JP7532856B2 - Immunochromatographic test piece and measurement method using the same - Google Patents

Description

The present invention relates to an immunochromatographic test piece for measuring a substance to be measured contained in a measurement sample by immunochromatography, and a measurement method using the same. More specifically, the present invention relates to an immunochromatographic test piece with high measurement accuracy, and a measurement method using the same.

In recent years, the term POCT has been gaining attention in the medical field. POCT stands for Point Of Care Testing, and refers to clinical testing performed by medical professionals at the patient's side. Unlike clinical testing performed in central laboratories at large hospitals, POCT tests provide instantaneous test results on the spot, and as such, POCT is becoming more widespread for a wide range of testing items.

A typical example of POCT is immunochromatography. Immunochromatography is an immunoassay in which a measurement sample is developed in a porous body by capillary action from one end of the porous body on which an antibody that specifically binds to the substance to be measured contained in the measurement sample is immobilized, and in the process, the labeled substance to be measured and the antibody bind through an antigen-antibody reaction, accumulating over time and producing a localized color to determine the presence or absence of the substance to be measured in the measurement sample.

The advantages of immunochromatography include rapid results, simple operation, and low cost. In-vitro diagnostics such as pregnancy tests and influenza diagnostics that take advantage of these advantages are in widespread use worldwide. Conventional immunochromatography generally involved visual judgment (qualitative evaluation), but in recent years, technology has been developed that quantifies the amount of the substance to be measured contained in the measurement sample using analytical equipment such as an immunochromatography reader that measures color intensity.

One of the methods for quantifying a substance to be measured using immunochromatography is the sandwich method using an antigen-antibody reaction. In the sandwich method, two types of antibodies with different epitopes for the substance to be measured are used. One antibody forms a test line as a capture antibody immobilized in a line on the surface of a porous body. The other antibody is used as a detection antibody (hereinafter also referred to as a test line detection reagent) sensitized with detection particles such as gold colloid, colored latex particles, and fluorescent particles in order to detect the test line. In addition, an antibody that specifically captures the test line detection reagent is immobilized in a line at a position different from the test line on the surface of the porous body to form a control line. The substance to be measured in the measurement sample is developed from one end (upstream side) of the porous body with the addition part (drop part) of the measurement sample solution of the immunochromatography test piece as the upstream side, moves while forming an immune complex with the test line detection reagent, comes into contact with the capture antibody on the test line, and is captured and colored. The free test line detection reagent that was not captured on the test line is captured by the capture antibody of the control line and colored. The amount of the substance being measured can be quantified by using an immunochromatography reader or other device to measure the intensity of these colors.

Conventional immunochromatography has a mechanism for capturing free test line detection reagent that is not captured on the test line with the capture antibody in the control line, so there was a problem that the color intensity of the control line changes depending on the concentration of the substance to be measured. For example, if a high concentration of the substance to be measured is present in the measurement sample, a large amount of the test line detection reagent forms an immune complex with the substance to be measured and is captured by the upstream test line, so the color intensity of the test line becomes high. In other words, there was a problem that the color intensity of the control line becomes low because there is a small amount of free test line detection reagent. On the other hand, if the substance to be measured is present in only a low concentration in the measurement sample, there is also a small amount of test line detection reagent that forms an immune complex with the substance to be measured, so a large amount of free test line detection reagent is captured by the control line, resulting in a problem that the color intensity of the control line becomes high.

In order to accurately measure the substance to be measured contained in the measurement sample by immunochromatography, an immunochromatographic test piece may be used in which the color intensity of the control line reflects the amount of the measurement sample (or test line detection reagent) developed into the porous body, and the color intensity of the control line may be used to correct for variations in the amount of the measurement sample developed into the porous body due to individual differences in the immunochromatographic test piece. In this case, it is desirable that when a constant amount of the measurement sample (or test line detection reagent) is developed into the porous body, the color intensity of the control line is always constant.

Patent Documents 1 and 2 disclose a technique that uses an immunochromatographic test piece having detection particles (hereinafter also referred to as control line detection reagent) labeled with a low molecular weight compound and a control line in which an antibody that specifically captures the low molecular weight compound is immobilized linearly on the surface of a porous body, and an antigen-antibody reaction proceeds independently between the test line and the control line, thereby stabilizing the color intensity of the control line without being affected by the concentration of the substance to be measured in the measurement sample. In the above invention, the control line reflects to some extent the amount of the measurement sample (or the test line detection reagent) flowing into the porous body, so that the substance to be measured contained in the measurement sample can be measured with a certain degree of accuracy by correcting the color intensity of the test line with the color intensity of the control line. However, since the test line and the control line are usually immobilized at different positions in the porous body, there is a difference in the time until the test line detection reagent is captured by the test line and the time until the control line detection reagent is captured by the control line, and the correction is insufficient.

International Publication No. 2017/065213 International Publication No. 2017/094825

The present invention aims to provide an immunochromatographic test piece for measuring a target substance contained in a measurement sample by immunochromatography, and a measurement method using the same. More specifically, the present invention aims to provide an immunochromatographic test piece with high measurement accuracy, and a measurement method using the same.

As a result of intensive research to solve the above problems, the inventors discovered that by using an immunochromatographic test piece in which a test line is placed downstream of a control line and a conjugation pad containing a test line detection reagent is placed downstream and a conjugation pad containing a control line detection reagent is placed upstream, the measurement accuracy is improved compared to conventional techniques, and the present invention was completed. The inventors discovered that by placing the conjugation pad containing the test line detection reagent downstream of the conjugation pad containing the control line detection reagent, the difference in the retention time until each reagent is captured by a capture substance such as an antibody is reduced, enabling more accurate correction, and the present invention was completed.

That is, representative aspects of the present invention are as follows.
(1) An immunochromatographic test piece in which a sample pad, a conjugation pad A, a conjugation pad B, a membrane, and an absorbent pad are arranged in this order,
Each of the members has a partial overlapping portion,
The membrane has a control line on the upstream side and a test line on the downstream side,
The conjugation pad A contains a control line detection reagent,
The conjugation pad B contains a test line detection reagent;
An immunochromatographic test strip used to quantify the target substance contained in a measurement sample.
(2) The immunochromatographic test piece according to (1), wherein the control line and the test line are spaced apart from each other by 1 mm or more and 15 mm or less.
(3) The immunochromatographic test piece according to (1) or (2), wherein the difference between the distance X from the upstream end of the conjugation pad A to the control line and the distance Y from the upstream end of the conjugation pad B to the test line is less than 10 mm.
(4) A method for quantifying a substance to be measured contained in a measurement sample using the immunochromatographic test piece described in any one of (1) to (3) above, by carrying out the following steps (i) to (iii) in order:
Step (i): A step of mixing a measurement sample with a diluent; Step (ii): A step of dropping the mixture of step (i) onto a sample pad and allowing it to spread on a test strip by capillary action; Step (iii): A step of measuring the measurement target substance based on the color intensity of the test line and the control line.

The immunochromatographic test strip of the present invention and the measurement method using the same allow independent antigen-antibody reactions to proceed in the test line and control line, stabilizing the color intensity of the control line and reducing the difference in residence time between the time until the test line detection reagent is captured in the test line and the time until the control line detection reagent is captured in the control line, thereby enabling highly accurate measurement of the substance to be measured contained in the measurement sample. In addition, by placing the control line upstream, it is possible to shorten the time until measurement is completed compared to using a normal test strip.

FIG. 1 is a (top) view showing an example of an immunochromatographic test piece. FIG. 1 is a side view showing an example of an immunochromatographic test piece.

In the present invention, the measurement sample is not particularly limited, but examples include biological samples such as blood, lymph, cerebrospinal fluid, sweat, urine, tears, saliva, skin, mucous membranes, and hair. In addition to whole blood, blood serum, blood cells, or plasma obtained by centrifuging blood can be used as the blood sample. Furthermore, the measurement sample is not limited to human-derived biological samples, and biological samples from mammals such as dogs, cats, and cows are also applicable.

In the present invention, the items to be measured are not particularly limited, but include, for example, HbA1c, C-peptide, insulin, m-aluminum, Cys-C, NGAL, Troponin1, D-dimer, NT-proBNP, CK-MB, Myoglobin, h-FABP, hs-CRP, PSA, AFP, CEA, FOB, Ferritin, PCT, CRP, SAA, ASO, hCG, LH, TSH, FSH, T3, T4, Vitamin D, etc., influenza A/B, Dengue, HBV, HCV, HIV, Syphilis, Malaria, H. Infectious diseases include H. pylori, Rota, Chlamydia, Strep A, Adeno, Zika, Chikungunya, and RSV.

In the present invention, the immunochromatographic test piece is arranged in the following order, with the application part (dropping part) of the measurement sample solution of the immunochromatographic test piece on the upstream side: a sample pad having the application part, conjugation pad A, conjugation pad B, a membrane, and an absorption pad. Next, the immunochromatographic test piece of the present invention will be described with reference to the drawings. In Figures 1 and 2, 1 indicates the sample pad, 2 indicates the conjugation pad A, 3 indicates the conjugation pad B, 4 indicates the membrane, 5 indicates the absorption pad, 6 indicates the backing sheet, 7 indicates the control line, 8 indicates the test line, and 9 indicates the adhesive sheet.

In Figures 1 and 2, the immunochromatographic test piece is in the form of a long, thin strip with a width of 3 to 5 mm (preferably about 4 mm) and a length of 40 to 100 mm (preferably about 60 mm). A control line 7 is formed on the upstream side of the membrane 4 of the immunochromatographic test piece, in which a capture substance that specifically binds to a low molecular weight compound described below is immobilized in a linear shape. Also, a test line 8 is formed on the downstream side, in which a capture substance that specifically binds to a substance to be measured is immobilized in a linear shape. Furthermore, the conjugation pad 2 of the immunochromatographic test piece carries a control line detection reagent composed of detection particles labeled with a low molecular weight compound, and the conjugation pad 3 carries a test line detection reagent composed of detection particles sensitized with a detection antibody that specifically binds to a substance to be measured.

In the present invention, the average particle diameter of the detection particles is not particularly limited, but is preferably 10 to 1000 nm, and more preferably 10 to 500 nm. If the average particle diameter of the detection particles is large, downstream development will be slow, and the measurement time will be long. In addition, the particles will be easily captured at any site on the membrane, and the background itself may become colored, making the color development at the test line and control line unclear. On the other hand, if the average particle diameter of the detection particles is small, in the case of a test line detection reagent, the amount of detection antibody that can be physically adsorbed or chemically bound will decrease, and the measurement sensitivity may decrease. In addition, in the case of a control line detection reagent, the amount of low molecular weight compounds that can be labeled will decrease, and correction may be insufficient.

In the present invention, the detection particles are not particularly limited, but colored particles and fluorescent particles can be used. Examples of colored particles include metal particles, latex particles, cellulose particles, etc. Examples of metal particles include gold colloids, silver colloids, platinum colloids, palladium colloids, gold nanorods, gold nanoplates, silver nanoplates, etc. Examples of latex particles include those made of materials such as polystyrene, polymethyl methacrylate, and acrylic acid polymers. Examples of fluorescent particles include those made of materials such as polystyrene, polymethyl methacrylate, polyvinyl toluene, and silica, and examples of fluorescent dyes include fluorescein and its derivatives, rhodamine and its derivatives, and cyanine and its derivatives.

Among these, it is preferable that the detection particles in the test line detection reagent and the detection particles in the control line detection reagent are both colored particles, and more preferably they are the same color. Here, the same color means that the maximum absorption wavelength is within ±50 nm. In addition, for example, if at least one of the two detection particles is a fluorescent particle, the immunochromato reader for detecting the fluorescence becomes large and expensive, which is not preferable from the viewpoint of POCT. In addition, if the two detection particles are different colors, for example, when measuring with a general light-emitting diode (hereinafter sometimes abbreviated as LED)-photodiode (hereinafter sometimes abbreviated as PD) immunochromato reader, multiple LED-PD combinations corresponding to the colors of the test line and control line are required, and the device becomes large and expensive, which is not preferable from the viewpoint of POCT.

In the present invention, the detection antibody in the test line detection reagent is not particularly limited as long as it can specifically bind to the substance to be measured. For example, if the substance to be measured is hCG, an anti-hCG antibody can be used, and if the substance to be measured is HbA1c, an anti-Hb antibody or an anti-HbA1c antibody can be used. The detection antibody may be a commercially available product or may be produced by a known method. In addition, the antibody may be a monoclonal antibody or a polyclonal antibody, and the molecular size is not particularly limited.

