JP5541748B2 - Immunochromatographic test device using avidin-biotin linked labeling reagent and use thereof - Google Patents

Description

  The present invention relates to an immunochromatographic test device.

  An immunochromatographic method combining a chromatographic method using a capillary phenomenon and an immunological method is widely used as a method capable of analyzing a test substance in a sample easily and in a short time.

  FIG. 1 shows a conventional general immunochromatographic test device. The sample addition pad 8, the development pad 3, the conjugate pad 2, the reaction membrane 1, and the absorption pad 4 are arranged in this order from the upstream (left end in the figure) to the downstream (right end in the figure) in the development direction. Are disposed on the backsheet 5. The conjugate pad is impregnated with a first affinity substance (labeling reagent) that is labeled with a labeling substance such as colloidal gold particles and specifically binds to the test substance. The reaction membrane has a test line 6 on which a second affinity substance (capture reagent) that specifically binds to a test substance at a site different from the first affinity substance is immobilized.

  Next, analysis of a test substance using this immunochromatographic test device will be described. When the test solution is supplied to the sample addition pad 8, it moves to the conjugate pad 2 via the development pad 3 by capillary action. Then, the labeling reagent impregnated in the conjugate pad is eluted in the test solution. The test solution in which the labeling reagent is dissolved is successively developed to the test line 6 on the reaction membrane 1 by capillary action. Here, if the test substance is contained in the sample, the complex of the labeling reagent and the test substance formed during the development is captured by the capture reagent immobilized on the test line. As a result, the test substance in the sample can be analyzed by determining the presence or absence or degree of signal generated by the captured labeling substance.

  In many cases, a control line 7 on which a substance (control reagent) that specifically binds to the labeling reagent but does not bind to the test substance is provided on the reaction membrane downstream in the development direction from the test line. It is used as an index for determining whether or not a test is possible. If a signal is detected on the control line, it means that the amount of test solution and labeling reagent required for the measurement has been successfully developed on the test line and the control line, but if the control line is not detected, The test is considered unsuccessful because the required amount of test solution and labeling reagent may not have developed.

  As a control reagent immobilized on the control line, an antibody against a labeling reagent has generally been used. For example, when a test substance-specific labeled mouse monoclonal antibody is used as the labeling reagent, an anti-mouse antibody is used as the control reagent.

  However, in tests using biological samples such as blood, interference-removing antibodies are often used because there are specimens having heterophilic antibodies against various animals such as human anti-mouse antibodies (HAMA). The interference-removing antibody is usually an unlabeled antibody of the same type as the labeled antibody specific for the test substance, and is used in excess compared to the labeled antibody. Therefore, when using the interference-removing antibody in the test device having the control mechanism as described above, there is a problem that the signal intensity of the control line is remarkably reduced by capturing the interference-removing antibody in the control reagent. Measures such as increasing the amount of immobilization and reducing the amount of interference-removing antibody used have been required.

On the other hand, Patent Documents 1 and 2 provide an immunochromatographic test device that prepares an independent control labeling substance that is not affected by an interference substance. For example, Patent Literature 1 describes an immunochromatographic test device in which a test substance-specific labeled antibody and labeled biotin are mixed as a labeling reagent and avidin is immobilized as a control reagent.
In addition, an immunochromatographic test device that binds a first affinity substance and a labeling substance using an avidin-biotin reaction has been reported so far for the purpose of increasing measurement sensitivity or manufacturing advantages. (Patent Documents 3 and 4). However, in all cases, avidin-biotin is reacted in the measurement process, and in the example of Patent Document 4, an immunochromatographic test device separately provided with a control labeling reagent is used. Patent Document 3 discloses that it is not preferable to react avidin (or biotin) -conjugated antibody and marker-labeled biotin (or avidin) in advance and react with the substance to be measured, because the measurement sensitivity decreases. (Paragraph 0011).

