Lateral flow assay (LFA) device and method

Abstract

A lateral flow assay (LFA) device (10) for testing the presence of a target analyte in a biofluid sample comprises a longitudinal backing (12) having a hybrid sample / conjugate pad (18), a testing membrane (26) downstream the hybrid sample / conjugate pad (18) and an absorbent pad (34) downstream the testing membrane (26) extending thereon. The hybrid sample / conjugate defines a first treated section (18a) comprising a protein precipitating agent (20), a second treated section (18b) comprising a target analyte specific conjugate agent (22), and a third treated section (18c) comprising a flow assisting agent (24). The biofluid sample is collected by the hybrid sample / conjugate pad which provides for laminar flow of the biofluid sample through the first, second and third treated sections in sequence. The test membrane comprises detection lines (28) comprising respective detection agent for detecting the presence of the target analyte in the biofluid sample flowing thereon from the hybrid sample / conjugate pad (18) and into the absorption pad (34).

Description

LATERAL FLOW ASSAY (LFA) DEVICE AND METHODCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority on United States Provisional Patent Application Serial Number 63 / 667,547 filed on July 3, 2024 and incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure generally relates to bodily fluid (biofluid) sample collection, testing and analysis. More specifically, but not exclusively the present disclosure relates to lateral flow assay (LFA) devices, systems, kits and methods.BACKGROUND

[0003] Biofluid sample collection, such as saliva sampling, is a common step for many analytical tests. This spans across a number of technical fields and is notably used in medical diagnostic tests. Human fluid samples may consist of a number of fluid types (saliva, blood, urine, mucus, etc.). Sample collection is conducted in a number of ways, whether by a medical professional sampling a patient or the patient being given the necessary tools to collect the sample themselves. This may be done with a swab, a container, or other similar devices. For example, a patient may be instructed to deposit urine into a container, which can then be transported to a laboratory for analysis. Samples are often analyzed using laboratory-grade tools and equipment, which are typically expensive. The common layperson does not usually have access to specialized equipment and isrequired to visit a clinic or hospital in order to receive a liquid sample test and analysis kit.

[0004] Point of care medicine is increasing the common person’s access to medical results. This field of medicine focuses on simple medical testing that can be conducted by a layperson in their home. At-home liquid sample collection devices will reduce the cost and wait time of a sample analysis, and additionally reduce the problems associated with the transportation and storage that is inherent to analyzing a sample using typical laboratory equipment and methods.

[0005] A common method of applying point of care medicine is through lateral flow assays (LFA) or lateral flow immunoassays, a known technology for the testing of the presence of a specific biological agent within the human body (i.e. Covid-19 test strips, pregnancy tests etc.). These tests are low-cost to manufacture, can be customized for very specific testing purposes, and do not require large liquid samples in order to generate a result. A lateral flow immunoassay is a simple diagnostic device that confirms the presence or absence of a specific analyte (biological agent). The principle of an LFA is based on the movement of a liquid sample across a polymeric strip with attached molecules (probes) that interact with the analyte, providing a signal that can be visually detected.

[0006] Immunoassays substantially share the same fundamental components: an analyte, basically the target biological agent to be detected (e.g. progesterone, cortisol, antigens, etc.); an analyte-specific capturing agent (e.g. antibody, aptamer, protein etc.) to capture or bind to the analyte; and a detectable label attached (or tagged) to the capturing agent (e.g. chromophore, fluorophore etc.). The analyte-specific capturing agent and the detectable label form a conjugate.

[0007] With reference to Figure 1 , a traditional LFA strip (A) is shown including a backing card (B) with a sample pad (C) mounted thereto at the upstream end (D) thereof for collecting a biofluid sample. The sample pad (C) partially overlies a downstream conjugate release pad (E). The conjugate release pad (E) is the second step in a lateral flow assay (LFA). It holds and preserves the detection agents, or conjugates. As the sample flows through the conjugate release pad, the conjugate is released into the sample and binds to the target analyte, if present. The sample and conjugate then flow together to the test membrane (F). The test membrane (F) is often a nitrocellulose membrane which includes the test line (G) and the control line (H). The test line (G) consists of immobilized proteins that can bind the conjugate to generate a signal that is correlated to the presence of the analyte in the sample. The control (H) line contains affinity ligands that will bind the conjugate with or without the analyte present in solution to confirm that the assay is working properly. Fluid is moved across the polymeric strip from the upstream end (D) towards the downstream end (I), as shown by arrow (J), to be collected at the absorbent or wicking pad (K).

[0008] Figure 2 shows the main steps of a traditional LFA architecture. At step (i), the liquid sample suspected of containing the analyte at the sample pad (C) begins to flow. At step (ii), the sample flows into the conjugate release pad (E) and conjugates (capturing agents with detectable tags) react with the sample. For example, labelled or tagged antibodies bind to the analyte and the resulting complexes are captured at the detection lines (i.e. the test line (G) and the control line (H)). At step (iii), the LFA reaction is complete, but unreacted analyte in the sample continues to flow towards the absorbent or wicking pad (K).OBJECTS

[0009] In an embodiment, an object of the present disclosure is to provide an LFA device for detecting the presence of a target analyte within a biofluid sample.

[0010] In an embodiment, an object of the present disclosure is to provide an LFA method providing for detecting the presence of a target analyte within a biofluid sampleSUMMARY

[0011] In accordance with an aspect of the present disclosure, there is provided a lateral flow assay (LFA) device for testing the presence of a target analyte in a biofluid sample, the LFA device comprising: a longitudinal backing defining an upstream end and a downstream end; a hybrid sample / conjugate pad for collecting the biofluid sample extending along the backing from the upstream end and defining a first treated section thereof comprising a protein precipitating agent, a second treated section thereof comprising a target analyte specific conjugate agent, and a third treated section thereof comprising a flow assisting agent; a testing membrane downstream the hybrid sample / conjugate pad and extending along the backing and comprising detection lines comprising respective detection agents; and an absorbent pad downstream the testing membrane and extending along the backing to the downstream end, wherein the biofluid sample when collected by the hybrid sample / conjugate pad is provided to flow therein through the first, second and third treated sections in sequence, the protein precipitating agent providing for precipitating non-target analyte proteins present in the biofluid sample, the conjugate agent providing for binding to the target analyte present in the biofluid sample to provide an analyte-conjugate complex, the flowassisting agent providing for assisting the biofluid sample to flow from the hybrid sample / conjugate pad along the testing membrane and into the absorption pad.

[0012] In an embodiment, the hybrid sample / conjugate pad provides for laminar flow of the biofluid sample therethrough.

