JPH0120382B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for automatically measuring enzyme immunity, and in particular, the present invention relates to a method for automatically measuring enzyme immunity.
This invention relates to an automatic enzyme immunoassay method that allows separation of (B-F separation).

Conventionally, enzyme immunoassay is a complicated measurement method, and although the enzyme activity measurement at the final stage can be easily performed automatically, it is difficult to separate B-F at the operational stage where B-F separation is performed. It was difficult to automate the entire process from a place where there was no such thing. Also,
Automatic biochemical analyzers are usually equipped with a cleaning device after the end of a measurement, but very few are equipped with a device that cleans the analyzer mid-way through analysis. Therefore, even in enzyme immunoassays, cleaning during the analysis had to be performed manually.

Furthermore, when performing enzyme immunoassay, the optimal time for antigen-antibody reaction between the solid phase, sample, and enzyme labeling reagent is determined depending on the type of substance to be measured in the sample and the type of enzyme labeling reagent used. However, depending on one measuring device, it may be set to 1.
If only one item can be measured, the operating rate of the measuring device will be poor, and installing and operating multiple measuring devices will require a large amount of expense and a large amount of space for installation.

An object of the present invention is to provide an automatic enzyme immunoassay method that can automatically perform B-F separation by washing during analysis and can arbitrarily set the antigen-antibody reaction time. .

The automatic enzyme immunoassay method of the present invention uses an enzyme as a labeling substance, and when measuring a specific substance in a sample through an antigen-antibody reaction, a reaction vessel containing a solid phase on which an antigen or antibody is immobilized is placed along a reaction line. From the time when the sample or enzyme labeling reagent is dispensed into the reaction vessel using the dispensing device, the solid phase and reaction vessel are washed by the cleaning device placed along the reaction line, leaving the solid phase in the reaction vessel. The antigen-antibody reaction time, which is defined as the time until the washing timing for performing B-F separation, can be changed by changing the arrangement position of the washing device. .

The invention will now be explained in detail with reference to the drawings.

FIG. 1 is a schematic diagram of the enzyme immunoreaction. 1 is
This is an immobilized solid phase in which antibody (or antigen) 2a is immobilized on a solid phase such as a polystyrene ball or glass beads (A). Antigen (or antibody) 2 for antibody (or antigen) 2a is placed on this immobilized solid phase 1.
Serum containing b or a sample corresponding thereto is added and reacted (B). Then, the antigen (or antibody) 2b reacts with the antibody (or antigen) 2a on the immobilized solid phase 1, and the antigen (antibody) - antibody (antigen)
A composite immobilized solid phase 3 is produced (C). An enzyme-labeled antibody (or antigen) 4, which is an enzyme-labeled antibody (or antigen) identical to the antibody (or antigen) 2a immobilized on the immobilized solid phase 1, is added to this complex-immobilized solid phase 3. React (D). This produces an enzyme-labeled antibody (antigen)-antigen (antibody)-antibody (antigen) complex-immobilized solid phase 5, which is a composite of the enzyme-labeled antibody (or antigen) 4 and the complex-immobilized solid phase 3. (E). Excess enzyme-labeled antibody (or antigen) 4 is removed, and the immobilized solid phase 5
Measure the enzyme activity above. This allows the content of antigen (or antibody) in the sample to be determined.

In the present invention, after producing the above-mentioned antigen (antibody)-antibody (antigen) complex-immobilized solid phase 3 and enzyme-labeled antibody (antigen)-antigen (antibody)-antibody (antigen) complex-immobilized solid phase 5, , by washing the solid phase and reaction tube while leaving the solid phase in the reaction tube.
To enable separation to be performed automatically.

It is also defined by the time between the timing (G) when a sample is dispensed into a reaction tube containing a solid phase and the antigen-antibody reaction is started, and the timing (I) when B-F separation is performed by washing. Measure the antigen-antibody reaction time defined by the time between the dispensing of the enzyme-labeled reagent into the reaction tube (J) and the time of B-F separation by washing (L). To make it possible to change the cleaning timing for B-F separation so that it becomes the optimum time according to the item.

