JP2020046436A - Simple membrane assay method and kit - Google Patents

Abstract

To provide a method for simply inspecting a specimen using a membrane assay method, capable of preventing occurrence of false positive and detecting a measuring object with high accuracy, and to provide a kit used in such a method.SOLUTION: A simple membrane assay method of a measuring object in a specimen sample uses an assay device including a membrane coupled with a capture reagent for capturing the measuring object. The method comprises the steps of: dropping the specimen sample on the membrane after filtering the specimen sample by using a filtration filter; and detecting existence of the measuring object in the specimen sample. An assay device comprises a simple membrane assay kit for inspecting existence of a measuring object in a specimen sample comprising: (1) a filtration filter; and (2) a membrane coupled with a capture substance for capturing the measuring object.SELECTED DRAWING: None

Description

  The present invention relates to a method for testing a sample using an assay device provided with a membrane, and more particularly to a simple membrane assay method that can be used clinically and a kit used in this method.

Recently, a simple test that detects or quantifies various measurement items such as infection with pathogens such as viruses and bacteria, the presence or absence of pregnancy, and blood sugar levels using antigen-antibody reactions and enzyme reactions in a short time of several minutes to tens of minutes Reagents or kits have been developed. Pathogen constituent proteins, human ciliary gonadotropin (hCG), blood sugar, 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. For example, simple test reagents for diagnosis of pregnancy are sold as OTC in 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. In many cases, these facilities are the first medical institutions visited by patients, and if the presence or absence of infection on a sample collected from the patient is determined on the spot, the treatment can be performed as soon as possible, so the treatment can be simplified. Test reagents are becoming increasingly important in medicine.
At present, as a simple inspection method, a membrane assay method, particularly an assay method using a membrane such as a membrane such as nitrocellulose or a filter, is generally known, and is roughly classified into a flow-through type and a lateral flow type membrane assay method. The former allows the solution containing the object to be measured to pass vertically through the film on which the substance to be measured is applied, and the latter allows the solution to spread in the horizontal direction. In any case, a membrane solid phase substance that specifically binds to the analyte, an analyte, a complex of a label substance that specifically binds to the analyte is formed on the membrane, and the label is detected or quantified. This is common in that detection or quantification of an object to be measured is performed.
However, in such a simple test method using a membrane assay using a membrane or a filter, in the analysis of a sample actually collected from a patient, a positive determination is made even though the analyte is not present in the sample. , So-called false positives may occur. If a false-positive reaction occurs when measuring the pathogen infection, it will give incorrect information about the disease, not only delaying the cause identification but also taking inappropriate measures and making the condition more serious. It can have serious consequences. Therefore, suppressing false positives is a very important issue from the viewpoint of the main purpose of using the simple test method. Conventionally, in order to solve this problem, a buffer containing a surfactant was used as a buffer for suspending or diluting the sample, or a device such as passing through a filter when adding a sample has been devised. It was not satisfactory.

  An object of the present invention is to provide a simple test method for a sample using a membrane assay method, to prevent the occurrence of false positives, to enable highly accurate detection or quantification of an analyte, and to use the method in such a method. It is to provide a kit to be performed.

In detecting and quantifying an analyte using a membrane assay, by filtering a sample sample that may contain the analyte through a filtration filter, the occurrence of false positives is greatly suppressed. Was found.
That is, an object of the present invention is to provide a simple membrane assay method for an analyte in a sample, which uses an assay device having a membrane to which a capture reagent is bound for capturing the analyte. The method is characterized in that the method is characterized in that the method is characterized in that the method is characterized in that the method is characterized in that the method is characterized in that the method is characterized in that the method is carried out by filtering the solution and then dropping the solution on a membrane to detect the presence of an object to be measured in the sample sample.
Another object of the present invention is to provide a simple membrane assay kit for inspecting the presence of an analyte in a specimen sample, including:
The problem is solved by (1) a sample sample filtration tube provided with a filtration filter, and (2) an assay device provided with a membrane bound with a capture substance for capturing an analyte.

In another embodiment of the present invention, the material of the filtration filter is a non-woven fabric, paper, glass fiber, silica fiber, nitrocellulose, cellulose ester, a mixture of nitrocellulose and cellulose ester, polyether sulfone, polysulfone, ethylene tetrafluoride resin. , Vinylidene fluoride resin, polycarbonate, polypropylene, polyamide, nylon 6,6, polyester, cotton, stainless steel fiber, and combinations thereof.
A preferred embodiment of the present invention is the above method or kit, wherein the filtration filter has a pore size or a retained particle size of 0.2 to 2.0 μm, particularly 0.2 to 0.6 μm.
Another preferred embodiment of the present invention is the above method or kit, wherein the filtration filter is a glass fiber filter, a nitrocellulose filter, or a combination of a glass fiber filter and a nitrocellulose filter.
Further, in another preferred embodiment of the present invention, the material of the membrane is a nonwoven fabric, paper, nitrocellulose, glass fiber, silica fiber, cellulose ester, polyether sulfone, polysulfone, tetrafluoroethylene resin, vinylidene fluoride resin, Selected from the group consisting of polycarbonate, polypropylene, polyamide, nylon 6,6, and a mixture of cellulose ester and nitrocellulose, and wherein the pore size or the retaining particle size of the membrane is greater than or equal to the pore size or the retaining particle size of the filtration filter; A method or kit as described above, wherein the thickness is between 3 and 10 μm.
A further preferred embodiment of the present invention is the above method or kit, wherein the material of the membrane is nitrocellulose and the pore size is 0.5 to 7 μm.
Another preferred embodiment of the present invention is the above method or kit, wherein the analyte in the sample sample is an influenza virus.
In addition, particularly preferred embodiments of the present invention are the above-described method and kit relating to a flow-through type or lateral flow type simple membrane assay method.

  According to the method of the present invention, the occurrence of false positives in a simple membrane assay method using an assay device provided with a membrane bound with a capture substance for capturing an analyte can be significantly reduced. This is considered for the following reasons. In a simple membrane assay using an assay device having a membrane to which a substance to be measured is bound, a swab is collected from a site where the substance is expected to be present, for example, from the patient's pharynx or nasal cavity. A sample from a secretion or excretion, such as nasal discharge or urine, containing the substance to be measured, and diluting with a buffer to prepare a sample for a membrane assay. In addition, in addition to the measured substance, components of cells, secretions, excretions, and the like, which have come off from the sample collection site, may be mixed into the sample. Since such contaminants contain various biological components including viscous substances such as proteoglycan and glycolipids, when the sample is added as it is to a membrane such as a membrane, a part of the components described above is partially removed from the membrane. It is believed that it adheres on or in the membrane. In particular, a membrane having a pore diameter or a retained particle diameter of 0.1 to 10 μm is often used as a membrane. When a component having a size comparable to the pore diameter or the retained particle diameter is added, pores in the membrane are reduced. It is conceivable to inhibit the migration of components in the plugging solution. It is considered that a nonspecific reaction occurs due to such a phenomenon, and a so-called false positive occurs in which a positive determination is made even though the analyte is not present in the sample. According to the method of the present invention, such false positives can be efficiently suppressed, and a highly reliable simple test method can be established.

It is a top view of a membrane assay device which is one embodiment of the present invention. FIG. 2 is a sectional view taken along the line I-I ′ of FIG. 1. 1 shows an embodiment of a sample sample filtration tube used in the present invention. FIG. 4 shows a perspective view of the distal end portion of the sample sample filtration tube of FIG. 3.

Hereinafter, the present invention will be described in detail.
(Simple membrane assay method)
The method of the present invention is a simple membrane assay method for an analyte in a sample sample using an assay device having a membrane to which a capture substance is bound for capturing the analyte, wherein the sample sample is filtered through a filter. The method is characterized in that it is dropped on the membrane after filtration using the filter, and the presence or absence of the analyte in the sample is detected or quantified.
As used herein, “simple membrane assay” refers to the presence or absence of an analyte in a sample sample using an assay device including a membrane on which a capture substance that specifically binds to the analyte is immobilized. This is a simple and easy method to inspect. Typically, the analyte is reacted with the capture substance and the labeled detection reagent to form a sandwich-like complex on the membrane, and the presence of the complex is detected by detecting the label. Is the way. Examples of the reaction between the analyte and the capture substance and the labeled detection reagent include an antigen-antibody reaction, a reaction between another receptor and a receptor, a specific binding reaction between biotin and avidin, a reaction between DNAs having complementary sequences, and the like. Is mentioned. In addition, the simple inspection method of the present invention is not limited in terms of the type of the object to be measured, the membrane, and the label as long as it is used for such a method.

  The membrane assay of the present invention preferably utilizes two types of membrane assays, namely, a flow-through type membrane assay or a lateral flow type membrane assay, because it is simple and quick. In the flow-through type membrane assay, a solution containing an analyte is passed vertically through a membrane coated with a capture reagent or a detection substance that specifically binds to the analyte. Forming a complex of a capture substance that specifically binds to a target substance, a target substance, and a label substance that specifically binds to the target substance on a membrane, and detecting or quantifying the label enables detection or detection of the target substance. Perform quantification. The lateral flow type membrane assay method differs from the flow-through type membrane assay method in that a solution containing the analyte is spread horizontally on the membrane using a similar membrane, but the principle of detection of the analyte is The same is true.

Hereinafter, an example of a more specific procedure for the method of the present invention will be described, and the present invention will be described.
(1) A sample sample collected from the pharynx or nasal cavity of a patient infected with a virus, a bacterium, or the like is suspended in a sample suspension described below.
(2) The suspension is put into a sample sample filtration tube provided with a filtration filter and filtered.
(3) The filtrate is dropped on a membrane to which a capture reagent that specifically binds to and captures the analyte in the assay device equipped with the membrane is attached, and the analyte is placed on the membrane. Let it be captured.
(4) A labeled detection reagent that specifically binds to the analyte is dropped onto the membrane to form a complex of capture reagent / analyte / labeled detection reagent.
(5) The presence or absence of the analyte in the sample is measured by detecting the presence of the complex with the labeled detection reagent in the complex.

(Filtration filter)
In the method of the present invention, a sample sample collected from a patient is suspended in a sample suspension and then filtered using a filtration filter. The pore size (diameter) or retained particle size of the filtration filter is 0.2 to 2.0 μm, preferably 0.2 to 0.6 μm. The pore size or retention particle size of the filtration filter is important for the effect of the present invention. If the pore size or retained particle size is too large, non-specific binding may occur on the membrane and false positives may be shown. Conversely, if it is too small, the filter itself will be clogged due to viscous substances and agglomerates present in the sample and filtration will not be possible, or the filter area will have to be considerably large, and it will be used for a simple inspection method It is inappropriate from the purpose. Therefore, the range of the pore size or the retained particle size of the filtration filter of the present invention is from 0.2 to 2.0 μm, and more preferably from 0.2 to 0.6 μm.
The filtration filter is not limited to one type, and may be a combination of several types having different materials, different pore sizes or different retention particle sizes. In this case, the filter having the smallest pore diameter or the retained particle diameter among those constituting the filter is the pore diameter or the retained particle diameter of the filter. Therefore, if at least one of the combinations has a pore diameter or a retained particle diameter in the range of 0.2 to 2.0 μm, there is no problem even if the other one exceeds the range.
In addition, by combining two or more of the same filters, there is obtained an advantage that a certain effect can be obtained even if a filter having a variation in pore diameter or retention particle diameter is used. Further, when a filter having insufficient strength is used, two or more filters can be stacked to increase the strength of the filter. However, depending on the type of the filter, there is also a disadvantage that the filter is easily clogged by stacking a plurality of filters, the pressure for filtration increases, and the simplicity is impaired.

The material of the filtration filter is nonwoven fabric, paper, glass fiber, silica fiber, nitrocellulose, cellulose ester, a mixture of nitrocellulose and cellulose ester, polyether sulfone, polysulfone, ethylene tetrafluoride resin, vinylidene fluoride resin, polycarbonate, polypropylene , Polyamide, nylon 6,6, polyester, cotton, stainless steel fiber, and the like, but are not limited thereto. Preferred are glass fiber and nitrocellulose.
Generally, a filtration filter is divided into a depth filter and a screen filter in terms of a collecting mechanism. The depth filter collects solids inside the filter, and the screen filter collects solids on the filter surface. Either mechanism can be suitably used.

