JP2000266752A - Immunochromatography measurement device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately measure an antigen in an aqueous sample solution quantitatively by finding a concentration of an object to be detected in the sample on the basis of a degree of coloring at a specific wavelength of an image provided by a correction means. SOLUTION: A specimen sample is added by a quantity (0.1-2 ml) decided in compliance with a shape into a sample introducing part in an immunochromatography device, and this is let stand still horizontally for 3-20 minutes for forming a color image in a determination part. The formed color image is photographed by means of an image sensor such as a CCD camera 6, and a background is corrected after chromatics and lightness of the provided image is regulated, so that the color image is emphasized. A coloring degree of the emphasized image is measured on an X-constituent and a Y-constituent, and an integral value of them (an integral value of the X-constituent and the Y-constituent) is found. Then, using information previously stored in a personal computer about a relative relationship between an integral value and a concentration of the object to be measured, and a concentration value corresponding to the integral value is found. In this way, a concentration can be found quantitatively by using the immunochromatography device.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a detecting device for detecting microorganisms in water and a diagnostic device for detecting a small amount of a marker substance in a body fluid. More specifically, the present invention is intended to improve the performance of immunochromatography that enables simple and rapid measurement.

[0002]

2. Description of the Related Art Immunochromatography is a measurement method that utilizes a substance (antibody) that specifically reacts with a substance to be measured as a material of a detection means, utilizing the high specificity of antigen-antibody reaction and detection sensitivity. . This method is currently widely used as a base technology for in vitro diagnostics. Among them, a pregnancy diagnostic agent is a typical immunochromatography device, the typical structure of which is described in, for example, US Pat.
No. 040.

[0003] The structure and operation of the simplest immunochromatography will be described below using a pregnancy tester as an example. FIG. 1 shows the structure of a porous carrier used in a typical immunochromatography device. The porous carrier 1 is
Nitrocellulose is most widely used because of its ease of immobilizing antibodies. However, if the whole is made of nitrocellulose, the flow rate of the sample is remarkably reduced. Therefore, it is preferable that only the judging section is made of nitrocellulose, and the other parts are made of glass filter paper or the like having a higher flow rate of the sample.
The sample introduction part 2 at one end of the carrier is constituted by a porous carrier such as glass filter paper, which has a property such that the sample is quickly absorbed, but the holding force is weak, and the sample quickly flows to the reaction part. I have. When the porous carrier is accommodated in a hollow case, the sample introduction section 2 is configured to be relatively long, and by exposing a part of the sample introduction section from the case, urine can be directly introduced into the sample introduction section. Alternatively, when the sample introduction part is also housed in the case, a hole can be made in the case, and the sample can be added to the sample introduction part from there.

[0004] The labeling section 3 is prepared by impregnating glass filter paper with an aqueous solution of an anti-hCG antibody adsorbed on colloidal gold or colored latex, and then freeze-drying. The determination unit 4 is prepared by dropping an aqueous solution of an antibody into an arbitrary shape on a nitrocellulose sheet, drying the solution, and washing the solution. Due to the very strong non-specific adsorption properties of nitrocellulose, antibodies are supported on nitrocellulose. After immobilizing the antibody, surface treatment with, for example, bovine serum albumin (BSA) or the like can be performed to prevent background color development due to non-specific adsorption during the detection reaction. This operation is called blocking. The liquid absorbing part 5 at the other end of the carrier is
In order to quickly absorb the excess sample, a material having excellent liquid absorbing power and liquid absorbing capacity and glass filter paper are used, for example.

[0005] As a sample, female urine is used in a predetermined amount (usually 0.1 to 2) according to the shape of the chromatography device.
ml) is added to the sample introduction part 2. The sample passes through the labeling section 3, passes through the determination section 4, and moves toward the liquid absorbing section 5. At this time, when the sample passes through the labeling section 3, the labeled antibody carried on the labeling section dissolves in the water of the sample and starts moving with the sample. Thereafter, the dissolved labeled antibody passes through the determination unit 4. Here, based on the specific binding reaction between the antigen and the antibody, if the determination is positive, the immobilized antibody and the labeled antibody react with each other via the antigen in the determination section 4, and the determination section is colored. If negative, no reaction occurs, and the labeled antibody passes through the determination unit 4 and is absorbed by the liquid absorbing unit 5. Thereby, the presence or absence of the antigen in the aqueous sample solution can be determined.

