WO2020166699A1

WO2020166699A1 - Immunochromatographic test piece, and measurement method using same

Info

Publication number: WO2020166699A1
Application number: PCT/JP2020/005780
Authority: WO
Inventors: 岡本　淳; 美穂松尾; 圭三米田
Original assignee: 東洋紡株式会社
Priority date: 2019-02-15
Filing date: 2020-02-14
Publication date: 2020-08-20
Also published as: JPWO2020166699A1; JP7533227B2

Abstract

[Problem] To provide an immunochromatographic test piece for quickly, and with high sensitivity, measuring a substance to be measured in a measurement sample by using immunochromatography, and a measurement method using the immunochromatographic test piece. [Solution] The present invention is an immunochromatographic test piece for quantifying a substance to be measured in a measurement sample, wherein: the immunochromatographic test piece is formed by arranging in a connected manner and in the following order a sample pad, a conjugation pad, a membrane and an absorbent pad; the membrane is provided with a control line and a test line that develop a color in response to an antigen-antibody reaction; the control line is positioned upstream of the test line; and the ratio of the average particle size of detection particles A captured on the test line and the average particle size of detection particles B captured on the control line is 3:1-100:1.

Description

Immunochromatographic test strip and measuring method using the same

The present invention relates to an immunochromatographic test strip for rapidly measuring a substance to be measured contained in a measurement sample by an immunochromatographic method and a measuring method using the same. More specifically, the present invention relates to an immunochromatographic test strip that is fast and has high measurement sensitivity, 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 medical staff beside a subject. Unlike the clinical test performed in the central laboratory of a large-scale hospital, etc., POCT is capable of instantly obtaining test results on the spot, so POCT is spreading in a wide range of test items.

A typical example of POCT is immunochromatography. The immunochromatography method is that from one end of a porous body on which an antibody that specifically binds to a measurement target substance contained in a measurement sample is immobilized, the measurement sample develops in the porous body by a capillary phenomenon and is labeled in the process. It is an immunoassay method for determining the presence or absence of a measurement target substance in a measurement sample by causing the measurement target substance and an antibody to bind with each other by an antigen-antibody reaction and accumulate over time and develop color locally.

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, which take advantage of these advantages, have become widespread worldwide. In the conventional immunochromatography method, visual judgment (qualitative evaluation) was generally used, but in recent years, the amount of the substance to be measured contained in the measurement sample was quantified using an analyzer such as an immunochromatographic reader that measures the color development intensity. Technology to do so is being developed.

One of the methods for quantifying the amount of a substance to be measured using the immunochromatography method is a sandwich method using an antigen-antibody reaction. The sandwich method uses two types of antibodies having different epitopes for the substance to be measured. One of the antibodies 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 may be 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 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 develops from one end (upstream side) of the porous body, moves while forming an immune complex with the test line detection reagent, and contacts the capture antibody on the test line to be captured and develop color. To do. The free test line detection reagent that is 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 a device such as an immunochromatographic reader for these color development intensities. Normally, the test line is placed upstream of the control line, and the color of the control line on the downstream side ensures that the sample has normally passed the test line. It may be placed upstream. The latter contributes to the shortening of the measurement time because the control line takes less time to develop color than the former.

In the conventional immunochromatography method, the mechanism that captures the free test line detection reagent, which was not captured on the test line, by the capture antibody on the control line, affects the concentration of the measurement target substance and changes the color intensity of the control line. 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. Will be higher. In other words, the amount of free test line detection reagent is small, so that the color intensity of the control line is low. On the other hand, when the measurement target substance is present in a low concentration in the measurement sample, the amount of the test line detection reagent that forms an immune complex with the measurement target substance also becomes small, so 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 is increased.

An immunochromatographic test strip in which the color intensity of the control line reflects the amount of the test sample (or test line detection reagent) developed on the porous body in order to accurately measure the measurement target substance contained in the measurement sample by the immunochromatographic method. In some cases, the color development intensity of the control line may be used to correct the variation in the spread amount of the measurement sample on the porous body due to the individual difference of the immunochromatographic test strip. In that case, when a certain amount of measurement sample (or test line detection reagent) is spread on the porous body, it is desired that the color intensity of the control line is always constant.

In

Patent Documents

1 and 2, a detection particle labeled with a low molecular weight compound (hereinafter sometimes referred to as a control line detection reagent) and an antibody that specifically captures the low molecular weight compound are lined on the surface of a porous body. By using an immunochromatographic test strip having a control line immobilized in a shape, an independent antigen-antibody reaction between the test line and the control line progresses, so that it is not affected by the concentration of the measurement target substance in the measurement sample, A technique capable of stabilizing the color intensity of the control line is disclosed. In the above invention, since the control line reflects the inflow amount of the measurement sample (or the test line detection reagent) into the porous body to some extent, the color intensity of the test line is corrected by the color intensity of the control line to obtain the measurement sample. The substance to be measured contained in the substance can be measured with a certain degree of accuracy. However, in Patent Document 1, since the test line is arranged on the upstream side of the control line, there is a problem that it takes time for the control line to develop color, that is, the measurement time becomes long. In addition, since it takes a short time for the test line detection reagent to reach the test line, the measurement sensitivity is slightly lowered. On the other hand, in Patent Document 2, since the control line is arranged on the upstream side of the test line, there is a certain effect in improving the measurement sensitivity, but the shortening of the measurement time is insufficient.

WO 2017/065213 WO2017/094825

An object of the present invention is to provide an immunochromatographic test strip for rapidly measuring a substance to be measured contained in a measurement sample by an immunochromatographic method and a measuring method using the same. More specifically, it is an object of the present invention to provide an immunochromatographic test strip that is rapid and has high measurement sensitivity, and a measurement method using the same.

