Systems, apparatus, and methods for amplifying signals from lateral flow assays.

Abstract

To provide a lateral flow assay device, system, and method.SOLUTION: The lateral flow assay device, system, and method detect analytes present in a sample in small amounts or at low concentrations by amplifying signals generated in a detection zone. Signals generated can exceed a detection threshold of a measurement system, increasing the sensitivity of the lateral flow assay device, system and method. In one aspect, the lateral flow device includes a signal-amplifying conjugate that binds to a complex that is bound to an immobilized capturing material in the detection zone, generates a chain reaction of binding events in the detection zone, resulting in amplification of the signal generated in the detection zone.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 686,606, filed on June 18, 2018, which is hereby incorporated by reference in its entirety.

[0002] The present disclosure generally relates to lateral flow devices, test systems, and methods. More particularly, the present disclosure relates to lateral flow assay devices that can measure analytes present in small or low - concentration samples by amplifying detection signals.

Background Art

[0003] Immunoassay systems, including lateral flow assays, provide reliable, inexpensive, small, rapid, and simple diagnostic tests. Lateral flow assays can quickly and accurately detect the presence or absence of a target analyte in a sample and, in some cases, quantify it. Advantageously, lateral flow assays are minimally invasive and can be used as point - of - care testing systems. A lateral flow assay generates a signal that can be measured to indicate the presence or amount of a target analyte in a sample. However, when the analyte is present in small amounts or at low concentrations in the sample, the signal generated by the labeled analyte captured in the test region of the lateral flow assay may not exceed the detection threshold of the measurement system that reads and processes the signal. As a result, the lateral flow assay may not reliably or accurately indicate the presence of the target analyte in the sample.

Summary of the Invention

[0004] Summary Accordingly, aspects of the present disclosure are to provide improved lateral flow assays capable of detecting the presence or amount of an analyte in a sample. Embodiments of the methods and systems described herein can measure the presence, and in some cases the amount or concentration, of an analyte even when the analyte is present in small amounts or low concentrations and would normally be expected to be generated at levels below the detection threshold of the measurement system. As a result, embodiments of the systems, apparatus, and methods described herein are highly sensitive to the target analyte, even at small amounts or low concentrations. Furthermore, embodiments of the methods and systems described herein can determine the absence of the target analyte in a sample with greater confidence than prior methods and systems. The absence of a detectable signal in embodiments of the present disclosure can reliably indicate the absence of the target analyte in the sample (in contrast to analytes present at such low concentrations that the generated signal is below the detection threshold of the system, even if they are actually present). Thus, embodiments of the present disclosure can reduce false negative readings. As described in detail below, the lateral flow assay described herein amplifies the signal generated in a test area, such as the capture zone of the lateral flow assay, to a level that would normally allow a measurement system to detect the target analyte present in small amounts or low concentrations in a sample.

[0005] The signals generated by the assays described herein are described herein in the context of optical signals generated by reflectivity labels (including, but not limited to, gold nanoparticle labels and latex particles of different colors). Embodiments of the disclosure are described herein by reference to “optical” signals, but it will be understood that any suitable material can be used for the labels to generate signals, including, but not limited to, fluorescent latex bead labels that generate fluorescent signals and magnetic nanoparticle labels that generate signals indicating changes in the magnetic field associated with the assay. The embodiments of the lateral flow assay described herein are particularly advantageous in diagnostic tests where the analyte of interest is present on the test strip in small amounts or at low concentrations. The analyte of interest may be present on the test strip at low concentrations for a number of reasons, including, but not limited to, the limited amount of analyte of interest in the sample applied to the test strip and the limited volume of the sample applied to the test strip. While embodiments of this disclosure are described in relation to the detection of the analyte of interest at low concentrations, it will be understood that this disclosure can also detect small amounts of the analyte of interest.

[0006] Some embodiments disclosed herein relate to lateral flow assays for detecting an analyte of interest in a sample. The lateral flow assay includes a first conjugate comprising a first label, an active substance configured to specifically bind to the analyte of interest, and a first binding partner; a second conjugate upstream of the first conjugate along the fluid flow path of the lateral flow assay, comprising a second label and a second binding partner configured to specifically bind to the first binding partner; and a detection zone downstream of the first and second conjugates along the fluid flow path of the lateral flow assay, comprising an immobilized capture substance (immobilized capture substance) that specifically binds to the analyte of interest. The first conjugate may be located in the sample receiving zone of the lateral flow assay, or in the first conjugate zone downstream of the sample receiving zone. The second conjugate may be located in the buffer receiving zone upstream of the sample receiving zone, or in the second conjugate zone downstream of the buffer receiving zone and upstream of the sample receiving zone. The first conjugate may be configured to be solubilized and move (collect) in the detection zone when a fluid sample is applied to the lateral flow assay. The second conjugate may be configured to be solubilized and move to the detection zone after the first conjugate has moved to the detection zone.

[0007] The active agent configured to specifically bind to the analyte of interest may be an antibody or antibody-conjugated fragment that specifically binds to the analyte of interest. The first and second binding partners may include binding pairs selected from the group consisting of antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid chain / complementary nucleic acid chain, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, biotin / avidin, receptor / ligand, and virus / cell receptor. The first and second binding partners may include a biotin / avidin binding pair, where avidin may include streptavidin or neutraavidin. The first and second binding partners may include an antigen / antibody binding pair, where the antigen may include a peptide or decapeptide. The analyte of interest may be a biological or environmental substance of interest. The analyte of interest may be an influenza virus. The influenza virus may be influenza A virus, influenza B virus, or influenza C virus. The immobilized capture agent may be an antibody or antibody-conjugated fragment that specifically binds to the analyte of interest. The test strip may include a nitrocellulose membrane. The lateral flow assay may also include a control zone containing an immobilized capture substance that specifically binds to the first conjugate.

[0008] The first and second labels may be selected from the group consisting of metal nanoparticles, blue latex beads, colored latex particles, colored latex beads, magnetic particles, carbon nanoparticles, quantum dots, up-converting phosphors, organic fluorophores, fiber dyes, enzymes, or liposomes. The first and second labels may contain gold nanoparticles. The first and second labels may be configured to produce an optical signal, a fluorescent signal, or a magnetic signal. A lateral flow assay may also include a housing containing a sample well positioned laterally above or upstream of a first conjugate, a buffer well positioned laterally above or upstream of a second conjugate zone, and a read window providing access to the detection zone. The buffer well, the second conjugate zone, the sample receiving well, and the first conjugate zone may be spatially separated along the fluid flow path of the lateral flow assay. Further embodiments disclosed herein relate to a method for preparing a lateral flow assay. The method includes applying a first conjugate in the sample receiving zone of a lateral flow test strip or to a downstream lateral flow test strip, and applying a second conjugate in a buffer receiving zone upstream of the sample receiving zone or to a downstream test strip. The first and second conjugates may be applied to the test strip simultaneously. The first and second conjugates may be applied to the test strip by air jet deposition.

[0009] Other embodiments described herein relate to a method for detecting an analyte of interest in a sample. The method comprises applying a sample to a lateral flow assay. The lateral flow assay includes a first conjugate comprising a first label, an active substance configured to specifically bind to the analyte of interest, and a first binding partner; a second conjugate located upstream of the first conjugate along the fluid flow path of the lateral flow assay, comprising a second label and a second binding partner configured to specifically bind to the first binding partner; and a detection zone located downstream of the first and second conjugates along the fluid flow path of the lateral flow assay, comprising an immobilized capture substance that specifically binds to the analyte of interest. The method also comprises binding the complex to the immobilized capture substance in the detection zone, the complex comprising the analyte of interest bound to the first conjugate. After binding the complex, the method comprises releasing the second conjugate and allowing it to flow along the fluid flow path of the lateral flow assay. The method further includes binding a second conjugate to the complex bound at the detection zone.

[0010] The method may include detecting the signal generated by the combined complex and the second conjugate in the detection zone. The method may also include binding the first conjugate, which was not bound to the analyte of interest, to the second conjugate that was bound to the complex in the detection zone. The first and second labels may be configured to generate an optical signal, a fluorescent signal, or a magnetic signal. After applying the sample to the lateral flow apparatus, the second conjugate may be released at intervals of 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes. Binding the complex to a capture substance immobilized in the detection zone may include labeling the target analyte with the first conjugate to form a complex, and then binding the complex to a capture substance immobilized in the detection zone. Releasing the second conjugate may include applying a buffer to the second conjugate or to a location upstream of the second conjugate in a lateral flow assay. Applying the buffer may include pouring the buffer into a buffer receiving well located lateral to the upper or upstream of the second conjugate. [Brief explanation of the drawing]

[0011] [Figure 1] An exemplary top view of the lateral flow apparatus described herein, including a buffer well, a sample well, and a reading window, is shown. [Figure 2] An exemplary assay test strip for the lateral flow signal amplification assay described in this disclosure is shown. [Figure 3] An exemplary method for amplifying the signal generated by the assay test strip described in this disclosure is schematically shown. [Figure 4]The results of the signal amplification reaction of a lateral flow apparatus implemented in accordance with this disclosure are shown, with the signal intensity expressed as a function of reading time for the control signal (without signal amplification conjugate - ◆) and the test signal (with signal amplification conjugate - ■). [Figure 5] The results of the signal amplification reaction of the lateral flow apparatus performed in accordance with this disclosure are shown, with the signal intensity as a function of analyte concentration, including the control signal of the assay performed without the signal amplification conjugate after 10 minutes (◆) and 20 minutes (▲), and the test signal with the signal amplification conjugate after 10 minutes (■) and 20 minutes (●). [Figure 6] A flow diagram is shown for an exemplary method using a lateral flow assay to detect a target analyte in a low-concentration sample. [Modes for carrying out the invention]

[0012] A typical lateral flow sandwich assay includes a conjugate pad (or labeling zone), a capture or test zone, and a test strip having an absorbent pad aligned along the fluid flow path, which typically does not necessarily need to be aligned along the longitudinal axis of the test strip. The test strip may include a sample receiving zone or well upstream of the conjugate pad. The conjugate pad typically contains a labeled analyte-specific antibody called a conjugate. The label on this conjugate may include an analyte-specific antibody bound to a detection molecule, such as gold nanoparticles, but is not limited to this label. The capture zone contains an immobilized capture material, such as an analyte-specific antibody immobilized on a carrier material, such as a nitrocellulose membrane. The absorbent pad assists the liquid sample applied to the conjugate pad (or sample receiving zone, if present) so that it flows through the carrier material into the capture zone. When the liquid sample is applied to the test strip (of the sample receiving zone or conjugate pad), the labeled analyte-specific antibody is rehydrated and made mobile. If a target antibody-specific analyte is present, the analyte binds to the labeled analyte-specific antibody, forming an analyte-antibody-detector complex that moves downstream through the carrier material into the test zone. In the test zone, the analyte of the analyte-antibody-detector complex interacts with the analyte-specific antibody immobilized in the capture zone to form an antibody-analyte-antibody-detector structure, typically called a sandwich. When a threshold number of these sandwich structures are formed in the capture zone, the signal generated by the detection molecule in the sandwich structure can be detected by the measurement system.

[0013] The measurement system can process the detected signal to determine the presence, and in some cases the amount, of the target analyte in the sample. If the target analyte is not present in the sample, the analyte-antibody-detector complex will not form, and consequently, no sandwich structure will form in the capture zone. In this example, there is no detection molecule in the capture zone, and the measurement system will show the correlation between the absence of a signal from the capture zone and the absence of the target analyte in the sample. If the target analyte is actually present in the sample, but only in very small amounts or at low concentrations, the number of sandwich structures formed in the capture zone may be very small. In some examples, the number of sandwich structures is so small that the signal produced by the detection molecule is below the detection threshold of the measurement system. In this case, the measurement system will show the correlation between the absence of a signal from the capture zone (or the absence of a signal above the system's detection threshold) and the absence of the target analyte in the sample, even though the target analyte is actually present in the sample, but only in very small amounts or at low concentrations. Therefore, typical lateral flow sandwich assays have limited sensitivity to target analytes present in small amounts or at low concentrations.

