JPH02242163A - Light-emitting-reagent injecting device of automatic immunity measuring apparatus - Google Patents

Abstract

PURPOSE:To maintain the stability of light emitting reagents and to make it possible to collect data stably by mixing and injecting a plurality of reagents immediately before the detection of light emission. CONSTITUTION:In a pump sucking step, a light-emission aiding reagent is sucked out of a tank 38 with a pump 34. Water is sucked out of a tank 41 with a pump 35. Light emitting reagents are sucked out of tanks 39 and 40 with pumps 36 and 37 at the same time. When a valve 33 is switched and a pump discharging step is set, the aiding reagent and the water reach specified temperature through a preheater 44 and are injected into a specified cartridge on a turntable 2. The light emitting reagents are injected into a cartridge of a detector 10 through a three-way joint 42 and a mixer 46. Therefore, the light emission reagents are injected and mixed immediately before the detection of the light emission.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an immunoassay device that measures components contained in samples such as serum or urine through an immune reaction using a solid phase reagent, and particularly relates to an immunoassay device that measures components contained in a sample such as serum or urine. The present invention relates to a luminescent reagent injection device for an automatic immunoassay device that mixes reagents and injects them into a sample.

[Conventional technology]

Enzyme immunoassay is a method that uses enzyme activity as a marker to determine the extent of antigen-antibody reaction, and then quantifies the amount of antigen or antibody. Sand-Deutsch method that does not compete with antibodies (or antigens), competitive reaction methods that determine the amount of unlabeled antigen by competing enzyme-labeled antigens (or antibodies) with unlabeled antigens (or antibodies), and various other methods. There is a method.

FIG. 4 is a diagram for explaining the principle of measurement by the Sand-Deutsch method, and FIG. 5 is a diagram for explaining the principle of measurement by the competitive method, in which 61 is a solid phase carrier, 62 is an antibody, 63 is an antigen to be measured, 64 is a labeling substance, 65 is an m-identifying antibody, 66 is a substrate,
67 indicates the product.

As shown in FIG. 4, in the Sand-Deutsch method, (1) First, the antibody 62 is made into a solid phase by binding using physical adsorption or chemical reaction to the surface of the solid phase carrier 61, and the antigen to be measured 63 is attached to this. Add samples containing.

(1) As a result, although there are various contaminant components in the sample, the antibody 62 and antigen 63 react specifically (first reaction), and the antigen to be measured 63 binds to the immobilized antibody 62; Contaminant components in the sample are discarded by washing.

(2) Next, an enzyme-labeled antibody 65 to which an enzyme is bound as a label 64 is added.

(2) As a result, an antigen-antibody reaction (second reaction) occurs, and the enzyme 1-recognizing antibody 65 binds to the antigen 63 bound to the immobilized antibody 62. In this case, since the enzyme-labeled antibody 65 is added in excess, a bound portion (b) formed by binding the antigen 63 and the antibody 65.

und , B) and the free part (fr
ee, F) can be done.

■ Therefore, by cleaning the free part (f
F) Discard the excess enzyme-labeled antibody.

That is, B/F separation is performed.

■ Next, perform an enzymatic reaction. Since the enzyme activity at this time is determined by the amount of enzyme-labeled antibody in the bound portion, the amount of enzyme-labeled antibody represents the amount of antigen bound to the immobilized antibody, that is, the amount of antigen to be measured.

In the competitive reaction method, as shown in Figure 5, (1) An antibody is immobilized on a solid phase, and an antigen to be measured and a labeled antigen in which a label is bound to the same antigen as the antigen to be measured are added.

(2) As a result, an antigen-antibody reaction occurs, and the antigen to be measured and the labeled antigen bind to the immobilized antibody in proportion to their respective amounts.

(2), (2) Next, B/F separation is performed in the same manner as the Sandersch method, and an enzymatic reaction is performed. Since the enzyme activity at this time is determined by the amount of enzyme-labeled antibody in the bound portion, the amount of the antigen to be measured can be determined from the amount of labeled antigen added using a calibration curve.

All of the above methods are so-called separation methods, and there are also non-separation methods that do not perform B/F separation.

