JPH02124464A - Immunological measuring method using magnetic marker - Google Patents

Abstract

PURPOSE:To shorten measuring time and to make it possible to perform measurement highly accurately without interference from sediment by mixing sample solution and magnetic marker grains, using a magnet for reacting solutions of both materials, and collecting the marker grains in a specified region. CONSTITUTION:A material which is specifically bonded to a material to be measured 6 or competes with said material 6 is fixed on the surface of a magnetic grain. Thus, a magnetic marker grain 5 is adjusted. Then, a sample and the grain 5 are mixed and made to react, and the material to be measured 6 and the grain 5 are bonded. Then reacting liquid is inputted in a measuring container (a microplate having a semi-spherical bottom surface) 1. A magnet 4 which is mounted on a supporting stage 3 is arranged beneath a well 2. The particle 5 is attracted with the magnet 4 and falls at a speed faster than a natural falling speed. The distribution patterns of the falled grains are considerably different based on whether the material to be measured 6 is present or not in the sample. When the distribution pattern is observed with the naked eyes or measured with an optical measuring instrument, the presence or absence of the material to be measured can be judged, and the quantity can be determined.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] 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 substance to be measured 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, Iflener, A.J., and Herva.
n, M, H.

A mixed aggregation method has been reported by (J, lm5uno
1. .

88.255 (1939)). This method has since been developed and established to the point where it is possible to determine blood type (Coombs, R.R.A. and Bedfor.
d,D, ,VoxSang,.

5.111 (1955); Coombs, R.R.
,^,at al,,Lafle13t,i.

461 (1956)). For example, USP No. 4,608.248, US
P 4.275. O5'i1 and USP 4.321
1.183 is mentioned.

Furthermore, USP No. 2,770.572 describes a method for improving the sensitivity of blood type determination on a solid surface by retaining an antibody that binds and reacts with an antigen in a sample on a dry solid surface in advance. A method is disclosed.

Furthermore, Rosenfled R.E. et al. reported an example of reacting red blood cell antigens and antibodies on a solid surface using the principle of mixed agglutination method (Par et al.
is.

Proc, 15Lh Cong, Intl, Soc, B
Load Transfusion, 27 (1976)
).

By the way, in the above conventional particle aggregation method and mixed aggregation 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, USP No. 4,297.104 discloses the following 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 the antigen in the sample and the antibody on the surface of the red blood cells are reacted in advance in the measurement container, an anti-1gG antibody against the antibody on the surface of the red blood cells and the wall of the container is added. 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.

[Problem to be solved by the invention]

The present invention was made in view of the above circumstances, and its first objective is to improve the immunological DI method using particle agglutination method,
The object of the present invention is to shorten the time required for determining an agglutination reaction without using marker particles having a particularly large specific gravity or particle size, and without performing centrifugation.

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.

Means for Solving CaWJ] The immunoassay method according to the present invention includes the following three steps.

In the first step, a sample solution and magnetic marker particles in which a substance that specifically binds to or competes with the substance to be measured is immobilized on magnetic particles are mixed to form a reaction solution for the two.

In the second step, 1. The magnetic marker particles are collected in the predetermined wall region by causing a magnet placed outside a predetermined wall region of the measurement container to act on the reaction solution contained in the measurement container of the illumination container. .

In the third step, the substance to be measured contained in the sample is measured based on the distribution state of the magnetic marker particles collected in the predetermined wall area.

In the present invention, it is desirable that a substance that specifically binds or competes with the substance to be measured be immobilized on the inner surface of a predetermined wall surface area of the measurement container in advance.

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.

In addition, since it is possible to selectively promote the sedimentation of only the magnetic marker particles, the magnetic marker
- Capable of forming sedimentation patterns of particles.

Therefore, highly sensitive measurements can be performed without removing naturally settling particles other than the substance to be measured in advance.

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 substances to be irradiated may be soluble or insoluble substances.

First, a magnetic marker is created by immobilizing a substance that specifically binds to or competes with the substance to be determined, such as a specific antibody or antigen, on the surface of known magnetic particles containing a magnetic material. Prepare particles. As magnetic particles, commercially available rDYNABEAS M-450J
(trade name manufactured by DYNAL), magnetic latex beads restapor J (RHONE-POULE
(trade name manufactured by NC Corporation) can be used.

Further, gelatin particles containing a magnetic material disclosed in Japanese Patent Application Laid-Open No. 59-19518 may 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 specific antibody or antigen on the surface of these magnetic particles.

Next, the sample and the magnetic marker particles are mixed and reacted to bond the substance to be measured contained in the sample to the magnetic marker particles.

Next, the above reaction solution is placed in a predetermined measurement container. Alternatively, the above reaction may be carried out in this /1 #1 constant vessel. The measurement container is not particularly limited.

However, when the magnetic marker particles move in a predetermined direction along the wall surface of the container, it is preferable to use a container having an inclined wall surface, such as a hemispherical or conical wall surface, so that the magnetic marker particles are converged in a predetermined area. . Such sloped walls may be on the sides of the container, but are preferably on the bottom. Preferred measurement containers include, for example, test tubes or microplates with hemispherical or conical bottoms.

Next, a magnet is placed on at least the inclined wall surface portion of the outer wall surface of the container. For example, as shown in Figure 1 (A) (6),
When using microplate 1 as a measurement container,
A magnet 4 placed on a support base 3 is placed below the hemispherical bottom of the well 2 . Note that FIG. 1(A) is a plan view, and FIG. 1(6) is a side view. In addition, the preparation of magnetic marker particles and the mixing of the magnetic marker particles and the sample are as follows:
The measurement may be carried out within this measurement container. When the magnet 4 is placed on the bottom of the measurement container in this manner, the magnetic marker particles are attracted to the magnet 4 and settle at a faster rate than natural settling.

The distribution pattern of the precipitated magnetic marker particles differs depending on whether the Δ11 constant substance is present or absent in the sample. Furthermore, the distribution pattern is different depending on whether a substance that binds to the Δ11 substance to be measured is immobilized on the magnetic marker particles or a case where a substance that competes with the analyte is immobilized on the magnetic marker particles.

First, a case will be described in which a magnetic marker substance in which a substance that forms condensates is immobilized on an IItal constant substance is used.

In this case, if the analyte is present in the sample, the second A
As shown in the figure, a plurality of sedimented magnetic marker particles 5
are combined with each other via the substance to be measured 6 to form an aggregate. Therefore, the magnetic marker particles 5 cannot move any further. Therefore, as shown in FIG. 2B, a uniform distribution pattern is formed on the spherical wall surface of the measurement container 1 (positive). On the other hand, if the substance to be measured is not present in the sample, the magnetic marker particles 5 will not be bonded to each other, and no aggregates will be formed. Therefore, the magnetic marker particles that have settled on the spherical wall surface of the container further move downward along the spherical wall surface and collect at the lowest part of the spherical wall surface, as shown in FIG. 3A. As a result, as shown in FIG. 3B, the magnetic marker particles 5 form a distribution pattern converging at the center of the spherical wall surface (negative). Therefore, by observing the distribution pattern of the precipitated magnetic marker particles with the naked eye or measuring it with an optical measuring device, it is possible to determine the presence or absence of the substance to be measured, and also to quantify the substance.

On the other hand, when using a magnetic marker substance immobilized with a substance that competes with the analyte (in other words, the same substance as the analyte), in addition to the magnetic marker particles, both the analyte and the magnetic marker particles A crosslinking substance capable of binding is added into the sample. If the analyte is not present in the sample, this crosslinking material will bind to the magnetic marker particles. Therefore, since the plurality of magnetic marker particles are bonded to each other via the crosslinking substance and aggregated, the sedimentation pattern becomes the same as that shown in FIGS. 2A and 2B (negative). On the other hand, if a large amount of the analyte is present in the sample, the crosslinking substance will bond to the analyte. Therefore, aggregation of magnetic marker particles via the crosslinking substance does not occur. Therefore, the sedimentation pattern at this time is 3A.
It will be the same as shown in the figure and Figure 3B (positive).

This method is known as an aggregation inhibition reaction. After all, when jIll determination is performed by an aggregation suppression reaction, the distribution pattern of the precipitated magnetic marker particles is opposite to that when a magnetic marker substance on which a substance that binds to the ΔIII substance is immobilized is used.

In the present invention, a substance that binds to or competes with the analyte in the sample may be immobilized on the wall of the measurement container in advance.

First, with reference to FIGS. 4A to 4C, a magnetic marker is prepared in which a capture substance 7 that binds to the substance to be measured in the sample is immobilized on the wall surface of the container 1, and a substance that binds to the substance to be measured is immobilized on the surface. A case where particles 5 are used will be explained.

In this case, a sample is first placed in the measurement container 1, incubated and reacted, and then the sample is removed and the container is washed. As a result, the substance to be measured present in the sample is bound to the capture substance 7 and captured. Next, the solution of the magnetic marker particles described above is placed in the container 1, and the magnet 4 is applied in the same manner as described above to form the magnetic marker particles 5.
to precipitate. The sedimented magnetic marker particles 5 are
As shown in the figure,
Since it binds to the constant substance 6, it does not move further downward along the wall of the container. Therefore, the sedimented magnetic marker particles 5 form a uniformly spread distribution pattern as shown in FIG. 2B (positive). On the other hand, when the substance to be measured 5 is not present in the sample, the precipitated magnetic marker particles 5 are not bonded to the wall surface of the container. Therefore,
As shown in Figure 4c, move to the center of the container wall, and
Similar to Figure B, a distribution pattern converged in the center of the spherical wall surface is formed (negative). In this case, since the magnetic marker particles 5 are added after the sample is removed and the container 1 is washed, the formation of the distribution pattern of the magnetic particles is not interfered with by other components in the sample. Therefore, it is possible to perform DI determination with higher sensitivity. Furthermore, in a positive reaction, magnetic particles are bonded to the wall of the container, so positive and negative can be determined more clearly. This also contributes to improving detection sensitivity.

Moreover, instead of washing the 4-1 fixed container 1 as described above, the reacted sample housed in the measurement container 1 may be diluted after magnetic marker particles are added and reacted. This also improves measurement sensitivity. However, in this case, the aggregation pattern of the magnetic marker particles when positive is not the state shown in FIG. 4A but the inclination angle shown in FIG. 4B.

On the other hand, a capture substance that binds to the analyte 71P1 is immobilized on the wall of the container 1, and a substance that competes with the analyte 6 (for example, the same substance as the analyte 6) is immobilized on the surface of the magnetic marker particles 5. In this case, the substance to be measured 6 and the magnetic marker particles 5 compete for the captured substance 7 on the wall of the container. Therefore, the distribution pattern of positive and negative results is opposite to that described above. That is, in a positive reaction, the capture substance 7 binds to the analyte 6 and becomes saturated. Therefore, magnetic marker particles 5
Since the particles are not captured, the distribution pattern is concentrated in the center of the spherical wall surface as shown in Fig. 4C. On the other hand, in a negative reaction, since the capture substance 7 on the wall of the container is not saturated, the magnetic marker particles 5 bind to the capture substance 7 and form a uniformly spread distribution pattern as shown in FIG. 4A.

