Immunochromatographic quantification method, immunochromatographic reader

Abstract

The present invention provides a highly accurate immunochromatographic quantification method and an immunochromatographic reader capable of carrying out this method. The immunochromatographic method uses a test plate 10 in a state where a sample is dropped onto one end of an immunochromatographic sheet 11 on the upstream side in the flow direction and developed, to determine the concentration of a substance to be measured contained in the sample. The method measures the amount of luminescence (L) of the test line 12 of the test plate 10 across the entire width direction W of the sheet, which is perpendicular to the flow direction R. T ) and the luminescence intensity of control line 13 (L C ) and measure the luminescence intensity (L T ) control line luminescence (L C ) to the ratio (L T / L C ) is calculated, and the ratio (L T / L C ) and the concentration of the substance to be measured, the calculated ratio (L T / L C ) to determine the concentration of the substance to be measured contained in the sample.

Description

[Technical Field]

[0001] The present invention relates to an immunochromatographic quantification method and an immunochromatographic reader. [Background technology]

[0002] Rapid immunochromatographic tests are becoming increasingly popular in the medical field, with demand for them increasing sharply, especially after the COVID-19 pandemic. These immunochromatographic tests have also been widely used for pregnancy testing, with emphasis placed on positive or negative results. This is because, in pregnancy testing, hormone levels change rapidly, so the accuracy of immunochromatographic tests is relatively less important, and more accurate tests are generally available at hospitals if necessary. However, when immunochromatographic tests are used to detect drug concentrations in samples due to drug abuse, or to determine the presence or severity of disease in medical settings, accuracy becomes important. However, currently, results are still shown as positive or negative, and are usually determined by whether or not a test line develops.

[0003] In conventional immunochromatographic tests, immunochromatographic test readers are designed to evaluate only the presence or absence of test line coloration, without taking into account the unevenness of the test line. Furthermore, the presence of a control line is not used in calculating the measured value, but only indicates that the test is valid. Furthermore, the presence of a test line indicates a positive result in a sandwich test and a negative result in a competitive test. To detect the color development of the test line, most readers subtract the average brightness of the background area (upstream and downstream of the test line) from the brightness of the test line, but this brightness of the background area is obtained without taking into account the non-uniformity in the flow direction.

[0004] As mentioned above, conventional methods detect only the presence (coloration) of a test line without considering the heterogeneity of the test line and its background area. While this method is sufficient for determining whether a patient is positive or negative for a disease, it lacks accuracy when quantitative measurements are required. For example, accurate and reliable measurements are essential for classifying the severity of a disease. Accurate quantitative measurements are especially important when the severity classification results affect treatment in clinical settings. Therefore, there is a need to develop new calculation methods to meet this need, as well as readers equipped with these new calculation methods that can accurately quantify the data.

[0005] For accurate quantification, it is desirable for the color development of the test line and control line to be uniform in each region. However, in addition to uneven binding of the detection antibody to the test line on the immunochromatography test paper (immunochromatography sheet), there are also hardware-related problems such as the state of application of reagents and imperfections in the structure and materials of the immunochromatography test kit itself, which can easily cause the flow of the sample to be uneven on the reagent paper. This non-uniformity phenomenon can occur anywhere, but the flow maintains the same direction and does not cause interference with other reagents moving in the same direction. In other words, if a certain part of the test line is darker than the other parts (indicating a large flow rate), the control line located relatively further back (downstream) will also tend to be darker.

[0006] In conventional methods, analysis is based on the area average brightness within a specific range of the test line alone, and this type of unevenness is one of the main causes of poor quantitative accuracy. Furthermore, due to technical limitations, the test line on an immunochromatographic test paper (immunochromatographic sheet) is not uniformly bound before or after the test line (upstream and downstream portions). Typically, there is a tendency for more antigen and antibody binding at the front end (upstream end), which first comes into contact with the reagent, and less at the rear end (downstream end). This can lead to errors in reading the values.

[0007] Literature relating to immunochromatographic quantification methods includes, for example, Patent Document 1. Patent Document 1 describes a method for processing and quantifying color images in immunochromatography or immunoconcentration, which eliminates the influence of background on the color image, enables accurate processing and quantification even for color images produced by label substances with different color tones, and enables an inexpensive system configuration that is also applicable to automatic determination of agglutination images by image analysis. The method described in Patent Document 1 is included in the immunochromatographic quantification method in which a sample is dropped onto one end of an immunochromatographic sheet on the upstream side in the flow direction, a test plate is used on which the sample is developed, and a calibration curve is used to determine the concentration of a substance to be measured contained in the sample.The characteristics of this method are as follows.

[0008] The color image formed on the test plate (color development device) is captured in full color, and the analysis range of the image data is specified. The column average of the pixel values ​​of the three primary colors of the raw data in the analysis range is calculated, and the moving average of the column averages of each pixel value is found, and the minimum color pixel value is calculated from the moving average. The background (background color) of the color image is corrected for the minimum pixel value, and the concentration of the sample analyte of unknown concentration is determined from the calibration curve showing the relationship between the pixel value after the correction process and the analyte of known concentration. In other words, in the method described in Patent Document 1, even in the case of a color image having a background (background color), correction is performed using a column moving average of pixel values ​​of the three primary colors of raw data in a specified analysis range in order to remove the influence of the background and make the image measurable. Furthermore, this method does not measure the amount of luminescence from the entire surface of the test plate. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Application Laid-Open No. 2000-338106 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a highly accurate immunochromatographic quantification method and an immunochromatographic reader capable of carrying out this method. [Means for solving the problem]

[0011] In order to solve the above problems, a first aspect of the present invention provides an immunochromatographic quantitative method having the following configurations (1) and (2). (1) This is an immunochromatographic quantitative method in which a sample is dropped onto one end of an immunochromatographic sheet on the upstream side in the flow direction, and a test plate on which the sample is developed is used to determine the concentration of a substance to be measured contained in the sample. (2) The amount of light emitted by the test line on the inspection plate (L T ) and the luminescence intensity of the control line (L C ) is measured, and the measured amount of luminescence is used to determine the amount of luminescence (L T ) control line luminescence (L C ) to the ratio (L T / L C ) is calculated, and the ratio (L T / L C ) and the concentration of the substance to be measured, the calculated ratio (L T / L C ) to determine the concentration of the substance to be measured contained in the sample.

[0012] A second aspect of the present invention provides an immunochromatographic quantitative method having the above configuration (1) and the following configuration (3). (3) Divide the entire sheet width direction, which is the direction perpendicular to the flow direction, into a plurality of regions, and measure the luminescence amount (L T ) and the luminescence intensity of the control line (L C ) is measured, and for each of the regions, the measured luminescence amount is used to calculate the luminescence amount (L T ) control line luminescence (L C) to the ratio (L T / L C ) is calculated, and the ratio (L T / L C ) and calculate the average value of the ratio (L T / L C ) and the concentration of the substance to be measured, the calculated ratio (L T / L C The concentration of the substance to be measured contained in the sample is calculated from the average value of the above values.

[0013] A third aspect of the present invention provides an immunochromatographic quantitative method having the above configuration (1) and the following configurations (4) to (15). (4) Dividing the entire sheet width direction, which is a direction perpendicular to the flow direction, into a plurality of regions, measuring the luminescence amount of the immunochromatographic sheet for each region, and creating a graph showing the change in the luminescence amount in the flow direction; (5) In the graph, a line where the amount of light emission is constant and continues to a portion where the amount of light emission is minimum in the portion upstream of the test line in the flow direction, and a line where the amount of light emission is constant and continues to a portion where the amount of light emission is minimum in the portion downstream of the test line in the flow direction, are connected by a straight line to form a first initial baseline; (6) A range that is 1.5 times or more the half-width of the peak showing the luminescence intensity of the test line in the graph, based on the first initial baseline, and in which the distances from the center point of the half-width to both ends of the range are the same, is set as a color value measurement range of the test line; (7) A region that is at least 0.5 times the dimension along the first initial baseline of the color value measurement range from the upstream end and downstream end of the color value measurement range of the test line is set as a background region, and the luminescence amount of the baseline in the background region of the test line on the graph is optimized by performing curve fitting using a line that indicates the luminescence amount of the background region on the graph; (8) The optimized baseline luminescence (L hT) is the luminescence intensity (L T ) minus the difference value (ΔL T ) is calculated as the color value of the test line, and (9) In the graph, a line where the amount of light emission is constant and continues to a portion where the amount of light emission is minimum in the portion upstream of the control line in the flow direction, and a line where the amount of light emission is constant and continues to a portion where the amount of light emission is minimum in the portion downstream of the control line in the flow direction, are connected by a straight line to form a second initial baseline; (10) A range that is 1.5 times or more the half-width of the peak showing the luminescence intensity of the control line in the graph based on the second initial baseline, and in which the distances from the center point of the half-width to both ends of the range are the same, is set as a color value measurement range of the control line; (11) A region that is at least 0.5 times the dimension along the second initial baseline of the color value measurement range from the upstream and downstream ends of the color value measurement range of the control line is set as a background region, and the luminescence intensity of the baseline in the background region of the control line on the graph is optimized by performing curve fitting using a line that indicates the luminescence intensity of the background region on the graph; (12) The optimized baseline luminescence (L hC ) is the luminescence intensity (L C ) minus the difference value (ΔL C ) is calculated as the color value of the control line, (13) For each of the regions, the difference value (ΔL C ) to the difference value (ΔL T ) ratio (ΔL T / ΔL C ) is calculated, (14) The ratio (ΔL T / ΔL C ) and calculate the average value. (15) The ratio (ΔL T / ΔL C ) and the concentration of the substance to be measured, the calculated ratio (ΔL T / ΔL C The concentration of the substance to be measured contained in the sample is calculated from the average value of the above values.

[0014] A fourth aspect of the present invention provides an immunochromatographic reader having the following configurations (21) to (24): (21) An immunochromatographic reader in which a sample is dropped onto one end of an immunochromatographic sheet on the upstream side in the flow direction, and a test plate on which the sample is developed is used to measure the concentration of a substance to be measured contained in the sample. (22) The test plate has a placement section for the test plate, a light irradiation section for irradiating the upper surface of the immunochromatographic sheet of the test plate placed in the placement section, and an luminescence amount acquisition section for acquiring the luminescence amount of reflected light from the test line and control line of the test plate across the entire width direction of the sheet, which is a direction perpendicular to the flow direction. (23) The light emission amount (L T ) and the luminescence intensity of the control line (L C ) to calculate the luminescence intensity of the test line (L T ) control line luminescence (L C ) to the ratio (L T / L C ) is calculated. (24) The ratio (L T / L C a calibration curve setting unit that sets a standard curve showing the relationship between the concentration of the substance to be measured and the concentration of the substance to be measured as a calibration curve; and a ratio (L T / L C ) and an output unit that outputs the concentration obtained by the concentration acquisition unit.

[0015] A fifth aspect of the present invention provides an immunochromatographic reader having the above configuration (21) and the following configurations (25) to (27). (25) The placement section of the test plate, the light irradiation section for the upper surface of the immunochromatographic sheet of the test plate placed in the placement section, and the entire sheet width direction, which is a direction perpendicular to the flow direction, are divided into a plurality of regions, and the amount of light emitted by the reflected light from the test line of the test plate (L T ) and the amount of light emitted from the control line (L C ) is acquired by a light emission amount acquisition unit. (26) The light emission amount (L T ) and the luminescence intensity (L C ) from the ratio of the two (L T / L C ) is calculated, and the ratio (L T / L C ) which is prepared in advance using a sample whose concentration of the substance to be measured is known, and T / L C The apparatus has a calibration curve setting unit that sets a standard curve showing the relationship between the average value of the above-mentioned values ​​and the concentration of the substance to be measured as a calibration curve. (27) Using the calibration curve set by the calibration curve setting unit, the ratio (L T / L C ) and an output unit that outputs the concentration obtained by the concentration acquisition unit.

