Immune analysis method, and immune analysis device

Abstract

To provide an immune analysis method and immune analysis device that do not need a cleaning process.SOLUTION: An immune analysis method according to the present invention has: a first process of reacting an analysis object substance 1, a first labelled molecule 3 having a first capture material 2 binding specifically to the analysis object substance 1, and a magnetic fine particle 5 having a second capture material 4 binding specifically to the analysis object substance 1 in the same solution; and a second process of putting the solution reacted in the first process to contact with a magnetic sensor 8 in which a second labelled molecule 7 binding specifically to the first labelled molecule 3 is fixed on a surface, and detecting intensity of a magnetic field induced by the magnetic fine particle 5 by the magnetic sensor 8 as it is.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to an immunoassay method and an immunoassay device. [Background technology]

[0002] Methods for quantitatively analyzing specific biological substances in samples are essential for investigating reactions and conditions in the body, and are widely used in clinical diagnosis, evaluation of drug efficacy and side effects, etc. Commonly used analytical methods include ELISA (Enzyme-Linked Immuno-Sorbent Assay) and ECL (Electro-Chemical Luminescence).

[0003] Additionally, there are analytical methods that use antibodies to specifically capture, label, and analyze analytes (immunoanalysis methods). In general, the bond formation between an antibody and an analyte such as a protein is an equilibrium reaction, and not only the binding process but also the dissociation process occurs simultaneously. For this reason, in immunoanalysis methods, it is common to select and use an antibody that is less likely to undergo the dissociation process than the binding process, that is, an antibody with a small dissociation constant (= dissociation rate constant / binding reaction rate constant). However, depending on the analyte, it may be difficult to select an antibody with a sufficiently small dissociation constant.

[0004] Furthermore, in recent years, for example, a technique described in Patent Document 1 (a method for detecting a target substance) has been proposed for an analytical method. The technique described in Patent Document 1 includes the following steps (a) to (e). (a) is a step of preparing a substrate having a nonspecific adsorption prevention film, a particle having a first ligand molecule that specifically captures a target substance, and a second ligand molecule having a site that specifically captures the target substance and a light irradiation binding site that binds to the nonspecific adsorption prevention film by light irradiation. (b) is a step of binding the first ligand molecule and the second ligand molecule to the target substance in a reaction medium to form a complex comprising the particle having the first ligand molecule, the target substance, and the second ligand molecule. (c) is a step of separating the complex by B / F separation. (d) is a step of binding the light irradiation binding site in the second ligand molecule contained in the complex to the nonspecific adsorption prevention film by light irradiation. (e) is a step of detecting the presence or absence or the amount of particles fixed to the nonspecific adsorption prevention film. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2009-288069 A Summary of the Invention [Problem to be solved by the invention]

[0006] However, in order to accurately quantify the analyte in any of the conventional analytical methods, including the target substance detection method described in Patent Document 1, a washing step (B / F separation step) was required to remove unreacted labeling materials and ligand molecules during analysis. Note that B / F separation means bound / free separation, and is synonymous with washing in a washing step. When these steps are performed, in analytical methods such as ELISA, it is impossible to avoid a dissociation reaction of a complex containing the analyte. In addition, in the target substance detection method described in Patent Document 1, a dissociation reaction of a complex (sandwich complex) inevitably occurs. As a result, all of the conventional analytical methods lead to a decrease in the final luminescence intensity and signal intensity, and the sensitivity is reduced, making it impossible to accurately quantify the analyte.

[0007] Furthermore, when detecting anti-drug antibodies produced in association with the administration of biopharmaceuticals such as antibody drugs, drugs are used as capture materials, but in many cases, it is not guaranteed that the drug has a sufficiently small dissociation constant. In addition, autoantibodies produced when a person is afflicted with cancer are expected to be useful as cancer-specific markers for early cancer diagnosis. To detect cancer-specific markers, tissue components derived from cancer are used as capture materials for autoantibodies. However, there is a large difference between individuals in the binding strength of autoantibodies to cancer tissues that serve as antigens, and this may result in low sensitivity in diagnosis. In other words, if a complex is formed using a drug or antibody that does not have a sufficiently small dissociation constant and then a washing process is performed, the capture material will dissociate from the complex. As a result, the luminescence intensity and signal intensity are reduced, and the sensitivity is reduced, making it impossible to accurately quantify the analyte.

[0008] An object of the present invention is to provide an immunoassay method and an immunoassay device that do not require a cleaning step. [Means for solving the problem]

[0009] The immunoassay method of the present invention comprises a first step of reacting an analyte, a first labeled molecule having a first capture material that specifically binds to the analyte, and a magnetic particle having a second capture material that specifically binds to the analyte in the same solution, and a second step of contacting the solution reacted in the first step with a magnetic sensor having a second labeled molecule fixed to its surface that specifically binds to the first labeled molecule, and detecting the strength of the magnetic field induced by the magnetic particle with the magnetic sensor. Effect of the Invention

[0010] According to the present invention, it is possible to provide an immunoassay method and an immunoassay device that do not require a washing step. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing the details of a first step in an immunoassay method. [Diagram 2] FIG. 2 is a schematic diagram showing the details of a second step in the immunoassay method. [Diagram 3] FIG. 1 is a schematic diagram showing a configuration of an immunoanalyzer. [Figure 4] A graph plotting magnetic signal intensity against antigen concentration (horizontal axis: antigen concentration (CEA concentration (pM)), vertical axis: magnetic signal intensity). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Next, one embodiment will be described with reference to the drawings as appropriate. In the drawings to be referred to, Fig. 1 is a schematic diagram showing the contents of a first step in an immunoassay method. Fig. 2 is a schematic diagram showing the contents of a second step in an immunoassay method.

[0013] [Immunological analysis method] The immunoassay method according to this embodiment (hereinafter, may be referred to as "this analysis method") has a first step and a second step. 1, the first step is a step of reacting an analyte 1, a first labeled molecule 3 having a first capture material 2 that specifically binds to the analyte 1, and a magnetic particle 5 having a second capture material 4 that specifically binds to the analyte 1 in the same solution. When these react in the solution in the first step, a sandwich complex 6 is produced. As shown in FIG. 2, the second step is a step in which the solution reacted in the first step is brought into contact with a magnetic sensor 8 having second labeled molecules 7 fixed to its surface that specifically bind to the first labeled molecules 3 (more specifically, the solution is brought into contact with the second labeled molecules 7 fixed to the surface of the magnetic sensor 8), and the strength of the magnetic field induced by the magnetic particles 5 is detected by the magnetic sensor 8.

[0014] The analyte 1 may be anything that can specifically bind to the first capture material 2 and the second capture material 4. Examples of such analyte 1 include, but are not limited to, tumor markers such as SCC, CXA-125, CEA, SLX, CYFRA, NSE, ProGRP, AFP, PIVKA-II, CA19-9, PSA, CA-125, CA15-3, NCC-ST-439, STN, Elastase I, and βHCG.

[0015] The first capture material 2 and the second capture material 4 are preferably capable of specifically binding to the analyte 1. The first capture material 2 and the second capture material 4 are preferably different in epitope (recognition site) for the analyte 1. The first capture material 2 and the second capture material 4 are preferably capable of forming a sandwich complex 6 (complex) composed of the first capture material 2, the analyte 1, and the second capture material 4. The first capture material 2 and the second capture material 4 are preferably, for example, a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody, a single-chain antibody, and a gene construct containing an antibody binding region. In addition, when the analyte 1 is an autoantibody, it is preferably a compound or tissue fragment that is a part of a component of a drug or tumor tissue, or a similar composition.

[0016] The first capture material 2 is modified in advance with the first labeled molecule 3. Examples of the first labeled molecule 3 include biotin and digoxigenin. The modification of the first capture material 2 with the first labeled molecule 3 can be performed, for example, by using a commercially available compound in which a functional group known to selectively react with an amino group, such as N-hydroxysuccinamide and biotin are linked by a carbon chain. When an antibody is used as the first capture material 2, the modification of the first labeled molecule 3 can be performed by cleaving the disulfide bond at the hinge site of the antibody with a reducing agent in advance, and using a commercially available compound in which the resulting sulfhydryl group, biotin, and maleimide group are linked by a carbon chain. When the first capture material 2 has a sulfhydryl group, the modification of the first labeled molecule 3 can be performed similarly by using a commercially available compound in which biotin and a maleimide group are linked by a carbon chain.

[0017] The second capture material 4 is fixed to the surface of the magnetic fine particles 5 in advance. The magnetic fine particles 5 can be made of fine particles of a magnetic material such as ferrite wrapped in a polymer such as dextran or lipid. Considering both dispersibility and detectability by the magnetic sensor 8, the magnetic fine particles 5 are preferably superparamagnetic. The particle size of the magnetic fine particles 5 is preferably selected from the viewpoints of dispersibility, superparamagnetism, reaction speed, etc., and is preferably 1 μm or less, more preferably 300 nm or less. The method of fixing the second capture material 4 to the surface of the magnetic fine particles 5 depends greatly on the surface coating material of the magnetic fine particles 5. For example, when using magnetic fine particles 5 coated with carboxymethyl dextran, a method of fixing them can be used in which carbodiimide is used as a linking reagent between the carboxyl group present on the surface of the magnetic fine particles 5 and the amino group of the second capture material 4. In this way, the binding reaction of these groups can be easily carried out, and the second capture material 4 can be easily fixed to the surface of the magnetic fine particles 5. As a method for immobilizing them, for example, when a lipid-coated magnetic particle 5 is used, a coupling reaction using cyanogen bromide as a linking agent can be used between the hydroxyl groups present on the surface of the magnetic particle 5 and the amino groups of the second capture material 4. In this case as well, as in the above, the binding reaction of these groups can be easily carried out, and the second capture material 4 can be easily immobilized on the surface of the magnetic particle 5.

[0018] The magnetic sensor 8 used in the second step may have any configuration as long as it can detect the magnetic fine particles 5. For example, a Hall element, a magnetoresistance effect element, a giant magnetoresistance effect (GMR) element, a tunnel magnetoresistance effect element, a SQUID (superconducting quantum interference) element, or the like can be used as the magnetic sensor 8. It is preferable that the surface of the magnetic sensor 8 is subjected to a surface treatment suitable for immobilizing the second labeled molecule 7. For example, it is preferable that a thin film of silicon oxide is vapor-deposited on the surface of the magnetic sensor 8, and a film having a strong adsorptive force is formed thereon using aminosilane or the like.

