JP4365329B2 - Simple assay device and method for detecting a plurality of objects to be detected - Google Patents

Description

  The present invention relates to a simple flow-through membrane enzyme immunoassay method for detecting an object to be detected in a specimen sample.

  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. In these reagents and kits, pathogen components, human chorionic gonadotropin, and the like are targets for detection or quantification. Many of the simple test reagents have the feature that they do not require special equipment, are easy to operate and are inexpensive, and 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, and if the presence or absence of infection is identified on the spot for samples collected from patients, treatment measures can be taken at an early stage. The importance in medicine is increasing.

  At present, membrane assay methods, particularly assay methods using membranes such as nitrocellulose and filters, are generally known as simple test methods, and are roughly classified into flow-through assay methods and lateral flow assay methods. . The former allows a solution containing an object to be detected to pass in a direction perpendicular to the film, and the latter allows the solution to be developed in the horizontal direction.

  In either case, a complex of a capture substance that specifically binds to the detected substance, a detected substance, and a labeled substance that specifically binds to the detected substance is formed on the membrane, and the label is detected or quantified. This is common in that the object to be detected is detected.

  In addition, in a simple test method using a membrane assay method, in the analysis of a sample actually collected from a patient, a so-called false positive may occur, in which a detected object is determined to be positive even though it is not present in the sample. is there. If a false positive reaction occurs when testing for a pathogen infection, it will give false information about the disease, which not only delays the identification of the cause, but also takes improper measures and makes the condition more serious. Can have serious consequences. Therefore, suppressing false positives is an extremely important issue from the main purpose of use of the simple test method.

  Conventionally, in order to solve this problem, a specimen is highly diluted, or a buffer containing bovine serum albumin, a surfactant, a basic amino acid, an inorganic salt, or the like is used as a solution for diluting the specimen (Patent Document 1). For example, 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). Have been devised.

  The flow-through type membrane enzyme immunoassay method using an enzyme for labeling is an excellent method for clear and easy determination of positive signals and suitable for high-sensitivity measurement. For each test item, a sample sample, an enzyme labeling reagent, a washing solution, Since the substrate solution needs to be dropped, the number of operation steps is large, which is a problem as a simple assay method.

  In general, acute respiratory tract infections occupy the most important position in clinical practice. Causative pathogens of febrile respiratory tract infection with cough are influenza A virus, influenza B virus, RS virus (RSV), rhinovirus, etc., and these viral infections occur mainly in the winter. It is said that if these viruses can be simultaneously and quickly measured with a single assay device with high sensitivity and the causative virus can be identified, it will be valuable information for clinicians and the quality of medical care will be greatly improved.

  In addition, the causative pathogens of infectious gastroenteritis in children are mostly viruses except in summer, and include rotaviruses, noroviruses, adenoviruses, and astroviruses. As well as respiratory tract infections are desired.

Below, an example of the operation procedure of the flow-through type assay method (flow-through type membrane enzyme immunoassay method) which measures one item with one assay apparatus using the enzyme labeled antibody described in patent document 2 is shown.
(1) A sample collected from a patient is suspended in a buffer solution to prepare a sample, filtered using a filtration filter, and dropped onto an assay device.
(2) An enzyme-labeled antibody is dropped.
(3) A cleaning solution is dropped.
(4) A substrate solution is dropped to cause a color reaction.
(5) The reaction stop solution is added dropwise.
(6) Judgment is made based on the presence or absence of coloring on the membrane.
As described above, the conventional method requires many steps until detection of an object to be detected, and takes an operation time.

JP 2003-279777 A JP 2004-28875 A

  Therefore, the present invention is a flow-through type membrane enzyme immunoassay device for detecting an analyte contained in a specimen sample, in order to identify what is the substance to be detected present in the specimen sample. It is an object of the present invention to provide a method for identifying a substance to be detected that shows a positive reaction by simultaneously testing a plurality of kinds of substances to be detected (measurement items) using an apparatus.

  As a result of diligent research, the present inventor added a specimen sample prepared by diluting with a buffer solution or the like to an enzyme labeling reagent part containing a plurality of enzyme labeling reagents capable of binding to a plurality of objects to be detected, and further adding a filtration filter. The above-mentioned object can be achieved by adding a plurality of capture reagents for capturing a plurality of analytes onto a membrane of an assay device having a membrane bound to a position distant from each other after being filtered. I found. Further, the present inventor uses an adapter (specimen sample supply device) or a specimen sample addition device equipped with one or both of an enzyme labeling reagent part and a filtration filter to prepare a specimen sample prepared by diluting with a buffer solution or the like. The inventors have found that the above object can be achieved by adding to an adapter (specimen sample supply device) or a sample sample adding device, filtering, and then adding to the membrane, thereby completing the present invention.

