JP3353978B2 - Optical specific affinity measuring method and specific affinity measuring device used therein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to, for example, an antigen-antibody reaction.
For measuring optical specific affinity for detecting chemiluminescence generated by a specific affinity reaction such as nucleic acid binding reaction and nucleic acid binding reaction, and a specific affinity measuring device used for the method.

[0002]

2. Description of the Related Art Measurement of biological substances is carried out as a daily test in the fields of environmental hygiene and medical care. In particular, in the medical field, many types of tests are performed at many facilities for the purpose of identifying a disease, determining a therapeutic effect on the disease, and the like. In particular, infectious diseases such as acquired immunodeficiency syndrome (AIDS), which have recently become public health problems,
Cancer-related substances, etc., which have been difficult to identify in the past, can be measured by using an antigen-antibody reaction, which is one of the reactions with a specific affinity substance. Diagnosis of infectious diseases represented by AIDS and the like can be detected using an antigen-antibody reaction.
It is also possible to measure A or RNA by a complementary nucleus binding to a characteristic portion of the nucleic acid.

[0003] The reaction between a nucleic acid and a complementary nucleic acid is one of the reactions using a specific affinity substance. In addition, a receptor reaction such as a reaction between insulin, which is one of the hormones, and an insulin receptor is also known. Have been.

There are many known analytical methods using the specific affinity reaction as described above, but all of them have in common that the amount of a substance bound to a specific affinity substance must be measured. That's what it means. As a method for measuring this substance, the fact that the specific affinity substance and the substance to be bound bind to each other and change the properties of the tracer bound to the substance itself or the substance to be bound is used. A homogeneous measurement method (homogeneous method) for determining the amount of a substance, and a method in which the complex of the specific affinity substance and the substance to be measured are made insoluble by some method, and then the substance not bound to the substance bound to the specific affinity substance B (bound) / F (Fre
e) It is broadly classified into a heterogeneous method requiring a separation operation.

[0005] Among these, the heterogeneous method is a method of adding a specific binding substance that binds to a complex with a specific affinity substance and a substance to be bound, and making the complex insoluble by making the complex a large molecule. Has been known for a long time, but is not widely used today due to lack of reliability.

In recent years, instead of this method, a specific affinity substance is previously bound to an insoluble substance, and the bound substance is separated from the unbound substance by separating the insoluble substance from the reaction solution. A method of performing B / F separation is general.

Further, a method using a substance other than the reaction tube such as a bead or a filter paper as an insoluble substance and a method using the reaction tube itself are known. Further, as a tracer, a method using a radioactive isotope has been generally used in the past. In recent years, an enzyme immunoassay (EIA) using an enzyme which is a non-radioactive substance has become widespread. In order to further increase sensitivity, a method using a luminescent substance as a tracer has been spreading recently. No. 245662.

[0008]

Some of the chemiluminescent substances give rise to an intense luminescence instantaneously in the presence of the corresponding substrate substance. In the method of emitting light after binding such a chemiluminescent substance as a tracer to the surface of an insoluble substance (for example, a reaction vessel or carrier particles), light is emitted only on the surface of the insoluble substance. It was difficult to measure. In particular, when a fine bead is used as the insoluble substance, the luminescence from the surface of the floating fine particles is reflected by the fine particles themselves. In addition, if the used fine particles are particles having a low reflection, a phenomenon called quenching occurs. On the contrary, it is difficult to accurately measure the light emission amount in any case.

[0009] It is an object of the present invention, the measurement accuracy high optical specificity parents can accurately detect the emitted light amount
An object of the present invention is to provide a method for measuring compatibility and a specific affinity measuring device used for the method.

[0010]

In order to achieve the above object, the invention of claim 1 is to provide a specific affinity binding complex obtained by using an insoluble substance with a first affinity binding compound after B / F separation.
In an optical immunoassay method, a reagent is added to dissociate the luminescence-inducing substance bound to the insoluble substance, and the second reagent is added to the luminescence-inducing substance, and the luminescence-inducing substance is chemiluminescent to detect the amount of light. After dissociating the luminescence-inducing substance, the insoluble substance in a floating state is made non-floating, and the insoluble substance and the luminescence-inducing substance are separated to cause the luminescence-inducing substance to emit chemiluminescence.