JP 2001-033454 A JP 2009-087551 A JP-A-10-68730 Special table 2010-512537 gazette

However, an increase in the amount of control reagent immobilized is physically limited, leading to increased costs. In addition, by suppressing the amount of interference-removing antibody used, the measurement result may be affected by heterophilic antibodies. On the other hand, in the test devices of Patent Documents 1 and 2, a labeling reagent must be separately prepared for the control reagent, which causes problems in complication and cost increase in reagent preparation. Further, the development of a control labeling reagent that is not involved in the reaction with the test substance may cause a nonspecific reaction on the test line.
Therefore, the present invention is not affected by endogenous components (such as heterophilic antibodies) or test reagent components (such as interference-removing antibodies) contained in the specimen, and it is necessary to prepare a control labeling reagent separately. It is an object of the present invention to provide an immunochromatographic test device having an improved control mechanism without any problem.

As a result of intensive studies, the present inventors used a labeling reagent in which a first affinity substance specific for a test substance and a labeling substance were linked using an avidin-biotin reaction, and used as a control reagent. It has been found that all of the above-mentioned drawbacks can be eliminated by using a biotin-containing one (biotin or biotinylated product).
The avidin-biotin reaction is commonly used in test methods such as immunochromatography, but the avidin-biotin interaction is rapidly formed, and once formed, the interaction has a very broad pH range. In a labeling reagent in which a sufficient amount of a labeling substance and a first affinity substance are linked using an avidin-biotin reaction, it is known that the temperature, organic solvent and other denaturing agents are not affected. It has not been thought that avidin capable of binding to the biotinylated product remains. However, the present inventors have unexpectedly found that avidin capable of binding to a biotinylated substance remains in the labeling reagent linked by the avidin-biotin reaction, and it is possible to use the biotinylated substance as a control reagent. . Furthermore, by using a labeling reagent linked by an avidin-biotin reaction, not only a good S / N ratio is exhibited, but also an immunochromatographic test device which has the further advantage of keeping the labeling reagent performance constant for each production lot. Has been found to be able to be produced, and the present invention has been completed.

That is, the present invention
[1] A test line on which a capture reagent that specifically reacts with a test substance is immobilized;
A control line on which a control reagent that specifically binds to the labeling reagent is immobilized;
An immunochromatographic test device comprising:
The labeling reagent is obtained by linking a first affinity substance and a labeling substance that specifically bind to a test substance via avidin and biotin,
The test reagent for immunochromatography, wherein the control reagent contains biotin,
[2] The immunochromatographic test device according to [1], wherein the control reagent is a biotinylated protein,
[3] The immunochromatographic test device according to [1] or [2], wherein the capture reagent is a second affinity substance, or a competitive substance of a test substance or an analog thereof,
[4] The immunochromatographic test device according to any one of [1] to [3], wherein the first affinity substance and / or the second affinity substance is an antibody.
[5] The immunochromatographic test device according to any one of [1] to [4], comprising an interference-removing antibody;
[6] A step of bringing a test substance into contact with a labeling reagent,
Capturing a complex of a test substance and a labeling reagent with a capture reagent that specifically binds to the test substance;
Capturing a test substance and an unreacted labeling reagent with a control reagent that specifically binds to the labeling reagent;
Detecting a labeling reagent captured with a capture reagent and a control reagent;
A method for analyzing a test substance containing
The labeling reagent is obtained by linking a first affinity substance and a labeling substance that specifically bind to a test substance via avidin and biotin,
The method for analyzing a test substance, wherein the control reagent contains biotin,
[7] Step of capturing the test substance with a capture reagent that specifically reacts with the test substance Step of contacting the label reagent with the complex of the test substance and the capture reagent The test substance and the unreacted labeling reagent Capturing with a control reagent that specifically binds to the labeling reagent,
Detecting a labeling reagent captured with a capture reagent and a control reagent;
A method for analyzing a test substance containing
The labeling reagent is obtained by linking a first affinity substance and a labeling substance that specifically bind to a test substance via avidin and biotin,
The method for analyzing a test substance, wherein the control reagent contains biotin,
[8] A step of contacting a test substance with a labeling reagent,
Capturing the unreacted labeling reagent with the test substance with a capture reagent that is a competitive substance of the test substance or its analog,
Capturing a test substance and an unreacted labeling reagent with a control reagent that specifically binds to the labeling reagent;
Detecting a labeling reagent captured with a capture reagent and a control reagent;
A method for analyzing a test substance containing
The labeling reagent is obtained by linking a first affinity substance and a labeling substance that specifically bind to a test substance via avidin and biotin,
The method for analyzing a test substance, wherein the control reagent contains biotin,
About.

