JPS62148858A - Automatic immunological measurement instrument - Google Patents

Abstract

PURPOSE:To make stable measurement with high accuracy as an automated system by successively disposing a conveyance route which makes the progressive and successive feed of test plates each having plural immunological reaction chambers opened upward at specified time intervals, sample dispenser, B/F separator, substrate dispenser and photometric instrument. CONSTITUTION:The conveyance route is usually constituted by the combination of a smooth conveyance of a specified length to be imposed with, for example, the test plates, and a conveyance mechanism and is provided with the devices A-D from the upper stream toward the down stream thereof. The sample dispenser A is disposed with a micropipette which is a device to inject a specified amt. of sample liquid into the test cups each having the prescribed antibody which possesses an enzyme label. The B/F separator B is used to clean, discharge and remove the residues of reaction and makes alternately injection, suction and discharge of a cleaning liquid plural times. The substrate dispenser C injects a substrate soln. into the cells in each of which the fixed phase antibody-antigen-enzyme labeling antibody complex is fixed. The photometric instrument D measures absorbancy or degree of fluorescent.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Application of the Invention) The present invention relates to an automatic immunoassay device used for automatically and continuously measuring a large number of analytes (samples).

[Background of the invention]

Detection of trace amounts of biological substances using immunological techniques, such as application as diagnostic kits, has recently attracted a lot of attention, and various attempts have been made. In particular, testing work is monotonous and tiring work, it is difficult to handle minute amounts of samples and reagents with high accuracy over a long period of time, and there is a possibility of errors due to individual differences among laboratory technicians, and Due to the shortage of laboratory technicians compared to the ever-increasing number of specimens, there is a growing demand for automatic measurement of immune reactions.

By the way, the method used as an immunological method is to label (
For example, PTA using a chemifluorescent substance as a label, RIA using a radioactive substance, EIA using an enzyme, etc. are known. Therefore, when automating these measurement systems, each immune reaction,
A 4N configuration is required that suitably employs and combines various configurations appropriate to problems specific to the nature of the sign, and also takes into consideration practical usability and cost.

[Purpose of the invention]

The present invention has been made from the above points of view. The present invention provides a suitable automatic immunoassay device.

Another object of the present invention is to provide an automatic immunoassay device that can continuously and efficiently measure and process a large number of samples.

[Summary of the invention]

The apparatus of the present invention, which has been made to achieve the above-mentioned object, is characterized by a transport path in which a test plate having a plurality of immunoreaction chambers opening upward is intermittently fed sequentially at regular intervals; From the upstream side to the downstream side, at least A: Sample dispensing device B that injects the sample to be measured into the immune reaction chamber; B/F separation device that removes reaction residues in the immune reaction chamber: Inside the immune reaction chamber It has a configuration in which devices A to D, which are a substrate dispensing device D for injecting a substrate solution and a photometric device for measuring optically detectable changes occurring in the substrate in the immunoreaction chamber, are arranged in sequence. .

The outline of the enzyme immunoassay processing system targeted by the device of the present invention is the sandwich method and the competition method.

There are some differences between antigen measurement and antibody measurement, but in this explanation, we will explain the case of antigen measurement using the Sand-Deutsch method. There is no essential difference in other measurement methods.

Generally, the first step is to immobilize an antibody on the surface of an insoluble carrier and react with the antigen in a specific specimen sample to form an immobilized antibody-antigen-enzyme-labeled antibody complex.The complex bound to the insoluble carrier is immunized. B: Remove excess sample while leaving it in the reaction chamber
The second step of /F separation, the third step of adding a substrate solution to the complex and applying the enzyme activity to the substrate, and the change that appears in the substrate simultaneously or independently of this, (For example, the change in which the substrate 4-methylumbelliferyl phosphate monoester is decomposed by the enzyme alkaline phosphatase into 4-methylumbelliferone, which can be detected by fluorescence) is optically measured. D: Further, the first step includes preparing a container etc. that provides a cell (reaction chamber) having a predetermined antibody according to the test item fixed on the surface of an insoluble carrier, injecting a sample into the cell,
It is necessary to perform operations such as injection of enzyme-labeled antibodies (which may be added to the cell in advance) simultaneously with or independently of the sample injection. It requires that.

