WO2021193680A1 - Bandelette réactive et dispositif comprenant une bandelette réactive - Google Patents

Bandelette réactive et dispositif comprenant une bandelette réactive Download PDF

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Publication number
WO2021193680A1
WO2021193680A1 PCT/JP2021/012116 JP2021012116W WO2021193680A1 WO 2021193680 A1 WO2021193680 A1 WO 2021193680A1 JP 2021012116 W JP2021012116 W JP 2021012116W WO 2021193680 A1 WO2021193680 A1 WO 2021193680A1
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Prior art keywords
test strip
substance
detected
sample
surfactant
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PCT/JP2021/012116
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English (en)
Japanese (ja)
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景吾 河野
駿 酒井
元喜 森田
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積水メディカル株式会社
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Priority to JP2022510579A priority Critical patent/JPWO2021193680A1/ja
Publication of WO2021193680A1 publication Critical patent/WO2021193680A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a test strip for detecting a substance to be detected and a device including the test strip.
  • Rapid assays such as dipstick assays and lateral flow assays have been used in numerous diagnostic and testing methods for many years.
  • a measurement method using a test strip for immunochromatography is widely used.
  • an antibody or an antigen against an antigen or an antibody to be detected is immobilized on an insoluble membrane carrier which is a chromatographic medium to prepare a detection unit which is a stationary phase.
  • a conjugate detection reagent
  • the substance to be detected and the conjugate, which is a mobile phase, are specifically reacted. Further, in the detection unit which is a stationary phase, the substance to be detected bound to the conjugate is specifically reacted with the antibody or antigen immobilized on the detection unit. Then, usually, a fluorescent substance, colloidal metal particles such as gold colloid, or color latex particles are used as the labeling substance.
  • the detection unit detects the presence or, in some cases, the amount of the substance to be detected in the sample from the signals derived from these labeled substances.
  • the rapid assay method tends to involve a non-specific reaction when a contaminant such as an heterophobic antibody is contained in the sample.
  • a contaminant such as an heterophobic antibody
  • the amount and properties of coexisting substances also vary from sample to sample. Self-aggregation of conjugates may occur due to the amount and properties of coexisting substances.
  • Such non-specific reactions may hinder accurate measurement and determination, which is a problem. Inability to make accurate measurements means that quantitative assays produce false highs or false lows, and qualitative assays produce false positives or false negatives. ..
  • Patent Document 1 discloses a porous solid phase for a binding assay, which comprises a specific surfactant before the addition of a measurement sample.
  • Patent Document 2 discloses a strip for chromatographic analysis, which comprises a means for capturing a target substance in a sample and a means for capturing a factor causing a false positive further upstream thereof.
  • the effect of suppressing the non-specific reaction may not be sufficient, and a further technique capable of efficiently suppressing the non-specific reaction has been required.
  • An object of the present invention is to be able to suppress a non-specific reaction in the measurement of the amount of a substance to be detected, or in the qualitative determination of presence / absence, that is, positive / negative, and to obtain an accurate measured value or determination result. It is to provide a test strip capable of and a device containing the test strip.
  • test strip that detects a substance to be detected by developing a sample that may contain the substance to be detected, and the test strip is a test strip.
  • Sample pad having a sample supply part for supplying a sample
  • insoluble membrane carrier having at least one detection part for capturing and detecting a substance to be detected.
  • a labeling unit is provided between the sample supply unit and the detection unit, in which a labeling unit that directly or indirectly binds to the substance to be detected and generates a signal in the detection unit is arranged, and the labeling unit is provided.
  • the test strip wherein a line-shaped surfactant portion containing a surfactant is provided between the detection portion and the detection portion.
  • the test strip according to ⁇ 1> wherein the antigen and the antibody or antigen that immunologically reacts with the substance to be detected, which are immobilized on the detection unit, are different from each other.
  • ⁇ 3> The test strip according to ⁇ 1> or ⁇ 2>, which is a lateral flow type.
  • ⁇ 4> The test strip according to any one of ⁇ 1> to ⁇ 3>, wherein the surfactant contained in the surfactant portion is a nonionic surfactant.
  • ⁇ 5> The test strip according to any one of ⁇ 1> to ⁇ 4>, wherein the substance to be detected is a substance of biological origin.
  • ⁇ 6> The substance derived from living organisms is troponin.
  • ⁇ 7> The test according to any one of ⁇ 1> to ⁇ 6>, wherein the sample that may contain the substance to be detected is at least one selected from the group consisting of blood, serum, and plasma. strip.
  • ⁇ 8> The test strip according to any one of ⁇ 1> to ⁇ 7>, wherein the sample that may contain a substance to be detected is a sample that has not been pretreated.
  • a labeled body-containing pad in which (3) a labeled body for generating a signal in the detection unit is arranged as the labeling part is included, and the surfactant part comprises.
  • the surfactant contained in the surfactant portion is a test strip containing a surfactant solution having a concentration equal to or higher than the critical micelle concentration. Described test strip.
