JP7828124B1 - Immunochromatographic quantification method, immunochromatographic reader - Google Patents

Abstract

The present invention provides a highly accurate immunochromatographic quantification method and an immunochromatographic reader capable of carrying out this method.
[Solution] This is an immunochromatographic quantitative method for determining the concentration of a substance to be measured contained in a sample, using a test plate 10 in a developed state after a sample has been dropped onto one end of the immunochromatographic sheet 11 on the upstream side in the flow direction. The luminescence intensity ( _LT ) of the test line 12 and the luminescence intensity ( _LC ) of the control line 13 on the test plate 10 are measured across the entire width direction W of the sheet, which is perpendicular to the flow direction R, and the ratio (LT/ _LC ) of the luminescence intensity ( _LT ) of the test line to the luminescence intensity ( _LC ) of the control line is calculated. The concentration of the substance to be measured contained in the sample is determined from the calculated ratio ( _LT / _LC ) using a calibration curve showing the relationship between the ratio ( _LT / _LC ) and the concentration of the substance to be measured, which is created from a sample with a known concentration of _the substance to be measured.
[Selected Figure] Figure 1

Description

The present invention relates to an immunochromatographic quantification method and an immunochromatographic reader.

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 on positive or negative results. This is because the rapid changes in hormone levels mean that the accuracy of immunochromatographic tests is relatively less important for pregnancy testing, and patients generally receive more accurate tests 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.

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 areas of the test line) from the brightness of the test line, but this brightness of the background area is obtained without taking into account non-uniformity in the flow direction.

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 disease severity classification. 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.

For accurate quantification, it is desirable for the color development of the test line and control line to be uniform in each region. However, due to uneven binding of the detection antibody to the test line on the immunochromatography test paper (immunochromatography sheet), as well as hardware-related problems such as the state of application of reagents and imperfect structure and materials of the immunochromatography test kit itself, the flow of the sample on the reagent paper is likely to be uneven.
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.

Conventional methods analyze the area average brightness within a specific range of the test line only, so this type of unevenness is one of the main causes of poor quantitative accuracy. Furthermore, due to technical limitations, the test line on an immunochromatography test paper (immunochromatograph sheet) is not uniformly bound before and after the test line (upstream and downstream portions). Typically, there is more antigen-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.

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 removes 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.

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 calculated, 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, in this method, the amount of luminescence from the entire surface of the test plate is not measured.

Japanese Patent Application Laid-Open No. 2000-338106

The objective of the present invention is to provide a highly accurate immunochromatographic quantification method and an immunochromatographic reader capable of carrying out this method.

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 luminescence intensity ( _LT ) of the test line and the luminescence intensity ( _{LC) of the control line of the test plate are measured across the entire width of the sheet, which is perpendicular to the flow direction, and the ratio (LT/LC ) of} the luminescence intensity of the test line ( _LT ) to the luminescence intensity of the control line ( _LC ) is calculated using the measured luminescence intensity. The concentration of the substance to be measured contained in the sample is determined from the calculated ratio ( _LT / _LC ) using a calibration curve showing the relationship between the ratio ( _LT / _LC ) and the concentration of the substance to be measured, which is prepared from a sample whose concentration of the substance to be measured is known.

A second aspect of the present invention provides an immunochromatographic quantitative method having the above configuration (1) and the following configuration (3).
(3) The entire width of the sheet, which is perpendicular to the flow direction, is divided into a plurality of regions, and the luminescence intensity ( _LT ) of the test line and the luminescence intensity ( _LC ) of the control line of the test plate are measured for each region. The measured luminescence intensity is used for each region to calculate the ratio ( _LT / _LC ) of the luminescence intensity of the test line ( _LT ) to the luminescence intensity of the control line ( _LC ). The average value of the ratios ( _LT / _LC ) for all regions is calculated, and the concentration of the substance to be measured contained in the sample is determined from the average value of the calculated ratios ( _LT / _LC ) using a calibration curve showing the relationship between the average value of the ratios ( _LT / _LC ) and the concentration of the substance to be measured, which is prepared from samples whose concentrations of the substance to be measured are known.

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) Calculating the difference (ΔL _T ) obtained by subtracting the luminescence amount (L _hT ) of the optimized baseline from the luminescence amount (L _T ) of the test line in the color value measurement range as the color value of the test line;
(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 end and downstream end 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) calculating the difference (ΔL _C ) obtained by subtracting the luminescence level (L _hC ) of the optimized baseline from the luminescence level (L _C ) of the control line as the color value of the control line;
(13) calculating the ratio (ΔL _T /ΔL _C ) of the difference value (ΔL _T ) of the test line to the difference value (ΔL _C ) of the control line for each of the regions;
(14) Calculating the average value of the ratio (ΔL _T /ΔL _C ) in all of the regions;
(15) Using a calibration curve showing the relationship between the average value of the ratio (ΔL _T /ΔL _C ) and the concentration of the substance to be measured, which is prepared from samples whose concentrations of the substance to be measured are known, the concentration of the substance to be measured contained in the sample is determined from the average value of the calculated ratio (ΔL _T /ΔL _C ).

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) A calculation unit is provided which calculates the ratio (LT/ _LC ) of the luminescence amount of the test line ( _LT ) to the luminescence amount of the control line ( _LC ) from the luminescence amount of the test _line ( _LT ) and the luminescence amount of the control line ( _LC ) acquired by the luminescence amount acquisition unit.
(24) The apparatus includes a calibration curve setting unit that sets, as a calibration curve, a standard curve showing the relationship between the ratio ( _LT / _LC ) and the concentration of the substance to be measured, which has been created in advance using a sample whose concentration of the substance to be measured is known; a concentration acquisition unit that obtains the concentration of the substance to be measured contained in the sample from the ratio ( _LT / _LC ) calculated by the calculation unit using the calibration curve set by the calibration curve setting unit; and an output unit that outputs the concentration obtained by the concentration acquisition unit.

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 test plate is provided with 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 a light emission amount acquisition section for dividing the entire sheet width direction, which is a direction perpendicular to the flow direction, into a plurality of regions and acquiring the light emission amount (L _T ) of the light reflected from the test line of the test plate and the light emission amount (L _C ) of the light reflected from the control line for each of the regions.
(26) The apparatus includes a calculation unit that calculates the ratio ( _LT / _LC ) between the luminescence amount ( _LT ) of the test line and the luminescence amount ( _LC ) of the control line acquired by the luminescence amount acquisition unit, and calculates the average value of the ratio ( _LT / _LC ) in all of the regions, and a calibration curve setting unit that sets a standard curve that shows the relationship between the average value of the ratio ( _LT / _LC ) and the concentration of the substance to be measured, which has been prepared in advance using a sample whose concentration of the substance to be measured is known, as a calibration curve.
(27) The apparatus includes a concentration acquisition unit that obtains the concentration of the substance to be measured contained in the sample from the average value of the ratio ( _LT / _LC ) calculated by the calculation unit using the calibration curve set by the calibration curve setting unit, and an output unit that outputs the concentration obtained by the concentration acquisition unit.

