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WO2009104369A1 - Procédé de détection d'un composant contenu dans le plasma et réactif et dispositif de détection destinés à être utilisés dans celui-ci - Google Patents

Procédé de détection d'un composant contenu dans le plasma et réactif et dispositif de détection destinés à être utilisés dans celui-ci Download PDF

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Publication number
WO2009104369A1
WO2009104369A1 PCT/JP2009/000537 JP2009000537W WO2009104369A1 WO 2009104369 A1 WO2009104369 A1 WO 2009104369A1 JP 2009000537 W JP2009000537 W JP 2009000537W WO 2009104369 A1 WO2009104369 A1 WO 2009104369A1
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WIPO (PCT)
Prior art keywords
particles
reagent
carrier particles
ethyl ester
aggregation
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PCT/JP2009/000537
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English (en)
Japanese (ja)
Inventor
純子 山田
修平 田中
純子 若井
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パナソニック株式会社
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Publication of WO2009104369A1 publication Critical patent/WO2009104369A1/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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form

Definitions

  • the present invention relates to a method for detecting components contained in plasma by pulse immunoassay method, and reagents and detection devices used therefor.
  • a "latex aggregation method" using latex particles on which an antibody or the like is immobilized is well known.
  • This method utilizes the fact that latex particles are aggregated due to an antigen-antibody reaction or the like between the component to be detected and an antibody on the surface of the latex particle, and the component to be detected can be detected simply from the degree of aggregation of latex particles. it can.
  • This agglutination reaction proceeds depending on the Brownian movement, and therefore takes some time.
  • a plurality of carrier particles are linearly arranged by applying an alternating voltage to carrier particles (such as latex particles having a diameter of 0.5 to 10 ⁇ m) to generate dielectric polarization (pearl "Chained” "pulse immunoassay” is known (see, for example, Patent Document 1).
  • the pulse immunoassay method can accelerate the agglutination reaction by positively contacting a plurality of carrier particles, so that the target component can be detected more rapidly and with higher sensitivity than the latex agglutination method that relies on Brownian motion. Can.
  • Detection by the latex agglutination method including pulse immunoassay method utilizes an agglutination reaction by specific binding such as antigen-antibody reaction, so nonspecific agglutination of carrier particles (agglutination by non-specific binding not through target component) When it occurs, the detection sensitivity decreases. Such nonspecific agglutination is not seen so much in standard solutions and buffers for biochemical measurement without plasma, and does not pose a major problem. On the other hand, in a solution containing plasma, nonspecific aggregation of carrier particles is remarkably observed, and there is a problem that detection sensitivity is significantly reduced.
  • Patent Document 2 10 mM to 2 M amino acid esters (arginine methyl ester, nitroarginine methyl ester, and the like) for suppressing nonspecific aggregation of carrier particles (latex particles having a diameter of 0.109 ⁇ m) on which an antibody etc. are immobilized.
  • Arginine ethyl ester, glycine ethyl ester, aspartic acid dimethyl ester, lysine methyl ester, lysine ethyl ester, glycine benzyl ester or glycine t-butyl ester) or 10 mM to 2 M polyamine (spermine or spermidine) is disclosed to be effective ing.
  • the conventional latex agglutination method detects the aggregation state of the carrier particles by the “turbidimetric method” that detects transmitted light (absorptivity) or the “specific method” that detects scattered light. It is common to more accurately detect the aggregation state of carrier particles using a microscope (see Patent Document 1).
  • Turbidimetric method that detects transmitted light (absorptivity)
  • specific method that detects scattered light. It is common to more accurately detect the aggregation state of carrier particles using a microscope (see Patent Document 1).
  • it is preferable to use small particles, as dispersion of the particles is dependent on Brownian motion whereas in pulse immunoassay methods, large carrier particles that can be distinguished by light microscopy are used There is a need to.
  • Patent Document 2 attempted to apply the technology described in Patent Document 2 to a pulse immunoassay method using carrier particles of about 2 ⁇ m in diameter that can be identified by an optical microscope.
  • the above-mentioned amino acid ester and polyamine promote the non-specific aggregation rather than suppressing it, and it was found that it can not be applied to the pulse immunoassay method (Comparative Example 1 described later) , 2).
  • the difference between the experimental results described in Patent Document 2 and the experimental results by the present inventor is considered to be 1) the difference in diameter of the carrier particles used, and 2) the difference in the method of measuring the degree of aggregation of the carrier particles. (Refer to the reference example mentioned later).
  • An object of the present invention is to provide a method for detecting a component to be detected by a pulse immunoassay method, and a reagent and a detection device used therefor, while preventing a decrease in detection sensitivity due to nonspecific aggregation of carrier particles.
  • the present inventors repeatedly studied and, surprisingly, in addition to the specific concentration of “arginine ethyl ester”, it further adds “plasma” which is generally considered to promote nonspecific aggregation. It was found that nonspecific aggregation of the carrier particles is suppressed only in the solution containing it.
