JPH07333229A - Automatic immunoassay device - Google Patents

Abstract

PURPOSE:To enhance the washing of a sample in order to enhance the separation accuracy of a bound label and a free label in immunoassay by connecting a disposable reaction tank for heterogenous immunoassay and a vacuum suction port part for excluding a residual soln. CONSTITUTION:A well 2 for holding a soln. for a reaction tank, the fibrous solid phase carrier 5 laid on the bottom surface of the well, the fine hole 4 piercing the bottom surface downwardly and a connection structure 10 are provided to a disposable reaction container piece 1. Treatments such as silane coupling treatment, biotin treatment, avidin treatment and the like are applied to the solid phase carrier 5 and an antibody and an antigen or a substance having the coupling property with a ligand having affinity with respect to them are provided and the connection structure with the fine hole and a vacuum device suction port part 7 is provided. The connection of the reaction tank piece 1 and the suction port part 7 is the fitting of taper surfaces 6, 6' and airtightness is good and the amt. of moisture remaining in a fiber aggregate 5 is easily controlled. By providing a plurality of holes along the peripheral surface of the holding well 2, not only the center part of the aggregate 5 but also the end part thereof can be efficiently washed.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to a heterogeneous substance using a fiber aggregate (hereinafter sometimes referred to as Fiber) as a solid phase carrier for qualitative and quantitative analysis of tumor markers, hormones, viral infectious diseases and the like. The present invention relates to an immunoassay method device.
More specifically, the present invention relates to a sample washing method for improving the accuracy of B / F separation in the immunoassay method and an automatic immunoassay apparatus.

[0002]

2. Description of the Related Art Conventionally, when performing B / F separation in an automatic immunoassay device using a disposable reaction vessel piece or plate with a solid support for a fiber carrier, the liquid is usually chromatographed or the fiber is placed at the bottom of the fiber. An absorber having a higher absorptivity is provided and absorbed. (Tokuhei 5-
59381, special table 5-500116, special table 5-03
772, JP-A-63-281053).

[0003]

However, such a conventional automatic immunoassay device has the following problems. The reaction solution is likely to remain inside the fiber and the replacement with the cleaning solution does not go smoothly, so a large amount of reagents will be mixed in the measured object, increasing the variation in the measured value and increasing the background during measurement. Invite.

[0004] Further, when an immune reaction is repeatedly carried out on the same solid phase and the target immune complex is cumulatively immobilized to obtain high sensitivity, the washing solution at the time of B / F separation of the previous reaction is , And remains in the fiber to dilute the next reaction reagent, which may reduce the Signal yield of the immune complex, which may be an obstacle. Here, “Signal” refers to a detectable signal emitted by the labeling substance.

Further, it is very difficult to remove the substance which is non-specifically adsorbed on the fiber carrier solid phase or the inner wall of the reaction solution holding well by the conventional method and apparatus, and the measured values greatly vary. As a result, the measurement background rises.

The present invention has been achieved as a result of various studies in order to solve such problems, and is an automatic immunoassay device using a fibrous carrier solid phase, particularly for an object to be measured. A configuration of a measuring device effective for cleaning and a suitable cleaning method by the device are presented.

[0007]

The automatic immunoassay device of the present invention is basically constructed by connecting a disposable reaction tank piece and a suction port of a vacuum suction device for removing residual liquid.

The disposable reaction vessel piece used in the present invention comprises a reaction solution holding well, a fibrous carrier solid phase laid on the bottom of the well, a plurality of pores penetrating from the bottom of the well, and the penetration. This is a reaction vessel piece with a connecting structure provided below the part, and glass fiber filter paper is used as the fibrous carrier in this structure, and the solid phase is a silane coupling treatment / biotin on the fibrous solid phase carrier. Conventional treatments such as treatment / avidin treatment, which have binding properties with antibodies, antigens, or ligands having an affinity for them, and those with pores provided along the periphery of the bottom surface of the well A device provided with a structure for connecting to the device suction port is suitable for the present invention.

The connection between the reaction tank piece and the suction port of the vacuum suction device is preferably a fitting connection, and particularly, male-female tapered surface fitting is suitable.

As a method for carrying out an immune reaction using the apparatus of the present invention and washing the object to be measured after the reaction is completed, various procedures can be carried out, but the following step is preferred.

(A) A step of introducing the sample and the labeled reagent solution simultaneously or sequentially into the reaction solution holding well in which the fibrous carrier solid phase is laid, and reacting with the fibrous carrier solid phase, (B) A step of suctioning and removing the residual reaction liquid under reduced pressure through the pores on the bottom of the well, (c) A step of introducing a cleaning liquid into the well, and (d) A vacuuming and removal of the cleaning liquid together with the residual reaction liquid. Step (e) A step of repeating the operations of steps (c) and (d) a plurality of times. In addition, process (b)
In (d) and (d), the liquid is removed under a gentle reduced pressure (0.5 atm or more, preferably 0.9 atm or more) until the solid phase is wet. In the step (e), the same operation is carried out, but it is preferable that only the final suction is carried out rapidly under a high reduced pressure (0.1 atm or less, preferably 0.05 atm or less). By such a two-step suction method, the physical separation of the reaction product from the solid phase can be suppressed.

[0012]

[Action and effect]

1, the connection between the reaction tank piece and the suction port is as shown in the figure.
Since the male and female type tapered surface fitting is performed, the airtightness is improved and the amount of water remaining in the fiber is easily controlled. That is, it becomes easy to keep the suction condition constant. If the suction condition is not kept constant, the reproducibility of the amount of the cleaning liquid remaining in the fiber deteriorates, and the final Signal (signal emitted by the labeling substance, which should be detected) amount varies. Further, if the suction pressure is not made sufficiently strong, the amount of the remaining cleaning liquid becomes large, the next reaction liquid becomes difficult to diffuse, and the amount of Signal decreases.

2. By providing a plurality of holes along the surface of the reaction solution holding well, it is possible to efficiently clean not only the central part of the fiber but also the end part thereof, and the cleaning solution can be reduced. In addition, since the suction condition is also kept constant, F
It is possible to suppress uneven distribution of the cleaning liquid in the iber.

3. Fib is a cleaning solution containing a surfactant.
Bubbles are generated when passing through the er, but in order to efficiently suck the bubbles as well, a shallow recess on the suction port contact surface creates a narrow gap between the reaction tank piece and the suction port fitting part. It was As a result, bubbles are less likely to remain on the bottom surface of the fitting portion of the reaction tank piece and return to the Fiber due to the capillary phenomenon is reduced, and the cleaning can be efficiently performed.

The interval is "narrow", not "contact / contact". This is because, in addition to the reason of cleaning efficiency, if they are brought into close contact with each other, the passage of the liquid is blocked because the reaction tank has a plurality of pores but only one suction port is provided at the center of the suction device. There is also a reason.

Since the cleaning efficiency is very good, the concentration of the reagent in the used cleaning liquid remaining in the suction portion is low. Therefore, the used residual cleaning liquid is less likely to be contaminated even if it returns to the cleaned Fiber together with the generated bubbles by the capillary phenomenon.

4. The reaction waste solution (excess reagent / washing solution) containing unreacted antigen or antibody can be quickly and surely removed by sucking from below the reaction tank piece having the Fiber with a vacuum pump or the like, and the reaction product Only the residue can be left, so the accuracy is improved. Further, the weaker the suction pressure is, the longer the immersion time of the cleaning liquid in the fiber is, and the better the cleaning effect is. Therefore, the pressure at the time of suction is weakened at the beginning and strengthened at the end. By performing such an operation, it is possible to suppress the immune complex trapped in the fiber from being dissociated from the fiber by a physical external force.

5. With the above method and structure, it is possible to perform efficient cleaning with a short time and a small amount of cleaning liquid. Examples will be described below.

[0019]

【Example】

Example 1 A standard curve was prepared using an AFP (α-fetoprotein) measuring system. 1. Materials and Methods An immune complex is formed in a solution using two kinds of antibodies sandwichable with an AFP antigen (each antibody is labeled with an acridinium derivative and biotin), and the Fiber is formed.
The immune complex was added to and the avidin / biotin reaction was caused in the Fiber to trap (immobilize) the immune complex in the Fiber.

Next, the cleaning jig and the avidin-bonded Fiber
Using the illustrated reaction piece in which r is set, the suction port, and the vacuum pump, a cleaning liquid (phosphoric acid Buf containing a surfactant is used).
The excess labeled antibody in Fiber was mechanically aspirated and removed by (fer). Specifically, the cleaning liquid is collected and suctioned,
Suction was repeated for a total of 10 times. The word "reserve" used here is "suction from the bottom, but since the suction pressure is weak, the amount of input liquid and the amount of discharge liquid do not change,
The amount of liquid in the well does not change ”. On the other hand, the cleaning liquid was chromatographed in the system which did not mechanically suck. Finally, the acridinium derivative was made to emit light by adding a luminescence induction buffer and a trigger.

2. Results Table 1 shows data of the system without mechanical suction, Table 2 shows data of the system with mechanical suction, and Table 3 shows a comparison between the two. As can be seen from these data, in the system that performs mechanical suction, there is little variation in the data and a highly sensitive calibration curve is obtained, whereas in the system that does not perform mechanical suction, the variation in the data is significantly large. And the sensitivity decreased.