Further, the invention of claim 3 provides a luminescence-inducing substance which is obtained by adding a first reagent to a specific affinity binding complex obtained by using an insoluble substance after B / F separation and binding to the insoluble substance. Is dissociated, the second reagent is added to the luminescence-inducing substance, and the luminescence-inducing substance is chemiluminescent to detect the amount of light. In this state, an insoluble substance separating means for separating the insoluble substance and the luminescence inducing substance was provided.

In these inventions, the amount of emitted light can be accurately detected, and the measurement accuracy can be improved. It is known that an aridinium compound emits light rapidly in the presence of hydrogen peroxide under alkaline conditions. However, since hydrogen peroxide is unstable under alkaline conditions, a method of mixing an alkali solution and a hydrogen peroxide solution immediately before use and adding this to a microbead to which an acridinium compound is bound has been used as an automation method. Is common. Of course, a method of adding the first reagent to an insoluble substance to which an acridinium compound is bound in advance, stirring the mixture thoroughly, and then adding the next reagent is well known as a semi-automated method.

At this time, it was noticed that by using an acridinium ester compound as an acridinium compound, the ester bond could be cleaved under acidity before causing the phenomenon of chemiluminescence. And the acridinium compound dissolved in the supernatant of the solution can emit light.

Specifically, a susceptible microinsoluble substance (magnetic beads) to which an acridinium ester compound is bound is attached.
), Add an acidic solution containing hydrogen peroxide, stir to dissociate the acridinium compound from the insoluble substance, collect the insoluble substance in one place with an electromagnet, add the alkaline solution to the supernatant, add the acridinium compound To emit light.
Then, the light emission amount is detected by the photodetector.

When magnetic particles are used, the reaction container can be easily washed and the amount of light can be easily measured while being magnetically attracted to the inner wall of the container. This is preferable because obstacles in measurement such as scattering and absorption can be effectively avoided. When using suspending fine particles that do not contain a magnetic fluid, those having a particle size that can be separated by an appropriate filter or a specific gravity that can be centrifuged may be used. In some cases, fine particles are used in the luminescence reaction process by using low specific gravity particles such as hollow particles, or by adjusting the reaction vessel to hold an appropriate high specific gravity liquid at the time of addition of the luminescent reagent. If it is made to float stably near the surface of the water without being substantially suspended in the liquid, it is possible to effectively eliminate the effects of particles during measurement without going through centrifugation or magnetic operation. This is preferable in that the amount of light can be measured from the lateral direction or from below.

In addition to the use of a reagent immobilized on fine particles, a reaction vessel made of a transparent member having an antibody or the like immobilized on the inner wall thereof, or a spherical or other material having a diameter or length of 0.5 mm or more. The same operation and effect can be obtained by using a test piece having the shape of On the other hand, when a reaction vessel having an antibody or the like immobilized thereon is used, the washing solution can be simply and sufficiently used because there is no danger of aspirating the solid phase such as fine particles during the immunoreaction including B / F separation. In addition, it is preferable in that the measurement can be performed without being disturbed by a solid phase such as fine particles in the detection step.

If necessary, the reaction and B / F separation were carried out smoothly by immobilizing an antibody or the like on the inner wall of the flow path in the middle of the tube and controlling the flow rate in the tube. Thereafter, the acridinium compound is caused to flow into the highly transparent measurement cell together with the above-mentioned acidic solution, so that the light receiving efficiency can be improved.

In the present invention, the solidified acridinium derivative is released from the fine particles or the wall surface of the reaction vessel and diffuses into the liquid to generate uniform light emission. Therefore, if necessary, vibration of an appropriate intensity is applied so as to agitate the liquid in the reaction vessel to accelerate diffusion, or an appropriate injection speed and
A treatment such as injecting a luminescent reagent at an injection angle may be performed. The principle of measurement may be a sandwich method or a competitive method. As a luminescence reagent for initiating a luminescence reaction of an acridinium derivative, hydrogen peroxide can be mentioned. Generally, the luminescence reaction by the acridinium derivative and hydrogen peroxide emits light slightly under a predetermined alkaline condition, but emits strong light under an acidic condition.