[0006]

As described above, the conventional immunochromatography device has been used only for visual qualitative judgment. This is because although the coloration in the determination part of immunochromatography has shading corresponding to the antigen concentration, a considerable amount of background occurs in the determination part, so it is necessary to accurately determine the antigen concentration from the degree of coloration. Because it was difficult. However, depending on the antigen to be measured, there are cases where it is desired to know the degree of positive or negative, or when it is meaningful to measure only after the concentration of the antigen is known. Therefore, there has been a demand for the development of an apparatus that enables quantitative measurement of an immunochromatographic device.

[0007] In view of the above, it is an object of the present invention to provide an immunochromatographic measurement device capable of accurately and quantitatively measuring an antigen in an aqueous sample solution.

[0008]

SUMMARY OF THE INVENTION In order to solve the above-mentioned problems, the present invention provides a color image formed on an immunochromatography device by dropping a sample specimen, with respect to an image obtained by using an image sensor to obtain brightness or brightness. Means for correcting saturation, or means for background correction for performing background removal processing on the image, or integrating the coloration at a specific wavelength of the image obtained by the above means with respect to the X component and the Y component. Provided is an immunochromatography apparatus having at least one of means for determining the concentration of a measurement target in a sample based on a value.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In recent years, in which image processing by a computer is performed very daily, it is seemingly self-evident that an image captured by a computer using an image sensor is arbitrarily enhanced. . However, in the immunochromatography targeted by the present invention, when a sample is actually added and operated, unevenness in color development due to non-uniformity in the flow of the sample, optical characteristics of the used antibody labeling agent, information on impurities, etc. However, the information and know-how that can be obtained for the first time by actually conducting immunochromatographic research are diverse. On the other hand, image processing is generally well-known by being installed in general-purpose software, but image mode conversion (grayscale, RGB, monochrome two-tone, etc.), level, tone curve, color balance, contrast There are numerous types such as, hue, saturation, brightness, color replacement, gradation inversion, averaging, and a wide variety of other special effect filters. If all these functions are installed, you can control the image freely.However, the more image processing, the more complicated the device, the longer the processing time and the higher the cost. It can be considered.

[0010] Therefore, rather than a simple combination of known techniques of immunochromatography and image processing, it is necessary to select and optimize the minimum and necessary functions based on sufficient R & D experience related to immunochromatography. Therefore, it is important to improve the reliability of data, and this is one point of the present invention.

In the present invention, an image sensor refers to a device that converts color chromatography into digital or analog electrical signals that can be captured by a computer. For example, a CCD camera or an image scanner can be used.

Further, the analyzing apparatus of the present invention performs correction of lightness / saturation and background correction on a color image, integrates the color degree of this image with respect to the X component and the Y component, and obtains the result. The values obtained correspond to the information on the correlation between the degree of coloration and the concentration, which is stored in advance, and the concentration of the object to be measured in the sample can be obtained.For example, a combination of a personal computer and a display for display It consists of.

The immunochromatography measuring apparatus of the present invention has the configuration shown in FIG. A sample sample is added to the sample introduction section of the immunochromatography device in an amount determined according to the shape (usually 0.1 ml to 2 ml), and left standing horizontally for 3 minutes to 20 minutes to form a color image on the determination section. Let it form. The formed color image is captured using an image sensor, for example, a CCD camera, and after adjusting the saturation and brightness of the obtained image, the background is corrected to enhance the color image. The degree of coloration of the emphasized image is measured for the X component and the Y component, the integrated value (integrated value for the X component and the Y component) is obtained, and the integrated value and the integrated value stored in the analyzer in advance are measured. Using the information on the correlation with the object concentration, a value of the concentration corresponding to the integral value is obtained. By doing so, using an immunochromatographic device,
The concentration of the measurement target in the sample can be quantitatively determined.

[0014]

The present invention will be described in more detail with reference to the following examples. In this example, a CCD camera was used as an image sensor, a personal computer was used as an analyzer, and Salmonella was used as an object to be measured.

A chromatographic device which had been subjected to a color reaction in advance was inserted into the imaging section. The chromatography device is positioned and fixed by a spring so as not to fluctuate in the imaging unit. At the same time, the position and the focus of the CCD camera are fixed so that the entire determination section of the chromatography device can be imaged. In addition, illumination that is optimized so that no shadow is reflected by the CCD camera is provided. In this embodiment, white LEDs arranged in a ring are used as the most suitable illumination. In this state, by clicking a button displayed in a window on the computer, a program was set up so that the following steps were performed. Here, the process is started by clicking on the computer screen, but the microswitch may be operated by inserting the chromatography device into the imaging unit, and a start signal may be transmitted to the computer. The process and the modules of the program will be described below in the order in which the data is processed.

