Quantitative analyte detection in lateral flow immunoassay

Abstract

A lateral flow immunochemical test system and method is provided. The system includes a tester substrate having at least two control lines and at least one test sample line. A test sample having an unknown amount of analyte can be deposited on the tester substrate, and the test sample can move along the tester substrate, contacting the at least two control lines and the at least one test sample line. A measurement device compares the at least one test sample line with the at least two control lines to provide a quantitative value for the amount of analyte present in the test sample.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Provisional Application No. 62 / 893,235, filed Aug. 29, 2019, and is incorporated herein by reference in its entirety, including the drawings.

Background Art

[0002] Lateral flow immunochemistry has been widely used in point - of - care testing, rapid home testing, and clinical settings for about 40 years. The first commercialization was a one - step pregnancy test (see, for example, Patent Document 1). Due to chromatography, flow, and material limitations, this technology is limited in related technologies to simply reporting the presence or absence of proteins or other analytes. When visually read, it is a comparison with a control line (e.g., luteinizing hormone (LH)), and the best that can be done is to determine whether the sample appears stronger or weaker than the control line. With the improvement of camera technology, readers based on pixel and / or camera technology have been developed, but such readers are extremely inconsistent and expensive.

[0003] Existing lateral flow immunochemistry testers use one control and compare the sample with the control regardless of the type of technology used in the tester. Such test methods are described in Patent Document 2, Patent Document 3, Patent Document 4, Patent Document 5, and Patent Document 6. Such testers and test methods compare the sample with a control line to determine the presence of an analyte (e.g., if the sample line is brighter than the reference line = absent, otherwise = present).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

[0005] Considering the limitations of related lateral flow immunochemical testers and test methods, there is a need in the art for an improved tester / reader and test method for lateral flow immunochemistry. [Means for Solving the Problems]

[0006] Embodiments of the present invention provide a novel lateral flow immunoassay system and method that address the drawbacks and limitations of related art systems and methods. The system includes a tester substrate having at least two control (known value) regions, each of which generates a control indicator line corresponding to a known value of an analyte, one having a lower known value than the other. The analyte is conjugated to a marker that can be measured by a measuring device. There is also at least one test sample region that generates a test sample line when contacted by the conjugated analyte in the test sample. A test sample having an unknown amount of conjugated analyte can be deposited on the tester substrate, for example, within a sample well in contact with the tester substrate. The test sample moves through the tester substrate by capillary action until it reaches at least two control regions and at least one test sample region, thereby generating at least two control lines and at least one test sample line. A reader, meter, or other measuring device can be used to compare at least one test sample line with at least two known value control lines, which are used as a reference to quantify the amount of analyte present in the test sample by comparing the test sample lines or can provide a reference. Embodiments of the systems and methods of the present invention are inexpensive and provide accurate and reproducible results.

[0007] For a more accurate understanding of the invention described above, the invention briefly described above will be described more specifically by reference to the specific embodiments of the invention shown in the accompanying drawings. The drawings presented are not to scale, and any reference to dimensions in the drawings or in the following description is specific to the disclosed embodiments. Variations in these dimensions for which the invention functions for its intended purpose are considered to be within the scope of the invention. These drawings are to show only representative embodiments of the invention and are not to be regarded as limiting the scope. The accompanying drawings are used to explain further specific examples and details of the invention.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 5

Figure 6A

Figure 6B

Figure 6C

Figure 6D

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Embodiments of the present invention provide a novel lateral flow immunoassay system and method that address the drawbacks of related art systems and methods. In lateral flow immunoassays where color and / or intensity are used as indicators, it is difficult to reproduce results for different tests because the color is slightly brighter or slightly darker, or the intensity varies between tests. Thus, tests in the related art typically provide binary results indicating the presence or absence of an analyte and do not provide quantitative results. When using colloidal gold as a color and / or intensity marker for an analyte, which is the most common color and / or intensity marker utilized in lateral flow chemical assays, it has previously been particularly difficult to produce reproducible and consistent results. Embodiments of the present invention address this problem by using a reference or known value control line for comparison with the test sample line.

[0010] In the following description, many terms are used in connection with embodiments of the present invention. The following definitions are provided to give a clear and consistent understanding of the specification and claims, including the scope of such terms.

[0011] As used herein, the term "proximal end" or "proximal direction" refers to the end of the test sample that is closest to, or in the vicinity of, the opening or "well" where the test sample is deposited on the tester substrate.

[0012] As used herein, the term "distal end" or "distal direction" refers to the end closest to the absorber, or the end farthest from where the test sample is deposited on the test strip.

[0013] When the term "substantially" or "about" is used herein in connection with a numerical value, the value is understood to be in the range of 95% to 105% of that value, i.e., the value is + / 5% of the stated value. For example, "about 1 kg" means 0.95 kg to 1.05 kg.

[0014] Embodiments of the use of the present invention include a tester substrate 100, and a test sample 160 is absorbed or taken in and moves, flows, or otherwise moves across or through the tester substrate. The tester substrate has at least two control regions, and the analyte binding values in each control region are known with high accuracy. The low control region 110 has a first known analyte binding value (e.g., 20 nanograms per milliliter (ng / ml)), and the high control region 120 has a known analyte binding value that is different from and higher than the analyte binding value of the low control region (e.g., 100 ng / ml). Although not necessarily required, the first and second known analyte binding values can be selected such that any test sample is expected (or highly likely, or very highly likely) to give a result that is between the high and low known analyte binding values. The test sample deposited on the tester substrate can move or be moved across the tester substrate so as to enter the low control region, the high control region, and the test sample region.

[0015] In one embodiment, the system includes a reader, a meter, or other measuring device 300, and a tester substrate 100 (e.g., a test strip, a test cassette, or a similar test substrate). The test substrate includes at least three regions that result in at least three lines. In one embodiment, one line is a low control region 110 used to establish a "low" reference where a low control line 115 is formed (based on a lower known analyte binding value), one is a test sample region 160 for a test sample that generates a test sample line 135 based on the amount of conjugate analyte 160, along with an associated marker bound thereto, for obtaining a test sample value 136, and one is a high control region 120 where a high control line 125 is formed and used to establish a "high" reference (based on a higher known analyte binding value). The three regions and the resulting lines formed therein by contact with the conjugate analyte are parallel or substantially parallel, but the embodiments are not limited thereto. The high and low references can be determined, respectively, by control regions that contain or have a predetermined known specific amount of an analyte binding substance 105, such as an antibody or an antigen, for example, such that a desired amount of conjugate analyte, such as conjugate antigen or conjugate antibody, binds to the analyte binding substance. In one embodiment, the ratio of the analyte binding substance to the conjugate analyte that binds to it is about 1:1. For example, typically, the ratio of conjugate antibody:bound antigen or conjugate antigen:bound antibody is 1:1. Of course, other ratios (e.g., 2:1, 1:2, 3:1, 1:3, or any other ratio including non-integer ratios) are also used.

[0016] In a further embodiment, the test sample region 130 has an effectively "unlimited" amount of an analyte binding substance 105, such as an antibody or an antigen, in the amount of conjugate analyte 160 that binds, by having a higher analyte binding value than is required to measure the analyte in any reasonably expected sample. If the analyte being tested is an antigen, the regions each have an antibody, and if the analyte being tested is an antibody, the regions each have an antigen.

[0017] In one embodiment, the test system includes a cassette 200 that includes at least a tester substrate 100 in which various regions are disposed, as shown, for example, in FIGS. 2A, 2B, 3A, 3B, 6C, and 6D, within a base 250 (moldable to form a case) that can secure the tester substrate. In one embodiment, the case secures the tester substrate 100 in its exact position and facilitates accurate measurement of low control lines 115, high control lines 125, and test sample lines 135 formed within a low control region 110, a high control region 120, and a test sample region 130, respectively. An example of a cover 210 and a base 250 is shown in FIGS. 2A and 2B. FIGS. 6C and 6D show the cover 210 and the base 250 of a modified embodiment of the cassette. The cover has an opening or well 215 at a proximal end 5 where a test sample can be placed, and the system is configured to allow the well to contact the tester substrate 100 directly, and to contact the tester substrate directly when a test sample is present therein. In one embodiment, the cover has an internal sheet or slot 225 that can be fitted to prevent the tester substrate 100 from moving within the cassette. An example of an internal slot within the case is shown in FIGS. 3A and 6C. The cover also has one or more windows 220 through which a measuring device 300 can view or utilize the control region and the test sample region. FIGS. 2A, 3A, and 6A show an embodiment of the cassette 200 having three windows. FIGS. 4A and 4B show a modified embodiment of the cassette having a single window. The base 250 can be attached to the cover 210 to enclose and secure the tester substrate in a predetermined position within the internal slot. In certain embodiments, the cover 210 can be snap-fastened with the base. In further certain embodiments, the cassette is engaged in cooperation with the measuring device 300 and one or more windows are positioned for proper alignment for the measuring device to utilize, measure, or read the high control line 125, the low control line 115, and the test sample line 135 thereon.

