JPH0827285B2 - Immunological measurement method using immobilized erythrocytes - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for measuring an antigen or an antibody present in a sample by an immunological agglutination reaction.

[Conventional technology]

The particle agglutination method has been conventionally known as a method for detecting an antigen or an antibody present in a sample based on an immunological agglutination reaction. In this method, specific marker particles having an antibody or an antigen that specifically binds to the substance to be measured immobilized on the surface are used. The marker particles are mixed with the sample in the measurement container to cause an immune reaction with the antigen or antibody contained in the sample, and then collected on a predetermined wall surface (for example, bottom surface) of the measurement container. The distribution pattern of the marker particles thus collected on the wall surface of the container is different depending on whether or not an immune reaction occurs with the substance to be measured in the sample. Therefore, positive or negative can be determined from the distribution pattern of the marker particles collected on the container wall surface.

Separately, a mixed coagulation method was reported by Wiener, AS and Herman, MH (J. Immunol., 36, 255 (1
939)). This method has since evolved and has been established by the time blood typing is possible (Coombs, RRA and Bedford, D., VoxSang, 5, 111 (1955); Coombs, RR).
A. et al., Lancet, i, 461 (1956)). As applied to the determination of various blood types, for example, USP 4,608,246, USP
4,275,053 and USP 4,328,183.

Also known is a method in which an antibody or the like that specifically binds to the substance to be measured is immobilized on the inner wall of the reaction vessel in advance (MPHA
Law). In this method, a substance (such as an antibody) that specifically binds to the substance to be measured is immobilized in advance on the inner wall of the reaction container. Then, after the substance to be measured is specifically bound to this antibody or the like, marker particles are added. The marker particles react with the substance to be measured bound to the antibody or the like immobilized on the inner wall of the container to form an aggregation pattern. MPHA like this
The method is described, for example, in USP 4,297,104.

Incidentally, most of the formation of the aggregation pattern is due to the spontaneous sedimentation of the marker particles, and it takes a lot of time to complete the pattern formation. In order to solve this problem, the inventors have proposed a method for forming a pattern in a short time by magnetic force using magnetic particles (Japanese Patent Application No. 1-184819, unpublished).

[Problems to be Solved by the Invention]

In the conventional particle aggregation method and mixed aggregation method,
Since the sedimentable particles other than the marker particles contained in the sample are sedimented, the determination accuracy is lowered. Therefore, in order to perform good determination, an operation such as washing the sample in order to remove the target sedimentable particles is required, and the operation is complicated.

In the MPHA method, as a method of binding a substance that specifically binds to a substance to be measured to the inner wall of the container, a method of directly binding to the inner wall of the container by physical adsorption is widely used.

However, this method may not form a normal pattern. For example, even if HBs is physically adsorbed directly on the inner wall of the container to measure the anti-HBs antibody in the sample, it is difficult to clearly distinguish the positive pattern from the negative pattern. Further, in the case where the substance is directly adsorbed on the inner wall of the container, it is necessary to select a container having a high adsorption ability. Further, the susceptibility of adsorption differs depending on the substance to be adsorbed, and it is necessary to select the material of the container depending on the substance.

On the other hand, in the chemical bonding method in which a substance is directly bound to the inner wall of the container by chemisorption, the chemical binding group of the antigen or antibody to be adsorbed is used to bind to the inner wall of the container. Therefore, it may impair the reactivity of the antigen or the antibody and cannot be applied to any substance.

Therefore, the first object of the present invention is to provide an immunological assay method capable of rapidly performing highly accurate measurement without being affected by a sample containing a sedimentable substance other than the substance to be measured. To do.

Further, the second object of the present invention, in the MPHA method of binding a substance that specifically binds to the substance to be measured to the inner wall of the container, does not change the property of the inner wall of the container, and impairs the reactivity of the substance to be adsorbed. And to provide a highly sensitive and highly stable immunological assay method in which the substance can be immobilized on the inner wall of the container without changing the material of the container depending on the type of the substance to be adsorbed. is there.

[Means for solving the problem]

The immunological measurement method of the present invention comprises a step of preparing a particle agglutination determination container in which immobilized red blood cells to which a substance that specifically binds to a substance to be measured is bound are immobilized on the inner wall, and a sample is placed in the particle agglutination determination container. A step of injecting a solution, a step of removing an unreacted substance from the particle agglutination determination container, a step of injecting a magnetic marker bound to a substance that binds to a substance to be measured into the particle agglutination determination container, and the particles A step of applying a magnetic field to the agglutination determination container to promote sedimentation of the magnetic marker particles, and based on the distribution state of the magnetic marker particles settled on the inner wall of the particle agglutination determination container, the measurement target contained in the sample solution Measuring the substance.

 Hereinafter, the method of the present invention will be described in detail.

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

The magnetic marker particle used in the present invention is a surface of a known magnetic particle containing a magnetic substance, which is a substance (hereinafter referred to as sensitizing substance B) that specifically binds to a substance to be measured, such as a specific antibody or antigen. It can be prepared by immobilizing on. As such magnetic particles,
Commercially available "DYNABEAS M-450" (trade name of DYNAL) and magnetic latex beads "estapor" (trade name of RHONE-POULENC) can be used. In addition, JP-A-59-1951
You may use the gelatin particle containing the magnetic body disclosed by the 61st bulletin. Furthermore, cells such as erythrocytes in which a ferromagnetic substance such as iron is incorporated may be used. As a method of immobilizing the sensitizing substance B (specific antibody or antigen) on the surface of these magnetic particles, various known methods can be used.

The particle aggregation determination container used in the present invention, when the sedimentable particles move in a predetermined direction along the container wall surface,
It is preferable to have an inclined wall surface that converges the sedimentable particles in a predetermined region, for example, a hemispherical or conical wall surface. Such a slope may be on the side of the container, but is preferably on the bottom. Examples of the preferable particle aggregation determination container include a test tube or a microplate having a hemispherical or conical bottom surface.

Next, an example of making a particle aggregation determination container in which immobilized red blood cells to which a substance that specifically binds to the substance to be measured (hereinafter referred to as sensitizing substance A) is immobilized is immobilized on the inner wall will be described with reference to the drawings.

1a to 1c are diagrams schematically showing a method for producing a particle aggregation determination container.

First, as shown in FIG. 1a, a substance 2 capable of specifically or nonspecifically binding erythrocytes such as human, sheep and chicken is immobilized on the inner surface of the particle agglutination determination container 1. Examples of such substances include poly-L-lysine, polyarginine, polyethyleneimine, various lectins, anti-erythrocyte antibodies and the like, which can be electrostatically bound.

Then, as shown in Fig. 1b, red blood cells 3 are placed in this container.
Is added to solidify the red blood cells 3 on the inner surface of the container. Immobilization of erythrocytes 3 is sufficiently performed by adding the erythrocyte suspension and then allowing it to stand still, but it is possible to further shorten the time by applying weak centrifugation.

Next, the red blood cells 3 immobilized on the inner wall of the container are fixed using a fixing reagent. As the immobilization reagent, a reagent conventionally used for immobilizing cells can be used, and in particular,
Glutaraldehyde, formalin and the like can be preferably used.

Then, the surface of this fixed erythrocyte is treated with tannic acid by a conventional method to facilitate the binding with protein. Then, a solution containing the sensitizing substance A 4 is placed in the container to bind the sensitizing substance A 4 to the surface of the immobilized red blood cells.

Alternatively, the sensitizer A may be first bound to the immobilized red blood cells, and then the immobilized red blood cells may be immobilized on the inner wall of the container. In this case, first, erythrocytes are fixed, the surface is treated with tannic acid, and then mixed with a solution containing the sensitizing substance A to bind the sensitizing substance A to the erythrocyte surface. Separately from this, the immobilized erythrocyte-capturing substance is immobilized on the inner wall of the container. Finally, the fixed red blood cells are added to the container to immobilize the fixed red blood cells on the inner wall of the container.

The immobilized erythrocyte-capturing substance that is immobilized in advance on the inner wall of the container is capable of binding to the immobilized erythrocyte at a site other than the sensitizing substance A. As shown in FIG. 2, when the immobilized erythrocytes 5 are bound to the inner wall of the container with the sensitizing substance A 4, the erythrocytes lose their reactivity with the substance to be measured. Further, the binding between the immobilized red blood cells and the immobilized red blood cell capturing substance may be a specific binding or a non-specific binding. Examples of the substance that forms a specific bond include an antigen or antibody that does not react with the sensitizing substance A but specifically reacts with immobilized erythrocytes, a lectin, and the like. In addition, examples of the substance that forms a non-specific bond include polyethyleneimine and poly-L-lysine that bind fixed erythrocytes by electrostatic binding.

After immobilizing the immobilized erythrocytes to which the sensitizing substance A is bound in a particle aggregation determination container, a sample solution is injected into this container. As shown in FIG. 3a, when the sample solution contains the substance 6 to be measured, the sensitizing substance A 4 bound to the surface of the immobilized red blood cells 3 and the substance 6 to be measured are bound to each other.

After reacting the sensitizing substance A and the substance to be measured, the unreacted substance is removed from the container 1. This is usually done by washing the container 1.

After removing the unreacted substances from the container 1, the magnetic marker particles 7 are injected into the container as shown in FIG. 3b, and the magnetic field formed by the magnet 9 is applied to promote the sedimentation of the magnetic marker particles 7. . As described above, the magnetic marker particles 7
A sensitizing substance B8 that specifically reacts with the substance to be measured is bound to. Therefore, the precipitated magnetic marker particles 7
Binds to the substance 6 to be measured fixed to the inner wall of the container via the immobilized red blood cells, as shown in FIG. 3c. That is, the fixed erythrocytes are distributed almost uniformly on the inner wall of the container on which the immobilized erythrocytes are immobilized. As a result, the distribution pattern of the magnetic marker particles spreads widely on the inner wall of the container, as shown in FIG. 4a.

When the substance to be measured is not contained in the sample solution, as shown in FIG. 3d, the substance that reacts with the subsequently added magnetic marker particles does not exist in the container. Therefore, the magnetic marker particles roll down the inner wall of the container without reacting, and gather at the lowest part of the container as shown in FIG. 3e. As a result, the distribution pattern of the magnetic marker particles is such that the particles are gathered in the center of the container, as shown in FIG. 4b.

As described above, in the method of the present invention, sedimentation of magnetic marker particles is promoted by the action of a magnetic field such as a magnet. Therefore, the measurement time can be significantly reduced. The magnet used here is not particularly limited and may be a permanent magnet or an electromagnet. The strength of the magnet can be appropriately selected according to the substance to be measured and the like.

〔Example〕

Example 1 Detection of Anti-HBs Antibody in Serum A. Preparation of Fixed Blood Cell Plate Wheat germ lectin (WGA) was dissolved in phosphate buffer (PBS) to a concentration of 100 μg / ml, and a U-bottom plate ( N
unc.Maxisorp) and WGA was adsorbed on the inner wall of the plate. Then, blocking was performed using bovine serum albumin (BSA) to prepare a WGA adsorption plate. Next, 0.7% P of O-type blood cell surge screen (Ortho)
A BS suspension was prepared and dispensed on the WGA adsorption plate.
The cells were allowed to stand at room temperature for 10 minutes to bind the blood cells to the inner wall of the well of the plate, and then washed 3 times with PBS. The blood cell binding plate was then loaded with 2.5 of glutaraldehyde (Sigma).
% PBS solution was dispensed and allowed to stand at room temperature for 1 hour to fix blood cells. Then, the wells were washed with PBS to remove glutaraldehyde. Finally, a solution of tannin (manufactured by Hanai Tesque Co., Ltd.) dissolved in PBS to 10 μg / ml was dispensed into each well in an amount of 50 μ, and incubated at room temperature for 1 hour to treat the immobilized erythrocytes with tannic acid. .

B. Antigen sensitization First, yeast recombinant HBs antigen (yHBs) was diluted to 10 μg / ml with PBS. This diluted solution was dispensed into the fixed hemocyte plate treated with tannic acid by 25 μm per well and incubated at room temperature for 1 hour to sensitize yHBs. After washing the wells, 0.3% BSA / PBS solution was dispensed and blocked. After further washing the wells, drain the water,
It was air-dried to prepare a yHBs sensitized plate.

C. Preparation of anti-human IgG-sensitized magnetic particles Anti-human IgG was sensitized to magnetic pair-encapsulated gelatin particles having a particle size of about 6 μm using 5 μg / ml of anti-human IgG (polyclonal antibody manufactured by Cappel). Then, blocking with BSA was performed to obtain anti-human IgG-sensitized magnetic particles.

D. Detection of HBs Antibody in Serum Using PBS containing 0.3% BSA, dilution series of HBs antibody positive sample and negative sample were prepared, respectively. These diluted solutions were dispensed on a yHBs-sensitized plate, incubated at 37 ° C for 10 minutes, and then washed 6 times with PBS. Then
The above-mentioned anti-human IgG-sensitized magnetic particles were dispensed into each well in an amount of 25 μm and attracted with a magnet for 3 minutes to form a pattern.

The results obtained are shown in Figures 4a and 4b. FIG. 4a shows the pattern obtained from the positive sample, and FIG. 4b shows the pattern obtained from the negative sample, which showed almost the same pattern up to the dilution ratio of 2 ⁻⁶ . As shown in the figure, the presence of HBs antibody can be clearly determined in a short time.

Example 2 Detection of IgG in Serum Anti-human IgG (manufactured by Cappel) was diluted with PBS to prepare a diluted solution having a concentration of 10 μg / ml, and this diluted solution was prepared in the same manner as in Example 1. 25 μl was dispensed into each well of fixed hemocyte plates treated with tannic acid. This at room temperature 1
Anti-human IgG was sensitized by incubating for a time. After sensitizing with anti-human IgG, the wells were washed,
A 0.3% BSA / PBS solution was dispensed for blocking. Then, the wells were further washed and air-dried to prepare an anti-human IgG sensitized plate.

A dilution series of a commercially available pooled serum (trade name "Suitrol N", manufactured by Nissui Pharmaceutical Co., Ltd.) was prepared with PBS containing 0.3% BSA, and the diluted series was dispensed on the anti-human IgG sensitized plate. After dispensing, 10 at 37 ℃
Incubate for min and wash 6 times with PBS. afterwards,
The anti-human IgG-sensitized magnetic substance-encapsulated particles were dispensed into each well in an amount of 25 μm, and aspirated with a magnet for 3 minutes to form a pattern.

The results obtained are shown in Table 1 below and Figures 5a to 5c. FIG. 5a when the dilution ratio of Suitororu is 2 ^-1, in the case of FIG. 5b is 2 ^-16, and the 5c diagram denotes a pattern without added Suitororu.

As is clear from these tables and figures, the shape of the formed pattern was different depending on the dilution ratio of styrene, and IgG in serum could be detected in a short time.

Comparative Example Detection of Anti-HBs Antibody in Serum Detection of anti-HBs antibody similar to that in Example 1 was carried out by a conventional method in which an antigen was directly bound to myoplate.

Yeast recombination, HBs antigen (yHBs) was diluted to 10 μg / ml with PBS, and each well was added to a U-bottom plate (Maxisorp manufactured by Nunc).
Dispense in 25μ aliquots and incubate at room temperature for 1 hour to yield yH
Bs was adsorbed on the inner wall of the well. Then, after washing the wells, 0.3% BSA / PBS solution was dispensed to perform blocking. After blocking, the wells were washed, drained, and air-dried to prepare a yHBs plate.

Then, a dilution series of HBs antibody positive sample and negative sample was prepared using PBS containing 0.3% BSA. Each dilution was dispensed on the yHBs plate and incubated at 30 ° C for 10 minutes. Then, it wash | cleaned 6 times with PBS.

Next, 5 pieces of gelatin beads containing a magnetic material with a particle size of about 6 μm
Sensitization was performed with 1 μg of anti-human IgG at μg / ml to prepare anti-human IgG-sensitized magnetic particles. The sensitized magnetic particles were dispensed into the plate in an amount of 25 μm per well and attracted with a magnet for 3 minutes.

However, in this case no pattern was formed that could clearly distinguish positive from negative.

〔The invention's effect〕

As described above, in the immunological measurement method of the present invention, magnetic marker particles are used, and a magnetic field is applied to promote the sedimentation of the magnetic marker particles. It is possible to form a distribution pattern of marker particles in a very short time. That is, the time required for the determination can be significantly reduced. Further, since the precipitation is promoted only by the magnetic marker particles, it is possible to form a pattern of the magnetic marker particles before other sedimentable particles are precipitated. Therefore, it is possible to perform highly sensitive and highly accurate measurement without previously removing the spontaneously sedimentable substance other than the substance to be measured.

Furthermore, in the immunological measurement method of the present invention, first, immobilized erythrocytes are immobilized on the inner wall of the container, and then a substance that specifically reacts with the substance to be measured is bound to the surface of the erythrocyte. The substance that specifically reacts with the substance is not directly bound to the inner wall of the container. Therefore, the property of the inner wall of the container is not changed, the reactivity of the substance that specifically reacts with the substance to be measured is not impaired, and the material of the container does not need to be changed according to the type of the substance.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1a to 1c are diagrams schematically showing a method for producing a particle aggregation determination container used in the immunological measurement method of the present invention, and FIG. In the preparation of the particle aggregation determination container used in the biological measurement method, a diagram schematically showing the case where the reactivity is impaired, FIGS. 3a to 3e are schematic diagrams showing the steps in the immunological measurement method of the present invention. 4a and 4b are diagrams showing the results in one embodiment of the present invention, and FIGS. 5a to 5c are diagrams showing the results in another embodiment of the present invention.

Claims (1)

[Claims] 1. A step of preparing a particle agglutination determination container in which immobilized red blood cells to which a substance that specifically binds to a substance to be measured is bound are immobilized on an inner wall, and a step of injecting a sample solution into the particle agglutination determination container. A step of removing an unreacted substance from the particle aggregation determination container, a step of injecting into the particle aggregation determination container a magnetic marker that binds a substance that binds to a substance to be measured, and a magnetic field in the particle aggregation determination container And a step of promoting the sedimentation of the magnetic marker particles, based on the distribution state of the magnetic marker particles settled on the inner wall of the particle aggregation determination container, the step of measuring the substance to be measured contained in the sample solution An immunological measurement method on fixed erythrocytes, comprising: