JP4286157B2 - Membrane assay - Google Patents

Description

  The present invention relates to a simple membrane assay for measuring an object to be measured in a specimen sample.

  Recently, a simple test that uses antigen-antibody reaction or enzyme reaction to detect or quantify various measurement items such as infection with pathogens such as viruses and bacteria, presence of pregnancy, blood glucose level in a few minutes to tens of minutes. Reagents or kits have been developed. Pathogen constituent proteins, human ciliary gonadotropin (hCG), blood glucose, and the like are targets for detection or quantification. Many of the simple test reagents are characterized by the fact that they do not require special equipment, are easy to operate, and are inexpensive. For example, simple test reagents for pregnancy diagnosis are sold as OTC at general pharmacies. Unlike other test reagents, simple test reagents for measuring infection with pathogens 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 specimens collected from patients are found to be infected on the spot, symptoms can be treated as soon as possible. The importance of test reagents in medicine is increasing.

  At present, membrane assay methods, particularly assay methods using membranes such as nitrocellulose and membranes such as filters, are generally known as simple test methods, including flow-through type (patent No. 2114531) and lateral flow type membrane assay. Laws (Japanese Patent No. 2890384) are roughly classified. The former allows a solution containing the object to be measured to pass through a film coated with a substance for detection on the object to be measured in the vertical direction, and the latter allows the solution to be developed in the horizontal direction. In either case, a membrane solid phase substance that specifically binds to the analyte, a analyte, or a complex of a label substance that specifically binds to the analyte is formed on the membrane to detect or quantify the label. This is common in that the object to be measured is detected or quantified.

  However, in such a simple test method using a membrane assay method using a membrane or a filter, in the analysis of a sample actually collected from a patient, it is determined that the object to be measured is positive even though it is not present in the sample. So-called false positives may occur. If a false positive reaction occurs when measuring the infection of a pathogen, it will give incorrect information about the disease, so it will not only delay the cause identification but also take inappropriate measures, making the medical condition more serious, etc. It can have serious consequences. Therefore, suppressing false positives is an extremely important issue in view of the main purpose of the simple test method.

  False positives are assumed to occur for the following reasons. In a simple membrane assay method using an assay device equipped with a membrane to which a capture substance is bound to capture the object to be measured, the wiping fluid is collected from the site where the object is expected to be present, such as the patient's pharynx or nasal cavity. In this case, a sample for membrane assay is prepared by floating in the buffer solution or collecting a part from secretions and excrement such as nasal discharge and urine containing the measurement object and diluting with buffer solution. In addition to the object to be measured, the cells, secretions, excrement components, and the like that have fallen from the specimen collection site may be mixed in the sample. Since such contaminants include various biological components including viscous substances such as proteoglucan and glycolipid, when the sample is added as it is onto a membrane such as a membrane, a part of the above components is membrane. It is thought that it adheres to the top or in the membrane. In particular, a membrane having a pore size or retained particle size of 0.1 to 15 μm is often used. When a component having a size comparable to this pore size or retained particle size is added, pores in the membrane are removed. It is conceivable to inhibit the movement of components in the plugging solution. It is considered that a non-specific reaction occurs due to such a phenomenon, and a so-called false positive occurs in which the object to be measured is determined to be positive even though it is not present in the sample.

  In order to solve the occurrence of false positives, it is effective to filter the specimen sample in advance and add it onto the membrane. Since the false positive causative substances in the specimen sample are removed by the filtration, it is possible to prevent these substances from adhering on the membrane of the assay device or causing them to cause false positives. The pore size or retained particle size of the sample filtration filter needs to be smaller than the pore size or retained particle size of the membrane. Therefore, a filter having a thickness of 0.1 to 10 [mu] m, particularly 0.2 to 2 [mu] m is often used. However, when filtration is performed with a filter having a pore size or a retained particle size within this range, substances in the sample may be clogged in the filter, making it impossible to add the assay device onto the membrane. One means for solving this problem is to devise the configuration of the sample filtration filter. By using a sample filtration filter obtained by stacking several small filters from a filter having a large pore diameter or retained particle diameter, it is possible to avoid clogging of the specimen sample during filtration. However, a new problem here is the adsorption of the object to be measured on the filter. Even if the size of the object to be measured is smaller than the pore size or the retained particle diameter of each filter constituting the sample filtration filter, if there is affinity with the filter material, a part of the object to be measured will be adsorbed, Sensitivity may be reduced. In particular, when different types of filters are stacked, even if adsorption occurs on one type of filter, the sensitivity is lowered. Moreover, since the degree of adsorption differs for each object to be measured, it is necessary to search for a filter that does not cause adsorption for each type. Another means is to increase the filtration area, but since the amount of sample specimen is limited, it cannot be increased without limitation. From the above, the problem of clogging has been a major obstacle to the construction of the kit measurement system.

Japanese Patent No. 2114531 Japanese Patent No. 2890384

  An object of the present invention is to provide a highly accurate detection of an object to be measured in a specimen inspection method using a membrane assay method by preventing occurrence of false positives without causing clogging of a filtration filter during filtration of a specimen sample. Alternatively, it is to provide a method that enables quantification.

  As a result of intensive studies, the inventors of the present application have found that the above object can be achieved by performing a shaking treatment before filtering the specimen sample, and have completed the present invention.