[0016] A sixth aspect of the present invention provides an immunochromatographic reader having the above configuration (21) and the following configurations (28) to (30). (28) The apparatus has a luminescence amount acquisition unit that divides the entire sheet width direction, which is the direction perpendicular to the flow direction, into multiple regions, measures the luminescence amount of the immunochromatographic sheet for each region, and creates a graph showing the change in the luminescence amount in the flow direction. (29) In the graph created by the luminescence amount acquisition unit, a line where the luminescence amount is constant and continues to a portion where the luminescence amount in the upstream side of the test line in the flow direction is at its lowest value, and a line where the luminescence amount is constant and continues to a portion where the luminescence amount in the downstream side of the test line in the flow direction is at its lowest value, are connected by a straight line to form a first initial baseline; a range that is 1.5 times or more the half-width of the peak showing the luminescence intensity of the test line in the graph based on the first initial baseline, and in which the distances from the center point of the half-width to both ends of the range are the same, is set as a color value measurement range of the test line; and a region that is 0.5 times or more the dimension along the first initial baseline of the color value measurement range from the upstream end and downstream end of the color value measurement range of the test line is set as a background region; and the luminescence amount of the baseline in the background region of the test line in the graph is optimized by performing curve fitting using a line that indicates the luminescence amount in the background region of the graph; and the luminescence amount (L hT ) is the luminescence intensity (L T ) minus the difference value (ΔL T) is calculated as the color value of the test line, and a second initial baseline is set by connecting with a straight line a line in the graph where the luminescence intensity is constant and continues to a portion where the luminescence intensity in the upstream side of the control line in the flow direction is at its lowest, and a line in the graph where the luminescence intensity is constant and continues to a portion where the luminescence intensity in the downstream side of the control line in the flow direction is at its lowest. A range that is 1.5 times or more the half-width of the peak showing the luminescence intensity of the control line in the graph based on the second initial baseline, and in which the distances from the center point of the half-width to both ends of the range are the same, is set as a color value measurement range of the control line, and a background region is set as a region that is 0.5 times or more the dimension along the second initial baseline of the color value measurement range from the upstream end and downstream end of the color value measurement range of the control line. Curve fitting is performed using a line that indicates the luminescence intensity in the background region of the graph, and the luminescence intensity of the optimized baseline (L hC ) is the luminescence intensity (L C ) minus the difference value (ΔL C ) was calculated as the color value of the control line, and the difference value (ΔL C ) to the difference value (ΔL T ) ratio (ΔL T / ΔL C ) is calculated, and the ratio (ΔL T / ΔL C ) and has a calculation unit that calculates the average value of (30) The ratio (ΔL T / ΔL C a calibration curve setting unit that sets a standard curve showing the relationship between the average value of the measured values ​​and the concentration of the substance to be measured as a calibration curve; and a ratio (ΔL T / ΔL C) and an output unit that outputs the concentration obtained by the concentration acquisition unit. [Effects of the Invention]

[0017] According to the present invention, a highly accurate immunochromatographic quantification method and an immunochromatographic reader are provided. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 2 is a plan view showing a test plate used in an embodiment of the present invention. [Figure 2] 1 is a flowchart illustrating a method according to a first embodiment of the present invention. [Figure 3] 4 is a flowchart illustrating a method according to a second embodiment of the present invention. [Figure 4] 1 is a graph illustrating the effect of the method according to the second embodiment of the present invention in comparison with a conventional method, in which the horizontal axis indicates the positions of the 18 regions. [Figure 5] 10 is a graph illustrating the effect of the method according to the second embodiment of the present invention in comparison with a conventional method, showing the R2 value and the agreement rate with the actually measured concentration. [Figure 6] 10 is a flowchart illustrating a method according to a third embodiment of the present invention. [Figure 7] This figure explains steps S31 to S33 of the flowchart in Figure 6, and includes (a) a plan view showing the state of the immunochromatographic sheet of the test plate, (b) a graph showing the change in brightness in the flow direction, and (c) a graph showing the optimized baseline with a dashed line. [Figure 8] FIG. 7 is a diagram illustrating step S32 in the flowchart of FIG. 6. [Figure 9] FIG. 7 is a diagram illustrating steps S33 and S34 of the flowchart in FIG. 6. [Figure 10] 7 is an example of a first graph set in step S37 of the flowchart in FIG. 6. [Figure 11]7 is an example of a second graph set in step S38 of the flowchart in FIG. 6. [Figure 12] Graph (a) shows the relationship between the luminescence intensity (LT) of the test line measured by a conventional method and the actual measured value (measured concentration) of the substance to be measured, and graph (b) shows the relationship between the average value (T / C) of ΔLT / ΔLC measured in step S35 of the method of the third embodiment and the actual measured value (measured concentration) of the substance to be measured. [Figure 13] FIG. 1 is a schematic cross-sectional view showing an example of an immunochromatographic reader capable of carrying out a method according to an embodiment of the present invention. [Figure 14] FIG. 14 is a schematic side view showing the immunochromatographic reader of FIG. 13. DETAILED DESCRIPTION OF THE INVENTION

[0019] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the following embodiments. In the following embodiments, technically preferable limitations are imposed for carrying out the present invention, but these limitations are not essential requirements for the present invention.

[0020] [First embodiment] <Configuration> The method according to the first embodiment of the present invention is an immunochromatographic quantitative method using the test plate 10 shown in FIG. 1, and has the procedure shown in FIG. As shown in Figure 1, in a test plate 10, a main portion of an immunochromatographic sheet 11 is exposed in a window portion 101 of a housing 100. A through-hole is formed in the housing 100 at one end on the upstream side of the flow direction R of the immunochromatographic sheet 11, and this through-hole serves as a sample dropping portion 102. A test line 12 is set upstream of the exposed portion of the immunochromatographic sheet 11, and a control line 13 is set downstream of it. The direction perpendicular to the flow direction R of the immunochromatographic sheet 11 is defined as the sheet width direction W.

[0021] In the method of the first embodiment, a test plate 10 is used in which a liquid sample has been dropped and developed on the dropping section 102, i.e., one end of the immunochromatographic sheet 11 on the upstream side in the flow direction, and the concentration of the substance to be measured contained in the sample developed on the immunochromatographic sheet 11 is determined according to the procedure shown in the flowchart of Figure 2.

[0022] As shown in FIG. 2, in the method of the first embodiment, first, the luminance (amount of light emitted) L of the test line 12 of the test plate 10 is measured over the entire width direction W of the sheet. T and the brightness (luminescence amount) of control line 13 L C Next, the luminance (amount of light emitted) L of the test line 12 is measured (step S11). T The brightness (luminescence amount) of the control line 13 is L C Ratio to (L T / L C ) is calculated (step S12). In the flowchart, the test line is referred to as the "T line" and the control line is referred to as the "C line." Next, the ratio (L T / L C ) and the concentration of the substance to be measured, the ratio (L T / L C ) to determine the concentration of the measurement target substance contained in the sample (step S13).

[0023] <Actions and Effects> In conventional methods, the concentration of the substance to be measured was determined from the amount of luminescence only in the center of the width of the test line. However, with this method, in addition to uneven binding of the detection antibody to the test line, factors such as the state of application of reagents and imperfections in the structure and materials of the immunochromatography test kit itself can cause uneven flow of the sample in certain places, resulting in unevenness in the color of certain parts of the test line being darker or lighter than other parts. This has a significant impact on the measured amount of luminescence of the test line, resulting in low quantitative accuracy.

[0024] In contrast, according to the method of the first embodiment, the amount of luminescence associated with the colored images of the test line and control line is measured across the entire area of ​​the immunochromatographic sheet in a direction perpendicular to the flow direction (width direction of the sheet), thereby enabling more precise quantification than conventional methods that use the amount of luminescence only in the central part of the width direction of the immunochromatographic sheet. Also, the luminescence intensity of the test line (L T ) but the luminescence intensity (L T ) to the control line (L T / L C The use of a control line reduces the influence of unevenness on the test line. This is because the sample flows in the same direction on the immunochromatographic sheet and does not interfere with other reagents moving in the same direction, so the unevenness pattern is similar on the test line and the control line.

[0025] [Second embodiment] <Configuration> The method according to the second embodiment of the present invention is an immunochromatographic quantitative method using the test plate 10 shown in FIG. 1, and has the procedure shown in FIG. In the method of the second embodiment, similarly to the method of the first embodiment, a test plate 10 is used in which a liquid sample has been dropped into the dropping section 102 and developed. Then, the concentration of the measurement target substance contained in the sample developed on the immunochromatographic sheet 11 is determined according to the procedure shown in the flowchart of FIG.

[0026] As shown in FIG. 3, in the method of the second embodiment, first, the entire width direction W of the immunochromatographic sheet 11 is divided into 18 regions, and the luminance (luminescence amount L T ) and the brightness of the control line 13 (luminescence amount L C Next, the luminance (luminescence amount L) of the test line 12 is measured for each region (step S21). T ) of the control line 13 (luminescence amount L C ) to the ratio (L T / L C ) is calculated (step S22). Next, the ratio in the whole area (L T / L C ) is calculated (step S23). Next, the ratio (L T / L C ) and the concentration of the substance to be measured, the ratio (L T / L C ) to determine the concentration of the substance to be measured contained in the sample (step S24).

[0027] <Actions and Effects> According to the method of the second embodiment, the entire width direction W of the immunochromatographic sheet 11 is divided into 18 (plural) regions, and the ratio (L T / L C ) and calculate the ratio (L T / L C ) is used to calculate the concentration, so the concentration of the substance to be measured can be measured accurately even when there is significant line unevenness or when there is a difference in the flow speed of the sample in the sheet width direction W.

[0028] Figures 4 and 5 show the luminescence intensity (L T 10A is a diagram comparing a conventional method for determining the concentration of a substance to be measured from a sample with a method according to a second embodiment. FIG. As shown in Figure 4(a), the entire sheet width direction W is divided into 18 (multiple) areas, and the luminescence intensity (L T ) varies greatly depending on the region. In contrast, as shown in Figure 4(b), the amount of light emitted by the test line in each of the 18 regions (L T ) control line luminescence (L C ) to the ratio (L T / L C ) shows little difference between regions. As shown in Figure 5(a), the conventional method had a large measurement error and a low agreement rate with the actually measured concentration. The average CV value was 20.4%. In contrast, as shown in Figure 5(b), the method of the second embodiment had a small measurement error and a significantly improved agreement rate with the actually measured concentration. The average CV value was 10.2%.

[0029] In addition, in medical immunochromatographic testing aimed at quantification (POCT: Point of Care Testing), an error range of around 10% is required, and therefore the method of the second embodiment can be used in medical immunochromatographic testing aimed at quantification. The greater the number of regions obtained by dividing the entire sheet width direction, the greater the degree to which non-uniformity due to differences in flow speed in the sheet width direction is eliminated, resulting in higher measurement accuracy. The number of regions should be 2 or more, but the more the better, for example, 15 or more is preferable, and 20 or more is even more preferable.

[0030] [Third embodiment] <Configuration> The method according to the third embodiment of the present invention is an immunochromatographic quantitative method using the test plate 10 shown in FIG. 1, and has the procedure shown in FIG. In the method of the third embodiment, similarly to the method of the first embodiment, a test plate 10 is used in which a liquid sample has been dropped into the dropping portion 102 and developed. Then, the concentration of the measurement target substance contained in the sample developed on the immunochromatographic sheet 11 is determined according to the procedure shown in the flowchart of FIG. As shown in FIG. 6, in the method of the third embodiment, first, the entire width direction of the sheet is divided into 18 regions, and the brightness is measured for each region in the entire flow direction to obtain a graph showing the change in brightness in the entire flow direction (step S31).

[0031] Next, from the graph obtained in step S31, the color value measurement ranges of the test line and control line are set to predetermined ranges for each region (step S32). Next, curve fitting is performed on each graph using a line indicating the luminance of a predetermined background region, and the baseline is optimized (step S33). Next, for each region, the color value measurement range H set in step S32 is T , H C The brightness of the test line is L T from the baseline luminance L of the test line optimized in step S33. hT The difference value ΔL minus T Calculate the brightness of the control line L C From the result, the brightness L of the baseline of the control line optimized in step S33 hC The difference value ΔL minus C is calculated (step S34).

