JP2013134174A - Method for measuring substance existing in two or more sorts of forms - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring a total amount of a measuring target substance capable of existing in a sample in two or more sorts of forms.SOLUTION: The method for measuring the total amount of the measuring target substance includes: a step (1) for bringing a specific standard substance into contact with the measuring target substance included in the sample; a step (2) for bringing a specific inhibitor into contact with the measuring target substance included in the sample before the step (1), simultaneously with the step (1) or after the step (1); a step (3) for bringing a specific solid-phase substance into contact with the measuring target substance included in the sample before the step (2), simultaneously with the step (2) or after the step (2); a step (4) for separating a component not coupled with the specific solid-phase substance from a component coupled with the specific solid-phase substance after the step (3); and a step (5) for detecting the specific standard substance included in any one of the component not coupled with the specific solid-phase substance and the component coupled with the specific solid-phase substance.

Description

  The present invention relates to a method for measuring the total amount of a substance to be measured that may be present in a sample in two or more types of forms.

  In recent years, a technique for measuring the abundance of substances contained in samples derived from blood or samples derived from rivers and lakes has been developed and put into practical use. For example, immunoassays that measure antigenic substances contained in blood-derived samples (hereinafter sometimes simply abbreviated as “blood samples”) using antibodies against them are now indispensable in the field of clinical diagnosis. Technology. For example, Prostate Specific Antigen (hereinafter sometimes abbreviated as “PSA”) is secreted from prostate epithelial cells, but is also secreted into the blood by prostate cancer cells. Therefore, PSA becomes positive in early prostate cancer, and its immunological measurement is widely used for diagnosis of prostate cancer, monitoring of the course after treatment, and the like.

  The substance to be measured may be present in the sample in two or more types of forms. For example, a substance to be measured may exist in a sample in a free form or may be present in one or more complexes by combining with other substances. In addition, the substance to be measured may not be present in a free form in the sample, but may be present as a combination of other substances to form two or more complexes. For example, PSA binds to α1-antichymotrypsin (ACT) in a form in which a PSA molecule alone is liberated in a blood sample (referred to as free PSA or free PSA, hereinafter sometimes abbreviated as “fPSA”). It exists in the form formed (hereinafter sometimes abbreviated as “PSA-ACT”) and the form formed by binding to macroglobulin. Among these, those bound to macroglobulin are in a form incorporated into the macroglobulin molecule, and therefore cannot be measured by an immunological method using an antibody against PSA. Accordingly, the amount of fPSA, the amount of PSA-ACT, or the total amount of fPSA and PSA-ACT (total PSA, hereinafter sometimes abbreviated as “tPSA”) can be measured immunologically.

  Hereinafter, the immunoassay will be described as an example. The measurement of total PSA is generally performed by an antibody that can specifically bind to both forms of fPSA and PSA-ACT, in other words, PSA forms a complex with ACT. Is also performed by immunoassay using an antibody recognizing a site of PSA in which binding to PSA is not inhibited. FIG. 1 is a conceptual diagram of a sandwich assay using antibodies that recognize different sites A and B on PSA (referred to as antibodies A and B, respectively). This site A and site B are sites where the reactivity with antibody A or antibody B is not affected even if PSA binds to ACT to form a complex.

  In the immunoassay as described above, both fPSA and PSA-ACT show the same measured value (signal intensity) with respect to the same number of moles. This is called “equimolar reactivity”. However, equimolar reactivity is established only after the immune reaction has reached equilibrium. If the immune response is terminated before the immune response reaches equilibrium, equimolar reactivity is not established and fPSA and PSA-ACT give different measurements (signal intensity) for the same number of moles (FIG. 2; non- Reprint of Patent Document 1).

PSA and PSA-ACT differ in size when viewed as a single molecule. Specifically, the molecular weight of PSA is about 33,000, but the molecular weight of PSA-ACT is about 101,000 (PSA is about 33,000 and ACT is about 68,000. The total is about 101,000). For this reason, the reaction rate of PSA-ACT and an antibody is low compared with the reaction rate of fPSA and an antibody. Therefore, when measuring equimolar fPSA and PSA-ACT, if the immune reaction is terminated before the immune reaction reaches equilibrium, the abundance of fPSA is apparently increased. Thus, since the reactivity with an antibody differs remarkably in fPSA and PSA-ACT, long reaction time is required to establish equimolar reactivity. If the immune reaction is terminated before equimolar reactivity is established in accordance with the desire in the clinical laboratory field to obtain measurement results as soon as possible after the start of measurement, the substances to be measured in different forms (different amounts) ) Is measured as a risk.

  In Patent Document 1, in the case of measuring PSA by immunoassay using a polyclonal antibody, an antibody capable of binding to both fPSA and PSA-ACT, and fPSA can be bound to the same polyclonal antibody, but PSA-ACT can be bound. It is disclosed that by masking the binding epitope of the latter antibody in fPSA with another antibody, the reactivity bias of the polyclonal antibody to fPSA and PSA-ACT can be reduced or eliminated when the antibody that cannot be used is included. ing.

  However, there is no known means for eliminating the difference in reactivity based on molecular weight as described above and achieving substantially equimolar reactivity.

Japanese Patent No. 3486413

R. T.A. McCorack et al. , Urology, 45 (5), 729-744 (1995). FIGURE 5

  The present invention provides a method for measuring the total amount of a substance to be measured by simply establishing a substantially equimolar reactivity with a substance to be measured that can be present in a sample in two or more types of forms. The purpose is to provide.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have determined that, in the measurement of PSA by a sandwich method using a solid phase antibody and a labeled antibody, each of fPSA and PSA-ACT and a solid phase antibody. It was found that substantially equimolar reactivity can be established for both forms of fPSA and PSA-ACT by coexisting antibodies that compete for binding, and the present invention was completed.

The present invention made in view of the above problems includes the following aspects:
[1]
A method for measuring the total amount of a substance to be measured that may be present in a sample in two or more types of forms,
Said two or more types of forms are (A) one or more types of complex forms bound to free and other substances, or (B) two or more forms bound to other substances. It is a form of the above kind of complex,
(A) A substance that specifically binds to all forms of the substance to be measured (label specific substance) bound by a detectable labeling substance (a) All forms and specifics of the substance to be measured bound to a water-insoluble carrier And (c) a substance that inhibits the binding between the whole form of the substance to be measured and the solid phase specific substance (inhibition specific substance), and the label specific substance and the solid phase The specific substance is capable of simultaneously binding to the measurement target substance at different sites, and includes the following steps (1) to (5), and measures the total amount of the measurement target substance: Method:
(1) A step of bringing a labeling specific substance into contact with a measurement target substance in a sample,
(2) before the step (1), simultaneously with the step (1), or after the step (1), bringing the inhibitory specific substance into contact with the measurement target substance in the sample;
(3) before the step (2), simultaneously with the step (2), or after the step (2), a step of bringing the solid phase specific substance into contact with the measurement target substance in the sample;
(4) After the step (3), separating the component that is not bound to the solid phase specific substance and the bound component;
(5) A step of detecting a label contained in either the component not bound to the solid phase specific substance or the bound component after the step (4).
[2]
The method according to [1], wherein the other substance is bound to one molecule per molecule of the complex.
[3]
The method according to [1] or [2], wherein the inhibition specific substance and the solid phase specific substance are substances that bind to the same site of the measurement target substance.
[4]
The method according to any one of [1] to [3], wherein the measurement target substance is a protein, and the label specific substance, the solid phase specific substance, and the inhibition specific substance are antibodies that bind to the measurement target substance.
[5]
The method according to any one of [1] to [4], wherein the step (3) is performed after the step (1) and the step (2).
[6]
The substance to be measured is prostate specific antigen,
Two or more types of forms are free prostate specific antigen and a complex of prostate specific antigen and α1-antichymotrypsin;
The label specific substance is a first anti-prostate specific antigen antibody to which the label substance is bound,
A solid phase specific substance is a second anti-prostate specific antigen antibody having a different antigen binding site from the first anti-prostate specific antigen antibody bound to the solid phase;
The method according to any one of [1] to [5], wherein the inhibitory specific substance is an antibody that competes with the second anti-prostate specific antigen antibody.
[7]
The method according to [6], wherein the inhibitory specific substance is the same antibody as the second anti-prostate specific antigen antibody.

  According to the present invention, it is possible to quickly and easily measure the total amount of a substance to be measured that can exist in a sample in two or more types of forms. Specifically, when measuring a substance to be measured by a sandwich method using a label specific substance and a solid phase specific substance, an inhibitory specific substance that competes for binding between the whole form of the target substance and the solid phase specific substance is used. Thus, substantially equimolar reactivity can be achieved before the reaction between the substance to be measured and the solid phase specific substance reaches equilibrium, and the total amount of the object to be measured can be measured quickly, simply and accurately. In addition, according to the present invention, in particular, before the measurement target substance and the solid phase specific substance are brought into contact with each other and reacted, the measurement target substance and the inhibitory specific substance are brought into contact with each other to react with each other. Substantially equimolar reactivity can be achieved even in the reaction system used.

FIG. 1 is a schematic diagram of the reaction between PSA-ACT and an antibody in a sandwich method using two monoclonal antibodies. FIG. 1 shows the positional relationship between each epitope (antibody binding site) on the PSA and the ACT binding site. FIG. 2 is a response curve in which the response to a solution containing fPSA and PSA-ACT alone in an equimolar concentration is plotted against the incubation time in a one-step sandwich radioimmunoassay (RIA) method. In the reaction with the antibody bound to the solid phase, PSA-ACT has a slower reaction than fPSA, and therefore the measurement result (signal intensity) is lower than that of fPSA after the same reaction time in the time zone where the equilibrium point is not reached. can get. FIG. 2 is a reprint from Non-Patent Document 1. FIG. 3 shows the difference between the reactivity (rate) of fPSA and the solid-phase antibody and the reactivity (rate) of PSA-ACT and the solid-phase antibody when fine particles having a particle diameter of about 3 μm are used as the solid phase. FIG. In the reaction with the antibody bound to the solid phase, PSA-ACT has a slower reaction than fPSA, and therefore the measurement result (signal intensity) is lower than that of fPSA after the same reaction time in the time zone where the equilibrium point is not reached. can get. The “reaction rate” on the vertical axis represents the relative value of the measurement result (signal intensity) when the measurement result (signal intensity) when the equilibrium point is reached is 100. FIG. 4 is a schematic diagram showing the influence of using an inhibitory specific substance (antibody). By using an inhibitory specific substance, a point where the measurement results (signal intensity) of fPSA and PSA-ACT coincide even after the same reaction time in a time zone where the equilibrium point is not reached occurs. FIG. 5 is a diagram showing the effect of using an inhibitory specific substance (antibody) when the microparticles are used as a solid phase and all specific substances and a sample are reacted simultaneously. The numbers in parentheses in the legend indicate the concentration (ng / mL). FIG. 6 shows the effect of using an inhibitory specific substance (antibody) when a microparticle is used as a solid phase, a labeled specific substance and an inhibitory specific substance are reacted first, and a solid phase specific substance is subsequently reacted with a sample. FIG. The numbers in parentheses in the legend indicate the concentration (ng / mL). FIG. 7 shows an inhibitory specific substance (in the case where a microparticle is used as a solid phase, a labeled specific substance and a solid phase specific substance are reacted first, an inhibitory specific substance is reacted later (delay time is 6 minutes), and a sample. It is a figure which shows the influence by using an antibody. The numbers in parentheses in the legend indicate the concentration (ng / mL). By delaying the contact between the solid phase specific substance and the sample, it is possible to match the measured values (signal intensity) of fPSA and PSA-ACT even when using a low concentration of inhibitory specific substance (antibody). FIG. 8 is a diagram showing that equimolar reactivity was established between fPSA and PSA-ACT by using an inhibitory specific substance (antibody).

  Hereinafter, the present invention will be described in detail.

  In the method of the present invention, the total amount of the substance to be measured that can be present in a sample in two or more types of forms is converted into a label specific substance, a solid phase specific substance, and an inhibitory specific substance (hereinafter referred to as a label specific substance, hereinafter). The solid phase specific substance and the inhibition specific substance are collectively referred to as “specific substance” in some cases.

  In the present invention, the “two or more types of forms” that can be taken by the substance to be measured means (A) a released form and one or more types of complex forms bound with other substances, or (B) refers to the form of two or more types of complexes to which other substances are bound.

That is, in the case of (A), the substance to be measured can be present in the sample in a released form and in the form of one or more kinds of complexes to which other substances are bound. In the case of (B), the substance to be measured does not exist in the free form, but may exist in the sample in the form of two or more kinds of complexes bound with other substances. The “form of a complex in which another substance is bound” refers to a form of a complex in which another substance is bound to a measurement target substance in a free form. The “other substance” refers to a substance different from the measurement target substance in a free form. The number of the other substances that bind to the measurement target substance in the released form may be one, or two or more, per one type of complex formed. In addition, the number of molecules of another substance that binds to the measurement target substance in a released form may be one molecule per complex formed, or two or more molecules. In the above (A), the type of the complex formed may be one, or two or more. In the above (B), the number of types of complexes formed may be two, or three or more.

  As the case where two or more types of complexes are formed, for example, the form of the first complex in which the measurement target substance is bonded to the other substance A and other forms different from the other substance A are used. The case where the form of the 2nd composite_body | complex couple | bonded with the substance B is formed is mentioned. In addition, in the case where two or more kinds of complexes are formed, the form of the first complex bound to the other substance C is different from the first complex in a binding mode. In the case of forming the form of the second complex bound to the substance C. The different binding modes of other substances include, for example, when the number of molecules of other substances per molecule of the first complex is different from that of the second complex, or the binding positions of other substances Is different. In the above, the case where two or more types of complexes are formed is exemplified as the case where two or more types of complexes are formed. However, in the above description, three or more types of complexes are formed. The above can also be applied mutatis mutandis.

  According to the method of the present invention, any substance that can be present in a sample in two or more types of forms as described above and can prepare specific substances described later for each form is particularly limited. The total amount can be measured.

  Examples of substances to be measured that can be measured in total by the method of the present invention include proteins such as prostate specific antigen (PSA), biopolymers such as hormones, sugar chains, and lipids, viruses, and pharmaceuticals. Organic / inorganic compounds can be exemplified.

  For example, in the case of prostate specific antigen (PSA), a form liberated in a blood-derived sample (fPSA) and a form of a complex bound to ACT (PSA-ACT) are mixed. In addition to the above forms, PSA is present in the sample in the form of a complex bound to macroglobulin. However, when an antibody against PSA is used as a specific substance described later, as described above, PSA bound to macroglobulin is It cannot be measured. In the present invention, a form that is not originally a measurement target in a certain measurement method is not included in a form that the measurement target substance can take in the measurement method. That is, for example, when an antibody against PSA is used as a specific substance, PSA is a substance to be measured that exists in a sample in a released form and in the form of one complex in which another substance is bound.

  In the present invention, the “total amount” of the substance to be measured refers to the total amount of all forms to be measured. In the present invention, the amount of the form that is not originally measured in a certain measurement method is not included in the total amount in the measurement method. That is, for example, when an antibody against PSA is used as a specific substance, the total amount of PSA is the total amount of fPSA and PSA-ACT, and the amount of PSA bound to macroglobulin is not included in the total amount.

  The term “can exist in two or more types of forms” as used in the present invention is not limited to the case where a certain substance naturally takes two or more types of forms. Or the case where it takes the form of the kind more than that is included. That is, the form that the measurement target substance can take may be a form that exists in nature, a form that does not exist in nature, or a combination thereof.

In the present invention, the “sample” is not particularly limited as long as it contains a substance to be measured. Samples include, for example, biological samples such as blood containing protein and hormones such as PSA, body fluids such as saliva, rivers, lakes and marshes containing proteins and chemicals, foods, pharmaceuticals, etc. Can be mentioned. Since the present invention utilizes a reaction in a liquid phase system, when measuring a sample that is not inherently liquid, the sample itself is suspended in a liquid, or a measurement target substance is added from the sample. The liquid obtained by extraction can be used as a sample. Extraction of the substance to be measured may be performed by a technique well known to those skilled in the art, for example. The sample may be used for measurement without special pretreatment, or may be used for measurement after pretreatment. For example, when an interfering substance that can inhibit the reaction between the specific substance and the measurement target substance is present in the sample, it is preferable to remove the interfering substance in advance.

  The label specific substance is composed of a label substance and a substance that specifically binds to the measurement target substance, the solid phase specific substance is composed of a solid phase and a substance that specifically binds to the measurement target substance, and the inhibition specific substance is Consists of substances that inhibit the binding between the substance to be measured and the solid phase specific substance. In the present invention, the “substance that specifically binds to the substance to be measured” refers to a substance that does not have binding properties (cross-reactivity) to substances other than the substance to be measured, Substances whose cross-reactivity does not substantially affect the measurement of the substance to be measured are included. “A substance that has cross-reactivity but does not substantially affect the measurement of the measurement target substance” refers to, for example, binding to a substance other than the measurement target substance but the binding is the measurement target It binds to a substance that is negligible compared to the binding to the substance or a substance other than the substance to be measured, and the binding is not negligible compared to the binding to the substance to be measured. There is no substance other than the substance to be measured in the sample, or even if it is present, the quantity is negligibly low compared to the quantity of the substance to be measured, or such is prior to the application of the present invention. Examples include substances that satisfy at least one of the following: substances other than the measurement target substance can be completely removed or can be removed to a negligibly low concentration.

  The “substance that specifically binds to the substance to be measured” constituting the specific substance needs to be able to bind to all forms of the substance to be measured that are present in the sample and whose total amount is to be measured. Here, “all forms” are all forms to be measured. In other words, even if it exists in a certain form in a sample, as long as it exists in that form and cannot bind any specific substance prepared, that form is included in the measurement target substance. Absent. For example, as described above, when measuring the total amount of PSA using an antibody against PSA as a specific substance, PSA bound to macroglobulin is not a measurement target, and the total amount of PSA is the total amount of fPSA and PSA-ACT. Means.

  Examples of the substance that specifically binds to the measurement target substance that constitutes the specific substance include a substance that specifically binds to the measurement target substance in a so-called “key and keyhole” relationship. Examples of combinations of substances having such a relationship include antigen and antibody, ligand and receptor, avidin and biotin, antibody and protein A, and the like. Examples of the ligand and receptor include an antibody and an Fc receptor. In addition, for example, as a substance that specifically binds to a measurement target substance, an inhibitor or the like when protease is used as the measurement target substance, and when a protein subunit is used as the measurement target substance, binds to the subunit. Other subunits are exemplified.

The label-specific substance used in the present invention is obtained by binding a detectable “label substance” and “substance that specifically binds to the substance to be measured”. Examples of the labeling substance include substances that themselves can be detected by optical detection or radioactivity detection, or substances that act on other substances to enable optical detection, for example. Specific examples of the labeling substance include enzymes, chemiluminescent substances, bioluminescent substances, radioisotopes, and colored substances. The labeling substance and the substance that specifically binds to the substance to be measured can be bound to each other, for example, by a chemical method such as covalent bonding, or by binding of affinity substances bound to both. In the present invention, a substance obtained by binding both in advance can be used as a label-specific substance, but both can also be bound by binding of affinity substances in the process of implementing the present invention. The “substance that specifically binds to the measurement target substance” constituting the label specific substance does not have to be a single substance, and may be a mixture of two or more kinds of substances. Specifically, for example, when an antibody is used as the substance, the antibody may be a monoclonal antibody or a polyclonal antibody that is a mixture of two or more kinds of antibodies.

  The solid phase specific substance used in the present invention is a substance obtained by binding a “water-insoluble solid phase” and a “substance that specifically binds to a measurement target substance”. By using the solid phase specific substance, the measurement target substance bound to the solid phase specific substance can be separated from the liquid phase by a so-called B / F (Bound / Free) separation operation. The solid phase can be prepared from various water-insoluble materials such as natural materials such as agarose and dextran, synthetic polymer materials such as polyethylene and polystyrene, and metal materials. The shape of the solid phase is not particularly limited, and may be, for example, a particle shape or a flat plate shape. Further, for example, the inner wall of a container for carrying out the present invention can be used as a solid phase. Among these, a substance capable of providing a relatively large surface area is preferable because it binds a large amount of a substance that specifically binds to the substance to be measured. The size of the solid phase is not particularly limited, but it is spherical and has a particle size of 0.1 μm to 100 μm in order to improve the reactivity with the substance to be measured and achieve the equimolar reaction within a short time. It is preferable to use a fine particle solid phase, and it is more preferable to use a fine particle solid phase having a particle diameter of 1 μm to 10 μm. The solid phase may contain a magnetic substance for stirring by magnetic force or B / F separation operation. That is, as the fine particle solid phase, for example, magnetic fine particles are preferable. Further, the “substance that specifically binds to the measurement target substance” constituting the solid phase specific substance does not have to be a single substance, and may be a mixture of two or more kinds of substances. Specifically, for example, when an antibody is used as the substance, the antibody may be a monoclonal antibody or a polyclonal antibody that is a mixture of two or more kinds of antibodies.

  The solid phase and the substance that specifically binds to the substance to be measured are bound to each other, for example, by a chemical method such as covalent bonding, physical adsorption, or by binding of affinity substances bound to both. Can do. In the present invention, a substance obtained by binding both in advance can be used as a solid phase specific substance, but both can also be bound by binding of affinity substances in the process of implementing the present invention.

  In the present invention, the label specific substance and the solid phase specific substance can be simultaneously bonded to the measurement target substance. That is, for example, the label-specific substance and the solid-phase specific substance bind to different positions of the measurement target substance, respectively.

The inhibitory specific substance used in the present invention is a substance that inhibits the binding between the whole form of the measurement target substance and the solid phase specific substance, that is, the “measurement target substance constituting the solid phase specific substance in binding to the measurement target substance” It is a substance that competes with "a substance that specifically binds to". As an inhibitory specific substance, for example, a substance that binds to a site in a measurement target substance to which a “substance that specifically binds to a measurement target substance” constituting a solid phase specific substance binds or binds to a measurement target substance Thus, a substance that sterically inhibits the binding between the “substance that specifically binds to the measurement target substance” constituting the solid phase specific substance and the measurement target substance can be used. As the inhibition specific substance, it is preferable to use a substance that binds to a site in the measurement target substance to which the “substance specifically binding to the measurement target substance” constituting the solid phase specific substance binds. That is, the inhibition specific substance and the solid phase specific substance are preferably substances that bind to the same site of the measurement target substance. Among these, the use of the same substance as the “substance that specifically binds to the substance to be measured” that constitutes the solid phase specific substance as the inhibition specific substance is particularly preferable for simplifying the preparation of the specific substance. In this case, if the preparation of the “substance that specifically binds to the measurement target substance” constituting the label specific substance and the solid phase specific substance is completed, the preparation of all the specific substances used in the present invention is completed. Because. Further, as is clear from the above description, the “substance that specifically binds to the substance to be measured” that constitutes the inhibitory specific substance does not have to be a single substance, and is a mixture of two or more kinds of substances. Also good. Specifically, for example, when an antibody is used as the substance, the antibody may be a monoclonal antibody or a polyclonal antibody that is a mixture of two or more kinds of antibodies.

  In the present invention, usually, the label specific substance and the inhibition specific substance can simultaneously bind to the measurement target substance. That is, for example, the label specific substance and the inhibition specific substance bind to different positions of the measurement target substance, respectively.

  When the “substance that specifically binds to the substance to be measured” used in the present invention is an antibody, the antibody constituting each specific substance may be a combination of polyclonal antibodies, or a combination of a polyclonal antibody and a monoclonal antibody. It may be a combination of monoclonal antibodies. Among these, the combination of monoclonal antibodies is preferable because once the hybridoma is established, the preparation of the antibody is easy and the reactivity is uniform.

  The “substance that specifically binds to the substance to be measured” used in the present invention may be prepared, for example, by a known method, or a commercially available product may be used.

  For example, an antibody against a measurement target substance uses as an immunogen the measurement target substance itself, a part of the measurement target substance, or a polynucleotide encoding the full length or a partial region of the measurement target substance when the measurement target substance is a protein. It can be obtained by immunizing animals. The animal used for immunization is not particularly limited as long as it has an antibody-producing ability, and may be a mammal such as a mouse, a rat, or a rabbit, which is usually used for immunization, or a bird such as a chicken.

  When a monoclonal antibody is used as the antibody, a hybridoma cell that produces a monoclonal antibody that recognizes the substance to be measured may be established to prepare the monoclonal antibody. Establishment of hybridoma cells can be performed by a known method. As an example, B cells are collected from an animal immunized by the above-described method, the B cells and myeloma cells are fused electrically or in the presence of polyethylene glycol, and hybridoma cells are selected using a HAT medium. Hybridoma cells producing a monoclonal antibody that recognizes the substance to be measured can be established by monocloning the cells by the limiting dilution method.

  As described above, the label-specific substance and the solid-phase specific substance can bind to the measurement target substance at the same time, while the solid-phase specific substance and the inhibitory specific substance can be alternatively selected. Only one of them can bind to the substance to be measured. And this invention is implemented by performing the following processes (1)-(5) using these specific substances.

  Step (1) is a step of bringing the labeling specific substance into contact with the measurement target substance in the sample.

  Step (2) is a step of bringing the inhibition specific substance into contact with the measurement target substance in the sample. This step (2) may be performed either before step (1), simultaneously with step (1), or after step (1).

  Step (3) is a step of bringing the solid phase specific substance into contact with the measurement target substance in the sample. This step (3) may be performed either before step (2), simultaneously with step (2), or after step (2).

  Step (4) is a step of separating the component not bound to the solid phase specific substance and the bound component. This step (4) is performed after the step (3).

  Step (5) is a step of detecting a label contained in either the component not bound to the solid phase specific substance or the bound component. This step (5) is performed after the step (4).

  The steps (1), (2), and (3) may be performed so that the measurement target substance in the sample and each specific substance come into contact with each other and a binding reaction can occur. In the steps (1), (2) and (3), for example, a predetermined amount of sample is added to and mixed with a solution containing a specific substance, or a solution containing a specific substance is fixed. It can be carried out by adding, mixing, etc. to an amount of sample. The conditions at that time are not particularly limited as long as a binding reaction between the substance to be measured and each specific substance can occur. For example, the components, pH, temperature, etc. that coexist in the reaction system depend on the type of substance to be measured, the type of each specific substance selected according to the substance to be measured, the mode of binding between each specific substance and the substance to be measured, etc. Can be selected and determined as appropriate. For example, when an antibody is used as a specific substance, the reaction temperature is 4 to 45 ° C., the pH is pH 4 to pH 9, and the salt concentration is 1 mM to 150 mM. Examples of coexistence of sodium, magnesium chloride and the like can be given. In order to carry out the above steps quickly, for example, an appropriate container is prepared, and a solution containing a label specific substance, a solid phase specific substance, an inhibitory specific substance, and other components is put therein in advance. In addition, the steps (1), (2), and (3) can be performed simultaneously by putting a certain amount of sample.

  The step (4) can be carried out by selecting an operation suitable for the solid phase selected in carrying out the present invention. For example, when a solid phase containing a magnetic substance is used, step (4) can be performed by tilting the container and decanting while collecting the solid phase on the side wall of the container with a magnet. For example, when the inner wall of the container is used as a solid phase or a plate-like solid phase is used, the step (4) can be performed by simple decantation. In step (4), in addition to the operation of simply separating the solid phase and the liquid phase as described above, it is preferable to perform an operation of washing the solid phase with an appropriate washing solution after the separation operation. The washing solution used for washing is not particularly limited as long as it does not release the binding between the substance to be measured and the specific substance. For example, from pH 5 containing a nonionic surfactant at a low concentration of about 0.01%. An example is the use of a Tris or phosphate buffer having a pH of about 8.

  Step (5) can be carried out by selecting an operation suitable for the labeling substance selected in carrying out the present invention. For example, when an enzyme is used as a labeling substance, a substance that can be converted into an optically detectable substance under the action of the enzyme is added, and the substance is optically detected after an enzyme reaction for a certain period of time. The label can be quantitatively detected by detecting the increase rate of the detectable substance within a certain time. For such detection, specifically, alkaline phosphatase (ALP) is used as a labeling substance, and 4-methylumbelliferyl phosphate (4-MUP), which is a substrate thereof, is added, and produced by ALP 4 -Detecting the production rate of methylumbelliferone (4-MU) can be exemplified. For example, when a chemiluminescent substance or a radioactive substance is used as a labeling substance, chemiluminescence intensity or radioactivity can be detected.

  In step (5), a label contained in a component not bound to the solid phase specific substance may be detected, or a label contained in a component bound to the solid phase specific substance may be detected. When detecting the label contained in the component bound to the solid phase specific substance, the amount of the detected label reflects the amount of the measurement target substance bound to the solid phase specific substance. When detecting a label contained in a component not bound to a solid phase specific substance, the difference between the amount of the detected label and the amount of label assumed from the amount of the label specific substance used is the solid phase specific substance. Reflects the amount of the analyte to be bound to.

In the following, data such as signal intensity obtained when detecting a labeled substance in this way may be described simply as “measured value” or “measured value (signal intensity)”.

  In the method of the present invention, when the solid phase specific substance and the inhibition specific substance contact and react with the measurement target substance, the measurement target substance competes between the solid phase specific substance and the inhibition specific substance as the reaction starts. . At this time, depending on the form of the substance to be measured, the substance having a smaller molecular weight binds to the inhibition specific substance with a higher reaction rate. That is, among the substances to be measured, those in the free form are bound to the inhibitory specific substance with a higher reaction rate than those in the form of the complex, and are bound to the substance having a low molecular weight to form the complex. Binds to an inhibitory specific substance with a reaction rate higher than that of a complex formed by binding to a substance having a higher molecular weight. Similarly, as described above, depending on the form of the substance to be measured, the substance having a smaller molecular weight binds to the solid phase specific substance with a higher reaction rate. Here, since the reactivity between the inhibitory specific substance and the measurement target substance is superior to the reactivity between the solid phase specific substance and the measurement target substance, the measurement is performed by competing the inhibitory specific substance with the solid phase specific substance. The difference in reactivity between the measurement target substance and the solid phase specific substance according to the form of the target substance is canceled, and substantially equimolar reactivity is achieved.

  Step (3) can be performed either before step (2), simultaneously with step (2), or after step (2). That is, there is no time limit other than the implementation before the step (4). The amount (concentration) of the inhibitory specific substance used in step (3) and the time from the contact of the inhibitory specific substance to the subsequent step (4) are two or more forms that can be taken by the substance to be measured. What is necessary is just to set suitably so that equimolar reactivity is materialized. For this purpose, after determining the substance to be measured, a sample containing a known concentration of the substance to be measured in each form is prepared, and for this sample, a substance that specifically binds to the substance to be measured, which constitutes the solid phase specific substance, is prepared. It is preferable to examine the amount, the reaction time, and the amount of the inhibitory specific substance to be used, and determine the details of the step (3). More specifically, in order to establish an apparent equimolar reactivity by coexisting an inhibitory specific substance, the amount (concentration) of the inhibitory specific substance to be coexisted is a measured value (signal intensity) of 2 or more. The total amount of the measurement target substance does not depend on the abundance ratio of the measurement target substance existing in the above form, in other words, irrespective of the abundance ratio of two or more forms that can be taken by the measurement target substance present in the sample. If the value does not change, adjust so that the same measurement result can be obtained. For this purpose, a curve showing the relationship between the concentration of the inhibition specific substance and the measured value (signal intensity) when measuring only a certain form of the measurement target substance using various concentrations of the inhibition specific substance, When measuring only the target substance in the form of, both of the same curves intersect at one point, that is, when the same concentration of inhibitory specific substance is used for the same number of moles of the target substance present in various forms In addition, it is sufficient that there is only one point that gives the same measurement value (signal intensity), and in the step (3), an inhibitory specific substance at that concentration may be used.

By using the amount of the inhibition specific substance as described above, equimolar reactivity can be established for two or more forms that the measurement target substance can take. On the other hand, by using an inhibitory specific substance, the final measured value (signal intensity) is lowered depending on the amount used. Therefore, it is important that the measured value (signal intensity) of the intersection point of the curves described above exists in the measurable range. In addition, although the measured value (signal intensity) at the intersection changes by increasing the amount of the solid phase specific substance, it is not practical to increase the solid phase specific substance indefinitely. It is preferable to examine the amount from an economical viewpoint. Here, as described above, the establishment of equimolar reactivity by using an inhibition specific substance is that the reactivity between the inhibition specific substance and the measurement target substance is superior to the reactivity between the solid phase specific substance and the measurement target substance. It depends. Therefore, the closer the rate of the solid-liquid phase reaction between the measurement target substance and the solid-phase specific substance is to the speed of the liquid phase-liquid phase reaction between the measurement target substance and the inhibition specific substance, the more the inhibition specific substance and the measurement target The superiority of the reaction of the substance is reduced and the need to use a larger amount of inhibitory specific substance. In other words, there are cases where equimolar reactivity can be easily established by using an inhibitory specific substance and cases where this is not the case. Specifically, when a microtiter plate or the inner wall of a container is used as the solid phase, the reaction between the inhibitory specific substance and the measurement target substance is significantly superior to the reaction between the solid phase specific substance and the measurement target substance. Therefore, equimolar reactivity can be easily established by using a small inhibitory specific substance. On the other hand, when fine particles, for example, fine particles having a particle size of about 1 μm to 10 μm are used as the solid phase, the superiority of the reaction between the inhibitory specific substance and the measurement target substance as described above is lowered, so that a relatively large amount of inhibition Since it is necessary to use a specific substance, and the measured value (signal intensity) is thereby reduced, it is relatively difficult to establish an equimolar reaction.

