JPH0643163A - Enzyme immunity automatic measurement method - Google Patents

Abstract

PURPOSE:To improve analysis capacity by dispensing a sample or an enzyme labeled reagent into a reaction container and then blowing air into a reaction liquid for accelerating reaction between the sample or the enzyme labeled reagent and a solid phase. CONSTITUTION:An immobilized solid phase 1 and a reagent or a buffer liquid are added from a large aperture part 6a of a U-shaped pipe 6 and then a certain amount of sample to be measured is added by a sample dispenser 7 and air is blown from a small aperture part 6b, thus generating an immobilized solid phase 3. Then, the solid phase 3 and the pipe 6 are washed. Then, an enzyme labeled antibody 4 is added from the large aperture part 6a by a dispenser 10, thus generating an immobilized solid phase 5. After that, the solid phase 5 and the pipe 6 are washed. Then, a reagent for measuring the enzyme activity on the solid phase 5 is added into the pipe 6 including the solid phase 5 by a reagent dispenser 11. Then, air is blown from the small aperture part 6b before agitation and reaction and then only the reaction liquid solution is transferred into a flow cell 13 by a suction tube 12. The absorbance of the reaction liquid solution is measured, thus obtaining the enzyme activity on the solid phase 5.

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme immunoassay method. [0002] Conventionally, in the enzyme immunoassay method, an enzyme is used as a labeling substance, and a solid phase on which an antigen or an antibody is immobilized is contained in order to measure a specific substance in a sample by an antigen-antibody reaction. After the sample or enzyme-labeled reagent is dispensed into the reaction vessel and the solid phase reacts with the sample or enzyme-labeled reagent, the substance bound by the reaction (Boun
d) Separation of substances not bound to (Free) (BF separation)
Then, the enzyme activity on the solid phase is measured to measure the antigen or antibody in the sample. In the conventional enzyme immunoassay method, since the solid phase is transferred from the reaction container to another container in order to carry out BF separation, the number of reaction containers required is increased. However, the BF separation cannot be performed efficiently and automatically, the measuring machine becomes large, and the solid phase transfer mechanism becomes complicated. Further, since the reaction between the solid phase and the sample or the enzyme-labeled reagent cannot be performed efficiently, there is a drawback that the reaction time becomes long and the processing ability becomes low. The present invention promotes the reaction between a sample dispensed in a reaction container or an enzyme-labeled reagent and a solid phase to improve the analytical ability, and performs BF separation after carrying out an antigen-antibody reaction by a washing device. It is an object of the present invention to provide an enzyme immunoassay method capable of automatically performing enzyme immunoassay by performing the method. According to the present invention, an enzyme is used as a labeling substance, and when a specific substance in a sample is automatically measured by an antigen-antibody reaction, a solid phase on which an antigen or an antibody is immobilized is used. After the sample or enzyme-labeled reagent is dispensed into the containing reaction vessel, air is blown into the reaction solution to accelerate the reaction between the solid phase that proceeds in the reaction vessel and the sample or enzyme-labeled reagent, and then the reaction vessel After injecting the cleaning liquid from the upper opening of the reactor and sucking and discharging the liquid in the reaction container to the outside of the reaction container to wash the solid phase and the reaction container while leaving the solid phase in the reaction container to remove unbound substances, the reaction It is characterized by measuring the subsequent enzyme activity. In the enzyme immunoassay method of the present invention as described above, after a sample or an enzyme labeling reagent is dispensed into a reaction container containing a solid phase, air is blown into the reaction solution to perform stirring. These reactions can be promoted, a sufficient reaction can be performed in a short reaction time, and BF separation can be performed by injecting a cleaning solution with the solid phase kept in the reaction container and sucking and discharging. Therefore, the number of reaction vessels used can be reduced. The present invention will now be described in detail with reference to the drawings.
