JPH0655084A - Reaction vessel for immunoassay - Google Patents

Abstract

PURPOSE:To provide the reaction vessel for immunoassay which can make immunoassay with higher sensitivity and efficiency by using relatively few samples and reagents. CONSTITUTION:This reaction vessel for immunoassay consists of reaction parts 12 and a moving body holding the plural reaction parts 12. Each of the reaction parts 12 is constituted by disposing two sheets of flat plate parts 21, 22 opposite to each other via two spacer parts 23, 24 disposed at both edges of the flat plate parts 21, 22, thereby forming a space 25 where the sample and reagent can be sucked and held by capillarity. The reaction parts 12 are so held and moved by the moving body as to face the flat plate parts 21.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a reaction container for immunoassay.

[0002]

2. Description of the Related Art Conventionally, as an immunoassay reaction container, for example, a microplate as described in US Pat. No. 4,303,616 has been used. In the immunoassay using a microplate, first, the antigen corresponding to the antibody to be measured and the antibody corresponding to the antigen are immobilized on the bottom of the well, and then the antibody to be measured or a sample containing the antigen is added. And cause an antigen-antibody reaction. Then, after adding a labeled antigen or labeled antibody and performing B / F separation, a substrate solution is added to cause an enzyme-substrate reaction, and the enzyme activity generated at this time is measured to detect the antigen or antibody. Quantify.

The enzyme activity can be generally measured by capturing the enzyme-substrate reaction between the enzyme-labeled antibody and the substrate using a luminescent substrate or a chromogenic substrate. When using a chemiluminescent substrate, a photoreceptor (eg, photomultiplier) above the open well.
Is installed and the intensity of chemiluminescence from the reaction part is measured.
Further, when a color-developing substrate that chemically develops color is used, the absorbance of the sample subjected to the immunoreaction can be measured.

Further, as a reaction container for immunoassay, JP-A-62-169054 and JP-A-62-16 are used.
No. 9055 uses a reaction vessel having a thin tubular shape with an inner diameter of 2 mm or less, and an antigen or antibody is immobilized on the inner wall, and particles immobilized with the antigen or antibody are placed in the capillary. It is described that an immunoassay is performed by adding a sample containing an antigen or an antibody and causing an antigen-antibody reaction.

In addition, the applicant of the present invention, in the gap between the two flat plates,
A reaction container for performing immunoassay utilizing an immunological agglutination reaction is proposed, in which a sample introduction path and a reaction part capable of sucking and holding a sample by a capillary phenomenon are formed (Japanese Patent Application No. 2-418978 and Japanese Patent Application No. 2-418979
issue).

[0006]

However, in the above-mentioned microplate, an antibody or an antigen (hereinafter referred to as a specific reaction substance) corresponding to the substance to be measured is immobilized on the bottom of the well. In order for an immune reaction to occur, it is necessary for the substance to be measured and the specific reaction substance to meet each other. However, in the microplate, since the specific reaction substance is immobilized only on the bottom of the well, the substance to be measured floating above the sample is unlikely to bind to the specific reaction substance. Therefore, it is necessary to lengthen the incubation time. In addition, extra samples and reagents necessary for measurement are also consumed.

[0007] Further, when the enzyme activity is measured by measuring chemiluminescence, the chemiluminescence emission intensity is weak, and therefore it is necessary to bring the photodetector as close as possible to the liquid surface of the sample to improve the measurement sensitivity. However, because of the structure of the microplate, the photodetector cannot be brought very close to the liquid level, and highly sensitive measurement cannot be achieved.

[0008] Further, in the above-mentioned thin tubular reaction container for immunoassay, when the specific reaction substance is immobilized on the inner wall surface, the inner diameter is relatively small, so that the substance to be measured and the specific reaction substance encounter each other. Is increased, and the amount of sample and reagent used can be reduced, which is excellent. However, when measuring the intensity of chemiluminescence with a photodetector, the inner diameter of the reaction vessel is 2 mm.
Below, the light-receiving surface of the photodetector is too large for the surface area of the sample that is emitting light. Therefore, a lot of noise is picked up in the measurement signal, and the S / N is remarkably reduced, so that highly sensitive immunoassay cannot be performed.