[0032] <Regarding steps S31 to S34> Figure 7(a) is a plan view showing the state of the immunochromatographic sheet 11 of the test plate 10, and the corresponding graphs (images obtained by averaging the graphs in each region) are shown in Figures 7(b) and 7(c). The solid lines in Figures 7(b) and 7(c) are graphs showing the change in brightness in the flow direction. As shown in FIG. 7(a), the test line 12 and the control line 13 tend to be strongly colored in the central portions 120, 130 in the flow direction, and weakly colored in the upstream portions 121, 131 and downstream portions 122, 132. In Fig. 7(b), the line L indicates the brightness of the background area used for curve fitting. kT ,L kC FIG. 7(c) shows the luminance L of the baseline of the test line and the control line optimized by curve fitting in step S33. hT ,L hC is shown.

[0033] In the method of this embodiment, the color value measurement range H of the test line 12 T , color value measurement range H of control line 13 C The distance k from the background region 14, 15 T (≧0.5H T), k C (≧0.5H C ) apart. Steps S31 to S34 will be specifically described with reference to FIGS. 8 and 9, which show the test lines. First, as shown in FIG. 8(a), in the graph obtained in step S31, a line B11 where the brightness (amount of light emitted) is constant and continues from the portion upstream of the test line in the flow direction where the brightness (amount of light emitted) is at its lowest value (for example, a line in the range where the lowest value appears and where that value is constant for a range of 10 consecutive pixels) and a line B12 where the brightness is constant and continues from the portion downstream of the test line in the flow direction where the brightness is at its lowest value are set.

[0034] Next, as shown in FIG. 8(b), the lines B11 and B12 are connected by a straight line to form a first initial baseline B1. The length (range) of lines B11 and B12 need only be 2 pixels, but as long as the brightness is constant in the flow direction, the longer (more pixels) the better; for example, 5 or more is preferable, and 10 or more is even more preferable.

[0035] Next, as shown in FIG. 8(c), a peak P , which indicates the luminescence intensity of the test line 12 in the graph, is calculated based on the first initial baseline B1. T The range of the half-width (full width at half maximum) FWHM of the test line is 1.5 times or more, and the distance from the center point C of the half-width to both ends of the range is the same. T Here, the width 1.6 times the half width (full width at half maximum) FWHM of the peak indicating the luminescence intensity of the test line 12 is set as the color value measurement range H T It is set to. In addition, in the graph obtained in step S31, a line where the brightness is constant and continues to the part upstream of the control line in the flow direction where the brightness is at its lowest value, and a line where the brightness is constant and continues to the part downstream of the control line in the flow direction where the brightness is at its lowest value are set, and both lines are connected by a straight line to form a second initial baseline.

[0036] Next, the peak indicating the luminescence intensity of the control line in the graph (P in Figure 7(b)) was calculated based on the second initial baseline. C The range of the color value measurement range H of the control line is 1.5 times or more of the half-value width of the control line. C Let's say. In step S33, first, as shown in FIG. 9(a), the color value measurement range H T From the upstream and downstream ends of the color value measurement range H T A region that is 0.5 times or more the dimension along the first initial baseline (B1 in FIG. 8) is set as the background region 14.

[0037] Next, as shown in FIG. 9(b), the line L indicating the luminance of the background region 14 in the graph is kT The brightness of the baseline in the background region of the test line of the graph is optimized by performing curve fitting using the following equation. Figure 9(c) shows the optimized brightness of the baseline in the background region of the test line (L hT ) is shown. Also, the color value measurement range of the control line is H C The area away from the upstream and downstream ends of the color value measurement range by 0.5 times or more of the dimension along the second initial baseline is set as the background area 15, and a line L indicating the brightness of the background area 15 in the graph is drawn. kC The baseline brightness in the background region of the control line of the graph is optimized by curve fitting using

[0038] In step S34, the optimized baseline luminance (LhT ) to the color value measurement range H T The brightness of the test line (L T ) minus the difference value (ΔL T ) is calculated as the color value of the test line. hC ) to the color value measurement range H C The brightness of the control line (L C ) minus the difference value (ΔL C ) is calculated as the color development value of the control line. In addition, the brightness of the test line L T The color value measurement range H T In other words, the average value of the brightness of each pixel in the shaded area in FIG. 9(c) (area average value of brightness) is used as the brightness L of the test line. T The brightness of the control line is L C Similarly, for the color value measurement range H C The average brightness value for each pixel within the image is used.

[0039] <About steps S35 and beyond> After step S34, for each region, ΔL T / ΔL C Calculate ΔL in all regions T / ΔL C The average value of these values ​​is calculated (step S35). Next, ΔL T / ΔL C It is determined whether the average value of is smaller than 1 (step S36). Next, ΔL T / ΔL C If the average value of is smaller than 1, proceed to step S37, and ΔL T / ΔL C The first graph, in which the average value of ΔL is linearly approximated, is set as the calibration curve. T / ΔL C If the average value is 1 or more, proceed to step S38, and ΔL T / ΔL CThe second graph, in which the average values ​​are logarithmically approximated, is set as the calibration curve.

[0040] FIG. 10 is an example of the first graph, and FIG. 11 is an example of the second graph. In these graphs, "ΔL T / ΔL C The average value of "T / C" is displayed as "T / C." The graph in Fig. 10 shows the range of "T / C≦1" in the graph in Fig. 12(c) described later, with the logarithmic scale of the X-axis changed to a normal scale. The graph in Fig. 11 shows the range of "T / C≧1" in the graph in Fig. 12(b). Next, using the calibration curve set in step S37 or step S38, ΔL calculated in step S35 T / ΔL C The concentration of the measurement target substance is calculated from the average value (step S39).

[0041] <Actions and Effects> The concentration of the standard sample of the substance to be measured is measured by the luminescence intensity (L T ) was measured by a conventional method for determining the concentration of the substance to be measured, and also by the method of the third embodiment. The agreement rate (%) of the calculated concentration measured by each method with the actual concentration measured by ELISA {= (calculated concentration / actual concentration) × 100} was then investigated. The results are shown in Table 1 below.