  That is, after adding a sample sample to an adapter (specimen sample supply device) or sample sample addition device equipped with an enzyme labeling reagent part and mixing the enzyme labeling reagent and the sample sample, the adapter (specimen sample supply device) or sample sample Filter using an addition device and drop multiple capture reagents for capturing multiple analytes onto the membrane of an assay device with a membrane bound to a remote location. A flow-through for simultaneous testing of a plurality of items consisting of a step of forming an immune complex consisting of a capture reagent-detected substance-enzyme labeling reagent, and subsequently dropping a substrate solution for the enzyme onto the membrane. The present invention is completed by finding that the substance to be detected in a specimen sample can be specified by the formula membrane enzyme immunoassay method. Led was.

That is, the aspects of the present invention are as follows.
[1] A plurality of capture reagents capable of binding to different analytes may bind to a membrane bound at a position distant from each other, a filtration filter located upstream of the membrane, and the different analytes located upstream of the filtration filter A method for detecting a plurality of analytes in a single assay using a flow-through membrane enzyme immunoassay device comprising an enzyme labeling reagent part containing a plurality of enzyme labeling reagents,
(i) A sample sample is added to the enzyme labeling reagent section, resulting in the formation of a sample sample and enzyme labeling reagent mixture, which is filtered through a filtration filter and added onto the membrane, and the detection target is captured by the capture reagent. Thus, an immune complex composed of a capture reagent-detected object-enzyme labeling reagent is formed, and then
(ii) dropping a substrate solution for the enzyme onto the membrane;
A method for detecting a plurality of analytes comprising a step in a single assay;
[2] The method according to [1], wherein the flow-through membrane enzyme immunoassay device integrally includes a sample sample supply device, and the sample sample supply device includes a filtration filter and / or an enzyme labeling reagent unit.
[3] A membrane in which a plurality of capture reagents capable of binding to different objects to be detected are bound to positions separated from each other, and an enzyme labeling reagent section including a filtration filter and / or a plurality of enzyme labeling reagents capable of binding to the different objects to be detected A method for detecting a plurality of analytes in a single assay using a flow-through membrane enzyme immunoassay device comprising a separate sample sample addition device comprising
(i) The sample sample is added to the sample sample addition device, and as a result, a mixture of the enzyme labeling reagent and the sample sample in the enzyme labeling reagent part included in the sample sample addition device is formed,
(ii) Add the sample sample and enzyme labeling reagent mixture from the sample sample addition device onto the membrane;
(iii) adding a substrate solution for the enzyme onto the membrane;
A method for detecting a plurality of analytes comprising a step in a single assay;
[4] Any one of [1] to [3], wherein the enzyme labeling reagent part including a plurality of enzyme labeling reagents capable of binding to different objects to be detected is made of a water-absorbing material impregnated with a plurality of enzyme labeling reagents and dried. A method for detecting a plurality of analytes in a single assay,
[5] A plurality of enzyme labeling reagents capable of binding to the detection target, wherein the detection target is an antigen or an antibody, and a capture reagent capable of binding to the detection target is an antibody or antigen binding to the detection target. Wherein the antibody or antigen that binds to the detection object is labeled with an enzyme, and a method for detecting a plurality of detection objects of any one of [1] to [4] in a single assay,
[6] A method for detecting a plurality of analytes according to any one of [1] to [5] in one assay, wherein the specimen sample is diluted with a buffer or physiological saline.
[7] A membrane in which a plurality of capture reagents capable of binding to different objects to be detected are bound to positions separated from each other, and an enzyme labeling reagent section including a filtration filter and / or a plurality of enzyme labeling reagents capable of binding to the different objects to be detected A flow-through membrane enzyme immunoassay device for detecting a plurality of objects to be detected in a single assay, including a specimen sample supply device including
[8] A membrane in which a plurality of capture reagents capable of binding to different objects to be detected are bound to positions separated from each other, and an enzyme labeling reagent section including a filtration filter and / or a plurality of enzyme labeling reagents capable of binding to the different objects to be detected A flow-through membrane enzyme immunoassay apparatus for detecting a plurality of analytes separately comprising a device for adding an analyte sample containing a single assay,
[9] The plurality of [7] or [8], wherein the enzyme labeling reagent portion including a plurality of enzyme labeling reagents capable of binding to different objects to be detected is made of a water-absorbing material impregnated with a plurality of enzyme labeling reagents and dried. A flow-through membrane enzyme immunoassay device for detecting an analyte in one assay, and
[10] A plurality of enzyme labeling reagents capable of binding to the detection target, wherein the detection target is an antigen or an antibody, and a capture reagent capable of binding to the detection target is an antibody or antigen binding to the detection target. Is a flow-through membrane for detecting a plurality of analytes of any one of [7] to [9] in one assay, wherein the antibody or antigen that binds to the analyte is labeled with an enzyme Enzyme immunoassay device.

