JPH04323560A - Magnetic particle labelling material for use in laser magnetic immunity measurement and adjusting method for subject - Google Patents

Abstract

PURPOSE:To provide a magnetic particle labelling material causing little non- singular reaction and a method of adjusting a subject in order to reduce and stabilize the controlled values of the material which pose problems when the material is actually applied in patient blood serum. CONSTITUTION:In magnetite particulates covered with dextran, a plurality of biotins are bonded to the surface of the dextran and avidin is bonded to other plural biotines. Because a subject is magnetically labelled through an avidin-biotin reaction, the time required for the reaction can be greatly shortened in comparison with when the subject is magnetically labelled through an antigen- antibody reaction. Application of the avidin-biotin reaction in a magnetically labelling reagent enhances not only the sensitivity of detection but also the stability of the reagent.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunoassay method that utilizes antigen-antibody reactions. More specifically, the present invention relates to a labeling material and a sample preparation method used in the laser magnetic immunoassay method previously invented by the present inventors.

0002

[Prior Art] Currently, the development of immunoassay methods using antigen-antibody reactions as an early detection method for new viral diseases such as acquired immunodeficiency syndrome, adult T-cell leukemia, etc., and various cancers is currently gaining popularity worldwide. It is driven by scale.

As conventionally known microimmunoassay methods, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), etc. have already been put into practical use. These methods use an antigen or antibody labeled with an isotope, an enzyme, or a fluorescent substance, respectively, and detect the presence or absence of an antibody or antigen that specifically reacts with the antigen or antibody.

[0004] The present inventors previously filed Japanese Patent Application No. 61-22456.
No. 7, No. 61-252427, No. 61-254164,
62-22062, 62-22063, 62-15
No. 2791, No. 62-152792, No. 62-18490
As No. 2, we have filed a patent application for an invention related to a laser magnetic immunoassay method and a measuring device. These new immunoassay methods are characterized by the fact that they use magnetic particles as labeling materials to detect, for example, the presence or absence of magnetically labeled analytes from interference fringes, making it possible to detect ultra-trace amounts of less than picograms without using isotopes. be. Based on the above-mentioned patent, the present inventors labeled magnetic fine particles with antigens or antibodies and detected viruses for the first time.

Regarding the dextran-coated magnetite particles related to the present invention, US Pat. No. 4,452,773 "
Magnetic iron-dextran m
There is an invention by Molday as "icrospheres". This invention uses magnetite fine particles as a core and coats dextran around it, and this dextran has a
It is a combination of protein A, antibodies, enzymes, etc. The present inventors improved the method for producing dextran-coated magnetite particles disclosed in the Molday patent, and invented a method for producing magnetic particles of any particle size. ``Production method'',
A patent application was filed as Japanese Patent Application No. 2-35925 titled ``Method for producing magnetic fine particles.''

[0006] The present inventors first bound protein A to dextran-coated magnetite particles produced by Molday's method or the present inventor's method, and added AIDS virus ( We carried out an experiment to detect a trace amount of HIV antigen by binding a monoclonal antibody against an HIV antigen, such as P24, and presented the results at the 37th Annual Meeting of the Japanese Society of Virology (November 1989). As a result, in the case of purified HIV antigen, a trace amount of virus antigen of 0.1 picogram/ml was successfully detected. However, when detecting HIV antigens from human serum using the method disclosed by Molday, the antibody bound to protein A easily dissociates and the dextran-coated magnetite particles directly bind to the microbeads, resulting in abnormal control values. A phenomenon occurred in which the value increased. Note that the control value is a signal value detected when no sample is added, and is so-called noise.

[0007] The avidin-biotin reaction involved in the present invention is well known and has conventionally been used for non-radioactive labeling of nucleic acids. In addition, enzyme immunoassay (EIA), fluorescent labeling immunoassay (
Although this method was also used in FIA), it is a new immunoassay method developed by the present inventors that uses magnetic fine particles as a label. That is, in the EIA method and the like, as is known as the ABC method, a pre-biotinylated enzyme and avidin are simultaneously added at the time of enzyme labeling.

[0008]

[Problems to be Solved by the Invention] The present invention has been made in view of the above circumstances, and aims to reduce and stabilize the control value, which is a problem when actually applied to patient serum. The purpose of the present invention is to provide a magnetic fine particle labeling material and a sample preparation method that cause less specific reaction.