  • ⁇ 12> Any of ⁇ 1> to ⁇ 11>, wherein the line width of the surfactant portion is 0.5 to 2 mm, and the line width is within the range of ⁇ 0.2 mm over the entire line. Described test strip. ⁇ 13> The test strip according to any one of ⁇ 1> to ⁇ 12>, wherein the surfactant contained in the surfactant portion is an alkyl glucoside-based surfactant.
  • the alkyl glucoside-based surfactant is n-octyl- ⁇ -D-glucoside, n-decyl- ⁇ -D-glucoside, n-dodecyl- ⁇ -D-glucoside, n-heptyl- ⁇ -D-.
  • the test strip according to ⁇ 13> which is at least one selected from the group consisting of thioglucosides and n-octyl- ⁇ -D-thioglucosides.
  • the alkyl glucoside-based surfactant is n-heptyl- ⁇ -D-thioglucoside, and the n-heptyl- ⁇ -D-thioglucoside solution is applied in a line to the test strip.
  • a surfactant portion on the line is formed, and the concentration of the n-heptyl- ⁇ -D-thioglucoside solution is 0.88% (w / v) or more, in ⁇ 14>. Described test strip.
  • the detection unit has a line shape with a line width of 0.5 to 2 mm, the line width of the detection unit is within a range of ⁇ 0.2 mm over the entire line, and the line-shaped detection unit and the above.
  • test strip of the present invention it is possible to suppress a non-specific reaction in the detection of a substance to be detected in a sample, and it is possible to obtain an accurate measured value or a judgment result.
  • FIG. 1 shows a side view of the structure of a test strip for immunochromatography before stacking.
  • the top view (part) of an embodiment of an insoluble membrane carrier is shown.
  • It is a graph which shows the measurement result of the substance to be detected by the test strip of the comparative example.
  • It is a graph which shows the bias between the measured value of the test strip of the comparative example, or the measured value of one Embodiment of this invention, and a known cTnI concentration.
  • Examples of the substance to be detected in the test strip of the present invention include physiologically active substances such as viruses and proteins.
  • the substances to be detected in the test strip of the present invention are as follows, for example.
  • Myocardial markers such as CK-MB, H-FABP, troponin (troponin I, troponin T), myoglobin; fibrin degradation products (eg D-dimer), soluble fibrin, TAT (thrombin-antithrombin complex), PIC (plasmin-plasmin) Coagulation / fibrinolysis markers such as inhibitor complex); Circulation-related markers such as oxidized LDL, BNP (cerebral sodium diuretic peptide); Metabolism-related markers such as adiponectin: CEA (cancer fetal antigen), AFP ( ⁇ -fetoprotein), Tumor markers such as CA19-9, CA125, PSA (Prostate Specific Antigen); Inflammatory markers such as CRP (C-Reactive Protein), IgA, IgG, I
  • Troponin is one of the proteins that make up muscle. Troponin forms a complex with troponin T, troponin I, and troponin C, and is responsible for the contraction regulation of myocardium and skeletal muscle. Troponin, which regulates myocardial contraction, is called myocardial troponin. Human myocardial troponin I and human myocardial troponin T have been utilized as markers for acute myocardial infarction.
  • troponin T or troponin I can be used as a substance to be detected, but troponin I is more preferably used as a substance to be detected, and human myocardial troponin I (cTnI) is covered. It is more preferable to use it as a detection substance.
  • the test strip of the present invention can also be used in nucleic acid chromatography.
  • Nucleic acid chromatography means chromatography that detects a specific nucleic acid sequence such as DNA or RNA in a sample.
  • the test strip of the present invention can be used for nucleic acid chromatography for the purpose of detecting allergens (buckwheat, eggs, etc.), gene polymorphisms, or specific bacteria, viruses, and the like.
  • a nucleic acid sequence complementary to at least a part of the detected sequence subjected to fluorescent labeling is fluorescently labeled.
  • a substance that captures the target nucleic acid sequence to form a complex using a nucleic acid aptamer or the like is used as a detection reagent, and a nucleic acid sequence different from the detection reagent complementary to at least a part of the detected sequence is used in the detection unit. Nucleic acid aptamers can be placed.
  • examples of the sample that may contain the substance to be detected include mainly biological substances and extracts obtained by extracting the substance to be detected from them.
  • Food samples typified by liquid beverages, semi-solid foods, solid foods, etc.
  • sample samples from the natural world such as soil, rivers, and seawater, and wiped samples from production lines or clean rooms in factories also contain substances to be detected. It can be used as a possible sample.
  • Specific examples of biological substances include blood (whole blood), serum, plasma, lymph, urine, feces, ascites, pleural effusion, and tissues / cells.
  • a body fluid particularly blood (whole blood), serum, or plasma as a sample that may contain a substance to be detected.
  • Samples that may contain substances to be detected include measurement sample components separated and fractionated from the whole blood by means of centrifugation, filtration, purification, etc., and measurement sample components extracted with an organic solvent, etc. , The measurement sample component solubilized by a surfactant or the like, the measurement sample component diluted with a buffer solution or the like, or the measurement sample component modified or modified by a chemical reaction or the like.
  • a sample in which nucleic acid is amplified in advance by a PCR method or the like can also be used.