Effect of presence or absence of mechanical suction on assay system
AFP (relationship between antigen concentration and luminescence)

[Table 1] Without suction

[0023]

[Table 2] With suction

[0024]

[Table 3] AFP STANDARD Aspiration (with) and (without) during cleaning

Example 2 Using a FER (ferritin) measurement system, BG (background: luminescence independent of immune complex, 0 ng / m 2)
l) and Signal (immune complex dependent luminescence, 5
00 ng / ml) was measured.

1. Materials and Methods An immune complex is formed in a solution using a FER antigen and two kinds of antibodies capable of sandwiching the antigen (each antibody is labeled with an acridinium derivative or biotin). However, BG has no antigen. After that, the immune complex is added to the fiber to which avidin is bound, and the avidin-biotin reaction is caused to cause the fiber to trap the immune complex. Next, using a cleaning jig, the illustrated reaction vessel piece in which the avidin-bound fiber is set, the suction port, and the vacuum pump, excess labeled antibody in the fiber is sucked and removed with the cleaning liquid. The cleaning method is the same as in Example 1.

During cleaning, air is leaked in the pipe to adjust the suction pressure, and the suction pressure is weakened. After washing, F
In order to always keep the amount of water remaining in the iber constant, suction is performed with a system that does not leak, that is, with a strong pressure. Also, as a control, a system that does not leak is placed.

Finally, the acridinium derivative is made to emit light by adding a luminescence induction buffer and a trigger to both.

2. Results Example of suppression of dissociation of immune complex by two-step suction method
(Signal, NSB) are shown in Table 5 (SN ratio). By setting the suction pressure at the initial stage to a weak pressure of 0.9 atm or more, it has become possible to obtain an SN ratio that is at least twice the current level.

[0030]

[Table 4] Inhibition of dissociation of immune complex Two-step aspiration

[0031]

[Table 5]

[Brief description of drawings]

FIG. 1 is a plan view of a disposable reaction tank piece.

FIG. 2 is a vertical sectional view taken along the line A-A ′ of the disposable reaction tank piece.

[Fig. 3] External view of the suction port of the vacuum suction device

FIG. 4 is a vertical sectional view of a main part of a connection structure of a disposable reaction tank piece and a suction port of a vacuum suction device.

[Explanation of symbols]

 1 Disposable Reaction Tank Piece 2 Reaction Solution Retaining Well 3 Well Bottom 4 Pore 5 Fibrous Carrier Solid Phase 6, 6'Fitting Tapered Surface 7 Decompression Suction Device Suction Port 8 Suction Port Shallow Dimple 8 ' Narrow gap at the time of connection 9 Suction port 10 Structure for connecting the reaction tank piece side

Claims (10)

[Claims] 1. An automatic immunoassay device comprising a disposable reaction tank piece (1) and a vacuum suction device suction port (7) for removing residual liquid. 2. A disposable reaction vessel piece (1) is provided with a reaction solution holding well (2), a fibrous carrier solid phase (5) laid on the well bottom surface (3), and below the well bottom surface (3). 2. The apparatus according to claim 1, which is a reaction vessel piece comprising a plurality of pores (4) penetrating to each other and a connecting structure (10) provided at a lower portion of the penetrating portion. 3. The device according to claim 2, wherein the fibrous carrier is a glass fiber filter paper. 4. Device according to claim 2, characterized in that the fibrous carrier solid phase (5) comprises means for binding antibodies, antigens or ligands having an affinity for them. 5. The method according to claim 2, characterized in that a plurality of pores (4) penetrating downward from the bottom surface (3) of the well are provided along the periphery of the bottom surface (3). Equipment. 6. The measure according to claim 1, characterized in that the connection is a mating connection. 7. Device according to claim 1, characterized in that the mating connection is a male-female connection with tapered surfaces (6, 6 '). 8. The device according to claim 6 or 7, wherein a shallow recess (8) is provided on the contact surface of the suction port portion (7), and a narrow interval (8 ') is formed at the time of fitting. . 9. (a) The reaction solution holding well (2) on which the fibrous carrier solid phase (5) is laid, is charged with the sample and the labeled reagent solution simultaneously or sequentially to form a fibrous carrier. A step of reacting with a carrier solid phase (5), (b) a bottom surface (3) of the well
Through the pores (4) of (4) to suction and remove the reaction residual liquid under reduced pressure, (c) the step of introducing the cleaning liquid into the well (2), (d) the cleaning liquid together with the reaction residual liquid, and the step (b). 9. The immune reaction and the washing of the object to be measured after the reaction are carried out by repeating the steps of (e) step (c) and (d) a plurality of times in the same manner. How to operate the device. 10. Only the final suction operation in the step (e) is set to 0.
10. The high pressure reduction of 1 atm or less, preferably 0.05 atm or less, and the other suction operation at 0.5 atm or more, preferably 0.9 atm or more.
How to operate the described device. [0001]