[0014] The apparatus, systems, and methods described herein overcome these and other drawbacks by amplifying the signal generated in the presence of the analyte of interest in a sample applied to a lateral flow assay. Advantageously, embodiments of the apparatus, systems, and methods described herein can amplify the signal generated in a test area, such as a trapping zone of a lateral flow assay, to a level above the detection threshold of a normal reader, even when the analyte of interest is present at low concentrations. The lateral flow apparatus, test system, and method described herein includes a signal amplification conjugate that binds to both (1) a first analyte-specific conjugate bound to an immobilized trapping material in the trapping zone, and (2) a second residual analyte-specific conjugate present in the trapping zone but not bound to the immobilized trapping material. This second, previously unbound residual analyte-specific conjugate (which has bound to the first signal amplification conjugate in the capture zone) contains additional binding sites that interact with and bind to the second signal amplification conjugate, and itself interacts with and binds to a third residual analyte-specific conjugate present in the capture zone in a multilevel chain reaction of binding events in the capture zone.

[0015] The presence of additional analyte-specific conjugates that bind to the capture zone, through interaction and coupling with signal amplification conjugates, can increase the intensity of the signal generated in the capture zone to a level exceeding the detection threshold of a typical measurement system. The presence of signal amplification conjugates that bind to the capture zone can also increase the intensity of the signal generated in the capture zone to a level exceeding the detection threshold of a measurement system. Embodiments of this disclosure improve the sensitivity of lateral flow assays by increasing the signal generated by the lateral flow assay to the typical detectable range rather than modifying the characteristics of the measurement system, and advantageously, the lateral flow assay described herein can be backward compatible with a typical lateral flow assay test system.

[0016] Without being bound by any particular theory, a signal amplification conjugate is generally thought to form a scaffold in which an additional analyte-specific conjugate, which would normally pass through the capture zone, can be captured and retained in the capture zone, resulting in the generation of an amplified signal in the capture zone. In addition to the detection molecule in the analyte-specific conjugate, the signal amplification conjugate may also contain a detection molecule, and the signal amplification conjugate that binds to the capture zone also generates a signal in the capture zone, thereby contributing to an increase in the intensity of the signal generated in the capture zone to a level above the detection threshold. Advantageously, embodiments of this disclosure do not rely on, or require, all or substantially all of the target analyte present in the sample to bind to both the analyte-specific conjugate and the immobilized capture material in the capture zone in order to amplify the signal in the capture zone. The advantage of this disclosure is that even if the analyte of interest bound to the analyte-specific conjugate and the capture material immobilized in the capture zone (for example, in the case of a sample with a low concentration of the analyte of interest) is at a very low level, some amount, in some cases very small amounts, of the analyte of interest can trigger a chain reaction of binding events in the capture zone, regardless of the presence or absence of the analyte of interest in the scaffold formed after binding with the capture material immobilized in the capture zone. Thus, embodiments of the systems, apparatus, and methods described herein can significantly improve the sensitivity of lateral flow assays to the analyte of interest present in a sample, even in trace amounts.

[0017] In addition to increasing the sensitivity of lateral flow assays to measure the presence or amount of target analytes in low-concentration samples, the apparatus, systems, and methods described herein can be advantageously implemented in a point-of-care setting without the use of highly specialized, and in some cases time-consuming, sample preparation techniques and detection systems typically used to analyze analytes in low-concentration samples, such as polymerase chain reaction (PCR). The embodiments of lateral flow apparatus described herein provide a user-friendly and easy-to-use format, rapidly generate a signal that can be detected by a standard reader, and can be implemented in a point-of-care setting rather than in an off-site laboratory. The description is intended to illustrate a test strip for measuring an analyte of interest in a test sample, even when present in low concentrations. Those skilled in the art will recognize that this example is illustrative and that various modifications and variations may be used in the lateral flow assay described herein. For example, the sample may contain one or more analytes of interest present in low concentrations, and the assay test strip may measure one or more analytes of interest. In each of the various iterations, the lateral flow assay includes the features described herein for amplifying the signal in a detection band for measuring an analyte in the test sample. If one or more target analytes are detected, for example, if there are multiple target analytes in the sample, the detection zone may include separate capture zones specific to each target analyte. For example, a sample may contain three target analytes: a first target analyte, a second target analyte, and a third target analyte. The detection zone of a lateral flow assay would therefore likely include three capture zones: a first capture zone specific to the first target analyte, a second capture zone specific to the second target analyte, and a third capture zone specific to the third target analyte.

[0018] Accordingly, this disclosure provides an assay test strip for measuring an analyte of interest in a test sample, even when present in low concentrations; a lateral flow assay including the test strip; a method for measuring an analyte of interest using the test strip described herein; and a method for preparing the assay test strip described herein. The embodiments of the lateral flow assay described herein are particularly advantageous in diagnostic tests where the target analyte is present in small amounts on the test strip. Embodiments of the present disclosure are described herein in relation to the small amount of analyte present on the test strip due to the analyte being present in low concentration in the sample, but it will be understood that the embodiments of the present disclosure are also applicable when the sample contains an amount of analyte capable of producing a signal above the detection threshold, but only a limited amount of the sample is applied to the test strip.

[0019] Various aspects of the apparatus, test system and method will be described more fully hereinafter in connection with 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 recognize that the scope of the present disclosure is intended to cover any aspect of the apparatus, test system and method disclosed herein, implemented independently or in combination with any other aspect of the present disclosure. For example, any number of the aspects described herein may be used to implement the apparatus or execute the method. Certain aspects are described herein, but many variations and permutations of these aspects are within the scope of the present disclosure. Although some benefits and advantages are mentioned, the scope of the present disclosure is not intended to be limited to particular benefits, uses or purposes. Rather, the aspects of the present disclosure are intended to be broadly applicable to different detection techniques and apparatus configurations, some of which are shown by way of example in the figures and the following description.

[0020] Conventional lateral flow device The lateral flow device described herein is an analytical device used in lateral flow chromatography. A lateral flow assay is an assay that can be performed with the lateral flow device described herein. The lateral flow device can be implemented on an assay test strip, but can also be applied to other forms, such as urine test strips, flow-through fractionation devices or microfluidic devices. In the test strip format, a fluid sample that is thought to contain or be likely to contain an analyte is placed on the sample receiving zone. The target analyte is labeled after contacting the test strip. The just-labeled target analyte then flows through the test strip (e.g., by capillary action). The test strip may be made of a medium such as paper, nitrocellulose, cellulose, fiber or nylon, or other materials through which the sample can flow.

[0021] Such assays are called sandwich assays. While the sandwich assays described herein are described in the context of reflective labels (gold nanoparticle labels and latex particles of different colors) that produce optical signals, it will be understood that the assays described herein may include latex bead labels configured to produce fluorescent signals, magnetic nanoparticle labels configured to produce magnetic signals, or any other labels configured to produce detectable signals. A sandwich-type lateral flow assay includes a labeled conjugate attached to a sample receiving zone of a solid substrate. After the sample is applied to the sample receiving zone, the labeled conjugate dissolves or solubilizes in the sample, where it recognizes and specifically binds to a first epitope on the analyte of the sample, forming a label-conjugate-analyte complex. This complex flows from the sample receiving zone through the solid substrate (sometimes called the “test line” or “capture zone”) along the liquid front to the detection zone, where an immobilized capture substance (e.g., an immobilized analyte-specific antibody) is placed. In some cases, where the analyte is a polymer or contains multiple identical epitopes in the same monomer, the labeled conjugate attached to the sample receiving zone may be the same as the capture material immobilized in the detection zone. The immobilized capture material recognizes and specifically binds to the epitope in the analyte, thereby capturing the label-conjugate-analyte complex in the detection zone. The presence of the labeled conjugate in the detection zone provides a detectable signal in the detection zone if the analyte is present in sufficient quantity. In one non-limiting example, gold nanoparticles are used to label the conjugate because they are relatively inexpensive, stable, and provide easily observable color representation due to the surface plasmon resonance attribute of the gold nanoparticles.

[0022] Detecting a signal generated in the detection band can indicate the presence of the target analyte in the sample. For example, if the signal exceeds the detection threshold of the measurement system, the system can detect its presence and, in some cases, quantify the analyte in the sample. However, the absence of any detectable signal in the detection band may indicate that the target analyte is not present in the sample or may be present below the detection limit. For example, if the sample does not contain any target analyte, the sample will remain solubilized of the labeled conjugate, and the labeled conjugate will still flow into the detection band. The labeled conjugate, however, will not bind to the immobilized conjugate in the detection band. Instead, the labeled conjugate will flow through the detection band, through the control line (if present), and, in some cases, to any absorption band. Some labeled conjugates will bind to the control substance attached to the control line, generating a detectable signal in the control line and indicating that the instrument is functioning correctly. If the analyte is present but in an amount below the detection limit, the label-conjugate-analyte complex will bind in the detection band but will not be detected. Under these circumstances, the absence of a detectable signal from the detection band means that the user cannot definitively confirm whether the analyte is absent from the sample or present in the sample below the detection limit of the measurement system.

[0023] Lateral flow signal amplifier as an example described in this disclosure The lateral flow assays, test systems, and methods described herein address the shortcomings of lateral flow assays, including their inability to distinguish between the absence of an analyte in a sample and the presence of an analyte in amounts below the detection limit. In addition to improving the quantitative measurement of analytes, embodiments of the assays, test systems, and methods of this disclosure can also significantly increase the sensitivity of the measurements to a standard reader, enabling quantitative measurement of the analyte in some cases.

[0024] Figure 1 shows an example of the lateral flow apparatus 100 described herein. Assay test strips are received in the housing of the apparatus 100. In this non-limiting example of the lateral flow apparatus 100, the housing comprises a buffer well 110, a sample well 120, and a reading window 130. The lateral flow apparatus 100 may be sized and shaped to facilitate use, deliver test results quickly, be portable, function well with an automated reader, be easily positioned, reduce the materials and costs used, or otherwise. The size and shape are therefore not limited to any particular size or shape and can be easily modified to suit the specific needs or requirements of a particular usage situation. Figure 2 shows an example of the assay test strip 101 described herein. The assay test strip 101 can be received or housed in the lateral flow device 100 of Figure 1. The assay test strip 101 as an example in this non-limiting embodiment includes a substrate having a sample receiving zone and a buffer receiving zone, a detection zone and an absorption pad, and an assay membrane. It will be understood that this disclosure is not limited to the assay test strip as an example, and other test strips having different characteristics can be implemented in accordance with this disclosure.

[0025] In the embodiment shown in Figure 2, the assay test strip 101 includes a back card 102, a conjugate pad 103 having a sample receiving zone 121 and a buffer receiving zone 111, a membrane having a detection zone 131, and a membrane 104 having an absorbent pad 105. The membrane 104 may be made of nitrocellulose material. The assay test strip 101 is housed in a lateral flow device 100, the buffer receiving zone 111 is accessible by a buffer well 110, the sample receiving zone 121 is accessible by a sample well 120, and the detection zone 131 is accessible by a reading window 130. The assay results may be measured in the reading window 130 by measuring the signal generated in the detection zone 131, if any. The back card 102 is any suitable material sufficient to support the assay test strip, and may be a waterproof layer such as a solid plastic, laminated sheet, or composite material. The absorbent pad 105 assists in promoting capillary action and fluid flow through the membrane and may include any suitable material for absorbing fluid, such as nitrocellulose, cellulose-derived materials, porous polyethylene pads, or glass fiber filter paper. The absorbent material may be treated with a surfactant to assist in the water absorption process.