Therefore, in the present invention, a cell (reaction chamber) is opened upward, and a test plate having this cell is sequentially and intermittently transported along a transport path at predetermined timing intervals, while performing appropriate process treatments. The device with the above-mentioned configuration was adopted in which continuous and automatic immunoassay processing is performed by moving the device to a certain position and stopping it.

According to such a configuration, by setting the intermittent feeding timing of the test plates, it is possible to automatically feed a large number of test plates continuously and through each process and perform measurements.

As described above, the intermittent feeding timing is determined based on the required time of the mechanism and device that takes the longest time to perform the required operation with the test plate stationary among the mechanisms and devices sequentially arranged along the conveyance path as described above. It can be determined based on the following.

In addition, the amount of feeding that is carried out intermittently is usually determined based on the arrangement interval of the reaction chambers on the test plate.In the present invention, the test plate used may be a known multi-titer plate, or a single plate. It may also be of the type with multiple fitting holes into which multiple test cups providing each reaction chamber are fitted. Particularly in the latter case, the advantage is that it is easy to extract and align multiple types of test cups filled with different types of antibodies prepared according to the test items of the subject according to the necessary test items. There is.

The conveyance path includes, for example, a smooth conveyance surface of a certain length on which the test plate is placed, and a conveyance mechanism (rotating belt, cam mechanism, etc.), and it is preferable to provide an appropriate temperature control device along the conveyance path in order to maintain an appropriate temperature for immune reactions and enzyme reactions.

The devices sequentially arranged from upstream to downstream of the transport path must include at least the devices A to D described above for the purpose of enzyme immunoassay.

The first sample dispensing device A contains a predetermined antibody immobilized on an insoluble carrier, such as beads in a test cup, and a predetermined antibody with an enzyme label loaded in the test cup separately. A micro and a bed, which are devices for injecting a certain amount of sample liquid into each test cup, are arranged in horizontal rows (!2 rows in a direction perpendicular to the feeding direction). It is preferable to use a method of scanning in the row direction. If the enzyme-labeled antibody is to be injected separately, the injection device may be placed downstream of the sample dispensing device.

The second B, /F separation device B is for washing and removing reaction residues from the inside of the cell, such as the test cup, which has been incubated for a predetermined period of time after the sample injection. Generally, a type is used in which a cleaning liquid is injected into the cell through a cleaning nozzle, and the cleaning liquid is sucked out using a suction nozzle.

Further, at this stage, it is preferable to alternately perform injection and suction and discharge of the cleaning liquid a plurality of times.

The third substrate dispensing device C dispenses the substrate into the cell from which the sample has been removed as described above and in which the immobilized antibody-antigen-enzyme-labeled antibody complex is immobilized on the surface of the insoluble carrier. A device for injecting solutions. This is a device used for measuring the fluorescence intensity of a general liquid injection device. When using a test plate having multiple rows of cells, which is a preferred embodiment of the device of the present invention,
It is preferable to have one photometer for each row of cells facing each other. In this way, the photometry operation, which takes a relatively long time for photometry and determines the timing of intermittent feeding of the test plate in the entire system, will be made more efficient, and the overall efficiency of the measurement operation will be improved. Because it will be.

(Example of the invention) The present invention will be explained below based on embodiments shown in the drawings.

FIGS. 1(a) and 1(b) show the external appearance of an example of the enzyme immunoassay device, which is the device of the present invention. An operation panel 2 is provided on the front. 3 is located below the measuring section 1.

This shows a test cup storage section in which a test plate can be raised and lowered between the test plate and the upper measuring section 1 using an elevator device to be described later. '4 is a base housing the control equipment, power mechanism, and reagent liquid container of the apparatus. Further, a disposal packet 70 for used test cups is attached to the side of the device.

FIG. 2 and FIG. 3 are diagrams showing the outline of the internal configuration of the measuring section of the device. A seal breaking device 1 is provided for tearing the upper opening seal of the test cup from the upstream to the downstream of the conveyance path in this example.
2. Test cup disposal devices 80 are provided above the conveyance path at predetermined intervals.

Reference numeral 50 in FIG. 3 indicates a sample loader. Feed in the direction of the arrow D: Attach the nozzle tip to the nozzle body and dispense a certain amount of the sample into a predetermined test cup for each subject, so that this can be repeated sequentially for each subject. controlled. In the figure, 16 indicates a test plate, and 17 indicates a test cup.