The present inventor has conducted extensive studies to solve the above-mentioned problems, and as a result, the detection in the test line detection reagent in which the control line is located upstream of the test line (on the sample pad side) and which is composed of the detection antibody and the detection particles A By using an immunochromatographic test strip in which the average particle size of the particles A is larger than the average particle size of the detection particles B in the control line detection reagent composed of the detection particles B labeled with a low molecular weight compound, it is faster and faster than the prior art. It was found that the measurement sensitivity was improved. This is because the average particle size of the detection particles A in the test line detection reagent captured by the test line located on the downstream side is the average particle size of the detection particles B in the control line detection reagent captured by the control line located on the upstream side. By making it larger than the diameter, it takes less time for the control line detection reagent to be captured on the control line, and more time for the test line detection reagent to be captured on the test line, resulting in quick and high It is speculated that sensitive measurements have become possible. Furthermore, the present inventor has found that the immunochromatographic reader can be miniaturized by making the detection particles A and the detection particles B have the same color, and completed the present invention.

That is, the representative present invention is as follows.
(1) An immunochromatographic test strip for quantifying a substance to be measured contained in a measurement sample, wherein the immunochromatographic test strip has a configuration in which a sample pad, a conjugation pad, a membrane, and an absorption pad are arranged in order. The membrane is provided with a test line and a control line that develop color by an antigen-antibody reaction, the control line is located upstream of the test line, and the average particle diameter of the detection particles A captured by the test line is large. And an average particle size ratio of the detection particles B captured by the control line is 3:1 to 100:1.
(2) The immunochromatographic test piece according to (1), wherein the detection particles A and the detection particles B are colored particles and have the same color.
(3) The immunochromatographic test piece according to (1) or (2), wherein the detection particles A are organic particles and the detection particles B are inorganic particles.
(4) The immunochromatographic test piece according to any one of (1) to (3), wherein the detection particles A are red cellulosic fine particles, and the detection particles B are red spherical gold fine particles.
(5) The immunochromatographic test piece according to any one of (1) to (3), wherein the detection particles A are blue-based cellulosic fine particles, and the detection particles B are blue-based plate-shaped gold fine particles.
(6) A method of using the immunochromatographic test piece according to any one of (1) to (5) to sequentially quantify a measurement target substance contained in a measurement sample through the following steps (i) to (iii).
Step (i): Step of mixing the measurement sample and the diluting solution Step (ii): Step of spotting the mixed solution of the step (i) on the sample pad Step (iii): From the coloring intensity of the test line and the control line Process to quantify the substance to be measured

The immunochromatographic test strip of the present invention and the measuring method using the same, the color development intensity of the control line is stabilized because the independent antigen-antibody reaction proceeds in the test line and the control line, and the control line detection reagent is captured by the control line. It is possible to measure the substance to be measured contained in the measurement sample quickly and with high sensitivity by shortening the time until the measurement is performed and increasing the time until the test line detection reagent is captured in the test line. it can.

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

In the present invention, the measurement sample is not particularly limited, but examples thereof include biological samples such as blood, lymph, spinal fluid, sweat, urine, lacrimal fluid, saliva, skin, mucous membrane, and hair. For blood, in addition to whole blood, serum, blood cells or plasma obtained by centrifugation of blood can be used as a sample. In addition, the measurement sample is not limited to a human-derived sample, and a biological sample derived from a mammal such as a dog, a cat, or a cow is also targeted.

In the present invention, the measurement items are not particularly limited, but for example, HbA1c, C-peptide, Insulin, m-Alumin, Cys-C, NGAL, TriponinI, 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., influenzaA/B, Dengue, HBV, HCV, HIV, Syphilis, Malaria, H.; Infectious disease items such as pylori, Rota, Chlamydia, StrepA, Adeno, Zika, Chikungunya, RSV and the like can be mentioned.

In the present invention, the constitution of the immunochromatographic test piece is such that the addition portion (dropping portion) of the measurement sample solution of the immunochromatographic test piece is on the upstream side, and the sample pad having the addition portion, the conjugation pad, the membrane, and the absorption pad are arranged in this order. Must have been Next, an example of the immunochromatographic test strip of the present invention will be described with reference to the drawings, but the present invention is not limited thereto. 1 and 2, 1 is a sample pad, 2 is a conjugation pad, 3 is a membrane, 4 is an absorbent pad, 5 is a backing sheet, 6 is a control line, 7 is a test line, and 8 is an adhesive sheet.

In the examples of FIGS. 1 and 2, the immunochromatographic test piece is in the form of an elongated strip 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 test line 7 in which a capture antibody that specifically binds to the substance to be measured is linearly immobilized is formed at a position approximately 15 mm from the upstream end of the membrane 3 of the immunochromatographic test strip. Further, a control line 6 in which a capture antibody that specifically binds to a low molecular weight compound described later is linearly immobilized is formed at a position of about 10 mm from the end. Furthermore, the conjugation pad 2 of the immunochromatographic test strip is composed of a test line detection reagent composed of a detection antibody that specifically binds to the substance to be measured and detection particles A, and a detection particle B labeled with a low molecular weight compound. A control line detection reagent is carried.

The average particle size of the detection particles A in the test line detection reagent and the average particle size of the detection particles B in the control line detection reagent are not particularly limited, but are preferably 10 to 1000 nm, more preferably 10 to 500 nm. If the average particle size of the detection particles is large, the downstream development becomes slow and the measurement time becomes long. In addition, it is easy to be captured on the membrane and the background itself is colored, so that the coloring on the test line and the control line becomes unclear. On the other hand, when the average particle size of the detection particles is small, in the case of the test line detection reagent, the amount of detection antibody that can be physically adsorbed or chemically bound decreases, and the measurement sensitivity decreases. Further, in the case of the control line detection reagent, the amount of the low molecular weight compound that can be labeled decreases, and the color development on the line becomes unclear.

