Sandwich-type assays using decreasing signal portions of dose response curve to measure analytes, including analytes at high concentration

Abstract

To solve the problem in which a sandwich lateral flow assay suffers from many disadvantages, including false negatives, inaccurately low results, and lack of resolution when an analyte of interest is present in a sample at high concentrations.SOLUTION: Sandwich-type lateral flow assay devices, systems and methods described herein measure the concentration of an analyte of interest in a sample, and can determine the precise concentration of the analyte when it is present at high concentrations. A signal of maximum intensity is generated when the concentration of the analyte of interest in the sample is zero. For low concentrations of the analyte, the lateral flow assays described herein generate signals that are the same as or substantially equivalent to the maximum intensity signal. High concentrations of the analyte of interest generate signals that are less than the maximum intensity signal. Lateral flow assays of the present disclosure solve drawbacks associated with the hook effect of sandwich-type lateral flow assays by eliminating the phase of the dose response curve where signals are increasing.SELECTED DRAWING: Figure 6A

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 62 / 526,051, filed June 28, 2017, which is incorporated herein by reference in its entirety. The present disclosure relates generally to lateral flow assay devices, test systems, and methods. More particularly, the present disclosure relates to lateral flow assay devices for determining the concentration of an analyte in a sample, including when the analyte of interest is present at high concentrations. [Background technology]

[0002] Immunoassay systems, including the lateral flow assays described herein, enable reliable, inexpensive, portable, rapid, and simple diagnostic testing. Lateral flow assays can rapidly and accurately detect, and in some cases, quantify, the presence or absence of an analyte of interest in a sample. Advantageously, lateral flow assays are minimally invasive and can be used as point-of-care testing systems. Lateral flow assays have been developed to detect a wide variety of medical or environmental analytes. In a sandwich-format lateral flow assay, a labeled antibody against the analyte of interest is deposited on a test strip in or near a sample-receiving zone. The labeled antibody may, for example, include a detection molecule or "label" attached to the antibody. When a sample is applied to the test strip, the analyte present in the sample binds to the labeled antibody, which flows along the strip to the capture zone. The immobilized antibody against the analyte binds to the labeled antibody-analyte complex. The antibody immobilized on the capture line can be different from the labeled antibody attached in or near the sample receiving zone. The captured complex is detected to determine the presence of the analyte. If the analyte is not present, the labeled antibody flows along the test strip but passes through the capture zone. The lack of signal at the capture zone indicates the absence of the analyte. However, sandwich lateral flow assays have many drawbacks, including false negatives, inaccurately low results, and a lack of resolution when the analyte of interest is present in high concentrations in the sample. Summary of the Invention

[0003] It is therefore an aspect of the present disclosure 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 high concentrations in the sample. Some embodiments disclosed herein relate to an assay test strip comprising: a flow path configured to receive a fluid sample; a sample receiving zone connected to the flow path; a capture zone; and a complex. The capture zone is connected to the flow path downstream of the sample receiving zone and comprises an immobilized capture agent specific to an analyte of interest. The complex is connected to the flow path in a first phase and configured to flow to the capture zone within the flow path in the presence of a fluid sample in a second phase. The complex comprises a label, an antibody or antibody fragment that specifically binds to the analyte of interest, and the analyte of interest. In some cases, the flow path is configured to receive a fluid sample containing an unlabeled analyte of interest, and the complex does not specifically bind to the unlabeled analyte of interest in the first or second phase. In some cases, the complex is configured to flow to the capture zone together with the unlabeled analyte of interest within the flow path in the second phase. In some examples, the complex is configured to bind to the immobilized capture agent in the capture zone in a third phase in competition with the unlabeled analyte of interest. In some cases, as the concentration of unlabeled target analyte increases in the fluid sample, the optical signal emitted from the complex bound to the immobilized capture agent in the capture zone decreases.

[0004] In some examples, the flow path is configured to receive a fluid sample that may or may not contain the analyte of interest. If the fluid sample does not contain the analyte of interest, the complexes specifically bind to all or substantially all of the immobilized capture agents in the capture zone in the second phase. In some cases, if the fluid sample does not contain the analyte of interest, the optical signal emitted from the complexes bound to the capture zone will be the maximum optical signal that can be emitted from the assay strip. If the fluid sample contains the analyte of interest, the optical signal emitted from the complexes bound to the capture zone will be less than the maximum optical signal. In some cases, the immobilized capture agent comprises an antibody or antibody fragment that specifically binds to the analyte of interest. In some examples, the complex is incorporated into the surface of the test strip in a first phase. In some cases, the complex is incorporated into the surface of the test strip by spraying a solution containing the complex onto the surface of the test strip and allowing the solution to dry. Fluid samples include blood, plasma, urine, sweat, or saliva samples. In one non-limiting example, the analyte of interest comprises C-reactive protein (CRP), and the complex comprises an anti-CRP antibody or fragment thereof bound to the CRP.

[0005] Other embodiments disclosed herein relate to a diagnostic test system including the above-described assay strip; a reader including a light source and a detector; and a data analyzer. In some cases, the data analyzer outputs an indication that the target analyte is absent from the fluid sample when the reader detects an optical signal from the assay strip that is the maximum optical signal of the dose-response curve for the strip. In one example, the data analyzer outputs an indication that the target analyte is present in a low concentration in the fluid sample when the reader detects an optical signal from the assay strip that is within 1% of the maximum optical signal. In another example, the data analyzer outputs an indication that the target analyte is present in a low concentration in the fluid sample when the reader detects an optical signal from the assay strip that is within 5% of the maximum optical signal. In yet another example, the data analyzer outputs an indication that the target analyte is present in a low concentration in the fluid sample when the reader detects an optical signal from the assay strip that is within 10% of the maximum optical signal. In a further example, the data analyzer outputs an indication of a high concentration of the target analyte in the fluid sample when the reader detects an optical signal from the assay strip that is 90% or less than 90% of the maximum optical signal. In yet another example, the data analyzer outputs an indication of the concentration of the target analyte in the sample when the reader detects an optical signal from the assay strip that is less than the maximum optical signal.

[0006] Further embodiments disclosed herein relate to a method for determining the concentration of a target analyte in a fluid sample. The method includes applying the fluid sample to the assay strip described above when the complex is coupled to a flow path in a first phase; dissociating the complex from the flow path; flowing the fluid sample and the complex in the flow path through a capture zone in a second phase; binding the complex to an immobilized capture agent in the capture zone; and detecting a signal from the complex bound to the immobilized capture agent in the capture zone. The detected signal can be an optical signal, a fluorescent signal, or a magnetic signal. In some cases, dissociating the complex includes solubilizing the complex with the fluid sample. In some cases, the fluid sample contains an unlabeled target analyte, and the complex does not specifically bind to the unlabeled target analyte in the first or second phase. In another example, the fluid sample contains an unlabeled target analyte, and the complex is configured to compete with the unlabeled target analyte to bind to the immobilized capture agent in the capture zone in a third phase. In one example, the fluid sample does not contain the analyte of interest and detecting comprises detecting a maximum optical signal of a dose-response curve of the test strip.

[0007] Optionally, the method includes determining that the concentration of the analyte in the fluid sample is zero. Optionally, the method further includes displaying an indication that the analyte of interest is not present in the fluid sample. In one example, the fluid sample contains the analyte of interest, and detecting includes detecting a signal from the test strip that is less than the maximum signal of a dose-response curve for the test strip. Optionally, the method further includes determining that the concentration of the analyte in the fluid sample is greater than zero. Optionally, the method further includes displaying an indication that the analyte of interest is present in the fluid sample. In one example, the method further includes determining that the detected signal is within 10% of the maximum optical signal; and displaying an indication that the analyte of interest is present in the fluid sample at a low concentration. In another example, the method further includes determining that the detected signal is 90% or less than 90% of the maximum signal; and displaying an indication that the analyte of interest is present in the fluid sample at a high concentration.

[0008] Additional embodiments disclosed herein relate to a method for manufacturing an assay test strip, the method including: connecting a sample receiving zone to a flow path configured to receive a fluid sample; connecting a capture zone to the flow path downstream of the sample receiving zone; and connecting a conjugate to the flow path. The conjugate includes a label, an antibody or antibody fragment that specifically binds to the analyte of interest, and the analyte of interest. Optionally, the analyte of interest includes C-reactive protein (CRP), and the antibody includes an anti-CRP antibody or an anti-CRP antibody fragment. In one example, the analyte of interest includes approximately 50 ng of CRP. In another example, the analyte of interest includes approximately 100 ng of CRP. Optionally, the method further includes immobilizing a capture agent specific for the analyte of interest to the capture zone. Optionally, connecting the conjugate to the flow path includes forming a bond between the conjugate and the flow path that is cleaved in the presence of the fluid sample in the flow path. In one example, connecting the conjugate includes spraying a solution containing the conjugate onto the surface of the sample receiving zone. In another example, the step of linking the complex comprises spraying a solution containing the complex onto the surface of the assay strip between the sample receiving zone and the capture zone. In a further example, the step of linking the complex comprises applying a fluid solution containing the complex to the surface of the assay strip; and allowing the fluid solution to dry. In yet another example, the step of linking the complex comprises incorporating the complex into the surface of the assay strip.

[0009] Optionally, the method further includes providing a solution containing the complex. Optionally, providing the solution includes mixing a first liquid containing the label and the antibody or antibody fragment with a second liquid containing the analyte of interest. In some examples, providing the solution further includes incubating the mixture of the first liquid and the second liquid for about 30 minutes. Optionally, connecting the complex to the flow path includes spraying the solution onto the surface of the assay strip. Further embodiments disclosed herein relate to assay strips made by the above methods. [Brief explanation of the drawings]