  That is, the present invention includes a process in which a sample sample containing a measurement object is filtered using a filtration filter and then dropped on an assay device including a membrane to which a capture reagent for capturing the measurement object is bound. There is provided a membrane assay method for detecting or quantifying the presence of an object to be measured in a specimen sample, wherein the specimen sample is shaken before being filtered with a filtration filter.

  By the method of the present invention, the occurrence of false positives can be prevented without causing clogging of the filtration filter during the filtration of the specimen sample, and an easy and reliable membrane assay method has been established. .

  As described above, the method of the present invention is a method of detecting or quantifying the analyte in the specimen sample by the membrane assay method after filtering the specimen sample with the filtration filter, and shaking the specimen sample before filtering with the filtration filter. It is characterized by processing.

Membrane Assay Method The method of the present invention uses an assay device including a filtration filter for filtering a sample sample and a membrane to which a capture substance for capturing the sample sample is bound. A membrane assay method in which a sample sample is stirred and then filtered using a filtration filter and dropped onto the membrane, so that the presence of an object to be measured in the sample sample does not clog the filtration filter. It is a method characterized by being able to detect or quantify. In particular, when the surfactant is contained in the specimen sample in an amount of 0.01 to 20 (w / v)%, the clogging suppressing effect becomes more remarkable.

  In this specification, “membrane assay” refers to the presence or absence of analyte in a sample using an assay device that includes a membrane on which a capture substance that specifically binds to the analyte is immobilized. It is a method to do. As a typical example, the presence of this complex is detected by reacting the analyte, the capture substance and the labeled detection reagent to form a sandwich complex on the membrane, and detecting the label. It is a method of detecting or quantifying. Examples of the reaction between the analyte and the capture substance and the labeled detection reagent include antigen-antibody reaction, other receptor-receptor reaction, biotin-avidin specific binding reaction, reaction between complementary DNA sequences, etc. Is mentioned. Of these, immunoassays are widely used for detection of influenza viruses, and the method of the present invention can be preferably applied to such immunoassays. In addition, the method of the present invention is not limited to the types of objects to be measured, membranes, and labels as long as they are used in such methods.

  The membrane assay method of the present invention preferably uses two types of membrane immunochromatography methods, that is, a flow-through membrane assay method or a lateral flow membrane assay method because it is simple and rapid. In the flow-through membrane assay method, a solution containing an object to be measured is passed in a direction perpendicular to a membrane coated with a capture reagent or detection substance that specifically binds to the object to be measured. By forming a complex of a capture substance that specifically binds to the target substance, the analyte, and a label substance that specifically binds to the analyte, and detecting or quantifying the label, Perform quantification. The lateral flow membrane assay method differs from the flow-through membrane assay method in that the same membrane is used and the solution containing the sample is spread horizontally with respect to the membrane. It is the same.

  Below, an example of a more specific procedure is shown about the method of this invention.

1. Flow-through type membrane assay (1) A specimen sample collected from the pharynx or nasal cavity of a patient infected with a virus or bacteria is suspended in a specimen suspension as described later. The specimen suspension preferably contains 0.01 to 20 (w / v)% surfactant. Alternatively, a labeled detection reagent may be added in advance to the suspension so that the analyte / labeled detection reagent complex is formed in the specimen suspension.
(2) The suspended liquid is put into a specimen sample filtration tube equipped with a filtration filter, and then stirred by hand or using a vortex mixer.
(3) The suspended liquid after stirring is filtered with a filtration filter.
(4) The filtrate is dropped on a membrane bound with a capture reagent that specifically binds to the analyte in the assay device equipped with the membrane and captures the analyte, and the analyte is placed on the membrane. Capture. However, in (1), when the complex of the analyte / labeled detection reagent is formed in advance in the sample suspension, the complex is captured on the membrane, or the sample suspension in (1) Add the labeled detection reagent to the membrane in the assay device so that the labeled detection reagent is dried and placed on the membrane so that it will dissolve when the filtrate is dropped on the membrane. In this case, the object to be measured and the labeled detection reagent are captured on the membrane while forming a complex.
(5) A labeled detection reagent that specifically binds to the analyte is dropped on the membrane to form a complex of capture reagent / analyte / labeled detection reagent. However, if the labeled detection reagent is previously added to the sample suspension in (1), or if the labeled detection reagent is dried and placed on the membrane in (4), this step is It is unnecessary.
(6) The presence or absence of the analyte in the specimen sample is measured by detecting the presence of the complex with the labeled detection reagent in the complex.

2. Lateral Flow Membrane Assay (1) A specimen sample collected from the pharynx or nasal cavity of a patient infected with a virus or bacteria is suspended in a specimen suspension as described later. The specimen suspension preferably contains 0.01 to 20 (w / v)% surfactant. Alternatively, a labeled detection reagent may be added in advance to the suspension so that the analyte / labeled detection reagent complex is formed in the specimen suspension.
(2) The suspended liquid is put into a specimen sample filtration tube equipped with a filtration filter, and then stirred by hand or using a vortex mixer.
(3) The suspended liquid after stirring is filtered with a filtration filter.
(4) The filtrate is separated from the capture reagent binding line of the membrane in the assay apparatus including a membrane in which a capture reagent that specifically binds to the measurement target and captures the measurement target is bound in a line. Drip into place. The filtrate spreads horizontally in the membrane, and upon reaching the capture reagent binding line, a capture reagent / measurement object complex is formed on the line. However, in (1), when a labeled detection reagent is previously added to the sample suspension, a complex of the capture reagent / measurement object / labeled detection reagent is formed on the capture reagent binding line. .
Alternatively, in (1), the labeled detection reagent is not added to the specimen suspension, and the labeled detection reagent is applied in advance between the position under the filtered droplet on the membrane or between the dropping position and the capture reagent binding line. If the reagent is dropped from the membrane in the assay device away from the capture reagent binding line, the labeled detection reagent and the analyte will form a complex in the membrane while reaching the capture reagent binding line. Then, a complex of capture reagent / measurement object / labeled detection reagent is formed.
(5) A labeled detection reagent that specifically binds to the analyte is dropped on the membrane to form a complex of capture reagent / analyte / labeled detection reagent. However, in (1), when a labeled detection reagent is added to the sample suspension in advance, or in (4), the label is placed under the filtered droplet on the membrane or between the dropping position and the capture reagent binding line position. This step is not necessary when using an assay device coated with a chemical detection reagent.
(6) The presence or absence of the analyte in the specimen sample is measured by detecting the presence of the complex with the labeled detection reagent in the complex.