[0042] [Table 1]

[0043] Also, Figure 12(a) shows the amount of light emitted by the test line (L T ) and the actual measured concentration of the substance to be measured by ELISA, and FIG. 12(b) is a graph showing the relationship between ΔL T / ΔL C 1 is a graph showing the relationship between the average value (T / C) of the above and the concentration of the substance to be measured actually measured by ELISA. From these results, it can be seen that the method of the third embodiment can effectively reduce the luminescence intensity (L T It can be seen that this method has superior quantitative accuracy and a higher agreement rate with the actual concentration measured by ELISA than the conventional method of determining the concentration of the substance to be measured from the ELISA.

[0044] In the method of the third embodiment, the peak P T , P C The range of 1.5 to 3 times the half width (full width at half maximum) FWHM of the test line and control line is the color development value measurement range H T , H C It is set to. Color value measurement range H T , H C If the range is set to 1.5 times the FWHM, approximately 92.3% of the analysis values ​​can be covered if the peak shape is normally distributed, and if the range is set to 3 times the FWHM, approximately 99% of the analysis values ​​can be covered if the peak shape is normally distributed, so it is thought that this will provide a statistically sufficient coverage of the variability in the measurement values.

[0045] Therefore, compared to a method in which the color value measurement range of the test line and control line is a fixed position on the immunochromatography sheet that is set in advance before measurement, this method reduces the risk of missing analytical values ​​or being affected by noise factors, enabling more scientific and reliable analysis. T , H C is preferably in the range of 1.5 to 3 times the FWHM.

[0046] Another conventional method is to use the areas immediately upstream and downstream of the test line in the flow direction as background areas, and calculate the concentration of the substance to be measured from the value obtained by subtracting the average brightness of both background areas from the brightness of the test line. However, with this method, the background values ​​obtained may not be uniform due to factors that affect the background reading, such as variations in lighting inside the reader and unevenness caused by gray spots or wet spots in the background on the test kit, resulting in insufficient measurement accuracy.

[0047] In contrast, in the method of the third embodiment, the color value measurement range H T , H C From the upstream and downstream ends of the color value measurement range H T , H C The range where the luminance is 0.5 times or more is set as the background regions 14 and 15, and the luminance of the baseline in the background regions 14 and 15 is optimized by performing curve fitting using a line that indicates the luminance of the background regions 14 and 15. Therefore, a sufficient distance is secured between the background regions 14, 15 and the test line 12 and control line 13. Therefore, according to the method of the third embodiment, accidental influences such as false detection can be eliminated, enabling more accurate measurements.

[0048] Additionally, curve fitting, which determines the optimal baseline brightness, is a technique for finding a mathematical curve that best fits the acquired data. Therefore, according to the method of the third embodiment, by optimizing the baseline brightness in the background regions 14 and 15, the influence of random errors on the analysis results can be eliminated, improving the consistency of the acquired data. Furthermore, major noise sources are reduced, improving the accuracy and reliability of the measurements.

[0049] In the method of this embodiment, the color value measurement range H T , H C From the upstream and downstream ends of the color value measurement range H T , H C The range where the image is 0.5 times or more is set as the background region 14, 15. That is, k T / H T ≥ 0.5, k C / H C ≧0.5. Also, the color value measurement range H T , HC From the upstream and downstream ends of the color value measurement range H T , H C It is preferable to set the background regions 14, 15 within a range where the value is 1.0 times or less. T / H T ≧0.5, 1.0≧k C / H C It is preferred that it is ≧0.5. In the methods of the first to third embodiments, if the sample does not contain the substance to be measured and no peak is detected on the test line, the concentration of the substance to be measured in the sample can be determined to be below the detection limit or to be zero.

[0050] [Immunochromatography Reader] Examples of embodiments of an immunochromatographic reader that can implement the first to third methods include those shown in Figures 13 and 14. Figure 13 is a cross-sectional view of the immunochromatographic reader of the embodiment with a test plate installed, taken along the X direction parallel to the flow direction R of the immunochromatographic sheet. Figure 14 is a side view of the immunochromatographic reader of the embodiment, taken along the Y direction parallel to the sheet width direction W.

[0051] As shown in Figure 13, the immunochromatographic reader 2 comprises a test plate placement section 21 consisting of a recess formed in the bottom of the housing 20, a light-emitting element (light irradiation section) 22 and an imaging element (light emission amount acquisition section) 23 installed above the placement section 21, an information processing base (calculation section, concentration acquisition section) 24, and an input / output panel (calibration curve setting section, output section) 25. 14, the housing 20 has a first portion 201 and a second portion 202 that have different dimensions in the Z direction, which is perpendicular to the X and Y directions. The dimension of the first portion 201 in the Z direction is larger than that of the second portion 202. A light emitting element 22, an imaging element 23, and an information processing board 24 are arranged in an upper part of a first portion 201 of the housing 20, and a plate insertion opening 27 is formed on a side surface of a lower part of the first portion 201. The plate insertion opening 27 is formed at a position continuous with the arrangement portion 21 in the first portion 201 of the housing 20. An input / output panel 25 is installed in an upper part of the second portion 202 of the housing 20.

[0052] The input / output panel 25 consists of an input section and an output section (display section), the input section includes a calibration curve setting section, and the output section outputs the concentration of the substance to be measured obtained by the information processing base (calculation section, concentration acquisition section) 24. The light emitting element (light emitting section) 22 irradiates the entire surface of the immunochromatographic sheet 11 of the test plate 10 placed in the placement section 21 with visible light. The image pickup element (light emission amount acquisition unit) 23 and the information processing board (calculation unit, concentration acquisition unit) 24 are configured to execute a program corresponding to each of the first to third methods.

[0053] In the device for carrying out the method of the first embodiment, the image pickup element (luminescence amount acquisition unit) 23 captures an image of the entire immunochromatographic sheet in the sheet width direction, and measures the luminance (L T ) and the brightness of the reflected light from the control line (L C ) is acquired (perform step S11). The information processing platform (calculation unit, density acquisition unit) 24 also calculates the luminance (L T ) and the brightness of the control line (L C ) to calculate the brightness of the test line (L T ) control line brightness (L C ) to the ratio (L T / L C ) is calculated (step S12 is performed). Furthermore, the calculated ratio (L T / L C ) to obtain the concentration of the measurement target substance contained in the sample (perform step S13).

[0054] In the device for carrying out the method of the second embodiment, the image sensor (luminescence amount acquisition unit) 23 divides the entire width of the immunochromatographic sheet into a plurality of regions, and measures the luminance (L T ) and the brightness of the reflected light from the control line (L C ) is acquired (step S21). In addition, the information processing platform (calculation unit, density acquisition unit) 24 calculates the luminance (L T ) and the brightness of the control line (L C ) to calculate the brightness of the test line (L T ) control line brightness (L C ) to the ratio (L T / L C ) and calculate the ratio (L T / L C ) is calculated. Furthermore, the calculated ratio (L T / L C ) to obtain the concentration of the measurement target substance contained in the sample (step S24).

[0055] In an apparatus for carrying out the method of the third embodiment, the image sensor (light emission amount acquisition unit) 23 divides the entire width of the immunochromatographic sheet into multiple regions, and obtains a graph showing the change in brightness in the flow direction for each region (step S31). Furthermore, the information processing infrastructure (calculation unit, concentration acquisition unit) 24 performs the processes of steps S32 to S39 to obtain the concentration of the measurement target substance contained in the sample. [others]

[0056] In the above embodiment, luminance is used as the amount of light emitted. That is, the three primary colors R, G, and B are converted into numerical values ​​from the image obtained by the imaging element, and the luminance (0.30*R+0.59*G+0.11*B) is calculated. However, instead of luminance, RGB values ​​or grayscale values, which are R / G / B directly converted into numerical data, can also be used as the amount of light emitted. [Explanation of symbols]

[0057] 10 Test Plate 11 Immunochromatography sheet 12 Test Line 120 Center of the test line 121 Upstream portion of the test line 122 Downstream portion of the test line 13 Control Line 130 Central part of the control line 131 Upstream portion of the control line 132 Downstream portion of the control line 14 Background area 15 Background area 100 Inspection Plate Housing 101 Housing window 102 Dripping part 2 Immunochromatography Reader 20 Immunochromatography Reader Housing 201 First part of housing 202 Second part of housing 21 Test plate placement section 22 Light emitting element (light emitting part) 23 Image sensor (light emission acquisition unit) 24 Information processing platform (calculation unit, concentration acquisition unit) 25 Input / output panel (calibration curve setting section, output section) 27 Plate insertion port

Claims

1. 1. An immunochromatographic quantitative method for determining the concentration of a substance to be measured contained in a sample by using a test plate in which a sample is dropped onto one end of an immunochromatographic sheet on the upstream side in a flow direction and the sample is developed, the method comprising: The entire sheet width direction, which is the direction perpendicular to the flow direction, is divided into a plurality of regions, and the luminescence amount (L T ) and the luminescence intensity of the control line (L C ) is measured, For each of the regions, the measured luminescence amount is used to calculate the luminescence amount (L T ) luminescence intensity of the control line (L C ) to the ratio (L T / L C ) is calculated, and the ratio (L T / L C ) and calculate the average value of The ratio (L T / L C ) and the concentration of the substance to be measured, the calculated ratio (L T / L C The concentration of the substance to be measured contained in the sample is determined from the average value of the values ​​of the measured values.

2. 1. An immunochromatographic quantitative method for determining the concentration of a substance to be measured contained in a sample by using a test plate in which a sample is dropped onto one end of an immunochromatographic sheet on the upstream side in a flow direction and the sample is developed, the method comprising: The entire sheet width direction, which is a direction perpendicular to the flow direction, is divided into a plurality of regions, For each of the regions, measuring the amount of luminescence of the immunochromatographic sheet and creating a graph showing the change in the amount of luminescence in the flow direction; a line in the graph where the amount of light emission is constant and continues to a portion where the amount of light emission is minimum in the upstream side of the test line in the flow direction, and a line in the graph where the amount of light emission is constant and continues to a portion where the amount of light emission is minimum in the downstream side of the test line in the flow direction, connected by a straight line to define a first initial baseline; a range that is 1.5 times or more the half-width of the peak that indicates the luminescence intensity of the test line in the graph, with the first initial baseline as a reference, and in which the distances from the center point of the half-width to both ends of the range are the same, is set as a color value measurement range of the test line; a background region is set to a region that is at least 0.5 times the dimension along the first initial baseline of the color value measurement range from the upstream end and downstream end of the color value measurement range of the test line, and the luminescence amount of the baseline in the background region of the test line on the graph is optimized by performing curve fitting using a line that indicates the luminescence amount of the background region on the graph; The optimized baseline luminescence (L ｈT ) is the luminescence amount (L T ) and the difference value (ΔL T ) is calculated as the color value of the test line, and a line in the graph where the amount of light emission is constant and continues from a portion where the amount of light emission is minimum in the upstream side of the control line in the flow direction, and a line in the graph where the amount of light emission is constant and continues from a portion where the amount of light emission is minimum in the downstream side of the control line in the flow direction, connected by a straight line to form a second initial baseline; a range that is 1.5 times or more the half-width of the peak showing the luminescence intensity of the control line in the graph, with the second initial baseline as a reference, and in which the distances from the center point of the half-width to both ends of the range are the same, is set as a color value measurement range of the control line; a background region is set to a region that is at least 0.5 times the dimension along the second initial baseline of the color value measurement range from the upstream end and downstream end of the color value measurement range of the control line, and the luminescence intensity of the baseline in the background region of the control line on the graph is optimized by performing curve fitting using a line that indicates the luminescence intensity of the background region on the graph; The optimized baseline luminescence (L ｈC ) to the luminescence intensity (L C ) and the difference value (ΔL C ) is calculated as the color value of the control line, For each of the regions, the difference value (ΔL C ) to the difference value (ΔL T ) ratio (ΔL T / ΔL C ) is calculated, The ratio (ΔL T / ΔL C ) and calculate the average value of The ratio (ΔL T / ΔL C ) and the concentration of the substance to be measured, the calculated ratio (ΔL T / ΔL C The concentration of the substance to be measured contained in the sample is determined from the average value of the values ​​of the measured values.

3. The calculated ratio (L T / L C ) is smaller than 1, the ratio (L T / L C ) is linearly approximated as the calibration curve, and the calculated ratio (L T / L C ) to determine the concentration of the substance to be measured contained in the sample from the average value of The ratio (L T / L C ) is greater than 1, the ratio (L T / L C ) average values ​​are logarithmically approximated, and the calculated ratio (L T / L C ) to determine the concentration of the substance to be measured contained in the sample from the average value of The ratio (L T / L C ) is 1, either of the two graphs is used as the calibration curve, and the calculated ratio (L T / L C 2. The immunochromatographic quantitative method according to claim 1, wherein the concentration of the substance to be measured contained in the sample is determined from the average value of the values ​​of the respective values.

4. The calculated ratio (ΔL T / ΔL C ) is smaller than 1, the ratio (ΔL T / ΔL C ) was used as the calibration curve to obtain a graph in which the average values ​​of T / ΔL C ) to determine the concentration of the substance to be measured contained in the sample from the average value of Said ratio (ΔL T / ΔL C ) is greater than 1, the ratio (ΔL T / ΔL C ) was used as the calibration curve to obtain a graph in which the average values ​​of the logarithmic approximation were obtained. T / ΔL C ) to determine the concentration of the substance to be measured contained in the sample from the average value of Said ratio (ΔL T / ΔL C ) is 1, either of the two graphs is used as the calibration curve, and the calculated ratio (ΔL T / ΔL C 3. The immunochromatographic quantitative method according to claim 2, wherein the concentration of the substance to be measured contained in the sample is determined from the average value of the values ​​of the respective values.

5. An immunochromatography reader in which a sample is dropped onto one end of an immunochromatography sheet on the upstream side in the flow direction, and a test plate on which the sample is developed is used to measure the concentration of a substance to be measured contained in the sample, a placement unit for the test plate; a light irradiating unit for irradiating an upper surface of the immunochromatographic sheet of the test plate placed in the placement unit; The entire sheet width direction, which is the direction perpendicular to the flow direction, is divided into a plurality of regions, and the amount of light emitted by the reflected light from the test line of the inspection plate (L T ) and the amount of light emitted from the control line (L C a light emission amount acquisition unit that acquires the light emission amount; The luminescence amount (L T ) and the luminescence amount (L C ) from the ratio of the two (L T / L C ) is calculated, and the ratio (L T / L C a calculation unit for calculating an average value of The ratio (L T / L C a calibration curve setting unit that sets a standard curve showing the relationship between the average value of the measured values ​​and the concentration of the substance to be measured as a calibration curve; The ratio (L T / L C a concentration acquisition unit that acquires the concentration of the measurement target substance contained in the sample from the average value of the measured values; an output unit that outputs the concentration obtained by the concentration acquisition unit; An immunochromatographic reader having the above structure.

6. An immunochromatography reader in which a sample is dropped onto one end of an immunochromatography sheet on the upstream side in the flow direction, and a test plate on which the sample is developed is used to measure the concentration of a substance to be measured contained in the sample, a placement unit for the test plate; a light irradiating unit for irradiating an upper surface of the immunochromatographic sheet of the test plate placed in the placement unit; a luminescence amount acquiring unit that divides the entire sheet width direction, which is a direction perpendicular to the flow direction, into a plurality of regions, measures the luminescence amount of the immunochromatographic sheet for each region, and creates a graph showing the change in the luminescence amount in the flow direction; a line in the graph created by the light emission amount acquisition unit, where the light emission amount is constant and continues to a portion where the light emission amount is minimum in the portion upstream of the test line in the flow direction, and a line in the graph in the graph, where the light emission amount is constant and continues to a portion where the light emission amount is minimum in the portion downstream of the test line in the flow direction, are connected by a straight line to form a first initial baseline; a range that is 1.5 times or more the half-width of the peak that indicates the luminescence intensity of the test line in the graph, with the first initial baseline as a reference, and in which the distances from the center point of the half-width to both ends of the range are the same, is set as a color value measurement range of the test line; a background region is set to a region that is at least 0.5 times the dimension along the first initial baseline of the color value measurement range from the upstream end and downstream end of the color value measurement range of the test line, and the luminescence amount of the baseline in the background region of the test line on the graph is optimized by performing curve fitting using a line that indicates the luminescence amount of the background region on the graph; The optimized baseline luminescence (L ｈT ) is the luminescence amount (L T ) and the difference value (ΔL T ) is calculated as the color value of the test line, and a line in the graph where the amount of light emission is constant and continues from a portion where the amount of light emission is minimum in the upstream side of the control line in the flow direction, and a line in the graph where the amount of light emission is constant and continues from a portion where the amount of light emission is minimum in the downstream side of the control line in the flow direction, connected by a straight line to form a second initial baseline; a range that is 1.5 times or more the half-width of the peak showing the luminescence intensity of the control line in the graph, with the second initial baseline as a reference, and in which the distances from the center point of the half-width to both ends of the range are the same, is set as a color value measurement range of the control line; a background region is set to a region that is at least 0.5 times the dimension along the second initial baseline of the color value measurement range from the upstream end and downstream end of the color value measurement range of the control line, and the luminescence intensity of the baseline in the background region of the control line on the graph is optimized by performing curve fitting using a line that indicates the luminescence intensity of the background region on the graph; The optimized baseline luminescence (L ｈC ) to the luminescence intensity (L C ) and the difference value (ΔL C ) is calculated as the color value of the control line, For each of the regions, the difference value (ΔL C ) to the difference value (ΔL T ) ratio (ΔL T / ΔL C ) is calculated, The ratio (ΔL T / ΔL C a calculation unit for calculating an average value of The ratio (ΔL T / ΔL C a calibration curve setting unit that sets a standard curve showing the relationship between the average value of the measured values ​​and the concentration of the substance to be measured as a calibration curve; The ratio (ΔL T / ΔL C a concentration acquisition unit that calculates the concentration of the measurement target substance contained in the sample from the average value of the measured values; an output unit that outputs the concentration obtained by the concentration acquisition unit; An immunochromatographic reader having the above structure.

Images