[0018] The system can further include an absorber 101 in contact with the tester substrate 100 at the distal end. In one embodiment, the absorber is part of the tester substrate or is incorporated as part of the tester substrate. In another embodiment, the absorber is a separate element from the tester substrate. The absorber 101 can be in direct physical contact with the tester substrate. The absorber can include any material, substance, or device that can take in, absorb, draw, immerse, or otherwise “drive” the test sample 160 to move across or through the tester substrate. When the test sample 160 is deposited in the well 215, the test sample is first absorbed by the tester substrate 100 by capillary action and as a result moves across the tester substrate until it reaches the absorber. The absorber can take in or absorb the test sample moving towards the distal end 10 of the tester substrate, thereby driving the capillary flow across the tester substrate and facilitating the movement of the test sample across the tester substrate in the direction from proximal 5 to distal 10.

[0019] In one embodiment, the test sample 160 contacts a low control region having a low analyte binding value to generate a low control line 115 that is measured to determine a "low" criterion, then it contacts the test sample region 130 where it can be measured to obtain a test sample value 136 to generate a test sample line, and finally, the test sample contacts a high control region having a high analyte binding value to generate a "high" reference line that is measured to determine a "high" criterion. The system can be configured. In an alternative embodiment, the test sample first contacts a high control region with a high analyte binding value to generate a "high" reference line, then contacts the test sample region where it generates a test sample line, and finally contacts a low control region with a low analyte binding value to generate a "low" reference line. The system can be reversed and configured. This reversed configuration is advantageous for competing analyte binding substances, and as the amount of analyte bound increases, the color and / or intensity becomes weaker or less distinct due to the reaction. Generally, during / after the test, the reference line expected to have a lighter color and / or lower intensity is closest to the well 215, and the other reference line expected to have a darker color and / or higher intensity during / after the test is closest to the absorber 101. If the analyte test is such that the color and / or intensity increases with the amount of analyte present, the low reference line will be closest to the well, or if the analyte test is such that the color and / or intensity decreases with the amount of analyte present (e.g., in the case of competitive binding), the high reference line will be closest to the well.

[0020] The system also includes a reading or measuring device 300. After a test sample is placed on a tester substrate, such as within a well, and contacts a low control region and a test sample region, and in some cases after the test sample reaches a high reference line, the tester substrate 100 can be inspected with a measuring device, either alone or as a cassette 200. For example, the tester substrate can be placed within a measuring device (e.g., a reflectance measuring device) that can calculate reflectance values, also known as analyte binding values, for each control line and test sample line 135, or alternatively, can engage in cooperation with the measuring device. The measuring device can be programmed to have a waiting period (e.g., 5 - 15 minutes, or 10 - 15 minutes, or 10 - 20 minutes), or a waiting period between any two of the enumerated values. The waiting period allows for the cessation of changes and stabilization by the appearance of the color and / or intensity of the control and test sample lines. After a predetermined waiting period, the measuring device can then determine the quantification of the analyte present in the test sample line by comparing it to the "high" and "low" reference lines for the extrapolated curve.

[0021] The comparison of the test sample line with the "high" and "low" reference lines can be performed using a first algorithm that can be executed by a processor (e.g., a microprocessor) of the reading device (the first algorithm can be stored (e.g., as code) in a storage medium (e.g., a (non-transitory) machine-readable medium) of the reading device). Alternatively, the processor and / or the storage medium may be on an external device (which can be considered part of the system or separate from the system) that is operably communicable with the reading device while measuring the amount of analyte present. The measuring device 300 can be calibrated by obtaining measurements from a plurality of analyte samples of various concentrations, each of which is known with very high accuracy. The second algorithm may be the same as or different from the first algorithm and is used to determine the extrapolation curve. In one embodiment, the extrapolation curve is a straight line and represents the linear relationship between the "high" and "low" references and their known analyte binding values. In a variant embodiment, the extrapolation curve is a curve and represents the non-linear relationship between the "high" and "low" references. The extrapolation curve, in combination with the first algorithm, is used to determine the quantification of the analyte present in the test sample. Alternatively, the extrapolation curve can be used, without the first algorithm or in combination with a third algorithm that may be the same as or different from the second algorithm, to determine the quantification of the analyte present in the test sample.

