WO2009104369A1

WO2009104369A1 - Method of detecting component contained in plasma and reagent and detection device to be used therein

Info

Publication number: WO2009104369A1
Application number: PCT/JP2009/000537
Authority: WO
Inventors: 純子山田; 修平田中; 純子若井
Original assignee: パナソニック株式会社
Priority date: 2008-02-22
Filing date: 2009-02-10
Publication date: 2009-08-27

Abstract

A method of detecting a component contained in plasma by the pulse immunoassay method while preventing a lowering in detection sensitivity caused by the nonspecific aggregation of carrier particles and a reagent which is to be used therein. A reagent containing carrier particles having a diameter of 0.5 to 100 μm, wherein a protein binding specifically to the component to be detected is immobilized on the surface thereof, and arginine ethyl ester serving as a nonspecific aggregation inhibitor in liquids including plasma is used in the pulse immunoassay method. More specifically, the above method comprises preparing a reagent, adding a specimen containing plasma to the reagent to give a mixture solution, applying an alternating voltage to the mixture solution to form pearl chains of the carrier particles, and, after stopping the application of the alternating voltage, observing the aggregation state of the carrier particles using an optical microscope or the like.

Description

Method of detecting component contained in plasma, reagent used therefor and detection device

The present invention relates to a method for detecting components contained in plasma by pulse immunoassay method, and reagents and detection devices used therefor.

As a method of detecting a component contained in a sample, 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.

As a method of promoting the above aggregation reaction, 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 μm) 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.

On the other hand, in

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 μm) 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.
Japanese Patent Application Laid-Open No. 7-83928 JP 2004-108850 A

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). Thus, in conventional latex agglutination methods, 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.

Based on this, the inventor attempted to apply the technology described in Patent Document 2 to a pulse immunoassay method using carrier particles of about 2 μm in diameter that can be identified by an optical microscope. However, regardless of the presence or absence of application of alternating current voltage, 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.

Then, 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. 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.

That is, the first of the present invention relates to the following reagents.

[1] A carrier particle having a diameter of 0.5 to 100 μm, 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.
[2] The reagent according to [1], wherein the diameter of the carrier particle is 0.5 to 10 μm.
[3] 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 reagent according to [1] or [2].
[4] The reagent according to any one of [1] to [3], wherein the protein is an antibody.

The second of the present invention relates to the following detection method.

[5] A method for detecting a component contained in plasma, which comprises: 0.5 to 100 μm 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.
[7] The detection method according to [5] or [6], wherein a concentration of the arginine ethyl ester in the mixed solution is 100 to 500 mM.
[8] The detection method according to any one of [5] to [7], wherein the diameter of the carrier particle is 0.5 to 10 μm.
[9] 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 detection method according to any one of [5] to [8].
[10] The detection method according to any one of [5] to [9], wherein the protein is an antibody.

The third of the present invention relates to the following detection device.

[11] 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 μm in diameter and arginine ethyl ester, immobilized on the surface of a protein to be bound thereto, in the flow path.
[12] The detection device according to [11], wherein the solid substance of the reagent is a lyophilizate of the reagent.
[13] The detection device according to [11], wherein the solid substance of the reagent is an air-dried product of the reagent.

According to the present invention, 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.

It is a schematic diagram of the device used in the Example, FIG. 1A is sectional drawing, FIG. 1B is a top view. It is a block diagram which shows the structure of the measuring apparatus used in the Example. 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, and FIG. 3C is a view after voltage application is stopped. It is a graph which shows the presence or absence of the aggregation suppression effect of each substance in the solution containing plasma. It is a graph which shows the relationship between the density | concentration of arginine ethyl ester and the degree of aggregation in the solution which does not contain plasma. It is a graph which shows the relationship between the density | concentration of arginine ethyl ester and the degree of aggregation in the solution containing plasma. It is a graph which shows the relationship between the density | concentration of arginine ethyl ester and the degree of aggregation in the solution which does not contain plasma. It is a graph which shows the measurement result of the simulated antigen by a pulse immunoassay method. It is a graph which shows the light absorbency in the visible light area | region of carrier particle suspension. It is a photograph which shows the optical microscope image of suspension (VII).

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 A first step of preparing a reagent of the present invention containing carrier particles of ̃100 μm and arginine ethyl ester, and 2) adding a sample containing plasma to the reagent of the present invention to prepare a mixed solution 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 And a fourth step of observing using a distribution meter or the like. In the present specification, "detection" 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.

In the first step, 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. Examples of 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 μm or more which can be identified by an optical microscope or the like, and is preferably 100 μm or less where dispersion by Brownian motion can easily occur, particularly 0.5 to 10 μm. 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 μm, 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. The optimum 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. In addition, 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. When the concentration in the mixed solution is less than 100 mM, nonspecific aggregation of carrier particles can not be sufficiently suppressed. On the other hand, if the 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. Examples of 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. In this case, 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. For example, 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). Although 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.

In addition, 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. As described above, by using 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).

Furthermore, 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.

In the second step, 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. For example, when 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. In addition, when 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.

In the third step, alternating voltage is applied to the mixed solution prepared in the second step to pearl chain 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 ). At this time, if a component to be detected is present in the mixed solution, the carrier particles bind to other carrier particles via this component, and a plurality of carrier particles aggregate (principle of pulse immunoassay method). In the detection method of the present invention, 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. In the examples described later, when arginine ethyl ester was added, 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). When a substance other than arginine ethyl ester was added, 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).

In addition, 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. Thus, by disposing the reagent of the present invention in advance in the flow channel, 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.

In the fourth step, after the application of the alternating voltage is stopped, the aggregation state of the carrier particles is observed using image recognition, a particle size distribution analyzer, or the like.

When the application of the alternating voltage is stopped, 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. At this time, 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. In addition, 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). For example, 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).

As described above, 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 μm, 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.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by these examples.

Example 1
[Effect to suppress nonspecific aggregation of arginine ethyl ester in solution containing plasma]
In this example, aggregation of the amino acid ester (arginine ethyl ester, glycine ethyl ester, lysine ethyl ester, arginine amide, asparagine amide, methionine amide, valine amide) and polyamine (spermidine) against nonspecific aggregation in a solution containing plasma It was confirmed whether it could function as an inhibitor. In this example, polystyrene particles on which an anti-HbA1c antibody was immobilized was used as a carrier particle. Moreover, the antigen was not added to the solution (sample) containing plasma.

1. Modification of carrier particles A suspension (1.5 × 10 ¹⁰ particles / mL) of polystyrene particles (Bangs Laboratories) with a diameter of 2.06 μm was treated with a test tube mixer (TRIO HM-1; As One Corporation) for 1 minute. It stirred above. Moreover, 20 mM glycine buffer (pH 8.6 (same below); Wako Pure Chemical Industries Ltd.) so that final concentration may be set to 0.21 mg / mL of anti-HbA1c antibody (F3A7: refer to Unexamined-Japanese-Patent No. 2003-344397). Solution to prepare an antibody solution. Then, the suspension of polystyrene particles after stirring was added to the antibody solution, and stirred at room temperature for 2 hours to prepare an antibody-modified particle suspension (6.0 × 10 ⁹ / mL). The antibody was immobilized on the surface of polystyrene particles by standing overnight at 4 ° C.

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.

2. Preparation of Reagent of the Present Invention, 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. As 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 μl of 20 mM glycine buffer to 5 μl of plasma (without antigen).

Just before the measurement, the plasma solution is added to the suspension for measurement (the reagent of the present invention), and 10 μL 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.).

3. Device fabrication 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 μm 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, and FIG. 1B is a plan view (cover glass and PET film not shown). As shown in FIG. 1A, 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. As shown in FIG. 1B, 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.

4. Measurement FIG. 2 is a block diagram showing the configuration of the measuring apparatus in the present embodiment. As shown in FIG. 2, a function generator (33120A; Agilent Technologies, Inc.) 200 and a high-speed bipolar power supply (4055; NF circuit design block, Inc.) 210 to control the voltage applied to the device 100 and its frequency and waveform. Are connected to the device 100. In addition, to observe the inside of the flow path 150 of the device 100, an inverted system microscope (IX70; Olympus Co., Ltd.) 220, a camera (C-5060; Olympus Co., Ltd.) 230 and a monitor (TH-15TA2; Matsushita Electric Industrial Co., Ltd.) ) Installed.