  The method of the present invention uses a membrane in which a plurality of capture reagents that can bind to different objects to be detected are bound to positions separated from each other and an enzyme label reagent part that includes a plurality of enzyme labeling reagents that can bind to the different objects to be detected. Therefore, a plurality of objects to be detected can be detected simultaneously with a small number of steps in one assay. Simultaneous detection of multiple analytes is possible, so it is possible to test multiple analyte species simultaneously in a single assay using a single assay device, and to easily identify what analytes show positive reactions. -It is possible to provide a reliable assay method that can be identified quickly and is easy to use. Furthermore, in the method of the present invention, by using an adapter (specimen sample supply apparatus) or a specimen sample addition device including a filtration filter and an enzyme labeling reagent part containing a plurality of enzyme labeling reagents that can bind to the different analytes. In addition, mixing of the detection object and the enzyme labeling reagent and filtration are efficiently performed almost simultaneously, and a highly sensitive, simple and rapid assay can be performed.

  The present invention is described in detail below.

  The method of the present invention is characterized in that it is possible to quickly and easily identify what a substance to be detected in a specimen sample is by simultaneously testing a plurality of items in one assay using a single assay device.

  In the method of the present invention, a membrane in which a plurality of capture reagents capable of binding to different analytes are bound to each other, a filtration filter located upstream of the membrane, and the different analytes located upstream of the filtration filter. A plurality of objects to be detected are detected in one assay using a flow-through type membrane enzyme immunoassay device including an enzyme labeling reagent part containing a plurality of enzyme labeling reagents that can bind. Further, an adapter including a membrane in which a plurality of capture reagents capable of binding to different objects to be detected are bound to positions separated from each other, a filtration filter, and an enzyme label reagent part including a plurality of enzyme labeling reagents capable of binding to the different objects to be detected A plurality of objects to be detected are detected in one assay using a (sample sample supply device) or a flow-through membrane enzyme immunoassay device including a sample sample addition device.

  An assay device including a membrane referred to in the present specification (also referred to as a membrane assay device) refers to a membrane in which capture reagents that specifically bind to a plurality of substances to be detected are immobilized at separate positions. It is preferably an assay device that uses a flow-through type membrane enzyme immunoassay method.

  Specifically, the flow-through type membrane enzyme immunoassay device is a device as shown in FIGS. 1 and 2, for example. The apparatus shown in FIG. 1 and FIG. 2 is an apparatus including a specimen sample addition device described later as a separate body.

  1 is a plan view of the apparatus, and FIG. 2 is a cross-sectional view taken along line II ′ of FIG. In FIGS. 1 and 2, a is a hole through which the sample passes through the bottom surface, having an opening for filtering and dropping the prepared sample using a sample sample addition device equipped with an enzyme labeling reagent unit. It is an adapter provided with (A hole and B hole). b is a membrane to which a capture reagent that specifically binds to an object to be detected is bound, and c is a member that absorbs liquid (liquid absorbing member).

  By increasing the number of holes through which the specimen sample on the bottom surface of the adapter a passes, it is possible to cope with more simultaneous items, and the number is about 1 to 5 items, for example, 2 items and 3 items. Although 4 items or 5 items are preferable, it is not limited to these.

  Examples of the material of the membrane in the membrane assay apparatus include nonwoven fabric, paper, nitrocellulose, glass fiber, silica fiber, cellulose ester, nylon 6, 6 and a group consisting of a mixture of cellulose ester and nitrocellulose, and particularly preferably nitro. Examples include microporous materials made from cellulose. Moreover, the mixture of the said cellulose ester and nitrocellulose can also be used suitably. The membrane has a pore size or retained particle size that is not less than the pore size or retained particle size of the filtration filter, and is preferably 1 to 12 μm, particularly preferably 3 to 5 μm. Moreover, although the thickness of a membrane is not specifically limited, Usually, it is about 100-200 micrometers.

  A capture reagent is immobilized (bound) on the membrane surface of the membrane assay device. Examples of the capture reagent include an antigen or an antibody that captures a target by specifically binding to the target by an antigen-antibody reaction to form an antigen-antibody complex. However, the capture reagent is not limited to an antigen or an antibody. -In the case of substances that can bind to each other in a receptor relationship, one can be used as a capture reagent to capture the other. Therefore, depending on the object to be detected, different capture antigens or antibodies that can bind to it may be used. For example, when the detected substance is a bacterium, virus, hormone, or other clinical marker, a polyclonal antibody or a monoclonal antibody that specifically reacts and binds to these can be used. The capture reagent may be solid-phased on the surface of the membrane corresponding to the hole in the bottom surface of the adapter a of the membrane device. By immobilizing in this way, the sample sample and / or enzyme labeling reagent added to the adapter a passes through the hole on the bottom surface of the adapter a and reaches the site where the capture reagent on the membrane is immobilized. The binding reaction occurs efficiently.

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

  In the present invention, multiple analytes can be detected simultaneously in a single assay. For this reason, on the membrane, a plurality of capture reagents capable of binding to the respective objects to be detected are bound. At this time, each capture reagent is bound apart on the membrane. Here, “separate binding” means that the sites where the respective capture reagents are bonded on the membrane do not overlap or come into contact with each other. In this case, the hole portion of the bottom surface portion of the adapter a of the membrane device corresponding to the solidified capture reagent site is also provided apart from the bottom surface portion of the adapter a.