Although the non-separation method is quick in measurement time and easy to operate, it has drawbacks such as low measurement sensitivity. In comparison, separation methods have high measurement sensitivity, but are extremely complex and cumbersome to operate, and are difficult to automate (previously, they were performed manually in most cases).

When the above immunoassay is automated, antibodies or antigens are immobilized on a solid phase such as polystyrene poles or glass beads, and the solid phase reagent is used as a solid phase reagent.

Since this solid phase reagent is unstable, it is usually kept in a container filled with a storage solution, and when performing enzyme immunoassay, the solid phase reagents are transferred one by one from the container to the reaction detection container. , sample dispensing, labeling reagent dispensing, B/F separation, and washing, and then the bound portion is transferred to the detector.

By the way, when measuring using an enzymatic reaction, for example, a dye solution is formed and the reaction is detected by measuring the color density with a colorimeter, but the detection sensitivity is low, so the immune reaction is difficult to detect. The chemiluminescence of the product is detected by injecting a luminescent reagent into the product. And for chemiluminescence detection,
There are two types: a flow cell type that measures the reaction product by continuously flowing it, and a batch type that measures each test tube using test tubes. Luminescence detection is also performed by placing a detection element such as a photomultiplier close to the luminescent reaction solution.

[Problem to be solved by the invention]

In automated immunoassays that detect such chemiluminescence,
For example, hydrogen peroxide (H,02) and an alkali are reacted and injected into a sample as a reagent, but there is a problem in that H3O2 and alkali react immediately and H3O2 disappears.

In this way, when multiple luminescent reagents are mixed and used, the luminescent reagents cannot maintain a stable state when mixed, resulting in data changes and making it impossible to stably collect data. There wasn't.

The present invention has been made in view of the above circumstances, and is an automatic immunization device that measures immune reactions using chemiluminescence.
Maintains the stability of the luminescent reagent and provides a stable amount of luminescent light.
The present invention relates to a luminescent reagent injection device for automatic immune reaction measurement that can stably collect data.

[Means to solve the problem]

To this end, the present invention provides an automatic immunoassay device that measures an immune reaction by injecting a luminescent reagent into an immune reaction product in a reaction tube and causing chemiluminescence, which includes a container each containing a plurality of luminescent reagents, and a container in each container. The present invention is equipped with a mixing means for mixing luminescent reagents, and an injection means for injecting the mixed reagents into a reaction tube, so that a plurality of reagents can be mixed and injected immediately before emitting light, and the luminescent reagent is injected by the luminescent reagent injection means. It is characterized by preliminary ejection.

[Effect]

The luminescent reagent injection device for automatic immunoreaction measurement of the present invention can stably hold the luminescent reagents by mixing the luminescent reagents stored separately just before luminescence detection, and furthermore, the luminescent reagents can be stably held during luminescence detection. By preliminarily discharging the reagents, the remaining substances in the injection part are washed away and their influence is eliminated, so that the stability of the data can be maintained.

〔Example〕

Examples will be described below based on the drawings.

FIG. 1 is a diagram showing one embodiment of the configuration of an automatic immunoassay device according to the present invention, and FIG. 2 is a diagram showing an embodiment of a cartridge used in the automatic immunoassay device according to the present invention. In the figure, ■ is a reaction turntable, 2 is a cartridge turntable, 3 is a reagent turntable, 4 is a sample turntable, 5 is a sample cup, 6 is a disposable chip,
7 to 9 are arm mechanisms, 10 is a detector, 11 is dust, 1
2 is the control processing section, 21 is the car) IJ body, 22 is the filter, 23 is the solid phase reagent, 24 is the discharge hole, 25 is the opening, 26 is the aluminum cap, 27 is the orimeter, and 28 is the tip. .