The ratio of the average particle size of the detection particles A to the average particle size of the detection particles B is preferably 3:1 to 100:1, more preferably 4:1 to 50:1, and more preferably 5:1 to 20:. 1 is more preferred. In particular, when the water absorption rate of the porous body is 75 to 180 seconds/4 cm, small detection particles spread faster downstream, and large detection particles spread later downstream. Therefore, if the ratio of the average particle size of the detection particles A and the average particle size of the detection particles B is 3:1 to 100:1, the test line detection reagent (that is, the detection particles A) is located on the downstream side of the test line. It takes a longer time to reach, that is, the time for the antigen-antibody reaction becomes longer, so that highly sensitive measurement becomes possible. Further, the time required for the control line detection reagent (that is, the detection particles B) to reach the control line on the upstream side becomes shorter, that is, the time until the control line develops color becomes shorter, which enables quick measurement. Become. When the ratio of the average particle size of the detection particles A and the average particle size of the detection particles B is small, the time until the test line detection reagent is captured in the test line is shortened, or the control line detection reagent is captured in the control line. Since it takes a long time until the measurement is completed, it becomes difficult to achieve both high-sensitivity measurement and quick measurement. Since the test line detection reagent undergoes an antigen-antibody reaction with the substance to be measured during development, a longer residence time contributes to higher sensitivity, but the control line detection reagent does not react during development. Therefore, the shorter the retention time, the faster it contributes to speeding up. On the other hand, if the ratio of the average particle size of the detection particles A and the average particle size of the detection particles B is large, for example, the detection particles A are larger than 1000 nm or the detection particles B are smaller than 10 nm, which is not preferable as described above.

The detection particles A and the detection particles B are not particularly limited, but colored particles or fluorescent particles can be used. Examples of the colored particles include metal particles, latex particles, cellulose particles and the like. Examples of metal particles include gold colloid, silver colloid, platinum colloid, palladium colloid, gold nanorods, gold nanoplates, and silver nanoplates. Examples of the latex particles include particles made of materials such as polystyrene, polymethylmethacrylate, and acrylic acid polymer. Examples of the fluorescent particles include polystyrene, polymethylmethacrylate, polyvinyltoluene, silica and the like, and examples of the fluorescent dye include fluorescein and its derivatives, rhodamine and its derivatives, cyanine and its derivatives and the like. be able to.

Among these, it is preferable that both the detection particles A and the detection particles B are colored particles, and it is more preferable that the detection particles A and the detection particles B have 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 detection particles A and the detection particles B is a fluorescent particle, the immunochromatographic reader becomes large and expensive for detecting fluorescence, which is not preferable from the viewpoint of POCT. When the detection particles A and the detection particles B have different colors, for example, in a general light emitting diode (hereinafter sometimes abbreviated as LED)-photodiode (hereinafter sometimes abbreviated as PD) immunochromatographic reader. In the case of measurement, it is necessary to combine a plurality of LED-PDs corresponding to each color of the test line and the control line, which makes the device large and expensive, which is not preferable from the viewpoint of POCT.

In order for the ratio of the average particle size of the detection particles A and the average particle size of the detection particles B to be 3:1 to 100:1, and for the same color, the detection particles A are organic particles and the detection particles B are Are preferably inorganic particles. Specifically, when both the detection particles A and the detection particles B are red, it is preferable that the detection particles A are cellulose particles or latex particles, and the detection particles B are spherical gold fine particles. Therefore, it is more preferable that the detection particles A are cellulose particles and the detection particles B are spherical gold fine particles. On the other hand, when both the detection particles A and the detection particles B are blue, the detection particles A are preferably cellulose particles or latex particles, and the detection particles B are preferably plate-shaped gold fine particles. From the viewpoint of highly sensitive measurement, it is more preferable that the detection particles A are cellulose particles and the detection particles B are plate-shaped gold fine particles. It should be noted that the gold fine particles exhibit a red color in a spherical shape and a blue color in a plate shape.

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, when the measurement target substance is hCG, an anti-hCG antibody can be used, and when the measurement target substance 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 separately produced by a known method. 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, and the operation is simple and the cost is low. Physical adsorption is more preferred. In addition, in order to improve the sensitization efficiency, a reactive active group may be introduced into the detection particles. The reactive active group is not particularly limited, but 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 low molecular weight 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 proven record in the field of protein labeling.

Preferably, the test line detection reagent and the control line detection reagent are blocked with a blocking protein. The blocking protein is not particularly limited, and Blocking Peptide Fragment (hereinafter sometimes abbreviated as BPF) of a microorganism-derived protein, bovine serum albumin (hereinafter sometimes abbreviated as BSA) of an animal-derived protein, and casein are preferable. .. The blocking protein may be a commercially available product, or may be separately produced by a known method. BPF is commercially available from Toyobo Co., Ltd. and is easily available.

The linearly immobilized capture antibody forming the test line 7 on the membrane 3 used in the present invention is not particularly limited as long as it can specifically bind to the substance to be measured. For example, when the measurement target substance is hCG, an anti-hCG antibody can be used, and when the measurement target substance is HbA1c, an anti-Hb antibody or an anti-HbA1c antibody can be used. It is preferable that the detection antibody and the capture antibody have different epitopes for the substance to be measured. The capture antibody may be a commercially available product or may be separately produced by a known method. Further, it may be a monoclonal antibody or a polyclonal antibody, and the molecular size is not particularly limited.

The linearly immobilized capture antibody forming the control line 6 on the membrane 3 used in the present invention 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 can be used, and when the low molecular weight compound is digoxigenin, an anti-digoxigenin antibody can be used. The capture antibody may be a commercially available product or may be separately produced by a known method. Further, it may be a monoclonal antibody or a polyclonal antibody, and the molecular size is not particularly limited.

The material of the sample pad 1 is not particularly limited as long as it can quickly absorb the measurement sample and then spread to the downstream conjugation pad, membrane, or absorbent pad. For example, cellulose filter paper or nonwoven 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. When the thickness is small, the flow of the measurement sample on the downstream side becomes non-uniform, and the measurement accuracy may decrease. On the other hand, if the thickness is large, the downstream expansion may be delayed and the measurement time may be long. In addition, the amount of measurement sample required for downstream development increases.