In the method of the present invention, the acridinium ester compound is rapidly diffused into a liquid by breaking an ester bond under strong oxidizing conditions and, in some cases, dissociating from a protein binding portion. Therefore, to provide strong acid conditions, known strong acidic compounds, for example, hydrochloric acid, sulfuric acid, nitric acid, etc.
The pH at which the above-mentioned diffusion action can be obtained may be appropriately selected. More specifically, when a solution containing 0.1 N (N) nitric acid is brought into contact with the support after the reaction, a sufficient amount of the acridinium ester compound is dissociated in about several seconds, and the tendency becomes substantially saturated. The effective pH under such strong acid conditions is 3 or less, preferably 2 or less. In the present invention, the kind of the acridinium ester compound is not particularly limited, even if various acridinium ester derivatives are used, since the ester bond is broken under a strong acid condition.

On the other hand, under such a strong acid condition, the luminescence reaction can be temporarily suspended as described above, so that the luminescence reaction can be started at a desired timing according to the timing of addition of the alkaline substance. However, in measuring the luminescence reaction, since the time from the addition of the alkaline substance to the emission of luminescence is extremely short, it is preferable to perform the addition operation of the alkaline substance in a dark environment where light is blocked. A preferable timing for shortening the analysis time is immediately after a sufficient amount of the acridinium ester compound is dissociated and diffused in the solution. Therefore, an optimal time may be set for various reaction conditions by experiments. In the dissociation reaction using a 0.1 N nitric acid solution, an alkaline substance may be added after a few seconds.

[0021]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention will be described below with reference to FIGS. FIG. 1 shows a main part of an embodiment of the present invention, and reference numeral 1 in the figure denotes an immunoassay apparatus for a biological substance using chemiluminescence (hereinafter, referred to as a measurement apparatus). The measuring device 1 is provided with a light emitting reaction section 2, in which a photomultiplier tube (hereinafter referred to as PMT) 3 as a light detecting means is inserted into a main body 4. The main body 4 is a closed box, in which a photometric cell housing chamber 6 and a PMT housing chamber 7 separated by a wall 5 are formed. A first window 8 is formed in the wall 5, and a first shutter 9 that can be opened and closed is formed in the window 8.
Is installed.

The PMT 3 is mounted on a socket 10 arranged outside the main body 4 and covered by a PMT holder 11. Further, the light receiving side of the PMT 3 faces the window 8. A signal line cable 12 is led out of the socket 10, and the PMT 3 is connected to the signal processing device 13 via the signal line cable 12. A high-voltage power supply cable 14 is also led out of the socket 10,
MT3 is connected to high voltage power supply 15. And PM
A high voltage (for example, -1000 V) is applied to T3 from the high-voltage power supply 15, and the PMT 3 outputs a pulse signal corresponding to the intensity of the incident light to the signal processing device 13. The signal processing device 13 has a pulse counting function, an arithmetic function, and the like.

A cell holder 16 is provided in the photometric cell storage chamber 6, and a photometric cell 17 as a sample container is freely attached to and detached from the cell holder 16. The photometric cell 17 is for containing a reagent or a sample to be analyzed.
The cell holder 16 is provided with a photometric cell sensor 18 for detecting the presence or absence of the photometric cell 17. Further, an electromagnet 19 for collecting magnetic particles in the photometric cell 17 is built in the cell holder 16.

First and second dispensing probes 20 and 21 (first and second reagent adding means) are provided in the photometric cell accommodating chamber 5.
And a stirring bar 22. Double dispensing probe 2
Numerals 0 and 21 are connected to the dispensing driving sources 23 and 24 and the first and second reagent containers 25 and 26, and dispens a predetermined reagent to the photometric cell 17. The dispensing drive sources 23 and 24 and the first and second reagent containers 25 and 26 are arranged outside the main body 4. Here, general equipment such as a syringe can be used as the dispensing drive sources 23 and 24.

The stirring rod 22 is connected to an elevating and rotating mechanism 27 disposed outside the main body 4. When the photometric cell 17 is attached to and detached from the cell holder 16, the stirring rod 22 is raised so as not to hinder the operation, and is lowered when the mixed solution of the reagent and the sample in the photometric cell 17 is stirred. To enter the photometric cell 17.

The main body 4 is provided with a second window 28 which is opened facing the photometric cell accommodating chamber 6.
A second shutter 29 that can be opened and closed is attached to 8. The second window 28 connects the photometric cell 17 to the photometric cell accommodation room 6.
Used to get in and out. The second shutter 29 prevents disturbance light from entering the photometric cell housing 6.

Open / close sensors 30 and 31 are attached to the second shutter 25 and the first shutter 9, respectively.
Each of these shutters 9,
29 is detected.

Next, a photometric method performed by the above-described measuring apparatus 1 will be described. Here, the separation of the immune complex generated as a result of the immune reaction and the unreacted antigen or antibody is carried out by immobilizing one of the antigen or antibody on magnetic particles (insoluble substance, magnetizable substance, beads), The following describes an example of an immune reaction system that forms an immune complex (specific affinity binding complex) on magnetic particles.

In this embodiment, an acridinium ester compound (a substance that induces luminescence) that causes an instantaneous luminous phenomenon is used as a luminescent substrate for chemiluminescence. Further, it is assumed that a series of immune reactions up to chemiluminescence has already been performed in the photometric cell 17 and B / F separation has been completed.

First, the second shutter 29 is opened, and the B / F
The photometric cell 17 after the separation is mounted on the photometric cell holder 16. Then, the second shutter 29 is closed to block disturbance light. At this time, the electromagnet 19 of the cell holder 16 is in the OFF state, and the stirring bar 22 is up. Further, the first shutter 9 is closed, and the high-voltage power supply 15 is in an OFF state. After the first and second shutters 9 and 29 are confirmed to be closed and the photometric cell 17 is confirmed to be mounted on the cell holder 16, the first dispensing probe 20 and the second A predetermined amount of a hydrogen peroxide solution as one reagent is added to the photometric cell 17.

This solution is adjusted to be acidic in order to maintain the stability of hydrogen peroxide, but because of this liquidity, the ester of an acridinium ester compound bound to the magnetic particles via an immune complex. The bond is dissociated and the aridinium compound diffuses into the supernatant of the hydrogen peroxide solution. In order to promote this reaction, the stirring rod 22 stirs the magnetic particles in the photometric cell 17 and the hydrogen peroxide solution.

Next, the first shutter 9 is opened, and PM
The high voltage power supply 15 of T3 is turned on. This is left for about 10 to 30 seconds until the output of PMT3 is stabilized. During this time,
When the electromagnet 19 is turned on, a magnetic force is applied to the magnetic particles in the photometric cell to collect the magnetic particles at the bottom of the photometric cell 17.

Thereafter, the photometric cell 17 is again placed in the cell holder 1.
6, a predetermined amount of an alkaline solution as a second reagent is added from the second dispensing probe 21 to the photometric cell 17. With the addition of the alkali solution, the acridinium compound diffused in the supernatant liquid in the photometric cell 17 emits light instantaneously. Therefore, the photometric operation by the PMT 3 is started in conjunction with the dispensing of the alkaline solution.

When measuring faint light, the output of the PMT 3 is a pulse output. Therefore, the time for counting pulses is determined in advance, the number of pulses within that time is counted, and the amount of weak light is calculated. This method is a photometric method called a photon counting method. In this embodiment, this method can be adopted.

When an acridinium compound is used as in this embodiment, the total light emission can be measured by photometry for about 10 seconds. After the photometric operation is completed, the first shutter 9 is closed, and the high-voltage power supply 15 is turned off. Prior to photometry, the electromagnet 19 is turned off. This is to prevent the magnetic force of the electromagnet 19 from becoming a noise source. Further, after it is confirmed that the first shutter 9 is closed and the high-voltage power supply 15 is turned off, the second shutter 24 is closed, and the photometric cell 17 is taken out.

