JPH0120383B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating bound and non-binding substances in an automatic enzyme immunoassay device that uses an enzyme as a labeling substance and measures a specific substance in a sample through an antigen-antibody reaction. be.

In enzyme immunoassay, substances bound by reaction (Bound) and substances not bound (Free)
However, it is difficult to perform this automatically, and automation has been hindered in this respect.

An object of the present invention is to enable enzyme immunoassay to be performed automatically by performing B-F separation using a washing device after antigen-antibody reaction.

The present invention uses an enzyme as a labeling substance to immobilize an antigen or antibody when separating bound substances and non-binding substances in an automatic enzyme immunoassay machine that automatically measures a specific substance in a sample through an antigen-antibody reaction. After dispensing the sample or enzyme-labeled reagent into a reaction container containing the solid phase and performing an antigen-antibody reaction, the reaction container is transported along the reaction line of the analyzer to the washing position, and the reaction container is placed in the washing position. The washing liquid is injected from the upper opening of the reaction vessel in the stopped state, and the liquid inside the reaction vessel is sucked and discharged outside the reaction vessel to wash the solid phase and the reaction vessel while leaving the solid phase inside the reaction vessel. It is characterized by this.

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 polystyrene balls or glass beads (A). Serum containing the antigen (or antibody) 2b against the antibody (or antigen) 2a or a sample corresponding thereto is added to the immobilized solid phase 1 and reacted (B). Then, antigen (or antibody) 2b
reacts with the antibody (or antigen) 2a on the immobilized solid phase 1 to produce an antigen (antibody)-antibody (antigen) complex immobilized solid phase 3 (C). This composite immobilized solid phase 3
Then, 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 and reacted (D). As a result, the enzyme-labeled antibody (antigen) - antigen (antibody) - which is a complex of the enzyme-labeled antibody (or antigen) 4 and the complex-immobilized solid phase 3
An antibody (antigen) complex immobilized solid phase 5 is produced (E).
Excess enzyme-labeled antibody (or antigen) 4 is removed, and the enzyme activity on the immobilized solid phase 5 is measured.
This allows the content of antigen (or antibody) in the sample to be determined.

In the present invention, after adding a sample or an enzyme labeling reagent to a reaction vessel containing the immobilized solid phase 1 and performing antigen-antibody reaction to generate the complex-immobilized solid phase 3 or 5, a washing liquid is supplied to the reaction vessel. Then, by suctioning and discharging the washing liquid, the solid phase 3 is added to the reaction vessel.
Alternatively, BF separation is performed with 5 remaining as it is.

FIG. 2 is a partial diagram showing an embodiment of an automatic measuring device employing the B--F separation 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, add a certain amount of the sample to be measured using the sample dispenser 7 (G), and
Air is blown in and stirred to form the above-mentioned immobilized solid phase 3.
is generated (H), and then the immobilized solid phase 3 and the U-shaped tube 6 are washed by applying a washing liquid supply device 8 to the large opening 6a and a washing liquid suction device 9 to the small opening 6b (I). Next, the enzyme-labeled antibody (or antigen) mentioned above
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 to produce the above-mentioned immobilized solid phase 5 (K). Thereafter, the immobilized solid phase 5 and the U-shaped tube 6 are washed using the washing liquid supply device 8 and the washing liquid suction device 9 (L). Next, a reagent for measuring the enzyme activity on the immobilized solid phase 5 is added into the U-shaped tube 6 containing the immobilized solid phase 5 using the reagent dispenser 11 (M).
After this, air is blown into the small opening 6b for stirring.
After the reaction (N), only 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 is removed by the cleaning liquid supply device 15 provided at the small opening 6b. Pour the cleaning liquid into the U-shaped tube 6.
After cleaning, it is removed by the suction device 14.

FIG. 3 is an overall view showing an embodiment of an automatic measuring machine employing the BF separation 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 mouth portion 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 solution simultaneously with 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 into the small mouth portion 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 leaving the solid phase.
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.

Next, the enzyme labeling reagent 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.

In the above example, the enzyme immunoreaction was carried out using a U-shaped tube, but the U-shaped tube is not limited to this. When the reaction solution is introduced into the flow cell, the solid phase is sucked out of the reaction container along with the reaction solution. It is possible to use a shape that prevents discharge, such as a funnel shape or a pipe with a mesh-like partition provided inside the pipe.

As described above, in the B-F separation method in the automatic enzyme immunoassay device of the present invention, after performing the antigen-antibody reaction, the washing liquid is supplied to the reaction container that is stopped at the washing position, and then the washing liquid is aspirated. B by leaving the solid phase in the reaction vessel by discharging it.
-F separation can be carried out easily and reliably, and therefore an apparatus for automatically measuring enzyme immunity can be realized.

[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 employing the B-F separation method according to the present invention, and Fig. 3 is a diagram showing the B-F separation method according to the present invention. FIG. 2 is a diagram showing in more detail the configuration of the automatic measuring device that was used. 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 A solid phase on which an antigen or antibody is immobilized is used to separate bound and non-bound substances in an automatic enzyme immunoassay machine that uses an enzyme as a labeling substance and automatically measures a specific substance in a sample through an antigen-antibody reaction. After dispensing a sample or an enzyme-labeled reagent into a reaction container containing a sample and causing an antigen-antibody reaction, the reaction container is transported along the reaction line of the analyzer to a cleaning position, and the reaction container is stopped at the cleaning position. , the solid phase and the reaction vessel are washed by injecting a cleaning liquid from the upper opening of the reaction vessel in a stopped state and sucking and discharging the liquid inside the reaction vessel to the outside of the reaction vessel, leaving the solid phase inside the reaction vessel. A method for separating bound and non-binding substances using an automatic enzyme immunoassay system.