JPH04273065A - Immunity analyzing method and device - Google Patents

Abstract

PURPOSE:To obtain a high-sensitivity immunity analyzing method for trace organic components. CONSTITUTION:Particles used as labelled elements, which are proportional to the quantity of measured objects 11, are trapped into a reactive solid phase 10 through specific reaction such as antigen antibody reaction, and then isolated to count the number of particles in isolated liquid so that the quantity of measured objects can be found. Measured liquid including isolated elements is guided into a flow cell 1 to detect the fluorescence of pulses which arise in passing the particles through laser flux radiated from the direction perpendicular to the flow, and the number of pulses is counted to measure the particles 21. As the measurement is made after the labelled elements are trapped once into the reactive solid phase, meaning that the particles are isolated, it is not affected by the labelled elements combined with the solid phase unspecifically. The particles as the labelled elements are counted to realize highly-linear detection even at a low concentration.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to an immunoassay method and an analyzer thereof, and in particular, after labeling an analyte such as an antigen, antibody, or hapten contained in a biological sample, the labeled substance is transferred to a flow cell. The present invention relates to an immunoassay method and an analyzer for the same.

0002

BACKGROUND OF THE INVENTION Analytical methods that utilize antigen-antibody reactions are known for measuring trace amounts of substances in biological samples such as blood. JP-A-1-18 which is one of the prior art of this kind.
No. 7459 describes an analysis method that combines antigen-antibody reaction and photoacoustic spectroscopy.

[0003] That is, this Japanese Patent Application Laid-Open No. 1-187459
In this issue, a sample containing an antigen to be analyzed is added to a reaction vessel with antibodies immobilized on its inner wall, unreacted samples are then washed away, and microparticles such as latex or inorganic oxides are bound. Through the steps of adding a solution containing antibodies, washing and removing excess antibodies, and adding a free solution, a suspended solution containing released microparticles is generated in a reaction container. Thereafter, the floating liquid is transferred to a photoacoustic cell, and a photoacoustic signal is obtained by intermittently irradiating laser light and detecting pressure changes within the photoacoustic cell.

On the other hand, Japanese Patent Laid-Open No. 63-214668 proposes a highly sensitive immunoassay method. In this prior art,
A labeled antibody, which is a combination of an antibody and a microcapsule containing a fluorescent substance, is used. No solid phase is used in this example. A microcapsule-labeled antibody is added to a sample containing test cells for immunoconjugation, followed by centrifugal washing. Inject the resuspended solution after centrifugal washing into the sheath flow cell of a flow cytometer. Fluorescence from the microcapsules is detected as the immune complex of cells and labeled antibody complex flows across the radar beam that illuminates the flow cell.

[0005] Although not related to immunoassay, examples of the use of sheath flow cells include, for example, Japanese Patent Laid-Open No. 2-80
There is one shown in No. 937. In this prior art, a sample container containing stained blood cells is transferred to a sampling position, and the blood cell suspension in the sample container is supplied by a pipetting nozzle to the inlet chamber of a sheath flow cell. The flow cell is irradiated with laser light and is configured to detect fluorescence and scattered light when stained cells cross the laser beam.

[0006]

[Problem to be solved by the invention] The above-mentioned JP-A-1-1
No. 87459 detects a photoacoustic signal and obtains a signal that depends on the concentration of the entire suspended liquid, making it difficult to measure with high precision when the concentration of the analyte is minute. When a labeled antibody is added to a reaction vessel equipped with a solid phase, the labeled antibody not only specifically binds to the antigen bound to the solid phase, but also nonspecifically adsorbs to the solid phase surface. There is a problem in that this non-specifically adsorbed labeled antibody cannot be removed by normal washing operations. However, when a sodium hydroxide solution is used as a label flotation liquid as in JP-A-1-187459, not only particles that are specifically bound but also particles that are non-specifically adsorbed are detected. Since the labeled substance is also released from the solid phase,
If a liquid to be measured containing these substances is measured by photoacoustic spectroscopy, the measurement error will inevitably increase.

[0007] The method shown in JP-A-63-214668 does not require the use of a solid phase and the amount of detection signal from each label is large, but it requires washing using a centrifuge and is cumbersome to operate. and is difficult to automate. Further, in JP-A-2-80937, no consideration is given to the application to immunoanalysis.

[0008] An object of the present invention is to provide an immunoassay method and an analyzer thereof that can obtain accurate and highly sensitive measurement results even if the amount of an analyte in a sample is minute.