Shaking treatment As described above, in the method of the present invention, the specimen sample is shaken before being filtered by the filtration filter. Here, the shaking treatment can be performed, for example, by shaking the container that holds the specimen sample by hand, or shaking the container with a vortex mixer. When shaking by hand, the amplitude (distance from the center to the end of the vibration) is about 5 cm to 30 cm and the period (time required for one round of shaking) is about 0.2 to 0.5 seconds. Preferably it is performed for about 2 seconds to 10 seconds. When the strength of shaking is expressed by average acceleration (the absolute value of acceleration when vibration is approximated to a uniform acceleration motion (only the direction of acceleration changes alternately 180 degrees)), 0.1 g or more (g is gravitational acceleration) ) Is preferable, and 0.2 g or more is more preferable. There is no particular upper limit, but the limit for normal humans is considered to be about 0.5 g. Moreover, even if it uses a vortex mixer, a shaking process can be performed simply. As the vortex mixer, an ordinary commercially available vortex mixer for a test tube or the like can be used. It is preferable to use a vortex mixer because it can be easily and easily shaken more strongly than when shaken strongly by hand. When shaking with a vortex mixer, the shaking treatment is preferably performed for 1 second or longer, more preferably for 2 seconds to 10 seconds.

Specimen sample Specimen sample for analysis by the test method of the present invention is a solution in which a pharynx or nasal wipe is suspended in an appropriate buffer, nasal aspirate, pharyngeal or nasal wash, stool suspension, plasma, serum , Urine, saliva, amniotic fluid, cerebrospinal fluid, pus, organ extracts, various tissue extracts, biological samples, food extracts, culture supernatants, clean water, sewage, lake water, river water, seawater, soil extracts, sludge Extracts and solutions obtained by diluting them with an appropriate buffer can be used, but are not limited thereto.

  The sample can be collected from a patient's pharynx, nasal cavity, etc. using a sample collection device such as a sterile cotton swab, or a nasal aspirate can be used. Suspend in suspension and perform assay. As the specimen suspension, buffers or the like usually used in sample detection or quantification methods by immunological techniques such as immunodiffusion method, enzyme immunoassay method, and aggregation method can be used.

  More specifically, physiological saline, phosphate buffered saline (PBS), gelatin-added PBS, bovine serum albumin (BSA) -added PBS, Good's buffer, calf infusion broth (VIB), heart-in Examples include, but are not limited to, fusion broth, Eagle's minimum essential medium (EMEM), and BMEM-added EMEM. Moreover, you may use the said buffers in combination of 2 or more types.

  In order to make the clogging suppression effect according to the present invention more prominent, the buffer solution may contain 0.01 to 20 (w / v)% of a surfactant. As the surfactant, Triton X-100 (trade name): polyethylene glycol mono-р-isooctylphenyl ether (Nacalai Tesque), Tween 20: polyoxyethylene sorbitan monolaurate (Nacalai Tesque), Tween 80 : Polyoxyethylene sorbitan monooleate (Nacalai Tesque), NP-40: Nonidet P-40 (Nacalai Tesque), Zwittergent: Zwittergent 3-14 (Calbiochem), SDS: Sodium dodecyl sulfate (Nacalai Tesque Co., Ltd.), CHAPS: 3-[(3-Colamidopropyl) dimethylammonio] propane sulfonic acid (Dojin Chemical Co., Ltd.) or a mixture of two or more of these can be used. ,this But it is not limited to.

  Furthermore, in addition to the above composition, basic amino acids and / or inorganic salts can also be added. By using a sample suspension containing at least one of a surfactant, basic amino acid, and inorganic salt, components other than the analyte in the sample can be bound to the membrane itself or on the membrane. This is preferable because nonspecific binding to the capture substance can be reduced.

Object to be measured The object to be measured in the present specification is not limited in any way, and may be any substance to be detected or quantified. Examples include viral viruses such as influenza virus, adenovirus, RS virus, HAV, HBc, HCV, HIV, EBV, Norwalk-like virus, Chlamydia trachomatis, Streptococcus, Bordetella pertussis, Helicobacter pylori, Leptospira, Treponema paridum , Bacterial antigens such as Toxoplasma gondii, Borrelia, anthrax, MRSA, mycoplasma lipid antigens, peptide hormones such as human cilia gonadotropin, steroids such as steroid hormones, physiologically active amines such as epinephrine and morphine, vitamin Bs, etc. Antibiotics such as vitamins, prostaglandins, tetracycline, toxins produced by bacteria, various tumor markers, pesticides, anti-E. Coli antibodies, anti-Salmonella antibodies, anti-staphylococcal antibodies, anti-Campylobacter antibodies, anti-tumor Ersch antibody, Vibrio parahaemolyticus antibody, anti-verotoxin antibody, anti-human transferrin antibody, anti-human albumin antibody, anti-human immunoglobulin antibody, anti-microglobulin antibody, anti-CRP antibody, anti-troponin antibody, anti-HCG antibody, anti-chlamydia Trachomatis antibody, anti-streptolysin O antibody, anti-Helicobacter pylori antibody, anti-β-glucan antibody, anti-HBe antibody, anti-HBs antibody, anti-adenovirus antibody, anti-HIV antibody, anti-rotavirus antibody, anti-influenza virus antibody, anti Examples include, but are not limited to, a parvovirus antibody, an anti-RS virus antibody, an anti-RF antibody, a nucleotide complementary to a nucleic acid component derived from a pathogenic microorganism, and the like.

Filtration filter In the method of the present invention, the specimen sample is filtered using a filtration filter after the shaking treatment. The pore size (diameter) or retained particle size of the filtration filter is usually 0.1 to 10 μm, preferably 0.2 to 2.0 μm, more preferably 0.2 to 0.6 μm.

  As long as the filter is not easy to adsorb the object to be measured, not only one type but also a combination of different materials, different pore diameters or different retained particle diameters may be combined. In that case, among the constituents of the filter, the one having the smallest pore diameter or retained particle diameter is the pore diameter or retained particle diameter of the filter.

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

Specimen Sample Storage Container A tube is preferable as a container for storing the sample sample when performing the shaking treatment, and the filtration filter is preferably attached to one end of the tube. A tube having a filter attached to one end is referred to as a “filter tube” in this specification. A method in which a specimen sample is placed in a filtration tube, filtered through a filtration filter attached to one end, and the filtrate is dropped onto a membrane in a membrane assay device is convenient and preferable. The schematic diagram of the specific example of this filtration tube is shown in FIG.3 and FIG.4. For example, as shown in FIG. 3, the filtration tube has a shape composed of a tip end portion d and a main body portion e, and a filtration filter f is provided inside the tip end portion as shown in FIG. A specimen sample is put in the main body part e, and a tip part d provided with a filtration filter f is attached to the main body part e. The specimen sample is filtered through the filtration filter f, and the filtrate is dropped onto the membrane 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 easily filtered and dropped by applying pressure inside the filter with the filter attached. Therefore, it is preferable.

Assay Device An assay device including a membrane (also referred to as a membrane assay device) is a device including a membrane on which a capture substance that specifically binds to a measurement object is immobilized. Such an assay device is preferably a device utilizing a flow-through membrane assay method or a lateral flow membrane assay method. A specific example of an assay device using a flow-through membrane assay is a device as shown in FIGS. 1 and 2, for example.

  FIG. 1 is a plan view of the apparatus, and FIG. 2 is a cross-sectional end view taken along the line II ′ of FIG. 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 substance that specifically binds to the object to be measured is bound, and c is a member that absorbs liquid.

  A specific example of an assay device using a lateral flow membrane assay is a device as shown in FIGS. 5 and 6, for example.

  5 is a plan view of the apparatus, and FIG. 6 is a cut end surface taken along the line I-I ′ of FIG. 5. 5 and 6, g is a pad installed for dropping the prepared specimen sample. h is a pad for absorbing the dropped solution. i is a membrane to which a capturing substance that specifically binds to the object to be measured is bound, and j and k indicate positions where the capturing substance that specifically binds to the object to be measured is bound in a line on i. l is a plastic backing sheet for fixing the member and increasing the strength.

Membrane The material of the membrane in the membrane assay device is not particularly limited, and preferably includes a microporous material made of nitrocellulose. Moreover, the mixture of a cellulose ester and nitrocellulose can also be used suitably. The pore diameter or retained particle diameter of the membrane is not less than the pore diameter or retained particle diameter of the filtration filter, preferably 0.1 to 15 μm, particularly preferably 0.5 to 7 μm. Moreover, although the thickness of a membrane is not specifically limited, Usually, it is about 100-200 micrometers.

Capture substance A capture substance for capturing a measurement object is a substance that binds to the measurement object by a specific reaction such as an antigen-antibody reaction to form a complex. Therefore, it is natural that the capture substance to be used differs depending on the object to be measured. In general, when the object to be measured is bacteria, viruses, hormones, other clinical markers, etc., they react specifically and bind to them. Polyclonal antibodies, monoclonal antibodies and the like. In addition, virus antigens, virus hollow particles, recombinant E. coli expressed protein, recombinant yeast expressed protein and the like can be mentioned. As a method for binding such a capturing substance to the above-described membrane surface, physical adsorption may be used, or chemical binding may be used. The membrane to which the capture substance is bound is prepared, for example, by adsorbing a solution obtained by diluting the capture substance in a buffer solution or the like and then drying the membrane.

Membrane assay kit The membrane assay kit used in the method of the present invention comprises at least (1) a filtration filter, and (2) an assay device provided with a membrane to which a capture substance for capturing an analyte is bound. The kit may further contain a washing solution composition and / or a labeled detection reagent, if necessary. Further, when the label of the labeled detection reagent is an enzyme label, it can contain an enzyme substrate, a reaction stop solution, etc., which will be described later.

