Test device

Abstract

The present invention provides test devices and methods for detecting the presence of an analyte in a sample. The device of the invention comprises a reagent zone, one or more capture zones and a detection zone. In the presence of a negative marker in the sample and / or the absence of a positive marker in the sample, the capture region can reduce the amount of labeled conjugates reaching the detection region, facilitating high sensitivity and improved specificity testing.

Description

[0001] This application is a divisional application with an application date of April 9, 2020, application number 202080034563.6, and invention name “Test Device”. Technical Field

[0002] The present invention provides an assay device for detecting the presence of an analyte in a sample. Background Art

[0003] See, for example, EP291194, which has developed and commercialized a simple lateral flow immunoassay device for detecting analytes in liquid samples. Such devices typically include a porous carrier comprising a dry, mobilisable labeled binding reagent capable of binding to the analyte in question, and a fixed binding reagent capable of binding to the analyte provided in the detection zone downstream of the labeled binding reagent. In the case of being in a wet state, the mobilisable labeled binding reagent is movable within the porous carrier. The fixed binding reagent is permanently fixed in the detection zone and is therefore immobile in a wet state. The presence of an analyte in the sample is indicated by the presence of a captured labeled binding reagent in the detection zone.

[0004] Alternatively, where the analyte of interest is a hapten, the immunoassay device may employ a competition reaction in which the labeled analyte or analyte analog competes with the analyte present in the sample for binding to an immobilized binding reagent at the detection zone. Alternatively, the test device may employ an inhibition reaction, providing an immobilized analyte or analyte analog at the detection zone, the test device also comprising a mobilizable labeled binding reagent for the analyte.

[0005] The test device is capable of detecting the presence and / or amount of more than one analyte. For example, in the case of a test for the presence of drugs of abuse, the device is capable of determining an entire group of drugs. Such lateral flow immunoassay devices are typically provided with a plurality of detection zones, which are provided on a single or multiple lateral flow carriers within the test device.

[0006] Digital devices have been developed that include optical detection means for identifying test results and display means for displaying the test results. A digital test reader used in combination with a test strip to determine the concentration and / or amount of an analyte in a liquid sample is referred to as a test device with an integrated digital test reader. An example of such a device is disclosed in EP1484601.

[0007] Light from a light source such as a light emitting diode (LED) is irradiated onto a portion of the porous carrier, and the reflected or transmitted light is detected by a light detector. Typically, the reader has more than one LED to illuminate various areas of the carrier, and a corresponding light detector is provided for each of the more LEDs. EP1484601 discloses an optical path arrangement for a lateral flow test strip digital reading device, which includes a baffle arrangement that reduces the possibility of the number of light detectors in the device.

[0008] The above-mentioned type of test technology has been applied to "self-test" pregnancy test devices. Generally, these devices are used by women who suspect that they may be pregnant. Therefore, they must be designed in a way that is easy to use (no medical or technical training is required) and can be discarded after a single use. The device is generally a lateral flow immunoassay device, and the test is usually initiated by contacting the sampling portion of the lateral flow test stick with the urine sample. The sampling portion of the test stick can be immersed in the urine sample in the container, or more generally, the user can urinate directly on the sampling portion. Then the test is performed without the user having to perform any further steps, and the results are displayed and read with the naked eye, or, in a digital device, the results are identified by a test result reading member and displayed to the user by a display such as a liquid crystal display (LCD).

[0009] These conventional pregnancy tests measure human chorionic gonadotropin (hCG) in a sample. hCG is produced by the developing embryo, and concentrations of hCG in the sample above a certain threshold will result in a positive (i.e., "pregnancy") result.

[0010] Improvements in pregnancy tests, especially self-diagnosed pregnancy tests, are necessary: ​​many women want to know whether they are pregnant as soon as possible. Confirming pregnancy as early as possible is beneficial because a positive pregnancy test can lead to important positive behavioral changes that promote maternal and fetal health. For example, pregnant women may be encouraged to adjust their diet, start taking pregnancy supplements, and avoid the risks to the developing fetus from drug and alcohol use and occupational exposure to mutagens / teratogens. Recently, Prior et al. (Obstetrics and Gynaecology, 2017, 129(4, 727-733)) examined the alcohol consumption of 5036 pregnant women and found that more than half of the women drank alcohol when trying to conceive, but most stopped drinking or at least reduced their drinking after obtaining a positive pregnancy test. They concluded that raising early pregnancy awareness is more effective than promoting abstinence in people trying to conceive, which may be applied to other important health information related to pregnancy behavior.

[0011] This has created a need for highly sensitive tests that can detect very low concentrations of hCG in samples such as urine during the early stages of pregnancy. However, one problem with using highly sensitive tests that measure hCG is that hCG is sometimes present in relatively low concentrations in urine for reasons other than pregnancy, so a highly sensitive pregnancy test may give a false positive result. For example, low levels of hCG may be produced in the pituitary gland in perimenopausal and menopausal women.

[0012] According to the World Health Organization, the generally accepted definitions of "menopause" and "perimenopause" are as follows:

[0013] Menopause (natural menopause) – defined as permanent menopause due to loss of ovarian follicle activity. Continuous amenorrhea for 12 months in the absence of other obvious pathological or physiological causes is considered natural menopause. Menopause occurs at the last menstrual period (FMP), which can only be determined one year or more after it has occurred.

[0014] Perimenopause - Perimenopause includes the period before menopause (when endocrine, biological, and clinical features begin to approach menopause) and the first year after menopause.

[0015] Therefore, for the time being, according to the above WHO definition, perimenopausal women are defined as women who are in the perimenopausal period. According to the above WHO definition, postmenopausal women are defined as women who have gone through menopause.

[0016] In the United States and most developed countries, women are starting families later. From 2000 to 2014, the average age of first-time mothers in the United States increased by 1.4 years, from 24.9 to 26.3 years (Mathews and Hamilton, National Center for Health Statistics, Data Brief 2016; No 232). Between 2000 and 2012, the first birth rates for women aged 35-39 and 40-44 increased by 24% and 35%, respectively (Mathews and Hamilton, National Center for Health Statistics, Data Brief 2014; No 152). Recently, data from the U.S. Department of Health and Human Services in 2017 (Brady et al, National Center for Health Statistics. Report No 004. May 2018) showed that the fertility rate of women in almost all age groups under 40 has declined, but the fertility rate of women in their early 40s has increased. The fertility rate for women aged 40-44 in 2017 was 11.6 births per 1,000 women, up 2% from 2016. Fertility rates for this age group have generally risen since 1982. According to one study, up to 10% of over-the-counter pregnancy tests are sold to women over 40 (Leavitt SA 2006, "A private little revolution: the home pregnancy test in American Culture". Bulletin of the History of Medicine 2006 80 ,317-45).

[0017] Perimenopause can begin in a woman's 30s and last up to 10 years (American College of Obstetricians and Gynocologists 2015, FAQ047), causing changes in hormone levels and possibly affecting ovulation, leading to irregular menstrual cycles. Menopause occurs at an average age of 51, but 1% of women experience it before age 40 (Haller-Kikkatalo et al, Human Reproduction 2015). 30(5), 1229-1238). Many women have little understanding of their fertility and are unaware that they are experiencing perimenopause / menopause. The interruption of menstrual cycles during perimenopause, or the absence of menstruation during menopause, can be mistaken for amenorrhea, leading to pregnancy testing.

[0018] Published literature reports elevated hCG levels during perimenopause, Snyder et al. (Clinical Chemistry 2005 51 ,1830-1835) found that 1.3% of the population they examined had serum hCG levels above 5mIU / ml.

[0019] During menopause, pituitary hCG production has been shown to increase (Odell and Griffin, New England Journal of Medicine 1987). 317, 1688-91), (Stenman et al, Journal of Clinical Endocrinology & Metabolism 1987 64, 730-6). As women approach menopause, the number of follicles available for recruitment in each cycle decreases, and more follicle-stimulating hormone (FSH) is needed to stimulate the remaining follicles. As a result, FSH levels increase, and in some women, this increase in FSH may continue into menopause. Increased FSH levels in perimenopausal and postmenopausal women result in the coproduction of small amounts of hCG in the pituitary gland.

[0020] As Snyder et al. (Clinical Chemistry 2005 51 ,1830-1835) and Alfthan et al. (Clinical Chemistry 1992 38, Several researchers from 1981-1987 have documented the production of hCG in postmenopausal women. Serum levels have been reported to be as high as 13 mIU / ml, with 6.7% of women having hCG concentrations above 5 mIU / ml (Snyder et al, 2005).

[0021] In order to be able to detect pregnancy as early as six days before the menstrual period, a highly sensitive hCG test is required. However, the risk of a highly sensitive pregnancy test that detects only low levels of hCG (i.e. a test that detects both pregnancy and hCG produced by the pituitary gland) is that it is also more likely to detect low levels of pituitary hCG and therefore give a false-positive result.

[0022] In a different context, in many countries, routine serum hCG testing is performed on almost all female patients of childbearing age before any medical intervention that could harm the developing fetus. The problem of elevated serum hCG levels due to “pituitary” hCG in perimenopausal and postmenopausal women is recognized. Snyder et al. (Clinical Chemistry 2005 51,1830-1835) examined the change in serum hCG concentration with age in nonpregnant women and investigated the use of serum FSH measurements as an aid in interpreting higher-than-expected hCG results. They suggested that the combination of serum hCG measurements, subject age, and serum FSH measurements could reduce or avoid "false-positive" pregnancy results. However, these researchers were not concerned with self-diagnosis pregnancy tests, especially not with the detection of pregnancy at a very early stage.

[0023] Attempts have been made to reduce the occurrence of false positive pregnancy results caused by the detection of non-pregnancy related hCG present in urine samples. For example, WO2016156981 discloses a test device for detecting pregnancy, comprising: a test member for measuring the absolute or relative amount of hCG in a subject sample; a test member for measuring the absolute or relative amount of FSH in a subject sample; a test member for measuring the absolute or relative amount of one or more progesterone metabolites in a subject sample. This concept is based on the following observations. First, after conception, since the woman is already pregnant, no further ovulation cycle is required. Therefore, after conception, the reduction in FSH inhibits folliculogenesis. Therefore, the expected hCG level of pregnant women is high and the FSH level is low. Secondly, during the first 10-12 weeks of pregnancy, the progesterone produced by the corpus luteum supports the endometrium, allowing the pregnancy to continue. Progesterone rises during the luteal phase of the menstrual cycle, but if there is no pregnancy, the level falls back to the baseline level (i.e., in the case of menstruation). However, if pregnancy occurs, the levels of progesterone (and its urinary metabolites) will remain elevated and will continue to rise throughout the pregnancy, so progesterone (and its urinary metabolites) can serve as an adjunct to hCG for additional confirmation of pregnancy. Therefore, WO2016156981 attempts to solve the problem of false positive pregnancy results by proposing a test device that, in addition to the hCG test, also includes a test for measuring FSH and one or more progesterone metabolites. The results of the FSH and progesterone metabolite tests are used to decide whether pregnancy is indicated based on a positive hCG result.

[0024] Reading and interpreting many test results to determine whether a positive result can be declared is laborious and leaves room for error, especially for typical users of such devices (who have not been trained to perform or interpret biochemical tests). WO2016156981 proposes using a digital test reader to reduce the burden on users. However, one disadvantage of this approach is that it increases the cost and complexity of the test device. Summary of the invention

[0025] The present invention provides an assay device for detecting the presence of an analyte in a sample. The device comprises an assay flow path, the flow path comprising:

[0026] (a) a reagent zone, the reagent zone comprising a mobilizable labeled conjugate, the labeled conjugate being configured to associate with the analyte, wherein the mobilizable labeled conjugate is also

[0027] i. configured to associate a first negative marker and / or

[0028] ii. configured to correlate positive markers, and / or

[0029] iii. comprising a positive marker or a positive marker conjugate or being configured to associate with a positive marker or a positive marker conjugate such that the positive marker or the positive marker portion of the conjugate is available for binding,

[0030] (b) one or more capture zones comprising an immobilized capture substance for capturing the labeled conjugate if the sample contains a negative marker and / or an immobilized capture substance for capturing the labeled conjugate if the sample does not contain a positive marker,

[0031] (c) a detection zone for capturing the labeled conjugate when the sample contains the analyte and is not captured by the capture zone,

[0032] wherein the one or more capture zones are downstream of the reagent zone and the detection zone is downstream of the one or more capture zones.

[0033] In case the reagent zone comprises option (ii) and option (iii), the positive marker of option (ii) is different from the positive marker or the positive marker portion of the positive marker conjugate of option (iii).

[0034] Also provided is an assay device for detecting the presence of an analyte in a sample, the assay device comprising an assay flow path, the flow path comprising:

[0035] (a) a reagent zone comprising a mobilizable labeled conjugate, wherein the labeled conjugate comprises a detectable label, wherein the detectable label is directly or indirectly linked to

[0036] (i) means for associating the labeled conjugate with the analyte and means for associating the labeled conjugate with a first negative marker, which may be present in the sample, and / or

[0037] (ii) means for associating the labeled conjugate with the analyte, wherein the labeled conjugate further comprises a positive marker or a positive marker conjugate, the positive marker portion of the positive marker conjugate being available for binding, and / or wherein a mobilizable positive marker or positive marker conjugate is separately provided in the reagent zone and is configured to associate with the labeled conjugate after being mobilized, so that when the labeled conjugate is associated with the positive marker or positive marker conjugate, the positive marker or the positive marker portion of the conjugate is available for binding, and / or

[0038] (iii) means for associating the labeled conjugate with the analyte and means for associating the labeled conjugate with a positive marker or a positive marker conjugate in the device or a positive marker that may be present in the sample, optionally, and wherein the reagent zone further comprises a mobilizable positive marker or a positive marker conjugate for immobilization downstream of the reagent zone,

[0039] (b) one or more capture zones, wherein:

[0040] (i) in the case where the reagent area comprises option (i),

[0041] at least one capture zone comprises an immobilized capture substance, wherein the capture substance is configured to capture a complex comprising the labeled conjugate and the first negative marker, wherein the capture of the complex is preferably achieved by a specific binding reaction with the first negative marker,

[0042] (ii) in the case where the reagent area comprises option (ii),

[0043] at least one capture zone comprises an immobilized capture substance, wherein the capture substance is configured to capture a labeled conjugate containing the positive marker or the positive marker conjugate or a labeled conjugate associated with the positive marker or the positive marker conjugate or the positive marker in the presence of the positive marker in the sample, wherein the capture of the labeled conjugate is preferably achieved by a specific binding reaction with the positive marker or the positive marker conjugate,

[0044] (iii) in the case where the reagent zone comprises option (iii),

[0045] At least one capture zone comprises an immobilized capture substance configured to capture a labeled conjugate comprising means for associating the labeled conjugate with a positive marker or a positive marker conjugate, wherein

[0046] The fixed capture substance comprises or consists of a fixed positive marker or a positive marker conjugate, and / or in case the reagent zone comprises a mobilizable positive marker or a positive marker conjugate, at least one capture zone comprises a fixed capture substance for positioning the mobilizable positive marker or positive marker conjugate in the capture zone, so that the positive marker or the positive marker part of the conjugate is available for binding, wherein the capture of the labeled conjugate is preferably achieved by a specific binding reaction with a means for associating the labeled conjugate with the positive marker or the positive marker conjugate,

[0047] (c) a detection zone, said detection zone comprising an immobilized binding reagent, said immobilized binding reagent being used to capture a complex comprising said labeled conjugate and said analyte, wherein the capture of the complex is preferably achieved by a specific binding reaction with said analyte,

[0048] wherein the or each capture zone is downstream of the reagent zone and the detection zone is downstream of the or each capture zone.

[0049] Embodiments of the present invention include test devices that include, but are not limited to, the following combinations of reagent zone and capture zone options:

[0050] Reagent Area Options Capture Zone Options (i) (i) (i) and (ii) (i) and (ii) (i), (ii) and (iii) (i), (ii) and (iii) (i) and (iii) (i) and (iii) (ii) (ii) (ii) and (iii) (ii) and (iii) (iii) (iii)

[0051] Additional embodiments of the invention are set forth in the dependent claims and the following description. In one embodiment, the test device is adapted to reduce the amount of labeled conjugate reaching the detection zone in the presence of one or more additional negative markers. In one embodiment, the test device is adapted to reduce the amount of labeled conjugate reaching the detection zone in the absence of one or more additional positive markers.

[0052] As discussed in further detail below, where the test device of the present invention includes an immobilized capture material for reducing the amount of labeled conjugate reaching the detection zone in the presence of a first negative marker, the test device may also include any suitable reagents and immobilized capture materials disclosed herein to reduce the amount of labeled conjugate reaching the detection zone in the presence of a second negative marker. This concept can be extended to reducing the amount of labeled conjugate reaching the detection zone in the presence of more negative markers.

