JPH04218775A - Immunoassay - Google Patents

Abstract

PURPOSE:To furnish a method of immunoassay wherein a large number of specimens are measured quickly in regard to a large number of measuring items and the results of inspection are obtained in a short time. CONSTITUTION:A method of immunoassay wherein a particle with which an antibody or an antigen is combined and a tagged antibody corresponding to a measuring item are provided in each tubular part of a vessel having at least two or more openings in the upper part and being formed to be tubular in the lower part, and wherein all reactions are completed in the tubular part of the aforesaid vessel, is furnished. Accordingly, not only operations for shift of a solid phase and others are dispensed with, but also a large number of measuring items can be met, and immunoassay can be expedited. The aforesaid vessel can be used for manual operations and also can be incorporated in an automated measuring apparatus and used. When this method of immunoassay is used for the automated measuring apparatus, in particular, it is very effective for reduction of a measuring time for one specimen.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method using a container having at least two openings at the top and a cylindrical shape at the bottom. Furthermore, the present invention relates to an immunoassay method for use in clinical tests by quantifying trace components in blood or body fluids.

0002

[Prior Art] Radioimmunoassay (hereinafter abbreviated as RIA) and enzyme immunoassay (hereinafter abbreviated as EIA) are highly sensitive measurement methods for detecting a target substance in a sample. As a method to remove this effect, bind/free (B
/F) A separation operation is used during the measurement. In order to perform this operation efficiently, there is a solid phase method that uses a solid phase bound to an antigen or antibody. Generally, RIA and EI by solid phase method
A includes steps such as reaction, washing, and detection during measurement, and in order to carry out this step, dispensing, diluting, stirring, and B/
Complex operations such as F separation and solid phase movement are performed repeatedly. Several improved techniques are known for its operation. One of these is the improvement of containers used for measurements. As an example of improving the container, a method has been reported in which a microplate, which is a dedicated container for containing magnetic particles, and a magnetic separation device containing a magnet that fits the plate are used (see JP-A-1-201156). As another example, a method is known that aims at automation and uses a container having multiple wells, such as a reaction well and a sample well, each having a fibrous matrix (Japanese Patent Laid-Open No. 63-2810
(See No. 53).

On the other hand, in RIA and EIA, automated measuring instruments have been developed for measuring large amounts of specimens. Various types of this equipment are known depending on the type of solid phase and label used in the measurement (see "Enzyme Immunoassay Method" by Eiji Ishikawa, Igaku Shoin, pages 180-207 (1987)).

0004

[Problem to be solved by the invention] Generally, R using a solid phase
IA and EIA require complicated operations such as dispensing, dilution, stirring, B/F separation, and movement of solid phase during measurement operations. As a means to solve this problem, methods using the aforementioned microplates and magnetic separation devices, and methods using containers with multiple wells are known, but the measurement time is long, ranging from 1 to 18 hours, and urgent tests This method has drawbacks such as the inability to perform measurements on a large amount of specimens quickly, and the inability to easily perform measurements using the same container for different detection systems, such as antigen detection systems or antibody detection systems. Furthermore, even when using the improved container described above,
Measuring instruments still required complicated measurement operations.

[0005]

[Means for Solving the Problems] The present invention uses a container having at least two openings at the top and a cylindrical shape at the bottom when measuring antigens or antibodies in serum or body fluids as the measurement target. The present invention aims to provide an immunoassay method that improves the conventional complicated and complex measurement operations and can handle multiple measurement targets for each detection system in a short time. This method is especially effective for immunoassays using automated measuring equipment.

The present invention can be employed in EIA and RIA. EIA and RIA are one-step or two-step solid-phase immunoassay methods using labeled substances,
Antigens or antibodies can be measured. For example, in a two-step antigen detection system, first, a sample is added to one cylindrical part and a solution collected from a cylindrical part containing another labeled antibody solution is mixed. A portion of this mixture was further added to the cylindrical part containing another antibody-bound solid phase, stirred, and reacted. After B/F separation, the amount of antigen bound to the solid phase was determined by reacting with the substrate. This is a method of measuring inside this cylindrical part. This measurement method is a method in which all the operations of each measurement process, such as dispensing, dilution, stirring, B/F separation, and measurement, are performed in a container having these three openings. The above-mentioned cylindrical part containing the antibody-bound solid phase contains the solid phase and reaction solution, and is longer downward than the other two parts and has a cylindrical shape with a large capacity to facilitate external detection. It is preferable that there be.

[0007] The number of openings in the container may be one having at least two openings, and is determined depending on the object to be measured, but the number is not limited and may be determined based on operability and convenience. I can do it. For example, two types of antibody-bound solid phases can be placed in separate cylindrical parts, and two measurements can be performed on one specimen at the same time. Furthermore, it is also possible to place the same type of antibody-bound solid phase in separate cylindrical parts and process two specimens at the same time. Furthermore, by using a container with four openings, the fourth cylindrical portion can be used as a sample dilution tank. The cylindrical part containing the antibody-bound solid phase described above is
In order to accommodate the solid phase and reaction solution and to facilitate detection from the outside, it is preferable to have a cylindrical shape that is longer downward than the other one or two cylindrical parts and has a large capacity. The container for carrying out the present invention can be made of plastic, such as polystyrene resin, acrylic resin, vinyl chloride resin, etc., or glass, but these are inexpensive and have low light transmittance. A material made of polystyrene resin, which is easy to handle, can be suitably used. Although this container has at least two openings, a protrusion or the like may be added to the portion other than the cylindrical portion as necessary to achieve the object of the present invention. Further, the container can be used by filling reagents and the like and sealing the top surface of the container. At this time, the top surface of the container is preferably flat. By this method, reagents, such as a solid phase or substrate bound to an antigen or antibody, can be placed in a container and stored with good stability until use. As the sealant, aluminum foil, various polymer films, etc. can be used alone or in a laminated form, and are torn with a breaker at the beginning of use.

[0008] Among the reagents used to carry out the present invention, the antigen- or antibody-binding solid phase is coated with a conventional EI on the inner wall of the cylindrical part.
Examples include those to which antigens or antibodies are bonded chemically or physically, as in A, and beads or particles to which antigens or antibodies are bonded in the same way. These beads are beads made of polystyrene and the like that are normally used in EIA. Furthermore, the particles are magnetic particles, such as magnetic substances, particles containing magnetic substances, and specifically, ferrite particles and the like.

[0009] Magnetic particles can be suitably used for measurements because the B/F separation operation can be easily performed by external magnetic force. At this time, by performing the B/F separation operation in the above-mentioned cylindrical part that is longer downward than other cylindrical parts, the magnetic force from the outside can be placed near the magnetic particles, and it can be carried out efficiently. .

[0010] The antigen and antibody bound to the solid phase can be determined depending on the substance to be measured in the sample. Antibodies can be used for drugs such as theophylline, phenytoin, valproic acid; small hormones such as thyroxine, estrogen,
Elastradiol; as a cancer marker, e.g. CEA
, AFP; for example, HIV, ATLA, HBV; as a polymeric hormone, for example, thyroid stimulating hormone (TSH),
Insulin; as a cytokine, e.g. IL-I, I
L-2, IL-6; various gross factors, such as EG
F, PDGF; further appropriate DNA and RN of the virus
It is an antibody against A. These antibodies may be monoclonal antibodies or polyclonal antibodies, and furthermore, F(ab')2, Fab', which are decomposition products of antibodies,
It may be Fab. The antigen may also be a virus, such as HIV, ATLA, HBV; DNA of the above-mentioned virus.
; high molecular weight hormones, such as insulin, TSH, etc.;

[0011] As a method of binding to the solid phase, a physical adsorption method or a chemical bonding method can be employed. The physical adsorption method is carried out by reacting the solid phase with an antigen or antibody in an appropriate buffer. Buffers used in this reaction include phosphate buffer, Tris-HCl buffer, carbonate buffer, and the like. The reaction can easily proceed by mixing both at 4°C to 37°C, preferably at room temperature, and the desired product can be obtained. Further, as the chemical bonding method, a carbodiimide method in the so-called peptide bonding method can be employed. Furthermore, as another chemical bonding method, a method performed in the presence of a divalent crosslinking reagent such as glutaraldehyde or cyanuric chloride can also be adopted ("Peptide Synthesis Method" Maruzen Co., Ltd. (published in 1975) and (See "Enzyme Immunoassay" Kyoritsu Shuppan Co., Ltd., "Protein Nucleic Acid Enzyme" Special Issue No. 31 (1987)).

[0012] In producing an enzyme-labeled antibody used in EIA, the antibody used is determined by the object to be measured, and may be an antibody that recognizes a different epitope or an antibody that recognizes the same epitope as the solid-phase bound antibody. Furthermore, antibodies against immunoglobulins can be used in the antibody detection system. In addition, the binding method of antibodies and enzymes can be produced using known methods of creating covalent bonds or non-covalent bonds (for example, "Protein Nucleic Acid Enzymes" Special Volume 31).
No., pp. 37-45 (1987)).

The enzymes used are peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, etc. In this case, it goes without saying that the substrate is suitable for the enzyme used, such as A
BTS, Luminol-H2 O2 (for peroxidase), p-nitrophenyl phosphate, methylumbelliphenyl phosphate, 3-(2'-pyro-haadamantane)-4-methoxy-4-(3''-phosphoryloxy) phenyl -1,2-dioxetane disodium salt (hereinafter abbreviated as AMPPD) (for alkaline phosphatase), p-nitrophenyl-β-o-galactose, methylumbelliphenyl-β-o-galactose
(for β-galactosidase), etc. can be used. The measurement is carried out by reacting at 4 to 40°C and measuring the amount of color, fluorescence, or luminescence produced using a detection device. These measurement methods are called colorimetric, fluorescent, and chemiluminescent enzyme immunoassays, respectively. Detection devices include spectrophotometers, photon counters, etc.
Photographic film or the like may also be used. Further measurements are
It is also possible to employ the so-called rate method in which the reaction is carried out while heating in the range of 4°C to 40°C.

[0014] In addition, RIA can be used instead of the above enzyme label.
This is done by labeling with a radioactive isotope such as 125I. Except for measuring radioactivity, the procedure is the same as for EIA described above.

[0015] Furthermore, radiolabeled antigens or antibodies can be easily prepared using Bolt-in Hunter reagent, which is already commercially available. For example, add this Bolt-in Hunter reagent to an antigen or antibody solution dissolved in a 0.1M sodium bicarbonate aqueous solution, and after 1 to 2 hours, remove unreacted Bolt-in Hunter reagent using a G-25 desalting column, etc. It can be prepared by In addition,
Radiolabeling of 125I can be easily performed by employing the chloramine T method or the iododine method. To perform an immune reaction, add the sample to the solid phase and heat at 4°C to 4°C.
The reaction is carried out at 0°C, preferably 20°C to 38°C, for 1 to 18 hours. After this, wash with distilled water or physiological saline, etc., add the radiolabeled antibody to the solid phase, and add the radiolabeled antibody to the solid phase at 4°C to 40°C.
Preferably, the reaction is carried out at 20° C. to 38° C. for 1 to 18 hours, washed with distilled water or physiological saline, and the radioactivity thereof is measured. A scintillation counter is used for measurement.

Although the present invention has been described above according to manual operations, each operation can be mechanized to assemble an automatic immunoassay device. As an automatic immunoassay device capable of implementing the present invention, for example, a device as shown in FIG. 7 can be considered. The apparatus shown in FIG. 7 performs a series of processes such as mixing, stirring, reacting, and measuring the sample, enzyme-labeled antibody, and substrate solution while the container having the opening moves from direction S to E in the reaction line section 111. This process completes the immunoassay without any manual intervention.

[0017]

[Operation] Next, the present invention will be explained in detail with reference to the drawings. FIG. 1 shows an example of the structure of a container. The container used here in carrying out the invention has openings 1, 2 and 3. The cylindrical part 8 having the opening 1 has a cylindrical shape that is longer downward than the other two parts, and is used to insert an antigen or antibody-bound solid phase for reaction, B/F separation, and detection. The cylindrical part 9 having the opening 2 is a cylindrical part for containing serum or a diluent to perform a reaction or dilution. This cylindrical part 9 is used for reaction in an antigen detection system in a specimen, and for dilution in an antibody detection system. Cylindrical part 10 having opening 3
is for containing an enzyme-labeled antibody in EIA or a radioisotope-labeled antibody in RIA.

FIG. 2 is an example showing the structure of another container. The container has openings 4 and 5, each with a first
A cylindrical part 11 having an action corresponding to the cylindrical parts 8 and 10 in the figure.
and 12. A container with two openings can be suitably used for measurement without performing a dilution operation.

FIG. 3 shows an example of the structure of a container devised to improve the efficiency of immunoassay. This container has 3
There are cylindrical portions 13, 15 and 14 having two openings, each having an action corresponding to the cylindrical portions 8 and 10 in FIG. This container can be used for simultaneous two serum measurements by filling the cylindrical parts 13 and 15 with the same type of antigen- or antibody-binding solid phase. Furthermore, by incorporating different types of antigen-binding solid phases, it can be used for simultaneous measurement of two items.

