WO2006011249A1 - 分析装置及び分析方法 - Google Patents
分析装置及び分析方法 Download PDFInfo
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- WO2006011249A1 WO2006011249A1 PCT/JP2004/017840 JP2004017840W WO2006011249A1 WO 2006011249 A1 WO2006011249 A1 WO 2006011249A1 JP 2004017840 W JP2004017840 W JP 2004017840W WO 2006011249 A1 WO2006011249 A1 WO 2006011249A1
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- granular
- ligand
- labeled
- ligands
- analyte
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Classifications
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
Definitions
- the present invention relates to an analysis apparatus and an analysis method such as an analytical test kit used for quantitative analysis, semi-quantitative analysis, or qualitative analysis of an analyte in a sample.
- Analytical methods for detecting or quantifying analytes (analytes) in samples using the specificity of immune reactions have been put into practical use for a long time.
- a porous membrane such as a membrane
- a flow-through method in which a sample or reagent flows in a filtration format perpendicular to the membrane surface or a lateral flow method in which a force is applied to the other end along the membrane surface.
- a lateral flow method in which a force is applied to the other end along the membrane surface.
- the most common immunochromatography method for detecting an analyte includes two types of specific binding substances (ligands such as antibodies) with different specific binding sites for the analyte.
- ligands such as antibodies
- Typical examples include a sandwich method for forming a complex and a competitive method in which an analyte is present in advance at a predetermined concentration with respect to a ligand and competes with the analyte in a sample.
- ligands such as antibodies
- Typical examples include a sandwich method for forming a complex and a competitive method in which an analyte is present in advance at a predetermined concentration with respect to a ligand and competes with the analyte in a sample.
- one is often insoluble in a porous membrane such as a camphor blank, and the other is directly or indirectly labeled as a means for detecting an analyte.
- various enzymes are used as labels. For indirect labeling, it may be necessary to visualize the label
- direct labeling does not require visual processing, and metal sols, non-metal sols, colored latex, dyes, pigments, chemiluminescent substances, fluorescent substances, etc. that can be directly observed visually are used. It has been.
- a detection method other than visual inspection a spectrophotometer (ultraviolet light, visible light, fluorescence) or the like is used.
- Japanese Laid-Open Patent Publication No. 4-351962 discloses a specific binding analysis method capable of performing semi-quantification without diluting a sample.
- This method uses an immunochromatographic technique.
- a specific substance is present in the measurement system and is measured as an index of the analyte by the presence of the specific substance. The result is the same as when the amount of the labeled substance is reduced and the sample containing the analyte is diluted (hereinafter referred to as “dilution effect”).
- the above-mentioned specific substance is disclosed as a regulating phase in JP-A-2002-328130 and JP-A-2002-328129.
- Japanese Patent Laid-Open No. 6-341989 discloses an art having one detection site in which the setting of the detectable analyte concentration (sensitivity) is changed stepwise.
- a so-called unit format is disclosed in which a plurality of ciestrips are arranged in parallel to form a single device.
- a plurality of detection sites are present on a single assembly strip, which is not possible with such a unit format, and the analyte in the sample and the label are labeled.
- a semi-quantitative method is disclosed in which a substance reacts sequentially with these detectors by chromatographic transfer.
- Japanese Patent Laid-Open No. 2004-85425 discloses a sandwich method on the upstream side of the judgment region and a prozone phenomenon in principle on the downstream side as a method for avoiding and semi-quantifying the prozone. A detection method combining no competitive methods is disclosed. The same disclosure is disclosed in Japanese Patent Laid-Open No. 2003-161733 and Japanese Patent Laid-Open No. 2002-122599.
- Japanese Patent Publication No. 2003-149244 discloses a reagent and method using a sulfate ester salt or sulfonate anionic surfactant as a prozone phenomenon inhibitor for immune reaction measurement.
- JP-A-2001-83153 discloses an immunochromatographic apparatus. With this device Are labeled Sequentially arranged in sections A, B, and C from the upstream, and labeled antibody that specifically binds to the analyte in section A and forms a sandwich complex only with the antibody immobilized in section B. , Another labeled antibody not related to Category B is placed. The latter labeled antibody binds only to the antibody immobilized in Category C, and the color of Category C is adjusted to be the same as that detected in Category B at the set concentration. Therefore, when the sample solution that contains the analyte is added, the coloring source (labeled antibody) in Category B and Category C differs depending on the labeled antibody in Category A. Measured at, and their comparative ability to quantify the concentration of the analyte!
