WO2016093134A1 - Dispositif d'analyse de groupage sanguin abo inverse et procédé d'analyse optique de groupage sanguin abo inverse - Google Patents

Dispositif d'analyse de groupage sanguin abo inverse et procédé d'analyse optique de groupage sanguin abo inverse Download PDF

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WO2016093134A1
WO2016093134A1 PCT/JP2015/083953 JP2015083953W WO2016093134A1 WO 2016093134 A1 WO2016093134 A1 WO 2016093134A1 JP 2015083953 W JP2015083953 W JP 2015083953W WO 2016093134 A1 WO2016093134 A1 WO 2016093134A1
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antigen
antibody
chip
abo blood
blood type
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PCT/JP2015/083953
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English (en)
Japanese (ja)
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寅彦 田中
茂之 宇野
誠 槇島
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学校法人日本大学
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Publication of WO2016093134A1 publication Critical patent/WO2016093134A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • 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
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells

Definitions

  • the present invention relates to an ABO blood type uranium inspection apparatus and an optical ABO blood type uranium inspection method.
  • a front test is a test that examines antigens on the surface of red blood cells.
  • an anti-A antibody that reacts with type A or AB and an anti-B antibody that reacts with type B or AB are individually added to red blood cells in the blood to be judged, and the red blood cells aggregate (that is, Whether or not red blood cells make a clump) is visually determined.
  • the anti-A antibody it is type A.
  • the anti-B antibody type B.
  • type AB When aggregation is observed with any antibody, type AB. When it is not seen, it is determined as O type.
  • the back test is a test for examining anti-A antibody and anti-B antibody in serum or plasma.
  • blood type determination A type erythrocytes (A antigen) and B type erythrocytes (B) are used for serum or plasma in blood to be determined (hereinafter, the case where plasma is used will be described as an example).
  • Antigen is added individually, and it is visually determined whether or not these red blood cells are aggregated.
  • anti-B antibodies are present in the blood of type A people
  • anti-A antibodies are present in the blood of type B people
  • anti-A antibodies and anti-B antibodies are present in the blood of type O people. Both exist.
  • type A is used. If aggregation is observed only in type A red blood cells, type B is used. If aggregation is observed in any type of red blood cells, type O is used. When aggregation is not seen, it is determined as AB type.
  • test results of the front test and the back test match. If the test results do not match, there are various possible causes, so blood type determination is suspended and scrutinized. Therefore, in order to smoothly determine the blood type, a quick and accurate front test method and back test method, and a high-sensitivity test apparatus used for these test methods are required.
  • Patent Document 1 discloses an optical waveguide biosensor in which a core region containing an antibody is exposed to the outside through an opening provided in a clad, and a biosensor system including the sensor. .
  • the light incident on the Mach-Zehnder core is branched into light propagating through the two cores in the propagation direction, and the light propagating through one of the cores contacts the opening, and then Once again, the light that has been propagated through the two cores interferes with each other.
  • the presence or absence of the antigen-antibody reaction that is, the antigen in the sample is detected. Presence / absence can be detected with high sensitivity.
  • a general method is to visually determine whether aggregation occurs on a glass slide or in a glass tube such as a test tube.
  • the procedure for stirring plasma and erythrocyte suspension affects the blood type determination result.
  • the aggregation of erythrocytes shows a complicated aspect in which the size of the aggregate varies from sample to sample. The judge visually observes such erythrocyte aggregation visually. Therefore, there has been a problem that a high level of skill is required for blood type determination.
  • the present invention has been made in view of the above circumstances, and can determine the presence or absence of an antigen-antibody reaction by a simple operation without requiring a high level of skill, and has a high S / N ratio.
  • An ABO blood type back examination apparatus and an optical ABO blood type back examination method using the ABO blood type back examination apparatus.
  • An ABO blood type uranium test apparatus includes a chip having a waveguide layer, an antigen fixing surface formed on the surface of the chip, one end fixed to the antigen fixing surface and the other end opened.
  • a determination unit for determining whether or not it has occurred.
  • one end of the antigen is fixed to the antigen fixing surface, that is, the surface of the chip by chemical bonding, and the antigen protrudes from the surface of the chip, and the other end of the antigen is the chip surface.