  The difficulty as described above can be overcome by securing the superiority of the reaction between the inhibition specific substance and the measurement target substance in the liquid phase-liquid phase. The superiority of the reaction between the inhibition specific substance and the measurement target substance is, for example, that the measurement target substance and the inhibition specific substance are brought into contact and reacted before the measurement target substance and the solid phase specific substance are brought into contact with each other. It is preferable to ensure by.

That is, an aspect of the present invention suitable for achieving equimolar reactivity in the situation described above using microparticles as the solid phase is generally described in the sample in two or more types of forms. A method for measuring the total amount of a substance to be measured that may be present, wherein the two or more types of forms are (A) one or more types of complexes to which a free form and other substances are bound. In the form, or (B) two or more kinds of complex forms bound to other substances, and (a) specifically bound to all forms of the substance to be measured bound to a detectable labeling substance. (B) a substance that specifically binds to all forms of the substance to be measured (solid phase specific substance) bound to the water-insoluble carrier, and (c) a substance that binds to all forms of the substance to be measured. Substances that inhibit the binding to phase-specific substances (inhibition-specific substances) ), And the label specific substance and the solid phase specific substance can simultaneously bind to the measurement target substance at different sites, and include the following steps (1) to (5): Features:
(1) A step of bringing a labeling specific substance into contact with a measurement target substance in a sample,
(2) before the step (1), simultaneously with the step (1), or after the step (1), bringing the inhibitory specific substance into contact with the measurement target substance in the sample;
(3) After the step (1) and the step (2), a step of bringing a solid phase specific substance into contact with a measurement target substance in a sample,
(4) After the step (3), separating the component that is not bound to the solid phase specific substance and the bound component;
(5) A step of detecting a label contained in either the component not bound to the solid phase specific substance or the bound component after the step (4).

  Specifically, a solid phase specific substance in which a sample, an inhibitory specific substance and a label specific substance are brought into contact with each other and reacted, and a substance that specifically binds to the measurement target substance is bound to the microparticle after a certain period of time. It is possible to exemplify reacting by delaying the contact with the sample. The delay time from the contact between the inhibitory specific substance and the sample to the contact between the solid phase specific substance and the sample depends on the reaction rate between the solid phase specific substance and the measurement target substance, and the inhibition specific substance and the measurement target substance. Although it may be determined by the balance of the reaction rate, etc., according to the knowledge of the present inventor, it is preferably 15 seconds to 20 minutes, more preferably 1 minute to 10 minutes. In this way, the superiority of the reaction between the inhibitory specific substance and the measurement target substance can be achieved by delaying the solid phase specific substance and bringing it into contact with the sample without contacting all the specific substances used in the present invention at once. Can be secured. Thereby, for example, even when the amount of the inhibitory specific substance used is small, or when a fine particle having a particle size of about 1 μm to 10 μm is used as the solid phase, equimolar reactivity can be established. The total amount can be measured in a short time.

The present invention may include a step of calculating the total amount of the substance to be measured based on the measured value (signal intensity) obtained as described above. When calculating the total amount of the substance to be measured, the method of the present invention is performed using a standard sample containing the substance to be measured whose concentration is known, and the correlation between the total amount of the substance to be measured and the measured value (signal intensity) obtained. Data may be acquired and the total amount of the measurement target substance present in the test sample may be calculated based on the correlation data. The correlation data is, for example, a calibration curve.

  The present invention also provides a reagent for measuring the total amount of the substance to be measured (also referred to as the measurement reagent of the present invention). The measurement reagent of the present invention is not particularly limited as long as it contains a label specific substance, an inhibition specific substance, and a solid phase specific substance. In the measurement reagent of the present invention, each specific substance and, if necessary, other components may be provided as a mixture, or may be provided separately or in any combination. For example, all or a part of each specific substance and, if necessary, other components may be dispensed into a reaction container. The amount of each specific substance and, if necessary, other components contained in the measurement reagent of the present invention is not particularly limited as long as the method of the present invention can be carried out. That is, the amount of each specific substance is the type of the measurement target substance, the type of each specific substance selected according to the measurement target substance, the mode of binding between each specific substance and the measurement target substance, and steps (1) to (3). May be appropriately set according to various conditions such as the order of the reaction and the reaction time. For example, when measuring the total amount of PSA in a blood sample using an antibody as a specific substance, 20 μL of a test sample containing 2 to 30 ng / mL PSA in terms of fPSA is used by binding to a solid phase. The amount of antibody may be 4 ng to 20 μg, and the amount of antibody used by binding to the labeling substance may be 2 ng to 0.2 μg. The amount of antibody used as an inhibitory specific substance may be an amount that can achieve equimolar reactivity, and may be, for example, 0.5 ng to 1 μg. The measurement reagent of the present invention may be provided as a kit for measuring the total amount of the substance to be measured. The kit for measuring the total amount of the substance to be measured is not particularly limited as long as it contains the measurement reagent of the present invention.

  Hereinafter, a mode in which PSA in a blood sample is a measurement target substance and the total amount of fPSA and PSA-ACT (total PSA) is measured (immunoassay) using an antibody as a specific substance will be described. As the antibody, an antibody that can specifically bind to both forms of fPSA and PSA-ACT is used. That is, the first antibody that recognizes the PSA moiety that is bound to the water-insoluble solid phase and is not covered by the ACT of PSA-ACT is used as the solid phase specific substance. A second antibody that recognizes the PSA moiety that is not covered by ACT is used. The first antibody and the second antibody do not interfere with each other in binding to PSA. A third antibody that competes with the first antibody is used as an inhibitory specific substance. The third antibody is preferably an antibody that binds to fPSA and PSA-ACT at the same position as the first antibody, and more preferably the same antibody as the first antibody. In the following description, the case where the same antibody as the first antibody is used as the inhibition specific substance will be exemplified. As a combination of the first antibody and the second antibody, a combination of a polyclonal antibody and a polyclonal antibody, a combination of a polyclonal antibody and a monoclonal antibody, and a combination of a monoclonal antibody and a monoclonal antibody can be used. Among these, a combination of a monoclonal antibody and a monoclonal antibody is particularly preferable because once the hybridoma is established, the preparation of the antibody is easy and the reactivity is uniform.

In the so-called conventional sandwich method in which fPSA or PSA-ACT is measured using a solid phase specific substance (solid phase antibody) and a label specific substance (labeled antibody), the reaction is performed as shown in FIGS. Since the reactivity between fPSA and each antibody and the reactivity between PSA-ACT and each antibody are kinetically different, fPSA and PSA-ACT cannot be measured equivalently in a short reaction. Most of the reaction between the labeled antibody and fPSA or PSA-ACT is a liquid phase-liquid phase reaction (partly a reaction with fPSA or PSA-ACT bound to a solid phase antibody), and generally labeled antibody Is excessively used with respect to the total amount of PSA, the difference in the reactivity of fPSA and PSA-ACT to the labeled antibody is slight, and the influence on the measurement result is considered to be relatively small. However, the reaction between the solid phase antibody and fPSA or PSA-ACT is carried out by solid phase-
Since it is a liquid phase reaction, the reaction is slow because it undergoes a process such as diffusion of the membrane, and the reactivity is significantly different between fPSA and PSA-ACT. This is because, for example, fine particles having a particle size of the order of μm are used as the solid phase, and the diffusion distance between the solid phase surface of fPSA and PSA-ACT is shortened by high density and uniform dispersion, thereby increasing the reactivity. Even so, it is difficult to completely eliminate the difference in reactivity between the solid phase antibody and fPSA or PSA-ACT. Therefore, conventionally, it has been extremely difficult to equalize the reactivity of fPSA and PSA-ACT with respect to a solid phase antibody without performing the reaction for a sufficient time until reaching the reaction equilibrium.

  According to the present invention, even if the reaction rate between fPSA and the solid phase antibody is different from the reaction rate between PSA-ACT and the solid phase antibody, the measured value (signal intensity) equivalent to achieving equimolar reactivity. Can be obtained. That is, the reactivity of fPSA with a solid-phase antibody is higher than that of PSA-ACT with a solid-phase antibody, but according to the present invention, the effect of offsetting the difference is an inhibitory specific substance (inhibitory antibody (added antibody). In other words, the apparent equimolar reactivity (pseudo equimolar reactivity) can be established. In the present invention, apparent equimolar reactivity is established by excluding a part of fPSA from the reaction with the solid phase antibody at a ratio reflecting the difference in reactivity with PSA-ACT. Specifically, the inhibitory antibody is allowed to coexist in a free form, and is bound to more fPSA than PSA-ACT in a manner that reflects the difference in reactivity between the solid phase antibody of fPSA and PSA-ACT. The bound fPSA and PSA-ACT can no longer bind to the solid phase antibody, and equimolar reactivity of fPSA and PSA-ACT is established. FIG. 4 schematically shows the relationship between the measurement value (signal intensity) and the inhibitory antibody concentration coexisting when such a reaction occurs.

  In the reaction vessel, a solid phase antibody composed of a solid phase and an antibody against PSA, a labeled antibody composed of a labeling substance and an antibody against PSA, and an inhibitory antibody (the same antibody as that constituting the solid phase antibody, The antibody is in a free form without being bound to a solid phase, and a certain amount of sample containing fPSA and PSA-ACT is added thereto. The inhibitory antibody inhibits the binding between the solid phase antibody and fPSA and the solid phase antibody and PSA-ACT. Therefore, when a sample is added and an immune reaction is started, fPSA and PSA-ACT are detected between the solid phase antibody and the inhibitory antibody. A scramble arises. At this time, the inhibitory antibody also shows a higher reactivity to fPSA than to PSA-ACT. As a result, while fPSA antagonized the reactivity superior to PSA-ACT to solid-phase antibody and the reactivity superior to PSA-ACT to inhibitory antibody, on solid phase (solid-phase antibody)-( In forming an immune complex called fPSA or PSA-ACT)-(labeled antibody), equimolar reactivity can be established between fPSA and PSA-ACT.

  Thus, in order to establish an apparent equimolar reactivity by coexisting an inhibitory specific substance, the amount (concentration) of the inhibitory antibody to be coexisted, the measured value (signal intensity) of total PSA is the amount of fPSA In other words, the same measurement result can be obtained if the total PSA amount does not change regardless of the abundance ratio of fPSA and PSA-ACT present in the sample. adjust. For this purpose, when measuring only fPSA using various concentrations of inhibitory antibody, a curve showing the relationship between the inhibitory antibody concentration and the measured value (signal intensity), and when measuring only PSA-ACT Both of the same curves in FIG. 1 intersect at one point, that is, the same measured value is obtained when the same concentration of inhibitory specific substance (antibody) coexists with the same mole number of fPSA and PSA-ACT. It is sufficient that one exists, and an inhibitory specific substance (antibody) at the concentration is used.

In the above, the solid phase antibody, the labeled antibody, and the inhibitory antibody are all put into the reaction vessel and the reaction with PSA is started. As described above, the inhibitory specific substance (inhibitory antibody) and the measurement target are exemplified. In order to ensure the superiority of the liquid phase-liquid phase reaction with the substance, the substance to be measured and the inhibitory specificity are reacted before the substance to be measured is brought into contact with the solid phase specific substance (solid phase antibody) and reacted. It is preferable to make a substance (inhibitory antibody) contact and to make it react. Specifically, a sample containing fPSA and PSA-ACT can be reacted with an inhibitory antibody and a labeled antibody in contact with each other first, and after a certain period of time, the solid phase antibody is delayed and brought into contact with the sample to be reacted. . The delay time from the contact between the inhibitory antibody and the sample and the contact between the solid phase antibody and the sample depends on the reaction rate between the solid phase antibody and fPSA and PSA-ACT, and between the inhibitory antibody and fPSA and PSA-ACT. Although it may be determined by the balance of the reaction rate, etc., according to the knowledge of the present inventor, it is preferably 15 seconds to 20 minutes, more preferably 1 minute to 10 minutes.

  Hereinafter, the present invention will be described more specifically based on examples.

Example 1
10 mg of magnetic fine particles (particle size: 2.8 μm; manufactured by Dynal) are included in the first antibody (manufactured by Tosoh; E test “TOSOH” II (PSA II)) that recognizes the PSA part that is not covered by ACT of PSA-ACT. ) 0.1 mg was bound according to a conventional method to obtain a solid phase antibody. Bovine small intestine alkaline phosphatase was added to a second antibody (made by Tosoh; included in E-test “TOSOH” II (PSA II)) that binds to a PSA moiety that is not coated with ACT without competing with the first antibody for the binding site. The bound product was used as an enzyme-labeled antibody. In addition to 400 ng of this solid phase antibody and 25 ng of enzyme-labeled antibody, 0.05 mL of a solution containing various concentrations of the same first antibody as the solid phase antibody (hereinafter referred to as added antibody) was added to the cup for immune reaction. And 20 μL of 2 ng / mL or 30 ng / mL fPSA was added as an antigen (1-step sandwich method). The immune reaction is carried out at 37 ° C. for 9 minutes, followed by washing to remove components that are not bound to the antibody on the solid magnetic particles, and a dioxetane-based chemiluminescent substrate is added thereto to change the luminescence intensity. Was measured. Next, PSA-ACT was added as an antigen in an equimolar amount with fPSA instead of fPSA (2 ng / mL or 30 ng / mL in terms of fPSA amount) and measured in the same manner. The results were plotted according to FIG.

  In the range where the concentration of added antibody is low, fPSA gives a higher signal with the same molar concentration of fPSA and PSA-ACT, but the signal intensity of fPSA decreases more preferentially due to the increase of added antibody concentration. It was revealed that equimolar reactivity can be achieved by approaching the signal intensity. However, a relatively high concentration of added antibody is required in order to completely match the signal intensities of the two. This is because, under these conditions, the solid-liquid reaction in the magnetic fine particles is relatively fast, so that the reaction superiority of the added antibody to the solid phase antibody is relatively low.

Example 2
No labeled antibody in advance in the immune reaction cup, the first reaction between the antigen, the solid phase antibody and the added antibody for 5 minutes, the intermediate B / F (Bound / Free) wash for 1 minute, and the second reaction with the labeled antibody Was performed for 3 minutes to examine the effect of the added antibody. As an antigen, 20 μL of 10 ng / mL or 30 ng / mL fPSA was used, or PSA-ACT was added in an equimolar amount with fPSA instead of fPSA (10 ng / mL or 30 ng / mL in terms of fPSA was added) and measured. . Other conditions are the same as those in Example 1. The results are shown in FIG. In the two-step sandwich method, as in FIG. 5 of the one-step sandwich method, it is clear that by increasing the added antibody concentration, the signal strengths of fPSA and PSA-ACT at the same molar concentration approach each other and equimolar reactivity can be achieved. Became. However, as in Example 1, a relatively high concentration of added antibody is required to make the signal intensities of the two completely coincide.

Example 3
In Examples 1 and 2, equimolar reactivity could be achieved by using the added antibody, but a relatively high concentration of added antibody was required to achieve equimolar reactivity. Therefore, in order to improve this point, it was examined to start a liquid-liquid reaction between the solid-phase antibody and the antigen with the antigen before the solid-liquid reaction between the antigen and the antigen was started.

  A solid phase antibody was not previously placed in the immune reaction cup, but the reaction between the antigen, the labeled antibody and the added antibody was started, and a liquid-liquid reaction was carried out for 6 minutes. Thereafter, a solid phase antibody was added, and a solid-liquid reaction was performed for 3 minutes. As an antigen, 20 μL of 2 ng / mL, 10 ng / mL, or 30 ng / mL of fPSA, or PSA-ACT instead of fPSA is equimolar with fPSA (2 ng / mL, 10 ng / mL, or 30 ng in terms of fPSA amount) / ML was added and measured. Other conditions are the same as those in Example 1. The results are shown in FIG.

  By performing a liquid-liquid reaction prior to the solid-liquid reaction, a single concentration (approximately 0.08 μg) is obtained in each case where the concentration of the antigen is significantly different, such as 2 ng / mL, 10 ng / mL, or 30 ng / mL. Equimolar reactivity could be achieved with the addition of the added antibody. Moreover, compared with the case of Example 1 and 2, the significant reduction of the addition antibody concentration required for achieving equimolar reactivity was implement | achieved.

  In such a case, the factors that determine the equimolar reaction point are the delay time from the start of the liquid-liquid reaction to the addition of the solid phase antibody and the amount of added antibody. In the case of using a general clinical laboratory automation device, the delay time is almost always fixed at a certain time in consideration of performance factors of other measurement items. Therefore, usually, reactivity can be adjusted by adjusting the amount of added antibody.

  Next, a measurement for directly confirming whether equimolar reactivity was achieved by the present invention was performed. Prepare a mixed sample in which the ratio of fPSA and PSA-ACT is changed so that the total PSA is a constant amount (15 ng / mL), add the added antibody at a concentration of 0.08 μg / mL, and other conditions The measurement results were obtained in the same manner as in 3. FIG. 8 plots the measured values (average of triplicate measurements ± standard deviation) at each fPSA ratio as a relative value to the signal intensity when the PSA-ACT ratio is 100% (fPSA ratio is 0%). The relative values of signal intensity at each fPSA ratio ranged from 94% to 103% on average. From the above, according to the present invention, it was confirmed that equimolar reactivity can be achieved regardless of the ratio between the free form of PSA and the complex form. That is, for example, the ratio between the free form of PSA and the complex form may differ depending on the patient specimen, but according to the present invention, it is confirmed that the total PSA can be measured regardless of the ratio of the free form and the complex form. It was done.

  According to the present invention, equimolar reactivity can be achieved in an immunoassay using a water-insoluble carrier for a substance to be measured that can be present in a sample in two or more types of forms such as PSA. Therefore, according to the present invention, it is possible to quickly and easily measure the total amount of the measurement target substance that can exist in the sample in two or more types of forms. That is, the present invention is useful for measuring PSA and the like.

Claims (7)

A method for measuring the total amount of a substance to be measured that may be present in a sample in two or more types of forms,
Said two or more types of forms are (A) one or more types of complex forms bound to free and other substances, or (B) two or more forms bound to other substances. It is a form of the above kind of complex,
(A) A substance that specifically binds to all forms of the substance to be measured (label specific substance) bound by a detectable labeling substance (a) All forms and specifics of the substance to be measured bound to a water-insoluble carrier And (c) a substance that inhibits the binding between the whole form of the substance to be measured and the solid phase specific substance (inhibition specific substance), and the label specific substance and the solid phase The specific substance is capable of simultaneously binding to the measurement target substance at different sites, and includes the following steps (1) to (5), and measures the total amount of the measurement target substance: Method:
(1) A step of bringing a labeling specific substance into contact with a measurement target substance in a sample,
(2) before the step (1), simultaneously with the step (1), or after the step (1), bringing the inhibitory specific substance into contact with the measurement target substance in the sample;
(3) before the step (2), simultaneously with the step (2), or after the step (2), a step of bringing the solid phase specific substance into contact with the measurement target substance in the sample;
(4) After the step (3), separating the component that is not bound to the solid phase specific substance and the bound component;
(5) A step of detecting a label contained in either the component not bound to the solid phase specific substance or the bound component after the step (4).   The method according to claim 1, wherein the other substance is bound to one molecule per molecule of the complex.   The method according to claim 1, wherein the inhibition specific substance and the solid phase specific substance are substances that bind to the same site of the measurement target substance.   The method according to any one of claims 1 to 3, wherein the measurement target substance is a protein, and the label specific substance, the solid phase specific substance, and the inhibition specific substance are antibodies that bind to the measurement target substance.   The method according to any one of claims 1 to 4, wherein the step (3) is performed after the step (1) and the step (2). The substance to be measured is prostate specific antigen,
Two or more types of forms are free prostate specific antigen and a complex of prostate specific antigen and α1-antichymotrypsin;
The label specific substance is a first anti-prostate specific antigen antibody to which the label substance is bound,
A solid phase specific substance is a second anti-prostate specific antigen antibody having a different antigen binding site from the first anti-prostate specific antigen antibody bound to the solid phase;
The method according to any one of claims 1 to 5, wherein the inhibitory specific substance is an antibody that competes with the second anti-prostate specific antigen antibody.   7. The method of claim 6, wherein the inhibitory specific substance is the same antibody as the second anti-prostate specific antigen antibody.