JP2019007982A - Protein detection device and protein detection method - Google Patents

Abstract

To provide a protein detection device capable of performing the detection with a high accuracy.SOLUTION: A protein detection device 1 uses an ELISA method. A reaction vessel 61 in which a solution dispersed with the number of magnetic beads immobilized with the primary antibody on the surface is stored. The reaction vessel includes a portion through which a transmissive light path TK passes. An optical mechanism 4 includes: a light emission part for irradiating irradiation light along the transmissive light pass TK; and a light reception part for receiving the irradiation light via the transmissive light path TK and measures a solution absorbance degree based on a light reception amount of the light reception part. A magnetic absorbance mechanism 6 allows magnetic beads to retreat from a portion through which the transmissive light path TK passes in the reaction vessel 61 so as to be held in the retreated portion by magnetically being adsorbed into the magnetic beads.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a protein detection apparatus and a protein detection method.

An ELISA (Enzyme-Linked Immunosorbent Assay) method is known as a method for measuring whether or not a specific protein is present in a biological sample.
In an apparatus for performing the ELISA method, a multiwell plate provided with a large number (for example, 96) of wells is generally used. That is, a measurement sample containing an antigen is added to the capture antibody immobilized on each well, and left for a predetermined time until the antigen binds to the immobilized capture antibody.

  On the other hand, an inspection apparatus using a reagent cartridge in which a solid phase capable of binding an antigen or an antibody is set in at least one recess, a sample holder, a fixture for the reagent cartridge and the measurement cuvette, a pipette device, and a measurement There has been proposed an inspection apparatus in which an optical unit for detecting an optical change in a cuvette, a cleaning unit for cleaning a pipette of a pipette device, and the like are laid out (see, for example, Patent Document 1).

Special table 2013-509572 gazette

  An object of the present invention is to provide a protein detection apparatus capable of detecting with high accuracy, and to provide a protein detection method capable of detecting with high accuracy.

  In order to achieve the above object, the invention of claim 1 is a protein detection apparatus using an ELISA method, in which a plurality of magnetic beads (83) having a primary antibody (81) immobilized thereon are dispersed on the surface. A reaction vessel (61) in which a light-transmitting portion (76; 761) through which a transparent light transmission path (TK) passes is provided, and a light-emitting unit that emits irradiation light along the transmission light path (76; 761) And an optical mechanism (4) for measuring the liquid absorbance based on the amount of light received by the light receiving part, and a light receiving part (15) for receiving the irradiation light via the transmitted light path. Magnetic attraction mechanism (6) for magnetically attracting the magnetic beads to retract the magnetic beads from the portion through which the transmission optical path passes in the reaction container and to retain the magnetic beads in the portion (77; 762) to be retracted in the reaction container When Providing protein detection device (1) comprising a.

In addition, although the alphanumeric character in a parenthesis represents the corresponding component etc. in embodiment mentioned later, this does not mean that this invention should be limited to those embodiment as a matter of course. The same applies hereinafter.
According to a second aspect of the present invention, the magnetic attraction mechanism may include an electromagnetic coil (6) that is turned on / off.

According to a third aspect of the present invention, the magnetic attraction mechanism includes a permanent magnet (6A) capable of changing a distance to the retracted portion and a drive member (94) for moving the permanent magnet closer to the retracted portion. And may be included.
The invention of claim 4 is a protein detection method using an ELISA method, which is a reaction vessel containing a liquid in which a large number of magnetic beads having a primary antibody immobilized on a surface thereof are dispersed, The reaction vessel having a portion through which a transmitted light path passes, and the irradiation light irradiated from the light emitting part of the optical mechanism along the transmitted light path is received by the light receiving part of the optical mechanism through the transmitted light path, and the light receiving part Measuring the liquid absorbance based on the amount of received light, wherein the magnetic bead is magnetically attracted to retract the magnetic bead from the portion of the reaction vessel through which the transmission light path passes. A protein detection method is provided that is configured to be held in a portion to be retracted.

  According to the first aspect of the present invention, the magnetically attracted magnetic beads are retracted from the portion through which the transmission optical path passes in the reaction vessel and are retained in the retracted portion, so that the irradiation light from the light emitting portion interferes with the magnetic beads. Thus, a protein detection device that can receive light at the light receiving unit and improve the detection accuracy by the optical mechanism, and thereby detect with high accuracy is provided. Further, when the reaction vessel is washed with the washing liquid, the magnetic beads can be magnetically attracted and held in the retreating portion.

According to the second aspect of the present invention, the magnetic beads can be easily retreated from the portion through which the transmission optical path passes or the retraction is released by turning on and off the electromagnetic coil.
According to a third aspect of the present invention, a strong magnetic field by a permanent magnet is applied to the retracted portion so that the magnetic beads are securely retracted from the portion through which the transmitted light path passes to the retracted portion. Can be held.