  • the present inventor further examines, by using a liquid containing “plasma” that is generally considered to promote nonspecific aggregation as a sample, “arginine ethyl ester” of a specific concentration.
  • a liquid containing “plasma” that is generally considered to promote nonspecific aggregation as a sample
  • “arginine ethyl ester” of a specific concentration By adding it, it discovered that the component for a detection object could be detected by a pulse immunoassay method, preventing the fall of the detection sensitivity by non-specific aggregation of a carrier particle, and completed the present invention.
  • the first of the present invention relates to the following reagents.
  • a carrier particle having a diameter of 0.5 to 100 ⁇ m which is a reagent for detecting a component contained in plasma by a pulse immunoassay method, and a protein that specifically binds to the component is immobilized on the surface thereof And a reagent containing arginine ethyl ester.
  • the carrier particles are latex particles, ceramic particles, silica particles, magnetic particles, metal particles, metal coated particles, bentonite, kaolin, gold colloid, red blood cells, white blood cells, platelets, gelatin, liposomes, pollen or microorganisms.
  • the second of the present invention relates to the following detection method.
  • a method for detecting a component contained in plasma which comprises: 0.5 to 100 ⁇ m diameter carrier particles having immobilized on its surface a protein that specifically binds to the component; and arginine ethyl ester Preparing a reagent containing the sample, adding a sample containing plasma to the reagent to prepare a mixed solution, and applying an AC voltage to the mixed solution to pearlize the carrier particles.
  • Detection method [6] The detection method according to [5], further including the step of observing the aggregation state of the carrier particles using image recognition or a particle size distribution meter after stopping the application of the AC voltage.
  • the carrier particles are latex particles, ceramic particles, silica particles, magnetic particles, metal particles, metal coated particles, bentonite, kaolin, gold colloid, red blood cells, white blood cells, platelets, gelatin, liposomes, pollen or microorganisms.
  • the third of the present invention relates to the following detection device.
  • a detection device for pulse immunoassay comprising a substrate, a pair of electrodes disposed on the substrate, and a flow path provided between the pair of electrodes, which is specific to a component to be detected
  • a detection device comprising a solid of a reagent containing carrier particles of 0.5 to 100 ⁇ m in diameter and arginine ethyl ester, immobilized on the surface of a protein to be bound thereto, in the flow path.
  • the solid substance of the reagent is a lyophilizate of the reagent.
  • the solid substance of the reagent is an air-dried product of the reagent.
  • components contained in plasma can be detected with high sensitivity by pulse immunoassay. This makes it possible to conduct a test by pulse immunoassay using blood or plasma as a sample.
  • FIG. 1A is sectional drawing
  • FIG. 1B is a top view
  • FIG. 3A is a schematic view showing the state of carrier particles in the flow channel
  • FIG. 3A is a view before voltage application
  • FIG. 3B is a view during voltage application
  • FIG. 3C is a view after voltage application is stopped.
  • the detection method of the present invention is a method of detecting a component contained in plasma by a pulse immunoassay method, and 1) a diameter of 0.5 where a protein which specifically binds to the component to be detected is immobilized on the surface
  • Step 3) Third step of applying an AC voltage to the mixed solution to pearl chain the carrier particles, 4) Stop the application of AC voltage, and then recognize the aggregation state of the carrier particles by image recognition or particle size
  • a fourth step of observing using a distribution meter or the like includes not only the meaning of examining the presence or absence of a specific substance, but also the meaning of measuring the concentration or amount of a specific substance.
  • a carrier particle in which a protein that specifically binds to a component to be detected is immobilized on the surface, and arginine ethyl ester that functions as a nonspecific aggregation inhibitor in a liquid containing plasma
  • the reagent of the present invention is prepared.
  • Carrier particles are not particularly limited as long as they can immobilize proteins on the surface, and latex particles, ceramic particles, silica particles, magnetic particles, metal particles, metal coated particles, bentonite, kaolin, gold colloid, red blood cells, White blood cells, platelets, gelatin, liposomes, pollen, microorganisms and the like can be used, among which latex particles are preferred.
  • latex particles include polystyrene latex particles, polyvinyl toluene latex particles, polymethacrylate latex particles, metal coated latex particles and the like.
  • the diameter (average particle diameter) of the carrier particles is preferably 0.5 ⁇ m or more which can be identified by an optical microscope or the like, and is preferably 100 ⁇ m or less where dispersion by Brownian motion can easily occur, particularly 0.5 to 10 ⁇ m. Is preferred. As described later, by using an optical microscope, Coulter counter or the like, it is possible to check the aggregation state of the carrier particles more accurately than the measurement using a spectrophotometer (see the reference example). When the diameter of the carrier particles exceeds 100 ⁇ m, the dispersion due to the Brownian movement is less likely to occur, so that it is difficult to observe the aggregation state in the fourth step.
