JP7181547B2 - Test strip for immunochromatography and method for detecting substance to be detected - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunochromatographic test strip for detecting a substance to be detected such as an influenza virus and a method for detecting a substance to be detected using the immunochromatographic test strip.

As one method for detecting a substance to be detected in a sample by an antigen-antibody reaction, a measurement method using an immunochromatographic test strip has been conventionally performed. Immunochromatography is performed by immobilizing an antibody or antigen against an antigen or antibody, which is a substance to be detected, on an insoluble membrane carrier, which is a chromatographic medium, to create a detection part, which is a stationary phase, and a conjugate (detection reagent). Using a label sensitized by an antibody or antigen that can bind to the substance to be detected as a mobile phase, the substance to be detected and the conjugate that is the mobile phase are specifically reacted, and further in the detection part that is the stationary phase , the substance to be detected bound to the conjugate is allowed to specifically react with the antibody or antigen immobilized on the detection part. Since colloidal metal particles such as colloidal gold or colored latex particles are usually used as labels, the presence of the substance to be detected in the sample and, in some cases, the amount thereof can be detected from the color in the detection section. .

As shown in Patent Document 1, the configuration of an immunochromatographic test strip includes a sample pad for supplying a sample, a conjugate pad for disposing a conjugate as a mobile phase, and a sample and conjugate. In general, an insoluble membrane carrier having a detection part for detection and an absorbent pad for absorbing the sample that has developed the insoluble membrane carrier are arranged as the body is developed. However, the immunochromatographic test strip having the above-described configuration has problems such that the conjugate is difficult to be released from the conjugate pad, so that it takes a long time to complete the reaction or the background tends to increase.

As described in Patent Document 2, a conjugate pad having a sample supply part and a linear conjugate part is included, and the lower surface of the sample supply part of the conjugate pad does not contact the upper surface of the insoluble membrane carrier, and the conjugate is Attempts have also been made to improve reaction completion time and sensitivity by means of immunochromatographic test strips characterized in that the lower surface of the conjugate portion of the gate pad is in contact with the upper surface of the insoluble membrane carrier. However, the technique described in Patent Document 2 requires 15 minutes from the addition of the biological sample to the sample supply unit to the determination, and sufficient sensitivity can be achieved even if the reaction completion time is further shortened. A technique that can ensure this has been desired.

U.S. Pat. No. 6,352,862 International Publication No. 2012/043746 pamphlet

An object of the present invention is to provide an immunochromatographic test strip that has a short reaction completion time and excellent sensitivity.

Specifically, the present invention is as follows.
<1> An immunochromatographic test strip that detects a substance to be detected by developing a sample that may contain the substance to be detected, comprising the following (1) and (2), (1) or In (2), a linear conjugate portion containing a conjugate in which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized on a label is provided between the sample supply portion and the detection portion. and the distance between the center line of the line of the conjugate portion and the center line of the line-shaped detection portion is 9 to 21 mm (1) containing a substance to be detected A sample pad (2) having a sample supply unit for supplying a possible sample, having at least one line-shaped detection unit on which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized, an insoluble membrane carrier,
<2> An antibody or antigen immobilized on the label that immunologically reacts with the substance to be detected, and an antibody or antigen that immunologically reacts with the substance to be detected immobilized on the detection part The immunochromatographic test strip according to <1>, which is different from the antigen,
<3> Immunochromatographic test strip according to <1> or <2>, which is a lateral flow type,
<4> The substance to be detected is an influenza virus, and the antibody or antigen that immunologically reacts with the substance to be detected immobilized on the label and the substance to be detected immunologically immobilized on the detection part The immunochromatographic test strip according to any one of <1> to <3>, wherein the antibody or antigen that specifically reacts is an anti-influenza virus monoclonal antibody,
<5> The immunochromatographic test strip according to any one of <1> to <4>, wherein the conjugate portion is provided on the sample pad,
<6> The immunochromatographic test strip according to any one of <1> to <5>, wherein the line-shaped conjugate portion has a line width of 2 to 8 mm,
<7> The signal intensity of the control capture reagent-immobilized part 5 minutes after the biological sample was supplied to the sample supply part was the same as that of the control capture reagent-immobilized part 15 minutes after the biological sample was supplied to the sample supply part. The immunochromatographic test strip according to any one of <1> to <6>, and the immunochromatographic test according to any one of <8><1> to <7>, which is 80% or more of the signal intensity of A method for detecting a substance to be detected using a strip, comprising steps (A) to (D), wherein all steps (A) to (D) are performed in less than 15 minutes. The detection method (A), characterized in that the sample supply unit and the substance to be detected are brought into contact,
(B) contacting the substance to be detected in the sample with the conjugate to form a first complex;
(C) contacting the first complex with an antibody or antigen that immunologically reacts with the substance to be detected and immobilized on the detection part to form a second complex; and (D ) Confirming the formation of said second complex by measuring the signal derived from the conjugate.
By configuring the immunochromatographic test strip as described above, it is possible to detect the substance to be detected in a short time after the biological sample is supplied to the sample supply unit.
In the present invention, immobilizing an antigen or antibody means physically or chemically supporting the antigen or antibody on a label or an insoluble membrane carrier.
In addition, the conjugate portion is arranged in a line in a direction orthogonal to the sample developing direction, that is, the line connecting the center of the sample supply portion of the sample pad and the center of the downstream end portion of the insoluble membrane support. is preferred.
Further, the term "detection" as used in the present invention includes not only qualitative detection, but also quantitative detection for substances to be detected that can be quantified.

In the above immunochromatographic test strip, an antibody or antigen that immunologically reacts with the substance to be detected immobilized on the label and an immunological reaction with the substance to be detected immobilized on the detection part Preferably, the antibody or antigen to be used is different. That is, the antibody or antigen that immunologically reacts with the substance to be detected immobilized on the label and the antibody or antigen that immunologically reacts with the substance to be detected immobilized on the detection part are , are preferably antibodies or antigens that each recognize a different site of the substance to be detected. Immobilization of another antibody or antigen on the label and the detection part will result in higher sensitivity.

According to the immunochromatographic test strip of the present invention, it is possible to detect a substance to be detected with high sensitivity in a short time (for example, 5 minutes) after supplying a biological sample to the sample supply section.

1 is a schematic diagram showing an example of the structure of an immunochromatographic test strip of the present invention. FIG. FIG. 1 shows a side view of the structure of an immunochromatographic test strip prior to lamination. FIG. 11 illustrates a top view of one embodiment of a sample pad;

(substance to be detected)
In the present invention, substances to be detected include viruses and physiologically active substances such as proteins that can generally be measured using an antigen-antibody reaction.
Examples of the virus include influenza viruses such as influenza A virus and influenza B virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, etc. Examples of the protein include human hemoglobin, Examples include hepatitis B virus antibody, hepatitis C virus antibody, human immunodeficiency virus antibody, and the like. Among them, it is preferable to use influenza virus as the substance to be detected, and it is more preferable to use influenza A virus and influenza B virus as the substance to be detected by forming a plurality of detection units (for example, two positions) to be described later.

(sample)
In the present invention, examples of samples that may contain the substance to be detected include substances mainly derived from living organisms such as body fluids, extracts obtained by extracting the substance to be detected from these substances, and the like. Substances derived from living organisms (living organisms) specifically include blood, urine, stool, nasal discharge and nasal aspirates derived from nostrils, nasal cavities, pharynx, nasopharynx, etc., sputum, and secretions collected as swab specimens. Fluid, saliva and the like can be mentioned. Above all, if the substance to be detected is an influenza virus, samples include nasal secretions and nasal aspirates derived from nostrils, nasal cavities, pharynx, nasopharynx, etc., and respiratory secretions collected as sputum and swab specimens. preferable. The biological (organism)-derived substance or its extract may be used as a sample as it is, or may be diluted with a diluent as appropriate and used as a sample. Moreover, you may use what filtered suitably as a sample.

(Antibody or antigen that immunologically reacts with the substance to be detected)
The antibody or antigen that immunologically reacts with the substance to be detected used in the present invention is an antibody or antigen that can bind to the substance to be detected. If is an antibody, the antigen is preferred. Antibodies or antigens that immunologically react with the substance to be detected are immobilized on the label and the detection section, which will be described later. Although the antibody or antigen immobilized on the label and the detection part may be the same, it is preferable that the label and the detection part are different. The substance to be detected bound to the conjugate in an immunochromatographic test strip obtained by using different antibodies or antigens immobilized on the label and antibodies or antigens immobilized on the detection part and the reaction between the antibody or antigen in the detection portion and the reaction between the substance to be detected bound to the conjugate and the unreacted conjugate can be suppressed from competing with each other, and the substance to be detected bound to the conjugate can increase the reactivity of the detection portion with antibodies or antigens, and as a result, the sensitivity of the immunochromatographic test strip can be improved. The term "different" refers to a different type of antibody, an antibody that recognizes a different epitope, and an antigen that has a different epitope.
Furthermore, monoclonal antibodies are preferable for the antibodies immobilized on the label and the detection part. The use of monoclonal antibodies can increase the specificity of the reaction.
When the substance to be detected is influenza virus, the antibody immobilized on the label and the detection part may be any antibody that can detect influenza virus, such as anti-influenza A virus monoclonal antibody and anti-influenza B virus monoclonal antibody. Anti-influenza virus monoclonal antibodies, such as antibodies, are preferred.
In addition to these whole antibody molecules, functional fragments of antibodies having antigen-antibody reaction activity are also treated as antibodies in the present invention. Functional fragments of antibodies include those obtained through the process of immunizing animals, those obtained using gene recombination techniques, and chimeric antibodies. Functional fragments of antibodies include, for example, F(ab') ₂ and Fab'. These functional fragments can be produced by treating the antibody with a proteolytic enzyme (eg, pepsin, papain, etc.).

(Label)
As the label used in the present invention, a known label conventionally used in immunochromatographic test strips can be used. For example, colloidal metal particles such as colloidal gold particles and colloidal platinum particles, color latex particles, magnetic particles, and the like are preferable, and colloidal gold particles are particularly preferable.
As for the particle size of the label, it is preferable to use a label having an appropriate particle size according to the label to be used. For example, when colloidal gold particles are used as the label, the particle size is preferably 20 to 70 nm, particularly preferably 45 to 65 nm. The above colloidal gold particles can be produced by a generally known method, for example, by dropping and stirring an aqueous solution of trisodium citrate into a heated aqueous solution of tetrachloroauric (III) acid.

(conjugate)
The conjugate used in the present invention is obtained by immobilizing an antibody or an antigen that immunologically reacts with the substance to be detected on the label as described above. When the substance to be detected is influenza virus, the conjugate is preferably colloidal gold particles on which an anti-influenza virus monoclonal antibody is immobilized.
Methods for immobilizing an antibody or antigen that immunologically reacts with a substance to be detected on a label include physical adsorption, chemical bonding, and the like, and immobilization by physical adsorption is common. For example, when immobilizing an anti-influenza virus monoclonal antibody on colloidal gold particles, the colloidal gold particles and the anti-influenza virus monoclonal antibody are usually added to a buffer solution and immobilized by physical adsorption. At this time, the antibody concentration is preferably adjusted to 20-100 μg/mL. The buffer and its pH are preferably, for example, 0.5-5 mM phosphate buffer (pH 6-8), 0.5-5 mM borate buffer (pH 8-9.5), and the like.
In addition, it is preferable to block the region on the label such as colloidal gold particles, to which the antibody or antigen is not bound, with BSA or the like.

(sample pad)
The sample pad used in the present invention is made of a pad-shaped porous material capable of developing a sample and holding a conjugate, and has a sample supply part in a part thereof. In addition, a porous material portion exists downstream of the sample supply section, and the sample supplied to the sample supply section develops the porous material to form the conjugate provided on the sample pad or the insoluble membrane carrier. It is preferably adapted to reach the part.
The sample supply part is a part that supplies a sample that may contain the substance to be detected, is formed in a part of the porous material, and is upstream of the sample pad.

(Conjugate part)
The conjugate part is a part containing a conjugate, and is formed in a line shape on the sample pad or the insoluble membrane carrier on the downstream side of the sample supply part. The line-shaped conjugate part is arranged in a line-like direction perpendicular to the sample development direction, that is, the line connecting the center of the sample supply part of the sample pad and the center of the downstream end of the insoluble membrane support described later. preferably. In other words, the linear conjugate parts are preferably arranged linearly in a direction perpendicular to the longitudinal direction of the sample pad and the insoluble membrane carrier. The conjugate part is preferably formed on the sample pad. In this case, it is not necessary that all of the downstream side of the conjugate part of the sample pad is a conjugate part, and further downstream of the conjugate part There may be portions of the porous material that do not contain conjugates.
The position of the conjugate part can be adjusted to an appropriate length by those skilled in the art, but the center line of the line-shaped conjugate part is arranged 10 to 23 mm downstream from the sample supply part. more preferably 10 to 21 mm downstream. Further, it is preferable that the center line of the line of the line-shaped conjugate portion is arranged at least 3 mm or more upstream from the downstream end of the sample pad. In this specification, the "center line" of the conjugate part or the detection part means the center line drawn in the direction perpendicular to the longitudinal direction of the sample pad, and the direction parallel to the longitudinal direction of the sample pad. It does not mean the center line drawn to
In this specification, the length of the line of the linear conjugate portion in the longitudinal direction of the sample pad is referred to as the "line width" of the conjugate portion. The line width of the line-shaped conjugate portion is sufficient to contain the amount of conjugate required for detection of the substance to be detected, and is preferably 2 to 8 mm, more preferably 3 to 7 mm. The line width of the line of the conjugate portion is preferably the same width throughout the line.
A part of the line-shaped conjugate part may protrude downstream and/or upstream. In this case, either the non-protruding portion or the protruding portion may have the above line width, but the non-protruding portion preferably has the above line width.

The sample pad is laminated with the insoluble membrane carrier so that the lower surface of the downstream end portion thereof contacts the upper surface of the insoluble membrane carrier, which will be described later. In the sample pad, the bottom surface of the sample supply part may or may not be in contact with the top surface of the insoluble membrane carrier. When the sample pad is provided with a conjugate portion, the lower surface of the conjugate portion may or may not be in contact with the upper surface of the insoluble membrane carrier. When the conjugate part is provided on the insoluble membrane carrier, the upper surface of the conjugate part may or may not be in contact with the lower surface of the insoluble membrane carrier, but preferably not in contact. When a sample that may contain a substance to be detected is supplied to the sample supply of the sample pad, the sample flows from the upstream sample supply through the conjugate-free porous material portion to the downstream side. It flows to the conjugate part. In the conjugate portion, the substance to be detected (eg, influenza virus) in the sample and the conjugate (eg, colloidal gold particles to which anti-influenza virus monoclonal antibody is immobilized) form a complex (aggregate).

Porous materials that make up the sample pad include pads made of non-woven fibers such as paper, cellulose mixtures, nitrocellulose, polyesters, acrylonitrile copolymers, glass, rayon, and the like. Among them, a pad made of glass fiber (a glass fiber pad) is preferable.

The total length of the sample pad, that is, the length from the upstream end to the downstream end of the sample pad, can be adjusted to an appropriate length by those skilled in the art. It can be adjusted to a length of 34 mm, 18-32 mm, or 20-30 mm.

(Insoluble membrane carrier)
The insoluble membrane carrier used in the present invention has at least one line-shaped detection portion on which an antibody or antigen that immunologically reacts with the substance to be detected is immobilized. The line-shaped detection section is arranged in a line in a direction perpendicular to the direction of sample development, that is, the line connecting the center of the sample supply section of the sample pad and the center of the downstream end of the insoluble membrane carrier, which will be described later. preferably In other words, the linear detectors are preferably arranged linearly in a direction perpendicular to the longitudinal direction of the insoluble membrane carrier. Antibodies or antigens that immunologically react with the substance to be detected can be immobilized on the insoluble membrane carrier by a conventionally known method. In the case of a lateral flow immunochromatographic test strip, immobilization is carried out as follows. Mechanism capable of preparing a liquid containing the above antibody or antigen at a predetermined concentration, and then moving the insoluble membrane carrier in a direction perpendicular to the longitudinal direction while ejecting the liquid from a nozzle at a constant speed. using a device having the above-mentioned liquid in a line perpendicular to the longitudinal direction of the insoluble membrane carrier, for example, a line with a line width of 1 to 5 mm and 2 to 4 mm. The insoluble membrane carrier is coated and dried. It can be immobilized by In this specification, the length of the line of the linear detection portion in the longitudinal direction of the insoluble membrane carrier is referred to as the "line width" of the detection portion. The line width of the lines of the detection section is preferably the same width over the entire line.
It is also conceivable that part of the line-shaped detection part protrudes downstream and/or upstream. In this case, either the non-protruding portion or the protruding portion may have the above line width, but the non-protruding portion preferably has the above line width.
The concentration of the antibody or antigen in the liquid is preferably 0.1-5 mg/mL, more preferably 0.5-2 mg/mL. In addition, the amount of antibody or antigen immobilized on the insoluble membrane carrier can be optimized by adjusting the ejection speed from the nozzle of the above device in the case of the lateral flow type, and is preferably 0.5 to 2 μL/cm. is.
In the measurement method using the lateral flow immunochromatographic test strip, the sample supplied from the portion of the sample pad that contacts the insoluble carrier moves in a parallel direction to the insoluble membrane carrier due to capillary action. This is a measurement method that expands as follows.
A solution containing the antibody or antigen at a predetermined concentration can be prepared by adding the antibody or antigen to a buffer solution. Examples of the buffer include commonly used buffers such as phosphate buffer, Tris buffer and Good's buffer. The pH of the buffer solution is preferably in the range of 6.0 to 9.5, more preferably 6.5 to 8.5, even more preferably 7.0 to 8.0. The buffer solution may further contain salts such as sodium chloride, stabilizers and preservatives such as sucrose, and preservatives such as proclin. Salts include those added for adjusting the ionic strength, such as sodium chloride, and those added for the purpose of adjusting the pH of the buffer, such as sodium hydroxide.
After immobilizing the antibody or antigen on the insoluble membrane carrier, a commonly used blocking agent may be made into a solution or vapor to coat the site other than the antibody or antigen immobilized site for blocking.
A control capture reagent conventionally used in immunochromatographic test strips may be immobilized on the insoluble membrane carrier. The control capture reagent is a reagent for ensuring the reliability of the assay, and captures the control reagent contained in the sample pad. For example, when the sample pad contains labeled KLH as a control reagent, an anti-KLH antibody or the like corresponds to the control capture reagent. The position at which the control capture reagent is immobilized can be appropriately selected to match the design of the assay system. It can be designed to be located at ~8mm. The position where the control capture reagent is immobilized relative to the center line of the line of the conjugate portion is located 12 to 35 mm, 14 to 30 mm, or 15 to 27 mm downstream from the center line of the line of the conjugate portion. be able to. Hereinafter, the position where the control capture reagent is immobilized on the insoluble membrane carrier may be referred to as the control capture reagent-immobilized portion.

