JPWO2015046293A1 - Test substance detection system - Google Patents

Abstract

A detection system for a test substance is bonded to a test substance via a site that is different from the binding site and a carrier modified with a first substance that can bind to the test substance via a binding site of the test substance. A first layer including a label modified with a second substance that can be applied, and a second layer that does not include a carrier and a label before applying an external attractive force. The complex bound to the test substance can move from the first layer to the second layer.

Description

  The present invention relates to a detection system for a test substance using a carrier and a label.

As a method for detecting a trace amount of a test substance in a sample, an ELISA (enzyme-linked immunosorbent assay) method, a MEIA (microparticle enzyme-based immunoassay) method, and the like are widely used.
These detection methods are methods for specifically and selectively capturing a test substance with an antibody. The test substance is captured with an antibody immobilized on a substrate or particle surface, and an enzyme or the like is labeled in advance. The antibody is reacted with the antibody to construct a sandwich structure.
Here, when the test substance is not contained in the sample, the labeled antibody remains because there is no test substance to be captured and bound, and is removed by washing the reaction container. That is, if a test substance exists in the sample, a sandwich structure composed of an immobilized antibody-test substance-labeled antibody is constructed. On the other hand, if there is no test substance, the labeled antibody remains and is removed by washing.
Using such a principle, the difference in the amount of the test substance in the sample is detected by capturing and binding the labeled antibody.
Therefore, when the enzyme remaining in the reaction vessel is finally colored, the presence or absence of the test substance can be determined or the amount of the test substance can be obtained.

However, in order to carry out these techniques, it is necessary to remove the unreacted labeled antibody. Washing requires a fluid handling step, and too much washing can remove the label to be detected.
However, if the washing is small, false positives due to unreacted labeled antibody may occur. Therefore, in these methods, since the cleaning operation needs to be performed a plurality of times, the operation time takes a long time, and the measurement operation is complicated.

  Therefore, a method of washing and removing unreacted labeled antibody by using magnetic particles as an antibody immobilization carrier and immobilizing the magnetic particles with a magnet is widely used. Cleaning operation time is shortened by fixing and cleaning with a magnet. However, the cleaning operation needs to be performed a plurality of times, and the operation may be complicated.

  In addition, a microchemical reaction method has been proposed in which a magnetic field is changed to a droplet containing magnetic particles to move the droplet and perform an operation necessary for a chemical reaction (see Patent Document 1). In the proposed method, fluid control elements such as pumps and valves are required by separating, moving, agitating, and washing droplets containing magnetic particles from the liquid due to fluctuations in the magnetic field. Therefore, it is possible to perform a chemical reaction operation and to perform a chemical reaction, a biochemical reaction, a biological interaction, and the like as a result. However, in the droplets cut out from the reaction solution by the magnetic field, a part of the reaction solution is cut off and remains together with the magnetic particles, and therefore, a plurality of operations may be required particularly in the cleaning operation.

Furthermore, a method for detecting a test substance by using a noble metal-coated magnetic particle and moving it by a magnetic field has been proposed (see Patent Document 2). In the proposed method, the complex formation with the test substance is performed under the influence of the magnetic field, the noble metal coated magnetic particles are moved by the magnetic field, and the coloration of the noble metal is detected. Detection is possible.
However, the noble metal-coated magnetic particles remain in the detection portion due to the magnetic field, so that there is a risk of false determination as a false positive when there is no test substance.

On the other hand, a method for detecting a test substance has also been proposed by separating particles from a liquid phase by centrifugation using particles and a precipitation reagent, and leaving detectable activity on the particles (see Patent Document 3). ). In the proposed method, separation and detection applicable to a clinical chemical analyzer can be performed by precipitating particles using a precipitation reagent and centrifuging them using a precipitation reagent.
However, the addition of the detection reagent after the addition of the precipitation reagent and the centrifugation is necessary, and the operation may be complicated.

Japanese Patent No. 4844263 Japanese Patent No. 4791867 Japanese Patent No. 3684454

  The present invention has been made in view of the above-described circumstances, and provides a test substance detection system capable of quickly and easily detecting a test substance in a sample.