The plasma solution was added to the suspension for measurement (the reagent of the present invention), and 1 μL of the mixed solution prepared was provided to the inlet of the channel of the device. 90 seconds after adding the plasma solution to the suspension for measurement (the reagent of the present invention), an AC voltage (100 kHz, 20 Vpp, square wave) 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.

As shown in FIG. 3, before applying the AC voltage, 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. In the present example, no antigen is present in the mixed solution, and therefore, if aggregation of carrier particles occurs, it is considered that all are due to nonspecific adsorption. When the application of the AC voltage is stopped, 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. In this example, carrier particles that exist alone are referred to as "dispersed particles", and particles in which two or more carrier particles are bound are referred to as "aggregated particles".

5. Results 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. When arginine ethyl ester was added, the higher the concentration of arginine ethyl ester, the lower the degree of aggregation, and when the final concentration was 200 to 500 mM, the degree of aggregation became less than 40%. On the other hand, when another substance (glycine ethyl ester, lysine ethyl ester, arginine amide, asparagine amide, methionine amide, valine amide, spermidine) is added, the degree of aggregation does not decrease so much and the degree of aggregation is less than 40%. There was no.

From the above, it was found that non-specific aggregation can be suppressed only when arginine ethyl ester is added to a solution (specimen) containing a high proportion (50%) of plasma so that the final concentration is 200 to 500 mM. On the other hand, it was also found that adding an amino acid ester or polyamine other than arginine ethyl ester to a solution (sample) containing plasma can not suppress nonspecific aggregation.

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.

1. Modification of Carrier Particles In the same manner as in Example 1, carrier particles similar to Example 1 were prepared.

2. Preparation of mixed solution Trehalose, arginine ethyl ester and carrier particles (made in 1 above) are added to glycine buffer (pH 8.6) to prepare a suspension for measurement, and 20 mM glycine buffer just before measurement 4.75 μL was added to prepare 10 μL of mixed solution (final concentration: glycine buffer 100 mM, trehalose 40%, arginine ethyl ester 0-200 mM, carrier particles 6 × 10 ⁸ / mL). The final concentration of arginine ethyl ester was either 0 mM, 5 mM, 10 mM, 50 mM, 100 mM, 150 mM, or 200 mM. The pH of the mixed solution was adjusted to pH 8.6 using sodium hydroxide.

3. Production of Device A device similar to Example 1 was produced according to the same procedure as Example 1 (see FIG. 1).

4. Measurement The same apparatus as in Example 1 was used for measurement (see FIG. 2).

Glycine buffer was added to the suspension for measurement, and 1 μL of the mixed solution prepared was provided to the inlet of the channel of the device. 90 seconds after adding the glycine buffer to the suspension for measurement, an AC voltage (100 kHz, 20 Vpp, square wave) was applied between the electrodes of the device for 60 seconds to pearlize the carrier particles. Before applying the voltage, while applying the voltage, 30 seconds after stopping the application of the voltage, the aggregation state of the carrier particles in the flow channel was observed, and the degree of aggregation at each time point was calculated (n = 3 (0-50 mM), n = 1 (100-200 mM)).

5. Results 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. As shown in FIG. 5, 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.

From the above, it was found that in a solution (specimen) free of plasma, arginine ethyl ester promotes nonspecific aggregation rather than showing an inhibitory effect on nonspecific aggregation.

The reason why 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.

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. . However, when arginine ethyl ester is added, the carrier particles become energetically difficult to disperse as arginine ethyl ester dehydrates the antibody (ie, salting out with arginine ethyl ester). As a result, 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).

On the other hand, 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.

When arginine ethyl ester is not present in a solution containing plasma, carrier particles are nonspecifically aggregated by plasma components (which are mainly considered to be proteins). However, when arginine ethyl ester is added, 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. As a result, when arginine ethyl ester is not added, the state is most difficult to disperse, and it is considered that nonspecific aggregation is suppressed as arginine ethyl ester is added up to a certain level (FIG. 4 and described later) See Figure 6). Here, if arginine ethyl ester is further added to a solution in which nonspecific aggregation is suppressed by arginine ethyl ester, arginine ethyl ester will be present in excess, as a result, as in the case of a solution containing no plasma. It is considered that non-specific aggregation occurs due to the salting out effect of arginine ethyl ester.