The method of binding the detection antibody and the detection particles in the test line detection reagent is not particularly limited, but sensitization by physical adsorption through hydrophobic bonds or chemical bonding through covalent bonds is preferable, and physical adsorption is more preferable because it is easy to operate and inexpensive. In addition, reactive active groups may be introduced into the detection particles to improve the sensitization efficiency. Examples of reactive active groups include, but are not limited to, carboxyl groups, amino groups, aldehyde groups, thiol groups, epoxy groups, and hydroxyl groups. Among these, carboxyl groups and amino groups are preferable. In the case of a carboxyl group, a covalent bond can be formed with the amino group of the ligand using a carbodiimide.

The low molecular weight compound in the control line detection reagent is not particularly limited, but is preferably biotin or digoxigenin, more preferably biotin, because they are relatively inexpensive, easy to obtain, and have a proven track record in the field of protein labeling.

The test line detection reagent and the control line detection reagent are preferably blocked with a blocking protein. The blocking protein is not particularly limited, and examples thereof include Blocking Peptide Fragment (hereinafter sometimes abbreviated as BPF), a protein derived from a microorganism, bovine serum albumin (hereinafter sometimes abbreviated as BSA), casein, and the like, which are proteins derived from animals. The blocking protein may be a commercially available product, or may be produced by a known method.

In the present invention, the capture substance immobilized on the control line 7 is not particularly limited as long as it can specifically bind to the low molecular weight compound of the control line detection reagent. For example, when the low molecular weight compound is biotin, an anti-biotin antibody or avidin can be used, and when the low molecular weight compound is digoxigenin, an anti-digoxigenin antibody can be used. The capture substance may be a commercially available product or may be produced by a known method, and the molecular size is not particularly limited. In addition, when the antibody is used, it may be a monoclonal antibody or a polyclonal antibody.

In the present invention, the capture substance immobilized on the test line 8 is not particularly limited as long as it can specifically bind to the substance to be measured. For example, if the substance to be measured is hCG, an anti-hCG antibody can be used, and if the substance to be measured is HbA1c, an anti-Hb antibody or an anti-HbA1c antibody can be used. It is preferable that the detection antibody and the capture substance have different binding sites for the substance to be measured. The capture substance may be a commercially available product or may be produced by a separately known method, and the molecular size is not particularly limited. In addition, in the case of an antibody, it may be a monoclonal antibody or a polyclonal antibody.

The material of the sample pad 1 is not particularly limited as long as it can rapidly absorb the measurement sample and then spread to the downstream conjugation pads 2 and 3, membrane 4, and absorption pad 5. Examples of the material include cellulose filter paper or nonwoven fabric, glass filter paper or nonwoven fabric, polyester filter paper or nonwoven fabric, and polyethylene filter paper or nonwoven fabric. Among these, cellulose filter paper is preferred. The thickness of the sample pad 1 is preferably 0.1 to 2.0 mm, and more preferably 0.2 to 1.0 mm. If the thickness is small, the flow of the measurement sample downstream may become uneven, resulting in a decrease in measurement accuracy. On the other hand, if the thickness is large, downstream spread may be slow and the measurement time may be long. In addition, a large amount of measurement sample is required for downstream spread.

The material of the conjugation pads 2 and 3 is not particularly limited as long as it can hold the control line detection reagent and the test line detection reagent in a dry state and can quickly release both detection reagents as the measurement sample develops downstream. Examples of such materials include cellulose filter paper or nonwoven fabric, glass filter paper or nonwoven fabric, polyester filter paper or nonwoven fabric, and polyethylene filter paper or nonwoven fabric. Among these, glass filter paper is preferred. The thickness of the conjugation pads 2 and 3 is preferably 0.1 to 2.0 mm, and more preferably 0.2 to 1.0 mm. If the thickness is small, the desired amount of the control line detection reagent and the test line detection reagent may not be held in a dry state. On the other hand, if the thickness is large, downstream development may be slowed and the measurement time may be long. In addition, the amount of measurement sample required for downstream development increases.

The material of the membrane 4 is not particularly limited as long as it can spread the measurement sample uniformly with high accuracy. Examples of the material include membranes made of cellulose, cellulose derivatives, nitrocellulose, cellulose acetate, polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, or nylon. Among these, membranes made of nitrocellulose are preferred.

The material of the absorbent pad 5 is not particularly limited as long as it can quickly absorb the measurement sample developed from the upstream and then hold it without backflowing, and examples of such materials include cellulose filter paper or nonwoven fabric, glass filter paper or nonwoven fabric, polyester filter paper or nonwoven fabric, and polyethylene filter paper or nonwoven fabric. Among these, cellulose filter paper is preferred. The thickness of the absorbent pad 5 is preferably 0.2 to 5.0 mm, and more preferably 0.5 to 2.0 mm. If the thickness is small, the measurement sample once absorbed in the absorbent pad may flow back to the membrane side depending on the amount of measurement sample dropped. On the other hand, if the thickness is large, the size of the immunochromatographic test piece and the housing case covering the immunochromatographic test piece will also become large, which is not preferable from the perspective of POCT.

The method of carrying the control line detection reagent on the conjugation pad 2 and the method of carrying the test line detection reagent on the conjugation pad 3 are not particularly limited, and can be prepared, for example, by uniformly applying, spraying or impregnating each detection reagent onto each pad, and then drying for a certain period of time in a thermostatic chamber at an appropriate temperature. The amount of the detection reagent applied is not particularly limited, but is preferably 5 to 50 μL per 1 cm of line length. Next, it is preferable to dry after the application. The drying temperature is not particularly limited, but is preferably 20°C to 80°C, and more preferably 20°C to 60°C. The drying time varies depending on the drying temperature, but is usually 5 to 120 minutes.

The method for immobilizing the capture substance forming the test line and the capture substance forming the control line in a linear shape on the membrane 4 is not particularly limited, and can be produced, for example, by applying a fixed amount of the capture substance forming the test line and the capture substance forming the control line to different positions on the line, and then drying for a fixed time at an appropriate temperature in a thermostatic chamber. The application amount of both capture substances is not particularly limited, but is preferably 0.1 to 2 μL per 1 cm of line length. Next, it is preferable to dry after the application. The drying temperature is not particularly limited, but is preferably 20°C to 80°C, and more preferably 20°C to 60°C. The drying time varies depending on the drying temperature, but is usually 5 to 120 minutes.

In the present invention, the immunochromatographic test strip is prepared by attaching the prepared membrane 4 near the center of the adhesive sheet 9, then attaching the conjugation pad 3 to the upstream end of the membrane 4 with a partial overlap, and then attaching the conjugation pad 2 to the upstream end of the conjugation pad 3 with a partial overlap. Similarly, the sample pad 1 is attached to the upstream end of the conjugation pad 2 with a partial overlap, and then the absorption pad 5 is attached to the downstream end of the membrane 4 with a partial overlap, and then cutting into strips of a certain width. The control line 7 and test line 8 may be prepared after the test strip is prepared, or they may be prepared before the test strip is prepared.

In the present invention, when each component constituting the immunochromatographic test piece (sample pad, conjugation pad A, conjugation pad B, membrane, and absorbent pad) is partially overlapped, the length of the overlapping portion is not particularly limited, but is preferably about 1 to 5 mm. Note that the overlapping portions are simply in contact with each other and are not bonded.

In the present invention, the control line 7 and the test line 8 are preferably spaced apart by 1 mm or more and 15 mm or less. If the distance is too close, the downstream side of the control line 7 and the upstream side of the test line 8 are close to each other, which reduces the measurement accuracy when reading the lines or requires a highly accurate reading device. If the distance is too far, the upstream control line is set close to the conjugation pad. In that case, the measurement is performed immediately after the sample (specimen) flows from the conjugation pad to the membrane, when the flow is not stable, and the measurement accuracy of the control line is reduced. Therefore, the distance is more preferably 2 mm or more and 10 mm or less, and even more preferably 3 mm or more and 10 mm or less.

In the present invention, by making the distance from the upstream end (upstream end) of conjugation pad A to the control line and the distance from the upstream end (upstream end) of conjugation pad B to the test line approximately equal, the difference between the migration time of the immune complex between the measurement target substance and the test line detection reagent (the time until the immune complex binds to the capture substance on the test line) and the migration time of the control line detection reagent (the time until the control line detection reagent is captured by the control line) becomes small, so that color development is stable and detection (quantification) with high accuracy is possible. Specifically, the distance X from the upstream end of conjugation pad A to the control line and the distance Y from the upstream end of conjugation pad B to the test line are preferably 15 to 35 mm, and more preferably 18 to 33 mm. In addition, the difference between X and Y is preferably less than 10 mm, more preferably less than 7 mm, even more preferably less than 5 mm, and even more preferably 2 mm or less.

In the present invention, the length of the conjugation pad A excluding the overlapping portion is preferably 2 mm to 15 mm, more preferably 3 mm to 15 mm. By setting the length of the conjugation pad A excluding the overlapping portion within the above range, it becomes easier to adjust each of the migration times without changing the overall length of the test piece (i.e., within a typical test piece length). In addition, detection can be performed using a general-purpose housing case or chromatograph reader.

The immunochromatographic test piece may be housed in a suitable plastic housing case having at least a first opening for dropping a measurement sample onto the sample pad 1, and a second opening for measuring the control line 7 and the test line 8 on the membrane 4.

In the present invention, it is more preferable to measure the concentration of the substance to be measured from a corrected value obtained by dividing the color intensity of the test line by the color intensity of the control line, taking into account the development spots of the measurement sample.

In the present invention, the method for measuring the test line and control line of the immunochromatographic test strip is not particularly limited, and a commercially available immunochromatographic reader may be used, or an immunochromatographic reader may be manufactured by a separately known method. The detection system is not particularly limited, but for example, an LED-FD, LED-CMOS, or LED-CCD can be used.

The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. The evaluation methods used in the specification are as follows.

Example 1
Preparation of test line detection reagent for hCG measurement
5.0 mg/mL anti-hCG-β monoclonal antibody (5014, Medix Biochemica) was adjusted to 1.0 mg/mL with distilled water (Otsuka distilled water, Otsuka Pharmaceutical Co., Ltd.). Next, 100 μL of 1.0 wt% cellulose particles (NanoAct (registered trademark), RE2: Dark Red, red, average particle size 340 nm, Asahi Kasei Corporation), 900 μL of 10 mM Tris buffer (204-07885, Wako Pure Chemical Industries, Ltd.) (pH 7.0), and 100 μL of the 1.0 mg/mL (0.1 wt%) anti-hCG-β monoclonal antibody were added to a 15 mL centrifuge tube and stirred with a vortex. Next, the mixture was placed in a low-temperature incubator (IN604, Yamato Scientific Co., Ltd.) adjusted to 37 ° C. and left to stand for 120 minutes. Next, 12 mL of a blocking solution (pH 8.0) consisting of 1.0 wt% casein (030-01505, manufactured by Wako Pure Chemical Industries, Ltd.) and 100 mM borate buffer (021-02195, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was left to stand for 60 minutes in a low-temperature incubator adjusted to 37 ° C. Next, using a centrifuge (MX-307, manufactured by Tommy Seiko Co., Ltd.), a rack-in rotor (TMA-300, manufactured by Tommy Seiko Co., Ltd.), and a rack (AR510-04, manufactured by Tommy Seiko Co., Ltd.), centrifugation was performed at 13,000 G for 15 minutes at 25 ° C. to precipitate the antibody-sensitized cellulose particles, and the supernatant was removed. Next, 12 mL of a washing solution (pH 10.0) consisting of 50 mM borate buffer was added, and the mixture was treated for 10 seconds with an ultrasonic disperser (UH-50, manufactured by SMT Co., Ltd.). Next, using a centrifuge, a rack-in rotor, and a rack, centrifugation was performed at 13,000 G for 15 minutes at 25 ° C. to settle the antibody-sensitized cellulose particles, and then the supernatant was removed. Next, 2.0 mL of a coating solution (pH 9.2) consisting of 15 wt % sucrose (196-00015, manufactured by Wako Pure Chemical Industries, Ltd.), 0.2 wt % casein, and 62 mM borate buffer was added, and the mixture was treated with an ultrasonic disperser for 10 seconds to prepare a test line detection reagent 1 for hCG measurement.