In Fig. 1, the cartridge turntable 2 is a table that stores and rotates an IJ tube (reaction detection container) as shown in Fig. 2, and has 10 removable cassettes. An example is shown in which 30 cartridges can be stored in each cassette. According to this, each section stores cartridges of the same immobilized antibody, so it is possible to store cartridges for 10 items (IJ).
can be prepared. The reagent turntable 3 is a rotating table that stores labeled antibody reagent bottles and disposable chips for dispensing the labeled antibodies.
That is, an example is shown in which reagent bottles for 10 items can be stored. The sample turntable 4 is a rotating table that stores a sample cup 5 containing a sample and a disposable chip 6 for dispensing the sample, and has two disposable chips 6 inside corresponding to each sample cup 5. An example is shown in which it can be stored.

The reaction turntable 1 is used to perform sample dispensing, addition of a labeled antibody, stirring reaction by vibration, washing, etc., as described above, while the cartridge of the cartridge turntable 2 is set and rotated by 1 position. . The arm mechanisms 7 to 9 are mechanisms for inserting and removing cartridges and dispensing reagents and samples between the reaction turntable 1, cartridge turntable 2, reagent turntable 3, and sample turntable 4, respectively. Circles a, b, and c show the locus of . Also, detection 5
Numeral 10 is for injecting a luminescent reagent into the cartridge after the reaction and detecting the amount of luminescence, and dust 11 is for discarding the cartridge after the amount of luminescence has been detected.

As shown in FIG. 2, the cartridge used in the automatic immunoassay device according to the present invention has a cartridge main body 21 having a cylindrical shape, a solid phase reagent 23 placed therein, and a filter 22 placed below it.
Further, a narrow discharge hole 24 is provided halfway below the filter 22, and an opening 25 at the upper end is closed with an aluminum cap 26. Solid phase reagent 2
3 has antibodies immobilized on the surface of granules with a diameter of several tens of μm. Since the antibodies and antigens in solid phase reagent 23 are proteins, they are easily decomposed, so they are treated with preservatives and buffers to maintain a constant pH. It is immersed in a preservation solution consisting of liquid, etc.

Further, there is an orimeter 27 cut in the part where the ejection hole 24 is provided, and by bending the tip end 28 at the orimeter 27, the cartridge can be easily removed from the cartridge body 21, and the ejection hole 24 can be passed through. be able to. The discharge hole 24 is formed with an extremely small diameter, and
Since the filter 22 is arranged, even when the tip 28 is removed from the car) IJ body 21, dispensed samples, reagents, washing water, etc. It is not easily discharged from the discharge hole 24, but is discharged by supplying pressurized air from the opening FA25 at the upper end or by suctioning from the discharge hole 24.

Therefore, the cartridge has an aluminum cap 2 at the top end.
Depending on the day, the solid phase reagent 23 is stored on the filter 22 with its lower end completely sealed by the tip 28 and stored in the cartridge turntable 2. and,
When this cartridge is transferred from the cartridge turntable 2 to the reaction turntable 1 by the arm mechanism 7, the tip portion 28 is removed in the dust 11, and the storage solution is discharged at the next position on the reaction turntable 1 for cleaning. ing.

Next, the operation of the automatic immunoassay device according to the present invention will be explained based on the flow system.

Figure 3 is the overall flow diagram, 31 is a drain tank,
32 is a compressor, 33 is an in-out switching valve, 34-37.51 and 53 are pumps, 38-41 are tanks, 42 is a three-way joint, 43 is a resistance pipe, 44 is a preheater, 45.52 and 54 are valves , 46 indicates a mixer.

As shown in FIG. 3, the flow system is connected to the reaction turntable 1 having 29 positions, starting from position 2, and flow systems for dispensing samples and reagents, washing, etc. At the base position (3), the cartridge is first set on the reaction turntable, and the reaction turntable is alternately rotated through two predetermined positions.

The cartridge after the reaction is transferred to the detector 10.

First, each flow system connected to the reaction turntable 1 will be explained in FIG.

The dray tank 31 is connected to each car of the reaction turntable 1)
This is for storing the storage solution and cleaning solution discarded from the +7 tube, and is connected to a drain path provided in an annular shape below each position of the reaction turntable 1. The compressor 32 is used for discarding storage liquid, cleaning immediately after that, and B/F.
It supplies pressurized air for cleaning during separation, disposal of samples and reagents left on the tip of the disposable chip, and cleaning.