Further, in the immunoassay reaction container used in the immunoassay utilizing the immunological agglutination reaction, the area of the reaction part is reduced in order to shorten the time required to precipitate the particles produced by the agglutination reaction. Is narrowing. However, narrowing the area of the reaction part is not preferable because it reduces the measurement sensitivity when such a reaction container is used for immunoassay utilizing chemiluminescence. Further, it is difficult to wash the microplate and the thin tubular reaction vessel due to their structure, and a special washing device is required for washing. Although the case where the chemiluminescence is measured to perform the immunoassay has been described above, there is a similar problem in the case where the immunoassay is performed by utilizing the color development reaction in the conventional immunoassay.

The present invention has been made in view of the above circumstances, and provides a reaction container for immunoassay which can perform immunoassay with higher sensitivity and efficiency by using relatively few samples and reagents. It is a thing.

[0011]

The present invention is a reaction container for immunoassay for measuring the reaction between a substance to be measured and a specific reaction substance in a sample, which comprises two flat plate portions. A reaction part formed by facing each other through two spacer parts arranged on both edges of the plate and forming a gap capable of sucking and holding the sample and the reagent by capillary action, and the reaction part on one plate. A reaction container for immunoassay, which is provided with a moving body which is held so as to face the section and moves the reaction section. Here, it is preferable that the moving body holds the reaction part such that the open end of the reaction part is located in a direction different from the moving direction of the reaction part.

Further, the moving body is configured to move the reaction section along an endless reaction line, and the light transmitted through the reaction section is guided to the inside of the reaction line. preferable.

[0013]

According to the container for immunoassay of the present invention, the two flat plate portions and the two spacer portions form a relatively thin and wide flat plate-shaped reaction portion. Thereby, chemiluminescence and chemical color development that occur inside the reaction section can be measured through the light-transmissive flat plate section. Therefore, since the light receiving surface of the photodetector can be arranged relatively close to the flat plate portion, a sufficient amount of received light or transmitted light can be obtained even with a small amount of sample. Further, since the light receiving surface of the photodetector and the area of the flat plate portion can be matched, the S / N can be increased.

Further, since the thickness of the gap is extremely thin, when the specific reaction substance is immobilized on the inner wall surface of the reaction vessel, the chances that the substance to be measured in the sample and the specific reaction substance come into contact with each other increase. The immunological reaction can be performed in a short time. Further, since the sample and the reagent are suctioned and held inside the reaction part by the capillary phenomenon, it is possible to easily supply the sample and the like and wash the reaction part. Further, since the reaction part is held by the moving body, automation of immunoassay becomes easier.

[0015]

Embodiments of the present invention will now be described in detail with reference to the drawings.

FIG. 1 is an explanatory view showing an example of the immunoassay reaction container of the present invention. In the figure, 11 is a moving body having a substantially rectangular parallelepiped shape. The moving body 11 is movable along the longitudinal direction. Six reaction parts 12 are held at predetermined intervals on the side surface of the moving body 11.

As shown in FIG. 2, the reaction section 12 includes two substantially rectangular flat plate portions 21 and 22 which are connected to each other via spacers 23 and 24 of substantially prismatic shapes arranged at both edges. The main surfaces are arranged substantially parallel to each other. Flat plate part 2
1, 22 and the spacer portions 23, 24 are bonded to each other with an adhesive, for example. With such a configuration, a gap 25 is formed inside the reaction section 12.

Here, the sizes and intervals of the flat plate portions 21 and 22 are set so that the sample and the reagent move efficiently in the gap 25 by the capillary phenomenon. Flat plate portions 21, 22
The size of the piece is preferably within the range of 10 to 50 mm,
The range of 10 to 30 mm is particularly preferable. Also, the flat plate portion 2
The distance between 1 and 22 is preferably in the range of 0.1 to 1 mm, for example. In the present embodiment, the flat plate portion 2 is used as the reaction portion 12.
1, 22 were 25 mm x 25 mm x 1 mm, and a reaction part with a spacing of 0.3 mm was used. When a sample or a reagent is supplied to the open end 12a above the reaction section 12 by a pipette or the like, the sample or the like is sucked and held in the gap 25 due to a capillary phenomenon.