Preparation of control line detection reagent 16 mg of D-biotin (04822-91, Nacalai Tesque) and 1,000 μL of dimethyl sulfoxide: DMSO (08904-14, Nacalai Tesque) were added to a 5 mL microtube and stirred with a vortex to obtain a D-biotin solution. Next, 200 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride: EDC (15022-86, Nacalai Tesque), 200 mg of N-hydroxysuccinimide: NHS (18948-02, Nacalai Tesque), and 1,000 μL of distilled water were added to a 5 mL microtube and stirred with a vortex to obtain an EDC/NHS solution. Next, 1,000 μL of the D-biotin solution and 1,000 μL of the EDC/NHS solution were mixed, and then placed in a low-temperature incubator adjusted to 25° C. and left to stand for 15 minutes to obtain a D-biotin/EDC/NHS solution. Next, 100 mg of bovine serum albumin:BSA (A7906, Sigma-Aldrich) and 1,000 μL of distilled water were added to a 5 mL microtube and stirred with a vortex to obtain a BSA solution. Next, 1,000 μL of the D-biotin/EDC/NHS solution and 1,000 μL of the BSA solution were mixed, and then placed in a low-temperature incubator adjusted to 25° C. and left to stand for 30 minutes to obtain a D-biotin-BSA solution. 100 μL of 1.0 wt % cellulose particles (NanoAct (registered trademark), RE2: Dark Red, red color, average particle size 340 nm, manufactured by Asahi Kasei Corporation), 900 μL of 10 mM Tris buffer solution (204-07885, manufactured by Wako Pure Chemical Industries, Ltd.) (pH 7.0), and 100 μL of the D-biotin-BSA solution were added to a 15 mL centrifuge tube and stirred with a vortex. Next, the tube was placed in a low-temperature incubator (IN604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 37° C. and allowed to stand for 120 minutes. Next, 12 mL of a blocking solution (pH 8.0) consisting of 1.0 wt% casein (030-01505, manufactured by Wako Pure Chemical Industries, Ltd.) and 100 mM borate buffer (021-02195, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was left to stand for 60 minutes in a low-temperature incubator adjusted to 37 ° C. Next, using a centrifuge (MX-307, manufactured by Tommy Seiko Co., Ltd.), a rack-in rotor (TMA-300, manufactured by Tommy Seiko Co., Ltd.), and a rack (AR510-04, manufactured by Tommy Seiko Co., Ltd.), centrifugation was performed at 13,000 G for 15 minutes at 25 ° C., and the D-biotin-BSA-sensitized cellulose particles were precipitated and the supernatant was removed. Next, 12 mL of a washing solution (pH 10.0) consisting of 50 mM borate buffer was added, and the mixture was treated for 10 seconds with an ultrasonic disperser (UH-50, manufactured by SMT Co., Ltd.). Next, using a centrifuge, a rack-in rotor, and a rack, centrifugation was performed at 13,000 G for 15 minutes at 25 ° C. to settle the D-biotin-BSA sensitized cellulose particles, and then the supernatant was removed. Next, 2.0 mL of a coating solution (pH 9.2) consisting of 15 wt % sucrose (196-00015, Wako Pure Chemical Industries, Ltd.), 0.2 wt % casein, and 62 mM borate buffer was added, and the mixture was treated with an ultrasonic disperser for 10 seconds to prepare control line detection reagent 1.

Preparation of membrane card for hCG measurement 5.0 mg/mL anti-hCG-α monoclonal antibody (6601, manufactured by Medix Biochemica) was adjusted to 0.5 mg/mL with distilled water. Next, 1.0 mg/mL anti-biotin polyclonal antibody (A150-111A, manufactured by BETHYL) was adjusted to 0.5 mg/mL with distilled water. Next, a 60 mm x 300 mm membrane card (Hi-Flow Plus 120 Membrane Cards, water absorption speed 120 sec/4 cm, HF120, Millipore) consisting of a 20 mm x 300 mm adhesive tape part on the upstream side, a 25 mm x 300 mm membrane in the center, and a 15 mm x 300 mm adhesive tape part on the downstream side was placed 15 mm from the upstream end of the membrane. Using a Bio Jet nozzle (BHQHR-XYZ, manufactured by BIODOT), the 0.5 mg/mL anti-hCG-α monoclonal antibody was applied in an amount of 1.0 μL/cm, and then dried for 30 minutes in a dryer (WFO-510, manufactured by Tokyo Rika Kikai Co., Ltd.) adjusted to 45 ° C. to form a test line with a line width of about 1 mm. Furthermore, using a dispensing platform and a Bio Jet nozzle, the 0.5 mg/mL anti-biotin polyclonal antibody was applied in an amount of 1.0 μL/cm at a position 10 mm from the upstream end of the membrane of the membrane card on which the test line was formed, and then dried for 30 minutes in a dryer adjusted to 45 ° C. to form a control line with a line width of about 1 mm, thereby preparing a membrane card 1 for hCG measurement.

Preparation of a conjugation pad for hCG measurement The hCG measurement control line detection reagent 1 was diluted 5-fold with the coating solution and applied to the entire surface of a 7 mm x 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, manufactured by Millipore). A dispenser platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT) were used for application, and the hCG measurement control line detection reagent was uniformly applied twice with an application amount of 10.7 μL/cm, and then dried for 30 minutes in a dryer adjusted to 45 ° C. to obtain a conjugation pad A1 for hCG measurement.
On the other hand, the test line detection reagent 1 was applied to the entire surface of a 10 mm x 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, manufactured by Millipore). A dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT) were used for application, and the test line detection reagent was uniformly applied twice with an application amount of 7.5 μL/cm, and then dried for 30 minutes in a dryer adjusted to 45 ° C. to prepare a conjugation pad B1 for hCG measurement.

Preparation of immunochromatographic test strip for hCG measurement The hCG measurement conjugation pad B1 was attached to the 20 mm x 300 mm adhesive tape part on the upstream side of the hCG measurement membrane card 1 so as to overlap with the membrane by 2 mm. Next, the hCG measurement conjugation pad A1 was attached to the upstream side so as to overlap with the conjugation pad B1 by 2 mm. Furthermore, a 9 mm x 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) was attached to the upstream side so as to overlap with the conjugation pad A1 by 2 mm. Next, a 17 mm x 300 mm absorbent pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) was attached to the 15 mm x 300 mm adhesive tape portion downstream of the hCG measurement membrane card so as to overlap the membrane by 2 mm. Next, the pad was cut into a strip of 4 mm width and 60 mm length using a guillotine cutting module (CM5000, manufactured by BIODOT) to prepare an immunochromatographic test piece 1 for hCG measurement.

Preparation of diluted hCG samples Commercially available hCG antigen (30-1132, Fitzgerald) was diluted with a diluent (pH 7.0) consisting of 50 mM potassium dihydrogen phosphate (28736-75, Nacalai Tesque) and 1.0 wt% BSA to prepare diluted hCG samples of 1 IU/L and 100 IU/L.