  The membrane assay device of the present invention may include a liquid absorbing member that absorbs the liquid that has passed through the membrane. The liquid absorbing member is included downstream of the membrane.

  The object to be detected in the present specification is not limited in any way, and includes, for example, any antigenic substance that can produce an antibody corresponding thereto. Examples include influenza A virus, influenza B virus, RS virus (RSV), rhinovirus, rotavirus, norovirus, adenovirus, astrovirus, HAV, HBs, HCV, HIV, EBV and other viral antigens, Chlamydia tracho Bacteria antigens such as Matis, Streptococcus, Bordetella pertussis, Helicobacter pylori, Leptospira, Treponema pallidum, Toxoplasma gondii, Borrelia, Legionella, Anthrax, MRSA, toxins produced by bacteria, mycoplasma lipid antigen, human chorionic Peptide hormones such as gonadotropins, steroids such as steroid hormones, bioactive amines such as epinephrine and morphine, vitamins such as vitamin B, antibiotics such as prostaglandins and tetracycline, and various tumors Manufacturers, agricultural chemicals, mention may be made of the environmental hormones. Moreover, the antibody with respect to these is also mentioned as a to-be-detected object.

  Specimens include, for example, pharyngeal or nasal swabs collected from a patient's pharynx or nasal cavity using a sterile cotton swab or other sample collection device, pharyngeal or nasal wash, nasal aspirate, saliva, serum, stool suspension, urine, A culture solution or the like can be used, but the specimen thus obtained is diluted with a buffer solution or the like for the assay. As a buffer used for dilution of the specimen, a buffer usually used in antigen detection or quantification by an immunological technique such as enzyme immunoassay or immunoagglutination can be used. More specifically, examples include, but are not limited to, Tris buffer and Good buffer. Further, physiological saline may be used.

  In addition, if the specimen has viscosity such as pharyngeal or nasal wipes, pharyngeal or nasal rinses collected from the patient's pharynx or nasal cavity using a specimen collection device such as a sterile cotton swab, the specimen should be diluted in a buffer solution that dilutes the specimen. The specimen floats. In this case, the buffer or the like is referred to as a specimen suspension buffer or the like.

  For the buffer for diluting the specimen, 0.2 to 10 (w / v) surfactant, animal serum albumin 1 to 5 (w / v)% such as bovine serum albumin (BSA), or an appropriate concentration By diluting with these buffers, components other than the detection target contained in the sample can be bound to the membrane itself or bound to the capture reagent on the membrane. Nonspecific binding can be reduced, which is preferable.

  As the surfactant, Triton X-100 (trade name): polyethylene glycol mono-р-isooctyl phenyl ether, Tween 20: polyoxyethylene sorbitan monolaurate, Tween 80: polyoxyethylene sorbitan monooleate, Nonidet P -40: Nonidet P-40, ZWITTERGENT 3-14: n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, CHAPS: 3-[(3-colamidopropyl) dimethylammonio Propanesulfonic acid, sodium dodecyl sulfate (SDS), or the like, or a mixture of two or more of these can be used, but is not limited thereto.

  An enzyme labeling reagent is a reagent in which a substance that specifically binds to a detected substance is labeled with an enzyme, and a substance that specifically binds to a detected substance is, for example, an antigen or antibody that specifically binds to the detected substance. is there. For example, when the detected substance is a virus or bacterial antigenic substance, it is an antibody against the viral antigen or bacterial antigen, which is labeled with an enzyme.

  As with the capture reagent, a polyclonal antibody, a monoclonal antibody, or the like that specifically reacts and binds to an object to be detected is used. The capture reagent and the enzyme labeling reagent are preferably those that bind to different parts of the detection target, but even if the capture reagent and the enzyme labeling reagent bind to the same part of the detection target, There is no problem when there are a plurality of the binding portions. For example, when the detection target is an antigen and the capture reagent and the enzyme labeling reagent are antibodies against the antigen, both antibodies are preferably antibodies that bind to different epitopes of the detection target. Are present (for example, when the antigen is a multimer having a plurality of the same subunits), both antibodies can be used even if they bind to the same epitope.

  Such an enzyme labeling reagent can detect 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. it can.

  Examples of the enzyme used for enzyme labeling include alkaline phosphatase and peroxidase. Labeling of an enzyme with an antigen or antibody can be performed by a known method.