FIG. 1 is a schematic diagram of enzyme immunoreaction. Reference numeral 1 is an immobilized solid phase in which an antibody (or antigen) 2a is immobilized on a solid phase such as polystyrene balls or glass beads (A). A serum containing an 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, the antigen (or antibody) 2b reacts with the antibody (or antigen) 2a on the immobilized solid phase 1 to generate an antigen (antibody) -antibody (antigen) complex-immobilized solid phase 3 (C). Antibodies (or antigens) immobilized on the solid phase 1 on which the complex is immobilized
An enzyme-labeled antibody (or antigen) 4 obtained by labeling an enzyme with the same antibody (or antigen) as 2a is added and reacted (D). Thus, the enzyme-labeled antibody (or antigen) 4
And an oxygen-labeled antibody (antigen) -antigen (antibody) -antibody (antigen) complex-immobilized solid phase 5 which is a complex of
Is generated (E). Excess enzyme-labeled antibody (or antigen) 4 is removed, and the enzyme activity on the immobilized solid phase 5 is measured. By this, the content of the antigen (or antibody) in the sample can be known. In the present invention, the sample or the enzyme labeling reagent is added to the reaction vessel containing the immobilized solid phase 1 and air is blown into the reaction solution to carry out an antigen-antibody reaction to cause the complex immobilized solid phase 3 or 5 to react. Is generated, the cleaning liquid is supplied to the reaction container, and then the cleaning liquid is sucked and discharged to perform BF separation while leaving the solid phase 3 or 5 in the reaction container. FIG. 2 is a partial view showing an embodiment of an automatic measuring machine for carrying out the enzyme immunoassay automatic measuring method of the present invention. In the U-shaped pipe 6 having the large mouth portion 6a and the small mouth portion 6b, the large mouth portion 6
An immobilized solid phase 1 that can be freely taken in and out from a and does not enter the foremost portion 6b is prepared. The immobilized solid phase 1 and the reagent or buffer solution are added from the large opening 6a of the U-shaped tube 6 (F). Next, a fixed amount of a sample to be measured is added by the sample dispenser 7 (G), and air is blown from the small opening 6b to stir to generate the immobilized solid phase 3 (H). After this, the large portion 6a
The cleaning liquid supply device 8 and the cleaning liquid suction device 9 are applied to the small portion 6b to clean the immobilized solid phase 3 and the U-shaped tube 6 (I). Next, the above-mentioned enzyme-labeled antibody (or antigen)
4 is added from the large opening portion 6a of the U-shaped tube 6 by the dispenser 10 (J), and air is blown from the small opening portion 6b to perform stirring to generate the above-mentioned immobilized solid phase 5 (K). After that, the immobilized solid phase 5 and the U-shaped tube 6 are cleaned by the cleaning liquid supply device 8 and the cleaning 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 by a reagent dispenser 11 (M). After that, air is blown from the small portion 6b to perform stirring to carry out the reaction (N), and then only the reaction solution is flown by the suction tube 12 to the flow cell 13
Transfer inside (O). Measure the absorbance of this reaction solution,
The enzyme activity on the immobilized solid phase 5 is determined. Thereby, the content of the antigen (or antibody) 2a in the sample can be known. Further, after the measurement of the absorbance, the immobilized solid phase 5 remaining in the U-shaped tube 6 is removed by the immobilized solid phase suction device 14 provided in the large mouth portion 6a, and the cleaning liquid supply device 15 provided in the small mouth portion 6b.
The cleaning liquid is supplied into the U-shaped tube 6 by means of, and after cleaning, it is removed by the suction device 14. FIG. 3 is a general view showing an embodiment of an automatic measuring machine for carrying out the enzyme-linked immunosorbent automatic measuring method according to the present invention in order to explain FIG. 2 in more detail. FIG. 3 is a view from above of a thermostat for holding a plurality of U-shaped tubes having a large mouth portion 6a and a small mouth portion 6b. A reaction tube turret 16 for moving the reaction tube is provided on the upper surface of the constant temperature bath, and the turret 16 is a ring-shaped disc 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 the sample dispenser 7 adds a fixed amount of sample. The sample dispenser 7 samples a sample from a sample cup 19 of a sampler 18 that rotates at a predetermined pitch in the direction of an arrow by vertically moving a syringe 20 and rotating and vertically moving a probe 21 so that a large mouth portion 6a of a U-shaped tube is formed. Dispense into. A