  • the diameter (average particle diameter) of the carrier particles is measured, for example, by observing using an optical microscope, measuring the electrical resistance using a Coulter counter, or measuring the amount of change in scattered light using a light scattering method. can do.
  • the amount (concentration) of carrier particles is preferably in the range of 1 ⁇ 10 6 to 1 ⁇ 10 11 particles / mL in the mixed solution prepared in the second step.
  • concentration of carrier particles in the mixed solution depends on the diameter of the carrier particles used.
  • the protein to be immobilized on the carrier particle is not particularly limited as long as it is a protein that specifically binds to the component to be detected, and antibodies, enzymes, coenzymes (eg, biotin), lectins, glycoproteins, nucleic acids, etc. are used. be able to.
  • proteins instead of proteins, organic compounds such as heme and porphyrins can also be immobilized on carrier particles.
  • the method for immobilizing these proteins on carrier particles is not particularly limited, and may be appropriately selected from methods known to those skilled in the art.
  • the concentration of arginine ethyl ester is preferably in the range of 100 to 500 mM, particularly preferably in the range of 200 to 300 mM, in the mixed solution prepared in the second step.
  • concentration in the mixed solution is less than 100 mM, nonspecific aggregation of carrier particles can not be sufficiently suppressed.
  • concentration in the mixed solution exceeds 500 mM, the conductivity of the mixed solution itself becomes high, and the dielectrophoretic force acting on the carrier particles becomes small when an AC voltage is applied in the third step. The efficiency of chaining is reduced.
  • the reagent of the present invention may contain any substance other than carrier particles and arginine ethyl ester depending on the purpose.
  • the optional substance include, for example, glycine, a protein such as bovine serum albumin (BSA), an inorganic salt such as sodium chloride, a saccharide such as trehalose, an organic compound, a lipid and the like, but it is not particularly limited.
  • the reagent of the present invention may be a liquid (suspension) containing a dispersion medium.
  • the dispersion medium is not particularly limited, but preferably is, for example, GOOD'S buffer such as phosphate buffer, glycine buffer, HEPES buffer, CHES buffer or TRIS buffer.
  • the reagent particles (suspension) of the present invention can be prepared by suspending carrier particles having a predetermined protein immobilized on their surface in these buffers and further dissolving arginine ethyl ester. (See Examples 1 and 2).
  • the pH of the reagent of the present invention is not particularly limited, it is preferably within the range of 6.0 to 11.0 in the mixed solution prepared in the second step, and will be within the range of 6.0 to 9.0. It is particularly preferred to The method of adjusting the pH is not particularly limited, and may be appropriately adjusted using hydrochloric acid or sodium hydroxide.
  • the reagent of the present invention may be a solid (solid substance) containing no dispersion medium, and may be, for example, one obtained by lyophilizing or air-drying the suspension prepared as described above. It is preferable that such a solid (a lyophilizate or an air-dried product of a suspension, etc.) maintains the performance as a reagent of the present invention and that it can be easily dissolved in a solution.
  • a medium that does not contain a dispersion medium it is possible to make the concentration of plasma in the mixed solution 100% in the second step, and the concentration of the test object is kept high without diluting the plasma. be able to. (See Example 3).
  • the reagent of the present invention may be an embodiment (so-called “kit") in which carrier particles and arginine ethyl ester are separated and mixed at the time of use.
  • a sample containing plasma is added to the reagent of the present invention prepared in the first step to prepare a mixed solution.
  • the sample containing plasma is not particularly limited, and examples thereof include whole blood, blood such as plasma and serum, and dilutions thereof.
  • the mixing method of the reagent of the present invention and the sample is not particularly limited, and may be appropriately selected according to the type (liquid or solid) of the reagent of the present invention.
  • the reagent of the present invention prepared in the first step is a suspension
  • an appropriate amount of plasma or the like may be added to the suspension and stirred, and the pH may be adjusted as necessary.
  • the reagent of the present invention is a solid (a lyophilizate of suspension, an air-dried product, etc.)
  • an appropriate amount of plasma etc. is added to the solid and stirred to suspend carrier particles and arginine ethyl ester Etc., and the pH may be adjusted as necessary.
  • the pH of the mixed solution is not particularly limited, but is preferably in the range of 6.0 to 11.0, and particularly preferably 6.0 to 9.0. This is because an antigen-antibody reaction is likely to occur.
  • the method of adjusting the pH is not particularly limited, and may be appropriately adjusted using hydrochloric acid or sodium hydroxide. Further, the temperature of the mixed solution is not particularly limited, but about 25 ° C. is preferable.
  • alternating voltage is applied to the mixed solution prepared in the second step to pearl chain the carrier particles.
  • the carrier particles When an external electric field is applied to the mixed solution, a dipole is induced in the carrier particles, and the interaction of the dipoles causes the carrier particles to migrate (dielectrophoresis), and the carrier particles align in parallel with the electric field direction (pearl chain ).
  • the carrier particles bind to other carrier particles via this component, and a plurality of carrier particles aggregate (principle of pulse immunoassay method).
  • non-specific aggregation is suppressed by adding arginine ethyl ester, so if the component to be detected does not exist in the mixed solution, the aggregation ratio of the carrier particles is suppressed to 40% or less It is possible.
  • the aggregation rate in a solution without an antigen was about 20 to 30%, so it was determined that the aggregation rate of less than 40% was not aggregated ( See Examples 1 and 2).
  • the aggregation rate was 40% or more, and the aggregation inhibitory effect of arginine ethyl ester was the highest among the tested substances.
  • the apparatus and device for applying an alternating voltage to the mixed solution and the method are not particularly limited, and may be the same as the apparatus and method used in the conventional pulse immunoassay method. For example, 1) preparing a detection device having a substrate made of glass or the like, a pair of electrodes disposed to face the substrate, and a flow path provided between the pair of electrodes; 2) Each electrode may be connected to an AC power supply; 3) An AC voltage may be applied between the electrodes after providing the mixed solution prepared in the second step into the flow path from the injection port (see Example).
  • a solid of the reagent of the present invention (such as a lyophilizate or an air-dried product of a suspension) may be disposed in the flow channel of the above-described detection device.
  • the second step can be completed only by providing a sample containing plasma from the inlet to the flow channel, and the detection of the present invention The method can be performed more simply.
  • the waveform of the AC voltage applied to the mixed solution may be a sine wave, a square wave, a square wave, a triangular wave or the like, and may be a continuous wave or a pulse wave.
  • the frequency is not particularly limited, but is preferably in the range of 10 kHz to 10 MHz.
  • the electric field strength of the AC voltage applied to the mixed solution is preferably in the range of 5 to 100 V (peak value) / mm. If the electric field strength is less than 5 V / mm, pearl chain formation hardly occurs and the agglutination reaction can not be sufficiently promoted. On the other hand, if the electric field strength is higher than 100 V / mm, electrolysis of the mixed solution is likely to occur and the detection sensitivity is lowered.
  • the aggregation state of the carrier particles is observed using image recognition, a particle size distribution analyzer, or the like.
  • the non-aggregated carrier particles disperse in the mixed solution by Brownian movement, but the carrier particles aggregated by specific binding are kept in the aggregated state. Therefore, detection of the component to be detected and measurement of the concentration can be performed by determining the ratio (aggregation degree) of aggregated carrier particles to all the carrier particles in the mixed solution.
  • the aggregation state of the carrier particles can be more accurately (one carrier particle (single body), and the like) by observing it using image recognition or a particle size distribution analyzer instead of measuring the absorbance using a spectrophotometer. It can be observed to the extent that it is possible to distinguish between aggregates of two carrier particles (see Reference Example).
  • the method of observing the aggregation state of the carrier particles may be observed using image recognition, a particle size distribution analyzer, or the like.
  • Observation of the aggregation state of the carrier particles by image recognition includes observation by an optical microscope.
  • observation of the aggregation state of the carrier particles using a particle size distribution analyzer includes observation using a Coulter counter or a light scattering method.
  • the type of the optical microscope and the Coulter counter is not particularly limited as long as the aggregation state of the carrier particles can be observed accurately enough to distinguish one carrier particle (single body) and an aggregate of the two carrier particles.
  • the method of calculating the degree of aggregation is not particularly limited and may be appropriately selected according to the observation method (apparatus).
  • the aggregation state of the carrier particles in the mixed solution is detected by a camera (CCD camera etc.) connected to an optical microscope.
  • the degree of aggregation may be calculated from the obtained image by using an image processing program (see Example 1).
  • the detection method of the present invention 1) uses a sample containing plasma, 2) adds arginine ethyl ester that suppresses nonspecific aggregation in a liquid containing plasma, 2) diameter (average particle size 3) using carrier particles in the range of 0.5 to 100 ⁇ m, and 3) observing the aggregation state of the carrier particles using an optical microscope, Coulter counter, etc.
  • the component to be detected can be detected while preventing the decrease.
  • Example 1 [Effect to suppress nonspecific aggregation of arginine ethyl ester in solution containing plasma]
  • aggregation of the amino acid ester arginine ethyl ester, glycine ethyl ester, lysine ethyl ester, arginine amide, asparagine amide, methionine amide, valine amide
  • polyamine spermidine
  • the suspension was centrifuged at 6200 rpm for 15 minutes using a centrifuge (Chibitan-R; Nippon Millipore Corporation), and then the supernatant was replaced with an aqueous solution for washing (20 mM glycine buffer, 0.1% BSA (Sigma)). did. This operation was repeated three times to prepare antibody particles on which carrier particles were immobilized.
  • Plasma Solution and Mixed Solution Trehalose (Wako Pure Chemical Industries, Ltd.), amino acid ester or polyamine, and carrier particles (prepared in the above 1) are added to glycine buffer and suspension for measurement
  • a liquid (reagent of the present invention) was prepared.
  • the amino acid ester any one of arginine ethyl ester, glycine ethyl ester, lysine ethyl ester, arginine amide, asparagine amide, methionine amide and valine amide (all by Sigma, so far) was used.
  • As a polyamine spermidine (Nacalai Tesque, Inc.) was used.
  • a plasma solution was prepared by adding 0.75 ⁇ l of 20 mM glycine buffer to 5 ⁇ l of plasma (without antigen).
  • the plasma solution is added to the suspension for measurement (the reagent of the present invention), and 10 ⁇ L of the mixed solution (final concentration: glycine buffer 100 mM, trehalose 5%, amino acid ester or polyamine 0 to 500 mM, carrier particles 6) ⁇ 10 8 cells / mL) were prepared.
  • the final concentrations of amino acid ester and polyamine were 0 mM, 100 mM, 200 mM, 300 mM, 400 mM and 500 mM for arginine ethyl ester, and 0 mM, 100 mM, 200 mM, 300 mM and 400 mM for other substances.
  • the pH of the mixed solution was adjusted to pH 8.6 using sodium hydroxide and hydrochloric acid (both Wako Pure Chemical Industries, Ltd.).
  • a gold thin film with a film thickness of 100 nm to be an electrode was formed by sputtering through a stencil mask (made of stainless steel) having an electrode pattern on a quartz glass substrate with a thickness of 1 mm. The distance between a pair of rectangular electrodes arranged so that the long sides face each other was 0.5 mm. Then, a 10 ⁇ m thick double-sided adhesive PET film having a flow path pattern is pasted on a glass substrate (and an electrode) so that the flow path portion is located between the electrodes, and 0.3 mm thick on this PET film A cover glass was attached to make a device.
  • FIG. 1 is a schematic view of the fabricated device
  • FIG. 1A is a cross-sectional view
  • FIG. 1B is a plan view (cover glass and PET film not shown).
  • the device 100 used in the present embodiment has a glass substrate 110, a pair of electrodes 120, a PET film 130, a cover glass 140, and a flow path 150 is formed between the pair of electrodes 120. It is done.
  • the flow path 150 has an inlet 160 and an outlet 170, and the pair of electrodes 120 is connected to an AC power supply 180.
  • FIG. 2 is a block diagram showing the configuration of the measuring apparatus in the present embodiment.
  • a function generator 33120A; Agilent Technologies, Inc.
  • a high-speed bipolar power supply 4055; NF circuit design block, Inc.
  • IX70 Olympus Co., Ltd.
  • a camera C-5060; Olympus Co., Ltd.
  • a monitor TH-15TA2; Matsushita Electric Industrial Co., Ltd.
  • the plasma solution was added to the suspension for measurement (the reagent of the present invention), and 1 ⁇ L of the mixed solution prepared was provided to the inlet of the channel of the device.
  • an AC voltage 100 kHz, 20 Vpp, square wave
  • an AC voltage is applied for 60 seconds between a pair of electrodes of the device to carry the carrier particles. Pearl chained. Thirty seconds after the application of the voltage was stopped, the aggregation of the carrier particles in the channel was observed.
  • FIG. 3 is a schematic view showing the state of antibody-modified carrier particles in the flow channel before and after voltage application;
  • FIG. 3A is before voltage application,
  • FIG. 3B is during voltage application (during pearl chain formation), and
  • FIG. 3C is voltage application It shows the situation after stopping.
  • the carrier particles 300 are completely dispersed by Brownian motion (see FIG. 3A), but by applying the AC voltage, the carrier particles 300 become pearl chained. , Contact with other carrier particles 300 (see FIG. 3B). At this time, if an antigen is present in the mixed solution, the carrier particles are bound to other carrier particles via the antigen, so that a plurality of carrier particles aggregate. On the other hand, if no antigen is present, the carrier particles should not bind to other carrier particles, but if nonspecific adsorption occurs, aggregation of the carrier particles will occur even in the absence of the antigen.
  • the non-aggregated antibody-modified carrier particles 300 are redispersed by Brownian motion, but the non-specifically adsorbed antibody-modified carrier particles 300 remain aggregated (see FIG. 3C). ). Therefore, in the present embodiment, the occurrence frequency of nonspecific adsorption was examined by calculating the degree of aggregation after stopping the application of the alternating voltage. The degree of aggregation was calculated by the following equation.
  • the image showing the aggregation state of the carrier particles was analyzed using image processing software ImageJ (US National Institutes of Health (NIH)) to calculate the degree of aggregation.
  • image processing software ImageJ US National Institutes of Health (NIH)
  • carrier particles that exist alone are referred to as “dispersed particles”
  • aggregated particles particles in which two or more carrier particles are bound
  • FIG. 4 is a graph showing the aggregation suppressing effect of each substance.
  • the vertical axis shows the type of substance added, and the horizontal axis shows the concentration of each substance.
  • the hatched segments indicate that the degree of aggregation is 40% or more, and the blackened segments indicate that the degree of aggregation is less than 40%.
  • the degree of aggregation is the degree of aggregation after applying an alternating voltage and once forming a pearl chain, and then stopping application of the alternating voltage and redispersing.
  • Comparative Example 1 [Effect of suppressing nonspecific aggregation of arginine ethyl ester in a solution not containing plasma] In this comparative example, it was confirmed whether arginine ethyl ester can function as an aggregation inhibitor against nonspecific aggregation even in a solution not containing plasma.
  • Example 3 Production of Device A device similar to Example 1 was produced according to the same procedure as Example 1 (see FIG. 1).
  • FIG. 5 is a graph showing the relationship between the concentration of arginine ethyl ester and the degree of aggregation.
  • the horizontal axis shows the concentration of arginine ethyl ester, and the vertical axis shows the degree of aggregation.
  • the higher the concentration of arginine ethyl ester the higher the degree of aggregation before and after voltage application, and if the final concentration is 50 mM or more, the degree of aggregation is 40% or more even before voltage application became.
  • arginine ethyl ester promotes nonspecific aggregation in a solution containing no plasma can be considered to be due to the following mechanism, although it is not limited thereto.
  • the carrier particles When arginine ethyl ester is not present in a solution that does not contain plasma, the carrier particles are likely to be dispersed energetically because the water molecules are hydrated to the antibody immobilized on the carrier particles. .
  • the carrier particles become energetically difficult to disperse as arginine ethyl ester dehydrates the antibody (ie, salting out with arginine ethyl ester).
  • arginine ethyl ester when arginine ethyl ester is not added, dispersion is most likely to occur, and as arginine ethyl ester is added, nonspecific aggregation is considered to occur (see FIG. 5).
  • arginine ethyl ester can suppress non-specific aggregation in a solution containing plasma, which is not limited thereto, but can be considered to be due to the following mechanism.
  • carrier particles are nonspecifically aggregated by plasma components (which are mainly considered to be proteins).
  • plasma components which are mainly considered to be proteins.
  • the arginine ethyl ester interacts with the antibody and plasma components on the surface of the carrier particle, and an electrostatic repulsion effect is formed and an optimal hydration structure is formed, so the carrier particle is energetically It becomes easy to disperse.
  • Example 2 [Effect to suppress nonspecific aggregation of arginine ethyl ester in solution containing plasma]
  • an antibody anti-myoglobin antibody
  • the suspension was centrifuged at 6200 rpm for 15 minutes using a centrifuge, and then the supernatant was replaced with a washing aqueous solution (glycine buffer 20 mM, BSA 0.1%). This operation was repeated three times to prepare antibody particles on which carrier particles were immobilized.
  • Plasma Solution and Mixed Solution Trehalose, arginine ethyl ester and carrier particles (as prepared in 1 above) were added to glycine buffer to prepare a suspension for measurement (reagent of the present invention) .
  • a plasma solution was prepared by adding 0.75 ⁇ l of 20 mM glycine buffer to 5 ⁇ l of plasma (without antigen).
  • plasma solution is added to the suspension for measurement (the reagent of the present invention), and 10 ⁇ L of mixed solution (final concentration: glycine buffer 100 mM, trehalose 5%, arginine ethyl ester 0-200 mM, carrier particles 6 ⁇ 10 8 cells / mL) were prepared.
  • the final concentration of arginine ethyl ester was either 0 mM, 100 mM or 200 mM.
  • the pH of the mixed solution was adjusted to pH 8.6 using sodium hydroxide and hydrochloric acid.
  • Example 3 Production of Device A device similar to Example 1 was produced according to the same procedure as Example 1 (see FIG. 1).
  • the plasma solution was added to the suspension for measurement (the reagent of the present invention), and 1 ⁇ L of the mixed solution prepared was provided to the inlet of the channel of the device.
  • an AC voltage 100 kHz, 20 Vpp, square wave
  • FIG. 6 is a graph showing the relationship between the concentration of arginine ethyl ester and the degree of aggregation.
  • the horizontal axis shows the concentration of arginine ethyl ester
  • the vertical axis shows the degree of aggregation.
  • the concentration of arginine ethyl ester is 200 mM
  • the degree of aggregation before and after the application of voltage becomes less than 20%.
  • Comparative Example 2 [Effect of suppressing nonspecific aggregation of arginine ethyl ester in a solution not containing plasma]
  • the same antibody (anti-myoglobin antibody) as in Example 2 was used to confirm whether arginine ethyl ester can function as an aggregation inhibitor against nonspecific aggregation in a solution containing no plasma.
  • Trehalose, arginine ethyl ester and carrier particles are added to glycine buffer to prepare a suspension for measurement, and 4.75 ⁇ l of 20 mM glycine buffer is added just before measurement. Then, 10 ⁇ L of mixed solution (final concentration: glycine buffer 100 mM, trehalose 40%, arginine ethyl ester 0-200 mM, carrier particles 6 ⁇ 10 8 / mL) was prepared. The final concentration of arginine ethyl ester was either 0 mM, 100 mM or 200 mM. The pH of the mixed solution was adjusted to pH 8.6 using sodium hydroxide.
  • Example 3 Production of Device A device similar to Example 1 was produced according to the same procedure as Example 1 (see FIG. 1).
  • FIG. 7 is a graph showing the relationship between the concentration of arginine ethyl ester and the degree of aggregation.
  • the horizontal axis shows the concentration of arginine ethyl ester, and the vertical axis shows the degree of aggregation.
  • concentration of arginine ethyl ester is higher, the degree of aggregation before and after the voltage application is higher.
  • Example 3 [Measurement by pulse immunoassay method using arginine ethyl ester]
  • arginine ethyl ester as an aggregation inhibitor against nonspecific aggregation to a sample (plasma)
  • polystyrene particles on which an anti-HbA1c antibody was immobilized was used as a carrier particle.
  • antigen home-made pseudo antigen
  • CGG conjugate in which 31 molecules of HbA1c epitope are bound to 1 molecule of chicken ⁇ -globulin (CGG) as a pseudo antigen as plasma antigen is added to plasma.
  • the solution was prepared.
  • the concentration of the mock antigen in the plasma solution was either 1.0 ⁇ 10 ⁇ 15 M, 6.7 ⁇ 10 ⁇ 12 M, 6.7 ⁇ 10 ⁇ 10 M, or 6.7 ⁇ 10 ⁇ 9 M.
  • a plasma solution containing a pseudoantigen is added to a lyophilized suspension (the reagent of the present invention) (10 ⁇ L of the suspension for measurement) to obtain a mixed solution (final concentration: glycine buffer) 100 mM, trehalose 5%, arginine ethyl ester 100-500 mM, carrier particles 6 ⁇ 10 8 / mL, pseudo antigen 1.0 ⁇ 10 ⁇ 15 to 6.7 ⁇ 10 ⁇ 9 M) were prepared.
  • the pH of the mixed solution was adjusted to pH 8.6 using sodium hydroxide and hydrochloric acid.
  • Example 3 Production of Device A device similar to Example 1 was produced according to the same procedure as Example 1 (see FIG. 1).
  • FIG. 8 is a graph showing the results of measurement of pseudoantigens by pulse immunoassay.
  • the horizontal axis shows the concentration of the mock antigen, and the vertical axis shows the degree of aggregation.
  • Circles ( ⁇ ) indicate the results when the concentration of arginine ethyl ester is 100 mM
  • diamonds indicate the results when the concentration of arginine ethyl ester is 200 mM
  • squares ( ⁇ ) indicate the concentration of arginine ethyl ester
  • the results at 300 mM are shown
  • the triangle ( ⁇ ) shows the results at a concentration of arginine ethyl ester of 400 mM
  • the inverse triangles ( ⁇ ) shows the results at a concentration of arginine ethyl ester of 500 mM.
  • arginine ethyl ester has an inhibitory effect on non-specific aggregation even in a mixed solution containing a high proportion (100%) of plasma, and measurement by a pulse immunoassay method is also performed in a system to which arginine ethyl ester is added. It turned out that it was possible.
  • FIG. 9 is a graph showing the absorbance of each suspension in the visible light region.
  • the horizontal axis indicates the wavelength, and the vertical axis indicates the absorbance.
  • FIG. 10 is a photograph showing an optical microscope image of the suspension (VII), and (B) is a photograph taken several seconds after the photograph of (A) was taken. In each of the photographs, particles of 1 ⁇ m in diameter (indicated by arrowheads of a and b) are scattered, and particles of 0.5 ⁇ m in diameter are present around the particles at a density sufficient to fill the gap.
  • particles with a diameter of 1 ⁇ m were moving in the direction of the arrow (see FIGS. 10A and 10B for comparison).
  • particles with a diameter of 0.5 ⁇ m and particles with a diameter of 1 ⁇ m can be easily distinguished, and a small number of particles in the suspension (I) containing particles with a diameter of 0.5 ⁇ m This aggregation can be detected even if it aggregates (even if aggregates occur at a concentration of 2.7 ⁇ 10 6 cells / mL).