- Japanese Patent Application Laid-Open No. 10-319018 discloses a method for avoiding the prozone phenomenon by adding an apparatus improvement.
- this method in order to prevent an excessive liquid sample or developing solvent from being supplied to the chromatograph, it is placed upstream from the site where the analyte is detected in the chromatograph, and the analyte can be detected.
- the prozone phenomenon is avoided by adopting a structure with a part that automatically cuts the flow path after a certain time.
- JP-A-8-94618 and JP-A-2000-292427 disclose that a chromatographic apparatus also includes a sample addition unit, a label antibody holding unit, and a determination region force. Different types of antibodies are immobilized at different positions in the determination region, and the antibodies that bind to the two different analytes are bound to the labeled antibody holders with labeled particles of different colors.
- a structure is disclosed in which a labeled antibody is held, and a sample that contains two types of analytes is added to the sample addition section and developed.
- two lines with different colors appear as a result of detection of the objects to be analyzed at different positions. This makes it possible to visually detect a plurality of analytes with different color tones at the same time using a single device, making it possible to make a clear determination without making a mistake.
- Japanese Unexamined Patent Application Publication Nos. 2002-303629 and 2004-132892 disclose these Unlike in Japanese Patent Application Laid-Open No. 8-94618 and Japanese Patent Application Laid-Open No. 2000-292427, two types of antibodies in the determination region are fixed at the same position and a plurality of analytes are simultaneously detected by one apparatus. Is disclosed. In this method, when a sample containing two types of analytes is developed, labeled antibodies of different colors are specifically bound to the same position in the determination region. As a result, depending on the abundance ratio of the two types of analytes, Since the coloring color, freshness, or brightness of the judgment part is different, this is measured by the difference in optical characteristics, that is, visually or spectroscopically at each absorption wavelength.
- Patent Document 1 discloses a label complex composition for immunochromatography that is useful for detecting an analyte in a wide range of samples by the immunochromatography method. This is because when a sample containing a labeled antibody and an analyte is developed against a stationary phase in which a specific binding substance that can bind to the analyte is immobilized on an immunochromatographic test strip, This is a solution that solves this problem because the complex between the labeled antibody and the analyte is formed when there is an excess of the product, and then the resulting complex forms aggregates and chromatographic development does not occur normally.
- a composition comprising two or more labeled complexes (labeled antibodies) in which an antibody against an analyte is bound to a labeling substance, and the detection sensitivity of these antibodies to the analyte is different from each other. Yes.
- This makes it possible to detect the analyte even when the amount of the analyte is small. Even if the amount of the analyte is large, the development of the complex becomes good, so that it is possible to detect the analyte in a wide range with high reliability.
- Patent Document 1 Japanese Unexamined Patent Publication No. 2003-107090
- the present invention provides a granular form in which a granular label and a ligand are bound, with the signal and particle size as the selection condition for the granular label, and the reactivity such as detection sensitivity as the selection condition for the ligand. It uses a labeled ligand and has the following characteristics.
- the analyzer according to the invention of claim 1 is a deviation from the following (A)-(G) in which one granular label is bound to one kind of ligand that specifically binds to the analyte. Two or more of these granular labeled ligands are supported on a water-absorbing material for developing an analyte in a state in which the reactivity in each concentration band of the analyte is regulated.
- the invention according to claim 2 is characterized in that two or more kinds of granular labeled ligands according to claim 1 are provided on the upstream side of the water-absorbing material that is infiltrated and developed by the sample liquid containing the analyte. It is supported in a state where it can be eluted, and a ligand or an analyte different from the ligand of the granular labeled ligand is immobilized on the downstream side.
- the invention according to claim 3 is such that the sample liquid addition part, the development part and the result measurement part are long so that the sample liquid added to the sample liquid addition part infiltrates from the development part toward the result measurement part.
- the two or more kinds of granular labeled ligands according to claim 1 are supported in a state in which they can be eluted by the sample liquid in the sample liquid addition part or the development part, and the result labeling part is provided with the granular label. It is characterized in that a ligand or an analyte different from the ligand of the ligand is immobilized.
- the invention described in claim 4 is arranged so that the sample liquid addition part, the permeation part, and the absorption part overlap so that the sample liquid added to the sample liquid addition part infiltrates from the permeation part to the absorption part.
- the two or more kinds of granular labeled ligands according to claim 1 are supported in the sample liquid addition section in a state in which they can be eluted by the sample liquid, and the ligand or analysis target is different from the ligand of the granular labeled ligand in the permeation section. An object is fixed.