FIG. 4 is a plan view showing an outline of the internal configuration of the test cup storage 3. test plate 16, rail 20.
A take-out device 19, which is moved in the horizontal x and y-axis directions by a support plate 20 and a support plate 21, takes out a predetermined test cup from the test cup shelves a to n, which are stored by type according to a predetermined order. It is assembled into a predetermined position of the plate 16 to form a test plate incorporating the necessary test cups before reaching the ascending elevator 5. Reference numeral 40 denotes a stopper for stopping the test plate 16 on the conveyance path 18; D: A stopper 40 is projected onto the conveyance path by a drive means (not shown) in order to time the loading of test cups and transfer to the ascending elevator 5, for example. , is provided so as to be evacuated outside the conveyance path when necessary.

The above operation control may be performed by a control circuit equipped with a microcomputer.
Reference numeral 60 shown in the drawings and FIG. 3 indicates control of each drive mechanism of the overall device of this example.

A main controller module with a built-in microcomputer for controlling input from the operation panel and displaying predetermined data, etc. D: Reference numeral 61 also controls the timing of each drive part according to commands from the main controller module 60. It shows a sub-controller module with a built-in microcomputer that performs control. An overview of the movements (operations) of the entire device performed by these control modules 60, 61 in this example is shown in the block diagram of FIG. 5(a).

S5 (b) is for explaining the outline of the system in which this example device is controlled by the main and sub-controller modules 60 and 61. A measurement order (or a measurement order read from the memory using a floppy disk) is input, and measurements of predetermined inspection items are performed according to this measurement order.

FIG. 6 is an expanded view of the structure of the conveyance path.D:
The conveyance path in this example includes an upper cover 22 forming a conveyance surface;
A magnet holder 23 that is in close contact with the lower surface of the upper cover 22 and has a plurality of grooves in the longitudinal direction. This magnet holder 2
A magnet bar 24 is fitted so as to be able to reciprocate in the longitudinal direction within the groove of No. 3, and has a large number of magnet pieces 25 fixed to its upper surface;
Temperature control plate 26 closely attached to the bottom surface of the magnet holder 23
, side plates 27 and 28 on both sides that support the above laminate on the sides.
D: One side plate 28 has a test plate conveyance bar 29, which will be described later, incorporated therein.

The magnet bar 24 in this example is used to stir or improve photometric accuracy by applying an oscillating magnetic field (usually several Hz) to the magnetic beads 34 in the test cup. In addition, in this figure, the temperature control plate 26 has a meandering groove, and the temperature is controlled by circulating constant temperature water in the groove. A planar heater may be installed on the surface between 26, and a temperature sensor may be installed in the stone plate 23 or the main plate 26 to directly control the temperature.

FIG. 7 is a diagram showing the test plate 16 on the transport path 7 and the transport mechanism. D: The test plate 16 of this example is
A thin PVC plate 16 is placed on the top surface of the main body 16a made of AfL.
b is coated to reduce heat dissipation. The test plate 16 has an appropriate number (for example, 5 x 5) of test cup mounting holes 16 that pass through the test plate 16 in the vertical direction.
c is formed. Further, on the side surface of the test plate 16 on the side plate 28 side, as shown in FIG. 7, serrated ratchet teeth 16d with a constant pitch are formed. It is adapted to engage with a ratchet pawl 30 of a reciprocating bar type conveying bar 29.

The conveyance bar 29 is provided so as to be reciprocated within a certain range by an air cylinder device (an eccentric cam mechanism may be used) 31. This allows the ratchet pawl 30 to move against the test plate 16. The test plate 16 is engaged with the ratchet teeth 16d, and the test plate 16 is sequentially and intermittently fed in the direction of the arrow on the conveyance cover 22. It goes without saying that the ratchet pawls 30 are assembled to the conveying bar 29 at appropriate intervals.

Reference numeral 35 denotes a return prevention pawl D: which is pivotally attached to the side plate 28 and engages with the ratchet @16d of the test plate to prevent the test plate from moving in the return direction.