As a modified example of the reaction part, as shown in FIG. 3 (A), the reaction part 31 in which a flat plate part and a spacer part are integrally molded, and as shown in FIG. A circular reaction portion 32 can be exemplified. In particular,
The latter well matches the shape of the light receiving surface of the photodetector, which is usually circular, and can efficiently detect the light emission. Furthermore, as shown in FIG. 4, foot portions 42 and 43 may be attached to both edge portions of the end portion of the reaction portion 41, respectively. The reaction section 41 can be placed in a gap because the end of the reaction section 41 can be floated from the work table by placing it on the work table or the like with the legs 42 and 43 facing down during the measurement operation. The sample and reagents that were allowed to flow do not flow out.

When the immunoassay is performed by measuring chemiluminescence, at least one of the flat plate portions 21 and 22 is made of a light transmissive material such as glass, polystyrene or vinyl chloride. Further, in order to further enhance the measurement sensitivity of light emission, the other flat plate portion may be made of a light-reflecting material such as aluminum, or the outer or inner surface of the other flat plate portion may be vapor-deposited with aluminum or the like. preferable. Further, in order to analyze a plurality of samples or measurement items simultaneously or successively, a plurality of reaction parts connected with a spacer interposed may be prepared and used.

As shown in FIG. 5, the reaction section 12 as described above is held so that one of the flat plate sections 21 and 22 and the main surface of the movable body 11 face each other. Reaction part 12
The open end portion of is held so as to face a direction substantially perpendicular to the moving direction of the moving body 11.

At this time, if any of the flat plate portions 21 and 22 is made of a light-reflecting material or the light-reflecting material is vapor-deposited as described above, this light-reflecting flat plate portion is used. The main surface of the moving body 11 is opposed to each other and held so that the light-transmissive flat plate portion faces the photomultiplier 51.

The moving body 11 is a photomultiplier 5
The main surface of the reaction unit 12 is arranged to be substantially parallel to the light receiving surface of No. 1 and moves along the longitudinal direction of the moving body 11 to sequentially detect the reaction unit 12 in front of the photomultiplier 51 at the detection position. Move to.

As shown in FIG. 6 (A), the reaction part is held by forming a light-transmitting window 62 in a predetermined region of the non-light-transmissive moving body 61, and the reaction part on one main surface of the moving body 61. It is also possible to hold 63 with the light reflecting layer 64 on the outside and arrange the photomultiplier 65 on the other main surface side of the moving body 61. Further, as shown in FIG. 6B, a light transmitting portion 67 made of a light transmitting material is provided in a predetermined region of the non-light transmitting moving body 66.
Is provided on one main surface of the moving body 66, and the flat plate portion 68 having the light reflecting layer 69 is provided outside the light reflecting layer 69 and at a predetermined distance from the light transmitting portion 67. Light part 6
It is also possible to arrange so that a gap 68a capable of absorbing and holding a sample or the like is formed between the seven by means of a capillary phenomenon, and the photomultiplier 65 can be arranged on the other main surface side of the moving body 66. In this case, since one of the flat plate portions is also used as the light transmitting portion 67, the material can be saved. The transparent window 62 and the transparent portion 67 are formed in a shape similar to the shape of the light receiving surface of the photomultiplier 65.

As described above, in the case where the reaction section and the photomultiplier 65 are provided on the opposite sides of the moving bodies 61 and 66, the sample and the reagent are placed on the open end 63a above the reaction section. In the case of automatically supplying to 68a, the arrangement and operation of the means for supplying the sample or reagent need not be disturbed by the photomultiplier 65. Next, the case where the measurement by the chemiluminescence reaction is performed by using the immunoassay reaction container 10 having such a configuration will be described.