(Filtration tube)
The above-mentioned filtration filter is preferably used by being attached to the tip of the sample sample filtration tube in the simple membrane assay method or kit of the present invention. That is, a simple and preferable method is to put a specimen sample suspended in a suspension in a filtration tube, filter through a filtration filter attached to the tip, and drop the filtrate onto a membrane in a membrane assay device. FIGS. 3 and 4 are schematic diagrams of one embodiment of the filtration tube. The filter tube has, for example, a shape including a distal end portion and a main body portion as shown in FIG. 3, and a filter is provided inside the distal end portion as shown in FIG. The specimen sample suspended in the suspension is put into the main body, and a tip provided with a filtration filter is attached to the main body. The specimen sample is filtered through a filtration filter, and the filtrate is dropped on a membrane assay device. It is preferable that the main body is made of a flexible material such as polyethylene or polyethylene terephthalate (PET) because the sample sample can be easily filtered by applying pressure to the inside by hand or the like with the filter attached. .

(Assay device)
An assay device provided with a membrane (also referred to as a membrane assay device) in the present specification is a device including a membrane on which a capture substance that specifically binds to an object to be measured is immobilized. Such an assay device is preferably a device utilizing a flow-through type membrane assay method or a lateral flow type membrane assay method. A specific example of an assay device utilizing a flow-through type membrane assay is, for example, a device as shown in FIGS.
FIG. 1 is a plan view of the device, and FIG. 2 is a sectional view taken along the line II ′ of FIG. In FIGS. 1 and 2, a is an adapter having an opening for dropping the prepared specimen sample and having holes (A hole and B hole) on the bottom surface through which the sample passes. b is a membrane to which a capture substance that specifically binds to the analyte is bound, and c is a member that absorbs liquid.

(Membrane)
As the material of the membrane in the membrane assay device, non-woven fabric, paper, nitrocellulose, glass fiber, silica fiber, cellulose ester, polyether sulfone, polysulfone, ethylene tetrafluoride resin, vinylidene fluoride resin, polycarbonate, polypropylene, polyamide, Examples include the group consisting of nylon 6,6 and a mixture of cellulose ester and nitrocellulose, particularly preferably a microporous material made from nitrocellulose. Further, a mixture of the cellulose ester and nitrocellulose can also be suitably used. The pore size or the retaining particle size of the membrane is preferably equal to or larger than the pore size or the retaining particle size of the filtration filter, and is preferably from 0.3 to 10 μm, particularly preferably from 0.5 to 7 μm. The thickness of the membrane is not particularly limited, but is usually about 100 to 200 μm.

(DUT)
The analytes referred to herein include influenza virus, adenovirus, respiratory syncytial virus, HAV, HBc, HCV, HIV, EBV, Norwalk-like virus and other viral antigens, Chlamydia trachomatis, streptococcus, B. pertussis, Helicobacter. Bacterial antigens such as H. pylori, leptospira, Treponema pallidum, Toxoplasma gondii, Borrelia, Bacillus anthracis, MRSA, peptide hormones such as mycoplasma lipid antigen, human ciliary gonadotropin, steroids such as steroid hormones, and bioactive amines such as epinephrine and morphine , Vitamins such as vitamin B, prostaglandins, antibiotics such as tetracycline, toxins produced by bacteria, various tumor markers, pesticides, anti-Escherichia coli antibodies, anti-Salmonella antibodies, anti-staphylococcal antibodies, anti-campylova Antibody, anti-C. Perfringens antibody, anti-V. 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 Examples include, but are not limited to, viral antibodies, anti-parvovirus antibodies, anti-RS virus antibodies, anti-RF antibodies, nucleotides complementary to nucleic acid components derived from pathogenic microorganisms, and the like.

  A sample sample to be analyzed by the test method of the present invention is a solution in which a pharyngeal or nasal swab is suspended in an appropriate buffer, nasal aspirate, stool suspension, plasma, serum, urine, saliva, amniotic fluid, Biological samples such as cerebrospinal fluid, pus, organ extracts, various tissue extracts, food extracts, culture supernatants, tap water, sewage, lake water, river water, seawater, soil extracts, sludge extracts and appropriate A solution diluted with a buffer can be used, but is not limited thereto. The buffer may contain 0.01 to 20% by mass of a surfactant. Examples of the surfactant include Triton X-100: polyethylene glycol mono-p-isooctylphenyl ether (Nacalai Tesque, Inc.), Tween 20: polyoxyethylene sorbitan monolaurate (Nacalai Tesque, Inc.), Tween 80: polyoxyethylene. Sorbitan monooleate (Nacalai Tesque), NP-40: Nonidet P-40 (Nacalai Tesque), Zwittergent: Zwitterg Pent 3-14 (Carbiochem), SDS: Sodium dodecyl sulfate (Nacalai Tesque) ), CHAPS: 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonic acid (Dojindo Co., Ltd.) or a mixture of two or more of these can be used, but is not limited thereto. Not done.

(Captured substance)
A capture substance for capturing an analyte is a substance that binds to the analyte by a specific reaction such as an antigen-antibody reaction to form a complex. Therefore, it is natural that the capture substance used differs depending on the analyte. However, in general, when the analyte is a bacterium, virus, hormone, or other clinical marker, it specifically reacts with and binds to these. Polyclonal antibodies, monoclonal antibodies and the like. Other examples include viral antigens, viral hollow particles, recombinant Escherichia coli expressed proteins, and recombinant yeast expressed proteins. The method of binding such a capture substance to the above-mentioned membrane surface may be physical adsorption or chemical binding. The preparation of the membrane to which the capturing substance is bound is performed, for example, by adsorbing a solution obtained by diluting the capturing substance in a buffer or the like onto the membrane, and then drying the solution.

(Simple membrane assay kit)
The simple membrane assay kit of the present invention is a kit used for the above-described simple membrane assay method of the present invention. The simple membrane assay kit of the present invention includes at least the following (i) and (ii).
(i) a filtration filter, and
(ii) An assay device provided with a membrane bound with a capture substance for capturing an analyte.
It is preferable that the filtration filter is further attached to the above-described sample sample filtration tube.
The kit may further include a sample suspension, a washing solution composition, and / or a labeled detection reagent, if necessary. When the label of the labeled detection reagent is an enzyme label, it may contain a substrate for an enzyme described later, a reaction stop solution, and the like.
If necessary, the kit may contain a negative control solution consisting only of a buffer for testing the activity of the kit, and a positive control consisting of a buffer containing an analyte such as an antigenic substance. Further, a sample collection device such as a sterilized cotton swab may be included.

(Sample suspension)
The sample can be collected, for example, from a patient's pharynx or nasal cavity using a sample collection device such as a sterile cotton swab, or a nasal aspirate can be used. Perform the assay in a suspension. As the sample suspension, buffers and the like usually used in a sample detection or quantification method by an immunological technique such as an immunodiffusion method, an enzyme immunoassay method, and an agglutination method can be used.
More specifically, physiological saline, phosphate buffered saline (PBS), PBS with gelatin, PBS with bovine serum albumin (BSA), 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 BSA-added EMEM. Further, two or more buffers may be used in combination.
Further, in addition to the above composition, a basic amino acid, an inorganic salt and / or a surfactant may be added. By using a sample suspension containing at least two types of basic amino acids, inorganic salts and surfactants, components other than the analyte contained in the sample can be bound to the membrane itself or trapped on the membrane. It is preferable because non-specific binding can be reduced.

(Detection reagent)
In the present specification, a detection reagent is a reagent that can specifically bind to an analyte and form a complex with the analyte. Further, the labeled detection reagent means a detection reagent that is labeled so as to be detectable by some means after forming a complex with the analyte. For example, when the analyte is an antigenic substance such as a virus, an antibody against the virus, such as an antibody labeled with an enzyme, may be mentioned. When labeled with an enzyme in this manner, the complex is detected by adding a substrate of the enzyme that generates a substance that can be detected by a colorimetric method or a fluorescent method by a reaction catalyzed by the enzyme. It can be carried out. The detection reagent before labeling may be the same as that described for the capture reagent. Examples of the label include an enzyme, a fluorescent label, a magnetic substance label, a radioisotope, a gold colloid, and a colored latex.
In the case of using the enzyme label, the enzyme used for example, alkali follower phosphatase, peroxidase, and glucose-6-phosphate dehydrogenase.

(Substrate)
When an enzyme label is used as the label of the labeled detection reagent, usually, a substrate for the enzyme that generates a substance that can be detected by a colorimetric method or a fluorescent method by a reaction catalyzed by the enzyme is used. Added. Specific examples include 5-bromo-4-chloro-3-indolyl phosphate (BCIP) / nitrotetrazolium blue (NBT), tetramethylbenzidine (TMB), and glucose-6-phosphate NAD +.

(Reaction stop solution)
If necessary, the kit of the present invention may further contain, for example, a reaction terminating solution for terminating the reaction between the enzyme and the substrate. Examples of such a reaction stopping solution include citric acid and sulfuric acid.

[Example 1]
1. Preparation of Monoclonal Antibody (1) Preparation of Anti-Influenza A Virus NP Monoclonal Antibody (Mouse) Spleen was excised from BALB / c mice immunized with purified influenza A virus antigen and maintained for a certain period of time, and the method of Keller et al. et al., Nature, vol. 256, p495-497 (1975)) and fused to mouse myeloma cells (P3 × 63).
The obtained fused cells (hybridomas) are maintained in a 37 ° C. incubator, and the cells are purified (monocloned) while confirming the antibody activity of the supernatant by ELISA using an influenza A virus NP antigen solid phase plate. went.
Each of the two obtained cell lines was intraperitoneally administered to BALB / c mice treated with pristane, and about 2 weeks later, antibody-containing ascites was collected. IgG was purified from the obtained ascites by the 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)
A spleen was excised from a BALB / c mouse immunized with a purified influenza B virus antigen and maintained for a certain period of time, and mouse myeloma was isolated by the method of Keller et al. (Kohler et al., Nature, vol. 256, p495-497 (1975)). Fused with cells (P3 × 63). The obtained fused cells (hybridomas) are maintained in an incubator at 37 ° C., and the cells are purified (monoclonalization) while confirming the antibody activity of the supernatant by ELISA using an influenza B virus NP antigen solid phase plate. Was done. Each of the two obtained cell lines was intraperitoneally administered to BALB / c mice treated with pristane, and about 2 weeks later, antibody-containing ascites was collected. IgG was purified from the obtained ascites by the ammonium sulfate fractionation method to obtain two kinds of purified anti-influenza B virus NP monoclonal antibodies.

2. Preparation of labeled anti-influenza antibody solution (1) Preparation of labeled anti-influenza A antibody solution After dialysis of 20 mg of one of the purified anti-influenza A virus NP monoclonal antibodies against 0.1 M acetate buffer (pH 3.8), 10 ′ of pepsin was added, and Fab ′ digestion treatment was performed at 37 ° C. for 1 hour. The treated solution was fractionated on an Ultrogel AcA44 column to obtain a purified anti-influenza A influenza F (ab ') ₂ 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 treated solution was fractionated on an Ultrogel AcA44 column to obtain an anti-influenza A influenza Fab ′ purified fraction, which was then concentrated to about 1 mL.
After dialyzing 1.5 mL of 10 mg / mL alkaline phosphatase against 50 mM borate buffer (50 mM boric acid (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 the mixture was treated at 30 ° C. for 1 hour. The solution after the treatment 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 type Fab ′ and maleimide-alkaline phosphatase were mixed at a protein ratio of 1: 2.3, and gently stirred at 4 ° C. for 20 hours to react, thereby obtaining alkaline phosphatase-labeled anti-influenza type A Fab ′. Was. The reaction solution was fractionated on an Ultrogel AcA44 column, and unreacted substances were removed to obtain purified alkaline phosphatase-labeled Fab ′.
Purified alkaline phosphatase-labeled Fab 'was converted 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, the mixture was filtered through a filter having a pore size of 0.22 μm. Then, a labeled anti-influenza A antibody solution was obtained.

(2) Preparation of labeled anti-influenza B antibody solution 20 mg of one of the purified anti-influenza B virus antibodies was dialyzed against 0.1 M acetate buffer (pH 3.8), and 10 mg of pepsin was added thereto. Fab 'digestion treatment was performed for hours. The treated solution was fractionated on an Ultrogel AcA44 column to obtain a purified anti-influenza B influenza F (ab ') ₂ 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 treated solution was fractionated on an Ultrogel AcA44 column to obtain a purified anti-influenza B influenza Fab 'fraction, and then concentrated to about 1 mL.
Concentrated anti-influenza B type Fab 'and maleimide-alkaline phosphatase prepared in 2- (1) were mixed at a protein ratio of 1: 2.3, and allowed to react with gentle stirring at 4 ° C for 20 hours to give alkaline phosphatase. A labeled anti-influenza B Fab ′ was obtained. The reaction solution was fractionated on an Ultrogel AcA44 column, and unreacted substances were removed to obtain purified alkaline phosphatase-labeled Fab ′.

  Purified alkaline phosphatase-labeled Fab 'was converted 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, the mixture was filtered through a 0.22 μm pore size filter. Then, a labeled anti-influenza B antibody solution was obtained.

3. Preparation of Membrane Assay Device for Influenza Virus Detection As the influenza virus detection membrane assay device, one having the same configuration as that shown in FIGS. 1 and 2 was used. As the membrane, a nitrocellulose membrane having a pore diameter of 3 μm (size 2 × 3 cm, thickness 125 μm) was used.
The solid phase was applied to the membrane by spotting the two antibody solutions onto a nitrocellulose membrane. In the A hole of the apparatus, 12 μL of a solution containing 0.2 mg / mL of the purified anti-influenza A virus virus NP monoclonal antibody which was not used for labeling was used. In the B hole, the purified anti-influenza B virus NP monoclonal antibody was used. 12 μL of a solution containing 1 mg / mL which was not used for labeling was filtered through a 0.22 μm pore size filter and spotted. As a buffer for diluting the antibody, 10 mM citrate buffer (pH 4.0) was used. After the spotting, the membrane was dried in a drying chamber at 45 ° C. for 40 minutes to prepare a membrane assay device for detecting influenza virus.

4. Detection of Influenza Virus (1) Detection by Membrane Assay A swab was collected from the nasal cavity of 115 patients clinically suspected of influenza virus infection using a sterile cotton swab, and 50 mM phosphate buffer (pH 7.0), 1 The sample was suspended in 1.5 mL of a solution having a composition of 0.5 M sodium chloride, 1 (W / V)% Triton X-100, and 0.5 (W / V)% bovine serum albumin to prepare a test sample. After the test sample was well suspended, 400 μL was divided into three equal parts in three sample sample filtration tubes, and the following three types of nozzles were attached to the tips of each tube. The nozzle (I) was loaded with a filter having a nitrocellulose membrane having a pore size of 0.45 μm interposed between two glass fibers having a pore size (retention particle size) of 0.67 μm, and the nozzle (II) was loaded with a pore size (retention particle size). A filter in which three glass fibers each having a diameter of 0.67 μm were stacked was loaded, and the nozzle (III) was not loaded with the filter.

  2. After the sample is filtered through each nozzle, 150 μL was added dropwise to each of the holes A and B of the membrane assay device prepared in the above, and the sample was allowed to stand until the sample was completely absorbed by the liquid absorbing member provided below the nitrocellulose membrane. Next, 180 μL of the labeled anti-influenza A antibody solution was added dropwise to the A hole and 180 μL of the labeled anti-B influenza antibody solution was added to the B hole, and the antibody solution was left until completely absorbed by the liquid absorbing member. did. 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, and 1 (W / V)% Triton X-100. Was added dropwise to each of the A-hole and the B-hole, and the mixture was allowed to stand until the cleaning liquid was completely absorbed by the liquid absorbing member. Subsequently, 250 μL of a BCIP / NBT substrate solution (manufactured by Sigma) was added to each of the A-hole and the B-hole to start a color reaction. After 10 minutes, the reaction was stopped by adding 150 μL of 100 mM citrate buffer solution (pH 3.0) to each of the A and B holes. Immediately after the reaction was stopped, holes A and B were observed vertically from above. If coloration was observed only in hole A, influenza A virus was positive; if coloration was observed only in hole B, influenza B virus was detected. Positive, influenza A and B viruses were positive when color development was observed in both AB holes, and negative in cases where color development was not observed in both AB holes.

(2) Detection by RT-PCR method The remainder of the test sample prepared in 5- (1) was used to confirm whether or not an influenza virus gene was present in the sample by RT-PCR method. The RT-PCR method was performed by the method of Shimizu (Infectious Diseases Magazine, Vol. 71, No. 6, p522-526).
(3) Comparison of Simple Membrane Assay and RT-PCR Method Tables 1 to 3 show comparisons of the judgment results of the simple 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 a false positive or a false negative is determined when a judgment different from the result of the RT-PCR method is made.

In the case of using the nozzle (I), the judgment completely coincided with the judgment by the RT-PCR method, and no false positive was observed. When the nozzle (II) was used, the false-positive incidence was 7% (8 out of 115 samples (in Table 2, the total number of false-positive samples enclosed in squares)). Matched the judgment. On the other hand, when the nozzle (III) was used, the false positive incidence rate was 83% (96 out of 115 samples (total of false positive samples enclosed in squares in Table 3)).
By the method and the kit of the present invention, generation of false positives can be prevented, and a highly reliable simple membrane assay method can be established.

A: Hole B: Hole a: Adapter b: Membrane c: Liquid absorbing member d: Filtration tube tip e: Filtration tube body f: Filtration filter

Claims (17)

  Using an assay device provided with a membrane bound with a capture reagent for capturing the analyte, a simple membrane assay method for the analyte in the sample sample, after filtering the sample sample using a filtration filter, A method comprising dropping the sample on a membrane and detecting or quantifying the presence of the analyte in the sample sample.   The material of the filtration filter is a nonwoven fabric, paper, glass fiber, silica fiber, nitrocellulose, cellulose ester, a mixture of nitrocellulose and cellulose ester, polyethersulfone, polysulfone, tetrafluoroethylene resin, vinylidene fluoride resin, polycarbonate, The method of claim 1, wherein the method is selected from the group consisting of polypropylene, polyamide, nylon 6,6, polyester, cotton, stainless steel fiber, and combinations thereof.   The method according to claim 1, wherein the filtration filter has a pore size or a retained particle size of 0.2 to 2.0 μm.   The method according to any one of claims 1 to 3, wherein the filtration filter is a glass fiber filter, a nitrocellulose filter, or a combination of a glass fiber filter and a nitrocellulose filter.   The material of the membrane is nonwoven fabric, paper, nitrocellulose, glass fiber, silica fiber, cellulose ester, polyether sulfone, polysulfone, ethylene tetrafluoride resin, vinylidene fluoride resin, polycarbonate, polypropylene, polyamide, nylon 6,6 and Being selected from the group consisting of a mixture of a cellulose ester and a nitrocellulose, and having a pore size or a retaining particle size of the membrane equal to or larger than the pore size or the retaining particle size of the filtration filter and 0.3 to 10 μm. A method according to any one of claims 1 to 4.   The method according to claim 5, wherein the material of the membrane is nitrocellulose and the pore size is 0.5 to 7 µm.   The method according to any one of claims 1 to 6, wherein the analyte is an influenza virus.   The method according to any one of claims 1 to 7, which is a flow-through or lateral flow membrane assay. A simple membrane assay kit for testing or quantifying the presence of an analyte in a sample sample, including:
An assay device including (1) a filtration filter, and (2) a membrane to which a capturing substance for capturing an analyte is bound. The kit of claim 9, further comprising:
(3) sample suspension (4) cleaning solution composition,
(5) a labeled detection reagent,
(6) a sample collection instrument and / or (7) a control solution.   The material of the filtration filter is a nonwoven fabric, paper, glass fiber, silica fiber, nitrocellulose, cellulose ester, a mixture of nitrocellulose and cellulose ester, polyethersulfone, polysulfone, tetrafluoroethylene resin, vinylidene fluoride resin, polycarbonate, The kit according to claim 9 or 10, wherein the kit is selected from the group consisting of polypropylene, polyamide, nylon 6,6, polyester, cotton, stainless steel fiber and a combination thereof.   The kit according to any one of claims 9 to 11, wherein the filtration filter has a pore size or a retention particle size of 0.2 to 2.0 µm.   The method according to any one of claims 9 to 12, wherein the filtration filter is a glass fiber filter, a nitrocellulose filter, or a combination of a glass fiber filter and a nitrocellulose filter.   The material of the membrane is nonwoven fabric, paper, nitrocellulose, glass fiber, silica fiber, cellulose ester, polyether sulfone, polysulfone, ethylene tetrafluoride resin, vinylidene fluoride resin, polycarbonate, polypropylene, polyamide, nylon 6,6 and Being selected from the group consisting of a mixture of a cellulose ester and a nitrocellulose, and having a pore size or a retaining particle size of the membrane equal to or larger than the pore size or the retaining particle size of the filtration filter and 0.3 to 10 μm. A kit according to any one of claims 9 to 13.   The kit according to claim 14, wherein the material of the membrane is nitrocellulose and the pore size is 0.5 to 7 µm.   The kit according to any one of claims 9 to 15, wherein the analyte is an influenza virus.   The kit according to any one of claims 9 to 16, which is a flow-through type or lateral flow type membrane assay kit.

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