Then, the above operation is repeated, and luminescence immunoassay is performed on a plurality of samples. In the measuring device 1 as described above, the magnetic particles are collected by the electromagnet 19 and settle at the bottom of the photometric cell 17, the magnetic particles in the supernatant liquid are separated from the acridinium compound, and the supernatant liquid becomes a homogenous substance. Become. Therefore, reflection on the surface of the magnetic particles can be prevented. Further, there is no need to use fine particles having low reflection in order to prevent quenching. As a result, weak chemiluminescence can be accurately detected, and measurement accuracy can be improved.

The following are specific experimental examples and results. 1. EXPERIMENTAL EXAMPLE First, an antibody (Mitsubishi Kasei) against β2-microglobulin (β2-MG) was treated with magnetic particles (DYNABEAD) having a particle size of 4.5 μm.
10 μl of 4 × 10 ⁸ magnetic reagents (particle concentration w / v%) immobilized on 4 × 10 ⁸ cells / ml in S.DYNA1 Co., Ltd. were applied to each well of a microplate (Nunc, flat bottom) at various concentrations of β2- M
10 μl of a PBS solution containing G (pH 7.4) and 5 μl each at room temperature.
Allowed to react for minutes. Next, the magnetic particles after the reaction were collected on one inner wall of the well by applying a magnet to the side of the well for 15 seconds, and washed twice with a buffer solution to obtain the first magnetic particles.
A second B / F separation was performed. Next, similarly, 100 μl of an anti-immunoglobulin antibody labeled with acridinium derivative-I (Dojindo) was injected, reacted at room temperature for 5 minutes, and then washed. A 0.1 N nitric acid solution containing about 0.6% hydrogen peroxide solution (A) and the same nitric acid solution containing no hydrogen peroxide solution (B) were dispensed into separate wells by 50 μl each. The microplate was moved into a dark box (photometry cell storage chamber 6 in FIG. 1) to be shielded from light, and 0.25 N sodium hydroxide was added to each of the wells to which solution A was added and the wells to which solution B was added. The luminescence reaction was performed by adding 50 μl of each solution, and 1 second, 2 seconds and 3 seconds after the luminescence reaction started.
The number of photons due to light emission after 2 seconds was counted by a universal counter (Hamamatsu Photonics) to obtain an integrated value. (i) First immune reaction 100 μl of a reaction buffer was added to the wells of the microplate, and 10 μl of a well-stirred suspension of magnetic particles and 10 μl of a standard solution for a calibration curve were added and reacted at room temperature for 5 minutes.

In order to adjust the stirring conditions, only the addition and removal of the test solution was repeated several times only at the time of addition of reagents and the like, and no stirring was performed during the reaction. (ii) B / F separation After the completion of the immune reaction, one lanthanum-based magnet was pressed against the side of the microplate well for about 15 seconds to collect the magnetic particles on one inner wall of the well, and then the test solution was collected. Removed with a micropipette.

Thereafter, 250 μl of the washing solution was injected so as to disperse the magnetic particles, and the magnet was immediately pressed for about 15 seconds to collect the magnetic particles on the inner wall of the well, and the washing solution was removed with a micropipette.

By repeating this operation twice thereafter, the B / F
Separated. (iii) Second immune reaction 100 μl of the labeled antibody was added to the wells after the B / F separation, and the mixture was stirred for 5 minutes at room temperature after stirring by sucking and discharging the test solution. (iv) B / F separation B / F separation was performed by washing three times with 250 μl of the washing solution in the same manner as in (ii). (v) Luminescence reaction A well in which 50 μl of luminescence reagent A solution was added and magnetic particles were uniformly dispersed was set in a dark box for photocounting,
50 μl of the luminescent reagent B solution was added by a dispensing unit (RD) set to dispense the reagent into the well. (vi) Measurement of the amount of emitted light Using a universal counter (manufactured by Hamamatsu Photonics), under the conditions of an applied voltage of 1250 V, a gate time of 10 ms, and the number of data: 512, synchronized with the RD in the item (v). A measurement was made. The measurement results were expressed as integrated values for 3 seconds from the start of light emission. 2. Measurement Results The integrated value of the count for 3 seconds is shown in Table 1 below and FIG.