[0010] [Figure 1A] FIG. 1 shows an exemplary sandwich-type lateral flow assay before and after a fluid sample is applied to a sample-receiving zone. [Figure 1B] FIG. 1 shows an exemplary sandwich-type lateral flow assay before and after a fluid sample is applied to a sample-receiving zone. [Figure 2] 1A and 1B are graphs showing exemplary dose-response curves for the lateral flow assays of FIGS. 1A and 1B. [Figure 3A] FIG. 1 shows an exemplary competitive lateral flow assay before and after a fluid sample is applied to a sample-receiving zone. [Figure 3B] FIG. 1 shows an exemplary competitive lateral flow assay before and after a fluid sample is applied to a sample-receiving zone. [Figure 4] 3C is a graph showing an exemplary dose-response curve for the competitive lateral flow assay of FIGS. 3A and 3B. [Figure 5A] 1A-1C show an exemplary lateral flow assay according to the present disclosure before and after a fluid sample is applied to a sample-receiving zone. [Figure 5B] 1A-1C show an exemplary lateral flow assay according to the present disclosure before and after a fluid sample is applied to a sample-receiving zone. [Figure 5C] 5C is a graph showing an exemplary dose-response curve for the lateral flow assay of FIGS. 5A and 5B. [Figure 6A] 1A and 1B, and an exemplary dose-response curve for a lateral flow assay according to the present disclosure. Analyte concentration is measured along the x-axis on a logarithmic scale. [Figure 6B] 6B is a graph showing the exemplary dose-response curve of FIG. 6A for a lateral flow assay according to the present disclosure, where the concentration of the analyte is measured along the x-axis on a non-logarithmic scale. [Figure 7A]1 is a table of experimental data correlating the concentration of CRP measured by a lateral flow assay with the concentration of CRP determined by ELISA according to one embodiment of the present disclosure. [Figure 7B] 1 is a graph representing experimental data correlating the concentration of CRP measured by a lateral flow assay with the concentration of CRP determined by ELISA according to one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0011] The devices, systems, and methods described herein accurately determine the amount of an analyte of interest in a sample, e.g., the concentration of the analyte in a known volume of sample. Advantageously, the lateral flow devices, test systems, and methods of the present disclosure accurately determine the amount of an analyte of interest in situations where the analyte of interest is present in the sample at elevated or "high" concentrations. The lateral flow assays described herein can generate a maximum intensity signal when the concentration of the analyte of interest in the sample is zero. The signals generated by assays of the present disclosure are described herein in the context of optical signals generated by reflectance-type labels (e.g., without limitation, gold nanoparticle labels). While embodiments of the present disclosure are described herein with reference to "optical" signals, it will be understood that the assays described herein generate a detectable signal using any suitable material for the label, including, without limitation, fluorescent latex bead labels that generate a fluorescent signal and magnetic nanoparticle labels that generate a signal indicative of changes in a magnetic field associated with the assay. For low concentrations of analyte, the lateral flow assays described herein generate an optical signal that is the same as or substantially equivalent to (within a limited range of variation from) the maximum intensity signal. Lateral flow assays according to the present disclosure produce less than maximal intensity signals for elevated or "high" concentrations of the analyte of interest.

[0012] According to the present disclosure, a labeled agent comprising a label-antibody-analyte complex is first incorporated into the surface of a lateral flow assay test strip, such as a conjugate pad. When a fluid sample is applied to the test strip, the label-antibody-analyte complex unbinds from the label zone and migrates to the capture zone of the test strip, along with the fluid sample and any target analyte (if present) in the sample. The label-antibody-analyte complex and the target analyte (if present) in the sample bind to the capture agent in the capture zone. If no target analyte is present in the sample to compete with the label-antibody-analyte complex, the capture agent binds completely to the label-antibody-analyte complex, generating a maximum intensity signal. If a low concentration of the target analyte is present in the sample, the label-antibody-analyte complex competes with a relatively small amount of unlabeled analyte to bind to the capture agent, resulting in a signal that is the same as or substantially equivalent to (within a limited range of variation from) the maximum intensity signal. When the analyte of interest is present in the sample at high concentrations, the label-antibody-analyte complex will compete with the relatively large amount of unlabeled analyte to bind to the capture agent, resulting in a less than maximal intensity signal. Without being bound by any particular theory, the addition of labeled analyte in the form of a label-antibody-analyte complex incorporated into the label zone masks the portion of the dose-response curve of a sandwich-type lateral flow assay when the signal increases (when the analyte concentration is low), thereby producing an improved dose-response curve that begins with a maximum intensity signal at zero concentration and then remains relatively constant (for low analyte concentrations) or decreases (for high analyte concentrations). The lateral flow assay of the present disclosure overcomes the drawbacks associated with the hook effect of sandwich-type lateral flow assays by eliminating the phase of the dose-response curve where the signal increases.

[0013] At high analyte concentrations, the signals generated by the lateral flow assays described herein include many advantageous features. In exemplary embodiments that generate optical signals, the signals generated at high analyte concentrations are easily detectable (e.g., have intensities within a range of optical signals typically distinguishable and well-spaced by conventional reading devices), do not overlap on a dose-response curve with signals generated at zero or low concentrations, and these signals can be used to calculate highly accurate concentration readings at high and very high concentrations. The lateral flow assay embodiments described herein avoid uncertainties associated with correlating a particular detection signal with the amount of analyte (especially at high analyte concentrations), e.g., the hook effect, which arises when reading sandwich-type lateral flow assays that generate a single optical signal corresponding to both low and high analyte concentrations. In contrast, lateral flow assays according to the present disclosure generate optical signals that clearly and unambiguously correspond to zero or low analyte concentrations (optical signals that are the same or substantially equivalent to the maximum intensity signal) or to high analyte concentrations (optical signals that are less than the maximum intensity signal). In some cases, a zero or low concentration may directly correlate with a normal or "healthy" level of the analyte in a subject, and a high concentration of the analyte may directly correlate with an abnormal or "unhealthy" level of the analyte in a subject.

[0014] Furthermore, embodiments of lateral flow assays according to the present disclosure correlate strongly with current gold standard assays for determining the amount of an analyte in a sample, such as enzyme-linked immunosorbent assays (ELISAs). Advantageously, it has been discovered that the concentration of CRP determined by embodiments of the lateral flow assays described herein correlates strongly with the concentration of CRP determined by ELISA. In one example described below, a 93% correlation was obtained between the concentration of CRP measured using an embodiment of an assay according to the present disclosure and the concentration of CRP determined by ELISA. The lateral flow assay embodiments described herein are particularly advantageous for diagnostic testing of analytes of interest that naturally occur at low concentrations in healthy individuals but rise to high concentrations in individuals with a disease state or disorder. Optical signals with relatively little variance from the maximum intensity signal are generated across a range of concentrations from zero to low, allowing operators to simply confirm the presence of the analyte at low concentrations (indicative of healthy levels) and not require specificity or resolution in the optical signal, but to generate easily detectable, high-resolution optical signals with greater variance from the maximum intensity signal when operators are attempting to confirm the presence of the analyte at high concentrations (indicative of an abnormal or diseased state), particularly when attempting to quantify the analyte whenever it occurs at high concentrations. The ability to precisely identify the exact concentration of the analyte of interest within a high concentration range allows operators to understand the stage or progression, such as the mild or severe stage, of a disease or other condition of interest.

[0015] Various aspects of lateral flow assays offer advantages over existing lateral flow assays. For example, in some embodiments, the lateral flow assays described herein do not require multiple test lines, instead possessing the ability to both accurately determine the concentration of an analyte and to determine whether the test functioned properly using only one capture line. Furthermore, in some embodiments, the lateral flow assays described herein can accurately determine elevated analyte concentrations in a sample without first requiring sample dilution. Additionally, in some embodiments, the amount of preformed label-antibody-analyte complex placed in the lateral flow assay can be varied to accommodate requirements for different analyte concentration ranges. Various aspects of the devices, test systems, and methods are described in detail below with reference to the accompanying drawings. However, the present disclosure may be embodied in many different forms. Based on the teachings herein, those skilled in the art will understand that the scope of the present disclosure is intended to cover any aspect of the devices, test systems, and methods disclosed herein, whether implemented independently of any aspect of the present disclosure or combined with any other aspect of the present disclosure. For example, a device may be implemented or a method may be performed using any number of the aspects described herein.

[0016] While specific embodiments are described herein, many variations and modifications of these embodiments are within the scope of the present disclosure. While certain benefits and advantages are described, it is not intended that the scope of the present disclosure be limited to any particular benefit, application, or purpose. Rather, aspects of the present disclosure are intended to be broadly applicable to a variety of detection techniques and device configurations, some of which are illustrated in the figures and description below. The detailed description and drawings are merely illustrative of the present disclosure rather than limiting, the scope of which is defined by the appended claims and their equivalents. The lateral flow devices described herein are analytical devices used in lateral flow chromatography. Lateral flow assays are assays that can be performed with the lateral flow devices described herein. Lateral flow devices can be implemented on test strips, although other formats may be suitable. In test strip formats, a test sample liquid suspected of containing an analyte flows through the test strip (e.g., by capillary action). The test strip may be made of a bibulous material such as paper, nitrocellulose, or cellulose. The sample fluid is received in a sample reservoir. The sample fluid is allowed to flow along the test strip to a capture zone, where the analyte (if present) interacts with a capture agent to indicate the presence and / or amount of the analyte. The capture agent can include an antibody immobilized in the capture zone.

[0017] Sandwich and competitive lateral flow assays Lateral flow assays can be performed in sandwich or competitive formats. While the sandwich and competitive assays described herein are described in the context of reflectance labels (e.g., gold nanoparticle labels) that generate an optical signal, it is understood that the assays also include latex bead labels configured to generate a fluorescent signal, magnetic nanoparticle labels configured to generate a magnetic signal, or other labels configured to generate a detectable signal. Sandwich lateral flow assays include a labeled antibody attached to a sample reservoir on a solid substrate. After the sample is applied to the sample reservoir, the labeled antibody dissolves in the sample, where it recognizes and binds to a first epitope on an analyte in the sample, forming a label-antibody-analyte complex. This complex flows along a fluid front from the sample reservoir through the solid substrate to a capture zone (called the "test line") where an immobilized antibody (sometimes called a "capture agent") is located. If the analyte is multimeric, or contains multiple identical epitopes on the same monomer, the labeled antibody attached to the sample reservoir can be the same as the antibody immobilized in the capture zone. The immobilized antibody recognizes and binds to an epitope on the analyte, thereby capturing the label-antibody-analyte complex in the capture zone. The presence of the labeled antibody in the capture zone results in a detectable optical signal in the capture zone. In one non-limiting example, gold nanoparticles are used to label the antibody because they are relatively inexpensive, stable, and provide an easily observable color indicator based on the surface plasmon resonance properties of the gold nanoparticles. In some cases, this signal provides qualitative information, such as whether the analyte is present in the sample. In some cases, this signal provides quantitative information, such as a measure of the amount of analyte in the sample.

[0018] 1A and 1B show an exemplary sandwich-type lateral flow device 10. The lateral flow device 10 includes a sample reservoir 12, a label zone 14, a capture zone 16, and a control line 18. FIGS. 1A and 1B show the lateral flow device 10 before and after a fluid sample 24 is applied to the sample reservoir 12. In the example shown in FIGS. 1A and 1B, the sample 24 includes an analyte of interest 26. The label zone 14, located in or near the sample reservoir 12, includes a labeled agent 28. In this exemplary sandwich-type lateral flow device, the labeled agent 28 includes an antibody or antibody fragment 30 bound to a label 32. A capture agent 34 is immobilized in the capture zone 16. A control agent 35 is immobilized in the control line 18.

[0019] When a fluid sample 24 is applied to the sample reservoir 12, the sample 24 solubilizes the labeled agent 28, which binds to the analyte 26, forming the label-antibody-analyte complex 20. Thus, in the exemplary sandwich-type lateral flow device 10, the label-antibody-analyte complex 20 is not formed until a fluid sample 24 containing the analyte of interest 26 is applied to the lateral flow device. Furthermore, in the exemplary sandwich-type lateral flow device 10, the analyte in the label-antibody-analyte complex 20 is the analyte from the fluid sample 24. As shown in FIG. 1B, this complex 20 flows through the test strip to the capture zone 16, where it is bound by a capture agent 34. The now-bound complex 20 (and specifically, the label 32 on the now-bound complex 20) emits a detectable optical signal at the capture zone 16.