  A first aspect of the present invention is a test substance detection system, a carrier modified with a first substance capable of binding to a test substance via a test substance binding site, a binding site, Includes a first layer containing a labeled substance modified with a second substance capable of binding to the test substance via different sites, and a second layer that does not contain a carrier and a labeled substance before applying an external attractive force. And a complex bound to the carrier and the test substance by external attraction is movable from the first layer to the second layer.

  According to a second aspect of the present invention, in the test substance detection system according to the first aspect, the first layer and the second layer are further separated, and the separation layer through which the complex bound to the carrier and the test substance can pass is provided. May be provided.

  According to a third aspect of the present invention, in the test substance detection system according to the first or second aspect, the system further includes a detection unit that detects a signal emitted from the second layer, and the label includes at least the test substance. It may be capable of emitting light and reacting in a bound state.

  According to a fourth aspect of the present invention, in the detection system for a test substance according to any one of the first to third aspects, the external attractive force may be selected from the group consisting of magnetic force, centrifugal force, and electrophoretic force. .

According to a fifth aspect of the present invention, in the detection system for a test substance according to any one of the first to fourth aspects, the first substance is selected from the group consisting of an antibody, a fragmented antibody, a complete antigen, and a hapten. May be.
According to a sixth aspect of the present invention, in the detection system for a test substance according to any one of the first to fifth aspects, the second substance is selected from the group consisting of an antibody, a fragmented antibody, a complete antigen, and a hapten. May be.

  According to a seventh aspect of the present invention, in the detection system for a test substance according to any one of the second to sixth aspects, the separation layer includes oil, a porous material, a polymer membrane, a polymer gel, and a high concentration solution. May be selected from the group consisting of

  According to an eighth aspect of the present invention, in the detection system for a test substance according to any one of the first to seventh aspects, the second layer includes oil, a porous material, a polymer film, a polymer gel, and a high concentration. It may be selected from the group consisting of solutions.

  According to a ninth aspect of the present invention, in the detection system for a test substance according to any one of the first to eighth aspects, the carrier includes a magnetic substance, a metal, an inorganic substance, an organic substance, a polymer, and a combination thereof. You may choose from the group which consists of a substance to contain.

  According to a tenth aspect of the present invention, in the detection system for a test substance according to any one of the first to ninth aspects, the label is a group consisting of a colorimetric substance, a luminescent substance, a redox substance, a nucleic acid, and an enzyme. You may choose from

  In an eleventh aspect of the present invention, in the detection system for a test substance according to any one of the first to tenth aspects, a substance that reacts with a labeling substance may be included in the second layer.

  According to a twelfth aspect of the present invention, in the test substance detection system according to the eleventh aspect, the substance that reacts with the label may be selected from the group consisting of a substrate, a chemiluminescent substance, and a redox substance.

  According to a thirteenth aspect of the present invention, in the detection system for a test substance according to any one of the third to twelfth aspects, the detection unit includes a first filter that transmits only light of a predetermined wavelength, and a first filter. And a detection unit that detects transmitted light, and the label may be labeled with a group consisting of a non-colored substance, a luminescent substance, and an enzyme.

  According to a fourteenth aspect of the present invention, in the detection system for a test substance according to any one of the third to twelfth aspects, the detection unit includes a light source that emits light including excitation light having a predetermined wavelength, and only the excitation light. A first filter that transmits light, an optical path member that irradiates the second layer with excitation light transmitted through the first filter, a second filter that removes excitation light, and a detection unit that detects light transmitted through the second filter, The label may be labeled with a fluorescent substance that emits fluorescence by excitation light.

  According to the first aspect of the present invention, in the first layer, the complex formed of the test substance, the carrier, and the label can be easily moved to the second layer by external attractive force. Only the carrier and the complex are moved by the external attractive force, and the unreacted label can be easily separated.

  In the second layer, a signal emitted from the labeled body is detected or a signal generated by a reaction with the labeled body is detected. The complex moved by the external attractive force can be detected without requiring a special operation.

  According to the above aspect of the present invention, it is possible to quickly and easily perform highly sensitive detection of a test substance in a sample.

1 is a schematic diagram of a test substance detection system according to a first embodiment of the present invention. It is a figure explaining the effect | action of the detection system of 1st embodiment of this invention. It is a figure explaining the effect | action of the detection system of 1st embodiment of this invention. It is a schematic diagram of the detection system of the test substance of 2nd embodiment of this invention. It is a figure explaining the effect | action of the detection system of 2nd embodiment of this invention. It is a figure explaining the effect | action of the detection system of 2nd embodiment of this invention. It is a figure which shows the test result of the reference example 1. It is a figure which shows the test result of Example 1. It is a figure which shows the test result of the reference example 2. It is a figure which shows the test result of Example 2.

(First embodiment)
A test substance detection system according to an embodiment of the present invention will be described. FIG. 1 is a schematic diagram of a detection system 1 of the present embodiment. 2A and 2B are diagrams for explaining the operation of the detection system 1.

  As shown in FIG. 1, the detection system 1 is a system that detects the presence or absence of a test substance 10 in a sample. In the present embodiment, any substance such as a biological substance or a synthetic substance can be used as the test substance 10. As the sample, for example, a sample solution derived from a living body such as blood, serum, or urine, a solution obtained by preparing these, and the like can be arbitrarily used.

The detection system 1 includes a first layer 4 including a carrier 2 and a label 3, a second layer 5, a separation layer 6, a magnetic force generation unit 7, and a detection unit 8.
The carrier 2 is selected from the group consisting of a magnetic substance, a metal, an inorganic substance, an organic substance, a polymer, and a substance containing a combination thereof.
An example of the carrier 2 is a magnetic material. Hereinafter, in this embodiment, a case where the carrier is a magnetic material will be described as an example. The magnetic body includes one or more magnetic materials or magnetizable particles of any suitable shape and is attracted by the magnetic force generator. The maximum diameter of the magnetic material is preferably 5 nm to 100 μm, more preferably 100 nm to 10 μm. The magnetic substance is surface-modified with a first substance 11 that binds to the test substance 10 via a binding site of the test substance 10. The method of modifying the magnetic material with the first substance 11 may not be surface modification.

  An example of a magnetic material is a magnetic polymer particle that includes a magnetic material and a polymer layer that covers the magnetic material. The magnetic polymer particles may be present by coating a plurality of magnetic particles on one polymer particle. As the magnetic particles covering the polymer particles, ferrite particles such as magnetite capable of generating fine particles in water are preferable. On the other hand, as magnetic particles other than ferrite, for example, fine particles of various magnetic metals or various magnetic compounds are used, and the characteristic magnetic properties of these magnetic particles can be used in various ways.

The first substance 11 is selected from the group consisting of an antibody, a fragmented antibody, a complete antigen, and a hapten, and is selected and adopted according to the test substance. The first substance 11 preferably has high specificity for the test substance 10. For example, the first substance easily binds to the test substance 10 in the sample, and hardly binds to a substance different from the test substance 10.
Moreover, it is preferable that the 1st substance 11 has low specificity with respect to the labeled body 3 and the 2nd substance 12 mentioned later.

The labeled body 3 is a labeling substance selected from the group consisting of a colorimetric substance, a luminescent substance, a redox substance, a nucleic acid, and an enzyme. Examples of the luminescent substance include a fluorescent molecule, a phosphorescent molecule, a chemiluminescent molecule, and an enzyme binding molecule. Etc. Examples of the detection method using nucleic acid as a labeling substance include nucleic acid detection methods such as immuno-PCR, Invader method (registered trademark), Taqman method, and fluorescent probe method. The labeled body 3 is modified with a second substance 12 that binds to the test substance 10 via a site different from the site to which the first substance 11 binds.
The second substance 12 is a substance different from the first substance 11 and binds to a site different from the binding site of the first substance 11 in the test substance 10. The second substance 12 is selected from the group consisting of an antibody, a fragmented antibody, a complete antigen, and a hapten, and is selected and adopted according to the test substance 10. The second substance 12 preferably has high specificity for the test substance 10. For example, the second substance 12 easily binds to the test substance 10 in the sample and hardly binds to a substance different from the test substance 10.
The second substance 12 preferably has low specificity for the carrier 2 and the first substance 11.

  The first layer 4 and the second layer 5 may have any shape as long as the carrier 2, the label 3 and the test substance 10 can be dispersed, and the substance is not particularly limited. For example, the first layer 4 and the second layer 5 can be an aqueous solution, oil, high-concentration solution, or the like. Moreover, a porous member etc. may be employ | adopted as the 1st layer 4 and the 2nd layer 5. FIG. For example, a polymer gel, a nonwoven fabric, a filter paper, a polymer membrane, a porous membrane, etc. can be used as the first layer and the second layer.

The separation layer 6 may have any shape as long as the first layer 4 and the second layer 5 can be separated, and the complex of the carrier, the labeled body, and the test substance 10 can pass through, and the substance is not particularly limited. Absent. For example, the separation layer 6 can be an aqueous solution, oil, high-concentration solution, or the like.
A porous member or the like may be employed as the separation layer 6. For example, the separation layer 6 may be a polymer gel, a nonwoven fabric, a filter paper, a polymer membrane, a porous membrane, or the like.

  The magnetic force generator 7 is provided under the second layer 5 in order to move the carrier 2 from the first layer 4 to the second layer 5. The magnetic force generator 7 is, for example, a permanent magnet or an electromagnet. The magnetic force generator 7 may be detachable under the second layer 5.

  The detection unit 8 detects the presence of the label 3. In the present embodiment, the detection unit 8 detects a signal emitted from the label 3 or a signal generated by a reaction with the label 3. Specifically, it includes a first filter that transmits only light of a predetermined wavelength and a detection unit that detects light transmitted through the first filter.

Next, the operation of the detection system 1 will be described together with the detection method of the test substance 10 using the detection system 1.
First, the sample to be detected for the presence or absence of the test substance 10 is mixed with the first layer 4 including the carrier 2 and the label 3.
When the test substance is contained in the sample, the carrier 2 and the test substance 10 are bonded via the first substance 11, and the label 3 and the test substance 10 are connected via the second substance 12. And combine. That is, the carrier 2 and the labeled body 3 are bound via the test substance 10 (see FIG. 2A (positive)).
When the test substance 10 is not contained in the sample, there is no substance that binds the carrier 2 and the labeled body 3, and therefore the carrier 2 and the labeled body 3 exist independently of each other (FIG. 2B). (Refer negative)).

Here, when the test substance 10 is bound to both the carrier 2 and the labeled body 3 (see FIG. 2A (positive)), the test substance 10, the carrier 2 and the labeled body 3 form a complex. It is formed and attracted to the magnetic force generator 7. As a result, the test substance 10 contained in the mixture spread in the first layer 4 is moved to the second layer 5 by the magnetic force from the magnetic force generator 7 together with the label 3 by the carrier 2 bonded to the test substance 10. To do.
On the other hand, when the carrier 2 and the label 3 are not bonded via the test substance 10 (see FIG. 2B (negative)), the carrier 2 is attracted by the magnetic force from the magnetic force generation unit 7. However, the marker 3 is present in the first layer 4 without being affected by the magnetic force.

  In addition, the detection unit 8 detects a signal emitted from the marker 3 attracted by the magnetic force generation unit 7 in the second layer 5. In addition, since the label | marker 3 which is not couple | bonded with the support | carrier 2 and the to-be-tested substance 10 is disperse | distributing to the 1st layer 4, it is not detected by the detection part 8.

  As described above, according to the detection system 1 of the present embodiment, a highly sensitive detection can be obtained quickly and easily without requiring a complicated washing operation or the like in the conventional immunoassay method.

  Next, another embodiment of the present invention will be described focusing on differences from the above-described embodiment.

(Second embodiment)
A test substance detection system 31 according to a second embodiment of the present invention will be described. FIG. 3 is a schematic diagram of the detection system 31 of the present embodiment. 4A and 4B are diagrams for explaining the operation of the detection system 31. FIG.

According to the present embodiment, the first layer 4 including the carrier 2, the labeled body 3, and the test substance 10 and the second layer 5 are present independently of each other without being mixed (see FIG. 3). . Further, the carrier 2 is attracted by the magnetic force of the magnetic force generator 7 and moves from the first layer 4 to the second layer 5.
However, when there is no magnetic force of the magnetic force generation part 7, the carrier 2 exists in the first layer 4. In addition, the label 3 and the test substance 10 exist in the first layer 4 regardless of the magnetic force of the magnetic force generation unit 7.

  Examples of the first layer 4 and the second layer 5 include a combination in which the first layer 4 is an aqueous solution and the second layer 5 is a high concentration solution.

Here, when the test substance 10 is bound to both the carrier 2 and the labeled body 3 (see FIG. 4A (positive)), the test substance 10, the carrier 2 and the labeled body 3 form a complex. It is formed and attracted to the magnetic force generator 7. As a result, the test substance 10 contained in the mixture spread in the first layer 4 is transferred to the second layer 5 by the magnetic force from the magnetic force generation unit 7 together with the label 3 by the carrier 2 bonded to the test substance 10. Moving.
On the other hand, when the carrier 2 and the label 3 are not bonded via the test substance 10 (see FIG. 4B (negative)), the carrier 2 is attracted by the magnetic force from the magnetic force generation unit 7. However, the marker 3 is present in the first layer 4 without being affected by the magnetic force.

  In addition, the detection unit 8 detects a signal emitted from the marker 3 attracted by the magnetic force generation unit 7 in the second layer 5. In addition, since the labeled body 3 which is not bound to the carrier 2 and the test substance 10 among the labeled bodies 3 is dispersed in the first layer 4, it is not detected by the detection unit.

(Third embodiment)
In the present embodiment, the means for moving the carrier 2 from the first layer 4 to the second layer 5 is different from the above-described embodiment. In this embodiment, the complex containing the test substance 10 to which the carrier and the carrier are bound is moved from the first layer 4 to the second layer 5 by centrifugal force or electrophoretic force. It is not accumulated by magnetic force. Therefore, the transferred carrier 2 and the composite can be dispersed in the second layer 5.

(Fourth embodiment)
In the present embodiment, the second layer 5 is formed in a state containing a substance that reacts with the label 3. Examples of the substance that reacts include a substrate, a chemiluminescent substance, and a redox substance. In the present embodiment, detection is performed only when the complex including the test substance 10 to which the label 3 is bound moves to the second layer 5 by detecting a signal due to the reaction between the label 3 and the substance. can do. That is, since detection can be performed regardless of the layer position, the detection apparatus can be simplified.

(Fifth embodiment)
In the present embodiment, the detection unit 8 detects fluorescence emitted from the marker 3. Specifically, the detection unit 3 includes a light source that emits light including excitation light having a predetermined wavelength, an optical path member that irradiates the second layer 5 with excitation light transmitted through the first filter, and a dichroic mirror (only excitation light is emitted). A first filter that transmits light and a second filter that removes excitation light), and a detection unit that detects light transmitted through the second filter. Further, in the present embodiment, the carrier 2 and the labeled body 3 moved to the second layer 5 by the magnetic force generation unit 7 are respectively subjected to magnetic detection and fluorescence detection, and the abundance ratio between the carrier 2 and the labeled body 3 is calculated. it can.
In this embodiment, the content of the test substance 10 can be quantified by making the usage amounts of the carrier 2 and the labeled body 3 known in advance.

(Sixth embodiment)
In this embodiment, an electrode and a sensor for electrochemically measuring an enzyme reaction are provided instead of the detection unit described in the above embodiment. The electrode in this embodiment can be formed of silver, gold, stainless steel, indium tin oxide (ITO), or the like.
Also in this embodiment, the carrier 2 moved to the second layer 5 by the magnetic force generator 7 can be quantified by a magnetic sensor. In the present embodiment, it is only necessary to provide at least one of the electrodes and sensors and the magnetic sensor.

As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the concrete structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included.
In addition, the constituent elements shown in the above-described embodiments can be combined as appropriate.

  Hereinafter, the present invention will be described in more detail based on examples. The present invention is not limited to these examples.

  In this example, a test substance detection experiment was performed using prostate specific antigen (PSA), which is a biomarker of prostate cancer, as the test substance. As the first substance, a monoclonal antibody (1H12) that recognizes PSA was used. As the second substance, a monoclonal antibody (5A6) that recognizes another site of PSA was used.

  First, a PSA antibody as the first substance was bound to magnetic beads having a particle diameter of 1 μm to obtain anti-PSA antibody-modified magnetic particles. Furthermore, anti-PSA antibody of the second substance was bound to horseradish peroxidase (HRP) to obtain an HRP-modified anti-PSA antibody. Similarly, the second substance anti-PSA antibody was bound to alkaline phosphatase (ALP) to obtain an HRP-modified anti-PSA antibody.

(Reference Example 1)
In Reference Example 1, PSA was detected using a 96-well plate using the above-described anti-PSA antibody-modified magnetic particles and HRP-modified anti-PSA antibody. First, anti-PSA antibody-modified magnetic particles were added to and mixed with an antigen solution (PSA concentration: 0 ng / mL, 1 ng / mL). After the mixed solution was transferred to each well of a 96-well plate, washing was performed 3 times using a 96-well plate magnet. Next, after adding and mixing the HRP-modified anti-PSA antibody, washing was similarly performed three times using a 96-well plate magnet. Further, 3,3 ′, 5,5′-tetramethylbenzidine (TMB) solution was added and mixed as a chromogenic substrate for HRP, 1% hydrochloric acid was added as a reaction stop solution, and the coloration of each well was determined by a plate reader. The results confirmed by (absorbance: 450 nm) are shown in FIG.

Example 1
In Example 1, as in Reference Example 1, PSA was detected using anti-PSA antibody-modified magnetic particles and HRP-modified anti-PSA antibody. First, anti-PSA antibody-modified magnetic particles and HRP-modified anti-PSA antibody were added to and mixed with an antigen solution (PSA concentration: 0 ng / mL, 1 ng / mL). Next, a mixed solution obtained by mixing a saturated sucrose solution and a TMB solution at a ratio of 1: 1 was transferred into a microtube to form a second layer. Further, an antigen, anti-PSA antibody-modified magnetic particles, and an HRP-modified anti-PSA antibody mixed solution were added to the tube to form a first layer. Centrifugation (6000 rpm) is applied to the microtube holding the first layer and the second layer, and after moving the magnetic particles and the composite from the first layer, the second layer solution is added to each well of the 96-well plate. FIG. 6 shows the result of confirming the coloration of each well with a plate reader (absorbance: 450 nm).

  From the results of Reference Example 1, a difference in absorbance was confirmed with respect to the presence or absence of the antigen (0 ng / mL, 1 ng / mL). Of PSA was confirmed.

  From the results of Example 1, the difference in absorbance was confirmed with respect to the presence or absence of the antigen (0 ng / mL, 1 ng / mL). The detection of PSA was confirmed by moving particles and complexes from one layer to the second layer.

(Reference Example 2)
In Reference Example 2, PSA was detected using a 96-well plate using the above-described anti-PSA antibody and ALP-modified anti-PSA antibody. First, blocking was performed using 5% skim milk on each well of a 96-well plate on which an anti-PSA antibody was immobilized, and washing was performed three times, followed by antigen solution (PSA concentration: 0 ng / mL, 0.01 ng). / ML, 0.1 ng / mL, 1 ng / mL) and mixed. Washing was performed 3 times after the reaction. Next, an ALP-modified anti-PSA antibody was added and mixed, and washing was performed three times after the reaction. Furthermore, after adding and mixing CSPD + Emerald as a chemiluminescence substrate of ALP, the result of having confirmed the chemiluminescence of each well with the plate reader is shown in FIG.

(Example 2)
In Example 2, PSA was detected using anti-PSA antibody-modified magnetic particles and ALP-modified anti-PSA antibody as in Reference Example 2. First, an anti-PSA antibody-modified magnetic particle and an ALP-modified anti-PSA antibody were added to and mixed with an antigen solution (PSA concentration: 0 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL). Next, a mixed solution in which a saturated sucrose solution and a CSPD + Emerald solution were mixed at a ratio of 1: 1 was transferred into a microtube to form a second layer. Further, an antigen, anti-PSA antibody-modified magnetic particles, and an ALP-modified anti-PSA antibody mixed solution were added to the tube to form a first layer. Centrifugation (6000 rpm) is applied to the microtube holding the first layer and the second layer, and after moving the magnetic particles and the composite from the first layer, the second layer solution is added to each well of the 96-well plate. FIG. 8 shows the result of confirming the chemiluminescence of each well with a plate reader.

  From the result of Reference Example 2, a change in chemiluminescence intensity was confirmed with respect to the antigen concentration, and high-sensitivity detection of PSA in the plate was confirmed using an anti-PSA antibody and an ALP-modified anti-PSA antibody.

  From the results of Example 2, a difference in chemiluminescence intensity was confirmed with respect to the antigen concentration, and particles were separated from the first layer to the second layer by centrifugation using anti-PSA antibody-modified magnetic particles and ALP-modified anti-PSA antibodies. And the sensitive detection of PSA by moving the complex was confirmed.

  As the above results show, antigen detection was confirmed by transferring particles and complexes from the first layer to the second layer, and washing was performed in the bead assay and plate assay in the background and signal. It was confirmed that it was equivalent to As a result, antigens can be detected quickly and easily without performing a complicated washing step in a normal immunoassay.

  DESCRIPTION OF SYMBOLS 1,31 ... Detection system, 2 ... Carrier, 3 ... Label body, 4 ... 1st layer, 5 ... 2nd layer, 6 ... 2nd layer, 7 ... Magnetic force generation part, 8 ... Detection part, 10 ... Test substance 11 ... 1st substance, 12 ... 2nd substance

Claims (14)

A carrier modified with a first substance capable of binding to the test substance via a binding site of the test substance, and a carrier capable of binding to the test substance via a site different from the binding site. A first layer comprising a label modified with two substances;
Before applying the external attractive force, the second layer that does not include the carrier and the label,
A test substance detection system in which a complex bound to the carrier and the test substance by an external attractive force can move from the first layer to the second layer.   2. The test substance detection system according to claim 1, further comprising a separation layer that separates the first layer and the second layer and allows a complex bound to the carrier and the test substance to pass therethrough. A detector for detecting a signal emitted from the second layer;
The test substance detection system according to claim 1, wherein the label is capable of emitting light and reacting at least in a state of being bound to the test substance.   The test substance detection system according to claim 1, wherein the external attractive force is selected from the group consisting of magnetic force, centrifugal force, and electrophoretic force.   The test substance detection system according to claim 1, wherein the first substance is selected from the group consisting of an antibody, a fragmented antibody, a complete antigen, and a hapten.   The test substance detection system according to any one of claims 1 to 5, wherein the second substance is selected from the group consisting of an antibody, a fragmented antibody, a complete antigen, and a hapten.   The test substance detection system according to any one of claims 2 to 6, wherein the separation layer is selected from the group consisting of oil, a porous material, a polymer membrane, a polymer gel, and a high-concentration solution.   The test substance detection system according to any one of claims 1 to 7, wherein the second layer is selected from the group consisting of oil, a porous material, a polymer film, a polymer gel, and a high-concentration solution. .   The detection of the test substance according to any one of claims 1 to 8, wherein the carrier is selected from the group consisting of a substance including a magnetic substance, a metal, an inorganic substance, an organic substance, a polymer, and a combination thereof. system.   The test substance detection system according to any one of claims 1 to 9, wherein the label is selected from the group consisting of a colorimetric substance, a luminescent substance, a redox substance, a nucleic acid, and an enzyme.   The test substance detection system according to claim 1, wherein the second layer includes a substance that reacts with the labeling body.   The test substance detection system according to claim 11, wherein the substance that reacts with the label is selected from the group consisting of a substrate, a chemiluminescent substance, and a redox substance. The detection unit includes a first filter that transmits only light of a predetermined wavelength, and a detection unit that detects light transmitted through the first filter,
The test substance detection system according to any one of claims 3 to 12, wherein the label is labeled with a group consisting of a non-color substance, a luminescent substance, and an enzyme. The detection unit irradiates the second layer with a light source that emits light including excitation light having a predetermined wavelength, a first filter that transmits only the excitation light, and the excitation light that has passed through the first filter. An optical path member, a second filter that removes the excitation light, and a detection unit that detects light transmitted through the second filter,
The test substance detection system according to claim 3, wherein the label is labeled with a fluorescent substance that emits fluorescence by the excitation light.

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