JPS6361954A - Method and apparatus for preparing sample liquid for analysis - Google Patents

Abstract

PURPOSE:To easily, surely and exactly complete a dilution system for a liquid to be examined by executing the dispensing, mixing and dilution of a diluting liquid with the same implements;. i.e., nozzles. CONSTITUTION:A slide base part 1 consists of a slide base 9, a pulse motor 10 for moving the base 9 back and force between prescribed positions, a belt 11 and a position detecting means 12. A nozzle section 2 moves the nozzles 13 upward and downward and to execute the suction and discharge of liquid. A suction and discharge pump section 3 consists of plural plunger pumps 19- connected via nozzle tubes to the respective nozzles 13- and a motor 20 and transmission means 21 to drive the pumps 19. An operation section 4 is provided with keys 22-25 for assigning the modes to instruct the actions of the respective stages, keys 26-30 for selecting items, a main switch 31, etc., and the operations thereof are checked and displayed on a display part 5. A memory and control section 6 consists of a CPU41 and outputs operation commands to the respective sections 1-3, 5 upon receipt of the input instructions from the operation section 4.

Description

[Detailed Description of the Invention] [Technical Field] The present invention is applicable to, for example, tests based on red blood cell agglutination reactions for diagnosing viral infections and malignant tumors, and various clinical tests using agglutination reactions using latex or microparticles. The present invention relates to a method and an apparatus for accurately and simply preparing a diluted sample solution for serving.

[Technical background]

Increase in the number of viral infections due to increased opportunities for human interaction within and outside the country and increased opportunities for blood transfusions and blood product injections 9 Technology for efficiently clinically testing these diseases in order to cope with the increasing incidence of cancer tumors associated with the aging of the population. development is desired. By the way, one way to determine whether or not a person is suffering from these diseases is by detecting certain trace components in the blood caused by viruses or tumors through antigen-antibody reactions.

For example, hepatitis B, which is a type of acute viral infection, has many virus carriers, and if it worsens, there is a risk of liver cirrhosis or liver cancer. HBc and HBe antigens and antibodies) are well known, and their detection is occupying an important position in the field of clinical testing.

This analysis is performed using the PHA method (passive hemagglutination reaction) or RPH method.
This is carried out using Method A (reverse passive hemagglutination reaction), in which the test solution is diluted stepwise (usually by 2 to the nth power) with a diluent (usually using a phosphate buffer) and a Add sensitized red blood cells of the species and check the presence or absence of agglutination at a certain dilution level (usually 16 times or 32 times) (screening test), or compare the degree of agglutination in the entire dilution series with the degree of agglutination in the series in which the reagent is added ( Confirmatory testing) is conducted to investigate the presence or absence of infection and the prognosis of infected patients.

[Prior art and its problems] Currently, the preparation of the above sample solution (dilution series) is
Using a standardized microplate with 12 wells, dispense the diluted solution (usually 25 μl or 50 μl) into the wells with a dropper or micropipette, collect the test solution (usually 25 μl or 50 μl), and mix by stirring. The dilution is carried out using a guilureator (a) as shown in FIG.

(Disadvantages of Prior Art) However, the conventional method has various drawbacks. First of all, since the dispensing of the diluent and the collection, stirring and dilution of the test liquid are performed using different tools (dromper and guilter), errors are likely to occur.

In addition, the Guilyutor (A) has a notch (C) at the tip of the rod-shaped body (B), and is configured to hold the test liquid or mixed diluted liquid in this notch (C) by capillary action. . The liquid that has accumulated in the notch (c) will fly out due to the rotation during stirring. Therefore, ■It requires skill to use, the amount brought in and the amount taken out is constant, and there are large individual differences.■
Serum has a higher viscosity than a diluted liquid, so the viscosity changes with dilution, and the higher the dilution rate, the greater the difference from the calculated dilution rate, and the degree of wetting varies depending on the dirt on the surface and cut, so the amount retained may vary. As the impurities change over time, it is necessary to periodically incinerate and remove impurities, and maintenance work such as pre-wetting, washing after use, and removing moisture with filter paper is required, which is time-consuming. There are many problems such as it is expensive, it is extremely hard and can damage the well wall and affect the agglutination reaction, and because the volume is constant, the dilution rate is determined by the initial ratio of buffer solution and test solution and cannot be changed. have.