  According to the fourth aspect of the invention, the magnetically attracted magnetic beads are retracted from the portion through which the transmission light path passes in the reaction vessel and retained in the retracted portion, whereby the irradiation light from the light emitting portion is applied to the magnetic beads. Provided is a protein detection method in which light is received by a light receiving unit without being obstructed and detection accuracy by an optical mechanism can be improved.

It is sectional drawing of schematic structure of the protein detection apparatus of one Embodiment of this invention. It is a top view of a cartridge and the supply system which supplies the liquid corresponding to each supply path of a cartridge is typically shown. It is a schematic diagram showing a cross section of a main part of a cartridge set in the apparatus main body and a supply system. It is an expanded sectional view of the principal part of a protein detection apparatus. It is a block diagram which shows the electric constitution of a protein detection apparatus. It is a flowchart of an antigen detection. (A) is a schematic cross-sectional view of a reaction vessel supplied with a primary antibody-containing solution, and (b) is an enlarged schematic view of the main part of magnetic beads. It is sectional drawing of the reaction container of a magnetic attraction state, and its periphery. It is an expanded sectional view of the principal part of the protein detection apparatus of another embodiment of the present invention, and shows the state where the permanent magnet was separated from the 2nd part (annular part) in the example using a movable permanent magnet for a magnetic attraction mechanism. ing. In the embodiment of FIG. 9, it is an expanded sectional view of the principal part of a protein detection apparatus, The permanent magnet has shown the state which adjoined to the 2nd part (annular part).

Embodiments of the present invention will be described with reference to the accompanying drawings.
FIG. 1 is a cross-sectional view of a protein detection apparatus 1 according to an embodiment of the present invention, and FIG. 2 is a plan view of a cartridge 60. First, referring to FIG. 2, the cartridge 60 includes a pair of translucent reaction vessels 61 for causing an antigen-antibody reaction, and a primary antibody-containing liquid containing a primary antibody (capture antibody) in each reaction vessel 61. A pair of primary antibody-containing liquid supply paths 62 to be supplied, a pair of first cleaning liquid supply paths 63 to supply the first cleaning liquid to each reaction container 61, and a secondary antibody (enzyme-labeled antibody) to each reaction container 61. And a secondary antibody-containing liquid supply path 64 for supplying the secondary antibody-containing liquids.

The cartridge 60 also has a second cleaning liquid supply path 65 for supplying a second cleaning liquid containing the second cleaning liquid to each reaction container 61, and a coloring substrate that is an enzyme substrate of the enzyme-labeled antibody labeling enzyme for each reaction container 61. A pair of chromogenic substrate-containing liquid supply paths 66 that respectively supply the chromogenic substrate-containing liquid that is included, and a sample supply path 67 that supplies a sample to each reaction vessel 61 are provided.
In the present embodiment, a description will be given in accordance with an example in which the cartridge 60 is set in the protein detection apparatus 1 in a state where each sample solution that will contain an antigen that is a protein to be detected is put in each reaction container 61. . Therefore, in this embodiment, the sample supply path 67 is not used.

  In addition, a sample solution containing an antigen that is a protein to be detected is placed in one reaction vessel 61 in advance, and a sample solution containing an antigen that is a protein to be detected is not put in the other reaction vessel 61 (that is, The cartridge 60 may be set in the protein detection apparatus 1 in a state where a blank liquid not included is charged. In addition, after the cartridge 60 is set in the protein detection apparatus 1, each different sample may be supplied through each sample supply path 67.

Further, the cartridge 60 has a common waste liquid storage unit 69 that stores waste liquid discharged from each reaction vessel 61 through a discharge path 68. An exhaust port 69 a communicating with an exhaust valve (not shown) is provided at the bottom of the waste liquid storage unit 69.
As shown in FIGS. 2 and 3, liquid insertion holes 62a, 63a, 64a, 65a, 66a, and 67a for inserting corresponding liquids are provided at one ends of the supply paths 62, 63, 64, 65, 66, and 67, respectively. Is provided.

  As shown in FIG. 3, each of the liquid insertion holes 62 a to 67 a is inserted through a convex portion 70 protruding from the bottom surface of the cartridge 60 and via a corresponding liquid path 71 and a corresponding tube 72 that pass through the cartridge receiving plate 12. Are connected to corresponding pumps (first pump 101, second pump 102, third pump 103, fourth pump 104, fifth pump 105, sixth pump 106 and seventh pump 107).

  Each pump (first pump 101 to seventh pump 107) is driven by a corresponding pump motor (first pump motor 201 to seventh pump motor 207) by an ECU 40 (Electronic Control Unit) as a control unit. ), The corresponding liquid is sucked from the corresponding tanks (first tank 301 to sixth tank 307) containing the corresponding liquid, and discharged to the corresponding supply paths 62 to 67 side.

