Inspection kit and inspection method

Abstract

To provide a method for simply and quickly measuring antibodies, antigens and genes, which can be detected by bonding to protein in blood or body fluid, and concentration of a medicine in blood or body fluid.SOLUTION: An inspection kit includes a qualitative analysis inspection kit based on color determination, and can quantitatively calculate an amount of antibodies. The inspection kit comprises: a capillary pipe for collecting blood or body fluid; a scale A for visually measuring an amount of blood or body fluid used for inspection; a scale B for visually determining the coloration of a determination portion where a detection target is detected; and a quantitative conversion sheet.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for simply and quickly quantifying the amount of an antibody or the concentration of a drug in blood or a body fluid.

[0002] For example, in the case of viruses, cervical cancer, etc., vaccination produces antibodies that are expected to produce immunity and thus provide preventive effects, but it is expected that by knowing whether sufficient immunity has been acquired or whether the vaccine effect is still in effect, it will be possible to easily determine whether or not to re-introduce the vaccine. Also, for example, anticancer drug therapy is performed as a cancer treatment, and it is expected that by knowing the concentration of the anticancer drug in the blood, it will be possible to adjust the timing and dosage of the anticancer drug.

[0003] 2. Description of the Related Art Conventionally, as a test kit for performing an immunoassay, a test kit that applies an immunochromatography method has been known.

[0004] Immunochromatography is an immunoassay that utilizes the property of a specimen slowly flowing over a porous test strip such as a cellulose membrane while dissolving the reagent (blood collection tube phenomenon), and this property is used in pregnancy diagnosis, influenza, etc. Also, as an example of a test kit using immunochromatography, a test kit is known in which a test strip with a labeled antibody and a capture antibody immobilized thereon is enclosed in a rectangular case having a sample dropping window and a detection window for visually observing the condition of the test strip, and when bacteria are detected in the specimen, the detection part develops color to determine the presence or absence of bacteria (Patent Document 1).

[0005] In addition, a method has been established to detect IgG antibodies and IgM antibodies, a type of immunoglobulin in the blood that reacts specifically to the nucleocapsid protein and spike protein of the virus (SARS-CoV-2) that causes novel coronavirus infection (COVID-19), and a test kit is known that can distinguish whether an antibody was produced by a vaccine or by natural infection (Patent Document 2). Furthermore, a method for measuring plasma proteins in whole blood using an immunochromatographic test strip is known, which is inexpensive and simple, but only describes a qualitative measurement based on color development. It is described that the target substance in a sample is preferably measured quantitatively, but does not specifically describe a method for quantitative measurement (Patent Document 3).

[0006] However, while conventional test kits can qualitatively determine, for example, whether a person is pregnant, infected with a bacteria, or has colon cancer, they cannot quantitatively determine, for example, whether a vaccine has acquired immunity and has a preventive effect. Therefore, a simple kit capable of such quantitative determination is desired.

[0007] Furthermore, in recent years, attention has been focused on TDM (therapeutic drug monitoring), i.e. "drug therapy monitoring" or "blood drug concentration monitoring." It is believed that the action of a drug depends on the blood concentration, but no test kit that can monitor quickly and easily is known. In particular, there is a demand for a method that can easily and quickly determine the blood concentration of anticancer drugs in anticancer drug therapy, as well as the blood concentration of drugs for epilepsy, multiple myeloma, acute leukemia, intrathecal dissemination, etc.

[0008] Conventionally, to quantify antibodies in blood, blood is drawn at a hospital or the like, centrifuged to obtain serum and plasma, and then analyzed, so it takes several days to determine the antibody amount. In addition, no method is known for easily and quickly quantifying the concentration of drugs in blood, urine, sweat, tears, etc. If it were possible to easily and quickly quantitatively determine the amount of antibodies and drugs in blood, urine, sweat, tears, etc., it would be possible to immediately determine the timing of vaccination and the effective administration timing of drugs, so there has been a demand for a method for easily and quickly quantifying the amount of antibodies and drugs in blood, urine, sweat, tears, etc. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent Publication No. 2014-23264 [Patent Document 2] Patent No. 6976621 [Patent Document 3] WO2014 / 119544 Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a test kit capable of easily and quickly measuring the concentrations of antibodies, antigens or genes that can be detected by binding to proteins in blood or body fluids, and drugs in blood or body fluids.

[0011] As a result of intensive research into solving the above problems, the inventors have found a quantitative relationship between the collected body fluid and the color produced by chromatography when performing tests using various types of chromatography, and have further investigated this relationship, which has led to the completion of the present invention. The present invention provides the following: (Configuration 1) A test kit including a qualitative analysis test kit by color determination, and capable of quantitatively calculating the amount of antibody, A tube for collecting blood or body fluid, scale A for visually measuring the amount of blood or body fluid used in the test, scale B for visually determining the color of the determination part where the detection target is detected, and a quantitative conversion sheet. A test kit comprising: (Configuration 2) The qualitative analysis test kit based on color determination is a test kit using an immunochromatography method. The test kit according to configuration 1, characterized in that (Configuration 3) The detection target is an antibody, an antigen, or a gene that can be detected by binding to a protein in the collected blood or body fluid, and a drug in the blood or body fluid. 3. The test kit according to claim 1 or 2. (Configuration 4) The quantitative conversion sheet is a matrix sheet including a vertical axis on which the amount of blood or body fluid read on scale A is recorded, and a horizontal axis on which the coloration is recorded on scale B, and the antibody amount is recorded at the intersection of the two scales, The antibody amount indicated at the intersection is the basic antibody amount obtained by substituting the horizontal axis value at the vertical axis value in the formula for calculating the antibody amount obtained from the calibration curve, and the specific antibody amount calculated based on the ratio of the basic antibody amount to the basic antibody amount. The test kit according to configuration 1, characterized in that

[0012] The qualitative analysis test kit based on color determination may be, for example, a commercially available qualitative analysis test kit using immunochromatography. Color determination also includes fluorescence, luminescence, and the like.

[0013] The blood may be whole blood, or serum or plasma may be used. In addition, examples of body fluids include, but are not limited to, saliva, urine, tears, sweat, and the like.

[0014] When collecting blood or a body fluid, a collector can be used. For blood, a puncture needle or the like can be used as the collector. Blood can be collected by inserting the puncture needle into a fingertip. Body fluids may also be collected using paper cups, test tubes, cotton swabs, etc.

[0015] The tube for collecting blood or body fluid is preferably a transparent or semi-transparent tube that does not easily allow air to enter. In addition, it is preferable that the diameter of the collected part does not change, but if a calibration curve described later can be drawn, even a tube whose diameter changes can be used. That is, the inner diameter of the tube is not particularly limited, and it is sufficient that an appropriate amount of collected blood or body fluid can be used for detection with a qualitative analysis kit using an immunochromatography method. Usually, the entire amount of blood or body fluid collected in the tube is used for detection with a qualitative analysis kit, so that the amount of blood or body fluid collected in the tube and the amount of blood or body fluid used for analysis are the same, as described later. In addition, a scale may be attached to the wall of the tube so that the collected amount can be read. Furthermore, it is preferable to use a capillary tube in which liquids, etc. are sucked by capillary action, such as a hematocrit tube. In addition, if liquids, etc. can be collected, a dropper, etc. can be used. Furthermore, when collecting blood, a tube with heparin attached to the wall of the tube can be used to prevent blood from coagulating.

[0016] Scale A may be any scale that allows the amount of blood or body fluid collected in a tube to be easily measured visually. For example, it is a scale with graduations as shown in FIG. 2. In FIG. 2, the unit of the graduations is cm. This is because the tube used in the test kit is fixed, and the collected amount (the amount used for analysis and the collected amount are the same) can be determined by indicating the amount of blood or the like in the tube by the height. If the inner diameter of the tube is constant in the tube, a calibration curve can be drawn as described later, so a scale with graduations as shown in FIG. 2 can be used as scale A. On the other hand, if the inner diameter of the tube varies depending on the location, the scale should be graduated in a manner according to the content volume. In this way, by incorporating a scale with graduations in advance in accordance with the tube for collecting blood or body fluid contained in the test kit into the test kit, the amount of collected blood or body fluid can be easily measured visually.

[0017] Scale B is a scale that can measure the degree of coloring of the test line (T line) of a qualitative analysis test kit. Normally, a qualitative analysis test kit judges the presence or absence of a target substance to be detected by the coloring of the T line. If the T line changes color, it indicates that the target substance is present in the blood or body fluid. However, it is expected that the greater the amount of the detection target, the greater the degree of coloring (shade of color) of the test line of the qualitative analysis test kit. The above-mentioned scale B can easily determine the coloring level of the T line by visually checking the relationship between the amount of the detection target and the coloring concentration. For example, scale B can be a scale in which a numerical value is assigned to each level of the shade of the coloring, as shown in FIG. 3. Then, the scale is used by comparing the coloring in the actual test with this scale and reading the numerical value indicating the color of the scale that corresponds to the coloring in the test.

[0018] The quantitative conversion sheet is a matrix sheet having a vertical axis on which the amount of blood or body fluid read on scale A is recorded, and a horizontal axis on which the amount of color read on scale B is recorded, and the antibody amount is recorded at the intersection of the two axes, The antibody amount shown at the intersection is the basic antibody amount obtained by substituting the horizontal axis value at a given vertical axis value into the calculation formula for the antibody amount determined from the calibration curve, and the specific antibody amount calculated based on the ratio to the basic antibody amount. Specifically, for example, as shown in FIG. 1, the vertical axis indicates the amount of blood (the height of the blood in the tube used in the test) as read on scale A, and the horizontal axis indicates the color value as read on scale B. The amount of antibody can be quantitatively determined by reading the value written at the intersection of scale A and scale B. The value for this quantitative determination is obtained from a calibration curve, which will be described later. Note that, for convenience, "blood" is written on the vertical axis in FIG. 1, but the same can be applied to body fluids. The indicators measured by Scale A and Scale B can be easily quantitatively determined using the quantitative conversion sheet shown in Figure 1. If the absolute amount of blood or body fluid dropped onto the qualitative analysis test kit is large, the color level of the T line will be high, and if the absolute amount of blood or body fluid dropped onto the kit is small, the color level of the T line will be low. In other words, to calculate the concentration of the target substance per unit volume, the amount of blood or body fluid to be dropped is measured in advance using Scale A, the color level of the T line is determined using Scale B, and the intersection of Scale A and Scale B on the quantitative conversion sheet is read to know the concentration of the target substance in the blood or body fluid. The quantitative conversion sheet is prepared by carrying out a general quantitative analysis of the detection target, creating a calibration curve, and calculating values. Furthermore, scale A, scale B and the quantitative conversion sheet may be separate sheets or may be integrated into one sheet.

[0019] The detection targets are antibodies, antigens, genes, or proteins that can be detected by binding to proteins in the collected blood or body fluids, and drugs in the blood or body fluids, such as anticancer drugs, antiepileptic drugs, enzymes (enzyme levels that are deficient due to genetic diseases), and vitamins.

[0020] The test kit of the present invention includes a tube for collecting blood or body fluid, scale A for visually measuring the amount of collected blood or body fluid, scale B for visually determining the color of the determination area where the target substance is detected, and a quantitative conversion sheet. Effect of the Invention

[0021] According to the present invention, it is possible to easily and quickly quantitatively determine the concentrations of antibodies, antigens, or genes that are detectable by binding to proteins in blood or body fluids, and of drugs in blood or body fluids. By being able to easily and quickly quantitatively determine the concentrations, it is possible to quickly determine the timing of vaccination and the timing of administration of therapeutic drugs such as anticancer drugs. [Brief description of the drawings]

[0022] [Figure 1] FIG. 13 is a diagram showing an example of a conversion table of the present invention. [Diagram 2] FIG. 2 is a diagram showing an example of scale A of the present invention. [Diagram 3] FIG. 13 is a diagram showing an example of scale B of the present invention. [Figure 4] FIG. 1 is a diagram showing an example of a calibration curve prepared using serum for preparing a conversion table of the present invention. [Diagram 5] FIG. 1 is a diagram showing an example of a calibration curve for creating a conversion table of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The following provides a detailed description of embodiments and examples of the test kit and test method of the present invention. However, the present invention is not limited to the following embodiments and examples, and can be practiced with appropriate modifications within the scope of the object of the present invention.

[0024] (Creating a calibration curve) In the present invention, a calibration curve is determined for preparing the quantitative conversion sheet. First, serum with known antibody levels (antibodies against the SARS-CoV-2 virus that causes COVID-19) was diluted to prepare test specimens with various concentrations of antibody levels. Each test specimen was measured to a size of 2 cm on scale A and soaked into the spreading membrane of a qualitative analysis test kit (COVID19Ab manufactured by Welsign). An appropriate amount of spreading liquid was then dropped onto the spreading membrane and left for about 15 minutes (until the antibodies in the blood and the labeling substance provided on the spreading membrane react sufficiently). After confirming that the C line (confirmation line) was colored, the degree of coloring of the T line was visually judged on scale B. The visual judgment was performed three to four times and the average was calculated. The results are shown in Table 1. After that, a regression analysis was performed using the concentration of the test specimen and the average degree of coloring read from scale B to create a calibration curve. The results are shown in Figure 4. Then, from this calibration curve, a calculation formula (1) was obtained to calculate the antibody amount (concentration) at the degree of coloring read from scale B. This calculation formula can be suitably used when confirming coloration using a qualitative analysis test kit that employs immunochromatography, but does not exclude the use of other chromatographic methods. y=53.594e 0.8073x ...Equation (1) (In the formula, x represents the degree of coloration, and y represents the amount of antibody in the test specimen.)

[0025] [Table 1]

[0026] (Creating a quantitative conversion sheet) The antibody amount (basic antibody amount) at each coloring degree of scale B when scale A is 2 cm was calculated from formula (1) and listed in the table. Furthermore, for the antibody amount (specific antibody amount) at each coloring degree when scale A is 0.5 cm to 4 cm, since it is inversely proportional to the antibody amount at 2 cm in terms of volume, for example, 1 / 2 times the antibody amount at 2 cm was listed in the 4 cm column, and twice the antibody amount at 2 cm was listed in the 1 cm column, and the table was filled in from 0.5 cm to 4 cm to complete the quantitative conversion sheet.

[0027] [Method for producing the test kit of the present invention including the quantitative conversion sheet] The test kit of the present invention can be obtained through the following steps. A scale A preparation step of preparing a scale by marking a scale that allows the length to be determined according to the length of the tube used for collecting blood or body fluid; a scale B creating step of creating a scale B by marking the scale within a range of 1 to 10±3 in accordance with the degree of color development of the qualitative analysis test kit; A calibration curve preparation step of collecting a test sample having a known antibody concentration in advance using the tube, reading the scale A, analyzing the test sample using a qualitative analysis test kit, reading the scale B, and preparing a calibration curve using the values ​​read; A formula calculation step of obtaining a formula for calculating the amount of antibody by substituting the degree of coloration from the obtained calibration curve; and A quantitative conversion sheet creation process in which the antibody amount at each coloration degree on scale B at any scale value on scale A is calculated from the obtained calculation formula, and the antibody amounts at other scale values ​​on scale A are also calculated from the obtained antibody amounts to obtain a quantitative conversion sheet. [Method of using the test kit of the present invention] The test kit of the present invention can be used as follows. Blood or bodily fluids are sampled and collected in a designated tube. The amount of collected blood or bodily fluid is measured using Scale A, and a designated qualitative analysis test kit is used to perform chromatographic analysis using immunochromatography or the like, visually checking the color of the T-line, and the value of the scale indicating the degree of coloration is read using Scale B. Then, using a quantitative conversion sheet, the amount of antibody is read at the intersection of the value of Scale A and the value of Scale B. This allows the amount of antibody to be known quantitatively. [Action and Effect] By using the test kit of the present invention, the quantitative amount of antibodies can be known using a normal qualitative analysis test kit, so that not only can negative / positive results be judged, but also the amount of antibodies can be confirmed. Since the amount of antibodies can be judged quickly and simply, the timing of vaccination can be quickly determined, and the timing of administration of therapeutic drugs such as anticancer drugs can also be quickly determined.