[0026] The fluid is configured to flow along the longitudinal axis of the assay test strip 101 from the conjugate pad 103 to the absorption pad 105. The components of the assay test strip 101 are described in relation to this direction of fluid flow. For example, the membrane 104 is downstream of the conjugate pad 103 and upstream of the absorption pad 105. For example, the sample receiving zone 121 is downstream of the buffer receiving zone 111 and upstream of the detection zone 131. The detection zone 131 of the membrane 104 includes an immobilized capture substance configured to specifically bind to the analyte of interest, if present in the sample. The membrane 104 may further include an additional detection zone for detecting one or more analytes of interest, and may include one or more control zones. The membrane 104 is transparent in the visible region and can minimize undesirable reading interference to accurately determine the presence and / or amount of the analyte of interest. The capture substance may be immobilized on or inside the membrane 104 by any suitable method including, for example, attachment, spraying, soaking, immersing, pouring, or injecting. For example, the capture substance may be attached to and immobilized on the membrane 104 by preparing a solution containing the capture substance and spraying the solution onto the membrane 104 using air jet technology. In another example, the capturing substance is attached by preparing a solution containing the capturing substance, injecting the solution, spraying the solution, formulating the solution as a powder or gel that is placed on or rubbed onto a test strip, or by any other suitable method.

[0027] The capture substance can be immobilized in any appropriate amount on the detection zone 131 of the assay test strip 101. Advantageously, embodiments of this disclosure can detect the presence and amount of low concentrations of the analyte of interest using certain types of capture substances and amounts typical of a standard lateral flow sandwich assay. In some embodiments, the immobilized capture substance is present in an amount ranging from approximately 0.1 to 20 μL / test strip. In this exemplary embodiment, the conjugate pad 103 is placed on a portion of the back card 102. When the assay test strip 101 is housed in the lateral flow device 100, the buffer receiving zone 111 is accessible by the buffer well 110 and is located laterally below the buffer well 110. The sample receiving zone 121 is accessible by the sample well 120 and is located laterally below the sample well 120. In some cases, the conjugate pad 103 may be fixed to the back card 102. The conjugate pad 103 may be made of any suitable material that enables fluid flow by means of a material such as fibers (including glass fibers), polyester, or other material that provides fluid flow by the conjugate pad 103. In this exemplary embodiment, the conjugate pad 103 includes an analyte-specific conjugate 200 attached to the sample receiving zone 121. Embodiments of the analyte-specific conjugate 200 may be referred to as the “first conjugate” described herein. The analyte-specific conjugate 200 is configured to solubilize when the fluid is received in the sample receiving zone 121. As described below in relation to Figure 3, the analyte-specific conjugate 200 includes a first label 210 (such as a detection molecule), a first binding partner 230, and an active substance 220 that specifically binds to the target analyte 221 (if present in the sample). Thus, the analyte-specific conjugate 200 is configured to specifically bind to the target analyte (if present) in the sample received in the sample receiving zone.

[0028] In one example, the analyte-specific conjugate 200 is placed on or inside the sample receiving zone 121. In another example, the analyte-specific conjugate 200 is placed on or inside a first conjugate zone located downstream of the sample receiving zone 121, such that the sample (and any target analyte, if present) added to the sample receiving zone 121 flows through the first conjugate zone and interacts with the analyte-specific conjugate 200. The analyte-specific conjugate 200 can be placed on or inside the assay apparatus 101 using any suitable method, including, for example, attaching, spraying, soaking, pouring, or injecting the analyte-specific conjugate onto or inside the sample receiving zone 121. For example, the analyte-specific conjugate can be attached by preparing a solution containing the analyte-specific conjugate and spraying the solution using air jet technology. In another example, the analyte-specific conjugate may be prepared in solution, and can be attached by injecting the solution, spraying the solution, formulating the solution as a powder or gel placed on or rubbed onto a test strip, or by any other suitable method.

[0029] The analyte-specific conjugate 200 can be provided in any suitable amount at the sample receiving zone 121 (or other suitable location) of the assay test strip 101. Advantageously, embodiments of this disclosure allow for the detection of the presence and amount of low concentrations of the target analyte using certain types of analyte-specific conjugate 200 and amounts typical of a standard lateral flow sandwich assay. In some embodiments, the analyte-specific conjugate is applied in amounts ranging from approximately 0.1 to 20 μL / test strip.

[0030] Embodiments of the systems, apparatus, and methods described herein include a signal amplification conjugate 250. Embodiments of the signal amplification conjugate 250 may be referred to as the “second conjugate” as described herein. As described below in relation to Figure 3, the signal amplification conjugate 250 includes a second label 211 and a second binding partner 231. The second binding partner 231 is configured to specifically bind to the first binding partner 230 of the analyte-specific conjugate 200. The signal amplification conjugate 250 is present or added upstream of the analyte-specific conjugate 200. In one example described in relation to Figure 2, the signal amplification conjugate 250 is added to the assay test strip 101 during the manufacturing process. In another example, the signal amplification conjugate 250 is added to the assay test strip 101 by an operator during the test event. Embodiments of this disclosure are not limited to the manner in which the signal amplification conjugate 250 is introduced into the lateral flow assay, and it will be understood that other examples of those described herein are also appropriate. In one example shown in Figure 2, the signal amplification conjugate is placed on or within the buffer receiving zone 111. In another example, the signal amplification conjugate 250 is placed on or within a second conjugate zone located downstream of the buffer receiving zone 111 and upstream of the sample receiving zone 121, such that the sample (and any target analyte, if present) added to the sample receiving zone 121 does not interact with the signal amplification conjugate 250 in the second conjugate zone. This spatial positioning also ensures that the buffer added to the buffer receiving zone 111 flows downstream through the second conjugate zone and solubilizes the signal amplification conjugate 250. The non-limiting implementation in Figure 2 does not rehydrate the signal amplification conjugate 250 using a sample solution, but it will be understood that other implementations are appropriate. For example, in another implementation of this disclosure, both the analyte-specific conjugate 200 and the signal amplification conjugate 250 can be rehydrated using a single solution (such as a sample solution). In this case, by applying the sample solution at different times, it is possible to ensure that the analyte-specific conjugate 200 moves down the channel to the detection zone, and then the signal amplification conjugate 250 moves down the channel to the detection zone of the test strip.

[0031] The signal amplification conjugate 250 can be placed on or inside the assay apparatus by any suitable method, including, for example, attaching, spraying, soaking, pouring, or injecting the signal amplification conjugate 250 onto or inside the buffer receiving zone 111 (or other suitable location). For example, the signal amplification conjugate 250 can be attached by preparing a solution containing the signal amplification conjugate 250 and spraying the solution using air jet technology. In another example, the signal amplification conjugate 250 may be attached by preparing a solution, injecting the solution, spraying the solution, formulating the solution as a powder or gel placed on or rubbed onto a test strip, or by any other suitable method.

[0032] It will be understood that the signal amplification conjugate 250 can be added to the assay test strip in an area other than the buffer receiving zone 111. For example, in one non-limiting embodiment, the lateral flow device 100 does not include a separate or dedicated location such as a buffer well or buffer receiving zone to receive the signal amplification conjugate 250 on the assay test strip 101. The signal amplification conjugate 250 can be added to the assay test strip 101 at any suitable location after the analyte-specific conjugate 200 has been solubilized in the sample receiving zone 121 and moved to the detection zone 131. Embodiments of the signal amplification conjugate 250 of this disclosure can be provided in any suitable amount in the buffer receiving zone 111 (or other suitable location) of the assay test strip 101. In some embodiments, the signal amplification conjugate 250 is attached in an amount ranging from about 0.1 to 20 μL / test strip. In some embodiments, the signal amplification conjugate 250 is attached to the assay test strip 101 in an amount of 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 μL / test strip.

[0033] The signal amplification conjugate 250 is placed on or added to the assay test strip 101 upstream of the location where the analyte-specific conjugate 200 is placed on or added to the assay test strip 101. The signal amplification conjugate 250 and the analyte-specific conjugate 200 can be placed on the assay test strip 101 simultaneously or at different times during the preparation process of the assay test strip 101, or by an operator performing an assay event using the assay test strip 101. In one non-limiting example, both the signal amplification conjugate 250 and the analyte-specific conjugate 200 are added to the assay test strip 101 simultaneously or substantially simultaneously, for example, by introducing or attaching both the signal amplification conjugate and the analyte-specific conjugate to the assay strip at the same time or nearly simultaneously, but at different locations on the assay test strip 101. In this example, the analyte-specific conjugate 200 is added on or placed downstream of the location where the signal amplification conjugate 250 is added and placed on the assay test strip 101. This spatial localization minimizes interactions between conjugates before the analyte-specific conjugate moves to the detection zone 131 and is bound there. In another non-limiting example, the signal amplification conjugate 250 is added to the assay test strip 101 before or after the addition of the analyte-specific conjugate 200, using any suitable method or technique, including those described herein, at any suitable location, but not limited to. For example, if the signal amplification conjugate 250 is added after the analyte-specific conjugate 200 has moved to the detection zone 131 and is bound there, it may be added at the same location, or substantially the same location, where the analyte-specific conjugate 200 was initially added.

[0034] In some embodiments, the lateral flow apparatus 100 includes the signal amplification conjugate 250 before the test event using the assay test strip 101, without including a separate or dedicated location such as a buffer well or buffer receiving zone. Alternatively, the signal amplification conjugate 250 is introduced into the assay test strip 101 by the operator during the test event. For example, the signal amplification conjugate 250 can be introduced into the assay test strip after the sample has been introduced into the assay test strip. In particular, the sample is first placed in the sample receiving zone, and the sample can be moved through the assay strip for an appropriate amount of time. The time may be sufficient for the analyte-specific conjugate 200 to solubilize, form a binding interaction with the target analyte in the sample (if present), move to the detection zone, and form a binding interaction with the immobilized capture substance in the detection zone. After incubation for an appropriate amount of time, the solution containing the signal amplification conjugate 250 is introduced into the lateral flow apparatus 100. The signal amplification conjugate 250 then moves through the assay test strip 101 to the detection zone 131.

[0035] The delay time before adding the sample and signal amplification conjugate 250 to the assay test strip 101 can be adjusted based on various parameters, including the fluid containing or suspected to contain the target analyte, the analyte-specific conjugate 200, the signal amplification conjugate 250, and the material properties of the assay test strip 101, although these parameters are not limited to the characteristics of the target analyte. Examples of delay times, but not limited to, include 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 12 minutes, and 15 minutes. In this example, the signal amplification conjugate 250 is not present on the assay test strip 101 at the start of the test event. Therefore, embodiments of the present disclosure for amplifying the signal of an analyte present at low concentrations, such as below the detection limit, can be achieved in various ways using the signal amplification conjugate 250, and include preparing a test strip with the signal amplification conjugate 250 present, or adding the signal amplification conjugate 250 in a later step.

[0036] Embodiments of the systems, apparatus, and methods described herein can detect the target analyte very rapidly. The total time from the start of the test event (defined as the time when the sample is added to the test strip) to the end of the test event (defined as the time when the measurement system detects a signal generated in the detection band, if any) is not limited but may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, and 15 minutes. Figure 3 schematically illustrates exemplary embodiments of the systems, apparatus, and methods described herein. Without being bound by any particular theory, the process by which the analyte-specific conjugate 200 conjugates with the analyte of interest 221, the trapping material 240 immobilized in the trapping zone 242, and the signal amplification conjugate 250 is now shown with reference to Figure 3, although it should be understood that this disclosure is not limited to the schematic diagram in Figure 3.

