JP2009069070A - Detecting method of target material, and kit for detection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detecting method of a target material for accurately and easily detecting a plurality of kinds of the target materials in an analyte in one reaction vessel in a solid phase hemagglutination method using an immunological method, and to provide a kit for detection used in the detecting method. <P>SOLUTION: This detecting method of the target material is a method for detecting the plurality of the kinds of the target materials in the analyte using particles for hemagglutination carrying a material specifically bondable to the target materials. This detecting method comprises (a) a process of solidifying the target material existing in the analyte in some cases on an inner surface partially including the bottom surface of the reaction vessel, and (b) a process of supplying a plurality of kinds of particles for hemagglutination at different sedimentation speeds to the reaction vessel obtained in the process (a) and then identifying the existence of hemagglutination, or (b') a process of supplying the plurality of kinds of particles for hemagglutination to the reaction vessel obtained in the process (a) and then identifying the existence of hemagglutination at a different timing. The kit for detection used in the detecting method is provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  INDUSTRIAL APPLICABILITY The present invention is a method for detecting a target substance for detecting a plurality of types of target substances in a specimen with high accuracy and convenience in a single reaction vessel using an immunological technique, and the detection method. The present invention relates to a detection kit.

  A detection method using an immunological technique such as an antigen-antibody reaction is widely used in order to detect a trace amount of a target substance from specimens such as blood containing various substances. For example, when particles carrying an antibody are mixed with an antigen, aggregation occurs as a result of an antigen-antibody reaction, and when particles carrying a substance that can specifically bind to a target substance are added to a specimen, There is a method for detecting whether or not a target substance is present in a specimen by identifying the presence or absence of particle aggregation. Especially in the field of immunoassay, when the target substance is an antibody or antigen, the target substance in the sample is captured and measured by binding the antigen or antibody that specifically binds to the target substance to the magnetic particles. How to do is widely known.

  One of the methods for detecting whether or not a specimen contains an antibody using antibody-sensitized magnetic particles to which an antibody is bound is a magnetic mixed passive agglutination test method (Magnetic-Mixed passive hemagglutination test; M- MPHA). The M-MPHA method is mainly performed to detect the presence or absence of antibodies or antigens in blood such as antiplatelet antibodies. Specifically, first, after coating human platelet antigen on the bottom of a reaction vessel such as a microplate or well, as a primary reaction, human serum as a specimen is added to the reaction vessel and reacted, Anti-platelet antibody binds to and captures human platelet antigen. Thereafter, B / F separation (removal of unbound serum component) is performed by washing, and as a secondary reaction, anti-human IgG antibody-sensitized magnetic particles or anti-human IgM antibody-sensitized magnetic particles are added, The presence or absence of aggregation resulting from the antigen-antibody reaction with the anti-platelet antibody captured in the reaction is identified. When IgG type antiplatelet antibody is present in human serum, antigen antibody between IgG type antiplatelet antibody captured in the reaction vessel and antihuman IgG antibody-sensitized magnetic particles added by secondary reaction Aggregation occurs due to the reaction. Aggregated particles are observed to be uniformly dispersed on the bottom surface of the reaction vessel, but particles that have not aggregated settle and settle on the lowest part of the reaction vessel. That is, in the M-MPHA method, since the presence or absence of aggregation can be identified by observing the aggregation pattern (aggregation particle distribution pattern), the presence or absence of the target substance in the sample can be easily detected.

  Usually, when both IgG type HLA antibody and IgM type HLA antibody are detected in human serum by M-MPHA method, an agglutination reaction using anti-human IgG antibody-sensitized magnetic particles, and anti-human IgM antibody sensitivity The aggregation reaction using the magnetic particles is performed in a separate reaction vessel. In general, detection of IgG type HLA antibodies is prioritized over detection of IgM type HLA antibodies. This is because the detection rate of the IgG type HLA antibody is higher. For this reason, in the field of clinical examinations, the M-MPHA method was first performed using IgG type HLA antibody, and it was determined that IgG type HLA antibody was negative without aggregation being observed. An M-MPHA method using an IgM type HLA antibody has been performed on sera suspected to exist, and the presence or absence of an IgM type HLA antibody has been confirmed. That is, in order to confirm the presence or absence of both the IgG type HLA antibody and the IgM type HLA antibody, it is necessary to carry out the M-MPHA method separately. There is a problem that it takes time and a sufficient amount of specimen cannot be tested when the amount of specimen is very small. Therefore, development of an agglutination reaction method that can rapidly detect a plurality of target substances using a small amount of specimen is desired.

Various methods have been disclosed for simultaneously performing agglutination reactions on a plurality of target substances. For example, (1) a reaction solution obtained by mixing a plurality of types of aggregating particles having different particle diameters for each type of target substance and a sample from a maximum interval sufficiently larger than the maximum particle diameter of the aggregating particles Injection into a tapered hollow member such as a dispensing nozzle with a gradually reduced gap to a minimum interval sufficiently smaller than the particle size, and agglomerates at different positions for each particle size of the particles for aggregation A method of detecting a plurality of target substances by separating them by a single operation is disclosed (for example, see Patent Document 1). In addition, (2) a plurality of types of aggregating particles are simultaneously added to the sample and reacted, and then the sample contains any one of a plurality of target substances by identifying the formed aggregation pattern Or a method of detecting whether a plurality of types are contained (for example, refer to Patent Document 2). In the method,
JP-A-6-11508 JP-A-3-2526

  However, when the agglutination reaction is performed simultaneously using a plurality of types of agglutination particles as in the methods (1) and (2) above, it may interfere with each other depending on the type of agglutination particles and the target substance. There's a problem. In particular, in the solid phase aggregation method in which the target substance is immobilized on the bottom surface of the reaction vessel as in the M-MPHA method, when two or more kinds of aggregation particles are present in the reaction vessel, the reaction vessel It is difficult to obtain a stable aggregation pattern by interfering with each other on the wall. For example, aggregating particles (negative particles) in which a target substance to be detected does not exist in a specimen entrain the aggregating particles (positive particles) in which the target substance is present, so that the positive particles are separated from the target substance. Without binding, it may deposit in the lowest part of the reaction vessel and form an agglomeration pattern close to false negatives. On the contrary, when the positive particles bound to the target substance entrain the negative particles, the negative particles are uniformly dispersed on the bottom surface of the reaction container, and an aggregation pattern close to false positive may be formed. For this reason, in the solid phase aggregation method, it is difficult to accurately detect a plurality of types of target substances in one reaction vessel.

  The present invention relates to a target substance for detecting a plurality of types of target substances in a sample in a single reaction container with high accuracy and in a solid phase aggregation method using an immunological technique such as the M-MPHA method. An object of the present invention is to provide a detection method for the above and a detection kit used in the detection method.

  As a result of diligent research to solve the above problems, the present inventors made use of the time difference until each aggregating particle settles to form an agglomeration pattern at different timings for each type of aggregating particle, The inventors have found that a stable aggregation pattern can be formed while preventing interference caused by different types of aggregation particles, and that a plurality of types of target substances can be detected with high accuracy, thereby completing the present invention.

That is, the present invention is a method for detecting a plurality of types of target substances in a specimen using aggregating particles carrying substances that can specifically bind to the target substance, and (a) in the specimen A step of solidifying an optionally present target substance on an inner surface including a bottom surface of the reaction vessel; and (b) a plurality of types of the aggregating particles in the reaction vessel obtained by the step (a), After supplying at different settling rates, the step of identifying the presence or absence of agglomeration, or (b ′) agglomeration after supplying a plurality of types of aggregating particles to the reaction vessel obtained by step (a) And a step of identifying the presence / absence of aggregation at different timings for each type of particles for use, and to provide a method for detecting a target substance.
Further, the present invention is characterized in that the target substance is an antibody or an antigen, and the substance that can specifically bind to the target substance is an antigen or an antibody that can specifically bind to the antibody or antigen. A method for detecting a target substance is provided.
Further, the present invention is characterized in that the weight ratio of any two kinds of particles of different kinds of agglomerating particles is 1.5 times or more of the large weight particles with respect to the small weight particles. A method for detecting a target substance is provided.
Further, according to the present invention, the ratio of the magnetic force held by any two types of particles of different types of agglomerating particles is 1.5 times or more that the particles with a large magnetic force are smaller than the particles with a small magnetic force. The present invention provides a method for detecting a target substance.
The present invention also provides a method for detecting a target substance, wherein a centrifugal force and / or a magnetic field is applied to the aggregating particles in the reaction vessel in the step (b) or the step (b ′). is there.
The present invention also provides a kit for detecting a plurality of types of target substances, each of which is specific to a reaction container capable of solidifying the target substance on an inner surface including a reaction container bottom as a part, and each target substance. There are provided a detection kit comprising a plurality of types of aggregating particles carrying a substance capable of binding chemically.
The present invention also provides a detection kit, wherein the aggregation particles are freeze-dried and have a reconstitution liquid for reconstitution of the aggregation particles.
Further, the present invention further includes a sample dilution solution for diluting the sample, and a cleaning solution for cleaning the reaction container after the target substance is immobilized on the bottom surface. A kit is provided.

By using the target substance detection method of the present invention, a plurality of types of target substances can be detected efficiently and with high accuracy in one reaction vessel. In the target substance detection method of the present invention, in order to form an aggregation pattern at different timings for each type of aggregation particles, different types of aggregation are performed even when the target substance is solid-phased in the reaction vessel. This is presumed to be able to prevent interference by the particles for use. In addition, if the amount of sample required in one reaction container is sufficient, multiple types of target substances can be detected. Therefore, even in a clinical test where the amount of sample is very small, detection cannot be missed and sufficient testing can be performed. it can. Furthermore, since the amount of reaction containers, reagents, and the like used can be reduced, it can be expected to reduce the cost of inspection.
In addition, by using the detection kit of the present invention, it is possible to easily detect a plurality of types of target substances by solid phase aggregation using an immunological technique.

  The target substance in the present invention is not particularly limited as long as it is a biological substance that is a detection target, and a substance that can specifically bind to it, and is a low molecular weight compound such as biotin. It may be a high molecular compound such as a nucleic acid, protein, or polysaccharide. The protein may be a protein composed only of amino acids, a glycoprotein, or a lipoprotein. However, specifically binding in the present invention means that it can bind specifically to the extent that it can be normally used for detection and purification of the target substance, and does not intersect with other substances at all. There is no need.