  • the antigen fixing surface that is, the surface of the chip by chemical bonding
  • the antigen protrudes from the surface of the chip
  • the other end of the antigen is the chip surface.
  • sample an appropriate amount of a sample (hereinafter simply referred to as “sample”) that contains selenium is brought into contact with the antigen, an antigen-antibody reaction between the anti-A antibody or anti-B antibody and the antigen occurs even with a small amount of sample. Therefore, changes in the dip value and dip wavelength of the waveguide mode spectrum distribution before and after the occurrence of the antigen-antibody reaction are detected with a high S / N ratio. Then, the detected information is analyzed by the determination unit, so that the presence or absence of the antigen-antibody reaction in the antigen can be determined quickly and accurately. In this way, an ABO blood type uranium test can be performed with a simple operation without requiring a high level of skill.
  • the antigen-immobilized surface can reduce non-specific reactions with substances other than the anti-A antibody and the anti-B antibody.
  • non-specific reaction between a substance other than anti-A antibody and anti-B antibody and an antigen on the antigen-fixing surface is reduced. Therefore, changes in the dip value, dip wavelength, etc. of the waveguide mode spectrum distribution before and after the occurrence of the antigen-antibody reaction are detected with a much higher S / N ratio.
  • an anti-A antibody recognizing substance that is specifically recognized by the anti-A antibody at the other end of the antigen or specific to the anti-B antibody It is an antigen having an anti-B antibody recognition substance (anti-B antibody epitope) recognized by
  • anti-A antibody specifically recognized by anti-A antibody
  • anti-B antibody epitope an antigen having an anti-B antibody recognition substance recognized by
  • “specifically recognized by anti-A antibody” means that anti-A antibody and anti-B antibody are further distinguished from each other and antibodies that react with other antigens are identified and bound only to anti-A antibody. . The same applies to the anti-B antibody.
  • the anti-A antibody recognizing substance or the anti-B antibody recognizing substance is disposed on the entire surface at a distance of the antigen from the chip surface.
  • the anti-A antibody or anti-B antibody in the sample quickly and efficiently recognizes the anti-A antibody recognition substance or anti-B antibody recognition substance, It binds specifically to these and an antigen-antibody reaction occurs. Therefore, the change in the waveguide mode spectrum distribution before and after the occurrence of the antigen-antibody reaction is detected at a higher S / N ratio than when no means for specifically binding is provided.
  • the length of the antigen can be easily adjusted and can be easily synthesized.
  • the anti-A antibody recognizing substance is a substance represented by the following chemical formula (1)
  • the anti-B antibody recognizing substance is represented by the following chemical formula (2 It is a substance represented by
  • R is an arbitrary structure.
  • R is an arbitrary structure.
  • the anti-A antibody recognizing substance or anti-B antibody recognizing substance similar to the sugar chain present on the erythrocyte membrane is provided at the tip of the antigen. Therefore, the anti-A antibody or anti-B antibody in the sample quickly and efficiently recognizes the anti-A antibody recognition substance or anti-B antibody recognition substance.
  • the chip includes silica
  • the antigen-immobilized surface is hydroxysuccinimide ester-dodecanedioic acid-triethoxysilane compound (C12Es) and methoxytriethylene glycol-triethoxy. It is composed of a silane compound (M3EG), and an amino group is arranged at the one end of the antigen.
  • C12Es hydroxysuccinimide ester-dodecanedioic acid-triethoxysilane compound
  • M3EG silane compound
  • an amino group is arranged at the one end of the antigen.
  • one end of the antigen is strongly fixed to the antigen fixing surface by the covalent bond between the amino group arranged at one end of the antigen and the silane compound C12Es.
  • M3EG prevents binding of irrelevant substances (for example, proteins other than anti-A antibody and anti-B antibody in plasma) to the antigen-immobilized surface. Therefore, a change in the dip value and dip wavelength of the waveguide mode spectrum distribution before and after the occurrence of the antigen-antibody reaction is detected with a high S / N ratio and with high reproducibility.
  • the chip includes a flat reflection layer, the waveguide layer in contact with the first plate surface of the reflection layer, and the first plate surface of the reflection layer.
  • a substrate in contact with the second plate surface on the back surface side of the chip, and the surface of the chip is a third plate surface on the back surface side of the surface in contact with the first plate surface of the reflective layer in the waveguide layer. It is characterized by.
  • an antigen fixing surface is provided on the third plate surface of the waveguide layer of the chip, and one end of the antigen is fixed to the antigen fixing surface, and the other end of the antigen is fixed. Is open.
  • the light is incident on the third plate surface of the waveguide layer from the outside of the chip, and the third layer of the reflection layer and the waveguide layer.
  • the wavelength distribution of the intensity of the light reflected from the plate surface and emitted from the chip can be acquired and used for the determination of the presence or absence of the antigen-antibody reaction.
  • the determiner may contact the sample with the antigen in an appropriate amount in a state where the taking of the guided mode spectrum distribution into the determination unit is started in advance.
  • a chip having a waveguide layer, an antigen fixing surface formed on the surface of the chip, one end fixed to the antigen fixing surface and the other end An incident step of making light incident on the chip using an apparatus comprising an antigen that has been released and has an antigen-antibody reaction with an anti-A antibody or an anti-B antibody; and A determination step of determining whether or not an antigen-antibody reaction has occurred in the antigen based on a wavelength distribution of intensity.
  • the determiner performs an incident step and starts taking in the waveguide mode spectrum distribution of the chip.
  • the determination step is performed.
  • the antigen-antibody reaction between the anti-A antibody or anti-B antibody and the antigen occurs as described above, but the nonspecific reaction between the substance other than the anti-A antibody or anti-B antibody and the antigen is reduced. Therefore, changes in the dip value and dip wavelength of the waveguide mode spectrum distribution before and after the occurrence of the antigen-antibody reaction can be detected with a high S / N ratio. In this way, the presence or absence of an antigen-antibody reaction in an antigen can be determined quickly and accurately by a simple operation, without requiring a high level of skill, and the ABO blood type back test can be performed optically. Can be implemented automatically.
  • an ABO blood type uranium test apparatus that can determine the presence or absence of an antigen-antibody reaction by a simple operation without requiring high skill, and has a high S / N ratio, and An optical ABO blood type uranium inspection method using this ABO blood type uranium inspection apparatus is provided.
  • FIG. 6 is a graph showing a waveguide mode spectrum distribution detected before (solid line) and after contacting (dashed line) the plasma of a type A subject with an antigen of an ABO blood type uranium test apparatus in an example of the present invention.
  • FIG. 6 is a graph showing a waveguide mode spectrum distribution detected before (solid line) and after contacting (dashed line) the plasma of a type A subject with an antigen of an ABO blood type uranium test apparatus in an example of the present invention. This is the result when the antigen is sugar chain antigen 20B (B-type sugar chain).
  • FIG. 7 is a graph showing a waveguide mode spectrum distribution detected before (solid line) and after contacting (broken line) the plasma of a B-type subject with the antigen of an ABO blood type uranium test apparatus in an example of the present invention. This is the result when the antigen is sugar chain antigen 20A.
  • FIG. 6 is a graph showing a waveguide mode spectrum distribution detected before (solid line) and after contacting (dashed line) the plasma of a type A subject with an antigen of an ABO blood type uranium test apparatus in an example of the present invention. This is the result when the antigen is sugar chain antigen 20A.
  • FIG. 7 is a graph showing a waveguide mode spectrum distribution detected before (solid line) and after contacting (broken line) the plasma of a B-type subject with the antigen of an ABO blood type uranium test apparatus in an example of the present invention. This is the result when the antigen is sugar chain antigen 20B.
  • FIG. 6 is a graph showing the waveguide mode spectrum distribution detected before (solid line) and after contacting (dashed line) the plasma of an O-type test subject with the antigen of an ABO blood type reverse blood test apparatus in an example of the present invention. This is the result when the antigen is sugar chain antigen 20A.
  • FIG. 6 is a graph showing the waveguide mode spectrum distribution detected before (solid line) and after contacting (dashed line) the plasma of an O-type test subject with the antigen of an ABO blood type reverse blood test apparatus in an example of the present invention. This is the result when the antigen is sugar chain antigen 20B.
  • FIG. 1 is a side view showing an ABO blood type back examination apparatus 1 of the present embodiment.
