the desired locations on the glass fibers. In this example, glass fibers were sprayed with 4 sequential runs of OD80 conjugate at a plotting rate of 1.65. mu.l/cm per spray.

The gold sol infused glass fibers were dried at 55 ℃ and stored overnight at room temperature in a sealed aluminum foil pouch together with a desiccant.

2.hCG assay:

gold sol impregnated/infused glass fiber tape for hCG assay was prepared in the same manner as above except that 3299 coated gold sol was used here (see section a.2) and the OD of the sol conjugate was OD 111. In this illustrated example, the glass fiber was sprayed with 2 sequential runs of gold conjugate at a plotting rate of 1.65. mu.l/cm.

3.FSH assay:

for the FSH assay, 3299 coated gold sol (see section a.2) was sprayed onto glass fibers at a feed rate of 1.65 μ l/cm in sequence using 2 of OD62 gold conjugates. The tape of the feedstock was dried and stored in the same manner as in the above example.

E. Assay strip configuration/generation: examples of Single strip assay chip constructs (one assay per chip/strip)

Method of producing a composite material

1.P3G determination:

the P3G assay components were assembled into one assay chip (strip) with the aid of a kinematic universal laminator module (universal laminationmodule) assembly unit.

The backing laminate (Ferrisgate corporation) was placed onto the moving card platen and a closed nitrocellulose strip with immobilized P3G-ovalbumin (section C.1) was affixed to the backing laminate at a predetermined location.

One strip of 5806 sol conjugate infused glass fibers (section d.1) was secured to the backing laminate with a 2mm overlap over the nitrocellulose strip.

A roller pad ensures good contact of all components of the chip with the backing laminate.

The strips were then cut into 6mm individual chips using a Biodot cutter and stored with desiccant in aluminum foil bags until ready for use.

2.hCG assay:

the hCG assay components were assembled in the same manner as the P3G assay, except that nitrocellulose strips fixed with 3468 (section c.2) and glass fibre strips impregnated with 3299 gold sol conjugate (section d.2) were used.

The strips were then cut into 6mm individual chips using a Biodot cutter and stored with desiccant in aluminum foil bags until ready for use.

3.FSH assay:

the FSH assay components were assembled in the same manner as the P3G assay, except that nitrocellulose strips immobilized with 5948 (section c.3) and glass fiber strips impregnated with 3299 gold sol conjugate (section d.3) were used.

The strips were then cut into 6mm individual chips using a Biodot cutter and stored with desiccant in aluminum foil bags until ready for use.

Example 9

In this example, further information is provided about an illustrative algorithm used in an apparatus according to the present invention.

The assay device of the present invention provides a higher pregnancy detection rate prior to the day of the expected menstrual period compared to conventional self-use pregnancy tests, while retaining specificity for pregnancy. The assay device of the present invention accomplishes this by: not only has a higher sensitivity towards hCG, but also FSH and one or more progesterone metabolites are measured in order to retain specificity for pregnancy at very low hCG concentrations; elevated hCG levels can be observed in some postmenopausal and perimenopausal women, which can produce false positive results in the case of an overly sensitive hCG test. FSH serves as a exclusion of pregnancy, as high levels are associated with peri-and postmenopausal states, while progesterone metabolites (e.g., P3G) serve as a break-in of pregnancy, as elevated levels are observed in pregnancy.

In a simple embodiment of this concept, the device may operate as outlined in table 2 below. In this example, the progesterone metabolite determined was P3G:

TABLE 2

The probability of envisaging instances where conflicting FSH and progesterone metabolite assays occur will be very low. The device will typically be programmed to declare an "not pregnant" result in such circumstances, and/or the user may be notified to test again at a later stage.

All tests were designated as assay tests and expressed in terms of the concentrations detected. The logic must be reversed for a competition assay for testing the intensity of a developed visible line, where the intensity of the line decreases with increasing analyte concentration.

Improved performance may potentially be achieved by using a wider algorithm: the importance (weight) of the level of FSH or progesterone metabolite level may depend on the level of hCG and/or the threshold for testing FSH and progesterone metabolite used may depend on the level of hCG. In a more complex case, the FSH threshold may depend on the hCG concentration and the progesterone metabolite threshold may depend on the FSH level (or vice versa).

This algorithm is schematically represented in fig. 6. Referring to the figure, the algorithm/logic tree relates to one embodiment of the test apparatus of the present invention comprising two lateral flow test strips. A first strip was used to perform an assay for hCG and a second strip was used to perform an assay for FSH and an assay for P3G.

In the logic tree, the assay result reader first examines the hCG assay result and compares the assay signal to a predetermined signal value corresponding to an hCG concentration of 2.5 mIU/ml. If the assay result is less than the 2.5mIU/ml threshold, the reader can immediately indicate that the test subject is "not pregnant" (following the "<" symbol in the figure). However, the outcome of the overall assay (i.e., "pregnant" or "not pregnant") may not necessarily be indicated to the user until the results of all three analyte assays have been determined. Alternatively, if the hCG concentration determined in the urine sample is equal to or greater than the 2.5mIU/ml threshold, the reader continues to check for FSH assay results (first test line or detection zone on the second assay strip). Since hCG concentrations above 2.5mIU/ml may be due to non-pregnancy related sources, an "early" determination of the overall outcome of the assay may not be likely based solely on hCG assay results.

The FSH assay has a threshold of 10 mIU/ml. If the determined FSH concentration in the sample is equal to or greater than the 10mIU/ml threshold, the assay device/reader declares the result of the test as "not pregnant". Conversely, if the FSH concentration in the sample is above the 10mIU/ml threshold, the assay device/reader will continue to check the P3G assay results.

The P3G assay results were read from a second test line or detection zone on a second lateral flow assay strip. A subject is declared "pregnant" if the P3G assay indicates a P3G concentration in the sample of less than 4 μ g/ml.

It will be clear to those skilled in the art that the exact signal value or threshold concentration selected for a particular embodiment will depend, at least in part, on the particular characteristics of the assays employed (e.g., the reagents, flow matrix, concentration of conjugate, etc.), such that the thresholds identified above may be slightly different in other embodiments, but the relative amounts will be generally the same.

Furthermore, the figure shows an assay device/reader that sequentially tests or checks the results of the hCG, FSH and P3G assays. It will be clear that the corresponding assay results may be examined in any order or substantially simultaneously. Furthermore, if the concentration of these analytes is very high (or low, where appropriate) above the relevant threshold concentration, an "early" determination of one or both assay results may enable a determination of the outcome of the overall assay result (i.e., "pregnant" or "not pregnant") without knowing the results of all three analyte assays.

Claims (35)

1. A test device for detecting pregnancy in a human female subject, the test device comprising: an assay device for measuring the absolute or relative amount of hCG in a sample from the subject;

an assay device for measuring the absolute or relative amount of FSH in a sample from the subject;

and an assay device for measuring the absolute or relative amount of a progesterone metabolite in a sample from the subject.

2. A test device according to claim 1, comprising one or more lateral flow test strips and/or microfluidics based assays.

3. A test device according to claim 1 or 2, comprising assay result reading means.

4. The test device of any of claims 1-3, further comprising: a microprocessor, ASIC or other computerized control device; and a measurement result display for displaying the result or outcome of the measurement to a user.

5. A test device according to any one of the preceding claims, comprising a digital memory device programmed with at least one predetermined signal value threshold for each analyte.

6. A test device according to claim 5, wherein the device comprises a microprocessor, ASIC or other programmable computer control means programmed with an algorithm to determine test results by comparing the analyte assay signal values with their respective predetermined thresholds.

7. A test device according to any one of the preceding claims, which is a point-of-care or "self-test" device.

8. A test device according to any one of the preceding claims, which is discarded after a single use.

9. The digital test device according to any of the preceding claims, which interprets the assay test results and displays a test outcome to the user.

10. A test device according to any one of the preceding claims, comprising one or more light sources to illuminate one or more microfluidic or lateral flow assay detection zones; and one or more light detectors to detect light reflected or transmitted by the detection zone.

11. A test device according to any one of the preceding claims, comprising at least two assays for hCG, one of the assays being a relatively high sensitivity assay and one of the assays being a relatively low sensitivity assay, such that the test device is capable of measuring hCG concentration over an extended range.

12. A test device according to claim 11, wherein the relatively high sensitivity assay and the relatively low sensitivity assay are on separate lateral flow test strips or separate microfluidic assay flow paths.

13. A test device according to claim 11, wherein the relatively high sensitivity assay and the relatively low sensitivity assay are on the same lateral flow test strip or on the same microfluidic assay flow path.

14. A test device according to any one of the preceding claims, wherein the progesterone metabolite detected by the progesterone metabolite assay comprises pregnanediol or a derivative thereof.