[0020] Labeled agents 28 that do not bind to analyte 26 pass through the capture zone 16 (because no analyte 26 binds to the capture agent 34 in the capture zone 16) and continue downstream through the lateral flow device 10. In a lateral flow assay including a control line 18 as illustrated herein, the attached control agent 35 captures labeled agents 28 that do not bind to analyte 26 and that pass through the capture zone 16 to reach the control line 18. In some embodiments, the control agent 35 captures agents 28 labeled with the Fc region of an antibody. In some embodiments, the control agent 35 captures agents 28 labeled with the Fab region of an antibody. This labeled agent 28 bound to the control line 18 emits a detectable optical signal that can be measured and used to indicate that the assay worked as intended (e.g., sample 24 flowed from the sample reservoir 12 and passed through the capture zone 16 as intended during normal operation of a lateral flow assay). One drawback of the exemplary sandwich-type lateral flow device 10 is that the strength of the signal generated in the control line 18 depends on the strength of the signal generated in the capture zone 16 (because the control agent 35 in the control line 18 does not bind to the analyte 26 in the capture zone 16 and then captures the labeled agent 28 delivered to the control line 18). For example, if a relatively large amount of analyte 26 binds in the capture zone 16, a relatively small amount of analyte 26 will pass through the capture zone 16 and become available to bind to the control agent 35 in the control line 18, resulting in a relatively weak intensity signal in the control line 18.

[0021] Lateral flow assays can provide qualitative information, such as information regarding the presence or absence of a target analyte in a sample. For example, detection of any measurable optical signal in the capture zone 16 can indicate that the target analyte is present in the sample (in some unknown amount). The absence of a measurable optical signal in the capture zone can indicate that the target analyte is absent or below the detection limit. For example, if the sample 24 did not contain the target analyte 26 (not shown), the sample 24 would still solubilize the labeled agent 28, which would still flow to the capture zone 16. However, the labeled agent 28 would not bind to the capture agent 34 in the capture zone 16. Instead, the labeled agent 28 would flow through the capture zone 16, pass through the control line 18, and, potentially, to any absorption zone. Some of the labeled agent 28 may bind to the control agent 35 deposited on the control line 18 and emit a detectable optical signal. In these circumstances, the absence of a measurable optical signal emanating from the capture zone 16 is an indication that the analyte of interest is not present in the sample 24, and the presence of a measurable optical signal emanating from the control line 18 is an indication that the sample 24 has traveled from the sample receiving zone 12, through the capture zone 16 and to the capture line 18, as intended during normal operation of a lateral flow assay.

[0022] Some lateral flow devices can provide quantitative information, such as a measurement of the amount of an analyte of interest in a sample. The quantitative measurement obtained from a lateral flow device can be the concentration of the analyte present in a given volume of sample. Figure 2 shows an exemplary quantitative measurement obtained from the sandwich-type lateral flow assay shown in Figures 1A and 1B. Figure 2 is a dose-response curve graphically illustrating the relationship between the intensity of the signal detected in the capture zone (measured along the y-axis) and the concentration of the analyte in the sample (measured along the x-axis). Examples of signals include optical, fluorescent, and magnetic signals.

[0023] As shown by the first data point of zero concentration in Figure 2, if a sample does not contain the analyte of interest, the concentration of analyte in the sample is zero, and no analyte binds to the labeled antibody to form a label-antibody-analyte complex. In this situation, no complex flows to the capture zone and binds to the capture antibody. Therefore, no detectable optical signal is observed in the capture zone, and the signal magnitude is zero. As the concentration of analyte in the sample increases from zero concentration, a signal is detected. As shown by the data points in Phase A, the signal increases as the concentration of analyte in the sample increases. This occurs because as the analyte concentration increases, the formation of label-antibody-analyte complexes increases. The capture agent immobilized in the capture zone binds to the complexes that flow into the capture zone, increasing in number, resulting 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.

[0024] In some cases, excess analyte is present in a sample when the concentration of analyte exceeds the amount of labeled agent available to bind to the analyte. In these situations, excess analyte not bound to the labeled agent competes with the labeled-antibody-analyte complex to bind to the capture agent in the capture zone. The capture agent in the capture zone binds unlabeled analyte (i.e., analyte not bound to the labeled agent) and the labeled-antibody-analyte complex. However, unlabeled analyte that binds to the capture agent does not emit a detectable signal. As the concentration of analyte in the sample increases in Phase B, an increasing amount of unlabeled analyte binds to the capture agent (instead of the labeled-antibody-analyte complex, which emits a detectable signal). As more unlabeled analyte binds to the capture agent instead of the labeled-antibody-analyte complex, the signal detected in the capture zone decreases, as shown by the Phase B data points. This phenomenon of increased detection signal in phase A and decreased detection signal in phase B is called the "hook effect." As the concentration of analyte increases in phase A, more analyte binds to the labeled drug, resulting in an increase in signal intensity. 飽和At this point, the labeled agent is saturated with analyte from the sample (e.g., all or nearly all of the available amount of labeled agent is bound to analyte from the sample) and the detected signal reaches a maximum value, Signal 最大 As the concentration of analyte in the sample continues to increase in Phase B, excess analyte beyond the labeled drug saturation point competes with the labeled drug-analyte to bind to the capture agent, resulting in a decrease in the detection signal.

[0025] The hook effect, also known as the "prozone effect," adversely affects lateral flow assays, especially in situations where the analyte of interest 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 result in false negative or incorrectly low results. Specifically, inaccurate results occur when a sample contains elevated levels of analyte that exceed the concentration of the labeled reagent deposited on the test strip. In this case, when the sample is placed on the test strip, the labeled reagent becomes saturated and not all of the analyte is labeled. Unlabeled analyte flows through the assay, binds in the capture zone, and competes to displace the labeled complex, thereby reducing the detectable signal. Therefore, a single detection signal corresponds to both low and high concentrations, and the device (or the device operator) cannot distinguish whether the optical signal corresponds to a low or high concentration. If the analyte level is sufficiently large, the analyte competes to displace the labeled complex, and no signal is observed in the capture zone, resulting in a false-negative test result.

[0026] 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 a sample compete with labeled target analytes for binding to a capture agent in the capture zone. Figures 3A and 3B show an exemplary competitive lateral flow assay 22. The lateral flow device 22 includes a sample reservoir 12, a label zone 14, and a capture zone 16. Figures 3A and 3B show the lateral flow device 22 before and after a fluid sample 24 is applied to the sample reservoir 12. In the example shown in Figures 3A and 3B, the fluid sample 24 contains a target analyte 26. The label zone 14, located in or near the sample reservoir 12, contains a labeled agent 29. In this exemplary competitive lateral flow device, the labeled agent 29 contains the target analyte 26 bound to a label 32. A capture agent 34 is immobilized in the capture zone 16.

[0027] A sample 24 containing unlabeled analyte 26 is applied to sample reservoir 12. Sample 24 solubilizes labeled agent 29. The unlabeled analyte 26 and labeled agent 29 in sample 24 flow together to capture zone 16, where both the unlabeled analyte 26 and labeled agent 29 from sample 24 bind to a capture agent 34 immobilized in capture zone 16. As shown in FIG. 3B, the labeled agent and unlabeled analyte 26 compete with each other to bind to a fixed amount of capture agent 34. The labeled agent 29 (and specifically the label 32 of the labeled agent 29) bound to capture agent 34 emits a detectable optical signal, whereas the unlabeled analyte 26 from sample 24 bound to capture agent 34 does not emit a detectable optical signal.

[0028] Detection of the optical signal from the capture zone 16 can provide qualitative or quantitative information about the target analyte 26. When the fluid sample 24 does not contain the analyte 26 (not shown), the sample 24 still solubilizes the labeled agent 29, which still flows into the capture zone 16. The capture agent 34 in the capture zone 16 binds to the labeled agent 29 (not competing with unlabeled analyte from the sample), resulting in a maximum or near-maximum detected optical signal. A maximum or near-maximum detected optical signal may also be detected when the sample 24 contains a very low or low concentration of analyte 26. 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 a maximum detected optical signal correlates with zero or low concentrations of analyte 26 in the sample 24.

[0029] As the concentration of unlabeled analyte 26 in sample 24 increases, the optical signal emitted and detected from capture zone 16 decreases. This is because competition for capture agent 34 increases with increasing analyte concentration in the sample, resulting in a progressive increase in the ratio of unlabeled analyte 26 bound to capture agent 34 relative to labeled agent 29 bound to capture agent 34. However, when the analyte is present in the sample at high or very high concentrations, the optical signal detected at capture zone 16 rapidly decreases to a low intensity signal. This rapid decrease in optical 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 analyte concentration, and in some cases, prevents the device from determining the analyte concentration at all. Competitive lateral flow devices such as those shown in FIGS. 3A and 3B are virtually incapable of accurately determining the exact concentration of the analyte of interest when the analyte is present at high concentrations (e.g., when the ratio of unlabeled analyte to labeled agent is high). Figure 4 shows a dose-response curve generated in an exemplary competitive lateral flow device, such as that described above with reference to Figures 3A and 3B. As shown in Figure 4, the dose-response curve for a competitive lateral flow assay shows a sharp decrease in signal with analyte concentration in the range of approximately 1 to 20 μg / mL. Because of the sharp decrease in the curve, resolution is poor and accuracy in determining the amount of analyte at high concentrations is reduced, making it impractical or virtually impossible in some cases to determine with any degree of accuracy the amount of analyte present at high concentrations in a sample.

[0030] Exemplary Lateral Flow Device for Accurately Quantifying Analytes Present at High Concentrations in a Sample The lateral flow assays, test systems, and methods described herein address these and other shortcomings of sandwich-type and competitive-type lateral flow assays, such as those shown in Figures 2A, 2B, 3A, and 3B. Figures 5A and 5B illustrate an exemplary lateral flow assay 100 capable of accurately measuring the amount of an analyte of interest present at high concentrations in a sample. Figure 5C is an exemplary dose-response curve graphically illustrating the relationship between the optical signal measured from the lateral flow assay 100, and in particular the magnitude of the optical signal detected in the capture zone (measured along the y-axis), and the analyte concentration in the sample applied to the assay (measured along the x-axis). While assays according to the present disclosure are described in the context of reflectance labels that generate an optical signal, it will be understood that assays according to the present disclosure may include labels of any suitable material configured to generate a fluorescent, magnetic, or other detectable signal. Lateral flow assay 100 includes a test strip 110 having a sample receiving zone 112, a label zone 114, and a capture zone 116. Figures 5A and 5B show lateral flow device 100 before and after a fluid sample 124 is applied to sample reservoir 112. In the illustrated example, label zone 114 is downstream of sample receiving zone 112 along the direction of sample flow 118 within test strip 110. In some cases, sample receiving zone 112 is disposed within and / or coextensive with label zone 114. A capture agent 134 is immobilized in capture zone 116.

