JP2004177402A - Method and apparatus for measuring specific bond reaction and reagent kit used therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and an apparatus for measuring a specific bond reaction in which a plurality of substances to be measured can be measured in one reaction container and to provide a reagent kit used therefor. <P>SOLUTION: In a step St1 shown in the figure, a reaction system which contains a sample 1 to be measured for the content of the substance 2 to be measured and magnetic particles 4 to which the specific bond substance 3 to be specifically bonded to the substance 2 to be measured is immobilized, is first built. In a step St2 shown in the figure, the optical characteristics of the reaction system is then measured. Then, if an aggregate complex 5 is generated in the step St1, a turbid occurs in the reaction system, and changes occur in a scattered light intensity, an amount of transmitted light, etc. Accordingly, the degree of the turbid of the reaction system can be estimated by measuring the scattered light intensity, the amount of the transmitted light, etc. In a step St3 shown in the figure, the aggregate complex 5 containing the substance 2 to be measured, the specific bond substance 3 and the magnetic particles 4 and magnetic particles (not shown) not forming the aggregate complex 5 are recovered by utilizing a magnetic force, and are removed from the reaction system. <P>COPYRIGHT: (C)2004,JPO

Description

  The present invention relates to a specific binding reaction measuring method capable of measuring the content of a substance to be measured which may be contained in a sample, a reagent kit used therefor, and a specific binding reaction measuring device.

  2. Description of the Related Art In the medical field, in order to diagnose various diseases and check the progress of disease states, it is widely practiced to measure the content of proteins characteristic of each disease present in human body fluids. For measuring the content of these proteins, an immunoreaction measurement method utilizing a reaction between an antibody that specifically recognizes a target protein as an antigen and an antigen (antigen-antibody reaction) is widely used. At present, methods utilizing various principles have also been developed for immunological reaction measurement methods.

  Among the various immunoreaction measurement methods described above, in order to measure the content of a substance to be measured in a sample, an aggregate of an antigen-antibody complex generated by an antigen-antibody reaction (hereinafter, referred to as an aggregate) is detected. Measurement methods such as immunoturbidimetry (hereinafter abbreviated as nephelometry), immunoturbidimetry (hereinafter abbreviated as nephelometry), and slide agglutination are well known. In the antigen-antibody reaction, the reaction system becomes turbid due to the formation of an aggregation complex. The degree of turbidity generated in the reaction system due to the formation of the aggregation complex depends on the amount of the antigen and the amount of the antibody. Based on this, the nephelometry and the nephelometry are methods of optically measuring the degree of turbidity occurring in the above-mentioned reaction system, and calculating the amount of the antigen or the amount of the antibody from the measured value. Specifically, the turbidimetric method is based on the change in the amount of light scattered in the reaction system, and the turbidimetric method is based on the change in the amount of transmitted light reduced by the scattering in the reaction system, and measures the aggregated complex. Is the way. Generally, in the above two measurement methods, the same reaction system can be used for the measurement. That is, a reaction system that can be measured by either one of the measurement methods can also be measured by the other measurement method. The slide agglutination method is a method in which turbidity or agglomerate generated in a reaction system due to the formation of an agglutination complex is visually determined on a slide glass or the like. In the slide agglutination method, the same reaction system as the nephelometry and the nephelometry can be used for the measurement. The above three methods are generally referred to as "homogeneous immunoreaction measurement methods" because the measurement is performed in a state where the antigen and the antibody are uniformly dispersed in the reaction system.

  In the medical field, the content of a plurality of types of substances to be measured is measured in order to diagnose various diseases and to check the progress of disease states. For example, in clinical tests, the content of total protein that can be contained in human urine as an indicator of the renal filtration capacity is determined by determining the content of acute glomerulonephritis, IgA nephropathy, renal tuberculosis, renal infarction, pyelonephritis, cystitis. It measures the content of red blood cells (hemoglobin) in human urine as an indicator of urinary tract inflammation such as urethritis, prostatitis, calculus and tumor (for example, see Non-Patent Document 1) ). Further, Patent Literature 1 listed below prepares a different reaction container for each of the analytes and individually performs measurement for each of the analytes as a homogeneous immunoreaction measurement method for measuring a plurality of analytes. A method is disclosed.

A method for measuring the content of a test substance in a sample by utilizing the reaction between the test substance contained in the sample and a specific binding substance that specifically binds to the test substance. In the present specification, this is referred to as “specific binding reaction measurement method”. The above-described method for measuring an immune reaction is also one of the methods for measuring a specific binding reaction.
JP-A-5-2024 Izumi Kanai, edited by Masamitsu Kanai, "Summary of Clinical Laboratory Methods," 31st Edition, Kanehara Publishing Co., Ltd., 1998, 156 pages, 179-180.

  However, in the above-mentioned conventional method, when measuring a plurality of types of substances to be measured, it is necessary to prepare the same number of reaction vessels as the number of substances to be measured, and a large amount of sample is required for the measurement.

  The present invention has been made in view of the above circumstances, and provides a specific binding reaction measuring method capable of measuring a plurality of substances to be measured in one reaction container, a reagent kit and a specific binding reaction measuring device used therefor. The purpose is to provide.

  The specific binding reaction measuring method of the present invention is a specific binding reaction measuring method for measuring the content of a test substance in a sample, wherein the sample and the specific binding substance specifically binding to the test substance are measured. (A) constructing a reaction system containing magnetic particles having immobilized thereon, and (b) measuring the optical characteristics of the reaction system; (C) removing the agglomerated complex containing from the reaction system using a magnetic force.

  According to the present invention, in the step (c) after the measurement, the magnetic particles existing in the reaction system and the aggregation complex containing the substance to be measured, the specific binding substance, and the magnetic particles utilize magnetic force. Is removed from the reaction system. In particular, in the present invention, since a magnetic force is used, no chemical influence is exerted on the reaction system. For this reason, using the reaction system after the measurement, other components contained in the sample can be measured by various measurement methods.

  Conventionally, when measuring two types of substances to be measured, it is necessary to prepare the same number of reaction vessels as the number of substances to be measured. However, according to the present invention, if there is one reaction vessel capable of constructing a reaction system, it is possible to measure two types of substances to be measured using the reaction vessel. Therefore, it is possible to make the sample required for the measurement very small. Therefore, for example, in the medical field, the amount of sample collected from a patient can be reduced, and the burden on the patient can be reduced.

  The optical characteristic may be scattered light intensity or transmitted light amount.

  The diameter of the magnetic particles is preferably in the range of about 0.05 to 2 μm.

  In the step (c), the magnetic particles remaining in the reaction system may be removed from the reaction system.

  Another specific binding reaction measuring method of the present invention is a specific binding reaction measuring method for measuring the contents of a first analyte and a second analyte in a sample, the method comprising: (A) constructing a reaction system including a magnetic particle on which a first specific binding substance that specifically binds to a substance to be measured is fixed, and after the step (a), (B) measuring the optical characteristics of step (b); and collecting (c) aggregating complexes containing the first substance to be measured, the first specific binding substance, and the magnetic particles using magnetic force. (D) adding a second specific binding substance that specifically binds to the second analyte to the reaction system; and after the step (d), the optical characteristics of the reaction system are added. Measuring step (e).

  According to the present invention, when measuring the second analyte, the change in the optical properties of the reaction system due to the analyte and the specific binding substance in the reaction system and the aggregation complex containing magnetic particles, It is eliminated by using magnetic force. Particularly, since the magnetic force is used, the reaction system is not chemically affected at all. For this reason, according to the method for measuring a specific binding reaction of the present invention, the reliability of the measured value is high even when the second analyte is measured.

  Conventionally, when measuring the contents of two types of substances to be measured, it is necessary to prepare two reaction vessels of the same type as the substance to be measured, but according to the present invention, a reaction system is constructed. If there is one reaction vessel capable of performing the measurement, it is possible to measure the contents of the two kinds of substances to be measured using the reaction vessel. Therefore, the sample required for measurement is very small. Therefore, for example, in the medical field, the amount of sample collected from a patient can be reduced, and the burden on the patient can be reduced.

  In the step (d), the reaction system preferably contains 2 to 6% by weight of polyethylene glycol.

  The second specific binding substance is preferably composed of an antigen or antibody that specifically binds to the second analyte, and nonmagnetic particles to which the antigen or antibody is immobilized. .

  Preferably, the diameter of the magnetic particles is in the range of about 0.05-2 μm, and the diameter of the non-magnetic particles is in the range of about 0.05-2 μm.

  The combination of the first analyte and the second analyte may be a combination of human hemoglobin and human albumin.

  In the step (c), the magnetic particles remaining in the reaction system may be collected.

  Still another specific binding reaction measuring method of the present invention is a specific binding reaction measuring method for measuring the content of n kinds of substances (n is an integer of 2 or more) in a sample. (A) constructing a reaction system including a magnetic particle on which a specific binding substance that specifically binds to one of the substances to be measured is immobilized, and after the step (a), Step (b) of measuring the optical characteristics of the reaction system, and removing the aggregated substance containing the one kind of the substance to be measured, the specific binding substance, and the magnetic particles from the reaction system by using a magnetic force. After the step (c) and the step (c), if the step (b) is less than the (n-1) th time, the step (d) of determining to proceed to the step (a) again; The reaction system specifically binds to one type of unmeasured analyte remaining in the reaction system. A step of adding a specific binding substance (e), after the step (e), and a step (f) for measuring an optical property of the reaction system.

  According to the present invention, the aggregated complex containing the substance to be measured, the specific binding substance, and the magnetic particles is removed by using the magnetic force after the measurement of the optical properties. Due to the use of magnetic force, there is no chemical effect on the reaction system. For this reason, even when measuring a plurality of substances to be measured, the influence of the aggregate formed beforehand is almost eliminated, so that the reliability of the measured value is high every time the optical characteristics are measured.

  In particular, conventionally, when measuring the content of two or more kinds of substances to be measured, it is necessary to prepare the same number of reaction vessels as the kinds of the substances to be measured. If there is one reaction vessel that can be constructed, the content of two or more kinds of substances to be measured can be measured using the reaction vessel. Therefore, the sample required for measurement is very small. Therefore, for example, in the medical field, the amount of sample collected from a patient can be reduced, and the burden on the patient can be reduced.

  In the step (c), the magnetic particles remaining in the reaction system may be removed from the reaction system.

  Still another specific binding reaction measuring method of the present invention is a specific binding reaction measuring method for measuring the content of n kinds of substances (n is an integer of 2 or more) in a sample, the method comprising: (A) constructing a reaction system containing a specific binding substance that specifically binds to one of the unmeasured substances to be measured, and after the step (a), optical characteristics of the reaction system And (b) adding a magnetic complex containing a bindable substance and magnetic particles to the aggregated complex containing the one kind of the substance to be measured and the specific binding substance to the reaction system ( c), a step (d) of removing the agglomerated complex to which the magnetic complex is bound from the reaction system using a magnetic force, and after the step (d), the step (b) is carried out by (n -1) Step (e) of judging to proceed to Step (a) again if less than the first time Adding a specific binding substance that specifically binds to one kind of unmeasured substance remaining in the reaction system to the reaction system; and (f) after the step (e), Measuring the optical properties of the reaction system (g).

  According to the present invention, the aggregated complex containing the substance to be measured and the specific binding substance is removed by using the magnetic force after the measurement of the optical properties. Due to the use of magnetic force, there is no chemical effect on the reaction system. For this reason, even when measuring a plurality of substances to be measured, the influence of the aggregate formed beforehand is almost eliminated, so that the reliability of the measured value is high every time the optical characteristics are measured.

  In particular, conventionally, when measuring the content of two or more kinds of substances to be measured, it is necessary to prepare the same number of reaction vessels as the kinds of the substances to be measured. If there is one reaction vessel that can be constructed, the content of two or more kinds of substances to be measured can be measured using the reaction vessel. Therefore, the sample required for measurement is very small. Therefore, for example, in the medical field, the amount of sample collected from a patient can be reduced, and the burden on the patient can be reduced.

  In the step (d), the magnetic complex not bound to the aggregated complex may be removed from the reaction system.

  The reagent kit of the present invention is a reagent kit for measuring the contents of a first analyte and a second analyte in a sample, and specifically for the first analyte. Magnetic particles to which the first specific binding substance to be bound are immobilized, and a second specific binding substance that specifically binds to the second substance to be measured.

  When the reagent kit of the present invention is used for the specific binding reaction measurement method, if there is one reaction container capable of constructing a reaction system, the contents of two or more kinds of the analytes are measured using the reaction container. be able to. Therefore, the sample required for measurement can be made very small.

  The diameter of the magnetic particles is preferably in the range of about 0.05 to 2 μm.

  The second specific binding substance is preferably composed of an antigen or antibody that specifically binds to the second analyte, and nonmagnetic particles to which the antigen or antibody is immobilized. .

  Another reagent kit of the present invention is a reagent kit for measuring the content of n kinds (n is an integer of 2 or more) of analytes in a sample, wherein One type of specific binding substance that specifically binds to one type of analyte; and specifically (n−1) types of analytes among the above n types of analytes. (N-1) types of magnetic particles to which specific binding substances to be bound are immobilized.

  When the reagent kit of the present invention is used for the specific binding reaction measurement method, if there is one reaction container capable of constructing a reaction system, the contents of two or more kinds of the analytes are measured using the reaction container. be able to. Therefore, the sample required for measurement can be made very small.

  Still another reagent kit of the present invention is a reagent kit for measuring the content of n kinds (n is an integer of 2 or more) of analytes in a sample, wherein Types of specific binding substances that specifically bind to each other, (n-1) types of analytes among the n types of analytes, and (n-1) types of specific binding substances corresponding thereto. And a magnetic composite including magnetic particles having the substance immobilized thereon.

  When the reagent kit of the present invention is used for the specific binding reaction measurement method, if there is one reaction container capable of constructing a reaction system, the contents of two or more kinds of the analytes are measured using the reaction container. be able to. Therefore, the sample required for measurement can be made very small.

  The specific binding reaction measurement device of the present invention includes a cell, a light source for irradiating light toward the cell, a photodetector for detecting scattered light or transmitted light from the reaction system, and magnetic particles in the cell. In the case where the magnetic particles are included, a removing means for removing the magnetic particles from the inside of the cell using a magnetic force is provided.

  According to the present invention, a specific binding reaction measuring apparatus which is very suitable for the above-described specific binding reaction measuring method of the present invention can be obtained.

  According to the present invention, a specific binding reaction measuring method capable of measuring two or more kinds of substances to be measured in one reaction vessel and reducing the number of samples required for the measurement, and a reagent kit and a measuring apparatus used therefor are provided. Can be provided.

  In the following embodiments, an immune reaction measurement method, which is one of the specific binding reaction measurement methods, will be described with reference to the drawings. For the sake of simplicity, components common to the embodiments are denoted by the same reference numerals.

(Embodiment 1)
FIG. 1 is a flowchart illustrating an immune reaction measurement method according to the present embodiment. FIGS. 2A to 2C are diagrams schematically showing a reaction system in each step of the immune reaction measurement method of the present embodiment.

  First, in a step St1 shown in FIG. 1, as shown in FIG. 2 (a), a sample 1 for which the content of the analyte 2 is to be measured and a specific bond which specifically binds to the analyte 2 A reaction system including the magnetic particles 4 having the substance 3 immobilized thereon is constructed. The sample 1 may be, for example, a body fluid such as blood or urine, or a mixture of these with a buffer solution. When the analyte 2 is an antigen, the specific binding substance 3 is an antibody, and when the analyte 2 is an antibody, the specific binding substance 3 is an antigen.

  As a result, when the analyte 2 is contained in the sample, as shown in FIG. 2B, the analyte 2 and the specific binding substance 3 react specifically with the analyte 2 by an antigen-antibody reaction. An aggregate 5 composed of the binding substance 3 and the magnetic particles 4 is produced. When the analyte 2 is not contained in the sample, the aggregate 5 does not occur due to the antigen-antibody reaction between the analyte 2 and the specific binding substance 3.

  Next, in step St2 shown in FIG. 1, the optical characteristics of the reaction system are measured. At this time, when the aggregated complex 5 is generated in the step St1, the reaction system becomes turbid, and the scattered light intensity and the transmitted light amount change. Therefore, the degree of turbidity of the reaction system can be estimated by measuring the intensity of scattered light or the amount of transmitted light. Here, it is preferable to measure the optical change amount of the reaction system, that is, the change amount of the scattered light intensity or the transmitted light amount, with reference to the sample 1 before measurement. When a reaction system is constructed by mixing the sample 1 with a buffer, the buffer may be used as a reference.

  Next, in step St3 shown in FIG. 1, the aggregated complex 5 composed of the substance to be measured 2, the specific binding substance 3, and the magnetic particles 4, and the magnetic particles (not shown) that do not form the aggregated complex 5 are separated. It is collected using a magnetic force (specifically, as shown in FIG. 2C, a magnet 6) and removed from the reaction system.

  In the method of measuring an immune reaction, the degree of turbidity generated in the reaction system due to the formation of an aggregation complex depends on the amount of the antigen and the amount of the antibody. Based on this, according to the present embodiment, it is possible to measure the optical characteristics of the above-mentioned reaction system and calculate the content of the substance 2 to be measured from the measured values.

  Furthermore, according to the present embodiment, after the measurement, the magnetic particles existing in the reaction system and the aggregate 5 composed of the substance 2 to be measured, the specific binding substance 3 and the magnetic particles 4 use the magnetic force. To remove from the reaction system. In the present embodiment, since the magnetic force is used, no chemical influence is exerted on the reaction system. Therefore, using the reaction system after the measurement, other components contained in the sample 1 can be measured by various measurement methods.

  Conventionally, when measuring two types of substances to be measured, it is necessary to prepare the same number of reaction vessels as the number of substances to be measured. However, according to the present embodiment, it is necessary to construct a reaction system. If there is one reaction container capable of performing the above, two kinds of substances to be measured can be measured using the reaction container. Therefore, according to the present embodiment, it is possible to make the sample required for measurement extremely small. Therefore, for example, in the medical field, the amount of sample collected from a patient can be reduced, and the burden on the patient can be reduced.

  Further, the magnetic particles are immobilized by inhibiting the antigen-antibody reaction under acidic conditions of pH 3.0 or less from the aggregated complex 5 removed from the reaction system and the magnetic particles (not shown) in the present embodiment. The recovered antibody or antigen can be recovered in a reactive state and reused. Alternatively, the antibody or antigen immobilized on the magnetic particles can be completely removed by treatment with a strong acid or alkali, and washing with a surfactant, and another antibody or antigen can be immobilized on the magnetic particles. is there.

  The magnetic particles 4 used in the immunoreaction measurement method of the present embodiment are preferably insoluble in the reaction system. Specific examples of the magnetic particles 4 include ferrite particles, ferrite colloid particles, and ferrite-containing latex particles. Such magnetic particles can be self-made. In particular, the number of bacteria is increased by culturing magnetic bacteria containing magnetic particles of 0.05 to 0.1 μm in the cells, crushing by a French press or the like, and collecting and separating the magnetic particles in the cells with a magnet. By taking it out, magnetic particles having a substantially uniform diameter can be obtained. A method for obtaining magnetic particles using magnetic bacteria is described in more detail in JP-A-2000-346843.

  Magnetic particles obtained from magnetic bacteria are covered with a lipid bilayer. Therefore, it has good dispersibility in an aqueous solution or the like, and is suitable for immobilizing a protein such as an antibody as a specific binding substance.

  The diameter of the magnetic particles 4 used in the present embodiment is changed from the viewpoint that it is easy to uniformly disperse the particles in an aqueous solution, and the amount of change in scattered light intensity or transmitted light amount for quantifying the aggregated complex generated by the antigen-antibody reaction. Is preferably 0.05 to 2 μm from the viewpoint that the detection of

  In the present embodiment, the magnet 6 is used to generate magnetic force for collecting magnetic particles, but the magnet 6 is more specifically a permanent magnet, an electromagnet, or the like.

  Any components known in the art may be added to the reaction system of the immune reaction measurement method according to the present embodiment, depending on the use and the like. For example, in order to reduce non-specific aggregation of each of the analyte 2 and the specific binding substance 3 due to self-aggregation, in this embodiment, Tween 20, octyl glucoside, sodium lauryl sulfate (SDS), sucrose Surfactants such as monolaurate or CHAPS may be added. The content of the surfactant in the reaction system is preferably 0.3% by weight or less, and more preferably 0.1% by weight or less, from the viewpoint of less inhibition of the antigen-antibody reaction.

  In the immunoreaction measurement method of the present embodiment, a change in scattered light intensity may be measured (nephelometry) as an optical property measured in step St2, and a change in the amount of transmitted light from the reaction system is measured (nephelometry). ).

  In the present embodiment, the substance to be measured is not particularly limited, and may be any substance that can be measured using an antigen-antibody reaction. For example, proteins, nucleic acids, lipids, bacteria, viruses, haptens and the like can be mentioned. In particular, the immunoreaction measurement method and reagent kit of the present embodiment are suitable for measurement of a protein to be measured, which has been conventionally measured using a antigen-antibody reaction in a clinical test. Examples of proteins include hormones such as LH (luteinizing hormone), FSH (follicle-stimulating hormone), hCG (chorionic gonadotropin), various immunoglobulin classes and subclasses, complement components, markers for various infectious diseases, CRP , Albumin, hemoglobin, rheumatoid factor, blood group antigens and the like.

  The antibody used in the present embodiment is not particularly limited as long as it can form an aggregated complex with the antigen by specifically binding to the antigen. For example, it may be any antibody class of IgG, IgM, IgE, IgA, IgD, or a mixture thereof. Further, any of a polyclonal antibody and a monoclonal antibody may be used, and a mixture thereof may be used. Of these, IgG antibodies are preferred because they have few nonspecific reactions, are relatively commercially available, and are easily available. There is no particular limitation on the animal species from which the antibody is derived, but antibodies derived from rabbits, goats and mice are preferred because they are relatively easily available and have many uses.

  In the present embodiment, as the specific binding reaction measurement method, an immunoreaction measurement method using an antigen-antibody reaction has been described, but an agglutination complex is formed using a reaction that produces a specific binding other than the antigen-antibody reaction. It is also possible to measure by performing the above. When utilizing a reaction that causes specific binding other than the antigen-antibody reaction, the combination of the analyte and the specific binding substance may be, for example, a combination of a ligand and a receptor, a single-stranded DNA (the analyte), Combination with various DNA fragments (specific binding substances) having a sequence complementary to single-stranded DNA, and the like.

  Examples of a combination of a ligand and a receptor include a combination of a molecule serving as a ligand and an allosteric protein having a plurality of binding sites for the molecule. When there is only one portion that binds to the allosteric protein in the molecule that becomes the ligand, if the molecule that becomes the ligand is immobilized on a magnetic particle, a non-magnetic particle, or the like other than the portion that binds to the allosteric protein, it becomes the ligand. An aggregated complex of the molecule and the allosteric protein can be formed.

  When a single-stranded DNA (substance to be measured) and various DNA fragments (specific binding substances) having a sequence complementary to the single-stranded DNA are used, the various DNA fragments are converted into a single-stranded DNA. What is necessary is just to immobilize to a magnetic particle in the state which can be complementarily bonded to DNA.

(Embodiment 2)
In the present embodiment, an immune reaction measuring method capable of measuring the contents of two types of substances to be measured will be described with reference to the drawings.

  FIG. 3 is a flowchart illustrating the immune reaction measurement method of the present embodiment. FIGS. 4A and 4B are diagrams schematically showing a reaction system in the immunological reaction measurement method of the present embodiment.

  First, in step St21 shown in FIG. 3, a sample whose content is to be measured for the first analyte and the second analyte is combined with a first analyte that specifically binds to the first analyte. And a magnetic particle on which the specific binding substance is immobilized. Examples of the sample include body fluids such as blood and urine as they are, or a mixture of these with a buffer. When the first analyte is an antigen, the first specific binding substance is an antibody. When the first analyte is an antibody, the first specific binding substance is an antigen.

  Thus, as shown in FIG. 4A, when the first analyte 2a is contained in the sample, the antigen-antibody of the first analyte 2a and the first specific binding substance 3a By the reaction, an aggregated complex 5a including the first substance to be measured 2a, the first specific binding substance 3a, and the magnetic particles 4a is generated. When the first analyte 2a is not contained in the sample, no aggregated complex 5a is generated by the antigen-antibody reaction between the first analyte 2a and the first specific binding substance 3a.

  Next, in step St22 shown in FIG. 3, the optical characteristics of the reaction system are measured. At this time, when the aggregate 5a is formed in the step St21, the reaction system becomes turbid, and the scattered light intensity and the transmitted light amount change. Therefore, the degree of turbidity of the reaction system can be estimated by measuring the intensity of scattered light or the amount of transmitted light. Here, it is preferable to measure the amount of optical change of the reaction system, that is, the amount of change in scattered light intensity or transmitted light amount, with reference to the sample before measurement. When a reaction system is constructed by mixing a sample with a buffer, the buffer may be used as a reference.

  Next, in step St23 shown in FIG. 3, the aggregated complex 5a including the first substance to be measured 2a, the first specific binding substance 3a, and the magnetic particles 4a, and the magnetic particles not forming the aggregated complex 5a And gather using magnetic force. At this time, the aggregated composite 5a and the magnetic particles may be removed from the reaction system, and the inside of the reaction vessel in which the reaction system is constructed so as not to hinder the measurement of the optical characteristics of the reaction system in step St25 described below. May be arranged in a concentrated manner.

  Next, in step St24 shown in FIG. 3, a second specific binding substance that specifically binds to the second analyte is added to the reaction system. For example, when the second analyte is an antigen, the second specific binding substance is an antibody, and when the second analyte is an antibody, the second specific binding substance is an antigen.

  Thus, when the second analyte is contained in the sample, the antigen-antibody reaction between the second analyte 2b and the second specific binding substance 3b as shown in FIG. 4B. Thus, an aggregate 4b composed of the second substance to be measured 2b and the second specific binding substance 3b is generated. Of course, when the second analyte 2b is not contained in the sample, the aggregated complex 4b does not occur due to the antigen-antibody reaction between the second analyte 2b and the second specific binding substance 3b.

  Next, in step St25 shown in FIG. 3, the optical characteristics of the reaction system are measured. At this time, if the aggregated composite 4b is formed in the above-described step St24, the reaction system becomes turbid, and the scattered light intensity and the transmitted light amount change. Therefore, the degree of turbidity of the reaction system can be estimated by measuring the intensity of scattered light or the amount of transmitted light. Here, it is preferable to measure the amount of optical change of the reaction system, that is, the amount of change in scattered light intensity or transmitted light amount, with reference to the sample before measurement. When a reaction system is constructed by mixing a sample with a buffer, the buffer may be used as a reference.

  In particular, in the above-described step St23, as shown in FIG. 4B, the aggregated composite 5a and the magnetic particles that do not form the aggregated composite 5a are collected using magnetic force. When measuring the optical characteristics of the reaction system, it is possible to measure changes in the scattered light intensity, the transmitted light amount, and the like caused only by the aggregated complex 4b.

  In particular, according to the present embodiment, when measuring the second substance to be measured 2b, the magnetic particles 4a existing in the reaction system, the substance to be measured 2a, the specific binding substance 3a, and the magnetic particles 4a The change in the optical characteristics of the reaction system due to the aggregated composite 5a made of is eliminated by utilizing magnetic force. Due to the use of magnetic force, there is no chemical effect on the reaction system. For this reason, according to the immune reaction measurement method of the present embodiment, the reliability of the measured value is high even when measuring the second substance to be measured 2b.

  Conventionally, when measuring the contents of two types of substances to be measured, it is necessary to prepare two reaction vessels that are the same as the type of the substance to be measured. However, according to the present embodiment, a reaction system is constructed. If there is one reaction vessel capable of performing this, it is possible to measure the contents of the two kinds of substances to be measured using the reaction vessel. Therefore, according to the present embodiment, the sample required for measurement is very small. Therefore, for example, in the medical field, the amount of sample collected from a patient can be reduced, and the burden on the patient can be reduced.

  Further, by inhibiting the antigen-antibody reaction under acidic conditions of pH 3.0 or less from the aggregated complex and the magnetic particles removed from the reaction system in the present embodiment, the magnetic particles can be immobilized on the antibody or antigen. Can be recovered in a reactive state and reused. Alternatively, treatment with a strong acid or alkali and washing with a surfactant can completely remove the antibody or antigen immobilized on the magnetic particles, and immobilize another antibody or antigen on the magnetic particles. It is.

  Next, a reagent kit used for the immunological reaction measurement method of the present embodiment will be described. This will be described with reference to FIGS.

  The reagent kit used in the immunoreaction measurement method of the present embodiment includes a magnetic particle 4a on which a first specific binding substance 3a that specifically binds to a first substance 2a is immobilized, and a second substance A second specific binding substance 3b that specifically binds to the measurement substance 2b.

  When the first analyte 2a is an antigen, the first specific binding substance 3a is an antibody. When the first analyte 2a is an antibody, the first specific binding substance 3a is an antigen. is there. When the second analyte 2b is an antigen, the second specific binding substance 3b is an antibody. When the second analyte 2b is an antibody, the second specific binding substance 3b is an antigen. is there.

  When the reagent kit of the present embodiment is used for the immunoreaction measurement method of the present embodiment, if there is one reaction container capable of constructing a reaction system, it is used to contain two types of substances to be measured. The amount can be measured. Therefore, if the reagent kit of the present embodiment is used, the sample required for the measurement can be made very small.

  In particular, as shown in FIG. 4 (b), when measuring the second substance to be measured 2b, the magnetic particles 4a, the substance to be measured 2a, the specific binding substance 3a, and the magnetic particles that were present in the reaction system. The change in the optical characteristics of the reaction system due to the aggregation complex 5a composed of 4a and 4a is eliminated by utilizing magnetic force. Due to the use of magnetic force, there is no chemical effect on the reaction system. For this reason, according to the immune reaction measurement method of the present embodiment, the reliability of the measured value is high even when measuring the second substance to be measured 2b.

  The magnetic particles 4a used in the immunoreaction measurement method and the reagent kit of the present embodiment are preferably insoluble in the reaction system. Specific examples of the magnetic particles 4a include ferrite particles, ferrite colloid particles, and ferrite-containing latex particles. Such magnetic particles can be self-made. In particular, the number of bacteria is increased by culturing magnetic bacteria containing magnetic particles of 0.05 to 0.1 μm in the cells, crushing by a French press or the like, and collecting and separating the magnetic particles in the cells with a magnet. By taking it out, magnetic particles having a substantially uniform diameter can be obtained. A method for obtaining magnetic particles using magnetic bacteria is described in more detail in JP-A-2000-346843.

  Magnetic particles obtained from magnetic bacteria are covered with a lipid bilayer. Therefore, it has good dispersibility in an aqueous solution or the like, and is suitable for immobilizing a protein such as an antibody as a specific binding substance.

  The diameter of the magnetic particles 4a used in the present embodiment is determined from the viewpoint that it is easy to disperse uniformly in an aqueous solution, and the amount of change in the intensity of scattered light or the amount of transmitted light for quantifying the aggregation complex generated by the antigen-antibody reaction. Is preferably 0.05 to 2 μm from the viewpoint that the detection of

  In the present embodiment, the magnet 6 is used to generate magnetic force for collecting magnetic particles, but the magnet 6 is more specifically a permanent magnet, an electromagnet, or the like.