The tank 38 is for storing the luminescent auxiliary reagent, and the tanks 39 and 40 are for storing the luminescent reagent, respectively.The in-out switching valve 33 and the pumps 34 to 37 are for storing the luminescent auxiliary reagent, the diluent, and the luminescent reagent. It is something. The diluting liquid and cleaning liquid were stored in tank 41! lliliquid is used. As this buffer solution, a mixture of a surfactant, sugar, etc. that promotes the immune reaction is used. Details of these injection methods will be described later.

In the cleaning process, the valve 45 is selectively opened and closed, and the buffer solution is injected from the tank 41 through the resistance tube 43, the bleed heater 44, and the valve 45 into the cartridges at the respective positions (1), (2), and (2). Then, the air valves are selectively opened and closed, and pressurized air is supplied from the compressor 32 to the cartridges at the respective positions (1), (2), and (2) through the air valves. In normal cleaning, cleaning solution (buffer solution)
injection and disposal with pressurized air four times.

In position ■, the sample dispensing flow system and the labeled antibody reagent dispensing flow system are connected so that the Sand-Deutsch method and the competitive reaction method can be applied, but these are each connected to a dedicated disposable from the sample cup or reagent bottle. It is inhaled and dispensed using a tip.

In this case, sample or reagent remains on the tip, so
A pressurized air flow system is connected to blow them out with pressurized air. In the sample dispensing system, this is the flow system of the valve 52. The tip of the disposable tip is inserted into the sample cup of the sample turntable 4, the sample is sucked by the sampling pump 51, and the sample is sucked into the position ■.
After dispensing into the cartridge, the system is switched to this flow system.

Similarly, when dispensing a reagent, insert the tip of the disbotib into the reagent bottle on the reagent turntable 3,
After the reagent pump 53 sucks and dispenses, the valve 54 switches to a pressurized air flow system. Additionally, since the amount of suction becomes unstable when the internal pressure increases, a valve for opening to the atmosphere is also provided.

Next, the rotation of the position of the reaction turntable 1 will be explained.

At position (2), the arm mechanism 7 operates to transport a new cartridge from the cartridge turntable 2, remove the tip, and set it.

Even if you remove the tip 28 shown in Figure 2 from the cartridge when setting a new cartridge in position ■, the storage solution inside will not be disposed of, so pressurized air is sent from the top opening of the cartridge in position ■. car) Discard the storage solution in the IJ.

In the next position (2), the cleaning valve is set at the upper opening of the IJ jig and cleaning is performed by repeatedly sending cleaning liquid and pressurized air alternately, for example, four times.

Next, at position ■, add the diluent. This is to make it easier to cause an immune reaction, since when blood, serum, urine, etc. are directly injected, various components may interfere with the immune reaction.

Then, at position ①, aspirate the sample from the sampling cup using the disposable tip and dispense it into the cartridge.

After that, the immune reaction (first reaction) is promoted by applying vibration and stirring each time it rotates one position at a time, and at position 0, water is supplied and water is drained using pressurized air, which is repeated four times for cleaning. Perform the B/F separation as described.

Here, we will explain in detail the operation after B/F separation.
First, rotate from position 0 to 20 positions in the forward direction, stop at position ■, and use the diluent as a buffer (
Buffer solution) is injected and further rotated in the forward direction of the ■0 position to advance to position ■, which is one position ahead of the previous position. After stirring by vibration, the tube is similarly rotated 20 positions in the forward direction, and once stopped at position (3), the labeled antibody is dispensed. The diluent preheats to stabilize the reaction temperature, has the effect of smoothing and promoting the immune reaction, and enhances the stirring effect when the next labeled reagent is dispensed. This is the operation in the case of the Sanderutsch method.

That is, by alternately performing an operation of rotating the pitch of 10 positions and an operation of rotating the pitch of 20 positions, the position is advanced one position at a time from the previous position. By doing so, it is possible to adopt an apparatus configuration in which the II identification reagent is dispensed at the sample dispensing position (2) even in the Sandersch method. the result,
By controlling the rotation operation so that the sample and the labeled antigen are simultaneously dispensed at position (3), immunoassays can be performed using the same flow system in the competitive reaction method as well.