[0053] In embodiments where both reagent zone option (ii) and reagent zone option (iii) are present, preferably, the positive marker of option (ii) is different from the positive marker of option (iii). For example, the positive marker of option (ii) may be a first positive marker, and the positive marker of option (iii) may be a second positive marker. This means that when both are present in the test device of the present invention, the positive marker conjugate of option (ii) or the positive marker of the device will be different from the positive marker conjugate of option (iii) or the positive marker of the device.

[0054] Where the test device of the invention includes an immobilized capture substance for reducing the amount of labeled conjugate reaching the detection zone in the absence of a first positive marker, the test device may also include any suitable reagents and immobilized capture substances disclosed herein to reduce the amount of labeled conjugate reaching the detection zone in the absence of a second positive marker. This concept can be extended to reducing the amount of labeled conjugate reaching the detection zone in the absence of more positive markers.

[0055] In one embodiment, the device does not include any means for reducing the amount of labeled conjugate reaching the detection zone in the absence of a positive marker. In one embodiment, the device does not include any means for reducing the amount of labeled conjugate reaching the detection zone in the presence of a negative marker.

[0056] The present invention also provides a method for conducting an experiment using the experimental device of the present invention, the method comprising:

[0057] (i) Apply the sample to the test device,

[0058] (ii) determining the test result at the detection zone without reference to any capture zone present in the device. DETAILED DESCRIPTION

[0059] The present invention is based in part on the recognition of the need for improving the specificity of analyte testing. In a conventional test device, a positive test result indicating the presence of a first state in a subject can be based on the identification of the presence of an analyte in a sample from the subject (e.g., by observing the accumulation of a detectable label associated with the analyte in the detection zone of the test device). However, in the case where the analyte is associated with more than one state, there is a risk that the presence of the analyte does not necessarily mean the presence of the first state in the subject. The present invention solves this problem by adjusting the ability of the test device to produce a detectable signal in the detection zone in response to the presence of the analyte in the sample using the presence or absence of an additional marker in the sample of the subject. The present invention contemplates the use of one or more negative markers in the sample to reduce the amount of labeled conjugates associated with the analyte that arrive in the detection zone and produce a signal. The present invention also contemplates the use of one or more positive markers in the sample to reduce the amount of labeled conjugates associated with the analyte that arrive in the detection zone and produce a signal. Using these two strategies alone or in combination reduces the risk of generating a signal (i.e., observing a positive test result) in the detection zone when the subject does not have the first state.

[0060] A non-limiting example of this principle is in the context of pregnancy testing. In this embodiment, the analyte indicating pregnancy (but may also be present in other states such as menopause or perimenopause) is hCG. According to the present invention, pregnancy negative markers such as FSH (increased in perimenopause or menopause) can be used to reduce the amount of labeled conjugates associated with hCG that reach the detection zone and generate signals. In the embodiment, the reagent zone can include a removable labeled conjugate (e.g., anti-hCG antibodies and anti-FSH antibodies can be included) that can be associated with both hCG and FSH. FSH capture zones (not visible to the user, or visible but not interpreted by the user, or not actually measured by the device) can be provided downstream of the reagent zone and upstream of the detection zone. In the case where the sample contains hCG but not FSH, the removable labeled conjugate is associated with hCG, passes through the capture zone and combines in the detection zone, so that a signal is observed in the detection zone, indicating that the subject is pregnant. In the case where the sample contains hCG and FSH (e.g., because the subject is in perimenopause), the removable labeled conjugate can be associated with hCG and FSH to form a complex. The complex is captured in the FSH capture zone and therefore cannot move to the detection zone. Therefore, although the sample contains hCG, the amount of labeled conjugate that reaches the detection zone and produces an observable signal is reduced relative to the presence of hCG but not FSH.

[0061] According to the present invention, a pregnancy positive marker such as P3G can be used to adjust the amount of labeled conjugates associated with the analyte that reaches the detection zone and generates a signal. For example, the reagent zone may include a removable labeled conjugate that can be associated with both hCG and P3G (for example, it may include anti-hCG antibodies and anti-P3G antibodies). A capture zone (not visible to the user, or visible but not interpretable by the user, or not actually measured by the device) may be provided downstream of the reagent zone and upstream of the detection zone and may include a P3G conjugate. The detection zone may include, for example, anti-hCG antibodies. In the case where the sample contains hCG and P3G, the removable labeled conjugate will be associated with hCG and P3G to form a complex to prevent the P3G conjugate in the capture zone from capturing the complex. The complex passes through the capture zone and is bound to the detection zone by the hCG in the sample, so that a signal is observed in the detection zone, indicating that the subject is pregnant. In the case where the sample contains hCG but not P3G, the removable labeled conjugate will be associated with hCG to form a complex. The P3G conjugate captures the labeled reagent in the capture zone, preventing it from continuing to enter the detection zone. Therefore, although the sample contains hCG, the amount of labeled conjugate that reaches the detection zone and produces an observable signal is reduced. In an optional arrangement, the P3G conjugate can be provided on a mobile conjugate instead, and a fixed capture substance (e.g., anti-P3G antibody) configured to capture P3G or P3G conjugate is provided in the capture zone. In this embodiment, in the case where the sample contains P3G, it will be captured by the capture zone, meaning that the labeled conjugate containing the P3G conjugate will not be captured in the capture zone and can continue to enter the detection zone, where it will react with the hCG in the sample and produce a signal. In the case where hCG is present in the sample but P3G is not present, the mobile conjugate will be captured by the fixed capture substance in the capture zone and cannot continue to enter the detection zone. Therefore, although the sample contains hCG, the amount of labeled conjugate that enters the detection zone and produces an observable signal is reduced relative to the case where both hCG and P3G are present.

[0062] One application of the present invention is to provide an improved high-sensitivity pregnancy test with improved specificity, allowing pregnancy to be determined earlier. This improved pregnancy test is particularly useful for perimenopausal or postmenopausal individuals because it can reduce the risk of obtaining a positive result due to a small amount of hCG from the pituitary gland in the test sample when these individuals are not pregnant. It should be understood that this principle can be applied to other analytes and markers, including those associated with other states. For example, one can envision a test for testing disease X based on identifying analyte A in a sample. Analyte A is also present in state Y. Negative marker N is present at a relatively high level in state Y, but is not present or is present at a relatively (significantly) low level in disease X. The test device of the present invention may include a movable labeled conjugate containing a binding reagent for analyte A and a binding reagent for negative marker N, a capture zone containing a fixed capture substance of a binding reagent for negative marker N, and a detection zone containing a fixed binding reagent for analyte A. In the presence of negative marker N and analyte A in the sample applied to the device (because the subject from which the sample was derived had condition Y but not disease X), N binds to the labeled conjugate to form a complex that is bound at the capture zone. Therefore, in the presence of negative marker N in the sample, the capture zone reduces the amount of labeled conjugate that reaches the detection zone and produces an observable signal in the presence of analyte A. Therefore, especially in the presence of condition Y and testing a sample containing analyte A, the risk of a positive result indicating the presence of disease X is reduced, and the specificity of the test is improved.

[0063] In another embodiment, an assay for testing for disease X may be based on the identification of analyte A in a sample. Analyte A is also present in state Y. Positive marker P is present at relatively high levels in disease X, but absent or at relatively (significantly) low levels in state Y. The test device of the present invention may include a removable labeled conjugate containing a binding reagent for analyte A and a binding reagent for positive marker P, a capture zone containing an immobilized positive marker or positive marker conjugate, and a detection zone containing an immobilized binding reagent for analyte A. In the presence of positive marker P and analyte A in the sample, the binding reagent for positive marker P will bind to the positive marker P in the sample, preventing the labeled conjugate from being captured by the immobilized positive marker or positive marker conjugate in the capture zone. The labeled conjugate will continue to enter the detection zone, where it is captured by the immobilized binding reagent for analyte A, thereby generating a signal indicating the presence of disease X in the detection zone. In the case where analyte A is present in the sample but positive marker P is not present, the binding reagent of the labeled conjugate for positive marker P will bind to the positive marker or positive marker conjugate immobilized in the capture zone, preventing the labeled conjugate from continuing to enter the detection zone. Therefore, in the case where analyte A is present in the sample but positive marker P is not present, the capture zone reduces the amount of labeled conjugate that reaches the detection zone and generates an observable signal.

[0064] The devices of the invention can be adapted to include mobilizable reagents and immobilized capture substances to reduce the amount of labeled conjugate reaching the detection zone in the presence of any amount of additional negative markers and / or in the absence of any amount of additional positive markers.

[0065] Test flow path

[0066] In one embodiment, the test device of the invention comprises a test flow path comprising a reagent zone, one or more capture zones and a detection zone. The or each capture zone is downstream of the reagent zone and the detection zone is downstream of the or each capture zone.

[0067] "Upstream" and "downstream" are used to describe the location of components of a test device with reference to the direction of flow of a sample applied to the device. If component A is upstream of component B and / or component B is downstream of component A, then a sample applied to the device (at a predetermined sample application location) will reach component A before reaching component B.

[0068] The dried binding reagent is provided in the flow path of the microfluidic device, or in the case of a lateral flow type device, on a porous carrier material, which is provided upstream of the porous carrier material containing the detection zone. The porous carrier material upstream can be macroporous. The macroporous carrier material should be low or non-protein binding, or should be easily blocked by reagents such as BSA or PVA to minimize nonspecific binding and promote the free movement of the labeled reagent after the macroporous carrier is wetted by the liquid sample. Where necessary, the macroporous carrier material can be pretreated with a surfactant or solvent to make it more hydrophilic and promote the rapid absorption of the liquid sample. In addition, one or more sugars (such as sucrose, trehalose) can be used to stabilize and help move the labeled reagent. These can be conveniently applied to the flow path and / or the porous carrier material as part of the solution, and the labeled reagent is applied to the porous carrier in the solution. Suitable materials for macroporous carriers include plastic materials (such as polyethylene and polypropylene) or other materials (such as paper or glass fiber). In the case where the labeled binding agent is labeled with a detectable particle, the pore size of the macroporous carrier may be at least ten times the maximum particle size of the labeled particle. A larger pore size may allow for better release of the labeled agent.

[0069] The test flow path may comprise a lateral flow porous support. The porous support material is usually a material with a high protein binding capacity, which is blocked with a reagent such as BSA or PVA after the reagent is applied to form a fixed capture and detection zone to minimize non-specific binding and promote the free movement of the labeled conjugate after the macroporous support is wetted with the liquid sample. Suitable materials that can be used as porous supports include nitrocellulose, acetate, cellulose or cellulose derivatives, polyesters, polyamides, polyolefins or glass fibers. The porous support may comprise nitrocellulose. It has the advantage that the binding reagent can be firmly fixed without prior chemical treatment. If the porous solid phase material comprises paper, for example, the immobilization of the antibody can be carried out by chemical coupling using such as CNBr, carbonyldiimidazole or trifluoroethanesulfonyl chloride. The test device may comprise a sample application zone. It is an area of ​​porous (usually absorbent) material to which an aqueous sample (such as urine) can be applied.

[0070] An alternative to the flow path of lateral flow tests is a "flow through" test device. In such a device, the sample flows essentially vertically rather than laterally. Preferably, the device comprises a sample application zone; one or more membranes containing one or more capture zones as described herein, arranged vertically or essentially vertically below the sample application zone; and a membrane containing a detection zone as described herein, arranged vertically or essentially vertically below one or more capture zone membranes. The sample is applied to the sample application zone and flows downward through the capture zone membrane or each capture zone membrane to reach the detection zone membrane. The test result is determined by observing or reading the detection zone, such as from the bottom surface of the flow device. The flow test device may or may not include a reagent zone as described above. If a reagent zone is provided, it is located in another membrane upstream (in a substantially vertical direction) of the capture zone membrane. Such a reagent zone may include any reagent described herein (e.g., a labeled conjugate) in a dry state. Alternatively, or in addition, a reagent such as a labeled conjugate described herein may be provided in a dry state near the capture zone membrane. Alternatively, the labeled conjugate may be provided separately and applied to the device before or after the sample is applied, or applied to the device together with the sample. For example, such reagents may be mixed with the sample before being applied to the device. The present invention includes a kit containing a flow-through test device as described herein, a labeled reagent as described herein, and optionally any other reagents as described herein (e.g., a reagent provided in a reagent zone).

[0071] Typically, the sample is applied by the user urinating directly on the sample application area. Alternatively, the sample application area can be immersed in a container containing the sample. In the case where the sample is blood, serum or plasma, the sample can be applied to the device using a pipette or other instrument, and then an elution buffer can be added to move the sample components along the test flow path.

[0072] The sample application zone is preferably upstream of the reagent zone.

[0073] An absorbent "sink" may be provided at the far, downstream end of the test flow path. The absorbent sink may preferably comprise a highly absorbent material, such as CF7 Whatman paper, and should provide sufficient absorptivity to remove any unbound label from near the detection zone. A solid phase porous material having a certain length extending out of the detection zone may serve as a substitute for such a sink. The advantage of providing a highly absorbent sink is that it removes or substantially removes excess labeled binding reagent from the test flow path, minimizing the amount of unbound labeled binding reagent near each area. Removing or reducing unbound labeled reagent near the detection zone (and control zone, if any) helps determine the correct test result.

[0074] Reagent Area

[0075] The reagent zone of the test device of the present invention comprises a mobile labeled conjugate which may contain a detectable label.

[0076] A "mobile" substance refers to a substance that is associated with a matrix (eg, a component of an assay flow path) in a dry state and that is mobile and capable of flowing along the matrix when in contact with a fluid.

[0077] The "label" in the labeled conjugate refers in the context to any substance that can produce a signal that can be detected by visual or instrumental means. Various labels applicable to the present invention include labels that can produce signals by chemical or physical means (e.g., optically detectable). Such labels include enzymes and substrates, chromogens, catalysts, fluorescent compounds, chemiluminescent compounds, electroactive substances, dye molecules, radioactive labels and particle labels. Particle labels may include magnetic or charged labels that can be detected by magnetic or electrochemical means. The label may be covalently attached to a binding agent. The label may be optically detectable. Preferred optically detectable labels include colloidal metal particle labels and dye particles hereinafter.

[0078] The label may include colloidal metal particles (e.g., gold, silver, platinum, silver enhanced gold sol, carbon sol or carbon nanoparticles), colloidal metalloid or non-metal particles (e.g., tellurium or selenium), or dyed or colored particles (e.g., polymer particles doped with dyes or dye sols). The dye may be any suitable color, such as blue. The dye may be fluorescent, or include quantum dots. Suitable fluorescent materials are well known to those skilled in the art. Dye sols may be prepared from commercially available hydrophobic dyes (e.g., Foron Blue SRP (Sandoz) and Resolin Blue BBLS (Bayer)). Suitable polymer labels may be selected from a range of synthetic polymers (e.g., polystyrene, polyvinyl toluene, polystyrene-acrylic acid and polyacrylaldehyde). The monomers used are typically water-insoluble and emulsified in an aqueous surfactant, thereby forming monomer micelles, and then polymerization is induced by adding an initiator to the emulsion to produce nearly spherical polymer particles. The ideal size range of such polymer particles is from about 0.05 μm to about 0.5 μm. Larger polymer particles can also be coupled to a porous membrane of appropriate size for use. According to an exemplary embodiment, the label is a gold colloid, preferably with an average particle diameter in the range of 0.02 μm to 0.25 μm. In one embodiment, the label is a blue latex. In one embodiment, the label is a gold sol. In one embodiment, the label is a 40 nm gold sol.

[0079] For the time being, the term "binding agent" refers to one of a binding pair (i.e., two different molecules, one of which binds to the second molecule by chemical and / or physical means). The two molecules are related in the sense that they bind to each other in a way that allows them to distinguish their binding partner from other test components with similar characteristics. The members of a binding pair may be referred to as a ligand and a receptor (anti-ligand), a binding pair member, and a binding pair partner, etc. The molecules may also be binding pair members of an aggregate of molecules; for example, an antibody and its corresponding antigen used in an immune complex of a secondary antibody may be considered a binding pair member of an immune complex. The binding agent is typically a specific binding agent. For example, a binding agent may specifically bind to the alpha subunit of hCG and the alpha subunit on FSH, the alpha subunit on luteinizing hormone (LH), or the alpha subunit on thyroid stimulating hormone (TSH). The specific binding agent may be a binding agent that binds to the beta subunit of hCG.

[0080] Binding reagents may consist of or include antibodies. As used herein, the term "antibody" includes complete antibodies and antigen-binding fragments thereof. Antigen-binding fragments can be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab, F(ab')2, and Fv fragments. Binding reagents may consist of or include aptamers. Binding reagents may consist of or include affimers. Binding reagents such as antibodies for use in the present invention may be obtained from various suppliers (e.g., MedixBiochemica Espoo, Finland).

[0081] In addition to antigen and antibody binding pair members, other binding pairs include, but are not limited to, biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, peptide sequences and antibodies specific for the sequence or the entire protein, polymeric acids and bases, dyes and protein binders, peptides and specific protein binders (e.g., ribonucleases, S-peptides and ribonuclease S-proteins), etc. In addition, specific binding pairs may include analogs of the original specific binding agents.

[0082] In some embodiments, the mobilizable labeled conjugate comprises means for associating the labeled conjugate with the analyte and means for associating the labeled conjugate with a first negative marker that may be present in the sample.

[0083] The member for associating the labeled conjugate with the analyte may include a binding agent or be composed of a binding agent. The binding agent may directly or indirectly bind to the analyte. In the case where the binding agent indirectly binds to the analyte, the removable analyte binding agent connected to the first binding partner may be provided separately from the labeled conjugate (e.g., in a reagent zone). In these embodiments, the labeled conjugate may be provided together with the second binding partner for the first binding partner. In this way, the binding agent for the analyte may be positioned at the labeled conjugate without being directly connected to the labeled conjugate. The first and second binding partners may be any suitable binding partners (e.g., antibody-antigen) that are specific to each other. Examples include binding agent pairs, such as biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies.

[0084] Either party in the binding pair can be connected to a labeled conjugate, while the other party is provided, for example, in a reagent zone or elsewhere along the flow path but upstream of the capture zone. This concept can be adapted to include more intermediate binding partners. This concept can be adapted to position other binding members disclosed herein at an ideal position on the assay flow path.

[0085] The means for associating the labeled conjugate with the analyte may comprise or consist of an antibody (eg, an antibody that binds an epitope of the analyte).

[0086] The member for associating the labeled conjugate with the first negative marker may include a binding agent or be composed of a binding agent. The binding agent may directly or indirectly bind to the first negative marker. In the case where the binding agent is indirectly bound to the first negative marker, the removable first negative marker binding agent connected to the first binding partner may be provided separately from the labeled conjugate (e.g., in a reagent zone). In these embodiments, the labeled conjugate may be provided together with the second binding partner for the first binding partner. In this way, the binding agent for the first negative marker may be positioned at the labeled conjugate, and not directly connected to the labeled conjugate. The first and second binding partners may be any suitable binding partners (e.g., antibody-antigen) that are specific to each other. Examples include binding agents to (e.g., biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies).

[0087] Either party in the binding pair can be connected to a labeled conjugate, while the other party is provided, for example, in a reagent zone or elsewhere along the flow path but upstream of the capture zone. This concept can be adapted to include more intermediate binding partners. This concept can be adapted to position other binding members disclosed herein at an ideal position on the assay flow path.

[0088] The means for associating the labeled conjugate with the first negative marker may comprise or consist of an antibody (eg an antibody that binds to an epitope of the first negative marker).

[0089] The component used to associate the labeled conjugate with the analyte and the component used to associate the labeled conjugate with the first negative marker can be the same. For example, the labeled conjugate can be directly or indirectly connected to an antibody that can bind to the analyte or the first negative marker. The antibody can bind to an epitope shared by the analyte and the first negative marker. The antibody can bind to a domain shared by the analyte and the first negative marker. In the case where the analyte is hCG and the first negative marker is FSH, the removable labeled conjugate can include an antibody that can bind to the α subunit of hCG. Both FSH and hCG are heterodimer molecules, containing α and β subunits. The α subunits of FSH and hCG are substantially the same, so the antibody for the α subunit can bind to both FSH and hCG. In some embodiments, the component used to associate the labeled conjugate with the analyte and the component used to associate the labeled conjugate with the first negative marker are different. For example, the labeled conjugate may be directly or indirectly linked to an antibody that binds to the analyte but not to the first negative marker, and also directly or indirectly linked to an antibody that binds to the first negative marker but not to the analyte.

[0090] In some embodiments, the removable labeled conjugate comprises a positive marker or a positive marker conjugate, wherein the positive marker portion of the positive marker conjugate can be used for binding. The positive marker or positive marker conjugate can be connected or otherwise associated with a component for associating the labeled conjugate with an analyte. In the case where the positive marker is a hapten, it is preferably coupled to another part such as a carrier to form a positive marker conjugate. A larger positive marker may or may not be coupled to another part. A larger (non-hapten) positive marker can be directly fixed on a removable labeled conjugate by a method such as physical absorption or chemical coupling. However, in the case where direct fixation has an adverse effect on the structure of the positive marker or its ability to participate in a binding reaction, it may be preferred to connect a non-hapten positive marker to another part. The positive marker may be a progesterone metabolite such as P3G. The component for associating the labeled conjugate with an analyte may be any component disclosed herein. The positive marker / positive marker conjugate can be coupled to a binding reagent for the analyte and directly or indirectly connected to the labeled conjugate in a manner discussed herein. For example, in the case where the analyte is hCG and the positive marker is a progesterone metabolite such as P3G, the progesterone metabolite such as P3G can be coupled to an anti-hCG antibody (e.g., an anti-α-hCG antibody). Alternatively or in addition, a removable positive marker or positive marker conjugate can be provided separately in the reagent zone. In this case, the removable positive marker or positive marker conjugate can be associated with the labeled conjugate after movement, so that when the labeled conjugate is associated with the positive marker or positive marker conjugate, the positive marker or the positive marker portion of the conjugate can be used for binding. In order to promote this association, the labeled conjugate and the positive marker / positive marker conjugate can each contain members of a binding pair, so that when the members of the binding pair are in contact with each other, the labeled conjugate is associated with the positive marker / positive marker. The first and second binding partners can be any suitable binding partners (e.g., antibody-antigen) that are specific to each other. In the case where the liquid contacts the positive marker or positive marker conjugate, the positive marker or positive marker conjugate is removable and can be associated with the labeled conjugate. Examples of binding pairs include biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies. Any partner can be connected to a labeled conjugate, and another partner (connected to a positive marker or a positive marker conjugate) is provided, such as in a reagent zone or elsewhere upstream of a capture zone along a flow path. This concept can be adapted to include more intermediate binding partners.

[0091] The positive marker conjugate comprises a positive marker portion. The positive marker portion may be bound by a binding agent such as an antibody. The positive marker conjugate may comprise a progesterone metabolite such as P3G or a portion thereof. The positive marker conjugate may comprise a protein carrier (e.g., BSA, ovalbumin, or mouse antibody).

[0092] In some embodiments, the removable labeled conjugate includes a component for associating the labeled conjugate with a positive marker or a positive marker conjugate. The component for associating the labeled conjugate with a positive marker / positive marker conjugate may include a binding agent or be composed of a binding agent. The binding agent may directly or indirectly bind to a positive marker or a positive marker conjugate. In the case where the binding agent indirectly binds to a positive marker or a positive marker conjugate, the removable binding agent for the positive marker / positive marker conjugate connected to the first binding partner may be provided separately from the labeled conjugate, such as in a reagent zone, or along the flow path but in other places upstream of the capture zone. In these embodiments, the labeled conjugate may be provided together with the second binding partner for the first binding partner. In this way, the binding agent for the positive marker / positive marker conjugate can be located at the labeled conjugate without being directly connected to the labeled conjugate. The first and second binding partners may be any suitable binding partners (e.g., antibody-antigen) specific to each other. Examples include biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies. Either one of the partners is linked to a labeled conjugate, while the other partner (linked to a binding reagent for the positive marker / positive marker conjugate) is provided, for example, in a reagent zone or elsewhere along the flow path but upstream of the capture zone. This concept can be adapted to include more intermediate binding partners.

[0093] The component for associating the labeled conjugate with the positive marker or the positive marker conjugate may comprise or consist of an antibody (e.g., an antibody that binds to an epitope of the positive marker). For example, in the case where the positive marker is P3G, the component for associating the labeled conjugate with the positive marker or the positive marker conjugate may comprise or consist of an anti-P3G antibody.

[0094] In some embodiments, the reagent zone further comprises a removable positive marker or a positive marker conjugate for being fixed on a capture zone such as the reagent zone downstream. This removable positive marker / positive marker conjugate can comprise a member in conjunction with right or be connected with a member in conjunction with right. Another member in conjunction with right is provided in the downstream of the reagent zone, such as the capture zone. Therefore removable positive marker / positive marker conjugate can be fixed on the capture zone, so that it can be associated with the member for being associated with positive marker / positive marker conjugate through labeling conjugate. In conjunction with right example comprises biotin and streptavidin, biotin and anti-biotin antibody, fluorescein and anti-fluorescein antibody. Any one in the partner can be connected to removable positive marker / positive marker conjugate, and another partner is provided, for example in the capture zone. This concept can be adapted to include more intermediate binding partners.

[0095] Capture Zone

[0096] The device of the present invention comprises one or more capture zones, located downstream of the reagent zone and upstream of the detection zone. The function of one or more capture zones is to ensure that the labeled conjugate detected in the detection zone can reliably indicate the presence of the first state in the subject from which the sample is derived. This function is achieved by: (i) capturing the labeled conjugate in the capture zone when the sample contains one or more negative markers (preferably markers indicating the absence of the first state), thereby reducing the amount of the labeled conjugate reaching the detection area, and / or (ii) unless the sample contains one or more positive markers (preferably markers indicating the presence of the first state), otherwise capturing the labeled conjugate in the capture zone, these positive markers thereby reducing the amount of the labeled reagent reaching the detection zone. Therefore, in the presence of negative markers and / or in the absence of positive markers, the capture zone will reduce the amount of the labeled conjugate reaching the detection zone, thereby reducing the possibility of the detection zone producing a detectable signal even in the presence of a small amount of analyte. This means that the possibility of a false positive (i.e., indicating the presence of the first state in the absence of the first state) is reduced, and the specificity of the test is improved. Advantageously, not only can the capture zone reduce the amount of labeled conjugate that reaches the detection zone, but the capture zone can also reduce the amount of analyte that reaches the detection zone, because the complex captured in the capture zone may also contain the analyte. In the case where the test sample is from a subject who does not have the first state, this dual effect significantly reduces the risk of detecting a signal in the detection zone. Preferably, the device does not include a capture zone, which includes a fixed capture material that can bind the analyte.

[0097] The capture zone may be designed to take advantage of the presence of one or more negative markers in the sample to reduce the amount of labeled conjugate (which may be associated with the analyte) that reaches the detection zone.

[0098] The capture zone can be designed so that one or more negative markers present in the sample reduce the amount of the labeled conjugate (which may be associated with the analyte) that reaches the detection zone. The capture zone may include a fixed capture material that is configured to capture a complex containing the labeled conjugate and the first negative marker. The capture of the complex is preferably achieved by a specific binding reaction with the first negative marker. Preferably, the binding reaction occurs at an epitope of the first negative marker that is different from the epitope used to bind the component associated with the first negative marker of the labeled conjugate that is movable, thereby forming a "sandwich". In the case of an analyte in the sample, the captured complex may further include the analyte. Preferably, the fixed capture material for the negative marker is configured not to capture a complex that does not contain the first negative marker.

[0099] One or more capture zones may include a fixed capture material specific to the first negative marker. In some embodiments, when a sample containing the first negative marker is applied to a test device, the first negative marker is associated with the labeled conjugate by a member for associating the labeled conjugate with the first negative marker, forming a first negative marker-labeled conjugate complex. In the case where there is also an analyte in the sample, the complex may further include the analyte, because the labeled conjugate can combine both the analyte and the negative marker. In the case where the complex arrives at a capture zone containing a fixed capture material configured to capture a complex containing the labeled conjugate and the first negative marker, the complex is captured and cannot flow to the detection zone. Therefore, even if there is an analyte in the sample, the presence of the first negative marker in the sample can prevent or reduce the total amount of the labeled conjugate that arrives in the detection zone and produces a detectable signal. This also effectively reduces the amount of the analyte that arrives in the detection zone, further reducing the signal of the detection zone in the sample containing the analyte and the negative marker.

[0100] The fixed capture material can directly bind to the first negative marker. The fixed capture material can bind to an epitope of the first negative marker that is not present in the analyte. The fixed capture material can comprise or consist of an antibody. The antibody can bind to an epitope of the first negative marker that is not present on the analyte. In the case where the first negative marker is FSH, the capture zone can comprise a fixed anti-FSH specific antibody (e.g., an anti-β-FSH antibody).

[0101] The capture zone can include a fixed capture material that indirectly binds the first negative marker. For example, in some embodiments, a movable first negative marker binding reagent that is specific to the first negative marker and connected to the first binding partner can be provided, such as in the reagent zone or along the flow path but in other places upstream of the capture zone. The movable first negative marker binding reagent can be combined with the epitope of the first negative marker that is not present in the analyte. The capture zone or each capture zone can provide a fixed second binding partner for the first binding partner. In this way, the movable first negative marker binding reagent can be located in the capture zone without being directly fixed to the flow path. The first and second binding partners can be any suitable binding partner (such as antibody-antigen) that is specific to each other. Examples include binding reagent pairs, such as biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies. Any member of the binding reagent pair can be fixed in the capture zone, and another member (connected to the binding reagent for negative markers) is provided in, for example, the reagent zone or along the flow path but in other places upstream of the capture zone. This concept can be adapted to include more intermediate binding partners. This concept can be adapted to locate other binding members disclosed herein at the desired position on the test flow path. In one embodiment, the reagent zone includes a removable anti-β-FSH antibody labeled with a first member (e.g., biotin) of the binding pair, and the capture zone includes a fixed second member (e.g., anti-biotin antibody or streptavidin) of the binding pair. In this way, the anti-β-FSH antibody can be located in the capture zone without being directly fixed in the capture zone. This concept can be adapted to the capture zone for other negative markers.

[0102] Alternatively, or additionally, the capture zone may be designed to take advantage of the absence of one or more positive markers in the sample to reduce the amount of labeled conjugate (which may associate with the analyte) that reaches the detection zone.

[0103] The presence of the positive marker in the sample reduces the likelihood that the labeled conjugate will be captured in the capture zone (eg, by occupying a positive marker binding site in the capture zone or the labeled conjugate).

[0104] In some embodiments, at least one capture zone comprises a fixed capture material, and the fixed capture material is configured to capture a positive marker or a labeled conjugate containing a positive marker / positive marker conjugate or associated with a positive marker / positive marker conjugate. The capture of the labeled conjugate is preferably achieved by a specific binding reaction with the positive marker / positive marker conjugate. The positive marker present in the sample is bound (and therefore may be occupied) by the binding site of the fixed capture material, so that the binding site is not available for capturing a labeled conjugate containing a positive marker / positive marker conjugate or associated with a positive marker / positive marker conjugate. The labeled conjugate containing a positive marker / positive marker conjugate or associated with a positive marker / positive marker conjugate can therefore continue to enter the detection zone (unless blocked by other capture members disclosed herein).

[0105] The positive marker in the sample can compete with the positive marker / positive marker conjugate of the device to bind in the capture zone. In the absence of a positive marker in the sample, the fixed capture material captures the positive marker / positive marker conjugate containing the device or the labeled conjugate associated with the positive marker / positive marker conjugate of the device, thereby preventing the labeled conjugate from continuing to enter the detection zone. The fixed capture material can directly bind to the positive marker or positive marker conjugate. The fixed capture material can contain antibodies or be composed of antibodies. In the case where the positive marker is P3G, the capture zone can contain fixed anti-P3G antibodies.

[0106] Capture zone can comprise the fixed capture material of indirect capture positive marker or positive marker conjugate.For example, in some embodiments, the removable binding reagent connected with the first binding partner for positive marker / positive marker conjugate is provided in such as reagent zone or along flow path but in other places upstream of capture zone.In such embodiment, this capture zone or each capture zone can be provided with the fixed second binding partner for the first binding partner.Like this, the removable binding reagent for positive marker / positive marker conjugate can be located in capture zone and not directly fixed to flow path.The first and second binding partners can be any suitable binding partners specific to each other.Example includes binding reagent pair, such as biotin and streptavidin, biotin and anti-biotin antibody, fluorescein and anti-fluorescein antibody.Any member of binding reagent pair can be fixed in capture zone, and another member is provided in such as reagent zone.This concept can be adapted to include more intermediate binding partners.

[0107] In one embodiment, the reagent zone includes a mobilizable anti-P3G antibody labeled with a first member of a binding pair (e.g., biotin), and the capture zone includes an immobilized second member of the binding pair (e.g., an anti-biotin antibody or streptavidin). In this way, the anti-P3G antibody can be located in the capture zone without being directly immobilized in the capture zone. This concept can be adapted to capture zones for other positive markers.

[0108] In some embodiments, at least one capture zone comprises a fixed capture material configured to capture a labeled conjugate, which contains a member for associating the labeled conjugate with a positive marker or a positive marker conjugate. The capture of the labeled conjugate is preferably achieved by a specific binding reaction with a member for associating the labeled conjugate with a positive marker or a positive marker conjugate. The fixed capture material may comprise a fixed positive marker or a positive marker conjugate or may be composed of a fixed positive marker or a positive marker. In the case where the positive marker is a hapten, it is preferably coupled to another part (such as a carrier) to form a positive marker conjugate. A larger positive marker may or may not be coupled to another part. A larger (non-hapten) positive marker may be directly fixed on the flow path. However, for example, in the case where direct fixation has an adverse effect on the structure of the positive marker or its ability to participate in a binding reaction, it may be preferred that a non-hapten positive marker be connected to another part. Alternatively or in addition, the reagent zone may comprise a removable positive marker or a positive marker conjugate. In this case, at least one capture zone comprises a fixed capture material for positioning a removable positive marker or positive marker conjugate in the capture zone, so that the positive marker or the positive marker part of the conjugate can be used for binding. In this way, the positive marker conjugate can be located in the capture zone without being directly fixed to the flow path. Using any binding reagent disclosed herein can achieve positioning the positive marker or the positive marker conjugate in the capture zone. In the case where the positive marker or the positive marker conjugate is (directly or indirectly) fixed in the capture zone, the removable labeled conjugate comprises a member for associating the labeled conjugate with the positive marker. In the case where there is no positive marker in the sample, the labeled conjugate is fixed or captured by the positive marker conjugate or the positive marker in the capture zone, preventing the captured labeled conjugate from continuing to enter the detection zone. In the case where there is a positive marker in the sample, it becomes associated with the labeled conjugate, so that the labeled conjugate is not fixed or captured by the positive marker conjugate / positive marker in the capture zone, and can continue to enter the detection zone. In the case where the positive marker is P3G, the device preferably includes a P3G conjugate. In one embodiment, the reagent zone includes a mobilizable P3G conjugate labeled with a first member of a binding pair (e.g., biotin), and the capture zone contains an immobilized second member of the binding pair (e.g., an anti-biotin antibody or streptavidin). In this way, the P3G / P3G conjugate can be located in the capture zone without being directly immobilized in the capture zone. This concept can be adapted to capture zones for other positive markers.

[0109] In one embodiment, the device is arranged so that the capture zone or each capture zone is not visible to the user during use. In one embodiment, the capture zone or each capture zone cannot be read by the test reading device. In one embodiment, the capture zone in the device is not visible to the user when the device is in use. In one embodiment, the device is provided as part of a test kit where one or more capture zones are fully or partially visible to the user, and the test kit also includes instructions for the user to ignore any signals generated in the capture zones and / or determine the test results based on the presence or absence of detection zone signals without reference to any capture zones. In one embodiment, the device is provided as part of a test kit, and the test kit includes accessories for shielding one or more capture zones or all capture zones present in the device.

[0110] In one embodiment, the capture zone may be visible to a user who may be instructed to capture an image or video of the device on a mobile device such as a mobile phone, which in turn interprets the test results by itself or through some other device connected directly or indirectly to the mobile device. In this case, the process / analysis of the derived test results does not include a reference or actual measurement capture zone.

[0111] Preferably, the test reading member cannot read the capture zone. In one embodiment, the test device does not include a test reading member. In one embodiment, the test device is incompatible with an external reading member. In one embodiment, the device does not include a member for reading or analyzing the capture zone or each capture zone. One or more capture zones can be shielded by any suitable member. For example, the test device may include a housing that shields the capture zone or each capture zone. In the case where the detection zone of the test device is designed to be read by a test reader and / or the test device includes a test reader for reading the detection zone, such a reader will be configured to read the detection zone and preferably is not configured to read or analyze any capture zone. For example, only a reading member for reading the detection zone can be provided. In this case, it may not be necessary to shield the capture zone because the reading member of the reader is usually aligned with the detection zone so the capture zone upstream of the detection zone cannot be read. Preferably, the test reader used with the test device includes a single reading member for reading the detection zone. In one embodiment, the test device does not include a test for measuring the amount of negative markers or positive markers.

[0112] In one embodiment, the test device is configured to allow only detection, measurement or observation of signals at the detection zone. In one embodiment, the test device is configured to prevent detection, observation and / or measurement of signals at the capture zone or each capture zone. In one embodiment, the capture zone or each capture zone is not suitable for detecting the presence of the labeled conjugate captured in the capture zone or measuring its amount. In one embodiment, the device does not include a member that enables the user to obtain test results from the capture zone. In one embodiment, the device does not include a member that enables the user to detect or analyze signals in the capture zone. Preferably, the test device is configured to enable the test results to be obtained only by detecting or observing the signal of the detection zone (that is, the region of the detection analyte). In the case of use, any capture zone does not need to be visible to the user or can be read by the test member, because the result of the test is determined only by viewing or analyzing the detection zone. It is not necessary to measure, view or explain any signal generated in the capture zone to obtain the test result. In addition, it is advantageous to shield the capture zone or each capture zone so that the user will not be confused or distracted by any signal generated at the capture zone.

[0113] Fixed capture materials can be arranged on the flow path in a variety of ways. For example, the flow path may include one or more regions containing fixed capture materials. The capture zone may include any fixed capture materials or fixed capture material combinations disclosed herein in the capture zone. For example, a fixed capture material for capturing a complex containing a first negative marker and a fixed capture material for capturing a complex containing a second negative marker may be provided in the same capture zone. For example, a binding agent specific to the first negative marker and the second negative marker may be fixed in the same capture zone (although they may be provided in different capture zones). A fixed capture material may be provided in the capture zone, the fixed capture material being configured to capture a positive marker or containing a positive marker / positive marker conjugate or a labeled conjugate associated with a positive marker / positive marker conjugate, and the capture zone includes a fixed capture material for capturing a complex containing the first negative marker and / or other negative markers. A fixed capture material containing or consisting of a positive marker / positive marker conjugate or used to position a positive marker / positive marker conjugate in a capture zone and a fixed capture material for capturing a complex containing a first negative marker and / or other negative markers can be provided in the same capture zone. The same capture zone may include a fixed capture material configured to capture a labeled conjugate in the absence of a first positive marker and a fixed capture material configured to capture a labeled conjugate in the absence of a second positive marker (or such capture materials may be provided in different capture zones). This concept can be adapted to fixed capture materials used with other negative and positive markers.

[0114] The fixed capture material can be arranged in one or more capture lines on the test flow path. The capture zone can contain 1, 2, 3, 4, 5 or more different capture lines. The capture lines can be arranged in parallel. The amount of the labeled conjugate captured by the capture zone can be controlled by adjusting the amount, position, arrangement and orientation of the fixed capture material. For example, by placing the fixed capture material further downstream, increasing the amount of the capture line and / or using a wider capture line, a higher capture efficiency can be achieved. In addition to the change in the size of the labeling reagent, the pore size of the porous carrier of the flow path can also be used to adjust the capture efficiency and thus change the sensitivity of the test of the analyte of interest. Generally, reducing the pore size of the porous carrier can improve the capture efficiency.

[0115] The sensitivity of the test to analytes, negative and positive markers can be controlled by methods known to those skilled in the art, including changing the amount of binding reagents and / or the amount of positive markers / positive markers associated with a removable labeled conjugate, and / or changing the amount of reagents in one or more capture zones, the amount of the capture zone in fact, and the amount of the labeled reagents used. Further modifications can be made by changing the affinity of the binding reagents used in the device. In embodiments involving positive marker conjugates, the relative amounts of the positive markers and carrier proteins used, for example, in the capture zone, can be modified. Therefore, the sensitivity of the test to any analyte and marker involved in the test can be modified.

[0116] Detection area

[0117] The test device comprises a detection zone containing an immobilized binding reagent for capturing a complex containing a labeled conjugate and an analyte. Capturing the complex in the detection zone is preferably achieved by a specific binding reaction with the analyte. Preferably, the binding reaction occurs at an epitope of the analyte that is different from the epitope to which the member used to associate the labeled conjugate with the analyte binds, thereby forming a "sandwich". Preferably, the detection zone is configured to capture the labeled conjugate only when the labeled conjugate is part of a complex that also contains the analyte.

[0118] The detection zone may comprise an immobilized binding reagent that directly binds to the analyte. The detection zone immobilized binding reagent may consist of or comprise an antibody specific for the analyte. In one embodiment, the immobilized binding reagent in the detection zone comprises or consists of an antibody that binds to an epitope of the analyte that is not present in the first negative marker.

[0119] The detection zone may include a fixed binding reagent for indirect binding of the analyte. For example, in some embodiments, a removable binding reagent connected to the first binding partner for the analyte can be provided in, for example, the reagent zone or along the flow path but in other places upstream of the detection zone. The removable binding reagent can bind to the epitope of the analyte not present in the first negative marker. A fixed second binding partner for the first binding partner can be provided in the detection zone. In this way, the removable binding reagent for the analyte can be located in the detection zone without being directly fixed to the flow path. The first and second binding partners can be any suitable binding partners (such as antibody-antigen) specific to each other. Examples include biotin and streptavidin, biotin and anti-biotin antibodies, fluorescein and anti-fluorescein antibodies. Either party in the partner can be fixed in the detection zone, while the other party is provided in, for example, the reagent zone or along the flow path but in other places upstream of the detection zone. This concept can be adapted to include more intermediate binding partners. In one embodiment, the reagent zone includes a mobilizable anti-β-hCG antibody labeled with a first member of a labeled binding pair such as biotin, and the detection zone contains an immobilized second member of the binding pair (e.g., an anti-biotin antibody or streptavidin). In this way, the anti-β-hCG antibody can be located in the detection zone without being directly immobilized in the detection zone. This concept can be adapted to other analytes.

[0120] In embodiments where the analyte is hCG, the detection zone may comprise an anti-hCG antibody (eg, an anti-β-hCG antibody).

[0121] The capture of the complex containing the labeled conjugate and the analyte at the detection zone will form a signal in the detection zone. The signal can be detected or checked, for example, visually or using a test reading component. The detection of the signal in the detection zone indicates the presence of the analyte in the sample, and the presence of the first state in the subject from which the sample is derived. Failure to detect a signal in the detection zone, or the detection of a signal level below a certain level when measured by the reading component, may indicate that the subject from which the sample is derived does not have the first state.

[0122] Where a reading member is used to determine the signal intensity of the detection zone, a set threshold may be used as part of an algorithm to determine the test result as a first state of positive or negative or in fact quantify the signal of the detection zone to indicate the amount of analyte in the sample.

[0123] For example, where the analyte is hCG, detection of a signal in the detection zone indicates that the subject from which the sample was derived is pregnant. If no signal is detected in the detection zone after applying the sample to the device, this indicates that the subject from which the sample was derived is not pregnant.

[0124] Negative and positive markers

[0125] The apparatus of the present invention is useful for situations where the analyte to be detected is associated with a state of interest in the subject ("first state"), but detection of the analyte itself is not sufficient to confirm the presence of the first state. For example, the analyte may also be associated with a second state. The analyte may indicate the presence of the second state.

[0126] In some embodiments, the analyte, negative marker, and positive marker are such that one or more of the following is true:

[0127] The presence of the analyte and the positive marker in the sample indicates the presence of the first state.

[0128] • The presence of a negative marker and / or the absence of the analyte in the sample indicates the absence of the first state.

[0129] The presence of the analyte in the sample and a negative marker indicates the absence of the first state.

[0130] The presence of the analyte and a negative marker in the sample indicates the presence of the second state.

[0131] • The presence of the analyte and / or the absence of the positive marker in the sample indicates the presence of the second state.

[0132] A negative marker may be a marker that indicates the absence of a first state. A negative marker may be a marker that indicates the presence of a second state. A positive marker may be a marker that indicates the presence of a first state. A positive marker may be a marker that indicates the absence of a second state.

[0133] In the case of a pregnancy test, the analyte may be hCG. A sample from a subject may contain hCG or hCG metabolites from the fetus, which may indicate pregnancy (a first state). Examples of hCG metabolites include nicked intact hCG, free β-hCG, nicked free β-hCG, free α-hCG, and betacore fragments.

[0134] However, hCG may also be present in a sample at low levels due to a secondary state, such as perimenopause or menopause. In this case, hCG is produced by the subject's pituitary gland rather than the embryo. Therefore, for example, in a conventional device using lateral flow, detection of low levels of hCG does not necessarily indicate that the subject is pregnant.

[0135] Negative markers

[0136] Negative markers are preferably markers indicating the absence of the first state in the subject. In the case of a pregnancy test, the first negative marker may be FSH or LH. Negative markers may be markers indicating female follicular development or other follicular growth or activity, such as FSH or metabolites of such markers. Examples of FSH and its metabolites include free β-FSH and proteolytic degradation products.

[0137] In some embodiments, the amount of the removable labeled conjugate that arrives at the detection zone (where the analyte of interest is detected) depends on the level of the first negative marker in the sample to adjust. In this case, the removable labeled conjugate can be coated with a reagent that directly or indirectly binds to both the analyte to be detected and the first negative marker. In the case of such a test with a sample containing analyte and the first negative marker, the amount of the removable labeled conjugate that arrives at the detection zone is reduced due to the presence of the negative marker. In such an embodiment, the presence of the negative marker causes the capture zone located upstream of the detection zone to capture the labeled conjugate, and the capture zone contains a fixed capture material that directly or indirectly captures the labeled conjugate, preferably by interacting with the first negative marker to capture. The amount of the removable labeled conjugate captured at the capture site may depend on the level of the first negative marker in the sample. In a sample that does not contain or contains a low level of the first negative marker but has a first amount of analyte to be detected, a relatively large amount of removable labeled conjugate will arrive at the detection zone and produce a first visible signal in the detection zone. Conversely, the presence of a certain level of the first negative marker in the sample and the first amount of the analyte to be detected will cause the mobile labeled conjugate to be captured in the capture zone and reduce the amount of mobile labeled conjugate reaching the detection zone, thereby reducing the signal generated in the detection zone, thereby adjusting the sensitivity of the test for the analyte to be detected.

[0138] In one embodiment, the analyte to be detected is hCG which is indicative of the presence of the first state, and the first negative marker is a marker which is indicative of the absence of the first state in the subject.

[0139] In some embodiments, the first negative marker can be FSH. FSH may be the result of perimenopause or postmenopause. In such embodiments, testing with a sample containing FSH as the first negative marker from a perimenopause or postmenopause woman will result in capturing a mobile labeled conjugate in a capture zone upstream of the detection zone, thereby reducing the amount of labeled reagents arriving in the detection zone and therefore reducing or eliminating the signal seen in the detection zone. Even if the sample from the perimenopause or postmenopause woman contains pituitary-derived hCG (except FSH), the presence of FSH will also cause the signal generated in the detection zone to reduce or eliminate, indicating that the result of the first state is negative.

[0140] The analyte and the first negative marker may share a common epitope. The analyte and the first negative marker may share a common structural feature (e.g., a subunit of the analyte). The analyte may be hCG and the first negative marker may be FSH. The first negative marker may be luteinizing hormone (LH). In a preferred embodiment, the first negative marker is FSH.

[0141] The mobilizable labeled conjugate may comprise a specific binding agent that binds to a common structural feature on the analyte and the first negative marker. For example, this may be an antibody to the alpha subunit of the analyte hCG that also binds to the alpha subunit of the first negative marker FSH. Testing using urine samples from perimenopausal or postmenopausal women that contain certain levels of FSH may result in some of the mobilizable labeled conjugate being captured by the anti-FSH capture zone, thereby reducing or eliminating the signal seen in the detection zone.

[0142] In other embodiments, the removable labeled conjugate may include a binding agent that specifically binds to the analyte and other binding agents that specifically bind to the first negative marker. The specific binding agent can be permanently fixed directly to the removable labeled conjugate by passive adsorption or covalent coupling. In other embodiments referred to as indirect binding mode, the specific binding agent can be indirectly bound to the removable labeled conjugate before or during the test. This can be achieved, for example, by encapsulating the removable labeled conjugate in a reagent such as avidin, streptavidin or avidin and biotinylating the specific binding agent. Before or after the test, the specific binding agent is associated with the removable labeled conjugate.

[0143] Positive markers

[0144] Positive markers are preferably markers indicating the presence of a first state in a subject, and are markers other than the analyte of interest. In the case of a pregnancy test, a positive marker is preferably a marker indicating the secretion or production of a maternal factor associated with female pregnancy maintenance, such as progesterone or a metabolite of such a marker, such as pregnanediol glucuronide (P3G). Other positive markers that can be used in a pregnancy test according to the present invention include subtypes of intact hCG, such as highly glycosylated hCG, nicked hCG; subunits of hCG, such as hCG β core fragments, β-hCG, α-hCG, nicked β-hCG; Early Pregnancy Factor (EPF), Early Conception Factor (ECF), follistatin (FST), activin A, inhibin A, inhibin B, Pro-Alpha C, and pregnancy-associated plasma protein A (PAPP-A).

[0145] The positive marker may be a small molecule called a hapten, which is too small to elicit an immune response in an attempt to generate a specific binding means or antibody in an animal. In the case of generating a specific binding means with a hapten, if used to generate antibodies in an animal, the hapten is usually coupled or linked to an immunogenic carrier such as a protein such as bovine serum albumin or ovalbumin. The resulting antibody can be used in a lateral flow assay to generate a competition or inhibition assay. For example, the antibody used for hapten generation can be coated on a removable labeled conjugate, and the hapten or an analog of the hapten can be coupled to a carrier that is usually a protein to form a hapten conjugate. The hapten conjugate can be fixed to an area on a porous carrier. In the case of testing in the absence of a hapten in the sample, the removable labeled conjugate binds to the fixed hapten conjugate, while testing in the presence of a hapten reduces the binding of the removable labeled conjugate to the fixed hapten conjugate.

[0146] Conversely, the mobilizable labeled conjugate can be coated with a hapten attached to a support, and antibodies for hapten generation can be immobilized on regions on the porous support. Haptens present in a sample applied to such a device occupy sites on the antibodies immobilized on the porous support and reduce binding to the mobilizable labeled conjugate coated in the hapten attached to the support.

[0147] This negative correlation is typical of a competition or inhibition type assay and is often employed when the analyte of interest is a hapten and / or is too small to bind two different antibodies simultaneously (such as in the case of progesterone metabolites).

[0148] The binding member specific to the hapten is usually produced in mice, and the monoclonal antibody is selected for testing. The specific binding member can be directly coated on the surface of the removable labeled conjugate by direct adsorption or chemical coupling. The specific binding member can be coated on the surface of the removable labeled conjugate by an indirect manner, including, for example, the above-mentioned avidin-biotin or biotin and avidin. This indirect binding can be formed in advance so that the specific binding tool is coated on the removable labeled conjugate before the test, or the specific binding member is combined with the removable labeled conjugate when the test is performed. In the latter case, the removable labeled conjugate can be coated in avidin, and the biotinylated specific binding member of the hapten can be provided as a separate reagent in the test.

[0149] The protein bound to the hapten can be directly immobilized on the porous support or immobilized by chemical coupling. The protein coupled to the hapten can be indirectly immobilized on the porous support and provided as a mobile reagent in the test, where it is immobilized on the porous support.

[0150] The positive marker conjugate preferably comprises a positive marker moiety. The positive marker moiety is preferably capable of being bound by a binding agent such as an antibody. The positive marker conjugate may comprise a progesterone metabolite, such as P3G, or a moiety thereof.

[0151] The removable labeled conjugate can directly bind to a positive marker or a positive marker conjugate. In some embodiments, the removable labeled conjugate can indirectly bind to a positive marker / positive marker conjugate. Here, the reagent zone can include a member for associating a removable labeled conjugate with a positive marker or a positive marker conjugate. The member for associating a removable labeled conjugate with a positive marker or a positive marker conjugate can include a binding agent. The binding agent can directly or indirectly bind to a positive marker or a positive marker conjugate. The member for associating a removable labeled conjugate with a positive marker or a positive marker conjugate can include an antibody that binds to an epitope (this can be an epitope on a carrier protein) of a positive marker conjugate or be composed of it. If the binding agent indirectly binds to a positive marker or a positive marker conjugate, it can bind to another binding agent that directly or indirectly binds to a positive marker or a positive marker conjugate provided in the reagent zone.

[0152] In some embodiments, the reagent zone further comprises a removable positive marker / positive marker conjugate for being fixed in the downstream of the reagent zone. This removable positive marker / positive marker conjugate can comprise or be connected to a member in conjunction with right. Another member in conjunction with right can be fixed in the downstream of the reagent zone, for example in the capture zone. Therefore, removable positive marker / positive marker conjugate can be fixed in the capture zone so that it can be combined with a member for associating a removable labeled conjugate with a positive marker / positive marker conjugate.

[0153] In some embodiments, where the analyte is hCG and the positive marker is P3G, P3G can be coupled to an anti-hCG antibody (eg, an anti-α-hCG antibody).

[0154] One or more capture zones may be located upstream of the detection zone. The removable labeled conjugate may be directly or indirectly coated in a specific binding member of the analyte of interest (such as an antibody against hCG used in pregnancy testing). In addition, the removable labeled conjugate may also be directly or indirectly coated in an antibody for a positive marker (e.g., an anti-P3G antibody in the case where the positive marker is a progesterone metabolite). In one embodiment, a porous carrier and a directly or indirectly fixed anti-hCG antibody are prepared in the detection zone, and a signal is provided in the detection zone by a typical sandwich test in the presence of hCG of pregnancy origin. In such an embodiment, the porous carrier may include one or more capture zones located upstream of the detection zone, the capture zone containing an immobilized hapten (P3G) coupled to a protein carrier (such as BSA). Alternatively, the specific binding member of P3G may be directly or indirectly fixed in the capture zone, and the P3G coupled to the carrier may be directly or indirectly coated on the removable labeled conjugate.

[0155] In samples from pregnant women, the level of the positive marker (P3G) increases along with the hCG level of pregnancy origin. In some embodiments, the presence of P3G reduces the binding of the mobile labeled conjugate in the capture zone, thereby allowing more labeled reagents to reach the detection zone, where the presence of hCG allows the mobile labeled conjugate to bind in the detection zone.

[0156] In contrast, samples from non-pregnant individuals contain significantly lower levels of positive markers (P3G). In some embodiments, applying such samples to the test device allows the mobile labeled conjugate to bind to the capture zone, reducing the amount of mobile labeled conjugate reaching the detection zone. Reducing the amount of mobile labeled conjugate reaching the detection zone reduces the signal generated by the detection zone, making a negative result of the pregnancy test more likely.

[0157] Analysis of urine samples of pregnant women shows that the level of positive markers (e.g., P3G) increases with the increase of hCG levels from pregnancy. However, in perimenopausal or postmenopausal women, the level of positive markers is relatively low. According to certain embodiments of the present invention, the lower level of positive markers present in urine samples from perimenopausal or postmenopausal women causes more mobile labeled conjugates to be bound to the capture zone, effectively reducing the amount of mobile labeled conjugates that reach the detection zone. Since the sample taken from perimenopausal or postmenopausal individuals contains pituitary-derived hCG, the signal seen in the detection zone in such embodiments is reduced as the amount of mobile labeled conjugates that arrive at the detection zone is damaged. In this case, it is more likely to report a negative result in the detection zone.

[0158] In some embodiments, when the analyte is hCG and the positive marker is a hapten such as P3G, P3G can be coupled to an anti-hCG antibody such as an anti-α-hCG antibody and a conjugate associated with a mobilizable labeled conjugate. Here, anti-α-hCG serves as a specific binding member for hCG (binding to a mobilizable labeled conjugate in the detection zone) and a specific binding member for a first negative marker (e.g., FSH), and in the presence of FSH, the labeled conjugate is bound to a capture zone containing a fixed capture substance for FSH. In addition, the anti-α-hCG-P3G conjugate on the mobilizable labeled conjugate is used to capture the mobilizable labeled conjugate in a capture zone containing a fixed capture substance of P3G in the absence of P3G in the sample.

[0159] The amount of labelled reagent reaching the detection zone may be further reduced by using more than one positive marker, using a porous carrier containing binding members for each positive marker.

[0160] In the case where the device of the present invention includes a positive marker or a positive marker conjugate, the positive marker or the positive marker portion of the conjugate can be an analog or variant of a naturally occurring positive marker. Any analog or variant can be used, provided that both the analog / variant and the naturally occurring marker can participate in the positive marker binding reaction described herein and compete for the relevant binding site.

[0161] Combinations of negative and positive markers and additional negative and / or positive markers

[0162] One or more negative markers and / or one or more specific binding members of positive markers can be provided in a single test device. This form further improves the ability to report negative results in the absence of the first state and to report positive results in the presence of the first state. In the case of pregnancy testing, this form further improves the ability to report negative results for non-pregnant samples and positive results in the case of true pregnancy. In this case, the removable labeled conjugate can be directly or indirectly coated with a specific binding member such as anti-α-hCG to bind hCG and negative marker FSH. The removable labeled conjugate can also be coated with a specific binding member (e.g., P3G) connected to a positive marker. In this embodiment, the porous carrier preferably includes a capture zone containing a specific binding reagent for FSH and an area of ​​one or more positive markers or positive marker conjugates (e.g., P3G coupled to BSA). The capture zone can contain separate zones for negative and positive markers, or the capture zone can include specific binding reagents that are co-fixed in the capture zone by mixing any proportion of reagents.

[0163] In such an embodiment, the sample taken from the pregnant individual with the positive marker of the elevated level is tested so that the amount of the mobile labeled conjugate captured in the capture zone is low, thereby allowing more mobile labeled conjugates to pass through the detection zone. Since the sample also contains the first negative marker FSH at a lower level, less mobile labeled conjugates are captured in the capture zone by interaction with FSH. This allows more mobile labeled conjugates to move to the detection zone and increases the possibility of generating signals in the detection zone due to the hCG of the pregnancy source present in the sample. Therefore, after checking the detection zone, the real pregnancy sample is more likely to provide a positive test result. On the contrary, the true negative pregnancy sample that is not from perimenopausal or postmenopausal women contains a relatively low level of positive markers, so that more mobile labeled conjugates are captured in the capture zone, reducing the amount of mobile labeled conjugates that arrive in the detection zone. Although the level of negative markers in such samples can change, the amount of negative markers is not important, because the hCG level of pregnancy source is low, so that the detection zone signal is reduced / no signal is formed. In the case of testing the sample from perimenopause or postmenopausal women, the relatively high level of negative marker FSH makes more movable labeled conjugates captured in the capture zone. In addition, due to the presence of relatively low level of positive marker P3G in the sample, more movable labeled conjugates are captured in the capture zone. In fact, the amount of the movable labeled conjugate arriving at the detection zone is reduced, increasing the possibility of reporting negative results in the case of the detection zone inspection. Because in certain embodiments, the movable labeled conjugate comprises anti-α antibody as a specific binding member, it will also be combined with the hCG of the pituitary source in the sample from perimenopause or postmenopausal women. Due to the presence of negative marker FSH in the sample, the movable labeled conjugate is captured in the capture zone, so some pituitary hCG will also be captured on the movable labeled conjugate, and will not move to the detection zone. Reduce the amount of pituitary derived hCG in the sample moving to the detection zone to further reduce the possibility of movable labeled conjugates being combined in the detection zone, increasing the possibility of reporting negative results in the detection zone when running individual samples from perimenopause or postmenopause.

[0164] Device of the present invention can be adapted to include components in the reagent zone and the capture zone, so that in the case of the presence of a second negative marker in the sample, the further capture of the labeled conjugate is promoted before the labeled conjugate reaches the detection zone. Any component described herein for capturing a complex containing a labeled conjugate and a first negative marker can be modified and included in the device of the present invention to capture a complex containing a labeled conjugate and a second negative marker. If the first negative marker is FSH, the second negative marker can be, for example, LH. If the first negative marker is LH, the second negative marker can be, for example, FSH.

[0165] Similarly, the device of the present invention can be adapted to include components in the reagent zone and the capture zone to facilitate further capture of the mobile labeled conjugate before the mobile labeled conjugate reaches the detection zone in the absence of a second positive marker in the sample (for example, in the case where the second positive marker is a hapten). In the absence of a positive marker, any component described herein for capturing a complex can be modified and included in the device of the present invention to capture a complex containing a mobile labeled conjugate in the absence of a second positive marker in the sample. If the first positive marker is P3G, the second positive marker may be another progesterone metabolite. If the first positive marker is a progesterone metabolite other than P3G, the second positive marker may be P3G. The device of the present invention can be further adapted to capture a complex in the presence of any amount of more negative markers and / or in the absence of any amount of more positive markers.

[0166] Relative position of the area

[0167] One or more capture zones are located downstream of the reagent zone and upstream of the detection zone. The detection zone can be located at the downstream end of the test flow path (e.g., a porous carrier such as nitrocellulose). The capture zone can be located anywhere between the reagent zone and the detection zone. The position can be adjusted as described herein to adjust the capture efficiency. In one example, the detection zone can be located at about 10 to 20 mm from the upstream end of a porous carrier (e.g., a nitrocellulose membrane). The capture zone can be located at about 3 to 15 mm from the upstream end of a porous carrier (e.g., a nitrocellulose membrane). In one embodiment, the detection zone is located at about 15 mm from the upstream end of a porous carrier (e.g., a nitrocellulose membrane), and the capture zone is located at about 8 mm from the upstream end of a porous carrier (e.g., a nitrocellulose membrane). The reagent zone can be provided on a separate porous carrier, located upstream of a porous carrier containing a capture zone and a detection zone. There is no inherent limitation in the spacing between the capture zone and the detection zone. However, in an embodiment where the test device is designed to be visually read by a user, the capture zone will be separated far enough from the detection zone so that the user observing the detection zone is invisible. Therefore, the size of the viewing window used to view the detection zone and the location of the detection zone beneath the window may be factors in determining the most appropriate location for the capture zone.

[0168] sample

[0169] Although urine is preferred as a sample matrix for home pregnancy testing, the sample can be any suitable body fluid, such as whole blood, plasma, serum, or urine. Urine samples are highly preferred because such samples are easily obtained and do not require invasive procedures. In addition, the use of urine samples facilitates self-testing by the user. Other samples can be collected using swabs, sanitary napkins, bandages, etc. and applied directly to the device, followed by an elution buffer to help move the sample components along the flow path. Before applying the sample to the test device, the sample can be collected and placed in an elution buffer as described above.

[0170] Runtime and Reader Properties

[0171] The result of the test can be determined by visual inspection of the detection zone by the user, typically after a set period of time after application of the sample to the device, or by the development and visual appearance of a control line or other end of the test indicator.

[0172] Generally speaking, in the case of a digital test device, the test result will be determined after a certain time (tE) has elapsed (usually, but not necessarily, determined by reference to the time that the sample is in contact with the sampling area of ​​the test device). If a test result reading device is provided, this may include some integral timing means to determine when tE is reached. The timing means may be automatically initiated by contacting the sample with the test device (e.g., by a liquid sample allowing an electric current to flow), or may be triggered by the user (e.g., pressing a switch, etc.) or by any other convenient means. The test reaction may conveniently reach equilibrium at tE, but this is not essential.

[0173] The reader can measure the analyte signal of the detection zone one or more times before tE. In the case where the signal of the detection zone is greater than the first threshold (the detection zone signal is higher than the set threshold for a positive result when the first threshold indicates tE), the early measurement of the detection zone signal can be used to output the final result to the user before tE. Similarly, if the signal of the detection zone is lower than the set threshold at any stage before tE, an early negative result can be output to the user before tE.

[0174] The tE endpoint may conveniently be determined by the reader with reference to a particular time point. (i.e., tE may be considered to occur a particular amount of time after the start of the assay, such as a particular interval after activation of the reader and / or insertion of the assay device into the reader and / or application of a sample to the assay device).

[0175] shell

[0176] In a preferred embodiment, the test device comprises a housing that holds most or all of the functional components and test reagents of the test. The housing is conveniently formed by a waterproof synthetic plastic material and is preferably substantially opaque. Sunscreen can be added to the plastic material to reach the required opacity level. Suitable synthetic plastic materials for forming the housing particularly include copolymers of polycarbonate, polystyrene, polystyrene, polyolefins, polypropylene, polyethylene and acrylonitrile. Ideally, the housing can be formed into two or more parts that are connected together with most (or all) of the remaining test device components contained in or between the assembly components of the housing. The parts of the housing can be connected and fastened by traditional fastening methods, such as snap-fit ​​action, or by plastic welding, etc.

[0177] In a preferred embodiment, the test device comprises a housing (usually having above-mentioned features), and wherein, the sample application portion or region extends beyond the housing to facilitate the application of urine sample to the sample application portion or region. Before use, the sample application portion or region extending beyond the housing can be covered by a removable cap. The cap can also preferably be formed by a synthetic plastic material, which can be opaque, transparent or translucent. Usually, once the sample is applied to the sample application area, the cap is replaced.

[0178] The housing may include a test window above the detection zone to facilitate viewing of the detection zone. The housing may obstruct a user's view of the or each capture zone.

[0179] In another example, no housing is provided. In such an embodiment, the test device components can be mounted on a support material such as polyester film to form a commonly known test paper. In this case, the capture area may be covered by an additional opaque cover in other ways, hiding the capture line from the user. In other cases, using such a test paper, the capture area is visible to the user, and the user is only instructed to interpret the results by observing the detection area. This test paper configuration without a housing can also be read by a reading member as described herein.

[0180] Other features

[0181] The test device may 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) a sample adequacy indicator (e.g., as described in WO2015 / 082646), a "flood" pad (e.g., as disclosed in WO2012 / 069610), use of sample flow as a test control (e.g., as disclosed in EP1,484,611; US6,194,222); "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., automatically starting after wetting the device with a sample, thereby completing a circuit); using a digital test progress meter (e.g., European Community Design Registration No. 1367304) or a "color-changing wick", etc., to give a visual indication that a sample has been applied to the test device and / or a visual indication that the test has begun (e.g., WO2003 / 058245). The test device will advantageously be presented to the consumer in a waterproof packaging which optionally further comprises a desiccant, such as a silica gel sachet.

[0182] The test flow path can be conveniently supported on some support or backing layer to provide mechanical strength and an appropriate degree of rigidity. The assay plus support can be conveniently referred to as and provided as a test paper for use without a housing, or actually placed in a housing as described herein to form what is commonly referred to as a test stick. The test stick or more test sticks can be adapted and configured to be inserted into a test device by a user, or the test stick or more test sticks can more preferably form an integral part of a test device, which is sold to consumers together with the test stick pre-inserted or loaded in the test device.

[0183] Reader

[0184] In a preferred embodiment, the test results can be read directly by the user in a manner known from traditional "self-test" pregnancy tests, such as by the user inspecting one or more windows covering the detection zone to determine the presence or absence of a detectable signal. In such an embodiment, the test device preferably does not include a test reader. In one embodiment, the test device of the present invention is not configured to be read by a test reader. Typically, in such user-reading devices, the user will directly inspect the detection zone of the lateral flow or microfluidic test. In other forms, the user can determine the results of the detection zone by reference to a colorimetric card or indicator. Conveniently, the test device can be provided with instructions or instructions for reading the test results (if the device does not interpret the test results for the user). For example, a printed colorimetric card can be provided to the user to facilitate the interpretation of such direct-read visual tests.

[0185] Alternatively, the detection zone can be read by a test result reading member referred to as a test result reader. In this case, the test result reader is preferably configured to read only the detection zone and is not configured to read or analyze any capture zone present in the test device. The test result reader can be external to the test device or integrated with the test device. The test result reader can advantageously include electronic components, especially digital electronic components, such as a microprocessor. The detection zone can be determined by an optical member, i.e., measuring the amount of light reflected and / or transmitted by the detection zone, wherein the optical labeled reagent tends to accumulate in a manner that is proportional (proportional or inversely proportional) to the analyte concentration in the sample. Alternatively, the detection zone can be read by, for example, magnetic or electrochemical measurements. Obviously, the mode of reading the test may depend on the properties of the label used to mark the test reagent. The generated data can be processed by an integrated microprocessor, and the test result can be output to the device itself by means of a liquid crystal screen or the like, or remotely transmitted to an external device such as a mobile phone for in-situ processing or transmitted to a central processing server via a network connection. The result may be reported on a mobile phone or other networked device.

[0186] The external test result reading means may comprise a dedicated result reading device (e.g., similar to the device described in EP 1 066 530). Alternatively, the external test result reader may be a "non-dedicated" device, such as a mobile phone or other portable electronic device (e.g., a tablet), preferably equipped with a camera, wherein the test result is read by measuring the signal intensity generated by the tag in the detection zone.

[0187] A test result reader, whether external or forming part of an integrated test device / test result reader, can read and interpret test data, or can transmit test data containing device operating characteristics, such as flow time and various outputs from the measurement system to a remote device for interpretation of analytical data, and the results obtained. The test data can be transmitted to the remote device in real time. The data can be transmitted via an Internet connection, or can be stored on a storage device (such as a "flash" drive, etc.) that is physically transmitted to the remote device, or the data can be transmitted by wireless communication means (such as Bluetooth, near field communication [NFC], etc.).

[0188] The microprocessor may control the operation of the optical readout or other test reading components and will conveniently be programmed with or able to access the relevant test signal thresholds for each analyte, compare the actual test signal values ​​to the predetermined thresholds, and interpret the test results to determine the result of the pregnancy test.

[0189] The test device may also include a test result display for displaying the test results to the user. Typically, the display will include an LCD, but other types of displays are possible (e.g., using "electronic ink"). In those embodiments where the test device (or more specifically, its components) requires a power source to operate, the test device will preferably be equipped with an integrated power source, such as a battery. Very small and inexpensive batteries are readily available on the market. The device may also be equipped with a switch to connect the integrated power source and start the device.

[0190] Combined pregnancy test sticks / test result reading devices with displays may be referred to as "digital pregnancy tests," and such digital test devices are commercially available and may be adapted with the benefit of this disclosure to provide test devices according to the present invention.

[0191] The microprocessor will ideally be programmed to cause the test result reading component to read the test results; interpret the test results; and display the conclusions to the user.

[0192] The components for reading the test results will preferably include 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 light detector is preferably a photodiode or a phototransistor. The light source illuminates a detection zone of the test, which tends to accumulate the labeled substance during the test in a manner that depends on the concentration of the analyte of interest in the sample applied to the test.

[0193] Devices may typically utilize a reference zone, which is a portion of the microfluidic or lateral flow assay flow path that is used to reference the readings obtained from the detection zone. The use of reference zones is well known to those skilled in the art and helps account for unbound labeled reagents that may be in the flow path and / or background staining of the flow path of the sample being tested.

[0194] In one embodiment, the light source emits light of different wavelengths at different times, and the light detector distinguishes the different wavelengths. Additionally or alternatively, an optical baffle (fixed or adjustable) can be used to control the area illuminated by a particular light source. More details of the optical arrangement that can be used are disclosed in, for example, EP 1,484,601, US 6,055,060 and US 5,889,585. For the avoidance of doubt, the term "light" as used herein is not intended to refer only to radiation in the portion of the electromagnetic spectrum visible to human observers, and includes, for example, ultraviolet and infrared radiation. However, components within the visible portion of the spectrum, components that are sensitive to the visible portion of the spectrum may be preferred and are selected according to the labeled reagent used.

[0195] The microprocessor or computerized control member may contain one or more stored analyte threshold values ​​to which the test result may be compared to allow the test result reading device to interpret the result and display an appropriate conclusion (e.g., pregnancy or non-pregnancy) to the user. The microprocessor or control member may be programmed with an algorithm to measure the test result, compare it to a predetermined threshold value, and display a conclusion.

[0196] Comparison

[0197] In some embodiments, it is desirable that the test device also include some kind of control function. This is conventional in self-testing devices to provide some indication that the test has been run correctly.

[0198] Typically, the control function will include the use of a control zone, and if enough samples have been applied to the sample application zone of the test device, the labeled reagent will tend to accumulate in the control. Typically, the labeled reagent is a labeled antibody or other reagent that is releasably deposited in a dry form on the upstream or proximal portion of the test paper, such as in the reagent zone, and moves when the sample is rehydrated, and is captured by the capture agent fixed in the control zone. The control indicates whether enough samples have been applied to the test device, and indicates that the test reagents have retained their binding properties to a reasonable extent, and that the labeled reagents have been moved to a sufficient degree. The labeled reagent used to form the control zone can be a second group of reagents for the first group of labeled reagents for detecting analytes. A mixture of a labeled reagent for detecting analytes and a separate labeled reagent group for forming the control zone can be used, and both groups participate in forming the control zone.

[0199] The embodiments described for each aspect of the present invention have been modified mutatis mutandis for the embodiments of each other aspect. The documents cited herein are incorporated by reference to the maximum extent permitted by law. The prior art documents mentioned herein are incorporated to the maximum extent permitted by law.

[0200] The various features of the present invention will now be further described by way of exemplary embodiments and with reference to the accompanying drawings, in which:

[0201] Figure 1A An embodiment of a test device (100) for detecting the presence of an analyte (101) in a sample is shown. The test device comprises a sample application zone (102), a reagent zone (104) downstream of the sample application zone, a capture zone (110) downstream of the reagent zone, a detection zone (108) downstream of the capture zone, a control zone (109) downstream of the detection zone, and a sink pad (105) downstream of the control zone. When a sample is applied to the sample application zone (102), it flows along a flow path of the test device in the direction indicated by arrow (103).

[0202] The reagent zone (104) comprises a test mobilizable labeled conjugate (106) and a control mobilizable labeled conjugate (107). The test mobilizable labeled conjugate (106) comprises a label (114) coupled to a binding reagent (113) (e.g., an antibody), and the test mobilizable labeled conjugate (106) binds to an analyte (101) or a first negative marker (112). The control mobilizable labeled conjugate (107) comprises a label (114) coupled to a binding member that is bound by a fixed binding member (116) at the control zone (109). The test mobilizable labeled conjugate can also be used as a control conjugate.

[0203] The capture zone (110) contains an immobilized capture substance (115) (eg, an immobilized antibody) specific for the first negative marker (112).

[0204] The detection zone (108) contains an immobilized binding reagent (111) (eg, an immobilized antibody) specific for the analyte (101).

[0205] A test window (117) is provided in the test device housing (not shown) above the detection zone (108) and control zone (109) to enable viewing by a user or reading by a test reader. The capture zone (110) is not visible through the test window (117) and is obscured by the test device housing.

[0206] Figure 1B Shows how to pass Figure 1A The test device shown in obtains a positive test result. In this case, the analyte (101) is present in the sample, but the first negative marker (112) is not present in the sample. The test movable labeled conjugate (106) and the control movable labeled conjugate (107) are moved by the sample and flow downstream from the reagent zone (104). The analyte (101) is bound by the binding reagent (113) of the test movable labeled conjugate (106) to form a complex. The complex is not captured in the capture zone (110) because the fixed capture material (115) at the capture zone (110) is specific to the first negative marker (112) and the first negative marker is not present in the sample. The fixed binding reagent (111) in the detection zone (108) binds to the analyte in the complex, thereby capturing the complex. The control movable labeled conjugate (107) is bound at the control zone (109). This results in the formation of a test line signal (118) and a control line signal (119), both of which are visible through the test window (117), indicating a positive result.

[0207] Figure 1C shows how, in the absence of an analyte (101) and a first negative marker (112) in a sample, Figure 1A A negative result is obtained with the test device of the present invention. The control movable labeled conjugate (107) is moved by the sample, flows downstream from the reagent zone (104) and binds at the control zone (109). The test movable labeled conjugate (106) is moved by the sample, but cannot bind at the detection zone (108) due to the absence of analyte (101). This causes only the control line signal (119) to be generated and visible to the user at the test window (117), indicating a negative result.

[0208] Figure 1D shows how, in the presence of an analyte (101) and a first negative marker (112) in a sample, Figure 1A A negative result is obtained by using a test device of the present invention. The test movable labeled conjugate (106) and the control movable labeled conjugate (107) are moved by the sample and flow downstream from the reagent zone (104). The first negative marker (112) and the analyte (101) are bound by the binding reagent (113) of the test movable labeled conjugate (106) to form a complex. The capture substance (115) fixed in the capture zone binds to the first negative marker (112) of the complex, so that the complex is captured at the capture zone (110). Since the capture zone (110) is upstream of the detection zone (108), the test movable labeled conjugate (106) is isolated before it reaches the detection zone (108). Therefore, although the analyte (101) is present in the sample, the accumulation of the test movable labeled conjugate (106) at the detection zone (108) is not enough to give a positive result. The control mobilizable labeled conjugate (107) is mobilized by the sample and binds to the control zone (109). This allows the user to see only the control line (119) at the test window (117), indicating a negative result. The capture zone (110) is blocked from view.

[0209] Figure 2A simplified schematic diagram of a test device of the type shown in Figure 1 is shown. The test device includes a reagent zone (1), a capture zone (5) downstream of the reagent zone (1), and a detection zone (7) downstream of the capture zone (5). The reagent zone (1) includes a removable labeled conjugate (2), and the removable labeled conjugate includes a detectable label (3) and a member (4a) for associating the labeled conjugate with an analyte and a member (4b) for associating the labeled conjugate with a first negative marker. The capture zone (5) includes a fixed capture material (6) that is capable of capturing a complex containing the labeled conjugate (2) and the first negative marker (10). The detection zone (7) includes a fixed binding member (8) for capturing a complex containing the labeled conjugate (2) and an analyte (9). In the presence of an analyte (9) and the absence of a first negative marker (10) (left figure), the member for associating the labeled conjugate with the analyte (4a) binds to the analyte to form a complex. The complex is captured at the detection zone (7) because the fixed binding member (8) at the detection zone (7) binds to the analyte (9) of the complex. In the presence of the analyte (9) and the first negative marker (10) (right figure), the member (4a) for associating the labeled conjugate with the analyte binds to the analyte, and the member (4b) for associating the labeled conjugate with the first negative marker binds to the first negative marker (10). It should be noted that in this embodiment, the member (4a) for associating the labeled conjugate with the analyte and the member (4b) for associating the labeled conjugate with the first negative marker are different (e.g., different antibodies). However, they can be the same, such as the same antibody that binds both the analyte and the first negative marker. A complex containing the labeled conjugate, the analyte (9) and the first negative marker (10) is formed. The complex is captured at the capture zone (5) because the fixed capture substance (6) binds to the first negative marker (10) of the complex. In this test device, the capture zone (5) is shielded from view, while the detection zone (7) is visible to the user or readable by a test reader.

[0210] Figure 3 Shows Figure 2 Schematic diagram of an assay device of the type shown, except that: (i) the mobilizable labeled conjugate (2) comprises a positive marker conjugate (4c) rather than means for associating the labeled conjugate with a first negative marker ( Figure 2In the embodiment of the present invention, the first positive marker (9) and the first positive marker (10a) are bound by the immobilized substance (6a) at the capture zone (5), so that the labeled conjugate (2) cannot be bound at the capture zone (5). The analyte (9) is used to bind the labeled conjugate to the analyte-associated component (4a) to form a complex. The complex passes through the capture zone (5) and binds at the detection zone (7) because the immobilized binding component (8) at the detection zone (7) binds to the analyte (9) of the complex. In the absence of the first positive marker (10a) and the presence of the analyte (9) (right figure), the analyte (9) is used to bind the labeled conjugate to the analyte-associated component (4a) to form a complex. The complex is bound at the capture zone (5) because the immobilized capture substance (6a) at the capture zone (5) binds to the positive marker conjugate (4c) of the complex.

[0211] Figure 4 Shows Figure 3 Schematic diagram of an assay device of the type shown, except that: (i) the mobilizable labeled conjugate (2) comprises means (4d) for associating the mobilizable labeled conjugate with a first positive marker or a first positive marker conjugate, rather than the positive marker conjugate ( Figure 3 (4c) in ), and (ii) the capture zone (5) comprises an immobilized first positive marker conjugate (6b) rather than an immobilized capture substance ( Figure 3In the presence of analyte (9) and first positive marker (10a) (left figure), first positive marker (10a) is used to bind the mobile labeled conjugate to the first positive marker or the first positive marker conjugate associated member (4d), so that the labeled conjugate (2) cannot bind at the capture zone (5). Analyte (9) is used to bind the labeled conjugate to the analyte associated member (4a) to form a complex. The complex passes through the capture zone (5) and binds at the detection zone (7) because the fixed binding member (8) of the detection zone (7) binds to the analyte (9) of the complex. In the absence of the first positive marker (10a) and the presence of analyte (9) (right figure), analyte (9) is used to bind the labeled conjugate to the analyte associated member (4a) to form a complex. The complex is bound in the capture zone (5) because the fixed first positive marker conjugate (6b) is bound to the member (4d) of the labeled conjugate (2) for associating the removable labeled conjugate with the first positive marker or the first positive marker conjugate. It should be understood that the embodiments shown in the figure can be combined so that, for example, both positive and negative markers affect the amount of the labeled conjugate (and the analyte present in the sample) reaching the detection zone and / or more negative and / or positive markers affect the labeled conjugate reaching the detection zone. For example, the removable labeled conjugate may include a binding reagent for the analyte, the first negative marker, and the first positive marker, and the capture zone may include a fixed capture substance for the first negative marker and a fixed first positive marker conjugate. In another embodiment, the removable labeled conjugate may include a binding reagent for the analyte, the first negative marker, and the first positive marker conjugate, and the capture zone may include a fixed capture substance for the first negative marker and a fixed capture substance for the first positive marker and the first positive marker conjugate.

[0212] Figure 5 Shows the use Figure 3 A modified version of the device shown in FIG. 1 shows indirect capture performed in the detection zone. In this embodiment, the reagent zone (1) also includes a removable binding reagent (8a) for the analyte connected to the first binding partner, which. The detection zone (7) includes a fixed second binding partner (8b) for the first binding partner. In this way, the removable binding reagent for the analyte can be located at the detection zone (7) without being directly fixed to the flow path.

[0213] In the presence of an analyte (9) and in the absence of a first negative marker (10) (left figure), the member (4a) for associating the labeled conjugate with the analyte binds to the analyte, thereby forming a complex. The mobilizable binding reagent (8a) for the analyte connected to the first binding partner is moved and flows to the detection zone (7), where the first binding partner is bound by the second binding partner (8b). The complex is captured in the detection zone (7) by the mobilizable binding reagent (8a) for the analyte, which is now located at the detection zone (7). In the presence of an analyte (9) and a first negative marker (10) (right figure), the member (4a) for associating the labeled conjugate with the analyte binds to the analyte (9), and the member (4b) for associating the labeled conjugate with the first negative marker binds to the first negative marker (10). A complex containing the labeled conjugate, the analyte (9) and the first negative marker (10) is formed. The complex is captured at the capture zone (5) because the immobilized capture substance (6) binds to the first negative marker (10) of the complex. The mobile binding reagent (8a) for the analyte linked to the first binding partner flows to the detection zone and is bound by the second binding partner (8b), but because the complex is captured at the capture zone (5), it is prevented from reaching the detection zone (7).

[0214] Figure 6 Shows Figure 4 A modified version of the device shown. In this embodiment, the reagent zone (1) also contains a removable first positive marker conjugate (6c) connected to the first binding partner. The capture zone (5) contains an immobilized second binding partner (6d) for the first binding partner. In this way, the removable first positive marker conjugate (6c) can be located in the capture zone (5) without being directly fixed to the flow path.

[0215] In the presence of a sample containing an analyte (9) and a first positive marker (10a) (left figure), the movable first positive marker conjugate (6c) connected to the first binding partner is moved and flows to the capture zone (5), where the first binding partner is bound by the immobilized second binding partner (6d). The member (4a) for associating the labeled conjugate with the analyte of the movable labeled conjugate (2) binds to the analyte (9) to form a complex. The member (4a) for associating the labeled conjugate with the first positive marker or the analyte of the movable labeled conjugate (2) binds to the first positive marker (10a), preventing the complex currently located in the capture zone (5) from being captured by the first positive marker conjugate (6c). The complex passes through the capture zone (5) and is bound in the detection zone (7) because the immobilized binding member (8) of the detection zone (7) binds to the analyte (9) of the complex.

[0216] In the presence of analyte (9) and the absence of first positive marker (10a) (right figure), the mobile first positive marker conjugate (6c) connected to the first binding partner is moved and flows to the capture zone (5), where the first binding partner is bound by the immobilized second binding partner (6d). The member (4a) for associating the labeled conjugate with the analyte of the mobile labeled conjugate (2) binds to the analyte (9) to form a complex. The complex is held in the capture zone (5) because the mobile first positive marker conjugate (6c) currently located in the capture zone (5) is bound by the member (4d) for associating the mobile labeled conjugate with the first positive marker or the first positive marker conjugate of the labeled conjugate (2).

[0217] Figure 7 is a graph showing the effect of P3G on the assay signal generated by labeled reagents coated with anti-P3G and anti-α-hCG, tested over a range of hCG concentrations on a porous support equipped with a capture zone with a P3G conjugate and an anti-β-hCG detection zone.

[0218] It should be noted that the positive marker conjugates shown in the figures may be replaced with the positive marker, for example, if the positive marker is a non-hapten marker that does not require conjugation.

[0219] Example

[0220] The invention will now be described with reference to the following non-limiting embodiments.

[0221] Example 1

[0222] This example describes the preparation of a pregnancy test strip with an anti-hCG detection zone and an anti-FSH capture zone for use with clinical urine samples and spiked urine standards for pregnant and peri- or post-menopausal women.

[0223] Production of testing reagents

[0224] Preparation of gold sol-labeled antibodies

[0225] Test Sol

[0226] 40 mL of 40 μg / mL mouse anti-α-hCG antibody solution in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 6.5 was added to 40 mL, 40 nm, A520 nm = OD5.0 gold sol (BBI International) and rapidly mixed with the gold sol on a magnetic stirrer at room temperature for 30 minutes. After 30 minutes, 836.5 μL, 52.6 mg / mL of β-casein solution in 50 mM carbonate buffer (pH 10.5) was added to the reaction mixture and continued to mix for another 30 minutes at room temperature. The final concentration of β-casein in the reaction mixture was 0.55 mg / mL. The anti-α-hCG antibody immobilized on the gold sol is able to bind to these hormones through the α subunits of hCG and FSH. The sol solution was poured into a Falcon tube (50 mL) and the solution was centrifuged (4000 rcf, 10 minutes, 15°C). Carefully remove the supernatant, add washing buffer (1 mL, 10 mM MES, pH 6.5, containing 0.1 mg / mL beta casein), vortex and sonicate the precipitated sol to resuspend. After resuspension, the solution is centrifuged (5000 rcf, 10 minutes, 15°C). Remove the supernatant and resuspend the sol in a small amount of storage buffer (10 mM MES pH 6.5, containing 0.1 mg / mL beta casein). Determine the final OD of the sol preparation by measuring the absorbance at 520 nm.

[0227] Control sol

[0228] 20 mL of 12.5 μg / mL Rabbit IgG antibody (Dako) solution in 20 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer pH 8.0 was added to 20 mL of 40 nm A520 nm = OD 5.0 ​​gold sol (BBI International) and rapidly mixed with the gold sol for 30 minutes at room temperature on a magnetic stirrer. After 30 minutes, 76 μL of 52.6 mg / mL beta casein solution in 50 mM carbonate buffer (pH 10.5) was added to the reaction mixture and continued to mix for another 30 minutes at room temperature. The final concentration of beta casein in the reaction mixture was 0.1 mg / mL. The sol solution was poured into a Falcon tube (50 mL) and the solution was centrifuged (4000 rcf, 10 minutes, 15° C.). Carefully remove the supernatant and add washing buffer (1mL 10mM MES pH6.5 containing 0.1mg / mL beta casein) to resuspend the sol. The precipitated sol is vortexed and ultrasonicated. After resuspending, the solution is centrifuged (5000rcf, 10 minutes, 15°C). Remove the supernatant and resuspend the sol in a small amount of storage buffer (10mM MES pH 6.5, containing 0.1mg / mL beta casein). Determine the final OD value of the sol preparation by measuring the absorbance at 520nm.

[0229] Preparation and fixation method of gold sol labeling binding reagent

[0230] The anti-hCG coated gold sol (test) conjugate and the rabbit IgG coated gold sol (control) conjugate were centrifuged in a centrifuge and the supernatant was removed. The obtained particles were vortexed and sonicated, and then reconstituted in the gold sol conjugate spray buffer to twice the OD of the required gold (in this embodiment, the test sol was 16OD / ml and the control sol was 10OD / mL). The test sol and the control sol were then mixed 1:1 to obtain a spray solution containing 8OD / ml test sol and 5OD / ml control sol. The gold sol conjugate spray buffer used in the following examples contained 100mM Tris pH 7.4, 20% w / v sucrose (Sigma) and 10% w / v bovine serum albumin (Proliant Biologicals SKU#68700). Glass fiber (GE Healthcare) was loaded onto the Biodot spray device. A Biodot spray device was provided to impregnate / perfuse the glass fiber with the test and control conjugates at the desired position on the glass fiber (6.5mm from the downstream edge). In this example, the glass fiber was sprayed with a single pass of the OD10 conjugate at a drawing rate of 1.7 μL / cm at a distance of 6.5 mm from the downstream edge of the glass fiber. In addition, a 0.56 M EDTA aqueous solution was sprayed at a drawing rate of 1.7 μl / mm from a distance of 16 mm from the downstream edge of the glass fiber. The gold sol-infused glass fiber was dried at 55°C and stored in a sealed foil bag with a desiccant at room temperature.

[0231] Preparation, localization and fixation of specific binding substances

[0232] Prior to immobilization on nitrocellulose, mouse anti-β-hCG antibody was diluted to 3 mg / mL in phosphate buffered saline (PBSA) containing 0.05% (w / v) sodium azide. Prior to immobilization on nitrocellulose, anti-β-FSH antibody was diluted to 1.5 mg / mL in PBSA. Prepare polyvinyl alcohol (PVA) blocking buffer (pH 9) (20 mM Tris base (Sigma), 1% w / v PVA (80% hydrolyzed PVA, 9-10K MW Sigma), 0.05% w / v Tween 20 (Sigma) and 150 mM NaCl (Sigma)). Prepare PVA blocking solution by adding 2% w / v sucrose (Sigma) and 2.5 ml of ethanol (Sigma) to 47.5 mL of PVA blocking buffer. Cut white backed nitrocellulose with pre-punched positioning holes to 4 mm (MDI) into 35 cm*40 mm strips. A Biodot plotter was provided to draw a single line of anti-β-hCG antibody to form a detection zone and anti-βFSH antibody to form a capture zone on a nitrocellulose strip and to draw goat anti-rabbit (Lampire) to form a control zone at a desired position. Anti-β-hCG antibody was deposited at a distance of 15 mm from the bottom (upstream end) of the nitrocellulose membrane at a concentration of 3 mg / ml and a drawing rate of 1 uL / cm, anti-FSH antibody was deposited at a concentration of 3 mg / mL, a drawing rate of 3 μL / cm, and goat anti-rabbit antibody was deposited at a concentration of 2 mg / mL, a drawing rate of 1 uL / cm, and 22 mm from the bottom (upstream end) of the nitrocellulose membrane. After drawing, the strip was dried at 55°C, blocked with PVA blocking solution, then dried at 65°C, and stored in a sealed aluminum foil bag with a desiccant at room temperature.

[0233] Test strip structure

[0234] The assay components are assembled into test strips with the help of a Kinesiology Universal Laminator Module Assembly Unit. A backing laminate (Lohman) is placed on the Kinesiology card press and enclosed nitrocellulose strips with fixed capture, detection and control zones are fixed to the predetermined positions on the backing laminate. Anti-α-hCG and rabbit IgG gold sol conjugate-infused glass fiber strips are fixed to the backing laminate with a 2 mm overlap on the nitrocellulose strip. A roller pad ensures that all components of the strip are in good contact with the backing laminate. The tape is then cut into 4 mm individual strips using an in-house rotary cutter and stored in a foil bag with desiccant until ready for use.

[0235] Preparation device

[0236] The test strips are assembled into a plastic housing component along with an absorbent sampler impregnated with a buffer to control the pH. The housing component contains a lower half with locating pins and an upper half with a window for viewing the lines on the nitrocellulose strip and a member that holds the absorbent sampler in contact with the test strip upstream of the reagent zone. The window position makes the test and control lines visible to the user, but the capture zone lines are hidden. Using the pins to locate the strip, the strip is placed in the lower housing portion. A 4×12 mm sink pad (GE Healthcare) is placed at the proximal end of the strip. A top cover is used to close the device.

[0237] Example 2

[0238] This example demonstrates an assay utilizing a negative pregnancy marker (FSH) in combination with the analyte of interest, hCG. The effect of a negative marker on the sensitivity of the hCG assay was demonstrated by positioning a capture zone containing anti-β-FSH antibodies upstream of an anti-β-hCG zone serving as the detection zone. The effect of the FSH capture zone on the amount of labeled particles that move to the detection zone as a function of FSH in the sample can thus be determined.

[0239] method

[0240] The device was prepared as described in Example 1. To test the effect of FSH concentration on the sensitivity of the test, non-pregnant urine samples were collected from internal volunteers. Urine was removed using anti-α-TSH antibodies to remove endogenous hCG and LH, and then a series of concentrations of hCG and FSH were added. The device was run by applying the spiked urine to the absorbent sampler of the test device. The intensity of the line in the detection zone was measured at 5 minutes using an internal camera system, and 40 non-professional users also scored the device for pregnancy or non-pregnancy. Non-professional users are non-SPD staff who have never seen this type of pregnancy test before. Each non-professional user visually scored 7 devices, which were presented to them in random order.

[0241] result

[0242] As the concentration of FSH in urine samples increased, the intensity of the line in the detection zone measured by the camera and the amount of the device scored as pregnancy by non-expert users both decreased, indicating the effect of FSH as a negative marker on the sensitivity of hCG detection. The results are shown in Table 1 below.

[0243]

[0244]

[0245] Table 1: Average line intensity measured by the internal camera system and the number of devices visually scored as positive for pregnancy test strips with an anti-hCG detection zone and an anti-FSH capture zone that ran mixed negative urine with hCG and FSH. NT—Not tested.

[0246] Example 3

[0247] This example demonstrates an assay utilizing a pregnancy negative marker (FSH) in combination with the analyte of interest, hCG, in clinical samples of pregnancy urine and pre- and post-menopausal urine containing pituitary hCG.

[0248] method

[0249] Device is prepared as described in Example 1. In order to test the impact of the test ability of the present invention on distinguishing hCG from pregnancy and pituitary hCG, urine samples collected from pregnancy and perimenopausal / postmenopausal women were used. 32 parts of pregnancy urine were selected, which were collected from the -5th day to the -3rd day relative to the expected menstrual period, and the hCG concentration range was 3-22mIU / mL. 50 parts of urine samples from perimenopausal and postmenopausal women were also selected, wherein, containing 5-10mIU / ml pituitary hCG. The device was run by applying urine to the absorptive sampler of the test device. The intensity of the line of the detection zone was measured at 5 minutes using an internal camera system, and the device was also scored as pregnancy or non-pregnancy by three non-professional users (SPD staff who do not use reading devices as part of their work).

[0250] result

[0251] As shown in Table 2 below, despite a maximum hCG concentration of up to 10 mIU / mL, none of the 50 perimenopausal / postmenopausal urines were called pregnant, whereas 90% of the pregnancy urines were called pregnant, including those with hCG concentrations as low as 3 mIU / ml. This demonstrates the effectiveness of using a capture zone with a negative marker in generating a sensitive pregnancy test that maintains high specificity for pregnancy in clinical samples.

[0252] Device scored for pregnancy Perimenopausal / Postmenopausal Urine 0 / 50 Pregnancy Urine 29 / 32

[0253] Table 2: Device volume scored as pregnancy by lay users when run with peri / postmenopausal urine samples containing 5-10 mIU / ml hCG and pregnancy urine samples from Day -5 to Day -3 (relative to expected menstrual period).

[0254] Example 4

[0255] This example demonstrates an assay utilizing a positive marker for pregnancy (P3G) in conjunction with the analyte of interest, hCG. The effect of a negative marker on the sensitivity of the hCG assay was demonstrated by positioning a capture zone with an anti-P3G-conjugate upstream of an anti-β-hCG zone that served as the detection zone. The effect of the P3G conjugate on the amount of particles moving to the detection zone as a function of P3G in the sample could therefore be determined. Prior to coating onto the latex particles, the blue latex particles were coated in the antibody mixture by mixing anti-α-hCG and anti-P3G antibodies together. In the test, the coated latex formulations were applied to the porous carrier in various mixtures of P3G (positive marker) and hCG (analyte of interest) to observe the effect of P3G levels on the detection zone signal in the assay.

[0256] Preparation of labeled reagents (latex coated with anti-α-hCG and anti-P3G)

[0257] 1 ml of 2% solid latex (400 nm in diameter) was washed by centrifugation (13000 rcf, 20°C, 4 minutes), the supernatant was removed, and the pellet was resuspended in 500 μl of 100 mM borate buffer (pH 8.5) to give a final concentration of 4% solid latex. The latex was heated at 46°C for about 30 minutes with gentle mixing. A mixture coating of 1200 μg / ml anti-α-hCG (mouse monoclonal) and 600 μg / ml anti-P3G (Fab, mouse monoclonal) was prepared in 100 mM borate buffer (pH 8.5). The antibody mixture was heated at 46°C for 30 minutes with gentle mixing.

[0258] The heated antibody mixture was added to the heated latex in equal volumes and mixed to give 1 ml of 2% solids latex plus 600 μg / ml anti-α-hCG and 300 μg / ml anti-P3G. 100 μl of 95% ethanol plus 0.5% sodium acetate was added to the mixture and the resulting mixture was incubated at 46°C with gentle mixing for 60 minutes.