  In the immunoreaction measurement method and the reagent kit of the present embodiment, the second specific binding substance 3b is, for example, an antigen or an antibody, but is not limited thereto. In particular, the second specific binding substance 3b is preferably a nonmagnetic particle on which an antigen or an antibody that causes an antigen-antibody reaction with the second analyte 2b is immobilized. Thus, the change range of the optical characteristics when measuring the second measured substance 2b (ie, step St25) is different from the change range of the optical properties when measuring the first measured substance 2a (ie, step St22). , Does not change much. For this reason, adjustment of the device used for measurement becomes easy. Another reason is that the progress of the antigen-antibody reaction of the second substance 2b is not affected at all by the magnetic force.

  Examples of the nonmagnetic particles include glass particles, graphite particles, colloidal gold particles, latex particles, and the like. Among them, latex particles are preferred in terms of high stability and easy availability of particles having various diameters, and particularly preferred are polystyrene latex particles having excellent protein adsorption.

  The above-mentioned non-magnetic particles are easy to disperse evenly in a solution, and detect changes in scattered light intensity and transmitted light amount for quantifying aggregated complexes generated by antigen-antibody reactions. Is required. Therefore, the diameter of the above-mentioned non-magnetic particles is preferably 0.05 to 2 μm, and from the viewpoint of making the measurement range using the magnetic particles 4a equal to the change range of the optical characteristics, the diameter of the magnetic particles 4a It is more preferable if the value is

  Any components known in the art may be added to the reaction system of the immune reaction measurement method according to the present embodiment, depending on the use and the like. For example, when the second specific binding substance 3b is not a nonmagnetic particle on which an antigen or an antibody is immobilized, polyethylene glycol may be added to the reaction system in step St24. In addition, in order to add polyethylene glycol to the reaction system in step St24, polyethylene glycol may be added to the reagent kit. The content of polyethylene glycol is preferably 2 to 6% by weight, more preferably 4% by weight, based on the reaction system. When polyethylene glycol having the above concentration is contained in the reaction system, non-specific aggregation due to self-aggregation of the second substance to be measured 2b and the second specific binding substance 3b is reduced, and the measurement sensitivity is improved.

  In steps St22 and St25, non-specific non-specific results due to self-aggregation of the first substance to be measured 2a, the first specific binding substance 3a, the second substance to be measured 2b, and the second specific binding substance 3b are obtained. In this embodiment, a surfactant such as Tween 20, octylglucoside, sodium lauryl sulfate (SDS), sucrose monolaurate, or CHAPS may be added to the reaction system to reduce aggregation. The content of the surfactant in the reaction system is preferably 0.3% by weight or less, and more preferably 0.1% by weight or less, from the viewpoint of less inhibition of the antigen-antibody reaction.

  In the immunoreaction measurement method of the present embodiment, a change in the intensity of scattered light may be measured (nephelometry) as the optical property measured in Step St22 and Step St25, and a change in the amount of transmitted light from the reaction system is measured ( Turbidimetry).

  In the present embodiment, the first measured substance 2a and the second measured substance 2b are not particularly limited as long as they can be measured using an antigen-antibody reaction. For example, proteins, nucleic acids, lipids, bacteria, viruses, haptens and the like can be mentioned. In particular, the immunoreaction measurement method and reagent kit of the present embodiment are suitable for measurement of a protein to be measured, which has been conventionally measured using a antigen-antibody reaction in a clinical test. Examples of proteins include hormones such as LH (luteinizing hormone), FSH (follicle-stimulating hormone), hCG (chorionic gonadotropin), various immunoglobulin classes and subclasses, complement components, markers for various infectious diseases, CRP , Albumin, hemoglobin, rheumatoid factor, blood group antigens and the like. For example, in the present embodiment, when the first analyte 2a and the second analyte 2b are human hemoglobin and human albumin, respectively, it is possible to provide a measurement result suitable for an initial screening for a kidney disease. This is because human hemoglobin in urine is an indicator of urinary tract inflammation such as acute glomerulonephritis, IgA nephropathy, renal tuberculosis, renal infarction, pyelonephritis, cystitis, urethritis, prostatitis, stone disease and tumors. The reason is that since human albumin in urine easily reflects a change in the total amount of protein in urine, renal function can be evaluated.

  The antibody used in the present embodiment is not particularly limited as long as it can form an aggregated complex with the antigen by specifically binding to the antigen. For example, it may be any antibody class of IgG, IgM, IgE, IgA, IgD, or a mixture thereof. Further, any of a polyclonal antibody and a monoclonal antibody may be used, and a mixture thereof may be used. Of these, IgG antibodies are preferred because they have few nonspecific reactions, are relatively commercially available, and are easily available. There is no particular limitation on the animal species from which the antibody is derived, but antibodies derived from rabbits, goats and mice are preferred because they are relatively easily available and have many uses.

  In the present embodiment, as the specific binding reaction measurement method, an immunoreaction measurement method using an antigen-antibody reaction has been described, but an agglutination complex is formed using a reaction that produces a specific binding other than the antigen-antibody reaction. It is also possible to measure by performing the above. When utilizing a reaction that causes specific binding other than the antigen-antibody reaction, the combination of the analyte and the specific binding substance may be, for example, a combination of a ligand and a receptor, a single-stranded DNA (the analyte), Combination with various DNA fragments (specific binding substances) having a sequence complementary to single-stranded DNA, and the like.

  Examples of a combination of a ligand and a receptor include a combination of a molecule serving as a ligand and an allosteric protein having a plurality of binding sites for the molecule. When there is only one portion that binds to the allosteric protein in the molecule that becomes the ligand, if the molecule that becomes the ligand is immobilized on a magnetic particle, a non-magnetic particle, or the like other than the portion that binds to the allosteric protein, it becomes the ligand. An aggregated complex of the molecule and the allosteric protein can be formed.

  When a single-stranded DNA (substance to be measured) and various DNA fragments (specific binding substances) having a sequence complementary to the single-stranded DNA are used, the various DNA fragments are converted into a single-stranded DNA. What is necessary is just to immobilize to magnetic particles, non-magnetic particles, etc. in the state which can be complementarily bonded to DNA.

(Embodiment 3)
In the present embodiment, an immunoreaction measurement method that is an extension of the immunoreaction measurement method of Embodiment 2 described above so that the content of various types of substances to be measured can be measured will be described with reference to the drawings.

  FIG. 5 is a flowchart illustrating the immune reaction measurement method of the present embodiment.

  First, in step St31 shown in FIG. 5, a sample whose content is to be measured for n kinds of substances (n is an integer of 2 or more) and one of unmeasured substances to be measured are specific. A reaction system comprising a magnetic particle on which a specific binding substance that binds specifically is immobilized is constructed. Examples of the sample include body fluids such as blood and urine as they are, or a mixture of these with a buffer. When the substance to be measured is an antigen, the specific binding substance is an antibody, and when the substance to be measured is an antibody, the specific binding substance is an antigen.

  Thus, when the analyte is contained in the sample, an antigen-antibody reaction between the analyte and the specific binding substance causes the same agglutination complex 5a as shown in FIG. Then, an aggregated complex composed of the substance to be measured, the specific binding substance, and the magnetic particles is generated. When the analyte is not contained in the sample, no aggregated complex is produced by the antigen-antibody reaction between the analyte and the specific binding substance.

  Next, in step St32 shown in FIG. 5, the optical characteristics of the reaction system are measured. At this time, if an aggregated complex is formed in step St31, turbidity occurs in the reaction system, and the scattered light intensity and the transmitted light amount change. Therefore, the degree of turbidity of the reaction system can be estimated by measuring the intensity of scattered light or the amount of transmitted light. Here, it is preferable to measure the amount of optical change of the reaction system, that is, the amount of change in scattered light intensity or transmitted light amount, with reference to the sample before measurement. When a reaction system is constructed by mixing a sample with a buffer, the buffer may be used as a reference.

  Next, in step St33 shown in FIG. 5, the aggregated complex comprising the substance to be measured, the specific binding substance, and the magnetic particles, and the magnetic particles not forming the aggregated complex are removed from the reaction system using magnetic force. I do.

  Next, in step St34 shown in FIG. 5, it is determined whether or not step St32 is the (n-1) th time. At this time, if the step St32 is the (n-1) -th time, the process proceeds to the step St35. If the step St32 is less than the (n-1) -th step, the process proceeds to the step St31 again. That is, the steps St31 to St33 are repeated until the above-mentioned step St32 becomes the (n-1) th time. This removes (n-1) types of analytes from the reaction system among the n types of analytes.

  Next, in step St35 shown in FIG. 5, a specific binding substance that specifically binds to one type of unmeasured substance remaining in the reaction system is added. At this time, for example, when the analyte is an antigen, the specific binding substance is an antibody, and when the analyte is an antibody, the specific binding substance is an antigen.

  Thereby, when the analyte is contained in the sample, the antigen-antibody reaction between the analyte and the specific binding substance causes the same agglutination complex 4b as shown in FIG. Then, an aggregated complex consisting of the substance to be measured and the specific binding substance is generated. Of course, when the substance to be measured is not contained in the sample, no aggregation complex is generated by the antigen-antibody reaction between the substance to be measured and the specific binding substance.

  Next, in step St36 shown in FIG. 5, the optical characteristics of the reaction system are measured. At this time, if an aggregated complex is formed in step St35, the reaction system becomes turbid, and the scattered light intensity, the transmitted light amount, and the like change. Therefore, the degree of turbidity of the reaction system can be estimated by measuring the intensity of scattered light or the amount of transmitted light. Here, it is preferable to measure the optical change amount of the reaction system, that is, the change amount of the scattered light intensity or the transmitted light amount, with reference to the sample before measurement. When a reaction system is constructed by mixing a sample with a buffer, the buffer may be used as a reference.

  In particular, in the above-described step St33, the aggregated composite and the magnetic particles that do not form the aggregated composite are collected using magnetic force. Therefore, in this step, when measuring the optical characteristics of the reaction system, In addition, changes in the intensity of scattered light and the amount of transmitted light due to only the aggregated complex can be measured.

  According to the present embodiment, the aggregate of the substance to be measured, the specific binding substance, and the magnetic particles is removed by using the magnetic force after the measurement of the optical characteristics. Due to the use of magnetic force, there is no chemical effect on the reaction system. For this reason, even when measuring a plurality of substances to be measured, the influence of the aggregate formed beforehand is almost eliminated, so that the reliability of the measured value is high every time the optical characteristics are measured.