[0037] The analyte-specific conjugate 200 comprises a first label 210, a first binding partner 230, and an active agent 220 that specifically binds to the analyte 221 (if present in the sample). The active agent 220, configured to specifically bind to the analyte 221, may include an antibody or antibody-binding fragment that specifically binds to the analyte 221. The analyte-specific conjugate 200 is attached to or added to the sample receiving zone 121 (or any other suitable location) of the assay test strip 101, for example, using the method described above in relation to Figure 2. The sample is added to the assay test strip 101, for example, at the sample receiving zone 121. The analyte-specific conjugate 200 is solubilized in the sample, and the active agent 220, if present in the sample, specifically binds to the analyte 221, forming an analyte-active agent-first label complex 225. The complex 225 contains an immobilized capture substance 240 configured to flow through the assay strip 101 to the detection zone 131 and specifically bind to the analyte 221. The complex 225 is captured in the detection zone 131 as shown in Figure 3.

[0038] After an appropriate amount of time has elapsed for the complex 225 to migrate and be captured in the detection zone 131, the signal amplification conjugate 250 described herein is moved downstream of the detection zone 131 along the assay test strip 101. The signal amplification conjugate 250 includes a second label 211 and a second binding partner 231. In one non-limiting embodiment, the signal amplification conjugate 250 is present on the assay test strip 101 before the sample is added (attached in any suitable way, including those described above in relation to Figure 2) and the buffer is applied to the signal amplification conjugate 250, thereby moving it downstream of the detection zone 131. In this embodiment, the previously attached signal amplification conjugate 250 attaches upstream of the sample receiving zone 121, for example, upstream of the buffer receiving zone 111 located upstream of the sample receiving zone 121. This spatial localization of the signal amplification conjugate 250 in the buffer receiving zone 111 upstream of the analyte-specific conjugate 200 in the sample receiving zone 121 ensures that when a sample is added to the sample receiving zone 121, the analyte-specific conjugate 200, rather than the signal amplification conjugate 250, becomes solubilized and mobile. In another non-limiting implementation, the signal amplification conjugate 250 is added to the assay test strip 101 after a suitable amount of time has elapsed and the sample has been added to the sample receiving zone. In this implementation, the buffer containing the signal amplification conjugate 250 may be added to the assay test strip 101 at the buffer receiving zone 111 (if present), the sample receiving zone 121, or any other suitable location on the assay test strip 101.

[0039] In both of these implementations, embodiments of the present disclosure allow the analyte-specific conjugate 200 to move to the detection zone 131 and bind to the capture material 240 before being exposed to the signal amplification conjugate 250 and forming a binding interaction. Advantageously, embodiments of the present disclosure allow the release timing of the signal amplification conjugate 250 to be aligned with the characteristics of a lateral flow assay, and the release to be activated only when the buffer is added to the system (either to the buffer receiving zone 111 containing the signal amplification conjugate 250, or to any suitable location where the buffer itself contains the signal amplification conjugate 250). These embodiments of the present disclosure also advantageously prevent interactions between the analyte-specific conjugate 200 and the signal amplification conjugate 250 from occurring at undesirable locations, primarily upstream of the detection zone 131, on the assay test strip 101. Therefore, embodiments of the present disclosure, which can time the release of the signal amplification conjugate 250 to an optimal time (after a sufficient amount of analyte-specific conjugate 200 has moved to and bound to the detection zone 131), are advantageous in that they maximize the occurrence of binding interactions between the analyte-specific conjugate 200 present in the detection zone (whether or not it is bound to the capture substance 240) and the signal-amplification conjugate 250 in the detection zone 131. This causes the signals generated by the conjugates 200 and 250 in the detection zone to aggregate and increase to a level above the detection threshold.

[0040] After the buffer is added to the assay strip 101, either by directly introducing the signal amplification conjugate 250 into the system or by solubilizing a signal amplification conjugate 250 already present in the system, a second series of binding events occurs due to the properties of the signal amplification conjugate 250 of this disclosure. As previously stated, the second binding partner 231 specifically binds to the first binding partner 230 of the analyte-specific conjugate 200. Without being bound by any particular theory, if the signal amplification conjugate 250 flows through the assay strip 101 to the detection zone 131, the second binding partner 231 of the signal amplification conjugate 250 is thought to specifically bind to the first binding partner 230 of the complex 225 bound to the capture substance 240, thereby capturing the signal amplification conjugate 250 in the detection zone 131. The second label 211 of the precisely bound signal amplification conjugate 250 generates a signal in the detection zone. In this way, the signal from a single analyte-specific conjugate 200 coupled to the detection band is amplified. In some embodiments, a single analyte-specific conjugate 200 of complex 225 binds to multiple signal-amplifying conjugates 250, which themselves contribute to the signal generated by the single analyte-specific conjugate 200 of complex 225. In some embodiments of this disclosure, even this first level of binding interaction between complex 225 and one or more signal-amplifying conjugates 250 is sufficient to increase the signal generated in the detection band above the threshold detection level.

[0041] Advantageously, embodiments of the present disclosure can generate a higher level of signal amplification than that possible with first-level binding interactions alone. In embodiments of the systems, apparatus, and methods described herein, the amplification of the signal generated in the detection zone is further enhanced by the ability of the coupled signal amplification conjugate 250 to bind to residual analyte-specific conjugates 200 that are present in the detection zone but not bound to the capture material 240. These residual analyte-specific conjugates 200 may normally pass through the detection zone 131 or remain scattered haphazardly in the detection zone in a typical lateral flow assay. In the presence of the signal amplification conjugate 250 described herein, these residual analyte-specific conjugates 200 can bind to a scaffold of conjugates retained in the detection zone 131 via the complex 225, and further contribute to the aggregation signal generated in the detection zone 131. The signal generated by embodiments of the present disclosure can increase exponentially in intensity. Accordingly, in some embodiments, the signal intensity generated in the detection band 131 can be increased by amplifying the signal by a factor of 2 to 100, for example, by a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100. It will be understood that this disclosure is not limited to these exemplary amplification levels, and other amplification levels are possible in embodiments of this disclosure.

[0042] Advantageously, the aforementioned chain reaction of binding events can occur even in the presence of very small amounts of the target analyte in the sample. Without being bound by any particular theory, it is conceivable that the residual analyte-specific conjugate 200 can form a binding interaction with the signal amplification conjugate 250, regardless of whether the residual analyte-specific conjugate 200 binds to the analyte in the sample or not. In other words, the binding interaction after the first interaction between complex 225 and signal amplification conjugate 250 reliably triggers a chain reaction of binding events in a local region (detection band), depending on this first interaction, but does not form a pair of binding conjugates 200, 250 that amplify the signal generated by complex 225, depending on the additional target analyte. An advantage of this disclosure is that the absence of a signal in the detection zone 131 can be correlated with high reliability to the presence of the target analyte in the sample. In the absence of the target analyte in the sample, the complex 225 does not form and does not bind to the capture material 240 in the detection zone 131. In the absence of a binding event, the signal amplification conjugate 250 does not bind in the detection zone 131 and passes through the detection zone 131 to the absorption pad 151.

[0043] As used herein, buffer refers to a solution used in a lateral flow assay that does not interfere with binding interactions, does not denature the analyte of interest and other components of the instrument, and maintains the neutrality intended to allow components to flow onto a test strip. Various buffers are known, and any variety of buffers may be used for the specific analytes, compounds, and components used in the lateral flow assays described herein. In some embodiments, the buffer is phosphate-buffered saline (PBS). Embodiments of this disclosure have been described in relation to a buffer receiving zone located upstream of a sample receiving zone, and a tracking buffer added to a buffer containing a signal amplification conjugate that is added to any suitable location on the assay test strip after the sample has been added. However, it will be understood that other methods of adding or releasing the buffer to the system are suitable and can be used in embodiments of this disclosure. For example, the tracking buffer can be pre-packaged in a small bag or other suitable structure and incorporated into the assay test strip upstream of the attached signal amplification conjugate and into the sample receiving well of the assay test strip. The tracking buffer can be released at an appropriate time after the sample has been added to the assay test strip, by rupturing the small bag using any suitable mechanism.

[0044] In some embodiments, the signal amplification conjugate 250 does not bind to other conjugates (such as the analyte-specific conjugate 200) until it reaches the detection zone 131. In some embodiments, the binding of the signal amplification conjugate 250 to other conjugates results in precipitation of the bound conjugate, causing smearing of the precipitated label across the test strip, thereby interfering with or confusing the results. As previously stated, embodiments of the present disclosure include features that prevent or minimize this precipitation of the bound conjugate upstream of the detection zone 131. These features ensure that the signal amplification conjugate 250 remains soluble in the solution and does not bind to the analyte-specific conjugate 200 before reaching the analyte-specific conjugate 200 of the complex 225 captured in the detection zone 131. In one example, an embodiment of the present disclosure includes attaching a signal amplification conjugate 250 to an assay test strip 101 upstream of an analyte-specific conjugate 200 in a sample receiving zone 121, and solubilizing the signal amplification conjugate 250 only after the analyte-specific conjugate has had the opportunity to flow first through a lateral flow device to a detection zone 131. In another example, an embodiment of the present disclosure includes adding the signal amplification conjugate 250 to a buffer applied to the assay test strip 101 only after the analyte-specific conjugate 200 has had the opportunity to flow first through a lateral flow device to a detection zone 131.

[0045] The first label 210 of the analyte-specific conjugate 200 may be the same label as the second label 211 of the signal amplification conjugate 250. In one non-limiting example, the first label 210 and the second label 211 may be colloidal gold labels. The colloidal gold labels may contain gold nanoparticles. The first label 210 and the second label 211 may contain any suitable labels that can provide a detectable signal. Thus, in some embodiments, the first label 210 and the second label 211 may be enzymes, colloidal gold particles (also called metal nanoparticles such as gold or silver nanoparticles), colored latex particles, radioisotopes, cofactors, ligands, chemiluminescent or fluorescent substances, protein-absorbing silver particles, protein-absorbing iron particles, protein-absorbing copper particles, protein-absorbing selenium particles, protein-absorbing sulfur particles, protein-absorbing tellurium particles, tellurium carbon particles, or protein-absorbing dye sacs or any other suitable labels.

[0046] As described above, the active agent 220 is an active agent that specifically binds to the analyte 221 and may include an antibody or antibody-binding fragment that specifically binds to the analyte 221. Therefore, the active agent 220 may also be an anti-analyte antibody. The first binding partner 230 of the analyte-specific conjugate 200 specifically binds to the second binding partner 231 of the signal amplification conjugate 250, and thus the first and second binding partners 230, 231 constitute a binding pair. This binding pair may include, for example, a binding pair selected from the group consisting of antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid chain / complementary nucleic acid chain, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, biotin / avidin, receptor / ligand, or virus / cell receptor. In one non-limiting example, the first binding partner 230 is avidin (or a derivative or analog thereof), and the second binding partner 231 is biotin (or a derivative or analog thereof). For example, the analyte-specific conjugate 200 may be biotinylated, and the signal amplification conjugate 250 may contain an anti-biotin antibody or a streptavidin-binding partner. In another non-limiting example, the binding pair is an antigen / antibody such as a peptide and an antibody. In some embodiments, the peptide is a decapeptide. In yet another non-limiting example, the binding pair is doxorubicin and anti-doxorubicin. In yet another non-limiting example, the binding pair is methotrexate and anti-methotrexate. In yet yet another non-limiting example, the binding pair is FITC and anti-FITC antibody. Embodiments of this disclosure may use any suitable first binding partner 230 that specifically binds to the second binding partner 231, and will be understood to be not limited to the examples given above.