  • the sample that may contain the substance to be detected may be pretreated such as dilution, extraction, and additive mixing, but may not be pretreated.
  • labeled substance means a substance that generates a signal in the detection unit when directly or indirectly bound to the substance to be detected.
  • the signal derived from the labeled substance may be measured according to a known method. For example, the absorbance or the intensity of reflected light can be measured. The signal may be confirmed visually or may be confirmed using a specific measuring instrument.
  • the labeling body in the present invention a known labeling body conventionally used for a test strip can be used.
  • colloidal metal particles such as gold colloidal particles and platinum colloidal particles, color latex particles, magnetic particles and the like are preferable, and gold colloidal particles are particularly preferable.
  • the labeled substance is preferably modified with a tag or the like, or an antibody or antigen that immunologically reacts with the substance to be detected is immobilized.
  • the particle size thereof is preferably 20 to 70 nm, particularly preferably 45 to 65 nm.
  • the above-mentioned colloidal gold particles can be produced by a generally known method, for example, by dropping and stirring an aqueous solution of trisodium citrate in a heated aqueous solution of tetrachloroauric (III) acid.
  • the test strip of the present invention is preferably a test strip for immunochromatography.
  • the labeled substance is preferably immobilized with an antibody or antigen that immunologically reacts with the substance to be detected, and the detection unit is immunologically immobilized with the substance to be detected. It is preferable that the antibody or antigen to react is immobilized.
  • a labeled substance on which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized may be referred to as a conjugate.
  • immobilizing an antigen or antibody means physically or chemically supporting the antigen or antibody on a labeled substance or insoluble membrane carrier.
  • the antibody that immunologically reacts with the substance to be detected immobilized on the label and the antibody that immunologically reacts with the substance to be detected immobilized on the detection unit are different. Is more preferable.
  • another thing means a different kind, and means an antibody which recognizes a different epitope.
  • the antibody immobilized on the label and the detection part is preferably a monoclonal antibody.
  • a monoclonal antibody By using a monoclonal antibody, the specificity of the reaction can be increased.
  • functional fragments of antibodies having antigen-antibody reaction activity are also included in the antibodies herein.
  • Functional fragments of the antibody include those obtained through an animal immunization step, those obtained using genetic recombination technology, and chimeric antibodies. Examples of the functional fragment of the antibody include F (ab') 2 , Fab'and the like. These functional fragments can be produced by treating the antibody with a proteolytic enzyme (eg, pepsin, papain, etc.).
  • a proteolytic enzyme eg, pepsin, papain, etc.
  • the antibody or antigen-immobilized label used in the test strip of the present invention that immunologically reacts with the substance to be detected that is, the conjugate is resistant to colloidal gold particles when the substance to be detected is troponin.
  • Those on which a troponin monoclonal antibody is immobilized are preferable.
  • Examples of a method for immobilizing an antibody or antigen that immunologically reacts with a substance to be detected on a labeled substance include physical adsorption, chemical bonding, and the like. It is generally fixed by physical adsorption.
  • the gold colloidal particles and the anti-troponin monoclonal antibody are usually added to a buffer solution and immobilized by physical adsorption.
  • the antibody concentration is preferably adjusted to 20 to 100 ⁇ g / mL.
  • the labeling portion on which the conjugate is arranged may be provided in a line on the sample pad described later, or may be provided between the sample pad and the insoluble membrane carrier as a label-containing pad described later.
  • sample pad In the test strip of the present invention, a pad-shaped porous material capable of holding a conjugate can be used as a sample pad.
  • the sample pad has a sample supply unit as a part thereof.
  • a porous material portion exists downstream of the sample supply section. The sample supplied to the sample supply section develops the porous material portion and reaches the labeling section.
  • the sample supply unit is a portion that supplies a sample that may contain a substance to be detected. This sample supply section is formed on a part of the porous material, and the sample supply section is on the upstream side of the sample pad.
  • the surfactant portion is a portion containing the surfactant.
  • the surfactant portion can be formed in a line on the sample pad or the insoluble membrane carrier on the downstream side of the labeling portion.
  • the labeled body-containing pad is provided separately from the sample pad, it can be formed on the insoluble membrane carrier downstream from the labeled body-containing pad.
  • the line-shaped surfactant portion is linear in the sample developing direction, that is, the line connecting the center of the sample supply portion of the sample pad and the center of the downstream end portion of the insoluble membrane carrier described later. It is preferably arranged.
  • the line-shaped surfactant portions are preferably arranged in a line shape in a direction perpendicular to the longitudinal direction of the sample pad and the insoluble membrane carrier.
  • the surfactant portion is preferably formed on an insoluble membrane carrier.
  • the position of the surfactant portion can be adjusted to an appropriate position by those skilled in the art, but the center line of the line of the line-shaped surfactant portion is 1 to 25 mm downstream from the downstream end portion of the labeling portion. It is preferably arranged on the side, and more preferably 10 to 20 mm downstream. Further, it is preferable that the center line of the line of the line-shaped surfactant portion is arranged at least 1 mm or more upstream from the upstream end portion of the detection portion.