suitable reagent or buffer may be dispensed at the same time as or before or after the dispensing of the sample. An air pump 22 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 completely react. Next, the unreacted liquid is discharged from the small portion 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, and BF separation is performed. The cleaning liquid is discharged by the liquid discharge pump 23. A disc (not shown) that moves up and down is provided on the upper surface of the small portion 6b, and when supplying and discharging air and liquid through the small portion 6b, the circular plate descends to connect the supply / discharge tube to the small portion 6b. And is arranged so as to rise when the turret 16 rotates. Next, the enzyme labeling reagent is dispensed by the dispenser 10.
Dispense from the large portion 6a. In the same manner as described above, air is blown from the air pump 22 into the small opening 6b to stir, and the liquid is discharged by the liquid discharge pump 23. The cleaning liquid is supplied from the large opening 6a by the cleaning pump 24, the solid phase and the U-shaped tube are cleaned while leaving the solid phase, and then the cleaning liquid is discharged by the liquid discharge pump 23. Next, the reagent dispenser 11 is used to
A reagent for measuring the enzyme activity is added from a, and air is blown from the air pump 22 into the small portion 6b and stirred. Then, the reaction solution is transferred to the flow cell 26 by the decompression pump 25 from the small portion 6b. The absorbance is measured by 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 pipe are discharged by the suction device 14 which can be hermetically connected to the large mouth portion 6a, and the cleaning liquid is supplied by the cleaning liquid supply device 15 attached to the small mouth portion 6b to wash the U-shaped pipe. The cleaning liquid is removed by the suction device 14. Although the U-shaped tube is used for the enzyme immunoreaction in the above-mentioned embodiment, the present invention is not limited to the U-shaped tube and various reaction vessels can be used. As described above, according to the present invention, in a state where the solid phase is contained in the reaction container, all the reactions and solid phases necessary for sample analysis can be obtained without changing the reaction container. It is possible to carry out a series of operations such as removal of unbound substances, and it is possible to reduce the number of reaction vessels required for analysis. This makes it possible to reduce the size of the enzyme immunoassay analyzer and from vessel to vessel. It is possible to omit the complicated solid phase transfer operation. Furthermore, in the present invention, the reaction between the solid phase and the sample or reagent is promoted by blowing air, so that the reaction time is shortened and the processing capacity per unit time can be improved.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of enzyme immunoreaction. FIG. 2 is a diagram showing sequential steps in the enzyme-immunoassay automatic measuring method according to the present invention. FIG. 3 is a diagram showing the configuration of an embodiment of an apparatus for carrying out the enzyme-immunoassay automatic measuring method of the present invention. [Explanation of symbols] 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-tube 7 sample Injection device 8 Wash water supply device 9 Wash water suction device 10 Dispenser 11 Reagent dispenser 12 Suction tube 13 Flow cell 14 Immobilized solid phase suction device 15 Wash water supply device for small mouth portion 16 Reaction tube turret 22 Air pump 23 Liquid discharge Pump 24 Cleaning pump

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location G01N 35/06 A 8310-2J

Claims (1)

[Claims] 1. When an enzyme is used as a labeling substance and a specific substance in a sample is automatically measured by an antigen-antibody reaction, the sample or the enzyme is placed in a reaction container containing a solid phase on which an antigen or antibody is immobilized. After the labeling reagent is dispensed, air is blown into the reaction solution to accelerate the reaction between the solid phase and the sample or enzyme labeling reagent that proceed in the reaction vessel, and then the washing solution is injected from the upper opening of the reaction vessel. To measure the enzyme activity after the reaction after removing the non-binding substances by washing the solid phase and the reaction container with the solid phase remaining inside the reaction container by suctioning and discharging the liquid in the reaction container outside the reaction container. An enzyme immunoassay method characterized by: