JPH04230859A - Apparatus and method for automatic immunoassay - Google Patents

Abstract

PURPOSE:To measure a large amount of a specimen over many items at the same time within a short time by performing the reaction of the specimen with a reaction solution and measurement while a plurality of reaction containers each composed of a doublet reaction tube receiving a solid phase and a labelled substance are moved. CONSTITUTION:When an apparatus is started, a reaction container moving part 32 arranges reaction containers 4 to the start end of a reaction line part 11 one at a time under the control of a CPU 24 and a suction distribution part 16a sucks a specimen 44 and an enzyme labelled substance to distribute them in the predetermined receiving part of each of the containers 4 and a stirring part 17a stirrs the specimen 44 and the enzyme labelled substance. Next, when each of the containers 4 reaches a magnetic separation part 18b, a reaction product and a non-reacted substance are separated and the non- reacted substance is removed in a washing part 19b and a measuring part 20 obtains luminous data from the reaction part while the CPU 24 refers to a calculation formula from a memory 23 and calculates a measuring result on the basis of the luminous data. By this method, specimen can be measured over many items at the same time within a short time.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an automatic immunoassay device for obtaining immunological information in a specimen and an immunoassay method using the device.

0002

[Prior Art] Immunoassay methods include radioimmunoassay methods (hereinafter referred to as "RIA methods") that use radioactive isotopes as labeling compounds.
That's what it means. ), enzyme immunoassay using various enzymes (hereinafter referred to as “
It is called "EIA Law." ) etc. Among them, the EIA method has excellent specificity and sensitivity, and is widely used in the field of clinical testing. Generally, the EIA method uses an antigen or antibody depending on the analyte to be measured, bound to a solid phase, and the analyte in the specimen is brought into contact with this. Next, a labeled substance such as an antigen or antibody that specifically reacts with the measurement target substance labeled with an enzyme, which is a labeled compound, is reacted and immobilized (bound), and washing is repeated to remove unreacted enzyme labeled substances (free). is completely removed (bind/free separation, hereinafter referred to as "B/F separation"). Thereafter, the enzyme activity of the labeled substance bound to the analyte in the sample is measured, and the analyte in the sample is quantitatively measured.

[0003] Therefore, in implementing the EIA method,
Very complicated operations such as dispensing, dilution, stirring, B/F separation, and movement of the solid phase were required.

[0004] Solid phases are generally used to carry out the EIA method, such as polystyrene beads, magnetic particles, and the inner wall of a reaction vessel. Among these, high sensitivity and
In order to perform B/F separation quickly, a method has been reported in which magnetic particles are used as the solid phase, and a magnetic separation device containing a special container and a magnet that fits the container is used to perform B/F separation (Japanese Unexamined Patent Publication No. 6
2-273453). Furthermore, partially automated measuring instruments have been developed to measure large amounts of samples, and various types are known depending on the solid phase and type of antibody label used ("Enzyme immunoassay", written by Eiji Ishikawa, Igaku Shoin 18
(See pages 0-207).

[0005]

[Problems to be Solved by the Invention] Even with the above-mentioned method using magnetic particles as a solid phase, measurement takes 1 to 18 hours, and measurement cannot be carried out in a short period of time. Moreover, even if partially automated measuring equipment is used, complicated operations are still required.

Furthermore, when measuring a large number of measurement items in a plurality of different measurement processes, no fully automatic measuring device has been developed that can measure a large number of samples simultaneously and in a short time of less than 30 minutes per item using the same device.

The present invention has been made in view of the above circumstances, and is intended to improve the conventional complicated and complicated measurement operations, speed up measurement and improve processing capacity, and support multi-item measurement. The purpose of the present invention is to provide an automatic immunoassay device and an immunoassay method using the device.