  The enzyme labeling reagent is included in the enzyme labeling reagent part. The enzyme labeling reagent part may be liquid or solid. In the case of a liquid, the enzyme labeling reagent solution may be placed in the enzyme labeling reagent part, and in the case of a solid, a solid impregnated with the enzyme labeling reagent or a freeze-dried product of the enzyme labeling reagent may be placed. By adding the specimen sample to the enzyme labeling reagent part, the enzyme labeling reagent and the specimen sample contained in the enzyme labeling reagent part are mixed to form a mixture. At this time, when the enzyme labeling reagent part is a solid, the enzyme labeling reagent in the solid is dissolved in the specimen sample to form a mixture of the enzyme labeling reagent and the specimen sample. In the case where the enzyme labeling reagent part is not impregnated with a solid and is a liquid or a lyophilized product, the enzyme labeling reagent itself added to the specimen sample addition device is referred to as an enzyme labeling reagent part. The enzyme labeling reagent part is provided upstream of the membrane and the filtration filter. Here, upstream means in the flow-through membrane enzyme immunoassay device, upstream in the flow of the sample or the like when the liquid moves after the liquid specimen sample or the like is added to the device. For example, in the apparatus of FIG. 1, a filtration filter and a solid or liquid enzyme labeling reagent part may be provided above or below the hole inside the adapter, and a filtration filter is provided below the hole inside the adapter. An enzyme labeling reagent part may be provided on the substrate. In this case, the enzyme labeling reagent is mixed with the specimen sample inside the adapter, passed through the filter through the hole, and then added to the membrane. When the enzyme labeling reagent portion is solid, the enzyme labeling reagent may be impregnated with a water-absorbing material. For example, a material such as a nonwoven fabric made of sponge and glass fiber is used, and a labeled antigen or antibody labeled with an enzyme is contained in a dry state. The enzyme labeling reagent portion may be dried by impregnating the water-absorbing material with a labeled antigen or antibody. The enzyme labeling reagent part may be used in advance or in the adapter a part at the time of assay.

  The filtration filter is provided upstream of the membrane and downstream of the enzyme labeling reagent part. The filtration filter may be used in advance or in the adapter a part at the time of assay. The specimen sample mixed with the enzyme labeling reagent is filtered through a filtration filter and added to the membrane. The filtration filter can be provided, for example, in an opening for dropping the specimen of the assay device. A specimen sample prepared by diluting a specimen collected from a patient with a buffer is mixed with an enzyme labeling reagent in an enzyme labeling reagent section and filtered through a filtration filter. The pore diameter (diameter) or the retained particle diameter of the filtration filter is 0.1 to 0.6 μm.

  The pore size or retained particle size of the filtration filter is extremely important for reducing non-specific reactions. If the pore size or retained particle size is too large, non-specific binding may occur on the membrane, which may indicate false positives. On the other hand, if it is too small, the filter itself is clogged due to viscous substances and agglomerates present in the specimen sample, which makes filtration impossible, or the filter area must be considerably widened. It is inappropriate for this purpose.

  The material of the filtration filter is not particularly limited, but is preferably glass fiber or nitrocellulose.

  As described above, in the assay device of the present invention, the adapter can include one or both of a filtration filter and an enzyme labeling reagent unit, and functions as a device for supplying a specimen sample to the assay device. In this regard, the adapter of the present invention can also be referred to as a specimen sample supply device. The assay device of the present invention is also a device that integrally includes an adapter (specimen sample supply device) that includes one or both of a filtration filter and an enzyme labeling reagent unit. It means to be attached to the device. In this respect, it can be said that the adapter (specimen sample supply device) including one or both of the filtration filter and the enzyme labeling reagent unit is used as an integral unit. Thus, the adapter may be present separately from the assay device prior to performing the assay.

  In the present invention, a specimen sample addition device including one or both of a filtration filter and an enzyme labeling reagent part may be used. The filtration filter and the enzyme labeling reagent unit may be included in either the sample sample addition device or the adapter (sample sample supply device). When both the filtration filter and the enzyme labeling reagent part are included in the adapter (specimen sample supply device), the sample sample addition device does not need to be included in the apparatus of the present invention.

  The sample sample addition device is a container-like device for adding a sample sample to an immunoassay apparatus equipped with a filtration filter, for example, a tube-like device (sample sample addition tube). In the simple assay method or kit of the present invention, the filtration filter is preferably used by being attached to the tip of the specimen sample addition device. That is, for example, a specimen sample prepared by diluting a specimen with a buffer solution is placed in a specimen sample addition device, a filtration filter equipped with an enzyme labeling reagent part is attached to the tip of the device, and the specimen sample and the enzyme labeling reagent part After the enzyme labeling reagent is mixed, it is filtered through a filtration filter, and the filtrate is dropped onto the membrane in the assay device, which is convenient and preferable.

  Schematic diagrams of one embodiment of a tubular sample sample addition device (sample sample addition tube) are shown in FIGS. 3 and 4, the enzyme labeling reagent part g is made of a solid impregnated with an enzyme labeling reagent. For example, the sample sample addition tube has a shape composed of a tip portion d and a body portion e as shown in FIG. 3, and an enzyme labeling reagent portion g and a filtration filter f are formed inside the tip portion d as shown in FIG. Is provided. A specimen sample prepared by diluting a specimen with a buffer solution is placed inside the main body e, and a tip part d having an enzyme labeling reagent part and a filtration filter f is attached to the main body e. After dissolving the enzyme labeling reagent in the enzyme labeling reagent part with the sample sample, the sample sample is filtered through the filtration filter f, and the filtrate is dropped onto the membrane of the assay device. When the main body e is made of a flexible material such as polyethylene or polyethylene terephthalate (PET), the specimen sample can be filtered easily and simply by applying pressure to the inside with a filter attached to the body e. This is preferable because it is possible.