FIG. 4 is an example showing the structure of another container. This container has four openings, each with a first
A cylindrical portion 16 having a function corresponding to the cylindrical portions 8 and 10 in the figure.
, 19 and 17. This container having the cylindrical portion 18 can be suitably used for simultaneous two-item measurement with dilution operation.

FIG. 5 shows an example of a configuration for performing a B/F separation operation when magnetic particles are used as the solid phase. 6 in the diagram
is a device that is placed adjacent to the cylindrical portion 8 and generates magnetic force, and may be a permanent magnet or an electromagnet.

FIG. 6 shows an example of a configuration for performing detection. In the figure, 7 is a detection device, and in EIA, it is a device for detecting the amount of color development, amount of fluorescence, or amount of luminescence depending on the substrate used. In RIA, it is a scintillation counter.

FIG. 7 shows an example of an automatic measuring device that can be used in carrying out the present invention.

[0024]

EXAMPLES The present invention will be explained in more detail with reference to reference examples, working examples, and measurement examples.

(Reference Example 1) Preparation of carboxylated ferrite particles Carboxylated ferrite particles were preliminarily washed in an ultrasonic cleaner (
It was washed five times with distilled water for 60 seconds each using a vat type (manufactured by Nippon Seiki Seisakusho). Add 50 ml of 3-aminopropyltriethoxysilane to 5 g of ferrite-coated particles manufactured by Nippon Paint Co., Ltd. (made of polystyrene with an average particle diameter of 0.3 μm), add 30 ml of glacial acetic acid, and react for 3 hours at room temperature.
After washing, it is obtained by reacting with glutaric anhydride. Glacial acetic acid was added dropwise with stirring under ice-cooling, and washed three times each with distilled water, methanol, and distilled water.
It was washed five times with 300 ml portions of 1M sodium bicarbonate solution. The reaction with glutaric anhydride was performed at 5 wt% (0
．． 2.85 g of glutaric anhydride was added to 100 ml of 1M sodium hydrogen carbonate solution, and reacted for 10 minutes. After the reaction was completed, the particles were washed three times with 300 ml of 0.1M sodium bicarbonate solution, and further washed five times with distilled water, and used as carboxylated ferrite particles.

(Reference Example 2) Preparation of anti-TSH-binding carboxylated ferrite particles Reference Example 1 was added to 5 ml of 20 mM phosphate buffer (pH 4.5).
50 mg of the carboxylated ferrite particles prepared above were dispersed, and 50 mg of water-soluble carbodiimide was added thereto. After incubating for 20 minutes at room temperature, remove the supernatant and remove the anti-TSH
5 ml of mouse IgG solution (1 mg/ml, 0.02 M phosphate buffer, pH 4.5) was added and stirred with an end-over-end mixer. After 2 hours, the particles were added to 2% BSA.
Solution (0.1M Tris-HCl, 1mM Mg
Cl2, pH 7.5) five times, and this was washed with the same BS
It was dispersed in solution A to obtain anti-TSH mouse IgG-bonded carboxylated ferrite particles.

(Example 1) Preparation of anti-TSH mouse IgG binding container Anti-TSH mouse IgG solution (
200 μl of 4 μg/ml, 10 mM phosphate buffer, pH 7.0) was added and left at room temperature for 18 hours. This cylindrical part 8
was washed 3 times with physiological saline and added with 2% BSA solution (0.1M
Tris-HCl, 1mM MgCl2, pH
7.6) was added to form an anti-TSH mouse IgG binding tube.

(Example 2) Preparation of anti-TSH mouse IgG-conjugated polystyrene beads 25 ml of anti-TSH mouse IgG solution (4 μg/ml, 1
100 1/8 inch polystyrene beads were soaked in 0mM phosphate buffer) and left overnight at room temperature. The beads were washed three times with physiological saline, and 25 ml of 2% BSA solution (same as above) was added to prepare anti-TSH mouse IgG-conjugated polystyrene beads.

(Example 3) Preparation of cylindrical part for TSH measurement (
3-1) Preparation of particle-type cylindrical part for TSH measurement Anti-TSH mouse IgG-binding ferrite particles prepared in Reference Example 2 (0.
04% particles) was placed in the cylindrical part 8 shown in FIG.
Mouse IgG-Fab solution (0.1M Tris-H
Cl, 0.1mM ZnCl2, 1mM MgC
l2, pH 7.5, 2% BSA, 500 ng/ml alkaline phosphatase-conjugated anti-TSH mouse IgG Fab
(containing) 100 μl was added. This cylindrical part was covered with aluminum foil whose adhesive surface was coated with polyvinyl chloride, and heat-sealed. This was used as a cylindrical part for particle type TSH measurement.

(3-2) Preparation of tube-shaped cylindrical part for TSH measurement Anti-TSH mouse IgG binding cylindrical part 1 prepared in Example 1
alkaline phosphatase-conjugated anti-TSH mouse IgG to
-Fab solution (0.1M Tris-HCl, 0.1
mM ZnCl2, 1mM MgCl2, pH
7.5, 2% BSA, 500 ng/ml alkaline phosphatase-conjugated anti-TSH mouse IgG Fab) 100
µl was added. This cartridge was covered with aluminum foil whose adhesive surface was coated with polyvinyl chloride and heat-sealed. This was used as a tube-shaped cylindrical part for TSH measurement.

(3-3) Preparation of bead-shaped cylindrical part for measuring TSH One anti-TSH mouse IgG-conjugated polystyrene bead prepared in Example 2 was placed in the cylindrical part 8 shown in FIG. 1, and 2% B
250 μl of SA solution was added. Next, alkaline phosphatase-conjugated anti-TSH mouse IgG-Fa was added to this cylindrical part 10.
b solution (0.1M Tris-HCl, 0.1mM
ZnCl2, 1mM MgCl2, pH 7.5
, 2% BSA, and 500 ng/ml alkaline phosphatase-conjugated anti-TSH mouse IgG Fab). This cylindrical part was covered with aluminum foil whose adhesive surface was coated with polyvinyl chloride, and heat-sealed. This was used as a bead-shaped cylindrical part for TSH measurement.

(Example 4) Preparation of HTLV-I antigen-binding ferrite particles Ferrite particles manufactured by Nippon Paint Co., Ltd. with a concentration of 10% and 800μ
HTLV-I antigen (400μg/
ml) and stirred overnight at room temperature using an end-over-end mixer. The particles were added to a 2% BSA solution (0.1M
Tris-HCl, 1mM MgCl2, pH 7
．． 5) and dispersed in the same BSA solution to obtain HTLV-I antigen-binding ferrite particles.

(Reference Example 4) Preparation of cylindrical part for detecting HTLV-I antibody The HTLV prepared in Example 3 was placed in the cylindrical part 8 shown in FIG.
-I antigen-binding ferrite solution (0.008%/particle/B
350 μl of SA solution was added. Next, in hole 3, add anti-human IgG mouse IgG-conjugated alkaline phosphatase 30
0 μl was added. This cylindrical part was covered with aluminum foil and heat-sealed to obtain the desired cylindrical part.

(Example 5) Measurement of TSH in each cylindrical part The seals of each cylindrical part 8, 9, and 10 were broken with an aluminum breaker, and samples containing 50 μl of TSH (0, 10 μl
U/ml) and 50μ of alkaline phosphatase conjugate bound to anti-TSHFab' in each tube 8.
l (conjugate concentration 0.5 μg/ml, 0.1M
Tris-HCl, 2% BSA, 1mM MgCl2
, 0.1mM ZnCl2, pH 7.5) were placed in each cylindrical part 9 and mixed.

(5-1) Measurement using the bead-type TSH measuring tube After 10 minutes, 30 μl of the mixed liquid in the tube 9 was added to the tube 1 containing the beads and stirred. After 10 minutes, the supernatant was aspirated off and washed four times with physiological saline. This cylindrical part 8 is filled with a substrate solution containing 100 μg/ml of AMPPD (0
．． 1M Tris-HCl, 1μM MgCl2
, 0.1mM ZnCl2, pH 9.8) 300μ
1 was added and reacted at room temperature. After reacting for 10 minutes, the reaction was measured using a luminometer (manufactured by Aloka). The results are shown in Figure 5.

(5-2) Measurement using the tube-shaped TSH measuring cylindrical part After 10 minutes, 30 μl of the mixed liquid that had entered the cylindrical part 9 was added to the cylindrical part 8 bound to the antibody and stirred. After 10 minutes, the supernatant was aspirated off and washed four times with physiological saline. Add 300 μl of the same AMPPD solution as in Example 5-1 to this cylindrical part 8.
was added, reacted at room temperature, and measured using a luminometer after 5 minutes. The results are shown in Figure 5.

