JP2014062820A - Membrane for immunochromatography test strip, test strip, and inspection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a membrane for an immunochromatography test strip, a test strip, and an inspection method that can shorten the time required detecting a substance to be detected and whose detection sensitivity quality is improved.SOLUTION: A membrane for an immunochromatography test strip is made of cellulose nitrate used for a chromatographic medium. The membrane has at least one substance selected among an alkane sulfonic acid sodium, an n-octanoyl-N-methylglutamine, polyoxyethylene(30)docosyl ether, and a glycerol externally added by 0.02 to 0.6 mass% with respect to membrane mass.

Description

  The present invention relates to an immunochromatographic test strip membrane, an immunochromatographic test strip, and an immunochromatographic test method.

In recent years, an immunoassay method for detecting a target substance comprising a specific antigen or antibody by utilizing a specific reaction between an antigen and an antibody thereto has been widely used as a means of biochemical examination.
Among these methods, there is an agglutination method in which a substance to be detected in a sample and an antibody or an antigen bound to a detection particle by a sensitization treatment are combined by an immune reaction, and the aggregation state of the detection particle generated thereby is measured. It is known as a simple method, and is generally used because the result can be visually judged.
Also known is a method of binding an antibody or antigen labeled with a labeling substance to a substance to be detected in a sample by an immune reaction and measuring the labeling substance in this binding state, and a radioisotope is used as the labeling substance. A radioimmunoassay method used, an enzyme immunoassay method using an enzyme, a fluorescent immunoassay method using a fluorescent substance, and the like are also employed.
These immunoassays require a reaction step between the substance to be detected and an antibody labeled with the labeling substance, and a separation step between the labeling substance that is bound to the substance to be detected and the labeled substance that is not bound. An immunochromatographic method is known as a method in which these steps are performed in a system comprising a stationary phase and a mobile phase that continuously flows in contact with the principle of chromatography by applying the principle of chromatography (Patent Document 1: Japanese Patent Publication No. Hokuhei). 7-13640).

As the immunoassay method, the sandwich method or the competitive method is known, but the following operation is performed when detecting the detection substance consisting of the antigen in the sample by the sandwich method by the immunochromatography method. Is called.
(1) An antibody that specifically binds to an antigen, which is a substance to be detected, is used as an immobilization reagent, and this immobilization reagent is applied to a predetermined portion of the chromatographic medium in a predetermined form. A reaction site is formed at the position of.
(2) On the other hand, by using an antibody that specifically binds to the substance to be detected as a detection reagent and labeling this detection reagent with a labeling substance such as an enzyme, or sensitizing the detection reagent to a labeling substance such as an insoluble carrier A labeled detection reagent is prepared.
(3) The developing solution constituting the mobile phase is developed on the chromatographic medium as the stationary phase together with the sample containing the substance to be detected and the labeled detection reagent.
By the above operation, at the reaction site formed in the chromatographic medium, the antigen as the substance to be detected is captured by binding with the antibody as the immobilization reagent fixed to the reaction site, and the antigen and the label are labeled. As a result of an antigen-antibody reaction occurring with an antibody that is a detection reagent, the reaction site includes an immobilized reagent (immobilized antibody), a substance to be detected (antigen), and a detection reagent (labeled antibody). When a target substance is present in the sample and the target substance is present in the sample, the target substance appears when the target substance appears indirectly due to the labeling substance binding to the reaction site. it can.

Such an immunochromatographic method is easy to operate and can be measured in a short time, and thus is widely used in clinical examinations and measurement tests in laboratories. In particular, it is widely used for clinical examinations and diagnoses that require rapid reporting, such as pregnancy diagnosis and influenza infection confirmation.
Generally, an enzyme or an insoluble carrier is used as a labeling substance for immunochromatography, but an insoluble carrier (such as colloidal metal particles or colored latex particles) that can be detected visually without requiring any special operation is labeled. By adopting as a substance, the utility value as a simple detection method which is a feature of the immunochromatography method is further increased. In recent years, many methods using the aggregation of colloidal gold particles as an index have been adopted.

As described above, the immunochromatography method has a great advantage that it has high sensitivity and a short inspection time. In order to increase sensitivity, there have been proposals such as improvement of detection reagents, improvement of developing solvents, and improvement of developing methods. In the improvement of the chromatographic development solution, for example, Patent Document 2 (Japanese Patent No. 4559510) discloses that detection sensitivity is improved by using a solution containing a polyvinyl-based water-soluble polymer and a nonionic surfactant. Has been. In Patent Document 3 (Japanese Patent Laid-Open No. 2009-150869), a test substance and a labeling substance modified with a first binding substance for the test substance are developed in a mixed state, and this is developed. Further, a method is disclosed in which the detection is performed by rotating 90 degrees and developing the developing solvent again instead of containing the amplification solution.
The present situation is that a simpler and more sensitive immunochromatographic test method including these methods is being sought.

Japanese Examined Patent Publication No. 7-13640 Japanese Patent No. 4559510 JP 2009-150869 A

  An object of the present invention is to provide a membrane for a test strip used in an immunochromatograph and an immunochromatographic test strip. Another object is to provide an immunochromatographic inspection method using this test strip.

  As a result of studying the sensitivity of the immunochromatography method and shortening the test time, the present inventors have found that the surface treatment of the membrane, which is a chromatographic medium, plays an important role, and determined the characteristics of the optimal immunochromatographic test strip. The headline and the present invention were completed.

That is, the present invention has the following configuration.
1. A membrane for immunochromatographic test strip made of nitrocellulose used as a chromatographic medium, the membrane comprising sodium alkanesulfonate, n-octanoyl-N-methylglutamine, polyoxyethylene (30) docosyl ether, glycerin A membrane for immunochromatographic test strips, wherein at least one substance is externally added so as to be 0.02 to 0.6% by mass with respect to the mass of the membrane. In the present specification, external addition means adding an additive to the membrane formed, and means an addition method excluding so-called internal addition in which the additive is added to the dope solution. And
2. 1. Alkanesulfonic acid alkyl group has a straight chain structure having 12 to 18 carbon atoms. The membrane for an immunochromatographic test strip as described.
3. 1. The membrane has a pore size (pore size) of 3.4 to 6.1 μm. Or 2. The membrane for immunochromatographic test strips described in 1.
4). 1. Membrane thickness is 100-150 μm ~ 3. The membrane for an immunochromatographic test strip according to any one of the above.
5. 1. ~ 4. An immunochromatographic test strip using the membrane for an immunochromatographic test strip described in any one of the above.
6). 5. An immunochromatographic test method using the described immunochromatographic test strip.
7). 5. The immunochromatographic labeling substance is a colloidal gold. The immunochromatographic inspection method as described.
8). 5. The substance to be detected is human chorionic gonadotropin. Or 7. The immunochromatographic inspection method as described.

  The present invention provides an immunochromatographic test strip membrane and an immunochromatographic test strip that can shorten the time until detection of a substance to be detected and have improved detection sensitivity. An immunochromatographic inspection method using a test strip is also provided. The use of this test strip enables accurate inspection and diagnosis.

The structure of a representative immunochromatographic test strip is shown. The test result which confirmed the influence of various addition components of an immunochromatograph is shown. The test result which confirmed the influence of the thickness of the membrane of an immunochromatograph is shown. The test result which confirmed the influence of the membrane pore diameter of an immunochromatograph is shown. The test result which confirmed the storage stability of the membrane of an immunochromatograph is shown.

Chromatographic medium In the present invention, the medium for immunochromatography plays the most important role. Reference numeral 3 in FIG. 1 is a developed portion of the chromatograph. The chromatographic medium, which is the development site of the immunochromatography, is an inactive film made of a microporous material that exhibits capillary action. A membrane made of nitrocellulose (hereinafter sometimes referred to as “nitrocellulose membrane”) is selected as having no reactivity with a detection reagent, an immobilization reagent, a substance to be detected and the like used in the chromatograph. Nitrocellulose-based membranes need only contain nitrocellulose as a main component. Nitrocellulose-based membranes, such as pure products and nitrocellulose-mixed products, should be used, but there are no problems with other materials. . As other materials, cellulose membranes, nylon membranes, porous plastic fabrics (polyethylene, polypropylene) and the like are used.

The nitrocellulose membrane preferably has a nitrogen content of less than 12.6% by weight, particularly preferably 11.0 to 12.2% by weight. The thickness of the nitrocellulose membrane is preferably 100 μm or more, particularly preferably 110 μm to 150 μm.
The nitrocellulose membrane is porous, so it can be backed by laminating impermeable films, or previously formed by laminating a nitrocellulose membrane on a film of impermeable polyethylene, etc. good. Although there is no restriction | limiting in particular in the thickness of this water-impermeable film, The thing of about 100 micrometers is preferable on handling.

The average pore diameter of the nitrocellulose membrane is preferably 3.4 to 6.1 μm when measured by the half dry method in accordance with ASTM E1294-89.
The nitrocellulose membrane thus selected further contains a substance that promotes capillary action. As such a substance, a substance that lowers the surface tension of the film surface and brings about hydrophilicity is desirable. For example, saccharides and amino acid derivatives, fatty acid esters, various synthetic surfactants, alcohols, and other substances that have amphiphilic action, have no effect on the movement of the substance to be detected on the immunochromatograph, and are marker substances ( Substances that do not affect the color development of, for example, colloidal gold are preferred. Such materials include sodium dodecylbenzene sulfonate (SDBS), sodium dodecyl sulfonate (SDS), C12-C18 alkyl sodium sulfonate (sodium alkane sulfonate), sodium dialkylsulfosuccinate, sodium cholate, n- Octanoyl-N-methylglutamine, CHAPS (3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonate), polyoxyethylene (10) nonylphenyl ether, polyoxyethylene (30) docosyl ether And glycerin.
Particularly preferred are sodium dodecyl sulfonate (SDS), C12-C18 alkyl sodium sulfonate (sodium alkane sulfonate), n-octanoyl-N-methylglutamine, polyoxyethylene (30) docosyl ether, and glycerin.
Optimally, sodium alkanesulfonate having a linear structure having an alkyl group of 12 to 18 carbon atoms is used.

The substance to be contained in the above medium is an aqueous solution having a concentration of 0.1 to 10%, and a nitrocellulose membrane is immersed in this solution, followed by vacuum drying or natural drying to obtain a chromatographic medium containing a desired substance. . In addition to the above-described method, the additive may be added to the formed membrane directly by adding an additive to the formed membrane, followed by drying, and the present invention is not limited to these methods. The content of the externally added surfactant is 0.02 to 0.6% by mass, preferably 0.1 to 0.6% by mass, based on the mass of the nitrocellulose membrane. Optimally, it is used at 0.1 to 0.4 mass%. When the amount is less than 0.02% by mass, sufficient sensitivity and an effect of shortening the detection time cannot be obtained, and storage stability is also lowered. When it exceeds 0.6% by mass, a nonspecific reaction occurs in a negative sample. The nitrocellulose membrane thus prepared is porous and exhibits capillary action.
The index of the capillary phenomenon can be confirmed by measuring the water absorption speed (water absorption time: capillary flow time). The water absorption rate affects detection sensitivity and inspection time. According to the present invention, in the case of a 40 mm long nitrocellulose membrane prepared by the above treatment, the water absorption time is 70 to 107 seconds.

  The form and size of the nitrocellulose membrane as the chromatographic medium used in the immunochromatographic test strip of the present invention are not particularly limited, and may be appropriate in terms of actual operation and observation of reaction results. In order to further simplify the operation, it is preferable to provide a support made of plastic or the like on the back surface of the chromatographic medium in which the reaction sites are formed on the surface. The properties of the support are not particularly limited, but when the measurement result is observed by visual judgment, the support preferably has a color that is not similar to the color caused by the labeling substance. Usually, it is preferably colorless or white.

Sample pad When the immunochromatograph described above is performed, the sample is dropped onto a sample pad corresponding to 1 in FIG. The sample pad may be any material as long as it is normally used for immunochromatography. As the sample pad 1, a glass fiber or cellulose film for absorbing and holding the sample is usually used.

Conjugate pad The conjugate pad contains in advance a labeling substance (marker substance) bound to an antibody that binds to the substance to be detected. When the substance to be detected moves in the conjugate pad, it binds to the antibody and is labeled. The conjugate pad is made of a glass fiber nonwoven fabric or a cellulose membrane.

An absorption pad for absorbing the developing solution can be installed at the end of the absorption pad chromatographic medium. The absorbent pad may be any pad that can absorb the developing solution, such as cellulose filter paper.
The sample pad, conjugate pad, and absorption pad can be used as long as they are membranes and non-woven fabrics used in conventional immunochromatographs.

