Lateral flow assays and methods for detecting high concentrations of analytes

Abstract

The sandwich lateral flow assay devices, kits, systems and methods described herein include antibody-conjugated ultra-large particles that bind to target analytes in a sample and are retained upstream of a capture zone when a fluid sample is applied to a lateral flow test device. Embodiments of the antibody-conjugated ultra-large particles allow for accurate determination of analyte concentrations in a sample, including analytes present at high and very high concentrations. The lateral flow assays of the present disclosure can address the disadvantages associated with the hook effect of sandwich lateral flow assays by eliminating the phase of the dose response curve at which the signal intensity decreases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 594,974, filed December 5, 2017, the entire contents of which are incorporated herein by reference. Technical Field

[0003] In general, the present disclosure relates to lateral flow assay devices, kits, testing systems and methods. More specifically, the present disclosure relates to lateral flow assay devices for determining the concentration of an analyte in a sample, including when the target analyte is present at high concentrations. Background Art

[0004] The immunoassay system including the lateral flow assay described herein provides a reliable, inexpensive, portable, fast and simple diagnostic test. The lateral flow assay can quickly and accurately detect the presence of a target analyte in a sample, and in some cases also quantify the target analyte. Advantageously, the lateral flow assay can be minimally invasive and can be used as a point-of-care testing system. Lateral flow assays have been developed to detect a variety of medical or environmental analytes. In a lateral flow assay in a sandwich format, labeled antibodies for the target analyte are deposited in or near a sample receiving area on a test strip. The labeled antibody may include, for example, a detector molecule or "label" that binds to the antibody. When the sample is applied to the test strip, the analyte present in the sample binds to the labeled antibody, which flows along the test strip to the capture zone, where the fixed antibody for the analyte binds to the labeled antibody-analyte complex. The antibody fixed on the capture line may be different from the labeled antibody deposited in or near the sample receiving zone. The captured complex is detected, and the presence of the analyte is determined. In the absence of the analyte, the labeled antibody flows along the test strip and passes through the capture zone. The lack of signal at the capture zone indicates that the analyte is not present. However, sandwich lateral flow assays have many disadvantages, including false negatives, inaccurate quantification, and lack of resolution when the target analyte is present at high concentrations in the sample. Summary of the invention

[0005]

[0013] Accordingly, one aspect of the present disclosure is to provide improved lateral flow assays that accurately measure the concentration of an analyte of interest in a sample, including when the analyte is present in a high concentration in the sample.

[0006] Some embodiments disclosed herein relate to a test strip, which includes a flow path configured to receive a fluid sample; a sample receiving area coupled to the flow path; a capture zone; a labeled antibody or fragment thereof; and an ultra-large particle in the flow path upstream of the capture zone. The capture zone is coupled to the flow path downstream of the sample receiving area, and it includes a fixed capture agent specific to the target analyte. The labeled antibody or fragment thereof is coupled to the flow path upstream of the capture zone that is specific to the target analyte. When the fluid sample is received on the test strip, the ultra-large particle is conjugated with the antibody or fragment thereof that is specific to the target analyte to form an antibody-conjugated ultra-large particle of a certain size and size to retain it upstream of the capture zone. In some embodiments, the flow path is configured to receive a fluid sample including a target analyte. In some embodiments, the labeled antibody or fragment thereof competes with the antibody-conjugated ultra-large particle to specifically bind to the target analyte. In some embodiments, the labeled antibody or fragment thereof is configured to flow to the capture zone in the flow path together with the bound target analyte when the fluid sample is received on the test strip. In some embodiments, the labeled antibody bound to the target analyte is captured at the capture zone and emits a detectable signal.

[0007] In some embodiments, the flow path is configured to receive a fluid sample that may or may not include a target analyte. In some embodiments, the antibody-conjugated super-large particles are specifically bound to a known amount of the target analyte, thereby retaining a known amount of the target analyte upstream of the capture zone.

[0008] In a further embodiment, the test strip is included in a control zone downstream of the capture zone. In some embodiments, the control zone includes an antibody specifically bound to a labeled antibody or fragment thereof, which does not bind to the target analyte and flows through the capture zone. In some embodiments, when the fluid sample does not include the target analyte, the labeled antibody or fragment thereof flows to the control zone and emits a light signal only in the control zone, which indicates that the target analyte is not present in the fluid sample. In some embodiments, the fixed capture agent includes an antibody or fragment thereof that is specific to the target analyte. In some embodiments, the antibody-conjugated super-large particles are integrated onto the surface of the test strip. In some embodiments, the super-large particles include gold particles, latex beads, magnetic beads, or silica beads. In some embodiments, the diameter of the super-large particles is about 1 μm to about 15 μm. In some embodiments, the fluid sample is selected from blood, plasma, urine, sweat, or saliva samples. In some embodiments, the target analyte includes C-reactive protein (CRP), and the antibody or fragment thereof conjugated to the super-large particle includes an anti-CRP antibody or fragment thereof that is bound to CRP.

[0009] Other embodiments disclosed herein relate to a kit comprising the above-described assay test strip, the test strip comprising a flow path configured to receive a fluid sample; a sample receiving area coupled to the flow path; a capture area coupled to the flow path downstream of the sample receiving area, and the capture area comprises a fixed capture agent specific for a target analyte; a labeled antibody or fragment thereof coupled to the flow path upstream of the capture area specific for the target analyte; and ultra-large particles conjugated to the antibody or fragment thereof specific for the target analyte to form antibody-conjugated ultra-large particles that are approximately 250 times the size of the labeled antibody or fragment thereof.

[0010] Other embodiments disclosed herein are directed to a diagnostic test system comprising the above-described assay test strip or kit; a reader comprising a light source and a detector; and a data analyzer.

[0011] Further embodiments disclosed herein relate to methods for determining the concentration of a target analyte in a fluid sample. The method includes applying a fluid sample to the above-mentioned assay test strip; combining an analyte present in the fluid sample with a labeled antibody or fragment thereof; combining an analyte present in the fluid sample with an antibody-conjugated super-large particle; allowing the fluid sample and the labeled antibody bound to the analyte to flow to the capture zone in the flow path, while the super-large particle conjugated to the antibody bound to the analyte does not flow to the capture zone in the flow path; combining the labeled antibody bound to the analyte with a capture agent fixed in the capture zone; detecting a signal from the labeled antibody bound to the analyte fixed in the capture zone; and determining the concentration of the analyte based on at least the detected signal. In some embodiments, the concentration is determined based on the detected signal and the amount of antibody-conjugated super-large particles on the assay test strip. In some embodiments, the detected signal is an optical signal, a fluorescent signal, or a magnetic signal. In some embodiments, the method further includes displaying an indication of the target analyte present in the fluid sample. In some embodiments, the method further includes displaying the amount of the target analyte in the fluid sample. In some embodiments, the method further includes displaying an indication that the target analyte is present in an elevated amount.

[0012] Additional embodiments disclosed herein relate to methods for determining the concentration of a target analyte in a fluid sample. In some embodiments, the method includes contacting a fluid sample with an ultra-large particle that has been conjugated to an antibody or fragment thereof that is specific to the target analyte to form an antibody-conjugated ultra-large particle; combining the target analyte in the fluid sample with the antibody-conjugated ultra-large particle; after binding, applying the fluid sample with the antibody-conjugated ultra-large particle to the assay test strip described herein; and causing the fluid sample and the labeled antibody to flow to the capture zone in the flow path. In some embodiments, if an excess of the target analyte remains unbound to the antibody-conjugated ultra-large particle, the excess target analyte is bound to the labeled antibody or fragment thereof and flows through the flow path to the capture zone, where it is bound to the fixed capture agent in the capture zone and a signal is emitted. In some embodiments, an assay test strip includes a flow path configured to receive a fluid sample, a sample receiving zone coupled to the flow path, a capture zone coupled to the flow path downstream of the sample receiving zone and comprising an immobilized capture agent specific for a target analyte, and a labeled antibody or fragment thereof coupled to the flow path upstream of the capture zone specific for the target analyte.

[0013] Some embodiments disclosed herein relate to methods for manufacturing assay test strips. In some embodiments, the method includes coupling a sample receiving area to a flow path configured to receive a fluid sample; coupling a capture zone to a flow path downstream of the sample receiving area; coupling a labeled agent to a flow path upstream of the capture zone, and coupling an ultra-large particle to the flow path. In some embodiments, the labeled agent includes a label and an antibody that specifically binds to a target analyte. In some embodiments, the ultra-large particle is conjugated to an antibody or fragment thereof that is specific to the target analyte to form an antibody-conjugated ultra-large particle.

[0014] In some embodiments, coupling the labeled agent to the flow path includes forming a bond between the labeled agent and the flow path, which breaks when there is a fluid sample in the flow path. In some embodiments, coupling the super-large particles includes spraying a solution including the super-large particles onto the surface of the sample receiving area. In some embodiments, coupling the super-large particles includes spraying a solution including the super-large particles onto the surface of the assay test strip between the sample receiving area and the capture area. In some embodiments, coupling the super-large particles includes applying a fluid solution including the super-large particles onto the surface of the assay test strip; and drying the fluid solution. In some embodiments, coupling includes integrating the super-large particles into the surface of the assay test strip.

[0015] In some embodiments, the method further includes fixing a capture agent specific to the target analyte on the capture zone. In some embodiments, the method further includes providing a solution including super-large particles, which are conjugated with antibodies or fragments thereof that are specific to the target analyte. In some embodiments, the target analyte includes a C-reactive protein (CRP) and a labeled agent, and the antibody-conjugated super-large particles include antibodies, and the antibodies include anti-CRP antibodies or fragments of anti-CRP antibodies. Still further embodiments disclosed herein relate to assay test strips manufactured by the above method. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A and 1B An example sandwich-type lateral flow assay is shown before and after applying a fluid sample to a sample receiving area.

[0017] Figure 2 Shown for Figure 1A and 1B Example dose response curve of a lateral flow assay.

[0018] Figure 3A and 3B An example competitive lateral flow assay is shown before and after applying a fluid sample to a sample receiving area.

[0019] Figure 4 Shown for Figure 3A and 3B Example dose response curve of a competitive lateral flow assay.

[0020] Figure 5A and 5B An example lateral flow assay according to a first embodiment of the present disclosure is shown before and after applying a fluid sample to a sample receiving area.

[0021] Figure 6 An example lateral flow assay according to a second embodiment of the present disclosure is shown, depicting application of a sample to the lateral flow assay.

[0022] Figure 7 The hook effect dose response curve is shown in Figure 2. Figure 5A and 5B Example dose response curve of a lateral flow assay. DETAILED DESCRIPTION

[0023] The devices, kits, systems and methods described herein accurately determine the amount of target analytes in a sample, such as the concentration of analytes in a sample of known volume. Advantageously, the lateral flow devices, test systems and methods according to the present disclosure accurately determine the amount of target analytes in a sample where the target analytes are present in an elevated or "high" concentration. The lateral flow assay described herein can improve the resolution of the signal indicating the concentration of the analyte. Embodiments of the devices, kits, systems and methods described herein may include lateral flow assays and ultra-large particles conjugated with a binding agent that is specific to the target analyte. The binding agent may include, for example, an analyte-specific antibody or a fragment thereof. As an example implementation throughout the present disclosure, the implementation of ultra-large particles is referred to as "antibody-conjugated ultra-large particles", but it should be understood that ultra-large particles according to the present disclosure can be conjugated with any suitable binding agent that is specific to the target analyte, such as, but not limited to, an analyte-specific antibody or a fragment thereof. In one embodiment, the antibody-conjugated ultra-large particles can be premixed with a sample suspected of including the target analyte and then added to the sample well of the lateral flow assay. In a second embodiment, the antibody-conjugated super-large particles can be incorporated into the lateral flow assay prior to adding a sample suspected of including the analyte of interest to the lateral flow assay.

[0024] According to the present disclosure, compared with the labeled agents commonly used in lateral flow assays (e.g., detector particles including colloidal gold as an example), the size of the antibody-conjugated super-large particles is relatively large. In a non-limiting example, the diameter of the embodiment of the antibody-conjugated super-large particles described herein may be about 10 μm, while the diameter of the commonly used labeled agents is generally about 40 nm. In contrast to the labeled agents that can easily move from the sample hole and pass through the membrane of the lateral flow assay when the fluid sample is added, the embodiment of the antibody-conjugated super-large particles described herein will not move through the membrane when the fluid sample is added to the lateral flow assay. In fact, the embodiment of the antibody-conjugated super-large particles described herein captures the analyte (if any) in the sample in the sample hole when the sample is applied and retains the captured analyte in the sample hole. As a result, the embodiment of the antibody-conjugated super-large particles described herein reduces the amount of analyte that can form a label-antibody-analyte complex and flow through the membrane to reach the capture zone of the lateral flow assay to produce a detectable signal. The reduced amount of label-antibody-analyte complex reaching the capture zone of the lateral flow assay results in a dose response curve that exhibits a single, ascending phase.