  According to the present invention, since the control reagent is not affected by the components of the test reagent (such as the interference removing antibody), an appropriate amount of the interference removing antibody can be used. For this reason, it is possible to avoid the influence of endogenous components (heterophilic antibodies and the like) contained in the specimen. Thereby, the certainty of the determination in a test line and a control line can be improved. In addition, since it is not necessary to prepare a control labeling reagent separately, a nonspecific reaction due to the control labeling substance in the test line does not occur, and an immunochromatographic test device can be manufactured easily and at low cost.

1 is a schematic cross-sectional view of a general immunochromatographic test device.

The present invention relates to an immunochromatographic test device for analyzing a test substance in a specimen and use thereof. In this specification, “analysis” includes both “measurement” for quantitatively or semi-quantitatively determining the amount of the test substance and “detection” for determining the presence or absence of the test substance. It is.
Hereinafter, although the form for implementing this invention is demonstrated, the test device of this invention can consist of the structure similar to a conventionally well-known immunochromatography test device except the structure of a labeling reagent and a target reagent. However, the present invention is not limited to the following embodiments.

There are two types of analysis using immunochromatographic test tools: competitive methods and non-competitive methods, and antigen detection systems and antibody detection systems. Those skilled in the art will select the appropriate test method according to the test substance. The present invention can be used. An example will be described in detail in the examples described later. In the case of an antigen detection system of the non-competitive method, a test substance-specific antibody (second affinity substance) is used as a capture reagent, and biotinylated BSA (biotinylated product) is used as a control reagent. ), And a test substance-specific antibody (first affinity substance) and a colloidal gold (label substance) linked by a streptavidin-biotin reaction as a labeling reagent. Test lines and control lines can be determined.
In the case of a non-competitive antibody detection system, for example, when the test substance is an allergen-specific IgE antibody, allergen (second affinity substance) is used as a capture reagent, and biotinylated BSA (biotinylated product) is used as a control reagent. Use a test line with a single labeling reagent by using a human IgE antibody-specific antibody (first affinity substance) and colloidal gold (labeling substance) linked by a streptavidin-biotin reaction as the labeling reagent. And the control line can be determined.
In the case of the competition method, for example, when the test substance is a hapten, a competition hapten is used as a capture reagent, biotinylated BSA (biotinylated product) is used as a control reagent, and a hapten-specific antibody (first affinity substance) is used as a labeling reagent. ) And gold colloid (labeling substance) linked by streptavidin-biotin reaction, the test line and the control line can be determined with one labeling reagent.

  The test device of the present invention comprises a test line on which a capture reagent that specifically reacts with a test substance is immobilized on a reaction membrane, and a control reagent that includes a first affinity substance that specifically binds to a labeling reagent. And a control line to which the test substance is immobilized, and the test solution presumed to contain the test substance passes through the test line and the control line in order of capillary action. Two or more test lines may be provided as necessary. Note that the test line and the control line are not limited to a line shape, and may be a circular shape, a polygonal shape, or the like.

  The test device of the present invention preferably further includes a sample addition pad for introducing the test solution upstream of the reaction membrane in the developing direction and an absorption pad for sucking up the test solution downstream of the reaction membrane. In the case where the test substance and the labeling reagent are bound on the test tool, it is preferable to provide a conjugate pad that holds the labeling reagent in a developable manner between the sample addition pad and the reaction membrane. For example, since the test reagent is not diluted because the labeling reagent is dried and held on the conjugate pad, the detection sensitivity is obtained by reacting the test substance with the first affinity substance or the second affinity substance at a high concentration. Can be raised. Further, if necessary, a development pad may be provided between the sample addition pad and the conjugate pad, and it is preferable that these members are disposed on an adhesive backsheet.

  The test solution used in the test device of the present invention includes not only a sample that may contain a test substance or a processed product thereof (for example, a lysate, a diluted product, etc.), but also various types of samples or processed products thereof. A mixture to which reagents (for example, a labeling reagent, an interference removing antibody, etc.) are added is also included. The specimen is not particularly limited as long as it may contain a test substance. Blood, serum, plasma, urine, pus, tears, sweat, saliva collected from animals (particularly humans) In addition to bodily fluids such as sputum and runny nose, or biological samples such as feces, organs, tissues, mucous membranes and skin, and swabs that are thought to contain them, culture fluids such as bacteria and viruses, suspensions, An extract etc. can be mentioned.

The labeling reagent used in the present invention includes one in which a first affinity substance that specifically binds to a test substance and a labeling substance are linked via avidin and biotin. Those skilled in the art can use the labeling substance by appropriately selecting from known substances, such as metal colloids such as gold, silver, platinum, and palladium, latex colored particles that are polymer polymer fine particles, and the like. And natural or synthetic resins and derivatives thereof, and liposomes and microcapsules containing dyes. In view of ease of handling and sensitivity, colloidal gold is particularly preferable. The particle size of the metal colloid is not particularly limited, but is preferably about 1 to 500 nm, and more preferably 5 to 100 nm, at which a particularly strong color tone can be obtained. The first affinity substance is not particularly limited as long as it is a substance that specifically binds to a test substance and can bind to avidin or biotin, and includes an antibody, an antigen, an aptamer, and the like. A person skilled in the art can determine appropriately according to the test substance. The antibody may be a monoclonal antibody or a polyclonal antibody, and may be used as an F (ab ′) ₂ , Fab ′ or Fab fragment by enzymatic treatment or chemical treatment.

  The labeling reagent used in the present invention is prepared by preparing an avidin (or biotin) -immobilized labeling substance and a biotin (or avidin) -binding first affinity substance, and then reacting both avidin and biotin. be able to.

  Avidin may be bound to either the labeling substance or the first affinity substance, but when binding to the labeling substance, after preparing the avidin-immobilized labeling substance and the biotinylated first affinity substance, It is preferable to connect both. Examples of avidin used in the present invention include egg white avidin and streptavidin, and analogs such as neutravidin. Streptavidin is particularly preferred because of less non-specific binding. There is no particular limitation as long as it has the ability to bind to biotin, and amino acid mutations, additions, deletions and the like can be appropriately performed.

  The avidin-immobilized labeling substance can be prepared by a known method such as physical adsorption, and any method may be used as long as it does not deactivate the biotin-binding ability of avidin. The degree of staining of the test line and control line varies depending on the amount of avidin fixed to the labeling substance, so depending on the type of labeling substance and avidin used, or the sensitivity and measurement range of the test substance, The reaction concentration can be set as appropriate.

The biotinylated first affinity substance can be prepared by a known method, and any method may be used as long as it does not deactivate the binding ability of the first affinity substance. Many biotinylation reagents such as those capable of binding biotin to the SH group, amino group, carboxyl group, or sugar chain of proteins are commercially available. When an antibody is used as the first affinity substance, the antibody species that can be biotinylated is not particularly limited, and (Whole) immunoglobulin (Ig) as a full length, F (ab ′) ₂ , Fab ′ that is an enzyme digest thereof. Alternatively, Fab fragments and the like can be used. In addition, a chimeric antibody obtained by chimerizing a protein into which biotin is introduced by recombinant gene manipulation can be used as a biotinylated antibody.

  In the labeling reagent used in the present invention, compared to a labeling reagent in which a labeling substance and a first affinity substance are directly bound, variation in reagent performance that occurs in each production lot can be kept constant, and storage stability can be improved. It becomes possible.

  In the present invention, the capture reagent immobilized on the test line includes a second affinity substance that specifically binds to the test substance, or a competitive substance of the test substance (or an analog thereof). For example, a general non-competitive method includes a second affinity substance that specifically binds to a test substance, and a competitive method includes a test substance (or an analog thereof). The competitive method is preferable because a low molecular weight compound or a hapten alone can be analyzed with a low antigenicity.

The second affinity substance is not particularly limited as long as it specifically binds to the test substance, and examples thereof include antibodies, antigens, aptamers and the like. The antibody may be used as an F (ab ′) ₂ , Fab ′ or Fab fragment by enzymatic treatment or chemical treatment. The second affinity substance may bind to the same site as the first affinity substance, but preferably binds specifically to the test substance at a different site. The competitive substance of the test substance (or an analog thereof) is not particularly limited as long as it specifically binds to the first affinity substance, and examples thereof include those having the same or similar structure as the test substance in the specimen. .

  The control reagent used in the present invention is not particularly limited as long as it contains biotin. Although biotin may be directly immobilized on the reaction membrane, it is desirable to use biotin bound to a support material. The support substance is preferably a protein, and the protein is not particularly limited as long as it is a protein that can react with a biotinylation reagent. The protein that can be used is preferably a protein that does not react with other substances during the measurement, and includes serum albumin, and bovine serum albumin (BSA) is particularly preferable. It is also possible to use biotin-introduced proteins prepared by recombinant gene manipulation or existing in nature. Examples of the biotinylation reagent include those similar to the preparation of a biotinylated first affinity substance. However, in the biotinylation reagent used for the preparation of the control reagent, the spacer length introduced into biotin has a biotin-binding first affinity. It is preferable to use the same or longer spacer length compared to the spacer length introduced into biotin used in the sex substance.

  The test device of the present invention uses an interference-removing antibody in order to avoid the influence of heterophilic antibodies that can cause misjudgment by reacting with a test substance-specific first affinity substance or second affinity substance. This is particularly effective when included. The interference-removing antibody is not particularly limited as long as it is a substance capable of reacting with a heterophilic antibody, but is preferably the same type of antibody as the first affinity substance and / or the second affinity substance. . For example, when the first affinity substance and / or the second affinity substance is a mouse monoclonal antibody, a mouse antibody is preferably used as the interference-removing antibody. The interference-removing antibody is preferably contained before the test substance comes into contact with the capture reagent and / or the labeled antibody. For example, it is preferably contained in any one or more of a test solution (labeling reagent, specimen processing reagent, etc.) or a member (sample addition pad, development pad, conjugate pad, etc.). A person skilled in the art can appropriately determine the difference in the test process.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

<< Example 1: Preparation and evaluation of immunochromatographic test device >>
(1) Preparation of labeling reagent (1-1) Preparation of streptavidin-immobilized gold colloid 10 mL of gold colloid solution (particle size) prepared with 5 mmol / L phosphate buffer pH 7.5 so that the absorbance at 520 nm is 10. Streptavidin (Wako Pure Chemical Industries, Ltd.) was mixed with 40 nm, Tanaka Precious Metals) so that the final concentration at the time of mixing was 0.1 mg / mL.
After the mixture was stirred for 1 hour, a polyethylene glycol (Mw. 20000, Wako Pure Chemical Industries) solution was added to a final concentration of 0.5%, and the mixture was further stirred for 1 hour. Subsequently, BSA (SIGMA) solution was added to a final concentration of 1%, and the mixture was stirred for 1 hour for blocking. This solution was centrifuged at 10000 × g for 10 minutes at 4 ° C. to remove the supernatant, and a dispersion buffer (5 mmol / L phosphate buffer pH 7.5 containing 1% BSA and 0.05% polyethylene glycol) was added. In addition, it was redispersed. Centrifugation was again performed at 4 ° C., 10,000 × g for 10 minutes, and the supernatant was removed and redispersed in a dispersion buffer to prepare a streptavidin-immobilized gold colloid solution.

(1-2) Preparation of Biotinylated Antibody (Capture Reagent) Anti-Mycoplasma pneumoniae 2 mg / mL F (ab ′) ₂ derived from mouse monoclonal antibody (MCM12) and SH group reducing agent (TCEP: PIERCE) at a final concentration of 0 After adding to 4 mmol / L to make Fab ′, maleimidated biotin reagent EZ-Link Maleimide-PEG2-Biotin (spacer length 29.1 mm, PIERCE) was adjusted to a final concentration of 0.4 mmol / L. And bound. This reaction product was buffer-exchanged to 50 mmol / L phosphate buffer pH 7.5 using an ultrafiltration membrane having a fractional molecular weight of 10,000 to prepare a biotinylated antibody.