Furthermore, a substance that competes with the substance to be measured 6 (for example, the same substance as the substance to be measured 6) may be immobilized on the wall surface of the container 1. In this case, the distribution pattern of the sedimented magnetic marker particles 5 is opposite to that in the case where a substance that binds to the substance to be measured 6 is immobilized on the bottom of the container. Details of the mechanism will be omitted.

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.

Furthermore, since the present invention can selectively promote only the sedimentation of magnetic marker particles, it is possible to perform measurements without being interfered with by sedimentary substances or insoluble substances other than the analyte contained in the sample. That is, by applying a magnet before or at the beginning of the precipitation of substances other than the analyte, a sedimentation pattern of magnetic marker particles is formed before the substances other than the analyte begin to settle. be able to. Similarly, even when the sample is a highly viscous solution or a solution with a higher specific gravity than the magnetic marker particles, it is possible to selectively promote sedimentation of the magnetic marker particles and perform good measurements.

Furthermore, in the present invention, since the magnetic marker particles are forcibly moved by the action of the magnet, the magnetic marker particles can be moved in a direction different from the sedimentation direction of substances other than the above-mentioned 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 magnetic marker particles can be collected on the side wall. This method also makes it possible to form a distribution pattern of magnetic marker particles without being influenced by substances other than the substance to be measured.

As mentioned above, he? #1 Since measurements can be performed without interference from sedimentation of substances other than the analyte, the method of the present invention can be applied to precipitated substances or insoluble substances other than the analyte, such as whole blood or chyle serum. It can also be effectively applied to samples containing 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 invention can be performed simultaneously with conventional measurements using non-magnetic marker particles. For example, it is possible to perform all measurements using magnetic marker particles in some wells of a 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 performing 411 determination of 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. Moreover, it is not limited to the disk shape shown in FIG. 1B, but may have any shape. Among these, it is particularly preferable to use a needle-shaped magnet 8 with a sharp tip, as shown in FIG. 5, for example. The reason is that when the magnetic marker particles 5 gather at the center of the bottom of the 1llllJ fixed container as shown in FIG. 3A or 4C, if the needle-shaped magnet 8 is used, the magnetic marker particles 5 can be focused in a narrower area. This is because it is possible. As a result, the difference between the positive pattern and the negative pattern becomes clearer, and all agreement can be improved. For the same reason, it is preferable for the wall surface of the well 2 to have a conical shape, as shown in FIG. 5, rather than a hemispherical shape.

When using a microplate in which a large number of wells 2 are formed as a 11PI constant container, a reaction accelerator as shown in FIG. 6 can be suitably used. As shown in the figure, this reaction promoting device has a support base 10 with a raised outer edge so that a microplate of 8×10 wells can be placed thereon without shifting its position. The support stand 10 includes
Circular recesses 11 are formed at positions corresponding to each well of the microplate. Inside the concave hole 11 is a needle-shaped ferrite magnet (magnetic flux density 2
200 Gauss) 12 are arranged. Using this reaction accelerator, the needle magnet 12 can be placed at the center of each well bottom simply by placing the microplate on the support stand 10. The tip of the ferrite magnet 12 may be either an N pole or an S pole.

FIG. 7 shows another example of a reaction accelerator that can be suitably used in the present invention. In this reaction accelerating device, a female thread is cut on the inner surface of a recessed hole 13 provided in a support base 10. A cylindrical magnet 14 having a male thread cut on its outer periphery is screwed into the recessed hole 13 . A (-) shaped groove 15 for inserting the tip of a driver is formed on the top surface of the head of the magnet 14, and the magnet 14 is screwed into the recessed hole 13 by being rotated by the driver. In this device, since the magnet 14 is screwed into the magnet 14, the height of the magnet head can be easily adjusted by controlling its rotation. Note that the groove 15 may have a (+) shape. Further, the head of the magnet 14 may be a north pole or a south pole.

In the present invention, the step of applying a magnet to promote sedimentation of the magnetic marker particles may be carried out with the 1-1 constant container standing still, or may be carried out while the measurement container is being transferred. It's okay.

〔Example〕

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

Example 1 <Creation of Ago type red blood cell solid plate U7 bottom microplate (manufactured by NUNC, serial number 46)
4394)! Concentration 10μg/1sfl using 0.01M phosphate buffer (PBS) with IK and p117.0.
100 μg of wheat germ lectin (WG^; manufactured by Seikagaku Corporation) prepared in 2008 was added to each well, and the mixture was treated at room temperature for 30 minutes. Then p) 0.01M PBS of 17.0
A WG^-treated plate was obtained by washing 5 times with 300 μg of each and drying at room temperature.

Separately, a surge screen (manufactured by 0rtho, manufacturing number 389837), which is human type 0 red blood cells having each blood type antigen in Table 1, was prepared, and this was diluted with physiological saline at a concentration of 0.3
diluted to %. This diluted surge screen,
Surge Screen was adsorbed onto the surface of each well of the microplate by adding 25 μg/well to the wG^-treated plate and allowing it to stand at room temperature for 10 minutes. Furthermore, pH containing 0.1% bovine serum albumin (BSA)
Using 200μp of 0.01M PBS of 7.0, 3
Washed twice.

Table 1 Donor number Genotype 41901R2R2 +: Blood antigen present - Blood antigen absent <B> Preparation of anti-human IgG sensitized magnetic latex beads 0.1M glycine/sodium hydroxide buffer (hereinafter referred to as glycine buffer) at pH 8.3 dark brown magnetic latex beads (trade name: res) with a particle size of 1μ
tapor J; manufactured by RIIONE-POULENC, serial number LMP 233) solution (concentration 0.4%)
) was prepared. In addition, using the same glycine buffer, anti-human IgG (manufactured by Capple, manufacturing number 0801-
0081) (concentration 1 μg/rrl) was prepared. Anti-human Ig was added to magnetic latex beads by mixing 2 ml of each solution and incubating for 1 hour at 37°C.
sensitized G. This sensitized latex was mixed with 2 ml of 9118.3 0.1M glycine buffer containing 1% BS^
After washing twice, blocking was performed to suppress non-specific aggregation by reacting in 2 mfl of the same buffer at 37°C for 300 minutes. Furthermore, 0.01% sarcosine) LN (surfactant manufactured by Nikko Chemicals);
sodlum N-1auroyl 5arcosl
nate) and o, 0.0% at pH 7.0 containing i% BSA.
Wash 3 times with 2 ml of 01M PBS, and add the same PB.
Stored in S4m, 77.

<C> Detection of irregular antibodies 60 Mg of Li5s solution was added to each well of the O-type red blood cell solid-phase plate prepared in <A>, and anti-antibodies were added as samples.
Antiserum of either anti-Fy1 or anti-i (0rth
(manufactured by Company O) was added thereto, and the mixture was reacted at 37° C. for 300 minutes. At the same time, as a negative control, one well received 30 mg of 0.01M PBS instead of antiserum.
was added and reacted in the same manner.

Next, 0.0 at pH 7.0. 200μg OLM PI35
After washing each well four times using the anti-human IgG magnetic latex beads, 25 μg/well of the anti-human IgG magnetic latex beads were added. Subsequently, as shown in FIG. 2(A) (6), the bottom of each well 7 is positioned above the disc-shaped ferrite magnet 5 disposed on the support base 4, and each well 7 is
The reaction was carried out at room temperature for 2 minutes while a magnet was applied in the vertical direction of the bottom surface of the sample. In addition, the diameter of the ferrite magnet 5 is 8
The weight and thickness are 3 nm, and the residual hourly density is 2000 Gauss.

By the above operation, anti-human IgG is added to the bottom of well 7.
The latex beads settled. When the precipitated anti-human IgG magnetic latex beads were uniformly spread and distributed on the bottom of the well, it was determined to be positive, and when they gathered at the center of the well, it was determined to be negative. The results are shown in Table 2. This result is
It matched with the red blood cell antigen shown in Table 1.

In the above detection operation, magnetic marker particles such as anti-human I
Since the sedimentation of the gG magnetic latex beads was promoted by a magnet, the time required for determination was shortened to one-tenth of that of conventional particle aggregation methods.

Note that, as understood from the above description, this example was carried out by the method described in FIGS. 4A and 4C. That is, a surge screen was immobilized on the well wall in advance as a capture substance 7 that binds to the test substance. In addition, the wells were washed after reacting the sample. Therefore, a stable and clear sedimentation distribution pattern was obtained, and the detection sensitivity was excellent.

Therefore, in order to evaluate the detection sensitivity in comparison with the conventional method,
The following comparative experiment was conducted.

<D> Sensitivity comparison with conventional method (a) Type O photo blood cells that react with 12 types of commercially available irregular antibodies shown in Table 3 were used with 3 types of type 0 red blood cells (No.

1 to 3) (manufactured by Ortho, serial number 3ssl19).

A 0.7% suspension was prepared according to the table. 25 μg of this diluted suspension was dispensed per well into a WGA-treated plate prepared in the same manner as in <A> above. After being left at room temperature for 10 minutes, the plate was washed with physiological saline to prepare a type O red blood cell solid-phase plate that reacts with each antibody.

On the other hand, each of the 12 types of irregular antibodies shown in Table 3 was sequentially diluted with physiological saline to 2 times, 4 times, 16 times, and so on.

66.7 μg LI for 100 μg of these dilutions
SS solution was added to prepare a series of diluted samples of each irregular antibody.

Next, using each diluted sample series prepared above,
The following experiment was conducted. First, 25μ diluted samples of each concentration.
g was dispensed into each well of the solid phase plate. After incubation at 37°C for 10 minutes, the cells were washed six times with physiological saline. Next, magnetic latex beads sensitized with the anti-human IgG antibody prepared in <8> were dispensed into each well at an amount of 25 μg/well, and then left on a magnet for 2 minutes in the same manner as described above. and formed a sedimentation pattern. Strongly positive (++) means that the precipitated anti-human 1gG magnetic latex beads are spread evenly and widely on the bottom of the well, positive (+) when they are spread uniformly on the bottom of the well, and positive (+) when the beads are slightly spread on the bottom of the well. Weak positive (±), and those gathered in the center of the well were determined to be negative (-). The results are shown in Table 4.

(b) As a comparative example for evaluating the measurement by the method of the present invention, as explained below, As
Based on the 5ay method (measurement was performed using the conventional method).

That is, after diluting the 12 types of irregular antibodies one-fold in the same manner as above, 0 AES was added to 60 μΩ of each dilution solution.
(Ortho AES200) A sample was prepared by adding 30 μfl. After adding 30μΩ of reactive surge screen shown in Table 2 to each sample,
and incubated for 15 minutes. After washing three times with 3 wN of normal saline, 80 μg of Coombs serum (manufactured by Ortho, serial number 1111858 old-1) was added. Subsequently, after centrifugation at +200 rpm for 30 seconds, a positive or negative determination was made based on the sedimentation pattern using the same criteria as above. The results are shown in Table 4.

From the results in Table 4, it can be seen that the assay sensitivity of the method according to the present invention is clearly higher than that of the conventional method.

Example 2 Detection of autoantibodies by direct antiglobulin test <A> Preparation of red blood cell solid-phase plate A vGA-treated plate is prepared by the same procedure as described in Example 1. Separately, red blood cells separated from the blood to be tested to which an anticoagulant has been added are washed with physiological saline.
By suspending this in physiological saline, 0.3%
Prepare a red blood cell suspension.

