WO2021083983A1

WO2021083983A1 - A lateral flow immunoassay, and uses thereof

Info

Publication number: WO2021083983A1
Application number: PCT/EP2020/080331
Authority: WO
Inventors: Aart Van Amerongen; Jeroen Veen; Jan WICHERS
Original assignee: Lifesure Limited
Priority date: 2019-10-29
Filing date: 2020-10-28
Publication date: 2021-05-06

Abstract

A lateral flow assay sensor strip for detecting Human chorionic gonadotropin (hCG)and other pregnancy-specific analytes, comprises a sample pad, a conjugate release pad in fluid communication with a distal end of the sample pad and comprising a non-immobilised conjugate comprising a hCG-specific primary antibody conjugated to a detectable label, a detection membrane in fluid communication with a distal end of the conjugate release pad and comprising at least one test zone comprising an immobilised hCG-binding ligand, and a control zone distal of the test zone comprising an immobilised ligand configured to bind the hCG-specific primary antibody, and an adsorbent pad in fluid communication with a distal end of the detection membrane. The detection membrane comprises a competitive analyte zone distal of the test zone comprising immobilised hCG. The assay may be employed to monitor a pregnancy, especially during the first trimester.

Description

TITLE

A Lateral Flow Immunoassay, and uses thereof

Field of the Invention

The present invention relates to a lateral flow assay sensor device. Also contemplated are methods of determining the levels of an analyte in a sample, especially Human chorionic gonadotropin (hCG), methods of detecting risk of an abnormal pregnancy in a pregnant woman, and systems including computer-implemented systems configured for performing the methods of the invention.

Background to the Invention

Human chorionic gonadotropin (hCG) is a glycoprotein secreted by placental trophoblasts cells during pregnancy (1). Placental HCG is excreted into urine as heterodimeric homomer, heterodimeric nicked HCG, free subunits (some nicked), and predominately as hCG beta core fragment (7). hCG has a number of known roles: the promotion of progesterone secretion by the corpus luteum in early pregnancy; the promotion of angiogenesis and vascular genesis in the uterine vasculature during pregnancy; it facilitates implantation; it promotes organ growth and differentiation in the foetus; it regulates maternal innate and adaptive immune responses allowing the acceptance of the foreign foetal antigen (3). Also, the expression of hCG is seen in several types of cancers, including: prostate cancer, colorectal cancer, lung adenocarcinoma, endometrial adenocarcinoma, breast cancer, cervical carcinoma and ovarian cancer, among others (3). hCG levels reach 25 mlU/ml 10 days after conception, increasing exponentially thereafter; doubling roughly every 2-3 days for the first 4 weeks of pregnancy, then the rate of increase slows reaching a peak of 30,000- 290,000 mlU/ml by week 8-10 before dropping to a lower steady state level for the remainder of the pregnancy(l). hCG is therefore very important for both pregnancy detection and monitoring (2). Slower than expected hCG increases indicate abnormal pregnancies such as ectopic pregnancies and declines usually indicate miscarriages (1). For example, Kirk et al (8), report the following for serial hCG samples taken at 48 hour intervals: > 66% increase indicates a normal intra-uterine pregnancy; < 66% increase indicates a probable ectopic pregnancy; > 13% decrease indicates a failed Pregnancy of Unknown Location; < 13% decrease indicates a failing PUL or possibly an ectopic pregnancy.

Miscarriage affects approximately 30% of biochemical pregnancies and 11-20% of clinically recognised pregnancies (6). After a diagnosis of miscarriage, women very frequently undergo significant psychological effects, which can last for a year (6). Currently hCG concentration measurement, in either serum or urine is carried out in laboratories. Therefore, a Point of Care (POC) or Point of Need (PON) test would be highly valuable to pregnant women wanting to monitor their pregnancies.

By far the most widely used method of commercial urine testing involves the immunochromatography double-antibody sandwich method and colloidal gold returning positive results at beta-HCG levels above 25 mlU/ml (1), or indeed at lower levels. These Lateral flow immunochromatographic assay (LFIA) strips (5) generally consist of a sample application pad, a conjugate pad, a membrane (e.g. nitrocellulose, cellulose), and an absorbent pad, as shown in figure A (comparative). LFAI strips in which the sample and conjugate pads are provided by proximal parts of the membrane are also known.

Reporter or detector antibodies conjugated with a contrast-providing reagent (also known as a label) are deposited but not immobilized on the conjugate pad. A fluid sample is applied to the sample pad and wicks down the length of the test strip. As the sample passes through the conjugate pad, the contrast reagent-reporter (label) antibody conjugates bind to the target analyte. Further along the strip, the target analyte also binds to capture antibodies immobilized in the test zone, resulting in the retention of the contrast label. Colour imparted to the test zone by the contrast label indicates the presence of target analyte in the sample. A control zone also tends to be included on the membrane, and this zone contains antibodies that bind to the reporter antibody. The absorbent pad ensures steady wicking of the sample fluid along the test strip. Many variations of this assay are possible including using a competitive assay format rather that a sandwich assay format. The list of materials used as a label in a LFIA is very vast but includes gold nanoparticles, coloured latex beads, magnetic particles, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, up converting phosphors, organic fluorophores, textile dyes, enzymes, liposomes and others (9). In the case of colour producing labels (gold, latex, carbon etc) the LFIAs can be read visually for qualitative results but for quantification, optical readers are used to measure the intensity of the colours produced at the test and control lines of the strip (9). Light absorption/reflection, fluorescence, electrical current/potential difference, chemilumenescence and magnetism among other methods can also be used to produce quantitative results depending on the label used (9).

The hook effect, or the prozone effect, is a type of interference which prevents “sandwich type” immuno-interactions being formed at the test lines, when both the capture and detection antibodies become saturated by the high analyte concentration, causing inaccurately low or false negative results. Classic quantitative and qualitative hCG lateral flow tests suffer at relatively high levels of hCG from the “hook effect” (4). As a result, there is not any LFIA test that can measure the full dynamic range of concentration of hCG present in blood or urine during pregnancy.

US2016/209101 describes a lateral flow assay device and method for quantitative measurement of an analyte that employs an assay strip having a test line, antigen line and control line. The methodology for quantitatively determining the level of the analyte (CRP is the analyte in the examples) notionally employs the intensities of the three lines, and a computational method that comprises entering the test line intensity into a quadratic equation configured to provide two output values for the amount of analyte, and then choosing the smaller of the two values if the antigen line intensity is greater than the control intensity, or choosing the higher of the two values if the antigen line intensity is lower than the control intensity. A problem with this method is that the sample has to be diluted 10-fold to avoid matrix interference, and this makes the method unsuitable for use in a home testing kit where from a practical point of view the assay is required to work with undiluted sample. In addition, the method would not work for analytes such as hCG where the control line intensity will always be greater than the antigen line intensity.

It is an object of the invention to overcome at least one of the above-referenced problems.

Summary of the Invention

The present invention addresses the need for a LFIA test that can measure the full dynamic range of concentration of hCG present in blood or urine during pregnancy, generally without the need to sample. This objective is achieved by providing a sandwich lateral flow assay having a conventional test and control indicia (line or spots), and in addition having a competitive analyte indicia (line or spots) comprising analyte directly bound to the membrane. The intensity of the three signals (referred to herein as a detectable label fingerprint) can be directly correlated with a level of hCG in the sample using a computational model such as a linear regression model. The Applicant has discovered that such a modified LFIA can accurately and reproducibly measure high levels of hCG in an undiluted blood or urine sample from a patient within the range appropriate for weeks 6-12 of pregnancy (i.e. 5000 - 300,000 mlU/ml hCG), and that these measurements can be employed to detect risk of an abnormal pregnancy in the woman in a home testing scenario, for example an ectopic pregnancy or miscarriage.

According to a first aspect of the present invention, there is provided a lateral flow assay sensor strip for detecting an analyte, comprising: a sample pad; a conjugate release pad in fluid communication with a distal end of the sample pad and comprising a non-immobilised conjugate comprising an analyte-specific primary antibody conjugated to a detectable label; a detection membrane in fluid communication with a distal end of the conjugate release pad and comprising a test zone comprising an immobilised analyte-binding ligand, and a control zone distal of the test zone comprising an immobilised ligand configured to bind the analyte-specific primary antibody; and an adsorbent pad in fluid communication with a distal end of the detection membrane, characterised in that the detection membrane comprises a competitive analyte zone distal or proximal of the test zone comprising immobilised analyte.

It will be appreciated that the conjugate release pad, sample pad, or sample pad and conjugate release pad, may be provided by proximal parts of the detection membrane. Thus, the sample and/or conjugate may be applied directly to the membrane. In one embodiment, the analyte is selected from Human chorionic gonadotropin (hCG), estrogen and progesterone.

In one embodiment, the immobilised analyte-binding ligand, immobilised ligand and immobilised analyte are provided as lines (typically three lines) or arrays of spots (typically three arrays of spots) spaced apart along the strip.

In one embodiment, the lateral flow assay sensor strip is for detecting hCG, and comprises a sample pad; a conjugate release pad in fluid communication with a distal end of the sample pad and comprising a non-immobilised conjugate comprising a hCG-specific primary antibody conjugated to a detectable label; a detection membrane in fluid communication with a distal end of the conjugate release pad and comprising at least one test zone comprising an immobilised hCG- binding ligand, and a control zone distal of the test zone comprising an immobilised ligand configured to bind the hCG-specific primary antibody; and an adsorbent pad in fluid communication with a distal end of the detection membrane, characterised in that the detection membrane comprises a competitive analyte zone distal or proximal of the test zone comprising immobilised hCG.

In one embodiment, the competitive analyte zone is disposed intermediate the test zone and the control zone.

In one embodiment, the analyte (i.e. hCG) is immobilised directly to the membrane in the competitive analyte zone. In one embodiment, the analyte in the competitive analyte zone is not conjugated to another protein, for example an antibody. In one embodiment, the membrane is nitrocellulose. Other cellulose-based membranes may also be employed, for example, the cellulose-based membranes disclosed in WO2011051562A1, WO2010128205 and W02008030546.

In one embodiment, the analyte (i.e. hCG)-binding ligand immobilised in the test zone is an analyte (i.e. hCG)-binding antibody.

In one embodiment, the ligand configured to bind the analyte (i.e. hCG)-specific primary antibody immobilised in the control zone is an IgG binding protein.

In one embodiment, the ligand configured to bind the analyte (i.e. hCG)-specific primary antibody immobilised in the control zone is an IgG binding antibody.

In one embodiment, the lateral flow assay sensor strip has a single test zone. In other embodiments, two or more test zones may be employed (for example 2, 3, 4, 5, 6, or 7). In this latter embodiment, the detectable label fingerprint comprises detected label at the test zones, control zone and competitive analyte zone.

In another aspect, the invention provides a method of determining the level of an analyte in a liquid sample, which method employs a lateral flow assay sensor strip according to the invention, the method comprising the steps of: applying an aliquot of the liquid sample to the sample pad, whereby the liquid sample is transferred by capillary action along a flow path defined by the sample pad, conjugate release pad, detection membrane, and adsorbent pad; detecting the level of the detectable label at the test zone, competitive analyte zone and control zone; and correlating the detected levels of detectable label with the level of analyte in the sample.

In one embodiment, the method is a method of determining the level of Human chorionic gonadotropin (hCG) in a liquid biological sample, although methods of determining the level of other analytes such as pregnancy-specific analytes (e.g estrogen or progesterone) are also envisaged.

In one embodiment, the correlation step comprises inputting the detectable label fingerprint into a computational model, in which the computational model is generated from reference detectable label fingerprints obtained from a calibration data set using known amounts of analyte, wherein the computational model is configured to output the determined level of hCG in the sample.

In one embodiment, the computational model is linear regression computational model.

In one embodiment, the linear regression computational model employs Principle Component Analysis (PCA).

In one embodiment, the detectable label is an optically detectable label, and wherein the detectable label fingerprint is detected using an optical detector. It will be appreciated that various types of detectable labels may be employed, for example, gold particles, magnetic particles, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, up converting phosphors, organic fluorophores, textile dyes, enzymes, liposomes and others (9).

In another aspect, the invention provides a method of monitoring a pregnancy, especially during the first trimester of pregnancy, in a pregnant woman, the method comprising the steps of monitoring pregnancy-specific analyte levels (i.e.hCG levels) in the pregnant woman over a measurement period during the first trimester of pregnancy, wherein the analyte levels are determined according to a method of the invention.

In another aspect, the invention provides a method of detecting risk of an abnormal pregnancy in a pregnant woman the method comprising the steps of detecting an abnormal rate of change in a pregnancy-specific analyte level (i.e. hCG levels) in the pregnant woman over a measurement period during the first trimester of pregnancy, wherein the analyte levels are determined according to a method of the invention.

Alternative pregnancy-specific analytes include estrogen and progesterone. Typically, the suitable time period is at least 1, 2, 3, 4, 5, 6 or 7 days. In one embodiment, the time points are separated by 1-14, 1-7, 1-5, 1-3, or 1-2 days.

In one embodiment, when the pregnancy-specific analyte is hCG, the abnormal rate of change is a decrease in hCG levels, no change in the hCG levels, or a rate of increase that is less than a 66% increase, during the measurement period, for example a 48 hour period. The literature indicates that < 66% increase indicates a probable ectopic pregnancy; > 13% decrease indicates a failed Pregnancy of Unknown Location; < 13% decrease indicates a failing PUL or possibly an ectopic pregnancy.

In another aspect, the invention provides a system suitable for determining the level of an analyte (such as estrogen, progesterone or Human chorionic gonadotropin (hCG)) in a liquid biological sample. The system typically comprises: a lateral flow assay sensor strip according to the invention; a detector configured to detect a detectable label fingerprint in the test zone of the lateral flow assay sensor strip, the detectable label fingerprint comprising detectable label at the test zone, competitive analyte zone and control zone; a comparison system configured to compare the detectable label fingerprint with one or more reference detectable label fingerprints; and a display system for displaying an output of the comparison step.

In one embodiment, the detector is an optical detector. In one embodiment, the detector is a real-time video reader configured to reads spots (or lines) of lateral flow assays from the start. As soon as sufficient information is acquired to calculate the slope of the signal intensity curve of a spot or line, this slope can be used to calculate quantitative results. Assessment of the slope is possible within 1 to 4 minutes, depending on the concentration of the analyte.

In one embodiment, the comparison system comprises a computational model configured for inputting the detectable label fingerprint, comparison of the detectable label fingerprint with one or more reference detectable label fingerprints corresponding with known amounts of analyte (i.e. hCG), and output of a content based in part of the comparison result.

In one embodiment, the one or more reference detectable label fingerprints are stored locally on the storage system.

In one embodiment, the one or more reference detectable label fingerprints are stored on a remote server and accessible through the internet.

The invention also provides a computer program which when executed on a computer causes the computer to perform a method of determining a level of an analyte in a liquid sample according to the invention or a method of detecting risk of an abnormal pregnancy in a pregnant woman according to the invention.

The invention also relates to a computer program recording medium storing a computer program according to the invention.

In one embodiment, the invention provides a method of determining the level of an analyte such as hCG in a liquid biological sample, which method employs a lateral flow assay sensor strip of the type comprising: a sample pad; a conjugate release pad in fluid communication with a distal end of the sample pad and comprising a non-immobilised conjugate comprising an analyte (i.e. hCG)- specific primary antibody conjugated to a detectable label; a detection membrane in fluid communication with a distal end of the conjugate release pad and comprising at least one test zone comprising an immobilised analyte (i.e.hCG)-binding ligand, and a control zone distal of the test zone comprising an immobilised ligand configured to bind the analyte (i.e. hCG)-specific primary antibody, and a competitive analyte zone distal or proximal of the test zone comprising immobilised analyte (i.e. hCG); and an adsorbent pad in fluid communication with a distal end of the detection membrane, the method comprising the steps of: applying an aliquot of the liquid biological sample to the sample pad, whereby the liquid biological sample is transferred by capillary action along a flow path defined by the sample pad, conjugate release pad, detection membrane, and adsorbent pad; detecting the level of the detectable label at the test zone, competitive analyte zone and control zone to provide a detectable label fingerprint; and correlating the detectable label fingerprint with the level of analyte (i.e. hCG) in the sample, characterised in that the correlation step comprises inputting the detectable label fingerprint into a linear regression computational model generated from reference detectable label fingerprints obtained from a calibration data set using known amounts of analyte (i.e. hCG), wherein the computational model is configured to output the determined level of analyte (i.e.hCG) in the sample.

In another aspect, the invention provides a system suitable for determining the level of an analyte such as Human chorionic gonadotropin (hCG) in a liquid biological sample, the system comprising: a lateral flow assay sensor strip comprising a sample pad, a conjugate release pad in fluid communication with a distal end of the sample pad and comprising a non-immobilised conjugate comprising an analyte-specific primary antibody conjugated to a detectable label, a detection membrane in fluid communication with a distal end of the conjugate release pad and comprising at least one test zone comprising an immobilised analyte-binding ligand, and a control zone distal of the test zone comprising an immobilised ligand configured to bind the analyte-specific primary antibody, and a competitive analyte zone distal or proximal of the test zone comprising immobilised analyte, and an adsorbent pad in fluid communication with a distal end of the detection membrane, a detector configured to detect a detectable label fingerprint in the test zone of the lateral flow assay sensor strip, the detectable label fingerprint comprising a level of the detectable label at the test zone, competitive analyte zone and control zone; a processor comprising a linear regression computational model generated from reference detectable label fingerprints obtained from a calibration data set using known amounts of analyte, in which the processor is configured to receive the detectable label fingerprint, input the detectable label fingerprint into the linear regression computational model, and generate an output comprising the determined level of analyte in the liquid biological sample; and a display system for displaying the output.

In another aspect, the invention provides a computer implemented method of determining the level of hCG in a liquid biological sample, which method employs a lateral flow assay sensor strip of the type comprising: a sample pad; a conjugate release pad in fluid communication with a distal end of the sample pad and comprising a non-immobilised conjugate comprising a hCG-specific primary antibody conjugated to a detectable label; a detection membrane in fluid communication with a distal end of the conjugate release pad and comprising at least one test zone comprising an immobilised hCG-binding ligand, and a control zone distal of the test zone comprising an immobilised ligand configured to bind the hCG-specific primary antibody, and a competitive analyte zone distal or proximal of the test zone comprising immobilised hCG; an adsorbent pad in fluid communication with a distal end of the detection membrane, wherein an aliquot of the liquid biological sample is applied to the sample pad, whereby the liquid biological sample is transferred by capillary action along a flow path defined by the sample pad, conjugate release pad, detection membrane, and adsorbent pad; and the level of the detectable label at the test zone, competitive analyte zone and control zone to provide a detectable label fingerprint is detected; characterised in that a processor is configured to perform the step of: correlating the detectable label fingerprint with the level of hCG in the sample, wherein by inputting the detectable label fingerprint into a linear regression computational model generated from reference detectable label fingerprints obtained from a calibration data set using known amounts of hCG, and the computational model is configured to output the determined level of hCG in the sample.

The computer implemented method above may be employed to determine the level of other analytes in a liquid biological sample.

Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

Brief Description of the Figures

Figure 1 (comparative): Lateral flow immunochromatographic assay (LFIA) strip consisting of a sample application pad, a conjugate pad, a membrane (e.g. nitrocellulose, cellulose), and an absorbent pad.

Figure 2: Sample image of Lateral Flow Strips after testing with 0 - 300 K mlU/ml b-hCG antigen dilutions, using three test lines (T1 p0.6, T2 p0.8 & T3 p 1.0) , a competitive antigen line (A p1.4) and a control line (C p1.8). The position of T1 is 0.6 cm from the start of the strip.

Figure 3: Curves fitted to the averaged experimental data points of three independent serial dilutions (R1 , R2 & R3) of b-hCG (1.25 - 300 K mlU/ml) for three test lines T1-T3, the competitive antigen (A) line and the control (C) line; the position of T1 is 0.6 cm from the start of the strip.

Figure 4: Sample image of Lateral Flow Strips after testing with 0 - 300 K mlU/ml b-hCG antigen dilutions, using three test lines (T1 p1.0, T2 p1.2 & T3 p1.4), a competitive antigen line (A p1.8) and a control line (C p2.2). The position of T1 is 1.0 cm from the start of the strip.

Figure 5: Curves fitted to the averaged experimental data points of three independent serial dilutions (R1,R2 & R3) of b-hCG (1.25 - 300 K mlll/ml) for three test lines T1-T3, the competitive antigen (A) line and the control (C) line; start position of T1 is 1 cm.

Figure 6: Relationship between added and predicted b-hCG concentrations expressed in a linear (A: upper panel) and a logarithmic way (B: lower panel).