  Further, if necessary, a negative control solution consisting only of a buffer for examining the activity of the kit, and a positive control consisting of a buffer containing an analyte such as an antigenic substance may be included. Furthermore, a sample collection device such as a sterile cotton swab may be included.

Detection reagent The detection reagent is capable of specifically binding to the analyte and forming a complex with the analyte. The labeled detection reagent is a detection reagent labeled so that it can be detected by some means after forming a complex with the object to be measured. For example, when the object to be measured is an antigenic substance such as a virus, an antibody against the virus and labeled with an enzyme or the like can be used. When labeled with an enzyme in this way, the complex can be detected by adding a substrate of the enzyme that produces a substance detectable by a colorimetric method or a fluorescence method by a reaction catalyzed by the enzyme. It can be carried out. Examples of the detection reagent before labeling are the same as those described for the capture reagent. Examples of the label include an enzyme, a fluorescent label, a magnetic label, a radioactive isotope, a metal colloid such as a gold colloid, a colored latex, and the like.

  When an enzyme label is used, examples of the enzyme used include alkaline phosphatase, peroxidase, and glucose-6-phosphate dehydrogenase.

Substrate When an enzyme label is used as a label for a labeled detection reagent, it is usually a substrate for the enzyme, and a substance catalyzed by the enzyme produces a substance detectable by a colorimetric method or a fluorescence method. Add. Specific examples include 5-bromo-4-chloro-3-indolyl phosphate (BCIP) / nitrotetrazolium blue (NBT), tetramethylbenzidine (TMB), and glucose-6-phosphate NAD +.

Reaction Stop Solution The kit of the present invention may further contain, for example, a reaction stop solution for stopping the reaction between the enzyme and the substrate, if necessary. Examples of such a reaction stop solution include citric acid and sulfuric acid.

  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 and Comparative Example 1
Detection of influenza virus by flow-through immunochromatographic assay Preparation of monoclonal antibody (1) Preparation of anti-influenza A virus NP monoclonal antibody (mouse) The spleen was excised from BALB / c mice that had been immunized with purified influenza A virus antigen and maintained for a certain period of time (Kohler et al. et al., Nature, vol, 256, p495-497 (1975)) and fused with mouse myeloma cells (P3 × 63). The obtained fused cells (hybridomas) are maintained in a 37 ° C. incubator, and purification of the cells (monocloning) is performed while confirming the antibody activity of the supernatant by ELISA using an influenza A virus NP antigen solid phase plate. went. 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 an ammonium sulfate fractionation method to obtain two types of purified anti-influenza A virus NP monoclonal antibodies.

(2) Anti-influenza B virus NP monoclonal antibody (mouse)
The spleen was excised from BALB / c mice immunized with purified influenza B virus antigen and maintained for a certain period of time, and mouse myeloma was obtained by the method of Keller et al., Nature, vol. 256, p495-497 (1975). Fused with cells (P3x63). The obtained fused cells (hybridomas) are maintained in an incubator at 37 ° C., and purification of the cells (monocloning) while confirming the antibody activity of the supernatant by ELISA using an influenza B virus NP antigen solid phase plate Went. 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 an ammonium sulfate fractionation method to obtain two types of purified anti-type B influenza virus NP monoclonal antibodies.

2. Preparation of labeled anti-influenza antibody solution (1) Preparation of labeled anti-influenza A antibody solution After dialyzing 20 mg of purified anti-influenza A virus NP monoclonal antibody with 0.1 M acetate buffer (pH 3.8), 10 mg of pepsin was added, and Fab ′ digestion treatment was performed at 37 ° C. for 1 hour. The treatment solution was fractionated with an Ultrogel AcA44 column to obtain an anti-influenza A influenza F (ab ′) ₂ purified fraction. 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 AcA44 column to obtain a purified anti-influenza A influenza Fab ′ fraction, and then concentrated to about 1 mL.

  After dialyzing 1.5 mL of 10 mg / mL alkaline phosphatase into 50 mM borate buffer (50 mM borate (pH 7.6), 1 mM magnesium chloride, 0.1 mM zinc chloride), N- (6-maleimidocaproyloxy) succinimide (EMCS; Dojin Chemical Co., Ltd.) 0.7 mg was 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 Fab ′ and maleimide-alkaline phosphatase were mixed at a protein ratio of 1: 2.3 and reacted by gently stirring at 4 ° C. for 20 hours to obtain alkaline phosphatase-labeled anti-influenza Fab ′. It was. The reaction solution was fractionated with an Ultrogel AcA44 column, and unreacted substances were removed to obtain purified alkaline phosphatase-labeled Fab ′.

  Purified alkaline phosphatase-labeled Fab ′ was changed to 4.5 (W / V)% bovine serum albumin, 5.25 (W / V)% polyethylene glycol 6000, 10 mM Tris-HCl buffer (pH 7.4), 150 mM sodium chloride, 1. After diluting to 1.0 μg / mL with a labeled antibody diluent having a composition of 5 (W / V)% Triton X-100, 1.5 mM magnesium chloride and 0.15 mM zinc chloride, it is filtered through a 0.22 μm pore size filter. Thus, a labeled anti-influenza A antibody solution was obtained.

(2) Preparation of labeled anti-influenza B antibody solution 20 mg of one type of purified anti-influenza B virus antibody was dialyzed with 0.1 M acetate buffer (pH 3.8), 10 mg of pepsin was added, Fab ′ digestion was performed over time. The treatment solution was fractionated with an Ultrogel AcA44 column to obtain an anti-type B influenza F (ab ′) ₂ purified fraction. 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 AcA44 column to obtain a purified anti-influenza B Fab ′ fraction, and then concentrated to about 1 mL.

  Concentrated anti-influenza B Fab ′ and maleimide-alkaline phosphatase prepared in 2- (1) were mixed at a protein ratio of 1: 2.3 and reacted with gentle agitation at 4 ° C. for 20 hours. Influenza B Fab ′ was obtained. The reaction solution was fractionated with an Ultrogel AcA44 column, and unreacted substances were removed to obtain purified alkaline phosphatase-labeled Fab ′.

  Purified alkaline phosphatase-labeled Fab ′ was changed to 4.5 (W / V)% bovine serum albumin, 5.25 (W / V)% polyethylene glycol 6000, 10 mM Tris-HCl buffer (pH 7.4), 150 mM sodium chloride, 1. After diluting to 1.0 μg / mL with a labeled antibody diluent having a composition of 5 (W / V)% Triton X-100, 1.5 mM magnesium chloride and 0.15 mM zinc chloride, it is filtered through a 0.22 μm pore size filter. Then, a labeled anti-B influenza antibody solution was obtained.

3. Production of Membrane Assay Device for Detection of Influenza Virus The membrane assay device for detection of influenza virus 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 on the membrane was obtained by spotting two antibody solutions onto a nitrocellulose membrane. In the apparatus A hole, 12 μL of a solution containing 0.2 mg / mL of the purified anti-influenza A virus NP monoclonal antibody not used for labeling, and in the hole B, out of the purified anti-influenza B virus NP monoclonal antibody 12 μL of a solution containing 1 mg / mL not used for labeling was filtered through a 0.22 μm pore size filter and spotted. As a buffer for diluting the antibody, a 10 mM citrate buffer (pH 4.0) was used. After spotting, the membrane was dried in a 45 ° C. drying room for 40 minutes to produce a membrane assay device for detecting influenza virus.

4). Detection of influenza virus (1) Detection by membrane assay The wipe was collected from the nasal cavity of 140 patients clinically suspected of influenza virus infection using a sterilized cotton swab, and 50 mM phosphate buffer (pH 7.0), 1 A test sample was prepared by floating in 1.0 mL of a solution having a composition of 5 M sodium chloride, 1 (W / V)% Triton X-100, 0.5 (W / V)% bovine serum albumin. Immediately divide 500 µL into two sample sample filtration tubes, and sandwich a nitrocellulose membrane with a pore size of 0.45 µm between two glass fibers with a pore size (retained particle size) of 0.67 µm at the tip of each tube. Loaded with a filter.

  The sample tube containing one of the bisected sample samples was shaken vigorously by hand 10 times (10 reciprocations) (amplitude 10 cm, period 0.4 seconds) (Example 1). 2. After shaking, immediately pass through a nozzle and filter, 150 μL was dropped into each of the A hole and B hole of the membrane assay device prepared in Step 1, and the sample was left until the sample was completely absorbed by the liquid absorbing member provided at the bottom of the nitrocellulose membrane. However, when the filtration filter was clogged in the middle, and 150 μL each could not be added to A hole and B hole, the subsequent assay was stopped, and it was determined that the measurement was impossible due to clogging. If the specimen sample can be added dropwise, then after adding 180 μL each of the labeled anti-influenza A antibody solution to the A hole and the labeled anti-B influenza antibody solution to the B hole, the antibody solution The liquid absorbing member was left until it was completely absorbed. Next, the adapter was removed, and a washing solution having a composition of 20 mM phosphate buffer (pH 7.0), 0.5 M sodium chloride, 5 (W / V)% arginine hydrochloride, 1 (W / V)% Triton X-100. Was added dropwise to each of the A hole and the B hole, and then allowed to stand until the cleaning solution was completely absorbed by the liquid absorbing member. Subsequently, 250 μL of BCIP / NBT substrate solution (manufactured by Sigma) was added dropwise to the A hole and the B hole, respectively, to initiate the color reaction. Ten minutes later, 100 μL of 100 mM citrate buffer (pH 3.0) was added dropwise to each of A hole and B hole to stop the reaction. Immediately after the reaction is stopped, A hole and B hole are observed from above, and if color is observed only in A hole, influenza A virus is positive. If color is observed only in B hole, influenza B virus is observed. When coloration was observed in both positive and AB holes, it was determined as positive for influenza A and B influenza viruses, and negative when no coloration was observed in both AB holes.

  The container containing the other specimen sample is filtered without passing through a filtration filter, and then assayed in the same manner as the other specimen sample described above. Infection was determined (Comparative Example 1).

(2) Detection by RT-PCR method Using the rest of the test sample prepared in 4- (1), it was confirmed by the RT-PCR method whether the influenza virus gene was present in the specimen. The RT-PCR method was performed by the method of Shimizu (Infectious Diseases Journal, Vol. 71, No. 6, p522-526).

(3) Comparison of Membrane Assay Method and RT-PCR Method Tables 1 and 2 show a comparison of the determination results of the membrane assay method of the present invention and the RT-PCR method described above. The RT-PCR method is known to be a measurement method with extremely high sensitivity and specificity, and when it was judged to be different from the result of the RT-PCR method, it was regarded as false positive or false negative.

※表内の数値は該当する検体数を示す。 Table 1 Comparison when shaking test sample (Example 1)

* The numbers in the table indicate the number of applicable samples.

※表内の数値は該当する検体数を示す。 Table 2 Comparison when the test sample is not shaken (Comparative Example 1)

* The numbers in the table indicate the number of applicable samples.