(5-3) After 10 minutes of measurement using the cylindrical part for measuring particle-type TSH, 30 μl of the mixed solution in the cylindrical part 9 was added to the cylindrical part 8 containing the antibody-binding particles, stirred, and then stirred for 10 minutes. I left it alone. This cylindrical portion 8 was brought into contact with a magnet with a surface magnetic field of 3000 Gauss to collect the ferrite particles, and the supernatant was drained by aspiration. After dispensing physiological saline into this cylindrical portion 8, the ferrite particles were collected by the aforementioned magnet, and the supernatant was drained by decantation. This operation was repeated four times. This cylindrical part 8 has the same AMP as in Example 5-1.
300 μl of PD solution was added and reacted at room temperature, and after 5 minutes, the amount of luminescence was measured using a luminometer. The results are shown in Figure 5.

(Example 6) Measurement of HTLV-I antibody using a cylindrical part for HTLV-I A hole was made in the seal of the cylindrical part 9 prepared in Example 3 with a seal breaker, and 20 μl of serum was poured into the cylindrical part. Add diluent (0.1M containing 10% NRS, 2% BSA)
Tris-HCl, 1mM MgCl2, 0.1m
180 μl of MZnCl2, pH 7.0) was added. 20 μl of this diluted serum was added to the HTLV-
The mixture was mixed with I antigen-binding particles, stirred, and then left at 37° C. for 10 minutes. A permanent magnet with a surface magnetic field of 3000 Gauss was brought into contact with the outside of the cylindrical portion 8 to concentrate the ferrite particles, and the supernatant was drained by aspiration. After this, 0.4%
400 μl of saline was added and stirred. This cylindrical part was magnetized by the above-mentioned magnet, and the supernatant was aspired and drained. This operation was repeated twice.

[0039] Anti-human IgG mouse IgG was added to this cylindrical part 8.
250 μl of bound alkaline phosphatase solution (300 ng/ml protein solution) was added and left for 10 minutes. This cylindrical part was brought into contact with a magnet with a surface magnetic field of 3000 Gauss to concentrate the ferrite particles, and the supernatant was drained by aspiration. After that, 400 μl of 0.4% saline was added and stirred. This cylindrical portion was brought into contact with the aforementioned magnet, and the supernatant was drained by aspiration. This operation was repeated four times. Add 30% of the same AMPPD solution as in Example 5-1 to this cylindrical part.
0 μl was added and allowed to react at room temperature for 5 minutes. After 5 minutes, the amount of light emitted for 5 seconds was measured using a luminometer. The results for each serum sample are shown in Table-1.

[Table 1]

(Example 7) Measurement of CA19-9 (7
-1) Preparation of CA19-9 antibody-bound particles Anti-CA19-9 monoclonal antibody (MCA) 2 mg as in Reference Example 2
were reacted and thoroughly washed to prepare anti-CA19-9MCA-bound ferrite particles (0.04% particles/2% BSA
, 0.1M Tris-HCl, 1mM MgCl
2, 0.1 mM ZnCl2, pH 7.5).

(7-2) Preparation of cylindrical part for particle type CA19-9 measurement 250 μl of the anti-CA19-9 mouse IgG-binding ferrite particles prepared in Example 7-1 were placed in the cylindrical part 1 shown in FIG.
1, and the alkaline phosphatase-conjugated anti-CA19-9 mouse IgG-Fab solution (0.5 μg/
ml) was added. The top of this was heat sealed with PET laminated aluminum foil.

(7-3) Measurement of CA19-9 In advance, the aluminum seal of the cylindrical part prepared in Example 7-2 was broken with a seal breaker, and 20 μl of serum was added to the cylindrical part 11 and stirred. Leave it for a minute. Next, a magnet with a surface magnetic field of 3000 Gauss is brought into contact with the outside of the tube of this cylindrical part 11 to collect the ferrite particles, and the supernatant is drained by decantation. After that, 0.2% saline is added. and stirred. Thereafter, the particles were magnetized in the same manner as described above, and the supernatant was removed. This operation is performed twice, and the labeled antibody solution 25 is removed from the cylindrical part 12.
0 μl was taken out and added to the cylindrical part 11 and stirred. After 10 minutes at room temperature, the ferrite particles were collected using the magnet described above and the supernatant was removed. Next, 400 μl of the washing solution was added, stirred, and magnetized again to remove the supernatant. This operation was repeated four times. Next, 300 μl of the same substrate solution as in Example 4 was added, stirred and left at room temperature for 5 minutes, and immediately the amount of luminescence for 5 seconds was measured using a luminometer (manufactured by Aloka). The results of each serum sample are shown in Table-2.