Reaction site On the chromatographic medium used in the immunochromatographic test strip of the present invention, a reaction site is formed in which a substance that specifically binds to the substance to be detected, for example, an antibody is immobilized as an immobilization reagent at an arbitrary position. . The immobilization reagent can be immobilized on the chromatographic medium by directly immobilizing the immobilization reagent on the chromatographic medium by physical or chemical means, and by physically or chemically immobilizing the immobilization reagent on fine particles such as latex particles. There is an indirect immobilization method in which these fine particles are chemically bound and captured and immobilized on a chromatographic medium.
As a method of directly immobilizing, physical adsorption may be used, or covalent bonding may be used. In the case of a nitrocellulose membrane, physical adsorption can be performed. In the covalent bond, cyanogen bromide, glutaraldehyde, carbodiimide and the like are generally used for activating the chromatographic medium, but any method can be used. As a method for immobilizing indirectly, there is a method in which an immobilization reagent is bound to insoluble fine particles and then immobilized on a chromatographic medium. The particle size of the insoluble fine particles can be selected such that it is captured by the chromatographic medium but cannot move, and is preferably fine particles having an average particle size of about 5 μm or less. Various particles used for antigen-antibody reaction are known as these particles, and these known particles can also be used in the present invention. For example, organic polymers such as organic polymer latex particles obtained by emulsion polymerization methods such as polystyrene, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer. Examples include fine particles of substances, fine particles such as gelatin, bentonite, agarose, and cross-linked dextran, inorganic oxides such as silica, silica-alumina, and alumina, and inorganic particles obtained by introducing functional groups into inorganic oxides by silane coupling treatment, and the like. . In the present invention, direct immobilization is preferred from the standpoint of ease of sensitivity adjustment. Various methods can be used to immobilize the immobilization reagent on the chromatographic medium. For example, various techniques such as a microsyringe, a pen with a control pump, and ink jet printing can be used. Although the form of the reaction site is not particularly limited, it can be fixed as a circular spot, a line extending perpendicularly to the developing direction of the chromatographic medium, a numeral, a letter, a symbol such as +, −, or the like.

  After immobilization of the immobilization reagent, the chromatographic medium can be subjected to a blocking process by a known method, if necessary, in order to prevent the accuracy of analysis from being reduced due to nonspecific adsorption. Generally, proteins such as bovine serum albumin, skim milk, casein, and gelatin are preferably used for the blocking treatment. After such blocking treatment, a surfactant such as Tween 20, Triton X-100, or SDS may be washed in combination with one or more if necessary.

Labeling substance The detection reagent used in the present invention is a substance that specifically binds to the substance to be detected, such as an antibody, and is labeled with a labeling substance. Generally, an enzyme or the like is used for labeling the detection reagent in the immunochromatography method, but an insoluble carrier is used as the labeling substance of the present invention because it is suitable for visually determining the presence of the substance to be detected. . In the present invention, a labeled detection reagent is prepared by sensitizing the detection reagent to an insoluble carrier.
The insoluble carrier as a labeling substance used in the present invention includes colloidal metal particles such as gold, silver and platinum, colloidal metal oxide particles such as iron oxide, colloidal nonmetal particles such as sulfur, and synthetic high Latex particles made of molecules and others can be used. In particular, colloidal gold is preferable because it is easy to detect.

  The insoluble carrier is a labeling substance suitable for visually determining the presence of the substance to be detected, and is preferably colored in order to facilitate the visual determination. The colloidal metal particles and the colloidal metal oxide particles themselves exhibit a specific natural color corresponding to the particle diameter, and the color can be used as a label.

Examples of colloidal metal particles and colloidal metal oxide particles that can be used as labeling substances in the present invention include colloidal gold particles, colloidal silver particles, colloidal platinum particles, colloidal iron oxide particles, and colloidal water. Examples thereof include aluminum oxide particles. In particular, the colloidal gold particles and the colloidal silver particles are preferable in that the colloidal gold particles are red and the colloidal silver particles are yellow in an appropriate particle size. These colloidal metal particles have an average particle diameter of 1 to 500 nm, more preferably within a range of 10 nm to 150 nm, more preferably 20 to 100 nm at which a particularly strong color tone is obtained.
For example, when colloidal gold particles are used as the colloidal metal particles, commercially available ones may be used. Alternatively, colloidal gold particles can be prepared by a conventional method, for example, a method of reducing chloroauric acid with sodium citrate.