Example 2
[Effect to suppress nonspecific aggregation of arginine ethyl ester in solution containing plasma]
In this example, even if an antibody (anti-myoglobin antibody) different from Example 1 was used, it was confirmed whether arginine ethyl ester can function as an aggregation inhibitor against nonspecific aggregation in a solution containing plasma.

1. Modification of Carrier Particles A suspension of polystyrene particles with a diameter of 2.06 μm (1.5 × 10 ¹⁰ / mL) was stirred using a test tube mixer for 1 minute or more. In addition, an anti-myoglobin antibody (home-made) was dissolved in 20 mM glycine buffer to a final concentration of 0.21 mg / mL to prepare an antibody solution. Then, the suspension of polystyrene particles after stirring was added to the antibody solution, and stirred at room temperature for 2 hours to prepare a suspension of polystyrene particles (6.0 × 10 ⁹ particles / mL). The antibody was immobilized on the surface of polystyrene particles by standing overnight at 4 ° C.

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.

2. Preparation of Reagent of the Present Invention, 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 μl of 20 mM glycine buffer to 5 μl of plasma (without antigen).

Just before measurement, plasma solution is added to the suspension for measurement (the reagent of the present invention), and 10 μL 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.

The plasma solution was added to the suspension for measurement (the reagent of the present invention), and 1 μL of the mixed solution prepared was provided to the inlet of the channel of the device. 90 seconds after adding the plasma solution to the suspension for measurement (the reagent of the present invention), an AC voltage (100 kHz, 20 Vpp, square wave) is applied for 60 seconds between the electrodes of the device to pearlize the carrier particles I did. Before applying the voltage, while applying the voltage, 30 seconds after stopping the application of the voltage, the aggregation state of the carrier particles in the flow channel was observed, and the degree of aggregation at each time point was calculated (n = 1).

5. Results 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, and the vertical axis shows the degree of aggregation. As shown in FIG. 6, the higher the concentration of arginine ethyl ester, the lower the degree of aggregation before and after voltage application, and when the final concentration is 100 mM or more, the degree of aggregation before and after voltage application is less than 40%. became. In particular, when the concentration of arginine ethyl ester is 200 mM, the degree of aggregation before and after the application of voltage becomes less than 20%.

From the above, even if the type of antibody is changed, arginine ethyl ester is high against nonspecific aggregation in a solution (specimen) containing a high proportion (50%) of plasma as in the result of Example 1. It turned out to show the suppression effect.

Comparative Example 2
[Effect of suppressing nonspecific aggregation of arginine ethyl ester in a solution not containing plasma]
In this comparative example, 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.

1. Modification of Carrier Particles In the same manner as in Example 2, carrier particles similar to Example 2 were prepared.

2. Preparation of mixed solution Trehalose, arginine ethyl ester and carrier particles (as prepared in 1 above) are added to glycine buffer to prepare a suspension for measurement, and 4.75 μl of 20 mM glycine buffer is added just before measurement. Then, 10 μL 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.

Glycine buffer was added to the suspension for measurement, and 1 μL of the mixed solution prepared was provided to the inlet of the channel of the device. 90 seconds after adding the glycine buffer to the suspension for measurement, an AC voltage (100 kHz, 20 Vpp, square wave) was applied between the electrodes of the device for 60 seconds to pearlize the carrier particles. Before applying the voltage, while applying the voltage, 30 seconds after stopping the application of the voltage, the aggregation state of the carrier particles in the flow channel was observed, and the degree of aggregation at each time point was calculated (n = 1).

5. Results 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. As shown in FIG. 7, as the concentration of arginine ethyl ester is higher, the degree of aggregation before and after the voltage application is higher.

From the above, even if the type of antibody is changed, as in the result of Comparative Example 1, in a solution (specimen) free of plasma, arginine ethyl ester does not show an aggregation inhibitory effect on nonspecific aggregation, Conversely, it was found to promote nonspecific aggregation.

[Example 3]
[Measurement by pulse immunoassay method using arginine ethyl ester]
In this example, after adding arginine ethyl ester as an aggregation inhibitor against nonspecific aggregation to a sample (plasma), it was confirmed whether or not measurement by the pulse immunoassay method can be appropriately performed. In this example, polystyrene particles on which an anti-HbA1c antibody was immobilized was used as a carrier particle. In addition, antigen (home-made pseudo antigen) was added to plasma (sample).

2. Preparation of Reagent of the Present Invention, Plasma Solution and Mixed Solution To glycine buffer (pH 8.6), add trehalose (reagent stabilizer, excipient), arginine ethyl ester and carrier particles (prepared in 1 above) 10 μL of the suspension for measurement (the reagent of the present invention) was prepared. The final concentration was 100 mM glycine buffer, 5% trehalose, 100 to 500 mM arginine ethyl ester, and 6 × 10 ⁸ carrier particles / mL. The pH of the measurement suspension (reagent of the present invention) was adjusted to pH 8.6 using sodium hydroxide and hydrochloric acid. Subsequently, the obtained suspension for measurement (the reagent of the present invention) was lyophilized using a vacuum freeze drier (Takara Co., Ltd.).

In addition, a CGG conjugate (see Japanese Patent Laid-Open No. 2003-344397; peptide research corporation) in which 31 molecules of HbA1c epitope are bound to 1 molecule of chicken γ-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.

Just before measurement, 10 μL of a plasma solution containing a pseudoantigen is added to a lyophilized suspension (the reagent of the present invention) (10 μL 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.

4. Measurement The measurement was carried out in the same manner as in Example 1 using the same apparatus as in Example 1 (see FIG. 2), and the degree of aggregation after voltage application was applied to samples in which the concentration of the pseudoantigen and the concentration of arginine ethyl ester were different. Calculated.

5. Results 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, and 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, and the inverse triangles (▼) shows the results at a concentration of arginine ethyl ester of 500 mM. As shown in FIG. 8, regardless of the concentration of arginine ethyl ester, it was observed that the degree of aggregation is low when the concentration of the pseudo antigen is low, and the aggregation degree is higher as the concentration of the pseudo antigen is higher. This means that even in a system to which arginine ethyl ester is added, the concentration of an object to be detected can be measured by a pulse immunoassay method. It was also observed that the higher the concentration of arginine ethyl ester, the more non-specific aggregation of the carrier particles is suppressed.

From the above, 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.

[Reference experiment]
[Comparison of spectrophotometer and light microscope for measurement accuracy of carrier particles]
In this experiment, a spectrophotometer (ultraviolet visible light spectrophotometer system) and an optical microscope (inverted system microscope) were compared for measurement accuracy of carrier particles.

1. Preparation of water suspension of carrier particles A water suspension (10% by weight) of polystyrene particles (Bangs Laboratories) was stirred using a test tube mixer for 1 minute or more. The polystyrene particles used had diameters of 2.06 μm, 1.04 μm, and 0.50 μm. Next, the suspension of polystyrene particles in water after stirring was diluted with ultrapure water, and mixed as needed to prepare suspensions of I to VII shown in the following table.

2. Measurement The absorbance of the suspension of the above I to VII was measured using a UV-visible spectrophotometer system (UV-1600 PC; Shimadzu Corporation). Further, the dispersion state of the above-mentioned suspensions I to VII was observed using an inverted system microscope.

3. Results 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.

As shown in FIG. 9, in the suspension containing particles of 0.5 μm in diameter (I: 1.8 × 10 ⁹ / mL), the dependency on the wavelength was observed, but the suspension containing particles of 1 μm in diameter was used. In the suspension (II: 2.7 × 10 ⁸ particles / mL) and the suspension containing particles 2 μm in diameter (III: 2.8 × 10 ⁷ particles / mL), no dependence on the wavelength was observed. In addition, even in the case of a suspension (IV) containing particles of 1 μm in diameter and particles of 2 μm in diameter, no dependence on the wavelength was observed. When the suspension (IV) is observed with an optical microscope, particles of 1 μm in diameter and particles of 2 μm in diameter can be easily distinguished, and it is confirmed that these particles are dispersed without aggregation. I was able to.