[0013] In an embodiment, the first and second treated sections are spaced apart by a non-treated section of the hybrid sample / conjugate pad positioned therebetween.

[0014] In an embodiment, the second and third treated sections are spaced apart by a non-treated section of the hybrid sample / conjugate pad positioned therebetween.

[0015] In an embodiment, the protein precipitating agent comprises polyphenol. In an embodiment, the polyphenol comprises tannin. In an embodiment, the tannin comprises tannic acid.

[0016] In an embodiment, the flow assisting agent comprises salts and surfactants.

[0017] In an embodiment, the conjugate agent comprises an anti-target- analyte antibody and a colloidal gold nanoparticle conjugated to the anti-target- analyte antibody.

[0018] In an embodiment, the conjugate agent further comprises streptavidin conjugated to the colloidal gold nanoparticle.

[0019] In an embodiment, the conjugate agent further comprises biotin conjugated to the colloidal gold nanoparticle. In an embodiment, the biotin is linked to the colloidal gold nanoparticle via a polyethylene glycol (PEG) spacer.

[0020] In an embodiment, the detections lines comprise a first line and a second line downstream the first line, the first line comprising a first line detection agent and the second line comprising a second line detection agent. In an embodiment, the first line is a test line and the second line is a control line. In an embodiment, the first line detection agent comprises the target analyte. In an embodiment, the first line detection agent comprises a target analyte molecule linked to a base protein linked to the testing membrane. In an embodiment, the target analyte molecule is linked to the base protein via a polyethylene glycol (PEG) spacer. In an embodiment, the base protein comprises bovine serum albumin (BSA).

[0021] In an embodiment, the detection lines comprise a first line and a second line downstream the first line, the second line comprising a second line detection agent comprising biotin for binding to the streptavidin. In an embodiment, second line detection agent comprises a base protein linked to the testing membrane, the biotin being linked to the base protein. In an embodiment, the biotin is linked to the base protein via a polyethylene glycol (PEG) spacer. In an embodiment, the base protein comprises bovine serum albumin (BSA).

[0022] In an embodiment, the detection lines comprise a first line and a second line downstream the first line, the second line comprising a second line detection agent comprising streptavidin for binding to the biotin. In an embodiment, second line detection agent comprises a base protein linked to the testing membrane, the streptavidin being linked to the base protein. In an embodiment, the streptavidin is linked to the base protein via a polyethylene glycol (PEG) spacer. In an embodiment, the base protein comprises bovine serum albumin (BSA).

[0023] In an embodiment, the first line is a test line and the second line is a control line.

[0024] In accordance with an aspect of the present disclosure, there is provided a lateral flow assay (LFA) device for testing the presence of a target analyte in a biofluid sample, the LFA device comprising: a longitudinal backing defining an upstream end and a downstream end; a pad assembly extending along the backing from the upstream end, the pad assembly providing for collecting the biofluid sample and releasing a target analyte specific conjugate agent for binding to the target analyte present in the biofluid sample to provide an analyte-conjugate complex, the conjugate agent comprising an anti-target-analyte antibody and an additional binding molecule respectively conjugated to a marker, wherein the additional binding molecule is selected form the group consisting of: a protein having a high affinity for a vitamin or a vitamin having a high affinity for a protein; a testing membrane downstream the pad assembly and extending along the backing and comprising a first detection line comprising a first detection agent comprising an a target analyte molecule and a second line downstream the first line, the second line comprising a second line detection agent selected from the group consisting of: a second line vitamin having a high affinity for the protein of the conjugate agent; or a second line protein having a high affinity for the vitamin of the conjugate agent; an absorbent pad downstream the testing membrane and extending along the backing to the downstream end, wherein the biofluid sample collected by the pad assembly when collected is provided to flow therein and therefrom along the testing membrane and into the absorption pad.

[0025] In an embodiment, the protein of the conjugate agent comprises streptavidin and the second line vitamin comprises biotin. In an embodiment, the vitamin of the conjugate agent comprises biotin and the second line protein comprises streptavidin.

[0026] In an embodiment, the marker comprises a colloidal gold nanoparticle.

[0027] In an embodiment, the pad assembly provides for laminar flow of the biofluid sample therethrough.

[0028] In an embodiment, the pad assembly comprises a hybrid sample / conjugate pad for collecting the biofluid sample extending along the backing from the upstream end and defining a first treated section thereof comprising a protein precipitating agent, a second treated section thereof comprising a target analyte specific conjugate agent, and a third treated section thereof comprising a flow assisting agent.

[0029] In an embodiment, the protein precipitating agent comprises polyphenol. In an embodiment, the polyphenol comprises tannin. In an embodiment, the tannin comprises tannic acid.

[0030] In accordance with an aspect of the present disclosure, there is provided a lateral flow assay (LFA) method for testing the presence of a target analyte in a biofluid sample, the LFA method comprising: collecting the biofluid sample on an elongated hybrid sample / conjugate pad defining a first treated section thereof comprising a protein precipitating agent, a second treated section thereof comprising a target analyte specific conjugate agent, and a third treated section thereof comprising a flow assisting agent; providing for laminar flow of the biofluid sample through the first, second and third treated sections in sequence, the protein precipitating agent providing for precipitating non-target analyte proteins present in the biofluid sample, the conjugate agent providing for binding to the target analyte present in the biofluid sample to provide an analyte-conjugate complex, the flow assisting agent providing for assisting the biofluid sample to flow from the hybrid sample / conjugate pad along a testing membrane downstream the hybrid sample / conjugate pad and into the absorption pad downstream the testing membrane.

[0031] In accordance with an aspect of the present disclosure, there is provided a lateral flow assay (LFA) method for testing the presence of a target analyte in a biofluid sample, the LFA method comprising: collecting the biofluid sample on a pad assembly; releasing a target analyte specific conjugate agent by the pad assembly for binding to the target analyte present in the biofluid sample to provide an analyte-conjugate complex, the conjugate agent comprising an anti- target-analyte antibody and an additional binding molecule respectively conjugated to a marker, wherein the additional binding molecule is selected from the group consisting of: a protein having a high affinity for a vitamin or a vitamin having a high affinity for a protein; providing the biofluid sample to flow from the pad assembly to a testing membrane comprising a first detection line comprising a first detection agent comprising an a target analyte molecule and a second line downstream the first line, the second line comprising a second line detection agent selected from the group consisting of: a second line vitamin having a high affinity for the protein of the conjugate agent; or a second line protein having a high affinity for the vitamin of the conjugate agent.

[0032] In an embodiment, the protein of the conjugate agent comprises streptavidin and the second line vitamin comprises biotin. In an embodiment, the vitamin of the conjugate agent comprises biotin and the second line protein comprises streptavidin.