  As a method for sensitizing the colloidal metal particles with the detection reagent used in the present invention, known methods such as physical adsorption and chemical bonding can be used. For example, in the detection reagent in which antibody is sensitized to colloidal gold particles, the antibody is unbound by adding bovine serum albumin solution after adding the antibody to a colloidally dispersed solution and physically adsorbing the antibody. Prepare by blocking the particle surface.

  In the actual immunochromatographic method, the detection reagent labeled with an insoluble carrier can be applied dispersed in the developing solution constituting the mobile phase, or the development of the mobile phase in the chromatographic medium constituting the stationary phase. The present invention can also be applied by being present on the movement path, that is, in a region between the end portion to which the mobile phase of the chromatographic medium is applied and the reaction site. When present on the chromatographic medium, it is preferable to support the detection reagent so that the detection reagent dissolves rapidly in the developing solution and can be freely moved by capillary action. In order to improve the re-solubility of the insoluble carrier sensitized with the detection reagent, it is applied to the site to be supported by adding sugars such as saccharose, maltose, lactose, sugar alcohols such as mannitol, or applying these substances. Can also be coated in advance. When the detection reagent is present on the chromatographic medium by application / drying or the like, it can be directly applied / dried to the chromatographic medium on which the immobilized reagent is immobilized, or another porous substance such as The detection reagent holding member may be formed by applying and drying on cellulose filter paper, glass fiber filter paper, nylon nonwoven fabric, and the like, and then arranged so as to be connected to the chromatographic medium on which the immobilized reagent is immobilized by a capillary.

Substance to be detected The substance to be detected to be detected by the method of the present invention is not particularly limited as long as a substance that specifically binds to the substance is present, and is not limited to proteins, peptides, nucleic acids, sugars (particularly the sugar portion of glycoproteins). , Sugar moieties of glycolipids, etc.), complex carbohydrates and the like. In the present invention, “specifically binds” means binding based on the affinity of a biomolecule. Such affinity-based binding includes antigen-antibody binding, sugar-lectin binding, hormone-receptor binding, enzyme-inhibitor binding, complementary nucleic acids and nucleic acid-nucleic acid binding proteins. And the like. Accordingly, when the substance to be detected has antigenicity, the substance that specifically binds to the substance to be detected can be exemplified by a polyclonal antibody or a monoclonal antibody. In addition, when the substance to be detected is a sugar, a lectin protein can be exemplified as a substance that specifically binds to the substance to be detected. Specific examples of the substance to be detected include carcinoembryonic antigen (CEA), HER2 protein, prostate specific antigen (PSA), CA19-9, α-fetoprotein (AFP), immunosuppressive acidic protein (IPA), CA15- 3, CA125, estrogen receptor, progesterone receptor, fecal occult blood, troponin I, troponin T, CK-MB, CRP, human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), syphilis antibody, Examples include, but are not limited to, influenza virus, human hemoglobin, chlamydia antigen, group A β streptococcal antigen, HBs antibody, HBs antigen, rotavirus, adenovirus, albumin, glycated albumin and the like.
The immunochromatographic test strip having the above-described configuration can be used as it is or in a housing shaped with plastic as necessary.

Samples containing the analyte to be detected include, for example, biological samples, that is, whole blood, serum, plasma, urine, saliva, sputum, nasal cavity or throat wipe, cerebrospinal fluid, amniotic fluid, nipple discharge, tears, sweat, skin In addition to leachate from milk, extracts from tissues, cells and stool, and the like, milk, eggs, wheat, beans, beef, pork, chicken, and other foods containing them, and the like.

Developing liquid In the present invention, a developing liquid may be used if necessary. The developing solution is a liquid that constitutes a mobile phase in the immunochromatography method, and moves along with a sample containing a substance to be detected and a labeled detection reagent on a chromatographic medium that is a stationary phase. Any developing solution may be used.

Hereinafter, the present invention will be described based on test examples and examples.
A test was conducted to select an optimal chromatographic medium in a system that measures human chorionic gonadotropin (hCG) as a substance to be detected. The hCG measurement system was performed under the following conditions.
1. Immunochromatographic test strip (1) Chromatographic medium An immunochromatographic test strip was prepared using a nitrocellulose membrane (manufactured by Toyo Roshi Kaisha, Ltd.) lined with a 100 μm thick hydrophobic polyester sheet as the chromatographic medium. did. In this strip tape, the sample pad, conjugate pad and absorption pad shown in FIG. 1 are installed according to a conventional method, the chromatographic migration distance is set to 40 mm, and the hCG recognition antibody is immobilized as the capture antibody, the labeling substance (marker) The control line (CL) on which the colloidal gold recognizing antibody was immobilized was used as a test strip. The average pore size of the nitrocellulose membrane was measured by peeling the nitrocellulose membrane from the polyethylene sheet and measuring the peeled nitrocellulose membrane with a porosimeter (product name: Palm Porometer CFP-200AEX, Porous MATERIALS, Inc.). It was calculated by the half dry method.
This test strip is cut in width and stored in accordance with a commercially available housing for an immunochromatograph.