  The enzyme labeling reagent part g is preferably attached to the tip part d of the specimen sample addition tube so as to pass through the filtration filter after the specimen sample contacts the enzyme labeling reagent part g as shown in FIG.

Below, an example of a more specific procedure is shown about the method of this invention, and this invention is demonstrated.
(1) A specimen sample is prepared by diluting a specimen collected from a patient with a buffer or the like, and added to the enzyme labeling reagent portion of the assay device. At this time, the enzyme-labeled reagent contained in the enzyme-labeled reagent part is dissolved by the sample sample, and the mixture of the detected substance and the enzyme-labeled reagent in the sample sample passes through the filtration filter downstream of the enzyme-labeled reagent part and enters the membrane. Added.
(2) A substrate solution is dropped to cause a color reaction.
(3) Judgment is made based on the presence or absence of coloring on the membrane.

Furthermore, an example of the specific procedure about the method using a filtration filter apparatus is shown, and this invention is demonstrated.
(1) A specimen sample is prepared by diluting a specimen collected from a patient with a buffer solution or the like, and using an adapter (specimen specimen supply apparatus) or a specimen specimen addition device equipped with a filtration filter and / or an enzyme labeling reagent section. Filter and drop into the assay device.
(2) A substrate solution is dropped to cause a color reaction.
(3) Judgment is made based on the presence or absence of coloring on the membrane.

  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 include 5-bromo-4-chloro-3-indolyl phosphate / nitrotetrazolium blue and tetramethylbenzidine.

  A so-called enzyme that uses coenzyme NADPH (nicotinamide adenine dinucleotide phosphate reduced form) as a substrate for alkaline phosphatase and amplifies the signal enzymatically with alcohol dehydrogenase and diaphorase in the presence of ethyl alcohol and tetrazolium salt. Cycling reaction reagents are commercially available (AmpliQ Dako), but enzyme cycling reaction reagents can also be suitably used.

  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 washing action, a surfactant generally used for enzyme immunoassay is added to the substrate solution, and the washing action is further increased to increase the non-specificity. Bonding can be reduced.

The concentration of the surfactant is preferably 0.02 to 1 (w / v)%.
When the determination is made with a predetermined reaction time, an operation of dropping the reaction stop solution is not necessary.
If the determination cannot be made within a predetermined reaction time, the reaction may be stopped by dropping a reaction stop solution and the determination may be made later.

      Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1. Production of anti-influenza virus monoclonal antibody (1) Anti-influenza A virus NP antibody BALB / c mice were immunized with influenza A virus antigen, and the spleen was removed from mice reared for a certain period of time, and the method of Keller et al. , Nature, vol, 256, p495-497 (1975)) and fused with mouse myeloma cells (P3 × 63). The obtained fused cells (hybridoma) were maintained in a 37 ° C. incubator, and cell purification was performed while confirming the antibody activity of the supernatant by ELISA using a plate on which influenza A virus NP antigen was immobilized (monocloning) ) The obtained two cell lines were each intraperitoneally administered to pristane-treated BALB / c mice, and about 2 weeks later, antibody-containing ascites was collected.
IgG was purified from the obtained ascites by affinity chromatography using a protein A column to obtain two types of purified anti-influenza A virus NP antibodies.

(2) Anti-B influenza virus NP antibody Two types of purified anti-B influenza virus NP antibodies were obtained in the same manner as in (1) using influenza B virus antigen.

(3) Anti-RS virus NP antibody Using the RS virus antigen, two types of purified anti-RS virus NP antibodies were obtained in the same manner as (1).

2. Preparation of enzyme-labeled anti-influenza virus antibody
(1) Preparation of enzyme-labeled anti-influenza A virus antibody One of the purified anti-influenza A virus NP antibodies was dialyzed with 45 mg of 0.1 M citrate buffer (pH 3.6), then 10 mg of pepsin was added, Fab ′ digestion was performed at 37 ° C. for 1 hour. The treated solution was fractionated with an Ultrogel AcA34 column to obtain a purified fraction of anti-influenza A virus NP antibody F (ab ′) ₂ . The fraction was concentrated to about 10 mg / mL, mixed with 0.1 M 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 a purified fraction of anti-influenza A virus NP antibody Fab ′, and then concentrated to about 1 mL.

  After dialyzing 1.5 mL of 10 mg / mL alkaline phosphatase into 50 mM borate buffer (pH 7.6) containing 1 mM magnesium chloride and 0.1 mM zinc chloride, 0.7 mg of N- (6-maleimidocaproyloxy) succinimide was added. Added and treated at 30 ° C. for 1 hour. The treated solution was fractionated on a Sephadex G-25 column, the first peak was collected to obtain maleimide-alkaline phosphatase, and then concentrated to about 1 mL.