[0022] Advantageously, since variations between the "high" and / or "low" reference lines between different tester substrates can be taken into account and / or adjusted, it is possible to suppress the influence of variations between tester substrates on the accuracy of the quantitative determination of the amount of analyte present in a particular test sample value. This is because the relationship between the colors and / or intensities of the known "high" and known "low" reference lines remains the same and is conformable or comparable to the extrapolation curve, so the test sample values are also conformable or comparable to the extrapolation curve. Thus, for any given set of measurements, the extrapolation curve is the same and the measurements are still reproducible and accurate.

[0023] In one embodiment, the extrapolation curve used to calculate the test sample value 136 of the test sample line 135 using the measuring device 300 can be obtained using an algorithm (e.g., the second algorithm described above, etc.) and calibration using a plurality (e.g., 2, 3, 4, 5, or more) of known quantities. For example, linear extrapolation can be assumed up to a certain level (high or low), at which point there can be a 10% reduction or decrease in slope (or another specific increase in slope), or a change to a certain inflection (second derivative) curve. Then, at another level, the curve changes again, and at another level (when three or more known quantities are used), the curve can be changed again, and this can be repeated as desired. A person skilled in the art can determine an appropriate method for calculating the extrapolation curve.

[0024] In one embodiment, the measuring device 300 is a disposable reflectometer. The term "disposable" is used in the normal context and means that the measuring device is intended to be used only once or for a short time (e.g., together with a test substrate provided with the instrument) and is made using inexpensive disposable materials.

[0025] The processor of the disposable reflectometer can be pre-calibrated, for example, using the above-described second algorithm and algorithms such as extrapolation curves. This data can be stored, for example, in the instrument's storage medium as software or code. Further, the disposable reflectometer can be part of a system or kit that includes a test strip or cassette with a set number or a predetermined number of tester substrates (e.g., 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, etc.). The reflectometer can be calibrated against the tester substrate attached to the reflectometer. In one embodiment, different disposable reflectometers can be specifically calibrated against a particular "lot" of tester substrates (i.e., each kit includes a disposable reflectometer and a set amount of tester substrates, and the disposable reflectometer is specifically calibrated against those tester substrates). The disposable reflectometer can be discarded after using the test substrates included in the kit.

[0026] In certain embodiments, in addition to the algorithm for calculating the test sample value 136 of the test sample line 135 based on the readings of the high control line 125 and the low control line 110, the measuring device 300 (e.g., a disposable reflectometer) can also be pre-programmed using a look-up table to correct for variations in the dose response as the level goes up or down. This can be based on, for example, a curve for increasing or decreasing the amount of analyte, or a complex curve relative to a straight line.

[0027] Colorimetry and spectrophotometric absorbance techniques have been used in other types of tests (e.g., glucose tests), but in the related art, they have not been applied to lateral flow antibody / antigen immunochemistry methods. The application of colorimetry and spectrophotometric absorbance to lateral flow antibody / antigen immunochemistry has not been previously considered possible, among other reasons, due to color changes that occur on the measuring device.

[0028] Embodiments of the present invention address this issue by using, in addition to the test sample line 135, two control lines that establish "high" and "low" criteria on the same tester substrate 100 that can all be measured simultaneously. Algorithms and / or extrapolation curves can be used to compare measured values thereto and increase / guarantee accuracy and reproducibility between tests. In one particular embodiment, the measuring device is pre-calibrated specifically for use with a particular group of tester substrates as part of the kit.

[0029] Assay tests using embodiments of the system and method of the present invention are based on regular binding (darker colors indicate more analyte) or competitive binding (lighter colors indicate more analyte). Due to the ability to generate a response measured at a control line for each known value of the analyte, embodiments of the present invention use at least two control lines (e.g., exactly two control lines). The advantage of the control lines is to provide a reference with a measurable response representing a specific known amount of analyte. The test line (and / or test area) need not be between the control lines (and / or control area), but in some embodiments, it can be. Ideally, the response is simple, inexpensive, and measurable with an accurate measuring device. The measuring device, e.g., a meter / reader, can be based on reflectance photometry, but embodiments are not limited to this. The technique of using at least two control lines described herein is effective when the marker conjugated or bound to the analyte (or other moiety) is visual (e.g., colloidal gold, latex), magnetic, fluorescent, or any other means suitable for identifying the result of the binding of the analyte involved, e.g., when the analyte is an antigen or an antibody. As long as the colors can be compared, the marker can bind to the analyte itself or to a moiety bound to the analyte (e.g., conjugate or part of the conjugate). The term "analyte binding" is used herein, but in each case, the moiety is either the analyte itself or a "partial binding" bound to the analyte (e.g., conjugate or part of the conjugate).

[0030] Generate at least two control lines having known partial binding values on the same tester substrate as the test sample line, and measure the results generated by these lines at the stable point of the reaction (or as the reaction rate, or by following the plot intercept) to generate two known reading values, and then the analyte in the test sample can be accurately quantified. Although not necessarily required, the quantification of the analyte in the test sample is preferably between the respective amounts of the analyte in the "low" control region 110 and the "high" control region 120. That is, the measurable amount of conjugate analyte 160 is preferably between the high and low standards derived from the "high" and "low" control lines.

[0031] When a calculated "reference" or "extrapolated" curve is generated (e.g., by a meter or reading device), it is possible to extend the calibration above and / or below the reference value in order to accurately plot both values smaller than the low reference value and / or larger than the high reference value. This is particularly accurate when the high and low references produce a linear calibration. However, even when the high and low references do not produce a linear calibration, the measuring device (reader or meter) can be pre-calibrated for use with a particular set, lot, or group of tester substrates 100. In one embodiment, the measuring device generates an accurate non-linear calibration curve such that reference values higher than the "high" control line and / or lower than the "low" control line are predicted with good accuracy from the programmed calibration curve, and can be calibrated by pre-testing with a plurality of samples of the analyte conjugate material having known values.

[0032] The same basic criteria for spectrophotometry or colorimetry can be used for visual readings (e.g., although embodiments are not necessarily limited thereto, the color intensity of a test sample is for an analyte of a quantitative value in the test sample, as low and / or high reference color intensities are for the values of the reference). Similar to magnetic, fluorescence, or other types of measurements (including vision), a reference curve (also referred to as an extrapolation curve) can be generated, whereby the measuring device (meter / reader) can be automatically calibrated during an actual test. Further, since the test sample line is generated on the same medium or tester substrate 100 as the high and low control lines, consistent and accurate results can be generated based on two or more control line measurements or readings performed simultaneously and using the same chemical substances (using the same meter / reader) at the same time. That is, variations between tester substrates 100 can be adjusted by embodiments of the present invention by providing high and low controls specific to each tester substrate that are compared to the extrapolation curve to calculate accurate test sample values obtained from the same tester substrate (e.g., conjugate).

[0033] Existing lateral flow immunoassay testers and test methods typically cannot produce accurate and reproducible results without using enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR), both of which are labor-intensive, time-consuming, and very expensive. Embodiments of the present invention provide accurate and reproducible quantitative results using a rapid and inexpensive system / method.

[0034] Tests based on color changes have been described in detail herein, but embodiments of the present invention can alternatively be used for tests based on any other means suitable for identifying the results of binding of magnetic, fluorescence, or the analyte involved (whether the analyte is an antigen or an antibody). The same principle applies, but the reading device / meter needs to be configured to read / analyze the appropriate characteristics (magnetic, fluorescence, etc.).

[0035] Some embodiments of the present invention have some aspects in common with the devices disclosed in Patent Document 7 and Patent Document 8, both of which are hereby incorporated by reference in their entirety.

