JPS61193073A - Method and instrument for immunological analysis - Google Patents

Abstract

PURPOSE:To make possible the reduction in the size of a device, the simplification of the constitution and the analysis of multiple terms by making analysis and measurement of the multiple terms by using one kind of sensitized solid phase and reagent. CONSTITUTION:A sample dispenser 22 is disposed to the stop position S4 of a reaction tube disk 21. The dispenser 22 dispenses selectively the sample from a sample cup 24 existing in the prescribed sample sucking position of a sampler 23 into U-tubes 20. A rack 23a existing in the sample dispensing position is intermittently moved toward an arrow S in synchronization with the suction operation of the dispenser 22. The sequential samples can be continuously fed to the sample dispensing position in the above-mentioned manner. The 1st reagent dispenser 25 is then disposed in the stop position S1 of the disk 21 to dispense selectively the 1st reagent 26b corresponding to the measuring item from the 1st reagents 26 of the same number as the number of the measuring items housed into the 1st reagent container 26a into the U-tubes 20. The 2nd reagent dispenser 27, the 3rd reagent dispenser 30, a carrier charger 31, a colorimeteric instrument 32, a carrier discharger 33 and a cleaner 34, etc. are further provided.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to an immunological analysis method and an apparatus thereof, and particularly to an immunological analysis method and apparatus suitable for analyzing multiple items.

[Prior art] As a conventional technology, for example, Japanese Patent Application Laid-Open No. 59-135366
An immunological analysis method using the same phase is disclosed in the publication. This involves immobilizing an antibody or antigen that directly makes a specific antigen-antibody reaction with the test substance on a solid phase, and then using this same phase (hereinafter, the solid phase on which a specific substance is immobilized is referred to as the sensitized solid phase). The test substance is placed in a test solution to bind to the sensitized solid phase. after that,
This is a method of labeling the analyte and determining the amount of the analyte by reacting a labeled antibody or labeled antigen that specifically undergoes an antigen-antibody reaction with the analyte and the analyte bound to the sensitized solid phase. .

[Problems to be solved by the invention] In conventional techniques, when performing multi-item analysis, a dedicated sensitized solid phase on which antigens or antibodies are immobilized for each measurement item is required; It is necessary to prepare the following types of sensitized solid phases, and it is necessary to have a space to accommodate the sensitized solid phases independently for each measurement item and a device to selectively take out the sensitized solid phases according to the measurement items. There was a problem that the device became large and complicated. The present invention was made in view of these problems, and provides an immunological analysis method that uses a common sensitized solid phase for all measurement items, and an immunological analysis device that is small and easy to configure. The purpose is to provide

Measures to solve problem E] FIG. 1A is a conceptual diagram of the first and second inventions.

This invention is a method for performing multiple immunological analyzes using a sensitized solid phase 3 in which a substance 1 is immobilized on a solid phase 2 in advance. As a reagent, an antibody 5 that specifically binds to the test substance 4 and specifically binds to the sensitized solid phase is used.

FIG. 1B is a conceptual diagram of the third invention.

This device is a device for carrying out the methods of the first and second inventions, and includes a carrier charging device @7 for charging a sensitized insoluble carrier in which substance 1 is immobilized in advance on an insoluble carrier, and a sample dispensing device @7. It consists of a bag @8 and a device 9 for dispensing a reagent that binds the sensitized insoluble carrier and the test substance in the sample.

[Production use] The first and second inventions will be explained with reference to FIG. 1A.

A reagent consisting of the antibody 5 and a sample containing the test substance 4 are added to the sensitized solid phase 3. Then, the test substance 4 and the reactive site 5a of the antibody 5 are reactively bonded, and the reactive site 5a of the antibody 5 is further bonded to the reactive site 5a of the antibody 5.
A reaction site 5b independent of the sensitized solid phase 3 reacts with the substance 1 of the sensitized solid phase 3. Or, conversely, the antibody 5 is first reactively bound to the substance 1, and the test substance 4 is reactively bound to the antibody 5 bound to the sensitized solid phase 3. Then, aggregated particles 6 are formed. The aggregated particles 6, that is, the analyte 4 bound to the sensitized solid phase 3 via the antibody 5, are quantitatively analyzed. When analyzing different measurement items, the same sensitized solid phase 3 is used, and the antibody 5 is changed depending on the test substance.

The third invention will be explained with reference to FIG. 1B.

In this immunological analyzer, a sensitizing insoluble carrier is charged into a reaction container by a carrier charging device 7. Next, the sample is dispensed into the reaction container by the sample dispensing device 8, and the reagent dispensing bag @9 dispenses a reagent that binds the analyte in the sample and the sensitized insoluble carrier into the reaction container. to initiate an immune response.

