JP4451637B2 - Specific binding reaction measurement method and reagent kit used therefor - Google Patents

Description

The present invention relates specifically binding reaction measurement method capable of measuring the content of a subject substance that may be contained in the sample, the reagent kit for use therein.

  In the medical field, in order to diagnose various diseases and examine the course of disease states, it is widely performed to measure the content of proteins characteristic of each disease present in human body fluids. For the measurement of the content of these proteins, an immune reaction measurement method using 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, immunological reaction measurement methods utilizing various principles have been developed.

  Among the various immune reaction measurement methods described above, an antigen-antibody complex aggregate (hereinafter referred to as an aggregate complex) generated by an antigen-antibody reaction is detected in order to measure the content of a substance to be measured in a sample. Measurement methods such as immunonephelometry (hereinafter abbreviated as “Nephelometry”), immunoturbidimetry (hereinafter referred to as “turbidimetry”), and slide agglutination are well known. In the antigen-antibody reaction, the reaction system becomes turbid due to the formation of an aggregated complex. The degree of turbidity generated in the reaction system due to the formation of the aggregate complex depends on the amount of antigen and the amount of antibody. Based on this, the specific gravity method and the turbidimetric method are methods for optically measuring the degree of turbidity generated in the above reaction system and calculating the amount of antigen or antibody from the measured value. Specifically, the Hiei method measures the aggregated complex based on the change in the amount of light scattered by the reaction system, and the nephelometry method measures the aggregated complex based on the change in the amount of transmitted light reduced by scattering in the reaction system. Is the method. Generally, in the above two measurement methods, the same reaction system can be used for the measurement. That is, the reaction system that can be measured by either one of the measurement methods can be measured by the other measurement method. The slide agglomeration method is a method for judging turbidity or agglomerates generated in the reaction system due to the formation of an agglomerated complex by visual observation on a slide glass or the like. Even in the slide agglutination method, the same reaction system as that of the specific gravity method and the turbidimetric method can be used for the measurement. The three measurement methods listed above are collectively referred to as “homogeneous immune reaction measurement methods” because the measurement is performed in a state where antigens and antibodies are uniformly dispersed in the reaction system.

  In the medical field, in order to diagnose various diseases and examine the course of medical conditions, the contents of a plurality of types of substances to be measured are measured. For example, in clinical examinations, the content of total protein that can be contained in human urine as an index of renal filtration ability is also measured by acute glomerulonephritis, IgA nephropathy, renal tuberculosis, renal infarction, pyelonephritis, cystitis The content of red blood cells (hemoglobin) that can be contained in human urine is measured as an index of inflammation of the urinary tract such as urethritis, prostatitis, calculus, and tumor (for example, see Non-patent Document 1). ). Furthermore, the following Patent Document 1 prepares a different reaction container for each substance to be measured as a homogeneous immune reaction measurement method for measuring a plurality of kinds of substances to be measured, and individually measures each substance to be measured. A method is disclosed.

The method of measuring the content of a substance to be measured in a sample by using the reaction between the substance to be measured contained in the sample and a specific binding substance that specifically binds to the substance to be measured. In the present specification, it is referred to as “specific binding reaction measurement method”. The above-described immune reaction measurement method is also one type of specific binding reaction measurement method.
JP-A-5-2024 Written by Izumi Kanai, edited by Masamitsu Kanai, “Procedure for Clinical Testing”, revised 31st edition, Kanehara Publishing Co., Ltd., 1998, pages 156-179-180.

  However, in the 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 samples are required for the measurement.

The present invention has been made in view of the above circumstances, it aims to provide specific binding reaction measurement method capable of measuring a plurality of the measured substance in one reaction vessel, a reagent kit for use therein And

Singular binding reaction measurement method of the present invention is a specific binding reaction measurement method for measuring the content of the first substance to be measured and the second substance to be measured in a sample, and the sample, the first A step (a) of constructing a reaction system comprising magnetic particles on which a first specific binding substance that specifically binds to a substance to be measured is immobilized; and after the step (a), After the step (b) of measuring the optical characteristics and the step (b), the aggregated complex containing the first substance to be measured, the first specific binding substance and the magnetic particles is utilized by using magnetic force. After the step (c), the step (d) of adding a second specific binding substance that specifically binds to the second substance to be measured to the reaction system, and the step (d) And (e) measuring the optical characteristics of the reaction system.

  According to the present invention, when measuring the second substance to be measured, the change in the optical characteristics of the reaction system due to the aggregated complex containing the substance to be measured, the specific binding substance and the magnetic particles in the reaction system, It is removed by using magnetic force. In particular, since the magnetic force is used, there is no chemical effect on the reaction system. For this reason, according to the specific binding reaction measuring method of the present invention, the reliability of the measured value is high when measuring the second substance to be measured.

  In addition, conventionally, when measuring the content of two kinds of substances to be measured, it is necessary to prepare two reaction vessels that are the same as the kinds of substances to be measured, but according to the present invention, a reaction system is constructed. If there is one reaction container that can be used, the content of two kinds of substances to be measured can be measured using the reaction container. Therefore, a very small amount of sample is required for measurement. 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 substance to be measured and nonmagnetic particles on which the antigen or antibody is immobilized. .

The diameter of the magnetic particles is 0 . In the range of 05～2Myuemu, the diameter of the non-magnetic particles, 0. It is preferable to be in the range of 05-2 μm.

  The combination of the first substance to be measured and the second substance to be measured may be a combination of human hemoglobin and human albumin.

In the step (c), the aggregated complex collected using magnetic force may be removed from the reaction system.

Another specific binding reaction measurement method of the present invention is a specific binding reaction measurement method for measuring the content of n kinds of substances to be measured (n is an integer of 2 or more) in a sample. A step (a) of constructing a reaction system comprising magnetic particles on which a specific binding substance that specifically binds to one type of the substance to be measured is immobilized, and after the step (a), After the step (b) of measuring the optical characteristics of the reaction system and the step (b), an aggregated complex containing the one kind of substance to be measured, the specific binding substance and the magnetic particles is utilized using magnetic force. If the step (b) is less than the (n-1) th after the step (c) and the step (c), it is determined to proceed to the step (a) again, and the step (b) ) is (n-1) th and was the case, the step of determining to proceed to step (e) (d), the reaction A step (e) of adding a specific binding substance that specifically binds to one type of unmeasured substance remaining in the reaction system to the system, and after the step (e), (F) measuring an optical characteristic.

  According to the present invention, the aggregated complex including 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. Because it uses magnetic force, it has no chemical effect on the reaction system. For this reason, even when measuring a plurality of substances to be measured, since the influence of the previously formed aggregate complex is almost eliminated, the reliability of the measured value is high every time when measuring the optical characteristics.

  In particular, conventionally, when measuring the content of two or more types of substances to be measured, it is necessary to prepare the same number of reaction vessels as the types of 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, a very small amount of sample is required for measurement. 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 aggregated complex collected using magnetic force may be removed from the reaction system.

