JP2012225726A - Sandwich immunoassay method and biosensor using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sandwich immunoassay method capable of determining false negative when a medical operator omits to set a specimen in error when preparing a specimen liquid, and to provide a biosensor using the sandwich immunoassay method.SOLUTION: In the biosensor configured by placing an optically transparent plate on which a recess is formed on a support to form a flow channel between the plate and the support, forming an injection/drainage port for successively injecting/draining a measurement solution including a specimen on one end of the flow channel and forming an exhaust port on the other end, an anti-IgE antibody capture part for capturing an IgE antibody included in the measurement solution and an allergen capture part for capturing a specific IgE antibody in the IgE antibody are arranged on the surface of the support between the liquid injection/drainage port side in the flow channel and the exhaust port.

Description

  The present invention relates to a detection apparatus for detecting a substance to be detected in a specimen by a sandwich immunoassay method and a method for detecting using the same.

  The present invention relates to patent applications related to the results of commissioned research by the national government (FY2008, FY2009 and FY2009, Japan Science and Technology Agency, Industry-Academia Innovation Acceleration Project “Advanced Measurement and Analysis Technology / Equipment Development”, Patent application subject to the application of Article 19 of the Industrial Technology Strengthening Act).

  One method for detecting the amount of a specific component in a specimen is a sandwich assay. The sandwich assay method is a method in which a substance to be detected is sandwiched and quantified by two kinds of substances, and since it can be detected quickly and with high sensitivity, it is widely used in measurement of chemical substances and clinical examinations. Among sandwich assay methods, a method of sandwiching a detected substance with an antibody and quantifying it is particularly called a sandwich immunoassay method.

  As a clinical test kit using the sandwich immunoassay method, there is a rapid measurement kit for diagnosing influenza. In this kit, a detection device based on a sample mixing container and a lateral flow is prepared. Here, the lateral flow is generally an immunoassay which is also called immunochromatography and is performed on a membrane. This detection apparatus is provided with a nitrocellulose membrane as a reaction field for performing an assay, and a plastic container is installed so as to cover the membrane. Furthermore, a capture reagent unit for visually judging the test result is prepared at one corner of the membrane. Diagnosis of influenza virus by this is performed as follows. First, the collected specimen and gold colloid-labeled anti-influenza virus antibody are mixed in a sample mixing container to obtain a specimen liquid. Thereafter, the sample liquid is added to a supply unit provided in the detection apparatus. The added specimen liquid is developed toward the water-absorbing carrier by capillary action. Finally, the presence or absence of human influenza virus in the specimen is detected by confirming the color change generated by the colloidal gold in the capture reagent part.

  However, in the detection of such a specimen, there is a problem of so-called false negative, in which it is determined that the substance to be detected is negative even though the substance to be detected is present in the specimen. If this false negative occurs, it not only delays the identification of the cause, but also causes serious medical accidents such as a serious medical condition caused by inappropriate measures. That is, preventing false negatives is a very important issue.

  Therefore, by immobilizing a recombinant influenza virus antigen that specifically reacts with the labeling reagent in the sample as a control part downstream of the capture reagent part of the nitrocellulose membrane, and confirming the color development of the control part Means for preventing false negatives are known by a method for discriminating that the liquid has reached the capture reagent part (see, for example, Patent Document 1).

JP 2007-333426 A

However, the above-described conventional configuration has a problem in that when a sampler prepares a sample liquid, a medical worker mistakenly forgets to put a sample, the negative is displayed, and an erroneous result (false negative) is generated.

  The present invention solves the above-mentioned conventional problems, and when a sampler prepares a sample solution, if a medical worker mistakenly forgets to insert a sample, a sandwich immunoassay method that can be determined as a false negative and a biosensor using the same The purpose is to provide.

In order to solve the above-mentioned conventional problems, the sandwich immunoassay method of the present invention and the biosensor using the same are obtained by placing an optically transparent plate having recesses on a support, and the plate and the support. In the biosensor having a flow passage formed between the two, and a pouring / draining port for sequentially pouring / discharging the measurement solution containing the sample at one end of the flow channel and an exhaust port at the other end, An anti-IgE antibody capturing part for capturing an IgE antibody contained therein, and an allergen capturing part for capturing a specific IgE antibody in the IgE antibody from the side of the water injection port in the flow path. It arrange | positions on the said support body surface between mouths, It is characterized by the above-mentioned.
The present invention also relates to a sandwich immunoassay method and a biosensor using the same, an anti-IgE antibody capturing part for capturing an IgE antibody contained in a measurement solution containing a specimen, and a specific IgE antibody among the IgE antibodies. An allergen-capturing section for capturing a label, an IgE-antibody capturing section for capturing a labeled anti-IgE antibody contained in the measurement solution, a labeled protein-capturing section for capturing a labeling enzyme contained in the measurement solution, and the measurement A plurality of sample solutions containing a sample are sequentially injected into an immune reaction system having a labeling enzyme capturing unit for capturing a detection reagent contained in the solution to cause an immune reaction, and then the enzyme activity is detected in the anti-IgE antibody capturing unit. Is detected and enzyme activity is detected in all of the IgE antibody capturing part, the labeled protein capturing part and the labeled enzyme capturing part, Is obtained is characterized in that the determined not to contain the analyte.

  According to the sandwich immunoassay method of the present invention and the biosensor using the same, it is possible to prevent the occurrence of false negatives if it can be determined whether or not the sample is correctly added to the sample solution.

Configuration diagram of biosensor in embodiment 1 of the present invention Exploded view of biosensor in embodiment 1 of the present invention The figure which shows the measurement procedure of the sandwich immunoassay method in Embodiment 1 of this invention Configuration diagram of biosensor in Examples 1 to 6 of the present invention The figure which shows the photometry image in Example 1 of this invention. The figure which shows the photometry image in Example 2 of this invention. The figure which shows the photometry image in Example 3 of this invention. The figure which shows the photometry image in Example 4 of this invention. The figure which shows the photometry image in Example 5 of this invention. The figure which shows the photometry image in Example 6 of this invention. Configuration diagram of conventional biosensor

  First, the prior art shown in FIG. 11 will be described. In the conventional capturing method, a colloidal gold-labeled anti-influenza virus antibody, which is a labeling reagent, can be captured as a control unit 1103 in the downstream region of the capture reagent unit 1102 provided on the nitrocellulose membrane 1101. Therefore, when the sample liquid 1109 containing the sample 1107 and the labeling reagent 1108 is injected from the supply unit 1104, the sample liquid 1109 is developed in the nitrocellulose membrane 1101 toward the water-absorbing carrier 1105 by capillary action. Finally, the presence or absence of the injection of the sample liquid 1109 is confirmed by confirming the change in color emitted by the gold colloid in the capture reagent unit 1102 and the control unit 1103. However, in the conventional configuration, since the determination is made only with the label component, it is determined as negative (false negative) and erroneously determined when the sample is not included.

Therefore, in this method, in order to completely prevent false negatives, not only the label component but also the sample component, such as the sample component, can be captured by the capture unit to capture the sample component. It was made possible to determine that the sample solution to be successfully developed in the biosensor.
Hereinafter, embodiments of a sandwich immunoassay method of the present invention and a biosensor using the same will be described in detail with reference to the drawings.

(Embodiment 1)
FIG. 1 shows a configuration diagram of a biosensor according to the first embodiment of the present invention.
The support body 101 in FIG. 1 is provided with a capturing part region 102 that can capture biomolecules. An upper cover 108 in which a flow path 111 is formed so as to cover the capturing part region 102 is connected thereto. One end of the upper cover 108 has an inlet / outlet port 109 for sequentially injecting / draining a plurality of types of sample solutions necessary for the measurement including the specimen in the flow path 111, and an exhaust port 110 at the other end. is doing. Furthermore, an allergen capture unit 103, an anti-IgE antibody capture unit 104, an IgE antibody capture unit 105, a labeled protein capture unit 106, and a label enzyme capture unit 107 are disposed in the capture unit region 102 in the channel 111. Is made up of. Here, the labeled product in the labeled protein capturing unit 106 is biotin, avidin, or streptavidin labeled product. Further, the labeled enzyme capturing unit 107 can capture an enzyme directly or an enzyme-labeled antibody.

In the present embodiment, a method for detecting a specific amount of IgE in a sample without causing false negatives by using each capture unit will be described as an example.
FIG. 2 is an exploded view of the biosensor shown in FIG. In the present embodiment, the support 201 has a length of 25 to 40 mm and a width of 40 to 80 mm. The upper cover 208 has the same dimensions as the support, the thickness in the vertical direction is 2 to 10 mm, and the thickness of the channel 211 to be formed is 0.1 to 0.5 mm. However, the dimension of each member for constituting the biosensor shown in FIG. 2 can be freely changed. The material of the upper cover can be used as long as it is transparent.

  The support 201 is made of a material capable of providing the capturing portion region 202. For example, synthetic resin, glass, metal, nitrocellulose, and vinylidene fluoride can be used. As the capture region 202, a material capable of capturing biomolecules can be used. For example, glass, polystyrene, gold coating, aluminum coating, nitrocellulose, vinylidene fluoride, cationic polymer coating, hydrophilic polymer coating, surface A chemical treatment and a porous support can be used. The surface chemical treatment includes silanization, amination, aldehyde formation, thiolation, epoxidation and active esterification. The upper cover can be made of transparent glass or a transparent resin material. Examples of the transparent resin include acrylic, polycarbonate, polystyrene, and PET (polyethylene terephthalate).

  The upper cover 208 in the upper diagram and the support 201 in the lower diagram were joined using the adhesive sheet 212 shown in the middle diagram of FIG. For the joining of the support 201 and the upper cover 208 for producing each trapping part, it is possible to use one that can form a sealed space so that the liquid injected into the flow channel does not leak. Examples of bonding methods other than the adhesive sheet include UV bonding, thermocompression bonding, and ultrasonic bonding.

  The production method and reason for each capturing part in the support 201 will be briefly described below. Details will be described later.

  The allergen capturing unit 203 is for specifically capturing IgE derived from each allergen, which is a target substance to be detected in a specimen, and is produced using a purified allergen. Here, IgG in the sample can also be bound, but since the labeled anti-IgE antibody, which is one of the sample solutions described later, does not bind to IgG, it does not affect the measurement by this sandwich immunoassay method. The combination of is omitted.

  The anti-IgE antibody capturing unit 204 is for capturing IgE in the specimen, and is produced using the anti-IgE antibody. Here, as the antibody captured by the anti-IgE antibody capturing part, any antibody that can capture any IgE is used as long as it is IgE. The capturing unit evaluates whether or not the sample is injected into the biosensor in each sample solution.

  The IgE antibody capture unit 205 is for specifically capturing the anti-IgE antibody in the sample solution, and is produced using the IgE antibody. This capture unit evaluates the presence or absence of anti-IgE antibodies in the biosensor in each sample solution.

  The labeled protein capturing unit 206 is for capturing the labeled enzyme in the sample solution, and is produced using the labeled protein. This capturing part evaluates the presence or absence of labeling enzyme injection into the biosensor in each sample solution. The protein to be captured by the labeled protein capturing unit is labeled with biotin or avidin or streptavidin labeled enzyme biotin or avidin or streptavidin in the sample solution.

  The label enzyme capture unit 207 is for capturing a detection reagent in a sample solution, and is produced using a label enzyme. This capturing part evaluates the presence or absence of a detection reagent in the biosensor in each sample solution.

  Each capturing part can be provided in the capturing part region 202 of the support located in the flow path 211 between the water inlet / outlet 209 and the exhaust outlet 210 of the upper cover 208.

  In addition, the allergen capturing unit 203 can capture at a plurality of locations even if one type of substance to be detected in the specimen is used. Further, when there are a plurality of types of substances to be detected in the specimen, it is possible to provide a capture unit corresponding to each. As an example of the allergen to be captured by the allergen capturing unit 203, a purified allergen extracted from cedar, mite, wheat or the like can be used.

  Similarly, capturing parts other than allergens can also be provided at a plurality of locations. In addition, it is preferable to capture at a location farther from the side of the water injection port 209 than the allergen capturing unit 203 because it can be determined that the liquid has been reliably injected into the channel 211. Furthermore, if it captures in the location near from the pouring outlet 209 side and the location far from it so that the allergen capture part 203 may be pinched | interposed, the mode of stirring can also be guessed from the value of the front and back capture parts. This is effective, for example, in a new flow path development stage where liquid feeding is not constant. In this way, each capturing part is produced.

  In addition, by providing the IgE antibody capturing unit 205, the labeled protein capturing unit 206, and the labeled enzyme capturing unit 207 in the biosensor, it is possible to detect defective injection of a sample solution other than the sample component, and search for a defective portion. be able to.

  In addition, if a biomolecule is captured in a capture part region other than the capture part, noise is generated. Therefore, a blocking process is performed so that the sample solution does not react with a place other than the capture part. Common blocking agents can be used, and examples include BSA, casein, skim milk, gelatin, normal goat serum, and artificial synthetic polymers.

A plurality of sample solutions necessary for the measurement including the specimen are sequentially injected into the biosensor thus prepared according to the flow chart shown in FIG. By detecting, it can be determined whether the sample solution processing performed correctly has been performed.
First, before describing the method for determining the injection of a specimen, it will be described that the allergen capturing unit 203 cannot be used for determining the injection of a specimen. This is because the antibody that can be captured by the allergen capturing unit 203 is the only IgE antibody that can bind to the captured allergen, which is specific to the captured allergen. That is, when a specimen that does not have an IgE antibody that can specifically bind to the allergen capture unit 203 is used, the allergen capture unit 203 may not confirm enzyme activity at all. For this reason, the allergen capturing unit 203 cannot accurately determine the presence or absence of the sample injection.

  Next, the anti-IgE antibody capturing unit 204 that can accurately determine the presence or absence of the sample injection will be described together with the reason. Here, explaining the IgE possessed by a person, the amount of IgE possessed by a person occupies about 0.004% of the total immune globulin, and the blood concentration occupies about 0.03 mg / dl. That is, in the case of an IgE antibody, in the anti-IgE antibody capturing unit 204 that captures an antibody that can be captured by any IgE antibody, the IgE that is always present in a human body as long as a specimen is injected. Can bind to antibodies. Therefore, it can be used for determination of the sample injection, and when the enzyme activity is detected by the anti-IgE antibody capturing unit 204, it is confirmed that the sample processing has been performed correctly by detecting the enzyme activity of the allergen capturing unit 203. In addition, the specific amount of IgE in the subject can be calculated.

  Next, a method for determining that the sample injection of the sample solution cannot be performed normally will be described. The determination is made when the enzyme activity is not detected in the sample solution in the anti-IgE antibody capturing unit 204 and the activity of the labeling enzyme is detected in the IgE antibody capturing unit 205, the labeled protein capturing unit 206, and the labeled enzyme capturing unit 207. It can be determined that the sample solution contains no specimen. Therefore, false negatives can be prevented. This is because the above-described anti-IgE antibody capture unit 204 is injected with the sample, and if the subsequent processing is performed correctly, the IgE antibody capture unit 205 is not limited to the flow chart S07. When the above process is normally performed, it can be determined by capturing a reagent having enzyme activity as a biosensor in advance.

  In other words, the IgE antibody capturing unit 205 has already captured the IgE antibody that is originally captured by performing the sample processing at the time of preparation of the biosensor. Therefore, the IgE antibody is contained in the biosensor regardless of the presence or absence of the sample. Can be involved in the processing after S07. Therefore, when the enzyme activity is not detected in the sample solution in the anti-IgE antibody capturing unit 204 and the activity of the labeling enzyme is detected in the IgE antibody capturing unit 205, the labeled protein capturing unit 206, and the labeled enzyme capturing unit 207, It can be determined that the sample solution contains no specimen. If the enzyme activity can be confirmed by the IgE antibody capture unit 205, the enzyme activity can be confirmed by the labeled protein capture unit 206 and the label enzyme capture unit 207, details of which will be described later.

  Hereinafter, the phenomenon that occurs when the first to seventh sample solutions are sequentially injected in accordance with the flowchart of FIG. 3 will be described in detail.

  First, a syringe is used to inject a first sample solution containing a specimen from a biosensor inlet / outlet (S01). Here, the first sample solution contains a specimen. Thereafter, the agitating operation is performed while moving the syringe up and down in the sealed space in the flow path, and the respective capture units and the specimen are reacted for a certain time (S02). Thereafter, the first sample solution is discarded from the pouring outlet (S03). The specimen that can be used here is whole blood or plasma or serum. Although it is possible to inject the sample directly into the biosensor, when a small amount of sample is used, it is diluted with a diluent and added. The dilution is carried out in consideration of the type and pH of the buffer used, the salt concentration, the surfactant concentration, the addition of the polymer compound, etc. so as not to inhibit the target immune reaction. As the diluent, generally known diluents can be used. For example, PBS buffer, TBS buffer, PBST buffer, TBST buffer, Can Get Signal Solution I (ToYoBo), Can Get Signal Solution II (ToY) Is mentioned.

  In the reaction with the first sample solution, in the anti-IgE antibody capture, the IgE antibody present in the sample is captured, and in the allergen capture unit, only IgE specific to the allergen present in the sample is captured.

  Next, a second sample solution is injected from the biosensor inlet / outlet using a syringe (S04). Here, the second sample solution contains a cleaning liquid. Thereafter, the agitating operation is performed while the syringe is moved up and down in the sealed space in the flow path, and the respective capture units and the second sample solution are reacted for a certain time (S05). Thereafter, the second sample solution is discarded from the pouring outlet (S06). As the cleaning liquid that can be used here, a generally known cleaning liquid can be used. For example, PBST buffer and TBST buffer can be used. The washing solution can remove non-specifically trapped components by using a higher salt concentration or a higher surfactant concentration than the diluted solution.

  In the reaction with the second sample solution, an unreacted specimen is removed and a non-specifically captured component is removed. By carrying out this step, noise can be reduced.

  Next, a third sample solution is injected using a syringe from the injection / drainage port of the biosensor (S07). Here, the third sample solution contains a labeled anti-IgE antibody. Here, the labeled product of the labeled anti-IgE antibody is a labeled product of biotin, avidin, or streptavidin. Thereafter, the agitating operation is performed while the syringe is moved up and down in the sealed space in the flow path, and the respective capture parts and the labeled anti-IgE antibody are reacted for a certain time (S08). Thereafter, the third sample solution is discarded from the pouring outlet (S09). Although it is possible to inject the labeled anti-IgE antibody directly into the biosensor, when a small amount of labeled anti-IgE antibody is used, it is diluted with a diluent and added. The diluent used when diluting the first sample solution can be used. The buffer type, pH, salt concentration, surfactant concentration, addition of polymer compound, and the like can be changed so as not to inhibit the immune reaction.

  In the reaction with the third sample solution, the IgE antibody captured by the allergen capture unit and the IgE antibody and IgE antibody capture unit captured by the anti-IgE antibody capture capture the anti-IgE antibody portion of the labeled anti-IgE antibody.

  Next, a fourth sample solution is injected using a syringe from the injection / drainage port of the biosensor (S10). Here, the fourth sample solution contains a cleaning solution. Thereafter, the agitating operation is performed while causing the syringe to move up and down in the sealed space in the flow path, and the respective capture units and the fourth sample solution are reacted for a certain time (S11). Thereafter, the fourth sample solution is discarded from the pouring outlet (S12). The cleaning liquid used here can be the same as the cleaning liquid used as the second sample solution. The second sample solution can be used by changing the reagent composition when it is desired to change the cleaning power.

  In the reaction with the fourth sample solution, unreacted labeled anti-IgE antibody is removed and non-specifically captured components are removed. By carrying out this step, noise can be reduced.

  Next, a fifth sample solution is injected from the biosensor inlet / outlet using a syringe (S13). Here, the fifth sample solution contains a labeling enzyme. Here, the label of the labeling enzyme is avidin, streptavidin, or biotin, and is capable of binding to the labeling part of the labeled anti-IgE antibody injected with the third sample solution. For example, if an avidin-labeled anti-IgE antibody is used as the labeled anti-IgE antibody, a biotin-labeled enzyme is used as the labeling enzyme. Examples of enzymes that can be used here include luciferase, peroxidase, alkaline phosphatase, glucose oxidase, glucose-6-phosphate dehydrogenase, and β-galactosidase. it can. Thereafter, the agitating operation is performed while the syringe is moved up and down in the sealed space in the flow path, and each capture unit and the labeling enzyme are reacted for a certain time (S14). Thereafter, the fifth sample solution is discarded from the pouring outlet (S15). Although it is possible to inject the labeling enzyme directly into the biosensor, when a small amount of the labeling enzyme is used, it is diluted with a diluent and added. The diluent used when diluting the first sample solution can be used. The type and pH of the buffer used so as not to inhibit the immune reaction, pH, salt concentration, surfactant concentration, addition of a polymer compound, etc. can also be used.

  In the reaction with the fifth sample solution, the labeled portion of the labeled anti-IgE antibody captured by the allergen capture unit, the labeled portion of the labeled anti-IgE antibody captured by the anti-IgE antibody capture, and the labeled anti-IgE antibody captured by the IgE antibody capture unit The labeling part of the IgE antibody and the labeling part of the labeling protein capturing part capture the labeling part of the labeling enzyme. As the protein to be captured as the labeled protein capturing section used here, a protein labeled with a labelable portion of the labeled anti-IgE antibody injected as the third sample solution is used.

  Next, a sixth sample solution is injected using a syringe from the pouring outlet of the biosensor (S16). Here, the sixth sample solution contains a cleaning liquid. Thereafter, the agitating operation is performed while the syringe is moved up and down in the sealed space in the flow path, and the respective capture units and the sixth sample solution are reacted for a certain time (S17). Thereafter, the sixth sample solution is discarded from the pouring outlet (S18). The cleaning liquid used here can be the same as the cleaning liquid used as the second sample solution. The second sample solution can be used by changing the reagent composition when it is desired to change the cleaning power.

In the reaction with the sixth sample solution, unreacted labeling enzyme is removed and non-specifically captured components are removed. By carrying out this step, noise can be reduced.
Next, a seventh sample solution is injected using a syringe from the bio-sensor inlet / outlet (S19). Here, the seventh sample solution contains a detection reagent. The detection reagent is a sample solution that includes a substrate component that reacts with an enzyme and that is adjusted in pH and the like so that the enzyme reaction is efficiently performed. Examples of substrates that can be used here include luciferin + ATP + magnesium, p-nitrophenyl phosphate (p-NPP), bromochloroindolyl phosphate / nitrobull-tetrazolium (BCIP / NBT), bromochloroindolyl phosphate / nitrobull-tetrazolium salt ( BCIP / INT), naphthol phosphate AS-TR / fast red RC, fuchsin, β-D-glucose + HRP + 3,3′-5,5′-tetramerbenzidine (TMB), glucose-6 Phosphoric acid + NADP, 2,2′-azinodi (3-ethylbenzothiazolyso 6-sulfonic acid) ammonium salt (ABTS), diaminobenzidine (DAB), o-phenylenediamine (OPD), 3,3′-5,5 '-Tetramerbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid (5 S), 3-amino-9-ethylcarbazole (AEC), 4-chloro-1-naphthol (4C1N), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside ( X-GAL), o-nitrophenyl-β-D-galactopyranoside (o-NPG), 4-methylumbelliferyl phosphate, 4-methylumbelliferyl-β-D-glucuronide, luminol, Mention may be made of tyramine and p-hydroxyphenylpropionic acid. Thereafter, the enzyme captured by each capturing unit reacts with the substrate in a sealed space in the flow path.

  A labeling enzyme captured by the anti-IgE antibody capture unit in the reaction with the seventh sample solution, a labeling enzyme captured by the allergen capture unit, a labeling enzyme captured by the IgE antibody capture unit, and a labeled protein capture unit The labeling enzyme captured by and each enzyme in the labeling enzyme capturing part react with the substrate. Here, the enzyme to be captured as the labeling enzyme capturing unit is the same as the enzyme part of the labeling enzyme poured as the seventh sample solution. As a result, the same substrate can be used in all the capture units.

  Next, the enzyme activity generated from each capture unit generated by the seventh reagent reaction is measured (S20). As a photometric method, for example, there is a method in which a capturing unit is photographed as a photometric image at a time using a CCD (Charge Coupled Devices) camera or the like.

  Next, the amount of light obtained from each capturing part of the photographed photometric image is calculated and determined (S21). In the determination method, as described above, when the enzyme activity is detected in the anti-IgE antibody capturing unit, it is determined that the sample solution processing including the specimen can be normally performed.

Here, a determination method when processing other than sample processing is incomplete will be described.
First, a method for determining that the labeled anti-IgE antibody injection of the sample solution cannot be performed normally will be described. This is because the enzyme activity is not detected in the sample solution in the anti-IgE antibody capturing unit 204 and the IgE antibody capturing unit 205, and the activity of the labeling enzyme is detected in the labeled protein capturing unit 206 and the labeled enzyme capturing unit 207. It can be determined that the sample solution does not contain a labeled anti-IgE antibody. This is because, in addition to the roles of the anti-IgE antibody capturing unit 204 and the IgE antibody capturing unit 205 described above, the labeled protein capturing unit 206 always has enzyme activity when the processing after the flow chart S13 is normally performed. It can be judged from. This is because the labeled protein capture unit 206 has already captured the anti-IgE antibody that is originally captured by performing the sample processing at the time of biosensor preparation, so that the sample to be injected as a sample solution and the labeled anti-IgE antibody This is because it can be involved in the processing after S13 regardless of the presence or absence. From this, it can be said that if the enzyme activity can be confirmed by the IgE antibody capturing unit 205, the enzyme activity can also be confirmed by the labeled protein capturing unit 206.

  Next, a method for determining that the labeling enzyme injection of the sample solution cannot be performed normally will be described. This is because when the enzyme activity is not detected in the sample solution in the anti-IgE antibody capturing unit 204, the IgE antibody capturing unit 205, and the labeled protein capturing unit 206, and the activity of the labeling enzyme is detected in the labeled enzyme capturing unit 207 It can be determined that the sample solution does not contain the labeling enzyme. This is because, in addition to the roles of the anti-IgE antibody capturing unit 204, the IgE antibody capturing unit 205, and the labeled protein capturing unit 206 described above, the labeled enzyme capturing unit 207 performs normal processing after the flow chart S19. It can be determined from the fact that it always has enzyme activity.

