JP2505088B2 - Reagent for solid-phase immunoassay - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a solid-phase immunoassay reagent used for diagnosing or determining various diseases and blood group typing by immunological reaction.

[0002]

2. Description of the Related Art A technique for coating monodisperse solid particles with a substance that specifically causes an antigen-antibody reaction with an antigen or antibody to be analyzed for immunological measurement has been known for a long time. Further, an agglutination method in which the carrier particles after the coating treatment are mixed with an unknown sample sample to examine the change in the dispersibility of the carrier particles is also a well-known technique.

Generally, the agglutination method includes a turbidimetric method for measuring the change in the amount of transmitted light with respect to carrier particles suspended in a reaction vessel, and a distribution form of carrier particles settled in the reaction vessel, that is, the bottom surface of the reaction vessel. A typical example is the microtiter method in which the distribution amount occupying is determined by the naked eye or image determination means. In the agglutination method, animal erythrocytes have been used exclusively for the reason that the particle size is almost uniform at first, but there are problems with the specificity for the antigen or antibody and the stability of the available amount. On the other hand, in recent years, the progress of particle synthesis technology has made it possible to artificially produce a large amount of particles having a uniform particle size, and it tends to be actively used as an alternative particle for red blood cells.

Since the carrier particles used in the agglutination method must sufficiently react with the specimen sample in the reaction vessel, how to optimize the sedimentation characteristics is the most important issue. For example, S. Hosaka et al. Immunological communicatio
n, 12, (5), 509 (1983), as carrier particles that can substitute for erythrocytes in the microtiter method, show a sedimentation rate almost equal to that of erythrocytes derived from sheep or chicken, and have a particle size of 2.0 to 4.0.
We have proposed acrylic synthetic particles of μm. Also,
Japanese Unexamined Patent Publication (Kokai) No. 62-115366 discloses a microtiter method using silica carrier particles having a high specific gravity of 2.0 or more and a particle size of less than 2.0 μm.

As described above, when artificial synthetic particles are used in the microtiter method as substitute particles for red blood cells, the artificial synthetic particles are designed to have a smaller particle size as the specific gravity increases. The microtiter method described above is suitable for a measurement target such as ABO blood group or HBs antibody that has relatively strong reactivity or is detected in a relatively large amount. The carrier particles are distributed directly on the surface of the hydrophobic wells.

[0006] On the other hand, the antigen or antibody to be measured in the specimen sample is subjected to U-bottom or V-based detection based on the immunological reaction.
The reaction method for binding to the well wall surface of a microtiter plate having a letter bottom and then binding carrier particles to this antigen or antibody is a solid-phase agglutination method (Solid agglut
ination). The solid phase aggregation method is
For example, rare blood cell antigens such as Rh (-) factor and HC
When detecting a measurement target such as an antigen or its antibody that causes an infectious disease such as V (hepatitis C virus) or the like, which has weak reactivity or its presence is in a very small amount, these measurement targets are well-bottomed. It is possible to wash and remove contaminants and the like in the measurement sample by indirectly binding carrier particles after binding to an antigen or antibody that specifically binds to the measurement target immobilized on the wall surface. It has certain advantages. Therefore, it is possible to form a distribution pattern similar to the agglutination reaction while avoiding the non-specific reaction. For example, I with short arms for immunological binding
In an antiglobulin test using a gG antibody as a sample, a solid phase agglutination method with reliable reactivity is carried out by previously coating carrier particles with an anti-IgG antibody having long arms.

Generally, red blood cells are used as carrier particles in the solid phase agglutination method. For example, U.S. Pat.
In the specification of the publication, the anti-D antibody immobilized on the bottom wall surface of the well of the U-bottom microplate is reacted with a sample blood sample (first reaction), and then red blood cells coated with anti-human IgG antibody are added. It describes a method for measuring Rh factor in blood, in which the distribution pattern of red blood cells is visually determined after spontaneously precipitating and reacting (second reaction). However, when red blood cells are used as such carrier particles, various antigens present on the surface of red blood cells are likely to cause non-specific reactions. Further, it has been difficult to obtain a large amount of red blood cells having a certain quality.

On the other hand, a solid-phase aggregation method using artificially synthesized particles as carrier particles has been proposed. For example, in JP-A-2-122266, latex particles (RH ) having a particle size of 1.7 μm in which a magnetic material is encapsulated as artificial synthetic particles are disclosed.
ONE-POULENC) and a particle size of 4.5 μm
U immobilized by using Tex particles (manufactured by DYNAL)
Human O type red blood cells immobilized in wells of a letter-bottomed microplate are reacted with a sample blood sample (first reaction), and then latex particles encapsulating the magnetic material coated with anti-human IgG are added and sedimented by a magnet. A method for measuring autoantibodies for visually determining the distribution pattern of the latex particles formed after the reaction (second reaction) is described.

[0009]

However, in the conventional solid phase aggregation method using artificial synthetic particles as carrier particles,
Formed after a predetermined reaction time , for example 2 to 60 minutes ,
The positive distribution pattern in which the carrier particles are uniformly spread on the bottom wall surface of the well changes gradually and temporally into a negative distribution pattern in which the carrier particles are gathered in a button shape at the center of the bottom wall surface of the well. For this reason, there is a problem that a positive sample is overlooked when the examiner makes a determination after a lapse of time longer than a predetermined reaction time , for example, 90 minutes to several hours . on the other hand,
Unlike the above-mentioned microtiter method, in the solid phase aggregation method in which the surface of the reaction vessel is covered with a hydrophilic protein such as an antigen, the behavior of carrier particles on the vessel surface after sedimentation is not clarified, which is preferable. The particle conditions are unknown.

The present invention has been made in view of the above points, and in a solid phase immunoassay applying a solid phase agglutination method using artificially synthesized particles, the precipitated carrier particles have a stable positive distribution pattern for a long time. It is an object of the present invention to provide a reagent for solid phase immunoassay that can be maintained at

[0011]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that the particle size of artificially synthesized particles is a molecular morphology of particles after sedimentation in a solid phase aggregation method, particularly a positive pattern. It was found to be closely related to the stability of. That is, the present inventors prepared immunoassay reagents consisting of various carrier particles having different particle size distributions, and observed the process of changing the positive pattern into a negative pattern with a microscope. It was discovered that, with the reagent whose pattern changed, the fine vibration of the carrier particles lasted for a long time during the positive pattern immediately after the predetermined reaction time. On the other hand, it was also confirmed that, in the reagent in which the positive pattern was stably maintained for a relatively long time and the negative pattern was changed on the next day, few carrier particles vibrated finely immediately after the predetermined reaction time. Therefore, we examined the particle size distribution of the carrier particles that compose each reagent for the reagents whose positive pattern was negatively patterned in a short time and those which were negatively patterned after a relatively long time. It was found that the higher the abundance ratio, the shorter the pattern changed to the negative pattern.

[0012] The present invention is based on such knowledge, and the substance to be measured in the specimen sample specifically bound to the antigen or antibody immobilized on the inner wall surface of the reaction vessel is By binding carrier particles coated with an antigen or antibody that specifically binds to the substance to be measured, the amount of the carrier particles occupying the surface area of the inner wall surface on which the antigen or antibody is immobilized is determined from the distribution amount of the carrier particles. A solid-phase immunoassay reagent used in a solid-phase immunoassay method for detecting the presence or absence of a substance to be measured.
Provided is a solid-phase immunoassay reagent characterized in that the abundance ratio of particles of 3.0 μm or less is 10.0% by volume or less.

Here, the particle size distribution of the carrier particles is 2.0
It is preferable that the abundance ratio of the particles having a size of μm or less is 3.0% by volume or less. It is more preferable that the average particle size is 6.0 μm or more. Hereinafter, the solid phase immunoassay reagent of the present invention will be described in more detail.

The carrier particles used in the present invention are synthetic particles derived from organic substances or inorganic substances such as dextran, polyacrylamide, gelatin, liposomes, cellulose, polystyrene, silica, carbon and magnetic metals. One of the factors that cause the above-mentioned inconvenient changes in the solid-phase agglutination reaction in the distribution morphology after sedimentation by these carrier particles is the balance between the intrinsic repulsive force of the particles and the van der Waals attractive force. The balance between repulsive force and attractive force is
It should be noted that this is a common problem for particles of all materials.