As the membrane constituting the insoluble membrane carrier used in the present invention, known membranes conventionally used as insoluble membrane carriers for immunochromatographic test strips can be used. Examples thereof include membranes composed of fibers made of polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, and ceramics. Specific examples include glass fiber filter paper and nitrocellulose membranes commercially available from Sartorius, Millipore, Toyo Roshi, Whatman, and the like. The average pore size or retained particle size of the insoluble membrane carrier is not limited to these, but can be 0.1 to 20 μm, more preferably 0.5 to 16 μm, more preferably 0.7 to 10 μm. is more preferred. These average pore diameters and retained particle diameters are values measured according to ASTM-F316-86.
The total length of the insoluble membrane carrier, that is, the length from the upstream end to the downstream end of the insoluble membrane carrier, can be adjusted to an appropriate length by those skilled in the art. It can be adjusted to a length of 15-40 mm, 18-35 mm, or 20-30 mm.

(Distance between the conjugate portion and the detection portion)
The immunochromatographic test strip of the present invention is characterized in that the distance between the center line of the conjugate portion and the center line of the detection portion is 9 to 21 mm. When the distance between the center line of the conjugate part and the center line of the detection part is larger than 21 mm, it takes a long time for the complex of the conjugate and the substance to be detected to develop in the detection part, and the reaction is completed. It takes longer to On the other hand, when the distance between the center line of the conjugate portion and the center line of the detection portion is less than 9 mm, the detection sensitivity is lowered. When there are multiple detectors, the distance between the center line of the most upstream detector and the center line of the conjugate part can be 9 to 21 mm. The distance between the centerline of the conjugate portion and the centerline of the detection portion can be, for example, 9 mm to 20.0 mm, 9 mm to 18.0 mm, or 10 mm to 18.0 mm. The distance between the center line of the conjugate portion and the center line of the detection portion is preferably 9 mm to 16.5 mm, more preferably 10.5 mm to 16.5 mm.

(absorbent pad)
In the immunochromatographic test strip of the present invention, an absorbent pad is preferably provided at the downstream end of the insoluble membrane carrier. The absorbent pad is a liquid-absorbent part that controls the development of the sample by absorbing the sample that has migrated and passed through the insoluble membrane carrier. As the absorbent pad, a known absorbent pad conventionally used in immunochromatographic test strips can be used, and for example, filter paper can be used.
The total length of the absorbent pad, that is, the length from the upstream end of the absorbent pad to the downstream end of the absorbent pad, can be adjusted to an appropriate length by those skilled in the art. , 20-100 mm, 20-80 mm, or 20-60 mm.

In the immunochromatographic test strip of the present invention, the signal intensity of the control capture reagent immobilized portion 5 minutes after the biological sample was supplied to the sample supply portion was 15 minutes after the biological sample was supplied to the sample supply portion. is preferably 80% or more of the signal intensity of the control capture reagent-immobilized portion in .