[0031] The labeled agent 128 is incorporated into the label zone 114. In lateral flow devices according to the present disclosure, such as the non-limiting examples described with reference to FIGS. 5A and 5B, the labeled agent 128 includes at least three components that bind together to form a complex: a label (detection molecule) 132, an analyte of interest 126, and an antibody or antibody fragment 130 specific for the analyte of interest 126. The labeled agent 128 is a label-antibody-analyte complex 128. In some cases, the labeled agent 128 is formed and applied to the test strip 110 by an operator prior to use of the test strip 110. For example, the labeled agent 128 can be incorporated into the label zone 114 during manufacture of the test strip 110. In another example, the labeled agent 128 is incorporated into the label zone 114 after manufacture but prior to application of a fluid sample to the test strip 110. The labeled agent 128 can be incorporated into the test strip 110 by a number of methods, which are discussed in more detail below.

[0032] Thus, in embodiments of the lateral flow device of the present disclosure, the label-antibody-analyte complex 128 is formed and incorporated into the test strip 110 before any fluid sample 124 is applied to the lateral flow device. In one non-limiting example, the label-antibody-analyte complex 128 is formed and incorporated onto the conjugate pad of the test strip 110 before any fluid sample 124 is applied to the lateral flow device. Furthermore, in embodiments of the lateral flow device of the present disclosure, the analyte in the label-antibody-analyte complex 128 is not the analyte from the fluid sample 124.

[0033] To perform a test using the test strip 110, a sample 124, which may or may not contain an analyte of interest 126, is applied to the sample-receiving zone 112. In the illustrated embodiment, in which the label zone 114 is downstream of the sample-receiving zone 112, the unlabeled analyte of interest 126 in the sample 124 then flows into the label zone 114 and comes into contact with the incorporated labeled agent 128. The sample 124 solubilizes the labeled agent 128. In one non-limiting example, the sample 124 dissolves the labeled agent 128. The bonds that held the labeled agent 128 to the surface of the test strip 110 within the label zone 114 are released, resulting in the labeled agent 128 no longer being incorporated onto the surface of the test strip 110. The labeled agent 128, along with any unlabeled analyte 126 in the sample 124, then migrate along the fluid front to the capture zone 116. The capture agent 134 in the capture zone 116 binds to the labeled agent 128 and, if present, the analyte 126 from the sample 124. Depending on the amount of unlabeled analyte 126 in the sample 124, the labeled agent 128 and the unlabeled analyte 126 compete with each other to bind to the capture agent 134 in the capture zone.

[0034] Thus, a lateral flow device according to the present disclosure has a labeled agent containing a label-antibody-analyte complex that binds to the label zone of the lateral flow device in a first phase (e.g., before applying a fluid sample to the lateral flow device) and then migrates through the test strip in a subsequent second phase (e.g., upon application of a fluid sample to the sample receiving zone). The labeled agent according to the present disclosure can bind to a capture agent in the capture zone in a third phase (e.g., after the fluid sample has flowed to the capture zone). Thus, the labeled agent described herein is initially located in a first region (e.g., the label zone) of the lateral flow device, then (when contacted with fluid) migrates with the fluid to another region of the lateral flow device downstream from the first region and then binds to the capture agent in the capture zone.

[0035] As described above, the fluid sample 124 solubilizes the labeled agent 128. In one embodiment, the analyte of interest 126 in the sample 124 does not, or does not substantially, interact with the labeled agent 128 during this process. Without being bound to any particular theory, in this implementation of the lateral flow device described herein, the unlabeled analyte of interest 126 does not conjugate, bind, or associate with the labeled agent 128 as the sample 124 flows through the label zone 114. This is in contrast to the sandwich-type lateral flow device described above with reference to FIGS. 1A and 1B, in which the labeled agent 28 binds to the unlabeled analyte of interest 26 as the sample 24 flows through the label zone 114. In another embodiment of the lateral flow device described herein, the analyte of interest 126 in the sample 124 interacts with the labeled agent 128 as the fluid sample 124 solubilizes the labeled agent 128. Without being bound to any particular theory, in this implementation, the capture agent 134 in the capture zone 116 may bind to at least some of the label-antibody-analyte complexes, where the analyte in the complex is the target analyte 126 introduced into the device via the sample 124.

[0036] When the target analyte 126 is not present in the sample 124 (not shown), the labeled agent 128 saturates the capture agents 134 in the capture zone 116 (e.g., each capture agent 134 molecule in the capture zone 135 binds to one labeled agent 128 that has flowed from the label zone 114). The labeled agent 128 captured in the capture zone 116 emits a detectable optical signal that is the maximum intensity signal obtainable from the lateral flow device 100. The optical signal detected in the capture zone 116 when the target analyte 126 is not present in the sample 124 is referred to herein as the "maximum intensity signal" because each available capture agent 134 binds to a labeled agent 128 in the capture zone 116. In the non-limiting example shown in FIG. 5C, the maximum intensity signal obtained when the concentration of the target analyte is zero is at or about 76 AU (arbitrary signal intensity units).

[0037] There are many ways to determine the maximum intensity signal of a lateral flow device 100. In one non-limiting example, the maximum intensity signal obtainable from a particular lateral flow device 100 can be empirically determined and stored in a look-up table. In some cases, the maximum intensity signal is determined empirically by testing lateral flow devices 100 of known characteristics and configuration, for example, by averaging the maximum intensity signals obtained when a zero or near-zero concentration of the analyte of interest is applied to a lateral flow device 100 of known specifications and configuration. In another non-limiting example, the maximum intensity signal obtainable from a particular lateral flow device 100 can be determined using logical calculations given the known specifications and configuration of the lateral flow device 100 (e.g., the amount and specific characteristics of the labeled agent 128 incorporated in the labeling zone 114).

[0038] Furthermore, although the term "maximum intensity signal" is used herein, it is understood that a signal that falls within a certain range of the expected maximum intensity can be considered substantially equivalent to the "maximum intensity signal." It is further understood that the term "maximum intensity signal" can refer to a maximum intensity optical signal, a maximum intensity fluorescent signal, a maximum intensity magnetic signal, or any other type of signal that occurs at maximum intensity. As one non-limiting example, a detected signal that is within 1% of the expected maximum intensity signal can be considered substantially equivalent to the expected maximum intensity signal. If the maximum intensity signal is at or about 76 AU, a detected signal that is within the range of about 75.24 AU to about 76.76 AU can be considered substantially equivalent to a 76 AU maximum intensity signal. As another example, in the non-limiting embodiment described with reference to FIGS. 5C, 6A, and 6B, a detected signal that is within 10% of the expected maximum intensity signal can be considered substantially equivalent to the expected maximum intensity signal. Thus, in the example shown in FIG. 5C , where the maximum intensity signal is at or about 76 AU, a detection signal within the range of about 68.4 AU to about 83.6 AU would be considered substantially equivalent to a maximum intensity signal of 76 AU. Other variations are permissible, and these examples are provided for illustrative purposes only. For example, in a lateral flow assay device according to the present disclosure, a detection signal within any suitable range of variation from the expected maximum intensity signal (e.g., without limitation, within 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 2.0%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14%, or 15% of the expected maximum intensity signal) would be considered substantially equivalent to the expected maximum intensity signal.

[0039] In cases such as those shown in FIGS. 5A and 5B, where the target analyte 126 is present in the sample 124, the labeled agent 128 from the labeling zone 114 and the analyte 126 from the sample flow to the capture zone 116, where they compete to bind to the capture agent 134. In one example, the target analyte 126 is present in the sample 124 at a low concentration. The target analyte 126 can be considered to be present in the sample 124 at a low concentration when the optical signal detected in the capture zone 116 is the same, substantially the same, and / or within a certain range of variation from the maximum intensity signal. In one non-limiting example, the target analyte 126 is considered to be present in the sample at a low concentration when the detected optical signal is within 5% of 76 AU (or within about 72.2 AU to about 79.8 AU). An optical signal within 5% of 76 AU correlates to a concentration of the target analyte between 0 and about 1 μg / mL; in this example, a concentration of 0 to about 1 μg / mL is considered to be a low concentration of the target analyte. In the non-limiting example shown in FIG. 5C , when the detected optical signal is within 10% of 76 AU (or within about 68.4 AU to about 83.6 AU), the target analyte 126 is considered to be present at a low concentration in the sample. An optical signal within 10% of 76 AU correlates with a target analyte concentration of 0 to about 10 μg / mL, which in this example is considered to be a low target analyte concentration. In cases where relatively little target analyte 126 is present in sample 124, the ratio of target analyte 126 from sample 124 bound to the capture agent relative to the labeled agent 128 bound to the capture agent is low. In such cases, the optical signal detected in the capture zone 116 is the same as or slightly less than the maximum intensity signal that would be detected if the target analyte 126 were not present in sample 124.

[0040] As the concentration of analyte 126 in sample 124 increases from about 1 μg / mL to 10 μg / mL, and then to concentrations of 20 μg / mL or greater, more analyte 126 becomes present in capture zone 116 and competes with and binds to labeled agent 128, capturing agent 134. This results in less labeled agent 128 binding in capture zone 134 as the concentration of analyte 126 increases, and the optical signal detected in capture zone 116 decreases. As shown in Figure 5C, in embodiments of lateral flow devices according to the present disclosure, the signal decrease with increasing concentration of the analyte of interest is advantageously gradual. As a result of this gradual decrease in detected signal, embodiments of lateral flow devices described herein advantageously allow the detector to accurately measure the signal with high resolution and the data analyzer to determine the concentration of the analyte of interest with high accuracy when the concentration is high. This is in contrast to the competitive lateral flow devices described above with reference to Figures 3A, 3B, and 4.

[0041] Additionally, the dose-response curve of a lateral flow device according to the present disclosure advantageously begins with a maximum intensity signal and then decreases from this maximum intensity signal. This advantageously means that the portion of the dose-response curve where the signal is decreasing does not have a signal with the same magnitude as the maximum intensity signal. Furthermore, because the signal at low concentrations of analyte in the sample is the same or substantially the same as the maximum intensity signal (e.g., these signals are considered to be substantially equivalent to the maximum intensity signal), there is a plateau of relatively constant value of the optical signal ("maximum intensity signal") for zero to low concentrations of analyte (as will be described in more detail below with reference to non-limiting examples). This advantageously means that the portion of the dose-response curve where the signal is decreasing does not have a signal with approximately the same magnitude as the maximum intensity signal. Thus, false negatives and inaccurately low readings are avoided in the embodiments of the lateral flow device described herein. This is in contrast to the sandwich-type lateral flow device described above with reference to FIGS. 1A, 1B, and 2, in which a high concentration of analyte in the sample generates a signal that is the same or approximately the same as the signal generated when the analyte concentration is low.

