JP2607102B2 - Assay method for substances with specific binding sites using a homogeneous system - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method in which two types of enzyme-labeled substances that catalyze a continuous series of reactions are brought close to each other via a test substance, and the enzymatic reaction in a sample is performed by a continuous enzyme reaction. The present invention relates to a homogeneous measurement method for determining the amount of a test substance.

[Conventional technology]

A method for simply measuring a trace component in a sample has been more strongly desired than before. Currently, methods for measuring trace components of serum include a method using an isotope as a labeling substance and a method using an enzyme as a labeling substance. The method using an isotope requires expensive reagents and special equipment, and furthermore, the effect of the isotope on the human body cannot be ignored and cannot be said to be a simple method. Enzyme immunoassay using an enzyme as a labeling substance does not have the above-mentioned drawbacks, but has operational problems, and particularly, the complicated steps of repeating washing increase the burden on the operator, and also require the measurement results to be obtained. This method requires 1 to 24 hours, and requires a dedicated and expensive apparatus to process a large amount of specimen. An improvement in the enzyme immunoassay was reported by Ullman et al. In 1976 as a homogeneous enzyme immunoassay that did not require washing. In this method, two kinds of enzyme-labeled substances that catalyze a continuous series of reactions are brought close to each other via a test substance, and the amount of the test substance is determined by a continuous enzyme reaction. For example,
Antigen is measured using both a carrier in which a specific antigen and glucose-6-phosphate dehydrogenase are chemically bound to cellulose beads and an enzyme-labeled antibody in which an antibody against the antigen and hexokinase are chemically bound. Method (Japanese
54-139794). That is, when the antigen is not contained in the reagent in which both are present, hexokinase and glucose-6-phosphate dehydrogenase come close to each other via the antigen-antibody reaction, and the degradation of glucose in the presence of the coenzyme is remarkable. NADP → NADPH is promoted. Since this change occurs in inverse proportion to the amount of antigen, NADPH can be quantified to determine the antigen concentration.

[Problems to be solved by the invention]

The present invention is based on such a technical background, and the measurement using a highly sensitive enzyme-labeled substance produced by a novel production method replacing the enzyme-labeled substance produced by the enzyme labeling method conventionally performed. A substance having a specific binding site in a homogeneous system that provides highly sensitive determination of the amount of a test substance in a sample by aggregating one or both of the two types of enzyme-labeled substances with the same type. It is intended to provide a measuring method of the above.

[Means for solving the problem]

The present inventors have conducted various studies in order to achieve the above object, and as a result, two types of enzyme labeling substances that catalyze a continuous series of reactions have been tested via a test substance having two or more specific binding sites. In a measuring method for determining the amount of a test substance by a continuous enzymatic reaction in close proximity, an insoluble carrier in which two different enzymes are separately used as the enzyme labeling substance and bound together with a specific binding partner corresponding to the test substance is used. In addition to finding a measurement method, in the measurement method, at or after the reaction of the test substance with the corresponding specific binding partner, two different enzymes are separately separated from the insoluble carrier together with the specific binding partner corresponding to the test substance. Or two different enzymes each separately bound to a specific binding partner corresponding to a test substance, or 1
Two types of enzymes consisting of an enzyme-labeled substance in which one type of enzyme is bound to an insoluble carrier together with a specific binding partner corresponding to the test substance, and one in which another different type of enzyme is bound to the specific binding partner corresponding to the test substance By aggregating one or both of the labeling substances of the same kind with each other, a method for measuring a substance having a specific binding site in a homogeneous system to obtain higher sensitivity has been found, and the present invention has been completed.

That is, according to the present invention, the two types of enzyme-labeled substances bind via a test substance having two or more specific binding sites,
Therefore, the first enzyme-labeled substance and the second enzyme-labeled substance come close to each other. Here, by adding the substrate of the first enzyme,
The reaction between the first enzyme and the second enzyme proceeds continuously, and a product of the second enzyme depending on the amount of the test substance is obtained as a signal. At this time, if the test substance does not exist, the reaction of the first enzyme and the reaction of the second enzyme do not proceed continuously, and a large signal cannot be obtained. The combination of the first enzyme and the second enzyme that can be used here requires the following conditions. That is, a substrate is added to the first enzyme, and the product obtained therefrom becomes a substrate for the second enzyme, and the product of the second enzyme gives a detectable signal. Such a combination of the first enzyme and the second enzyme is, for example, the first enzyme.
Those shown in the table can be used.