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 of the color value measurement range along the first initial baseline from the upstream end and downstream end of the color value measurement range of the test line is set as a background region; and curve fitting is performed using a line that shows the luminescence amount in the background region of the graph, thereby optimizing the luminescence amount of the baseline in the background region of the test line in the graph, and the optimized luminescence amount (L _hT ) from the luminescence amount (L _T ) of the test line in the color value measurement range _, ) is calculated as the color value of the test line, and a second initial baseline is defined 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 defined as a color value measurement range of the control line, and a background region is defined 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 the 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, thereby optimizing the luminescence intensity of the baseline in the background region of the control line in the graph, and the optimized luminescence intensity (L _hC The apparatus has a calculation unit that calculates the difference value (ΔL _{C ) by subtracting the luminescence amount (ΔL C )} of the control line from the luminescence amount (L _C ) of the control line as the color development value of the control line, calculates the ratio (ΔL _T /ΔL C ) of the difference value (ΔL _T ) of the test line to the difference value (ΔL _C ) of the control line for each region, and calculates the average value of the ratios (ΔL _T /ΔL _C ) for all the regions.
(30) The apparatus includes a calibration curve setting unit that sets, as a calibration curve, a standard curve showing the relationship between the average value of the ratio (ΔL _T /ΔL _C ) and the concentration of the substance to be measured, which is created from samples whose concentrations of the substance to be measured are known; a concentration acquisition unit that uses the calibration curve set by the calibration curve setting unit to determine the concentration of the substance to be measured contained in the sample from the average value of the ratio (ΔL _T /ΔL _C ) calculated by the calculation unit; and an output unit that outputs the concentration obtained by the concentration acquisition unit.

The present invention provides a highly accurate immunochromatographic quantification method and immunochromatographic reader.

FIG. 2 is a plan view showing a test plate used in an embodiment of the present invention. 1 is a flowchart illustrating a method according to a first embodiment of the present invention. 4 is a flowchart illustrating a method according to a second embodiment of the present invention. 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, in which the horizontal axis indicates the positions of the 18 regions. 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 measured concentration. 10 is a flowchart illustrating a method according to a third embodiment of the present invention. 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. FIG. 7 is a diagram illustrating step S32 in the flowchart of FIG. 6. FIG. 7 is a diagram illustrating steps S33 and S34 of the flowchart in FIG. 6. 7 is an example of a first graph set in step S37 of the flowchart of FIG. 6. 7 is an example of a second graph set in step S38 of the flowchart of FIG. 6. Graph (a) shows the relationship between the amount of luminescence (L _T ) 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 ΔL _T /ΔL _C measured in step S35 of the method according to the third embodiment and the actual measured value (measured concentration) of the substance to be measured. 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. FIG. 14 is a schematic side view showing the immunochromatographic reader of FIG. 13.

Embodiments of the present invention will be described below, but the present invention is not limited to the embodiments described below. While the embodiments described below have technically preferable limitations for implementing the present invention, these limitations are not essential requirements for the present invention.

[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.

In the method of the first embodiment, a test plate 10 is used in which a liquid sample has been dropped and spread 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 spread on the immunochromatographic sheet 11 is determined according to the procedure shown in the flowchart of Figure 2.

2, in the method of the first embodiment, first, the luminance (luminescence amount) L _T of the test line 12 and the luminance (luminescence amount) L _C of the control line 13 on the test plate 10 are measured across the entire sheet width direction W (step S11). Next, the ratio (L _T /L _C ) of the luminance (luminescence amount) L _T of the test line 12 to the luminance (luminescence amount) L _C of the control line 13 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, using a calibration curve showing the relationship between the ratio ( _LT / _LC ) and the concentration of the substance to be measured, which was created using a sample whose concentration of the substance to be measured is known in advance, the concentration of the substance to be measured contained in the sample is determined from the ratio ( _LT / _LC ) calculated in step S12 (step S13).

<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.

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 over the entire area of the immunochromatographic sheet in a direction perpendicular to the flow direction (the 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.
Furthermore, by using the ratio of the luminescence intensity of the test line ( _LT ) to the control line ( _LT / _LC ) instead of the luminescence intensity of the test line ( _LT ), the influence of unevenness in the test line is reduced. This is thought to be because the flow of the sample on the immunochromatographic sheet maintains the same direction and does not cause interference with other reagents moving in the same direction, resulting in similar patterns of unevenness on the test line and the control line.

[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 spread thereon. Then, the concentration of the measurement target substance contained in the sample spread on the immunochromatographic sheet 11 is determined according to the procedure shown in the flowchart of FIG.