Yet another specific binding reaction measurement method of the present invention is a specific binding reaction measurement method for measuring the content of n kinds (n is an integer of 2 or more) of a substance to be measured in a sample, A step (a) of constructing a reaction system including a specific binding substance that specifically binds to one type of substances to be measured; and after the step (a), the optical characteristics of the reaction system are After the step (b) of measuring and the step (b), a magnetic complex including a substance capable of binding and a magnetic particle is added to the aggregate complex including the one kind of substance to be measured and the specific binding substance. Step (c) to be added to the reaction system, Step (d) for collecting the aggregated complex to which the magnetic complex is bound using magnetic force, and Step (b) after Step (d) (n-1) when the a was less than th, it is determined to proceed to again step (a), the above step If b) is a (n-1) th, the step of determining to proceed to step (f) (e), to the reaction system, one of a measured substance unmeasured remaining in the reaction system A step (f) of adding a specific binding substance that specifically binds to the step, and a step (g) of measuring the optical characteristics of the reaction system after the step (e).

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

  In particular, conventionally, when measuring the content of two or more types of substances to be measured, it is necessary to prepare the same number of reaction vessels as the types of 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, a very small amount of sample is required for measurement. 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 aggregated complex collected using magnetic force may be removed from the reaction system.

The reagent kit of the present invention is a reagent kit for measuring the content of n kinds of substances to be measured (n is an integer of 2 or more) in a sample, and one kind of the above n kinds of substances to be measured. Specifically binding to one kind of specifically binding substance that specifically binds to the substance to be measured and the remaining (n-1) kinds of substances to be measured among the n kinds of substances to be measured. (N-1) types of magnetic particles each having a specific binding substance immobilized thereon.

  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 specific binding reaction measurement methods, will be described with reference to the drawings. For the sake of simplicity, components common to the respective embodiments are denoted by the same reference numerals.

(Embodiment 1)
FIG. 1 is a flowchart showing the immune reaction measurement method of the present embodiment. 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 step St1 shown in FIG. 1, as shown in FIG. 2A, the sample 1 to be measured for the content of the substance 2 to be measured and the specific binding that specifically binds to the substance 2 to be measured. A reaction system including the magnetic particles 4 on which the substance 3 is immobilized is constructed. Examples of the sample 1 include body fluids such as blood and urine as they are, or a mixture of these with a buffer solution. When the substance to be measured 2 is an antigen, the specific binding substance 3 is an antibody. When the substance to be measured 2 is an antibody, the specific binding substance 3 is an antigen.

  Thus, when the substance to be measured 2 is contained in the sample, the substance to be measured 2 and the specific substance are specifically detected by the antigen-antibody reaction between the substance to be measured 2 and the specific binding substance 3 as shown in FIG. An aggregated complex 5 composed of the binding substance 3 and the magnetic particles 4 is generated. When the substance to be measured 2 is not contained in the sample, the aggregated complex 5 due to the antigen-antibody reaction between the substance to be measured 2 and the specific binding substance 3 does not occur.

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

  Next, in step St3 shown in FIG. 1, the aggregated complex 5 composed of the substance 2 to be measured, the specific binding substance 3 and the magnetic particles 4 and the magnetic particles (not shown) not forming the aggregated complex 5 are obtained. The magnetic force (specifically, as shown in FIG. 2 (c), magnet 6) is used for recovery and removal from the reaction system.

  In the immune reaction measurement method, the degree of turbidity generated in the reaction system due to the formation of the aggregated complex depends on the amount of antigen and the amount of antibody. Based on this, according to the present embodiment, the optical characteristics of the reaction system described above can be measured, and the content of the substance to be measured 2 can be calculated from the measured value.

  Furthermore, according to the present embodiment, after the measurement, the magnetic particles existing in the reaction system and the aggregated complex 5 composed of the substance 2 to be measured, the specific binding substance 3 and the magnetic particles 4 utilize magnetic force. Is removed from the reaction system. In this embodiment, since the magnetic force is used, there is no chemical influence on the reaction system. For this reason, it is possible to measure the other components contained in the sample 1 by various measurement methods using the reaction system after the measurement.

  In addition, conventionally, when measuring two types of substances to be measured, it is necessary to prepare the same number of reaction containers as the number of substances to be measured, but according to this embodiment, a reaction system is constructed. If there is one reaction container capable of performing the above, it is possible to measure two types of substances to be measured using the reaction container. Therefore, according to this embodiment, it is possible to make the sample required for 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.

  Also, the magnetic particles are immobilized by inhibiting the antigen-antibody reaction under acidic conditions of pH 3.0 or lower from the aggregated complex 5 and magnetic particles (not shown) removed from the reaction system in the present embodiment. The collected 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 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 immune reaction 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 made by themselves. In particular, cultivate magnetic bacteria containing 0.05-0.1 μm magnetic particles in the cells to increase the number of bacteria, crush it with a French press, etc., collect the magnetic particles in the cells with a magnet and separate them By taking out, magnetic particles having a substantially uniform diameter can be obtained. A method for obtaining magnetic particles using magnetic bacteria is described in detail in JP-A No. 2000-346843.

  Magnetic particles obtained from magnetic bacteria are covered with a lipid bilayer membrane. For this reason, the dispersibility in aqueous solution etc. is favorable, and it is suitable also for immobilizing proteins, such as an antibody which is a specific binding substance.

  The diameter of the magnetic particles 4 used in this embodiment is easy to uniformly disperse in an aqueous solution, and the amount of change in scattered light intensity or transmitted light amount for quantifying the aggregated complex produced by the antigen-antibody reaction. It is preferable that it is 0.05-2 micrometers from a viewpoint that detection of this is possible.

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

  Any component known in the art may be added to the reaction system of the immune reaction measurement method of the present embodiment, depending on its use and the like. For example, in order to reduce non-specific aggregation due to self-aggregation of each of the substance to be measured 2 and the specific binding substance 3, in this embodiment, the reaction system includes Tween 20, octyl glucoside, sodium lauryl sulfate (SDS), sucrose. A surfactant 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 that there is little inhibition of the antigen-antibody reaction.

  In the immune reaction measurement method of the present embodiment, the change in scattered light intensity may be measured as the optical characteristic to be measured in step St2 (Nippon method), and the change in the amount of transmitted light from the reaction system is measured (turbidimetric method). )

  In the present embodiment, the substance to be measured is not particularly limited as long as it 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 immune reaction measurement method and reagent kit of the present embodiment are suitable for measurement of a protein that is a measurement object that has been conventionally measured using an 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 of various infectious diseases, CRP , Albumin, hemoglobin, rheumatoid factor, blood group antigen and the like.

  The antibody used in the present embodiment is not particularly limited as long as it can form an aggregated complex with an antigen by specifically binding to the antigen. For example, any antibody class of IgG, IgM, IgE, IgA, and IgD may be used, or a mixture thereof. Moreover, any of a polyclonal antibody and a monoclonal antibody may be sufficient, and a mixture thereof may be sufficient. Of these, IgG antibodies are preferred because they have few non-specific reactions, are relatively commercially available, and are easily available. The species of animal from which the antibody is derived is also not particularly limited, but antibodies derived from rabbits, goats, and mice are relatively easy to obtain and are preferred because there are many examples of use.

  In the present embodiment, the immunoreaction measurement method using an antigen-antibody reaction has been described as a specific binding reaction measurement method. However, an agglutination complex is generated using a reaction that produces specific binding other than the antigen-antibody reaction. It is also possible to measure by. When utilizing a reaction that causes specific binding other than the antigen-antibody reaction, the combination of the substance to be measured and the specific binding substance is, for example, a combination of a ligand and a receptor, single-stranded DNA (substance to be measured), and this Examples thereof include combinations with various DNA fragments (specific binding substances) having a sequence complementary to single-stranded DNA.

  As an example of a combination of a ligand and a receptor, a combination of a molecule serving as a ligand and an allosteric protein having a plurality of binding sites for the molecule can be mentioned. If there is only one part that binds to the allosteric protein in the molecule that becomes the ligand, it becomes the ligand if the molecule that becomes the ligand is immobilized on a magnetic particle, non-magnetic particle, etc., other than the part that binds to the allosteric protein. Aggregated complexes of molecules and allosteric proteins can be generated.

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

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

  FIG. 3 is a flowchart showing the immune reaction measurement method of the present embodiment. 4 (a) and 4 (b) are diagrams schematically showing a reaction system in the immune reaction measurement method of the present embodiment.

  First, in step St21 shown in FIG. 3, the sample to be measured for the contents of the first substance to be measured and the second substance to be measured and the first that specifically binds to the first substance to be measured. A reaction system including magnetic particles on which the specific binding substance 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 solution. When the first substance to be measured is an antigen, the first specific binding substance is an antibody, and when the first substance to be measured is an antibody, the first specific binding substance is an antigen.

  As a result, as shown in FIG. 4A, when the first substance to be measured 2a is contained in the sample, the antigen antibody between the first substance to be measured 2a and the first specific binding substance 3a. By the reaction, an aggregate complex 5a composed of the first substance to be measured 2a, the first specific binding substance 3a, and the magnetic particles 4a is generated. When the first substance to be measured 2a is not contained in the sample, the aggregated complex 5a due to the antigen-antibody reaction between the first substance to be measured 2a and the first specific binding substance 3a does not occur.

  Next, in step St22 shown in FIG. 3, the optical characteristics of the reaction system are measured. At this time, when the aggregated composite 5a is generated in the above-described step St21, the reaction system is turbid, and the scattered light intensity, the transmitted light amount, and the like are changed. Therefore, the degree of turbidity in the reaction system can be estimated by measuring the scattered light intensity 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 the measurement. Further, when a reaction system is constructed by mixing a sample with a buffer solution, the buffer solution may be used as a reference.

  Next, in step St23 shown in FIG. 3, the aggregated complex 5a composed of 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 collect them using magnetic force. At this time, the aggregated complex 5a and the magnetic particles may be removed from the reaction system, and the reaction system in which the reaction system is constructed so as not to hinder measurement of optical characteristics of the reaction system in step St25 described later. You may concentrate and arrange in a part of.

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

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

  Next, in step St25 shown in FIG. 3, the optical characteristics of the reaction system are measured. At this time, when the aggregated composite 4b is generated in the step St24, the reaction system is turbid, and the scattered light intensity, transmitted light amount, and the like are changed. Therefore, the degree of turbidity in the reaction system can be estimated by measuring the scattered light intensity 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 the measurement. Further, when a reaction system is constructed by mixing a sample with a buffer solution, the buffer solution 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 such as the intensity of scattered light and the amount of transmitted light due to only the aggregated composite 4b.

  In particular, according to this embodiment, when measuring the second substance to be measured 2b, the magnetic particles 4a that existed 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 complex 5a made of is removed by using magnetic force. Because it uses magnetic force, it has no chemical effect on the reaction system. For this reason, according to the immune reaction measuring method of this embodiment, when measuring the 2nd to-be-measured substance 2b, the reliability of a measured value is high.

  Conventionally, when measuring the contents of two kinds of substances to be measured, it is necessary to prepare two reaction vessels that are the same as the kinds of substances to be measured. According to this embodiment, a reaction system is constructed. If there is one reaction container that can be used, the content of two kinds of substances to be measured can be measured using the reaction container. Therefore, according to the present embodiment, a very small amount of sample is required for measurement. 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 removed from the reaction system and the magnetic particles in the present embodiment, the magnetic particles are immobilized on the immobilized 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. It is.

  Next, a reagent kit used in the immune reaction measurement method of this embodiment will be described. This will be described with reference to FIGS. 4 (a) and 4 (b).

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

  When the first substance to be measured 2a is an antigen, the first specific binding substance 3a is an antibody, and when the first substance to be measured 2a is an antibody, the first specific binding substance 3a is an antigen. is there. Further, when the second substance to be measured 2b is an antigen, the second specific binding substance 3b is an antibody, and when the second substance to be measured 2b is an antibody, the second specific binding substance 3b is an antigen. is there.

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

  In particular, as shown in FIG. 4B, when measuring the second substance to be measured 2b, the magnetic particles 4a that existed in the reaction system, the substance to be measured 2a, the specific binding substance 3a, and the magnetic particles The change in the optical characteristics of the reaction system due to the aggregated complex 5a composed of 4a is eliminated by using magnetic force. Because it uses magnetic force, it has no chemical effect on the reaction system. For this reason, according to the immune reaction measuring method of this embodiment, when measuring the 2nd to-be-measured substance 2b, the reliability of a measured value is high.

  The magnetic particles 4a used in the immune reaction measurement method and 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 made by themselves. In particular, cultivate magnetic bacteria containing 0.05-0.1 μm magnetic particles in the cells to increase the number of bacteria, crush it with a French press, etc., collect the magnetic particles in the cells with a magnet and separate them By taking out, magnetic particles having a substantially uniform diameter can be obtained. A method for obtaining magnetic particles using magnetic bacteria is described in detail in JP-A No. 2000-346843.

  Magnetic particles obtained from magnetic bacteria are covered with a lipid bilayer membrane. For this reason, the dispersibility in aqueous solution etc. is favorable, and it is suitable also for immobilizing proteins, such as an antibody which is a specific binding substance.

  The diameter of the magnetic particles 4a used in the present embodiment is from the viewpoint that it can be easily dispersed uniformly in an aqueous solution, and the amount of change in scattered light intensity or transmitted light amount for quantifying the aggregated complex produced by the antigen-antibody reaction. It is preferable that it is 0.05-2 micrometers from a viewpoint that detection of this is possible.

