JP3541232B2 - Method for measuring the degree of coloration of immunochromatographic test strips - Google Patents

Description

[0001]
[Industrial applications]
The present invention relates to a method for measuring the degree of coloration of an immunochromatographic test strip.
[0002]
[Prior art]
An immunochromatographic test piece is a technique in which an antibody that causes an antigen-antibody reaction with a specific substance (test substance) in a sample is fixed at a predetermined position in a developing layer, and a predetermined color is generated when the target substance spreads at that position. Things.
[0003]
The use of the immunization method allows for the quantification of a smaller amount of concentration as compared with a normal color test method (such as a normal urine test piece). That is, in the color test method, the measurement limit is about mg / dl, and the measurement at a concentration lower than that cannot be performed. However, the immunization method can measure 1/100 to 1/1000 of that. Therefore, depending on the concentration, some of them can be measured only by the immunoassay.
[0004]
Hereinafter, the immunization method will be described using a pregnancy reagent as an example. However, it is clear that the present invention is not limited to this.
[0005]
Pregnancy is determined based on whether human chorionic gonadotropin (hCG) is detected in urine. Since the amount is very small, an immunization method is used. According to this principle, an anti-hCG monoclonal antibody is immobilized at a predetermined position on a test piece, and anti-hCG antibody-binding particles are applied to hCG and a sample application portion (immersion portion). When urine containing hCG is applied to the sample application portion, the anti-hCG antibody-bound particles and hCG bind there to form a complex. This complex binds to the anti-hCG monoclonal antibody and the color is established at that location.
[0006]
Since the position where the anti-hCG monoclonal antibody is fixed is known in advance, if the color is developed at that position (by visual observation), it is determined to be positive (pregnancy). The color itself varies depending on the color of the anti-hCG antibody-bound particles.
[0007]
This immunization method is also suitable when combined with the paper chromatography method. The paper chromatography method utilizes the fact that various substances in a solution penetrate from a dropped (or immersed) position to a predetermined position. For example, in the above example, the sample application section and the determination section are different places, the sample and the target component penetrate from the sample application section to the determination section, and only the target component is fixed in the determination section. It is. According to this method, the color of the judging portion is hardly influenced by the color of the dropped sample itself or other components, and the judgment becomes easier.
[0008]
As described above, the presence or absence (qualitative) of a very small amount of a component can be determined by using the immunization method. Further, since the anti-hCG antibody-bound particles and the anti-hCG monoclonal antibody bind very selectively, components other than the target component can be determined. There is no possibility of measuring erroneously. Therefore, a very reliable determination can be made.
[0009]
[Problems to be solved by the invention]
However, the above-described immunization method has the following disadvantages.
When the immunochromatographic test strip is visually determined, if the concentration of the target test substance is near the cutoff level for discriminating between positive and negative, the reproducibility of the determination becomes poor and individual differences appear. Even when the determination can be made surely, there are many cases where it is desired to know the degree of positive or negative, and knowing the degree is useful for diagnosis. If we try to do this visually, there is a method to determine it by comparison with a color sample, but it is more difficult for the naked eye to determine the degree of coloration, as opposed to the presence or absence of coloration in the determination of positive and negative. And the reproducibility of the judgment is worse than the judgment of positive and negative, and individual differences appear, and it is unlikely that a useful result in diagnosis will be obtained.
[0010]
On the other hand, if the immunochromatographic test strip is judged by the apparatus, poor reproducibility in visual judgment and individual differences should be improved. As a method of realizing the apparatus, the reflectance of the colored portion is measured using a conventional reflectometer for a colored test piece. However, this method measures a uniformly colored test piece, and only measures the reflectance of a spot having a diameter of about 3 mm. This method assumes that the lightness (reflectance) of the spot is correlated with the density. However, in the immunochromatography method, there is no such a relationship, and even when the concentration of the target substance increases, the reflectance of the spot portion hardly changes, and the area for coloring often increases. In this case, the concentration cannot be obtained.