[0025] Thus, the embodiments described herein address the disadvantages associated with the hook effect of conventional sandwich-type lateral flow assays by reducing or completely eliminating the phase of the dose response curve where the signal is decreasing. Advantageously, the single, ascending phase exhibited by the lateral flow assay of the present disclosure can be used to determine the amount of analyte in a sample with very high accuracy and reliability, and specifically to determine the amount of analyte when the analyte is present in high or very high concentrations. In some embodiments described below, very accurate quantitative measurements are achieved because the antibody-conjugated ultra-large particles according to the present disclosure can reliably retain a predictable, known amount of bound target analyte upstream of the capture zone. In addition, compared to conventional lateral flow assays that require up to 10 to 15 minutes to generate a detectable signal and generate a test result, the devices, systems, and methods of the present disclosure include lateral flow assays that can generate a detectable signal and generate a test result in as little as 2 minutes.

[0026] Embodiments of the present disclosure are described with reference to lateral flow devices. In some embodiments, the lateral flow device is implemented on a test strip, but other forms may be suitable. In the test strip format, a test sample fluid suspected of containing an analyte flows through the strip (e.g., by capillary action). The strip can be made of any suitable material, including but not limited to absorbent materials, such as paper, nitrocellulose, and cellulose. The sample fluid is received at a sample pool. The sample fluid can flow along the strip to a capture zone, where the analyte (if present) interacts with a capture agent to indicate the presence, absence, and / or amount of the analyte. The capture agent may include an antibody fixed in the capture zone.

[0027] Embodiments of the present disclosure are described with reference to optical signals generated at the capture zone of a lateral flow device, but other forms of detectable signals may be implemented in accordance with the present disclosure. Signals generated by assays according to the present disclosure may include optical signals generated by reflective labels (such as, but not limited to, gold nanoparticle labels), fluorescent signals generated by fluorescent latex bead labels, magnetic field signals generated by magnetic nanoparticle labels (which generate signals indicating magnetic field changes associated with the assay), or any other suitable signals.

[0028] According to the present disclosure, the lateral flow assay includes a target analyte suspected to be present in the sample with a specific labeled agent. The labeled agent is initially integrated on the surface of the lateral flow assay test strip at the sample well (or sample pool area), such as integrated on the conjugate pad. In some embodiments, the labeled agent is a labeled antibody or fragment thereof. In some embodiments, the labeled agent is labeled with a label that emits a detectable signal, such as a metal nanoparticle, such as a gold nanoparticle, a colored latex, a fluorescent particle, or other labels that emit a detectable signal.

[0029] When the fluid sample is applied to the test strip at the sample pool, the agent of the mark is dissolved in the fluid sample, and competes with the super-large particles of the antibody conjugate to bind the target analyte in the sample. The analyte combined by the super-large particles of the antibody conjugate is retained with a known amount (based on the amount and binding capacity of the super-large particles of the antibody conjugate applied to the test strip), and will not flow through the test strip. On the contrary, the analyte combined by the agent of the mark forms a marker-agent-analyte complex, and advances to the capture zone of the test strip together with the liquid sample. In some embodiments, the capture agent is an antibody or its fragment that is specific to the target analyte. The marker-agent-analyte complex is combined with the capture agent in the capture zone, and the signal of the binding capacity of the super-large particles of the antibody conjugate is generated to indicate that the amount of the analyte exceeds the conjugation of the antibody. The result is a single-phase dose response curve, wherein the signal intensity increases with the increase of analyte concentration (exceeding the super-large particles of the antibody conjugate).

[0030] The super-large particles according to the antibody conjugation of the present disclosure can be applied to the test strip in a variety of ways. In a non-limiting example described in detail below, before the sample is applied to the test strip, the super-large particles of the antibody conjugation are initially integrated in the sample pool or the labeling area on the surface of the test strip. In another non-limiting example, the sample is contacted with a certain amount of super-large particles of the antibody conjugation, and then the sample mixed with the super-large particles of the antibody conjugation is applied to the test strip. The super-large particles of the antibody conjugation are pre-integrated into the test strip (or contacted with the sample) with a known amount, so that the target analyte of a known amount is combined with the super-large particles of the antibody conjugation. When the sample is applied to the test strip, the fluid leading edge will carry the agent of the labeling combined with the target analyte to the capture zone, but the analyte combined with the super-large particles of the antibody conjugation will not move through the test strip, and remain in the appropriate position upstream of the capture zone (in the region initially integrated into the test strip, or in the sample well where it is deposited when the sample is applied to the test strip). In some cases, the optical signal detected at the capture zone is compared to a dose response curve specific to the test device and a known amount of antibody-conjugated ultra-large particles to determine the amount of the target analyte. In other cases, the dose response curve of a conventional lateral flow sandwich assay is adjusted by the amount of analyte retained by the antibody-conjugated ultra-large particles (which can be determined using the amount of antibody-conjugated ultra-large particles used during the test and the conjugation ratio) to directly calculate the amount of the target analyte.

[0031] Without being bound by any particular theory, the antibody-conjugated super-large particles, whether pre-integrated on the lateral flow device or contacted with the sample and then applied to the lateral flow device, reduce the amount of analyte captured by the labeled agent and detected in the capture zone by binding the analyte through the antibody-conjugated super-large particles, thereby improving the resolution of the rising phase of the dose response curve and generating a single-phase dose response curve by removing the second phase from the conventional sandwich-type lateral flow assay dose response curve. The lateral flow assay of the present disclosure solves the shortcomings associated with the hook effect of the sandwich-type lateral flow assay by eliminating the phase of the dose response curve where the signal is decreasing. Additionally, the resolution of the signal in the rising portion of the dose response curve of the present disclosure is greatly improved compared to the portion where the signal is increasing in the conventional sandwich-type dose response curve.

[0032] When analyte is in high concentration, the signal generated by the lateral flow assay described herein includes many advantageous features. In example embodiments, the signal generated when analyte is in high concentration is easily detected (e.g., they have an intensity within the signal range that can be usually distinguished and well-spaced by conventional readers), they will not overlap with the signal generated at zero or low concentration on the dose response curve, and they can be used to calculate the high-precision concentration readings at high concentration or even very high concentration. The embodiment of the lateral flow assay described herein avoids the uncertainty associated with the amount of specific detection signal and analyte (especially high concentration of analyte), such as the uncertainty occurring in reading sandwich lateral flow assays, which generates a single optical signal corresponding to both low and high concentrations of analyte due to the hook effect. On the contrary, the lateral flow assay according to the present disclosure generates a light signal that clearly and unambiguously corresponds to zero or low concentrations of analyte or high concentrations of analyte. In some cases, zero or low concentrations may be directly correlated to normal or "healthy" levels of the analyte in a subject, while high concentrations of the analyte may be directly correlated to abnormal or "unhealthy" levels of the analyte in a subject.

[0033] Embodiments of lateral flow assays described herein are particularly advantageous in diagnostic tests for target analytes that naturally exist at low concentrations in healthy individuals but are elevated to high concentrations in individuals with disease conditions or disorders. Relatively few or no signals are detected that are associated with a range from zero to low concentrations, in which case the operator is only attempting to confirm that the analyte is present at low concentrations (an indicator of a healthy level), and no specificity or resolution of the signal is required, because the signal is still present at zero or close to zero until the binding capacity of the antibody-conjugated super-large particles reaches saturation. Once the antibody-conjugated super-large particles become saturated, a high-resolution signal that is easy to detect is generated in a dose response curve of a single rising phase, in which case the operator is attempting to confirm that the analyte is present at high concentrations (an indicator of an abnormal or diseased condition), specifically when the target analyte is present at high concentrations, attempting to quantify the target analyte. When the target analyte is in a high concentration range, the ability to accurately pinpoint the exact concentration of the target analyte can also allow the operator to determine the stage or progression of the disease or other conditions of the subject, such as a mild stage or a severe stage.

[0034] The present invention describes a kit comprising a lateral flow assay and an antibody-conjugated super-large particle; a system comprising a lateral flow assay, an antibody-conjugated super-large particle and a reader; and a method for determining the amount of an analyte in a sample using a lateral flow assay. Various aspects of the lateral flow assay provide advantages over existing lateral flow assays. For example, in some embodiments, the lateral flow assay described herein can accurately determine the elevated analyte concentration in a sample without first diluting the sample. In addition, in some embodiments, the amount of the antibody-conjugated super-large particle placed in the lateral flow assay or placed in a sample and then applied to the lateral flow assay can be varied to accommodate the requirements of different analyte concentration ranges.

[0035] Aspects of the device, test system and method will be described more fully below with reference to the accompanying drawings. However, the present disclosure can be embodied in many different forms. Based on the teachings of this article, it will be appreciated by those skilled in the art that the scope of the present disclosure is intended to cover any aspect of the device, test system and method disclosed herein, whether implemented independently of the present disclosure or implemented in combination with any other aspect of the present disclosure. For example, using any number of aspects set forth herein, a device can be implemented or a method can be put into practice.

[0036] Although specific aspects are described herein, many variations and permutations of these aspects fall within the scope of the present disclosure. Although some benefits and advantages are mentioned, the scope of the present disclosure is not intended to be limited to specific benefits, uses or purposes. On the contrary, aspects of the present disclosure are intended to be widely applicable to different detection techniques and device configurations, some of which are illustrated by way of examples in the accompanying drawings and the following description. The detailed description and drawings are merely illustrative of the present disclosure and are not intended to be limiting, and the scope of the present disclosure is limited by the appended claims and their equivalents.

[0037] Sandwich and competitive lateral flow assays

[0038] Lateral flow assays can be performed in the form of sandwich or competition. The determination of sandwich and competitive forms described herein will be described in the context of reflective markers (e.g., gold nanoparticle markers) that generate optical signals, but it should be understood that the determination may include latex bead markers configured to generate fluorescent signals, magnetic nanoparticle markers configured to generate magnetic signals, or any other markers configured to generate detectable signals. Sandwich-type lateral flow assays include labeled antibodies that are deposited at a sample pool on a solid substrate. After the sample is applied to the sample pool, the labeled antibodies dissolve in the sample, and then the antibodies recognize and bind to the first epitope of the analyte in the sample to form a marker-antibody-analyte complex. The complex flows from the sample pool through a solid substrate along the liquid front to a capture zone (also referred to herein as a "test line"), where a fixed antibody (sometimes also referred to as a "capture agent") is located in the capture zone. In some embodiments, the fixed antibody can be the same antibody type as the labeled antibody. For example, in some cases where the analyte is a polymer or contains multiple identical epitopes on the same monomer, the labeled antibody deposited at the sample pool can be the same antibody type as the antibody fixed in the capture zone. In some embodiments, the immobilized antibodies may be of different antibody types and recognize different epitopes or recognition sites on the analyte. The immobilized antibodies recognize and bind to the epitope on the analyte, thereby capturing the label-antibody-analyte complex at the capture zone.

[0039] The presence of the antibody labeled at the capture zone provides a detectable light signal at the capture zone. In a non-limiting example, metal nanoparticles (gold, silver, copper, platinum, cadmium, palladium or its composite material) are used to label antibodies because they are relatively cheap, stable and provide an easily observable color indication based on the surface plasmon resonance properties of metal nanoparticles. In some cases, the signal provides qualitative information, such as whether there is an analyte in the sample. In some cases, the signal provides quantitative information, such as the measurement of the amount of analyte in the sample.

[0040] Figure 1A and 1BAn example sandwich-type lateral flow device 10 is shown. The lateral flow device 10 includes a sample reservoir 12, a label zone 14, a capture zone 16, and a control line 18. Figure 1A and 1B The lateral flow device 10 is shown before and after applying a fluid sample 24 to the sample reservoir 12. Figure 1A and 1B In the example shown, sample 24 includes target analyte 26. Label zone 14 in or near sample reservoir 12 includes labeled agent 28. In this example sandwich-type lateral flow device, labeled agent 28 includes antibody or antibody fragment 30 bound to label 32. Capture agent 34 is immobilized in capture zone 16. Control agent 35 is immobilized on control line 18.

[0041] When the fluid sample 24 is applied to the sample reservoir 12, the sample 24 dissolves the labeled agent 28, and the labeled agent 28 binds to the analyte 26, which forms the label-antibody-analyte complex 20. Therefore, in the example sandwich-type lateral flow device 10, the label-antibody-analyte complex 20 is not formed until after the fluid sample 24 containing the target analyte 26 is applied to the lateral flow device. Further, in the example sandwich-type lateral flow device 10, the analyte in the label-antibody-analyte complex 20 is the analyte from the fluid sample 24. Figure 1B As shown, the complex 20 flows through the test strip to the capture zone 16, where it is bound by the capture agent 34. The now bound complex 20 (and specifically, the label 32 on the now bound complex 20) emits a detectable light signal at the capture zone 16.

[0042] The labeled agent 28 that is not bound to any analyte 26 passes through the capture zone 16 (where no analyte 26 is bound to the capture agent 34) and continues to flow down the lateral flow device 10. In a lateral flow assay that includes a control line 18 as described herein, an immobilized control agent 35 captures the labeled agent 28 that is not bound to the analyte 26 and passes through the capture zone 16 to the control line 18. In some embodiments, the control agent 35 captures the labeled agent 28 at the Fc region of an antibody. In some embodiments, the control agent 35 captures the labeled agent 28 at the Fab region of an antibody. The labeled agent 28 bound at the control line 18 emits a detectable light signal that can be measured and used to indicate that the assay is operating as expected (e.g., the sample 24 flows from the sample pool 12 and through the capture zone 16 as expected during normal operation of the lateral flow assay).

[0043] Lateral flow assay can provide qualitative information, such as information about whether there is a target analyte in the sample. For example, detecting any measurable light signal at capture zone 16 can indicate that the target analyte (with some unknown amount) is present in the sample. The absence of any measurable light signal in the capture zone can indicate that the target analyte is not present in the sample or is below the detection limit. For example, if sample 24 does not contain any target analyte 26 (not shown), sample 24 will still dissolve the labeled agent 28, and the labeled agent 28 will still flow to capture zone 16. However, the labeled agent 28 will not be in conjunction with the capture agent 34 at capture zone 16. On the contrary, it will flow through capture zone 16, flow through control line 18, and flow to an optional absorption zone in some cases. Some labeled agents 28 will be in conjunction with the control agent 35 deposited on the control line 18 and emit a detectable light signal. In these cases, the lack of a measurable light signal emanating from capture zone 16 indicates the absence of the target analyte in sample 24, and the presence of a measurable light signal emanating from control line 18 indicates that sample 24 traveled from sample receiving zone 12 through capture zone 16 and to capture line 18 as expected during normal operation of the lateral flow assay.

[0044] Some lateral flow devices can provide quantitative information, such as a measurement of the amount of an analyte of interest in a sample. A quantitative measurement obtained from a lateral flow device can be the concentration of an analyte present in a given volume of a sample. Figure 2 Shows from Figure 1A and 1B Example quantitative measurements obtained in a sandwich-type lateral flow assay are shown. Figure 2 is a dose response curve that graphically illustrates the relationship between the intensity of the signal detected at the capture zone (measured along the y-axis) and the concentration of analyte in the sample (measured along the x-axis).Example signals include optical signals, fluorescent signals, and magnetic signals.

[0045] like Figure 2 As shown in the first data point at zero concentration, if the sample does not contain any target analyte, the concentration of the analyte in the sample is zero, and no analyte binds to the labeled agent to form a marker-antibody-analyte complex. In this case, no complex flows to the capture zone and binds to the capture antibody. Therefore, no detectable light signal is observed in the capture zone, and the signal amplitude is zero.

[0046] As the concentration of analyte in the sample increases from zero concentration, a signal is detected. As shown in the data points in phase A, the signal increases as the concentration of analyte in the sample increases. This occurs because the formation of the label-antibody-analyte complex increases as the analyte concentration increases. The capture agent immobilized in the capture zone binds to the increased number of complexes flowing to the capture zone, which results in an increase in the signal detected in the capture zone. In phase A, the signal continues to increase as the concentration of analyte in the sample increases.

[0047] In some cases, if the analyte concentration of the sample exceeds the amount of the agent of the labeling that can bind analyte, there is an excess of analyte. In this case, the excess analyte of the agent that is not labeled competes with the label-antibody-analyte complex to bind the capture agent in the capture zone. The capture agent in the capture zone will be combined with unlabeled analyte (in other words, the analyte that is not combined with the agent of labeling) and label-antibody-analyte complex. However, the unlabeled analyte combined with the capture agent will not emit a detectable signal. Along with phase B, the analyte concentration in the sample increases, and the amount of the unlabeled analyte combined with the capture agent (instead of the label-antibody-analyte complex that emits a detectable signal) also increases. Along with the unlabeled analyte of the increased amount is combined with the capture agent---instead of label-antibody-analyte complex, the signal detected in the capture zone is reduced, as shown in the data point in phase B.

[0048] This phenomenon that the detected signal increases during phase A and decreases in phase B is called the "hook effect". As the analyte concentration in phase A increases, more analyte binds to the labeled agent, which results in an increase in signal intensity. sat " point, the labeled agent is saturated with the analyte in the sample (e.g., the available amount of labeled agent has all or almost all bound to the analyte in the sample), and the detected signal has reached a maximum value Signal max As the concentration of analyte in the sample continues to increase in phase B, the detected signal decreases because excess analyte above the saturation point of the labeled agent competes with the labeled agent-analyte for binding to the capture agent.

[0049] The hook effect, also known as the "prozone effect", adversely affects lateral flow assays, particularly when the target analyte is present in the sample at a concentration in phase B. The hook effect can lead to inaccurate test results. For example, the hook effect can lead to false negatives or inaccurate low results. Specifically, inaccurate results occur when the sample contains an elevated level of analyte, which exceeds the concentration of the labeled agent deposited on the test strip. In this case, when the sample is placed on the test strip, the labeled agent is saturated, and not all analytes are labeled. Unlabeled analytes flow through the assay and bind at the capture zone, outcompeting the labeled complex, thereby reducing the detectable signal. Therefore, the device (or the operator of the device) cannot distinguish whether the optical signal corresponds to a low concentration or a high concentration, because the detected signal corresponds to both a low concentration and a high concentration. If the analyte level is high enough, the analyte will completely outcompete the labeled complex, and no signal is observed in the capture zone, resulting in a false negative test result.

[0050] Inaccurate test results can also result from competitive lateral flow assays. In contrast to sandwich lateral flow assays, in competitive lateral flow assays, unlabeled target analytes from the sample compete with labeled target analytes for binding to the capture agent at the capture zone. Figure 3A and 3B An example competitive lateral flow assay 22 is shown. The lateral flow device 22 includes a sample reservoir 12, a labeling zone 14, and a capture zone 16. Figure 3A and 3B The lateral flow device 22 is shown before and after applying a fluid sample 24 to the sample reservoir 12. Figure 3A and 3B In the example shown, the fluid sample 24 includes a target analyte 26. The labeling zone 14 in or near the sample reservoir 12 includes a labeled agent 29. In this example competitive lateral flow device, the labeled agent 29 includes the target analyte 26 bound to a label 32. A capture agent 34 is immobilized in the capture zone 16.

[0051] A sample 24 including an unlabeled analyte 26 is applied to the sample cell 12. The sample 24 dissolves the labeled agent 29. The unlabeled analyte 26 and the labeled agent 29 in the sample 24 flow together to the capture zone 16, where both the unlabeled analyte 26 and the labeled agent 29 from the sample 24 bind to the capture agent 34 immobilized in the capture zone 16. Figure 3B As shown, the labeled agent and the unlabeled analyte 26 compete with each other to bind to a fixed amount of capture agent 34. The labeled agent 29 (specifically, the label 32 in the labeled agent 29) bound to the capture agent 34 emits a detectable light signal, while the unlabeled analyte 26 derived from the sample 24 and bound to the capture agent 34 does not emit a detectable light signal.

[0052] Detection of the optical signal from the capture zone 16 can provide qualitative or quantitative information about the target analyte 26. In the case where the fluid sample 24 does not include any analyte 26 (not shown), the sample 24 will still dissolve the labeled agent 29 and the labeled agent 29 will still flow to the capture zone 16. The capture agent 34 in the capture zone 16 will bind to the labeled agent 29 (without competing with any unlabeled analyte in the sample), resulting in a detected optical signal of maximum intensity or close to maximum intensity. In the case where the sample 24 includes a very low or low concentration of analyte 26, an optical signal of maximum intensity or close to maximum intensity can also be detected. This is because the ratio of unlabeled analyte 26 bound to the capture agent 34 is low relative to the labeled agent 29 bound to the capture agent 34. Therefore, it may be difficult to determine whether the optical signal detected at maximum intensity should be associated with zero or low concentrations of analyte 26 in the sample 24.

[0053] As the concentration of unlabeled analyte 26 in the sample 24 increases, the detected light signal emitted from the capture zone 16 decreases. This is because as the concentration of analyte in the sample increases, competition for the capture agent 34 increases, and the ratio of unlabeled analyte 26 bound to the capture agent 34 relative to the labeled agent 29 bound to the capture agent 34 will gradually increase. However, if the analyte is present in the sample at a high or very high concentration, the light signal detected at the capture zone 16 decreases rapidly to a low amplitude signal. This rapid decrease in light signal intensity as the concentration of analyte in the sample increases to high and very high concentrations makes it difficult (if not impossible) to accurately determine the concentration of the analyte, and in some cases makes it impossible for the device to determine the concentration of the analyte at all. When the target analyte is present in high concentrations (e.g., when the ratio of unlabeled analyte to labeled agent is high), such as Figure 3A and 3B The competitive lateral flow device shown in FIG. 1 is not practically capable of accurately determining the exact concentration of the target analyte.

[0054] Figure 4 As shown in the example above reference Figure 3A and 3B Dose response curves were generated in the example competitive lateral flow device described above. Figure 4 As shown, the dose response curve of the competitive lateral flow assay shows a sharp drop in signal over the range of analyte concentrations from about 1 to 20 μg / mL. Due to the sharp drop in the curve, the resolution is poor, which reduces the accuracy of determining the amount of analyte at high concentrations, and in some cases, it is impractical or almost impossible to determine the amount of analyte present at high concentrations in the sample with any accuracy.

[0055] Example of a lateral flow device for accurate quantification of analytes present at high concentrations in a sample

[0056] The lateral flow devices, kits, test systems and methods described herein address both sandwich and competitive lateral flow assays (e.g. Figure 1A , 1B , those shown in 3A and 3B) these and other disadvantages, such as Figure 1A , 1B , those shown in 3A and 3B. Figure 5A and 5B An example lateral flow assay 100 is shown that can accurately measure the amount of a target analyte present in a sample, including analytes present in high concentrations. Figure 7 is an example dose response curve that graphically illustrates the optical signal measured from the lateral flow assay 100, and specifically the relationship between the signal amplitude detected at the capture zone (measured along the y-axis) and the analyte concentration in the sample applied to the assay (measured along the x-axis).

[0057] In some embodiments, for example Figure 5A and 5B As shown, lateral flow assay 100 can include a test strip having a sample receiving zone 112, a label zone 114, and a capture zone 116. In some embodiments, lateral flow assays can also include a control zone 118. Figure 5A and 5B The lateral flow device 100 is shown before and after applying a fluid sample 124 to the sample reservoir 112. In the example shown, the label zone 114 is downstream of the receiving zone 112 along the direction of sample flow within the test strip. In some cases, the sample receiving zone 112 is located within and / or coextensive with the label zone 114.

[0058] The labeling zone 114 in or near the sample reservoir 112 includes a labeled agent 128. The labeled agent 128 may include an antibody or antibody fragment 130 bound to a label 132. A capture agent 134 is immobilized in the capture zone 116. In some cases, the antibody 130 or a fragment thereof bound to the label 132 is the same type of antibody as the capture agent 134. When they are the same type of antibody or fragment thereof, both the antibody or fragment thereof 130 bound to the label 132 and the capture agent 134 recognize and bind to the same epitope of the analyte 126. In other cases, the antibody 130 or a fragment thereof bound to the label 132 is a different type of antibody or fragment thereof than the capture agent 134. When they are different types of antibodies or fragments thereof, the antibody or fragment thereof 130 bound to the label 132 recognizes and binds to an epitope of the analyte 126 that is different from the epitope recognized and bound by the capture agent 134.

[0059] In some embodiments, a lateral flow assay according to the present disclosure includes a control line 118 downstream of the capture zone 116. A control agent 135 is immobilized in the control zone 118. The control agent 135 is specific for a labeled agent that is not bound to an analyte. In some cases, the control agent 135 is an antibody or a fragment thereof. The labeled agent 128 that is not bound to any analyte 126 passes through and flows through the capture zone 116 (if there is an excess of the labeled agent 128 that is not bound to the analyte 126) and continues to flow down the lateral flow device 100. In a lateral flow assay including a control line 118 (such as shown herein), the immobilized control agent 135 binds to the labeled agent 128 that is not bound to the analyte 126 and reaches the control line 118 through the capture zone 116. In some embodiments, the control agent 135 captures the labeled agent 128 at the Fc region of the antibody. In some embodiments, the control agent 135 captures the labeled agent 128 at the Fab region of the antibody. The labeled agent 128 bound at the control line 118 emits a detectable light signal that can be measured and used to indicate that the assay is operating as expected (e.g., the sample 124 flows from the sample reservoir 112 and through the capture zone 116 as expected during normal operation of the lateral flow assay). In one embodiment, the lateral flow assay 100 includes multiple capture zones (including at least one capture zone 116 according to the present disclosure, which is configured to capture the labeled agent 128), each capture zone is configured to indicate the presence, absence and / or concentration of a different target analyte; and a single control line 118, which is configured to indicate that the sample flows through the multiple capture zones as expected.