The material of the conjugation pad 2 should be one that can hold the test line detection reagent and the control line detection reagent in a dry state, and can rapidly release both of the detection reagents as the measurement sample is developed downstream. There is no particular limitation, and examples thereof 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. Of these, glass filter paper is preferable. The thickness of the conjugation pad 2 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 the test line detection reagent and the control line detection reagent in a dry state. On the other hand, if the thickness is large, the downstream expansion may be delayed and the measurement time may be long. In addition, the amount of measurement sample required for downstream development increases.

The material of the membrane 3 is not particularly limited as long as it can accurately and uniformly spread the measurement sample, and examples thereof include cellulose, cellulose derivatives, nitrocellulose, cellulose acetate, polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride. , Or a nylon membrane. Among these, a nitrocellulose membrane is preferable.

The material of the absorbent pad 4 is not particularly limited as long as it can quickly absorb the measurement sample that has been developed from the upstream and then hold it so that it does not backflow. , 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 absorbent pad 4 is preferably 0.2 mm to 5.0 mm, more preferably 0.5 mm to 2.0 mm. When 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, when the thickness is large, the size of the immunochromatographic test piece and the housing case covering the immunochromatographic test piece also becomes large, which is not preferable from the viewpoint of POCT.

The method of supporting the test line detection reagent and the control line detection reagent on the conjugation pad 2 is not particularly limited, and for example, the test line detection reagent and the control line detection reagent are mixed at a constant ratio, and then the mixed solution is mixed. Can be produced by uniformly applying, spraying or impregnating a conjugation pad on the conjugation pad, and then drying at a suitable temperature for a certain time in a constant temperature bath. The coating amount of the mixed solution is not particularly limited, but is preferably 5 μL to 50 μL per 1 cm line length. Then, it is preferable to dry after the application. 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 minutes to 120 minutes.

The method of linearly immobilizing the capture antibody forming the test line and the capture antibody forming the control line on the membrane 3 is not particularly limited. For example, the capture antibody forming the test line and the control line are formed. It can be prepared by applying a fixed amount of each of the capture antibodies to different positions on the line and then drying at a suitable temperature for a fixed time in a constant temperature bath. The coating amount of both capture antibodies is not particularly limited, but 0.1 μL to 2 μL per 1 cm line length is preferable. Then, it is preferable to dry after the application. 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 minutes to 120 minutes.

In the present invention, in the immunochromatographic test piece, the above-prepared membrane 3 is attached to the vicinity of the center of the adhesive sheet 8, and then the conjugation pad 2 is attached to one end of the membrane 3 so as to partially overlap with it, and then the sample pad 1 is partially overlapped and pasted on the end opposite to the overlapping of the conjugation pad 2 with the membrane 3, and then the absorbent pad 4 is partially overlapped and pasted on the other end of the membrane 3, It can be manufactured by cutting it into a strip having a constant width. The test line 7 and the control line 6 may be prepared after producing the test piece, or may be prepared before producing the test piece.

The immunochromatographic test strip has at least a first opening for dropping a measurement sample on the sample pad 1 and a second opening for measuring the test line 7 and the control line 6 on the membrane 3 It may be housed in a plastic housing case.

In the present invention, the concentration of the substance to be measured is more preferably measured from a 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 measuring method of the test line and control line of the immunochromatographic test piece is not particularly limited, and a commercially available immunochromatographic reader may be used, or the immunochromatographic reader may be manufactured by a separately known method. The detection system is not particularly limited, but for example, LED-FD, LED-CMOS, 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 methods in the specification are as follows.

<1. Shape of detection particles>
Each of the detection particles was observed with a scanning electron microscope (SEM, S-4800 manufactured by Hitachi Ltd.) and classified into a [1] sphere, a spheroid, and a plate that is a spherical [2] prism that is an ellipsoid.

<2. Average particle size of detected particles>
Each particle was observed with a scanning electron microscope (SEM), and when the shape was [1] spherical, the diameter of 100 particles was measured and the average diameter (average particle diameter) was calculated. When the shape was a [2] plate shape, the maximum length of the plane portion of 100 particles was measured, and the average length (average particle diameter) was calculated.

(Example 1)
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 (Otsuka distilled water, manufactured by Otsuka Pharmaceutical Co., Ltd.). did. Then, 1.0 wt% cellulose particles (NanoAct (registered trademark), RE2: Dark Red, red, average particle size 340 nm, manufactured by Asahi Kasei) 100 μL, 10 mM Tris buffer (204-07885, manufactured by Wako Pure Chemical Industries, Ltd.) ) (PH 7.0) (900 μL) and the above 1.0 mg/mL (0.1 wt%) of anti-hCG-β monoclonal antibody (100 μL) were added to a 15 mL centrifuge tube and vortexed. Then, it was placed in a low temperature incubator (IN604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 37° C. and left standing for 120 minutes. Then, 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. In addition, it was further left to stand for 60 minutes in a low temperature incubator adjusted to 37°C. Then, using a centrifuge (MX-307, manufactured by Tommy Seiko), a rack in rotor (TMA-300, manufactured by Tommy Seiko), and a rack (AR510-04, manufactured by Tommy Seiko), centrifuge at 13,000 G. The reaction was carried out at 25°C for 15 minutes to precipitate the antibody-sensitized cellulose particles, and then the supernatant was removed. Then, 12 mL of a washing solution (pH 10.0) consisting of 50 mM borate buffer was added and treated with an ultrasonic disperser (UH-50, 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 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 (196-00001, manufactured by Wako Pure Chemical Industries, Ltd.), 0.2 wt% casein, and 62 mM borate buffer was added, and ultrasonic dispersion was performed. The sample was treated with a machine for 10 seconds to obtain a test line detection reagent 1 for hCG measurement.

(2) Preparation of control line detection reagent D Biotin (04822-91, manufactured by Nacalai Tesque, Inc.) 16 mg and dimethyl sulfoxide: DMSO (08904-14, manufactured by Nacalai Tesque, Inc.) 1,000 μL were added to a 5 mL microtube and vortexed. The mixture was stirred with to obtain a D biotin solution. 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 by 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 left standing for 15 minutes to obtain a D biotin/EDC/NHS solution. Next, 100 mg of Bovine serum album:BSA (A7906, manufactured by Sigma-Aldrich) and 1,000 μL of distilled water were added to a 5 mL microtube, and the mixture was vortexed to obtain a BSA solution. Then, 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 standing for 30 minutes to obtain a D biotin-BSA solution. Then, 13.5 mL of a spherical colloidal gold solution (OD ₅₂₀ =1.0) (EMGC40, red, average particle size=40 nm, manufactured by BBI Solutions), and 50 mM potassium dihydrogen phosphate (28736-75, Nacalai Tesque, Inc.) 1.5 mL (manufactured by the company) (pH 7.0) was added to a 50 mL centrifuge tube, and the mixture was stirred lightly. Then, 1.5 mL of the D-biotin-BSA solution was added, and after lightly stirring, the mixture was placed in a low temperature incubator adjusted to 25° C. and allowed to stand for 10 minutes. Next, 500 μL of 1.0 wt% PEG 20,000 (168-11285, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was lightly stirred, then 1,000 μL of 10 wt% BSA was added and gently stirred. Then, using a centrifuge, a rack in rotor, and a rack, centrifugation at 8,000 G was performed at 25° C. for 15 minutes to precipitate the D biotin-sensitized gold colloid, and then the supernatant was removed. Next, a colloidal gold preservation solution (pH 8.2) consisting of 0.05 wt% PEG20,000, 150 mM sodium chloride (198-01675, manufactured by Wako Pure Chemical Industries, Ltd.), 1.0 wt% BSA, and 20 mM Tris buffer. ) 30 mL was added and the mixture was treated with an ultrasonic disperser for 10 seconds. Then, using a centrifuge, a rack in rotor, and a rack, centrifugation at 8,000 G was performed at 25° C. for 15 minutes to precipitate the D biotin-sensitized gold colloid, and then the supernatant was removed. Then, a gold colloid coating solution (pH 8.2) consisting of 0.05 wt% PEG20,000, 37.5 mM sodium chloride, 0.25 wt% BSA, 2.5 wt% sucrose, and 20 mM Tris buffer was OD. ₅₂₀ was added to 3.75 and the mixture was treated with an ultrasonic disperser for 10 seconds to obtain a control line detection reagent 1.

(3) Preparation of hCG Measurement Membrane Card 5.0 mg/mL of anti-hCG-α monoclonal antibody (6601, manufactured by MedixBiochemica) was prepared to 0.5 mg/mL with distilled water. Then, 1.0 mg/mL of anti-biotin polyclonal antibody (A150-111A, manufactured by BETHYL) was prepared to 0.5 mg/mL with distilled water. Next, a 60 mm x 300 mm membrane card (Hi-Flow Plus 120 Membrane Cards) consisting of a 20 mm x 300 mm adhesive tape part on the upstream side, a 25 mm x 300 mm membrane part in the center, and a 15 mm x 300 mm adhesive tape part on the downstream side. , Water absorption rate 120 sec/4 cm, HF120, manufactured by Millipore) 15 mm from the upstream side of the membrane part, a dispensing platform (XYZ3060, manufactured by BIODOT) and a Bio Jet nozzle (BHQHR-XYZ, manufactured by BIODOT). 0.5 mg/mL of the anti-hCG-α monoclonal antibody was applied at a coating amount of 1.0 μL/cm, and then dried with a dryer (WFO-510, manufactured by Tokyo Rika Kikai Co., Ltd.) adjusted to 45° C. After drying for a minute, a test line having a line width of about 1 mm was formed. Furthermore, at a position 10 mm from the upstream side of the membrane portion of the membrane card on which the test line was formed, 0.5 μg/mL of the anti-biotin polyclonal antibody was added at 1.0 μL/cm 2 using a dispensing platform and a Bio Jet nozzle. Then, the hCG measurement membrane card 1 was obtained by forming a control line having a line width of about 1 mm by applying a coating amount of 1 and then drying for 30 minutes with a dryer adjusted to 45°C.

(4) Preparation of conjugation pad for hCG measurement The test line detection reagent for hCG measurement and the control line detection reagent were mixed at a ratio of 1:1 (volume ratio). Next, using a dispensing platform and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT) on the entire surface of a 10 mm×300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, manufactured by Millipore), 15 μL/ After being applied twice uniformly with a coating amount of cm, it was dried for 30 minutes with a dryer adjusted to 45° C. to obtain a hCG measurement conjugation pad 1.

(5) Preparation of Immunochromatographic Test Piece for hCG Measurement The hCG measurement conjugation pad was attached to the 20 mm×300 mm adhesive tape portion on the upstream side of the hCG measurement membrane card so as to overlap with the membrane portion by 5 mm. Then, a 20 mm×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 by 5 mm. Then, a 20 mm×300 mm absorption 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 with the membrane portion by 5 mm. Then, a guillotine-type cutting module (CM5000, manufactured by BIODOT) was used to cut into strips having a width of 4 mm and a length of 60 mm to obtain an immunochromatographic test piece 1 for hCG measurement.

(6) Preparation of diluted hCG sample Commercially available hCG antigen (30-1132, manufactured by Fitzgerald) was diluted with a diluent (pH 7.0) consisting of 50 mM potassium dihydrogen phosphate and 1.0 wt% BSA, 1 IU/L and 100 IU/L diluted hCG samples were prepared.

(7) Evaluation of immunochromatographic test piece for hCG measurement: evaluation of rapidity The immunochromatographic test piece for hCG measurement was placed on a horizontal table. Then, 100 μL of a diluting solution (pH 7.0) consisting of 50 mM potassium dihydrogen phosphate and 1.0 wt% BSA was dispensed with a micropipette and gently dropped on the sample pad at 0 seconds to be 25° C. Then, the reflection absorbance (mAbs) of the control line on the membrane is measured with an immunochromatographic reader (C10060-10, measurement mode: Gold Colloid, Line, manufactured by Hamamatsu Photonics) over time from 0 seconds to 10 minutes. did. As an index of promptness, the measurement time at the time when the reflection absorbance ≧50 mAbs at which the control line is visible is calculated: t (second) is calculated, and t≦60 seconds is ◎(excellent), 60 seconds<t≦90 seconds Was evaluated as ◯ (good), and 90 seconds<t was evaluated as × (bad). The rapidity of the immunochromatographic test piece for hCG measurement of Example 1 was ◎ at t=50 seconds, which means that rapid measurement was possible. Table 1 shows the obtained evaluation results.

(8) Evaluation of immunochromatographic test piece for hCG measurement: evaluation of sensitivity The immunochromatographic test piece for hCG measurement was placed on a horizontal table. Then, 100 μL of the diluted hCG sample (1 IU/L, 100 IU/L) was sampled with a micropipette and allowed to stand still at 25° C. with the time point at which it was gently dropped on the sample pad set at 0 seconds, and the control line on the membrane and The reflection absorbance (mAbs) of the test line was measured over time from 0 seconds to 10 minutes using an immunochromatographic reader, and the measurement time at the time when the reflection absorbance of the control line exceeded 50 mAbs: 5 minutes after t (seconds) The reflection absorbance of the control line and the test line of was used as the measured value. The experimental procedure was performed for each of the diluted hCG samples (1 IU/L, 100 IU/L) at N=10. As an index of sensitivity, N=10 average value of the reflection absorbance (mAbs) of the test line of the diluted hCG sample (100 IU/L) and the reflection absorbance (mAbs) of the test line of the diluted hCG sample (1 IU/L) of N=10 Difference from average value: Δ(mAbs) was calculated, and 300 Abs≦Δ was evaluated as excellent (excellent), 200≦Δ<300 mAbs was evaluated as ○ (good), and Δ<200 mAbs was evaluated as × (bad). The immunochromatographic test piece for hCG measurement of Example 1 had a sensitivity of Δ=320 mAbs, which was ⊚, indicating that highly sensitive measurement was possible. Table 1 shows the obtained evaluation results.

(Examples 2 to 7)
Gold colloid solution spherical the particles detected in the control line detection reagent _{(OD 520 = 1.0) (EMGC40} , red, average particle diameter = 40 nm, BBI Solutions Inc.) instead of a spherical colloidal gold solution _{(OD 520} = 1.0) (EMGC5, red, average particle size=5 nm, manufactured by BBI Solutions) (Example 2), spherical gold colloid solution (OD ₅₂₀ =1.0) (EMGC10, red, average particle size=10 nm, BBI Solutions (manufactured by BBI Solutions) (Example 3), spherical gold colloidal solution (OD ₅₂₀ =1.0) (EMGC20, red, average particle size=20 nm, manufactured by BBI Solutions) (Example 4), spherical gold colloids Liquid (OD ₅₂₀ =1.0) (EMGC60, red, average particle size=60 nm, manufactured by BBI Solutions, Inc.) (Example 5), spherical gold colloid liquid (OD ₅₂₀ =1.0) (EMGC80, red, average) Particle size=80 nm, manufactured by BBI Solutions (Example 6), spherical gold colloidal solution (OD ₅₂₀ =1.0) (EMGC100, red, average particle size=100 nm, manufactured by BBI Solutions) (Example 7) Immunochromatographic test pieces 2 to 7 for hCG measurement were prepared and evaluated in the same manner as in Example 1 except that was used. Table 1 shows the obtained evaluation results.

(Example 8)
(1) Preparation of Test Line Detection Reagent for hCG Measurement Instead of cellulose particles (NanoAct (registered trademark), RE2: Dark Red, red, average particle size 340 nm, manufactured by Asahi Kasei Co., Ltd.) as detection particles in the test line detection reagent, cellulose was used. A test line detection reagent 8 for measuring hCG was prepared in the same manner as in Example 1 except that particles (NanoAct (registered trademark), BL2: Dark Navy, blue, average particle size 365 nm, manufactured by Asahi Kasei Corporation) were used.

(2) Preparation of control line detection reagent A D-biotin-BSA solution was obtained in the same manner as in Example 1. Then, 14.7 mL of a plate-shaped colloidal gold solution (OD ₆₁₀ =1.0) (Au-WPPLC1-C, blue, average particle size=45 nm, manufactured by Dainippon Paint Co., Ltd.), and 50 mM potassium dihydrogen phosphate ( 300 μL of pH 7.5) was added to a 50 mL centrifuge tube and stirred gently. Then, 1.5 mL of the D-biotin-BSA solution was added, and after lightly stirring, the mixture was placed in a low temperature incubator adjusted to 25° C. and allowed to stand for 1 hour. Then, 750 μL of 0.5 wt% PEG 6,000 (169-09125, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and after lightly stirring, the mixture was placed in a low temperature incubator adjusted to 25° C. and allowed to stand for 10 minutes. Then, 1,500 μL of 2.0 wt% BSA was added, and after lightly stirring, the mixture was placed in a low temperature incubator adjusted to 25° C. and left standing for 10 minutes. Then, using a centrifuge, a rack-in rotor and a rack, centrifugation at 8,000 G was performed at 25° C. for 10 minutes to precipitate the D biotin-sensitized gold colloid, and then the supernatant was removed. Then, 15 mL of a gold colloid storage solution (pH 8.2) consisting of 0.05 wt% PEG 6,000, 150 mM sodium chloride, 1.0 wt% BSA, and 20 mM Tris buffer was added, and the mixture was ultrasonically dispersed for 10 seconds. Processed. Then, using a centrifuge, a rack-in rotor and a rack, centrifugation at 8,000 G was performed at 25° C. for 10 minutes to precipitate the D biotin-sensitized gold colloid, and then the supernatant was removed. Next, a gold colloid coating solution (pH 8.2) consisting of 0.05 wt% PEG 6,000, 37.5 mM sodium chloride, 0.25 wt% BSA, 2.5 wt% sucrose, and 20 mM Tris buffer was OD. ₆₁₀ was added to be 3.75, and the mixture was treated with an ultrasonic disperser for 10 seconds to obtain a control line detection reagent 8.

