JPH03189560A - Immunological measurement method using magnetic marker particle - Google Patents

Abstract

PURPOSE:To shorten the time required for decision of an agglutination reaction and to allow measurement with high accuracy by using a method for accelerating the settling of magnetic marker particles by a magnet. CONSTITUTION:The suspension of the magnetic marker particles 5 formed by immobilizing a material which bonds specifically to a material to be measured to magnetic particles 4 is injected into a particle agglutination deciding vessel 1 immobilized with a magnetic particle capturing material 2 which can bond to the particles 4 in the section exclusive of the parts bonded to the material to be measured on the inside wall. A magnetic field is then impressed to settle the particles 5. The particles 5 are captured on the inside wall of the vessel 1 by the bonding of the particles 4 and the capturing material 2. The particles 5 which are not captured are removed from the inside of the vessel. A sample soln. is in succession injected into the vessel 1 to cause reaction to bond the material to be measured to the particles 5 captured on the inside wall of the vessel. The material which is not bonded is removed from the vessel 1. Further, the suspension of the paticles 5 is injected into the vessel 1. The magnetic field is impressed to accelerate the settling of the particles 5. The material to be measured contained in the sample soln. is measured in accordance with the agglutination patterns of the particles 5 settled on the inside wall of the vessel 1.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for determining antigens or antibodies present in a sample by immunological agglutination reactions.

[Conventional technology]

Particle agglutination methods are conventionally known as methods for detecting antigens or antibodies present in samples based on immunological agglutination reactions. This method uses specific marker particles on whose surface an antibody or antigen that specifically binds to the target m11 substance is immobilized. The marker particles are mixed with a sample in a measurement container, subjected to an immune reaction with the antigen or antibody contained in the sample, and then collected on a predetermined wall surface (for example, the bottom surface) of the measurement container. The marker particles thus collected on the wall of the container have different distribution patterns depending on whether or not an immune reaction has occurred with the substance to be measured in the sample.

Therefore, positivity or negativity can be determined from the distribution pattern of marker particles collected on the wall of the container.

Separately, Wlener, A. S., and Herma.
n, M, H.

A mixed aggregation method has been reported by (J, I uno 1
．． .

36.255 (1939)) This method has since been developed and established to the point where it is possible to determine blood types (CooIlbs, R.R.A., and Bedfor.
d,D,,VoxSang,.

5.111 (1955); Cooibs, R.R.
, A. et al., Lancet.

i, 461 (195B)). For example, USP No. 4,608,246, USP No. 4.275.053 and USP 4,328 as applied to determination of various blood types.
．． No. 183 is mentioned.

Additionally, a method is known in which an antibody, etc. that specifically binds to the substance to be measured is immobilized on the inner wall of the reaction container in advance (M
PHA method). In this method, a substance (such as an antibody) that specifically binds to a substance to be measured is immobilized on the inner wall of a reaction vessel in advance. After specifically binding the substance to be measured to this antibody or the like, marker particles are added. The marker particles react with the substance to be measured bound to the inner wall of the container to form an aggregation pattern. The M PHA method is, for example, USP
No. 4,297,104, etc.

By the way, the conventional particle aggregation method, mixed aggregation method and M
In the PIIA method, since sedimentable particles other than marker particles contained in the sample settle, the determination accuracy decreases. Therefore, in order to make a good judgment, operations such as washing the sample are required to remove these spontaneously settling particles.

Furthermore, most of the conventional particle aggregation methods and mixed aggregation methods described above use a method in which marker particles are collected on the bottom surface of the container by natural sedimentation after an immune reaction is performed in a predetermined container.

Therefore, there is a problem in that it takes a long time for marker particles, especially unreacted particles, to uniformly settle on the bottom surface and form a pattern. For example, in the "Anti-1gG Cell Kit (trade name)" for anti-platelet antibody detection sold by Olympus Optical Co., Ltd., the applicant of this case, it takes four
It takes ~6 hours.

If the sedimentation rate is simply to be increased, the specific gravity and particle size of the marker particles may be increased. However, since the specific gravity and particle size of the marker particles must be selected within a range that does not make the sedimentation pattern of the particles rough, this method cannot be expected to significantly shorten the measurement time.

On the other hand, the aforementioned USP No. 4,297,104 discloses a method using centrifugation in order to shorten the time required to form a sedimentation pattern of red blood cells used as marker particles. In this method, antibodies (IgG) are immobilized on the bottom surface of a measurement container having an axis of symmetry and on the surface of red blood cells used as marker particles. After reacting the antigen in the sample with the antibody on the surface of red blood cells in the measurement container in advance, 1g of anti-antibody against the surface of red blood cells and the antibody on the wall of the container is
Add G antibody. Next, by performing a first centrifugation process, red blood cells, which are marker particles, are attached to the wall surface of the container via the added anti-1gG. After the centrifugation is temporarily stopped, a second centrifugation process is performed to collect red blood cells that have not reacted with the antigen in the sample to the bottom of the container to form a sedimentation pattern.

The above centrifugation method is effective in promoting aggregation and sedimentation of marker particles due to immune reaction. However, centrifugation has the following drawbacks.

First, it is difficult to handle measurement containers smoothly in a series of operations from sampling to reaction, determination, disposal, etc. In particular, when performing centrifugation, the axis of symmetry of the measurement container must be precisely aligned with the direction in which centrifugal force acts, which is not so easy.

The second drawback is that analysis regarding different items is difficult. That is, it is necessary to change the sedimentation conditions for analysis regarding different items, but it is difficult to set multiple degrees and timings of centrifugation for each sample in the same centrifugation process. Therefore, it is necessary to perform centrifugation with different conditions set for each analysis item, or to use a plurality of centrifuges with different conditions set for each analysis item.

The third drawback is that when automating the analysis process, the use of a centrifuge necessitates a large-sized device.

In addition to the above problems, conventional M P )! Method A has a problem in that the reaction vessel is not very versatile. In other words, the substance that should be immobilized on the inner wall of the container in advance, that is, the substance that specifically binds to the substance to be measured, differs depending on the type of substance to be measured. I have to prepare.

[Problem to be solved by the invention]

The present invention was developed in view of the above circumstances, and its first objective is to improve an immunoassay method based on particle agglutination, without using marker particles with particularly large specific gravity or particle size, and without using centrifugation. The objective is to shorten the time required to determine an agglutination reaction without performing any processing.

A second object of the present invention is to provide an immunoassay method that can perform highly accurate measurements even on samples containing precipitable substances other than the substance to be measured, without being hindered by the precipitable substances. It is to be.

A third object of the present invention is to provide an immunological standard method that allows measurements to be carried out using the same reaction vessel even if the types of hep+determinants are different.

[Means to solve the problem]

The immunoassay method according to the present invention includes the following eight steps.

In the first step, a suspension of magnetic marker particles in which a substance that specifically binds to the analyte is immobilized on magnetic particles is added to a suspension of magnetic marker particles that can bind to the magnetic particles at a site other than the binding site with the analyte. Particle-trapping substances are injected into a particle aggregation determination container that is immobilized on the inner wall.

In the second step, a magnetic field is applied to cause the magnetic marker particles to settle, and the magnetic particles of the settled magnetic marker particles are combined with the magnetic particle capturing substance to cause the magnetic marker particles to be attached to the inner wall of the particle aggregation determination container. to be captured.

In the third step, the uncaptured magnetic macer particles are removed from the container.

In the fourth step, the sample solution is injected into the particle aggregation determination container and reacted, thereby binding the substance to be measured to the magnetic marker particles captured on the inner wall of the container.

In the fifth step, the substance that is not bound to the magnetic marker particles is removed from the particle aggregation determination container.

In the sixth step, the suspension of magnetic marker particles is injected into a container for determining particle aggregation.

In the seventh step, a magnetic field is applied to promote sedimentation of the magnetic marker particles.

In the second step, the substance to be measured contained in the sample solution is measured based on the aggregation pattern of the magnetic marker particles that have settled on the inner wall of the particle aggregation determination container.

In the present invention, in the first step, the sample solution may be injected simultaneously with the suspension of magnetic marker particles. In this case, the second to sixth steps described above are unnecessary.

In the method of the present invention, magnetic particles containing a magnetic substance are used as marker particles. Then, using a suitable magnet,
The sedimentation rate of the magnetic marker particles is increased. As a result, a sedimentation pattern of marker particles can be formed in a significantly shorter time than in conventional particle aggregation methods, and the time required for determination can be shortened.

Furthermore, since only the sedimentation of the magnetic marker particles can be selectively promoted, a sedimentation pattern of the magnetic marker particles can be formed before other sedimentable particles sediment.

Therefore, highly sensitive measurements can be performed without previously removing naturally settling particles other than the target substance.

In addition, in the method of the present invention, the magnetic particle trapping substance previously immobilized on the inner wall of the particle aggregation determination container only needs to be able to bind to the magnetic particles constituting the magnetic marker particles. That is, this magnetic particle capturing substance does not need to specifically bind to the target substance.

Therefore, the particle aggregation determination container used in the present invention has high versatility and can be used for measuring a wide range of substances to be measured.

The method of the present invention will be explained in detail below.

The substances to be measured in the present invention include, for example, antigenic substances such as viruses, bacteria, or various proteins, and antibodies against these antigenic substances. These target substances may be soluble substances or may be insoluble substances.

The magnetic marker particles used in the present invention immobilize a substance that specifically binds to the substance to be measured (hereinafter referred to as a sensitizer), such as a specific antibody or antigen, on the surface of known magnetic particles containing a magnetic substance. Prepared by oxidation. As magnetic particles, commercially available rDYNABEAs M-450J
(DYNAI, product name manufactured by the company) Magnetic latex beads r estapor J (PH0NE-POULE
(trade name manufactured by NC Corporation) can be used. Further, gelatin particles containing a magnetic material disclosed in JP-A-59-195113 may also be used. Furthermore, cells such as red blood cells incorporating a ferromagnetic material such as iron powder may also be used. Various known methods can be used to immobilize the sensitizer (specific antibody or antigen) on the surface of these magnetic particles.

The particle aggregation determination container used in the present invention is not particularly limited, except that a magnetic particle trapping substance capable of binding to the magnetic particles at a site other than the sensitizer is immobilized on its inner wall in advance. However, when the magnetic marker particles move in a predetermined direction along the container wall/surface, it is difficult to use an inclined wall surface, such as a hemispherical or conical wall surface, that causes the magnetic marker particles to converge in a predetermined area. is preferred.

Preferably, such an inclined wall surface may be located on the side surface of the container, or preferably on the bottom surface. A preferred particle aggregation determination container includes, for example, a test tube or microplate having a hemispherical or conical bottom surface, with the capturing substance immobilized on the inner wall thereof.

As the magnetic particle trapping substance, a substance that can bind to the magnetic particles at a site other than the sensitizing substance is used.

Various modes are possible for binding the magnetic particle trapping substance and the magnetic particles. FIG. 1 shows a case where the two are directly coupled. In the figure, 1 is a container for determining particle aggregation. A magnetic particle trapping substance 2 is immobilized on the bottom surface of the container. Into this container, magnetic marker particles 5 are introduced, in which only the sensitizing substance 3 that specifically binds to the substance to be measured is immobilized on the surface of the magnetic particles 4 . In this case, the capture substance 2 must be able to bind directly to the surface of the magnetic particles 4. The mode of binding may be specific or non-specific. Examples of the capture substance 2 that achieves specific binding include substances such as antigens, antibodies, and lectins that do not react with the sensitizer 3 but specifically react with the magnetic particles 4 . On the other hand, the capture substance 2 that achieves non-specific binding
Examples of the material include polyethyleneimine or poly-L-lysine that binds to the magnetic particles 4 by electrostatic bonding. Non-specific binding with the magnetic particles 4 can also be achieved by using a substance that non-specifically binds by hydrophobic bonding, coordinate bonding, or magnetic force, or by using antibodies and lectins with a wide range of reactivity. In this way, the magnetic particles 4 are directly bonded to the capture substance 2 and fixed to the bottom surface of the container 1, so that the magnetic particles 4
The sensitizer 3, which has been immobilized in advance, remains exposed in a free state.

FIG. 2 shows another embodiment in which the capture substance 2 is bound to the magnetic particles 4 via the link substance 6. In this case, in addition to the sensitizing substance 3 that specifically binds to the measurement substance, a link substance 6 that binds to the capture substance 2 is immobilized on the surface of the magnetic particles 4 in advance. When such a magnetic particle marker is introduced into the container 1, the capture substance 2 binds to the magnetic particles 4 via the link substance 6, thereby fixing the magnetic marker particles 5 to the inner wall of the container. Therefore, the sensitizer 3 remains free and exposed. Note that the bond between the link substance 6 and the capture substance 2 may be a specific bond or a non-specific bond, as described above.

This embodiment of FIG. 2 is particularly useful when there is no capture substance 2 that can bind directly to the magnetic particles 4.

In the embodiment of FIG. 2, examples of using specific binding between the capture substance 2 and the link substance 2 include the following case. That is, while a BSA antibody is preliminarily immobilized on the inner wall of the particle aggregation determination container 1 as the capture substance 2, BSA may be immobilized on the magnetic particles 4 as the link substance 6. When binding the sensitizer 3 to the magnetic particles 4, blocking with BSA is often performed. Therefore, this example has the advantage that BSA can be immobilized as the link substance 3 without any special operation.

Similar examples include antigen-antibody reactions such as protein A-1gG, gelatin-gelatin antibody, and lectin-sugar.

Next, with reference to FIGS. 3 to 9, a procedure for carrying out the measuring method according to the present invention according to the embodiment shown in FIG. 1 will be described.

First, as shown in FIG. 3, a particle aggregation determination container 1 having a capture substance 2 immobilized on its inner wall is prepared. This container 1
A suspension of magnetic marker particles 5 in which a sensitizing substance 3 is previously immobilized on the surface of magnetic particles 4 is added to the solution.

Next, as shown in FIG. 4, a magnet 10 is placed on the bottom of the container 1.
Place. When the magnet 10 is placed on the bottom of the measurement container in this manner, the magnetic marker particles 5 are attracted to the magnet 10 and settle at a faster rate than natural sedimentation. The sedimented magnetic marker particles 5 are immobilized on the inner wall of the container by the binding of the magnetic particles 4 and the capture substance 2. Therefore, the sensitizer 3 remains free and exposed as shown.

Next, after washing the container 741 to remove the remaining unbound magnetic marker particles 5, as shown in FIG. 5, a sample solution containing the substance to be measured 7 is added and reacted.

As shown in FIG. 6, the substance to be measured 7 specifically binds to the sensitizer 4 of the magnetic marker particles immobilized on the bottom of the container.

Next, as shown in FIG. 7, secondary magnetic marker particles 5
′ is added. As the secondary magnetic marker particles 5', the same particles as the previously added magnetic particles 5 can be used. Further, as long as the sensitizing substance 3 is the same as the magnetic marker particles 5 used previously, magnetic marker particles using different magnetic particles may be used.

Next, as shown in FIG. 8, a magnet 10 is placed on the bottom of the container 1. As a result, the secondary magnetic marker particles 5' are attracted to the magnet 10 and settle at a faster rate than natural settling. The precipitated secondary magnetic marker particles 5' are fixed to the inner wall of the container due to the combination of the sensitizing substance 3 and the substance to be measured 7. As a result, the secondary magnetic marker particles 5' form a uniformly spread positive distribution pattern on the bottom surface of the container 1, as shown in FIG. In other words, it is possible to detect the presence of the analyte in the sample based on the positive distribution pattern as shown in FIG.

On the other hand, if the substance 7 to be measured does not exist in the sample, the substance 7 to be measured does not exist on the inner wall of the container 1 at the stage shown in FIG. 7, that is, when the secondary magnetic marker particles 5' are added. . Therefore, as shown in FIG. 10, when the secondary magnetic particle marker 5' is caused to settle by acting on the magnet 10, the secondary magnetic marker particle 5' moves downward along the spherical wall surface.
As shown in the figure, it gathers at the lowest part of the spherical wall surface. As a result, as shown in FIG. 11, the magnetic marker particles 5 form a negative distribution pattern converging at the center of the spherical wall surface. Therefore, from the fact that a negative distribution pattern as shown in FIG. 11 is formed, it is possible to detect that the analyte is not present in the sample.

As described above, according to the present invention, the presence or absence of the substance to be measured is determined by observing the distribution pattern of the precipitated secondary magnetic marker particles 5- with the naked eye or by measuring it with an optical measuring device, and The substance can be quantified.

Alternatively, as shown in FIG. 12, the magnetic marker particles 5 and the sample may be simultaneously added into the container 1 in which the capture substance 2 is immobilized, and then the sedimentation may be promoted using the magnet 10. In this case as well, the analyte 7 is present in the sample.
, the same type of bond as shown in FIG. 8 is formed, and the magnetic particles form the same positive distribution pattern as shown in FIG. 9. That is, first, the first layer of magnetic marker particles 5 is bonded and immobilized on the inner wall of the container. continue,
By immobilizing the second and third layers of magnetic marker particles 5 on the second layer via the non-measurable substance 7,
A positive pattern is formed.

On the other hand, if the substance to be measured 7 is not present in the sample, the first layer and the third layer of magnetic marker particles are not formed, so that the same negative distribution pattern as shown in FIG. 11 is formed.

Further, even when adopting the embodiment explained in FIG. 2, the measurement can be performed using the same procedure as described above.

As described above, in the present invention, magnetic marker particles are used in the particle aggregation method, and a magnet is applied to promote sedimentation of the magnetic marker particles. Therefore, the time required to form a sedimentation pattern of magnetic particles can be significantly shortened without increasing the specific gravity or particle size of the marker particles or without performing centrifugation. Therefore, the measurement time can be significantly shortened without the sedimentation pattern becoming rough and without the drawbacks that occur when centrifugation is used.

Further, according to the procedure shown in FIGS. 3 to 9, the container is cleaned at the stage shown in FIG. 6, when the substance to be measured 7 is bound to the magnetic marker particles 5. Therefore, like whole blood or chyle serum, etc.
It can also be effectively applied to samples containing precipitable substances or insoluble substances other than the target substances. That is, good measurement results can be obtained without preliminarily separating precipitated substances or insoluble substances other than the substance to be measured and purifying the sample.

Even when the procedure explained in FIG. 10 is used, the present invention can selectively promote only the sedimentation of the magnetic marker particles 5, so that it is not interfered with by sedimentary substances or insoluble substances other than the analyte contained in the sample. It is possible to perform measurements without any That is, by applying a magnet before or at the beginning of the precipitation of substances other than the analyte to be measured, a sedimentation pattern of the magnetic marker particles 5 is formed before the substances other than the analyte to be measured are precipitated. can do. Similarly, even when the sample is a highly viscous solution or a solution with a higher specific gravity than the magnetic marker particles 5, it is possible to selectively promote the sedimentation of the magnetic marker particles 5 and perform good measurements. .

Furthermore, in the present invention, since the magnetic marker particles 5 are forcibly moved by the action of the magnet, the magnetic marker particles 5 can be moved in a direction different from the sedimentation direction of substances other than the substance to be measured. For example, when using a test tube as a measurement container, by arranging a magnet on the side wall, substances other than the substance to be measured can settle to the bottom by natural sedimentation, and the magnetic marker particles 5 can be collected on the side wall. This method also allows the distribution pattern of the magnetic marker particles 5 to be formed without being influenced by substances other than the substance to be measured.

As mentioned above, the method of the present invention allows measurement to be performed without being disturbed by sedimentation of substances other than the analyte, so that the method of the present invention can be used for samples with sedimentation of substances other than the analyte, such as whole blood or chyle serum. It can also be effectively applied to samples containing substances or insoluble substances. That is, good measurement results can be obtained without preliminarily separating precipitated substances or insoluble substances other than the substance to be measured and purifying the sample.

Furthermore, the method of the present invention does not require that the capture substance 2 immobilized on the inner wall of the particle aggregation determination container 1 has specificity for the substance to be measured 7.

Therefore, it can be used commonly for multiple substances to be measured.
Unlike the conventional M P II A method, there is no problem of poor versatility of the reaction vessel.

Additionally, the method of the invention can be performed simultaneously with conventional measurements using non-magnetic marker particles. for example,
It is possible to perform measurements using magnetic marker particles in some wells of the microplate, while simultaneously performing measurements using non-magnetic marker particles in other wells. That is, even if a magnet is used to promote sedimentation of magnetic marker particles in some wells, it does not have the same effect on non-magnetic marker particles in other wells.

This is advantageous for measuring multiple items at the same time.

The magnet used in the present invention is not particularly limited, and may be a permanent magnet or an electromagnet. The strength of the magnet is appropriately selected depending on the substance to be measured. Further, the shape of the magnet may be any shape other than a disk shape.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 <ALU-shaped bottom microplate (“Ma” manufactured by NUNC)
xisorpJ, catalog number 4692B4) was prepared.

On the other hand, polyethyleneimine (manufactured by Hanui Chemical Co., Ltd.) was diluted with physiological saline (0.9% NaCI) to prepare a 1.0% polyethyleneimine solution. This polyethyleneimine was dispensed in an amount of 50 μΩ into each well of the microplate, and incubated at room temperature for 30 minutes. Thereafter, the plate was washed five times with 200 μg/well of physiological saline and dried at room temperature to obtain a microplate having a polyethyleneimine coating layer. This polyethyleneimine coating layer acts as the magnetic particle trapping substance 2 described above.

<B> Anti-human IgG (manufactured by CPL, catalog number 31867) on gelatin beads (particle size -5 μm) containing magnetic material.
) Sensitized beads were prepared by sensitizing. These sensitized beads act as the magnetic marker particles 5 described above.

<C> The 0.5% sensitized bead suspension prepared above was dispensed at 50 μg/well into the microplate having the polyethyleneimine coating layer described above. By applying a magnet for 3 minutes, the sensitized beads are attracted to the bottom and allowed to settle, and then washed twice with 200 μg/well of physiological saline. As a result, the sensitized beads could be uniformly immobilized on the bottom surface of the well in the state shown in FIG. 4.

<D> Human IgG (manufactured by ICN) was diluted with PBS (phosphate buffered saline), and 0.1 to 0.01 ug/
A positive sample solution of 16 degrees was prepared. This positive sample solution was dispensed at 25 μl/well into a microplate treated with C>. Further, PBS was used as a negative sample, and 25 μg/well of this was dispensed.

After 10 min incubation at 37°C, 200 μp
/well was washed 6 times with physiological saline.

Next, 25 μg/well of the 0.5% sensitized bead suspension prepared in <B> was dispensed again. This was placed on a magnet for 3 minutes to sediment the sensitized beads and form a distribution pattern. As a result, a positive pattern as shown in FIG. 9 was obtained in the well injected with the positive sample, and a negative pattern as shown in FIG. 11 was obtained in the well injected with the negative sample. These results revealed that the present invention enables immunological measurement of human IgG.

In this example, the time required from the addition of the sensitized beads in C> to the determination was approximately 16 minutes, excluding washing time.

Example 2 Detection of human IgG without washing <A> By substantially the same procedure as described in Example 1, a microplate whose inner wall was coated with polyethyleneimine was prepared. However, the concentration of the polyethyleneimine solution is 0. C was set at 11%. In addition, the number of times of washing with physiological saline was three times.

<B> 0.5% of the same sensitized beads prepared in Example 1
A solution, a positive sample containing human IgG, and a human IgG
Human IgG was assayed in the following manner using a negative sample containing no .

First, 50 Mg/well of the sensitized bead suspension and 25 μρ/well of the positive sample were dispensed into the same wells of the microplate prepared above. Also,
The sensitized bead suspension at 50 μρ/well and the negative sample at 25 μΩ/well were dispensed into the same wells of the microplate. After gently mixing the two components dispensed into each well, they were incubated at 37°C for 10 minutes.

Each well was placed on a magnet to promote sedimentation and form a distribution pattern. As a result, a positive pattern as shown in FIG. 9 was obtained in the well injected with the positive sample, and a negative pattern as shown in FIG. 11 was obtained in the well injected with the negative sample. This result shows that the measurement method of the present invention can be carried out even if the sensitized beads and the sample are added at the same time without washing.

Note that in this example, the pattern could be determined in about 13 minutes, and the measurement time could be shortened by 3 minutes compared to Example 1.

Example 3 <ALU-shaped bottom microplate (rM manufactured by NUNC)
axisorpJ, catalog number 489264) was prepared.

On the other hand, anti-BSA antibody (anti-BSA serum albumin antibody 2B
ET) was diluted with p++-9.5 O, 01M carbonate buffer to prepare a solution with a concentration of 10 Mg/n+I.

This anti-BSA antibody solution was dispensed in an amount of 50 μΩ into each well of the microplate, and incubated at room temperature for 60 minutes. Then 0.01M PB at pH-7.0
A microplate with an anti-BSA antibody immobilized on its inner wall was obtained by washing the plate 5 times with S at 200 μg/well and drying at room temperature. This anti-BSA antibody is
It acts as the magnetic particle trapping substance 2 described above.

<B> Anti-human IgG (manufactured by CPL, catalog number 31867) on gelatin beads (particle size -5 μm) containing magnetic material.
) to 1. Sensitized beads were prepared by sensitizing with czg/ml and blocking with BSA. 20 The sensitized beads act as the magnetic marker particles 5 described above. 0.01M citric acid containing 0.2% BSA-0,
A suspension of the above-mentioned sensitized beads was prepared using a mild isotonic solution (pH-6) of 02M phosphoric acid.

<C> Dilute human IgG (manufactured by ICN) with PBS,
A positive sample solution with a concentration of 0.01-0.1 μg/ml was prepared. In addition, PBS itself was used as a negative sample. Using these positive and negative samples, human IgG was assayed as follows.

First, 30 Mg/well of the sensitized bead suspension and 25 μg/well of the positive sample were dispensed into the same wells of the microplate prepared above. Also,
30 μΩ/well of the sensitized bead suspension and 25 μg/well of the negative sample were dispensed into the same wells of the microplate.

After incubation for 10 minutes at 37°C, each well was placed on a magnet to promote sedimentation and form a distribution pattern. As a result, a positive pattern as shown in FIG. 9 was obtained in the well injected with the positive sample, and a negative pattern as shown in FIG. 11 was obtained in the well injected with the negative sample.

The pattern formation mechanism in this example is as follows. That is, the sensitized beads are immobilized on the well wall by binding with the blocking BSA (!:) present on the surface of the sensitized beads and the anti-BSA antibody on the well wall.
Since the SA antibody is neutralized by BSA contained in the bead suspension, the well wall surface becomes blocked with BSA.

Then, on the immobilized sensitized beads, human IgG further reacts with other sensitized beads to form a positive pattern or a negative pattern.

Note that in this example as well, as in Example 2, the pattern could be determined in a short time of about 13 minutes.

〔Effect of the invention〕

As detailed above, according to the present invention, by using a method of promoting the sedimentation of magnetic marker particles using a magnet, immunological studies can be performed more quickly than in the case of natural sedimentation, and more easily than in the case of sedimentation using centrifugal force. It is possible to carry out targeted measurements. Furthermore, since there are more opportunities for particles to come into contact with each other than in conventional particle mixing and aggregation methods, a stable distribution pattern can be formed quickly and with high sensitivity. In addition, since there is no need to change the capture substance immobilized on the inner wall of the container depending on the target non-measurable substance, significant effects can be obtained, such as the ability to measure various test items in the same test container. be able to.

[Brief explanation of drawings]

FIG. 1 is a diagram showing an example of a particle aggregation determination container and magnetic marker particles used in the present invention, FIG. 2 is a diagram showing another example, and FIGS. 3 to 11 are diagrams showing one example of the present invention. FIG. 12 is a diagram for explaining a procedure for carrying out an immunological measurement method according to another embodiment of the present invention. DESCRIPTION OF SYMBOLS 1... Particle aggregation determination container, 2... Magnetic particle capture substance, 3... Sensitizing substance, 4... Magnetic particles, 5... Magnetic marker particles, 6... Link substance, 7... ...Non-measurable substance, 10...Magnet

Claims (2)

[Claims] (1) A magnetic particle capture substance that can bind a suspension of magnetic marker particles in which a substance that specifically binds to the analyte is immobilized to the magnetic particles at a site other than the binding site with the analyte. is injected into a particle aggregation determination container in which is immobilized on the inner wall, applying a magnetic field to sediment the magnetic marker particles, and binding the magnetic particles of the sedimented magnetic marker particles with the magnetic particle capturing substance. , capturing the magnetic marker particles on the inner wall of the particle aggregation determination container; removing the uncaptured magnetic marker particles from the container; and injecting the sample solution into the particle aggregation determination container and causing a reaction. , a step of binding a substance to be measured to the magnetic marker particles captured on the inner wall of the container; a step of removing the substance not bound to the magnetic marker particles from the particle aggregation determination container; and suspending the magnetic marker particles. A step of injecting a liquid into a container for determining particle aggregation, a step of applying a magnetic field to promote sedimentation of magnetic marker particles, and a step of injecting a sample solution into a container for determining particle aggregation based on the aggregation pattern of the magnetic marker particles that have settled on the inner wall of the container for determining particle aggregation. 1. An immunological measurement method using magnetic marker particles, comprising the step of measuring a substance to be measured contained therein. (2) A suspension of magnetic marker particles in which a substance that specifically binds to the analyte is immobilized on the magnetic particles and a sample solution can be bonded to the magnetic particles at a site other than the binding site with the analyte. a step of injecting a particle capture substance into a particle aggregation determination container having an immobilized particle on the inner wall; a step of applying a magnetic field to the reaction solution to promote sedimentation of the magnetic marker particles; 1. An immunoquantitative measurement method using magnetic marker particles, comprising the step of measuring a substance to be measured contained in a sample solution based on an aggregation pattern of the magnetic marker particles.