(test strip for immunochromatography)
An immunochromatographic test strip is preferably placed on a solid support such as a plastic adhesive sheet. The solid phase support is composed of a material that does not impede capillary flow of the sample and conjugate. Also, an immunochromatographic test strip may be immobilized on a solid support with an adhesive or the like. In this case, the components of the adhesive and the like are also composed of substances that do not interfere with the capillary flow of the sample and conjugate. It is also possible to laminate a polyester film or the like for the purpose of increasing the mechanical strength of the insoluble membrane carrier and preventing evaporation (drying) of water during the assay. The immunochromatographic test strip is placed in an appropriate container (housing) in consideration of the size of the immunochromatographic test strip, the method and position of adding the sample, the position of the detection part of the insoluble membrane carrier, the method of detecting the signal, etc. It can be stored and mounted for use, and such a stored and mounted state is called a "device."
In addition, the immunochromatographic test strip of the present invention includes a sample pad and an insoluble membrane carrier, and may further include other reagents and components depending on measurement conditions and samples. Other reagents include, for example, blocking agents that prevent non-specific reactions, and other configurations include, for example, additional pads for removing components unnecessary for measurement in the sample.

(Manufacturing method of test strip for immunochromatography)
The manufacturing method of the immunochromatographic test strip is not particularly limited, but the manufacturing procedure is to apply the conjugate liquid in a line to a portion of the pad-shaped porous material (the portion to be the conjugate portion). , drying to prepare a sample pad, and contacting the sample pad with an insoluble membrane carrier having a detection part to form an immunochromatographic test strip.
For example, the method for producing an immunochromatographic test strip of the present invention comprises the following steps (1) to (3).
(1) A conjugate solution containing a conjugate in which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized on a labeled substance is applied to a portion of a pad-shaped porous material that serves as a sample pad. A step of forming a sample pad by applying in a line and drying to form a conjugate portion, and using a part other than the conjugate portion as a sample supply portion (2) Immunologically against a substance to be detected Step (3) of preparing an insoluble membrane carrier having at least one line-shaped detection part on which a reacting antibody or antigen is immobilized; on the upstream side of the insoluble membrane carrier prepared in (2), the A step of laminating the sample pad so that the distance between the center line of the line of the conjugate part and the center line of the line-shaped detection part is 9 to 21 mm. The concentration of the conjugate is appropriately adjusted according to the amount of conjugate to be contained in the conjugate portion.
The conjugate liquid is applied to a part of the pad-like porous material that will be the sample pad (the part that will be the conjugate part) using a nozzle or the like that can discharge the liquid at a constant speed. Thereafter, the sample pad is obtained by drying by heat drying, natural drying, or the like.
The sample pad has a sample supply part, but the sample supply part is a porous material part that does not contain a conjugate. can be placed in
The method for forming the insoluble membrane carrier, the method for laminating the sample pad and the insoluble membrane carrier, and the like are as described above. When the conjugate portion is provided on the insoluble membrane carrier, the conjugate liquid can be applied to the insoluble membrane carrier in the same manner as the sample pad.

(Method for detecting substance to be detected using immunochromatographic test strip)
The method for detecting a substance to be detected of the present invention comprises the following steps (A) to (D), and is characterized in that all of the steps (A) to (D) are performed in less than 15 minutes. .
(A) contacting the sample supply unit with the substance to be detected;
(B) contacting the substance to be detected in the sample with the conjugate to form a first complex;
(C) contacting the first complex with an antibody or antigen that immunologically reacts with the substance to be detected and immobilized on the detection part to form a second complex; and (D ) Confirming the formation of said second complex by measuring the signal derived from the conjugate.
All of the steps (A) to (D) are preferably performed for 2 minutes or more and less than 15 minutes, more preferably 5 minutes or more and less than 15 minutes, even more preferably 5 minutes or more and less than 10 minutes, most preferably 5 minutes or more and less than 7 minutes. The method for detecting a substance to be detected of the present invention is characterized in that the substance to be detected can be detected with sufficient sensitivity even if steps (A) to (D) are performed in less than 15 minutes.

(others)
The immunochromatographic test strip of the present invention can be prepared by appropriately modifying or altering the methods described in the Examples.
A signal derived from the conjugate may be measured according to a known method, for example, absorbance or intensity of reflected light may be measured.

EXAMPLES Next, the present invention will be specifically described with reference to examples, but these are not intended to limit the scope of the present invention.