FIG. 9 is a plan view showing the upstream side of the conveyance path 7 and the opening of the ascending elevator. A push-in air cylinder device 32 is provided for causing this. 32a is an air cylinder;
32b is a cylinder piston, and 32C is a test plate pushing piece fixed to the piston.

33 is a drive motor for reciprocating the magnet bar 24 in the direction of the arrow at a predetermined frequency; D: reciprocating the magnet bar 24 via an eccentric cam mechanism;

FIG. 10 is a plan view showing the downstream side of the conveyance path 7 and the opening of the descending elevator. It is designed to be lowered to 3.

In this example, a gate 36 that supports both sides of the lower surface of the test plate to be accommodated is arranged in the descending elevator part, and when necessary, the gate 36 is moved to the air cylinder device 37.
Then, the test plate is moved away from the support position of the elevator so that the test plate can be lowered.

In the automated apparatus configured as described above, first, a test plate, on which a predetermined number and combination of test cups are assembled and mounted, is pushed up from the ascending elevator 5 to an upstream position of the conveyance path 7. Next, the test plate is pushed into the conveyance path 7 by the pushing cylinder device 32, and thereafter the test plate is conveyed downstream by a constant length at predetermined timing intervals by the intermittent conveyance means having the ratchet pawl 31.

A summary of the above-mentioned overall movement is shown in Figure 5 (a) as mentioned above.
This is illustrated by a block diagram.

Next, each device arranged along the conveyance path 7 will be explained.

In this example, the test plate is first stored and stopped at the position of the seal pre-cooling J device 8, and a plurality of test cups in a horizontal row (a row perpendicular to the feeding direction) are moved downward by a vertically movable wedge. Press the seal on the top of the test cup to break it. This seal breaking is done simultaneously by placing one wedge for each row of test cups, and then repeating the same operation for the next row of test cups. One pitch of the intermittent conveyance of the test plate is matched with the interval in the conveyance direction of the rows of test cups mounted on the test plate.

The test cups with broken seals are then placed in the sample dispensing device 9, and a predetermined sample is sequentially injected into each test cup using nozzles that are scanned in the row direction. The test cup 17 of this example has magnetic beads 34 to which an antibody is bound on the surface and another enzyme-labeled antibody loaded inside the test cup 17. A specific reaction of phased antibody-antigen-enzyme-labeled antibody is performed. This reaction needs to be carried out for a certain period of time. Therefore, in this example, as shown in FIG.
is given an incubation area of a predetermined length.

FIG. 11 is a diagram showing a specific configuration example of the sample dispensing device 9 of this example described above.
A pipette 91 with a nozzle tip 14 removably attached to its lower end, which is replaced for each sample, is connected at its upper end to an air pressure supply/discharge device (not shown) through an air pipe 92, and the pipette 91 is connected to a pulse motor. 93, the sample suction 1 is moved up and down via the cam mechanism 94.
Dispensing 1 etc. are performed sequentially.

The sample dispensing device 9 of this example uses one pipette 91 to dispense samples into test cups 17 on test plates 16 arranged in multiple rows.D: For this purpose, the pipette 91 are guided by guide rods 95, 95 and scanned by a scanning drive mechanism (not shown).

Note that 96 is a removal lever that is rotated downward about a fulcrum 97 by a cylinder device (not shown) in order to remove the nozzle tip.

The test plate loaded with the test cup that has undergone the sufficient reaction after passing through the incubation area is transferred and stopped intermittently at the position of the B/F separator 10, and while being intermittently fed, one test cup is prepared for each horizontal row of test cups. Separate B/F and wash by suctioning and discharging the cleaning liquid using the nozzle provided! The conditions are met.

A specific example of the configuration of the B/F separation device of this example is shown in FIG. Note that this example is mechanically related to and integrated with the substrate dispensing device 11 in the next stage.

In this example, two pairs of cleaning tubes 101 and 102 consisting of a cleaning liquid injection tube and a discharge tube are supported by a support punch 104, and the support punch 104 is further supported by a support frame 103 so as to be movable up and down. Further, a substrate solution reservoir block 105 having a temperature control function and a substrate dispensing tube 106 are fixed to the support frame 103.

Then, this support punch 104 is placed on the support frame 103 with respect to the test cup 17 on the test plate 16 that is intermittently fed.
After moving to a predetermined position by moving in the a-a direction in FIG.