Evaluation of hCG immunochromatographic test strip: Evaluation of accuracy The hCG immunochromatographic test strip 1 was placed on a horizontal table. Next, 100 μL of the diluted hCG sample (1 IU/L and 100 IU/L) was dispensed with a micropipette, gently dropped onto the sample pad, and allowed to stand at 25° C. for 10 minutes. Next, the reflection absorbance (mAbs) of the control line and test line on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Gold Colloid, Line, manufactured by Hamamatsu Photonics). The experimental procedure was performed with N=10 for each of the diluted hCG samples (1 IU/L and 100 IU/L). As an index of precision, the reflection absorbance (mAbs) of the test line of the diluted hCG samples (1 IU/L and 100 IU/L) was divided by the reflection absorbance (mAbs) of the control line to calculate the corrected values (1 IU/L and 100 IU/L) respectively, and then the CV (1 IU/L and 100 IU/L) (%) of the obtained corrected values (1 IU/L and 100 IU/L) at N=10 was calculated respectively. Furthermore, the average value of CV (1 IU/L) (%) and CV (100 IU/L) (%) was calculated: CV (%), and CV≦2% was evaluated as ◎ (excellent), 2%<CV≦3% was evaluated as ○ (good), 3%<CV≦5% was evaluated as △ (average), and 5%<CV was evaluated as × (bad). The accuracy of the immunochromatographic test strip 1 for measuring hCG in Example 1 was rated as ⊚ with a CV of 1.8%, indicating that highly accurate measurement was possible. The obtained evaluation results are shown in Table 1.

Evaluation of immunochromatographic test strip for measuring hCG: Evaluation of sensitivity The immunochromatographic test strip for measuring hCG 1 was placed on a horizontal table. Next, 100 μL of the diluted hCG sample (1 IU/L and 100 IU/L) was dispensed with a micropipette, gently dropped onto the sample pad, and allowed to stand at 25° C. for 10 minutes. Next, the reflection absorbance (mAbs) of the control line and test line on the membrane was measured using an immunochromatographic reader (C10060-10, measurement mode: Gold Colloid, Line, manufactured by Hamamatsu Photonics). The experimental procedure was performed with N=10 for each of the diluted hCG samples (1 IU/L and 100 IU/L). As an index of sensitivity, the difference between the average value of the reflection absorbance (mAbs) of the test line of the diluted hCG sample (100 IU/L) (N=10) and the average value of the reflection absorbance (mAbs) of the test line of the diluted hCG sample (1 IU/L) (N=10): Δ(mAbs) was calculated, and 300Abs≦Δ was evaluated as ◎ (excellent), 200≦ΔL=<300mAbs as ○ (good), and Δ<200mAbs as × (bad). The sensitivity of the immunochromatographic test strip 1 for measuring hCG in Example 1 was ◎ at ΔL=450mAbs, and a highly sensitive measurement was possible. The evaluation results obtained are shown in Table 1.

Example 2
Preparation of conjugation pad for hCG measurement
The hCG measurement control line detection reagent 1 was diluted 5-fold with the coating solution and applied to the entire surface of a 5 mm x 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, manufactured by Millipore). A dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT) were used for application, and the hCG measurement control line detection reagent 1 was uniformly applied twice with an application amount of 15 μL/cm, and then dried for 30 minutes in a dryer adjusted to 45 ° C. to obtain a conjugation pad A2 for hCG measurement.
On the other hand, a conjugation pad B2 for measuring hCG was prepared in the same manner as in Example 1.

Preparation of immunochromatographic test strip for hCG measurement In the same manner as in Example 1, the membrane, the conjugation pad B2 for hCG measurement, and the conjugation pad A2 for hCG measurement were attached to the adhesive tape, and then a sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) measuring 11 mm x 300 mm was attached to the upstream side of the conjugation pad A2 for hCG measurement so as to overlap the conjugation pad A2 by 2 mm. Except for this, an immunochromatographic test strip 2 for hCG measurement was prepared in the same manner as in Example 1 and evaluated in the same manner as in Example 1. The evaluation results obtained are shown in Table 1.

Example 3
Preparation of conjugation pad for hCG measurement
The hCG measurement control line detection reagent 1 was diluted 5-fold with the coating solution and applied to the entire surface of a 12 mm x 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, manufactured by Millipore). A dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT) were used for application, and the hCG measurement control line detection reagent was uniformly applied twice with an application amount of 6.3 μL/cm, and then dried for 30 minutes in a dryer adjusted to 45 ° C. to obtain a conjugation pad A3 for hCG measurement.
On the other hand, a conjugation pad B3 for measuring hCG was prepared in the same manner as in Example 1.

Preparation of immunochromatographic test strip for hCG measurement In the same manner as in Example 1, the membrane, the conjugation pad B3 for hCG measurement, and the conjugation pad A3 for hCG measurement were attached to the adhesive tape, and then a sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) measuring 4 mm x 300 mm was attached to the upstream side of the conjugation pad A3 for hCG measurement so as to overlap the conjugation pad A3 by 2 mm. Except for this, an immunochromatographic test strip 3 for hCG measurement was prepared in the same manner as in Example 1 and evaluated in the same manner as in Example 1. The evaluation results obtained are shown in Table 1.

Example 4
Preparation of membrane cards for hCG measurement
A membrane card 4 for hCG measurement was prepared in the same manner as in Example 1, except that a test line having a line width of about 1 mm was formed at a position 20 mm from the upstream end of the membrane.

Preparation of conjugation pads for hCG measurement Conjugation pads A4 for hCG measurement and conjugation pads B4 for hCG measurement were prepared in the same manner as in Example 3.

Preparation of immunochromatographic test strip for hCG measurement The hCG measurement conjugation pad B4 was attached to the 20 mm x 300 mm adhesive tape part on the upstream side of the hCG measurement membrane card 4 so as to overlap with the membrane by 2 mm. Next, the hCG measurement conjugation pad A4 was attached to the upstream side so as to overlap with the conjugation pad B4 by 2 mm. Furthermore, except that a 4 mm x 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) was attached to the upstream side so as to overlap with the conjugation pad A4 by 2 mm, the hCG measurement immunochromatographic test strip 4 was prepared in the same manner as in Example 1 and evaluated in the same manner as in Example 1. The obtained evaluation results are shown in Table 1.

Example 5
Preparation of membrane cards for hCG measurement
A membrane card 5 for hCG measurement was prepared in the same manner as in Example 1, except that a test line having a line width of about 1 mm was formed at a position 13 mm from the upstream end of the membrane.