[0041]

[Table 1]

[0042]

[Table 2]

From these results, it is worrisome that the count number in the 0 ng / ml standard solution is slightly large.
It was confirmed that the calibration curve up to the standard solution showed linearity at a reaction time of 10 minutes.

Since the 200 ng / ml standard solution was lower than the expected count, this standard solution was
A dilution series was prepared by doubling the dilution with a standard solution of 00 ng / ml, and the linearity thereof was examined. The results are shown in Table 2 and FIG. 5 described above. It is better than the previous study, but still 200ng
It was found that / ml was measured to be low. From this, it is considered that under the present measurement conditions, a sample of about 200 ng / ml is in an antigen-excess state.

As a supplementary material, FIG. 6 shows the change over time of the luminescence phenomenon with each standard solution. This graph shows the number of counts per millisecond every 10 milliseconds.
It takes about one second for the light emission phenomenon, which indicates that the RD dispensing the reagent B solution takes about one second from the start to the actual dispensing of the reagent.

From the change over time of the luminescence phenomenon and the results of the measurements so far, it has been clarified that the measurement of the present reagent can be performed by the integrated value of the photo count for about 1 second from the dispensing of the luminescence reagent B solution. 3. Additional Consideration Since AE, which is a luminescent substance, is considered to dissociate from the surface of the magnetic particles and dissolve in the solution when reagent A solution is added,
When the magnetic particles are uniformly dispersed after the addition of the liquid (Case 1)
And when magnetic particles are forcibly collected on the bottom surface (Case 2)
The authors examined which is more advantageous for measuring the amount of emitted light. The results of the study are shown in Table 3 below.

[0047]

[Table 3]

As a result of the study, it was found that the number of measurement counts increased by about 10% when the magnetic particles were collected on the bottom surface.
Although no clear result was obtained with respect to reproducibility, it seemed that collecting magnetic particles on the bottom surface was slightly better.

The present invention can be variously modified without departing from the gist. For example, in the present embodiment, the electromagnet 19 is disposed in the main body 4 and the magnetic particles are collected in the main body 4, but the present invention is not limited to this, and the operation of the magnetic collection is not limited to this. May be performed outside the main body 4. That is, since the electromagnet 19 becomes a noise source to the PMT 3, it is desirable to keep the distance between the PMT 3 and the electromagnet 19 as far as possible or to provide an electromagnetic shield between them. Therefore, by arranging the electromagnet 19 outside the main body 4 and performing magnetic flux collection outside the main body 4, noise can be further reduced.

In this case, the first dispensing probe 20 is also disposed outside the main body 4, and the operations from the addition of the hydrogen peroxide solution to the subsequent stirring and the magnetic collection of the magnetic particles are performed on the main body 4. It is conceivable that the operation is performed outside and only the operation after the operation of adding the alkali solution is performed in the main body 4.

Although the electromagnet 19 is provided in this embodiment, if the magnetic particles can be collected on the wall surface of the photometric cell 17 using gravity or other force, the electromagnet 19 becomes unnecessary.

Further, as shown in FIG. 7, for example, a curved portion may be formed in the photometric cell 41 and a stirring section 42 may be provided.
That is, the stirring part 42 is continuous with the vertical side surface 43,
The reagent is dispensed along side 43. And the reagent is
First dispensing probe 20 or second dispensing probe 21
Is dispensed from an appropriate angle direction. At this time,
The reagent is injected along the side surface 43 of the cell 41. The reagent after the injection reaches the bottom portion 45 from the side surface 43 via the curved surface 44 and finally reaches the B / F already contained.
Since the solid phase particles after separation are efficiently involved and the chance of contact is increased, a sufficient stirring effect can be obtained without using the stirring rod 22. This is particularly preferable in that the number of parts in the shielding structure as shown in FIG. 1 is reduced. Also, unlike stirring by vibration, the positioning structure of the photometric cell 41 is simplified, so that the entire photometric device is reduced in size.

[0053]

As described above, the first aspect of the present invention is:
The specific affinity binding complex obtained using the insoluble substance,
After the B / F separation, the first reagent is added to dissociate the luminescence-inducing substance bound to the insoluble substance, and the second reagent is added to the luminescence-inducing substance.
In an optical specific affinity measurement method in which a reagent is added and chemiluminescence is caused by the luminescence-inducing substance to detect the amount of light, after the luminescence-inducing substance is dissociated, the insoluble substance in a floating state is changed to a non-floating state, and the insoluble substance is removed. And the luminescence inducing substance are separated from each other to cause the luminescence inducing substance to emit chemiluminescence.

Further, the invention of claim 3 relates to a luminescence-inducing substance which is obtained by adding a first reagent to a specific affinity binding complex obtained using an insoluble substance after B / F separation and binding to the insoluble substance. were dissociated, for detecting the amount of light added to the light emission inducing substance of the second reagent is a chemiluminescent onto light emission inducing substance specific affinity
The insolubility measuring device is provided with an insoluble substance separating means for dissociating the insoluble substance in a floating state by dissociation from the luminescence inducing substance, and separating the insoluble substance from the luminescence inducing substance. According to these inventions, the amount of emitted light can be accurately detected, and the measurement accuracy can be improved.

[Brief description of the drawings]

FIG. 1 is a configuration diagram showing a main part of an embodiment of the present invention.

FIG. 2 is a cross-sectional view showing a light emitting reaction section.

FIG. 3 is a flowchart showing an immunoassay method according to one embodiment of the present invention.
Chart.

FIG. 4 is a graph showing experimental results.

FIG. 5 is a graph showing experimental results.

FIG. 6 is a graph showing experimental results.

7A and 7B show a modified example of the photometric cell, wherein FIG. 7A is a front view and FIG. 7B is a side view.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Immunoassay device, 3 ... Photomultiplier tube, 13 ... Signal processing device, 17 ... Photometry cell, 19 ... Electromagnet (insoluble substance separation means, magnetism collection means), 22 ... Stirring bar, 25 ... Hydrogen peroxide solution (No. 1 reagent), 26 ... alkaline solution (second reagent).

Continuation of the front page (56) References JP-A-5-255264 (JP, A) JP-A-4-301764 (JP, A) JP-A-7-12731 (JP, A) JP-A-2-245662 (JP) JP-A-5-18971 (JP, A) JP-A-5-18970 (JP, A) JP-A-4-102062 (JP, A) JP-A-61-41966 (JP, A) (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 33/543 G01N 33/532 G01N 21/75-21/83

Claims (4)

(57) [Claims] A first reagent is added to a specific affinity binding complex obtained using an insoluble substance after B / F separation to dissociate a luminescence inducing substance bound to the insoluble substance, In the optical specific affinity measurement method in which the second reagent is added to the luminescence inducing substance and the luminescence inducing substance is chemiluminescent to detect the amount of light, the luminescence inducing substance is dissociated and then floated. The insoluble substance in the state is in a non-floating state,
An optical specific affinity measurement method, comprising separating the insoluble substance and the luminescence inducing substance and causing the luminescence inducing substance to chemiluminescent. 2. The optical device according to claim 1, wherein the insoluble substance is brought into a non-floating state by a magnetic force.
Specific affinity measurement method. 3. A B-F separation and a first reagent are added to the specific affinity binding complex obtained using the insoluble substance to dissociate the luminescence inducing substance bound to the insoluble substance, In a specific affinity measuring apparatus for detecting the amount of light by adding a second reagent to the luminescence-inducing substance and causing the luminescence-inducing substance to emit chemiluminescence, the insoluble substance dissociated from the luminescence-inducing substance and in a floating state is not suspended. A specific affinity measuring device, wherein the apparatus is in a state, and provided with an insoluble substance separating means for separating the insoluble substance and the luminescence inducing substance. 4. The apparatus according to claim 3, wherein the insoluble substance is a magnetizable substance, and the insoluble substance separating means is a magnetic flux collecting means for making the insoluble substance non-floating using a magnetic force. Specific affinity measurement device.