In a measurement method in which two types of enzyme-labeled substances that catalyze a continuous series of reactions are brought close to each other through a test substance having two or more specific binding sites to determine the amount of the test substance by a continuous enzymatic reaction, When the test substance is present, the two types of enzyme-labeled substances bind to the test substance specifically and in a concentration-dependent manner. That is, a proximity state of two types of enzyme-labeled substances is formed via the test substance depending on the amount of the test substance. Since the amount of signal obtained by adding the substrate depends on the amount of the nearby enzyme, the test substance can be quantified. At this time, a more clear signal can be obtained by removing the (primary) product generated from the first enzyme-labeled substance that does not participate in the reaction. For example, when an enzyme that produces hydrogen peroxide is used as the first enzyme labeling substance, the third substance shown in Table 2 may be added as a removing method.

Table 2 Catalase Cytochrome C oxidase Ascorbic acid Ascorbic acid derivative Dartathione cysteine N-acetylcysteine dithiothreitol However, the signal amount obtained by this measurement method is relatively small, and in many cases, satisfactory measurement sensitivity cannot be obtained. High sensitivity of the system was a major issue. Therefore, the present inventors believe that increasing the amount of an enzyme that forms a proximity state in response to one molecule of a test substance in response to the above-described measurement principle is effective for signal amount amplification, and hypothesized this hypothesis. The following method was tried based on it.

First, instead of the conventional enzyme-labeled substance consisting of two kinds of enzyme-labeled antibodies, two different enzymes are separately bound as enzyme labeling substances to an insoluble carrier together with a specific binding partner corresponding to the test substance. This is a method using two kinds of enzyme labeling substances.

Since a large number of enzymes are bound on one carrier, once a reaction between the test substance and the corresponding specific binding partner occurs, many enzymes are involved in this, and therefore,
It is expected that the amount of nearby enzymes formed by a certain amount of the test substance will increase.

In the second method, one or both of the first enzyme-labeled substance and the second enzyme-labeled substance are used during the reaction of the test substance with the corresponding specific binding partner or after the reaction. This is a method of aggregation. As a result, the enzyme (labeled substance) that could not participate in the reaction with the test substance in the conventional method also participates in this, that is, the amount of the enzyme approaching via the test substance is secondarily amplified. Thus, measurement with higher sensitivity becomes possible.

The method of aggregating two different enzyme-labeled substances into the same kind is performed by using an anti-enzyme antibody against each enzyme, an anti-protein antibody against the protein to be used, or an anti-specific binding partner antibody against the specific binding partner corresponding to the test substance. It is carried out by using any one of the coagulants or two or more thereof.

For example, the first enzyme-labeled substance is glucose oxidase (hereinafter referred to as GOD), the protein used is bovine serum albumin, and the specific binding partner corresponding to the test substance is obtained from a mouse. The second
Enzyme labeling enzyme is peroxidase (hereinafter POD)
Assuming that the protein used is casein and that the specific binding partner corresponding to the test substance is that obtained from a rabbit, the first enzyme-labeled substance is an anti-GOD antibody, Aggregation can be achieved by using any one of anti-bovine serum albumin antibody or anti-mouse specific binding partner antibody, or two or more thereof, and the second enzyme labeling substance is an anti-POD antibody, anti-casein antibody or anti-rabbit specific binding partner antibody Aggregation can be achieved by using any one of them or two or more of them.

Alternatively, one enzyme-labeled substance can be aggregated by immobilizing biotin directly or indirectly on one insoluble carrier and adding avidin.

The sensitivity can be further increased by adding the substances shown in Table 2 in order to remove products generated from the first enzyme-labeled substance not related to aggregation.

Furthermore, forming one or both of the first enzyme-labeled substance and the second enzyme-labeled substance into a nodular form by specifically aggregating the same kind with each other is insoluble. Regardless of whether the substance is a label consisting of an enzyme immobilized on a carrier, the effect is not related at all. Therefore, in forming one or more of the first enzyme-labeled substance and the second enzyme-labeled substance of the present invention in the form of a nodule by specifically aggregating the same kind with each other, 2
The two types of enzyme-labeled substances include: two different types of enzymes, each separately, and an enzyme-labeled substance bound to an insoluble carrier together with a specific binding partner corresponding to the test substance. Enzyme-labeled substance bound to the specific binding partner corresponding to the test substance: One enzyme is tested together with the specific binding partner corresponding to the test substance, an enzyme-labeled substance bound to an insoluble carrier, and another different enzyme is tested. The objective can be achieved with any of the enzyme-labeled substances bound to the specific binding partner corresponding to the substance.

In addition, the method of aggregating the enzyme-labeled substance is not limited to the above-described method, and any method can be used regardless of a chemical or physical method as long as the same kind of enzyme-labeled substance can be aggregated.

As described above, according to the present invention, two kinds of enzyme-labeled substances that catalyze a continuous series of reactions are brought close to each other via a test substance having two or more specific binding sites, and the amount of the test substance is determined by a continuous enzyme reaction. In this case, first, the test substance sample, the first enzyme-labeled substance, and the second enzyme-labeled substance are mixed at 37 ° C. to cause primary aggregation, and then or simultaneously, one of the two different enzyme-labeled substances is used. On the other hand, a coagulant for coagulating one or both of the same types is added. Thereafter, substrates for giving a quantitative signal are added, and the mixture is incubated for a certain period of time, and the absorbance is measured. This can be applied to a previously prepared calibration curve to determine the amount of the test substance.

As the insoluble carrier used in the present invention, for example, polystyrene, styrene-butadiene copolymer, styrene-divinylbenzene, polyvinyl toluene, polyvinyl chloride, polyethylene, polypropylene or a latex partially having a carboxyl group, a hydroxyl group or an amino group or the like in them Known compounds such as synthetic polymer compounds such as particles, porous glass, silica gel, alumina, and activated carbon can be used.
An enzyme-labeled substance bound to an enzyme using these carriers together with a specific binding partner corresponding to the test substance is obtained as follows. For example, after a specific binding partner corresponding to a test substance is chemically bound to an enzyme, this is bound to an insoluble carrier. At that time, if necessary, albumin, casein,
Treat with a suitable blocking agent such as skim milk and gelatin. This method is described in Japanese Patent Application No.
It is described in detail in 62-123907. Otherwise,
The insoluble carrier and the specific binding partner corresponding to the test substance are bound by a known covalent bonding method or physical adsorption method, and then mixed with the enzyme. Of course, the enzyme, the specific binding partner, and the insoluble carrier may be bound to each other in any order or may be bound simultaneously. If the enzyme has a hydrophobic moiety such as an alkyl chain or a phenyl group on the molecular surface, it can be directly used for binding. Bonding is performed after the introduction of a sex substituent. This method is described in detail in Japanese Patent Application No. 61-103830 filed earlier by the present inventors.

Examples of the test substance in the present invention include an antigen and an antibody in a sample, or a substance having two or more specific binding sites. For example, IgG, IgM, IgA, Ig as antigens
Immunoglobulin E etc., alpha-FP, CRP, CEA, transferrin, proteins such as beta _{2-microglobulin,} HBs antigen, viral antigens such as rubella antigen, antibody, anti-HBs antibody,
Anti-viral antibodies such as anti-HBc antibody, anti-HBe antibody, anti-HTL antibody, anti-HIV antibody, anti-rubella antibody, anti-mycoplasma antibody, and other substances having two or more specific binding sites include concanavalin A and PHA. Examples include immunoglobulin-binding cell proteins such as lectin and protein A.

In the present invention, when the test substance is an antigen, when the test substance is an antigen, the corresponding antibody is enzyme-labeled as a specific binding partner corresponding to the test substance, and when the test substance is a specific antibody, the corresponding antibody is used. Enzyme is labeled as a specific binding partner corresponding to the test substance, and if the test substance has two or more other specific binding sites, the partner that specifically binds to it corresponds to the test substance. Enzyme labeling as a specific binding partner. The enzyme labeling may or may not be performed via an insoluble carrier.

 Hereinafter, the present invention will be described with reference to examples.

Example 1 Preparation of peroxidase-labeled anti-HBs antibody-bound latex 5 mg of peroxidase was dissolved in 1 ml of 0.3 M sodium bicarbonate, 0.2 ml of 1% 2-dinitrofluorobenzene was added, and the mixture was stirred for 1 hour, and then 1 ml. 0.06M sodium periodate is added, and the mixture is stirred for 30 minutes. Further, 1 ml of 0.16M ethylene glycol is added, and the mixture is stirred for 1 hour. This is dialyzed overnight in a carbonate buffer (pH 9.5). Add this to the carbonate buffer (p
Anti-HBs monoclonal antibody (5mg / ml) dissolved in H9.5) 1
After reacting for 3 hours at room temperature (PBS dialysis), the mixture was subjected to gel filtration with Sephacryl S-200 and peroxidase-labeled anti-HBs.
Obtain antibodies. This peroxidase-labeled antibody (250 μg / m
l) and 1 ml of a 0.2% latex suspension having a particle size of 0.22 μm were mixed, allowed to stand at 37 ° C. for 3 hours, centrifuged at 14,000 rpm × 20 minutes, and the precipitated portion was subjected to 50 mM Tris buffer solution containing 0.5% casein ( After suspending at pH 8.0) and blocking for 1 hour, the mixture was washed with the same solution to obtain a peroxidase-labeled anti-HBs antibody-bound latex.

Example 2 Preparation of glucose oxidase-labeled anti-HBs antibody-bound latex 180 mg of glucose oxidase was dissolved in 4 ml of PBS. After leaving the mixture at 30 ° C. for 30 minutes and performing gel filtration on Sephadex G-25, a glucose oxidase active fraction is collected. To this is added 400 μm of 100 mM dithiothreitol, and after standing for 20 minutes, gel filtration is performed again to obtain SPDP glucose oxidase. Next, 1.5 ml of anti-HBs monoclonal antibody (25 mg / ml)
And 150 mM of 20 mM PDP, left for 30 minutes, and gel-filtered with Sephadex 25 to obtain SPDP anti-HBs monoclonal antibody. An equal amount of the SPDP glucose oxidase thus obtained and an SPDP anti-HBs monoclonal antibody are mixed, left overnight, and then subjected to gel filtration to perform glucose oxidase-labeled anti-HBs.
Obtain s antibody. This glucose oxidase-labeled antibody (2
5ml / ml) 1ml and 0.2% latex suspension 0.2% particle size 1
After mixing at 37 ° C. for 3 hours, the mixture was centrifuged at 14000 rpm × 20 minutes, and the precipitate was resuspended in 50 mM Tris-HCl buffer (pH 8.0) containing 0.5% casein, and blocked for 1 hour. Centrifuge again at 14000 rpm × 20 minutes, wash with the same solution,
A glucose oxidase-labeled anti-HBs antibody-bound latex is obtained.

Example 3 Measurement of HBs Antigen Each of the POD-labeled anti-HBs antibody-bound latex and the GOD-labeled anti-HBs antibody-bound latex prepared in Examples 1 and 2 was 0.01%.
100% Tris buffer (containing 0.5% casein)
After mixing 10 μm of serum containing HBs antigen and standing at 37 ° C. for 30 minutes, glucose 33 mM, 4-aminoantipyrine 0.82 mM,
2 ml of a substrate solution consisting of 2 mM TOOS and 13000 U / ml catalase was added, and the change in absorbance at 550 nm at 37 ° C. was measured. The result is shown in FIG. As is clear from this, the determination of the test substance became possible.

Example 4 Measurement of HBs Antigen Each of the POD-labeled anti-HBs antibody-bound latex and the GOD-labeled anti-HBs antibody-bound latex prepared in Examples 1 and 2 was 0.01%.
100% Tris buffer (containing 0.5% casein)
After mixing 10 μm of serum containing HBs antigen and standing at 37 ° C. for 10 minutes, 10 μl of Tris buffer containing anti-GOD antibody was added, and
After mixing at 37 ° C for 2 minutes, 2 ml of a substrate solution consisting of 33 mM glucose, 0.82 mM 4-aminoantipyrine, 2 mM TOOS, and 13000 U / ml catalase was added, and the change in absorbance at 550 nm at 37 ° C was measured. The result is shown in FIG. As is clear from this, the test substance could be quantified with high sensitivity.

Example 5 Measurement of HBs Antigen Each of the POD-labeled anti-HBs antibody-bound latex and the GOD-labeled anti-HBs antibody-bound latex prepared in Examples 1 and 2 was 0.01%.
100% Tris buffer (containing 0.5% casein)
After mixing 10 μm of serum containing HBs antigen and leaving at 37 ° C. for 10 minutes, 10 μl of Tris buffer containing anti-GOD antibody and anti-POD antibody was added, and mixed for 20 minutes at 37 ° C., glucose 33 mM, 4-aminoantipyrine 0.82 mM , TOOS2mM, catalase 13000U / ml
2 ml of a substrate solution was added, and the change in absorbance at 550 nm at 37 ° C. was measured. The result is shown in FIG. As is clear from this, the test substance could be quantified with high sensitivity.

Example 6 Preparation of POD-labeled anti-CRP antibody-bound latex A POD-labeled anti-CRP antibody-bound latex was prepared in the same manner as in Example 1 using a purified anti-CRP goat antibody.

Example 7 Preparation of GOD-labeled anti-CRP antibody-bound latex A GOD-labeled anti-CRP antibody-bound latex was prepared in the same manner as in Example 2 using a purified anti-CRP goat antibody.

Example 8 Measurement of CPR CRP was measured according to the method of Example 3 using the POD-labeled anti-CRP antibody-bound latex and the GOD-labeled anti-CRP antibody-bound latex prepared in Examples 6 and 7. The result is shown in FIG. As is clear from this, CRP could be measured with high sensitivity.

Example 9 Measurement of HBs antigen POD-labeled anti-HBs antibody and GOD-labeled anti-HB prepared in Examples 1 and 2
Mix s antibody 100μ and serum 10μ containing HBs antigen, 37 ° C
After 10 minutes, add 10 μl of Tris buffer containing anti-GOD antibody and anti-POD antibody, mix for 20 minutes at 37 ° C., add 2 ml of the substrate solution used in Example 3, and change the absorbance at 550 nm at 37 ° C. Was measured. FIG. 3 shows the results.

As is clear from FIG. 3, the determination of the test substance became possible.

Example 10 Preparation of Hexonase (HK) -Labeled Anti-HBs Antibody-Bound Latex An HK-labeled anti-HBs antibody was prepared by the SPDP method described in Example 2, and an HK-labeled anti-HBs antibody-bound latex was prepared by the method described above.

Example 11 Preparation of Glucose-6-Phosphate Dehydrogenase (G6PDH) -Labeled Anti-HBs Antibody-Binding Latex A G6PDH-labeled anti-HBs antibody was prepared by the SPDP method described in Example 2, and the G6PDH-labeled anti-HBs antibody was prepared by the method described above. A bonded latex was made.

Example 12 Measurement of HBs Antigen The HK-labeled anti-HBs antibody-bound latex and the G6PDH-labeled anti-HBs antibody-bound latex prepared in Examples 10 and 11, respectively, were used.
Tris buffer containing 100% 01% and serum containing HBs antigen 10
After mixing 0μ and leaving at 37 ° C for 10 minutes, add 10μ of Tris buffer containing anti-HK antibody and anti-G6PDH antibody, and
And 2 ml of a substrate solution consisting of 40 mM glucose, 3 mM ATP, 3 mM NAD ⁺ , 3.0 U / ml phosphofructokinase, and 1.5 U / ml isomerase were added, and the change in absorbance at 340 nm at 37 ° C was measured. The result is shown in FIG.

As is clear from FIG. 4, the determination of the test substance became possible.

Example 13 Preparation of peroxidase / HBs antigen-bound latex The activated POD prepared in Example 1 and immunoglobulin obtained from normal rabbit serum were processed in the same manner as in Example 1 to prepare peroxidase-labeled normal rabbit immunoglobulin. did. 1 ml of peroxidase-labeled normal rabbit immunoglobulin and 1 ml of purified HBs antigen were mixed, 2 ml of 0.2% latex was mixed, and shaking was performed for 1.5 hours.
Centrifuge for 20 minutes, then remove the sedimented part with 0.5% casein
After suspending in 0 mM Tris buffer (pH 8.0) and blocking for 1 hour, washing was performed to prepare a peroxidase / HBs antigen-bound latex in which peroxidase and HBs antigen were independently bound to latex.

Example 14 Preparation of Glucose Oxidase / HBs Antigen-Binding Latex The activated GOD prepared in Example 2 and immunoglobulin obtained from normal rabbit serum were treated in the same manner as in Example 2.
GOD-labeled normal rabbit immunoglobulin was prepared. Further, according to Example 13, a glucose oxidase / HBs antigen-bound latex in which glucose oxidase and HBs antigen were independently bound to latex was produced.

Example 15 Measurement of Anti-HBs Antibody Mixing 100 μl of Tris buffer containing 0.01% each of POD / HBs antigen-bound latex and GOD / HBs antigen-bound latex prepared in Examples 13 and 14, and 10 μm of serum containing anti-HBs antibody After 10 minutes at 37 ° C, add 10μ of Tris buffer containing anti-GOD antibody and anti-POD antibody, mix for 20 minutes at 37 ° C,
2 ml of the substrate solution used in Example 3 was added, and the change in absorbance at 550 nm at 37 ° C. was measured. The results are shown in FIG.

As is clear from FIG. 5, the test substance was quantified with high sensitivity.

Example 16 Measurement of HBs antibody Latex bound to GOD-labeled anti-HBs antibody prepared in Example 2
100μ and POD-labeled anti-HBs antibody 100μ prepared in Example 1
(1U / ml) and 10μm serum containing HBs antigen and mix for 10 minutes
After leaving at 37 ° C, add anti-GOD antibody and anti-POD antibody 100μ,
After standing again for 20 minutes, the substrate solution used in Example 3 was added and 37
The change in absorbance at 550 nm was measured at 550C. The result is the sixth
Shown in the figure. As is clear from the results, the test substance could be measured with high sensitivity.

[Brief description of the drawings]

FIG. 1 shows the third embodiment in the third, fourth and fifth embodiments of the present invention.
FIG. 4, FIG. 4 and FIG. 6 show Embodiments 9 and 12 of the present invention, respectively.
FIG. 2 shows the relationship between the HBs antigen concentration and the absorbance measured in Examples 1 and 2, and FIG. 2 shows the results measured in Example 8 of the present invention.
FIG. 5 shows the relationship between the CRP concentration and the absorbance, and FIG. 5 shows a calibration curve showing the relationship between the anti-HBs antibody concentration and the absorbance measured in Example 15 of the present invention. The symbols in FIG. 1 have the following meanings.

Claims (4)

(57) [Claims] A method for determining the amount of a test substance by a continuous enzymatic reaction by bringing two kinds of enzyme-labeled substances, which catalyze a continuous series of reactions, into close proximity via a test substance having two or more specific binding sites. Wherein two different enzymes are separately used as the enzyme labeling substances, and two enzyme labeling substances bound to an insoluble carrier together with a specific binding partner corresponding to the test substance are used. Of a substance having a specific binding site. 2. A method for determining the amount of a test substance by a continuous enzymatic reaction by bringing two types of enzyme-labeled substances, which catalyze a continuous series of reactions, into close proximity via a test substance having two or more specific binding sites. In the above, two kinds of different enzymes are separately used as the enzyme labeling substance, and an enzyme labeling substance bound to an insoluble carrier together with a specific binding partner corresponding to the test substance is used, and the test substance and the specific binding corresponding thereto are used. A method for measuring a substance having a specific binding site in a homogeneous system, wherein one or both of the two types of enzyme-labeled substances are agglutinated with each other during or after the reaction with a partner. 3. A method for determining the amount of a test substance by bringing two enzyme-labeled substances catalyzing a continuous series of reactions into close proximity via a test substance having two or more specific binding sites, and performing a continuous enzymatic reaction. In the above, two kinds of different enzymes are separately used as the enzyme labeling substance, an enzyme labeling substance bound to a specific binding partner corresponding to the test substance is used, and the test substance and a specific binding partner corresponding thereto are used. A method for measuring a substance having a specific binding site in a homogeneous system, wherein one or both of the two enzyme labeling substances are agglutinated with each other during or after the reaction. 4. A measuring method for determining the amount of a test substance by a continuous enzymatic reaction by bringing two types of enzyme-labeled substances catalyzing a continuous series of reactions into close proximity via a test substance having two or more specific binding sites. In the above, an enzyme-labeled substance in which one kind of enzyme is bound to an insoluble carrier together with a specific binding partner corresponding to the test substance, and another different enzyme is a specific binding partner corresponding to the test substance. And using the enzyme-labeled substance bound to the test substance and, during or after the reaction of the test substance with the corresponding specific binding partner, aggregating one or both of the two types of enzyme-labeled substances with each other. A method for measuring a substance having a specific binding site by using a homogeneous system.