  Concentrated anti-influenza A virus NP antibody Fab ′ and maleimide-alkaline phosphatase were mixed at a protein ratio of 1: 2.3 and reacted with gentle agitation at 4 ° C. for 20 hours to give an alkaline 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 ′ contains 0.15 M sodium chloride, 4% (W / V) bovine serum albumin, 1.5 mM magnesium chloride, 0.15 mM zinc chloride, 1% (W / V ) Diluted with a labeled antibody diluent consisting of Triton X-100, 25 mM Tris buffer (pH 8.0) containing 0.09% sodium azide, then filtered through a 0.22 μm pore size filter and enzyme labeled An anti-influenza A virus antibody was obtained.

(2) Preparation of enzyme-labeled anti-B influenza virus antibody An enzyme-labeled anti-B influenza virus antibody was obtained in the same manner as in (1) for one type of purified anti-B influenza virus NP antibody.

(3) Preparation of enzyme-labeled anti-RS virus antibody An enzyme-labeled anti-RS virus antibody was obtained in the same manner as in (1) for one type of purified anti-RS virus NP antibody.

Production of assay devices for detecting type A and type B influenza viruses (for two items) The assay device for detecting influenza viruses was the same as that shown in FIGS. 1 and 2. As the membrane, a nitrocellulose membrane (size 2 × 3 cm, thickness 125 μm) having a pore diameter of 3 μm was used. The solid phase of the capture antibody on the membrane was obtained by spotting two types of antibody solutions on the nitrocellulose membrane. In the A-hole of the apparatus, a purified anti-influenza A virus NP antibody that was not used for labeling was diluted with purified water so as to be contained in 1 mg / mL, and filtered through a 0.22 μm pore size filter. Of purified anti-influenza B virus NP antibody that was not used for labeling was diluted with purified water to contain 1 mg / mL and filtered through a 0.22 μm pore size filtration filter. 3 μL of each was spotted. After spotting, drying was performed in a 45 ° C. drying cabinet for 40 minutes to produce an assay device for detecting influenza virus. Next, a specimen sample addition device equipped with an enzyme labeling reagent part and a filtration filter was produced with the same configuration as that shown in FIGS. The enzyme-labeled reagent part is mixed with an enzyme-labeled anti-influenza A virus antibody and an enzyme-labeled anti-influenza B virus antibody at an optimal concentration, impregnated in a polyvinyl sponge having a diameter of 9 mm and a thickness of 4 mm, and dried. The sample was added to the tip of the sample sample addition tube.

Detection of influenza A and B viruses As specimen samples, specimens collected from the nasal cavity using a sterile cotton swab were used as buffer solutions (Triton X-100 1 (w / v)%, bovine serum albumin 4 (w / v)%, Prepared by diluting with sodium chloride 0.15M, sodium azide 0.09 (w / v)% 50 mM Tris buffer, pH 8.0),
Nasal wiping fluid determined as positive for influenza A virus by PCR: sample 1
Nasal wiping fluid determined as positive for influenza B virus by PCR: Specimen sample 2
Nasal wipes determined to be negative for influenza A and B influenza viruses by PCR: Specimen sample 3
Was used.
These specimen samples were prepared in the specimen sample addition tube main body containing a buffer solution.

Test Attach the tip of the sample sample addition tube with the enzyme labeling reagent containing the enzyme labeling reagent to the sample sample addition tube body containing the prepared sample sample, dissolve the enzyme labeling reagent with the sample sample, and filter It filtered with the filter, 150 microliters was dripped at the adapter opening part of the assay apparatus, and it left still until the sample was absorbed into the liquid absorption member with which the lower part of the nitrocellulose membrane was equipped.

  Next, the adapter was removed, and the substrate solution (5-bromo-4-chloro-3-indolyl phosphate 0.15 mg / mL, nitrotetrazolium blue 0.3 mg / mL, magnesium chloride 5 mM, Triton X-100 0.1 250 μL of 0.1 M Tris buffer (pH 9.5) containing (w / v)% and sodium azide 0.09 (w / v)% was added dropwise to initiate the color reaction.

  10 minutes later, when observed from the top vertically, if the coloration was observed only on the membrane in the A-hole part, the influenza A virus was positive; if the coloration was observed only on the B-hole membrane, the influenza B virus was positive; When color development was not recognized in the membranes of both A and B holes, it was judged as negative.

Results When specimen sample 1 positive for influenza A virus was used as the specimen sample, the membrane in the A-hole part was positive and the membrane in the B-hole part was negative, confirming that the influenza A virus could be detected specifically.

When the specimen sample 2 positive for influenza B virus was used as the specimen sample, it was confirmed that the membrane in the A-hole part was negative and the membrane in the B-hole was positive, so that the influenza B virus could be detected specifically.
In addition, when A-type and B-type influenza virus-negative specimen sample 3 was used as the specimen sample, both the A-hole part and the B-hole membrane were negative.
The obtained results are shown in Table 1.

  As shown in Table 1, it can be seen that influenza virus types A and B can be specifically detected simultaneously.