  In particular, conventionally, when measuring the contents of two or more types of substances to be measured, it is necessary to prepare the same number of reaction vessels as the number of types of the substances to be measured. If there is one reaction vessel capable of constructing the above, the content of two or more kinds of substances to be measured can be measured using the reaction vessel. Therefore, according to the present embodiment, the sample required for measurement is very small. Therefore, for example, in the medical field, the amount of sample collected from a patient can be reduced, and the burden on the patient can be reduced.

  Further, by inhibiting the antigen-antibody reaction under acidic conditions of pH 3.0 or less from the aggregated complex and the magnetic particles that are removed from the reaction system in the present embodiment, the magnetic particles can be used to fix the immobilized antibody or antigen. Can be recovered in a reactive state and reused. Alternatively, treatment with a strong acid or alkali and washing with a surfactant can completely remove the antibody or antigen immobilized on the magnetic particles, and immobilize another antibody or antigen on the magnetic particles. It is.

  Next, a reagent kit used for the immunological reaction measurement method of the present embodiment will be described.

  The reagent kit used in the immunoreaction measurement method according to the present embodiment includes a magnetic particle on which a specific binding substance that specifically binds to each of the (n-1) kinds of substances to be measured is immobilized, and the remaining one kind is used. And a specific binding substance that specifically binds to the unmeasured analyte.

  When the reagent kit of the present embodiment is used for the immunoreaction measurement method of the present embodiment, if there is one reaction container capable of constructing a reaction system, two or more types of the analytes can be used using the same. The content can be measured. Therefore, if the reagent kit of the present embodiment is used, the sample required for the measurement can be made very small.

  As the magnetic particles used in the immunoreaction measurement method and the reagent kit of the present embodiment and the magnet 6 for collecting the magnetic particles, exactly the same as those described in the second embodiment can be used.

  Further, in the immunoreaction measurement method and the reagent kit of the present embodiment, the specific binding substance to be added in step St35 shown in FIG. 5 generates an antigen-antibody reaction with one type of unmeasured analyte remaining in the reaction system. It is preferable that the antigen or antibody is immobilized non-magnetic particles. As a result, the change range of the optical characteristics in the measurement of the substance to be measured in the step St36 does not significantly change from the change range of the optical properties in the measurement of the (n-1) types of the substances to be measured in the step St32. For this reason, adjustment of the device used for measurement becomes easy.

  As the non-magnetic particles, exactly the same ones as described in the second embodiment can be used. That is, the diameter of the non-magnetic particles is preferably 0.05 to 2 μm, and more preferably the same as the diameter of the magnetic particles.

  Any components known in the art may be added to the reaction system of the immune reaction measurement method according to the present embodiment, depending on the use and the like. For example, when the specific binding substance is not a nonmagnetic particle on which an antigen or an antibody is immobilized, polyethylene glycol may be added to the reaction system in step St35. Further, in order to add polyethylene glycol to the reaction system in step St35, polyethylene glycol may be added to the reagent kit. The content of polyethylene glycol is preferably 2 to 6% by weight, more preferably 4% by weight, based on the reaction system. When polyethylene glycol having the above concentration is contained in the reaction system, non-specific aggregation due to self-aggregation of the substance to be measured or the specific binding substance is reduced in step St36, and the measurement sensitivity is improved.

  In step St32 and step St36, in order to reduce non-specific aggregation of each analyte and each specific binding substance due to self-aggregation, in the present embodiment, Tween 20, octyl glucoside, sodium lauryl sulfate are added to the reaction system. A surfactant such as (SDS), sucrose monolaurate or CHAPS may be added. The content of the surfactant in the reaction system is preferably 0.3% by weight or less, and more preferably 0.1% by weight or less, from the viewpoint of less inhibition of the antigen-antibody reaction.

  In the immunoreaction measurement method of the present embodiment, a change in scattered light intensity may be measured (nephelometry) as the optical property measured in Step St32 and Step St36, and a change in the amount of transmitted light from the reaction system is measured ( Turbidimetry).

  In the present embodiment, each of the n kinds of substances to be measured is not particularly limited, and may be any substance that can be measured using an antigen-antibody reaction. For example, proteins, nucleic acids, lipids, bacteria, viruses, haptens and the like can be mentioned. In particular, the immunoreaction measurement method and reagent kit of the present embodiment are suitable for measurement of a protein to be measured, which has been conventionally measured using a antigen-antibody reaction in a clinical test. Examples of proteins include hormones such as LH (luteinizing hormone), FSH (follicle-stimulating hormone), hCG (chorionic gonadotropin), various immunoglobulin classes and subclasses, complement components, markers for various infectious diseases, CRP , Albumin, hemoglobin, rheumatoid factor, blood group antigens and the like.

  The antibody used in the present embodiment is not particularly limited as long as it can form an aggregated complex with the antigen by specifically binding to the antigen. For example, it may be any antibody class of IgG, IgM, IgE, IgA, IgD, or a mixture thereof. Further, any of a polyclonal antibody and a monoclonal antibody may be used, and a mixture thereof may be used. Of these, IgG antibodies are preferred because they have few nonspecific reactions, are relatively commercially available, and are easily available. There is no particular limitation on the animal species from which the antibody is derived, but antibodies derived from rabbits, goats and mice are preferred because they are relatively easily available and have many uses.

  In the present embodiment, as the specific binding reaction measurement method, an immunoreaction measurement method using an antigen-antibody reaction has been described, but an agglutination complex is formed using a reaction that produces a specific binding other than the antigen-antibody reaction. It is also possible to measure by performing the above. When utilizing a reaction that causes specific binding other than the antigen-antibody reaction, the combination of the analyte and the specific binding substance may be, for example, a combination of a ligand and a receptor, a single-stranded DNA (the analyte), Combination with various DNA fragments (specific binding substances) having a sequence complementary to single-stranded DNA, and the like.

  Examples of a combination of a ligand and a receptor include a combination of a molecule serving as a ligand and an allosteric protein having a plurality of binding sites for the molecule. When there is only one portion that binds to the allosteric protein in the molecule that becomes the ligand, if the molecule that becomes the ligand is immobilized on a magnetic particle, a non-magnetic particle, or the like other than the portion that binds to the allosteric protein, it becomes the ligand. An aggregated complex of the molecule and the allosteric protein can be formed.

  When a single-stranded DNA (substance to be measured) and various DNA fragments (specific binding substances) having a sequence complementary to the single-stranded DNA are used, the various DNA fragments are converted into a single-stranded DNA. What is necessary is just to immobilize to magnetic particles, non-magnetic particles, etc. in the state which can be complementarily bonded to DNA.

(Embodiment 4)
In the present embodiment, another method for measuring an immune reaction that can measure the contents of various types of substances to be measured will be described with reference to the drawings.

  FIG. 6 is a flowchart illustrating the immune reaction measurement method according to the present embodiment. FIGS. 7A and 7B are diagrams schematically illustrating a reaction system in the immune reaction measurement method of the present embodiment.

  First, in step St41 shown in FIG. 6, a sample whose content is to be measured for n kinds of substances (n is an integer of 2 or more) and one of unmeasured substances to be measured are specific. A reaction system containing a specific binding substance that binds specifically is constructed. Examples of the sample include body fluids such as blood and urine. When the substance to be measured is an antigen, the specific binding substance is an antibody, and when the substance to be measured is an antibody, the specific binding substance is an antigen.

  As a result, as shown in FIG. 7A, when one kind of the substance to be measured 2a is contained in the sample, the substance to be measured is caused by an antigen-antibody reaction between the substance to be measured 2a and the specific binding substance 3a. An aggregate 5a composed of 2a and the specific binding substance 3a is generated. When the test substance 2a is not contained in the sample, the aggregate 5a does not occur due to the antigen-antibody reaction between the test substance 2a and the specific binding substance 3a.

  Next, in step St42 shown in FIG. 6, the optical characteristics of the reaction system are measured. At this time, if an aggregated complex is formed in the step St41, the reaction system becomes turbid, and the scattered light intensity and the transmitted light amount change. Therefore, the degree of turbidity of the reaction system can be estimated by measuring the intensity of scattered light or the amount of transmitted light. Here, it is preferable to measure the amount of optical change of the reaction system, that is, the amount of change in scattered light intensity or transmitted light amount, with reference to the sample before measurement. When a reaction system is constructed by mixing a sample with a buffer, the buffer may be used as a reference.

  Next, in step St43 shown in FIG. 6, a substance capable of binding to the aggregation complex comprising the one kind of the substance to be measured and the specific binding substance (for example, an antibody capable of binding to an antibody contained in the aggregation complex) And a magnetic composite comprising magnetic particles are added to the reaction system. As a result, as shown in FIG. 7 (b), the magnetic complex 8 composed of the antibody 7 and the magnetic particles 4c binds to the aggregation complex 5c.

  Next, in step St44 shown in FIG. 6, the aggregated complex to which the magnetic composite has been bonded and the magnetic composite that has not been bonded to the aggregated composite are removed using magnetic force. As a result, as shown in FIG. 7 (b), the aggregated complex 5c is attracted to and removed by the magnetic force together with the magnetic complex 8, and another substance to be measured 2b remains in the reaction system.

  Next, in a step St45 shown in FIG. 6, it is determined whether or not the step St42 is the (n-1) th time. At this time, if the above step St42 is the (n-1) th time, the process proceeds to step St46. If the above step St42 is less than the (n-1) th step, the process proceeds to step St41 again. That is, the steps St41 to St44 are repeated until the above-mentioned step St42 becomes the (n-1) th time. This removes (n-1) types of analytes from the reaction system among the n types of analytes.

  Next, in step St46 shown in FIG. 6, a specific binding substance that specifically binds to one type of unmeasured analyte remaining in the reaction system is added. At this time, for example, when the analyte is an antigen, the specific binding substance is an antibody, and when the analyte is an antibody, the specific binding substance is an antigen.

  Thereby, when the analyte is contained in the sample, the antigen-antibody reaction between the analyte and the specific binding substance causes the same agglutination complex 4b as shown in FIG. Then, an aggregated complex consisting of the substance to be measured and the specific binding substance is generated. Of course, when the substance to be measured is not contained in the sample, no aggregation complex is generated by the antigen-antibody reaction between the substance to be measured and the specific binding substance.

  Next, in step St46 shown in FIG. 6, the optical characteristics of the reaction system are measured. At this time, if an aggregated complex is formed in step St45, the reaction system becomes turbid, and the scattered light intensity, the transmitted light amount, and the like change. Therefore, the degree of turbidity of the reaction system can be estimated by measuring the intensity of scattered light or the amount of transmitted light. Here, it is preferable to measure the amount of optical change of the reaction system, that is, the amount of change in scattered light intensity or transmitted light amount, with reference to the sample before measurement. When a reaction system is constructed by mixing a sample with a buffer, the buffer may be used as a reference.

  In particular, in the above-mentioned step St44, since the aggregated composite and the magnetic composite are collected using magnetic force, in this step, when measuring the optical characteristics of the reaction system, scattering by only the aggregated composite is performed. Changes in light intensity and transmitted light amount can be measured.

  According to the present embodiment, the aggregate of the substance to be measured and the specific binding substance is removed by using the magnetic force after the measurement of the optical properties. Due to the use of magnetic force, there is no chemical effect on the reaction system. For this reason, even when measuring a plurality of substances to be measured, the influence of the aggregate formed beforehand is almost eliminated, so that the reliability of the measured value is high every time the optical characteristics are measured.

  In particular, conventionally, when measuring the contents of two or more types of substances to be measured, it is necessary to prepare the same number of reaction vessels as the number of types of the substances to be measured. If there is one reaction vessel capable of constructing the above, the content of two or more kinds of substances to be measured can be measured using the reaction vessel. Therefore, according to the present embodiment, the sample required for measurement is very small. Therefore, for example, in the medical field, the amount of sample collected from a patient can be reduced, and the burden on the patient can be reduced.

  Further, the magnetic complex removed from the reaction system in the present embodiment inhibits the antigen-antibody reaction under acidic conditions of pH 3.0 or less, so that the magnetic particles immobilize the antibody or antigen immobilized thereon. It can be collected and reused as it is. Alternatively, treatment with a strong acid or alkali and washing with a surfactant can completely remove the antibody or antigen immobilized on the magnetic particles, and immobilize another antibody or antigen on the magnetic particles. It is.

  Next, a reagent kit used for the immunological reaction measurement method of the present embodiment will be described.

  The reagent kit used in the immunological reaction measurement method of the present embodiment includes a specific binding substance that specifically binds to each of n kinds of analytes, (n-1) kinds of analytes, and a specific substance corresponding thereto. A magnetic complex comprising a substance (eg, a secondary antibody capable of binding to an antibody contained in the aggregate) which can bind to each of the aggregates comprising the binding substance, and magnetic particles having the substance immobilized thereon And

  When the reagent kit of the present embodiment is used for the immunoreaction measurement method of the present embodiment, if there is one reaction container capable of constructing a reaction system, two or more types of the analytes can be used using the same. The content can be measured. Therefore, if the reagent kit of the present embodiment is used, the sample required for the measurement can be made very small.

  As the magnetic particles used in the immunoreaction measurement method and the reagent kit of the present embodiment and the magnet 6 for collecting the magnetic particles, exactly the same as those described in the second embodiment can be used.

  The magnetic complex capable of specifically binding to the aggregated complex used in the present embodiment includes an antibody that specifically binds to the antibody used to generate each aggregated complex and the formation of the aggregated complex. An antibody that specifically binds to a different place from the antibody used for the above, or an antibody or the like that specifically binds to a specific structure of the aggregation complex is immobilized on magnetic particles.

  In the immunoreaction measurement method and the reagent kit of the present embodiment, the specific binding substance to be added in step St46 shown in FIG. 6 causes an antigen-antibody reaction with one type of unmeasured analyte remaining in the reaction system. It is preferable that the antigen or antibody is immobilized non-magnetic particles. As a result, in the step St47, the change range of the optical characteristics in the measurement of the substance to be measured does not significantly change from the change range of the optical properties in the measurement of the (n-1) types of the substances to be measured in the step St42. For this reason, adjustment of the device used for measurement becomes easy.

  As the non-magnetic particles, exactly the same ones as described in the second embodiment can be used. That is, the diameter of the non-magnetic particles is preferably 0.05 to 2 μm, and more preferably the same as the diameter of the magnetic particles.

  Any components known in the art may be added to the reaction system of the immune reaction measurement method according to the present embodiment, depending on the use and the like. For example, when the specific binding substance is not a nonmagnetic particle on which an antigen or an antibody is immobilized, polyethylene glycol may be added to the reaction system in step St46. In addition, in order to add polyethylene glycol to the reaction system in step St46, polyethylene glycol may be added to the reagent kit. The content of polyethylene glycol is preferably 2 to 6% by weight, more preferably 4% by weight, based on the reaction system. When polyethylene glycol having the above concentration is contained in the reaction system, non-specific aggregation due to self-aggregation of the substance to be measured or the specific binding substance is reduced in step St47, and the measurement sensitivity is improved.

  In step St42 and step St47, in order to reduce non-specific aggregation due to self-aggregation of each analyte and each specific binding substance, in the present embodiment, Tween 20, octyl glucoside, sodium lauryl sulfate, A surfactant such as (SDS), sucrose monolaurate or CHAPS may be added. The content of the surfactant in the reaction system is preferably 0.3% by weight or less, and more preferably 0.1% by weight or less, from the viewpoint of less inhibition of the antigen-antibody reaction.

  In the immunoreaction measurement method of the present embodiment, a change in the intensity of scattered light may be measured (nephelometry) as the optical property measured in Step St42 and Step St47, and a change in the amount of transmitted light from the reaction system is measured ( Turbidimetry).

  In the present embodiment, each of the n kinds of substances to be measured is not particularly limited, and may be any substance that can be measured using an antigen-antibody reaction. For example, proteins, nucleic acids, lipids, bacteria, viruses, haptens and the like can be mentioned. In particular, the immunoreaction measurement method and reagent kit of the present embodiment are suitable for measurement of a protein to be measured, which has been conventionally measured using a antigen-antibody reaction in a clinical test. Examples of proteins include hormones such as LH (luteinizing hormone), FSH (follicle-stimulating hormone), hCG (chorionic gonadotropin), various immunoglobulin classes and subclasses, complement components, markers for various infectious diseases, CRP , Albumin, hemoglobin, rheumatoid factor, blood group antigens and the like.

  The antibody used in the present embodiment is not particularly limited as long as it can form an aggregated complex with the antigen by specifically binding to the antigen. For example, it may be any antibody class of IgG, IgM, IgE, IgA, IgD, or a mixture thereof. Further, any of a polyclonal antibody and a monoclonal antibody may be used, and a mixture thereof may be used. Of these, IgG antibodies are preferred because they have few nonspecific reactions, are relatively commercially available, and are easily available. There is no particular limitation on the animal species from which the antibody is derived, but antibodies derived from rabbits, goats and mice are preferred because they are relatively easily available and have many uses.

  In the present embodiment, as the specific binding reaction measurement method, an immunoreaction measurement method using an antigen-antibody reaction has been described, but an agglutination complex is formed using a reaction that produces a specific binding other than the antigen-antibody reaction. It is also possible to measure by performing the above. When utilizing a reaction that causes specific binding other than the antigen-antibody reaction, the combination of the analyte and the specific binding substance may be, for example, a combination of a ligand and a receptor, a single-stranded DNA (the analyte), Combination with various DNA fragments (specific binding substances) having a sequence complementary to single-stranded DNA, and the like.

  Examples of a combination of a ligand and a receptor include a combination of a molecule serving as a ligand and an allosteric protein having a plurality of binding sites for the molecule. When there is only one portion that binds to the allosteric protein in the molecule that becomes the ligand, if the molecule that becomes the ligand is immobilized on a magnetic particle, a non-magnetic particle, or the like other than the portion that binds to the allosteric protein, it becomes the ligand. An aggregated complex of the molecule and the allosteric protein can be formed.

  When a single-stranded DNA (substance to be measured) and various DNA fragments (specific binding substances) having a sequence complementary to the single-stranded DNA are used, the various DNA fragments are converted into a single-stranded DNA. What is necessary is just to immobilize to magnetic particles, non-magnetic particles, etc. in the state which can be complementarily bonded to DNA.

(Embodiment 5)
In the present embodiment, a measuring apparatus for performing the method for measuring an immune reaction according to the first to fourth embodiments will be described with reference to the drawings.

  First, the measuring device 100 will be described with reference to FIG. FIG. 8 is a schematic diagram illustrating a configuration of the measuring apparatus 100 according to the present embodiment.

  As shown in FIG. 8, the measuring apparatus 100 of the present embodiment includes a light source 101, a cell (reaction vessel) 102, a cell holder 103 holding the cell 102, and a photodetector 104 detecting light from the cell 102. , Removing means 108.

  The light source 101 is arranged so that emitted light is directed to the cell 102.

  The light detector 104 detects scattered light or transmitted light from the reaction system built in the cell 102.

  The removing means 108 is for collecting and removing magnetic particles, magnetic composites, aggregated composites, and the like from within the reaction system by using magnetic force in the first to fourth embodiments. In the measuring apparatus 100, the removing means 108 includes a permanent magnet 105, a cover 106 for protecting the permanent magnet 105, and an arm 107 provided with the permanent magnet 105 and the cover 106.

  Here, the operation of the removing means 108 will be described with reference to FIG. FIG. 9 is a diagram illustrating an arrangement relationship between the cell 102 and the removing unit 108 in the measuring apparatus 100 according to the present embodiment.

  As shown in FIG. 9, the arm 107 can lower the permanent magnet 105 and the cover 106, insert the permanent magnet 105 and the cover 106 into the inside of the cell 102, raise the arm 107, and pull up the arm 107 from the inside of the cell 102. Therefore, the permanent magnet 105 and the cover 106 are introduced into the reaction system, and the magnetic particles, the magnetic composite, the aggregated composite, and the like in the reaction system adhere to the surface of the cover 106. Next, when the turbidity of the reaction system has almost disappeared, the reaction system is pulled out of the cell 102. Thus, magnetic particles, magnetic composites, aggregated composites, and the like can be collected and removed from the reaction system. The magnetic particles, the magnetic composite, the aggregated composite, and the like attached to the cover 106 may be removed by a mechanical method. For example, after the permanent magnet 105 is pulled out from the cover 106, the collected magnetic particles attached to the cover 106 may be removed by washing or the like, or the cover 106 may be made disposable. When an electromagnet is used instead of the permanent magnet 105, collection and removal of the magnetic particles may be performed by turning on and off the generation of the magnetic force of the electromagnet.

  Next, the measuring device 200 will be described with reference to FIG. FIG. 10 is a schematic diagram illustrating a configuration of the measuring device 200 of the present embodiment.

  As shown in FIG. 10, the measuring apparatus 200 of the present embodiment detects a light source 201, a cell (reaction vessel) 202 having an outlet 202a, a cell holder 203 holding the cell 202, and light from the cell 202. It includes a photodetector 204 and a removing unit 208.

  The light source 201 is arranged so that emitted light is directed to the cell 202.

  The light detector 204 detects scattered light or transmitted light from a reaction system built in the cell 202.

  The removing means 208 is for collecting and removing magnetic particles, magnetic composites, aggregated composites, and the like from within the reaction system by using magnetic force in the first to fourth embodiments. In the measuring device 200, the removing means 208 includes an electromagnet 205, a container 206 for storing the discharge from the outlet 202a of the cell 202, and a valve 207 which is connected to the outlet 202a of the cell 202 and can be opened and closed. Is provided.

  Here, the operation of the removing means 208 will be described with reference to FIG. FIG. 11 is a diagram illustrating an arrangement relationship between the cell 202 and the removing unit 208 in the measuring apparatus 200 according to the present embodiment.

  The valve 207 is connected to the outlet 202a of the cell 202 as shown in FIG. When the electromagnet 205 is energized in this state, the magnetic particles, the magnetic composite, the aggregated composite, and the like in the reaction system in the cell 202 move from the outlet 202 a to the container 206 through the valve 207. Next, when the turbidity of the reaction system has almost disappeared, the valve 207 is closed, and the power supply to the electromagnet 205 is stopped. Thus, magnetic particles, magnetic composites, aggregated composites, and the like can be collected and removed from the reaction system. Note that the magnetic particles, the magnetic composite, the aggregated composite, and the like stored in the container 206 may be removed by washing. Alternatively, the container 206 may be disposable.

  Note that, in the measurement device 200, a discharge pipe connected to the cell 202 may be provided instead of the container 206.

  Further, in the measuring device 200, a removing means 208 'shown in FIG. As shown in FIG. 12, the removing means 208 ′ includes a permanent magnet 105, an arm 107 holding the permanent magnet 105, a container 206 for storing the discharge from the outlet 202 a of the cell 202, A valve 207 that is connected to the outlet 202a and that can be opened and closed is provided.

  The arm 107 can move the permanent magnet 105 up and down as shown in FIG. Therefore, the permanent magnet 105 is brought close to the container 206, and the magnetic particles, the magnetic composite, the aggregated composite, and the like in the reaction system are discharged into the container 206. Next, when the turbidity of the reaction system has almost disappeared, the valve 207 is closed, and the permanent magnet 105 is moved away from the container 206. Thus, magnetic particles, magnetic composites, aggregated composites, and the like can be collected and removed from the reaction system. Note that the magnetic particles, the magnetic composite, the aggregated composite, and the like stored in the container 206 may be removed by washing. Alternatively, the container 206 may be disposable.

  When the removing means 108 and 208 'including the permanent magnet 105 are used, when measuring the optical characteristics of the reaction system, the permanent magnet 105 is kept in a standby position away from the cell 202, and the cell 202 It is preferable to operate the removing means 108 and 208 'so that the magnetic particles, the magnetic composite, the aggregated composite and the like can be collected and removed.

  When the removing unit 108 including the electromagnet 205 is used, the electromagnet 205 can be arranged near the cell 202. This is because the electromagnet 205 hardly generates a magnetic force when no current is supplied. Therefore, when measuring the optical characteristics of the reaction system, a magnetic force is not generated due to non-energization, but a magnetic force is generated by energization after the measurement is completed, and the magnetic particles, magnetic composite, aggregated composite, etc. in the cell 202 are collected. Preferably, the removal means 108 and 208 'are operated so that they can be removed. In any of the above cases, the influence of the magnetic force on the antigen-antibody reaction can be reduced as much as possible when measuring the optical characteristics of the reaction system.

  At this time, it is preferable that the inside of the container 206 is filled in advance with the same buffer as the reaction system. Thus, the change in the volume of the reaction system in the cell 202 can be reduced. Further, since the change in the volume of the reaction system in the cell 202 is small, only the volume of the reagent added in the measurement needs to be considered when measuring the optical characteristics of the reaction system. Therefore, it becomes easy to estimate the concentration of the substance to be measured from the measured values of the optical characteristics.

  Hereinafter, embodiments of the present invention will be described in detail. The following examples do not limit the present invention.

  In this example, the first analyte in the second embodiment was human hemoglobin, the second analyte was human albumin, the first specific binding substance was an anti-human hemoglobin antibody, and the second specific binding was The method of constructing the reagent when the substance is an anti-human albumin antibody and the second specific binding substance is not immobilized on non-magnetic particles will be described.

  In addition, pure water filtered with Milli-Q SP TOC (manufactured by Millipore) was used for the preparation of the buffer solution and the like shown in this example. In addition, reagents such as salts and buffers not particularly described were obtained from Wako Pure Chemical Industries, and polyethylene glycol 6,000 used a primary reagent, and the other reagents used a special reagent.

  An anti-human hemoglobin antibody as the first specific binding substance and an anti-human albumin antibody as the second specific binding substance were prepared as follows.

  First, an anti-human albumin antibody was prepared by purifying an IgG fraction using anti-serum collected from a rabbit immunized with human albumin (manufactured by Wako Pure Chemical Industries) using protein A column chromatography. The protein A-immobilized gel packed in the column was manufactured by Amersham Pharmacia. The equilibration buffer used for the purification had a composition of 1.5 M glycine, 3.0 M NaCl, pH 8.9, and the elution buffer had a composition of 0.1 M citric acid, pH 4.0. One used.

Purification was performed as follows. After equilibrating the column by flowing an equilibration buffer 5 times the gel volume packed in the column, the antiserum containing 10 to 20% of the antibody in the total binding volume of the column is doubled in volume with the equilibration buffer. After dilution and flowing over the column, the antibody in the serum was bound to protein A. Subsequently, the equilibration buffer was allowed to flow until serum components not adsorbed to protein A did not come out of the column, and the column was washed. Subsequently, an elution buffer was applied to the column to elute the antibody bound to protein A. The eluted antibody fraction was placed in a dialysis tube having a molecular weight cut off of 10,000, and about 100-fold volume of 0.05M 3- (N-morpholino) propanesulfonic acid (manufactured by Dojin, hereinafter abbreviated as mops), 0.15M NaCl , 0.04% by weight of NaN ₃ , pH 7.4, was dialyzed several times to replace the buffer components. Subsequently, the antibody concentration was estimated by measuring the absorbance at 280 nm, and adjusted with the same buffer solution used for dialysis to give an antibody concentration of 3.0 mg / ml, which was used as an anti-human albumin antibody solution. The antibody concentration is not particularly limited to this. Although the prepared antibody solution can be stored at room temperature, it is preferably stored at a lower temperature, more preferably at 4 ° C., from the viewpoint of preventing denaturation of the antibody.

The anti-human hemoglobin antibody is obtained by purifying an IgG fraction using protein G column chromatography from antiserum collected from a goat immunized with human hemoglobin (SIGMA, Cat. No. H-7379), immobilizing the fraction on magnetic particles. Prepared. The protein G-immobilized gel packed in the column used for the purification of the IgG fraction was manufactured by Amersham Pharmacia. The equilibration buffer used for the purification was 0.02 M Na ₂ HPO ₄ —NaH ₂ PO ₄ , pH 7.0, and the elution buffer was 0.1 M glycine, pH 2.7. The composition was used. The composition of the buffer used for the dialysis was 0.05 M mop, 0.15 M NaCl, 0.04 wt% NaN ₃ , pH 7.4. As for the purification operation by column chromatography and the method of replacing the buffer by dialysis, the same method as in the preparation of the anti-human albumin antibody was used.

  Subsequently, the antibody concentration was estimated by measuring the absorbance at 280 nm, and adjusted with the same buffer as used in the dialysis to adjust the antibody concentration to 3.0 mg / ml, which was immobilized on the magnetic particles of the anti-human hemoglobin antibody. Anti-human hemoglobin antibody solution.

  Although magnetic particles can be prepared, commercially available magnetic particles can also be used. In this example, Polystyrene Superparamagnetic Microspheres, 1-2 μm (Cat. No. 18190) manufactured by Polysciences, which is a polystyrene latex particle containing iron having a particle diameter of 1 to 2 μm, was used. The immobilization of the antibody on the magnetic particles was performed as follows.

0.5 ml of 2.5% by weight magnetic particles was placed in a microtube, and 0.5 ml of 0.05 M mopse, 0.04% by weight of NaN ₃ , pH 7.4 buffer was added thereto to suspend the magnetic particles, This was centrifuged at 12,000 rpm for 30 minutes using a centrifuge (manufactured by Tommy Seiko, model number MRX-150), and the magnetic particles were sedimented to remove the supernatant.

Subsequently, 1 ml of a buffer solution of 0.05 M mop, 0.04% by weight of NaN ₃ , pH 7.4 was added to suspend the magnetic particles, and the suspension was centrifuged at 12,000 rpm for 30 minutes to precipitate the magnetic particles. The supernatant was removed. The suspension of the magnetic particles and the sedimentation by centrifugation were repeated again.

Further, 0.9 ml of a buffer solution of 0.05 M mopse, 0.04 wt% NaN ₃ , pH 7.4 was added to suspend the magnetic particles, and 0.1 ml of the anti-human hemoglobin antibody against the magnetic particles prepared above The solution was added. This was stirred overnight at room temperature with a rotator (manufactured by TAITEC, model number RT-50). After stirring, the mixture was centrifuged at 12,000 rpm for 30 minutes, and the magnetic particles were again sedimented to remove the supernatant. A buffer having a composition of 0.05 M mopse, 1% by weight of sodium caseinate, 0.04% by weight of NaN ₃ and a pH of 7.4 was added to suspend the magnetic particles, and the suspension was stirred with a rotator for 30 minutes. After stirring, the mixture was centrifuged at 12,000 rpm for 30 minutes to precipitate magnetic particles. The suspension of the magnetic particles and the sedimentation by centrifugation were repeated a total of three times to block the surface of the magnetic particles on which the antibody was not immobilized.

Further, a buffer having a composition of 0.05 M mop, 0.15 M NaCl, 0.04 wt% NaN ₃ , 5 vol% glycerol, and pH 7.4 was added to suspend the magnetic particles. The antibody concentration and the magnetic particle concentration used for immobilization are not limited to the above. Although the prepared antibody solution can be stored at room temperature, it is preferably stored at a lower temperature, more preferably at 4 ° C., from the viewpoint of preventing denaturation of the antibody.

In addition, in order to measure human hemoglobin, a first buffer having a composition of 0.05 M mop, 0.04% by weight of NaN ₃ , pH 7.4 was prepared as a first buffer added to the reaction system, and human albumin was measured. As a second buffer to be added to the reaction system, a composition having a composition of 0.05 M mop, 8.6% by weight of polyethylene glycol 6,000, 0.04% by weight of NaN ₃ , and pH 7.4 was prepared. Each prepared buffer was stored at room temperature.

  By combining the magnetic particles to which the anti-human hemoglobin antibody (first specific binding substance) configured as described above is immobilized, an anti-human albumin antibody (second specific binding substance), and each buffer, An immunoreaction reagent can be configured.

  The method of using the above-configured reagent will be described below. First, a sample containing two kinds of analytes (human hemoglobin and human albumin), magnetic particles on which an anti-human hemoglobin antibody (first specific binding substance) is immobilized, and a first buffer are mixed. To construct a reaction system. Next, the optical characteristics of the reaction system were measured. After completion of the measurement, an aggregated complex composed of human hemoglobin (first analyte), an anti-human hemoglobin antibody (first specific binding substance) and magnetic particles, and a magnetic particle not forming an aggregated complex Was collected by magnetic force to eliminate the turbidity of the reaction system.

  Subsequently, an anti-human albumin antibody (second specific binding substance) solution and a second buffer were mixed in the reaction system. Thereafter, the optical characteristics of the reaction system were measured. By the above procedure, the concentration of the substance to be measured in the sample could be known.

  Although not shown in this example, an anti-albumin antibody (second specific binding substance) may be immobilized on non-magnetic particles such as latex or colloidal gold. For example, as a method of immobilizing the particles on latex particles, the method used for the magnetic particles described above can be applied as it is.

  In this example, the particle surface on which the antibody was not immobilized was blocked with sodium caseinate, but gelatin, skim milk or the like can be used instead. The concentration used may be the same as for sodium caseinate.

  Further, in this example, in order to construct a reaction system, a buffer was prepared for each substance to be measured. However, each reaction system was constituted by one type of buffer to which a water-soluble polymer such as polyethylene glycol was added. May be.

  Further, the buffer component and the pH of the antibody solution are not limited to the above composition and pH as long as they do not adversely affect the immune reaction due to the binding between the antigen and the antibody.

  In order to remove hemoglobin contained in red blood cells, a hemolytic agent such as potassium chloride may be added to the reaction system, or a solution containing the hemolytic agent may be added to the reagent kit.

  As described above, two kinds of substances to be measured could be measured in one reaction container by the immunoreaction measurement method and the reagent kit of the present invention.

  INDUSTRIAL APPLICABILITY The specific binding reaction measuring method, the reagent kit and the specific binding reaction measuring device of the present invention are useful for diagnosing various diseases and examining the progress of disease states. For example, if the configuration of the present invention is configured to measure human hemoglobin and human albumin from one sample, it is useful for diagnosis of kidney disease.

FIG. 1 is a flowchart illustrating an immune reaction measurement method according to one embodiment of the present invention. FIGS. 2A to 2C are diagrams schematically showing a reaction system in each step of the immunoreaction measurement method shown in FIG. FIG. 3 is a flowchart illustrating an immune reaction measurement method according to another embodiment of the present invention. FIGS. 4A and 4B are diagrams schematically showing a reaction system in the immunoreaction measurement method shown in FIG. FIG. 5 is a flowchart showing a method for measuring an immune response according to still another embodiment of the present invention. FIG. 6 is a flowchart illustrating a method for measuring an immune response according to still another embodiment of the present invention. FIGS. 7A and 7B are diagrams schematically showing a reaction system in the immunoreaction measurement method shown in FIG. FIG. 8 is a schematic diagram illustrating a configuration of a measurement device according to one embodiment of the present invention. FIG. 9 is a diagram showing a positional relationship of a part of the measuring device of FIG. FIG. 10 is a schematic diagram illustrating a configuration of a measurement device according to another embodiment of the present invention. FIG. 11 is a diagram showing an arrangement relationship of a part of the measuring device of FIG. FIG. 12 is a diagram showing a positional relationship of a part of the measuring device of FIG.

Explanation of reference numerals

1 Sample 2 Substance to be measured 2a First substance to be measured 2b Second substance to be measured 3 Specific binding substance 3a First specific binding substance 3b Second specific binding substance 4, 4a, 4c Magnetic particles 4b, 5, 5a 5c Aggregation complex 6 Magnet 7 Antibody 8 Magnetic complex 100, 200 Measurement device 101, 201 Light source 102, 202 Cell 103, 203 Cell holder 104, 204 Photodetector 105 Permanent magnet 106 Cover 107 Arm 108, 208 Removal means 202a Outlet 205 Electromagnet 206 Container 207 Valve

Claims (20)

A specific binding reaction measurement method for measuring the content of the analyte in the sample,
Constructing a reaction system including the sample and magnetic particles on which a specific binding substance that specifically binds to the substance to be measured is immobilized;
(B) measuring the optical properties of the reaction system;
Removing (c) an agglomerated complex containing the substance to be measured, the specific binding substance, and the magnetic particles from the reaction system by using a magnetic force;
And a method for measuring a specific binding reaction. The method for measuring a specific binding reaction according to claim 1,
The method for measuring a specific binding reaction, wherein the optical characteristic is a scattered light intensity or a transmitted light amount. The method for measuring a specific binding reaction according to claim 1,
The method for measuring a specific binding reaction, wherein the diameter of the magnetic particles is in a range of about 0.05 to 2 μm. The method for measuring a specific binding reaction according to claim 1,
A method for measuring a specific binding reaction, wherein the magnetic particles remaining in the reaction system are removed from the reaction system in the step (c). A specific binding reaction measurement method for measuring the contents of a first analyte and a second analyte in a sample,
(A) constructing a reaction system including the sample and magnetic particles on which a first specific binding substance that specifically binds to the first analyte is immobilized;
(B) measuring the optical characteristics of the reaction system after the step (a);
A step (c) of collecting an agglutinated complex including the first substance to be measured, the first specific binding substance, and the magnetic particles by using a magnetic force;
(D) adding a second specific binding substance that specifically binds to the second analyte to the reaction system;
After the step (d), a step (e) of measuring the optical characteristics of the reaction system;
And a method for measuring a specific binding reaction. The method for measuring a specific binding reaction according to claim 5,
In the above step (d), a specific binding reaction measuring method, wherein the reaction system contains 2 to 6% by weight of polyethylene glycol. The method for measuring a specific binding reaction according to claim 5,
The second specific binding substance is composed of an antigen or an antibody that specifically binds to the second substance to be measured, and a non-magnetic particle having the antigen or antibody immobilized thereon. Reaction measurement method. The method for measuring a specific binding reaction according to claim 7,
The diameter of the magnetic particles is in the range of about 0.05-2 μm;
The method for measuring a specific binding reaction, wherein the diameter of the nonmagnetic particles is in a range of about 0.05 to 2 μm. The method for measuring a specific binding reaction according to claim 5,
The method for measuring a specific binding reaction, wherein the combination of the first analyte and the second analyte is a combination of human hemoglobin and human albumin. The method for measuring a specific binding reaction according to claim 5,
A method for measuring a specific binding reaction, wherein the magnetic particles remaining in the reaction system are collected in the step (c). A specific binding reaction measurement method for measuring the contents of n kinds of substances (n is an integer of 2 or more) in a sample,
Constructing a reaction system including the sample and magnetic particles on which a specific binding substance that specifically binds to one of unmeasured substances to be measured is immobilized;
(B) measuring the optical characteristics of the reaction system after the step (a);
(C) removing the aggregated complex containing the one kind of the analyte, the specific binding substance, and the magnetic particles from the reaction system by using a magnetic force;
After the step (c), if the step (b) is less than the (n-1) th step, a step (d) of determining to proceed to the step (a) again;
(E) adding, to the reaction system, a specific binding substance that specifically binds to one type of unmeasured analyte remaining in the reaction system;
After the step (e), a step (f) of measuring the optical characteristics of the reaction system;
And a method for measuring a specific binding reaction. The method for measuring a specific binding reaction according to claim 11,
A method for measuring a specific binding reaction, wherein the magnetic particles remaining in the reaction system are removed from the reaction system in the step (c). A specific binding reaction measurement method for measuring the contents of n kinds of substances (n is an integer of 2 or more) in a sample,
(A) constructing a reaction system including the sample and a specific binding substance that specifically binds to one of unmeasured substances to be measured;
(B) measuring the optical characteristics of the reaction system after the step (a);
A step (c) of adding a magnetic complex containing a bindable substance and magnetic particles to a reaction system to an aggregate complex comprising the one kind of the analyte and the specific binding substance;
(D) removing the agglomerated complex to which the magnetic complex is bound from the reaction system by using a magnetic force;
After the step (d), if the step (b) is less than the (n-1) th step, a step (e) of determining to proceed to the step (a) again;
(F) adding to the reaction system a specific binding substance that specifically binds to one type of unmeasured analyte remaining in the reaction system;
A step (g) of measuring the optical properties of the reaction system after the step (e);
And a method for measuring a specific binding reaction. The method for measuring a specific binding reaction according to claim 13,
A method for measuring a specific binding reaction, wherein the magnetic complex not bound to the aggregate complex is removed from the reaction system in the step (d). A reagent kit for measuring the contents of a first analyte and a second analyte in a sample,
Magnetic particles on which a first specific binding substance that specifically binds to the first analyte is fixed;
A second specific binding substance that specifically binds to the second analyte,
A reagent kit comprising: The reagent kit according to claim 15,
The reagent kit, wherein the diameter of the magnetic particles is in a range of about 0.05 to 2 μm. The reagent kit according to claim 15,
The reagent kit, wherein the second specific binding substance is composed of an antigen or an antibody that specifically binds to the second analyte and non-magnetic particles to which the antigen or the antibody is immobilized. . A reagent kit for measuring the content of n kinds of substances to be measured in a sample (n is an integer of 2 or more),
One kind of specific binding substance that specifically binds to one kind of the analytes among the n kinds of analytes;
(N-1) kinds of magnetic particles each having immobilized specific binding substances specifically binding to the remaining (n-1) kinds of the test substances among the n kinds of the test substances,
A reagent kit comprising: A reagent kit for measuring the content of n kinds of substances to be measured in a sample (n is an integer of 2 or more),
N types of specific binding substances that specifically bind to the n types of analytes,
A substance capable of binding to an aggregating complex comprising (n-1) kinds of analytes among the n kinds of analytes and a corresponding (n-1) kinds of specific binding substances; A magnetic composite comprising immobilized magnetic particles,
A reagent kit comprising: Cells and
A light source for emitting light toward the cell;
A light detector for detecting scattered light or transmitted light from the reaction system,
When magnetic particles are contained in the cell, using magnetic force, removing means for removing the magnetic particles from the cell,
A specific binding reaction measurement device comprising:

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