The carrier particles can be synthesized by various known polymerization methods, coacervate method, microcapsule method and the like. For example, a suitable method for producing gelatinous carrier particles by the coacervate method is described in Japanese Patent Laid-Open No.
No. 4-25060. In such a coacervate method, a technique for controlling the particle size of synthetic particles is already known, and reaction conditions such as pH may be changed when forming the coacervate. A method of introducing a magnetically responsive substance such as a magnetic material into synthetic particles is also known, and a solution containing a magnetic material may be added at least at the initial stage of particle formation. The amount of the magnetic substance enclosed at this time can also be adjusted by a known technique. The carrier particles preferably have a spherical shape and have a particle size of 1 μm to 20 μm.
It is synthesized so as to be in the range of m. In the synthesis of carrier particles,
Depending on the synthesis conditions and the like, various types of atypical particles (such as a daruma shape and a Kompei-sugar shape) may be produced, but these can also be effectively used as long as they do not adversely affect the reagent performance. The carrier particles made of a colorless material can be dyed with a known dye such as a reactive dye, a cationic dye or a basic dye to a visually or optically recognizable level. Particle size 20-30
It should be noted that carrier particles having a particle size of more than μm have a low yield based on the volume occupied after synthesis. Also, 30
Particles having a size of μm or more are not preferable in that the number of sparse voids generated in the distribution after sedimentation hinders detection.

In the particle size distribution of the carrier particles synthesized as described above, when the particles having a particle diameter of 3.0 μm or less exceed 10% by volume, the known classification treatment or sieving treatment is performed to obtain 10.0% by volume or less, It can be adjusted to preferably 5.0% by volume or less. In addition, particles with a particle size of 2.0 μm or less
If the particle size distribution exceeds 3.0% by volume, it can be adjusted to 3.0% by volume or less in the same manner. As a means for measuring the particle size, a known device such as a Coulter meter or a microscope can be used, but a particle sedimentation meter is preferable from the viewpoint of simplicity. According to the sedimentation meter, it is possible to know the particle size calculated as spherical particles regardless of the shape of the particles themselves. There are volume average and number average as criteria for showing particle size,
Since the area occupied by the wall surface of the container is important in the aggregation pattern, it is preferable to discuss the particle size based on the volume average.

The carrier particle concentration in the reagent solution when the immunoassay reagent of the present invention is added to a predetermined reaction vessel for measurement is in the range of 0.01 to 10% by weight, and particularly,
0.1-1.0% by weight is preferred.

The dispersion medium for suspending the carrier particles is p
The H is about 5.0 to 8.5, and if necessary, additives such as a non-specific reaction inhibitor, a surfactant, a freeze / drying protective agent, and an antioxidant can be added.

As the reaction container, for example, a microtiter plate having a plurality of wells can be used. The bottom shape of the well of the microtiter plate is
Either V-shape or U-shape may be used as long as unreacted carrier particles can settle to form a button-shaped focused negative pattern. Further, the bottom surface may be inclined only to one side.

To immobilize the antigen or antibody on the inner wall surface of the reaction container, the affinity of protein is used, or a naturally-occurring agglutinin such as lectin or an adhesive derived from a chemical substance is used. It can be achieved by incubating or centrifuging.

The coating of the antigen or antibody on the surface of the carrier particles is carried out by, for example, a method utilizing surface charge, a method via a cross-linking agent, a method using a natural adhesive such as tannic acid, a specific method such as biotin-avidin reaction. It is carried out by a method utilizing a bond.

The types of the antigen or antibody immobilized on the inner wall surface of the reaction container and the antigen or antibody coated on the carrier particles differ depending on the substance to be measured. For example, when typing ABO blood group or irregular antibody or platelet antibody, erythrocytes of known ABO type or erythrocyte or platelet prototype of known Rh serotype are respectively on the inner wall surface of the reaction vessel. Known platelets are immobilized and carrier particles are coated with anti-IgG antibody. When detecting the presence of antigens such as infectious diseases virus or cancer-specific proteins or antibodies against these antigens, the respective antibodies or antigens are immobilized on the inner wall surface of the reaction vessel, and the carrier particles are immobilized on the carrier particles. Coat an anti-IgG antibody.
The coated carrier particles are completed by refrigerating or freezing in a dispersion medium or by freeze-drying.

The concentration of the antigen or antibody to be immobilized is very important because it is related to the reaction sensitivity. For example, when cells are used as the antigen or antibody, the cell concentration is preferably 0.2 × 10 ⁶ cells / cm ^{2 to} 1.2 × 10 ^6.
Cell / cm ² .

