JP2024059028A - Test kits and testing methods - Google Patents

Abstract

[Problem] The object of the present invention is to provide a method for easily and quickly measuring the concentration of an antibody, antigen or gene that can be detected by binding to a protein in blood or body fluid, and a drug in blood or body fluid. [Solution] This test kit includes a qualitative analysis test kit using color determination and can quantitatively calculate the amount of an antibody, and is characterized in that it includes a capillary 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 portion where the target substance is detected, and a quantitative conversion sheet. [Selected Figure] Figure 1

Description

The present invention relates to a method for easily and quickly quantifying the amount of antibodies and drug concentrations in blood and body fluids.

For example, when it comes to viruses, cervical cancer, etc., vaccination produces antibodies that are expected to acquire immunity and provide preventive effects, but it is expected that knowing whether sufficient immunity has been acquired or whether the vaccine's effectiveness is still sustained will make it easier to decide whether or not to re-take the vaccine. Also, for example, anticancer drug therapy is used to treat cancer, and it is expected that knowing the concentration of the anticancer drug in the blood will allow adjustments to be made to the timing and dosage of the anticancer drug.

Conventionally, test kits that apply immunochromatography are known as test kits that perform immunoassays.

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. One example of a test kit using immunochromatography is a test kit in which a test strip with a labeled antibody and a capture antibody immobilized thereon is enclosed in a rectangular case with a sample drop 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 changes color to determine the presence or absence of bacteria (Patent Document 1).

In addition, a method has been established to detect IgG antibodies and IgM antibodies, which are types of immunoglobulins in the blood that specifically react to the nucleocapsid protein and spike protein of the virus (SARS-CoV-2) that causes the novel coronavirus infection (COVID-19), and a test kit is known that can distinguish whether an antibody has been 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).

However, while conventional test kits can make qualitative determinations, such as determining whether or not a person is pregnant, whether or not a person has a bacterial infection, and whether or not a person has colon cancer, they cannot quantitatively determine, for example, whether a vaccine has provided immunity and has a preventive effect. For this reason, there is a demand for a simple kit that can make such quantitative determinations.

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 allows for rapid and simple monitoring 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 treatment, as well as the blood concentration of drugs for epilepsy, multiple myeloma, acute leukemia, intrathecal dissemination, etc.

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

Patent Publication 2014-23264 Patent No. 6976621 WO2014/119544

The object of the present invention is to provide a test kit that can easily and quickly measure the concentration of an antibody, antigen, or gene that can be detected by binding to a protein in blood or a body fluid, and the concentration of a drug in blood or a body fluid.

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 test kit comprising: 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 portion where the target substance is detected; and a quantitative conversion sheet.
(Configuration 2)
The test kit according to the above-mentioned embodiment 1, wherein the qualitative analysis test kit based on color determination is a test kit using an immunochromatography method.
(Configuration 3)
The test kit according to configuration 1 or 2, characterized in that 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.
(Configuration 4)
The quantitative conversion sheet is a matrix sheet including a vertical axis on which the amount of blood or body fluid is indicated by a value read on scale A, and a horizontal axis on which the amount of color is indicated by a value read on scale B, and the amount of antibody is indicated at the intersection of the two lines,
The antibody amount indicated at the intersection is a basic antibody amount obtained by substituting the horizontal axis value at a predetermined vertical axis value into a calculation formula for the antibody amount obtained from the calibration curve, and a specific antibody amount calculated based on the ratio of the basic antibody amount to the basic antibody amount.

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

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.

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.

The tube for collecting blood or body fluid is preferably a transparent or semi-transparent tube that does not easily allow air to enter. It is also preferable that the diameter of the collected part does not change, but if a calibration curve, which will be described later, can be drawn, a tube whose diameter changes can also be used. In other words, 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, as will be described later, are the same. Also, 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 in by capillary action, such as a hematocrit tube. Also, if liquids, etc. can be collected, a dropper, etc. can be used. Furthermore, when collecting blood, using a tube with heparin attached to the wall of the tube can prevent blood from clotting.

Scale A may be any scale that allows the amount of blood or body fluid collected in the tube to be easily measured visually. For example, it may be a scale marked with a scale as shown in FIG. 2. In FIG. 2, the scale is in 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, a calibration curve can be drawn as described later, so a scale marked with a scale 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 marked in a manner according to the content volume. In this way, by incorporating a scale marked 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.

Scale B is a scale that can measure the degree of coloring of the test line (T line) of a qualitative analysis test kit. Usually, a qualitative analysis test kit judges the presence or absence of a target substance to be detected by the coloring of the T line. The coloring of the T line indicates the presence of the target substance in 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 the shade of the coloring is assigned a numerical value for each level, 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.