  When the test specimen was shaken (Example 1), it completely coincided with the determination by the RT-PCR method, and no unmeasurable specimen was found. When the sample was not shaken (Comparative Example 1), the measurable sample was completely consistent with the determination by the RT-PCR method, but the measurement failure rate was 11% (16 samples out of 140).

Example 2 and Comparative Example 2
Detection of influenza virus by lateral flow immunochromatographic assay Preparation of monoclonal antibody (1) Preparation of anti-influenza A virus NP monoclonal antibody (mouse) [Example 1] (1) It produced similarly to the method of description.

(2) Anti-influenza B virus NP monoclonal antibody (mouse)
[Example 1] (2) It produced similarly to the method of description.

2. Preparation of labeled anti-influenza A antibody (1) Preparation of colloidal gold antibody 10 mL of colloidal gold was taken and adjusted to pH 7.0 with 100 mM potassium carbonate. One type of anti-influenza A virus NP monoclonal antibody was dialyzed with 2 mM boric acid solution, centrifuged and purified, and then prepared with 2 mM boric acid solution to a concentration of 100 μg / mL. An amount of the prepared anti-influenza A virus NP monoclonal antibody having a final concentration of 10 μg / mL was added to the colloidal gold with sufficient stirring. After 5 minutes, 1 mL of 10% BSA was added and gently stirred with a rotator for 10 minutes. The whole amount was transferred to a centrifuge tube and centrifuged at 14,000 rpm for 30 minutes at 4 ° C. After centrifugation, the supernatant is aspirated and discarded, and the precipitated gold colloid and sensitized anti-influenza A virus NP monoclonal antibody contain a final concentration of 10 mM Tris-HCl buffer, 1% BSA, 150 mM sodium chloride. Float with 1 mL of solution.

(2) Drying of colloidal gold antibody The colloidal gold antibody prepared in the previous section was formed into a 10 mm × 300 mm polystyrene non-woven fabric at a concentration and speed of 8.0 OD ₅₂₀ , 10 μL / cm using a positive pressure spray device (BioJet, BioJet). Sprayed. Subsequently, it was dried under reduced pressure in a vacuum apparatus for 1 hour to obtain a dry anti-A type gold colloid antibody pad. At the time of use, it was cut at intervals of 10 mm × 7 mm.

3. Preparation of labeled anti-influenza B antibody (1) Preparation of colloidal gold antibody Take 10 mL of colloidal gold and adjust the pH to 7.0 with 100 mM potassium carbonate. One of the anti-influenza B virus NP monoclonal antibodies was dialyzed with 2 mM boric acid solution, centrifuged and purified, and then prepared with a 2 mM boric acid solution to a concentration of 100 μg / mL. An amount of 10 μg / mL final concentration of the prepared anti-influenza B virus NP monoclonal antibody is added to the colloidal gold with sufficient stirring. After 5 minutes, 1 mL of 10% BSA was added and gently stirred with a rotator for 10 minutes. The whole amount was transferred to a centrifuge tube and centrifuged at 14,000 rpm for 30 minutes at 4 ° C. After centrifugation, the supernatant is aspirated and discarded, and the precipitated gold colloid and sensitized anti-influenza B virus NP monoclonal antibody contain a final concentration of 10 mM Tris-HCl buffer, 1% BSA, 150 mM sodium chloride. Float with 1 mL of solution.

(2) Drying of colloidal gold antibody The colloidal gold antibody prepared in the previous section was formed into a 10 mm × 300 mm polystyrene non-woven fabric at a concentration and speed of 8.0 OD ₅₂₀ , 10 μL / cm using a positive pressure spray device (BioJet, BioJet). Sprayed. Subsequently, it was dried under reduced pressure in a vacuum apparatus for 1 hour to obtain a dry anti-B type gold colloidal antibody pad. At the time of use, it was cut at intervals of 10 mm × 7 mm.

4). Production of Flow-through Membrane Assay Device for Detection of Influenza Virus A lateral flow membrane assay device for detection of influenza virus having the same configuration as that shown in FIGS. 5 and 6 was used. Membrane i consists of a nitrocellulose membrane (PuraBind, manufactured by Whatman, size 5 × 50 mm, thickness 200 μm) having a pore diameter of 5 μm, and purified at a position of j 15 mm away from one end thereof (this end is the downstream end). A 2.0 μL solution containing 1 mg / ml of an anti-influenza B virus NP monoclonal antibody that was not used for labeling was applied in a line (width 1.0 mm), and purified anti-type A at a position 18 k away. 2.0 μL of a solution containing 0.2 mg / ml of an influenza virus NP monoclonal antibody not used for labeling was applied in a line (width 1.0 mm). At that time, 10 mM citrate buffer (pH 4.0) was used as a buffer for diluting the solid phase antibody, and 0.22 μm filtration was performed immediately before solid phase. After coating, drying was performed for 40 minutes in a 45 ° C. drying room.

  Next, in order to fix the member and increase the strength, a plastic backing sheet 1 (manufactured by BioDot) was adhered to the side opposite to the antibody-coated surface of the membrane.

  Next, a cellulose / glass fiber pad (WF1.5, manufactured by Whatman, 7 mm × 10 mm, thickness 1.4 mm) was pasted at a position 23 mm away from the downstream end of the membrane to obtain a sample dropping pad g.

