JPH04242167A - Indirect agglutination reaction method using magnetic particle carrier - Google Patents

Abstract

PURPOSE:To facilitate judgment of an agglutination image with an agglutination image, especially a negative image, made clear by preventing unclearness of the agglutination image that will be caused by a magnetic coupling between magnetic particle carriers as caused by a magnetic force applied on the magnetic particle carrier. CONSTITUTION:A ferromagnetic material 2 is arranged on an angular ferrite magnet 1 and a measuring container is placed on the ferromagnetic material 2 to applying a magnetic force on a magnetic particle carrier with in the measuring container. Thereafter, the measuring container is taken off from the apparatus and the magnetic force applied on the magnetic particle carrier is cut off to observe and judge an agglutination image.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological particle agglutination analysis method using a magnetic particle carrier.

0002

[Prior Art] In particle agglutination reactions using immunological reactions, indirect agglutination methods such as the microtiter method are inexpensive and simple, so they have been used in the field of immunological testing methods.
This is a widely used measurement method. This method uses red blood cells,
The interparticle agglutination reaction, which occurs due to the immunological reaction between antigens or antibodies physically or chemically bound to the surface of polymer particles or gelatin particles, and the antibodies or antigens in the specimen sample, is carried out using a microplate, etc. Measurement is performed based on the state of agglomeration of particles at the bottom of the measurement container. This takes advantage of the fact that when the particles aggregate, the ease with which they roll (or slide) on the inner wall surface of the measurement container decreases. Alternatively, as disclosed in Japanese Unexamined Patent Application Publication No. 64-69954, antigens, antibodies, etc. are bound in advance to the inner wall surface of the measurement container, and the reaction between the particles and the inner wall surface of the measurement container is stimulated. Some are in use. These methods can be said to be excellent in that they not only allow measurement with a small amount of sample, but also allow processing of many samples at the same time. However, in the above-mentioned indirect agglutination reaction method, the signal due to the antigen-antibody reaction is measured based on the difference in the agglutination images of particles at the bottom of the measurement container, so the measurement time depends on the sedimentation speed of the particles and the rolling speed at the bottom of the measurement container. Much depends. Therefore, a method has been considered that uses a magnetic particle carrier in which the particles are magnetized, and accelerates the operation of sedimentation by gravity by applying a magnet (JP-A-2-107968, JP-A-2-107968, -124464 publication, JP-A-2-210
Publication No. 262).

[0003] However, the immunoassay methods disclosed by these methods have problems in terms of measurement sensitivity and ease of determining aggregation images, and cannot be said to be satisfactory. Generally, when a magnetic force is applied to magnetic particles, the magnetic particles become magnetized, and the magnetic particles are connected to each other in a chain shape along magnetic lines of force through magnetic coupling. Such a phenomenon also occurs, for example, when a magnet is brought close to iron powder in the air, but in this case, the difference in specific gravity between the iron powder and the air is large, so the magnet is strongly influenced by gravity. However, when magnetic particles, which have a lower specific gravity than iron powder, are added to an aqueous solvent system whose specific gravity is greater than that of air, the influence of gravity becomes relatively small, causing the magnetic particles to line up more orderly along the lines of magnetic force. The following problems that arise due to the properties of magnetic particles remain to be solved. When a magnetic force is applied to a magnetic particle carrier,
The magnetic particle carriers are connected in a chain shape along the magnetic field lines through magnetic coupling, and this inhibits the original immunological reaction between the magnetic particle carriers or the reaction between the magnetic particle carriers and the wall surface of the measurement container. Because it acts on the shape, it tends to reduce measurement sensitivity. Since chains of magnetic particle carriers generated by applying magnetic force are magnetized in substantially the same direction, magnetic repulsion prevents the chains of magnetic particle carriers from coming closer to each other beyond a certain extent. Therefore, the negative image spreads out, and the difference between the negative image and the weakly positive image becomes smaller. When trying to determine the agglomerated image of magnetic particle carriers by applying magnetic force, the magnetic particle carriers are connected in a chain shape along the lines of magnetic force through magnetic coupling. Therefore, when observing an agglomerated image, most of the magnetic particle carriers fall into the blind spot and the color becomes lighter, making it difficult to judge.

[0004] Indirect aggregation reaction methods such as the present method are methods in which the agglutination image is determined based on the color distribution of the particle carrier, and therefore, measurement will be hindered if the color cannot be clearly determined. For a positive image, it is sufficient that the particle carriers are uniformly distributed, so it is not a big problem even if the color of the particle carriers cannot be distinguished.
If the distribution state of particle carriers cannot be clearly determined from the color of a negative image or a weakly positive image, measurement will be hindered. This can be done by simply making the magnetic particle carrier darker in color.
This is a difficult problem to solve using methods such as increasing the concentration of magnetic particle carriers. However, when trying to promote the formation of an agglomerated image of the magnetic particle carriers on the inner wall surface of the measurement container by applying a magnetic force to the magnetic particle carriers, the magnetic particle carriers become magnetized at least temporarily. Combining is simply unavoidable.

[0005]

[Problems to be Solved by the Invention] In view of the above-mentioned current situation, the present inventors have proposed the following problems caused by magnetic coupling between magnetic particle carriers in an indirect aggregation reaction method using magnetic particle carriers. With the aim of resolving the shortcomings in measurement based on phenomena, the present invention has been completed through extensive research.

The first problem is that the magnetic coupling between the magnetic particle carriers inhibits the reaction between the magnetic particle carriers or between the magnetic particle carriers and the inner wall surface of the measurement container, which is the original immunological reaction. The objective is to prevent the resulting decrease in measurement sensitivity as much as possible and to enable measurement in a short time. The second problem is that chains of magnetic particle carriers (magnetic particle carriers connected in a chain shape along the lines of magnetic force) generated by applying magnetic force become magnetized in the same direction, and the chains repel each other. This is to suppress the spread of aggregated images (especially negative images) that occurs when the object cannot be approached beyond a certain level.

[0007] The third problem is that when a magnetic force is applied to determine an agglomerated image, the magnetic particle carriers form a chain along the lines of magnetic force and most of the magnetic particle carriers enter the blind area. The purpose of the present invention is to solve the problem of not being able to determine the distribution state clearly enough.

[0008]

[Means for Solving the Problems] In order to solve the above problems, the present invention is characterized in that, in an indirect aggregation reaction method using magnetic particle carriers, the magnetic vector acting on the magnetic particle carriers is temporally changed. Provides an indirect agglutination reaction method. Changing the magnetic vector over time means changing the strength of the magnetic force acting on the magnetic particle carrier (magnetic vector size)
Alternatively, it means changing one or both of the directions of magnetic force (direction of magnetic vector) over time.

[0009] The above-mentioned "temporally changing" means that the magnetic vector applied to the magnetic particle carrier is changed at least once during the period from when the magnetic force starts acting on the magnetic particle carrier until the aggregation image is determined. It means to cause. The most important feature of the present invention is that the magnetic vector acting on the magnetic particle carrier is changed over time.

By changing the strength of the magnetic force acting on the magnetic particle carrier, it is possible to reduce the decrease in measurement sensitivity caused mainly by applying the magnetic force, and also to prevent the magnetic particle carrier from being chained along the lines of magnetic force. This has the effect of preventing the distribution of the magnetic particle carriers from becoming unclear due to the formation of a shape and standing up. When the magnetic force acting on the magnetic particle carriers is blocked or sufficiently weakened, the magnetic particle carriers are demagnetized and the magnetic bonds between the magnetic particle carriers are dissociated. It is thought that the above effects can be obtained by this.

[0011] By temporally changing the direction of the magnetic force of the magnetic particle carrier, there is an effect of preventing the negative image from spreading mainly by applying the magnetic force. This is thought to be because the chains of magnetic particle carriers are magnetized in the same direction, and the phenomenon in which these chains cannot get closer to each other due to magnetic repulsion is alleviated by temporal changes in the direction of magnetic force. It will be done. Alternatively, it is also considered that the magnetic bond between the magnetic particle carriers is temporarily dissociated when the direction of the magnetic force acting on the magnetic particle carriers is changed.

To further explain the change in the strength of the magnetic force acting on the magnetic particle carrier, the strength of the magnetic force may be changed continuously, intermittently, or stepwise. Examples of this include changing the strength of the magnetic force at regular intervals, continuously changing the strength of the magnetic force within a certain range at regular intervals, or temporarily changing the magnetic force acting on the magnetic particle carrier. Examples include shutting off the system temporarily or for a while. Furthermore, this also includes applying a magnetic force to the magnetic particle carrier to promote the formation of an aggregated image, and then cutting off the magnetic force by, for example, lowering it from a magnetic generator, and then determining the aggregated image.

[0013] The blocking of magnetic force does not necessarily mean that the magnetic force is completely blocked, but may be weakened to the extent that it does not have a sufficient effect on the magnetic particle carrier. That is, it is only necessary to weaken the magnetic particle carriers to such an extent that they do not line up in a chain shape along the lines of magnetic force. The maximum value of the magnetic force at this time differs depending on the content of the ferromagnetic substance in the magnetic particle carrier, the size of the magnetic particle carrier, and the like. As a method for temporally changing the strength of the magnetic force acting on the magnetic particle carrier, for example, by changing the strength of the magnetic force generated by the magnetic generator used, or by moving the magnetic generator or the measuring container. It can also be achieved by

To further explain the changing of the direction of the magnetic force acting on the magnetic particle carrier, the direction of the magnetic force may be changed continuously or intermittently. Examples include a method using an alternating magnetic field or a method of rotating the direction of magnetic force. Examples of methods for temporally changing the direction of magnetic force acting on magnetic particle carriers include:
This can be achieved by temporally changing the direction of the magnetic force generated by the magnetism generator used, or by moving the magnetism generator or the measurement container.

The strength and direction of the magnetic force acting on the magnetic particle carrier may be changed simultaneously or at different times. The magnetic force that acts on the magnetic particle carrier can be determined regardless of whether a nonuniform magnetic vector is used or a uniform magnetic vector is used, when considering the time axis (i.e., when considering a certain moment of continuous time). good. Here, the method using a nonuniform magnetic vector is a method in which magnetic particle carriers are directly attracted by magnetic force. In addition, the method using a uniform magnetic vector takes advantage of the fact that by applying a uniform magnetic vector to a magnetic particle carrier, the magnetic particle carrier becomes more strongly influenced by gravity, and this method is used to form an agglomerated image. This is a way to promote this.

When applying a non-uniform magnetic vector to a magnetic particle carrier, changing the strength of the magnetic force also changes the direction of the magnetic force. The indirect agglutination reaction method using magnetic particle carriers of the present invention is as follows: 1) A reaction between a test substance in a sample solution and a particle carrier on which a substance having an affinity for the test substance is immobilized is carried out at the bottom of a measurement container. In the indirect aggregation reaction determined as an aggregation image of particle carriers, a step of using a magnetic particle carrier as the particle carrier, reacting the test substance with the magnetic particle carrier, and simultaneously or subsequently applying a magnetic force to the magnetic particle carrier. 2) a step of mixing a sample solution and a magnetic particle carrier immobilized with a substance that competes with the analyte in the sample solution to form a reaction solution for the two; a step of applying a magnetic force to the reaction solution contained in the measurement container on the magnetic particle carriers in the measurement container; or 3) preparing a magnetic particle carrier in which a ferromagnetic substance is sealed inside red blood cells, and combining this with the sample solution. react,
At the same time or subsequently, the agglutination image or agglutination value formed by applying magnetic force to the magnetic particle carrier reacts with the test substance (e.g., red blood cell surface antigens such as ABO blood group antibodies or irregular antibodies) in the sample solution. This method involves changing the magnetic vector over time in the process from the time when each magnetic force starts to act until the agglutination image is observed or determined.

In either case, it is possible to use a measurement container in which a substance that has an affinity for the test substance or a substance that competes with the test substance is immobilized on the inner wall of the measurement container in advance. When a substance that has an affinity for the test substance is immobilized on the inner wall of the measurement container in advance, after adding the sample solution to the measurement container, a step of reacting the test substance in the sample solution with the measurement container and washing are performed. Either or both of the steps may be performed.

When a substance that competes with the analyte is immobilized on the magnetic particle carrier or the inner wall surface of the measurement container in advance for measurement, conversely, if a substance that competes with the analyte is immobilized on the inner wall surface of the measurement container or the magnetic particle carrier and has an affinity for the analyte. It is desirable to immobilize the substance or add a substance having an affinity for the test substance to the diluted sample solution or the dispersion of the magnetic particle carrier. In the present invention, for convenience, the case where the magnetic particle carrier spreads on the inner wall surface of the measurement container is expressed as positive, and the case where it converges on a part of the inner wall surface of the measurement container is expressed as negative. However, if a substance that competes with the test substance is immobilized on the magnetic particle carrier or the inner wall surface of the measurement container beforehand and then measured, the expressions of the positive image and negative image may be reversed.

The magnetic particle carrier is a ferromagnetic material (ferromagnetic metal such as iron, cobalt, nickel, etc. or an alloy containing these), or a nonmagnetic material containing a sufficient amount of the ferromagnetic metal or ferromagnetic alloy. (including those containing non-magnetic materials in ferromagnetic metals or ferromagnetic alloys), or magnetic particle carriers molded into particles alone, or with a ferromagnetic material as the core and its surface as polystyrene, silica gel, Magnetic particle carriers coated with gelatin, acrylamide, etc.; magnetic particle carriers made by dispersing finely powdered ferromagnetic material in polystyrene, silica gel, gelatin, acrylamide, etc. and forming them into particles; or polystyrene, silica gel,
A magnetic particle carrier in which a core of particles such as gelatin or acrylamide is coated with a ferromagnetic material, or a magnetic particle carrier prepared by encapsulating a ferromagnetic material in red blood cells or microcapsules can be used. The magnetic particle carrier may be colored by coating or dispersing or encapsulating a dye in the particles. Magnetic particle carriers become magnetized while being subjected to external magnetic force;
It is desirable that the material has the property of quickly demagnetizing by blocking external magnetic force. The particle diameter of the magnetic particle carrier is 0.01 to 100 μm, preferably 0.5 to 10 μm.
It is m. The magnetic particle carrier has a specific gravity of 1 to 10, preferably 1 to 2. Such magnetic particle carriers include, for example, solid phase carriers that are already used in the art as carriers for immunomagnetic separation in enzyme immunoassays, fluoroimmunoassays, chemiluminescence immunoassays, radioimmunoassays, etc., or carriers for cell separation. It is possible to adopt without any restrictions.

Sample solutions include human or animal blood, serum, plasma, urine, spinal fluid, saliva, sweat, ascites, amniotic fluid, human or animal cell or organ extracts, and human or animal fecal extracts. , cell or bacterial culture solution,
Any liquid such as extracts, plant extracts, or diluted solutions of these can be targeted. The test substance may be any substance that has been conventionally measured by immunoassay or can be measured. For example, CRP (C-reactive protein), α
-Fetoprotein, CEA, RF (rheumatoid factor)
, anti-mycoplasma antibodies, various hormones, allergens,
Examples include reagin, IgE, albumin, hemoglobin, virus-related antigens and antibodies, other infectious disease-related antigens and antibodies, and various blood type-related antigens and antibodies. Measurement items that may require an emergency, especially in clinical testing settings, such as ABO
Blood type antibodies, irregular antibodies, hepatitis B virus-related antigen antibodies (HBs antigen, HBs antibody, HBc antibody, HBe antigen, HBe antibody, Pre-S1 antigen, Pre-S1 antibody, Pre-S2 antigen, Pre-S2 antibodies, etc.), hepatitis C virus (HCV)-related antigens and antibodies, AIDS virus (HCV),
IV) Related antigen-antibodies, Treponema pallidum-related antibodies, adult T-cell leukemia virus (HTLV-I)-related antigens and antibodies are particularly preferred.

[0021] A substance that has an affinity for a test substance is, for example, when the test substance is used as an antigen for measurement, an antibody against the test substance, and when the test substance is an antibody against a certain antigen, It is an antigen that reacts with the antibody that is the test substance. Additionally, if the test substance is a sugar chain, a lectin that reacts with the sugar chain can be used, or conversely, if the test substance is a lectin, a sugar chain that reacts with the lectin can be used. . That is, a substance having an affinity for a test substance refers to a substance that reacts with the test substance.

[0022] A substance that competes with the test substance refers to a substance that competitively inhibits the reaction between the test substance and a substance that has an affinity for the test substance. That is, it is the test substance itself or a substance immunologically similar to the test substance. For example, when the test substance is an antigen and the substance that has affinity for the test substance is an antibody against that antigen, the substance that competes with the test substance is the antigen itself, the epitope of the antigen, or It is a substance that has epitope analogues. When the test substance is an antibody and the substance that has affinity for the test substance is an antigen for that antibody, the substances that compete with the test substance are the antibody itself and the same epitope on the antigen recognized by the antibody. or an antibody that recognizes an epitope in the vicinity thereof, or a fragment having an antigen recognition site of these antibodies.

The type, material, and shape of the measurement container are not limited in any way as long as it is a container commonly used in indirect aggregation reactions. For example, glass, polystyrene,
These include test tubes, microplates, trays, etc. made of polyvinyl chloride, polymethacrylate, etc. The bottom surface of the area (well) that accommodates the sample in the measurement container is U-shaped or V-shaped.
It is desirable to have a shape that slopes from the center of the bottom surface to the periphery, such as a mold or UV type. Although some measurement containers, such as test tubes, have only one portion for accommodating a sample, for convenience, they will all be referred to as wells.

[0024] The method of immobilizing a substance that has an affinity for the test substance or a substance that competes with the test substance on the magnetic particle carrier or the inner wall surface of the measurement container is not particularly limited, and may be any known method. is preferably adopted. For example, physicochemical methods such as hydrophobic bonding, hydrophilic adsorption, and covalent bonding may be used. Furthermore, if necessary, it may be treated with a masking agent such as BSA, casein, gelatin, ovalbumin, or various surfactants.

Various aqueous solutions can be used as the magnetic particle carrier dispersion or sample solution dilution used in the immunoassay of the present invention. For example, with aqueous solvents such as purified water, physiological saline, and various buffer solutions, various proteins,
Various saccharides, various animal serums, various preservatives, various surfactants, various synthetic high molecular compounds, various natural high molecular compounds, various synthetic low molecular compounds, etc. can be added. When measuring a substance that has an affinity for the test substance or a substance that competes with the test substance immobilized on the inner wall surface of the measurement container, it is particularly desirable to use a solution containing casein.

[0026] In the immunoassay method of the present invention, the sample solution can be diluted with a diluent in the measurement container or in a separate container for measurement. It is also possible to determine the concentration of the test substance as the agglutination value by serially diluting the sample solution with a diluent. For serial dilution, a diluter, which is commonly used in conventional passive hemagglutination reactions, etc., can also be used.

[0027]

[Action] By temporally changing the magnetic vector that acts on the magnetic particle carrier, it is possible to minimize the decrease in measurement sensitivity caused by applying magnetic force, and to form a particle aggregation image in a short time. It became so. In addition, magnetic particle carriers are connected in a chain shape, and this chain stands up when a negative image cannot be converged or when determining an agglomerated image, preventing many of the magnetic particle carriers from entering a blind spot. Negative image) can now be clearly determined.

[0028]

[Examples] The present invention will be explained below according to Examples and Comparative Examples, but the present invention is not limited to these Examples in any way. Example 1 Effect of Magnetic Blocking (1) Measurement of HBs Antigen Purified HBs antigen (manufactured by Meiji Dairy Co., Ltd.) was used in Comparative Example 1 (described below).
1) 2.0ng/ml, 1.0 in diluent A shown in
ng/ml, 0.5ng/ml, 0.25ng/ml,
0.13ng/ml, 0.063ng/ml, 0.03
Prepare each solution diluted to 1 ng/ml, and apply each of lanes A, B, C, D, E, F, and G of lanes 1 and 2 of the anti-HBs antibody-binding microplate in (3) of Comparative Example 1. 25 μl of each solution was added to each well in turn. In addition, in the 2 H wells of lanes 1 and 2, (
1) Add 25 μl of diluted solution A shown in 1). 25 μl of the anti-HBs antibody-bound magnetic particle carrier suspension of (2) in Comparative Example 1 was added to each well of this microplate, and after stirring for about 1 minute with a microplate mixer, the microplate was transferred to Comparative Example 2 described below. Device 1 shown in (1) of
, and a magnetic force was applied for 5 minutes. When unloaded from the apparatus, the color of the magnetic particle carriers rapidly appeared, and the state of distribution of the magnetic particle carriers could be determined. After about 1 to 2 minutes, the aggregation image of each well was observed and determined.

As shown in FIG. 7, the case where the image is uniformly spread on the well bottom surface is positive (+), and the case where the image is not completely spread on the well bottom surface and is slightly converged as shown in FIG. As shown in Figure 9, a case where the magnetic particle carrier is almost converged at the center of the well bottom is considered negative (-).
). The results are shown in Table 1.

[0030]

[Table 1]

As shown in Table 1, good results were obtained. In the above operation, even if the square ferrite magnet 1 of the apparatus 1 is pulled out from the apparatus without removing the microplate from the apparatus, an agglomerated image appears rapidly, and the state of distribution of the magnetic particle carriers can be determined, similar to Table 1. The results were obtained. It has been confirmed that at least when determining an agglutinated image, it becomes easier to determine an agglutinated image by blocking or sufficiently weakening the magnetic force.

Example 2 Intermittent magnetic force and HBs antigen measurement (1) Magnetic generator manufacturing device 2: As shown in FIG. 2, a square ferrite magnet 1 with a size of 100 x 100 x 10 mm is placed on a horizontal plate (experimental table). with the N pole facing upward, and place the
Iron plates 2 and 5 measuring 80x150x2mm were inserted and placed horizontally. The distances between the magnetic poles of the magnet 1 and the iron plates 2 and 5 are 60 mm and 100 mm, respectively. The measuring container is placed approximately in the center on the iron plate 2 below. (2) Measurement of HBs antigen Purified HBs antigen (manufactured by Meiji Dairies Co., Ltd.) was diluted with diluent A to 2.0
ng/ml, 1.0ng/ml, 0.5ng/ml, 0
．． 25ng/ml, 0.13ng/ml, 0.063n
Prepare each solution diluted to be 0.031 ng/ml and 0.031 ng/ml, and compare lanes A, B, C, D, and E of lanes 1 and 2 of the anti-HBs antibody-binding microplate in (3) of Comparative Example 1.
, F, and G, 25 μl of each solution was sequentially added to each well. In addition, 25 μl of diluted solution A shown in (1) of Comparative Example 1 was added to the 2 H wells of lanes 1 and 2.
Added one by one. 25 μl of the anti-HBs antibody-bound magnetic particle carrier suspension of (2) of Comparative Example 1 was added to each hole, and after stirring for about 1 minute using a microplate mixer, the plate was placed on the device 2 and subjected to magnetic force for 5 minutes. However, the rectangular ferrite magnet 1 of the device 1 was pulled out for 10 seconds out of 30 seconds to reduce the time during which magnetic force was applied to 2/3. Then, the microplate was taken down from the apparatus, and the aggregation image of each well was observed after about 1 to 2 minutes.

As shown in FIG. 7, the case where the image is uniformly spread on the well bottom surface is positive (+), and as shown in FIG. As shown in Figure 9, a case where the magnetic particle carrier is almost converged at the center of the well bottom is considered negative (-).
). The results are shown in Table 2.

[0034]

[Table 2]

A slight increase in measurement sensitivity was confirmed compared to when the magnetic force was applied continuously for 5 minutes using the device 2 described above. Furthermore, although data is not shown, the agglutination image (negative image) when the same operation was performed continuously for 2/3 of 5 minutes, that is, 3 minutes and 20 seconds using apparatus 2, was not as clear as in this example. . This example showed that by intermittently applying a magnetic force to a magnetic particle carrier, more sensitive measurement becomes possible.

Example 3 Effect of SN reversal (1) Measurement of HBs antigen Purified HBs antigen (manufactured by Meiji Dairy Co., Ltd.) was used in (1) of Comparative Example 1.
2.0nml, 1.0ng/ml with diluent A shown in
, 0.5ng/ml, 0.25ng/ml, 0.13n
g/ml, 0.063ng/ml, 0.031ng/m
Prepare each solution diluted so that it becomes 1.
) Lane 1 of the anti-HBs antibody-bound microplate,
2 in each of 2 wells of A, B, C, D, E, F, and G of 2.
5 μl of each solution was added in sequence. Furthermore, 25 μl of diluted solution A shown in (1) of Comparative Example 1 was added to the two H wells of lanes 1 and 2. Comparative example 1 (2)
Add 25 μl of anti-HBs antibody-bound magnetic particle carrier suspension to each hole, stir for about 1 minute with a microplate mixer, then place on device 2 shown in Example 2 (1) and apply magnetic force for 5 minutes. Ta. However, the square ferrite magnet 1 of device 2
was pulled out of the device every 15 seconds and S and N were turned over. In addition, in order to remove the magnet, reverse S and N, and place them again,
Each time it took about 5 seconds, the actual time the magnetic force was applied was about 3 minutes and 30 seconds. Thereafter, the microplate was taken down from the apparatus, and the aggregation image of each well was observed after about 1 to 2 minutes.

As shown in FIG. 10, the case where the image is uniformly spread over the well bottom surface is positive (+), and as shown in FIG. The results were determined to be weakly positive (±), and the results were determined to be negative (−) if an image showing a small convergence of magnetic particle carriers at the center of the bottom of the well as shown in FIG. 12 was determined. The results are shown in Table 3.

[0038]

[Table 3]

Although it was confirmed that the measurement sensitivity was lower than the results of Examples 1 and 2, the negative image converged particularly well compared to Examples 1 and 2, so the determination was very easy. This negative image was equivalent to the negative image shown in Comparative Example 1.
According to this example, the SN of the magnetic force acting on the magnetic particle carrier is
It was shown that by inverting the negative image, the negative image converges to a smaller size and the determination of the aggregated image becomes clearer. Example 4 Measurement using alternating magnetic field (1) Magnetism generator manufacturing device 3: As shown in Fig. 3, a 180 x 150 x 2 mm iron plate 2 is placed horizontally on a horizontal plate (experiment table), and a Place the electromagnet 8 on top and another iron plate 5 on top of it.
was mounted horizontally with a paper support 10 interposed therebetween. The electromagnet 8 used here is a solenoid made by wrapping an enameled wire 9 of 0.26 mm φ approximately 3,000 times around a 20 mm φ x 100 mm cylindrical iron core, and a voltage of 80 V AC at 50 Hz is applied to both ends of the enameled wire 9. I applied it. At this time, electromagnet 8
The magnetic force at both ends was about 220 Gauss (AC), and the magnetic force inside the device where the measurement container was placed was about 9 Gauss (AC). Place the measuring container approximately in the center on the lower iron plate 2. (2) Measurement of HBs antigen Purified HBs antigen (manufactured by Meiji Dairy Products Co., Ltd.) was used as described in (2) of Example 1.
2.0 ng/ml, 1.0 ng/ml in diluent A shown in
ml, 0.5ng/ml, 0.25ng/ml, 0.1
3ng/ml, 0.063ng/ml, 0.031ng
Prepare each solution diluted so that it is /ml, and add each of lanes A, B, C, D, E, F, and G of lanes 1 and 2 of the anti-HBs antibody-binding microplate in (3) of Example 1. 25 μl of each solution was added to each well in turn. Also,
In the 2 H wells of lanes 1 and 2, (1) of Comparative Example 1 was added.
25 μl of diluted solution A shown in 2 was added. Comparative Example 1 (
2) 25 μl of anti-HBs antibody-bound magnetic particle carrier suspension
was added to each well, and after stirring for about 1 minute using a microplate mixer, the plate was placed on the device 3 shown in (1) above, and a magnetic force was applied for 10 minutes. Then, the microplate was taken down from the apparatus, and the aggregation image of each well was observed after about 1 to 2 minutes.

As shown in FIG. 10, the case where the image is uniformly spread over the well bottom surface is positive (+); as shown in FIG. The results were determined to be weakly positive (±), and the results were determined to be negative (−) if an image showing a small convergence of magnetic particle carriers at the center of the bottom of the well as shown in FIG. 12 was determined. The results are shown in Table 4.

[0041]

[Table 4]

From the results of both Examples 1 and 2, negative images were clearly observed. This negative image was converged to the same extent as Comparative Example 1 in which sedimentation was performed using gravity. This example shows that the magnetic force acting on the magnetic particle carrier can be measured using an alternating magnetic field, and also shows that the negative image becomes clearer when an alternating magnetic field is used. Comparative Example 1 HBs antigen measurement by gravity sedimentation (1) Preparation of diluent A 15 grams of sodium caseinate (manufactured by Wako Pure Chemical Industries, Ltd.) was added to
The mixture was added to 3 liters of 05% NaN3-100mM NaCl-50mM Tris-HCl buffer (pH 8) and dissolved. Hereinafter, diluent A refers to the one having the above composition. (2) Preparation of anti-HBs antibody-bound magnetic particle carrier Magnetic particle carrier (Dynabeads M-450 uncoat
ed, manufactured by Dynal) and an anti-HBs monoclonal antibody (manufactured by Sinotest) and reacted at 37°C for 30 minutes. After adding about 20 times the volume of diluent A and reacting, washing with diluent A and redispersing with diluent A so that the magnetic particle carrier is about 0.1%, anti-HBs An antibody-bound magnetic particle carrier suspension was prepared. (3) Preparation of anti-HBs antibody-bound microplate anti-H
Bs monoclonal antibody (manufactured by Sinotest) at 0.0
It was diluted with isotonic phosphate buffer (PBS) containing 5% NaN3, and 50 μl was dispensed into all wells of a 96-well U-shaped microplate (manufactured by Nunc). After reacting at room temperature or 37°C, remove the liquid from all wells by suction and dilute solution A1.
Add 00-200 μl to all wells and leave again at room temperature or 3
The reaction was carried out at 7°C. Thereafter, all wells were washed with purified water to prepare an anti-HBs antibody-bound microplate. (4) Measurement of HBs antigen Purified HBs antigen (manufactured by Meiji Dairies Co., Ltd.) was diluted with diluent A to 2.0
ng/ml, 1.0ng/ml, 0.5ng/ml, 0
．． 25ng/ml, 0.13ng/ml, 0.063n
Prepare each solution diluted to be 0.031 ng/ml and 0.031 ng/ml, and apply the A, B, C, D, E, F,
25 μl of each solution was sequentially added to each of 2 wells of G. In addition, in the H 2 wells of lanes 1 and 2,
25 μl each of diluted solution A shown in (1) above was added. Anti-HBs antibody-bound magnetic particle carrier suspension 2 of (2) above
5 μl was added to each well, stirred for about 1 minute using a microplate mixer, then left at room temperature for about 2 hours, and the aggregation image was observed and judged.

As shown in FIG. 4, the case where the image is uniformly spread on the well bottom surface is positive (+), and the case where the image is not completely spread on the well bottom surface and is slightly converged as shown in FIG. 5 is positive (+). The image is weakly positive (±), and the image where the magnetic particle carrier is completely converged at the center of the bottom of the well as shown in Figure 6 is negative (
−). The results are shown in Table 5.

[0044]

[Table 5]

The results were very clear for negative images, weakly positive images, and positive images, but it took a long time to judge the aggregated image. Comparative Example 2 Agglomerated image in a state where the magnetic force is not changed over time (1) Magnetism generator manufacturing device 1: As shown in Figure 1, a square shape with a size of 100 x 100 x 10 mm is placed on a horizontal plate (experimental table). Place a ferrite magnet 1 with the N pole facing upward, and place it in a groove 4 of 50 mm height in a styrofoam support column 3 of 180 x 150 x 2 m.
A steel plate 2 of 2 m was inserted and placed horizontally. The measurement container is
Place it approximately in the center on this iron plate 2. The magnetic poles of this square ferrite magnet 1 are on a 100x100mm surface, and the magnetic flux density on the magnetic pole surface is about 250 Gauss at the center and about 1 Gauss at the four corners.
It was 100 Gauss. (2) Measurement of HBs antigen Purified HBs antigen (manufactured by Meiji Dairies Co., Ltd.) was used as described in (1) of Comparative Example 1.
2.0 ng/ml, 1.0 ng/ml in diluent A shown in
ml, 0.5ng/ml, 0.25ng/ml, 0.1
3ng/ml, 0.063ng/ml, 0.031ng
Prepare each solution diluted so that it is /ml, and add each of lanes A, B, C, D, E, F, and G of lanes 1 and 2 of the anti-HBs antibody-binding microplate in (3) of Comparative Example 1. 25 μl of each solution was added to each well in turn. Also,
In the 2 H wells of lanes 1 and 2, (1) of Comparative Example 1 was added.
25 μl of diluted solution A shown in 2 was added. In each well of this microplate, anti-HBs of (2) of Comparative Example 1 was added.
25 μl of the antibody-bound magnetic particle carrier suspension was added, and after stirring for about 1 minute using a microplate mixer, the microplate was placed on the device 1, and magnetic force was applied for 5 minutes. Thereafter, the aggregated image formed in each well of the microplate was observed. Although there was a difference between the negative and positive images, the colors were so pale that the distribution of the magnetic particle carriers within each well could not be clearly confirmed. For this reason, we were unable to publish a diagram of the agglutination image and a table of the determination results.

[0046]

As described above, according to the present invention, it has become possible to clearly judge agglutinated images, especially negative images. By changing the strength of the magnetic force over time, it has the effect of preventing a decrease in measurement sensitivity mainly due to the application of magnetic force, and also has the effect of preventing the distribution of magnetic particle carriers from becoming unclear. This has been confirmed. In addition, it has been confirmed that changing the direction of magnetic force over time has the effect of preventing the spread of negative images, which occurs when magnetic particle carriers are magnetized in the same direction by applying magnetic force. Ta.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory diagram showing a magnetism generating device that can be used in the present invention. FIG. 2 is an explanatory diagram showing a magnetism generating device that can be used in the present invention. FIG. 3 is an explanatory diagram showing a magnetism generating device that can be used in the present invention. FIG. 4 is an explanatory diagram showing a positive image (+) observed from directly above the well after 2 hours of sedimentation by gravity alone. The image is shown uniformly spread over the bottom of the well. [Figure 5] The image of the weakly positive image (±) observed from directly above the well after 2 hours when sedimentation was performed only by gravity.
FIG. The image shows a slightly converged image that cannot be completely spread out on the well bottom surface. FIG. 6 is an explanatory diagram showing a negative image (-) observed from directly above the well after 2 hours of sedimentation by gravity alone. This shows an image completely focused on the bottom of the well. FIG. 7 is an explanatory diagram showing a positive image (+) observed from directly above the well 1 to 2 minutes after the magnetic force was applied and the magnetic force was interrupted. The image is shown uniformly spread over the bottom of the well. FIG. 8 is an explanatory diagram showing a weakly positive image (±) observed from directly above the well, 1 to 2 minutes after the magnetic force was applied and the magnetic force was interrupted. The image shows a slightly converged image that cannot be completely spread out on the well bottom surface. FIG. 9 is an explanatory diagram showing a negative image (-) observed from directly above the well, 1 to 2 minutes after the magnetic force is applied and the magnetic force is interrupted. The image is almost converged on the bottom of the well. [Figure 10] After applying magnetic force, cut off the magnetic force and
FIG. 2 is an explanatory diagram showing a positive image (+) after 30 minutes when observed from directly above the well. The image is shown uniformly spread over the bottom of the well. [Figure 11] After applying magnetic force, cut off the magnetic force and
FIG. 2 is an explanatory diagram showing a weakly positive image (±) observed from directly above the well after 30 minutes. The image shows a slightly converged image that cannot be completely spread out on the well bottom surface. [Figure 12] After applying magnetic force, cut off the magnetic force and
FIG. 3 is an explanatory diagram showing a negative image (-) after 30 minutes when observed from directly above the well. It shows a small converged image on the bottom of the well. [Explanation of symbols] 1 Square ferrite magnet 2 Iron plate 3 Styrofoam support column 4 50 mm height groove 5 Iron plate 6 70 mm height groove 7 110 mm height groove 8 Electromagnet 9 Enameled wire 10 Paper support column

Claims (4)

[Claims] 1. An indirect aggregation reaction method using a magnetic particle carrier, characterized in that a magnetic vector acting on the magnetic particle carrier is temporally changed. 2. The indirect aggregation reaction method according to claim 1, wherein the temporal change in the magnetic vector is a temporal change in the strength of the magnetic force acting on the magnetic particle carrier. 3. The indirect aggregation reaction method according to claim 1, wherein the temporal change in the magnetic vector is a temporal change in the direction of the magnetic force acting on the magnetic particle carrier. 4. Claim 1, wherein the temporal change in the magnetic vector is interruption of the magnetic force in a process from the time when a magnetic force is first applied to the magnetic particle carrier until the aggregation image of the magnetic particle carrier is determined. The indirect agglutination reaction method described.