  • the ABO blood type back examination apparatus 1 includes a chip 2, an antigen fixing surface 10, a prism 16, an antigen 20, a light source unit 30, and a determination unit 40.
  • the fixed end 20 a of the antigen 20 is fixed to the antigen fixing surface 10.
  • the open end 20b of the antigen 20 is open without being fixed to any component on the channel 60 side. When the fixed end 20a is one end, the open end 20b is the other end.
  • the antigen 20 includes a sugar chain antigen 20A and a sugar chain antigen 20B.
  • the chip 2 includes a flat reflective layer 4, a waveguide layer 6 in contact with the first plate surface 4 a of the reflective layer 4, and a substrate in contact with the second plate surface 4 b on the back side of the first plate surface 4 a of the reflective layer 4. 8 and.
  • the reflective layer 4, the waveguide layer 6, and the substrate 8 are configured to include silica (SiO 2 ) as a main component.
  • the reflective layer 4 is made of silicon
  • the waveguide layer 6 and the substrate 8 are made of pure SiO 2 .
  • the width dimension and thickness dimension of the reflective layer 4, the waveguide layer 6, and the substrate 8 are set in consideration of the wavelength band of light propagating to the chip 2 and the type of waveguide mode.
  • the prism 16 is provided in contact with the second surface 2 b of the chip 2. Light incident at an angle ⁇ from the thickness direction of the prism 16 (that is, the direction of the arrow D1) is guided to the chip 2, reflected and refracted, and emitted to the outside of the prism 16.
  • the antigen immobilizing surface 10 is a surface formed by an active molecule for immobilizing the antigen 20 on the first surface 2a (surface) of the chip 2 and an inactive molecule that prevents the binding of non-specific molecules. 2 is formed with a predetermined thickness on the first surface 2a.
  • the first surface 2 a of the chip 2 is a third plate surface 6 a on the back surface side of the surface 6 b in contact with the first plate surface 4 a of the reflective layer 4 in the waveguide layer 6. According to the antigen fixed surface 10, non-specific reaction with anti-A antibody P A and anti-B antibodies P B other than the material in the sample (e.g. protein, lipids, etc.) is reduced.
  • the antigen-immobilizing surface 10 is represented by a hydroxysuccinimide ester-dodecanedioic acid-triethoxysilane compound (C12Es) represented by the following chemical formula (3) and a chemical formula (4) shown below.
  • C12Es hydroxysuccinimide ester-dodecanedioic acid-triethoxysilane compound
  • M3EG methoxytriethylene glycol-triethoxysilane compound
  • M3EG on the antigen-immobilizing surface 10 functions as a nonspecific adsorption-suppressing molecule, the occurrence of a nonspecific reaction on the antigen-immobilizing surface 10 is suppressed.
  • C12Es on the antigen fixing surface 10 is covalently bonded to an amino group arranged at the fixed end 20a of the antigen 20, the fixed end 20a of the antigen 20 is strongly fixed to the antigen fixing surface 10.
  • C12Es is a silane compound having an active group that reacts with a compound having an amino group.
  • M3EG is an inactive silane compound.
  • Antigen 20 when in contact with potentially sample comprising at least one of the anti-A antibody P A and anti-B antibodies P B, anti-A antibody P A and anti-B antibodies P B specifically bind to sugar chains It is an antigen.
  • Antigen 20 is a sugar chain antigen 20A to anti-A antibody recognition substance R A is disposed to the open end 20b anti-A antibody P A specifically binds to recognize, specifically the open end 20b to the anti-B antibody P B and a recognizable anti-B antibodies recognizing substance R carbohydrate antigen 20B which B is disposed.
  • Each anti-A antibody recognition substance R A and anti-B antibodies recognizing substance R B is a substance which recognizes and specifically binds to their corresponding anti-A antibody P A and anti-B antibodies P B is.
  • sugar chain antigens 20A and 20B when the sugar chain antigens 20A and 20B are not distinguished from each other, they are referred to as the antigen 20, and when the sugar chain antigen 20A and the sugar chain antigen 20B are distinguished from each other, they are referred to as the sugar chain antigens 20A and 20B.
  • An amino group is arranged at the fixed end 20a of the antigen 20 (that is, the right end of the page).
  • Open end 20b of the carbohydrate antigen 20A i.e., the plane of the left end
  • the open end 20b of the carbohydrate antigens. 20B, galactose represented by the chemical formula (6) shown below as an anti-B antibody recognizing substance R B (Gal), galactose (Gal) and fucose (Fuc) is arranged.
  • the difference between the anti-A antibody recognizing substance R A and anti-B antibodies recognizing substance R B is a portion surrounded by a double circle in the formula. That is, the tip portion of the anti-A antibody recognition substance R A contrast is GalNAc, the tip of the anti-B antibodies recognizing substance R B is Gal.
  • R is an arbitrary structure.
  • R is an arbitrary structure.
  • the sugar chain antigens represented by the following chemical formulas (7) and (8) are used in this embodiment.
  • the sugar chain antigens represented by the chemical formulas (7) and (8) are examples of the sugar chain antigens 20A and 20B having the structures of the chemical formulas (5) and (6)
  • the sugar chain antigens 20A and 20B are represented by the chemical formula (7).
  • (8) is not limited to the sugar chain antigen.
  • n is an integer of 1 or more.
  • n is an integer of 1 or more.
  • the amino group disposed at the fixed end 20a is covalently bonded to C12Es included in the antigen fixing surface 10. Therefore, the fixed end 20 a of the antigen 20 is stably bonded to the antigen fixing surface 10.
  • GalNAc arranged at the open end 20b of the sugar chain antigen 20A and Gal arranged at the open end 20b of the sugar chain antigen 20A do not bind to either C12Es or M3EG of the antigen fixing surface 10, and the chip 2 Has been released from.
  • the sugar chain antigens 20A and 20B are arranged so as to protrude from the first surface 2a of the chip 2.
  • carbohydrate antigens 20A, 20B anti-A antibody recognition substance R A and anti-B antibodies recognizing substance R B disposed at the open end 20b of the from the first surface 2a, carbohydrate antigens 20A, corresponding to the length of 20B It is arranged all over the distance.
  • the anti-A antibody recognizing substance RA is anti-A if the anti-A antibody P A and the anti-B antibody P B are present in the sample when the sample such as plasma contacts the antigen 20. and distinguishing the antibody P a and anti-B antibodies P B, which specifically binds only the anti-a antibody.
  • anti-B antibodies recognizing substance R B when the same sample is in contact, and distinguish the anti-A antibody P A and anti-B antibodies P B, that specifically bind only with anti B antibody P B.
  • the physiological anti-A antibody recognizing substance RA includes a sphingo having a structure of N-acetylgalactosamine (GalNAc), galactose (Gal), and fucose (Fuc) represented by the chemical formula (5) at one end of the molecule. Examples include glycolipids and glycoproteins.
  • the sugar chain antigens 20A and 20B may be disposed, or both may be disposed.
  • the sample such as blood plasma contains both anti-A antibody P A and anti-B antibodies P B (i.e., when the blood type determination subject is type O) There is. Even in such a case, a possible recognizes and specifically binds to the respective anti-A antibody P A and anti-B antibodies P each anti-B antibody recognition substance B R B and anti-B antibodies recognizing substance R B Therefore, the first surface 2a of the chip 2 may be divided into two or more areas, and the sugar chain antigens 20A and 20B may be individually fixed in each area.
  • Formula (7) as shown in (8), between the amino group of the anti-A antibody recognizing substance R A or anti-B antibody recognition substance R B and the open end 20b of the fixed end 20a of the antigen 20, the intermediate The part is interposed.
  • This intermediate part is a chemical bond or peptide bond (CONH) of oxygen (O) and methylene group (CH 2 ) having a predetermined length.
  • CONH chemical bond or peptide bond
  • O oxygen
  • CH 2 methylene group
  • the length of the antigen 20 is determined by the “predetermined length”, these lengths are not particularly limited.
  • n may be about 8.
  • the light source unit 30 is configured to irradiate the chip 2 with light.
  • a white light source or a white LED that can emit visible light having a wavelength of 300 nm to 800 nm can be used as the light source unit 30.
  • the determination unit 40 determines whether an antigen-antibody reaction has occurred in the antigen 20 based on the waveguide mode spectrum distribution incident on the chip 2 and emitted from the chip 2.