(2) Sample pad Using a 30 mm × 300 mm glass fiber pad manufactured by Millipore (SureWick, trade name, manufactured by Millipore), pH 8.5 containing 1 wt% Tween 20 and 1 wt% bovine serum albumin. Was infiltrated with 0.25M Tris-HCl buffer solution and vacuum-dried overnight.

(3) Preparation of labeling substance solution To 500 μL of colloidal gold solution (Tanaka Kikinzoku Kogyo Co., Ltd .: LC 40 nm), add 100 μL of phosphate buffer (pH 7.5) so that the concentration is 40 μg / mL with 5 mM phosphate buffer. 100 μL of diluted anti-hCG antibody was added and allowed to stand at room temperature for 10 minutes. Next, 100 μL of 50 mM phosphate buffer (pH 7.5) containing 10% by weight bovine serum albumin was added, and 50 μL of 50 mM phosphate buffer (pH 7.5) containing 1% polyethylene glycol (average molecular weight 20000). And left at room temperature for 10 minutes. Then, after sufficient stirring, the mixture is centrifuged at 8000 × g for 15 minutes, the supernatant is removed, and 100 μL of phosphate buffer (pH 7.5) containing 1% by weight of bovine serum albumin is added to prepare a labeling substance solution. did.

(4) Conjugate pad A conjugate pad was prepared using the labeling substance solution prepared above. Pipette a mixed solution of the above labeled substance solution (anti-hCG antibody-binding colloidal gold solution) and 600 μL of purified water onto a Millipore 15 mm × 300 mm glass fiber pad (SureWick, trade name, manufactured by Millipore). Was dripped into the pad and soaked into the pad. This was vacuum-dried overnight to form a conjugate pad.

(5) Preparation of detection line and control line on chromatographic media a. Antibody solution for detection line Anti-hCG antibody was diluted with 5 mM phosphate buffer (pH 7.5) to a concentration of 0.4 mg / mL to obtain an antibody solution.
A. Antibody solution for control line Goat anti-mouse IgG antibody was diluted with 5 mM phosphate buffer (pH 7.5) to a concentration of 1 mg / mL to obtain an antibody solution for control line.
C. Spotting on chromatographic media a. A. The antibody solution was spotted on a nitrocellulose membrane as a chromatographic medium using an inkjet dispensing apparatus XYZ3050 manufactured by BioDot, and dried at 50 ° C. overnight.

2. Preparation of detected substances The detected substances used when evaluating test strips are as follows.
hCG reagent (standard substance) was dissolved in urine of a healthy male and diluted to 50 mIU / mL. This was defined as a positive specimen. The same urine to which no hCG reagent was added was used as a negative sample.

3. Immunochromatographic conditions 1. The immunochromatograph was developed using the sheet-shaped test strip prepared in (1), cut to a width of 5 mm with a cutter and stored in a housing. 2. 120 μL of the sample was dropped onto the sample pad, and the red color of the colloidal gold in the detection line and control line that appeared on the immunochromatographic test strip after 5 minutes, 10 minutes, and 30 minutes was measured using an immunochromatography reader. The detection sensitivity and measurement time were evaluated. If the measured value is 15 mAbs or more, a red line can be visually confirmed.