[0047] An advantage of the embodiments of this disclosure is that the second binding partner 231 of the signal amplification conjugate 240 can be selected to specifically bind to the first binding partner 230 of the analyte-specific conjugate 200 without interfering with the binding of the analyte-specific antibody of the analyte-specific conjugate 200 to the analyte-specific conjugate 200. In one example, different proteins / peptides can co-bind with the analyte-specific antibody on the first label 210 to form the analyte-specific conjugate 200. In another example, a different protein / peptide can be biotinylated first and then co-bind with the analyte-specific conjugate on the first label 210 to form the analyte-specific conjugate 200. In a further example, a small molecule such as biotin is added to the analyte-specific antibody and subsequently bound to the first label 210 of the analyte-specific conjugate 200, and the signal amplification conjugate 250 contains a small molecule-specific binding protein / antibody. In this example, the small molecule added to the analyte-specific antibody is selected so as not to interfere with the binding of the analyte to the analyte-specific antibody in the analyte-specific conjugate 200. In yet another example, a protein / peptide, rather than biotin, may be able to bind to another molecule that contains another antibody or binding protein. The added protein / peptide, biotin, or other molecule bound to the first label 210 can therefore provide a binding site for the antibody to this protein / peptide, biotin, or other molecule in the signal amplification conjugate 250 without interfering with the analyte-specific binding interaction between the target analyte 221 and the analyte-specific antibody bound to the first label 210.

[0048] In some embodiments, the lateral flow apparatus includes one or more control zones. The control zone may be in the detection zone or separate from the detection zone. In some embodiments, the control zone may be a positive control zone and may contain small molecules that bind to proteins such as bovine serum albumin (BSA). A positively labeled control antibody that specifically binds to the small molecules may be attached to the conjugate pad. When the positively labeled control antibody is rehydrated in the liquid sample, it flows into the positive control zone, binds to the small molecules, and forms a semi-sandwich. In some embodiments, the positive control zone includes an immobilized capture substance that specifically binds to an analyte-specific conjugate, and an analyte-specific conjugate that does not bind to the target analyte passes through the detection zone (test line) and specifically binds to the immobilized capture substance in the control line, generating a signal indicating proper functioning of the lateral flow apparatus. It will be understood that any suitable control zone can be implemented in embodiments of this disclosure. Embodiments of the reader and data analyzer disclosed herein can process signal measurements obtained from the control band to correct signals measured in the detection band or to warn the operator that the test is invalid.

[0049] As used herein, “analyte” generally refers to the substance to be detected. In some embodiments, the analyte is found in a sample at low concentrations, such as below the detection limit of the measurement system. Some analytes are normally present in a sample at high concentrations, but may be present at low concentrations in a particular sample under test due to a specific disorder, a phase of the disorder, or processing of the sample. For example, an analyte may be present in a sample at low concentrations in the early stages of a disease or disorder, followed by the peak stages of the disease or disorder. Embodiments of this disclosure will be understood to be applicable to lateral flow assays that measure any analyte present or suspected to be present in a sample in small amounts, or when there is low sensitivity to measure such analytes.

[0050] The analytes may include antigenic substances, haptens, antibodies, and combinations thereof. Examples of analytes, but not limited to, include toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs (including drugs administered for therapeutic purposes and drugs administered illegally), drug intermediates or by-products, bacteria, viral particles, and metabolites or antibodies of any of the aforementioned substances. In some embodiments, the target analyte is an influenza virus, such as influenza A, influenza B, or influenza C virus. Additional analytes may be included for the target organism or environmental substance.

[0051] In some embodiments, the sample may contain one or more target analytes, and therefore the lateral flow apparatus may be configured to detect one or more target analytes. To detect one or more target analytes, the lateral flow apparatus described in this disclosure may include one or more analyte-specific conjugates 200, each analyte-specific conjugate 250 specifically binding to a target analyte. For example, for a desired number of target analytes present in or expected to be present in the sample, a first analyte-specific conjugate 200A may specifically bind to a first target analyte 221A, a second analyte-specific conjugate 200B may specifically bind to a second target analyte 221B, a third analyte-specific conjugate 200C may specifically bind to a third target analyte 221C, and so on. In some embodiments, each analyte-specific conjugate that specifically binds to a target analyte does not specifically bind to any other target analytes. Furthermore, in some embodiments, each analyte-specific conjugate may have the same or different labels as each other. For example, each analyte-specific conjugate may include a unique label that is different from any other labels of the analyte-specific conjugates. In addition, if there are one or more target analytes, the lateral flow apparatus may include one or more capture lines, each capture line containing an immobilized capture substance that specifically binds to one particular target analyte. For example, for a desired number of target analytes to be analyzed in a test sample, a first capture substance 240A immobilized in capture zone 242A specifically binds to the first target analyte 221A, a second capture substance 240B immobilized in capture zone 242B specifically binds to the second target analyte 221B, and a third capture substance 242C immobilized in capture zone 242C specifically binds to the third target analyte 221C. In some embodiments where the lateral flow apparatus is configured to measure one or more target analytes, the apparatus further includes a signal amplification conjugate 250. The signal amplification conjugate 250 may include one binding partner of a binding pair that specifically binds to another binding partner of the binding pair. In some embodiments, the signal amplification conjugate binds to a first analyte-specific conjugate, a second analyte-specific conjugate, a third analyte-specific conjugate, etc., for a desired number of target analytes, thereby amplifying the signal for each of the target analytes. In some embodiments, the signal amplification conjugate binds to one, two, three or more analyte-specific conjugates, thereby amplifying the signal only for a desired number of analytes, for example, only for target analytes that are known or suspected to be present in the sample at low concentrations.

[0052] It should be understood that since target analytes are often present in samples at various concentrations, signal amplification is not required for each target analyte, but only for target analytes that are known or suspected to be present at low concentrations, such as concentrations below the detection limit of the measurement system. Accordingly, in some embodiments, a first analyte-specific conjugate that specifically binds to a first analyte present at low concentrations includes a binding partner, while a second analyte-specific conjugate that specifically binds to a second analyte present at concentrations above the detection limit does not include a binding partner. Thus, the signal amplification conjugate binds to the first analyte-specific conjugate but not to the second analyte-specific conjugate, thereby amplifying the signal of the first target analyte. Various iterations of this disclosure can be carried out, for example, by amplifying any desired signal, including signals below the detection limit of the measurement system. The following non-limiting examples illustrate the features of the lateral flow apparatus, test systems, and methods described herein and are not intended to limit the scope of this disclosure in any way.

[0053] (Example 1) Preparation of lateral flow assay according to the present invention The following examples describe the preparation of the lateral flow assay described in this disclosure for measuring an analyte present in a sample at low concentrations. In this non-limiting example, the analyte of interest is influenza A ("Flu A"). Test strips were prepared with a conjugate pad having a sample receiving zone and a buffer receiving zone. Analyte-specific conjugates were prepared in solution, and the solution was sprayed onto the sample receiving zone by air jet deposition. In short, anti-Flu A antibody and biotin were incubated with gold nanoparticles to form analyte-specific conjugates. The analyte-specific conjugates were deposited onto the conjugate pad in the sample receiving zone at a rate of 7 μL / test strip by spraying the solution containing the analyte-specific conjugates with an air jet. The conjugate pad was heated to dry the analyte-specific conjugates onto the conjugate pad. The amount of anti-Flu A antibody deposited onto the conjugate pad used to formulate the analyte-specific conjugates was approximately 260 ng per test strip.

[0054] The signal amplification conjugate was prepared in solution, and the solution was sprayed onto a buffer receiving zone by air jet attachment. In short, antibiotin was incubated with gold nanoparticles to form a signal amplification conjugate. The signal amplification conjugate was attached to the conjugate pad in the buffer receiving zone at a rate of 2 μL / test strip by spraying the solution containing the signal amplification conjugate with an air jet. The conjugate pad was heated to dry the signal amplification conjugate onto the conjugate pad. The amount of antibiotin antibody attached to the conjugate pad used to formulate the signal amplification conjugate was approximately 74 ng per test strip. Furthermore, a test strip with a detection band was prepared. The detection band contained an immobilized capture substance that specifically binds to Flu A. In this example, an anti-Flu A antibody was attached to the detection band at a concentration of 2.4 mg / mL per 0.75 μL / cm. In this example, the detection band also included a positive control capture band. The positive control capture band was prepared to ensure that the assay functioned correctly. In this example, the positive control capture band contained immobilized bovine serum albumin derivatized with antibiotin (BSA-antibiotin). The immobilized BSA-biotin captured the signal amplification conjugate present in the test strip rehydrated with buffer and flowed into the positive control band, indicating that the assay was functioning correctly. The signal amplification conjugate was captured in the positive control line, and the positive control signal indicated that the assay was functioning correctly. The test strip was placed in a lateral flow apparatus having a housing that held the test strip. The lateral flow apparatus included a buffer well placed above a buffer receiving zone, a sample well placed above a sample receiving zone, and a reading window placed above a detection zone, as shown in Figure 1.

[0055] (Example 2) Amplification of low-concentration analyte signals The following examples demonstrate the use of the lateral flow assay described in Example 1 to detect low concentrations of Flu A in a sample. The lateral flow assay, as prepared in Example 1, was contacted with a sample containing a low concentration of Flu A. In this non-limiting example, the Flu A antigen was a diluted recombinant Flu A nucleoprotein expressed in Escherichia coli (E. coli) and was applied as crude cell lysate. As a control, the lateral flow assay prepared in Example 1, lacking a signal amplification conjugate, was used. Samples containing Flu A were added to the sample wells and control lateral flow apparatus. The generated signals were measured at various time points, as shown in Figure 4 and Table 1.

[0056] [Table 1]

[0057] Samples that did not contain the analyte of interest were also applied to lateral flow assays prepared with various amounts of the signal amplification conjugate described in this disclosure. In particular, amounts of 0.5, 1, 1.5, and 3 μL of the signal amplification conjugate were attached to test strips and compared to control strips, with readings obtained at 10 and 20 minutes. The results are shown in Table 2, and were measured twice at each time point. Samples that did not contain the analyte of interest were attached to assay test strips to verify that no signal was generated in the test line when the analyte of interest was not present in the sample. As shown in Table 2 below, the signal in the test line remained negative for all samples that did not contain the analyte of interest (signal intensities of 0 AU or substantially 0 AU were measured in the test line). Therefore, any signal of 1 AU or less can be considered equivalent to a 0 AU measurement as system noise. Furthermore, as shown in Table 2 below, the intensity of the system noise measurement did not increase with increasing amounts of the signal amplification conjugate. Without being bound by any particular theory, it is assumed that a signal amplification conjugate will not amplify a signal in the absence of the target analyte because there is no initial signal to amplify (in other words, the absence of the analyte in the sample means that no sandwich structure is formed in the test line, and therefore there is no sandwich structure present to generate the signal that will be amplified by the signal amplification conjugate).

[0058] [Table 2]

[0059] Finally, the amount of Flu A varied in the samples before they were placed in the lateral flow apparatus. In particular, samples with Flu A in amounts of 0%, 0.04%, 0.1%, 0.2%, and 0.5% (expressed as a percentage of the antigen concentration in the sample) were prepared, and the samples were attached to lateral flow apparatuses and controlled lateral flow apparatuses without any signal amplification conjugates. Signal intensity was read at 10 and 20 minutes. The results are shown in Figure 5 and Table 3.

[0060] [Table 3]

[0061] Advantageously, the lateral flow assay described herein amplifies the signal of an analyte present in a sample at low concentrations, such as below the detection limit, thereby enabling reliable measurement of the analyte present in the sample at low concentrations. In addition to determining the presence of a target analyte in a sample, embodiments of this disclosure can also be used to quantify the amount of a target analyte present in a sample. For example, a solution containing a known amount of a target analyte can be applied to an assay test strip prepared according to an embodiment of this disclosure. A measurement system can measure the intensity of the signal generated by the test strip, and a dose-response curve can be constructed using this data. After performing a test event using a test strip prepared according to an embodiment of this disclosure, the measurement system can measure the signal generated by the test strip and compare this test signal with the signal plotted on the dose-response curve to determine the amount of the target analyte in the test sample. It will be understood that other methods for quantifying the amount of a target analyte are possible using embodiments of this disclosure.

[0062] Method for detecting a target analyte in a sample using a lateral flow assay described herein. Figure 6 shows an exemplary method 600 for detecting a target analyte in a low-concentration sample described herein using a lateral flow assay. Method 600 begins with step 605, in which the lateral flow assay described herein is provided. In step 610, the sample is applied to the sample well of the lateral flow apparatus. In some embodiments, applying a sample to a sample well involves bringing the sample into contact with the lateral flow assay. A sample can be brought into contact with the lateral flow assay by external application, such as by 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 that holds the sample. In some embodiments, the sample may be poured, immersed, sprayed, placed, or brought into contact with the sample reservoir. The method then proceeds to step 615, in which the application of the sample in the sample well solubilizes the analyte-specific conjugate ("first conjugate") present in the sample receiving zone or the first conjugate zone downstream of the sample receiving zone. The analyte-specific conjugate includes a first label, a first binding partner, and an active agent that specifically binds to the analyte of interest. As previously stated, the apparatus may include a second analyte-specific conjugate, depending on the number of analytes being analyzed, which includes a label, a binding partner, and an active agent that specifically binds to a second analyte of interest, and the apparatus may include a third analyte-specific conjugate or additional analyte-specific conjugate, which includes a label, a binding partner, and an active agent that specifically binds to a third analyte of interest. In some embodiments, the analyte-specific conjugate may not include a binding partner, since the particular analyte of interest may not require amplification due to the expected or typical concentration of the particular analyte of interest in the sample.

[0063] An analyte-specific conjugate (or possibly one or more analyte-specific conjugates) can be integrated into the sample receiving zone by physical or chemical bonding. After the sample is added to the sample well, the sample solubilizes the analyte-specific conjugate, releasing the bond that holds the analyte-specific conjugate to the conjugate pad. Next, in step 620, the analyte of interest (if present in the sample) is labeled with an analyte-specific conjugate. If present in the sample, the analyte-specific conjugate binds to the analyte of interest and forms a complex. It should be understood that steps 615 and 620 can occur substantially simultaneously. The method then moves to step 625, in which the complex moves along the fluid channel of the assay apparatus and, in the detection zone of the lateral flow assay, binds to the immobilized capture material to form a sandwich structure. It will also be understood that the implementation of the present disclosure may form a sandwich structure in the detection zone in many different ways. For example, steps 615, 620 and 625 may be replaced with step 615A, in which the analyte of interest and the analyte-specific conjugate flow separately into the detection zone, the analyte of interest binds to the immobilized capture material in the detection zone, and then the analyte-specific conjugate binds to the analyte of interest that has just been bound to it, forming an antibody-analyte-antibody-first label structure, commonly referred to as a sandwich.

[0064] After the complex is bound to the immobilized capture material (or forms a sandwich structure), the method then proceeds to step 630, in which the buffer is applied to the buffer well (or other suitable location) of the apparatus. In step 635, the buffer solubilizes the signal amplification conjugate ("second conjugate") which is located downstream of the buffer receiving zone. The signal amplification conjugate includes a second binding partner which specifically binds to the first binding partner of the second label and analyte-specific conjugate. The signal amplification conjugate may be integrated into the buffer receiving zone (or other suitable location) by physical or chemical binding. After the buffer is added to the buffer well, the buffer solubilizes the signal-amplification conjugate, releasing the binding that holds the signal amplification conjugate to the conjugate pad of the buffer receiving zone (or other suitable location). It will be understood that steps 630 and 635 may be replaced by alternative methods to add the signal amplification conjugate to the lateral flow assay.

[0065] The method then moves to step 640, in which the signal amplification conjugate flows into the detection band along with the buffer. In the detection band, the second binding partner forms a binding pair with the first binding partner of the analyte-specific conjugate of the complex bound to the capture substance in the detection band. As previously stated, the first binding interaction between the signal amplification conjugate and the complex bound to the capture substance in the detection band can amplify the signal generated in the detection band to a level above the threshold detection level. Thus, in some exemplary implementations, the method then moves to step 650, in which the amplified signal is detected in the detection band. Embodiments of the method of the present disclosure may also include the following binding interaction as described above: A chain reaction of binding events occurs according to the present disclosure, in which the interaction of a series of first and second binding pairs causes the accumulation of an analyte-specific conjugate and a signal amplification conjugate that form in the detection band. The accumulation of conjugates in the detection band amplifies the signal generated in the detection band, and in some cases, the signal is amplified exponentially. This chain reaction of binding events is shown in step 645 of Figure 6, in which the unbound first conjugate present in the detection band binds to the signal amplification conjugate that has become bound to the complex in the detection band.

[0066] Next, moving to step 650, the method includes detecting the signal generated in the detection zone. The detection zone may include capture zones that capture each complex (one or more target analytes are detected and / or quantified). For example, the detection zone may include a first capture zone for capturing a first complex, a second capture zone for capturing a second complex, and a third capture zone for capturing a third complex. A first immobilized capture material in the first capture zone binds to the first analyte (if present) and the first complex. If the first complex binds to the first immobilized capture material in the first capture zone and a signal amplification conjugate binds thereto, a first amplified signal is detected. The first amplified signal may include an optical signal as described herein. The signal generated in the detection zone can be detected using any suitable measuring system in step 650, including, but not limited to, visual inspection of the instrument and optical detection using an optical reader. The signal detected in step 650 may correlate to the presence or amount of the target analyte in the sample. In some embodiments, the sample is obtained from a source including environmental or biological sources. In some embodiments, the sample is thought to have one or more target analytes, including one or more target analytes present at low concentrations, such as below the detection limit of the measurement system. In some embodiments, the sample is thought to have no target analytes. In some embodiments, the sample is obtained and analyzed to verify the presence or absence of multiple analytes. In some embodiments, the fluid sample is blood or plasma. In some embodiments, the fluid sample is applied in an amount of 50 to 100 μL. In some embodiments, the detected signal is an optical signal, a fluorescence signal, or a magnetic signal.

[0067] Exemplary Test System Including the Lateral Flow Assay as Described in This Disclosure The lateral flow assay test systems described herein may include a lateral flow assay apparatus (including, but not limited to, test strips), a system housing including ports configured to receive all or part of the apparatus, a reader including a light source and a photodetector, a data analyzer, and combinations thereof. The system housing may be made of any one of a wide variety of materials, including plastic, metal, or composite materials. The system housing forms a protective enclosure for the components of the diagnostic test system. The system housing also specifies a container for mechanically registering the test strips with the reader. The container may be designed to receive any one of a wide variety of different types of test strips. In some embodiments, the system housing is a compact device capable of performing lateral flow assays in a variety of environments, including benches, fields, homes, or facilities for domestic, commercial, or environmental applications.

[0068] The reader may include one or more optoelectronic components for optically inspecting an exposed area of ​​the detection band of a test strip, and may detect multiple capture bands in the detection band. In some embodiments, 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 within a specific wavelength range or light having a specific polarization. For example, if the label is a fluorescent label such as a quantum dot, the light source is designed to illuminate the exposed area of ​​the capture band of the test strip with light in a wavelength range that induces fluorescence emission from the label. Similarly, the photodetector may be designed to selectively capture light from the exposed area of ​​the capture band. For example, if the label is a fluorescent label, the photodetector is designed to selectively capture light within the wavelength range of fluorescence emitted by the label or with light of a specific polarization. On the other hand, if the label is a reflective type label, the photodetector is designed to selectively capture light within the wavelength range of light emitted by the light source. Finally, 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 visually or by a spectrophotometer that detects the color from the color-producing substrate. 125 Analysis can be performed using radiation detectors for detecting radiation, such as gamma detectors for detecting I, or fluorophotometers for detecting fluorescence in the presence of light of a specific wavelength. When an enzyme-binding assay is used, quantitative analysis of the amount of the target analyte can be performed using a spectrophotometer. The lateral flow assay described herein can be robotically automated or performed, and, if desired, signals from multiple samples can be detected simultaneously. Furthermore, multiple signals can be detected for multiple target analytes, including cases where the labels of each target analyte are the same or different.

[0069] A data analyzer processes signal measurements obtained by a reader. Generally, a data analyzer can be implemented in any computing or processing environment, including digital electrical circuits 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 integrated into the housing of a diagnostic test system. In other embodiments, the data analyzer is located in a separate device, such as a computer, which can communicate with the diagnostic test system by wired or wireless connection. The data analyzer may also include circuitry for transferring results by wirelessly connecting to an external device for data analysis or review of results.

[0070] In general, result indicators may include one of a wide variety of different mechanisms that indicate one or more results of an assay test. In some implementations, a result indicator includes, for example, one or more lights (e.g., light-emitting diodes) that are activated to indicate the completion of the assay test. In other implementations, a result indicator includes an alphanumeric display (e.g., a two or three-letter light-emitting diode array) to indicate the results of the assay test. The test systems described herein may include a power supply to provide power to the active components of the diagnostic test system, including a reader, data analyzer, and results indicator. Power supply may be provided, for example, by a replaceable battery or a rechargeable battery. In other embodiments, the diagnostic test system may be powered by an external host device (e.g., a computer connected by a USB cable).

[0071] Characteristics of an exemplary lateral flow system The lateral flow apparatus described herein includes an apparatus housing. Any lateral flow apparatus housing described herein includes a top housing or a base housing and may be made of any suitable material, including, for example, vinyl, nylon, polyvinyl chloride, polypropylene, polystyrene, polyethylene, polycarbonate, polysulfane, polyester, urethane, or epoxy. The housing may be prepared by any suitable method, including, for example, injection molding, compression molding, transfer molding, blow molding, extrusion molding, foam molding, thermoforming, casting, lamination, or printing. The lateral flow apparatus described herein may include, but is not limited to, sample wells into which a fluid sample is introduced to a test strip, such as an immunochromatography test strip, present in the lateral flow apparatus. In one example, the sample may be introduced into the sample well by external application, such as using a dropper or other applicator. The sample may be poured or squeezed into the sample well. In another example, the sample well may be directly immersed in the sample, such as when the test strip is immersed in a container that holds the sample.

[0072] The lateral flow apparatus described herein may include, but is not limited to, buffer wells into which a buffer is introduced to a test strip, such as an immunochromatography test strip, present in the lateral flow apparatus. In one example, the buffer may be introduced to the buffer well by external application, such as using a dropper or other applicator. The buffer may be poured or squeezed into the buffer well. In another example, the buffer well may be directly immersed in the buffer, such as when a test strip is immersed in a container holding the buffer. The lateral flow apparatus described herein may include a solid support or substrate. Suitable solid supports include, but are not limited to, nitrocellulose, well walls of reaction trays, multiwell plates, test tubes, polystyrene beads, magnetic beads, membranes, and fine particles (such as latex particles). Any suitable porous material with sufficient porosity that is accessible by analyte-specific conjugates and signal amplification conjugates and has appropriate surface affinity for immobilized capture material can be used in the lateral flow apparatus described herein. For example, the porous structure of nitrocellulose has excellent absorption and absorption quality for a wide variety of substances, such as immobilized capture material. Nylon also has similar characteristics and is suitable. Microporous structures are as useful as materials having a gel structure in a hydrated state.