[0009] Another object of the present invention is to provide an immunoassay method in which contamination of a particle label due to non-specific adsorption that does not bind to an analyte is extremely reduced in a sample liquid supplied from a reaction container to a flow cell. and to provide such equipment.

[0010] Another object of the present invention is to provide an immunoassay method and an apparatus therefor that can measure a plurality of analytes using a plurality of reaction vessels having the same type of reaction solid phase.

[0011]

[Means for Solving the Problems] According to the present invention, the amount of a labeled substance by an antigen-antibody reaction is measured not as a concentration but as the number of labeled substances. Furthermore, in order to distinguish and measure labeled antibodies nonspecifically adsorbed to the solid phase, we selectively released bound labeled substances through antigen-antibody reactions without releasing nonspecifically adsorbed labeled antibodies. Afterwards, measure this.

[0012] As the label in the present invention, it is preferable to use fluorescent microparticles. The immunoassay method based on the present invention is generally operated through the following steps. That is, a sample is supplied to a reaction vessel having a solid phase bound to a receptor, the analyte in this sample is bound to the receptor, and after removing the unreacted sample from the reaction vessel, fluorescent particles are added to the reaction vessel. The labeled receptor labeled with is supplied to the reaction vessel, the labeled receptor is bound to the solid phase via the analyte, and the excess labeled receptor is removed from the reaction vessel, and then the labeled receptor is released into the reaction vessel. providing a free reagent containing an agent;
Introducing a liquid to be measured containing fluorescent particles released from a solid phase into a flow cell, detecting fluorescence based on the fluorescent particles passing through the flow cell, counting the number of detected particles, and counting the number of particles counted. Based on this, the concentration of the analyte in the sample is calculated.

A preferred embodiment of the present invention also includes the following operations. Specifically, a sample is supplied to a reaction vessel having a solid phase to which a receptor is bound via a nucleic acid, an analyte in the sample is immunoconjugated to the receptor, and a labeled receptor is introduced into the reaction vessel. The labeled receptor is bound to the solid phase via the supplied analyte, and after removing excess labeled receptor from the reaction vessel, a restriction enzyme-containing solution is supplied to the reaction vessel to bind the labeled receptor to the solid phase. The method includes cleaving the nucleic acid present, introducing a liquid to be measured containing the labeled substance released from the solid phase into a flow cell, and counting the number of the labeled substances passing through the flow cell.

[0014]

[Action] When the analyte is an antigen or hapten, an antibody that can specifically bind to it is used as the receptor bound to the solid phase; , an antigen capable of specifically binding thereto is used.

[0015] After a biological sample is supplied to a reaction vessel equipped with a solid phase and an analyte is bound to the solid phase, a labeled antibody capable of immunologically binding to the analyte is added to the reaction vessel. Thereafter, the liquid containing excess labeled antibody is removed from the reaction vessel by suction and discharge, but such washing and removal operations do not remove the labeled antibody nonspecifically adsorbed to the solid phase surface. Remains in the reaction vessel.

[0016] By using a release reagent solution containing a specific label flotation agent, the antigen on the solid phase can be released without substantially releasing (dissociating) the labeled antibody nonspecifically adsorbed to the solid phase surface. Antibody binding (immune binding) can be selectively dissociated. Therefore, by supplying the floating reagent solution, the immunoconjugated label is selectively released into the solution in the reaction container. One particular label release agent is a chaotropic ion in neutral solution. As a chaotropic ion, C
Cl3COO-, SCN-, CF3COO-, etc. can be used.

Nucleic acids for binding antibodies or antigens as receptors to a solid phase here include oligonucleotides. The antibody or antigen on the solid phase side in the reaction container is bound to the solid phase via a double-stranded nucleic acid when a sample is supplied.

[0018] When performing multi-item analysis, a large number of reaction vessels having solid phases of the same type are prepared, and at the start of the analysis operation, a unique receptor assigned to each analyte is bound to the solid phase. That is, single-stranded nucleic acids of the same form are immobilized on the solid phase in each reaction vessel in advance, and during analysis operations complementary single-stranded nucleic acids with receptors corresponding to various analytes are immobilized. are added to reaction vessels corresponding to each type of analyte as assigned, and bind various receptors by forming double strands (base pairs) on the solid phase in each reaction vessel. . As a result, a group of reaction vessels for multi-item analysis that can subsequently undergo immunoreaction with various analytes is prepared.

