KR101495665B1 - Method for magnetic transfer multi-fluorescence immunoassay - Google Patents

Abstract

The present invention relates to an immunodiagnosis method using a magnetic. More specifically, since a target antigen bound to a magnetic assembly of the present invention in a clinical specimen directly moves by the magnetic, time occupied on a test is reduced. Accordingly, the present invention can be used for an immunodiagnosis method and an immunodiagnosis device.

Description

[0001] The present invention relates to a magnetic multi-fluorescence immunoassay

The present invention relates to a fluorescence multiple immunoassay method using magnetism.

In the field of clinical testing, various types of diseases are diagnosed by using biological samples (blood, urine, etc.). As such diagnostic methods, various measurement methods have been developed and used. Representative methods of such a measurement method include biochemical assay using an enzyme reaction or immunoassay using an antigen-antibody reaction. In recent years, it is required to precisely measure components in living body samples, and immunoassay methods using an antigen-antibody reaction with high specificity are widely used.

Immunoassays include radioimmunoassay (RIA) for detecting signals using radioactive isotopes according to their detection principles and methods, enzyme-linked immunosorbent assay (ELISA) using signal amplification using enzymes, (EIA), fluorescence antibody (FA) detection using fluorescence, and chemiluminescence immunoassay (CLIA) using chemiluminescence. In addition, the labeling substance Depending on the method of use and the type of substrate, various classification is possible.

Among the various immunoassays, enzyme immunoassay has excellent sensitivity, specificity, rapidity and reproducibility of reactions, and has advantages of versatility that can be used for detecting various kinds of antigens and antibodies by the same manipulation when different antigens or antibodies are used . Enzyme immunoassays can be subdivided into three categories: Direct ELISA, Indirect ELISA, and Sandwich ELISA, depending on how the antibody is used.

Specifically, in the direct enzyme immunoassay, when an enzyme linked to an enzyme immobilized on a solid surface is bound (an enzyme-linked Antibody), the enzyme catalyzes the substrate reaction to generate a signal. In indirect enzyme immunoassay, the primary antibody binds specifically to the antigen, and the secondary antibody to which the enzyme is attached recognizes and binds to the main antibody. In the ideal state, the enzyme linked to the secondary antibody catalyzes the reaction of the substrate. Finally, sandwich enzyme immunoassay, which is the most widely used form, uses two antibodies with different epitopes for one antigen to be detected and shows high selectivity for the antigen to be detected, Is also being used a lot.

However, the enzyme immunoassay has a disadvantage in that a washing time is required for at least 2 hours to remove unbound substances after an antigen-antibody reaction time and an antigen-antibody reaction. In addition, the absorbance of a marker or the like is measured for the detection of a substance to be inspected. However, there is a disadvantage in that it is impossible to detect a trace amount of absorbance.

Accordingly, the present inventors have shortened the time required for the experiment while maintaining the merits of the ELISA and have attempted to detect a very small amount of the target antigen. As a result, the magnetic binders are directly moved by the magnets, And detecting a marker bound to the antigen using a photomultiplier tube fluorescence detector, thereby completing the present invention.

SUMMARY OF THE INVENTION

1) a first moving step of moving a magnetic binding body including a magnetic bead and a first antibody to a first well containing a sample using a magnet;

2) a first binding step in which the magnetic binding substance and a target antigen in the sample bind to each other to form a magnetic binding substance-antigen complex;

3) a second moving step of moving the magnetic binding body-antigen complex to a second well containing a marker using a magnet;

4) a second binding step of binding the magnetic binding body-antigen complex to the marker to form a magnetic binding body-antigen-marker complex; And

5) detecting the magnetic binding body-antigen-marker complex.

Another object of the present invention is to provide

A well portion including a well into which a sample is dispensed;

A magnet portion composed of a magnetic bar and a magnetic bar cap capable of covering the magnetic bar;

A fluorescence detector capable of detecting fluorescence of a marker bound to a specimen in the sample; And

And a moving unit capable of moving the well to the fluorescence detection unit.

It is another object of the present invention to provide a method for performing single or multiple immunoassays,

Characterized in that a magnet for realizing the magnet portion and the fluorescence detecting portion in one automated apparatus is used.