[0036] The present invention includes, but is not limited to, the following exemplary embodiments. Embodiment 1 A tester substrate configured to obtain quantification of an analyte in a test sample, at least one test sample region containing a partial binding substance that binds to the analyte or to a portion that binds to the analyte, and at least two control regions, each control region contains a different known amount of the partial binding substance, and when contacted with the analyte in the test sample, binds to the corresponding known amount of the portion in the test sample, and by obtaining a measurement of the amount of the portion that binds to each control region, a reference is provided for comparison with the measurement obtained for the amount of the portion that binds to the test sample region, and the tester substrate is configured to be able to determine the quantification of the analyte in the test sample. Embodiment 2 The tester substrate according to Embodiment 1, wherein a visual, magnetic, or fluorescent marker that forms a test sample line when the portion binds to the at least one test sample region and forms a control line in each of the at least two control regions, and to which the portion conjugates. Embodiment 3 The tester substrate according to any one of Embodiments 1 to 2, wherein the line is measured to obtain the quantification of the analyte bound to each of the at least one test region and the at least two control regions. Embodiment 4 The tester substrate according to any one of Embodiments 2 to 3, further comprising the marker and the analyte in a ratio of 1:1. Embodiment 5 The tester substrate according to any one of Embodiments 2 to 4, wherein the measuring device is configured to utilize colorimetry or spectroscopic colorimetry techniques to measure the marker. Embodiment 6: The tester substrate according to any one of Embodiments 1 to 5, wherein the analyte in the test sample first contacts the control region having a larger amount of the partial binding substance among the at least two control regions. Embodiment 7: The tester substrate according to any one of Embodiments 2 to 6, wherein the amount of the marker present in each of the at least one test sample region and the at least two control regions is measured to obtain the quantification of the bound portion in each of the at least one test region and the at least two control regions. Embodiment 8: A kit for obtaining the quantification of an analyte in a test sample, comprising at least one tester substrate, and a measuring device that utilizes a calibration curve, wherein the tester substrate has at least one test sample region containing a partial binding substance and at least two control regions each containing a different known amount of the partial binding substance. When the analyte contacts the partial binding substance in the at least one test sample region, a test sample line is formed. When the analyte contacts the partial binding substance in the at least two control regions, control region lines are formed in each of them. The measuring device measures each of the control region lines, compares the measured values with the calibration curve to determine the high and low standards on the calibration curve corresponding to the known amounts of the partial binding substance present in each control region, measures the test sample line, and compares the test sample line with the high and low standards to determine the amount of the portion bound to the partial binding substance in the test sample region. Embodiment 9: The kit according to Embodiment 8, further comprising a predetermined number of tester substrates, wherein the measuring device is configured to measure a predetermined number of tester substrates. Embodiment 10: The kit according to any one of Embodiments 8 to 9, wherein each of the tester substrates is fixed in a cassette. Embodiment 11: The kit according to Embodiment 10, which is engaged in cooperation with the measuring device. Embodiment 12: The kit according to any one of Embodiments 8 to 11, wherein the analyte is conjugated to a visual, magnetic, or fluorescent marker. Embodiment 13: A method for determining the amount of an analyte in a test sample, comprising: depositing the test sample onto a tester substrate according to any one of Embodiments 1 to 7 (or at least one tester substrate of the kit according to any one of Embodiments 8 to 12); contacting the test sample with a first of the at least two control regions to form a first control line; contacting the test sample with the at least one test sample region to form a test sample line; contacting the test sample with a second of the at least two control regions to form a second control line; using a measuring device to measure the first control line, the test sample line, and the second control line; fitting the first control line and the second control line to a calibration curve programmed into the measuring device; comparing the measured value of the test sample line with the fitted first control line and the fitted second control line to determine the amount of the portion bound to the test sample region, thereby providing the amount of the analyte in the test sample. Embodiment 14: The portion is conjugated to a visual, magnetic, or fluorescent marker that forms the test sample line, the first control line, and the second control line, and the method further comprises measuring, by the measuring device, the amount of the marker present in the test sample line, the first control line, and the second control line, according to Embodiment 13. Embodiment 15: The tester substrate is fixed within the cassette. The method further includes engaging the cassette in cooperation with the measuring device, the method according to any of embodiments 13-14. Embodiment 16 The measuring device utilizes colorimetry or spectrophotometry to measure the test sample line, the first control line, and the second control line, the method according to any of embodiments 13-15. Embodiment 17 The amount of the partial binding substance in the first control region is greater than the amount of the partial binding substance in the second control region, the method according to any of embodiments 13-16.

[0037] Embodiments of the present invention and many of their advantages are understood from the following examples given by way of illustration. The following examples illustrate some of the methods, uses, embodiments, and variations of the present invention. Of course, the present invention is not to be limited thereby. Many changes and modifications can be made with respect to the present invention.

[0038] Example 1 A lateral flow immunoassay tester substrate (e.g., a test strip or a test cassette) for testing vitamin D includes a "low" reference line and a "high" reference line, and a test sample line is disposed therebetween. The test for vitamin D is based on competitive binding, and it should be noted that this means that the color reaction weakens as the amount of the analyte increases (similar to many drug or small molecule tests). The "high" reference line contains a known amount of 100 ng / ml of vitamin D, and the "low" reference line contains a known amount of 20 ng / ml of vitamin D. Antibody capture in the control line reacts to a specific amount of substance conjugated to the same conjugate as the antibody against vitamin D. Colloidal gold is used as a color marker, and each antibody capture control line (providing measurements for the high reference, test sample, and low reference) captures a proportional representative amount of colored particles. By measuring the reflectance of the two control lines, an accurate quantification of the vitamin D present in the test sample line therebetween can be calculated with clinical test accuracy.

[0039] Example 2 To test for vitamin D in a sample, a disposable reflectometer is calibrated (using an algorithm) for 20 lateral flow immunochemical test cassettes. Each test cassette includes a low control line that, when measured, provides a low reference value, and a high control line that, when measured, provides a high reference value, along with a test sample line disposed therebetween. The high control line contains a known amount of 100 ng / ml of vitamin D, and the low control line is for a known amount of 20 ng / ml of vitamin D. During testing, the test cassette can be arranged such that the high control line is closer to the well of the cassette than the low control line, and the low control line is closer to the absorber than the high control line. Using each test cassette, one test is performed to determine the quantification of vitamin D present in the sample. The disposable reflectometer can be discarded after 20 test cassettes have been used.

[0040] Example 3 A lateral flow immunochemical tester substrate (e.g., a test strip or test cassette) for testing D-dimer (a protein that determines coagulation function) includes a low control line that provides a value for a low reference and a high control line that provides a value for a high reference, with a test sample line disposed therebetween. Note that the test for D-dimer is not based on competitive binding, and thus, in this example, due to the amount of gold captured on each line, as the amount of bound-analyte increases, the chromogenic reaction becomes stronger. The high reference line contains a known amount of 300 ng / ml (or possibly 330 ng / ml) of D-dimer, and the low reference line contains a known amount of 100 ng / ml of D-dimer. By measuring the reflectance of the two controls and comparing / fitting the results to an extrapolation curve, the exact quantification of D-dimer present in the test sample can be calculated (through measurement of the test sample line) with clinical laboratory accuracy.

[0041] Example 4 Calibrate a disposable reflectometer (using an algorithm) for 20 lateral flow immunoassay cassettes and test for D-dimer. Each test cassette includes a control line providing a low standard and a control line providing a high standard, with a test sample line disposed therebetween. The high standard control line is for a known amount of 300 ng / ml (or possibly 330 ng / ml) of D-dimer, and the low standard control line is for a known amount of 100 ng / ml of D-dimer. During testing, the test cassette can be arranged such that the high standard control line is closer to the absorber than the low standard control line, whereby the low standard control line is closer to the well within the cassette than the high standard line. Each test cassette performs one test to determine the quantification of D-dimer present in the sample. The disposable reflectometer can be discarded after 20 test cassettes have been used.

[0042] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes are suggested to those skilled in the art in light of them. They are intended to be included within the spirit and scope of this specification.

[0043] All patents, patent applications, provisional applications, and publications mentioned or cited herein are hereby incorporated by reference in their entirety, including their drawings and tables, to the extent that they do not conflict with the explicit disclosure of this specification.

Claims

1. A kit for obtaining quantification of an analyte in a test sample, comprising: at least one tester substrate; a measuring device that utilizes a calibration curve; wherein the tester substrate includes at least one test sample region containing a partial binding substance that competitively binds to a portion of the analyte or a conjugate portion bound to the analyte; and at least two control regions each containing a different known amount of the partial binding substance; when the analyte contacts the partial binding substance in at least one test sample region, a test sample line is formed, and when the analyte contacts the partial binding substance in the at least two control regions, control region lines are formed respectively; wherein the measuring device measures each of the control region lines, compares the measured values with a calibration curve to determine a high standard and a low standard on the calibration curve corresponding to the known amounts of the partial binding substance present in each control region, measures the test sample line, and compares the test sample line with the high standard and the low standard to determine the amount of the portion bound to the partial binding substance in the test sample region; the portion conjugates to a visual, magnetic, or fluorescent marker that forms a test sample line when the portion binds to the at least one test sample region and forms a control line for each of the at least two control regions; the analyte in the test sample first contacts a control region having a greater amount of the partial binding substance among the at least two control regions; further includes a plurality of tester substrates in a predetermined number; the measuring device is configured to measure the plurality of tester substrates in the predetermined number; and each of the tester substrates is fixed in a cassette. A kit.

2. The kit according to claim 1, wherein the cassette is engaged in cooperation with the measuring device.

Images