[0060] Embodiments of the lateral flow assay 100 according to the present disclosure also include oversized particles 145. Figure 5A and 5B In the illustrated embodiment, the super-large particle 145 is located in the labeling zone 114, but other locations upstream of the capture zone 116 may be suitable. For example, the super-large particle 145 may be located in the sample receiving zone 112 upstream of the labeling zone 114. According to the present disclosure, the super-large particle 145 is conjugated with a binding agent 141 that is specific for the analyte 126. The binding agent 141 may include, for example, an analyte-specific antibody 141 or a fragment thereof. The super-large particle 145 conjugated with the analyte-specific antibody 141 forms an antibody-conjugated super-large particle 148. As will be described in detail below, the size of the antibody-conjugated super-large particle 148 is significantly larger than the labeled agent 128, and therefore Figure 5A and 5B The features shown in are not drawn to scale. Additionally, although explained in the context of antibody-conjugated ultra-large particles 148 Figure 5A and 5BExamples are provided, but it will be appreciated that the ultra-large particles according to the present disclosure may be conjugated to any suitable binding agent specific for the target analyte (such as, but not limited to, an analyte-specific antibody or fragment thereof).

[0061] The super-large particle 145 can be made of any suitable material and can have any suitable size / dimension to substantially maintain its position and resist flow to the capture zone 116 when the sample is applied to the lateral flow device 100. In some embodiments, the super-large particle 145 is a silicon particle, a latex particle, a magnetic particle, a gold particle, or another particle of a size sufficient to resist movement and flow through the assay test strip to reach the capture zone 116. The super-large particle 145 may include, for example, latex beads, magnetic beads, silicon beads, gold beads, or beads formed by another suitable material. In a non-limiting example, the diameter of the super-large particle 145 is about 10 μm, and the diameter of the labeled agent 128 is about 40 nm. Therefore, the implementation of the super-large particle described herein can have a diameter of 250 times the diameter of the conventional labeled agent. Other sizes are suitable. For example, the diameter of the super large particle 145 can be 1 to 15 μm, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 μm, or a diameter within the range defined by any two of the above values. The super large particle 148 has a sufficient size so that it cannot move through the lateral flow device 100, but instead remains fixed at the labeling zone 114. In some cases, the super large particle 145 does not move when the sample is applied to the lateral flow device 100. In some cases, the super large particle 145 can move a small amount relative to its initial position (for example, in the current non-limiting example, the super large particle 145 can slightly shift or change position within the labeling zone 114). However, when the sample is applied to the lateral flow device 100, the super large particle 145 does not flow to the capture zone 116 with the fluid front like the labeled agent 128. The oversized particles 145 can be incorporated into the lateral flow assay 100 in a variety of ways as described in detail below.

[0062] exist Figure 5A and 5BIn the illustrated embodiment, the binding agent 141 conjugated to the super-large particle 145 is an antibody or fragment thereof that is specific for the analyte 126. In some cases, the antibody 141 or fragment thereof can be the same antibody type as the antibody 130 and / or the capture agent 134 that binds to the label 132. In other cases, the antibody 141 or fragment thereof can be a different antibody type than the antibody 130 and / or the capture agent 134 that binds to the label 132. Therefore, in some embodiments, the antibody 141, the antibody 130, and the capture agent 134 have the same antibody type for the analyte 126, and recognize and bind to the same epitope on the analyte 126. In other embodiments, the antibody 141, the antibody 130, and the capture agent 134 have different antibody types for the analyte 126, and recognize and bind to different epitopes on the analyte.

[0063] According to the present disclosure, the binding agent 141 is conjugated to the very large particle 145 in a specific known ratio. The conjugation ratio can be adjusted according to the specific very large particle 145, the specific target analyte, the test parameters of the lateral flow device, or other parameters. In some embodiments, the binding agent 141 is present on the very large particle 145 in a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1, or in a range defined by any two of the above values. In a non-limiting example, the ratio of the binding agent 141 to the very large particle 145 represents the binding capacity of the antibody-conjugated very large particle 148. For example, a ratio of 1:1 means that the antibody-conjugated super-large particle 148 has the ability to bind a single target analyte in the sample, and a ratio of 100:1 means that the antibody-conjugated super-large particle 148 has the ability to bind up to 100 target analytes in the sample. In another non-limiting example, the conjugation ratio of the binding agent 141 to the super-large particle 145 is not directly related to the binding ability of the antibody-conjugated super-large particle 148. For example, this may occur when the binding efficiency is less than 100%, such as when less than 100% of the antibody conjugated to the super-large particle 145 is bound to the target analyte in the sample. In still another non-limiting example, the ratio of the binding agent 141 present on the super-large particle 145 is less than 1:1, as when the super-large particle 145 has a high density of functional groups relative to the conjugate antibody. In this case, the amount of antibody required to form the antibody-conjugated super-large particle 148 is reduced. Therefore, in this example, the ratio of binder 141 to super-large particles 145 can be, for example, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100, or an amount within the range defined by any two of the above values.

[0064] In some advantageous embodiments of the present disclosure, the ratio of binding agent 141 to super large particles is carefully adjusted and quantified in order to determine the binding capacity of a specific antibody-conjugated super large particles 148 for a test event on the lateral flow device 100. In addition, the amount of antibody-conjugated super large particles 148 initially integrated onto the surface of the lateral flow device 100 is carefully adjusted and quantified so that the total binding capacity of the antibody-conjugated super large particles 148 integrated onto the surface of the lateral flow device 100 is predetermined. In a non-limiting example of the present disclosure, the antibody-conjugated super-large particles 148 are integrated into the surface of the lateral flow 100 in an amount to bind 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 or 50 μg / mL of analyte or an amount of target analyte within a range defined by any two of the above values. Thus, the antibody-conjugated super-large particles 148 can be integrated into the labeling zone 114 in a known amount and can be used to capture a known maximum amount of analyte when the analyte is present in a sample applied to the sample pool 112.

[0065] When the fluid sample 124 is applied to the sample pool 112, the sample 124 dissolves the labeled agent 128 and the antibody-conjugated super-large particles 148. The labeled agent 128 binds to the analyte 126 to form a label-antibody-analyte complex 120. The labeled agent 128 competes with the antibody-conjugated super-large particles 148, and the antibody-conjugated super-large particles 148 also bind to the analyte 126 to form an analyte-antibody-super-large particle complex 140. The label-antibody-analyte complex 120 flows through the lateral flow device 100 to the capture zone 116, where it is bound by the capture agent 134 and emits a detectable signal. However, the analyte-antibody-super-large particle complex 140 does not flow through the lateral flow device 100. Instead, the complex 140 is retained in the label zone 114, thereby retaining a certain amount of bound analyte 126 and preventing the bound analyte 126 from flowing through the lateral flow device to the capture zone 116. In some embodiments in which the antibody-conjugated super-large particles 148 have a known binding capacity for the analyte 126, a known amount of the analyte 126 is retained in the label zone 114 when the fluid sample 124 is applied to the lateral flow device 100. For example, in some embodiments, the antibody-conjugated super-large particles 148 according to the present disclosure are capable of retaining an amount of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 μg / mL of the analyte, or an amount of the analyte 126 within a range defined by any two of the above values, in the label zone 114.

[0066] A multiplex assay testing for the presence, absence and / or amount of a plurality of different target analytes may include a lateral flow assay according to the present disclosure on the same test strip as one or more sandwich lateral flow assays described in references 1A and 1B (e.g., the lateral flow assays described in references 1A and 1B above). Figure 5A and 5B In such multiplex assays, a control line may advantageously be included on the test strip to confirm that the sample has flowed through the control zone 116.

[0067] Now refer to Figure 7 Advantages of the lateral flow device 100 are described. By maintaining a known amount of analyte 126 within the label zone 114, the lateral flow assay 100 is able to detect elevated concentrations of analyte 126 in a sample 124 in a single phase format. Figure 7 The dose response curve of FIG. 1 graphically illustrates the relationship between the signal intensity detected at the capture zone (measured in arbitrary signal intensity units along the y-axis) and the concentration of the analyte in the sample (in this non-limiting example, C-reactive protein (CRP), measured in μg / ml along the x-axis). Figure 7As shown in , the dose response curve of a lateral flow assay including antibody-conjugated super-large particles 148 according to the present disclosure (square data points) is compared with the dose response curve of a typical sandwich-type assay that does not include antibody-conjugated super-large particles 148 (circled data points).

[0068] like Figure 7 As shown in the examples of , the dose response curve of the sandwich assay shows that the signal intensity increases very sharply with small increases in concentration in the range of 0.1 to 5 μg / mL of C-reactive protein (CRP), and reaches a maximum signal intensity at about 5 μg / mL of CRP. The dose response curve then decreases as free, unbound analyte competes with the label-antibody-analyte complex in the capture zone, resulting in the above reference. Figure 2 In contrast, the dose response curve of the assay comprising antibody-conjugated ultra-large particles according to the present disclosure shows a gradual, monophasic increase in optical signal intensity as CRP concentration increases, which allows accurate determination of CRP concentration over a wide range of concentrations, and notably at elevated concentrations, such as at concentrations greater than 5 μg / mL, the concentration at which a typical sandwich-type assay reaches maximum signal intensity.

[0069] exist Figure 7 In the example shown, the binding capacity of the antibody-conjugated super-large particles is about 1 μg / mL. For CRP concentrations less than 1 μg / mL (in this example, the binding capacity of the antibody-conjugated super-large particles), the dose response curve of the assay containing the antibody-conjugated super-large particles emits a signal at or near zero, after which the signal gradually increases, allowing accurate determination of the analyte concentration when the analyte is present in an amount greater than 5 μg / mL.

[0070] The amount of binding agent and the binding capacity of the binding agent can be precisely adjusted for a specific assay. For example, in a sample where the analyte concentration is known or suspected to be greater than a given amount, a certain amount of antibody-conjugated ultra-large particles of known binding capacity can be incorporated into the lateral flow device to produce a dose response curve similar to that of Figure 7 As shown, the concentration of the analyte can be accurately determined. This is particularly advantageous in cases where the sample is suspected to have a high or very high concentration of the analyte. Alternatively or in addition, the binding capacity of the antibody-conjugated super-large particles can be precisely adjusted to capture a given amount of analyte, thereby allowing the operator to very accurately quantify the amount of analyte in the sample applied to the lateral flow device.

[0071] The antibody-conjugated super-large particles 148 can be applied to the test strip in many different ways. Figure 5A and 5BIn the embodiment of the present disclosure shown in , antibody-conjugated super-large particles 148 are preformed and integrated into the test strip before any fluid sample 124 is applied to the lateral flow device 100. In a non-limiting example, antibody-conjugated super-large particles 148 are formed and integrated into the conjugate pad before the fluid sample 124 is applied. In one non-limiting aspect, antibody-conjugated super-large particles 148 are applied to the marking area 114 by spraying a solution of antibody-conjugated super-large particles 148 with a jet technique. In another non-limiting aspect, a solution comprising antibody-conjugated super-large particles 148 is deposited by pouring a solution, spraying a solution, formulating the solution as a powder or gel placed on or rubbed on a test strip, or any other suitable method of applying antibody-conjugated super-large particles 148.

[0072] In some embodiments, after deposition, by heating or blowing on the conjugate pad, the antibody-conjugated super-large particles 148 are dried on the test strip surface after deposition. Other mechanisms of drying the antibody-conjugated super-large particles 148 on the test strip surface are suitable. For example, vacuum or freeze drying can also be used to dry the antibody-conjugated super-large particles 148 on the conjugate pad. In some cases, the antibody-conjugated super-large particles 148 are not added to the solution before deposition, but are directly applied to the test strip. Any suitable method can be used to directly apply the antibody-conjugated super-large particles 148, which includes but is not limited to applying compression or vacuum pressure to the antibody-conjugated super-large particles 148 on the test strip surface, and / or applying the antibody-conjugated super-large particles 148 in the form of freeze-dried particles to the surface of the test strip. The labeled agent 128 can be similarly deposited on the test strip using the same or similar techniques as described for depositing the antibody-conjugated super-large particles 148.

[0073] Now refer to Figure 6 Another non-limiting example of the present disclosure is described. Figure 5A and 5B In contrast to the illustrated embodiment, the antibody-conjugated super-large particles 148 are not incorporated onto the surface of the lateral flow device 100 prior to applying the sample 126 having or suspected of having the target analyte. Instead, the antibody-conjugated super-large particles 148 are applied to the lateral flow device 100 simultaneously with the sample. Figure 6 As shown, a fluid sample 124 to be tested for the presence, absence or amount of a target analyte is added to the antibody-conjugated super-large particles 148 in a separate container 101. The fluid sample is mixed to integrate the antibody-conjugated super-large particles 148 within the fluid sample and to allow the antibody-conjugated super-large particles 148 to bind to the analyte 126 (if present). If the analyte 126 is present in the fluid sample 124, it binds to the antibody-conjugated super-large particles 148 to form an analyte-antibody-super-large particle complex 140.