In order to accomplish the above object, the present invention provides a method for preparing a magnetic bead, comprising: 1) a first moving step of moving a magnetic binding body including a magnetic bead and a first antibody to a first well containing a sample using a magnet;

2) a first binding step in which the magnetic binding substance and a target antigen in the sample bind to each other to form a magnetic binding substance-antigen complex;

3) a second moving step of moving the magnetic binding body-antigen complex to a second well containing a marker using a magnet;

4) a second binding step of binding the magnetic binding body-antigen complex to the marker to form a magnetic binding body-antigen-marker complex; And

5) a step of detecting the magnetic binding body-antigen-marker complex.

The present invention also relates to a method of measuring a sample comprising: a well portion comprising a well into which a sample is dispensed; A magnet portion including a magnetic bar and a magnetic bar cap covering the magnetic bar; a fluorescence detecting portion capable of detecting fluorescence of a marker bound to the specimen in the sample; And a moving unit capable of moving the well to the fluorescence detection unit.

The present invention provides an immunoassay method using a magnetic binding substance, so that the objective antigen in the sample bound to the magnetic binding substance is directly moved by the magnet, thereby shortening the time required for the examination. In addition, since the specimen is moved, each antibody and a marker for a plurality of target antigens can be used to be useful for multiple tests.

FIG. 1 is a diagram illustrating an ELISA of a general enzyme immunoassay,
FIG. 2 is a graphical illustration of an experiment of fluorescence multiple immunoassay using magnetic,
FIG. 3 is a diagram illustrating a test flow in which the present invention is manually implemented,
4 is a device diagram of a magnetic fluorescence multiple immunoassay.

Hereinafter, terms used in the present invention will be described.

The term " magnetic bead " used in the present invention means small particles having magnetic particles inside and coated with silica and various functional groups on the outside.

The term " antibody " as used herein also means an immunoglobulin molecule that is immunologically reactive with a particular antigen. A form produced by genetic engineering, an antibody fragment having an antigen binding ability, but it means a monoclonal antibody for the purpose of the present invention.

The term " first antibody " used in the present invention means an antibody that specifically binds to a target antigen and binds to the magnetic bead.

The term " second antibody " used in the present invention means an antibody that specifically binds to a target antigen and binds to a marker.

The term " magnetic binding body " used in the present invention means a binding body in which the first antibody binding specifically to the magnetic bead is bound to the magnetic bead.

The term " magnetic binding body-antigen complex " used in the present invention means a complex in which the magnetic binding body and the antigen are bound.

The term " magnetic binding body-antigen-marker complex " used in the present invention means a complex in which the magnetic binding body-antigen complex and the marker are combined.

The term " marker " as used herein also means a complex comprising a marker binding to a second antibody and a second antibody.

The term "EDC [N-ethyl-N '- (dimethlaminopropyl) carbodiimide hydrochloride" hereinafter referred to as EDC) coupling "used in the present invention means that a carboxyl group is functionalized on the surface of a magnetic bead or the like .

Hereinafter, the present invention will be described in detail.

The present invention

1) a first moving step of moving a magnetic binding body including a magnetic bead and a first antibody to a first well containing a sample using a magnet;

2) a first binding step in which the magnetic binding substance and a target antigen in the sample bind to each other to form a magnetic binding substance-antigen complex;

3) a second moving step of moving the magnetic binding body-antigen complex to a second well containing a marker using a magnet;

4) a second binding step of binding the magnetic binding body-antigen complex to the marker to form a magnetic binding body-antigen-marker complex; And

5) a step of detecting the magnetic binding body-antigen-marker complex.

The most commonly used ELISA method is a method in which a target antigen specifically binds to a first antibody (main antibody) immobilized on a surface as shown in FIG. 1, and a second antibody After recognizing and binding the first antibody-antigen complex, the fluorescence of the marker is finally detected and the target antigen is detected.

Specifically, the present invention relates to a method for detecting a target antigen by moving a magnetic binding body to a first well containing a sample using an external magnet as shown in FIG. 2, detecting a target antigen with a magnetic binding body attached to the magnet, Antigen complex is transferred to a plate containing a washing solution to block unreacted residues on the surface of the magnetic binding substance through washing. Thereafter, the magnetic binding body-antigen complex attached to the magnet is transferred to a second well containing the marker to form a magnetic binding body-antigen-marker complex.

In addition, the present invention may further include a washing step in which the magnetic binding body-antigen complex is moved to a plate containing a washing solution using a magnet before the step 3), but the washing step is not limited thereto.

In addition, the present invention may further include, before step (5), washing the magnetic binding body-antigen-marker complex with a plate containing a washing solution by using a magnet, but the present invention is not limited thereto.

The washing step may be performed using buffer solutions such as Tris-HCL, Tris borate EDTA, Tris-buffered saline (PBS), phosphate buffered saline (PBS), and Bovine serum albumin (BSA) (-COOH) remaining in the magnetic beads. However, the present invention is not limited thereto and can be achieved by any known buffer solution known to those skilled in the art.

In addition, the marker of the present invention may include, but is not limited to, a second antibody that binds to the magnetic binding complex-antigen complex.

In addition, the detection step of the present invention may be characterized by measuring the fluorescence value of the magnetic binding body-antigen-marker complex, but is not limited thereto.

The fluorescence value may be measured using a fluorescence spectrophotometer, a spectrophotometer, a fluorescence spectrophotometer, an ultraviolet-visible spectrophotometer, a microplate reader or the like, but is not limited thereto.

Further, the magnetic beads of the present invention is neutral carboxyl group (-COOH) on the surface of magnetic beads, an ester group (-COO ^-), a neutral amine group (-NH _2), neutral ion of an amine group (-NH ^-, or NH ₃ ^-), neutral thiol (-SH) and ion (-S neutral thiol group ^- but can contain any of the functional groups selected from the group consisting of a), and the like.

In the magnetic bead combination of the present invention, the magnetic beads may be selected from the group consisting of antibodies, streptavidin-biotin bonds, avidin-biotin bonds, EDC couplings, sulfhydrylamine couplings, Ni-NTA-histidine bonds, However, the present invention is not limited thereto.

In addition, the marker of the present invention may be a fluorescent substance or an ultraviolet (UV) marker which binds to the second antibody. Specifically, the fluorescent substance or ultraviolet (UV) markers may be selected from the group consisting of FITC (Fluorescein isothiocyanate), PE (phycoerythrin), TRITC (tetramethyl-rhodamine isothiocyanate), Cy3 (Cyanine 3), Cy5 (Cyanine 5) and Rhodamine But it is not limited thereto.

In addition, the present invention provides a method for measuring a sample comprising: a well portion including a well to which a sample is dispensed;

A magnet portion composed of a magnetic bar and a magnetic bar cap capable of covering the magnetic bar;

A fluorescence detector capable of detecting fluorescence of a marker bound to a specimen in the sample; And

And a moving unit capable of moving the well to the fluorescence detection unit.

In addition, the wells of the present invention are composed of 6 well plate, 12 well plate, 18 well plate, 24 well plate, 36 well plate, 48 well plate, 96 well plate, 128 well plate, 256 well plate and 384 well plate And a well plate selected from the group consisting of:

In the wells of the present invention, the sample is dispensed in one row, the magnetic coupling substance is dispensed in the two columns, the washing solution is dispensed in the columns 3 and 5, the labeled complex is dispensed in the four columns, But the present invention is not limited thereto.

In addition, the present invention provides a fluorescence multiple immunoassay apparatus that uses magnet for realizing the magnet unit and the fluorescence detection unit as one automated apparatus, but is not limited thereto.

In a specific example of the present invention, 10 μl magnetic beads (7-12 × 10 ⁹ beads / ml) were mixed with EDC [N-ethyl-N '- (dimethlaminopropyl) -carbodiimide hydrochloride, (-NH ₂ ) of the first thrombin-binding antibody to the carboxyl group (amide binding) by providing a carboxyl group (-COOH) as a functional group on the surface of the magnetic bead by the EDC coupling method The absorbance at 260 nm was measured by a spectrometer, and the binding of the magnetic binding substance was confirmed.

Also, in a specific embodiment of the present invention, in order to block unreacted functional groups formed on the surface of the magnetic beads after the EDC solution treatment, it is preferable that after the preparation of the magnetic binding body, after the preparation of the magnetic binding body- (0.1% BSA [bovime serum albumin] and 0.05% Tween 20) were added to each step of each antigen-marker complex, and the mixture was incubated at room temperature (200 rpm) .

Further, in a specific embodiment of the present invention, the present inventors used a second thrombin monoclonal antibody in 15 ml of FITC (BD Biosciences) in a 0.5 ml of borate buffer (pH 8.5, 0.1 M) , USA), and the mixture was reacted at room temperature in a dark room for 2 hours. The solution was purified by desalting column, and absorbance was measured at 280 nm and 496 nm to determine the concentration of the second thrombin As a result of checking whether the monoclonal antibody binds with FITC, a marker could be obtained.