[0011]
Therefore, it is conceivable to sequentially move an optical measurement system (or a test piece) for measuring the reflectance at the spot and obtain the reflectance one-dimensionally. From this one-dimensional reflectance data group, it is conceivable to determine the overall coloration degree.
[0012]
However, this not only has the disadvantage that the apparatus becomes complicated and takes a long time for measurement, but it is still a reflectometer having an area for the spot itself. Even if the spot is narrowed down to about 1 mm, it is not enough. Only resolution can be obtained.
[0013]
Furthermore, even if a one-dimensional reflectance data group is obtained, the chromatographic image of the test piece is not the same quality as a chromatographic image of a gas chromatograph, etc. There is a problem peculiar to the immunochromatographic test piece, such as image distortion due to undulation, and it is impossible to implement such an apparatus as described above.
[0014]
Therefore, there has been a demand in the art for a simple immunization method that allows easy quantification and also enables automation for measuring a large number of samples.
[0015]
[Means for Solving the Problems]
In view of such a current situation, the present inventor has completed the method of the present invention as a result of earnest research, and the feature of the method is to quantitatively or qualitatively determine the concentration of a specific substance in a sample. Analyzing the colored immunochromatographic test strip using an image sensor, obtaining a gradation image corresponding to the brightness of each pixel of the image sensor, and then correcting the brightness distortion of the gradation image. And noise removal processing to obtain the coloration degree by obtaining the area or height of the coloration portion of the image-converted gradation image.
[0016]
The present invention is a method for determining the degree of coloration of an immunochromatographic test strip, and converting the degree of coloration to the concentration of a target substance can be performed in another arithmetic unit even if performed in the same apparatus. May be input by inputting the degree of the operation.
[0017]
Because of the immunochromatography method, it is natural that the test strip is coated with particles to which the antibody is immobilized and which binds to the test substance with the antibody. These particles are components that develop color and are usually applied to the sample application (dropping, dipping, etc.) portion. It is natural that the antibody and the antibody-bound particles differ depending on the target substance. Those which cause an antigen-antibody reaction and those which form a binding complex. In a multi-item simultaneous measurement in which a plurality of different target substances are simultaneously measured, each antibody-bound particle that reacts with the target substance is mixed and applied to a sample dropping portion, and each antibody that reacts similarly is determined. The parts are fixed in different places.
[0018]
In the present invention, the degree of coloration is automatically read by a machine without visual observation. The reading method will be described. An antibody is usually immobilized in a band on the colored portion of the immunochromatographic test strip. Therefore, the coloration is also band-shaped. In the multi-item simultaneous measurement, a plurality of band-like colors are shown at intervals. Using an optical system having an image sensor, an image is taken including the colored portion of the immunochromatographic test piece.
[0019]
The optical system usually includes an illumination light source, a lens, and a filter. The image of the immunochromatographic test piece illuminated forms an image on the image sensor by the lens. Therefore, an electric signal corresponding to the brightness of each portion of the test piece is obtained at each pixel of the image sensor, and the electrical signal corresponding to the brightness of each pixel of the image sensor is input to a signal processing circuit or a computer. Obtain a gradation image. The filter uses a wavelength having a large spectral absorption characteristic in a colored portion, and is inserted between the light source and the test piece or between the test piece and the image sensor. When a light source having a specific wavelength such as an LED is used, or when a color image sensor having a spectral function is used, a filter is unnecessary. The position resolution of the image sensor usually has a resolution of several tens of μm to several hundreds of μm in terms of dimensions on a test piece.
[0020]
A gradation image is a group of pixels having gradations. The gradation is a stage of brightness, which is determined by the resolution at which an analog signal is converted into a digital signal, and if converted by 8 bits, becomes 256 gradations.
[0021]
As an image sensor, a CCD is most suitable in terms of price and performance. As described above, the image sensor may be a color image sensor or a monochrome image sensor. Further, a two-dimensional image sensor such as a video camera or a one-dimensional image sensor may be used.
[0022]
In the case of imaging with a one-dimensional image sensor, a gradation image corresponding to a chromatographic image can be obtained by imaging in a direction perpendicular to the band-like coloration. When the image is picked up by the two-dimensional image sensor, a two-dimensional gradation image is naturally obtained. However, this image can be considered as a plurality of two-dimensionally collected one-dimensionally picked-up gradation images. Therefore, the subsequent processing can be simplified by taking the average value of the pixels arranged in the belt-shaped color direction and converting it to a one-dimensional gradation image. Thereby, in the case where the coloration is uneven, or in the case where there is dust or a scratch on the test piece surface, the effect can be reduced as compared with the case where the image is captured by the one-dimensional image sensor. Even when imaging is performed with a one-dimensional image sensor, a two-dimensional gradation image can be obtained by performing imaging a plurality of times while moving the image sensor or the test piece in a belt-shaped color direction.
[0023]
Next, image conversion and image measurement will be described. Here, image conversion refers to brightness distortion correction processing and noise removal processing. The brightness distortion means that when an ND paper or the like having a uniform scattering surface is imaged, the gradation of each pixel of the obtained gradation image is not uniform and distortion occurs. The brightness distortion appears when the distribution of the irradiation light amount and the distribution of the irradiation angle component on the test piece surface of the illumination optical system are not uniform. Further, in the imaging optical system, the brightness of the corresponding pixel decreases as the distance from the optical axis deviates from the brightness of the pixel on the optical axis of the lens on the image sensor surface. Further, brightness unevenness appears due to a variation in sensitivity between pixels of the image sensor and a bias in the image of the gain of the signal processing circuit. Making the brightness of each pixel of a gradation image having such brightness distortion uniform is referred to as brightness distortion correction processing. Generally, correction is performed by multiplying each pixel by a coefficient obtained in advance to convert the pixel into a gradation image without distortion.
[0024]
The noise removal processing is generally a smoothing processing. In the case where the above-described two-dimensional gradation image is converted into a one-dimensional gradation image, the average value of the pixels arranged in the band color direction is calculated. This is the averaging process to be obtained. Moving average processing is suitable for a one-dimensional gradation image in a direction perpendicular to the band-like coloration.
As a special noise removal process, there is a selective noise removal process. In a two-dimensional gradation image, pixels arranged in a band-like color direction should normally show the same gradation. However, if there is a clear coloration unevenness, or a clear test piece surface dust or scratch, This is not the case. By performing a statistical calculation such as obtaining a standard deviation for the gradation of a pixel in a band-shaped color direction, the degree of noise can be known, and for a large noise, a pixel indicating a gradation that is outside the allowable deviation value. Can be selectively removed, so that the effect can be greatly reduced.
[0025]
Determining the area or height of the colored portion means determining the baseline of the figure in the gradation image and determining the height or area of the peak. The obtained numerical value is used as a color index. Of course, in the multi-item simultaneous measurement, the coloration index corresponding to each item is obtained. The color index obtained here corresponds to the color intensity of the color portion of the test piece, which generally almost reflects the concentration of the target substance. Therefore, usually, the concentration of the target substance is quantitatively or qualitatively determined from a calibration curve of the concentration and the coloration index determined in advance.
[0026]
As described above, imaging using an image sensor having a high resolution makes it possible to measure an immunochromatographic test specimen as a chromatographic image, which was difficult with a conventional reflectometer, and furthermore, it is possible to obtain an image of the obtained image. By performing the image conversion and the image measurement of the toned image, the disturbance of the chromatographic image can be corrected and reduced, and the color index corresponding to the color degree can be obtained. Further, even in simultaneous measurement of multiple items that are confusing and susceptible to errors, the measurement can be performed quickly and accurately.