[0019] The second labeled molecule 7 immobilized on the surface of the magnetic sensor 8 specifically binds to the first labeled molecule 3. In other words, the second labeled molecule 7 may be selected appropriately depending on the first labeled molecule 3. For example, when biotin is used as the first labeled molecule 3, avidin or streptavidin may be selected as the second labeled molecule 7. Furthermore, when digoxigenin is used as the first labeled molecule 3, an anti-digoxigenin antibody may be selected as the second labeled molecule 7.

[0020] As described above, in the second step, the solution reacted in the first step is brought into contact with the magnetic sensor 8 on the surface of which the second labeled molecule 7 that specifically binds to the first labeled molecule 3 is immobilized, and the magnetic field strength induced by the magnetic fine particles 5 is detected by the magnetic sensor 8 as it is. The magnetic sensor 8 can obtain an output signal proportional to the amount of the second labeled molecule 7, the first labeled molecule 3, the first capture material 2, the analyte 1, and the magnetic fine particles 5 immobilized on the surface of the magnetic sensor 8 via the second capture material 4. Here, "as it is" means that after the solution reacted in the first step is brought into contact with the magnetic sensor 8, the contact state between them is maintained in the solution without performing a washing step (B / F separation). In other words, in this analysis method, after forming a sandwich complex 6 in a solution in the first step, the solution is brought into contact with the magnetic sensor 8 in the second step, and the signal of the magnetic sensor 8 is detected in the solution without performing a washing step. In this way, since washing is not performed, a dissociation reaction in the sandwich complex 6 is unlikely to occur, and the final emission intensity and signal intensity are unlikely to decrease. Therefore, the present analytical method is less susceptible to a decrease in sensitivity and can accurately quantify the analyte 1. The reason why the present analytical method can accurately quantify the analyte 1 even though it does not include a washing step will be explained later.

[0021] The magnetic field caused by the magnetic particles 5 can be generated by applying a magnetic field using an external magnetic field generating device 10. The external magnetic field generating device 10 can be, for example, a Helmholtz coil in which two identical coils are arranged to have the same central axis.

[0022] An example of this analytical method will now be described in detail. Analysis of the analyte 1 can be carried out as follows. First, in the first step, as shown in Fig. 1, a sample containing an analyte 1 in a buffer solution is mixed with a first capture material 2 previously modified with a first labeling molecule 3, and a magnetic particle 5 previously immobilized on the surface with a second capture material 4 to prepare a mixed solution 9. The buffer solution may be, for example, a phosphate buffer prepared to have a predetermined pH, such as pH 7.0. The mixed solution 9 may contain a blocking agent, such as bovine serum albumin or mouse IgG, to suppress non-specific reactions.

[0023] The mixture 9 is placed in a reaction vessel and reacted at a predetermined temperature such as room temperature or 37° C. for a predetermined time such as 30 minutes or 60 minutes. In order to increase the reaction efficiency, it is preferable to use a stirring means such as a shaker for the reaction. After the reaction is completed, a sandwich complex 6 is formed as shown in FIG.

[0024] Next, in the second step, as shown in Fig. 2, the mixed solution 9 containing the sandwich complex 6 is brought into contact with a magnetic sensor 8 having a surface on which a second labeled molecule 7 that specifically binds to the first labeled molecule 3 is immobilized, for a predetermined time and in a predetermined environment, without purification or washing. Alternatively, in the second step, the mixed solution 9 containing the sandwich complex 6 can be placed in a predetermined container, and the magnetic sensor 8 can be immersed therein. Furthermore, in the second step, the mixed solution 9 containing the sandwich complex 6 can be dripped onto the surface of the magnetic sensor 8 as droplets, and allowed to stand in a sufficiently humid environment for a predetermined time.

[0025] As described above, the combination of the second labeled molecule 7 that specifically binds to the first labeled molecule 3 can be, for example, a combination of biotin and avidin or streptavidin, or a combination of digoxigenin and an anti-digoxigenin antibody. As a method for immobilizing the second labeled molecule 7 on the surface of the magnetic sensor 8, for example, the surface of the magnetic sensor 8 is coated in advance with aminopropylsilane or poly(L-lysine), and then a solution containing the second labeled molecule 7 is brought into contact with the surface. In this way, the second labeled molecule 7 is immobilized on the surface of the magnetic sensor 8 by physical adsorption.

[0026] In this analysis method, the amount of the second labeled molecule 7 fixed on the surface of the magnetic sensor 8 is preferably at least 1 / 10 of the total amount of the first labeled molecule 3, more preferably 1 / 4 or more, and even more preferably 1 / 2 or more. In this way, high sensitivity detection is possible. More preferably, the amount of the second labeled molecule 7 fixed on the surface of the magnetic sensor 8 is greater than the amount of the first labeled molecule 3. The unreacted first capture material 2 that is not involved in the preparation of the sandwich complex 6 is also fixed on the surface of the magnetic sensor 8 through the bond between the first labeled molecule 3 and the second labeled molecule 7. If the amount of the second labeled molecule 7 is less than the amount of the first labeled molecule 3, the sandwich complex 6 may be less than the amount required for detection by the magnetic sensor 8. This is because the unreacted first capture material 2 modified with the first labeled molecule 3 has a smaller molecular size than the sandwich complex 6, and therefore may be preferentially captured by the second labeled molecule 7 present on the surface of the magnetic sensor 8. Therefore, in order to obtain high sensitivity, it is preferable that the amount of the second labeled molecule 7 fixed on the magnetic sensor 8 is greater than the amount of the first labeled molecule 3. The upper limit of the amount of the second labeled molecule 7 immobilized per unit area of ​​the magnetic sensor 8 is determined by the area of ​​the magnetic sensor 8 and the size (occupancy area) of the second labeled molecule 7. For example, when the length of one side of the second labeled molecule 7 is about 6 nm, the upper limit of the amount of the second labeled molecule 7 immobilized per unit area of ​​1 μm 2 Among them, the calculation is 1000nm × 1000nm ÷ 36nm 2 =The upper limit is approximately 27,800.