  The sample in the present invention is not particularly limited as long as it contains a biological component for measuring the presence or absence of the target substance, and may be a sample collected from a living organism, and a cultured cell was prepared. It may be a sample. In particular, it is preferably a biological sample used for clinical examinations. Examples of the biological sample include blood, bone marrow fluid, lymph fluid, urine, ascites, exudate, amniotic fluid, and organ washing fluid. The biological sample may be a sample collected from a living organism or may be a prepared sample. The preparation method is not particularly limited as long as it does not impair the target substance contained in the biological sample. Usually, the preparation method used for the biological sample can be performed. . Examples of the preparation method include dilution using physiological saline and the like, separation or concentration of a target substance, and the like.

  In particular, in the method for detecting a target substance of the present invention, the target substance and a substance that can specifically bind to the target substance carried by the aggregation particles (hereinafter sometimes referred to as a target substance-binding substance) are antigens. It is preferable that the antibody has a relationship with the antibody. For example, when the target substance is an antigen, the target substance-binding substance is preferably an antibody against the target substance, and when the target substance is an antibody, the target substance-binding substance may be an antigen of the target substance. preferable. It is more preferable that the target substance is an antigen and the target substance-binding substance is an antibody against the target substance, because the aggregation particles are well aggregated. The antibody used as the target substance-binding substance may be an unpurified antibody or a polyclonal antibody, but is preferably a purified monoclonal antibody from the viewpoint of detection and quantification accuracy.

  The aggregating particles of the present invention are particles that carry a target substance-binding substance and are aggregated by binding to the target substance via the target substance-binding substance. The material for the aggregation particles is not particularly limited as long as it is a material that is usually used as a carrier in the aggregation reaction. Examples of the aggregating particles include particles formed from biological components such as gelatin, gum arabic, polystyrene, latex, agarose, dextran, cellulose, polyacrylamide, kaolin, activated carbon powder, ceramic, and red blood cells. Moreover, the particle | grains which consist of multiple types of materials may be sufficient like the coacervate of gelatin and gum arabic.

  The particles for aggregation of the present invention may be magnetic particles. Examples of the magnetic particles include particles obtained by adding magnetic materials such as ferricolloid and iron powder to the particles of the materials listed above as aggregating particles, and particles formed only from the magnetic material.

  The particle size of the aggregating particles of the present invention can be appropriately determined in consideration of the particle material, particle weight, aggregation efficiency, and the like. For example, in the case of coagulate particles of gelatin and gum arabic, it is preferably about 4 to 20 μm. Since the specific surface area is sufficient, the aggregation efficiency is good, and the size is sufficiently large, a clear aggregation pattern without bleeding suitable for visual observation can be formed. This is because it has advantages such as being easy to form.

  The method for supporting the target substance binding substance on the aggregation particles is not particularly limited as long as the target substance binding substance can be bound to at least the surface of the aggregation particles. For example, the target substance-binding substance may be physically adsorbed on the surface of the aggregation particles, or may be chemically bonded to a functional group on the surface of the aggregation particles. In the case of chemical bonding, the target substance-binding substance can be bonded by a method suitable for each functional group. For example, EDAC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride) method in which carboxylic acid and amino group are bonded with water-soluble carbodiimide, EDAC and N-hydroxysuccinimide (NHS) are mixed in advance. There are a method of binding a carboxylic acid and an amino group, a method of cross-linking amino groups using a bipolar linker, a method of binding an activated aldehyde group or tosyl group and a functional group in the acceptor, etc. . In the case of magnetic particles having no functional group on the surface of the aggregating particle, the surface of the aggregating particle may be coated with a functional group.

  The present invention is a method for detecting a plurality of types of target substances in a specimen using a substance capable of specifically binding to the target substance, that is, aggregating particles carrying the target substance-binding substance, comprising: (a) A step of immobilizing a target substance optionally present in the specimen on an inner surface partially including a bottom surface of the reaction vessel; and (b) a plurality of types of the aggregation in the reaction vessel obtained by the step (a). Supplying particles with different sedimentation speeds, and (b ′) supplying a plurality of types of the particles for aggregation to the reaction vessel obtained by step (a) And a step of identifying the presence or absence of aggregation at different timings for each type of aggregating particles.

  Specifically, first, as a step (a), a target substance optionally present in a specimen is solid-phased on an inner surface including a reaction vessel bottom as a part. The solid phase immobilization method is not particularly limited as long as the solid phase can be obtained without impairing the binding activity of the target substance with the target substance binding substance. Any method used in the above can be used. For example, the target substance may be directly adsorbed directly, and after coating the surface of the reaction vessel with a substance capable of binding the target substance such as lectin, the target substance is bound to the surface of the reaction vessel through the coating. You may let them. The immobilization site of the target substance is an inner surface including the container bottom as a part.

  The reaction vessel used in the present invention is preferably a reaction vessel having a bottom surface inclined in a concentric shape such as a hemispherical shape or a conical shape. By using a reaction vessel having a bottom portion on the bottom surface instead of a flat bottom reaction vessel, unaggregated agglomerated particles that do not bind to the target substance can be deposited on the bottom portion of the bottom surface. As shown, it is possible to clearly identify the aggregation pattern when the target substance is present in the specimen and when it is not present. An example of such a reaction vessel is a microplate having a U-shaped or V-shaped well on the bottom.