- the invention according to claim 5 is the analyzer according to any one of claims 1 to 4, wherein the concentration band of the analysis object has a band accompanied by a prozone phenomenon, One or more of the plurality of granular labeled ligands whose responsiveness in the band is adjusted is characterized in that a signal is emitted in the band accompanied by the prozone phenomenon.
- the invention according to claim 6 is the analyzer according to any one of claims 1 to 4, wherein the concentration band of the analysis object has a concentration that is 100 to 1 million times the detection limit concentration force. It is characterized by a [0024]
- the invention according to claim 7 is the analyzer according to any one of claims 1 to 6, characterized in that the different signals in the particulate marker are color differences.
- the invention according to claim 8 is the analyzer according to any one of claims 1 to 6, characterized in that the different signals in the particulate marker are different absorption wavelengths. .
- the invention according to claim 9 is the analyzer according to any one of claims 1 to 8, wherein the particulate marker is a combination of a signal substance that emits a signal and colorless particles. It is characterized by.
- the invention according to claim 10 is the analyzer according to claim 9, characterized in that the signal substance is an enzyme.
- the invention described in claim 11 is the analyzer according to claim 9, wherein the signal substance is a dye or a fluorescent dye.
- the invention according to claim 12 is the analyzer according to claim 9, characterized in that the colorless particles are white latex or silica particles.
- the invention according to claim 13 is the analyzer according to any one of claims 1 to 8, characterized in that the particulate label is a colored latex particle or a metal colloid.
- the invention according to claim 14 is the analyzer according to any one of claims 1 to 8, characterized in that the ligand that specifically binds to the analyte is an antibody.
- the analysis method of the invention described in claim 15 is the following (A)-(G) in which one granular label is bound to one kind of ligand that specifically binds to the analyte. And using two or more kinds of granular labeled ligands whose reactivity in each concentration band of the analyte is adjusted, and the concentration of the analyte is determined based on the signal of the granular labeled ligand reacted with the analyte. It is characterized by measuring.
- the granular label is selected based on the signal and the particle diameter, the ligand is selected based on the reactivity, and the selected granular label is combined with the ligand.
- the analyte can be easily or semi-quantified in a wide concentration range.
- the present invention provides granular labels that emit the same force or different signals and have the same or different particle diameters, and bind one of the granular labels to one ligand. It uses two or more types of granular labeled ligands, and the granular labeled ligands can be adjusted so as to have reactivity in each concentration band with respect to an analyte that can exist in a wide range of concentrations. Features. In two or more kinds of granular labeling ligands, one ligand having the same reactivity or a ligand having different reactivities is used.
- the analyzer of the present invention is a particulate label of any one of the following (A)-(G) in which one kind of particulate label is bound to one kind of ligand that specifically binds to the analyte.
- Two or more kinds of ligands are characterized in that they are supported on a water-absorbing material for developing an analyte in a state in which the reactivity in each concentration band of the analyte is adjusted.
- the granular labeled ligand used in the present invention is a granular labeled ligand in which one type of ligand is combined with a granular label that emits the same or different signals. Two or more kinds of these granular labeled ligands are used. Each of the granular labeled ligands is adjusted to have different reactivity, and thus when using a granular label that emits the same signal, when analyte is present in the sample, When the analyte is at a low concentration, only a highly reactive, granular labeled ligand forms a Santiago complex at the measurement site, so that a low concentration of analyte can be detected.
- the analyte concentration in the concentration band can be semi-quantified or quantified.
- the granular markers emit different signals, it is possible to easily determine whether the analyte is in the low concentration region or the high concentration region because the signals are different.
- the intensity of the granular label signal in each concentration range the analyte concentration within that concentration range can be semi-quantified or quantified.
- ligands having the same reactivity or different reactivities can be used. There are the following methods for preparing granular labeled ligands having different reactivities using one ligand having the same reactivity.
- a granular labeled ligand to which a certain amount of ligand is bound is combined with a regulating substance such as an inactive substance, an inhibitory substance, or a sensitizing substance in stages, or at the same time or afterwards. It regulates the reactivity of the labeled ligand.
- a regulating substance such as an inactive substance, an inhibitory substance, or a sensitizing substance in stages, or at the same time or afterwards. It regulates the reactivity of the labeled ligand.
- a regulating substance such as an inactive substance, an inhibitory substance, or a sensitizing substance in stages, or at the same time or afterwards. It regulates the reactivity of the labeled ligand.
- a regulating substance such as an inactive substance, an inhibitory substance, or a sensitizing substance in stages, or at the same time or afterwards. It regulates the reactivity of the labeled ligand.
- the regulator ushi serum albumin, casein, skim milk,
- This method results in the force that belongs to the above (1) and (2).
- a condition at the time of binding for example,
- the reactivity of the particulate labeled ligand can be adjusted by adjusting the pH in a timely manner.
- the reactivity of the granular labeled ligand can also be adjusted by using various preparation conditions of the granular labeled ligand.
- the particle size of the granular label and the pore size of the porous support are combined in a combination of the granular label ligand and another ligand or analyte immobilized on a water-absorbing material such as -trocellulose. It utilizes the relationship with roughness (space size within the support) (hereinafter referred to as particle size effect). For example, when a granular label having a particle size large enough not to be clogged when flowing through the space of the porous support is used, the ligand or the analyte on the fixed base side is used.
- the probability of collision (contact) on the light is increased, and a large number of granular labeled ligand complexes are formed there, resulting in an apparently high reactivity.
- the probability is small, and whether a granular labeled ligand complex is formed on the ligand or analyte on the fixed side. Or less, resulting in a less reactive appearance.
- it is a method that utilizes the fact that the reactivity changes depending on whether the passage resistance (through resistance) is high or low.
- ligands having different reactivities are prepared. For example, if the ligand is an antibody, a plurality of types are obtained using a normal antibody production method, and ones with different reactivity (sensitivity) are selected from these, or in the case of antibodies with the same reactivity, The reactivity (sensitivity) of one antibody is changed by partial decomposition by heating or enzyme treatment. Then, the ligands having different reactivity (sensitivity) obtained as described above are combined with particulate labels having different forces or the same signal. As a result, particulate labeled ligands having different or identical signals and different reactivities (sensitivities) can be obtained.
- the immunochromatography method (lateral flow method) can be used as long as the immunoassay method is used.
- a test piece such as a nitrocellulose membrane in which a ligand that is different from the particulate labeled ligand is immobilized is used. From one end of this test piece, a mixture of a granular labeled ligand to be evaluated for reactivity and a sample solution containing an analyte at a predetermined concentration is developed to form a sandwich complex on the fixed part of the membrane, and a granular composite is formed. Measure the presence / absence and strength of the sign signal. By measuring these at various analyte concentrations, a band having reactivity of the granular labeled ligand in a wide concentration band becomes clear.
- sample liquid in the present invention examples include body fluids such as urine, blood, hemolyzed blood, plasma, serum, saliva, sweat, and tears, and other substances to be analyzed for environmental sample power such as food, food, or soil.
- An extract may be mentioned.
- analyte or biologically modified product thereof in the present invention examples include proteins, glycoproteins, antibodies, enzymes, polysaccharides, cells, bacteria and viruses, drugs, chemical substances, and the like.
- the ligand in the present invention is a substance that specifically binds to a substance having a specific structure, and includes an antigen, an antibody, a nucleic acid sequence fragment, an effector molecule, a receptor molecule, an enzyme and its inhibitor, avidin, and piotin. , Sugar chain compounds, lectins and the like.
- the signal in the present invention is measured by absorption or emission at a specific wavelength, and examples thereof include a color visually confirmed in the visible range.
- radioactivity, luminescence, phosphorescence, fluorescence, etc. can be used.
- the bond in the present invention refers to an ionic bond, a covalent bond, a coordinate bond, or the like in physical adsorption and chemical bond.
- the granular labeling substance in the present invention has a signal itself, such as metal colloids such as gold colloid and selenium colloid, colored latex particles colored with dye pigments, fluorescent latex particles colored with fluorescent pigments, Other examples include colored lipid vesicles (ribosomes) and vesicles.
- a signal such as metal colloids such as gold colloid and selenium colloid, colored latex particles colored with dye pigments, fluorescent latex particles colored with fluorescent pigments, Other examples include colored lipid vesicles (ribosomes) and vesicles.
- examples of substances that do not have a signal themselves include white metal colloids, silica particles, white latex, lipid vesicles (ribosomes) and vesicles, and these include signal substances (various dyes, pigments, pigments, By attaching or encapsulating a fluorescent dye or the like, a granular label having a signal can be obtained.
- the analysis apparatus of the present invention uses a water-absorbing material that is infiltrated and developed by a sample liquid containing an analysis target, and the above-described granular labeled ligand is dissolved by the sample liquid upstream of the water-absorbing material. While supported in a possible state, the structure is such that a ligand or analyte different from the ligand of the granular labeled ligand is fixed on the downstream side, and this analyzer can be used for the immunoassay method,
- the Imnoassey method includes a lateral flow method and a slow-through method.