[0027] In the second step, the solution (mixture 9) containing the sandwich complex 6 is brought into contact with the magnetic sensor 8, and the amount of the magnetic particles 5 fixed on the surface of the magnetic sensor 8 is calculated as it is. The amount of the magnetic particles 5 is calculated by using a reading device 11 for reading the output of the magnetic sensor 8 and a control device 12 for calculating and storing the output, and the amount of the magnetic particles 5 fixed on the surface of the magnetic sensor 8, that is, the amount of the sandwich complex 6 is calculated. As described above, the magnetic sensor 8 can obtain an output signal proportional to the amount of the magnetic particles 5 attached to the surface. When a giant magnetoresistance sensor is used as the magnetic sensor 8, a magnetic field is applied to the magnetic sensor 8 and the solution containing the magnetic particles 5 in contact with it. The magnetic field can be applied by the external magnetic field generator 10 described above. The external magnetic field generator 10 magnetizes the magnetic particles 5 having superparamagnetism, and the strength of the induced magnetic field is detected by the magnetic sensor 8.

[0028] The strength of the magnetic field attenuates in proportion to the cube of the distance. The magnetic sensor 8 can detect only the magnetic field of the magnetic particles 5 that are present within approximately 500 nm from the surface of the magnetic sensor 8 and remain there for a predetermined time. In other words, the unreacted magnetic particles 13 that are not contained in the sandwich complex 6 and are dispersed in the solution do not remain in one place even if they are temporarily present within 500 nm, so they are not detected by the magnetic sensor 8. Therefore, in this analysis method, the amount of the sandwich complex 6 can be accurately quantified without washing and removing the unreacted magnetic particles 13 in the second step. When a giant magnetoresistance sensor is used as the magnetic sensor 8, the resistance value of the magnetic sensor 8 is measured by the reading device 11, and the measured value measured by the reading device 11 is input into the control device 12, where calculations, data storage, display, etc. are performed. The control device 12 can be a general computer or a dedicated control device developed for measurement by the magnetic sensor 8.

[0029] The conditions for detecting the sandwich complex 6 with the magnetic sensor 8 are as follows. The number of molecules of the first capture material 2 having the first label molecule 3 is a, the number of molecules of the analyte 1 is b, and the area of ​​the magnetic sensor 8 is f (μm 2), the number of molecules of the second label molecule 7 is g, and the minimum density of magnetic particles at which the magnetic sensor 8 can detect the magnetic particles 5 immobilized on its surface is h (particles / μm 2 The reaction efficiency of the formation of sandwich complex 6 in solution is 100%. The binding rate constant is 10 5 ~10 6 (M -1 s -1 ) as the first capture material 2 and the second capture material 4, the capture material concentration during the reaction is 1 nM or more, and the reaction time is about 1 hour, it is easy to achieve a high reaction efficiency of nearly 100% for the formation of the sandwich complex 6. The immobilization density of the sandwich complex 6 immobilized on the magnetic sensor 8 is given by b×g / a / f. If this immobilization density is higher than the detection sensitivity of the magnetic sensor 8 (the minimum magnetic particle density h), the sandwich complex 6, i.e., the analyte 1, can be quantified and detected.

[0030] For this purpose, the ratio of the second labeled molecules 7 (number of molecules g) to the first captured material 2 (number of molecules a) having the first labeled molecules 3 should be set to satisfy g / a>h×f / b, i.e., b×g / a / f>h. For example, when the analyte 1 is detected with a detection sensitivity of 10 fM (number of molecules b=10×10 -15 x6x10 23 x400x10 -6 =24×10 5 The volume of the reaction solution is 400 μL, the area of ​​the magnetic sensor 8 is 100 μm × 120 μm, and the detection sensitivity (the magnetic particle density (number / unit area of ​​the magnetic sensor 8) at which the magnetic sensor 8 can detect the magnetic particles 5 fixed on its surface)) is 0.1 particles / μm 2 The concentration of the first capture material 2 having the first label molecule 3 was set to 1 nM (number of molecules a=1×10 -9 x6x10 23 x400x10 -6 =24×10 10 pcs), the number of molecules g of the second labeled molecule 7 is 1.2 × 10 8If streptavidin is used as the second labeled molecule 7 and biotin is used as the first labeled molecule 3, the number of streptavidin molecules fixed on the magnetic sensor 8 having an area of ​​100 μm×120 μm is about 3×10 8 This makes it possible to easily achieve a detection sensitivity of 10 fM.

