JP2021162467A - Immunochromatographic test piece and measurement method using the same - Google Patents

Abstract

To provide an immunochromatographic test piece for measuring the measurement object substance included in a measurement sample with good accuracy by an immunochromatography, and a measurement method using the same.SOLUTION: The present invention is an immunochromatographic test piece for quantifying the measurement object substance included in a measurement sample, said test piece comprising a sample pad, a conjugation pad A, a conjugation pad B, a membrane, and an absorption pad which are contiguously arranged in order, each of these members being partially overlapped. The membrane has a control line on the upstream side and a test line on the downstream side, the conjugation pad A includes a control line detection reagent, and the conjugation pad B includes a test line detection reagent.SELECTED DRAWING: None

Description

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

In recent years, the word POCT has been attracting attention in the medical field. POCT is an abbreviation for Point Of Care Testing, and refers to a clinical test performed by a healthcare professional near a subject. Unlike clinical tests performed in a central laboratory of a large-scale hospital, POCT is becoming widespread in a wide range of test items because test results can be obtained instantly on the spot.

A typical example of POCT is the immunochromatography method. The immunochromatography method is a method in which a measurement sample is developed into the porous body by a capillary phenomenon from one end of a porous body on which an antibody that specifically binds to a substance to be measured contained in the measurement sample is immobilized, and is labeled in the process. This is an immunoassay method for determining the presence or absence of a substance to be measured in a measurement sample by accumulating the substance to be measured and an antibody by an antigen-antibody reaction and accumulating with the passage of time and locally developing a color.

Advantages of the immunochromatography method include quick results, easy operation, and low cost. In-vitro diagnostic agents such as pregnancy test agents and influenza diagnostic agents that take advantage of these advantages are widely used worldwide. In the conventional immunochromatography method, visual judgment (qualitative evaluation) is common, but in recent years, the amount of the substance to be measured contained in the measurement sample is quantified by using an analyzer such as an immunochromatographic reader that measures the color development intensity. Technology is being developed.

As one of the methods for quantifying the substance to be measured by using the immunochromatography method, there is a sandwich method using an antigen-antibody reaction. In the sandwich method, two types of antibodies having different epitopes are used for the substance to be measured. One antibody forms a test line as a capture antibody linearly immobilized on the surface of the 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, the antibody that specifically captures the test line detection reagent is linearly immobilized 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 (dropping part) of the measurement sample solution of the immunochromatographic test piece on the upstream side, and the test line detection reagent and the immune complex are developed. It moves while forming, and on the test line, it comes into contact with the capture antibody and is captured and develops color. The free test line detection reagent that was not captured on the test line is captured by the capture antibody on the control line and develops color. The amount of the substance to be measured can be quantified by using an apparatus such as an immunochromatographic reader for these color development intensities.

In the conventional immunochromatography method, the free test line detection reagent that was not captured on the test line is captured by the capture antibody on the control line, so the color development intensity of the control line changes depending on the concentration of the substance to be measured. It was a problem to do. For example, when 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 test line on the upstream side, so that the color development intensity of the test line is high. Will be higher. That is, there is a problem that the color development intensity of the control line is lowered because the amount of the free test line detection reagent is small. On the other hand, when the substance to be measured is present in a low concentration in the measurement sample, the amount of test line detection reagent forming an immune complex with the substance to be measured is also small, so that a large amount of free test line detection reagent is captured by the control line. As a result, there is a problem that the color development intensity of the control line becomes high.

In order to accurately measure the substance to be measured contained in the measurement sample by the immunochromatography method, the color development intensity of the control line reflects the amount of development of the measurement sample (or test line detection reagent) into the porous body. In some cases, the color development intensity of the control line corrects the variation in the amount of the measured sample developed into the porous body due to the individual difference of the immunochromatographic test piece. In that case, when a certain amount of measurement sample (or test line detection reagent) is developed on the porous body, it is desired that the color development intensity of the control line is always constant.

In Patent Documents 1 and 2, detection particles labeled with a low molecular weight compound (hereinafter, also referred to as a control line detection reagent) and an antibody that specifically captures the low molecular weight compound are linearly formed on the surface of the porous body. By using an immunochromatographic test piece having an immobilized control line, the antigen-antibody reaction proceeds independently between the test line and the control line, so that the control line is not affected by the concentration of the substance to be measured in the measurement sample. A technique capable of stabilizing the color development intensity of the antibody is disclosed. In the above invention, since the control line reflects the inflow amount of the measurement sample (or test line detection reagent) into the porous body to some extent, the measurement sample is measured by correcting the color development intensity of the test line with the color development intensity of the control line. The substance to be measured contained therein can be measured with a certain degree of accuracy. However, since the test line and control line are usually immobilized at different positions on the porous body, the time required for the test line detection reagent to be captured on the test line and the time required for the control line detection reagent to be captured on the control line. The correction was insufficient because there was a difference in the above.

International Publication No. 2017/06513 International Publication No. 2017/094825

An object of the present invention is to provide an immunochromatographic test piece for measuring a substance to be measured contained in a measurement sample by an immunochromatographic method, and a measurement method using the same. Specifically, it is an object of the present invention to provide an immunochromatographic test piece having high measurement accuracy and a measurement method using the same.

As a result of diligent research to solve the above problems, the present inventor has placed the test line on the downstream side of the control line, placed the conjugation pad containing the test line detection reagent on the downstream side, and the conjugation containing the control line detection reagent. We have found that the measurement accuracy is improved as compared with the prior art by using the immunochromatographic test piece in which the gatement pad is arranged separately on the upstream side, and completed the present invention. By arranging the conjugation pad containing the test line detection reagent separately on the downstream side of the conjugation pad containing the control line detection reagent, the residence time until each reagent is captured by a capturing substance such as an antibody can be obtained. We have found that the difference is reduced and more accurate correction is possible, and completed the present invention.

That is, a typical invention is as follows.
(1) An immunochromatographic test piece in which each member of a sample pad, a conjugation pad A, a conjugation pad B, a membrane, and an absorption pad is sequentially arranged in an articulated manner.
Each of the above members has a partially overlapped 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 and contains a control line detection reagent.
The conjugation pad B contains a test line detection reagent.
An immunochromatographic test piece for quantifying the substance to be measured contained in the measurement sample.
(2) The immunochromatographic test piece according to (1), wherein the control line and the test line are separated by 1 mm or more and 15 mm or less.
(3) 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, (1) or (1) or ( The immunochromatographic test piece according to 2).
(4) Using the immunochromatographic test piece according to any one of (1) to (3), the substance to be measured contained in the measurement sample is quantified through the following steps (i) to (iii) in order. Method.
Step (i): Mixing the measurement sample and the diluent Step (ii): Dropping the mixed solution of step (i) onto the sample pad and developing it on the test piece by capillarity Step (iii): Test The process of measuring the substance to be measured from the color development intensity of the line and control line

In the immunochromatographic test piece of the present invention and the measurement method using the same, the color development intensity of the control line is stabilized because the independent antigen-antibody reaction proceeds at the test line and the control line, and the test line detection reagent is captured at the test line. By reducing the difference between the time required for the measurement and the residence time until the control line detection reagent is captured by the control line, the substance to be measured contained in the measurement sample can be measured with high accuracy. In addition, by placing the control line on the upstream side, it is possible to shorten the time required to complete the measurement as compared with the case of using a normal test piece.

It is a (top) view which shows an example of an immunochromatographic test piece. It is a (side) view which shows an example of an immunochromatographic test piece.

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

In the present invention, the measurement items are not particularly limited, but for example, HbA1c, C-peptide, Insulin, m-Allumin, 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, VitaminD, etc., influenza A / B, Dengue, HBV, HCV, HIV, Cyfilis, Malaria, H. et al. Infectious disease items such as Helicobacter pylori, Rota, Chlamydia, StrepA, Adeno, Zika, Chikungunya, RSV and the like can be mentioned.

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

In FIGS. 1 and 2, the lateral flow test piece has an elongated strip shape having 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 in which a capture substance that specifically binds to a small molecule compound, which will be described later, is linearly immobilized is formed on the upstream side of the membrane 4 of the immunochromatographic test piece. In addition, a test line 8 is formed on the downstream side in which a trapping substance that specifically binds to the substance to be measured is linearly immobilized. Further, the conjugation pad 2 of the immunochromatographic test piece contains a control line detection reagent composed of detection particles labeled with a small molecule compound, and the conjugation pad 3 contains a detection antibody that specifically binds to the substance to be measured. A test line detection reagent composed of sensitized detection particles is carried.

In the present invention, the average particle size of the detected particles is not particularly limited, but is preferably 10 to 1000 nm, more preferably 10 to 500 nm. If the average particle size of the detected particles is large, the deployment to the downstream becomes slow and the measurement time becomes long. In addition, it becomes easy to be captured by an arbitrary part on the membrane, and the background itself develops color, which may obscure the color development on the test line and the control line. On the other hand, if the average particle size of the detected particles is small, in the case of the test line detection reagent, the amount of detected antibody that can be physically adsorbed or chemically bonded decreases, and the measurement sensitivity may decrease. Further, in the case of the control line detection reagent, the amount of the small molecule compound that can be labeled decreases, and the 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 the colored particles include metal particles, latex particles, and cellulose particles. Examples of the metal particles include gold colloid, silver colloid, platinum colloid, palladium colloid, gold nanorod, gold nanoplate, and silver nanoplate. 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, polyvinyltoluene, and silica, and examples of fluorescent dyes include fluorescein and its derivatives, rhodamine and its derivatives, cyanine and its derivatives, and the like. be able to.

Among these, the detection particles in the test line detection reagent and the detection particles in the control line detection reagent are both preferably colored particles, and more preferably the same color. Here, the same color means that the maximum absorption wavelength is within ± 50 nm. Further, for example, if at least one of the two detection particles is a fluorescent particle, the immunochromatographic reader becomes large and expensive to detect the fluorescence, which is not preferable from the viewpoint of POCT. Further, when both detected particles have different colors, for example, when measuring with an immunochromatographic reader of a general light emitting diode (hereinafter, may be abbreviated as LED) -photodiode (hereinafter, may be abbreviated as PD), A combination of a plurality of LEDs-PDs corresponding to each color of the test line and the control line is required, which makes the device 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 method known separately. Further, it 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 it is preferable to sensitize by physical adsorption by a hydrophobic bond or chemical bond by a covalent bond, which is easy to operate and costly. Cheap physisorption is more preferred. In addition, in order to improve the sensitization efficiency, a reactive active group may be introduced into the detected particles. The reactive active group is not particularly limited, and examples thereof include a carboxyl group, an amino group, an aldehyde group, a thiol group, an epoxy group, and a hydroxyl group. Among these, a carboxyl group and an amino group are preferable. In the case of a carboxyl group, carbodiimide can be used to form a covalent bond with the amino group of the ligand.

The small molecule compound in the control line detection reagent is not particularly limited, but biotin or digoxigenin is preferable, and biotin is more preferable because it is relatively inexpensive, easily available, and has a track record in the field of protein labeling and the like.

The test line detection reagent and the control line detection reagent are preferably blocked by a blocking protein. The blocking protein is not particularly limited, and includes a microorganism-derived protein Blocking Peptide Fragment (hereinafter, may be abbreviated as BPF), an animal-derived protein, bovine serum albumin (hereinafter, may be abbreviated as BSA), casein, and the like. Can be mentioned. As the blocking protein, a commercially available product may be used, or a separately known method may be used for production.

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 small molecule compound of the control line detection reagent. For example, when the low molecular weight compound is biotin, it is an anti-biotin antibody or avidin, and when the low molecular weight compound is digoxigenin, an anti-digoxigenin antibody can be used. As the trapping substance, a commercially available product may be used, or the trapping substance may be produced by a method known separately, and the molecular size is not particularly limited. Further, in the case of an antibody, it may be either a monoclonal antibody or a polyclonal antibody.

In the present invention, the trapping 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. As the trapping substance, a commercially available product may be used, or the trapping substance may be produced by a method known separately, and the molecular size is not particularly limited. Further, in the case of an antibody, it may be either a monoclonal antibody or a polyclonal antibody.

The material of the sample pad 1 is not particularly limited as long as it can be rapidly absorbed into the measurement sample and then expanded to the downstream conjugation pads 2 and 3, the membrane 4, and the absorption pad 5, and is made of, for example, polyethylene. Examples include filter paper or non-woven fabric, glass filter paper or non-woven fabric, polyester filter paper or non-woven fabric, polyethylene filter paper or non-woven fabric. Of these, cellulose filter paper is preferable. The thickness of the sample pad 1 is preferably 0.1 to 2.0 mm, more preferably 0.2 to 1.0 mm. If the thickness is small, the flow of the measurement sample in the downstream becomes non-uniform, and the measurement accuracy may decrease. On the other hand, if the thickness is large, the deployment to the downstream may be delayed and the measurement time may be long. In addition, the amount of measurement sample required for downstream deployment increases.

The materials of the conjugation pads 2 and 3 can hold the control line detection reagent and the test line detection reagent in a dry state, and can rapidly release both detection reagents as the measurement sample is developed downstream. The material is not particularly limited, and examples thereof include cellulose filter paper or non-woven fabric, glass filter paper or non-woven fabric, polyester filter paper or non-woven fabric, and polyethylene filter paper or non-woven fabric. Among these, glass filter paper is preferable. The thickness of the conjugation pads 2 and 3 is preferably 0.1 to 2.0 mm, more preferably 0.2 to 1.0 mm. If the thickness is small, it may not be possible to keep the target amount of control line detection reagent and test line detection reagent in a dry state. On the other hand, if the thickness is large, the deployment to the downstream may be delayed and the measurement time may be long. In addition, the amount of measurement sample required for downstream deployment increases.

The material of the membrane 4 is not particularly limited as long as the measurement sample can be developed accurately and uniformly, and for example, cellulose, cellulose derivative, nitrocellulose, cellulose acetate, polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride. , Or a membrane made of nylon. Among these, a membrane made of nitrocellulose is preferable.

The material of the absorption pad 5 is not particularly limited as long as it can quickly absorb the measurement sample developed from the upstream and then hold it so as not to flow back. For example, a cellulose filter paper or a non-woven fabric, or a glass filter paper. Alternatively, a non-woven fabric, a polyester filter paper or a non-woven fabric, a polyethylene filter paper or a non-woven fabric can be mentioned. Of these, cellulose filter paper is preferable. The thickness of the absorption pad 5 is preferably 0.2 to 5.0 mm, more preferably 0.5 to 2.0 mm. If the thickness is small, the measurement sample once absorbed by the absorption pad may flow back to the membrane side depending on the amount of the measurement sample dropped. On the other hand, if the thickness is large, the size of the immunochromatographic test piece and the housing case that covers the immunochromatographic test piece also becomes large, which is not preferable from the viewpoint of POCT.

The method of supporting the control line detection reagent on the conjugation pad 2 and the method of supporting the test line detection reagent on the conjugation pad 3 are not particularly limited, and for example, each detection reagent is uniformly applied to each pad. After spraying or impregnating, it can be produced by drying in a constant temperature bath at an appropriate temperature for a certain period of time. The amount of the detection reagent applied is not particularly limited, but is preferably 5 to 50 μL per 1 cm of line length. Then, it is preferable to dry after the coating. The drying temperature is not particularly limited, but is preferably 20 ° C to 80 ° C, 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 of linearly immobilizing the capture substance forming the test line and the capture substance forming the control line on the membrane 4 is not particularly limited, and for example, the capture substance forming the test line and the control line are formed. It can be produced by applying a certain amount of the trapping substance to each line at different positions and then drying it in a constant temperature bath at an appropriate temperature for a certain period of time. The amount of both trapping substances applied is not particularly limited, but is preferably 0.1 to 2 μL per 1 cm of line length. Then, it is preferable to dry after the coating. The drying temperature is not particularly limited, but is preferably 20 ° C to 80 ° C, 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, in the immunochromatographic test piece, the prepared membrane 4 is attached near the center of the pressure-sensitive adhesive sheet 9, and then the conjugation pad 3 is partially overlapped and attached to the upstream end of the membrane 4, and then the conjugation is performed. A part of the gated pad 2 is overlapped and attached to the upstream end of the conjugation pad 3. Similarly, the sample pad 1 is partially overlapped and attached to the upstream end of the conjugation pad 2, and then the absorption pad 5 is partially overlapped and attached to the downstream end of the membrane 4. It can be produced by cutting it into strips of a certain width. The control line 7 and the test line 8 may be prepared after the test piece is prepared, or may be prepared before the test piece is prepared.

In the present invention, the length of the overlapping portion when a part of each member (sample pad, conjugation pad A, conjugation pad B, membrane, absorption pad) constituting the immunochromatographic test piece is partially overlapped is not particularly limited. However, it is preferably about 1 to 5 mm. It should be noted that the overlapping portions are not adhered to each other, only the members are in contact with each other.

In the present invention, the control line 7 and the test line 8 are preferably separated from each other 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, and the measurement accuracy is lowered at the time of line reading, or a highly accurate reading device is required. If the distance is too far, the control line on the upstream side will be set close to the conjugation pad. Then, the measurement is performed in a state where the flow is not stable immediately after the sample (sample) flows from the conjugation pad to the membrane, and the measurement accuracy of the control line is lowered. Therefore, the distance is more preferably 2 mm or more and 10 mm or less, and further 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 the conjugation pad A to the control line equal to the distance from the upstream end (upstream end) of the conjugation pad B to the test line. , Movement time of the immune complex between the substance to be measured and the test line detection reagent (time until the immune complex binds to the capture substance on the test line) and movement time of the control line detection reagent (control line detection reagent controls) Since the difference from the time until it is captured by the line) becomes small, the color development is stable and highly accurate detection (quantification) is possible. Specifically, 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 are preferably 15 to 35 mm, more preferably 18 to 33 mm. The difference between X and Y is preferably less than 10 mm, more preferably less than 7 mm, still 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 or more and 15 mm or less, and more preferably 3 mm or more and 15 mm or less. By setting the length of the conjugation pad A excluding the overlapping portion to the above range, it becomes easy to adjust each movement time without changing the total length of the test piece (that is, within the general test piece length). In addition, detection can be performed using a general-purpose housing case or a chromatographic reader.

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

In the present invention, it is more preferable to measure the concentration of the substance to be measured from the correction value obtained by dividing the color development intensity of the test line by the color development intensity of the control line in consideration of the development unevenness of the measurement sample.

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

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

(Example 1)
Preparation of test line detection reagent for hCG measurement 5.0 mg / mL anti-hCG-β monoclonal antibody (5014, manufactured by Medix Biochemica) was prepared to 1.0 mg / mL with distilled water (distilled water from Otsuka, manufactured by Otsuka Pharmaceutical Co., Ltd.). bottom. Next, 1.0 wt% cellulose particles (NanoAct®, RE2: Dark Red, red, average particle diameter 340 nm, manufactured by Asahi Kasei Corporation) 100 μL, 10 mM Tris buffer (204-07858, manufactured by Wako Pure Chemical Industries, Ltd.) ) (PH 7.0) 900 μL 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 vortexed. Then, it was placed in a low temperature incubator (IN604, manufactured by Yamato Kagaku 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 boric acid buffer (021-02195, manufactured by Wako Pure Chemical Industries, Ltd.) was added. In addition, it was allowed to stand for 60 minutes in a low temperature incubator adjusted to 37 ° C. Then, using a centrifuge (MX-307, manufactured by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, manufactured by Tomy Seiko Co., Ltd.) and a rack (AR510-04, manufactured by Tomy Seiko Co., Ltd.), centrifuge 13,000 G. The mixture was carried out at 25 ° C. for 15 minutes to allow the antibody-sensitized cellulose particles to precipitate, and then the supernatant was removed. Next, 12 mL of a cleaning solution (pH 10.0) consisting of 50 mM boric acid buffer was added, and the mixture was treated with an ultrasonic disperser (UH-50, manufactured by SMT) for 10 seconds. Then, using a centrifuge, a rack-in rotor and a rack, centrifugation at 13,000 G was performed at 25 ° C. for 15 minutes to precipitate 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 boric acid buffer was added and ultrasonically dispersed. The process was performed for 10 seconds on the machine to prepare a test line detection reagent 1 for hCG measurement.

Preparation of control line detection reagent D biotin (04822-91, manufactured by Nacalai Tesque) 16 mg and dimethyl sulfoxide: DMSO (08904-14, manufactured by Nacalai Tesque) 1,000 μL were added to a 5 mL microtube and stirred with a vortex. , D biotin solution was obtained. Next, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride: EDC (15022-86, manufactured by Nacalai Tesque) 200 mg, N-hydroxysuccinimide: NHS (18948-02, manufactured by Nacalai Tesque) 200 mg, And 1,000 μL of distilled water was added to a 5 mL microtube and stirred with vortex to obtain an EDC / NHS solution. Then, 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 allowed to stand for 15 minutes to obtain a D biotin / EDC / NHS solution. Then, 100 mg of Bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) and 1,000 μL of distilled water were added to a 5 mL microtube and stirred with 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 allowed to stand for 30 minutes to obtain a D-biotin-BSA solution. 1.0 wt% cellulose particles (NanoAct®, RE2: Dark Red, red, average particle size 340 nm, manufactured by Asahi Kasei Corporation) 100 μL, 10 mM Tris buffer (204-07858, manufactured by Wako Pure Chemical Industries, Ltd.) ( pH 7.0) 900 μL and 100 μL of the D-biotin-BSA solution were added to a 15 mL centrifuge tube and vortexed. Then, it was placed in a low temperature incubator (IN604, manufactured by Yamato Kagaku 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 boric acid buffer (021-02195, manufactured by Wako Pure Chemical Industries, Ltd.) was added. In addition, it was allowed to stand for 60 minutes in a low temperature incubator adjusted to 37 ° C. Then, using a centrifuge (MX-307, manufactured by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, manufactured by Tomy Seiko Co., Ltd.) and a rack (AR510-04, manufactured by Tomy Seiko Co., Ltd.), centrifuge 13,000 G. The mixture was carried out at 25 ° C. for 15 minutes to settle the D biotin-BSA sensitized cellulose particles, and then the supernatant was removed. Next, 12 mL of a cleaning solution (pH 10.0) consisting of 50 mM boric acid buffer was added, and the mixture was treated with an ultrasonic disperser (UH-50, manufactured by SMT) for 10 seconds. Then, using a centrifuge, a rack-in rotor and a rack, centrifugation at 13,000 G was performed at 25 ° C. for 15 minutes 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, manufactured by Wako Pure Chemical Industries, Ltd.), 0.2 wt% casein, and 62 mM boric acid buffer was added and ultrasonically dispersed. The process was carried out with a machine for 10 seconds to prepare a control line detection reagent 1.

Preparation of membrane card for hCG measurement 5.0 mg / mL anti-hCG-α monoclonal antibody (6601, manufactured by MedixBiochemica) was prepared to 0.5 mg / mL with distilled water. Then, 1.0 mg / mL anti-biotin polyclonal antibody (A150-111A, manufactured by BETHYL) was prepared to 0.5 mg / mL with distilled water. Next, a 60 mm × 300 mm membrane card (Hi-Flow Plus 120 Membrane Cards) composed of a 20 mm × 300 mm adhesive tape portion on the upstream side, a 25 mm × 300 mm membrane in the center, and a 15 mm × 300 mm adhesive tape portion on the downstream side. A dispensing platform (XYZ3060, manufactured by BIODOT) and a Bio Jet nozzle (BHQHR-XYZ, manufactured by BIODOT) are used at a position 15 mm from the upstream end of the membrane having a water absorption rate of 120 seconds / 4 cm, HF120, manufactured by Millipore). , The 0.5 mg / mL anti-hCG-α monoclonal antibody was applied at a coating amount of 1.0 μL / cm, and then dried in a dryer (WFO-510, manufactured by Tokyo Rika Kikai Co., Ltd.) adjusted to 45 ° C. for 30 minutes. Then, a test line having a line width of about 1 mm was formed. Further, using a dispensing platform and a BioJet nozzle at a position 10 mm from the upstream end of the membrane of the membrane card on which the test line was formed, 1.0 μL / cm of the 0.5 mg / mL anti-biotin polyclonal antibody was used. After coating with a coating amount, it was dried for 30 minutes in a dryer adjusted to 45 ° C. to form a control line having a line width of about 1 mm, thereby producing a membrane card 1 for hCG measurement.

Fabrication of Conjugation Pad for hCG Measurement A 7 mm x 300 mm junction 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. bottom. Using a dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT), the hCG measurement control line detection reagent was uniformly applied twice at a coating amount of 10.7 μL / cm, and then 45. It was dried for 30 minutes in a dryer adjusted to ° 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 × 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, manufactured by Millipore). Using a dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT), the test line detection reagent was uniformly applied twice at a coating amount of 7.5 μL / cm, and then adjusted to 45 ° C. The hCG measurement conjugation pad B1 was prepared by drying in the dryer for 30 minutes.

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

Preparation of diluted hCG sample A diluted solution (pH 7) of a commercially available hCG antigen (30-1132, manufactured by Fitzgerald), 50 mM potassium dihydrogen phosphate (28736-75, manufactured by Nacalai Tesque), and 1.0 wt% BSA. Diluted with 0.0) to prepare 1 IU / L and 100 IU / L diluted hCG samples.

Evaluation of hCG measurement immunochromatographic test piece: Evaluation of accuracy The hCG measurement immunochromatographic test piece 1 was placed on a horizontal table. Then, 100 μL of the diluted hCG sample (1 IU / L and 100 IU / L) was separated by a micropipette, gently added dropwise to the sample pad, and allowed to stand at 25 ° C. for 10 minutes. Then, the reflected absorbance (mAbs) of the control line and the 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 on the diluted hCG samples (1 IU / L and 100 IU / L) at N = 10, respectively. As an index of accuracy, a correction value (1 IU / L and 100 IU / L) obtained by dividing the reflected absorbance (mAbs) of the test line of the diluted hCG sample (1 IU / L and 100 IU / L) by the reflected absorbance (mAbs) of the control line. ), Then CV (1 IU / L and 100 IU / L) (%) at N = 10 of the obtained correction values (1 IU / L and 100 IU / L) are calculated, and further CV (1 IU / L) is calculated. ) (%) And CV (100 IU / L) (%): Calculate CV (%), CV ≦ 2% is ⊚ (excellent), 2% <CV ≦ 3% is ○ (good), 3% <CV ≦ 5% was evaluated as Δ (average), and 5% <CV was evaluated as × (bad). The accuracy of the immunochromatographic test piece 1 for hCG measurement of Example 1 was CV = 1.8%, which was ⊚, and high-precision measurement was possible. The obtained evaluation results are shown in Table 1.

Evaluation of hCG measurement immunochromatographic test piece: Evaluation of sensitivity The hCG measurement immunochromatographic test piece 1 was placed on a horizontal table. Then, 100 μL of the diluted hCG sample (1 IU / L and 100 IU / L) was separated by a micropipette, gently added dropwise to the sample pad, and allowed to stand at 25 ° C. for 10 minutes. Then, the reflected absorbance (mAbs) of the control line and the 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 on the diluted hCG samples (1 IU / L and 100 IU / L) at N = 10, respectively. As an index of sensitivity, N = 10 average value of the reflected absorbance (mAbs) of the test line of the diluted hCG sample (100 IU / L) and the reflected absorbance (mAbs) of the test line of the diluted hCG sample (1 IU / L). Difference from N = 10 average value: Δ (mAbs) was calculated, and 300 Abs ≦ Δ was evaluated as ⊚ (excellent), 200 ≦ ΔL = <300 mAbs was evaluated as ◯ (good), and Δ <200 mAbs was evaluated as × (bad). The sensitivity of the immunochromatographic test piece 1 for hCG measurement of Example 1 was ΔL = 450 mAbs, which was ⊚, and highly sensitive measurement was possible. The obtained evaluation results are shown in Table 1.

(Example 2)
Fabrication of Conjugation Pad for hCG Measurement A 5 mm × 300 mm fusion 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. bottom. 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 1 was uniformly coated twice at a coating amount of 15 μL / cm, and then at 45 ° C. The hCG measurement conjugation pad A2 was obtained by drying in a dryer adjusted to the above for 30 minutes.
On the other hand, the hCG measurement conjugation pad B2 was produced in the same manner as in Example 1.

Preparation of immunochromatographic test piece for hCG measurement In the same manner as in Example 1, after attaching the membrane, hCG measurement conjugation pad B2, and hCG measurement conjugation pad A2 to the adhesive tape, the hCG measurement conjugation pad A2 An immunochromatographic test piece for hCG measurement in the same manner as in Example 1 except that a sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) of 11 mm × 300 mm was attached to the upstream side so as to overlap the conjugation pad A2 by 2 mm. 2 was prepared and evaluated by the same method as in Example 1. The obtained evaluation results are shown in Table 1.

(Example 3)
Fabrication of Conjugation Pad for hCG Measurement A 12 mm × 300 mm fusion 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. bottom. Using a dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT), the hCG measurement control line detection reagent was uniformly applied twice at a coating amount of 6.3 μL / cm, and then 45. It was dried for 30 minutes in a dryer adjusted to ° C. to obtain a conjugation pad A3 for hCG measurement.
On the other hand, the hCG measurement conjugation pad B3 was produced in the same manner as in Example 1.

Preparation of immunochromatographic test piece for hCG measurement In the same manner as in Example 1, after attaching the membrane, the hCG measurement conjugation pad B3, and the hCG measurement conjugation pad A3 to the adhesive tape, the hCG measurement conjugation pad A3 An immunochromatographic test piece for hCG measurement in the same manner as in Example 1 except that a 4 mm × 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) was attached to the upstream side so as to overlap the conjugation pad A3 by 2 mm. 3 was prepared and evaluated by the same method as in Example 1. The obtained evaluation results are shown in Table 1.

(Example 4)
Preparation of hCG measurement membrane card The hCG measurement membrane card 4 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 hCG measurement conjugation pad The hCG measurement conjugation pad A4 and the hCG measurement conjugation pad B4 were produced in the same manner as in Example 3.

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

(Example 5)
Preparation of hCG measurement membrane card The hCG measurement membrane card 5 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 hCG measurement conjugation pad The hCG measurement conjugation pad A5 and the hCG measurement conjugation pad B5 were produced in the same manner as in Example 2.

Preparation of hCG measurement immunochromatographic test piece The hCG measurement conjugation pad B5 was attached to a 20 mm × 300 mm adhesive tape portion on the upstream side of the hCG measurement membrane card 5 so as to overlap the membrane by 2 mm. Next, the hCG measurement conjugation pad A5 was attached to the upstream side so as to overlap the conjugation pad B5 by 2 mm. Further, an 11 mm × 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) is attached to the upstream side so as to overlap the conjugation pad A5 by 2 mm, and the same as in Example 1 is used for hCG measurement immunochromatography. Test piece 5 was prepared and evaluated by the same method as in Example 1. The obtained evaluation results are shown in Table 1.

(Example 6)
Preparation of membrane card for hCG measurement Except that a control line with a line width of about 1 mm was formed at a position 5 mm from the upstream end of the membrane, and a test line with a line width of about 1 mm was formed at a position 10 mm from the upstream end of the membrane. A membrane card 6 for hCG measurement was produced in the same manner as in Example 1.

Preparation of hCG measurement conjugation pad In the same manner as in Example 1, hCG measurement conjugation pad A6 and hCG measurement conjugation pad B6 were produced.

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

(Example 7)
Preparation of membrane card for hCG measurement Except that a control line with a line width of about 1 mm was formed at a position 7 mm from the upstream end of the membrane, and a test line with a line width of about 1 mm was formed at a position 22 mm from the upstream end of the membrane. A membrane card 7 for hCG measurement was produced in the same manner as in Example 1.

Preparation of Conjugation Pad for hCG Measurement A 17 mm × 300 mm fusion 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. bottom. Using a dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT), the hCG measurement control line detection reagent was uniformly applied twice at a coating amount of 4.4 μL / cm, and then 45. It was dried for 30 minutes in a dryer adjusted to ° C. to obtain a hCG measurement conjugation pad A7.
On the other hand, the test line detection reagent 1 was applied to the entire surface of a 6 mm × 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, manufactured by Millipore). Using a dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT), the test line detection reagent was uniformly applied twice at a coating amount of 12.5 μL / cm, and then adjusted to 45 ° C. The hCG measurement conjugation pad B7 was prepared by drying in the dryer for 30 minutes.

Preparation of hCG measurement immunochromatographic test piece The hCG measurement conjugation pad B7 was attached to a 20 mm × 300 mm adhesive tape portion on the upstream side of the hCG measurement membrane card 7 so as to overlap the membrane by 2 mm. Next, the hCG measurement conjugation pad A7 was attached to the upstream side so as to overlap the conjugation pad B7 by 2 mm. Further, a 3 mm × 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) is attached to the upstream side so as to overlap the conjugation pad B7 by 2 mm, and an immunochromatography for hCG measurement is performed in the same manner as in Example 1. A test piece 7 was prepared and evaluated by the same method as in Example 1. The obtained evaluation results are shown in Table 1.

(Example 8)
Preparation of Membrane Card for hCG Measurement A control line with a line width of about 1 mm was formed at a position 10 mm from the upstream end of the membrane, and a test line with a line width of about 1 mm was formed at a position 11.5 mm from the upstream end of the membrane. A membrane card 8 for hCG measurement was produced in the same manner as in Example 1 except for the above.

Preparation of hCG measurement conjugation pad The hCG measurement conjugation pad A8 and the hCG measurement conjugation pad B8 were produced in the same manner as in Example 2.

Preparation of hCG measurement immunochromatographic test piece The hCG measurement conjugation pad B8 was attached to a 20 mm × 300 mm adhesive tape portion on the upstream side of the hCG measurement membrane card 8 so as to overlap the membrane by 2 mm. Next, the hCG measurement conjugation pad A8 was attached to the upstream side so as to overlap the conjugation pad B8 by 2 mm. Further, an 11 mm × 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) is attached to the upstream side so as to overlap the conjugation pad A8 by 2 mm, and the same as in Example 1 is used for hCG measurement immunochromatography. Test piece 8 was prepared and evaluated by the same method as in Example 1. The obtained evaluation results are shown in Table 1.

(Example 9)
Preparation of hCG measurement membrane card 9 The hCG measurement membrane card 9 was prepared in the same manner as in Example 5.

Preparation of hCG measurement conjugation pad In the same manner as in Example 2, hCG measurement conjugation pad A9 and hCG measurement conjugation pad B9 were produced.

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

(Example 10)
Preparation of membrane card for hCG measurement Except that a control line with a line width of about 1 mm was formed at a position 5 mm from the upstream end of the membrane, and a test line with a line width of about 1 mm was formed at a position 15 mm from the upstream end of the membrane. A membrane card 10 for hCG measurement was produced in the same manner as in Example 1.

Preparation of hCG measurement conjugation pad The hCG measurement conjugation pad A10 and the hCG measurement conjugation pad B10 were produced in the same manner as in Example 3.

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

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

Preparation of Conjugation Pad for hCG Measurement The test line detection reagent 1 for hCG measurement and the control line detection reagent 1 are mixed at a ratio of 5: 1 and a 12 mm × 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, manufactured by Millipore). ) Was coated with a mixed solution of the test line detection reagent 1 for hCG measurement and the control line detection reagent 1. A dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT) are used for coating, and 7.5 μL / cm of the mixed solution of the test line detection test 1 for hCG measurement and the control line detection reagent 1 is applied. After uniformly applying the amount twice, it was dried for 30 minutes in a dryer adjusted to 45 ° C. to prepare a conjugation pad C for hCG measurement.

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

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

Preparation of hCG measurement conjugation pad The hCG measurement conjugation pad A12 and the hCG measurement conjugation pad B12 were produced in the same manner as in Example 1.

Preparation of hCG measurement immunochromatographic test piece The hCG measurement conjugation pad B12 was attached to a 20 mm × 300 mm adhesive tape portion on the upstream side of the hCG measurement membrane card 12 so as to overlap the membrane by 2 mm. Next, the hCG measurement conjugation pad A12 was attached to the upstream side so as to overlap the conjugation pad B12 by 2 mm. Further, an immunochromatography for hCG measurement is performed in the same manner as in Example 1 except that a 9 mm × 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP002000, manufactured by Millipore) is attached to the upstream side so as to overlap the conjugation pad A12 by 2 mm. A test piece 12 was prepared 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, the substance to be measured was determined by using the conjugation pad A impregnated with the control line detection reagent and the conjugation pad B impregnated with the test line detection reagent. An immunochromatographic test piece that can be quantified with high accuracy has been obtained. In Example 7, the measurement accuracy was lower than that in other Examples, probably because the distance between the test line and the control line was slightly different. Further, in Example 8, probably because the distance between the test line and the control line is too close, the reading of the measuring device is insufficient, resulting in a low measurement accuracy. On the other hand, in Comparative Example 1, the measurement accuracy was low probably because the conjugation pad impregnated with both the test line detection reagent and the control line detection reagent was used. Further, in Comparative Example 2, the measurement accuracy was low probably because the distance from the upstream end of the conjugation pad A to the test line was considerably larger than the distance from the upstream end of the conjugation pad B to the control line.

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

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

Claims (4)

This is an immunochromatographic test piece in which each member of the sample pad, the conjugation pad A, the conjugation pad B, the membrane, and the absorption pad is arranged in this order.
Each of the above members has a partially overlapped 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 and contains a control line detection reagent.
The conjugation pad B contains a test line detection reagent.
An immunochromatographic test piece for quantifying the substance to be measured contained in the measurement sample. The immunochromatographic test piece according to claim 1, wherein the control line and the test line are separated by 1 mm or more and 15 mm or less. The first or second claim, 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. Immunochromatographic test piece. A method for quantifying a substance to be measured contained in a measurement sample by sequentially passing through the following steps (i) to (iii) using the immunochromatographic test piece according to any one of claims 1 to 3.
Step (i): Mixing the measurement sample and the diluent Step (ii): Dropping the mixed solution of step (i) onto the sample pad and developing it on the test piece by capillarity Step (iii): Test The process of measuring the substance to be measured from the color development intensity of the line and control line

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