The step of measuring the substance to be measured using the thus prepared solid-phase immunoassay reagent and the reaction vessel in which the antigen or antibody is solid-phased is based on a known solid-phase agglutination method. Is done. First, a predetermined amount of a specimen sample is poured into a reaction container (for example, a well of a microplate) and incubated at room temperature to 37 ° C. As a result, the substance to be measured in the specimen sample binds to the antibody or antigen immobilized on the well wall surface (first reaction). Next, phosphate buffer (hereinafter,
Contaminants in the wells are removed by washing with a buffer solution such as PBS) or purified water or physiological saline. next,
A predetermined amount of the reagent for solid phase immunoreaction of the present invention is added, stirred, and then incubated at room temperature to 37 ° C. for a predetermined reaction time.
During this incubation period, the carrier particles are bound to the substance to be measured bound to the wall surface of the well, and when the substance to be measured is not present, the incubation is performed so that the carrier particles are gathered locally on any of the wall faces of the well. Therefore, it is preferable that the bottom surface of the reaction container is inclined. Although the inclined shape is not particularly limited, it may have a converging portion where unreacted particles locally collect (second reaction). After this,
The presence or absence of the substance to be measured is determined by visually observing the morphology of the distribution pattern of carrier particles formed on the bottom wall surface of the well. At this time, by relatively comparing the distribution amount of carrier particles occupying the surface area of the well wall surface immobilized with the antibody or antigen,
If the carrier particles are spread evenly on the wall surface of the well, it is determined that the substance to be measured is present (positive), and if it is localized, it is determined that the substance to be measured is not (negative).

[0025]

The function of the reagent for solid phase immunoreaction of the present invention is such that the carrier particles coated with an antigen or antibody that specifically binds to the substance to be measured have a particle size distribution of 10.0 μm or less when the abundance ratio of the particles is 3.0 μm or less. % Or less. Therefore, the particle size that vibrates finely for a long time when bonded to the substance to be measured is 3.0
Since only a few particles having a size of μm or less are present, the positive pattern thus formed is stably maintained for a long time.

[0026]

EXAMPLES Examples of the present invention will be described in detail below. Reference example 1

First, in order to confirm that the change of the distribution pattern from positive to negative occurs regardless of the specific gravity of the carrier particles, two kinds of carrier particles having the same average particle diameter but different materials are previously prepared. Human IgG, the substance to be measured
A microplate on which red blood cells coated with was immobilized was prepared and a solid phase agglutination method was performed.

The IgG-coated erythrocyte-immobilized microplate, anti-human IgG-sensitized magnetic carrier particles and anti-human IgG-sensitized magnetic particles used in this reference example were prepared as follows. <Preparation of IgG coated erythrocyte immobilized microplate>

U-shaped bottom microplate (NUNC, NO.
464394) and prepare a concentration of 10 μg with 0.01 M PBS.
100 μl of wheat germ lectin (hereinafter referred to as WGA; manufactured by Seikagaku Kogyo Co., Ltd.) adjusted to 1 / ml was added to each well and treated at room temperature for 30 minutes. After washing with PBS, it was dried at room temperature, and WGA was attached to the well wall surface.

On the other hand, the mixing ratio of Coombs control (manufactured by Ortho), which is red blood cells coated with human IgG, and O-type human red blood cells was adjusted, and the ratio of Coombs control was 60% (V / V) with physiological saline. , 50% (V / V), 40% (V
/ V), 30% (V / V), 20% (V / V), 10% (V / V), 5% (V /
V), 0% (V / V), adjust the red blood cell concentration to 0.7% with physiological saline, and add 25 μl of each of these to the above WGA
It was dispensed to the treated microplate and incubated at room temperature for 10 minutes. Unbound red blood cells were washed away with physiological saline to obtain an IgG-coated red blood cell immobilized microplate. <Preparation of anti-human IgG-sensitized magnetic carrier particles>

Magnetic latex particles (estapor; rhone.) Having an average particle size of 1.8 μm in 0.1 M glycine-sodium hydroxide (hereinafter referred to as glycine buffer) (pH 8.7).
A magnetic latex particle solution containing 0.4% (W / V) of NO. M1-180 / 20 manufactured by Poulenc Co., Ltd. was prepared. A glycine buffer solution (concentration: 10 μg / ml) of an anti-human IgG goat antibody (manufactured by CAPPEL) was mixed with the latex particle solution in an amount of 500 μl and incubated at room temperature for 30 minutes. Next, 0.2% bovine serum albumin (hereinafter referred to as BSA; manufactured by Boehringer Mannheim) and 0.14M Nacl
It was washed with 0.02 M glycine buffer containing the above, further suspended in 2 ml of the same glycine buffer, and stored under refrigeration. <Preparation of anti-human IgG-sensitized magnetic particles>

5% (W / W) of magnetic gelatin particles having an average particle diameter of 1.8 μm (manufactured by Olympus Optical Co., Ltd., specific gravity 1.12)
V) 0.01M PB except the supernatant of 500 μl of particle suspension
It was washed twice with S (pH 7.0). Next, add 1 ml of PBS solution of tannic acid (concentration: 10 μg / ml) and incubate at 37 ℃ for 30
Incubated for minutes. Furthermore, 0.01M PBS (p
H7.0) and washed twice. Then, an anti-human IgG goat antibody (manufactured by CAPPEL) in PBS (concentration: 5.0 μg /
2 ml) was added and incubated at 37 ° C. for 1 hour. Furthermore, 0.01M PBS containing 0.1% BSA (pH
After washing 3 times with 7.43), the particles were suspended in the same PBS at a particle concentration of 0.4% (W / V). <Formation of distribution pattern>

To each well of the above-prepared IgG-coated erythrocyte-immobilized microplate, 25 μl of two kinds of anti-human IgG-sensitized magnetic particles having different materials were dispensed and shaken. Immediately after shaking, a plurality of cylindrical ferrite magnets (diameter 6 mm, 2800 gauss) arranged in a matrix so that the microplates are aligned with the intervals between the wells
It was placed close to one side and allowed to stand. Regarding the distribution pattern of the carrier particles, first, the first judgment was made at 2 minutes after standing, and then the second judgment was made when the magnet was moved away and left standing for 24 hours. Table 1 shows the results.

[0034]

[Table 1] As shown in Table 1, it was confirmed that the negative patterning of the positive pattern in the solid phase immunoassay similarly occurred between carrier particles made of different materials. In the above particles
Positive patterns can be determined up to 30-60 minutes later
However, after 90 minutes, it shifts to a false positive pattern.
Show a trend. Example 1

The distribution patterns of the solid-phase immunoassay reagents consisting of four types of carrier particles having the same specific gravity and the same material but different particle size distributions were observed and compared as follows. IgG immobilized microplate and anti-human I used in this example
GG-sensitized magnetic particles were prepared as follows. <Preparation of IgG-immobilized microplate>

U-shaped bottom microplate (Minc
cro well Module 2 × 8 well roundbolloom High bindi
ng Capacity NO. 469264) in each well, human IgG
(Seikagaku, ICN, product number 64-145) 0.01M
Diluted with PBS (pH7.43), 15.6μg / ml, 7.8μ
g / ml, 3.9 μg / ml, 1.95 μg / ml, 0.98 μg / ml,
Prepared 0.49μg / ml, 0.24μg / ml, 0.0μg / ml and dispensed 50μl per well,
Incubated for minutes. 0.01M including 0.05% tween 20
The wells were washed 3 times with PBS (pH 7.43). It was drained, air-dried and then refrigerated. <Preparation of anti-human IgG-sensitized magnetic particles>

Four types of magnetic gelatinous particles having a particle size distribution as shown in Table 2 and having the same magnetic substance encapsulation amount but different average particle sizes are described in Japanese Patent Application Laid-Open No. 64-25060. Was prepared as a reference. In preparation,
First, gelatin particles having an average particle size of about 3 to 4 μm are obtained by the coacervation method, and then they are set in a centrifuge in a state of being suspended with pure water (or PBS) as a dispersion medium, and the centrifugation speed is appropriately adjusted. Particle groups having various particle sizes were collected while controlling. The volume average particle size distribution of these magnetic gelatinous particles is shown as an abundance ratio, which is the average of three measurements. In addition, for the measurement of particle size distribution, automatic particle size analyzer CAPA
-700 (Horiba) was used. The concentration of the particle sample used for the measurement is 0.33% (W / V). The specific gravity of these particles is 1.17, the average particle size by volume average is,
1.8 μm (particle A), 3.2 μm (particle B), 4.0 μm
(Particle C) and 4.4 μm (Particle D).

The sensitization treatment of the particles A, B, C and D was performed as follows. 200 μl of a particle solution suspended at a particle concentration of 5% (W / W) was mixed with 0.01M PBS (pH7.43) to 3 times.
Washed twice. Tannic acid in PBS (concentration 10 μg / m
1 ml) was added and incubated at 37 ° C for 30 minutes.
After washing twice with 0.01 M PBS (pH 7.43), 1 ml of an anti-human IgG goat antibody (manufactured by CAPPEL) in PBS (concentration 25 μg / ml) was added and incubated at 37 ° C. for 1 hour. After that, it was washed twice with 0.01M PBS (pH7.43), and then 0.1% bovine serum albumin (BSA), 0.05%
0.25% in 0.01M PBS (pH7.43) containing NaN ₃ (W /
The particles were suspended at the particle concentration of (W). <Formation of distribution pattern>

Into each well of the above-prepared IgG-immobilized microplate, 25 μl of each of the prepared anti-human IgG-sensitized magnetic particles A to D was dispensed per well, shaken, and allowed to stand naturally. It was Anti-human Ig precipitated after 120 minutes and 24 hours after being allowed to stand naturally
The distribution pattern of the G-sensitized magnetic particles was observed and judged.

Similarly, cylindrical ferrite magnets in which IgG-immobilized microplates immediately after dispensing and shaking various anti-human IgG-sensitized magnetic particles A to D are individually arranged so as to have an interval between each well It was allowed to stand approximately 5 mm above (diameter 6 mm, 2800 gauss). The determination was performed by observing the distribution pattern of the anti-human IgG-sensitized magnetic particles that had settled at 3 minutes after standing and 24 hours after leaving the magnet . The results are shown in Tables 2 and 3.

[0041]

[Table 2]

[0042]

[Table 3]

As shown in Tables 2 and 3, the order in which the distribution pattern of the precipitated carrier particles was likely to change from the positive pattern to the negative pattern was the order of particles A, B, C and D. Particle C could be judged up to 120 minutes later, but
After time, it showed a negative tendency. That is, the particle size is 3.0 μm
It was confirmed that the distribution pattern was more stable as the abundance ratio of the following particles, particularly particles having a particle size of 2.0 μm or less was smaller. Further, according to Table 2, the particle size is 34.1 μm to 8.0 μm.
The distribution pattern tended to become more stable as the abundance ratio of the m carrier particles increased. Furthermore, interestingly, similar results were obtained when the magnetic substance-encapsulated particles were rapidly sedimented with a magnet or spontaneously. This suggests that the stability of the sedimentation pattern is significantly influenced by the particle size or particle size distribution of the carrier particles, not by the sedimentation velocity of the carrier particles. Example 2 The following test was conducted in order to investigate in more detail the relationship between the abundance ratio of carrier particles having a particle size of 4.1 μm to 8.0 μm and the stability of the sedimentation pattern found in Example 1. <Preparation of IgG-immobilized microplate>

The dilution concentration of human IgG was 500 μg / ml,
250 μg / ml, 125 μg / ml, 62.5 μg / ml, 31.3 μg
/ Ml, 15.6 μg / ml, 7.8 μg / ml, 0 μg / ml, and an IgG-immobilized microplate was prepared in the same manner as in Example 1. <Preparation of anti-human IgG-sensitized magnetic particles>

Specific gravity 1.12 and particle size 4.0 to 8.0 μm
As shown in Table 4, the abundance ratios of the carrier particles up to the above were prepared in the same manner as in Example 1 such that magnetic substance-encapsulated gelatinous particles E, F and G having different particle size distributions by volume average were prepared. The average particle size of these particles is 3.1 μm for particle E and 4.
1 μm, particle G is 6.1 μm.

The sensitizing treatment of the particles E, F and G is as follows. Particle suspension with a particle concentration of 5% (W / V)
Remove 500 μl of supernatant and remove 0.01M PBS (pH7.43)
It was washed twice with. Tannic acid in PBS (concentration 10 μg /
1 ml) was added and incubated at 37 ° C. for 30 minutes. After washing twice with 0.01M PBS (pH7.43),
1 ml of a PBS solution (concentration: 10 μg / ml) of an anti-human IgG goat antibody (manufactured by CAPPEL) was added, and the mixture was incubated at 37 ° C. for 1 hour. 0.01M PBS containing 0.1% BSA (pH
The particles were suspended in the same PBS washed three times with 7.43) so that the particle concentration was 0.4% (W / V). <Formation of distribution pattern>

The prepared anti-human IgG-sensitized magnetic particles E, F and G were dispensed into each well of the prepared IgG-immobilized microplate in an amount of 25 μl per well, shaken, and then sealed with a tape. Each well was sealed and left to stand naturally at room temperature. 120 minutes, 24 hours, and 2
After a lapse of weeks, the distribution pattern of the precipitated anti-human IgG-sensitized magnetic particles was visually observed and judged. The results are shown in Table 5.
Shown in

[0048]

[Table 4]

[0049]

[Table 5]

As can be seen from Tables 4 and 5, particles E which account for about 80% by volume of the whole have a particle size of 1.0 to 5.0 μm and particle E of the same particle size of 2.0 to 6.0 μm. With the particle F in the range of, the stability of the particle F was obviously better. In addition, a particle F having a particle size of 2.0 to 7.0 μm and a particle G having a particle size of 5.0 to 6.0 μm occupies about 70% by volume of the entire particle. The stability of G was obviously better. That is, the particle F is
After a few hours, it shifted to a false positive pattern. In particular, the particle G
Considering that the result of satisfying the long-term stability of the distribution pattern of the aggregated particles was obtained in the particle size distribution of No. 1 and each particle size distribution of the particles C and D that was stable in Example 1, it is suitable for the solid phase aggregation method. The carrier particles have a particle size distribution of 5.0 to 6.
It was found that particles containing 0 μm particles in an amount of 15% by volume or more, preferably 20% by volume or more are preferable. Also, comparing particles F and G, particles having a particle size of 6.0 to 8.0 μm are
It has been found that those containing 10% by volume or more, preferably 20% by volume or more are preferable. However, the carrier particles used for the reagent for solid phase immunoassay of the present invention are not particularly limited to those having the above-mentioned particle size distribution. Example 3

In the above Examples 1 and 2, the particle size is 1.0 to 1
In the carrier particles having a particle size distribution within the range of 0.0 μm, it was observed that the larger the average particle size, the better the result. Therefore, next, in a carrier particle having a particle size distribution in which the particle size is in the range of 1.0 to 20.0 μm, Example 2
The stability of the distribution pattern of aggregated particles was examined in the same manner as in. That is, the four types of carrier particles H to K shown in Table 6 below are all particles having a particle size of 10.0 μm or more.
It exceeds 15.0% by weight. However, here, no sieving or classification treatment such as centrifugation was performed.

According to the same procedure as in the preparation of the anti-human IgG-sensitized magnetic particles of Example 2, the volume average particle size was 6.0 to 8.0 μm.
Particles H containing 44 to 53% by volume of particles within the range of m (average volume particle size 7.6 μm) and 68 to 74% by volume of particles within the range of particle size 6.0 to 10.0 μm. Among them, particle I (average volume particle diameter 8.8 μm) containing 33 to 58% by volume of particles within the particle diameter range of 8.0 to 10.0 μm, particle diameter 8.
Particles J containing 60 to 75% by volume of particles in the range of 0 to 14.0 μm, of which 35 to 56% by volume of particles in the range of particle diameter of 8.0 to 12.0 μm (average volume particle Diameter 1
0.2 μm) and particles within a particle size range of 10.0 to 16.0 μm in an amount of 64 to 69% by volume, of which particle size of 10.0 to 1
Particles K (average volume particle diameter 11.7 μm) containing particles in the range of 4.0 μm at 47 to 53% by volume were obtained. Table 6 shows the particle size distribution of the obtained particles H to K. In addition, particles H to K
The feature of the particle size distribution is that the particle size range is such that the volume average ratio is large and the total particle size reaches 50% by volume.
Table 7 shows the 50 volume% occupied area and volume average particle diameter of each particle. The particles A, C and G described later are also shown.

[0053]

[Table 6]

[0054]

[Table 7]

These four types of particles H to K and Example 1
With respect to the particles A, C, and G prepared in ~ 2, the same sensitization treatment and distribution pattern formation as in Example 2 were performed. However, the tannic acid concentration was set to 2 μg / ml and the anti-human IgG antibody concentration was set to 10 μg / ml to weakly set the reactivity. The results are shown in Table 8.

[0056]

[Table 8]

According to Table 8, in the solid phase aggregation method, particles having an average particle size of 6 μm or more, or particles having a particle size distribution such that particles having a particle size of 5 μm occupy more than half of all particles (particles G and H). , I, J, K) have been shown to be able to form stable distribution patterns over long periods of time. In contrast, as used in conventional indirect aggregation,
It was difficult to form a stable distribution pattern with particles having an average particle size of less than 5 μm, particularly 3 μm or less (particles A and C). From the above results, in the solid phase aggregation method,
It has been found that preferable carrier particles are particles having an average particle size of 6 μm or more, or particles having a particle size distribution in which particles having a particle size of 5 μm or more account for more than half of the whole. However, the particle size distribution of the particles produced by the coacervation method is relatively wide, and the closer the average particle size is to 5 μm, the more
Since it is highly possible that a large number of particles having a particle size of less than m, particularly 3 μm or less are contained, as shown in Table 7, the average particle size is 7.5 or less.
By using particles prepared to have a particle size of ˜8.5 μm or more, preferably 8.5 to 9.0 μm or more, the optimum average particle size can be obtained by further considering the recovery efficiency during synthesis and the clarity of the distribution pattern. The diameter is 8.5 to 15 μm. As a result, it is possible to provide a reagent for solid phase immunoreaction capable of forming a more stable distribution pattern without performing a removing operation with a sieve or the like. Example 4 Detection of anti-IgM antibody using particles I and K

Using the particles I and K prepared in Example 3,
Sensitization treatment and formation of a distribution pattern were performed in the same manner as in Example 3 except that IgM (manufactured by Cosmo Bio) as the solid phase antigen and anti-human IgM antibody (manufactured by CAPPEL) were used as the particle-sensitizing antibody. It was The results are shown in Table 9.

[0059]

[Table 9]

According to Tables 8 and 9, particles I and K having a particle size distribution such that particles having a particle size of 7.5 μm or more occupy more than half of the whole are different analytes (IgG, IgM).
It was found that the solid-phase agglutination method for A. can form a stable distribution pattern for a long period of time. Example 5 Detection of Irregular Antibody Using Particles I and K The particles I and K prepared in Example 3 were used to detect an irregular antibody as follows. <Preparation of erythrocyte immobilized microplate with known antigen>

First, in the same manner as in Reference Example 1, WGA was coated on the wells of a microplate. Next, a commercially available screening red blood cell having a known antigenicity (SurgeScreen, manufactured by Ortho Co., Ltd.) diluted to 1% by volume with PBS was dispensed in 25 μl per well,
It was allowed to stand at room temperature for 10 minutes. Further, unbound erythrocytes were washed off with physiological saline to obtain microplates on which erythrocytes of known antigen were immobilized. <Preparation of anti-human IgG-sensitized magnetic carrier particles> In the same manner as in Reference Example 1, particles I and K were sensitized with an anti-human IgG antibody. <Detection of anti-D antibody>

25 μl of the LISS solution was dispensed into each well of an erythrocyte-immobilized microplate of known antigen. Next, two types of anti-D serum and normal human serum (negative serum) containing no anti-D antibody were dispensed into separate wells. On the other hand, the anti-D serum was serially diluted as shown in Table 10, and sample solutions of various concentrations were dispensed into separate wells.

After this, after incubation at 37 ° C. for 10 minutes, the wells were washed with physiological saline. Particles I and K after sensitization with anti-human IgG antibody were dispensed at 25 μl per well, and a magnet was placed under the microplate to form a distribution pattern in 3 minutes.
The stability of the distribution pattern was evaluated by standing for a period of time. Table 10 shows the results.

[0064]

[Table 10] As is clear from Table 10, it was confirmed that a distribution pattern having the same stability can be formed even for an actual blood sample.

[0065]

INDUSTRIAL APPLICABILITY The solid-phase immunoassay reagent of the present invention is obtained by lowering the abundance ratio of particles having a particle size that causes a change from a positive pattern to a negative pattern in the solid-phase agglutination method below a predetermined abundance ratio. In addition, it is possible to maintain the distribution form of particles that are stable and positive for a long period of time. As a result, it is possible to prevent the examiner from overlooking the positive pattern in the solid-phase immunoassay, which brings about an effect of improving the measurement accuracy.

Claims (3)

(57) [Claims] 1. A substance to be measured in a specimen sample that specifically binds to an antigen or antibody immobilized on the inner wall surface of a reaction container,
By binding carrier particles coated with an antigen or an antibody that specifically binds to the substance to be measured, the distribution amount of the carrier particles occupying the surface area of the inner wall surface on which the antigen or the antibody is immobilized A solid-phase immunoassay reagent used in a solid-phase immunoassay method for detecting the presence or absence of the substance to be measured, wherein the abundance ratio of particles having a particle size of 3.0 μm or less is 10.0 in the particle size distribution of artificially synthesized carrier particles. A reagent for solid phase immunoassay characterized by being less than or equal to volume%. 2. The reagent for solid phase immunoassay according to claim 1, wherein the particle size distribution of the carrier particles is such that the abundance ratio of particles having a particle size of 2.0 μm or less is 3.0% by volume or less. 3. The particle size distribution of carrier particles is such that the average particle size is 6 μm.
The above is the reagent for solid phase immunoassay according to claim 1 or 2.