WO2006043614A1

WO2006043614A1 - Membrane immunoassay method

Info

Publication number: WO2006043614A1
Application number: PCT/JP2005/019271
Authority: WO
Inventors: Shosaku Motoda; Takashi Miyazawa
Original assignee: Denka Seiken Co., Ltd.
Priority date: 2004-10-20
Filing date: 2005-10-20
Publication date: 2006-04-27
Also published as: JP2006118936A

Abstract

It is intended to provide an easy-to-use and highly reliable assay method, whereby the number of detection procedure steps can be decreased in a method of testing a sample with the use of a flow-through type membrane immunoassay method, and a kit to be used in this method. Namely, a simplified flow-through type membrane immunoassay method for detecting a subject in a test sample with the use of an assay apparatus provided with a membrane to which a scavenger antibody or antigen for capturing the subject to be detected is bound, characterized by comprising filtering the test sample by using a filter and then dropping, allowing the scavenger antibody or antigen on the membrane to capture the subject to be detected, dropping an enzyme-labeling reagent, which comprises an enzyme-labeled substance capable of reacting with the detection subject, onto the membrane carrying the detection subject having been captured, and detecting the presence of the detection subject in the test sample without resorting to a washing step; and a kit for detecting the presence of a subject in a test sample.

Description

Membrane Enzymno Atssay Method Technical Field

[0001] The present invention relates to a simple flow-through membrane enzyme immunoassay method for detecting an object to be detected in a specimen sample and a kit used in this method. Background art

[0002] Recently, simple test reagents and kits have been developed that perform various tests in a short time, such as the presence or absence of infection with pathogens such as viruses and bacteria, and the presence or absence of pregnancy, using antigen-antibody reactions and enzyme reactions. Yes. In these reagents and kits, pathogen components, human chorionic gonadotropins, and the like are targets for detection or quantification. Many of the simple test reagents do not require special equipment and are easy to operate and inexpensive. For example, simple test reagents for pregnancy diagnosis are sold at general pharmacies. Also, unlike other test reagents, simple test reagents for testing pathogen infection are widely used in general hospitals and clinics in addition to large hospitals and medical test centers. These facilities are often the first medical institutions visited by patients. Patient power Collected specimens can be treated at an early stage if the presence or absence of infection is identified on the spot. The importance of reagents in medicine is increasing.

[0003] At present, as a simple inspection method, a membrane assembly method, in particular, an assembly method using a membrane such as -trocellulose or a membrane such as a filter is generally known, and a flow-through method and a lateral flow method are used. Broadly divided into laws. The former allows the solution containing the object to be detected to pass in a direction perpendicular to the membrane, and the latter allows the solution to be developed in the horizontal direction.

[0004] In either case, a label is detected by forming a complex of a capture substance that specifically binds to the detection target, a detection target, and a labeling substance that specifically binds to the detection target on the membrane. Quantification means that the detection of an object to be detected is common in terms of quantification.

[0005] In addition, in the simple test method based on the membrane assay method, the patient force is determined to be positive even though the sample to be detected is not present in the sample, based on the analysis of the sample actually collected. Well, so-called false positives may occur. If a false positive reaction occurs when testing for a pathogen infection, it will give false information about the disease, so it will not only delay the identification of the cause, but will take inappropriate measures and make the condition more serious. It can have serious consequences. Therefore, suppressing false positives is a very important issue from the main purpose of simple test methods.

[0006] Conventionally, in order to solve this problem, a specimen is highly diluted, or a buffer containing a surfactant, a basic amino acid, an inorganic salt, or the like is used as a solution for diluting the specimen (see Patent Document 1). In addition, a buffer solution containing a surfactant, a basic amino acid, an inorganic salt, or the like is used as a cleaning solution (see Patent Document 1), and a filter is passed when adding a sample (see Patent Document 2). Ingenuity has been made!