[0033] As used herein, the term “sample” refers to a volume of a specimen (e.g., a biofluid) taken for testing or analysis.

[0034] As used herein, the term “biofluid” refers to any biological fluid originating from a subject or patient. The biofluid can be, for example, saliva, sweat, tears, interstitial fluid, blood, urine, or the like. The biofluid can include water, an analyte, and one or more additional components.

[0035] As used herein, the term “analyte” refers to a substance whose chemical constituents are being identified and measured. The analyte can be a key biomarker for a certain medical condition that can be used to diagnose and / or monitor a subject’s health and wellness.

[0036] Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of illustrative embodiments thereof, given by way of example only with reference to the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In the appended drawings:

[0038] Figure 1 is a schematic representation of a prior art LFA device;

[0039] Figure 2 is a schematic representation of the architecture of the prior art LFA device of Figure 1 and of the assay steps;

[0040] Figure 3 is a schematic representation of the LFA device in accordance with a non-restrictive illustrative embodiment of the present disclosure;

[0041] Figure 4 is cross-sectional view of a device for collecting, transferring and testing biofluids including the LFA device of Figure 3 positioned therein in accordance with a non-restrictive illustrative embodiment of the present disclosure;

[0042] Figure 5 is a table showing the technical specifications of the absorbent hybrid sample / conjugate pad of the LFA device of Figure 3 in accordance with a non-restrictive illustrative embodiment of the present disclosure;

[0043] Figure 6 is a schematic representation of a conjugate in the hybrid sample and conjugate pad of the LFA device of Figure 3 in accordance with a non- restrictive illustrative embodiment of the present disclosure;

[0044] Figure 7A is a schematic representation of the test agent on the first or test line of the testing membrane of the LFA device of Figure 3 reacting with an analyte-conjugate complex in accordance with a non-restrictive illustrative embodiment of the present disclosure;

[0045] Figure 7B is a schematic representation of the test agent on the first or test line of the testing membrane of the LFA device of Figure 3 reacting with an analyte-conjugate complex in accordance with another non-restrictive illustrative embodiment of the present disclosure;

[0046] Figure 8A is a schematic representation of the control agent on the second or control line of the testing membrane of the LFA device of Figure 3 reacting with an analyte-conjugate complex in accordance with a non-restrictive illustrative embodiment of the present disclosure;

[0047] Figure 8B is a schematic representation of the control agent on the second or control line of the testing membrane of the LFA device of Figure 3 reacting with an analyte-conjugate complex in accordance with another non-restrictive illustrative embodiment of the present disclosure;

[0048] Figure 8C is a schematic representation of the control agent on the second or control line of the testing membrane of the LFA device of Figure 3 reacting with an analyte-conjugate complex in accordance with a further non-restrictive illustrative embodiment of the present disclosure;

[0049] Figure 9 is a schematic representation of the progressive and sequential laminar flow of a sample volume of biofluid though through components of the LFA device of Figure 3 at three time stamps (a), (b) and (c) illustrating assay steps in accordance with a non-restrictive illustrative embodiment of the present disclosure;

[0050] Figure 10 is a table showing technical specifications of the absorbent or wicking pad of the LFA device of Figure 3 in accordance with a non-restrictive illustrative embodiment of the present disclosure; and

[0051] Figure 1 1 is a graphic illustrating the intensity ratio signaled by the test of the present LFA device based on analyte concentration in the tested sample in accordance with a non-restrictive illustrative embodiment of the present disclosure.DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0052] Generally stated and in accordance with an aspect of the present disclosure, there is a lateral flow assay (LFA) device for testing the presence of a target analyte in a biofluid sample comprising a longitudinal backing having a hybrid sample / conjugate pad, a testing membrane downstream the hybrid sample / conjugate pad and an absorbent pad downstream the testing membrane respectively extending along the length of the backing. The hybrid sample / conjugate defines a first treated section comprising a protein precipitating agent, a second treated section comprising a target analyte specific conjugate agent, and a third treated section comprising a flow assisting agent. The biofluid sample is collected by the hybrid sample / conjugate pad which provides for laminar flow of the biofluid sample through the first, second and third treated sections in sequence. The protein precipitating agent precipitates non-target analyte proteins present in the biofluid sample. The conjugate agent binds to the target analyte present in the biofluidsample providing an analyte-conjugate complex. The flow assisting agent assists the biofluid sample to flow from the hybrid sample / conjugate pad along the testing membrane and into the absorption pad. The test membrane comprises detection lines comprising respective detection agents for detecting the presence of the target analyte.

[0053] With reference to Figure 3, there is shown a lateral flow assay (LFA) testing device 10 in accordance with a non-limiting illustrative embodiment of the present disclosure.

[0054] In a non-restrictive illustrative embodiment, the LFA device 10 can be used in conjunction with a device 1 (shown in Figure 4) for collecting, transferring and testing biofluids as described, for example, in International Patent Application Number PCT / CA2024 / 050960 filed on July 19th, 2024 and incorporated herein by reference in its entirety. In the example of Figure 4, device 1 comprises a housing 3 defining upstream 5 and downstream 7 ends and a passage 9 therebetween. An opening 11 at the upstream end 3 is in fluid communication with passage 9. A transfer element 13 is movably connected to the housing 3 along the passage 9 and comprises an upstream portion 15 outwardly protruding from the upstream opening 1 1 . A collection element 17 is positioned at the upstream portion 15 of the transfer element 13 for collecting biofluid. Compression elements 19 are positioned along the passage 9 for compressing the collection element 17 to release the absorbed biofluid. A downstream portion 21 of the transfer element 13 protrudes outwardly of the housing via a downstream opening 23 at downstream end 7. Imparting a movement to the transfer element 13 in a downstream direction by pulling on the downstream portion 23 moves the collection element 17 with collected biofluid into the housing 3 (as shown by arrow 25) via the upstream opening 1 1 providing engagement of the collection element 17 by the compression elements 19 forcompression thereof and concurrent release of the biofluid onto the testing device 10 interfacing with the collection element 19 during compression.

[0055] Returning to Figure 3, device 10 comprises a longitudinal backing 12 such as a backing card. The longitudinal backing 12 defines an upstream end 14 and a downstream end 16. In one non-limiting example, the backing card 12 is a strip of about 66 mm wide and about 30 cm long and holds the assay components provided herein together with an adhesive.

[0056] Device 10 comprises a hybrid sample / conjugate element 18 in the form of a pad mounted at the upstream end 14 of the backing 12. In a non-limiting example, the biofluid sample will be collected on pad 18 directly from a collection element. In one embodiment, the pad 18 is thick, with low density and high- absorptivity. The material of the pad 18 that is of low density and high absorptivity. The material for pad 18 is suitable for sample application, it does not crumble / disintegrate upon sample application. A non-limiting example of the material that can be used for the hybrid pad 18 is detailed in the table shown in Figure 5. Typically, the sample and conjugate pads are separate elements as shown in Figure 1. In the present disclosure pad 18 is both a sample collecting and conjugate releasing element. Traditional LFA conjugate pads allow for fast, even release of the conjugate whereas a thick highly absorptive material provides for slow release of the conjugate. In an embodiment, pad 18 is spongy, containing a large amount of air providing a much greater surface area for sample flow, thereby slowing down flow. The sponge is large enough to hold a large volume of saliva (which means a large amount of analyte). Therefore pad 18 provides for containing a large volume of saliva as well as slowing down the flow of the conjugate.

[0057] The hybrid sample conjugate pad 18 comprises three separate and spaced apart treated sections 18a, 18b and 18c.

[0058] The first treated section 18a contains a protein precipitating agent 20. In an embodiment, the protein precipitating agent comprises a polyphenol. In an embodiment, the polyphenol is a tannin. In an embodiment, the tannin comprises tannic acid. The role of the protein precipitating agent is to remove unwanted proteins from the saliva sample thereby increasing the relative presence of the target analyte. The second treated section 18b is downstream the first section 18b and spaced apart therefrom, defining an untreated pad section 18i therebetween. Section 18b contains a conjugate 22. The conjugate 22 is analyte specific. The third treated section 18c is downstream the second section 18b and spaced apart therefrom defining untreated pad section 18ii therebetween. Section 18c contains a flow assisting agent 24 such as salts and surfactants.

[0059] In an embodiment, the present disclosure provides for tannic acid pretreatment separate from the salts and surfactants. Tannic acid causes surfactants to polymerize, and it itself polymerizes in the presence of NaCI. By including the tannic acid 20 at the beginning of the test in section 18a, it acts on salivary proteins first to avoid unwanted reactions with the salt and surfactant 24 in section 18c. Polyphenolic compounds can precipitate proteins in a manner similar to tannic acid. Other clarifying or precipitating agents (e.g. flocculants) are generally too harsh on the assay chemistry. For example, these other agents denature saliva proteins but also denature the antibodies on a gold nanoparticle-antibody conjugate (discussed further below), rendering the assay useless. Moreover, these other agents cause gold aggregation due to their strong charge, for example SDS (strong negative charge) or quaternary ammonium compounds (strong positive charge). A sodium dodecyl sulfate (SDS) can influence the aggregation of gold nanoparticles (AuNPs) in solution. While SDS is often used as a stabilizing agent to prevent aggregation due to its ability to adsorb onto the surface of nanoparticles and create a steric or electrostatic barrier, it can also induce aggregation under certainconditions. The effect of SDS on AuNP aggregation depends on factors like SDS concentration, solution pH, and the presence of other ions.

[0060] In accordance with a non-limiting embodiment and with reference to Figure 6, the conjugate 22 comprises a gold nanoparticle-antibody conjugate (“GC”) formed by a colloidal gold nanoparticle passively conjugated to monoclonal mouse anti-progesterone antibodies and streptavidin proteins. In an embodiment, the antibody was raised against Progesterone-1 1 -HS-BSA. HS is hemisuccinyl. BSA is bovine serum albumin. In one example, the colloidal gold nanoparticle has a diameter of about 40nm. Of course, other conjugates can be provided for coupling to other analytes and these are within the realm of the skilled artisan.

[0061] In an embodiment, the flow assisting agent 24 comprises 2.5M NaCI, 5% (v / v) Tween-20, 0.5M HEPES, pH 7.4, sprayed at 12 uL / cm. These agents serve to standardize sample pH (HEPES) and to further condition the saliva sample (Tween-20, NaCI).

[0062] The separation of the salts and surfactants from the tannic acid solution is achieved by spraying each agent in separate steps and at separate locations on the pad 18.

[0063] Indeed, agents 20, 22 and 24 are liquids sprayed onto their appropriate sections 18a, 18b and 18c.

[0064] A testing membrane 26 is positioned on the backing 12 downstream the hybrid pad 18 and includes the detection lines 28, namely a first line 30 and a second line 32 downstream the first line 30. In an embodiment, the first line 30 is a test line comprising a test line agent and the second line 32 is a control line comprising a control line agent. In an embodiment, the testing membrane 26 is anitrocellulose membrane. In an embodiment, the nitrocellulose membrane is a UniSart GN 95 (Sartorius) membrane of about 25mm in width.

[0065] In an embodiment test line agent comprises a target analyte molecule similar to the target analyte in the user sample. In an embodiment, the target analyte molecule is linked to a base protein that is linked to the testing membrane.

[0066] In a non-limiting example and with reference to Figures 3, 6 and 7A, the target analyte molecule is progesterone (P4) and the base protein is bovine serum albumin (BSA). As such, the test line agent is Progesterone-3-CMO-BSA. The position of BSA conjugation (3-CMO) was formulated for efficient performance with the capturing antibody of conjugate 22 (provided in the non-limiting example shown in Figure 6). Thus, BSA is conjugated to progesterone at the 3 position of GMO. Commonly with progesterone, it can be either conjugated with BSA at the 3 position or the 1 1 position. Free progesterone in saliva competes (for binding to the conjugate GC) with progesterone-3-CMO-BSA immobilized on the test line area 30. In the context of "Progesterone-3-CMO-BSA", the "3" indicates that the carboxymethyl oxime group (GMO) is attached to the 3rd carbon atom of the progesterone molecule. This specific attachment point is crucial for the function and use of this modified progesterone molecule in various assays and studies.

[0067] In an embodiment, the control line agent comprises biotin and the conjugate agent comprises streptavidin (see Figure 8A). In an embodiment, the control line agent comprises streptavidin and the conjugate agent comprises biotin (see Figures 8B and 8C). In an embodiment, the streptavidin or the biotin can be linked to the gold colloidal nanoparticle via a PEG spacer. In an embodiment, the control line agent comprises a base protein to which the streptavidin or the biotin are directly linked or linked thereto via a PEG spacer. In an embodiment, the base protein comprises BSA.

[0068] In an embodiment and with reference to Figures 3, 6 and 8A, the control line agent is Biotin-PEGi2-BSA. Streptavidin binds to Biotin-PEGi2-BSA on the control line area 32. The use of biotin-streptavidin binding provides efficacious assay sensitivity and is especially applicable for the complex saliva sample matrix. The binding between the streptavidin and biotin is greater than the binding between an antigen and an antibody.