After that, the immune reaction (second reaction) is promoted by stirring with vibration while rotating alternately with the pitch of the 1O position and the pitch of the 20 position in the same way as the first reaction up to position 0, and then washed again at position ■. Perform B/F separation by

Then, a luminescence auxiliary reagent for intensifying luminescence is added at position (2), and the cartridge is transferred to the detector 10 at the initial position (2). The detector 10 measures the amount of luminescence immediately after adding the luminescence reagent to the cartridge.

In the above operation, for example, it stays at position ■ for 12 seconds, rotates in the forward direction at a pitch of 20 positions, stays at position ■ for 12 seconds, then rotates to position ■ at a pitch of 10 positions, and rotates for 24 seconds. If the position is advanced from position (2) to position (2) by l positions, at position (2), the cartridge is first transferred to the detector 10 in 12 seconds, and a new cartridge is brought from the cartridge turntable 2 and set. In this case, the first reaction takes about 3 minutes, the second reaction takes about 5 minutes, and the immune reaction measurement for one sample can be performed in about 10 minutes in total, which is approximately 150 tests)/
A measurement speed of hr can be achieved.

In addition, when measuring multiple items with the 1 sample, at position 2, solid phase reagent cartridges corresponding to each measurement item are set on the cartridge turntable, and the same sample is dispensed into those cartridges. Furthermore, reagents corresponding to the measurement items are dispensed.

Then, the amount of light emitted is measured by the detector 10, and the process ends.

If you need to measure again, use the remaining disposable tip to dispense the sample. For this purpose, two disposable chips are stored for each sample in the sample turntable shown in FIG. Re-measurement is required not only when the measured value shows an abnormal value that significantly deviates from the preset range, but also when a judgment value is given for each measurement item in advance as a secondary screening, and the next measurement item is determined based on the judgment result. may be set. For example, in a serum hepatitis test, first a test for HBS antigen is performed, and if the result is positive, a test for HBE or HBS antigen is performed. In addition, the reason why disposable tips are used is because the range is very wide in the case of immunoassays, and complete cleaning cannot be done with conventional pipettes.

Next, a method for injecting a luminescent reagent will be described in detail.

As mentioned above, the tank 38 contains the luminescence auxiliary reagent, and the tank 3
Reference numerals 9 and 40 each house a luminescent reagent, and send the luminescent auxiliary reagent, diluent, and luminescent reagent through the in-out switching valve 33 and pumps 34 to 37. A buffer solution stored in the tank 41 is used as the diluting solution and the washing solution. As this buffer solution, a mixture of a surfactant, sugar, etc. that promotes the immune reaction is used. The tank 41 has a sealed structure and is configured to supply pressurized air from the compressor 32 and apply pressure to feed the liquid.
The cleaning liquid is controlled to have a stable predetermined temperature and flow rate through a resistance tube 43, a break heater 44, and a valve 45, thereby facilitating the reaction and stabilizing the reaction.

Similarly, for luminescent reagents, by adding a heater to the mixer 46, it is possible to stabilize the temperature during the luminescent reaction.

For example, in the pump suction step with the in-out switching valve 33 in the state shown, the pump 34 suctions the luminescent auxiliary reagent from the tank 38, the pump 35 suctions the diluent (water) from the tank 41, and the pump 36.
37 simultaneously aspirates luminescent reagent from tanks 39 and 40. Then, when the in-out switching valve 33 switches (the upper half shifts to the right) and enters the pump discharge process, the luminescent auxiliary reagent and the diluent are heated to a predetermined temperature through the bleed heater 44, and are heated to the cartridges in positions ■ and ■, respectively. is injected into. Further, the luminescent reagent is mixed through a three-way joint 42 and a mixer 46,
injected into the cartridge of the detector IO.

Incidentally, the luminescent reagent varies depending on the labeling substance, but for example, in the case of A cridinium, it is a mixture of hydrogen peroxide and an alkali, and in the case of Luminol, it is a mixture of hydrogen peroxide and Fe ions. ,
In the case of dioxetone (1,2-D 1oxejone), a mixture of hydrogen peroxide and a fluorescent substance is used,
Since these react in a short time, just before detecting the luminescence, the three-way joint 42,
They are injected simultaneously through mixer 46. Since the luminescent reagent is mixed immediately before detection in this manner, the luminescent reagent can be maintained in a stable state.

Note that when starting operation after stopping the operation of the apparatus for a relatively long time, the luminescent reagent left in the tube beyond the three-way joint 42 has deteriorated, so It is necessary to discard the luminescent reagent beforehand. To do this, move the luminescent reagent supply pipette to a drain placed at a position different from the detection position, operate the pumps 36 and 37 and the in-out switching valve 33 to spit out the remaining luminescent reagent, and then A new luminescent reagent may be injected into the cartridge.

The above description is an example of the present invention, and many modifications can be made to its implementation. For example, although the above explanation was about mixing two reagents, three or more reagents may be used. In addition, if two or more types of samples exist in the same cartridge, separate dispensing nozzles are prepared to detect them separately, and each reagent can be detected individually by injecting each reagent at a different time. can.

Furthermore, when there is one type of measurement target and other background, the first luminescent reagent is dispensed to cause the background substances other than the target substance to emit light, and then the second reagent is dispensed. Note that it is also possible to detect the luminescence of the target luminescent substance, which allows background to be removed.

In addition, the dispensing nozzle, detector lid, and cartridge chuck are integrated to form an arm, and this arm serves as a third
As shown in the figure, the luminescent reagent is preliminarily discharged into the hole part of the reaction turntable where there is no cartridge at the timing just before the cartridge to be detected is moved, and the cartridge to be analyzed is then transferred to the detector and placed in a predetermined position. Data stability can be maintained by dispensing the luminescent reagent and detecting luminescence at this timing.

〔Effect of the invention〕

As described above, according to the present invention, a plurality of luminescence reagents are prepared and mixed and injected into the sample immediately before luminescence detection, so that the stability of the reagents is obtained, and as a result, a stable amount of luminescence light is obtained. can get. In addition, by preliminarily discharging the luminescent reagent, the influence of residual substances can be removed and accurate measurements can be made. Furthermore, by injecting different reagents at different times using separate dispensing nozzles, it is possible to detect multiple components and remove backgrounds other than the target substance1, making it possible to collect stable data.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the configuration of an embodiment of an automatic immunoassay device according to the present invention, FIG. 2 is a diagram showing an embodiment of a cartridge used in the automatic immunoassay device according to the present invention, and FIG. The overall flow diagram, FIG. 4 is a diagram for explaining the principle of measurement by the Sand-Deutsch method, and FIG. 5 is a diagram for explaining the principle of measurement by the competition method. 1... Reaction turntable, 2... Cartridge turntable, 3... Reagent turntable, 4...
Sample turntable, 5...Sample cup, 6
...Disposable chip, 7-9...Arm mechanism, IO
Detector, 11 Dust, 12 Control processing unit, 21 Cartridge body, 22 Filter, 23 Solid phase reagent, 24 Discharge hole, 25・
- Opening, 26... Aluminum cap, 27... Orimeter, 28... Tip. Figure 2 Applicant JEOL Co., Ltd. Agent Patent attorney Masanobu Hirukawa (5 others) Procedural amendment (method) Description of the case 1999 Patent Application No. 062753 2゜ Title of invention Automatic immunoassay device Relationship to the case of person making 3° correction of luminescent reagent injection device

Claims (2)

[Claims] (1) In an automatic immunoassay device that measures an immune reaction by injecting a luminescent reagent into an immune reaction product in a reaction tube and causing chemiluminescence, there are containers each containing a plurality of luminescent reagents, and a luminescent reagent in each container. a mixing means for mixing;
1. A luminescence reagent injection device for an automatic immunoassay device, comprising an injection means for injecting mixed reagents into a reaction tube, and configured to mix and inject a plurality of reagents immediately before emitting light. (2) The luminescent reagent injection device for an automatic immunoassay device according to claim 1, characterized in that the luminescent reagent is preliminarily discharged by the luminescent reagent injection means.