[0259] 110 μl of 200 mg / ml BSA (Proliant Biologicals, reagent grade) was added to deionized water to block the latex and mixed at 46°C for 30 minutes. After blocking in BSA, the latex was washed and the supernatant was removed by three centrifugation steps (discarded), and the pellet was resuspended in 10 mM borate buffer (pH 8.5) after each centrifugation step. The supernatant was removed after the last centrifugation step and the latex was resuspended in 800 μl of a buffer containing 10% BSA and 20% sucrose in 100 mM Tris pH 8.5 (final suspension buffer) to produce about 2% solid latex. The latex was further diluted 1+7 in the final resuspension buffer before use.

[0260] A porous carrier containing an immobilized area for anti-β-hCG (detection area) and an immobilized area for P3G-conjugate as a capture area was prepared.

[0261] A nitrocellulose membrane (MDI 12 μm) was used as a porous support on which the capture zone and the separate detection zone were deposited.

[0262] As described in Example 1, PBSA containing 3 mg / ml of anti-β-hCG was applied as one zone (detection zone) to a nitrocellulose membrane 10 mm from the proximal end of the test strip using a Biodot plotter. P3G-conjugate (prepared by linking P3G to a mouse monoclonal antibody as a carrier protein) at a protein concentration of 3 mg / ml was deposited 5 mm from the proximal end of the test strip upstream of the detection zone as the capture zone. After the drying step, the membrane was sealed in PVA and dried and then stored in a foil bag with a desiccant. The resulting membrane was cut into 4 mm wide test strips, each with a capture zone 5 mm from the proximal end of the test strip and a detection zone 10 mm from the proximal end of the test strip.

[0263] Tests Anti-P3G and anti-α-hCG latex particles were coated on a porous support prepared with a capture zone for the P3G conjugate and an anti-β-hCG zone as a detection zone.

[0264] The test procedure or "assay" involves preparing a mixture (sample) of coated latex plus a standard buffer (sample) containing varying levels of P3G and hCG (5 μl of diluted latex plus 50 μl of standard buffer) and applying the mixture to a nitrocellulose membrane containing a capture zone and a detection zone. This is accomplished by dipping the proximal end of the test strip into a pool or mixture containing the coated latex and a buffer standard containing varying levels of P3G and hCG. A paper sink material should be used as a sink at the distal end of the test strip (approximately 20 mm downstream of the detection zone). Once the mixture on the bottom of the test strip has dried, the unbound latex is washed away by applying a buffer (50 μl PBSA plus 0.1% ovalbumin) to the bottom of the test strip and passing it through the sink to the end of the test strip. After washing, the signal generated by the detection zone is measured on an optical reading system (image capture system) and the intensity of the signal generated by the detection zone is reported as a number, with the higher the signal intensity in the detection zone, the larger the number.

[0265] To test the effect of the P3G assay on the detection zone response, latex preparations were tested in a buffer containing 0 μg / ml P3G but with varying levels of hCG. This was repeated in a standard buffer containing 75 ug / ml P3G but also with varying levels of hCG.

[0266] The latex preparation is mixed with 0μg / mlP3GThe test strips were mixed with standard buffer containing different levels of hCG (0, 2.5 and 10 mIU / ml hCG) and had a capture zone of P3G conjugate and anti-β-hCG as the detection zone, and were held vertically while immersed in the mixture, allowing the mixture to move through the membrane into a water tank applied at the far end. The test strips were washed as described above and the readings of the detection zone were taken on the imaging system.

[0267] The above procedure was repeated, this time mixing the latex preparation with a standard buffer containing 75 μg / ml P3G and 0, 2.5 and 10 mIU / ml hCG. The above procedure was followed and the detection area was read on the in-house camera system.

[0268] result

[0269] The latex tested with 0 μg / ml P3G in the assay resulted in the majority of the labeled reagent being captured in the capture zone, (the anti-P3G on the labeled reagent bound to the P3G-conjugate in the capture zone). This significantly reduced the amount of labeled reagent that moved to the detection zone, resulting in a reduced response to hCG seen in the detection zone. The presence of 75 μg / ml P3G in the assay reduced the capture of labeled reagent in the capture zone, (due to the P3G in the standard buffer binding to the anti-P3G on the labeled reagent, preventing the labeled reagent from binding to the P3G-conjugate zone) allowing more labeled reagent to move to the detection zone, where a significantly stronger signal was seen in the presence of hCG (see Figure 7 ). Obviously, the level of the positive marker in the test will affect the response of the detection zone to hCG, depending on the amount of positive marker (in this case P3G) in the assay.

[0270] Example 5

[0271] This example demonstrates an assay utilizing a negative marker of pregnancy (FSH) in combination with the analyte of interest, hCG. The effect of the negative marker on the sensitivity of the hCG assay in clinical urine samples was demonstrated by positioning a capture zone containing anti-β-FSH antibodies upstream of the anti-β-hCG zone as the detection zone. The effect of the FSH capture zone on the assay signal generated in the detection zone was shown using peri- and post-menopausal clinical urine samples with physiological levels of hCG and FSH and compared to a standard prepared by adding hCG to a pool of urine depleted of FSH and hCG.

[0272] method

[0273] Device is prepared as described in Example 1. In order to test the influence of negative markers on the sensitivity of hCG test in clinical samples, urine samples collected from perimenopausal / postmenopausal women were tested. In addition, standard products were prepared by adding known concentrations of hCG to a urine pool (FSH and hCG depleted as described in Example 2) that FSH and hCG are depleted. A total of 200 perimenopausal samples and 200 postmenopausal samples were tested. The hCG levels in these samples were measured by a clinical analyzer (DELFIA). In addition, 25 devices used 5mIU / ml hCG to be added to the mixed urine that hCG and FSH are depleted. Other 25 devices used 10mIU / ml hCG to be added to the mixed urine that hCG and FSH are depleted. Device was run by applying urine to the absorptive sampler of the test device. The intensity of the hCG line in the detection zone was measured 5 minutes after sample addition using an internal camera system and the device was visually scored as either pregnant or non-pregnant by two technicians observing the hCG line result, either a visible line (indicating a positive, therefore pregnancy result) or an absence of a line, (indicating a negative, therefore non-pregnancy result). The order in which clinical samples and standards were tested was randomized and the technicians reading the tests were blinded to the randomization to eliminate any technician reading bias.

[0274] result

[0275] As shown in Table 3 below, 92% of the devices using 5mIU / ml hCG spiked mixed urine and 100% of the devices using 10mIU / ml hCG spiked mixed urine were visually assessed as pregnant by the technicians. Despite including hCG concentrations as high as 8.8mIU / ml hCG, all 200 perimenopausal and 200 postmenopausal urine samples were visually assessed as not pregnant by two technician readers. Such hCG levels seen in perimenopausal and postmenopausal urine may increase false positive results in traditional pregnancy tests, which are designed to detect hCG as low as 5mIU / ml.

[0276]

[0277] Table 3: hCG concentration range and visual scores for perimenopausal and postmenopausal urine samples used in the device and hCG standards prepared by adding FSH and hCG depleted negative urine pools.

[0278] Figure 8The signal intensity of the hCG test line in the detection zone, as measured by the internal camera system, is shown, plotted against the hCG concentration. For the spiked standards in the absence of FSH, the signal intensity increases with increasing hCG concentration. Regarding the peri- and post-menopausal samples containing hCG and FSH, it is clear that the presence of FSH in the sample reduces the amount of marker that reaches the hCG test zone as the marker binds in the capture zone. This results in a decrease in the signal in the hCG detection zone, thus providing a true result in the hCG detection zone as the results for these peri- and post-menopausal samples are negative for pregnancy.

Claims

1. An assay device for detecting the presence of an analyte in a sample, the device comprising an assay flow path, the flow path comprising: (a) a reagent zone, the reagent zone comprising a mobilizable labeled conjugate, the labeled conjugate comprising a detectable label, the detectable label being directly or indirectly linked to a means for associating the labeled conjugate with the analyte, wherein the labeled conjugate further comprises a positive marker or a positive marker conjugate, the positive marker portion of the positive marker conjugate being capable of binding, (b) one or more capture zones, wherein: at least one capture zone comprises an immobilized capture substance, the capture substance being configured to capture the positive marker in the presence of the positive marker in the sample, or to capture a labeled conjugate containing or associated with the positive marker or the positive marker conjugate, (c) a detection zone comprising an immobilized binding reagent for capturing a complex comprising the labeled conjugate and the analyte, wherein the or each capture zone is downstream of the reagent zone and the detection zone is downstream of the or each capture zone.

2. An assay device for detecting the presence of an analyte in a sample, the device comprising an assay flow path, the flow path comprising: (a) a reagent zone comprising a mobilizable labeled conjugate, the labeled conjugate comprising a detectable label, the detectable label being directly or indirectly linked to a means for associating the labeled conjugate with the analyte, wherein a mobilizable positive marker or a positive marker conjugate is separately provided in the reagent zone and configured to associate with the labeled conjugate after mobilization, such that the positive marker portion of the conjugate is available for binding in the event that the labeled conjugate associates with the positive marker or positive marker conjugate, (b) one or more capture zones, wherein: at least one capture zone comprises an immobilized capture substance, the capture substance being configured to capture the positive marker in the presence of the positive marker in the sample, or to capture a labeled conjugate containing or associated with the positive marker or the positive marker conjugate, (c) a detection zone comprising an immobilized binding reagent for capturing a complex comprising the labeled conjugate and the analyte, wherein the or each capture zone is downstream of the reagent zone and the detection zone is downstream of the or each capture zone.

3. The test device according to claim 1 or 2, wherein: The reagent zone comprises a mobilizable positive marker binding reagent specific for the positive marker, the mobilizable positive marker binding reagent is linked to a first binding partner, and the capture zone comprises an immobilized second binding partner for the first binding partner.

4. A test device according to any one of the preceding claims, wherein: The capture of the labeled conjugate in the capture zone is achieved by a specific binding reaction with the positive marker or the positive marker conjugate on the labeled conjugate.

5. A test device according to any one of the preceding claims, wherein: The one or more capture zones comprise an immobilized capture substance that specifically binds to the positive marker or positive marker conjugate.

6. A test device according to any one of the preceding claims, wherein: The mobilizable labeled conjugate comprises a P3G conjugate, and the capture zone comprises an immobilized anti-P3G antibody.

7. A test device according to any one of the preceding claims, wherein: The mobilizable labeled conjugate comprises an anti-α-hCG antibody coupled to P3G, and the capture zone comprises an immobilized anti-P3G antibody.

8. A test device according to any one of the preceding claims, wherein: The mobilizable labeled conjugate comprises P3G coupled to a support, and the capture zone comprises an immobilized anti-P3G antibody.

9. An assay device for detecting the presence of an analyte in a sample, the device comprising an assay flow path, the flow path comprising: (a) a reagent zone comprising a mobilizable labeled conjugate, the labeled conjugate comprising a detectable label, the detectable label being directly or indirectly linked to means for associating the labeled conjugate with the analyte or means for associating the labeled conjugate with a positive marker that may be present in the sample or a positive marker or a positive marker conjugate in the device, (b) one or more capture zones, wherein: at least one capture zone comprises an immobilized capture substance configured to capture a labeled conjugate comprising means for associating the labeled conjugate with a positive marker or a positive marker conjugate, wherein the immobilized capture substance comprises or consists of an immobilized positive marker or a positive marker conjugate, (c) a detection zone comprising an immobilized binding reagent for capturing a complex comprising the labeled conjugate and the analyte, wherein the or each capture zone is downstream of the reagent zone and the detection zone is downstream of the or each capture zone.

10. An assay device for detecting the presence of an analyte in a sample, the device comprising an assay flow path, the flow path comprising: (a) a reagent zone, the reagent zone comprising a mobilizable labeled conjugate, the labeled conjugate comprising a detectable label, the detectable label being directly or indirectly linked to means for associating the labeled conjugate with the analyte and means for associating the labeled conjugate with a positive marker that may be present in the sample or a positive marker or a positive marker conjugate in the device, and wherein the reagent zone further comprises a mobilizable positive marker or a positive marker conjugate for immobilization downstream of the reagent zone, (b) at least one capture zone, wherein said at least one capture zone comprises an immobilized capture substance for positioning said mobilizable positive marker or positive marker conjugate in said capture zone, thereby making the positive marker portion of said conjugate available for binding, (c) a detection zone comprising an immobilized binding reagent for capturing a complex comprising the labeled conjugate and the analyte, wherein the or each capture zone is downstream of the reagent zone and the detection zone is downstream of the or each capture zone.

11. The test device according to claim 10, wherein: The reagent zone comprises a mobilisable positive marker or a positive marker conjugate linked to a first binding partner and the or each capture zone comprises an immobilised second binding partner for the first binding partner.

12. The test device according to any one of claims 9 to 11, wherein: Capture of the labelled conjugate in the or each capture zone is achieved by a specific binding reaction with means for associating the labelled conjugate with the positive marker or positive marker conjugate on the labelled conjugate.

13. A test device according to any one of claims 9 to 12, wherein: The means for associating the labeled conjugate with a positive marker comprises or consists of a binding agent, such as an antibody that specifically binds to the positive marker or the positive marker conjugate.

14. A test device according to any one of claims 9 to 13, wherein: The mobilizable labeled conjugate comprises an anti-P3G antibody, and the capture zone comprises an immobilized P3G conjugate.

15. A test device according to any one of the preceding claims, wherein: Capture of the complex in the detection zone is achieved through a specific binding reaction with the analyte.

16. A test device according to any one of the preceding claims, wherein: The device is arranged so that the or each capture zone is not visible to a user in use.

17. A test device according to any one of the preceding claims, wherein: The or each capture zone is not readable by an assay reading member.

18. A test device according to any one of the preceding claims, wherein: The means for associating the labeled conjugate with the analyte comprises or consists of a binding reagent, such as an antibody that binds to an epitope of the analyte.

19. A test device according to any one of the preceding claims, wherein: At least one capture zone comprises a first line or region of immobilized capture material and one or more additional lines or regions of immobilized capture material located downstream of the first line or region of immobilized capture material.

20. A test device according to any one of the preceding claims, wherein: The device comprises a housing which obscures the view of the or each capture zone to a user.

21. A test device according to any one of the preceding claims, wherein: The detection zone comprises an immobilized binding reagent that specifically binds to the analyte.

22. A test device according to any one of the preceding claims, wherein: The immobilized binding reagent in the detection zone does not bind to the positive marker.

23. A test device according to any one of the preceding claims, wherein: The immobilized binding reagent in the detection zone comprises or consists of an antibody.

24. A test device according to any one of the preceding claims, wherein: The reagent zone includes a mobilizable binding reagent that specifically binds to the analyte, the mobilizable binding reagent is linked to a first binding partner, and the detection zone contains an immobilized second binding partner for the first binding partner.

25. A test device according to any one of the preceding claims, wherein: The positive marker indicates the presence of the first condition.

26. The test apparatus according to claim 25, wherein: The first state is pregnancy.

27. A test device according to any one of the preceding claims, wherein: The analyte is hCG and / or wherein the positive marker is a progesterone metabolite, such as P3G.

28. A test device according to any one of the preceding claims, wherein: The mobilizable labeled conjugate comprises an anti-α-hCG antibody.

29. A test device according to any one of the preceding claims, wherein: The detection zone contains anti-β-hCG antibodies.

30. A test device according to any one of the preceding claims, wherein: The label is gold sol or latex.

31. An assay device according to any preceding claim, adapted to reduce the amount of labelled conjugate reaching the detection zone in the absence of one or more additional positive markers.

32. An assay device for detecting the presence of an analyte in a sample, the device comprising an assay flow path, the flow path comprising: (a) a reagent zone comprising a mobilizable labeled conjugate configured to associate with the analyte, wherein the mobilizable labeled conjugate i. configured to be associated with a positive marker or a positive marker conjugate, and / or ii. comprising a positive marker or a positive marker conjugate, thereby making the positive marker or the positive marker portion of the conjugate available for binding, (b) one or more capture zones containing immobilized capture substances for capturing labeled conjugates if the sample does not contain a positive marker, (c) a detection zone for capturing the labeled conjugate if the sample contains the analyte and the labeled conjugate is not captured by the capture zone, in, The one or more capture zones are downstream of the reagent zone, and the detection zone is downstream of the one or more capture zones.

33. The test apparatus according to claim 32, wherein: In case the reagent zone comprises option (i) and option (ii), the positive marker of option (i) is different from the positive marker or the positive marker portion of the positive marker conjugate of option (ii).

34. A test device according to any one of the preceding claims, wherein: In the absence of the positive marker in the sample, the capture zone reduces the amount of the labeled conjugate that reaches the detection zone, and in the presence of the analyte, a detectable signal is formed in the detection zone.

35. A test device according to any one of the preceding claims, wherein: Detection of a signal in the detection zone indicates the presence of the analyte in the sample and indicates the presence of a first condition in the subject from which the sample originated.

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