Dispense 25 μg of the above red blood cell suspension into each well of the above wCA-treated plate and let stand at room temperature for 10 minutes. Then put 0.2% gelatin, o. OIM
Wash once with 200 μg of PBS.

<B> Preparation of anti-rabbit IgG sensitized magnetic latex beads Instead of the anti-human IgG used in Example 1, anti-rabbit I
gG (Klrkegaard & Perry L.
laboratories.

Inc., product number 5O-0115-16) was used. Other than that, magnetic latex beads sensitized with anti-rabbit IgG were prepared in the same manner as in Example 1.

<C> Detection of autoantibodies Rabbit-derived Coombs serum (manufactured by 0rtho) was added to the above <
Add 25μ to each well of the red blood cell solid-phase plate prepared in A>.
Dispense 1 g each and allow to react at room temperature for 10 minutes. pH7.0
0. 3 washing steps using 200 μg of OfM PBS
After repeating the procedure several times, 25 μg of the anti-heron IgG magnetic latex beads prepared in <8> are added to each well.

Subsequently, in the same manner as in Example 1, a magnet is applied to cause the magnetic latex beads to settle and form a distribution pattern on the bottom of the well for determination.

In this embodiment as well, the time required for determination is reduced to one to several tenths of the conventional method.

Further, as in the case of Example 1, high detection sensitivity can be obtained because the method described in FIGS. 4A and 4C is used.

Example 3 Detection of HBs antigen (Part 1) <A> Creation of anti-HBs antibody solid-phase plate Rabbit anti-HBs antibody purified with an affinity column was incubated at pH 7.0.
in 0.0 IM phosphate buffered saline (PBS) at a concentration of 10 μg.
/mj! Prepare to. This is U7 bottom microplay)
(Manufactured by NUNC, serial number 484H4)
00 μg each and incubated at 37° C. for 1 hour to adsorb onto the microplate. Subsequently, the plate is washed three times using 250 μg each of 0.01M PBS containing poy and o. Next, 0.2% bovine serum albumin (BSA) containing 0.2% bovine serum albumin (BSA) was added. Add 200 μg of OIN PBS to each well and react at 37° C. for 1 hour to perform blocking. Furthermore, 0.05%Tve
25% of 0.01M PBS, pH 7.0 containing en20
After repeating the washing operation three times using 0 μg/well, the wells are dried at room temperature and stored at 4°C.

<a> Preparation of anti-HBs antibody-sensitized magnetic latex beads Using 91111.3 (1,114 glycine buffer), prepare the same magnetic latex bead solution (concentration 0.4%) used in Example 1. In addition, using the same glycine buffer, a solution of anti-Has antibody (concentration 2.5 μg/rr
Prepare l). Mix 2ml/1l of each solution,
Incubate at 7°C for 1 hour and add llB to magnetic latex beads.
sensitize with s antibody. After washing the sensitized magnetic latex beads three times with 2 mg of 0.1 M glycine buffer on p+is, a containing 1% BS^, blocking is performed by reacting for 300 minutes at 37°C in 2 mΩ of the same buffer. . Subsequently, 0.01 MPBS at pH 7,s containing 0.05% Tveen20 and 0.1% BSA
Washed three times with 2mρ each, and the same PBS 4mf!
Add and save.

<C> Detection of tlBs antigen To each well of the anti-HBs antibody solid-phase plate prepared in <A>, 25 μg of HBs antigen-positive serum is added as a sample as a stock solution and reacted for 15 minutes at room temperature. At the same time, one well was added as a control.
Add 25 μg of Bs antigen-negative serum as a stock solution and react in the same manner. Added blood? Discard R and further adjust to pH 7,
5 of 0. Wash each well once with 200 μg of OIM PBS. Next, the anti-HBs antibody-sensitized magnetic latex beads prepared in step B> are added in an amount of 25 Mg/well. Subsequently, the magnetic latex beads are allowed to settle in the same manner as in Example 1 to form a distribution pattern.

In this example as well, the time required for determination is a fraction of the conventional method.
~ Shortened to several tenths of a second.

Further, as in the case of Example 1, high detection sensitivity can be obtained because the method described in FIGS. 4A and 4C is used.

Example 4 Detection of 118s antigen (Part 2) <A> Preparation of anti-11Bs antibody solid-phase plate An anti-11Bs antibody solid-phase plate is prepared in the same manner as in Example 3.

<B> Preparation of anti-11Bs antibody-sensitized magnetic latex beads Anti-11Bs antibody-sensitized magnetic latex beads are prepared in the same manner as in Example 3.

<C> Detection of HBs antigen As in Example 3, HBs antigen was added as a sample to each well of the anti-HBs antibody solid-phase plate prepared in <A>.
25 μg of s antigen positive serum is added as a stock solution. In addition, 25 μg of HBs antigen-negative serum is added as a control to one well as a control. Next, <B
Add the anti-HBg antibody-sensitized magnetic latex beads prepared above in an amount of 25 μI/well, and react for 15 minutes at room temperature.

Then mouth, 05% Tveen 20 and 0.1% BS
Add 150 μg of 0.01 M PBS containing A in pH 7.5 to each well. Subsequently, in the same manner as in Example 1, a magnet is activated to cause the magnetic latex beads to settle on the bottom of the well to form a distribution pattern.

In this example as well, the time required for determination is a fraction of the conventional method.
It will be shortened to a few tenths of a second.