[0074] The fluid sample 125 containing the complex 140 is then applied to the lateral flow device 100 at the sample reservoir 112. The lateral flow device 100 may be a conventional sandwich-type lateral flow assay device (e.g., as described above with reference to Figure 1A and 1B The fluid sample travels to the labeling zone 114, where it dissolves the labeled agent 128. When the fluid front moves to the labeling zone 114, the antibody-conjugated super-large particles 148 remain in the sample pool 112. When the sample 125 is applied to the sample pool 112, the oversize of the antibody-conjugated super-large particles 148 makes the antibody-conjugated super-large particles 148 resist moving from their initial position in the sample pool 112 where they were initially deposited. At the same time, in the labeling zone 114, the labeled agent 128 binds to the unbound analyte 126 that was not bound to the antibody-conjugated super-large particles 148 when the antibody-conjugated super-large particles 148 were mixed with the sample 124 before being applied to the device 100. The labeled agent 128 then moves with the fluid front and travels to the capture zone 116, where it is captured by the capture agent 134 and generates a detectable signal.

[0075] In some cases, the detectable signal emitted at the capture zone is compared to a dose response curve specific to the test device and a known amount of antibody-conjugated super-large particles in container 101 to determine the amount of target analyte. In other cases, the dose response curve of the lateral flow sandwich assay 100 is adjusted by the amount of analyte retained by the antibody-conjugated super-large particles (which can be determined using the amount of antibody-conjugated super-large particles used during the test and the conjugation ratio) to directly calculate the amount of target analyte.

[0076] Embodiments of the present disclosure that contact a sample with antibody-conjugated ultra-large particles include many advantages. Figure 6 The embodiments of the present disclosure described herein use any conventional sandwich-type lateral flow assay test strips and can detect precise, highly accurate amounts of target analytes even when high or extremely high concentrations of the target analytes are present in the sample. Figure 6 The described embodiments of the present disclosure allow the operator the flexibility to apply the antibody-conjugated super-large particles in an amount and conjugation ratio selected for the specific test the operator desires to perform on the lateral flow device. Thus, once the parameters of the test event (e.g., the characteristics of the sample to be tested) are known, the operator can adjust the amount of antibody-conjugated super-large particles and / or the conjugation ratio of the antibody-conjugated super-large particles used during the test. Figure 6The embodiments of the present disclosure described can be advantageously packaged in a kit format, wherein the lateral flow test device and the antibody-conjugated super-large particles are packaged in separate containers. This can result in the kit having a longer shelf life than the kit in which the antibody-conjugated super-large particle reagent of the embodiments of the present disclosure is pre-integrated on a test strip.

[0077] Example lateral flow kit for quantification of analytes present at high concentrations in a sample

[0078] Embodiments of the present disclosure include a kit comprising a lateral flow device and antibody-conjugated ultra-large particles. The kit may also include a reader device configured to read a signal from the lateral flow device and output a test result. In a non-limiting example, the kit includes a lateral flow device as described herein. Figure 5A and 5B The lateral flow device. The lateral flow device includes an agent with a label specific to the target analyte in the labeling zone, a capture zone including a capture agent with a specificity to the target analyte, and an antibody-conjugated super-large particle (such as but not limited to the agent with a label in the labeling zone) integrated upstream of the capture zone on the lateral flow device. In some embodiments, the lateral flow device may further include a control zone, which includes a control agent with a specificity to the labeled agent that is not bound to the target analyte. The kit may include instructions for use, which include instructions for the operator to apply the sample to be tested to the sample pool of the lateral flow device. The instructions for use can guide the operator to insert the lateral flow device into the reader (during development or after the development time is completed) to detect the light signal generated on the lateral flow device.

[0079] In another non-limiting example, the test kit includes a lateral flow device and a separately packaged antibody-conjugated super-large particle. In this example, the lateral flow device can be a conventional sandwich-type lateral flow device. In this example, the lateral flow device includes a marker having a specific label in the labeling zone for the target analyte, a capture zone including a capture agent having a specific label for the target analyte, but does not include the antibody-conjugated super-large particle pre-integrated into the lateral flow device. On the contrary, the antibody-conjugated super-large particle is packaged separately with the lateral flow device in the test kit. In a non-limiting example, in a separate package, the super-large particle is provided in a lyophilized form. The test kit can include instructions for use, which include instructions for the operator: open the packaging of the antibody-conjugated super-large particle, add the antibody-conjugated super-large particle to the sample to be tested, optionally mix the sample, optionally allow the incubation sample to make the antibody-conjugated super-large particle bind to the analyte present in the sample, and then apply the sample containing the antibody-conjugated super-large particle to the lateral flow device. The instructions for use may direct the operator to insert the lateral flow device into the reader (either during development or after the development time is complete) to detect the optical signal generated on the lateral flow device. Example lateral flow assay for accurate quantitation of analytes present at high concentrations in a sample Law

[0080] Embodiments of the present disclosure include methods for quantifying analytes present in high concentrations in samples. In an example method, the method begins with providing a lateral flow device according to the present disclosure. The lateral flow device includes a labeled agent and an antibody-conjugated super-large particle integrated on the lateral flow device before applying the sample. Next, the sample to be tested is applied to the lateral flow assay at the sample pool. After the sample is applied to the sample pool, the antibody-conjugated super-large particle binds the analyte present in the sample (if present), competing with the labeled agent to bind the analyte. The analyte not bound to the antibody-conjugated super-large particle in the sample is combined with the labeled agent to form a label-agent-analyte complex. Next, the label-agent-analyte complex flows to the capture zone and is combined by the capture agent, and the analyte bound by the antibody-conjugated super-large particle is retained in the label zone (or other positions initially integrated on the test strip). The method proceeds to the next step, wherein the label-agent-analyte complex sends a detectable signal in the capture zone. Next, the detectable signal is read by human observation or with a reader device. The detected signal can be related to the presence, absence or concentration of the analyte in the sample. In one example, the concentration of the analyte is determined empirically by comparing the intensity of the detectable signal to a dose response curve specific to the lateral flow device and the known amount and binding capacity of the antibody-conjugated super-large particles pre-integrated on the lateral flow device.

[0081] In another example method, the method begins by providing a conventional lateral flow device (e.g., Figure 1A and 1B The method is to add the antibody-conjugated super-large particles of known binding capacity to the sample to be tested (or alternatively, by adding the sample to be tested to the antibody-conjugated super-large particles). Optionally, the method may include mixing the sample and / or incubating the mixture, during which the analyte of a known amount is combined with the antibody-conjugated super-large particles in the mixture. The method continues to the next step, wherein the sample including the antibody-conjugated super-large particles now combined with the analyte is applied to the sample pool of the lateral flow device. Next, the analyte not combined by the antibody-conjugated super-large particles is combined with the labeled agent in the labeling zone to form a label-agent-analyte complex. The method enters the next step, in which the label-agent-analyte complex flows to the capture zone and is captured by the capture agent, while the analyte combined by the antibody-conjugated super-large particles is retained in the sample pool and does not flow through the lateral flow device. The method continues to the next step, wherein the label-agent-analyte complex emits a detectable signal in the capture zone. Next, the detectable signal is read by human observation or a reader device. The detected signal can be related to the presence, absence or concentration of the analyte in the sample. In one example, the concentration of the analyte is determined empirically by comparing the intensity of the detectable signal to a dose response curve specific to the lateral flow device and the known amount and binding capacity of the separately packaged antibody-conjugated super-large particles initially mixed with the sample.

[0082] In some embodiments, the lateral flow device can be used to determine multiple target analytes in a single sample or multiple samples in a multiplex assay format. Some embodiments of the multiplex assay include detecting specific subspecies of the target analyte. For example, the sample may contain one or more subspecies of the target analyte. The sample may first be treated with an agent to remove one or more specific subspecies of the target analyte, thereby reducing the concentration of the target analyte. For example, the sample may contain three subspecies of the target analyte. The sample may be premixed with antibody-conjugated ultra-large particles containing antibodies to remove the first subspecies of the target analyte. The antibody-conjugated ultra-large particles that bind to the first subspecies of the target analyte are removed from the sample. The sample containing the remaining two subspecies of the target analyte is now applied to the lateral flow device. Then, a universal antibody that binds to all subspecies of the target analyte is used as a labeled agent to recognize and bind to the remaining two subspecies of the target analyte.

[0083] The following non-limiting examples illustrate features of the lateral flow devices, testing systems, and methods described herein and are in no way intended to limit the scope of the present disclosure.

[0084] Example 1

[0085] 1. Preparation of Lateral Flow Assays for Quantitation of Elevated Protein Concentrations

[0086] The following example describes the preparation of a lateral flow assay as described herein for quantifying an analyte of interest. In this non-limiting example, the analyte of interest is a protein (C-reactive protein (CRP)) that is present at elevated or high concentrations in serum samples.

[0087] CRP is a protein found in plasma. In response to inflammation, the level of CRP increases. Therefore, CRP is a marker of inflammation, which can be used to screen for inflammation. Elevated levels of CRP in the serum of a subject can be associated with inflammation, viral infection, and / or bacterial infection in the subject. Normal levels of CRP in healthy human subjects are about 1 μg / mL to about 10 μg / mL. During mild inflammation and viral infection, the concentration of CRP ranges from 10-40 μg / mL; during active inflammation and bacterial infection, the concentration of CRP is 40-200 μg / mL; in severe bacterial infection and burn cases, the concentration of CRP is greater than 200 μg / mL. Measuring and plotting CRP levels can be used to determine disease progression or treatment efficacy.

[0088] The assay prepared according to this non-limiting example can be used to determine the precise concentration of CRP (the target analyte) in a serum sample, even if the concentration is higher than the normal level of CRP in healthy human subjects (about 1 μg / mL to about 10 μg / mL). The assay includes a labeled agent and very large particles conjugated to antibodies, and avoids several disadvantages of sandwich-type lateral flow assays, including those associated with the hook effect.

[0089] In order to prepare the assay, anti-C-reactive protein (anti-CRP) antibodies were incubated with gold nanoparticles to form labeled anti-CRP antibodies. The labeled antibodies were deposited onto the conjugate pad (labeled area) at 1.8 μL / test strip by spraying the labeled antibody solution with an air jet. The conjugate pad was heated to dry the complex to the conjugate pad.

[0090] Antibody-conjugated super-large particles are prepared by first conjugating anti-CRP antibodies to super-large particles. Magnetic particles (10 μm) with active surface N-hydroxysuccinimide (NHS) groups for conjugation are washed with equilibrium buffer and removed from the buffer by applying a magnetic field. 200 μg of anti-CRP is placed in 60 μL of PBS buffer. The antibody solution is added to the settled particle pellet and mixed at room temperature for 1 hour. Super-large particles conjugated with antibodies are removed from the buffer by applying a magnetic field. The antibody-conjugated super-large particles are washed with quenching buffer and incubated at room temperature for 1 hour. The quenching buffer is removed and the particles are resuspended in 100 μL of PBS and stored at 4 ° C.

[0091] The antibody-conjugated super-large particles can be stored for later use, such as being mixed with the sample to be tested before the sample is applied to the lateral flow assay, or being directly placed on the lateral flow assay before applying the sample. The antibody-conjugated super-large particles can be directly applied to the lateral flow assay during the manufacture of the lateral flow assay, just before the sample is applied to the lateral flow assay, or at any other suitable time. In the example of applying the antibody-conjugated super-large particles to the lateral flow assay before applying the sample, the solution containing the antibody-conjugated super-large particles is deposited on the conjugate pad (labeling area) by spraying the solution with an air jet. The conjugate pad is heated to dry the complex to the conjugate pad.

[0092] In this example, anti-CRP antibody was deposited in the capture zone at 2 mg / mL. Goat anti-mouse antibody was deposited in the control zone at 2 mg / mL.

[0093] Example 2

[0094] Quantification of high concentrations of C-reactive protein using a lateral flow assay

[0095] Due to the hook effect, for example, Figure 1A and 1B Those sandwich-type lateral flow assays described are generally not suitable for quantifying CRP concentrations when CRP is present at elevated levels in a sample. In order to determine elevated concentrations, it was previously necessary to serially dilute the sample, which resulted in an inefficient and laborious process that was susceptible to operator error. However, using the lateral flow devices, kits, test systems and methods described herein, CRP concentrations above healthy levels can be accurately, reliably and rapidly quantified.

[0096] The lateral flow assay of the super-large particles not including the antibody conjugation pre-integrated on the test strip described in Example 1 is used for the following examples. Antibody-conjugated super-large particles with conjugated antibodies thereon are prepared in storage buffer. CRP samples are prepared with different concentrations including 0, 0.1, 0.5, 1, 5, 20, 40, 100, 150 μg / mL. Next, 20 μL of antibody-conjugated super-large particles are placed in a tube, and the storage buffer is removed by applying a magnetic field. 40 μL of CRP solution is added to a tube containing antibody-conjugated super-large particle pills (using a separate tube for each CRP sample), and the solution is mixed for about 30 seconds by stirring. The sample from each tube is applied to the sample pool of a conventional sandwich type lateral flow device, and the test result is read at about two minutes.

[0097] Similarly, samples not treated with antibody-conjugated ultra-large particles were prepared at the above concentrations. 40 μL of each sample was applied to the sample cell of a conventional sandwich-type lateral flow device, and the test results were read at approximately ten minutes.

[0098] According to the advantageous features of the present disclosure, the amount of the antibody-conjugated super-large particles used to bind CRP is carefully considered to ensure that it is combined with the necessary amount of CRP to provide an optimal signal range in the capture zone, which causes a single rising phase dose response to occur in the test system, thereby being able to quantitatively increase the level of CRP. Insufficient amounts of antibody-conjugated super-large particles (or insufficient anti-CRP conjugated to super-large particles) can lead to a sharp rise phase of the dose response curve, which can lead to insufficient resolution for determining CRP concentration. Excessive antibody-conjugated super-large particles (or excessive anti-CRP conjugated to super-large particles) result in no or very low signal intensity until the antibody-conjugated super-large particles are saturated, at which point the signal intensity increases, making it difficult to determine the analyte concentration within a lower analyte concentration range. Therefore, the amount of antibody-conjugated super-large particles added to the sample or pre-deposited on the lateral flow device can be advantageously changed to adapt to the requirements of different analyte concentration ranges.

[0099] A lateral flow assay comprising antibody-conjugated super-large particles deposited in a suitable position upstream of the capture zone prior to sample application may also be similarly used. In this case, the sample does not need to be mixed or pre-incubated with the antibody-conjugated super-large particles, but instead is directly applied to the lateral flow assay comprising the antibody-conjugated super-large particles pre-integrated on the lateral flow assay surface.

[0100] The results of Example 2 are Figure 7 , which shows the resulting dose response curves for the lateral flow assay with antibody-conjugated super-large particles (line with square data points) and the lateral flow assay without antibody-conjugated super-large particles (conventional sandwich lateral flow assay; line with circular data points). The test results are also described in Table 1.

[0101] Table 1: Comparison of traditional sandwich-type lateral flow assays and the lateral flow assays of the present disclosure

[0102]

[0103] As shown in Table 1, the signal intensity of the sample without antibody-conjugated super-large particles increases sharply from 0 to 5 μg / mL concentration. When the amount is greater than 5 μg / mL, the signal decreases as the concentration increases. In addition, the signal (33.14AU) observed at 0.1 μg / mL is similar to the signal (31.55) observed at 100 μg / mL. Therefore, if a sample with a signal intensity of about 31-33AU is obtained, it is unclear whether the signal corresponds to a healthy amount (about 0.1 μg / mL) or an unhealthy amount (100 μg / mL) of CRP.

[0104] In contrast, the lateral flow assay according to the present disclosure allows the concentration of CRP to be accurately determined at a concentration greater than 5 μg / mL. This is particularly advantageous in the present embodiment (where the target analyte is CRP, and when there is an inflammatory or disease condition, the concentration of the CRP rises to greater than 10 μg / mL). In contrast to sandwich-type lateral flow assays that do not include antibody-conjugated super-large particles (where CRP concentrations greater than 5 μg / mL cannot be determined), lateral flow assays with antibody-conjugated super-large particles allow the concentration to be accurately determined by providing a single ascending phase dose response curve with sufficient resolution to accurately quantify the CRP concentration. Embodiments of the lateral flow assay described herein allow the user to confidently determine that the CRP concentration in the tested subject is higher than normal levels. When a test according to the present disclosure is performed and it is shown that the CRP concentration is greater than a healthy level (e.g., greater than 10 μg / mL), the information can be associated with inflammation, viral infection, and / or bacterial infection conditions.

[0105] Further, the ability to accurately pinpoint the exact concentration of CRP in the subject under test can allow the test results to be associated with a specific type of disease condition. For example, a concentration between 10 μg / mL and 20 μg / mL may be associated with mild inflammation, while a concentration between 40 μg / mL and 200 μg / mL may be associated with a bacterial infection. In addition, the ability to accurately pinpoint the exact concentration of CRP in the subject under test can allow the test results to be associated with the stage of the disease. For example, a concentration between 40 μg / mL and 200 μg / mL may be associated with a mild bacterial infection, while a concentration greater than 200 μg / mL may be associated with a severe bacterial infection. These embodiments are illustrative and are not intended to limit the scope of the present disclosure.

[0106] Furthermore, the lateral flow devices described herein quantify elevated concentrations of analytes in a sample in a single assay without the need to dilute the sample. Figure 1A , 1B , 3A and 3B require dilution of samples containing high concentrations of analytes; otherwise, the signals in the high concentration portion of the dose response curve are indistinguishable. The lateral flow assays of the present disclosure are able to determine even small differences in elevated analyte concentrations based on a single signal obtained in the capture zone after one test.

[0107] Embodiments of the lateral flow devices herein (including super-large particles) can be advantageously combined with conventional sandwich-type lateral flow devices (excluding super-large particles) to expand the concentration range that can be accurately and precisely tested. Figure 7As shown in, in some embodiments of the lateral flow device described herein, the resolution of the signal may be low at very low analyte concentrations. In the case where the concentration of the target analyte is very low, the lateral flow device comprising super-large particles may still indicate the presence or absence of the target analyte, but may not provide the best quantitative measurement of very low concentration analytes. In the case where the target analyte of unknown concentration may fall within a larger concentration range, the sample can be divided into two parts. The first sample portion can be mixed with super-large particles and applied to the first lateral flow device (or as described herein, applied to the first lateral flow device with integrated super-large particles). The second sample portion can be applied to the second conventional lateral flow device (without super-large particles). The test result of the first lateral flow device combined with the test result of the second lateral flow device can provide the operator with a very accurate actual concentration measurement value of the target analyte in a very wide possible concentration range.

[0108] Methods of diagnosing conditions using lateral flow assays according to the present disclosure

[0109] Some embodiments provided herein relate to methods for diagnosing medical conditions using lateral flow assays. In some embodiments, the method includes providing a lateral flow assay as described herein and an antibody-conjugated super-large particle (packaged separately or pre-integrated on a lateral flow assay). In some embodiments, the method includes receiving a sample at a sample pool of a lateral flow assay.

[0110] In some embodiments, the sample is derived from a source including an environment or a biological source. In some embodiments, the sample is suspected of having a target analyte. In some embodiments, the sample is not suspected of having a target analyte. In some embodiments, the sample is obtained and analyzed to verify the absence or presence of an analyte. In some embodiments, the sample is obtained and analyzed to quantify the analyte in the sample. In some embodiments, the amount of the analyte in the sample is less than the normal value present in a healthy subject, is at or near the normal value present in a healthy subject, or is higher than the normal value present in a healthy subject.

[0111] In some embodiments, receiving a sample at a sample well of a lateral flow assay includes contacting the sample with the lateral flow assay. The sample can be contacted with the lateral flow assay by introducing the sample into the sample well using an external application of a dropper or other applicator. In some embodiments, the sample well can be directly immersed in the sample, such as when the test strip is immersed in a container containing the sample. In some embodiments, the sample can be poured, dripped, sprayed, placed, or otherwise contacted with the sample well.

[0112] The labeled agent in the embodiment of the present disclosure includes an antibody and a label, and can be deposited on a conjugate pad (or labeling zone) in or downstream of the sample pool. The labeled agent can be integrated on the conjugate pad by physical binding or chemical bonding. The binding particles can also be integrated on the conjugate pad by physical binding or chemical bonding, or can be added to the sample before the sample is applied to the lateral flow device. The antibody-conjugated super-large particles recognize and bind to the analyte in the sample to form an analyte-antibody-super-large particle complex (the antibody-conjugated super-large particles are first added to the sample, or the sample is added to the lateral flow device on which the antibody-conjugated super-large particles are integrated). After the sample is added to the sample pool, the sample dissolves the labeled agent, thereby releasing the binding that keeps the labeled agent on the conjugate pad. The labeled agent recognizes and binds to the analyte that is not bound by the antibody-conjugated super-large particles, thereby forming a label-agent-analyte complex. The sample including the marker-agent-analyte complex flows through the lateral flow assay along the fluid front to the capture zone, while the analyte-antibody-ultra-large particle complex remains at the location where the antibody-conjugated ultra-large particles were initially deposited on the test strip (either during application of the sample to the test strip or directly integrated onto the surface of the test strip prior to application of the sample).

[0113] The capture agent fixed at the capture zone is combined with the label-agent-analyte complex. When the label-agent-analyte complex is combined with the capture agent in the capture zone, a detectable signal from the label is emitted. The signal may include a light signal as described herein. When there is a low concentration of analyte in the sample (for example, at or below the level of a healthy level), the antibody-conjugated super-large particles bind all or substantially all analytes in the sample, and no signal is detected in the capture zone. Under the analyte concentration (for example, higher than the level of a healthy value) that rises, the signal intensity detected increases in an amount proportional to the amount of the analyte in the sample. The detected signal and the antibody-conjugated super-large particles (including a known amount of bound analytes) are compared with the value on the dose response curve of the target analyte, and the concentration of the analyte in the sample is determined.

[0114] In some embodiments, there is an analyte in an elevated concentration. The elevated concentration of an analyte may refer to an analyte concentration that is higher than a healthy level. Therefore, the elevated concentration of an analyte may include a concentration of an analyte that is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200% or more higher than a healthy level. In some embodiments, the target analyte includes C-reactive protein (CRP), which is present in the serum of a healthy individual in an amount of about 1 to about 10 μg / mL. Thus, elevated concentrations of CRP in a sample include 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 μg / mL or more. The level at which a target analyte is considered elevated may vary depending on the specific target analyte.

[0115] In some embodiments, when determining that the analyte is present in the sample at an elevated concentration, the subject is diagnosed with a disease. In some embodiments, when determining that the concentration of CRP is 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 μg / mL or higher, a diagnosis of infection is made. In some embodiments, when determining that the concentration is greater than 200 μg / mL, such as 400-500 μg / mL, a diagnosis of a severe bacterial infection is produced.

[0116] In some embodiments, when a sample is placed on a lateral flow device described herein, a detected signal is determined at the capture zone, and the detected signal is combined with the binding capacity of the antibody-conjugated ultra-large particles to determine the concentration of the analyte in the sample. In some embodiments, when the test system determines a certain signal intensity, the test system outputs a test result. The test result may include the concentration of the analyte in the sample and / or a verbal or written indication that the test result is "normal" or "within normal levels" or that the test result is "not within normal levels", "elevated", "very high", or other indications.

[0117] Additional Embodiments of the Disclosure

[0118] Embodiments of the present disclosure have been described with reference to the oversized particles conjugated with a binding agent. As described herein, the oversized particles are significantly larger than other components of the lateral flow device that flows through the test strip when a fluid sample is applied. For example, the oversized particles described herein can be significantly larger than the agent of the label, and the agent size and size of the label are designed to dissolve and move from the labeling area to the capture area when the fluid sample is applied to the sample well. On the contrary, when the fluid sample is applied, the oversized particles according to the present disclosure resist movement, and remain in substantially the same position despite the fluid flowing through the test strip. Although the implementation of the oversized particles is described herein with reference to their significantly large size, it should be understood that according to the present disclosure, other particles that resist movement through the test strip can be implemented. For example, inert particles can be conjugated with a binding agent in the manner described herein with reference to the oversized particles, and applied to the lateral flow device. In a first non-limiting embodiment, the size of the inert particles is the same or smaller than the agent of the label as described herein. By way of example, magnetic particles can be conjugated with a binding agent to form a magnetic inert particle conjugated with an antibody of the same size or even smaller than the agent of the label. In this embodiment, by applying magnetic force to magnetic inert particles, the magnetic inert particles of antibody conjugation can be kept in a suitable position upstream of the capture zone, thereby preventing it from moving downstream to the capture zone with the fluid sample. The magnetic force can be a magnetic field applied to the test strip above or below or inside during sample application. In a second non-limiting embodiment, the size of the inert particles can be less than, about the same as, or slightly larger than the agent of the mark, but significantly denser than the agent of the mark. For example, the inert particles can be denser than the agent of the mark by 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or another multiple. As a result, due to this significant increase in density, when the sample is applied to the test strip, the inert particles resist the movement of the test strip through the capture zone from the initial position.

[0119] Other features in addition to or in lieu of increased density (such as, but not limited to, surface features that stick to or hook onto the nitrocellulose fibers of the conjugate pad) may also be suitable for ensuring that the antibody-conjugated particles do not move through the test strip. Features that increase the coefficient of friction of the antibody-conjugated particles may also be implemented. Therefore, the following embodiments explaining the present disclosure in the context of oversized particles should not be limited to oversized particles.

[0120] In addition, according to the present disclosure, other structures other than or in place of the oversized particles can be implemented so that a certain amount of target analyte is maintained in the sample hole and / or the labeling zone so that a portion of the analyte in the sample does not flow to the capture zone. In a non-limiting example, a filter is placed in the fluid flow path between the sample hole and the capture zone (e.g., at or near the labeling zone) to capture the oversized particles that happen to start moving toward the capture zone and are bound to the analyte. The filter can be a size exclusion filter that allows fluids and particles of a selected size or smaller to pass through, but does not allow particles greater than a selected size to pass through. The size of the through hole in the filter can be designed to allow the labeled agent and fluid sample bound to the analyte to flow through the filter to reach the capture zone, but does not allow oversized particles to flow through the filter, thereby maintaining the target analyte upstream of the filter. Other implementations are also suitable.