First, a specific reaction substance such as an antibody or an antigen is immobilized on the inner surfaces of the flat plates 21, 22 by physical adsorption or the like. Next, the sample is injected into the reaction section 12. After that, incubation is performed to react the substance to be measured in the sample with the specific reaction substance, and the substance to be measured is bound to the specific reaction substance. Then, the inside of the reaction part 12 is washed with a buffer solution.

After washing, an enzyme-labeled antibody or an enzyme-labeled antigen is added, incubation is carried out, and washing with a buffer solution is carried out again. After this, a chemiluminescent substrate is added and incubation is performed. Then, the luminescence intensity due to the reaction between the enzyme and the chemiluminescent substrate is measured. The emission intensity is measured by the photodetector 70 shown in FIG. That is, the photodetector 7
1 (for example, photomultiplier 51 shown in FIG. 5)
After amplifying the photoelectron pulse obtained from
It is passed through a discriminator 73 and compared with a preset reference voltage, and a pulse within a certain level range is output to discriminate the wave height. And discriminator 7
The output pulse of 3 is passed through a waveform shaping circuit 74 to perform waveform shaping, and is guided to a counter 75 to perform photon counting. Then, for example, the number of pulses per second is counted and displayed or printed as a measured value. here,
The immunoassay reaction container 10 is detachably attached to the photodetector 70 so that it can be replaced.

The chemiluminescent assay is carried out by moving the moving body 11 to sequentially move the reaction section 12 to the front of the photodetector 71, and continuously perform the sample held in each reaction section 12. Since the luminescence intensity measured in this manner depends on the concentration of the substance to be measured, the concentration of the substance to be measured in the sample can be quantitatively obtained from the luminescence intensity.

FIG. 8 shows a calibration curve obtained by measuring 100 μl of a sample containing mouse IgG antigens at various concentrations according to the above-mentioned immunoassay method. Here, the two-step sandwich method was used. Further, as the labeling reagent,
A mouse anti-human IgG antibody labeled with alkaline phosphatase was used, and AMPPD (dioxetane derivative) was used as a chemiluminescent substrate. The antigen-antibody reaction was carried out at room temperature for 60 minutes in the first incubation, 60 minutes in the second incubation, and 20 minutes in the enzyme-substrate reaction.

As a comparative example, the same sample 100 is used.
FIG. 9 shows a calibration curve obtained by measuring the mouse IgG concentration in the same manner as in this example except that the conventional micro titer plate was used for μl.

From these results, the immunoassay reaction container 1
It was confirmed that 0 has the simple operability as described above, enables efficient analysis, and obtains the same sensitivity as the conventional microplate-based measurement method.

Further, FIG. 10 shows a reaction container 10 for immunoassay.
The calibration curve which measured HBs antigen on the same measurement conditions using is shown. The HBs antigen is a Tris buffer (pH
Diluted to various concentrations according to 7.6). Both the solid phase antibody and the labeling antibody are mouse anti-human HBs antibodies. In this case, it was confirmed that highly sensitive detection of 1 to 10 ng / ml can be realized.

As described above, the immunoassay reaction container 10
According to the method, chemiluminescence generated inside the reaction part 12 can be measured via the flat plate parts 21 and 22. Therefore, the photodetector 7
Since one light receiving surface can be arranged relatively close to the flat plate portions 21 and 22, a sufficient amount of received light or transmitted light can be obtained even with a small amount of sample. Further, the light receiving surface of the photodetector 71 and the flat plate portion 2
Since the areas of 1 and 22 can be adapted, the S / N can be increased.

Since the thickness of the gap is extremely thin, when the specific reactive substance is immobilized on the inner surfaces of the flat plate portions 21 and 22, the substance to be measured in the sample and the specific reactive substance come into contact with each other. And the reaction can be carried out in a short time.

Further, since the sample and the reagent are suctioned and held inside the reaction section 12 by the capillary phenomenon, it is possible to easily supply the sample and the like and wash the reaction section 12. Furthermore, since the reaction unit 12 is held by the moving body 11, automation of immunoassay becomes easier.