[Preparation of anti-influenza virus monoclonal antibody]
The anti-influenza A virus monoclonal antibody and the anti-influenza B virus monoclonal antibody used in the following tests were obtained by using recombinant influenza nucleoprotein as an antigen, immunizing mice, and using a method commonly used by those skilled in the art to produce monoclonal antibodies. was obtained using

[Test strip preparation example 1] Preparation of immunochromatographic test strip of the present invention 1) Preparation of colloidal gold particle-labeled anti-influenza virus monoclonal antibody (conjugate) Anti-influenza A virus monoclonal antibody and anti-influenza B virus monoclonal antibody were prepared. Antibody concentrations and buffer conditions were prepared as shown below. 1 mL of each antibody solution was added to 20 mL of 1 OD/mL colloidal gold particle solution, and the mixture was stirred at room temperature for 10 minutes. 2 mL of 10% bovine serum albumin (BSA) aqueous solution was added to the colloidal gold particles-anti-influenza A virus monoclonal antibody mixture and the colloidal gold-anti-influenza B virus monoclonal antibody mixture, and the mixture was further stirred for 5 minutes. , 10°C and 10,000 rpm for 45 minutes to obtain a sediment (conjugate). Conjugate Dilution Buffer (manufactured by Scripps) was added to the obtained conjugate to suspend the conjugate.

2) Preparation of sample pad The conjugate prepared in 1) above is mixed with a 1.33% casein, 4% sucrose solution (pH 7.5) so as to have an OD/mL of 8 to 20 OD/mL to prepare a conjugate solution. A glass fiber pad having a total length of 28 mm was impregnated so as to form a line with a line width of 4 mm at a position 13 mm, 15 mm, 17 mm or 19 mm from the upstream end of the pad. It was dried by heating at 70°C for 30 minutes to obtain a sample pad containing the conjugate portion.

3) Preparation of insoluble membrane carrier (antibody-immobilized membrane) on which anti-influenza virus monoclonal antibody is immobilized On one end of the short side of a nitrocellulose membrane (pore size 8 μm), an anti-influenza antibody adjusted to 0.75 mg/mL with phosphate buffer Influenza A virus monoclonal antibody, the following anti-influenza B virus monoclonal antibody adjusted to 1.0 mg/mL, and anti-mouse IgG antibody adjusted to 0.75 mg/mL were applied in a line perpendicular to the longitudinal direction of the insoluble membrane carrier. applied in a shape. Then, it was dried at 70°C for 45 minutes to obtain an antibody-immobilized membrane. When the test strip was assembled, the lines were coated in the order of influenza A virus monoclonal antibody (g), anti-influenza B virus monoclonal antibody (h), and control antibody (i) from the upstream side. The centerline of the influenza A virus monoclonal antibody line and the centerline of the control antibody line were 6 mm.

4) Preparation of immunochromatographic test strip Each of the sample pads prepared in 2) was placed so that the distance between the center line of the line of the conjugate part and the center line of the influenza A virus monoclonal antibody line was 15.5 mm and 13.5 mm, respectively. , 11.5 mm, or 9.5 mm to prepare an immunochromatographic test strip 1.

[Test strip preparation example 2] Preparation of immunochromatographic test strip of the present invention Eight glass fiber pads with a total length of 28 mm were used, and the distance from the upstream end of the glass fiber pad was 11 mm, 13 mm, 15 mm, 17 mm, 19 mm, and 21 mm. , the conjugate solution was impregnated so as to form a line with a line width of 4 mm at a position of 23 mm or 25 mm, and the distance between the center line of the line of the conjugate part and the center line of the influenza B virus monoclonal antibody line is 20.5 mm, 18.5 mm, 16.5 mm, 14.5 mm, 12.5 mm, 10.5 mm, 8.5 mm, or 6.5 mm, respectively. A graphic test strip 2 was prepared.

[Example 1]
Using the immunochromatographic test strips prepared in Test Strip Preparation Examples 1 and 2, an influenza virus detection test was performed.
1. Test method (1) sample
Influenza virus type A antigens and influenza virus type B antigens were each diluted with PBS (pH 7.4) containing 2% BSA to 1/160 to 1/2560 to prepare mock samples. (2) Procedure Dispense 135 μL of the simulated sample prepared in (1) above into a test tube, insert the immunochromatographic strip, and after 2 (or 2.5), 5, 7 (or 7.5), 10, and 15 minutes , test line A or B on the antibody-immobilized membrane of the detection portion, and the color development intensity of the control line were measured using an immunochromatographic reader (manufactured by Hamamatsu Photonics).
2. Test Results Tables 1 to 3 show the test line A coloring intensity, the control line coloring intensity, and the ratio (%) of the coloring intensity to the 15-minute control line at each measurement time point of the immunochromatographic device 1 . All numerical values are average values of N=2.

Tables 4 to 6 show the test line B color intensity, the control line color intensity, and the 15-minute color intensity ratio (%) of the control line at each measurement time point for immunochromatographic strip 2. All numerical values are average values of N=2.