- a sample liquid adding part, a developing part, and a result measuring part are provided in order along the length direction, and the sample liquid added to the sample liquid adding part passes through the developing part.
- a structure in which a sample solution adding part, a developing part and a result measuring part are arranged so as to infiltrate the result measuring part is used.
- the result measurement unit has a standard that specifically binds to the analyte.
- a ligand different from the ligand of the ligand is immobilized, and the labeled ligand is supported on the sample solution addition part or the development part in a state where it can be eluted by the sample liquid.
- the analyzer used for the flow-through method includes a sample liquid addition part, a permeation part, and an absorption part, and the sample liquid added to the sample liquid addition part is infiltrated into the absorption part through the permeation part.
- a structure in which the liquid addition part, the permeation part, and the absorption part are stacked is used.
- a ligand different from the ligand of the labeled ligand, which specifically binds to the analyte, is immobilized on the permeation section, and the labeled ligand is supported on the sample solution addition section in a state where it can be eluted by the sample solution.
- the hydrophilic material used in the development part and the permeation part in the above structure may be any water-absorbing material that easily absorbs an aqueous solution such as a sample solution, an analyte, a labeled ligand, or an immobilized material. Any material that can secure and adjust the time required for sufficient reaction with the ligand can be used. Specific examples include porous membranes such as -trocellulose, nylon, and cellulose acetate, crosslinked dextran, cellulose filter paper, and glass fiber paper. [0060] As the material for the sample solution addition portion, the same hydrophilic material as described above can be used.
- Examples of the material for the absorption section include pulp, high molecular water-absorbing sheet, glass fiber paper, and the like as long as they can rapidly absorb the sample liquid.
- FIG. 1 and FIG. 2 show a test piece as an analyzer used in the lateral flow method.
- the test piece 10 is not a combination of the absorbent body 2, the judgment paper 3, the reagent paper 5, and the sample liquid adding section 6, but each of these members is fixed on the mount 1 having an adhesive surface on one side.
- Reagent paper 5 is arranged at the lower end of the judgment paper 3 with a slight overlap so that the liquid infiltrates and communicates capillary-wide over the entire width
- the sample liquid addition unit 6 is arranged so as to overlap the reagent paper 5. It is installed.
- the absorbent body 2 is disposed on the top of the judgment paper 3 with a slight overlap so that the liquid similarly infiltrates and communicates in a capillary manner.
- the absorber 2 acts as an absorbent for the sample liquid flowing from the sample liquid addition member 6 through the reagent paper 5 and the determination paper 3 by capillary action.
- Reagent paper 5 includes visible granular A-labeled first antibody 7 (see Fig. 6) and granular B-labeled first antibody 8 (see Fig. 8), both of which have the same antibody and differ only in the granular label. These reagents can freely move in the direction of the absorber 2 through the judgment paper 3 in the presence of the sample solution.
- the granular A-labeled first antibody 7 has a structure in which the granular label 7b is bound to the ligand antibody 7a.
- the granular B-labeled first antibody 8 is bound to the ligand antibody 8a.
- the first antibody 7a and the first antibody 8a are the same antibody.
- the second antibody 9 having a specific binding site different from that of the first antibodies 7a and 8a is immobilized on the judgment line portion 4 in the judgment paper 3.
- one end of the sample solution addition member 6 of the test piece 10 is attached to the antigen (analyte).
- a sample solution (not shown) containing
- the sample solution absorbed in the sample solution addition member 6 instantaneously infiltrates into the reagent paper 5, and the granular A-labeled first antibody 7 and the granular B-labeled first antibody 8 carried therein are dissolved in the sample solution. Or disperse and move toward judgment paper 3 together with the sample liquid.
- the particulate labeled first antibody can bind to the antigen while moving toward the determination paper 3.
- the antigen of the bound complex binds to the second antibody 9 immobilized on the determination line part 4 and sandwiches there as shown in FIG. Captured as a complex.
- granular A-labeled first antibody 7 is adjusted for high sensitivity
- granular A-label is adjusted to blue latex particles
- granular B-labeled first antibody 8 is adjusted for low sensitivity
- granular B The label is prepared in red latex particles.
- a blue line derived from the granular A-labeled first antibody 7 appears in the judgment line portion 4, and a schematic diagram as shown in FIG. 6 is obtained.
- the granular A-labeled first antibody 7 adjusted for high sensitivity is not captured as a sandwich complex due to the prozone phenomenon.