Note that, unlike Example 3, in this example, the sample was not discarded and the wells were not washed after reacting with the magnetic marker particles. However, in this example, after reacting with magnetic marker particles according to the method explained in FIG. 4B and FIG. This problem can be easily avoided without sacrificing operability. The problem of fats 90S1tlV8 due to non-specific agglutination reaction can be reduced by increasing the dilution factor. Furthermore, since the reaction between the sample and the magnetic marker particles has already proceeded before dilution, there is almost no decrease in sensitivity due to dilution. Therefore, stable and clear patterns can be formed even with high concentration serum.

Example 5 Detection of llBs antigen in whole blood sample <A> Preparation of anti-HBs antibody solid-phase plate An anti-tlBs antibody solid-phase plate is prepared in the same manner as in Example 3.

<B> Preparation of anti-11Bs antibody-sensitized magnetic latex beads at pH 8.3. A solution of the same magnetic latex beads (concentration 0.4%) as used in Example 1 is prepared using IM glycine buffer. Also, using the same glycine buffer, a solution (concentration 1 μg/rtl) of rabbit anti-HBs antibody purified using an affinity column is prepared. 2 ml of each solution was mixed and incubated at 37° C. for 1 hour to sensitize the magnetic latex beads with the HBs antibody. The sensitized magnetic latex beads were added to a pH solution containing 1% BSA.
8. Wash with 2rrl of 0.1M glycine buffer in 3.
After repeating the reaction, blocking is performed by reacting in 2rrl of the same buffer at 37°C for 300 minutes.

Furthermore, it was washed three times with 2 ml each of 0.01M PBS containing 0.01% "Sarcosinate LN" (trade name of a surfactant manufactured by Nikko Chemicals) and 0.1% BSA, and the same PI354rrl was used. Add and save.

<C> Detection of HBs antigen HBs antigen negative blood to which an anticoagulant had been added was prepared.

The blood to which HBs antigen (manufactured by Midori Juji Co., Ltd.) of subtype .ad is added is used as a positive sample, and the blood to which it is not added is used as a negative sample.

First, 0.01% sarcosinate LNJ and 5% dextran (molecular weight 2000.0
p including 00~aoo, ooo, manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.)
Add 100 μg of 0.01M Pt3S in H7.5. Subsequently, 5 μg of the above positive or negative sample is added to each well. Furthermore, 25 μp of the anti-11Bs antibody-sensitized magnetic latex bead solution prepared in <B> is added to each well and reacted for 5 minutes at room temperature.

Next, in the same manner as in Example 1, a magnet is applied to the microplate to cause the anti-llBs antibody-sensitized magnetic latex beads to settle on the bottoms of the wells. By observing the distribution pattern formed by sedimentation from below the microplate, the presence of HBs antibodies can be detected and confirmed.

Example 6 Determination of ABO blood type A
10 wheat germ lectin (WGA; manufactured by Seikagaku Corporation) prepared at a concentration of 10 μg/mfl using
0 μl was added and treated at room temperature for 30 minutes. Then p
0.0114 P of H7,0! A VGA-treated plate was obtained by washing five times using 300 μg of 38 and drying at room temperature.

Add 25 μg of a 0.7% saline suspension of type A red blood cells to each well of the vGA-treated plate prepared above, leave it at room temperature for 10 minutes, and then add 200 μg of physiological saline to each well.
A type A red blood cell solid-phase plate was prepared by washing each well three times. Similarly, a type B red blood cell solid phase plate and a type 0 red blood cell solid phase plate were prepared.

<B> Preparation of antibody-sensitized magnetic latex beads 9118.
A solution of the same magnetic latex beads as used in Example 1 (50-fold dilution) was prepared using 0.1M glycine buffer of No. 5. In addition, using the same glycine buffer, a solution of anti-human IgM antibody (manufactured by KPL) (concentration 20 μg/
m#) was prepared. Magnetic latex beads were sensitized with 1 gM anti-Human antibody by mixing 500 μg of each solution and incubating for 30 minutes at room temperature. The sensitized magnetic latex beads were treated with 0.2% BSA and 0.2% BSA.
Washing was performed twice with 2 ml of 0.02 M glycine buffer, pH 8.5, containing 14 M NaCl. For cleaning,
Centrifugal washing at 3000 rpm was used. Thereafter, sensitized magnetic latex beads were suspended in 2 ml of the same buffer solution, and blocked by incubation at room temperature for 30 minutes, and then stored under refrigeration.

<C> Determination of ABO blood type Using the type A red blood cell solid-phase plate prepared in <A>, inject commercially available anti-A serum C0rth into some wells.
25 μg of a diluted physiological saline solution (manufactured by O Company, production number 5A9BOA-1) was dispensed. In addition, a diluted solution of commercially available anti-B serum (manufactured by Ortho, serial number 5B445A-1) in physiological saline was added to several other wells.
It was dispensed in μg portions. After incubation at 37° C. for 10 minutes, the cells were washed five times with 200 μl/well of saline. Next, add 25μ of the anti-human IgM antibody-sensitized magnetic latex bead suspension prepared in <B> to each well.
g was added at a time. Furthermore, the magnetic latex beads were allowed to stand for 2 minutes on a magnet to form a sedimentation pattern, and positive (+) or negative (-) was determined from the sedimentation pattern. In addition, the same procedure as above was carried out using the type B red blood cell solid phase plate and the type 0 red blood cell solid phase plate prepared in <A>. The results are shown in Table 5.

Table 5 As mentioned above, in the assay using the type A red blood cell solid-phase plate, a positive reaction occurred only for the anti-A serum sample.

In addition, in assays using type B red blood cell solid-phase plates, anti-B
Positive reactions occurred only for serum samples. On the other hand, 0
In the assay using type red blood cell solid-phase plate, no positive reaction occurred against either the anti-B serum sample or the anti-B serum sample.

This result shows that ABOB liquid type can be determined by the method of the present invention.