As shown in FIG. 2, common supply paths 62 to 65 that communicate with one reaction container 61 and corresponding supply paths 62 to 65 that communicate with the other reaction container 61 share a common tank 301 to 305. A common liquid is supplied from the pumps 101 to 105 through a tube 72 that branches into a bifurcated way.
In the present embodiment, the sample supply path 67 is not used, but when the sample supply path 67 is used (when the sample is supplied to the reaction container 61 after the cartridge 60 is set), the sample supply leading to one reaction container 61 is performed. Each of the different samples is supplied to each of the channel 67 and the sample supply channel 67 communicating with the other reaction vessel 61 from the tanks 306 and 307 via the different tubes 72 by the different pumps 106 and 107. become. The other ends of the supply paths 62 to 67 are connected to a common supply path 75 that communicates with the corresponding reaction vessel 61.

  As shown in FIG. 3, the convex portion 70 is fitted in a concave portion 73 provided on the upper surface 12 a of the cartridge receiving plate 12. A space between the lower surface of the cartridge 60 and the upper surface 12a of the cartridge receiving plate 12 is sealed by a sealing member 74 such as an O-ring fitted around the base end of the convex portion 70. Thereby, the corresponding liquid can be inserted from each liquid path 71 into the corresponding liquid insertion holes 62a to 67a without leakage.

  As shown in FIGS. 7A and 7B, the primary antibody (capture antibody) 81 is immobilized on the surface of countless magnetic beads 83 dispersed in the primary antibody-containing liquid 82 supplied into the reaction vessel 61. It is phased. In the magnetic bead 83, the primary antibody 81 is solid-phased on the surface 83a of the magnetic bead 83 formed of a synthetic resin material containing magnetic powder. The diameter of a magnetic bead is 0.1-10 micrometers in diameter, for example, More preferably, it is 0.1-3 micrometers. Referring to FIG. 2, the primary antibody-containing liquid 82 is supplied from the first tank 301 to the reaction vessel 61 through the tube 72, the primary antibody-containing liquid supply path 62 and the common supply path 75.

  As shown in FIG. 4 which is an enlarged cross-sectional view, the reaction vessel 61 has a bottom wall 76 as a first part through which a transmission light path TK, which will be described later, and a conical taper-shaped circumference as a second part surrounding the bottom wall 76. And a side wall 77. The cartridge 60 is entirely formed of a resin such as an acrylic resin, a polyethylene resin, and a polypropylene resin so as to be transparent. However, only the reaction vessel 61 may be translucent, or only a part of the reaction vessel 61 (for example, the bottom wall 76 in FIG. 3 or the central portion of the bottom wall 76) may be translucent.

  Referring to FIG. 1, a protein detection device 1 includes a device main body 3 having a holding portion 2 that is, for example, a recess, on which a cartridge 60 is detachably mounted, an optical mechanism 4 supported by the device main body 3, and a device main body. 3, an electromagnetic coil 6 as a magnetic attraction mechanism supported by the apparatus main body 3, and an ECU 40 as a control unit that controls operations of the optical mechanism 4, the vibration mechanism 5, and the electromagnetic coil 6. (Electronic Control Unit).

  The apparatus main body 3 includes a base 7, front and rear side frames 8 fixed to the base 7, a first support plate 9 supported by the side frame 8, and a first support plate 9 disposed below the first support plate 9. A second support plate 10 facing the support plate 9, a cartridge receiving plate 12 fixed on the first support plate 9 and having a holding portion 2 formed of a recess for receiving and holding the cartridge 60 on the upper surface 12 a, and the holding portion 2 The cartridge holding plate 13 is detachably fixed to the upper surface 12a of the cartridge receiving plate 12 so as to hold the cartridge 60 held by the cartridge.

The optical mechanism 4 serves as a first light emitting element that emits the first irradiation light B (blue light) along the transmission light path PL that passes through the reaction container 61 of the cartridge 60 held by the holding unit 2 of the cartridge receiving plate 12. It includes a blue LED 14 and a single light receiving element 15 for detection that is provided at the end of the transmitted light path TK and receives the first irradiation light.
The absorbance of the blue light B (first irradiation light) emitted from the blue LED 14 (first light emitting element) changes due to the color change of the chromogenic substrate (reactant thereof) due to the enzyme reaction described later.

The detection light receiving element 15 is fixed to the mounting recess 13b on the upper surface 13a of the cartridge pressing plate 13 so as to block the opening of the insertion hole 16 through which the cartridge pressing plate 13 is inserted (corresponding to the end of the transmitted light path PL). .
The optical mechanism 4 includes an optical path forming block 17 supported by an arm 30 fixed to the base 7. The arm 30 is interposed between the lower surface 17 a of the optical path forming block 17 and the base 7. Further, the upper surface 17 b of the optical path forming block 17 runs along the lower surface 10 a of the second support plate 10. An optical path forming cylinder 31 is fitted in an optical path forming hole in the optical path forming block 17. A part of the optical path forming cylinder 31 protrudes from the upper surface 17 b of the optical path forming block 17 through the insertion hole of the second support plate 10.

The blue LED 14 as the first light emitting element is fixed to the lower surface 17 a of the optical path forming block 17. Further, the first side surface 17c of the optical path forming block 17 is a second light emitting element that emits red light R (second irradiation light) whose absorbance does not change due to a color change of a chromogenic substrate (reaction product) by an enzyme reaction described later. Red LED 18 is fixed.
The optical path forming block 17 is disposed on the transmission optical path TK, transmits blue light B (first irradiation light) from the blue LED 14 (first light emitting element), and transmits light from the red LED 18 (second light emitting element). A dichroic mirror 19 that guides red light R (second irradiation light) incident from a direction orthogonal to the optical path TK to the downstream side of the transmission optical path TK is fixed.

  The only light receiving element 20 for correction is disposed on the second side surface 17 d of the optical path forming block 17. A half mirror 21 disposed downstream of the dichroic mirror 19 on the transmission optical path TK is fixed to the optical path forming block 17. The half mirror 21 reflects a part of the blue light B (first irradiation light) from the blue LED 14 and part of the red light R (second irradiation light) from the red LED 18, which is an optical path different from the transmission optical path TK. The light is guided to the correction light receiving element 20 through HK.

The ECU 40 has a function of detecting the degree of antigen-antibody reaction as the degree of color change by the chromogenic substrate based on the detection results of the detection light receiving element 15 and the correction light receiving element 20.
Referring to FIG. 4, the vibration mechanism 5 transmits an ultrasonic wave from the ultrasonic oscillator 23 fixed to the ultrasonic oscillator 23 and the ultrasonic oscillator 23 as an excitation source held by the holder 22. And an ultrasonic horn 24. The ultrasonic horn 24 is formed of a cylindrical member having a center hole 24a through which a part of the transmitted light path TK passes, and has an annular shape surrounding a part of the transmitted light path TK. A conical tapered transmission surface 24 b is formed at the end of the ultrasonic horn 24 along the conical tapered peripheral side wall 77 of the reaction container 61 of the cartridge 60.

A holding recess 22 b for holding the ultrasonic oscillator 23 is formed on the upper surface 22 a of the holder 22. A compression coil spring 25 as an elastic member for elastically supporting the holder 22 is interposed between the lower surface 22c of the holder 22 and the upper surface 10b of the second support plate 10 opposed thereto. The compression coil spring 25 elastically supports the excitation mechanism 5 via the holder 22.
A fixed end 25 a of the compression coil spring 25 is fixed to a locking portion made of, for example, a convex portion of the upper surface 10 b of the second support plate 10. The movable end 25 b of the compression coil spring 25 is locked to a locking portion 27 made of, for example, a recess on the lower surface 22 c of the holder 22.

It is preferable that a plurality (three in this embodiment) of the compression coil springs 25 are provided at equal intervals on the circumference centered on the transmission optical path TK. Due to expansion / contraction deformation and collapse deformation of the compression coil spring 25, variations in dimensional errors among the components are absorbed, and the transmission surface 24b of the vibration exciting mechanism 5 reliably comes into surface contact with the peripheral side wall 77 of the reaction container 61 of the cartridge 60. .
The center hole of the holder 22 is fitted to the upper end of the optical path forming cylinder 31 that forms the transmitted optical path TK so as to be slidable in the longitudinal direction of the optical path forming cylinder 31.

  The electromagnetic coil 6 serving as a magnetic attraction mechanism is disposed in an annular shape so as to surround the ultrasonic horn 24 of the vibration mechanism 5 and is controlled on and off by the ECU 40. The electromagnetic coil 6 that has been turned on magnetically attracts the magnetic beads dispersed in the antibody-containing liquid contained in the reaction vessel 61, thereby retracting the magnetic beads from the bottom wall 76 (first portion) and surrounding wall 77. Hold on (second part). As a result, the magnetic beads are retracted from the transmitted light path TK, and the detection accuracy by the optical mechanism 4 is improved. Further, the magnetic beads are held on the peripheral side wall 77 (second portion) at the stage of cleaning the reaction vessel 61 with the cleaning liquid supplied from the cleaning liquid supply path 64.

  FIG. 5 is a block diagram showing an electrical configuration of the protein detection apparatus 1. As shown in FIG. 5, a detection light receiving element 15 and a correction light receiving element 20 are connected to the ECU 40. The ECU 40 includes AD converters 41 and 42 that AD convert the detection signals input from the light receiving elements 15 and 20, respectively. The ECU 40 is connected to an operation unit 43 including an operation switch such as a YES switch 44a that is manually operated by an operator. An operation signal from the operation unit 43 is input to the ECU 40.

The ECU 40 also includes an ultrasonic oscillator 23 as a vibration source, an electromagnetic coil 6 as a magnetic attraction mechanism, and a display unit 44 such as a liquid crystal display unit that displays a message display to the operator and a result of antigen detection. The blue LED 14 (first light emitting element) and the red LED 18 (second light emitting element) of the optical mechanism 4 and the pump motors 201 to 207 are connected.
The ECU 40 includes a drive unit 45 for driving the ultrasonic oscillator 23, a drive unit 46 for driving the electromagnetic coil 6, a drive unit 47 for driving the display unit 44, a drive unit 48 for driving the blue LED 14, and a red LED 18. , And drive units 401 to 407 for driving the pump motors 201 to 207, respectively.

ECU40 outputs the signal which drives the pump motor 201-207 corresponding to each process to the corresponding drive parts 401-407. In addition, the ECU 40 includes a color change detection unit 50 that receives a signal from the detection light receiving element 15 and the correction light receiving element 20 via the corresponding AD converters 41 and 42 and detects a color change.
The ECU 40 includes a process control unit 51 that inputs a signal from the operation unit 43 and controls the inspection process. The process control unit 51 controls the ultrasonic oscillator 23, the electromagnetic coil 6, and the display unit 44 via corresponding drive units 45 to 47 in accordance with signals from the operation unit 43. In addition, the ECU 40 operates the color change detection unit 50 in accordance with a signal from the process control unit 51. The color change detection unit 50 outputs drive signals to the corresponding drive units 48 and 49 so as to drive the blue LED 14 and the red LED 18 in the color change detection stage.

Next, based on the flowchart of FIG. 6 showing the operation of the ECU 40, the detection process using the protein detection device 1 will be described sequentially. In this embodiment, the ELISA method used in the detection step will be described based on a so-called sandwich method in which a sandwich structure of primary antibody + antigen + secondary antibody (enzyme-labeled antibody) is presented. There may be.
In step S <b> 1, the sample liquid containing the antigen (detection target protein) is charged into one reaction container 61, and the cartridge 60 in which the blank liquid not containing the antigen is charged into the other reaction container 61 is held in the holding unit 2 of the apparatus main body 3. The display unit 44 displays a message with a content requesting that the user set to “ON” and a message with a content requesting that the YES switch 43a be turned on after setting. The message is, for example, “Set cartridge and press YES switch after setting”.

In response to the message, the operator sets the cartridge 60 in the holding portion 2 of the cartridge receiving plate 12 with the cartridge pressing plate 13 removed (corresponding to a cartridge setting step).
In step S2, the operator waits for the operator to press the YES switch 43a. When the input of turning on the YES switch 43a is confirmed in step S2 (YES in step S2), the process proceeds to step S3.

  In step S3, the optical mechanism 4 is driven. Specifically, the blue LED 14 (first light emitting element) and the red LED 18 (second light emitting element) are driven alternately. At this time, when the blue LED 14 is driven, feedback control is performed so that the amount of blue light B received from the blue LED 14 by the detection light receiving element 15 is constant. Further, when the red LED 18 is driven, feedback control is performed so that the amount of red light R received from the red LED 18 by the correction light receiving element 20 is constant.

When the blue LED 14 is driven, the detected received light amount of the blue light (first irradiation light) B from the blue LED 14 by the detection light receiving element 15 is stored as the reference value B0. When the red LED 18 is driven, the detected light reception amount of the red light (second irradiation light) R from the red LED 18 by the correction light receiving element 20 is stored as the reference value R0.
In step S4, the first pump 101 is driven, and the primary antibody-containing liquid 82 stored in the first tank 301 is supplied to the reaction vessel 61 as shown in FIG. 7A (primary antibody supply step). Equivalent). The primary antibody 81 is preliminarily immobilized on the surface of a large number of magnetic beads 83 contained in the primary antibody-containing solution 82.

  Next, in step S5, for example, the electromagnetic coil 6 is turned off (or the electromagnetic coil 6 is turned on and the magnetic beads 83 are magnetically attracted and held on the peripheral side wall 77 as shown in FIG. 8). The ultrasonic oscillator 23 of the mechanism 5 is driven for a predetermined time, and the reaction vessel 61 is vibrated. That is, the ultrasonic vibration of the ultrasonic oscillator 23 of the vibration mechanism 5 is propagated to the reaction container 61 via the ultrasonic horn 24 to vibrate the reaction container 61, and the antigen is transferred to the antibody 81 in the reaction container 61. Promotes antigen-antibody reaction to bind.

  Next, in step S6, the electromagnetic coil 6 is turned on, the magnetic beads 83 are magnetically attracted and held on the peripheral side wall 77 as shown in FIG. 8, and in step S7, the second pump 102 (second pump motor 202 is selected). And the ultrasonic oscillator 23 of the vibration mechanism 5 are driven to feed the first cleaning liquid to the reaction vessel 61 and to vibrate the reaction vessel 61. That is, the ultrasonic oscillator of the vibration mechanism 5 is turned on in a state where the electromagnetic coil 6 as the magnetic attraction mechanism is turned on and the magnetic beads are held on the peripheral side wall 77 (second portion) of the reaction vessel 61 by the magnetic attraction force. 23 is propagated to the reaction vessel 61 through the ultrasonic horn 24, and the reaction vessel 61 is vibrated to facilitate the cleaning of the reaction vessel 61 (corresponding to the first cleaning step).

By performing the first washing step, only the antigen bound to the primary antibody 81 immobilized on the magnetic beads 83 is left. The first cleaning liquid in the reaction container 61 is discharged to the waste liquid storage unit 69 through the discharge path 68 as waste liquid together with the antigen that has not reacted.
Next, in step S8, the third pump 103 (third pump motor 203) is driven, and a secondary antibody-containing liquid containing a secondary antibody that is an enzyme-labeled antibody labeled with an enzyme is supplied to the reaction vessel 61 (two). Corresponds to the secondary antibody-containing solution supply step).

Next, in step S9, for example, the ultrasonic oscillator 23 of the vibration mechanism 5 is driven for a predetermined time while the electromagnetic coil 6 is turned off (or can be turned on), and the reaction vessel 61 is vibrated.
That is, the vibration promotes that the secondary antibody (enzyme-labeled antibody) recognizes the antigen bound to the primary antibody 81 (capture antibody) in the reaction container 61 and binds to the recognized antigen (antigen recognition). Equivalent to the process).

  Next, in step S10, with the electromagnetic coil 6 turned on, the fourth pump 104 (fourth pump motor 204) and the ultrasonic oscillator 23 of the vibration mechanism 5 are driven to send the second cleaning liquid to the reaction vessel 61. While being fed, the reaction vessel 61 is vibrated. That is, with the electromagnetic coil 6 as the magnetic attraction mechanism turned on and the magnetic beads are held on the peripheral side wall 77 (second portion) of the reaction vessel 61 as shown in FIG. 5 is propagated to the reaction vessel 61 through the ultrasonic horn 24 to vibrate the reaction vessel 61 and promote cleaning of the reaction vessel 61 (in the second washing step). Equivalent).

By performing the second washing step, only the secondary antibody bound to the antigen bound to the primary antibody 81 immobilized on the magnetic beads 83 is left. The second washing liquid in the reaction container 61 is discharged as waste liquid to the waste liquid storage unit 69 through the discharge path 68 together with excess secondary antibody that has not been bound to the antigen.
Next, in step S11, the fifth pump 105 (fifth pump motor 205) is driven, and a chromogenic substrate-containing liquid containing a chromogenic substrate that is an enzyme substrate of the labeling enzyme is supplied from the fifth tank 305 to the reaction vessel 61. (Corresponding to the chromogenic substrate supply step).

Next, in step S12, for example, the ultrasonic oscillator 23 of the vibration mechanism 5 is driven for a predetermined time while the electromagnetic coil 6 is turned off (or can be turned on), and the reaction vessel 61 is vibrated. Using the chromogenic substrate, an enzyme reaction is performed in the reaction vessel 61, and the enzyme reaction is promoted by shaking. The reaction product from the enzyme reaction is colored yellow, for example.
Next, in step S13, the optical mechanism 4 is driven to detect the amount of received light B and L after color development. Specifically, the electromagnetic coil 6 as the magnetic attraction mechanism is turned on, and the magnetic beads 83 are retracted from the bottom wall 76 (first portion) of the reaction vessel 61 by the magnetic attraction force as shown in FIG. That is, the optical mechanism 4 is driven in a state in which it is retracted from the transmitted light path TK and held on the peripheral side wall 77 (second portion).

  The blue LED 14 (first light emitting element) and the red LED 18 (second light emitting element) are driven alternately and stored each time the amount of received light B, L is measured. Then, the detected light reception amount Bn of the blue light (first irradiation light) B from the blue LED 14 by the detection light receiving element 15 in the measurement n (n is predetermined) is stored. Further, the detected light reception amount Rn of the red light (second irradiation light) R from the red LED 18 by the correction light receiving element 20 at the nth measurement is stored.

When the blue LED 14 is driven, feedback control is performed so that the amount of blue light B received from the blue LED 14 by the detection light receiving element 15 is constant. Further, when the red LED 18 is driven, feedback control is performed so that the amount of red light R received from the red LED 18 by the correction light receiving element 20 is constant.
Next, in step S14, the degree of color change An (corresponding to change in absorbance) at the n-th measurement is calculated based on the following equation (1).

An = (Bn / B0) / (Rn / R0) (1)
Next, in step S15, the detected color change degree An is compared with a threshold value F1. If it is determined in step S15 that the detected color change degree An is greater than or equal to the threshold value F1 (An ≧ F1) (YES in step S15), the process proceeds to step S16 and the display unit 44 displays For example, after displaying that the antigen has been detected, such as “An antigen has been detected”, the processing is terminated.

In step S15, when it is determined that the detected degree An of the color change is less than the threshold value F1 (An <F1) (NO in step S15), the process proceeds to step S17 and the display unit 44 displays, for example, After displaying that the antigen is not detected, such as “An antigen was not detected”, the process is terminated.
According to the present embodiment, the reaction container 61 can be vibrated to promote the antigen-antibody reaction and the cleaning while the cartridge 60 is held in the holding unit 2 of the apparatus main body 3, so that the inspection efficiency is improved. Greatly improved. In addition, since the excitation mechanism 5 is arranged in an annular shape surrounding the transmission optical path TK, it is possible to achieve downsizing.

  Further, at the stage of detecting the color change, the magnetically attracted magnetic beads 83 are retracted from the first part (bottom wall 76) through which the transmission optical path TK passes and are retained on the second part (circumferential side wall 77). The light received from the light emitting part (the blue LED 14 as the first light emitting element, the red LED 18 as the second light emitting element) is received by the light receiving part (detecting light receiving element 15) without being obstructed by the magnetic beads 83, Color change can be detected with high accuracy. Further, when the reaction vessel 61 is cleaned with the cleaning liquid, the magnetic beads 83 can be magnetically attracted and held on the second portion (the peripheral side wall 77).

Further, the magnetic beads 83 can be easily retracted from the first portion (bottom wall 76) or the retracting can be released easily by turning on and off the electromagnetic coil 6 as a magnetic attraction mechanism.
Further, by placing the conical taper-shaped transmission surface 24b of the ultrasonic horn 24 of the vibration mechanism 5 along the conical taper-shaped peripheral side wall 77 of the reaction vessel 61, the transmission surface 24b having a large area can be obtained even though it is small. Can be vibrated.

In addition, since the vibration mechanism 5 is elastically supported by the elastic member (compression coil spring 25), the transmission surface 24b of the ultrasonic horn 24 is reliably faced to the peripheral side wall 77 of the reaction vessel 61 regardless of variations in dimensional accuracy. It is possible to increase the inspection efficiency by making sufficient contact and sufficient vibration to improve reaction efficiency and cleaning efficiency.
Further, in the optical mechanism 4, the first light emitting element (blue LED 14) and the second light emitting element (red LED 18) are caused to emit light alternately and intermittently, so that the first irradiation light (blue light B) and the second irradiation light ( Since the red light R) is received by the common detection light-receiving element 15 and the common correction light-receiving element 20, the structure can be simplified and the apparatus can be miniaturized.

In addition, using the formula (1), the change in the amount of received light of the first irradiation light (blue light B) whose absorbance changes due to color change and the reception of the second irradiation light (red light R) whose absorbance does not change due to color change. A color change is detected based on a comparison with the change in quantity. Therefore, the influence of disturbance can be suppressed, color change can be detected with high accuracy, and accurate antigen detection can be performed.
9 and 10 show another embodiment of the present invention. This embodiment shown in FIGS. 9 and 10 is mainly different from the first embodiment of FIG. 4 in that, in the embodiment of FIG. 4, an electromagnetic coil 6 that is on / off controlled is used as the magnetic attraction mechanism. In the second embodiment, as shown in FIGS. 9 and 10, a magnetic attraction mechanism including an annular permanent magnet 6A and a solenoid 94 as a driving member for displacing the permanent magnet 6A is used.

Specifically, the center portion 761 of the bottom wall 76A constitutes a first portion through which the transmitted light path TK passes, and the annular portion 762 surrounding the center portion 761 in the bottom wall 76A constitutes a second portion. The permanent magnet 6A can be inserted through the ultrasonic horn 24, the ultrasonic oscillator 23, and the holder 22 and can change the distance to the annular portion 762 (second portion) of the bottom wall 76A.
Further, the permanent magnet 6A is moved to a position close to the second portion (annular portion 762) as shown in FIG. 12 and a position separated from the second portion (annular portion 762) as shown in FIG. The ECU 40 controls the drive of the solenoid 94 so that the control is performed.

The solenoid 94 includes a control rod 95 that can be expanded and contracted, and the control rod 95 connects the permanent magnet 6 </ b> A through the first connection arm 96 and the second connection arm 97 so as to be integrally movable. The first connecting arm 96 is inserted through a longitudinal groove 98 through which the optical path forming cylinder 31 is inserted, and the second connecting arm 97 is disposed in the optical path forming cylinder 31 and the holder 22.
In the components of the embodiment of FIGS. 9 and 10, the same reference numerals as those of the embodiment of FIG. 4 are attached to the same components as those of the embodiment of FIG.

  According to the present embodiment, a strong magnetic field by the permanent magnet 6A is applied to the second portion (the annular portion 672 of the bottom wall 76A), so that the magnetic bead 83 is securely attached to the first portion (the center portion of the bottom wall 76A). 761) and can be retained in the second portion (the annular portion 672 of the bottom wall 76A). Although not shown as a modification of the embodiment in FIGS. 9 and 10, an electric motor that is driven and controlled by the ECU may be used as the drive member instead of the solenoid. In this case, the output (linear motion) of a motion conversion mechanism (for example, a ball screw mechanism) that converts rotation of the electric motor into linear motion is transmitted to the permanent magnet. For example, by rotating and driving a screw shaft whose axial movement is restricted by an electric motor, the ball nut whose rotation is restricted and whose axial movement is allowed is moved in the axial direction, and is moved integrally with the ball nut in the axial direction. Displace possible permanent magnets.

The present invention is not limited to each of the above-described embodiments, and the first irradiation light may be irradiation light of a color other than blue light as long as the absorbance changes with color change. In addition, as the second irradiation light, irradiation light of a color other than red light may be used as long as it is irradiation light whose absorbance does not change due to a color change.
Further, the degree of color change An ′ (corresponding to the change in absorbance) may be calculated based on the following formula (2) which is a common logarithm formula.

An '=-log {(Bn / B0) / (Rn / R0)} (2)
In the embodiment, each process is automatically performed. In the process of supplying various liquids, the operator manually supplies the liquid according to instructions displayed on the display unit, and then a feed switch. It is also possible to press and wait for the next instruction.
Further, as at least a part of the display unit, when an antigen is detected, a lamp that displays that fact by lighting may be used.

  DESCRIPTION OF SYMBOLS 1 ... Protein detection apparatus, 2 ... Holding part, 3 ... Apparatus main body, 4 ... Optical mechanism, 5 ... Excitation mechanism, 6 ... Electromagnetic coil (magnetic attraction mechanism), 6A ... Permanent magnet, 94 ... Solenoid (drive member), DESCRIPTION OF SYMBOLS 7 ... Base, 8 ... Side frame, 9 ... 1st support plate, 10 ... 2nd support plate, 11 ... Cartridge receiving plate, 12 ... Cartridge pressing plate, 14 ... Blue LED (1st light emitting element), 15 ... For detection Light receiving element, 16 ... insertion hole, 17 ... light forming block, 18 ... red LED (second light emitting element), 19 ... dichroic mirror, 20 ... light receiving element for correction, 21 ... half mirror, 22 ... holder, 23 ... ultrasonic wave Oscillator, 24 ... ultrasonic horn, 24a ... center hole, 24b ... transmission surface, 25 ... compression coil spring (inertial member), 31 ... optical path forming cylinder, 40 ... ECU (control unit), 43 ... operation unit, 43a ... YES switch, 44 Display unit 50 ... Color change detection unit 51 ... Process detection unit 60 ... Cartridge 61 ... Reaction vessel 62 ... Primary antibody-containing liquid supply path 63 ... First washing liquid supply path 64 ... Secondary antibody-containing liquid supply , 65 ... second cleaning liquid supply path, 66 ... chromogenic substrate-containing liquid supply path, 67 ... sample supply path, 62a to 67a ... liquid insertion hole, 68 ... discharge path, 69 ... waste liquid storage part, 70 ... convex part, 71 ... Liquid channel, 72 ... tube, 75 ... common supply channel, 76 ... bottom wall (first portion), 77 ... circumferential side wall (second portion), 76A ... bottom wall, 761 ... center portion (first portion), 762 ... circular part (second part), 81 ... primary antibody, 82 ... primary antibody-containing liquid, 83 ... magnetic beads, 83a ... surface, 101-107 ... first to seventh pumps, 201-207 ... first to seventh Pump motor, 301, 307 ... first to seventh tanks, B ... blue light (first Shako), R ... red light (second irradiation light), TK ... transmission optical path, HK ... reflection optical path

Claims (4)

A protein detection apparatus using an ELISA method,
A reaction vessel containing a liquid in which a large number of magnetic beads having a primary antibody immobilized on its surface are dispersed, and having a portion through which a transparent light transmission path passes, along the transmission light path. An optical mechanism for measuring the liquid absorbance based on the amount of light received by the light receiving unit, including a light emitting unit that emits irradiation light, and a light receiving unit that receives the irradiation light through the transmission light path;
A protein detection apparatus comprising: a magnetic attraction mechanism that magnetically attracts the magnetic beads to retract the magnetic beads from a portion through which the transmission optical path passes in the reaction container and retains the magnetic beads in a portion to be retracted in the reaction container.   The protein detection apparatus according to claim 1, wherein the magnetic attraction mechanism includes an electromagnetic coil that is turned on and off.   The protein detection apparatus according to claim 1, wherein the magnetic attraction mechanism includes a permanent magnet that can change a distance to the retracted portion, and a drive member that moves the permanent magnet closer to the retracted portion. Protein detection device. A protein detection method using an ELISA method,
A reaction vessel containing a liquid in which a large number of magnetic beads having a primary antibody immobilized on its surface are dispersed, and having a portion through which a transparent light transmission path passes, along the transmission light path. Receiving the irradiation light emitted from the light emitting unit of the optical mechanism by the light receiving unit of the optical mechanism through the transmission optical path, and measuring the liquid absorbance based on the amount of light received by the light receiving unit,
In this step, the magnetic beads are magnetically attracted to retract the magnetic beads from the portion through which the transmission optical path passes in the reaction vessel, and hold the protein in the portion to be withdrawn in the reaction vessel. Detection method.

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