[0073] Further examples of useful solid supports include natural polymer carbohydrates and their synthetic modifications, crosslinking or substitution derivatives, e.g., agar, agarose, crosslinked alginic acid, substituted and crosslinked guar gum, particularly nitrile acids and carboxylic acids, mixed cellulose esters, and cellulose esters having cellulose ethers; nitrogen-containing natural polymers such as proteins and derivatives including crosslinked or modified gelatin; natural hydrocarbon polymers such as latex and rubber; polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and its partially hydrolyzed derivatives, polyacrylamide, polymethacrylic acid, copolymers and terpolymers of the aforementioned polycondensates, e.g., polyester, polyamide and poly Examples include synthetic polymers that can be prepared with a suitable porous structure, such as vinyl polymers containing urethane or other polymers such as polyepoxides; porous inorganic materials such as alkaline earth metal and magnesium sulfates or carbonates, including barium sulfate, calcium sulfate, calcium carbonate, alkali and alkaline earth metal silicates, aluminum and magnesium; and aluminum or silicon oxides such as clay, alumina, talc, kaolin, zeolite, silica gel or glass (these materials can be used as filters together with the aforementioned polymer materials); and mixtures or copolymers of the aforementioned classes, such as graft copolymers obtained by initiating the polymerization of synthetic polymers into existing natural polymers.

[0074] The lateral flow apparatus described herein may include porous solid supports such as nitrocellulose in the form of sheets or pieces. The thickness of such sheets or pieces can vary over a wide range, for example, about 0.01–0.5 mm, about 0.02–0.45 mm, about 0.05–0.3 mm, about 0.075–0.25 mm, about 0.1–0.2 mm, or about 0.11–0.15 mm. The pore size of such sheets or pieces can also vary over a wide range, for example, about 0.025–15 μm, more specifically about 0.1–3 μm, but the pore size is not intended to be a limiting factor in the selection of the solid support. The flow velocity of the solid support can also vary over a wide range where applicable, for example, about 12.5 to 90 sec / cm (i.e., 50 to 300 sec / 4cm), about 22.5 to 62.5 sec / cm (i.e., 90 to 250 sec / 4cm), about 25 to 62.5 sec / cm (i.e., 100 to 250 sec / 4cm), about 37.5 to 62.5 sec / cm (i.e., 150 to 250 sec / 4cm), or about 50 to 62.5 sec / cm (i.e., 200 to 250 sec / 4cm). In certain embodiments of the apparatus described herein, the flow velocity is about 35 sec / cm (i.e., 140 sec / 4cm). In other specific embodiments described herein, the flow velocity is about 37.5 sec / cm (i.e., 150 sec / 4cm).

[0075] The surface of a solid support can be activated by a chemical treatment that induces covalent bonding of the active material (e.g., an immobilized conjugate) to the support. As described below, the solid support may include a conjugate pad. Many other suitable methods can be used to immobilize the active material (e.g., an immobilized trapping material) onto the solid support, and such methods include, but are not limited to, ionic interactions, hydrophobic interactions, and covalent interactions. Unless physically restricted, the solid support may be used in any suitable shape such as a film, sheet, strip, or plate, or the solid support may be coated, bonded, or laminated to a suitable inert carrier such as paper, glass, plastic film, or cloth.

[0076] The lateral flow apparatus described herein may include a conjugate pad made of a membrane or other type of material including a trapping substance. The conjugate pad may be made of cellulose acetate, nitrocellulose, 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., mixtures of cellulose acetate and nitrocellulose), nylon (e.g., polyamide, hexamethylenediamine and nylon 66), polypropylene, PVDF, high-density polyethylene (HDPE) + nucleating agent "aluminum dibenzoate" (DBS) (e.g., 80u0.024HDPE DBS(Porex)), and HDPE. The lateral flow apparatus described herein is used for low-susceptibility samples, such as those having low concentrations of analytes or samples with common concentrations of analytes but present in low doses. "Susceptibility" refers to the proportion of actual positives correctly identified as positive (e.g., the proportion of infected, latent, or symptomatic subjects correctly identified as having a disease). Susceptibility 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 apparatus described herein can accurately measure multiple target analytes in many different types of samples. Samples can include lesions or cultures obtained from any source, as well as from biological and environmental samples. Biological samples may be obtained from animals (including humans) and may include fluids, solids, tissues, and gases. Samples may be processed before being applied to the lateral flow apparatus of this disclosure. In a first non-limiting example, a whole blood sample may be processed to obtain plasma or serum, and plasma and serum may be applied to the lateral flow apparatus described herein. In a second non-limiting example, a sample containing cells may be processed using one or more sample preparation steps, such as a cell lysis step that releases intracellular proteins for detection, but is not limited to these. The processed sample may be applied to the lateral flow apparatus described herein. Examples of biological samples include urine, saliva, and blood products such as plasma and serum. Such examples, however, are not intended to limit the types of samples to which this disclosure can be applied.

[0078] The lateral flow apparatus described herein may include labels. Labels can take many different forms, comprising molecules or compositions that are bound to or can be bound to an analyte, analyte analogue, detection reagent, or binding partner that can be detected 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, radioisotopes, cofactors, ligands, chemiluminescent or fluorescent substances, protein-absorbing silver particles, protein-absorbing iron particles, protein-absorbing copper particles, protein-absorbing selenium particles, protein-absorbing sulfur particles, protein-absorbing tellurium particles, protein-absorbing carbon particles, and protein-binding disacks. Adhesion of compounds (e.g., detection reagents) to the labels may be by electrostatic bonding or a combination of bonding and interaction, such as covalent bonding, absorption processes, anaerobic and / or chelation, and / or may involve binding groups. The term "specific binding partner" or "binding partner" refers to a member of a molecular pair that interacts through specific, non-covalent interactions that depend on the three-dimensional structure of the molecules involved. Common specific binding partner pairs include antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid chain / complementary nucleic acid chain, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, biotin / (strept)avidin, receptor / ligand, or virus / molecular receptor, or various combinations thereof.

[0079] As used herein, the terms “immunoglobulin” or “antibody” refer to proteins that bind to a specific antigen. Immunoglobulins include, but are not limited to, polyclonal, monoclonal, chimeric, and humanized antibodies, Fab fragments, and F(ab')2 fragments, and include immunoglobulins of the following classes: IgG, IgA, IgM, IgD, IgE, and secretory immunoglobulin (sIg). Immunoglobulins generally consist of two identical heavy chains and two light chains. However, the terms “antibody” and “immunoglobulin” also include single-chain and double-chain antibodies. As used herein, the term “antibody” refers to the antibody as a whole or any fragment thereof, including its bound fragments. Therefore, when referring to a labeled antibody that specifically binds to the analyte of interest, the term refers to the labeled antibody or its fragment that specifically binds to the analyte of interest. Similarly, when referring to a capture substance or antibody, the term refers to the capture antibody or its fragment that specifically binds to the analyte of interest. The antibodies, testing apparatus, and methods in the lateral flow apparatus described herein may include polyclonal antibodies. Polyclonal antibodies for measuring any target analytes disclosed herein may include, but are not limited to, antibodies produced from serum by active immunization of one or more rabbits, goats, sheep, chickens, ducks, guinea pigs, mice, donkeys, camels, rats, and horses. The antibodies, testing apparatus, and methods in the lateral flow apparatus described herein may include monoclonal antibodies. Antibodies for binding to the target analyte are known in the art or can be readily expressed by methods known in the art.

[0080] The lateral flow apparatus described herein includes an immobilized capture substance. The immobilized capture substance includes an active agent that can bind to an analyte, including a free (unlabeled) analyte and / or a labeled analyte (such as an analyte bound to an analyte-specific conjugate, as described herein). The immobilized capture substance includes (i) the analyte of interest bound by an analyte-specific conjugate, (ii) the free analyte, or (iii) an unlabeled specific binding partner that is specific to an accompanying specific binding partner that is itself specific to the analyte, such as in an indirect assay. As used herein, “accompanying specific binding partner” is a specific binding partner that binds to the specific binding partner of the analyte. For example, the accompanying specific binding partner may include another antibody, such as an antibody specific to a goat anti-human antibody.

[0081] The lateral flow apparatus described herein may include a “detection area” or “detection zone” which is an area containing one or more capture areas or capture zones, and which is an area capable of detecting detectable signals. The lateral flow apparatus described herein may include a “capture zone” or “capture area” which is an area of ​​the lateral flow apparatus to which the capture material is immobilized. The lateral flow apparatus described herein may include one or more capture zones. In some cases, different capture materials are immobilized in different capture zones (such as a first immobilized capture material in a first capture zone and a second immobilized capture material in a second capture zone). Multiple capture zones may be positioned relative to each other on the lateral flow substrate in any way, for example, the first capture zone may be distal or proximal to a second (or other) capture zone along the fluid flow path, or vice versa. Alternatively, the first and second (or other) capture zones may be aligned along an axis perpendicular to the fluid flow path such that the fluid contacts them simultaneously or approximately simultaneously.

[0082] The lateral flow apparatus described herein includes an immobilized capture material, which is immobilized such that its movement is restricted while the lateral flow apparatus is operating normally. For example, the movement of the immobilized capture material is restricted before and after the fluid sample is applied to the lateral flow apparatus. Immobilization of the immobilized capture material can be achieved by physical means such as shielding, electrostatic interactions, hydrogen bonding, bioaffinity, covalent interactions, or a combination thereof. The lateral flow apparatus described in this disclosure can measure living organisms. Living organisms include chemical or biochemical compounds produced by organisms, including prokaryotic cell lines, eukaryotic cell lines, mammalian cell lines, microbial cell lines, insect cell lines, plant cell lines, mixed cell lines, naturally occurring cell lines, or synthetically modified cell lines. Living organisms may include macromolecules such as proteins, polysaccharides, lipids, and nucleic acids, as well as small molecules such as primary metabolites, secondary metabolites, and natural products.

[0083] The descriptions, specific examples, and data are provided for illustrative purposes only, illustrating exemplary embodiments, and are not intended to limit the various embodiments of this disclosure. Various changes and modifications in this disclosure will be evident to those skilled in the art from the descriptions and data contained herein and are therefore considered to be part of the various embodiments of this disclosure. Another aspect of the present invention may be as follows: [1] A lateral flow assay for detecting a target analyte in a sample, A first conjugate comprising a first label, an active substance configured to specifically bind to the target analyte, and a first binding partner, A second conjugate located upstream of the first conjugate along the fluid flow path of a lateral flow assay, wherein the second conjugate includes a second label and a second binding partner configured to specifically bind to the first binding partner, and Along the fluid flow path of the lateral flow assay, a detection zone located downstream of the first and second conjugates, wherein the detection zone includes an immobilized capture substance that specifically binds to the analyte of interest. Lateral flow assay including [2] The assay according to [1], wherein the first conjugate is located in the sample receiving zone of the lateral flow assay, or the first conjugate is located in a first conjugate zone downstream of the sample receiving zone. [3] The assay according to [1], wherein the second conjugate is located in a buffer receiving zone upstream of the sample receiving zone, or the second conjugate is located in a second conjugate zone downstream of the buffer receiving zone and upstream of the sample receiving zone. [4] The assay according to [1], wherein when a fluid sample is applied to the lateral flow assay, the first conjugate is solubilized and moves to the detection zone. [5] The assay according to [4], wherein the second conjugate is solubilized and moves to the detection zone after the first conjugate has moved to the detection zone. [6] The assay according to [1], wherein the active substance configured to specifically bind to the target analyte is an antibody or antibody-binding fragment that specifically binds to the target analyte. [7] The assay according to [1], wherein the first binding partner and the second binding partner comprise binding pairs selected from the group consisting of antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid chain / complementary nucleic acid chain, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, biotin / avidin, receptor / ligand, and virus / cell receptor. [8] The assay according to [1], wherein the first binding partner and the second binding partner each contain a biotin / avidin binding pair, and the avidin each contains streptavidin or neutraavidin. [9] The assay according to [1], wherein the first binding partner and the second binding partner each comprise an antigen / antibody binding pair, and the antigen is a peptide or decapeptide.

[10] The assay according to [1], wherein the target analyte is the target biological or environmental substance.

[11] The assay according to [1], wherein the target analyte is influenza virus.

[12] The assay according to

[11] , wherein the influenza virus is influenza A virus, influenza B virus, or influenza C virus.

[13] The assay according to [1], wherein the immobilized capture substance is an antibody or antibody-conjugated fragment that specifically binds to the target analyte.

[14] The assay according to [1], wherein the test strip comprises a nitrocellulose membrane.

[15] The assay according to [1], further comprising a control zone containing an immobilized capture substance that specifically binds to the first conjugate.

[16] The assay according to [1], wherein the first and second labels are selected from the group consisting of metal nanoparticles, blue latex beads, metal nanoparticles, colored latex particles, colored latex beads, magnetic particles, carbon nanoparticles, quantum dots, upconverting phosphors, organic fluorophores, fiber dyes, enzymes, or liposomes.

[17] The assay according to [1], wherein the first label and the second label contain gold nanoparticles.

[18] The assay according to [1], wherein the first label and the second label are configured to generate an optical signal, a fluorescent signal, or a magnetic signal.

[19] Housing including a sample well located on the upper side or upstream of the first conjugate, a buffer well located on the upper side or upstream of the second conjugate zone, and a reading window that provides access to the detection zone. The assay according to [3], further comprising:

[20] The assay according to

[19] , wherein the buffer well, the second conjugate zone, the sample receiving well, and the first conjugate zone are spatially separated along the fluid flow path of the lateral flow assay.

[21] Applying the first conjugate to the lateral flow test strip in or downstream of the sample receiving zone of the lateral flow test strip, and The second conjugate is applied to the test strip in the buffer receiving zone upstream of the sample receiving zone or downstream of it. A method for performing the lateral flow assay described in [1] above, including the following:

[22] The method according to

[21] , wherein the first conjugate and the second conjugate are applied to the test strip simultaneously.

[23] The method according to

[21] , wherein the first conjugate and the second conjugate are applied to the test strip by air jet adhesion.

[24] A method for detecting a target analyte in a sample, A first conjugate comprising a first label, an active substance configured to specifically bind to the target analyte, and a first binding partner, A second conjugate located upstream of the first conjugate along the fluid flow path of a lateral flow assay, wherein the second conjugate comprises a second label and a second binding partner configured to specifically bind to the first binding partner, and Along the fluid flow path of the lateral flow assay, a detection zone located downstream of the first and second conjugates, wherein the detection zone includes an immobilized capture substance that specifically binds to the analyte of interest. Apply the sample to a lateral flow assay that includes, In the detection zone, the complex is bound to the immobilized capture substance, wherein the complex includes the target analyte bound to the first conjugate. After the composite is bound, the second conjugate is released and allowed to flow along the fluid channel of the lateral flow assay. The second conjugate is coupled to the composite that is coupled in the detection band. A method that includes this.

[25] The method according to

[24] , further comprising detecting the signal generated by the complex and the second conjugate coupled in the detection band.

[26] The method according to

[25] , further comprising binding the first conjugate that was not bound to the target analyte to the second conjugate that is bound to the complex in the detection band.

[27] The method according to

[24] , wherein the first label and the second label are configured to generate an optical signal, a fluorescence signal, or a magnetic signal.

[28] The method according to

[24] , wherein after applying the sample to the lateral flow apparatus, the second conjugate is released at 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes.

[29] In the detection zone, the composite is bound to the immobilized capture material. The target analyte is labeled with the first conjugate to form a complex, and The composite is bound to the immobilized capture substance in the detection zone. The method according to

[24] , including the method described above.

[30] The method according to

[24] , wherein releasing the second conjugate includes applying a buffer to the second conjugate or to the location of the lateral flow assay upstream of the second conjugate.

[31] The method according to

[30] , wherein applying the buffer solution includes pouring the buffer solution into a buffer receiving well located on the upper side or upstream of the second conjugate.

Claims

1. A lateral flow assay apparatus for detecting a target analyte in a sample, A first conjugate comprising a first label, an active substance configured to specifically bind to the target analyte, and a first binding partner, A second conjugate located upstream of the first conjugate along the fluid flow path of a lateral flow assay apparatus, wherein the second conjugate comprises a second label and a second binding partner configured to specifically bind to the first binding partner, the second binding partner being an antibody against the first binding partner, and the first label and the second label being the same, and Along the fluid flow path of the lateral flow assay apparatus, a detection zone located downstream of the first and second conjugates, wherein the detection zone includes an immobilized capture substance that specifically binds to the target analyte. This is a lateral flow assay apparatus that includes, Here, the second conjugate is configured to be solubilized and move to the detection zone after the first conjugate has moved to the detection zone, and when a buffer solution is applied to the second conjugate or to a position on the lateral flow assay apparatus upstream of the second conjugate, the second conjugate is configured to be solubilized and move to the detection zone. Lateral flow assay apparatus.

2. The assay apparatus according to claim 1, wherein the first conjugate is located in the sample receiving zone of the lateral flow assay apparatus, or the first conjugate is located in a first conjugate zone downstream of the sample receiving zone.

3. The assay apparatus according to claim 1, wherein the second conjugate is located in a buffer receiving zone upstream of the sample receiving zone, or the second conjugate is located in a second conjugate zone downstream of the buffer receiving zone and upstream of the sample receiving zone.

4. The assay apparatus according to claim 1, wherein when a fluid sample is applied to the lateral flow assay apparatus, the first conjugate is solubilized and moves to the detection zone.

5. The assay apparatus according to claim 1, wherein the active substance configured to specifically bind to the target analyte is an antibody or antibody-binding fragment that specifically binds to the target analyte.

6. The assay apparatus according to claim 1, wherein the first binding partner is an antigen or hapten, and the second binding partner is an antibody against the antigen or hapten.

7. The assay apparatus according to claim 1, wherein the target analyte is a target biological or environmental substance.

8. The assay apparatus according to claim 1, wherein the target analyte is influenza virus.

9. The assay apparatus according to claim 8, wherein the influenza virus is influenza A virus, influenza B virus, or influenza C virus.

10. The assay apparatus according to claim 1, wherein the immobilized capture substance is an antibody or antibody-conjugated fragment that specifically binds to the target analyte.

11. The assay apparatus according to claim 1, comprising a test strip containing a nitrocellulose membrane.

12. The assay apparatus according to claim 1, further comprising a control zone containing an immobilized capture substance that specifically binds to the first conjugate.

13. The assay apparatus according to claim 1, wherein the first and second labels are selected from the group consisting of metal nanoparticles, blue latex beads, colored latex particles, colored latex beads, magnetic particles, carbon nanoparticles, quantum dots, upconverting phosphors, organic fluorophores, fiber dyes, enzymes, or liposomes.

14. The assay apparatus according to claim 1, wherein the first label and the second label contain gold nanoparticles.

15. The assay apparatus according to claim 1, wherein the first label and the second label are configured to generate an optical signal, a fluorescence signal, or a magnetic signal.

16. The assay apparatus according to claim 1, further comprising a housing including a sample well located lateral upper or upstream of a first conjugate zone, a buffer well located lateral upper or upstream of a second conjugate zone, and a reading window accessible to the detection zone.

17. The assay apparatus according to claim 16, wherein the buffer well, the second conjugate zone, the sample well, and the first conjugate zone are spatially separated along the fluid flow path of the lateral flow assay apparatus.

18. Applying the first conjugate to the lateral flow test strip in or downstream of the sample receiving zone of the lateral flow test strip, and The second conjugate is applied to the lateral flow test strip in the buffer receiving zone upstream of the sample receiving zone or downstream of it. A method for manufacturing a lateral flow assay apparatus according to claim 1, including the method described in claim 1.

19. The method according to claim 18, wherein the first conjugate and the second conjugate are applied simultaneously to the lateral flow test strip.

20. The method according to claim 18, wherein the first conjugate and the second conjugate are applied to the lateral flow test strip by air jet adhesion.

21. A method for detecting a target analyte in a sample, A first conjugate comprising a first label, an active substance configured to specifically bind to the target analyte, and a first binding partner. A second conjugate located upstream of the first conjugate along the fluid flow path of a lateral flow assay apparatus, wherein the second conjugate comprises a second label and a second binding partner configured to specifically bind to the first binding partner, the second binding partner being an antibody against the first binding partner, and the first label and the second label being the same, and Along the fluid flow path of the lateral flow assay apparatus, a detection zone located downstream of the first and second conjugates, wherein the detection zone contains an immobilized capture substance that specifically binds to the target analyte. Applying the sample to a lateral flow assay apparatus including, In the detection zone, a complex is bound to the immobilized capture substance, wherein the complex includes the target analyte bound to the first conjugate. After the complex is bound, the second conjugate is released and allowed to flow along the fluid channel of the lateral flow assay apparatus, wherein the release of the second conjugate includes applying a buffer to the second conjugate or to a location on the lateral flow assay apparatus upstream of the second conjugate. The second conjugate is coupled to the composite that is coupled in the detection band. A method that includes this.

22. The method according to claim 21, further comprising detecting the signal generated by the composite and the second conjugate coupled in the detection band.

23. The method according to claim 22, further comprising binding a first conjugate not bound to the target analyte to the second conjugate bound to the complex in the detection zone.

24. The method according to claim 21, wherein the first label and the second label are configured to generate an optical signal, a fluorescence signal, or a magnetic signal.

25. The method according to claim 21, wherein the second conjugate is released after the sample is applied to the lateral flow assay apparatus, at intervals of 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes.

26. In the detection zone, the composite is bound to the immobilized capture material. The target analyte is labeled with the first conjugate to form a complex, and The composite is bound to the immobilized capture substance in the detection zone. The method according to claim 21, including the method described in claim 21.

27. The method according to claim 22, wherein applying the buffer comprises pouring the buffer into a buffer receiving well located on the upper side or upstream of the second conjugate.

28. A sample receiving zone comprising a first label, an active substance configured to specifically bind to the target analyte, and a first conjugate containing a first binding partner, A buffer receiving zone located upstream of the sample receiving zone, wherein the buffer receiving zone includes a second conjugate comprising a second label and a second binding partner configured to specifically bind to the first binding partner, wherein the first label and the second label are the same. A detection zone located downstream of the sample receiving zone, wherein the detection zone includes an immobilized capture substance that specifically binds to the target analyte. Assay test strips, including

29. The assay test strip according to claim 28, wherein the assay test strip comprises a back card, a conjugate pad, a nitrocellulose membrane, and an absorbent pad.

30. Assay test strip according to claim 28, A reader including a light source and a detector, and Data analyzer A diagnostic testing system that includes this.

31. The diagnostic testing system according to claim 30, wherein the reader is configured to obtain an optical signal at the detection zone on the assay test strip.

32. The diagnostic test system according to claim 31, wherein the data analyzer is configured to process the optical signal obtained by the reader.

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