JP2012159405A - Cartridge and automatic analysis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve such a problem that an automatic inspection device suitable for point of care testing does not support an inspection other than a general biochemistry item in general.SOLUTION: In a cartridge that integrally has a reagent cuvette, a photometry cuvette, and a reaction well, a surface layer of target protein or a surface layer having high affinity with the protein is formed in an internal surface of the reaction well. The surface layer is formed as a solid phased layer of an antibody or an antigen.

Description

  The present invention relates to a cartridge having a surface layer having a high affinity for a target protein in a sample on an inner wall surface. The present invention also relates to a method for automatically analyzing a sample using the cartridge.

  At present, clinical examination apparatuses that can obtain test results quickly, easily, and with high accuracy are being used in clinical practice. This type of test, also called point-of-care testing (POCT), aims to quickly test the specimen “at the place” when the need for the test occurs. There is an apparatus disclosed in Patent Document 1 as a small automatic analyzer suitable for this type of inspection at present.

JP 2006-220494 A

  However, devices and cartridges such as those described in Patent Document 1 are specialized in the inspection of general biochemical items (for example, diabetes, arteriosclerosis, kidney / liver disease, etc.). The test items do not have immune responses such as tests and diabetes tests.

  Therefore, the present inventor proposes a dedicated cartridge that enables these inspections and an automatic analysis method optimized for the inspection even in an automatic analysis apparatus suitable for point-of-care testing.

  Specifically, a cartridge having a reagent cuvette, a photometric cuvette, and a reaction well integrally having a surface layer of the target protein or a surface layer having a high affinity for the protein on the inner wall surface of the reaction well is proposed. .

  Further, when the type of the cartridge mounted on the rotor is read from the information presentation unit and the mounted cartridge has a surface layer of the target protein or a surface layer having a high affinity for the protein on the inner wall surface of the reaction well, the cartridge is We propose a method to automatically activate the examination mode of the immune response to be used.

  According to the present invention, the use range of point-of-care testing can be further expanded.

  Problems, configurations, and effects other than those described above will be clarified by the following description of embodiments.

The perspective view which shows the example of a 1st cartridge. FIG. 3 is a side sectional view of the first cartridge. FIG. 5 is a conceptual diagram illustrating an example of a first cartridge manufacturing method. The side view of the 1st cartridge. The perspective view which shows the example of a 2nd cartridge. The sectional side view of the 2nd cartridge. The figure which shows the example of whole structure of an automatic analyzer. The figure which shows the plane structure of the rotor vicinity of an automatic analyzer. The block diagram which shows the control system of an automatic analyzer. The flowchart which shows the inspection mode automatic start procedure. The figure explaining inspection mode automatic start conditions. The figure explaining inspection mode automatic start conditions. The figure explaining the probe control conditions at the time of aspiration.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. The embodiment of the present invention is not limited to the embodiments described later, and various modifications can be made within the scope of the technical idea.

<Inspection method (principle)>
In this embodiment, an automatic analyzer for point-of-care testing capable of analyzing or testing not only general biochemical items but also immunological test items will be described. The automatic analyzer uses an enzyme-linked immunosorbent assay (ELISA) method to detect and quantify the concentration of an antibody or antigen contained in a sample.

  The ELISA method is a test method that combines the two basic reactions of antigen-antibody reaction and enzyme-substrate reaction, and quantifies the target antigen by detecting the color density and fluorescence intensity of the enzyme antibody that has reacted with the target antigen in the sample. is there. More specifically, an antibody or a fragment thereof that binds to a certain antigen protein, or an antigen protein or a fragment thereof is immobilized on a solid phase carrier, and an immunological reaction with the antibody or the antigen protein is enzymatically detected. Is the method.

  As the ELISA method, a sandwich method, a direct method, a competitive method and the like are known, but any method can be used in the present invention.

  In the sandwich method, a first antibody (solid-phased antibody) immobilized on a solid phase carrier is bound to an antigen, and then a second antibody that recognizes an epitope different from the first antibody is added to bind to the antigen. When the second antibody is a labeled antibody, the label is used. When the second antibody is an unlabeled primary antibody, the labeled third antibody (secondary antibody) is used. This is a method for detecting an antigen with high sensitivity.