[0042] Advantageously, in embodiments of the lateral flow device described herein, the labeled agent 128 can be pre-formulated to contain a known amount of the target analyte before application to the conjugate pad. In some embodiments, a known concentration of the target analyte is incubated with an antibody or antibody fragment and a labeled molecule in a reaction vessel separate from the test strip. During incubation, the target analyte conjugates, binds, or associates with the antibody and labeled molecule to form the labeled agent 128 described above. After incubation, the labeled agent 128 is added directly to a solution at a precise, known concentration or isolated, and excess free CRP is removed before spraying onto the conjugate pad. The solution containing the labeled agent 128 is applied to the test strip, such as the label zone 114 described above. During application, the labeled agent 128 is incorporated into the surface of the test strip. In one non-limiting example, the labeled agent is incorporated onto the conjugate pad of the test strip. Advantageously, the labeled agent 128 physically binds to the surface of the test strip and remains chemically stable until the operator applies a fluid sample to the test strip, after which the labeled agent 128 is released from the test strip and flows with the fluid sample as described above.

[0043] In some embodiments, the labeled agent 128 is deposited in an amount ranging from about 0.1 to 20 μm per test strip, or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μm per test strip in the labeling zone.

[0044] The solution containing the labeled agent 128 can be applied to the test strip in many different ways. In one example, the solution is applied to the labeling zone 114 by spraying the solution with an air jet technique. In another example, the solution containing the labeled agent 128 is applied by pouring the solution, spraying the solution, formulating the solution as a powder or gel that is placed or rubbed onto the test strip, or other suitable methods of applying the isolated labeled agent 128. In some embodiments, the labeled agent 128 is dried on the surface of the test strip after application by heating or blowing air over the conjugate pad. Other mechanisms for drying the labeled agent 128 on the surface of the test strip are suitable. For example, vacuum or freeze-drying can also be used to dry the labeled agent 128 on the conjugate pad. In some cases, the isolated labeled agent 128 is not added to a solution before application, but instead is applied directly to the test strip. The labeled agent 128 may be applied directly using any suitable method, including, but not limited to, applying compression or vacuum pressure to the labeled agent 128 on the surface of the test specimen and / or applying the labeled agent 128 in the form of lyophilized particles to the surface of the test specimen.

[0045] The lateral flow assay embodiment shown in FIGS. 5A and 5B does not need to include a control line or zone configured to verify that the sample applied to the sample receiving zone 112 flows to the capture zone 116 as intended. Under normal operating circumstances, as the sample flows through the capture zone 116, some detectable signal will always be emitted from the capture zone 116. This is true even when the analyte of interest is present in the sample at extremely low concentrations, because the lateral flow devices of the present disclosure have a dose-response curve that remains at or near a maximum intensity signal at low concentrations. Therefore, the absence of a detectable signal in the capture zone 116 after the sample is applied to the sample receiving zone 112 can be used as an indicator that the lateral flow assay did not operate as intended (e.g., the sample did not flow to the intended capture zone 116, or, as another example, the immobilized capture agent 134 in the capture zone is defective or faulty). Thus, a further advantage of embodiments of lateral flow devices according to the present disclosure is the ability of the capture zone to function as a control line, thereby allowing a separate control line to be omitted entirely from the test strip, although it is understood that control lines may be included in embodiments of lateral flow devices described herein for a variety of purposes, including, but not limited to, as observation lines for normalizing noise or detecting interference from analytes in serum.

[0046] In some cases, a lateral flow assay according to the present disclosure includes a control line, such as a control line similar to control line 18 described above with reference to FIGS. 1A and 1B. In one embodiment (not shown), the lateral flow assay includes a control line containing a capture reagent that emits a signal having an intensity independent of the intensity of the signal generated by the labeled agent 128 in the capture zone 116. In one embodiment, the lateral flow assay includes multiple capture zones (including at least one capture zone 116 configured to capture a labeled agent 128 according to the present disclosure), each capture zone configured to indicate the presence or absence and / or concentration of a different analyte of interest, and a single control line that indicates that the sample has flowed through the multiple capture zones as intended. In contrast to control line 18 described above with reference to FIGS. 1A and 1B, the intensity of the signal emitted from the control line in this implementation may not be related to or dependent on the intensity of the signal emitted from any of the capture zones. Embodiments including a control line may also be advantageous in instances where the capture zone 116 emits a signal of relatively low intensity when the analyte of interest is present in a sample at very high concentrations. In such cases, the signal emitted from the capture zone 116 may be of insufficient intensity to confirm that the assay worked as intended (e.g., that the sample flowed past the capture zone 116 as intended).

[0047] Additionally, a multiplex assay testing for the presence and / or amount of multiple different analytes of interest may include a lateral flow assay according to the present disclosure (as described above with reference to FIGS. 5A and 5B) on the same test strip as one or more sandwich-type lateral flow assays described above with reference to FIGS. 1A and 1B. In such multiplex assays, a control line is not required for the lateral flow assay according to the present disclosure, but a control line may still be advantageously included on the test strip to confirm that the sample has flowed through the control zone associated with the sandwich-type lateral flow assay. This option of including a control line for one assay and omitting a control line for an assay according to the present disclosure may be particularly beneficial in multiplex assays where the number of lines or zones that can be placed on the test strip is limited. The following non-limiting examples illustrate features of the lateral flow devices, test systems, and methods described herein and are not intended to limit the scope of the present disclosure in any way. [Example]

[0048] (Example 1) Preparation of a lateral flow assay to quantify elevated protein concentrations The following example describes the preparation of a lateral flow assay for quantifying an analyte of interest described herein. In this non-limiting example, the analyte of interest is C-reactive protein (CRP), a protein present in serum samples at elevated or high concentrations. CRP is a protein found in plasma. CRP levels increase in response to inflammation. Therefore, CRP is an inflammation marker that can be used to screen for inflammation. Elevated CRP levels in a subject's serum can correlate with inflammation, viral infection, and / or bacterial infection in the subject. Normal levels of CRP in healthy human subjects range from about 1 μg / mL to about 10 μg / mL. CRP concentrations range from 10 to 40 μg / mL during mild inflammation and viral infection, 40 to 200 μg / mL during active inflammation and bacterial infection, and greater than 200 μg / mL in severe bacterial infection and burns. Measuring and charting CRP levels can be useful in determining disease progression or the effectiveness of treatment.

[0049] An assay prepared according to this non-limiting example can be used to determine the precise concentration of CRP (the analyte of interest) in serum samples, even if it exceeds the normal level of CRP in healthy human subjects (approximately 1 μg / mL to approximately 10 μg / mL). This assay includes a labeled agent, including an antibody-label-CRP complex, which avoids some of the drawbacks of sandwich-type lateral flow assays, including those associated with the hook effect. To prepare the assay, anti-C-reactive protein (anti-CRP) antibody was incubated with gold nanoparticles to form a labeled anti-CRP antibody. The labeled antibody was then incubated with CRP to form a complex of the labeled antibody bound to CRP. The complex was deposited onto the conjugate pad (labeled zone) at a volume of 1.8 μL per test strip by spraying a solution containing the complex with an air jet. The conjugate pad was heated to dry the complex onto the conjugate pad.

[0050] The amount of antibody-labeled-CRP conjugate attached to the conjugate pad was carefully considered to ensure the necessary amount of conjugate provided an optimal range of optical signal at the capture zone to allow quantification of elevated levels of CRP in the test system. Attaching an excessive amount of conjugate to the conjugate pad shifts the dose-response curve, resulting in excessively high quantifiable CRP concentrations (potentially generating an optical signal for very high concentrations of CRP, if present, but not for moderate to high concentrations). Attaching an insufficient amount of conjugate to the conjugate pad shifts the dose-response curve in the other direction, resulting in an unquantifiable signal for very high CRP concentrations. Table 1 shows the results of experiments conducted to determine the optimal amount of antibody-labeled-CRP conjugate attached to the conjugate pad. The amount of preformed labeled-antibody-analyte conjugate attached to the conjugate pad can be varied to accommodate requirements for different analyte concentration ranges.

[0051] [Table 1]

[0052] In this example, the optimal amount of antibody-labeled-CRP conjugate added to the conjugate pad results in 50 ng of CRP deposited on the conjugate pad, corresponding to a signal of 70.06 AU. At this amount, the ratio of unlabeled CRP in the sample to the antibody-labeled-CRP conjugate generates a strong optical signal above the optimal range of unlabeled CRP concentrations when they compete to bind to the capture agent in the capture zone, thereby allowing adequate resolution of the signal and accurate quantification of elevated CRP concentrations in the sample. Advantageously, depositing 50 ng of CRP on the conjugate pad (by depositing an appropriate amount of antibody-labeled-CRP conjugate on the conjugate pad) results in a ratio of unlabeled CRP to labeled agent (antibody-labeled-CRP conjugate) that can be seen in the portion of the dose-response curve of a sandwich-type assay where the optical signal is decreasing (e.g., phase B in Figure 2). This ratio of unlabeled CRP to labeled agent (antibody-label-CRP complex) allows the lateral flow assay of this example to mask the portion of the dose-response curve of a sandwich-type assay where the optical signal is increasing (e.g., phase A in FIG. 2 ). Without being bound by any particular theory, it is believed that the lateral flow assay embodiment of this example, by adding an optimal amount (50 ng in this example) of CRP to the conjugate pad, effectively masks the portion of the sandwich-type lateral flow assay where the optical signal is increasing by using only the portion of the dose-response curve that shows a decrease in signal intensity (the portion of the curve that shows the "hook effect"), thereby avoiding the disadvantages described above with reference to FIGS. 1A, 1B, and 2 . In this example, anti-CRP antibody was attached to the capture zone at 2 mg / mL, and goat anti-mouse antibody was attached to the control zone at 2 mg / mL.

[0053] (Example 2) Quantification of high-level C-reactive protein using a lateral flow assay Due to the hook effect, sandwich-type lateral flow assays such as those described above with reference to Figures 1A and 1B are generally inappropriate for quantifying CRP concentrations when CRP is present at elevated levels in a sample. Determining elevated concentrations previously required serial dilutions of the sample, an inefficient and tedious process. However, the lateral flow devices, test systems, and methods described herein allow for accurate, reliable, and rapid quantification of CRP concentrations above healthy levels.