Preparation of conjugation pads for hCG measurement Conjugation pads A5 and B5 for hCG measurement were prepared in the same manner as in Example 2.

Preparation of immunochromatographic test strip for hCG measurement The hCG measurement conjugation pad B5 was attached to the 20 mm x 300 mm adhesive tape part on the upstream side of the hCG measurement membrane card 5 so as to overlap with the membrane by 2 mm. Next, the hCG measurement conjugation pad A5 was attached to the upstream side so as to overlap with the conjugation pad B5 by 2 mm. Furthermore, except that a sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, Millipore) of 11 mm x 300 mm was attached to the upstream side so as to overlap with the conjugation pad A5 by 2 mm, the hCG measurement immunochromatographic test strip 5 was prepared in the same manner as in Example 1 and evaluated in the same manner as in Example 1. The obtained evaluation results are shown in Table 1.

Example 6
Preparation of membrane cards for hCG measurement
A membrane card 6 for hCG measurement was prepared in the same manner as in Example 1, except that a control line with a line width of approximately 1 mm was formed 5 mm from the upstream end of the membrane, and a test line with a line width of approximately 1 mm was formed 10 mm from the upstream end of the membrane.

Preparation of conjugation pads for hCG measurement Conjugation pads A6 and B6 for hCG measurement were prepared in the same manner as in Example 1.

Preparation of immunochromatographic test strip for hCG measurement The hCG measurement conjugation pad B6 was attached to the 20 mm x 300 mm adhesive tape part on the upstream side of the hCG measurement membrane card 6 so as to overlap with the membrane by 2 mm. Next, the hCG measurement conjugation pad A6 was attached to the upstream side so as to overlap with the conjugation pad B6 by 2 mm. Furthermore, except that a 9 mm x 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) was attached to the upstream side so as to overlap with the conjugation pad A6 by 2 mm, the hCG measurement immunochromatographic test strip 6 was prepared in the same manner as in Example 1 and evaluated in the same manner as in Example 1. The obtained evaluation results are shown in Table 1.

(Example 7)
Preparation of membrane cards for hCG measurement
A membrane card 7 for hCG measurement was prepared in the same manner as in Example 1, except that a control line approximately 1 mm wide was formed 7 mm from the upstream end of the membrane, and a test line approximately 1 mm wide was formed 22 mm from the upstream end of the membrane.

Preparation of a conjugation pad for hCG measurement A 17 mm x 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, manufactured by Millipore) was coated with the hCG measurement control line detection reagent 1 diluted 5 times with the coating solution over the entire surface. A dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT) were used for coating, and the hCG measurement control line detection reagent was uniformly coated twice with a coating amount of 4.4 μL/cm, and then dried for 30 minutes in a dryer adjusted to 45 ° C. to obtain a conjugation pad A7 for hCG measurement.
On the other hand, the test line detection reagent 1 was applied to the entire surface of a 6 mm x 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, manufactured by Millipore). A dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT) were used for application, and the test line detection reagent was uniformly applied twice with an application amount of 12.5 μL/cm, and then dried for 30 minutes in a dryer adjusted to 45 ° C. to prepare a conjugation pad B7 for hCG measurement.

Preparation of immunochromatographic test strip for hCG measurement The hCG measurement conjugation pad B7 was attached to the 20 mm x 300 mm adhesive tape part on the upstream side of the hCG measurement membrane card 7 so as to overlap with the membrane by 2 mm. Next, the hCG measurement conjugation pad A7 was attached to the upstream side so as to overlap with the conjugation pad B7 by 2 mm. Furthermore, except that a 3 mm x 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) was attached to the upstream side so as to overlap with the conjugation pad B7 by 2 mm, the hCG measurement immunochromatographic test strip 7 was prepared in the same manner as in Example 1 and evaluated in the same manner as in Example 1. The obtained evaluation results are shown in Table 1.

(Example 8)
Preparation of membrane cards for hCG measurement
A membrane card 8 for hCG measurement was prepared in the same manner as in Example 1, except that a control line approximately 1 mm wide was formed 10 mm from the upstream end of the membrane, and a test line approximately 1 mm wide was formed 11.5 mm from the upstream end of the membrane.

Preparation of conjugation pads for hCG measurement Conjugation pads A8 for hCG measurement and conjugation pads B8 for hCG measurement were prepared in the same manner as in Example 2.

Preparation of immunochromatographic test strip for hCG measurement The hCG measurement conjugation pad B8 was attached to the 20 mm x 300 mm adhesive tape part on the upstream side of the hCG measurement membrane card 8 so as to overlap with the membrane by 2 mm. Next, the hCG measurement conjugation pad A8 was attached to the upstream side so as to overlap with the conjugation pad B8 by 2 mm. Furthermore, except that a sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, Millipore) of 11 mm x 300 mm was attached to the upstream side so as to overlap with the conjugation pad A8 by 2 mm, the hCG measurement immunochromatographic test strip 8 was prepared in the same manner as in Example 1 and evaluated in the same manner as in Example 1. The obtained evaluation results are shown in Table 1.

Example 9
Preparation of membrane cards for hCG measurement
A membrane card 9 for measuring hCG was prepared in the same manner as in Example 5.

Preparation of conjugation pads for hCG measurement Conjugation pads A9 for hCG measurement and conjugation pads B9 for hCG measurement were prepared in the same manner as in Example 2.

Preparation of immunochromatographic test strip for hCG measurement The hCG measurement conjugation pad B9 was attached to the 20 mm x 300 mm adhesive tape part on the upstream side of the hCG measurement membrane card 9 so as to overlap with the membrane by 2 mm. Next, the hCG measurement conjugation pad A9 was attached to the upstream side so as to overlap with the conjugation pad B9 by 2 mm. Furthermore, except that a 4 mm x 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) was attached to the upstream side so as to overlap with the conjugation pad A9 by 2 mm, the hCG measurement immunochromatographic test strip 9 was prepared in the same manner as in Example 1 and evaluated in the same manner as in Example 1. The obtained evaluation results are shown in Table 1.

Example 10
Preparation of membrane cards for hCG measurement
A membrane card 10 for measuring hCG was prepared in the same manner as in Example 1, except that a control line having a width of approximately 1 mm was formed 5 mm from the upstream end of the membrane, and a test line having a width of approximately 1 mm was formed 15 mm from the upstream end of the membrane.

Preparation of conjugation pads for hCG measurement Conjugation pad A10 for hCG measurement and conjugation pad B10 for hCG measurement were prepared in the same manner as in Example 3.

Preparation of immunochromatographic test strip for hCG measurement The hCG measurement conjugation pad B10 was attached to the 20 mm x 300 mm adhesive tape portion on the upstream side of the hCG measurement membrane card 10 so as to overlap with the membrane by 2 mm. Next, the hCG measurement conjugation pad A10 was attached to the upstream side so as to overlap with the conjugation pad B10 by 2 mm. Furthermore, except that a 4 mm x 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) was attached to the upstream side so as to overlap with the conjugation pad A10 by 2 mm, the hCG measurement immunochromatographic test strip 10 was prepared in the same manner as in Example 1 and evaluated in the same manner as in Example 1. The obtained evaluation results are shown in Table 1.

(Comparative Example 1)
Preparation of membrane cards for hCG measurement
A membrane card 11 for measuring hCG was prepared in the same manner as in Example 10.

Preparation of a conjugation pad for hCG measurement The hCG measurement test line detection reagent 1 and the control line detection reagent 1 were mixed at a ratio of 5:1, and the mixture of the hCG measurement test line detection reagent 1 and the control line detection reagent 1 was applied to the entire surface of a 12 mm x 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, manufactured by Millipore). A dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT) were used for application, and the mixture of the hCG measurement test line detection reagent 1 and the control line detection reagent 1 was uniformly applied twice with an application amount of 7.5 μL / cm, and then dried for 30 minutes in a dryer adjusted to 45 ° C. to prepare a conjugation pad C for hCG measurement.

Preparation of immunochromatographic test strip for hCG measurement The conjugation pad C for hCG measurement was attached to the 20 mm x 300 mm adhesive tape part on the upstream side of the membrane card 11 for hCG measurement so as to overlap with the membrane by 2 mm. Next, the immunochromatographic test strip 11 for hCG measurement was prepared in the same manner as in Example 1, except that a sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) of 9 mm x 300 mm was attached to the upstream side so as to overlap with the conjugation pad C by 2 mm, and was evaluated in the same manner as in Example 1. The obtained evaluation results are shown in Table 1.

(Comparative Example 2)
Preparation of membrane cards for hCG measurement
A membrane card 12 for hCG measurement was prepared in the same manner as in Example 1, except that a control line with a line width of approximately 1 mm was formed 15 mm from the upstream end of the membrane, and a test line with a line width of approximately 1 mm was formed 10 mm from the upstream end of the membrane.

Preparation of conjugation pads for hCG measurement Conjugation pads A12 for hCG measurement and conjugation pads B12 for hCG measurement were prepared in the same manner as in Example 1.

Preparation of immunochromatographic test strip for hCG measurement The hCG measurement conjugation pad B12 was attached to the 20 mm x 300 mm adhesive tape part on the upstream side of the hCG measurement membrane card 12 so as to overlap with the membrane by 2 mm. Next, the hCG measurement conjugation pad A12 was attached to the upstream side so as to overlap with the conjugation pad B12 by 2 mm. Furthermore, except that a 9 mm x 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) was attached to the upstream side so as to overlap with the conjugation pad A12 by 2 mm, the hCG measurement immunochromatographic test strip 12 was prepared in the same manner as in Example 1 and evaluated in the same manner as in Example 1. The obtained evaluation results are shown in Table 1.

As is clear from the results in Table 1, in Examples 1 to 10, by using conjugation pad A impregnated with a control line detection reagent and conjugation pad B impregnated with a test line detection reagent, an immunochromatographic test strip capable of quantifying the substance to be measured with high accuracy was obtained. In Example 7, the measurement accuracy was lower than in the other Examples, possibly because the distance between the test line and the control line was slightly farther. In Example 8, the measurement accuracy was lower, possibly because the distance between the test line and the control line was too close, resulting in insufficient reading by the measuring device. On the other hand, in Comparative Example 1, the measurement accuracy was low, possibly because a conjugation pad impregnated with both the test line detection reagent and the control line detection reagent was used. In Comparative Example 2, the measurement accuracy was low, possibly because the distance from the upstream end of conjugation pad A to the test line was significantly greater than the distance from the upstream end of conjugation pad B to the control line.

By using the immunochromatographic test strip of the present invention, the target substance contained in the measurement sample can be measured quickly, easily, inexpensively, and with high accuracy, which will greatly contribute to industry.

1: Sample pad 2: Conjugation pad A
3: Conjugation Pad B
4: Membrane 5: Absorbent pad 6: Backing sheet 7: Control line 8: Test line 9: Adhesive sheet

Claims (4)

An immunochromatographic test piece in which a sample pad, a conjugation pad A, a conjugation pad B, a membrane, and an absorbent pad are arranged in this order,
Each of the members has a partial overlapping portion,
The membrane has a control line on the upstream side and a test line on the downstream side,
The conjugation pad A carries a control line detection reagent composed of detection particles labeled with a low molecular weight compound,
The conjugation pad B carries a test line detection reagent composed of detection particles sensitized with a detection antibody that specifically binds to a substance to be measured,
A capture substance that specifically binds to the low molecular weight compound is immobilized in a linear manner on the control line,
A capture substance that specifically binds to the measurement target substance is immobilized in a linear shape on the test line,
the detection particles in the control line detection reagent and the detection particles in the test line detection reagent are at least one selected from the group consisting of colored particles and fluorescent particles;
The difference between the distance X from the upstream end of the conjugation pad A to the control line and the distance Y from the upstream end of the conjugation pad B to the test line is less than 10 mm;
The control line and the test line are spaced apart from each other by 1 mm or more and 15 mm or less.
An immunochromatographic test strip used to quantify the target substance contained in a measurement sample. The immunochromatographic test piece according to claim 1 , wherein the low molecular weight compound is biotin or digoxigenin. 3. The immunochromatographic test strip according to claim 1, wherein the detection particles in said control line detection reagent and the detection particles in said test line detection reagent are both colored particles and have the same color. A method for quantifying a substance to be measured contained in a measurement sample using the immunochromatographic test piece according to any one of claims 1 to 3, by carrying out the following steps (i) to (iii) in order:
Step (i): A step of mixing a measurement sample with a diluent; Step (ii): A step of dropping the mixture of step (i) onto a sample pad and allowing it to spread on a test strip by capillary action; Step (iii): A step of measuring the measurement target substance based on the color intensity of the test line and the control line.