[0031] [Immunology analyzer] Next, the immunoanalyzer 100 will be described with reference to Fig. 3. Elements already described will be given the same reference numerals, and descriptions thereof may be omitted. Fig. 3 is a schematic diagram showing the configuration of the immunoanalyzer 100. Fig. 3 shows the planar configuration of the immunoanalyzer 100 installed on the horizontal XY plane as viewed from above (Z direction). The X and Y directions are mutually perpendicular directions constituting the horizontal plane, and here, the X direction corresponds to the width direction of the immunoanalyzer 100, and the Y direction corresponds to the length direction of the immunoanalyzer 100. The Z direction is a vertical direction perpendicular to the X and Y directions, and corresponds to the height direction of the immunoanalyzer 100. In addition, Fig. 3 shows the radial direction R of the reagent disk 112 and the circumferential direction C of the reagent disk 112 on the horizontal plane.

[0032] As shown in FIG. 3, the immunoassay analyzer 100 includes a reaction section 200, a detection section 300, a washing section 400, and an input section 500.

[0033] The reaction section 200 reacts an analyte 1 with a first labeled molecule 3 having a first capture material 2 that specifically binds to the analyte 1, and a magnetic particle 5 having a second capture material 4 that specifically binds to the analyte 1 in the same solution (mixture 9). The detection unit 300 brings this solution into contact with a magnetic sensor 8 having second labeled molecules 7 fixed to its surface, which specifically bind to the first labeled molecules 3, and detects the strength of the magnetic field induced by the magnetic particles 5 with the magnetic sensor 8. The washing section 400 is provided adjacent to the reaction section 200 or the detection section 300, and removes the first labeled molecules 3 and magnetic particles 5 that are not bound to the analyte 1 by injecting and removing a washing liquid. The input unit 500 is an input device through which a user inputs various information and performs operations on the immunoanalyzer 100. When the immunoanalyzer 100 has a control computer 123 as shown in Fig. 3, a keyboard and a mouse correspond to the input unit 500. When the immunoanalyzer 100 has a touch panel or the like that can display a screen and directly input information, the input unit 500 corresponds to the touch panel or the like.

[0034] These elements of the immunoanalyzer 100 are similar to those of conventional immunoanalyzers. An example of a conventional immunoanalyzer is the automatic analyzer described in International Publication No. 2019 / 159609. The basic configuration, operation, and the like of the immunoanalyzer 100 can be referenced to the automatic analyzer described in the publication.

[0035] The immunoanalyzer 100 is provided with a selection section (not shown) in the input section 500 that allows the selection of a mode in which no cleaning is performed for the cleaning section 400. The selection section may be a switch or a button, or may be something like a menu button displayed at a predetermined position on the screen. When a mode that does not involve washing is selected at the input section 500, the immunoassay analyzer 100 detects a signal from the magnetic sensor 8 in the detection section 300 while the solution is in solution, without injecting and removing the washing liquid. In other words, the immunoassay device 100 of this embodiment can perform general immunoassays that involve a washing step, but when using a capture material that does not have a sufficiently small dissociation constant (e.g., a drug, an antibody, a composition of biological tissue, etc.), a mode that does not involve washing can be selected to perform the above-mentioned immunoassay method without involving washing.

[0036] An example of the immunoanalyzer 100 will be described in more detail with reference to FIG. As shown in Figure 3, the immunoanalyzer 100 has a control computer 123, a rack transport section 120, a rack transport line 118, a sample dispensing mechanism 103, an incubator (reaction disk) 104, a transport mechanism 106, a holding member 107, a reaction vessel stirring mechanism 108, a waste hole 109, a reagent disk 112, a reagent dispensing mechanism 114, a magnetic microparticle stirring device 115, a reaction vessel transport mechanism 116, and a detection unit 117.

[0037] The control computer 123 controls each mechanism based on the analysis request information of the immunoanalyzer 100 to realize each process for analysis. The processes include a dispensing process and a washing process. As described above, the control computer 123 also provides an interface for the user. In other words, the control computer 123 corresponds to the input unit 500.

[0038] A sample (analyte 1) to be analyzed by the immunoanalyzer 100 is contained in a sample container 102. The sample container 102 is loaded on a rack 101 and then loaded into the immunoanalyzer 100. A rack transport unit 120 is a mechanism for loading and unloading the rack 101 between the outside and the immunoanalyzer 100. The rack transport unit 120 is also provided with a power-on instruction unit 121 and a power-off instruction unit 122 for the immunoanalyzer 100. The power-on instruction unit 121 and the power-off instruction unit 122 are buttons that are input by a user who operates the immunoanalyzer 100. Note that the display unit of the control computer 123 may be provided with menu buttons corresponding to the power-on instruction unit 121 and the power-off instruction unit 122.

[0039] The rack 101 carried in by the rack transport unit 120 is moved by the rack transport line 118 to a sample dispensing position near the sample dispensing mechanism 103. The incubator 104 can accommodate a plurality of reaction vessels 105 on its circumferential portion. The incubator 104 can perform a rotational motion that moves each of the reaction vessels 105 arranged in the circumferential direction to a predetermined position.

[0040] The transport mechanism 106 is movable in each of the three axial directions of X, Y, and Z. The transport mechanism 106 is a mechanism for transporting the sample dispensing tip and the reaction vessel 105. The transport mechanism 106 moves within a range including a holding member 107 for holding the sample dispensing tip and the reaction vessel 105, a reaction vessel stirring mechanism 108 for stirring the reaction vessel 105, a disposal hole 109 for disposing of the sample dispensing tip or the reaction vessel 105, a sample dispensing tip mounting position 110, and a predetermined portion of the incubator 104.

[0041] A plurality of unused reaction vessels 105 and unused sample dispensing tips are held on the holding member 107. The unused reaction vessels 105 are provided on their inner surfaces with magnetic sensors 8 having the above-mentioned second labeled molecules 7 immobilized on their surfaces.

[0042] First, the transport mechanism 106 moves above the holding member 107, descends to grip an unused reaction vessel 105, and then ascends. Then, the transport mechanism 106 moves above a predetermined position in the incubator 104, descends, and places the reaction vessel 105 at the predetermined position in the incubator 104.

[0043] Next, the transport mechanism 106 again moves above the holding member 107, descends to grip an unused sample dispensing tip, and then ascends. The transport mechanism 106 then moves above the sample dispensing tip mounting position 110, descends, and places the sample dispensing tip at the sample dispensing tip mounting position 110. In order to prevent contamination, the sample dispensing tip is mounted on the tip of the nozzle (probe) when the sample dispensing mechanism 103 dispenses a sample, and is discarded when dispensing of the sample is completed.

[0044] The sample dispensing mechanism 103 can rotate on a horizontal plane and move up and down in the vertical direction (Z direction). The sample dispensing mechanism 103 rotates to move above the sample dispensing tip mounting position 110, then descends and presses the sample dispensing tip into the tip of the nozzle. The sample dispensing mechanism 103 with the sample dispensing tip mounted on the tip of the nozzle moves above the sample container 102 placed on the rack 101, then descends and aspirates a predetermined amount of sample held in the sample container 102. The sample dispensing mechanism 103 that has aspirated the sample moves above the incubator 104, then descends and dispenses the sample into an unused reaction container 105 held in the incubator 104. When the sample is dispensed, the sample dispensing mechanism 103 moves above the disposal hole 109 and discards the used sample dispensing tip from the disposal hole 109.

[0045] The reagent disk 112 has a disk shape and rotates. A plurality of reagent bottles 113 are placed on the reagent disk 112. The reagent disk 112 rotates around a vertical central axis on a horizontal plane. As a result, the reagent bottles 113 arranged on the reagent disk 112 move in the circumferential direction C and are transported to predetermined positions according to the process.

[0046] The reagent disk 112 is capable of accommodating reagent bottles 113, each set including three container sections 113a. The number of container sections 113a is not limited to three. An openable and closable lid may be provided at the opening of the container section 113a. The openable and closable lid can be set arbitrarily. Examples of such a lid include a lid connected to the main body by a hinge, and a lid having a screw-type cap structure.

[0047] The reagent bottle 113 is configured as a set of one container 113a containing a solution (magnetic particle solution) including magnetic particles 5, and two containers 113a containing reagents. One of the reagents is a solution including the first labeled molecule 3 having the first capture material 2 described above.

[0048] When a cleaning liquid is used, at least one of the two container sections 113a that contain the reagent can contain a cleaning liquid such as physiological saline or phosphate buffer. When a cleaning liquid is used, all three container sections 113a in one set of reagent bottles 113 can contain a cleaning liquid, making the reagent bottles 113 dedicated to cleaning liquid. In this way, the device can be made more compact. When a cleaning liquid is used, the cleaning liquid can be contained in a dedicated cleaning liquid bottle (not shown) provided outside the reagent disk 112. In this way, the reagent bottles 113 can contain reagents other than the cleaning liquid, so that more samples can be analyzed.

[0049] In a typical immunoassay in which a sample is reacted with a magnetic particle solution and then washed with a washing solution at a predetermined stage, the sample dispensing mechanism 103 attaches an unused sample dispensing tip to the tip of the nozzle, aspirates a predetermined amount of washing solution from a reagent bottle 113 or a container section 113a that contains the washing solution, and discharges the washing solution into a target reaction container 105. After washing, the sample dispensing mechanism 103 sucks up the washing solution used for washing and discards it in a waste liquid container (not shown), and discards the used sample dispensing tip from a disposal hole 109.

[0050] A cover (not shown) is provided on the top of the reagent disk 112 to prevent dust and the like from entering and to keep the space including the reagent disk 112 warm or cold at a predetermined temperature. That is, the space including the reagent disk 112 also functions as a warming box or a cold box. In this embodiment, since the reagent dispensing mechanism 114 or the magnetic particle stirrer 115 accesses the reagent bottle 113 in the region 113b, it is preferable to provide an opening in the cover at the region 113b. When an openable lid is provided at the opening of the container section 113a, it is preferable to provide a mechanism (reagent container lid opening / closing mechanism; not shown) that can open and close the lid according to the openable lid at the opening of the cover. This eliminates the need for a rotation operation of the reagent disk 112 that may be performed between the opening and closing operation of the lid of the container section 113a and the reagent suction operation, and the time required for the dispensing process and the washing process can be shortened.