[0019] In a reaction vessel in which nucleic acid is bound to a solid phase,
After the analyte in the sample is immunoconjugated to the solid phase, a receptor with a labeled substance is further bound, but in this case, a label releasing agent for detaching the labeled substance from the solid phase is used. Restriction enzymes are used. Restriction enzymes include restriction endonuclease, phosphogesterase, pyrophosphatase,
Hydrolytic enzymes such as peptidase and esterase are preferably used, but many other known restriction enzymes can also be used. Such restriction enzymes cleave the double strands of nucleic acids at predetermined sites.

[0020] The solid phase in the reaction container is used to direct the inner wall surface of the reaction container such as a test tube or microplate, or the surface of a spherical body such as a glass ball contained in the reaction container, to the receptor to be bound. The surface has been treated to enable chemical bonding.

Fluorescent microparticles are suitable as the label. The fluorescent particles may be obtained by forming a fluorescent material layer on the surface of Stex particles or inorganic particles, or may be obtained by forming particles of a mixture of a particle-forming composition and a fluorescent material. Fluorescence is detected by a flow cytometer and the number of particles passing through the flow cell is counted, but the particle size of the fluorescent particles in this case is between 10-5 and 10-5.
It is 3mm. Fluorescent substances that can be used for the fluorescent particles are known substances such as fluorescein, coumarin derivatives, rhodamine derivatives, dansyl, umbelliferone, etc.

[0022] When the nucleic acid is cleaved to release the labeled substance, the labeled substance-containing body guided to the flow cell contains a part of the cleaved nucleic acid, the receptor on the solid phase side, the analyte, and the labeled receptor. are in a combined state. On the other hand, nucleic acids connected to receptors on the solid phase to which the analyte has not been bound are also cleaved by restriction enzymes and guided to the flow cell, but since no label is bound to such free substances, , no fluorescence is detected in the flow cell, resulting in virtually no measurement error.

[0023] In conventional concentration measurements, the bound amount is measured as a statistical average value, which not only lacks detection sensitivity at low concentrations but also increases the sensitivity of interfering factors contained in samples, etc. Due to this influence, the measured values vary from sample to sample and do not give accurate measured values. In addition, when attempting to re-dissociate the antigen-antibody bond and measure it in order to avoid the effects of non-specific adsorption, the composition of the dissociating solution does not match the enzyme reaction conditions, resulting in new problems such as the inability to directly measure the dissociated sample. Challenges also arise. The dissociation and separation operations cause dilution of the measurement solution, raising the actual lower limit of quantification.

[0024] In the method of counting particles, since it is possible to measure even one particle, sufficient detection sensitivity can be obtained for measuring low concentrations of antigen. That is, each particle provides an amplified signal. Since the number of particles bound by an antigen-antibody reaction is measured using the particles as a label, the counted value directly reflects the amount of antigen.

[0025] In the method of labeling fine particles and counting them one by one, even if interfering factors cover the particle surface and reduce or amplify the fluorescence intensity, signals within a preset constant level are measured. By doing so, the influence of interfering factors can be avoided and accurate counting can be achieved. Measurement system noise also has no effect. Since the absolute number can be measured, there is no effect of dilution,
Furthermore, since the measurement can be performed even with the composition of the dissociation liquid as it is, no new consideration is required during measurement.

[0026] In the atmosphere, there is a large amount of dust of several microns or less, and this dust is mixed into the measurement liquid as foreign particles. In the method of irradiating a sample with light and measuring the light scattered by the sample, target labeled particles and foreign particles cannot be distinguished, and the amount of foreign particles increases the background. On the other hand, in the method of measuring fluorescence, there are no foreign particles with strong fluorescence in the normal environment, so measurements can be made without miscounting. Therefore, a method of measuring particles one by one using fluorescent particles enables highly sensitive detection.

[0027] In the case of a nucleic acid, for example, DNA, constituent dioxyribonucleotides are linked by 3',5'-phosphodiethyl ester bonds. On the other hand, proteins that form the main part of antibodies are polymerized amino acids through polypeptide bonds. Therefore, since the binding mode of nucleic acids is completely different from that of proteins, the possibility of denaturing or decomposing proteins is extremely small under reaction conditions that cleave nucleic acids. In particular, if an endonuclease called a restriction enzyme is used, it is possible to cleave only a specific base sequence in a nucleic acid, so only the nucleic acid portion can be selectively cleaved without cutting the protein portion.

[0028] Therefore, it is possible to bind an antibody to a solid phase via a nucleic acid, capture the antigen and labeled antibody through an antigen-antibody reaction, and after washing, selectively cleave the antibody at the nucleic acid portion. At this time, only the labeled substance involved in the target antigen-antibody reaction can be removed from the solid phase without denaturing or decomposing the antibody or enzyme. Labeled substances that are nonspecifically adsorbed without being caused by antigen-antibody reactions are directly attached to the solid phase without intervening the nucleic acid, and therefore are not released by the action of restriction enzymes. This allows the amount of antigen to be measured while eliminating the influence of non-specific adsorption, and does not impair quantitative performance. Among enzymes that cut DNA (deoxyribonucleic acid), restriction enzymes have the function of recognizing and cutting only specific base sequences. This base sequence, called a recognition cleavage sequence, is unique to each restriction enzyme. Therefore, if at least one recognition cleavage sequence of the restriction enzyme to be used is inserted into the base sequence of the nucleic acid or oligonucleotide used for binding, the restriction enzyme can be used to cleave. A base sequence that satisfies this condition can be freely set.

[0029]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A first embodiment of the present invention will be described with reference to FIGS. 1 to 5.

FIG. 1 is a schematic diagram showing the overall configuration of an immunoanalyzer according to a first embodiment. The turntable 32 can place two rows of containers, with a plurality of sample containers 33 containing blood samples arranged in a circle on the inner periphery, and a plurality of sample containers 33 containing a solid phase on the outer periphery. Reaction container 34
are arranged in a circle. A single strand of complementary oligonucleotide is immobilized on the solid phase of the reaction vessel 34, as will be described later. This turntable 32 rotates a desired sample container 33 and reaction container 34 by a pulse motor 92 whose operation is controlled by a controller 46.
It is rotated to stop at the suction position by the pipetting nozzle 43. The array of reaction vessels is configured to move within the thermostatic chamber.

Various reagent liquid containers necessary for analysis operations are placed on the reagent table 35. That is, containers 90a, 90b, 90c containing fixing antibody solutions corresponding to various analysis items A, B, and C, respectively, and containers 36a, 3 containing fluorescent particle-labeled antibody solutions corresponding to various analysis items.
6b, 36c, and a free reagent solution storage container 3 containing restriction enzymes.
8 and other necessary buffer containers etc. are placed on the reagent table 35.
is maintained. The pulse motor 93, whose operation is controlled by the controller 46, rotates the reagent table 35 by a desired angle, and positions the specified reagent container at the required timing so as to stop at the suction position by the nozzle 43.

The automatic pipetting mechanism 39 has a movable arm 42
Pipetting nozzle 43 and arm 42 attached to
A rotation drive unit 45 for horizontally rotating the arm 42, a vertical drive unit 44 for moving the arm 42 up and down, a syringe pump 40 connected to the nozzle 43 via a tube 41, and a cleaning liquid tank 37 that also serves as extrusion liquid. ing. Arm 42
With this operation, the pipetting nozzle 43 moves around two suction positions on the turntable 32, the reagent suction position on the reagent table 35, the injection chamber entrance 48 of the flow cell 47, and the top of the nozzle cleaning tank 94 as a radius of rotation. It can rotate and can be lowered and raised in each position.

The internal structure of the sheath flow cell 47 is similar to that used in known flow cytometers, but a reagent injection chamber similar to that shown in Japanese Patent Application Laid-Open No. 2-80937 is provided above. It is opened. Therefore, the nozzle 43 can penetrate into the injection chamber inlet 48 and discharge the liquid to be measured into the sheath flow cell 47 . The sheath liquid in the sheath liquid tank 6 is fed at a constant flow rate by the liquid feed pump 9, and flows along the inner wall of the flow cell 47 to the drain liquid reservoir 95.
is discharged. The liquid to be measured introduced into the flow cell is
Flows through the center of the sheath fluid flow.

The laser light source 49 has an oscillation wavelength of 488 nm.
The beam width of this laser beam is expanded by a beam expander 50, and then narrowed by a lens 51 and irradiated onto the sheath flow cell 47 so as to focus on the flow of the liquid to be measured. . A microscope objective lens 52 is used to collect the fluorescence from the flow cell 47.
is used. A spatial filter 54 and a wavelength selection filter 5 are installed in front of the photomultiplier 53 as a photoelectric detector.
5 to remove scattered light and Raman light. After the output of the photomultiplier 53 is amplified by the preamplifier 18, it is amplified by the linear amplifier 19, and noise is removed by the lower limit wave height discriminator 20a and the upper limit wave height discriminator 20b. Thereafter, a counter 56 integrates the pulses between the two threshold values.

A high voltage is applied to the photomultiplier 53 via a transformer 96 and a high voltage power supply 97. Sample number, counting results, calibration curve, fluorescence measurement histogram,
etc. are output to the display 57, printer 22, and floppy disk 58. It can also communicate with a personal computer via the interface 59.

A diagram of signal processing is shown in FIG. A pulse train 82 is obtained through a circuit 20 that discriminates the output 80 of the preamplifier based on the pulse height value. Counter 56
Accumulate with . In the other signal processing path, the output 80 is passed through a smoothing circuit 84, the number of pulses is converted into a voltage change to obtain a signal 85, and the voltage change is measured with a voltmeter 86. This smoothing measurement can also be performed, for example, by connecting a pen recorder and reading the recorded values.

An example of the analysis items by the analyzer in FIG. 1 is as follows:
Carcinoembryonic antigen CEA (CarcinoEmbryoni)
c Antigen), digoxin, and α-fetoprotein AFP. In this case, the immobilization antibody liquid container 90a on the reagent table 35 contains a liquid containing an anti-CEA antibody bound to a single-stranded oligonucleotide, and the immobilization antibody liquid container 90b contains a similarly bound anti-digoxin antibody. Further, the fixing antibody liquid container 90c contains a liquid containing a similarly bound anti-AFP antibody. Further, the labeled antibody liquid containers 36a, 36b, and 36c contain liquids containing an anti-CEA antibody, an anti-digoxin antibody, and an anti-AFP antibody, which are labeled with fluorescent fine particles, respectively.

Since the immune binding process for each analysis item is similar, the analysis operation process in the reaction vessel will be explained here using CEA as an example. The reaction vessels 34 on the turntable 32 all have the same configuration before starting the analysis operation. That is, as shown in Figure 3,
The inner wall of the glass reaction vessel 34 forms a solid phase 10, and a single-stranded oligonucleotide 15 is immobilized on the solid phase. Since the analysis item is specified by the type of immobilizing antibody added after the start of the operation, the same oligonucleotide 15 is immobilized in each of the reaction vessels 34 before the start of the operation. Oligonucleotide 15 is attached to a solid phase having amino groups as surface active groups.

On the other hand, the antibody solution container 90a contains restriction enzyme H.
A solution containing an immobilizing antibody in which an anti-CEA antibody is bound to a synthetic oligonucleotide having a base sequence AGGCCT that can be cleaved with ae I by maleimide thiol coupling is prepared. Here, A, G, T, and C are constituent units of nucleotides, and indicate, in order, an adenine base, a guanine base, a thymine base, and a cytosine base.

When the analysis operation is started, the nozzle 43 separates the antibody from the antibody liquid container 90a and discharges it into the corresponding reaction container 34, thereby converting the reaction container into C.
Specify that it is for EA analysis. By this, Figure 3
As shown, oligonucleotide 17 possessed by anti-CEA antibody 16 binds to oligonucleotide 15 having a complementary strand on the solid phase side. Therefore, this reaction vessel 34 is
Can recognize EA.

[0041] When a reaction vessel subjected to the above treatment is prepared before the start of the analysis operation, the reaction vessel can have a reaction solid phase that allows each analysis item to be recognized. Other analysis items can be specified by simply changing the solid phase antibody solution.

Next, a sample separated from the sample container 33 by the nozzle 43 is added to the reaction container 34 prepared in this manner. As shown in FIG. 4(a), CEA 11 in the sample binds to the anti-CEA antibody 16 immobilized on the solid phase 10. After this reaction is carried out for 15 minutes, the unreacted sample is sucked in from the reaction vessel 34 through the nozzle 43 and discharged into the cleaning tank 94. Next, reaction vessel 34
A buffer solution is added to the solution, which is suctioned and discharged through the nozzle 43, and such a washing operation is repeated several times.

Next, as shown in FIG. 4(b), a solution containing an anti-CEA antibody 24 labeled with latex particles 21 containing a coumarin derivative as a fluorescent substance,
A fixed amount of reagent is sucked in through the nozzle 43 from the container 36a on the reagent table 35 and discharged into the corresponding reaction container 34. The reaction vessel 34 maintains the reaction on a turntable for a certain period of time (15 minutes) at a predetermined temperature (37° C.). As a result, the labeled anti-CEA antibody 24 is bound to CEA11, and the solid phase 10
Fixed. At this time, as shown in FIG. 4(c),
Labeled anti-CEA antibody 24 that did not immunobind to CEA11
A part of a is directly adsorbed on the surface of the solid phase 10. The liquid containing excess labeled antibody in the reaction container 34 is sucked in by the nozzle 43 and discharged into the cleaning tank 94 . Then nozzle 43
A cleaning operation is repeated in which the cleaning liquid is discharged into the reaction container, and the cleaning liquid is further suctioned and discharged.

Next, by rotating the reagent table 35, the free reagent liquid container 38 is positioned at the suction position, and the container 3
A predetermined amount of the solution containing the restriction enzyme Hae I 25 in 8 is sucked in by the pipetting nozzle 43 and discharged into the corresponding reaction container 34 on the turntable 32 . Figure 4
As shown in (c) and (d), within the reaction vessel, the double strands of the oligonucleotide pair bound to the solid phase 10 are cleaved at a predetermined cleavage site, and free substances are released into the liquid within the reaction vessel 34. floats. A certain amount of time (15 minutes) is required for such a release reaction. When the base pair of the nucleic acid is cut midway by the restriction enzyme 25, two types of educts are present in the reaction vessel. One of them is sign 2
The other is a fluorescently detectable free body consisting of an antibody 24 with 1, an antigen 11, an immobilizing antibody 16, and a nucleic acid fragment, and the other is an immobilizing antibody that remains on the solid phase without being immunoconjugated. It is a free substance consisting of an antibody and a fragment of a nucleic acid that cannot be detected by fluorescence. The liquid to be measured containing these educts is sucked in by the pipetting nozzle 43 and discharged into the sheath flow cell 47 . During the release reaction using the restriction enzyme 25, the labeled antibody 24a directly adsorbed on the solid phase 10 is not released and is therefore not mixed into the liquid to be measured.

FIG. 5 shows an example of a calibration curve obtained by using latex particles 21 containing a coumarin derivative with a particle size of 0.1 μm and using a CEA standard sample instead of the above-mentioned sample. The quantitative linearity was high, especially at low concentrations, and there was no bending of the calibration curve at low concentrations, which is the case with detection methods using enzyme reactions. If one particle corresponds to one molecule and the detection limit is determined, the absolute amount is 10-
22 mol, the concentration was 10-18 mol/l or less.

In the analyzer shown in FIG. 1, even when a plurality of types of analysis items are to be analyzed, the reaction vessels 34 placed on the turntable 32 may be equipped with the same type of solid phase. If the relationship between the sample and the analysis item is input to the controller (computer) 46 from the input device, the controller 46
controls the operation of each part of the analyzer to measure the necessary analysis items in the desired order. For example, the measurement items are A, A, B.
, C, B in order, the immobilizing antibody is 90a
, 90a, 90b, 90c, and 90b are injected into each reaction vessel in this order and immobilized. The controller 46 also controls the samples to be dispensed so that they are measured in any order. Note that the sample should be diluted if necessary. After the sample reaction, the reaction container 34 is washed, and then the labeled antibodies 36a, 36b,
Dispense 36c. The labeled antibodies were measured for measurement item A, respectively.
Labels are bound to antibodies corresponding to B and C, so
The labeled antibodies 36a, 36a, 36b, 36c, 36b are also automatically selected by the controller 46 in the order of measurement items A, A, B, C, B specified at the beginning.

Next, a second embodiment based on the present invention is shown in FIG.
~Explained with reference to FIG. The immunoanalyzer shown in FIG. 6 uses the inner wall of a microplate as a solid phase,
An X, Y moving mechanism is used in place of the turntable in the first embodiment. In FIG. 6, a reaction microplate 61, a sample microplate 62, a labeled antibody 63, a free solution 64, and a washing solution 65 are placed on the top of the apparatus. The reagent section was kept cool by a cooler 66, and the reaction section was kept at 37° C. by a warmer 67. In addition, the reaction area and the labeled antibody 63 could be shaken using a vibrator 68.

The oscillation wavelength of the light source 69 is 632.8 nm.
A He-Ne laser light source was used. A light shielding plate 70 was provided in the photometry section to prevent external light from entering.

The laser beam was shaped by a prism 71 and focused by a lens 72. Fluorescence was collected by a lens 52, passed through a spatial filter 54 and an interference filter 55, and then detected by a semiconductor photodetector 73. The signal was amplified by an amplifier 74, selected by a pulse height discriminator 20, and then inputted to a counter 56. Data processing was performed by a computer 46, and an interface 59 was connected to enable communication with external equipment.