  In the competition method, an antigen (solid-phased antigen) immobilized on a solid phase carrier is bound to a first antibody, and then the first antibody is used in the case of a labeled antibody. In the case of an unlabeled primary antibody, the target antigen is detected with high sensitivity using a second antibody (secondary antibody) that is labeled and bound to the antigen.

  For details of the measurement method of the ELISA method, refer to a well-known document (Japanese Clinical Pathology Society, “Special Issue on Clinical Pathology No. 53, Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, 1983, Ishikawa. "Enzyme Immunoassay" edited by Eiji et al., 3rd edition, School of Medicine, 1987, edited by Tsuneaki Kitagawa et al. Immunoassay ", Kodansha Scientific, 1974) and the like.

<Cartridge>
Hereinafter, an example of the structure of a newly proposed cartridge for quantifying a small amount of allergen (antigen) contained in a sample with an automatic analyzer will be described. As will be described later, two types of cartridges are prepared to automate the inspection. Hereinafter, these two types of cartridges are referred to as a first cartridge and a second cartridge.

  A structural example of the first cartridge 101 will be described with reference to FIGS. FIG. 1 is a perspective view of the first cartridge 101 as viewed obliquely from above. FIG. 2 is a side cross-sectional view showing the first cartridge 101 cut away at the position of the broken line in FIG. FIG. 3 is a diagram conceptually illustrating an example of a method for manufacturing the first cartridge 101. FIG. 4 is a side view of the first cartridge 101 as viewed from the direction of FIG.

  The first cartridge 101 is made of polypropylene, for example, and is manufactured by integral molding. The cartridge 101 shown in FIG. 1 includes three containers, a photometric cuvette 102, a reagent cuvette 103, and a reaction well 104. These three containers are arranged in a line, and the photometric cuvette 102 is arranged in the center thereof. In the case of FIG. 1, the cartridge 101 is composed of three containers, but a structure in which the reagent cuvette 103 is divided into two containers may be employed.

  In the case of this embodiment, 200 μl of the secondary antibody reagent necessary for the allergen (antigen) test is injected only into the reagent cuvette 103, and nothing is injected into other containers. An immobilized antibody (primary antibody) layer 107 that captures the target allergen (antigen) is formed on the inner wall surface of the reaction well 104. In the case of this embodiment, the bottom surface of the reaction well 104 is processed into a downwardly convex conical shape. That is, it has a hollow shape whose diameter decreases from the outer peripheral side toward the center.

  FIG. 2 shows an image of forming the immobilized antibody layer 107. In the case of FIG. 2, the immobilized antibody layer 107 is formed so as to cover the bottom surface of the inner wall surface. However, the immobilized antibody layer 107 may cover not only the bottom surface (a part of the inner wall surface) but also the entire inner wall surface. As a surface treatment method used for generating the solid-phased antibody layer 107, a UV treatment method, a plasma treatment method, a polymer treatment method, or the like can be used. As shown in FIG. 3, a single reaction well 108 in which the immobilized antibody layer 107 is formed on the inner surface may be formed by fitting into the reaction well 104 of the cartridge 101.

  Incidentally, the upper surface of the cartridge 101 after shipment is hermetically sealed with a film. Inspection items that can be inspected with the reagent cartridge are printed on the surface of the film. A panel 105 is arranged on the back side of the reagent cuvette 103, and a seal 106 printed with a dot code (two-dimensional code) as shown in FIG. The seal 106 serving as an information presentation unit includes (1) date of manufacture and expiration date, (2) serial number, (3) test use, (4) specimen type, and (5) required amount of sample and dispensing timing. (6) Necessary amount and dispensing timing of reagent, (7) Photometric method, (8) Photometric timing, (9) Main / sub wavelength of photometry, (10) Conversion formula from absorbance to concentration, etc. Has been recorded.

  A structural example of the second cartridge 111 will be described with reference to FIGS. FIG. 5 is a perspective view of the second cartridge 111 as viewed obliquely from above. FIG. 6 is a side cross-sectional view showing the second cartridge 111 cut away at the position of the broken line in FIG.