[0054] The lateral flow assay prepared in Example 1 was contacted with samples containing various concentrations of CRP, as shown in the last column of Table 2 below. In this example, the amount of antibody-labeled-CRP complex added to the conjugate pad resulted in 100 ng of CRP attached to the conjugate pad. A sandwich-type lateral flow assay, as described above with reference to Figures 1A and 1B, was contacted with the same samples shown in the middle column of Table 2. As noted above, the sandwich-type lateral flow assay referenced in the middle column contained only labeled antibody attached to the conjugate pad (no CRP attached to the conjugate pad via the antibody-labeled-CRP complex). Fluid samples were prepared by adding the amount of CRP shown in the first column of Table 2 to 30 μL of human serum. The sample was received on the lateral flow assay and chased 15 seconds later with 45 μL of HEPES buffer. After 10 minutes, the optical signal was measured. All samples were run in sextuplicate, and the average values ​​are reported in Table 2. Figure 6A shows the dose-response curves obtained for a lateral flow assay using a labeled antibody attached to a conjugate pad (solid line with diamonds) and a lateral flow assay using an antibody-label-CRP complex attached to a conjugate pad (dashed line with squares), where the analyte concentration is measured along the x-axis on a logarithmic scale. Figure 6B shows the exemplary dose-response curve of Figure 6A for a lateral flow assay using an antibody-label-CRP complex, where the analyte concentration is measured along the x-axis on a non-logarithmic scale.

[0055] [Table 2]

[0056] Figure 6A highlights the significant difference between a sandwich lateral flow assay with the hook effect and a lateral flow assay according to the present disclosure. In a sandwich lateral flow assay with the hook effect, a CRP concentration greater than 10 μg / mL (1.00 on the logarithmic scale) generates an optical signal of the same intensity as a CRP concentration less than 10 μg / mL. In contrast, a lateral flow assay according to the present disclosure allows for accurate determination of CRP concentrations at concentrations greater than 10 μg / mL. This is particularly advantageous in this example, where the analyte of interest is CRP and CRP is elevated to concentrations greater than 10 μg / mL in the presence of inflammation or a disease state. The lateral flow assay embodiments described herein allow a user to reliably determine that a subject's CRP concentration is above normal levels. If a test according to the present disclosure is performed and shows a CRP concentration higher than healthy levels (e.g., greater than 10 μg / mL), this information can be correlated with an inflammatory, viral infection, and / or bacterial infection state.

[0057] Furthermore, because the exact concentration of CRP in a test subject can be accurately determined, test results can be correlated with specific types of disease states. For example, a concentration of 10 μg / mL to 20 μg / mL may correlate with mild inflammation, while a concentration of 40 μg / mL to 200 μg / mL may correlate with bacterial infection. Furthermore, because the exact concentration of CRP in a test subject can be accurately determined, test results can be correlated with disease stages. For example, a concentration of 40 μg / mL to 200 μg / mL may correlate with mild bacterial infection, while a concentration greater than 200 μg / mL may correlate with severe bacterial infection. These examples are illustrative and are not intended to limit the scope of the present disclosure.

[0058] The lateral flow assay devices, systems, and methods disclosed herein offer additional advantages. For example, the lateral flow assays disclosed herein enable reliable quantification of analytes in a sample by utilizing the portion of the dose-response curve that exhibits the Hook effect. As shown in Figure 6A, concentrations of CRP below healthy levels (approximately 10 μg / mL or less) result in a signal within 10% of the maximum intensity of 76 AU (76.20 AU to 70.29 AU). Thus, low-concentration samples produce a plateau of relatively constant signal (in this case, within 10% of the maximum intensity signal of 76 AU). In embodiments of the lateral flow assays disclosed herein, this overlap in the optical signal at low CRP concentrations is not a drawback because low concentrations of CRP are always present in healthy subjects, and the test does not need to be highly sensitive to low levels of CRP.

[0059] Instead, the lateral flow assay of the present disclosure is advantageously particularly sensitive to analytes of interest present at high concentrations. High concentrations of analytes generate signals that are not on or near a plateau—in this case, a signal less than about 70 AU. Lateral flow assays generate signals that gradually decrease as CRP concentrations exceed healthy levels (above 10 μg / mL), and in this case, the signals are easily detectable (have distinguishable signal intensities and sufficient spacing), and do not overlap with other signals on the dose-response curve. This eliminates uncertainty in determining the amount of analyte at a particular detection signal, such as in sandwich-type lateral flow assays, which generate signal values ​​that can correspond to multiple analyte amounts due to the hook effect. In such situations, the user cannot determine whether the analyte concentration is low or high, creating uncertainty for diagnostic purposes. In contrast, lateral flow assays of the present disclosure generate signals that clearly and unambiguously correspond to zero or low analyte concentrations (signals that are the same as or substantially equivalent to the maximum intensity signal) or high analyte concentrations (signals that are less than the maximum intensity signal). These signals can then be directly correlated to normal levels of the analyte (zero or low concentrations of the analyte) or non-normal levels of the analyte (high concentrations of the analyte). Furthermore, the lateral flow devices described herein quantify elevated analyte concentrations in a sample in a single assay without the need for sample dilution. In contrast, the assays described with reference to Figures 1A, 1B, 3A, and 3B require dilution of samples containing high concentrations of analyte, otherwise signals in the high-concentration portion of the dose-response curve cannot be distinguished. The lateral flow assays of the present disclosure can determine even small differences in elevated analyte concentrations based on a single signal obtained at the capture zone after a single test.

[0060] (Example 3) CRP concentrations measured using the disclosed lateral flow assay correlate highly with ELISA assays Furthermore, embodiments of lateral flow assays according to the present disclosure correlate strongly with current gold standard assays for determining the amount of an analyte in a sample, such as enzyme-linked immunosorbent assays (ELISAs). Advantageously, it has been discovered that the concentrations of CRP determined by embodiments of the lateral flow assays described herein correlate strongly with the concentrations of CRP determined by ELISA. Figure 7A is a table summarizing the concentrations of CRP in various serum samples measured using a lateral flow assay according to the present disclosure and the concentrations of CRP in the same serum samples measured using ELISA. Figure 7B is a chart correlating the CRP concentrations obtained according to the present disclosure with the CRP concentrations measured by ELISA. As shown in Figure 7B, a 93% correlation was obtained between the concentrations of CRP measured using embodiments of the assay according to the present disclosure and the concentrations of CRP determined by ELISA.

[0061] Methods of diagnosing disease conditions using lateral flow assays according to the present disclosure Some embodiments provided herein relate to methods of diagnosing a medical condition using a lateral flow assay. In some embodiments, the method includes providing a lateral flow assay described herein. In some embodiments, the method includes receiving a sample in a sample reservoir of the lateral flow assay. In some embodiments, the sample is obtained from a source, including the environment or a biological source. In some embodiments, the sample is suspected of having the analyte of interest. In some embodiments, the sample is not suspected of having the analyte of interest. In some embodiments, the sample is obtained and analyzed to establish the presence or absence of the analyte. In some embodiments, the sample is obtained and analyzed for the amount of the analyte in the sample. In some embodiments, the amount of the analyte in the sample is less than, at or near, or greater than the normal value present in a healthy subject.

[0062] In some embodiments, receiving the sample in a sample reservoir of a lateral flow assay includes contacting the sample with the lateral flow assay. The sample may be contacted with the lateral flow assay by introducing the sample into the sample reservoir by external application, such as when using a dropper or other applicator. In some embodiments, the sample reservoir may be directly immersed in the sample, such as when a test strip is immersed in a container holding the sample. In some embodiments, the sample may be poured, dripped, sprayed, placed, or otherwise contacted with the sample reservoir. In embodiments of the present disclosure, the labeled agent comprises an antibody, a label, and an analyte of interest and can be attached to a conjugate pad (or label zone) within the sample reservoir or downstream thereof. The labeled agent can be incorporated into the conjugate pad by physical or chemical bonding. After sample is added to the sample reservoir, the sample solubilizes the labeled agent and releases the bond holding the labeled agent to the conjugate pad. The sample, containing the analyte (if present) and the labeled agent, flows along a liquid front through the lateral flow assay to the capture zone. The capture agent immobilized in the capture zone binds the analyte (if present) and the labeled agent. When the labeled agent binds to the capture agent in the capture zone, a signal from the label is detected. The signal can include an optical signal as described herein. When a low concentration of analyte is present in the sample (e.g., at or below a healthy level), a maximum intensity signal is detected in the capture zone. At elevated concentrations of analyte (e.g., above a healthy level), the intensity of the detected signal decreases by an amount proportional to the amount of analyte in the sample. The detected signal is compared to a value on a dose-response curve for the analyte of interest to determine the concentration of the analyte in the sample.

[0063] In some embodiments, the analyte is present at an elevated concentration. An elevated concentration of an analyte refers to a concentration of the analyte that is above a healthy level. Thus, an elevated concentration of an analyte is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, or above a healthy level. In some embodiments, the analyte of interest comprises C-reactive protein (CRP), which is present in serum of healthy individuals in an amount of about 1 to about 10 μg / mL. Thus, elevated concentrations of CRP in a sample include amounts of 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 greater. The level at which an analyte of interest is considered elevated may vary depending on the particular analyte of interest.

[0064] In some embodiments, if the analyte is determined to be present at an elevated concentration in the sample, the subject is diagnosed with a particular disease. In some embodiments, a diagnosis of infection is made when the concentration of CRP is determined to be 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 greater. In some embodiments, a determination of a concentration greater than 200 μg / mL, for example, 400-500 μg / mL, leads to a diagnosis of severe bacterial infection.

[0065] Exemplary Test Systems Including Lateral Flow Assays According to the Present Disclosure The lateral flow assay test systems described herein may include a lateral flow assay test device (e.g., without limitation, a test strip), a housing including a port configured to receive all or a portion of the test device, a reader including a light source and a photodetector, a data analyzer, or combinations thereof. The housing may be made of any of a wide variety of materials, including plastic, metal, or composite materials. The housing forms a protective enclosure for the components of the diagnostic test system. The housing also defines a receptacle that mechanically mates the test strip with the reader. The receptacle can be designed to accept any of a variety of types of test strips. In some embodiments, the housing is a portable device that enables the ability to perform lateral flow assays in a variety of environments, including outdoors, indoors, or within an institution, on a bench, etc., for home, commercial, or environmental use.

[0066] The reader may include one or more optoelectronic components for optically examining 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 label used by the test strip, the light source may be designed to emit light in a specific wavelength range or with a specific polarization. For example, if the label is a fluorescent label such as a quantum dot, the light source may be designed to illuminate the exposed area of ​​the capture zone of the test strip with light in a wavelength range that causes fluorescence emission from the label. Similarly, the photodetector may be designed to selectively capture light from the exposed area of ​​the capture zone. For example, if the label is a fluorescent label, the photodetector may be designed to selectively capture light within the wavelength range of the fluorescence emitted by the label or light with a specific polarization. On the other hand, if the label is a reflective label, the photodetector may be designed to selectively capture light within the wavelength range of light emitted from the light source. For these purposes, 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 detected by visual observation or by a spectrophotometer; an emission counter for detecting the emission, e.g. 125 The color from the chromogenic substrate can be detected and analyzed using a gamma counter for detecting I; or a fluorometer for detecting fluorescence in the presence of specific wavelengths of light. When enzyme-linked assays are used, a spectrophotometer can be used to perform quantitative analysis of the amount of the analyte of interest. The lateral flow assays described herein can be automated or performed robotically, if desired, to simultaneously detect signals from multiple samples. Furthermore, multiple signals can be detected in multiplexed assays in which two or more analytes of interest are detected, identified, or quantified.

