JP2008185529A - Method and device for detecting substance to be detected - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To rapidly perform the separation operation of a reacted substance and an unreacted substance with high reaction efficiency and to easily identify the signal from a label substance after reaction. <P>SOLUTION: A color developing substance is bonded to a bead carrier by utilizing the specific bond of the substance to be detected in a sample and a bonding substance for recognizing the substance to be detected and, after the color developing substance bonded to the color developing substance bonded to the bead carrier is separated by sedimenting the bead carrier on the bottom part of a container as a sediment by centrifugal separation, the signal originating from the color developing substance in the sediment observed from the outside of the container is used as an index to detect the substance to be detected. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a detection method of a test substance using centrifugation and a detection device used in the detection method.

  A bead carrier is known as one type of carrier used when competitively or non-competitively detecting a test substance in a sample. The bead carrier is advantageous in that the reaction efficiency is better than that of a flat plate such as a 96-well plate, and is used, for example, in the aggregation method. In the agglutination method, a specific binding substance that recognizes a test substance is immobilized on a bead carrier such as polystyrene beads, and the test substance is detected using the aggregation of the bead carrier accompanying the binding of the test substance as an index. For example, in Patent Document 1, an antigenic substance in a test sample is adsorbed or bound to insoluble carrier particles, and after reacting with a monoclonal antibody that specifically reacts with the antigenic substance, the monoclonal antibody is selectively selected. An aggregation immunoassay method has been proposed in which a second antibody to be bound is further reacted to selectively aggregate insoluble carrier particles.

On the other hand, methods other than the agglutination method described above are also known as methods for detecting a test substance using a bead carrier. For example, in Patent Document 2, after performing a specific binding reaction using beads, a method of measuring a target substance in a sample by detecting a fluorescent or luminescent labeling substance bound to the beads, For example, a measurement method is described in which a bead is sedimented on the bottom surface by, for example, centrifugation, irradiated with excitation light while being held on the bottom surface, and the generated fluorescence or luminescence is measured.
JP-A-6-167495 JP 2004-028862 A

  However, in the agglutination method described in Patent Document 1 described above, an antigen that performs a specific reaction needs to have one or more recognition sites. For this reason, there is a problem that a low-molecular biomaterial such as a hormone that cannot take a plurality of recognition sites cannot be detected.

  Moreover, although the above-mentioned Patent Document 2 describes that the beads are settled on the bottom surface by centrifugation, it does not describe the use of a coloring material such as gold colloid particles as a labeling material. In the technique described in Patent Document 2, a luminescent substance or a fluorescent substance is used as a labeling substance. However, there is a disadvantage that a large apparatus is required to read a signal derived from the luminescent substance or the fluorescent substance.

  The present invention has been proposed in view of such a conventional situation, has high reaction efficiency, can perform a separation operation of a reactant and an unreacted substance quickly, and can be performed from a labeled substance after reaction. It is an object of the present invention to provide a method for detecting a test substance capable of easily discriminating a signal. The present invention also relates to a detection device suitable for use in the detection method.

  In order to solve the above-mentioned problems, the detection method according to the present invention uses a specific binding between a test substance in a sample and a binding substance that recognizes the test substance to bind the coloring substance to the bead carrier. The sediment observed from the outside of the container after separating the colored substance bound to the bead carrier from the uncolored substance by separating the bead carrier as a sediment at the bottom of the container by centrifugation. The test substance is detected using a signal derived from the color developing substance therein as an index.

  In the detection method as described above, the labeling color substance bound to the bead carrier and the unreacted labeling color substance are separated by centrifugation, so the respective sedimentation coefficients are set appropriately for the bead carrier and the color substance. By doing so, these simple separations are realized in a short time. Further, by using a bead carrier as a carrier, the reaction efficiency is higher than that of a flat carrier, and therefore the time required for detection is shortened. Furthermore, since a coloring substance that can be visually discriminated is used as the labeling substance, it is effective for quick and simple detection.

  In addition, the detection device according to the present invention includes a reaction unit in which a reaction solution is accommodated, and a transparent separation detection unit that is open at a bottom surface of the reaction unit and has an inner diameter narrower than the reaction unit, and the separation The reaction solution is not contained in the detection unit.

  In order to centrifuge a small amount of sample, a conical microtube is generally used. However, the place where the sediment accumulates is indefinite, and the sediment spreads on the wall. The density of the chromogenic material may decrease, and the signal intensity derived from the chromogenic material may decrease.

  On the other hand, in the detection device of the present invention, since the container in which the separation detection unit is formed is used, when the detection device is centrifuged after the reaction between the sample and the reaction solution, the bead carrier having a large sedimentation coefficient has a narrow inner diameter. The labeling color substance that settles on the separation detection unit and binds to the bead carrier is accumulated at a high concentration on the bottom surface of the transparent separation detection unit. On the other hand, since the sample and the reaction solution are reacted in the reaction part having a larger inner diameter than the separation detection part, sufficient reaction efficiency is ensured. In addition, since no reaction solution is present in the separation detection unit during the reaction, the reaction can be performed in the reaction unit having a wide inner diameter, and the entire reaction solution can contribute to the reaction with the sample. For this reason, the reaction efficiency between the reaction solution and the sample becomes higher. Therefore, it is possible to reduce both the reaction time of the sample and the reaction solution and the time required for the centrifugation, and to improve the color signal intensity derived from the color developing substance in the sediment.

  According to the present invention, the presence / absence or concentration of a test substance can be quickly, easily and clearly known. In addition, the device structure can be simplified because washing for separating the reactants from the unreacted materials is not required, such as ELISA.

  Hereinafter, a detection method and a detection device to which the present invention is applied will be described in detail with reference to the drawings.

  First, the coloring substance is bound to the bead carrier using specific binding between the test substance in the sample and the binding substance that recognizes the test substance. Next, the bead carrier is sedimented as a sediment to the bottom of the container by centrifugation, thereby separating the coloring substance bound to the bead carrier and the unreacted coloring substance. Next, the test substance is detected using a signal derived from the coloring substance in the sediment observed from the outside of the container as an index.

  In the present invention, for example, any substance such as a small molecule such as a hormone or a virus such as a phage can be used as a test substance. As the binding substance that recognizes the test substance, a substance that specifically binds to the test substance may be appropriately selected. Examples of the combination of the test substance and the binding substance include an antigen-antibody combination. The antibody used as the binding substance may be either a monoclonal antibody or a polyclonal antibody. In addition, as the combination of the test substance and the binding substance, the effect of the present invention can be obtained in the same manner as the antigen-antibody combination as long as it is a substance that specifically binds to the test substance. Examples include nucleic acid-nucleic acid, nucleic acid-nucleic acid binding protein, lectin-sugar chain, or receptor-ligand combination. The order of the test substance-binding substance relationship may be reversed.

  As the color former, a known color substance used as a labeling substance can be used without limitation. For example, metal fine particles such as gold, platinum, silver, and colloidal particles thereof can be used. Among these, colloidal gold particles are preferable because a clear color can be obtained.

As the bead carrier, a bead carrier having a larger sedimentation coefficient than the labeling coloring substance can be used. In order to shorten the time required for the centrifugation, it is preferable to use a bead carrier having a sufficiently larger sedimentation coefficient than the labeling coloring material. Specifically, it is preferable to use a bead carrier having a sedimentation coefficient larger than 10 times the sedimentation coefficient of the coloring substance. For example, it is preferable to use a bead carrier with s (sedimentation coefficient) = 1 × 10 ⁻¹¹ or more.

However, if the sedimentation coefficient of the bead carrier is made larger than that of the color developing substance, inconvenience such as sedimentation due to its own weight in the reaction solution occurs. For this reason, it is preferable that the bead carrier is kept floating in the reaction solution. In addition, since the reaction efficiency when the test substance is bound to the bead carrier using specific binding is increased, a bead carrier that performs Brownian motion in the reaction solution is more preferable. (Sedimentation coefficient) = 1 × 10 ⁻⁹ or less bead carrier.

  As a material constituting the bead carrier, a material used for this kind of bead carrier can be used without limitation. For example, latex beads such as polystyrene, silica beads, glass beads and the like can be exemplified. In particular, the bead carrier is preferably made of a transparent material. When a bead carrier made of a transparent material is used, the color developing substance in the sediment can be seen through the bead carrier, so that a strong color intensity can be obtained and the color development can be easily confirmed. A bead carrier composed of a transparent material having a particle size of 70 nm or less is a preferable material because its aggregate is also transparent.

  From the viewpoint of improving reaction efficiency, the particle size of the bead carrier is preferably 5 μm or less. This is because if the particle size exceeds 5 μm, the efficiency of a specific binding reaction such as an antigen-antibody reaction is poor, and the reaction time cannot be shortened. For example, in the case of polystyrene, the thickness is preferably 500 nm to 5 μm, and more preferably 1 μm to 3 μm. In the case of silica beads having a specific gravity greater than that of polystyrene, the thickness is preferably 50 nm to 100 nm.

  Either the competitive method or the sandwich method may be used as the step of binding the coloring substance to the bead carrier by using the specific binding between the test substance in the sample and the binding substance that recognizes the test substance.

  The conditions for centrifugation may be such that the bead carrier to which the labeling substance is bound by the specific binding reaction can be separated from the unreacted coloring substance label-binding substance. Such centrifugation conditions vary as appropriate depending on the amount of sample and the centrifugation time, and can be, for example, 2000 to 6000 g.

  In order to distinguish a signal derived from a coloring substance in the sediment, it is easiest to use visual observation. For example, a known reading device such as an absorptiometer or a densitometer may be used to discriminate a signal derived from a coloring substance. In this case, too, for example, when a signal derived from a luminescent labeling substance or a fluorescent labeling substance is read. In comparison, there is an advantage that a small and simple device can be used.

  Hereinafter, as an embodiment of the detection method of the present invention, a detection method using a competition method will be described with reference to FIG. The competition method is an effective method when a low-molecular compound having no plurality of recognition sites is used as a test substance.

  First, a sample solution containing a test substance and a solution containing an antibody labeled with colloidal gold particles (gold colloid labeled antibody) are prepared. The antibody used for the colloidal gold labeled antibody is a monoclonal antibody that recognizes a test substance. These solutions are mixed and reacted. When an antigen as a test substance is contained in the sample solution, the test substance and the colloidal gold labeled antibody are specifically bound by the antigen-antibody reaction.

  On the other hand, a solution containing a bead carrier (polystyrene beads) on which a test substance is immobilized is prepared. The solution containing the bead carrier on which the test substance is immobilized is mixed with the solution after the antigen-antibody reaction. Then, the unreacted substance with the test substance in the gold colloid-labeled antibody specifically binds to the test substance on the bead carrier by the antigen-antibody reaction and binds to the bead carrier. On the other hand, the colloidal gold-labeled antibody (gold colloid-labeled antibody-test substance complex) already bound with the test substance does not react with the test substance on the bead carrier and remains dispersed in the solution. .

  When the subsequent solution is centrifuged under appropriate conditions (eg, 3250 g, 15 seconds), the colloidal gold-labeled antibody-test substance complex that is not bound to the bead carrier remains dispersed in the supernatant, while The colloidal gold-labeled antibody bound to the bead carrier via the test substance settles together with the bead carrier at the bottom of the container, and these are separated. This sediment is observed from the outside of the container, for example, visually. At this time, since the competitive method is used, the higher the concentration of the test substance in the sample solution, the smaller the amount of colloidal gold particles in the sediment and the weaker the color development intensity. Therefore, the test substance in the sample solution can be detected qualitatively using the color development intensity of the obtained sediment as an index. Further, by obtaining the correlation between the test substance concentration and the color development intensity in advance, the test substance in the sample solution can be quantitatively detected.

  Hereinafter, as a second embodiment of the detection method of the present invention, a detection method using a sandwich method will be described with reference to FIG.

  First, as in the first embodiment, a sample solution containing a test substance and a solution containing a gold colloid-labeled antibody are prepared and mixed.

  On the other hand, a second specific binding substance (second antibody) that specifically binds to the test substance and binds to the antigen at a recognition site different from the colloidal gold labeled antibody is prepared. A solution containing the immobilized bead carrier is prepared. The solution containing the bead carrier on which the second antibody is immobilized is mixed with the solution after the antigen-antibody reaction. The colloidal gold labeled antibody and the bead carrier may be reacted with the sample at the same time. Then, the gold colloid labeled antibody (gold colloid labeled antibody-test substance complex) to which the test substance is bound specifically binds to the second antibody on the bead carrier by the antigen-antibody reaction, and binds to the bead carrier. On the other hand, the unreacted colloidal gold labeled antibody does not react with the second antibody on the bead carrier, and is maintained in a dispersed state in the solution.

  When this is centrifuged under appropriate conditions (for example, 3250 g, 15 seconds), the colloidal gold-labeled antibody not bound to the bead carrier remains dispersed in the supernatant, while the second antibody is passed through the bead carrier. The bound gold colloid-labeled antibody-test substance complex settles together with the bead carrier at the bottom of the container. This precipitate is observed, for example, visually from the outside of the container. Since the sandwich method is used, the higher the concentration of the test substance in the sample solution, the greater the amount of colloidal gold particles in the sediment and the stronger the color development intensity. Therefore, the test substance in the sample solution can be detected qualitatively using the color development intensity of the obtained sediment as an index. Further, by obtaining the correlation between the test substance concentration and the color development intensity in advance, the test substance in the sample solution can be quantitatively detected.

Hereinafter, a detection device suitable for use in the above-described detection method will be described.
FIG. 3 shows an example of the detection device of the present invention. The detection device includes a transparent container 10 made of a transparent synthetic resin or the like, and a reaction solution 20 accommodated in the transparent container 10.

  The transparent container 10 includes a reaction unit 11 having an open top surface and a separation detection unit 12 having an opening on the bottom surface of the reaction unit 11. The reaction unit 11 and the separation detection unit 12 are integrally formed of a hydrophobic resin. ing. From the opening of the reaction part 11 to the middle part, it has a substantially cylindrical shape, and the bottom surface has a substantially arc shape to the extent that the centrifugal movement of the bead carrier is not hindered, and the reaction solution 20 is held and held. By making the bottom face substantially arc-shaped, the outer dimension with respect to the volume of the reaction portion 11 is reduced, and the reaction efficiency can be increased.

  The separation detection unit 12 is formed to have a tapered cross section whose inner diameter is narrower than that of the reaction unit 11 and whose diameter becomes narrower toward the bottom surface. Further, the separation detection unit 12 is transparent, and the internal state is visible from the outside. At the open end of the separation detection unit 12, the reaction solution 20 is held inside the reaction unit 11 due to surface tension, and does not enter the separation detection unit 12. Thereby, in the detection device before the centrifugal treatment, the reaction solution 20 is accommodated and held inside the reaction unit 11, but is prevented from entering the separation detection unit 12. The separation detection unit 12 is filled with a gas such as air.

  The reaction solution 20 includes a bead carrier and a binding substance that recognizes a test substance and is labeled with a coloring substance. A test substance, a binding substance that recognizes the test substance, and the like are immobilized on the bead carrier according to the detection method.

  A method for detecting a test substance in a sample using the above detection device will be described with reference to FIG. Here, the sandwich method is used. First, a detection device in which a reaction solution is accommodated in the reaction part is prepared. The reaction solution includes a colloidal gold labeled antibody that recognizes an antigen as a test substance and a bead carrier on which a second binding substance (second antibody) that recognizes the test substance is immobilized. The second antibody immobilized on the bead carrier recognizes the test substance and binds to the test substance at a recognition site different from the colloidal gold labeled antibody. On the other hand, the separation detection unit is filled with air.

  A sample solution containing a test substance is introduced from the opening of the detection device, and the reaction solution and the sample solution are mixed. As a result, an antigen-antibody reaction occurs, and the gold colloid-labeled antibody specifically binds to the second antibody on the bead carrier via the test substance. On the other hand, the unreacted colloidal gold labeled antibody does not react with the second antibody on the bead carrier, and is maintained in a dispersed state in the reaction solution.

  The detection device is then set in a centrifuge and centrifuged under appropriate conditions (eg, 3250 g, 15 seconds). Thereby, the air inside the separation detection unit is removed by centrifugal force, and the reaction solution accommodated and held in the reaction unit enters the separation detection unit. Then, the gold colloid-labeled antibody-test substance complex bound to the bead carrier via the second antibody settles together with the bead carrier at the bottom of the separation detection unit. On the other hand, the colloidal gold labeled antibody that is not bound to the bead carrier remains in the supernatant.

  After centrifugation, the sediment that has settled on the bottom surface of the separation detection unit is observed, for example, visually from the outside of the transparent container. Since the sandwich method is used, the higher the concentration of the test substance in the sample solution, the greater the amount of colloidal gold particles in the sediment and the stronger the color development intensity. Therefore, the test substance in the sample solution can be detected qualitatively using the color development intensity of the obtained sediment as an index. Further, by obtaining the correlation between the test substance concentration and the color development intensity in advance, the test substance in the sample solution can be quantitatively detected.

  In the detection device of the present invention, as shown in FIG. 5, as shown in FIG. 5, separation promotion that promotes separation of the labeling color substance bound to the bead carrier and the unreacted labeling color substance inside the separation detection unit 12. The liquid 21 may be accommodated. Examples of the separation promoting liquid 21 include a PBS buffer containing a surfactant such as 0.05% Tween 20 or the like. By storing such a separation promoting liquid 21 in the separation detecting unit 12, pseudo-aggregation between the bead carriers and adsorption to the side of the container are prevented, so that the labeling coloring substance and the unreacted labeling coloring substance Can be efficiently separated. When accommodating the separation promoting liquid 21 in the separation detection unit 12, the reaction solution 20 and the separation promoting liquid 21 are mixed by interposing a gas such as air between the reaction solution 20 held in the reaction unit 11. Can be prevented. For this reason, it is preferable that the separation promoting liquid 21 accommodated in the separation detection unit 12 is about 2/3 or less of the volume of the separation detection unit 12.

  Further, the detection device of the present invention may have a configuration in which an isolation film 13 is formed between the reaction unit 11 and the separation detection unit 12 as shown in FIG. The separation membrane 13 functions to reliably block the flow of the solution between the reaction unit 11 and the separation detection unit 12 before the centrifugation process. Thereby, the reaction solution 20 is prevented from entering the separation detection unit 12, and the reaction between the sample and the reaction solution proceeds only in the reaction unit 11, so that the reaction efficiency can be increased. On the other hand, when the centrifugal process is performed, the separation membrane 13 is broken by the centrifugal force. As a result, the reaction solution accommodated in the reaction unit 11 flows into the separation detection unit 12, and a substance having a large sedimentation coefficient settles in the separation detection unit 12. Be made.

  Furthermore, in the detection device including the isolation film 13, the separation promoting liquid 20 may be accommodated in the separation detection unit 12 as shown in FIG. In this case, as in the case of the detection device in which the separation film 13 does not exist, it is possible to efficiently separate the labeling coloring material and the unreacted labeling coloring material. When the separation accelerating liquid 21 is accommodated in the separation detection unit 12 with the separation membrane 13, a gas such as air is interposed between the separation solution 13 and the reaction solution 20 held in the reaction unit 11. The isolation film 13 is easily broken, and the reaction solution 20 and the separation promoting liquid 21 can be reliably mixed.

(Example 1)
In Example 1, an attempt was made to detect cortisol, which is a type of hormone, using a competition method.
First, a solution (OD = 6) containing an anti-cortisol antibody modified with colloidal gold particles having a particle size of 40 nm is prepared (OD = 6), and this gold colloid-labeled antibody solution is added to a transparent conical tube in a volume of 5 μl. I put them one by one.

  On the other hand, a cortisol solution having a cortisol concentration of 3000 ng / ml, 300 ng / ml, 30 ng / ml, 3 ng / ml, 0.3 ng / ml was prepared as a sample solution, and 5 μl of each cortisol solution was placed in each tube. The solution was stirred by pipetting to cause an antigen-antibody reaction.

  Next, a polystyrene bead carrier having a particle diameter of 2 μm modified with cortisol was prepared, and 10 μl of the solution containing this bead carrier was placed in each tube and stirred to cause a reaction.

  Finally, the entire tube was set in a centrifuge and centrifuged at 3250 g for 15 seconds to precipitate the bead carrier at the bottom of the tube. A photograph of the tube after centrifugation is shown in FIG. As shown in the figure, it was confirmed that the red coloration of the precipitate became deeper as the antigen (cortisol) concentration decreased. That is, if the antigen concentration in the sample solution is high, the colloidal gold-labeled antibody does not bind to the bead carrier and the color becomes light. If the antigen concentration is low, the colloidal gold-labeled antibody binds to the bead carrier and the color becomes dark. Was confirmed.

  Here, the precipitate when the sample solution having a cortisol concentration of 0.3 ng / ml was collected and observed with a scanning electron microscope (SEM). A SEM observation photograph is shown in FIG. It is observed that gold colloid particles are bound to the surface of a polystyrene bead carrier having a particle size of 2 μm.

(Example 2)
During surgery such as primary aldosteronism, the catheter position may be confirmed by the blood cortisol concentration. For example, while moving the catheter inserted into the vein from the thigh toward the adrenal gland, venous blood sampling and blood cortisol concentration measurement were repeated, and the catheter tip was positioned appropriately to raise the cortisol concentration value. It may be an indicator of this. Since these series of operations need to be completed within a limited operation time, there is a demand for measuring the cortisol concentration in a short time. Moreover, it must be possible to discriminate a slight concentration difference such as 100 ng / ml and 300 ng / ml as the cortisol concentration. Therefore, in this embodiment, the applicability of the present invention to the above specific example was examined.

  As a sample solution, cortisol solutions having cortisol concentrations of 75 ng / ml, 150 ng / ml, and 300 ng / ml were prepared. Further, a silica bead carrier having a particle size of 50 nm was used as the bead carrier. Except for these, cortisol in the sample solution was detected in the same manner as in Example 1. An appearance photograph of the tube after centrifugation is shown in FIG.

  FIG. 10 shows that the coloration of the precipitates is completely different between the cortisol concentration of around 100 ng / ml and around 300 ng / ml. Therefore, the present example shows the applicability to the case as described above.

(Example 3)
In Example 3, it was examined whether or not the sample could be detected by the sandwich method. In this example, detection of human chorionic gonadotropin (hCG) was attempted.

  First, as sample solutions, solutions having hCG antigen concentrations of 100 ng / ml, 10 ng / ml, 1 ng / ml, 0.1 ng / ml, 0.01 ng / ml are prepared, and 10 μl of each concentration of the sample solution is conical. In a tube. Next, a solution containing a polystyrene bead (particle size: 2 μm) carrier on which the anti-hCG antibody was immobilized was prepared, and 5 μl of this solution was placed in each tube and stirred to cause a reaction.

  On the other hand, a solution (OD = 3) containing a colloidal gold labeled antibody obtained by modifying a colloidal gold particle having a particle size of 40 nm to an anti-hCG antibody was prepared. The antibody used for the colloidal gold labeled antibody recognizes a different site on hCG from the anti-hCG antibody immobilized on the polystyrene bead carrier, and specifically recognizes the α subunit of hCG. 5 μl of this colloidal gold labeled antibody solution was put into the tube and stirred by pipetting to react. Then, it left still for 3 minutes.

  Finally, the tube was set in a centrifuge and centrifuged at 3250 g for 15 seconds to settle the bead carrier to the bottom of the tube. A photograph of the tube after centrifugation is shown in FIG. As shown in the figure, it was confirmed that the red color of the sediment became deeper as the hCG concentration increased.

Example 4
In Example 4, hCG was detected using the sandwich method in the same manner as in Example 3 using a detection device having the structure shown in FIG. 3 instead of the conical tube. In the reaction part, a reaction solution containing a polystyrene bead (particle size 2 μm) carrier on which an anti-hCG antibody is immobilized and a colloidal gold labeled antibody is held and held. On the other hand, the separation detection unit is filled with air.

  10 μl each of the hCG antigen-containing sample solution prepared in Example 3 was placed in the reaction part of the device and reacted. After the reaction, the detection device was set in a centrifuge and centrifuged at 3250 g for 15 seconds to precipitate the bead carrier at the bottom of the separation detection unit. A photograph of the separation detector after centrifugation is shown in FIG. It was confirmed that the color depth and color tone of the sediment changed depending on the hCG concentration of the sample solution.

(Example 5)
In this example, the material and particle size of beads used as a carrier were examined. A solution containing polystyrene (PS) beads having a particle size of 400 nm, a solution containing polystyrene (PS) beads having a particle size of 200 nm, and a solution containing silica beads having a particle size of 50 nm were prepared. These solutions were put into a container constituting the detection device used in Example 4 and subjected to centrifugal treatment, so that beads were allowed to settle at the bottom of the container. A photograph of the separation detector after centrifugation is shown in FIG.

  From the figure, the sediment of the polystyrene beads was white, but the sediment of the silica beads was transparent. In the detection method of the present invention, the test substance is detected qualitatively or quantitatively based on the color development derived from the color development substance bound to the bead carrier constituting the sediment. Therefore, it was shown that the use of transparent silica beads as the bead carrier is more preferable.

It is a schematic diagram for describing one embodiment of the detection method of the present invention, (a) is a mixing step of mixing a sample solution and a colloidal gold labeled antibody, (b) is a binding step of binding to a bead carrier, c) shows a centrifugation step. It is a schematic diagram for demonstrating other embodiment of the detection method of this invention, (a) is the mixing process of mixing a sample solution and a gold colloid labeled antibody, (b) is the coupling | bonding process couple | bonded with a bead support | carrier. (C) shows a centrifugation step. It is a schematic sectional drawing which shows an example of the detection device of this invention. It is a schematic diagram for demonstrating the detection method using the detection device of this invention, (a) shows the reaction process of a sample solution and a reaction solution, (b) shows the centrifugation process. It is a schematic sectional drawing which shows the 2nd example of the detection device of this invention. It is a schematic sectional drawing which shows the 3rd example of the detection device of this invention. It is a schematic sectional drawing which shows the 4th example of the detection device of this invention. 2 is a photograph showing the results of Example 1. FIG. 2 is a SEM photograph observing the surface of a bead carrier precipitated in Example 1. FIG. 6 is a photograph showing the results of Example 2. 6 is a photograph showing the results of Example 3. 6 is a photograph showing the results of Example 4. 6 is a photograph showing the results of Example 5.

Explanation of symbols

  11 Reaction unit, 12 Separation detection unit, 13 Separation membrane, 20 Reaction solution

Claims (14)

  Using a specific binding between a test substance in a sample and a binding substance that recognizes the test substance, a coloring substance is bound to the bead carrier, and the bead carrier is sedimented as a sediment to the bottom of the container by centrifugation. After separating the coloring substance bound to the bead carrier and the non-binding coloring substance by the above, the test substance is detected using the signal derived from the coloring substance in the sediment observed from the outside of the container as an index. A method for detecting a test substance, comprising:   The method for detecting a test substance according to claim 1, wherein the sedimentation coefficient of the bead carrier is larger than the sedimentation coefficient of the coloring substance.   3. The method for detecting a test substance according to claim 2, wherein the sedimentation coefficient of the bead carrier is 10 times larger than the sedimentation coefficient of the coloring substance.   The method for detecting a test substance according to claim 1, wherein the bead carrier has a particle size of 5 μm or less.   The method for detecting a test substance according to claim 1, wherein the bead carrier is made of a transparent material.   6. The method for detecting a test substance according to claim 5, wherein the particle size of the bead carrier is 70 nm or less.   The method for detecting a test substance according to any one of claims 1 to 6, wherein the coloring substance is gold colloidal particles. A reaction portion having an upper surface and a bottom surface and containing a reaction solution;
An opening at the bottom of the reaction unit, and a transparent separation detection unit formed with an inner diameter narrower than the reaction unit,
The detection device, wherein the reaction solution is not contained in the separation detection unit.   The detection device according to claim 8, wherein the separation detection unit contains air.   The detection device according to claim 9, wherein the reaction solution is held in the reaction part by surface tension at an opening of the separation detection part.   The liquid flow between the reaction unit and the separation detection unit is blocked by a separation membrane, and the separation membrane is broken by the centrifugal force generated when the detection device is centrifuged, and the reaction stored in the reaction unit The detection device according to claim 8, wherein the solution is formed so as to flow into the separation detection unit.   The detection device according to any one of claims 8 to 11, wherein a bottom surface of the reaction portion has a substantially arc shape.   The reaction solution contains a solution containing a binding substance that recognizes a test substance contained in a sample and is labeled with a coloring substance and a bead carrier. The detection device according to claim 1.   The detection device according to claim 13, wherein a separation promoting liquid that promotes separation of the labeling coloring substance bound to the bead carrier and the unreacted labeling coloring substance is accommodated in the separation detection unit.

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