  • the "center line” of the surfactant portion means a center line drawn in a direction perpendicular to the longitudinal direction of the sample pad, and is drawn in a direction parallel to the longitudinal direction of the sample pad. It does not mean the center line to be drawn.
  • the length of the line-shaped surfactant portion in the longitudinal direction of the sample pad is referred to as the "line width" of the surfactant portion.
  • the line width of the line-shaped surfactant portion may be such that the amount of the surfactant required for obtaining the effect of the present invention can be contained.
  • the line width is, for example, 0.5 to 10 mm, 0.5 to 5 mm, or 0.5 to 2 mm.
  • the line width of the line-shaped surfactant portion is preferably substantially the same width over the entire line (that is, the width is ⁇ 1 mm, ⁇ 0.5 mm, or ⁇ 0.2 mm over the entire line). It is also conceivable that a part of the line-shaped surfactant portion protrudes to the downstream side and / or the upstream side. In this case, either the non-protruding portion or the protruding portion may have the above-mentioned line width, but it is preferable that the non-protruding portion has the above-mentioned line width.
  • the surfactant in the entire line-shaped surfactant portion, and as long as the effect of the present invention can be obtained, there may be a portion in the line that does not contain the surfactant.
  • the line does not contain a surfactant
  • an effect can be obtained by arranging the surfactant in a limited region and evenly so as to be able to elute against the movement of the substance to be detected and the labeled substance. It is considered that such a configuration prevents the generation of agglomerates that cause false low and false high values or false negatives and false positives, and as a result, accurate measured values can be obtained.
  • Examples of the surfactant used in the present invention include cationic surfactants, anionic surfactants, amphoteric surfactants, and nonionic surfactants.
  • Examples of the cationic surfactant include alkylamine salts and quaternary ammonium salts.
  • Examples of the anionic surfactant include cole acids, alkyl sulfates, polyoxyethylene alkyl ether sulfates, alkylbenzene sulfonates and the like.
  • Examples of the amphoteric surfactant include alkyl betaine, alkyl amine oxide, and colamide.
  • Nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyalkylene derivatives, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitol fatty acid esters, glycerin fatty acid esters, polyoxyethylene fatty acid esters, and polyoxy. Examples thereof include ethylene-hardened castor oil, polyoxyethylene alkylamine, fatty acid alkanolamide, alkylimidazoline, alkyl glucoside, alkylmannoside, alkylmaltoside, and trehalose compound.
  • alkyl glucoside-based surfactant means a nonionic surfactant in which a sugar molecule and a higher alcohol are glycosidic bonded.
  • glucosidic bond has a broad meaning, and for example, S-glucoside bond (thioglucoside bond) and N-glycoside bond are also included in "glucosidic bond”.
  • sugar molecule a monosaccharide molecule is preferable, and glucose is more preferable.
  • the higher alcohol is preferably a C6 to C22 alkyl alcohol.
  • the alkyl alcohol can be linear or branched chain, preferably linear. Further, the number of OH groups contained in the alkyl alcohol is preferably 1.
  • Specific alkyl glucoside-based surfactants include n-octyl- ⁇ -D-glucoside (CAS No. 29836-26-8) and n-decyl- ⁇ -D-glucoside (CAS No. 58846-77-8). ), N-dodecyl- ⁇ -D-glucoside (CAS No. 59122-55-3), n-heptyl- ⁇ -D-thioglucoside (CAS No. 85618-20-8), and n-octyl- ⁇ - Examples thereof include D-thioglucoside (CAS No. 85618-21-9). Most preferred is n-heptyl- ⁇ -D-thioglucoside.
  • the concentration of the surfactant solution applied in a line to the test strip is, for example, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0. .5 mass by volume or more, or 1 mass by volume or more.
  • the line-shaped surfactant portion can be produced on the insoluble membrane carrier as follows. Prepare a surfactant part solution containing the surfactant part at a predetermined concentration. Next, the liquid is discharged from the nozzle in the longitudinal direction of the insoluble membrane carrier by using a device having a mechanism capable of moving the liquid in a direction perpendicular to the longitudinal direction of the insoluble membrane carrier while discharging the liquid at a constant speed. It is applied to the insoluble membrane carrier in a line shape in a direction perpendicular to the vertical direction, for example, in a line shape having a line width of 0.5 to 10 mm, 0.5 to 5 mm, or 0.5 to 2 mm, and dried.
  • the sample pad is laminated with the insoluble membrane carrier so that the lower surface of the downstream end thereof contacts the upper surface of the insoluble membrane carrier described later.
  • the lower surface of the sample supply section may or may not be in contact with the upper surface of the insoluble membrane carrier.
  • the sample pad is provided with a labeling portion
  • the lower surface of the labeling portion may or may not be in contact with the upper surface of the insoluble membrane carrier.
  • a more porous material may be provided between the sample pad and the label-containing pad or between the label-containing pad and the insoluble membrane so that the sample can reach the insoluble membrane from the sample supply part by capillary action.
  • the upstream and downstream ends may be appropriately laminated.