Example 7 Cross-matching test <A> Creation of a homogeneous plate for type AB blood cells Using red blood cells from a person whose blood type is A and red blood cells from a person whose blood type is B, A A type B red blood cell solid phase plate and a type B red blood cell solid phase plate were prepared.

<B> Preparation of antibody-sensitized magnetic latex beads A suspension of anti-human IgM antibody-sensitized magnetic latex beads was prepared in the same manner as in Example 6.

<C> Crossover test of type A and type B. Using the type A red blood cell solid-phase plate prepared in <A>, some wells were filled with serum collected from a person with blood type A, and some of the wells were filled with serum collected from a person with blood type A. 25 μg of serum collected from a person with blood type B was added to each well. 10 at 37℃
After incubation for minutes, the cells were washed five times with 200 μp/well of saline. Next, 25 μp of the anti-human IgM/antibody-sensitized magnetic latex bead suspension prepared in step B was added to each well. Furthermore, the magnetic latex beads were allowed to stand for 2 minutes on a magnet to form a sedimentation pattern, and positive (+) or negative (-) was determined from the sedimentation pattern.

In addition, the same procedure as above was carried out using the type B red blood cell solid-phase plate prepared in <A>. The results are shown in Table 6.

Table 6 As mentioned above, a positive reaction occurred with the combination of type A red blood cells and type B human blood 18, as well as the combination of type B human red blood cells and type A human serum.

This revealed that a cross-matching test was possible. Furthermore, in conjunction with the fact that irregular antibodies can be detected in Example 1, it is possible to determine overall blood transfusion compatibility.

In addition, since it is possible to detect irregular antibodies in Example 1, it is possible to detect anti-human antibodies! gG antibody-sensitized magnetic particles and anti-human I
A cross-compatibility test is also possible by using gM antibody-sensitized magnetic particles individually or in combination.

Example 8 <A> Preparation of ABO12B Blood Cell Solid Phase Plate In the same manner as in Example 6, a type A red blood cell solid phase plate, a type B red blood cell solid phase plate, and a type 0 red blood cell solid phase plate were prepared.

<B> Preparation of antibody-sensitized magnetic latex beads 9H8,5
0. The same solution of magnetic latex beads used in Example 1 (50x dilution) using IM glycine buffer.
was prepared. In addition, a solution (concentration 20 μg/mN) of anti-A monoclonal antibody (manufactured by Olympus Optical Industries, Ltd.) was prepared using the same glycine buffer. each solution 5
Magnetic latex beads were sensitized with the anti-A monoclonal antibody by mixing 00 μg each and incubating at room temperature for 30 minutes. The sensitized magnetic latex beads were washed twice with 2 ml of 0.02M glycine buffer at pH 8.5 containing 0.2% BSA and O, 14M NaCl. Centrifugal washing of the intestines at 3000 rpm was used for washing. Thereafter, sensitized magnetic latex beads were suspended in 2 ml of the same buffer solution, and blocked by incubation at room temperature for 30 minutes, and then stored under refrigeration.

Anti-B monoclonal antibody-sensitized magnetic latex beads were prepared in the same manner as described above, using an anti-B monoclonal antibody (manufactured by Olympus Optical Industries, Ltd.) instead of the anti-A monoclonal antibody.

<C> Determination of ABO blood type Using the type A red blood cell solid-phase plate prepared in <A> above,
Add 25μ of the anti-A monoclonal antibody-sensitized magnetic latex bead suspension prepared in step B to some of the wells.
P portions were dispensed. Also, some other wells contained anti-B
A suspension of monoclonal antibody-sensitized magnetic latex beads was dispensed in 25 μg portions. Furthermore, the magnetic latex beads were allowed to stand for 2 minutes on a magnet to form a sedimentation pattern, and positive (+) or negative (-) was determined from the sedimentation pattern. In addition, the same procedure as above was carried out using the type B red blood cell solid phase plate and the type O red blood cell solid phase plate prepared in A>. The results are shown in Table 7.

Table 7 As described above, determination of ABO blood type was also possible by using anti-A monoclonal antibody-sensitized magnetic latex beads and anti-B monoclonal antibody-sensitized magnetic latex beads.

Example 9 <A> Preparation of anti-A monoclonal antibody-sensitized magnetic gelatin beads 200 μg of a 5% saline suspension of colored magnetic gelatin beads (manufactured by Olympus Optical Industries, Ltd.) was added to PBS.
Centrifugal washing with 2sII (2000g, 5 minutes)
Washed twice with Add 2mj? of tannic acid PBS solution (concentration 20ug/m#) to these colored magnetic gelatin beads.
and incubated for 30 minutes at 37°C. Subsequently, centrifugal washing (2000 g) using 2 mjl of PBS.

Washed twice for 5 minutes).

Anti-A monoclonal antibody (manufactured by Olympus Optical Industries, Ltd.) was diluted with PBS to prepare a solution with a concentration of 10 μg/m II. 2 ml of this solution was added to the above magnetic gelatin beads treated with tannic acid. 6 at 37℃
After incubating for 0 minutes to sensitize the magnetic gelatin beads with anti-A monoclonal antibody, they were washed three times by centrifugal washing (1500 g, 5 minutes) using PBS 2-g.

The sensitized magnetic gelatin beads were mixed with 0.5% BSA and 06
The suspension was suspended in 4 ml of pH 6 phosphate-citrate buffer containing 0.5% sodium azide. After performing blocking treatment by incubating at 37°C for 30 minutes, it was stored in a refrigerator.