[0121] Example test systems including lateral flow assays according to the present disclosure

[0122] Lateral flow assay test system as described herein can include lateral flow assay test device (such as but not limited to test strip), including a housing configured to receive a port of all or part of the test device, a reader including a light source and a photodetector, a data analyzer and a combination thereof. The housing can be made of any of a variety of materials, including plastic, metal or composite material. The housing forms a protective housing for diagnostic test system components. The housing also defines a receiver that mechanically registers the test strip relative to the reader. The receiver can be designed to receive any of a variety of different types of test strips. In some embodiments, the housing is a portable device that allows the ability to perform lateral flow assays in a variety of environments, including on a workbench, on the scene, at home or in a facility for home, commercial or environmental applications.

[0123] The reader may include one or more optoelectronic components for optically inspecting the exposed area of ​​the capture zone of the test strip. In some implementations, the reader includes at least one light source and at least one photodetector. In some embodiments, the light source may include a semiconductor light emitting diode, and the photodetector may include a semiconductor photodiode. Depending on the nature of the marker used by the test strip, the light source may be designed to emit light within a specific wavelength range or light with a specific polarization. For example, if the marker is a fluorescent marker, such as a quantum dot, the light source will be designed to illuminate the exposed area of ​​the capture zone of the test strip with light within a wavelength range that induces fluorescence emission from the marker. Similarly, the photodetector may be designed to selectively capture light from the exposed area of ​​the capture zone. For example, if the marker is a fluorescent marker, the photodetector will be designed to selectively capture light within the wavelength range of the fluorescence emitted by the marker or light with a specific polarization. On the other hand, if the marker is a reflective marker, the photodetector will be designed to selectively capture light within the wavelength range of the light emitted by the light source. To this end, the photodetector may include one or more optical filters that define the wavelength range or polarization axis of the captured light. The signal from the label can be analyzed using: visual observation or a spectrophotometer to detect the color of a chromogenic substrate; a radiation counter to detect radiation, such as for detecting 125 1 gamma counter; or a fluorometer to detect fluorescence in the presence of light of certain wavelengths. When using an enzyme-linked assay, a spectrophotometer can be used to perform quantitative analysis of the amount of the target analyte. If desired, the lateral flow assay described herein can be automated or automatically performed, and signals from multiple samples can be detected simultaneously. In addition, multiple signals can be detected in a multiplex assay, wherein more than one target analyte can be detected, identified or quantified.

[0124] The data analyzer processes the signal measurements obtained by the reader. Generally, the data analyzer can be implemented in any computing or processing environment, including in digital electronic circuits or computer hardware, firmware or software. In some embodiments, the data analyzer includes a processor (e.g., a microcontroller, a microprocessor or an ASIC) and an analog-to-digital converter. The data analyzer can be incorporated into the housing of the diagnostic test system. In other embodiments, the data analyzer is located in a separate device (e.g., a computer), which can communicate with the diagnostic test system by wired or wireless connection. The data analyzer may also include a circuit for transmitting the result to an external source via a wireless connection to perform data analysis or view the result.

[0125] Generally, the result indicator can include any of a variety of different mechanisms for indicating one or more results of the assay test. In some implementations, the result indicator includes one or more lights (e.g., light emitting diodes) that are activated to indicate, for example, completion of the assay test. In other implementations, the result indicator includes an alphanumeric display (e.g., a two or three character light emitting diode array) for presenting the assay test results.

[0126] The test systems described herein may include a power source that provides power to the active components of the diagnostic test system including the reader, data analyzer, and result indicator. The power source may be implemented, for example, by a replaceable battery or a rechargeable battery. In other embodiments, the diagnostic test system may be powered by an external host device (e.g., a computer connected via a USB cable).

[0127] Features of an Example Lateral Flow Device

[0128] Lateral flow devices as described herein may include a sample pool (also referred to as a sample receiving area), where a fluid sample is introduced into a test strip, such as, but not limited to, an immunochromatographic test strip present in the lateral flow device. In one example, the sample can be introduced into the sample pool by external application such as using a dropper or other applicator. The sample can be poured or put into (express) the sample pool. In another example, the sample pool can be directly immersed in the sample, such as when the test strip is immersed in a container containing the sample.

[0129] Lateral flow devices as described herein may include solid supports or substrates. Suitable solid supports include, but are not limited to, walls of nitrocellulose, reaction tray holes, porous plates, test tubes, polystyrene beads, magnetic beads, membranes, and microparticles (e.g., latex particles). Any suitable porous material with sufficient porosity to allow labeled agents to enter and with suitable surface affinity to fix capture agents can be used in lateral flow devices as described herein. For example, the porous structure of nitrocellulose has excellent absorption and adsorption qualities for a variety of reagents (e.g., capture agents). Nylon has similar properties and is also suitable. Microporous structures are useful, and materials with gel structures in a hydrated state are also useful.

[0130] Further examples of useful solid supports include: natural polymeric carbohydrates and synthetically modified, cross-linked or substituted derivatives thereof, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gum, cellulose esters (particularly with nitric and carboxylic acids), mixed cellulose esters and cellulose ethers; nitrogen-containing natural polymers, such as proteins and derivatives, including cross-linked or modified gelatin; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers that can be prepared with an appropriate porous structure, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and its partially hydrolyzed derivatives, polyacrylamide, polyvinyl acetate ... Amines, polymethyl acrylates, copolymers and terpolymers of the above condensation polymers, such as polyesters, polyamides and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials, such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali and alkaline earth metals, aluminum and magnesium; oxides or hydrates of aluminum or silicon, such as clay, alumina, talc, kaolin, zeolites, silica gel or glass (these materials can be used as filters together with the above polymer materials); and mixtures or copolymers of the above types, such as graft copolymers obtained by initiating polymerization of synthetic polymers on existing natural polymers.

[0131] Lateral flow devices as described herein may include a porous solid support in the form of a sheet or strip, such as nitrocellulose. The thickness of such a sheet or strip may vary within wide limits, for example, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, from about 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2 mm, or from about 0.11 to 0.15 mm. The pore size of such a sheet or strip may similarly vary within wide limits, for example, from about 0.025 to 15 microns, or more specifically from about 0.1 to 3 microns; however, pore size is not intended to be a limiting factor in selecting a solid support. If applicable, the flow rate of the solid support can also vary within wide limits, such as from about 12.5 to 90 sec / cm (e.g., 50 to 300 sec / 4 cm), about 22.5 to 62.5 sec / cm (e.g., 90 to 250 sec / 4 cm), about 25 to 62.5 sec / cm (e.g., 100 to 250 sec / 4 cm), about 37.5 to 62.5 sec / cm (e.g., 150 to 250 sec / 4 cm), or about 50 to 62.5 sec / cm (e.g., 200 to 250 sec / 4 cm). In specific embodiments of the devices described herein, the flow rate is about 35 sec / cm (e.g., 140 sec / 4 cm). In other specific embodiments of the devices described herein, the flow rate is about 37.5 sec / cm (e.g., 150 sec / 4 cm).

[0132] The surface of the solid support can be activated by a chemical process that causes the agent (e.g., capture agent) to be covalently attached to the support. As described below, the solid support can include a conjugate pad. Many other suitable methods can be used to fix the agent (e.g., capture agent) to the solid support, including but not limited to ionic interactions, hydrophobic interactions, covalent interactions, etc.

[0133] Unless otherwise physically constrained, the solid support may be used in any suitable shape, such as a membrane, sheet, strip or plate, or may be coated or bonded or laminated to a suitable inert carrier such as paper, glass, plastic film or fabric.

[0134] The lateral flow device described herein may include a conjugate pad, such as a membrane or other type of material, which contains a capture agent. The conjugate pad can be cellulose acetate, cellulose nitrate, polyamide, polycarbonate, glass fiber, membrane, polyethersulfone, regenerated cellulose (RC), polytetrafluoroethylene (PTFE), polyester (e.g., polyethylene terephthalate), polycarbonate (e.g., 4,4-hydroxy-diphenyl-2,2'-propane), alumina, mixed cellulose esters (e.g., a mixture of cellulose acetate and cellulose nitrate), nylon (e.g., polyamide, hexamethylenediamine and nylon 66), polypropylene, PVDF, high-density polyethylene (HDPE) + nucleating agent "aluminum dibenzoate" (DBS) (e.g., 80u 0.024 HDPE DBS (Porex)) and HDPE. Examples of conjugate pads also include (Polyethylene terephthalate), (Polyethylene terephthalate), (cellulose acetate and cellulose nitrate), (cellulose acetate and cellulose nitrate), Whatman #12-S (rayon), (aluminum oxide), (alumina), Sartorius (cellulose acetate, e.g. 5 μm) and Whatman Standard 17 (bonded glass).

[0135] Lateral flow devices described herein are highly sensitive to target analytes present in high concentrations in samples. As described above, when the amount of unlabeled target analytes present in the sample is sufficient to compete with the labeled compound to bind to the capture agent in the capture zone, resulting in a detected signal on the negative slope portion of the dose response curve (e.g., on the "hook effect" portion of the dose response curve of a conventional sandwich-type lateral flow assay or on the negative slope portion of the dose response curve of a lateral flow assay according to the present disclosure), high concentrations are present. "Sensitivity" refers to the proportion of actual positives correctly identified as such (e.g., the percentage of infected, latent or symptomatic subjects correctly identified as suffering from a condition). Sensitivity can be calculated as the sum of the number of true positives divided by the number of true positives and the number of false negatives.

[0136] Lateral flow devices as described herein can accurately measure target analytes in many different kinds of samples. Samples can include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans) and include fluids, solids, tissues, and gases. Biological samples include urine, saliva, and blood products, such as blood plasma, serum, etc. However, these examples should not be construed as limiting the sample types applicable to the present disclosure.

[0137] In some embodiments, the sample is an environmental sample for detecting an analyte in an environment. In some embodiments, the sample is a biological sample from a subject. In some embodiments, the biological sample may include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculation, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menstruation, pus, sebum, vomitus, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, lavage fluid from the sinus cavity, bronchopulmonary aspirate or other lavage fluid.

[0138] As used herein, "analyte" generally refers to a substance to be detected. For example, analytes may include antigenic substances, haptens, antibodies, and combinations thereof. Analytes include, but are not limited to, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs (including those used for therapeutic purposes as well as those used for illicit purposes), drug intermediates or by-products, bacteria, virus particles, and metabolites or antibodies to any of the above. Specific examples of some analytes include ferritin; creatine kinase MB (CK-MB); human chorionic gonadotropin (hCG); digoxin; phenytoin; phenobarbital; carbamazepine; vancomycin; gentamicin; theophylline; valproic acid; quinidine; luteinizing hormone (LH); follicle stimulating hormone (FSH); estradiol, progesterone; C-reactive protein (CRP); lipocalin; IgE antibodies; cytokines; TNF-related apoptosis-inducing ligand (TRAIL); vitamin B2 microglobulin; interferon-gamma induced protein 10 (IP-10); glycosylated hemoglobin (Gly Hb); cortisol; digitalis poisoning; N-acetylprocainamide (NAPA); procainamide; rubella antibodies, such as rubella IgG and rubella IgM; toxoplasmosis antibodies, such as toxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM); testosterone; salicylates; acetaminophen; hepatitis B virus surface antigen (HBsAg); antibodies to hepatitis B core antigen, such as anti-hepatitis B core antigen IgG and IgM (anti-HBC); human immunodeficiency virus 1 and 2 (HIV 1 and 2); human T-cell leukemia virus 1 and 2 (HTLV); hepatitis B e antigen (HBeAg); antibodies to hepatitis B e antigen (anti-HBe); influenza virus; thyroid stimulating hormone (TSH); thyroxine (T4); total triiodothyronine (total T3); free triiodothyronine (free T3); carcinoembryonic antigen (CEA); lipoproteins, cholesterol and triglycerides; and alpha-fetoprotein (AFP). Drugs of abuse and controlled substances include, but are not intended to be limited to, amphetamines; methamphetamines; barbiturates, such as amobarbital, secobarbital, pentobarbital, phenobarbital and pentobarbital; benzodiazepines analytes, such as chlordiazepoxide and diazepam; cannabinoids, such as hashish and marijuana; cocaine; fentanyl; LSD; methaqualone; opioids, such as heroin, morphine, codeine, hydromorphone, hydrocodone, methadone, oxycodone, oxymorphone, and opium; phencyclidine; and propoxyhene. Additional analytes may be included for the purpose of targeting biological or environmental substances.