- the red granular B-labeled first antibody 8 adjusted for low sensitivity is captured as a sandwich complex, and a red line appears in the judgment line section 4, resulting in a schematic diagram as shown in FIG.
- the judgment line part 4 is mixed with blue granular A-labeled first antibody 7 and red granular B-labeled first antibody 8, The mixed color is a blue-purple to red-purple line.
- Figure 7 schematically illustrates this. Thereby, the concentration of the antigen in the sample solution can be quantified or semi-quantified.
- FIGS. 3 to 5 are flow-through analyzers
- FIG. 3 is a perspective view exploded in the vertical axis direction
- FIG. FIG. 5 is a perspective view exploded in the vertical axis direction
- the reagent paper 21 as the sample liquid adding portion is in contact with the surface so that the liquid is infiltrated in a capillary manner up and down on the determination paper 23 that is the permeation portion.
- an absorber 24 as an absorbing portion that acts as an absorbing material such as a sample liquid penetrating through the reagent paper 21 and then the judgment paper 23 due to a capillary phenomenon from the sample liquid addition portion comes into contact with the surface. It is piled up.
- Reagent paper 21 has two types of visible granular A-labeled first antibody 7 and particles with different granular labels.
- the B-labeled first antibody 8 is supported, and these reagents 25 can freely move to the absorber 24 through the judgment paper 23 in the presence of the sample solution.
- the granular A-labeled first antibody 7 has a structure in which the granular label 7b is bound to the ligand antibody 7a.
- the granular B-labeled first antibody 8 and as shown in FIG. 8, it has a structure in which the granular label 8b is bound to the antibody 8a that is a ligand.
- the first antibody 7a and the first antibody 8a are the same antibody.
- the second antibody 9 having a specific binding site different from that of the first antibodies 7a and 8a is immobilized on the judgment line portion 4 in the judgment paper 3.
- the above laminate is filled into a housing 27 having an open top surface. Then, using a dropper or the like from above the reagent paper 21 in the sample solution addition section, a sample solution (not shown) that may contain an antigen (analyte) is applied.
- the sample solution applied to the sample solution addition section instantaneously infiltrates into the reagent paper 21, and the granular A-labeled first antibody 7 and granular B-labeled first antibody 8 carried thereon are dissolved or dispersed in the sample solution. Then, it penetrates downward toward the judgment paper 23 together with the sample liquid.
- the particulate labeled first antibody can bind to the antigen while moving to the determination paper 23 by force.
- the antigen of the bound complex granular labeled first antibody-antigen
- the second antibody 9 fixed to the spot part 22, and there is a sandwich complex. Captured.
- the granular A-labeled first antibody 7 is adjusted for high sensitivity
- the granular A label is adjusted to blue latex particles
- the granular B-labeled first antibody 8 is adjusted for low sensitivity
- granular B label is prepared in red latex particles.
- the amount of antigen in the sample solution is small
- a blue spot derived from the granular A-labeled first antibody 7 appears in the determination spot portion 22, and a schematic diagram as shown in FIG. 6 is obtained.
- the antigen in the sample solution is present in a large excess
- the granular A-labeled first antibody 7 adjusted for high sensitivity is not captured as a sandwich complex by the prozone phenomenon.
- the red granular B-labeled first antibody 8 adjusted for low sensitivity is captured as a sandwich complex, and a red spot appears in the judgment spot portion 22, resulting in a schematic diagram as shown in FIG.
- the judgment spot part 22 is mixed with blue granular A-labeled first antibody 7 and red granular B-labeled first antibody 8, and the mixture The color is blue-purple power.
- Figure 7 schematically illustrates this.
- the concentration of the antigen in the sample solution can be quantified or semi-quantified.
- an antigen is hCG
- an anti-hCG monoclonal antibody is used as a first antibody and a second antibody that form a sandwich with the hCG. It is possible to use granular dyes, colloidal gold or colored latex as a label. In this embodiment, the description has been made in relation to the sandwich reaction. However, it can be changed to a competitive reaction if necessary.
- test pieces and reagents are not limited to this.
- the selection of the water-absorbing material (porous support) used for the determination is important for creating a granular labeled ligand with different reactivity (detection sensitivity) using one ligand with the same reactivity. It is.
- the adjustment to increase the sensitivity utilizes the particle size effect described above, for example, with respect to the coarseness of the porous support (especially the judgment paper) (spatial area in the support).
- a porous support having a degree of coarseness that does not clog even though the particulate labeling ligand flows through the space is selected.
- the adjustment to lower sensitivity is sufficiently small compared to the pore size of the judgment paper! / Choose a particulate marker with a soot particle size! ⁇ .
- the particulate marker may be selected so that the particle diameter is 50 times to 100 times the particle diameter of the judgment paper.
- the granular labeling ligand adjusted to high sensitivity and the granular labeling ligand adjusted to low sensitivity together with the judgment paper selected by doing so into the analyzer, an analyte present at a low concentration or high concentration can be obtained. It is possible to measure with one instrument and one analytical test.
- Materials for the judgment paper include paper, glass fiber, nitrocellulose, and nylon membrane. And so on. These are readily available from s & s, Millipore, Whatman, Paul, etc. with various pore sizes (nominal pore sizes) as is well known to those skilled in the art. In most cases, the larger the pore size, the faster the liquid infiltration rate (wicking speed or deployment speed), and the wicking speed can be selected as a guideline.
- Examples of the granular label that can be used include gold colloid, selenium colloid, ribosome, dye sol, and colored latex particles.
- colloidal gold and colored latex particles are used as the granular label.
- Colored latex particles that are uniform and have various particle sizes can be easily obtained from Bangs Laboratories, Inc., Seradyn, Inc., JSR Corporation, Merck (also the manufacturer).
- Materials include polystyrene, polyvinyltoluene, styrene acrylate copolymer, styrene butadiene copolymer, etc., and there are functional groups that can be covalently bonded to those that can be physically adsorbed on the particle surface. It can be selected appropriately depending on the ligand.
- This suspension is mixed with 730 g of anti- ⁇ hCG mouse monoclonal antibody, and the mixed suspension is stirred with a rotary mixer at room temperature for 4 hours.
- This suspension is centrifuged at 12000 rpm for 1 hour in a microcentrifuge and the supernatant is decanted.
- Disperse the resulting latex particles in 0.5 mM weight-containing 25 mM HEPES (pH 7.0) buffer solution containing bovine serum albumin 1. Stir with a rotary mixer for 1 hour at room temperature.
- This suspension is microcentrifuge Centrifuge at 12000 rpm for 1 hour, discard the supernatant by decanting, and wash the settled particles three times with 25 mM HEPES (pH 7.0) buffer solution 1. Oml by the above washing method. Finally, disperse in 25 mM HEPES (pH 7.0) buffer containing 0.5% by weight bovine serum albumin and store at 4 ° C until use.
- This suspension is mixed with 700 g of anti- ⁇ hCG mouse monoclonal antibody, and the mixed suspension is agitated by a rotary mixer at room temperature for 4 hours.
- This suspension is centrifuged at 6000 rpm for 30 minutes in a microcentrifuge, and the supernatant is discarded by decantation.
- Disperse the resulting latex particles with 0.5% by weight in 25 mM MES (pH 6.1) buffer containing bovine serum albumin 1.
- Disperse in Oml and stir with a rotary mixer at room temperature for 1 hour.
- pH of colloidal gold of 40 nm is adjusted to 6.0 with lOO mM potassium carbonate aqueous solution, and 2 ml of anti-13-hCG mouse monoclonal antibody is added to 100 ml while stirring. Stir the mixture slowly for 5 minutes, then add 5 ml casein to the mixture. Similarly, gently stir for 5 minutes. The resulting mixture is purified by centrifugation at 5500G for 45 minutes at 4 ° C. Resuspend the pelleted colloidal gold colloid labeled anti- ⁇ -hCG mouse monoclonal antibody pellet in distilled water to 5 ml and store at 4 ° C until use.
- a milk-white polyester mount 1 (250 m thick, 125 mm long, 18 Omm wide, manufactured by Lintec Co., Ltd.), one side of which is coated with adhesive, is fixed on the desk with the adhesive side facing up.
- paste judgment sheet 3 created in (3) above.
- the reagent paper 5 prepared in (4) above is pasted in parallel with the one end.
- the sample solution addition part 6 of 30mm in length and 200mm in width, which also has the power of the trade name “Benize (manufactured by Asahi Kasei)”, is pasted so as to partially cover the reagent paper 5.
- Adsorber 2 is bonded to a glass fiber filter paper having a length of 40 mm and a width of 200 mm made of “AP-20 Millipore” at a position 32 mm from the other end in parallel with the other end.
- the judgment paper 3 and the absorber 2 are connected with an overlap of about 2 mm.
- a protective cover 12 made of a transparent polypropylene film (Lintec Co., Ltd.) 110 mm long and 200 mm wide is pasted with the adhesive side down.