[0051] The reagent dispensing mechanism 114 is capable of rotating on a horizontal plane and moving up and down in a vertical direction. The reagent dispensing mechanism 114 moves by rotating above the area 113b (opening of the cover), and then descends, immersing the tip of the nozzle (probe) in the reagent (including the case where the reagent is a cleaning liquid) in the container section 113a, and aspirates a predetermined amount of the reagent. Next, the reagent dispensing mechanism 114 moves by rotating above a predetermined position of the incubator 104, and ejects the reagent into the reaction container 105. As shown in FIG. 3, the rotational trajectory of the reagent dispensing mechanism 114 is set so as to pass over a plurality of openings of one reagent bottle 113 at a predetermined position.

[0052] The magnetic microparticle stirrer 115 can also rotate on a horizontal plane and move up and down in a vertical direction. The position of the suction port of the container that contains the magnetic microparticle solution is position 113c. Therefore, the magnetic microparticle stirrer 115 moves by rotating above position 113c, and then descends, immersing the tip of the magnetic microparticle stirrer 115 in the magnetic microparticle solution in the reagent bottle 113 to stir the solution. Then, while the magnetic microparticle stirrer 115 is stirring the magnetic microparticle solution, the reagent can be dispensed by the reagent dispensing mechanism 114.

[0053] The reaction vessel 105 into which the sample, reagent, and magnetic microparticle solution have been discharged is moved to a predetermined position by the rotation of the incubator 104, and is transported to the reaction vessel stirring mechanism 108 by the transport mechanism 106. The reaction vessel stirring mechanism 108 applies a rotational motion to the reaction vessel 105, thereby stirring and mixing the sample and reagent in the reaction vessel 105. As a result, a reaction liquid (solution, mixed liquid 9) is generated in the reaction vessel 105.

[0054] That is, the reaction vessel 105 , incubator 104 , reagent disk 112 , reagent bottle 113 , sample dispensing mechanism 103 , reagent dispensing mechanism 114 , magnetic particle stirrer 115 , etc. in the immunoassay analyzer 100 correspond to the reaction section 200 . In addition, when washing and removing unreacted magnetic microparticles 13 that are not contained in the sandwich complex 6 and first labeled molecules 3 having unreacted first capture materials 2 that are dispersed in the solution in the reaction vessel 105, the reagent bottle 113 and washing solution bottle used during washing, the reaction vessel 105, the incubator 104, the reagent disk 112, the sample dispensing mechanism 103, the reagent dispensing mechanism 114, etc. correspond to the washing section 400.

[0055] After stirring, the reaction vessel 105 is returned to a predetermined position in the incubator 104 by the transport mechanism 106. The reaction vessel transport mechanism 116 transports the reaction vessel 105 between the incubator 104 and the detection unit 117. The reaction vessel transport mechanism 116 grasps the reaction vessel 105 and moves up, and transports the reaction vessel 105 to the detection unit 117 by a rotational motion. The reaction vessel 105 is analyzed in the detection unit 117. In other words, the detection unit 117 corresponds to the detection section 300.

[0056] As described above, when the mode without washing is selected in the input section 500, the immunoassay device 100 reacts the analyte 1, the first labeled molecule 3 having the first capture material 2 that specifically binds to the analyte 1, and the magnetic particle 5 having the second capture material 4 that specifically binds to the analyte 1 in the same solution in the reaction section 200 (first step), to generate a sandwich complex 6. Thereafter, the immunoassay device 100 brings the solution into contact with a magnetic sensor 8 having a second labeled molecule 7 that specifically binds to the first labeled molecule 3 immobilized on its surface, and transports the solution to the detection unit 117 by the reaction vessel transport mechanism 116 without washing in the washing section 400. Note that, as one aspect of "without washing," for example, the sample dispensing mechanism 103 does not perform any of the following operations: attaching an unused sample dispensing tip to the tip of the nozzle, aspirating a predetermined amount of washing liquid from the reagent bottle 113 or vessel section 113a that contains the washing liquid, and discharging the washing liquid into the target reaction vessel 105. Then, the detection unit 117 applies a magnetic field using the external magnetic field generating device 10, and detects the strength of the magnetic field induced by the magnetic fine particles 5 contained in the sandwich complex 6 using the magnetic sensor 8 (second step).

[0057] After the analysis, the reaction vessel 105 is returned to the incubator 104 by the reaction vessel transport mechanism 116. Thereafter, the reaction vessel 105 is moved by the transport mechanism 106 from the incubator 104 to above the disposal hole 109, and is disposed of through the disposal hole 109.

[0058] As the configuration described above, the immunoanalysis method and immunoanalysis device 100 according to the present embodiment do not require a washing step. Therefore, even when the sandwich complex 6 is formed using a drug, antibody, biological tissue composition, or the like that does not have a sufficiently small dissociation constant (i.e., the binding force is not sufficiently strong), the immunoanalysis method and immunoanalysis device 100 according to the present embodiment can prevent the capture material, such as a drug or antibody, from dissociating from the sandwich complex 6. Therefore, the present analysis method and immunoanalysis device 100 are unlikely to cause a decrease in luminescence intensity or signal intensity, and high sensitivity can be obtained, so that the analyte can be accurately quantified. EXAMPLES

[0059] Next, a specific example of the present analysis method and the immunoassay device 100 will be described. In this embodiment, a system for detecting CEA, a tumor marker, was constructed and carried out. Anti-CEA antibody 5909 manufactured by Medix Biochemica was used as the capture antibody, which was the first capture material 2. The capture antibody was biotinylated using a biotinylation kit (LK10 manufactured by Dojindo Chemical Industries, Ltd.) (biotin corresponds to the first labeling molecule 3). Anti-CEA antibody 5905 manufactured by Medix Biochemica was used as the detection antibody, which was the second capture material 4. Magnetic particle labeling kit (catalog #130-105-805) manufactured by Miltenyi was used as the magnetic particle 5, and the detection antibody 5905 was fixed to the surface of the magnetic particle 5 according to the protocol recommended by Miltenyi. A GMR chip manufactured by MagArray was used as the magnetic sensor 8.