[0008]

[Means for Solving the Problems] In order to solve the above object, the present invention provides a sample storage section for storing a plurality of specimens, a reagent storage section for storing a washing liquid and a diluent, and a reaction system having an opening formed in the upper part. At least two reaction tubes are connected, one of which contains a solid phase bound to either an antigen or an antibody, and one of the reaction tubes other than that reaction tube contains the antigen or antibody labeled with a labeling compound. a reaction container that stores a labeled substance; a reaction container storage section that stores a plurality of the reaction containers; a reaction line section that can perform measurements; a reaction container moving section that moves and arranges the reaction containers from the reaction container storage section one by one in accordance with measurement items near the starting point of the reaction line section; a suction and dispensing unit that aspirates and dispenses the specimen contained in the specimen storage unit and the labeled substance contained in the reaction container at a predetermined position into the other reaction tube to obtain a mixed solution of the specimen and the labeled substance; a stirring section for stirring the reaction vessel containing the mixed liquid obtained by the suction dispensing section; and a stirring section for separating reactants and unreacted substances bound to the solid phase in the mixed liquid stirred by the stirring section. a washing section that removes unreacted substances separated by this separation section, a measurement section that measures information generated by the labeled compound bound to the solid phase, and outputs the measurement results measured by this measurement section. an output section to
The apparatus is characterized in that it has a discharge section that discharges the reaction vessel in which the reaction and measurement have been completed near the end point of the reaction line section, and a control section that controls the operation of each section.

[0009] The reaction tube used in this device can contain a solid phase bound to either an antigen or an antibody. The solid phase is a solid phase used in an immunoassay device, and examples include various polystyrene beads and various magnetic particles (see, for example, Japanese Patent Application No. 1-252051). Furthermore, it is also possible to use the inner wall of the reaction tube as a solid phase without adding a solid phase to which the antigen or antibody is bound. Among these solid phases, magnetic particles can be suitably used because they have a wide reaction area and can support rapid measurement, and B/F separation can be performed using a simple device. For measurements using magnetic particles, a magnetic separation section can be used as the separation section.

[0010] Examples of the labeling compound that binds to and labels the antigen or antibody contained in the reaction container include enzymes,
Examples include fluorescent substances, luminescent substances, and radioactive isotopes. Among these, examples of enzymes include alkaline phosphatase, peroxidase, calactosidase, and glucose oxidase. Examples of fluorescent substances include fluorescein and rhodamine. Examples of the luminescent substance include acridinium ester, luminol, isoluminol, and the like. As a radioactive isotope, for example, iodine 1
Examples include 31, iodine 125, carbon 14, and trideuterium.

[0011] When using an enzyme as a labeling compound,
A reagent storage section containing a substrate solution for each enzyme can be used. Further, a dispensing stirring section for dispensing the substrate liquid from the reagent storage section can be added after the washing section for performing B/F separation.

Furthermore, when the labeling compound is a fluorescent substance or a luminescent substance, an initiator can be contained in the reagent container to initiate the reaction. As the dispensing stirring section, the same device as in the case of using the substrate liquid can be used.

It goes without saying that the substrate solution dispensed from the reagent container should be one suitable for the various enzymes mentioned above, such as ABTS, Luminol-H2O, etc.
2 (for peroxidase), p-nitrophenyl phosphate, methylumbelliphenyl phosphate, 3-(
2'-spiro-adamantane)-4-methoxy-4-(
3″-phosphoryloxy)phenyl-1,2-dioxetane disodium salt (for alkaline phosphatase)
, p-nitrophenyl-β-O-galactose, methylumbelliphenyl-β-O-galactose (for β-galactosidase), and the like can be used.

When a radioactive isotope is used as a labeled compound, there is no need for a substrate to be dispensed, and the measurement can be carried out directly in the measuring section. The measuring section can be determined depending on the type of labeled compound, and a spectrophotometer, fluorometer, photon counter, scintillation counter, etc. can be used, respectively.

[0015] When measuring using an enzyme as a labeling compound, the reaction line is heated at 20 to 40°C, preferably at 30°C.
It is preferable to add a device to maintain the temperature in the range of 6 to 38°C.

[0016]

[Operation] The operation of the invention having the above structure will be explained.

[0017] In the apparatus according to claim 2, since the solid phase is a magnetic particle bound to either an antigen or an antibody, it is possible to magnetically separate reactants and unreacted substances. Based on the operation control of each part of the apparatus by the control part, each part performs a predetermined process, and the measurement result measured by the measurement part is automatically output.

[0018] Regarding the method according to claim 5, the method according to claim 1
Predetermined processing is performed by controlling the operation of each part of the apparatus by the described control unit, and immunoassay can be performed efficiently.

[0019]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A device 1 according to an embodiment of the present invention and a measuring method using the device 1 will be described in detail below with reference to the drawings.

FIG. 1 is an external perspective view of the device 1 of this embodiment, and FIG.
1 shows a schematic configuration diagram of this device 1. FIG.

The apparatus 1 includes an input section 2 equipped with a start button 2a, a measurement method selection button 2b, etc., an output section 3 for outputting measurement results, and a mixing and stirring section for mixing and stirring a sample 44, an enzyme label, and a substrate solution. , a processing section 10 that performs a series of processes such as reaction, measurement, etc., a supply section 30 that supplies a reaction container 4 to this processing section 10, a control section 5 that controls each section of this device 1, and a case main body 1a. However, the overall shape is approximately a rectangular parallelepiped.

The processing section 10 is equipped with a motor (not shown) and has a plurality of reaction line sections 11 capable of moving the reaction container 4 in the direction A and performing reactions, measurements, etc. at each predetermined position on the movement path L. Specimen storage unit 1 that accommodates the specimen 44 of
2, and a chip accommodating section 13 that accommodates a plurality of chips 13a.
Then, the chip 13a is taken out from the chip accommodating section 13, a predetermined amount of the sample 44 accommodated in the sample accommodating section 12 is sucked into the tip 13a by an appropriate pump means, and the reaction line section 1
1 is dispensed into the moving reaction vessel 4, and the enzyme label is further transferred from the enzyme label storage section 7b or 7c of the reaction vessel 4 containing the enzyme label to the other reaction tube of the reaction vessel 4 using an appropriate pump means. Suction and dispensing unit 16 that obtains a mixed liquid by suctioning and dispensing
a, a stirring unit 17 that stirs the mixed liquid, and a magnetic separation unit 18 that magnetically separates reactants and unreacted substances in the mixed liquid;
A washing section 19 that washes and removes unreacted substances, a suction dispensing stirring section 16b that dispenses the substrate liquid from the reagent storage section 15 that accommodates the substrate liquid, and luminescence information generated by the reaction between the reactant and the substrate liquid. It has a measurement section 20 that measures by an optical method, and a discharge section 21 that is disposed near the terminal end E of the reaction line section 11 and discharges the reaction container 4.

[0023] The control unit 5 controls each part of the apparatus 1 and stores a one-step assay method, a two-step assay method, a delay reaction assay method, a two-step assay method using a diluent, and other enzyme immunoassay programs. memory 22
and a memory 2 that stores information such as calculation formulas for obtaining immune information based on the measurement information measured by the measurement unit 20.
3, and a CPU 24 that controls each part of this device 1. The control section 5 is also capable of executing an enzyme immunoassay based on the selection operation of the measurement method selection button 2b of the input section 2.

[0024] The motor of the reaction line section 11 is operated by the CPU
Under the control of 24, the reaction vessel 4 is moved in steps, and in order to ensure that each part performs the specified operation, the reaction vessel 4 is kept at a predetermined position for a certain period of time, for example, 30 seconds. Drive to operate.

The agitation section 17 provides agitation for a predetermined period of time to the reaction vessel 4 containing the liquid mixture under the control of the CPU 24 to ensure sufficient agitation.

The supply section 30 includes a reaction container storage section 31 that stores a plurality of reaction containers 4, and a reaction container moving section 32 that moves and arranges the reaction containers 4 one by one near the starting end S of the reaction line section 11. and remove the reaction container 4 from the reaction container storage section 31.
It has a reaction container take-out section 33 that takes out individual containers and moves them to a reaction container moving section 32.

FIGS. 3 and 4 are views of the reaction vessel 4, with FIG. 3 showing a plan view and FIG. 4 showing a side sectional view.

This reaction vessel 4 includes a long reaction tube 7a having an opening 6a formed in its upper part, and two short reaction tubes 7b connected to this reaction tube 7a having openings 6b and 6c formed in their upper parts.
7c. The short reaction tubes 7b and 7c are designed to contain necessary amounts of other necessary chemicals, and the long reaction tube 7
Magnetic particles bound to either an antigen or an antibody are housed in the chamber a, and the sample 44 and the enzyme label are dispensed to form a mixed solution, which enables optical measurement. By making this reaction tube 7a longer than the other reaction tubes 7b and 7c, the mixed liquid can be easily stirred without scattering from the container, and optical measurements can be easily performed. ing.

Next, the operation and method of using the apparatus 1 having the above structure will be explained with reference to FIGS. 5 to 8.

The one-step measurement method shown in FIG. 5 will be explained.

First, the operator presses the selection button 2b and start button 2a of the input section 2 (S1). Select button 2 in input section 2
b. When the start button 2a is pressed, the CPU 24 reads the corresponding program from the program memory 22, controls each part of this device 1, and executes the processing described below. The reaction container moving section 32 moves and arranges the reaction containers 4 taken out by the reaction container takeout section 33 one by one near the starting end S of the reaction line section 11 (S2). Suction dispensing part 16a
Under the control of the CPU 24, the sample 44 is aspirated and dispensed into the storage section 7a of the reaction container 4, and further into the storage section 7c of the reaction container 4.
The enzyme labeled substance is aspirated and dispensed into the storage section 7a of the reaction container 4 into which the specimen 44 has been dispensed (S3). Next, the first stirring section 17a performs stirring under the control of the CPU 24 (S
4). Next, when the reaction container 4 reaches the second magnetic separation section 18b, the reactants and unreacted substances are separated, the unreacted substances are removed by the second washing section 19b (S11), and the substrate is dispensed and stirred. (S12), the measurement unit 20 obtains luminescence information about the reactant, and this luminescence information is output to the CPU 24. Then, the CPU 24 searches the memory 23 for a calculation formula, etc., and obtains a measurement result based on the transmitted light emission information (S
13). This result is output to the output section 3, and the output section 3 prints out the immunity information (S14). On the other hand, the reaction container 4 in which the measurement has been completed is discharged by the transfer section 21 near the terminal end E of the reaction line section 11 (S15).

Next, the two-step measurement method shown in FIG. 6 will be explained.

[0033] After the same processing as shown in steps S1 and S2 shown in FIG.
Under the control of the PU 24, only the specimen 44 is aspirated and dispensed into the storage section 7a of the reaction container 4 (S5). Subsequently, the first stirring section 17a performs stirring under the control of the CPU 24 (S6). Next, when the reaction container 4 reaches the first magnetic separation section 18a,
Reactants and unreacted substances are separated, and the unreacted substances are removed by the first washing section 19a (S7). Subsequently, the enzyme labeled substance is dispensed and stirred by the suction dispensing stirring section 16b (S8). After that, the processes of steps S9 to S15 shown in FIG. 5 are performed.

Next, the delay reaction measurement method shown in FIG. 7 will be explained.

When the steps S1 to S4 shown in FIG. 5 are performed and the reaction container 4 reaches the suction dispensing stirring section 16b,
The contents are dispensed from the storage section 7b of the reaction container 4 to the storage section 7c of the reaction container 4 (S51). After that, the processes of steps S9 to S15 shown in FIG. 5 are performed.

Next, a two-step measurement method using a diluent shown in FIG. 8 will be explained.

When steps S1 and S2 shown in the two-step measurement method shown in FIG. 6 are performed, the diluent is dispensed from the diluting section 25 (S61), and the sample 44 is dispensed (S5).
, only the specimen 44 is aspirated and dispensed into the storage section 7a of the reaction container 4 (S62). Subsequently, the first stirring section 17a
Stirring is performed under the control of 24 (S63). Next, when the reaction container 4 reaches the first magnetic separation section 18a, the reactants and unreacted substances are removed (S7), and then the suction dispensing stirring section 16b
The enzyme label is dispensed and stirred (S8). After that, the processes of steps S9 to S15 shown in FIG. 5 are performed.

[0037] As described above, the above-mentioned Example Apparatus 1 uses a reaction vessel with three reaction tubes, so the three reaction tubes can be used appropriately depending on the types of reaction liquids, specimens, etc. to be mixed. This makes it possible to measure a wide variety of items.

Note that the present invention is not limited to the above embodiments,
It can be implemented in various ways without changing its gist. For example, the reaction vessel may have two or more reaction tubes connected in series. Alternatively, a dispersion solution in which antibodies are bound to magnetic particles may be used.

[0039]

[Effects of the Invention] According to the present invention described in detail above, immunoassays are automatically performed on multiple items simply by operating the start button, and the measurement results are printed out. , an automatic immunoassay device that can measure a large number of samples simultaneously and in a short time using the same device, speeding up measurements and improving the ability to measure multiple items simultaneously, and immunoassay using this device. A measurement method can be provided.

[Brief explanation of the drawing]

FIG. 1 is an external perspective view of an apparatus according to an embodiment of the present invention.

FIG. 2 is a schematic configuration diagram of this device.

FIG. 3 is a plan view of a reaction vessel of this apparatus.

FIG. 4 is a side sectional view of the reaction vessel shown in FIG. 3.

FIG. 5 is a flowchart for explaining a one-step measurement method using this device.

FIG. 6 is a flowchart of the two-step measurement method of this device.

FIG. 7 is a flowchart of a delay reaction measurement method using this device.

FIG. 8 is a flowchart of a two-step measurement method using a diluted solution of this device.

[Explanation of symbols]

1 Automatic immunoassay device 3 Output section 4 Reaction container 5 Control section 11 Reaction line section 12 Sample storage section 15 Reagent solution storage section 16a Suction dispensing section 16b Suction dispensing stirring section 16c Dispensing stirring section 17, 17a, 17b Stirring section 18, 18a, 18b Magnetic separation section 19, 19a
, 19b Cleaning section 20 Measuring section 21 Discharging section 44 Sample

Claims (8)

[Claims] [Claim 1] A specimen storage unit that stores a plurality of specimens;
A reagent container containing a washing solution and a diluent, and at least two reaction tubes each having an opening at the top are connected, one of which contains a solid phase bound to either an antigen or an antibody, and the reaction is carried out. a reaction container containing a labeled substance, such as an antigen or an antibody labeled with a labeled compound, in one of the reaction tubes other than the reaction tube; a reaction container storage section for storing a plurality of the reaction containers; and moving the reaction container. and a reaction line section capable of reacting and measuring the sample and the reaction solution at each predetermined position on the movement route; and a reaction line section that stores the reaction containers one by one from the reaction container storage section according to the measurement item. a reaction vessel moving unit that is moved and arranged near the starting point of the reaction line unit, and a reaction vessel moving unit that aspirates and separates the sample contained in the sample storage unit and the labeled substance contained in the reaction vessel at a predetermined position of the reaction line unit into the other reaction tube. an aspiration/dispensing section for obtaining a mixed solution of a sample and a label by dispensing the mixture; a stirring section for stirring the reaction container containing the mixed solution obtained by the aspiration/dispensing section; A separation section that separates reactants and unreacted substances bound to the solid phase in the mixed solution, a washing section that removes the unreacted substances separated by this separation section, and a labeled compound bound to the solid phase. a measurement unit that measures information;
an output section that outputs the measurement results measured by the measurement section; a discharge section that discharges the reaction vessel in which the reaction and measurement have been completed near the end of the reaction line section; and a control section that controls the operation of each section. An automatic immunoassay device comprising: 2. The automatic immunoassay device according to claim 1, wherein the solid phase is a magnetic particle and the separation section is a magnetic separation section. 3. The automatic immunoassay device according to claim 1, wherein the labeled compound is an enzyme. 4. The automatic immunoassay device according to claim 1, wherein the measuring section is a measuring section using an optical method. 5. An immunoassay method using the automatic immunoassay device according to claim 1. 6. An immunoassay method using the apparatus according to claim 2. 7. An immunoassay method using the apparatus according to claim 3. 8. An immunoassay method using the apparatus according to claim 4.