3, in the method of the second embodiment, first, the entire immunochromatographic sheet 11 is divided into 18 regions in the sheet width direction W, and the luminance (luminescence amount L _T ) of the test line 12 and the luminance (luminescence amount L _C ) of the control line 13 are measured for each region (step S21). Next, the ratio (L _T /L _C ) of the luminance (luminescence amount L _T ) of the test line 12 to the luminance (luminescence amount L _C ) of the control line 13 is calculated for each region (step S22).
Next, the average value of the ratio (L _T /L _C ) in the entire region is calculated (step S23).
Next, using a calibration curve showing the relationship between the average value of the ratio ( _LT / _LC ) and the concentration of the substance to be measured, which was created using samples whose concentrations of the substance to be measured are known in advance, the concentration of the substance to be measured contained in the sample is determined from the ratio ( _LT / _LC ) calculated in step S23 (step S24).

<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 (multiple) regions, the ratio ( _LT / _LC ) is calculated for each region, and the concentration is determined from the average value of the ratios ( _LT / _LC ) for all regions. Therefore, even if line unevenness is more pronounced or there is a difference in the flow speed of the sample in the width direction W of the sheet, the concentration of the substance to be measured can be measured with high accuracy.

4 and 5 are diagrams comparing a conventional method (a) for determining the concentration of a substance to be measured from the amount of luminescence (L _T ) of the test line with the method (b) of the second embodiment.
As shown in Figure 4(a), when the entire sheet width direction W is divided into 18 (plural) regions and measurements are taken for each region, the luminescence amount ( _LT ) of the test line varies greatly among regions. In contrast, as shown in Figure 4(b), the ratio ( _LT / _LC ) of the luminescence amount ( _LT ) of the test line to the luminescence amount ( _LC ) of the control line for each of the 18 regions varies little among regions.
As shown in Figure 5(a), the conventional method had a large measurement error and a low agreement rate with the actual measured concentration. The average CV 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 actual measured concentration. The average CV was 10.2%.

In addition, in medical immunochromatographic testing (POCT: Point of Care Testing) aimed at quantification, 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 may be two or more, but the more the number, the more preferable, for example, 15 or more is preferable, and 20 or more is even more preferable.

[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 section 102 and spread thereon. Then, the concentration of the measurement target substance contained in the sample spread 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 across the entire flow direction to obtain a graph showing the change in brightness across the entire flow direction (step S31).

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, in each color value measurement range H _T , H _C set in step S32, a difference value ΔL _T is calculated by subtracting the luminance L _hT of the baseline of the test line optimized in step S33 from the luminance L _T of the test line, and a difference value ΔL C is calculated by subtracting the luminance L _hC of the baseline of the control line optimized in step _S33 from the luminance L _C of the control line (step S34).

<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 Figures 7(b) and 7(c) show corresponding graphs (images obtained by averaging the graphs in each region). 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. 7A, the test line 12 and the control line 13 tend to be strongly colored in the central portions 120 and 130 in the flow direction, and weakly colored in the upstream portions 121 and 131 and the downstream portions 122 and 132.
7(b) shows lines _LkT and _LkC indicating the luminance of the background region used in curve fitting, and FIG. 7(c) shows the luminance _LhT and _LhC of the baselines of the test line and control line optimized by curve fitting in step S33.

In the method of this embodiment, the color value measurement range H _T of the test line 12 and the color value measurement range H _C of the control line 13 are set to ranges separated from the background regions 14, 15 by distances k _T (≧0.5H _T ) and k _C (≧0.5H _C ).
Steps S31 to S34 will be specifically described with reference to FIGS. 8 and 9 showing the test lines.
First, as shown in FIG. 8A, 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 a range where the lowest value appears and where that value is constant for a range of 10 consecutive pixels) is set, 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 is set.

Next, as shown in FIG. 8B, 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 (the more pixels) the better; for example, 5 or more is preferable, and 10 or more is even more preferable.

8(c), the range HT for measuring the color value of the test line is set to a range that is 1.5 times or more the half-width (full width at half maximum) FWHM of the peak _PT indicating the luminescence intensity of the test line 12 in the graph, with the first initial baseline B1 as the reference, and in which the distances from the center point C of the half-width to both ends of the range are the same. Here, the range _HT for measuring the color value of the test line is set to a width that is 1.6 times the half-width (full width at half maximum) _FWHM of the peak indicating the luminescence intensity of the test line 12.
In addition, in the graph obtained in step S31, a line where the brightness is constant and continues from the portion 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 from the portion 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.

Next, the range H C of the control line color value measurement is defined as the range 1.5 times or more the half-width of the peak (P _C in Figure 7(b)) showing the luminescence intensity of the control line in the graph, based on the second initial baseline, and in which the distance from the center point of the half-width to both ends of the range is _the same.
In step S33, first, as shown in FIG. 9(a), an area that is at least 0.5 times the dimension along the first initial baseline (B1 in FIG. 8) of the color value measurement range H _T from the upstream and downstream ends of the color value measurement range H _T of the test line is set as the background area 14.

Next, as shown in Figure 9(b), the luminance of the baseline in the background region of the test line on the graph is optimized by curve fitting using a line _LkT that represents the luminance of the background region 14 on the graph. Figure 9(c) shows the luminance of the optimized baseline ( _LhT ) in the background region of the test line.
In addition, 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 _Hc of the control line is set as background region 15, and curve fitting is performed using line _Lkc , which indicates the brightness of background region 15 in the graph, to optimize the brightness of the baseline in the background region of the control line in the graph.

In step S34, the difference value (ΔL _T ) obtained by subtracting the luminance (L _{h T} ) of the optimized baseline from the luminance (L _T ) of the test line in the color value measurement range H _T is calculated as the color value of the test line. Also, the difference value (ΔL _C ) obtained by subtracting the luminance (L _{h C} ) of the optimized baseline from the luminance (L _C ) of the control line in the color value measurement range H _C is calculated as the color value of the control line.
The luminance _L of the test line is the average value of the luminance for each pixel within the color value measurement range _H. In other words, the average value of the shaded area in Figure 9(c) (average area value of luminance) is taken as the luminance _L of the test line. Similarly, the luminance _L of the control line is the average value of the luminance for each pixel within the color value measurement range _H.

<Steps S35 and after>
After step S34, ΔL _T /ΔL _C is calculated for each region, and the average value of ΔL _T /ΔL _C for all regions is calculated (step S35).
Next, it is determined whether the average value of ΔL _T /ΔL _C is smaller than 1 (step S36).
Next, if the average value of ΔL _T /ΔL _C is smaller than 1, the process proceeds to step S37, and a first graph prepared in advance in which the average value of ΔL _T /ΔL _C is linearly approximated against the concentration of the substance to be measured is set as the calibration curve. If the average value of ΔL _T /ΔL _C is 1 or greater, the process proceeds to step S38, and a second graph prepared in advance in which the average value of ΔL _T /ΔL _C is logarithmically approximated against the concentration of the substance to be measured is set as the calibration curve.

Fig. 10 is an example of the first graph, and Fig. 11 is an example of the second graph. In these graphs, "average value of ΔL _T /ΔL _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 below, 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, the concentration of the substance to be measured is calculated from the average value of ΔL _T /ΔL _C calculated in step S35 (step S39).

<Actions and Effects>
The concentrations of the standards of the substances to be measured were measured by a conventional method in which the concentrations of the substances to be measured are determined from the luminescence amount (L _T ) of the test line, 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) x 100} was then examined. The results are shown in Table 1 below.

FIG. 12(a) is a graph showing the relationship between the luminescence intensity (L _T ) of the test line measured by the conventional method and the actual measured concentration of the substance to be measured by ELISA, and FIG. 12(b) is a graph showing the relationship between the average value (T/C) of ΔL _T /ΔL _C measured in step S35 of the method of the third embodiment and the actual measured concentration of the substance to be measured by ELISA.
These results show that in both the high concentration range of dilution ratios of 64 times or less and the low concentration range of dilution ratios of 128 times or more, the method of the third embodiment has superior quantitative accuracy and a higher agreement rate with the actual measured concentration by ELISA than the conventional method of determining the concentration of the substance to be measured from the luminescence intensity (L _T ) of the test line.