  • porous material constituting the sample pad and the label-containing pad examples include a pad made of non-woven fibers such as paper, cellulose mixture, nitrocellulose, polyester, acrylonitrile copolymer, glass, rayon and the like. Of these, a pad made of glass fiber (a pad made of glass fiber) is preferable.
  • the total length of the sample pad that is, the length from the upstream end to the downstream end of the sample pad can be adjusted to an appropriate length by those skilled in the art. Specifically, it can be adjusted to 5 to 40 mm, 10 to 30 mm, or 10 to 20 mm.
  • the length from the end on the upstream side of the sample pad to the end on the downstream side of the labeled body-containing pad may be adjusted to an appropriate length, but it can be adjusted within the above range. preferable.
  • the insoluble membrane carrier that can be used in the present invention has at least one detection unit for capturing and detecting the substance to be detected.
  • the detection unit is preferably line-shaped. It is preferable that an antibody or antigen that immunologically reacts with the substance to be detected is immobilized in the detection unit.
  • the detection part is in a line shape, it is arranged in a line shape in a direction orthogonal to the sample development direction, that is, the line connecting the center of the sample supply part of the sample pad and the center of the downstream end portion of the insoluble membrane carrier. Is preferable.
  • the line-shaped detection unit is preferably arranged in a line shape in a direction perpendicular to the longitudinal direction of the insoluble membrane carrier.
  • Immobilization of an antibody or antigen that immunologically reacts with a substance to be detected on an insoluble membrane carrier can be carried out by a conventionally known method.
  • immobilization can be performed as follows. A mechanism capable of preparing a liquid containing the above antibody or antigen at a predetermined concentration and then moving the liquid in a direction perpendicular to the longitudinal direction of the insoluble membrane carrier while discharging the liquid from a nozzle at a constant speed.
  • the above liquid is applied to the insoluble membrane carrier in a line shape in a direction perpendicular to the longitudinal direction of the insoluble membrane carrier, for example, in a line shape having a line width of 0.5 to 5 mm and 0.5 to 2 mm. It can be immobilized by drying.
  • the length of the line of the line-shaped detection unit in the longitudinal direction of the insoluble membrane carrier is referred to as the "line width" of the detection unit. It is preferable that the line width of the line of the detection unit is substantially the same over the entire line (that is, the width is ⁇ 0.2 mm over the entire line). It is also conceivable that a part of the line-shaped detection unit protrudes to the downstream side and / or the upstream side.
  • either the non-protruding portion or the protruding portion may have the above-mentioned line width, but it is preferable that the non-protruding portion has the above-mentioned line width.
  • the distance between the line-shaped detection portion and the surfactant portion on the line is preferably 2 mm to 12 mm, more preferably 2 to 10 mm, further preferably 3 to 8 mm, and 4 to 7 mm. Is more preferable, and 4.5 to 6.5 mm is most preferable.
  • the "distance between the line-shaped detection part and the surfactant part on the line” is a straight line equilibrium with respect to the longitudinal direction of the insoluble membrane carrier, and the line-shaped detection part and the surfactant part on the line. Means the shortest length when tied.
  • the concentration of the antibody or antigen in the above solution is preferably 0.1 to 5 mg / mL, more preferably 0.5 to 3 mg / mL.
  • the sample supplied from the portion of the sample pad that comes into contact with the insoluble carrier moves in a direction parallel to the longitudinal direction of the insoluble membrane carrier due to the capillary phenomenon. It is a measurement method of the method developed as described above.
  • the test strip of the present invention can be used not only in the lateral flow method but also in a measurement method (dipstick method) in which the test strip is immersed in a sample extract.
  • a liquid containing the above antibody or antigen at a predetermined concentration can be prepared by adding the antibody or antigen to the buffer solution.
  • the type of the buffer solution include commonly used buffer solutions such as a phosphate buffer solution, a Tris buffer solution, and a Good's buffer solution.
  • the pH of the buffer solution is preferably in the range of 6.0 to 9.5, more preferably 6.5 to 8.5, and even more preferably 7.0 to 8.0.
  • the buffer solution may further contain salts such as sodium chloride, stabilizers such as sucrose, preservatives, preservatives such as proclin, and the like.
  • blocking can be performed by further coating a normally used blocking agent in the form of a solution or vapor to cover a portion other than the site where the antibody or antigen is immobilized.
  • the insoluble membrane carrier may be immobilized with a control capture reagent conventionally used in a test strip for later later chromatography.
  • the control capture reagent is a reagent for ensuring the reliability of the assay, and captures the control reagent contained in the sample pad.
  • an anti-KLH antibody or the like corresponds to the control capture reagent.
  • the position at which the control capture reagent is immobilized can be appropriately selected to suit the design of the assay system, and is, for example, 2 to 15 mm, 2 to 10 mm, or 3 from the center line of the detection unit located most upstream. It can be designed to be located ⁇ 8 mm downstream.
  • the position where the control capture reagent is immobilized may be referred to as a control capture reagent immobilization unit.
  • the control capture reagent immobilization portion is preferably line-shaped.
  • the definition of the preferable line width range and the line width is the same as that of the detection unit.