<a> Typing of red blood cell antigen A 0.2% diluted suspension of type A red blood cells was prepared using a pH 6 phosphate-citrate isotonic buffer containing 0.5% BSA. To 50 μg of this type A blood cell suspension, 50 μg of the anti-A monoclonal antibody-sensitized magnetic gelatin bead suspension prepared in <A> above was added, and the mixture was placed in a well of a microplate with a hemispherical bottom at 37°C. Incubated for 10 minutes. Furthermore, the magnetic latex beads were allowed to stand for 3 minutes on a magnet to form a sedimentation pattern, and positive (+) or negative (-) was determined from the sedimentation pattern. Also,
The same procedure as above was carried out using type B red blood cells and type O red blood cells instead of type A red blood cells. The results are shown in Table 8.

Table 8 As described above, the presence of type A red blood cells could be determined in about 13 minutes after adding the anti-A monoclonal antibody-sensitized magnetic gelatin beads.

〔Effect of the invention〕

As detailed above, the immunoassay method of the present invention uses magnetic marker particles and uses a magnet to increase the sedimentation rate of the magnetic marker particles, so it is more effective than conventional particle aggregation methods. A sedimentation pattern of marker particles can be formed in a significantly short period of time, and therefore the time required for determination can be significantly shortened.

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

Therefore, it is possible to carry out highly sensitive measurements without previously removing naturally settling particles other than the substance to be measured.

[Brief explanation of drawings]

FIG. 1A and FIG. 1B are diagrams showing an example of how magnets are arranged when a microplate is used as a measurement container. FIG. 2A and FIG. 2B are diagrams showing the sedimentation distribution pattern due to a positive reaction of magnetic marker particles on which a substance that binds to the analyte is immobilized. FIGS. 3A and 3B are diagrams showing the sedimentation distribution pattern due to a negative reaction of magnetic marker particles on which a substance that binds to the AP1 constant substance is immobilized. Figure 4A shows the first sedimentation distribution pattern due to a positive reaction of magnetic marker particles on which a substance that binds to the analyte is immobilized when using a measurement container with a substance that binds to the analyte immobilized on the wall surface. FIG. Figure 4B shows a second sedimentation distribution pattern due to a positive reaction of magnetic marker particles immobilized with a substance that binds to the analyte when using a measurement container with a substance that binds to the analyte immobilized on the wall surface. FIG. Figure 4C is a diagram showing the sedimentation distribution pattern due to the negative reaction of magnetic marker particles immobilized with a substance that binds to the analyte when using a measurement container on which the substance that binds to the analyte is immobilized. It is. FIG. 5 is a diagram showing another example of a measurement container and a magnet that can be used in the present invention. FIG. 6 is a diagram showing an example of a reaction promoting device that can be suitably used in the present invention. FIG. 7 is a diagram showing another example of a reaction accelerator that can be suitably used in the present invention. 1... Microplate, 2... Well, 4...
Magnet, 5... Magnetic marker particle, 6... Measured substance, 7... Captured substance, 8... Needle magnet, 10... Support base, 11... Recessed hole, 12...・Conical magnet, 13
...Concave hole with female thread, 14...Cylindrical magnet with male thread, 15...Groove

Claims (13)

[Claims] (1) A step of mixing a sample solution and magnetic marker particles in which a substance that specifically binds to or competes with the substance to be measured is immobilized on magnetic particles to form a reaction solution for both; and a measurement container. collecting the magnetic marker particles on the predetermined wall surface area by causing a magnet placed outside the predetermined wall surface area of the measurement container to act on the reaction solution contained in the measurement container; An immunological measurement method using magnetic marker particles, comprising the step of measuring the substance to be measured contained in the sample based on the distribution state of the magnetic marker particles collected in a region. (2) An immunoassay according to claim 1, characterized in that a substance that specifically binds to or competes with the substance to be measured is immobilized on at least the inner surface of the predetermined wall area in advance. Method. (3) The immunoassay method according to claim 2, wherein the reaction solution of the sample and magnetic marker particles is diluted. (4) On the inner surface of at least a predetermined wall area of the measurement container,
A step of pre-immobilizing a substance that specifically binds to or competes with the analyte substance; After adding a sample solution to the measurement container and causing a reaction, removing the sample and washing the measurement container. a step of adding a solution of magnetic marker particles in which a substance that specifically binds to or competes with the analyte to be measured is immobilized on magnetic particles to the washed measurement container; collecting the magnetic marker particles in the predetermined wall region by causing a magnet placed outside the predetermined wall region of the measurement container to act on the contained magnetic marker particles; An immunological measurement method using magnetic marker particles, comprising the step of measuring the substance to be measured contained in the sample based on the distribution state of the magnetic marker particles collected in the sample. (5) The immunoassay method according to any one of claims 1 to 4, wherein the predetermined wall area is a bottom surface of the measurement container. (6) The immunoassay method according to any one of claims 1 to 5, wherein the predetermined wall surface area is a hemispherical wall surface. (7) The immunoassay method according to any one of claims 1 to 5, wherein the predetermined wall surface area is a conical wall surface. (8) A support base having an upper flat surface on which a plate on which a plurality of reaction vessels are arranged can be placed, and a plurality of magnets provided on the support base, each of the plurality of magnets including:
A reaction promoting device for use in immunological measurements, which is provided at a position corresponding to each of the reaction vessels when the plate is placed on a support stand. (9) The reaction promoting device according to claim 8, wherein the magnet is needle-shaped. (10) The reaction promoting device according to claim 8, wherein the magnet is columnar. (11) Claims 8 to 10, wherein a male thread is cut on the outer periphery of the magnet, and the magnet is attached to the support base by being screwed into a female thread formed on the support base.
The reaction accelerator according to any one of the above. (12) The reaction promoting device according to claim 11, wherein the head of the magnet is provided with a −-shaped or +-shaped groove. (13) The reaction promoting device according to any one of claims 8 to 12, wherein the outer edge of the support base is raised to stabilize the state in which the plate is placed.