(1-3) Preparation of Labeling Reagent The biotinylated antibody obtained in (1-2) was mixed with 10 mL of the streptavidin-immobilized gold colloid solution (OD _{520 nm} = 9) obtained in (1-1). Add to a concentration of 0.14 mg / mL, stir at 25 ° C. for 1 hour, leave at 4 ° C. for 20 hours, centrifuge at 4 ° C., 10,000 × g for 10 minutes to remove the supernatant and disperse The working buffer was added and redispersed. Centrifugation was again performed at 4 ° C., 10000 × g for 10 minutes, and the supernatant was removed and re-dispersed in a dispersion buffer to obtain a labeling reagent.

(2) Preparation of Control Reagent A 12 mg / mL BSA (SIGMA) biotinylated reagent EZ-Link NHS-PEG12-Biotin (spacer length 56 mm, PIERCE) that reacts with amino groups is adjusted to a final concentration of 3.2 mmol / L. Then, the solution was exchanged with a 50 mmol / L phosphate buffer (pH 7.5) using an ultrafiltration membrane with a molecular weight cut off of 50,000, and used as a control reagent.

(3) Preparation of immunochromatographic test device (3-1) Preparation of conjugate pad The labeling reagent prepared in (1) was prepared with a dispersion buffer so that OD _{520 nm} = 4.5. This was impregnated with 10 μL of a 5 mm × 6 mm glass fiber pad (Millipore) and dried overnight at room temperature to prepare a conjugate pad.

(3-2) Preparation of reaction membrane An anti-Mycoplasma pneumoniae mouse monoclonal antibody (MCM19) solution prepared to be 2 mg / mL as a test line on a 30 mm × 6 mm nitrocellulose membrane (Millipore), 2 mg / ml as a control line The control reagent (2) prepared to be mL was applied in a line shape having a width of about 1 mm using a coating machine (BioDot) and dried to prepare a reaction film. The control line was provided at a position 9 mm from the downstream end of the nitrocellulose membrane, and the test line was provided at a position 14 mm from the downstream end of the nitrocellulose membrane.

(3-3) Assembly of immunochromatographic test device Each member was affixed on a 69 mm x 6 mm backsheet (Nichiei Kako) as shown in FIG. First, the reaction film prepared in (3-2) was attached. Furthermore, the conjugate pad prepared in (3-1), a development pad composed of a 17 mm × 6 mm glass fiber pad (Millipore), a 10 mm × 6 mm sample addition pad (Nihon Pole), a 26.7 mm × 6 mm absorption pad ( NIPPON POLE) was stacked and pasted to prepare an immunochromatographic test device.

(4) Mycoplasma pneumoniae solution was prepared as a sample for evaluation and evaluation of immunochromatographic test devices . The cultured Mycoplasma pneumoniae was suspended in the extract so as to be 10 ⁴ to 10 ⁷ CFU / mL, and used as a positive specimen. A negative sample for evaluation was prepared that does not contain Mycoplasma pneumoniae. The composition of the extract is 0.1 mmol / L phosphate buffer pH 7.5, 0.15 mmol / L NaCl, 1% Triton X-100, 1% BSA.

120 μL of each specimen was dropped on each immunochromatographic test device prepared in (3), and staining of the test line and the control line after 15 minutes was determined. The results are shown in Table 1. The degree of staining was evaluated semi-quantitatively by comparing “+” to “+++++ (6+)” from weak to strong staining with a color sample for determination. “-” Indicates a negative reaction.
With the test device of the present invention, it was confirmed that the test line and the control line were judged with one labeling reagent over a concentration range of 10 ⁴ to 10 ⁷ CFU / mL. It has been found useful because it can be used in a concentration range of 10 ⁴ to 10 ⁷ CFU / mL which is usually required as a detectable range.

<< Example 2: Confirmation of effect when using interference-removing antibody >>
A sample treatment pad (Nihon Pole) was dipped in 0-10 mg / mL normal mouse antibody (MaK33 IgG1: Roche) and then dried to prepare a sample addition pad containing an interference removing antibody. As an evaluation sample, 10 ⁵ CFU / mL of Mycoplasma pneumoniae bacterial solution was used. Otherwise, an immunochromatographic test device was prepared in the same manner as in Example 1, and the visibility of the test line and the control line at the time of determination was evaluated.

The results are shown in Table 2. The degree of staining was evaluated semi-quantitatively by comparing “+” to “+++++ (6+)” from weak to strong staining with a color sample for determination.
Even when the interference-removing antibody, the capture antibody and the labeled antibody were mouse antibodies of the same subclass, no decrease in the control line was observed. It was confirmed that the test device of the present invention can avoid the effects of heterophilic antibodies and interference-removing antibodies.

With the test device of the present invention, the test line and the control line can be determined with high certainty with one labeling reagent. In addition, since it is not necessary to prepare a control labeling reagent separately, it is possible to manufacture an immunochromatographic test device easily and at low cost without causing non-specific reaction due to the control labeling substance in the test line. It is useful above.
As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.

1 ... Reaction membrane; 2 ... Conjugate pad; 3 ... Development pad;
4 ... Absorbing pad; 5 ... Back sheet; 6 ... Test line;
7 ... control line; 8 ... sample addition pad.

Claims (8)

A test line on which a capture reagent that specifically reacts with a test substance is immobilized;
A control line on which a control reagent that specifically binds to the labeling reagent is immobilized;
An immunochromatographic test device comprising:
The labeling reagent is obtained by linking a first affinity substance and a labeling substance that specifically bind to a test substance via avidin and biotin,
An immunochromatographic test device, wherein the control reagent contains biotin.   The immunochromatographic test device according to claim 1, wherein the control reagent is a biotinylated protein.   The immunochromatographic test device according to claim 1 or 2, wherein the capture reagent is a second affinity substance, or a competitive substance of a test substance or an analog thereof.   The immunochromatographic test device according to any one of claims 1 to 3, wherein the first affinity substance and / or the second affinity substance is an antibody.   The immunochromatographic test device according to any one of claims 1 to 4, comprising an interference-removing antibody. A step of bringing a test substance into contact with a labeling reagent,
Capturing a complex of a test substance and a labeling reagent with a capture reagent that specifically binds to the test substance;
Capturing a test substance and an unreacted labeling reagent with a control reagent that specifically binds to the labeling reagent;
Detecting a labeling reagent captured with a capture reagent and a control reagent;
A method for analyzing a test substance containing
The labeling reagent is obtained by linking a first affinity substance and a labeling substance that specifically bind to a test substance via avidin and biotin,
A method for analyzing a test substance, wherein the control reagent contains biotin. A step of capturing a test substance with a capture reagent that reacts specifically with the test substance A step of contacting a labeling reagent with a complex of the test substance and the capture reagent Labeling the test substance and an unreacted labeling reagent Capturing with a control reagent that specifically binds to the reagent;
Detecting a labeling reagent captured with a capture reagent and a control reagent;
A method for analyzing a test substance containing
The labeling reagent is obtained by linking a first affinity substance and a labeling substance that specifically bind to a test substance via avidin and biotin,
A method for analyzing a test substance, wherein the control reagent contains biotin. A step of bringing a test substance into contact with a labeling reagent,
Capturing the unreacted labeling reagent with the test substance with a capture reagent that is a competitive substance of the test substance or its analog,
Capturing a test substance and an unreacted labeling reagent with a control reagent that specifically binds to the labeling reagent;
Detecting a labeling reagent captured with a capture reagent and a control reagent;
A method for analyzing a test substance containing
The labeling reagent is obtained by linking a first affinity substance and a labeling substance that specifically bind to a test substance via avidin and biotin,
A method for analyzing a test substance, wherein the control reagent contains biotin.

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