  The second cartridge 111 is also made of polypropylene and is manufactured by integral molding. The outer diameter of the second cartridge 111 and the outer dimension of the first cartridge 101 are the same. The cartridge 111 shown in FIG. 5 includes two containers, a reagent cuvette 112 and a reagent cuvette 113. Similarly, a panel 105 is arranged on the back side of the reagent cuvette 113, and a seal 106 printed with a dot code (two-dimensional code) is attached to the surface thereof.

  In the case of this embodiment, 850 μl of a washing buffer solution is injected into the reagent cuvette 112. On the other hand, 250 μl of a coloring reagent is injected into the reagent cuvette 113. Again, the upper surface of the cartridge 111 after shipment is hermetically sealed with a film.

<Automatic analyzer>
FIG. 7 shows an example of the overall configuration of the automatic analyzer used in this embodiment, and FIG. 8 shows a plan view of the vicinity of the rotor of the automatic analyzer. FIG. 9 shows a block diagram of a control system of the automatic analyzer.

  The automatic analyzer is connected to a commercial power source, and the automatic analyzer is turned on by turning on a power switch (not shown). The main control unit 200 controls the operation of the entire automatic analyzer. The main control unit mainly includes a CPU, a ROM, a RAM, and a real time clock. The ROM is used as a storage area for the control program of the automatic analyzer, and the RAM is used as a primary storage area.

  The automatic analyzer has one probe 201 used for sucking and discharging the solution. The probe 201 is attached in the vertical direction from the tip of the arm 202. Aspiration, discharge, positioning, temperature management, and the like by the probe 201 are controlled by the probe driving unit and the arm driving unit. The probe driving unit and the arm driving unit include a suction / discharge control unit, an arm rotation control unit, an arm vertical control unit, and a probe heater control unit.

  The suction / discharge control unit includes a suction / pressure pump 203b that generates a negative pressure or a positive pressure at the tip opening of the probe 201, and a suction / discharge control circuit 203a that controls driving of the pump. The arm rotation control unit includes a stepping motor 204b that rotationally drives the arm 202, and an arm rotation control circuit 204a that drives and controls the motor. The stepping motor 204b rotates the rotation axis of the arm 202 in both clockwise and counterclockwise directions. Here, the length of the arm 202 gives the turning radius.

  The arm vertical control unit includes a stepping motor 205b that drives the arm 202 in the vertical direction and an arm vertical control circuit 205a that drives and controls the motor. The stepping motor 205b moves the rotation axis of the arm 202 up and down in the vertical direction. The probe heater control unit includes a probe heater 206b serving as a heat source for the probe 201 and a probe heater control circuit 206a that controls the amount of heat generated by the probe heater 106b in accordance with the detected temperature of the probe 201. When not used for inspection, the probe 201 is stored in the housing 207.

  The main control unit 200 controls the positioning of the probe 201, suction / discharge of the solution, and disposal through these individual control units.

  The automatic analyzer includes sample holders 208a to 208d that hold sample containers (not shown). The automatic analyzer shown in FIG. 7 can hold four sample containers at a time. These holders are disposed on the rotation radius 209 of the probe 201. A cleaning tank 210 for cleaning the inner surface and the outer surface of the probe 201 is also disposed on the rotation radius 209. The cleaning tank 210 is connected to the pump unit 211 through a pipe. Pure water or cleaning water used for cleaning is pumped from the water tank 212 by the pump unit 211. The waste water is sent to a waste liquid tank (not shown).

  The automatic analyzer has a rotor 220 that can hold a plurality of first cartridges 101, second cartridges 111, and other cartridges in the circumferential direction. The rotor 220 in this embodiment is disk-shaped, and 40 cartridge holders 221 (holes into which cartridges can be fitted are formed) are arranged along the outer periphery thereof.

  In FIG. 8, the position in the radial direction of the rotor 220 of the container constituting each cartridge in a state where the first cartridge 101 and the second cartridge 111 are mounted on the rotor 220 is indicated by a broken line. In FIG. 8, three circles are drawn. As shown in FIG. 8, each circle intersects the rotation radius 209 of the probe 201 at the position of three black circles. In this way, the position where the probe 201 intersects differs for each container into which the solution is taken out and put in. Therefore, when the solution is taken in and out, the rotational positions of the probe 201 and the rotor 220 are controlled according to the position of the target container.

