JP2010216947A - Immunological assay method and inspection disc in the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an immunological assay method whereby considerable shortening of the measurement time and high sensitivity at least one order of magnitude greater than that of a conventional method is achieved, while exploiting the advantages of immunological assay methods, namely ease of measuring operations, high accuracy and low costs, and also to provide an inspection disc usable in the same. <P>SOLUTION: Disclosed are: a competitive immunological assay method in which an antigen is immobilized on micro-beads and the size (volume) of a micro-chamber that is a reaction field and the total volume of the micro-beads that are maintained in the micro-chamber are kept in a fixed range; and an inspection disc used in this method. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a quantitative analysis of a biological sample by an antigen-antibody reaction, and relates to an immunoassay (immunoassay) for accurately quantifying an antigen and a test disk used for the immunoassay.

  An immunoassay (immunoassay), also called an immunoassay, is a biological sample that applies the specific binding reaction of a specific antibody (antigen) with a specific antigen (antibody). This method is useful for various situations such as the onset of an infectious disease or cancer or observation of its prognosis.

  Since immunoassay has the advantages of good measurement sensitivity, generally high measurement accuracy, relatively simple measurement operation and low cost analysis, various methods have been developed. Among these, those in which the labeling reagent is a radioisotope (radioimmunoassay) or an enzyme (enzyme immunoassay), and the measurement principle is based on a competitive principle or non-competitive principle (sandwich binding principle) are typical. This is a valid test method.

  In the immunoassay using the above enzyme as a label, for example, in ELISA (Enzyme-Linked Immunosorbent Assay), measurement (assay) using a microtiter plate is widely performed. This method has the advantage that multiple analytes can be measured simultaneously, but not only for antigen-antibody reaction time, but also for immobilization (adsorption) of antigen or antibody in the well of a microtiter plate and pre-measurement treatment after the reaction. , Each requires one hour or more, and the total time required for measurement takes several hours.

  In order to solve the problem of long measurement time when using a microtiter plate, we also propose a measurement method called an antigen-antibody reaction in a narrow channel with a small cross-sectional area called microchannel. (Non-Patent Document 1). However, the method of Non-Patent Document 1 has another problem that a large number of samples cannot be measured at the same time.

  In order to solve the above problem by using microchannels, the present inventor has developed a test disk having a large number of microchannels and microbeads on which antibodies are immobilized in an ELISA, which is based on the non-competitive binding principle. It applied to the measuring method (sandwich method) (nonpatent literature 2). Thereby, the measurement time was shortened to about 30 minutes to about 1 hour.

Sato K. Kimura H .; Kitamori T. "Determination of Carcinoembryonic Antigen in Human Seraby Integrated Bed-Bed Immunoassay in a Microchip for Cancer Diagnosis", Anal Che. 2001, 73, p. 1213-1218 N. Matsunaga, S.M. Furutani, I.K. Kubo, “CENTRIFUGAL FLOW DEVICE FOR HIGH-THROUGHPUT DETECTION OF CANCER MARKER CEA”, ECS Transactions, 2008, 16 (11), p. 123-128

  However, the assay method of Non-Patent Document 2 requires a two-step antigen-antibody reaction, so that the measurement time has not been shortened yet. In order to shorten the measurement time, it is conceivable to increase the surface area of the reaction field of the antigen-antibody reaction. From the viewpoint of the concentration of the antibody to be immobilized, a microchamber (detection part) that is a reaction field is suitable. It was difficult for Non-Patent Document 2 to set a large size (volume). On the other hand, there is a growing need for more sensitive and accurate measurements (tests) than ever before, in order to quickly capture the slightest changes in the body and enable effective diagnosis or treatment at an early stage. Yes.

  Therefore, the object of the present invention is to greatly reduce the measurement time while taking advantage of the immunoassay such as the ease of measurement operation, high accuracy and low cost, and further, the sensitivity is more than one digit higher than the conventional method. It is to provide an immunoassay and a test disc that can be used for it.

  In order to solve the above-mentioned problems, the present inventors have conducted extensive research and changed the measurement principle to the competitive binding method (hereinafter referred to as the competitive method), and implements the following method and the method thereof. An inspection disc was developed. Specifically, an antigen is immobilized on a microbead instead of an antibody, and preferably the size (volume) of a microchamber (detection unit) as a reaction field and the microbead held (filled) in the microchamber. The above-mentioned problems were solved and the present invention was completed by making the total volume of the above range constant.