  This is because the labeling enzyme capturing unit 207 has already captured the labeling enzyme that is originally captured by executing the labeling enzyme injection solution at the time of biosensor preparation. This is because it can be involved in the processing after S19 irrespective of the presence or absence of the IgE antibody and the labeling enzyme. From this, it can be said that if the enzyme activity can be confirmed by the IgE antibody capture unit 205, the enzyme activity can also be confirmed by the label enzyme capture unit 207. Further, if the enzyme activity can be confirmed by the labeled protein capturing unit 206, it can be said that the enzyme activity can also be confirmed by the labeled enzyme capturing unit 207.

  Next, a method for determining that the sample solution detection reagent injection has not been performed normally will be described.

  This is because when the enzyme activity is not detected in the sample solution in the anti-IgE antibody capturing unit 204, the IgE antibody capturing unit 205, the labeled protein capturing unit 206, and the labeled enzyme capturing unit 207, the sample solution contains no detection reagent. It can be determined. This can be determined from the fact that the enzyme activity cannot be confirmed even from the labeled enzyme capture unit 207 having the enzyme activity whenever the above-described processing after the flow chart S19 is normally performed.

  Finally, when it is determined that normal processing has been completed, the enzyme activity of the allergen capture unit is detected, calculated as a specific amount of IgE in the subject, and displayed as an analysis result (S22). When normal processing cannot be performed, a defective process is displayed according to the determination.

  As described above, in this method, a capture unit corresponding to each sample solution is provided, and before displaying the measurement result of the substance to be detected, the enzyme activity of each capture unit is determined to confirm that normal processing has been performed. Therefore, false emission due to forgetting to put the sample can be prevented.

  Experiments were performed using human serum as the measurement solution. The biosensor produced for the experiment is shown in FIG.

  As the above-described biosensor support, a glass substrate 401 (GenTel, PATH Protein Microarray Slide) in which the nitrocellulose 402 treatment was performed in the capture region was used. On the support, squirrel allergen 403 is captured as an allergen capturing part, anti-IgE antibody f1974 404 (manufactured by Biomatrix Laboratory) is captured as an anti-IgE antibody capturing part, and human IgE antibody 405 (as an IgE antibody capturing part) Yamasa Shoyu Co., Ltd.) was captured, biotinylated BSA406 was captured as the labeled protein capturing portion, and luciferase-labeled anti-IgE antibody 407 (manufactured by Kikkoman Corp.) was captured as the labeled enzyme capturing portion.

  In the application solution to the Suierergen 403 capturing part, glycerol, BSA and borate buffer were added to cedar pollen antigen SBP (Seikagaku Corporation) and mixed. Glycerol was added as a reproducibility improver at the time of coating because it can suppress variations during coating due to its own viscosity. BSA and borate buffer were added as structure stabilizers when the cedar pollen antigen SBP was in solution. The mixture for application was adjusted so that cedar pollen antigen SBP was diluted 1.5 times by mixing glycerol, BSA (bovine serum albumin) and borate buffer. The adjusted coating liquid mixture was coated on a support using Nano-Plotter NP2.1 (GeSiM) so that the coating amount per spot was 4.5 nL. The coated sample was left to be fixed at 30 ° C. for 8 hours in a tapper with a wet cloth. In this way, an allergen capturing part was produced.

  The anti-IgE antibody f1974 404 capture unit, the human IgE antibody 405 capture unit, the biotinylated BSA406 capture unit, and the luciferase-labeled anti-IgE antibody 407 supplemental unit were also coated in the same manner as the Squire allergen 403 capture unit. At that time, in the coating solution to the anti-IgE antibody f1974 404 capturing part, glycerol, BSA and borate buffer solution were added to the anti-IgE antibody f1974 (manufactured by Biomatrix Laboratories, Inc., f1974 is Clone No.). The mixture was mixed and adjusted so that anti-IgE antibody f1974 was diluted 60-fold. Further, in the coating solution to the human IgE antibody 405 capturing part, human IgE (Yamasa Soy Sauce Co., Ltd.) was mixed with glycerol, BSA and boric acid so that human IgE was diluted 6000 times. In addition, in the coating solution to the biotinylated BSA406 capturing part, glycerol, BSA and boric acid were mixed with a biotinylated BSA solution (manufactured by SIGMA) adjusted to 250 μg / mL, and the biotinylated BSA solution was 40 times. It was adjusted to be diluted. In the application solution to the supplement part of the luciferase-labeled anti-IgE antibody 407, luciferase (manufactured by Kikkoman Corporation) is added to the anti-IgE antibody f0822 (manufactured by Biomatrix Laboratories, Inc., f0822 is Clone No.). Glycerol, BSA, and boric acid were mixed with the bound anti-IgE antibody f0822-labeled luciferase so that the anti-IgE antibody f0822-labeled luciferase was diluted 10-fold. The luciferase-labeled anti-IgE antibody 407 supplement can capture luciferase directly, but it affects the variability of the enzyme and lowers the ability to recognize the substrate. The thing which bound the thing was caught.

  As described above, the adhesive sheet 412 is cut in accordance with the width of the support on the capturing surface of the support that captured each sample, and the flow path 411 and the pouring / draining port 409 processed on the upper cover side. Cut according to the shape of the exhaust port 410, stick one side of the adhesive surface to the immobilization surface of the support, and paste the acrylic plate with the post-processed channel shape on the remaining surface as an upper cover 408 made of transparent resin Thus, a biosensor was produced.

  Next, measurement was performed using this biosensor. First, N101 (Nippon Yushi Co., Ltd.) was filled in the biosensor as a blocking agent. The blocking treatment was performed while standing at 37 ° C. for 1 h. This blocking agent is a capture part that is exposed other than the Squirergen 403 capture part, the anti-IgE antibody f1974 404 capture part, the human IgE antibody 405 capture part, the biotinylated BSA406 capture part, the labeling enzyme or the enzyme-labeled antibody supplement part. Since it binds to the region, carbohydrates, lipids and hormones including proteins contained in the sample are allergen capture part, anti-IgE antibody f1974 404 capture part, human IgE antibody 405 capture part, biotinylated BSA406 capture part, luciferase label It can be adsorbed to other than the anti-IgE antibody 407 supplement, and it can be prevented that the subsequent processing causes noise and adverse effects.

  Next, the step of reacting the sample with the Squire allergen 403 capture unit and the anti-IgE antibody f1974 404 capture unit was performed. A first sample solution in which 42 μL of Can Get Signal I (manufactured by ToYoBo) was mixed as a diluent with 18 μL of serum was developed and reacted in the biosensor. After stirring in the channel by pipetting for 5 minutes, the first sample solution was excluded from the biosensor. Can Get Signal I was added to increase the reaction efficiency. By this step, the cedar-derived IgE antibody specifically binds to the capture part of Sugi allergen 403, and IgE in the diluent specifically binds to the capture part of anti-IgE antibody f1974 404. Join.

  Subsequently, the inside of the flow path was cleaned using the second sample solution. The purpose was to remove the first sample solution remaining on the wall surface in the biosensor, and to remove the non-specifically captured substance from the capturing part of the Suierergen 403 and the anti-IgE antibody f1974 404. By this step, noise can be reduced. As a cleaning agent, a solution to which 50 mM Tris-HCl (pH 7.8), 0.45 M NaCl, and 0.1% Tween 20 (manufactured by Tokyo Chemical Industry Co., Ltd.) was added was used in the biosensor. After stirring in the channel by pipetting for 1 minute, the cleaning agent was excluded from the biosensor.

  Next, the anti-IgE antibody was added to the IgE antibody and the human IgE antibody 405 capture unit bound to the capture unit of Swirlergen 403 and the anti-IgE antibody f1974 404 capture reaction. In this example, biotin-labeled anti-IgE antibody clone No. An antibody solution containing f0822 (manufactured by Biomatrix Laboratories) was used. Biotin-labeled anti-IgE antibody clone No. A third sample solution in which 55.5 μL of Can Get Signal I (ToYoBo) was mixed with 4.5 μL of f0822 was developed and reacted in the biosensor. After stirring in the channel by pipetting for 5 minutes, the third sample solution was excluded from the biosensor. Can Get Signal I was added to increase the reaction efficiency. By this step, anti-IgE antibody clone No. 1 in the antibody solution was detected against IgE present in the Squire allergen 403 capture unit, anti-IgE antibody f1974 404 capture unit and human IgE antibody 405 capture unit. f0822 specifically binds.

  Subsequently, the inside of the flow path was cleaned using the fourth sample solution. Conducted for the purpose of removing the third sample solution remaining on the inner wall of the biosensor and the substance that non-specifically binds to the capture unit of Suierergen 403, the capture unit of anti-IgE antibody f1974 404 and the capture unit of human IgE antibody 405 did. By this step, noise can be reduced. As the cleaning agent, 50 mM Tris-HCl (pH 7.8), 0.45 M NaCl, and 0.1% Tween 20 were added to the biosensor and used. After stirring in the channel by pipetting for 1 minute, the cleaning agent was excluded from the biosensor.

  Next, streptavidin-labeled luciferase (Kicko) was used as a labeling enzyme for the anti-IgE antibody and biotinylated BSA406 capture unit bound to the Suierergen 403 capture unit, anti-IgE antibody f1974 404 capture unit, human IgE antibody 405 capture unit. -Mann Co., Ltd.) was added and reacted. A fifth sample solution prepared by mixing 54 μL of a luciferase diluted solution (manufactured by Kikkoman Co., Ltd.) with 6 μL of 1000 × streptavidin-labeled luciferase was developed and reacted in the biosensor. After stirring in the channel by pipetting for 5 minutes, the fifth sample solution was excluded from the biosensor. Can Get Signal I was added to increase the reaction efficiency. Through this step, streptavidin in the enzyme solution specifically binds to the biotin structures present in the Squire allergen 403 capture unit, anti-IgE antibody f1974 404 capture unit, human IgE antibody 405 capture unit and biotinylated BSA406 capture unit. .

  Subsequently, the inside of the flow path was cleaned using the sixth sample solution. Removal of the fifth sample solution remaining on the wall surface in the biosensor and removal of substances that non-specifically bind to the Squire allergen 403 capture unit, anti-IgE antibody f1974 404 capture unit, IgE antibody capture unit and biotinylated BSA406 capture unit We carried out for the purpose. By this step, noise can be reduced. As the cleaning agent, 50 mM Tris-HCl (pH 7.8), 0.45 M NaCl, and 0.1% Tween 20 were added to the biosensor and used. After stirring in the channel by pipetting for 1 minute, the cleaning agent was excluded from the biosensor.

  Next, the luciferase and the luciferase-labeled anti-IgE antibody 407 supplemental part bound to the Squire allergen 403 capture part, the anti-IgE antibody f1974 404 capture part, the human IgE antibody 405 capture part, and the biotinylated BSA406 capture part A luciferase luminescence substrate solution (manufactured by Kikkoman Corp.) of Intellilite AB containing luciferin, ATP, and magnesium ions as a substrate was added as a seventh sample solution, and a luminescence reaction was performed. 60 μL of 1 × Luciferase luminescent substrate solution was developed in the biosensor. By this step, the luminescence reaction by luciferase in the capture part of squier allergen 403, capture part of anti-IgE antibody f1974 404, capture part of human IgE antibody 405, capture part of biotinylated BSA406, supplement part of luciferase-labeled anti-IgE antibody 407 Occur.

  Next, the amount of luminescence was measured. After the seventh sample solution is developed in the biosensor, the immobilization surface of the biosensor is quickly fixed on the stage facing the CCD camera, and the amount of luminescence is photographed for 3 minutes. A photometric image in which the integrated value of luminance for 3 minutes was mapped was generated.

  Then, in the image analysis step, pixels corresponding to each spot in the biosensor were specified, and the luminance value was used as a measurement value.

  FIG. 5 is a diagram showing a photometric image obtained by mapping the integrated values of the luminance of each pixel for 3 minutes output in the photometric process as described in the flowchart of FIG. The larger the luminance value, the more white it is displayed. A broken line indicated by reference numeral 501 indicates the shape of the flow path near the pouring / draining port. In the actual photometric image, the channel portion cannot be confirmed, but is shown here for easy understanding. 502 corresponds to the light emission spot of the capture part of Suierergen 403, 503 corresponds to the light emission spot of the capture part of anti-IgE antibody f1974 404, 504 corresponds to the light emission spot of the capture part of human IgE antibody 405, and 505 corresponds to biotinylated BSA406. 506 corresponds to the light emission spot of the capturing part, and 506 corresponds to the light emission spot of the supplement part of the luciferase-labeled anti-IgE antibody 407.

  Referring to FIG. 5, since light emission is confirmed at 503, 504, 505, and 506, it indicates that the processing in each step has been performed normally. Therefore, allergy can be measured.

  Next, the luminance value obtained from 502 was calculated to be 1492514.

  Here, a calibration curve equation was prepared using a specimen having a known cedar IgE concentration and using a biosensor coated with only a cedar allergen. The calibration curve obtained as a result was y = 25276X + 129261. Luminance value 1492514 obtained from the allergen capture part was input to this calibration curve, and the cedar IgE value in serum was calculated. When this numerical value was applied to a calibration curve formula y = 25276X + 129261, it could be converted to 53.93 UI / mL. This was 5 when expressed as an allergy score of 0-7. The allergic score when the same serum was measured with DiaPack2000 (Nippon Chemifa Co., Ltd.) as an assay machine was also 5. From this, it was confirmed that there was a correlation on the score. This indicates that the measurement was correct.

  In addition, since the correlation was obtained, the anti-IgE antibody f1974 404 capturing part, the human IgE antibody 405 capturing part, the biotinylated BSA406 capturing part, and the luciferase-labeled anti-IgE antibody 407 supplemental part were compared with the Squire allergen 403 capturing part. It was also confirmed that it did not act as a reaction inhibitor.

  The same process as in Example 1 was performed without intentionally adding only the specimen. FIG. 6 is a diagram showing a photometric image obtained at that time. The larger the luminance value, the more white it is displayed. A broken line indicated by reference numeral 601 indicates the shape of the flow path near the pouring / draining port. In the actual photometric image, the channel portion cannot be confirmed, but is shown here for easy understanding. A circular dotted line indicated by 602 corresponds to the light emission spot position of the Sigeria allergen 403 capturing portion, and a circular dotted line indicated by 603 corresponds to the light emission spot position of the anti-IgE antibody f1974 404 capturing portion. In the portion indicated by the dotted circle, the luminance cannot be confirmed at the visual level, but the portion corresponding to the capture position is surrounded by a circular dotted line for easy understanding. 604 corresponds to the luminescent spot of the human IgE antibody 405 capture part, 605 corresponds to the luminescent spot of the biotinylated BSA406 capture part, and 606 corresponds to the luminescent spot of the luciferase-labeled anti-IgE antibody 407 supplement part.

  In FIG. 6, the luminance could not be confirmed at 602. Normally, the specimen is treated as a specimen that does not hold the cedar IgE antibody, but luminance cannot be observed at 603. On the other hand, since the luminance can be confirmed at 604, 605, and 606, it can be determined that the sample is not included. This prevented false negatives.

  The same process as in Example 1 was performed with a sample solution intentionally containing no anti-IgE antibody f0822. FIG. 7 is a diagram showing a photometric image obtained at that time. The larger the luminance value, the more white it is displayed. The broken line shown by 701 has shown the flow-path shape of the pouring / draining port vicinity. In the actual photometric image, the channel portion cannot be confirmed, but is shown here for easy understanding. A circular dotted line indicated by 702 corresponds to the light emission spot position of the capturing part of Suierergen 403, and a circular dotted line indicated by 703 corresponds to the light emission spot position of the anti-IgE antibody f1974 404 capturing part. The dotted line corresponds to the light emission spot position of the human IgE antibody 405 capturing part. In the portion indicated by the dotted circle, the luminance cannot be confirmed at the visual level, but the portion corresponding to the capture position is surrounded by a circular dotted line for easy understanding. 705 corresponds to the luminescent spot of the biotinylated BSA406 capturing part, and 706 corresponds to the luminescent spot of the luciferase-labeled anti-IgE antibody 407 supplement part.

  In 702, 703 and 704, the luminance cannot be confirmed. On the other hand, since the luminance can be confirmed at 705 and 706, it can be determined that the anti-IgE antibody f0822 is not contained in the sample solution.

  The same steps as in Example 1 were performed with a sample solution intentionally containing no streptavidin-labeled luciferase. FIG. 8 is a diagram showing a photometric image obtained at that time. The larger the luminance value, the more white it is displayed. The broken line shown by 801 has shown the flow path shape of the pouring / draining port vicinity. In the actual photometric image, the channel portion cannot be confirmed, but is shown here for easy understanding. A round dotted line indicated by 802 corresponds to the light emission spot position of the capturing part of Suierergen 403, a round dotted line indicated by 803 corresponds to the light emission spot position of the capturing part of anti-IgE antibody f1974 404, and The dotted line corresponds to the light emission spot position of the human IgE antibody 405 capture portion, and the round dotted line indicated by 805 corresponds to the light emission spot of the biotinylated BSA406 capture portion. In the portion indicated by the dotted circle, the luminance cannot be confirmed at the visual level, but the portion corresponding to the capture position is surrounded by a circular dotted line for easy understanding. Reference numeral 806 corresponds to a light emission spot of a luciferase-labeled anti-IgE antibody 407 supplement.

  In 802, 803, 804 and 805, the luminance cannot be confirmed. On the other hand, since the luminance can be confirmed at 806, it can be determined that the sample solution does not contain streptavidin-labeled luciferase.

  The same process as in Example 1 was carried out with a sample solution intentionally containing no detection reagent. FIG. 9 is a diagram showing a photometric image obtained at that time. The larger the luminance value, the more white it is displayed. A broken line indicated by reference numeral 901 indicates the shape of the flow path near the pouring / draining port. In the actual photometric image, the channel portion cannot be confirmed, but is shown here for easy understanding. A round dotted line indicated by 902 corresponds to the light emission spot position of the capturing part of Suierergen 403, a circular dotted line indicated by 903 corresponds to the light emission spot position of the capturing part of the anti-IgE antibody f1974 404, and the circle indicated by 904 The dotted line corresponds to the luminescence spot position of the human IgE antibody 405 capture part, the round dotted line indicated by 905 corresponds to the luminescence spot of the biotinylated BSA406 capture part, and 906 represents the luminescence of the luciferase-labeled anti-IgE antibody 407 supplement part. Corresponds to a spot. In the portion indicated by the dotted circle, the luminance cannot be confirmed at the visual level, but the portion corresponding to the capture position is surrounded by a circular dotted line for easy understanding.

  Since luminance cannot be obtained from anywhere in the photometric image, it can be determined that the detection reagent is not contained in the sample solution.

  The same steps as in Example 1 were performed. However, unlike Example 1, a specimen having a cedar IgE score of 0 by a reference machine was used. FIG. 10 is a diagram showing a photometric image obtained at that time. The larger the luminance value, the more white it is displayed. A broken line indicated by reference numeral 601 indicates the shape of the flow path near the pouring / draining port. In the actual photometric image, the channel portion cannot be confirmed, but is shown here for easy understanding. A circular dotted line indicated by reference numeral 602 corresponds to the light emission spot position of the capturing part of the Squire allergen 403. In the portion indicated by the dotted circle, the luminance cannot be confirmed at the visual level, but the portion corresponding to the capture position is surrounded by a circular dotted line for easy understanding. 603 corresponds to the luminescent spot position of the anti-IgE antibody f1974 404 capturing part, 604 corresponds to the luminescent spot of the human IgE antibody 405 capturing part, 605 corresponds to the luminescent spot of the biotinylated BSA406 capturing part, 606 This corresponds to the light emission spot of the supplemented portion of the z-labeled anti-IgE antibody 407.

At 602, almost no luminance can be confirmed. The luminance value was 3856.
On the other hand, the luminance can be confirmed in the anti-IgE antibody capturing part. From this, it can be determined that the processing has been normally performed, and therefore, the calibration curve formula y = 25276X + 129261 was converted to 0.153 UI / mL. This corresponds to 0 in the score display.
Therefore, this sample can be determined as a sample having a score of 0 for cedar IgE.
From the results of Examples 1 to 6 above, it can be determined whether or not the serum has been correctly injected. Moreover, when measurement is not possible even with serum, it can be determined which process has not been successfully performed.

  The sandwich immunoassay method and biosensor using the same according to the present invention have the ability to discriminate false positives by identifying whether a sample is correctly added, and to detect a substance to be detected in the sample by the sandwich immunoassay method. It is useful as a detection device for detection and a detection method using the detection device.

101, 201 Support 102, 202 Capture part region 103, 203 Allergen capture part 104, 204 Anti-IgE antibody capture part 105, 205 IgE antibody capture part 106, 206 Labeled protein capture part 107, 207 Labeled enzyme capture part 108, 208 Cover
109, 209, 409 Inlet / outlet port 110, 210, 410 Exhaust port 111, 211, 411 Flow path 212, 412 Adhesive sheet 401 Glass substrate 402 Nitrocellulose 403 Sgia allergen 404 Anti-IgE antibody f1974
405 Human IgE antibody 406 Biotin-labeled BSA
407 Luciferase labeled anti-IgE antibody 408 Transparent resin top cover
501, 601, 701, 801, 901, 1001 Shape of the flow path near the drainage port 502, 1002 Luminescence spot by the Shear allergen 403 trapping part 503, 1003 Luminescence spot by the anti-IgE antibody f1974 404 504, 604, 1004 Human IgE antibody 405 Emission spot by the capture unit 505, 605, 705, 1005 Emission spot by the biotin-labeled BSA406 capture unit 506, 606, 706, 806, 1006 Luminescence spot by the luciferase-labeled anti-IgE antibody f0822 407 capture unit 602, 702, 802, 902 Luminescence spot by Swirl allergen 403 capture unit 603, 703, 803, 903 Luminescence spot by anti-IgE antibody f1974 404 704, 804, 904 By human IgE antibody 405 capture unit Luminescent spot 805, 905 Luminescent spot by biotin-labeled BSA406 capturing part 906 Luciferase-labeled anti-IgE antibody f0822 407 Luminescent spot by capturing part 1101 Nitrocellulose membrane 1102 Capture reagent part 1103 Control part 1104 Supply part 1105 Absorbing carrier 1106 Sample liquid 1107 Specimen 1108 Labeling reagent 1109 Specimen solution

Claims (11)

An optically transparent plate in which a recess is formed is placed on a support, a flow path is formed between the plate and the support, and a measurement solution containing a sample is sequentially poured and discharged at one end of the flow path. In a biosensor having a water inlet and outlet and an exhaust port at the other end,
An anti-IgE antibody capture unit for capturing IgE antibody contained in the measurement solution;
A biosensor in which an allergen capturing part for capturing a specific IgE antibody in the IgE antibody is disposed on the surface of the support between the pouring / draining port side and the exhaust port in the channel. further,
An IgE antibody capturing part for capturing the labeled anti-IgE antibody contained in the measurement solution;
A labeled protein capturing part for capturing the labeling enzyme contained in the measurement solution;
The label enzyme capturing part for capturing the detection reagent contained in the measurement solution is disposed on the surface of the support body between the water injection port side and the exhaust port in the channel. Biosensor. The biosensor according to claim 2, wherein the anti-IgE antibody capturing part does not bind to the labeled anti-IgE antibody. The biosensor according to claim 1, wherein the specimen is whole blood, plasma, or serum. The said labeling enzyme comprises luciferase, peroxidase, alkaline phosphatase, glucose oxidase, glucose-6-phosphate dehydrogenase or β-galactosidase. The biosensor described. The substrate of the measurement solution was luciferin + ATP + magnesium, p-nitrophenyl phosphate (p-NPP), bromochloroindolyl phosphate / nitrobull-tetrazolium (BCIP / NBT), bromochloroindolyl phosphate / nitrobull-tetrazolium salt (BCIP) / INT), naphthol phosphate AS-TR / fast red RC, fuchsin, β-D-glucose + HRP + 3,3′-5,5′-tetramerbenzidine (TMB), glucose-6-phosphate + NADP, 2,2′-azinodi (3-ethylbenzothiazolyso-6-sulfonic acid) ammonium salt (ABTS), diaminobenzidine (DAB), o-phenylenediamine (OPD), 3,3′-5,5 ′ -Tetramerbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid (5A S), 3-amino-9-ethylcarbazole (AEC), 4-chloro-1-naphthol (4C1N), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside ( X-GAL), o-nitrophenyl-β-D-galactopyranoside (o-NPG), 4-methylumbelliferyl phosphate, 4-methylumbelliferyl-β-D-glucuronide, luminol, The biosensor according to claim 2, comprising either tyramine or p-hydroxyphenylpropionic acid. Anti-IgE antibody capture unit for capturing IgE antibody contained in measurement solution containing specimen, allergen capture unit for capturing specific IgE antibody in IgE antibody, and labeled anti-IgE antibody contained in measurement solution An immunity comprising an IgE antibody capturing part for capturing a protein, a labeled protein capturing part for capturing a labeling enzyme contained in the measurement solution, and a labeled enzyme capturing part for capturing a detection reagent contained in the measurement solution In the reaction system,
After sequentially injecting multiple sample solutions including specimens and causing an immune reaction,
When no enzyme activity is detected in the anti-IgE antibody capture unit and enzyme activity is detected in all of the IgE antibody capture unit, the labeled protein capture unit, and the labeled enzyme capture unit, the sample solution contains the sample A sandwich immunoassay method for determining that no is contained. When the enzyme activity is detected by the anti-IgE antibody capture unit,
The sandwich immunoassay method according to claim 7, wherein an enzyme activity of the allergen capture unit is detected and calculated as a specific amount of IgE in a subject. When enzyme activity is not detected in the anti-IgE antibody capture unit and the IgE antibody capture unit and enzyme activity is detected in all of the labeled protein capture unit and the label enzyme capture unit, the label is added to the sample solution. The sandwich immunoassay method according to claim 7, wherein it is determined that no anti-IgE antibody is contained. When the enzyme activity is not detected in all of the anti-IgE antibody capture unit, the IgE antibody capture unit and the labeled protein capture unit, and the enzyme activity is detected in the label enzyme capture unit, the sample solution is used for the labeling. The sandwich immunoassay method according to claim 7, wherein it is determined that no enzyme is contained. When enzyme activity is not detected in all of the anti-IgE antibody capturing unit, the IgE antibody capturing unit, the labeled protein capturing unit, and the labeled enzyme capturing unit, it is determined that the sample solution does not contain a detection reagent. The sandwich immunoassay method according to claim 7.