  The rotor 220 is controlled by a rotor driving unit. The rotor drive unit includes a stepping motor 222b that can rotationally drive the rotor 220 in both clockwise and counterclockwise directions, and a rotor control circuit 222a that controls the motor. The rotor 220, the arm 202, the probe 201, the housing 207, and the sample holders 208a to 208d are covered with an openable / closable case (not shown). A case open detection switch (not shown) and a case lock mechanism 223 are attached in the vicinity of the lid. When the case is opened, the case open detection switch is turned on, and a detection signal is sent to the main control unit 200. Further, the case lock mechanism 223 is operated by the control from the main control unit 200, and the case can be locked.

  In addition, the automatic analyzer includes an information reading unit 224 that reads information on the cartridge mounted on the rotor 220 from the cartridge seal 106. The information reading unit 224 includes an image sensor and a processor. Information read from the information reading unit 224 is given to the main control unit 200 and stored in the storage device 225. In addition, the storage device 225 also stores analysis results, patient IDs, and analysis dates. The storage device 225 is also used for storing past analysis results.

  Further, the automatic analyzer includes a light source 226 for measuring absorbance, an optical system 227, and a photodetector 228. These devices are used to measure the absorbance of the solution to be examined injected into the photometric cuvette 102. For example, a tungsten lamp is used as the light source 226. The light source 226 is basically lit while the automatic analyzer is powered on. However, the light source 226 is turned off if the unused state continues for a predetermined time after the power is turned on. The optical system 227 includes a rotary wavelength selection filter, a lens, and a slit. The main control unit 200 can rotate the rotary wavelength selection filter to place a desired filter on the optical axis. The wavelengths that can be selected by the rotary wavelength selection filter are, for example, 340 nm, 380 nm, 415 nm, 450 nm, 480 nm, 508 nm, 546 nm, 576 nm, 600 nm, 620 nm, 660 nm, 700 nm, and 800 nm. The photodetector 228 includes a photodiode and an amplifier. The amount of light detected by the photodiode is output as a voltage and amplified by an amplifier. The amplified analog voltage value is converted into digital data by the A / D converter and is taken into the main control unit 200.

  The automatic analyzer is provided with a display / operation unit 230. The display / operation unit 230 includes a touch panel sensor, an LCD unit, a start button, and a stop button. By using the display / operation unit 230, the user can recognize messages and analysis results, and can give instructions to the main control unit 200. The start button is used to manually indicate the start of the analysis operation. The stop button is used to interrupt the processing operation during the analysis operation.

  An external printer 231 is connected to the automatic analyzer via a printer I / F circuit. The external printer 231 is an option and is used for printing out analysis results.

<Inspection operation>
Next, an allergen (antigen) test operation using the automatic analyzer will be described. The following operations are executed through the control of the main control unit 200.

  First, the power switch is turned on by the user. This on operation enables the main control unit 200 to operate. The main control unit 200 that has become operable displays a main menu on the display / operation unit 230.

  In this state, the user removes a main body cover (not shown) from the apparatus main body, and mounts a cartridge necessary for the inspection on the rotor 220. Since the automatic analyzer according to the embodiment can be used not only for the immune reaction test but also for the general biochemical test, it is possible to mount a cartridge according to the test purpose. The automatic analyzer activates the processing routine shown in FIG. 10 while the case is open after the power is turned on. Note that the processing routine shown in FIG. 10 is executed for each cartridge until completion of the mounting of the cartridge is instructed.

  The main control unit 200 uses the information reading unit 224 to monitor the mounting of the cartridge on the rotor 220. The main control unit 200 repeats the same determination process until it is determined that the output of the information reading unit 224 is cartridge information (step S1). If it is confirmed that the information is read from the cartridge, the main control unit 200 identifies the type of cartridge mounted on the rotor 220 (step S2).

  The main control unit 200 determines the type of the loaded cartridge from the read information such as the inspection application and the reagent type. In the case of this embodiment, it is determined whether or not the cartridge 101 for immune reaction is attached (step S3). That is, it is determined whether or not the first cartridge 101 in which the immobilized antibody layer 107 is formed on the inner wall surface of the reaction well 104 is mounted as shown in FIG. If a positive result is obtained, the main control unit 200 automatically sets the test mode of the apparatus to the immune reaction test automatically (step S4). Specifically, the IgE inspection mode specified by the inspection application, the type of reagent, and the like written on the seal 106 is set. This setting eliminates the need for user settings. In addition, erroneous setting by the user can be avoided.

  As shown in FIG. 12, this setting can be based on the detection condition that the first cartridge 101 and the second cartridge 111 are mounted on two adjacent cartridge holders 221. This is because the presence of the second cartridge 111 is indispensable for the immune reaction test, as will be described later. The first cartridge 101 and the second cartridge 111 are only required to be mounted, and the first cartridge 101 and the second cartridge 111 are not necessarily mounted to the two adjacent cartridge holders 221. good.

  On the other hand, when a negative result is obtained in the determination process of step S3, the main control unit 200 automatically sets the inspection mode to the general biochemical inspection (step S5).

  Next, the user attaches a sample container containing a measurement sample (for example, blood) to any of the sample holders 208a to 208d. Thereafter, the user attaches the main body cover and presses the start button of the display / operation unit 230. Thereafter, the measurement items are displayed on the screen of the display / operation unit 230. When the user who confirmed the measurement presses the measurement execution button, the measurement is started. In the case of this embodiment, specific IgE analysis as an allergen test is performed by the following procedure.

(1) The serum sample 50 μl and the secondary antibody reagent 120 μl in the reagent cuvette 103 of the first cartridge 101 are sucked by the probe 201 and discharged to the reaction well 104 of the first cartridge 101.
(2) 15 minutes after the process (1), the entire solution in the reaction well 104 of the first cartridge 101 is sucked by the probe 201 and discharged into the reagent cuvette 103 of the first cartridge 101.

(3) 200 μl of the washing buffer in the reagent cuvette 112 of the second cartridge 111 is sucked by the probe 201 and discharged to the reaction well 104 of the first cartridge 101.
(4) The entire solution in the reaction well 104 of the first cartridge 101 is sucked by the probe 201 and discharged to the cleaning tank 210 of the apparatus.
(5) Repeat the operation of process (3) -process (4) two more times.

(6) 180 μl of the coloring reagent in the reagent cuvette 113 of the second cartridge 111 is sucked by the probe 201 and discharged to the reaction well 104 of the first cartridge 101.
(7) Five minutes after the process (6), 175 μl of the solution in the reaction well 104 of the first cartridge 101 is sucked by the probe 201 and discharged to the photometric cuvette 102 of the first cartridge 101.

(8) The absorbance is measured, for example, at a main wavelength of 450 nm / subwavelength of 620 nm.
(9) The specific absorbance is obtained by substituting the measured absorbance into the absorbance-concentration conversion equation stored in the seal 106.
(10) The measurement result is displayed on the display / operation unit 230.

  The above is the content of the specific IgE inspection process realized in an automatic analyzer suitable for point-of-care testing.

  Note that it is necessary to avoid a situation in which the probe 201 comes into contact with the bottom surface of the reaction well 104 and is damaged when the whole amount of the solution is sucked in the processing (2) and the processing (4). In general, the automatic analyzer is equipped with a function of detecting the liquid level by detecting a change in capacitance between the probe 201 and the ground potential. In this embodiment, however, the reaction well 104 has a function of improving safety. The probe 201 is automatically stopped at a target position determined within 3 mm upward from the bottom surface of the probe. The target position is determined in consideration of cartridge mounting errors and manufacturing errors. The arm vertical control circuit 205a detects that the target position has been reached by monitoring the drive amount of the arm 202 in the vertical direction. By mounting the stop function, non-contact between the tip of the probe 201 and the bottom surface of the reaction well 104 is ensured. After the automatic stop, a gap is formed between the tip of the probe 201 and the bottom surface of the reaction well 104, but all the solution can be sucked using the surface tension of the solution.

  Incidentally, when not only the cartridge 101 for the immune reaction but also the cartridge for the general biochemical test are mounted on the automatic analyzer, the general biochemical test is also performed in parallel with the above-described immune reaction test. .

<Other forms>
In addition, this invention is not limited to the form example mentioned above, Various modifications are included. For example, in the embodiment described above, automatic analysis of specific IgE has been described as an example. However, automatic analysis of total IgE, automatic analysis of infectious diseases and cancer (such as chlamydia and PSA), automatic detection of thyroid hormones (such as TSH and FT4) It can be applied to analysis and other immune response tests.

  The above-described embodiments are described in detail for easy understanding of the invention, and the actual product is not necessarily provided with all the configurations described. Moreover, it is possible to replace a part of a certain form example with the structure of another form example, and it is also possible to add the structure of another form example to the structure of a certain form example. Moreover, it is also possible to add, delete, or replace another structure with respect to a part of structure of each form example.

  Moreover, you may implement | achieve some or all of each structure, a function, a process part, a process means, etc. which were mentioned above as an integrated circuit or other hardware, for example. Each of the above-described configurations, functions, and the like may be realized by the processor interpreting and executing a program that realizes each function. That is, it may be realized as software. Information such as programs, tables, and files for realizing each function can be stored in a memory, a hard disk, a storage device such as an SSD (Solid State Drive), or a storage medium such as an IC card, an SD card, or a DVD.

  Control lines and information lines indicate what is considered necessary for the description, and do not represent all control lines and information lines necessary for the product. In practice, it can be considered that almost all components are connected to each other.

DESCRIPTION OF SYMBOLS 101 ... 1st cartridge 102 ... Photometric cuvette 103 ... Reagent cuvette 104 ... Reaction well 105 ... Panel 106 ... Seal 107 ... Solid-phased antibody layer 111 ... 2nd cartridge 112 ... Reagent cuvette 113 ... Reagent cuvette

Claims (12)

In a cartridge integrally having a reagent cuvette, a photometric cuvette, and a reaction well,
An inner wall surface of the reaction well has a surface layer of a target protein or a surface layer having a high affinity for the protein. The cartridge according to claim 1, wherein
The cartridge is characterized in that the surface layer is a solid phase layer of an antibody or an antigen. The cartridge according to claim 2, wherein
The surface layer is formed by fitting a surface layer of a target protein or a container having an inner wall surface with high affinity with the protein into the reaction well. The cartridge according to claim 2, wherein
The cartridge is characterized in that the bottom surface of the reaction well has an inner wall surface that is recessed so that the diameter decreases from the outer peripheral side toward the center. The cartridge according to claim 2, wherein
The surface layer is formed by UV treatment. The cartridge according to claim 2, wherein
The cartridge is characterized in that the surface layer is formed by plasma treatment. The cartridge according to claim 2, wherein
The cartridge is characterized in that the surface layer is formed by polymer treatment. One probe used for sucking and discharging liquid, a probe driving unit for driving the probe, a sample holder for holding a plurality of sample containers, and a rotor for holding a plurality of cartridges having information presentation units in the circumferential direction A rotor driving unit that rotationally drives the rotor, an information reading unit that reads cartridge information from the information presentation unit, an optical measurement unit that includes a light source and a photodetector, and information read by the information reading unit In an automatic analysis method of an automatic analyzer comprising a storage unit and a control unit that controls each part of the device,
The control unit is
Based on the information read from the information reading unit, it is determined whether or not a first cartridge including a reaction well having a surface layer of a target protein or a surface layer having a high affinity for the protein on the inner wall surface is attached to the rotor. And steps to
And a step of automatically activating an immune reaction test mode using the first cartridge when the mounting is confirmed. The automatic analysis method according to claim 8,
In addition to mounting the first cartridge, the control unit activates and controls the inspection mode when it is detected that a second cartridge containing a cleaning buffer solution is mounted on the rotor. An automatic analysis method characterized by that. The automatic analysis method according to claim 9,
The automatic analysis method, wherein the control unit activates and controls the inspection mode when the first and second cartridges are mounted adjacent to each other on a rotor. The automatic analysis method according to claim 8,
The controller controls the lowering of the tip of the probe when sucking a solution from a cartridge including a reaction well having a surface layer of a target protein or an inner wall surface having a high affinity with the protein, which is attached to the rotor. An automatic analysis method comprising: automatically stopping at a predetermined height position that does not contact the bottom surface within 3 mm from the bottom surface of the reaction well. The automatic analysis method according to claim 11,
The automatic analysis method, wherein the bottom surface of the reaction well has an inner wall surface that is recessed from the outer peripheral side so as to decrease in diameter toward the center.

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