  That is, according to the first invention of the present invention, 2 to 100 microchannels are formed, and in each of the microchannels, a pre-reaction sample supply unit, an antigen-antibody reaction detection unit, and a post-reaction An immunity for detecting a measurement object by a competitive binding principle using a disk-shaped test container, which has a sample discharge section, and further holds a microbead on which an antigen is immobilized. A clinical assay is provided. In this application, the term “microchannel” refers to the entire basic configuration including the supply unit, the detection unit, and the discharge unit in principle, and does not include the supply unit, the detection unit, and the discharge unit. It is simply referred to as “flow path”.

  According to the second invention, it is preferable that the antigen immobilized on the microbead is a tumor marker.

  According to the third invention, the tumor marker is immobilized on the microbeads with an aqueous solution of the tumor marker having a concentration of 1 μg / mL to 20 μg / mL, and the measurement object can be detected by the competitive binding principle. Is preferred.

According to the fourth invention, it is preferable that one volume of the detection unit is 0.001 to 0.1 mm ³ .

  According to the fifth invention, there is provided an immunoassay method wherein the reaction time of the antigen-antibody reaction is 1 to 5 minutes.

  According to the sixth invention, 2 to 100 microchannels are formed, and a pre-reaction sample supply unit, an antigen-antibody reaction detection unit, and a post-reaction sample discharge unit are formed in each of the microchannels. And a test disk for use in an immunoassay method, in which microbeads on which an antigen is immobilized are held in the detection section.

  According to the seventh invention, the antigen immobilized on the microbeads of the examination disk is preferably a tumor marker.

According to the eighth invention, it is preferable that the volume of one of the detection units is 0.001 to 0.1 mm ³ .

  According to the immunoassay method and test disk of the present invention, the measurement time of the immunoassay can be greatly shortened. Furthermore, the biological sample can be quantified with higher sensitivity and accuracy than the conventional method by one digit or more.

It is process drawing which shows roughly the measurement flow of the immunoassay of this invention. (A) is a figure which shows typically an example of the test | inspection disc of this invention by a top view. (B) is a schematic diagram conceptually showing one microchannel. In a single microchannel, a cross-sectional view of the single microchannel schematically shows a state in which an antigen-antibody reaction is performed by holding a microbead on which an antigen is immobilized in a detection unit (microchamber). is there. (A) is the figure which represented roughly sectional drawing which looked at the vicinity of one detection part (microchamber) from the side. (B) is the figure which showed roughly sectional drawing of the arrow direction of the IVb-IVb line | wire of (a), in order to show the volume V3 part of a detection part. It is a figure which shows typically another example of the test | inspection disc of this invention which has the 2nd supply part connected with the some detection part in top view. FIG. 3 is a graph showing the relationship between the concentration of an antigen (tumor marker) to be measured and chemiluminescence intensity (CL intensity; Chemiluminescence intensity) in the first embodiment.

  In the following, embodiments of the present invention will be described with reference to the drawings, with respect to important points for solving the problems and features that are different from the prior art.

  In the immunoassay method of the present invention, an antigen is immobilized on microbeads and held in a microchamber which is a reaction field and a detection part to cause an antigen-antibody reaction. When an antibody is immobilized on a carrier such as a bead (solid phase) or the like, the molecular form (structure) of the antibody has a common structure called a Y-shaped heterotetramer. It is established and has no particular difficulty. However, in the case of immobilizing an antigen, since the antigen has molecules having various structures and properties, there is a difficulty in immobilizing the antigen on the carrier because the method needs to be devised for each target antigen.

  Therefore, a method of immobilizing an antibody on a carrier and labeling an antigen and performing an immunoassay by a competitive method can be considered, but for example, when an antigen is labeled with an enzyme, there is a problem that the reaction efficiency with the enzyme is low. is there. Furthermore, there is a disadvantage that the cost is high when an antigen such as a tumor marker is used as a labeled antigen as a reagent. Therefore, in order to take advantage of this assay method with high accuracy and low cost, a competition method by immobilizing an antigen was completed.

  However, more than the above difficulties, when an antigen is immobilized, an antibody having a relatively large molecular weight must approach the surface of the microbead and react with the immobilized antigen without steric hindrance. There was a need to overcome the difficulties. Details of the embodiments of the method and apparatus for solving these difficulties will be described below.

  That is, in the present invention, the test disc 1 as shown in FIG. 2 is used as an example, and the immunoassay by the competition method in antigen immobilization is performed according to the flow shown in FIG. Preferably, highly accurate measurement can be performed in a short time by setting the size of the microchamber 3 as a detection unit within a predetermined range.

  There are various antigens such as bacteria and viruses that enter the living body from the outside, and tumor markers that are released into the blood due to tumors such as cancer. A tumor marker is preferable as the antigen in terms of the immobilization ability to the microbeads 7 of the present invention and the significance of microanalysis in prognostic observation after surgery.

  Tumor markers include CEA (carcinoembryonic antigen), STN (sialyl Tn antigen), TPA (tissue polypeptide antigen), PSA (prostate specific antigen), AFP (α-fetoprotein) and erbB-2 (HER2 protein code) Gene) and the like. Glycoprotein systems such as CEA, AFP and PSA are preferred in view of the suitability of the reaction with the antibody on the microbeads 7.

  The measurement procedure of the immunoassay according to the present invention will be described with reference to the flow chart of the measurement flow shown in FIG. First, an antigen is immobilized on a plurality of microbeads 7 by a carbodiimide method or the like (S1). The antigen to be immobilized (immobilized antigen 8) is preferably a tumor marker for the reasons described above.

  The microbeads 7 of the present invention may be in the form of a sphere, polyhedron, egg, rugby ball, or a sphere having a large number of dimples on the surface, such as a golf ball, from the supply unit 2 to the detection unit 3. From the viewpoint of fluidity and efficient progress of the antigen-antibody reaction on the surface of the microbead 7, a sphere (including not only a true sphere but also a substantially sphere) is preferable. In addition, when the microbead 7 is a sphere, the diameter is 1 μm or more and 100 μm or less, and 5 μm or more is preferable in terms of effectively holding the detection unit 3. In addition, the diameter is preferably 50 μm or less and more preferably 30 μm or less in terms of immobilizing the antigen 8 so that the surface density is suitable for antigen-antibody reaction. Even when the microbeads 7 have a shape other than a sphere, the size is preferably approximately the same as the above size.

  The material of the microbead 7 is not particularly limited as long as the antigen is immobilized, and glass, resin such as polystyrene or polycarbonate, metal such as gold or silver, and metal compound such as stainless steel can be used. . Resin-made microbeads are preferable because they are inexpensive and lightweight.

  The number of microbeads 7 used for antigen immobilization is determined to be several thousand to several tens of thousands per detection part of the test disk 1. The specific number per detection unit is determined by the relative relationship between the volume of the microbead 7 and the size (volume) of the detection unit 3. From the standpoint of improving the reaction rate and high sensitivity, the total volume of the microbeads is preferably 5% or more, more preferably 10%, and even more preferably 25% or more of the volume of the detection unit 3. On the other hand, since it is preferable to incubate in the antigen-antibody reaction as will be described later, the total volume of the microbeads is 70% or less of the detection part volume in terms of the contact efficiency between the sample solution and the microbeads during the incubation. Preferably, it is 50%, more preferably 40% or less.

  Note that the volume of one detection unit 3 is a value obtained by multiplying the area of the part connected to the flow paths 5 and 6 and having a width larger than the flow paths 5 and 6 by the height (depth) of the part. . 4A is a cross-sectional view of the vicinity of one detection unit 3 as viewed from the side of the inspection disk. FIG. 4A is a cross-sectional view taken along the line IVb-IVb in FIG. 4 (the vicinity of the one detection unit 3 is shown). FIG. 4B shows a cross-sectional view as viewed from the upper surface of the inspection disk. The volume of one detection unit 3 is indicated by V3 in FIG. Although the detection unit 3 is open and connected to the flow paths 5 and 6, the volume V3 is a volume up to the boundary between the detection unit 3 and the flow paths 5 and 6.

From the above, the detection unit 3 of the present invention preferably has a volume of 0.001 to 0.1 mm ³ in terms of holding a suitable number of microbeads for the antigen-antibody reaction. In order to secure this volume, the width of the detection unit 3 is about 2 to 10 times the width of the flow path 5 or 6, and the length of the detection unit 3 is about 100 to 3000 μm. The depth is preferably 1 to 2 times that of the flow path 5 from the supply unit 2 to the detection unit 3 in that the antigen-antibody reaction is efficiently performed on the microbead surface. The shape of the detection unit 3 is not particularly limited as long as the volume can be secured. For example, the detection unit 3 may take various shapes such as a substantially rectangular parallelepiped or a top view as shown in FIG.

  The number of microbeads is determined so that the total volume of the microbeads held in the detection unit 3 and the volume of the detection unit 3 have the above-described relative relationship, and an appropriate suspension is measured from the suspension whose concentration is measured by a hemocytometer. Prepare a concentration of microbead suspension. An antigen is added to the suspension and immobilized while incubation (S1). Specific examples of operations will be described in detail in the first embodiment described later.

  The antigen-immobilized microbead suspension prepared in this way is injected into the microchannel from the microchannel supply section 2 of the test disk 1 with a micropipette or the like, and the antigen-immobilized microbeads are centrifuged, for example, The detection unit 3 is filled with force (S3). When using an inspection disk having a plurality of microchannels as shown in FIG. 2A, the suspension is injected into each supply unit 2 and filled in each detection unit 3. At this time, in order for the antigen-antibody reaction in each microchannel to proceed uniformly, the ratio of the total volume of microbeads held in each detector 3 to the volume of the detector 3 is within the range shown above. Therefore, it is desirable that all the detection units 3 have substantially the same level.

  An example of the test disk is shown in FIG. 2, and it is preferable in terms of multi-sample simultaneous testing that the test disk 1 includes a large number of microchannels (including the supply unit 2, the detection unit 3, and the discharge unit 4). . The number of the microchannels is preferably 20 or more, more preferably 40 or more on one test disk, in order to improve the effectiveness of the simultaneous multi-analyte test. On the other hand, 100 or less is preferable, and 80 or less is more preferable in that the practical size of the inspection disk and the inspection operation are not hindered.

  In addition, from the viewpoint of convenience in which a test (measurement) sample or the like can be sent and filled into the detection unit 3 by centrifugal force, the plurality of microchannels formed on the test disk are symmetrically or equally positioned. It is preferably formed in the inspection disc. For example, as shown in FIG. 2 (a), it is preferable to arrange in a substantially point-symmetric form with the center of the inspection disk 1 as the symmetry point.

FIG. 2A is a schematic view when the inspection disk is viewed from above, and a large number of microchannels are simply described. FIG. 2 (b) schematically shows one micro flow path, where the supply section 2 and the discharge section 4 are open to the atmosphere, but the detection section 3 and the flow paths 5 and 6 are located inside the inspection disk. Is formed. Each of these portions has a certain space (volume) so that the microbead suspension and the sample solution can flow and be held. In addition, as for the supply part 2 and the discharge | emission part 4, the area of the upper surface part of the opening part has a preferable 0.5-3 mm < ² > from the point of easy supply and discharge | emission of a sample liquid etc. In this case, the volume of the supply unit and the discharge unit is a respective 0.5 mm ³ to 5 mm ³ or so. As described above, each microchannel is opened to the atmosphere only by the sample supply unit 2 and the discharge unit 4, and the channels 5 and 6 and the detection unit 3 are formed inside the main body of the inspection disk 1. .

  In the inspection disk of the present invention, the width of the flow path 5 from the supply unit 2 to the detection unit 3 is 50 to 200 μm, and the depth is 30 to 150 μm. Or when the cross section of this flow path 5 is circular, the diameter is 30-200 micrometers. Alternatively, the cross section of the flow path 5 may have an elliptical shape with the same cross sectional area. However, since it is necessary for the microbeads 7 to pass therethrough, the width, depth, or diameter of the channel 5 is determined within these ranges in consideration of the diameter of the microbeads 7.

  On the other hand, the width of the flow path 6 from the detection unit 3 to the discharge unit 4 is 50 to 200 μm and the depth is 2 to 10 μm. However, since it is necessary for the microbeads 7 to stop at the detection unit 3 and not flow out in the direction of the discharge unit 4, the width of the flow path 6 from the detection unit 3 to the discharge unit 4 depends on the diameter of the microbead 7. Determine within the range of

  FIG. 2 (a) shows a case where each microchannel has one supply unit 2, one detection unit 3 and one discharge unit 4, but there are 2 to 4 microchannels. You may have a supply part. Specifically, a flow path is formed in a form branched from a flow path 5 connecting the supply section 2 and the detection section 3 in FIG. 2, and a second, third, or fourth supply section is formed at the end of the flow path. May be. Alternatively, a plurality of flow paths may be formed so that the second, third, and fourth supply units are directly connected to the detection unit 3.

  As another form of the inspection disk, as schematically shown in FIG. 5, separately from the supply unit 2 (a) corresponding to the supply unit 2 of FIG. You may provide the supply part 2 (b) which can be supplied. The form of the inspection disk shown in FIG. 5 is preferable in that the cleaning S7 and the luminescent substrate injection S8 in FIG. 1 can be performed quickly. The number of detection units 3 that can simultaneously supply the sample solution and the like from the supply unit 2 (b) is preferably 2 to 20 in terms of being able to uniformly supply the sample solution, the cleaning solution, the luminescent substrate, and the like, and in terms of efficiency. 4 or more are more preferable. More preferably, it is 8 or more.

  While preparing the antigen-immobilized microbeads, the antibody is labeled with an enzyme (S2) and the antigen to be measured is mixed to prepare a measurement sample solution (S4). After the antigen-immobilized microbeads are filled in the detection unit 3, the measurement sample solution is injected from the supply unit 2, and similarly sent to the detection unit 3 by centrifugal force to detect that the antigen-immobilized microbeads are filled. Part 3 is filled with the measurement sample solution (S5). After the antigen-immobilized microbeads and the measurement sample solution are brought into contact in this manner, an antigen-antibody reaction is performed by incubating for a predetermined time (S6).

  As the types of antibodies that can be used in the present invention, IgG, IgM, IgA, IgD, and IgE can be used. Of course, the antibodies that are specifically used for the measurement are antibodies that react with the antigen to be measured. Selected.

  FIG. 3 conceptually shows the internal state of the detection unit 3 in which the antigen-antibody reaction takes place. A microbead 7 having an antigen immobilized on the surface (immobilized antigen 8) is held. Since the depth of the flow path 6 connected from the detection section 3 to the discharge section 4 is smaller than the diameter of the microbead 7, the microbead 7 does not flow out toward the flow path 6 but stops at the detection section 3. The measurement sample solution containing the antibody 10 labeled with the enzyme and the measurement target antigen 9 is sent to the detection unit 3 as described above, and the enzyme labeled antibody 10 and the immobilized antigen 8 or the measurement target antigen 9 are sent. An antigen-antibody reaction is competitively performed between the two (FIG. 3).

  In the present embodiment, the amount of the enzyme-labeled antibody 10 is less than the amount that binds (reacts) with the measurement target antigen 9 and all of the immobilized antigen 8 on the microbead 7, and thus the measurement target antigen 9 and the immobilized antigen 8. And competitive binding occurs in the form of binding the enzyme-labeled antibody 10. Since the amount of the enzyme-labeled antibody 10 that binds to the immobilized antigen 8 varies with the regularity depending on the amount (concentration) of the antigen 9 to be measured in the sample, the enzyme-labeled antibody 10 that binds to the immobilized antigen 8. Is detected by emission intensity analysis or the like, the amount of antigen to be measured in the sample can be quantified.

  In measuring the amount of the enzyme-labeled antibody 10 bound to the immobilized antigen 8, after the antigen-antibody reaction by incubation, a buffer solution is injected from the supply unit 2 to wash the channels 5 and 6 and the detection unit 3 (S 7). ). The purpose of washing is to discharge and remove the binding substance between the measurement target antigen 9 and the enzyme-labeled antibody 10, the unreacted measurement target antigen 9 and other substances that become obstacles during detection. These discharges are discharged from the discharge part 4 of the microchannel.

After the washing, an aqueous solution containing p-iodophenol, luminol, and hydrogen peroxide (H ₂ O ₂ ) is injected from the supply unit 2 as a luminescent reagent to cause the enzyme-labeled antibody 10 bound to the immobilized antigen 8 to emit light ( S8) The emission intensity of the emitted light is measured with an imaging analyzer (S9). The above measurement (assay) operation is performed by changing the concentration of the antigen 9 to be measured, and a calibration curve regarding the relationship between the antigen concentration and the luminescence intensity is created. In creating the calibration curve, the test disk may be changed for each concentration of the antigen 9 to be measured, and the concentration of the antigen to be measured is changed for each microchannel using a test disk having a plurality of microchannels. Measurement may be performed with one inspection disk. In terms of time saving and efficiency, it is preferable to perform measurement that can create a calibration curve with a single inspection disk.

  The actual measurement target sample whose antigen concentration is unknown is measured by the same measurement flow as described above, and the obtained luminescence intensity is plotted on the calibration curve, and the antigen concentration is read to determine the antigen concentration in the unknown sample. Can be determined.

  With respect to the antigen-antibody reaction described above, a few minutes, specifically, 2 minutes or more within 10 minutes is sufficient. In consideration of the time lag due to simultaneous measurement in a plurality of microchannels on one inspection disk, 3 minutes or more is preferable.

  As an example of the present invention, immunoassay was performed using CEA (carcinoembryonic antigen) as a tumor marker as an antigen, and a goat polyclonal antibody labeled with horseradish peroxidase (HRP) as an enzyme-labeled antibody. . In this example, a calibration curve obtained when the antigen-antibody reaction was performed for 5 minutes is shown in FIG. Note that a calibration curve having almost the same plot was drawn even when the antigen-antibody reaction was 10 minutes and 15 minutes.

  As is clear from FIG. 6, there is a clear difference in luminescence intensity between the measurement target antigen concentration of 0.9 pg / mL and 9 pg / mL. This is because the measurement target antigen concentration in the sample is within this range. It can also be measured. As described above, according to the present invention, extremely sensitive measurement can be performed in a short time and with high accuracy.

  As a material of the test disc used in the immunoassay of the present invention, any material such as a polymer material such as a thermoplastic resin or a thermosetting resin, ceramics, glass, silicon or metal can be used. Glass, silicon, a polymer material, or a combination thereof is preferable in terms of hermeticity and fine workability. As the polymer material, for example, siloxanes such as polydimethylsiloxane (PDMS) can be used. Note that these materials are preferably transparent in order to visually or optically observe the state of the microchannel and the microchamber (detection unit).

  The following method can be mentioned as a manufacturing method of the said inspection disk. For example, it can be manufactured by adhering a sheet or plate of the above-mentioned material having a flow path and a microchamber pattern and a flat sheet or plate having a sample solution supply part and a discharge part. Further, the supply unit and the discharge unit may be formed on a sheet or plate having a flow path and a microchamber pattern. Alternatively, the inspection disk is manufactured by pasting together patterns (for example, substantially plane-symmetric shapes) created by dividing the flow path, microchamber, supply unit, and discharge unit patterns into two sheets or plates. May be. In terms of being suitable for optical observation and analysis, the thickness of the inspection disk is preferably 1 mm to 3 mm.

  A sheet having a pattern such as a flow path and a micro chamber may be formed by pressing with a mold or pouring a fluid material such as a thermoplastic resin into the mold. A pattern of a mold may be formed, and a fluid material such as a thermoplastic resin may be poured onto the pattern, or may be formed directly on a sheet or plate by machining or etching using photolithography. .

<Embodiment 1>
Hereinafter, preferred embodiments will be described in more detail with reference to the drawings.

Production of Inspection Disk First, a microchannel mold was produced using a silicon wafer having a diameter of 4 inches. A thin film photoresist (SU-8 2005) was spin-coated on a silicon wafer at 2000 rpm for 30 seconds, and spread. This was heated on a hot plate at 65 ° C. for 1 minute and at 95 ° C. for 2 minutes, and then set on a mask aligner together with a designed photomask, UV (350 nm) irradiation was applied for 90 seconds, and the thin film photoresist was photocured. . The silicon wafer was then heated at 65 ° C. for 1 minute and 95 ° C. for 1 minute.

  A thick film photoresist (SU-850) was dropped onto the silicon wafer, and rotated to 2000 rpm for 30 seconds with a spin coater to spread it to a uniform thickness (50 μm). Furthermore, after heating at 65 ° C. for 6 minutes and at 95 ° C. for 20 minutes on a hot plate, it is set on the mask aligner together with the designed photomask and UV (350 nm) irradiation is performed for 90 seconds, then at 65 ° C. for 1 minute and 95 ° C. For 5 minutes. Thereafter, the resist was immersed in a SU-8 developer to remove excess unpolymerized resist, and dried with dry nitrogen gas to complete a mold.

  A prepolymer of polydimethylsiloxane (PDMS) (Silgard 184 manufactured by Dow Corning) was poured into this mold and cured by heat. After the cured PDMS sheet is taken out from the mold and formed so that the supply unit and the discharge unit are open to the atmosphere, smooth glass having the same shape as the sheet is laminated on the sheet surface on which the recesses are formed. The inspection disk (diameter 100 mm, thickness 1.8 mm) provided with 64 microchannels shown in FIG.

The upper surface area of the openings of the supply part 2 and the discharge part 4 of the inspection disk produced in this embodiment is about 1 mm ² . Further, the “width × depth” of the flow channel 5 is “100 μm × 56 μm”, the volume of the detection unit 3 is 0.020 mm ³ (average width × length × depth = 350 μm × 1000 μm × 56 μm), “Width × depth” is “100 μm × 5 μm”.

Preparation of antigen-immobilized microbeads The tumor marker “Carcinoembryonic antigen (CEA)” was used as an antigen. CEA was immobilized on spherical polystyrene microbeads having a diameter of 10 μm by the carbodiimide method as follows. After 0.2 mL of the polystyrene microbead suspension, 0.75 mL of EDC solution (1 mg / mL) and 0.05 mL of ultrapure water were mixed, the mixture was slowly stirred at room temperature for 1 hour, and then centrifuged at 2000 rpm for 10 minutes to obtain a supernatant. The solution was removed. Further, 200 μL of CEA solution (9 μg / mL) was mixed, and the mixture was allowed to react with stirring slowly in a cold room (4 ° C.) overnight. After centrifuging this reaction suspension at 2000 rpm for 10 minutes, the supernatant solution was removed, 1 mL of blocking agent solution (400 ng / mL) was added, and the mixture was slowly stirred for 1.5 hours in an incubator (37 ° C.). After centrifuging the bead suspension at 2000 rpm for 10 minutes, the supernatant was removed and the beads were collected. 1 mL of TBST buffer (20 mM Tris-HCl, 0.15 M NaCl, and 0.05% Tween; pH 7.6) was added to the precipitated beads and resuspended. Thereafter, the washing operation for collecting the microbeads by centrifugation at 2000 rpm for 10 minutes was performed three times, and the same washing operation as described above was performed twice with 0.1 M phosphate buffer (PB; pH 7.0). PB was added to the beads to prepare 0.1 mL of a suspension. The CEA-immobilized microbead suspension was stored in a refrigerator until the immunoassay was performed.

CEA immunoassay according to the present invention (preparation and verification of calibration curve)
1.5 μL of the CEA-immobilized microbead suspension is injected from the supply unit 2 of the inspection disk 1 produced by the above method, and sent to the detection unit 3 by centrifugal force (3000 rpm, 30 seconds). The detection unit 3 was held. In the present embodiment, the ratio of the total volume of the antigen-immobilized microbeads to the volume of the detection unit 3 is filled and held so that each detection unit is 26 ± 0.5%.

A sample solution containing the CEA to be measured and a 1000-fold diluted HRP-labeled goat polyclonal antibody are mixed at 1: 1, and 1 μL of the measurement sample solution, which is the mixed solution, is injected from the supply unit 2 and centrifuged (3000 rpm, 30 seconds). Then, the solution was sent to the detector 3. After feeding, the antigen-antibody reaction was performed while incubating at room temperature. The CEA concentrations to be measured were 0.9, 9, 90, 900, and 9000 (pg / mL). In addition, measurement was also performed on PB containing neither CEA nor HRP-labeled goat polyclonal antibody. This measurement was performed on one inspection disk using 3 to 5 flow paths (n number: 3 to 5) for one kind of concentration. In addition, three types of incubation time of 5, 10 or 15 minutes were carried out for comparison.

  After incubation, inject 2 μL each of 5 times with TBST buffer and 2 times with PB from the supply unit 2, and wash the channels 5 and 6, the detection unit 3, and the microbeads 7 by centrifugal force (3000 rpm, 30 seconds). (The cleaning liquid was discharged from the discharge section 4). This operation was repeated twice to remove the binding substance between CEA and the HRP-labeled goat polyclonal antibody in the measurement sample solution, unreacted CEA, and other substances that interfere with detection.

After washing, 1 μL of a luminescent substrate aqueous solution (p-iodophenol 0.4 mM, luminol 0.375 mM, and H ₂ O ₂ 0.75 mM) is injected from the supply unit 2, and is issued to the detection unit by centrifugal force (3000 rpm, 30 seconds). The solution was fed and reacted with the labeled enzyme of HRP-labeled goat polyclonal antibody reacted with CEA immobilized on microbeads 7. As a result, the labeled enzyme present on the microbeads 7 emits light, and the chemiluminescence intensity (CL intensity) was measured with an imaging analyzer for 5 minutes. When an antigen-antibody reaction is performed under each condition of the CEA concentration to be measured, the amount of binding between CEA immobilized on the surface of the microbead 7 and the antibody is different, and a difference in chemiluminescence intensity appears.

  If there are few measurement target CEAs, there are many enzyme-labeled antibodies that react with CEA immobilized on the microbead surface. Conversely, if there are many measurement target CEAs, there are many enzyme-labeled antibodies that react with the measurement target CEA. Fewer enzyme-labeled antibodies react with the immobilized CEA. Therefore, the smaller the measurement target CEA, the stronger the emission intensity of the detection unit 3 (microbead holding portion), and the smaller the measurement object CEA, the weaker the emission intensity.

  The relationship between the CEA concentration in the sample and the emission intensity is shown in FIG. Although it is a measurement result when the antigen-antibody reaction time is 5 minutes, when the reaction time is 10 minutes or 15 minutes, almost the same plots and lines are obtained. In this embodiment, the antigen-antibody reaction time is 5 minutes. Minutes were shown to be sufficient. In FIG. 6, the luminescence intensity at 9000 pg / ml is the same as the background luminescence intensity when the PB containing neither CEA nor HRP-labeled goat polyclonal antibody was fed to the detector 3 and then the luminescent substrate aqueous solution was fed. It was shown that there is almost no difference. In addition, the measurement time from the injection of CEA-immobilized microbeads into the inspection disk to the measurement of the luminescence intensity is about 15 minutes, and the time is reduced to 1/2 to 1/4 of the method according to Non-Patent Document 2. It was.

  In addition, there is a clear difference in luminescence intensity between the CEA concentration in the sample of 0.9 pg / mL and 9 pg / mL, which indicates that measurement can be performed even if the concentration of CEA in the sample is within this range. ing. In this regard, the known samples with different CEA concentrations were measured several times, and all were confirmed to be plotted on the calibration curve in FIG. From the above, it has been proved that the present invention can perform highly sensitive measurement in a short time and with high accuracy.

  The reason why high-precision measurement can be performed in such a short time is not clear, but the ratio of the volume of the microbead to the volume of the microchamber, which is the detection unit, is specified, and very small microbeads are used. One reason is that the surface area, which is the reaction field, was increased. Further, it is considered that the concentration of the antigen-antibody reaction on the microbead surface can be reduced to 1/10 or less of the method of Non-Patent Document 2 due to these reasons. That is, because the concentration of the antigen and antibody on the microbead surface has decreased, the antibody with a relatively large molecular weight can proceed smoothly with the immobilized antigen without undergoing steric hindrance. It seems to be.

  By enabling high-sensitivity and rapid immunoassay, it can be applied to early detection of cancer metastasis, for example, by early and accurate diagnosis of various diseases such as cancer or infectious diseases, and follow-up of prognosis. .

1 Inspection disk 2, 2a, 2b Supply unit 3 Detection unit (microchamber)
4 Discharge parts 5 and 6 Channel 7 Microbead 8 Immobilized antigen 9 Antigen to be measured 10 Enzyme-labeled antibody 11 Another type of test disk

Claims (8)

  2 to 100 microchannels are formed, and each of the microchannels has a pre-reaction sample supply unit, an antigen-antibody reaction detection unit, and a post-reaction sample discharge unit. An immunoassay method in which a measurement target is detected by a competitive binding principle using a disc-shaped test container in which immobilized microbeads are held in the detection unit.   2. The immunoassay method according to claim 1, wherein the antigen immobilized on the microbead is a tumor marker.   The immunity according to claim 1 or 2, wherein the tumor marker is immobilized on the microbead with an aqueous solution of the tumor marker having a concentration of 1 µg / mL to 20 µg / mL, and the measurement target is detected by a competitive binding principle. Test. The immunoassay according to any one of claims 1 to ³ , wherein one volume of the detection unit is 0.001 to 0.1 mm ³ .   The immunoassay method according to any one of claims 1 to 4, wherein a reaction time of the antigen-antibody reaction is 1 to 5 minutes.   2 to 100 microchannels are formed, and each of the microchannels has a pre-reaction sample supply unit, an antigen-antibody reaction detection unit, and a post-reaction sample discharge unit. A test disk used for immunoassay, in which immobilized microbeads are held in the detection section.   The test disc used for the immunoassay according to claim 6, wherein the antigen immobilized on the microbead is a tumor marker. The test disk used for the immunoassay according to claim 6 or 7, wherein one volume of the detection unit is 0.001 to 0.1 mm ³ .

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