Immunochromatography for hCG measurement was performed in the same manner as in Example 1 except that the hCG measurement test line detection reagent 8 was used in place of the hCG measurement test line detection reagent 1, and the control line detection reagent 8 was used in place of the control line detection reagent 1. Test piece 8 was prepared and evaluated. Table 1 shows the obtained evaluation results. In addition, with the change of detection particles from red to blue, the measurement mode of the immunochromatographic reader was also changed from Gold Colloid to Latex.

(Example 9)
Instead of the plate-like gold colloidal solution (OD ₆₁₀ =1.0) (Au-WPPLC1-C, blue, average particle size=45 nm, manufactured by Dainippon Paint Co., Ltd.) in the plate-like shape, the detection particles in the control line detection reagent were used. HCG was performed in the same manner as in Example 8 except that a gold colloidal solution (OD ₆₆₀ =1.0) (Au-WPPLC3-C, blue, average particle size=100 nm, manufactured by Dainippon Paint Co., Ltd.) (Example 9) was used. The immunochromatographic test piece 9 for measurement was prepared and evaluated. Table 1 shows the obtained evaluation results.

(Example 10)
(1) Preparation of test line detection reagent for hCG measurement 5.0 mg/mL of anti-hCG-β monoclonal antibody was added to 50 mM potassium dihydrogen phosphate (166-04255, manufactured by Wako Pure Chemical Industries, Ltd.) (pH 7.0). 1.0 wt/mL of 10 wt% latex particles (Estapor (registered trademark) K030, red, average particle size = 300 nm, manufactured by Merk millipore) with 50 mM potassium dihydrogen phosphate (pH 7.0). Were prepared respectively. Next, 100 μL of the 1.0 wt% latex particles and 100 μL of the 1.0 mg/mL anti-hCG-β monoclonal antibody were added to a 15 mL centrifuge tube and stirred by vortex. Then, it was placed in a low temperature incubator adjusted to 25° C. and left standing for 60 minutes. Next, 12 mL of a blocking solution consisting of 1.0 wt% Bovine serum albumin:BSA was added, and the mixture was allowed to stand for 60 minutes in a low temperature incubator adjusted to 25°C. Then, using a centrifuge, a rack-in rotor and a rack, centrifugation at 8,000 G was performed at 25° C. for 15 minutes to precipitate antibody-sensitized latex particles, and then the supernatant was removed. Next, 12 mL of a cleaning solution (pH 7.0) consisting of 1.0 wt% Bovine serum albumin:BSA and 50 mM potassium dihydrogen phosphate was added, and the mixture was treated with an ultrasonic disperser for 10 seconds. Then, using a centrifuge, a rack-in rotor and a rack, centrifugation at 8,000 G was performed at 25° C. for 15 minutes to precipitate antibody-sensitized latex particles, and then the supernatant was removed. Then, 2 mL of a coating solution (pH 7.0) consisting of 1.0 wt% Bovine serum albumin:BSA and 50 mM potassium dihydrogen phosphate was added, and treated with an ultrasonic disperser for 10 seconds, and a test line detection reagent for hCG measurement. Got 10.

An hCG measurement immunochromatographic test piece 10 was prepared and evaluated in the same manner as in Example 1 except that the hCG measurement test line detection reagent 10 was used in place of the hCG measurement test line detection reagent 1. Table 1 shows the obtained evaluation results.

(Examples 11 and 12)
Instead of the membrane card (Hi-Flow Plus 120 Membrane Cards, water absorption rate 120 seconds/4 cm, HF120, manufactured by Millipore), the membrane card (Hi-Flow Plus 75 Membrane Cards, water absorption rate 75 seconds/4 cm, HF75, Millipore) was used. (Example 11), a membrane card (Hi-Flow Plus 180 Membrane Cards, water absorption speed 180 seconds/4 cm, HF180, manufactured by Millipore) (Example 12) (Example 12) The immunochromatographic test pieces 11 and 12 for measurement were produced and evaluated. Table 1 shows the obtained evaluation results.

(Example 13)
Instead of 5.0 mg/mL anti-hCG-β monoclonal antibody as the detection antibody in the test line detection reagent, 8.57 mg/mL anti-HbA1c monoclonal antibody (HbA1c Antibody, OAMA02329, manufactured by AVIVA SYSTEM BIOLOGY) was used as a test line. Example except that a 3.6 mg/mL anti-Hb monoclonal antibody (HBA1 Antibody, OAMA02326, AVIVA SYSTEM BIOLOGY) was used as the capture antibody forming the antibody instead of 5.0 mg/mL anti-hCG-α monoclonal antibody. An immunochromatographic test strip 13 for HbA1c measurement was prepared in the same manner as in 1.

Then, a commercially available HbA1c standard substance, HbA1c measurement performance evaluation sample (QRM HbA1c 2007-1, manufactured by the Institute of Standards for Testing Medicine, L1, L5) was added to 50 mM PBS (162-19321, manufactured by Wako Pure Chemical Industries, Ltd.) , 1.0 wt% polyoxyethylene (20) sorbitan monolaurate: Tween (registered trademark) 20 (166-21213, manufactured by Wako Pure Chemical Industries, Ltd.), 1.0 wt% polyoxyethylene (10) octyl phenyl ether : HbA1c (%) was diluted to 1,000 times with a diluent (pH 7.4) consisting of TritonX (registered trademark)-100 (160-24751, manufactured by Wako Pure Chemical Industries, Ltd.), and L1=5. A diluted HbA1c sample (L1, L5) of 01%, L5=11.26% was obtained. The immunochromatographic test piece 13 for HbA1c measurement was evaluated in the same manner as in Example 1 except that the diluted HbA1c samples (L1, L5) were used instead of the diluted hCG samples (1 IU/L, 100 IU/L). Table 1 shows the obtained evaluation results.