[0027]
【Example】
Next, a test piece and a measuring device used in the method of the present invention will be described based on an embodiment shown in the drawings. FIG. 1 shows an example of a test piece 1 used in the present invention, in which (a) is a plan view and (b) is a cross-sectional view. The whole is covered with plastic, and windows are provided in the sample dropping unit 2 and the determination unit 3 so that the sample absorbing part is exposed. The sample dropping portion 2 is a non-absorbent nonwoven fabric 4, followed by an antibody fixing portion (judgment portion) of a filter paper 5, and an antibody is fixed to a predetermined band. In this case, a check antibody for checking that the sample has reached the antibody is also fixed on the right side. If the right side is colored, it is confirmed that the sample has passed through the left fixing portion. In addition, antibody binding particles that form a complex with the target substance are applied to the sample dropping portion. The reason for using such a test piece is to facilitate application to an automatic device and to accelerate development and absorption.
[0028]
Next, an example of an apparatus used in the method of the present invention will be described. This device continuously and automatically measures the concentration of a specific trace component in a sample contained in a sample container. Here, an example will be described in which hCG for pregnancy diagnosis is quantified. The apparatus includes a display, a key panel, a printer, a CCD, a CPU, a test piece introduction unit, and a light source. These devices themselves do not need to be special, and are similar to conventional automatic urine analyzers, and the present invention itself does not have such features. However, the difference is that an image sensor is provided instead of the conventional light receiving element.
[0029]
Next, measurement and data processing of this device will be described. As a result of the reaction, a thin red-purple line appears near the center of the judgment window only in the positive sample. In the optical system, an LED of a specific wavelength (565 nm) that senses red-violet color is used for illumination, and measurement is performed by a CCD image sensor. The procedure is as follows.
(1) Reading a reaction image A test image is two-dimensionally imaged by a CCD image sensor. The light intensity of each pixel is calculated on ten lines (width 0.05 mm) in the horizontal direction (perpendicular to the color band) to obtain ten one-dimensional gradation images. By multiplying each pixel by a coefficient obtained by imaging ND paper of N9.0, ten one-dimensional gradation images in which lightness distortion has been corrected are obtained. The ten images are averaged to form one one-dimensional gradation image.
The moving average is taken and noise is removed. The noise-removed image is used as a read reaction image. This reaction image is shown in FIG. The downward peak on the left side of this graph is a hCG coloring peak, and the peak on the right side is a check peak. In this case, since the peak also appears on the right side, the sample has reached a predetermined position.
(2) The position of the judgment window is searched from the analysis read reaction image. Since the line of red purple appears in the position of ± 2mm from the center of the decision window if there is hCG in the urine, the center -2mm position of the determination window and S _0, the position of 2mm and E ₀ from there. The area of a portion surrounded by a straight line connecting S ₀ and E ₀ and a reaction line is determined. Then, the _{S 0} is moved 0.1mm increments, sequentially calculates the same area. The maximum value of the determined 21 areas is defined as the displacement of this sample. According to this method, even a gentle peak can be reliably determined.
[0030]
Until now, it was only possible to determine the presence or absence of hCG in the determination of pregnancy, but with the device of the present invention, it was possible to sufficiently quantify it.
[0031]
【The invention's effect】
According to the method of the present invention described in detail above, the measurement (reading) of the degree of coloration is collectively captured as an image, so that it can be performed instantaneously. Further, the determination of a trace component, which is an advantage of immunochromatography, can be easily, automatically, reliably performed, and quantified without requiring skilled skills. Further, since the data is read as an image and then subjected to data processing, an accurate value can be read with excellent resolution.
[Brief description of the drawings]
FIG. 1 shows an example of a test piece used in the present invention, in which (a) is a plan view and (b) is a cross-sectional view.
FIG. 2 is a graph showing absorption.
[Explanation of symbols]
1 Test piece 2 Dropping window 3 Judgment window 4 Nonwoven fabric 5 Filter paper

Claims (1)

An analysis method for quantitatively or qualitatively determining the concentration of a specific substance in a sample, in which an image of a colored immunochromatographic test piece is taken using an image sensor, and the image is taken in correspondence with the brightness of each pixel of the image sensor. A tone image is obtained, and then a brightness distortion correction process and a noise removal process are performed on the tone image, and the area or height of the color portion of the image-converted tone image is determined to obtain the color degree. Characteristic method for measuring the degree of coloration of immunochromatographic test strips.

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