  • each carrier particle is aggregated in the measurement of absorbance by a spectrophotometer, but using the optical microscope, each carrier particle is aggregated It can be seen that it can be detected up to
  • the detection method and detection reagent of the present invention are useful as a method of performing a test by pulse immunoassay using blood or plasma as a sample and a reagent used therefor.

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Abstract

L'invention porte sur un procédé de détection d'un composant contenu dans le plasma à l'aide du procédé d'immuno-analyse à impulsion, tout en empêchant une diminution de la sensibilité de détection provoquée par l'agrégation non spécifique de particules porteuses et d'un réactif qui doit être utilisé dans ledit procédé. Un réactif contenant des particules porteuses ayant un diamètre de 0,5 à 100 µm, une protéine se liant de façon spécifique au composant devant être détecté étant immobilisée sur la surface de celles-ci, et de l'ester éthylique d'arginine servant d'inhibiteur d'agrégation non spécifique dans des liquides, y compris le plasma, est utilisé dans le procédé d'immuno-analyse à impulsion. De façon plus spécifique, le procédé ci-dessus consiste à préparer un réactif, ajouter un échantillon contenant du plasma au réactif pour obtenir une solution de mélange, appliquer une tension alternative à la solution de mélange de façon à former des chaînes de perles des particules porteuses, et, après l'arrêt de l'application de la tension alternative, observer l'état d'agrégation des particules porteuses à l'aide d'un microscope optique ou similaire.
PCT/JP2009/000537 2008-02-22 2009-02-10 Procédé de détection d'un composant contenu dans le plasma et réactif et dispositif de détection destinés à être utilisés dans celui-ci WO2009104369A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2287609A1 (fr) * 2008-05-09 2011-02-23 ARKRAY, Inc. Procédé de production de particule porteuse insoluble, particule porteuse insoluble, réactif de mesure, outil d'analyse d'échantillon et procédé turbidimétrique immunologique
CN105203747A (zh) * 2015-09-22 2015-12-30 宁波瑞源生物科技有限公司 一种精氨酸对标记抗体荧光粒子的封闭方法
JP2017222654A (ja) * 2011-07-19 2017-12-21 中外製薬株式会社 アルギニンアミドまたはその類似化合物を含む安定なタンパク質含有製剤

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004108850A (ja) * 2002-09-17 2004-04-08 Eiken Chem Co Ltd 蛋白質の安定化方法
WO2004036194A1 (fr) * 2002-08-02 2004-04-29 Nec Corporation Puce et appareil d'analyse
WO2004111649A1 (fr) * 2003-06-16 2004-12-23 Pulse-Immunotech Corporation Procede de mesure de substance a affinite
WO2007037410A1 (fr) * 2005-09-30 2007-04-05 Pulse-Immunotech Corporation Methode d'analyse d'une substance avec affinite dans un echantillon et qui consiste notamment a detruire l'ingredient de cellule sanguine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004036194A1 (fr) * 2002-08-02 2004-04-29 Nec Corporation Puce et appareil d'analyse
JP2004108850A (ja) * 2002-09-17 2004-04-08 Eiken Chem Co Ltd 蛋白質の安定化方法
WO2004111649A1 (fr) * 2003-06-16 2004-12-23 Pulse-Immunotech Corporation Procede de mesure de substance a affinite
WO2007037410A1 (fr) * 2005-09-30 2007-04-05 Pulse-Immunotech Corporation Methode d'analyse d'une substance avec affinite dans un echantillon et qui consiste notamment a detruire l'ingredient de cellule sanguine

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2287609A1 (fr) * 2008-05-09 2011-02-23 ARKRAY, Inc. Procédé de production de particule porteuse insoluble, particule porteuse insoluble, réactif de mesure, outil d'analyse d'échantillon et procédé turbidimétrique immunologique
EP2287609A4 (fr) * 2008-05-09 2011-05-04 Arkray Inc Procédé de production de particule porteuse insoluble, particule porteuse insoluble, réactif de mesure, outil d'analyse d'échantillon et procédé turbidimétrique immunologique
US9182391B2 (en) 2008-05-09 2015-11-10 Arkray, Inc. Method of producing insoluble carrier particles, insoluble carrier particles, measurement reagent, specimen analyzing tool, and immunoturbidimetric assay
JP2017222654A (ja) * 2011-07-19 2017-12-21 中外製薬株式会社 アルギニンアミドまたはその類似化合物を含む安定なタンパク質含有製剤
US10898572B2 (en) 2011-07-19 2021-01-26 Chugai Seiyaku Kabushiki Kaisha Stable protein-containing preparation containing argininamide or analogous compound thereof
CN105203747A (zh) * 2015-09-22 2015-12-30 宁波瑞源生物科技有限公司 一种精氨酸对标记抗体荧光粒子的封闭方法

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