[0060] On the surface of the 10 mm square substrate of the chip, multiple giant magnetoresistance sensors with an area of ​​100 μm × 120 μm are provided. After (3-aminopropyl)triethoxysilane (Sigma-Aldrich 440140) is evaporated onto the sensor surface, a phosphate buffer solution of streptavidin (invitrogen, S888) is spotted onto the magnetic sensor, thereby immobilizing streptavidin, which is the second labeling molecule 7, on the surface of the magnetic sensor 8.

[0061] Lyphocheck Lv2 from Bio-Rad was used as the CEA sample solution. It was diluted with a standard control serum (Roche's cal1 control reagent for TSH assay) to the specified concentration. Phosphate buffer containing 1% bovine serum albumin was used as the reaction buffer. The reaction volume was 400 μl. The concentrations of the biotinylated capture antibody and magnetic particles with detection antibody were each 1 nM, and four types of antigen CEA concentrations were prepared: 0 pM, 0.1 pM, 1 pM, and 10 pM.

[0062] The capture antibody, antigen, and magnetic particles with detection antibody were placed in a reaction tube and reacted at 37°C for 60 minutes while stirring. Immediately after the reaction, the reaction solution was poured into the chip placed in a designated container, and the output of the giant magnetoresistance sensor was read out using the MagArray magnetic signal measuring device (i.e., measurement was started without washing or B / F separation). The same experiment was performed four times independently (Experiment 1 to Experiment 4).

[0063] When the change in the output signal intensity over time was monitored, the signal intensity increased immediately after reading in each experiment, the increase in the signal intensity was suppressed after about 20 to 30 minutes, and the signal intensity was saturated after about 40 minutes. This saturated value was used as the signal intensity. The signal intensity at a CEA concentration of 0 pM was subtracted from the signal intensity at CEA concentrations of 0.1 pM, 1 pM, and 10 pM, and the signal intensity was plotted against the antigen concentration, as shown in Figure 4. As shown in Figure 4, in the case of 0.1 pM, the signal intensity varied greatly, but linearity was good. From this, it was confirmed that the immunoassay method shown in this example can realize quantitative analysis of CEA, which is a tumor marker, while not requiring a washing step.

[0064] Although the immunoanalysis method and the immunoanalysis device according to the present invention have been described in detail above through the embodiments and examples, the gist of the present invention is not limited to these, and various modifications are included. For example, the above-mentioned embodiments have been described in detail to easily explain the present invention, and the present invention is not necessarily limited to those having all of the configurations described. In addition, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. In addition, it is possible to add, delete, or replace a part of the configuration of each embodiment with another configuration. [Explanation of symbols]

[0065] 1. Analyte 2 First capture material 3 First label molecule 4 Second capture material 5. Magnetic particles 7 Second labeled molecule 8 Magnetic Sensors 100 immunoanalyzer 200 Reaction unit 300 Detector 400 Cleaning section 500 Input section

Claims

1. a first step of reacting an analyte with a first labeled molecule having a first capture material that specifically binds to the analyte, and a magnetic particle having a second capture material that specifically binds to the analyte in the same solution; a second step of contacting the solution reacted in the first step with a magnetic sensor having a surface on which a second labeled molecule that specifically binds to the first labeled molecule is immobilized, and detecting the strength of the magnetic field induced by the magnetic nanoparticles in this state with the magnetic sensor; The immunoassay method comprises:

2. The number of molecules of the first capture material having the first label molecule is a, the number of molecules of the target substance to be analyzed is b, and the area of ​​the magnetic sensor is f (μm ２ , the number of molecules of the second labeled molecule is g, and the minimum density of magnetic particles at which the magnetic sensor can detect the magnetic particles immobilized on its surface is h (particles / μm ２ 2. The immunoassay method according to claim 1 , wherein b×g / a / f>h satisfies the relational expression:

3. 3. The immunoassay method according to claim 1, wherein the magnetic particles have superparamagnetic properties, and the magnetic sensor is a giant magnetoresistance sensor.

4. a reaction section for reacting, in the same solution, a target substance, a first labeled molecule having a first capture material that specifically binds to the target substance, and a magnetic particle having a second capture material that specifically binds to the target substance; a detection unit that brings the solution into contact with a magnetic sensor having a surface onto which a second labeled molecule that specifically binds to the first labeled molecule is immobilized, and detects the strength of a magnetic field induced by the magnetic nanoparticles with the magnetic sensor; a washing section that is provided adjacent to the reaction section or the detection section and that removes the first labeled molecules and the magnetic particles that are not bound to the analyte by injecting and removing a washing solution; An input unit capable of selecting a mode in which the cleaning unit does not perform cleaning, When a mode that does not involve washing is selected in the input section, the detection section detects a signal from the magnetic sensor while the solution is in solution, without injecting and removing the washing solution.

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