Furthermore, in a specific example of the present invention, the inventors of the present invention have found that, in order to detect the magnetic conjugate-antigen complex, The magnetic binding substance (10 μl, 7-12 × 10 ⁹ beads / ml) was mixed with the PBS solution, and the solution was allowed to react at 37 ° C for 10 minutes while stirring (200 rpm) , And a magnetic binder-antigen complex.

In addition, in a specific example of the present invention, the present inventors measured the magnetic binding body-antigen (thrombin) complex and the marker of Example 2 in a PBS solution at 37 DEG C for 10 After incubation for a few minutes, the mixture was irradiated with visible light at 488 nm, and visible light at 520 nm was measured. As a result, the target antigen (thrombin) could be detected by detecting the magnetic binding agent-antigen-marker complex.

(HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis C virus (HCV) in order to perform fluorescence multiple immunoassay Antigen complexes were prepared by binding each fluorescent substance to each antigen (specifically, the fluorescent substance for HAV was labeled with 'Cy3', HBV (Cy3: 544 nm, FAM: 492 nm, and Fluo-4: 492 nm) specific for each marker fluorescent material were used for the fluorescence detection After irradiation of visible light (590 nm for Cy3, 518 nm for FAM, and 518 nm for Fluo-4), fluorescence multiple immunization was performed on the three antigens to detect the target antigen.

Further, in a specific example of the present invention, the inventors of the present invention conducted experiments in order to realize a fluorescence multiple immunoassay in an automated instrument, in which a well of a sixth row in which a buffer was dispensed was moved to the back surface of an FGM using an XY stepper Luminescence values of the marker conjugated with the specimen were measured using a fluorescence optical apparatus mounted on the back side of the instrument, and the target antigen could be detected using an automated fluorescence multiple immunoassay.

Therefore, the immunoassay method of the present invention can directly collect the target antigen in the specimen by the movement of the magnetic substance, so that the experiment time can be greatly shortened and can be usefully used in the immunoassay method.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

≪ Example 1 > Preparation of magnetic coupling body

In order to detect an antigen using magnetism, the inventors of the present invention have found that a first thrombin monoclonal antibody is bound to a magnetic bead by EDC [N-ethyl-N '- (dimethlaminopropyl) -carbodiimide hydrochloride / RTI >

First, 10 ㎕ magnetic beads (7-12 x 10 ⁹ beads / ml) were prepared by washing with 0.01 M NaOH aqueous solution and deionized water. After washing, the cleaning solution was exposed on the magnet for 1 minute to separate the magnetic beads.

Next, 500 μl of EDC solution (20 mg / ml) was added to the magnetic beads so as to form a carboxyl group on the surface of the magnetic beads, and the mixture was incubated at 4 ° C for 30 minutes in a shaking incubator (200 rpm).

After incubation, the unreacted EDC solution was removed and the remaining magnetic beads were resuspended in 1000 μl of PBS (phosphate buffered saline), mixed with a first thrombin monoclonal antibody (Abcam (ab8926), UK) (200 rpm).

After the binding, the mixture of the magnetic beads and the first thrombin monoclonal antibody was exposed on the magnet for 1 minute to remove unbound antibody. (The amount of bound antibody was analyzed by measuring the absorbance at 260 nm with a spectrometer.) After that, the antibody-bound magnetic beads were washed three times with 1000 μl of PBS (10 mM, pH 7.4).

After washing, 1000 μl of a buffer solution (0.1% BSA [bovime serum albumin] and 0.05% Tween 20) was added to the reactor to protect unreacted carboxyl groups on the surface of the magnetic beads, and the mixture was stirred at room temperature (200 rpm). The solution was then exposed to a magnet to separate the magnetic binding-antigen complex

After the incubation, the mixture was resuspended in 1000 μl of PBS (10 mM, pH 7.4) solution to prepare a magnetic binding substance capable of binding to the antigen.

≪ Example 2 > Preparation of markers

The present inventors produced a 'marker' for detecting the magnetic binding body-antigen complex detected in the <Experimental Example 1> by the following method.

A 'marker' was prepared by combining a second thrombin monoclonal antibody ([5G9] (ab7199)) with Fluorescein isothiocyanate (hereinafter FITC).

Specifically, the second thrombin monoclonal antibody was added to a solution containing 15 μl of FITC (BD Biosciences, USA) in 0.5 ml of a borate buffer (pH 8.5, 0.1 M) For 2 hours.