In the method of the third embodiment, the color value measurement ranges _{H T} and H _C of the test line and control line are set to a range of 1.5 to 3 times the half width (full width at half maximum) FWHM of the peaks P _T and P _C , which indicate the luminescence intensity of the test line 12 and the control line 13.
If the color value measurement range H _T and H _C is set to a range 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 considered that the variation in the measurement values can be statistically sufficiently covered.

Therefore, compared to a method in which the color value measurement ranges for the test line and control line are set to fixed positions on an immunochromatographic sheet that are preset before measurement, this method reduces the risk of missing analytical values or being affected by noise factors, enabling more scientific and reliable analysis. Therefore, it is preferable that the color value measurement ranges H _T and H _C be set to a range of 1.5 to 3 times the FWHM.

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.

In contrast, in the method of the third embodiment, the ranges from the upstream and downstream ends of the color value measurement ranges H _T and H _C of the test line 12 and the control line 13 to 0.5 times or more of the color value measurement ranges H _T and H _C are set as background regions 14 and 15, and curve fitting using a line indicating the luminance of the background regions 14 and 15 is performed to optimize the baseline luminance in 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.

Furthermore, 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 is eliminated, improving the consistency of the acquired data. Furthermore, major noise factors are reduced, improving the accuracy and reliability of the measurements.

In the method of this embodiment, the background regions 14, 15 are set to ranges from the upstream and downstream ends of the color value measurement ranges H _T and H _C that are 0.5 times or more the color value measurement ranges H _T and H _C , i.e., k _T /H _T ≧0.5, k _C /H _C ≧0.5. It is also preferable to set the background regions 14, 15 to ranges from the upstream and downstream ends of the color value measurement ranges H _T and H _C that are 1.0 times or less the color value measurement ranges _{H T} and H _C , i.e., 1.0≧k _T /H _T ≧0.5, 1.0≧k C /H _C ≧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.

[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.

As shown in Figure 13, the immunochromatographic reader 2 includes 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 image sensor (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.
The light emitting element 22, the image pickup element 23, and the information processing board 24 are arranged in the upper part of the first part 201 of the housing 20, and a plate insertion opening 27 is formed in the side of the lower part of the first part 201. The plate insertion opening 27 is formed in a position continuous with the arrangement section 21 in the first part 201 of the housing 20. An input/output panel 25 is installed in the upper part of the second part 202 of the housing 20.

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 platform (calculation unit, concentration acquisition unit) 24 are configured to execute a program corresponding to each of the first to third methods.

In the device for carrying out the method of the first embodiment, the image sensor (light emission acquisition unit) 23 captures an image of the entire width of the immunochromatographic sheet, and acquires the luminance (L _T ) of the reflected light from the test line on the test plate and the luminance (L _C ) of the reflected light from the control line (step S11).
The information processing platform (calculation unit, concentration acquisition unit) 24 also calculates (step S12) the ratio (LT/ _{LC) of the test line luminance (LT} ) to the control line luminance ( _LC ) from the test line luminance ( _LT ) and control line luminance ( _LC ) acquired by the image sensor (luminescence amount acquisition unit) 23. Furthermore, the concentration of the substance to be measured contained in the sample is obtained from the calculated ratio ( _{LT / LC} ) using the calibration curve set in the input unit (calibration curve setting unit) of the input/ _output panel 25 (step S13).

In the device for carrying out the method of the second embodiment, the image sensor (light emission acquisition unit) 23 divides the entire width of the immunochromatographic sheet into multiple regions, and acquires the luminance (L _T ) of the reflected light from the test line on the test plate and the luminance (L _C ) of the reflected light from the control line for each region (step S21).
Furthermore, in the information processing platform (calculation unit, concentration acquisition unit) 24, the ratio (LT/LC) of the test line luminance ( _LT ) to the control line luminance ( _LC ) for each region is calculated from the test line luminance ( _LT ) and control line luminance ( _LC ) acquired by the image sensor ( _luminescence amount acquisition unit) 23, and the average value of the ratios ( _LT / _LC ) for all regions is calculated. Furthermore, the concentration of the substance to be measured contained in the sample is obtained from the average value of the calculated ratios ( _LT / _LC ) using the calibration curve set in the input unit (calibration curve setting unit) of the input/output panel 25 ₍ step S24).

In an apparatus for carrying out the method of the third embodiment, the image sensor (light emission 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 platform (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]

In the above embodiment, luminance is used as the amount of light emitted. In other words, the three primary colors R, G, and B are converted into numerical values from the image obtained by the image sensor, and luminance (0.30 * R + 0.59 * G + 0.11 * B) is calculated. However, in addition to luminance, it is also possible to use RGB values, which are R/G/B directly converted into numerical data, or grayscale values, as the amount of light emitted.

10 Test plate 11 Immunochromatographic sheet 12 Test line 120 Central portion of test line 121 Upstream portion of test line 122 Downstream portion of test line 13 Control line 130 Central portion of control line 131 Upstream portion of control line 132 Downstream portion of control line 14 Background area 15 Background area 100 Housing of test plate 101 Window portion of housing 102 Dropping portion 2 Immunochromatographic reader 20 Housing of immunochromatographic reader 201 First portion of housing 202 Second portion of housing 21 Placement portion of test plate 22 Light-emitting element (light-emitting portion)
23 Image sensor (light emission amount 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 (6)

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, comprising:
The entire sheet width direction, which is a direction perpendicular to the flow direction, is divided into a plurality of regions, and the luminescence amount (L _T ) of the test line and the luminescence amount (L _C ) of the control line of the test plate are measured for each region;
For each of the regions, the ratio ( _LT / _{LC) of the luminescence level at the test line (LT) to the luminescence level at the control line (LC ) is} calculated using the measured luminescence level, and the average value of the ratio ( _LT / _LC ) for all of the regions is calculated.
An immunochromatographic quantitative method in which the concentration of the substance to be measured contained in the sample is determined from the calculated average value of the ratio ( _LT / _LC ) using a calibration curve that is prepared from samples in which the concentration of the substance to be measured is known and that shows the relationship between the average value of the ratio ( _LT / _LC ) and the concentration of the substance to be measured. 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, 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 difference (ΔL _T ) obtained by subtracting the luminescence amount (L _hT ) of the optimized baseline from the luminescence amount (L _T ) of the test line in the color value measurement range is calculated as the color value of the test line;
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 difference (ΔL _C ) obtained by subtracting the luminescence level (L _hC ) of the optimized baseline from the luminescence level (L _C ) of the control line is calculated as the color value of the control line;
For each of the regions, the ratio (ΔL _T /ΔL _C ) of the difference value (ΔL _T ) of the test line to the difference value (ΔL _C ) of the control line is calculated;
Calculating the average value of the ratio (ΔL _T /ΔL _C ) in all of the regions;
An immunochromatographic quantitative method in which the concentration of the substance to be measured contained in the sample is determined from the average value of the calculated ratio (ΔL _T /ΔL _C ) using a calibration curve that is prepared from samples in which the concentration of the substance to be measured is known and that shows the relationship between the average value of the ratio (ΔL _T /ΔL _C ) and the concentration of the substance to be measured. If the average value of the calculated ratio (L _T /L _C ) is smaller than 1, a graph in which the average value of the ratio (L _T /L _C ) is linearly approximated against the concentration of the substance to be measured is used as the calibration curve, and the concentration of the substance to be measured contained in the sample is determined from the average value of the calculated ratio (L _T /L _C );
If the average value of the ratio ( _LT / _LC ) is greater than 1, a graph in which the average value of the ratio ( _LT / _LC ) is logarithmically approximated against the concentration of the substance to be measured is used as the calibration curve, and the concentration of the substance to be measured contained in the sample is determined from the calculated average value of the ratio ( _LT / _LC );
2. The immunochromatographic quantitative method according to claim 1, wherein, when the average value of the ratio ( _LT / _LC ) is 1 , one of the two graphs is used as the calibration curve, and the concentration of the substance to be measured contained in the sample is determined from the average value of the calculated ratio ( _LT / _LC ). If the calculated average value of the ratio (ΔL _T /ΔL _C ) is smaller than 1, a graph in which the average value of the ratio (ΔL _T /ΔL _C ) is linearly approximated against the concentration of the substance to be measured is used as the calibration curve, and the concentration of the substance to be measured contained in the sample is determined from the average value of the calculated ratio (ΔL _T /ΔL _C );
If the average value of the ratio (ΔL _T /ΔL _C ) is greater than 1, a graph obtained by logarithmically approximating the average value of the ratio (ΔL _T /ΔL _C ) versus the concentration of the substance to be measured is used as the calibration curve, and the concentration of the substance to be measured contained in the sample is determined from the average value of the calculated ratio (ΔL _T /ΔL _C );
3. The immunochromatographic quantitative method according to claim 2, wherein, when the average value of the ratio (ΔL _T /ΔL _C ) is 1 , one of the two graphs is used as the calibration curve, and the concentration of the substance to be measured contained in the sample is determined from the average value of the calculated ratio (ΔL _T /ΔL _C ). 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 section 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 light emission amount acquiring unit that divides the entire sheet width direction, which is a direction perpendicular to the flow direction, into a plurality of regions and acquires, for each region, the light emission amount (L _T ) of the light reflected from the test line of the test plate and the light emission amount (L _C ) of the light reflected from the control line;
a calculation unit that calculates the ratio ( _LT / _LC ) between the luminescence amount ( _LT ) of the test line and the luminescence amount ( _LC ) of the control line acquired by the luminescence amount acquisition unit, and calculates the average value of the ratio ( _LT / _LC ) in all of the regions;
a calibration curve setting unit that sets, as a calibration curve, a standard curve that shows the relationship between the average value of the ratio ( _LT / _LC ) and the concentration of the substance to be measured, which has been prepared in advance using samples whose concentrations of the substance to be measured are known;
a concentration acquisition unit that obtains the concentration of the measurement target substance contained in the sample from the average value of the ratio ( _LT / _LC ) calculated by the calculation unit using the calibration curve set by the calibration curve setting unit;
an output unit that outputs the concentration obtained by the concentration acquisition unit;
An immunochromatographic reader having the above structure. 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 section 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 difference (ΔL _T ) obtained by subtracting the luminescence amount (L _hT ) of the optimized baseline from the luminescence amount (L _T ) of the test line in the color value measurement range is calculated as the color value of the test line;
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 difference (ΔL _C ) obtained by subtracting the luminescence level (L _hC ) of the optimized baseline from the luminescence level (L _C ) of the control line is calculated as the color value of the control line;
For each of the regions, the ratio (ΔL _T /ΔL _C ) of the difference value (ΔL _T ) of the test line to the difference value (ΔL _C ) of the control line is calculated;
a calculation unit that calculates an average value of the ratio (ΔL _T /ΔL _C ) in all of the regions;
a calibration curve setting unit that sets, as a calibration curve, a standard curve that shows the relationship between the average value of the ratio (ΔL _T /ΔL _C ) and the concentration of the substance to be measured, which is prepared from samples in which the concentration of the substance to be measured is known;
a concentration acquisition unit that uses the calibration curve set by the calibration curve setting unit to determine the concentration of the measurement target substance contained in the sample from the average value of the ratio (ΔL _T /ΔL _C ) calculated by the calculation unit;
an output unit that outputs the concentration obtained by the concentration acquisition unit;
An immunochromatographic reader having the above structure.