  • a detection unit such as a photo detector for detection and a computer (not shown) for analyzing changes in the waveguide mode spectrum distribution are provided.
  • the optical ABO blood type uranium test method of this embodiment includes an initial state, an incident step, a cleaning step, and a determination step.
  • FIG. 1 illustrates light reflected by the antigen fixing surface 10 among the reflected light. Further, according to this step, when light is incident on the chip 2, the light propagates inside the waveguide layer 6 in the plate surface direction (arrow D ⁇ b> 2 direction).
  • the detection unit of the determination unit 40 detects the waveguide mode spectrum distribution of the light emitted from the chip 2 in the above-described initial state before bringing the sample into contact with the antigen 20.
  • the PBS on the antigen 20 is removed and the sample is brought into contact.
  • the contact time is, for example, about 5 minutes to 10 minutes.
  • This step for example, if it contains anti-A antibodies P A in the sample, after contacting the sample to carbohydrate antigens 20A, anti-A antibody anti-A antibody P A is out carbohydrate antigens 20A antigen 20 It specifically binds to the recognition substance RA .
  • anti-B antibody P B in the sample anti-B antibody P B specifically binds to an anti-B antibody recognition substance R B antigen 20.
  • FIG. 2 is a graph showing an example of a waveguide mode spectrum distribution of the light emitted from the chip 2.
  • the ABO blood type back test apparatus 1 measures the intensity of light emitted from the chip 2. In an example of this embodiment, the intensity of this light is measured as a reflectance. If at least one of the anti-A antibody P A and the anti-B antibody P B is bound to the antigen 20, the sample before contacting the sample with the antigen 20 as shown in FIG.
  • the wave mode spectrum distribution X changes in the wavelength direction (ie, the direction of the black arrow shown in FIG.
  • the blood type of the sample can be determined by analyzing the change amount of the waveguide mode spectrum distribution by the computer of the determination unit 40.
  • the change threshold is set to 1.5 nm, for example, and if the change is greater than 1.5 nm, an antigen-antibody reaction in the antigen 20 occurs. Can be determined.
  • this threshold value is not limited to 1.5 nm, and is preferably set in advance in consideration of the result of a test using a sample such as a blood sample.
  • the fixed end 20 a of the antigen 20 is fixed to the surface of the chip 2 by the antigen fixing surface 10.
  • the open end 20b of the antigen is separated from the first surface 2a of the chip 2 by a predetermined length, and is arranged over the entire surface in a state of being aligned. Therefore, a small amount of sample can be obtained by bringing an appropriate amount of sample into contact with the antigen 20 in the state in which the light incident on the chip 2 and emitted from the chip 2 is started to be taken into the determination unit 40 of the waveguide mode spectrum distribution.
  • the ABO blood type blood test apparatus 1 includes a chip 2, a plurality of light source units 30, optical paths 52, 54, 64, 66, collimator lens arrays 56, 62, and a polarizing plate. 58 and the determination unit 40 may be integrated into an ABO blood type back examination apparatus 1 ′.
  • sugar antigens 20A and 20B or other antigens / antibodies are selectively arranged on the chip 2 as the antigen 20 (not shown) in contact with the microchannel 68, and the microchannel 68 is arranged. If the sample introduced into is brought into contact, multiple types of blood tests can be performed simultaneously.
  • An ABO blood type back blood test apparatus 1 shown in FIG. 1 and a visible light source capable of emitting visible light of 300 nm to 800 nm were prepared as the light source unit 30.
  • the sugar chain antigens 20A and 20B the sugar chain antigens represented by the chemical formulas (7) and (8) were used.
  • PBS was brought into contact with the antigen 20 of the ABO blood type back examination apparatus 1, and the waveguide mode spectrum distribution in the initial state was measured. Thereafter, PBS was removed, and about 20 ⁇ L of the plasma of a subject with blood type A was brought into contact with antigen 20. And the antigen 20 was wash
  • FIG. 4A and FIG. 4B show before the plasma of the subject A is brought into contact with the antigen 20, that is, after the antigen 20 is brought into contact with the antigen 20 and the antigen 20 is washed with the PBS in the initial state (solid line) in PBS. It is a graph which shows the waveguide mode spectrum distribution detected by (broken line).
  • FIG. 4A shows the results when the antigen 20 is a sugar chain antigen 20A.
  • FIG. 4B shows the results when the antigen 20 is a sugar chain antigen 20B.
  • the plasma contains anti-A antibodies P A is an anti-A antibody P A to specifically recognize and bind to anti-A antibody recognition substance R A carbohydrate antigen 20A.
  • the plasma contains anti-B antibodies P B is an anti-B antibody P B to specifically recognize and bind to anti-B antibodies recognizing substance R B carbohydrate antigens 20B.
  • the waveguide The wavelength change amount of the dip of the mode spectrum distribution was about 1.5 nm.
  • the wavelength change amount of the dip of the waveguide mode spectrum distribution was about 8 nm.
  • 5A and 5B are graphs showing the waveguide mode spectrum distribution detected after the initial state (solid line) and plasma after contacting the antigen 20 and washing the antigen 20 with PBS (broken line).
  • FIG. 5A shows the results when the antigen 20 is a sugar chain antigen 20A.
  • FIG. 5B shows the results when the antigen 20 is a sugar chain antigen 20B. As shown in FIG.
  • the plasma type B subjects contains only anti-A antibody P A
  • the antigen-antibody reaction has occurred.
  • the wavelength change amount of the dip of the waveguide mode spectrum distribution was about 5 nm.
  • the antigen-antibody reaction hardly occurred, and the wavelength change amount of the dip of the waveguide mode spectrum distribution was about 1.5 nm.
  • FIGS. 6A and 6B are graphs showing the waveguide mode spectrum distribution detected after the initial state (solid line) and plasma after contacting the antigen 20 and washing the antigen 20 with PBS (broken line).
  • FIG. 6A shows the results when the antigen 20 is a sugar chain antigen 20A.
  • FIG. 6B shows the results when the antigen 20 is the sugar chain antigen 20B. As shown in FIG.
  • the threshold value of the wavelength change amount of the dip of the waveguide mode spectrum distribution is 1 If the wavelength change of the dip of the guided mode spectrum distribution is 1.5 nm or less, there is no antigen-antibody reaction (ie, negative), and if it is greater than 1.5 nm, there is an antigen-antibody reaction (ie, positive). Then, the correct determination result was obtained in any blood type.
  • the ABO blood type uranium test apparatus 1 it was confirmed that an ABO blood type uranium test can be performed with a simple operation without requiring a high level of skill.
  • the present invention performs blood type back test quickly and easily, for example, in an emergency site such as a large-scale disaster or accident, or in an ambulance. It can be widely used as a portable blood test tool used in the field.
  • ABO blood type back testing apparatus 2 ... chip 6 ... waveguide layer 8 ... substrate 10 ... antigen fixed surface 20 ... antigens 20A, 20B ... carbohydrate antigen P A ... anti-A antibody P B ... anti-B antibody R A ... Anti-A antibody recognition substance R B ... Anti-B antibody recognition substance

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Abstract

La présente invention concerne un dispositif d'analyse de de groupage sanguin ABO inverse (1) qui est pourvu : d'une puce (2), dans laquelle est formée une couche guide d'ondes (6) ; d'une surface d'immobilisation d'antigène (10), qui est formée sur une première surface (2a) de la puce (2) ; d'un antigène (20), dont une extrémité d'immobilisation (20a) est immobilisée sur la surface d'immobilisation d'antigène (10) et une extrémité ouverte (20b) est maintenue non immobilisée, et qui peut provoquer une réaction antigène-anticorps avec un anticorps anti-A (PA) ou un anticorps anti-B (PB) ; d'une section de détermination (40), dans laquelle il est déterminé si une réaction antigène-anticorps se produit ou non au niveau de l'antigène (20) sur la base de la distribution des longueurs d'onde de l'intensité de la lumière qui pénètre dans la puce (2) et qui est émise à travers la puce (2).
PCT/JP2015/083953 2014-12-10 2015-12-03 Dispositif d'analyse de groupage sanguin abo inverse et procédé d'analyse optique de groupage sanguin abo inverse WO2016093134A1 (fr)

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JP2009085714A (ja) * 2007-09-28 2009-04-23 National Institute Of Advanced Industrial & Technology 酸化膜を用いた光導波モードセンサー及びその製造方法
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