Detailed Description of the Invention

All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

Definitions and general preferences

Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps. In the context of diagnosis an analyte levels as employed herein, the term subject (which is to be read to include "individual", "animal", "patient" or "mammal" where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human.

As used herein, the term “lateral flow assay sensor strip” primarily refers to the sandwich lateral flow assays of the type shown in Figure A that generally comprise a strip of capillary flow materials having a longitudinal dimension and a capillary flow path along the longitudinal dimension. The strip generally comprises discrete overlapping sections including a sample pad, a conjugate release pad, a detection membrane (i.e. cellulose or a cellulose derivative such as nitrocellulose), and an adsorbent pad. The sample pad is generally formed from a wicking material, the details of which will be known to those skilled in the art, although it may also be provided by a proximal part of the detection membrane. The conjugate release pad generally comprises a conjugate dispersed within a dissolvable matrix (often salt or sugar) that releases the conjugate upon dissolution by the sample fluid. The conjugate comprises an analyte-specific antibody conjugated to a detectable label.

The conjugate release pad may also be provided by a proximal part of the detection membrane. Further description of the components of the sections of the lateral flow assay are not provided herein, but will be known to a person skilled in the art and are described in the literature referenced below and in EP0349215.

As used herein, the term “analyte” primarily refers to hCG or its metabolites, but may also refers to other ligands present in biological and non-biological fluids such as environmental or industrial samples. The biological fluids of interest to the present invention are primarily blood and blood derivatives (serum, plasma, etc) and urine, but may also include other biological fluids such as saliva, sweat, cerebrospinal fluid, semen, and lymph. Pregnancy- specific analytes include hCG, estrogen and progesterone. In one embodiment, the liquid biological sample employed in the methods of the invention, is undiluted (especially when the sample is urine). This provides an advantage insofar as the user is not required to dilute the sample, and makes the test suitable as a home-use test. In one embodiment, the sample is diluted no more than 2, 3, 4, 5, 6, 7, 8, or 9 fold.

As used herein, the term ”hCG” refers to Human chorionic gonadotropin (hCG), a glycoprotein secreted by placental trophoblasts cells during pregnancy (1). Placental HCG is excreted into urine as heterodimeric hormone, heterodimeric nicked HCG, free subunits (some nicked), and predominately as hCG beta core fragment (7). The term hCG includes the heterodimeric hormone, and its derivatives (i.e. heterodimeric nicked hCG, free subunits (some nicked), and hCG beta core fragment).

As used herein, the term “conjugate” refers to an analyte-specific antibody, or analyte- specific antibody fragment, conjugated to a detectable label. Generally, the antibody is a monoclonal antibody, but may also be a polyclonal antibody. When the analyte is a human analyte, the antibody is generally a non-human antibody, for example a mouse or goat antibody that is specific for the analyte in question. The purpose of the conjugate is to bind to any analyte in the sample, and travel along the flow path in the strip to the detection membrane where the conjugate binds to the test line (via the analyte part of the conjugate binding to the immobilised antibody in the test zone).

As used herein, the term “detectable label” refers to a label that can be detected (for example, some emit a signal that is detectable), for example an optical, fluorescent, luminescent, magnetic, or electrical signal. Examples of detectable labels useful for the present invention include gold nanoparticles, coloured latex beads, magnetic particles, carbon nanoparticles, selenium nanoparticles, silver nanoparticles, quantum dots, up converting phosphors, organic fluorophores, textile dyes, enzymes, liposomes and others (9).

As used herein, the term “test zone” refers to an area of the detection membrane containing a binding ligand for the analyte. Generally, the binding ligand for the analyte is provided along a line (i.e. test line) that is generally perpendicular to the longitudinal axis of the test strip. Generally, the binding ligand for the analyte is an analyte-specific antibody (i.e. capture antibody). When the analyte is hCG, the binding ligand is generally a hCG- specific antibody. The detection membrane may contain one or more test zones, and each may contain different concentrations of analyte-binding ligand. As used herein, the term “control zone” refers to an area of the detection membrane, generally distal of the test zone, containing a binding ligand for the analyte-binding ligand. Generally, the binding ligand is provided along a line (i.e. control line) that is generally perpendicular to the longitudinal axis of the test strip. Generally, the binding ligand for the capture (analyte-specific) antibody is an antibody, for example an anti-lgG antibody or antibody-binding protein (i.e. Protein A).

As used herein, the term “competitive analyte zone” refers to an area of the detection membrane containing analyte. Generally, the analyte is provided along a line (i.e. test line) that is generally perpendicular to the longitudinal axis of the test strip, or provided as a series or pattern of dots. Generally, the binding ligand for the analyte is an analyte-specific antibody. The analyte generally binds directly to the membrane, for example the nitrocellulose, and is typically not immobilised to the membrane via a binding partner.

As used herein, the term “abnormal pregnancy” refers to an ectopic pregnancy or a miscarriage.

As used herein, the term “detectable label fingerprint” refers to the combination of the intensities of the detectable label at the test zone, control zone and competitive analyte zone. Generally, the detectable label is an optical label that can be read by a reader, for example an optical label scanner, and then converted into a set of detectable label intensity values. However, the intensities may be non-optical intensities, for example magnetic or electrical intensities, or the like. These values can be input into a computational model which is configured to provide a hCG level output. The computational model may be a linear regression model, that may employ Principle Component Analysis (PCA).

The invention also provides a system suitable for determining the level of an analyte such as Human chorionic gonadotropin (hCG) in a liquid sample, typically a biological sample. The system or kit typically comprises a lateral flow sensor strip according to the invention having a detection membrane comprising a test zone, control zone, and competitive analyte zone. The system or kit comprises a detector configured to detect a detectable label fingerprint in the test zone of the lateral flow assay sensor strip, the detectable label fingerprint comprising detectable label at the test zone, competitive analyte zone and control zone;

The system or kit also comprises a storage system (optional) and a comparison system. These functional modules can be executed on one, or multiple, computers, or by using one, or multiple, computer networks. The determination system has computer executable instructions to provide e.g., sequence information in computer readable form.

The information determined in the determination system can be read by the storage system. As used herein the “storage system” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of an electronic apparatus suitable for use with the present invention include a stand-alone computing apparatus, data telecommunications networks, including local area networks (LAN), wide area networks (WAN), Internet, Intranet, and Extranet, and local and distributed computer processing systems. Storage devices also include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage media, magnetic tape, optical storage media such as CD-ROM, DVD, electronic storage media such as RAM, ROM, EPROM, EEPROM and the like, general hard disks and hybrids of these categories such as magnetic/optical storage media. The storage system is adapted or configured for having recorded thereon detectable label fingerprint information. Such information may be provided in digital form that can be transmitted and read electronically, e.g., via the Internet, on diskette, via USB (universal serial bus) or via any other suitable mode of communication.

The storage system may have reference detectable label fingerprint information stored thereon. As used herein, "stored" refers to a process for encoding information on the storage device. In one embodiment the reference data stored in the storage device to be read by the comparison module is compared, e.g., comparison of a test detectable label fingerprint with a set or panel of reference detectable label fingerprints.

The “comparison system” can use a variety of available software programs and formats for the comparison operative to compare query growth response fingerprint with reference growth response fingerprints and identify a “match”. In one embodiment, the comparison module is configured to use pattern recognition techniques to compare information from one or more entries to one or more reference data patterns. The comparison module may be configured using existing commercially-available or freely-available software for comparing patterns, and may be optimized for particular data comparisons that are conducted. The comparison module provides computer readable information related to the genotype of the sample. Preferably, the comparison system employs a computational model for comparison purposes.

The comparison module, or any other module of the invention, may include an operating system (e.g., UNIX) on which runs a relational database management system, a World Wide Web application, and a World Wide Web server. World Wide Web application includes the executable code necessary for generation of database language statements (e.g., Structured Query Language (SQL) statements). Generally, the executables will include embedded SQL statements. In addition, the World Wide Web application may include a configuration file which contains pointers and addresses to the various software entities that comprise the server as well as the various external and internal databases which must be accessed to service user requests. The Configuration file also directs requests for server resources to the appropriate hardware--as may be necessary should the server be distributed over two or more separate computers. In one embodiment, the World Wide Web server supports a TCP/IP protocol. Local networks such as this are sometimes referred to as "Intranets." An advantage of such Intranets is that they allow easy communication with public domain databases residing on the World Wide Web (e.g., the GenBank or Swiss Pro World Wide Web site). Thus, in a particular preferred embodiment of the present invention, users can directly access data (via Hypertext links for example) residing on Internet databases using a HTML interface provided by Web browsers and Web servers.

The comparison module typically provides a computer readable comparison result that can be processed in computer readable form by predefined criteria, or criteria defined by a user, to provide a content based in part on the comparison result that may be stored and output as requested by a user using a display system.

In one embodiment of the invention, the content based on the comparison result is displayed on a computer monitor. In one embodiment of the invention, the content based on the comparison result is displayed through printable media. The display module can be any suitable device configured to receive from a computer and display computer readable information to a user. Non-limiting examples include, for example, general-purpose computers such as those based on Intel PENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC, Hewlett-Packard PA-RISC processors, any of a variety of processors available from Advanced Micro Devices (AMD) of Sunnyvale, California, or any other type of processor, visual display devices such as flat panel displays, cathode ray tubes and the like, as well as computer printers of various types.

In one embodiment, a World Wide Web browser is used for providing a user interface for display of the content based on the comparison result. It should be understood that other modules of the invention can be adapted to have a web browser interface. Through the Web browser, a user may construct requests for retrieving data from the comparison module. Thus, the user will typically point and click to user interface elements such as buttons, pull down menus, scroll bars and the like conventionally employed in graphical user interfaces.

Exemplification

The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

The new product is a POC/PON device that can fully quantify the levels of hCG in the urine of pregnant women being able to cope with the full dynamic range required during pregnancy. The device is a modification of a Lateral Flow Immuno Assay (LFIA), as described above. As well as one or more test lines and a control line, a Competitive Antigen Line has been introduced along the test strip, after the test line or lines. The Competitive Antigen Line consists of a layer of hCG (or one of its metabolites) fixed at a point along the strip.

The biological sample, containing the analyte of interest (hCG or one of its metabolites), is applied to the sample pad. The fluid then travels to the conjugate pad. The conjugate binds to the analyte and travels along the strip where it binds to analyte specific antibodies that have been fixed at the test line or lines. The fluid then moves to the Competitive Antigen Line and the control line. Unbound conjugate will attach and become fixed to the Competitive Antigen Line. The control line will also bind to unbound conjugate.

