JPH02236455A - Luminous reagent injector of automatic immunoassay device - Google Patents

Abstract

PURPOSE:To stably induce the chemical reaction relating to light emission by adjusting luminous reagents to a prescribed temp. before the reagents are injected into reaction tubes. CONSTITUTION:The luminous reagents are preserved in tanks 39, 40 and are mixed by a 3-way joint 42 and a mixer 46 just before the injection into a cartridge in order to inject the same in a stable state into the cartridge in the case of, for example, 2-liquids mixing. A temp. regulating means is added to, for example, the mixer 46 and the luminous reagents are regulated to the prescribed temp. prior to the injection into the cartridge so that the luminous reagents can be injected always in the specified temp. state into the cartridge. The heater is provided in proximity to the mixer 46 or the mixer 46 is disposed in a hot bath adjusted to the specified temp. and the luminous reagents are preheated to the prescribed temp. as the temp. regulating means. Conversely, cooling and heating means may be provided to the mixer 46 and the luminous reagents may be precooled to the prescribed low temp.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an immunoassay device that measures components contained in a sample such as serum or urine by an immune reaction using a solid-phase reagent. The present invention relates to a luminescence reagent injection device for an automatic immunoassay device that injects a sample 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. Sandwich 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.

FIG. 5 is a diagram for explaining the measurement principle by the sandwich method, and FIG. 6 is a diagram for explaining the measurement principle 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 labeled substance, 65 is a labeled antibody, 66 is a substrate,
67 indicates the product.

In the sandwich method, as shown in FIG. Add samples containing.

■ As a result, although there are various impurity 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. , the impurities in the sample are discarded by washing.

(2) Next, an enzyme-labeled antibody θ5 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-labeled antibody 85 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 (bound N, hereinafter referred to as B) generated by binding of the antigen 63 and the antibody 65
The free part that is not bound to (freev, hereinafter F
) can be done.

■ Therefore, by cleaning the free part (t
ree, 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 FIG. 6, (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 in the sandwich method, and an enzyme 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, conventionally being carried out manually in most cases.

When the above-mentioned immunoassay is automated, antibodies or antigens are immobilized on a solid phase such as polystyrene balls 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 enzyme 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 it is difficult to detect the reaction. The chemiluminescence of the reaction product is detected by pouring a luminescent reagent into the reaction product. and,
There are two types of chemiluminescence detection: a flow cell type that continuously flows the reaction product and measures it, and a batch measurement type that measures each test tube using test tubes. Therefore, in any type of chemiluminescence, luminescence detection is performed by installing a detection element such as a photomultiplier close to the site of the luminescence reaction solution [Problem to be solved by the invention] Automatic detection of such chemiluminescence In immunoassays, various combinations of samples and luminescent reagents can be considered, but even if the same luminescent reagent is used for the same sample, the amount of luminescent light detected may not be constant. That is, there was a problem that data could not be collected stably.

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

[Means for Solving the Problems] 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. It is characterized by comprising means for adjusting the temperature of the luminescent reagent to a predetermined temperature before injecting the luminescent reagent into the luminescent reagent.

[Effect]

The luminescent reagent injection device of the automatic immunoassay device of the present invention adjusts the luminescent reagent to a predetermined temperature by insulating, preheating, or postcooling before injecting the luminescent reagent into the reaction tube, so that the chemical Since reactions can occur stably, data stability can be maintained.

〔Example〕

Examples will be described below based on the drawings.

FIG. 1 is a diagram showing the configuration of one embodiment of the automatic immunoassay device according to the present invention, and FIG. 2 is a diagram showing one embodiment of the car 1-Uge used in the automatic immunoassay device according to the present invention. It is.

In the figure, 1 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 θ are arm mechanisms, 10 is a detector, 11 is dust, 12 is a control processing unit, 21 is a cartridge body, 22
23 is a solid phase reagent, 24 is a discharge hole, 25 is an opening, 26 is an aluminum cap, 27 is an orifice, and 28 is a tip.