[0069] In an embodiment and the locations of the streptavidin and the biotin can be flipped in the sense that the conjugate contains the biotin and the control line agent contains the streptavidin. The foregoing is shown in Figure 8B, where the conjugate agent 22’ is shown comprising the colloidal gold nanoparticle (GC) conjugated to the antibody and further conjugated to biotin. The control line 32’ comprises a base protein (e.g. BSA) linked to the testing membrane 26 with streptavidin linked thereto. In an embodiment, the streptavidin is linked to the BSA via a PEG spacer. Thus, in this non-limiting example, the control line 32’ comprises Streptavidin-PEGi2-BSA.

[0070] Turning the Figure 8C, there is no PEG spacer between the streptavidin and the BSA and as such, the control line 32” comprises Streptavidin- BSA. In this non-limiting example, the conjugate agent 22” comprises a biotin linked to the gold colloidal nanoparticle via a PEG spacer.

[0071] The PEG12 spacer provides increased assay sensitivity by facilitating the binding of streptavidin and biotin. The control line 32 or 32’ captures excess gold nanoparticle-antibody conjugates that were not captured by the test line 30, regardless of interaction with progesterone from the user. The control line agent captures the gold nanoparticle-antibody conjugates with a stronger affinity than the test line agent. This provides for a more complete LFA reaction, yielding more accurate results. In an embodiment, the range of the PEG (polyethylene glycol)spacer may be between 4 and 30 (i.e., PEG4 to PEG30). In an embodiment, the range of the PEG spacer may be between 4 and 12 (i.e., PEG4 to PEG12). In an embodiment, the range of the PEG spacer may be between 4 and 1 1 or 4-10 or 4- 9, or 4-8, or 4-7, or 4-6. In an embodiment, the PEG spacer is PEG4. In an embodiment, the PEG spacer is PEGs. In an embodiment, the PEG spacer is PEGe. In an embodiment, the PEG spacer is PEG?. In an embodiment, the PEG spacer is PEGs. In an embodiment, the PEG spacer is PEG9. In an embodiment, the PEG spacer is PEG10. In an embodiment, the PEG spacer is PEG11. In an embodiment, the PEG spacer is PEG12.

[0072] In an embodiment and with reference to Figures 3, 6, and 7B the test line 30 includes a PEG spacer between the base line and the hormone. Thus, the test line 30 comprises in this example Progesterone-3-CMO-PEGi2-BSA. The PEG spacer avoids for the hormones to be too densely packed or crowded near the base line which hinders the antibodies from accessing all of the hormones. This is due to steric hinderance where the antibodies repel each other. As such, by adding the PEG spacer, the hormones are provided with more freedom to bind with the antibodies thereby increasing sensitivity of the LFA. In an embodiment, the range of the PEG spacer may be between 4 and 30 (i.e., PEG4 to PEG30). In an embodiment, the range of the PEG spacer may be between 4 and 12 (i.e., PEG4 to PEG12). In an embodiment, the range of the PEG spacer may be between 4 and 1 1 or 4-10 or 4-9, or 4-8, or 4-7, or 4-6. In an embodiment, the PEG spacer is PEG4. In an embodiment, the PEG spacer is PEGs. In an embodiment, the PEG spacer is PEGe. In an embodiment, the PEG spacer is PEG?. In an embodiment, the PEG spacer is PEGs. In an embodiment, the PEG spacer is PEG9. In an embodiment, the PEG spacer is PEG10. In an embodiment, the PEG spacer is PEG11. In an embodiment, the PEG spacer is PEG12.

[0073] An absorbent pad 34 is positioned at the downstream end 16 of the backing 12. A typical cotton absorbent pad is used, and non-limiting examples are provided in the table shown in Figure 10. In one non-limiting example, the absorbent pad 34 comprises Ahlstrom™ grade 222. The absorbent pad provides for continuously pulling sample across the device 10 in the downstream direction.

[0074] In a traditional LFA test (Figures 1 and 2) the sample pulls all of the reagents (agents having reacted with the sample) with it which leaves behind uncaptured analytes (see step ill in Figure 2). In contrast, the spongy material of pad 18 provides for the sample to flow through the assay in a manner analogous to a chromatography column advantageously providing for greater sensitivity. Moreover, the tannic acid acts on the saliva sample, which is a complex mixture of proteins and salts along with the target analyte, so that unwanted proteins are precipitated out of the saliva sample providing a purer sample allowing for a one step LFA test. Tannic acid precipitates out large proteins that are in the mucus but does not destroy hormones or antibodies thereby “purifying” the sample.

[0075] The control line 32 of the present LFA system is not a control line in the traditional sense. Traditionally, a control line is a constant indicator of flow, whereas in the present LFA system, there are two detection lines 30, 32 that are changing with respect to each other. The second detection line 32 is based on a biotin-streptavidin interaction. This provides greater sensitivity. The first line 30 is related to the analyte and changes based on how much of the analyte is in the sample whereas the second line 32 is not directly related to the analyte concentration but changes based on how much analyte is captured by the first line 30. Indeed, the first line 30 starts out darker and gets fainter as it captures more analyte, whereas the second line 32 starts out fainter and gets darker (as the first line 30 captures more analyte). Therefore, there is an inverse relationship between lines 30 and 32. In the competitive LFA system provided herein, the first line 30includes a first agent that mimics the target analyte, therefore if there is very little analyte in the user sample, the conjugate 22 will bind to analyte-like molecules on the first line 30 making the line darker. As the conjugate 22 is captured by the first line 30 it will not flow to the second line 32 to be captured thereby and as such, this second line 32 will remain faint. If the target analyte is present, the analyte will bind to conjugate 22 preventing the conjugate 22 from binding to the analyte-like molecule on the first line 30 and as such, the first line 30 will be fainter. The second line 32 captures the analyte-conjugate complex via the biotin-streptavidin interaction making the line 32 darker as the concentration of the captured analyte-conjugate complex increases. As such, the detection lines 28 provided herein allow for a more accurate test result via the inverse confirmatory relationship between the lines 30, 32 as described above.

[0076] Turning to Figure 9, the sample flow within device 10 will be further described. A sample volume SV is shown at step (a) being transferred from a collection element to device 10. Indeed, the sample volume SV is a continuous flow but since this flow is advantageously laminar (slow, no mixing) it can be broken up into many small volumes (SVi, SV2, SV3, SV4 etc.) There are generally two types of fluid flow, laminar flow and turbulent flow. In laminar flow, the fluid flows in smooth layers or lamina. This occurs when adjacent fluid layers slide smoothly over one another with mixing between layers or lamina occurring predominantly on a molecular level by diffusion. Turbulent flow is characterized by fluctuations of the velocity of the fluid in both space and time. Mixing of two or more substances in turbulent flow conditions generally proceeds faster than under laminar flow conditions.

[0077] The pad 18 configuration (density, absorptivity, thickness, sponge-like structure filled with holes) advantageously provides for a continued laminar flow of the sample with delayed / gradual release of the conjugate. In a traditional LFA, mostof the conjugate is released at once, in the present system the conjugate is advantageously slowly released being provided with the opportunity of interacting and binding with a greater sample volume and therefore more analyte.

[0078] At step (b) the sample begins flowing through pad 18 and the target analyte in the sample reacts with tagged capturing agent (i.e. the conjugate) of the LFA device 10. Unlike traditional LFA systems where the sample moves quickly pulling the conjugate with it, in the present LFA device 10, the conjugate 22 advantageously stays intact and section 18b acts as a “mini-incubator”. Sensitivity is increased because at section 18b, the conjugate is moving slower than the sample, so a relatively high local concentration of conjugate is continuously present at section 18b for interaction with the sample volumes that continue to flow. At section 18b, the conjugate is advantageously also provided with a sufficient volume of “purified” saliva and with sufficient time to bind with the analyte and the pad 18 configuration eventually provides for successively releasing analyte-conjugate complexes to section 18c and then to the testing membrane 26. Therefore, by the time analyte-conjugate complex volumes are captured by the detection lines 28, enough sample volume and reaction time has been provided within pad 18 thereby maximizing the concentration of the target volume and optimizing the detection test. In step (b) of Figure 9, a sample volume SV4 is advantageously experiencing the same high local concentration of conjugate at section 18b that was experienced by SVi.

[0079] As previously explained, the first section 18a provides an advantageous protein precipitating agent, a non-limiting example being tannic acid, that purifies the sample by removal of unwanted proteins, the second section 18b provides the conjugate agent that binds to the analyte in the sample and the third section 18c provides flow assisting agents that provide for the reacted sample (containing analyte-conjugate complexes) to flow though the rest of the LFA device10. The agents “stay in place” in so far as they flow very slowly with the sample. Indeed, the laminar flow ensures that the sample moves through the agents 20, 22 and 24 rather than with the agents which results in a longer reaction time that is much more sensitive. The foregoing is provided by the spongy pad 18 which contains a large amount of air providing a much greater surface area for sample flow and as such the sample is not forced to carry the reagents (agents having reacted with the sample) with it in a quick manner but flows slowly through the spongy material and through the agents rather than with the agents as shown in step (b).

[0080] Moreover, by the time the purified sample analyte bound to the conjugate reaches section 18c, the tannic acid will not have reacted with the salts and surfactants 24 because it has already reacted with proteins in the saliva and is relatively inactive by the time it encounters the conjugate and then the salts and surfactants Hence, the action of each agent 20, 22 and 24 at each separate section 18a, 18b and 18c benefits from their slow release from the pad 18.

[0081] The present LFA test provides for a significant change in the detection lines 28 with a small amount of analyte as shown in the graphic of Figure 11 where the intensity ratio between the detection lines is very responsive to low concentrations of the analyte (e.g. progesterone). The detection line intensity is visually read and / or captured and the intensity ratio therebetween is calculated by a calibration curve providing a test result. The inverse relationship between the detection lines advantageously provides for a higher resolution in the assay as two directly related parameters are provided to determine the ratio between the two lines. The greater the ratio, the greater the concentration of the target analyte.

[0082] With reference to step (c) of Figure 9, the absorbent pad 34 pulls through the sample volume SV which is plentiful at the beginning of the process butis reduced as more sample volume is absorbed by the absorbent pad 34 and by this time the absorbent pad 34 begins to pull through the reagents (the agents 20, 22, 24 having reacted with the sample) in the pad 18 that have reacted with the analyte as provided herein (i.e. reacted sample volume or RSV) in sequential small volumes as they are released in this manner by the pad 18 and it is at this point that line development begins to show at the testing membrane 26. Line development begins as soon as the first RSV hits test line 30, yet due to the fact that the flow out of pad 18 of agents 20, 22 of pad 18, the line development is much slower than a standard LFA in which agents are released quickly with sample flow. As such, in each RSV there is only a small amount of agents 20, 22, 24, we see very slow line development when comparing to a traditional LFA line development.

[0083] In light of the description herein and in accordance with an embodiment of the present disclosure, there is provided a lateral flow assay (LFA) device for testing the presence of a target analyte in a biofluid sample.

[0084] With reference to Figure 3, the LFA device 10 comprises a longitudinal backing 12 , a pad assembly 18, a testing membrane 26 and an absorbent pad 34.

[0085] The longitudinal backing 12 defines an upstream end 16 and a downstream end 16.

[0086] The pad assembly 18 extends along the backing 12 from the upstream end 14. The pad assembly 18 providing for collecting the biofluid sample and releasing a target analyte specific conjugate agent for binding to the target analyte present in the biofluid sample to provide an analyte-conjugate complex.

[0087] With reference to Figures 6, and 7A-8C, the conjugate agent 22, 22’, or 22” comprises an anti-target-analyte antibody and an additional binding molecule respectively conjugated to a marker.

[0088] In an embodiment, the additional binding molecule is selected form the group consisting of a protein having a high affinity for a vitamin or a vitamin having a high affinity for a protein.

[0089] With reference to Figures 3 and 7A-8C, the testing membrane 26 is downstream the pad assembly 18 extending along the backing 12 and comprising a first detection line (30, 30’, 30”) comprising a first detection agent comprising a target analyte molecule and a second line downstream the first lin. The second line comprises a second line detection agent selected from the group consisting of second line vitamin having a high affinity for the protein of the conjugate agent or a second line protein having a high affinity for the vitamin of the conjugate agent.

[0090] In an embodiment, the target analyte molecule is similar to the target analyte. Indeed, the target analyte is naturally occurring and the target analyte molecule is industrially produced.

[0091] The absorbent pad 34 is downstream the testing membrane 26 and extending along the backing 12 to the downstream end 16.

[0092] When the biofluid sample is collected by the pad assembly 18 it flows therein and therefrom along the testing membrane 26 and into the absorption pad 34.

[0093] In an embodiment, the protein of the conjugate agent comprises streptavidin and the second line vitamin comprises biotin.

[0094] In an embodiment, the vitamin of the conjugate agent comprises biotin and the second line protein comprises streptavidin.

[0095] In an embodiment, the marker comprises a colloidal gold nanoparticle.

[0096] In an embodiment, the pad assembly 18 provides for laminar flow of the biofluid sample therethrough.