15. A test device according to any one of the preceding claims, wherein the progesterone metabolite detected by the progesterone metabolite assay comprises a glucuronide.

16. A test device according to any one of the preceding claims, wherein the progesterone metabolite detected by the progesterone metabolite assay comprises pregnanediol-3-glucuronide.

17. A test device according to any one of the preceding claims, wherein the test device is programmed with a lower hCG threshold and an upper hCG threshold such that an hCG assay result below the lower hCG threshold is interpreted as meaning that the subject is not pregnant; and an hCG assay result above the upper threshold is interpreted as meaning that the subject is pregnant, regardless of the results of the assays for the other two analytes; and an hCG assay result between the lower threshold and the upper threshold is interpreted as pregnant or not pregnant depending on the FSH assay result and the progesterone metabolite assay result.

18. A test device according to any one of claims 1 to 16, wherein the test device is programmed with a lower hCG threshold and an upper hCG threshold such that an hCG assay result below the lower hCG threshold is interpreted as meaning that the subject is not pregnant; and an hCG assay result above the upper threshold is interpreted to mean that the subject is pregnant, undergoing confirmation of the results of the assay for the other two analytes.

19. A test device according to claim 17 or 18, further comprising at least one further hCG threshold.

20. A test device according to any one of the preceding claims, adapted and configured to display an approximate gestational age of a pregnancy of a pregnant subject.

21. A test device according to claim 20, wherein the device displays gestational age as intervals of 1-2 weeks, 2-3 weeks or 3+ weeks, as appropriate, for a pregnant subject.

22. A test device according to any one of the preceding claims, further comprising a control function to indicate whether the device is functioning correctly.

23. A test device according to any one of the preceding claims, comprising a moisture impermeable housing containing most or all of the components of the test device; and a sample application region on a sample application member that protrudes beyond the housing to allow application of a sample.

24. A test device according to any one of the preceding claims, comprising a common sample application zone such that a sample applied to the common sample application zone enters two or more different flow paths, wherein at least one different analyte assay is located on a respective one of the two or more different flow paths.

25. A test device according to any one of claims 1-23, wherein assays for each of hCG, FSH and one progesterone metabolite are all provided on a single, common flow path.

26. A test device according to any one of the preceding claims, additionally comprising an assay means for measuring the absolute or relative amount of luteinizing hormone and/or an assay means for measuring the absolute or relative amount of human placental lactogen.

27. A test device according to claim 1 or 2 or any of claims 7-8, 11-16 or 22-25 as dependent on claim 1 or 2, wherein the test device is a combination of a disposable visual reading device and a separate permanent assay reading device.

28. A test device in combination with a separate durable assay reading device, according to claim 27, wherein the separate assay reading device comprises a camera.

29. A test device in combination with a separate assay reading device according to claim 27 or claim 28, wherein the separate assay reading device comprises a smartphone or other digital handheld reading device.

30. A method of detecting pregnancy in a human female subject, the method comprising the steps of: contacting a sample from the subject with a test device according to any one of the preceding claims.

31. An electronically programmable device programmed to analyse the results of assays for hCG, FSH and a progesterone metabolite performed on a sample from a human female and to determine from the analysis of one or more of the assays whether the subject is pregnant and/or the gestational age of the pregnancy.

32. The apparatus of claim 31, wherein the analysis comprises comparing a measured signal value for a particular analyte with at least one corresponding stored threshold signal value.

33. A test device according to any one of claims 1-29 or a method according to claim 30, which provides at least one, preferably two or more, more preferably three or more and most preferably all of features (i) - (vii) as set out below:

(i) sensitivity of 99% or higher;

(ii) a specificity of 99% or more;

(iii) (iii) the ability to achieve (i) and (ii) even on the test day early in pregnancy (before the day of expected menstruation);

(iv) (iii) enable (i) and (ii) even when testing/using the assay device once at a single point in time (i.e. on a single day);

(v) the assay device/method does not require any external information (e.g. the age of the female or any history of the subject, such as previous measurements of hCG, FSH or progesterone); and

(vi) (iii) is capable of achieving (i) and (ii) even when the subjects comprise perimenopausal and/or postmenopausal women.

34. An electronic programmable device according to claim 31 or 32 and further according to any of claims 1-25.

35. A test device according to any of claims 1-29, substantially as hereinbefore described and with reference to the accompanying drawings.