C. Purpose of the present invention In view of the above, the present invention aims to easily, reliably, and accurately complete a dilution series of a test solution without being affected by the properties of the test solution or the level of skill of the examiner. do. Another object of the present invention is to provide a method for preparing a sample liquid in which the dilution ratio can be easily changed. Another object of the present invention is to provide a sample liquid preparation device that is easy to operate and maintain, operates reliably, has a simple structure, and can be obtained at low cost.

These objectives are achieved by performing the dispensing and mixing of the diluent and the guillusion with the same device or nozzle, instead of a combination of dromper and guilluter. This nozzle is connected via a tube to a pump such as a plunger pump or a tube pump that sucks and discharges minute amounts accurately, and is controlled by a microcomputer.

〔Example〕

Hereinafter, the present invention will be explained in detail by taking the PHA method as an example and based on embodiments shown in the drawings.

Figures 1 to 3 show an example of a sample preparation device that embodies the method of the present invention; Figure 1 is a configuration diagram of the main mechanical parts;
The figure is an overall block diagram, and FIG. 3 is a flow path diagram.

(Structure) This device consists of a slide base part (1), a nozzle part (2)
.

It is composed of a suction/discharge pump section (3), an operation section (4), a display section (5), and a storage/control section (6).

The slide base part (1) includes a slide base (9) on which a microplate (7) and a slow tE liquid container (8) are placed in a predetermined position, and a drive that reciprocates the slide base (9) between predetermined positions. a pulse motor (10) as a source, a bell (11) as a transmission means and a position detection means (12)
Consisting of The position detection means (12) includes, for example, a photocoupler and a baffle plate. A space may be provided on the slide base (9) to place a test liquid and a blood cell reagent (both paths shown), and a rack and pinion may be used instead of the belt (11).

The nozzle part (2) moves the nozzle (13) up and down and sucks and discharges liquid.
(Hard or 12 (hard, 10 depending on the structure of the microplate (7)) nozzles (13)... are supported in a row by a nozzle holder (14), and the nozzle holder (14) is supported. Connect the nozzle arm (15) to the motor (1
6), it is configured to be moved up and down by an eccentric cam (17) that rotates. Incidentally, reference numeral (42) in the figure is a position detection means for detecting the normal position of the eccentric cam. Eccentric cam (17
) instead of the nozzle (I) using a hydraulic cylinder or other means.
The vertical movement of 3) may also be performed. By the action of the nozzle part (2), each well (71) of the microplate (7)...
- Dispense the diluent solution (81), or stir and dilute the test solution and buffer solution (81) that are dropped separately. It is also possible to collect and drip test fluids and blood cell reagents.

The suction/discharge pump section (3) includes a plurality of plunger pumps (19) connected to each of the nozzles (13) through nozzle tubes (18), and the plunger pumps (19). Drive motor (pulse motor') (2
0) and a transmitter l2 (21) consisting of a rack and pinion.
It becomes more. A tube pump may be used instead of the plunger pump (19), and a belt may be used instead of the rank and pinion. In the figure, reference numeral (43) is a position detection means for detecting the normal position of the rack and thus the plunger pump (I9).

The operation unit (4) has mode designation keys (22) and ( 23)・(
24), (25), and keys for selecting items (number of dilution steps, initial magnification, etc.) (26), (27), (28)
・(29)・(30) and a main switch (31), etc., and this operation is displayed on the display section (5) for confirmation. Reference numerals (32) to (40) are LEDs for confirmation display.