<Operation Process of Apparatus> 1) Image Capture The CCD camera captures an image of the determination unit and transmits an image signal as an analog signal in NTSC format. Here, in the case of signal transmission, using a digital format can maintain high image quality because there is no deterioration due to DA-AD conversion, but the transmission speed is generally higher in an analog format. The transmission method should be determined based on the cost and performance of each component constituting the device, and largely depends on the product performance at the time of development.

The signal sent from the CCD camera passes through a still image capture board and is sent to a computer.
The handling of signals in a computer is, of course,
It depends on the OS used. Here, Microsoft Windows95 was used. The image signal is stored in a memory as a matrix in which one data corresponds to one pixel of the CCD camera. Since the image data is large, the data is first stored in the storage device. In the present embodiment, the number of data transmitted to the computer is determined by an external device such as a hard disk in order to operate the device under the restriction of the performance of the capture board and the camera and the memory. In the configuration of this embodiment, the horizontal direction 80
0 data and 600 data in the vertical direction are transmitted. Further, in this embodiment, since the screen setting of the OS is set to 24-bit color, data of 24 bits, that is, 3 bytes is transmitted for one pixel. In a development environment of a so-called high-level language such as VisualBasic of MicroSoft, for example, such data is 600000 from 00000 to FFFFFF.
It is represented by a hexadecimal digit. Here, it is possible to decompose the data into R, G, and B data by extracting a hexadecimal number from the lower two digits at a time. For example, FF0000 is red, 00FF00 is green, 0000
FF is blue, 000000 is black, FFFFFF is white, and so on. In this case, each color has 256 gradations.

2) Preview A sub-window for displaying images is provided on the operation window,
The captured image is displayed in the sub window. This allows the operator to intuitively confirm that the color development of the chromatography device has been successfully taken. Here, in order to reduce the size of the program, the data of each pixel (pixel) of the image displayed in the preview may be used as it is in the subsequent processing, but in the present embodiment, the preview and the data for information processing are used. Was treated differently. As a result, the speed of display and the compactness of the display area are realized by setting the preview screen to the minimum number of pixels for confirming an image. On the other hand, for processing, a data array with no loss of image information
It can be used for calculations without being conscious of the user.

3) Background processing A threshold was set in advance, each data was compared with this value, and data below the threshold was treated as white. It is effective to increase the concentration of the labeled antibody in order to increase the sensitivity in immunochromatography, but on the other hand, if the concentration is increased, the background develops a color, which may cause erroneous judgment in visual confirmation. By performing the background processing, it is possible to improve the sensitivity while preventing the erroneous determination. That is, a threshold value is set in advance, and data is compared with this threshold value one by one. Data indicating a density lower than the threshold is corrected to white data. This allowed the elimination of the effects of overall coloring, which were not based on the original immune response. In the present embodiment, a predetermined value is used as the threshold value. However, in the determination unit of the chromatography device, an area in which it is known that coloration is not performed is set in advance, and coloring at this location is determined by the threshold value. And the same operation as above can be performed.

4) Color Tone Processing In addition to the method of expressing colors using the three primary colors of light, that is, R, G and B, hue (H), saturation (S), and brightness (B) Can also be expressed as In order to perform the color tone processing of the present embodiment, a method of expressing in H, S, and B is effective.
Then, after the data transmitted from the camera was decomposed into three elements of R, G, and B by the above-described method, the data was passed through a routine for converting the data into H, S, and B values. Hue is usually 0 for red
Hereinafter, the degree is represented by the angle of a circle that advances in the order of yellow, green, cyan, blue, and magenta by 60 degrees. Here, since the color image in this embodiment has a hue very close to magenta, a range of ± 30 degrees is set around magenta, that is, around 330 degrees, and only data within this range is set. It was targeted for subsequent processing. Next, the sample with the deepest color was created in advance, and after capturing this image, the optimal values were obtained by mutually examining the saturation and lightness coefficients so that the color was 100% and the white color was 0%. Was experimentally determined and the data was factored in the program to enhance the saturation and brightness.