[0007] The flow-through type membrane enzyme immunoassay method using an enzyme for labeling is an excellent method for high-sensitivity measurement. As described above, the sample is diluted or a washing solution is used. This is a problem as a simple assembly method with many detection operation steps.

[0008] An example of the operation procedure of the flow-through type assay method (flow-through type membrane enzyme immunoassay method) using an enzyme-labeled antibody described in Patent Document 2 is shown below.

[0009] (1) A specimen sample is prepared by suspending a specimen collected from a patient in a buffer solution, filtered using a filtration filter, and dropped onto an accessory device.

[0010] (2) An enzyme-labeled antibody is added dropwise.

[0011] (3) A cleaning solution is dropped.

[0012] (4) A substrate solution is dropped to cause a color reaction.

[0013] (5) The reaction stop solution is added dropwise.

(6) Judgment is made based on the presence or absence of coloration on the membrane.

[0015] As described above, the conventional method requires many steps to detect the detection target.

Patent Document 1: Japanese Patent Laid-Open No. 2003-279577

Patent Document 2: JP 2004-28875 A

Disclosure of the invention

Problems to be solved by the invention Accordingly, an object of the present invention is to reduce an operation process for detecting an object to be detected contained in a sample sample in the flow-through type membrane zymno assay method. A further object of the present invention is to provide a method for accurately and rapidly detecting an object in a specimen sample, and a kit therefor. Specifically, it is an object to provide a method and a kit that do not require the washing step of the present invention.

Means for solving the problem

[0017] As a result of earnest research, the inventors of the present application have found that the above object can be achieved by stopping the use of the cleaning liquid and omitting the cleaning step, and have completed the present invention.

That is, the inventor of the present application reduces the nonspecific reaction by filtering a specimen sample using a filtration filter device, and adds a surfactant and Z or urine serum albumin to the specimen diluent. A capture antibody for capturing non-specific reactions and adding a surfactant to the substrate solution to provide a washing effect, and further reducing the filter filter pore size and increasing the membrane pore size. Alternatively, a sample sample containing a target substance is filtered on an assembly device equipped with a membrane to which an antigen is bound using a filtration filter device, and then dropped, and the target substance is captured by the capture antibody or antigen on the membrane to be detected. An enzyme-labeled reagent in which a substance that binds to the target substance is labeled with an enzyme is dropped on the membrane on which the target substance has been trapped, and the capture antibody or antigen target substance. It was found that the washing step can be omitted in the flow-through type membrane enzyme immunoassay method, which is an operation step of forming a complex and subsequently dropping the substrate for the enzyme. The present invention has been completed.

[0019] Aspects of the present invention are as follows.

[0020] [1] A sample sample diluted with a buffer solution was filtered on a membrane of an assembly device equipped with a membrane to which a capture antibody or a capture antigen was bound to capture an object to be detected, using a filtration filter device. It is dropped later to capture the target to be captured by the capture antibody or capture antigen, and an enzyme labeling reagent that binds to the target is dropped on the membrane on which the target is captured, and the capture antibody or capture antigen is detected. A flow-through type membrane enzyme that does not include a step of forming an immunocomplex of a labeling reagent and subsequently dripping a substrate solution for the enzyme onto the membrane and washing with a washing solution. Munoassei Law,

[2] The method according to [1], wherein the buffer for diluting the specimen contains a surfactant at a final concentration of 0.2 to 10 (w / v)%,

[3] The method according to [1] or [2], wherein serum albumin is contained in a buffer solution for diluting the specimen at a final concentration of 1 to 5 (w / v)%,

[4] The method according to any one of [1] to [3], wherein the pore size of the filtration filter is 0.1 to 0.6 m.

[5] The method according to any one of [1] to [4], wherein the membrane has a pore size of 3 to 12 μm,

[6] The method according to any one of [1] to [5], wherein the enzyme used for labeling is alkaline phosphatase,

[7] The method according to any one of [1] to [6], wherein a surfactant is contained in the enzyme substrate solution in an amount of 0.02 to: L (w / v)%.

[8] The method according to any one of [1] to [7], characterized in that it is a detectable influenza virus,

[9] A group force consisting of a pharyngeal or nasal wipe, pharyngeal or nasal wash and nasal aspirate is also selected [1]-[8]

[10] A flow-through membrane zymno assay kit for detecting the presence of an analyte in a sample without using a wash solution, including:

(1) A filtration filter having a pore diameter of 0.1 to 0.6 m,

(2) A membrane attached with a capture antibody or antigen for capturing an object to be detected, and comprising a membrane having a pore diameter of 3 to 12 m,

(3) Surfactant at a concentration such that the final concentration after sample dilution is 0.2 to 10 (w / v)% and Z or the final concentration after sample dilution is 1 to 5 (w / v)% A sample dilution buffer containing serum albumin at a concentration such that

(4) enzyme-labeled antibody or antigen, and

(5) 0.02 ~: A substrate solution containing L (w / v)% surfactant.

The invention's effect [0021] With the method and kit of the present invention, it was possible to reduce the number of detection operation steps, and to establish an easy-to-use and highly reliable assembly method.

[0022] This specification includes the contents described in the specification and Z or drawings of Japanese Patent Application No. 2004-305836, which is the basis of the priority of the present application.

Brief Description of Drawings

FIG. 1 is a plan view of a flow-through type membrane zymno assay device according to an embodiment of the present invention.

FIG. 2 is a cross-sectional view taken along the line I Γ in FIG.

FIG. 3 is a view showing an embodiment of a specimen sample filtration tube used in the present invention.

FIG. 4 is a perspective view of the distal end portion of the specimen sample filtration tube of FIG. 3.

Explanation of symbols

[0024] A: hole

B: Hole

a: Adapter

b: membrane

c: Liquid absorbing member

d: Filtration tube tip

e: Filtration tube body

f: Filtration filter

BEST MODE FOR CARRYING OUT THE INVENTION

[0025] The present invention is described in detail below.

[0026] Flow-through type membrane zymno assay method

The method of the present invention is a simple assembly method for an object to be detected in an analyte sample using an assembly device equipped with a capture antibody or an antigen-bound membrane for capturing the analyte. After being filtered using a filtration filter, the solution is dropped on the membrane to capture the target to be captured by the capture antibody or antigen on the membrane, and the enzyme reacts with the target on the membrane where the target is captured. Drop the labeling reagent and capture antibody or Is a flow-through membrane enzyme that forms an immune complex of an antigen to be detected and an enzyme labeling reagent, and then drops the substrate for the enzyme onto the membrane on which the immune complex has been formed. This is characterized in that the cleaning process is omitted in accordance with the Immunoassay method.

[0027] Hereinafter, the present invention will be described with reference to an example of a more specific procedure for the method of the present invention.

[0028] (1) A specimen sample is prepared by diluting a specimen collected from a patient with a buffer solution, filtered using a filtration filter, and dropped on an accessory device.

[0029] (2) An enzyme labeling reagent is dropped.

[0030] (3) A substrate solution is dropped to cause a color reaction.

[0031] (4) Judgment is made based on the presence or absence of coloring on the membrane.

[0032] When determining the presence or absence of color development, a reaction stop solution may be used as necessary.

[0033] When the determination is made at a predetermined reaction time, an operation of dropping the reaction stop solution is necessary! /, But the reaction is stopped by dropping the reaction stop solution at the predetermined reaction time. Use the reaction stop solution when judging by

[0034] In this specification! /, An assembly device equipped with a membrane (also referred to as a membrane assembly device) is a device comprising a membrane on which a capture antibody that specifically binds to an object to be detected is immobilized. It is. Such an accessory device is preferably an accessory device that uses a flow-through membrane enzyme control method.

[0035] The flow-through type membrane zymno assay device is, for example, a device as shown in Figs.

FIG. 1 is a plan view of the apparatus, and FIG. 2 is a cross-sectional view taken along the line I Γ of FIG. In FIGS. 1 and 2, a is an adapter having an opening for dropping the prepared specimen sample and having holes (A hole and B hole) through which the sample passes on the bottom surface. b is a membrane to which a capture antibody that specifically binds to an object to be detected is bound, and c is a member that absorbs liquid.

[0037] The material of the membrane in the membrane assembly device includes non-woven fabric, paper, nitrocellulose, glass fiber, silica fiber, cellulose ester, nylon 6, 6 and a group force composed of a mixture of cellulose ester and -trocellulose. The ones that are selected are listed and are particularly preferred An example is a microporous material made of trocellulose. In addition, a mixture of the cellulose ester and -trocellulose can also be suitably used. The membrane has a pore size or retained particle size that is not less than the pore size of the filtration filter or the retained particle size, and is preferably 3 to 12 μm, particularly preferably 3 to 5 μm. The thickness of the membrane is not particularly limited, but is usually about 100 to 200 / ζ πι.

[0038] On the membrane surface of the membrane assembly device, an antibody or an antigen that captures the detection target by binding specifically to the detection target by an antigen-antibody reaction to form an antigen-antibody complex is solid-phased. The Therefore, it is natural that the capture antibody to be used differs depending on the substance to be detected. However, if the object to be detected is bacteria, viruses, hormones, other clinical markers, etc., the polyclonal antibody that specifically reacts and binds to these. Examples thereof include antibodies and monoclonal antibodies. Examples of the antigen include a purified natural antigen or a recombinant antigen.

[0039] The method for binding such a capture antibody to the above-described membrane may be physical adsorption or chemical binding. The membrane to which the capture antibody is bound is prepared, for example, by applying a solution obtained by diluting the capture antibody with a buffer or the like to the membrane and then drying it.

[0040] The membrane assembly device of the present invention may include a liquid absorbing member that absorbs the liquid that has passed through the membrane.

[0041] The sample collected from the patient is diluted with a buffer solution to obtain a sample sample, which is filtered using a filtration filter. The pore size (diameter) or retained particle size of the filtration filter is 0.1-0.

It is.

[0042] The pore size or retained particle size of the filtration filter is extremely important in omitting the above-described washing step. If the pore size or retained particle size is too large, non-specific binding may occur on the membrane, indicating false positives. On the other hand, if it is too small, the filter itself will be clogged due to viscous substances and agglomerates present in the sample, and there is a force that makes filtration impossible! It is inappropriate because of its purpose of use in simple inspection methods.

[0043] The material of the filtration filter is not particularly limited, but is preferably glass fiber or -trosse It is roulose.

[0044] The filtration filter 1 may be provided in an opening for dropping the specimen of the assembly device. However, in the simple assembly method or kit of the present invention, the filtration filter is used by being attached to a specimen sample filtration device. It is preferable. The specimen sample filtration device is a container-like device having a single filtration filter, for example, a tubular device (a specimen sample filtration tube). For example, a method in which a filtration filter is attached to the tip of a filtration tube, a sample diluted with a buffer solution is poured into the filtration tube, filtered through a filtration filter attached to the tip, and the filtrate is dropped onto a membrane in an accessory device Is convenient and preferable.

A schematic view of one embodiment of this filtration tube is shown in FIGS. For example, as shown in FIG. 3, the filtration tube has a shape composed of a tip part d and a body part e, and a filtration filter f is provided inside the tip part as shown in FIG. Place the specimen sample diluted with the buffer solution in the body part e, and attach the tip part d with the filter f to the body part e. The specimen sample is filtered through the filtration filter f, and the filtrate is dropped on the membrane of the accessories. If the main body e is made of a flexible material such as polyethylene or polyethylene terephthalate (PET), the sample can be filtered easily by applying pressure to the inside with the filter attached. ,preferable.

[0046] The object to be detected in the present specification is not limited in any way, and may be any antigenic substance capable of producing an antibody corresponding thereto. Examples include virus antigens such as influenza virus, adenovirus, RS virus, HAV, HBs, HCV, HIV, EBV, Norwalk-like virus, Chlamydia 'Tracomatis, Streptococcus, Bordetella pertussis, Helicopactor' H. pylori, Leptovira, Treponema ' Paridam, Toxoplasma gondii, Borrelia, Legionella spp., Bacillus anthracis, MRSA and other bacterial antigens, toxins produced by bacteria, mycoplasma lipid antigens, peptide hormones such as gonadotropin from human chorionic steroids, steroid hormones such as steroids, and epinephrine And bioactive amines such as morphine, vitamins such as vitamin B, prostaglandins, antibiotics such as tetracycline, various tumor markers, agricultural chemicals, environmental hormones, and the like.

[0047] Further, in the present specification, the detected object may be an antibody, for example, influenza virus. Virus, adenovirus, RS virus, HAV, HBs, HCV, HIV, EBV, Norwalk virus, etc. Examples include antibodies against bacterial antigens such as Gondi, Borrelia, Legionella, Bacillus anthracis, MRSA, toxins produced by bacteria, and mycoplasma lipid antigens.

[0048] The specimen may be, for example, a pharynx or nasal cavity wipe collected from a patient's pharynx or nasal cavity, a sterile swab, a pharyngeal or nasal rinse, a nasal aspirate, saliva, serum, stool suspension Liquid, urine, culture solution, etc. can be used, but the specimen obtained in this way is diluted in a buffer solution and subjected to assay. As a buffer solution used for dilution of the specimen, a buffer solution or the like usually used in antigen detection or quantification by an immunological technique such as Enzyme Immunoassay or immunoagglutination can be used. More specifically, the force includes Tris buffer solution, Good buffer solution and the like, but is not limited thereto.

[0049] In addition, if the specimen has a pharynx or nasal cavity, the pharynx collected using a specimen collection device such as a sterilized cotton swab! /, If the specimen has viscosity such as nasal wipes, liquid, pharynx or nasal rinse, etc. The sample floats in the buffer that dilutes. In this case, the buffer is referred to as a sample suspension buffer.

[0050] The buffer for diluting the specimen contains animal-derived serum albumin such as surfactant 0.2 to 10 (w / v)%, ushi serum albumin (BSA) at the final concentration when the specimen is added. 1 to 5 (w / v)%, or an appropriate concentration of inorganic salt, etc. can be contained, and by diluting with these buffers, components other than the detection target contained in the sample The binding to the membrane itself is preferable because non-specific binding to the capture antibody on the membrane can be reduced.

[0051] As the surfactant, Triton X-100 (trade name): polyethylene glycol mono-p isooctyl phenol ether, Tween 20: polyoxyethylene sorbitan monolaurate, Tween 80: polyoxyethylene sorbitan monooleate, Nonidet P—40: Nodette P—40, ZWITTERGENT 3—14: n—Tetradecyl N, N Dimethyl-3—Ammonia 1 Propanesulfonate, CHAPS: 3 — [(3 colamidopropyl) dimethylammonium -O] Propanesulfonic acid, sodium dodecyl sulfate (SDS), etc., or a mixture of two or more of these can be used, but is not limited thereto. [0052] The enzyme labeling reagent is an enzyme labeling reagent in which a substance that specifically binds to an object to be detected is labeled with an enzyme. The substance that specifically binds to the detection target is not limited, but an antigen or antibody that specifically binds to the detection target is desirable. For example, when the detected substance is a viral or bacterial antigen, it is an antibody against the viral antigen or bacterial antigen, which is labeled with an enzyme. Further, when the detection target is an antibody, it is an antibody to which the antibody binds or an antibody against the antibody, which is labeled with an enzyme. As described above, a polyclonal antibody, a monoclonal antibody, or the like that specifically reacts with and binds to an object to be detected is used.

[0053] Such an enzyme labeling reagent detects a complex by adding a substrate solution of the enzyme that generates a substance detectable by a colorimetric method or a fluorescence method by a reaction catalyzed by the enzyme. Can go out.

[0054] Examples of the enzyme used for enzyme labeling include alkaline phosphatase and peroxidase. The substance can be labeled with an enzyme by a known method.

[0055] The substrate solution is a substrate for the enzyme used as a label for the enzyme labeling reagent, and generates a substance detectable by a colorimetric method or a fluorescence method by a reaction catalyzed by the enzyme. . Specific examples thereof include 5-bromo-4-chloro-tri-indolyl phosphate trotetrazolium blue and tetramethylbenzidine.

[0056] When the substrate solution is dropped on the membrane, most of the substrate solution passes through the membrane within a short time and is absorbed by the liquid absorbing member, and the substrate solution remaining on the membrane undergoes a color reaction by the labeling enzyme. . Therefore, since the substrate solution that passes through the membrane exhibits a cleaning action, a surfactant generally used in Enzymno Assay is added to the substrate solution to further perform the cleaning action. To reduce non-specific binding.

[0057] The concentration of the surfactant is preferably 0.02 to: L (w / v)%.

[0058] The present invention also includes an accessory kit for performing the above method.

[0059] The accessory kit of the present invention preferably includes at least the following 1 to 5, each having the above-mentioned characteristics.

[0060] 1 filtration filter,

2 Provided with a capture antibody or antigen-bound membrane to capture the analyte Atsy equipment,

3 Sample dilution (floating) buffer,

4 Enzyme labeling reagent,

5 Substrate solution

The filtration filter is preferably attached to a specimen sample filtering device such as the specimen sample filtration tube described above.

[0061] In addition, if necessary, a negative control that also has a buffer solution for testing the activity of the kit, a positive control such as a buffer solution containing a detection target, and a reaction stop solution can be included. . In addition, it may include a sample collection device such as a sterile swab used to collect pharyngeal or nasal wipes.

Example

[0062] Hereinafter, the present invention will be described more specifically based on examples.

However, the present invention is not limited to the following examples.

[0064] Example 1

1. Production of anti-influenza virus monoclonal antibodies

(1) Anti-influenza A virus NP antibody

BALBZc mice were immunized with influenza A virus antigens, spleens were removed from mice reared for a certain period of time, and mice were obtained by the method of Keller et al., Nature, vol, 256, p495-497 (19 75)). Fused with myeloma cells (P3 X 63). The obtained fused cells (hybrid cells) are maintained in a 37 ° C incubator, and the antibody activity of the supernatant is confirmed by ELISA using a plate with a solid phase of influenza A virus NP antigen.匕匕 (monocloning) was performed. The obtained two cell lines were each intraperitoneally administered to pristane-treated BALB Zc mice, and about 2 weeks later, antibody-containing ascites was collected.

[0065] IgG was purified from the obtained ascites by a affinity chromatography method using a protein A column to obtain two types of purified anti-influenza A virus NP antibodies.

[0066] (2) Anti-B influenza virus NP antibody

Two types of purified anti-type B using influenza B virus antigen in the same way as (1) INFNORE ENZYNORES NP antibody was obtained.

[0067] 2. Preparation of enzyme-labeled anti-influenza virus antibody

(1) Production of enzyme-labeled anti-influenza A virus antibody

One of the purified anti-influenza A virus NP antibodies! 45 mg was dialyzed with 0.1 M citrate buffer (PH3.6), and then supplemented with pepsin lOmg, 1 at 37 ° C. Time, Fab 'digestion treatment was performed. The treated solution was fractionated on an Ultrogel AcA34 column to obtain a purified fraction of anti-A-type influenza virus NP antibody F (ab ′) 2. The fraction was concentrated to about 1 OmgZmL, mixed with 0.1M mercaptoethylamine at a volume ratio of 10: 1, and reduced at 37 ° C. for 90 minutes. The treatment solution was fractionated with an Ultrogel AcA34 column to obtain an anti-influenza A influenza virus NP antibody Fab and purified fraction, and then concentrated to about 1 mL.

[0068] lOmgZmL alkaline phosphatase 1. Dialyze 5mL against 50mM borate buffer (pH7.6) containing ImM magnesium chloride and 0. ImM zinc chloride, then N- (6-maleimidocaproyloxy) succinimide. 7 mg was added and treated at 30 ° C for 1 hour. The treated solution was fractionated on a Sephadex G-25 column, and the first peak was collected to obtain maleimide-alkaline phosphatase, and then concentrated to about 1 mL.

[0069] Concentrated anti-influenza A virus NP antibody Fab and maleimide-alkaline phosphatase were mixed at a protein ratio of 1: 2.3 and allowed to react with gentle stirring at 4 ° C for 20 hours. A phosphatase-labeled anti-influenza A virus NP antibody Fab was obtained. Further, the reaction solution was fractionated with an Ultrogel AcA34 column, and unreacted substances were removed to obtain a purified alkaline phosphatase labeled anti-influenza A virus NP antibody Fab. Purified alkaline phosphatase labeled anti-influenza A virus NP antibody Fab, 0.1M sodium chloride, 4% (WZV) urine serum albumin, 1.5mM magnesium chloride, 0.15mM zinc chloride, l% (WZV ) Triton X-100, diluted with a labeled antibody diluent consisting of 25 mM Tris buffer (pH 8.0) containing 0.09% sodium azide, filtered through a 0.22 m pore size filter, An enzyme-labeled anti-influenza A virus antibody was obtained.

[0070] (2) Preparation of enzyme-labeled anti-influenza B virus antibody

For one of the purified anti-influenza B virus NP antibodies, an enzyme-labeled anti-influenza B virus antibody was obtained in the same manner as (1). [0071] Fabrication of influenza device for influenza virus detection

As an assembly device for detecting influenza virus, a device having the same configuration as that shown in FIGS. 1 and 2 was used. As the membrane, a trocellulose membrane (size 2 × 3 cm, thickness 125 m) having a pore diameter of 3 m was used. The solid phase of the capture antibody on the membrane was performed by spotting two antibody solutions onto the -trocellulose membrane. In the A-hole of the device, purified anti-influenza A virus NP antibody that was not used for labeling was diluted with purified water so as to be contained in lmgZmL, and filtered through a 0.22 μm pore size filter. Dilute the purified anti-influenza B virus NP antibody, which was used for labeling, with purified water to contain lmg / mL, and add 0.2 μm pore size to the B hole. 12 L of each filtered with a filter was spotted. After spotting, drying was performed in a 45 ° C drying cabinet for 40 minutes to produce an influenza virus detection assembly.

[0072] Detection of influenza virus

As samples, influenza A virus and influenza B virus cultured in hatched chicken eggs were used.

[0073] Influenza virus buffer for specimen suspension (Triton X—100 l (w / v)%, urine serum albumin 4 (w / v)%, sodium chloride 0.15M, sodium azide 0.09 ( Using 50 mM Tris buffer solution (pH 8.0) containing w / v)%, the sample was diluted 10-fold in a sample sample filter tube to prepare a sample sample.

[0074] 150 μL each of the prepared specimen sample was dropped into the A-hole and B-hole of the Atsey device, and was allowed to stand until the sample was completely absorbed by the liquid absorbing member provided at the bottom of the nitrocellulose membrane.

[0075] Next, 180 L each of the enzyme-labeled anti-type influenza virus antibody is dropped into the hallway and the enzyme-labeled anti-type B influenza virus antibody is dropped into the hall B, and the enzyme-labeled antibody is placed on the liquid absorbing member. Let stand until completely absorbed.

[0076] Next, the adapter was removed, and the substrate solution (5 bromo-4 chloro-3 indolyl phosphate 0.15 mgZmL, nitrotetrazolium blue 0.3 mg / mL, sodium chloride 5 mM, Triton X-100 0.1% (w / v)%, sodium azide contained in 0.09 (w / v)% 0.1M A tris buffer solution, ρΗ9.5), was added dropwise to each of A hole and B hole in an amount of 250 μL to initiate the color reaction.

[0077] After 10 minutes, the hall and hall were observed from above, and if color development was observed only in the A hole, it was positive for influenza A virus, and if color development was observed only in the B hole, it was type B If the influenza virus was positive and no color was observed in both A and B holes, it was judged negative.

[0078] Comparative study

At the same time, a comparison test using a conventional detection procedure using a cleaning solution was performed and compared.

[0079] Results

When influenza A virus was used as a specimen, A-hole was positive up to an influenza A virus concentration of 10 ⁵ (pfo / mL), B-holes were all negative, and it was confirmed that influenza A virus could be detected specifically. It was.

[0080] In addition, when influenza B virus was used as a specimen, B-hole was positive up to a B-type influenza virus concentration of 10 ⁵ (pfo / mL), all A-holes were negative, and the influenza B virus was specifically detected. It was confirmed that it could be detected.

[0081] The results of the method of the present invention and the comparative method were consistent.

[0082] Tables 1 and 2 show the obtained results.

[table 1]

Detection of influenza A virus Virus concentration (pfu / mL)

1 0 ⁶ 1 0 ⁵ 1 0 ⁴

Meiji A Hall + + One

Hall B

Hall A + +

B Hall criteria

+: Blue coloring

No blue coloration

[¾2]

Detection of influenza B virus Virus concentration (piu / niL)

1 0 ⁶ 1 0 ⁵ 1 0

Method A Hall

Hall B + +

Hall A

Hall B + criteria

+: Blue coloring

No blue coloration

[0083] As shown in Tables 1 and 2, it can be seen that influenza virus can be specifically detected with high sensitivity.

[0084] All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims

[1] A sample sample diluted with a buffer solution is filtered on a membrane of an assembly unit equipped with a membrane to which a capture antibody or a capture antigen is bound to capture an object to be detected. The target substance is captured by a capture antibody or capture antigen, and an enzyme labeling reagent that binds to the target substance is dropped onto the membrane on which the target object is captured, and the capture antibody or capture antigen is detected. The body is then formed, and then the substrate solution for the enzyme is dropped onto the membrane.

V, a flow-through membrane zymno assay method that does not include a cleaning step.

[2] The method according to [1], wherein the buffer for diluting the specimen contains a surfactant at a final concentration of 0.2 to 10 (w / v)%.

[3] The method according to claim 1 or 2, wherein the serum albumin is contained at a final concentration of 1 to 5 (w / v)% in the buffer for diluting the specimen.

[4] The method according to any one of claims 1 to 3, wherein the pore size of the filtration filter is 0.1 to 0.6 m.

[5] The method according to any one of [1] to [4], wherein the membrane has a pore size of 3 to 12 μm.

[6] The enzyme according to claim 1 to 5, wherein the enzyme used for labeling is alkaline phosphatase.

V, the method according to item 1 above.

[7] The method according to any one of [1] to [6], wherein a surfactant is contained in the enzyme substrate solution in an amount of 0.02 to L (w / v)%.

[8] The method according to any one of [1] to [7], wherein the detected substance is an influenza virus.

[9] The method according to any one of [1] to [8], wherein the specimen is selected as a group force that also has a pharyngeal or nasal wiping solution, a pharyngeal or nasal washing solution, and a nasal suction fluid force.

(1) A filtration filter having a pore diameter of 0.1 to 0.6 m,

(2) A membrane to which a capture antibody or antigen for capturing an object to be detected is bound, Assembly equipment with a membrane with a pore size of 3-12 m,

(4) enzyme-labeled antibody or antigen, and

(5) 0.02 ~: A substrate solution containing L (w / v)% surfactant.