(1) Detection sensitivity of the test line In immunochromatographic test strip 1, the distance between the center line of test line A and the line of the conjugate part is 15.5 mm, 13.5 mm, 11.5 mm, or 9.5 mm, respectively. Also, when the measurement was performed 5 minutes after supplying the sample to the sample supplying section, a sufficient measured intensity could be confirmed (see Table 1).
In immunochromatographic test strip 2, when the distance between the center line of test line B and the line of the conjugate portion is 20.5 mm, 18.5 mm, 16.5 mm, 14.5 mm, 12.5 mm, or 10.5 mm, Sufficient measured intensity could be confirmed when the measurement was performed 5 minutes after supplying the sample to the sample supplying section. On the other hand, when the distance between the center line of test line B and the line of the conjugate portion is 8.5 mm or 6.5 mm, respectively, the color development intensity is measured 5 minutes after supplying the sample to the sample supply unit. was less than 40, a sufficient measurement intensity could not be confirmed, resulting in a decrease in detection sensitivity (see Table 4).
(2) Detection sensitivity of control line In immunochromatographic device 1, the distance between the center line of test line A and the line of the conjugate part is 15.5 mm, 13.5 mm, 11.5 mm, or 9.5 mm, respectively. When the measurement was performed 5 minutes after supplying the sample to the sample supplying section, it was possible to confirm a color development intensity ratio of 80% or more for 15 minutes (see Table 3).
In the immunochromatographic device 2, the distance between the center line of the test line B and the line of the conjugate portion is 20.5 mm, 18.5 mm, 16.5 mm, 14.5 mm, 12.5 mm, 10.5 mm, 8.5 mm, or 6.5 mm. Also in the case of (2), a color development intensity ratio of 80% or more for 15 minutes could be confirmed when measured 5 minutes after supplying the sample to the sample supplying section (see Table 6).

According to the immunochromatographic test strip of the present invention, it is possible to provide an immunochromatographic test strip having a short reaction completion time and excellent sensitivity.

(a) plastic adhesive sheet (b) film (c) absorbent pad (d) antibody-immobilized membrane (e) sample pad (f) conjugate part (g) test line A
(h) Test line B
(i) control line (D) distance between the center line of the line of the conjugate portion and the center line of the detection portion (W) line width of the conjugate portion

Claims (7)

An immunochromatographic test strip for detecting a substance to be detected by developing a sample that may contain the substance to be detected, comprising the following (1) and (2): (1) or (2) A line-shaped conjugate having a width of 2 to 8 mm containing a conjugate in which an antibody or an antigen that immunologically reacts with the substance to be detected is immobilized on the labeled body between the sample supply part and the detection part in A test strip for immunochromatography, characterized in that a portion is provided, and the distance between the center line of the line of the conjugate portion and the center line of the line-shaped detection portion is 9 to 21 mm.
(1) A sample pad having a sample supply unit that supplies a sample that may contain a substance to be detected (2) A linear antibody or antigen that immunologically reacts with the substance to be detected is immobilized An insoluble membrane carrier having at least one detection part of The antibody or antigen immobilized on the label that immunologically reacts with the substance to be detected and the antibody or antigen immobilized on the detection part that immunologically reacts with the substance to be detected , the immunochromatographic test strip according to claim 1, which is different. The immunochromatographic test strip according to claim 1 or 2, which is of lateral flow type. The substance to be detected is influenza virus, and the antibody or antigen that immunologically reacts to the substance to be detected immobilized on the label and the substance to be detected that is immobilized to the detection part immunologically reacts. The immunochromatographic test strip according to any one of claims 1 to 3, wherein the antibody or antigen to be tested is an anti-influenza virus monoclonal antibody. The immunochromatographic test strip according to any one of claims 1 to 4, wherein the conjugate portion is provided on the sample pad. The signal intensity of the control capture reagent immobilized portion 5 minutes after supplying the biological sample to the sample supply portion is the signal intensity of the control capture reagent immobilized portion 15 minutes after the biological sample is supplied to the sample supply portion. 80% or more of the immunochromatographic test strip according to any one of claims 1 to 5 . A method for detecting a substance to be detected using the immunochromatographic test strip according to any one of claims 1 to 6 , comprising steps (A) to (D), wherein the steps (A) to (D) ) are all performed in less than 15 minutes.
(A) contacting the sample supply unit with the substance to be detected;
(B) contacting the substance to be detected in the sample with the conjugate to form a first complex;
(C) contacting the first complex with an antibody or antigen that immunologically reacts with the substance to be detected and immobilized on the detection portion to form a second complex;
(D) confirming the formation of the second complex by measuring the signal derived from the conjugate

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