  For example, when the target substance is an anti-platelet antibody, the specimen is sometimes present in the specimen at the bottom of the reaction container by supplying the specimen to a reaction container coated with a platelet antigen such as lectin and allowing to stand for a certain period of time. The target substance can be immobilized. In addition, you may use a commercially available reaction container coated with platelet antigen. Thereafter, the specimen is removed from the reaction container, and components that have not been solid-phased in the specimen are removed. The reaction vessel may be further cleaned using an appropriate cleaning solution. The washing solution is not particularly limited as long as it is a solution that can wash the reaction vessel without damaging the target substance. For example, physiological saline, PBS (phosphate buffered saline, pH 7.4), HEPES buffer etc. Further, it may be a cleaning liquid containing a surfactant such as Tween 20 or Triton X-100.

  Next, as the step (b), after supplying a plurality of types of the aggregating particles to the reaction vessel obtained in the step (a) with different settling rates, the presence or absence of aggregation is identified. Aggregation occurs when the particles for aggregation supplied to the reaction vessel settle and bind to the target substance solid-phased on the bottom surface of the reaction vessel. By differentiating the settling speed for each type of aggregating particle, even when multiple types of aggregating particles are supplied at one time, the time when the type of aggregating particle settles down to the bottom of the reaction vessel, that is, Since the formation time of the aggregation pattern can be shifted, interference by other types of aggregation particles can be minimized. In this way, by sequentially identifying the aggregation patterns obtained as a result of the aggregation at different times for each type of aggregation particles, it is possible to accurately detect a plurality of types of target substances in one reaction vessel.

  For example, the sedimentation speed can be varied by increasing the weight ratio of the particles for aggregation. The weight of the aggregating particles can be changed by adjusting the particle diameter or forming the aggregating particles using a material having an appropriate specific gravity. The sedimentation rate may be varied depending on both the particle diameter and specific gravity. In addition, when magnetic particles are used as the aggregating particles, the difference in the magnetic force held by the aggregating particles causes a difference in the magnetizing speed, so that the settling speed can be varied. The magnetic force held by the aggregating particles can be changed by adjusting the amount of the magnetic substance contained in the aggregating particles.

  The settling speed of each aggregating particle is suitably set so as to have a settling speed difference that causes formation of an agglomerated pattern with another type of aggregating particle after the aggregating pattern formation with one type of aggregating particle is completed. It is preferable to adjust. When the aggregating particles are settled by gravity, the weight ratio of any two types of particles of different types of aggregating particles is 1.5 times or more of the large weight particles to the small weight particles. It is preferable that it is 2 times or more. Similarly, in the case of sedimentation by magnetic force, the ratio of the magnetic force held by any two types of particles of different types of agglomerating particles is 1.5. It is preferably 2 times or more, and more preferably 2 times or more. Magnetic particles and nonmagnetic particles may be used. Alternatively, the weight ratio or the magnetic substance content may be varied for each type of aggregating particle so that the aggregating particle having a smaller particle size has a higher sedimentation rate.

  In the method for detecting a target substance of the present invention, after step (a), as step (b ′), after supplying a plurality of types of the particles for aggregation to the reaction container obtained in step (a), aggregation is performed. The presence or absence of aggregation may be identified at different timings for each type of particle. The aggregating particles may be supplied all at once or sequentially for each type. When supplying at different timings for each type of aggregating particles, it is possible to appropriately determine the timing to supply in consideration of the sedimentation speed of each aggregating particle so that the timings of aggregation pattern formation do not overlap. . For example, when there is a sufficient difference in the settling speed of each aggregating particle due to the weight ratio or the magnetic force ratio to be retained being 1.5 times or more, the case where a plurality of types of aggregating particles are supplied at one time Even if it exists, the formation time of an aggregation pattern can be shifted for every kind of particle for aggregation. On the other hand, even if there is not a sufficient difference in the settling speed of each aggregating particle, agglomeration pattern by one type of aggregating particle is formed, and then another type of aggregating particle is supplied to agglomerate. The formation time of the aggregation pattern can be shifted for each kind of particles.

  By applying a centrifugal force or a magnetic field to the aggregation particles in the reaction vessel, the aggregation particles can be quickly settled. In addition, since the difference in the sedimentation speed due to the weight ratio or the magnetic force ratio can be made more prominent, the presence or absence of aggregation due to each aggregation particle can be accurately identified. Therefore, when highly accurate detection is performed in a shorter time, the ratio of weight and magnetic force for each type of aggregating particle is increased, and the sedimentation speed of each aggregating particle is increased by applying centrifugal force or magnetic field. It is preferable. Both centrifugal force and magnetic field may be applied to the aggregating particles in the reaction vessel. In addition, for example, in a gel column agglutination method such as MTS (microtyping system), the weight ratio of each type of agglomeration particles is adjusted so that the moving speed due to centrifugal force is different, thereby providing a time difference in agglomeration pattern formation. May be. In addition, a centrifugal force can be applied to the aggregating particles in the reaction container by centrifuging the reaction container in a centrifuge. Moreover, a magnetic field can be applied to the coagulating particles in the reaction vessel by installing a magnet in the lower part of the bottom of the bottom of the reaction vessel. The strength of the centrifugal force and magnetic field applied to the aggregating particles is appropriately determined in consideration of the respective sedimentation speeds of the aggregating particles to be used so that they can settle to the bottom of the reaction vessel at different timings depending on the type of the aggregating particles. Can be set.

  Further, by using different color tone particles for each type of aggregation particles, it is possible to easily identify the aggregation pattern by each aggregation particle. The color tone of the aggregating particles can be appropriately changed by incorporating a pigment or the like into the aggregating particles. In addition, the color tone can be changed for each type of aggregating particles by using materials having different color tones.

  Identification of the presence or absence of agglomeration by various aggregating particles may be performed at each time point when each agglomeration pattern is formed, may be performed once every predetermined time, or may be performed intermittently or continuously multiple times. Also good. In particular, it is also possible to acquire a video image continuously from the start of formation of the aggregation pattern using an imaging device such as a CCD, analyze the pattern change, and accurately determine the dynamics of various aggregation particles.

  Two types of target substances A and B in a specimen are divided into two types of aggregating particles having different weights and magnetic forces (antibody a-sensitized aggregating particles 2 carrying antibody a that specifically binds to target substance A, and target FIG. 1 shows a schematic diagram of an aggregation pattern formed when detection is performed using antibody b-sensitized aggregation particles 3) carrying an antibody b that specifically binds to substance B. 1A to 1F, the left diagram is a cross-sectional view of the reaction vessel 1, and the right diagram is a plan view of the reaction vessel 1. 1 (a) and 1 (b) are schematic diagrams of an aggregation pattern previously formed by antibody a-sensitized aggregation particles 2 having a large weight and magnetic force, and FIGS. FIG. 4 is a schematic diagram of an aggregation pattern formed by antibody a-sensitized aggregation particles 2 and antibody b-sensitized aggregation particles 3 thereafter.

  The weight and magnetic force of the antibody a-sensitized agglutinating particle 2 are set to be twice the weight and magnetic force of the antibody b-sensitized agglutinating particle 3, and target substances A and B optionally present in the specimen are immobilized on the bottom surface. After simultaneously supplying antibody a-sensitized aggregation particles 2 and antibody b-sensitized aggregation particles 3 to a reaction vessel 1 which is a U-shaped well, a magnet is placed under the bottom surface of the reaction vessel 1 to form a magnetic field. Is added, the antibody a-sensitized aggregation particles 2 having a large weight and magnetic force are first settled to form an aggregation pattern. When the target substance A is present in the sample, that is, when the sample is positive for antibody a, the target substance A is solid-phased on the bottom surface of the reaction container 1, so FIG. As shown in the figure, the antibody a-sensitized agglutination particles 2 form a substantially uniform thin aggregate layer on the entire bottom surface, and as shown in the right figure of FIG. An agglomeration pattern is formed as if the color of the aggregating particles 2 is lightly dyed. On the other hand, when the target substance A is not present in the sample, that is, when the sample is antibody a-negative, the target substance A is not solid-phased on the bottom surface of the reaction vessel 1, so FIG. b) As shown in the left figure, the antibody a-sensitized agglutinating particles 2 are deposited on the lowest part of the bottom surface of the reaction container 1, and as shown in the right figure of FIG. Is formed such that the antibody a-sensitized agglutinating particles 2 are deeply dyed in the color tone.

  After the aggregation pattern by the antibody a-sensitized aggregation particles 2 is formed, the antibody b-sensitized aggregation particles 3 having a small weight and magnetic force settle on the bottom surface of the reaction vessel 1 to form an aggregation pattern. When the specimen is antibody a-positive and antibody b-positive, both the target substance A and the target substance B are solid-phased on the bottom surface of the reaction vessel 1, and as shown in the left figure of FIG. The antibody b-sensitized agglutinating particles 3 form a substantially uniform thin agglomerated layer on the entire bottom surface, and as shown in the right figure of FIG. Thus, an aggregation pattern is formed in which the color tone of the antibody b and the color tone of the antibody b-sensitized aggregation particles 3 are lightly dyed. On the other hand, when the specimen is antibody a positive and antibody b negative, the target substance A is immobilized on the bottom surface of the reaction vessel 1, but the target substance B is not immobilized. (D) As shown in the left figure, antibody b sensitized aggregation particles 3 are deposited on the lowest part of the bottom surface of the reaction vessel 1, and as shown in the right figure of FIG. Thus, an aggregation pattern is formed in which the lowest portion is darkly stained in the color tone of the antibody b-sensitized aggregation particles 3 and the other regions are lightly stained in the color tone of the antibody-a sensitized aggregation particles 2.

  When the sample is antibody a-negative and antibody b-positive, the target substance A in the reaction container 1 is not solid-phased and the target substance B is solid-phased. As shown in FIG. 1A, a substantially uniform thin aggregate layer is formed on the entire bottom surface by the antibody b-sensitized aggregation particles 3, and as shown in the right figure of FIG. An aggregation pattern is formed in which the color tone of the antibody a-sensitized agglutinating particles 2 is darkly stained and the other region is lightly stained in the color tone of the antibody-b sensitized agglutinating particles 3. Further, when the sample is antibody a negative and antibody b negative, neither the target substance A nor the target substance B is solid-phased on the bottom surface of the reaction vessel 1, and therefore, the left figure in FIG. Thus, the antibody b-sensitized agglutinating particles 3 are deposited on the lowest part of the bottom surface of the reaction container 1, and only the lowest part of the bottom surface of the reaction container 1 has antibody a sensation as shown in the right figure of FIG. An agglomeration pattern is formed in which the color tone of the aggregating particles 2 and the color tone of the antibody b-sensitized agglomerating particles 3 are darkly dyed.

  The detection kit of the present invention is a kit for detecting a plurality of types of target substances, and comprises a reaction vessel capable of solidifying the target substance on the bottom surface, and a substance capable of specifically binding to each target substance. A plurality of types of aggregating particles carried. By using such a detection kit, a plurality of types of target substances can be detected from a specimen with high accuracy and ease. The aggregation particles contained in the detection kit are not particularly limited as long as they do not impair the binding activity with the target substance, and may be in the form of a slurry using an appropriate buffer. It may be dried. The aggregating particles are preferably freeze-dried because they are suitable for long-term storage and are less likely to impair the binding activity with the target substance. In addition, when the aggregation particles are freeze-dried, it is also preferable that the detection kit has a reconstitution liquid for reconstitution of the aggregation particles. The reconstitution liquid is not particularly limited as long as it is a solution that can reconstitute lyophilized aggregating particles without impairing the binding activity with the target substance. Examples of the restoration liquid include physiological saline and PBS. Furthermore, it is preferable to have a sample dilution solution for diluting the sample and a cleaning solution for cleaning the reaction container after the target substance is solid-phased on the bottom surface. As the sample dilution solution, the solutions mentioned in the cleaning liquid can be used.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

(Synthesis example 1) Production of anti-human IgG antibody-sensitized magnetic particles Anti-human IgG antibody-sensitized magnetic particles, which are particles for aggregation carrying antibodies against human IgG antibodies, were prepared.
First, 0.6 g of gum arabic (manufactured by Semba Gakka Kogyo Co., Ltd.) is dissolved in EtOH / H ₂ O (2/1), Tween 20 and ferricolloid W10 (manufactured by Taiho Kogyo Co., Ltd.) are added, and the pH is adjusted with NaOH. After that, a 4% gelatin aqueous solution (manufactured by Nippi Industry Co., Ltd.) heated to 40 ° C. was added and mixed. Next, acetic acid was slowly added while stirring so that the pH was about 5 to prepare a coacervate having an average diameter of 4 to 5 μm.
The produced coacervate was cooled to 10 ° C. or lower and gelled. Thereafter, 2 mL of glutaraldehyde (manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred, and then allowed to stand overnight at room temperature to crosslink the coacervate.
The crosslinked coacervate was washed with pure water and then adjusted to a 20% (V / V) slurry with pure water. 10 mL of the slurry was collected, and 10 mL of N-hydroxysuccinimide (manufactured by Nacalai Tesque) 0.01 g / mL and EDAC (manufactured by Sigma Chemical) 0.01 g / mL aqueous solution were added to the precipitate. It was made to react with. After the reaction, the coacervate was washed, and a rabbit anti-human IgG antibody (manufactured by Jackson) dissolved in 0.1 M citrate phosphate buffer (pH 5) to a concentration of 5 μg / mL was reacted at room temperature. Anti-human IgG antibody-sensitized magnetic particles were obtained by washing with PBS (pH 7.2) containing 0.1% BSA (bovine serum albumin). The obtained particles had a brown color due to the ferricolloid W10 contained therein.

(Synthesis Example 2) Production of Anti-Human IgM Antibody-Sensitized Magnetic Particles Anti-human IgM antibody-sensitized magnetic particles, which are particles for aggregation carrying antibodies against human IgM antibodies, were produced.
First, 0.6 g of gum arabic (manufactured by Senba Gakka Kogyo Co., Ltd.) is dissolved in EtOH / H ₂ O (2/1). After that, a 4% gelatin aqueous solution (manufactured by Nippi Industry Co., Ltd.) heated to 40 ° C. was added and mixed. Next, acetic acid was slowly added while stirring so that the pH was about 4, thereby preparing a coacervate having an average diameter of 9 to 10 μm.
The prepared coacervate was washed with pure water, 1.5 g of sodium carbonate, 0.26 g of green pigment Green ES4BD (manufactured by Nippon Kayaku Co., Ltd.) and 3.0 g of sodium chloride were added and stirred overnight. On the next day, the coacervate was further washed with pure water, and then adjusted to a slurry of 20% (V / V) with pure water. 10 mL of the slurry was collected, and 10 mL of N-hydroxysuccinimide (manufactured by Nacalai Tesque) 0.01 g / mL and EDAC (manufactured by Sigma Chemical) 0.01 g / mL aqueous solution were added to the precipitate. For 2 hours. After the reaction, the coacervate was washed, a rabbit anti-human IgM antibody (manufactured by Jackson) dissolved in 0.1 M citrate phosphate buffer (pH 5) to 5 μg / mL was added, and reacted at room temperature. Anti-human IgM antibody-sensitized magnetic particles were obtained by washing with PBS (pH 7.2) containing 0.1% BSA (bovine serum albumin). The obtained particles had a green color due to the contained pigment.