Note 1. The control unit (6) consists of a CPU (41) containing dilution and other programs and a clock, and the operation unit (4).
Each section receives input instructions from (1), (2), (3),
(5) Outputs the operation command. In this example, the nozzle (13
) and the amount of liquid aspirated (from 25μ to 50μ) and the number of dilution stages (4 stages.

8 steps, 12 steps), initial magnification (2x, 4x), etc. are predetermined, but a program can be created so that these can be changed and set as desired.

In addition, in the above example, a structure is adopted in which the slide base (9) on which the microplate (7) is placed is moved, but it is also possible to support the nozzle part (2) with a slide base etc. and fix the microplate (7) etc. It is also possible to have a structure in which

(operation) Next, a method for preparing a sample liquid using the illustrated apparatus will be explained.

First, settings are made using the operation section (4) according to the purpose of measurement. For example, in the PHA method (hepatitis B HB, e antibody measurement using passive hemagglutination reaction) screening test,
8x12 (solid (10x12 also available) wells (7
1) A microplate (7) with ... is placed horizontally or horizontally, and four wells (71) in one row are used for one test liquid. This is a positive test based on whether the sample solution diluted 16 times (dilution factor 2 in the first well) or 32 times (dilution factor 4 in the first well) aggregates. This is to determine whether the test is negative.

Then, turn on the main switch (31) and select key (30).
) is used in 4 stages (LED (33) lights up) 1 selection key (27
), use the 1st to 4th wells (71) (L E D (
35) lit).

Initial magnification 2 (LED (32) lights up) with selection key (26)
Select each. Next, a new microplate (7)
is fixed at a predetermined position on the slide base (9), and press the mode designation key (dropper/buffer injection) (22). Then, the slide base (9) moves toward the nozzle part (2) and temporarily stops at a position where the buffer solution (81) of the buffer solution container (8) comes under the wings of the nozzle (13). This stop position is controlled by the number of pulses from the home position detected by the position detection means (I2). In this state, the tip of the nozzle (13) is lowered, sucks in a predetermined amount (25μ) of the buffer solution (81), and rises. Next, the slide base (9) moves in the opposite direction so that the nozzle (13) comes to the position of the well (7I) in the first stage, and after stopping, the nozzle (13) descends and is located in the same stage. BtE solution (81) in all wells (7I)
Dispense (dispense). Repeat the same operation for 2nd to 4th
25μ of the buffer solution (81) is also dispensed into each well (7I) of the row. A phosphate buffer is usually used as the buffer (81).

Next, after separately injecting 25 centimeters of a different test solution into each well (71) in the first stage using a micropipette, press the mode designation key (23) (for stirring/dilution). Then, the nozzle (13) enters the first stage well (71).
Repeat inhalation and exhalation several times while moving up and down at this position. By this operation, the test liquid and the buffer solution (81) are uniformly stirred. (If the inhalation amount at this time is 25 μl, the operation command will be easier.) In this state, make sure that the first sample is diluted twice.
50 pl of sample solution is obtained.

Finally, 25μl of this diluted liquid is sucked up and the second stage well (71) is moved to just below the nozzle (13), where 25μl of the diluted liquid is discharged.
Similarly, inhale and exhale are repeated and stirred to obtain a 4-fold diluted sample solution. Similarly, in the fourth well (71), 1
50 μl of a 6-fold diluted sample solution is obtained. 2 of them
5 is aspirated and drained into the waste container (84) of the buffer container (8).

With the above steps, each of the four wells is filled with 25 .mu.i of the sample solution diluted to 4'@, and the dilution operation (sample solution adjustment) is completed.

After that, incubate at 37 degrees for about 1 hour to absorb non-specific reactions, and then add H to the fourth well (7I).
A 25 μm sample of a suspension of BeAg-sensitized sheep red blood cells (trade name: e-cell, manufactured by Kokusai Reagents) in phosphate buffer is collected with a micropipette, added, and shaken for about 10 seconds to mix. After that, incubate at 37 degrees for more than 3 hours,
Observe the aggregate image. Judgment is made by comparing with the agglutination image of a negative control. For the negative control, use a normal buffer solution, put 25 μl into each row of 4 wells (71) in any one of the microplates (7), and add the blood cell reagent (sensitized blood cell suspension) in the same way. It is.