[0097] In an embodiment, the pad assembly 18 comprises a hybrid sample / conjugate pad for collecting the biofluid sample extending along the backing 12 from the upstream end 14 and defining a first treated section 18a thereof comprising a protein precipitating agent 20, a second treated section 18b thereof comprising a target analyte specific conjugate agent 22, and a third treated section 18c thereof comprising a flow assisting agent 24.

[0098] The various features described herein can be combined in a variety of ways within the context of the present disclosure so as to provide still other embodiments. As such, the embodiments are not mutually exclusive. Moreover, the embodiments discussed herein need not include all of the features and elements illustrated and / or described and thus partial combinations of features can also be contemplated. Furthermore, embodiments with less features than those described can also be contemplated. It is to be understood that the present disclosure is not limited in its application to the details of construction and parts illustrated in the accompanying drawings and described hereinabove. The disclosure is capable of other embodiments and of being practiced in various ways. It is also to be understood that the phraseology or terminology used herein is for the purpose of description and not limitation. Hence, although the present disclosure has been provided hereinabove by way of non-restrictive illustrative embodiments thereof, it can be modified, without departing from the scope, spirit and nature thereof and of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A lateral flow assay (LFA) device for testing the presence of a target analyte in a biofluid sample, the LFA device comprising: a longitudinal backing defining an upstream end and a downstream end; a hybrid sample / conjugate pad for collecting the biofluid sample extending along the backing from the upstream end and defining a first treated section thereof comprising a protein precipitating agent, a second treated section thereof comprising a target analyte specific conjugate agent, and a third treated section thereof comprising a flow assisting agent; a testing membrane downstream the hybrid sample / conjugate pad and extending along the backing and comprising detection lines comprising respective detection agents; and an absorbent pad downstream the testing membrane and extending along the backing to the downstream end, wherein the biofluid sample when collected by the hybrid sample / conjugate pad is provided to flow therein through the first, second and third treated sections in sequence, the protein precipitating agent providing for precipitating non-target analyte proteins present in the biofluid sample, the conjugate agent providing for binding to the target analyte present in the biofluid sample to provide an analyte-conjugate complex, the flow assisting agent providing for assisting the biofluid sample to flow from the hybrid sample / conjugate pad along the testing membrane and into the absorption pad.

2. The LFA device according to claim 1 , wherein the hybrid sample / conjugate pad provides for laminar flow of the biofluid sample therethrough.

3. The LFA device according to any one of claims 1 or 2, wherein the first and second treated sections are spaced apart by a non-treated section of the hybrid sample / conjugate pad positioned therebetween.

4. The LFA device according to any one of claims 1 to 3, wherein the second and third treated sections are spaced apart by a non-treated section of the hybrid sample / conjugate pad positioned therebetween.

5. The LFA device according to any one of claims 1 to 4, wherein the protein precipitating agent comprises polyphenol.

6. The LFA device according to claim 5, wherein the polyphenol comprises tannin.

7. The LFA device according to claim 6, wherein the tannin comprises tannic acid.

8. The LFA device according to any one of claims 1 to 7, wherein the flow assisting agent comprises salts and surfactants.

9. The LFA device according to any one of claims 1 to 8, wherein the conjugate agent comprises an anti-target-analyte antibody and a colloidal gold nanoparticle conjugated to the anti-target-analyte antibody.

10. The LFA device according to claim 9, wherein the conjugate agent further comprises streptavidin conjugated to the colloidal gold nanoparticle.

11. The LFA device according to claim 9, wherein the conjugateagent further comprises biotin conjugated to the colloidal gold nanoparticle.

12. The LFA device according to claim 1 1 , wherein the biotin is linked to the colloidal gold nanoparticle via a polyethylene glycol (PEG) spacer.

13. The LFA device according to any one of claims 1 to 9, wherein the detections lines comprise a first line and a second line downstream the first line, the first line comprising a first line detection agent and the second line comprising a second line detection agent.

14. The LFA device according to claim 13, wherein the first line is a test line and the second line is a control line.

15. The LFA device according to any one of claims 13 or 14, wherein the first line detection agent comprises the target analyte.

16. The LFA device according to claim 15, wherein the first line detection agent comprises a target analyte molecule linked to a base protein linked to the testing membrane.

17. The LFA device according to claim 16, wherein the target analyte molecule is linked to the base protein via a polyethylene glycol (PEG) spacer.

18. The LFA device according to any one of claims 16 or 17, wherein the base protein comprises bovine serum albumin (BSA).

19. The LFA device according to claim 10, wherein the detectionlines comprise a first line and a second line downstream the first line, the second line comprising a second line detection agent comprising biotin for binding to the streptavidin.

20. The LFA device according to claim 19, wherein second line detection agent comprises a base protein linked to the testing membrane, the biotin being linked to the base protein.

21. The LFA device according to claim 20, wherein the biotin is linked to the base protein via a polyethylene glycol (PEG) spacer.

22. The LFA device according to any one of claims 20 or 21 , wherein the base protein comprises bovine serum albumin (BSA).

23. The LFA device according to any one of claims 1 1 or 12, wherein the detection lines comprise a first line and a second line downstream the first line, the second line comprising a second line detection agent comprising streptavidin for binding to the biotin.

24. The LFA device according to claim 23, wherein the second line detection agent comprises a base protein linked to the testing membrane, the streptavidin being linked to the base protein.

25. The LFA device according to claim 24, wherein the streptavidin is linked to the base protein via a polyethylene glycol (PEG) spacer.

26. The LFA device according to any one of claims 24 or 25, wherein the base protein comprises bovine serum albumin (BSA).

27. The LFA device according to any one of claims 19 to 25, wherein the first line is a test line and the second line is a control line.

28. A lateral flow assay (LFA) device for testing the presence of a target analyte in a biofluid sample, the LFA device comprising: a longitudinal backing defining an upstream end and a downstream end; a pad assembly extending along the backing from the upstream end, the pad assembly providing for collecting the biofluid sample and releasing a target analyte specific conjugate agent for binding to the target analyte present in the biofluid sample to provide an analyte-conjugate complex, the conjugate agent comprising an anti-target-analyte antibody and an additional binding molecule respectively conjugated to a marker, wherein the additional binding molecule is selected form the group consisting of:- a protein having a high affinity for a vitamin or- a vitamin having a high affinity for a protein; a testing membrane downstream the pad assembly and extending along the backing and comprising a first detection line comprising a first detection agent comprising a target analyte molecule and a second line downstream the first line, the second line comprising a second line detection agent selected from the group consisting of:- a second line vitamin having a high affinity for the protein of the conjugate agent; or- a second line protein having a high affinity for the vitamin of the conjugate agent; an absorbent pad downstream the testing membrane and extending along the backing to the downstream end,wherein the biofluid sample collected by the pad assembly when collected is provided to flow therein and therefrom along the testing membrane and into the absorption pad.

29. The LFA device according to claim 28, wherein the protein of the conjugate agent comprises streptavidin and the second line vitamin comprises biotin.

30. The LFA device according to claim 29, wherein the vitamin of the conjugate agent comprises biotin and the second line protein comprises streptavidin.

31. The LFA device according to any one of claims 28 to 30, wherein the marker comprises a colloidal gold nanoparticle.

32. The LFA device according to any one of claims 28 to 31 , wherein the pad assembly provides for laminar flow of the biofluid sample therethrough.

33. The LFA device according to any one of claims 28 to 32, wherein the pad assembly comprises a hybrid sample / conjugate pad for collecting the biofluid sample extending along the backing from the upstream end and defining a first treated section thereof comprising a protein precipitating agent, a second treated section thereof comprising a target analyte specific conjugate agent, and a third treated section thereof comprising a flow assisting agent.

34. The LFA device according to claim 33, wherein the protein precipitating agent comprises polyphenol.

35. The LFA device according to claim 34, wherein the polyphenol comprises tannin.

36. The LFA device according to claim 35, wherein the tannin comprises tannic acid.

37. A lateral flow assay (LFA) method for testing the presence of a target analyte in a biofluid sample, the LFA method comprising: collecting the biofluid sample on an elongated hybrid sample / conjugate pad defining a first treated section thereof comprising a protein precipitating agent, a second treated section thereof comprising a target analyte specific conjugate agent, and a third treated section thereof comprising a flow assisting agent; providing for laminar flow of the biofluid sample through the first, second and third treated sections in sequence, the protein precipitating agent providing for precipitating non-target analyte proteins present in the biofluid sample, the conjugate agent providing for binding to the target analyte present in the biofluid sample to provide an analyte-conjugate complex, the flow assisting agent providing for assisting the biofluid sample to flow from the hybrid sample / conjugate pad along a testing membrane downstream the hybrid sample / conjugate pad and into the absorption pad downstream the testing membrane.

38. The LFA method according to claim 37, wherein the first and second treated sections are spaced apart by a non-treated section of the hybrid sample / conjugate pad positioned therebetween.

39. The LFA method according to any one of claims 37 or 38,wherein the second and third treated sections are spaced apart by a non-treated section of the hybrid sample / conjugate pad positioned therebetween.

40. The LFA method according to any one of claims 37 to 39, wherein the protein precipitating agent comprises polyphenol.41 . The LFA method according to claim 40, wherein the polyphenol comprises tannin.

42. The LFA method according to claim 41 , wherein the tannin comprises tannic acid.

43. The LFA method according to any one of claims 37 to 42, wherein the flow assisting agent comprises salts and surfactants.

44. The LFA method according to any one of claims 37 to 43, wherein the conjugate agent comprises an anti-target-analyte antibody and a colloidal gold nanoparticle conjugated to the anti-target-analyte antibody.

45. The LFA method according to claim 44, wherein the conjugate agent further comprises streptavidin conjugated to the colloidal gold nanoparticle.

46. The LFA method according to claim 44, wherein the conjugate agent further comprises biotin conjugated to the colloidal gold nanoparticle.

47. The LFA device according to claim 46, wherein the biotin is linked to the colloidal gold nanoparticle via a polyethylene glycol (PEG) spacer.

48. The LFA method according to any one of claims 37 to 44, wherein the detections lines comprise a first line and a second line downstream the first line, the first line comprising a first line detection agent and the second line comprising a second line detection agent.

49. The LFA method according to claim 48, wherein the first line is a test line and the second line is a control line.

50. The LFA method according to any one of claims 48 or 49, wherein the first line detection agent comprises the target analyte.

51. The LFA method according to claim 50, wherein the first line detection agent comprises a target analyte molecule linked to a base protein linked to the testing membrane.

52. The LFA method according to claim 51, wherein the target analyte molecule is linked to the base protein via a polyethylene glycol (PEG) spacer.

53. The LFA method according to any one of claims 51 or 52, wherein the base protein comprises bovine serum albumin (BSA).

54. The LFA method according to claim 45, wherein the detection lines comprise a first line and a second line downstream the first line, the second line comprising a second line detection agent comprising biotin for binding to the streptavidin.

55. The LFA method according to claim 54, wherein the secondline detection agent comprises a base protein linked to the testing membrane, the biotin being linked to the base protein.

57. The LFA method according to claim 56, wherein the biotin is linked to the base protein via a polyethylene glycol (PEG) spacer.

58. The LFA method according to any one of claims 56 or 57, wherein the base protein comprises bovine serum albumin (BSA).

59. The LFA method according to any one of claims 46 or 47, wherein the detection lines comprise a first line and a second line downstream the first line, the second line comprising a second line detection agent comprising streptavidin for binding to the biotin.

60. The LFA method according to claim 59, wherein second line detection agent comprises a base protein linked to the testing membrane, the streptavidin being linked to the base protein.61 . The LFA method according to claim 60, wherein the streptavidin is linked to the base protein via a polyethylene glycol (PEG) spacer.

62. The LFA method according to any one of claims 60 or 61 , wherein the base protein comprises bovine serum albumin (BSA).

63. The LFA method according to any one of claims 48 to 62, wherein the first line is a test line and the second line is a control line.

64. A lateral flow assay (LFA) method for testing the presence of atarget analyte in a biofluid sample, the LFA method comprising: collecting the biofluid sample on a pad assembly; releasing a target analyte specific conjugate agent by the pad assembly for binding to the target analyte present in the biofluid sample to provide an analyte-conjugate complex, the conjugate agent comprising an anti-target- analyte antibody and an additional binding molecule respectively conjugated to a marker, wherein the additional binding molecule is selected form the group consisting of:- a protein having a high affinity for a vitamin or- a vitamin having a high affinity for a protein; providing the biofluid sample to flow from the pad assembly to a testing membrane comprising a first detection line comprising a first detection agent comprising a target analyte molecule and a second line downstream the first line, the second line comprising a second line detection agent selected from the group consisting of:- a second line vitamin having a high affinity for the protein of the conjugate agent; or- a second line protein having a high affinity for the vitamin of the conjugate agent.

65. The LFA method according to claim 64, wherein the protein of the conjugate agent comprises streptavidin and the second line vitamin comprises biotin.

66. The LFA device according to claim 64, wherein the vitamin of the conjugate agent comprises biotin and the second line protein comprises streptavidin.

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