  Next, a cellulose / glass fiber pad (WF 1.5, manufactured by Whatman, 10 mm × 20 mm, thickness 1.5 mm) was pasted so that 5 mm on the downstream end side of the membrane overlapped to make an absorption pad h.

5. Detection of influenza virus (1) Detection by membrane assay The wipe was collected from the nasal cavity of 90 patients clinically suspected of influenza virus infection using a sterile cotton swab, and 20 mM Tris-HCl buffer (pH 8.0), 0 A solution having the following composition: 0.6 M sodium chloride, 1 (W / V)% Triton X-100, 2.0 (W / V)% L-arginine hydrochloride, 1.0 (W / V)% bovine serum albumin. After floating in 9 mL, the mixture was allowed to stand for 3 minutes, and then divided into two 400 μL aliquots of two specimen sample filtration tubes. A test sample was prepared by adding the dry anti-A type gold colloid pad (10 mm × 7 mm) and the dry anti-B type gold colloid pad (10 mm × 7 mm) prepared in (2) one by one. A filter having a nitrocellulose membrane having a pore diameter of 0.45 μm sandwiched between two glass fibers having a pore diameter (retained particle diameter) of 0.67 μm was loaded at the tip of each tube.

  The sample tube containing one of the bisected samples was vigorously shaken by hand 10 times (10 reciprocations) (amplitude 10 cm, period 0.4 seconds) (Example 2). 3. After shaking, immediately pass through a nozzle and filter, 150 μL was dropped on the sample dropping pad of the membrane assay device prepared in the above. 10 minutes later, the positions of j and k in FIGS. 5 and 6 were observed from above, and if color development was observed only at position j, influenza B virus was positive, and color development was observed only at position k. In the case of influenza A virus positive, color development was observed at both positions j and k, positive for influenza A and B influenza viruses, negative when neither color was observed at positions j, k It was determined.

  The container with the remaining sample sample is mixed with the sample and the colloidal gold antibody, filtered through a filtration filter, and then assayed in the same manner as the other sample sample described above. (Comparative Example 2).

(2) Detection by RT-PCR method Using the rest of the test sample prepared in 5- (1), it was confirmed by RT-PCR method whether an influenza virus gene was present in the sample. The RT-PCR method was performed by the method of Shimizu (Infectious Diseases Journal, Vol. 71, No. 6, p522-526).

(3) Comparison of Membrane Assay Method and RT-PCR Method Tables 3 and 4 show a comparison of the determination results of the membrane assay method of the present invention and the RT-PCR method described above. A false positive or false negative was considered when the determination was different from the result of the RT-PCR method.

※表内の数値は該当する検体数を示す。 Table 3 Comparison of test samples when shaken (Example 2)

* The numbers in the table indicate the number of applicable samples.

表内の数値は該当する検体数を示す。 Table 4 Comparison when the test sample is not shaken (Comparative Example 2)

The numbers in the table indicate the number of corresponding samples.

  When the test specimen was shaken (Example 2), it completely coincided with the determination by the RT-PCR method, and no unmeasurable specimen was found. When the sample was not shaken (Comparative Example 2), the measurable sample completely coincided with the determination by the RT-PCR method, but the unmeasureable occurrence rate was 8% (7 samples out of 90 samples).

It is a top view of the flow-through type membrane assay apparatus which is one embodiment of this invention. FIG. 2 is an end view taken along the line II ′ of FIG. 1. 1 shows 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. It is a top view of the lateral flow type membrane assay apparatus which is one embodiment of this invention. FIG. 6 is an end view taken along the line I-I ′ of FIG. 5.

Explanation of symbols

A Hole B Hole a Adapter b Membrane c Liquid absorbing member d Filtration tube tip e Filtration tube body f Filtration filter g Sample dropping pad h Absorption pad i Nitrocellulose membrane j Anti-B influenza virus NP monoclonal antibody application line k Anti-A Influenza virus NP monoclonal antibody application line l Backing sheet

Claims (11)

  Measure the sample in the sample sample, including the process of filtering the sample sample containing the sample to be measured using a filtration filter and then dropping the sample on the assay device having a membrane to which the capture reagent for capturing the sample to be measured is bound. In a membrane assay method for detecting or quantifying the presence of an object, the sample is shaken before being filtered with a filtration filter.   The method according to claim 1, further comprising the step of adding a labeling reagent for detection or quantification into the specimen sample before the shaking treatment.   3. The sample according to claim 1, wherein the specimen sample is accommodated in a tube, the filter is provided at one end of the tube, and the shaking treatment is performed in a state where the specimen sample is accommodated in the tube. Method.   The method according to any one of claims 1 to 3, wherein the specimen sample is a wiping material collected from a patient suspended in a buffer solution, or an aspirate or washing solution collected from a patient.   The method according to claim 4, wherein the specimen sample is a pharynx or nasal cavity wipe suspended in a buffer solution, a nasal aspirate, or a nasal cavity or pharyngeal wash.   The method according to any one of claims 1 to 5, wherein the detection or quantification of the analyte is performed by an immunoassay.   The method according to claim 1, wherein the object to be measured is an influenza virus.   The method according to any one of claims 1 to 7, wherein a surfactant is contained in the specimen sample in an amount of 0.1 to 20 (W / V)%.   The method according to any one of claims 1 to 8, which is a flow-through type or lateral flow type membrane assay method.   The method according to any one of claims 1 to 9, wherein the filter has a pore size of 0.1 µm to 10 µm. The method according to claim 10, wherein the filter has a pore size of 0.2 μm to 2 μm.

Images