If you press the mode designation key (25) (for cleaning) at any time, the cleaning solution container (82) or (83) of the buffer container (8) will come directly below the nozzle (13), so you can aspirate the cleaning solution here. The nozzle tip is cleaned by discharging it into a waste liquid container (84). Note that the nozzle (I3) is also automatically cleaned at the final stage of other modes. In addition, the mode designation key (24) is for collecting the sample liquid, and if you have this key, you can use the multi-hole mode selection key (24) on the slide base (9). ,!
! ! A test liquid container (not shown) is placed there, and each test liquid is suctioned into the first well (71) using the nozzle (13) instead of manually.
) can be injected.

Further, in the above example, only the first to fourth wells (71) were used, but depending on the number of test liquids, the selection key (28) and (
29) to move to the 5th to 8th rows (L E D (36)
(LED (37) lit) and the 9th to 12th stages (LED (37) lit) can also be used in the same way. In this case as well, press the selection key (30) to use 4 stages (LED (38) lights up). The movement of the nozzle part (2), slide base part (1), etc. is the first stage.
The 5th and 9th steps have the same movement. Furthermore, if the amount to be collected from each machine is 50 μt, it is preferable to operate the selection key (26) and adopt the 50 μ-poor method (LED (34) lights up: 1st stage is 2-fold dilution).

To perform a measurement using a sample solution diluted 32 times, press the selection key (
2G) to turn on the LED (33).

In this way, the buffer solution (81) is placed in the first stage well (71).
) to the other wells (71) at 25° intervals. The above-mentioned 75 formation may be carried out three times with each 25 μl, but it can also be carried out at -degrees. The test solution is the same (25 μl, so 100 samples are obtained by diluting it 4 times in the first stage, so 50 μt, which is half of that amount)
After discarding , you can handle it in the same way as in the previous example.

Next, a confirmation test using the PHA method will be similarly explained. The confirmation test is performed more precisely on the test liquid determined to be positive or false positive in the screening, and two series of 8-stage (8-well) wells (71) are used for each test liquid. Similarly, one series of 8 columns each is used for positive and negative controls. To make the dilution ratio of the 8th stage 256, the first stage is 2 times diluted, and to make it 512 times, the first stage is diluted 4 times.

In this case, press the selection keys (27) and (28) to
and use of the 5th to 8th stages (LEDs (35) and (36) are lit), and press the selection key (30) to specify the 8th stage (LED
(39) Lighting).

Others are 2 and 4 according to the above. ..., 256 times or 4,8. ...A series of 25 μt sample solutions diluted 512 times is created. For all of this, add 25μ each of buffer in the first column and HBeAg reagent in the second column.
After shaking and mixing for about 10 seconds, incubate at 37 degrees for 1 hour. Subsequently, the HBeAg-sensitized sheep red blood cell suspension was
Add 5 μl each, mix by shaking for about 10 seconds, incubate at 37 degrees for 3 hours or more, and observe the aggregation image. The judgment is
Observe whether aggregation is inhibited in the series in which the buffer solution is added and in the series in which the HBeAg reagent is added. If there is no instrumental difference between the two, it is negative, if there is a difference of 2 or more tubes, it is positive, and if there is a difference of 1 tube, the test is repeated. If the test is reproducible, it is considered positive.

(Modified example) The above is an HB method using the PHA method using red blood cell agglutination reaction.
Although the explanation has been given using e-antibody measurement as an example, the present invention is also applicable to measurement of HBe antigen by RPHA method, measurement of HBc, HBd, HBs antigens/antibodies, and tumor markers (tumor-specific proteins) based on hemagglutination reaction. It can be used in the same way by changing the reagents when carrying out this procedure. Furthermore, it can also be used in various clinical tests that utilize agglutination reactions using latex or microparticles.