[Table 2]

(Example 8) Preparation of cylindrical part for particle type CA19-9 measurement (8-1) Anti-CA19-9 prepared in Example 7-1
250 μl of mouse IgG-bound ferrite particles were placed in the cylindrical parts 13 and 15 shown in FIG.
350 μl of solution b (0.5 μg/ml) was added. The top of this was heat sealed with PET laminated aluminum foil.

(8-2) Measurement of CA19-9 in advance,
The aluminum seal of the cylindrical part prepared in Example 8 was broken with a seal breaker, and 20 μl of each of the two types of serum were added to the cylindrical parts 13 and 15, stirred, and left at room temperature for 10 minutes. Next, a magnet with a surface magnetic field of 3000 Gauss is brought into contact with the outside of the tube of the cylindrical portions 13 and 15 to collect the ferrite particles, and the supernatant is drained by decantation.
．． 2% saline was added and stirred. Thereafter, the particles were magnetized in the same manner as described above, and the supernatant was removed. This operation is performed twice, and 250 μl of the labeled antibody solution is transferred from the cylindrical portion 14 to the cylindrical portions 13 and 15.
and stirred. After 10 minutes at room temperature, the ferrite particles were collected using the magnet described above and the supernatant was removed. Next, wash liquid 400
μl was added, stirred, magnetized again, and the supernatant was removed. This operation was repeated four times. Next, add 3 ml of the same substrate solution as in Example 4.
00 μl was added, stirred and left at room temperature for 5 minutes, and the amount of luminescence was immediately measured for 5 seconds using a luminometer (manufactured by Aloka). The results of each serum sample are shown in Table-3.

[Table 3]

(Example 9) Preparation of HBs antigen-binding ferrite particles 800 μl of 5% concentration of ferrite particles manufactured by Nippon Paint Co., Ltd.
HBs antigen (400 μg/ml)
2 ml was added and stirred overnight at room temperature using an end-over-end mixer. The particles were added to a 2% BSA solution (0.1M Tr
is-HCl, 1mM MgCl2, pH 7.5)
The particles were washed twice and dispersed in the same BSA solution to obtain HBs antigen-binding ferrite particles.

(Reference Example 5) Preparation of a cylindrical part for detecting particle-type HTLV-I and HBs antibodies The HTL prepared in Example 3 was placed in the cylindrical part 16 shown in FIG.
VI antigen-binding ferrite solution (0.008%/particle/
350 μl of BSA solution was added. Furthermore, the HBs antigen-binding ferrite solution prepared in Example 9 (
0.008%/particle/BSA solution) 350 μl was added. Next, 300 μl of anti-human IgG mouse IgG-conjugated alkaline phosphatase was placed in hole 17. This cylindrical part was covered with aluminum foil and heat-sealed to obtain the desired cylindrical part.

(Example 10) HTLV-I and HB
s Antibody Measurement Make holes in the seals of the cylindrical parts 16, 17, 18, and 19 prepared in Reference Example 5 with a seal breaker, put 20 μl of serum in the cylindrical part 18, and add diluent (10% NRS, 2%
0.1M Tris-HCl with BSA, 1mM
MgCl2, 0.1mM ZnCl2, pH 7
．． 0) Added 180 μl. 20 μl of this diluted serum was mixed with the HTLV-I antigen-binding particles and HBs antigen-binding particles contained in the cylindrical parts 16 and 17, stirred, and left at 37° C. for 10 minutes. A permanent magnet with a surface magnetic field of 3000 Gauss was brought into contact with the outside of the cylindrical portions 16 and 19 to concentrate the ferrite particles, and the supernatant was drained by aspiration. After this, 400 μl of 0.4% saline was added and stirred. This cylindrical part was magnetized by the above-mentioned magnet, and the supernatant was aspired and drained. This operation was repeated twice.

[0048] Anti-human I in the cylindrical parts 16 and 19
gG mouse IgG-conjugated alkaline phosphatase solution (3
00 ng/ml protein solution) was added to each plate in an amount of 250 μl and left for 10 minutes. The surface magnetic field of this cylindrical part is 3000
The ferrite particles were brought into contact with a Gaussian magnet to collect the magnetic field, and the supernatant was drained by aspiration. After this 0.4%
400 μl of saline was added and stirred. This cylindrical portion was brought into contact with the aforementioned magnet, and the supernatant was drained by aspiration. This operation was repeated four times. Example 5-1
300 μl of the same AMPPD solution was added to each and reacted at room temperature for 5 minutes. After 5 minutes, the amount of light emitted for 5 seconds was measured using a luminometer. Table the results for each serum sample.
4.

[Table 4]

(Example 11) Apparatus that can be used to carry out the present invention An example of an apparatus that can be used to carry out the present invention will be described with reference to FIG. In the figure, 102 is an input section equipped with a start button, a measurement method selection button, etc., 103 is an output section that outputs the measurement results, and 110 is the mixing, stirring, and reaction of the sample 144, enzyme-labeled antibody, and substrate solution. , a processing unit that performs a series of processing such as measurement, and 130 is this processing unit 110.
105 is a control unit that controls each part of this apparatus, and 101 is a case body.

[0050] The processing section 110 is a motor (not shown).
a reaction line section 111 that is equipped with a reaction line section 111 that moves the reaction container 104 in the direction A and that can perform reactions, measurements, etc. at each predetermined position in this moving route L; A chip accommodating part 113 that accommodates the chip 113a, and a chip 1 from the chip accommodating part 113.
13a is taken out, a predetermined amount of the sample 144 accommodated in the sample storage section 112 is sucked into the tip 113a by an appropriate pump means, and the reaction vessel 1 is moved through the reaction line section 111.
04 and further contains the enzyme-labeled antibody.
From the cylindrical part containing the enzyme-labeled antibody No. 04 to the reaction vessel 10
a suction and dispensing section 116a for aspirating and dispensing the enzyme labeled substance into the other reaction tube of No. 4 using an appropriate pump means to obtain a mixed solution;
A stirring section 117 that stirs the mixed liquid, a magnetic separation section 118 that magnetically separates reactants and unreacted substances in the mixed liquid, a washing section 119 that washes and removes unreacted substances, and accommodates a substrate liquid. a suction dispensing stirring section 116b that dispenses the substrate liquid from the reagent storage section 115; and a measuring section 1 that measures luminescence information generated by the reaction between the reactant and the substrate liquid by an optical method.
20, and a discharge part 121 which is arranged near the terminal end E of the reaction line part 111 and discharges the reaction container 104.

[0051] The control unit 105 is a program that controls each part of the apparatus 101 and stores a one-step measurement method, a two-step measurement method, a delay reaction measurement method, a two-step measurement method using a diluent, and other enzyme immunoassay programs. A memory 122, a memory 123 that stores information such as calculation formulas for obtaining immune information based on measurement information measured by the measurement section 120, and a CPU that controls each part of this device.
124. The control unit 105 is also capable of executing an enzyme immunoassay based on the selection operation of the measurement method selection button of the input unit 102.

[0052]

Effects of the Invention The method of the present invention detects trace components in a specimen by immunoreaction using a container having at least two openings at the top and a cylindrical shape at the bottom. According to the present invention, it is possible to easily and quickly measure multiple measurement targets of each detection system, and it can be applied to automated measurement equipment, making a great contribution to the field of clinical testing. do.

[Brief explanation of the drawing]

1 is a sectional view of a container with three openings; FIG.

FIG. 2 is a cross-sectional view of a container with two openings.

FIG. 3 is a cross-sectional view of a container with three openings used for simultaneous two-serum measurement or simultaneous two-item measurement.

FIG. 4 is a cross-sectional view of a container with four openings.

FIG. 5 shows an example of a configuration when performing a B/F separation operation.

[Explanation of symbols]

6 is a magnetic force generator.

FIG. 6 shows an example of a configuration when performing a detection operation.

[Explanation of symbols]

7 is a detection device.

FIG. 7 is a schematic configuration diagram of an apparatus that can be used in implementing the present invention.

FIG. 8 shows the measurement results of TSH by luminescence method using particle-type, tube-type, and bead-type solid phases.

Claims (9)

[Claims] Claim 1: Having at least two openings in the upper part,
An immunoassay method that uses a container with a cylindrical shape at the bottom. 2. The method according to claim 1, wherein at least one cylindrical portion is longer downwardly than the other cylindrical portions. 3. The method according to claim 1, wherein at least one of the cylindrical parts is for containing an antigen- or antibody-binding solid phase. 4. The method according to claim 1, wherein at least one of the cylindrical portions is for containing serum or a diluent. 5. The method according to claim 1, wherein one of the cylindrical parts is for containing a labeled antibody. 6. The method according to claim 3, wherein the solid phase is a magnetic particle. 7. The method according to claim 3, wherein the antigen or antibody is bound to the inner wall of the container. 8. The method according to claim 3, wherein the solid phase is beads. 9. The method according to claim 1, which is an enzyme immunoassay.