In Fig. 1, the cartridge turntable 2 is a table that stores and rotates disposable cartridges (reaction detection containers) as shown in Fig. 2, and is composed of 10 removable cassettes. 30
An example is shown in which 2 cartridges can be stored. According to this, since cartridges of the same immobilized antibody are stored in each section, cartridges for 10 items can be prepared. The reagent turntable 3 is a rotating table that stores reagent bottles of labeled antibodies and disbo chips for dispensing the labeled antibodies, and an example is shown in which reagent bottles for 10 types, that is, 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 labeled antibodies, stirring reaction by vibration, washing, etc., as described above, with the cartridges of the cartridge turntable 2 set therein and rotating one unit at a time. It is. 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. The circle a1 b1 c shows the locus of . Further, the detector 10 is for injecting a luminescent reagent into the cartridge after reaction and detecting the amount of luminescence, and the 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.

In addition, there is an orimeter 27 cut in the part where the discharge hole 24 is provided, and by bending the tip 28 at the orimeter 27, it can be easily removed from the cart lisso main body 21. It can be penetrated. The discharge hole 24 is formed with an extremely small diameter, and
Since the filter 22 is disposed, when the cartridge is used, the tip 28 is attached to the cartridge body 21.
Even when removed from the drain hole 24, dispensed samples, reagents, washing water, etc. are not easily discharged from the drain hole 24.
By supplying pressurized air from the opening 25 at the upper end,
Alternatively, it can be discharged by suctioning through the discharge hole 24.

Therefore, the cartridge has an aluminum cap 2 at the top end.
6, the solid phase reagent 23 is stored on the filter 22 with the lower part 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 bombs, 38-41 are tanks, 42 is a three-way girointo, 43 is a resistance tube, 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 with 29 positions, with the positive scene (2) as the base point, 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 drain tank 31 is for storing the storage solution and cleaning solution discarded from each cartridge of the reaction turntable 1, and is connected to a drain path provided in an annular shape below each positron of the reaction turntable 1. The hump presser 32 supplies pressurized air for discarding the storage solution, cleaning immediately after it, cleaning during B/F separation, disposing of the sample or reagent remaining at the tip of the disbochip, 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. 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. 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 preheater 44, and the vanoleb 45 into the cartridges of positions 1, 2, [phase], and 0, respectively. Then, the air valves are selectively opened and closed, and pressurized air is supplied from the compressor 32 to the respective Posisilon cartridges ①, 0, and @ through the air valves. In normal cleaning, injection of a cleaning solution (buffer solution) and disposal using pressurized air are performed four times.

In the positive chain ■, the sample dispensing flow system and the labeled antibody reagent dispensing flow system are connected so that the sandwich method and competitive reaction method can be applied. It is inhaled and dispensed using a disbo 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 Disbochip into the reagent bottle of the reagent turntable 3,
After inhalation and dispensing with the reagent pump 53, 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 cylinder of the reaction turntable 1 will be explained.

At position 1, the arm mechanism 7 operates to transport a new cartridge from the cartridge turntable 2, remove the leading end, 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 ■. Discard the storage solution in the cartridge.

Next, cleaning is performed by setting the cleaning valve at the upper opening of the cartridge and repeatedly sending the cleaning liquid and pressurized air alternately, for example, four times.

Next, at position 1, 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, in the positive position ■, aspirate the sample from the sampling cup with a disposable tip and dispense it into the cartridge.

After that, the immune reaction (first reaction) is promoted by applying vibrations and stirring each time each ponosibon is rotated, and cleaning is repeated four times by supplying water with position 0 and draining with pressurized air. The above-described B/F separation is performed.

Here, we will explain in detail the operation after B/F separation.
First, rotate forward for 20 positions from position [phase], stop at point ■, inject the diluent (buffer) as a buffer, and rotate forward for another 10 positions until position ■ is one position ahead of the previous position ■. Proceed until. After stirring by vibration,
Rotate the body in the forward direction, stop at position (2), and dispense the labeled antibody. The diluent has the effect of stabilizing the reaction temperature by preheating, facilitating and promoting the immune reaction, and enhances the stirring effect when the next labeling reagent is dispensed. This is the operation for the sandwich method.

That is, the operation of rotating the pitch of the 10th position and the operation of rotating the pitch of the 20th position are performed alternately, and the position is advanced one position at a time from the previous position. In this way, even in the sandwich method, it is possible to adopt an apparatus configuration in which the labeling reagent is dispensed using the positilon (2) for dispensing the sample. As a 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 even in the competitive reaction method.

Thereafter, the immune reaction (second reaction) is promoted by stirring and applying vibration while rotating alternately with the pitch of the 10th position and the pitch of the 20th position in the same way as the first reaction until the position ν. B/F separation is performed again by washing with O.

Then, a luminescence auxiliary reagent for intensifying luminescence is added at position O, and the cartridge is transferred to the detector 10 at the initial position (3). The detector 10 measures the amount of luminescence immediately after adding the luminescence reagent to the cartridge.

In the above operation, for example, it remains at position 1 position ■ for 12 seconds, rotates in the forward direction at a pitch of 20 positions, remains stationary at position O for 12 seconds, then rotates at position O for 10 positions until position ■ ■. Then, for 24 seconds, the cartridge is moved one position at a time from position ■ to position one ■, and so on.At position ■, the cartridge is first transferred to the detector 10 in 12 seconds, and a new cartridge is transferred to the cartridge turntable. You will need to bring it from step 2 and set it up. In this case, the first reaction takes about 3 minutes, the second reaction takes about 5 minutes, and the immune reaction measurement of one sample can be performed in about 10 minutes in total, and about 150 tests/
A measurement speed of hr can be achieved.

In addition, when measuring multiple items with one sample, the cartridges of solid phase reagents corresponding to each measurement item are set on the cartridge turntable in Posin Nikki, and the same sample is dispensed into those cartridges. Then, 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. To this end, the sample turntable shown in FIG. 1 stores two disbo chips corresponding to each sample. 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 primary screening, and the next measurement item is determined based on the judgment result. In some cases, it is 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 a disposable Disbo tip is used is because the range is very wide in the case of immunoassay, and conventional pipettes cannot completely wash the tip.

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
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 bombs 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 preheater 44, and a valve 45, thereby facilitating the reaction and stabilizing the reaction.

For example, in the case of a two-liquid mixture, the luminescent reagents are placed in separate tanks 39 in order to inject them into the cartridge in a stable state.
, save it to 40. Immediately before injection into the cartridge, the mixture is mixed in a three-way inlet 42 and a mixer 46.

For example, a temperature adjustment means is added to the mixer 46 to adjust the temperature of the luminescent reagent to a predetermined temperature prior to injection into the cartridge, so that the luminescent reagent can be always injected into the cartridge at a constant temperature.

As the temperature adjustment means, it is preferable to preheat the luminescent reagent to a predetermined temperature by providing a heater close to the mixer 46 or placing the mixer 46 in a hot bath adjusted to a constant temperature.

Conversely, the mixer 46 may be provided with a cooling means to pre-cool the luminescent reagent to a predetermined low temperature. In this case, in addition to the stability of the light emission, the detector 10
Since the reaction results in low-temperature luminescence, it also has the effect of eliminating background luminescence. Furthermore, in this case,
If the tanks 39 and 40 for storing the luminescent reagent are also stored in a cold room, the stability of the luminescent reagent can be further improved.

In addition to adjusting the luminescent reagent to a predetermined temperature,
Adjusting the cartridge side to a predetermined temperature is also effective. For this purpose, for example, the temperature may be adjusted before transferring the cartridge to the detector 10. Specifically, the luminescent auxiliary reagent added at position O of the reaction turntable 1 and the diluent added prior to injection of the luminescent reagent are maintained at a predetermined temperature, and the additives at the predetermined temperature are It is preferable to keep the cartridge at a predetermined temperature.

In addition, B/F is set at position O of reaction turntable number 1.
If you perform separation and leave only the solid phase carrier in the cartridge, then use a method that does not add luminescence auxiliary reagents or diluents.
Since the luminescent reaction temperature depends on the temperature of the luminescent reagent in terms of heat capacity, the luminescent reaction temperature can be sufficiently controlled by adjusting the luminescent reagent to a predetermined temperature.

Next, a more preferred embodiment of the luminescence detector of the present invention will be described with reference to FIG.

FIG. 4 is a diagram showing the configuration of an embodiment of the luminescence detector 10 in a state where a reaction tube 51 is transferred to the luminescence detector 10, 52 is a dispensing nozzle, 53 54 is a detection element such as a photomultiplier, 54 is a cartridge indicating means, and 55 is a motor.

In this embodiment, the cartridge 51 is first inserted into the luminescence detector 1.
0 in the cartridge indicating means 54. In this state, the motor 55 connected to the instruction means 54 is rotated to rotate the cartridge 51. While the cartridge 51 is being rotated, a luminescent reagent is supplied into the cartridge 51 from the dispensing nozzle 52, and the sample and the luminescent reagent in the cartridge 51 are stirred and mixed. Then, the detection element 53 detects luminescence from the time when the supply of the luminescent reagent is started.

In this case, stirring efficiency can be improved by making the rotation axis of the cartridge indicating means 54 eccentric to the rotation axis of the motor 55.

In addition, the stirring means is not limited to the motor 55 shown in FIG. 4, but also stirring with a stirring bowl, or by inserting a stirring magnetic material into the cartridge 51, and by magnetic induction, the magnetic material is transferred from the outside without contact. Various means can be used, such as stirring by rotating the body.

In this way, by detecting luminescence while stirring the reaction solution in Cartoliso, the concentration distribution of the reaction solution becomes uniform and a uniform reaction can occur, making data collection even more stable. . This is particularly effective when detecting luminescence using liquids that are difficult to mix, or when detecting luminescence in which the amount of luminescence changes depending on the mixing ratio.

In the above embodiments, the case where two liquids are mixed as luminescent reagents has been described, but it goes without saying that one reagent or three or more reagents may be mixed. Furthermore, the luminescent reagents in multiple dishes may be supplied at different times, that is, without being mixed before being supplied to the cartridge.

If the luminescent reagent is an IM, the three-way girointo 42 and mixer 4θ shown in FIG. 3 are basically unnecessary, but
In short, it goes without saying that a means for adjusting the temperature of the luminescent reagent may be provided in a part of the flow system between the time when the luminescent reagent is taken out from the cartridge 39 or 40 and the luminescent reagent is supplied to the cartridge.

〔Effect of the invention〕

As described above, according to the present invention, the chemical reaction related to luminescence can be stably carried out by adjusting the luminescent reagent to a predetermined temperature (heat retention, preheating, postcooling, etc.) before injecting the luminescent reagent into the reaction tube. As a result, a stable amount of emitted light can be obtained, and data collection can be performed stably.

Furthermore, by injecting the luminescent reagent into the reaction tube while stirring and performing luminescence detection, the mixed concentration of the sample and luminescent reagent can be made uniform, and a more stable amount of luminescent light can be obtained for stable data collection. It becomes possible.

[Brief explanation of drawings]

FIG. 1 is a diagram showing an 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 car used in the automatic immunoassay device according to the present invention. Fig. 3 is the overall flow diagram, and Fig. 4 is one of the luminescence detectors according to the present invention.
FIG. 5 is a diagram for explaining the principle of measurement by the sandwich method, and FIG. θ is a diagram for explaining the principle of measurement by the competitive method. 1... Reaction turntable, 2... Cartridge turntable, 3... Reagent turntable, 4...
Sample turntable, 5...Sample cup, 6
...Disposable chip, 7-9...Arm mechanism, 10
...Detector, 11...Dust, 12...Control processing unit, 21...Cartridge body, 22-...Filter 23...Solid phase reagent, 24...Extraction hole, 25...・
- Opening, 26... Aluminum cap, 27... Orimeter, 28... Tip. Applicant JEOL Co., Ltd. Agent Patent Attorney Hideo Sugai (5 others) Figure 2 Figure 4

Claims (1)

[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, the luminescent reagent is heated to a predetermined temperature before being injected into the reaction tube. A luminescent reagent injection device for an automatic immunoassay device, characterized in that it is equipped with a means for adjusting.