  • the distance between the line-shaped capture reagent immobilization portion and the surfactant portion on the line is preferably 6 mm to 16 mm, more preferably 6 to 14 mm, and 7 to 12 mm. Is more preferable, 8 to 11 mm is further preferable, and 8.5 to 10.5 mm is most preferable.
  • the "distance between the line-shaped capture reagent immobilization portion and the surfactant portion on the line” is a straight line equilibrium with respect to the longitudinal direction of the insoluble membrane carrier, and is a line-shaped capture reagent immobilization portion and on the line. It means the shortest length when tied to the surfactant part of.
  • the membrane constituting the insoluble membrane carrier used in the present invention a known membrane that has been conventionally used as an insoluble membrane carrier for test strips can be used.
  • membranes composed of fibers made of polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, and ceramics.
  • Specific examples thereof include glass fiber filter paper and nitrocellulose membrane commercially available from Sartorius, Merck, Toyo Filter Paper, Whatman and the like.
  • the average pore size or reserved particle size of the insoluble membrane carrier is not limited to these, but 0.1 to 20 ⁇ m can be used, more preferably 0.5 to 16 ⁇ m, and 0.7 to 10 ⁇ m. Is more preferable.
  • one of 70 to 300 seconds can be used, preferably 90 to 200 seconds, and even more preferably 120 to 180 seconds.
  • the flow rate means the number of seconds required for water to be developed on one end of the insoluble membrane carrier and the development to proceed by 4 cm.
  • the total length of the insoluble membrane carrier that is, the length from the upstream end to the downstream end of the insoluble membrane carrier can be adjusted to an appropriate length by those skilled in the art, but specifically, The length can be adjusted to 15-40 mm, 18-35 mm, or 20-30 mm.
  • the “labeled body-containing pad” is obtained by impregnating a material suitable for the labeled body-containing pad described later with a conjugate and drying the conjugate.
  • the labeled substance-containing pad has a function of forming a complex between the conjugate and the substance to be detected when the sample passes through the labeled substance-containing pad.
  • the label-containing pad may be arranged so as to be in contact with the porous membrane on which the specific binding substance is immobilized.
  • a sample that is placed in contact with the sample pad and has passed through the sample pad by capillary flow is received, and the sample is subsequently transferred by capillary flow to a 3rd pad that is in contact with the sample pad on a surface different from the contact surface. It may be arranged as follows.
  • Suitable materials for the labeled pad contain include, but are not limited to, paper, cellulose mixtures, nitrocellulose, polyesters, acrylonitrile copolymers, glass fibers or non-woven fibers such as rayon. Preferably, a glass fiber pad is used.
  • the labeled substance-containing pad is labeled with a "control reagent" for ensuring the reliability of the immunochromatographic detection method, for example, an antibody or a labeled substance that does not react with the sample component labeled with the labeled substance. It can contain highly antigenic proteins such as KLH (keyhole limpet hemocyanin). These control reagents are components (substances) that are unlikely to be present in the sample and can be appropriately selected.
  • the absorption pad is a site having liquid absorbability that controls the development of the sample by absorbing the sample that has moved and passed through the insoluble membrane carrier.
  • a known absorption pad conventionally used for a test strip is used, and for example, filter paper can be used. It is considered that a person skilled in the art can adjust the total length of the absorption pad, that is, the length from the upstream end to the downstream end of the absorption pad to an appropriate length. The length can be adjusted to 5 to 100 mm, 20 to 80 mm, or 20 to 60 mm.
  • Test strip The test strip is preferably placed on a solid phase support such as a plastic adhesive sheet.
  • the solid phase support is composed of a substance that does not interfere with the capillary flow of the sample and the conjugate.
  • the test strip may be fixed on the solid phase support with an adhesive or the like. In this case, even the components of the adhesive are composed of a substance that does not interfere with the capillary flow of the sample and the conjugate.
  • a polyester film or the like as a top film for the purpose of increasing the mechanical strength of the insoluble membrane carrier and preventing evaporation (drying) of water during the assay.
  • test strip of the present invention includes a sample pad and an insoluble membrane carrier, and may further contain other reagents and configurations depending on the measurement conditions and the sample.
  • Other reagents include, for example, blocking agents that prevent non-specific reactions, and other configurations include, for example, additional pads for removing components that are not needed for measurement in the sample.
  • a 3rd Pad can be provided between the sample pad and the porous membrane to allow the substance to be detected or the complex containing the substance to be detected to smoothly develop the insoluble membrane carrier.
  • the method for producing a test strip for immunochromatography of the present invention has the following steps (1) to (4).
  • An antibody-immobilized porous membrane is attached to the backing sheet, and a coating portion is arranged on the upstream side of the development in the order of the surfactant part, the antibody that binds to the substance to be detected, and then the control antibody.
  • the surfactant portion is provided by applying or spraying the surfactant solution in a line perpendicular to the sample development direction.
  • the concentration of the surfactant solution is preferably a concentration equal to or higher than the critical micelle concentration.
  • the term "detection” includes not only qualitative detection but also quantitative detection of a substance to be detected that can be quantified.