(Comparative Examples 1 and 2)
Gold colloid solution spherical the particles detected in the control line detection reagent _{(OD 520 = 1.0) (EMGC40} , red, average particle diameter = 40 nm, BBI Solutions Inc.) instead of a spherical colloidal gold solution _{(OD 520} = 1.0) (EMGC2, red, average particle size=2 nm, manufactured by BBI Solutions) (Comparative Example 1), spherical gold colloid solution (OD ₅₂₀ =1.0) (EMGC200, red, average particle size=200 nm, Immunochromatographic test strips 14 and 15 for hCG measurement were prepared and evaluated in the same manner as in Example 1 except that BBI Solutions Co., Ltd. (Comparative Example 2) was used. Table 2 shows the obtained evaluation results.

(Comparative example 3)
(2) Preparation of control line detection reagent A D-biotin-BSA solution was obtained in the same manner as in Example 1. Next, 10 wt% latex particles (Estapor (registered trademark) K030, red, average particle diameter=300 nm, manufactured by Merk millipore) were prepared to 1.0 wt% with 50 mM potassium dihydrogen phosphate (pH 7.0). .. Next, 100 μL of the 1.0 wt% latex particles and 100 μL of the D biotin-BSA solution were added to a 15 mL centrifuge tube, and the mixture was vortexed. Then, it was placed in a low temperature incubator adjusted to 25° C. and left standing for 60 minutes. Next, 12 mL of a blocking solution consisting of 1.0 wt% Bovine serum albumin:BSA was added, and the mixture was allowed to stand for 60 minutes in a low temperature incubator adjusted to 25°C. Then, using a centrifuge, a rack-in rotor, and a rack, centrifugation at 8,000 G was performed at 25° C. for 15 minutes to precipitate D-biotin-sensitized latex particles, and then the supernatant was removed. Next, 12 mL of a cleaning solution (pH 7.0) consisting of 1.0 wt% Bovine serum albumin:BSA and 50 mM potassium dihydrogen phosphate was added, and the mixture was treated with an ultrasonic disperser for 10 seconds. Then, using a centrifuge, a rack-in rotor, and a rack, centrifugation at 8,000 G was performed at 25° C. for 15 minutes to precipitate D-biotin-sensitized latex particles, and then the supernatant was removed. Then, 2 mL of a coating solution (pH 7.0) consisting of 1.0 wt% Bovine serum albumin:BSA and 50 mM potassium dihydrogen phosphate was added, and treated with an ultrasonic disperser for 10 seconds to obtain a control line detection reagent 16. ..

An immunochromatographic test strip 16 for hCG measurement was prepared and evaluated in the same manner as in Example 1 except that the control line detection reagent 16 was used in place of the control line detection reagent 1. Table 2 shows the obtained evaluation results.

(Comparative example 4)
An immunochromatographic test strip 17 for hCG measurement was prepared and evaluated in the same manner as in Comparative Example 3 except that the hCG measurement test line detection reagent 1 was used in place of the hCG measurement test line detection reagent 1. Table 2 shows the obtained evaluation results.

As shown in Comparative Examples 1, 3 and 4, when the ratio of the average particle size of the detection particles A captured on the test line to the average particle size of the detection particles B captured on the control line is smaller than 3:1. Since the time until the control line detection reagent is captured by the control line becomes long, the time until the reflection absorbance ≧50 mAbs, which is a level at which the control line that is an index of rapidity is visible, is long: t (second) is long. And the measurement time became longer. Actually, in the case where the measurement was carried out 5 minutes after the measurement time: t (second) when the reflection absorbance of the control line exceeded 50 mAbs as in the present invention, the measurement time was delayed by about 1 minute.

Further, as shown in Comparative Example 2, when the ratio of the average particle size of the detection particles A captured on the test line and the average particle size of the detection particles B captured on the control line is larger than 100:1, detection is performed. Since the average particle size of the particle B is as small as 2 nm, the amount of low-molecular compounds that can be labeled is reduced, and as a result, the control line becomes unclear, and the reflectance is ≧50 mAbs, which is the level at which the control line, which is an index of rapidity, can be visually recognized. Until: It was difficult to determine t (seconds). Therefore, evaluation of sensitivity has not been carried out.

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

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

Claims

An immunochromatographic test strip for quantifying a measurement target substance contained in a measurement sample, wherein the immunochromatographic test strip has a configuration in which a sample pad, a conjugation pad, a membrane, and an absorption pad are arranged in sequence, and The membrane comprises a test line and a control line that develop color by an antigen-antibody reaction, the control line is located upstream of the test line, and the average particle size of the detection particles A captured by the test line and the control An immunochromatographic test strip in which the ratio of the average particle diameters of the detection particles B captured in the line is 3:1 to 100:1.
The immunochromatographic test strip according to claim 1, wherein the detection particles A and the detection particles B are colored particles and have the same color.
The immunochromatographic test piece according to claim 1 or 2, wherein the detection particles A are organic particles, and the detection particles B are inorganic particles.
The immunochromatographic test strip according to claim 1, wherein the detection particles A are red cellulose-based fine particles, and the detection particles B are red spherical gold fine particles.
The immunochromatographic test strip according to any one of claims 1 to 3, wherein the detection particles A are blue cellulose-based fine particles, and the detection particles B are blue plate-shaped gold fine particles.
A method for quantifying a substance to be measured contained in a measurement sample, using the immunochromatographic test strip according to claim 1 through the following steps (i) to (iii) in order.
Step (i): Step of mixing the measurement sample and the diluting solution Step (ii): Step of spotting the mixed solution of the step (i) on the sample pad Step (iii): From the coloring intensity of the test line and the control line Process to quantify the substance to be measured