Preparation of A, B type influenza virus, RS virus (RSV) detection assay device (for 3 items) 3 holes (A), B type influenza virus, and RS virus are provided in the adapter with 3 holes. An assay device for detecting (A hole: influenza A virus, B hole: influenza B virus, C hole: RS virus) was prepared in the same manner as described above. Next, a specimen sample addition device equipped with an enzyme labeling reagent part and a filtration filter was produced with the same configuration as described above. The enzyme-labeled reagent part is prepared by mixing an enzyme-labeled anti-influenza A virus antibody, an enzyme-labeled anti-type B influenza virus antibody and an enzyme-labeled anti-RS virus antibody at an optimal concentration, followed by drying in the same manner as described above. It was prepared by attaching to the part.

Detection of A-type, B-type influenza virus, and RS virus
Nasal wiping fluid determined to be positive for influenza A virus by PCR: Specimen sample 4
Nasal wiping fluid determined to be positive for influenza B virus by PCR method: Specimen sample 5
Nasal wipes determined to be positive for RS virus by PCR method: Specimen sample 6
Nasal wiping fluid determined to be A-type, B-type influenza virus negative and RS virus negative by PCR method: specimen sample 7
Was used.

The test was performed as described above.

Results When specimen sample 4 positive for influenza A virus is used as the specimen sample, the membrane in the A-hole part is positive and the membranes in the B-hole part and C-hole part are negative, so that the influenza A virus can be detected specifically. It was confirmed.

  When the specimen sample 5 positive for influenza B virus was used as the specimen sample, the membrane in the B-hole part was positive and the membranes in the A-hole part and C-hole part were negative, confirming that the influenza B virus could be detected specifically. It was.

  When specimen sample 6 positive for RS virus was used as the specimen sample, the membrane in the C-hole part was positive and the membranes in the A-hole part and B-hole part were negative, confirming that RS virus could be detected specifically.

In addition, when A-type, B-type influenza virus negative and RS virus negative sample samples 7 were used as sample samples, the membranes of the A-hole part, B-hole part and C-hole part were negative.
The obtained results are shown in Table 2.

  As shown in Table 2, it can be seen that influenza virus type A, type B and RS virus can be specifically detected simultaneously.

It is a top view of the flow-through type membrane enzyme immunoassay device which is one embodiment of the present invention. FIG. 2 is a cross-sectional view taken along the line I-I ′ of FIG. 1. It is a figure which shows one embodiment of the sample sample addition tube used in this invention. FIG. 4 is a perspective view of the distal end portion of the specimen sample addition tube of FIG. 3.

Explanation of symbols

A: Hole B: Hole a: Adapter b: Membrane c: Liquid absorbing member d: Sample sample addition tube tip
e: specimen sample addition tube body f: filtration filter g: enzyme labeling reagent part

Claims (5)

(a) a membrane in which a plurality of capture reagents capable of binding to different analytes are bound to positions separated from each other; and
(b) An analyte sample supply adapter having an opening for dropping the analyte sample and a plurality of holes through which the sample passes and is added to the membrane, and a plurality of enzyme labeling reagents capable of binding to the different analytes Including a sample sample supply adapter including an enzyme labeling reagent part and a filtration filter,
The specimen sample supply adapter is used to attach a plurality of objects to be detected that are attached upstream of the membrane so that a plurality of capture reagent binding sites bound to the membrane correspond to a plurality of holes of the specimen sample supply adapter, respectively. Flow-through type membrane enzyme immunoassay device for detection in the above assay. Different enzyme-labeled reagent portion containing a plurality of enzyme-labeled reagent capable of binding to the object to be detected, a plurality of the detected object according to claim 1 Symbol mounting comprising a plurality of enzyme-labeled reagent is impregnated with a water absorbing material was dried 1 Flow-through membrane enzyme immunoassay device for detection in a single assay. The detection object is an antigen or an antibody, the capture reagent that can bind to the detection object is an antibody or antigen that binds to the detection object, and a plurality of enzyme labeling reagents that can bind to the detection object include the detection object. 3. A flow-through membrane enzyme immunoassay device for detecting a plurality of analytes according to claim 1 or 2 in one assay, wherein an antibody or an antigen that binds to the analyte is labeled with an enzyme. A method for detecting a plurality of analytes in one assay using the flow-through membrane enzyme immunoassay device according to any one of claims 1 to 3 ,
(i) The sample sample is added to an enzyme labeling reagent part included in the sample sample supply adapter or the sample sample addition device, and as a result, a sample sample and an enzyme labeling reagent mixture are formed, and the mixture is filtered through a filtration filter. By adding to the capture reagent on the membrane from multiple holes of the sample sample supply adapter and capturing the target to be captured by the capture reagent, an immune complex consisting of the capture reagent, the target, and the enzyme labeling reagent is formed. Then
(ii) dropping a substrate solution for the enzyme onto the membrane;
A method for detecting a plurality of objects to be detected in a single assay. The method for detecting a plurality of analytes according to claim 4 , wherein the specimen sample is diluted with a buffer or physiological saline and used in one assay.

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