The colour intensity of all the test line(s), the control and the Competitive Antigen Line is measured optically. By using at least one test line, the Competitive Antigen Line and the control line a linear regression model results in an algorithm that can be used to measure the analyte concentration of unknown samples.

The device allows for the serial measurement of the concentration of the analyte in biological samples, in a non-laboratory context thus, over a very broad range of concentrations, thus facilitating the monitoring of a pregnancy or hCG producing cancers.

Data/Results

The following results were achieved using an antibody- carbon conjugate (a different label could be used, gold for example). The Competitive Antigen Line was placed between the last test line and the control line. The colour intensity of the test lines, the Competitive Antigen Line and control line were measured by scanning the dry lateral flow strips using a flatbed scanner and generating pixel grey values. A different optical method could also be used to measure the colour intensity at each of the lines.

Approach

Three test lines of goat PAb anti^-hCG (500 ng/pL) were sprayed on Merck Millipore HF- 135 starting at 0.6 or 1,0 cm from the origin. A competitive antigen line of 1 pg/pl b-hCG was applied at position 1.4 or 1.8 cm, respectively, and a goat anti-mouse IgG control line (200 ng/pL) was applied at either position 1.8 cm or position 2.2 cm. Strips were developed with net 0.5 pL carbon conjugate in standard running buffer (100 mM borate, 1%(w/v) BSA, 0.05%(v/v) Tween-20, pH 9). Three independently and freshly prepared b-hCG antigen dilution ranges (0 - 300 K mlU/ml b-hCG) were tested and scored for signal development. After air drying of the strips, signals were scanned with the Epson flatbed scanner and data were subsequently processed, ending up with dose response curves.

Table 1 : The Pixel Grey Values of the control line, the competitive antigen line and the three test lines (T1, T2 & T3), after testing with 0 - 300 K mlU/ml b-hCG antigen dilutions, repeated three times (R1 , R2 & R3). The position of T1 is 0.6 cm from the start of the strip.

Table 2: The Pixel Grey Values of the control line, the competitive antigen line and the three test lines (T1 , T2 & T3), after testing with 0 - 300 K mlll/ml b-hCG antigen dilutions, repeated three times (R1 , R2 & R3). The position of T1 is 1.0 cm from the start of the strip.

Statistical analysis of the results

A machine learning algorithm was developed in Python by using a linear fit model. Hereto, two of the three sets of membranes were used that had been run with the three independently and freshly prepared b-hCG antigen dilution ranges. The third set was used to test the developed model and to predict b-hCG concentrations by a Principle Component Analysis (PCA).

Firstly, the model was developed by using all three test lines, the competitive antigen line and the control line. Secondly, only the second test line was used and thirdly, only the first test line was used. The third approach yielded the best fit upon predicting the b-hCG concentrations of the third run. Only data of 5,000 mlll/mL b-hCG and more were used in this third approach, which is the range that will be of interest for a quantitative b-hCG test to be used between week 6 and 12 of pregnancy.

The data of the training data cross validation RMS error were 0.0725 and 0.0407 for mean and standard deviation, respectively. The Test data RMS error was 0.09, which is close to the cross-validation RMS error of 0.07, indicating a good fit of the third run data to the model. This was further supported by a high Variance score of 0.96.

In the following table the added and predicted b-hCG concentrations are depicted. In addition, the relative error data are indicated.

Table 3: Added and predicted b-hCG concentrations

By using one of the test lines, the competitive antigen line and the control line a linear regression model can be fit to the data. The resulting algorithm can be used to predict b- hCG concentrations in unknown samples. Equivalents

The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

References:

(1) Jing fan, Mandy Wang, Chengyin Wang & Yu Cao. Advances in human chorionic goanadotropin detection technologies: a review. Bioanalysis (2017) 9(19), 209-229.

(2) Ruth McChesney, Allen J. Wilcox, John F.O’ Connor, Clarice R. Weinberg, Donna D. Baird, John P. Schlatterer, D. Rober McConnaughey, Steven Birken and Robert E. Canfield. Intact HCG, free HCG beta-subunit and HCG beta-core fragment: longtitudinal patterns in urine during early pregnancy. Human Reproduction Vol. 20, No. 4 pp 928-935, 2005.

(3) Helene Heidegger and Udo Jeschke. Human Chorionic Gonadotropin (hCG)-An endocrine regulator of gestation and cancer. Int. J Mol. Sci. 2018, 19(5), 1502

(4) Robert D. Nerenz, Haowei Song and Ann M. Gronowski. Screening Method to evaluate Point-of-Care Human Chorionic Gonadotropin (hCG) devices for susceptibility to the hook effect by hCG beta core fragment: Evaluation of 11 devices

(5) Eleonara Petryayeva & Walter Algar. Toward Point-of Care Diagnostics with Consumer Electronic Devices: the expanding role of Nanoparticles

(6) Cecilia Bottomley & Tom Bourne. Diagnosing miscarriage. Best Practice and Research Clinical Obstetrics and Gynaecology 23 (2009) 463-477

(7) Steven Birken, Galina Kovalevskaya, John O Connor. Metabolism of hCG and hl_H to multiple urinary forms. Molecular and Cellular Endocrinology 125 (1996) 121-131

(8) E. Kirk, C. Bottomley and T. Bourne. Diagnosing ectopic pregnancy and current concepts in the management of pregnancy of unknown location. Human Reproduction Update, Vol. 20, No.2 pp. 250-261,2014

(9) Muhammad Sajin, Abdel-Nasser Kawde, Muhammad Daud. Designs, formats and applications of lateral flow: A literature review. Journal of Saudi Chemical Society.

Claims

CLAIMS:

1. A method of determining the level of hCG in a liquid biological sample, which method employs a lateral flow assay sensor strip of the type comprising: a sample pad; a conjugate release pad in fluid communication with a distal end of the sample pad and comprising a non-immobilised conjugate comprising a hCG-specific primary antibody conjugated to a detectable label; a detection membrane in fluid communication with a distal end of the conjugate release pad and comprising at least one test zone comprising an immobilised hCG- binding ligand, and a control zone distal of the test zone comprising an immobilised ligand configured to bind the hCG-specific primary antibody, and a competitive analyte zone distal or proximal of the test zone comprising immobilised hCG; and an adsorbent pad in fluid communication with a distal end of the detection membrane, the method comprising the steps of: applying an aliquot of the liquid biological sample to the sample pad, whereby the liquid biological sample is transferred by capillary action along a flow path defined by the sample pad, conjugate release pad, detection membrane, and adsorbent pad; detecting the level of the detectable label at the test zone, competitive analyte zone and control zone to provide a detectable label fingerprint; and correlating the detectable label fingerprint with the level of hCG in the sample, characterised in that the liquid biological sample is undiluted and the correlation step comprises inputting the detectable label fingerprint into a linear regression computational model generated from reference detectable label fingerprints obtained from a calibration data set using known amounts of hCG, wherein the computational model is configured to output the determined level of hCG in the sample.

2. A method according to Claim 1, in which the computational model is linear regression computational model that optionally employs Principle Component Analysis (PCA).

3. A method according to Claim 1 or 2, in which the detectable label is an optically detectable label, and wherein the detectable label fingerprint is detected using an optical detector.

4. A method according to any of Claims 1 to 3, in which the liquid biological sample is undiluted urine.

5. A method of monitoring pregnancy in a pregnant woman, the method comprising the steps of detecting hCG levels in the pregnant woman over a measurement period during the first trimester of pregnancy, wherein the hCG levels are determined according to a method of any of Claims 1 to 4.

6. A method according to Claim 5, in which the measurement period is 1-3 days.

7. A method according to Claim 5 or 6, in which detection of a decrease in hCG levels, or no change in the hCG levels, during the measurement period is indicative of an abnormal pregnancy.

8. A method according to Claim 6, in which detection of an increase in hCG levels of less than 33% per day during the measurement period is indicative of an abnormal pregnancy.

9. A method according to Claim 8, in which the measurement period is 48 hours and in which detection of an increase in hCG levels during the 48 hour period of less that 66% is indicative of an abnormal pregnancy.

10. A system suitable for determining the level of Human chorionic gonadotropin (hCG) in a liquid biological sample, the system comprising: a lateral flow assay sensor strip comprising a sample pad, a conjugate release pad in fluid communication with a distal end of the sample pad and comprising a non-immobilised conjugate comprising a hCG-specific primary antibody conjugated to a detectable label, a detection membrane in fluid communication with a distal end of the conjugate release pad and comprising at least one test zone comprising an immobilised hCG-binding ligand, and a control zone distal of the test zone comprising an immobilised ligand configured to bind the hCG-specific primary antibody, and a competitive analyte zone distal or proximal of the test zone comprising immobilised hCG, and an adsorbent pad in fluid communication with a distal end of the detection membrane, a detector configured to detect a detectable label fingerprint in the test zone of the lateral flow assay sensor strip, the detectable label fingerprint comprising a level of the detectable label at the test zone, competitive analyte zone and control zone; a processor comprising a linear regression computational model generated from reference detectable label fingerprints obtained from a calibration data set using known amounts of hCG, in which the processor is configured to receive the detectable label fingerprint, input the detectable label fingerprint into the linear regression computational model, and generate an output comprising the determined level of hCG in the liquid biological sample; and a display system for displaying the output.

11. A computer implemented method of determining the level of hCG in a liquid biological sample, which method employs a lateral flow assay sensor strip of the type comprising: a sample pad; a conjugate release pad in fluid communication with a distal end of the sample pad and comprising a non-immobilised conjugate comprising a hCG-specific primary antibody conjugated to a detectable label; a detection membrane in fluid communication with a distal end of the conjugate release pad and comprising at least one test zone comprising an immobilised hCG- binding ligand, and a control zone distal of the test zone comprising an immobilised ligand configured to bind the hCG-specific primary antibody, and a competitive analyte zone distal or proximal of the test zone comprising immobilised hCG; an adsorbent pad in fluid communication with a distal end of the detection membrane, wherein an aliquot of the liquid biological sample is applied to the sample pad, whereby the liquid biological sample is transferred by capillary action along a flow path defined by the sample pad, conjugate release pad, detection membrane, and adsorbent pad; and the level of the detectable label at the test zone, competitive analyte zone and control zone is detected to provide a detectable label fingerprint; characterised in that a processor is configured to perform the steps of: receiving the detectable label fingerprint; inputting the detectable label fingerprint into the linear regression computational model; generating an output comprising the determined level of hCG in the liquid biological sample; and optionally, graphically display the determined level of hCG on a display system.