[0139] Lateral flow devices as described herein may include labels. Labels may take many different forms, including molecules or compositions that can be combined with or can be combined with analytes, analyte analogs, detector reagents or binding partners that can be detected by spectroscopy, photochemistry, biochemistry, immunochemistry, electricity, optics or chemical means. Examples of labels include enzymes, colloidal metal particles (also referred to as metal nanoparticles, including, for example, gold, silver, copper, palladium, platinum, cadmium or its composite material), colored latex particles, radioisotopes, cofactors, ligands, chemiluminescence or fluorescent agents, protein-adsorbed silver particles, protein-adsorbed iron particles, protein-adsorbed copper particles, protein-adsorbed selenium particles, protein-adsorbed sulfur particles, protein-adsorbed tellurium particles, protein-adsorbed carbon particles and protein-coupled dye capsules. Compounds (e.g., detector reagents) and labeling can be attached by covalent bonds, adsorption processes, hydrophobic bonds and / or electrostatic bonds (such as chelates) or combinations of these bonds and interactions and / or may involve linking groups.

[0140] The term "specific binding partner (or binding partner)" refers to members of a pair of molecules that interact through specific, non-covalent interactions that depend on the three-dimensional structure of the molecules involved. Typical specific binding partner pairs include antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid strand / complementary nucleic acid strand, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, biotin / (streptavidin), receptor / ligand and virus / cell receptor or various combinations thereof.

[0141] As used herein, the term "immunoglobulin" or "antibody" refers to a protein that binds to a specific antigen. Immunoglobulins include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, Fab fragments, F(ab')2 fragments, and include the following classes of immunoglobulins: IgG, IgA, IgM, IgD, IgE, and secretory immunoglobulins (sIg). Immunoglobulins typically contain two identical heavy chains and two light chains. However, the terms "antibody" and "immunoglobulin" also encompass single-chain antibodies and double-chain antibodies.

[0142] The lateral flow device described herein includes a labeled agent. The labeled agent can be specific to the analyte. In some embodiments, the labeled agent can be an antibody or fragment thereof that has been conjugated, bound or associated with a label.

[0143] The lateral flow device according to the present disclosure includes a capture agent. The capture agent includes a fixed agent capable of binding to an analyte, which includes a label-agent-analyte complex. The capture agent includes an unlabeled specific binding partner, which has specificity for the following (i) target label-agent-analyte complex, (ii) label-agent-analyte complex or unbound analyte (such as in a competitive assay) or (iii) an auxiliary specific binding partner, which itself has specificity for the analyte, such as in an indirect assay. As used herein, an "auxiliary specific binding partner" is a specific binding partner that binds to the specific binding partner of the analyte. For example, an auxiliary specific binding partner may include an antibody that is specific for another antibody, such as a goat anti-human antibody. The lateral flow device described herein may include a "capture zone", which is an area of ​​a fixed capture reagent of the lateral flow device. The lateral flow device described herein may include more than one capture zone, for example, a "primary capture zone", a "secondary capture zone", etc. In some cases, different capture reagents are fixed in primary, secondary and / or other capture zones. On a lateral flow substrate, multiple capture zones can have any orientation relative to each other; for example, a primary capture zone can be distal or proximal to a secondary (or other) capture zone along a fluid flow path, or vice versa. Alternatively, a primary capture zone and a secondary (or other) capture zone can be aligned along an axis perpendicular to the fluid flow path so that the fluid contacts the capture zones simultaneously or approximately simultaneously.

[0144] The lateral flow device according to the present disclosure includes a fixed capture agent so that the movement of the capture agent is restricted during the normal operation of the lateral flow device. For example, before and after the fluid sample is applied to the lateral flow device, the movement of the fixed capture agent is restricted. The fixation of the capture agent can be accomplished by physical means, such as barriers, electrostatic interactions, hydrogen bonds, bioaffinity, covalent interactions, or combinations thereof.

[0145] Lateral flow devices according to the present disclosure can include multiple assays. Multiple assays include assays that can detect, identify, and in some cases quantify a variety of different target analytes. For example, in a multiple assay device, there can be primary, secondary, or more capture zones, each of which is specific for one of the multiple target analytes.

[0146] Lateral flow devices according to the present disclosure can detect, identify and quantify biological preparations. Biological preparations include chemical or biochemical compounds produced by living organisms, which may include prokaryotic cell lines, eukaryotic cell lines, mammalian cell lines, microbial cell lines, insect cell lines, plant cell lines, mixed cell lines, naturally occurring cell lines or synthetic engineered cell lines. Biological preparations may include macromolecules, such as proteins, polysaccharides, lipids and nucleic acids and small molecules, such as primary metabolites, secondary metabolites and natural products.

[0147] It should be understood that this specification, specific examples and data, although indicating exemplary embodiments, are given by way of illustration and are not intended to limit the various embodiments of the present disclosure. From this specification and the data contained therein, various changes and modifications within the present disclosure will become apparent to those skilled in the art and are therefore considered to be part of the various embodiments of the present disclosure.

Claims

1. A test strip comprising: a flow path configured to receive a fluid sample; a sample receiving area coupled to the flow path; a capture zone coupled to the flow path downstream of the sample receiving zone and comprising an immobilized capture agent specific for a target analyte; a labeled antibody or fragment thereof coupled to the flow path upstream of the capture zone that is specific for the target analyte; and The ultra-large particles in the flow path upstream of the capture zone are conjugated with antibodies or fragments thereof specific to the target analyte when the assay test strip receives the fluid sample to form antibody-conjugated ultra-large particles of a certain size and dimension to be retained upstream of the capture zone, the antibody-conjugated ultra-large particles are configured to bind to the target analyte in an amount sufficient to generate a single ascending phase dose response curve, wherein the diameter of the ultra-large particles is 1 μm to 15 μm.

2. The assay test strip of claim 1, wherein the flow path is configured to receive a fluid sample comprising the target analyte, and wherein the labeled antibody or fragment thereof and the antibody-conjugated super-large particles compete for specific binding to the target analyte.

3. The assay test strip of claim 2, wherein the labeled antibody or fragment thereof is configured to flow in the flow path to the capture zone together with the bound target analyte when the assay test strip receives the fluid sample.

4. The assay test strip of claim 3, wherein the labeled antibody bound to the target analyte is captured at the capture zone and emits a detectable signal.

5. The assay test strip of claim 1, wherein the flow path is configured to receive a fluid sample that may or may not include a target analyte, and wherein the antibody-conjugated ultra-large particles specifically bind to a known amount of the target analyte, thereby retaining a known amount of the target analyte upstream of the capture zone.

6. The assay test strip of claim 1, further comprising a control zone downstream of the capture zone, wherein the control zone comprises an antibody that specifically binds to the labeled antibody or fragment thereof, the labeled antibody or fragment thereof does not bind to the target analyte and flows through the capture zone.

7. The assay test strip according to claim 6, wherein When the fluid sample does not include the target analyte, the labeled antibody or fragment thereof flows to the control area and emits a light signal only in the control area, indicating that the target analyte is not present in the fluid sample.

8. The assay test strip of claim 1, wherein the immobilized capture agent comprises an antibody or fragment thereof specific for the target analyte.

9. The assay test strip of claim 1, wherein the antibody-conjugated super-large particles are integrated onto the surface of the test strip.

10. The assay test strip of claim 1, wherein the oversized particles comprise gold particles, latex beads, magnetic beads, or silica beads.

11. The assay test strip of claim 1, wherein the fluid sample is selected from a blood, plasma, urine, sweat, or saliva sample.

12. The assay test strip of claim 1, wherein the target analyte comprises C-reactive protein, and the antibody or fragment thereof conjugated to the ultra-large particle comprises an anti-C-reactive protein antibody or fragment thereof that binds to the C-reactive protein.

13. A kit comprising: An assay test strip comprising: a flow path configured to receive a fluid sample; a sample receiving area coupled to the flow path; a capture zone coupled to the flow path downstream of the sample receiving zone and comprising an immobilized capture agent specific for the target analyte; and a labeled antibody or fragment thereof coupled to the flow path upstream of the capture zone that is specific for the target analyte; Ultra-large particles are conjugated to an antibody or a fragment thereof specific to the target analyte to form antibody-conjugated ultra-large particles, the ultra-large particles are 250 times the size of the labeled antibody or a fragment thereof, and the antibody-conjugated ultra-large particles are present in an amount sufficient to generate a single ascending phase dose response curve, wherein the diameter of the ultra-large particles is 1 μm to 15 μm.

14. A diagnostic test system comprising: The assay test strip of claim 1 or the kit of claim 13; a reader including a light source and a detector; and Data analyzer.

15. A method for determining the concentration of a target analyte in a fluid sample, comprising: applying said fluid sample to the assay test strip of claim 1; binding an analyte present in the fluid sample to a labeled antibody or fragment thereof; binding an analyte present in the fluid sample to the antibody-conjugated super-large particles; Allowing the fluid sample and the labeled antibody bound to the analyte to flow to the capture zone in the flow path, while the ultra-large particles conjugated to the antibody bound to the analyte do not flow to the capture zone in the flow path; binding the labeled antibody bound to the analyte to the immobilized capture agent in the capture zone; detecting a signal from the labeled antibody bound to the analyte immobilized in the capture zone; and The concentration of the analyte is determined based at least on the detected signal.

16. The method of claim 15, wherein the concentration is determined based on the detected signal and the amount of the antibody-conjugated super-large particles on the assay test strip.

17. The method of claim 15, wherein the detected signal is an optical signal, a fluorescent signal, or a magnetic signal.

18. The method of claim 15, further comprising displaying an indication of the target analyte present in the fluid sample.

19. The method of claim 15, further comprising displaying the amount of the target analyte in the fluid sample.

20. The method of claim 15, further comprising displaying an indication that the target analyte is present in an elevated amount.

21. A method for determining the concentration of a target analyte in a fluid sample, comprising: contacting the fluid sample with super-large particles that have been conjugated to an antibody or fragment thereof specific for the target analyte to form antibody-conjugated super-large particles, the antibody-conjugated super-large particles being present in an amount sufficient to generate a single ascending phase dose response curve, wherein the super-large particles have a diameter of 1 μm to 15 μm; combining the target analyte in the fluid sample with the antibody-conjugated super-large particles; After binding, the fluid sample with the antibody-conjugated super-large particles is applied to an assay test strip, the assay test strip comprising: a flow path configured to receive a fluid sample, a sample receiving area coupled to the flow path, a capture zone coupled to the flow path downstream of the sample receiving zone and comprising an immobilized capture agent specific for the target analyte, and a labeled antibody or fragment thereof coupled to the fluid path upstream of the capture zone that is specific for the target analyte; The fluid sample and the labeled antibody are allowed to flow in the flow path to the capture zone, wherein if the excess target analyte is not bound to the antibody-conjugated super-large particles, the excess target analyte is bound to the labeled antibody or a fragment thereof and flows through the flow path to the capture zone, where it is bound to the fixed capture agent in the capture zone and emits a signal.

22. A method of making an assay test strip comprising: coupling the sample receiving area to a flow path configured to receive a fluid sample; coupling a capture zone to a flow path downstream of the sample receiving zone; coupling a labeled agent to the fluid path upstream of the capture zone, wherein the labeled agent comprises a label and an antibody that specifically binds to the target analyte; and The ultra-large particles are coupled to the flow path, the ultra-large particles are conjugated with antibodies or fragments thereof specific to the target analyte to form antibody-conjugated ultra-large particles, the antibody-conjugated ultra-large particles are present in an amount sufficient to generate a single ascending phase dose response curve, wherein the diameter of the ultra-large particles is 1 μm to 15 μm.

23. The method of claim 22, further comprising immobilizing a capture agent specific for the target analyte on the capture zone.

24. The method of claim 22, wherein coupling the labeled agent to the flow path comprises forming a bond between the labeled agent and the flow path, the bond being broken when a fluid sample is present in the flow path.

25. The method of claim 22, wherein coupling the oversized particles comprises spraying a solution comprising the oversized particles onto the surface of the sample receiving area.

26. The method of claim 22, wherein coupling the oversized particles comprises spraying a solution comprising the oversized particles onto the surface of the assay test strip between the sample receiving area and the capture area.

27. The method of claim 22, wherein coupling the ultra-large particles comprises: applying a fluid solution including the oversized particles to a surface of the assay test strip; and The fluid solution is dried.

28. The method of claim 22, wherein coupling comprises integrating the ultra-large particles into the surface of the assay test strip.

29. The method of claim 22, further comprising providing a solution comprising ultra-large particles conjugated to an antibody or fragment thereof specific for the target analyte.

30. The method of claim 22, wherein the target analyte comprises C-reactive protein and the labeled agent, and the antibody-conjugated ultra-large particles comprise an antibody comprising an anti-C-reactive protein antibody or a fragment of an anti-C-reactive protein antibody.

31. An assay test strip manufactured by the method of any one of claims 22-30.

32. The assay test strip of any one of claims 1-12, wherein the oversized particles have a diameter of 10 µm to 15 µm.

33. The kit of claim 13, wherein the super-large particles have a diameter of 10 µm to 15 µm.

34. The diagnostic test system of claim 14, wherein the oversized particles have a diameter of 10 μm to 15 μm.

35. The method of claim 15, wherein the oversized particles have a diameter of 10 µm to 15 µm.

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