[0009]

[Means for Solving the Problems] The magnetic fine particle labeling material for laser magnetic immunoassay of the present invention is magnetite fine particles coated with dextran, and has a plurality of avidins (as defined in claim 1) on the surface of the dextran. A solution to the above problem is to combine biotin (corresponding to claim 2) or a plurality of biotins (corresponding to claim 2). Furthermore, the biotin-bound dextran magnetite particles can further bind avidin to the biotin (corresponding to claim 3). In this case, the magnetic particle labeling material is composed of avidin-biotin-dextran-magnetite. The magnetic fine particle labeling material is used in a suspended state in a suitable buffer to prevent self-aggregation. As the buffer solution, for example, B
A HEPES solution to which 1% SA (bovine albumin) is added is preferred. Further, the magnetic fine particle labeling material can be lyophilized and stored for a long period of time. Note that it is desirable to completely remove avidin or biotin that is not bonded to the dextran-coated magnetite particles from the magnetic fine particle labeling material. This is because if unreacted avidin or biotin remains, it will interfere with the magnetic labeling of the specimen with the magnetic fine particle labeling material.

In order to exhibit the performance of the magnetic fine particle labeling material of the present invention, the following sample preparation method is preferred.

[0011] As described in claim 4, this sample preparation method involves reacting a sample with a microbead suspension to which a first specific antibody against an antigen to be detected has been bound in advance, and depositing the sample on the surface of the microbeads. The first step is to capture the
a second step in which the captured specimen is further reacted with a second specific antibody for the specimen to which biotin has been bound in advance, and the specimen is sandwiched between the first and second specific antibodies; Afterwards, a dispersion suspension of the magnetic fine particle labeling material for laser magnetic immunoassay is added, and the biotin in the second specific antibody and avidin in the magnetic fine particle labeling material are reacted, and the particles captured by the microbeads are reacted with each other. The method includes at least a third step of magnetically labeling only the specimen, and a fourth step of separating and removing the magnetic fine particle labeling material that did not react in the third step.

Alternatively, as described in claim 5, in the above sample preparation method, avidin is added alone after the second step to bind the avidin to biotin bound to the second specific antibody; A third step of reacting avidin bound to biotin in the second specific antibody with biotin in the magnetic particle labeling material according to claim 2, and magnetically labeling only the specimen captured by the microbeads; It may also include at least a fourth step of separating and removing the magnetic fine particle labeling material that did not react in the third step.

[0013]

EXAMPLES The magnetic fine particle labeling material and sample preparation method will be explained in detail below based on examples.

(Example 1) Dextran-coated magnetite fine particles were produced by the following method. Dextran 125
g, urea 50g was dissolved in 11 pure water, FeCl3 6
0.5g of H2O, 0.7g of FeCl24H2O
Add 3g (corresponding to Fe3+:Fe2+ = 1:2 (molar ratio)) to prepare a starting solution, put it in a sealable container, for example, a Teflon container, and soak it in an oil bath to give a solution of 100 to 110 It was heated at ℃ for about 2 hours. By heating,
Urea decomposed to generate ammonia and dextran-coated magnetite (Fe3O4) was created in solution. This solution was subjected to ultrafiltration (filter: for 100,000 molecular weight fraction) to make it suitable for the subsequent operation, and the solution was filtered to about 8 to 10
It was concentrated twice to obtain an aqueous solution containing dextran-coated fine particles. Particle size measurement of the magnetite fine particles using X-rays confirmed that these fine particles had a diameter of 100 angstroms. In addition, by applying dynamic light scattering measurement, it was confirmed that the average particle diameter of the fine particles containing the dextran coating layer was 100 nm (1000 angstroms), and that they had a particle size distribution close to monodisperse. .

Next, in order to bind avidin and biotin to these fine particles, the following operation was performed as a preliminary step. To 100 ml of the above concentrated aqueous solution, 100 ml of acetic acid-sodium acetate buffer adjusted to pH 6.5 was added, then cooled to 4°C, and while applying ultrasonic vibration, 2 g of an oxidizing agent (NaIO4) was added. Allowed time to react. By this operation, some of the bonds in dextran are cleaved. Hereinafter, this solution will be referred to as a solution containing oxidized dextran-coated magnetite fine particles. The aforementioned X-rays,
When the particle sizes of the magnetite and oxidized dextran-coated fine particles were measured by dynamic scattering measurement, no change was observed in the particle size of the magnetite itself. In addition, although the particle size of the coated fine particles also shows a slight tendency to decrease, (
For example, the average particle diameter changed from 100 nm to 95 nm) and remained at roughly the same value. After oxidation treatment, excess NaIO
In order to remove 4 etc. and further concentrate at the same time, ultrafiltration was applied again and concentrated 4 to 5 times. For this reason, compared to when the fine particles were first produced, they are about 20 to 30 times more concentrated. Such a high concentration is required to increase the rate at which avidin and biotin can be added next.

Using the thus produced oxidized dextran-coated magnetite fine particles as a raw material, three types of magnetic fine particle labeling materials for laser magnetic immunoassay of the present invention, which will be described below, were obtained.

(1) Method for producing avidin-bound dextran-coated magnetite fine particles. (Corresponding to claim 1)

001
8] To 1 ml of the oxidized dextran-coated fine particle solution, add 1 ml of boric acid-borax buffer (pH between 7.5 and 8.5 may be selected), then add several mg of avidin and mix.
The addition reaction was allowed to proceed with shaking for ~2 hours. This was stored overnight at 4°C, and then several mg of NaBH4 was added at 4°C for reduction treatment. Excess reducing reagent was removed by further dialysis overnight with HEPES buffer. Avidin that did not participate in the addition reaction (referred to as free avidin) was then removed by centrifugation or gel filtration to produce avidin-bound dextran-coated magnetite microparticles.

(2) Method for producing biotin-bound dextran magnetite particles. (Corresponding to claim 2)

[0020]
Bicarbonate buffer (NaHCO3 + Na2 CO3) (
After adjusting the pH by adding 1 ml of (pH 8.5), add biotin hydrazide dissolved in DMF (dimethylformamide) (for example, add 0.1 ml of 10 mg/ml biotin hydrazide), and add 1 ml of 10 mg/ml biotin hydrazide at room temperature. Shake for 1 hour and store at 4°C overnight. Add NaBH4 to this at 4℃.
The mixture was stabilized by reduction treatment and dialyzed overnight with HEPES to remove free biotin. In this way, biotin-bound dextran magnetite microparticles were produced.

(3) Method for producing avidin-biotin-bound dextran-coated magnetite fine particles. (Corresponding to claim 3)

[0022] An avidin reagent is added to the biotin-bound dextran magnetite microparticle solution prepared by the above method to cause an avidin-biotin reaction, and free avidin is removed by centrifugation or gel filtration to coat the avidin-biotin bound dextran. Fine particles were produced.

(Example 2) After adding avidin or biotin to dextran-coated magnetite fine particles, it is necessary to remove free avidin or free biotin by some method. , which is important when used for detection. On the other hand, experiments conducted by the inventors revealed that a portion of the bound avidin, biotin, and dextran can be separated by centrifugation or gel filtration. For this reason, it became necessary to adopt a bonding form that is difficult to peel off. These matters are shown below as Example 2.

FIG. 1 shows avidin-bound dextran-coated magnetite fine particles (abbreviated as avidin-magnetite).
When biotin-bound dextran-coated magnetite particles (abbreviated as biotin-magnetite) are centrifuged, what percentage of the initially bound avidin (or biotin) remains after centrifugation, relative to the applied centrifugal force? This shows whether or not there are any. Specifically, the concentration of avidin (or biotin) contained in the supernatant after centrifugation was measured and subtracted from the initial value to determine the remaining amount. The concentration of avidin was determined from protein absorption at 280 nm, and the concentration of biotin was determined by a biotin analysis method using a HABA-avidin complex. This result shows that biotin-magnetite exfoliates more rapidly than avidin-magnetite. In the case of avidin-magnetite, this degree of peeling does not pose a practical problem for later use (usually, the rotational speed for removing free avidin is 15,000 rpm). With biotin-magnetite, this is thought to reflect the instability of the fine particles themselves, and care must be taken when using them.

Gel filtration method, which is considered to be a milder fraction than centrifugation (gel: Pharmacia Sephacryl S-10)
FIG. 2 shows the results when free avidin and biotin were removed using 0. The magnetite curve in FIG. 2 is a plot of values measured by chemical analysis of iron versus elution amount. In addition, glycerol indicated by an arrow in the figure has a relatively small molecular weight (M
W:92) is a reagent added to clarify the elution position. The figure shows an example of avidin, and it can be seen that free avidin is completely removed. On the other hand, biotin continues to elute endlessly even after the free biotin elution peak, and shows elution in a lower molecular weight range than the glycerol position. This phenomenon does not normally occur. It is possible that some of the biotin or dextran is also detached during passage through the gel.

Next, FIG. 3 shows the results of reacting biotin-magnetite with avidin and then removing free avidin. In this case, it was found that free avidin was removed to some extent, and the detachment of biotin was further prevented by the added avidin.

Furthermore, FIG. 4 shows the amount of avidin bound to the amount of avidin introduced. This was determined by performing gel filtration (gel: Pharmacia Sephacryl S-300), measuring the amount of free avidin, and subtracting it from the input amount. It is observed that the amount of avidin effectively bound is significantly smaller in the case of avidin-biotin-magnetite than in the case of avidin-magnetite.

As a result of subsequent experiments conducted by the present inventors,
These three types of magnetite, namely avidin-conjugated dextran-coated magnetite, avidin-biotin-conjugated dextran-coated magnetite, and biotin-conjugated dextran-coated magnetite, could all be used successfully in subsequent detection experiments. However, when considering the effective use of reagents and the stability of microparticles, it can be said that avidin-bonded dextran-coated magnetite is the most desirable.

Based on the knowledge obtained by the present inventors from the research on the method for removing free biotin and avidin using the above-mentioned centrifugation method and gel filtration method, in the case of biotin-bound dextran-coated magnetite, free When removing biotin, it is preferable to use the dialysis method described in Example 1 rather than a method that applies external force such as centrifugation or gel filtration. Furthermore, when biotin-bound dextran-coated magnetite is used as avidin-biotin-bound dextran-coated magnetite, the step of removing free biotin in advance can be omitted, and a method can be used in which free biotin and avidin are simultaneously removed after adding avidin. .

(Example 3) The magnetic fine particle labeling material of the present invention produced by the method of Example 1 was applied to antigen detection of influenza virus. Details are as follows.

Specimen preparation method (corresponding to claim 4): Type B influenza virus (B/Chaing Rai/3/
85) with normal human serum to achieve a virus concentration of 100/
Adjusted to ml.

After activating acrylic resin microbeads (Tresphere, Toray Industries, Inc.) with a particle size of 2 μm, they were reacted with anti-influenza rabbit serum at 35° C. for 30 minutes.
After washing with PBS, a 0.2% microbead suspension in 1% BSA-PBS was prepared. Through this adjustment, the influenza antibody (first specific antibody) is immobilized on the surface of the microbeads.

[0033] 25μ of the influenza virus suspension
1 and 25 μl of the microbead suspension were reacted at 35° C. for 2 hours to capture influenza virus on the surface of the microbeads.

Add 10 μl of 0.1 mg/ml of biotinylated antibody (second specific antibody) to which biotin has been bound in advance,
React at 35°C for 1 hour. Thereafter, unreacted biotinylated antibodies are subjected to B/F separation by centrifugation.

Furthermore, the magnetic fine particle labeling material of the present invention 0.1
Add 10 μl of mg/ml and react at 35° C. for 30 minutes. After the reaction, the unreacted magnetic particle labeled material is centrifuged to B/
F Separate. Add 25 μl of HE to the precipitated microbeads.
After resuspending with PES buffer, it is used for measurement.

FIG. 5 shows a schematic diagram of how viruses are magnetically labeled in the above sample preparation step. (a) is the antigen capture step, (
b) shows the binding state in the biotinylated antibody reaction step, and (c) shows the binding state in the magnetic fine particle labeled material reaction step. Although FIG. 5 shows an example of magnetite fine particles coated with avidin-bonded dextran as the magnetic fine particle labeling material, magnetite fine particles coated with avidin-biotin-bound dextran are also used. That is, in any case, since avidin is bound to the surface of the magnetic fine particle labeling material, it can specifically bind to a biotinylated antibody bound to a virus by a known avidin-biotin reaction. Viruses captured on the microbeads in this way are magnetically labeled.

On the other hand, when using biotin-bound dextran-coated magnetite particles as the magnetic particle labeling material (corresponding to claim 5), in the above sample processing method, a biotinylated antibody (second specific antibody) to which biotin has been bound in advance is used.
After the unreacted biotinylated antibody was subjected to B/F separation by centrifugation, avidin was added alone to bind the biotinylated antibody bound to the virus, and then biotin-bound dextran-coated macnetite microparticles were added. All you have to do is react.

Specimen measurement method: Measurement was carried out using the interferometry method invented in Japanese Patent Application No. 184902/1984 entitled "Laser Magnetic Immunoassay Method and Apparatus".

[0039] A total amount of 25 μl of the sample after sample preparation is put into the well of the test container into which 350 μl of pure water has been injected.

The test container is inserted into the gradient magnetic field generator, and a magnetic field of about 8 k Gauss is applied to magnetically concentrate the sample at one point on the water surface of the well.

[0041] A 5 mW He-Ne laser is irradiated obliquely to this concentration point at an incident angle of 30 degrees, the interference fringes in the reflected light beam are received by a white screen, and the center light intensity of the interference fringes is measured using a CCD camera and an image processing device. Measure.

Results: Table 1 below shows that 100 viruses were detected in the method of the present invention using three types of magnetic fine particle labeling materials.
The values of the center light intensity of interference fringes and the negative control are shown.

[0043] The negative control is always used in the field of immunoassays, and is the name given to a control sample to which no specimen is added. This control sample was subjected to the same procedures as the specimen at the same time. The lower the negative control value, the better.

[0044]

[Table 1]

[0045] In a comparative example, influenza antibody-protein A-conjugated dextran was coated using a method previously carried out by the present inventors and presented, for example, at the 37th General Meeting of the Japanese Society for Virology (November 1989, Osaka, Lecture No. 439). These are the results when magnetite fine particles were used. This table 1
Therefore, it was confirmed that the negative control value of the present invention was lower than that of the comparative example, and that a virus concentration of 100 cells/ml could be reliably detected.

[0046]

[Effects of the Invention] As detailed above, the use of the magnetic fine particle labeling material used in the laser magnetic immunoassay of the present invention is less likely to cause non-specific adsorption to microbeads, so the negative control is low and stable even in human serum. be. The reason for this is that compared to the method of directly binding antibodies to dextran-coated magnetite, which was previously invented by the present inventors, in the present invention, the specimen is magnetically labeled by an avidin-biotin reaction, so the conventional antigen-antibody reaction is not possible. The reaction time can be significantly reduced compared to when magnetic labeling is used. In Example 3, the magnetic labeling reaction time was set at 30 minutes, but 15 minutes is also sufficient. Generally, the longer the time, the more likely non-specific reactions occur, so it is advantageous to use the avidin-biotin reaction of the present invention to suppress non-specific reactions.

Furthermore, the material of the present invention is versatile because the same material can be used even if the target virus changes. That is, in the case of the conventional method of directly binding an antibody to dextran-coated magnetite, it was necessary to prepare a magnetic fine particle labeling material for each virus. However, in the case of the present invention, it is only necessary to prepare a biotinylated antibody for each virus. This difference is extremely effective when put to practical use as a diagnostic agent. It is also extremely effective when actually producing diagnostic reagents. This is because, according to the experience of the present inventors, when an antibody is directly bonded to dextran-coated magnetite, if the type and properties of the antibody change, the antibody titer and aggregation property of the resulting magnetic fine particle labeling material (referred to as a magnetic labeling reagent) will change. changes, and its stability as a reagent over time, especially its aggregation properties, changes significantly. Therefore, producing many types of magnetic labeling reagents requires a long development period and cost. This point is a particular problem of magnetic labeling reagents compared to enzyme labeling reagents used in conventional enzyme immunoassays. On the other hand, in the case of biotinylated antibodies, it is much easier to produce a wide variety of biotinylated antibodies than magnetic labeling reagents, and a variety of biotinylated antibodies are actually commercially available.

As described above, by using the method of the present invention, a highly versatile reagent can be provided.

By the way, in the case of the EIA method using the avidin-biotin reaction, the enzyme contains biotin (molecular weight = 244
) are combined. The reason for this is that the molecular weight of these labeling materials is small, and avidin, which has a large molecular weight (molecular weight =
66000) is disadvantageous. In the case of the magnetic labeling reagent of the present invention, as described in Example 2, binding avidin with a large molecular weight to dextran is more advantageous than binding biotin because the magnetic labeling reagent becomes more stable. As described above, the avidin-biotin reaction itself has been known, but the present inventors have found that when applied to magnetic labeling reagents, it is possible to improve not only the detection sensitivity but also the stability of the reagent. This was revealed for the first time through research.

Now, in the case of influenza virus, conventional hemagglutination methods could not detect the virus unless the virus concentration was 10 million cells/ml or more, but according to the method of the present invention using the materials of the present invention, the conventional hemagglutination method could not detect the influenza virus. Viruses can now be detected with 100,000 times more sensitivity. Currently, the present inventors are conducting research on high-sensitivity detection of HIV (AIDS virus) antigen.
We have also succeeded in detecting extremely small amounts of p24 core antigen. Therefore, since the virus can be detected directly from the patient without culturing the virus, it is extremely effective for early diagnosis of various infectious diseases. Note that the method of the present invention is not limited to the detection of viral antigens described in the Examples, but can also be applied to antibody tests that are currently widely practiced. When applied to antibody testing,
Taking advantage of its high detection sensitivity, it is possible to detect, for example, IgE antibodies with small amounts of antibodies in blood in a short period of time, making it applicable to allergy diagnosis.

Furthermore, it can be applied not only to antigen-antibody reactions, but also to the measurement of various hormones such as peptide hormones, various enzymes, vitamins, drugs, etc., to which the RIA method has conventionally been applied. Therefore, precise measurements that could conventionally only be carried out using the RIA method in limited facilities can now be carried out widely and quickly in general environments. If accurate measurements such as screening tests for various viruses and cancers could be widely implemented in common situations such as mass medical examinations, early diagnosis of cancer or viral diseases, etc. would be possible, and effective early treatment would be possible. This makes it possible to implement As described above, the effects of the present invention in the fields of medicine and medical care are immeasurable.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the residual percentage of avidin (or biotin) relative to the centrifugal force applied when avidin-magnetite and biotin-magnetite are centrifuged.

FIG. 2 is an elution curve of avidin-magnetite and biotin-magnetite obtained by gel filtration.

FIG. 3 is an elution curve of free avidin and magnetite after reacting biotin-magnetite with avidin.

FIG. 4 is a graph showing the relationship between the amount of avidin input and bound avidin during the production of avidin-magnetite and avidin-biotin-magnetite.

FIG. 5 is a schematic diagram of the binding state in which the virus is magnetically labeled in the sample preparation step in the third embodiment of the present invention;
(a) shows the binding state in the antigen capture step, (b) in the biotinylated antibody reaction step, and (c) in the magnetic fine particle labeled material reaction step.

Claims (5)

[Claims] 1. A magnetic particle labeling material for laser magnetic immunoassay, comprising magnetite particles coated with dextran, and a plurality of avidins bonded to the surface of the dextran. 2. A magnetic particle labeling material for laser magnetic immunoassay, comprising magnetite particles coated with dextran, and a plurality of biotins bonded to the surface of the dextran. 3. The magnetic particle labeling material for laser magnetic immunoassay according to claim 2, wherein the biotin is bound to avidin. 4. A first step of reacting a sample with a microbead suspension to which a first specific antibody against an antigen to be detected has been bound in advance, and capturing the sample on the surface of the microbeads; further reacting the specimen with a second specific antibody against the specimen to which biotin has been bound in advance;
a second step of sandwiching the specimen with a first and second specific antibody, and adding a suspended suspension of the magnetic fine particle labeling material according to any one of claims 1, 2, and 3 after the second step. , a third step in which biotin in the second specific antibody and avidin in the magnetic fine particle labeling material are reacted to magnetically label only the specimen captured by the microbeads, and a reaction in the third step; A method for preparing a specimen, comprising at least a fourth step of separating and removing the magnetic fine particle labeling material that was not used. 5. A first step of reacting a sample with a suspension of microbeads to which a first specific antibody against an antigen to be detected has been bound in advance, and causing the sample to be captured on the surface of the microbeads; further reacting the specimen with a second specific antibody against the specimen to which biotin has been bound in advance;
a second step of sandwiching the specimen with the first and second specific antibodies; after the second step, adding avidin alone to bind the avidin to biotin bound to the second specific antibody; A third method of magnetically labeling only the specimen captured by the microbeads by reacting the avidin bound to biotin in the second specific antibody and the biotin in the magnetic particle labeling material according to claim 2. and a fourth step of separating and removing the magnetic fine particle labeling material that did not react in the third step.