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

  In the immune reaction measurement method and 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 antibody that causes an antigen-antibody reaction with the second substance to be measured 2b is immobilized. Thus, the change range of the optical characteristics when measuring the second substance to be measured 2b (that is, step St25) is the same as the change range of the optical characteristics when measuring the first substance to be measured 2a (that is, step St22). , Not much change. For this reason, the adjustment of the apparatus used for a measurement becomes easy. It is also preferable that the progress of the antigen-antibody reaction of the second substance to be measured 2b is not affected at all by the magnetic force.

  Examples of nonmagnetic particles include glass particles, graphite particles, gold colloid particles, and latex particles. Among these, latex particles are preferable because they are highly stable and particles having various diameters are easily available, and polystyrene latex particles excellent in protein adsorptivity are particularly preferable.

  The above non-magnetic particles can be easily dispersed uniformly in a solution, and the amount of scattered light and the amount of transmitted light can be detected to quantify the aggregate complex produced by the antigen-antibody reaction. It is required to be. Accordingly, the diameter of the above-mentioned nonmagnetic particles is preferably 0.05 to 2 μm, and the diameter of the magnetic particles 4a is the same from the viewpoint of making the measurement range when using the magnetic particles 4a equal to the change range of the optical characteristics. Is more preferable.

  Any component known in the art may be added to the reaction system of the immune reaction measurement method of the present embodiment, depending on its use and the like. For example, when the second specific binding substance 3b is not a nonmagnetic particle on which an antigen or 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, nonspecific aggregation due to self-aggregation of each of the second substance to be measured 2b and the second specific binding substance 3b is reduced, and the measurement sensitivity is improved.

  Further, in step St22 and step St25, non-specific 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, respectively. In order to reduce aggregation, in this embodiment, a surfactant such as Tween 20, octyl glucoside, sodium lauryl sulfate (SDS), sucrose monolaurate or CHAPS may be added to the reaction system. 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 that there is little inhibition of the antigen-antibody reaction.

  In the immune reaction measurement method of the present embodiment, the change in scattered light intensity may be measured as an optical characteristic to be measured in steps St22 and St25 (comparison method), and the change in the amount of transmitted light from the reaction system is measured ( Turbidimetric method).

  In this embodiment, the 1st to-be-measured substance 2a and the 2nd to-be-measured substance 2b are not specifically limited, What is necessary is just a substance which 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 immune reaction measurement method and reagent kit of the present embodiment are suitable for measurement of a protein that is a measurement object that has been conventionally measured using an 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 of various infectious diseases, CRP , Albumin, hemoglobin, rheumatoid factor, blood group antigen and the like. For example, in the present embodiment, when the first substance to be measured 2a and the second substance to be measured 2b are human hemoglobin and human albumin, respectively, measurement results suitable for initial screening for kidney disease can be provided. This is because human hemoglobin in the urine is an index of acute glomerulonephritis, IgA nephropathy, renal tuberculosis, renal infarction, pyelonephritis, cystitis, urethritis, prostatitis, urinary tract inflammation, stone disease and tumor This is because human albumin in urine easily reflects changes in the total amount of protein in urine, so that 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 an antigen by specifically binding to the antigen. For example, any antibody class of IgG, IgM, IgE, IgA, and IgD may be used, or a mixture thereof. Moreover, any of a polyclonal antibody and a monoclonal antibody may be sufficient, and a mixture thereof may be sufficient. Of these, IgG antibodies are preferred because they have few non-specific reactions, are relatively commercially available, and are easily available. The species of animal from which the antibody is derived is also not particularly limited, but antibodies derived from rabbits, goats, and mice are relatively easy to obtain and are preferred because there are many examples of use.

  In the present embodiment, the immunoreaction measurement method using an antigen-antibody reaction has been described as a specific binding reaction measurement method. However, an agglutination complex is generated using a reaction that produces specific binding other than the antigen-antibody reaction. It is also possible to measure by. When utilizing a reaction that causes specific binding other than the antigen-antibody reaction, the combination of the substance to be measured and the specific binding substance is, for example, a combination of a ligand and a receptor, single-stranded DNA (substance to be measured), and this Examples thereof include combinations with various DNA fragments (specific binding substances) having a sequence complementary to single-stranded DNA.

  As an example of a combination of a ligand and a receptor, a combination of a molecule serving as a ligand and an allosteric protein having a plurality of binding sites for the molecule can be mentioned. If there is only one part that binds to the allosteric protein in the molecule that becomes the ligand, it becomes the ligand if the molecule that becomes the ligand is immobilized on a magnetic particle, non-magnetic particle, etc., other than the part that binds to the allosteric protein. Aggregated complexes of molecules and allosteric proteins can be generated.

  When using single-stranded DNA (substance to be measured) and various DNA fragments (specific binding substances) having a sequence complementary to the single-stranded DNA, the various DNA fragments are converted into single-stranded DNA. What is necessary is just to fix | immobilize to a magnetic particle, a nonmagnetic particle, etc. in the state which can couple | bond with DNA complementarily.

(Embodiment 3)
In the present embodiment, an immune reaction measurement method that is an extension of the immune reaction measurement method of the second embodiment so that the contents of various types of substances to be measured can be measured will be described with reference to the drawings.

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

  First, in step St31 shown in FIG. 5, a sample that is to be measured for the content of n kinds of substances to be measured (n is an integer of 2 or more) and one kind of substances to be measured that are not measured are unique. A reaction system including a magnetic particle on which a specific binding substance that binds to the target 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 solution. 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, when the substance to be measured is contained in the sample, the antigen-antibody reaction between the substance to be measured and the specific binding substance is equivalent to the aggregated complex 5a shown in FIG. As a result, an aggregated complex composed of the substance to be measured, the specific binding substance, and the magnetic particles is generated. When the substance to be measured is not contained in the sample, an aggregate complex due to the antigen-antibody reaction between the substance to be measured and the specific binding substance does not occur.

  Next, in step St32 shown in FIG. 5, the optical characteristics of the reaction system are measured. At this time, in the case where an aggregated complex is generated in the step St31, the reaction system becomes turbid, and the scattered light intensity, transmitted light amount, and the like are changed. Therefore, the degree of turbidity in the reaction system can be estimated by measuring the scattered light intensity 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 the measurement. Further, when a reaction system is constructed by mixing a sample with a buffer solution, the buffer solution may be used as a reference.

  Next, in step St33 shown in FIG. 5, the aggregated complex composed of 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. To do.

  Next, in step St34 shown in FIG. 5, it is determined whether the step St32 is the (n-1) th time. At this time, if the process St32 is the (n-1) th, the process proceeds to process St35. When the process St32 is less than the (n-1) th process, the process proceeds to the process St31 again. That is, steps St31 to St33 are repeated until the step St32 is the (n-1) th. As a result, (n−1) kinds of substances to be measured among n kinds of substances to be measured are removed from the reaction system.

  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 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, when the substance to be measured is contained in the sample, the antigen-antibody reaction between the substance to be measured and the specific binding substance is equivalent to the aggregated complex 4b shown in FIG. As a result, an aggregated complex composed 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, an aggregate complex due to the antigen-antibody reaction between the substance to be measured and the specific binding substance does not occur.

  Next, in step St36 shown in FIG. 5, the optical characteristics of the reaction system are measured. At this time, in the case where an aggregated complex is generated in step St35, turbidity occurs in the reaction system, and the scattered light intensity, transmitted light amount, and the like change. Therefore, the degree of turbidity in the reaction system can be estimated by measuring the scattered light intensity 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 the measurement. Further, when a reaction system is constructed by mixing a sample with a buffer solution, the buffer solution may be used as a reference.

  In particular, since the aggregated complex and the magnetic particles not forming the aggregated complex are collected using magnetic force in the above-described step St33, in this step, when measuring the optical characteristics of the reaction system, In addition, it is possible to measure changes in scattered light intensity, transmitted light amount, and the like due to only the aggregate complex.

  According to this embodiment, the aggregated complex composed of the substance to be measured, the specific binding substance, and the magnetic particles is removed by using the magnetic force after measuring the optical characteristics. Because it uses magnetic force, it has no chemical effect on the reaction system. For this reason, even when measuring a plurality of substances to be measured, since the influence of the previously formed aggregate complex is almost eliminated, the reliability of the measured value is high every time when measuring the optical characteristics.

  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 substances to be measured. If there is one reaction container capable of constructing, the content of two or more kinds of substances to be measured can be measured using the reaction container. Therefore, according to the present embodiment, a very small amount of sample is required for measurement. 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 removed from the reaction system and the magnetic particles in the present embodiment, the magnetic particles are immobilized on the immobilized 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. It is.

  Next, a reagent kit used in the immune reaction measurement method of this embodiment will be described.

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

  If the reagent kit of the present embodiment is used in the immune reaction measurement method of the present embodiment, if there is one reaction container capable of constructing a reaction system, two or more kinds of substances to be measured can be used by using it. The content can be measured. Therefore, if the reagent kit of this embodiment is used, the sample required for measurement can be made very small.

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

  Further, in the immune reaction measurement method and reagent kit of this embodiment, the specific binding substance added in step St35 shown in FIG. 5 causes an antigen-antibody reaction with one kind of unmeasured substance remaining in the reaction system. Nonmagnetic particles to which antigens or antibodies are immobilized are preferred. As a result, the change range of the optical characteristics in the measurement of the substance to be measured in step St36 does not change so much as the change range of the optical characteristics in the measurement of the (n−1) kinds of measurement substances in step St32. For this reason, the adjustment of the apparatus used for a measurement becomes easy.

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

  Any component known in the art may be added to the reaction system of the immune reaction measurement method of the present embodiment, depending on its use and the like. For example, when the specific binding substance is not a nonmagnetic particle on which an antigen or antibody is immobilized, polyethylene glycol may be added to the reaction system in step St35. In addition, 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.

  Further, in this embodiment, in order to reduce non-specific aggregation due to self-aggregation of each substance to be measured and each specific binding substance in step St32 and step St36, in the present embodiment, the reaction system includes tween 20, octylglucoside, 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 that there is little inhibition of the antigen-antibody reaction.

  In the immune reaction measurement method of the present embodiment, the change in scattered light intensity may be measured as an optical characteristic to be measured in steps St32 and St36 (comparative method), and the change in the amount of transmitted light from the reaction system is measured ( Turbidimetric method).

  In the present embodiment, each of the n types 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 immune reaction measurement method and reagent kit of the present embodiment are suitable for measurement of a protein that is a measurement object that has been conventionally measured using an 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 of various infectious diseases, CRP , Albumin, hemoglobin, rheumatoid factor, blood group antigen and the like.

  The antibody used in the present embodiment is not particularly limited as long as it can form an aggregated complex with an antigen by specifically binding to the antigen. For example, any antibody class of IgG, IgM, IgE, IgA, and IgD may be used, or a mixture thereof. Moreover, any of a polyclonal antibody and a monoclonal antibody may be sufficient, and a mixture thereof may be sufficient. Of these, IgG antibodies are preferred because they have few non-specific reactions, are relatively commercially available, and are easily available. The species of animal from which the antibody is derived is also not particularly limited, but antibodies derived from rabbits, goats, and mice are relatively easy to obtain and are preferred because there are many examples of use.

  In this embodiment, an immune reaction measurement method using an antigen-antibody reaction has been described as a specific binding reaction measurement method. However, an aggregate complex is generated using a reaction that produces specific binding other than the antigen-antibody reaction. It is also possible to measure by. When utilizing a reaction that causes specific binding other than the antigen-antibody reaction, the combination of the substance to be measured and the specific binding substance is, for example, a combination of a ligand and a receptor, single-stranded DNA (substance to be measured), and this Examples thereof include combinations with various DNA fragments (specific binding substances) having a sequence complementary to single-stranded DNA.

  As an example of a combination of a ligand and a receptor, a combination of a molecule serving as a ligand and an allosteric protein having a plurality of binding sites for the molecule can be mentioned. If there is only one part that binds to the allosteric protein in the molecule that becomes the ligand, it becomes the ligand if the molecule that becomes the ligand is immobilized on a magnetic particle, non-magnetic particle, etc., other than the part that binds to the allosteric protein. Aggregated complexes of molecules and allosteric proteins can be generated.

  When using single-stranded DNA (substance to be measured) and various DNA fragments (specific binding substances) having a sequence complementary to the single-stranded DNA, the various DNA fragments are converted into single-stranded DNA. What is necessary is just to fix | immobilize to a magnetic particle, a nonmagnetic particle, etc. in the state which can couple | bond with DNA complementarily.

(Embodiment 4)
In the present embodiment, another immune reaction measurement method capable of measuring the contents of many types of substances to be measured will be described with reference to the drawings.

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

  First, in step St41 shown in FIG. 6, it is specific to a sample to be measured for the content of n types of substances to be measured (n is an integer of 2 or more) and one type of unmeasured substances to be measured. A reaction system including a specific binding substance that binds to the target. 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.

  Thus, as shown in FIG. 7 (a), when one kind of 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 complex 5a composed of 2a and the specific binding substance 3a is generated. When the sample 2a is not contained in the sample, the aggregated complex 5a due to the antigen-antibody reaction between the sample 2a and the specific binding substance 3a does not occur.

  Next, in step St42 shown in FIG. 6, the optical characteristics of the reaction system are measured. At this time, in the case where an aggregated complex is generated in Step St41, turbidity is generated in the reaction system, and the scattered light intensity, transmitted light amount, and the like are changed. Therefore, the degree of turbidity in the reaction system can be estimated by measuring the scattered light intensity 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 the measurement. Further, when a reaction system is constructed by mixing a sample with a buffer solution, the buffer solution may be used as a reference.

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

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

  Next, in step St45 shown in FIG. 6, it is determined whether the step St42 has been (n-1) th. At this time, if the process St42 is the (n-1) th, the process proceeds to process St46. When the step St42 is less than the (n-1) th time, the process proceeds to the step St41 again. That is, steps St41 to St44 are repeated until the step St42 is the (n-1) th. As a result, (n−1) kinds of substances to be measured among n kinds of substances to be measured are removed from the reaction system.

  Next, in step St46 shown in FIG. 6, 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 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, when the substance to be measured is contained in the sample, the antigen-antibody reaction between the substance to be measured and the specific binding substance is equivalent to the aggregated complex 4b shown in FIG. As a result, an aggregated complex composed 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, an aggregate complex due to the antigen-antibody reaction between the substance to be measured and the specific binding substance does not occur.

  Next, in step St46 shown in FIG. 6, the optical characteristics of the reaction system are measured. At this time, in the case where an aggregated complex is generated in step St45, turbidity is generated in the reaction system, and the scattered light intensity, transmitted light amount, and the like are changed. Therefore, the degree of turbidity in the reaction system can be estimated by measuring the scattered light intensity 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 the measurement. Further, when a reaction system is constructed by mixing a sample with a buffer solution, the buffer solution may be used as a reference.

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

  According to this embodiment, the aggregate complex composed of the substance to be measured and the specific binding substance is removed by using magnetic force after the measurement of the optical characteristics. Because it uses magnetic force, it has no chemical effect on the reaction system. For this reason, even when measuring a plurality of substances to be measured, since the influence of the previously formed aggregate complex is almost eliminated, the reliability of the measured value is high every time when measuring the optical characteristics.

  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 substances to be measured. If there is one reaction container capable of constructing, the content of two or more kinds of substances to be measured can be measured using the reaction container. Therefore, according to the present embodiment, a very small amount of sample is required for measurement. 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 addition, the magnetic complex removed from the reaction system in the present embodiment inhibits the antigen-antibody reaction under an acidic condition of pH 3.0 or lower, so that the immobilized antibody or antigen is made reactive. It can also be collected 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. It is.

  Next, a reagent kit used in the immune reaction measurement method of this embodiment will be described.

  The reagent kit used in the immune reaction measurement method of the present embodiment includes a specific binding substance that specifically binds to each of n kinds of substances to be measured, (n-1) kinds of substances to be measured, and specifics corresponding thereto. A magnetic complex comprising a substance (for example, a secondary antibody capable of binding to an antibody contained in the aggregated complex) capable of binding to each of the aggregated complexes composed of the binding substance, and magnetic particles on which the substance is immobilized. And including.

  If the reagent kit of the present embodiment is used in the immune reaction measurement method of the present embodiment, if there is one reaction container capable of constructing a reaction system, two or more kinds of substances to be measured can be used by using it. The content can be measured. Therefore, if the reagent kit of this embodiment is used, the sample required for measurement can be made very small.

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

  The magnetic complex that can be specifically bound to the aggregated complex used in this embodiment includes an antibody that specifically binds to the antibody used to generate each aggregated complex, and the generation of the aggregated complex. An antibody that specifically binds to a site different from the antibody used in the above, or an antibody that specifically binds to a specific structure of the aggregated complex is immobilized on magnetic particles.

  In the immune reaction measurement method and reagent kit of this embodiment, the specific binding substance added in step St46 shown in FIG. 6 causes an antigen-antibody reaction with one type of unmeasured substance remaining in the reaction system. Nonmagnetic particles to which antigens or antibodies are immobilized are preferred. As a result, in step St47, the change range of the optical characteristics in the measurement of the substance to be measured does not change so much as the change range of the optical characteristics in the measurement of the (n−1) types of measurement substances in step St42. For this reason, the adjustment of the apparatus used for a measurement becomes easy.

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

  Any component known in the art may be added to the reaction system of the immune reaction measurement method of the present embodiment, depending on its use and the like. For example, when the specific binding substance is not a non-magnetic particle on which an antigen or antibody is immobilized, polyethylene glycol may be added to the reaction system in step St46. Further, in step St46, polyethylene glycol may be added to the reagent kit in order to add polyethylene glycol to the reaction system. 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 the self-aggregation of the substance to be measured or the specific binding substance is reduced in step St47, and the measurement sensitivity is improved.

  Further, in this embodiment, in order to reduce non-specific aggregation due to self-aggregation of each substance to be measured and each specific binding substance in step St42 and step St47, in this embodiment, the reaction system includes tween 20, octylglucoside, 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 that there is little inhibition of the antigen-antibody reaction.

  In the immune reaction measurement method of the present embodiment, the change in scattered light intensity may be measured as an optical characteristic to be measured in steps St42 and St47 (comparison method), and the change in the amount of transmitted light from the reaction system is measured ( Turbidimetric method).

  In the present embodiment, each of the n types 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 immune reaction measurement method and reagent kit of the present embodiment are suitable for measurement of a protein that is a measurement object that has been conventionally measured using an 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 of various infectious diseases, CRP , Albumin, hemoglobin, rheumatoid factor, blood group antigen and the like.

  The antibody used in the present embodiment is not particularly limited as long as it can form an aggregated complex with an antigen by specifically binding to the antigen. For example, any antibody class of IgG, IgM, IgE, IgA, and IgD may be used, or a mixture thereof. Moreover, any of a polyclonal antibody and a monoclonal antibody may be sufficient, and a mixture thereof may be sufficient. Of these, IgG antibodies are preferred because they have few non-specific reactions, are relatively commercially available, and are easily available. The species of animal from which the antibody is derived is also not particularly limited, but antibodies derived from rabbits, goats, and mice are relatively easy to obtain and are preferred because there are many examples of use.

  In the present embodiment, the immunoreaction measurement method using an antigen-antibody reaction has been described as a specific binding reaction measurement method. However, an agglutination complex is generated using a reaction that produces specific binding other than the antigen-antibody reaction. It is also possible to measure by. When utilizing a reaction that causes specific binding other than the antigen-antibody reaction, the combination of the substance to be measured and the specific binding substance is, for example, a combination of a ligand and a receptor, single-stranded DNA (substance to be measured), and this Examples thereof include combinations with various DNA fragments (specific binding substances) having a sequence complementary to single-stranded DNA.

  As an example of a combination of a ligand and a receptor, a combination of a molecule serving as a ligand and an allosteric protein having a plurality of binding sites for the molecule can be mentioned. If there is only one part that binds to the allosteric protein in the molecule that becomes the ligand, it becomes the ligand if the molecule that becomes the ligand is immobilized on a magnetic particle, non-magnetic particle, etc., other than the part that binds to the allosteric protein. Aggregated complexes of molecules and allosteric proteins can be generated.

  When using single-stranded DNA (substance to be measured) and various DNA fragments (specific binding substances) having a sequence complementary to the single-stranded DNA, the various DNA fragments are converted into single-stranded DNA. What is necessary is just to fix | immobilize to a magnetic particle, a nonmagnetic particle, etc. in the state which can couple | bond with DNA complementarily.

(Embodiment 5)
In the present embodiment, a measurement apparatus for performing the immune reaction measurement method of Embodiments 1 to 4 will be described with reference to the drawings.

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

  As shown in FIG. 8, the measurement apparatus 100 according to the present embodiment includes a light source 101, a cell (reaction vessel) 102, a cell holder 103 that holds the cell 102, and a photodetector 104 that detects light from the cell 102. Removal means 108.

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

  The photodetector 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 the reaction system using the 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 measurement apparatus 100 of the present embodiment.

  As shown in FIG. 9, the arm 107 can lower the permanent magnet 105 and the cover 106 to be inserted into the cell 102, and can be lifted and pulled up from the cell 102. Therefore, the permanent magnet 105 and the cover 106 are introduced into the reaction system, and the magnetic particles, the magnetic complex, the aggregated complex, and the like in the reaction system are adhered to the surface of the cover 106. Next, when the turbidity of the reaction system is almost eliminated, the reaction system is pulled up from the cell 102. This makes it possible to collect and remove magnetic particles, magnetic complexes, aggregated complexes, and the like from the reaction system. Note that the magnetic particles, the magnetic composite, the aggregated composite, and the like attached to the cover 106 may be detached by a mechanical method. For example, after the permanent magnet 105 is pulled out from the cover 106, the recovered magnetic particles attached to the cover 106 may be removed by washing or the like, or the cover 106 may be made disposable. In the case where an electromagnet is used instead of the permanent magnet 105, magnetic particles may be collected and removed by ON / OFF of the magnetic force generation of the electromagnet.

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

  As shown in FIG. 10, the measurement apparatus 200 according to the present embodiment detects light from a light source 201, a cell (reaction vessel) 202 including a discharge port 202 a, a cell holder 203 that holds the cell 202, and light from the cell 202. A photodetector 204 and a removing means 208 are provided.

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

  The photodetector 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 the reaction system using the magnetic force in the first to fourth embodiments. In the measuring apparatus 200, the removing means 208 includes an electromagnet 205, a container 206 for storing discharge from the discharge port 202a of the cell 202, and a valve 207 that is connected to the discharge port 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 measurement apparatus 200 of the present embodiment.

  The valve 207 is connected to the discharge port 202a of the cell 202 as shown in FIG. When the electromagnet 205 is energized in this state, the magnetic particles, magnetic complex, aggregated complex, and the like in the reaction system in the cell 202 move from the discharge port 202a to the container 206 through the valve 207. Next, when the turbidity of the reaction system is almost eliminated, the valve 207 is closed and the energization to the electromagnet 205 is stopped. This makes it possible to collect and remove magnetic particles, magnetic complexes, aggregated complexes, and the like from the reaction system. Note that the magnetic particles, magnetic composite, 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 measuring apparatus 200, a discharge pipe connected to the cell 202 may be provided instead of the container 206.

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

  As shown in FIG. 12, the arm 107 can move the permanent magnet 105 up and down. Therefore, the permanent magnet 105 is brought close to the container 206 to discharge the magnetic particles, magnetic complex, aggregated complex, and the like in the reaction system 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. This makes it possible to collect and remove magnetic particles, magnetic composites, aggregated composites, and the like from the reaction system. The magnetic particles, magnetic composite, aggregated composite, etc. stored in the container 206 may be removed by washing. Alternatively, the container 206 may be disposable.

  When using the removing means 108 and 208 ′ including the permanent magnet 105, when measuring the optical characteristics of the reaction system, the permanent magnet 105 is kept waiting in a place away from the cell 202, and after the measurement is finished, the cell 202 It is preferable to operate the removing means 108 and 208 ′ so that the magnetic particles, the magnetic complex, the aggregated complex and the like can be collected and removed.

  In the case where the removing means 108 including the electromagnet 205 is used, the electromagnet 205 can be disposed near the cell 202. This is because the electromagnet 205 hardly generates a magnetic force when it is not energized. Therefore, when measuring the optical characteristics of the reaction system, no magnetic force is generated by deenergization, and magnetic force is generated by energization after the measurement is completed, and the magnetic particles, magnetic complex, aggregated complex, etc. in the cell 202 are collected. It is preferable to operate the removal means 108 and 208 ′ so that they can be removed. This is because in any of the above cases, when measuring the optical characteristics of the reaction system, the influence of the magnetic force on the antigen-antibody reaction can be reduced as much as possible.

  At this time, it is preferable to fill the inside of the container 206 with the same buffer as the reaction system in advance. As a result, 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. In addition, the Example shown below does not limit this invention.

  In this example, the first substance to be measured in the second embodiment is human hemoglobin, the second substance to be measured is human albumin, the first specific binding substance is an anti-human hemoglobin antibody, and the second specific binding is performed. The reagent composition method in the case where the substance is an anti-human albumin antibody and the second specific binding substance is not immobilized on non-magnetic particles is shown.

  In addition, the pure water filtered with Milli-Q SP TOC (made by Millipore) was used for preparation of the buffer solution etc. which were shown to the present Example. In addition, reagents such as salts and buffering agents not specifically described were obtained from Wako Pure Chemical Industries, Ltd., polyethylene glycol 6,000 was a first grade reagent, and other reagents were special grade reagents.

  The anti-human hemoglobin antibody as the first specific binding substance and the 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 protein A column chromatography from antiserum collected from a rabbit immunized with human albumin (manufactured by Wako Pure Chemical Industries). The protein A-immobilized gel packed in the column was Amersham Pharmacia. The equilibration buffer used for purification was 1.5M glycine, 3.0M NaCl, pH 8.9, and the elution buffer was 0.1M citric acid, pH 4.0. I used something.

Purification was performed as follows. Flow the equilibration buffer 5 times the gel volume packed in the column to equilibrate the column, then double the volume of antisera containing 10-20% of the total binding capacity of the antibody with the equilibration buffer. The antibody in the serum was bound to protein A by diluting and flowing through the column. Subsequently, the equilibration buffer was allowed to flow until no serum component that did not adsorb to protein A came out of the column, and the column was washed. Subsequently, an elution buffer was passed through the column to elute the antibody bound to protein A. The eluted antibody fraction was put into a dialysis tube having a molecular weight cut off of 10,000, and about 100 times volume 0.05M 3- (N-morpholino) propanesulfonic acid (manufactured by Dojin, hereinafter abbreviated as MOPS), 0.15M NaCl. The buffer component was replaced by dialyzing several times with a buffer solution having a composition of 0.04 wt% NaN ₃ , pH 7.4. Subsequently, the antibody concentration was estimated by measuring the absorbance at 280 nm and adjusted with the same buffer as that used in dialysis to make the antibody concentration 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 and more preferably stored at 4 ° C. from the viewpoint of preventing antibody denaturation.

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

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

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

0.5 ml of 2.5 wt% magnetic particles are placed in a microtube, and 0.5 ml of 0.05 M mops, 0.04 wt% NaN ₃ , pH 7.4 buffer is added to suspend the magnetic particles, This was centrifuged at 12,000 rpm for 30 minutes using a centrifuge (Model number MRX-150, manufactured by Tommy Seiko Co., Ltd.), the magnetic particles were precipitated, and the supernatant was removed.

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

Furthermore, a buffer solution having a composition of 0.05 M mops, 0.15 M NaCl, 0.04 wt% NaN ₃ , 5 vol% glycerol, pH 7.4 was added to suspend the magnetic particles. The antibody concentration and 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 and more preferably stored at 4 ° C. from the viewpoint of preventing antibody denaturation.

Further, in order to measure human hemoglobin, a first buffer solution added to the reaction system is prepared with a composition of 0.05 M mops, 0.04 wt% NaN ₃ , pH 7.4, and human albumin is measured. As a second buffer solution to be added to the reaction system, a composition of 0.05 M mops, 8.6 wt% polyethylene glycol 6,000, 0.04 wt% NaN ₃ , pH 7.4 was prepared. Each prepared buffer was stored at room temperature.

  By combining the magnetic particles with the anti-human hemoglobin antibody (first specific binding substance) immobilized as described above, the anti-human albumin antibody (second specific binding substance), and each buffer solution, A reagent for immune reaction can be constituted.

  A method of using the reagent configured as described above is shown below. First, a sample containing two kinds of substances to be measured (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. The reaction system was constructed. Next, the optical characteristics of the reaction system were measured. After completion of the measurement, an aggregate complex composed of human hemoglobin (first substance to be measured), an anti-human hemoglobin antibody (first specific binding substance) and magnetic particles, and a magnetic particle that does not form an aggregate complex Were collected by magnetic force to eliminate turbidity in 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, the anti-albumin antibody (second specific binding substance) may be immobilized on nonmagnetic particles such as latex or gold colloid. For example, the method used for the magnetic particles described above can be applied as it is to the latex particles.

  In this example, the surface of the particles 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 that for sodium caseinate.

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

  Furthermore, the buffer component and 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 of the antigen and the antibody.

  Further, in order to take out 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, with the immune reaction measurement method and reagent kit of the present invention, two types of substances to be measured can be measured in one reaction container.

  The specific binding reaction measurement method, the reagent kit and the specific binding reaction measurement apparatus of the present invention are useful for diagnosing various diseases and examining the course 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 diseases.

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

Explanation of symbols

DESCRIPTION OF SYMBOLS 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 Measuring device 101, 201 Light source 102, 202 Cell 103, 203 Cell holder 104, 204 Photo detector 105 Permanent magnet 106 Cover 107 Arm 108, 208 Removal means 202a Exhaust Outlet 205 Electromagnet 206 Container 207 Valve

Claims (11)

A specific binding reaction measurement method for measuring the contents of a first substance to be measured and a second substance to be measured in a sample,
(A) constructing a reaction system comprising the sample and a magnetic particle on which a first specific binding substance that specifically binds to the first substance to be measured is immobilized;
After the step (a), a step (b) of measuring optical characteristics of the reaction system;
After the step (b), a step (c) of collecting an aggregated complex containing the first substance to be measured, the first specific binding substance and the magnetic particles using a magnetic force;
Adding a second specific binding substance that specifically binds to the second substance to be measured to the reaction system (d);
A step (e) of measuring optical characteristics of the reaction system after the step (d);
A specific binding reaction measurement method comprising: In the specific binding reaction measuring method according to claim 1,
In the step (d), the specific binding reaction measurement method, wherein the reaction system contains 2 to 6% by weight of polyethylene glycol. In the specific binding reaction measuring method according to claim 1,
The second specific binding substance is composed of an antigen or antibody that specifically binds to the second substance to be measured, and non-magnetic particles on which the antigen or antibody is immobilized. Reaction measurement method. In the specific binding reaction measuring method according to claim 3,
The diameter of the magnetic particles is 0 . In the range of 05-2 μm,
The non-magnetic particles have a diameter of 0 . A specific binding reaction measurement method in a range of 05 to 2 μm. In the specific binding reaction measuring method according to claim 1,
The specific binding reaction measurement method, wherein the combination of the first substance to be measured and the second substance to be measured is a combination of human hemoglobin and human albumin. In the specific binding reaction measuring method according to claim 1,
A method for measuring a specific binding reaction, wherein the aggregated complex collected using magnetic force in the step (c) is removed from the reaction system. A specific binding reaction measurement method for measuring the content of n kinds of substances to be measured (n is an integer of 2 or more) in a sample,
(A) constructing a reaction system comprising the sample and a magnetic particle on which a specific binding substance that specifically binds to one type of substance to be measured is immobilized;
After the step (a), a step (b) of measuring optical characteristics of the reaction system;
After the step (b), a step (c) of collecting the aggregated complex containing the one kind of substance to be measured, the specific binding substance, and the magnetic particles by using magnetic force;
After the step (c), when the step (b) is less than the (n-1) th time, it is determined to proceed to the step (a) again , and the step (b) is the (n-1) th time. If so, step (d) for determining to proceed to step (e) ,
Adding to the reaction system a specific binding substance that specifically binds to one kind of unmeasured substance remaining in the reaction system (e);
A step (f) of measuring optical characteristics of the reaction system after the step (e);
A specific binding reaction measurement method comprising: In the specific binding reaction measuring method according to claim 7,
A method for measuring a specific binding reaction, wherein the aggregated complex collected using magnetic force in the step (c) is removed from the reaction system. A specific binding reaction measurement method for measuring the content of n kinds of substances to be measured (n is an integer of 2 or more) in a sample,
(A) constructing a reaction system comprising the sample and a specific binding substance that specifically binds to one type of unmeasured substance to be measured;
After the step (a), a step (b) of measuring optical characteristics of the reaction system;
After the step (b), a step of adding a magnetic complex containing a substance capable of binding and a magnetic particle to the aggregated complex composed of the one kind of substance to be measured and the specific binding substance (c) )When,
Collecting the aggregated complex to which the magnetic complex is bound using a magnetic force (d);
After the step (d), when the step (b) is less than the (n-1) th time, it is determined to proceed to the step (a) again , and the step (b) is the (n-1) th time. If so, the step (e) for judging to proceed to the step (f) ,
Adding to the reaction system a specific binding substance that specifically binds to one type of unmeasured substance remaining in the reaction system (f);
After the step (e), measuring the optical properties of the reaction system (g),
A specific binding reaction measurement method comprising: The specific binding reaction measurement method according to claim 9,
A method for measuring a specific binding reaction, wherein the aggregated complex collected using magnetic force in the step (d) is removed from the reaction system. A reagent kit for measuring the content of n kinds of substances to be measured (n is an integer of 2 or more) in a sample,
N kinds of specific binding substances that specifically bind to the n kinds of substances to be measured,
Of the n kinds of substances to be measured, a substance capable of binding to an aggregate complex composed of (n-1) kinds of substances to be measured and (n-1) kinds of specific binding substances corresponding thereto; A magnetic composite composed of immobilized magnetic particles;
A reagent kit.

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