[0028] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples in any way. (Examples 1 to 4) (Blood collection) Use a puncture needle (e.g., Nipro LS Lancet 28G 1.0 mm 11-166, manufactured by Nipro) to draw blood from the fingertip. Kneading the finger from the base to the tip will help blood come out. When enough blood has come out, collect it in a tube (e.g., 2-454-21 hematocrit capillary tube heparinized, manufactured by AS ONE). (Blood volume measurement) Next, to confirm the amount of blood collected in the tube, use scale A to visually measure the height (cm) of the collected volume. (Determination of the amount of detection target) The collected blood is soaked into the spreading membrane of the immunochromatography qualitative analysis test kit (Welsign COVID19Ab). Then, an appropriate amount of spreading liquid is dropped onto the spreading membrane. Leave it for about 15 minutes (until the antibodies in the blood and the labeling substance on the spreading membrane have sufficiently reacted). Confirm that the C line has changed color, and judge the degree of coloration of the T line using Scale B. (Quantitative determination using quantitative conversion sheet) Using the quantitative conversion sheet shown in Figure 1, which was created using a previously prepared calibration curve, the amount of antibody was quantified by reading the value at the intersection between the amount of blood read on scale A and the color determination read on scale B. The results are shown in Table 2. Furthermore, a graph of the verification quantitative analysis results and the amount of antibody read from the quantitative conversion sheet is shown in Figure 5.

[0029] Example 5 (Urine collection) Urine is collected in a paper cup and then collected in a tube (e.g., 2-454-21 hematocrit capillary tube, heparinized, manufactured by AS ONE). (Measurement of collected urine volume) Next, to check the amount of urine collected in the tube, visually measure the height (cm) of the collected volume using scale A. (Determination of the amount of detection target) The collected urine is soaked into the spreading membrane of the immunochromatographic qualitative analysis test kit (Welsign COVID19Ab). Then, an appropriate amount of spreading liquid is dropped onto the spreading membrane. Leave it for about 15 minutes (until the amount of drug in the urine and the labeling substance on the spreading membrane have sufficiently reacted). Confirm that the C line has changed color, and judge the degree of coloration of the T line using Scale B. (Quantitative determination using quantitative conversion sheet) Using the quantitative conversion sheet shown in Figure 1, which was created using a previously prepared calibration curve, the amount of antibody was quantified by reading the value at the intersection between the amount of blood read on scale A and the color determination read on scale B. The results are shown in Table 2. Furthermore, a graph of the verification quantitative analysis results and the amount of antibody read from the quantitative conversion sheet is shown in Figure 5.

[0030] [Table 2]

Claims

1. A test kit that includes a qualitative analysis test kit by color determination and can quantitatively calculate the amount of antibody, A tube for collecting blood or body fluid, a scale A for visually measuring the amount of blood or body fluid used in the test, a scale B for visually determining the color of the determination part where the detection target is detected, and a quantitative conversion sheet. A test kit comprising:

2. The qualitative analysis test kit based on color determination is a test kit using an immunochromatography method. The test kit according to claim 1,

3. The detection target is an antibody, an antigen, or a gene that can be detected by binding to a protein in the collected blood or body fluid, and a drug in the blood or body fluid.

3. The test kit according to claim 1 or 2.

4. The quantitative conversion sheet is a matrix sheet including a vertical axis on which the amount of blood or body fluid read on scale A is recorded, and a horizontal axis on which the amount of coloring read on scale B is recorded, and the amount of antibody is recorded at the intersection of the two scales, The antibody amount indicated at the intersection is the basic antibody amount obtained by substituting the horizontal axis value at the vertical axis value in the formula for calculating the antibody amount obtained from the calibration curve, and the specific antibody amount calculated based on the ratio of the basic antibody amount to the basic antibody amount. The test kit according to claim 1,

Images