In addition, in order to measure a wide range of dilution ratios with a small number of sample solutions, we can create a dilution series of 3 to the n power, or to measure more precisely in a narrower range of dilution ratios, we can make dilution series of 2/3 n times. You can also freely create a multiplication dilution series.

As for the device, a sample liquid container and a sensitized blood cell reagent container are placed on the slide base (1), and these are connected to the same nozzle (
13) may be dispensed into the wells (71).

Further, in the case of the embodiment, the nozzle (13) is a disposable type in which the nozzle tip is replaced, and if the number of specimens is small, the tip can be left unused. Of course, a fixed nozzle may also be used. The drive of the nozzle (I3) is as follows:
A nozzle holder (IIO) and a nozzle arm (I5) may be provided for each nozzle (13) and may be moved up and down individually.

〔effect〕

As described in detail above, the method of the present invention involves the use of a series of stepwise diluted sample solutions used for analysis using antigen-antibody reactions.
Adjustment is performed using one nozzle. Therefore, the same amount of inhalation and ejection of the diluted liquid, dilution by stirring and mixing with the test liquid, collection, etc., is carried out using the same nozzle, compared to the conventional method that uses a micropipette and diluter. This ensures extremely accurate measurements with no individual or instrumental differences. In addition, since suction and discharge are performed using a plunger pump or the like, the collection and mixing ratio of the diluent and test liquid can be freely selected, increasing the degree of freedom. Furthermore, there is no need to maintain it like a diluter, which greatly improves work efficiency. Furthermore, unlike stirring using a diluter, this method is extremely superior in that it does not accelerate the reaction by scratching the inner walls of the wells of the microplate and does not cause errors due to scattering of the sample liquid.

The device of the present invention is an embodiment of the above method, and has an extremely simple structure, yet can easily, accurately, and quickly perform dispensing of a buffer solution, stirring/mixing with a test solution, and dilution.

[Brief explanation of the drawing]

1 to 3 show an example of the apparatus of the present invention, in which FIG. 1 is a schematic front view of the main parts, FIG. 2 is a block diagram, and FIG. 3 is a flow path diagram. Figure 4 is a plan view of the microplate.
FIG. 5 is a perspective view of the diluter. 1...Slide base part 2...Nozzle part 3...Suction/exhaust pump part 4...Operation part 5...Display part 6... ...Storage/control unit 7...Microplate 71...Well 8...Buffer container 81...Buffer solution 9...Slide base 13... ... Nozzle 19 ... Plunger pump patent applicant Kokusai Reagent Co., Ltd. Patent issuer
Applicant Teramex Co., Ltd. No. 30 Figure 4 Figure 5

Claims (1)

[Claims] 1. After dispensing a predetermined amount of diluent into the required number of wells in each row of the microplate using a nozzle connected to a suction pump, the nozzle or other means is used to dispense a predetermined amount of diluent into the required number of wells in each row of the microplate. Dispense a predetermined amount of the test liquid into one well, then repeat suction and discharge several times with the nozzle to stir and mix the diluted liquid and test liquid, and then aspirate a predetermined amount of the mixed liquid into the second well. A method for preparing a sample solution for analysis, characterized by diluting it by injecting it into a well and stirring and mixing in the same manner, and subsequently inhaling, mixing and diluting each well in sequence to create a dilution series. 2. A slide base part that places a container for diluting solution etc. together with the microplate and moves it intermittently between predetermined positions, and dispenses, stirs, and dilutes each solution into each well of the microplate by suctioning and discharging the liquid. A nozzle unit, a suction pump unit that causes the nozzle to suck and discharge, an operation unit that has keys for operating instructions for each process and selecting items, and outputs a confirmation message to the display unit based on input signals from the operation unit. 1. A device for preparing a sample liquid for analysis, comprising: a storage/control section that outputs operation commands to various sections according to the instructions;