[Example] Figures 2A and 2B show an example of the immune reaction used in the first and second inventions.

As the solid phase, an insoluble carrier 13, which is a bead made of glass or synthetic resin, is used. In addition, as a substance to be immobilized in advance on a solid phase (hereinafter referred to as an immobilized substance), Fc fragment of 1 (+-G) of a certain animal species (CryStall
An antibody or protein A that specifically reacts with iZable fragment (hereinafter referred to as Fc) is used. Also,
The immobilized substance and the insoluble carrier are collectively referred to as the sensitized insoluble carrier. The antibody that binds to the immobilized substance and further binds to the test substance (hereinafter referred to as the first antibody) is immunized with the test substance to an animal species that has the same origin as IgG with which the immobilized substance specifically reacts. This is an antibody obtained by The antibody (hereinafter referred to as the second antibody) that binds to the test substance (yA labeling substance 4jJ I) is a conventional labeled antibody, and an enzyme-labeled antibody using an enzyme as the labeling substance is used.

Next, the immune reaction process will be explained. Figure 2 A shows test substance 1.
The case where 0 is analyzed is shown.

Immobilized substance 12 that specifically reacts with Fc of first antibody 11
The test substance 10 and Fab
fragment (ant igenbinding f
The first antibody 11 that specifically reacts with the ragment (hereinafter referred to as Fab) and the test substance 10 are added and allowed to react at a constant temperature for a certain period of time, followed by washing and B-F separation. l"re
e particles are removed and the immobilized substance 12 is left on the insoluble carrier 13.
The first antibody 11 and the test substance 10 are bonded to each other via. After that, the second antibody 14, which is an enzyme-conjugated antibody that has the property of binding to the test substance, is added and allowed to react at a constant temperature for a certain period of time, followed by 8.F separation.Finally, a coloring reagent is added to allow it to bind to oxygen. , the test substance 10 is quantified by colorimetrically measuring the remaining liquid. FIG. 2B shows the immune reaction when measuring substance 16 different from substance 10 in FIG. 2A. If the test substance is different, the Fab of the first antibody 11 and the second antibody 14 are changed to the first antibody 17 and the second antibody 18 in correspondence with the test substance 16, respectively. However, even if the measurement items are different, the first antibody 1
7 has the same FC as the first antibody 11, so the sensitized insoluble carrier 13 can be used in common.

Next, FIGS. 3A and 3B show an embodiment of an enzyme immunological automatic analyzer implementing the present invention.

As shown in FIG. 3B, the reaction vessel uses 25 U-shaped tubes 20 having a large diameter portion 20a and a small diameter portion 20b, and these are held at equal intervals on the same circumference of a reaction tube disk 21. The reaction tube disk 21 is intermittently rotated at a predetermined pitch (eg, 15 seconds) in the direction shown by the arrow. The stopping positions of the U-shaped tube 20 due to the intermittent rotation of the reaction tube disk 21 are indicated by symbols 81 to S25.

In this example, the sample dispensing station 6 22 is placed at the stop position S4 of the reaction tube disk 21. The sample dispensing device W122 selectively dispenses the sample from the sample cup 24 located at a predetermined sample suction position of the sampler 23 into the U-shaped tube 20.

That is, the sample dispensing device f122 aspirates the necessary amount of sample according to the measurement item using a control device (not shown), and performs seeding and dispensing into the U-shaped tubes 20 corresponding to the number of measurement items. As the sampler 23, a large number of racks 23a each holding 10 sample cups 24 are held side by side, and the racks in the left row are sequentially moved downward in FIG. Sample cup 2 after pouring
The rack in the right column holding number 4 is moved upward. The rack 23a at the sample dispensing position is intermittently moved in the direction of arrow S in synchronization with the suction operation of the sample dispensing device @22. When the dispensing of all the samples held in this rack 23a is completed, this rack 23a is sent to the bottom of the rack row on the right, and the rack at the bottom of the row on the left is next moved to the sample dispensing position. Sent. In this way, successive samples can be delivered to the sample dispensing position in succession.

Next, the first reagent dispensing device 25 is placed at the stop position S1 of the reaction tube disk 21. The first reagent dispensing device selectively dispenses first reagents 26 corresponding to the measurement items into the U-shaped tube 20 from the same number of first reagents 26 as the number of measurement items stored in the first reagent container 26a. . As this first reagent 26, the first antibody 5.11.17 described above is used. The second reagent dispensing device 27 is placed at the stop position 183. The second reagent dispensing device 27 dispenses a second reagent 28 corresponding to the measurement item from the same number of second reagents 28 as the number of measurement items stored in the second reagent container 28a.
b is selectively dispensed into the U-shaped tube 20. This second reagent 28
The second antibody 14.18 described above is used as the second antibody 14.18. Also,
At the stop position @S2, there is a third tube for selectively dispensing the coloring reagent 29.
The reagent dispensing device 30 is arranged. Further, at the stop position S1, a carrier loading device 31 is arranged for selectively loading one sensitized insoluble carrier 13 into the large opening 20a of the O-tube. The carrier 13 has a size that allows it to be easily taken in and taken out from the large opening 20a of the U-shaped tube 20, but does not enter the small opening 20b, and the above-mentioned immobilizing substance 12 is immobilized on the surface of the carrier 13 in advance. In addition, a colorimetric device 32 is arranged at the stop position @ 320, and the reaction liquid contained in the U-shaped tube 20 is transferred to the colorimetric device 32.
At the stop position MS23, there is a carrier discharge device @33 for selectively taking out and discharging the carrier 13 housed in the U-shaped tube 20. 8
A cleaning device 34 for selectively injecting and discharging a cleaning solution such as ion-exchange water, immunoassay buffer, and physiological saline to perform B-F separation and cleaning the U-shaped tube 20 is disposed at each of the cleaning devices 25 .

Next, the operation of the automatic enzyme immunoanalyzer of the present invention shown in FIG. 3 will be explained with reference to FIGS. 4 and 5.

In this embodiment, analysis is carried out by the sandwich method, and for each sample, the reaction tube disk 21 rotates three times to complete the analysis. That is, B-F
Separation is performed twice and cleaning is performed once for repeated use of the zero-shaped tube. For this reason.

Sample dispensing, 1st. Second. The dispensing of the third reagent, the loading and unloading of the carrier 13, the supply to the colorimetric device, etc. are performed once while the reaction tube disk 21 moves three pitches. However, since cleaning is performed three times during the analysis as described above, it is performed at each pitch of movement of the reaction tube disk 21. In addition, in order to operate in this way, the number of U-shaped tubes 20 loaded on the reaction tube disk 21 is 3n+1 or 3n when n is 12.3°...
It needs to be +2. This morning, the number of U-shaped pipes 20 was 2.
There are 5 pieces, 3n+1 (n=8).

In the first rotation of the reaction tube disk 21, one carrier 13 is first introduced into the U-shaped tube 20 at the stop position S1 from the carrier input device @31 through its large opening 20a, as shown in FIG.

At this stop position $1, at the same time, the first reagent dispensing device 25 dispenses a predetermined amount of the first reagent 26b made of the first antibody and corresponding to the measurement item. After this reaction tube 20 is fed three pitches, at a stop position S4, a predetermined amount of sample is dispensed by the sample dispensing device 22 according to the measurement item.

At the end of the first rotation, the reaction tube reaches a stop position 825, where it is cleaned by the cleaning device 34 and the first B-F separation is performed. In FIG. 5, the timing of the operation performed on the sample is indicated by diagonal lines downward to the left.

Next, the reaction tube disk 21 enters the second rotation, and the stop position S
In step 3, a predetermined amount of the second reagent 28b (composed of a second reagent (labeled antibody)) corresponding to the measurement item is dispensed into the U-shaped tube 2o by the second reagent dispensing device 27, and the second reaction is started. be done. At the last stop position S25 of this second rotation, the second B-F separation is performed by the cleaning device 34.

Furthermore, the reaction tube disk 21 enters the third rotation, and at the stop position S2, a third reagent dispensing device 30 is inserted into this U-shaped tube.
Two coloring reagents are dispensed in predetermined amounts, and a third reaction is started.

When the stop position @S20 is reached, the test liquid in the U-shaped tube 20 is sucked by the pump 32a of the colorimetric device 32 and transferred to the colorimetric cell 32b.
where a colorimetric measurement using light of a predetermined wavelength is performed. Next, after rotating three pitches, at the stop position @S23, the carrier 13 remaining in the U-shaped tube is removed by the carrier discharge device @33. At the stop position 825 after the third rotation R, the U-shaped tube 2o is cleaned by the cleaning device 34 and used repeatedly for analysis of the next sample. In FIG. 5, the operation timing for the next sample is indicated by diagonal lines downward to the right.

The cleaning device 34 cleans the large opening 20a of the U-shaped tube 20.
This can be carried out by intermittently injecting the cleaning liquid in a shower-like manner and by suctioning and discharging it from the small opening 20b using a drainage pump.

As shown in FIG. 4, the cleaning device 34 includes a cleaning liquid tank 34.
A1 cleaning liquid supply pump 34b1 nostle 34C1 drainage pump 34d1 drainage tank 346, etc. are provided. The carrier loading device 31 also includes a hopper 31a for storing a large number of carriers 13, and a gate device 131b for separating and supplying the carriers 13 one by one from the hopper, as shown in FIG.
etc. are provided.

Generally, the carrier 13 is placed in the hopper 31 in a state moistened with a buffer solution.
It is held within a. Roughly, the carrier discharging device 33 lowers the nozzle 33a to the large opening 20a of the U-shaped tube 20, and removes the carrier 13 by adsorbing it to the tip of the nozzle using the suction force of the pump 33b, or inserts the arm into the large opening of the U-shaped tube. It is possible to lower the carrier 13 and take it out by grasping it. As shown in FIG. 4, the colorimetric device 32
The coloring reaction liquid in the O-tube is suctioned into the colorimetric cell 32b from 0b using the pump 32a, and the light from the light source 32c is received by the photodetector 32d through the colorimetric cell 32b to perform colorimetric measurement. Further, the U-shaped tube 20 is immersed in a constant temperature bath 35.

As described above, the analysis operation for one sample ends when the reaction tube disk 21 rotates three times, but in this example, sample dispensing, first . Second. The reaction tube disk 21 is used for dispensing the third reagent, loading and discharging the carrier, and performing colorimetric measurements.
moves 3 pitches and rotates once, and the reaction tube disk 2
1 has 25 (3 x 8 + 1) U-shaped tubes 20 installed at equal intervals, so for example, it can be placed in the stopping position! When the sample dispensing device 22 operates at is 4, the U-tubes in position will shift by one for each revolution of the reaction tube disk 21. This situation can be said for all operations performed once for 3 pitches, so sample dispensing is performed once for 3 pitches.
It can be done continuously, once every pitch. Therefore, sample ID control, analysis result processing, etc. can be performed at regular intervals, making various controls easier.

In this example, an antibody that specifically binds to the FC of the first antibody or protein 8 was used as the immobilization substance.
However, the present invention is not limited to this, and any substance that specifically binds to the first antibody can be used as the immobilization substance. Also,
In addition to insoluble carriers typified by pieces of glass beads, plastics, synthetic resins, etc., reaction tubes and the like can also be used as the solid phase.

[Effects of the Invention] Since it is not necessary to prepare the same number of types of sensitized solid phases as the number of measurement items, the analyzer can be downsized and its configuration can be simplified. Furthermore, multiple types of analysis can be performed using one type of sensitized solid phase.

[Brief explanation of the drawing]

Figure 1 △ is a conceptual diagram of the first and second inventions, Figure 1B is a conceptual diagram of the invention of Figure 3, Figure 2 is a diagram showing the immune reaction of the present invention, Figure 3 is a diagram of the invention of the present invention. FIG. 4 is an explanatory diagram of the operation of the analyzer shown in FIG. 3, and FIG. 5 is a diagram showing the operation timing of the analyzer shown in FIG. 3. 4.10.17... Test object 25... First reagent dispensing device 5.11.17... First antibody 26...
First reagent 1.12... Immobilized substance 27... Second reagent dispensing device 2.13... Solid phase/carrier 28.
...Second reagent 3...Sensitized solid phase 29...Coloring reagent 6...Agglutinated particles 30...Third reagent dispensing device 14.18...Second antibody 31...Carrier injection Device 20...U-shaped tube 32...Colorimetric device 21...Reaction tube disk 33...Carrier discharge 1f122...Sample dispensing device 34...Cleaning device 23...Sampler 24...・Sampler cup patent applicant Olympus Optical Industry Co., Ltd. Figure 1 (A) (B) 111-f---J Figure 2 (A) (B)

Claims (1)

[Claims] 1. A method for immunologically analyzing a test substance in a sample by performing an immune reaction using a solid phase; Immunological analysis method for analytical measurement. 2. The immunological analysis method according to claim 1, which uses a reagent that independently shares the ability to bind to a sensitized solid phase and the ability to specifically bind to a test substance. . 3. Claim 2, characterized in that antibodies having at least some of the fragments are used as reagents.
Immunological analysis method described in section. 4. A method for immunologically analyzing a test substance in a sample by performing an immune reaction using a solid phase, which is characterized by reactively binding a sensitized solid phase to a test substance via an antibody. scientific analysis method. 5. A carrier charging means for charging a sensitizing insoluble carrier into a reaction container, a sample dispensing means for dispensing a sample into the reaction container, and a carrier charging means for dispensing a sample into the reaction container, a carrier that binds to the sensitizing insoluble carrier and is specific to the test substance in the sample. reagent dispensing means for dispensing a reagent that binds to the reaction container into the reaction container.