  • a signal derived from a labeled substance or a conjugate is detected even though the substance to be detected is absent or substantially absent in the sample, and is judged to be positive. (So-called false positives), and despite the presence of a considerable amount of the substance to be detected in the sample, the signal derived from the labeled substance or conjugate is not detected and is judged to be negative (so-called false negatives). ) Is included.
  • the measured value fluctuates to a low value (pseudo-low value) or a high value (pseudo-high value).
  • test strip for immunochromatography of the present invention can be prepared by appropriately modifying or modifying the method described in Examples.
  • % means mass volume%.
  • Test Strip for Immunochromatography which is an embodiment of the test strip of the present invention, a test was conducted to confirm the nonspecific reaction inhibitory effect.
  • sample pad 20 mM MOPS (pH 7.2) containing 0.5% glucose and 2% polybrene was prepared and used as a sample pad pretreatment solution.
  • a glass fiber pad (Lydall) was appropriately cut to a required size, and the sample pad pretreatment solution was impregnated in a volume 1.5 times the volume of the pad.
  • a sample pad dried at 70 ° C. for 45 minutes in a dry oven was used.
  • the anti-cTnI monoclonal antibody was set at a position 9 mm from the upstream end of the nitrocellulose membrane to 1 ⁇ L / cm using an immunochromatographic dispenser “XYZ3050” (BIO DOT), and applied in a line to form a test line. bottom.
  • the line width was about 0.7 mm.
  • An anti-KLH polyclonal antibody was similarly applied to a downstream position at a distance of about 4 mm from the position of the test line to form a control line.
  • the line width was about 0.7 mm.
  • a surfactant solution was prepared by diluting n-heptyl- ⁇ -D-thioglucoside to 1.0% by mass by mass.
  • This surfactant solution was applied in a line at a position 3.5 mm from the upstream end of the membrane carrier so as to be 1 ⁇ L / cm, and dried.
  • the line width was about 1 mm. Therefore, the distance between the detection part (test line) and the surfactant part is 5.5 mm.
  • the membrane was dried at 70 ° C. for 45 minutes in a dry oven to obtain an antibody-immobilized membrane.
  • test strip for immunochromatography An antibody-immobilized porous membrane (b) is attached to the backing sheet (a), and (j) a surfactant portion, an anti-cTnI antibody (c), and then an anti-antibody are placed on the upstream side of the development.
  • the coating part was arranged in the order of the KLH antibody (d).
  • a polysulfone porous membrane (3rd Pad) (e) was mounted on the membrane.
  • the labeled body-containing pad (f) prepared in 3) above is placed and mounted, and the sample pad (g) prepared in 2) above is placed and mounted so as to overlap the labeled body-containing pad, and the opposite end is placed.
  • An absorption pad (h) was arranged and attached to the body.
  • each component was cut into a superposed structure to prepare a test strip 1 for immunochromatography.
  • Test Strip 2 Preparation of Test Strip for Immunochromatography of Comparative Example
  • the procedure of Comparative Example was the same as that of Comparative Example except that the line-shaped surfactant portion was not prepared.
  • a test strip 2 for immunochromatography was prepared.
  • the horizontal axis is a value measured by the E test “TOSOH” II (cTnI3) manufactured by Tosoh Corporation, which is an in vitro diagnostic drug for detecting myocardial troponin I (cTnI).
  • TOSOH TOSOH II
  • cTnI3 myocardial troponin I
  • the immunochromatographic test strip 1 of the example reduced the deviation of the measured value from the E test “TOSOH” II (cTnI3).
  • the correlation coefficient (R value) indicating the correlation of the measured values with the E test "TOSOH” II (cTnI3) was 0.897 in the test strip 2 for immunochromatography of the comparative example, but the immunochromatography of the example. In the test strip 1 for use, it was 0.935. Therefore, by having a line-shaped surfactant portion, a more accurate measured value was obtained. In addition, more accurate measured values were obtained for the divergent samples (plots in black diamonds in FIGS.
  • FIG. 5 is a graph showing the ratio between the measured value of the immunochromatographic test strip 2 of the comparative example or the measured value of the immunochromatographic test strip 1 of the example and the measured value of “TOSOH” II (cTnI3).
  • FIGS. 6 and 7 are diagrams showing a comparison with the measured values by Architect High Sensitive Troponin IST (manufactured by Abbott Japan Co., Ltd.), which is an in vitro diagnostic drug for detecting myocardial troponin I (cTnI).
  • Example 2 Confirmation of appearance of the dissociated sample after deployment
  • the dissociated sample black diamond in FIGS. 3 to 7) showing a measured value significantly different from the measured value by the E test “TOSOH” II (cTnI3) in Example 1.
  • the expanded state of the conjugate was observed 15 minutes after the addition to the upstream end of the sample pad of the test strips 1 and 2 for immunochromatography. The results are shown in FIG. On test strip 2, delay in conjugate deployment was observed as background redness before the test line. On test strip 1, the conjugate deployment delay was reduced and the test line was darker than test strip 2. On test strip 1, it is believed that most of the conjugates reached the test line and were captured.
  • Example 3 Confirmation test 2 of non-specific reaction inhibitory effect The same test as in Example 1 was performed using 13 different plasma sample solutions. In Example 3, it was compared with the value measured by the architect high sensitive troponin IST. The results are shown in FIGS. 9 and 10.
  • the correlation coefficient (R value) indicating the correlation of the measured values with the architect high sensitive troponin IST was 0.797 in the test strip 2 for the later lateral flow of the comparative example, but for the later of the example. On test strip 1, it was 0.845. Therefore, by having a line-shaped surfactant portion, a more accurate measured value was obtained.
  • the immunochromatographic test strip 3 of Comparative Example was prepared in the same procedure as the immunochromatographic test strip prepared in Test Example 1 except that the test strip was sprayed in a shape and applied to the entire antibody-immobilized membrane.
  • An immunochromatographic test strip 4 prepared by the same procedure was also prepared except that the surfactant solution was not applied to the antibody-immobilized membrane.
  • the immunochromatographic test strip 4 was prepared and tested in contrast to the lateral flow test strip 3 to verify the effect of applying the surfactant over the entire antibody-immobilized membrane.
  • the correlation coefficient (R value) indicating the correlation between the measured value and the architect was 0.879 (FIG. 11).
  • the correlation coefficient (R value) indicating the correlation of the measured value with the architect was 0.864 (FIG. 12).
  • the correlation coefficient between the values of both immunochromatographic test strips was 0.984. Therefore, it was found that even if the surfactant was applied to the entire antibody-immobilized membrane, the correlation coefficient was hardly improved.
  • test strip for immunochromatography of the present invention it is possible to provide a test strip for immunochromatography having a short reaction completion time and excellent accuracy.

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Abstract

La présente invention aborde le problème de la fourniture d'une bandelette réactive pour immunochromatographie, la bandelette réactive ayant un temps d'achèvement de réaction court et une excellente précision.　Cette bandelette réactive détecte une substance à détecter, par le développement d'un échantillon qui est susceptible de contenir la substance à détecter, la bandelette réactive comprenant (1) un tampon d'échantillon ayant une unité d'alimentation d'échantillon qui fournit un échantillon, et (2) un support de membrane insoluble ayant au moins une unité de détection qui capture et détecte la substance à détecter, une unité de marquage, dans laquelle une substance de marquage qui se lie directement ou indirectement à la substance à détecter et génère un signal dans l'unité de détection est disposée, est disposée entre l'unité d'alimentation d'échantillon et l'unité de détection, et une unité de tensioactif en forme de ligne comprenant un tensioactif est disposée entre l'unité de marquage et l'unité de détection. Le problème peut être résolu par la bandelette réactive.
PCT/JP2021/012116 2020-03-25 2021-03-24 Bandelette réactive et dispositif comprenant une bandelette réactive WO2021193680A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060076249A1 (en) * 2004-09-09 2006-04-13 Bernhard Meisegeier Measuring device and measuring method for detecting analytes
WO2007069673A1 (fr) * 2005-12-14 2007-06-21 Denka Seiken Co., Ltd. Procede de detection immunochromatographique pour staphylocoque presentant une multiresistance aux medicaments, et kit de diagnostic
JP2009258095A (ja) * 2008-03-18 2009-11-05 Hitachi Chem Co Ltd クロマトグラフィー分析用ストリップの製造方法
JP2010513858A (ja) * 2006-12-15 2010-04-30 キンバリー クラーク ワールドワイド インコーポレイテッド ラテラルフローアッセイデバイス及び当該デバイスを含む吸収性物品
WO2015054546A1 (fr) * 2013-10-10 2015-04-16 Song Diagnostic Research Llc. Dosages à écoulement latéral améliorés
JP2018512882A (ja) * 2015-04-24 2018-05-24 メサ バイオテック，インク． 流体検査用カセット
JP2020020724A (ja) * 2018-08-02 2020-02-06 積水メディカル株式会社 イムノクロマト用試験片及びイムノクロマト検出キット

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060076249A1 (en) * 2004-09-09 2006-04-13 Bernhard Meisegeier Measuring device and measuring method for detecting analytes
WO2007069673A1 (fr) * 2005-12-14 2007-06-21 Denka Seiken Co., Ltd. Procede de detection immunochromatographique pour staphylocoque presentant une multiresistance aux medicaments, et kit de diagnostic
JP2010513858A (ja) * 2006-12-15 2010-04-30 キンバリー クラーク ワールドワイド インコーポレイテッド ラテラルフローアッセイデバイス及び当該デバイスを含む吸収性物品
JP2009258095A (ja) * 2008-03-18 2009-11-05 Hitachi Chem Co Ltd クロマトグラフィー分析用ストリップの製造方法
WO2015054546A1 (fr) * 2013-10-10 2015-04-16 Song Diagnostic Research Llc. Dosages à écoulement latéral améliorés
JP2018512882A (ja) * 2015-04-24 2018-05-24 メサ バイオテック，インク． 流体検査用カセット
JP2020020724A (ja) * 2018-08-02 2020-02-06 積水メディカル株式会社 イムノクロマト用試験片及びイムノクロマト検出キット

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