Output can be performed not only to a printer but also to an IC memory card 75. Initial settings of the device
It was stored in the C card, making startup easier.

Particles containing fluorescein and having particle sizes of 0.1 and 0.2 μm were used as fluorescent fine particles.

Deaerators 76 and 77 were installed to prevent air bubbles contained in the sheath liquid from affecting the air bubbles.

[0053] In order to feed the sheath liquid and the measurement liquid, a pneumatic pump using pressurized air may be used instead of using the syringe pump 40. In this case, there is an effect of reducing pulsating flow.

An example of immunoassay of CEA using the analyzer shown in FIG. 6 using a labeled antibody prepared by binding an antibody to commercially available fluorescent fine particles will be explained with reference to the schematic diagram shown in FIG.

[0055] Fluorescent particles with carboxyl groups introduced onto the surface (containing fluorescein; particle size 0.1 μm, Mol
ecular Probe Inc. ) 23 was conjugated with anti-CEA antibody 24 using water-soluble carbodiimide.

[0056] Anti-CEA antibody 26 was bound to a polystyrene microplate 61 to serve as a reaction solid phase.

[0057] Before the measurement operation, a standard solution of CEA was prepared and designated as sample 27. Sample 27 was added to each well 28 of microplate 61 on which anti-CEA antibody 26 had been immobilized and left at room temperature for a certain period of time. After washing, labeled antibody 29 labeled with fluorescent particles 23 was added and allowed to react for a certain period of time. After thorough washing, chaotropic ion release solution 64 was added,
Fluorescent particles 31 captured on the microplate were released by antigen-antibody reaction. A liquid containing liberated fluorescent particles was used as a liquid to be measured, and the number of particles in the liquid to be measured was counted. CEA
An example of a calibration curve for concentration is shown in FIG. 8a.

For comparison, horseradish peroxidase (POD), an enzyme, was used as a label.
A calibration curve was created by a method of detecting the amount of POD by reacting with a fluorescent substrate. In this case, the labeled substance (
POD) was released and transferred to another microplate and then subjected to enzymatic reaction for measurement (shown in Figure 8b), and the case where the enzymatic reaction and fluorescence measurement was performed in the microplate without being released (shown in Figure 8c). went.

Clearly, the result a according to the method of the present invention is
It can be seen that the background is low and the detection sensitivity at low concentrations is high.

[0060] In the particle counting device explained in Example 1,
We focused only on the pulse component of the signal obtained by the photomultiplier tube and counted the number of pulses. We have already explained that samples with a low particle number density give highly quantitative measurements, but in samples with a high particle number density, two or more particles pass through the laser beam at the same time, making it difficult to count them. Dropping leads to a decrease in the counting rate. On the other hand, in a highly concentrated sample, the generated fluorescence intensity increases, so even if the fluorescence intensity, that is, the signal, is smoothed with a certain time constant and its DC component is measured, a measured value proportional to the concentration will be given. therefore,
If both the number of pulses and the DC component value are measured simultaneously, the concentration range to be measured can be expanded. By employing these methods, we were able to increase the sensitivity of immunoassay by approximately two orders of magnitude.

[0061]

[Effects of the Invention] According to the present invention, accurate and highly sensitive measurement results can be obtained even if the amount of the analyte in the sample is minute, thereby greatly contributing to immunoassays.

[Brief explanation of the drawing]

FIG. 1 is a schematic configuration diagram of an embodiment of the present invention.

FIG. 2 is a diagram illustrating two systems of signal processing in the apparatus of FIG. 1;

FIG. 3 is a schematic diagram showing an example of immobilization of a receptor on a solid phase.

FIG. 4 is a schematic diagram for explaining the reaction state inside the reaction container.

FIG. 5 is a diagram showing an example of a calibration curve obtained by the apparatus of FIG. 1;

FIG. 6 is a schematic configuration diagram of another embodiment of the present invention.

7 is a diagram for explaining the operation when measuring CEA with the apparatus shown in FIG. 6. FIG.

FIG. 8 is a diagram comparing the results of measuring CEA standard samples.

[Explanation of symbols]

10...Solid phase, 11,27...Analyte (CEA), 15
, 17... Oligonucleotide, 16, 26... Solid phase receptor (anti-CEA antibody), 21... Fluorescent latex particle, 2
3, 31... Fluorescent particle, 24, 24a, 29, 63... Labeled antibody, 25... Restriction enzyme, 32... Turntable, 33
...Sample container, 35...Reagent table, 36a, 36b
, 36c... Labeled antibody liquid container, 38... Free reagent liquid container, 4
2... Movable arm, 43... Pipetting nozzle, 46...
Controller, 47... Sheath flow cell, 48... Injection chamber inlet, 56... Counter, 61, 62... Microplate, 90a, 90b, 90c... Antibody liquid container for fixation, 94...
Nozzle cleaning tank.

Claims (16)

[Claims] Claim 1: (a) supplying a sample to a reaction vessel having a solid phase to which a receptor is bound, and allowing the analyte in the sample to bind to the receptor; (b) discharging the unreacted sample into the reaction vessel; (c) supplying a labeled receptor labeled with fluorescent particles to the reaction vessel after removal from the reaction vessel to bind the labeled receptor to the solid phase via the analyte; (c) excess label; (d) providing a release reagent comprising a labeled release agent to the reaction vessel after the receptor is removed from the reaction vessel;
introducing a liquid to be measured containing fluorescent particles released from the solid phase into a flow cell; (e) detecting fluorescence based on the fluorescent particles passing through the flow cell and counting the number of detected particles; and (f) ) An immunoassay method characterized by calculating the concentration of an analyte in the sample based on the number of counted particles. 2. The immunoassay method according to claim 1, wherein the receptor is an antibody, and the analyte is an antibody or a hapten. 3. In the immunoassay method according to claim 1, the fluorescent particles are latex particles or inorganic particles with a fluorescent substance layer formed on their surfaces, or a mixture of a particle-forming composition and a fluorescent substance. An immunoassay method characterized in that the is made into particles. 4. The immunoassay method according to claim 1, wherein the particle size of the fluorescent particles is 10-5-10-3 mm. 5. The immunoassay method according to claim 1, wherein the free reagent contains a chaotropic ion. 6. An immunoassay apparatus for performing the analysis operation according to claim 1. 7. (a) supplying a sample to a reaction vessel having a solid phase to which a receptor is bound via a nucleic acid, and allowing an analyte in the sample to immunoconjugate to the receptor; (b) )
supplying a labeled receptor to the reaction vessel and binding the labeled receptor to the solid phase via the analyte; (c
) after removing excess labeled receptor from the reaction vessel, supplying a restriction enzyme-containing solution to the reaction vessel to cleave the nucleic acid; (d) a liquid to be measured containing the labeled substance released from the solid phase; and (e) counting the number of the labeled substances passing through the flow cell. 8. The immunoassay method according to claim 7, wherein the nucleic acid has base pairs. 9. The immunoassay method according to claim 7, wherein the nucleic acid is an oligonucleotide. 10. The immunoassay method according to claim 7, comprising:
The immunoassay method is characterized in that the solid phase is made of an inner wall of the reaction vessel or a spherical body housed in the reaction vessel, which has been surface-treated to enable chemical bonding to the receptor. 11. An immunoassay device for performing the analysis operation according to claim 7. (12) (a) preparing a plurality of reaction vessels each having a solid phase to which one of nucleic acids capable of forming a pair is bound;
(b) supplying a first receptor bound to the other nucleic acid capable of complementary binding to the one nucleic acid to a first reaction vessel of the plurality of reaction vessels; the receptor is capable of specifically binding to the first analyte in the sample; (c) the same receptor as the other nucleic acid is bound and specifically binds to the second analyte; (d) supplying a sample to the first and second reaction vessels, and (d) supplying a sample to the first and second reaction vessels; An immunoassay method characterized by proceeding with an immune reaction of the first analyte in a reaction vessel, and proceeding with an immune reaction of the second analyte in the second reaction vessel. 13. The immunoassay method according to claim 12, further comprising supplying a labeled receptor to the reaction vessel after the immune reaction, and removing excess labeled receptor from the reaction vessel.
An immunoassay method comprising: supplying a restriction enzyme-containing solution to the reaction vessel; and introducing a solution containing a labeled substance released from the solid phase into a flow cell as a liquid to be measured. 14. An immunoassay analyzer that performs the analysis operation according to claim 13. 15. Binding a particle label to a solid phase in a reaction vessel via a nucleic acid, supplying a restriction enzyme to the reaction vessel to cleave the nucleic acid, and binding a part of the nucleic acid after the cleavage. An immunoanalysis method characterized by guiding particle labels containing particles to a flow cell. 16. A reaction vessel having a solid phase on the inner wall,
A reaction vessel in which a receptor is bound to the solid phase via a nucleic acid.