Thereafter, the solution was purified with a desalting column, and the absorbance at 280 nm and 496 nm was measured to confirm binding of the second thrombin monoclonal antibody to FITC. As a result, a marker could be obtained.

<Experimental Example 1> Detection of magnetic binding body-antigen complex

The present inventors collected the antigen (thrombin) using the magnetic binding substance prepared in the above <Example 1> according to the following experiment.

Specifically, 10 μl of the magnetic binding substance of Example 1 (7-12 × 10 ⁹ beads / ml) was added to a PBS solution containing 1 ng / ml to 1 μg / ml of thrombin (Abcam (ab48626) ). For detection of the target antigen thrombin, the solution was subjected to a binding reaction with stirring (200 rpm) at 37 DEG C for 10 minutes.

After the binding reaction, a PBS solution containing the magnetic binding substance and the antigen (thrombin) was exposed to magnetism at room temperature to isolate the magnetic binding substance-antigen complex.

The supernatant of the solution was transferred to a separate tube to measure the amount of thrombin captured.

The amount of collected thrombin was measured by Micro BCA method.

The magnetic complex-thrombin (antigen) complex was then washed five times with 1000 μl of PBS (10 mM, pH 7.4) solution.

After washing, 1000 μl of a buffer solution (0.1% BSA [bovime serum albumin] and 0.05% Tween 20) was added to the reactor to protect unreacted carboxyl groups on the surface of the magnetic beads, and the mixture was stirred at room temperature (200 rpm). After incubation, the solution was exposed to a magnet for one minute to remove the buffer solution.

After resuspension in 1000 占 퐇 of a solution of PBS (10 mM, pH 7.4), a magnetic coupling-antigen complex was obtained using a magnet.

<Experimental Example 2> Detection of magnetic coupling agent-antigen-marker complex

The present inventors conducted the following experiment to detect a magnetic binding body-antigen-marker complex.

Specifically, the above-mentioned magnetic binding substance-antigen (thrombin) complex and the marker of Example 2 were incubated in a PBS solution at 37 ° C for 10 minutes, and then visible light of 488 nm was irradiated to obtain visible light of 520 nm Measured

As a result, the target antigen (thrombin) could be detected by detecting the magnetic binding body-antigen-marker complex.

Experimental Example 3: Fluorescence multiple immunoassay

The present inventors performed the following fluorescence multiple immunoassay to perform fluorescence multiple immunoassay.

In order to simultaneously detect three types of antigens, hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV), the same method as in Examples 1 and 2, Antigen complexes.

HBV Ab1) and antibody 2 (hereinafter referred to as HBV Ab2), hepatitis B virus (HBV) antibody 1 (hereinafter referred to as HBV Ab1) and antibody 2 (hereinafter referred to as HBV Ab2) HAV Ab1, HBV Ab1 and HCV Ab1 were each bound to magnetic beads to prepare a magnetic binding body, and HAV (humanized antibody) Ab2, HBV Ab2 and HCV Ab2 were bound to Cy3 (HAV Ab2), FAM (HBV Ab2) and Fluo-4 (HCV Ab2), respectively.

For fluorescence multiple immunoassay, blood samples were dispensed in one 96-well plate, and each of the magnetic binders in the second column was dispensed in the same amount of 0.1 mg / ml, respectively. In the third column, 1000 μl (Cy3-HAV Ab2, FAM-HBV Ab2, and Fluo-4-HCV Ab2) were dispensed in the same amount of 0.1 mg / ml each in the column 4, Was dispensed with the same washing solution as in the third column. In column 6, the buffer was dispensed.

Specifically, the two rows of magnetic binding bodies were moved to a row of the divided samples using a magnet, and then reacted for 10 minutes to bind the magnetic binding body and the antigen. At this time, to accelerate the antigen-antibody reaction, heating was carried out through an aluminum block mounted on the bottom at 37 ° C., and the plastic bar cap was moved up and down with the magnetic bar on the upper part to promote the binding reaction. Thereafter, a row of magnetic bond-antigen complexes was moved in three rows using a magnet and washed in a washing solution for 2 minutes to block the carboxyl residue on the surface of the magnetic beads. Thereafter, the magnetic bead-antigen complex was transferred to four rows using a magnet and reacted for 10 minutes. As a result, the magnetic conjugate-antigen complex and the marker were bound to each other. Thereafter, the magnetic coupling agent-antigen-marker conjugate was transferred to five columns using a magnet, and then washed in a washing solution for 2 minutes, thereby removing impurities.

The washed column-bound magnetic binder-antigen-marker complex of the fifth column was put into six-row buffers using a magnet again. The samples contained in the 6 columns were put into a microplate fluorescence detector and fluorescence values were measured. Specifically, hepatitis A was irradiated with visible light at 544 nm and measured at 590 nm. For hepatitis B, visible light at 492 nm was irradiated and 518 nm was measured. For hepatitis C, 485 nm visible light was irradiated and 518 nm Were measured.

As a result, it was possible to detect the target antigen by performing fluorescence multiple immunization by detecting the magnetic binding substance-antigen-marker complex for the three antigens (hepatitis A, B and C).

<Experimental Example 4> Detection of antigens using a fully automated fluorescence multiple immunoassay

The inventors of the present invention conducted the following experiment to implement the fluorescence multiple immunoassay in an automated instrument.

Specifically, the wells in the sixth column of Experimental Example 3 were moved to the back surface of a fully automated fluorescence multiple immunoassay using an XY stepper motor, and then the specimen Fluorescence values of the combined markers were measured.

As a result, the target antigen could be detected using an automated fluorescence multiple immunoassay instrument.

Claims (13)

1) a first moving step of moving a magnetic binding body including a magnetic bead and a first antibody to a first well containing a sample using a magnet;
2) a first binding step in which the magnetic binding substance and a target antigen in the sample bind to each other to form a magnetic binding substance-antigen complex;
3) a second moving step of moving the magnetic binding body-antigen complex to a second well containing a marker using a magnet;
4) a second binding step in which the magnetic binding body-antigen complex and the indicator bind to each other to form a magnetic binding body-antigen-marker complex; And
5) A method for immunoassay comprising the step of detecting the magnetic binding body-antigen-marker complex.
The immunoassay method according to claim 1, further comprising, before step 3), moving the magnetic coupling-antigen complex to a plate containing a washing liquid by using a magnet.
The immunoassay method according to claim 1, further comprising, before step (5), washing the magnetic binding substance-antigen-marker complex with a magnet using a plate containing a washing solution.
The method of claim 1, wherein the marker comprises a second antibody that binds to the magnetic binding complex-antigen complex.
2. The immunoassay method according to claim 1, wherein the detecting step measures the fluorescence value of the magnetic binding body-antigen-marker complex.
The magnetic bead of claim 1, wherein the magnetic bead
Neutral carboxyl group (-COOH) on the surface of magnetic beads, an ester group (-COO ^-), a neutral amine group (-NH _2), neutral ion of an amine group (-NH ^-, or NH ₃ ^-), Immunoassay method, comprising: any one of functional groups selected from the group consisting of: ^- a neutral thiol group (-SH), ion (-S) of the neutral thiol group.
The magnetic recording medium according to claim 1,
Wherein the magnetic beads are conjugated with an antibody to a streptavidin-biotin bond, an avidin-biotin bond, an EDC coupling, a sulfhydrylamine coupling, a Ni-NTA-histidine bond or an amide bond.
5. The method of claim 4, wherein the marker is a fluorescent substance or an ultraviolet (UV) marker that binds to the second antibody.
The method according to claim 8, wherein the fluorescent substance or ultraviolet (UV) marker is selected from the group consisting of FITC (Fluorescein isothiocyanate), PE (phycoerythrin), TRITC (tetramethyl-rhodamine isothiocyanate), Cy3 (Cyanine 3), Cy5 Rhodamine). &Lt; / RTI &gt;
A well portion including a well into which a sample is dispensed;
A magnet portion composed of a magnetic bar and a magnetic bar cap capable of covering the magnetic bar;
A fluorescence detector capable of detecting fluorescence of a marker bound to a specimen in the sample; And
And a moving unit capable of moving the well to the fluorescence detecting unit.
11. The method of claim 10, wherein the wells are comprised of a 6 well plate, a 12 well plate, an 18 well plate, a 24 well plate, a 36 well plate, a 48 well plate, a 96 well plate, a 128 well plate, a 256 well plate, Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
12. The method according to claim 11, wherein the sample is dispensed in one row of the wells, the magnetic coupling substance is dispensed in the second row, the washing solution is dispensed in the third and fifth columns, the marker complex is dispensed in the fourth row, Wherein the solution is dispensed.
11. The method of claim 10, wherein the fluorescence multiple immunoassay
And a magnet for realizing the magnet portion and the fluorescence detecting portion as one automated device.

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