[0067] The complex assay can include, for example, a virus differentiation assay.For example, the complex lateral flow assay described herein can detect the presence of one or more viral proteins in a sample from a subject suffering from or suspected of suffering from a viral infection (e.g., exhibiting influenza-like symptoms).In some embodiments, the complex lateral flow assay for this purpose can detect elevated levels of CRP and low levels of TRAIL and IP-10.

[0068] The data analyzer processes the signal measurements obtained by the reader. Generally, the data analyzer may be implemented in any computing or processing environment, including digital electronic circuitry or computer hardware, firmware, or software. In some embodiments, the data analyzer includes a processor (e.g., a microcontroller, microprocessor, or ASIC) and an analog-to-digital converter. The data analyzer can be incorporated within the housing of the diagnostic test system. In other embodiments, the data analyzer is located in a separate device, such as a computer, that can communicate with the diagnostic test system via a wired or wireless connection. The data analyzer may also include circuitry for transferring results via a wireless connection to an external source for data analysis or evaluation of the results.

[0069] Generally, the result indicator may include any one of a wide variety of mechanisms for indicating one or more results of an 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, the completion of an assay test. In other embodiments, the result indicator includes an alphanumeric display (e.g., a two- or three-character light emitting diode array) for presenting the assay test results. The test systems described herein may include a power supply that provides power to the active components of the diagnostic test system, including the reader, data analyzer, and result display. The power supply may be implemented, for example, by a replaceable or 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).

[0070] Exemplary Lateral Flow Device Functions The lateral flow devices described herein can include a sample reservoir (also referred to as a sample-receiving zone) where a fluid sample is introduced to a test strip, such as, but not limited to, an immunochromatographic test strip, present in the lateral flow device. In one example, the sample may be introduced to the sample reservoir by external application, such as with a dropper or other applicator. The sample may be injected or squeezed into the sample reservoir. In another example, the sample reservoir may be directly immersed in the sample, such as when a test strip is immersed in a container that holds the sample.

[0071] The lateral flow devices described herein may include a solid support or substrate. Suitable solid supports include, but are not limited to, nitrocellulose, the walls of wells in a reaction tray, multiwell plates, test tubes, polystyrene beads, magnetic beads, membranes, and microparticles (such as latex particles). Any suitable porous material with sufficient porosity to allow access by labeled agents and suitable surface affinity for immobilizing capture agents can be used in the lateral flow devices described herein. For example, the porous structure of nitrocellulose provides excellent absorption and adsorption properties for various reagents, such as capture agents. Nylon also has similar properties and is suitable. Microporous structures are useful, as are materials with a gel structure in a hydrated state.

[0072] Further examples of useful solid supports include natural polymeric carbohydrates and their synthetically modified cross-linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gum, cellulose esters, particularly nitrates and carboxylic acids, mixed cellulose esters, and cellulose ethers; nitrogen-containing natural polymers such as proteins and derivatives, including cross-linked gelatin or modified gelatin; natural hydrocarbon polymers such as latex and rubber; polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and their partially hydrolyzed derivatives, polyacrylamide, polymethacrylate, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or Examples of suitable porous materials include synthetic polymers, such as vinyl polymers, which can be prepared with suitable porous structures, including 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; and aluminum or silicon oxides or hydrates, such as clay, alumina, talc, kaolin, zeolites, silica gel, or glass (these materials may be used as filters in conjunction with the above polymeric materials); and mixtures or copolymers of the above classes, such as graft copolymers obtained by initializing the polymerization of a synthetic polymer onto an existing natural polymer.

[0073] The lateral flow devices described herein can include a porous solid support, e.g., nitrocellulose, in the form of a sheet or strip. The thickness of such sheets or strips can vary within a wide range, e.g., about 0.01 to 0.5 mm, about 0.02 to 0.45 mm, about 0.05 to 0.3 mm, about 0.075 to 0.25 mm, about 0.1 to 0.2 mm, or about 0.11 to 0.15 mm. The pore size of such sheets or strips can also vary within a wide range, e.g., about 0.025 to 15 microns, or more specifically, about 0.1 to 3 microns. However, pore size is not intended to be a limiting factor in the selection of the solid support. The flow rate of the solid support, if applicable, can vary within a range of, for example, about 12.5-90 s / cm (i.e., 50-300 s / 4 cm), about 22.5-62.5 s / cm (i.e., 90-250 s / 4 cm), about 25-62.5 s / cm (i.e., 100-250 s / 4 cm), about 37.5-62.5 s / cm (i.e., 150-250 s / 4 cm), or about 50-62.5 s / cm (i.e., 200-250 s / 4 cm). In certain embodiments of the devices described herein, the flow rate is about 35 s / cm (i.e., 140 s / 4 cm). In other certain embodiments of the devices described herein, the flow rate is about 37.5 s / cm (i.e., 150 s / 4 cm).

[0074] The surface of the solid support can be activated by a chemical process that results in covalent bonding of an agent (e.g., a capture reagent) to the support. As described below, the solid support can include a conjugate pad. Many other suitable methods can be used to immobilize an agent (e.g., a capture reagent) to the solid support, including, but not limited to, ionic interactions, hydrophobic interactions, covalent interactions, etc. Unless physically limited, a solid support may be used in any suitable form such as a film, sheet, strip, plate, or may be coated on or adhered or laminated to a suitable inert carrier such as paper, glass, plastic film, or fabric.

[0075] The lateral flow devices described herein may include a conjugate pad, such as a membrane or other type of material, containing a capture reagent. The conjugate pad may be made of 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), aluminum oxide, 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) plus nucleating agent "aluminum dibenzoate" (DBS) (e.g., 80 u 0.024 HDPE DBS (Porex)), and HDPE.

[0076] The lateral flow devices described herein are highly sensitive to target analytes present in high concentrations in a sample. As described above, when unlabeled target analytes are present in a sample in sufficient amounts to compete with the labeled compound and bind to the capture agent in the capture zone, a high concentration is present and a detection signal is obtained in the negatively sloping portion of the dose-response curve (e.g., the "hook effect" portion of the dose-response curve for a conventional sandwich-type lateral flow assay or the negatively sloping portion of the dose-response curve for the lateral flow assay of the present disclosure). "Sensitivity" refers to the proportion of actual positives that are correctly identified as such (e.g., the proportion of infected, latent, or symptomatic subjects that are correctly identified as having a disease state). Sensitivity can be calculated as the number of true positives divided by the sum of the number of true positives and the number of false negatives.

[0077] The lateral flow devices described herein can accurately measure analytes of interest in many different types 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 can include liquids, solids, tissues, and gases. Biological samples include urine, saliva, and blood products, such as plasma and serum. However, such examples should not be construed as limiting the types of samples applicable to the present disclosure.

[0078] In some embodiments, the sample is an environmental sample for detecting an analyte in the environment. In some embodiments, the sample is a biological sample from a subject. In some embodiments, the biological sample can include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculate, female ejaculate, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menstruation, pus, sebum, vomit, vaginal secretions, mucosal secretions, stool water, pancreatic juice, lavage fluid from a sinus cavity, bronchopulmonary aspirate, or other lavage fluid.

[0079] 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 administered therapeutically and illicitly), drug intermediaries or by-products, bacteria, virus particles, and metabolites or antibodies of any of the above substances.Some specific examples of analytes include ferritin; creatinine kinase MB (CK-MB); human chorionic gonadotropin (hCG); digoxin; phenytoin; phenobarbitol; 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-inducible protein 10 (IP-10); glycated hemoglobin (Gly Hb); cortisol; digitoxin; N-acetylprocainamide (NAPA); procainamide; antibodies to rubella, e.g., rubella IgG and rubella IgM; antibodies to toxoplasmosis, e.g., toxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM); testosterone; salicylate; acetaminophen; hepatitis B virus surface antigen (HBsAg); antibodies to hepatitis B core antigen, e.g., anti-hepatitis B core antigen IgG and IgM (anti-HBC); human immunodeficiency virus (HIV) These include human T-cell leukemia virus 1 and 2 (HIV1 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); carcinoembryoic antigen (CEA); lipoproteins, cholesterol, and triglycerides; and alpha-fetoprotein (AFP).Drugs of abuse and controlled substances include, but are not limited to, amphetamines, methamphetamines, barbiturates such as amobarbital, secobarbital, pentobarbital, phenobarbital, and barbital, benzodiazepines such as librium and valium, cannabinoids such as cannabis and marijuana, cocaine, fentanyl, LSD, methaqualone, opiates such as heroin, morphine, codeine, hydromorphone, hydrocodone, methadone, oxycodone, oxymorphone, and opium, phencyclidine, and propoxyphen. Additional analytes may be included for biological or environmental targets.

[0080] The lateral flow devices described herein may include labels. Labels can take many different forms, including molecules or compositions that are bound to or can bind to an analyte, analyte analog, detection reagent, or binding partner, and that are detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Examples of labels include enzymes, colloidal gold particles (also called gold nanoparticles), colored latex particles, emissive isotopes, cofactors, ligands, chemiluminescent 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-bound dye capsules. Attachment of a compound (e.g., a detection reagent) to the label can be via covalent bonding, adsorption processes, hydrophobic and / or electrostatic bonding (such as in chelates), or these bonds and interactions, and / or may include a linking group.

[0081] The term "specific binding partner (or binding partner)" refers to a member of a pair of molecules that interact by specific, non-covalent interactions that depend on the three-dimensional structure of the molecules involved. Typical pairs of specific binding partners include antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid strand / complementary nucleic acid strand, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, biotin / (strept)avidin, receptor / ligand, and viral / cellular receptor, or various combinations thereof. As used herein, the term "immunoglobulin" or "antibody" refers to a protein that binds to a specific antigen. Immunoglobulins include polyclonal, monoclonal, chimeric, and humanized antibodies, Fab fragments, F(ab')2 fragments, and include, but are not limited to, the following classes: IgG, IgA, IgM, IgD, IbE, and secretory immunoglobulin (sIg). Immunoglobulins generally contain two identical heavy chains and two light chains. However, the terms "antibody" and "immunoglobulin" also encompass single-chain and two-chain antibodies.

[0082] The lateral flow devices described herein include a labeled agent. Optionally, the labeled agent includes a detection agent capable of binding to a detection agent. The labeled agent may be specific to the analyte. In some embodiments, the labeled agent may be an antibody or fragment thereof conjugated, bound, or associated with the detection agent. In embodiments of lateral flow assays according to the present disclosure, the labeled agent may be an antibody or fragment thereof conjugated, bound, or associated with the detection agent and the analyte of interest, forming a label-antibody-analyte complex.

[0083] Lateral flow devices according to the present disclosure include a capture agent. The capture agent comprises an immobilized agent capable of binding to an analyte, including free (unlabeled) analyte and / or labeled analyte. The capture agent includes (i) a labeled analyte of interest, (ii) a labeled or unlabeled analyte, such as in a competitive assay, or (iii) an unlabeled specific binding partner specific for an auxiliary specific binding partner that is itself specific 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, the auxiliary specific binding partner may include another antibody, e.g., an antibody specific for a goat anti-human antibody. The lateral flow devices described herein may include a "capture region," which is an area of ​​the lateral flow device where a capture reagent is immobilized. The lateral flow devices described herein may include two or more capture regions, e.g., a "primary capture region," a "secondary capture region," etc. In some cases, different capture reagents are immobilized in the primary, secondary, and / or other capture regions. Multiple capture regions can have any orientation relative to one another on a lateral flow substrate. For example, a primary capture region can be distal or proximal to a secondary (or other) capture region along the fluid flow path, or vice versa. Alternatively, the primary and secondary (or other) capture regions can be aligned along an axis perpendicular to the fluid flow path, such that the fluid contacts the capture regions simultaneously or nearly simultaneously.

[0084] Lateral flow devices according to the present disclosure include a capture agent that is immobilized such that movement of the capture agent is restricted during normal operation of the lateral flow device. For example, movement of the immobilized capture agent is restricted before and after a fluid sample is applied to the lateral flow device. The immobilization of the capture agent can be achieved by physical means such as a barrier, electrostatic interaction, hydrogen bonding, bioaffinity, covalent interaction, or a combination thereof. Lateral flow devices according to the present disclosure can include multiplex assays, including assays capable of detecting, identifying, and possibly quantifying multiple different analytes of interest. For example, in multiplex assay devices, there can be primary, secondary, or more capture regions, each specific for one of the multiple analytes of interest.

[0085] Lateral flow devices according to the present disclosure can detect, identify, and in some cases quantify biologics. Biologicals include chemical or biochemical compounds produced by living organisms, which may include prokaryotic, eukaryotic, mammalian, microbial, insect, plant, mixed, naturally occurring, or synthetically engineered cell lines. Biologicals may include large macromolecules such as proteins, polysaccharides, lipids, and nucleic acids, as well as small molecules such as primary metabolites, secondary metabolites, and natural products. It should be understood that the description, specific examples, and data, while indicating exemplary embodiments, are given by way of illustration and are not intended to limit various embodiments of the present disclosure. Various changes and modifications within the present disclosure will become apparent to those skilled in the art from the description and data contained herein and, therefore, are considered to be part of various embodiments of the present disclosure.

Claims

1. 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, the capture zone comprising an immobilized capture agent specific for an analyte of interest; a complex configured to be coupled to the flow path in a first phase and to flow in the flow path to the capture zone in the presence of the fluid sample in a second phase, signs, an antibody or antibody fragment that specifically binds to the analyte of interest, and the analyte of interest, and a complex containing 12. An assay test strip comprising:

2. 10. The assay test strip of claim 1, wherein the flow path is configured to receive a fluid sample containing an unlabeled analyte of interest, and the complex does not specifically bind to the unlabeled analyte of interest in the first phase or the second phase.

3. 3. The assay strip of claim 2, wherein the complex is configured to flow in the flow path to the capture zone along with the unlabeled analyte of interest in the second phase.

4. 4. The assay strip of claim 3, wherein the complex is configured to bind to the immobilized capture agent in the capture zone in a third phase in competition with the unlabeled analyte of interest.

5. 5. The assay test strip of claim 4, wherein the optical signal emitted from complexes bound to the immobilized capture agent in the capture zone decreases as the concentration of unlabeled target analyte increases in the fluid sample.

6. 2. The assay test strip of claim 1, wherein the flow path is configured to receive a fluid sample that may or may not contain a target analyte, and wherein the complex specifically binds to all or substantially all of the immobilized capture agents in the capture zone in the second phase when the fluid sample does not contain the target analyte.

7. 7. The assay test strip of claim 6, wherein when the fluid sample does not contain the analyte of interest, the optical signal emitted from the complex bound to the capture zone is the maximum optical signal that can be emitted from the assay test strip.

8. 8. The assay strip of claim 7, wherein if the fluid sample contains an analyte of interest, the optical signal emitted from the complex bound to the capture zone is less than the maximum optical signal.

9. 10. The assay strip of claim 1, wherein the immobilized capture agent comprises an antibody or antibody fragment that specifically binds to the analyte of interest.

10. 10. The assay strip of claim 1, wherein the complex is incorporated into the surface of the strip in a first phase.

11. 10. The assay strip of claim 1, wherein the conjugate is incorporated into the surface of the strip by spraying a solution containing the conjugate onto the surface of the strip and allowing the solution to dry.

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

13. 10. The assay strip of claim 1, wherein the analyte of interest comprises C-reactive protein (CRP) and the conjugate comprises an anti-CRP antibody or fragment thereof bound to the CRP.

14. The assay strip of claim 1; a reader including a light source and a detector; Data analyzer and 1. A diagnostic testing system comprising:

15. 10. The diagnostic test system of claim 1, wherein the data analyzer outputs an indication that the target analyte is absent from the fluid sample when the reader detects an optical signal from the assay strip that is a maximum optical signal of the dose-response curve for the strip.

16. 16. The diagnostic test system of claim 15, wherein the data analyzer outputs an indication that there is a low concentration of a target analyte in the fluid sample when the reader detects an optical signal from the assay strip that is within 1% of the maximum optical signal.

17. 16. The diagnostic test system of claim 15, wherein the data analyzer outputs an indication that there is a low concentration of a target analyte in the fluid sample when the reader detects an optical signal from the assay strip that is within 5% of the maximum optical signal.

18. 16. The diagnostic test system of claim 15, wherein the data analyzer outputs an indication that there is a low concentration of a target analyte in the fluid sample when the reader detects an optical signal from the assay strip that is within 10% of the maximum optical signal.

19. 16. The diagnostic test system of claim 15, wherein the data analyzer outputs an indication that there is a high concentration of a target analyte in the fluid sample when the reader detects an optical signal from the assay strip that is 90% or less than 90% of the maximum optical signal.

20. 16. The diagnostic test system of claim 15, wherein the data analyzer outputs an indication of the concentration of the analyte of interest in the sample when the reader detects an optical signal from the assay strip that is less than the maximum optical signal.

21. 1. A method for determining the concentration of an analyte of interest in a fluid sample using an assay strip, the assay strip comprising: 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, the capture zone comprising an immobilized capture agent specific for the analyte of interest; and a complex coupled to the flow path in a first phase and configured to flow in the flow path to the capture zone in a second phase in the presence of the fluid sample, the complex comprising a label, an antibody or antibody fragment that specifically binds to the analyte of interest, and the analyte of interest, the method comprising: applying the fluid sample to the assay strip when the complex is coupled to the flow path in the first phase; decoupling the composite from the flow path; flowing the fluid sample and the complexes in the flow path to the capture zone in the second phase; allowing the complex to bind to the immobilized capture agent in the capture zone; detecting a signal from the complex bound to the immobilized capture agent in the capture zone; A method comprising:

22. 22. The method of claim 21, wherein the detection signal is an optical signal, a fluorescent signal, or a magnetic signal.

23. 22. The method of claim 21, wherein the step of delinking the complex comprises solubilizing the complex in the fluid sample.

24. 22. The method of claim 21, wherein the fluid sample comprises an unlabeled analyte of interest, and the complex does not specifically bind to the unlabeled analyte of interest in the first phase or the second phase.

25. 22. The method of claim 21, wherein the fluid sample contains an unlabeled target analyte, and the complex is configured to bind to the immobilized capture agent in the capture zone in a third phase in competition with the unlabeled target analyte.

26. 22. The method of claim 21, wherein the fluid sample does not contain the analyte of interest and detecting comprises detecting a maximum signal in a dose-response curve of the test strip.

27. 27. The method of claim 26, further comprising determining that the concentration of the analyte in the fluid sample is zero.

28. 28. The method of claim 27, further comprising displaying an indication that the analyte of interest is not present in the fluid sample.

29. 22. The method of claim 21, wherein the fluid sample contains an analyte of interest, and detecting comprises detecting a signal from the test strip that is less than a maximum signal of a dose-response curve for the test strip.

30. 30. The method of claim 29, further comprising determining that the concentration of the analyte in the fluid sample is greater than zero.

31. 31. The method of claim 30, further comprising displaying an indication that the analyte of interest is present in the fluid sample.

32. determining that the detected signal is within 10% of the maximum optical signal; providing an indication that the analyte of interest is present in the fluid sample at a low concentration; 30. The method of claim 29, further comprising:

33. determining that the detected signal is 90% or less than 90% of the maximum signal; providing an indication that the analyte of interest is present in the fluid sample at a high concentration; 30. The method of claim 29, further comprising:

34. 1. A method of manufacturing an assay strip, comprising: connecting the sample receiving zone to a flow path configured to receive the fluid sample; connecting a capture zone to the flow path downstream of the sample receiving zone; A step of connecting a complex to the flow channel, wherein the complex comprises: signs, an antibody or antibody fragment that specifically binds to the analyte of interest, and the analyte of interest and A method comprising:

35. 35. The method of claim 34, wherein the analyte of interest comprises C-reactive protein (CRP) and the antibody comprises an anti-CRP antibody or a fragment of an anti-CRP antibody.

36. 36. The method of claim 35, wherein the target analyte comprises about 50 ng of CRP.

37. 36. The method of claim 35, wherein the analyte of interest comprises about 100 ng of CRP.

38. 35. The method of claim 34, further comprising immobilizing a capture agent specific for the analyte of interest in the capture zone.

39. 35. The method of claim 34, wherein coupling the complex to the flow path comprises forming a bond between the complex and the flow path that is cleaved in the presence of a fluid sample in the flow path.

40. 35. The method of claim 34, wherein the step of ligating the complex comprises spraying a solution containing the complex onto the surface of the sample-receiving zone.

41. 35. The method of claim 34, wherein the step of ligating the complex comprises spraying a solution containing the complex onto the surface of the assay strip between the sample-receiving zone and the capture zone.

42. The step of linking the complex comprises: applying a fluid solution containing the complex to a surface of the assay strip; drying the fluid solution; 35. The method of claim 34, comprising:

43. 35. The method of claim 34, wherein the step of linking the complex comprises incorporating the complex into a surface of the assay strip.

44. 35. The method of claim 34, further comprising providing a solution comprising the complex.

45. 45. The method of claim 44, wherein the step of providing a solution comprises mixing a first liquid containing the label and the antibody or fragment of the antibody with a second liquid containing the analyte of interest.

46. 46. ​​The method of claim 45, wherein the step of providing the solution further comprises incubating the mixture of the first liquid and the second liquid for about 30 minutes.

47. 45. The method of claim 44, wherein the step of coupling the complex to the fluid path comprises spraying the solution onto the surface of the assay strip.

48. An assay test strip made by the method of any one of claims 34 to 47.

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