Example 1
The target substance in the specimen was detected by applying a magnetic field to the aggregation particles in the reaction vessel. Human IgG type HLA antibody and human IgM type HLA antibody as target substances, human IgG type HLA antibody positive serum, human IgM type HLA antibody positive serum, human IgG type HLA antibody and human IgM type HLA antibody positive serum, human HLA antibody negative Four types of sera were used as specimens.
First, a platelet antigen solid phase plate (anti-PLT / Olivio / MPHA II antigen plate, manufactured by Olympus) was washed five times with a washing solution (physiological saline containing 0.05% Tween 20). Each well of the platelet antigen solid phase plate was supplied with 25 mL of each serum diluted 4 times with a specimen diluent (saline containing 1% gelatin) and incubated at 37 ° C. for 30 minutes. The target substance contained in the serum was immobilized. Thereafter, the diluted serum in the wells was discarded, and the wells were washed 5 times with a washing solution (physiological saline containing 0.05% Tween 20).
After supplying 25 μL each of the anti-human IgG antibody-sensitized magnetic particles synthesized in Synthesis Example 1 and the anti-human IgM antibody-sensitized magnetic particles synthesized in Synthetic Example 2 to the wells, The mixture was allowed to stand on a magnetic plate for 3 minutes to form an aggregation pattern with anti-human IgG antibody-sensitized magnetic particles. Thereafter, the platelet antigen solid phase plate was removed from the magnet plate, and the formed aggregation pattern was observed to determine the presence or absence of the first aggregation. Further, the platelet antigen solid phase plate was again returned to the magnet plate and allowed to stand for 3 minutes to form an aggregation pattern with anti-human IgM antibody-sensitized magnetic particles. Thereafter, the platelet antigen solid phase plate was removed from the magnet plate, the formed aggregation pattern was observed, and the presence or absence of the second aggregation was determined. In addition, as a control, the same operation was performed on a well supplied with only anti-human IgG antibody-sensitized magnetic particles and a well supplied with only anti-human IgM antibody-sensitized magnetic particles, and an aggregation pattern formed. Was observed.

  Table 1 shows the results of the determination of the presence or absence of aggregation in each serum. In the table, “+” indicates that aggregation was observed, and “−” indicates that aggregation was not observed. In the table, the color name next to “+” indicates the color tone of the observed aggregation. Is described. In the table, “IgG antibody positive serum” is a human IgG type HLA antibody positive serum, “IgM antibody positive serum” is a human IgM type HLA antibody positive serum, “IgG + IgM antibody positive serum” is a human IgG type HLA antibody and Human IgM type HLA antibody positive sera and “antibody negative serum” represent human HLA antibody negative sera, respectively.

  As shown in Table 1, in human IgG type HLA antibody positive sera, aggregation was observed in the first determination and no change in aggregation pattern was observed in the second determination, whereas human IgM type HLA antibody In the positive serum, no aggregation was observed in the first determination, and aggregation was observed in the second determination. On the other hand, in human IgG type HLA antibody and human IgM type HLA antibody positive serum, aggregation was observed in the first determination, and change in color of aggregation was observed in the second determination, whereas human HLA antibody In the negative serum, no aggregation was observed both in the first and second rounds. From these results, aggregating particles with different particle diameters, that is, aggregating particles with different weight and magnetic substance amount, are used to apply a magnetic field to each aggregating particle, thereby providing a difference in the settling speed of each aggregating particle. Thus, it is clear that a plurality of types of target substances can be detected in one reaction vessel by shifting the formation timing of the aggregation pattern.

(Example 2)
The target substance in the specimen was detected by applying centrifugal force to the aggregating particles in the reaction vessel.
In the same manner as in Example 1, diluted serum was supplied to each well of the platelet antigen solid phase plate to immobilize the target substance, and then each well was washed.
After supplying 25 μL of the anti-human IgM antibody-sensitized magnetic particles synthesized in Synthesis Example 2 to the well, the platelet antigen solid phase plate was centrifuged at 27 × g (medium-sized centrifuge, about 300 rpm) for 1 minute, Aggregation patterns were formed by human IgM antibody-sensitized magnetic particles. Thereafter, the platelet antigen solid phase plate was removed from the centrifuge, and the formed aggregation pattern was observed to determine the presence or absence of the first aggregation. Furthermore, 25 μL of the anti-human IgG antibody-sensitized magnetic particles synthesized in Synthesis Example 1 were supplied to the wells, and then the platelet antigen solid phase plate was centrifuged again at 27 × g (medium size centrifuge, about 300 rpm) for 1 minute. Then, an aggregation pattern of anti-human IgG antibody-sensitized magnetic particles was formed. Thereafter, the platelet antigen solid phase plate was removed from the centrifuge, and the formed aggregation pattern was observed to determine the presence or absence of the second aggregation. In addition, as a control, the same operation was performed on a well supplied with only anti-human IgG antibody-sensitized magnetic particles and a well supplied with only anti-human IgM antibody-sensitized magnetic particles, and an aggregation pattern formed. Was observed.

  Table 2 shows the results of the determination of the presence or absence of aggregation in each serum. In the table, the color name next to “+”, “−”, “+” and the description of serum are the same as in Table 1. As shown in Table 2, in human IgG type HLA antibody-positive serum, no aggregation was observed in the first determination, whereas aggregation was observed in the second determination, whereas in human IgM type HLA antibody-positive serum Aggregation was observed in the first determination, and no change in the aggregation pattern was observed in the second determination. On the other hand, in human IgG type HLA antibody and human IgM type HLA antibody positive serum, aggregation was observed in the first determination, and change in color of aggregation was observed in the second determination, whereas human HLA antibody In the negative serum, no aggregation was observed both in the first and second rounds. From these results, it is clear that a plurality of types of target substances can be detected in one reaction container by adjusting the timing of supplying the aggregating particles to the reaction container and shifting the formation time of the aggregation pattern. .

(Example 3)
The target substance in the specimen was detected by allowing the particles for aggregation in the reaction vessel to settle naturally.
In the same manner as in Example 1, diluted serum was supplied to each well of the platelet antigen solid phase plate to immobilize the target substance, and then each well was washed.
25 μL each of the anti-human IgG antibody-sensitized magnetic particles synthesized in Synthesis Example 1 and the anti-human IgM antibody-sensitized magnetic particles synthesized in Synthesis Example 2 were supplied to the wells, and the platelet antigen solid phase plate was placed at room temperature. The mixture was allowed to stand for 1 to 2 hours in a wet state to form an aggregation pattern with anti-human IgG antibody-sensitized magnetic particles. Then, the formed aggregation pattern was observed and the presence or absence of the 1st aggregation was determined. Further, the platelet antigen solid phase plate was allowed to stand for 1 hour in a room temperature wet state to form an aggregation pattern with anti-human IgM antibody-sensitized magnetic particles. Then, the formed aggregation pattern was observed and the presence or absence of the 1st aggregation was determined. In addition, as a control, the same operation was performed on a well supplied with only anti-human IgG antibody-sensitized magnetic particles and a well supplied with only anti-human IgM antibody-sensitized magnetic particles, and an aggregation pattern formed. Was observed.

  Table 3 shows the results of the determination of the presence or absence of aggregation in each serum. In the table, the color name next to “+”, “−”, “+” and the description of serum are the same as in Table 1. As shown in Table 3, in human IgG type HLA antibody positive sera, aggregation was observed in the first determination, and no change in aggregation pattern was observed in the second determination, whereas human IgM type HLA antibody In the positive serum, no aggregation was observed in the first determination, and aggregation was observed in the second determination. On the other hand, in human IgG type HLA antibody and human IgM type HLA antibody positive serum, aggregation was observed in the first determination, and change in color of aggregation was observed in the second determination, whereas human HLA antibody In the negative serum, no aggregation was observed both in the first and second rounds. From these results, by using agglomerated particles having different particle diameters, that is, agglomerated particles having different weights, by setting a difference in the sedimentation speed of each aggregating particle and shifting the formation time of the agglomerated pattern, It is clear that multiple types of target substances can be detected in the reaction vessel.

By using the detection method of the target substance of the present invention and the detection kit used in the detection method,
Because it is possible to detect multiple types of target substances in a single reaction container easily and with high accuracy, the amount of sample is small, and clinical tests that require processing multiple samples with high accuracy and speed, etc. It is possible to use in the field.

Two types of target substances A and B in a specimen are divided into two types of aggregating particles having different weights and magnetic forces (antibody a-sensitized aggregating particles 2 carrying antibody a that specifically binds to target substance A, and target FIG. 6 is a diagram schematically showing an aggregation pattern obtained when detection is performed using antibody b-sensitized aggregation particles 3) carrying an antibody b that specifically binds to substance B. 1A to 1F, the left diagram is a cross-sectional view of the reaction vessel 1, and the right diagram is a plan view of the reaction vessel 1. FIGS. 1 (a) and 1 (b) show an aggregation pattern previously formed by antibody a-sensitized aggregation particles 2 having a large weight and magnetic force. FIG. 1 (a) shows a case where the specimen is positive for antibody a. b) is a schematic diagram of the aggregation pattern when the specimen is antibody a-negative. FIGS. 1C to 1F show the aggregation pattern formed by the antibody a-sensitized aggregation particle 2 and the antibody b-sensitized aggregation particle 3 after that, and (c) shows that the specimen is antibody a positive and antibody When b is positive, (d) is when the sample is antibody a positive and antibody b negative, (e) is when the sample is antibody a negative and antibody b positive, (f) is when the sample is antibody a negative and It is a schematic diagram of each aggregation pattern in the case of antibody b negative.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Reaction container, 2 ... Antibody a sensitized aggregation particle, 3 ... Antibody b sensitized aggregation particle

Claims (8)

A method for detecting a plurality of types of target substances in a specimen using aggregating particles carrying substances that can specifically bind to the target substances,
(A) immobilizing a target substance optionally present in the specimen on an inner surface including a reaction vessel bottom as a part;
(B) a step of identifying the presence or absence of agglomeration after supplying a plurality of types of the aggregating particles to the reaction vessel obtained in step (a) with different settling rates;
Or
(B ′) After supplying a plurality of types of the aggregation particles to the reaction vessel obtained in the step (a), for each type of the aggregation particles, identifying the presence or absence of aggregation at different timings;
A method for detecting a target substance, comprising:   The target according to claim 1, wherein the target substance is an antibody or an antigen, and the substance that can specifically bind to the target substance is an antigen or an antibody that can specifically bind to the antibody or antigen. Substance detection method.   3. The weight ratio of any two types of particles of different types of agglomerating particles per particle is such that a large weight particle is 1.5 times or more of a small weight particle. Of detecting a target substance of a substance.   The ratio of the magnetic force held by any two types of particles of different types of agglomerating particles is 1.5 times or more of particles having a large magnetic force compared to particles having a small magnetic force. Item 4. A method for detecting a target substance according to any one of Items 1 to 3.   The method for detecting a target substance according to claim 1, wherein in the step (b) or the step (b ′), a centrifugal force and / or a magnetic field is applied to the aggregating particles in the reaction vessel.   A kit for detecting a plurality of types of target substances, a reaction container capable of solidifying the target substance on the inner surface including a reaction vessel bottom as a part, and a substance capable of specifically binding to each target substance. And a plurality of types of agglutinating particles supporting the detection kit.   The detection kit according to claim 6, wherein the aggregation particles are freeze-dried and have a restoration liquid for restoring the aggregation particles.   The detection according to claim 6 or 7, further comprising: a sample dilution solution for diluting the sample; and a cleaning solution for cleaning the reaction container after solidifying the target substance on the bottom surface. For kit.

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