Test example 1
Selection of substances to be included in the chromatographic medium for immunochromatographic tests Various substances that may affect the surface tension of the chromatographic medium and that do not affect the target substance, such as protein or detection antibody, are searched for. Were selected as candidate substances.
Anionic surfactant (sodium alkylbenzenesulfonate, sodium alkanesulfonate, sodium dialkylsulfosuccinate (OTP)), cationic surfactant (cholate), amphoteric surfactant (n-octanoyl-N-methylglutamine ( Mega-8)), CHAPS (3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonate), polyoxyethylene (10) nonylphenyl ether, polyoxyethylene (30) docosyl ether, Alcohol (glycerin) was selected and evaluated. In addition, sodium dobesilbenzenesulfonate (SDBS) was selected as sodium alkylbenzenesulfonate. As sodium alkane sulfonate, sodium dodecyl sulfonate (SDS) and coconut oil fatty acid sodium sulfonate (hereinafter referred to as sodium alkane sulfonate mixture) containing fatty acids having 12, 16, and 18 carbon atoms were selected.
Each substance was made into a 0.1% aqueous solution, a nitrocellulose membrane (manufactured by Toyo Filter Paper Co., Ltd., membrane layer thickness of about 140 μm) lined with a hydrophobic polyethylene sheet having a thickness of 100 μm was dipped and then vacuum dried. In the chromatographic medium. It was confirmed by mass measurement that each substance contained in the produced membrane was 0.2 mass% with respect to the mass of the nitrocellulose membrane (concentration of external additive solution). Using this nitrocellulose membrane, hCG was detected by immunochromatography.
The measurement was performed using three test strips per sample, and indicated by an average value and a standard deviation value (SD). The detection line was measured using an immunochromatographic reader at 5 minutes (group of graph 1), 10 minutes (group 2 of graph), 15 minutes (group 3 of graph), 30 minutes (group 4 of graph). The color intensity was expressed as an average value and a standard deviation value (SD) of three samples.
Table 1 shows the measurement time when immunochromatographic inspection was performed with various substances contained in the chromatographic medium. Moreover, evaluation evaluation of measurement sensitivity is shown in FIG.

From the test results shown in Table 1, as for various substances, sodium alkanesulfonate, sodium cholate, n-octanoyl-N-methylglutamine, and CHAPS were excellent in the migration speed of the chromatograph (time during which CL develops color). Three types of polyoxyethylene (10) nonylphenyl ether, polyoxyethylene (30) docosyl ether) and alcohol (glycerin) required 5 minutes or more for color development of the control line.
The measurement sensitivity is as shown in FIG.
Two substances of sodium alkanesulfonate (SDS and sodium alkanesulfonate), n-octanoyl-N-methylglutamine (Mega-8), polyoxyethylene (30) docosyl ether, and glycerin are 5 minutes later. It became clear that the measurement intensity of the detection line observable with the naked eye was reached.
From the above results, the chromatographic medium of the immunochromatographic test strip includes two substances of sodium alkanesulfonate (a mixture of SDS and sodium alkanesulfonate), n-octanoyl-N-methylglutamine (Mega-8), polyoxyethylene ( 30) It has been clarified that the measurement sensitivity can be improved and the measurement time can be shortened by containing at least one of four substances of docosyl ether and glycerin.

Test example 2
Evaluation of influence of thickness of nitrocellulose membrane used for immunochromatographic examination Effect of the thickness of the nitrocellulose membrane (manufactured by Toyo Roshi Kaisha, Ltd.) lined with a hydrophobic sheet having a thickness of 100 μm on the detection time and sensitivity of the chromatograph as the medium for chromatography described in (1) No. Three test strips were used for evaluation.
Select four kinds of nitrocellulose membranes (with the same pore diameter) with membrane thickness (film thickness) of 93 μm, 120 μm, 131 μm, and 140 μm (No. 11-14), and the detection sensitivity and detection of the above-mentioned 2 to-be-detected substances Time was measured. The measurement results of the time required until the control line develops are shown in Table 2 below. The color intensity of the detection line is shown in FIG.

  This test revealed that the thickness of the nitrocellulose membrane affects the color intensity of the chromatograph. It has been clarified that the membrane thickness obtained by subtracting the thickness of the water-impermeable layer having a thickness of 100 μm or more is effective in shortening the measurement time and improving the sensitivity.

Test example 3
Evaluation of influence of average pore size of nitrocellulose membrane used for immunochromatographic examination The average pore size of the nitrocellulose membrane (thickness: about 130 μm, manufactured by Toyo Roshi Kaisha, Ltd.) lined with a hydrophobic sheet having a thickness of 100 μm as the chromatographic medium described in (1) is the detection time of the chromatograph. And the effect on sensitivity. Three test strips were used for evaluation.
The average pore diameter of the membrane was measured using the porosimeter method, and was classified into 3.4 μm (small), 4.7 μm (medium), and 6.1 μm (large) (No. 15 to 17). The effects on chromatographic detection time and color intensity were evaluated.
Table 3 shows the time until the control line develops color, and FIG. 4 shows the measurement result of the color intensity of the detection line.

  From Table 3, the larger average pore diameter of the membrane contributes to shortening the water absorption time. However, from FIG. 4 where the detection sensitivity and detection time of the chromatograph were actually measured, the average pore diameter of the membrane was in the range of 3.4 to 6.1 μm (small to large) for the purpose of sufficient detection sensitivity and detection time reduction. It became clear that it can be used.

Test example 4
Evaluation of influence of substance content in nitrocellulose membrane used for immunochromatographic examination The chromatographic detection of the substance content in the nitrocellulose membrane (manufactured by Toyo Roshi Kaisha, Ltd.) lined with a hydrophobic polyester sheet having a thickness of 100 μm as the chromatographic medium described in (1) The effects on time and sensitivity were evaluated.
A sodium alkanesulfonate mixture was used as the substance in the nitrocellulose membrane, and the content was 0.02% by mass, 0.2% by mass, 0.4% by mass, 0.6% by mass, and 1% by mass with respect to the mass of the nitrocellulose membrane. A chromatographic medium externally added so as to be in mass% was prepared in the same manner as in Test Example 1, and the detection sensitivity and detection time of the sample 2 were measured. The measurement results of the time required until the control line develops are shown in Table 4 below. Table 5 shows the presence or absence of color development by visual judgment of the detection line. For visual color determination, + that can confirm the red line of the gold nanoparticle labeling reagent +, + + that can be confirmed strongly and clearly + can be confirmed, but it is ± that the red color is very thin, and the red line cannot be confirmed The thing was-.

  From Table 4, it was confirmed that the detection time was reduced with any surfactant content. Further, from Table 5, it was confirmed that non-specific false positives occurred at a content of 1.0% by weight or more.

Test Example 5
Evaluation of the effect of substance content in the nitrocellulose membrane used for immunochromatographic tests on storage stability Storage that the content of the substance in the nitrocellulose membrane (manufactured by Toyo Roshi Kaisha, Ltd.) lined with a hydrophobic polyester sheet having a thickness of 100 μm as a medium for chromatography described in (1) affects the sensitivity The impact on stability was evaluated.
Sodium alkanesulfonate is used as the substance in the nitrocellulose membrane, and the content is 0.02% by mass, 0.1% by mass, 0.2% by mass, and 0.3% by mass with respect to the mass of the nitrocellulose membrane. A chromatographic medium externally added to was prepared by the same method as in Test Example 1, and the detection sensitivity of the above-mentioned specimen 2 was measured immediately after the creation of the chromatographic medium and under anaerobic conditions at 50 ° C. for 2 weeks. FIG. 5 shows the measurement result of the color intensity of the detection line 5 minutes after the sample was developed on the chromatograph.

  As shown in FIG. 5, the content of the surfactant is preferably 0.1% by mass or more based on the results of the storage stability test at 50 ° C. for 2 weeks.

1. Sample pad 2. 2. Conjugate pad 3. Chromatographic medium Detection line (antibody for detection)
5. Absorption pad 6. Impermeable membrane

Claims (8)

  A membrane for immunochromatographic test strip made of nitrocellulose used as a chromatographic medium, the membrane comprising sodium alkanesulfonate, n-octanoyl-N-methylglutamine, polyoxyethylene (30) docosyl ether, glycerin A membrane for immunochromatographic test strips, wherein at least one substance is externally added so as to be 0.02 to 0.6% by mass with respect to the mass of the membrane.   2. The membrane for an immunochromatographic test strip according to claim 1, wherein the alkyl group of sodium alkanesulfonate has a linear structure having 12 to 18 carbon atoms.   The membrane for immunochromatographic test strips according to claim 1 or 2, wherein the membrane has an average pore size of 3.4 to 6.1 µm.   The membrane for an immunochromatographic test strip according to any one of claims 1 to 3, wherein the membrane has a thickness of 100 to 150 µm.   The immunochromatographic test strip using the membrane for immunochromatographic test strips in any one of Claims 1-4.   An immunochromatographic test method using the immunochromatographic test strip according to claim 5.   The immunochromatographic test method according to claim 6, wherein the labeling substance in the immunochromatograph is a gold colloid.   The immunochromatographic test method according to claim 6 or 7, wherein the substance to be detected is human chorionic gonadotropin.

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