According to such an example, sufficient washing is carried out, so that there is substantially no residual free enzyme-labeled antibody, which is effective in improving measurement accuracy.

Reference numeral 108 indicates the support punch 104 by a driving means (not shown).
a guide rod that guides the movement when moving in the direction;
Reference numeral 107 indicates an air cylinder device for moving the support punch 104 in the bb direction.

FIG. 13 shows a flowchart of the control operation in the sub-controller module 61 that drives and controls the B/F separation device 10 described above.

As described above, the substrate solution is injected into each of the row test cups, and the enzyme reaction is started.

The test cup injected with the substrate solution is transferred to the photometric device 12 and stopped intermittently for one period, the state of change occurring in the substrate is optically measured, and the antigen concentration in the sample is assayed based on this photometric information.

As this photometric device, a known photometric device used for establishing an immune reaction can be used. Figure 14 is a diagram schematically showing the principle configuration of such a photometric device.D: For convenience of illustration, the figure shows the light coming out from side to side with respect to the test cup, but in reality In this example device, this is performed from top to top.

In this figure, 121 is a pulse voltage power supply circuit with a built-in clock pulse generator, and 122 is a fluorescent tube to which a pulse voltage is applied. It is lit intermittently by pulse voltages of different predetermined frequencies. The light from this fluorescent tube 122 is irradiated into the test cup 17 through an excitation side filter 123 and a condensing lens 124. The above constitutes a light projection system.

Fluorescence emitted from the test cup 17 to the outside is received by a light receiving sensor 127 through a condensing lens 125 and a fluorescence side filter 126, and an electrical signal obtained according to the characteristics of this light receiving sensor 127 is synchronously detected via an amplifier 128. 129. The synchronous detector 129 receives the clock pulse signal from the clock pulse generator of the pulse voltage power supply circuit 121 and synchronously detects the signal from the amplifier 128. Effects of external light, dark current, and offset components can be effectively removed. The detected signal is manually input to a voltmeter (not shown) and measured, for example, as the amount of change in fluorescence intensity over a certain time range, and is used as data for calculating the amount of antigen-antibody combination in the sample container.

The above constitutes the light receiving measurement system.

Note that in this example, one photometer is provided for each row of test cups in accordance with the number of rows of test cups, but if there is a restriction on the arrangement space, it may be necessary to install one photometer for each row in the direction of the rows of test cups. After one photometry, the photometry unit is shifted by one test cup in the row direction and a second photometry is performed, so that the photometry of all the test cups in the row is doubled.
Dosing may be done in batches.

The test plate for which photometry has been completed is transported to the test plate storage section 3 by a descending elevator, and is loaded, for example, on a shelf for which verification has been completed.

In this example, the test cup 17 on the test plate 16 that has been moved to the position of the descending elevator 6 is automatically pulled out from the test plate 16 by the cup disposal device 80, and the test cup 17 on the disposal chute 81 is placed in the disposal packet 70. I try to let it slide and fall into place.

Such a cup discarding device 80 may be provided with, for example, a fork-shaped cup suspension means, so that each test cup 17 enters the groove between the fork-shaped portions as the test plate 16 is moved, and is suspended by the fork. An example of such a system is that the test plate can be suspended, and then the test plate is automatically uncoupled by moving the test plate downward, leaving only the test cup in the suspended state. The test cup left suspended by the fork-like suspension means is pushed out by the next test plate and dropped into the waste packet 70 when the next test plate is transferred.

According to such a configuration, a plurality of test plates can be
It has the advantage that it can be conveyed continuously with almost no intervals, and the processing efficiency is extremely high.

In addition, as in this example, a vibrating magnet bar is provided on the lower surface of the transport path, and the magnetic beads 34 in the test cup are constantly vibrated at low frequencies. Validation has benefits D: There is an effect of making it suitable for high precision measurement.

Another advantage is that the loading of the test cup onto the test plate can be done automatically in the test cup storage based on pre-entered data.
As a device with a high degree of completeness as a system, its practical usefulness is extremely high.

It goes without saying that the present invention is not limited to the embodiments described above, but can be implemented in various modified embodiments.

For example, it may be advantageous to configure the B/F aliquoting device and the substrate dispensing device separately. This is illustrated, for example, by FIG. That is, FIG. 15 shows an example in which the base 7i solution is dispensed into the test cup 17 located opposite the photometric device 12 in the apparatus shown in FIG. 12. In this way, changes in the fluorescence of the substrate in the dispensed solution can be measured from the initial stage, which is particularly useful for measuring highly concentrated analytes.

〔Effect of the invention〕

As mentioned above, the automatic immunoassay according to the present invention enables stable and highly accurate measurements.Furthermore, it is possible to efficiently measure a large number of samples through continuous processing, and its practical usefulness is extremely high.

[Brief explanation of drawings]

1(a) and 1(b) are diagrams showing the external configuration of an exemplary device according to the present invention, FIG. 2 is a front view showing an outline of the internal configuration of the measuring section of the device, and FIG. 3 is similar to FIG. 4 is a schematic diagram of the cross-sectional configuration taken along the line II-II of FIG. 2, FIG. ) is a simple explanatory diagram of the control system, Figure 6 is an exploded view of the configuration of the transport path, Figure 7 is a partial perspective view to explain the test plate transport mechanism, and Figure 8 is a part of the test plate. 9 is a detailed plan view of the upstream portion of the conveyance path, FIG. 10 is a detailed plan view of the downstream portion of the conveyance path, FIG. 11 is a perspective view showing an example of the configuration of the sample dispensing device, and FIG. The figure shows an overview of the configuration of the device for B/F separation and substrate dispensing - an example; Figure 13 shows the B/F separation,
Flow chart explaining the operation of the substrate dispensing device, No. 14
The figure is a schematic diagram for explaining an example of a fluorescence detection device, and FIG. 15 is a diagram for explaining another example of a substrate dispensing device. 1: Measuring section 2: Operation panel 3: Test cup storage 4: Base 5.6: Elevator vertical path 7: 112 feed path 8: Seal break device 9: Sample dispensing device 10: B/F separation device 11
: Substrate dispensing device 12: Fluorescence measurement device 13: Sample cup 14: Nozzle tip 15: Cup holder 16: Test plate 16a: Main body 16b: PVC plate 16c: Test cup installation hole 16d: Ratchet teeth 17: Test cup 18: @ Feeding path 19 extraction device 2o: Rail 21: Support plate 22. Upper cover 23 = magnet holder 24: magnet bar 25 = magnet piece 26
: Temperature control plate 27, 28: Side plate 29: Test plate transport bar 30: Ratchet claw 31: Air cylinder device 32 Niji cylinder device W 32 a: Air cylinder 3
2b Niji cylinder piston 32c: Test plate pushing piece 33: Drive motor 34: Magnetic bead 35: Take-back prevention claw 36: Gate 37: Air cylinder device 40: Stop collar 5o: Sample loader 60: Main control module 61: Sub-control module 70, Disposal packet 80: Cup disposal device 81: Disposal chute 91; Pipette 92: Air pipe 93: Pulse motor 94: Cam mechanism 95 Guide rod 96: Removal lever 97: Fulcrum 101: Washing nozzle 102: Washing nozzle 103: Drain pipe 104: Support Punch 105: Substrate solution reservoir block 106: Substrate dispensing tube 107: Air cylinder device 108 Ni guide rod 121: Pulse voltage power supply circuit 122: Fluorescent tube 123: Excitation side filter 124: Condensing lens 125: i-light lens 126: Fluorescence side filter 127: Light receiving sensor 128: Amplifier 129: Synchronous detector Fig. 1 (α (b) Fig. 2 Fig. 5 (α) Fig. 13 Fig. 14 Fig. 1?1 Fig. 15 Procedure amendment book Showa 1/1999 R U to Tsukiyu

Claims (1)

[Scope of Claims] A transport path in which a test plate having a plurality of immune reaction chambers opened upward is sequentially and intermittently fed at regular time intervals; An automatic immunoassay device comprising the following devices A, B, C, and D. A: Sample pipetting device for injecting the sample to be measured into the immune reaction chamber B: B/F separation device for removing reaction residues in the immune reaction chamber C: Substrate pipetting device D for injecting the substrate solution into the immune reaction chamber : Photometric device for measuring optically detectable changes in the substrate within the immune reaction chamber.