Next, another embodiment of the moving body will be described. First, as shown in FIG. 11, a substantially cylindrical moving body 76
The one that holds the reaction portion 77 at a predetermined interval on the outer surface can be cited. The moving body 76 rotates about the central axis and moves the reaction part 77 in the circumferential direction. In this case, by providing the moving body 76 with a light-transmitting window or a light-transmitting portion and disposing the photomultiplier inside the moving body 76 as described above, there is an advantage that the entire apparatus can be downsized.

Using such a moving body 76,
The case where the sample is held will be described. here,
As the reaction portion 77, for example, a polystyrene flat plate portion having a thickness of 1 mm and a size of 25 mm and facing each other with an interval of about 0.3 mm is used. As a sample, 100 μl of a solution (pH 7.4) in which 0.3% bovine serum albumin (BSA) was added to Tris buffer was used. First, the sample was dropped on the upper open end of the reaction part 77 using a drip pipette to hold the sample in the reaction part 77. After that, the moving body 76 was rotated while gradually increasing the number of rotations, and the holding state of the sample was observed. As a result, 1 per minute
It was found that the sample did not scatter up to 500 rotations. However, since the holding capacity of the sample varies depending on the material of the flat plate portion, the gap distance, the inclination angle of the moving body, or the moving speed, it is preferable to appropriately change the conditions or the supply means for the sample or the arrangement of the photodetector. .

Further, as shown in FIG. 12, an endless belt-shaped moving body 81 which holds the reaction portion 82 on the outer side surface thereof and moves the reaction portion 82 laterally may be used. good. In this case, the open ends 82a, 8 of the moving body 82
Since 2b is oriented horizontally, the open ends 82a, 8
A sample or the like can be supplied from either 2b.

Further, as shown in FIG. 13, a disk-shaped moving body 91 may be used, in which reaction portions 92 are arranged at a predetermined interval on the peripheral portion thereof. Here, the open ends of the reaction parts 92 are arranged so as to face the center and the outer periphery of the moving body 91, respectively. The moving body 91 rotates about the rotation shaft 93 and rotates the reaction section 92 in the circumferential direction. in this case,
Since it is preferable that the multiplier can be arranged above to receive light and the width is not wide in the vertical direction, even if the photomultiplier above or below is arranged, the size of the apparatus is not so large. In addition, a container 10 for measuring an immune reaction
By disposing the dispensing means (not shown) for dispensing the sample and the photodetector on the upper side or the lower side of the moving body 91, respectively, it is possible to set the layout so that the operations are not disturbed.

In such a configuration shown in FIG. 13, first,
Relatively slow speed, eg 0.2-2.0 per minute
The movable body 91 is rotated once, and a predetermined amount of the sample, the reagent or the cleaning liquid is sequentially and continuously dispensed to each reaction part 92 by the dispensing means at the position where the sample is dispensed. Next, after the incubation for a required time, the rotation speed is increased to drain from the end portion on the outer peripheral side of the reaction section 92 and drained into a drainage container (not shown). As shown in FIG. 13, when the reaction part 92 is arranged so that the flat plate part is horizontal, the reaction part 92 is arranged below the moving body 91, and the reaction part 92 is moved from the tip part of the pipette of the dispensing means. The sample can be injected or spotted in the gap. Furthermore, if the upper and lower surfaces of the moving body 91 are both reflective surfaces and the reaction portions 92 with the flat plate portions horizontal are arranged on the upper and lower surfaces, the processing efficiency is doubled.

Further, as shown in FIG. 14, the disk portion 10
1 is a moving body 100 having an inclined portion 102 that is inclined outward at the peripheral edge of the reaction unit 1.
A radial arrangement of 3 may be used. Here, the reaction part 1
The open ends of 03 are arranged so as to face the center and the outer periphery of the moving body 100, respectively. Inclined part 102 Reaction part 10
A light-transmitting region 104 is formed in a region corresponding to 3. The translucent region 104 may be either the translucent window 62 or the transmissive portion 67 shown in FIGS. 6A and 6B. The inclination angle of the inclined portion 102 maintains the balance between the gravity of the sample and the centrifugal force due to the rotation of the moving body 100 in order to prevent the sample and the like held inside the reaction portion 103 from dripping. To be set. In this case, the drive means 106 is connected to the center of the disc portion 101 via the rotary shaft 105,
Further, the rotation control means 107 is electrically connected to the driving means 106. Further, the multiplier 108 is arranged below the inclined portion 102. The rotation control unit 107 controls the driving unit 106 to rotate the moving body 100 at a constant speed so that the reaction unit 102 can be sequentially passed over the light receiving surface of the multiplier 108.

Here, the holding state of the sample varies depending on the volume and viscosity of the sample, the distance between the flat plate portions, and the inclination angle of the inclined portion 102. Therefore, depending on these conditions and the arrangement of the sample supplying means and the light detecting means. It is preferable to appropriately change the rotation speed.

[0043]

As described above, according to the immunoassay reaction container of the present invention, a small amount of sample or reagent can be used.
The immunoassay can be performed with high sensitivity and efficiency. As a result, for example, the amount of sample collected from the patient can be reduced, so that the pain to the patient can be reduced. It is particularly preferable when collecting blood from an infant. In addition, since the reaction part is held on the moving body, it is easy to automate the immunoassay, so that a large amount of sample can be processed in a short time.

[Brief description of drawings]

FIG. 1 is an explanatory view showing an example of a reaction container for immunoassay of the present invention.

FIG. 2 is an explanatory view showing an example of a reaction part of the immunoassay reaction container of the embodiment.

FIG. 3A and FIG. 3B are explanatory views showing another embodiment of the reaction part.

FIG. 4 is an explanatory view showing another embodiment of the reaction section.

FIG. 5 is an explanatory view showing the measurement of chemiluminescence using the reaction container for immunoassay of the present invention.

6 (A) and 6 (B) are explanatory views showing a reaction container for immunoassay of the present invention.

FIG. 7 is a block diagram showing a photodetector used for immunoassay using the immunoassay reaction container of the present invention.

FIG. 8 is a characteristic diagram showing a calibration curve of mouse IgG antigen obtained using the reaction container for immunoassay of the present invention.

FIG. 9 is a characteristic diagram showing a calibration curve of mouse IgG antigen obtained using a conventional microplate.

FIG. 10 is a characteristic diagram showing a calibration curve of HBs antigen obtained using the reaction container for immunoassay of the present invention.

FIG. 11 is an explanatory view showing another embodiment of the moving body of the reaction container for immunoassay of the present invention.

FIG. 12 is an explanatory view showing another embodiment of the moving body of the reaction container for immunoassay of the present invention.

FIG. 13 is an explanatory view showing another embodiment of the moving body of the reaction container for immunoassay of the present invention.

FIG. 14 is an explanatory view showing another embodiment of the moving body of the reaction container for immunoassay of the present invention.

[Explanation of symbols]

10 ... Reaction container for immunoassay, 11 ... Moving body, 12 ... Reaction part, 21, 22 ... Flat plate part, 23, 24 ... Spacer part,
25 ... Gap.

Claims (3)

[Claims] 1. A reaction container for immunoassay for measuring a reaction between a substance to be measured and a specific reaction substance in a sample, wherein two flat plate portions are arranged at both edges of the flat plate portion. A reaction part, which is made to face each other through two spacer parts and has a gap capable of sucking and holding the sample and the reagent by a capillary phenomenon, and the reaction part is held so as to face one flat plate part. And a moving body for moving the reaction part, which is a reaction container for immunoassay. 2. The reaction for immunoassay according to claim 1, wherein the moving body holds the reaction part such that the open end of the reaction part is located in a direction different from the moving direction of the reaction part. container. 3. A moving body is configured to move the reaction part along an endlessly formed reaction line and guide light transmitted through the reaction part to the inside of the reaction line. The reaction container for immunoassay according to claim 1.