Also, not only a suspension (V) containing particles of 0.5 μm in diameter and particles of 1 μm in diameter but also the same weight, and a suspension containing particles of 0.5 μm in diameter and 1 μm in diameter at a ratio Also in VI), no dependence on the wavelength was observed. However, in the suspension (VII) containing particles of 0.5 μm in diameter and particles of 1 μm in diameter at a ratio of 99: 1, wavelength dependency almost similar to that of the suspension (I) was observed. The concentration of particles of 0.5 μm in diameter and particles of 1 μm in diameter in this suspension (VII) was 1.8 × 10 ⁹ / mL and 2.7 × 10 ⁶ / mL, respectively.

When the absorbance at a wavelength of 650 nm of the suspensions (I) and (VII) was measured, there was a difference of 0.0064 as shown in the following table, but this was not a significant difference. From this, even if a small number of particles aggregate in the suspension (I) containing particles with a diameter of 0.5 μm (even if aggregates occur at a concentration of 2.7 × 10 ⁶ / mL), the spectrophotometric It is speculated that this aggregation can not be detected by measurement with a meter.

On the other hand, when the suspension (VII) is observed by an optical microscope, particles of 0.5 μm in diameter and particles of 1 μm in diameter can be easily distinguished, and these particles are dispersed without aggregation. I was able to confirm. 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 μm in diameter (indicated by arrowheads of a and b) are scattered, and particles of 0.5 μm in diameter are present around the particles at a density sufficient to fill the gap. In addition, particles with a diameter of 1 μm (indicated by arrowheads of a and b) were moving in the direction of the arrow (see FIGS. 10A and 10B for comparison). Thus, by observing with an optical microscope, particles with a diameter of 0.5 μm and particles with a diameter of 1 μm can be easily distinguished, and a small number of particles in the suspension (I) containing particles with a diameter of 0.5 μm This aggregation can be detected even if it aggregates (even if aggregates occur at a concentration of 2.7 × 10 ⁶ cells / mL).

From the above, it is difficult to detect whether or not 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

This application claims the priority based on Japanese Patent Application No. 2008-041885 filed on February 22, 2008. The contents described in the application specification and drawings are all incorporated herein by reference.

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.

[Explanation of the code]
100 Device 110 Glass Substrate 120 Electrode 130 PET Film 140 Cover Glass 150 Channel 160 Inlet 170 Outlet 180 AC Power Supply 200 Function Generator 210 Bipolar Power Supply 220 Optical Microscope 230 Camera 240 Monitor 300 Carrier Particles

Claims

A reagent for detecting components contained in plasma by pulse immunoassay,
A carrier particle having a diameter of 0.5 to 100 μm, on the surface of which a protein that specifically binds to the component is immobilized;
Arginine ethyl ester,
Containing reagents.
The reagent according to claim 1, wherein the diameter of the carrier particle is 0.5 to 10 μm.
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. Item 2. The reagent according to item 1.
The reagent according to claim 1, wherein the protein is an antibody.
A method of detecting a component contained in plasma, comprising:
Providing a reagent comprising carrier particles of 0.5 to 100 μm in diameter immobilized on the surface of a protein that specifically binds to the component, and arginine ethyl ester;
Adding a sample containing plasma to the reagent to prepare a mixed solution;
Applying an alternating voltage to the mixed solution to pearlize the carrier particles;
Detection methods including:
The detection method according to claim 5, further comprising the step of observing the aggregation state of the carrier particles by using an image recognition or a particle size distribution meter after stopping the application of the AC voltage.
The detection method according to claim 5, wherein the concentration of arginine ethyl ester in the mixed solution is 100 to 500 mM.
The detection method according to claim 5, wherein the diameter of the carrier particles is 0.5 to 10 μm.
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. A detection method according to item 5.
The detection method according to claim 5, wherein the protein is an antibody.
A detection device for a pulse immunoassay method, comprising: a substrate, a pair of electrodes disposed on the substrate, and a flow channel provided between the pair of electrodes,
A solid of a reagent containing 0.5 to 100 μm in diameter of carrier particles immobilized on the surface of a protein that specifically binds to a component to be detected, and arginine ethyl ester is contained in the channel Detection device.
The detection device according to claim 11, wherein the solid substance of the reagent is a lyophilizate of the reagent.
The detection device according to claim 11, wherein the solid substance of the reagent is an air-dried product of the reagent.