FIG. 2 is a partial diagram showing an example of a measuring machine that implements the automatic measuring method of the present invention.

U-shaped tube 6 with a large opening 6a and a small opening 6b
First, an immobilized solid phase 1 is prepared which can be freely taken in and taken out from the large opening 6a and which does not enter the small opening 6b. The immobilized solid phase 1 and reagent or buffer are added through the large opening 6a of the U-shaped tube 6 (F). Next, a predetermined amount of the sample to be measured is added using the sample dispenser 7 (G), and air is blown into the sample through the small opening 6b for stirring to generate the above-mentioned immobilized solid phase 3 (H). After this, large mouth part 6
The immobilized solid phase 3 and the U-shaped tube 6 are washed by applying a washing liquid supply device 8 to a and a washing liquid suction device 9 to the small opening 6b (I). Next, the above-mentioned enzyme-labeled antibody (or antigen), that is, the enzyme-labeled reagent 4, is added to the large opening 6a of the U-shaped tube 6 using the dispenser 10 (J), and air is blown into the small opening 6b for stirring.
The above-mentioned immobilized solid phase 5 is produced (K). After this,
The immobilized solid phase 5 and the U-shaped tube 6 are washed by the washing liquid supply device 8 and the washing liquid suction device 9 (L). Next, in the U-shaped tube 6 containing the immobilized solid phase 5,
A reagent for measuring the enzyme activity on the immobilized solid phase 5 is added using the reagent dispenser 11 (M). After that, air is blown in through the small opening 6b for stirring, and after the reaction is carried out (N), the reaction solution is transferred into the flow cell 13 by the suction tube 12 (O). The absorbance of this reaction solution is measured to determine the enzyme activity on the immobilized solid phase 5. This makes it possible to know the content of antigen (or antibody) 2a in the sample. After the absorbance measurement, the immobilized solid phase 5 remaining in the U-shaped tube 6 is removed by the immobilized solid phase suction device 14 provided at the large opening 6a, and then removed by the cleaning liquid supply device 15 provided at the small opening 6b. A cleaning liquid is supplied into the U-shaped tube 6, and after cleaning, it is removed by the suction device 14.

FIG. 3 is an overall view showing an apparatus for carrying out the automatic measurement method according to the present invention, in order to explain FIG. 2 in more detail.

FIG. 3 is a top view of a thermostatic chamber holding a plurality of U-shaped tubes each having a large opening 6a and a small opening 6b. A reaction tube turret 16 for moving the reaction tubes is provided on the upper surface of the thermostatic chamber, and this turret 16 is a ring-shaped disk that rotates at a predetermined pitch in the direction of the arrow. A fixed amount of solid phase is supplied from the immobilized solid phase supply device 17 to the large mouth portion 6a. Next, the turret 16 is rotated and a fixed amount of sample is added by the sample dispenser 7. This sample dispenser 7
The sample is dispensed from the sample cup 19 of the sampler 18, which rotates at a predetermined pitch in the direction of the arrow, into the large opening 6a of the U-shaped tube by the vertical movement of the syringe 20 and the rotation and vertical movement of the probe 21. It can also be configured to dispense an appropriate reagent or buffer at the same time as or before or after dispensing the sample. 22 is an air pump that sends air to the small opening 6b of the U-shaped tube. Air is blown from the small opening 6b to stir the immobilized solid phase and the sample to cause a complete reaction. Next, the unreacted liquid is discharged from the small opening 6b by the liquid discharge pump 23. At the same time, a cleaning liquid is supplied from the large opening 6a by the cleaning pump 24, and the solid phase and the U-shaped tube are cleaned while the solid phase remains.
- Perform F separation. The cleaning liquid is discharged by a liquid discharge pump 23. A vertically movable disc (not shown) is provided on the upper surface of the mouth part 6b, and when air and liquid are supplied and discharged through the mouth part 6b, this disc descends and the supply/discharge tube is connected to the mouth part 6b. The turret 16 is arranged so that it rises when it rotates. Therefore, B-
The washing position for F separation, that is, the timing of B--F separation can be changed.

Next, the enzyme-labeled antibody is dispensed from the large mouth portion 6a using the dispenser 10. Air is blown into the mouth part 6b from the air pump 22 and stirred in the same manner as described above,
The liquid is discharged by the liquid discharge pump 23. Supplying cleaning liquid from the large mouth part 6a by the cleaning pump 24,
After washing the solid phase and the U-shaped tube while leaving the solid phase, the washing liquid is discharged by the liquid discharge pump 23.

Next, a reagent for measuring enzyme activity is added from the large opening 6a using the reagent dispenser 11, and air is blown into the small opening 6b from the air pump 22 for stirring. Next, the reaction solution is transferred to the flow cell 26 from the small opening 6b by the vacuum pump 25. Absorbance is measured using this flow cell 26. 27 is a light source,
28 is a filter, and 29 is a light receiving element.

After the measurement, the contents of the U-shaped tube are discharged by a suction device 14 that can be tightly connected to the large opening 6a, and the contents of the U-shaped tube are removed from the small opening 6b.
After cleaning the U-shaped tube by supplying the cleaning liquid by the cleaning liquid supply device 15 attached to the holder, the cleaning liquid is removed by the suction device 14.

Although the enzyme immunoreaction was carried out using a U-shaped tube in the above example, it is not limited to the U-shaped tube. It is possible to use a shape that prevents the liquid from being suctioned and discharged from the container, such as a funnel shape or a tube with a mesh-like partition provided inside the tube.

As described above, the automatic enzyme immunoassay method of the present invention is equipped with a cleaning device that can automatically perform B-F separation by cleaning the solid phase while leaving it in the reaction vessel during the analysis. Therefore, reactants and unreacted products can be easily separated, and the entire process can be operated automatically. Furthermore, since this cleaning device has a simple structure, it can be selectively installed at any position during the reaction steps from sample dispensing to final colorimetric determination and other optical measurements, and it is also removable. be. In other words, by changing the timing of B-F separation by washing, it is possible to set the optimal antigen-antibody reaction time according to the measurement item, and one measuring device can be used efficiently. It is also economically advantageous.

[Brief explanation of drawings]

Fig. 1 is a schematic diagram of the enzyme immunoreaction, Fig. 2 is a diagram showing the sequential steps in the automatic measurement method according to the present invention, and Fig. 3 shows the configuration of the measuring machine that implements the automatic measurement method shown in Fig. 2. It is a figure shown in more detail. 1... Immobilized solid phase, 2... Antigen (antibody), 3... Antigen-antibody complex immobilized solid phase, 4... Enzyme-labeled antibody, 5
...Enzyme-labeled antibody-antigen-antibody complex immobilized solid phase,
6... U-shaped tube, 7... Sample dispenser, 8... Washing water supply device, 9... Washing water suction device, 10... Dispensing device, 11...
Reagent dispenser, 12... Suction tube, 13... Flow cell, 14... Immobilized solid phase suction device, 15... Small mouth washing water supply device, 16... Reaction tube turret, 2
2...Air pump, 23...Liquid discharge pump, 24...Washing pump.

Claims (1)

[Claims] 1. When measuring a specific substance in a sample by an antigen-antibody reaction using an enzyme as a labeling substance, a reaction container containing a solid phase on which an antigen or antibody is immobilized is transported along a reaction line. From the timing of dispensing the sample or enzyme-labeled reagent into the reaction vessel using the dispensing device, the solid phase and reaction vessel are washed by the cleaning device placed along the reaction line while leaving the solid phase in the reaction vessel. Therefore, an automatic enzyme immunoassay method is characterized in that the setting of the antigen-antibody reaction time, which is defined as the time until the washing timing for performing B-F separation, is changed by changing the arrangement position of the washing device.