5) Meaning of Data The data processed by the above operation is a set of data that has a value from 0 to 100% at this stage, and is meaningless on inspection. First, all data values were summed. This processing corresponds to integrating a variable called density appearing in the image of the determination unit in the X-axis and Y-axis directions of the image. Finally, a sample in which the concentration was changed for each object to be measured was measured, and the correlation between the experimentally obtained concentration and the value obtained by the integration processing in this section was obtained to obtain a coefficient. By multiplying this coefficient by the integral value of the image of the sample sample of unknown concentration, the image information could be converted to the concentration of the measurement object. Note that the above operation is performed only in a computer program. In practice, the operator simply inserts the chromatographic device and clicks a button on the screen to display the measured sample concentration on the screen. Is displayed.

<Measurement of Salmonella> The results of actual measurement of Salmonella are shown below.

1) Correlation between the integrated value of coloration and the concentration of Salmonella The concentrations of Salmonella were 0, 105, 106, and 1, respectively.
After adding 250 μl of the 07, 108, and 109 cells / ml sample to the sample introduction unit 6 and allowing it to stand in a horizontal state for 10 minutes, the degree of coloration of the judgment unit was measured by the above-described method. The correlation with the concentration of Salmonella in the sample was determined. The result is shown in FIG.

2) Measurement of sample sample of unknown concentration A sample sample of unknown concentration was measured using the correlation described above. FIG. 4 shows the degree of coloration (integral value) of the determination part measured for each sample and the concentration of Salmonella determined from the value.

[0025]

As described above, the use of the apparatus of the present invention enables quantitative measurement by immunochromatography conventionally used only for qualitative judgment. In addition, by performing image enhancement suitable for immunochromatography, weak points of chromato (a weak color development in a low concentration region and a background color development) can be compensated, and as a result, its performance can be improved. This makes it possible to measure many substances that have been difficult to measure by immunochromatography, for example, various markers in blood, etc., because of background, coloration, and the like.

[Brief description of the drawings]

FIG. 1 is a perspective view showing a conventional immunochromatography device.

FIG. 2 is a diagram showing a configuration of an immunochromatography measurement device of the present invention.

FIG. 3 is a graph showing the relationship between the degree of coloration and the concentration of Salmonella in a determination unit.

FIG. 4 is a diagram showing the results of measurement of Salmonella concentration using a sample sample of unknown concentration.

 ──────────────────────────────────────────────────の Continued on the front page (72) Inventor Masato Hirai 1006 Kadoma Kadoma, Osaka Pref.Matsushita Electric Industrial Co., Ltd. Term (reference) 2G045 AA28 BA11 CB21 FA11 FA29 FB03 FB06 GC12 JA01 JA02 JA04 4D017 AA01 AA11 BA03 CA11 DA03 EA05 EB10

Claims (5)

[Claims] 1. A means for correcting the brightness and saturation of an image obtained by using an image sensor to pick up a color image formed on an immunochromatography device by dropping a sample, and subjecting the image to background removal processing. Immunochromatography comprising at least one of means for background correction to be performed, and means for determining the concentration of a detection target in a sample based on the degree of coloration at a specific wavelength of an image obtained by the correction means. measuring device. 2. The method according to claim 1, further comprising the steps of: integrating a color degree of an image corrected by said brightness and saturation correction means and said background correction means with respect to an X component and a Y component;
The immunochromatography measurement device according to claim 1, wherein the concentration of the measurement target in the sample is determined. 3. The method according to claim 1, wherein information indicating the relationship between the density of the object to be measured and the measured value of the degree of coloration is stored therein, and the density of the object to be measured corresponding to the degree of coloration is obtained using the information. The immunochromatographic measurement device according to claim 1 or 2, wherein: 4. An area where coloration based on an immune reaction does not occur, that is, comparison areas 1 and 2, is set on the upstream side and the downstream side of the flow of the sample with reference to the coloration section.
3. The immunochromatography measurement apparatus according to claim 1, wherein the background of the sample is compared with time to determine the flow state of the sample, and measurement of the concentration of the detection target is started. 5. In addition to a region where coloration occurs due to an immune reaction via the measurement target, there is a region where the color is formed when the sample passes regardless of the presence or absence of the measurement target. 3. The immunochromatography measurement apparatus according to claim 1, wherein the flow state of the sample is sufficiently determined by observing the chromaticity over time, and the measurement of the concentration of the detection target is started.