- Fig. 9 shows this laminated state, and this laminated sheet is sequentially cut into strips by a rotary cutter at an interval of 8 mm in width so as to cross each member at right angles. This completes approximately 20 analyzers and stores them in a desiccant bag at room temperature until used in analytical tests.
- 0.1 wt% bovine serum albumin physiological saline solution containing hCG at concentrations of 0, 25, 500, 1000, and 1000000 iu / L was prepared in each test tube. After immersing in a test tube containing hCG of each concentration for about 3 seconds to the position of about 20 mm from one end of the sample solution addition part 6 of the analyzer created as described above, it is pulled up and placed on a horizontal desk, and the judgment line The presence or absence of color development in part 4 and the color tone were observed. The results are shown in Table 1.
- both the high-sensitivity blue latex labeled anti- ⁇ hCG mouse monoclonal antibody and the low-sensitivity red latex labeled anti- ⁇ hCG mouse monoclonal antibody are used.
- the hCG concentration increases in the order of 25 iu 500 iu / L and lOOOiuZL
- the color tone of the judgment line part changes from light blue to dark to blue-violet, maintaining semiquantitativeness, and hCG concentration is maintained.
- the judgment line was red, and it was easily distinguished from the 25iuZL, 500iuZL, and lOOOiuZL concentration ranges by color.
- the concentration of the analyte in the sample solution can be quantified by obtaining the relationship between the color tone of the judgment line and the hCG concentration in advance.
- FIG. 1 is a perspective view showing a state before use of an analyzer used in a lateral flow method.
- FIG. 2 is a perspective view showing a state after use of the analyzer used in the lateral flow method.
- FIG. 3 is an exploded perspective view of an analyzer used for a flow-through method.
- FIG. 4 is a cross-sectional view showing a state before use of the analyzer used in the flow-through method.
- FIG. 5 is a cross-sectional view showing a state after use of the analyzer used in the flow-through method.
- FIG. 6 is a schematic diagram showing a state where blue lines or spots derived from granular A-labeled first antibody appear.
- FIG. 7 is a schematic diagram showing a state where a blue-purple line or spot appears by mixing a blue color derived from a granular A-labeled first antibody and a red color derived from a granular B-labeled first antibody.
- FIG. 8 is a schematic diagram showing a state in which a red line or spot derived from granular B-labeled first antibody appears.
- FIG. 9 is an exploded perspective view of an analyzer used in an example before creation. Explanation of symbols
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WO2014084260A1 (ja) * | 2012-11-28 | 2014-06-05 | 古河電気工業株式会社 | イムノクロマトグラフィー、これに用いられる検出装置および試薬 |
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JP4486059B2 (ja) * | 2006-05-25 | 2010-06-23 | デンカ生研株式会社 | 免疫測定用ラテックス組成物 |
JP4399492B2 (ja) * | 2007-11-30 | 2010-01-13 | 森永乳業株式会社 | 試験容器、試験片、試験キットおよび試験方法 |
JP4980944B2 (ja) | 2008-02-12 | 2012-07-18 | 富士フイルム株式会社 | 免疫学的測定方法 |
JP5100541B2 (ja) * | 2008-07-04 | 2012-12-19 | 古河電気工業株式会社 | 標識粒子として、蛍光粒子と着色粒子とを含有するイムノクロマト法用コンジュゲートパッド、それを用いたイムノクロマト法用テストストリップおよび検査方法 |
JP2011047923A (ja) * | 2009-07-27 | 2011-03-10 | Panasonic Corp | リポソーム組成物及びその製造方法並びにそれを用いたアナライトの分析方法 |
JP5132664B2 (ja) * | 2009-12-07 | 2013-01-30 | 富士フイルム株式会社 | イムノクロマトグラフ方法 |
JP5798720B2 (ja) * | 2010-03-30 | 2015-10-21 | 積水メディカル株式会社 | ヒトc反応性タンパク質(crp)測定用イムノクロマト試薬 |
KR101858765B1 (ko) * | 2010-03-31 | 2018-05-16 | 세키스이 메디칼 가부시키가이샤 | 면역 크로마토그래피를 이용한 측정 방법, 면역 크로마토그래피용 테스트 스트립 및 면역 크로마토그래피용 측정 시약 키트 |
JP2014055773A (ja) * | 2010-12-08 | 2014-03-27 | Kureha Corp | 抗pskポリクローナル抗体並びにそれを用いたpskの免疫学的分析方法およびpskの免疫学的分析用キット |
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JPH11326326A (ja) * | 1998-05-13 | 1999-11-26 | Dainippon Printing Co Ltd | 免疫検査体 |
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