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 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 Figure 1, the vertical axis represents 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 represents 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 determined from a calibration curve, which will be described later. Note that, for convenience, "blood" is written on the vertical axis in Figure 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 on 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 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, thereby knowing 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.

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. Examples of drugs include anticancer drugs, antiepileptic drugs, enzymes (enzyme levels that are deficient due to genetic diseases), and vitamins.

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 portion where the detection target is detected, and a quantitative conversion sheet.

According to the present invention, it is possible to easily and quickly quantitatively determine the concentration of an antibody, antigen, or gene that can be detected by binding to a protein in blood or body fluid, and a drug in blood or body fluid. By being able to easily and quickly quantitatively determine the concentration, it is possible to quickly determine the timing of vaccination and the timing of administration of a therapeutic drug such as an anticancer drug.

FIG. 13 is a diagram showing an example of a conversion table of the present invention. FIG. 2 is a diagram showing an example of scale A of the present invention. FIG. 13 is a diagram showing an example of scale B of the present invention. FIG. 1 is a diagram showing an example of a calibration curve prepared using serum for preparing the conversion table of the present invention. FIG. 1 is a diagram showing an example of a calibration curve for creating a conversion table of the present invention.

The following provides a detailed explanation of embodiments and examples of the test kit and test method of the present invention, but the present invention is not limited to the following embodiments and examples, and can be modified as appropriate within the scope of the object of the present invention.

(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 antibody levels. Each test specimen was measured to a length of 2 cm using 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 the specimen was left for about 15 minutes (until the antibodies in the blood reacted sufficiently with the labeling substance provided on the spreading membrane). After confirming that the C line (confirmation line) was colored, the degree of coloring of the T line was visually judged using 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.)

(Creating a quantitative conversion sheet)
The antibody amount (basic antibody amount) at each degree of coloration 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 degree of coloration 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 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.

[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 for preparing a scale by marking marks that allow the determination of length according to the length of a 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 corresponding to 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 for calculating the antibody amount at each coloration degree on scale B at any scale value on scale A from the obtained calculation formula, and further calculating the antibody amounts at other scale values on scale A from the obtained antibody amounts, thereby obtaining 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 body fluid is sampled and collected in a designated tube. The amount of collected blood or body fluid is measured using Scale A, and a designated qualitative analysis test kit is used to perform chromatographic analysis using an immunochromatography method 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 antibody amount at the intersection of the value of Scale A and the value of Scale B is read. This allows the antibody amount 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.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.
(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, the height (cm) of the collected volume is visually measured using scale A.
(Determination of the amount to be detected)
The collected blood is soaked into the developing membrane of a qualitative analysis test kit (COVID19 Ab manufactured by Welsign) using the immunochromatography method. An appropriate amount of developing solution is then dripped onto the developing membrane. It is left for about 15 minutes (until the antibodies in the blood and the labeling substance provided on the developing 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 a 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 numerical 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.

Example 5
(Urine Collection)
Urine is collected in a paper cup and then collected in a tube (for example, 2-454-21 hematocrit capillary tube, heparinized, manufactured by AS ONE).
(Measurement of collected urine volume)
Next, in order to confirm the amount of urine collected in the tube, the height (cm) of the collected volume is visually measured using scale A.
(Determination of the amount to be detected)
The collected urine is soaked into the developing membrane of a qualitative analysis test kit (COVID19 Ab manufactured by Welsign) using the immunochromatography method. An appropriate amount of developing solution is then dropped onto the developing membrane. The mixture is left for about 15 minutes (until the amount of drug in the urine and the labeling substance provided on the developing 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 a quantitative conversion sheet)
Using a quantitative conversion sheet shown in Figure 1 prepared using a previously prepared calibration curve, the amount of antibody was quantified by reading the numerical 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.

Claims (4)

A test kit that includes a qualitative analysis test kit by color determination and can quantitatively calculate the amount of antibody,
A test kit comprising: 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 portion where the target substance is detected; and a quantitative conversion sheet. 2. The test kit according to claim 1, wherein the qualitative analysis test kit based on color determination is a test kit using an immunochromatography method. 3. The test kit according to claim 1 or 2, characterized in that 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. The quantitative conversion sheet is a matrix sheet consisting of a vertical axis on which the amount of blood or body fluid read on scale A is written, and a horizontal axis on which the coloration is written on scale B, and the antibody amount is written at the intersection of the two.
The antibody amount indicated at the intersection above is the basic antibody amount obtained by substituting the horizontal axis value at the specified vertical axis value into the calculation formula for 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 above.
The test kit described in claim 1, characterized by: